Edible bio-ink, prolamin microspheres, cultured meat and manufacturing method thereof

Abstract

The invention relates to edible bio-ink, prolamin microspheres, cultured meat and a manufacturing method thereof. The edible bio-ink comprises prolamin microspheres loaded with cells and an ink solution containing gelatin and alginate, the edible bio-ink for three-dimensional printing of cultured meat is formed, and the particle size of the prolamin microspheres ranges from 150 m to 500 m.

Description

Technical Field

[0001] This invention relates to the food industry, specifically to edible bio-inks, cell-loaded prolysin microspheres, cultured meat, and methods for manufacturing the same. Background Technology

[0002] Cultured meat, also known as cell-cultured meat or lab-grown meat, is hailed as an environmentally friendly and innovative meat production method that utilizes animal stem cells instead of feeding livestock. In practice, the process of cultured meat production typically includes cell isolation and screening, large-scale cell expansion, tissue maturation, and food processing into the final product. It is estimated that one kilogram of animal meat contains approximately 10... 11 One major obstacle to the large-scale commercialization of cultured meat is how to obtain large quantities of seed cells, such as skeletal muscle satellite cells (PSCs) and adipose-derived mesenchymal stem cells (ADSCs), in a highly economical manner. Summary of the Invention

[0003] This application relates to an edible bio-ink. The edible bio-ink comprises prolamin microbeads loaded with cells and an ink solution containing gelatine and alginate, forming an edible bio-ink for 3D printing cultured meat, wherein the particle size of the prolamin microbeads ranges from greater than 150 µm to 500 µm.

[0004] On the other hand, this application relates to a method for producing an edible bio-ink, comprising providing an ink solution including gelatin and alginate, and mixing cell-loaded gliadin microspheres with the ink solution to form an edible bio-ink for 3D printing cultured meat, wherein the particle size of the gliadin microspheres ranges from greater than 150 µm to 500 µm.

[0005] On the other hand, this application relates to a method for manufacturing cultured meat, comprising the following steps: culturing cells on gliadin microspheres to form cell-loaded gliadin microspheres; incorporating the cell-loaded gliadin microspheres into an ink solution comprising gelatin and alginate to form bio-ink; and extruding the bio-ink using a 3D printer to form cultured meat, wherein the particle size of the gliadin microspheres ranges from greater than 150 µm to 500 µm.

[0006] On the other hand, this application relates to a method for manufacturing alcohol-soluble protein microspheres, comprising mixing alcohol-soluble protein stock solution and an oil phase at a 1:1 v / v ratio to form a pre-emulsified mixture, homogenizing the pre-emulsified mixture to form an ethanol / water-in-oil emulsion, and heating the ethanol / water-in-oil emulsion to remove ethanol to form alcohol-soluble protein microspheres, wherein the particle size of the alcohol-soluble protein microspheres ranges from greater than 150 µm to 500 µm.

[0007] In another aspect, this application provides an alcohol-soluble protein microsphere comprising alcohol-soluble protein microspheres manufactured by the methods described above. Attached Figure Description

[0008] In the accompanying drawings, the same reference numerals generally denote the same parts in different views. The drawings are not necessarily drawn to scale, but generally focus on illustrating the principles of various embodiments. In the following description, various embodiments of this application will be described in conjunction with the following drawings.

[0009] Figure 1 This is a schematic diagram of a method for manufacturing cultured meat according to an embodiment of this application.

[0010] Figure 2A The image shows a confocal laser scanning microscopy (CLSM) image of an oil-in-water (water / ethanol) emulsion prepared according to an embodiment of this application, wherein the aqueous phase and the oil phase (volume ratio 1:1) are stained with fluorescein isothiocyanate FITC (green) and Nile Red (red), respectively.

[0011] Figure 2B and 2C The image shows a scanning electron microscope (SEM) image of dried corn gliadin microspheres with a particle size range of 100 µm to 200 µm.

[0012] Figure 2D Histograms showing the particle size distribution of dried and hydrated zein microspheres are presented.

[0013] Figure 2E The hydration level and particle size variation of dried and hydrated zein microspheres are shown (data presented as mean ± standard deviation, n = 3, two-tailed t-test).

[0014] Figure 2F A CLSM image of hydrated zein microspheres exhibiting autofluorescence is shown.

[0015] Figures 2G to 2HThis image shows representative surface morphology images of dried and hydrated zein microspheres measured using atomic force microscopy (AFM).

[0016] Figure 2I The surface roughness and Young's modulus of dried and hydrated zein microspheres are shown (data presented as mean ± standard deviation, n = 5, two-tailed t-test).

[0017] Figure 3A The example shown illustrates that the particle size range of zein microspheres prepared according to one embodiment of this application is 100 µm-200 µm.

[0018] Figure 3B The example shown illustrates that the particle size range of zein microspheres prepared according to one embodiment of this application is 200 µm-300 µm.

[0019] Figure 3C The example shown is a zein microsphere with a particle size range of 300 µm-400 µm prepared according to one embodiment of this application.

[0020] Figure 3D The example shown illustrates that the particle size range of zein microspheres prepared according to one embodiment of this application is 400 µm-500 µm.

[0021] Figure 3E The example shown is a zein microsphere with a particle size range of >500µm prepared according to one embodiment of this application.

[0022] Figure 3F The particle size distribution of zein microspheres prepared according to one embodiment of this application is shown.

[0023] Figure 4A This shows the number of C2C12 cells on zein microspheres over 12 days.

[0024] Figure 4B-4C Images of CLSM on C2C12 cultured on zein microspheres after 2 and 4 days are shown.

[0025] Figure 4D The image shows a CLSM image of C2C12 myoblasts on zein microspheres 7 days after differentiation (scale bar: 100 µm; nuclei and cytoskeleton stained with Hoechst 33342 (blue) and phalloidin (red), respectively).

[0026] Figure 4E-4FThe relative gene expression levels of C2C12 on 2D tissue plates and zein microspheres are shown (data are presented as mean ± standard deviation, n = 4, one-way ANOVA).

[0027] Figure 4G-4I Western blot analysis and protein expression quantification of GAPDH on 2D tissue plates and zein microspheres are shown (data presented as mean ± standard deviation, n = 6, one-way ANOVA).

[0028] Figure 4J SEM images showing the surface morphology changes of C2C12 cells after 3, 6, and 12 days of culture are shown.

[0029] Figure 5A This diagram shows porcine skeletal muscle satellite cells (PSCs) cultured on zein microspheres in a rotating flask bioreactor.

[0030] Figure 5B The number of PSC cells on zein microspheres over 9 days is shown (data presented as mean ± standard deviation, n = 3).

[0031] Figure 5C The image shows a CLSM image of PCS on zein microspheres after 6 days of culture (scale bar: 100 µm; cell nuclei and cytoskeleton stained with Hoechst 33342 (blue) and phalloidin (red), respectively).

[0032] Figure 5D Immunostaining (red fluorescence, scale bar: 100 µm) of myosin heavy chain (MYHC) on PSCs on zein microspheres after differentiation.

[0033] Figure 5E-5H The expression of myogenic genes in PSCs on 2D tissue plates and zein microspheres is shown (data are presented as mean ± standard deviation, n = 4, one-way ANOVA).

[0034] Figure 6A The number of 3T3-L1 cells on zein microspheres over 11 days is shown (data presented as mean ± standard deviation, n = 3).

[0035] Figures 6B-6CThe image shows CLSM images of 3T3-L1 on zein microspheres before and after adipogenesis differentiation (scale bar: 100 µm; nuclei, cytoskeleton and lipid droplets stained with Hoechst 33342 (blue), phalloidin (red) and BODIPY 493 / 503 (green), respectively).

[0036] Figure 6D The image shows 3T3-L1 on 2D tissue plates and 3D zein microspheres after differentiation, stained with Oil Red O (scale bar: 50 µm).

[0037] Figure 6E Transcriptomic analysis of 3T3-L1 cells after differentiation on 2D tissue plates and 3D zein microspheres is shown, with volcano plots revealing differentially expressed genes between the two groups.

[0038] Figure 6F Transcriptome analysis of 3T3-L1 cells after differentiation on 2D tissue plates and 3D zein microspheres is shown, enriching the KEGG pathway.

[0039] Figure 6G Transcriptome analysis and gene ontology function enrichment analysis of 3T3-L1 cells after differentiation on 2D tissue plates and 3D zein microspheres are shown.

[0040] Figure 6H-6K The volcano plot shows differentially expressed genes corresponding to actin cytoskeleton regulation, focal adhesion, fatty acid biosynthesis, and fatty acid metabolism.

[0041] Figure 7A This diagram illustrates adipogenesis and the maturation of adipogenesis precursor cells on zein microspheres.

[0042] Figure 7B The number of porcine adipose-derived mesenchymal stem cells (ADSCs) on zein microspheres over 12 days is shown (data presented as mean ± standard deviation, n = 3).

[0043] Figures 7C-7D Images of ADSC on zein microspheres before and after adipogenesis differentiation are shown in CLSM (scale bar: 100 µm; nuclei, cytoskeleton, and lipid droplets were stained with Hoechst 33342 (blue), phalloidin (red), and BODIPY 493 / 503 (green), respectively).

[0044] Figure 7E Bright-field image showing ADSCs differentiated on zein microspheres (scale bar: 100 µm).

[0045] Figure 7F-7K The relative expression of lipogenesis-related genes on 2D tissue plates and 3D zein microspheres is shown (data are presented as mean ± standard deviation, n = 4, one-way ANOVA).

[0046] Figure 7L Principal component analysis (PCA) of pork fat and cultured meat is shown.

[0047] Figure 7M The relative percentages of fat and lipid composition in pork and cultured meat are shown.

[0048] Figure 8A An optical image of a 3D scaffold printed using GA-Z20 ink is shown.

[0049] Figure 8B CLSM images of PSC and ADSC cells in a printed single fiber are shown, stained with live cells (green) / dead cells (red).

[0050] Figure 8C The image shows a photograph of a cultured meat prototype, including a CLSM image of cells on printed zein microspheres.

[0051] Figure 8D The nutritional components of pork belly and cultured meat are shown.

[0052] Figure 8E The textural profile analysis (TPA) of pork belly and cultured meat is shown (data are presented as mean ± standard deviation, n = 4, two-tailed t-test).

[0053] Figure 8F The viscosity versus shear rate curves for GA and GA-Z inks are shown.

[0054] Figure 8G The relationship between storage modulus (G') and loss modulus (G'') of GA and GA-Z20 inks at 4°C and 30°C is shown in the amplitude scanning test (constant frequency of 1 Hz).

[0055] Figure 9A Optical images of d-ADCS-GA-Z fibers are shown, including zein microspheres (particle size >150 µm) cultured in ADSC cells (scale bar: 100 µm).

[0056] Figure 9B Optical images of printed PSC-GA-Z fibers are shown, including zein microspheres (particle size >150 µm) cultured in PSC cells (scale bar: 100 µm). Detailed Implementation

[0057] The specific details and embodiments of this application will be described in detail below with reference to the accompanying drawings, which illustrate possible implementations of this application. These embodiments are described in detail to enable those skilled in the art to implement this application. Other embodiments may be used, and structural and logical changes may be made without departing from the scope of this application. The various embodiments are not necessarily mutually exclusive, as some embodiments may be combined with one or more other embodiments to form new embodiments.

[0058] Features described in one embodiment may be applied accordingly to the same or similar features in other embodiments, even if not explicitly described in those other embodiments. Furthermore, additions, combinations, and / or substitutions to the feature descriptions may also be applied accordingly to the same or similar features in other embodiments.

[0059] The articles “a,” “one,” and “the” used with respect to features or elements include references to one or more features or elements.

[0060] In various embodiments, the term "about" or "approximately" when applied to numerical values ​​covers the value and a reasonable difference, such as within + / - 10% of the value.

[0061] As used herein, the term “and / or” includes any and all combinations of one or more of the listed related items.

[0062] Plant-derived edible microspheres for cultured meat production show promise but remain underdeveloped. Using microspheres in suspension culture within bioreactors could be a way to make meat cultivation greener and more economically attractive. Edible microspheres based on polysaccharides, such as dextran (Cytodex 1®), alginate, and rice grains, can be used to produce cultured meat. However, these microspheres have poor cell affinity and require specific coatings to promote cell adhesion.

[0063] In one aspect, this application provides an edible bio-ink comprising cell-loaded prolysin microspheres and an ink solution containing gelatin and alginate, forming an edible bio-ink for 3D printing cultured meat.

[0064] On the other hand, this application provides a method for producing edible bio-ink. The method includes providing an ink solution comprising gelatin and alginate; and mixing cell-loaded gliadin microspheres with the ink solution to form an edible bio-ink for 3D printing cultured meat. In some embodiments, the mixing includes stirring at 100 rpm for 5 minutes.

[0065] Preferably, the mass ratio of gelatin to alginate is 9:1.

[0066] In some embodiments, each milliliter of ink solution contains 0.01 g to 0.1 g of alcohol-soluble protein microspheres. Preferably, each milliliter of ink solution contains 0.03 g to 0.05 g of alcohol-soluble protein microspheres.

[0067] In some embodiments, the edible bio-ink is an edible plant-based bio-ink.

[0068] Preferably, the concentration of the ink solution is 14 w / v%.

[0069] According to one aspect, this application provides a method for manufacturing cultured meat. For example... Figure 1 As shown, the method includes culturing cells (105, 107) on gliadin microspheres 103 via suspension culture 300 to form cell-loaded gliadin microspheres; incorporating the cell-loaded gliadin microspheres into an ink solution 400 comprising gelatin and alginate to form bio-ink (110, 112); and extruding the bio-ink using a 3D printer to form cultured meat 500.

[0070] Cell 105 includes muscle stem cells, while cell 107 includes adipose stem cells. After inducing cell differentiation by adding differentiation culture medium, cell 105 forms mature muscle cells 105a; cell 107 forms mature adipocytes 107a.

[0071] In some embodiments, the cell culture step requires 6 to 14 days to form differentiated cells, and then the cell-loaded prolysin microspheres are incorporated into the ink solution.

[0072] Glycoprotein microspheres loaded with cells are incorporated into ink solution 400 to form bio-ink 110; glycoprotein microspheres loaded with cells 107 are incorporated into ink solution 400 to form bio-ink 112.

[0073] Preferably, the ink solution 400 comprises gelatin and alginate.

[0074] In some embodiments, the particle size of the prolysin microspheres may be greater than 150 micrometers (µm). Furthermore, the particle size of the prolysin microspheres is also less than or equal to 500 µm. In some embodiments, the particle size range of the prolysin microspheres is greater than 150 µm to 500 µm. In some embodiments, in a dry state, the particle size range of the prolysin microspheres is 100 µm to 200 µm. In some embodiments, under aqueous conditions, the particle size range of the prolysin microspheres is greater than 150 µm to 300 µm.

[0075] In some embodiments, after being cultured in aqueous conditions, the particle size of the cell-loaded prolysin microspheres ranges from greater than 150 µm to 300 µm.

[0076] In some embodiments, the cells include porcine muscleatellite cells and differentiated adipose-derived stem cells.

[0077] In some embodiments, the cells also include mouse myoblasts (C2C12) and mouse preadipocytes (3T3-L1). Specifically, for mouse preadipocytes (3T3-L1), on day 5 of culture, the gliadin microspheres loaded with mouse preadipocytes showed 7.58 × 10⁻⁶ mg gliadin microspheres. 4 Live cell density per cell.

[0078] On the other hand, this application provides a method for manufacturing alcohol-soluble protein microspheres, which includes mixing alcohol-soluble protein stock solution and an oil phase at a ratio of 1:1 v / v to form a pre-emulsified mixture, homogenizing the pre-emulsified mixture to form an ethanol / water-in-oil emulsion, and heating the ethanol / water-in-oil emulsion to remove ethanol to form alcohol-soluble protein microspheres, wherein the particle size of the alcohol-soluble protein microspheres ranges from greater than 150 µm to 500 µm.

[0079] Preferably, the prolysin microspheres comprise zein microspheres.

[0080] Preferably, the oil phase comprises lecithin in caprylic / capric triglycerides (GTCC).

[0081] In one example, various cell types can adhere to, proliferate, and differentiate within the prolysin microspheres.

[0082] The prolysin microspheres may further include differentiation-inducing factors, growth factors, growth stimulants, physiologically active substances, or peptides or polysaccharides that serve as cell-binding mediators, for inducing differentiation or proliferation of transplanted cells.

[0083] Adipose-derived stem cells and muscle cells can be implanted into the prolysin microspheres.

[0084] Cells or adipocytes are used for three-dimensional culture.

[0085] The following detailed examples illustrate the method for preparing prolysin microspheres according to embodiments of the present invention, and the use of bio-ink prepared according to the method for manufacturing cell-cultured meat.

[0086] Example 1

[0087] 1.1 Preparation method of zein microspheres

[0088] Zeadin powder was dissolved in an ethanol-water solution (70% v / v) at 50°C with stirring (500 rpm) to prepare a zeadin stock solution (30% w / v). Lecithin was dissolved in food-grade caprylic / capric triglycerides (GTCC) at 50°C with stirring (500 rpm) to obtain a clear solution (5% w / v), which served as the oil phase solution. To prepare zeadin microspheres with a target particle size of 100 µm to 200 µm per kilogram, the zeadin stock solution (3.3 L) and the oil phase solution (3.3 L) were mixed with stirring (500 rpm) for pre-emulsification. The mixture was then emulsified using a high-speed homogenizer at 8000 rpm for 3 min and rapidly transferred to a 20 L flask. Under reduced pressure (100 kPa), ethanol was removed by rotary evaporation at 50°C and 140 rpm for 5 h, allowing zein microspheres to gradually precipitate. The precipitated zein microspheres were then sequentially screened through 200 µm and 100 µm meshes to achieve the desired particle size. The screened zein microspheres were washed several times with n-hexane and then dried in an oven at 70°C for 48 h to obtain dried zein microspheres with a particle size ranging from 100 µm to 200 µm, with a yield >90%. To prepare zein microspheres with different particle sizes (100–500 µm), the concentration of the zein stock solution, lecithin concentration, the ratio of aqueous to oil phases, the homogenization rate, and the rotary evaporation conditions could be optimized. To visualize the prepared emulsion solution, the aqueous and oil phases were stained by adding fluorescein isothiocyanate (FITC, 0.1 mg / mL) and Nile Red (0.1 mg / mL), respectively, and fluorescence imaging was performed using a confocal laser scanning microscope (Nikon A1, Japan).

[0089] 1.2 Characterization of zein microspheres

[0090] The morphology of dried zein microspheres coated with platinum was observed using a scanning electron microscope (SEM, Quanta 250, FEI, USA). The diameter of the dried zein microspheres was measured from the corresponding SEM images. The degree of swelling of the zein microspheres in aqueous solution was assessed by measuring the weight change before and after immersion in deionized water at 25 °C for 12 hours. The diameter of hydrated zein microspheres was measured using optical images taken with an inverted microscope (Eclipse 2, Nikon, Tokyo, Japan). The density of the zein microspheres was determined using a gas specific gravity bottle (AccuPyc II 1340, Anton Paar, Graz, Austria). The surface properties of the dried and hydrated zein microspheres were measured using an atomic force microscope (AFM, Dimension icon®, Bruker, Massachusetts, USA) in peak force impact mode. Samples were mounted on mica slides, and dried and hydrated zein microspheres were scanned at a frequency of 1 Hz using RTESA-150® and ScanAsyst-Air® probes (Brook). The spring constants of the probes were individually calibrated by the manufacturer. The raw data were processed using NanoScope Analysis software (v1.80, Book) and averaged over six unique indentations to obtain topographic images, roughness, and Young's modulus of the zein microsphere surface.

[0091] 1.3 Cell Culture Protocol

[0092] The growth medium for mouse C2C12 and 3T3-L1 cells was high-glucose DMEM containing 10% FBS and 1% P / S. DMEM / F-12 containing 10% FBS, 1% P / S, and 5 ng / mL bFGF was used to culture porcine skeletal muscle satellite cells (PSCs) and porcine adipose-derived mesenchymal stem cells (ADSCs). Cells were cultured at 37 °C in a 5% CO2 incubator and passaged every 3 days.

[0093] To induce myogenesis in muscle stem cells, C2C12 and PSCs were cultured in 6-well plates to 80% confluence. Then, on days 7 and 4, C2C12 and PSCs were cultured in differentiation medium (DMEM supplemented with 2% horse serum and 1% P / S). The adipogenic differentiation medium contained INS (1 µg / mL). -1 IBMX (0.5 mM), DEX (1 µM), and ROS (2 µM) were used to induce adipogenic differentiation of 3T3-L1 and ADSC cells for 4 and 5 days, respectively. Subsequently, cells were cultured in maintenance medium (containing 1 µg / mL)... -1 They were cultured in the INS growth medium for 2 and 3 days.

[0094] 1.43D Cell Culture

[0095] Prior to cell seeding, zein microspheres were sterilized under UV light for 2 hours and then immersed in growth medium to obtain fully hydrated microspheres. Zein microspheres (500 mg) were dispersed in 75 mL of growth medium in sterile 125 mL spin-on flasks mounted on a 3D FloTrx-miniSPIN platform (Beijing CytoNiche Biotechnology Co., Ltd.) connected to a rotation rate controller. An appropriate cell suspension was added to the spin-on flask. The seeding densities for C2C12, PSC, 3T3-L1, and ADSC were 5 x 10⁻⁶ cells / mL. 5 2.7 x 10 6 2.4 x 10 6 and 2.2 x 10 6 To initiate cell attachment, a cyclic stirring program was used in the roller flask (40 rpm for 5 minutes, 0 rpm for 55 minutes; 24 cycles). In another example, the cell numbers for C2C12, PSCs, 3T3-L1, and ADSCs were 1 x 10⁻⁶ cells. 6 1 x 10 6 2 x 10 6 2 x 10 6 The flasks were stirred under an attachment program (35 rpm for 5 minutes, then 0 rpm for 115 minutes). Subsequently, for C2C12 and PSC, the stirring program was switched to a constant speed of 40 rpm. For 3T3-L1 and ADSC, during the proliferation phase, the stirring program was initially set to a constant speed of 40 rpm, and then switched to a cyclic program (30 rpm for 5 minutes, then 0 rpm for 115 minutes) during the differentiation and maintenance phases.

[0096] During culture, cell number on zein microspheres was determined by CCK-8 assay. For fluorescence imaging of cells, zein microspheres were fixed with 4% paraformaldehyde (PFA) at 4 °C for 20 min. After three washes with phosphate-buffered saline (PBS), nuclei were stained with Hoechst 33342 for 10 min. Cells were then permeabilized with 0.1% Triton X-100 for 5 min and the cytoskeleton was stained using an F-actin staining kit. Lipid droplets were stained with BODIPY 493 / 503 (5 mM) for 30 min at room temperature. PSCs were stained for MYHC using rabbit anti-MYHC as the primary antibody and Alexa Fluor 594 goat anti-rabbit IgG as the secondary antibody. Cell morphology was then captured using a confocal laser scanning microscopy (CLSM, Nikon A1RHD25, Tokyo, Japan).

[0097] 1.5 RT-qPCR and transcriptomics analysis

[0098] Cells were cultured on zein microspheres until the cell number reached its maximum according to the proliferation curve. Myogenesis or adipogenesis was then induced using the appropriate differentiation medium. Total RNA was extracted using Trizol reagent, and mRNA was reverse transcribed into cDNA using the iScript cDNA Synthesis Kit (Biorad, CA, USA) on a thermal cycler (Bio-Rad, T100 thermal cycler, CA, USA). Relative gene expression levels were then detected using specific primer pairs on a GoTaq qPCR Master Mix with a QuantStudio 3 qPCR system (Thermo Fisher Scientific, MA, USA). The method quantifies gene expression and normalizes it to the expression of housekeeping genes (GAPDH). Primers were synthesized by Shanghai Sangon Biotech Co., Ltd., and their sequences are shown in Table 1.

[0099] Table 1 Primer sequences used in RT-qPCR detection.

[0100]

[0101] RNA sequencing was performed on differentiated 3T3-L1 samples cultured on 2D tissue plates and 3D zein microspheres. RNA purity was determined using a NanoPhotometer spectrophotometer (IMPLEN, CA, USA), and RNA integrity was assessed using the RNA nano 6000 assay kit with an Agilent 2100 Bioanalyzer (Agilent Technologies, CA, USA). Libraries were prepared using the NEBNext Ultra™ RNA library preparation kit, which included mRNA purification, cDNA synthesis, and fragment size selection. The sequencing libraries were then clustered and sequenced on the Illumina platform to generate 150 bp paired-end reads. Reads were mapped to a reference genome using HISAT2 (version 2.0.5), and gene expression levels were quantified using StringTie. Differential expression analysis between the two groups was performed using the DESeq2 R package (version 1.16.1), with an adjusted p-value < 0.05 considered differentially expressed. Gene Ontology (GO) and KEGG enrichment analyses were performed using clusterProfiler (version 3.4.4).

[0102] 1.6 Western blot and Oil Red O staining

[0103] Cells prepared on ice were lysed using RIPA lysis buffer on two-dimensional tissue plates and three-dimensional zein microspheres. After electrophoretic separation, proteins were transferred to polyvinylidene fluoride (PVDF, 0.2 µm) membranes. The membranes were blocked with blocking buffer for 30 min at room temperature and then incubated overnight with primary antibody at 4°C. After washing three times with Tris-buffered saline (TBST) containing Tween 20, the PVDF membranes were incubated with secondary antibody at room temperature for 1 h. Protein bands were then visualized on a chemiluminescence imaging system with enhanced chemiluminescence substrates (ChemiDoc XRS+, Bio-Rad, CA, USA). To further analyze the adipogenic differentiation of 3T3-L1 cells, they were fixed with 4% PFA and stained with lipids using an Oil Red O staining kit. Accumulated lipids were then observed using an inverted microscope (Eclipse 2, Nikon, Tokyo, Japan).

[0104] 1.7 Lipidomics Analysis

[0105] Lipidomics analysis was performed by Wuhan Metawell Biotechnology Co., Ltd. Lipids from differentiated ADSCs on pig stomach and zein microspheres were extracted using the Folch method. Samples were then analyzed using a liquid chromatography-electrospray ionization-mass spectrometry / mass spectrometry system (LC-MS / MS, QTRAP 6500+, SCIEX) and identified based on a customized Metware database.

[0106] 1.8 Assembly of Marble-Patterned Cultured Meat via 3D Bioprinting

[0107] Zein microspheres were added to an edible gelatin / alginate (GA, 9:1 mass ratio) ink solution (14 w / v%) to provide a microsphere-loaded bio-ink. Sodium alginate powder was first dissolved in phosphate-buffered saline at 70°C and stirred for 30 minutes with a mechanical stirrer (750 rpm). After cooling to 50°C, gelatin was added at the same rate, and stirring was continued for 20 minutes until a clear, viscous solution was obtained. After degassing at 37°C for 30 minutes, hydrated zein microspheres were added to the ink solution and stirred at 100 rpm for 5 minutes to produce a bio-ink with uniformly distributed zein microspheres. Different amounts of zein microspheres (0.15, 0.2, and 0.25 g, dry basis) were uniformly dispersed in a gelatin / alginate solution (5 mL) by stirring to form GA-Z ink for printability testing.

[0108] The rheological properties of the zein microsphere-loaded ink were characterized using a rheometer (MCR102e, Anton Paar, Graz, Austria) equipped with PP25 parallel plates (50 mm in diameter). Viscosities of the GA and GA-Z inks were recorded as a function of shear rate, calculated from 10 s⁻¹ at 30°C. -1 Logarithmically increasing to 100 s -1 While maintaining a constant frequency of 1 Hz, amplitude scans from 0.1% and 100% were measured at 30°C and 4°C, respectively. Storage modulus (G') and loss modulus (G'') were recorded as functions of strain (%).

[0109] The prepared bio-ink was transferred to a 5 mL syringe fitted with a conical needle (600µm nozzle size) and loaded into a desktop 3D bioprinter (Biomaker 1, Beijing Shangpu Biotechnology Co., Ltd., China). In printing tests, a multi-layered open-pore structure with a square pore shape (2.5 mm line spacing, 10 layers) was used as a model to verify the ink's printability and optimize printing parameters, including cartridge temperature (27 to 33°C) and extrusion rate (0.3 to 2.3 mm). 3The printing speed was set to 0.7 to 8.7 mm / s. The substrate temperature was set to 4 °C to promote the coagulation of deposited fibers. The printed structures were observed and imaged using a stereomicroscope (EZ4HD, Leica Microsystems, Wetzlar, Germany). After optimization, zein microspheres of PSCs cultured for 6 days and ADSCs cultured for 14 days were collected through a 40 µm cell filter and then mixed with sterile gelatin / alginate solution to form PSCs-GA-Z ink and d-ADSCs-GA-Z ink, respectively, which were then molded into pre-defined scaffolds under optimized printing conditions.

[0110] After staining with calcein AM and propidium iodide dye (Shanghai Beyotime Biotechnology Co., Ltd., China) at 37°C for 30 minutes, cell viability in the printed structures was examined by fluorescence imaging of live and dead cells. Subsequently, marbled cultured meat prototypes were constructed using the developed PSCs-GA-Z and d-ADSCs-GA-Z bioinks via a dual-nozzle 3D bioprinter (Biomaker2i, Beijing Shangpu Biotechnology Co., Ltd., China). Lycopene and beetroot extract were added to the PSCs-GA-Z bioink to mimic the color of muscle tissue.

[0111] 1.9 Characteristics of Cultured Meat

[0112] Textural profile analysis (TPA) of printed cultured meat and fresh pork belly was performed at 25 °C using a texture analyzer equipped with a cylindrical probe (approximately 15 mm in diameter) (Shanghai Tianba Instruments Co., Ltd., China). Samples were placed in petri dishes and compressed to 30% of their maximum height at a rate of 60 mm / min, with 10-second intervals between consecutive compression cycles. Hardness, elasticity, cohesiveness, and chewiness were calculated based on the corresponding compression profiles.

[0113] The crude protein and fat contents of pork belly and printed cultured meat samples were determined using the Kjeldahl method and acid hydrolysis method, respectively. The carbohydrate content was calculated by subtracting the protein, fat, water, and ash contents from the total mass. Hydrochloric acid (6 mol L⁻¹) was used for determination. -1 ) and sodium hydroxide (6 mol L) -1 After hydrolysis, the amino acid composition of freeze-dried cultured meat and pork belly samples was analyzed using an automated amino acid analyzer (1260 InfinityII, Agilent Technologies, California, USA).

[0114] For flavor analysis, pork belly and embodied meat samples were placed in an oven preheated to 200 °C for 10 minutes. Volatile compounds were detected using headspace solid-phase microextraction gas chromatography-time-of-flight mass spectrometry (HS-SPME-GC-TOF-MS, Pegasus BT, LECO Corp., Michigan, USA).

[0115] 1.10 Statistical Analysis

[0116] All data are expressed as mean ± standard deviation. Each measurement was performed at least three times. Statistical significance was determined using one-way or two-way analysis of variance (ANOVA) and Tukey's multiple comparison test. p < 0.05 p < 0.01, p < 0.001 p<0.0001, GraphPad Prism 9.5.1).

[0117] Example 2

[0118] 2.1 Preparation and characterization of zein microspheres

[0119] Kilogram-scale edible corn protein microspheres with sizes or diameters ranging from 100 μm to 200 μm were prepared via an emulsion-template evaporation method to meet the scalability, sustainability, cost-effectiveness, and food safety requirements of cultured meat production. Utilizing the unique solubility of corn protein in 70% ethanol solution, food-grade caprylic / capric triglycerides (GTCC) containing soybean lecithin were used as the oil phase to prepare an ethanol / water-in-oil emulsion via high-speed homogenization. The emulsion structure was observed using confocal laser scanning microscopy (CLSM), and the ethanol-water phase and oil phase were stained with fluorescein isothiocyanate (FITC) and Nile Red, respectively. Figure 2A As shown, an oil-in-ethanol / water-in-oil biemulsion was formed, with lecithin and corn gluten acting as emulsifiers to stabilize the biemulsion. The mixture was filtered through sieves of different pore sizes, washed with n-hexane, and dried to prepare corn gluten microspheres of the desired particle size. Figure 2B-2CAs shown in the scanning electron microscope (SEM) images, smooth-surfaced spherical corn protein microspheres can still be fabricated on a kilogram scale despite the formation of a dual emulsion as a template. This fabrication method can be attributed to the demulsification effect of the oil-in-water phase during the slow precipitation of corn protein. Other studies have shown that rapid removal of ethanol after emulsification can form porous corn protein microspheres, as corn protein plays a crucial role in maintaining the stability of the dual emulsion. Furthermore, the size and yield of corn protein microspheres can be controlled by adjusting various emulsion properties, primarily determined by the water / oil phase ratio, corn protein and lecithin concentrations, homogenization rate and time, and evaporation conditions (including rotation speed, pressure, temperature, and loading). With optimization, corn protein microspheres of different sizes (100 μm to 500 μm) can be produced. Figures 3A-3F ), and the output can exceed 90%.

[0120] In one example, zein microspheres with an average size of 123.5 μm ± 14.2 µm were used in subsequent steps. Although insoluble in water, zein swells under aqueous conditions, exposing its hydrophilic portion to the surface. The degree of hydration of the zein microspheres was assessed by measuring changes in weight and particle size before and after immersion in water for 12 hours. Figure 2D-2E As shown, the weight and particle size of the hydrated zein microspheres increased to 405% ± 30% and 192.1 μm ± 37.4 µm, respectively. The hydrated zein microspheres exhibited strong fluorescence under aqueous conditions. Figure 2F The excitation wavelength was 488 nanometers (nm), which is attributed to the binding of pigments such as lutein on zein. The surface properties of dried and hydrated zein microspheres were further investigated using atomic force microscopy (AFM). The results showed that the arithmetic mean surface roughness (Ra) increased significantly by approximately 100-fold from 0.27 nm ± 0.05 nm to 31.12 nm ± 7.10 nm after hydration. Figure 2I ). Figure 2G The dried zein microspheres show a smooth surface, while Figure 2H The results show that the surface of hydrated zein becomes moderately rough, which is beneficial for cell adhesion and proliferation. Since water is the most effective plasticizer for protein materials, the surface stiffness of hydrated zein microspheres, expressed as Young's modulus, significantly decreased from 535.2 MPa ± 60.5 MPa to 4.4 MPa ± 2.5 MPa compared to the dry material. Figure 2I The results are consistent with previous studies, indicating that the tensile strength of the alcohol-soluble protein scaffold decreased by approximately 100-fold after hydration.

[0121] 2.2 Scalable Expansion of Muscle Stem Cells on Zein Glycol Microspheres

[0122] First, C2C12 mouse myoblasts were seeded in a 125 mL rotary flask bioreactor to assess their growth and differentiation behavior and to investigate the feasibility of using the developed zein microspheres for scalable expansion of skeletal muscle stem cells in the bioreactor. Prior to cell seeding, the zein microspheres were sterilized by exposure to UV light and hydrated by immersion in complete culture medium. On day 1, an intermittent stirring program (40 rpm for 5 minutes, 0 rpm for 55 minutes; 24 cycles) was used to promote cell adhesion after mixing the suspended cells with the zein microspheres in the rotary flask. Subsequently, stirring was maintained at a constant speed of 40 rpm to ensure uniform dispersion and suspension of the zein microspheres in the culture medium. At different culture days, the cell count on the zein microspheres was monitored using a colorimetric cell counting kit-8 (CCK-8) assay, and recorded as the number of cells per milligram of microspheres. The growth curve is shown below. Figure 4A As shown. Figure 4B CLSM images showed that C2C12 cells were able to adhere to and proliferate on the surface of zein microspheres in a diffuse manner, with a high adhesion rate (109%), indicating that zein has excellent cell affinity. Cells proliferated exponentially on the zein microspheres until they covered the entire surface. Figure 4C The cell number peaked on day 5, increasing approximately 16-fold. From day 6 onwards, under the regulation of muscle regulatory factors, C2C12 cells on zein microspheres underwent myoblastic differentiation induced by serum starvation. Until day 8, the cell number continued to decrease by approximately 40%, consistent with the growth behavior of C2C12 cells on porous gelatin microspheres. This significant cell loss is likely due to apoptosis, cell contraction, and mechanical damage during myoblastic differentiation. After serum starvation, F-actin staining revealed elongated myotubular morphologies at the bridging cell junctions between microspheres. Figure 4D (As indicated by the white arrow in the middle), this shows that muscle micro-tissue has formed on the zein microspheres.

[0123] In addition, the expression of genes and proteins related to muscle formation was examined to compare the differentiation behavior of C2C12 cells on 2D tissue plates and 3D zein microspheres. Figure 4EAs shown, real-time quantitative polymerase chain reaction (RT-qPCR) results revealed that after 7 days of differentiation on 2D tissue plates, the expression of myoblast determination protein 1 (MYOD), an early marker of myogenesis, was significantly upregulated, while MYOD, a transcription factor promoting muscle generation and proliferation, was downregulated on 3D microspheres. Furthermore, myopoietin (MYOG), a key transcription factor for terminal differentiation and fusion of myoblasts into myotubes, showed approximately 11-fold increased expression on 3D microspheres compared to cells grown on 2D plates, even before serum starvation. Transcriptional analysis indicated that C2C12 cells underwent a spontaneous myogenic process on zein microspheres. Additionally, muscle-specific proteins, including desmin and myosin heavy chain (MYHC), were detected in both 2D and 3D environments, with their expression increasing more than 2-fold after 7 days of serum starvation, indicating myotube fusion and maturation. Figure 4G-4I After isolating C2C12 cells on days 3, 6, and 12, the surface morphological changes of zein microspheres were visualized using SEM imaging. As shown in Figure 4J, with prolonged culture time, the zein microspheres exhibited a porous structure from the surface to the core, which may be due to digestion by proteases secreted by the cells.

[0124] To verify the potential of incorporating edible zein microspheres into cultured meat products, specifically zein microspheres already covered with cells, this application used the same culture parameters (including inoculation density and agitation program) as C2C12 cells to culture PSCs ( Figure 5A ).like Figure 5B As shown in the growth curve of PSCs, PSCs attached to the surface of zein microspheres and proliferated exponentially from day 1 to day 6, reaching an 8-fold increase on day 6, which was defined as the expansion phase. Similarly, when the growth medium was changed to differentiation medium containing 2% horse serum, a significant decrease in cell number was observed starting from day 7. After 6 days of inoculation and culture on zein microspheres, Figure 5C CLSM images of PSCs show that PSCs cover the entire surface of the microspheres, migrate from one microsphere to another through intercellular interactions, and eventually form microtissues (indicated by white arrows) through bridging connections between zein microspheres, which facilitates fusion into myotubes during differentiation. Furthermore, biomarkers at different myoblastic stages were quantified to confirm whether PSCs could differentiate into mature myotubes. Despite a decrease in cell number, myoblastic induction of PSCs on zein microspheres resulted in a slow but significant increase in the gene expression levels of MYOG and MYHC. Figure 5E-5F This was confirmed by immunofluorescence staining of MYHC. Figure 5D).

[0125] 2.3. Expansion and maturation of mammalian adipocytes on zein microspheres

[0126] Besides muscle protein, fat content is also a key component affecting the taste, texture, juiciness, and appearance of meat. Therefore, 3T3-L1 mouse preadipocytes and porcine adipose-derived mesenchymal stem cells were used as precursors for adipose tissue production to evaluate the compatibility of developing zein microspheres for adipose tissue production. Specifically, on day one, mouse 3T3-L1 cells were seeded onto zein microspheres in a rotating flask using an intermittent stirring program to ensure sufficient contact between the cells and the microsphere surface for adhesion / attachment. By monitoring cell numbers during the proliferation phase (with constant stirring at 40 rpm), 3T3-L1 cells exhibited exponential expansion, reaching a viable cell density of 7.58 × 10⁶ cells per milligram of zein microspheres by day 5. 4 The number of cells increased approximately 16 times compared to day 1. Figure 6A The culture medium was then replaced with a differentiation medium containing adipogenesis inducers, including insulin (INS), 3-isobutyl-1-methylxanthine (IBMX), dexamethasone (DEX), and rosiglitazone (ROS). These inducers activate the peroxisome proliferation-activating receptor γ (PPARγ) pathway and CCAAT / enhancer-binding protein (C / EBP), inducing preadipocyte differentiation. Figure 7A Cell growth curves showed that adipocytes maintained a high density during differentiation, and the cell number did not decrease significantly. Furthermore, under the regulation of these adipogenic inducers, intracellular lipid droplets were detected in 3T3-L1 cells attached to zein microspheres using CLSM imaging, and their morphology clearly changed from spindle-shaped or star-shaped to round. Figure 6B -C), during which time stirring is switched to intermittent periods starting with long periods of stillness (30 rpm for 5 minutes, 0 rpm for 115 minutes) to prevent cells from detaching from the microspheres. During the maintenance phase, densely dispersed small lipid droplets can aggregate into larger droplets, and differentiated cells can aggregate into cell clusters, forming adipose tissue-like structures. Figure 6D ).

[0127] Transcriptome analysis was performed on differentiated 3T3-L1 cells cultured for 12 days on 2D tissue plates and zein microspheres to further elucidate the differences in cellular behavior between preadipocytes grown on 2D tissue plates and 3 microspheres. Overall, as Figure 6EAs shown in the volcano plot, compared to 3T3-L1 cells grown on zein microspheres, 5001 and 4409 genes were significantly upregulated and downregulated, respectively, in 3T3-L1 cells grown on 2D plates used as a control. KEGG enrichment (Fig. 6F) and Gene Ontology (GO) enrichment analyses were also performed. Figure 6G Both studies indicated that differentially expressed genes (DEGs) between 3D and 2D cultured cells were primarily located in ribosomes, the mitochondrial respiratory chain, focal adhesion, and the actin cytoskeleton, and appeared to be involved in the regulation of translation, cell adhesion and cell shape, energy metabolism, and lipid metabolism. Preadipocytes cultured in a 3D environment exhibited a more rounded morphology, greater energy demand, and reproduced the cellular and molecular phenotypes of in vivo adipocytes, with improved adipogenesis. More specifically, almost all DEGs in the ribosomal, oxidative phosphorylation, and proteasome pathways were upregulated, indicating increased metabolic activity and energy consumption in 3T3-L1 cells grown on zein microspheres. Figure 6H-6K The volcano diagrams of the various pathways shown reveal that changes in cell shape result from cytoskeleton remodeling through increased expression of cytoplasmic actin γ and β (ACTG 1 and ACTB) and myosin (MYL9 and MYL12A) and decreased expression of cell adhesion molecules such as integrin α6 (INTAG 6), laminin family members (LAMA5, LAMC1, LA microspheres 2, LA microspheres 4, and LAMAB1), and vesicle protein (VCL). Furthermore, in 3T3-L1 cells differentiated on zein microspheres, mitochondrial fatty acid elongation and polyunsaturated fatty acid biosynthesis are enhanced, manifested by increased expression of fatty acid elongase (ELOVL3), acetyl-CoA acyltransferases (ACAA1A, ACAA1B, and ACAA2), and acyl-CoA dehydrogenase (ACADL). Transcriptome analysis revealed that the different morphologies of preadipocytes on 2D plates and zein microspheres are mainly regulated by the reorganization of the cytoskeleton and adhesion proteins (such as laminin), thereby affecting lipid biosynthesis and metabolic efficiency, possibly through the regulation of ribosome and mitochondrial respiration-related genes.

[0128] Porcine adipose-derived stem cells were seeded onto zein microspheres to produce cultured meat products containing fat. The growth curve is shown below. Figure 5B As shown. Similar to 3T3-L1 cells, porcine ADSCs attached to and proliferated on zein microspheres from day 1 to day 6, during which time the number of cells per milligram of zein microspheres reached a peak of 2.64 × 10⁻⁶. 4The number of cells increased approximately 6-fold compared to the initial seeded cell count. Although a slight decrease in cell number was observed after treatment with the lipogenesis mixture, the cell density remained at approximately 2 × 10⁻⁶ cells from day 7 onwards. 4 Cells / mg indicates that ADSCs in the system are sufficiently expanded and differentiated. Figures 7C-7D As shown in the CLSM images, ADSCs in the amplification phase diffuse in a spindle-shaped pattern on the surface of zein microspheres, identified by staining the nucleus and cytoskeleton with blue and red fluorescent dyes. Following differentiation, the cells become round and accumulate unilocular lipid droplets (green fluorescence), a characteristic of mature adipocytes. Optical images further reveal that after adipogenesis differentiation, round adipocytes can aggregate between zein microspheres to form cell clusters, with densely distributed lipid droplets. Figure 7E Furthermore, transcriptional analysis revealed that PPARγ, perilipin-1 (PLIN1, a surface protein that maintains lipid droplets), fatty acid-binding protein (FABP4), acetyl-CoA carboxylase (ACC), fatty acid synthase (FAS), and adiponectin (ADIPOQ) were significantly upregulated after 3 days of treatment with the lipogenesis mixture, revealing high efficiency in lipogenesis. Figure 7F-7K Interestingly, during the maintenance phase (D5 to D8), genes related to fatty acid biosynthesis, including FAS, ACC, and PPARγ, were downregulated, while genes related to fatty acid metabolism, such as FABP4, PLIN1, and ADIPOQ, remained upregulated compared to pre-differentiation (D0) levels. This suggests that ADSCs mature on zein microspheres during the maintenance phase (without lipogenesis inducers).

[0129] Subsequently, lipids secreted by ADSCs on zein microspheres were extracted using the Folch method, and their content and composition were characterized by lipidomics analysis and compared with lipids extracted from fresh pork adipose tissue. Figure 7L As shown, principal component analysis (PCA) revealed no significant difference between lipids extracted from ADSC and pork fat (p = 0.4). Lipids in pork fat were mainly composed of glycerides (90%) and fatty acids (8%), while lipids in ADSC were mainly composed of fatty acids (30%), glycerides (25%), sphingolipids (31%), and glycerophospholipids (14%).

[0130] 2.4. Developing bio-inks loaded with zein microspheres using 3D bioprinting and assembling marbled cultured meat.

[0131] This paper utilizes 3D bioprinting technology to assemble cell-grown zein microspheres into cultured meat products, producing cultured meat structures with realistic meat texture characteristics using gelatin / alginate (GA-Z) ink containing zein microspheres. This application verifies the feasibility of using gelatin and alginate as the base ink materials, as both materials exhibit good printability. To achieve continuous printing with GA-Z ink, it is necessary to optimize the nozzle size, zein microsphere loading density, and printing parameters. According to one example, this application selected a tapered tip with an opening size of 600 µm for printing tests because using a tapered tip with an opening size at least three times larger than the average size of the loaded microspheres allows for the deposition of continuous filaments without clogging. Three different amounts of zein microspheres (0.15, 0.2, and 0.25 g, dry basis) were uniformly dispersed in a gelatin / alginate solution (9:1, 14 wt%, 5 mL), namely GA-Z15, GA-Z20, and GA-Z25 inks, to optimize the microsphere density. The rheological properties of the inks, which play a crucial role in determining printability, were then investigated. The GA inks exhibited typical shear-thinning behavior, where viscosity decreased with increasing shear rate. Figure 8F Due to interparticle friction, the addition of zein microspheres will reduce the ink viscosity in the low shear rate range (0.1-10 s). -1 The amplitude decreased by approximately 30%, regardless of the amount of microspheres added, while the ink maintained its shear-thinning behavior. Amplitude scan results ( Figure 8G Further analysis showed that the addition of zein microspheres had little effect on the flowability (30°C) and gelling properties (4°C) of GA ink, as the storage modulus (G') and loss modulus (G'') almost overlapped within the tested shear strain range. After optimizing printing parameters (including ink feed rate, print speed, cartridge temperature, and substrate temperature), GA-Z15 and GA-Z20 inks exhibited good printability and high fidelity in deposited fibers. Conversely, when using GA-Z25 ink, discrete fiber structures frequently appeared due to nozzle tip clogging caused by the high density of zein microspheres. After fixing the structure in a calcium chloride solution, the GA-Z20 ink printed structures exhibited clear pore structures and fiber stacking, with fiber widths of 1.01 mm ± 0.14 mm and pore diameters of 1.44 mm ± 0.19 mm, indicating good extrudability and print fidelity. The number of zein microspheres in the deposited fiber monolayer was calculated to be an average areal density of 600 to 900 zein microspheres per square centimeter. Overall, considering the cell loading density and extrudability of the ink, this application uses GA-Z20 ink for subsequent studies.

[0132] Subsequently, cell-loaded zein microspheres were incorporated into the printed cultured meat constructs. Specifically, PSCs grown on zein microspheres and differentiated ADSCs were collected after 6 and 11 days, respectively, in spin-dip flasks and added to sterilized GA ink to form cell-grown PSC-GA-Z and d-ADCS-GA-Z bioinks. The cell-grown zein microspheres were filtered through a fiber mesh (300µm pore size) to remove large aggregates, with the addition amount proportional to the GA-Z20 ink to ensure printability. The printing fibers ( Figures 9A-9B The PSC and ADSC viability on the zein microspheres remained high (>90%) because most cells remained attached to the microspheres, protecting against shear stress during printing. Furthermore, by importing the designed marbling meat model into a dual-nozzle 3D bioprinter, centimeter-scale 3D-printed cultured meat was successfully constructed, mimicking the structural and sensory characteristics of real marbling meat. Figure 8C The cultured meat was achieved using d-ADSC-GA-Z and PSC-GA-Z bio-inks (using lycopene and beetroot extract as fat and muscle components, respectively). CLSM images of the printed structures, obtained by pre-staining PSCs and differentiated ADSCs grown on zein microspheres, showed that the cellular structures of the PSCs and ADSCs on the zein microspheres remained intact. Numerous secreted lipid droplets remained within the ADSC cells without leakage. Some cells and cell aggregates, especially differentiated ADSCs, may detach from the zein microspheres during preparation but may remain trapped in the GA gel network after printing.

[0133] like Figure 8DAs shown, the nutritional composition of the printed marbled cultured meat was compared with that of fresh pork belly purchased from the local market. The results confirmed that the marbled cultured meat had a high water content (85.53% ± 0.14%), which can be attributed to the hydrogel properties of the bio-ink and the low loading density of the zein microspheres, while also containing all three essential nutrients similar to those found in pork. The high protein content in the cultured meat product is attributed to the zein and gelatin encapsulated in the microspheres and bio-ink, while the carbohydrates in the cultured meat mainly come from alginate in the bio-ink. Amino acid composition analysis (Table 2) showed that the cultured meat product contained all essential amino acids, with relative proportions very close to those of the pork belly sample (dry basis). Compared to the pork sample, the cultured meat product had significantly higher levels of glutamic acid, glycine, proline, and alanine, which are derived from zein and gelatin. Notably, the cellular biomass in the cultured meat can supplement the amino acids lacking in zein and gelatin, such as lysine and tryptophan, thereby improving its nutritional value. The fat content (3.8%) in cultured meat products originates from lipids secreted by differentiated ADSCs and their cellular components, but this fat content is lower than that of pork belly (17.2%). In principle, the protein and fat content in marbling cultured meat can be customized by using a custom-designed meat model with a specific muscle-to-fat ratio, thereby optimizing the nutritional characteristics of cultured meat products.

[0134] Table 2. Amino acid composition of pork belly and cultured meat.

[0135]

[0136]

[0137] Textural profile analysis (TPA) was performed on marbling cultured meat and fresh pork belly using a continuous compression test. Figure 8EThe results showed that the firmness and elasticity of the cultured meat were similar to those of pork belly, but the differences were not significant. However, the cultured meat was noticeably chewier than pork due to its greater viscosity, attributed to the gel structure of the printed cultured meat. Furthermore, after baking the cultured meat and pork belly samples at 200°C for 10 minutes, volatile compounds were detected by headspace solid-phase microextraction gas chromatography-mass spectrometry (HS-SPME-GCMS) for flavor analysis and comparison. As listed in Table 3, the detected volatile compounds were classified according to their chemical structure and functional groups, and their flavor descriptions were labeled according to the American Flavor and Extract Manufacturers Association (FEMA). Specifically, the fatty and green flavors primarily originated from aldehydes, including octanal, heptanal, and hexanal; alcohols, such as 2-nonen-1-ol; and ketones, such as 2,3-octanedione. These compounds were detected in relatively high amounts in both the marbled cultured meat and pork samples and are considered representative flavor compounds produced by lipid oxidation degradation and hydrolysis. These results demonstrate the significant contribution of lipids to the flavor of roasted meat and the necessity of incorporating differentiated ADSCs into cultured meat products. The charred and roasted nutty flavors in the samples primarily originate from heterocyclic compounds, such as pyrazines, which are typical flavor compounds resulting from Maillard reactions between amino acids in proteins and reducing sugars in polysaccharides. Interestingly, fruity esters were detected in the cultured meat samples but not in the pork samples. Conversely, the cultured meat samples lacked almond, malt, butter, cream, and garlic flavors, likely due to differences in amino acid and sugar composition between the cultured and pork samples.

[0138] Table 3 Comparison of volatile compounds in pork belly and marbling cultured meat [mean ± standard deviation].

[0139]

[0140]

[0141]

[0142]

[0143] -ND indicates that it was not detected.

[0144] Compared to traditional 3D bioprinted cultured meat, the method described in this application offers several advantages. As shown in Table 4, 1) zein is a purely plant-based edible material with high nutritional value and low production costs; 2) zein is insoluble in aqueous solutions, thus eliminating the need for further cross-linking in cell culture applications; 3) zein exhibits high cell affinity, supporting the growth of various cell types without surface modification or the use of additional highly toxic cross-linking agents; 4) zein microspheres, as a plant protein, can be embedded in cultured meat products to enhance their protein content. Furthermore, the estimated production cost of zein microspheres is lower than that of commercially available microspheres, making them particularly suitable for cultured meat applications. The prepared bio-ink solution also eliminates the need for additional cell separation steps, thereby minimizing food safety concerns and reducing costs.

[0145] Table 4 Comparison of commercially available microspheres and developed corn gliadin microspheres

[0146]

[0147]

[0148] Product prices are determined by the manufacturers.

[0149] Furthermore, microtissues formed from differentiated muscle or fat cells can be directly textured into the final product without further culturing. Zein microspheres protect cells from mechanical damage during printing and enhance the nutritional value of the final product. Additionally, the stiffness of the gelatin / alginate scaffold can be precisely adjusted by changing the alginate concentration and cross-linking degree, thereby influencing the differentiation of bovine myoblasts and ADSCs. It is feasible to develop gelatin / alginate inks using this strategy for printing fibrous gels with stiffness closely resembling the texture characteristics of muscle and fat tissue. Utilizing the customizability of 3D / 3D printing technology, the ratio of muscle to fat content in printed marbled meat can be precisely controlled by designing marbled meat models, thereby adjusting the nutritional composition of cultured meat products in terms of protein, fat, and carbohydrate content. Finally, the addition of differentiated ADSCs to printed marbled cultured meat plays a crucial role in enhancing the flavor of the product, as lipids participate in the generation of various flavor compounds during thermal processing. In summary, the bio-ink and its zein microspheres provided in this application have commercial potential for the large-scale production of cellular tissues for cultured meat production and for assembling cultured meat with customizable sensory characteristics.

Claims

1. Edible bio-ink, characterized in that, include: cell-loaded prolysin microspheres; and An ink solution containing gelatin and alginate forms an edible bio-ink for 3D printing cultured meat, wherein the gliadin microspheres have a particle size range of greater than 150 µm to 500 µm.

2. The edible bio-ink as described in claim 1, characterized in that, The cells include porcine muscle satellite cells and differentiated adipose-derived stem cells.

3. The edible bio-ink as described in claim 2, characterized in that, The cells also include mouse myoblasts (C2C12) and mouse preadipocytes (3T3-L1).

4. The edible bio-ink as described in claim 1, characterized in that, The mass ratio of the gelatin to the alginate is 9:

1.

5. The edible bio-ink as described in claim 1, characterized in that, Each milliliter of ink solution contains 0.01 g to 0.1 g of alcohol-soluble protein microspheres.

6. The edible bio-ink as described in claim 1, characterized in that, The edible bio-ink is an edible plant-based bio-ink.

7. The edible bio-ink as described in claim 1, characterized in that, The prolysin microspheres are corn prolysin microspheres.

8. The edible bio-ink as described in claim 1, characterized in that, The concentration of the ink solution is 14 w / v%.

9. A method for producing edible bio-ink, comprising: Ink solutions containing gelatin and alginate are available; as well as Glycol microspheres loaded with cells are mixed with the ink solution to form an edible bio-ink for 3D printing cultured meat, wherein the particle size of the glycol microspheres ranges from greater than 150 µm to 500 µm.

10. The method as described in claim 9, characterized in that, The mixing process involves stirring at 100 rpm for 5 minutes.

11. The method as described in claim 9, characterized in that, The cells include porcine muscle satellite cells and differentiated adipose-derived stem cells.

12. The method as described in claim 11, characterized in that, The cells also include mouse myoblasts (C2C12) and mouse preadipocytes (3T3-L1).

13. The method as described in claim 9, characterized in that, The mass ratio of the gelatin to the alginate is 9:

1.

14. A method for manufacturing cultured meat, comprising the following steps: Cells were cultured on prolyl microspheres to form cell-loaded prolyl microspheres; The cell-loaded prolysin microspheres were incorporated into an ink solution comprising gelatin and alginate to form a bio-ink; The bio-ink was then extruded using a 3D printer to form cultured meat, wherein the gliadin microspheres had a particle size ranging from greater than 150 µm to 500 µm.

15. The method as described in claim 14, characterized in that, The cell culture step also includes inducing cell differentiation loaded in prolysin microspheres.

16. The method as described in claim 14 or 15, characterized in that, The cell culture step requires 6 to 14 days to form differentiated cells, and then the cell-loaded prolysin microspheres are incorporated into the ink solution.

17. The method as described in claim 14, characterized in that, The cells include porcine muscle satellite cells and adipose-derived stem cells.

18. The method as described in claim 17, characterized in that, The cells also include mouse myoblasts (C2C12) and mouse preadipocytes (3T3-L1).

19. The method of claim 14, wherein on day 5 after culturing mouse preadipocytes 3T3-L1, the cell-loaded glioprotein microspheres show a viable cell density of 7.58 × 10⁻⁶ per milligram of glioprotein microspheres. 4 Each cell.

20. A method for manufacturing alcohol-soluble protein microspheres, comprising: The alcohol-soluble protein stock solution and the oil phase were stirred and mixed at a ratio of 1:1 v / v to form a pre-emulsified mixture; The pre-emulsified mixture was homogenized to form an ethanol / water-in-oil emulsion; and The ethanol / water-in-oil emulsion is heated to remove the ethanol and form alcohol-soluble protein microspheres. The particle size range of the prolysin microspheres is greater than 150 µm to 500 µm.

21. The method as described in claim 20, characterized in that, The prolysin microspheres include zein microspheres.

22. The method as described in claim 21, characterized in that, The oil phase includes lecithin in caprylic / capric triglycerides.

23. The method as described in claim 20, characterized in that, The prolysin microspheres are loaded with cells.

24. Glycol protein microspheres manufactured by the method of any one of claims 20-23, having a particle size greater than 150 µm to 500 µm.

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