Konjac glucomannan-pea protein isolate-based scaffold and preparation method and application thereof

Abstract

This application discloses a scaffold based on konjac glucomannan and pea protein isolate, its preparation method, and its application, belonging to the fields of tissue engineering scaffolds and cultured meat technology. The scaffold is a three-dimensional porous scaffold formed by compounding konjac glucomannan and pea protein isolate under alkaline heat-induced conditions to form a hydrogel, followed by freeze-drying. The preparation method includes steps such as mixing and dissolving KGM and PPI, adding an alkaline reagent to induce gelation, molding, and freeze-drying. The scaffold possesses non-animal origin, high porosity (>70%), low degradation rate (<10% after 30 days), and excellent biocompatibility, effectively promoting the adhesion, proliferation, directional alignment, and myogenic differentiation of myoblasts. The resulting cultured meat closely resembles real meat in texture. The scaffold material in this application is widely available, cost-effective, and simple to prepare, possessing enormous application potential in the field of cultured meat.

Description

Technical Field

[0001] This application belongs to the field of tissue engineering scaffolds and cell cultured meat technology, specifically relating to a scaffold based on konjac glucomannan-pea protein isolate, its preparation method and application. Background Technology

[0002] With global population growth and increasing demand for meat, traditional livestock farming faces resource pressures and environmental challenges. Cultured meat, as an emerging meat production technology, produces edible meat tissue by differentiating animal stem cells in vitro, offering a new approach to solving future meat supply problems.

[0003] In the production of cultured meat, the development of three-dimensional (3D) scaffolds is a crucial step. Scaffolds act as a support network, enabling cell attachment and facilitating the transport of oxygen and nutrients to form structured tissue. Ideal cultured meat scaffolds should possess good biocompatibility, suitable mechanical strength, edibility, and be of non-animal origin to meet ethical and cost requirements.

[0004] Currently, a variety of materials are used to construct scaffolds. For example, animal-derived proteins such as collagen and gelatin support cell adhesion, but they suffer from limited availability, high cost, and contradict the original intention of non-animal-derived cultured meat. Plant-derived proteins, such as pea protein isolate (PPI), have attracted attention due to their high nutritional value and low allergenicity, but scaffolds constructed from these single materials often have inferior texture properties. Konjac glucomannan (KGM) is a natural high-molecular-weight polysaccharide with good gelling and film-forming properties, but pure KGM gels have low cell compatibility, which is detrimental to cell adhesion.

[0005] Therefore, how to effectively combine plant proteins with polysaccharides to develop a non-animal-derived edible scaffold with both excellent mechanical properties and biocompatibility remains a technical problem that urgently needs to be solved in this field. Summary of the Invention

[0006] 1. The problem to be solved The technical problem to be solved by this application is to overcome one of the defects of existing cell culture meat scaffold materials being animal-derived and having poor performance of single materials (such as poor cell compatibility of pure konjac glucomannan and insufficient mechanical strength of pure pea protein isolate), and to provide a non-animal-derived, biocompatible, mechanically stable and edible composite porous scaffold.

[0007] 2. Technical Solution To solve the above problems, the technical solution adopted in this application is as follows: This application provides a scaffold based on konjac glucomannan-pea protein isolate, the scaffold comprising konjac glucomannan and pea protein isolate, wherein the mass ratio of konjac glucomannan to pea protein isolate is 1:2 to 1:8.

[0008] Furthermore, in the aforementioned scaffold based on konjac glucomannan and pea protein isolate, the mass ratio of konjac glucomannan to pea protein isolate is 1:2 to 1:6.

[0009] Furthermore, in the aforementioned scaffold based on konjac glucomannan and pea protein isolate, the mass ratio of konjac glucomannan to pea protein isolate is 1:4 to 1:6.

[0010] Furthermore, in the aforementioned scaffold based on konjac glucomannan and pea protein isolate, the mass ratio of konjac glucomannan to pea protein isolate is 1:6.

[0011] Furthermore, the aforementioned scaffold based on konjac glucomannan-pea protein isolate has a porosity greater than 70%. Even further, the aforementioned scaffold based on konjac glucomannan-pea protein isolate has a porosity greater than 80%.

[0012] Furthermore, the degradation rate of the above-mentioned scaffold based on konjac glucomannan-pea protein isolate was less than 10% after soaking in phosphate buffer (pH=7.2) for 30 days.

[0013] Furthermore, the degradation rate of the above-mentioned scaffold based on konjac glucomannan-pea protein isolate is less than 5% after soaking in phosphate buffer (pH=7.2) for 14 days.

[0014] Furthermore, the scaffold based on konjac glucomannan-pea protein isolate has a swelling ratio greater than 1500%.

[0015] This application also provides a method for preparing the above-mentioned scaffold based on konjac glucomannan-pea protein isolate. The method involves compounding konjac glucomannan and pea protein isolate under alkaline heat-induced conditions to form a hydrogel, and then freeze-drying it to form a three-dimensional porous scaffold.

[0016] Furthermore, the preparation method of the above-mentioned scaffold based on konjac glucomannan-pea protein isolate includes the following steps: S1: Mix konjac glucomannan and pea protein isolate in a certain proportion, add them to a solvent, heat and stir until completely dissolved to obtain a konjac glucomannan-pea protein isolate mixed solution; S2: Add an alkaline reagent to the mixed solution obtained in S1, stir quickly to mix well, and obtain a gelation precursor solution; S3: Inject the gelation precursor solution obtained in S2 into the mold and let it stand to form a hydrogel; S4: The hydrogel obtained in S3 was pre-frozen and then freeze-dried to obtain a (porous) scaffold based on konjac glucomannan-pea protein isolate.

[0017] Furthermore, in S1 above, the solvent is deionized water.

[0018] Furthermore, in S1 above, the heating and stirring temperature is 70~90℃, and the time is 2~6 hours.

[0019] Furthermore, in S1 above, the heating and stirring temperature is 80°C, and the time is 4 hours.

[0020] Furthermore, in S2 above, the alkaline reagent is one or more of sodium carbonate, sodium bicarbonate, or sodium hydroxide, and its final concentration in the mixed solution is 0.05~0.2 M. As a further explanation of this application, the role of the alkaline reagent is to induce the deacetylation of konjac glucomannan, forming a thermally irreversible gel.

[0021] Furthermore, in S2 above, the final concentration of the alkaline reagent in the mixed solution is 0.1 M.

[0022] Furthermore, in S3 above, standing is done at room temperature for 0.5 to 2 hours.

[0023] Furthermore, in the above S4, pre-freezing includes: pre-freezing at 4°C for 10 to 15 hours, and then freezing at -20°C for 20 to 30 hours.

[0024] Furthermore, in the above S4, pre-freezing includes: pre-freezing at 4°C for 12 hours, and then freezing at -20°C for 24 hours.

[0025] Furthermore, in S4 above, the freeze-drying time is 24-72 hours. Even further, in S4 above, the freeze-drying time is 48 hours.

[0026] This application also provides the application of the above-mentioned scaffold based on konjac glucomannan-pea protein isolate in the preparation of cell-cultured meat products.

[0027] Furthermore, the aforementioned cell-cultured meat includes cell-cultured muscle.

[0028] Furthermore, the aforementioned cells include animal myoblasts.

[0029] This application also provides a cell-cultured meat product comprising the aforementioned scaffold based on konjac glucomannan-pea protein isolate.

[0030] Furthermore, the aforementioned cell-cultured meat includes cell-cultured muscle.

[0031] 3. Technical Effects Compared with the prior art, the advantages of this application are as follows: (1) The scaffold based on konjac glucomannan-pea protein isolate provided in this application, its preparation method and application, have the following characteristics: Non-animal origin: This application uses konjac glucomannan (plant source) and pea protein isolate (plant source) as raw materials, completely avoiding the use of animal-derived materials, which meets the ethical requirements and sustainable development concept of the cell cultured meat industry; Excellent biocompatibility: By combining konjac glucomannan with pea protein isolate, the poor cell adhesion of the KGM scaffold alone is overcome; the introduction of pea protein isolate provides cells with more adhesion sites, and the composite scaffold significantly promotes the adhesion, proliferation, and orientation of C2C12 myoblasts. Figure 3 , Figure 4 ); Suitable physicochemical properties and mechanical strength: A gel network is formed by the thermal alkali-induced deacetylation of KGM, and the network is further developed through hydrogen bonding between KGM and PPI and possible Maillard reactions. Figure 1 (FT-IR analysis) constructed a stable three-dimensional network structure; the obtained scaffold has high porosity (>70%), good swelling performance (>1500%), and low degradation rate (<10% after 30 days), which can provide stable physical support for long-term cell culture; Supporting myoblastic differentiation: The scaffolds prepared in this application, particularly the scaffold with a KGM to PPI mass ratio of 1:6 (KP3), have a porous structure (average pore size >150 μm) and mechanical properties that are more conducive to the fusion of C2C12 cells to form myotubes, and highly express myoblast-related proteins (Myosin) and genes (…). MyHC , MYH4 () Figure 5 , Figure 6 The resulting cell-cultured meat showed no significant difference in texture characteristics (such as chewiness, cohesion, and elasticity) from real pork tenderloin. Figure 7 ).

[0032] (2) The preparation method of the scaffold based on konjac glucomannan-pea protein isolate provided in this application is simple and cost-controllable: the preparation process of this application does not involve organic solvents or toxic crosslinking agents, is easy to operate, has a wide range of raw material sources, is low in cost, and is easy to scale up. Attached Figure Description

[0033] Figure 1The figures shown are physicochemical performance test results of different ratios of KGM / PPI stents in the embodiments of this application, where: A is a top view of the porous stent; B is a side view of the porous stent; C is the expansion rate of the porous stent; D is the porosity of the porous stent; E is the degradation rate of the porous stent; and F is the FT-IR of the porous stent (from top to bottom, they are PPI, KGM-P, KP4, KP3, KP2, KP1, and KGM).

[0034] Figure 2 These are scanning electron microscope (SEM) images and average pore sizes of the raw materials and KGM / PPI porous scaffolds with different ratios in the embodiments of this application, where: APD is the average pore size, and 500 μm and 200 μm are scale bars.

[0035] Figure 3 This application presents the cell viability of C2C12 cells on KGM / PPI scaffolds at different ratios in the embodiments of this application. Specifically: A shows the staining of live / dead cells of C2C12 cells on the scaffold after 24 hours of culture; colors: green: calcein-AM, live cells; red: propidium iodide, dead cells (scale bar: 750 μm); B shows the staining of live / dead cells on the porous scaffold after 7 days of culture (scale bar: 750 μm); C shows a magnified image of live / dead cells on a portion of the porous scaffold after 7 days of culture (scale bar: 250 μm); D shows the cell adhesion rate 24 hours after cell seeding on the scaffold; E shows the live / dead cell count 24 hours after cell seeding on the porous scaffold; and F shows the live / dead cell count of C2C12 cells 7 days after seeding on the porous scaffold.

[0036] Figure 4 This is a scanning electron microscope image of C2C12 cells cultured for 7 days on KGM / PPI scaffolds with different ratios in the embodiments of this application.

[0037] Figure 5 The images shown in this application are immunofluorescence staining images (A) and statistical graphs (B) of fusion index and maturity index of C2C12 cells differentiated at different times on KP1 and KP3 scaffolds. The horizontal axes of B and C in the figures are KP1-DD1, KP3-DD1, KP1-DD4, KP3-DD4, KP1-DD7, and KP3-DD7, respectively.

[0038] Figure 6The figures shown in this application are Western Blot (A, B) results of C2C12 cells differentiated on KP1 and KP3 scaffolds at different times, and qRT-PCR results 7 days after differentiation (C). In the figures, the x-axis of B is C2C12-D2, KP1-DD1, KP1-DD4, KP1-DD7, KP3-DD1, KP3-DD4, and KP3-DD7, respectively, and the x-axis of C is C2C12-D2, KP1-DD7, and KP3-DD7, respectively.

[0039] Figure 7 These are texture profile analysis (TPA) results of different groups of scaffolds and cultured meat in the embodiments of this application, where: A is the appearance image; B is the texture comparison of the blank scaffold, namely hardness, adhesiveness, chewiness, adhesion, cohesion, elasticity and resilience, with the horizontal axis being KGM, KP1, KP2, KP3 and KP4 respectively; C is the texture comparison with pork tenderloin, namely hardness, adhesiveness, chewiness, adhesion, cohesion, elasticity and resilience, with the horizontal axis being Pork, KP1-D14, KP3-D14, CM1 and CM3 respectively. Detailed Implementation

[0040] The present application will be further described below with reference to specific embodiments.

[0041] It should be noted that terms such as "upper", "lower", "left", "right", and "middle" used in this specification are only for clarity of description and are not intended to limit the scope of implementation. Changes or adjustments to their relative relationships, without substantially altering the technical content, should also be considered as within the scope of this application.

[0042] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains; the term “and / or” as used herein includes any and all combinations of one or more of the associated listed items.

[0043] Unless otherwise specified in the examples, the procedures should be performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0044] As used herein, the term “about” is used to provide for the flexibility and imprecision associated with a given term, measure, or value. Those skilled in the art can readily determine the degree of flexibility for a particular variable. As used herein, the term “at least one of…” is intended to be synonymous with “one or more of…”. For example, “at least one of A, B, and C” explicitly includes only A, only B, only C, and combinations thereof.

[0045] Concentration, amount, and other numerical data may be presented in range format herein. It should be understood that such range format is used solely for convenience and brevity and should be flexibly interpreted to include not only the values ​​explicitly stated as the limits of the range, but also all individual values ​​or subranges encompassed within the range, as if each value and subrange were explicitly stated. For example, a range of values ​​from about 1 to about 4.5 should be interpreted to include not only the explicitly stated limits of 1 to 4.5, but also individual numbers (such as 2, 3, 4) and subranges (such as 1 to 3, 2 to 4, etc.). The same principle applies to ranges that describe only a single value, such as "less than about 4.5," which should be interpreted to include all the values ​​and ranges described above. Furthermore, this interpretation should apply regardless of the breadth of the range or characteristic described.

[0046] In this application, konjac glucomannan (KGM, purity ≥95%, viscosity >15000 mPa) and pea protein isolate (PPI, purity ≥90%) were purchased from Shanghai Maclean Biochemical Co., Ltd.

[0047] Example 1 This embodiment provides a scaffold based on konjac glucomannan-pea protein isolate and its preparation method.

[0048] In this embodiment, the mass ratios of konjac glucomannan (KGM) and pea protein isolate (PPI) in the scaffold are 1:2, 1:4, 1:6 and 1:8, respectively.

[0049] Preparation methods include: Table 1. Raw material ratio of each group of stents

[0050] Konjac glucomannan (KGM) and pea protein isolate (PPI) were mixed in the proportions shown in Table 1, added to 100 mL of deionized water, and stirred in an 80°C water bath at 400 rpm for 4 hours until completely dissolved to obtain a konjac glucomannan-pea protein isolate mixed solution.

[0051] Weigh anhydrous sodium carbonate (Na2CO3) and add it to the mixed solution to make its final concentration in the mixed solution 0.1 M. Dissolve it with a small amount of hot water and then quickly add it to the above konjac glucomannan-pea protein isolate mixed solution and stir quickly and evenly to obtain the gelation precursor solution.

[0052] Immediately inject the gelation precursor solution into each 24-well cell culture plate at a rate of 1 mL and allow it to stand at room temperature for 1 hour to form a hydrogel.

[0053] 24-well cell culture plates were pre-frozen at 4°C for 12 hours, then transferred to a -20°C freezer for 24 hours, and finally freeze-dried in a freeze dryer for 48 hours to obtain cylindrical porous scaffolds based on konjac glucomannan-pea protein isolate. The scaffolds were named according to the KGM to PPI mass ratio: KGM group (1:0), KP1 group (1:2), KP2 group (1:4), KP3 group (1:6), and KP4 group (1:8). Their macroscopic appearance is as follows: Figure 1 China A and Figure 1 As shown in B.

[0054] Example 2 This embodiment provides performance characterization of each group of stents obtained in Embodiment 1.

[0055] (1) Porosity determination The porosity of the stent was measured using the ethanol displacement method. Under isothermal conditions, the total mass of the specific gravity bottle filled with ethanol is denoted as W1. The mass of W... s The dry stent was completely immersed in ethanol and ultrasonically degassed until the ethanol completely filled the pores of the stent. Then, the specific gravity bottle was refilled with ethanol, and the total mass was recorded as W2. The stent immersed in ethanol was removed, and the remaining ethanol and the specific gravity bottle were weighed as W3. The porosity was calculated using the following formula: Porosity = [(W2)] W3 W s ) / (W1 W3)]×100%.

[0056] The results are as follows Figure 1 As shown in Figure D, the porosity of all scaffolds is greater than 70%, and the porosity of the konjac glucomannan-pea protein isolate complex group (KP3-KP4) is significantly higher than that of the pure KGM group.

[0057] (2) Determination of swelling rate Weigh the lyophilized support (Wd), immerse it in PBS, remove it at different time points, blot the surface moisture with filter paper, and weigh it again (Ws). Swelling rate (%) = (Ws-Wd) / Wd × 100%.

[0058] The results are as follows Figure 1 As shown in Figure C, all stents reached swelling equilibrium within 12 hours, with a swelling rate of >1500%.

[0059] (3) Degradation rate determination The scaffold was immersed in PBS (pH=7.2) for 24 h and weighed as W0. It was then placed in a 37℃ incubator and weighed at 7, 14, and 30 days, respectively. The degradation rate (%) = (W0-Wt) / W0×100%.

[0060] The results are as follows Figure 1As shown in Figure E, all stents exhibited a degradation rate of less than 5% within 14 days.

[0061] (4) Fourier transform infrared (FT-IR) analysis FT-IR analysis of lyophilized samples was performed using the KBr pellet method.

[0062] The results are as follows Figure 1 As shown in Figure F, compared to KGM powder (KGM-P), KGM gel exhibits a higher concentration at 1732 cm⁻¹. -1 The disappearance of the characteristic acetyl peak at the acetyl group indicates successful deacetylation. The red shift of the OH peak in the composite scaffold and the change in the peak position of the amide I band confirm the formation of hydrogen bonds between KGM and PPI.

[0063] (5) Scanning electron microscopy analysis The lyophilized samples were analyzed using scanning electron microscopy.

[0064] The results are as follows Figure 2 As shown, after freeze-drying, both KGM and KGM-PPI composite scaffolds formed relatively uniform honeycomb-like porous three-dimensional network structures, which are highly suitable for cell growth and proliferation. The average pore size (APD) of different scaffold groups was measured. The results showed that, except for group KP2 which showed no significant difference from KGM, the average pore size of the other groups (KP1, KP3, KP4) was significantly higher than that of the KGM group. Figure 2 As shown, the microstructure of the KGM, KP1, and KP3 scaffolds was observed at a higher magnification (scale bar 200 μm). Compared with the KGM group, it can be clearly seen that the pores of KP1 and KP3 are more three-dimensional and clearer.

[0065] Example 3 This embodiment provides a biocompatibility evaluation of the scaffolds obtained in Example 1.

[0066] (1) Cell inoculation and culture Place the sterilized scaffold in a 6-well plate and seed each well with 100 μL of C2C12 cell suspension (containing 2×10⁶ cells). 5 Cells were incubated at 37°C for 4 hours to promote cell attachment, followed by the addition of 400 μL of growth medium (DMEM + 10% FBS + 1% penicillin-streptomycin). The medium was changed every 2 days, and the culture was carried out for 7 days.

[0067] (2) Cell adhesion rate 24 hours after cell seeding, the culture medium was collected and the number of unattached cells was counted. Cell adhesion rate (%) = (total number of seeded cells - number of unattached cells) / total number of seeded cells × 100%.

[0068] The results are as follows Figure 3As shown in Figure D, the cell adhesion rate on each scaffold exceeded 80%, with the composite group (KP1-KP4) showing an even higher adhesion rate.

[0069] (3) Cell viability and morphology Cell viability was observed using Calcein-AM / PI staining. The scaffolds were stained on day 1 and day 7 of culture, and observed under a fluorescence microscope.

[0070] The results are as follows Figure 3 China A and Figure 3 As shown in Figure B, the number of live cells (green) on each scaffold far exceeded the number of dead cells (red), indicating that the scaffolds were non-cytotoxic. On day 7, the cell density on the composite groups (KP1-KP4) scaffolds was significantly higher than that on the KGM group, and the cells were more evenly distributed. Scanning electron microscopy (SEM) Figure 4 Further analysis showed that the cells grew well on the composite scaffold, spreading out and arranging in a certain direction.

[0071] Example 4 This embodiment provides the application of the scaffold provided in this application in myogenic differentiation.

[0072] In this embodiment, the cells are C2C12 cells.

[0073] In this embodiment, KP1 and KP3 scaffolds with good biocompatibility were selected for differentiation experiments.

[0074] After 7 days of proliferation culture, the scaffold was replaced with differentiation medium (DMEM / F-12 + 2% horse serum + 1% penicillin-streptomycin), and half of the medium was changed every 2 days. The culture was continued for another 7 days.

[0075] (1) Immunofluorescence staining Scaffolds were harvested on days 1, 4, and 7 of differentiation. After fixation, permeabilization, and blocking, the scaffolds were incubated with Myosin primary antibody, followed by incubation with fluorescent secondary antibody, phalloidin (F-actin), and DAPI. The scaffolds were then observed using a laser confocal microscope.

[0076] The results are as follows Figure 5 As shown, both scaffolds support myotube formation. On day 7 of differentiation, the myotube fusion index (82.56%) and maturity index (77.89%) on the KP3 scaffold were significantly higher than those on the KP1 scaffold.

[0077] (2) Western Blot analysis Total protein was extracted from cells at different differentiation time points, and the expression of Myosin was detected.

[0078] The results are as follows Figure 6 China A and Figure 6As shown in Figure B, the expression level of Myosin protein increased significantly with the extension of differentiation time, and the expression level in the KP3 group was higher than that in the KP1 group.

[0079] (3) qRT-PCR analysis Total RNA was extracted from cells 7 days after differentiation, and myoblast-related genes were detected. MyoD , MyoG , MyHC , MYH4 The expression.

[0080] The results are as follows Figure 6 As shown in Figure C, compared with proliferating cells cultured on a 2D scaffold, the expression of myoblast genes in cells differentiated on the scaffold was significantly upregulated, and in the KP3 group... MyHC and MYH4 The expression level of the group was significantly higher than that of the KP1 group.

[0081] Example 5 This embodiment provides the application of the scaffold of this application in the preparation of cultured meat.

[0082] TPA tests were performed on KP1 and KP3 scaffolds without cell inoculation, and cell-cultured meat (named CM1 and CM3, respectively) formed after 14 days of cell inoculation (7 days of proliferation culture and 7 days of differentiation). Fresh pork tenderloin was used as a control.

[0083] The results are as follows Figure 7 As shown, the scaffolds (CM1, CM3) after cell culture showed no significant difference from pork tenderloin in terms of chewiness, cohesion, elasticity, and resilience, indicating that their textural properties are close to those of real meat.

[0084] Example 6 This embodiment provides an exploration of the formation mechanism of the konjac glucomannan-pea protein isolate composite scaffold in this application.

[0085] Based on the experimental results of the above embodiments, the composite stent formation mechanism of this application is as follows: Figure 7 As shown, under hot alkaline conditions, the acetyl groups on the KGM molecular chain are removed (deacetylation), increasing intermolecular entanglement and forming a physically cross-linked network. Simultaneously, the hydroxyl groups on the KGM chain interact with the amino groups on the PPI molecular chain (e.g., hydrogen bonding, possibly including preliminary Maillard reactions), forming a more compact and stable three-dimensional network structure. The introduction of PPI not only acts as a cross-linking enhancer but also provides cells with more adhesion recognition sites.

[0086] In summary, this application successfully constructed a composite porous scaffold based on konjac glucomannan and pea protein isolate. This scaffold is simple to prepare, uses safe and edible raw materials, possesses good physicochemical properties and excellent biocompatibility, and can effectively support the adhesion, proliferation, and directed differentiation of myoblasts, forming cell-cultured meat with a texture similar to real meat. Among these, the scaffold with a KGM to PPI mass ratio of 1:6 (KP3) exhibits superior pore structure and differentiation-promoting performance, showing broad application prospects.

[0087] The above description is merely a preferred embodiment of this application and is not intended to limit the scope of protection of this application. Any simple modifications, alterations, or equivalent changes made to the above embodiments based on the technical essence of this application shall still fall within the protection scope of the technical solution of this application.

Claims

1. Konjac glucomannan-pea protein isolate based scaffold, characterized in that, The scaffold comprises konjac glucomannan and pea protein isolate, wherein the mass ratio of konjac glucomannan to pea protein isolate is 1:2 to 1:

8.

2. The scaffold based on konjac glucomannan-pea protein isolate according to claim 1, characterized in that, The mass ratio of konjac glucomannan to pea protein isolate is 1:2 to 1:6, or 1:4 to 1:6, or 1:

6.

3. The scaffold based on konjac glucomannan-pea protein isolate according to claim 2, characterized in that, The porosity of the scaffold is greater than 70%; and / or the degradation rate after immersion in phosphate buffer for 30 days is less than 10%.

4. The scaffold based on konjac glucomannan-pea protein isolate according to claim 3, characterized in that, The scaffold has a porosity greater than 80% and / or a degradation rate of less than 5% after immersion in phosphate buffer for 14 days.

5. The method for preparing the scaffold based on konjac glucomannan-pea protein isolate according to any one of claims 1-4, characterized in that, The method involves compounding konjac glucomannan and pea protein isolate under alkaline heat-induced conditions to form a hydrogel, which is then freeze-dried to form a three-dimensional porous scaffold.

6. The method for preparing the scaffold based on konjac glucomannan-pea protein isolate according to claim 5, characterized in that, The preparation method includes the following steps: S1: Mix konjac glucomannan and pea protein isolate in a certain proportion, add them to a solvent, heat and stir until completely dissolved to obtain a konjac glucomannan-pea protein isolate mixed solution; S2: Add an alkaline reagent to the mixed solution obtained in S1, stir and mix well to obtain a gelation precursor solution; S3: Inject the gelation precursor solution obtained in S2 into the mold and let it stand to form a hydrogel; S4: The hydrogel obtained in S3 was pre-frozen and then freeze-dried to obtain a scaffold based on konjac glucomannan-pea protein isolate.

7. The method for preparing the scaffold based on konjac glucomannan-pea protein isolate according to claim 6, characterized in that, In step S1, the heating and stirring temperature is 70~90℃, and the time is 2~6 hours; In S2, the alkaline reagent is one or more of sodium carbonate, sodium bicarbonate, or sodium hydroxide, and its final concentration in the mixed solution is 0.05~0.2 M. In step S3, standing is done at room temperature for 0.5 to 2 hours; In step S4, pre-freezing includes: pre-freezing at 4°C for 10-15 hours, followed by freezing at -20°C for 20-30 hours; the freeze-drying time is 24-72 hours.

8. The application of the scaffold based on konjac glucomannan-pea protein isolate as described in any one of claims 1-4 in the preparation of cell-cultured meat products.

9. The application according to claim 8, characterized in that, The cells include animal myoblasts.

10. A cell-cultured meat product, characterized in that, The cell-cultured meat product includes the scaffold based on konjac glucomannan-pea protein isolate as described in any one of claims 1-4.

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