CN117179282A - Preparation method and application of edible plant hydrogel bracket - Google Patents

Abstract

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

Description

Preparation method and application of edible plant hydrogel bracket

Technical Field

The invention relates to the technical field of biological materials and food science, in particular to a preparation method and application of an edible plant hydrogel bracket.

Background

Meat is the most predominant source of animal proteins in the human diet. As the global population continues to grow, the contradiction between the growing demand for meat consumption and the limited productivity of meat products is increasingly prominent. According to the federal grain and farming organization (FAO) forecast, the global meat product demand would be expected to increase by 73% by 2050. However, the existing meat production and supply chain has a number of hidden problems. In addition, as the scale of livestock production continues to expand, many problems such as environmental pollution, water resource shortage, increased greenhouse gas emission, land ecological damage, and increased animal epidemic disease are increasingly prominent. Therefore, the development of a green and efficient sustainable meat production mode has important significance for relieving the current insufficient production efficiency of the meat industry and various social problems such as public health, environmental damage, animal welfare and the like.

The cell culture meat is based on the proliferation and differentiation characteristics of animal stem cells, is cultured in vitro by utilizing a tissue engineering technology, and is assembled into a meat product with similar appearance, texture, taste and nutrition to the traditional meat by means of a bracket system. Compared with the traditional livestock production, the method has remarkable sustainability, environmental protection and animal welfare benefits, and is considered as an emerging food production technology with great prospect.

The traditional meat mainly consists of skeletal muscle, intramuscular fat (IMF) and the like, but the related research of cell culture meat at home and abroad is mainly focused on muscle stem cells and cell culture meat thereof, and the research of the related culture meat of IMF cells needs to be carried out urgently. IMF is also a key component of meat, directly determines the "color, flavor" of meat, is rich in high-quality unsaturated fatty acid, and has high nutritive value. When the IMF content is more than 8, snowflake-like 'snowflake pork' is formed, and the tenderness, flavor and juiciness of the snowflake-like 'snowflake pork' are improved remarkably. Thus, snowflake pork is a terminology for high-end pork. However, at present, more than 90% of the domestic pig breeds belong to external lean type pig breeds, the lean meat percentage is high, the growth speed is high, the IMF content is low, and the meat quality is general. Therefore, the in-vitro production of high-quality snowflake pork through the large-scale culture of IMF cells has important significance by combining with the advanced cell culture meat biosynthesis technology.

In the in vitro culture process of meat, the scaffold material plays an important role in simulating the structure and environment of extracellular matrix in natural tissues, on one hand, provides sufficient attachment surface, growth space and stable mechanical support for cells, and on the other hand, serves as artificial extracellular matrix, so that the response of cells to endogenous and exogenous stimuli is closer to the natural behavior of cells in vivo. The biological scaffold suitable for producing the cell culture meat needs to be a continuous through porous structure to simulate the functions of blood vessels in natural tissues, and provide nutrition sources and substance metabolism channels for the growth and development of cells. The porosity of the scaffold can directly influence the cell culture effect, and proper micropores are beneficial to nutrient exchange and can adsorb enough specific proteins to meet the growth requirement of cells. However, too much porosity is detrimental to maintaining mechanical properties. In addition, the cell scaffold should have good cell compatibility, thermal stability, separability/degradability or compatibility, low cost, wide sources, etc. However, at present, a certain amount of animal-derived extracellular matrix needs to be added into most scaffold materials to ensure good cell adhesion, such as gelatin, collagen, RGD peptide and the like, and the components generally have the defects of complex extraction process, high cost, unsustainability and the like, so that the scaffold materials are difficult to apply to commercial production of cell culture meat. In addition, in order to maintain the mechanical strength and structural stability of the scaffold material, crosslinking agents having a certain edible toxicity such as glutaraldehyde, EDC/NHS and the like are required to be used in most scaffolds. Moreover, the related scaffold materials currently existing are mainly used for skeletal muscle cell culture and the like. Therefore, there is an urgent need for an edible safe scaffold which has simple preparation method, controllable cost and no animal-derived components, and particularly provides a proper in vitro growth environment for proliferation and differentiation of fat cells such as pig FAPs, so as to produce fat tissues and snowflake pork with similar characteristics to the traditional IMF.

Disclosure of Invention

The invention aims to provide a preparation method and application of an edible plant hydrogel stent, and the prepared stent has high biosafety and good cell adhesion and compatibility.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides a preparation method of an edible plant hydrogel bracket, which comprises the following steps:

1) Dissolving kappa-carrageenan and konjac gum in water to obtain a mixed solution, and standing overnight to form gel;

2) And adding a potassium chloride solution into the gel, and performing ionic crosslinking to obtain the edible plant hydrogel scaffold.

Preferably, the mass ratio of the kappa-carrageenan to the konjac gum is 2-5:5-8.

Preferably, the mass-volume ratio of the total mass of the kappa-carrageenan and the konjak gum in the mixed solution to water is 0.5-1 g/100 mL.

Preferably, the temperature of the dissolution in step 1) is 90 to 98 ℃.

Preferably, the temperature of the standing overnight in the step 1) is 2-5 ℃.

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

Preferably, the duration of the ionic crosslinking in the step 2) is 5-15 min.

The invention also provides the edible plant hydrogel bracket obtained by the preparation method.

The invention also provides application of the edible plant hydrogel bracket in snowflake pork culture.

Preferably, the snowflake pork culture is to co-culture the edible plant hydrogel scaffold with pig FAPs.

According to the invention, the 3D bracket with a stable structure is obtained through intermolecular interaction and further ionic crosslinking between carrageenan and konjac gum, the preparation method is simple and environment-friendly, the production cost of cell culture meat is greatly reduced, and the 3D bracket does not contain any animal-derived component and toxic crosslinking agent and has good food safety. The crosslinked bracket has higher porosity and good cell compatibility, can promote proliferation and differentiation of FAPs cells, and is suitable for production of edible IMF cultured meat, IMF-enriched cell cultured snowflake pork, other cell cultured meat and other related researches in the tissue engineering field.

Drawings

FIG. 1 is a macroscopic view of the whole edible plant hydrogel scaffold material obtained in example 1;

FIG. 2 is a graph showing the results of measuring the degradation rate of the edible plant hydrogel scaffold in example 2;

FIG. 3 is a graph showing the toxicity measurement results of the edible plant hydrogel scaffolds of example 3;

FIG. 4 shows the proliferation potency assay results of 3D culture of FAPs cells in example 3;

FIG. 5 is a freeze-dried (top) and scanning electron microscope image (bottom) of example 3;

FIG. 6 is a photograph of a red dye of a lipid drop Nile taken by a fluorescent inverted microscope of comparative example 1;

FIG. 7 is a chart showing FAPs nile red staining and lipid drop area and coverage statistics for comparative example 1;

FIG. 8 shows the results of the measurement of the triglyceride content in comparative example 1.

Detailed Description

The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.

Example 1

Preparation of edible plant hydrogel scaffold

Weighing the following components in percentage by mass at normal temperature: 0. 9: 1. 8: 2. 7: 3. 6: 4. 5: 5. 4: 6. 3: 7. 2:8, adding deionized water into the kappa-carrageenan powder and the konjak gum powder, and stirring uniformly to prepare a mixed solution with the total gum concentration of 0.5% and 1% (w/v). And (3) placing the mixed solution into a water bath kettle at 95 ℃ for water bath dissolution, fully stirring to obtain a well mixed solution, transferring the well mixed solution into a 24-hole cell culture plate, and standing overnight at 4 ℃ for preliminary gel formation. Slowly adding 1% (w/v) potassium chloride solution, standing for 10min to gel the scaffold completely, and obtaining edible plant hydrogel scaffold as shown in figure 1.

Example 2

The surface of the edible plant hydrogel blocks obtained in example 1 was blotted with filter paper, and the initial weight of each set of hydrogel blocks was weighed with an electronic balance and recorded as W ₀ . Will bePlacing the hydrogel block in 24-well plate at 37deg.C and 5% CO ₂ Is immersed in DMEM medium (1.5 ml/well) and incubated for 15 days. After the incubation, the scaffolds were dried in the same way, and their weights were measured and recorded as W _t Each group was provided with 4 replicates.

The degradation rate is calculated by the following formula: degradation rate (%) = [ (W) ₀ -W _t )/W _t ]×100％

As shown in FIG. 2, after the DMEM is soaked for 15 days, the total mass loss percentage of the 0.5% hydrogel scaffold is less than 40%, and the degradation rate of the 1% hydrogel scaffold is less than 30%. And the degradation rate of the bracket is obviously reduced along with the increase of the total gum concentration.

Example 3

Biological evaluation of edible plant hydrogel scaffold materials

3.1 cytotoxicity test of edible plant hydrogel scaffolds

Referring to the preparation method of example 1, the gel solution which was not subjected to the ionic crosslinking was steam sterilized at 121℃under high temperature and high pressure for 15min, and the potassium chloride solution was filtered through a 0.22 μm filter membrane to ensure sterility. The ionomer scaffolds were prepared as in example 1, washed with PBS buffer and then immersed in DMEM complete medium containing 15% fetal calf serum at 37deg.C with 5% CO ₂ The extracts were prepared by co-incubation for 24h under conditions.

The density is taken to be 2 multiplied by 10 ⁴ cell/ml FAPs cell suspension was seeded in 96-well plates, 100. Mu.l of the extract was added to each well, and the solution was changed every 2 days. Cytotoxicity was determined using CCK-8 kit for 1 day, 2 days, 3 days, 4 days of culture. Mu.l of CCK-8 solution was added to each well and incubated for 3h in a constant temperature incubator at 37 ℃. The aspirated liquid was placed in a completely new 96 well plate and absorbance (OD) at 450nm was measured using a fully automated microplate reader.

The results are shown in FIG. 3: the proliferation curve of FAPs cells in different stent leaching solutions within 4 days can be seen that each group of cells proliferate well, namely, all materials do not influence the normal growth of the cells, and the FAPs have good biocompatibility. The larger increases between days 1-2 and 3-4 may be due to the decreased secretion of certain growth factors by the cells themselves in the original medium by replacement of fresh extract after day 2, resulting in a slow proliferation. Analysis of the OD450 values found 0.5%2: 8. 1%2:8 and 1%3: the proliferation effect of 7 groups of cells is optimal.

3.2FAPs cell 3D culture and proliferation Capacity determination

FAPs cells were incubated at 37℃with 5% CO ₂ When the cell confluence rate reaches about 70%, 1ml trypsin is added to digest until the cell morphology tends to ellipse and floats from the bottom of the culture dish, and an equal volume of growth medium is added to mix uniformly to stop digestion. Transferring the cells into a 5ml centrifuge tube, centrifuging at 800rpm/min for 4min to obtain cell precipitate, discarding the supernatant, and adding a growth medium for resuspension.

1% (w/v) sterile potassium chloride solution was added to the cell culture plate to prepare a cell suspension (2X 10) ⁴ cell/ml) to sterile gel solution in a volume ratio of 1:2, uniformly mixing, transferring into a potassium chloride solution by using a 1ml pipette (the gun head is inserted below the liquid level), and crosslinking for 10min to form a cell/scaffold complex. Discarding the remaining potassium chloride solution, washing with PBS once, and immersing in DMEM growth medium containing 15% fetal calf serum at 37deg.C and 5% CO ₂ Culturing under the condition, and changing liquid every 2 days.

CCK-8 detection was performed on days 2, 4, 6, 8, respectively, as follows: 3D culture of FAPs cells was performed in 96-well cell culture plates, 100. Mu.l of growth medium and 10. Mu.l of CCK-8 solution were added to each well, and incubated in a constant temperature incubator at 37℃for 4 hours. The aspirated liquid was placed in a completely new 96 well plate and absorbance (OD) at 450nm was measured using a fully automated microplate reader.

The results are shown in FIG. 4: FAPs cells subjected to 3D culture in the gel always have a continuous increasing trend in quantity, the proliferation rate is stable in 0-4 days, and the proliferation rate is remarkably increased in 4-6 days, wherein 1% and 3:7 and 1%2: the cell number of group 8 increased significantly faster than the other groups, and the OD values measured on day 8 of each group were significantly different from that measured on day 2 of each group. Comprehensive degradation rate, pore structure, scaffold toxicity and cell proliferation rate analysis found 1%5: 5. 1%4:6 and 1%3: the physical property and biological evaluation of the group 7 scaffolds are better, and excellent environments can be provided for the growth and proliferation of cells.

3.3 morphology observations of stents

1%5 screened in 3.2: 5. 1%4:6 and 1%3: and (5) putting the 7 groups of gels into a vacuum freeze dryer for freeze drying for 24 hours to form the bracket material with the directionally arranged pore canals. And (3) brittle fracture of the bracket by liquid nitrogen, metal spraying treatment on the section, and observing the pore structure by a scanning electron microscope. And (3) scanning images under the conditions of continuous scanning mode and high voltage of 5kV, photographing the microscopic surface of the sample and collecting data.

The results are shown in FIG. 5: the bracket is white, light and fluffy after freeze drying; and microscopic observation of the pore structure of the support material by a scanning electron microscope shows that the support material has a loose and porous three-dimensional structure in the transverse and longitudinal sections. As the proportion of carrageenan increases, the internal structure of the hydrogel becomes tighter and the pore structure becomes smaller. The pore diameters are distributed between 200 and 800 mu m, and the large pores and the small pores are alternately distributed, so that the double-crosslinked carrageenan-konjac gum interpenetrating polymer network is formed.

Example 4

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

FAPs cell suspension (1×10) ⁷ cell/ml) to sterile gel solution in a volume ratio of 1:2, uniformly mixing, transferring into a sterile potassium chloride solution by using a 1ml pipette (the tip of the pipette is inserted below the liquid level), and crosslinking for 10min until the gel is completely solidified. Discarding the remaining potassium chloride solution, washing with PBS once, and immersing in DMEM complete medium containing 15% fetal calf serum at 37deg.C and 5% CO ₂ Culturing for 24 hours under the condition. Sucking the growth medium, and adding into lipid-forming differentiation medium, wherein the components are as follows: 10. Mu.g/ml insulin, 500. Mu.M 3-isobutyl-1-methylxanthine, 1. Mu.M dexamethasone and 10. Mu.M rosiglitazone were added to DMEM complete medium containing 10% FBS at 37℃in 5% CO ₂ Is cultured in a cell culture box for 5 days, and the differentiation medium is replaced every 48 hours. After 5 days of induced differentiation, the differentiation medium was aspirated and replaced with maintenance medium, which consisted of: adding 10 μg/mL insulin and 10 μM rosiglitazone to DMEM complete medium containing 10% FBS at 37deg.C, 5% CO ₂ Cell incubator of (a)The maintenance medium was changed every 48h for 9 days.

In order to better illustrate the advancement of the technical scheme, compared with the sodium alginate gel scaffold in the prior art, the performance difference of the sodium alginate gel scaffold and the sodium alginate gel scaffold in the aspect of promoting FAPs cell differentiation and biocompatibility is analyzed.

Comparative example 1

Preparation and 3D culture of sodium alginate gel scaffold

And weighing sodium alginate powder with a certain mass by using an electronic balance, and dissolving the sodium alginate powder in deionized water to prepare sodium alginate solution with the concentration of 2% (w/v). And (3) placing the sodium alginate solution into a water bath kettle at 95 ℃ for water bath dissolution, and fully stirring to obtain a well-mixed solution. Sterilizing the gel solution with high temperature and high pressure steam at 121deg.C for 15min to reach the sterility standard. A mass of calcium chloride powder was weighed and dissolved in deionized water, filtered through a 0.22 μm filter to ensure sterility, and transferred to a cell culture plate. The cell suspension and the sterile gel solution are mixed according to the volume ratio of 1:2, uniformly mixing, transferring into a calcium chloride solution by using a 1ml pipette (the gun head is inserted below the liquid level), and crosslinking for 10min to form a cell/scaffold complex. Discarding the remaining calcium chloride solution, washing with PBS once, and immersing in DMEM growth medium containing 15% fetal calf serum at 37deg.C and 5% CO ₂ Culturing under the condition. The cell proliferation and differentiation protocols were the same as those of the carrageenan/konjac gum group of example 4.

Nile red staining identification

After 3D culture differentiation of FAPs cells in example 4 and comparative example 1 was completed, the maintenance cell culture solution in each well was discarded, the cells were fixed in 4% paraformaldehyde for 20min, the fixed solution was discarded, and the cells were washed 1 time with PBS buffer. 1mg/ml nile red stock solution (prepared by absolute ethanol) was diluted with PBS to working solution (1:200), incubated with cells/scaffolds for 4h in the dark for labeling lipid droplets; the nile red working solution was discarded, and the nuclei were labeled by incubation with 5. Mu.g/ml DAPI dye at room temperature for 2h in the dark. The fluorescent staining reagent was blotted and washed 2 times with PBS buffer, red excitation light waves (Ex/em=495/635 nm) and DAPI laser/emission filters (Ex/em=364/454 nm) were selected, lipid droplet size and distribution were observed under a fluorescent microscope, and the appropriate area was selected for imaging by adjusting the field of view. Lipid droplet coverage, total lipid droplet area, and average lipid droplet area were counted using ImageJ software.

The results show that: most FAPs cells were clearly observed to have differentiated under fluorescence microscopy and exhibited a pellet morphology, demonstrating the feasibility of three-dimensional culture and induced differentiation based on hydrogel media (FIG. 6). The fluorescence photograph was further quantitatively analyzed at 1%3: the lipid drop area and lipid drop coverage of the differentiated cells cultured in the 7-group scaffolds were significantly higher than those of the 2% sodium alginate scaffolds (fig. 7).

Triglyceride content detection

After 3D culturing and differentiation of FAPs cells in example 4 and comparative example 1 was completed, the maintenance cell culture solution in each well was discarded, 200. Mu.l of the lysate was added for sufficient homogenization, and the mixture was centrifuged at 4℃for 10 minutes, and the supernatant was transferred to a new centrifuge tube for subsequent measurement.

The content of triglycerides in the cell/scaffold complex was determined using a triglyceride detection kit (beijing plaril) according to the manufacturer's instructions.

In addition, protein concentration in the homogenate was detected using BCA protein assay kit (thermosusher), and triglyceride concentration was normalized to total protein concentration.

The results show that: compared with 2% sodium alginate scaffolds, FAPs subjected to 3D differentiation in carrageenan/konjac gum scaffolds had higher triglyceride content, with 1%3:7 groups significantly different from sodium alginate groups, further demonstrating that carrageenan/konjac gum-based scaffolds have good biocompatibility, capable of providing a good environment for in vitro culture of IMF (fig. 8).

The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.

Claims (10)

1. A method for preparing an edible plant hydrogel scaffold, which is characterized by comprising the following steps:

1) Dissolving kappa-carrageenan and konjac gum in water to obtain a mixed solution, and standing overnight to form gel;

2) And adding a potassium chloride solution into the gel, and performing ionic crosslinking to obtain the edible plant hydrogel scaffold.

2. The preparation method according to claim 1, wherein the mass ratio of the kappa-carrageenan to the konjac gum is 2-5:5-8.

3. The preparation method according to claim 2, wherein the mass-volume ratio of the total mass of kappa-carrageenan and konjac gum to water in the mixed solution is 0.5-1 g/100 ml.

4. A process according to claim 3, wherein the temperature of dissolution in step 1) is 90-98 ℃.

5. The method according to claim 4, wherein the temperature of the standing overnight in step 1) is 2 to 5 ℃.

6. The method according to claim 1, wherein the concentration of the potassium chloride solution is 10 to 20g/L.

7. The method according to claim 1, wherein the ionic crosslinking in step 2) is carried out for a period of 5 to 15 minutes.

8. An edible plant hydrogel scaffold obtainable by the method of any one of claims 1 to 7.

9. Use of the edible plant hydrogel scaffold of claim 8 in snowflake pork culture.

10. The use according to claim 9, wherein the snowflake pork culture is a co-culture of edible plant hydrogel scaffolds with porcine FAPs.