Edible protein filaments, edible food products and methods of making same

Abstract

The invention relates to a method for producing edible protein filaments, said method comprising the steps of:-preparing a first composition (130) comprising:-animal protein from non-human cultured animal cells, and-a multivalent ion or polyelectrolyte; -preparing a second composition (140),-said second composition comprising said multivalent ions when the step of preparing said first composition (130) comprises the addition of said polyelectrolyte, or-said second composition comprising said polyelectrolyte when the step of preparing said first composition (130) comprises the addition of said multivalent ions; and-contacting (160) said first composition with said second composition to form an edible protein filament, said first composition being at a temperature of at most 65 DEG C.

Description

Technical Field

[0001] The present invention relates to the field of food products. In particular, the present invention relates to the field of meat substitutes. In particular, the present invention relates to meat based on cultured cells. In particular, the present invention can provide new edible protein filaments, which allow the production of edible products with a meat-like texture. Such edible products with a meat-like texture can be considered as substitutes for traditional meat. Background Art

[0002] From 2020 to 2050, the world population is estimated to increase by 2 billion people (United Nations, 2015). Humanity will face major challenges, among which is food production, including meat production. Traditional meat production is a resource-intensive process that generates a huge environmental footprint. Domestic animals are raised in agricultural environments that require large amounts of fresh water, feed, land and other resources (Mark J Post: “Cultured meat from stem cells: Challenges and prospects” Meatscience, Elsevier Science, GB, vol 92, no 3, 3 April 2012, pages 297-301). As a result, food production has been identified as being responsible for approximately 26% of global greenhouse gas (GHG) emissions, with livestock and fisheries accounting for 31%. Reductions in traditional meat consumption worldwide could lead to significant reductions in greenhouse gas emissions associated with climate change (Martin and Brandão, 2017. Evaluating the environmental consequences of Swedish food consumption and dietary choices. Sustainability, 9(12), 2227), especially if reductions were made in countries where meat consumption is high or rising (Stoll-Kleemann and Schmidt, 2017, Reducing meat consumption in developed and transition countries to counter climatechange 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 meatsubstitutes among Swedish consumers; Appetite 167 (2021) 105643.). In addition, animal welfare is receiving increasing attention. For example, the European Union has implemented various laws on animal welfare since 1986.Various directives set out rules for the protection of laying hens (in 1986 and 1988), calves and pigs (in 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 technologies). In particular, cell technologies are developing rapidly in response to new consumer demands.

[0004] Crucial to increasing people's willingness to consume meat alternatives is that, as a substitute, the product should mimic the aesthetic and sensory qualities of meat, such as size, appearance, flavor, mouthfeel, 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 texturization techniques to improve the sensory and taste of these products are constantly improving, meat analogs are still different from traditional meat in terms of mouthfeel and flavor (Samard and Ryu, 2019; A comparison of physicochemical characteristics, texture, and structure of meat analogue and meats. Journal of the Science of Food and Agriculture, 99(6), 2708–2715). Additionally, consumers have reported being unfamiliar with how to prepare meals using meat substitutes, making it more difficult and time-consuming to prepare satisfying, tasty meals using such products than 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 to be one of the most important qualities of meat analogs (Sha and 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 analogs. For example, it has been proposed to combine oat and pea proteins as a practical 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). It has also been proposed to use biosurfactant-based emulsions for the production of 3D printed foods (Shahbazi et al. 2021. Construction of 3D printed reduced-fat meat analogue 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, it is suggested to replace oil with biopolymer surfactants to produce fibrous, 3D printed low-fat meat analogs, in which the printed reduced-fat constructs present the desired sensory profile. However, for many meat analogs, fats rich in satisfactory flavors are still lacking in the products. The combination of biopolymer surfactants and hydrocolloids cannot simulate the typical full set of sensory properties of animal-derived meat, i.e., texture, taste and aroma. It has also been proposed to produce extruded food products containing cultured animal cells (WO2022047263). However, the resulting extruded food has little resemblance to what consumers expect from meat products, especially if the desired appearance is the raw product and the flavor retained within the meat slice.

[0006] The use of scaffolds to culture myoblasts has also been proposed, and scaffolds prepared from non-mammalian biomaterials such as salmon gelatin, alginate, agarose, and glycerol have been demonstrated for use in in vitro meat formulations. However, the transition of such technologies to large-scale manufacturing presents regulatory issues and significant technical barriers.

[0007] Therefore, there is an important need to find satisfactory alternatives to animal slaughter and intensive meat production methods in order to produce edible food products with a pleasant flavor, texture and cooking behavior that corresponds to what is expected from meat. In particular, there is a need for edible protein strands that can be used in meat analog production to simulate the texture of conventional meat. The texture expected from animal meat before and after refined cooking is desirable so that consumers can cook the product in the same manner as conventional meat and experience the same taste quality. Summary of the invention

[0008] For the purpose of providing a basic understanding of the present invention, a simplified overview of selected aspects, embodiments and examples of the present invention is set forth below. However, this overview does not constitute a broad overview of all aspects, embodiments and examples of the present invention. Its sole purpose is to present selected aspects, embodiments and examples of the present invention in a concise form, as an introduction to a more detailed description of aspects, embodiments and examples of the present invention after this overview.

[0009] The present invention aims to overcome the shortcomings of the prior art. In particular, the present invention provides a method for producing edible protein filaments, the method comprising the following steps:

[0010] - preparing a first composition, the first composition comprising:

[0011] o Animal protein derived from cultured non-human animal cells, and

[0012] oMultivalent ions or polyelectrolytes;

[0013] -Preparing the second composition

[0014] o when the first composition preparation step comprises adding a polyelectrolyte, the second composition comprises multivalent ions, or

[0015] o when the first composition preparation step comprises adding multivalent ions, the second composition comprises a polyelectrolyte; and

[0016] - contacting a first composition with a second composition to form edible protein filaments, the first composition being at a temperature below 65°C.

[0017] Such edible protein filaments have a texture similar to that of meat fibers (eg, when assembled in an edible product, they may have a texture similar to that of a rump steak filet).

[0018] Furthermore, unlike the high moisture extrudates commonly used to construct plant-based products, protein filaments according to the present invention can exhibit a great variety of shapes and sizes. Furthermore, the process according to the present invention can produce three-dimensional products and larger filaments containing flavors desired by consumers.

[0019] In particular, the method according to the invention allows the production of edible protein filaments and therefore edible food products with a raw aspect. With such advantages, the consumer can still cook the product in the same way as conventional meat and experience the same organoleptic qualities. The method is scalable and allows the production of filaments with a higher range of fats and proteins (compared to extrudates) and without oxidation during processing.

[0020] Therefore, the edible protein filaments according to the present invention can be considered as a building block and one of the steps to achieve the production of edible products that constitute an alternative to conventional meat products. Such edible protein filaments can be processed to simulate a wide variety of meat fibers, including but not limited to beef, scallops, crab meat, chicken breast, duck breast, tuna meat, salmon meat, and have a matching meaty or fishy taste. In fact, the presence of cultured cells or their extracts introduces a complex and desired flavor into the protein filaments, and this can be adjusted according to the type of cultured cells used.

[0021] Advantageously, the first composition comprises at least 10% total protein by weight compared to the total wet weight of the first composition. As shown in the examples, such protein concentrations in the first composition can improve the mechanical properties of the silk, such as breaking strain, breaking load or tensile stress.

[0022] In addition, the first composition advantageously comprises at least 5% of non-human animal protein relative to the total protein weight, the animal protein being derived from non-human cultured animal cells. In particular, the first composition comprises at least 0.5% by weight of animal protein from non-human cultured animal cells compared to the total wet weight of the first composition. In fact, as shown in the examples, such concentrations can improve the appearance, texture or flavor of the fracture strain, fracture load, tensile stress, Young's modulus or meat.

[0023] According to other optional features of the edible food product according to the present invention, it may optionally include one or more of the following features, alone or in combination:

[0024] - The first composition is not heated at a temperature of 60°C or above for 10 minutes or more. In fact, as shown in the examples, when the first composition is heated at a high temperature for a duration of at least 10 min, the properties of the edible protein filaments may be degraded. Preferably, the first composition is not heated at a temperature of 50°C or above for 10 minutes or more.

[0025] - Animal cells are selected from cells from the animal kingdom, preferably, the animal cells are selected from mammal cells, Aves cells, Actinopterygii cells, Malacostraca cells, and combinations thereof; for example, mammal cells may be Bovidae cells, Cervidae cells, Leporidae cells, or Suidae cells; Aves cells may be Antidae cells. or Phasianidae cells; Actinopterygian cells may be Gadidae cells, Merlucciidae cells, Pleuronectidae cells, Salmonidae cells or Scombridae cells; Mollusca cells may be Palaemonidae cells, and Mollusca cells may be Cephalopoda cells or Bivalvia cells. The above cells are particularly suitable for producing edible protein filaments according to the method of the present invention, and are therefore suitable for preparing edible food products.

[0026] - 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, fibro-adipogenic progenitors, muscle cells such as skeletal muscle cells, cardiac cells or smooth muscle cells; myoblasts, myocytes, 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. The above cells are particularly suitable for producing edible protein filaments according to the method of the present invention, and are therefore suitable for preparing edible food products. Preferably, non-human animal cells include cells selected from the following: stem cells, muscle cells, fibroblasts, adipocytes, erythrocytes, and combinations thereof.

[0027] - The animal protein includes myofibrillar protein, preferably at least 0.25% by weight compared to the total weight of the protein. In fact, as shown in the examples, when edible protein threads are made only with plant proteins that do not contain myofibrillar protein or contain a small amount of myofibrillar protein, the properties of the edible protein threads may be degraded.

[0028] -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 the protein. Preferably, the extracellular matrix non-human animal molecules are selected from the following: collagen, elastin, fibronectin, laminin, heparan sulfate, chondroitin sulfate, keratan sulfate, hyaluronic acid or a combination thereof. In fact, as shown in the examples, when the first composition comprises elastin and / or collagen, the properties of the edible protein filaments are improved. In particular, elastin and / or collagen should be added in soluble form.

[0029] - The first composition has a dry content of at least 12 wt%. Preferably, the first composition has a dry content of at least 15 wt%, more preferably a dry content of at least 20 wt%.

[0030] - The polyelectrolyte is a thermostable gel-forming polyelectrolyte. In fact, as shown in the examples, the use of a thermostable gel-forming polyelectrolyte improves the properties of the edible protein filaments. In fact, the polyelectrolyte is advantageously chosen among polyelectrolytes that are able to complex with multivalent ions to form a thermostable gel.

[0031] The polyelectrolyte is a polysaccharide. As polyelectrolyte according to the invention, polysaccharides are particularly suitable for producing edible protein threads according to the method of the invention and thus for preparing edible food products.

[0032] - polyelectrolytes are selected from the group consisting of low methoxy pectin, low methoxy pectin derivatives, high methoxy pectin, high methoxy pectin derivatives, alginate, alginate derivatives, xanthan gum, xanthan gum derivatives, chitosan, chitosan derivatives, ionic carboxymethylcellulose derivatives, ionic pullulan derivatives, ionic dextran derivatives, ionic starch derivatives, or combinations thereof. The above polysaccharides are particularly suitable for producing edible protein filaments according to the process of the present invention and are therefore suitable for preparing edible food products.

[0033] - The first composition or the second composition, preferably the first composition, further comprises: peptidase, α-galactosidase, alkaline protease, thermolysin, pepsin, trypsin, chymotrypsin, asparaginase, elastase, subtilisin, glucose oxidase, laccase, transglutaminase, pectinesterase, sortase, tyrosinase, oxidoreductase such as lysyl oxidase and peroxidase, genipin, riboflavin, monoamine oxidase or a combination thereof, preferably further comprises laccase, transglutaminase or a combination thereof. As shown in the examples, in the method of the present invention, the use of enzymes can improve the mechanical properties of the edible protein filaments.

[0034] - The first composition further comprises plant protein, algae protein and / or fungal protein. Preferably, the plant protein is selected from the following: sunflower protein, soy protein, pea protein, rape protein, mung bean protein, chickpea protein, faba bean protein, lentil protein, seaweed protein, potato protein, quinoa protein, nut protein, wheat protein, winged bean protein, bambara bean protein, dulse protein, mesquite bean protein, duckweed protein, winged bean protein, dry broad bean protein, dry cowpea protein, lupin protein, jackfruit protein, amaranth protein, millet protein, oat protein, chia protein, hemp seed protein and rice protein or a combination thereof.

[0035] - The first composition comprises at least 10% total protein by weight compared 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 from non-human cultured animal cells is necessary to obtain the desired properties of the edible protein filaments. Therefore, preferably, the first composition comprises at least 5% non-human animal protein from non-human cultured animal cells by weight compared to the total weight of protein in the first composition.

[0036] - The preparation of the first composition further comprises the addition of fat, preferably vegetable fat or fermented fat. In particular, it comprises the addition of at least 1% fat by weight compared to the total wet weight of the first composition. As shown in the examples, the addition of fat in the first composition improves the properties of the edible protein filaments.

[0037] - The preparation of the first composition includes homogenization, preferably the homogenization is carried out at least 100 rpm for a period of at least 30 seconds. Homogenization improves the properties of the edible protein filaments. In addition, when combined with a fat addition, it can be used to create an emulsion, which can improve the properties of the edible protein filaments produced.

[0038] -The preparation of the first composition includes adding disrupted non-human cultured animal cells and / or intact non-human cultured animal cells. Therefore, the preparation of the first composition may include adding disrupted non-human cultured animal cells and / or intact non-human cultured animal cells. Preferably, the preparation of the first composition includes adding non-human intact cultured animal cells.

[0039] - The first composition comprises:

[0040] o at least 10% by weight of total protein relative to the total wet weight of the first composition, including at least 5% non-human animal protein relative to the weight of total protein, and at least 0.25% by weight of myofibrillar protein relative to the total weight of protein, and

[0041] o At least 0.1 % by weight of polyelectrolyte relative to the total wet weight of the first composition.

[0042] - The first composition comprises:

[0043] o at least 10% total protein by weight relative to the total wet weight of the first composition, including at least 5% non-human animal protein relative to the weight of total protein, said animal protein being derived from non-human cultured animal cells, and

[0044] o At least 0.1 % by weight of polyelectrolyte relative to the total wet weight of the first composition.

[0045] - The first composition comprises:

[0046] o at least 10% total protein by weight relative to the total wet weight of the first composition, including at least 5% non-human animal protein relative to the weight of total protein, said animal protein being derived from non-human cultured animal cells, and

[0047] o at least 0.1% by weight of a polyelectrolyte relative to the total wet weight of the first composition, and

[0048] o At least one cross-linking molecule.

[0049] The present invention also relates to a method for manufacturing an edible food product. In particular, the method for manufacturing an edible food product comprises the step of producing edible protein filaments according to the method of the present invention. In addition, the method for manufacturing an edible food product may comprise the step of assembling one or more of the manufactured edible protein filaments into an edible food product.

[0050] The present invention also relates to edible protein filaments obtainable from the method according to the present invention. In particular, the present invention also relates to edible protein filaments obtainable from the method according to the present invention. Preferably, the edible protein filaments according to the present invention comprise animal protein and a polyelectrolyte forming a heat-resistant gel, the animal protein being a cultured non-human animal cell protein.

[0051] The present invention also relates to edible food products comprising edible protein filaments according to the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] The foregoing and other objects, features and advantages of the present invention will become more apparent from the following detailed description when taken in conjunction with the accompanying drawings, in which:

[0053] Figure 1 is a schematic diagram of a method of producing an edible food product having a meat-like texture according to an embodiment of the present invention.

[0054] Figure 2 It is a photo of edible protein shreds.

[0055] Figure 3 is a photo of an edible food product.

[0056] Several aspects of the present invention are disclosed with reference to flowchart illustrations and / or block diagrams of methods and apparatus.

[0057] In the figures, when present, flow charts and / or block diagrams illustrate the architecture, functionality, and possible implementations of devices or systems or methods according to several embodiments of the present invention.

[0058] In some implementations, the functions associated with the blocks may appear in a different order than that indicated in the drawings. For example, two blocks shown in succession may be executed substantially simultaneously, or the blocks may sometimes be executed in reverse order, depending on the functions involved. DETAILED DESCRIPTION

[0059] Following is a description of example embodiments of the invention.

[0060] As used herein, the expression "protein filaments" may relate to long, thin strands of material comprising at least 5% protein by weight, preferably at least 7.5%, more preferably at least 10% protein by weight compared to the wet weight of the filaments. Preferably, the protein filaments have 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 filaments have a diameter of at most 2 mm, more preferably at most 1 mm, even more preferably at most 0.5 mm.

[0061] As used herein, the expression "edible protein filaments" may relate to protein filaments suitable for animal consumption, and preferably protein filaments suitable for human consumption.

[0062] As used herein, the expression "edible product" or "edible food product" may relate to a product suitable for animal consumption, and preferably a product suitable for human consumption. The edible product according to the invention may be a ready-to-eat (i.e. finished) food product or an intermediate in the production chain of a finished food product. As will be described below, the edible product according to the invention may be produced in the following forms: snacks, which may be pressed, fried and / or baked; sauces; spreads; pastas; pastes; converted meat analogs or food specialty foods, such as sausages or cured sausages, pate or foie gras; meat dough; soups; smoothies; seafood; unconverted meat analogs, such as "meat-like" products.

[0063] In the following description, the term "meat" may refer to any edible part of an animal, such as animal tissue taken from slaughtered animals. Thus, meat may refer to liver or other visceral tissue, adipose tissue, muscle tissue conventionally found in animals. Dead animals may refer to all species of the animal kingdom except humans, and preferably to all edible species, such as non-human vertebrates, for example livestock, fish, birds; insects; crustaceans, for example shrimp, prawns, crabs, crayfish and / or lobsters; mollusks, for example octopus, squid, cuttlefish, scallops, snails. Thus, for example, the present invention allows the production of edible products with a meat-like texture, such as products imitating foie gras, marbled beef or salmon meat.

[0064] As used herein, the expression "by weight" generally refers to the weight of a component compared to the weight of another component or the entire composition, and may be considered wet weight or dry weight. Preferably, percentages are disclosed with reference to wet weight.

[0065] As used herein, the expressions "cultivated cells" or "cultured cells" are used interchangeably. They may refer to cells that are multiplied and / or grown, preferably in a controlled environment, using a culture medium. It refers in particular to cells whose growth is controlled by humans, for example in an industrial process, as opposed to cells from conventional meat that have been multiplied in a living organism or cells that grow in a natural environment, such as forest-grown mushrooms. For proteins, cultured cells may refer to cells belonging to the animal kingdom, but may also refer to cells of the bacteria kingdom, the green plant kingdom (Viridiplantae) and the fungi kingdom (Fungi), for example to provide additional protein or fat. Cultured cells may be derived from cells of any origin, such as cells from a biopsy, from stem cells or corresponding to the stem cells themselves. More specifically, protein filaments based on cultured cells may refer to protein filaments that are mainly composed of proteins from cultured cells. For example, the cultured cell-based protein filaments contain at least 5% by weight of protein from cultured cells, preferably at least 10% by weight of protein from cultured cells, more preferably 20% by weight of protein from cultured cells, and even more preferably 30% by weight of protein from cultured cells, compared to the total weight of the protein.

[0066] As used herein, the expression "extract of cultured cells" may refer to any fraction of ruptured cells or to any purified or partially purified biomaterial recovered from ruptured cells, such as cultured cell protein extracts. Ruptured cells may be cells with partially or completely destroyed cell walls. In protein filaments, the extract of cultured cells may include ruptured cells and / or biomaterials recovered from ruptured cells. The extract of cultured cells may be obtained, for example, by separating and purifying the biomaterials recovered from ruptured cells. Therefore, the extract may be obtained, for example, after at least a drying step, a precipitation step, or a solvent extraction step. Complete cultured animal cells may refer to cultured animal cells with intact membranes such as those that can be evaluated by microscopy.

[0067] As used herein, the term "polyelectrolyte" may refer to a macromolecule having (a large number of) charged groups covalently attached to it when dissolved in a polar solvent such as water. In general, polyelectrolytes may have various types of such groups. Homogeneous polyelectrolytes have only one type of charged group, i.e. g. only carboxylate groups.

[0068] As used herein, the expression "myofibrillar protein" may refer to actin, myosin, tropomyosin, troponin, α-actinin, connectin, titin, connexin, C protein, M protein, desmin, or a combination thereof.

[0069] As used herein, the expression "heat-resistant gel" may refer to a gel formed, for example, by a polyelectrolyte and a multivalent ion, which is not liquid at a temperature below 80°C. Preferably, it refers to a gel that is not liquid at a temperature below 100°C. As used herein, the expression "heat-resistant gel-forming polyelectrolyte" may refer to a polyelectrolyte that will form a heat-resistant gel when combined with a suitable multivalent ion. In particular, the heat-resistant gel-forming polyelectrolyte is combined with a multivalent ion-forming agent that can complex with the polyelectrolyte to form a heat-resistant complex. For example, when the second composition comprises a polyelectrolyte, the first composition comprises a multivalent ion, or when the second composition comprises a multivalent ion, the first composition comprises a polyelectrolyte.

[0070] As used herein, the expression "biodegradable polymer" may refer to a polymer that can be broken down or degraded into harmless products by biological activities of living organisms such as microorganisms.

[0071] As used herein, the expression "fermentation-obtained fat" or "fermented fat" may refer to lipid molecules produced, for example, by microbial fermentation in a growth reactor. The lipid molecules obtained by fermentation may be chemically identical to fats produced by plants or animals.

[0072] As used herein, the term "flavor" is generally a product quality that affects taste and / or aroma. Thus, "meat-like flavor" may refer to a flavor that is close to or approximates the flavor of a related conventional meat product.

[0073] As used herein, the term "texture" may be considered as "a combination of rheological and structural (geometric and surface) properties of a food product perceptible by mechanical, tactile and, where appropriate, visual and auditory receptors" as defined in 2008 by the International Organization for Standardization (ISO, 2008, Sensory Analysis-Vocabular, Vols. 1-107, p. 5492). Thus, "meat-like texture" may refer to the textural and structural (geometric and surface) properties of a food product that are close to or approximate the texture of a related conventional meat product (i.e., a meat product derived from the slaughter of an animal). An edible food product according to the present invention having a meat-like texture and a meat-like flavor may be considered an alternative to meat products.

[0074] As used herein, the term "about" can allow for a certain degree of variability in a value or range, for example, within 10%, within 5%, or within 1% of the stated value or limit of the stated range.

[0075] As used herein, the term "substantially" refers to most or almost all, 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 even more. Thus, a composition having cells that are substantially intact refers to a composition comprising at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, 99.99% of intact cells.

[0076] As mentioned above, producing tasty meat-like products without animal slaughter is a challenge both for animal welfare and the environment. In addition to animal welfare or environmental protection, it seems necessary to produce food products that correspond to consumer expectations by exhibiting qualities close to those of conventional products, in particular by producing protein strands similar to those found in meat.

[0077] A new method has been developed for producing edible protein filaments that can have a meat-like texture without including tissue from slaughtered animals.

[0078] Thus, according to a first aspect, the present invention is directed to a method 100 of producing edible protein filaments.

[0079] The manufacturing method 100 according to the present invention preferably allows for the production of edible protein filaments that may have a meat-like texture.

[0080] like Figure 1 As shown, the method includes the following steps: preparing a first composition 130, preparing a second composition 140, and contacting the first composition with the second composition 160 to form edible protein filaments.

[0081] The method for manufacturing edible protein filaments or edible food products may 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, ultrasonic treatment, 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, gas conditioning, drying technology (freeze drying, membrane), fermentation, homogenization, mincing, grinding, chopping, salting, tumbling, pickling, brine injection. In particular, the method according to the present invention may also include additional steps, such as: culturing 110 non-human animal cells, processing 120 non-human animal proteins, adding food additives 150, transforming 170 edible protein filaments and / or conditioning 180 edible food products.

[0082] like Figure 1 As shown in , the method according to the present invention may include the step of culturing 110 non-human animal cells.

[0083] In particular, this step is designed to produce an animal protein source without having to slaughter the animal.

[0084] Animal cells can be selected, for example, from all non-human cells that can be found in animals that are normally bred, hunted or fished. Thus, cultured non-human animal cells can be selected from: avian cells, bovine cells, seafood cells, porcine cells, sheep cells. Throughout the text, when animal cells are mentioned in this text, they obviously refer to non-human animal cells.

[0085] Preferably, the animal cell is selected among cells from the animal kingdom, in particular the animal cell is selected among mammalian cells, avian cells, ray-finned fish cells, malacian cells, mollusca cells and combinations thereof.

[0086] For example, and in a non-limiting manner, a mammal cell can be a Bovidae cell, a Cervidae cell, a Leporidae cell, or a Suidae cell; an Aves cell can be an Antidae cell or a Phasianidae cell; an Actinopterygian cell can be a Gadidae cell, a Merlucciidae cell, a Pleuronectidae cell, a Salmonidae cell, or a Scombridae cell; a Malacrustacean cell can be a Palaemonidae cell, and a Mollusca cell can be a Cephalopoda cell or a Bivalvia cell.

[0087] Preferably, the non-human animal cells include bovine cells, cervical cells, lagomorph cells, porcine cells, duck cells, pheasant cells, cod cells, hake cells, flatfish cells, salmonid cells, scombroid cells and / or longarm shrimp cells.

[0088] More preferably, the non-human animal cells include bovine cells, porcine cells, duck cells, pheasant cells, cod cells, hake cells, salmon cells, and scombroid cells.

[0089] Even more preferably, the non-human animal cells include bovine cells, porcine cells, duck cells, pheasant cells, scombroid cells.

[0090] Regardless of their origin, several types of cells can be used in this culturing step 110 .

[0091] For example, non-human 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 or smooth muscle cells; myoblasts, myocytes, 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.

[0092] Preferably, the non-human animal cells include cells selected from the group consisting of stem cells, muscle cells, fibroblasts, adipocytes, erythrocytes, and combinations thereof.

[0093] There are many methods for culturing cells. These methods are often laboratory methods, but there are also many methods suitable for large-scale production and human consumption. In the following, we describe some suitable methods for culturing non-human cells in the context of the present invention.

[0094] During the step of culturing 110 non-human animal cells, the cells may be cultured in suspension or adherently.

[0095] Advantageously, regardless of their origin, the cultured non-human animal cells can be considered as cells grown in a culture medium. Advantageously, the culture medium does not contain fetal bovine serum or growth factors. The culture medium can preferably be gradually supplemented with a hydrolysate, such as a plant hydrolysate or a yeast hydrolysate. Such serum-free culture media allow the need for animal-derived components to be reduced or eliminated.

[0096] Optionally, such as during cell colony expansion, various culture medium formulations are used to enable the ability to maintain self-renewal. As explained, the culture medium formulation can be improved from conventional culture medium without the need for animal replacement selection of fetal bovine serum or bovine serum, and no growth factor is required. Instead, the culture medium can include plant hydrolyzates or yeast hydrolyzates. Examples of plant-based formulations include soy-based and plant hydrolyzate-based culture medium formulations. Some culture medium formulations can further include at least one component for enhancing the nutritional content of cultured cells.

[0097] Furthermore, the culture medium contains all components and nutrients necessary for cell development, such as salts, glucose, water, salt minerals, and amino acids.

[0098] In one embodiment, the culture medium may comprise a scaffold.The cells may be cultured in an incubator set at, for example, a temperature of 37°C, 5% CO2, a pH of 7, and a humidity of at least 95%.

[0099] In addition, the step of culturing 110 non-human animal cells may include a step of differentiating the cultured cells. There are many methods for cell differentiation. These methods are often laboratory methods, but there are also many methods suitable for large-volume production and human consumption. In the following, we describe some suitable 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 texture in a specific way, more particularly related to a specific edible product.

[0100] Differentiation of non-human embryonic stem cells

[0101] The cells in the first composition can be derived from the differentiation of non-human embryonic stem cells. Differentiation includes the sum of the processes in which undifferentiated or non-specialized cells obtain their functions. Stem cells can be separated from embryos and cultured using specific culture media to achieve cell proliferation and maintain an undifferentiated state. This is particularly advantageous for reaching sufficient cell density or cell number. In one embodiment, the culture medium formulation uses a synthetic serum-free culture medium.

[0102] Next, embryonic stem cells can be induced to differentiate into, for example, hepatocytes, fibroblasts, keratinocytes, myocytes or adipocytes. Differentiation can be triggered by exposure to specific factors (such as growth factors or proteins) and / or culture conditions (such as shear stress). For example, in order to obtain hepatocytes, specific growth factors such as activin A, WNT, FGF or BMP are preferably used; or other components that have an effect on differentiation, such as insulin, transferrin, selenium, rapamycin, KOSR or sodium butyrate, non-human embryonic stem cells are induced into cells of definitive endoderm. Next, preferably with specific factors such as HGF, FGF, FGF and BMP, the cells of the final endoderm are specialized into liver endoderm cells, followed by hepatoblasts. By inducing differentiation with a combination of factors (preferably such as HGF, oncostatin M, dexamethasone and TGF-β), hepatoblasts are differentiated into hepatocytes. Next, the differentiated hepatocytes can be cultured and expanded to the desired number of cells.

[0103] Differentiation of non-human induced pluripotent stem cells.

[0104] The cells in the first composition may be derived from differentiation of non-human induced pluripotent stem cells.

[0105] Episomal reprogramming strategies, such as that of avian dermal fibroblasts isolated from geese, ducks, or chickens, can be employed to generate induced pluripotent stem cells from fibroblasts without the use of classical viral reprogramming techniques.

[0106] Optimized culture medium substrates and culture medium formulations can be used to culture induced pluripotent stem cells to achieve sustained cell proliferation and maintenance of a dedifferentiated state. The culture medium formulation preferably uses a synthetic serum-free culture medium. Preferably, the cells are cultured in a pathogen-free cell culture system. Then, pluripotent stem cells can be triggered to differentiate into specific lineages, such as hepatocytes, and expanded to a desired number of cells.

[0107] Transdifferentiated non-human isolated cells.

[0108] The cells in the first composition can be derived from transdifferentiated non-human isolated cells. Transdifferentiation refers to, preferably in one step, differentiation from a differentiated cell type to another differentiated cell type. Transdifferentiation can involve the following method: without forming a multipotent intermediate cell, modifying the differentiation phenotype or developmental potential of the cell; that is, it does not require the cell to first dedifferentiate (or reprogram), and then differentiate into another cell type. On the contrary, without experiencing a phenotype with a lower degree of differentiation, the cell type is only "converted" from one cell type to another cell type. Transdifferentiation can also include a first step in which a first cell with a first cell fate is subjected to conditions to generate a second cell (i.e., a cell with a lower degree of differentiation) that can differentiate into a second cell fate; and a second step in which a cell with a lower degree of differentiation is subjected to conditions that trigger differentiation into a cell (e.g., a hepatocyte) with a second cell fate.

[0109] 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 disclosed above and cultured in a medium comprising a basal medium, antibiotics, non-essential amino acids and a reducing agent, serum, minerals and growth factors.

[0110] Immortalized mature non-human myocytes or hepatocytes.

[0111] The cells in the first composition can be selected from immortalized mature non-human differentiated cells. Classical techniques such as transformation or spontaneous immortalization can be used to immortalize cells by sequential passage of cells until spontaneous mutations leading to immortalization. For example, in the case of hepatocytes, the immortalized mature non-human hepatocytes are characterized by the fact that the cells can divide indefinitely. Mature avian hepatocytes can be separated from duck liver, goose liver or chicken liver. Immortalized myocytes or hepatocytes can be expanded to the desired number of cells and grown in culture medium.

[0112] Differentiated cells derived from differentiation of non-human progenitor cells.

[0113] The cells in the first composition can be selected from differentiated cells derived from non-human progenitor cells. Progenitor cells can be grown using optimized culture medium substrates and culture medium formulations to obtain sustained cell proliferation and maintenance of a pluripotent state. The culture medium formulation can include a synthetic serum-free culture medium. In the case of satellite cells, due to specific and well-known differentiation factors, they are induced to differentiate into mature skeletal muscle cells and expand to the desired number of cells.

[0114] like Figure 1 As shown in , the method according to the present invention may include the step of processing 120 the animal protein.

[0115] In particular, this step is designed to prepare animal proteins, especially those produced during the cell culture step, so that they are suitable for subsequent steps.

[0116] For example, the animal proteins used in the present invention can be brought into the composition within intact cultured cells. Alternatively, they can be brought into the composition together with cultured cells that have been ruptured. In addition, the animal proteins can be extracted from cultured cells, such as ruptured cultured cells.

[0117] Thus, after the culturing step, the cultured cells may remain substantially intact, or they may have been substantially disrupted (e.g. by homogenization, extrusion, mixing, blending or melt-blowing, electrospinning, centrifugal spinning, blow-spinning).

[0118] When the cultured cells have been ruptured, the method according to the invention may comprise a step of extracting a specific compound after rupture. For example, the method according to the invention may comprise a step of extracting a protein from the cultured cells. The protein may be purified or separated into protein fractions according to specific physicochemical properties.

[0119] like Figure 1 As shown in FIG. 1 , the method 100 for producing edible protein filaments according to the present invention includes the step of preparing a first composition 130 .

[0120] In particular, this step is designed to determine the main composition of the protein filaments and their mechanical and sensory properties. Thus, such a step contributes to solving the problem solved by the present invention.

[0121] The dry content of the first composition can have a significant impact on the mechanical properties of protein filaments obtained with the first composition.

[0122] Thus, preferably, the first composition has a dry content of at least 12 wt%. Preferably, the first composition has a dry content of at least 15 wt%, more preferably a dry content of at least 20 wt%. For example, the first composition has a dry content of at most 90 wt%. Preferably, the first composition has a dry content of at most 85 wt%, more preferably at most 80 wt%.

[0123] In addition, since the composition is the basis for forming protein silk, it is preferably included in a significant amount of protein. Therefore, the first composition may include at least 10% protein by weight compared to the total wet weight of the first composition. Preferably, the first composition may include at least 15% protein by weight compared to the total wet weight of the first composition, more preferably at least 20% by weight compared to the total wet weight of the first composition, and even more preferably at least 25% protein by weight. The first composition may include up to 40% protein by weight compared to the total wet weight of the first composition. Preferably, the first composition may include up to 38% protein by weight compared to the total wet weight of the first composition, more preferably up to 35% by weight compared to the total wet weight of the first composition, and even more preferably up to 30% protein by weight. Therefore, the first composition may include 10% to 40% protein by weight compared to the total wet weight of the first composition. Preferably, the first composition may comprise 15% by weight to 38% by weight, more preferably 20% by weight to 35% by weight, even more preferably 25% by weight to 30% by weight of protein compared to the total wet weight of the first composition.

[0124] Notably, the first composition 130 comprises animal proteins that have been obtained from non-human cultured animal cells.

[0125] Animal protein

[0126] Contrary to conventional methods of producing protein silk, the animal proteins used herein are not proteins produced from slaughtered animals, but rather proteins produced from non-human animal cells grown in a cell culture facility. When animal proteins are mentioned in this application, they are obviously non-human animal proteins.

[0127] As already described, the animal protein can be brought into the first composition in the form of whole or disrupted cultured cells or also in the form of extracted protein.

[0128] Therefore, the step of preparing the first composition 130 may include adding cultured cell extracts, 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 cell culture (suspended or adherent, preferably suspended), or an extract of the cells, preferably a protein extract of the cells.

[0129] Preferably, the first composition comprises cultured non-human animal cells. The cultured non-human animal cells may include disrupted cultured cells and / or intact cultured cells.

[0130] In particular, the first composition may contain at least 1% cultured non-human animal cells by dry weight compared to the total wet weight of the first composition. Preferably, before the contacting 160 step, the first composition may contain at least 2% cultured non-human animal cells by dry weight compared to the total wet weight of the first composition, more preferably at least 4% by dry weight compared to the total wet weight of the first composition, and even more preferably at least 8% cultured non-human animal cells by dry weight.

[0131] In particular, the first composition may contain at least 30% cultured non-human animal cells by dry weight compared to the total wet weight of the first composition. Preferably, before the contacting 160 step, the first composition may contain up to 28% cultured non-human animal cells by dry weight compared to the total wet weight of the first composition, more preferably up to 25% by dry weight, even more preferably up to 22% cultured non-human animal cells by dry weight compared to the total wet weight of the first composition.

[0132] In particular, the first composition may comprise 1% to 30% of cultured non-human animal cells by dry weight compared to the total wet weight of the first composition. Preferably, the first composition may comprise 2% to 28% of cultured non-human animal cells by dry weight compared to the total wet weight of the first composition, more preferably 4% to 25% by dry weight, even more preferably 8% to 22% by dry weight of cultured non-human animal cells compared to the total wet weight of the first composition.

[0133] 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 the international standard ISO 1442:1997. When implementing the method of producing an edible product or ingredient, the origin of the ingredient is generally known. Therefore, a person skilled in the art implementing the present invention will be able to know whether he is adding a protein that is or is not an animal protein from non-human cultured animal cells. Animal protein from non-human cultured animal cells is, for example, a protein produced by non-human animal cells grown in a bioreactor or fermentation reactor.

[0134] In particular, the first composition may contain at least 1% by dry weight of a cultured non-human animal cell extract compared to the total wet weight of the first composition. Preferably, before the contacting 160 step, the first composition may contain at least 2% by dry weight of a cultured non-human animal cell extract compared to the total wet weight of the first composition, more preferably at least 4% by dry weight, even more preferably at least 8% by dry weight of cultured non-human animal cells compared to the total wet weight of the first composition.

[0135] In particular, the first composition may contain at least 30% by dry weight of the cultured non-human animal cell extract compared to the total wet weight of the first composition. Preferably, before the contacting 160 step, the first composition may contain up to 28% by dry weight of the cultured non-human animal cell extract compared to the total wet weight of the first composition, more preferably up to 25% by dry weight, even more preferably up to 22% by dry weight of the cultured non-human animal cell extract compared to the total wet weight of the first composition.

[0136] In particular, the first composition may contain 1% to 30% of the cultured non-human animal cell extract by dry weight compared to the total wet weight of the first composition. Preferably, before the contacting 160 step, the first composition may contain 2% to 28% of the cultured non-human animal cell extract by dry weight compared 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 of the cultured non-human animal cell extract.

[0137] The concentration of protein, particularly animal protein, in the first composition can affect the texture of the protein filaments.

[0138] The first composition may contain at least 0.5% by weight of animal protein from non-human cultured animal cells compared to the total wet weight of the first composition. Preferably, the first composition may contain at least 1% by weight of animal protein from non-human cultured animal cells compared to the total wet weight of the first composition, more preferably at least 2% by weight, even more preferably at least 4% by weight of animal protein from non-human cultured animal cells compared to the total wet weight of the first composition.

[0139] The first composition may contain up to 30% by weight of animal protein from non-human cultured animal cells compared to the total wet weight of the first composition. Preferably, the first composition may contain up to 28% by weight of animal protein from non-human cultured animal cells compared to the total wet weight of the first composition, more preferably up to 25% by weight, even more preferably up to 22% by weight of animal protein from non-human cultured animal cells compared to the total wet weight of the first composition.

[0140] Therefore, the first composition may contain 0.5% to 30% by weight of animal protein from non-human cultured animal cells compared to the total wet weight of the first composition. Preferably, the first composition may contain 1% to 28% by weight of animal protein from non-human cultured animal cells compared to the total wet weight of the first composition, more preferably 2% to 25% by weight, even more preferably 4% to 22% by weight of animal protein from non-human cultured animal cells compared to the total wet weight of the first composition.

[0141] The first composition may contain at least 5% by weight of animal protein from non-human cultured animal cells compared to the total weight of protein in the first composition. Preferably, the first composition may contain at least 10% by weight of animal protein from non-human cultured animal cells compared to the total weight of protein in the first composition, 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, at least 60% by weight of animal protein from non-human cultured animal cells compared to the total weight of protein in the first composition.

[0142] The first composition may contain up to 100% by weight of animal protein from non-human cultured animal cells compared to the total weight of protein in the first composition. Preferably, the first composition may contain up to 90% by weight of animal protein from non-human cultured animal cells compared to the total weight of protein in the first composition, more preferably up to 80% by weight of animal protein from non-human cultured animal cells compared to the total weight of protein in the first composition.

[0143] Therefore, the first composition may contain 5% to 100% by weight of animal protein from non-human cultured animal cells compared to the total weight of protein in the first composition. Preferably, the first composition may contain 10% to 100% by weight of animal protein from non-human cultured animal cells compared to the total weight of protein in the first composition, more preferably 20% to 100% by weight, even more preferably 30% to 100% by weight, 40% to 90% by weight, 50% to 100% by weight, 50% to 90% by weight of animal protein from non-human cultured animal cells compared to the total weight of protein in the first composition.

[0144] Preferably, the amount of animal protein can be measured using Kjeldahl titration according to international standard ISO-937:1978. When implementing the method of producing an edible product or ingredient, the origin of the ingredient is usually known. Therefore, a person skilled in the art implementing the present invention will be able to know whether he is adding a protein that is or is not an animal protein from non-human cultured animal cells.

[0145] The concentration of the protein can also be considered with respect to the concentration of the polyelectrolyte. This ratio can be controlled to optimize the behavior of the protein filaments.

[0146] Thus, the first composition may comprise animal protein (from cultured cells) and a polyelectrolyte capable of forming a heat-resistant gel at concentrations such that the ratio of the weight of animal protein to the weight of polyelectrolyte is at least 5, preferably at least 10, more preferably at least 15, and even more preferably at least 20.

[0147] However, the first composition may comprise animal protein and heat-resistant gel-forming polyelectrolyte in concentrations such that the ratio of the weight of animal protein to the weight of polyelectrolyte is at most 200, preferably at most 100, more preferably at most 80, even more preferably at most 60.

[0148] Thus, the first composition may comprise animal protein and polyelectrolyte at concentrations such that the ratio of animal protein weight to polyelectrolyte weight is 5 to 200, preferably 10 to 100, more preferably 15 to 80, even more preferably 20 to 60.

[0149] In the context of the present invention, the presence of specific molecules, especially proteins, may be most important for improving the final protein silk quality. The amount of specific molecules, especially proteins, can be measured by techniques well known to those skilled in the art such as enzyme-linked immunosorbent assay or mass spectrometry.

[0150] For example, the animal protein may include heat shock proteins.

[0151] Preferably, the animal protein comprises myofibrillar protein. For example, the myofibrillar protein may comprise actin, myosin, tropomyosin, troponin, actin, connectin, titin, connexin, C protein, M protein, desmin or a combination thereof.

[0152] Preferably, the myofibrillar protein is selected from the group consisting of actin, desmin, myosin, troponin, titin, chaperone or a combination thereof. More preferably, the myofibrillar protein is selected from the group consisting of actin, desmin, myosin, troponin or a combination thereof. Even more preferably, the myofibrillar protein is selected from the group consisting of actin, myosin or a combination thereof.

[0153] The first composition may comprise at least 0.25% by weight of myofibrillar proteins compared to the total weight of proteins, preferably at least 0.5% by weight of myofibrillar proteins compared to the total weight of proteins, more preferably at least 1% by weight of myofibrillar proteins, even more preferably at least 2% by weight of myofibrillar proteins.

[0154] The first composition may comprise up to 70% by weight of myofibrillar proteins compared to the total weight of proteins, preferably up to 60% by weight of myofibrillar proteins compared to the total weight of proteins, more preferably up to 50% by weight of myofibrillar proteins, even more preferably up to 40% by weight of myofibrillar proteins.

[0155] Thus, the first composition may comprise 0.25% to 70% by weight of myofibrillar proteins compared to the total weight of protein, preferably 0.5% to 60% by weight of myofibrillar proteins compared to the total weight of protein, more preferably 1% to 50% by weight of myofibrillar proteins, even more preferably 2% to 40% by weight of myofibrillar proteins.

[0156] The concentration of myofibrillar protein in the first composition can also be considered with respect to the concentration of polyelectrolyte. The ratio can be controlled to optimize the behavior of protein filaments. Therefore, the first composition can include myofibrillar protein (from cultured cells) and polyelectrolyte at a concentration such that the ratio of myofibrillar protein weight to polyelectrolyte is at least 2.5, preferably 5, more preferably 7.5, and even more preferably 10. However, the first composition can include myofibrillar protein and polyelectrolyte at a concentration such that the ratio of myofibrillar protein weight to polyelectrolyte weight 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 include myofibrillar protein and polyelectrolyte at a concentration such that the ratio of myofibrillar protein weight to polyelectrolyte weight 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 like polyelectrolyte.

[0157] In particular, the first composition may advantageously comprise a specific myofibrillar protein. For example, the first composition may comprise troponin, preferably at least 0.0125% by weight of troponin compared to the total weight of proteins in the first composition, preferably at least 0.025% by weight, more preferably at least 0.05% by weight, even more preferably at least 0.1% by weight of troponin compared to the total weight of proteins in the first composition.

[0158] The first composition may comprise up to 3.5% by weight of troponin compared to the total weight of protein in the first composition, preferably up to 3% by weight, more preferably up to 2.5% by weight, even more preferably up to 2% by weight of troponin compared to the total weight of protein in the first composition.

[0159] Thus, the first composition may comprise 0.0125% to 3.5% by weight of troponin compared to the total weight of protein in the first composition, 0.025% to 3% by weight compared to the total weight of protein in the first composition, more preferably 0.05% to 2.5% by weight; even more preferably 0.1% to 2% by weight of troponin.

[0160] Troponin may be produced by cultured animal cells. However, it may also be a recombinant troponin produced by cultured cells, which may be plant cells, insect cells or microbial cells.

[0161] In the context of the present invention, the presence of certain specific molecules can improve the protein filaments produced. In particular, the presence of extracellular matrix animal molecules (ECM) such as ECM polysaccharides, ECM proteoglycans or ECM proteins can improve the protein filaments produced. ECM proteins are particularly preferred. For example, the first composition can include at least 0.1% by weight of extracellular matrix animal molecules compared to the gross weight of the protein. Preferably, it includes at least 0.5% by weight of extracellular matrix animal molecules compared to the gross weight of the protein, more preferably at least 1% by weight compared to the gross weight of the protein, and even more preferably at least 1.5% by weight of extracellular matrix animal molecules. When the first composition includes several extracellular matrix animal molecules, the cumulative concentration is to assess the amount of extracellular matrix animal molecules in the first composition.

[0162] Such molecules may be added to the first composition via cultured cells known to produce such molecules, preferably cultured animal cells, such as fibroblasts.

[0163] The first composition may comprise collagen, preferably at least 1% by weight of collagen compared to the total weight of protein in the first composition, preferably at least 2% by weight, more preferably at least 3% by weight, even more preferably at least 4% by weight of collagen compared to the total weight of protein in the first composition.

[0164] The first composition may comprise up to 16% by weight of collagen compared to the total weight of protein in the first composition, preferably up to 14% by weight, more preferably up to 12%, even more preferably up to 10% by weight of troponin compared to the total weight of protein in the first composition.

[0165] Thus, the first composition may comprise 1% to 16% collagen by weight compared to the total weight of protein in the first composition, 2% to 14% by weight compared to the total weight of protein in the first composition, more preferably 3% to 12% by weight; even more preferably 4% to 10% collagen by weight.

[0166] The collagen may be collagen produced by cultured animal cells. However, it may also be recombinant collagen produced by cultured cells, which may be plant cells, insect cells or microbial cells.

[0167] The first composition may comprise elastin and / or tropoelastin, preferably at least 0.065% by weight of elastin and / or tropoelastin compared to the total weight of protein in the first composition, preferably at least 0.13% by weight, more preferably at least 0.2% by weight, even more preferably at least 0.25% by weight of elastin and / or tropoelastin compared to the total weight of protein in the first composition.

[0168] The first composition may comprise up to 7.5% by weight of elastin and / or tropoelastin compared to the total weight of proteins in the first composition, preferably up to 7% by weight, more preferably up to 6.5% by weight, even more preferably up to 6% by weight of troponin compared to the total weight of proteins in the first composition.

[0169] Thus, the first composition may comprise from 0.065% to 7.5% by weight of elastin and / or tropoelastin compared to the total weight of protein in the first composition, from 0.13% to 7% by weight compared to the total weight of protein in the first composition, more preferably from 0.2% to 6.5% by weight; even more preferably from 0.25% to 6% by weight of elastin and / or tropoelastin.

[0170] Preferably, the elastin and / or tropoelastin are produced by cultured animal cells. However, they may also be recombinant elastin and / or tropoelastin produced by cultured cells, which may be plant cells, insect cells or microbial cells, preferably in soluble form.

[0171] As already described, the first composition may comprise animal proteins from non-human cultured animal cells. However, the first composition may comprise proteins from other origins.

[0172] In particular, the step of preparing the first composition 130 may include adding non-animal proteins, such as plant proteins, microbial proteins, algae proteins and / or fungal proteins. As mentioned in the embodiments, adding some non-animal proteins can be used to adjust the viscoelastic properties or flow properties of the first composition containing non-human animal cultured cells.

[0173] In a specific embodiment, the first composition further comprises plant protein, preferably, the plant protein can be selected from the following items: sunflower protein, soy protein, pea protein, rape protein, mung bean protein, chickpea protein, fava bean protein, lentil protein, algae protein, potato protein, quinoa protein, nut protein, wheat protein, winged bean protein, bambara bean protein, dulse protein, mesquite beans protein, duckweed protein, dry fava bean protein, dry cowpea protein, lupin protein, jackfruit protein, amaranth protein, millet protein, oat protein, chia protein, hemp seed protein and rice protein or a combination thereof.

[0174] Preferably, the step of preparing the first composition 130 may comprise the addition of non-animal proteins from at least two different sources. For example, the first composition comprises vegetable proteins from at least two different plants. In fact, the combination of several origins allows to improve the properties of edible protein threads and to establish an amino acid profile optimized for human nutrition. More preferably, the step of preparing the first composition 130 may comprise the addition of m-pulse proteins and cereal proteins.

[0175] The first composition 130 also includes multivalent ions or polyelectrolytes.

[0176] As described below and shown in the examples, the method 100 for producing edible protein filaments according to the present invention uses complementary multivalent ions and polyelectrolytes and when combined produces a heat-resistant gel.

[0177] Typically, when the first composition includes one of a multivalent ion or a polyelectrolyte, the second composition includes the other. The multivalent ion and the polyelectrolyte can then be combined during the step of contacting 160 the first composition with the second composition.

[0178] Therefore, if Figure 1 As shown in FIG. 1 , the method 100 for producing edible protein filaments according to the present invention includes the step of preparing a second composition 140 .

[0179] In particular, this step is designed to prepare a second composition that is complementary to the first composition so as to form protein filaments having a desired texture when the two compositions are contacted. Thus, such a step helps to solve the problem solved by the present invention.

[0180] In particular, when the step of preparing the first composition 130 includes adding a polyelectrolyte, the step of preparing the second composition 140 includes adding a multivalent ion to the second composition, or when the step of preparing the first composition 130 includes adding a multivalent ion, the step of preparing the second composition 140 includes adding a polyelectrolyte to the second composition.

[0181] As stated above, polyelectrolytes and multivalent ions within the meaning of the present invention are complementary components which, when combined, form a heat-resistant gel.

[0182] It should be noted that when the first composition comprises a polyelectrolyte, the first composition may still comprise one or several multivalent ions, but such multivalent ions may not be able to form a heat-resistant gel with the polyelectrolyte. Their nature and / or their concentration should not allow the formation of a heat-resistant gel with the polyelectrolyte.

[0183] Polyelectrolytes

[0184] As mentioned above, a polyelectrolyte suitable for forming a heat-resistant gel may be added to the first composition or the second composition. It is interesting that very small amounts of polyelectrolytes can modify the properties of the silk.

[0185] Thus, the first composition or the second composition may comprise at least 0.01% polyelectrolyte by weight compared to the total wet weight of the relevant composition. Preferably, the first composition or the second composition may comprise at least 0.02% polyelectrolyte by weight compared to the total wet weight of the relevant composition; more preferably at least 0.05% by weight, even more preferably at least 0.1% polyelectrolyte by weight compared to the total wet weight of the relevant composition.

[0186] In particular, when the first composition comprises a polyelectrolyte, the first composition may comprise at least 0.1% by weight of the polyelectrolyte compared to the total wet weight of the first composition. Preferably, the first composition may comprise at least 0.2% by weight of the polyelectrolyte compared to the total wet weight of the first composition; more preferably at least 0.3% by weight, even more preferably at least 0.4% by weight of the polyelectrolyte compared to the total wet weight of the first composition. When the concentration of the polyelectrolyte is mentioned herein, it preferably refers to the concentration of the polyelectrolyte salt.

[0187] The first composition or the second composition may comprise up to 15% polyelectrolyte by weight compared to the total wet weight of the relevant composition. Preferably, the first composition or the second composition may comprise up to 10% polyelectrolyte by weight compared to the total wet weight of the relevant composition; more preferably up to 5% by weight, even more preferably up to 3% polyelectrolyte by weight compared to the total wet weight of the relevant composition. In particular, as shown in the examples, the first composition or the second composition may comprise less than 2% polyelectrolyte by weight compared to the total wet weight of the relevant composition. More particularly, the first composition comprises less than 2% polyelectrolyte, such as alginate, by weight compared to the total wet weight of the first composition.

[0188] The first composition or the second composition may contain 0.01% to 15% polyelectrolyte by weight compared to the total wet weight of the relevant composition. Preferably, the first composition or the second composition may contain 0.02% to 10% polyelectrolyte by weight compared to the total wet weight of the relevant composition; more preferably 0.05% to 5% by weight, even more preferably 0.1% to 3% polyelectrolyte by weight compared to the total wet weight of the relevant composition.

[0189] As mentioned above, the polyelectrolyte is a heat-resistant gel-forming polyelectrolyte, especially when complexed with multivalent ions. It is also preferred that the polyelectrolyte is not a heat-activatable gel-forming polyelectrolyte. For example, the polyelectrolyte does not require heat treatment at a temperature above 80°C, preferably above 60°C, to be activated.

[0190] Furthermore, since it is intended for human consumption, the polyelectrolyte is digestible by the human digestive system.

[0191] For example, the polyelectrolyte is a cation (e.g. chitosan) or an anion (e.g. alginate). Preferably, when the polyelectrolyte is an anionic polyelectrolyte, the polyvalent ions are polyvalent cations. In addition, when the polyelectrolyte is a cationic polyelectrolyte, the polyvalent ions are polyvalent anions. More preferably, the polyelectrolyte is an anionic polysaccharide.

[0192] It is not necessary that all monomers of the polyelectrolyte carry ionizable groups. For example, the polyelectrolyte is a polymer having at least 10%, preferably at least 20%, more preferably at least 30%, and even more preferably at least 40% of its monomers carrying ionizable groups. Preferably, the ionizable groups are selected from amine groups, carboxyl groups, sulfate groups or phosphate groups.

[0193] For example, a polyelectrolyte is a polymer having at least 10%, preferably at least 20%, more preferably at least 30% of the monomers that can be negatively charged, for example all monomers can be negatively charged. Preferably, the negatively charged monomers contain 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.

[0194] For example, a polyelectrolyte is a polymer having at least 10%, preferably at least 20%, more preferably at least 30%, even more preferably at least 40% of the monomers which may be positively charged, for example all monomers may be positively charged. Preferably, the positively chargeable monomers contain at least one cationic group. Preferably, the cationic group is an amine.

[0195] Preferably, the polyelectrolyte is a high molecular weight polyelectrolyte. For example, the polyelectrolyte has a molecular weight of at least 2 kg.mol -1 Preferably, the polyelectrolyte has an average molar mass of at least 10 kg.mol -1More preferably, the polyelectrolyte has an average molar mass of at least 20 kg.mol -1 Even more preferably, the polyelectrolyte has an average molar mass of at least 30 kg.mol -1 The average molar mass.

[0196] For optimum texture, the molecular weight of the polyelectrolyte should not be too high. For example, a polyelectrolyte with a molecular weight of at most 3000 kg.mol -1 Preferably, the polyelectrolyte has an average molar mass of at most 2500 kg.mol -1 More preferably, the polyelectrolyte has an average molar mass of at most 2000 kg.mol -1 Even more preferably, the polyelectrolyte has an average molar mass of at most 1500 kg.mol -1 The average molar mass.

[0197] Thus, for example, a polyelectrolyte has 2 kg.mol -1 Up to 3000 kg.mol -1 Preferably, the polyelectrolyte has an average molecular weight of 10 kg.mol -1 Up to 2500 kg.mol -1 More preferably, the polyelectrolyte has an average molar mass of 20 kg.mol -1 Up to 2000 kg.mol -1 Even more preferably, the polyelectrolyte has an average molar mass of 30 kg.mol -1 Up to 1500 kg.mol -1 The average molar mass.

[0198] The average molecular weight of the polyelectrolyte can be measured by size exclusion chromatography coupled with light scattering. Preferably, it can be measured by following the instructions of international standard ISO 16014-5:2019.

[0199] Several polyelectrolytes can be used in the present invention. However, some polyelectrolytes are preferred. Preferably, the polyelectrolyte is selected from: low methoxy pectin, low methoxy pectin derivatives, high methoxy pectin, high methoxy pectin derivatives, alginate, alginate derivatives, xanthan gum, xanthan gum derivatives, chitosan, chitosan derivatives, ionic carboxymethyl cellulose derivatives, ionic pullulan derivatives, ionic dextran derivatives, ionic starch derivatives, or a combination thereof.

[0200] More preferably, the polyelectrolyte is selected from alginate, alginate derivatives, low methoxy pectin, low methoxy pectin derivatives, chitosan, chitosan derivatives or combinations thereof.

[0201] Even more preferably, the polyelectrolyte is selected from alginate, low methoxyl pectin, chitosan, or a combination thereof.

[0202] Multivalent ions

[0203] As described above, in the method according to the present invention, multivalent ions are combined with polyelectrolytes to form a heat-resistant gel.

[0204] The polyvalent ion can be a polyvalent anion or a polyvalent cation. For example, the polyvalent ion is selected from the following: calcium, magnesium, iron, manganese, copper, zinc, carbonate, oxalate, sulfate, sulfite, phosphate or a combination thereof. Preferably, the polyvalent ion is selected from the following: calcium, magnesium, iron, manganese, copper, zinc, carbonate, oxalate, sulfate, phosphate or a combination thereof.

[0205] The multivalent ions can be obtained in the form of dissolved salts. For example, the counterions can be selected from: calcium, magnesium, sodium, ammonium, chloride, gluconate, lactate, sulfate, carbonate, phosphate, or a combination thereof. When the concentration of a multivalent ion is mentioned herein, it preferably refers to the concentration of the multivalent ion itself.

[0206] The multivalent ion is present in the first composition or the second composition. It may be present in a concentration of at least 0.001% by weight compared 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 compared to the wet weight of the composition.

[0207] The maximum concentration of the multivalent ion is generally guided by the solubility of the multivalent ion. For example, the multivalent ion may be present in the first composition or the second composition in a concentration of up to 3% by weight compared to the wet weight of the composition, preferably up to 2% by weight compared to the wet weight of the composition, more preferably up to 1% by weight, and even more preferably up to 0.5%.

[0208] Thus, the multivalent ion may be present in the first or second composition at a concentration of 0.001 to 3% by weight compared to the wet weight of the composition. Preferably, the multivalent ion is present in the first or second composition at a concentration of 0.002% to 2% by weight compared to the wet weight of the composition; more preferably 0.005% to 1% by weight compared to the wet weight of the composition, even more preferably 0.01% to 0.5% by weight.

[0209] The first composition and the second composition can be prepared in various ways to improve the efficiency of protein filament formation. The drawings and this specification should not be understood to limit the present invention to the following embodiments: wherein the first composition is prepared before the second composition. It is known to those skilled in the art that within the scope of the present invention, the second composition can also be prepared before or simultaneously with the first composition.

[0210] The step of preparing the first composition or the second composition may include adjusting the pH of any of the compositions. For example, the first composition may have a pH between 2 and 12, preferably between 3 and 11, more preferably between 4 and 10, and even more preferably between 5 and 9. For example, the second composition may have a pH between 2 and 12, preferably between 3 and 11, more preferably between 4 and 10, and even more preferably between 5 and 9.

[0211] 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 filaments produced.

[0212] Preferably, the first composition is not heated at a temperature of 65°C or above for a duration of more than 10 minutes, the first composition is not heated at a temperature of preferably 60°C or above, more preferably 55°C or above, even more preferably 50°C or above for a duration of more than 10 minutes.

[0213] For example, the temperature of the first composition during its preparation step is below 65°C, preferably below 60°C, more preferably below 55°C, even more preferably below 50°C.

[0214] Preferably, the second composition is not heated at a temperature above 85°C for a duration of more than 10 minutes, the second composition is not heated at a temperature preferably above 75°C, more preferably above 65°C, even more preferably above 55°C for a duration of more than 10 minutes.

[0215] For example, the temperature of the second composition during its preparation step is at most 85°C, preferably at most 75°C, more preferably at most 65°C, even more preferably at most 55°C.

[0216] The first composition and the second composition can also undergo physical treatment, such as blending, mixing, homogenizing, sieving or ultrasonic treatment. Preferably, the step of preparing the first composition or the second composition can include blending or homogenizing or emulsifying or stirring steps. In particular, these steps are designed to homogenize or mix all components together. In addition, when present, these steps can cause at least part of the animal cells of the culture to rupture. The step of preparing the first composition can include rupturing non-human animal cell membranes. Therefore, the homogenizing step can be blending. For example, when the first composition or the second composition has been supplemented with other ingredients such as plant materials or food additives, it can be used. Preferably, homogenizing or blending or emulsifying or stirring steps are carried out with a high-speed mixer or with a homogenizer (such as a rotor-stator homogenizer, a cutter or a colloid mill).

[0217] Homogenization can be performed for at least 30 seconds, preferably at least 1 min, more preferably at least 2 min (e.g., at least 10 min). The homogenization step can be performed for up to 1 hour, preferably up to 45 min, more preferably up to 30 min; even more preferably up to 10 min (e.g., up to 2 min). Thus, homogenization can be performed for 30 seconds to 60 min, preferably for 1 min to 45 min, more preferably for 2 min to 30 min (e.g., for about 10 min).

[0218] Homogenization can be carried out 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 up to 30 000 rpm, preferably up to 25 000 rpm, more preferably up to 20 000 rpm; for example, at up to 15 000 rpm. Thus, homogenization can be carried out at 100 rpm to 30 000 rpm, preferably at 1000 rpm to 25 000 rpm, more preferably at 2000 rpm to 20 000 rpm; even more preferably at 5000 rpm to 15000 rpm. Homogenization preferably induces an emulsion, more preferably a microemulsion, in the first composition.

[0219] Supplements

[0220] The first composition or the second composition, preferably the first composition can further comprise a cross-linking molecule, such as a peptidase, α-galactosidase, alkaline protease, thermolysin, pepsin, trypsin, chymotrypsin, asparaginase, elastase, subtilisin, glucose oxidase, laccase, transglutaminase, pectinesterase, sortase, tyrosinase, oxidoreductase such as lysyl oxidase and peroxidase, genipin, riboflavin, monoamine oxidase or a combination thereof, preferably further comprising laccase, transglutaminase or a combination thereof. Such molecules can serve as natural cross-linking molecules and help to regulate the physical properties of protein silk.

[0221] As mentioned in the examples, the presence of supplemented fat such as non-animal fat in the first composition can improve the properties of the protein filaments produced. Therefore, advantageously, the step of preparing the first composition further comprises adding fat, such as non-animal fat. Preferably, the supplemented fat is vegetable fat or fermented fat.

[0222] For example, the first composition may comprise at least 1% by weight of supplemented fat, preferably vegetable fat or fermented fat, compared to the total wet weight of the first composition. Preferably, at least 2% by weight of supplemented fat, more preferably at least 5% by weight, even more preferably at least 10% by weight of supplemented fat, compared to the total wet weight of the first composition. However, the amount of supplemented fat should not be too high. For example, the first composition may comprise at most 50% by weight of supplemented fat, compared to the total wet weight of the first composition. Preferably, at most 40% by weight of supplemented fat, more preferably at most 30% by weight, even more preferably at most 25% by weight of supplemented fat, compared to the total wet weight of the first composition.

[0223] Thus, the first composition may comprise 1% to 50% by weight of supplemented fat compared to the total wet weight of the first composition. Preferably, 2% to 40% by weight of supplemented fat compared to the total wet weight of the first composition, more preferably, 5% to 30% by weight, even more preferably 10% to 25% by weight of supplemented fat compared to the total wet weight of the first composition.

[0224] For example, the first composition may further comprise vegetable fat, preferably, the vegetable fat comprises fat or oil extracted from edible plant matter (including flowers, fruits, stems, leaves, roots, germs and seeds), more preferably, the vegetable fat comprises fat or oil extracted from oilseeds or fruits; in particular, the vegetable fat includes fat extracted from rapeseed (rapeseed), castor, coconut, linseed, allanblackia, olive, sunflower, soybean, peanut, illipe, cottonseed, shea, palm, avocado, safflower, sesame, lemon, grape seed, macadamia, almond, sal, kokum or mango, or a combination thereof.

[0225] In particular, the vegetable fat used according to the present invention may be chosen from olive oil, palm oil, sunflower oil, avocado oil, almond oil or a combination thereof.

[0226] The fat of fermentation can comprise the fat or the oil extracted from the cell cultivated in the process of anaerobic aerobic fermentation or aerobic fermentation process, particularly relating to cultivating oil-producing microorganism or animal cell (not comprising human cell).For example, fermented fat can comprise from following fat: cyanobacteria, microalgae, yeast, fungi such as filamentous fungi, bacterium or do not comprise the animal cell such as adipocyte of the cultivation of human cell.Preferably, fermented fat comprises from oil-producing yeast, such as the fat or the oil extracted by Rhodosporidium toruloides, Lipomyces starkeyi and Yarrowia lipolytica.

[0227] Preferably, the supplemented fat consists mainly of triglycerides (at least 60% triglycerides by weight compared to the total weight of the fat). More preferably, the supplemented fat comprises fatty acids, such as fatty acids in the form of triglycerides, selected from the group consisting of butyric acid, isobutyric acid, isovaleric acid, caproic acid, caprylic acid, nonanoic 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.

[0228] like Figure 1 As shown in, the method according to the present invention may include the step of adding food additives 150. Food additives can be added to the first composition, the second composition or both. In particular, this step is designed to improve the flavor, texture, appearance or shelf life of protein silk. The first composition or the second composition may include food additives between 0.01% and 25% by weight compared to the total wet weight of the relevant composition, preferably food additives between 0.01% and 10% by weight compared to the total wet weight of the relevant composition. Accordingly, protein silk may include food additives between 0.01% and 10% by weight compared to the total weight of silk, preferably food additives between 0.01% and 4% by weight compared to the total weight of silk.

[0229] The food additive may be selected from seasonings, flavoring additives, texturizing additives, food coloring agents, preservative additives, or combinations thereof.

[0230] The food additive may be, for example, a flavoring, a mineral salt, a flavoring additive, a wetting agent additive, a texturizing additive, a defoaming agent, an emulsifying additive, a solidifying agent, a gelling agent, a stabilizing additive, a thickening agent, a food coloring agent, a preservative additive, or a combination thereof.

[0231] The seasoning may, for example, be selected from: salt; pepper; aromatic herbs and / or spices including rosemary, sage, mint, oregano, parsley, thyme, bay leaf, cloves, basil, chives, marjoram, nutmeg, cardamom, chili, cinnamon, fennel, fenugreek, ginger, saffron, vanilla and coriander; alcohol including wines such as Jurançon, sauterne or pacherenc, spirits such as cognac or armagnac; or any combination thereof.

[0232] The flavoring additives may, for example, be selected from: flavor enhancers, sweeteners, or any combination thereof.

[0233] The texturizing additive may for example be selected from: a filler or thickener, a desiccant, a curing agent or any combination thereof. For example, the first composition may further comprise a texturizing additive such as carrageenan, pectin, pullulan, dextran, starch.

[0234] The preservative additive may, for example, be selected from: an antimicrobial agent, a pH adjuster, or any combination thereof.

[0235] The food colorant can be selected from, for example, colorants extracted from natural sources, such as carotene, anthocyanins, tomatoes (lycopene), beetroot (betacyanins and betaxanthins), or mixtures thereof. The edible food product can comprise between 0.01% and 4% of a food colorant compared to the total weight of the edible food product.

[0236] In particular, in a first embodiment, as shown in the examples, the first composition may comprise:

[0237] at least 10% by weight of total protein relative to the total wet weight of the first composition, including at least 5% by weight of non-human animal protein relative to the weight of total protein, said animal protein being derived from non-human cultured animal cells, and

[0238] - at least 0.1 % by weight of polyelectrolyte relative to the total wet weight of the first composition.

[0239] Preferably, as shown in the examples, the first composition may comprise:

[0240] at least 10% by weight of total protein relative to the total wet weight of the first composition, including at least 5% by weight of non-human animal protein relative to the weight of total protein, said animal protein being derived from non-human cultured animal cells, and

[0241] - at least 0.1% by weight of polyelectrolyte relative to the total wet weight of the first composition, and

[0242] at least 1% of vegetable fat or fat obtained by fermentation relative to the total wet weight of the first composition.

[0243] Preferably, the first composition may comprise:

[0244] at least 10% by weight of total protein relative to the total wet weight of the first composition, including at least 5% by weight of non-human animal protein relative to the weight of total protein, said animal protein being derived from non-human cultured animal cells, and

[0245] - at least 0.1% by weight of polyelectrolyte relative to the total wet weight of the first composition, and

[0246] - At least one cross-linking molecule.

[0247] Preferably, as shown in the examples, the first composition may comprise:

[0248] at least 10% by weight of total protein relative to the total wet weight of the first composition, including at least 5% by weight of non-human animal protein relative to the weight of total protein, said animal protein being derived from non-human cultured animal cells, and

[0249] - at least 0.1% by weight of polyelectrolyte relative to the total wet weight of the first composition, and

[0250] - at least one cross-linking molecule, and

[0251] at least 1% of vegetable fat or fat obtained by fermentation relative to the total wet weight of the first composition.

[0252] Even more preferably, as shown in the examples, the first composition may comprise:

[0253] at least 10% by weight of total protein relative to the total wet weight of the first composition, including at least 5% by weight of non-human animal protein relative to the weight of total protein, said animal protein being derived from non-human cultured animal cells,

[0254] - at least 0.001% by weight of multivalent ions relative to the total wet weight of the first composition, and

[0255] - at least one cross-linking molecule, and

[0256] - at least 1% of vegetable fat or fat obtained by fermentation relative to the total wet weight of the first composition, and

[0257] At least one extracellular matrix non-human animal molecule, preferably at least 0.1% by weight of extracellular matrix animal molecules compared to the total weight of the protein.

[0258] More preferably, the first composition may comprise:

[0259] o at least 10% by weight of total protein relative to the total wet weight of the first composition, including at least 5% non-human animal protein relative to the weight of total protein (the animal protein being derived from non-human cultured animal cells), and at least 0.25% by weight of myofibrillar protein relative to the total weight of protein, and

[0260] - at least 0.1 % by weight of polyelectrolyte relative to the total wet weight of the first composition.

[0261] More preferably, as shown in the examples, the first composition may comprise:

[0262] o at least 10% by weight of total protein relative to the total wet weight of the first composition, including at least 5% non-human animal protein relative to the weight of total protein (the animal protein being derived from non-human cultured animal cells), and at least 0.25% by weight of myofibrillar protein relative to the total weight of protein, and

[0263] - at least 0.1% by weight of polyelectrolyte relative to the total wet weight of the first composition, and

[0264] at least 1% of vegetable fat or fat obtained by fermentation relative to the total wet weight of the first composition.

[0265] Even more preferably, the first composition may comprise:

[0266] at least 10% by weight of total protein relative to the total wet weight of the first composition, including at least 5% by weight of non-human animal protein relative to the weight of total protein, said animal protein being derived from non-human cultured animal cells, and

[0267] - at least 0.1% by weight of polyelectrolyte relative to the total wet weight of the first composition;

[0268] - The ratio of myofibrillar protein weight to polyelectrolyte is at least 2.5.

[0269] In particular, in the second embodiment, as shown in the examples, the first composition may comprise:

[0270] at least 10% by weight of total protein relative to the total wet weight of the first composition, including at least 5% by weight of non-human animal protein relative to the weight of total protein, said animal protein being derived from non-human cultured animal cells, and

[0271] - at least 0.001 % by weight of multivalent ions relative to the total wet weight of the first composition.

[0272] Preferably, as shown in the examples, the first composition may comprise:

[0273] at least 10% by weight of total protein relative to the total wet weight of the first composition, including at least 5% by weight of non-human animal protein relative to the weight of total protein, said animal protein being derived from non-human cultured animal cells, and

[0274] - at least 0.001% by weight of multivalent ions relative to the total wet weight of the first composition, and

[0275] at least 1% of vegetable fat or fat obtained by fermentation relative to the total wet weight of the first composition.

[0276] More preferably, as shown in the examples, the first composition may comprise:

[0277] o at least 10% by weight of total protein relative to the total wet weight of the first composition, including at least 5% non-human animal protein relative to the weight of total protein (the animal protein being derived from non-human cultured animal cells), and at least 0.25% by weight of myofibrillar protein relative to the total weight of protein, and

[0278] - at least 0.001 % by weight of multivalent ions relative to the total wet weight of the first composition.

[0279] Even more preferably, as shown in the examples, the first composition may comprise:

[0280] o at least 10% by weight of total protein relative to the total wet weight of the first composition, including at least 5% of non-human animal protein relative to the weight of total protein (the animal protein being derived from non-human cultured animal cells), and at least 0.25% by weight of myofibrillar protein relative to the total weight of protein,

[0281] - at least 0.001% by weight of multivalent ions relative to the total wet weight of the first composition, and

[0282] - At least one cross-linking molecule.

[0283] Preferably, as shown in the examples, the second composition is an aqueous composition.

[0284] In particular, in a first embodiment, as shown in the examples, the second composition may comprise:

[0285] - at least 0.001 % by weight of multivalent ions relative to the total wet weight of the second composition.

[0286] Preferably, the second composition may comprise:

[0287] - at least 0.001% by weight of multivalent ions relative to the total wet weight of the second composition, and

[0288] - At least one cross-linking molecule.

[0289] In particular, in the second embodiment, as shown in the examples, the second composition may comprise:

[0290] - at least 0.01 % by weight of polyelectrolyte relative to the total wet weight of the first composition.

[0291] Preferably, the second composition may comprise:

[0292] - at least 0.01% by weight of polyelectrolyte relative to the total wet weight of the first composition, and

[0293] - At least one cross-linking molecule.

[0294] like Figure 1 As shown in , the method 100 for producing edible protein filaments according to the present invention includes a step of contacting a first composition with a second composition 160. In particular, this step is designed to form edible protein filaments. More specifically, several edible protein filaments can be prepared at the same time.

[0295] Advantageously, as shown in the examples, when contacting the second composition, the temperature of the first composition is less than 65° C. Preferably, when contacting the second composition, the temperature of the first composition is less than 60° C., more preferably less than 55° C., even more preferably less than 50° C. This can improve the texture, handleability or appearance of the edible protein filaments.

[0296] Furthermore, once prepared, the first composition should not be heat treated at a temperature of 65°C or higher, preferably 60°C or higher, more preferably 55°C or higher, even more preferably 50°C or higher.

[0297] For example, the temperature of the first composition used during the contacting 160 step is between 1°C and 65°C, preferably between 3°C and 60°C, more preferably between 3°C and 55°C, even more preferably between 3°C and 50°C.

[0298] It has been shown that the second composition can be used at higher temperatures without effect on the quality of the edible protein filaments produced. Thus, the temperature of the second composition when in contact with the first composition can be above 35° C. However, the temperature of the second composition when in contact with the first composition should preferably be at most 85° C., more preferably at most 75° C., even more preferably at most 65° C.

[0299] The step of contacting the first composition with the second composition 160 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 of contacting the first composition with the second composition 160 is performed by wet spinning or dry-jet wet spinning, bath-assisted 3D printing. More preferably, the step of contacting the first composition with the second composition 160 is performed by wet spinning or dry-jet wet spinning.

[0300] In particular, the step of contacting 160 the first composition with the second composition may comprise impregnating the filaments made of the first composition with the second composition, and preferably immersing the filaments made of the first composition in a bath made of the second composition. Preferably, a flow may be generated in the second composition (e.g. by a vortex collector) and / or the filaments may be wound (which may advantageously be done so that they are straightened).

[0301] In particular, the contacting 160 step includes immersing the first composition into the second composition and forming edible protein filaments in a bath made from the second composition.

[0302] The step of contacting 160 the first composition with the second composition may be performed during at least 10 ms, preferably during at least 100 ms, more preferably during at least 500 ms, even more preferably during at least 1 second.

[0303] The step of contacting 160 the first composition with the second composition may be performed during at most 20 minutes, preferably during at most 10 minutes, more preferably during at most 5 minutes, even more preferably during at most 4 minutes.

[0304] Thus, the step of contacting 160 the first composition with the second composition may be performed during 10 ms to 20 min, preferably during 100 ms to 10 minutes, more preferably during 500 ms to 5 minutes, even more preferably during 1 second to 4 minutes.

[0305] like Figure 1 As shown in , the method according to the present invention may include the step of converting 170 edible protein filaments.

[0306] In particular, this step is designed to prepare the edible protein filaments so that they are suitable for subsequent steps, in particular for making an edible food product that can be used as an edible meat substitute. In addition, the step of converting the edible protein filaments may also include combining the edible protein filaments with another food matrix.

[0307] For example, the protein filaments can undergo further processing, such as soaking, crushing, squeezing, weaving, cutting, chopping, grinding, mixing, shredding, squeezing, dosing, molding, pressing, 3D printing, extrusion baking or cooking steps, such as smoking, baking, frying, surface treatment, coating or also combined with other ingredients to produce edible food products that simulate known conventional meat products. In particular, the edible product can be a meat substitute product that is intended to imitate known meat products (whether converted or not).

[0308] Furthermore, the method according to the invention may comprise the step of combining the protein filaments with another food matrix, such as a fat matrix, a protein matrix, a carbohydrate matrix, a vegetable matrix and / or a fiber matrix.

[0309] like Figure 1 As shown in , a method according to the present invention may include the step of conditioning 180 an edible food product comprising edible protein shreds.

[0310] In particular, this step is designed to obtain an edible food product having meat-like properties, such as meat-like organoleptic properties, in particular meat-like texture and flavour.

[0311] According to the present invention, the step of conditioning 180 the edible food product may include steps such as affecting the water content, shape, texture, flavor or even shelf life of the edible food product.

[0312] For example, according to the present invention, the step of conditioning 180 the edible food product may include steps such as drying, desiccation, dehydration, freeze drying, filtering, or a combination thereof.

[0313] For example, according to the present invention, the step of conditioning 180 the edible food product may include steps such as sterilization or pasteurization.

[0314] Finally, according to the present invention, the step of conditioning 180 the edible food product may include steps such as cooling, refrigerating, deep freezing, packaging or a combination thereof.

[0315] In another aspect, the present invention relates to edible protein filaments obtainable from a process according to the present invention. Preferably, the edible protein filaments have been obtained by a process according to the present invention.

[0316] In particular, the edible protein filaments include non-human animal protein, which is cultured non-human animal cell protein. In addition, the edible protein filaments may include a heat-resistant gel formed by a polyelectrolyte combined with a suitable multivalent ion.

[0317] Thus, the present invention preferably relates to edible protein filaments obtainable from the process according to the present invention, said edible protein filaments comprising animal protein, which is cultured non-human animal cell protein, and forming a thermostable polyelectrolyte.

[0318] Advantageously, the edible protein shreds have an improved meat-like flavor and / or meat-like texture compared to edible protein shreds made solely from plant protein.

[0319] Several preferred or unpreferred embodiments have been described previously in conjunction with the method according to the invention for producing the protein silk of the invention. Therefore, the protein silk according to the invention may comprise each of the above-mentioned features associated with the method according to the invention and any of the above-mentioned steps thereof, either individually or in combination.

[0320] We have presented in detail the general and preferred features of each component of the protein filaments and edible food products according to the present invention. These embodiments apply to both edible protein filaments and edible food products according to the present invention. Some are mentioned below to emphasize their importance.

[0321] Edible protein filaments according to the present invention may have been produced from a first composition comprising one of a multivalent ion or a polyelectrolyte and a second composition comprising the other.

[0322] In particular, edible protein filaments according to the present invention can be produced from a first composition comprising a polyelectrolyte or from a first composition comprising a multivalent ion.

[0323] Advantageously, the edible protein filaments have been produced from cultured non-human animal cells. Therefore, as described in detail below, the edible protein filaments according to the present invention may comprise intact cultured cells, ruptured cultured cells and / or extracts of cultured cells (such as ruptured cultured cells), the cells being cultured cells from organisms of the animal kingdom excluding humans. However, considering preferred embodiments of the method according to the present invention for producing edible protein filaments, the edible protein filaments may not contain intact cultured cells.

[0324] The protein filaments may comprise at least 0.25% myofibrillar protein by weight compared to the total weight of the protein, preferably at least 0.5% myofibrillar protein by weight compared to the total weight of the protein in the protein filaments, more preferably at least 1% myofibrillar protein by weight, and even more preferably at least 2% myofibrillar protein by weight. The protein filaments may comprise up to 70% myofibrillar protein by weight compared to the total weight of the protein, preferably up to 60% myofibrillar protein by weight compared to the total weight of the protein in the protein filaments, more preferably up to 50% myofibrillar protein by weight, and even more preferably up to 40% myofibrillar protein by weight. Therefore, the protein filaments may comprise 0.25% to 70% myofibrillar protein by weight compared to the total weight of the protein, preferably 0.5% to 60% myofibrillar protein by weight compared to the total weight of the protein in the protein filaments, more preferably 1% to 50% myofibrillar protein by weight, and even more preferably 2% to 40% myofibrillar protein by weight.

[0325] The concentration of myofibrillar protein in protein filaments can also be considered with respect to the concentration of polyelectrolyte. The ratio can be controlled to optimize the behavior of protein filaments. Therefore, protein filaments can include myofibrillar protein (from cultured cells) and polyelectrolyte at a concentration such that the ratio of myofibrillar protein weight to polyelectrolyte is at least 2.5, preferably 5, more preferably 7.5, and even more preferably 10. However, protein filaments can include myofibrillar protein and polyelectrolyte at a concentration such that the ratio of myofibrillar protein weight to polyelectrolyte weight is at most 100, preferably at most 50, more preferably at most 40, and even more preferably at most 30. Therefore, protein filaments can include myofibrillar protein and polyelectrolyte at a concentration such that the ratio of myofibrillar protein weight to polyelectrolyte weight 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 like polyelectrolyte.

[0326] The protein filaments may comprise at least 0.1% polyelectrolyte by weight compared to the total wet weight of the protein filaments. Preferably, the protein filaments may comprise at least 0.2% polyelectrolyte by weight compared to the total wet weight of the protein filaments; more preferably at least 0.3% by weight, even more preferably at least 0.4% polyelectrolyte by weight compared to the total wet weight of the protein filaments.

[0327] The protein filaments may comprise up to 15% polyelectrolyte by weight compared to the total wet weight of the protein filaments. Preferably, the protein filaments may comprise up to 10% polyelectrolyte by weight compared to the total wet weight of the protein filaments, more preferably up to 5% by weight compared to the total wet weight of the protein filaments, even more preferably up to 3% polyelectrolyte by weight.

[0328] The protein filaments may contain 0.1% to 15% polyelectrolyte by weight compared to the total wet weight of the protein filaments. Preferably, the protein filaments may contain 0.3% to 10% polyelectrolyte by weight compared to the total wet weight of the protein filaments, more preferably 0.4% to 5% by weight, even more preferably 0.5% to 3% polyelectrolyte by weight compared to the total wet weight of the protein filaments.

[0329] The protein filament may comprise troponin, preferably at least 0.0125% troponin compared to the total weight of protein in the protein filament, preferably at least 0.025%, more preferably at least 0.05%, even more preferably at least 0.1% troponin compared to the total weight of protein in the protein filament.

[0330] The protein filaments may comprise collagen, preferably at least 1% collagen compared to the total weight of protein in the protein filaments, preferably at least 2%, more preferably at least 3%, even more preferably at least 4% collagen compared to the total weight of protein in the protein filaments.

[0331] The protein filaments may comprise elastin and / or tropoelastin, preferably at least 0.065% elastin and / or tropoelastin compared to the total weight of protein in the protein filaments, preferably at least 0.13%, more preferably at least 0.2%, even more preferably at least 0.25% elastin and / or tropoelastin compared to the total weight of protein in the protein filaments.

[0332] As already described, the method according to the invention may comprise adding supplementary fat, preferably vegetable fat or fat obtained by fermentation, to the edible protein threads.Thus, the edible protein threads according to the invention may comprise fat, preferably vegetable fat or fat obtained by fermentation.

[0333] For example, the edible protein filaments according to the present invention comprise at least 1% fat by weight compared to the total wet weight of the edible protein filaments. Preferably, at least 2% of the supplemented fat by weight compared to the total wet weight of the edible protein filaments, more preferably, at least 5% by weight compared to the total wet weight of the edible protein filaments, and even more preferably at least 10% fat by weight. However, the amount of fat should not be too high. For example, the edible protein filaments may comprise up to 50% fat by weight compared to the total wet weight of the edible protein filaments. Preferably, up to 40% of the supplemented fat by weight compared to the total wet weight of the protein filaments, more preferably, up to 30% by weight compared to the total wet weight of the edible protein filaments, and even more preferably up to 25% fat by weight.

[0334] Thus, the edible protein filaments may, for example, contain 1% to 50% fat by weight compared to the total wet weight of the edible protein filaments. Preferably, 2% to 40% supplemented fat by weight compared to the total wet weight of the edible protein filaments, more preferably, 5% to 30% by weight compared to the total wet weight of the edible protein filaments, even more preferably 10% to 25% fat by weight.

[0335] The edible protein filaments according to the present invention preferably have a shape and / or mechanical properties that allow them to simulate the behavior of conventional meat from slaughtered animals when cooked and tasted.

[0336] For example, the edible protein filaments according to the present invention have a length of at least 2 mm, preferably at least 10 mm, more preferably at least 50 mm, even more preferably at least 100 mm.

[0337] For example, the edible protein filaments according to the present invention have a diameter of at most 4 mm, preferably at most 2 mm, more preferably at most 1 mm, even more preferably at most 0.5 mm.

[0338] Advantageously, the edible protein shreds according to the present invention may be considered an ingredient as an alternative to conventional meat products from slaughtered animals or as an alternative to conventional meat products from slaughtered animals themselves.

[0339] Edible protein filaments can be, for example, ready-to-eat food products that can be consumed directly, or ready-to-eat food products that can be consumed directly after processing steps (e.g., crushing, flattening, weaving, cutting, grinding, mixing, shredding, extruding, dosing, molding, pressing, 3D printing, extrusion baking or cooking steps, such as smoking, baking, frying, surface treatment and / or coating) and / or cooking steps. Edible protein filaments can also be intermediate products used in combination with other products to produce ready-to-eat food products. In particular, edible protein filaments can be alternatives to meat from slaughtered animals, which are intended to imitate conventional meat products (e.g., steak, sausage, meat sauce ...). As shown in the examples, compared with edible protein filaments made from vegetable protein, edible protein filaments according to the present invention can show improved meat-like texture and / or meat-like flavor.

[0340] In another aspect, the present invention relates to a method for making an edible food product. In particular, the method for making an edible food product comprises using an edible protein filament according to the present invention. As already mentioned, such edible food products can be considered as edible meat substitutes.

[0341] In a preferred embodiment, a method of making an edible food product comprises assembling one or more strands of edible protein filaments produced according to the present invention into an edible food product.

[0342] In particular, the method for manufacturing an edible food product may comprise the use of a combination of edible protein filaments according to the present invention produced from a first composition comprising polyelectrolytes and / or edible protein filaments according to the present invention produced from a first composition comprising multivalent ions. In a preferred embodiment, the method for manufacturing an edible food product comprises the use of a combination of edible protein filaments according to the present invention produced from a first composition comprising polyelectrolytes and edible protein filaments according to the present invention produced from a first composition comprising multivalent ions. The protein filaments in the combination may comprise protein filaments that differ from one another due to their composition (e.g., protein, cells, fat, or any other component) or their shape or size (e.g., different diameters).

[0343] In a preferred embodiment, the method of making an edible food product comprises: a) producing one or several edible protein threads according to the present invention; and b) assembling the one or several produced edible protein threads into an edible food product.

[0344] The assembly of the manufactured edible protein filaments may include assembling the manufactured edible protein filaments into a structure that mimics the texture of meat. The process includes collecting and arranging the protein filaments and further processing them through various treatments to produce a meat-like texture and smoothness.

[0345] In one embodiment of the present invention, assembling protein filaments into a meat-like texture comprises one or more of the following steps, which may be performed in any different order:

[0346] ● Collect and stretch protein filaments to orient the proteins longitudinally, similar to the orientation of muscle fibers in meat;

[0347] ● organization of protein filaments in parallel or entangled configurations, followed by a lamination process in which the filaments are compacted;

[0348] ● The protein filaments are layered on top of each other, with each layer positioned to contribute to the overall meat-like structure.

[0349] ● chemically and / or enzymatically and / or physically cross-linking protein filaments to enhance the textural properties and integrity of the assembled protein;

[0350] ● applying mild heat and pressure to the protein filaments to improve protein-protein interactions; and / or

[0351] ●Using looms to weave protein filaments into fabric-like structures, designed to replicate the interwoven nature of muscle fibers in meat;

[0352] Furthermore, when assembling edible protein filaments, it would be relevant to use an adhesive-like composition such as a fat matrix and / or a connective tissue analog to improve the mechanical and organoleptic properties of the edible product.

[0353] The edible food product according to the invention is preferably a processed food product. In fact, it does not exist in the meat of slaughtered animals per se and is preferably produced by a combination of edible substances from different biological sources (for example, a combination of proteins from the animal kingdom and fats from the vegetable kingdom, or a combination of proteins from the vegetable kingdom and / or the animal kingdom and / or the fungal kingdom and / or the bacterial kingdom and fats from the vegetable kingdom and / or the animal kingdom and / or the fungal kingdom and / or the bacterial kingdom).

[0354] Food manufacturing 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, ultrasonic treatment / 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 technology (freeze drying, membrane), fermentation, homogenization, mincing, grinding, chopping, salting, tumbling, pickling, brine injection.

[0355] In another aspect, the present invention relates to an edible food product obtainable from the manufacturing method according to the present invention. Preferably, the edible food product has been obtained by a method according to the present invention.

[0356] In particular, the edible food product comprises edible protein filaments according to the present invention.

[0357] In particular, the edible food product may include a combination of edible protein filaments according to the invention produced from a first composition comprising polyelectrolytes and / or additional edible protein filaments according to the invention produced from a first composition comprising multivalent ions. In a preferred embodiment, the edible food product includes a combination of edible protein filaments according to the invention produced from a first composition comprising polyelectrolytes and edible protein filaments according to the invention produced from a first composition comprising multivalent ions. Such a combination is particularly interesting because it can enrich the mouthfeel, since protein filaments do not have exactly the same mouthfeel.

[0358] Without further description, it is believed that one of ordinary skill in the art can use the foregoing description and the following illustrative examples to make and utilize the products of the present invention and practice the claimed methods. Therefore, the following examples specifically point out the preferred embodiments of the present invention and should not be construed as limiting the remainder of the present disclosure in any way.

[0359] In another aspect, the present invention is directed to a system for producing edible protein filaments, and in particular edible food products comprising edible protein filaments.

[0360] System according to the present invention may include a first composition container that can hold a first composition, which comprises protein. The first composition container may be made of food grade plastics, ceramics, glass, silicones, stainless steel or a combination thereof. The first composition container may be associated with a temperature controller that is configured to measure and modify the temperature of the first composition. The first composition container may be associated with a pressure controller that is configured to measure the pressure in the first composition container.

[0361] The system according to the present invention may include a second composition container capable of accommodating the second composition. The second composition container may be made of food grade plastic, ceramic, glass, silicones, stainless steel or a combination thereof. The second composition container may be associated with a temperature controller configured to measure and modify the temperature of the second composition.

[0362] The system according to the invention comprises a contacting device. Advantageously, the contacting device is arranged to enable the first composition to be contacted with the second composition to form edible protein filaments, preferably to form edible protein filaments from the first composition. Advantageously, the contacting device has one or several holes connected to the container of the first composition, the one or several holes being arranged to allow the first composition to be contacted with the second composition when in the form of filaments. The hole may have a diameter of less than 4 mm. 2 , preferably less than 3.5mm 2 , preferably less than 2 mm 2 , preferably less than 1 mm 2 , even more preferably less than 0.5 mm 2 The hole can have an irregular or definite shape, such as rectangular, circular, oval, ...

[0363] In one embodiment, the contact device comprises a plurality of pins connected to the pumping device associated with the first composition container. In addition, several pins can be connected to a common chamber, or each pin can be connected to an independent chamber. The pins can be arranged parallel to each other, and they can all show the same pattern (circular, rectangular, oval, irregular) or present different patterns. Preferably, the pins show different patterns, so as to obtain the irregular part of the fiber embedded in the fat, therefore providing more natural aspect for the food product.

[0364] The system according to the present invention may include a production container capable of accommodating the protein filaments embedded in the second composition. The production container may be made of food grade plastic, ceramic, glass, silicones, stainless steel or a combination thereof. The production container may be associated with a temperature controller configured to measure and change the temperature of the edible food product.

[0365] Preferably, the system according to the present invention may further comprise a cooling device. Preferably, the system according to the present invention may further comprise a roller.

[0366] Preferably, the system according to the present invention may further include a spooler.

[0367] Preferably, the system according to the present invention may further comprise a cutting device or a slicer device or a packaging device.

[0368] Example

[0369] The present invention is further described in detail by reference to the following experimental examples. These examples are provided for illustrative purposes only and are not intended to be limiting unless otherwise stated. Therefore, the present invention should never be construed as being limited to the following illustrative examples, but should be construed as covering any and all variations that become apparent due to the teachings provided herein.

[0370] Materials and methods

[0371] Chemicals

[0372] Sodium Alginate Food Grade Powder, E 401

[0373] LMC Pectin E440

[0374] Gelatin n°CAS 9000-70-8

[0375] Chitosan n°CAS 9012-76-4

[0376] Calcium chloride n° CAS 10043-52-4

[0377] NaCl

[0378] Vegetable fats and / or fats obtained by fermentation can be obtained by mechanical or chemical extraction from seeds or other parts of the fruit. It can then be purified (if necessary), refined or chemically altered. Many commercial references are available, such as CremoFLEX ® L and CremoFLEX ® A mixture of E.

[0379] Transglutaminase (TGase) powder - 125 U / g

[0380] Soy protein solution 20 wt%

[0381] Elastin - Sigma

[0382] Recombinant Collagen - Sigma

[0383] Cell culture and preparation

[0384] Bovine cells are obtained from biopsies or cultured cells. Cultured cells are bovine embryonic stem cells originally isolated from bovine embryos and adapted to grow in suspension in serum-free and growth factor-free medium. These cells are characterized by their property of growing in suspension on a larger scale in bioreactors.

[0385] Bovine cells were cultured at 37°C with constant agitation at 50 rpm and CO 2 Injected into a 30 L stainless steel bioreactor at adjusted pH 7.1. Four days after inoculation, cells were harvested from the bioreactor, subjected to two-step centrifugation, and the dried pellet was weighed. Harvested cell samples can be subjected to protein dosage determination using the Bradford method.

[0386] The moisture content is measured and can be adjusted. The cells can be used as is. However, additional steps, such as protein extraction, can be performed on the harvested cultured cells.

[0387] Preparation of the first and second compositions

[0388] The first composition and the second composition are prepared by mixing the ingredients following the mixing composition.

[0389] The first composition was homogenized using a homogenizer (8000 rpm, 60 s).

[0390] Contacting a first composition and a second composition to produce edible protein filaments

[0391] Protein filaments were spun using a syringe pump (Chemyx), a 10 mL syringe equipped with an 18G (0.8 mm) or 14G (1.6 mm) needle, or a spinneret comprising several holes. The injection rate was fixed at 5 mL / min.

[0392] Mechanical Testing

[0393] The protein filaments were evaluated by tensile testing using a texture meter (Ametek LS1 equipped with a 10 kg load cell). Briefly, the protein filaments were stretched until failure, and the force was recorded as a function of the elongated distance. At failure, the measured force dropped dramatically.

[0394] The extension rate was 100 mm / min and for each diameter, 6 samples were measured.

[0395] Sensory evaluation

[0396] The food samples were anonymized before tasting. In addition, panelists were provided with water to rinse their mouths between samples and crackers to reset the flavor receptors.

[0397] Panelists evaluate the overall connection and meat-like sensory properties of edible food product samples by tasting. For example, to evaluate beef substitutes, panelists evaluate beef flavor and texture (tenderness, cohesiveness, oiliness, and juiciness).

[0398] Evaluation of the effect of protein content in the first composition on protein silk

[0399] The purpose of this experiment was to illustrate the effect of protein content on the mechanical properties of silk.

[0400] A first composition was prepared by mixing the ingredients in Table 1 below.

[0401] Table 1. Ingredients of the first composition (all ingredients except water are expressed as dry weight / total weight of the composition)

[0402]

[0403] The second composition was a coagulation bath comprising 0.5% w / w calcium chloride dissolved in distilled water, thus 0.18% multivalent ion: calcium.

[0404] The first composition is used in a spinneret to produce several streams of the first composition in contact with a second composition that serves as a coagulation bath.

[0405] Results and Conclusions

[0406] Protein filaments prepared with a first composition having a protein content of 10.56 wt% (A2), 13.20 wt% (A3) or 17.6 wt% (A4) had significantly improved breaking strain, breaking load and tensile stress compared to protein filaments having a protein content of 7.1 wt% (A1).

[0407] In particular, protein filaments prepared with the first composition having a protein content of 17.6 wt % (A4) had +6% breaking strain, +37% breaking load, and +36% tensile stress, compared to protein filaments having a protein content of 10.56 wt % (A2).

[0408] Therefore, when forming protein filaments according to the invention, it should preferably be used a first composition having at least 10 wt% protein compared to the total wet weight of the first composition and preferably at least 13 wt%, more preferably at least 17 wt% protein compared to the wet weight of the composition.

[0409] Evaluation of the effect of protein origin in the first composition on protein filaments

[0410] The aim of this experiment was to evaluate the effect of protein origin on the mechanical properties of protein filaments using tensile testing.

[0411] A first composition was prepared by mixing the ingredients in Table 2 below.

[0412] Table 2. Ingredients of the first composition (all ingredients except water are expressed as weight / total weight of the composition)

[0413]

[0414] The vegetable protein used in the first composition is soy protein.

[0415] The second composition was a coagulation bath comprising 0.5% w / w calcium chloride dissolved in distilled water.

[0416] The first composition is used in a spinneret to cause a stream of the first composition to contact a second composition that serves as a coagulation bath.

[0417] After incubation at 37 °C for 1 h, the mechanical properties of the protein filaments were evaluated by tensile testing using a texturometer.

[0418] Results and Conclusions

[0419] The mechanical properties of the protein filaments are shown in Table 3 below.

[0420] Table 3. Results from tensile testing

[0421]

[0422] The results of the tensile tests were normalized to the values ​​obtained for protein filaments made with vegetable protein only (B0).

[0423] Protein filaments prepared with animal protein (B80; B40) from cells or cell extracts have a higher resistance to traction (tensile test) than protein filaments prepared with soy protein under the same conditions. When animal protein (B40) from cells or cell extracts is used in combination with vegetable protein (resulting in a myofibrillar protein concentration above 4%), the observed improvement is a factor of 2, and when the concentration of animal protein (B80) from cells or cell extracts is above 15%, the observed improvement is at least a factor of 3.

[0424] Furthermore, in the method of the present invention, the combination of animal protein from cultured cells and plant protein improves the tensile properties of the silk compared to plant protein alone (B0 vs. B40).

[0425] This indicates that the protein silk prepared according to the method of the present invention has much higher desirable mechanical properties than silk made only from plant protein.

[0426] Furthermore, the addition of some plant proteins can be used to adjust the viscoelastic properties or flow properties of the first composition comprising non-human animal cultured cells.

[0427] Evaluation of the Effect of Temperature on Protein Silk

[0428] The purpose of this experiment was to determine the maximum temperature that the first composition could be subjected to prior to spinning at atmospheric pressure and the effect of the elevated temperature of the second composition.

[0429] A first composition was prepared by mixing the ingredients in Table 4 below.

[0430] Table 4. Composition of the first composition (all ingredients except water are expressed as dry weight / total weight of the composition)

[0431]

[0432] The second composition was a coagulation bath comprising 0.5% w / w calcium chloride dissolved in distilled water.

[0433] The first composition is used in a spinneret to cause a stream of the first composition to contact a second composition that serves as a coagulation bath.

[0434] To evaluate the effect of the temperature of the first composition on the process of the invention, the first composition was incubated for 10 minutes at different temperatures: at room temperature (RT), 55° C., 60° C. or 65° C. The first composition was used in a spinneret to induce a 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.

[0435] To evaluate the effect of the temperature of the second composition in the coagulation bath on the method of the present invention, the second composition was incubated at 65°C, 75°C and 85°C for 10 minutes, and protein filaments were produced by contacting the first composition (at room temperature) with the second composition (the latter being a calcium coagulation bath). The protein filaments were incubated in the second composition for 1 minute before harvesting.

[0436] After harvesting the fibers from the calcium coagulation bath, their quality / integrity was evaluated.

[0437] Results and Conclusions

[0438] When the first composition was at 55°C and 60°C, spinning was possible, but the protein filament morphology was not as homogeneous as that at RT, and some lumps and irregularities could be observed on the surface of the protein filaments.

[0439] When the first composition was incubated at 65°C, spinning was impossible. The number of clumps increased significantly, blocking the needle and producing discontinuous protein filaments. Therefore, the first composition should not be subjected to heat treatment at 65°C or higher for 10 minutes.

[0440] Regarding the second composition, the silk produced in the coagulation baths at 65°C and 75°C had a similar appearance to the silk obtained at RT (no lumps), but the color was slightly faded. The protein silk formed in the second composition at 85°C was acceptable, but began to have a lumpy texture on the outside.

[0441] Evaluation of the Effects of Polyelectrolyte Properties on Protein Silk

[0442] The purpose of this experiment was to determine the effect of the properties of the polyelectrolyte on the protein filaments.

[0443] A first composition was prepared by mixing the ingredients in Table 5 below.

[0444] Table 5. Composition of the first composition (all ingredients except water are expressed as dry weight / total weight of the composition)

[0445]

[0446] With the exception of D4, in which the second composition was a coagulation bath comprising disodium hydrogen phosphate dissolved in distilled water at 1 mol / L, the second composition was a coagulation bath comprising 1.5% w / w calcium chloride dissolved in distilled water.

[0447] The first composition is used in a spinneret to cause a stream of the first composition to contact a second composition that serves as a coagulation bath.

[0448] After incubation at 37 °C for 1 h, the mechanical properties of the protein filaments were evaluated by tensile testing using a texturometer.

[0449] Results and Conclusions

[0450] It was possible to form good and heat-resistant protein filaments with alginate (D1), LMC pectin (D2) and chitosan (D4), whereas gelatin (D3) produced protein filaments that melted completely when cooked during the subsequent steps.

[0451] The mechanical properties of the protein filaments are shown in Table 6 below. The results of the tensile tests were normalized based on the values ​​obtained for protein filaments prepared with alginate (D1). Tensile testing was not performed on D3 because these protein filaments melted during cooking.

[0452] Table 6. Results from tensile testing

[0453]

[0454] Therefore, the interaction between cationic polyelectrolytes and polyanions or anionic polyelectrolytes and polycations plays a role in the method according to the present invention. This emphasizes the importance of using ion-mediated gelation (rapid polymerization when contacting two solutions) to obtain silk that can maintain its structure when cooked. Compared with other alternatives of conventional meat products from slaughtered animals, this is most important to ensure good acceptance by consumers who can cook and eat the edible food products from the present invention.

[0455] Evaluation of the effects of extracellular matrix (ECM) molecules on protein filaments

[0456] The purpose of this experiment was to determine the effect of the presence of extracellular matrix (ECM) molecules on the properties of protein filaments using tensile strength measurements.

[0457] A first composition was prepared by mixing the ingredients in Table 7 below.

[0458] Table 7. Composition of the first composition (all ingredients except water are expressed as dry weight / total weight of the composition)

[0459]

[0460] Sol: soluble

[0461] The second composition was a coagulation bath comprising 0.5% w / w calcium chloride dissolved in distilled water.

[0462] The first composition is used in a spinneret to produce several streams of the first composition in contact with a second composition that serves as a coagulation bath.

[0463] After incubation at 37 °C for 1 h, the mechanical properties of the protein filaments were evaluated by tensile testing using a texturometer.

[0464] Results and Conclusions

[0465] The mechanical properties of the protein filaments are shown below in Table 8. The results of the tensile tests were normalized based on the values ​​obtained for protein filaments made with animal protein from cultured cells (E0) without the addition of ECM proteins.

[0466] Table 8. Results from tensile testing

[0467]

[0468] As shown in the table above, the addition of elastin as a powder in the first composition has no detrimental effect on the strain at break, but can reduce the load at break and the tensile stress (E1p).

[0469] However, when elastin is present in the first composition (E1s), it can improve the breaking load, tensile stress and Young's modulus by more than 75%.

[0470] Furthermore, the presence of collagen in soluble form in the first composition improves the breaking load, tensile stress and Young's modulus of the produced filaments.

[0471] This suggests that ECM molecules, particularly ECM proteins, contribute to the tensile strength of the protein filaments when present in the first composition.

[0472] Therefore, the use of non-human animal cultured cells with increased concentrations in ECM molecules and in particular in proteins may help to increase the stiffness and elasticity of protein filaments produced according to the present invention.

[0473] Evaluation of the effect of cross-linking molecules on protein filaments

[0474] The purpose of this experiment was to determine the effect of adding transglutaminase to the first composition using a tensile test.

[0475] A first composition was prepared by mixing the ingredients in Table 9 below.

[0476] Table 9. Composition of the first composition (all ingredients except water are expressed as dry weight / total weight of the composition)

[0477]

[0478] The second composition was a coagulation bath comprising 1.5% w / w calcium chloride dissolved in distilled water.

[0479] The first composition is used in a spinneret to produce several streams of the first composition in contact with a second composition that serves as a coagulation bath.

[0480] The first half of the sample was incubated at 37°C for 1 h to allow transglutaminase activity, while the second half was incubated at 4°C for 1 h to prevent transglutaminase activity.

[0481] After incubation, the mechanical properties of protein filaments were evaluated by tensile testing using a texturometer.

[0482] Results and Conclusions

[0483] The mechanical properties of the protein filaments are shown below in Table 10. The results of the tensile tests were normalized based on the values ​​obtained with incubation at 4°C (thus not benefiting from the activity of transglutaminase) (F4).

[0484] Table 10. Results from tensile testing

[0485]

[0486] As shown in the table above, the addition of transglutaminase to the first composition in combination with incubation at 37°C (F37) had no effect on the breaking strain of the protein filaments according to the invention, but significantly improved the breaking load, tensile stress and Young's modulus.

[0487] Therefore, the use of enzymes as cross-linking agents in the first composition helps to increase the hardness and elasticity of the produced protein filaments.

[0488] Evaluation of the effect of alginate concentration on protein filaments in the second composition

[0489] The purpose of this experiment was to evaluate the effect of the alginate concentration in the second composition.

[0490] A first composition was prepared by mixing the ingredients in Table 11 below.

[0491] Table 11. Ingredients of the first composition (all ingredients except water are expressed as wet weight / total weight of the composition)

[0492]

[0493] The second composition was a coagulation bath comprising 0.5% or 2% w / w sodium alginate dissolved in distilled water.

[0494] Results and Conclusions

[0495] Regarding the concentration of calcium chloride, silk was obtained for both concentrations 0.03% (G1) and 0.3% (G2).

[0496] Regarding the concentration of alginate in the second composition, filaments were obtained at 0.5% alginate or 2% alginate in the second bath. Since the 2% alginate bath produced some irregular morphology on the filaments, a lower concentration was recommended.

[0497] This method allows the production of protein filaments with desired mechanical properties. In addition, the protein filaments produced with polyelectrolytes in the second composition have a gel-like texture on the outside, which can help simulate the extracellular matrix. This can also improve texture and mouthfeel.

[0498] Cross-linking ability and properties of edible silk with respect to protein content and alginate content in the first composition evaluate

[0499] Using tensile strength measurements, the purpose of this experiment was to compare protein filaments produced with varying concentrations of protein and alginate, and to determine the ability of increased alginate content in the first composition to overcome low protein content and produce an edible food product with meat-like properties.

[0500] A first composition was prepared by mixing the ingredients in Table 12 below.

[0501] Table 12. Ingredients of the first composition (all ingredients except water are expressed as dry weight / total weight of the composition)

[0502]

[0503] The second composition was a coagulation bath comprising 1.5% w / w calcium chloride dissolved in distilled water.

[0504] After incubation at 37° C. for 1 hour, the protein filaments were then incubated during 10 min at 70° C. The mechanical properties of the protein filaments were evaluated by tensile testing using a texturometer.

[0505] Results and Conclusions

[0506] It was possible to form protein filaments of good appearance and heat resistance from the first compositions H1, H2 and H4, whereas the protein filaments formed with the first composition H3 were not optimal.

[0507] The mechanical properties of the protein filaments are shown in Table 13 below. The results of the tensile test are normalized based on the values ​​obtained for samples made with at least 10% protein and less than 2% alginate (H1). The protein filament H3 formed with the first composition could not be evaluated by the tensile test because the measured signal was below the detection limit. Surprisingly, the protein filaments obtained from composition H2 have slightly improved mechanical properties compared to the protein filaments obtained from composition H1.

[0508] Table 13. Results from tensile testing

[0509]

[0510] As shown in the table above, the results of the tensile tests indicate that the low protein content cannot be compensated for by increasing the concentration of alginate in the first composition in order to prepare a meat alternative option. The first composition H4 had a detrimental effect on the samples in terms of breaking load, tensile stress and Young's modulus.

[0511] In addition, the protein filaments produced with the first composition H1 (or composition H2) were very smooth and regular, while the protein filaments produced with the first composition H4 showed irregular and blocky shapes. In addition, sample H4 was too jelly-like and rubbery (which is consistent with the high strain at break) to be comparable to the fibers of conventional meat. Therefore, the appearance and texture of the protein filaments became better when having a higher protein content and a lower alginate concentration.

[0512] Forming an edible product comprising edible protein filaments

[0513] As already described, Figure 2 The protein filaments shown in can be used to produce Figure 3 The edible food product shown in .

[0514] In addition to those described above, the present invention may be the subject of many variations and applications. In particular, unless otherwise specified, the different structural and functional features of each of the above-described embodiments should not be considered as being combined with each other and / or tightly and / or inseparably connected, but rather as being simply juxtaposed. In addition, the structural and / or functional features of the various embodiments described above may be the subject of all or part of any different juxtaposition or any different combination.

[0515] Evaluation of the effect of protein content and origin in the first composition on the sensory properties of the product

[0516] The aim of this experiment was to evaluate the effect of protein origin and content on the organoleptic properties of edible food products according to the invention, as well as the overall liking and similarity compared to the organoleptic characteristics of conventional meat.

[0517] A first composition was prepared by mixing the ingredients in Table 14 below.

[0518] Table 14. Ingredients of the first composition (all ingredients except water are expressed as dry weight / total weight of the composition)

[0519]

[0520] The second composition was a coagulation bath comprising 0.5% w / w calcium chloride dissolved in distilled water.

[0521] Results and Conclusions

[0522] Similarity to conventional meat texture, flavor, and overall liking were rated by a trained sensory panel (n=16) on a 1-9 scale, with 1 being least like or least similar to conventional meat and 9 being most like or most similar to conventional meat.

[0523] The sensory evaluation of the edible food product samples according to the present invention is shown in Table 15 below.

[0524] Table 15. Results from sensory evaluation

[0525]

[0526] The similarity of the samples to conventional meat was equally assessed by the panel without any significant differences.

[0527] The panelists found that the edible food product according to the present invention with the highest protein content from cells or cell extracts (Sample J3) provided the best texture and flavor. In addition, Sample J3 was described as having comparable texture and better flavor than an edible food product with a high protein content from plants but a low protein content from cells or cell extracts (Sample J4). These results indicate that the origin of the protein has a noticeable effect on flavor, particularly texture, for consumers.

[0528] In addition, between two edible food products with the same protein content, but one with more protein from cells or cell extracts (sample J2) versus the other with primarily plant-based protein (sample J1), sample J2 outperformed sample J1 in flavor and texture. Sample J5, which had an overall low amount of protein, exhibited low similarity values ​​to regular meat texture and flavor.

[0529] Thus, edible food products according to the present invention comprising filaments made from high animal protein content from cells or cell extracts induce the generation of meat-like texture and flavor within the edible food product, further demonstrating that such edible food products have similar organoleptic properties to conventional meat.

Claims

1. A method for producing edible protein silk, the method The following steps are involved: - preparing a first composition (130), the first composition comprising: o Animal protein derived from non-human cultured animal cells, and oMultivalent ions or polyelectrolytes; - preparing a second composition (140), o When the first composition (130) preparation step comprises adding the polyelectrolyte, the second composition comprises the multivalent ion, or o when the first composition (130) preparation step includes adding the multivalent ion, the second composition comprises the polyelectrolyte; and - contacting (160) the first composition with the second composition to form edible protein filaments, the first composition being at a temperature below 65°C.

2. The method of claim 1, wherein the first composition is not heated at a temperature of 60°C or above for 10 minutes or more.

3. The method according to claim 1 or 2, wherein the non-human cultured animal cells are selected from cells from the animal kingdom, preferably, the animal cells are selected from mammalian cells, avian cells, ray-finned fish cells, mollusc cells and combinations thereof; for example, the mammalian cells may be bovine cells, cervidae cells, lagomorpha cells or porcine cells; the avian cells may be duck cells or pheasant cells; the ray-finned fish cells may be cod cells, hake cells, flatfish cells, salmonid cells or scombroid cells; the mollusc cells may be longarm shrimp cells, and the mollusc cells may be cephalopod cells or Bivalvia cells, and wherein 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 or smooth muscle cells; myoblasts, myocytes, 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.

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

5. The method according to any one of claims 1 to 4, 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. The method according to any one of claims 1 to 5, wherein the polyelectrolyte is a heat-resistant gel-forming polyelectrolyte, especially when complexed with the multivalent ion, preferably the polyelectrolyte is a polysaccharide.

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

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

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

10. The method according to any one of claims 1 to 9, wherein the preparation of the first composition comprises homogenization, preferably the homogenization is performed at at least 100 rpm for a period of at least 30 seconds. 11 . The method according to any one of claims 1 to 10 , wherein the preparation of the first composition comprises adding disrupted non-human cultured animal cells and / or intact non-human cultured animal cells.

12. The method according to any one of claims 1 to 11, wherein the first composition comprises: at least 10% by weight of total protein relative to the total wet weight of the first composition, including at least 5% by weight of non-human animal protein relative to the weight of total 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 the first composition.

13. A method of making an edible food product, wherein The following steps are involved: - producing edible protein filaments according to the method of any one of claims 1 to 12; and - Assembling one or more of the manufactured edible protein threads into an edible food product.

14. Edible protein filaments obtainable from a process according to any one of claims 1 to 12, said edible protein filaments comprising animal protein and a thermostable gel-forming polyelectrolyte, said animal protein being cultured non-human animal cell protein.

15. An edible food product comprising the edible protein filaments according to claim 14.

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