Preparation method and application of edible plant hydrogel scaffold

Abstract

The application provides a preparation method of an edible plant hydrogel scaffold and application thereof, and belongs to the field of biomaterials and food science and technology. The preparation method of the edible plant hydrogel scaffold provided by the application comprises the following steps: dissolving kappa-carrageenan and konjac gum in water to obtain a mixed solution, standing overnight to form a gel; and adding a potassium chloride solution into the gel to perform ion crosslinking to obtain the edible plant hydrogel scaffold. The 3D scaffold with stable structure is obtained through the intermolecular interaction between the carrageenan and the konjac gum and further ion crosslinking, the preparation method is simple and environment-friendly, the prepared scaffold has high biological safety, and has good cell adhesion and compatibility.

Description

Technical Field

[0001] This invention relates to the fields of biomaterials and food science and technology, and in particular to a method for preparing an edible plant hydrogel scaffold and its application. Background Technology

[0002] Meat is the primary source of animal protein in the human diet. With the continuous growth of the global population, the contradiction between the increasing demand for meat and the limited production capacity of meat products is becoming increasingly prominent. According to the Food and Agriculture Organization of the United Nations (FAO), global demand for meat products is projected to increase by 73% by 2050. However, the existing meat production supply chain has many hidden dangers. Furthermore, with the continuous expansion of livestock production, problems such as environmental pollution, water shortages, increased greenhouse gas emissions, land ecological damage, and an increase in animal diseases are becoming increasingly prominent. Therefore, developing a green, efficient, and sustainable meat production method is of great significance for alleviating the current inefficiency of meat production and addressing numerous social problems such as public health, environmental damage, and animal welfare.

[0003] Cultured meat is based on the proliferation and differentiation characteristics of animal stem cells. It involves in vitro culture using tissue engineering techniques and assembly into meat products with an appearance, texture, taste, and nutritional value similar to traditional meat using a scaffold system. Compared to traditional livestock production, it offers significant sustainability, environmental benefits, and animal welfare advantages, and is considered a promising emerging food production technology.

[0004] Traditional meat is mainly composed of skeletal muscle and intramuscular fat (IMF), but current research on cell-cultured meat, both domestically and internationally, primarily focuses on muscle stem cells and their cultured form. Research on IMF-related cultured meat is urgently needed. IMF is a key component of meat, directly determining its color, aroma, and flavor. It is rich in high-quality unsaturated fatty acids and has high nutritional value. When the IMF content is greater than 8, it forms "snowflake pork," resembling snowflakes, significantly improving its tenderness, flavor, and juiciness. Therefore, snowflake pork is synonymous with high-end pork. However, currently, over 90% of pig breeds raised in my country are imported lean-type breeds with high lean meat percentages and fast growth rates, but lower IMF content and generally lower meat quality. Therefore, combining advanced cell-cultured meat biosynthesis technology with large-scale IMF cell culture to achieve the in vitro production of high-quality snowflake pork is of great significance.

[0005] In the in vitro culture of meat, scaffold materials play a crucial role in mimicking the structure and environment of the extracellular matrix in natural tissues. On one hand, they provide cells with ample attachment surface, growth space, and stable mechanical support; on the other hand, they act as an artificial extracellular matrix, enabling cells to respond to endogenous and exogenous stimuli in a manner closer to the natural behavior of cells in vivo. Suitable bioscaffolds for cell-cultured meat production need to be continuous, porous structures to mimic the function of blood vessels in natural tissues, providing nutrient sources and metabolic pathways for cell growth and development. The porosity of the scaffold directly affects the cell culture effect; appropriate micropores facilitate nutrient exchange and can adsorb sufficient specific proteins to meet the cell growth requirements. However, excessive porosity is detrimental to maintaining mechanical properties. Furthermore, cell scaffolds should possess good cell compatibility, thermal stability, separability / degradability or edibility, low cost, and wide availability. However, most current scaffold materials require the addition of a certain amount of animal-derived extracellular matrix to ensure good cell adhesion, such as gelatin, collagen, and RGD peptides. These components typically suffer from complex extraction processes, high costs, and unsustainability, making them difficult to apply to the commercial production of cell-cultured meat. Furthermore, to maintain the mechanical strength and structural stability of the scaffold material, most scaffolds require the use of cross-linking agents with certain food toxicity, such as glutaraldehyde and EDC / NHS. Moreover, existing scaffold materials are primarily used for skeletal muscle cell culture. Therefore, there is an urgent need for an edible and safe scaffold that is simple to prepare, cost-effective, and free of animal-derived components, especially to provide a suitable in vitro growth environment for the proliferation and differentiation of adipocytes such as porcine fat grafts (FAPs), thereby producing adipose tissue and marbled pork with characteristics similar to traditional in vitro fat grafts (IMFs). Summary of the Invention

[0006] The purpose of this invention is to provide a method for preparing an edible plant hydrogel scaffold and its application. The prepared scaffold has high biosafety and good cell adhesion and compatibility.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0008] This invention provides a method for preparing an edible plant hydrogel scaffold, the method comprising the following steps:

[0009] 1) Dissolve κ-carrageenan and konjac gum in water to obtain a mixed solution, let stand overnight to form a gel;

[0010] 2) Add potassium chloride solution to the gel to perform ionic cross-linking and obtain an edible plant hydrogel scaffold.

[0011] Preferably, the mass ratio of κ-carrageenan to konjac gum is 2-5:5-8.

[0012] Preferably, the total mass ratio of κ-carrageenan and konjac gum to water in the mixed solution is 0.5–1 g: 100 mL.

[0013] Preferably, the dissolution temperature in step 1) is 90–98°C.

[0014] Preferably, the temperature for overnight standing in step 1) is 2-5°C.

[0015] Preferably, the concentration of the potassium chloride solution is 10-20 g / L.

[0016] Preferably, the duration of ionic crosslinking in step 2) is 5 to 15 minutes.

[0017] The present invention also provides an edible plant hydrogel scaffold obtained by the preparation method described above.

[0018] This invention also provides the application of edible plant hydrogel scaffolds in the cultivation of marbled pork.

[0019] Preferably, the snowflake pork culture involves co-culturing edible plant hydrogel scaffolds with pig FAPs.

[0020] This invention utilizes the intermolecular interactions and further ionic cross-linking between carrageenan and konjac gum to obtain a structurally stable 3D scaffold. The preparation method is simple and environmentally friendly, significantly reducing the production cost of cell-cultured meat. Furthermore, it contains no animal-derived components or toxic cross-linking agents, ensuring excellent food safety. The cross-linked scaffold exhibits high porosity and good cell compatibility, promoting the proliferation and differentiation of FAP (fermented protein polymerase) cells. It is suitable for the production of edible IMF-cultured meat, IMF-rich cell-cultured marbled pork, and other cell-cultured meats, as well as related research in the field of tissue engineering. Attached Figure Description

[0021] Figure 1 This is a macroscopic view of the edible plant hydrogel scaffold material obtained in Example 1;

[0022] Figure 2 This is a graph showing the degradation rate of the edible plant hydrogel scaffold as determined in Example 2.

[0023] Figure 3 The results of the toxicity test of the edible plant hydrogel scaffold in Example 3 are shown.

[0024] Figure 4 The results show the proliferation capacity of FAPs cells cultured in 3D in Example 3;

[0025] Figure 5 The images shown are of freeze-drying (top) and scanning electron microscope (bottom) of Example 3;

[0026] Figure 6 This is a photograph of Nile Red stained lipid droplets taken with a fluorescence inverted microscope in Comparative Example 1;

[0027] Figure 7 The results of Nile Red staining and lipid droplet area and coverage of FAPs in Comparative Example 1 are as follows;

[0028] Figure 8 The results show the triglyceride content determination in Comparative Example 1. Detailed Implementation

[0029] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0030] Example 1

[0031] Preparation of edible plant hydrogel scaffolds

[0032] Weigh out κ-carrageenan powder and konjac gum powder at mass ratios of 10:0, 9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, and 2:8 at room temperature, add deionized water and stir until homogeneous to prepare mixed solutions with total gel concentrations of 0.5% and 1% (w / v). Dissolve the mixed solutions in a 95℃ water bath, stirring thoroughly to obtain a well-mixed solution, and transfer it to a 24-well cell culture plate. Incubate overnight at 4℃ to initially form a gel. Slowly add 1% (w / v) potassium chloride solution and allow to stand for 10 minutes to allow the scaffold to completely gel and solidify, obtaining an edible plant hydrogel scaffold. Figure 1 As shown.

[0033] Example 2

[0034] The surface moisture of the edible plant hydrogel blocks obtained in Example 1 was absorbed using filter paper. The initial weight of each group of hydrogel blocks was measured using an electronic balance and recorded as W0. The hydrogel blocks were placed in 24-well plates and incubated for 15 days in DMEM medium (1.5 ml / well) at 37°C and 5% CO2. After incubation, the supports were dried using the same method, and their weight was measured and recorded as W. t Four replicates were set up for each group.

[0035] The degradation rate is calculated using the following formula: Degradation rate (%) = [(W0 - W] t ) / W t ]×100%

[0036] The results are as follows Figure 2 As shown, after 15 days of DMEM immersion, the total mass loss percentage of the 0.5% hydrogel scaffold was less than 40%, and the degradation rate of the 1% hydrogel scaffold was less than 30%. Moreover, the degradation rate of the scaffold decreased significantly with the increase of the total gel concentration.

[0037] Example 3

[0038] Biological evaluation of edible plant hydrogel scaffold materials

[0039] 3.1 Cytotoxicity test of edible plant hydrogel scaffolds

[0040] Following the preparation method of Example 1, the uncrosslinked gel solution was sterilized by high-temperature and high-pressure steam sterilization at 121°C for 15 min, and the potassium chloride solution was filtered through a 0.22 μm filter membrane to ensure sterility. Each crosslinked scaffold was prepared using the method described in Example 1. After rinsing with PBS buffer, the scaffolds were immersed in DMEM complete medium containing 15% fetal bovine serum and incubated at 37°C and 5% CO2 for 24 h to prepare the extract.

[0041] Take a density of 2×10 4 FAP cell suspension at a concentration of [cell / ml] was seeded into 96-well plates, with 100 μl of extraction medium added to each well. The medium was changed every 2 days. Cytotoxicity was assessed using a CCK-8 assay kit at 1, 2, 3, and 4 days of culture. 10 μl of CCK-8 solution was added to each well, and the plates were incubated at 37°C for 3 hours. The liquid was then aspirated and transferred to fresh 96-well plates, and the absorbance (OD) at 450 nm was measured using an automated microplate reader.

[0042] The results are as follows Figure 3 As shown, the proliferation curves of FAPs cells in different scaffold extracts over 4 days indicate that all groups of cells proliferated well, meaning that none of the materials affected normal cell growth and demonstrated good biocompatibility. The larger increases on days 1-2 and 3-4 are likely due to the replacement of the extract with fresh extract after day 2, which reduced certain growth factors secreted by the cells themselves in the original culture medium, thus slowing proliferation. Analysis of OD450 values ​​revealed that the 0.5% 2:8, 1% 2:8, and 1% 3:7 groups showed the best cell proliferation effects.

[0043] 3.2 FAPs cell 3D culture and proliferation assay

[0044] FAP cells were cultured at 37°C in a 5% CO2 incubator until the cell confluence reached approximately 70%. 1 ml of trypsin was added for digestion until the cells became elliptical and floated to the surface. An equal volume of growth medium was then added and mixed thoroughly to terminate the digestion. The cells were transferred to 5 ml centrifuge tubes and centrifuged at 800 rpm for 4 minutes to obtain the cell pellet. The supernatant was discarded, and the pellet was resuspended in growth medium for later use.

[0045] Add 1% (w / v) sterile potassium chloride solution to the cell culture plate, and add the cell suspension (2×10⁻⁶). 4The cell / ml sample was mixed with sterile gel solution at a volume ratio of 1:2 and transferred to potassium chloride solution using a 1ml pipette (with the pipette tip inserted below the liquid surface). Crosslinking was performed for 10 minutes to form a cell / scaffold complex. The remaining potassium chloride solution was discarded, and the sample was washed once with PBS. It was then immersed in DMEM growth medium containing 15% fetal bovine serum and cultured at 37°C and 5% CO2, changing the medium every 2 days.

[0046] CCK-8 assays were performed on days 2, 4, 6, and 8, following these steps: FAPs cells were cultured for 3D in 96-well cell culture plates. 100 μl of growth medium and 10 μl of CCK-8 solution were added to each well, and the plates were incubated at 37°C for 4 hours. The liquid was then aspirated and placed in a fresh 96-well plate, and the absorbance (OD) at 450 nm was measured using an automated microplate reader.

[0047] The results are as follows Figure 4 As shown, FAPs cells cultured in the gel under 3D conditions exhibited a continuous growth trend in number. The proliferation rate was relatively stable from 0 to 4 days, but increased significantly from 4 to 6 days. The cell number increase rate was significantly faster in the 1% 3:7 and 1% 2:8 groups than in other groups. The OD values ​​of all groups on day 8 after inoculation were significantly different from those on day 2. Analysis of degradation rate, pore structure, scaffold toxicity, and cell proliferation rate revealed that the scaffolds in the 1% 5:5, 1% 4:6, and 1% 3:7 groups had better physical properties and biological evaluation, providing an excellent environment for cell growth and proliferation.

[0048] 3.3 Observation of stent morphology

[0049] The gels selected in section 3.2 (1% 5:5, 1% 4:6, and 1% 3:7) were freeze-dried in a vacuum freeze dryer for 24 hours to form scaffold materials with oriented pores. The scaffolds were then subjected to liquid nitrogen brittle fracture, and the fracture surfaces were sputtered with gold. The pore structure was observed using a scanning electron microscope (SEM). Images were taken of the microscopic surface of the samples and data were collected under continuous scanning mode and a high voltage of 5 kV.

[0050] The results are as follows Figure 5 As shown, the scaffolds, after freeze-drying, exhibit a white, lightweight, and fluffy texture. Scanning electron microscopy (SEM) revealed the pore structure of the scaffold material, showing a loose, porous three-dimensional structure in both cross-sections. With increasing carrageenan content, the internal structure of the hydrogel became more compact, and the pore size decreased. The pore size was mostly distributed between 200-800 μm, with alternating large and small pores forming a double-crosslinked carrageenan-konjac gum interpenetrating polymer network.

[0051] Example 4

[0052] Differentiation of FAPs cells in carrageenan / konjac gum 3D culture system

[0053] FAPs cell suspension (1×10) 7 The cell / ml) and sterile gel solution were mixed at a volume ratio of 1:2 and transferred to sterile potassium chloride solution using a 1ml pipette (pipette tip submerged below the liquid surface). Crosslinking was carried out for 10 minutes until complete gelation and solidification. The remaining potassium chloride solution was discarded, and the cells were washed once with PBS. Then, the cells were immersed in DMEM complete medium containing 15% fetal bovine serum and cultured at 37°C and 5% CO2 for 24 hours. The growth medium was aspirated, and adipogenic differentiation medium was added. The composition of the adipogenic differentiation medium was: 10 μg / ml insulin, 500 μM 3-isobutyl-1-methylxanthine, 1 μM dexamethasone, and 10 μM rosiglitazone added to DMEM complete medium containing 10% FBS. The cells were cultured in a cell culture incubator at 37°C and 5% CO2 for 5 days, with the differentiation medium replaced every 48 hours. Five days after induction of differentiation, the differentiation medium was removed and replaced with maintenance medium, which consisted of 10 μg / mL insulin and 10 μM rosiglitazone added to DMEM complete medium containing 10% FBS. The cells were cultured in a cell culture incubator at 37°C and 5% CO2 for 9 days, with the maintenance medium being replaced every 48 hours.

[0054] To better illustrate the advancements of this technical solution, it was compared with existing sodium alginate gel scaffolds to analyze the performance differences between the two in promoting FAP cell differentiation and biocompatibility.

[0055] Comparative Example 1

[0056] Preparation and 3D culture of sodium alginate gel scaffold

[0057] A certain mass of sodium alginate powder was weighed using an electronic balance and dissolved in deionized water to prepare a 2% (w / v) sodium alginate solution. The sodium alginate solution was dissolved in a 95°C water bath and thoroughly stirred to obtain a well-mixed solution. The gel solution was sterilized by high-temperature autoclaving at 121°C for 15 min to achieve sterility. A certain mass of calcium chloride powder was weighed and dissolved in deionized water, filtered through a 0.22 μm filter membrane to ensure sterility, and transferred to a cell culture plate. The cell suspension and sterile gel solution were mixed at a volume ratio of 1:2 and transferred to the calcium chloride solution using a 1 ml pipette (pipette tip submerged below the liquid surface). Crosslinking was performed for 10 min to form a cell / scaffold complex. The remaining calcium chloride solution was discarded, and the cells were washed once with PBS, then immersed in DMEM growth medium containing 15% fetal bovine serum and cultured at 37°C and 5% CO2. The cell proliferation and differentiation protocols were consistent with those of the carrageenan / konjac gum group in Example 4.

[0058] Nile Red Staining Identification

[0059] After the FAPs cells in Example 4 and Comparative Example 1 underwent 3D culture and differentiation, the maintenance cell culture medium in each well was discarded, and the cells were fixed by immersion in 4% paraformaldehyde for 20 min. The fixative was then discarded, and the cells were washed once with PBS buffer. 1 mg / ml Nile Red stock solution (prepared with anhydrous ethanol) was diluted with PBS to prepare a working solution (1:200), and incubated with the cells / scaffold in the dark for 4 h to label lipid droplets. The Nile Red working solution was discarded, and 5 μg / ml DAPI staining solution was added and incubated at room temperature in the dark for 2 h to label cell nuclei. The fluorescent staining reagent was removed, and the cells were washed twice with PBS buffer. Using a red excitation wavelength (Ex / Em = 495 / 635 nm) and a DAPI laser / emission filter (Ex / Em = 364 / 454 nm), the size and distribution of lipid droplets were observed under a fluorescence microscope, and the field of view was adjusted to select appropriate areas for imaging. The lipid droplet coverage, total lipid droplet area, and average lipid droplet area were statistically analyzed using ImageJ software.

[0060] The results showed that most FAPs cells had clearly differentiated under a fluorescence microscope and exhibited a clustered cell spheroid morphology, indicating that three-dimensional culture and differentiation induction based on hydrogel media is feasible. Figure 6 Further quantitative analysis of the fluorescence image revealed that cells cultured and differentiated in the 1% 3:7 scaffold had significantly higher lipid droplet area and lipid droplet coverage than those cultured in the 2% sodium alginate scaffold. Figure 7 ).

[0061] Triglyceride content detection

[0062] After the FAPs cells in Example 4 and Comparative Example 1 were cultured and differentiated in 3D, the maintenance cell culture medium in each well was discarded, 200 μl of lysis buffer was added and homogenized thoroughly, centrifuged at 4°C for 10 min, and the supernatant was transferred to a new centrifuge tube for subsequent measurements.

[0063] According to the manufacturer's instructions, the triglyceride content in the cell / scaffold complex was determined using a triglyceride assay kit (Beijing Pulilai).

[0064] In addition, the protein concentration in the homogenate was detected using the BCA Protein Analyzer (Thermofisher) kit, and the triglyceride concentration was normalized to the total protein concentration.

[0065] The results showed that FAPs differentiated in 3D on carrageenan / konjac gum scaffolds had higher triglyceride content compared with 2% sodium alginate scaffolds. The 1% 3:7 group showed a significant difference from the sodium alginate group, further confirming that carrageenan / konjac gum-based scaffolds have good biocompatibility and can provide a favorable environment for the in vitro culture of IMFs. Figure 8 ).

[0066] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The application of an edible plant hydrogel scaffold in the cultivation of marbled pork, characterized in that, The preparation method of the edible plant hydrogel scaffold includes the following steps: 1) Dissolve κ-carrageenan and konjac gum in water to obtain a mixed solution, let stand overnight to form a gel; 2) Add potassium chloride solution to the gel to perform ionic cross-linking and obtain an edible plant hydrogel scaffold; The mass ratio of κ-carrageenan to konjac gum is 2~5:5~8; The total mass ratio of κ-carrageenan and konjac gum to water in the mixed solution is 0.5~1g:100mL.

2. The application according to claim 1, wherein the snowflake pork culture involves co-culturing edible plant hydrogel scaffolds with pig FAPs.

3. The application according to claim 1, characterized in that, The dissolution temperature described in step 1) is 90~98℃.

4. The application according to claim 1, characterized in that, The temperature for overnight standing as described in step 1) is 2~5℃.

5. The application according to claim 1, characterized in that, The concentration of the potassium chloride solution mentioned in step 2) is 10~20g / L.

6. The application according to claim 1, characterized in that, The duration of ionic crosslinking in step 2) is 5~15 min.

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