Casein source 3D printing bio-ink and preparation method of biological scaffold and cell culture meat

Abstract

The invention provides a casein source 3D printing bio-ink and a preparation method of a biological scaffold and cell culture meat. The bio-ink is prepared from the following raw materials in parts by weight: 15.6 parts of gelatin; 2.6 parts of sodium alginate; 4.2 parts of casein; 52.5 parts of a sodium hydroxide deionized water solution with a pH value of 8; and 87.5 parts of a sterile 1 * PBS buffer solution. The ink is subjected to extrusion type 3D printing and calcium chloride crosslinking to obtain a biological scaffold; c2C12 cells are inoculated on the support, and cell culture meat is obtained through proliferation-differentiation culture. The problems that existing 3D printing bio-ink is single in performance and poor in component adaptability, a biological scaffold material is insufficient in mechanical stability, and nutritional ingredients are deficient are solved. The prepared bio-ink has excellent mechanical properties and printing adaptability, and provides key technical support for industrial preparation of cell culture meat.

Description

Casein-derived 3D printing bio-inks and bio-scaffolds, and methods for preparing cell-cultured meat. Technical Field

[0001] This invention relates to the field of three-dimensional bioprinting technology, specifically to a casein-derived 3D printing bio-ink and bio-scaffold, and a method for preparing cell-cultured meat. Background Technology

[0002] In vitro cell-cultured meat, as an innovative protein supply solution, has become a research hotspot in the field of food science and technology due to its advantages such as being unrestricted by natural conditions, low resource consumption, and environmental friendliness. Compared with traditional meat, the core raw materials of cell-cultured meat are mostly nutrient-rich natural proteins. These raw materials usually contain abundant essential amino acids and bioactive peptide sequences, which not only meet the human body's nutritional needs for protein but also provide key biochemical support for cell growth, ensuring the nutritional balance and edible value of the cultured meat.

[0003] The core quality of cell-cultured meat depends on the performance of the biological scaffold. This scaffold needs to provide muscle cells (such as myoblasts) with stable attachment sites, efficient nutrient transport channels, and a suitable growth microenvironment. Its biocompatibility, mechanical stability, and nutrient supply capacity directly determine the efficiency of cell proliferation and differentiation, thus affecting the texture, taste, and nutritional quality of the final cultured meat. Currently, 3D printing technology has become the mainstream method for preparing biological scaffolds due to its ability to precisely control the three-dimensional structure of the scaffold. Bio-inks, as the core raw material carrier for 3D printing, are crucial in their formulation and performance.

[0004] However, existing 3D printing bio-inks still have many shortcomings: natural polysaccharide inks have low mechanical strength and lack cell adhesion sites; natural protein inks suffer from poor thermal stability and insufficient solubility; composite inks often face the dilemma of poor component compatibility and uneven dispersion, and most inks do not fully consider the specific nutritional (such as amino acid) requirements of muscle cell growth. Some products also pose cytotoxic risks due to the addition of chemical cross-linking agents, or their poor porosity and rheological properties affect nutrient transport and structural stability. Therefore, developing a 3D printing bio-ink with good component compatibility, excellent mechanical and printing properties, and the ability to provide rich nutritional support to cells (meeting amino acid supply requirements), along with establishing efficient scaffold preparation and cell culture processes, is key to breaking through the current technological bottlenecks in cell-cultured meat and promoting its large-scale application. Summary of the Invention

[0005] Based on this, the purpose of this invention is to provide a casein-derived 3D printing bio-ink and bio-scaffold, as well as a method for preparing cell-cultured meat, to solve the core problems in the prior art: First, existing bio-inks suffer from poor component compatibility and uneven dispersion, resulting in poor mechanical and rheological properties, making it difficult to meet the core technical requirements of 3D printing such as "smooth extrusion - stable molding - precise structure," and lacking the nutritional support (such as essential amino acids and bioactive peptides) required for myoblast growth, thus failing to fully meet the specific needs of cell growth; Second, existing bio-scaffolds suffer from insufficient mechanical stability and are prone to structural failure during subsequent processing, and the swelling rate is unbalanced, either resulting in excessive swelling leading to a loose structure or insufficient swelling hindering the exchange of nutrients and metabolic waste; Third, existing inks and scaffolds lack compatibility, lacking both the nutritional basis for promoting myoblast adhesion and proliferation, and their physical microenvironment (such as elasticity and pore structure) cannot simulate the natural growth conditions of muscle tissue, ultimately leading to low cell differentiation efficiency and difficulty in preparing cell-cultured meat products with good texture and taste.

[0006] To address the aforementioned technical problems, one objective of this invention is to provide a casein-based 3D printing bio-ink, which is prepared from the following raw materials in parts by weight: gelatin: 15.6 parts; sodium alginate: 2.6 parts; casein: 4.2 parts; sodium hydroxide deionized water solution at pH=8: 52.5 parts; sterile 1×PBS buffer solution: 87.5 parts.

[0007] Based on the same inventive concept, this invention also provides a method for preparing casein-based 3D printing bio-ink, comprising the following steps: S11: Add casein to a sodium hydroxide deionized water solution with pH=8, and mechanically stir at 50°C for 3 hours to fully disperse the casein and obtain a casein dispersion; S12: Add sodium alginate to a sterile 1×PBS buffer solution, and mechanically stir at 50°C for 30 minutes to completely dissolve the sodium alginate, then add gelatin, and mechanically stir at 50°C for 30 minutes to obtain a sodium alginate / gelatin mixed solution; S13: Add the casein dispersion obtained in S11 and the sodium alginate / gelatin mixed solution obtained in S12 to a reaction vessel, and mechanically stir for 10-15 minutes to ensure that all components are fully and evenly mixed to obtain casein-based 3D printing bio-ink.

[0008] Preferably, in S11 and S12, the mechanical stirring rate is 150-250 rpm; in S13, the mechanical stirring rate is 200-300 rpm.

[0009] The present invention also provides a method for printing biological scaffolds using casein-derived 3D printing bio-ink, comprising the following steps: S21: The bio-ink is filled into a 5 mL syringe of an extrusion 3D printing device, and 3D printing is performed according to a pre-set biological scaffold model of 24 mm × 24 mm × 1.1 mm, with the printing speed set to 2 mm / s, the extrusion speed to 0.35 mm³ / s, the nozzle temperature to 24.12-24.44℃, the platform temperature to 4℃, and the line spacing to 1.2 mm; S22: The printed scaffold is immersed in a 3% calcium chloride deionized water solution for crosslinking for 5 min, and then washed 3 times with sterile 1×PBS buffer solution to obtain the biological scaffold.

[0010] This invention also provides a method for preparing cell-cultured meat, comprising the following steps: S31: cutting a biological scaffold into cylinders with a diameter of 13 mm, sterilizing it by soaking it in 75% ethanol solution for 30 min, and then washing it three times with sterile 1×PBS buffer solution; S32: placing the biological scaffold prepared in S31 in a cell culture plate, and adding C2C12 cells at a ratio of 1×10⁻⁶ cells / cm². 6 Cells were seeded onto the biological scaffold at a density of 100 μL / well; S33: The seeded culture plate was transferred to a cell culture incubator. Cells were first cultured in proliferation medium. After the cells reached the pre-differentiation density, the medium was replaced with differentiation medium and cultured until the cells differentiated and matured to obtain cell-cultured meat.

[0011] Preferably, in step S33, the environmental parameters of the incubator are: relative humidity 95%-100%, temperature 37℃, and CO2 concentration 5%.

[0012] Preferably, in S33, the proliferation medium is a DMEM high-glucose medium containing 10% fetal bovine serum and 1% penicillin-streptomycin mixture; the differentiation medium is a DMEM high-glucose medium containing 2% horse serum and 1% penicillin-streptomycin mixture.

[0013] Preferably, in S33, the proliferation medium is replaced every 48 hours during the culture process. When the cells proliferate to the pre-differentiation density, the medium is replaced with the differentiation medium, and the medium is replaced every 48 hours to induce cell differentiation and maturation.

[0014] The above-mentioned one or more technical solutions of the present invention have the following technical effects: 1) By adding casein aqueous solution to sodium alginate-gelatin mixed solution, the casein source composite bio-ink formed significantly improves the viscosity and modulus of bio-ink, enhances the mechanical properties of bio-ink, and the formed bio-ink has good biological activity, which can provide a favorable biochemical environment for cells and support cell growth and proliferation.

[0015] 2) By adjusting the casein content, the scaffold swelling rate can be precisely controlled. The casein-based scaffold has a swelling rate of 64.65%, which is similar to that of the casein-free scaffold (66.54%), maintaining a moderate swelling level of 60-70%. This ensures efficient exchange of nutrients and metabolic waste while avoiding structural loosening caused by excessive swelling, thus providing a balanced physical microenvironment for three-dimensional cell growth.

[0016] 3) Casein is rich in essential amino acids and bioactive peptide sequences, which can promote cell adhesion and functional expression. Combined with the sequence of gelatin and the biocompatibility of sodium alginate, the composite scaffold can efficiently support the growth of C2C12 myoblasts. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below.

[0018] Figure 1. Viscosity variation curves of bio-ink at different shear rates; Figure 2. Storage modulus (G') and loss modulus (G'') of bio-ink as a function of strain; Figure 3. Storage modulus (G') and loss modulus (G'') of bio-ink as a function of frequency; Figure 4. Comparison of bio-scaffold structures, where Figure 4a shows the bio-scaffold structure printed with sodium alginate-gelatin composite bio-ink; Figure 4b shows the bio-scaffold structure printed with casein-derived 3D-printed bio-ink; Figure 5. Swelling rates of bio-scaffolds printed with bio-inks of different components; Figure 6. Laser confocal microscopy fluorescence staining observations of C2C12 cells on different bio-scaffolds on day 8, where Figure 6a shows the C2C12 cells on the bio-scaffold printed with sodium alginate-gelatin composite bio-ink. Figure 6 shows the fluorescence staining observation of C2C12 cells on a biological scaffold printed with casein-derived 3D-printed bio-ink on day 8 of proliferation under laser confocal microscopy. Figure 7 shows the fluorescence staining observation of C2C12 cells on different biological scaffolds on day 8 of differentiation under laser confocal microscopy. Specifically, Figure 7a shows the fluorescence staining observation of C2C12 cells on a biological scaffold printed with sodium alginate-gelatin composite bio-ink on day 8 of differentiation under laser confocal microscopy; Figure 7b shows the fluorescence staining observation of C2C12 cells on a biological scaffold printed with casein-derived 3D-printed bio-ink on day 8 of differentiation under laser confocal microscopy; Figure 8 shows the appearance of the cells cultured on the biological scaffold printed with casein-derived 3D-printed bio-ink after baking. Detailed Implementation

[0019] This invention provides a method for preparing casein-derived 3D printing bio-ink, bio-scaffold, and cell-cultured meat, which solves the problems of existing 3D printing bio-inks having limited performance and poor component compatibility, and bio-scaffold materials having poor performance and insufficient nutritional components.

[0020] To address the aforementioned technical problems, one objective of this invention is to provide a casein-based 3D printing bio-ink, which is prepared from the following raw materials in parts by weight: gelatin: 15.6 parts; sodium alginate: 2.6 parts; casein: 4.2 parts; sodium hydroxide deionized water solution at pH=8: 52.5 parts; sterile 1×PBS buffer solution: 87.5 parts.

[0021] Based on the same inventive concept, this invention also provides a method for preparing casein-based 3D printing bio-ink, comprising the following steps: S11: Add casein to a sodium hydroxide deionized water solution with pH=8, and mechanically stir at 50°C for 3 hours to fully disperse the casein and obtain a casein dispersion; S12: Add sodium alginate to a sterile 1×PBS buffer solution, and mechanically stir at 50°C for 30 minutes to completely dissolve the sodium alginate, then add gelatin, and mechanically stir at 50°C for 30 minutes to obtain a sodium alginate / gelatin mixed solution; S13: Add the casein dispersion obtained in S11 and the sodium alginate / gelatin mixed solution obtained in S12 to a reaction vessel, and mechanically stir for 10-15 minutes to ensure that all components are fully and evenly mixed to obtain casein-based 3D printing bio-ink.

[0022] In S11 and S12, the mechanical stirring rate is 150-250 rpm; in S13, the mechanical stirring rate is 200-300 rpm.

[0023] The present invention also provides a method for printing biological scaffolds using casein-derived 3D printing bio-ink, comprising the following steps: S21: The bio-ink is filled into a 5 mL syringe of an extrusion 3D printing device, and 3D printing is performed according to a pre-set biological scaffold model of 24 mm × 24 mm × 1.1 mm, with the printing speed set to 2 mm / s, the extrusion speed to 0.35 mm³ / s, the nozzle temperature to 24.12-24.44℃, the platform temperature to 4℃, and the line spacing to 1.2 mm; S22: The printed scaffold is immersed in a 3% calcium chloride deionized water solution for crosslinking for 5 min, and then washed 3 times with sterile 1×PBS buffer solution to obtain the biological scaffold.

[0024] This invention also provides a method for preparing cell-cultured meat, comprising the following steps: S31: cutting a biological scaffold into cylinders with a diameter of 13 mm, sterilizing it by soaking it in 75% ethanol solution for 30 min, and then washing it three times with sterile 1×PBS buffer solution; S32: placing the biological scaffold prepared in S31 in a cell culture plate, and adding C2C12 cells at a ratio of 1×10⁻⁶ cells / cm². 6 Cells were seeded onto the biological scaffold at a density of 100 μL / well; S33: The seeded culture plate was transferred to a cell culture incubator. Cells were first cultured in proliferation medium. After the cells reached the pre-differentiation density, the medium was replaced with differentiation medium and cultured until the cells differentiated and matured to obtain cell-cultured meat.

[0025] In S33, the environmental parameters of the incubator are: relative humidity 95%-100%, temperature 37℃, and CO2 concentration 5%.

[0026] In S33, the proliferation medium is a DMEM high-glucose medium containing 10% fetal bovine serum and 1% penicillin-streptomycin mixture; the differentiation medium is a DMEM high-glucose medium containing 2% horse serum and 1% penicillin-streptomycin mixture.

[0027] In S33, the proliferation medium is replaced every 48 hours during the culture process. When the cells proliferate to the pre-differentiation density, the medium is replaced with the differentiation medium, and the medium is replaced every 48 hours to induce cell differentiation and maturation.

[0028] The technical solution of the present invention will now be clearly and completely described with reference to the accompanying drawings. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

[0029] Throughout this specification, unless otherwise specified, the terminology used herein should be understood as having the meaning commonly used in the art. Therefore, 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 invention pertains. In the event of any conflict, this specification shall prevail.

[0030] Example 1: A method for preparing casein-derived 3D printing bio-ink, comprising the following steps: S1: Add 12.8 g of casein to 160 mL of pH=8 deionized sodium hydroxide aqueous solution, and mechanically stir at 150-250 rpm for 3 h at 50℃ to fully disperse the casein, obtaining a casein dispersion; S2: Add 2.6 g of sodium alginate to 87.5 mL of sterile 1×PBS buffer solution, and mechanically stir at 150-250 rpm for 30 min at 50℃ to completely dissolve the sodium alginate, then add 15.6 g of gelatin, and mechanically stir at 150-250 rpm for 30 min at 50℃ to obtain a sodium alginate / gelatin mixed solution; S3: Add 52.5 mL of casein dispersion and 87.5 mL of sodium alginate / gelatin mixed solution to a reaction vessel, and mechanically stir at 200-300 rpm for 10-15 minutes. The process involves mixing each component thoroughly and evenly to obtain casein-based 3D printing bio-ink.

[0031] Example 2: A method for preparing a biological scaffold, comprising the following steps: S1: The casein-derived 3D printing bio-ink prepared in Example 1 is filled into a 5 mL syringe of an extrusion 3D printing device. A biological scaffold model of 24 mm × 24 mm × 1.1 mm is pre-set, with the printing speed set to 2 mm / s, the extrusion speed to 0.35 mm³ / s, the nozzle temperature to 24.12-24.44℃, the platform temperature to 4℃, and the line spacing to 1.2 mm for 3D printing; S2: The printed scaffold is immersed in a 3% calcium chloride deionized water solution for crosslinking for 5 min, then washed three times with sterile 1×PBS buffer solution, and cut into cylinders with a diameter of 13 mm using a cutter to obtain the 3D printed biological scaffold.

[0032] Example 3: A method for preparing cell-cultured meat, comprising the following steps: S1: The biological scaffold prepared in Example 2 is sterilized by immersing in 75% ethanol solution for 30 min, followed by washing three times with sterile 1×PBS buffer solution; S2: The biological scaffold prepared in S1 is placed in a cell culture plate, and the environmental parameters of the cell culture incubator are set as follows: relative humidity 95%-100%, temperature 37℃, CO2 concentration 5%; C2C12 cells are prepared at a density of 1×10⁻⁶ cells / cm². 6S3: A cell suspension of cells / ml is seeded onto the surface of the biological scaffold at a volume of 100 μL / well; S4: The seeded culture plate is transferred into a cell culture incubator with the above-set parameters. Cell proliferation culture is first performed using proliferation medium. When the cells proliferate to the pre-differentiation density, the medium is replaced with differentiation medium for induction differentiation culture until the cells differentiate and mature, thus obtaining cell-cultured meat; wherein, the proliferation medium is DMEM high-glucose medium containing 10% fetal bovine serum and 1% penicillin-streptomycin mixture; the differentiation medium is DMEM high-glucose medium containing 2% horse serum and 1% penicillin-streptomycin mixture; the proliferation medium is changed every 48 hours during the proliferation culture period, and after being replaced with differentiation medium, the differentiation medium is changed every 48 hours to continuously induce cell differentiation and maturation.

[0033] Comparative Example 1: A method for preparing sodium alginate / gelatin bio-ink, comprising the following steps: S1: 2.6 g of sodium alginate is added to 140 mL of sterile 1×PBS buffer solution, and mechanically stirred at 150-250 rpm for 30 min at 50℃ to completely dissolve the sodium alginate; S3: 15.6 g of gelatin is added to S1, and mechanically stirred at 150-250 rpm for 30 min at 50℃ to obtain sodium alginate / gelatin bio-ink.

[0034] Comparative Example 2: A method for preparing a biological scaffold, comprising the following steps: S1: The bio-ink prepared in Comparative Example 1 is filled into a 5 mL syringe of an extrusion 3D printing device. The biological scaffold model is pre-set to be 24 mm × 24 mm × 1.1 mm. The printing speed is set to 2 mm / s, the extrusion speed to 0.35 mm³ / s, the nozzle temperature to 24.12-24.44℃, the platform temperature to 4℃, and the line spacing to 1.2 mm. 3D printing is then performed. S2: The printed scaffold is immersed in a 3% calcium chloride deionized water solution for crosslinking for 5 min. Then it is washed three times with sterile 1×PBS buffer solution and cut into cylinders with a diameter of 13 mm to obtain a 3D printed biological scaffold.

[0035] Comparative Example 3: A method for preparing cell-cultured meat, comprising the following steps: S1: The biological scaffold prepared in Comparative Example 2 is sterilized by immersing in 75% ethanol solution for 30 min, followed by washing three times with sterile 1×PBS buffer solution; S2: The biological scaffold prepared in S1 is placed in a cell culture plate, and the environmental parameters of the cell culture incubator are set as follows: relative humidity 95%-100%, temperature 37℃, CO2 concentration 5%; C2C12 cells are prepared into a density of 1×10⁻⁶ cells / cm². 6S3: A cell suspension of cells / ml is seeded onto the surface of the biological scaffold at a volume of 100 μL / well; S4: The seeded culture plate is transferred into a cell culture incubator with the above-set parameters. Cell proliferation culture is first performed using proliferation medium. When the cells proliferate to the pre-differentiation density, the medium is replaced with differentiation medium for induction differentiation culture until the cells differentiate and mature, thus obtaining cell-cultured meat; wherein, the proliferation medium is DMEM high-glucose medium containing 10% fetal bovine serum and 1% penicillin-streptomycin mixture; the differentiation medium is DMEM high-glucose medium containing 2% horse serum and 1% penicillin-streptomycin mixture; the proliferation medium is changed every 48 hours during the proliferation culture period, and after being replaced with differentiation medium, the differentiation medium is changed every 48 hours to continuously induce cell differentiation and maturation.

[0036] Test Example 1: Rheological Properties Testing The rheological properties of the casein-derived 3D printing bio-ink prepared in Example 1 and the bio-ink prepared in Comparative Example 1 were tested. A PP25 probe was used in the rheometer, with a plate spacing of 1 mm and a test temperature of 24℃. The viscosity of the bio-ink was measured within a shear rate range of 0.1-100 s⁻¹ to study its shear thinning behavior. The linear viscoelastic region was determined by amplitude scanning experiments (constant frequency of 10 rad / s, strain range of 0.1-100%). The dynamic viscoelastic properties of the samples were evaluated by frequency scanning (oscillation frequency range of 1-100 rad / s, constant strain of 1%).

[0037] As shown in Figure 1, the apparent viscosity of all bio-ink systems decreases with increasing shear rate, exhibiting significant shear-thinning characteristics and demonstrating typical pseudoplastic fluid behavior. This shear-thinning phenomenon is beneficial for the material's flowability during extrusion, while maintaining high viscosity under low shear conditions to preserve the shape stability after printing, thus meeting the core requirements of extrusion 3D printing.

[0038] As shown in Figure 2, the casein-containing bio-ink system exhibits a storage modulus (G') greater than the loss modulus (G'') across the entire strain range, indicating that the material exhibits gel-like characteristics, possessing good elasticity and structural stability. After printing, it can form stable filaments, demonstrating an enhanced cross-linked network structure. Comparative Example 1, exhibiting a rheological characteristic of G'≈G'', with similar values ​​for storage and loss moduli, indicates that this ink system is a highly viscoelastic fluid, lacking the ability to form a stable three-dimensional network structure and failing to maintain a stable filament morphology after extrusion.

[0039] As shown in Figure 3, the casein-containing bio-ink system consistently exhibits a storage modulus (G') greater than the loss modulus (G'') within the tested frequency range. Furthermore, G' shows extremely weak frequency dependence in the mid-to-high frequency region (almost unchanging with frequency), exhibiting plateau-like characteristics. This indicates that the material forms a stable three-dimensional gel network structure with shape retention over long timescales, ensuring the stability and accuracy of the printed structure. Comparative Example 1 shows critical gel characteristics where G'≈G'', with similar storage and loss moduli, indicating weaker network stability. These results are consistent with amplitude scanning data, further confirming that casein-derived bio-inks possess excellent extrudability (shear thinning), shape fidelity (high G' / G''), and structural stability (frequency independence).

[0040] The above analysis shows that by introducing casein into the gelatin-sodium alginate system, a composite bio-ink suitable for 3D printing has been successfully constructed, and its rheological properties can meet the core requirements of "smooth extrusion, stable shape, and precise structure".

[0041] Test Example 2: Morphological Analysis of Biological Scaffolds Morphological analysis was performed on the biological scaffolds prepared in Example 2 and Comparative Example 2, as shown in Figure 4. The morphological parameters of the scaffolds in Figure 4 were quantitatively analyzed using ImageJ software. To ensure the representativeness and accuracy of the measurements, three different regions were randomly selected from each scaffold, and three independent fields of view were randomly selected from each region for measurement (n = 9). The printability index was calculated using the following formula.

[0042] Printability Index = L 2 / 16A 2 (L is the perimeter, A is the area) Table 1 Morphological evaluation of bio-ink scaffolds

[0043] Printability index is an important parameter for evaluating the morphological fidelity of 3D bioprinted scaffolds, with a theoretical optimal value of 1.0. When the printability index is close to 1, it indicates that the scaffold has good shape retention ability. The printability index of Comparative Example 2 is 0.91±0.03, while the printability index of the casein-derived bio-ink scaffold is 1.01±0.02, which is very close to the ideal value of 1.0. This indicates that the casein-derived bio-ink has excellent rheological properties and outstanding shape fidelity, and can accurately maintain the preset geometry after printing, effectively avoiding filament diffusion and collapse.

[0044] Test Example 3: Swelling Test of Biological Scaffolds The biological scaffolds prepared in Example 2 and Comparative Example 2 were subjected to a swelling test. The specific steps are as follows: First, the dry weight (W0) of the scaffold was accurately weighed. Then, it was immersed in ultrapure water and incubated at 37°C for 40 min until it reached swelling equilibrium. After swelling, the scaffold was removed, excess solution on the surface was absorbed with filter paper, and the wet weight (W0) was accurately weighed again. s All samples were tested in triplicate in each group. All experimental data are expressed as mean ± standard deviation. A p-value < 0.05 was considered statistically significant, and NS indicated no significant difference (p ≥ 0.05). The swelling rate was calculated using the following formula: Swelling rate = (W... s As shown in Figure 5, the swelling ratios of the bioscaffolds printed with casein-based 3D printing bio-ink and sodium alginate-gelatin bio-ink were 64.65% and 66.54%, respectively. The decrease in swelling ratio with increasing casein content is attributed to the formation of a dense cross-linked network between casein and the sodium alginate-gelatin system through ion bridging, hydrophobic interactions, and hydrogen bonds, effectively limiting the penetration of water molecules. However, the bioscaffolds printed with sodium alginate-gelatin bio-ink and casein-based 3D printing bio-ink maintained a moderate swelling ratio of 60-70%. This swelling characteristic ensures efficient exchange of nutrients and metabolic waste, creating an ideal physical microenvironment for three-dimensional cell growth.

[0045] Test Example 4: Texture Analysis of Bio-ink Scaffolds Texture analysis was performed on the bio-scaffolds prepared in Example 2 and Comparative Example 2. The rheometer was set to Total Texture Analysis (TPA). The pre-test, mid-test, and post-test speeds were all 0.5 mm / s, the contact force was 8 gf, the deformation was 20%, the test pause time was 2 s, and the test temperature was 25℃. To ensure experimental repeatability, three parallel samples of each bio-ink scaffold were selected for testing. The results are expressed as mean ± standard deviation. The test results are shown in Table 2.

[0046] Table 2. Texture analysis of bio-ink scaffolds

[0047] Texture analysis results showed that the introduction of casein significantly improved the mechanical properties of the bio-ink scaffold: compared with the casein-free scaffold in Comparative Example 2, the casein-added scaffold was more than twice as stiff; adhesiveness and chewiness also increased significantly with increasing casein content, while elasticity showed no significant difference among the three groups. Although both casein and sodium alginate are negatively charged at neutral pH, they formed a denser composite network structure through Ca²⁺-mediated ion bridging, hydrophobic interactions, and hydrogen bonding with gelatin, significantly improving the mechanical strength and structural stability of the scaffold.

[0048] Test Example 5: Cell Proliferation and Differentiation Characteristics Test. To evaluate the cell compatibility of the bioscaffold and its impact on myoblast function, cell-cultured meat samples prepared in Example 3 and Comparative Example 3 at "day 8 of proliferation culture" and "day 8 of differentiation culture," respectively, were subjected to immunofluorescence detection. The detection method is as follows: F-actin-specific fluorescent dye was used to label the cytoskeleton, and DAPI fluorescent dye was used to label the cell nuclei. The cell distribution density, morphological characteristics, and spatial extension of C2C12 cells on bioscaffolds printed with sodium alginate / gelatin bio-ink and bioscaffolds printed with casein-derived 3D printing bio-ink were observed using a laser confocal microscope.

[0049] The results of laser confocal microscopy observation are shown in Figures 6-7: 1) Day 8 of proliferation (Figure 6): C2C12 cells on the bioscaffolds printed with sodium alginate / gelatin bio-ink and casein-derived 3D printing bio-ink were evenly distributed, had high growth density, and fully extended cytoskeleton, exhibiting typical myoblast spindle morphology, indicating that both types of scaffolds can provide good attachment sites and growth microenvironment for cell proliferation; 2) Day 8 of differentiation (Figure 7): Cells on the bioscaffolds printed with sodium alginate / gelatin bio-ink and casein-derived 3D printing bio-ink both showed highly efficient myogenic differentiation characteristics—cells formed continuous myotube-like structures, and the cytoskeleton was arranged regularly, confirming that both types of scaffolds can support the functional differentiation of myoblasts.

[0050] In summary, the bio-ink scaffold of the present invention has good cell compatibility, which can not only efficiently support the proliferation of C2C12 myoblasts, but also promote their myogenic differentiation, fully verifying the adaptability of the composite bio-ink to the growth requirements of muscle cells.

[0051] Test Example 6: Texture Analysis of Cell-Based Meat Texture analysis was performed on the cell-based meat prepared in Example 3 and Comparative Example 3, as well as commercially available pork tenderloin. The texture analyzer was set to Total Texture Analysis (TPA). The speed for the pre-test, mid-test, and post-test was 0.5 mm / s, the contact force was 8 gf, the deformation was 20%, the pause time was 2 s, and the test temperature was 25℃. To ensure experimental repeatability, three parallel samples of cell-based meat from each proportion were tested. The results are expressed as mean ± standard deviation. The test results are shown in Table 3.

[0052] Table 3 Texture analysis of pork tenderloin and cell-derived meat

[0053] Texture results showed that, in terms of firmness, Example 3 was not significantly different from pork tenderloin, while Comparative Example 3 was significantly lower than both groups, indicating that the addition of casein effectively improved the firmness of the cell-cultured meat, bringing it to the level of natural pork. The results for adhesiveness and chewiness were consistent with the firmness trend; Example 3 was not significantly different from pork tenderloin, while Comparative Example 3 was lower. In terms of elasticity, cohesion, and resilience, there were no significant differences among the three groups, indicating that the cell-cultured meat in both groups had similar elastic characteristics to pork tenderloin. In summary, Example 3 showed no significant difference from pork tenderloin in any of the texture indicators, and its overall texture characteristics were significantly better than Comparative Example 3, demonstrating that the addition of casein effectively improved the texture quality of the cell-cultured meat, enabling it to better simulate the taste of natural pork and possessing certain application potential.

[0054] Test Example 7: Amino Acid Composition Analysis of Cell-Based Meat. High-performance liquid chromatography (HPLC) was used to analyze the amino acid composition of cell-based meat cultured in Comparative Example 3 and Example 3, as well as commercially available pork tenderloin. Appropriate amounts of sample were weighed and added to 2.0 mL of a 6 mol / L hydrochloric acid solution containing 0.1% phenol. After nitrogen purging and sealing, the solution was hydrolyzed at 110℃ for 24 h. 1.0 mL of the hydrolysate was dried under N2, reconstituted, and then derivatized. After filtration through a 0.22 μm organic membrane, the solution was analyzed using HPLC. The test results are shown in Table 4.

[0055] Table 4. Amino acid composition analysis of pork tenderloin and cell-celled meat (unit: g amino acids / 100 g protein)

[0056] The results of amino acid analysis are shown in Table 4. Both groups of cell-cultured meat contained 18 amino acids, and the essential amino acids were complete. Regarding non-essential amino acids, the glycine (10.58 g / 100 g protein) and hydroxyproline (5.21 g / 100 g protein) content in Comparative Example 3 was significantly higher than that in pork tenderloin, consistent with the characteristic of gelatin being rich in these amino acids. The glycine and hydroxyproline content (5.68 g / 100 g protein and 1.10 g / 100 g protein) in Example 3 were close to the level of pork tenderloin, while the glutamic acid content (18.54 g / 100 g protein) was close to that of pork tenderloin (19.33 g / 100 g protein) and higher than that of Comparative Example 3 (16.18 g / 100 g protein), contributing to the good umami characteristics of the cell-cultured meat. Regarding essential amino acids, the contents of leucine (9.38 g / 100 g protein), isoleucine (3.00 g / 100 g protein), and phenylalanine (5.00 g / 100 g protein) in Example 3 were higher than those in pork tenderloin (7.00 g / 100 g protein, 2.95 g / 100 g protein, and 3.62 g / 100 g protein). Furthermore, the contents of all six essential amino acids in Example 3 were significantly higher than those in Comparative Example 3. This indicates that the addition of casein effectively improved the amino acid composition of cell-cultured meat and further enhanced its nutritional value.

[0057] Test Example 8: Flavor Analysis of Cell-Based Meat. The volatile flavor components of cell-based meat cultured in Comparative Example 3 and Example 3, as well as pork tenderloin, were analyzed using gas chromatography-high-throughput time-of-flight mass spectrometry (GC-MS / MS). The cultured cell-based meat was washed three times with PBS to remove residual culture medium and then baked at 180°C, as shown in Figure 8. A suitable amount of sample was placed in a headspace vial, and volatile components were extracted using solid-phase microextraction (SPME). The extracted components were then analyzed using the instrument. The test results are shown in Table 5.

[0058] Table 5. Analysis of volatile flavor components in pork tenderloin and cell-celled meat

[0059] Meat aromas are generated from Maillard reactions, lipid thermal oxidation, and degradation reactions of precursor substances (such as carbohydrates, lipids, and amino acids) during heat treatment. As shown in Table 5, a total of 23 volatile flavor compounds related to pork were identified.

[0060] The oxidative degradation and hydrolysis of lipids produce various aldehydes, ketones, and acids, such as heptanal, nonanal, and 2-heptenal. These compounds impart the basic fatty aroma flavor to cell-cultured meat. The contents of hexanal, heptanal, and nonanal in cell-cultured meat are all lower than those in pork tenderloin, which is related to insufficient lipid accumulation and weaker lipid oxidation in cell-cultured meat. However, the contents of 2-heptenal and 2-n-butylpropenal in Example 3 are significantly higher than those in pork tenderloin and Comparative Example 3. Both are products of unsaturated fatty acid oxidative degradation, possessing fatty and grassy aromas, which help enrich the fatty aroma flavor profile of cell-cultured meat.

[0061] Among the ketones, 2,3-octanedione was mainly produced by lipid oxidation and was not detected in either group of cell-cultured meat, indicating insufficient lipid oxidation and a lack of creamy aroma. A small amount of acetophenone was detected in Example 3, but not in pork tenderloin, contributing some floral and sweet aroma. The contents of 1-pentanol and 1-hexanol in both groups of cell-cultured meat were lower than in pork tenderloin, consistent with insufficient lipid accumulation in the cell-cultured meat. 1-Octen-3-ol showed a significant increase in Example 3 compared to Comparative Example 2, and to some extent approached the flavor characteristics of pork tenderloin.

[0062] In summary, compared with sodium alginate / gelatin bio-ink for printing bio-scaffolds, the bio-scaffolds printed with casein-derived 3D printing bio-ink described in this invention have superior cell meat culture characteristics due to the introduction of casein. The cultured pork tenderloin cell meat is close to the nutritional value and quality of real pork tenderloin.

Claims

1. A casein-based 3D printing bio-ink, characterized in that, The casein-derived 3D printing bio-ink is prepared from the following raw materials in parts by weight: gelatin: 15.6 parts; sodium alginate: 2.6 parts; casein: 4.2 parts; sodium hydroxide deionized water solution at pH=8: 52.5 parts; sterile 1×PBS buffer solution: 87.5 parts.

2. The method for preparing casein-derived 3D printing bio-ink according to claim 1, characterized in that, The process includes the following steps: S11: Casein is added to a deionized sodium hydroxide aqueous solution with pH=8 and mechanically stirred at 50℃ for 3 h to fully disperse the casein and obtain a casein dispersion; S12: Sodium alginate is added to a sterile 1×PBS buffer solution and mechanically stirred at 50℃ for 30 min to completely dissolve the sodium alginate. Gelatin is then added and mechanically stirred at 50℃ for 30 min to obtain a sodium alginate / gelatin mixed solution; S13: The casein dispersion obtained in S11 and the sodium alginate / gelatin mixed solution obtained in S12 are added to a reaction vessel and mechanically stirred for 10-15 min to ensure that all components are fully mixed and homogeneous, thus obtaining a casein-based 3D printing bio-ink.

3. The method for preparing casein-derived 3D printing bio-ink according to claim 2, characterized in that: In S11 and S12, the mechanical stirring rate is 150-250 rpm; in S13, the mechanical stirring rate is 200-300 rpm.

4. A method for printing biological scaffolds using the casein-derived 3D printing bio-ink according to claim 1, characterized in that, The process includes the following steps: S21: The bio-ink is loaded into a 5 mL syringe of an extrusion 3D printing device, and 3D printing is performed according to a pre-set 24 mm × 24 mm × 1.1 mm bio-scaffold model, with the printing speed set to 2 mm / s, the extrusion speed to 0.35 mm³ / s, the nozzle temperature to 24.12-24.44℃, the platform temperature to 4℃, and the line spacing to 1.2 mm; S22: The printed scaffold is immersed in a 3% calcium chloride deionized water solution for crosslinking for 5 min, and then washed 3 times with sterile 1×PBS buffer solution to obtain the bio-scaffold.

5. A method for preparing cell-cultured meat, characterized in that, The procedure includes the following steps: S31: The biological scaffold prepared by the method described in claim 4 is cut into cylinders with a diameter of 13 mm, sterilized by soaking in 75% ethanol solution for 30 min, and then washed three times with sterile 1×PBS buffer solution; S32: The biological scaffold prepared in S31 is placed in a cell culture plate, and C2C12 cells are cultured at a ratio of 1×10⁻⁶ cells / mL. 6 Cells were seeded onto the biological scaffold at a density of 100 μL / well; S33: The seeded culture plate was transferred to a cell culture incubator. Cells were first cultured in proliferation medium. After the cells reached the pre-differentiation density, the medium was replaced with differentiation medium and cultured until the cells differentiated and matured to obtain cell-cultured meat.

6. The method for preparing cell-cultured meat according to claim 5, characterized in that: In S33, the environmental parameters of the incubator are: relative humidity 95%-100%, temperature 37℃, and CO2 concentration 5%.

7. The method for preparing cell-cultured meat according to claim 5, characterized in that: In S33, the proliferation medium is a DMEM high-glucose medium containing 10% fetal bovine serum and 1% penicillin-streptomycin mixture; the differentiation medium is a DMEM high-glucose medium containing 2% horse serum and 1% penicillin-streptomycin mixture.

8. The method for preparing cell-cultured meat according to claim 5, characterized in that: In S33, the proliferation medium is replaced every 48 hours during the culture process. When the cells proliferate to the pre-differentiation density, the medium is replaced with the differentiation medium, and the medium is replaced every 48 hours to induce cell differentiation and maturation.

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