Cultivated cell products

Abstract

A hybrid food product comprises: a substance of plant origin; and cultivated non-human fat cells, wherein the cultivated non-human cells comprise differentiated non-human fat cells.

Description

[0001] Cultivated cell products

[0002] Field of the invention

[0003] The present invention relates to a hybrid food product and to a method for making such a food product.

[0004] Background of the invention

[0005] According to the most recent United Nations estimations, the current world population is 7.9 billion in July 2022 [https: / / www.worldometers.info / es / poblacion-mundial / #ref-1] and it is expected to reach 10 billion around the year 2056. This increase will be heterogeneously distributed around the globe, with nine countries covering half the projected growth of the global population in the next 30 years, including India, Nigeria, Pakistan, Egypt, and the United States of America. Population and economic growth are major drivers of increased meat consumption. According to the United Nations Food and Agricultural Organization (FAO, https: / / www.oecd- ilibrary.org / agriculture-and-food / oecd-fao-agricultural-outlook-2022-2031_f1 b0b29c-en), an estimated growth of 15% in global meat consumption is projected by 2031 . On the other hand, the correlation between income growth and higher meat consumption is clearly demonstrable at lower income rates but once consumers reach an adequate standard of living, they become more sensitive to environmental, ethical, and animal welfare and health concerns.

[0006] For this reason, there is a growing interest in finding alternative protein sources which ideally will be sustainable and will contain the nutrients normally provided by meat in the human diet which can then be used in meat substitute products.

[0007] However, current meat substitute products, such as plant-based meat substitutes, cannot offer tastes similar to animal meat, and therefore have not been widely accepted by consumers.

[0008] There is therefore a need in the art for meat substitute products that closely mimic the characteristics and tastes of animal meat, for example improved flavor, texture, appearance and nutritional value.

[0009] Summary of the invention

[0010] The present invention relates to a meat substitute product, in particular a hybrid food product, and to a process for making such a product, for example based on cultivated meat.

[0011] Cultivated meat has arisen as an alternative to traditional animal agriculture that aims to produce the adipose tissues and skeletal muscle tissues that normally comprise animal meats, except using in vitro tissue and biological engineering techniques.

[0012] It has been found that hybrid foodstuffs comprising cultivated non-human cells, in particular differentiated cultivated fat cells, such as differentiated cultivated fat cells differentiated from pluripotent stem cells, may be used in the production of hybrid products which have properties which closely mimic those of animal meat. According to the invention, there is thus provided, A hybrid food product which comprises: a substance of plant origin; and cultivated non-human cells, wherein the cultivated non-human cells comprise differentiated non-human fat cells.

[0013] The invention also provides a method for the preparation of a hybrid food product which method comprises combining: a substance of plant origin; and cultivated non-human cells, wherein the cultivated non-human cells comprise differentiated non-human fat cells, thereby to produce a hybrid food product.

[0014] Description of the invention

[0015] The invention relates to a hybrid food product. A hybrid food product of the invention comprises a substance of plant origin and cultivated non-human cells. The cultivated non-human cells typically comprise differentiated fat cells which may be derived from pluripotent stem cells.

[0016] The term “cultivated cell (or cells)” is used interchangeably with “cultured cell (or cells)” and refers to any non-human cell or cells that are generated, for example by cell culture, in vitro or ex vivo.

[0017] A hybrid food product of the invention is one which comprises substance of plant origin and an amount of cultivated non-human cells.

[0018] A hybrid food product thus comprises: a portion comprising a substance of plant origin; and a portion comprising an amount of cultivated non-human cells. A hybrid food product of the invention may comprise one or more substances other than a substance of plant origin and an amount of cultivated non-human cells

[0019] A hybrid food product of the invention will typically have one or more organoleptic properties of a meat food product, for example a pork food product, a beef food product, a poultry food product or a fish or seafood product. A hybrid food product of the invention will typically have one or more nutritional properties of a meat food product, for example a pork food product, a beef food product, a poultry food product or a fish or seafood product.

[0020] “Organoleptic property” means a sensory characteristic of the hybrid food product, including taste, look or aroma and feel. Thus, the incorporation of an amount of differentiated cultivated cells into a hybrid food product of the invention may provide that an organoleptic property of a hybrid food product of the invention is improved compared to a hybrid food product comprising no differentiated cultivated cells.

[0021] Herein, a substance of plant origin means any non-meat substance. Such a substance may be one conventionally referred to as vegetarian and / or vegan. For the purposes of this invention, a substance of plant origin is taken to include microbial substances, fungal substances and the like. The substance of plant origin may comprise one or more plant based proteins (which includes, for example, proteins of microbial origin such as yeast proteins). Plant based proteins suitable for the use in food products are known to the skilled person in the art. A hybrid food product of the invention may be referred to as cultivated meat or a cultivated meat product.

[0022] A hybrid food product of the invention may comprise up to about 30% (w / w) of the cultivated non-human cells. For example, a hybrid food product of the invention may comprise up to about 1 % of the cultivated non-human cells, up to about 5% (w / w) of the cultivated non-human cells, up to about 10% (w / w) of the cultivated non-human cells, up to about 15% (w / w) of the cultivated non- human cells, up to about 20% (w / w) of the cultivated non-human cells or up to about 25% (w / w) of the cultivated non-human cells.

[0023] All or substantially all of the cultivated non-human cells in a hybrid food product of the invention may be differentiated.

[0024] A hybrid food product of the invention may comprise: differentiated cells; and nondifferentiated cells and / or partially differentiated cells. Accordingly, a hybrid food product of the invention may comprise: up to about 29% differentiated cells; and up to about 1 % non-differentiated and / or partially differentiated cells; up to about 28% differentiated cells; and up to about 2% non-differentiated and / or partially differentiated cells; up to about 25% differentiated cells; and up to about 5% non-differentiated and / or partially differentiated cells; up to about 20% differentiated cells; and up to about 10% non-differentiated and / or partially differentiated cells; up to about 15% differentiated cells; and up to about 15% non-differentiated and / or partially differentiated cells; up to about 10% differentiated cells; and up to about 20% non-differentiated and / or partially differentiated cells; up to about 5% differentiated cells; and up to about 25% non-differentiated and / or partially differentiated cells; or up to about 1 % differentiated cells; and up to about 29% non-differentiated and / or partially differentiated cells;

[0025] In a hybrid food product of the invention, the cultivated non-human cells may comprise cells which are derived from pluripotent stem cells. The cultivated non-human cells may comprise fully differentiated cells derived from pluripotent stem cells. The cultivated non-human cells may also comprise pluripotent stem cells or partially-differentiated derivatives thereof.

[0026] As used herein, the term "pluripotent stem cells" includes embryonic stem cells, embryo- derived stem cells, induced pluripotent stem cells and somatic cells, regardless of the method by which the pluripotent stem cells are derived. Accordingly, in certain embodiments the pluripotent stem cell is selected from the group consisting of embryonic stem cells, induced pluripotent stem cells, embryonic cell lines, and somatic cell lines. In certain embodiments, the pluripotent stem cells are epiblast-derived stem cells (EpiSC). In certain embodiments, pluripotent stem cells express one or more markers selected from the group consisting of: OCT-4, Sox2, Klf4, c-MYC, Nanog, Lin28, alkaline phosphatase, SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81. Exemplary pluripotent stem cells can be generated using, methods known in the art. "Induced pluripotent stem cells" (iPS cells or iPSC) can be produced by protein transduction of reprogramming factors in a somatic cell.

[0027] The cultivated non-human cells and pluripotent stem cells used in the invention can be from any species. Typically, they are animal cells, such as mammalian cells.

[0028] Embryonic stem cells have been successfully derived in, for example, mice, multiple species of non-human primates, and humans, and embryonic stem-like cells have been generated from numerous additional species. Thus, one of skill in the art can generate embryonic stem cells and embryo-derived stem cells from any species, including but not limited to, human, non-human primates, rodents (mice, rats), ungulates (cows, sheep, etc.), dogs (domestic and wild dogs), cats (domestic and wild cats such as lions, tigers, cheetahs), rabbits, hamsters, gerbils, squirrel, guinea pig, goats, elephants, panda (including giant panda), pigs, raccoon, horse, zebra, marine mammals (dolphin, whales, etc.) and the like.

[0029] Similarly, iPS cells can be from any species. These iPS cells have been successfully generated using mouse and human cells. Furthermore, iPS cells have been successfully generated using embryonic, fetal, newborn, and adult tissue. Accordingly, one can readily generate iPS cells using a donor cell from any species. Thus, one can generate iPS cells from any species, including but not limited to, human, non-human primates, rodents (mice, rats), ungulates (cows, sheep, etc.), dogs (domestic and wild dogs), cats (domestic and wild cats such as lions, tigers, cheetahs), rabbits, hamsters, goats, elephants, panda (including giant panda), pigs, raccoon, horse, zebra, marine mammals (dolphin, whales, etc.) and the like.

[0030] In certain embodiments, the cultivated non-human cells and pluripotent stem cells for use in the invention may be an animal cell. In certain embodiments the pluripotent stem cell for use in the invention is from an edible animal species.

[0031] Preferably, the cultivated non-human cells or pluripotent stem cells for use in the invention are from a livestock or poultry animal. Livestock species include but are not limited to domestic cattle, pigs, sheep, goats, lamb, camels, water buffalo and rabbits. Preferably, the cultivated non- human cells or pluripotent stem cells used in the invention are of porcine or bovine origin.

[0032] Preferably, the pluripotent stem cell for use in the invention is a porcine or a bovine pluripotent stem cell. Preferably, a porcine pluripotent stem cell. In certain embodiments, the stem cell for use in the invention is a porcine epiblast stem cell (pEpiSCs). Preferably, a bovine pluripotent stem cell. In certain embodiments, the stem cell or use in the invention is a bovine epiblast stem cell (bEpiSCs).

[0033] Poultry species include but are not limited to domestic chicken, turkeys, ducks, geese and pigeons. In certain embodiments, the cells originate from common game species such as wild deer, gallinaceous fowl, waterfowl and hare. Preferably a pluripotent stem cell according to the invention, or for use in the invention, is not a human cell.

[0034] A hybrid food product of the invention may comprise cultivated non-human fat cells of a single cell type or single cell lineage. That is to say, the cultivated non-human fat cells may comprise cells of more than one species and / or more than two types of cell from the same species. A hybrid food product of the invention may comprise non-human cultivated muscle cells. Cultivated muscle cells may be provided, for example, as set out in WO2024170696 and WO2024170702. In one embodiment, the non-human cultivated muscle cells are EpiSCs comprising the gene combination MYOD and MYOG. In one embodiment, the non-human cultivated muscle cells are EpiSCs comprising the gene combination MYOD and PAX7.

[0035] A hybrid food product of the invention may optionally not comprise or substantially not comprise cultivated muscle cells.

[0036] Non-human cells in a hybrid food product of the invention may comprise cultivated porcine cells or cultivated bovine cells. Non-human cells in a hybrid food product of the invention may comprise cultivated porcine cells and cultivated bovine cells.

[0037] A hybrid product of the invention may not comprise or substantially not comprise adult stem cells or cells derived therefrom.

[0038] A hybrid product of the invention may not comprise or substantially not comprise primary cells or cells derived therefrom.

[0039] A non-human fat cell of a hybrid food product of the invention may be a cell that is capable of lipid accumulation such as a fat cell (adipocytes) or precursor cells thereof (such as fibro- adipogenic progenitor cells, lipoblasts, adipoblasts and, pre-adipocytes) or liver cells (hepatocyte) or precursor cells thereof (such as pre-adipocytes, hepatoblasts and pre-heptatocytes). Thus, the cell may be one which is partially or fully differentiated into an mature adipocyte or may be one which is capable of being differentiated into a mature adipocyte such as a pluripotent stem cell.

[0040] Cultivated differentiated adipocytes may be provided, for example, as set out in W02024084082. In one embodiment, Cultivated differentiated adipocytes are EpiSCs comprising the gene combination PPARgamma and CEBPalpha.

[0041] A hybrid food product of the invention optionally does not or substantially does not comprise connective tissue cells other than adipose cells. That is to say, a hybrid food product of the invention may optionally not comprise or substantially not comprise cells of connective tissue, such as fibroblasts, macrophages, mast cell and leucocytes.

[0042] A hybrid food product of the invention may comprise from about 0.5% (w / w) to about 99.5% (w / w) of a substance of plant origin. Typically, a hybrid food product of the invention may comprise about 99.0% (w / w) of a substance of plant origin, for example about 95% (w / w) of a substance of plant origin, about 90% (w / w) of a substance of plant origin, about 85% (w / w) of a substance of plant origin, about 80% (w / w) of a substance of plant origin, about 75% (w / w) of a substance of plant origin or about 70% (w / w) of a substance of plant origin.

[0043] A hybrid food product of the invention comprises a substance of plant origin and cultivated non-human cells. In certain embodiments, the hybrid food is or further comprises an edible composition for human or non-human consumption.

[0044] The hybrid food product may in addition minerals, synthetic substances, flavoring substances (such as for examples herbs and spices).

[0045] A hybrid food product of the present invention may comprise one or more of minerals, synthetic substances, flavors, texture enhancers, nutritional additives, preservatives, and fats. In an aspect of the invention, the flavors are selected from one or more of essential oils, oleoresin (ESO), enzymes (ENZ), natural substances and extractives (NAT), non-nutritive sweetener (NNS), nutritive sweetener (NUTRS), herbs, spices, natural seasonings & flavorings (SP), and synthetic flavors (SY / FL), fumigant (FUM), artificial sweeteners and yeast extract. In another aspect of the present invention, the texture enhancers are selected from one or more of pureed plant material, guar gum, cellulose, hemicellulose, lignin, beta glucans, soy, wheat, maize and rice isolates and beet fiber, pea fiber, bamboo fiber, plant derived fiber, plant derived gluten, carrageenan, xanthan gum, lecithin, pectin, agar, alginate, natural polysaccharides, grain husk, calcium citrate, calcium phosphates, calcium sulfate, magnesium sulfate and salts. In another aspect of the present invention, the nutritional additives are selected from one or more of trace elements, bioactive compounds, endogenous antioxidants, A, B-complex, C, D, E vitamins, zinc, thiamin, riboflavin, selenium, iron, niacin, potassium, phosphorus, omega-3, omega-6, fatty acids, magnesium, protein, amino acids salt, creatine, taurine, carnitine, carnosine, ubiquinone, glutathione, choline, glutathione, lipoic acid, spermine, anserine, linoleic acid, pantothenic acid, cholesterol, Retinol, folic acid, dietary fiber and amino acids. In yet another aspect of the present invention, the fats are selected from one or more of saturated, monounsaturated, polyunsaturated fats, corn oil, canola oil, sunflower oil, safflower oil, olive oil, peanut oil, soybean, flax seed oil, sesame oil, canola oil, avocado oil, seed oils, nut oils, safflower and sunflower oils, palm oil, coconut oil, omega-3, fish oil, lard, butter, processed animal fat, adipose tissue, cellular agriculture derived fat essential oil and oleoresin. In another aspect of the present invention, the preservative and / or antioxidant is selected from one or more of: sodium salt, chloride salt, iodine salt. Nitrates, nitrosamines. butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), sodium benzoate, potassium benzoate and benzene ascorbic acid, citric acid, potassium, monosodium glutamate (MSG), sulphur dioxide, sulphites, antibiotics. It is noted here that any one additive, flavor, texture enhancer, nutrient additive, fat / oil and / or preservative / antioxidant may supply more than one attribute to food product of the present invention.

[0046] A hybrid food product of the invention may comprise a water immiscible portion. The oil immiscible portion may comprise oil portion comprising such as a vegetable oil.

[0047] A hybrid food product of the invention optionally is free from or substantially free from animal growth hormones and / or antibiotics.

[0048] A hybrid food product of the invention may be a vegetarian product. A hybrid food product of the invention may be a vegan product. A hybrid food product of the invention may be a meat substitute product. A hybrid food product of the invention may be halal. A hybrid food product of the invention may be kosher.

[0049] The invention also relates to a method for the preparation of a hybrid food product which method comprises combining: a substance of plant origin; and cultivated non-human cells, wherein the cultivated non-human cells comprise differentiated non-human fat cells, thereby to produce a hybrid food product. In yet a further aspect, the invention provides for a method of producing a food product, the method comprising combining the non-human cells as described herein with an edible composition for human consumption or non-human consumption. In certain embodiments, the food product is cultured meat.

[0050] A reference herein to a patent document or other matter which is given as prior art is not to be taken as an admission that that document or matter was known or that the information it contains was part of the common general knowledge as at the priority date of any of the claims.

[0051] The disclosure of each reference set forth herein is incorporated herein by reference in its entirety.

[0052] The present invention is further illustrated by the following Examples which should not be construed as limiting the scope of the invention:

[0053] Description of the figures

[0054] Figures 1 a and 1 b show the results of a sensory comparison of undifferentiated cultivated porcine fat cells, cultivated differentiated porcine cells and a pork fat mimic of porcine origin. In the legend: “cultivated fat mimic” = pork fat mimic of porcine origin (non-cultivated); “cultivated pork fat” = differentiated cultivated porcine fat cells; and “undifferentiated” = undifferentiated cultivated porcine cells. The results show that differentiated cultivated porcine fat cells are closer to pork fat than undifferentiated cultivated porcine cells.

[0055] Examples

[0056] Example 1 . Preparation of undifferentiated cells and differentiated fat cells from pluripotent stem cells

[0057] Cultivated porcine cells, undifferentiated cells and differentiated adipocytes, were generated as set out W02024084082.

[0058] Example 2. Sensory evaluation of undifferentiated cultivated porcine cells vs differentiated cultivated porcine fat cells

[0059] A sensory evaluation of undifferentiated cultivated porcine cells and differentiated cultivated porcine fat cells was caried out.

[0060] Samples were prepared by mixing 1 ,5 g cells with 0,1 g salt and 5,6 g water in 50 ml Erlenmeyer tubes with screw top. The mixture was heated “au bain Marie” in a pan with boiling water for 20 minutes. The screw tops from the Erlenmeyer tubes were not completely closed.

[0061] The two test products were evaluated on smell to see if any differences could be determined using a panel of evaluators. In this experiment, differences were observed between the three test products as follows:

[0062] - The differentiated cultivated porcine fat cells were meaty, animalic, with some rancid fat. - The undifferentiated cultivated porcine cells were weak in odour, not very meaty, not very specific, with some off note.

[0063] Example 3. Sensory evaluation of undifferentiated cultivated porcine cells vs differentiated cultivated porcine fat cells

[0064] In this evaluation, samples were prepared as in Example 2, except that the samples were tasted, so the salt content was reduced to 0,5%. Accordingly, 8,0 g cells were mixed with 0,2 g salt and 31 ,8 g water. The samples were prepared in larger 250 ml fermentation bottles with screw tops and placed in boiling water for 20 minutes

[0065] The evaluation was carried out based on odour and on taste and the samples were coded, so no information was given on which bottle contained what type of cells.

[0066] The overall conclusion was that the differentiated cultivated porcine fat cells are stronger and more specific “pork” than the undifferentiated cultivated porcine cells which in general were judged as old, milky, fatty and non-specific.

[0067] Differentiated cells were evaluated to taste of fat and fatty acids.

[0068] Undifferentiated cells were evaluated to be salty and milky.

[0069] It was concluded from the evaluations in Examples 2 and 3 that the differentiated cultivated porcine fat cells are more meaty, fatty and specific than the undifferentiated cultivated porcine cells.

[0070] Example 4. Sensory evaluation of undifferentiated cultivated cells vs cultivated differentiated porcine fat cells

[0071] A further sensory evaluation of undifferentiated cultivated porcine cells and differentiated cultivated porcine fat cells, but this time tasted alongside a pork fat mimic of porcine origin, was carried out. In this evaluation, 2 sensory training sessions were carried out prior to the cultivated product tasting: (1) to decide on the most relevant descriptors; and (2) to optimize the dilution ration (cultivated porcine cells / water) for the tasting.

[0072] The tasting evaluation was performed by 6 trained panel members and an expert flavourist. 3 samples, each containing 50% test product diluted in water, containing salt at 6g / kg and MSG at 0,1g / kg. The following were tested: cultivated porcine fat cells - differentiated; cultivated porcine cells - undifferentiated; and pork fat mimic - porcine origin (non-cultivated). Total sample size was c.a. 60g for the cultivated porcine samples and c.a. 120g for the mimic sample. Samples were kept warm (c.a. 80°C) and distributed, unidentified, one-by one in 3 different sequences in order to minimize as much as possible any bias.

[0073] The results of the sensory evaluation are set out in Figures 1 a and 1 b. All 3 samples were quite distinguishable from each other in terms of taste, texture and appearance. The main descriptors responsible from driving differences between the samples were: meaty, fatty, pork, sweet, umami and dry.

[0074] The undifferentiated cultivated porcine cells had a jelly structure and the smell reminded the flavourist of cooked milk with a strong sulfurolFI and slight notes of 2-methyl-3-furanthiol (typically associated with pork meat). The differentiated cultivated porcine fat cells had a meatier, fattier character with a slight note of pork and also there was a note of 2-methyl-3-furanthiol. The results show that differentiated cultivated porcine fat cells are closer to pork fat than the undifferentiated cultivated porcine cells.

[0075] Embodiments

[0076] The present invention provides at least the following numbered statements / embodiments:

[0077] 1 . A hybrid food product which comprises: a substance of plant origin; and cultivated non-human cells, wherein the cultivated non-human cells comprise differentiated non-human fat cells.

[0078] 2. A hybrid food product according to embodiment 1 , wherein the differentiated non-human fat cells are derived from pluripotent stem cells.

[0079] 3. A hybrid food product according to embodiment 1 or 2, which comprises up to about 30 % (w / w) of the cultivated non-human fat cells.

[0080] 4. A hybrid food product according to any one of the preceding embodiments, wherein the cultivated non-human cells comprise pluripotent stem cells and / or partially-differentiated derivatives thereof.

[0081] 5. A hybrid food product according to any one of the preceding embodiments, wherein the cultivated non-human cells are of a single cell type or single cell lineage.

[0082] 6. A hybrid food product according to any one of the preceding embodiments, which hybrid food product comprises non-human cultivated muscle cells. 7. A hybrid food product according to any one of the embodiments, which does not comprise non-human cultivated muscle cells.

[0083] 8. A hybrid food product to any one of the preceding cembodiments, wherein the cultivated non-human cells comprise cultivated porcine fat cells or cultivated bovine fat cells.

[0084] 9. A hybrid food product according to any one of the preceding embodiments, which does not substantially comprise connective tissue.

[0085] 10. A hybrid food product according to any one of the preceding embodiments, which comprises from about 70% to about 99.5% (w / w) of a substance of plant origin and about 0.5% to about 30% (w / w) cultivated non-human cells.

[0086] 11. A hybrid food product according to any one of the preceding embodiments, which is substantially free of growth hormones or antibiotics.

[0087] 12. A method for the preparation of a hybrid food product which method comprises combining: a substance of plant origin; and cultivated non-human cells, wherein the cultivated non-human cells comprise differentiated non-human fat cells, thereby to produce a hybrid food product.

Claims

Claims1 . A hybrid food product which comprises: a substance of plant origin; and cultivated non-human cells, wherein the cultivated non-human cells comprise differentiated non-human fat cells.

2. A hybrid food product according to claim 1 , wherein the differentiated non-human fat cells are derived from pluripotent stem cells.

3. A hybrid food product according to claim 1 or 2, which comprises up to about 30 % (w / w) of the cultivated non-human fat cells.

4. A hybrid food product according to any one of the preceding claims, wherein the cultivated non-human cells comprise pluripotent stem cells and / or partially-differentiated derivatives thereof.

5. A hybrid food product according to any one of the preceding claims, wherein the cultivated non-human cells are of a single cell type or single cell lineage.

6. A hybrid food product according to any one of the preceding claims, which hybrid food product comprises non-human cultivated muscle cells.

7. A hybrid food product according to any one of the claims, which does not comprise non- human cultivated muscle cells.

8. A hybrid food product to any one of the preceding claims, wherein the cultivated non-human cells comprise cultivated porcine fat cells or cultivated bovine fat cells.

9. A hybrid food product according to any one of the preceding claims, which does not substantially comprise connective tissue.

10. A hybrid food product according to any one of the preceding claims, which comprises from about 70% to about 99.5% (w / w) of a substance of plant origin and about 0.5% to about 30% (w / w) cultivated non-human cells.11 . A hybrid food product according to any one of the preceding claims, which is substantially free of growth hormones or antibiotics.

12. A method for the preparation of a hybrid food product which method comprises combining: a substance of plant origin; andcultivated non-human cells, wherein the cultivated non-human cells comprise differentiated non-human fat cells, thereby to produce a hybrid food product.

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