A kind of flavone-three-dimensional stent synergistic regulation based on bovine cell culture meat and its construction method

Abstract

The application discloses a kind of based on flavone-three-dimensional support synergic regulation and control cattle cell culture meat and its construction method, belong to cell culture meat and tissue engineering technical field.The application first applies quercetin and 3,2'-dihydroxy flavone respectively in the proliferation stage and differentiation stage of bovine muscle stem cells, and is combined with the edible three-dimensional porous support constructed by konjac glucomannan and sodium alginate, establishes a kind of phased, synergic culture meat construction system;Quercetin promotes bovine muscle stem cell proliferation;3,2'-dihydroxy flavone promotes myogenic differentiation;Konjac glucomannan and sodium alginate support provide biomimetic three-dimensional microenvironment.Experiments prove that the method of the application can significantly improve the efficiency of myotube formation, the degree of tissue culture meat and product weight, and improve the nutritional quality of culture meat, with the advantages of low cost, edible, high food safety and easy industrial production, etc., which provides a new technical scheme for the construction of cell culture meat.

Description

Technical Field

[0001] This invention belongs to the field of cell-cultured meat and tissue engineering technology, and particularly relates to a bovine cell-cultured meat based on flavonoid-three-dimensional scaffold synergistic regulation and its construction method. Background Technology

[0002] Cultured meat is a novel food production technology that obtains edible muscle tissue by culturing animal cells in vitro. It offers advantages such as reduced environmental impact, less animal welfare controversy, and precise nutritional control. Currently, the cultured meat industry still faces challenges including low cell expansion efficiency, insufficient myogenic differentiation, imperfect tissue structure, and high culture costs. Muscle stem cells (MuSCs), as crucial seed cells in cultured meat, directly influence the yield and quality of the cultured meat due to their proliferation capacity and myogenic differentiation efficiency.

[0003] In existing technologies, growth factors, bioactive small molecules, and tissue engineering scaffolds are commonly used to regulate the function of muscle stem cells. However, the method using growth factors is costly and has poor stability. On the other hand, three-dimensional porous scaffolds, as an important carrier for constructing cell-cultured meat tissue, can simulate the natural extracellular matrix microenvironment and improve cell adhesion, migration, and myogenic differentiation efficiency. However, the scaffold materials currently used for cultured meat mainly include collagen, gelatin, and synthetic polymers, but these have problems such as high cost, limited availability, or inedibility.

[0004] Therefore, constructing high-quality, low-cost cultured meat has become an important research direction in the field of tissue engineering technology. Summary of the Invention

[0005] This invention provides a method for constructing bovine cell-cultured meat based on the synergistic regulation of flavonoids and a three-dimensional scaffold. It aims to address technical problems in existing technologies, such as low proliferation efficiency of bovine muscle stem cells, lack of a biomimetic three-dimensional microenvironment, inedible or high-cost scaffold materials, and lack of precise stage-based regulation. The method of this invention constructs a low-cost, edible three-dimensional culture system with good cell affinity, and achieves stage-based synergistic regulation of bovine muscle stem cell proliferation and myogenic differentiation, thereby improving the construction efficiency and quality of cell-cultured meat.

[0006] The technical solution of this invention is implemented as follows:

[0007] A method for constructing bovine cell-cultured meat based on the synergistic regulation of flavonoids and a three-dimensional scaffold includes the following steps:

[0008] S01. Preparation of three-dimensional porous scaffold: Konjac glucomannan (KGM) and sodium alginate (SA) were mixed and cross-linked and freeze-dried to prepare an edible three-dimensional porous scaffold;

[0009] S02. Cell seeding: Bovine muscle stem cells are seeded onto the three-dimensional porous scaffold;

[0010] S03. Regulation of proliferation stage: During the cell proliferation culture stage, quercetin (QUE) is added to the culture system to promote the proliferation of bovine muscle stem cells;

[0011] S04. Differentiation Stage Regulation: During the myoblastic differentiation stage, 3,2'-dihydroxyflavone (3,2'-DHF) is added to the culture system to promote the myoblastic differentiation of bovine MuSCs and form three-dimensional cell cultured meat tissue.

[0012] In some embodiments, in step S03, the concentration of quercetin added is 400 nM.

[0013] In some embodiments, in step S04, the concentration of the added 3,2'-dihydroxyflavone is 10 μM.

[0014] In some embodiments, the preparation of the three-dimensional porous scaffold specifically includes: mixing a 2% (w / v) konjac glucomannan solution and a sodium alginate solution at a volume ratio of 1:1 to obtain a composite solution; freezing and setting the composite solution, then performing displacement crosslinking with a 2% (w / v) CaCl2-ethanol solution, and finally freeze-drying to obtain the three-dimensional porous scaffold. The natural polysaccharide materials used in the preparation of the three-dimensional porous scaffold of this invention, such as konjac glucomannan and sodium alginate, have good biocompatibility and edibility; a purely plant-derived, edible three-dimensional culture scaffold is constructed by mixing konjac glucomannan and sodium alginate. The prepared three-dimensional porous scaffold has a highly interconnected pore structure, good swelling performance, good degradation performance, good compressive mechanical properties, and good cell compatibility; furthermore, this three-dimensional porous scaffold can effectively support the adhesion, proliferation, and myogenic differentiation of bovine muscle stem cells, improving the food safety and industrial application potential of the culture system.

[0015] Preferably, the mixing is performed by magnetic stirring at room temperature for 30 min; the CaCl2-ethanol solution is prepared from 75% ethanol; the pre-freezing in the pre-freezing and shaping process is pre-freezing in a -20℃ refrigerator for 12 h; the crosslinking time is 24 h; and the freeze-drying conditions are freeze-drying at -50℃ and a vacuum degree ≤10 Pa for 48 h.

[0016] In some embodiments, the method for preparing bovine muscle stem cells includes the following steps: taking bovine fetal longissimus dorsi muscle tissue, washing it with PBS and then cutting it into small pieces; digesting it sequentially with 0.2% type I collagenase for 70 min and 0.25% trypsin for 20-30 min; collecting the cells by centrifugation after filtration; purifying the bovine muscle stem cells using differential adhesion and culturing them at 37°C and 5% CO2; and passage culturing them when the cell confluence reaches 80% to obtain bovine muscle stem cells.

[0017] In some embodiments, the bovine muscle stem cells are cultured for 4 days during the proliferation phase, with quercetin added for 24 hours on day 1 of the proliferation phase; and for 7 days during the differentiation phase, with 3,2'-dihydroxyflavone added for 24 hours on day 1 of the differentiation phase. Quercetin promotes the proliferation of bovine muscle stem cells during the cell proliferation phase, and 3,2'-dihydroxyflavone promotes the myogenic differentiation of bovine muscle stem cells during the myogenic differentiation phase. Through the 4-day proliferation phase and 7-day differentiation phase, combined with the synergistic treatment of quercetin and 3,2'-dihydroxyflavone, precise phased regulation of bovine muscle stem cell proliferation and myogenic differentiation is achieved, improving the efficiency and tissue composition of cultured meat.

[0018] In some embodiments, the three-dimensional porous scaffold has a highly interconnected pore structure with a pore size ranging from 100 to 300 μm.

[0019] In some embodiments, the bovine muscle stem cells are seeded at a density of 1.0 × 10⁶ per scaffold. 5 Each cell.

[0020] This invention also provides bovine cell-cultured meat prepared by the above-described construction method.

[0021] This invention is the first to apply quercetin and 3,2'-dihydroxyflavone to the proliferation and differentiation stages of bovine muscle stem cells, respectively, and combines them with an edible three-dimensional porous scaffold constructed from konjac glucomannan and sodium alginate to establish a staged and synergistic cultured meat construction system. Specifically, quercetin promotes bovine muscle stem cell proliferation; 3,2'-dihydroxyflavone promotes myoblast differentiation; and the three-dimensional porous scaffold of konjac glucomannan and sodium alginate provides a three-dimensional growth microenvironment. This system can significantly improve myotube formation efficiency, the degree of tissue formation in cultured meat, and product weight, while also improving the nutritional quality of cultured meat.

[0022] Compared with the prior art, the technical solution of the present invention has the following advantages:

[0023] 1. This invention achieves precise phased regulation of bovine muscle stem cell proliferation and myogenic differentiation through 4-day proliferation culture and 7-day differentiation culture, combined with quercetin and 3,2'-dihydroxyflavone treatment, thereby improving the efficiency and tissue quality of cell-cultured meat construction.

[0024] 2. The natural polysaccharide materials used in the preparation of the three-dimensional porous scaffold of this invention, such as konjac glucomannan and sodium alginate, have good biocompatibility and edibility. By mixing konjac glucomannan and sodium alginate, a pure plant-derived, edible three-dimensional culture scaffold is constructed, which improves the food safety and industrial application potential of the culture system.

[0025] 3. This invention achieves a synergistic effect between flavonoids (quercetin and 3,2'-dihydroxyflavone) and a three-dimensional porous scaffold, significantly improving the weight of cultured meat and myotube formation ability. Furthermore, the two flavonoids mentioned above have the characteristics of low cost, high safety, and multi-target regulation, which can precisely regulate the proliferation and myogenic differentiation of bovine muscle stem cells on a three-dimensional porous scaffold.

[0026] 4. This invention reduces dependence on expensive growth factors and provides a low-cost, easily industrialized cell-cultured meat production solution. Attached Figure Description

[0027] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0028] Figure 1 This is a schematic diagram of the division of culture stages and the timeline of flavonoid treatment in the cell-cultured meat of this invention.

[0029] Figure 2 These are SEM images of the macroscopic morphology and microstructure of the KGM / SA three-dimensional porous scaffold in this embodiment of the invention.

[0030] Figure 3 This is a swelling rate curve of the KGM / SA three-dimensional porous scaffold in an embodiment of the present invention;

[0031] Figure 4 This is a degradation rate curve of the KGM / SA three-dimensional porous scaffold in an embodiment of the present invention;

[0032] Figure 5 This is a curve showing the compressive mechanical properties of the KGM / SA three-dimensional porous scaffold in an embodiment of the present invention.

[0033] Figure 6 This is an evaluation diagram of the proliferation capacity of bovine MuSCs in a three-dimensional porous scaffold in an embodiment of the present invention, where A is the CCK-8 detection result, B is the EdU staining result, and C is the Pax7 immunofluorescence staining result.

[0034] Figure 7 This is a fluorescence staining evaluation image of the myogenic differentiation ability of bovine MuSCs in a three-dimensional porous scaffold in an embodiment of the present invention;

[0035] Figure 8 This is an image showing the RT-qPCR detection results of the myogenic differentiation ability of bovine MuSCs in a three-dimensional porous scaffold in an embodiment of the present invention;

[0036] Figure 9 The figures show the construction and morphological observation results of the cell-cultured meat in this embodiment of the invention; where A: macroscopic morphology of the cell-cultured meat during the culture process, with the right side being a magnified image showing the enrichment of surface cell clusters; B: staining of cell nuclei in the cell-cultured meat, with blue representing cell nuclei; scale bar: 200 μm.

[0037] Figure 10 The images show the SEM morphological observation results of bovine MuSCs cultured on three-dimensional scaffolds during the proliferation and differentiation stages in this embodiment of the invention; where A: SEM of the cell proliferation phase (GM); B: SEM of the cell myogenic differentiation phase (DM); scale bars: 100μm, 20μm.

[0038] Figure 11 This is an image showing the immunofluorescence characterization results of cell cultured meat during the proliferation and differentiation stages in an embodiment of the present invention; where A: IF, detecting Pax7 positive cells, with red and blue representing Pax7 protein and cell nucleus, respectively; B: IF, detecting MyHC positive myotubes, with red and blue representing MyHC protein and cell nucleus, respectively; scale bar: 200μm, 50μm.

[0039] Figure 12 This figure shows the quantitative evaluation results of flavonoids on myoblast differentiation in a cell-cultured meat culture system according to embodiments of the present invention; where A: mRNA expression levels of myoblast differentiation marker genes (MyoD1, MyoG, MyHC) detected by RT-qPCR under 2D and 3D culture conditions (both with added flavonoids); B: mRNA expression levels of myoblast differentiation marker genes in the control group and flavonoid-treated group under 3D culture conditions detected by RT-qPCR; C: weight comparison of cell-cultured meat in the control group (without added flavonoids) and flavonoid-treated group under 3D culture conditions. N=3, ****P ≤ 0.0001, *P ≤ 0.05

[0040] Figure 13Figure 1 shows the macroscopic morphology and weight characterization results of the cultured meat in this embodiment of the invention; where A: Appearance comparison between cultured meat and blank scaffold; B: Weight statistics of cultured meat and blank scaffold; N=3, **P < 0.001.

[0041] Figure 14 The figures show the comparison of moisture content and morphology before and after drying between cell-cultured meat and natural beef in this embodiment of the invention; where A: morphological observation of adult bovine longissimus dorsi muscle, fetal bovine longissimus dorsi muscle, cell-cultured meat, and 3D blank scaffold before and after drying; B: comparison of moisture content between adult bovine longissimus dorsi muscle, fetal bovine longissimus dorsi muscle, cell-cultured meat, and 3D blank scaffold; N = 3, ****P≤0.0001, ***P≤0.001, **P≤0.01, *P≤0.05.

[0042] Figure 15 The diagram shows the nutritional composition analysis results in an embodiment of the present invention; where A: Nutritional composition table of cell-cultured meat; B: Nutritional composition table of 3D scaffold; C: Nutritional composition table of fetal bovine muscle tissue.

[0043] Figure 16 This is a graph showing the comparison of amino acid content between cultured meat and fetal bovine longissimus dorsi muscle in an embodiment of the present invention.

[0044] Figure 17 This is a graph showing the comparison of amino acid content between cultured meat and 3D blank scaffolds in an embodiment of the present invention. Detailed Implementation

[0045] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. In addition, the technical solutions of the various embodiments can be combined with each other, but this must be based on the ability of those skilled in the art to implement them. When the combination of technical solutions is contradictory or cannot be implemented, it should be considered that such combination of technical solutions does not exist and is not within the scope of protection claimed by the present invention.

[0046] Currently, existing bovine cell-cultured meat technologies suffer from several technical problems, including low proliferation efficiency of bovine muscle stem cells, lack of a biomimetic three-dimensional microenvironment, inedible or costly scaffold materials, and a lack of precise, phased control. To address these issues, this invention proposes a bovine cell-cultured meat method based on the synergistic regulation of flavonoids and a three-dimensional scaffold.

[0047] Example 1: Isolation and Culture of Bovine Muscle Stem Cells (MuSCs)

[0048] Bovine fetal longissimus dorsi muscle tissue was collected, washed with PBS, and then minced. The cells were digested sequentially with 0.2% type I collagenase for 70 min and 0.25% trypsin for 20-30 min. After filtration, the cells were collected by centrifugation. Bovine muscle stem cells were purified using differential adhesion and cultured at 37℃ and 5% CO2. When the cell confluence reached 80%, the cells were passaged to obtain bovine muscle stem cells.

[0049] Example 2: Preparation and characterization of konjac glucomannan-sodium alginate (KGM-SA) three-dimensional porous scaffold

[0050] Preparation and performance characterization of the three-dimensional porous scaffold in this embodiment: The three-dimensional porous scaffold was prepared by freeze-drying using konjac glucomannan and sodium alginate as raw materials. The microstructure was observed by scanning electron microscopy, and the swelling rate, degradation rate and compressive mechanical properties were measured to systematically evaluate its suitability as a cell culture substrate.

[0051] This embodiment provides an edible three-dimensional porous scaffold and its preparation method. Konjac glucomannan powder (purity ≥95%) and sodium alginate powder (purity ≥95%) were weighed separately and prepared into solutions with a mass concentration of 2% (w / v) using deionized water. The two solutions were mixed at a volume ratio of 1:1 and magnetically stirred at room temperature for 30 min to obtain a konjac glucomannan / sodium alginate composite solution. The composite solution was dispensed into 24-well plates, 0.5 g per well, and pre-frozen at -20℃ for 12 h. After pre-freezing, 1 mL of a 2% (w / v) CaCl2-ethanol solution (prepared with 75% ethanol) was added to each well, and crosslinking was performed at room temperature for 24 h. After crosslinking, the solution was discarded, and the scaffold was washed 5 times with PBS. The scaffold was placed in a freeze dryer and freeze-dried at -50℃ and a vacuum degree ≤10 Pa for 48 h to obtain a konjac glucomannan-sodium alginate (KGM-SA) three-dimensional porous scaffold.

[0052] In this embodiment, cell colonization was observed by DAPI staining; cell adhesion and morphological changes at different culture stages were observed by scanning electron microscopy (SEM); cell proliferation and differentiation status were verified by Pax7 and MyHC immunofluorescence staining; the expression level of myogenic differentiation marker genes was detected by RT-qPCR, and the weight of cultured meat was measured to systematically evaluate the synergistic differentiation-promoting effect of flavonoids and three-dimensional scaffolds.

[0053] The macroscopic morphology and microstructure of three-dimensional porous scaffolds are as follows: Figure 2 As shown, the konjac glucomannan-sodium alginate three-dimensional porous scaffold, after freeze-drying, presents a regular disc shape, approximately 2 cm in diameter and 0.5 cm in thickness. It is milky white to pale yellow in color, lightweight, and exhibits good integrity. Figure 2A). Scanning electron microscopy (SEM) observations showed that ( Figure 2 (B) The scaffold exhibits a typical three-dimensional porous network structure with uniformly distributed and interconnected pores. The pore size is mainly distributed in the range of 100-300 μm, which is suitable for cell adhesion, migration, and three-dimensional growth. The scaffold pores are irregular polygonal in shape, interconnected to form a continuous three-dimensional space. The directional growth of ice crystals during freeze-drying forms interconnected channels, providing pathways for the efficient transport of nutrients and metabolic waste, while ensuring the scaffold's porous structure and good material exchange capacity.

[0054] Swelling performance is an important indicator for evaluating the water absorption capacity and structural stability of a scaffold. The swelling rate test results of the three-dimensional porous scaffold prepared in this embodiment are as follows: Figure 3 As shown, in the initial soaking stage, the scaffold rapidly absorbed water, with the swelling rate quickly increasing from 0 to approximately 600%. After 12 hours, the rate of increase in swelling rate slowed significantly, eventually stabilizing at 700-720%, reaching a water absorption-release equilibrium. Within 24 hours to 5 days, the swelling rate increased by less than 5%, indicating that the scaffold structure was stable and would not expand or disintegrate after long-term soaking. After 5 days of soaking, the scaffold volume increased to 7-8 times, but the overall shape remained intact without any fragments falling off, further demonstrating the hydrogen bond cross-linking between konjac glucomannan and sodium alginate and the Ca2+ cross-linking... 2+ Ionic cross-linking is sufficient to maintain structural stability and can provide a stable three-dimensional support environment for cell-cultured meat.

[0055] The degradation performance of the scaffold directly affects its application in cultured meat. An ideal scaffold should maintain structural stability during the early stages of cell growth (0-7 days) to support cell adhesion and differentiation, while gradually degrading in the later stages (7-15 days). The degradation rate of the three-dimensional porous scaffold prepared in this example is as follows: Figure 4 As shown, the scaffold degradation rate was slow in the first 6 days, gradually increasing from 0% to 3%-5%. After 6 days, the degradation rate accelerated, reaching 6%-8% on day 9, 9%-10% on day 12, and 10%-12% on day 15. Throughout the degradation process, the scaffold volume slightly decreased, but the overall structure remained intact, without complete disintegration. These results indicate that the scaffold can maintain structural stability in the early stages of cell culture, meeting the needs of cell adhesion and growth, while moderate degradation in the later stages is beneficial for tissue maturation and nutrient transport.

[0056] Mechanical properties are a key indicator for evaluating whether a scaffold can provide adequate mechanical support for cells. A scaffold that is too soft cannot provide sufficient mechanical signals to cells, while one that is too stiff will restrict cell migration and differentiation. The compressive mechanical properties of the three-dimensional porous scaffold prepared in this embodiment are as follows: Figure 5As shown, compression tests of the scaffolds after soaking for 3, 6, 9, 12, and 15 days revealed no significant difference in compressive stress (P > 0.05) among the scaffolds soaked for different times, with the average compressive modulus remaining between 3.5 and 4.5 kPa, within the mechanical range of natural muscle tissue. After soaking for 3–15 days, the compressive strength of the scaffolds showed no significant change, indicating that the cross-linked network of the scaffolds remained stable in the hydrated state and would not undergo mechanical property degradation due to long-term culture. This is crucial for the preparation of cell-cultured meat requiring a 7–14 day culture cycle.

[0057] Example 3: Verification of the proliferation and differentiation capacity of bovine muscle stem cells in a three-dimensional scaffold

[0058] Bovine muscle stem cells were administered at a ratio of 1.0 × 10⁻⁶. 5 The density of individual scaffolds was seeded onto the scaffolds prepared in Example 2.

[0059] 3.1 Regulation of proliferation phase: During the cell proliferation culture phase, quercetin was added to the culture system to promote the proliferation of bovine muscle stem cells; the culture time during the proliferation phase was 4 days, of which quercetin was added and treated for 24 hours on the first day of the proliferation phase.

[0060] 3.2 Differentiation Stage Regulation: During the myogenic differentiation stage, 3,2'-dihydroxyflavone was added to the culture system to promote the myogenic differentiation of bovine muscle stem cells. The culture time during the differentiation stage was 7 days, with 3,2'-dihydroxyflavone added on the first day of the differentiation stage for 24 hours to form three-dimensional cell cultured meat tissue.

[0061] Specifically, regarding the regulation of the proliferation phase: quercetin (QUE) was dissolved in ethanol to prepare a stock solution. Experimental results showed that 400 nM QUE had the best effect on promoting the proliferation of bovine MuSCs. Adding 400 nM QUE during the proliferation culture phase significantly improved: cell viability; EdU positivity rate; Cyclin D1 expression; CDK2 expression; and PCNA expression.

[0062] Differentiation stage regulation: A stock solution was prepared by dissolving 3,2'-DHF in DMSO. Experimental results showed that 10 μM 3,2'-DHF significantly promoted myoblastic differentiation of bovine MuSCs. After adding 10 μM 3,2'-DHF during the differentiation stage: the myotube fusion index was significantly increased; MyoD1 expression, MyOG expression, and MyHC expression were upregulated. Transcriptome results showed that differentially expressed genes were significantly enriched in the "actin cytoskeleton organization" pathway after 3,2'-DHF treatment.

[0063] The following is an evaluation of the proliferation and myogenic differentiation capacity of bovine muscle stem cells in a three-dimensional porous scaffold:

[0064] Cell proliferation assessment: To evaluate the effect of three-dimensional scaffolds on the proliferation of bovine MuSCs, this study used the CCK-8 assay to detect cell viability under two-dimensional (2D) culture conditions (control group, seeded in 2D culture dishes) and three-dimensional (3D) scaffold culture conditions (experimental group). The results are as follows: Figure 6 As shown, the cell viability of the three-dimensional scaffold culture group was significantly higher than that of the 2D culture group (P<0.001), with an increase of 75%. Figure 6 A). EdU assay results showed that the proportion of cells in the DNA synthesis phase (S phase) of the three-dimensional (3D) scaffold cultured cells (A). Figure 6 B). Meanwhile, immunofluorescence results showed ( Figure 6 C) The significant proportion of Pax7-positive cells indicates that the three-dimensional scaffold can effectively maintain the stemness characteristics of bovine MuSCs, and the cells maintain their myoblastic differentiation potential while proliferating.

[0065] Evaluation of myogenic differentiation capacity of bovine MuSCs in a three-dimensional scaffold: To evaluate the myogenic differentiation capacity of bovine MuSCs in a three-dimensional scaffold, this study used MyHC immunofluorescence staining to observe myotube formation and detected the expression of myogenic differentiation marker genes by RT-qPCR. Immunofluorescence results are shown below. Figure 7 As shown, MyHC staining revealed distinct green fluorescence, with some cells exhibiting a linear multinucleated arrangement forming typical multinucleated myotubes. RT-qPCR results are as follows... Figure 8 As shown, compared with two-dimensional culture (2D), three-dimensional scaffold culture (3D) significantly upregulated the expression of myoblast differentiation marker genes, with the mRNA expression levels of MyoD1, MyoG, and MyHC increasing by 2.3-fold, 2.1-fold, and 3.0-fold, respectively (P < 0.0001). These results indicate that the three-dimensional scaffold can provide a suitable microenvironment, significantly promoting myoblast differentiation and myotube formation in bovine MuSCs, laying the foundation for the construction of cell-cultured meat.

[0066] Example 4: Construction and Effect Evaluation of the Flavonoid-Scaffold Synergistic System

[0067] This embodiment employs a staged flavonoid-assisted strategy, such as... Figure 1 As shown, synergistic regulation is carried out during the construction of cell-cultured meat.

[0068] Specific methods: Bovine muscle stem cells were seeded onto a konjac glucomannan-sodium alginate three-dimensional porous scaffold. A phased control protocol was used to construct cultured meat: proliferation culture lasted 4 days, with the first day treated with medium containing 400 nM quercetin for 24 hours, followed by continued culture in conventional complete medium; myogenic differentiation culture lasted 7 days, with the first day treated with medium containing 10 μM 3,2'-dihydroxyflavone for 24 hours, followed by induction differentiation medium to complete the remaining culture time. After a total of 11 days of culture, cultured meat was successfully constructed.

[0069] Macroscopic morphological observation results as follows Figure 9 As shown, the cultured meat is disc-shaped, approximately 2 cm in diameter and 0.5 cm thick, with a pale pink to flesh-colored appearance. Obvious cell clusters are visible on the surface, and the overall morphology is uniform and the structure is intact. Further microscopic characterization was performed using nuclear staining. Figure 9 A) The results showed that a large number of clearly identifiable DAPI cell nuclei were present inside the cultured meat, confirming that the cells successfully colonized and proliferated within the scaffold. Figure 9 (B) Under normal light, the cultured meat exhibits a continuous three-dimensional network structure, with cell nuclei evenly distributed in the scaffold pores. The cells are firmly attached to the scaffold, indicating that the culture system can effectively support cell growth in a three-dimensional environment, and the construction of cell-cultured meat has been successfully achieved.

[0070] The microstructure and cellular functional status of cultured meat at different stages of culture are characterized, including scanning electron microscopy (SEM) observations as follows: Figure 10 As shown, the morphological changes of cells at different culture stages. During the proliferation phase (GM, 4 days of culture), bovine MuSCs spread out and adhered to the surface of the three-dimensional scaffold wells, with obvious cell adhesion visible. Figure 10 A); During the differentiation phase (DM, 7 days of culture), cell adhesion on the scaffold surface is further enhanced, cell density increases, cell aggregation occurs in some areas, and myotube-like structures can be observed. Figure 10 B). Immunofluorescence (IF) results further confirmed the cell proliferation and differentiation status: during the proliferative phase (GM), Pax7 staining showed a clear red fluorescent signal, and Pax7-positive cells were prominent, indicating that the cells maintained good muscle stemness. Figure 11 A); During differentiation (DM), MyHC staining showed significant red fluorescence, distributed in a linear and network-like pattern, highly overlapping with multinucleated structures, confirming successful formation of multinucleated myotubes and normal expression of myosin. Figure 11 B). The above results indicate that bovine MuSCs can successfully complete the entire process of adhesion, proliferation, differentiation, and myotube formation in a flavonoid-assisted three-dimensional culture system.

[0071] Quantitative Validation and Evaluation of the Effect of the Flavonoid-Scaffold Synergistic System on Myoblast Differentiation: To systematically evaluate the effect of the combination of three-dimensional scaffolds and flavonoids on cultured meat, this study detected the expression levels of myoblast differentiation marker genes (MyOD1, MyOG, MyHC) and the weight of cultured meat under different culture conditions. RT-qPCR results are shown below. Figure 12 As shown, under the condition of adding combined flavonoids, the expression levels of MyOD1, MyOG, and MyHC in the 3D scaffold culture group were significantly higher than those in the 2D planar culture group (P≤0.0001). Figure 12 A) indicates that the three-dimensional microenvironment can more effectively promote myoblastic differentiation of bovine MuSCs. Further comparison of the role of flavonoids in the 3D culture system ( Figure 12 (B) The expression levels of myogenic differentiation marker genes in the flavonoid-treated group were significantly higher than those in the untreated group (P ≤ 0.0001), with MyoG showing the most significant increase; simultaneously, the cultured meat weight in the flavonoid-treated group (approximately 420 mg) was significantly higher than that in the control group (approximately 400 mg) (P ≤ 0.05). Figure 12 C). The above results indicate that both the three-dimensional scaffold and flavonoids can independently promote myoblastic differentiation of bovine MuSCs, and the combination of the two has a synergistic effect, effectively enhancing muscle tissue synthesis and accumulation while strengthening myoblastic differentiation signals.

[0072] The results of this embodiment indicate that flavonoids and three-dimensional scaffolds have a synergistic effect, significantly improving cell differentiation efficiency and muscle tissue accumulation.

[0073] Example 5: Nutritional quality evaluation of cell-cultured meat

[0074] The cell-cultured meat prepared in Example 4 (cultured for 11 days) was collected for quality analysis.

[0075] Analysis of the quality and moisture properties of cell-cultured meat: Quality comparison analysis, etc. Figure 13 As shown, the cultured scaffold is significantly fuller and denser in texture than the blank scaffold in terms of macroscopic morphology. Figure 13 A). Weight results showed that the weight of the cultured meat (approximately 460 mg) was significantly higher than that of the blank scaffold (approximately 340 mg) (P<0.001), representing an increase of approximately 1.36 times (36% increase). Figure 13 (B) This increase in quality primarily originates from the cells themselves and their secreted extracellular matrix, synthesized muscle proteins, and intracellular water. Water content measurements are shown below. Figure 14 As shown, the water content of cell-cultured meat is approximately 95%, significantly higher than that of adult bovine longissimus dorsi muscle (72%) and fetal bovine longissimus dorsi muscle (90%), and close to the water content of the three-dimensional scaffold (94%). Figure 14 The higher water content in cell-cultured meat is related to the short culture time and incomplete cell maturation. However, the 36% increase in mass indicates that the cells did indeed synthesize and accumulate muscle protein, rather than simply adsorbing water. High water content has a positive effect on the tenderness and juiciness of meat products. In the future, protein content can be further increased by extending the culture time and optimizing culture conditions, making the dry matter ratio closer to that of natural meat.

[0076] Nutritional analysis of cell-cultured meat: Nutritional composition results are as follows Figure 15 As shown, the crude protein content of cell-cultured meat was as high as 42.6%, significantly higher than that of three-dimensional scaffold (22.02%) and fetal bovine muscle (27.04%), with increases of 93.6% and 57.6%, respectively. Figure 15(AC) This result indicates that the cells successfully synthesized and accumulated a large amount of muscle protein on the scaffold, validating the aforementioned gene expression and protein level results. The crude fat content of the cell-cultured meat was 12.85%, and the energy was 576 kJ / 100g, which is close to that of chicken breast (crude fat of about 10-15% and energy of about 500-600 kJ / 100g), meeting the standards for healthy meat (low fat and high protein). The carbohydrate content was 17.6 mg / g, close to that of the three-dimensional scaffold (18.2 mg / g), mainly derived from KGM / SA natural edible polysaccharide material, possessing dietary fiber properties and being safe and harmless to the human body. The ash content was low (0.721 mg / g), and mainly derived from minerals in the culture medium, with no risk of heavy metal contamination; furthermore, the carbohydrates were derived from KGM / SA, possessing dietary fiber properties. In addition, amino acid composition analysis showed... Figure 16 and Figure 17 As shown, the cell-cultured meat contains all 17 amino acids, including all 8 essential amino acids required by the human body. The amino acid composition ratio is close to that of fetal bovine muscle. Although the total amino acid content is lower than that of natural muscle due to the shorter culture time, it is significantly higher than that of the blank three-dimensional scaffold, proving that the cells successfully synthesized muscle protein. In summary, the crude protein content of the cell-cultured meat constructed in this study is as high as 42.6%, significantly higher than that of the three-dimensional scaffold (22.02%) and fetal bovine muscle (27.04%); the crude fat content is 12.85%, meeting the standards for low-fat lean meat; and the amino acid composition contains all 17 amino acids, including all 8 essential amino acids required by the human body, with a composition ratio close to that of natural beef. It exhibits excellent performance in protein content, amino acid composition, and safety, possessing advantages such as high protein content, complete amino acid profile, low fat, moderate energy, and high safety, demonstrating its potential as a novel healthy protein source.

[0077] The above test results show that

[0078] (1) Development of edible natural polysaccharide three-dimensional scaffold: using KGM and SA as raw materials, performance tests (swelling rate, degradation rate, compression mechanical properties) have confirmed that it is all-natural, edible, low-cost and has excellent physicochemical properties.

[0079] (2) Verification of the cell affinity of the three-dimensional scaffold: The porous structure significantly promotes the adhesion and activity of bovine MuSCs during the proliferation period, as well as the formation of myotubes and tissue construction during the differentiation period.

[0080] (3) Flavonoid-scaffold synergistic differentiation promotion: Adding 400 nM quercetin (QUE) during the proliferation phase and adding 10 µM 3,2'-DHF during the differentiation phase significantly improved cell differentiation efficiency, and the synergistic effect was better than simple 2D or three-dimensional culture.

[0081] (4) Nutritional quality evaluation:

[0082] Weight and moisture: The cultured meat weighed 1.36 times that of the blank scaffold and had a moisture content of 95% (higher than natural beef (72%) and fetal bovine muscle (90%).

[0083] Key nutrients: Contains 17 amino acids (8 essential amino acids), with a composition close to that of natural beef; crude fat 12.85% (within the lean meat range); carbohydrates are derived from KGM / SA natural polysaccharides, making them safe for consumption.

[0084] In summary, this invention successfully constructs a method for bovine cell-cultured meat based on the synergistic regulation of flavonoids and a three-dimensional scaffold. This method achieves precise, staged regulation of bovine muscle stem cell behavior by applying proliferation-promoting quercetin and differentiation-promoting 3,2'-dihydroxyflavone in stages to an edible KGM-SA three-dimensional porous scaffold culture system. Compared with existing technologies, this invention significantly improves myotube formation efficiency, the degree of tissue formation in cultured meat, and product weight, while also improving the nutritional quality of the cultured meat. It provides a low-cost, edible, and cell-compatible three-dimensional culture system, offering a new technical solution for the industrial production of cell-cultured meat.

[0085] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for constructing bovine cell-cultured meat based on the synergistic regulation of flavonoids and a three-dimensional scaffold, characterized in that, Includes the following steps: S01. Preparation of three-dimensional porous scaffold: Konjac glucomannan and sodium alginate were mixed and cross-linked and freeze-dried to prepare an edible three-dimensional porous scaffold; S02. Cell seeding: Bovine muscle stem cells are seeded onto the three-dimensional porous scaffold; S03. Regulation of proliferation stage: During the cell proliferation culture stage, quercetin is added to the culture system to promote the proliferation of bovine muscle stem cells; S04. Differentiation Stage Regulation: During the myogenic differentiation stage, 3,2'-dihydroxyflavone is added to the culture system to promote the myogenic differentiation of bovine muscle stem cells and form three-dimensional cultured meat tissue.

2. The construction method according to claim 1, characterized in that, In step S03, the concentration of quercetin added is 400 nM.

3. The construction method according to claim 1, characterized in that, In step S04, the concentration of the added 3,2'-dihydroxyflavone is 10 μM.

4. The construction method according to claim 1, characterized in that, In step S01, the preparation of the three-dimensional porous scaffold specifically includes: mixing konjac glucomannan solution with a mass concentration of 2% and sodium alginate solution at a volume ratio of 1:1 to obtain a composite solution; after pre-freezing and shaping the composite solution, it is subjected to displacement crosslinking with a mass concentration of 2% CaCl2-ethanol solution, and finally freeze-drying to obtain the three-dimensional porous scaffold.

5. The construction method according to claim 4, characterized in that, The mixing was performed by magnetic stirring at room temperature for 30 minutes. The CaCl2-ethanol solution was prepared from 75% ethanol; The pre-freezing in the pre-freezing and shaping process refers to pre-freezing in a -20°C refrigerator for 12 hours. The crosslinking time is 24 hours; The freeze-drying conditions are -50℃ and vacuum degree ≤10Pa for 48 hours.

6. The construction method according to claim 1, characterized in that, In step S02, the method for preparing bovine muscle stem cells includes the following steps: taking bovine fetal longissimus dorsi muscle tissue, washing it with PBS and then cutting it into small pieces; digesting it sequentially with 0.2% type I collagenase for 70 min and 0.25% trypsin for 20-30 min; collecting the cells by filtration and centrifugation; purifying the bovine muscle stem cells using differential adhesion and culturing them at 37℃ and 5% CO2; and passage culturing them when the cell confluence reaches 80% to obtain bovine muscle stem cells.

7. The construction method according to claim 1, characterized in that, The bovine muscle stem cells were cultured for 4 days during the proliferation phase, with quercetin added for 24 hours on the first day of the proliferation phase; and for 7 days during the differentiation phase, with 3,2'-dihydroxyflavone added for 24 hours on the first day of the differentiation phase.

8. The construction method according to claim 1, characterized in that, The three-dimensional porous scaffold has a highly interconnected pore structure with a pore size ranging from 100 to 300 μm.

9. The construction method according to claim 1, characterized in that, The seeding density of the bovine muscle stem cells was 1.0 × 10⁶ per scaffold. 5 Each cell.

10. Bovine cell-cultured meat prepared by the construction method according to any one of claims 1-9.

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