Edible microcarrier for cell culture meat with fish gelatin / fish collagen as substrate and preparation method

Abstract

The invention belongs to the technical field of cell culture meat, and particularly relates to an edible microcarrier for cell culture meat with fish gelatin / fish collagen as a substrate and a preparation method thereof.The preparation method comprises the steps that fish gelatin / fish collagen is dissolved in water to prepare a fish gelatin aqueous solution, an oil-phase solvent is added and mixed to be uniform, an emulsifier is added, and the fish gelatin aqueous solution is prepared; the preparation method comprises the following steps: slowly dropwise adding a cross-linking agent solution into an emulsion system in a stirring process, adding the emulsion into a phase separation solution, carrying out phase separation, after the phase separation is complete, carrying out reduced pressure suction filtration to obtain frozen emulsion particles, putting the frozen emulsion particles into a freeze dryer, and carrying out freeze drying and the like.

Description

Technical Field

[0001] The present invention belongs to the technical field of cell cultured meat, and in particular relates to an edible microcarrier for cell cultured meat based on fish glue / fish collagen and a preparation method thereof. Background Art

[0002] Cell cultured meat is a meat analog produced through tissue engineering technology. Its preparation is mainly divided into four steps: first, the isolation of animal primary cells; then, these cells are expanded in a bioreactor to produce a large number of cell clusters; then, in order to create a muscle-like tissue structure, these cell clusters are cultured on a scaffold to differentiate them into muscle tissue; finally, the muscle tissue is further processed through food processing and other steps to better imitate the flavor, texture and nutritional value of traditional meat. Among them, the use of bioreactors to expand cells to obtain cell clusters is the core step, which greatly affects the production efficiency and cost of cell cultured meat. The way of cell expansion in bioreactors is three-dimensional cell culture, and three-dimensional cell culture cannot be separated from the use of carriers. Microcarriers are the first choice for cell carriers used in the cell expansion process in bioreactors due to their high specific surface area and suitability for dynamic culture systems.

[0003] Microcarriers are mainly microspheres with a diameter of 200-300 microns, and their synthesis methods mostly use the emulsion method. The specific process is to first mix a certain concentration of raw material aqueous solution with a hydrophobic solvent in a certain proportion, add an emulsifier and a cross-linking agent, and obtain a w / o type emulsion (oil-in-water type emulsion) after a certain period of reaction; then separate the aqueous phase from the emulsion system through a certain phase separation method (commonly known as demulsification), and obtain the microcarrier product through post-treatment. According to existing technical information, the raw materials for microcarrier synthesis mainly include synthetic polymer materials (polypropylene, polystyrene, polylactic acid, etc.) and natural polymer materials (polysaccharides, polypeptides, natural fibers, plant proteins, etc.). Among them, synthetic polymer materials are not suitable for the preparation of microcarriers for cell culture meat due to their inedibility, while some edible substances in natural polymer materials such as chitosan, gelatin, plant proteins, etc. have been used in the preparation of edible microcarriers.

[0004] There are also microcarrier synthesis methods that use the reverse emulsion method: first, a certain concentration of raw material (polysaccharide, gelatin, plant protein, etc.) aqueous solution is mixed with a hydrophobic solvent (such as toluene, petroleum ether, mineral oil, etc.) in a certain proportion, and then a certain amount of emulsifier is added to the mixed system, so that the entire system forms a uniform w / o type emulsion (oil-in-water type emulsion) under stirring conditions, and finally a cross-linking agent is added to the already formed emulsion system, and the final microcarrier emulsion is obtained after a period of reaction. After that, the microcarrier particles are separated from the emulsion system by a certain phase separation method (demulsification), and the microcarrier product is obtained through a series of post-processing steps such as washing and freeze drying.

[0005] However, there are obvious defects in the selection of raw materials in the preparation process of microcarriers. First, for polysaccharides (chitosan, dextran, etc.), most of them have low solubility in aqueous solution. Some sugars even need to add other substances to dissolve. For example, chitosan generally needs to be dissolved in 0.1-0.2M acetic acid solution, and even if it is dissolved, its final mass fraction is only 1%-2%. The low solubility of polysaccharides will inevitably bring the following defects: 1. It is difficult to increase the yield of microcarriers; 2. The reduction in the concentration of raw material aqueous solution may cause the final microcarrier particles to have a hollow structure, and the later freeze-drying process will damage the microcarrier particle structure; 3. The addition of cosolvents such as acetic acid has a great impact on the cell compatibility and food safety of microcarriers.

[0006] Second, gelatin is a raw material that is widely used in the preparation of microcarriers. Although it has good cell compatibility and edibility, and is highly soluble in water, the swelling of gelatin materials is more obvious, which makes it easy to deform when used in liquid culture media. If you want to prevent gelatin microcarriers from swelling, you need to increase the degree of cross-linking between gelatin molecules during preparation and use more cross-linking agents. However, the increase in the amount of cross-linking agents will inevitably bring food safety risks.

[0007] Third, for plant protein substances (silk fibroin, grain protein, etc.), the number of active functional groups (amino, hydroxyl, etc.) in their molecular structure is relatively small. If a certain structural strength is to be achieved, a large amount of cross-linking agents must be used, which also increases the food safety risk of cell-cultured meat.

[0008] In addition, in the process of preparing microcarriers using the traditional emulsion method, the use of emulsifiers is essential because the raw material aqueous solution needs to be dispersed in the oil phase solvent. Currently commonly used emulsifiers include Span 60, Span 80, etc., and their dosage is about 1% of the total emulsion volume. Although the amount of emulsifier used is less than that of other substances, it is easily adsorbed on the surface of the microcarrier and cannot be completely removed by subsequent washing, so it is very easy to remain. At present, most food emulsifiers are considered safe, but some people still experience symptoms such as nausea, vomiting, and abdominal pain after ingestion. In addition, long-term intake of emulsifiers may cause cardiovascular irritation and increase the risk of diseases such as hyperlipidemia and diabetes. Therefore, the use of emulsifiers is very likely to cause food safety risks in cell-cultured meat. Summary of the invention

[0009] In order to solve the above technical problems, the present invention provides an edible microcarrier for cell cultured meat based on fish glue / fish collagen and a preparation method, which is safe to eat. The microcarrier can not only avoid the food safety risks of cell cultured meat caused by the use of emulsifiers to a great extent, but also has a simple preparation process and easy to obtain raw materials.

[0010] The present invention solves the above technical problems by providing a method for preparing an edible microcarrier for cell cultured meat based on fish glue / fish collagen, comprising the following steps:

[0011] (1) dissolving fish glue / fish collagen in water to prepare a fish glue aqueous solution, and adding an oil phase solvent to mix evenly; wherein the volume ratio of the fish glue aqueous solution to the oil phase solvent is 0.2 / 1 to 0.5 / 1;

[0012] (2) Add emulsifier Span 60 or Span 80 and emulsify by mechanical stirring;

[0013] (3) slowly adding a crosslinking agent solution dropwise to the emulsion system under mechanical stirring; wherein the crosslinking agent is a polylysine aqueous solution, and the mass fraction of the polylysine aqueous solution is 0.1 to 0.3;

[0014] The mass fraction of polylysine aqueous solution in the range of 0.1 to 0.3 can ensure that the cross-linking between the fish glue molecular chains is relatively sufficient. Lower than 0.1 will lead to insufficient cross-linking, resulting in unstable physical structure of the final microcarrier particles; higher than 0.3 will cause certain waste and increase unnecessary preparation costs.

[0015] (4) adding the emulsion to the phase separation liquid under magnetic stirring to perform low-temperature phase separation, and after the phase separation is complete, filtering under reduced pressure to obtain frozen emulsion particles; the low temperature is -20°C to -80°C;

[0016] (5) The frozen emulsion particles are placed in a freeze dryer and freeze-dried to obtain microcarrier particles.

[0017] The essence of fish glue is a protein with complex composition. Its molecular structure contains a large number of hydrophilic functional groups (amino, hydroxyl, carboxyl, thiol, etc.) and hydrophobic functional groups (methyl, ethyl, isobutyl, benzyl, etc.). Therefore, fish glue has strong amphiphilicity and can be well dispersed in the oil phase solvent to form a uniform emulsion in the presence of trace emulsifiers or even in the absence of emulsifiers.

[0018] In the optimized solution, the mass fraction of the fish glue / fish collagen aqueous solution in step (1) is 20% to 50%.

[0019] Furthermore, the volume fraction of the emulsifier in step (2) to the oil phase solvent is 1‰ to 2‰.

[0020] The temperature during mechanical stirring in step (2) is 30° C., the stirring time is 40 min, and the stirring speed is 600 rpm.

[0021] Furthermore, the slow dripping rate in step (3) is less than 5 mL / min, and the stirring rate when the cross-linking agent is dripped is 600 rpm.

[0022] Furthermore, in step (4), the speed of adding the emulsion into the phase separation liquid is less than 5 mL / s, the temperature of the phase separation liquid is -20-80°C, and the time required for complete phase separation is 5-10 minutes.

[0023] The rotation speed of the magnetic stirring is 1000 rpm; the pore size of the filter paper for vacuum filtration is less than 50 μm.

[0024] In the step (4), the phase separation agent is one or more of n-hexane, n-pentane and isopentane.

[0025] Furthermore, in step (5), the freeze-drying temperature is ≤-40°C, the vacuum degree is ≤5Pa, and the freeze-drying time is 24-48h.

[0026] The step (2) is replaced by a step of directly performing an emulsification operation at a mechanical stirring speed without adding an emulsifier.

[0027] In the step (2) of not adding an emulsifier, the stirring temperature is 30-40° C., the stirring time is 50-60 min, and the stirring speed is 600 rpm.

[0028] The invention discloses a carrier prepared by a method for preparing edible microcarriers for cell cultured meat based on fish glue / fish collagen.

[0029] The oil phase solvent is one or more of toluene, petroleum ether or mineral oil.

[0030] The phase separation liquid is one or more of n-hexane, n-pentane and isopentane.

[0031] The emulsifier is Span 60 or Span 80.

[0032] The present invention aims at various defects existing in current raw materials such as polysaccharides, gelatin, and plant proteins, and selects fish glue / fish collagen as the main raw material to prepare edible microcarriers. First, compared with polysaccharides, the solubility of fish glue in water is 10 to 20 times that of polysaccharides, and no co-solvent is needed to help it dissolve; compared with gelatin-like substances, there is no technical data reporting that fish glue has swelling properties; compared with plant proteins, fish glue molecules have a large number of active functional groups, which can easily react physically or chemically with cross-linking agents.

[0033] The present invention selects fish glue (or fish collagen) as the main raw material to prepare edible microcarriers. The solubility of fish glue in water is 10 to 20 times that of polysaccharides, and no co-solvent is needed to help it dissolve; in addition, no technical data has yet reported that fish glue has swelling properties; fish glue molecules have a large number of active functional groups, which can easily react physically or chemically with cross-linking agents.

[0034] The cross-linking principle of fish glue and polylysine is mainly physical cross-linking: because fish glue molecules have a large number of active functional groups such as hydroxyl and amino groups, they can form strong hydrogen bonds with the amino groups on the polylysine molecular chain, thus achieving a cross-linking effect. During the cross-linking reaction, the viscosity of the entire emulsion system will increase slightly.

[0035] The biggest difference between the microcarrier preparation method of the present invention and the traditional preparation method is that after using fish glue as the main raw material, the amount of emulsifier used is greatly reduced, and even a preparation method without adding emulsion can be achieved.

[0036] The physical structure of the microcarrier in the present invention is porous loose particles with a diameter of about 200 microns. The microcarrier has stable chemical properties and can maintain a stable structure for a long time in water or a culture medium containing serum. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 The present invention is a flow chart of the preparation method of edible microcarriers for cell cultured meat based on fish glue / fish collagen.

[0038] Figure 2 The following are scanning electron microscope photos of the microcarriers prepared in Example 1 and Example 2 of the present invention.

[0039] Figure 3 This is a surface scanning electron microscope photograph of the microcarrier prepared in Example 2-5 of the present invention.

[0040] Figure 4 This is a statistical chart of cell survival rate of the fish gelatin-based microcarriers and chicken myoblasts cultured for three days in the present invention.

[0041] Figure 5 This is a scanning electron microscope photograph of the microcarrier surface obtained after cross-linking with glutaraldehyde in the present invention. DETAILED DESCRIPTION

[0042] The present invention is further described below in conjunction with specific embodiments:

[0043] Example 1

[0044] Preparation process Figure 1As shown, specifically, 25g of fish glue / fish collagen is taken and added to 100mL of deionized water to prepare a fish glue aqueous solution with a mass fraction of 20%; secondly, 20mL of fish glue aqueous solution is mixed with 40mL of toluene and added to a 200mL three-necked flask, followed by adding 50μL of emulsifier Span 80, so that the whole system is stirred at 600rpm and 30°C for 40min. Then prepare 5mL of polylysine solution with a mass fraction of 10%, and add it dropwise to the emulsion system at a rate of 1mL / min to complete crosslinking. After crosslinking is completed, the emulsion is added to 250mL of n-hexane at a temperature of minus 20°C at a rate of 2mL / s for phase separation. After phase separation is completed, the frozen emulsion particles are separated by vacuum filtration, and then the frozen emulsion particles are placed in a freeze dryer with a cold trap temperature of minus 50°C and a vacuum degree of 2-3Pa. After 36h, take it out to finally obtain microcarrier particles.

[0045] The essence of fish glue is a complex protein, similar to the commonly used emulsifiers on the market. Its molecular structure contains a large number of hydrophilic functional groups (amino, hydroxyl, carboxyl, thiol, etc.) and hydrophobic functional groups (methyl, ethyl, isobutyl, benzyl, etc.). Therefore, fish glue has strong amphiphilicity and can be well dispersed in the oil phase solvent to form a uniform emulsion in the presence of trace emulsifiers or even in the absence of emulsifiers. The microcarriers prepared by this microemulsion and emulsion-free system can reduce or even eliminate the food safety risks of cell cultured meat brought by microcarriers.

[0046] In addition, fish glue molecules have a large number of polar functional groups (amino, hydroxyl, carboxyl, thiol, etc.), which can form strong hydrogen bonds with the amino groups in polylysine molecules. Therefore, polylysine, an edible polypeptide, is used as a cross-linking agent.

[0047] Example 2

[0048] Take 25g of fish glue (fish collagen) and add it to 100mL of deionized water to prepare a 20% fish glue aqueous solution; secondly, mix 20mL of fish glue aqueous solution with 80mL of toluene and add it to a 200mL three-necked flask, and stir the whole system at 600rpm and 30℃ for 40min. Then prepare 5mL of polylysine solution with a mass fraction of 10%, and add it dropwise to the emulsion system at a rate of 1mL / min to complete crosslinking. After crosslinking is completed, the emulsion is added to 250mL of n-hexane at a temperature of minus 20℃ at a rate of 2mL / s for phase separation. After phase separation is completed, the frozen emulsion particles are separated by vacuum filtration, and then the frozen emulsion particles are placed in a freeze dryer with a cold trap temperature of minus 50℃ and a vacuum degree of 2-3Pa. Take it out after 36h to finally obtain microcarrier particles.

[0049] Example 3

[0050] Take 25g of fish glue (fish collagen) and add it to 100mL of deionized water to prepare a 20% fish glue aqueous solution; secondly, mix 20mL of fish glue aqueous solution with 80mL of toluene and add it to a 200mL three-necked flask, and stir the whole system at 600rpm and 30℃ for 40min. Then prepare 5mL of polylysine solution with a mass fraction of 10%, and add it dropwise to the emulsion system at a rate of 1mL / min to complete crosslinking. After crosslinking is completed, the emulsion is added to 250mL of n-hexane at a temperature of minus 40℃ at a rate of 2mL / s for phase separation. After phase separation is completed, the frozen emulsion particles are separated by vacuum filtration, and then the frozen emulsion particles are placed in a freeze dryer with a cold trap temperature of minus 50℃ and a vacuum degree of 2-3Pa. Take it out after 36h to finally obtain microcarrier particles.

[0051] Example 4

[0052] Take 25g of fish glue (fish collagen) and add it to 100mL of deionized water to prepare a 20% fish glue aqueous solution; secondly, mix 20mL of fish glue aqueous solution with 80mL of toluene and add it to a 200mL three-necked flask, and stir the whole system at 600rpm and 30℃ for 40min. Then prepare 5mL of polylysine solution with a mass fraction of 10%, and add it dropwise to the emulsion system at a rate of 1mL / min to complete crosslinking. After crosslinking is completed, the emulsion is added to 250mL of n-hexane at a temperature of minus 60℃ at a rate of 2mL / s for phase separation. After phase separation is completed, the frozen emulsion particles are separated by vacuum filtration, and then the frozen emulsion particles are placed in a freeze dryer with a cold trap temperature of minus 50℃ and a vacuum degree of 2-3Pa. Take it out after 36h to finally obtain microcarrier particles.

[0053] Example 5

[0054] Take 25g of fish glue (fish collagen) and add it to 100mL of deionized water to prepare a 20% fish glue aqueous solution; secondly, mix 20mL of fish glue aqueous solution with 80mL of toluene and add it to a 200mL three-necked flask, and stir the whole system at 600rpm and 30℃ for 40min. Then prepare 5mL of polylysine solution with a mass fraction of 10%, and add it dropwise to the emulsion system at a rate of 1mL / min to complete crosslinking. After crosslinking is completed, the emulsion is added to 250mL of n-hexane at a temperature of minus 80℃ at a rate of 2mL / s for phase separation. After phase separation is completed, the frozen emulsion particles are separated by vacuum filtration, and then the frozen emulsion particles are placed in a freeze dryer with a cold trap temperature of minus 50℃ and a vacuum degree of 2-3Pa. Take it out after 36h to finally obtain microcarrier particles.

[0055] Example 6

[0056] Other contents are as in Example 1, wherein the volume ratio of the fish glue aqueous solution to the oil phase solvent is 0.2 / 1, the mass fraction of the added polylysine is 30%, the oil phase solvent is petroleum ether, and the phase separation liquid is n-pentane.

[0057] Example 7

[0058] Other contents are as in Example 1, wherein the volume ratio of the fish glue aqueous solution to the oil phase solvent is 0.3 / 1, the mass fraction of the added polylysine is 40%, the oil phase solvent is mineral oil, and the phase separation liquid is isopentane.

[0059] Example 8

[0060] Other contents are the same as in Example 1, wherein the volume ratio of the fish glue aqueous solution to the oil phase solvent is 0.4 / 1, the mass fraction of the added polylysine is 50%, the oil phase solvent is toluene, and the phase separation liquid is n-pentane.

[0061] Example 9

[0062] Other contents are the same as in Example 1, wherein the volume ratio of the fish glue aqueous solution to the oil phase solvent is 0.5 / 1, the mass fraction of the added polylysine is 35%, the oil phase solvent is toluene, and the phase separation liquid is isopentane.

[0063] Test 1

[0064] The other contents are the same as in Example 1, and control groups 1, 2 and 3 are set to prepare microcarriers, wherein the fish gelatin material is replaced with chitosan in control group 1, and the volume ratio of the emulsifier to the oil phase agent is 1-2%;

[0065] Control group 2 was to replace the fish glue material with gelatin, and the volume ratio of the emulsifier to the oil phase agent was 1%;

[0066] Control group 3 is a control group in which the fish gelatin material is replaced with silk fibroin, and the volume ratio of the emulsifier to the oil phase agent is 0.5-1%.

[0067] The microcarriers in Example 1 and Example 2 were examined by electron microscopy. The results are as follows: Figure 2 and as shown in Table 1.

[0068] from Figure 2 It can be seen from the electron microscope photos in that when fish gelatin (fish collagen) is used as the raw material, microcarrier spherical particles with a diameter of 100 to 200 μm can be obtained with or without adding a trace amount of emulsifier.

[0069] Table 1 The amount of emulsifier used in preparing microcarriers from different raw materials and the particle size of the obtained microcarriers

[0070]

[0071] It can be seen from Table 1 that, referring to existing literature and technical data, for chitosan, gelatin and silk fibroin, three commonly used raw materials for preparing microcarriers, the amount of emulsifier used when the final microcarrier particle size is 200 μm is greater than the amount of emulsifier used in the previous Example 1 and this Example 2.

[0072] Test 2

[0073] The cell proliferation kit (CCK-8) was used to characterize the cytotoxicity of fish gelatin-based microcarriers in chicken myoblasts.

[0074] First, prepare the microcarrier dispersions of Examples 2 to 5 respectively, and then 4 The cells were seeded in four 96-well plates at a density of 10 cells per well. After culturing for 24, 48, and 96 hours, the four microcarrier dispersions prepared in advance at 2 mg / mL were added to each well of the corresponding plate. After 2 hours of co-culture, 2 μL of CCK-8 reagent was added to each well. After incubation at 37°C for 2 hours, the absorbance was read using an enzyme reader and the cell survival rate was calculated. The results are shown in Figure 3 and Figure 4 shown.

[0075] like Figure 3 As shown, in Examples 2-5, as the phase separation temperature decreases (-20°C, -40°C, -60°C and -80°C), the microcarrier surface gradually changes from a porous structure to a non-porous structure. This phenomenon shows that the microcarrier surface morphology can be changed by adjusting the phase separation temperature, thereby obtaining microcarrier particles with different surface structures.

[0076] like Figure 4 As shown, chicken myoblasts were co-cultured with fish gelatin-based microcarriers for three days, and the cell survival rate was tested every day. The cell survival rate was higher than 80%, and the cell survival rate increased every day compared with the previous day. On the third day, the cell survival rate was significantly higher than that of the previous two days, indicating that the cells proliferated. The results show that the prepared fish gelatin-based microcarriers have good cell compatibility and no obvious cytotoxicity.

[0077] Test 3

[0078] The other conditions were the same as those in Example 1, wherein the volume ratios of the fish gelatin aqueous solution to the oil phase solvent were 0.2 / 1, 0.3 / 1, 0.4 / 1 and 0.5 / 1, respectively, and the particle size variation data of the microcarriers finally prepared were shown in Table 2 below:

[0079] Table 2 Changes in microcarrier particle size under different volume ratios of isinglass aqueous solution to oil phase solvent

[0080]

[0081] According to the data in Table 2 above, it can be seen that the average particle size of the microcarriers prepared under different volume ratios of isinglass aqueous solution to oil phase solvent is about 200 μm, and increases slowly with the increase of the ratio of isinglass aqueous solution.

[0082] Test 4

[0083] A control group was set up, and other conditions were the same as those in Example 1, wherein the cross-linking agent was replaced with glutaraldehyde. The obtained microcarriers were examined by electron microscopy, and the scanning electron microscopic photograph of the microcarrier surface is as follows: Figure 5 .

[0084] In the process of microcarrier preparation, the choice of cross-linking agents is very limited. The most commonly used chemical cross-linking agent is glutaraldehyde. The use of glutaraldehyde will cause obvious cytotoxicity of microcarriers and also cause food safety risks.

[0085] from Figure 5 It can be seen that the surface of the microcarrier obtained after cross-linking with glutaraldehyde does not have obvious pores, and the surface morphology cannot be regulated by adjusting the phase separation temperature; while the surface of the microcarrier obtained after cross-linking with polylysine has obvious pores, and the pore size can be adjusted by changing the phase separation temperature, such as Figure 3 shown.

[0086] When fish glue material is used in the present invention, polylysine aqueous solution is needed as a cross-linking agent to achieve good results. It also shows that the selection of any fish glue material is not simply deduced or easily thought of, and requires the coordination of an overall solution to achieve the purpose of the invention.

[0087] The above implementation / test examples are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. A method for preparing edible microcarriers for cell cultured meat based on fish glue / fish collagen, characterized in that: The following steps are involved: (1) dissolving fish glue / fish collagen in water to prepare a fish glue aqueous solution, and adding an oil phase solvent to mix evenly; wherein the volume ratio of the fish glue aqueous solution to the oil phase solvent is 0.2 / 1 to 0.5 / 1; (2) Add emulsifier Span 60 or Span 80 and emulsify by mechanical stirring; (3) slowly adding a crosslinking agent solution to the emulsion system under mechanical stirring; wherein the crosslinking agent is a polylysine aqueous solution, and the mass fraction of the polylysine aqueous solution is 0.1 to 0.3; (4) adding the emulsion in step (3) to the phase separation liquid under magnetic stirring to perform low-temperature phase separation, and after the phase separation is complete, filtering under reduced pressure to obtain frozen emulsion particles; the low-temperature temperature is -20°C to -80°C. (5) The frozen emulsion particles are placed in a freeze dryer and freeze-dried to obtain microcarrier particles.

2. The method for preparing an edible microcarrier for cell cultured meat based on fish gelatin / fish collagen according to claim 1, characterized in that: The mass fraction of the fish glue / fish collagen aqueous solution in step (1) is 20% to 50%. The method for preparing an edible microcarrier for cell cultured meat based on fish glue / fish collagen as claimed in claim 1, characterized in that the volume fraction of the emulsifier in the oil phase solvent in the step (2) is 1‰ to 2‰.

3. The method for preparing an edible microcarrier for cell cultured meat based on fish glue / fish collagen according to claim 1, characterized in that: The temperature during the mechanical stirring was 30° C., the stirring time was 40 min, and the stirring speed was 600 rpm.

4. The method for preparing an edible microcarrier for cell cultured meat based on fish gelatin / fish collagen according to claim 1, characterized in that: The slow addition rate in step (3) is less than 5 mL / min, and the stirring rate when the cross-linking agent is added is 600 rpm.

5. The method for preparing an edible microcarrier for cell cultured meat based on fish glue / fish collagen as claimed in claim 1, characterized in that: In step (4), the speed of adding the emulsion into the phase separation liquid is less than 5 mL / s, and the time required for complete phase separation is 5 to 10 minutes.

6. The method for preparing an edible microcarrier for cell cultured meat based on fish gelatin / fish collagen according to claim 1, characterized in that: The rotation speed of the magnetic stirring is 1000 rpm; the pore size of the filter paper for vacuum filtration is less than 50 μm.

7. The method for preparing an edible microcarrier for cell cultured meat based on fish gelatin / fish collagen according to claim 1, characterized in that: In the step (4), the phase separation agent is one or more of n-hexane, n-pentane and isopentane.

8. The method for preparing an edible microcarrier for cell cultured meat based on fish gelatin / fish collagen as claimed in claim 1, characterized in that: The step (2) is replaced by a step of directly performing an emulsification operation at a mechanical stirring speed without adding an emulsifier.

9. The method for preparing an edible microcarrier for cell cultured meat based on fish gelatin / fish collagen according to claim 8, characterized in that: In the step (2) of not adding an emulsifier, the stirring temperature is 30-40° C., the stirring time is 50-60 min, and the stirring speed is 600 rpm.

10. A carrier prepared by a method for preparing edible microcarriers for cell cultured meat based on fish glue / fish collagen.

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