A starch-based edible scaffold and its preparation method and application

Abstract

The application discloses a starch-based edible scaffold and a preparation method and application thereof, and belongs to the technical field of cell scaffold materials. The edible starch is used as a base material, and a semi-interpenetrating network (Semi-IPN) hydrogel scaffold is constructed through heat-induced self-assembly, and the scaffold has good biocompatibility and edible safety. The preparation method can accurately control the elastic modulus and mechanical properties of the scaffold, so that the scaffold is suitable for providing tensile stress stimulation for fish myoblasts. Thanks to the above-mentioned characteristics, the scaffold can not only effectively support the adhesion and proliferation of fish myoblasts, but also significantly promote the directional differentiation of the fish myoblasts, and form a biomimetic muscle tissue. The application provides an innovative and performance-controllable cell scaffold solution for producing fish culture meat products which do not need to be separated from the scaffold and can be directly eaten.

Description

Technical Field

[0001] This invention belongs to the field of food biotechnology, specifically relating to a starch-based edible scaffold, its preparation method, and its application. Background Technology

[0002] Cell-cultured meat technology offers a revolutionary solution for the sustainable acquisition of animal protein, but its transition from the laboratory to the dining table still faces a core bottleneck: a lack of suitable cell scaffold materials. An ideal muscle tissue culture scaffold needs to meet multiple requirements: excellent biocompatibility to support seed cell adhesion, proliferation, and directed differentiation into mature muscle fibers; suitable and controllable mechanical properties to simulate the dynamic mechanical microenvironment in vivo, actively inducing cells to oriented along stress directions and achieve functional maturation; complete edibility and safety, so that complex and expensive separation or removal steps are unnecessary in the final product; and industrialization characteristics including wide availability, low cost, and easy process scaling.

[0003] However, existing edible scaffold technologies for the application of cultured meat are constrained by two major bottlenecks: the contradiction between mechanical properties and structural stability, and the asynchrony between degradation rate and cell growth. On the one hand, natural polymer scaffolds such as pure gelatin and alginate lack mechanical strength under physiological culture conditions, making them prone to collapse or deformation. This not only results in a final product that is too soft and lacks realistic muscle texture and chewiness, but also makes it impossible for them to maintain a fixed three-dimensional shape in large-scale bioreactors. On the other hand, natural materials such as natural starch gel and gelatin usually have defects such as poor mechanical properties and uncontrollable degradation behavior. Summary of the Invention

[0004] To address the problem of insufficient mechanical strength in existing edible scaffold technologies, which cannot meet the requirements of stretch culture, this invention provides a method for preparing a starch-based edible scaffold. This invention utilizes a one-pot process to form a stable and uniform semi-interpenetrating network between starch and gelatin, significantly improving the mechanical strength of the edible scaffold. The elastic modulus is no less than 5.76 kPa, the tensile strength is no less than 2.67 kPa, and the elongation at break is no less than 59.74%. This effectively matches the mechanical properties of soft tissue, providing a foundation for cells to withstand dynamic tensile stress.

[0005] The technical solution of the present invention is as follows: (1) Preparation of gelatinized starch solution: Mix starch with deionized water, heat and stir to completely gelatinize the starch to obtain gelatinized starch solution, and cool to 50-60℃ for later use; Starch is completely gelatinized at high temperature, allowing its molecular chains to fully unwind and disperse, forming a dynamic, linear starch molecular chain solution. Simultaneously, gelatin is kept in a dissolved state above its gelation temperature but below its denaturation temperature (55°C). Ensuring that both are in a highly fluid, ungelatinized linear molecular chain state when mixed is a thermodynamic and kinetic prerequisite for achieving molecular-level interpenetration.

[0006] (2) Preparation of gelatin-enzyme mixture: Dissolve fish gelatin in deionized water at a temperature not higher than 60°C to prepare a gelatin solution, then add transglutaminase and glycerol to it, mix well to obtain a mixture; (3) Blending and ultrasonic dispersion: The gelatinized starch solution obtained in step (1) is mixed with the gelatin-enzyme mixture obtained in step (2) to obtain a blend. Before the blend undergoes significant gelation, it is immediately subjected to ultrasonic treatment to form a uniform pre-assembled system. The ultrasonic power is 100W-500W and the treatment time is 5 min-30 min. Generally, the gelatinized starch solution and the gelatin-enzyme mixture are ultrasonically treated within 3 min after blending.

[0007] After mixing and before gelation, the cavitation bubbles and strong shear forces generated by ultrasonic treatment physically break down the aggregates and initially formed microphase regions of starch and gelatin molecular chains. This significantly enhances the motion and collision frequency of the molecular chains, driving them closer and entangled at the nanoscale. This mechanism damages the α-1,4 and α-1,6 glycosidic bonds in starch molecules, leading to a decrease in molecular weight and disruption of molecular structure, thereby increasing molecular mobility and reactivity. (4) Thermal induction molding and cross-linking: The pre-assembled system obtained in step (3) is placed in a constant temperature environment of 37-55℃ for thermal induction self-assembly and enzymatic cross-linking reaction for 3-12 hours, so that the system is solidified into a hydrogel with a three-dimensional network structure. After washing, the starch-based edible scaffold is obtained. After ultrasonic treatment to form a uniform pre-assembled body, the gelatin is immediately cross-linked with TG enzyme. This can simultaneously achieve the highest catalytic efficiency of transglutaminase, maintain the natural conformation of fish gelatin, and inhibit the orderly rearrangement of starch, thereby creating the best thermodynamic and kinetic conditions for forming a uniform, stable, and highly elastic semi-interpenetrating network. The gelatin network is solidified in situ, thereby permanently locking its interpenetrating topology with the starch chain.

[0008] Furthermore, the starch is selected from at least one of corn starch, potato starch, tapioca starch, wheat starch, sweet potato starch, rice starch, and pea starch.

[0009] Further, the heating temperature in step (1) is 95°C, the stirring speed is 300 rpm, and the stirring time is 30 min.

[0010] Furthermore, in step (2), the concentration of glycerol in the mixture is 1% (w / v), and the amount of glutamine transaminase added is 30 U / g fish gelatin powder.

[0011] Furthermore, in step (3), the volume ratio of the gelatinized starch solution to the gelatin-enzyme mixture is 1:4 - 4:1. The concentration of starch in the mixture is 1.5-6% (w / v), and the concentration of gelatin is 3% (w / v).

[0012] Further, in step (4), the pre-assembled system is injected or poured into a mold, the shape of which is a strip or sheet with biomimetic muscle texture.

[0013] Furthermore, the starch-based edible scaffold is stored at 4°C.

[0014] This invention also provides a starch-based edible scaffold prepared by the above method and its application in fish cell stretching culture. The fish cells can be large yellow croaker myoblasts, rainbow trout myoblasts, eel myoblasts, etc.

[0015] The starch-based edible scaffold prepared by this method is suitable for fish cell culture in the temperature range of 15-30°C.

[0016] The advantages of this invention are: 1. By constructing a semi-interpenetrating network structure in which a linear starch molecular network and a gelatin network cross-linked with TG enzyme interpenetrate each other, and by combining this with ultrasonic treatment to optimize network uniformity, effective control over the scaffold's elastic modulus (e.g., within the range of 2-20 kPa), tensile strength, and elongation at break can be achieved. This structure significantly improves the mechanical properties of pure starch gel, enabling it to withstand and transmit dynamic tensile stress.

[0017] 2. The scaffold is made entirely from food-grade raw materials such as starch, fish gelatin, glycerol, and TG enzyme, ensuring complete edibility without the need for additional separation.

[0018] 3. It provides a biomimetic mechanical microenvironment for large yellow croaker myoblasts, effectively promoting directed differentiation. The three-dimensional porous structure of the scaffold facilitates cell migration and material exchange, and its suitable mechanical properties can effectively transfer external tensile stress to the cells, thereby inducing cells to arrange and mature in an orderly manner along the stress direction, forming a tissue structure that is closer to natural muscle.

[0019] 4. The preparation process is simple, the morphology is controllable, and it has good application potential. This method is based on mature gelatinization and enzymatic cross-linking technology, with mild conditions and no need for complex equipment. By using molds, scaffolds of specific shapes such as strips and sheets can be easily prepared, and the raw material cost is low, making it easy to scale up production. Attached Figure Description

[0020] Figure 1 This is a flowchart illustrating the preparation process of the starch-based edible scaffold used for the stretch culture of large yellow croaker cells according to the present invention. Figure 2 (a) is a scanning electron microscope (SEM) image of Example 1; (b) is a scanning electron microscope (SEM) image of Example 2; (c) is a scanning electron microscope (SEM) image of Example 3; (d) is a scanning electron microscope (SEM) image of Comparative Example 1; and (e) is a scanning electron microscope (SEM) image of Comparative Example 2. Figure 3 (a) is the pore size distribution diagram of Example 1, (b) is the pore size distribution diagram of Example 2, (c) is the pore size distribution diagram of Example 3, and (d) is the pore size distribution diagram of Comparative Example 1. Figure 4 (a) is the differential scanning calorimetry (DSC) diagram of Example 1, (b) is the differential scanning calorimetry (DSC) diagram of Example 2, (c) is the differential scanning calorimetry (DSC) diagram of Example 3, and (d) is the differential scanning calorimetry (DSC) diagram of Comparative Example 1. Figure 5 (a) is the Fourier transform infrared (FTIR) spectrum of Example 1, (b) is the Fourier transform infrared (FTIR) spectrum of Example 2, (c) is the Fourier transform infrared (FTIR) spectrum of Example 3, and (d) is the Fourier transform infrared (FTIR) spectrum of Comparative Example 1. Figure 6 (a) is the swelling curve of Example 1, (b) is the swelling curve of Example 2, (c) is the swelling curve of Example 3, and (d) is the swelling curve of Comparative Example 1. Figure 7 (a) is the degradation curve of Example 1, (b) is the degradation curve of Example 2, (c) is the degradation curve of Example 3, and (d) is the degradation curve of Comparative Example 1. Figure 8 (a) is the tensile strain-stress curve of Example 1, (b) is the tensile strain-stress curve of Example 2, (c) is the tensile strain-stress curve of Example 3, and (d) is the tensile strain-stress curve of Comparative Example 1. Figure 9 The image shows a fluorescence micrograph of the proliferation of large yellow croaker myoblasts cultured on a starch-based edible scaffold for 5 days in Example 4. The image shows that the cells grew well on the scaffold surface, demonstrating the biocompatibility of the scaffold. Figure 10 This is a microstructure diagram of the scaffold prepared using corn starch as a substrate in Example 5 after double fluorescent staining; Figure 11This is a diagram of the apparatus used in Example 6 to dynamically stretch and culture large yellow croaker myoblasts on a corn starch-based edible scaffold. Figure 12 This is a staining image of myotubes formed by the differentiation of large yellow croaker myoblasts before and after stretching on a corn starch-based edible scaffold in Example 6; Figure 13 Fluorescence micrograph of rainbow trout myoblasts cultured on a starch-based edible scaffold for 5 days in Example 7; Figure 14 This is a staining image of the myotubes formed by rainbow trout myoblasts before and after stretching on a corn starch-based edible scaffold in Example 7. Figure 15 The dynamic rheological properties of Example 8. Detailed Implementation

[0021] The following examples are provided to further illustrate the present invention and are intended to explain the invention, not to limit its scope. Unless otherwise specified, all terms are parts by weight and weight percentages.

[0022] Unless otherwise specified, the raw materials used in this invention are all conventional commercially available products; unless otherwise specified, the methods used in this invention are all conventional methods in the field.

[0023] In this invention, the culture temperature of large yellow croaker myoblasts on starch-based edible scaffolds is 27°C, and that of rainbow trout myoblasts on starch-based edible scaffolds is 18°C.

[0024] In this invention, % (w / v) means g / 100ml.

[0025] The embodiments of the present invention will be further described below with reference to several examples.

[0026] It should be understood that the described embodiments are merely some, not all, of the embodiments in this application. All other embodiments obtained by those skilled in the art based on the embodiments in this application without inventive effort are within the scope of protection of this application.

[0027] The terminology used in the embodiments of this application is for the purpose of describing particular embodiments only and is not intended to be limiting of this application. The singular forms “a,” “the,” and “the” used in the embodiments of this application and the appended claims are also intended to include the plural forms unless the context clearly indicates otherwise.

[0028] Example 1 (1) Preparation of gelatinized starch solution: Weigh 4 g of potato starch, add 50 mL of deionized water, place in a 95℃ water bath, and continuously stir magnetically at a speed of about 400 rpm for 30 min to obtain a transparent and uniform starch solution, and cool to about 55℃ for later use.

[0029] (2) Preparation of gelatin-enzyme mixture: Weigh 1.2 g of fish gelatin powder, add it to 25 mL of deionized water at 55℃, and stir gently until completely dissolved to obtain a gelatin solution. Then add 0.4 g of glycerol and 36 U of transglutaminase to the solution, stir evenly, and obtain gelatin-enzyme mixture.

[0030] (3) Blending and ultrasonic dispersion: The gelatinized starch solution obtained in step (1) and the gelatin-enzyme mixture obtained in step (2) are mixed at 55°C at a volume ratio of 15:25 and stirred evenly with a glass rod. After standing for 3 minutes, the mixture is placed in an ultrasonic cell disruptor and ultrasonically treated for 10 minutes at a power of 300W.

[0031] (4) Thermal induction molding and cross-linking: The ultrasonically treated blend liquid is rapidly injected into a rectangular strip silicone mold and placed in a constant temperature oven at 55°C for 4 hours to allow the system to solidify into a hydrogel with a three-dimensional network structure.

[0032] (5) Post-processing: Remove the solidified hydrogel from the mold, wash it with phosphate buffer (PBS), and store it at 4°C to obtain the required starch-based edible scaffold.

[0033] The morphology, structure, and stability of the prepared samples are characterized as follows: Morphological observation: such as Figure 2 In (a), the sample was freeze-dried, adhered to conductive adhesive, sputtered with gold, and observed using a scanning electron microscope (SEM). The porosity distribution was then calculated. Figure 3 (a) in the middle.

[0034] Structural observation: such as Figure 4 (a) Figure 5 (a) Thermogravimetric analysis (DSC) and Fourier transform infrared spectrum (FTIR) of the test sample.

[0035] Structural stability observation: such as Figure 6 (a) and Figure 7 In (a), the sample was placed in PBS to test its swelling and degradation properties.

[0036] Mechanical property observation: such as Figure 8 In (a), a tensile test is performed on the sample until the sample breaks, and the tensile curve is obtained.

[0037] The experimental results show that the scaffold has an interconnected three-dimensional porous structure with a pore size distribution of 30 μm-150 μm and a porosity of 57.30%. Its elastic modulus was measured to be 5.99 kPa, tensile strength to be 4.00 kPa, and elongation at break to be 59.74% using a mechanical testing instrument. This range of mechanical properties effectively matches the mechanical characteristics of soft tissue, providing a basis for cells to withstand dynamic tensile stress.

[0038] Example 2 (1) Preparation of gelatinized starch solution: Weigh 4 g of cassava starch, add 50 mL of deionized water, place in a 95℃ water bath, and continuously stir magnetically at a speed of about 400 rpm for 30 min to obtain a transparent and uniform starch solution, and cool to about 55℃ for later use.

[0039] (2) Preparation of gelatin-enzyme mixture: Weigh 1.2 g of fish gelatin powder, add it to 25 mL of deionized water at 55℃, and stir gently until completely dissolved to obtain a gelatin solution. Then add 0.4 g of glycerol and 36 U of transglutaminase to the solution, stir evenly, and obtain gelatin-enzyme mixture.

[0040] (3) Blending and ultrasonic dispersion: The gelatinized starch solution obtained in step (1) and the gelatin-enzyme mixture obtained in step (2) are mixed at 55°C at a volume ratio of 15:25 and stirred evenly with a glass rod. The mixture is then immediately placed in an ultrasonic cell disruptor and ultrasonically treated at 300W for 10 min.

[0041] (4) Thermal induction molding and cross-linking: The ultrasonically treated blend liquid is rapidly injected into a rectangular strip silicone mold and placed in a constant temperature oven at 55°C for 4 hours to allow the system to solidify into a hydrogel with a three-dimensional network structure.

[0042] (5) Post-processing: Remove the solidified hydrogel from the mold, wash it with phosphate buffer (PBS), and store it at 4°C to obtain the required starch-based edible scaffold.

[0043] The morphology, structure, and stability of the prepared samples are characterized as follows: Morphological observation: such as Figure 2 In (b), the sample was freeze-dried, adhered to conductive adhesive, sputtered with gold, and observed using a scanning electron microscope (SEM). The porosity distribution was then calculated. Figure 3 (b) in the middle.

[0044] Structural observation: such as Figure 4 (b) Figure 5 (b) Thermogravimetric analysis (DSC) and Fourier transform infrared spectrum (FTIR) of the test sample.

[0045] Structural stability observation: such as Figure 6 (b) and Figure 7 In (b), the sample was placed in PBS to test its swelling and degradation properties.

[0046] Mechanical property observation: such as Figure 8 In (b), a tensile test is performed on the sample until the sample breaks, and the tensile curve is obtained.

[0047] The experimental results show that the scaffold has an interconnected three-dimensional porous structure with a pore size distribution of 35 μm-115 μm and a porosity of 51.66%. Its elastic modulus was measured to be 6.48 kPa, tensile strength to be 2.67 kPa, and elongation at break to be 68.82% using a mechanical testing instrument. This range of mechanical properties effectively matches the mechanical characteristics of soft tissue, providing a basis for cells to withstand dynamic tensile stress.

[0048] Example 3 (1) Preparation of gelatinized starch solution: Weigh 4 g of corn starch, add 50 mL of deionized water, place in a 95℃ water bath, and continuously stir magnetically at a speed of about 400 rpm for 30 min to obtain a transparent and uniform starch solution, and cool to about 55℃ for later use.

[0049] (2) Preparation of gelatin-enzyme mixture: Weigh 1.2 g of fish gelatin powder, add it to 25 mL of deionized water at 55℃, and stir gently until completely dissolved to obtain a gelatin solution. Then add 0.4 g of glycerol and 36 U of transglutaminase to the solution, stir evenly, and obtain gelatin-enzyme mixture.

[0050] (3) Blending and ultrasonic dispersion: The gelatinized starch solution obtained in step (1) and the gelatin-enzyme mixture obtained in step (2) are mixed at 55°C at a volume ratio of 15:25 and stirred evenly with a glass rod. The mixture is then immediately placed in an ultrasonic cell disruptor and ultrasonically treated at 300W for 10 min.

[0051] (4) Thermal induction molding and cross-linking: The ultrasonically treated blend liquid is rapidly injected into a rectangular strip silicone mold and placed in a constant temperature oven at 55°C for 4 hours to allow the system to solidify into a hydrogel with a three-dimensional network structure.

[0052] (5) Post-processing: Remove the solidified hydrogel from the mold, wash it with phosphate buffer (PBS), and store it at 4°C to obtain the required starch-based edible scaffold.

[0053] The morphology, structure, and stability of the prepared samples are characterized as follows: Morphological observation: such as Figure 2 In step (c), the sample was freeze-dried, adhered to conductive adhesive, sputtered with gold, and observed using a scanning electron microscope (SEM). The porosity distribution was then calculated. Figure 3 (c) in the middle.

[0054] Structural observation: such as Figure 4 (c) Figure 5 (c) Thermogravimetric analysis (DSC) and Fourier transform infrared spectrum (FTIR) of the test sample.

[0055] Structural stability observation: such as Figure 6 (c) and Figure 7 (c) In this case, the sample was placed in PBS to test its swelling and degradation properties.

[0056] Mechanical property observation: such as Figure 8 In step (c), a tensile test is performed on the sample until the sample breaks, and the tensile curve is obtained.

[0057] The experimental results show that the scaffold has an interconnected three-dimensional porous structure with a pore size distribution of 50 μm-200 μm and a porosity of 56.17%. Its elastic modulus was measured to be 5.76 kPa, tensile strength to be 4.00 kPa, and elongation at break to be 77.75% using a mechanical testing instrument. This range of mechanical properties effectively matches the mechanical characteristics of soft tissue, providing a basis for cells to withstand dynamic tensile stress.

[0058] Example 4: Growth of large yellow croaker myoblasts on an edible starch-based scaffold. Edible scaffolds of different starch types (potato starch, tapioca starch, and corn starch) were first soaked in 75% alcohol for 6 hours under ultraviolet light, then washed three times with PBS for 10 minutes each time. Then, 1×10⁻⁶ ppm of PBS was used as the scaffolds. 5 A suspension of large yellow croaker myoblasts was seeded onto a scaffold and cultured in DMEM high-glucose medium for 5 days. The cells were then stained with the live cell dye Calcein-AM / PI and observed under a fluorescence microscope. Results are as follows: Figure 9 As shown, large yellow croaker myoblasts grew well on edible scaffolds made of potato starch, tapioca starch, and corn starch. The large yellow croaker myoblasts exhibited high survival rates, normal spreading morphology, and formed a dense cell layer on the scaffold surface. These results fully demonstrate that the starch-based edible scaffold provided by this invention has excellent and universal biocompatibility, effectively supporting the adhesion and proliferation of large yellow croaker myoblasts.

[0059] Example 5: Validation of Semi-Interpenetrating Network Hydrogel The corn starch-based edible scaffold prepared in Example 3 was sliced ​​into thin pieces. Gelatin and starch were stained with 0.013% (w / v) Rhodamine B and 0.35% (w / v) fluorescein isothiocyanate, respectively. Cover slips were carefully added to avoid air bubbles. After staining in the dark for 30 min, the samples were washed three times with PBS and then observed. Figure 10 As shown in the fluorescence microscopy image, the green (starch phase) and red (gelatin phase) fluorescence signals are clearly observed to interweave and penetrate each other in three-dimensional space, forming a continuous and interpenetrating interpenetrating network topology. This result directly provides intuitive morphological evidence that starch molecular chains and gelatin molecular chains interpenetrate to form a semi-interpenetrating network hydrogel, which is the microscopic basis for its high elasticity and controllable degradation.

[0060] Example 6: Traction differentiation of large yellow croaker myoblasts on an edible starch-based scaffold. The corn starch-based edible scaffold (2 cm × 9 cm) prepared in Example 3 was first soaked in 75% alcohol for 6 h under ultraviolet light, then washed three times with PBS for 10 min each time, and then... 6 Large yellow croaker myoblast suspension was seeded onto a scaffold and subjected to periodic stretching culture (10% constant strain) in a stretching chamber filled with DMEM high-glucose medium. The control group was placed in the same system but without mechanical stimulation. After 3 days of culture, the cells were differentiated in F12 low-serum medium for 4 days, followed by desmin immunofluorescence and DAPI nuclear staining, and observation under a fluorescence microscope. Figure 12 As shown, traction promotes the differentiation of myoblasts in large yellow croaker, increasing the myotube fusion index from 6.74% to 40.35%, and improving the terminal differentiation efficiency of large yellow croaker myoblasts by approximately 6 times. The cell nuclei elongate along the traction direction, and the myotubes develop a certain orientation, while the myotubes in the untraction group are randomly arranged. This indicates that traction stimulation can promote the differentiation efficiency of large yellow croaker myoblasts and simultaneously complete effective mechanical signal transduction, guiding cells to perform spatial biomimetic assembly.

[0061] Example 7: Growth of rainbow trout myoblasts on an edible starch-based scaffold. The edible corn starch scaffold prepared in Example 3 was first soaked in 75% alcohol for 6 hours under ultraviolet light, then washed three times with PBS for 10 minutes each time. Then, a solution containing 1×10⁻⁶ ppm was added. 5 A suspension of rainbow trout myoblasts was seeded onto a scaffold and cultured in L-15 medium for 5 days. The cells were then stained with the live cell dye Calcein-AM / PI and observed under a fluorescence microscope. Results are as follows: Figure 13 As shown, rainbow trout myoblasts grew well on an edible corn starch scaffold. All rainbow trout myoblasts exhibited high survival rates, normal spreading morphology, and formed a dense cell layer on the scaffold surface. This result fully demonstrates that, under suitable physiological temperature conditions, cold-water rainbow trout myoblasts on the starch-based scaffold provided by this invention exhibit equally excellent adhesion, spreading, and proliferation capabilities as warm-water large yellow croaker myoblasts. The differentiation of rainbow trout before and after traction is shown in the figure. Figure 14As shown, traction stimulation can promote the differentiation efficiency of rainbow trout myoblasts and simultaneously facilitate effective mechanical signal transduction, guiding cells to perform spatial biomimetic assembly. The biocompatibility of this invention is independent of cell type or culture conditions in specific fish species. The RGD integrin recognition site provided by fish gelatin satisfies the common molecular requirements for cell adhesion; the three-dimensional porous interconnected structure provides a universal physical space for cell growth and material exchange; and the controllable mechanical properties enable effective activation of conserved force signaling pathways. This can provide a universal growth microenvironment supporting the in vitro expansion of myoblasts from various fish species.

[0062] Example 8 The difference from Example 3 is that the final concentration of starch in the blend is 1.5% (w / v). The prepared edible scaffold is labeled S-1.5.

[0063] The difference from Example 3 is that the final concentration of starch in the blend is 6% (w / v). The prepared edible scaffold is labeled S-6.

[0064] The edible scaffold was first soaked in 75% alcohol under UV light for 6 hours, then washed three times with PBS for 10 minutes each time. 1×10 5 Large yellow croaker myoblasts were seeded onto an S-6 scaffold and cultured at 27°C for 7 days. 1×10... 5 Rainbow trout myoblasts were seeded onto S-1.5 scaffolds and cultured at 18°C ​​for 7 days. After culture, cell morphology was observed using Calcein-AM / PI staining, and amplitude scanning of S-1.5 and S-6 scaffolds was performed using a rheometer. Results ( Figure 15 The results showed that the energy storage modulus of the S-6 scaffold was significantly higher than that of the S-1.5 scaffold, indicating that under the same gelatin network, increasing the starch concentration can effectively enhance the overall mechanical strength of the semi-interpenetrating network.

[0065] Evaluation after 5 days of culture showed that both groups of cells adhered well and proliferated normally under their respective conditions. This example demonstrates that increasing starch concentration can enhance network strength to adapt to high-temperature (27°C) culture, while moderately reducing the concentration is more conducive to cell growth at low temperatures (18°C). This provides a clear process path for customizing scaffold performance to suit the physiological temperatures of different fish species, demonstrating the designability and wide temperature adaptability potential of the material platform of this invention.

[0066] Comparative Example 1 This comparative example aims to investigate the role of starch components in the construction of semi-interpenetrating networks. Its preparation method differs from that of Example 3 (corn starch) in that step (1) of preparing the gelatinized starch solution is completely omitted; only a gelatin-enzyme mixture is used for cross-linking and curing. The preparation steps are as follows: Weigh 1.2 g of fish gelatin powder and add it to 40 mL of deionized water at 55°C. Stir gently until completely dissolved. Add 0.4 g of glycerol and 36 U of transglutaminase to the gelatin solution and stir until homogeneous. Pour the resulting mixture into a rectangular silicone mold and place it in a 55°C incubator for 4 hours to allow for full cross-linking and curing. Post-treatment is the same as in Example 1 to obtain a pure gelatin cross-linked gel.

[0067] The morphology, structure, and stability of the prepared samples are characterized as follows: Morphological observation: such as Figure 2 In step (d), the sample was freeze-dried, adhered to conductive adhesive, sputtered with gold, and observed using a scanning electron microscope (SEM). The porosity distribution was then calculated. Figure 3 (d) in the middle.

[0068] Structural observation: such as Figure 4 (d) Figure 5 (d) Thermogravimetric analysis (DSC) and Fourier transform infrared spectrum (FTIR) of the test sample.

[0069] Structural stability observation: such as Figure 6 (d) and Figure 7 (d) In this case, the sample was placed in PBS to test its swelling and degradation properties.

[0070] Mechanical property observation: such as Figure 8 In step (d), a tensile test is performed on the sample until the sample breaks, and the tensile curve is obtained.

[0071] FTIR and XRD analysis showed that a semi-interpenetrating network hydrogel was not successfully prepared in this embodiment, and its maximum tensile strain and maximum tensile stress were 39.06% and 2.0 kPa, respectively, indicating poor mechanical properties. Comparison with Example 3 shows that the construction of a semi-interpenetrating network is crucial for the scaffold to achieve excellent mechanical properties.

[0072] Comparative Example 2 This comparative example aims to verify the importance of ultrasonic treatment for the formation of a uniform semi-interpenetrating network. The preparation method differs from Example 3 in that ultrasonic dispersion is not performed; the preparation steps are as follows: The raw materials and formula of this comparative example are exactly the same as those of Example 3, except that the ultrasonic treatment in step (3) is omitted. Specifically, the gelatinized corn starch base liquid and the prepared gelatin-enzyme mixture are mixed at 55°C at a volume ratio of 15:25, and then manually stirred with a glass rod for 2 minutes until macroscopically uniform. The mixture is then poured into a mold and allowed to stand at 55°C for 4 hours to solidify. The subsequent steps are the same as in Example 3.

[0073] The comparative sample was difficult to mold, and macroscopic inhomogeneity was visible after demolding, indicating significant phase separation. Scanning electron microscopy (SEM) observations are as follows: Figure 2 As shown in (e). Similar to Example 3 ( Figure 2 c) Compared to a uniform porous structure, the sample exhibits a severely heterogeneous internal pore structure, with obvious phase separation regions and starch agglomeration, failing to form a coherent three-dimensional network. Due to these severe structural defects, the sample is brittle and easily broken, making it impossible to conduct a complete tensile test to obtain an effective stress-strain curve, indicating that it completely lacks the mechanical integrity required to support dynamic mechanical culture. This comparative example, in direct comparison with Example 3, demonstrates that omitting ultrasonic treatment prevents the starch and gelatin phases from achieving uniform dispersion and pre-assembly at the molecular / nanoscale, leading to macroscopic phase separation, microstructural defects, and complete failure of mechanical properties.

[0074] This is because starch is a polysaccharide with a backbone rich in hydroxyl groups and strong hydrophilicity; gelatin is a protein composed of amino acids, with both hydrophilic and hydrophobic regions on its molecular chain. The significant differences in their molecular conformation, polarity, and chain flexibility in solution lead to a weak entropy increase during mixing, which cannot effectively counteract the positive mixing enthalpy change caused by poor interfacial interactions. Consequently, the system thermodynamically tends towards phase separation. Furthermore, starch forms a rigid physical gel with crystalline regions as crosslinking points through hydrogen bond rearrangement, while gelatin forms a thermally reversible physical gel through helical coiling and hydrogen bonding.

[0075] In solution, the two polymer chains have high viscosity, extensive entanglement, and slow diffusion. If simply mixed, the molecular chains do not have enough time and energy to penetrate and interpenetrate each other's network regions, ultimately forming an "island structure" or macroscopic phase separation, rather than molecular-level interpenetration.

[0076] Comparative Example 3 The difference from Example 3 is that the gelatin is cross-linked and cured before being added to the gelatinized starch solution. The specific steps are as follows: (1) Prepare gelatinized starch solution: Weigh 4 g of corn starch, add 50 mL of deionized water, place it in a 95°C water bath, and continuously stir magnetically at a speed of about 400 rpm for 30 min to obtain a transparent and uniform starch solution, and cool it to about 55°C for later use.

[0077] (2) Preparation of gelatin-enzyme mixture: Weigh 1.2 g of fish gelatin powder, add it to 25 mL of deionized water at 55℃, and stir gently until completely dissolved to obtain a gelatin solution. Then add 0.4 g of glycerol and 36 U of transglutaminase to the solution, stir evenly, and obtain gelatin-enzyme mixture. Pour the gelatin-enzyme mixture into a rectangular silicone mold, place it in a constant temperature oven at 55℃ and let it stand for 4 h to solidify the system.

[0078] (3) Blending and ultrasonic dispersion: The gelatinized starch solution obtained in step (1) and the solidified gelatin obtained in step (2) are placed in an ultrasonic cell disruptor and ultrasonically treated for 10 min at a power of 300W.

[0079] It was found that gelatinized starch and gelatin could not be mixed and crosslinked, and the two remained separated. This is because after the gelatin is fully crosslinked and cured, the dense network forms a three-dimensional network that physically hinders the subsequent diffusion and penetration of starch molecular chains, leading to macroscopic phase separation.

[0080] Comparative Example 4 The difference from Example 3 is that the gelatinization temperature of the starch is 60°C.

[0081] It was found that starch gelatinization was incomplete, and some starch granules could not be fully dissolved. The residual starch granules not only disrupt the integrity and uniformity of the semi-interpenetrating network structure, resulting in poor local mechanical properties of the network, but also introduce interfering factors at the cell culture scale, which is not conducive to long-term cell culture.

[0082] Comparative Example 5 The difference from Example 3 is that the gelatinized starch solution was mixed with the gelatin-enzyme mixture and allowed to stand for 5 minutes before being sonicated. The results showed that the mechanical strength of the resulting edible scaffold was significantly reduced. Due to severe structural defects, the sample was easily broken, and a complete tensile test could not be performed to obtain an effective stress-strain curve, indicating that it completely lacked the mechanical integrity required to support dynamic mechanical culture.

[0083] This is because the formation of the gelatin network is uneven and incomplete, the material system is not uniform, and it cannot be subjected to uniform force, which makes the edible scaffold easy to break and unsuitable for use in dynamic cell stretching culture.

[0084] The above embodiments describe in detail the structure, features, and effects of the present invention. The above description is only a preferred embodiment of the present invention. Any changes made in accordance with the concept of the present invention, or equivalent embodiments modified to have equivalent changes, shall still fall within the scope of protection of the present invention if they do not exceed the scope covered by the specification.

Claims

1. A method for preparing a starch-based edible scaffold, characterized in that, Includes the following steps: (1) Preparation of gelatinized starch solution: Mix starch with deionized water, heat and stir to completely gelatinize the starch to obtain gelatinized starch solution, and cool to 50-60℃ for later use; (2) Preparation of gelatin-enzyme mixture: Dissolve fish gelatin in deionized water at a temperature not higher than 60°C to prepare a gelatin solution, then add transglutaminase and glycerol to it, mix well to obtain a mixture; (3) Blending and ultrasonic dispersion: The gelatinized starch solution obtained in step (1) is mixed with the gelatin-enzyme mixture obtained in step (2) to obtain a blend; after mixing, it is ultrasonically treated within 3 minutes to form a uniform pre-assembled system; the ultrasonic power is 100W-500W and the treatment time is 5 min-30 min. (4) Thermal induction molding and cross-linking: The pre-assembled system obtained in step (3) is placed in a constant temperature environment of 37-55℃ for 3-12 hours to solidify the system into a hydrogel with a three-dimensional network structure. After cleaning, the starch-based edible scaffold is obtained.

2. The method according to claim 1, characterized in that, The starch is selected from at least one of corn starch, potato starch, cassava starch, wheat starch, sweet potato starch, rice starch, and pea starch.

3. The method according to claim 1, characterized in that, The heating temperature in step (1) is 95℃, the stirring speed is 300 rpm, and the stirring time is 30 min.

4. The method according to claim 1, characterized in that, In step (2), the concentration of glycerol in the mixture is 1g / 100ml, and the amount of transglutaminase added is 30 U / g fish gelatin powder.

5. The method according to claim 1, characterized in that, In step (3), the volume ratio of the gelatinized starch solution to the gelatin-enzyme mixture is 1:4 - 4:1; the concentration of starch in the mixture is 1.5-6g / 100ml, and the concentration of gelatin is 3g / 100ml.

6. The method according to claim 1, characterized in that, In step (4), the pre-assembled system is injected or poured into a mold, the shape of which is a strip or sheet with biomimetic muscle texture.

7. The method according to claim 1, characterized in that, The starch-based edible scaffold is stored at 4°C.

8. A starch-based edible scaffold prepared by the method of claim 1.

9. The application of the starch-based edible scaffold as described in claim 8 in fish cell stretching culture.

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