Edible beefsteak scaffold based on vegetable protein and construction method of edible beefsteak scaffold

Abstract

The invention discloses an edible beefsteak scaffold based on vegetable protein and a construction method thereof, and belongs to the fields of tissue engineering, material science, food science and biology. The edible beefsteak scaffold is a 3D printing scaffold based on vegetable protein, combined with gellan gum and sodium alginate, and is a cell culture meat bionic beefsteak composed of a bovine fibroblast and bovine muscle satellite cell co-culture system dynamically constructed by using a rotary culture system. The selected materials are all edible protein and polysaccharide, have high biocompatibility and safety, and are wide in source; the mode of dynamically constructing the bionic beefsteak can effectively reduce the formation of local necrotic areas, and the bionic beefsteak has excellent physical characteristics, has texture structures and flavor characteristics similar to those of real beefsteak, is sterile in surface, and can significantly reduce food sanitation and safety problems. The scheme provided by the invention has remarkable advantages in the aspects of food safety, texture regulation and control, sensory simulation and the like, and provides important technical support for the field of cell culture meat.

Description

Technical Field

[0001] This invention belongs to the fields of tissue engineering, materials science, food science and biology, and relates to an edible steak scaffold based on plant protein and its construction method, particularly to an edible cell-cultured meat biomimetic steak scaffold based on plant proteins such as mung bean protein isolate, pea protein isolate and peanut protein isolate, combined with gellan gum and sodium alginate and its construction method. Background Technology

[0002] With population growth, increased personal economic benefits, and rising living standards, the demand for meat is increasing year by year. However, global livestock production capacity is nearing saturation, and traditional livestock farming will struggle to meet the growing consumption and nutritional needs. Furthermore, traditional livestock production exposes problems such as resource waste, environmental pollution, the spread of animal diseases, and ethical concerns. Therefore, the meat production industry urgently needs an environmentally friendly and ethical meat alternative to address the current meat crisis.

[0003] Cell-cultured meat technology is a novel interdisciplinary technology based on cell biology and tissue engineering. It obtains complete cultured meat through cell-scaffold culture and tissue shaping processes and is considered one of the most promising solutions to future meat production challenges and consumption dilemmas. This technology simulates the three-dimensional microenvironment of natural muscle tissue using tissue-engineered scaffolds, providing adhesion sites for cells to support their directional alignment, proliferation, and differentiation. Bio-3D printing technology, with its advantages of high precision, strong material adaptability, personalized customization, and reproducible production, has become a key technology for constructing tissue-engineered scaffolds and has been widely applied in the field of regenerative medicine. It has also opened up new avenues for manufacturing cell-cultured meat scaffolds that simulate real meat.

[0004] Plant proteins such as mung bean protein isolate (MBPI), pea protein isolate (PPI), and arachis hypogaea protein isolate (AHPI) have become candidate materials for cell culture scaffolds due to their wide availability, low cost, rich amino acid composition, high nutritional value, and non-animal origin. However, single plant protein-based bioinks suffer from poor mechanical properties and rheological instability in 3D printing, making it difficult to achieve high-precision bioprinting. Sodium alginate (Alg) and gellan gum (GG) are two low-cost natural polysaccharides. Alginate has good biocompatibility, and its rapid ionic cross-linking properties help maintain the structural stability of the scaffold after molding; gellan gum's shear-thinning properties can significantly improve the printability of bioinks. The introduction of both can enable bioinks to have both higher printing precision and interlayer adhesion strength. The fibrous network of plant proteins, the flexible network of sodium alginate, and the rigid segments of gellan gum together form an interpenetrating network structure, which can significantly improve the biomechanical properties of the scaffold. This not only provides a mechanical microenvironment that matches muscle tissue for cell growth, but also lays the foundation for simulating the textural properties of real meat.

[0005] The generation of natural muscle tissue is complex, requiring the synergistic action of multiple cell types. Bovine fibroblasts (FIBs) are the main cells for connective tissue generation, while bovine satellite cells (BSCs) proliferate and differentiate into myotubes and muscle fibers. Co-culturing these cells promotes stable cell proliferation and differentiation. The resulting connective and muscle tissues mimic the complex structure of collagen and myofiber in natural muscle, thereby improving the texture and mouthfeel of cultured meat products. In tissue engineering applications, while biomimetic scaffolds can mimic the structural characteristics of the extracellular matrix and provide a suitable microenvironment for cell growth, prolonged static culture can affect mass transfer within the scaffold, leading to hypoxic / nutrient-deficient necrotic zones and hindering further tissue development. Rotation systems of culture (RSOCs), due to their unique low-shear microgravity environment and efficient mass transfer characteristics, have been proven to effectively maintain the long-term culture requirements of tissue constructs, providing a powerful solution to the mass transfer problem in the process of constructing biomimetic steaks from cell culture.

[0006] Sodium alginate and gellan gum have been widely recognized and applied in the field of tissue engineering. However, edible tissue-engineered composite steak scaffolds based on plant proteins such as mung bean protein isolate, pea protein isolate, and peanut protein isolate have not yet been reported. This invention constructs a 3D-printed tissue-engineered scaffold based on the above-mentioned plant proteins combined with a certain proportion of gellan gum and sodium alginate, and utilizes RSOC to dynamically construct a cell-cultured biomimetic steak composed of a co-culture system of bovine fibroblasts and bovine muscle satellite cells. Testing shows that this biomimetic steak exhibits good mechanical strength, effectively avoids localized cell necrosis, possesses the sensory and textural characteristics of real steak, and has a sterile surface, ensuring food safety and providing technical support for commercial applications. Summary of the Invention

[0007] To address the problems existing in the prior art, this invention provides an edible steak scaffold based on plant protein and its construction method. The invention aims to establish a method for constructing an edible steak scaffold based on plant protein and its application in food engineering. The edible cell-cultured meat biomimetic steak prepared by this method has good mechanical strength, real steak sensory and textural characteristics, can effectively avoid local cell necrosis areas, has a sterile surface, and ensures food safety.

[0008] To achieve the above objectives, the technical solution of the present invention is as follows:

[0009] An edible steak scaffold based on plant protein, wherein the edible steak scaffold is based on plant protein, combined with gellan gum and sodium alginate 3D printed scaffold, and dynamically constructed using a rotary culture system to form a cell culture meat biomimetic steak composed of a co-culture system of bovine fibroblasts and bovine muscle satellite cells.

[0010] Furthermore, the plant protein includes mung bean protein isolate (MBPI), pea protein isolate (PPI), peanut protein isolate (AHPI) or other plant proteins, preferably mung bean protein isolate (MBPI).

[0011] A method for constructing an edible steak scaffold based on plant protein, comprising the following steps:

[0012] The first step, the preparation of 3D printing ink, includes the following steps:

[0013] Step 1.1: Add plant protein to distilled water and stir to dissolve at 20-100℃ and 50-300 rpm to obtain a plant protein solution;

[0014] Step 1.2: After centrifuging the plant protein solution at 500-5000 rpm for 5-50 min to remove air bubbles, add gellan gum GG and stir to dissolve at 20-100℃ and 50-300 rpm to obtain a mixture; wherein, add the corresponding amount to each portion of plant protein solution.

[0015] Step 1.3: Add sodium alginate Alg to the mixture and stir to dissolve at 20-100℃ and 50-300rpm. Centrifuge at 500-5000rpm for 5-50min to remove air bubbles to obtain fat block ink. Add beetroot food coloring agent to the fat block ink to obtain muscle block ink.

[0016] Furthermore, in the first step, 1-20g of plant protein (step 1.1), 1-5g of gellan gum GG (step 1.2), and 0.5-3g of sodium alginate Alg (step 1.3) are added to every 100mL of distilled water.

[0017] Furthermore, in step 1.3, the beetroot food coloring agent added to the fat block ink has a mass-volume fraction of 1-5%.

[0018] Furthermore, in step 1.3, when the plant protein is mung bean protein isolate (MBPI), the fat block ink is MBPI / GG / Alg, abbreviated as MGA; when the plant protein is pea protein isolate (PPI), the fat block ink is PPI / GG / Alg, abbreviated as PGA; and when the plant protein is peanut protein isolate (AHPI), the fat block ink is AHPI / GG / Alg, abbreviated as HGA.

[0019] The second step, 3D printing of the steak support, includes the following steps:

[0020] Step 2.1: Load the ink cartridges containing the muscle block ink and fat block ink prepared in Step 1 into the bio-3D printer, install the printing needle with an inner diameter of 100-1200μm, set the steak support model size to 10-100mm×10-100mm×2-20mm on the computer, the material filling spacing to 0.2-2mm, the material layer thickness to 0.1-1mm, adjust the nozzle temperature to 10-40℃, the platform temperature to 5-30℃, the extrusion pressure to 0.1-1.0MPa, and the printing speed to 1-50mm / s, and run the printer to print the support.

[0021] Furthermore, in step 2.1, when the plant protein is mung bean protein isolate (MBPI), the scaffold is an MGA scaffold; when the plant protein is pea protein isolate (PPI), the scaffold is a PGA scaffold; and when the plant protein is peanut protein isolate (AHPI), the scaffold is an HGA scaffold.

[0022] Step 2.2: At room temperature, the printed scaffold is placed in a CaCl2 solution for cross-linking for 1–24 h, and then freeze-dried to obtain a steak scaffold.

[0023] Furthermore, the CaCl2 solution in step 2.2 has a mass-volume fraction of 1-10%.

[0024] Furthermore, in step 2.2, the freeze-drying temperature is -20 to -50°C, and the time is 6 to 48 hours.

[0025] The third step involves dynamically co-culturing to construct a biomimetic steak made from cell-cultured meat, which includes the following steps:

[0026] Step 3.1: After sterilizing the steak supports prepared in step 2 with 75% alcohol and ultraviolet light, inoculate each support with 1–10 × 10⁻⁶ cells / mL. 6 Bovine fibroblasts and bovine muscle satellite cells were added to complete culture medium and statically cultured at 37°C and 5% CO2 for 6–48 h.

[0027] Step 3.2: Transfer the steak scaffold inoculated with cells from Step 3.1 to a rotary culture system for dynamic culture. Set the rotation speed to 10-100 rpm and culture dynamically for 1-28 days while ensuring that the steak scaffold does not move and only the culture medium flows, thereby constructing a cell cultured meat biomimetic steak.

[0028] The edible cell-cultured meat biomimetic steak prepared using the above method has good mechanical strength, the sensory and textural properties of real steak, can effectively avoid local cell necrosis areas, has a sterile surface, and ensures food safety.

[0029] The advantages of this invention compared to the prior art are as follows:

[0030] (1) The edible steak scaffold prepared in this invention is a 3D-printed scaffold based on plant proteins such as mung bean protein isolate, pea protein isolate, and peanut protein isolate, combined with gellan gum and sodium alginate. All three plant proteins, gellan gum, and sodium alginate are edible proteins and polysaccharides with high biocompatibility and biosafety, and are widely available. The use of bio-3D printing technology enables the rapid and precise construction of steak scaffolds, laying the foundation for the large-scale production of edible steak scaffolds.

[0031] (2) The edible steak scaffold prepared by this invention is beneficial for the co-culture of FIB and BSC, promotes cell adhesion and proliferation, and can effectively improve the efficiency of nutrient delivery in the microenvironment of RSOC system and enhance cell metabolism. On day 7, the content of glucose consumed and lactic acid produced by metabolism in the dynamic culture environment (14.62±0.89mmol / L and 9.56±0.55mmol / L, respectively) is significantly lower than and higher than that in the static culture conditions (16.56±0.70mmol / L and 6.50±0.14mmol / L, respectively), which can reduce the formation of local necrotic areas.

[0032] (3) The edible steak scaffold prepared by this invention has excellent physical properties, with a porosity of 76.33±2.82%, good hydrophilicity, a water contact angle of 44.68±2.30° at 0s, and a swelling rate of 477.52±102.71%. This structured cell-cultured steak exhibits juiciness similar to real steak. At the same time, this cell-cultured steak has good mechanical properties, with a compression modulus of 1.20±0.16MPa, which is not significantly different from that of natural steak (compression modulus of 1.24±0.25MPa).

[0033] (4) The cell-cultured beef steak prepared by this invention has a similar texture and flavor characteristics to real beef steak. Its appearance and sensory characteristics before and after frying are highly similar to those of real beef steak, and its surface is sterile (the bacterial colony count on the surface of real beef steak is 7.05 × 10⁻⁶). 5 (CFU / g) can significantly reduce food hygiene and safety issues.

[0034] In summary, the solution proposed in this invention has significant advantages in terms of food safety, texture control, and sensory simulation, providing important technical support for the field of cell-cultured meat. Attached Figure Description

[0035] Figure 1 The surface morphology of three types of steak supports 3D printed: MGA, PGA, and HGA; Figure (a) shows the MGA of Example 1; Figure (b) shows the PGA of Example 2; Figure (c) shows the HGA of Example 3.

[0036] Figure 2 The cross-sectional morphology of three steak supports (MGA, PGA, and HGA) printed by 3D printing is shown in Figure 1. (a) shows the MGA of Example 1; (b) shows the PGA of Example 2; and (c) shows the HGA of Example 3.

[0037] Figure 3 The porosity of the three types of steak supports (MGA, PGA, and HGA) 3D printed in Examples 1-3;

[0038] Figure 4 The water contact angles of the 3D-printed MGA, PGA, and HGA steak supports in Examples 1-3;

[0039] Figure 5 The swelling properties of the 3D-printed steak supports of MGA, PGA and HGA in Examples 1-3;

[0040] Figure 6 The water absorption rates of the three types of steak supports (MGA, PGA, and HGA) 3D printed in Examples 1-3 are shown.

[0041] Figure 7 The images show the liveness / death staining of FIB cells cultured on three types of steak scaffolds (MGA, PGA, and HGA) on day 7. Figure (a) shows MGA from Example 1; Figure (b) shows PGA from Example 2; and Figure (c) shows HGA from Example 3.

[0042] Figure 8 SEM characterization of FIB cells on three types of 3D-printed steak scaffolds: MGA, PGA, and HGA; Figure (a) shows MGA in Example 1; Figure (b) shows PGA in Example 2; Figure (c) shows HGA in Example 3;

[0043] Figure 9 CCK detection for different time periods of co-culturing BSC and FIB on MGA steak supports in Example 1;

[0044] Figure 10 The staining results for cell life and death after 7 days of static and dynamic culture on an MGA steak scaffold in Example 1;

[0045] Figure 11 The concentration of lactic acid produced by cells in Example 1 during static and dynamic culture on an MGA steak scaffold for 7 days;

[0046] Figure 12 The concentration of glucose consumed by cells in Example 1 during 7 days of static and dynamic culture on an MGA steak scaffold;

[0047] Figure 13 Figure 1 shows the structural design and printing example of the steak support in Example 1; Figure (a) is a picture of the actual steak; Figure (b) is a 3D printed steak structure designed by AutoCAD; Figure (c) is a 3D printed steak support; Figure (d) is a 3D printed steak assembly mold designed by AutoCAD; Figure (e) is a picture of the actual 3D printed steak assembly mold; Figure (f) shows steak supports of different thicknesses printed by 3D.

[0048] Figure 14The images show the cell-cultured steak prepared in Example 1 using the ROSC system and its appearance before and after frying. Figure (a) shows the assembly process of the structured steak (a1: 3D printed steak scaffold; a2: culturing the scaffold inoculated with cells in the RSOC system; a3: cell-cultured meat after 7 days of culture; a4: cross-linking assembly of cell-cultured meat in a mold); Figure (b) shows the actual structured steak after cell culture (b1: front; b2: side; b3: back); Figure (c) shows the actual structured steak after frying (c1: front; c2: side; c3: back).

[0049] Figure 15 This refers to the moisture loss after frying the real steak and cultured meat in Example 1;

[0050] Figure 16 The compression modulus of the real steak, cultured meat, and simple scaffold in Example 1;

[0051] Figure 17 The figures show the bacterial colony distribution on the surfaces of real steak, cultured meat, and simple scaffold in Example 1; Figure (a) shows real steak (natural meat); Figure (b) shows cultured meat; Figure (c) shows simple scaffold.

[0052] Figure 18 This study analyzed the number of bacterial colonies on the surfaces of real steak, cultured meat, and simple scaffolds in Example 1. Detailed Implementation

[0053] The present invention will now be described in further detail with reference to specific embodiments and accompanying drawings. It should be understood that these descriptions are only for further illustrating the features and advantages of the present invention, and not for limiting the scope of the patent claims of the present invention. Those skilled in the art can make some non-essential improvements and adjustments based on the content of the present invention.

[0054] Example 1

[0055] (1) Weigh 1g of MBPI, add 100mL of distilled water, and stir to dissolve at 20℃ and 50rpm.

[0056] (2) The obtained solution was centrifuged at 500 rpm for 5 min to remove air bubbles, and 1 g of GG was added. The solution was stirred and dissolved at 20℃ and 50 rpm.

[0057] (3) Weigh and add 0.5g of Alg, stir and dissolve at 20℃ and 50rpm, centrifuge at 500rpm for 5min to remove air bubbles to obtain MGA fat block ink, and add 1% beetroot food coloring to obtain MGA muscle block ink.

[0058] (4) Insert the ink cartridges containing MGA muscle block ink and MGA fat block ink into the bio 3D printer, install the printing needle with an inner diameter of 100μm, set the steak support model size to 10mm×10mm×2mm, the material filling spacing to 0.2mm, the material layer thickness to 0.1mm, adjust the nozzle temperature to 10℃, the platform temperature to 5℃, the extrusion pressure to 0.1MPa, and the printing speed to 1mm / s, and run the printer to print the MGA support.

[0059] (5) The printed MGA bracket was placed in a CaCl2 solution with a mass volume fraction of 1% for cross-linking for 1 hour, and then freeze-dried at -20℃ for 6 hours to obtain the MGA steak bracket.

[0060] (6) After sterilizing the MGA steak supports prepared in step (5) with 75% alcohol and ultraviolet light, each support is inoculated with 1×10⁻⁶ mol / L. 6 Bovine fibroblasts and bovine muscle satellite cells were added to complete culture medium and statically cultured at 37°C and 5% CO2 for 6 hours.

[0061] (7) The MGA steak scaffold inoculated with cells in step (6) was transferred to a rotary culture system for dynamic culture. The rotation speed was set to 10 rpm. The scaffold was dynamically cultured for 1 day under the condition that the scaffold did not move and only the culture medium flowed, thus constructing the MGA cell culture meat biomimetic steak.

[0062] Example 2

[0063] (1) Weigh 10g of PPI, add 100mL of distilled water, and stir to dissolve at 50℃ and 100rpm.

[0064] (2) The obtained solution was centrifuged at 2000 rpm for 20 min to remove bubbles, and 2 g of GG was added. The solution was stirred and dissolved at 50℃ and 100 rpm.

[0065] (3) Weigh and add 1g of Alg, stir and dissolve at 50℃ and 100rpm, remove bubbles by centrifugation at 2000rpm for 20min to obtain PGA fat block ink, and add 2% beetroot food coloring to obtain PGA muscle block ink.

[0066] (4) Insert the ink cartridges containing PGA muscle block ink and PGA fat block ink into the bio 3D printer, install the printing needle with an inner diameter of 600μm, set the steak support model size to 50mm×50mm×10mm, the material filling gap to 1mm, the material layer thickness to 0.5mm, adjust the nozzle temperature to 20℃, the platform temperature to 10℃, the extrusion air pressure to 0.5MPa, the printing speed to 20mm / s, and run the printer to print the PGA support.

[0067] (5) The printed PGA scaffold was placed in a CaCl2 solution with a mass volume fraction of 5% for cross-linking for 12 hours, and then freeze-dried at -30℃ for 24 hours to obtain the PGA steak scaffold.

[0068] (6) After sterilizing the PGA steak supports prepared in step (5) with 75% alcohol and ultraviolet light, inoculate each with 5×10 6 Bovine fibroblasts and bovine muscle satellite cells were added to complete culture medium and statically cultured at 37°C and 5% CO2 for 24 hours.

[0069] (7) The PGA steak scaffold inoculated with cells in step (6) was transferred to a rotary culture system for dynamic culture. The rotation speed was set to 50 rpm. The scaffold was dynamically cultured for 14 days under the condition that the scaffold did not move and only the culture medium flowed, thus constructing a PGA cell culture meat biomimetic steak.

[0070] Example 3

[0071] (1) Weigh 20g of AHPI, add 100mL of distilled water, and stir to dissolve at 100℃ and 300rpm.

[0072] (2) The obtained solution was centrifuged at 5000 rpm for 50 min to remove air bubbles, and 5 g of GG was added. The solution was stirred and dissolved at 100℃ and 300 rpm.

[0073] (3) Weigh and add 3g of Alg, stir and dissolve at 100℃ and 300rpm, remove bubbles by centrifugation at 5000rpm for 50min to obtain HGA fat block ink, and add 5% beetroot food coloring to obtain HGA muscle block ink.

[0074] (4) Insert the ink cartridges containing HGA muscle block ink and HGA fat block ink into the bio 3D printer, install the printing needle with an inner diameter of 1200μm, set the steak support model size to 100mm×100mm×20mm, the material filling gap to 2mm, the material layer thickness to 1mm, adjust the nozzle temperature to 40℃, the platform temperature to 30℃, the extrusion pressure to 1.0MPa, the printing speed to 50mm / s, and run the printer to print the HGA support.

[0075] (5) The printed HGA scaffold was placed in a CaCl2 solution with a mass-volume fraction of 10% for cross-linking for 24 hours, and then freeze-dried at -50℃ for 48 hours to obtain the HGA steak scaffold.

[0076] (6) After sterilizing the HGA steak supports prepared in step (5) with 75% alcohol and ultraviolet light, each support is inoculated with 1×10⁻⁶ mol / L. 7Bovine fibroblasts and bovine muscle satellite cells were added to complete culture medium and statically cultured at 37°C and 5% CO2 for 48 hours.

[0077] (7) The HGA steak scaffold inoculated with cells in step (6) was transferred to a rotary culture system for dynamic culture. The rotation speed was set to 100 rpm. The scaffold was dynamically cultured for 28 days under the condition that the scaffold did not move and only the culture medium flowed, thus constructing HGA cell culture meat biomimetic steak.

[0078] Performance testing and characterization:

[0079] (1) Macroscopic and microscopic morphology of the three types of stents, MGA, PGA and HGA, prepared in Examples 1-3;

[0080] Macroscopic and surface microscopic morphology of three types of steak supports are as follows: Figure 1 The cross-sectional micromorphology is as follows Figure 2 As shown in the figure. Observation of the three steak scaffolds reveals that the scaffolds have a uniform porous structure, and the rough surface morphology provides adhesion sites for cells, which is conducive to cell adhesion, proliferation, and extension on the scaffold surface. Figure 1 Furthermore, the cross-sectional view reveals the interconnected pore structure of the support structure. Figure 2 It can provide growth space for cells, endow the scaffold with excellent water absorption properties, construct a biomimetic three-dimensional structure, and provide a microenvironmental condition similar to in vivo growth for cell proliferation and growth.

[0081] (2) Performance of the three types of stents, MGA, PGA and HGA, prepared in Examples 1-3;

[0082] The physical properties of the three types of steak supports, such as porosity, water contact angle, swelling rate, and water absorption rate, are as follows: Figure 3 , Figure 4 , Figure 5 and Figure 6 As shown. The porosities of the three steak supports obtained by this invention are 70.29±1.29%, 75.69±0.45%, and 76.33±2.82%, respectively. Figure 3 This is beneficial for the scaffold's moisturizing properties and provides ample space for cell proliferation; it also exhibits excellent hydrophilicity, with water contact angles of 44.68±2.30°, 47.78±2.25°, and 48.09±1.42° at 0s, respectively. Figure 4 This causes it to swell rapidly. Figure 5 The three steak scaffolds prepared in this invention exhibited high water absorption rates of 477.52±102.71%, 431.14±14.26%, and 376.78±27.71%, respectively, providing a favorable scaffold wetting microenvironment for cells. The biocompatibility of the three steak scaffolds prepared in this invention is as follows: Figure 7 and Figure 8As shown, the excellent physical properties of the scaffold give it good biocompatibility, which is beneficial to cell adhesion and promotes cell proliferation.

[0083] (3) Co-culture of BSC and FIB on the MGA steak support prepared in Example 1;

[0084] CCK assays of BSC and FIB co-cultured on MGA steak supports for different time periods are shown below. Figure 9 As shown, co-culture of cells promotes cell adhesion, proliferation, and growth more effectively than single-cell culture. Therefore, the MGA steak scaffold prepared in this invention is beneficial for the co-culture of FIB and BSC cells. In a rotary culture bioreactor, RSOC dynamic culture, compared to the cell cluster necrosis area indicated by red fluorescence signals in a static culture system, allows for effective convection and diffusion, promoting the transport of nutrients around and within the scaffold, providing stable nutrients and oxygen to the cells, resulting in better cell growth. Figure 10 As shown.

[0085] The metabolic state of cells in static and RSOC dynamic culture systems, respectively, is as follows: Figure 11 and Figure 12 As shown, the efficient diffusion and high mass transfer efficiency in the RSOC dynamic system accelerate the uptake of nutrients and the excretion of metabolic products by cells, forming a continuous and efficient metabolic cycle system. Therefore, compared with the static culture system, the RSOC dynamic culture used in this invention produces higher concentrations of lactate in cells (9.56±0.55 mmol / L and 6.50±0.14 mmol / L, respectively). Figure 11 ), and the remaining low concentrations of glucose (14.62±0.89 mmol / L and 16.56±0.70 mmol / L, respectively) Figure 12 ).

[0086] (4) Construction and characterization of structured cell culture beef steak in Example 1;

[0087] Example diagram of the structural design and printing of the steak support. Figure 13 As shown, a partitioned modeling strategy was adopted, dividing the fat portion into 3 independent blocks and the muscle portion into 5 blocks. AutoCAD was used to design 3D printing scaffold models for different blocks. Muscle blocks were printed using muscle block ink, and fat blocks were printed using fat block ink. The construction process of the structured cell cultured beef steak and its appearance before and after frying are shown in the figure. Figure 14As shown, a porous steak scaffold was prepared using a partitioned printing strategy. BSC and FIB cells were seeded on the scaffold and dynamically cultured in an RSOC system. Subsequently, cross-linking and assembly were performed to form a cell-cultured steak. The morphology and sensory characteristics of the steak before and after frying were highly similar to those of real steak, and the moisture loss after frying was significantly higher (p < 0.05) than that of real steak. Figure 15 This demonstrates that the cell-cultured beef steak prepared according to the present invention has juiciness similar to that of real beef steak. Simultaneously, the cell-cultured beef steak prepared according to the present invention exhibits good mechanical properties. Figure 16 The compressive modulus of the cultured beef was 1.20 ± 0.16 MPa, which was not significantly different from that of natural beef steak (compressive modulus 1.24 ± 0.25 MPa), demonstrating the technical feasibility of this invention in simulating the chewing characteristics of real beef steak. Furthermore, the cell-cultured beef steak produced by this invention exhibited sterile surface conditions. Figure 17 (Note: The text contains some inconsistencies and unclear grammatical structures, which have been omitted from the translation.) The actual bacterial count on the surface of a steak can reach 7.05 × 10⁻⁶. 5 CFU / g ( Figure 18 This demonstrates that the cell-cultured beef steak prepared by this invention has significant advantages in microbial safety indicators compared to real beef steak, and exhibits excellent food hygiene and safety reliability.

[0088] The above embodiments and descriptions illustrate the basic principles and main features of the present invention, as well as its advantages. Those skilled in the art should understand that the present invention is not limited to the above embodiments; the embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications made without departing from the spirit and scope of the invention are all within the protection scope of the present invention.

Claims

1. An edible steak support based on plant protein, characterized in that, The edible steak scaffold described is a cell-cultured meat biomimetic steak composed of bovine fibroblasts and bovine muscle satellite cells, dynamically constructed using a rotary culture system based on plant protein, combined with gellan gum and sodium alginate in a 3D-printed scaffold.

2. The edible steak support based on plant protein according to claim 1, characterized in that, The plant proteins include mung bean protein isolate (MBPI), pea protein isolate (PPI), peanut protein isolate (AHPI), or other plant proteins.

3. The edible steak support based on plant protein according to claim 1, characterized in that, The plant protein is preferably mung bean protein isolate (MBPI).

4. A method for constructing an edible steak scaffold based on plant protein as described in any one of claims 1-3, characterized in that, Includes the following steps: The first step, the preparation of 3D printing ink, includes the following steps: Step 1.1: Add plant protein to distilled water and stir to dissolve under certain conditions to obtain a plant protein solution; Step 1.2: After centrifuging the plant protein solution to remove air bubbles, add gellan gum GG and stir to dissolve under certain conditions to obtain a mixture; wherein, add the corresponding amount to each portion of plant protein solution. Step 1.3: Add sodium alginate Alg to the mixture, stir and dissolve under certain conditions, remove air bubbles by centrifugation to obtain fat block ink, add beetroot food coloring agent to fat block ink to obtain muscle block ink. The second step, 3D printing of the steak support, includes the following steps: Step 2.1: Insert the ink cartridges containing the muscle block ink and fat block ink prepared in the first step into the bio-3D printer to print the support frame. Step 2.2: At room temperature, the printed scaffold is placed in a CaCl2 solution for cross-linking for 1–24 h, and then freeze-dried to obtain a steak scaffold. The third step involves dynamically co-culturing to construct a biomimetic steak made from cell-cultured meat, which includes the following steps: Step 3.1: After sterilizing the steak scaffold prepared in the second step, inoculate it with bovine fibroblasts and bovine muscle satellite cells, add complete culture medium, and statically culture it under certain conditions; Step 3.2: Transfer the steak scaffold from which cells were inoculated in Step 3.1 to a rotary culture system for dynamic culture to construct a cell-cultured meat biomimetic steak.

5. The method for constructing an edible steak scaffold based on plant protein according to claim 4, characterized in that, In the first step, 1-20g of plant protein, 1-5g of gellan gum GG, and 0.5-3g of sodium alginate Alg are added to every 100mL of distilled water; in step 1.3, the mass-volume fraction of beetroot food coloring added to the fat block ink is 1-5%.

6. The method for constructing an edible steak scaffold based on plant protein according to claim 4, characterized in that, In the first step: In step 1.1, the mixture is stirred and dissolved at 20–100°C and 50–300 rpm. In step 1.2, the mixture is stirred and dissolved at 20–100°C and 50–300 rpm; the centrifugation speed is 500–5000 rpm and the time is 5–50 min. In step 1.3, the mixture is stirred and dissolved at 20–100°C and 50–300 rpm; the centrifugation speed is 500–5000 rpm and the time is 5–50 min.

7. The method for constructing an edible steak scaffold based on plant protein according to claim 4, characterized in that, In step 2.1, the specific parameters of the bio-3D printer are as follows: install a printing needle with an inner diameter of 100-1200μm, set the steak support model size on the computer to 10-100mm×10-100mm×2-20mm, the material filling spacing to 0.2-2mm, the material layer thickness to 0.1-1mm, adjust the nozzle temperature to 10-40℃, the platform temperature to 5-30℃, the extrusion pressure to 0.1-1.0MPa, and the printing speed to 1-50mm / s.

8. A method for constructing an edible steak scaffold based on plant protein according to claim 4, characterized in that, The CaCl2 solution in step 2.2 has a mass-volume fraction of 1-10%; the freeze-drying temperature is -20 to -50°C; and the time is 6-48 hours.

9. A method for constructing an edible steak scaffold based on plant protein according to claim 4, characterized in that, In step 3.1, the steak support is sterilized by alcohol and ultraviolet light; inoculated with 1–10 × 10 6 Bovine fibroblasts and bovine muscle satellite cells were statically cultured at 37°C and 5% CO2 for 6–48 h.

10. A method for constructing an edible steak scaffold based on plant protein according to claim 4, characterized in that, In step 3.2, the dynamic culture specifically involves setting the rotation speed to 10-100 rpm and dynamically culturing for 1-28 days while ensuring that the steak support does not move and only the culture medium flows.

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