Chitosan-based edible microcarriers prepared by low-temperature confined phase separation and pH-triggered solidification and a preparation method thereof

Abstract

The present application relates to the field of cell culture meat and bioengineering technology, in particular to a kind of chitosan-based edible microcarrier prepared by low-temperature limited phase separation and pH triggered solidification and a preparation method thereof.By the continuous process combination of "acid complex droplet formation-low-temperature limited phase separation-basic trigger diffusion-pH gradient solidification-fractionation oil removal", chitosan-based composite microcarrier preparation is realized without additional crosslinking agent.The method does not rely on chemical crosslinking agent, enzyme crosslinking agent or pre-formed scaffold for microcarrier construction, but utilizes the acid-base response characteristics of chitosan: soluble in acidic environment, quickly gelatinized in neutral and weakly alkaline environment.The preparation method of the present application is safe, low in cost and easy to scale up, and can provide a new scheme for the three-dimensional expansion of adherent cells in cell culture meat.

Description

Technical Field

[0001] This invention relates to the fields of cell-cultured meat and bioengineering technology, specifically to a chitosan-based edible microcarrier prepared by low-temperature confined phase separation and pH-triggered solidification, and its preparation method. Background Technology

[0002] Cultured meat is a novel manufacturing method that utilizes the in vitro expansion, differentiation, and tissue processing of animal cells to obtain meat products. It holds significant promise for alleviating resource pressures on traditional livestock and aquaculture, reducing reliance on animal slaughter, and improving the controllability of food production. As research on cultured meat progresses from laboratory validation to large-scale production, the efficient and low-cost acquisition of large quantities of edible animal cells has become a key technological challenge limiting the field's development. Specifically, the large-scale expansion of adherent stem cells, muscle cells, adipocytes, and other tissue cells typically requires the use of microcarriers in stirred bioreactors to provide a three-dimensional attachment interface. Therefore, developing microcarrier materials and preparation processes suitable for cultured meat production is crucial for the industrialization of cultured meat.

[0003] Existing commercially available microcarriers are mostly made from polystyrene, cross-linked dextran, gelatin, collagen, or other synthetic and semi-synthetic materials. While these microcarriers can meet some cell culture requirements, they still have significant shortcomings in the application of cultured meat. On the one hand, some microcarrier materials are inedible or lack food properties, requiring separation of cells from the microcarrier after culture through enzymatic hydrolysis, filtration, sedimentation, centrifugation, or other methods. This not only increases production steps and equipment costs but may also cause cell damage and product loss. On the other hand, to maintain the structural stability of the microcarrier during culture, existing preparation methods often require the use of chemical or enzymatic cross-linking agents. Chemical cross-linking agents may pose residual risks and biocompatibility issues, while enzymatic cross-linking, although relatively mild, is more expensive, has limited reaction conditions, and requires high system control in large-scale preparation. Therefore, microcarriers for cultured meat applications not only need to have cell adhesion support capabilities but should also be edible, have low residual risks, low cost, and be feasible for process scale-up.

[0004] In recent years, edible microcarriers have gradually become an important research direction in the field of cell-cultured meat. Natural polysaccharides, plant proteins, animal-derived proteins, and food-grade composite materials are widely used in microcarrier construction, such as gelatin, alginate, chitosan, soy protein, pea protein, and pumpkin seed protein. These materials have certain advantages in terms of source safety, food processing suitability, and cell culture compatibility. However, from the perspective of current technological development, the innovation focus of many edible microcarrier solutions mainly concentrates on material replacement or component blending, that is, introducing a certain natural polymer, plant protein, or polysaccharide component into the microcarrier system to improve cell adhesion, nutritional properties, or edibility. While such strategies can improve the food applicability of microcarriers to a certain extent, if the formation and stability of microcarriers still rely on traditional cross-linking, precipitation, or material blending logic, it is difficult to fundamentally solve problems such as cross-linking agent residue, process complexity, insufficient structural controllability, and insufficient stability in scale-up preparation.

[0005] Chitosan, a natural polysaccharide obtained by deacetylation of chitin, is widely available, biocompatible, biodegradable, and low-cost, and has been used in food, medicine, tissue engineering, and cell culture scaffolds. The chitosan molecular chain contains numerous amino groups. Under acidic conditions, these amino groups are protonated, allowing chitosan to dissolve and form a positively charged polymer solution. When the pH rises to neutral or weakly alkaline conditions, the amino groups gradually deprotonate, weakening intermolecular electrostatic repulsion and enhancing hydrogen bonding, hydrophobic interactions, and chain entanglement, thus transforming chitosan from a solution state to a gel or solid state. This pH-responsive characteristic provides a theoretical basis for constructing chitosan-based microcarriers without added cross-linking agents.

[0006] However, directly utilizing the pH changes of chitosan to prepare microcarriers suitable for cell-cultured meat still faces several challenges. Adding an acidic chitosan solution directly to an alkaline aqueous solution easily results in gel particles with uneven size, irregular morphology, rapid surface hardening, and insufficient internal solidification. Adding alkaline substances directly to an emulsion system can easily lead to droplet rupture, particle adhesion, or structural collapse due to sudden local pH changes. Furthermore, chitosan itself has limited adhesion and support capabilities for some cells; relying solely on chitosan may not meet the demands for efficient adhesion and continuous expansion in cell-cultured meat production. Therefore, how to stably form spherical and uniformly solidify an acidic chitosan solution without introducing chemical cross-linking agents, while also ensuring cell adhesion support, is a key issue that needs to be addressed in the preparation of chitosan-based edible microcarriers.

[0007] In the prior art, CN118546869A discloses a scheme related to edible microcarriers, which mainly uses edible materials such as plant proteins to prepare microcarriers through an emulsification-crosslinking method. However, this scheme still relies on crosslinking agents to achieve microcarrier stabilization and does not involve the pH-responsive curing mechanism of chitosan. CN116536257A discloses related content of chitosan-based microcarriers, but its spheroidization mechanism mainly revolves around material compounding and conventional curing methods, without disclosing the complete process path of "acidic chitosan droplets undergoing low-temperature phase separation in an oil-phase confined environment, followed by pH gradient curing induced by diffusion from an alkaline triggering agent." Therefore, the prior art has not fully solved the process problem of "how acidic chitosan droplets can achieve in-situ curing without crosslinking in an oil-phase confined environment through a controlled pH gradient." Especially in the context of microcarriers for cell-cultured meat, microcarriers need to simultaneously meet multiple requirements, including edibility, structural stability, controllable particle size, scalable preparation, low residue risk, and compatibility with cell culture. Relying solely on single-material replacement or conventional compounding is insufficient to establish a stable, clear, and industrially viable technological path. Therefore, there is an urgent need to establish a new method for preparing chitosan-based edible microcarriers. Summary of the Invention

[0008] The technical problem this invention aims to solve is that existing microcarriers for cell-cultured meat mostly rely on chemical cross-linking, enzymatic cross-linking, or material compounding to achieve structural stability, which easily leads to problems such as cross-linking agent residue, high preparation costs, complex process scale-up, and high pressure for food safety evaluation. At the same time, simply replacing plant proteins, natural acid polymers, or other edible materials to form microcarriers often makes it difficult to clearly distinguish them from existing edible microcarrier technologies.

[0009] To address the aforementioned issues, this invention proposes a microcarrier preparation pathway centered on a pH-driven, non-crosslinking curing process. Through a continuous process combination of "acidic composite droplet formation—low-temperature confined phase separation—alkaline trigger diffusion—pH gradient curing—graded purification and oil removal," chitosan-based composite microcarriers can be prepared without the need for external crosslinking agents. This method does not rely on chemical crosslinking agents, enzyme crosslinking agents, or pre-formed scaffolds for microcarrier construction. Instead, it utilizes the acid-base responsiveness of chitosan: it is soluble in acidic environments and rapidly gels in neutral and weakly alkaline environments. First, an acidic chitosan solution is dispersed into micron-sized droplets. Low-temperature treatment enhances stability and structural controllability. Then, an alkaline trigger diffusion forms a pH gradient from the outside in, allowing the droplets to solidify in situ in the oil phase, resulting in a structurally stable, tunable-size, edible microcarrier that supports cell adhesion. This invention's preparation method is safe, low-cost, and easily scalable, providing a new solution for the three-dimensional expansion of adherent cells in cultured meat.

[0010] To achieve the above objectives, the present invention specifically implements the following technical solution: a method for preparing chitosan-based edible microcarriers by low-temperature confined phase separation synergistic pH-triggered curing, comprising the following steps: (1) Preparation of oil phase dispersion system: Water-in-oil emulsifier is added to the oil phase medium and fully dissolved under stirring conditions to obtain a continuous oil phase; the mass volume concentration of water-in-oil emulsifier in the continuous oil phase is 0.5% to 8.0%, more preferably 1.0% to 5.0%. The role of the continuous oil phase is to disperse the acidic chitosan composite solution into micron-sized droplets and maintain the stability of droplet boundaries during subsequent low-temperature treatment and alkaline trigger diffusion, so as to avoid droplet co-aggregation, breakage or premature coagulation.

[0011] (2) Preparation of acidic chitosan-based mother liquor: Chitosan is added to an acidic co-solvent and stirred and dissolved at 20–60°C to obtain a clear or semi-transparent acidic chitosan solution; the concentration of the acidic co-solvent is 0.2%–3.0%, more preferably 0.5%–1.5%. The mass-volume concentration of chitosan in the acidic mother liquor is 0.5%–4.0%, more preferably 1.0%–2.5%. In this step, the amino groups on the chitosan molecular chains are protonated, and the molecular chains are fully extended and dispersed in the aqueous phase, providing a reversible response basis for subsequent pH-triggered curing.

[0012] (3) Preparation of functional composite dispersion: Plant protein, plant polysaccharide, edible fiber, cell adhesion promoting components or combinations thereof are added to the acidic chitosan-based mother liquor obtained in step (2) to form a chitosan-based composite dispersion; the total mass ratio of the plant protein, plant polysaccharide, edible fiber, and cell adhesion promoting components to chitosan is 0.05:1 to 2.0:1, more preferably 0.1:1 to 1.0:1. In this step, plant protein is not the only innovation, but is introduced as a cell adhesion and nutritional property regulating component to improve the problem of insufficient support for cell adhesion by chitosan alone. Among them, the plant protein is first dispersed in deionized water or weakly acidic aqueous solution, and after centrifugation at 2000 to 8000 rpm for 3 to 15 min to remove insoluble components, it is then added to the acidic chitosan mother liquor to improve the homogeneity of the composite aqueous phase.

[0013] (4) Formation of acidic composite aqueous droplets: The chitosan-based composite dispersion from step (3) is added to the continuous oil phase from step (1), and stirred and emulsified at 200–1200 rpm for 5–90 min to disperse the composite aqueous phase into acidic microdroplets. Preferably, the stirring speed is 300–700 rpm and the emulsification time is 15–60 min. The droplet size can be adjusted by controlling the volume ratio of the aqueous phase to the oil phase, the emulsifier concentration, the stirring speed, and the emulsification time, thereby adjusting the microcarrier particle size. The volume ratio of the aqueous phase to the oil phase is 1:3–1:30, more preferably 1:5–1:15. The particle size of the acidic composite aqueous droplets is preferably 50–1000 μm, more preferably 100–500 μm. The key to this step is that the chitosan composite material is first confined by the oil phase in the form of flowable droplets, rather than undergoing crosslinking or gelation first, thus enabling the acquisition of a controllable spherical precursor without the use of a crosslinking agent.

[0014] (5) Low-temperature confined phase separation treatment: The emulsion system of acidic composite aqueous droplets formed in step (4) is cooled to -30 to 10 ℃ and maintained at low temperature for 0.2 to 6 h to obtain a low-temperature emulsion system; preferably, the cooling endpoint is -25 to 0 ℃, more preferably -20 to -5 ℃; the low-temperature maintenance time is 0.5 to 4 h. During the low-temperature treatment, the viscosity of the aqueous phase inside the droplet increases, the mobility of chitosan molecular chains decreases, and plant proteins and other functional components undergo confined phase separation or micro-region enrichment inside the droplet, thereby improving the morphological stability of the droplet in the subsequent alkaline triggering process. Low-temperature confined phase separation can also slow down the rate at which the alkaline trigger enters the droplet, so that the solidification process gradually advances from the surface of the droplet to the center, avoiding instantaneous alkalization that leads to particle collapse, agglomeration or structural inhomogeneity.

[0015] (6) Alkaline trigger diffusion-induced pH gradient solidification: An alkaline trigger is added to the low-temperature emulsification system of step (5), causing the alkaline trigger to enter the acidic composite aqueous droplet from the continuous phase, inducing the chitosan in the droplet to change from a protonated dissolved state to a deprotonated gel state, thereby forming a chitosan-based composite microcarrier. The alkaline trigger is sodium hydroxide, potassium hydroxide, sodium carbonate, sodium bicarbonate, ammonia, triethanolamine, arginine, lysine, histidine, or a combination thereof; preferably an alkaline trigger acceptable for food processing or biocompatibility. The concentration of the alkaline trigger is 0.05–3.0 mol / L, more preferably 0.2–1.5 mol / L. The volume ratio of the alkaline trigger to the acidic composite aqueous phase is 0.1:1–3:1, more preferably 0.5:1–1.5:1. After adding the alkaline trigger, the system is continuously stirred at -20–30 °C for 0.5–12 h, preferably 1–5 h. In this step, the alkaline trigger does not participate in covalent cross-linking as a cross-linking agent. Instead, it adjusts the pH value inside and outside the droplet to deprotonate the chitosan amino groups, enhancing intermolecular hydrogen bonds, hydrophobic interactions, and chain entanglement, thereby forming a stable three-dimensional network. The resulting microcarriers are free of chemical cross-linking agent residues and are suitable for use in cell-cultured meat applications.

[0016] (7) Graded purification and degreasing treatment: After solidification, the emulsion system is collected by sieving, static sedimentation or low-speed centrifugation to collect microcarrier particles; then food-grade alcohol-water solution, deionized water, buffer solution or combination thereof are used for multiple rounds of cleaning to remove oil phase, emulsifier, free basic trigger, unbound protein and acidic cosolvent residue.

[0017] (8) Drying and sterilization: The purified microcarriers are stored directly in wet form, or freeze-dried, vacuum-dried or air-dried at low temperature to form dry microcarriers.

[0018] Furthermore, the water-in-oil emulsifier mentioned in step (1) is Span 80, Span 85, PGPR or a combination thereof; the oil phase medium is liquid paraffin, n-hexane, vegetable oil, medium-chain triglycerides or a combination thereof.

[0019] Furthermore, the degree of deacetylation of the chitosan in step (2) is 75% to 95%, and the viscosity-average molecular weight is 50 to 500 kDa; the acidic co-solvent is acetic acid, lactic acid, citric acid, malic acid, dilute hydrochloric acid solution or a combination thereof.

[0020] Further, in step (3), the plant protein is pumpkin seed protein, soy protein isolate, pea protein, peanut protein, sunflower seed protein, rice protein, or a combination thereof; preferably, pumpkin seed protein. The plant polysaccharide is pectin, alginate, gum arabic, xanthan gum, guar gum, dextran, carboxymethyl cellulose, or a combination thereof. The edible fiber is cellulose or its derivatives, inulin, resistant starch, lignin, or a combination thereof. The cell adhesion promoting component is one or more of the following: polypeptides containing the arginine-glycine-aspartic acid (RGD) sequence from natural or recombinant sources, collagen, gelatin, fibronectin, laminin, hyaluronic acid, and chondroitin sulfate. The mass ratio of the added plant protein, plant polysaccharide, edible fiber, and cell adhesion promoting component to chitosan is 0.05:1 to 2.0:1, more preferably 0.1:1 to 1.0:1.

[0021] Further, in step (7), the sample is first washed 1 to 5 times with a 30%–95% ethanol aqueous solution, then washed 3 to 10 times with deionized water or phosphate buffer until the pH of the washing solution is close to neutral. If necessary, a short wash of 1 to 2 times with 0.01%–0.5% food-grade nonionic surfactant can be used, followed by thorough rinsing with plenty of deionized water. The purification steps are controlled at 4–30 °C to avoid deformation of the microcarrier or excessive loss of functional components.

[0022] Furthermore, in step (8), when freeze-drying is used, the microcarriers are equilibrated in a 1%–10% solution of trehalose, sucrose, mannitol, glucose, or a combination thereof for 0.5–4 h before freeze-drying to reduce pore structure collapse during the drying process. Subsequently, they are pre-frozen at -20–-80 °C for 2–24 h, followed by freeze-drying for 12–72 h. The dried microcarriers can be treated by ultraviolet irradiation, moist heat sterilization, radiation sterilization, ethanol soaking followed by sterile water replacement, or a combination thereof. For cell-cultured meat applications, radiation sterilization or mild moist heat sterilization is preferred to reduce the impact of residual organic solvents and the sterilization process on the material structure.

[0023] The chitosan-based edible microcarriers prepared by the above method using low-temperature confined phase separation and pH-triggered curing are spherical, near-spherical, or irregularly spherical, with an average particle size of 50–1000 μm, preferably 100–500 μm; they have micropores, wrinkles, or rough structures on their surface or inside; the microcarriers are based on a deprotonated chitosan physical gel network, with plant proteins or other edible functional components distributed in the gel network as a composite phase; the microcarriers do not contain residual chemical cross-linking agents such as glutaraldehyde, epichlorohydrin, genipin, or carbodiimide cross-linking agents, nor do they rely on enzymatic cross-linking steps such as transglutaminase or tyrosinase to obtain the stability of the main structure.

[0024] This invention also provides the application of the above-mentioned chitosan-based edible microcarriers in cultured meat. The microcarriers can be used for attachment culture, three-dimensional expansion, co-culture, or subsequent tissue processing of stem cells, muscle stem cells, adipose-derived stem cells, mesenchymal-like cells, fibroblast-like cells, or tissue cells derived from fish, poultry, livestock, or other edible animals. Preferably, the microcarriers are used for the three-dimensional expansion of fish muscle stem cells or adipose-derived stem cells. Since the microcarriers themselves are composed of edible materials and the solidification process does not introduce chemical cross-linking agents, they can be used together with cells as raw materials for cultured meat in subsequent processing steps after culture, thereby reducing the complexity of cell-scaffold separation.

[0025] Compared with the prior art, the present invention has the following beneficial effects.

[0026] (1) The core innovation of this invention lies in the pH-driven non-crosslinking curing process, rather than simply replacing a certain plant protein or natural polymer. By forming acidic chitosan droplets in the oil phase and then inducing pH gradient curing through diffusion with an alkaline trigger, the microcarriers are formed and stabilized in one step, avoiding the use of chemical crosslinking agents or enzyme crosslinking agents.

[0027] (2) The present invention improves droplet stability and curing uniformity through a low-temperature confined phase separation process. The low-temperature treatment enables acidic composite droplets to obtain higher viscoelasticity and phase separation microstructure before curing, which can reduce droplet breakage, adhesion and collapse during alkaline triggering process, and improve the sphericity and particle size controllability of microcarriers.

[0028] (3) The present invention forms a solidified interface that progresses from the outside to the inside through diffusion control of an alkaline trigger. The solidification process is mild, adjustable, and does not rely on covalent crosslinking. By adjusting the type, concentration, addition method, temperature, and stirring time of the alkaline trigger, the mechanical stability, pore structure, swelling behavior, and cell culture performance of the microcarrier can be adjusted.

[0029] (4) The microcarriers prepared by this invention have good food safety properties. Since the main structure is derived from the chitosan pH deprotonated physical gel network, it does not require chemical cross-linking agents such as glutaraldehyde, epichlorohydrin, and genipin, nor does it require high-cost enzymatic cross-linking steps, thus making it more suitable for cell culture meat raw material systems.

[0030] (5) The preparation process of this invention is simple and suitable for scale-up. The whole process mainly includes emulsification and dispersion, low temperature treatment, alkaline triggering solidification and washing and purification. The equipment requirements are low, the parameters can be scaled up, and it is easy to carry out large-scale production through a stirred emulsification reactor, an online alkali addition device, a low temperature circulation system and a continuous screening and washing equipment. Attached Figure Description

[0031] Figure 1Images of the two microcarriers in Examples 1 and 2 are shown below. (a) is an optical microscope image of the pumpkin seed protein-chitosan composite edible microcarrier (denoted as PSP-CS MC); (b) is an optical microscope image of the chitosan pH-cured microcarrier (denoted as CS MC) without the addition of pumpkin seed protein; (c) is a particle size distribution map of the pumpkin seed protein-chitosan composite edible microcarrier; and (d) is an infrared spectrum of the two microcarriers.

[0032] Figure 2 Images of chitosan-based composite microcarriers prepared with different alkaline triggers and concentrations in Examples 3 and 4 are shown. Among them, (a), (b), and (c) are optical microscope images of chitosan-based composite microcarriers prepared with 100 mL of 0.5 mol / L, 1.0 mol / L, and 1.5 mol / L sodium hydroxide solutions as alkaline triggers, respectively; and (d) is an optical microscope image of chitosan-based composite microcarriers prepared with 50 mL of 1.0 mol / L sodium bicarbonate solution as an alkaline trigger.

[0033] Figure 3 These are SEM images of composite microcarriers with different pore structures prepared by adjusting the phase separation temperature in Example 5; wherein, the phase separation temperatures in (a), (b), (c), and (d) are 0 ℃, -10 ℃, -20 ℃, and -30 ℃, respectively.

[0034] Figure 4 These are microscopic images of chitosan-based composite microcarriers prepared under pH-driven conditions with plant protein type substitution in Example 6; where (a), (b), (c), and (d) are respectively edible microcarriers of pumpkin seed protein-chitosan composite (denoted as PSP-CS MC), edible microcarriers of pea protein-chitosan composite (denoted as PPI-CS MC), edible microcarriers of soybean protein-chitosan composite (denoted as SPI-CS MC), and edible microcarriers of chickpea protein-chitosan composite (denoted as CPI-CS MC).

[0035] Figure 5 This is the particle size distribution diagram in Example 6; wherein, pumpkin seed protein-chitosan composite edible microcarrier (denoted as PSP-CS MC), pea protein-chitosan composite edible microcarrier (denoted as PPI-CS MC), soybean protein-chitosan composite edible microcarrier (denoted as SPI-CS MC), and chickpea protein-chitosan composite edible microcarrier (denoted as CPI-CSMC).

[0036] Figure 6This refers to the biocompatibility verification results in Example 6; among them, pumpkin seed protein-chitosan composite edible microcarrier (denoted as PSP-CS MC), pea protein-chitosan composite edible microcarrier (denoted as PPI-CS MC), soybean protein-chitosan composite edible microcarrier (denoted as SPI-CS MC), and chickpea protein-chitosan composite edible microcarrier (denoted as CPI-CS MC).

[0037] Figure 7 This is a magnified microscope image of the prepared product of the chitosan-based composite microcarrier in Example 7.

[0038] Figure 8 This is an image showing the effect of chitosan-based composite microcarriers used in the three-dimensional culture of fish muscle stem cells in Example 8; wherein, the edible microcarrier is a pumpkin seed protein-chitosan composite (denoted as PSP-CS MC), and the chitosan pH-fixed microcarrier without added pumpkin seed protein (denoted as CS MC).

[0039] Figure 9 These are the absorbance values ​​of the chitosan-based composite microcarriers in Example 8; wherein, the pumpkin seed protein-chitosan composite edible microcarrier (denoted as PSP-CS MC) and the chitosan pH-cured microcarrier without added pumpkin seed protein (denoted as CSMC).

[0040] Figure 10 These are microscopic images of chitosan microcarriers cured at room temperature pH without low-temperature confinement treatment, as described in Comparative Example 1. (a) shows emulsified acidic droplets, and (b) shows an optical microscopic image of the microcarriers prepared by direct curing at room temperature.

[0041] Figure 11 Images of chitosan particles prepared by direct mixing and precipitation of alkaline solution in Comparative Example 2 are shown. (a) is a schematic diagram of the operation; (b), (c), and (d) are microscopic images of the chitosan particles.

[0042] Figure 12 The images are of the microcarriers mentioned in Comparative Example 3. (a) and (b) are scanning electron microscope images of edible chitosan microcarriers prepared by low-temperature confined phase separation and pH curing and chitosan microcarriers prepared by crosslinking agent, respectively. (c) and (d) are microscope images of edible chitosan microcarriers prepared by low-temperature confined phase separation and pH curing and chitosan microcarriers prepared by crosslinking agent, respectively.

[0043] Figure 13 This is a microscopic image of a conventional emulsified microcarrier in Comparative Example 4, in which only plant protein was used to replace the complex components.

[0044] Figure 14The images are microscopic and scanning electron microscope (SEM) images of the microcarriers prepared by Comparative Example 5 using only three steps: preparation of the oil phase, preparation of the aqueous phase of the chitosan composite solution, and phase separation. (a) and (b) are optical microscopic and SEM images of the microcarriers prepared by only three steps: preparation of the oil phase, preparation of the aqueous phase of the chitosan composite solution, and phase separation, respectively.

[0045] Figure 15 Examples 1 and 3, 4 show the effect of using the microcarriers described in these examples for three-dimensional culture of fish muscle stem cells. E1 is a pumpkin seed protein-chitosan composite edible microcarrier prepared by low-temperature confinement phase separation and pH gradient solidification. CE1 is a room-temperature pH solidified microcarrier without low-temperature confinement treatment. CE3 is a chitosan composite microcarrier prepared using a chemical cross-linking agent. CE4 is a conventional emulsified microcarrier in which only plant protein is used to replace the composite components. CE5 is a microcarrier prepared by only three steps: preparing an oil phase, a chitosan composite solution aqueous phase, and phase separation.

[0046] Figure 16 The results are the absorbance values ​​of the microcarriers described in Example 1 and Comparative Examples 1, 3, and 4 for three-dimensional culture of fish muscle stem cells; E1 is the pumpkin seed protein-chitosan composite edible microcarrier prepared by low-temperature confinement phase separation and pH gradient solidification; CE1 is the room temperature pH solidified microcarrier without low-temperature confinement treatment; CE3 is the chitosan composite microcarrier prepared using a chemical crosslinking agent; CE4 is the conventional emulsified microcarrier in which only plant protein is replaced with the composite components; and CE5 is the microcarrier prepared by only three steps: preparing the oil phase, the aqueous phase of the chitosan composite solution, and phase separation.

[0047] Figure 17 The results show the cell adhesion rates of the microcarriers described in Example 1 and Comparative Examples 1, 3, and 4 for three-dimensional culture of fish muscle stem cells; E1 is the edible pumpkin seed protein-chitosan composite microcarrier prepared by low-temperature confinement phase separation and pH gradient solidification; CE1 is the room-temperature pH solidified microcarrier without low-temperature confinement treatment; CE3 is the chitosan composite microcarrier prepared using a chemical crosslinking agent; CE4 is the conventional emulsified microcarrier in which only plant protein is replaced with the composite components; and CE5 is the microcarrier prepared by only three steps: preparing the oil phase, the aqueous phase of the chitosan composite solution, and phase separation.

[0048] Figure 18The results are for the cell density of the microcarriers described in Example 1 and Comparative Examples 1, 3, and 4 for three-dimensional culture of fish muscle stem cells; E1 is a pumpkin seed protein-chitosan composite edible microcarrier prepared by low-temperature confinement phase separation and pH gradient solidification; CE1 is a room-temperature pH solidified microcarrier without low-temperature confinement treatment; CE3 is a chitosan composite microcarrier prepared using a chemical cross-linking agent; CE4 is a conventional emulsified microcarrier in which only plant protein is replaced with the composite components; and CE5 is a microcarrier prepared by only three steps: preparing an oil phase, a chitosan composite solution aqueous phase, and phase separation.

[0049] Figure 19 The images show the microcarrier preparations with the water-to-oil volume ratio adjusted in Comparative Example 6. (a), (b), and (c) are images of the emulsion droplet state when the water-to-oil volume ratio is 1:5, 1:10, and 1:20, respectively. (d) is a microcarrier particle size diagram under the three water-to-oil volume ratio conditions.

[0050] Figure 20 The images show the microcarrier preparations with adjusted emulsifier concentrations in Comparative Example 6. (a) is an optical image of the product prepared when the emulsifier concentration is 0.3%, and (b) is a microcarrier particle size diagram under three emulsifier concentration conditions.

[0051] Figure 21 The images show the microcarrier preparations under different stirring speeds in Comparative Example 6. (a) is an optical image of the product prepared at a stirring speed of 1200 rpm, and (b) is a microcarrier particle size diagram under three stirring speed conditions.

[0052] Figure 22 The images show the microcarrier preparations with adjusted emulsification time in Comparative Example 6. (a), (b), and (c) are images of the state of the emulsion droplets when the emulsification time is 3 min, 60 min, and 90 min, respectively. (d) is a microcarrier particle size diagram under the three emulsification time conditions. Detailed Implementation

[0053] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention. Unless otherwise specified, the reagents, methods, and instruments used in the present invention are conventional reagents, methods, and instruments in this technical field.

[0054] Unless otherwise specified, the reagents and materials used in the following examples are all commercially available, and the experimental methods and detection methods involved are all conventional experimental methods and detection methods that are already in the prior art.

[0055] Example 1: Preparation of pumpkin seed protein-chitosan composite edible microcarrier by pH gradient solidification.

[0056] This embodiment illustrates a method for preparing microcarriers using pumpkin seed protein as the functional composite phase and chitosan pH-driven solidification as the main spheroidization mechanism.

[0057] Weigh 10 g of Span 80 and add it to 400 mL of n-hexane. Stir at 600 rpm for 20 min at room temperature to fully dissolve the emulsifier and obtain a water-in-oil continuous oil phase. Liquid paraffin or food-grade vegetable oil can also be used instead of n-hexane; when using vegetable oil, the amount of emulsifier can be appropriately increased to 12-18 g to ensure the stability of droplet dispersion.

[0058] Weigh 1.0 g of chitosan and add it to 50 mL of 0.5% acetic acid aqueous solution. Stir at 35 °C for 4 h until a uniform, transparent or translucent acidic chitosan mother liquor is formed. The degree of deacetylation of the chitosan used should be 85%–95%, and the viscosity-average molecular weight should be 100–300 kDa. If complete dissolution of chitosan is difficult, the temperature can be raised to 45 °C, but should not exceed 60 °C to avoid material degradation or abnormal viscosity changes.

[0059] Separately weigh 1.0 g of pumpkin seed protein powder and add it to 25 mL of deionized water. Stir at room temperature for 30 min to ensure thorough dispersion. Then centrifuge at 5000 rpm for 10 min to remove insoluble coarse particles, and use the supernatant as the pumpkin seed protein dispersion. Slowly add the pumpkin seed protein dispersion to the acidic chitosan mother liquor and stir at 300 rpm for 20 min to obtain a pumpkin seed protein-chitosan acidic composite aqueous phase. In this composite aqueous phase, chitosan is in a protonated dissolved state, and pumpkin seed protein, as a functional nutrient and cell adhesion aid, is uniformly dispersed in the aqueous phase.

[0060] The acidic composite aqueous phase was slowly added dropwise to the prepared continuous oil phase, maintaining a stirring speed of 400 rpm during the addition. After the addition was complete, stirring was continued for 30 minutes to allow the acidic composite aqueous phase to form stable micron-sized droplets in the oil phase. No crosslinking agents or alkaline triggers were added during this stage to maintain the acidic flow state of the aqueous droplets. If droplet co-aggregation occurred during emulsification, the Span 80 concentration could be appropriately increased or the stirring speed could be increased to 450–500 rpm; if the particle size was too small, the stirring speed could be reduced to 300–350 rpm.

[0061] After emulsification, a low-temperature circulation system was activated to slowly cool the emulsion system to -15°C at a rate of approximately 1–3 °C / min, and stirring was continued at -15°C for 3.5 h. During the low-temperature treatment, the acidic composite droplets were confined by the oil phase, the viscosity of the aqueous phase inside the droplets increased, the movement of chitosan molecular chains was restricted, and pumpkin seed protein formed micro-regions within the droplets, which is beneficial for the formation of a more stable particle structure during subsequent solidification. This low-temperature stage is an important process step in this invention, unlike conventional preparation methods that involve direct crosslinking or direct precipitation after room-temperature emulsification.

[0062] After the cryogenic confinement treatment, 50 mL of 1.0 mol / L sodium hydroxide solution was slowly added to the emulsion system. The preferred method of addition was dropwise or in batches, with the addition time controlled between 5 and 30 minutes to avoid sudden pH changes that could cause instantaneous hardening of the microsphere surface, insufficient internal solidification, or particle adhesion. After adding the sodium hydroxide solution, stirring was continued at -15 to 10°C for 1 hour to allow the alkaline trigger to gradually enter the acidic composite droplets. The refrigeration system was then turned off, and the system was slowly raised to room temperature, with stirring continuing for 1.5 hours. As the alkaline trigger diffused into the droplets, the pH value inside the droplets gradually increased. The protonated amino groups on the chitosan molecular chains gradually deprotonated, and intermolecular hydrogen bonds, hydrophobic interactions, and chain entanglement were enhanced. The acidic droplets gelled from the outside in, ultimately forming a pumpkin seed protein-chitosan composite microcarrier. The curing process is essentially a pH-driven physical gelation process, which does not involve chemical cross-linking agents such as glutaraldehyde, genipin, epichlorohydrin, or carbodiimide, nor does it rely on enzymatic cross-linking agents such as transglutaminase.

[0063] After solidification, the emulsion system was poured onto a 100 μm sieve for wet sieving to collect the microcarrier particles. The particles were first washed three times with 75% ethanol aqueous solution for 5 min each time to remove most of the oil phase and emulsifier; then washed twice with 50% ethanol aqueous solution, followed by six washes with plenty of deionized water until the pH of the washing solution was close to 7.0–7.5. If necessary, a short wash of 0.05% Tween 80 aqueous solution for 3 min could be added after the first ethanol wash to enhance the oil removal effect, but this must be followed by thorough replacement with deionized water to avoid surfactant residue. The final morphology of the wet pumpkin seed protein-chitosan composite microcarrier is shown in the attached figure. Figure 1 As shown in Figure (a), by attaching Figure 1 Fourier transform infrared spectroscopy analysis of (d) in the figure shows that the pumpkin seed protein complex is effective.

[0064] The wet microcarriers were equilibrated in a 5% trehalose solution for 1 h, then pre-frozen at -40 °C for 12 h, and finally freeze-dried for 36 h to obtain dry composite microcarriers. Before use, they can be treated with UV irradiation for 30 min, soaking in 75% ethanol followed by sterile water replacement, or irradiation sterilization. The resulting microcarriers, after rehydration, are spherical or near-spherical, possessing a certain degree of surface roughness and internal porous structure, and can be used for the three-dimensional expansion of adherent cells in cultured meat.

[0065] Example 2: Preparation of chitosan pH-cured microcarriers without the addition of pumpkin seed protein.

[0066] This embodiment is used to illustrate that the main spheroidization mechanism of the present invention comes from the pH-driven solidification of chitosan, rather than necessarily relying on pumpkin seed protein.

[0067] Weigh 9 g of Span 80 and add it to 300 mL of n-hexane, stirring to form a continuous oil phase. Weigh 0.5 g of chitosan and add it to 50 mL of 0.5% acetic acid aqueous solution, stirring at 35 °C until completely dissolved to obtain an acidic chitosan solution. Add the acidic chitosan solution to the continuous oil phase and emulsify at 550 rpm for 30 min to form acidic chitosan droplets. Then cool the system to -25 °C and maintain for 3.5 h. Afterward, slowly add 50 mL of 1.5 mol / L sodium hydroxide solution and continue stirring for 3 h to allow the acidic chitosan droplets to undergo pH-triggered solidification.

[0068] After solidification, the mixture was sieved, washed with ethanol and deionized water, and freeze-dried to obtain chitosan pH-cured microcarriers, as shown in the attached image. Figure 1 As shown in Figure (b), this microcarrier can form stable particles, but its cell adhesion support and surface functionality may be weaker compared to microcarriers composed of pumpkin seed protein. This embodiment demonstrates that pumpkin seed protein in this invention is mainly used to improve cell adhesion and nutritional properties, rather than being a necessary condition for achieving microcarrier sphericity and solidification; the key to microcarrier formation lies in the pH gradient solidification process induced by the diffusion of acidic chitosan droplets through an alkaline trigger.

[0069] Example 3: Preparation of chitosan-based composite microcarriers with different degrees of curing by adjusting the concentration of alkaline triggering agent.

[0070] This embodiment illustrates how adjusting the curing degree and structural stability of microcarriers can be achieved by controlling the concentration and addition method of the alkaline trigger agent.

[0071] Weigh 18 g of Span 80 and add it to 800 mL of liquid paraffin. Stir at 700 rpm for 30 min to fully dissolve it, obtaining a continuous oil phase. Weigh 1.2 g of chitosan and add it to 100 mL of 1.0% acetic acid aqueous solution. Stir at 40 ℃ for 5 h to obtain an acidic chitosan mother liquor. Weigh 1.5 g of pumpkin seed protein and disperse it in 40 mL of deionized water. Centrifuge at 5000 rpm for 10 min, collect the supernatant, and add it to the acidic chitosan mother liquor. Stir until homogeneous to obtain a composite aqueous phase.

[0072] The composite aqueous phase was added to the continuous oil phase and stirred at 450 rpm for 25 min to form acidic composite droplets. The system was then cooled to -20 °C and maintained at this temperature for 3 h. After the low-temperature maintenance, the system was divided into three groups, and 100 mL of 0.5 mol / L, 1.0 mol / L, and 1.5 mol / L sodium hydroxide solutions were added to each group, respectively. All three groups were added dropwise over a period of 20 min. After the addition was complete, the system was stirred at -20 °C for 1 h, and then at room temperature for 2 h.

[0073] After solidification, the microcarriers were sieved, washed with ethanol and deionized water, freeze-dried, and sterilized according to the method in Example 1. The obtained microcarriers are shown in the attached figure. Figure 2 Figures (a), (b), and (c) show the results. The results indicate that at lower concentrations of the alkaline trigger, the microcarriers solidify more gently, resulting in softer particles and a higher degree of swelling after rehydration. Higher concentrations of the alkaline trigger increase the degree of chitosan deprotonation and improve the structural stability of the microcarriers; however, excessively high concentrations may lead to rapid surface solidification, insufficient internal pH adjustment, or particle adhesion. Therefore, the optimal concentration of the alkaline trigger is controlled within the range of 0.5–1.5 mol / L, which is more suitable for obtaining composite microcarriers that combine sphericity, structural stability, and suitability for cell culture.

[0074] Example 4: Preparation of edible chitosan-based composite microcarriers using food-grade weak base triggering agents.

[0075] This embodiment illustrates that food-grade or more biocompatible weakly alkaline triggering agents can be used to replace strong alkaline solutions to further improve the safety properties of microcarriers for food applications.

[0076] Weigh 12 g of Span 80 and add it to 350 mL of food-grade medium-chain triglycerides. Stir at 600 rpm for 20 min at room temperature to obtain a continuous oil phase. Weigh 0.75 g of chitosan and add it to 50 mL of 1.0% lactic acid aqueous solution. Stir at 40℃ for 4 h to obtain a chitosan-lactic acid solution. Weigh 0.5 g of pumpkin seed protein and disperse it in 15 mL of deionized water. After centrifugation to remove insoluble matter, add the supernatant to the chitosan-lactic acid solution to obtain an acidic composite aqueous phase.

[0077] The acidic aqueous composite phase was added to the continuous oil phase and stirred at 350 rpm for 50 min to form acidic droplets with larger particle sizes. The emulsion system was then cooled to -25 °C and maintained for 3.5 h. After the low-temperature treatment, 50 mL of 1.0 mol / L sodium bicarbonate solution was added to the system, with the addition time controlled at 30 min. Since sodium bicarbonate is less basic than sodium hydroxide, its pH increase after entering the droplets is more gradual. Therefore, after the addition, stirring was continued at -10 to 10 °C for 2 h, followed by stirring at room temperature for 3 h to ensure sufficient deprotonation and solidification within the droplets.

[0078] After solidification, the system was sieved to collect the microcarriers. The morphology of the sieved microcarriers is shown in the attached figure. Figure 2 As shown in Figure (d), the mixture was first washed three times with a 50% ethanol aqueous solution, then washed eight times with deionized water until the pH of the washing solution was neutral. Subsequently, freeze-drying was performed. Compared to a strong base-triggered system, a weak base-triggered system has a slower solidification rate and a softer particle structure, making it suitable for applications with high food safety requirements or stricter control over residual risks during cell culture. This embodiment illustrates that the key to this invention is not limited to a single alkaline substance, but rather lies in the process mechanism of using an alkaline trigger to diffuse into acidic chitosan droplets, initiating a pH gradient solidification process.

[0079] Example 5: Preparation of composite microcarriers with different pore structures by controlling the low-temperature phase separation temperature.

[0080] This embodiment illustrates the effect of low-temperature confined phase separation temperature on the stability of the pore structure and morphology of microcarriers.

[0081] Pumpkin seed protein-chitosan acidic composite aqueous phase and continuous oil phase were prepared according to the method in Example 1. After adding the composite aqueous phase to the continuous oil phase, emulsification was carried out at 420 rpm for 40 min to obtain stable acidic composite droplets. The emulsion system was then divided into four groups, cooled to 0 °C, -10 °C, -20 °C, and -30 °C respectively, and maintained at these temperatures for 3 h. Then, an equal volume of 1.0 mol / L sodium hydroxide solution was added to each group, and pH-triggered curing was performed under the same stirring conditions.

[0082] After curing, microcarriers were obtained under different low-temperature treatment conditions through sieving, washing, and freeze-drying. The freeze-dried microcarriers were then sputter-coated with gold, and the surface pore structure was observed using scanning electron microscopy. The results are attached. Figure 3 As shown in the figures, the phase separation temperatures in diagrams (a), (b), (c), and (d) are 0 °C, -10 °C, -20 °C, and -30 °C, respectively. The 0 °C treatment group exhibits a lower degree of phase separation within the droplets, resulting in a relatively dense internal structure of the microcarrier after solidification. The -10 °C to -20 °C treatment groups can form a more uniform surface roughness structure and internal pores, which is beneficial for cell adhesion and nutrient exchange. If the cooling rate is too rapid in the -30 °C treatment group, some droplets may experience ice crystal-induced structural rupture or non-uniform particle surfaces. Therefore, the preferred low-temperature confined phase separation temperature is -25 to 0 °C, more preferably -20 to -5 °C. This embodiment illustrates that the low-temperature process is not a simple cooling step, but rather a structural control step coupled with subsequent pH-curing, which can be used to adjust the porosity, surface morphology, and rehydration stability of the microcarrier.

[0083] Example 6: Preparation of pH-driven composite microcarriers under plant protein type substitution conditions.

[0084] This embodiment illustrates that the present invention is applicable to different plant protein composite systems, but the core is still the pH-driven non-crosslinking curing process.

[0085] Weigh out 0.5 g each of pumpkin seed protein, soy protein isolate, pea protein, and chickpea protein, and disperse them in 20 mL of deionized water. Centrifuge at 5000 rpm for 10 min and collect the supernatant. Weigh out 1.0 g of chitosan, dissolve it in 50 mL of 1.0% acetic acid aqueous solution, divide it into four equal parts, and add the above four plant protein dispersions to each part to obtain four groups of plant protein-chitosan acidic aqueous complexes.

[0086] Each composite aqueous phase was added to a continuous oil phase containing Span 80 and emulsified at 400 rpm for 30 min, then cooled to -15 °C and maintained for 3 h. Subsequently, 1.0 mol / L sodium hydroxide solution was added for pH-triggered curing, which lasted for 3 h. After curing, the mixtures were sieved, washed, and dried.

[0087] Appendix Figure 4 Figures (a), (b), (c), and (d) show edible microcarriers made from pumpkin seed protein and chitosan, pea protein and chitosan, soybean protein and chitosan, respectively. Figures 4-6 The results showed that the four plant protein composite microcarriers had a concentrated particle size distribution and good biocompatibility. Different plant proteins could be introduced into the chitosan pH-curing system as composite functional phases. However, the solubility, isoelectric point, hydrophobicity, and particle dispersion of different proteins affected the surface morphology of the microcarriers and thus the cell adhesion effect. Pumpkin seed protein, due to its good dispersibility and cell affinity potential, was suitable as a preferred composite component. This embodiment further illustrates that the present invention is not based on "pumpkin seed protein as the sole material selection," but rather on a holistic technical solution combining plant protein-adjustable composite water with a pH gradient curing process.

[0088] Example 7: Scale-up preparation of chitosan-based composite microcarriers.

[0089] This embodiment illustrates that the method of the present invention is suitable for transformation from laboratory-scale testing to large-scale preparation.

[0090] 6 L of food-grade vegetable oil and 180 g of Span 80 were added to a 10 L jacketed stirred reactor and stirred at 300 rpm for 30 min at 25 °C to obtain a continuous oil phase. Separately, 120 g of chitosan was added to 1.5 L of a 1.0% acetic acid aqueous solution in an aqueous phase preparation tank and stirred at 40 °C for 6 h to obtain an acidic chitosan mother liquor. 60 g of pumpkin seed protein was dispersed in 0.5 L of deionized water, and after centrifugation or filtration to remove insoluble matter, it was mixed with the acidic chitosan mother liquor to obtain a composite aqueous phase.

[0091] The composite aqueous phase was added to the oil phase in the reactor using a peristaltic pump at a rate of 100–300 mL / min, while the stirring speed in the reactor was controlled at 250–500 rpm. After the composite aqueous phase was added, emulsification continued for 40 min. Subsequently, the jacketed low-temperature circulation was started to cool the system to -10 °C and maintain this temperature for 4 h. Then, 6 L of 1.0 mol / L sodium carbonate or sodium hydroxide solution was slowly added using a metering pump over a period of 60–120 min. After the alkaline trigger was added, the system was stirred at low temperature for 1 h, and then heated to room temperature and stirred for 3 h to allow the composite droplets to fully solidify.

[0092] After solidification, the reaction system was separated by a continuous sieve to collect the microcarriers, and then sequentially washed with 70% ethanol aqueous solution, 50% ethanol aqueous solution, and deionized water. Washing continued until the effluent pH reached 7.0–7.5 and the oil phase residue was significantly reduced. The wet microcarriers were then collected. The morphology of the product is shown in the attached figure. Figure 7 As shown, the wet microcarriers can be used directly for cell culture, or they can be freeze-dried after equilibration with a 5% trehalose solution. This embodiment demonstrates that the process of the present invention does not rely on complex cross-linking reaction equipment, and is mainly achieved through emulsification, low temperature, alkali addition, and washing, facilitating continuous or semi-continuous scale-up.

[0093] Example 8: Chitosan-based composite microcarriers for three-dimensional culture of fish muscle stem cells.

[0094] This embodiment illustrates that the microcarriers prepared in this invention can be used for three-dimensional attachment and amplification of meat-related cells in cell culture.

[0095] The pumpkin seed protein-chitosan composite microcarrier prepared in Example 1 was sterilized, washed three times with sterile PBS, and then pre-wetted with complete culture medium for 12 h. The microcarrier was added to a cell culture flask or stirring flask at a concentration of 1–5 mg / mL. Fish muscle stem cells were digested into a single-cell suspension and cultured at a concentration of 2 × 10⁻⁶ cells / mL. 4 ~1×10 5 Cells were seeded at a density of cells / mL into a culture system containing microcarriers. In the initial stage of seeding, intermittent stirring was used to promote uniform cell adhesion, i.e., stirring for 3-5 min and then letting stand for 20-30 min, repeated 6-10 times; then the culture was switched to continuous low-speed stirring at 30-60 rpm.

[0096] During the culture process, half of the culture medium was replaced every 48 hours. Cell adhesion and proliferation on the microcarriers were evaluated by microscopic observation, live / dead cell staining, and CCK-8 quantitative assay. Results are attached. Figure 8 and attached Figure 9As shown, the composite microcarriers provide a three-dimensional adhesion interface for cells, allowing cells to attach and proliferate on the microcarrier surface and around its pore structure. Compared to chitosan-based microcarriers alone, the microcarriers combined with plant proteins are more effective in improving initial cell adhesion. After culture, the microcarriers can be collected along with the cells and used as raw materials for subsequent texturization or food processing of cultured meat, thereby reducing the need for scaffold removal.

[0097] Comparative Example 1: Room temperature pH-cured microcarriers without low-temperature confinement treatment.

[0098] To illustrate the role of the low-temperature confined phase separation step, acidic composite droplets were prepared according to the formulation in Example 1. However, after emulsification, no low-temperature treatment was performed; instead, a 1.0 mol / L sodium hydroxide solution was directly added at room temperature for solidification. The remaining washing and drying steps were the same. (See attached image) Figure 10 Figure (a) shows acidic composite droplets, and Figure (b) shows microcarriers prepared by pH curing at room temperature.

[0099] Compared to Example 1, the room-temperature direct curing group was more prone to localized rapid solidification of droplets, particle adhesion, and morphological inhomogeneity during alkali addition. Some particles exhibited insufficient surface roughness or inhomogeneous internal structures. This indicates that the low-temperature confined phase separation step can reduce droplet flowability, improve the diffusion process of the alkali trigger, and make the pH curing interface more stable, thereby improving the microcarrier spheroid quality and structural uniformity. The cell experiment procedures were consistent with those in Example 8, and the results are attached. Figure 15-18 As shown, the room temperature pH solidified microcarrier (labeled CE1) without low-temperature confinement treatment had lower cell adhesion rate and proliferation trend than the low-temperature confinement pH solidified pumpkin seed protein-chitosan composite edible microcarrier (labeled E1).

[0100] Comparative Example 2: Chitosan particles were prepared by direct mixing and precipitation of alkaline solution.

[0101] To illustrate the importance of confined droplet formation in the oil phase, an acidic chitosan solution was directly added dropwise to an aqueous sodium hydroxide solution, causing the chitosan to precipitate and gel. The results are shown in the attached figure. Figure 11 As shown, this method can form chitosan gel particles, but the particles have irregular morphology, wide particle size distribution, and are prone to adhesion. Furthermore, it is difficult to form a uniform microcarrier structure suitable for three-dimensional cell culture, making it unsuitable for cell culture. Compared to this invention, direct aqueous precipitation lacks an oil-phase confined droplet template and also lacks low-temperature phase separation and pH gradient diffusion processes, thus making it difficult to obtain edible microcarriers with controllable particle size and stable morphology.

[0102] Comparative Example 3: Chitosan composite microcarriers were prepared using chemical cross-linking agents.

[0103] To illustrate the advantages of the crosslink-free curing process of this invention, a chemical crosslinking agent was added to the chitosan composite aqueous phase for crosslinking and curing according to conventional methods. (See attached document.) Figure 12 (a) and (b) are scanning electron microscope images of edible chitosan microcarriers prepared by low-temperature confined phase separation and pH curing, and chitosan microcarriers prepared by crosslinking agent, respectively; (c) and (d) are microscope images of edible chitosan microcarriers prepared by low-temperature confined phase separation and pH curing, and chitosan microcarriers prepared by crosslinking agent, respectively.

[0104] Biocompatibility verification and cell proliferation experiments were conducted on chitosan composite microcarriers (labeled CE3) prepared with chemical cross-linking agents. Figure 15-18 The results showed that chemical cross-linking can improve the stability of particle structure, but there is a risk of cross-linking agent residue. Under the same experimental conditions, the cell viability, adhesion rate, and proliferation trend of the CE3 group were lower than those of the group without cross-linking agent, requiring more stringent washing, residue detection, and food safety evaluation in the future. Compared with this comparative example, this invention achieves physical solidification through pH deprotonation of chitosan without introducing chemical cross-linking agents, making it more suitable for the preparation of edible scaffold materials for cell cultured meat.

[0105] Comparative Example 4: Conventional emulsified microcarriers in which only plant protein was replaced with plant protein as the composite component.

[0106] To demonstrate that this invention is not simply based on the substitution of pumpkin seed protein for other edible microcarriers, conventional emulsification-crosslinking methods were used to prepare microcarriers (labeled CE4) with pumpkin seed protein as the main matrix material. (See attached image.) Figure 13 Microscopic images of the microcarriers show that, compared to the preparation of chitosan-based edible microcarriers by low-temperature confined phase separation and pH-triggered curing, microcarriers prepared by conventional emulsification-crosslinking methods suffer from poor uniformity, structural collapse, and poor dispersibility. This type of microcarrier morphology is not conducive to cell culture. (See attached image) Figure 15-18 The key technical focus of conventional emulsification-crosslinking methods lies in the plant protein material itself and the crosslinking stabilization process, while the key technical focus of this invention lies in the pH gradient-based crosslink-free curing of acidic chitosan droplets through low-temperature confined phase separation and diffusion with an alkaline trigger. The two methods differ in their spheroidization mechanism, curing method, and key process control points. The pumpkin seed protein of this invention exists only as a functional composite phase and should not be simply understood as "replacing existing microcarrier materials with pumpkin seed protein."

[0107] Comparative Example 5: Microcarriers were prepared by only three steps: preparing the oil phase, the aqueous phase of the chitosan composite solution, and phase separation.

[0108] To illustrate the importance of the subsequent low-temperature confined phase separation synergistic pH-triggered curing steps in this invention, only three steps were used to prepare the microcarrier (labeled CE5): oil phase, chitosan composite solution aqueous phase, and phase separation. Low-temperature induced phase separation can control the separation scale between the polymer-enriched phase and the oil phase, thereby forming interconnected pores. The pH-triggered curing step can promote the gelation of the chitosan material and "lock in" the pore structure and overall structure formed by phase separation. Removing these steps results in the chitosan-based microcarrier exhibiting... Figure 14 The shape shown is uncontrollable, and the pore structure is missing or out of control, with problems such as agglomeration and clumping. Compared with the pH gradient solidification method for preparing chitosan composite edible microcarriers obtained in Example 1, this method, which only involves three steps—preparing the oil phase, the aqueous phase of the chitosan composite solution, and phase separation—is not only difficult to replicate batch by batch, but also unfavorable for subsequent cell culture (see appendix). Figure 15-18 ).

[0109] Comparative Example 6: Microcarriers were prepared by adjusting the volume ratio of the aqueous phase to the oil phase, the concentration of the emulsifier, the stirring speed, and the emulsification time.

[0110] To illustrate the specific effects of four factors—the volume ratio of aqueous to oil phase, emulsifier concentration, stirring speed, and emulsification time—on the morphology and particle size of microcarriers, a single-factor experimental method was employed. The volume ratios of aqueous to oil phase were set at 1:5, 1:10, and 1:20 to cover different emulsion systems ranging from oil-dominant to relatively increased aqueous phase concentration. The emulsifier concentrations in the continuous oil phase were set at 0.3%, 4%, and 8% to investigate the stabilizing effect of the emulsifier on the droplet interface under low, medium, and high concentration gradients. Stirring speeds of 150 rpm, 700 rpm, and 1200 rpm were selected to represent low, medium, and high shear conditions, respectively. Emulsification times were set at 3 min, 60 min, and 90 min to examine the effects of short-time emulsification, full emulsification, and long-time emulsification on the droplet breakup and coalescence equilibrium. In all the above experiments, except for the single factor being investigated, all other operating conditions and steps remained constant, and corresponding microcarriers were prepared accordingly.

[0111] The experimental results are attached. Figure 19 As shown, when the volume ratio of the aqueous phase to the oil phase is 1:5, the droplets exhibit poor stability and tend to merge, resulting in a larger average particle size of the prepared microcarriers. As the proportion of the aqueous phase decreases (or the proportion of the oil phase increases), specifically from 1:5 to 1:10 and then to 1:20, the average particle size of the microcarriers gradually decreases. This is because increasing the volume fraction of the oil phase reduces the number of aqueous droplets per unit volume of oil phase, decreasing the probability of droplet collisions and merging. Simultaneously, the emulsifier is more fully adsorbed at the oil-water interface, facilitating the formation of smaller emulsion droplets, ultimately resulting in microcarriers with smaller particle sizes after solidification.

[0112] As attached Figure 20 As shown, when the emulsifier concentration is 0.3%, the prepared microcarriers have the largest average particle size and a wide particle size distribution, with some microspheres exhibiting adhesion. When the emulsifier concentration increases to 4%, the average particle size of the microcarriers decreases significantly, the particle size distribution narrows, and the microspheres disperse well. When the emulsifier concentration continues to increase to 8%, the particle size of the microcarriers decreases further, but the rate of decrease slows down. This is because increasing the emulsifier concentration reduces the interfacial tension between oil and water, which helps the aqueous droplets break into smaller droplets under shear. At the same time, the adsorption rate of the emulsifier on the surface of the newly formed droplets accelerates, effectively preventing droplet coalescence. However, when the emulsifier concentration exceeds a certain value, the interfacial tension has already dropped to a low level, and further increasing the emulsifier concentration has limited effect on refining the particle size.

[0113] As attached Figure 21 As shown, the average particle size of the prepared microcarriers was largest at a stirring speed of 150 rpm, but the microspheres were irregular in shape and had poor sphericity. As the stirring speed increased to 700 rpm, the average particle size of the microcarriers decreased significantly, the sphericity of the microspheres improved markedly, and the surface became smooth. When the stirring speed was further increased to 1200 rpm, the particle size of the microcarriers continued to decrease, and the particle size distribution became more concentrated. This is because the increased stirring speed increases the shear force of the system, making the aqueous phase more easily broken into fine droplets in the oil phase. However, it should be noted that a higher stirring speed is not always better; excessively high stirring speeds can cause the formed microspheres to break down due to excessive shear force, affecting the quality of the microcarriers.

[0114] As attached Figure 22 As shown, when the emulsification time is 3 min, the droplet diameter is relatively large, and the average particle size of the prepared microcarriers is relatively large. Some droplets enter the solidification stage before they can be fully broken up. When the emulsification time is extended to 60 min, the droplet diameter decreases, and the average particle size of the microcarriers decreases. When the emulsification time is further extended to 90 min, the droplet diameter is relatively small, and the particle size of the microcarriers does not change much compared to that at 60 min. The reason for this is that droplet breakage is dominant in the early stage of emulsification. As the emulsification time is extended, the droplet size gradually decreases and tends to reach equilibrium. When the emulsification time is long enough, the breakage and merging of droplets reach a dynamic equilibrium, and further extending the emulsification time has little effect on further refining the particle size.

[0115] All aspects, embodiments, and features of this invention should be considered illustrative in all respects and not limiting of the invention; the scope of the invention is defined only by the claims. Other embodiments, modifications, and uses will become apparent to those skilled in the art without departing from the spirit and scope of the invention as claimed.

[0116] In the preparation method of this invention, the order of the steps is not limited to the listed order. For those skilled in the art, variations in the order of the steps without creative effort are also within the scope of protection of this invention. Furthermore, two or more steps or actions can be performed simultaneously.

[0117] Finally, it should be noted that the specific embodiments described herein are merely illustrative examples of the invention and are not intended to limit the implementation of the invention. Those skilled in the art can make various modifications or additions to the described specific embodiments or use similar methods to replace them; it is neither necessary nor possible to exemplify all embodiments here. However, these obvious variations or modifications derived from the essential spirit of the invention still fall within the scope of protection of the invention, and interpreting them as any additional limitation would contradict the spirit of the invention.

Claims

1. A method for preparing chitosan-based edible microcarriers by low-temperature confined phase separation and pH-triggered curing, characterized in that... Includes the following steps: (1) Preparation of oil phase dispersion system: Add water-in-oil emulsifier to oil phase medium and dissolve it completely under stirring conditions to obtain continuous oil phase; the mass volume concentration of water-in-oil emulsifier in continuous oil phase is 0.5% to 8.0%; (2) Preparation of acidic chitosan-based mother liquor: Chitosan is added to an acidic co-solvent and stirred and dissolved at 20-60°C to obtain a clear or semi-transparent acidic chitosan solution; the concentration of the acidic co-solvent is 0.2%-3.0%, and the mass-volume concentration of chitosan in the acidic mother liquor is 0.5%-4.0%; (3) Preparation of functional composite dispersion: Add plant protein, plant polysaccharide, edible fiber, cell adhesion promoting component or combination thereof to the acidic chitosan-based mother liquor obtained in step (2) to form chitosan-based composite dispersion; the mass ratio of plant protein to chitosan is 0.05:1 to 2.0:1; (4) Forming acidic composite aqueous droplets: Add the chitosan-based composite dispersion from step (3) to the continuous oil phase from step (1), and stir and emulsify at 200-1200 rpm for 5-90 min. The volume ratio of the aqueous phase to the oil phase is 1:3-1:

30. (5) Low-temperature confined phase separation treatment: The emulsion system of acidic composite aqueous droplets formed in step (4) is cooled to -30 to 10 °C and maintained at low temperature for 0.2 to 6 h to obtain a low-temperature emulsion system; (6) Alkaline trigger diffusion-induced pH gradient solidification: An alkaline trigger is added to the low-temperature emulsification system of step (5). The alkaline trigger is sodium hydroxide, potassium hydroxide, sodium carbonate, sodium bicarbonate, ammonia, triethanolamine, arginine, lysine, histidine or a combination thereof. The concentration of the alkaline trigger is 0.05 to 3.0 mol / L, and the volume ratio of the alkaline trigger to the acidic composite aqueous phase is 0.1:1 to 3:

1. After adding the alkaline trigger, the system is continuously stirred at -20 to 30 ℃ for 0.5 to 12 h. (7) Graded purification and degreasing treatment: After solidification, the emulsion system is collected by sieving, static sedimentation or low-speed centrifugation to collect microcarrier particles; then, food-grade alcohol-water solution, deionized water, buffer solution or combination thereof are used for multiple rounds of cleaning. (8) Drying and sterilization: The purified microcarriers are stored directly in wet form, or freeze-dried, vacuum-dried or air-dried at low temperature to form dry microcarriers.

2. The preparation method according to claim 1, characterized in that: The water-in-oil emulsifier mentioned in step (1) is Span80, Span85, PGPR or a combination thereof; the oil phase medium is liquid paraffin, n-hexane, vegetable oil, medium-chain triglycerides or a combination thereof.

3. The preparation method according to claim 1, characterized in that: The degree of deacetylation of the chitosan in step (2) is 75% to 95%, and the viscosity-average molecular weight is 50 to 500 kDa; the acidic co-solvent is acetic acid, lactic acid, citric acid, malic acid, dilute hydrochloric acid solution or a combination thereof.

4. The preparation method according to claim 1, characterized in that: In step (3), the plant protein is pumpkin seed protein, soy protein isolate, pea protein, peanut protein, sunflower seed protein, rice protein or a combination thereof; the plant polysaccharide is pectin, alginate, gum arabic, xanthan gum, guar gum, dextran, carboxymethyl cellulose or a combination thereof.

5. The preparation method according to claim 1, characterized in that: Step (7) First wash with 30% to 95% ethanol aqueous solution 1 to 5 times, then wash with deionized water or phosphate buffer 3 to 10 times until the pH of the washing solution is neutral; the purification steps are carried out at 4 to 30 ℃.

6. The preparation method according to claim 1, characterized in that: When using freeze-drying in step (8), the microcarrier is placed in a solution of 1% to 10% trehalose, sucrose, mannitol, glucose or a combination thereof for equilibration for 0.5 to 4 hours before freeze-drying. Then, it is pre-frozen at -20 to -80 °C for 2 to 24 hours and then freeze-dried for 12 to 72 hours.

7. A chitosan-based edible microcarrier prepared by low-temperature confined phase separation and pH-triggered curing according to any one of claims 1-6, characterized in that: The microcarriers are spherical, near-spherical, or irregularly spherical, with an average particle size of 50–1000 μm; they have micropores, wrinkles, or rough structures on their surface or inside; the microcarriers are based on a deprotonated chitosan physical gel network, with plant proteins or other edible functional components distributed in the gel network as a composite phase; the microcarriers do not contain glutaraldehyde, epichlorohydrin, genipin, or carbodiimide chemical cross-linking agents, nor do they rely on enzymatic cross-linking steps of transglutaminase or tyrosinase to obtain the stability of the main structure.

8. The application of the chitosan-based edible microcarrier as described in claim 7 in cultured meat.

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