阅读说明：本技术 由培养的肌肉细胞制成的干燥的食物制品 (Dried food product made from cultured muscle cells ) 是由 弗朗索瓦丝·苏珊娜·马尔加 于 2015-02-05 设计创作，主要内容包括：本发明涉及由培养的肌肉细胞制成的干燥的食物制品。本发明公开了一种可食用的、脱水的食物制品,所述食物制品包括：培养的动物肌肉细胞,所述培养的动物肌肉细胞与植物源水凝胶组合；其中,所述培养的动物肌肉细胞和所述植物源水凝胶被制成脱水的材料片。本发明还公开了一种构造成小吃薄片的可食用的、脱水的食物制品,一种制作可食用的食物制品的方法,以及一种将可食用的食物制品制成小吃薄片的方法。(The present invention relates to dried food products made from cultured muscle cells. The present invention discloses an edible, dehydrated food product comprising: cultured animal muscle cells in combination with a plant-derived hydrogel; wherein the cultured animal muscle cells and the plant-derived hydrogel are formed into a sheet of dehydrated material. An edible, dehydrated food product configured as a snack chip, a method of making an edible food product, and a method of forming an edible food product into a snack chip are also disclosed.)

1. An edible, dehydrated food product, the food product comprising:

cultured animal muscle cells in combination with a plant-derived hydrogel;

wherein the cultured animal muscle cells and the plant-derived hydrogel are formed into a sheet of dehydrated material.

2. The food article of claim 1, further comprising a flavorant.

3. The food product of claim 1, wherein the cultured animal muscle cells and the plant-derived hydrogel are distributed throughout the sheet of material.

4. The food product of claim 1, wherein dehydrated cultured animal muscle cells having a diameter between about 5 μ ι η and 30 μ ι η are distributed throughout the sheet of material.

5. The food product of claim 1, wherein the cultured animal muscle cells are derived from one or more of beef, veal, pork, chicken, or fish.

6. The food product of claim 1, wherein the cultured animal muscle cells comprise one or more of skeletal muscle cells, satellite cells, smooth muscle cells, and cardiac muscle cells.

7. The food product of claim 1, wherein the plant-derived hydrogel comprises pectin.

8. The food product of claim 1, wherein the plant-derived hydrogel comprises Low Methyl (LM) esterified pectin.

9. An edible, dehydrated food product configured as a snack chip, the food product comprising:

an edible body formed as a sheet of material, the edible body comprising dehydrated cultured animal muscle cells and a plant-derived polysaccharide; and

a fragrance, which is a perfume,

wherein the cultured animal muscle cells and the plant-derived polysaccharide are distributed throughout the sheet of material.

10. The food product of claim 9, wherein the edible body has a diameter greater than 10 times its thickness.

11. The food product of claim 9, wherein the cultured animal muscle cells are derived from one or more of beef, veal, pork, chicken, or fish.

12. The food product of claim 9, wherein the cultured animal muscle cells comprise one or more of skeletal muscle cells, satellite cells, smooth muscle cells, and cardiac muscle cells.

13. The food product of claim 9, wherein the plant-derived hydrogel comprises pectin.

14. The food product of claim 9, wherein the plant-derived polysaccharide comprises Low Methyl (LM) esterified pectin.

15. A method of making an edible food product, the method comprising:

combining cultured muscle cells and a plant-derived hydrogel to form a mixture; and

dehydrating the mixture to form a sheet of edible material.

16. The method of claim 15, further comprising adding a fragrance.

17. The method of claim 15, wherein the plant-derived hydrogel comprises an edible microcarrier on which the cultured muscle cells are grown.

18. The method of claim 15, wherein combining comprises combining cultured muscle cells grown on an edible microcarrier with the plant-derived hydrogel to form the mixture.

19. The method of claim 15, wherein combining comprises adding a flavorant to the mixture of muscle cells and plant-derived hydrogel.

20. The method of claim 15, wherein combining comprises combining one or more of cultured skeletal muscle cells, satellite cells, smooth muscle cells, and cardiac muscle cells with the plant-derived hydrogel to form the mixture.

21. The method of claim 15, wherein combining comprises combining cultured muscle cells with a plant-derived hydrogel and a calcium chloride solution, and gelling the plant-derived hydrogel.

22. The method of claim 15, wherein combining comprises combining the cultured muscle cells with a plant-derived hydrogel, a fragrance, and a calcium chloride solution.

23. The method of claim 15, further comprising harvesting the cultured cells from a tissue culture chamber and washing the cells prior to combining with the plant-derived hydrogel.

24. The method of claim 15, further comprising spreading the mixture on a surface and gelling the plant-derived hydrogel prior to dewatering.

25. The method of claim 15, wherein dewatering comprises dewatering the mixture to make a brittle sheet.

26. A method of forming an edible food product into a snack chip, the method comprising:

combining cultured muscle cells and a plant-derived hydrogel to form a mixture;

spreading the mixture on a surface;

allowing the mixture to solidify; and

dehydrating the mixture to form a sheet.

Technical Field

Described herein are edible (e.g., suitable for human consumption) food products made from a dehydrated mixture of cultured cells and a carrier (e.g., a hydrogel), and methods of making and using the edible food products to make engineered meat products.

Background

The human body requires proteins for growth and maintenance. In addition to water, proteins are the most abundant molecules in the body. According to the Dietary Reference Intake guidelines (diet Reference Intake guidelines) in the united states and canada, women aged 19-70 years need to consume 46 grams of protein per day, while men aged 19-70 years need to consume 56 grams of protein per day to avoid deficiency. However, the recommended amount is appropriate for a sedentary person who is free of disease. Protein deficiency can lead to decreased or retarded intelligence and prevalence of diseases such as quasishieoko (kwashiorkor). Protein deficiency is a serious problem in developing countries, especially in countries affected by war, famine and overpopulation. Animal sources of protein, such as meat, are often sources that supplement substantially all of the essential amino acids in sufficient proportions.

The nutritional benefits of meat are diminished by potentially associated environmental degradation. According toFood and agriculture organization of the United nations(FAO) Entitled Long-term Shadow-Environmental problems and Options for animal husbandry (2006), which is one of the biggest contributors to Environmental degradation worldwide, and the practice of modern farm animals for food generally results in air and water pollution, land degradation, climate change, and loss of biodiversity. The production and consumption of meat and other animal sources of protein is also associated with the disappearance of rainforests and the extinction of species. Therefore, there is a need for a solution to the need for an alternative to meat produced by live animals.

Food items such as chips (e.g., potato chips, crisps, puffs, cookies, jerky, etc.) are a popular snack food in the united states. Commercially available flakes typically contain high levels of fat and sodium, suggesting high caloric intake. Excessive consumption can cause increased health risks, such as hypertension. For example, potato flakes contain a high heating value, typically 150-. Baked potato slices, advertised as a healthier alternative to traditional fried slices, typically contain 120 calories, 18 of which are derived from fat at the same serving size. When combined with a negative lifestyle, high caloric intake can cause obesity, hypertension, and peripheral arterial disease. In addition, conventional potato flakes typically contain a high content of sodium in the range of 7% to 8% of the daily recommended value based on a 2000 calorie diet; this is a surprising amount considering that an ounce typically consists of less than 15 flakes. It has been reported that high levels of sodium cause the development of symptoms such as hypertension, which can lead to an increased risk of heart disease. Fried potato flakes often contain high amounts of fat and saturated fat, with a serving weight of 10 to 11 grams of fat per serving, of which 3 grams is saturated fat (a 2000 calorie based diet represents 15% to 17% of the daily recommendation). High fat content can pose a serious health risk, as high fat intake can lead to the formation of arterial plaque, increasing the propensity for heart disease and stroke. Similarly, consumption of excess fat on a normal basis may increase the risk of diabetes and obesity.

There is a need for a snack food, especially one that can resemble the widely popular chips, i.e., a high protein, high fiber, and high calcium, low fat snack food. Although so-called "meat slices" have been proposed in the past, such products have proven to be expensive, lack taste and potentially high in protein and also high in sodium and fat, which prevents these products from being an effective substitute for traditional slices. It is also important that such "meat slices" have been made from growing and slaughtered animals in the same manner as most commercially available meat. As mentioned above, this is not only environmentally problematic, but can cause ethical and ethical issues for the consumer.

For example, crisp meat based snacks (crisp meats) similar to potato flakes or similar to other carbohydrate based snacks are described, for example, in U.S. patent No.3,497,363, which proposes a crisp fried meat snack formed by deep frying freeze-dried pieces of meat. The freeze-drying is said to be critical to the crunchy, chewable nature of the flakes. Freeze-drying can be quite expensive on a commercial scale, and deep-frying increases the fat content of the chip, resulting in an expensive, high-fat snack. U.S. patent No.3,512,993 proposes mixing meat or seafood with water and 50/50 mixtures of potato and corn starch to form a dough that is cooked and sliced under pressure. The resulting slices are dried and deep-fried before consumption. Frying imparts a crunchy texture to the chip, rather than the "hard, horny texture" of the dried chip. The product has a high content of fat (with the proposed fat content of 30% -40%) and starch, which makes the chip less desirable for those who control caloric and carbohydrate intake from snack foods. Others have proposed methods for drying sausage slices to make snack foods without having to fry the slices. Such methods are set forth, for example, in U.S. patent No.6,383,549 and U.S. patent application publications 2003/0113433 and 2004/0039727. However, most of these methods are not well suited for commercial scale production of inexpensive snack foods and are limited to home-scale batch production volumes or expensive specialty products.

A sheetable dehydrated food product is described which addresses the above-mentioned deficiencies.

Disclosure of Invention

The present invention relates to food products that can be made from cultured cells combined (e.g., mixed) with a dehydrated hydrogel (e.g., a plant-derived polysaccharide or polysaccharide-based hydrogel, such as pectin). The food product can be made into any conventional dry food product including, but not limited to, potato chips, cookies, bars, oatmeal, doughs, jerky, and the like. Although the embodiments described herein illustrate food products and methods of forming the resulting food products into slices similar to traditional potato chips, the invention described herein is also applicable to the production of other food products. The resulting product can be a snack food, such as a chip, which is made without harm to the animal, and has high protein and low fat. The food product can provide a healthy, gluten-free, protein, fiber, and calcium rich snack that can resemble the texture of traditional potato chips (e.g., crispness and/or friability), but is not fried or baked to remove fat, particularly saturated fat. Also described herein are methods of making food products, such as flakes, from cultured cells (e.g., muscle cells from an animal).

For example, described herein is an edible, dehydrated food product comprising cultured animal muscle cells in combination with a plant-derived hydrogel, wherein the cultured animal muscle cells and plant-derived hydrogel are formed into a sheet of dehydrated material, and a flavorant.

Generally, cultured cells are mixed with a hydrogel, followed by coagulation or gelation and dehydration. The mixture may be homogeneous (e.g., relatively homogeneous) or heterogeneous, e.g., the cells may clump. The cells may be non-adherent when mixed, or they may be clustered, for example in small clusters or clumps. Thus, the mixture may comprise individual cells and/or clusters of cells distributed in a hydrogel, which is then dehydrated. In the final food product, the cultured animal muscle cells and the plant-derived hydrogel can be distributed throughout the sheet of material.

The flavor can be coated on and/or within the food article (contained within the mixture of cultured cells and hydrogel). The perfume may be added before, during or after dehydration.

For example, snack foods such as chips are described herein. The edible snack chip can include: cultured animal muscle cells in combination with a plant-derived hydrogel; and a flavor, wherein the cultured animal muscle cells and the plant-derived hydrogel are made into a dehydrated sheet of material.

Any suitable cultured animal muscle cell can be used. For example, cultured muscle cells (myocytes) can be derived from one or more of the following: beef, veal, pork, chicken, and fish. The cultured animal muscle cells can include one or more of: skeletal muscle cells, smooth muscle cells, and cardiac muscle cells (or mixtures thereof). The cultured cells may be all muscle cells or most of the cultured muscle cells. For example, the cellular component of the food product can have greater than 70% muscle cells, greater than 80% muscle cells, greater than 85% muscle cells, greater than 90% muscle cells, greater than 95% muscle cells, greater than 98% muscle cells, greater than 99% muscle cells, and the like.

Any suitable hydrogel, particularly a plant-derived polysaccharide, may be used. For example, the plant-derived polysaccharide may comprise pectin. The plant-derived polysaccharide (polysaccharide-based hydrogel) may be a Low Methyl (LM) esterified pectin. Typically, the plant-derived hydrogel is a hydrogel derived from a non-animal source. For example, the plant-derived hydrogel may be extracted from a plant source, purified, or otherwise obtained from a plant source. The plant-derived hydrogel may also be a hydrogel of natural products identified as plants. Although plant-derived hydrogels can be identified as being from plant sources, the direct source of plant-derived hydrogels used in the food products described herein can be synthetic, e.g., the plant-derived hydrogels can be synthetic or refined. Any of the plant-derived hydrogels described herein may also be replaced by and/or mixed with a non-plant-derived hydrogel.

For example, an edible, dehydrated food product as described herein may include: cultured animal muscle cells in combination with a plant-derived hydrogel; and (optionally) a fragrance; wherein the cultured animal muscle cells and the plant-derived hydrogel are formed into a sheet of dehydrated material.

In the food product, the cultured animal muscle cells and the plant-derived hydrogel can be distributed throughout the sheet of material such that a cross-section through the sheet of material has discrete muscle cells (cultured muscle cells) that can have a diameter of about 2 μm to 50 μm (e.g., 2 μm to 40 μm, 2 μm to 35 μm, 5 μm to 50 μm, 5 μm to 40 μm, 5 μm to 30 μm, etc.) and can be distributed throughout the cross-section. The cultured muscle cells can be identified morphologically and by the expression of markers of their muscle proteins, which are visible on ultrastructures. For example, cultured animal muscle cells and plant-derived hydrogels can be distributed throughout a sheet of material such that a cross-section through the sheet of material has a pattern of dehydrated cultured muscle cells. The cultured muscle cells may be relatively intact, even after dehydration, and their origin as cultured cells may be confirmed by one or more markers, for example by identifying patterns of muscle proteins such as actin and myosin within the dehydrated cultured muscle cells. Thus, even in a dehydrated food product, a cross-section through the food product will exhibit a unique pattern resulting from the use of a mixture of cultured cells and plant-derived hydrogel.

Any of the food products ("slices") described herein may also include an edible microcarrier on which cultured animal muscle cells are grown.

In the context of food products, the term "dehydrated" or "dehydrated" as used herein may refer to removing water from a food product, particularly removing a majority of the water from a food product as compared to a non-dehydrated form of the food product, such that the water content of the food product is less than, e.g., 70% (e.g., less than 65%, less than 75%, less than 80%, less than 85%, less than 90%, less than 95%, etc.).

In some variations, an edible, dehydrated food product configured as a snack chip comprises: cultured animal muscle cells in combination with a plant-derived hydrogel (e.g., a polysaccharide); and a fragrance, wherein the cultured animal muscle cells and the plant-derived hydrogel are arranged into a sheet of dehydrated material, wherein clusters of muscle proteins distributed throughout the sheet of material have a diameter of between 2 μ ι η and 50 μ ι η (e.g., 2 μ ι η to 40 μ ι η,2 μ ι η to 30 μ ι η,2 μ ι η to 20 μ ι η,5 μ ι η to 50 μ ι η,5 μ ι η to 40 μ ι η,5 μ ι η to 30 μ ι η, etc.).

The edible food products described herein can also be made or shaped into a readily consumable form and can resemble conventional snack foods (e.g., potato flakes, bars, pretzels (pretzels), etc.). For example, the thickness (or height) may typically be much smaller than the surface dimensions (e.g., breadth, diameter, etc.) such as width and length. In some variations, the edible body of the sheet may have a diameter (e.g., surface diameter/sheet diameter) that is greater than ten times its thickness.

Methods of making edible food products are also described. For example, described herein is a method of making an edible food product comprising combining cultured muscle cells and a plant-derived hydrogel to form a mixture; and dehydrating the mixture to form a sheet of edible material.

For example, the method can be used to make edible snack foods. For example, a method of making a snack chip can comprise: combining cultured muscle cells with a plant-derived hydrogel to form a mixture; and dehydrating the mixture to form a sheet.

Any of these methods may further comprise adding a fragrance. The flavor can be added during the mixing step or can be mixed with any of the components (e.g., cultured cells) prior to the mixing step. For example, the combining step can include adding a flavorant to a mixture of muscle cells and the plant-derived hydrogel. The flavor may be added after mixing. For example, the fragrance can be added before, during, or after dehydration. The flavor can be applied to the food article.

Typically, the combining step can include combining one or more of cultured skeletal muscle cells, smooth muscle cells, and cardiac muscle cells with the plant-derived hydrogel to form the mixture. As noted above, any suitable cell type may be included.

In some variations, additional components may also be mixed with the cells and the hydrogel. For example, combining can include combining cultured muscle cells and a plant-derived hydrogel with a calcium chloride solution. Calcium chloride solutions can help to gel the hydrogel, and calcium can also be added to food products, which can be beneficial. For example, combining can include combining cultured muscle cells with a plant-derived hydrogel, a fragrance, and a calcium chloride solution.

Any of these methods may include the steps of: harvesting the cultured cells from the tissue culture chamber, and washing the cells prior to combining with the plant-derived hydrogel. The cultured cells may be washed by repeatedly rinsing and spinning (e.g., centrifugation) to clump the cultured cells and remove the wash solution. In some variations, the cells may be harvested and/or washed in a solution comprising a flavorant.

In any of the methods described herein, the cells may be live, dead, or dried immediately prior to mixing. Thus, the washing and/or mixing can reduce cell activity without affecting the quality (e.g., taste, texture, nutritional content) of the final food product. However, in some variations, the cells may remain viable until dehydration.

Any of the methods described herein can further comprise spreading the mixture onto a surface (e.g., a mold) and gelling the plant-derived hydrogel prior to dewatering. This dewatering step may be performed on the same surface (e.g., mold) or they may be transferred to a different surface. Any suitable die (including coated dies) may be used. For example, the mold may be a flat surface (e.g., foil, polymer, paper, etc.). In some variations, the die may be adapted for use with a dehydration engine. For example, the mold may be thermally conductive and/or vented or water permeable.

In some variations, a method of making a snack chip can comprise: combining cultured muscle cells, a plant-derived hydrogel, and a fragrance to form a mixture; and dehydrating the mixture to make a brittle (e.g., friable) sheet.

For example, a method of making an edible food product may comprise: combining cultured muscle cells with a plant-derived hydrogel to form a mixture; and dehydrating the mixture to form a sheet of edible material. The plant-derived hydrogel can be configured as an edible microcarrier (which can also include a polypeptide that includes a cell attachment motif) on which cultured muscle cells are grown. Alternatively or additionally, in some variations, combining comprises combining cultured muscle cells grown on an edible microcarrier with a plant-derived hydrogel to form a mixture.

Combining may include combining the cultured muscle cells with a plant-derived hydrogel and a calcium chloride solution, and allowing the plant-derived hydrogel to solidify (e.g., gel). Any of these methods may include spreading (e.g., pouring, coating, spraying, etc.) the mixture on a surface and gelling the plant-derived hydrogel prior to dehydration.

Additionally, in any of these methods, dewatering can include dewatering the mixture to make a brittle (e.g., friable) sheet.

For example, described herein is a method of forming an edible food product into a snack chip, the method comprising: combining cultured muscle cells with a plant-derived hydrogel to form a mixture; spreading the mixture in the form of a layer on a surface; allowing the mixture to solidify; and dehydrating the mixture to form a sheet.

Drawings

The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:

fig. 1A-1D illustrate components that may be combined to make the food products described herein. Fig. 1A shows a mass of cultured muscle cells (approximately 5 hundred million cells), which is shown in more detail in fig. 1B. Fig. 1C shows a flavoured vegetable soup (including flavour) and fig. 1D shows a 4% pectin solution.

Fig. 2A and 2B illustrate the formation of a food article configured as a sheet. In fig. 2A, a food product is made by combining the components shown in fig. 1A-1D and a calcium chloride solution; the mixture is spread onto a mold, gelled, and then dehydrated. Fig. 2B shows the resulting dehydrated food product.

Fig. 3a1 and 3a2 show top and side views, respectively, of one configuration of a food article configured as a chip as described herein. Fig. 3B1 and 3B2 show top and side views, respectively, of another configuration of a food article configured as an oval shaped slice. Fig. 3C1 and 3C2 show top and side views, respectively, of another configuration of a food article configured as a thin triangular sheet.

Fig. 4 shows a cross-section through a dewatered flake as described herein. The sections have been stained with anti-alpha Smooth Muscle Actin (SMA) antibody. The SMA appears as a darker, somewhat rounded (cellular) shape. This histology demonstrates the characteristic distribution of muscle proteins (e.g., actin) within the cultured cells that were flaked, which shows a pattern of dehydrated cultured muscle cells mixed with an animal-based hydrogel.

Fig. 5 is a table showing compositional analysis of one embodiment of a dried food product (flake) as described herein, showing a high percentage (e.g., greater than 50%) of protein. The combination may also indicate the presence of an animal-based hydrogel and shows that in this embodiment, the dehydrated food product has a water content of less than 5% (e.g., 4.01% in this embodiment) for the body of the dehydrated food product.

Detailed Description

Generally, food products made from cultured cells, particularly cultured muscle cells (myocytes) grown in vitro, without the need for additional manipulation of the animal of origin are described herein. The cultured cells can be grown using a medium that is not derived from an animal source (e.g., plant-derived, yeast-derived, single cell-derived, etc.). In addition, the food products described herein can be made by combining cultured cells with a hydrogel to form a mixture, gelling the mixture, and dehydrating the resulting mixture to form an edible food product. The shape and/or taste of the edible food product can be manipulated to determine the type of food product being made, including flakes, cookies, bars, oatmeal, dough, and the like. One or more flavors and/or enhancers may be added before, during, or after combining and dehydrating the cultured cells and hydrogel.

In general, any suitable method of culturing cells may be used, including culturing on a surface, solution, bioreactor, or the like. The cells in culture are typically muscle cells, such as non-human muscle cells, although other cell types may be used. The cells may originate from any suitable source. For example, suitable cells can be derived from a mammal, such as antelope, bear, beaver, bison, boar (bone), camel, reindeer, cow, deer, elephant, elk, fox, giraffe, goat, hare, horse, northgoat, kangaroo, lion, llama, moose, boar (peccaray), pig, rabbit, seal, sheep, squirrel, tiger, whale, yak, and zebra, or a combination thereof. In some embodiments, suitable cells are derived from birds, such as chickens, ducks, emus, geese, grouse, ostriches, pheasants, pigeons, quail and turkeys, or combinations thereof. In some embodiments, suitable cells are derived from reptiles, such as turtles, snakes, crocodiles, and alligators, or combinations thereof. In some embodiments, suitable cells are derived from fish, such as anchovies, bass (bass), catfish, carp, cod, eel, flatfish, globefish, grouper, haddock, halibut, herring, mackerel, mahi, tuna, orange roughy, perch, pike, pollack, salmon, sardine, shark, snapper, sole (sole), swordfish, tilapia, trout, tuna, and whitefish, or combinations thereof. In some embodiments, suitable cells are derived from crustaceans, such as crabs, crayfish, lobsters, prawns, and shrimps, or combinations thereof. In some embodiments, suitable cells are derived from molluscs, such as abalone, clams, conchs, mussels, oysters, scallops and snails, or combinations thereof. In some embodiments, suitable cells are derived from cephalopods, such as cuttlefish, octopus, and squid, or combinations thereof. In some embodiments, suitable cells are derived from insects such as ants, bees, beetles, butterflies, cockroaches, crickets, brides (damselfly), dragonflies (dragonfly), earwigs, fleas, flies, grasshoppers, mantises, mayflies, moths, silverfish, termites, wasps, or combinations thereof. In some embodiments, suitable cells are derived from a non-arthropod invertebrate (e.g., a worm), such as a flatworm, a tapeworm, a trematode, a nematode, a roundworm, a hookworm, an annelid worm (e.g., an earthworm, an annelid, etc.), or a combination thereof. The cultured cells may be native or modified (e.g., transgenic).

Typically, the cultured cells can be grown to a sufficient density, harvested, and then washed prior to combining with the other components of the food product mixture, including the hydrogel. Washing can remove the medium, and can be in water (including buffer solution, such as PBS) washing. Cultured cells may be repeatedly clumped (e.g., by centrifugation) and washed for washing. In some variations, the cells may be cultured using microcarriers, particularly edible microcarriers. As will be described in more detail below, the edible microcarrier may be an edible plant-derived polysaccharide that may also include a polypeptide that includes a cell attachment motif. Plant-derived hydrogels for cells grown without edible microcarriers may also be used in combination with cells grown on edible microcarriers as described herein; alternatively, in some variations, no additional hydrogel is added, and only edible microcarriers and cells can be used to make the sheet.

As noted above, any suitable hydrogel may be used. Typically, the hydrogel must be edible (e.g., safe for human consumption). The hydrogel may comprise a polysaccharide capable of cross-linking, such as pectin. For example, one class of polysaccharides that can be used is Low Methyl (LM) esterified pectin, an abundant plant derivative that has been used in food.

Any food product described herein can be referred to as a dried food product or a dehydrated food product.

Any of the food articles described herein can include one or more flavorants. The term "perfume" may refer to both natural and artificial varieties. This is intended to include "natural flavors" such as those defined by title 21 of the U.S. code of Federal Regulations (u.s.code of Federal Regulations), namely essential oils, oleoresins, essences or extracts, protein hydrolysates, distillates, or any broil, heat or enzymatic hydrolysate containing flavor ingredients derived from spices, fruit or juice, vegetable or vegetable juice, edible yeast, herbs, bark, sprouts, roots, leaves, or any other edible part of a plant, meat, seafood, poultry, eggs, dairy products, or fermentation products thereof, whose primary function in food is flavor rather than nutrition (21CFR 101.22).

Fragrances may also include "artificial fragrances", particularly chemically synthesized compounds of natural fragrances that do not necessarily meet the above-mentioned specifications. Artificial flavors may include chemical compounds found in "natural flavors".

In addition, "flavors" may also be a general term used to refer to agents that impart taste, flavoring aromatics, and sensory factors. Taste is the sensation that is processed by receptors on the tongue and generally includes salty, sweet, sour and bitter tastes. Flavoring aromatics are those flavoring volatiles that are emitted when food is bitten, chewed, drunk, and swallowed, and are sensed by olfactory receptors. Sensory factors in flavored languages describe the sensation perceived in the mouth, on the tongue, or in the nasal passages (or anywhere in the oral/nasal cavity). These sensations can be independent and distinct from salty, sweet, sour and bitter tastes, and from the myriad of flavoring aromatics perceived by olfactory receptors. The compounds that produce these sensations vary in volatility, but many are susceptible to vapor phase transfer. Such sensory factors include the pungency of the smoke, the astringency of fruits, the coolness of mints, or the hot pungency of peppers. More specifically, the flavorant may enhance or alter the taste or odor, or both, of the article. The change may enhance a desired taste or flavor, or mask an undesired taste or odor. It will be appreciated that in most applications, the perfume is non-toxic and ingestible.

The fragrance may comprise a flavoring aroma, but some components of the fragrance do not have olfactory stimulating properties. For example, flavorings, including artificial sweeteners, some spices and seasonings, while lacking olfactory stimulating properties, are still useful flavors in practicing the present invention. Certain spices or mixtures of spices used to season packaged snack foods, including representative examples such as potato flakes, corn flakes, barbeque flakes, cheese crackers, and others, can be seasoned using a uniform and non-uniform combination of solid or particulate spices and seasonings such as spicy barbeque flavors. They have the property of enhancing the flavor (taste) and thus are useful as spices in the manufacturing process together with other spices commonly used as flavouring in foods.

The following is not a comprehensive list, but merely representative of some commonly used taste flavors, as well as some flavors that produce a sensation. Examples of flavors that impart taste and sensation include artificial sweeteners, glutamate, glycinate, guanylate, inosinate, ribonucleoside, and organic acids (including acetic, citric, malic, tartaric), polyphenols, and the like.

This list is merely an example of common flavoring aromatics. There are thousands of molecular compounds that can be combined or used alone to form a particular desired scent. Some representative examples of common flavoring aromatics include isoamyl acetate (banana), cinnamaldehyde (cinnamon), ethyl propionate (fruity), limonene (orange), ethyl- (E, Z) -2, 4-decanedioic acid ester (pear), allyl caproate (pineapple), ethyl maltol (sugar, cotton candy), methyl salicylate (wintergreen), and mixtures thereof.

Any of the food products described herein can further comprise one or more fortifiers. The fortifier may be a vitamin, mineral, etc., including any suitable micronutrient. Examples may include, but are not limited to, essential trace elements, vitamins, co-vitamins (co-vitamin), essential fatty acids, essential amino acids, photosynthetic nutrients (phototonuents), enzymes, and the like.

As described above, any of the food articles and methods of making them can be formed into any suitable form factor. For example, the food product can be configured as a flake (e.g., potato flake, corn tortilla, or crisp form factor). As used herein, a chip may generally refer to a thin piece of food (typically consumed by hand), which is often made crisp by frying, baking, or drying, and is typically consumed as a snack or as part of a meal.

Examples

In one embodiment, the method of making a dehydrated food product from cultured cells is suitable for producing flakes having a high animal protein content.

Edible flakes are prepared by dehydrating a mixture of animal cells and a hydrogel solution, such as a plant-derived polysaccharide. An example of a polysaccharide that can be used is low methyl esterified pectin (an abundant plant derivative that has been used in food). To season the flakes, specially prepared vegetable soups and/or seasonings may be added. The mixture can be spread over a mold (e.g., a parchment mold) and then dehydrated, such as in a food dehydrator, to enhance flavor and achieve crispness.

Fig. 1A-1D illustrate components that can be combined to make a food product. In this example, the ingredients in the composition entering each slice included approximately 5 hundred million cells (shown as clumped in fig. 1A and 1B), 800 microliters of dressing soup (fig. 1C), and 300 microliters of a 4% plant-derived hydrogel solution (shown in fig. 1D, in this example, polypeptide pectin). As shown in fig. 2A, the addition of calcium may cause the pectin to gel after the mixture is spread onto a surface such as a parchment paper mold. After the hydrogel is allowed to set, dehydration may be performed. Dehydration can be performed at 60 ℃ for 19 hours. The final product was a crisp, tasty flake (shown in fig. 2B).

The food product (which may be referred to herein as a chip, edible snack chip, cultured cell snack chip, etc.) can have any shape, including the shape of a conventional "chip," including square, rectangular, triangular, oval, circular, etc., and can be flat, curved, or arcuate. The shape may be formed by a dehydration process (e.g., on a mold). Once dehydrated, the shape can be removed from the substrate (mold surface) and further processed. Further processing may include the addition of additional flavoring agents, including the addition of salt, sugar, etc., or edible coatings.

For example, figures 3a 1-3C 2 illustrate examples of shapes of snack chips of cultured cells that can be made as described herein. In example 1, any suitable size and shape may be formed as described herein. In this embodiment, the shape is planar (but could also be curved or bent); more complex shapes may also be formed. For example, figure 3a1 shows that the fabricated cultured cell snack chip has a somewhat irregular shape that appears ridged, similar to a traditional ridged potato chip. Fig. 3a2 shows a side view of this shape. Similarly, figure 3B1 shows a snack chip with cultured cells in an oval shape, while figure 3B2 shows a side view of the chip. Fig. 3C1 shows a sheet having a triangular shape and a relatively thin cross-section (fig. 3C 2). As mentioned, the snack chip of cultured cells can be formed of any size (surface diameter, breadth, length, etc.) and thickness, including a size and thickness that can be easily eaten by hand. For example, the snack chip of cultured cells can have a size (e.g., average surface diameter or in some variations median surface diameter) of between about 1cm and 15cm (e.g., between about 2cm and 10cm, etc.) and a thickness of between about 0.1mm and 10mm (e.g., between 0.5mm and 5mm, between 0.5mm and 4mm, between 0.5mm and 3mm, between 0.5mm and 2mm, etc.).

The cultured muscle cells may be combined with a plant-derived hydrogel solution immediately prior to dehydration or they may be cultured using a hydrogel. In some variations, as described above, the cells may be cultured on edible microcarriers, which may be made of polysaccharides (which may also include polypeptides having cell attachment motifs) and/or other edible materials. The cells may be muscle cells from established cell lines, including immortalized muscle cells, or they may be primary cultures, or they may be mixtures of these.

For example, cells cultured in vitro can be harvested to make food products. For example, in the sample slices shown above, a 5 billion cell-yielding culture can be removed from a multi-layered cell culture chamber (CellStack culture chamber) (i.e., 3000 ten thousand newton smooth muscle cells are seeded and cultured for 5 days). Cells may be washed by centrifugation to pellet the cells and washing with PBS. The PBS was then removed. In this example, 800 microliters of specially prepared soup (spicy sauce) can be added to the cells, and 300 microliters of pectin solution (4% in distilled water) warmed at 70 ℃ can also be added. The cell, soup and pectin mixture may be mixed by vortexing or the like (e.g., using an Eppendorf combbitip). Then, approximately 50 microliters of calcium chloride solution (0.5M in water) can be added and the mixture mixed again (e.g., using an Eppendorf combbitip for homogenization). Some air may be incorporated in this step.

Similar to that shown in fig. 2A-2B, the mixture can then be distributed into a pseudo-parchment mold that has been sprayed with 50 microliters of calcium chloride solution (0.5M in water). The pectin was allowed to gel at room temperature for 5 minutes. Thereafter, the mold and gel may be placed in a dehydration engine at 60 ℃ for 19 hours to dehydrate the food product and then to flake it. The sheet can then be removed from the mould (pseudo parchment).

The method can be scaled up and/or automated to make multiple sheets. As mentioned, the method can also be modified to make other food products.

The methods of making snack chips of cultured cells described herein generally result in chips that are structurally different from existing food products. This is evident when the ultrastructural properties of the resulting flakes were examined. For example, a cross section through a cultured cell snack chip shows dehydrated cultured muscle cells within a matrix of dehydrated hydrogel. The identity of cultured muscle cells can be confirmed by staining for identification markers, including protein markers (e.g., actin, myosin, etc.). For example, in the cross-section through the dried edible food product shown in fig. 4, the ultrastructures illustrate the remaining cell shape (e.g., cultured muscle cells) distributed within the dehydrated hydrogel. Typically, in a dehydrated sheet, the ultrastructures may show discrete and dehydrated cultured cells (or clusters of cells) expressing a specific marker, such as an animal protein marker. These cells are typically mixed with a plant-derived hydrogel. These flakes may also be referred to as animal protein-containing flakes. Visualization may show different patterns of cultured cells (or dehydrated remnants of cultured cells) within the plant-derived hydrogel. Cultured muscle cells may still be evident (and moderately intact) in the dehydrated preparation.

In fig. 4, a cross-section through one example of a dried sheet made as described herein shows smooth muscle actin (detected by reaction with an alpha smooth muscle actin antibody). The pattern of discrete cell-shaped bodies in the dehydrated sample is a feature of the culturing step for making the sheet, i.e., mixing the cultured cells with the hydrogel, and then dehydrating the sheet-like mixture.

The composition of the edible food product made into a chip snack can also be unique compared to other edible food products that are not made from cultured cells mixed with a hydrogel and then dehydrated. For example, the table of fig. 5 shows the results of a nutritional analysis of a flake made as described herein, and shows that the flake has a high protein content of approximately 70% in this example, but more generally the protein content can be between about 40% and 90% (e.g., between about 40% and 80%, between about 45% and 90%, between about 45% and 80%, between about 50% and 90%, between about 50% and 80%, between about 60% and 90%, between about 60% and 80%, greater than 50%, greater than 55%, greater than 60%, greater than 65%, greater than 70%, greater than 75%, greater than 80%, etc.).

Edible microcarrier

As noted above, any of the food products described herein can use cultured cells grown on edible microcarriers (including microbeads). The edible microcarrier may be made of one animal product-free material or a plurality of animal product-free materials, meaning that the material or materials originate from a non-animal (including vegetable) source. Edible microcarriers can be generally made of edible (nutritional and/or high volume consumption is safe to digest) materials as well as materials with cell attachment domains or motifs. In some variations, the edible microcarrier may be made at least in part by a cross-linked structure of a polysaccharide and a polypeptide comprising a cell attachment motif (such as RGD). As a specific example, edible microcarriers may be made by cross-linking pectin (e.g. thiol-modified pectin, PTP) and RGD containing polypeptides (such as cardosin).

Any of the edible microcarriers described herein can further include additional (supplemental) materials, including flavors (additives to enhance flavor), additives to enhance the appearance and/or nutritional value of the edible microcarrier, and the resulting food product (e.g., chip) is made using the edible microcarrier. These additives (e.g., perfumes) can be used in place of or in addition to perfumes added before or after dewatering.

For example, the edible microcarrier may comprise an edible microsponge and/or an edible microbead. These microcarriers may be porous (e.g. sponge-like) or smooth. Edible microcarriers used to form engineered meat can be made into microbeads/microparticles for use in bioreactors and can have diameters between about 3mm and about 0.02mm (e.g., between about 2mm and about 0.05mm, between about 1mm and 0.1mm, between about 1mm and 0.3mm, etc.). For example, the diameter of the beads may be about 0.5 mm. The size may represent an average size or a median size, or a maximum/minimum size. The shape of the microcarrier may be regular (e.g. spherical, circular, etc.) or irregular, e.g. spherical, cubical, etc.; any of these shapes may be porous.

The edible microcarrier may be made by any suitable process, including molding, extrusion, injection, infusion, etc. of the material from which the edible microcarrier is formed. Edible, highly porous microcarriers that can be used in cell culture techniques utilizing bioreactors and that can hold an integral part of the final engineered edible product (e.g., chip) can be made of edible animal-free materials, including cultured animal cells that can be derived from long-term culture or can be removed without killing the animal. Such edible microcarriers can be prepared by forming components, such as polysaccharides and polypeptides, into a cross-linked hydrogel, lyophilizing the cross-linked hydrogel, and shaping (e.g., cutting) the lyophilized gel to the appropriate size.

One example of a method of forming edible microcarriers includes forming the major components (polysaccharides and polypeptides) of the microcarriers. For example, one class of polysaccharides that can be used is Low Methyl (LM) esterified pectin, an abundant plant derivative that has been used in food. For example, the LM esterified pectin used may be derivatized to form a thiol modified pectin (PTP) which is 100% edible and digestible. Thiol functional groups are found in garlic and onion. One class of polypeptides that can be used includes cardosin. cardosin is an aspartic protease extractable from Cynara cardunculus L and containing a cell-binding RGD motif that promotes cell attachment. For example, cardosin may be derivatized with its cysteine to introduce new thiol groups. cardosin has been used by the food industry, especially in cheese making. In other variations, the cardosin can be replaced (or supplemented) by another polypeptide, including a synthetic peptide, with an edible RGD sequence.

In some variations, the PTP and derivatized cardosin may be crosslinked through oxidative disulfide bond formation. In this example, the PTP-cardosin hydrogel may be crosslinked under mild conditions using glutathione disulfide (in oxidized form) (GSSG) obtained by bubbling air into a solution of higher glutathione (GSH, e.g. such as healthy food storage grade glutathione). Additional additives (e.g., flavors, nutrients, colors, etc.) may also be added.

The hydrogel may then be shaped or formed. For example, microsponges (1mm to 5mm thick) can be formed by pouring a hydrogel solution into a mold and allowing crosslinking to continue overnight in air, then lyophilized and cut to the desired size (larger sponges for tissue engineering applications, pieces of about 0.5mm for bioreactor applications).

For mass production of microbeads, a coaxial air-flow type bead-making apparatus may be used. For example, the beads may be composed of modified cross-linked pectin and cardosin hydrogels (e.g., PTP-cardosin hydrogels). In one variant, the method for making microcarriers of PTP and cardosin can be carried out by the following steps: (1) derivatization of pectin at two modification levels (e.g., 10%, 25%) by using cystamine followed by reduction to form pectin-thiopropionamide (PTP); (2) introduction of new thiol groups (e.g. cardosin a) by derivatization of cardosin; (3) GSSG crosslinked hydrogels in the form of plates using PTP and thiolated cardosin (pH, concentration, etc. can be optimized for hydrogel formation; additives such as colorants, nutrients, etc. can also be included), and hydrogels can be lyophilized; (4) beads were formed using a bead generator, such as a Nisco co coaxial air-flow bead generator, and GSSG hydrogel spheres were lyophilized to obtain microcarriers.

In use, microcarriers can be used to culture cells, such as smooth muscle cells, in large quantities to make edible materials (e.g., flakes). As noted above, in general, other cell types can be used on microcarriers in addition to (or instead of) muscle cells, including satellite cells and the like.

For example, a microcarrier as described herein can be seeded with muscle cells (e.g., smooth muscle cells) and then cultured. In particular, the cells and microcarriers may be cultured in a bioreactor. The resulting culture can be grown to the desired level and can be used directly to make sheets without the need to separate or remove microcarriers. The sheet may be made as described above, where (instead of combining only cells and hydrogel) microcarriers on which cultured cells have been grown are combined with hydrogel and then dehydrated. In some variations, the microcarrier with cells can be applied (e.g., poured, sprayed, etc.) directly onto a forming surface (such as a mold) without the addition of a hydrogel prior to dehydration; for example, microcarriers on the surface may be cultured such that the cells and/or microcarriers are at least partially fused. For example, microcarriers with cells can be cultured on a surface for a certain amount of time (e.g., 4 hours, 12 hours, 18 hours, 24 hours, 48 hours, 3 days, etc.), or can be dehydrated immediately.

In some variations, microcarriers with cells (which cells can grow to density, including confluency on microcarriers) can be added along with a solution of additives including perfume and hydrogel (e.g., a plant-derived polysaccharide) similar to that shown and described in example 1 above. The mixture may then be dehydrated.

For example, the edible microcarriers and cultured cells can be flaked after incubating in a bioreactor for a suitable time to allow the cells to grow and multiply (e.g., 12 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, etc.) on the microcarriers to form the cellularized microcarriers. Typically, a cellularized microcarrier is a (e.g. edible, animal product-free) microcarrier to which cells (e.g. muscle cells) have been attached and grown. As mentioned, cells on the microcarriers may grow to confluence, although this is not required. In addition, the cells may be fused on and/or in the surface of the microcarriers. The cellularized microcarriers may be at least partially fused.

In these embodiments, the body of the food article may also include a microcarrier, which may be visualized (e.g., in the case of magnification).

For example, in variations where an edible microcarrier is used, the method of making a sheet as described herein may comprise culturing a plurality of muscle cells on the edible animal product-free microcarrier in suspension to form a plurality of cellularized microcarriers. The cellularized microcarriers may be mixed with a plant-derived hydrogel, and in some variations with additives such as perfume. The mixture of the cellularized microcarriers and hydrogel (with or without additional additives) can then be placed, poured, sprayed or otherwise applied onto a substrate (e.g., a mold or other surface) suitable for use in a dehydration engine. The mixture may then be dehydrated as described above. Additional flavors/additives (e.g., salt, etc.) can then be added, and the flakes can then be further processed and/or packaged.

The cells may be cultured, typically using any of the edible microcarriers described herein in suspension, including in a bioreactor. For example, the cells may be seeded into a culture medium with an edible microcarrier, and the cells may be allowed to contact, attach, and grow on a suitable edible microcarrier. For example, culturing may include culturing a plurality of muscle cells on an edible, animal product-free microcarrier comprising a hydrogel with a thiol-modified pectin (PTP) and cardosin. In some variations, culturing comprises culturing the plurality of muscle cells on an edible animal product-free microcarrier, wherein the animal product-free microcarrier comprises a flavoring agent, a coloring agent, and a nutrient enrichment agent.

In some variations, the cellularized microcarriers are covered with cells (e.g., greater than 50% covered, greater than 60% covered, greater than 70% covered, greater than 80% covered, greater than 90% covered, covered to cell confluence). As described in u.s.8703216, previously incorporated by reference in its entirety, the cells used may be of one type or multiple types, including muscle cells, among others. Microcarriers that are covered with cells to a suitable degree (e.g. > 50%, > 60%, > 70%, > 80%, > 90% etc.) may be referred to as cellularized microcarriers.

Although the use of edible microcarriers as described herein is optional, it may provide some advantages over traditionally cultured cells. For example, the cells used in the edible sheets described herein (which may include, for example, smooth muscle cells, satellite cells, fibroblasts, adipocyte progenitor cells, etc.) are generally anchorage dependent, requiring a surface for attachment. Current cell culture methods may use flasks, tubes, and/or plates (e.g., cellStacks or superframes) to provide a surface to which cells may attach and grow, which may result in a manually labor intensive process, and may require enzymes to detach cells from the surface and large amounts of media to harvest the cells. Most materials are disposable, thus generating waste; as culture progresses, expansion of cells is typically achieved by seeding more plates with a greater number of layers.

The microcarriers described herein may provide a large surface area/volume for cell attachment, especially if the microcarriers are microporous or macroporous. The initial step of cell expansion may include mixing the cells and microcarriers in a mini-bioreactor. Cells are attached and propagated on microcarriers that are maintained in suspension. When maximum growth is achieved, the microcarriers can be collected and used to inoculate a larger volume bioreactor, or used directly if grown in sufficient quantities. The cells do not have to be separated from the microcarriers, since the microcarriers described herein are edible, thereby eliminating the use of enzymes and the risk of damaging the cells. The process is time-saving and easy to scale up. Industrial bioreactors can achieve large volumes (e.g., greater than 1000L) in less space than traditional cell culture incubators.

The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. For example, as used herein, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will be further understood that the terms "comprises" and/or "comprising," when used in this specification, specify the presence of stated features, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, steps, operations, elements, components, and/or groups thereof. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items, and may be abbreviated as "/".

As used herein in the specification and claims, including as used in the examples and unless otherwise indicated, all numbers may be understood as if prefaced by the word "about" or "approximately", even if the term does not expressly appear. When describing amplitude and/or position, the phrase "about" or "approximately" may be used to indicate that the described value and/or position is within a reasonably expected range of values and/or positions. For example, a numerical value may have a value that is +/-0.1% of the value (or range of values), a value that is +/-1% of the value (or range of values), a value that is +/-2% of the value (or range of values), a value that is +/-5% of the value (or range of values), a value that is +/-10% of the value (or range of values), and the like. Any numerical range recited herein is intended to include all sub-ranges subsumed therein.

Although a number of exemplary embodiments have been described above, any of a number of modifications may be made to the various embodiments without departing from the scope of the invention as set forth in the claims. For example, in alternative embodiments, the order in which a plurality of described method steps are performed may be changed from time to time, and in other alternative embodiments, one or more steps may be skipped altogether. Optional features of various apparatus and system embodiments may be included in some embodiments and not in others. Accordingly, the foregoing description is provided primarily for the purpose of illustration and should not be construed as limiting the scope of the invention as set forth in the claims.

The examples and illustrations included herein show, by way of example and not by way of limitation, specific embodiments in which the subject matter may be practiced. As noted, other embodiments may be utilized and derived therefrom, such that structural and logical substitutions and changes may be made without departing from the scope of this disclosure. Such embodiments of the inventive subject matter may be referred to herein, individually or collectively, by the term "invention" merely for convenience and without intending to voluntarily limit the scope of this application to any single invention or inventive concept if more than one is in fact disclosed. Thus, although specific embodiments have been illustrated and described herein, any arrangement calculated to achieve the same purpose may be substituted for the specific embodiments shown. This disclosure is intended to cover any and all adaptations or variations of various embodiments. Combinations of the above embodiments, and other embodiments not specifically described herein, will be apparent to those of skill in the art upon reviewing the above description.