Carrier suitable for preparing cell culture meat and application thereof

Abstract

The invention relates to the field of cell culture meat preparation, and particularly discloses a carrier suitable for preparing cell culture meat, and the carrier is selected from any one of the following components: a) the carrier comprises edible fungus mycelia; and b) the carrier comprises edible fungus mycelia and a natural high polymer material, and the mass ratio of the mycelia solid matter to the natural high polymer material is (2-3): (0.4-15). The mycelium carrier provided by the invention is edible and low in cost, has a flexible processing attribute, can form a spherical microcarrier, a sheet carrier and a filamentous three-dimensional bracket, and is suitable for multi-dimensional cell culture such as 2D and 3D. The technology has the remarkable advantages that the mycelium carrier is good in biocompatibility, rich in source, easy to amplify and low in cost, and is expected to be applied to amplification of a cell culture meat process and biological manufacturing of whle-cut whole meat and the like.

Description

Technical Field

[0001] The present invention relates to the field of cell-cultured meat preparation, and in particular to an edible carrier suitable for preparing cell-cultured meat and its application. Background Art

[0002] In recent years, the research and development and production of cell-cultured meat has been steadily expanding, encompassing meat cultured from mammalian, poultry, and fish cells. Related products have also begun to be sold internationally. Cell-cultured meat, a traditional meat alternative, is primarily composed of animal cells, produced through sterile in vitro culture. It has attracted significant attention due to its efficient and energy-efficient production process. The cell-cultured meat industry has also experienced rapid growth. However, since most cells exhibit adherent properties, meaning they must adhere to a wall before they can grow, divide, and proliferate, this characteristic makes direct suspension culture and scaling of cells in bioreactors difficult.

[0003] Based on the cell adhesion characteristics, a series of cell culture carriers have been derived. By utilizing their structural characteristics, they provide a support surface and growth and proliferation space for cell growth, which is conducive to the scale-up of cell culture production. In recent years, with the development of science and technology, more and more microcarriers have been produced and applied in the field of medical research and development. Common commercial microcarriers include Cytodex1, 2, and 3 with cross-linked dextran as the matrix, Cytopore with cellulose as the matrix, gelatin microcarriers with protein as the matrix, and Cytoline with polymer synthetic materials as the matrix. Although these cell culture carriers are widely used in the medical field, most of them are not edible, their production and processing technology is relatively complex, and it is necessary to prevent the problem of chemical reagent residues. In industrial production, a large amount of wastewater and carbon emissions will also be generated. Therefore, the existing carriers are limited in the development and application of the culture and research and development processes of cell cultured meat. Summary of the Invention

[0004] In order to solve the above technical problems, the present application provides a non-animal mycelium edible carrier that can be used for cell meat culture. Mixing the mycelium with other edible materials can improve the mechanical properties of the non-animal mycelium as a cell culture carrier. The mechanical properties are better than those of pure mycelium materials and the plasticity is strong.

[0005] The present application provides a carrier suitable for preparing cell-cultured meat, wherein the carrier is selected from any one of the following: a) the carrier comprises edible fungus mycelium; b) the carrier comprises edible fungus mycelium and natural polymer materials, and the mass ratio of the mycelium solids to the natural polymer materials is (2 to 3): (0.4 to 15).

[0006] The present application also provides a method for preparing the above-mentioned carrier, which comprises mixing edible fungus mycelium material with a support material to obtain the carrier, wherein the support material contains a natural polymer material.

[0007] The present application also provides a cell-cultured meat, comprising the above-mentioned carrier and non-human animal cells cultured and grown thereon.

[0008] The present application also provides a device for cell culture and / or cell culture meat preparation, comprising a cell culture vessel and the above-mentioned carrier located in the cell culture vessel.

[0009] The present application also provides the use of the above-mentioned carriers and devices in cell culture and / or preparation of cell-cultured meat.

[0010] The present application also provides a method for preparing the above-mentioned cell-cultured meat, using the above-mentioned device to culture non-human animal cells.

[0011] The carrier proposed in this specification has the following beneficial effects, including but not limited to: (1) The preparation process of the edible mycelium carrier is simple, the cost is low, it is edible, and it is suitable for scale-up production. (2) When the edible mycelium carrier is used as a cell carrier in the production process of cell culture meat, the mycelium provides it with good cell compatibility, which helps the cells adhere, proliferate and aggregate. There is no need to consider operations such as digestion and removal of the carrier, which not only helps to increase the yield of cell meat, but also reduces the tedious operations such as cell digestion and collection, thereby reducing the risk of contamination, and at the same time reduces the use of reagents such as digestive enzymes and the problem of residual inedible materials. (3) In terms of mechanical properties, the mechanical properties of the carrier after mixing with other edible polymer support materials are significantly enhanced, and it can provide a normal support space for cell attachment. At the same time, it can achieve no obvious deformation under stirring conditions during the entire culture cycle, providing a relatively stable external environment for cell growth. The mycelium carrier is suitable for scale-up production of cell meat. (4) The mycelium filamentous three-dimensional scaffold described in this application has a wider application scenario than the sheet carrier and can be produced on a large scale. Moreover, its unique spinning characteristics can more realistically imitate the texture of chicken in the subsequent productization process, which is beneficial to the research and development and production of the final product. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] The present application will be further described in terms of exemplary embodiments, which will be described in detail with reference to the accompanying drawings. These embodiments are non-limiting, and include:

[0013] Figure 1 The original morphology of the Morchella mycelium microcarrier shown in some embodiments of the present application;

[0014] Figure 2 The compatibility of different mycelium microcarrier cells shown in some embodiments of the present application;

[0015] Figure 3 The compatibility of Morchella mycelium microcarrier cells shown in some embodiments of the present application;

[0016] Figure 4 The results of amplified culture of Pleurotus ostreatus mycelium in a small stirred-tank reactor using microcarriers according to some embodiments of the present application are shown;

[0017] Figure 5 This is a growth curve of Morchella mycelium in a fermentation tank according to some embodiments of the present application;

[0018] Figure 6 The mycelium cellulose sheet carrier and cell culture results shown in some examples of the present application;

[0019] Figure 7 The mycelium chitosan sheet carrier and cell culture results shown in some examples of the present application;

[0020] Figure 8 The mycelium soy protein isolate filamentous three-dimensional scaffold and cell culture results shown in some embodiments of the present application;

[0021] Figure 9 The pure mycelium sheet carrier according to some embodiments of the present application;

[0022] Figure 10 The soy protein isolate filamentous three-dimensional scaffold according to some embodiments of the present application;

[0023] Figure 11 The cellulose sheet-like carrier according to some embodiments of the present application;

[0024] Figure 12 A chitosan and soy protein isolate mixed sheet carrier according to some embodiments of the present application;

[0025] Figure 13 This is a pure mycelium film according to some embodiments of the present application. DETAILED DESCRIPTION

[0026] To more clearly illustrate the technical solutions of the embodiments of this specification, the following briefly describes the drawings required for describing the embodiments. Obviously, the drawings described below are merely examples or embodiments of this specification. Those skilled in the art can apply this specification to other similar scenarios based on these drawings without inventive effort. Unless otherwise apparent from the context or otherwise noted, the same reference numerals in the figures represent the same structure or operation.

[0027] As used in this specification and claims, unless the context clearly indicates otherwise, the words "a," "an," "an," and / or "the" do not refer to the singular but also include the plural. Generally speaking, the terms "comprises" and "include" only indicate the inclusion of the steps and elements specifically identified, and these steps and elements do not constitute an exclusive list. A method or apparatus may also include other steps or elements.

[0028] Flowcharts are used throughout this specification to illustrate the operations performed by systems according to embodiments of this specification. It should be understood that preceding or following operations do not necessarily need to be performed in exact order. Instead, the steps may be processed in reverse order or simultaneously. Furthermore, other operations may be added to these processes, or one or more operations may be removed from these processes.

[0029] Edible fungi are a delicious, high-protein, low-fat food. Their mycelium acts as both a transmitter and accumulator of nutrients, with a nutrient composition not much different from that of the fruiting body, making them an edible biological material. During liquid fermentation, edible fungus mycelium tends to form a spherical shape, with intertwined branches and internal pores. It settles in the fermentation tank without stirring, but flows with the water when stirred. Overall, this conforms to the microcarrier characteristics of low density and large specific surface area. If mycelium material could be used in the production of cell-cultured meat, it would not only provide nutrition, but also eliminate the purification process required to digest microcarriers, while avoiding the use of harmful chemical and biological reagents, thereby promoting the development of the cell-cultured meat industry.

[0030] Attempts to use mycelium as a carrier for cell culture revealed good cellular compatibility and high plasticity. However, pure mycelium exhibits weak mechanical properties, prone to deformation, and unable to sustain a full cell culture cycle. Therefore, the present inventors attempted to blend mycelium with other edible materials to improve its mechanical properties. Furthermore, they explored alternative carrier forms besides spherical microcarriers, such as sheet-like carriers and filamentous three-dimensional scaffolds.

[0031] The present application provides a carrier suitable for preparing cell-cultured meat, wherein the carrier is selected from any of the following: a) the carrier comprises edible fungus mycelium; b) the carrier comprises edible fungus mycelium and a natural polymer material, wherein the mass ratio of the mycelium solids to the natural polymer material is (2-3):(0.4-15). This carrier has the advantages of being edible, low cost, and simple to prepare.

[0032] In some embodiments, preferably, the mass ratio of the mycelium solids to the natural polymer material can be (2.231-2.6): (0.498-13). In some embodiments, preferably, the mass ratio of the mycelium solids to the natural polymer material can be (2.3-2.55): (1-11). In some embodiments, preferably, the mass ratio of the mycelium solids to the natural polymer material can be (2.35-2.50): (3-9). In some embodiments, preferably, the mass ratio of the mycelium solids to the natural polymer material can be (2.4-2.45): (5-7). In some embodiments, more preferably, the mass ratio of the mycelium solids to the natural polymer material can be 2.4:0.498.

[0033] In some embodiments, the edible fungi may include at least one of Morchella, Pleurotus ostreatus, Sulphur Fungus, Acanthopanax gloeosporioides, Pleurotus eryngii, Boletus edulis, King oyster mushroom, Ganoderma lucidum, Pleurotus eryngii, Cordyceps militaris, Pleurotus eryngii, Enoki mushroom, and Shiitake mushroom. In some embodiments, preferably, the edible fungi may be Pleurotus ostreatus, Sulphur Fungus, or Morchella. In some embodiments, more preferably, the edible fungi may be Morchella.

[0034] In some embodiments, the natural polymer material may include at least one of plant protein, chitosan, chitosan oligosaccharide, wheat flour, or cellulose. In some embodiments, preferably, the natural polymer material may be plant protein, chitosan, or cellulose. In some embodiments, more preferably, the plant protein may be soy protein isolate, the cellulose may be bacterial cellulose, and the chitosan may be mushroom chitosan.

[0035] In some embodiments, the chitosan cps may be 200 to 1000. For example, the chitosan cps may be about 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950 or 1000. Any range characterized by a combination of the above end values is also included, which will not be repeated here. In some embodiments, preferably, the chitosan cps may be 700 to 900. In some embodiments, more preferably, the chitosan cps may be 800.

[0036] In some embodiments, the natural polymer material is edible.

[0037] In some embodiments, the carrier can be a spherical microcarrier, a sheet-like carrier, or a filamentous three-dimensional scaffold. In some embodiments, preferably, the spherical microcarrier has a diameter of 100 to 300 μm. For example, the spherical microcarrier diameter can be approximately 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, or 300 μm. Any range characterized by a combination of the above end values is also included, which will not be repeated here. In some embodiments, more preferably, the spherical microcarrier diameter can be 200 μm.

[0038] In some embodiments, the sheet carrier can preferably be a 0.6-1 cm square sheet. For example, the sheet carrier can be approximately 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, or 1 cm square sheet. Any range characterized by combinations of the aforementioned values is also included and will not be further described here. In some embodiments, the sheet carrier can more preferably be a 0.8 cm square sheet.

[0039] In some embodiments, preferably, the diameter of the filamentous three-dimensional scaffold can be 500 to 1000 μm. For example, the diameter of the filamentous three-dimensional scaffold can be approximately 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1000 μm. Any range characterized by a combination of the above end values is also included, which is not repeated here. In some embodiments, more preferably, the diameter of the filamentous three-dimensional scaffold can be 800 μm.

[0040] In some embodiments, the sheet carrier may have a thickness of 0.5 to 5 mm. For example, the sheet carrier may have a thickness of approximately 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, or 5 mm. Any range characterized by a combination of the aforementioned values is also included and will not be further described herein.

[0041] In some embodiments, the carrier can be a sterile carrier.

[0042] The present application also provides a method for preparing the above-mentioned carrier, which comprises mixing edible fungus mycelium material with a support material to obtain the carrier, wherein the support material contains a natural polymer material.

[0043] In some embodiments, the edible fungus mycelium material can be prepared by the following steps: breaking the edible fungus mycelium, washing it, and removing water to obtain the edible fungus mycelium material.

[0044] In some embodiments, the edible fungus mycelium material can be prepared by the following steps: crushing the edible fungus mycelium, rinsing with water, filtering with a gauze, and squeezing out the water to obtain the edible fungus mycelium material.

[0045] In some embodiments, the solid content of the edible fungus mycelium material may be 2.3-2.6 w / w%.

[0046] In some embodiments, the support material is solid or has a fluidized state with a solids content of 0.1 to 2 w / w%. For example, the support material may have a fluidized state with a solids content of approximately 0.1, 0.25, 0.5, 0.75, 1, 1.25, 1.5, 1.75, or 2 w / w%. Any range characterized by a combination of the above values is also included and is not further described here. In some embodiments, preferably, the support material may have a fluidized state with a solids content of 0.5 w / w%.

[0047] In some embodiments, when the support material is in a fluid state, the mass ratio of the edible fungus mycelium material to the support material may be (0.5-1.5): (0.5-1.5). In some embodiments, when the support material is in a fluid state, the mass ratio of the edible fungus mycelium material to the support material may be (0.75-1.25): (0.75-1.25). In some embodiments, when the support material is in a fluid state, the mass ratio of the edible fungus mycelium material to the support material may be (1-1.25): (1-1.25). In some embodiments, preferably, when the support material is in a fluid state, the mass ratio of the edible fungus mycelium material to the support material may be 1:1.

[0048] In some embodiments, when the support material is in a solid state, the mass ratio of the edible fungus mycelium material to the support material may be (95-100): (11-15). In some embodiments, when the support material is in a solid state, the mass ratio of the edible fungus mycelium material to the support material may be (96-99): (12-14). In some embodiments, when the support material is in a solid state, the mass ratio of the edible fungus mycelium material to the support material may be (97-98): (13-14). In some embodiments, preferably, when the support material is in a solid state, the mass ratio of the edible fungus mycelium material to the support material may be 97:13.

[0049] In some embodiments, after the edible fungus mycelium material is mixed with the support material, the following steps are further performed:

[0050] The mixture of edible fungus mycelium material and support material is spread in a mold, and after removing moisture, a sheet-shaped carrier is obtained.

[0051] In some embodiments, after the edible fungus mycelium material is mixed with the support material, the following steps are further performed:

[0052] The mixed liquid is spread in a mold, dried, and then cut into square slices to form a sheet carrier. In some embodiments, the mold can be a silicone mold. In some embodiments, the drying conditions can be 60°C for 4-6 hours.

[0053] In some embodiments, after the edible fungus mycelium material is mixed with the support material, the following steps are further performed:

[0054] a Wet spinning to obtain filaments;

[0055] b. Cleaning and chopping the filaments to obtain a carrier in the form of a filamentous three-dimensional scaffold.

[0056] Wet spinning is a technology in which the spinning solution passes through the spinning machine spinneret and precipitates into fibers in the coagulation liquid.

[0057] In some embodiments, the carrier needs to be soaked in PBS and sterilized before use. In some embodiments, the PBS soaking time can be 1 to 24 hours. In some embodiments, the sterilization can be high temperature and high pressure sterilization. In some embodiments, the sterilization conditions can be 121°C for 20 to 45 minutes.

[0058] The present application also provides a cell-cultured meat, comprising the above-mentioned carrier and non-human animal cells cultured and grown thereon.

[0059] In some embodiments, the non-human animal is any one of a rodent, an artiodactyl, a perissodactyl, a lagomorph, or a primate. In some embodiments, preferably, the non-human animal is any one of a chicken, a pig, a cow, a sheep, a fish, a duck, a goose, a rabbit, a turkey, a pigeon, a quail, a horse, a donkey, or a deer.

[0060] In some embodiments, the non-human animal cells include at least one of myocytes, fibroblasts, hepatocytes, osteoblasts, adipocytes, endothelial cells, hematopoietic stem cells, hepatic hematopoietic stem cells, bone marrow stem cells, adipose fibroblasts, adipose stem cells, mesenchymal stem cells, bone marrow stromal cells, muscle side group cells, blood-derived mesenchymal precursor cells, muscle precursor cells, circulating skeletal stem cells, multipotent adult progenitor cells, mesoderm progenitor cells or spinal cord progenitor cells. In some embodiments, preferably, the non-human animal cells are fibroblasts. In some embodiments, the fibroblasts can be chicken fibroblasts, porcine myocardial fibroblasts, porcine skin fibroblasts, porcine kidney fibroblasts, bovine ear fibroblasts, large bovine skin fibroblast-like cells, bovine dermal fibroblasts, sheep lung fibroblasts or sheep skin fibroblasts.

[0061] The present application also provides a device for cell culture and / or cell culture meat preparation, comprising a cell culture vessel and the above-mentioned carrier located in the cell culture vessel.

[0062] In some embodiments, the cell culture vessel can be a low-adhesion cell culture vessel.

[0063] In some embodiments, the cell culture vessel can be any one of a cell culture dish, a cell culture flask, or a cell culture plate.

[0064] The present application also provides the use of the above-mentioned carriers and devices in cell culture and / or preparation of cell-cultured meat.

[0065] The present application also provides a method for preparing the above-mentioned cell cultured meat, using the above-mentioned device to culture non-human animal cells. In some embodiments, the cell culture medium can be changed every other day. In some embodiments, the cell culture density can be 2-3*10 5 pieces / mL.

[0066] The experimental methods in the following examples, unless otherwise specified, are all conventional methods. The experimental materials used in the following examples, unless otherwise specified, were purchased from conventional biochemical reagent companies. The quantitative experiments in the following examples were all repeated three times, and the results were averaged.

[0067] Example 1 - Preparation of spherical mycelium microcarriers and cell compatibility testing

[0068] Take out the preserved edible fungi species, such as Morchella eximia, Pleurotus ostreatus, Laetiporus sulphureus (Fr.) Murrill, Oudemansiella raphanipies, Stropharia rugoso-annulata Farlow, Boletus aereus (Boletus aereus, Boletus luridus), Pleurotus eryngii, Ganoderma lucidum (Leyss.ex Fr.) Karst), Pleurotus nebrodensis, Cordyceps militaris (L.ex Fr.) Link., Agaricus bisporus var. bisporus, Flammulina avelutiper (Fr.) Sing, and Lentinus edodes. S1 High-Density Liquid Culture Preparation: In a clean bench, transfer the solid culture to a sterile mixing cup. Add a small amount of enriched potato liquid culture medium (formula: 200g / L potato, 10g / L sucrose, 10g / L glucose, 4g / L yeast extract, 1g / L potassium dihydrogen phosphate, 0.5g / L magnesium sulfate). Crush until no visible particles are present. Transfer to a conical flask and culture in a shake flask at 22-25°C for 5-7 days to produce a high-density liquid culture. S2 Inoculation: Transfer the liquid culture to a sterile mixing cup and crush until no visible granular mycelium is present. Filter to remove large mycelium clumps. After filtering through 100- and 300-mesh sterile sieves to remove large mycelium clumps, inoculate the culture into a liquid shake flask at a 1:10 inoculation ratio. Preparation of S3 spherical mycelium microcarriers: Culture on a shaker at 23-25°C and 150-180 rpm for 4-7 days to obtain a large amount of spherical mycelium. Filter to remove the culture medium and large pieces of mycelium. Wash the culture medium remaining on the surface with deionized water, soak in deionized water, and sterilize in a high-pressure sterilizer at 121°C for 20 minutes to obtain the desired spherical mycelium microcarriers.

[0069] The mycelial microcarriers prepared using this method, including those from Morchella, Pleurotus ostreatus, Pleurotus sulphureus, Acanthopanax nigra, Pleurotus eryngii, Boletus edulis, King oyster mushroom, Ganoderma lucidum, Pleurotus eryngii, Cordyceps militaris, Pleurotus eryngii, Enoki mushroom, and Lentinus edodes, are mostly spherical in shape, approximately 100-300 μm in diameter, with a fluffy surface and interior, ideal for cell attachment and growth. The Morchella mycelial microcarriers have a diameter of 200 μm. The liquid culture medium used in the preparation process is all food-grade, and drinking water or deionized water is used to ensure the edible nature of the mycelial microcarriers.

[0070] The mycelium microcarriers were placed in a cell culture medium and soaked for 24 hours. The soaked mycelium microcarriers were added to a 12-well plate, followed by the culture medium and then chicken embryonic fibroblasts. After repeated blowing, the plates were placed in an incubator for static culture. Samples were taken and observed every 48 hours. Fibroblasts grew on a variety of mycelium microcarriers. The growth status was ranked from best to worst: Morel, Pleurotus ostreatus, Sulfur Fungus, Black-skinned Acanthopanax, Wrinkled Globe Oyster Mushroom, Boletus, King Oyster Mushroom, Ganoderma Lucidum, Pleurotus eryngii, Cordyceps militaris, White Jade Mushroom, Enoki Mushroom, Lentinus edodes, etc. Figure 2 Morchella mycelium microcarriers have the best cell compatibility. On the first day, cells were densely attached and slightly spread. On the third day, cells covered the entire surface and interior of the microcarriers. They were stained with PI dye, incubated in the dark for 5 minutes, and photographed. Figure 3 .

[0071] Dynamic experiments have been conducted on Pleurotus ostreatus mycelium microcarriers, on a larger scale than compatibility experiments. This supports the theory of large-scale cell culture. However, the cell attachment effect is slightly inferior to that of Morchella.

[0072] Example 2 - Experimental experiment on amplified culture of cells in a small stirred reactor with microcarriers of Pleurotus ostreatus mycelium

[0073] The Pleurotus ostreatus mycelium microcarriers were placed in a cell culture solution and soaked for 24 hours. The soaked mycelium microcarriers were added to a small stirred reactor, followed by culture solution and then chicken embryonic fibroblasts. After repeated blowing, the cells were dynamically cultured in an incubator. Samples were taken every 24 hours for observation, and CCK8 dye was used to detect the cell status and cell density. Each group was sampled twice for repeatability testing. The fibroblasts grew well on the Pleurotus ostreatus mycelium microcarriers. On the first day, the cells were densely attached and slightly spread. On the third day, the cells covered the entire surface and interior of the microcarrier. After 7 days of culture, the cells were still growing and entered the plateau phase on the ninth day. Stain with PI dye, incubate in the dark for 5 minutes, and take pictures. Figure 4 .

[0074] OD value is proportional to cell density, and the cell growth trend is shown in Figure 4 .

[0075] Example 3 - Mass Preparation of Morchella Mycelium

[0076] The solid plate of Morchella spp. was aseptically transferred to a liquid shake flask and cultured in a constant temperature shaker for 3-5 days. After the strain grew to a millet porridge-like consistency, it was inoculated in a 15L fermentation tank at a ratio of 1:10. After 3 days of high-density fermentation, the mycelium was filtered and collected, and washed with deionized water several times to remove the residual culture medium on the surface. Pure Morchella spp. mycelium was obtained. After sampling and drying, the mycelium dry weight yield was measured to be 15.05g / L, that is, the average growth efficiency was 5.01g / (L·day). The growth curve is shown in FIG. Figure 5 .

[0077] Through step-by-step scale-up, this high-density fermentation process can be scaled up to 5000L, achieving a dry weight yield of 25g / L. All raw materials used in mycelium preparation are food-grade, and all water used is deionized. The harvested mycelium has a sweet and chewy texture, similar to, yet distinct from, animal meat.

[0078] The mycelium used in the sheet-like carrier and the filamentous three-dimensional scaffold of the present invention is prepared by the same fermentation process and equipment as in this embodiment.

[0079] Example 4 - Preparation of mycelial cellulose sheet carrier and cell culture method

[0080] Mycelium Preparation: Crush the fermented Morchella mycelium and rinse thoroughly with drinking water or deionized water. Filter through a 300-mesh screen and squeeze out as much water as possible to obtain the desired mycelium. A 2g sample was dried and weighed to obtain a dry weight of 0.048g, indicating a moisture content of 97.6% before drying and a solids content of 2.4%.

[0081] Preparation of edible support material: 0.5 g of bacterial cellulose was weighed and dissolved in 100 ml of deionized water to prepare a cellulose solution with a bacterial cellulose solid content of 0.5%.

[0082] Preparation of mycelium cellulose sheet carrier: Mix the mycelium material and the edible support material in a mass ratio of 1:1 and evenly break them up. Spread the mixed material flatly in a silicone mold with a thickness of about 3 mm. Place it in a 60°C oven and dry it for 4-6 hours. After drying, cut it into 0.8 cm square slices to obtain the sheet carrier.

[0083] Moist heat sterilization: Soak the sheet carrier in PBS for 1 hour, then place it in a high-temperature autoclave for sterilization (parameters: 121°C, 45 minutes)

[0084] Cell culture: Fibroblasts were used as the cell type. Adherent cells were verified using a 24-well low-adhesion plate. Two to three sheet carriers were placed in each well. The cell seeding density was 3.0*10^5 cells / well. 2 ml of culture medium was added to each well. The medium was changed every other day, with 1 ml of medium changed per well.

[0085] Comparative conclusion: Compared with the pure mycelium material (Comparative Example 1), the cell density of D7 is higher and the cells can be fully spread on the carrier. Figure 6 The cell growth cycle could be extended to 11 days, during which time the cells continued to grow without apoptosis. However, pure mycelium sheet carriers were unable to extend the cell culture cycle, with cells beginning to apoptosis on day 5, and the carrier surface was no longer completely covered by cells. At day 5, the number of cells in the mycelium-cellulose mixed carrier was approximately 1.5 times that of the pure mycelium carrier.

[0086] Compared to the pure cellulose sheet carrier (Comparative Example 3), the mycelium-cellulose hybrid material showed a higher cell count, approximately three times that of the pure cellulose sheet carrier, better spreading, and a longer cell growth cycle. The cells aggregated and grew into spheres on the pure cellulose sheet carrier, indicating that the pure cellulose sheet carrier could not support the growth of large numbers of cells attached.

[0087] The mycelium cellulose sheet carrier is better than the pure mycelium sheet carrier and the pure cellulose sheet carrier.

[0088] Example 5 - Preparation of mycelium chitosan sheet carrier and cell culture method

[0089] Mycelium Preparation: Crush the fermented Morchella mycelium and rinse thoroughly with drinking water or deionized water. Filter through a 300-mesh screen and squeeze out as much water as possible to obtain the desired mycelium. Take 1.8 g of the mycelium and dry it to obtain a dry weight of 0.046 g, indicating a moisture content of 97.4% before drying and a solids content of 2.6%.

[0090] Preparation of edible support material: 0.5 g of 800 cps mushroom chitosan was weighed and dissolved in 100 ml of deionized water to prepare a solution with a chitosan solid content of 0.5%.

[0091] Preparation of mycelium chitosan sheet carrier: Mix the mycelium material and the edible support material in a mass ratio of 1:1 and evenly break them up. Spread the mixed material flatly in a silicone mold with a thickness of about 3 mm. Place it in a 60°C oven and dry it for 4-6 hours. After drying, cut it into 0.8 cm square slices to obtain the sheet carrier.

[0092] Moist heat sterilization: Soak the sheet carrier in PBS for 1 hour, then place it in a high-temperature autoclave for sterilization (parameters: 121°C, 45 minutes)

[0093] Cell culture: Fibroblasts were used as the cell type. Adherent cells were verified using a 24-well low-adhesion plate. Two to three sheet carriers were placed in each well. The cell seeding density was 3.0*10^5 cells / well. 2 ml of culture medium was added to each well. The medium was changed every other day, with 1 ml of medium changed per well.

[0094] Comparative conclusion: The mycelium chitosan mixed material has similar cell compatibility to the pure mycelium material (Comparative Example 1). The cells can spread completely on the carrier and proliferate effectively. Figure 7 Both carriers can support cells for a complete 5-7 day culture cycle. The hybrid material exhibits superior mechanical properties to pure mycelium materials during preparation, exhibits greater toughness, and exhibits lower shrinkage during drying, resulting in a higher yield of sheet carriers.

[0095] Comparisons between the mycelium-chitosan hybrid and the chitosan and soy protein isolate mixed sheet carrier (Comparative Example 4) showed a higher cell count, approximately twice that of the soy protein isolate mixed sheet carrier, better spreading, and superior performance. The chitosan solution itself was unable to form, and after mixing with soy protein isolate and drying, the porosity was too high, making cell attachment somewhat difficult. Cells only attached normally after three days, extending the cell growth cycle and causing a decline in cell status.

[0096] The cell compatibility of mycelium is significantly improved compared with chitosan materials, and the performance of the hybrid material is better.

[0097] Example 6 - Preparation of mycelial soy protein isolate filamentous three-dimensional scaffold and cell culture method

[0098] Mycelium Preparation: Crush the fermented Morchella mycelium and rinse thoroughly with drinking water or deionized water. Filter through a 300-mesh screen and squeeze out as much water as possible to obtain the desired mycelium. A 2.0g sample was dried and weighed to obtain a dry weight of 0.046g, indicating a moisture content of 97.7% before drying and a solids content of 2.3%.

[0099] Preparation of mycelium soy protein isolate filamentous three-dimensional scaffold: Soy protein isolate is mixed into the crushed mycelium material to prepare a 13% soy protein isolate + mycelium mixed liquid, and then wet spinning is performed to obtain filaments with a diameter of 0.5-1 mm.

[0100] Moist heat sterilization: After thoroughly washing the filamentous carrier with deionized water, slightly chop it into small pieces, soak it in PBS overnight, and place it in a high-temperature autoclave for sterilization (parameters: 121°C, 45 minutes).

[0101] Cell culture: Fibroblasts were used as cell types, and adherent cells were verified using a 24-well low-adhesion plate. Two to three filamentous three-dimensional scaffolds were placed in each well. The cell seeding density was 3.0*10^5 cells / well. 2 ml of culture medium was added to each well. The medium was changed every other day, with 1 ml of medium being changed per well.

[0102] Comparison conclusion: Compared with the pure soy protein isolate filamentous three-dimensional scaffold (Comparative Example 2), the mycelium effectively increased the cell compatibility of soy protein isolate. The cell attachment and spreading effect was better, and the cell proliferation was more obvious. Figure 8 .

[0103] Comparative Example 1 - Preparation of pure mycelium sheet carrier and cell culture method

[0104] Mycelium Preparation: Crush the fermented Morchella mycelium and rinse thoroughly with drinking water or deionized water. Filter through a 300-mesh screen and squeeze out as much water as possible to obtain the desired mycelium. Dried 2.2 g of the mycelium was weighed to obtain a dry weight of 0.050 g, indicating a moisture content of 97.7% before drying and a solids content of 2.3%.

[0105] Preparation of pure mycelium sheet carrier: The mycelium material is spread flat in a silicone mold with a thickness of about 3 mm, placed in a 60°C oven to dry for 4-6 hours, and then cut into 0.8 cm square slices and sterilized by wet heat.

[0106] Moist heat sterilization: Soak the sheet carrier in PBS for 1 hour, then place it in a high-temperature autoclave for sterilization (parameters: 121°C, 45 minutes)

[0107] Cell culture: Fibroblasts were used as cell types, and adherent cells were verified using a 24-well low-adhesion plate. 2-3 sheet carriers were placed in each well. The cell seeding density was 3.0*10^5 cells / well. 2 ml of culture medium was added to each well. The medium was changed every other day, with 1 ml of medium per well. Cell culture results are shown in Figure 9 .

[0108] Comparative Example 2 - Preparation of soy protein isolate filamentous three-dimensional scaffold and cell culture method

[0109] Preparation of soy protein isolate filamentous three-dimensional scaffolds: Weigh 8g of soy protein isolate and dissolve it in 100ml of deionized water to make an 8% soy protein isolate solution for wet spinning. Fibers with a diameter of 0.5-1mm were obtained.

[0110] Moist heat sterilization: After thoroughly washing the filamentous carrier with deionized water, slightly chop it into small pieces, soak it in PBS overnight, and place it in a high-temperature autoclave for sterilization (parameters: 121°C, 45 minutes).

[0111] Cell culture: Fibroblasts were used as cell types, and adherent cells were verified using a 24-well low-adhesion plate. 2-3 sheet carriers were placed in each well. The cell seeding density was 3.0*10^5 cells / well. 2 ml of culture medium was added to each well. The medium was changed every other day, with 1 ml of medium per well. Cell culture results are shown in Figure 10 .

[0112] Comparative Example 3 - Preparation of cellulose sheet carrier and cell culture method

[0113] Preparation of cellulose sheet carrier: Weigh 0.5g of bacterial cellulose and dissolve it in 100ml of deionized water to create a cellulose solution with a 0.5% solids content. Spread the solution onto a silicone mold to a thickness of approximately 3mm. Dry in a 60°C oven for 4-6 hours. After drying, cut the solution into 0.8cm square slices and sterilize them by wet heat.

[0114] Moist heat sterilization: Soak the sheet carrier in PBS for 1 hour, then place it in a high-temperature autoclave for sterilization (parameters: 121°C, 45 minutes)

[0115] Cell culture: Fibroblasts were used as the cell type. Adherent cells were verified using a 24-well low-adhesion plate. Two to three sheet carriers were placed in each well. The cell seeding density was 3.0*10^5 cells / well. 2 ml of culture medium was added to each well. The medium was changed every other day, with 1 ml of medium changed per well.

[0116] The cells showed aggregated growth and the number of cells was low. Figure 11 .

[0117] Comparative Example 4 - Preparation of Chitosan and Soy Protein Isolate Mixed Sheet Carrier and Cell Culture Method

[0118] Preparation of a chitosan and soy protein isolate mixed sheet carrier: Weigh 3g of 20cps mushroom chitosan and 10g of soy protein isolate and dissolve them in 100ml of deionized water to create a mixed solution with a chitosan solids content of 3% and an SPI concentration of 10%. Spread the solution onto a silicone mold to a thickness of approximately 3mm and dry it in a 60°C oven for 4-6 hours. After drying, cut it into 0.8cm square slices and sterilize it with moist heat.

[0119] Moist heat sterilization: Soak the sheet carrier in PBS for 1 hour, then place it in a high-temperature autoclave for sterilization (parameters: 121°C, 45 minutes)

[0120] Cell culture: Fibroblasts were used as the cell type. Adherent cells were verified using a 24-well low-adhesion plate. Two to three sheet carriers were placed in each well. The cell seeding density was 3.0*10^5 cells / well. 2 ml of culture medium was added to each well. The medium was changed every other day, with 1 ml of medium changed per well.

[0121] Cell culture results are shown in Figure 12 .

[0122] Comparative Example 5-Preparation of pure mycelium membrane

[0123] Preparation of pure mycelium membrane: The pure mycelium of Morchella edulis was cultured statically by the interfacial culture method. The mycelium membrane was obtained at the air-liquid interface. The pure mycelium membrane was obtained after being thoroughly washed with deionized water. Figure 13 .

[0124] Characteristics of pure mycelium membrane after wet heat sterilization: After wet heat sterilization, the mycelium membrane shrinks and has poor mechanical strength, making it unable to be used as a carrier to support cell culture. Figure 13 .

[0125] Characteristics of pure mycelium membrane after drying: After drying the pure mycelium membrane at 60℃ overnight (see Figure 11 ) The mechanical strength is enhanced, but the volume is compacted and the toughness is reduced. The toughness and volume are irreversible after rehydration, and cell culture testing cannot be performed.

[0126] Pure mycelium membrane is not suitable for direct use as a cell culture carrier.

[0127] While the basic concepts have been described above, it will be apparent to those skilled in the art that the detailed disclosure is merely illustrative and does not limit this specification. Although not explicitly stated herein, various modifications, improvements, and revisions to this specification may be made by those skilled in the art. Such modifications, improvements, and revisions are suggested in this specification and remain within the spirit and scope of the exemplary embodiments of this specification.

[0128] This specification also uses specific terms to describe the embodiments of this specification. For example, "one embodiment," "an embodiment," and / or "some embodiments" refer to a feature, structure, or characteristic associated with at least one embodiment of this specification. Therefore, it should be emphasized and noted that references to "one embodiment," "an embodiment," or "an alternative embodiment" two or more times in different locations in this specification do not necessarily refer to the same embodiment. Furthermore, certain features, structures, or characteristics of one or more embodiments of this specification may be appropriately combined.

[0129] In some embodiments, numbers are used to describe the quantity of components and attributes. It should be understood that such numbers used in the description of the embodiments are modified by the modifiers "about", "approximately" or "substantially" in some examples. Unless otherwise stated, "about", "approximately" or "substantially" indicate that the numbers are allowed to vary by ±20%. Accordingly, in some embodiments, the numerical parameters used in the description and claims are approximate values, which may change according to the required characteristics of individual embodiments. In some embodiments, the numerical parameters should take into account the specified significant digits and adopt the general method of retaining digits. Although the numerical domains and parameters used to confirm the breadth of their range in some embodiments of this specification are approximate values, in specific embodiments, the settings of such numerical values are as accurate as possible within the feasible range.

[0130] Finally, it should be understood that the embodiments described in this specification are intended only to illustrate the principles of the embodiments of this specification. Other variations may also fall within the scope of this specification. Therefore, by way of example and not limitation, alternative configurations of the embodiments of this specification may be considered consistent with the teachings of this specification. Accordingly, the embodiments of this specification are not limited to the embodiments explicitly described and illustrated in this specification.

Claims

1. A carrier suitable for preparing cell-cultured meat, wherein the carrier is selected from any one of the following: a) the carrier comprises edible fungus mycelium; b) The carrier comprises edible fungus mycelium and natural polymer materials, and the mass ratio of the mycelium solids to the natural polymer materials is (2-3): (0.4-15).

2. The carrier according to claim 1, wherein The edible fungi include at least one of morels, oyster mushrooms, sulfur mushrooms, black-skinned chicken mushrooms, wrinkled shiitake mushrooms, boletus, king oyster mushrooms, ganoderma lucidum, white lingzhi mushrooms, cordyceps militaris, white jade mushrooms, enoki mushrooms, and shiitake mushrooms. Preferably, the edible fungi are oyster mushrooms, sulfur mushrooms, or morels. More preferably, the edible fungi are morels. And / or, the natural polymer material comprises at least one of plant protein, chitosan, chitosan oligosaccharide, wheat flour or cellulose. Preferably, the natural polymer material is plant protein, chitosan or cellulose. More preferably, the plant protein is soy protein isolate, the cellulose is bacterial cellulose, and the chitosan is mushroom chitosan. And / or, the natural polymer material is edible; And / or, the mass ratio of the mycelium solids to the natural polymer material is (2.231-2.6): (0.498-13), preferably, the mass ratio of the mycelium solids to the natural polymer material is 2.4:0.498; And / or, the carrier is a spherical microcarrier, a sheet carrier or a filamentous three-dimensional scaffold. Preferably, the spherical microcarrier has a diameter of 100 to 300 μm, the sheet carrier is a 0.6 to 1 cm square sheet, and the filamentous three-dimensional scaffold has a diameter of 500 to 1000 μm. More preferably, the spherical microcarrier has a diameter of 200 μm, the sheet carrier is a 0.8 cm square sheet, and the filamentous three-dimensional scaffold has a diameter of 800 μm. And / or, the sheet carrier has a thickness of 0.5 to 5 mm; And / or, the carrier is a sterile carrier.

3. The method for preparing a carrier according to claim 1 or 2, comprising mixing edible fungus mycelium material with a support material to obtain the carrier, wherein the support material contains an edible natural polymer material.

4. The preparation method according to claim 3, wherein The edible fungus mycelium material is prepared by the following steps: The edible fungus mycelium is crushed, washed and dehydrated to obtain the edible fungus mycelium material; and / or, the solid content of the edible fungus mycelium material is 2.3-2.6 w / w%; and / or, the support material is in a solid state or a fluid state with a solid content of 0.1 to 2 w / w%, preferably, the support material is in a fluid state with a solid content of 0.5 w / w%; and / or, when the support material is in a fluid state, the mass ratio of the edible fungus mycelium material to the support material is (0.5-1.5):(0.5-1.5), preferably, the mass ratio of the edible fungus mycelium material to the support material is 1:1; And / or, when the support material is in a solid state, the mass ratio of the edible fungus mycelium material to the support material is (95-100):(11-15), preferably, the mass ratio of the edible fungus mycelium material to the support material is 97:13; And / or, after the edible fungus mycelium material is mixed with the support material, the following steps are required: The mixture of edible fungus mycelium material and support material is spread in a mold and after removing the moisture, a sheet carrier is obtained; And / or, after the edible fungus mycelium material is mixed with the support material, the following steps are required: a Wet spinning to obtain filaments; b. washing and chopping the filaments to obtain a carrier in the form of a filamentous three-dimensional scaffold; And / or, the carrier needs to be soaked in PBS and sterilized before use.

5. Cell-cultured meat, comprising the carrier according to claim 1 or 2 and non-human animal cells cultured and grown thereon.

6. The cell-cultured meat according to claim 5, wherein The non-human animal is any one of rodents, artiodactyls, perissodactyls, lagomorphs or primates; preferably, the non-human animal is any one of chicken, pig, cow, sheep, fish, duck, goose, turkey, pigeon, quail, rabbit, horse, donkey or deer.

7. The cell-cultured meat according to claim 5, wherein The non-human animal cells include at least one of muscle cells, fibroblasts, hepatocytes, osteoblasts, adipocytes, endothelial cells, hematopoietic stem cells, liver hematopoietic stem cells, bone marrow stem cells, adipose fibroblasts, adipose stem cells, mesenchymal stem cells, bone marrow stromal cells, muscle side group cells, blood-derived mesenchymal precursor cells, muscle precursor cells, circulating skeletal stem cells, multipotent adult progenitor cells, mesoderm progenitor cells, and spinal cord progenitor cells. Preferably, the non-human animal cells are fibroblasts.

8. A device for cell culture and / or cell culture meat preparation, comprising a cell culture vessel and the carrier according to claim 1 or 2 located in the cell culture vessel.

9. The device according to claim 8, wherein The cell culture vessel is a low-adhesion cell culture vessel; And / or, the cell culture vessel is any one of a cell culture dish, a cell culture flask, a cell culture plate or a bioreactor.

10. Use of the carrier according to claim 1 or 2, or the device according to claim 8 and 9 in cell culture and / or preparation of cell-cultured meat.

11. The method for preparing cell-cultured meat according to any one of claims 5 to 7, wherein the non-human animal cells are cultured using the apparatus according to claim 9 or 10.

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