Edible microcarrier for producing cell culture meat and production method thereof

Abstract

The invention discloses an edible microcarrier for cell culture meat production and a production method thereof, and belongs to the technical field of cell culture meat production. The production method comprises the following steps: slowly adding a first mixed solution containing chitosan and lactose into a second mixed solution containing glycerol and egg white freeze-dried powder, mixing, and carrying out an emulsification reaction to obtain an emulsion; and carrying out phase separation treatment and drying treatment on the emulsion to obtain the edible microcarrier. According to the production method of the edible microcarrier for producing the cell culture meat, provided by the invention, edible chitosan is used as a main framework material, lactose is used as a cross-linking agent, glycerol is used as an oil phase, egg white freeze-dried powder is used as an emulsifier, ethanol is used as a phase separating agent, and production conditions of emulsification, phase separation and drying are controlled to prepare the edible microcarrier for producing the cell culture meat. The continuous, controllable and amplified preparation process of the microcarrier is realized, the uniform average particle size and stable structure of the microcarrier are ensured in the process, and the requirements of food safety standards are met at the same time.

Description

Technical Field

[0001] This invention relates to the field of cell-cultured meat production technology, specifically to an edible microcarrier for cell-cultured meat production and its production method. Background Technology

[0002] Cultured meat is a novel food production method that obtains edible tissues by culturing animal cells in vitro. Its core lies in providing a three-dimensional support environment for cell attachment, growth, and differentiation. Microcarriers, as important support materials in cultured meat systems, can significantly improve the specific surface area utilization of cells and support high-density cell culture in suspension systems. As the cultured meat industry gradually develops towards large-scale and engineered production, the technology for mass production of microcarriers has become one of the key factors restricting its industrial application.

[0003] Currently, the main methods for preparing microcarriers include emulsion template method, phase separation method, spray drying method, gel drop method, and microfluidic preparation method. These methods all use natural polymer materials as the main matrix and form microcarriers with certain porous structures and spherical morphologies through physical or chemical cross-linking. Emulsion template method is currently the most widely used method. Its basic principle is to disperse an aqueous polymer solution in an oil phase system, forming a water-in-oil emulsion under the action of an emulsifier. By adding a crosslinking agent, the dispersed droplets are solidified and shaped. Finally, after washing, solvent removal, and drying, microcarrier particles are obtained. Phase separation method usually uses temperature-induced or non-solvent-induced phase separation of polymer solution under controlled conditions to generate spherical or irregular particles. Spray drying method uses an atomizing device to atomize polymer solution into tiny droplets, which are rapidly dehydrated during hot air drying to form particles. Gel drop preparation method is mostly used to prepare gel carriers. The polymer solution is dropped into a coagulating liquid containing crosslinking agents or ions to achieve spheroidization and solidification. Microfluidic preparation method relies on the flow field of microfluidic chip to precisely control the droplet generation and solidification process, which can obtain microcarriers with uniform average particle size and concentrated distribution.

[0004] While existing microcarrier preparation technologies have been applied in laboratories and some pilot-scale trials, several limitations remain in achieving large-scale production of edible microcarriers. First, most microcarrier preparation processes rely on laboratory dispersion systems, depending on small-volume emulsification equipment or small magnetic stirring systems. This leads to uneven energy distribution and difficulty in consistently controlling the average emulsion particle size, resulting in wide particle size distribution and irregular morphology. Second, traditional cross-linking methods often employ highly efficient but potentially toxic chemical cross-linking agents such as glutaraldehyde and formaldehyde. Although these provide good structural stability, they fail to meet food-grade safety standards, and residue issues severely limit their application in the edible field. Furthermore, traditional solvent systems often rely on organic solvents, which not only increase post-processing burdens but may also lead to volatile organic compound (VOC) contamination and food safety hazards.

[0005] In terms of large-scale production, current production models generally lack continuity and controllability. Common methods such as dispersion and stirring, spray drying, or freeze-drying to form pellets are prone to problems such as uneven phase separation and changes in system viscosity leading to emulsion instability during scale-up, affecting batch-to-batch consistency of products.

[0006] Furthermore, some preparation processes still rely on organic solvents or industrial-grade emulsifiers, which not only affects edibility but also makes it difficult to meet the safety requirements of food-grade production environments. The National Food Safety Standard for the Use of Food Additives (GB2760-2024), which officially came into effect on February 8, 2025, imposes stricter requirements on the scope of use, residue limits, and safety assessments of process aids. Chemical crosslinking agents (such as glutaraldehyde), surfactants (such as Span and Tween series), and non-edible oil phase systems commonly used in traditional microcarrier preparation all face replacement or strict restrictions. More importantly, with the implementation of the new regulations in the National Food Safety Standard for the Use of Food Additives (GB 2760), stricter restrictions are placed on the types and amounts of food-grade aids, emulsifiers, and crosslinking agents, further increasing the difficulty of achieving complete food-grade production while maintaining structural properties.

[0007] Therefore, how to establish a stable, controllable, and scalable microcarrier production method by rationally selecting edible additives and mild cross-linking systems while meeting food safety standards has become a core issue that urgently needs to be addressed in this field. Summary of the Invention

[0008] The purpose of this invention is to provide an edible microcarrier for the production of cell-cultured meat and its production method, addressing the problems of inedible raw materials, unstable production processes, and difficulties in scale-up in existing microcarrier preparation methods. This production method uses an emulsion method to prepare microcarriers, achieving large-scale production by controlling reaction conditions, ensuring that the microcarriers have uniform average particle size, stability, and edibility, thus providing a new technical route for the engineering production of cell-cultured meat.

[0009] This invention is achieved through the following technical solution: This invention provides a method for producing edible microcarriers for cell-cultured meat production, comprising the following steps: Chitosan was dissolved in glacial acetic acid aqueous solution to prepare a chitosan solution with a concentration of 0.1 mol / L to 0.5 mol / L; Add 0.01 mol to 0.1 mol of lactose to the chitosan solution and stir thoroughly to obtain the first mixture. 0.01 mol to 0.05 mol of glycerol was mixed evenly with egg white freeze-dried powder to obtain a second mixture; the mass ratio of the egg white freeze-dried powder to the chitosan was (2 / 9 to 1 / 3): 1. The first mixture is slowly added to the second mixture and mixed and emulsified to obtain an emulsion; The emulsion is subjected to phase separation and drying processes to obtain the edible microcarrier.

[0010] More specifically, in the method for producing edible microcarriers for cell-cultured meat production, the egg white freeze-dried powder includes: egg white freeze-dried powder or duck egg white freeze-dried powder.

[0011] More specifically, in the method for producing edible microcarriers for cell-cultured meat production, the preparation of the freeze-dried egg white powder includes: After refrigerating egg whites or duck egg whites at 0~5℃ for 10h~20h, freeze-dry them at -50℃~-30℃ under a vacuum of 0.5Pa~4Pa for 40h~50h. The freeze-dried egg white powder was ground and graded using a 300-mesh sieve to obtain the freeze-dried egg white powder.

[0012] More specifically, in the method for producing edible microcarriers for cell-cultured meat, the emulsification reaction conditions include: an emulsification speed of 120 rpm to 130 rpm; an emulsification temperature of 50°C to 60°C; and an emulsification time of 30 min to 50 min.

[0013] More specifically, in the method for producing edible microcarriers for cell-cultured meat production, the phase separation process includes: adding the emulsion to a cooled ethanol solution for low-temperature phase separation to obtain microcarrier solid particles.

[0014] Further specifying, in the method for producing edible microcarriers for cell-cultured meat production, the temperature of the cooled ethanol is -28℃ to -32℃; the volume ratio of the emulsion to the ethanol is 1:(20~25).

[0015] Further specifying, in the method for producing edible microcarriers for cell-cultured meat production, the phase separation conditions include: emulsion flow rate of 10 mL / min to 20 mL / min; phase separation rotation speed of 300 rpm to 400 rpm; and stirring time of 30 min to 40 min.

[0016] More specifically, in the method for producing edible microcarriers for cell-cultured meat, the drying conditions include: under a vacuum of 1 Pa to 4 Pa, an initial temperature of -40°C to -45°C, freeze-drying for 70 to 75 hours, followed by a heating rate of 1.25°C / hour to -10°C to -15°C, for a total freeze-drying time of 90 to 100 hours.

[0017] More specifically, in the method for producing edible microcarriers for cell-cultured meat production, the average particle size of the edible microcarriers is 200µm to 300µm.

[0018] This invention provides an edible microcarrier for cell-cultured meat production, wherein the edible microcarrier is prepared by the above-described production method for edible microcarriers for cell-cultured meat production.

[0019] Compared with the prior art, the present invention has the following advantages and beneficial effects: This invention provides a method for producing edible microcarriers for cell-cultured meat production. Using edible chitosan as the main framework material, lactose as a cross-linking agent, glycerol as the oil phase, freeze-dried egg white powder as an emulsifier, and ethanol as a phase-separating agent, the lactose is fully dissolved and forms a non-covalent network structure with hydrogen bonds with chitosan, enhancing the stability of the emulsion interface. The egg white emulsifier is adsorbed on the surface of the emulsion droplets to prevent droplet fusion, providing a uniform microcarrier particle basis for subsequent low-temperature phase separation. This production method achieves continuous, controllable, and scaled-up microcarrier preparation by controlling the production conditions of emulsification, phase separation, and drying, while ensuring uniform average particle size and structural stability of the microcarriers, and meeting food safety standards. Attached Figure Description

[0020] To more clearly illustrate the technical solutions of the exemplary embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly described below. It should be understood that the following drawings only show some embodiments of the present invention and should not be considered as a limitation of the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort. In the drawings: Figure 1 Scanning electron microscope image of the edible microcarrier provided in Example 1 of the present invention; Figure 2 The results provided by this invention show the effect of emulsification speed on the average particle size of edible microcarriers. Figure 3 The results of the influence of emulsification temperature on the average particle size of edible microcarriers provided by the present invention; Figure 4 The results of the influence of the amount of freeze-dried egg white powder added on the average particle size of edible microcarriers provided by the present invention; Figure 5 The results of this invention on the effect of phase separation temperature on the average particle size of edible microcarriers; Figure 6 The results of the influence of emulsion flow rate on the average particle size of edible microcarriers provided by the present invention; Figure 7 The cell survival rate results of chicken myoblasts co-cultured with edible microcarriers provided in Example 2 of the present invention; Figure 8 The cell adhesion results of chicken myoblasts co-cultured with edible microcarriers provided in Example 2 of the present invention. Detailed Implementation

[0021] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments and accompanying drawings. The illustrative embodiments and descriptions of this invention are for explanation only and are not intended to limit the invention. Unless otherwise specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0022] A method for producing edible microcarriers for cell-cultured meat production includes the following steps: Chitosan was dissolved in glacial acetic acid aqueous solution to prepare a chitosan solution with a concentration of 0.1 mol / L to 0.5 mol / L; Add 0.01 mol to 0.1 mol of lactose to the chitosan solution and stir thoroughly to obtain the first mixture. 0.01 mol to 0.05 mol of glycerol was mixed evenly with egg white freeze-dried powder to obtain a second mixture; the mass ratio of the egg white freeze-dried powder to the chitosan was (2 / 9 to 1 / 3): 1. The first mixture is slowly added to the second mixture and mixed and emulsified to obtain an emulsion; The emulsion is subjected to phase separation and drying processes to obtain the edible microcarrier.

[0023] This invention provides a method for producing edible microcarriers for cell-cultured meat production. Using edible chitosan as the main framework material, lactose as a cross-linking agent, glycerol as the oil phase, freeze-dried egg white powder as an emulsifier, and ethanol as a phase-separating agent, the lactose is fully dissolved and forms a non-covalent network structure with hydrogen bonds with chitosan, enhancing the stability of the emulsion interface. The egg white emulsifier is adsorbed on the surface of the emulsion droplets to prevent droplet fusion, providing a uniform microcarrier particle basis for subsequent low-temperature phase separation. This production method achieves continuous, controllable, and scaled-up microcarrier preparation by controlling the production conditions of emulsification, phase separation, and drying, while ensuring uniform average particle size and structural stability of the microcarriers, and meeting food safety standards.

[0024] The key to the effectiveness of freeze-dried egg white powder as an emulsifier lies in its protein component. Proteins exhibit hydrophilic-hydrophobic amphoteric properties in aqueous solutions, allowing them to bind to the oil phase (glycerol) through their hydrophobic portion and to the aqueous phase (chitosan solution) through their hydrophilic portion. This forms an interfacial film between the oil and aqueous phases, promoting stable emulsion formation. Through this interfacial activity, freeze-dried egg white powder effectively reduces the surface tension at the oil-water interface, helping to uniformly disperse the oil and aqueous phases and form a stable emulsion, thus preventing droplet aggregation and stratification. Furthermore, the porous structure of freeze-dried egg white powder provides excellent interfacial adsorption capacity and prevents droplet aggregation.

[0025] Glycerol is a lipid-soluble molecule, while chitosan dissolves in glacial acetic acid to form a homogeneous aqueous solution. In this emulsion system, glycerol, as the oil phase, is not directly miscible with the chitosan aqueous solution. Instead, it forms a stable emulsion through the emulsifier, lyophilized egg white powder, ensuring the separation of the oil and aqueous phases and forming a stable emulsion structure. Glycerol, existing as the oil phase, maintains its dispersion with the aqueous chitosan solution through emulsification, avoiding solution instability caused by direct mixing.

[0026] The mechanism by which lactose acts as a cross-linking agent on chitosan is based on the intermolecular forces between lactose and chitosan. Lactose molecules contain multiple hydroxyl groups, which, in aqueous solution, form a non-covalent cross-linked network structure with the amino (–NH2) and hydroxyl (–OH) groups on chitosan molecules through hydrogen bonding. This network enhances the structural stability and morphological integrity of the microcarrier particles while maintaining the material's edibility and biocompatibility. By controlling the lactose concentration, reaction temperature, and stirring conditions, the density and uniformity of the hydrogen-bonded network can be optimized, thereby controlling the pore structure, particle size distribution, and mechanical stability of the microcarrier.

[0027] The present invention provides a method for producing edible microcarriers for cell-cultured meat production. By adjusting the reaction temperature and stirring rate, it can achieve efficient energy distribution, promote emulsion formation and cross-linking reactions, and effectively regulate the rate and extent of lactose cross-linking, ensuring uniform particle size distribution and structural stability of the microcarrier particles. The stability of the oil-water interface is controlled by using freeze-dried egg white powder as an emulsifier, keeping the emulsion stable during emulsification and cross-linking processes and preventing uneven particle size or emulsion breakage.

[0028] The present invention provides a method for producing edible microcarriers for cell-cultured meat production. This method selects edible raw materials and food-grade emulsifiers, cross-linking agents, or auxiliary agents to establish a green, large-scale preparation route for microcarriers, thereby avoiding the residue of inedible components. Glycerol, lactose, glacial acetic acid, and ethanol all belong to Appendix C, Table C.1 of the National Food Safety Standard for the Use of Food Additives (GB 2760-2024), which lists "processing aids that can be used in various food processing processes without residue limitations." All raw materials comply with food safety standards, ensuring that the final prepared microcarriers are completely edible.

[0029] The present invention provides a method for producing edible microcarriers for cell-cultured meat production. The resulting microcarriers can be widely used in cell-cultured meat production, have good cell adhesion, proliferation and biocompatibility, and are suitable for cell culture systems of different scales.

[0030] More specifically, the egg white freeze-dried powder includes: egg white freeze-dried powder or duck egg white freeze-dried powder.

[0031] More specifically, the preparation of the egg white freeze-dried powder includes: After refrigerating egg whites or duck egg whites at 0~5℃ for 10h~20h, freeze-dry them at -50℃~-30℃ under a vacuum of 0.5Pa~4Pa for 40h~50h. The freeze-dried egg white powder was ground and graded using a 300-mesh sieve to obtain the freeze-dried egg white powder.

[0032] Further specifying, the emulsification reaction conditions include: an emulsification speed of 120 rpm to 130 rpm; an emulsification temperature of 50°C to 60°C; and an emulsification time of 30 min to 50 min.

[0033] More specifically, the phase separation process includes: adding the emulsion to a cooled ethanol solution for low-temperature phase separation to obtain microcarrier solid particles.

[0034] Further specified, the temperature of the cooled ethanol is -28℃ to -32℃; the volume ratio of the emulsion to the ethanol is 1:(20~25).

[0035] Further specifying, the phase separation conditions include: emulsion flow rate of 10 mL / min to 20 mL / min; phase separation rotation speed of 300 rpm to 400 rpm; and stirring time of 30 min to 40 min.

[0036] Further specified, the drying conditions include: under a vacuum of 1 Pa to 4 Pa, an initial temperature of -40°C to -45°C, freeze-drying for 70 to 75 hours, followed by a heating rate of 1.25°C / hour to -10°C to -15°C, for a total freeze-drying time of 90 to 100 hours.

[0037] More specifically, the average particle size of the edible microcarrier is 200µm to 300µm.

[0038] To further illustrate the present invention, the following describes an edible microcarrier for cell-cultured meat production and its production method in conjunction with embodiments. However, it should be understood that these embodiments are implemented under the premise of the technical solution of the present invention, and provide detailed implementation methods and specific operating procedures. They are only for further illustrating the features and advantages of the present invention, and are not intended to limit the scope of the claims of the present invention. The scope of protection of the present invention is not limited to the following embodiments.

[0039] Example 1: The method for producing edible microcarriers for cell-cultured meat production provided in this embodiment includes: (1) Dissolve chitosan in glacial acetic acid aqueous solution to prepare a chitosan solution with a concentration of 0.1 mol / L; (2) Add 0.01 mol of lactose to the chitosan solution and stir thoroughly to obtain the first mixture; (3) Mix 0.01 mol of glycerol with the freeze-dried egg white powder to obtain a second mixture; the mass ratio of the freeze-dried egg white powder to the chitosan is 2 / 9:1; The freeze-dried egg white powder includes: freeze-dried egg white powder or freeze-dried duck egg white powder.

[0040] The preparation of the freeze-dried egg white powder includes: refrigerating egg white or duck egg white at 0°C for 10 hours, then freeze-drying it at -50°C under a vacuum of 0.5 Pa for 40 hours; grinding the freeze-dried egg white powder and grading it using a 300-mesh sieve to obtain the freeze-dried egg white powder.

[0041] (4) The first mixture is slowly added to the second mixture and mixed and emulsified to obtain an emulsion; The emulsification reaction conditions include: an emulsification speed of 120 rpm; an emulsification temperature of 50°C; and an emulsification time of 30 min.

[0042] (5) The phase separation process includes: adding the emulsion to a cooled ethanol solution for low-temperature phase separation to obtain microcarrier solid particles.

[0043] The temperature of the ethanol is -28°C; the volume ratio of the emulsion to the ethanol is 1:20.

[0044] The phase separation conditions include: emulsion flow rate of 10 mL / min; phase separation rotation speed of 300 rpm; and stirring time of 30 min.

[0045] (6) Dry the microcarrier solid particles to obtain edible microcarriers; The drying conditions include: under a vacuum of 1 Pa, an initial temperature of -40°C, freeze-drying for 70 hours, followed by a heating rate of 1.25°C / h to -10°C, for a total freeze-drying time of 90 hours.

[0046] Example 2: The method for producing edible microcarriers for cell-cultured meat production provided in this embodiment includes: (1) Dissolve chitosan in glacial acetic acid aqueous solution to prepare a chitosan solution with a concentration of 0.3 mol / L; (2) Add 0.05 mol of lactose to the chitosan solution and stir thoroughly to obtain the first mixture; (3) Mix 0.03 mol of glycerol with the freeze-dried egg white powder to obtain a second mixture; the mass ratio of the freeze-dried egg white powder to the chitosan is 5 / 18:1; The freeze-dried egg white powder includes: freeze-dried egg white powder or freeze-dried duck egg white powder.

[0047] The preparation of the freeze-dried egg white powder includes: refrigerating egg white or duck egg white at 3°C ​​for 15 hours, then freeze-drying it at -40°C under a vacuum of 2 Pa for 45 hours; grinding the freeze-dried egg white powder and grading it using a 300-mesh sieve to obtain the freeze-dried egg white powder.

[0048] (4) The first mixture is slowly added to the second mixture and mixed and emulsified to obtain an emulsion; The emulsification reaction conditions include: an emulsification speed of 125 rpm; an emulsification temperature of 55°C; and an emulsification time of 40 min.

[0049] (5) The phase separation process includes: adding the emulsion to a cooled ethanol solution for low-temperature phase separation to obtain microcarrier solid particles.

[0050] The temperature of the cooled ethanol is -30°C; the volume ratio of the emulsion to the ethanol is 1:22.

[0051] The phase separation conditions include: emulsion flow rate of 15 mL / min; phase separation rotation speed of 350 rpm; and stirring time of 35 min.

[0052] (6) Dry the microcarrier solid particles to obtain edible microcarriers; The drying conditions include: under vacuum Pa, an initial temperature of -42℃, freeze-drying for 72 hours, followed by a heating rate of 1.25℃ / h to -12℃, for a total freeze-drying time of 92 hours.

[0053] Example 3: The method for producing edible microcarriers for cell-cultured meat production provided in this embodiment includes: (1) Dissolve chitosan in glacial acetic acid aqueous solution to prepare a chitosan solution with a concentration of 0.5 mol / L; (2) Add 0.1 mol of lactose to the chitosan solution and stir thoroughly to obtain the first mixture; (3) Mix 0.05 mol of glycerol with the freeze-dried egg white powder to obtain a second mixture; the mass ratio of the freeze-dried egg white powder to the chitosan is 1 / 3:1; The freeze-dried egg white powder includes: freeze-dried egg white powder or freeze-dried duck egg white powder.

[0054] The preparation of the freeze-dried egg white powder includes: refrigerating egg white or duck egg white at 5°C for 20 hours, then freeze-drying it at -30°C under a vacuum of 4Pa for 50 hours; grinding the freeze-dried egg white powder and grading it using a 300-mesh sieve to obtain the freeze-dried egg white powder.

[0055] (4) The first mixture is slowly added to the second mixture and mixed and emulsified to obtain an emulsion; The emulsification reaction conditions include: an emulsification speed of 130 rpm; an emulsification temperature of ~60℃; and an emulsification time of 50 min.

[0056] (5) The phase separation process includes: adding the emulsion to a cooled ethanol solution for low-temperature phase separation to obtain microcarrier solid particles.

[0057] The temperature of the ethanol is -32°C; the volume ratio of the emulsion to the ethanol is 1:25.

[0058] The phase separation conditions include: emulsion flow rate of 20 mL / min; phase separation rotation speed of 400 rpm; and stirring time of 40 min.

[0059] (6) Dry the microcarrier solid particles to obtain edible microcarriers; The drying conditions include: under a vacuum of 4 Pa, an initial temperature of -45°C, freeze-drying for 75 hours, followed by a heating rate of 1.25°C / hour to -15°C, for a total freeze-drying time of 100 hours.

[0060] Example 1: Preparation of edible microcarriers The method for producing edible microcarriers for cell-cultured meat production provided in this example includes: (1) Preparation of emulsifier egg white lyophilized powder: 50g of fresh egg white (or duck egg white) was refrigerated at 4℃ for 12 hours to remove air bubbles. Then the egg white was evenly divided into trays and placed in a freeze dryer for pre-freezing at -40℃ and a vacuum of 2Pa for about 48 hours to ensure that the moisture was completely removed and a loose powder structure was formed.

[0061] The freeze-dried egg white powder is ground and graded through a 300-mesh sieve to obtain a uniform and easily soluble freeze-dried protein powder, which can be directly used for emulsion emulsification, providing good interfacial adsorption capacity and preventing droplet aggregation.

[0062] (2) Preparation of edible microcarriers: Select a 20L reactor, dissolve 36g of edible chitosan in 1L of 2% glacial acetic acid aqueous solution, then add 18g of lactose, and stir to fully dissolve the lactose and form a preliminary network with hydrogen bonds with the chitosan, which provides stability for the microcarrier particles. Select a 20L reactor, add 2.5L of glycerol and 10g of freeze-dried egg white powder to the reactor and mix well; A mixed solution containing chitosan and lactose was slowly added to a mixed solution containing glycerol and lyophilized egg white powder, while the stirring speed in the reactor was maintained at 120 rpm, the temperature at 50°C, and the emulsion stirring time at 40 min. Lactose forms a non-covalent network structure with chitosan through hydrogen bonding, enhancing the interfacial stability of the emulsion, while the egg white emulsifier is adsorbed on the surface of the emulsion droplets, preventing droplet fusion and providing a uniform microcarrier particle basis for subsequent low-temperature phase separation.

[0063] Select a 50L reactor, add 20L of ethanol, and cool to -30℃. After the emulsion preparation in the previous step is completed, slowly add it to the 50L reactor for low-temperature phase separation with ethanol. The bottom of the reactor is equipped with a 50-mesh filter tray, connected to a 50L reservoir ball for collecting the filtrate, which can be vacuum filtered. The emulsion is added using a peristaltic pump at a flow rate of 20mL / min to ensure sufficient contact between the emulsion and the phase separating agent and the formation of microcarrier particles. After the emulsion is completely added, continue stirring for 30 minutes at a speed of 300rpm. Then, start the vacuum pump to filter and remove the liquid, collecting the remaining solid particles.

[0064] The collected solid particles were placed in a freeze dryer under a vacuum of 2 Pa and an initial temperature of -40°C. After 72 hours of freeze-drying, the temperature was slowly increased to -10°C at a rate of 1.25°C / h to prevent particle collapse. The total freeze-drying time was 92 hours. After freeze-drying, the microcarriers were gently washed 2-3 times with deionized water to remove residues, and then dried at low temperature until the moisture content was stable to obtain edible microcarriers.

[0065] Electron scanning was performed on the edible microcarriers prepared in Example 1, and the results are shown in [Figure 1]. Figure 1 As shown. From Figure 1 It can be seen that the edible microcarrier has a uniform average particle size and stable structure, with an average particle size of 200-300µm.

[0066] Experimental Example: Effect of a Single Factor on the Average Particle Size of Edible Microcarriers 1.1 Effect of emulsification speed on the average particle size of edible microcarriers Referring to the production method of edible microcarriers for cell-cultured meat production provided in Example 1, the emulsification speed was set to 50 rpm, 60 rpm, 70 rpm, 80 rpm, 90 rpm, 100 rpm, 110 rpm, 120 rpm, 130 rpm, 140 rpm, 160 rpm, 170 rpm, 180 rpm, 190 rpm, and 200 rpm, respectively. The average particle size of the edible microcarriers was measured, and the results are shown in […]. Figure 2 As shown.

[0067] from Figure 2 It can be seen that the average particle size is stable at 200-300µm when the emulsification speed is 120 rpm. Therefore, the emulsification speed should be maintained at 120-130 rpm during implementation.

[0068] 1.2 Effect of emulsification temperature on the average particle size of edible microcarriers Referring to the production method of edible microcarriers for cell-cultured meat production provided in Example 1, the emulsification temperatures were set to 25℃, 30℃, 35℃, 40℃, 45℃, 50℃, 55℃, 60℃, 65℃, 70℃, 75℃, and 80℃, respectively. The average particle size of the edible microcarriers was measured, and the results are shown in [Figure 1]. Figure 3 As shown.

[0069] from Figure 3 It can be seen that when the emulsification temperature is 50~60℃, the average particle size of edible microcarriers is about 200-300µm. Therefore, the temperature should be controlled at 50~60℃ during the implementation process.

[0070] 1.3 Effect of Egg White Freeze-Dried Powder Addition on Average Particle Size of Edible Microcarriers Referring to the production method of edible microcarriers for cell-cultured meat production provided in Example 1, the amount of freeze-dried egg white powder added was set to 2g, 4g, 6g, 8g, 10g, 12g, 14g, 16g, 18g, and 20g, respectively. The average particle size of the edible microcarriers was measured, and the results are shown in [Figure 1]. Figure 4 As shown.

[0071] from Figure 3 It can be seen that when the amount of freeze-dried egg white powder added is about 8g~12g, the average particle size of the edible microcarriers is 200-300 µm. Therefore, 8g~12g should be added during the implementation process. 1.4 Effect of phase separation temperature (temperature for cooling ethanol) on the average particle size of edible microcarriers Referring to the production method of edible microcarriers for cell-cultured meat production provided in Example 1, the ethanol cooling temperatures were set to -70℃, -60℃, -50℃, -40℃, -60℃, -20℃, and -10℃, respectively. The average particle size of the edible microcarriers was measured, and the results are shown in [Figure 1]. Figure 5 As shown.

[0072] from Figure 5 It can be seen that when the phase separation temperature is between -28℃ and -32℃, the particle size of edible microcarriers is 200-300µm. Therefore, the temperature should be maintained between -28℃ and -32℃ during the implementation process.

[0073] 1.5 Effect of emulsion flow rate on the average particle size of edible microcarriers Referring to the production method of edible microcarriers for cell-cultured meat production provided in Example 1, the emulsion flow rate was set to 5 mL / min, 10 mL / min, 15 mL / min, 20 mL / min, 25 mL / min, and 30 mL / min, respectively. The average particle size of the edible microcarriers was measured, and the results are shown in [Figure 1]. Figure 6 As shown.

[0074] from Figure 6 It can be seen that when the emulsion flow rate is 10 mL / min to 20 mL / min, the particle size of the edible microcarriers is 200-300 µm. Therefore, the flow rate should be maintained at 10 mL / min to 20 mL / min during the implementation process.

[0075] Example 2: Co-culture of chicken myoblasts with microcarriers The edible microcarriers prepared in Example 1, which were pre-sterilized and pre-wetted with culture medium, were added to a 500 mL cell transfer flask (2 g / L). After adding an appropriate amount of culture medium, the flask was incubated at 37°C with 5% CO2 for 30 min to equilibrate. Chicken myoblasts in the logarithmic growth phase were taken, digested with trypsin, and a single-cell suspension was prepared, adjusting the cell concentration to approximately 1 × 10⁻⁶. 5Cells / mL. The cell suspension was then added to the culture system containing the microcarriers, ensuring thorough mixing. Initially, the cells were cultured statically or with intermittent low-speed shaking (approximately 30 rpm) for 4–6 hours to promote cell attachment. During culture, cell counts were performed daily, and half of the culture medium was replaced with fresh medium to maintain nutritional stability. Chicken myoblasts were co-cultured with the microcarriers for three days, with cell viability tested daily. Results are shown below. Figure 7 As shown in the figure. 1 mL of liquid was collected daily by centrifugation, and the microcarriers were stained with a live cell dye. Cell adhesion was observed using a fluorescence microscope. The results are shown in the figure. Figure 8 As shown from Figure 7 As can be seen, the cell survival rate was above 80% each day, and the cell survival rate increased each day compared to the previous day. Furthermore, on the third day, the cell survival rate was significantly higher than the previous two days, indicating that the cells had proliferated.

[0076] from Figure 8 As can be seen, after staining and observation under a fluorescence microscope, the cells exhibited a distinct spindle shape and good proliferation status. This result indicates that the prepared microcarrier has good cell compatibility and no obvious cytotoxicity.

[0077] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above description is only a specific embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for producing edible microcarriers for cell-cultured meat production, characterized in that, Includes the following steps: Chitosan was dissolved in glacial acetic acid aqueous solution to prepare a chitosan solution with a concentration of 0.1 mol / L to 0.5 mol / L; Add 0.01 mol to 0.1 mol of lactose to the chitosan solution and stir thoroughly to obtain the first mixture. 0.01 mol to 0.05 mol of glycerol was mixed evenly with egg white freeze-dried powder to obtain a second mixture; the mass ratio of the egg white freeze-dried powder to the chitosan was (2 / 9 to 1 / 3):

1. The first mixture is slowly added to the second mixture and mixed and emulsified to obtain an emulsion; The emulsion is subjected to phase separation and drying processes to obtain the edible microcarrier.

2. The method for producing edible microcarriers for cell-cultured meat production according to claim 1, characterized in that, The freeze-dried egg white powder includes: freeze-dried egg white powder or freeze-dried duck egg white powder.

3. The method for producing edible microcarriers for cell-cultured meat production according to claim 2, characterized in that, The preparation of the freeze-dried egg white powder includes: After refrigerating egg whites or duck egg whites at 0~5℃ for 10h~20h, freeze-dry them at -50℃~-30℃ under a vacuum of 0.5Pa~4Pa for 40h~50h. The freeze-dried egg white powder was ground and graded using a 300-mesh sieve to obtain the freeze-dried egg white powder.

4. The method for producing edible microcarriers for cell-cultured meat production according to claim 1, characterized in that, The emulsification reaction conditions include: emulsification speed of 120 rpm to 130 rpm; emulsification temperature of 50℃ to 60℃; and emulsification time of 30 min to 50 min.

5. The method for producing edible microcarriers for cell-cultured meat production according to claim 1, characterized in that, The phase separation process includes: adding the emulsion to a cooled ethanol solution for low-temperature phase separation to obtain microcarrier solid particles.

6. The method for producing edible microcarriers for cell-cultured meat production according to claim 5, characterized in that, The temperature of the cooled ethanol is -28℃ to -32℃; the volume ratio of the emulsion to the ethanol is 1:(20~25).

7. The method for producing edible microcarriers for cell-cultured meat production according to claim 5, characterized in that, The phase separation conditions include: emulsion flow rate of 10 mL / min to 20 mL / min; phase separation rotation speed of 300 rpm to 400 rpm; and stirring time of 30 min to 40 min.

8. The method for producing edible microcarriers for cell-cultured meat production according to claim 1 or 5, characterized in that, The drying conditions include: under a vacuum of 1 Pa to 4 Pa, an initial temperature of -40°C to -45°C, freeze-drying for 70 to 75 hours, followed by a heating rate of 1.25°C / hour to -10°C to -15°C, for a total freeze-drying time of 90 to 100 hours.

9. The method for producing edible microcarriers for cell-cultured meat production according to claim 1, characterized in that, The average particle size of the edible microcarrier is 200µm~300µm.

10. An edible microcarrier for cell-cultured meat production, characterized in that, The edible microcarrier is prepared by the production method of the edible microcarrier for cell-cultured meat production according to any one of claims 1-9.

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