Avian stem cells for the production of a food product

Abstract

To provide alternative meat substitutes to meet current food issues i.e. a healthy and safe food product, having a nutritional profile level similar to meat products derived from animals, while having a reduced environmental footprint and avoiding the slaughter of animals.SOLUTION: There is provided a food product comprising avian stem cells and at least one food component.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a food product comprising avian stem cells and at least one food ingredient. [Background technology]

[0002] In recent years, the food industry has faced several challenges that have prompted a rethinking of future production methods. The main challenge is to meet the increasing food demand from a growing population while reducing the negative environmental impact of production. Among the sectors involved, the meat industry is one of the most affected, as global meat demand is expected to double by 2050. Therefore, supplying the world population with protein is one of the major issues for the future.

[0003] Conventional farming methods use 60% of the land and animal feed crops, account for 15-20% of greenhouse gas emissions, and slaughter 60 billion animals annually. By 2050, this number is expected to increase to 110 billion.

[0004] Furthermore, the evolution of antibiotic resistance, which reduces the possibility of treatment in case of infectious diseases, is of great concern from the perspective of human and animal health, which also contributes to the loss of consumer confidence in these traditional agricultural methods.

[0005] Alternative solutions to livestock already exist, such as the consumption of algae, insects in snacks or as animal feed, or more traditionally the use of plant-based meat.

[0006] Plant-based meat, a meat alternative made from restructured plant proteins, is gaining increasing consumer interest. For example, WO 2015 / 161099 describes such plant-based meat structured protein products. Sales of plant-based meat in the United States increased 6% in 2017 and 24% in 2018, while sales of animal meat in the United States increased 2% in 2018.

[0007] However, plant-based meat does not have the same nutritional profile as its animal-based counterpart. In particular, relying solely on plant protein as a protein source can result in deficiencies in certain essential amino acids, especially lysine. Plant-based meat can also be deficient in important minerals such as iron and zinc, as well as certain vitamins such as vitamin B12.

[0008] Therefore, there is a need to develop alternative meat substitutes to meet current food problems, i.e. healthy and safe foods, that have a similar nutritional profile level to animal-derived meat products while reducing the environmental footprint and avoiding animal slaughter. Summary of the Invention

[0009] The present invention arises from the inventors' unexpected discovery that food products obtained by at least one step of in vitro culture of avian stem cells provide high quality proteins.

[0010] The present invention therefore relates to a food product, in particular a manufactured food product, comprising avian stem cells and at least one food ingredient.

[0011] The present invention also relates to the use of avian stem cells and at least one food ingredient for the production of a food product.

[0012] The present invention also relates to a method for preparing or manufacturing a food product, comprising the step of mixing avian stem cells with at least one food ingredient.

[0013] The present invention also relates to a food product susceptible to or obtainable by such a method. DETAILED DESCRIPTION OF THE INVENTION

[0014] As a premise, as intended herein, the term "comprising" has the meaning of "including" or "containing," which states that when an object "comprises" one or more elements, it means that other elements besides those mentioned may also be included in the object. In contrast, when an object is said to "consist of" one or more elements, the object is limited to the listed elements and cannot include elements other than those mentioned.

[0015] Furthermore, as intended herein, the term "cultured meat" is considered equivalent to the terms "synthetic meat," "clean meat," "in vitro meat," or "cell-based meat."

[0016] Obtaining avian stem cells Preferably, the avian stem cells in the food product according to the present invention are avian embryonic stem cells isolated from embryos. Also preferably, the avian stem cells according to the present invention are continuous diploid avian cell lines. Preferably, the avian stem cells according to the present invention, in particular the avian embryonic stem cells, consist essentially of undifferentiated cells. Preferably, the avian stem cells according to the present invention, in particular the avian embryonic stem cells, consist of at least 50% undifferentiated cells. More preferably, the avian stem cells according to the present invention, in particular the avian embryonic stem cells, consist of at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% undifferentiated cells.

[0017] Preferably, the avian stem cells according to the invention, in particular the avian embryonic stem cells, are obtained by a method comprising at least one step of in vitro culture. Preferably, the avian stem cells according to the invention, in particular the avian embryonic stem cells, are obtained by a method comprising the following steps: a) Isolation, culture, and propagation of avian stem cells, particularly avian embryonic stem cells, in a complete medium containing. Preferably, the complete medium according to the present invention contains factors that allow the proliferation of avian stem cells, particularly embryonic avian stem cells. Preferably, the culture and propagation of avian stem cells, particularly avian embryonic stem cells, according to the present invention is carried out in the presence of a feeder layer supplemented with animal serum. Optionally, the complete medium may contain additives such as amino acids (i.e., glutamine, non-essential amino acids, etc.), sodium pyruvate, β-mercaptoethanol, vitamins, protein hydrolysates of non-animal origin (i.e., yeastolate, plant hydrolysates such as soy, wheat, etc.). b) Passaging by modifying the culture medium to obtain total withdrawal of said factors, said feeder layer and said serum, and optionally said additives, and further obtaining adherent or suspension avian stem cells, in particular avian embryonic stem cells, capable of proliferating for a long period in a basal medium in the absence of exogenous growth factors, feeder layer and animal serum.

[0018] The term "avian" as used herein is intended to refer to any species, subspecies, or genus of organisms in the taxonomic order "ava," including, but not limited to, chickens, turkeys, ducks, geese, quail, pheasants, parrots, finches, hawks, crows, ostriches, emus, and cassowaries. As used herein, the terms "avian," "bird," "avian class," or "ava" are intended to have the same meaning and are used interchangeably. In a preferred embodiment, "bird" refers to any animal in the taxonomic order: - "Anseriformes" (i.e., ducks, geese, swans, and allies). The Anseriformes order includes about 150 species of birds in three families: Anhimidae (screamers), Anseranatidae (Magpie-goose), and Anatidae, including over 140 species of waterfowl, especially ducks, geese, and swans. All species in this order are highly adapted for life on the surface of water. All have webbed feet for efficient swimming (although some have since become primarily terrestrial). The term includes various lineages of ducks, such as Pekin and Muscovy ducks. - "Galliformes" (i.e. chickens, quail, turkeys, pheasants and allies). Galliformes is an order of birds that includes chickens, turkeys, quail and pheasants. There are about 256 species found worldwide. The term includes various strains of Gallus gallus or chickens, such as S86N, VaIo, White Leghorn, Brown Leghorn, Sussex, New Hampshire, Rhode Island, Australorp, Minorca, Amrox, California Gray, East Lansing, Italian-Partridge-colored, Marans, Barred Rock, Cou Nu Rouge (CNR), GF30, and ISA, as well as turkeys, pheasants, quail, and other commonly kept strains of poultry. - "Columbiformes" (i.e. pigeons and allies). The bird order Columbiformes includes the very widespread doves and pigeons.

[0019] Preferably, the avian embryonic stem cells according to the present invention are stem cells that have the advantage that they are obtained by culturing part or all of a very early embryo (e.g., the blastula stage). These embryonic stem cells preferably exhibit all the properties of stem cells in vitro and exhibit the intrinsic ability in vivo to contribute to embryonic morphogenesis and to participate in germline colonization when reimplanted in any way into a recipient embryo. Primordial germ cells (PGCs), the progenitors of sperm or oocytes that develop after sexual maturation, are pluripotent embryonic stem cells and constitute a subtype of embryonic stem cells.

[0020] In a preferred embodiment, the avian embryonic stem cells of the present invention are chicken embryonic stem cells, preferably selected from the group consisting of chicken lines DF-1, S86N, VaIo, White Leghorn, Brown Leghorn, Sussex, New Hampshire, Rhode Island, Australorp, Minorca, Amrox, California Gray, East Lansing, Italian-Partridge-colored, Marans, Barred Rock, Cou Nu Rouge (CNR), GF30, and ISA.

[0021] In another preferred embodiment, the avian embryonic stem cells according to the present invention are duck embryonic stem cells. In a more preferred embodiment, the duck embryonic stem cells according to the present invention are preferably selected from the group consisting of Pekin or Muscovy strains. Duck embryonic stem cells can also be derived from mulard embryos. As is well known to those skilled in the art, mulard embryos can be obtained by crossing a male Muscovy duck (Cairina moschata) with a female mallard duck (Anas platyrhynchos).

[0022] Preferably, the avian embryonic stem cells of step a) according to the present invention are isolated from freshly laid fertilized eggs, i.e. at the developmental stage called oviposition, which corresponds to the following developmental stage according to the Eyal-Giladi classification: -Muscovy duck: Phase VII; - Guinea fowl: Periods VII-VII; -Turkey: stages VII-VIII; -Pekin duck: Stage VIII; -Chicken: Stage X; - Japanese Quail: 1st stage; - Goose: Stage XL.

[0023] Preferably, Pekin duck embryonic stem cells are obtained by dissociating embryos near stage VIII (egg-laying) of the Eyal-Giladi classification. If the eggs collected at the egg-laying stage are not sufficiently developed to harvest embryonic stem cells, the eggs may be further incubated for several hours to one or two days to allow the embryos to mature.

[0024] Preferably, Muscovy duck embryonic stem cells are obtained by dissociating embryos around stage VII (egg-laying) of the Eyal-Giladi classification.

[0025] Preferably, chicken embryonic stem cells are obtained by dissociating embryos around stage X (egg-lay) according to the Eyal-Giladi classification.

[0026] Alternatively, the avian embryonic stem cells of step a) according to the present invention are harvested from embryos before egg laying. The main limitations encountered before egg laying are the fact that eggs must be surgically removed from hens and that the amount of embryonic stem cells per embryo is not very critical. Those skilled in the art will be able to define the time frame before egg laying that allows avian embryonic stem cells to be harvested.

[0027] Alternatively, the avian embryonic stem cells of step a) according to the present invention can be collected from avian embryos from the time of egg-laying to hatching. Those skilled in the art will be able to define the time frame after egg-laying during which avian embryonic stem cells can be collected. However, it is preferred that avian stem cells, especially avian embryonic stem cells, are not collected after egg-laying, especially from the formed embryo.

[0028] According to one embodiment of the present invention, the avian embryonic stem cells in step a) of the present invention can be a population of embryonic stem cells enriched in primordial germ cells (PGCs). Preferably, the avian embryonic stem cells are purified primordial germ cells. In avian species, primordial germ cells arise from the central region of the blastoderm. They then migrate to the anterior extraembryonic region, the genital crescent, until they are collected by the vasculature and reach the genital ridges between days 2.5 and 5 of embryonic development. They then settle in the genital ridges, where they ultimately differentiate into oocytes or spermatocytes. The method for isolating PGCs from donor avian embryos can be easily performed by those skilled in the art. According to one embodiment, PGCs are collected from embryonic blood collected from the dorsal aorta of stage 12-14 chick embryos according to the Hamburger & Hamilton classification (Hamburger & Hamilton (1951) J. Morpholog. 88:49-92). In another preferred embodiment, PGCs are harvested from the reproductive crescent by mechanical dissection of the chick embryo or gonads, however, other methods for isolating PGCs are known and can alternatively be used by those skilled in the art.

[0029] "Passage" refers to the transfer of cells from one culture vessel to another, with or without dilution. It is understood that whenever cells are transferred from one vessel to another, a certain portion of the cells may be lost, and thus, dilution of the cells may occur, whether intentional or unintentional. This term is synonymous with the term "subculture." Passage number is the number of times cells in a culture growing either in suspension or adherent have been subcultured or passed into a new vessel.

[0030] The term "diploid" refers to cells of the invention that have two copies (2n) of each chromosome, usually one from the mother and one from the father.

[0031] The cell line of the present invention is "continuous" because it has the property of being cultured in vitro for a long period of time. Advantageously, the cells of the present invention can be propagated for at least 50 generations, at least 75 generations, at least 100 generations, at least 125 generations, at least 150 generations, at least 175 generations, at least 200 generations, or at least 250 generations. 250 generations does not constitute a time limit, since the resulting cells are still viable and can be passaged for further passage.

[0032] The factor enabling proliferation of avian stem cells, in particular avian embryonic stem cells, in step a) of the present invention is preferably selected from the group consisting of insulin growth factor 1 (IGF-1), ciliary neurotrophic factor (CNTF), interleukin 6 (IL-6), interleukin 6 receptor (IL-6R), stem cell factor (SCF) and fibroblast growth factor (FGF).

[0033] The complete medium of step a) according to the present invention is preferably a "basal medium". The basal medium according to the present invention is preferably a medium having a classical medium formulation that allows at least cell survival and, even better, cell proliferation by itself. Examples of basal media according to the present invention are BME (basal Eagle's medium), MEM (minimal Eagle's medium), medium 199, DMEM (Dulbecco's modified Eagle's medium), GMEM (Glasgow's modified Eagle's medium), DMEM-HamF12, Ham-F12 and Ham-F10, Iscove's modified Dulbecco's medium, McCoy's 5A medium, RPMI 1640, GTM3, EX-CELL EBx GRO-I serum-free medium (SAFC Biosciences) or HyClone CDM4 avian medium. Preferably, the basal medium according to the present invention contains inorganic salts such as CaCl2, KCl, NaCl, NaHCO3, NaH2PO4, and MgSO4; amino acids; vitamins such as thiamine, riboflavin, folic acid, and D-calcium pantothenate; and other components such as glucose, β-mercaptoethanol, and sodium pyruvate. Preferably, the basal medium according to the present invention is a synthetic medium. Furthermore, the basal medium according to the present invention may be supplemented with additives selected from the group consisting of 0.1 to 5 mM L-glutamine, preferably 2 to 3 mM L-glutamine; 0.05 to 2 mM sodium pyruvate, preferably 0.1 to 1 mM sodium pyruvate; 0.1 to 2.5% non-essential amino acids, preferably about 1% non-essential amino acids; 0.1 to 2.5% vitamins, preferably about 1% vitamins; 0.05 to 5 mM β-mercaptoethanol, preferably about 0.16 mM β-mercaptoethanol; and protein hydrolysates of non-animal origin.

[0034] During the establishment process of avian stem cells, particularly avian embryonic stem cells, according to the present invention, the cells are preferably cultured on a layer of feeder cells. Preferably, the feeder cells are animal cells or cell lines cultured for the purpose of culturing avian stem cells, particularly avian embryonic stem cells. Alternatively, the feeder cells can be replaced with an extracellular matrix and associated growth factors. Hereinafter, the term "feeder matrix" refers to either feeder cells or extracellular matrix. As used herein, the feeder matrix is ​​preferably constructed according to procedures known in the art. Preferably, the feeder matrix is ​​preconditioned. The term "preconditioned" refers to a feeder matrix that has been cultured in the presence of a medium for a period of time prior to the deposition of avian stem cells, particularly avian embryonic stem cells, for example, a period of time sufficient for the feeder matrix to initiate and establish production of growth factors or other factors. Typically, the feeder matrix is ​​preconditioned by culturing it alone for 1 to 2 days before contacting and depositing avian stem cells, particularly avian embryonic stem cells, with the feeder matrix. The feeder cells preferably comprise mouse fibroblasts. STO fibroblasts are preferred, although primary fibroblasts are also suitable. It is contemplated that feeder matrices comprising cells from other mouse species (e.g., rat); other mammalian species (e.g., ungulate, bovine, porcine); or avian species (e.g., Gallinacea, chicken, turkey, duck, goose, quail, pheasant) may also be used. In another embodiment, the feeder cells of the present invention can be transfected with an expression vector that allows the constitutive expression of growth factors, such as avian SCF, in STO cells. Thus, the feeder may produce the factors in a soluble form and / or attached to the plasma membrane of the cells. Therefore, the culture process of the present invention may optionally include establishing a monolayer of feeder cells. The feeder cells are mitotically inactivated using standard techniques.For example, the feeder cells may be exposed to X-rays or gamma irradiation (e.g., 4000 Rad gamma irradiation) or may be treated with mitomycin C (e.g., 10 μg / ml for 2-3 hours). The monolayer may optionally be cultured to about 80% confluency, preferably to about 90% confluency, and more preferably to about 100% confluency. While the configuration of feeder cells as a monolayer is the preferred configuration for culture, any suitable configuration is considered to be within the scope of the present invention. Thus, for example, layers, monolayers, clusters, aggregates, or other associations or groupings of feeder cells are considered to be within the scope of the present invention, and particularly within the meaning of the term "matrix."

[0035] In one embodiment, animal serum is added to the medium in step a) according to the present invention. The animal serum preferably used is fetal animal serum. Fetal bovine serum is preferred. Serum from other animal species (e.g., chicken, horse, pig, ungulate, etc.) can also be used. The final concentration of animal serum in the medium is preferably between about 1% and 25%, preferably between 5% and 20%, more preferably between 8% and 12%.

[0036] In another embodiment, the culture medium of step a) according to the present invention is not supplemented with animal serum or is animal serum-free.

[0037] The medium changes in step b) of the process for obtaining avian stem cells, in particular avian embryonic stem cells, according to the present invention, to obtain a gradual or complete weaning of growth factors, serum and feeder layers can be carried out simultaneously, sequentially or separately. The weaning order can be chosen, for example, from the following: -feeder layer / serum / growth factors; -feeder layer / growth factors / serum; - Serum / growth factors / feeder layers; - Serum / feeder layer / growth factors; -Growth factors / serum / feeder layers; -Growth factors / feeder layers / serum.

[0038] In a preferred embodiment, the weaning order is growth factors / feeder layer / serum. In a preferred embodiment, weaning of additives such as sodium pyruvate, non-essential amino acids (NNEA), vitamins, yeastolate, etc., occurs after weaning of the feeder layer and before weaning of serum. Preferably, weaning of yeastolate occurs after weaning of sodium pyruvate, NNEA, and vitamins.

[0039] Avian stem cell culture The avian stem cells according to the present invention, in particular avian embryonic stem cells, established by the method disclosed above have the property of growing without feeder cells, preferably in a serum-free medium, meaning that it does not contain exogenous growth factors and animal serum.

[0040] The avian stem cells according to the invention, in particular avian embryonic stem cells, preferably duck and chicken embryonic stem cells, can be cultured in vitro for a considerable period of time. Advantageously, a suspension of avian stem cells, in particular avian embryonic stem cells, obtained by the method according to the invention disclosed above can be propagated for at least 50 generations, at least 75 generations, at least 100 generations, at least 125 generations, at least 150 generations, at least 175 generations, at least 200 generations, at least 250 generations.

[0041] Preferably, the avian stem cells, particularly avian embryonic stem cells, of the present invention are capable of indefinite proliferation in serum-free medium.

[0042] The term "serum-free medium" (SFM) according to the present invention preferably refers to a ready-made cell culture medium that does not require the addition of animal serum to allow cell survival and cell growth. This medium does not need to be chemically defined and may contain hydrolysates of various origins, such as plants or yeast. Preferably, the SFM is of "non-animal origin," meaning that it does not contain components of animal or human origin. In SFM, natural serum proteins are preferably replaced with recombinant proteins. Alternatively, the SFM medium according to the present invention is preferably a protein-free and / or chemically defined medium. SFM medium preferably offers several advantages: (i) regulatory compliance of such a medium; (ii) optimization of the purification process; and (iii) better reproducibility in the process due to a better-defined medium.Examples of commercially available SFM media are VP SFM (InVitrogen Ref 11681-020, Cat. 2003), Opti Pro (InVitrogen Ref 12309-019, Cat. 2003), Episerf (InVitrogen Ref 10732-022, Cat. 2003), Pro293S-CDM (Cambrex Ref 12765Q, Cat. 2003), LC17 (Cambrex Ref BESP302Q), Pro CHO5-CDM (Cambrex Ref 12-766Q, Cat. 2003), HyQ SFM4CHO (Hyclone Ref SH30515-02), HyQ SFM4CHO-Utility (Hyclone Ref SH30516.02), HyQ PF293 (Hyclone Ref SH30356.02), HyQ PF Vero (Hyclone Ref SH30352.02), Excell 293 Medium (SAFC Biosciences ref 14570-1000M), Excell 325PF CHO Protein-Free Medium (SAFC Biosciences ref 14335-1000M), Excell VPRO Medium (SAFC Biosciences ref 14560-1000M), Excell 302 Serum-Free Medium (SAFC Biosciences ref 14312-1000M), Excell 65319, Excell 65421, Excell 65625, Excell 65626, Excell 65627, Excell 65628, Excell 65629 (JRH Biosciences), Excell MDCK SFM (SAFC-Biosciences Ref. 14581C), Excell MDCK Prod (Ref. M3678), Gene Therapy Medium 3 (Animal Component Free) (SIGMA-Aldrich, ref. G-9916 or Excell GTM-3) (hereafter referred to as G9916 medium), HYQ CDM4HEK-293 (Hyclone Ref. SH30859), HYQ SFM4HEK-293 (HYCLONE Ref. SH30521), AEM (InVitrogen), EX-CELL EBx GRO-I Serum-Free Medium (SAFC Biosciences), CDM4 Avian Medium (Hyclone).

[0043] The serum-free medium may be supplemented with at least one component selected from the group consisting of amino acids, lipids, fatty acids, cholesterol, vitamins, carbohydrates, protein hydrolysates of non-animal origin, and mixtures thereof.

[0044] By way of example, the amino acids added to the medium may be selected from the group consisting of asparagine and glutamine, or a mixture thereof.

[0045] By way of example, the carbohydrate added to the medium may be selected from the group consisting of D-glucose, D-sucrose and D-galactose, or a mixture thereof. According to a more preferred embodiment, the carbohydrate added is D-glucose.

[0046] By way of example, the lipid is selected from the group consisting of cholesterol, steroids, and fatty acids such as palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, linolenic acid, and derivatives thereof, or mixtures thereof.

[0047] The culture medium may contain auxiliary substances such as buffer substances like sodium bicarbonate, oxidative stabilizers, stabilizers to counteract mechanical stress, or protease inhibitors.

[0048] The culture vessel according to the present invention is preferably selected from the group consisting of a continuous stirred tank bioreactor, a Wave™ bioreactor, a Bello™ bioreactor, a spinner flask, a flask and a cell factory. According to a preferred embodiment, the culture vessel is a continuous stirred tank bioreactor allowing for control of temperature, aeration, pH and other controlled conditions, and equipped with suitable inlets for introducing cells, sterile oxygen, various media for culture, and outlets for removing cells and media, as well as means for stirring the media within the bioreactor.

[0049] food Preferably, the food ingredient according to the present invention is selected from the group consisting of antifoaming agents, emulsifiers, solidifying agents, gelling agents, humectants, mineral salts, stabilizers, thickeners, and texturizing agents.

[0050] Preferably, the antifoaming agent is selected from the group consisting of polyethylene glycol and triethyl citrate.

[0051] Preferably, the emulsifier is selected from lecithin, sorbian monostearate and ammonium slats of phosphatidic acid.

[0052] The solidifying agent is preferably selected from the group consisting of calcium chloride, calcium gluconate and calcium sulfate.

[0053] The gelling agent is preferably selected from the group consisting of agar, calcium alginate and carrageenan.

[0054] The humectant is preferably selected from the group consisting of glycerin, glycerol, lactitol and polyethylene oxide, and the mineral salt is cupric sulfate.

[0055] Preferably, the stabilizer is selected from the group consisting of xanthan gum, guar gum, and bleached starch.

[0056] The thickening agent is preferably selected from the group consisting of tannin, sodium alginate and pectin.

[0057] Preferably, the texturing agent is selected from the group consisting of phosphates, sodium tripolyphosphate, sodium hexametaphosphate and sodium pyrophosphate.

[0058] In one embodiment of the invention, the food product according to the invention further comprises at least one supplementary food ingredient, preferably selected from the group consisting of vitamins, minerals, fiber, vegetable oils, fatty acids, amino acids, flavorings, colorants, antioxidants, sweeteners, seasonings, acidifiers, preservatives, isolating agents, flavor enhancers, sugar, flour, prebiotics, salt, water and antimicrobial agents.

[0059] Preferably, the vitamins are selected from the group consisting of B vitamins, including niacin, vitamin C and vitamin E.

[0060] The flavoring agent is preferably selected from the group consisting of oleoresins and water-soluble resins.

[0061] The coloring agent is preferably selected from the group consisting of curcumin, brilliant blue, tartrazine and ferrous gluconate.

[0062] Preferably, the antioxidant is selected from the group consisting of nitrates, nitrites, butylated hydroxyanisole, ascorbyl palmitate, and calcium ascorbate.

[0063] Preferably, the sweetening agent is selected from the group consisting of sorbitol, alitame, aspartame, saccharin, calcium saccharin, and corn syrup.

[0064] The flavoring agent is preferably selected from the group consisting of acetic acid, citric acid and fumaric acid.

[0065] The acidifying agent is preferably lactic acid.

[0066] The preservative is preferably selected from the group consisting of sodium nitrate, benzoic acid, sodium benzoate, tocopherol, ascorbic acid, niacin, riboflavin and thiamine.

[0067] Preferably, the sequestrant is potassium gluconate.

[0068] The seasonings are preferably selected from the group consisting of spices or oleoresins extracted therefrom, herbs, vegetables, essential oils, sodium nitrate, water, salt, sugar, and flavorings.

[0069] Preferably, the antibacterial agent is selected from the group consisting of lactic acid, citric acid, acetic acid, sodium diacetate, acidified sodium chloride or calcium sulfate, cetylpyridinium chloride, activated lactoferrin, sodium or potassium lactate, or a bacteriocin such as herring.

[0070] Preferably, the food product according to the present invention does not contain any animal components other than avian stem cells.

[0071] It is clear that the food product according to the invention is neither a bird / bird part nor an egg, in particular not a fresh fertilized egg or a fertilized egg such as a balut, and more generally it is clear that the food product according to the invention is not a naturally occurring product but a manufactured product.

[0072] Moreover, the avian stem cells, in particular avian embryonic stem cells, contained in the food product according to the invention preferably consist of at least 60%, at least 70%, at least 80%, at least 90, at least 95% undifferentiated cells.

[0073] Preferably, the food product according to the invention comprises less than 50%, 40%, 30%, 20%, 10%, 5%, 2% or 1% differentiated cells of avian origin (calculated as number of differentiated cells of avian origin / total number of cells of avian origin (differentiated + undifferentiated)).

[0074] Preferably, the food product according to the present invention is suitable for human and / or non-human animal consumption.

[0075] Preferably, the food product according to the present invention has organoleptic properties similar to those of a meat product. Methods for determining organoleptic properties are well known in the art, such as the use of panelists or artificial means. More preferably, the food product according to the present invention has organoleptic properties similar to those of a meat product selected from the group consisting of beef, buffalo, bison, veal, goat, ham, horse, kangaroo, lamb, moose, mutton, pork, bacon, rabbit, venison, chicken, duck, emu, goose, guinea fowl, ostrich, partridge, pheasant, pigeon, quail, and turkey.

[0076] Preferably, the food product according to the invention comprises 5 to 30% (dry w / w) protein.

[0077] Preferably, the food product according to the invention comprises 1 to 5% (dry w / w) lipids.

[0078] Preferably, the food product according to the present invention contains at least 1% (dry w / w) of a mineral such as iron.

[0079] Preferably, the food product according to the invention comprises at least one essential amino acid selected from phenylalanine, leucine, methionine, lysine, isoleucine, valine, threonine, tryptophan and histidine, more preferably at least 2, 3, 4, 5, 7 and 8 essential amino acids.

[0080] Food products according to the present invention are preferably in unit form of at least 5 grams, at least 10 grams, at least 15 grams, at least 25 grams, at least 50 grams, at least 70 grams, at least 80 grams, at least 90 grams, at least 100 grams.

[0081] Also preferably, the food product according to the invention is in the form of a fresh product, a dried product, a frozen product, a cooked, pickled or smoked product or incorporated into a processed food, in particular in a form selected from the group consisting of soups, stews, sausages, spreads, purees, biscuits, dry granules, tablets, capsules, powders, pasta boxes, pizzas, ready meals, sandwiches or nuggets.

[0082] The food product according to the invention can be used as a dietary supplement, especially a protein supplement, preferably for the elderly, or as a sports or training supplement. Preferred forms of the dietary supplement are in liquid form, especially shaken, or in powder form.

[0083] The food products according to the invention are preferably packaged in commercial packaging suitable for distribution to the end consumer, which may be any suitable packaging known to those skilled in the art, such as bags, boxes, cans, cartons, coated paper, flexible packaging, pallets, wrappers, trays, bottles, glass containers, cups, envelopes, pizza boxes, microwave boxes, pasta boxes, jars, etc.

[0084] Food preparation Preferably, the food product according to the invention is prepared by a method comprising the step of mixing avian stem cells according to the invention, in particular avian embryonic stem cells, with at least one food ingredient according to the invention.

[0085] In one embodiment, the method for preparing a food product according to the present invention further comprises at least one step of culturing in vitro avian stem cells according to the present invention, in particular avian embryonic stem cells, in a medium. Preferably, the method for preparing a food product according to the present invention comprises the following steps: - at least one step of culturing avian stem cells, in particular avian embryonic stem cells, in vitro in a culture medium; - harvesting avian stem cells; - mixing said avian stem cells with at least one food ingredient according to the invention to obtain a food product.

[0086] Preferably, the process of culturing avian stem cells, in particular avian embryonic stem cells, does not result in substantial differentiation of the cells, i.e. results in less than 50%, 40%, 30%, 20%, 10%, 5%, 2% or 1% differentiated cells (calculated as number of differentiated cells / total number of cells (differentiated + undifferentiated)).

[0087] The step of culturing avian stem cells, in particular avian embryonic stem cells, in vitro in a medium according to the present invention can be carried out before the incorporation of a food ingredient according to the present invention or after the incorporation of a food ingredient according to the present invention.

[0088] In one embodiment of the invention, the method further comprises the addition of at least one additional food ingredient according to the present invention.

[0089] Preferably, the method further comprises at least one step of food processing to provide the food in a consumable form. The food processing step according to the present invention can be carried out by any means known to those skilled in the art for transforming food. By way of example, the food processing step can be selected from the group consisting of solidifying, pressing, drying, freeze-drying, freezing, boiling, cooking, smoking, irradiating, homogenizing, cooking under pressure, canning, pasteurizing, and packaging.

[0090] Preferably the food product is processed into a fresh product, a dried product, a frozen product, a cooked, pickled or smoked product or incorporated into a processed food product, in particular a form selected from the group consisting of soups, stews, sausages, spreads, purees, biscuits, dry granules, tablets, capsules, powders, pasta boxes, pizzas, ready meals, sandwiches or nuggets.

[0091] The invention is further illustrated by the following non-limiting examples. [Example]

[0092] Example 1 1. Method Duck embryonic stem cells are stored in cryovials at -80°C. Cells are thawed and used to seed 3 L Erlenmeyer flasks in serum-free liquid HyClone CDM4 avian medium (GE Healthcare) supplemented with 2.5 mM l-glutamine. Cells are cultured at 37°C in a 7.5% CO2 atmosphere using an orbital shaker (150 rpm). Cells are then pelleted in 1 L bottles by centrifugation (3 x 100 g).

[0093] 250 g of the harvested cells are then mixed with 1 / 2 an egg and 1 / 2 a tablespoon of flour, processed into balls, and cooked in a preheated oven at 180°C until the internal temperature of the balls reaches 70°C to obtain the food product. Similar meatballs are prepared with minced duck meat.

[0094] 2.Results To evaluate the nutritional quality of the product and compare it with the nutritional profile of duck meatballs.

[0095] The cell-based meatballs of the present invention have a similar nutritional profile to duck meatballs, particularly with regard to protein content.

[0096] Example 2 Cells are prepared as described in Example 1.

[0097] Prepare the following food (nuggets): TIFF2025134719000001.tif40170

[0098] After being fried, the food product according to the invention exhibits a protein content similar to that of the control, but provides organoleptic properties closer to that of traditional chicken nuggets than the control.

Claims

1. A food product comprising avian stem cells and at least one food ingredient.

2. 10. The food product of claim 1, wherein the avian stem cells are isolated from an embryo.

3. 3. The food product according to claim 1 or 2, wherein the avian stem cells are derived from ducks and / or chickens.

4. 4. Food product according to any one of claims 1 to 3, wherein the avian stem cells have been obtained by a method comprising at least one step of in vitro culture.

5. The food product according to claim 1 , which does not contain any animal components other than avian stem cells.

6. 6. The food product of claim 1, wherein the food ingredient is selected from the group consisting of antifoaming agents, emulsifiers, solidifying agents, gelling agents, humectants, mineral salts, stabilizers, thickeners, and texturizing agents.

7. 7. The food product of claim 1, further comprising at least one supplemental food ingredient selected from the group consisting of vitamins, minerals, fiber, fatty acids, amino acids, flavorings, colorings, antioxidants, sweeteners, seasonings, acidifying agents, preservatives, isolating agents, flavor enhancers, sugars, prebiotics, salt, water, and antimicrobial agents.

8. 8. The food product of claim 1, having organoleptic properties similar to those of meat products, in particular meat products selected from the group consisting of beef, buffalo, bison, veal, goat, ham, horse, kangaroo, lamb, moose, mutton, pork, bacon, rabbit, venison, chicken, duck, emu, goose, guinea fowl, ostrich, partridge, pheasant, pigeon, quail, and turkey.

9. 9. The food product of any one of claims 1 to 8, in a unit form of at least 15 grams, at least 25 grams, at least 50 grams, at least 70 grams, at least 80 grams, at least 90 grams, or at least 100 grams.

10. 10. A food product according to any one of claims 1 to 9 in the form of a fresh product, a dried product, a frozen product, a cooked, pickled or smoked product or incorporated into a processed food, in particular in a form selected from the group consisting of soups, stews, sausages, spreads, purees, biscuits, dry granules, tablets, capsules, powders, pasta boxes, pizzas, ready meals, sandwiches or nuggets.

11. 11. The food product of any one of claims 1 to 10, packaged in a commercial packaging suitable for distribution to end consumers.

12. Use of avian stem cells and at least one food ingredient for the production of a food product.

13. 13. Use according to claim 12, wherein the avian stem cells are as defined in claims 2 to 5.

14. 1. A method for preparing a food product, the method comprising the step of mixing avian stem cells with at least one food ingredient.

15. 15. The method of claim 14, further comprising at least one prior step of culturing avian stem cells in vitro in a culture medium.

16. 16. The method of claim 14 or 15, comprising: - at least one step of culturing avian stem cells in vitro in a culture medium; - Harvesting avian stem cells; - mixing said avian stem cells with at least one food additive, Getting food.

17. 17. The method of any one of claims 14 to 16, wherein the culture medium is free of animal components such as animal serum.

18. 18. The method according to any one of claims 14 to 17, further comprising at least one step of processing the avian stem cells.

19. 19. The method according to any one of claims 14 to 18, wherein the avian stem cells are as defined in claims 2 to 5.

20. 20. A food product obtainable or obtainable by the method of any of claims 14 to 19.

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