Method for preparing an edible nutritional composition and its use

Abstract

The present invention relates to (i) culturing proliferating non-human animal cells of interest in vitro until the cells have expanded in number by at least twofold; (ii) harvesting the cells to obtain cell-derived material; (iii) preserving the cell-derived material from the cells harvested in step (ii) against spoilage. Furthermore, the present invention refers to an edible nutritional composition obtained from such a method, and to its use for providing a well-defined nutritional composition.

Description

[Technical field]

[0001] The present invention relates to a method for preparing an edible nutritional composition comprising the steps of: (i) culturing in vitro proliferating non-human animal cells of interest until the cell number of said cells has increased by at least two-fold; (ii) harvesting the cells to obtain cell-derived material; and (iii) preserving the cell-derived material from the cells harvested in step (ii) against spoilage. Furthermore, the present invention refers to an edible nutritional composition obtained from such a method, and its use to provide a well-defined nutritional composition.

[0002] There is an increasing global demand for edible products that provide nutritional value, such as high protein content and healthy nutrient content. This demand is often met by products of slaughtered animals, such as meat. Meat often contains a relatively high content of valuable nutrients, such as vitamins (such as vitamin B1 (thiamine), vitamin B3 (niacin), vitamin B6, and vitamin B12), meat-specific amino acids such as lysine, methionine, isoleucine, tryptophan, selenium, and pantothenic acid that are not present in plant-based edible nutritional compositions. In addition, liver tissue often contains a high content of the nutrients vitamin A, copper, collagen, polylysine, and hyaluronic acid.

[0003] However, there are increasing ethical and economic constraints and concerns when using meat from slaughtered animals. Undesirable substances such as xenobiotics, hormones and their precursors, and high calorie components are inevitably present in meat from animals. Nutrient content, including macronutrients (e.g., proteins, fatty acids, and carbohydrates, and dietary fiber) and micronutrient composition (e.g., minerals, vitamins), cannot be controlled and cannot be adjusted as needed. Furthermore, meat production is often associated with high greenhouse gas production and environmental pollution (e.g., eutrophication), and the need for large areas. With a world population exceeding 7 billion and growing, it is hardly possible or even desirable to supply everyone with meat.

[0004] As a result, there is great interest in products that can replace the meat of slaughtered animals.

[0005] In 2013, a burger based on cell culture material from cultivated bovine muscle cells was introduced. Solid meat substitutes that mimic parts of animal muscle tissue are also considered. In this context, it is considered to produce pork meat from either skeletal muscle cells or from pig embryo cells cultured as stem cells, matured into muscle fibers, and combined with a scaffold to obtain a meat-like product (see Kurt et al., Austin Food Sci, 2021, 6(1): 1041). Obtaining cell culture-derived meat from chicken, duck, beef, and pork cells has been found to have multiple advantages, such as providing clean and safe meat (see Hong et al., Food Sci. of Anim. Resour., 2021, 41(3): 355-372). However, preparing such materials has been laborious and cost-prohibitive. 3D molding is energy- and resource-intensive, typically requiring a scaffold structure.

[0006] Moreover, solid meat-like products are not suitable for individuals with problems chewing and / or swallowing, such as the elderly and dysphagic patients, for whom general protein shakes, often derived from specific purified proteins or milk, such as high-protein milk protein concentrates, are known (see Sikand et al., J. Diary Sci., 2011, 94:6194-6202).

[0007] If a patient is unable to ingest any food or adequately due to a medical condition associated with difficulty chewing and / or swallowing, such as dysphagia, surgery that prevents eating, or loss of appetite, enteral feeding (e.g., nasogastric feeding (i.e., via a tube through the nose to the stomach or small intestine, also called nasogastric intubation), gastrostomy (i.e., via a tube through the skin to the stomach), jejunostomy (i.e., via a tube through the skin to the jejunum), or other types of tube feeding using such protein compositions may be considered. In such cases, the composition administered may contain proteins, carbohydrates (sugars), fats, vitamins, and minerals and is given via a tube to the stomach or small intestine. Exemplary compositions are described in Malone, 2005 ("Enteral Formula Selection: A Review of Selected Product Categories", Nutrition Issues in Gastroenterology, Series #28, ed. Carol Rees Parrish, Practical Gastroenterology).

[0008] Protein compositions that can be used for the aforementioned purposes have the disadvantage that their nutritional content is not meat-like and does not contain valuable nutrients such as minerals, essential amino acids (e.g., lysine, isoleucine, tryptophan, etc.), and vitamins, which are typical for meat. Rather, such protein compositions known for the above-referenced purposes are typically derived from milk (e.g., containing milk, skim milk, milk protein concentrate, casein, caseinates, lactalbumin), or from plant materials (e.g., including soy protein isolate, whey protein concentrate) or their hydrolysates or isolates, as described in Malone, 2005. This may not be optimal for human and animal nutrition.

[0009] Furthermore, the compositions known in the art have the disadvantage that many subjects have intolerance to soy, wheat, or dairy products, which are the main protein sources in the protein compositions known in the art, which is of particular interest in the context of elderly people and enteral nutrition.

[0010] There remains an unmet need for edible products that can replace meat and provide beneficial and controllable nutritional content, while at the same time being efficiently prepared.

[0011] Surprisingly, it has been found that edible nutritional compositions can be efficiently prepared based on cell cultures which can be preserved against spoilage in subsequent steps and optionally the cell-derived material of step (iii) can be mixed with one or more further consumable ingredients.

[0012] One aspect of the present invention is (i) culturing proliferating non-human animal cells of interest in vitro until said cells have expanded in number by at least twofold; (ii) harvesting the cells to obtain cell-derived material; (iii) preserving cell-derived material from the cells harvested in step (ii) against spoilage; (iv) optionally mixing the cell-derived material of step (iii) with one or more further consumable ingredients selected from the group consisting of one or more vitamins, one or more minerals, one or more aroma compounds, one or more food colorants, one or more fibers, ethanol, acetic acid, carbonic acid, and combinations of two or more thereof.

[0013] The edible nutritional composition obtained (or obtained) from such a method may have potential advantages such as, for example, supporting weight loss, enhancing metabolic rate, detoxifying the body, and improving organ function. The content of nutrients such as macronutrients (e.g., proteins, fatty acids, and carbohydrates, and dietary fiber) and micronutrient compositions (e.g., minerals, vitamins, amino acids) may be well controlled and adjustable as needed. By using controlled laboratory environmental conditions, undesirable materials such as xenobiotics (e.g., drug residues such as antibiotics), hormones and their precursors, pathogens, and high-calorie ingredients may be largely avoided. Thus, particularly clean conditions may be achieved in the preparation and edible nutritional composition. The ability to use chemically and biochemically defined cell culture media and cells allows for a stable and reliable production that is broadly independent of external factors. The aforementioned advantages are superior compared to natural animal-derived products such as meat. Moreover, the edible nutritional composition of the present invention is prepared in a particularly efficient manner.

[0014] The edible nutritional composition obtained (or obtained) from such a method may be suitable for nutrition of the elderly, as well as for enteral feeding (also enteral feeding, e.g. nasogastric feeding, e.g. nasogastric intubation, gastrostomy, jejunostomy, or other types of tube feeding, etc.).

[0015] The edible nutritional composition may contain cell-derived materials from animal cells (non-human) to design beverages that cater to various demographics and dietary preferences. This process requires only a small number of cells as raw material. This may greatly conserve resources and is believed to be more sustainable. Less land / space is required to prepare 1 kg of cell biomass of the edible nutritional composition of the present invention than to prepare 1 kg of cell biomass of animal meat. Less water is consumed. In addition, water recycling and efficient waste management are possible.

[0016] The release of unwanted emissions, such as methane gas emissions, is typically significantly less than that released during the same amount of production of animal-derived cellular products, such as meat.

[0017] The methods of the present invention can be scaled up to any desired extent.

[0018] Individual needs can be met and digestion of the edible nutritional composition is more effective (easier) than a comparable piece of meat. The nutrient content can be freely adjusted to the nutritional needs of the individual. The edible nutritional composition can allow a highly efficient or optimal route for absorbing valuable nutritional components, superior to other routes of administration.

[0019] Utilization and resorption can be improved. This can have beneficial effects in the medical field. In this context, prebiotics and drugs (such as ribonucleic acid-based drugs such as silencer RNA (siRNA)) can also be added and applied to consumers.

[0020] The term "edible" as used in the context of the present invention, as set forth herein, may be understood as generally understood in the art as being consumable without (substantial) health risk when consumed in appropriate amounts. Preferably, "edible" refers to an ingredient that is suitable for animal, particularly human, food consumption. This may also be interpreted in the context of official regulatory agency approval.

[0021] (Non-human) animal cells and their extracellular matrix (ECM) components, optionally together with further consumable ingredients (including "additives"), can be prepared as cell-based beverages from clean and controlled bioprocessing, as a new form of nutritional consumable / supplement, adapted to individual lifestyles as well as clinical and therapeutic requirements.

[0022] As used herein, the terms "beverage" and "drink" may be understood interchangeably in the broadest sense generally understood in the art as a liquid or syrupy, orally consumable composition, which may or may not contain alcohol.

[0023] Optionally, the edible nutritional composition may comprise an ethanol / water mixture. In certain embodiments of the present invention, the edible nutritional composition may comprise 0-80 wt%, 0-40 wt%, 0-6 wt%, 1-15 wt%, 5-15 wt%, or 10-40 wt% ethanol, based on the total composition. Optionally, the edible nutritional composition may comprise a water / fat emulsion. In preferred embodiments, the edible nutritional composition may comprise at least 0.5 wt%, at least 1 wt%, at least 2 wt%, at least 5 wt%, at least 10 wt%, or at least 25 wt% cell-derived material, based on the total weight of the composition.

[0024] The methods of the invention may also be considered as bioprocessing or cellular agriculture methods. The methods of the invention may be considered as being based on cultured cells as consumables. The methods of the invention may provide cell-based nutrition.

[0025] The taste of the edible nutritional composition can be sweet, salty, bitter, sour, umami, or a combination of two or more thereof.

[0026] (i) culturing a proliferating non-human animal cell of interest; The term "cell of interest" as used herein can be understood as one or more cell types intended to be used in the process. Preferably, the material derived from these cells should be contained in the edible nutritional composition. The cell of interest can be any non-human animal cell.

[0027] In a preferred embodiment, the cell of interest is Muscle cells or their precursor cells, in particular (muscle) satellite cells; Adipocytes or their precursor cells; Hepatocytes or their precursor cells; Induced pluripotent stem cells (iPSCs); non-human embryonic stem cells; Or a combination of two or more of them.

[0028] The cells of interest may be obtained by any means. When using primary cells, tissue pieces may be obtained from donor animals or animal-derived materials (e.g., eggs, placenta (reproductive tissue), skin, and / or hair, etc.). Optionally, they are washed and / or cut into small pieces. Optionally, the cells may be at least partially separated from each other by shearing forces and / or enzymes (e.g., collagenase I and / or dispase and / or (chymo)trypsin) and / or hypertonic solutions (e.g., 1 mM sodium citrate in potassium chloride / sodium chloride with a final osmolarity of 570 mOsm / kg). Optionally, the cells may be further treated with a complexing agent (e.g., ethylenediaminetetraacetic acid (EDTA) and / or sodium citrate). Optionally, the cells may be suspended in a suspension medium (e.g., phosphate-buffered saline (PBS)). Optionally, one or more antibiotics (e.g., penicillin, streptomycin, etc.) may be added. Optionally, the cells may be subjected to a centrifugation / resuspension cycle to wash. The cells may be added to suitable culture conditions.

[0029] Adult stem cells, such as satellite cells, can be obtained by any means. For example, such adult stem cells can be obtained as described in Schmidt et al. (Cellular and Molecular Life Sciences, 2019, 76:2559-2570), Hindi et al. (Science Signaling, 2013, 6(272):DOI:10.1126 / scisignal.2003832), Choi et al. (Food Sci.Anim.Resour., 2020, 40(5):852-859), and / or Nie et al. (PlosONE, 2014, 9(1):e88012). This can optionally include cellular factors, such as, for example, Pax3 and / or Pax7 for satellite cells, MyoD and / or Myf5 for myoblasts. Muscle cells can optionally be detected by myogenin. Myotubes may be myosin heavy chain (MyHC) positive.

[0030] Additionally, mature myotubes may be multinucleated, which can be detected using nuclear specific dyes such as DAPI and Hoechst. Optionally, magnetic activated cell sorting (MACS) can be used to separate cells from each other and / or isolate specific desired cell types (such as CD29 positive satellite cells, identifiable by immunostaining with CD29 and / or Pax7, using CD29 as a cell marker).

[0031] The cells (e.g., satellite cells) may optionally be subjected to ice-cooling as described in Benedetti et al. (Skeletal Muscle, 2021, 11(7):doi.org / 10.1186 / s13395-021-00261-w), and therefore ice-cold buffer (e.g., ice-cold PBS) may optionally be added to the flask when passaging the cells.

[0032] In a preferred embodiment, the cells of interest are or include muscle cells or their precursor cells, in particular (muscle) satellite cells. For example, bovine satellite cells can be cultured under conditions described in the art (see Skrivergaard et al., Int. J. Mol. Sci., 2021, 22:8376). In one embodiment, cells such as muscle cells or their precursor cells (e.g., (muscle) satellite cells), or other cell types, can be harvested from skeletal muscle tissue. This is exemplified in the experimental section below.

[0033] In further embodiments, the cells may originate from eggs (e.g., avian eggs, such as chicken eggs). Egg cells may be harvested before hatching (e.g., chicken embryos no older than about 21 days). This allows for obtaining cells at any developmental stage, such as embryonic stem cells, embryonic germ stem cells, progenitor cells, and fully developed functional cells.

[0034] In further embodiments, the cells may originate from bone marrow. From bone marrow, large amounts of mesenchymal stem cells can be obtained. For example, mesenchymal stem cells can differentiate into muscle cells, adipocytes, osteoblasts, neurons, and chondrocytes. Mesenchymal stem cells can secrete one or more growth factors / cytokines that are beneficial for the culture of cells. In further embodiments, the cells may originate from poultry feathers, such as, for example, follicular cells, which may have similar properties to mesenchymal stem cells.

[0035] In further embodiments, cells may be harvested from any organ / tissue of an organism from any kingdom of life, such as, for example, organs or tissues of one or more animals, one or more plants, one or more fungi, one or more protists, one or more eubacteria, and / or one or more archaea, for culturing and growing the cells and obtaining the macro- and micronutrients. Such cells may be added to the edible nutritional composition of the present invention.

[0036] The cells of interest can be primary cells or cells of long-term cell culture. In one embodiment, the cells of interest are primary cells. In another embodiment, the cells of interest are long-term cell cultures.

[0037] Primary cells may originate from a biopsy or autopsy. A cell culture may be established. The long-term cell culture may be a (optionally commercially available) cell bank. The long-term cell culture may include an immortalized cell line. Optionally, induced pluripotent stem cells (iPSCs) and / or non-human embryonic stem cells may be induced to form the desired cell type. Such steps may also be included in the present invention. In one embodiment, the cells of interest are somatic mature cells. Animal slaughter may be reduced or avoided. This may be desirable for ethical reasons and from an animal welfare perspective.

[0038] The cells of interest may originate from any non-human animal species ("donor"). For example, the cells of interest may be derived from mammals (mammals) (e.g., sheep, cow / beef, goat, pig, buffalo, deer, horse, camel, rabbit, etc.), birds (poultry, avian cells) (e.g., duck, chicken, goose, turkey, goose, pigeon, adult chicken, etc.), reptiles (e.g., alligator, crocodile, snake, etc.), fish (e.g., salmon), or insects (e.g., mealworms, bee larvae, etc.). Avian cells have been used for the production of vaccines, which may allow for the obtaining of serum-free production cells in suspension on a relatively large scale and scalable from commercial sources.

[0039] In one embodiment, the cells of interest may originate from a ruminant, which may be selected from the group consisting of, for example, bovine, ovine, caprine, deer, and buffalo cells. In one embodiment, the cells of interest may originate from a non-ruminant, such as, for example, a porcine cell. In one embodiment, the cells of interest may originate from a pseudoruminant, which may be selected from the group consisting of, for example, horse, camel, and rabbit cells. The cells of interest may originate from any non-human animal species ("donor").

[0040] In one embodiment, the cell of interest may be a primary cell of mammalian (mammalian), avian, reptile, fish, or insect origin. In another embodiment, the cell of interest may be a cell from a long-term cell culture of a mammal, bird, reptile, fish, or insect. In one embodiment, the cell used in the context of the present invention is a primary mammalian cell (also a mammalian cell). In one embodiment, the cell used in the context of the present invention is from a mammalian cell culture (also a mammalian cell culture).

[0041] The cells may optionally be stored as cell stocks in any condition available for such purposes, such as in a refrigerator (e.g., 1-10°C, e.g., about 4°C), a freezer (e.g., -30 to 0°C, e.g., about -20°C), a high-performance freezer (e.g., -90 to -70°C, e.g., about -80°C), or in liquid nitrogen (N2). Optionally, the frozen stocks may or may not contain dimethyl sulfoxide (DMSO).

[0042] Optionally, different cell types and / or cells of different species may be combined with each other, which may optimize the nutrient content of the edible nutritional composition.

[0043] For example, cells may be combined with each other, whereby cells of at least two different cell types may be combined with each other, which may optionally be of the same or different species origin. For example, the following cells may be combined with each other, whereby cells of at least two different species, such as; Cells from two or more different bird species, Two or more different mammalian cells, Cells from two or more different fish species, Cells from two or more reptile species, Cells from two or more different insect species, cells of at least one avian species and at least one mammalian species; cells from at least one bird species and at least one fish species; cells from at least one bird species and at least one reptile species; cells of at least one bird species and at least one insect species; at least one mammalian cell and at least one fish cell; cells from at least one mammalian species and at least one reptilian species; at least one mammalian cell and at least one insect cell; cells from at least one fish species and at least one reptile species, or Cells from at least one fish species and at least one insect species may be combined with each other.

[0044] The ratio of the cell numbers of the two cell types can be in any range, for example, the ratio of the cell numbers can be in the range of 1:1 to 1:1000, 1:1.5 to 1:500, 1:2 to 1:200, 1:3 to 1:100, 1:5 to 1:50, or 1:5 to 1:10.

[0045] For example, cells may be combined with each other, whereby cells of at least three different cell types may be combined with each other, which may optionally be of the same or different species of origin. For example, cells may be combined with each other, whereby cells of at least three different species, such as; Cells from three or more different bird species, Three or more different mammalian cell lines, Cells from three or more species of fish, Three or more species of reptile cells, Cells from three or more different insect species, Cells from at least two avian species and at least one mammalian species; Cells from at least two bird species and at least one fish species, Cells from at least two bird species and at least one reptile species, Cells from at least two bird species and at least one insect species, Cells from at least two mammalian species and at least one avian species, Cells from at least two mammalian species and at least one fish species, cells from at least two mammalian species and at least one reptilian species; at least two mammalian and one insect cell lines; Cells from at least two fish species and at least one bird species, Cells from at least two fish species and at least one mammalian species; Cells from at least two species of fish and at least one species of reptile, Cells from at least two fish species and at least one insect species, Cells from at least two reptile species and at least one bird species, cells from at least two reptilian species and at least one mammalian species; Cells from at least two species of reptiles and at least one species of fish; Cells from at least two reptile species and at least one insect species, Cells from at least two insect species and at least one bird species, Cells from at least two insect species and at least one mammalian species, Cells from at least two insect species and at least one fish species, or Cells from at least two insect species and at least one reptile species may be combined with each other.

[0046] The ratio of cell numbers of the three cell types can be in any range. For example, the ratio of cell numbers can be in the range of 1:1:1, 2:1:1, 2:2:1, 3:2:1, 4:2:1, 4:2:1, 4:3:1, 4:4:1, 4:2:1, 4:1:1, 4:3:2, 4:4:2, 5:1:1, etc.

[0047] Such cells of different species origin may be of the same cell origin or of different cell origin. In one embodiment, all the cells are of the same cell type origin. In another embodiment, the cells are of different cell types origin. In one embodiment, all the cells are muscle cells (particularly (muscle) satellite cells). For example, poultry and mammalian muscle cells (particularly (muscle) satellite cells) may be mixed. For example, poultry and fish muscle cells (particularly (muscle) satellite cells) may be mixed. For example, pork and salmon muscle cells (particularly (muscle) satellite cells) may be mixed. For example, beef and salmon muscle cells (particularly (muscle) satellite cells) may be mixed. For example, pork and beef muscle cells (particularly (muscle) satellite cells) may be mixed. For example, pork, salmon and beef muscle cells (particularly (muscle) satellite cells) may be mixed (e.g., in a 1:1:1 ratio).

[0048] The cells can be cultured under any conditions suitable for cell proliferation. In a preferred embodiment, the cells are cultured until the cell number of the cells is increased by at least 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, or 25-fold.

[0049] Preferably, the cells of interest are cultured under sterile and controlled conditions to prevent contamination and provide beneficial growth conditions. External factors such as temperature, pH, and humidity can be controlled. Cultivation may be performed in laboratory-scale incubators, or on an industrial scale. For mammalian cells, cell growth conditions may be, for example: 37°C, about 5% CO2 by weight, about 95% air, and about 85-95% relative humidity. Culture conditions may be adapted to the respective cells of interest. For avian cells, the culture temperature may be 40-42°C. For example, on a laboratory scale, cells may be cultured in plastic containers such as glass or plastic Petri dishes, glass flasks, or plastic flasks. Typically, the pH range is in the cell physiological range (pH 7.0-7.4). Optionally, the cell culture device may be pre-coated (e.g., with proteins such as collagen (type I), gelatin, etc.), which may improve the initial attachment of the cells.

[0050] Bioreactors may also be used to culture cells. This may be any type of bioreactor. For example, suitable bioreactors may be stirred tank bioreactors, wave bioreactors, airlift bioreactors, packed bed bioreactors, vertical wheel bioreactors, or hollow fiber bioreactors.

[0051] The method of the present invention can be scaled up to any desired extent. The bioreactor can have any size, such as less than 10 L, 10-50 L, 20-100 L, 50-250 L, 100-1000 L, or more than 1000 L in volume.

[0052] Culture conditions (e.g., pH, gas, temperature, etc.) can be controlled. An advantage of using a bioreactor over traditional 2D monolayer cultures can be the ability to optionally increase cell density up to 109 cells / mL cell culture medium.

[0053] Cells may be cultured as suspension cells, adherent cells, semi-adherent cells, or a combination thereof. Optionally, for adherent cells, the surface may be treated by plasma treatment, collagen coating, etc. to improve the initial attachment of cells and allow them to reach a proliferative state (division and increase in size) or a terminally differentiated or quiescent state to achieve a cell monolayer for the subsequent growth step (attachment to the surface).

[0054] Any suitable cell culture medium suitable for the respective cell type may be used. Typically, cell culture media suitable for the growth of animal cells include one or more carbon sources, such as sugars. Optionally, the cell culture medium may include supplements, such as amino acids, fatty acids, vitamins, minerals, growth factors, trace elements, etc. A person skilled in the art will be aware of cell culture media suitable for multiple cell types. For example, for (muscle) satellite cells, B8 medium and Beefy-9 medium may be used, which contain minimal components such as DMEM / F-12 medium as a basal medium, in combination with recombinant insulin, L-ascorbic acid 2-phosphate, recombinant transferrin, sodium selenite, NRG1, FGF2, TGF-β1, HEPES, and bicarbonate, and may be modified according to the specific growth conditions.

[0055] Optionally, medium components such as glucose, amino acids, metabolites: lactate, ammonia, active growth factors: FGF2 and / or TFG-b1 can be measured and controlled during culture.

[0056] The cell culture medium may or may not contain fetal bovine serum (FBS) (or fetal calf serum (FCS)). The cell culture medium may or may not contain animal-derived serum. In a preferred embodiment, step (i) is performed in the absence of animal-derived serum. Fetal bovine serum (FBS) is commonly used at 5-20% by weight in conventional cell culture to reduce the total cost of cell culture. The disadvantages of using FBS are multiple, including the slaughter of fetal bovine animals, the possibility of contamination originating from animal sources such as bacteria and pathogens, and high processing and storage costs.

[0057] As mentioned above, the beneficial technical effect of the present invention may be that it can control the ingredient content in edible nutritional composition.Therefore, in a preferred embodiment, the method comprises adjusting the content ratio and type of cells cultured in step (i) to obtain a desired defined nutrient content, in particular a desired defined protein content.Therefore, depending on the type of cells used as the desired cell, the type and content range of the desired protein and optionally its metabolites (e.g. peptides and amino acids) may be adjusted.

[0058] (ii) harvesting the cells Step (ii) of harvesting cells to obtain cell-derived material can be carried out by any means known in the art. If cells are cultured as adherent or semi-adherent cells, step (ii) of harvesting cells to obtain cell-derived material can include detaching cells from the substrate. This can be done mechanically (e.g., by scratching) or assisted by one or more enzymes (e.g., trypsin) as generally known in the art. A cell suspension can be obtained.

[0059] In one embodiment, step (ii) comprises administering a solution containing a citrate (e.g., sodium citrate) to the cells to be harvested. In one embodiment, the solution containing a citrate (e.g., sodium citrate) is a buffer, in particular phosphate buffered saline (PBS). In one embodiment, the citrate is a water-soluble citrate. In one embodiment, the citrate is sodium citrate. In one embodiment, the solution containing a citrate (e.g., sodium citrate) is administered to the cells for 2-20 minutes, 5-10 minutes, or 10-15 minutes, and then the cells are washed off. Thereby, the cells may be detached from their substrate, especially if the cells grew adherently (at least partially or completely) in step (i). The concentration of the citrate (e.g., sodium citrate) may be adjusted. In one embodiment, the concentration of citrate (e.g., sodium citrate) may range from 0.01 to 10 mM, or 0.05 to 5 mM, or 0.1 to 4 mM, or 0.2 to 3 mM, or 0.5 to 5 mM, or (about 1 mM) of citrate ion (including fully deprotonated citric acid, hydrogen citrate, and dihydrogen citrate). In one embodiment, sodium citrate may be used in the range of 0.01 to 10 mM, or 0.05 to 5 mM, or 0.1 to 4 mM, or 0.2 to 3 mM, or 0.5 to 5 mM, or (about 1 mM).

[0060] In one embodiment, the step (ii) of harvesting the cells further comprises at least partially separating the cells from the cell culture medium. This can be achieved by any means. In a preferred embodiment, the step (ii) comprises removing the cell culture medium. In such a step, based on the total amount of cell culture medium present at the end of the culturing step (i), at least 10% by weight, at least 25% by weight, at least 50% by weight, at least 70% by weight, at least 80% by weight, at least 90% by weight, at least 95% by weight, at least 98% by weight, or at least 99% by weight, or (essentially) all of the cell culture medium present at the end of the culturing step (i), can be removed.

[0061] At least partially removing the cell culture medium from the cells can be accomplished by any means. In a preferred embodiment, step (ii) comprises removing the cell culture medium by centrifugation, filtration, dialysis, or rinsing of the adherent cells. Such a step may optionally comprise an optional washing step with one or more buffers. Such a washing step may also remove residuals and metabolites. In a preferred embodiment, step (ii) comprises removing the cell culture medium by centrifugation and an optional washing step with one or more buffers.

[0062] Optionally, the removed cell culture medium or its water may be recycled. Optionally, this may be used in the process as the cell culture medium in step (i). Waste management may be used to further use the nutrients contained in the spent cell culture medium. Thus, optionally, nutrients that have not yet been consumed may also be recycled to step (i).

[0063] In an alternative embodiment, step (ii) comprises retaining and pasteurizing at least a portion of the cell culture medium.

[0064] Optionally, the cells may be redissolved in a buffer solution or the like. Preferably, one or more of the buffering agents contained in such a buffer solution are consumable at a moderate concentration. Optionally, such a buffer solution may contain a buffering agent selected from the group consisting of histidine, acetate, citrate, glycine, phosphate, or tris(hydroxymethyl)aminomethane (TRIS).

[0065] Optionally, the step (ii) of harvesting cells to obtain cell-derived material comprises the further step of rendering at least a portion of the cells non-viable, in particular destroying the cells. This can be achieved by any means, such as, for example, freeze-thaw cycles, freeze-drying, applying ultrasound, mechanical stress (such as French press or homogenizer), etc. This can provide cell-derived material containing dead cell-derived material. Such dead cell-derived material can include cell membranes and their fragments, as well as internal parts of cells, such as cell lysates containing cytoplasm and / or cell organelles.

[0066] In a preferred embodiment, the released protein is preferably at least partially dissolved in the edible nutritional composition. Proteins may be susceptible to precipitation or even unstable near the isoelectric point, so it is preferred to avoid such a pH range. In a preferred embodiment, the method of the present invention does not involve discarding certain parts of the cell. In a preferred embodiment, the cell-derived material and the final edible nutritional composition include the cell membrane and its fractions, as well as the interior parts of the cell, such as the cell lysate, including the cytoplasm and / or cell organelles. In a preferred embodiment, the cell-derived material (and the final edible nutritional composition) includes (essentially) the entire contents of the cell.

[0067] In a preferred embodiment, the cells of interest may be muscle cells. The protein of interest may be a protein present in the edible nutritional composition, which may be a myofibrillar protein. Myofibrillar proteins are the most common proteins in muscle tissue and have an isoelectric point around pH 5.5. Therefore, it is preferable to avoid a pH range of about 5.5 (e.g., pH 5-6, preferably pH 5.2-5.8, particularly pH 5.4-5.6).

[0068] Optionally, the cell-derived material may or may not be sonicated (e.g., as described in Chen et al., Ultrasonics Sonochemistry, 2021, 76:105652).

[0069] A buffer for maintaining a protein in solution may optionally contain additives that stabilize the solution. For example, such a buffer may contain a salt (e.g., sodium chloride) for stabilizing a protein (e.g., myofibrillar protein). Optionally, the mass ratio of salt (e.g., sodium chloride) / dried cell-derived material may be set at 100:4-4.5 to ensure the solubility of the protein in the liquid. Alternatively or additionally, a stabilizer such as tripolyphosphate, tetrasodium pyrophosphate, tetrapotassium pyrophosphate, sodium tripolyphosphate, or potassium tripolyphosphate, or a combination of two or more thereof, may be added to increase the solubility of the protein (e.g., myofibrillar protein).

[0070] In an alternative embodiment, viable cells may be harvested. This may be optionally achieved by culturing the cells in suspension culture. The cells, including the cell culture medium, may further be used as cell-derived material.

[0071] In an alternative embodiment, viable cells may be harvested by applying moderate centrifugal force or filtration at low shear stress to cells grown in suspension culture or adherent cells previously detached by gentle means such as enzymes. Cells may be harvested as described in Nienow et al., (Biochemical Engineering Journal, 2014, 85:79-88).

[0072] In one embodiment, the cells or fragments thereof represent cell-derived material that has been separated from the medium and is free of cell culture medium, while in another embodiment, the cell-derived material still contains at least a portion of the cell culture medium.

[0073] (iii) preserving the cell-derived material against spoilage; Step (iii) of preserving the cell-derived material from spoilage can be carried out by any means. For example, the cell-derived material can be freeze-dried, fermented, pickled, smoked, or dried to avoid microbial growth. Alternatively or additionally, the product can be heated and reducing sugars such as glucose, lactose, and maltose can be optionally added to achieve the Maillard reaction during heating. Alternatively or additionally, a preservative (e.g., benzoic acid) can be optionally added.

[0074] In a preferred embodiment, step (iii) comprises processing the cell-derived material into a powder. This can be achieved by any means known in the art. In a preferred embodiment, processing the cell-derived material into a powder is obtained by at least partially removing the liquid content. This can be achieved by any means known in the art. Processing the cell-derived material into a powder can be achieved by lyophilization or another drying step. In a preferred embodiment, processing the cell-derived material into a powder is obtained by lyophilization.

[0075] Lyophilization is understood in the broadest sense and may include (classical) freeze-drying or spray freeze-drying. Lyophilization may be carried out with or without prior homogenization of the suspension. The freeze-drying procedure has been generally known to those skilled in the art for several decades (see US Pat. No. 3,964,174). Freeze-drying is also suitable for preserving meat-based products such as ham while essentially maintaining their nutritional content (see Ma et al., Saudi Journal of Biological Sciences, 2018, 25:724-732). For example, the conditions described in Ma et al. or US Pat. No. 3,964,174 may be used in the context of the present invention.

[0076] When performing a freeze-drying step, cryopreservatives and freeze-drying preservatives may be added optionally. The cryopreservatives and freeze-drying preservatives may include, for example, an agent selected from the group consisting of trehalose, sucrose, sorbitol, dextran, sodium chloride, disodium sulfate, diammonium sulfate, glycerol, polyphenols, proline, glycine, glutamic acid, histidine, arginine, 4-hydroxyproline, L-serine, β-alanine, lysine, hydrochloride, sarcosine, or γ-aminobutyric acid, and combinations of two or more thereof. Furthermore, in such a freeze-drying step, one or more bulking agents may be added optionally. These may improve the mechanical properties and appearance of the final freeze-dried product. The bulking agent may be optionally selected from the group consisting of mannitol, lactose, and combinations thereof.

[0077] In a preferred embodiment, the cell culture medium is at least partially removed from the cell-derived material harvested in step (ii). In an alternative preferred embodiment, the cell-derived material from the cells harvested in step (ii) still contains cell culture medium and is a liquid or pasty composition.

[0078] In a preferred embodiment, step (iii) comprises pasteurization. Pasteurization may be understood in the broadest sense as a method of treating an edible nutritional composition with mild heat, usually below 150°C (e.g., 45-150°C, 50-125°C, 55-100°C, or 60-95°C), to prevent microbial growth. Pasteurization may be intended to destroy or inactivate microorganisms and / or enzymes that contribute to spoilage or disease risk. In a preferred embodiment, step (iii) comprises pasteurization, in which the cell-derived material from the cells harvested in step (ii) still contains cell culture medium and is a liquid or pasty composition.

[0079] For example, the storing step (iii), which includes lyophilization or drying, may also optionally include the step of adding one or more antioxidants, such as, for example, vitamin D, vitamin E, protein hydrolysates, sodium sulfite, or a combination of two or more thereof, which may be added to prevent oxidation.

[0080] Optionally, the lyophilized or dried product (which may be, for example, a powder or foam) may be milled to form a homogenous powder. Any such milling process may be applied using a mill.

[0081] Optionally, the resulting dry powder can be the final product (edible nutritional composition).Alternatively or additionally, such powder can be redissolved or resuspended in a suitable liquid, such as water, a consumable aqueous buffer, or a mixture thereof, including ethanol.Thus, in a preferred embodiment, the edible nutritional composition is a beverage composition, step (iii) comprises processing the cell-derived material into a powder, and the method comprises the further step of suspending the powder in an aqueous liquid, in particular mineral water or tap water.

[0082] Alternatively or additionally, such powders may be further processed into granules.

[0083] The product (e.g., powder, or liquid, or gel) resulting from step (iii) may optionally be sealed. The product may be prepared and / or subsequently processed at or below room temperature (i.e., below 20° C.), at room temperature (e.g., (approximately) 20° C.), or at elevated temperature (above 20° C., e.g., 60-120° C. for faster dissolution). The product may optionally be sterilized (e.g., pasteurization, heating, irradiation with ultraviolet (UV) or X-rays, gamma rays, etc.).

[0084] In a preferred embodiment, the method comprises the steps of: (a) fermenting an edible nutritional composition; (b) smoking the edible nutritional composition; (c) soaking the food product with a composition comprising an edible nutritional composition, preferably in combination with one or more further ingredients, such as at least 1% by weight of the composition of sodium chloride, at least 1% by weight of the composition of one or more sugars, at least 1% by weight of the composition of acetic acid, at least 0.5% by weight of the composition of ethanol, or at least 5% by weight of the composition of one or more edible oils; and / or; (d) thickening the edible nutritional composition to obtain a syrup, in particular by adding sugar and / or other sweeteners to obtain a sweet syrup.

[0085] As used herein, fermenting can be understood in the broadest sense as any kind of metabolic process that produces a chemical change in an organic substrate contained in the composition through the action of enzymes. Such enzymes can optionally originate from, be added to, and / or be present in the form of microorganisms and / or be secreted from such. In one embodiment, fermentation can be used to increase umami taste. In one embodiment, fermentation can be used to extend shelf life. In one embodiment, the fermented edible nutritional composition can contain at least 0.5% by weight, at least 1% by weight, at least 2% by weight, at least 5% by weight, at least 10% by weight of cell-derived material based on the total weight of the composition after the fermenting step.

[0086] In preferred embodiments, the edible nutritional composition comprises a substantial amount of killed material derived from cells of interest. In preferred embodiments, the edible nutritional composition comprises killed material derived from cells of interest and live cells of interest in a mass ratio of at least 2:1, at least 3:1, at least 4:1, at least 5:1, at least 10:1, at least 50:1, at least 100:1, or killed material derived from cells that are (essentially) free of live cells.

[0087] The thickening step may be carried out by any means. It may involve partial removal of water and / or addition of further ingredients with thickening properties, such as, for example, one or more sugars, one or more other (poly)saccharides (e.g., starch, resistant starch, agar, pectin, inulin, fructan, raffinose, polydextrose), one or more thickening sweeteners, one or more thickening agents (e.g., cellulose, hemicellulose, carboxymethylcellulose, carboxyethylcellulose, carboxypropylcellulose, chitin, beta-glucan, raw guar gum, xanthan gum, lignin, polyuronides, alginic acid or a salt thereof (e.g., sodium alginate), or a combination of two or more thereof).

[0088] Thickening of edible nutritional compositions may improve swallowing properties and, when adjusted to the desired extent, may prevent aspiration (see Nishinary et al., npj Science of Food, 2019, 3:5). Mixing with a thickening agent (such as sodium alginate) may optionally be used to improve the solubility of proteins (such as myofibrillar proteins) (see Li et al., Food Biophysics, 2020, 15:113-121). Optionally, thickening of soluble aggregates of myofibrillar proteins may also be obtained by mixing and heating with gallic acid (see Chen et al., J. Agric. Food Chem., 2020, 68:11535-11544). In contrast to these methods, in a preferred embodiment of the present invention, the myofibrillar proteins are not purified or separated from other proteins or components of the cultured cells. This allows the entire nutrient content of the cells to be obtained.

[0089] In the context of the present invention, dead material derived from the cells of interest may be understood as material originally contained in the cells of interest harvested in step (ii) (optionally no longer present in viable cells). In the context of the present invention, cell viability may be understood as the ability to grow under suitable conditions.

[0090] In a preferred embodiment, the edible nutritional composition comprises: (a) a beverage composition, particularly a beverage composition having a mass ratio of killed material derived from cells of interest to viable cells of interest of at least 2:1 or greater; (b) aqueous solutions and water-suspendable powders or granules, particularly those having a mass ratio of killed material derived from the cells of interest to viable cells of interest of at least 2:1 or more; or (c) A liquid or pasty composition comprising a cell culture medium and live and / or dead cells, preferably wherein the cell-derived material from step (ii) comprises the cell culture medium in which the cells were cultured in the preceding step (i), in particular wherein the liquid or pasty composition is pasteurized.

[0091] In alternative embodiments, the cells of interest remain (essentially) viable in the product of interest, for example, greater than 50% or greater than 75% of the cells may remain viable, based on the total cells harvested in step (ii).

[0092] and (iv) the optional step of combining one or more additional consumable ingredients. As noted above, the edible nutritional composition may optionally further comprise one or more additional consumable ingredients selected from the group consisting of one or more vitamins, one or more minerals, one or more aroma compounds, one or more food colorants, one or more fibers, ethanol, acetic acid, carbonic acid, and combinations of two or more thereof. Ingredients may also be referred to interchangeably as components, additives, and the like.

[0093] This may allow to compensate for common micronutrient deficiencies of consumers (such as iron, vitamin A, and iodine, or zinc, folic acid, vitamin B12, other vitamin B, vitamin C, vitamin D, calcium, selenium, and fluoride). In one embodiment, this may allow to compensate for micronutrient deficiencies of elderly consumers. In one embodiment, this may allow to compensate for micronutrient deficiencies of consumers with chewing and / or swallowing problems, and perhaps even consumers suffering from dysphagia. Thus, nutrients can be efficiently supplemented to consumers who have difficulty chewing and / or swallowing large amounts of meat. The edible nutritional composition may also be optionally used for enteral nutrition (such as nasogastric feeding, e.g. nasogastric intubation, gastrostomy, jejunostomy, or other types of tube feeding).

[0094] The vitamin can be any vitamin known in the art.For example, the vitamin that can be added is selected from the group consisting of vitamin A, vitamin B (e.g., vitamin B1, vitamin B2, vitamin B3, vitamin B5, vitamin B6, folic acid, vitamin B12), vitamin C, vitamin D, and metabolic precursors, and combinations of two or more thereof.The vitamin or its metabolic precursors can be of natural or synthetic origin (same structure as natural or artificial).They can be commercially available.

[0095] Preferably, the edible nutritional composition comprises a total of 20% by weight or less, 10% by weight or less, 5% by weight or less, 2% by weight or less, or 1% by weight or less of vitamins, based on the total weight of the edible nutritional composition.

[0096] Minerals can be any nutrient that can have nutritional value for consumers. Typically and preferably, minerals include ions or complexes of metals, such as metals selected from the group consisting of iron, copper, calcium, zinc, and combinations of two or more thereof. Furthermore, minerals can be selenium, iodine, and / or fluoride. Minerals may be commercially available. Preferably, the edible nutritional composition includes minerals in a total amount of 20% by weight or less, 10% by weight or less, 5% by weight or less, 2% by weight or less, or 1% by weight or less, based on the total mass of the edible nutritional composition.

[0097] The aroma compound may be any edible compound known in the art that modifies flavor or taste. Thus, the flavor or taste is rather freely selectable and may optionally include non-meat flavors, even exotic flavors. Preferably, the aroma compound has a noticeable effect on the taste of the edible nutritional composition when applied in small amounts. Preferably, the edible nutritional composition contains aroma compounds in a total amount of 20% by weight or less, 10% by weight or less, 5% by weight or less, 2% by weight or less, or 1% by weight or less, based on the total mass of the edible nutritional composition. The aroma compound may be of natural or synthetic origin (structure is identical to natural or structure is artificial). For example, the aroma compound may be menthol, furaneol hexyl, cinnamaldehyde, isovaleraldehyde, anisaldehyde, cuminaldehyde, glutamic acid, fructone, ethyl methylphenylglycidate, dihydrojasmone, oct-1-en-3-one, 2-acetyl-1-pyrroline, 6-acetyl-2,3,4,5-tetrahydropyridine, delta-octalactone, massoialactone, diacetylacetoin, nerolin, and combinations of two or more thereof. Many aroma compounds are known to those skilled in the art and may be commercially available.

[0098] Food coloring is any edible dye known in the art that changes the color of food, also called food coloring. Preferably, the food coloring has a noticeable effect on the color of the edible nutritional composition when applied in small amounts. The coloring can be freely selected and can optionally be a non-meat coloring.

[0099] The edible nutritional composition may have any texture that can be defined by adding to the edible nutritional composition.For example, one or more polypeptides of the extracellular matrix (e.g., collagen, elastin, etc.) may be added to the edible nutritional composition as texturing and / or nutrients.The nutritional value may be equivalent to meat or may be superior to meat.

[0100] Preferably, the edible nutritional composition comprises a total amount of food colorings of 20% by weight or less, 10% by weight or less, 5% by weight or less, 2% by weight or less, or 1% by weight or less, based on the total mass of the edible nutritional composition. The food colorings may be of natural or synthetic origin (either identical in structure to natural or artificial in structure). For example, food colorings include beetroot juice, beta-carotene, quinoline yellow, Ponceau 4R, patent blue V, green S, brilliant blue FCF, citrus red 2, orange B indigotine, fast green FCF, erythrosine, allura red AC, tartrazine, sunset yellow FCF, or a combination of two or more thereof. The food colorings may even be fluorescent, such as fluorescein. Many food colorings are known to those skilled in the art and may be commercially available.

[0101] Fiber is any edible fiber, which may also be referred to as dietary fiber. Typically, fiber in the context of the present invention is a plant-derived food ingredient that cannot be completely degraded by human digestive enzymes. For example, fiber may originate from legumes, whole grains, and cereals, vegetables, fruits, nuts, or seeds. For example, fiber may include or consist of cellulose, hemicellulose, chitin, pectin, resistant starch, or a combination of two or more thereof. Fiber may also include inulin, beta-glucan, raw guar gum, xanthan gum, fructan, lignin, polyuronides, alginic acid or a salt thereof (e.g., sodium alginate), agar, carrageenan, raffinose, polydextrose, or a combination of two or more thereof. Many fibers are known to those skilled in the art and may be commercially available.

[0102] Based on the manufacturing process of the present invention, and as an ingredient of the combined finished food product, proteins of the extracellular matrix (ECM), such as collagen type I, hyaluronic acid, pyorhinin, vinculin, laminin, fibronectin, etc., may be included in the composition.

[0103] The edible nutritional composition may optionally further comprise one or more ingredients as described in Malone, 2005 ("Enteral Formula Selection: A Review of Selected Product Categories", Nutrition Issues in Gastroenterology, Series #28, ed. Carol Rees Parrish, Practical Gastroenterology). The content ranges may optionally be similar to those taught in Malone, 2005, but preferably replace milk-derived and / or plant-derived protein sources with cell-derived materials as (preferably the main or only) protein sources. Preferably, the edible nutritional composition comprises cell-derived materials as protein sources, and preferably does not comprise milk-derived and / or plant-derived protein sources, in particular does not comprise (essentially) milk-derived and does not comprise (essentially) plant-derived protein sources.

[0104] In one embodiment, the edible nutritional composition may comprise ethanol, optionally in addition to one or more of the further ingredients described herein. In certain embodiments of the present invention, the edible nutritional composition may comprise a total amount of 0-80% by weight, 0-40% by weight, 0-6% by weight, 1-15% by weight, 5-15% by weight, or 10-40% by weight of ethanol, based on the total weight of the edible nutritional composition.

[0105] In one embodiment, the edible nutritional composition may comprise acetic acid, optionally in addition to one or more of the further ingredients described herein. In certain embodiments of the present invention, the edible nutritional composition may comprise a total amount of acetic acid of 0-20% by weight, 0-6% by weight, 1-15% by weight, 5-15% by weight, or 10-20% by weight, based on the total weight of the edible nutritional composition.

[0106] In one embodiment, the edible nutritional composition may contain carbonation, optionally in addition to one or more of the further ingredients described herein. In particular embodiments of the present invention, the total amount of carbonation may be 0-5% by weight, 0.1-2% by weight, 0.2-1% by weight, 0.3-0.8% by weight, or 0.4-0.7% by weight, based on the total mass of the edible nutritional composition. These contents refer to a fresh product, preferably provided in an (essentially) sealed container (e.g., a bottle). The presence of carbonation may provide an effervescent beverage. In one embodiment, the edible nutritional composition may be fermented in such a container (e.g., a bottle), i.e. the container may optionally function as a fermenter.

[0107] Optionally, the edible nutritional composition may include one or more sugars, optionally in addition to one or more further components described herein. For example, one or more sugars such as saccharose, glucose, fructose, trehalose, sucrose, sorbitol, dextran, etc. This may improve the solubility of the cell-derived material at different pH values ​​and / or may also improve the taste. For example, the edible nutritional composition may include 0-80% by weight, 0-40% by weight, 0-6% by weight, 1-15% by weight, 5-15% by weight, or 10-40% by weight of the total amount of sugars, based on the total mass of the edible nutritional composition.

[0108] Optionally, the edible nutritional composition may include one or more amino acids, optionally in addition to one or more additional ingredients described herein. For example, it may include one or more natural amino acids. This may supplement a consumer in need thereof. For example, the edible nutritional composition may include 0-80% by weight, 0-40% by weight, 0-6% by weight, 1-15% by weight, 5-15% by weight, or 10-40% by weight of amino acids based on the total mass of the edible nutritional composition.

[0109] Optionally, the edible nutritional composition may include one or more polypeptides (or proteins) in addition to one or more additional ingredients described herein. For example, the edible nutritional composition may include one or more polypeptides of natural origin (e.g., obtained from milk, bacteria, plant-based expression systems, etc.), which may supplement consumers who require amino acids. For example, the edible nutritional composition may include 0-80% by weight, 0-40% by weight, 0-6% by weight, 1-15% by weight, 5-15% by weight, or 10-40% by weight of the total amount of polypeptides, based on the total mass of the edible nutritional composition.

[0110] Optionally, the edible nutritional composition may contain one or more fats (also lipids) and / or fatty acids, optionally in addition to one or more of the further ingredients described herein. For example, it may contain one or more fats and / or fatty acids of natural origin (e.g., obtained from plants, fish, and / or milk). It may contain essential fatty acids. It may contain unsaturated fatty acids or polyunsaturated fatty acids. It may supplement consumers who require fatty acids, especially essential fatty acids. For example, the edible nutritional composition may contain 0-80% by weight, 0-40% by weight, 0-6% by weight, 1-15% by weight, 5-15% by weight, or 10-40% by weight of fats and / or fatty acids, based on the total mass of the edible nutritional composition.

[0111] Optionally, the edible nutritional composition may optionally contain one or more electrolytes in addition to one or more additional components described herein.Its osmotic pressure may be hypotonic, isotonic or hypertonic compared to human blood (herein understood as the osmotic pressure of aqueous saline solution consisting of water and 0.9% by weight of sodium chloride).The osmotic pressure may be adapted and adjusted according to the total content of dissolved components in the edible nutritional composition.

[0112] Optionally, the edible nutritional composition may comprise pharma- ceutical active ingredients, such as, for example, adaptogens, growth factors, polyphenols, antibodies or antigen-binding fragments thereof, small molecule drugs, prebiotics, and / or nucleic acids (e.g., ribonucleic acid (RNA) (e.g., siRNA), deoxyribonucleic acid (DNA)), optionally in addition to one or more of the further ingredients mentioned herein.

[0113] In one embodiment, the edible nutritional composition may also be suitable for enteral administration, such as enteral feeding (nasogastric feeding, e.g., nasogastric intubation, gastrostomy, jejunostomy, or other types of tube feeding).

[0114] In an alternative embodiment, the edible nutritional composition may be suitable for parenteral administration, optionally in addition to one or more of the further components described herein.In an embodiment, the edible nutritional composition may be suitable for intravenous injection / infusion.In this case, the edible nutritional composition may be filtered.The edible nutritional composition may be suitable for treating either cellular dehydration or hypervolemia.

[0115] Optionally, the edible nutritional composition may include one or more juices, in addition to one or more additional ingredients described herein. For example, the edible nutritional composition may include one or more fruit juices or vegetable juices.

[0116] The pH of the edible nutritional composition may be adjusted to a desired range. Preferably, the pH is essentially neutral or acidic. In one embodiment, the pH is within the range of pH 2-9, pH 2-8, pH 3-7, pH 4-7, pH 5-7, or pH 4-6. The pH may be adjusted by edible acids, bases, buffers, and / or acidity regulators.

[0117] In a preferred embodiment, a high quality nutritional liquid product can be obtained according to individual requirements. The edible nutritional composition can be adjusted to the lifestyle requirements of an individual, so that its content range can be adapted to the desires of a particular consumer or consumer group. This can be considered as a "customizable food" and / or "personalized food".

[0118] The edible nutritional composition may be a cell-based beverage, which may also be considered an "animal drink" or a "cell drink", which may also be understood as a "clean meat", i.e., an animal-like nutritional source that is not obtained as meat from slaughtered animals.

[0119] In a preferred embodiment, step (iv) comprises at least 0.01% by weight of the total edible nutritional composition of at least one food colorant; at least 0.2% by weight of carbon dioxide, based on the total edible nutritional composition, to obtain an effervescent beverage; At least 5% by volume of ethanol based on the total edible nutritional composition; At least 0.01% by weight of the total edible nutritional composition of at least one medicamentously active ingredient; or This includes adding a combination of two or more.

[0120] In one embodiment, step (iv) comprises adding at least 0.01%, at least 0.02%, at least 0.05%, at least 0.1% or at least 0.5% by weight of at least one food colorant based on the total edible nutritional composition. Preferably, step (iv) does not comprise a total content of food colorants greater than 5%, greater than 2% or greater than 1% by weight based on the total edible nutritional composition.

[0121] In one embodiment, step (iv) comprises adding at least 0.1%, at least 0.2%, at least 0.3%, 0.1-2%, 0.2-1%, 0.3-0.8%, or 0.4-0.7% carbonation by weight based on the total edible nutritional composition, which may provide a sparkling beverage.

[0122] In one embodiment, step (iv) comprises adding at least 3% by volume of ethanol based on the total edible nutritional composition. In one embodiment, step (iv) comprises adding at least 5% by volume of ethanol based on the total edible nutritional composition. It may comprise adding 5-15% or 10-40% by weight of ethanol in total based on the total mass of the edible nutritional composition.

[0123] In one embodiment, step (iv) comprises adding at least 0.01%, at least 0.02%, at least 0.1%, or at least 0.5% by weight of at least one pharma- ceutical active ingredient, based on the total edible nutritional composition, which may be, for example, an adaptogen, a growth factor, a polyphenol, an antibody or an antigen-binding fragment thereof, a small molecule drug, a prebiotic, or a nucleic acid, such as ribonucleic acid (RNA) (e.g., siRNA), deoxyribonucleic acid (DNA), etc.

[0124] Optionally, the edible nutritional composition may be sealed.For example, the edible nutritional composition may be packaged in a container such as a bottle, a sealed glass, a sealed beaker, or a liquid packaging board.If the edible nutritional composition is a powder or granule, it may be packaged in a cardboard box and / or sealed with a film.

[0125] It will be appreciated that the edible nutritional compositions obtained (or obtained) from the method of the present invention also have particularly advantageous technical properties.

[0126] Thus, a further aspect of the present invention refers to an edible nutritional composition obtainable (or obtained) from the method of the present invention.

[0127] It will be understood that the definitions and embodiments made in the context of the methods of the present invention apply mutatis mutandis to the edible nutritional compositions resulting from such methods.

[0128] As mentioned above, the edible nutritional composition may have any form. In a preferred embodiment, the edible nutritional composition comprises: (a) Beverage composition; (b) a powder or granular composition; (c) a gel; or (d) a frozen or partially frozen composition; Here, the edible nutritional composition may optionally form part of the filling of a capsule or may optionally form part of a beverage, a dairy product, a non-dairy creamer, a sauce or a bakery product.

[0129] The beverage composition may be in liquid or pasty form, preferably in liquid form.

[0130] The term "gel" as used herein may be understood in the broadest sense and may include gels per se, any type of jelly composition, capsule fillings, bakery product fillings, pudding forms, and the like.

[0131] The edible nutritional composition may be a frozen or partially frozen composition, such as ice cubes, frozen yogurt, soft ice, slush, and the like.

[0132] A further aspect of the present invention refers to the use of the edible nutritional composition of the present invention for providing a consumer with a well-defined nutritional composition, in particular a well-defined protein composition.

[0133] It will be understood that the definitions and embodiments made in the context of the method of the present invention and the edible nutritional composition obtained from such method also apply mutatis mutandis to their use. Administering a clearly defined protein composition to the consumer can optionally be personalized nutrition. Thus, the nutritional composition can be optimized for the individual.

[0134] The edible nutritional composition of the present invention is useful for application to patients and / or the elderly. In particular, the edible nutritional composition of the present invention is particularly useful for application to patients and / or the elderly as a drink. This also allows consumption by people with problems in chewing and / or swallowing. The present invention also refers to the use of the edible nutritional composition of the present invention for enteral nutrition (e.g., nasogastric feeding, such as nasogastric intubation, gastrostomy, jejunostomy, or other types of tube feeding).

[0135] As mentioned above, many subjects have intolerance to soybean, wheat or dairy products, which are the main protein sources in protein compositions known in the art.The edible nutritional composition of the present invention makes it possible to avoid such drawbacks.Therefore, the present invention also refers to the use of the edible nutritional composition of the present invention for the nutrition of subjects who have intolerance to soybean, wheat or dairy products.Therefore, the present invention also refers to the use of the edible nutritional composition of the present invention for the enteral nutrition of subjects who have intolerance to soybean, wheat or dairy products.

[0136] The edible nutritional compositions of the present invention may be an alternative for subjects who are unable or poorly able to digest proteins derived from meat and / or milk, eggs, one or more plant sources (e.g., soy), one or more bacterial sources, fish, and / or similar sources, or combinations thereof, but who require optimal nutrition through specific, personalized nutrition.

[0137] The edible nutritional composition can be used to provide sufficient nutrition to patients suffering from swallowing problems, such as dysphagia, which is listed as a pathological condition in R13 of the International Statistical Classification of Diseases and Related Health Problems (ICD), 10th Edition (ICD-10). Thus, the present invention also refers to the edible nutritional composition of the present invention for use in a method for treating a patient suffering from dysphagia with a well-defined nutritional composition. In other words, the present invention also refers to a method for treating a patient suffering from dysphagia with a well-defined nutritional composition, in which the patient is administered a sufficient amount of the edible nutritional composition of the present invention. Preferably, the patient is orally administered the edible nutritional composition.

[0138] A further aspect refers to the edible nutritional composition of the present invention for use in a method of treating a patient suffering from dysphagia, wherein the patient is preferably administered the edible nutritional composition by enteral feeding (e.g. nasogastric feeding, e.g. nasogastric intubation, gastrostomy, jejunostomy, or other type of tube feeding), by swallowing a drinkable edible nutritional composition, or a combination thereof.

[0139] It will be understood that the definitions and embodiments made in the context of the methods of the invention and the edible nutritional compositions resulting from such methods also apply mutatis mutandis to their medical uses.

[0140] In other words, the present invention also refers to a method of treating a patient suffering from dysphagia, in which the patient is administered a sufficient amount of an edible nutritional composition by enteral feeding (e.g., nasogastric feeding, e.g., nasogastric intubation, gastrostomy, jejunostomy, or other types of tube feeding), by swallowing a drinkable edible nutritional composition, or a combination thereof.

[0141] The edible nutritional compositions may also be consumed by gastrostomy patients, patients who have had their stomach volume reduced by surgery, and patients with reduced ability to chew and swallow (including dental prostheses).

[0142] The edible nutritional composition may also be consumed as a lifestyle product. Thus, the present invention also refers to the use of the edible nutritional composition as a lifestyle product.

[0143] The invention is further described in the following examples, the scope of which is defined by the claims. [Brief description of the drawings]

[0144] [Figure 1] Figure 1 shows the detachment of porcine muscle cells CD29++Bio8.1, passage 9 / 10, from collagen-coated 6-well plates using different reagents. Figure 1A shows non-detached, i.e. adherent cells (t=0 min). Figure 1B shows cells after treatment with PBS containing Ca2+ and Mg2+ (t=15 min). Figure 1C shows cells after treatment with PBS without Ca2+ and Mg2+ (t=15 min). Figure 1D shows cells after treatment with 1 mM sodium citrate in 270 mOsm / L PBS without Ca2+ and Mg2+ (t=15 min). Figure 1E shows cells after treatment with 1 mM sodium citrate in 570 mOsm / L PBS without Ca2+ and Mg2+ (t=15 min). Figure 1F shows cells after treatment with TrypLE (t=15 min). Scale bars represent 100 μm. [Diagram 2] Attachment of CD29++ porcine muscle cells (CD29++Bio8.1 passage 14 (P14)) to Cytodex3 microcarriers. Cells were cultured in surface water-repellent 6-well plates seeded with Cytodex3 microcarriers at a cell-to-beads ratio of 13. Culture medium DMEM / F-12 + 20% FBS. Figure 2A shows cells before incubation (t = 0 h). Figure 2B shows cells after incubation at 37 °C, 5% CO2 (t = 28 h). Figure 2C shows cells after incubation at 37 °C, 5% CO2 (t = 48 h). Scale bars represent 100 μm. [Diagram 3]Figure 3 shows the attachment of porcine muscle cells CD29++Bio8.1P14 after 48 hours of incubation at 37°C and 5% CO2. Different cell / bead ratios (C / B) were compared with each other and DAPI was used to stain the nuclei of the cells. Culture in surface water-repellent 6-well plates with Cytodex3 microcarriers was used. Culture medium was DMEM / F-12 + 20% FBS. Figure 3A shows cells using a cell / bead ratio of 3. Figure 3B shows cells using a cell / bead ratio of 7. Figure 3C shows cells using a cell / bead ratio of 13. Scale bars represent 100 μm. [Figure 4] Different cell / bead ratios (C / B) and different detachment solutions are shown. Cells were cultured in surface-repellent 6-well plates using Cytodex3 microcarriers. The culture medium was DMEM / F-12 + 20% FBS. Figure 4A shows porcine myocytes CD29++Bio8.1P14 after 48 hours of incubation at 37°C before detachment (C / B approx. 7). Figure 4B shows porcine myocytes CD29++Bio8.1P14 incubated for 48 hours at 37°C and detached using 15 mM sodium citrate in 570 mOsm / L PBS without Ca2+ and Mg2+ (C / B approx. 7) before resuspension. Figure 4C shows porcine myocytes CD29++Bio8.1P14 after the detachment procedure using 15 mM sodium citrate in 570 mOsm / L PBS without Ca2+ and Mg2+ after resuspension (C / B approx. 7). FIG. 4D shows porcine myocytes CD29++Bio8.1P14 after the detachment procedure using 1 mM sodium citrate in 570 mOsm / L PBS without Ca2+ and Mg2+ after resuspension (C / B approx. 8). FIG. 4E shows porcine myocytes CD29++Bio8.1P14 after the detachment procedure using only PBS without Ca2+ and Mg2+ after resuspension (C / B approx. 13). FIG. 4F shows porcine myocytes CD29++Bio8.1P14 after the detachment procedure using 1 mM sodium citrate in PBS without Ca2+ and Mg2+ after resuspension (C / B approx. 10). Scale bars represent 100 μm. [Diagram 5]Dose-response curves of doxorubicin (Figure 5A, IC50=0.079 μM, n=3 / SD, R2=0.9483, Sy.x=8.153) in HepG2 cells by neutral red assay (Figure 5B, IC50=0.116 μM, n=4 / SD, R2=0.9660, Sy.x=7.001) using CellTiter Glo 2.0. [Figure 6] The linearity of CellTiterGlo 2.0 with increasing amounts of cell lysate is shown, depending on the amount of cell lysate and on both high (Figure 6A, n=3 / SD) and low (Figure 6B, n=4 / SD) numbers of HepG2 cells.

[0145] Working Example Example 1 Harvesting skeletal muscle tissue for satellite cell isolation and cryopreservation (mammalian cell example) 1. Tissue collection (TCB) Approximately 2 g pieces of skeletal muscle tissue are harvested from ruminant (cattle, sheep, goats, deer, water buffalo, etc.), non-ruminant (e.g. pigs, etc.), or pseudoruminant (horse, camel, rabbit, etc.) animals. After washing with preservation medium (phosphate-buffered saline (PBS) on ice supplemented with antibiotics) in a 15 mL tube, the material is transferred to 10 mL of freshly prepared preservation medium for further processing.

[0146] 2. Tissue Processing / Digestion (CLS) Each muscle tissue is cut into small pieces (approximately 1-3 mm3) using a sterile scalpel. It is then subjected to enzymatic digestion with collagenase I and dispase, followed by a rinsing step with PBS supplemented with 50x diluted Pen / Strep solution (2x the usual concentration, commercially available 100x solution: 5000 units / mL penicillin G and 5 mg / mL streptomycin), and plated onto collagen type I coated 6-well plates. Triturate each well using a 1000 µL pipette to obtain single cells and cell clusters. A 40 µm cell sorter can be used instead to obtain single cell populations. Centrifuge the cell suspension at 300 x g for 5 min. Remove the supernatant and resuspend the pellet in 8 mL of PBS supplemented with antibiotics (1% penicillin / streptomycin solution) and pool in a 15 mL tube. After centrifugation at 300×g for 3 minutes, the cell pellet is optionally resuspended in 4.5 mL of DMEM / F-12 supplemented with 5% fetal bovine serum (FBS) and antibiotics and pipetted into three wells of a 12-well plate (1.5 mL / well).

[0147] 3. Cell Culture / Expansion (CLS) Heterogeneous cell populations from muscle tissue are cultured under conditions of 37 °C, 5% CO, 95% air, and 85-95% humidity.

[0148] The surface of the culture well is coated with collagen type I for initial attachment of the cells. During culture in DMEM / F-12 supplemented with 5% FBS and antibiotics, the heterogeneous cell population is either in a proliferative phase (dividing and increasing in size) as a monolayer (attached to the surface), or in a differentiated or quiescent phase.

[0149] 4. Cell purification / satellite cells 4.1. Adult stem cell culture Cellular purification of satellite cells can be obtained as described by Schmidt et al. (Cellular and Molecular Life Sciences, 2019, 76:2559-2570) or Hindi et al. (Science Signaling, 2013, 6(272):DOI:10.1126 / scisignal.2003832). The development of satellite cells through myoblasts and myocytes to myotubes can be monitored using muscle-specific biomarkers. These include Pax3 and Pax7 for satellite cells, and MyoD and Myf5 for myoblasts. Myocytes can be detected by myogenin. Myotubes are myosin heavy chain (MyHC) positive. Furthermore, mature myotubes are multinucleated, which can be detected using nuclear-specific dyes such as DAPI and Hoechst. The expression of desmin, another muscle-specific marker, increases during myogenesis and can be detected at the onset of the myoblast stage.

[0150] 4.2.MACS (magnetic activated cell sorting) with CD29 (surface marker) Further processing by MACS may include the method described by Choi et al. (Food Sci. Anim. Resour., 2020, 40(5):852-859). CD29 positive satellite cells are sorted using the MACS cell separation system (Miltenyi Biotec, Bergisch Gladbach, Germany) to purify the population. When the cells reach 80-90% confluency, dissociate the cells using TrypLE™ Express (Gibco) or Accutase (Innovative Cell Technologies, Inc.), or 1 mM hypertonic (adjusted to a final osmolarity of 570 mOsm / kg with potassium chloride) sodium citrate solution (Nie et al., (PlosONE, 2014, 9(1):e88012). React the dissociated satellite cells with anti-CD29 antibody (MAB17783, Novus Biologicals) and anti-mouse IgG microbeads (1:5, Miltenyi Biotec). Sort CD29-positive cells with an MS column (capacity, 1x107 magnetically labeled cells, Miltenyi Biotec) according to the manufacturer's instructions. Confirm the identification of the sorted CD29-positive cells as muscle stem cells by immunostaining with CD29 and Pax7.

[0151] 4.3. Ice treatment The cells are optionally subjected to ice-cold treatment as described by Benedetti et al. (Skeletal Muscle, 2021, 11(7):doi.org / 10.1186 / s13395-021-00261-w). Satellite cells are more sensitive to cold than any other cell population in the treated cell suspension and detach from the surface faster. The harvested or cultured heterogeneous cell population is applied to a non-coated T-25 flask and incubated at 37 °C for 1 h (plating number 1). The supernatant containing the non-adherent cells is collected in a new 15 mL tube. Fresh growth medium (DMEM supplemented with 10% FBS and antibiotics) is added to the flask containing the adherent cells, which are mainly fibroblasts and myogenic cells in a later developmental stage such as myoblasts and myocytes.

[0152] Centrifuge the Falcon tube at 180xg for 10 min, discard the supernatant, and resuspend the pelleted cells in 3 mL (3 mL for T-25 and 6 mL for T-75) of growth medium. Repeat the plating procedure (plating no. 2). Collect the supernatant containing the non-adherent cells in a new 15 mL Falcon tube. Add fresh growth medium to the flask containing the adherent cells. Centrifuge the 15 mL tube at 180xg for 10 min, discard the supernatant, and resuspend the pelleted cells in the desired amount of growth medium. Plate the resulting cells onto a T-25 or T-75 flask coated with 0.1% gelatin and incubate overnight at 37 °C. The next day, wash the flask three times with 5 mL (for T-25 and 10 mL for T-75) of PBS. Add 5 mL (for T-25) and 10 mL (for T-75) of ice-cold PBS to the flask. Keep the flask on ice (approximately 0°C) for 30 minutes, shaking gently by hand (swirling) from time to time. Collect the supernatant containing the detached cells in a 15mL tube and centrifuge at 180xg for 10 minutes. Discard the supernatant and resuspend the cells in 3mL or less of growth medium for a T-25. Seed the resuspended cells in a vessel coated with 0.1% gelatin and incubate at 37°C. At each passage, an ice-cooling step (after plating number 2) is optional.

[0153] 5. Cell preservation / cryopreservation for cell banking 5.1.When DMSO is included Single-cell suspensions are collected from 2D or 3D cultures after a washing process with PBS and either enzymatic digestion (collagenase) or chelating treatment (ethylenediaminetetraacetic acid (EDTA), sodium citrate). Centrifuge the suspensions at 180 x g for 5 min and discard the supernatant.

[0154] The pellet is resuspended in 10% (v / v) DMSO and 90% (v / v) culture medium and dispensed into 1 mL / cryotube at 4°C. The resulting tubes are stored at -80°C (Mr. Frosty (Nalgene, Merck)). After 24 hours or more, the tubes are transferred to a liquid N2 tank. When the cells are to be used, they are thawed from storage. The vials are kept thawed in liquid N2 in a Dewar vessel. Thawing may be performed in a pre-warmed (37°C) water bath immediately after removal from the liquid N2. When 80% liquefaction of the cryopreservation medium is observed, 1 mL of pre-warmed medium is added to the cryotube. The cells may be further processed by gently pipetting and adding 8 mL of basal medium (4°C or on ice) to the 15 mL tube. The suspension is centrifuged (4°C), plated in culture dishes, and cultured in an incubator.

[0155] 5.2. DMSO-free Single cell suspensions are collected from 2D or 3D cultures after a washing process with PBS and either enzymatic digestion (collagenase) or chelating treatment (EDTA, sodium citrate). Centrifuge the suspension at 180xg for 5 min and discard the supernatant. For 5x105-5x106 cells, resuspend the cell pellet in 200 µL of DMSO-free freezing medium, StemCellKeep (Abnova). Transfer the cryotube to a liquid N2 chamber (-196 °C). Thawing from storage is performed by maintaining the vial to be thawed in liquid N2 in a Dewar vessel. Immediately after removal from liquid N2, add pre-warmed (37 °C) medium (1 mL) directly to the vial. Thaw by gently pipetting the solution and add to 9 mL of basal medium (4 °C or on ice) in a 15 mL tube. The suspension is centrifuged (4° C.), plated onto culture dishes, and cultured in an incubator.

[0156] Example 2 Harvesting and culturing avian muscle cells Other non-ruminant animals (e.g., poultry, birds, turkeys, ducks, geese, chickens, pigeons, etc.) can also be used as a source of cells. Their skeletal muscles are harvested in the same manner as above. Avian cells are used for vaccine production (scalable suspension serum-free vaccine production). Cultivation of avian cells is comparable to that of mammalian cells described above, except that the culture temperature can be slightly higher than that of mammalian cells due to their natural body temperature (chickens: approx. 41-42°C, newly hatched chickens approx. 40°C).

[0157] Example 3 Harvesting and culturing non-muscle avian cells Bird (poultry) eggs and feathers can be used as cell sources. From chicken eggs (pre-hatched chicken embryos, approximately 21 days old), embryonic stem cells, embryonic germ stem cells, progenitor cells, fully developed / functional cells are harvested at any developmental stage. Chicken embryonic stem cells can be multiplied indefinitely and differentiated into any cell type desired. Cells are also harvested from bone marrow. Large amounts of mesenchymal stem cells can be harvested from bone marrow. Mesenchymal stem cells can differentiate into muscle cells, adipocytes, osteoblasts, neurons, and chondrocytes. Mesenchymal stem cells can also secrete growth factors / cytokines that are beneficial for cell culture. Cells can also be harvested from poultry feathers. Follicular cells can be harvested from feathers. Follicular cells have similar properties to mesenchymal stem cells.

[0158] Example 4 Harvesting and culturing non-muscle avian cells For cell growth and culture to obtain macro- and micronutrients, cells can be taken from any organ / tissue from any kingdom of life, including animals, plants, fungi, protists, eubacteria, and archaea.

[0159] Example 5 Use Example: Protein / selenium-rich cell-based composition (water containing pork muscle and fat cell-based solution) The main component of pork muscle is myofibers, which are composed of muscle cells with white adipose tissue. For cells and extracellular matrix (ECM) freshly harvested from a bioreactor, 10% (w / v) pork muscle cells (approximately 1 billion cells, 10 g) contain an aqueous 100 mL solution with high protein (1.7 g, 3.4% DV (reference daily intake in percent, i.e. percent of the recommended daily intake)) and high selenium (0.24 mg, 4.5% DV), with a low amount of carbohydrate (0 g). The recommended daily fluid intake for a healthy adult male is 3.7 L. By replacing half of the 1.8 L of fluid with water containing pork muscle cell-based solution, 30.6 g of protein (61% DV) and 4.32 mg of selenium (81% DV) can be reached. The amount of saturated fatty acids (14.2 g per 1.8 L, 70% DV) is adjustable.

[0160] Example 6 Use Examples: Protein / vitamin B12 / D-rich cell-based composition (water containing salmon muscle and fat cell-based solution)

[0161] Applying the same back calculations as in Example 1, one can expect 36.9 g protein (73.8% DV), 4.74 mg vitamin B12 (311% DV), and 19.63 mg vitamin D (97.2% DV) from 1.8 L of salmon cell-based beverage.

[0162] Example 7 Use Example: Cell-based composition (water and beef muscle and fat cell-based solution) rich in protein, vitamin B12, zinc and iron A 1.8L serving of beef cell-based beverage is expected to provide 35g protein (70.2% DV), 3.55mg vitamin B12 (147.6% DV), 8.19mg zinc (73.8% DV), and 3.58mg iron (19.8% DV).

[0163] When the three examples above (pork, salmon, and beef) are mixed in a 1:1:1 ratio, 1.8 L of cell-based beverage will provide 34.08 g protein (68.4% DV), 42.12 mg selenium (76.8% DV), 4.092 mg vitamin B12 (170.4% DV), 6.54 mg vitamin D (32.4% DV), 4.284 mg zinc (39% DV), and 8 mg iron (10.8% DV). This cell-based blended beverage can be supplemented with iron, vitamin C, dietary fiber, and flavorings to be consumed as a functional food as well as tailored to an individual's lifestyle needs. Optionally, additives such as local produce, fruits, and vegetables may be added to improve / control nutritional value.

[0164] Example 8 Use Example: Hepatocyte-Based Composition A goose liver cell-based drink (1.8 L) rich in vitamin A, vitamin B12 and copper has been shown to contain 16.8 mg (1861% DV) of vitamin A, 97.2 μg (4050% DV) of vitamin B12 and 13.5 mg (1505% DV) of copper, exceeding the DV (%) by more than 15 times that of a healthy adult male. Thus, such a liver cell-based drink can be consumed in a small amount of 120 mL (100% DV of copper) as a recommendation. Hepatocytes can be a valuable source of these dietary supplements for other cell-based compositions.

[0165] Example 9. Use Examples: Cell-Based Compositions Containing Different Cell Types The cell-based beverage is prepared by mixing pork, salmon, and beef muscle cells in a 1:1:1 mass ratio. This is compared to an equivalent composition containing an equivalent content of only one cell type. The resulting nutritional content is shown in the table below. [Table 1] [Table 2] [Table 3]

[0166] Example 10. Cell Culture and Detachment We investigated how CD29++ sorted (CD29 antibody magnetic activated cell sorting, MACS, twice) porcine muscle cells detach from cell culture 6-well plates (collagen coated and non-coated) using various phosphate buffered saline (PBS), e.g., PBS with or without Ca2+, Mg2+, and sodium citrate at isotonic or hypertonic osmolarity. Furthermore, CD29++ porcine muscle cells were tested for adhesion and detachment to Cytodex3 microcarriers using pre-tested detachment solutions.

[0167] 1. Materials and Methods 1.1. Reagents and consumables The reagents and consumables used in the experiments performed are listed below: All chemicals and reagents, as well as inocula, were prepared under a clean hood. DMEM / F-12 without glutamine, without Hepes (VWR, LOT / product number 392-0409P), Glutamax™-I (100x) (Life Technologies Corporation, LOT / product number 2390275), Fetal bovine serum (FBS) (Sigma, lot number / product number 0001650386), TrypLE™ Express (1x) (TrypLE) (recombinant enzyme, Life Technologies Corp., LOT / product no. 12604013); PBS without Mg2+, Ca2 and phenol red (w / o) (Hi-Media Laboratories Pvt. Ltd., Lot No. / Item No. TL1006), PBS containing Mg2+ and Ca2 (PBSw / Mg2+, Ca2), without phenol red (Hi-Media Laboratories Pvt. Ltd., Lot No. / Item No. TL1023), Glucose monohydrate (NeoLab, lot number / product number TC208-1KG), KCl (Biofroxx, lot number / product number 1617KG001), Trisodium citrate dihydrate (Sigma, lot number / product number W302600), Acridine orange propidium iodide stain (Aligned Genetics Inc., lot number / product number APOBAK1001), 6-well culture plate (neoLab, lot number / product number GF-0074), Calfskin collagen (Sigma, lot number C8919), Cytodex3 microcarrier beads (Cytiva, LOT / product number GEHE17_0485-02_P) can be used as a solid support, and Double distilled water (H2Odd).

[0168] The PBS solution can be isotonic (about 270 Osm / L) or can have an osmolarity of 570 mOsm / L. Unless otherwise stated, PBS has an osmolarity of 270 Osm / L.

[0169] 1.2. Further equipment CO2 incubator, microscope, Luna-fl™ dual fluorescent cell counter

[0170] 2. Preparation of sodium citrate solutions / PBS with different osmolalities A 1 mM hypertonic sodium citrate solution was prepared by dissolving 0.2941 g of trisodium citrate dihydrate and 11.487 g of KCl in Ca, Mg-free PBS. The volume of the solution was adjusted to 1 liter with PBS. According to Nie et al., ("Scalable Passaging of Adherent Human Pluripotent Stem Cells", PLoS ONE, 2014, 9(1):e88012.doi:10.1371 / journal.pone.0088012), the osmolarity of the solution was estimated to be 570 mOsm / L when PBS was used instead of distilled water. The preparation of sodium citrate solutions with different osmolarity was performed by dissolving a small amount of KCl in PBS and subtracting the osmolarity provided by PBS (approximately 270 mOsm / L). The amount of sodium citrate was adjusted respectively.

[0171] 3. Incubation of porcine muscle cells (CD29++) in 6-well plates To investigate the detachment of CD29++ porcine muscle cells using sodium citrate / PBS and other reagents, cells were first incubated in 6-well plates (collagen-coated and non-coated). For this, CD29++ porcine muscle cells of passages 10 and 11 with viabilities of 99.30% and 98.60% were used to seed the wells at 15000 cells / cm2. The wells were then filled up to 2 mL with DMEM / F12 + 20% FBS and incubated for 72 h at 37 °C, 5% CO2 (incubation time depending on the seeding density) until 80-90% confluency was reached.

[0172] Each detachment was performed in biological replicates. The experimental set-up using 6-well plates containing the detachment medium (Rdetach) and detachment time (tincubation) was as follows: 1.Rdetach=H2Odd,tincubation=15 minutes; 2.Remove=PBS containing Ca2+ and Mg2+, incubation=15 min; 3.Remove=PBS without Ca2+ and Mg2+, tincubation=15 min; 4.Remove=TrypLE, tincubation=15min; 5.Remove=1mM sodium citrate / 270mOsm / L PBS, tincubation=15min; 6.Remove=1mM sodium citrate / 570mOsm / L PBS, tincubation=15min;

[0173] 4.6 Example of removal procedure from a well plate Detachment of CD29++ porcine muscle cells in 6-well plates is performed as follows: (i) Aspirate the cell culture medium; (ii) Wash the cells twice with 2 mL of the solution that you plan to use for detachment (in the case of TrypLE treatment, wash with PBS without Ca2+ and Mg2+) (pre-warm the solution to 37 °C); (iii) add 2 mL of the respective stripping solution (0.5 mL for TrypLE) and incubate at 37°C, 5% CO2 for 15 min (the stripping solution is pH 7.2 and pre-heated to 37°C); (iv) add 2 mL of complete culture medium (DMEM / F12 + 20% FBS); (v) Resuspend / wash the cells from the surface by gently pipetting up and down (10 times) (optional); (vi) taking photographs of the detached cells / aggregates; (vii) Transfer the cell suspension from the 6-well plate to a 15 mL Falcon tube and centrifuge at 300 x g for 5 minutes; (viii) removing the supernatant and resuspending the cells in approximately 0.4 mL of culture medium; (ix) Cell viability is measured using a Luna-fl™ dual fluorescent cell counter with acridine orange / propidium iodide.

[0174] 5. Cultivation of CD29++Bio 8.1 porcine muscle cells on Cytodex3 in 6-well plates To investigate the attachment of CD29++Bio 8.1 porcine muscle cells to Cytodex3 microcarriers, passage 14 porcine muscle cells (viability 98.60%) were seeded on Cytodex3 microcarriers in a water-repellent 6-well plate. The seeding density was adjusted to obtain different cell-to-bead ratios, as shown in the following figure. The 6-wells were incubated at 37°C, 5% CO2 for 48 hours. DMEM / F-12+20%FBS was used as the medium. The total volume of one well was 2 mL. The attachment of the cells was monitored by taking microscopic pictures. CD29++Bio was added to Cytodex3 microcarriers in a water-repellent 6-well plate. 8.1 The experimental setup for adhering porcine muscle cells is shown below. Different cell to bead ratios (RatioCell / Bead) ranging from about 3 to 13 are tested for cell attachment. Detachment of cells with the corresponding detachment reagent (detachment medium, Rdetach) is as follows: 1.RatioCell / Bead=13.33, Rdetach=PBS without Ca2+ and Mg2+; 2.RatioCell / Bead=10.00, Rdetach=1 mM sodium citrate / PBS without Ca2+ and Mg2+. 2.RatioCell / Bead=8.00, Rdetach=1mM Sodium Citrate / 570mOsm / L PBS without Ca2+ and Mg2+. 3.RatioCell / Bead=8.00, Rdetach=1mM Sodium Citrate / 570mOsm / L PBS without Ca2+ and Mg2+. 4.RatioCell / Bead=6.67, Rdetach=15mM Sodium Citrate / 570mOsm / L PBS without Ca2+ and Mg2+. 5. RatioCell / Bead=5.00, Rdetach=hypotonic solution (8g / L NaCl, 0.4g KCl, 1g / L glucose / H2Odd (according to Lai et al., "ESR studies on membrane fluidity of Chinese hamster ovary cells grown on microcarriers and in suspension", Exp. Cell Res., 1980, 130:437-442); and 6.RatioCell / Bead=3.00, Rdetach=TrypLE.

[0175] Detachment of cells from Cytodex3 was performed as follows: (i) Tilt the plate and aspirate off as much of the culture medium as possible; (ii) Wash the cells with 1–2 mL of PBS without Ca2+ and Mg2+; (iii) Add 2 mL of each stripping reagent (preheated to 37°C, pH 7.2). (iv) incubate at 37°C, 5% CO2 for 15 minutes; (v) Wash the cells off the microcarriers by gently shaking the plate or gently pipetting up and down.

[0176] The results are shown in Figure 1. It can be seen that cells can be detached using PBS that does not contain Mg2+ or Ca2+, especially when sodium citrate is added.

[0177] 6. Viability of Cells Detached from Collagen-coated 6-well Plates The viability of cells detached using each solution of sodium citrate in PBS is shown in Table 3 below. [Table 4]

[0178] As a result, it is recognized that the addition of a citrate salt, such as sodium citrate, can improve cell detachment.

[0179] Additionally, for comparison, the detachment of CD29++ porcine muscle cells was tested on non-coated 6-well plates. For this, the use of H2Odd was replaced by the use of 1 mM sodium citrate (SC) in tap water. Additionally, standard deviations (SD) are shown. [Table 5]

[0180] As a result, it is observed that the addition of a citrate salt such as sodium citrate can improve cell detachment. Even the simple addition of a citrate salt such as sodium citrate can result in significant cell detachment. This is evident from a comparison of water and water containing 1 mM sodium citrate (see the first column of Tables 3 and 4).

[0181] 7. Attachment of CD29++ porcine muscle cells to Cytodex3 microcarriers For visualization experiments, CD29++Bio8.1P14 cells were seeded onto Cytodex3 microcarriers. A cell / bead ratio of 13 was used. Results before, during and after incubation are shown in Figure 2. Using a cell / bead ratio of 13, after 48 hours of incubation, cells were confluent on the Cytodex3 microcarriers.

[0182] Additionally, different cell / bead ratios were compared to each other. The results before, during, and after incubation are shown in Figure 3. When cells were stained using DAPI, the cell density on the microcarriers was most visualized at a cell / bead ratio of 13 (C / B13).

[0183] 8. Detachment of CD29++Bio 8.1 Porcine Muscle Cells from Cytodex3 Microcarriers CD29++Bio8.1P14 was incubated for 48 hours at 37° C. and then detached from the Cytodex3 microcarriers. The results are shown in FIG.

[0184] 9. Conclusion 9.1. Detachment from culture plate Detachment of cells (exemplified as CD29++ porcine muscle cells, passages P10 and P11) using various buffers (exemplified as PBS buffer base) showed that cell attachment appears to be possibly dependent on Ca2+ ions. In the examples, the highest viability could be achieved (here: 95.65% ± 2.15%) by using a hyperosmolarity buffer (exemplified here as 570 mOsm / L PBS) containing citrate (exemplified here as 1 mM sodium citrate). In contrast, when detaching from non-coated 6-well plates, a viability of 95.95% ± 1.45% was reached by using only PBS without Ca2+ and Mg2+. In summary, no significant difference was observed in detachment between collagen-coated and non-collagen-coated surfaces. Without being bound by this theory, it is believed that the difference in cell numbers is due to TrypLE detaching cells from the surface as single cells, whereas sodium citrate and PBS or a combination of both (1 mM sodium citrate / PBS without Ca2+ and Mg2+ or 15 mM sodium citrate / PBS without Ca2+ and Mg2+) detach cells from the surface as larger aggregates. When comparing microscopic images, it was observed that essentially the entire surface of all six wells was detached from cells during detachment with PBS alone or the combination of sodium citrate and PBS without Ca2+ and Mg2+.

[0185] 9.2.Detachment from Cytodex3 Microcarriers Attachment of cells (exemplified as CD29++Bio8.1 porcine muscle cells) was observed on solid carriers (exemplified as Cytodex3 microcarriers). Wells containing a cell-to-bead ratio of about 13 became confluent after 48 hours of incubation at 37° C. and 5% CO2. For CD29++Bio8.1, a suitable cell-to-bead ratio can be qualitatively determined to be 7 or higher. Detachment of CD29++Bio8.1 was performed using 15 mM sodium citrate / 570 mOsm / L PBS without Ca2+ and Mg2+. CD29++ porcine muscle cells were detached from Cytodex3 microcarriers using 15 mM sodium citrate / 570 mOsm / L PBS solution without Ca2+ and Mg2+. It is expected that it is scientifically reasonable that cells such as CD29+ porcine muscle cells can attach to and be detached from different microcarriers such as Cytodex1.

[0186] Example 11 Cell pulp processing The protein solubilization behavior of freeze-dried meat cell-containing material (exemplified as chicken meat cell-containing material) was carried out under different conditions.

[0187] 1. Materials and Methods The freeze-dried chicken meat samples were pulverized in a mortar and stored in a 250 mL glass beaker. Acetic acid (CAS7647-01-0), Sodium acetate (CAS127-09-3), Monosodium phosphate (CAS10028-24-7), Disodium phosphate (CAS13472-35-0), and Sodium chloride (CAS 7647-14-5).

[0188] The solubility of freeze-dried meat was tested using the following factors and concentrations based on previous analyses: 0.2-1 M NaCl, 2-10 wt% glucose, pH range 4-8 (0.05 M).

[0189] A trial version of DesignExpert13 software was used to create the matrix and the design is full factorial23 with two replicates and three center points as shown in Table 5. [Table 6] [Table 7] [Table 8] [Table 9]

[0190] Solubilization of Chicken Powder To test protein solubility, as an example, a meat powder concentration of 100 mg / mL was chosen based on literature reports. Approximately 500 mg of powder was homogenized in 5 mL of solution according to Table 5 (see above). The solution was vortexed briefly and homogenized in a tube rotator for 1 h. A tube rotator Stuart Tube Rotator SB3 with a speed of 14 rpm was used for homogenization. After homogenization, the tubes were centrifuged and 500 μL of supernatant was collected for Bradford assay. A 5810R Eppendorf centrifuge was used for centrifugation at a relative centrifugal force (RCF) of 3214 at 4°C for 10 min.

[0191] Bradford assay The Quick Start™ Bradford protein assay was performed in a 96-well plate. The procedure was performed according to the instruction manual of the assay kit manufacturer (Bio Rad). The samples were appropriately diluted to meet the protein limit detection concentration requirement of the Bradford assay. The protein concentration results were further analyzed. The results are shown in Table 9.

[0192] 2.Results [Table 10]

[0193] 3. Conclusion Results are presented with 95% confidence intervals. NaCl concentration can affect the solubility of cell-containing material.

[0194] Example 12. Biocompatibility and lack of toxicity The cell-based material according to the invention was guaranteed to be biocompatible and therefore ingestible without any toxic effects. For this purpose, a cellular assay was established to evaluate the toxicity of the composition according to the invention (here "liquid meat") at various stages of production. Instead of standard 3T3 rodent cells, human HepG2 cells isolated from liver tissue, commonly used for testing drug metabolism and hepatotoxicity, were used. Two different assays were evaluated to measure cytotoxicity and cell viability.

[0195] The Neutral Red assay is an established method that has been validated by EURL ECVAM in the context of 3T3 cells. It is a cellular assay based on Eurodine dye that stains lysosomes in viable cells.

[0196] The CellTiter Glo assay is a viability assay commercialized by Promega. This cellular assay quantifies the amount of ATP, which can be correlated with cell viability.

[0197] Both assays were set up using doxorubicin, a potent chemotherapeutic agent that intercalates with DNA and inhibits topoisomerase III, an enzyme essential for DNA transcription.

[0198] As a next step, the toxicity of a reconstituted freeze-dried cell lysate using the initial manufacturing step of the composition according to the invention (here "liquid meat") was tested. This sample was not further processed and therefore contained a large amount of insoluble cell fraction.

[0199] 1. Materials and Methods cell culture HepG2 cells were provided by ATCC (HB-8065) ​​and cultured in DMEM supplemented with 10% FBS and grown in an incubator set at 37°C and 5% CO2. For both assays, 5,000 cells / well were seeded using only the center 32 wells of a 96-well plate. HepG2 cells inherently aggregate, which prevented proper measurement of cell volume and led to variability between assays. Various concentrations of doxorubicin were added using IDOT 18 hours after cells were seeded at a cell volume of 0.2 μL. Cells were processed 2 days after compound addition.

[0200] Growth curve time points were measured every 3 hours and 4 images were recorded per well using an IncuCyte.

[0201] Neutral Red Assay Neutral Red Assay Kit-Cell Viability / Cytotoxicity (ab234039, Abcam) was used. All solutions from the kit were aliquoted and stored at -20°C as recommended. On the day of the experiment, aliquots of 20 mM doxorubicin, washing solution, lysate, and neutral red staining solution were thawed at room temperature (RT) and prepared according to the manufacturer's instructions. Cell media was removed and cells were washed with 200 μL 1x washing solution. After adding 150 μL 1x neutral red staining solution, cells were stained for 2 h in an incubator. Cells were washed with 250 μL 1x washing solution and air-dried for approximately 10 min until no liquid was visible on the plate. After adding 150 μL 1x lysate, plates were shaken at 500 rpm for 20 min at room temperature. The 96-well plate was then read on a microplate reader at OD540nm.

[0202] Cell Titer Glo Assay The CellTiter-Glo 2.0 cell viability assay (G9242, Promega) was used. The solution was thawed and stored at 4 °C as recommended. The cell media was removed and the cells were washed twice with 100 μL PBS. After the final wash, 50 μL PBS was added, followed by 50 μL CellTiter-Glo 2.0 reagent. The 96-well plate was mixed on an orbital shaker for 2 min and incubated at room temperature for 10 min. Luminescence was recorded on a microplate reader using an integration time of 1.000 ms. In both assays, dose-response experimental data were fitted to the equation "Log(agonist) vs response - variable slope (four parameters)" using Prism. For the dose-response curve of the Cell TiterGlo assay, two data points were removed as outliers.

[0203] 2.Results The Neutral Red assay showed an IC50 of 0.079 μM, whereas CellTiter Glo2.0 showed an IC50 of 0.116 μM. This is higher than the expected literature value of 0.69 μM given in the Neutral Red Assay Instructions using HepG2 cells, but it is not unexpected for a cellular assay to show this degree of variation. The results are shown in Figure 5.

[0204] As a consequence, toxicity could not be assessed using the Neutral Red assay, since the particles interfered with the Eurodine dye. CellTiter Glo was more successful and allowed to assess cell viability up to 100 μg of cell lysate, depending on the cell number (see FIG. 6). No toxic effects were observed. If the insoluble fraction constitutes an essential feature of the composition according to the invention (here "liquid meat") and is not removed by filtration or centrifugation, the CellTiter Glo assay can be used, provided that the cell number in the assay is carefully adjusted and the final cell lysate concentration is less than 100 μg.

[0205] In summary, we have set up two assays with different methodologies to assess toxicity, but both can be used to assess the toxicity of small molecules without major adjustments: the former cannot detect toxicity in unpurified cell lysates, whereas the latter shows compatibility with cell lysates of up to 100 μg protein when carefully adjusted.

Claims

1. 1. A method for preparing an edible nutritional composition comprising: (i) culturing proliferating non-human animal cells of interest in vitro until the cells have expanded in number by at least twofold; (ii) harvesting the cells to obtain cell-derived material; (iii) preserving the cell-derived material from the cells harvested in step (ii) against spoilage; (iv) optionally mixing the cell-derived material of step (iii) with one or more further consumable ingredients selected from the group consisting of one or more vitamins, one or more minerals, one or more aroma compounds, one or more food colorants, one or more fibers, ethanol, acetic acid, carbonic acid, and combinations of two or more thereof.

2. The method of claim 1, wherein step (i) is a culturing step carried out in the absence of animal-derived serum.

3. 3. The method of claim 1 or 2, wherein step (iii) comprises processing the cell-derived material into a powder, preferably wherein the processing of the cell-derived material into a powder is obtained by at least partially removing the liquid content, in particular wherein the processing of the cell-derived material into a powder is obtained by freeze-drying.

4. 3. The method of claim 1 or 2, wherein step (iii) comprises pasteurization, and preferably the cell-derived material from the cells harvested in step (ii) still contains cell culture medium and is a liquid or pasty composition.

5. The edible nutritional composition comprises: (a) a beverage composition, in particular a beverage composition having a mass ratio of killed material derived from said cells of interest to viable cells of interest of at least 2:1 or more; (b) aqueous solutions and water-suspendable powders or granules, in particular those having a mass ratio of killed material derived from said cells of interest to viable cells of interest of at least 2:1 or more; or (c) a liquid or pasty composition comprising a cell culture medium and living and / or dead cells, preferably wherein the cell-derived material from step (ii) contains the cell culture medium in which the cells were cultured in the preceding step (i), in particular said liquid or pasty composition being pasteurized.

6. The target cell is Cardiomyocytes or their precursor cells, in particular satellite cells; Adipocytes or their precursor cells; Hepatocytes or their progenitor cells; Induced pluripotent stem cells; Non-human embryonic stem cells; The method according to any one of claims 1 to 5, wherein the method is a combination of two or more thereof.

7. The method according to any one of claims 1 to 6, wherein the cells of interest are primary cells.

8. The method according to any one of claims 1 to 6, wherein the cells of interest are cells in a long-term cell culture.

9. 9. The method according to any one of claims 1 to 8, comprising adjusting the proportion and type of cells cultured in step (i) to obtain a defined nutrient content of interest, in particular a defined protein content of interest.

10. 10. The method of claim 1 or 9, wherein step (ii) comprises administering to the cells to be harvested a solution containing citrate, in particular sodium citrate.

11. The method according to claim 10, wherein the solution containing citrate is a buffer solution, in particular phosphate buffered saline (PBS).

12. 12. The method of claim 10 or 11, wherein the solution containing the citrate is administered to the cells for 2-20 minutes, 5-10 minutes, or 10-15 minutes before washing the cells off.

13. The method according to any one of claims 1 to 12, wherein step (ii) comprises removing the cell culture medium, preferably by centrifugation, filtration, dialysis or rinsing of the adherent cells, in particular by centrifugation and an optional washing step with one or more buffers.

14. 13. The method of any one of claims 1 to 12, wherein step (ii) comprises retaining and pasteurizing at least a portion of the cell culture medium.

15. Step (iv) at least 0.01% by weight, based on the total edible nutritional composition, of at least one food colorant; at least 0.2% by weight carbonation, based on the total edible nutritional composition, to obtain a sparkling beverage; At least 5% by volume of ethanol based on the total edible nutritional composition; At least 0.01% by weight, based on the total edible nutritional composition, of at least one medicamentously active ingredient; or The method of any one of claims 1 to 14, comprising adding a combination of two or more.

16. 16. The method according to any one of claims 1 to 15, wherein the edible nutritional composition is a beverage composition, step (iii) comprises processing the cell-derived material into a powder, and the method comprises the further step of suspending the powder in an aqueous liquid, in particular mineral water or tap water.

17. (a) fermenting the edible nutritional composition; (b) smoking the edible nutritional composition; (c) soaking a food product with a composition comprising said edible nutritional composition, preferably in combination with one or more further ingredients, such as at least 1% by weight of said composition of sodium chloride, at least 1% by weight of said composition of one or more sugars, at least 1% by weight of said composition of acetic acid, at least 0.5% by weight of said composition of ethanol, or at least 5% by weight of said composition of one or more edible oils; and / or; (d) thickening said edible nutritional composition to obtain a syrup, in particular by adding sugar and / or other sweeteners to obtain a sweet syrup; The method according to any one of claims 1 to 16.

18. Preferably, the resulting nutritional composition comprises: (a) Beverage composition; (b) a powder or granular composition; (c) a gel; or (d) characterized in that the composition is frozen or partially frozen; An edible nutritional composition obtainable from the method according to any one of claims 1 to 17, wherein said edible nutritional composition optionally forms part of the filling of a capsule, or optionally forms part of a beverage, a dairy product, a non-dairy creamer, a sauce or a bakery product.

19. 20. The edible nutritional composition of claim 18 for use in a method of treating a patient suffering from dysphagia.

20. 20. The edible nutritional composition for use according to claim 19, wherein the patient is administered the edible nutritional composition by enteral feeding, by swallowing a beverage edible nutritional composition, or a combination thereof.

21. 20. A method of treating a patient suffering from dysphagia, comprising administering to said patient, via enteral nutrition, a sufficient amount of the edible nutritional composition of claim 18.

22. 22. The method of claim 21, wherein the patient is administered the edible nutritional composition by enteral feeding, swallowing a beverage edible nutritional composition, or a combination thereof.

23. 20. Use of an edible nutritional composition according to claim 18 for providing a consumer with a well-defined nutritional composition, in particular a well-defined protein composition.

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