CN114438014A

CN114438014A - Edible chitosan glutenin bionic-oriented cell culture meat biological scaffold

Info

Publication number: CN114438014A
Application number: CN202210096358.3A
Authority: CN
Inventors: 陈双双; 向宁
Original assignee: Shanghai Shiwei Biotechnology Co ltd
Current assignee: Shanghai Shiwei Biotechnology Co ltd
Priority date: 2022-01-26
Filing date: 2022-01-26
Publication date: 2022-05-06

Abstract

The invention discloses an edible chitosan glutenin bionic-oriented cell culture meat biological scaffold which is obtained by mixing and dissolving glutenin and chitosan, forming in a directional mold, freeze-drying and then crosslinking. The edible chitosan vital gluten bionic oriented cell culture meat biological scaffold provided by the invention has the advantages that the related raw materials are non-animal protein, so that the edible chitosan vital gluten bionic oriented cell culture meat biological scaffold not only has good cell compatibility and biosafety, but also can promote cell adhesion growth. The prepared biological scaffold has a directional bionic orientation structure, can simulate the texture of natural muscle tissue, and brings good taste to cell culture meat. The edible chitosan vital gluten bionic oriented cell culture meat biological scaffold of the embodiments of the invention has rich porosity, which effectively improves the permeability of cells. Therefore, the invention provides a healthier and more effective biological scaffold for the field of cell culture meat and bioengineering.

Description

Edible chitosan glutenin bionic-oriented cell culture meat biological scaffold

Technical Field

The invention belongs to the field of food, and particularly relates to an edible chitosan glutenin bionic oriented cell culture meat biological scaffold.

Background

The cell culture meat forms a meat product with the flavor and taste of real muscle in vitro through cell culture and tissue engineering technology. The technology is based on sterile environment and quality control, and can avoid the food safety problem caused by antibiotics and hormones; the technology is released from the passive dependence on the growth cycle of animals, is expected to produce meat products more efficiently, and solves the problem of grain crisis. In addition, the technology avoids animal slaughtering and more compounds the health concept of animal protection. The cell culture meat mainly comprises cells and biological scaffolds. The biological scaffold needs to meet the performance requirements of edibility and promotion of cell adhesion growth. Furthermore, there is a significant directional texture due to the natural muscle tissue. In order to make the cell culture meat show natural muscle texture, the preparation of the microstructure with bionic orientation is of great significance.

At present, the preparation of the oriented microstructure can be realized by the techniques of oriented electrostatic spinning, wet extrusion spinning and the like. However, these biological scaffolds have the problems of insufficient cell permeability and the like, which leads to severe limitation on the increase of the thickness of the scaffold, and cannot realize the molding processing of a 3D three-dimensional structure; and because cells are difficult to permeate the inside of the bracket, the cell ratio in the prepared cell culture meat is low, and the cell ratio is difficult to break through 70%. Increasing the pore structure of biological scaffolds is an important way to increase cell permeability. Therefore, a new technique for preparing edible biological scaffolds is needed to solve the challenges of porosity and biocompatibility.

The directional freeze-drying is a method for directionally forming an ice crystal template by taking a temperature gradient as a drive, and a 3D integrated porous aerogel can be formed after freeze-drying. The prepared material has abundant micro-nano holes, and provides a required space for the osmotic growth of cells. In addition, due to the directional formation of ice crystals, the ordered arrangement of the microfibers of the scaffold material may be induced in the direction of ice crystal formation. Therefore, the technology is expected to directly prepare the three-dimensional cell culture meat with bionic muscle oriented textures in vitro. CN113208059A and CN113215090A utilize the technology to prepare gelatin-based and collagen derivative oriented biological scaffolds which can be used as biological scaffolds for cell culture meat. However, gelatin and collagen still belong to animal proteins and are limited in source, which is contrary to the vision of the cell culture meat industry.

Disclosure of Invention

The invention aims to overcome at least one defect of the prior art and provide an edible chitosan vital gluten bionic oriented cell culture meat biological scaffold.

The technical scheme adopted by the invention is as follows:

in a first aspect of the present invention, there is provided:

an edible chitosan glutenin bionic oriented cell culture meat biological scaffold, which comprises the following steps:

1) fully mixing glutenin, chitosan and other edible proteins with water, and adjusting the pH of the mixed solution to 9-10 to obtain mixed protein solution, wherein the addition amount of the other edible proteins is 0-500% of the sum of the glutenin and the chitosan by mass;

2) transferring the mixed protein liquid into a directional bracket die, freezing the mixed protein liquid within 10min, demolding, and freeze-drying to obtain an unfixed biological bracket;

3) and thermally crosslinking the unfixed biological scaffold to obtain the bionic oriented biological scaffold.

In some examples of the cell culture meat biological scaffold, the mass ratio of the glutenin to the chitosan is (3-8): (2-7).

In some examples of cell culture meat scaffolds, glutenin and chitosan are dissolved in an acetic acid solution and then mixed with other materials.

In some examples of cell culture meat bioscaffolds, the oriented scaffold molds are snap frozen in a mixture of ethanol and liquid nitrogen.

In some examples of the cell culture meat bioscaffold, the additional edible protein is selected from at least one of soy protein isolate, potato protein, zein.

In some examples of the cell culture meat bioscaffold, the temperature of the thermal crosslinking is 90-150 ℃.

In some examples of the cell culture meat bioscaffold, the chitosan is selected from shrimp shell-derived and/or mushroom-derived chitosan.

In some examples of the cell culture meat bioscaffold, the chitosan has a viscosity in the range of 20-800 cps.

In some examples of the cell culture meat bioscaffold, the bioscaffold has a tenacity of not less than 10000g per square centimeter of the blocky scaffold, as measured by a texture analyzer TPA test.

In a second aspect of the present invention, there is provided:

a cell culture meat obtained by culturing animal cells on a bioscaffold having a biomimetic color in the range of light yellow to brown as described in the first aspect of the present invention.

In some examples of the cell culture meat, the animal cell is selected from the group consisting of a myoblast, a muscle satellite cell, a fibroblast, an adipocyte precursor cell, an adult stem cell, and a pluripotent stem cell. Including but not limited to muscle cells, liver cells, osteoblasts, fibroblasts, adipoblasts, dentin cells, adult neuronal progenitor cells, neural stem cells, a plurality of multipotent stem cells from the lower anterior brain region of the ventricles, ependymal neural stem cells, hematopoietic stem cells, hepatic hematopoietic stem cells, bone marrow stem cells, adipose fibroblasts, adipose stem cells, stem cells that produce a plurality of islet cells, pancreatic-derived multipotent islet stem cells, mesenchymal stem cells, placental cells, bone marrow stromal cells, muscle side population cells, bone marrow-derived recovered cells, blood-derived mesenchymal precursor cells, bone marrow-derived side population cells, muscle precursor cells, circulating skeletal stem cells, neural progenitor cells, multipotent adult progenitor cells, mesodermal progenitor cells, spinal cord progenitor cells, and spore-like cells, and any combination thereof.

The invention has the beneficial effects that:

the edible chitosan vital gluten bionic oriented cell culture meat biological scaffold provided by the invention has the advantages that the related raw materials are non-animal protein, so that the edible chitosan vital gluten bionic oriented cell culture meat biological scaffold not only has good cell compatibility and biosafety, but also can promote cell adhesion growth. The prepared biological scaffold has a directional bionic orientation structure, can simulate the texture of natural muscle tissue, and brings good taste to cell culture meat.

The edible chitosan vital gluten bionic oriented cell culture meat biological scaffold of the embodiments of the invention has rich porosity, which effectively improves the permeability of cells. Therefore, the invention provides a healthier and more effective biological scaffold for the field of cell culture meat and bioengineering.

Drawings

FIG. 1 shows the results of mechanical property test of the stent in TPA mode;

FIG. 2 is the color change of the bioscaffold after lyophilization, thermal crosslinking fixation, and cell culture;

FIG. 3 is a graph of cell live-dead staining at 5d after inoculation of C2C12 cells on a cell culture meat bioscaffold;

FIG. 4 is a graph of viable cell death staining at 12d after cell culture meat scaffolds were seeded with C2C12 cells.

Detailed Description

In a first aspect of the present invention, there is provided:

an edible chitosan glutenin biomimetic oriented cell culture meat biological scaffold, a preparation method thereof comprises the following steps:

Can be prepared by adding edible alkali or acid such as NaOH and Na into protein solution₂CO₃And the like to adjust the pH of the mixed protein solution. Other edible proteins refer to edible proteins other than glutenins, preferably proteins of non-animal origin, more preferably vegetable proteins.

In some examples of the cell culture meat biological scaffold, the mass ratio of the glutenin to the chitosan is (3-8): (2-7). Thus, the composite material has good strength and can promote cell adhesion growth.

In some examples of cell culture meat scaffolds, glutenin and chitosan are dissolved in an acetic acid solution and then mixed with other materials. This allows for more uniform mixing.

In some examples of cell culture meat bioscaffolds, the oriented scaffold molds are snap frozen in a mixture of ethanol and liquid nitrogen. Thus, the process temperature of the freeze-drying process can be effectively regulated and controlled to realize the adjustment of the pore structure.

In some examples of the cell culture meat bioscaffold, the additional edible protein is selected from at least one of soy protein isolate, potato protein, corn protein to meet the customized design of mouthfeel and nutritional composition.

In some examples of the cell culture meat bioscaffold, the thermal crosslinking temperature is 90-150 ℃. By controlling the temperature and time of the thermal crosslinking reaction, the color of the bracket can be further adjusted to be closer to the natural meat.

In some examples of the cell culture meat bioscaffold, the chitosan is selected from shrimp shell-derived and/or mushroom-derived chitosan. The chitosan source of the sources is stable, and particularly the chitosan source of mushroom can completely get rid of the dependence of aquaculture.

In some examples of the cell culture meat bioscaffold, the bioscaffold has a tenacity of not less than 10000g per square centimeter of the blocky scaffold, as measured by a texture analyzer TPA test. This has a fibrous feel more similar to natural meat.

In a second aspect of the present invention, there is provided:

In some examples of cell culture meat, the animal cells are selected from myoblasts, muscle satellite cells, fibroblasts, adipocytes, adipogenic precursor cells, adult stem cells, and pluripotent stem cells. Including but not limited to muscle cells, liver cells, osteoblasts, fibroblasts, adipoblasts, dentin cells, adult neuronal progenitor cells, neural stem cells, a plurality of multipotent stem cells from the lower anterior brain region of the ventricles, ependymal neural stem cells, hematopoietic stem cells, hepatic hematopoietic stem cells, bone marrow stem cells, adipose fibroblasts, adipose stem cells, stem cells that produce a plurality of islet cells, pancreatic-derived multipotent islet stem cells, mesenchymal stem cells, placental cells, bone marrow stromal cells, muscle side population cells, bone marrow-derived recovered cells, blood-derived mesenchymal precursor cells, bone marrow-derived side population cells, muscle precursor cells, circulating skeletal stem cells, neural progenitor cells, multipotent adult progenitor cells, mesodermal progenitor cells, spinal cord progenitor cells, and spore-like cells, and any combination thereof.

The technical scheme of the invention is further explained by combining the embodiment.

In the following examples, unless otherwise specified, the bottom of the freeze-drying mold used was a metal copper plate and the upper layer was a fluorine-containing material PTFE.

The glutenin solution is obtained by dissolving glutenin in acid, and removing insoluble substances.

The percentages are by mass unless otherwise specified.

Example 1

Preparing a biological scaffold:

solution preparation:

1) preparing 5% wheat gluten with 1% food grade glacial acetic acid, and stirring at 1000rpm overnight; after stirring, the mixture was centrifuged at 5000rpm for 5 minutes. Taking the supernatant fluid, namely the glutenin solution;

2) 2.5% shrimp chitosan (chitosan source is medium molecular weight provided by Sigma-Aldrich, its degree of deacetylation > 95%) was formulated with 1% food grade glacial acetic acid;

3) mixing the glutenin solution and the chitosan solution according to the mass ratio of 1:1 to form a uniform scaffold solution; the soybean dietary fiber is added into the solution, and the concentration is 0.1 mg/mL.

The bionic biological scaffold is prepared by directional freeze-drying through an ice crystal template method, and the preparation method comprises the following steps:

1) pouring the scaffold solution into a self-made mold, placing the mold into an ethanol liquid nitrogen mixed solution at the temperature of-80 ℃, standing for 20min to completely freeze the solution, and freeze-drying water by a freeze dryer to obtain an unfixed biological scaffold;

2) and (5) fixing after the sample is freeze-dried. The specific method comprises thermal crosslinking, and steaming lyophilized scaffold in 100 deg.C water for 15 min; or steaming and baking at 140 deg.C for 10min in a steaming and baking integrated machine;

3) after fixation was completed, the acid and base were removed with PBS and adjusted to neutral.

Mechanical properties of the biological scaffold:

texture measuring instruments are commonly used in food industry and scientific research. Scaffold mechanical property testing was performed in full Texture analysis (TPA, also secondary mastication experiment) mode using a ta.xt Plus Texture Analyser Texture analyzer.

When TPA test is carried out, a disc-shaped probe is used, the sectional area of the probe is larger than the area of a sample, and the running track of the probe is as follows:

1) after the initial position of the probe returns to zero, firstly pressing the probe to a test sample at the speed of 1 mm/s;

2) compressing the sample at the test rate for a certain distance (5mm) after contacting the sample surface; and then back to the trigger point for compression;

3) after a certain time, continuously compressing downwards for the same distance; and then returns to the probe pre-test position at a post-test rate. The mechanical property of the biological scaffold can be obtained.

TPA test results are shown in fig. 1, and it can be found that the oriented freeze-dried scaffolds have anisotropy. For example, the chewiness of the scaffold, i.e. the energy required to chew the solid food product into a swallowable state, is only 20% of the vertical direction along the freeze drying direction. This anisotropic and biomimetic behavior can provide a mouthfeel very close to that of natural muscle tissue.

Example 2

Preparing a biological scaffold:

2) preparing a 5% peanut protein solution by using 0.5% food-grade hydrochloric acid, and stirring at 1000rpm overnight to completely dissolve peanut protein to obtain a peanut protein solution;

3) preparing 5% mushroom source chitosan (chitosan source is food grade mushroom chitosan, viscosity is 100 cps) with 1% food grade glacial acetic acid to obtain chitosan solution;

4) mixing the glutenin solution, the peanut protein and the chitosan solution according to the mass ratio of 1:1:1 to form a uniform scaffold solution;

5) the bionic biological scaffold is prepared by directional freeze-drying through an ice crystal template method, and the preparation method comprises the following steps: the scaffold solution was poured into the home-made mold. Placing in the mixture of ethanol and liquid nitrogen at the temperature of minus 80 ℃. Standing for 20min to completely freeze the solution. Freeze-drying the water content by a freeze dryer to obtain an unfixed biological scaffold;

6) and (5) fixing after the sample is freeze-dried. The specific method comprises thermal crosslinking, and steaming lyophilized scaffold in 100 deg.C water for 15 min; or steaming and baking at 140 deg.C for 10min in a steaming and baking integrated machine;

7) after fixation was completed, the acid and base were removed with PBS and adjusted to neutral.

Color change of biological scaffold:

natural meat products have typical haematochrome. Therefore, there is also a need for cell culture meat industries that provide more simulated colors. For this purpose, the present example uses a common optical camera to record the color change of the bioscaffold after lyophilization, thermal crosslinking fixation, and cell culture.

As a result, as shown in FIG. 2, a typical Maillard reaction occurred between glutenin and chitosan during thermal crosslinking. Thus, the color formed brown. After cell attachment, the prepared cell culture meat shows a structure and color very close to those of natural meat.

Example 3

Preparing a biological scaffold:

2) preparing 5% mushroom source chitosan (chitosan source is food grade mushroom chitosan, viscosity is 300 cps) with 1% food grade glacial acetic acid to obtain chitosan solution;

3) mixing the glutenin solution and the chitosan solution according to the mass ratio of 1:1 to form a uniform scaffold solution;

4) the bionic biological scaffold is prepared by directional freeze-drying through an ice crystal template method, and the preparation method comprises the following steps: the scaffold solution was poured into the home-made mold. Placing in the mixture of ethanol and liquid nitrogen at the temperature of minus 80 ℃. Standing for 20min to completely freeze the solution. Freeze-drying the water content by a freeze dryer to obtain an unfixed biological scaffold;

5) and (5) fixing after the sample is freeze-dried. The specific method comprises thermal crosslinking, and steaming lyophilized scaffold in 100 deg.C water for 15 min; or steaming and baking at 140 deg.C for 10min in a steaming and baking integrated machine;

6) after fixation was completed, the acid and base were removed with PBS and adjusted to neutral.

Cell culture:

1) mouse myoblast C2C12 with a cell passage no greater than 6 was cultured in high-glucose medium/fetal bovine serum (DMEM/FBS) medium containing 10% FBS and 1% triantibody. The cells were cultured at 37 ℃ in 5% CO₂An incubator. After the cells reached 80% coverage in the culture dish, the cell dispersion was obtained by trypsinization and centrifugation and resuspended. The number of scattered cells was 10 by counting⁵cells/mL solution is ready for use.

2) The biomimetic scaffold prepared in this example was sterilized by soaking in 75% medical alcohol. Meanwhile, the system is placed in an ultraviolet sterilizing lamp for sterilization treatment. The cells were then washed 5 times with PBS in a biosafety cabinet for 5min each time. After removal of PBS, the cell suspension was seeded onto the bioscaffold. Cells were allowed to grow adherently. The cells were cultured at 37 ℃ in 5% CO₂An incubator.

3) Cell characterization: after the cells are cultured for 5 days, adding Calcein AM/PI detection working solution with proper volume. Typically, 100. mu.l are added to 96-well plates, 250. mu.l to 24-well plates, 500. mu.l to 12-well plates, and 1ml to 6-well plates. Incubate at 37 ℃ for 30min in the dark. After the incubation, the staining effect was observed under a fluorescence microscope (Calcein AM is green fluorescence, Ex/Em =494/517 nm; PI is red fluorescence, Ex/Em =535/617 nm).

4) To follow the adherent growth of the cells over a longer time frame, the incubation time was extended to 12 days and the staining procedure was repeated.

The experimental results (fig. 3 and fig. 4) show that all cells exhibit green fluorescence, which indicates that the survival rate of the cells exceeds 95%, and the adhesion density of the cells on the scaffold is high, and the experimental results show that the directional freeze-dried scaffold has good cell compatibility and can promote the adhesion growth of the cells. Compared with the morphology of the cells on day 12, the bionic scaffold has obvious oriented growth, which shows that the bionic scaffold prepared by the invention has good promotion effect on the formation of muscle tissues.

The foregoing is a more detailed description of the invention and is not to be taken in a limiting sense. It will be apparent to those skilled in the art that simple deductions or substitutions without departing from the spirit of the invention are within the scope of the invention.

Claims

1. An edible chitosan glutenin bionic oriented cell culture meat biological scaffold, which comprises the following steps:

2. The cell culture meat bioscaffold of claim 1, wherein: the weight and dosage ratio of the glutenin to the chitosan is (3-8): (2-7).

3. The cell culture meat bioscaffold of claim 1, wherein: dissolving glutenin and chitosan with acetic acid solution, and mixing with other materials; and/or

The chitosan is selected from chitosan derived from shrimp shell and/or mushroom.

4. A cell culture meat bioscaffold according to any one of claims 1-3 wherein: and (3) placing the directional bracket mould in a mixed solution of ethanol and liquid nitrogen for quick freezing.

5. A cell culture meat bioscaffold according to any one of claims 1-3 wherein: the other edible protein is at least one selected from soy protein isolate, potato protein, and corn protein.

6. A cell culture meat bioscaffold according to any one of claims 1-3 wherein: the temperature of the thermal crosslinking is 90-150 ℃.

7. A cell culture meat bioscaffold according to any one of claims 1-3 wherein: the viscosity range of the chitosan is 20-800 cps.

8. A cell culture meat bioscaffold according to any one of claims 1-3 wherein: the toughness of the block-shaped scaffold of each square centimeter of the biological scaffold tested by a texture analyzer TPA is not less than 10000 g.

9. A cell culture meat obtained by culturing animal cells on a bioscaffold, characterized in that: a bioscaffold for use in cell culture according to any one of claims 1 to 8 having a biomimetic colour in the range of light yellow to brown.

10. The cell culture meat of claim 9, wherein: the animal cells are selected from myoblasts, muscle satellite cells, fibroblasts, adipocytes, adipocyte precursor cells, adult stem cells and pluripotent stem cells. Including but not limited to muscle cells, liver cells, osteoblasts, fibroblasts, adipoblasts, dentin cells, adult neuronal progenitor cells, neural stem cells, a plurality of multipotent stem cells from the lower anterior brain region of the ventricles, ependymal neural stem cells, hematopoietic stem cells, hepatic hematopoietic stem cells, bone marrow stem cells, adipose fibroblasts, adipose stem cells, stem cells that produce a plurality of islet cells, pancreatic-derived multipotent islet stem cells, mesenchymal stem cells, placental cells, bone marrow stromal cells, muscle side population cells, bone marrow-derived recovered cells, blood-derived mesenchymal precursor cells, bone marrow-derived side population cells, muscle precursor cells, circulating skeletal stem cells, neural progenitor cells, multipotent adult progenitor cells, mesodermal progenitor cells, spinal cord progenitor cells, and spore-like cells, and any combination thereof.