A method for preparing a 3D printed starch-based gel scaffold for cultured meat production

Abstract

The present invention discloses a method for preparing a 3D printed starch-based gel scaffold for cultured meat production, comprising: adding starch to water and stirring until mixed evenly to obtain a starch suspension, fully gelatinizing the starch suspension in a water bath, and cooling to room temperature; then adding sodium alginate and gelatin to the starch suspension, stirring evenly and letting it stand to allow the starch gel to age to a certain extent; finally adding an appropriate amount of GDL and nano-calcium carbonate particles and stirring evenly, and then homogenizing to remove the bubbles immersed in the mixing process. The gel scaffold of the present invention is used as a scaffold for cultured meat, and the sodium alginate-GDL-calcium ions, gelatin, and starch provided are all safe and non-toxic ingredients, and the calcium ion content is also within a suitable range. It is a commonly used ingredient in cultured meat scaffolds, and as a food product, it has a part that can ensure food safety.

Description

Technical Field

[0001] The present invention relates to the technical field of preparing starch-based gel scaffolds for cultured meat, and in particular to a method for preparing a 3D printed starch-based gel scaffold for cultured meat production. Background Art

[0002] Meat is an indispensable part of the human table because it is rich in nutrients such as protein, fat, and vitamins. Global meat consumption remains high. At the same time, the contradiction between traditional meat production methods that rely on animal husbandry and resources and the environment (production resources, greenhouse gases, and environmental pollution) is becoming increasingly prominent. In addition, the prevalence of animal diseases in recent years has brought severe challenges to the safe production of meat, so there is an urgent need to develop green and efficient meat production methods. Cultured meat, as one of the methods for producing artificial meat, has been favored by researchers because the taste and texture of the product are not much different from those of farmed meat. However, the lack of scaffold materials for cultured meat is one of the key bottlenecks that currently restrict the widespread application of artificial meat in the food field.

[0003] At present, the production of cultured meat scaffolds generally uses various scaffolds obtained through the natural gel process; there are few reports on the production of cultured meat scaffolds using food 3D extrusion printing technology; compared with traditional 3D printing technology, biological 3D printing technology is strictly different in terms of both technical level and ultimate goal; biological 3D printing requires precise control of the position, combination and interaction of biological materials, biological cells and growth factors in the overall 3D structure, which is essentially biologically active and achieves functions close to, the same as, or even superior to those of target tissues or biological organs. GDL is slowly hydrolyzed in the system to release H + , lowering the pH in the system, allowing the nano-calcium carbonate particles to slowly release calcium ions, thereby slowly cross-linking the calcium ions with sodium alginate to obtain a uniform gel. This rule is now used in combination with 3D printing to apply it to the manufacture of cultured meat scaffolds. While improving the problem of gel uniformity, the pore size distribution of the gel scaffold is also improved. Therefore, in response to the above situation, we make full use of starch-based gels with different calcium ion contents, and perform / not perform 3D printing on them to obtain 3D printed gel scaffolds / gel scaffolds, and develop a 3D printed starch-based gel scaffold for cultured meat. Summary of the invention

[0004] The purpose of the present invention is to provide a method for preparing a 3D printed starch-based gel scaffold for cultured meat production. The method not only has a simple preparation process, but also the obtained starch-based gel scaffold has certain mechanical strength, suitable microstructure, good water stability, suitable swelling characteristics, certain metabolic activity and cell differentiation ability.

[0005] The specific technical solution according to the present invention is as follows:

[0006] A method for preparing a starch-based gel scaffold based on 3D printing, comprising:

[0007] (1) Add appropriate amount of distilled water to starch and stir thoroughly until there are no particles to form a starch-based gel suspension.

[0008] (2) placing the starch suspension in a sealed water bath to fully gelatinize the starch suspension to form a starch gel; and cooling the starch-based gel after the water bath to room temperature.

[0009] (3) Sodium alginate and gelatin are added to the starch gel, stirred evenly, and allowed to stand for 18 to 30 hours (most preferably 24 hours) to allow the starch to age to a certain extent, thereby obtaining a starch-based gel.

[0010] (4) Add appropriate amount of GDL (gluconolactone) and nano calcium carbonate particles into the starch-based gel, stir evenly, and remove bubbles during the mixing process by homogenization.

[0011] (5) A portion of the starch-based gel obtained in (4) was naturally gelled for 24 hours, and then freeze-dried and water-annealed to obtain a common starch gel scaffold.

[0012] (6) A portion of the starch-based gel obtained in (4) was 3D printed, and after 24 hours of gelation, a 3D printed starch-based gel scaffold was obtained by freeze drying and water annealing.

[0013] The present invention forms starch gel by gelatinization of starch through water bath treatment, and after cooling to room temperature, sodium alginate and gelatin are added, and after uniform stirring, the mixture is allowed to stand for a certain period of time to allow the starch to age to a certain extent, and then appropriate amounts of GDL and nano-calcium carbonate particles are uniformly stirred and then homogenized to remove bubbles; the starch-based gel has suitable rheological properties through the gelatinization of starch, and then the self-supporting performance of the starch-based gel is enhanced by the crystal structure generated by the short-term regeneration of starch; and then GDL is hydrolyzed to reduce the pH in the system, so that the nano-calcium carbonate particles are prompted to slowly release calcium ions, so that the sodium alginate is uniformly cross-linked with the calcium ions, so that the scaffold becomes a starch-based gel cell culture meat scaffold suitable for 3D printing.

[0014] In step (1), the mass ratio of starch to water is 5:70-90.

[0015] In step (2), the gelatinization time is 30 to 90 minutes, and the gelatinization temperature is 70 to 80°C.

[0016] In step (3), the mass ratio of sodium alginate to gelatin is 1:0.5-2.

[0017] In step (4), the molar ratio of GDL to nano calcium carbonate is 1-3:1 (more preferably 2:1). The homogenizing speed is 6000-10000 rpm.

[0018] In step (6), the gelling time is 18 to 30 hours, the freeze drying time is 36 to 60 hours, the water annealing temperature is 116 to 126° C., and the water annealing time is 10 to 30 minutes. 3D printing is performed using a 3D food printer, and the filling rate of the 3D printing is set at 25%-75% (more preferably 50%).

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] (1) The GDL used in the present invention can reduce the pH in the system through hydrolysis, thereby prompting the nano-calcium carbonate particles to slowly release calcium ions, thereby allowing the sodium alginate and calcium ions to be uniformly cross-linked.

[0021] (2) In the present invention, by adjusting the calcium ion concentration, GDL is hydrolyzed to release hydrogen ions, which react with carbonate ions to release gas, resulting in a decrease in the number of pores in the formed gel scaffold and an increase in the pore area, which is beneficial to the proliferation and differentiation of C2C12 cells.

[0022] (3) In the present invention, the starch-based gel scaffold is manufactured by 3D printing technology, and the gel scaffold with a filling rate of 50% has a significant improvement in the proliferation and differentiation of C2C12 cells compared with the ordinary starch-based gel scaffold.

[0023] (4) The gel scaffold of the present invention can be used as a scaffold for cultured meat. The present invention can differentiate mouse myoblasts into tissues with myotubes in a low-cost and high-efficiency method. The sodium alginate-GDL-calcium ions, gelatin, and starch provided by the present invention are all safe and non-toxic ingredients. The calcium ion content is also within a suitable range. They are commonly used ingredients in cultured meat scaffolds and can ensure food safety as a food product. The scaffold has a certain thickness and elasticity, so the scaffold can provide a certain degree of taste, providing support for the production scaffold of cell meat. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Table 1 Starch-based gel formulations with different calcium ion contents.

[0025] Figure 1 The rheological properties of starch-based gels with different calcium ion contents. A, B, C, and D are the viscosity curve, storage modulus, loss coefficient, and loss modulus of starch-based gels, respectively.

[0026] Figure 2The textural properties of starch-based gels with different calcium ion contents. A is the gelation process of starch-based gels of different proportions within 0-24 hours; B, C, D, E, F and G are the hardness, viscosity, elasticity, chewiness, adhesiveness and cohesion of starch-based gels within 0-24 hours.

[0027] Figure 3 The compression modulus and filling rate of the gel scaffolds of various embodiments and comparative examples are shown in FIG. (The compression modulus of 1-5% is calculated by the formula E=σ / ε, where E is the compression modulus, σ is the stress, and ε is the strain; the slope in the figure represents the compression modulus).

[0028] Figure 4 The water stability of the gel scaffolds in various embodiments and comparative examples in 0-7 days.

[0029] Figure 5 The swelling properties of the gel scaffolds of Example 1, Example 2, Example 3, Example 4, Example 5 and Comparative Example 1 are shown.

[0030] Figure 6 The cross-sectional SEM (A), pore frequency (B), and pore area distribution (C) of the gel scaffolds of Example 1, Example 2, Example 3, Example 4, Example 5, and Comparative Example 1 are shown.

[0031] Figure 7 The proliferation of C2C12 cells on the gel scaffolds of Example 1, Example 2, Example 3, Example 4, Example 5 and Comparative Example 1 on the 1st day, the 4th day and the 7th day (A); the cell viability of the gel scaffolds of Example 1, Example 2, Example 3, Example 4, Example 5 and Comparative Example 1 within 7 days using the PrestoBlue detection method (B).

[0032] Figure 8 The immunostaining differentiation culture medium of C2C12 cells on the gel scaffold of Example 1, Example 2, Example 3, Example 4, Example 5 and Comparative Example 1 shows the myotube differentiation after 7 days.

[0033] Fig. 9 It is a schematic diagram of the principle of the preparation method of the present invention based on 3D printing starch-based gel scaffold. DETAILED DESCRIPTION

[0034] The present invention is further explained below in conjunction with specific examples. The present invention is implemented according to the technical solution of the invention, and detailed implementation schemes and operating steps are given, but the protection scope of the present invention is not limited to the following examples. The experimental methods in the following examples without specifying specific conditions are usually based on conventional conditions.

[0035] Example 1

[0036] A method for preparing a 3D printed starch-based gel scaffold for cultured meat production, the specific steps are as follows:

[0037] (1) Add 5 g of starch to 80 mL of distilled water and stir thoroughly until there are no particles to form a starch-based gel suspension.

[0038] (2) The starch suspension is sealed and placed in a water bath at 80° C. for 30 minutes to form a starch gel after sufficient gelatinization, and the starch-based gel after the water bath is cooled to room temperature (25° C.).

[0039] (3) Add 1 g of sodium alginate and 1 g of gelatin to the starch gel, stir evenly, and let stand for 24 hours to allow the starch to age to a certain extent, thereby obtaining a starch-based gel.

[0040] (4) 0.375 g of GDL (gluconolactone) and 0.075 g of nano calcium carbonate particles were added to the starch-based gel, stirred evenly, and homogenized at 10,000 rpm to remove bubbles introduced during the mixing process.

[0041] (5) The SSC3 starch-based gel obtained in (4) was 3D printed at a filling rate of 50%. After 24 hours of gelation, the gel scaffold of 3D printing Example 1 was obtained by freeze drying for 48 hours and annealing in water at 121° C. for 15 minutes.

[0042] like Figure 1 As shown in the figure, the obtained SSC3 gel has shear thinning and solid-like behavior; and through the slow hydrolysis of GDL, sodium alginate and Ca 2+ Slow cross-linking produces uniform, stable starch-based gels, such as Figure 2 As shown in the figure, the hardness, viscosity, chewiness and adhesiveness of the starch-based gel gradually increased with the increase of gel time within 24 hours, while its elasticity and cohesion did not change significantly; the SSC3 gel was subjected to 3D printing, freeze drying and water annealing to obtain the scaffold of Example 1; Figure 3 As shown, the compression modulus of the stent in Example 1 reaches 246.76Pa, which is high in strength. Fig. 9 As shown, the printed sample is close to the ideal model. And the bracket of Example 1 has strong water stability ( Figure 4 ) and 466.23±14.33% swelling capacity ( Figure 5 );Depend on Figure 5 It can be seen that the pore size distribution of the scaffold in Example 1 is uniform, with about 45.45% of the pore size distributed in the range of 80-120 μm and accounting for about 42.99% of the pore area; 4 After C2C12 myocytes were inoculated into the scaffold of Example 1, Figure 6It can be seen that the cells proliferated stably from 1d, 4d to 7d, and their number increased from 33331.67±634.00, 44727.17±734.50 to 64169.67±915.00. After 7d of differentiation, immunofluorescence staining revealed that C2C12 muscle cells differentiated, and the differentiation was the best, indicating that the scaffold in Example 1 best supported the differentiation of C2C12 muscle cells.

[0043] Example 2

[0044] A method for preparing a 3D printed starch-based gel scaffold for cultured meat production, the specific steps are as follows:

[0045] (1) Add 5 g of starch to 80 mL of distilled water and stir thoroughly until there are no particles to form a starch-based gel suspension.

[0046] (2) The starch suspension is sealed and placed in a water bath at 80° C. for 30 minutes to form a starch gel after sufficient gelatinization, and the starch-based gel after the water bath is cooled to room temperature (25° C.).

[0047] (3) Add 1 g of sodium alginate and 1 g of gelatin to the starch gel, stir evenly, and let stand for 24 hours to allow the starch to age to a certain extent, thereby obtaining a starch-based gel.

[0048] (4) 0.500 g of GDL (gluconolactone) and 0.100 g of nano calcium carbonate particles were added to the starch-based gel, stirred evenly, and homogenized at 10,000 rpm to remove bubbles introduced during the mixing process.

[0049] (5) The SSC4 gel obtained in (4) was 3D printed at a filling rate of 50%. After 24 hours of gelation, the 3D printed gel scaffold of Example 2 was obtained by freeze drying and annealing in water at 121°C for 15 minutes.

[0050] like Figure 1 As shown in the figure, the obtained SSC4 gel has shear thinning and solid-like behavior; and through the slow hydrolysis of GDL, sodium alginate and Ca 2+ Slow cross-linking produces uniform, stable starch-based gels, such as Figure 2 As shown in the figure, the hardness, viscosity, chewiness and adhesiveness of SSC4 gel gradually increased with the increase of gel time within 24 hours, while its elasticity and cohesion did not change significantly; the SSC4 gel was subjected to 3D printing, freeze drying and water annealing to obtain the gel scaffold of Example 2; Figure 3 As shown, the compression modulus of the stent of Example 2 reaches 113.55 Pa; and the stent of Example 2 has good water stability ( Figure 4 ) and 234.64±9.69% swelling capacity ( Figure 5 );Depend on Figure 5 It can be seen that the pore size distribution of the scaffold in Example 2 is relatively uniform, with about 39.29% of the pore size distributed in the range of 120-160 μm and accounting for about 48.41% of the pore area; 4 After C2C12 muscle cells were inoculated onto the scaffold of Example 2, Figure 6 It can be seen that the cells proliferated stably from 1d, 4d to 7d, and their number increased from 32509.67±640.00, 46595.17±652.50 to 62438.17±870.50. After 7d of differentiation, immunofluorescence staining showed that C2C12 muscle cells differentiated and the differentiation was good, indicating that the scaffold of Example 2 can support the differentiation of C2C12 muscle cells.

[0051] Example 3

[0052] A method for preparing a 3D printed starch-based gel scaffold for cultured meat production, the specific steps are as follows:

[0053] (1) Add 5 g of starch to 80 mL of distilled water and stir thoroughly until there are no particles to form a starch-based gel suspension.

[0054] (2) The starch suspension is sealed and placed in a water bath at 80° C. for 30 minutes to form a starch gel after sufficient gelatinization, and the starch-based gel after the water bath is cooled to room temperature (25° C.).

[0055] (3) Add 1 g of sodium alginate and 1 g of gelatin to the starch gel, stir evenly, and let stand for 24 hours to allow the starch to age to a certain extent, thereby obtaining a starch-based gel.

[0056] (4) 0.250 g GDL (gluconolactone) and 0.050 g nano calcium carbonate particles were added to the starch-based gel, stirred evenly, and homogenized at 10,000 rpm to remove bubbles introduced during the mixing process.

[0057] (5) The SSC2 gel obtained in (4) was 3D printed at a filling rate of 50%. After 24 hours of gelation, the 3D printed gel scaffold of Example 3 was obtained by freeze drying and annealing in water at 121°C for 15 minutes.

[0058] like Figure 1 As shown in the figure, the obtained SSC2 gel has shear thinning and solid-like behavior; and through the slow hydrolysis of GDL, sodium alginate and Ca 2+ Slow cross-linking produces uniform and stable SSC2 gels, such as Figure 2As shown in the figure, the hardness, viscosity, chewiness and adhesiveness of SSC2 gel gradually increased with the increase of gel time within 24 hours, while its elasticity and cohesion did not change significantly; the SSC2 gel was subjected to 3D printing, freeze drying and water annealing to obtain the gel scaffold of Example 3; Figure 3 As shown, the compression modulus of the embodiment 3 bracket reaches 83.48Pa; and the embodiment 3 bracket has general water stability ( Figure 4 ) and 521.88±43.15% swelling capacity ( Figure 5 );Depend on Figure 5 It can be seen that the pore size distribution of the scaffold in Example 3 is relatively concentrated, with about 98.27% of the pore size distributed in the range of 40-120 μm and accounting for about 97.86% of the pore area; 4 After C2C12 myocytes were seeded onto the scaffold of Example 3, Figure 6 It can be seen that the cells proliferated stably from 1d, 4d to 7d, and their number increased from 30999.67±277.00, 45613.17±626.50 to 61289.67±266.00. After 7d of differentiation, immunofluorescence staining revealed that C2C12 muscle cells differentiated well, indicating that the scaffold of Example 3 supports the differentiation of C2C12 muscle cells.

[0059] Example 4

[0060] A method for preparing a 3D printed starch-based gel scaffold for cultured meat production, the specific steps are as follows:

[0061] (1) Add 5 g of starch to 80 mL of distilled water and stir thoroughly until there are no particles to form a starch-based gel suspension.

[0062] (2) The starch suspension is sealed and placed in a water bath at 80° C. for 30 minutes to form a starch gel after sufficient gelatinization, and the starch-based gel after the water bath is cooled to room temperature (25° C.).

[0063] (3) Add 1 g of sodium alginate and 1 g of gelatin to the starch gel, stir evenly, and let stand for 24 hours to allow the starch to age to a certain extent, thereby obtaining a starch-based gel.

[0064] (4) 0.125 g of GDL (gluconolactone) and 0.025 g of nano calcium carbonate particles were added to the starch-based gel, stirred evenly, and homogenized at 10,000 rpm to remove bubbles introduced during the mixing process.

[0065] (5) The SSC1 gel obtained in (4) was 3D printed at a filling rate of 50%. After 24 hours of gelation, the 3D printed gel scaffold of Example 4 was obtained by freeze drying and annealing in water at 121°C for 15 minutes.

[0066] like Figure 1 As shown, SSC1 gel has shear thinning and solid-like behavior; and through the slow hydrolysis of GDL, sodium alginate and Ca 2+ Slow cross-linking produces a uniform, stable gel, such as Figure 2 As shown in the figure, the hardness, viscosity, chewiness and adhesiveness of SSC1 gel gradually increased with the increase of gel time within 24 hours, while its elasticity and cohesion did not change significantly; the SSC1 gel was subjected to 3D printing, freeze drying and water annealing to obtain the gel scaffold of Example 4; Figure 3 As shown, the compression modulus of the stent of Example 4 reaches 37.39 Pa; and the water stability of the stent of Example 4 is poor ( Figure 4 ) and 755.22±37.58% swelling capacity ( Figure 5 );Depend on Figure 5 It can be seen that the pore size distribution of the scaffold in Example 4 is concentrated, with about 61.44% of the pore size distributed in the range of 80-120 μm and accounting for about 8.29% of the pore area; 4 After C2C12 myocytes were seeded onto the scaffold of Example 4, Figure 6 It can be seen that the cells proliferated stably from 1d, 4d to 7d, and the number increased from 29473.67±1023.00, 36043±851.50 to 51067±679.50. After 7d of differentiation, immunofluorescence staining revealed that C2C12 muscle cells differentiated, and the differentiation was not good, indicating that the scaffold of Example 4 did not support the differentiation of C2C12 muscle cells.

[0067] Example 5

[0068] A method for preparing a 3D printed starch-based gel scaffold for cultured meat production, the specific steps are as follows:

[0069] (1) Add 5 g of starch to 80 mL of distilled water and stir thoroughly until there are no particles to form a starch-based gel suspension.

[0070] (2) The starch suspension is sealed and placed in a water bath at 80° C. for 30 minutes to form a starch gel after sufficient gelatinization, and the starch-based gel after the water bath is cooled to room temperature (25° C.).

[0071] (3) Add 1 g of sodium alginate and 1 g of gelatin to the starch gel, stir evenly, and let stand for 24 hours to allow the starch to age to a certain extent, thereby obtaining a starch-based gel.

[0072] (4) The starch-based gel obtained in (3) was uniformly stirred and homogenized at 10,000 rpm to remove bubbles introduced during the mixing process.

[0073] (5) The SS gel obtained in (4) was 3D printed at a filling rate of 50%. After 24 hours of gelation, the 3D printed gel scaffold of Example 5 was obtained by freeze drying and annealing in water at 121°C for 15 minutes.

[0074] like Figure 1 As shown in the figure, SS gel has shear thinning and solid-like behavior; and through the slow hydrolysis of GDL, sodium alginate and Ca 2+ Slow cross-linking produces a uniform, stable gel, such as Figure 2 As shown in the figure, the hardness, viscosity, chewiness and adhesiveness of the SS gel gradually increased with the increase of gel time within 24 hours, while its elasticity and cohesion did not change significantly; the SS gel was subjected to 3D printing, freeze drying and water annealing to obtain the gel scaffold of Example 5; Figure 3 As shown, the compression modulus of the stent of Example 5 reaches 28.34 Pa; and the water stability of the stent of Example 5 is average ( Figure 4 ) and 518.56.22±28.97% swelling capacity ( Figure 5 );Depend on Figure 5 It can be seen that the pore size distribution of the scaffold in Example 5 is concentrated, with about 67.95% of the pore size distributed in the range of 120-160 μm and accounting for about 74.63% of the pore area; 4 After C2C12 myocytes were inoculated onto the scaffold of Example 5, Figure 6 It can be seen that the cells proliferated stably from 1d, 4d to 7d, and the number increased from 31875.17±306.50, 33211±397.00 to 46532.17±267.50. After 7d of differentiation, immunofluorescence staining revealed that C2C12 muscle cells differentiated, and the differentiation was poor, indicating that the scaffold in Example 5 did not support the differentiation of C2C12 muscle cells.

[0075] Example 6

[0076] A method for preparing a 3D printed starch-based gel scaffold for cultured meat production, the specific steps are as follows:

[0077] (1) Add 5 g of starch to 80 mL of distilled water and stir thoroughly until there are no particles to form a starch-based gel suspension.

[0078] (2) The starch suspension is sealed and placed in a water bath at 80° C. for 30 minutes to form a starch gel after sufficient gelatinization, and the starch-based gel after the water bath is cooled to room temperature (25° C.).

[0079] (3) Add 1 g of sodium alginate and 1 g of gelatin to the starch gel, stir evenly, and let stand for 24 hours to allow the starch to age to a certain extent, thereby obtaining a starch-based gel.

[0080] (4) 0.375 g of GDL (gluconolactone) and 0.075 g of nano calcium carbonate particles were added to the starch-based gel, stirred evenly, and homogenized at 10,000 rpm to remove bubbles introduced during the mixing process.

[0081] (5) The SSC3 gel obtained in (4) was 3D printed at a filling rate of 25%. After 24 hours of gelation, the 3D printed gel scaffold of Example 6 was obtained by freeze drying and annealing in water at 121°C for 15 minutes.

[0082] like Figure 1 As shown in the figure, SSC3 gel has shear thinning and solid-like behavior; and through the slow hydrolysis of GDL, sodium alginate and Ca 2+ Slow cross-linking produces a uniform, stable gel, such as Figure 2 As shown in the figure, the hardness, viscosity, chewiness and adhesiveness of SSC3 gel gradually increased with the increase of gel time within 24 hours, while its elasticity and cohesion did not change significantly; the SSC3 gel was subjected to 3D printing, freeze drying and water annealing to obtain the gel scaffold of Example 6; Figure 3 As shown, the compression modulus of the bracket of Example 6 is 131.34 Pa, the strength is relatively low, and the support is insufficient, and the printed product deviates from the ideal model.

[0083] Example 7

[0084] A method for preparing a 3D printed starch-based gel scaffold for cultured meat production, the specific steps are as follows:

[0085] (1) Add 5 g of starch to 80 mL of distilled water and stir thoroughly until there are no particles to form a starch-based gel suspension.

[0086] (2) The starch suspension is sealed and placed in a water bath at 80° C. for 30 minutes to form a starch gel after sufficient gelatinization, and the starch-based gel after the water bath is cooled to room temperature (25° C.).

[0087] (3) Add 1 g of sodium alginate and 1 g of gelatin to the starch gel, stir evenly, and let stand for 24 hours to allow the starch to age to a certain extent, thereby obtaining a starch-based gel.

[0088] (4) 0.375 g of GDL (gluconolactone) and 0.075 g of nano calcium carbonate particles were added to the starch-based gel, stirred evenly, and homogenized at 10,000 rpm to remove bubbles introduced during the mixing process.

[0089] (5) The SSC3 gel obtained in (4) was 3D printed at a filling rate of 75%. After 24 hours of gelation, the 3D printed gel scaffold of Example 7 was obtained by freeze drying and annealing in water at 121°C for 15 minutes.

[0090] like Figure 1 As shown in the figure, SSC3 gel has shear thinning and solid-like behavior; and through the slow hydrolysis of GDL, sodium alginate and Ca 2+ Slow cross-linking produces a uniform, stable gel, such as Figure 2 As shown in the figure, the hardness, viscosity, chewiness and adhesiveness of SSC3 gel gradually increased with the increase of gel time within 24 hours, while its elasticity and cohesion did not change significantly; the SSC3 gel was subjected to 3D printing, freeze drying and water annealing to obtain the gel scaffold of Example 7; Figure 3 As shown, the compression modulus of the bracket of Example 7 is 107.95 Pa, the strength is relatively low, and the printed product deviates from the ideal model.

[0091] Comparative Example 1

[0092] A method for preparing a starch-based gel scaffold for cultured meat production, the specific steps are as follows:

[0093] (1) Add 5 g of starch to 80 mL of distilled water and stir thoroughly until there are no particles to form a starch-based gel suspension.

[0094] (2) The starch suspension is sealed and placed in a water bath at 80° C. for 30 minutes to form a starch gel after sufficient gelatinization, and the starch-based gel after the water bath is cooled to room temperature (25° C.).

[0095] (3) Add 1 g of sodium alginate and 1 g of gelatin to the starch gel, stir evenly, and let stand for 24 hours to allow the starch to age to a certain extent, thereby obtaining a starch-based gel.

[0096] (4) 0.375 g GDL (gluconolactone) and 0.075 g nano calcium carbonate particles were added to the starch-based gel, stirred evenly, and homogenized at 10,000 rpm to remove bubbles introduced during the mixing process.

[0097] (5) The SSC3 gel obtained in (4) was placed in a 96-well plate and allowed to gel naturally for 4 h. The gel scaffold of Comparative Example 1 was obtained by freeze drying and annealing in water at 121° C. for 15 min.

[0098] like Figure 1 As shown in the figure, SSC3 gel has shear thinning and solid-like behavior; and through the slow hydrolysis of GDL, sodium alginate and Ca 2+ Slow cross-linking produces a uniform, stable gel, such as Figure 2As shown in the figure, the hardness, viscosity, chewiness and adhesiveness of SSC3 gel gradually increased with the increase of gel time within 24 hours, while its elasticity and cohesion did not change significantly; the SSC3 gel was freeze-dried and water-annealed to obtain the comparative example 1 scaffold; Figure 3 As shown, the compression modulus of the bracket of comparative example 1 reaches 113.55Pa; and the water stability of the bracket of comparative example 1 is average ( Figure 4 ) and 1128.00±52.00% swelling capacity ( Figure 5 );Depend on Figure 5 It can be seen that the pore size distribution of the scaffold in comparative example 1 is concentrated, with about 43.23% of the pore size distributed in the range of 120-160 μm and accounting for about 74.63% of the pore area; 4 After C2C12 muscle cells were inoculated into the scaffold of comparative example 1, Figure 6 It can be seen that the cells proliferated stably from 1d, 4d to 7d, and their number increased from 28612.17±549.50, 35278.50±366.50 to 58020.67±13.00. After 7d of differentiation, immunofluorescence staining showed that C2C12 muscle cells differentiated, and the differentiation was poor, indicating that the comparative example 1 scaffold did not support the differentiation of C2C12 muscle cells.

[0099] According to the above embodiments, comparative examples and Table 1, Figure 1 , Figure 2 , Figure 3 , Figure 4 , Figure 5 , Figure 6 , Figure 7 , Figure 8It can be seen from the comparison that starch-based gel scaffolds with different calcium ion contents and 3D printing processes will change the texture characteristics of each gel and the compression modulus, water stability, swelling, pore size distribution, pore area, and cell proliferation and differentiation of the gel scaffold; among them, the starch gel obtained by 0.375g GDL, 0.075g nano calcium carbonate particles, 1g sodium alginate, 1g gelatin, 5g starch, and 80mL distilled water is subjected to a 3D printing process with a filling rate of 50%, and the compression modulus of the generated starch-based gel scaffold is the best, which is 246.76Pa; the filling rate of the 3D printing process will affect the compression modulus of the gel scaffold. The higher / lower the compression modulus, the closer / farther the taste of the cultured meat generated is to muscle tissue. Its water stability capacity is between the embodiments and the comparative examples, and the swelling is 466.23±14.33%, which will ensure the integrity of the scaffold during cell inoculation, proliferation, and differentiation. The pore size distribution between 80-120μm will better support the proliferation and differentiation of cells; while the pore size that is too large / small will seriously affect the growth of cells; within 1d, 4d to 7d after inoculating C2C12 myoblasts on the scaffold, the number of cells increased from 33331.67±634.00, 44727.17±734.50 to 64169.67±915.00; and after 7d of differentiation culture, the differentiation was optimal. Therefore, when the starch-based gel is 3D printed to obtain a gel scaffold, its compression modulus is 246.76Pa, the swelling capacity is 466.23±14.33%, and the pore size of the gel scaffold is mostly 80-120um. After inoculating C2C12 myoblasts, the proliferation and differentiation of cells on the scaffold are optimal.

[0100] Table 1 Starch-based gel formulations with different calcium ion contents.

[0101]

[0102] Note: The molar ratio of nano-calcium carbonate particles to GDL was always controlled at 1:2.

Claims

1. A method for preparing a 3D printed starch-based gel scaffold for cultured meat production, characterized in that: include: (1) Add water to 5 g of starch and stir thoroughly until there are no particles to form a starch suspension; (2) The starch suspension is sealed in a water bath, fully gelatinized, and cooled to form a starch gel; (3) Add 1 g of sodium alginate and 1 g of gelatin to the starch gel, stir evenly, and let stand to allow the starch to age, thereby obtaining a starch-based gel; (4) adding GDL and 0.05 g or 0.075 g or 0.100 g of nano calcium carbonate particles into the starch-based gel, stirring evenly, and removing bubbles in the mixing process by homogenization to obtain a homogenized starch-based gel; The molar ratio of GDL to nano calcium carbonate is 2:1; (5) The homogenized starch-based gel obtained in step (4) is subjected to 3D printing and gelation, and then freeze-dried and water annealed to obtain a 3D printed starch-based gel scaffold for cultured meat production, wherein the filling rate of the 3D printing is set at 50%.

2. The method for preparing a 3D printed starch-based gel scaffold for cultured meat production according to claim 1, characterized in that: In step (1), the mass ratio of starch to water is 5:70-90.

3. The method for preparing a 3D printed starch-based gel scaffold for cultured meat production according to claim 1, characterized in that: In step (2), the gelatinization time is 30-90 minutes and the gelatinization temperature is 70-80°C.

4. The method for preparing a 3D printed starch-based gel scaffold for cultured meat production according to claim 1, characterized in that: In step (3), the standing time is 18h~30h.

5. The method for preparing a 3D printed starch-based gel scaffold for cultured meat production according to claim 1, characterized in that: In step (4), the homogenization speed is 6000-10000 rpm.

6. The method for preparing a 3D printed starch-based gel scaffold for cultured meat production according to claim 1, characterized in that: In step (5), the gelling time is 18 to 30 hours, the freeze drying time is 36 to 60 hours, the water annealing temperature is 116 to 126° C., and the water annealing time is 10 to 30 minutes.

7. The method for preparing a 3D printed starch-based gel scaffold for cultured meat production according to claim 1, characterized in that: In step (5), 3D printing is performed using a 3D food printer.

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