Edible chitosan glutenin biomimetic oriented cell culture meat biological scaffold

Abstract

The present invention discloses an edible chitosan gluten biomimetic oriented cell culture meat bioscaffold, which is obtained by mixing and dissolving glutenin and chitosan, forming in a directional mold, freeze-drying, and then cross-linking. The raw materials involved in the edible chitosan gluten biomimetic oriented cell culture meat bioscaffolds of some examples of the present invention are all non-animal protein, which not only has good cell compatibility and biosafety, but also promotes cell adhesion and growth. The prepared bioscaffold has a directional biomimetic oriented structure, which can simulate the texture of natural muscle tissue and bring a good taste to cell cultured meat. The edible chitosan gluten biomimetic oriented cell culture meat bioscaffolds of some examples of the present invention contain rich porosity, which effectively improves the permeability of cells. Therefore, the present invention will provide a healthier and more effective bioscaffold for the field of cell cultured meat and bioengineering.

Description

Technical Field

[0001] The invention belongs to the field of food, and in particular relates to an edible chitosan glutenin bionic-oriented cell culture meat biological scaffold. Background Art

[0002] Cell cultured meat uses cell culture and tissue engineering technology to form meat products with the flavor and taste of real muscles in vitro. This technology is based on a sterile environment and quality control, which can avoid food safety issues caused by antibiotics and hormones; this technology is freed from passive dependence on the growth cycle of animals, and is expected to produce meat products more efficiently and solve the problem of food crisis. In addition, this technology avoids animal slaughter and is more in line with the healthy concept of animal protection. Cell cultured meat is mainly composed of cells and biological scaffolds. Among them, the biological scaffold must meet the performance requirements of edibility and promoting cell adhesion and growth. In addition, due to the obvious oriented texture of natural muscle tissue. In order to make cell cultured meat show natural muscle texture, it is of great significance to prepare microstructures with bionic orientation.

[0003] At present, the preparation of oriented microstructures can be achieved through oriented electrospinning, wet extrusion spinning and other technologies. However, these bioscaffolds have problems such as insufficient cell permeability, which leads to serious limitations in increasing the thickness of the scaffolds and the inability to achieve 3D structure molding and processing; and because it is difficult for cells to penetrate the interior of the scaffold, the proportion of cells in the prepared cell-cultured meat is low and difficult to exceed 70%. Improving the pore structure of the bioscaffold is an important way to increase cell permeability. Therefore, a new preparation technology for edible bioscaffolds is urgently needed to solve the challenges of porosity and biocompatibility.

[0004] Directed freeze-drying is a method that uses temperature gradient as a driving force to form an ice crystal template in a directional manner. After freeze-drying, a 3D integrated porous aerogel can be formed. The prepared material has abundant micro-nano pores, which provide the required space for the infiltration and growth of cells. In addition, due to the directional formation of ice crystals, the microfibers of the scaffold material can be induced to be arranged in an orderly manner in the direction of ice crystal formation. Therefore, this technology is expected to directly prepare three-dimensional cell-cultured meat with bionic muscle-oriented texture in vitro. CN113208059A and CN113215090A used this technology to prepare gelatin-based and collagen derivative oriented biological scaffolds, which can be used as biological scaffolds for cell-cultured meat. However, gelatin and collagen are still animal proteins with limited sources, which is contrary to the vision of the cell-cultured meat industry. Summary of the invention

[0005] The object of the present invention is to overcome at least one deficiency of the prior art and to provide an edible chitosan gluten biomimetic oriented cell culture meat biological scaffold.

[0006] The technical solution adopted by the present invention is:

[0007] The first aspect of the present invention provides:

[0008] An edible chitosan glutenin biomimetic oriented cell culture meat biological scaffold, the preparation method of which comprises the following steps:

[0009] 1) fully mixing glutenin, chitosan, other edible proteins and water, adjusting the pH of the mixed solution to 9-10, and obtaining a mixed protein solution, wherein the amount of the other edible proteins added is 0-500% of the sum of the mass of glutenin and chitosan;

[0010] 2) transferring the mixed protein solution into a directional scaffold mold, freezing the mixed protein solution within a period of no more than 10 minutes, and freeze-drying after demolding to obtain an unfixed biological scaffold;

[0011] 3) thermally cross-linking the unfixed biological scaffold to obtain a biomimetic oriented biological scaffold.

[0012] In some examples of cell culture meat bioscaffolds, the mass ratio of glutenin to chitosan is (3-8): (2-7).

[0013] In some examples of cell cultured meat bioscaffolds, glutenin and chitosan are dissolved in acetic acid solution and then mixed with other raw materials.

[0014] In some examples of cell cultured meat bioscaffolds, the oriented scaffold mold is placed in a mixture of ethanol and liquid nitrogen for quick freezing.

[0015] In some examples of cell cultured meat bioscaffolds, the other edible protein is selected from at least one of soy protein isolate, potato protein, and corn protein.

[0016] In some examples of cell culture meat bioscaffolds, the temperature of the thermal cross-linking is 90-150°C.

[0017] In some examples of cell culture meat bioscaffolds, the chitosan is selected from shrimp shell-derived and / or mushroom-derived chitosan.

[0018] In some examples of cell culture meat bioscaffolds, the viscosity of the chitosan ranges from 20 to 800 cps.

[0019] In some examples of cell culture meat bioscaffolds, the toughness of the bioscaffold per square centimeter of block scaffold is not less than 10,000 g as measured by a texture analyzer TPA test.

[0020] The second aspect of the present invention provides:

[0021] A cell cultured meat is obtained by culturing animal cells on a biological scaffold. The biological scaffold used for cell culture is as described in the first aspect of the present invention and has a bionic color ranging from light yellow to brown.

[0022] In some examples of cell cultured meat, the animal cells are selected from myoblasts, muscle satellite cells, fibroblasts, adipocytes, fat precursor cells, adult stem cells and pluripotent stem cells. The cells include but are not limited to myocytes, hepatocytes, osteoblasts, fibroblasts, adipocytes, odontoblasts, adult neuronal progenitor cells, neural stem cells, multiple multipotent stem cells from the subventricular forebrain region, ependymal neural stem cells, hematopoietic stem cells, hematopoietic stem cells, bone marrow stem cells, adipose fibroblasts, adipose stem cells, stem cells that produce multiple islet cells, pancreatic multipotent islet stem cells, mesenchymal stem cells, placental cells, bone marrow stromal cells, muscle side group cells, bone marrow-derived recovered cells, blood-derived mesenchymal precursor cells, bone marrow-derived side group cells, muscle precursor cells, circulating skeletal stem cells, neural progenitor cells, multipotent adult progenitor cells, mesoderm progenitor cells, spinal cord progenitor cells and spore-like cells and any combination thereof.

[0023] The beneficial effects of the present invention are:

[0024] The edible chitosan gluten biomimetic oriented cell culture meat bioscaffolds of some examples of the present invention, the raw materials involved are all non-animal source proteins, not only have good cell compatibility and biosafety, but also can promote cell adhesion and growth. The prepared bioscaffold has a directional biomimetic oriented structure, can simulate the texture of natural muscle tissue, and bring a good taste to the cell cultured meat.

[0025] The edible chitosan gluten biomimetic oriented cell culture meat biological scaffolds of some embodiments of the present invention contain rich porosity and effectively improve the permeability of cells. Therefore, the present invention will provide a healthier and more effective biological scaffold for the field of cell culture meat and bioengineering. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 It is the test result of mechanical properties of the stent under TPA mode;

[0027] Figure 2 It is the color change of the bioscaffold after freeze-drying, thermal cross-linking fixation, and cell culture;

[0028] Figure 3 This is the cell live-death staining image of the cell cultured meat bioscaffold after inoculation of C2C12 cells for 5 days;

[0029] Figure 4This is a cell live-death staining image of the cell culture meat bioscaffold 12 days after inoculation of C2C12 cells. DETAILED DESCRIPTION

[0030] The first aspect of the present invention provides:

[0031] An edible chitosan glutenin biomimetic oriented cell culture meat biological scaffold, the preparation method of which comprises the following steps:

[0032] 1) fully mixing glutenin, chitosan, other edible proteins and water, adjusting the pH of the mixed solution to 9-10, and obtaining a mixed protein solution, wherein the amount of the other edible proteins added is 0-500% of the sum of the mass of glutenin and chitosan;

[0033] 2) transferring the mixed protein solution into a directional scaffold mold, freezing the mixed protein solution within a period of no more than 10 minutes, and freeze-drying after demolding to obtain an unfixed biological scaffold;

[0034] 3) thermally cross-linking the unfixed biological scaffold to obtain a biomimetic oriented biological scaffold.

[0035] The pH of the mixed protein solution can be adjusted by adding edible alkali or acid, such as NaOH, Na2CO3, etc. to the protein solution. Other edible proteins refer to edible proteins other than glutenin, preferably proteins from non-animal sources, and more preferably plant proteins.

[0036] In some examples of cell culture meat biological scaffolds, the mass ratio of glutenin and chitosan is (3-8): (2-7). This has both good strength and good promotion of cell adhesion and growth.

[0037] In some examples of cell cultured meat bioscaffolds, glutenin and chitosan are dissolved in acetic acid solution before mixing with other raw materials. This allows for a more uniform mixing.

[0038] In some examples of cell culture meat bioscaffolds, the oriented scaffold mold is placed in a mixture of ethanol and liquid nitrogen for quick freezing. This can effectively control the process temperature of the freeze-drying process to achieve the adjustment of the pore structure.

[0039] In some examples of cell cultured meat bioscaffolds, the other edible proteins are selected from at least one of soy protein isolate, potato protein, and corn protein to meet the personalized design of taste and nutritional ingredients.

[0040] In some examples of cell cultured meat biological scaffolds, the temperature of the thermal crosslinking is 90-150° C. By controlling the temperature and time of the thermal crosslinking reaction, the color of the scaffold can be further adjusted to make it closer to natural meat.

[0041] In some examples of cell culture meat bioscaffolds, the chitosan is selected from shrimp shell and / or mushroom chitosan. The chitosans from these sources are stable, especially the chitosan from mushrooms, which can completely get rid of the dependence on aquaculture.

[0042] In some examples of cell culture meat bioscaffolds, the viscosity of the chitosan ranges from 20 to 800 cps.

[0043] In some examples of cell cultured meat bio-scaffolds, the bio-scaffold has a toughness of no less than 10,000 g per square centimeter of block scaffold tested by a texture analyzer TPA, which makes the bio-scaffold have a fiber feel closer to that of natural meat.

[0044] The second aspect of the present invention provides:

[0045] A cell cultured meat is obtained by culturing animal cells on a biological scaffold. The biological scaffold used for cell culture is as described in the first aspect of the present invention and has a bionic color ranging from light yellow to brown.

[0046] In some examples of cell cultured meat, the animal cells are selected from myoblasts, muscle satellite cells, fibroblasts, adipocytes, fat precursor cells, adult stem cells and pluripotent stem cells. The cells include but are not limited to myocytes, hepatocytes, osteoblasts, fibroblasts, adipocytes, odontoblasts, adult neuron progenitor cells, neural stem cells, multiple multipotent stem cells from the subventricular forebrain region, ependymal neural stem cells, hematopoietic stem cells, hematopoietic stem cells, bone marrow stem cells, adipose fibroblasts, adipose stem cells, stem cells that produce multiple islet cells, pancreatic multipotent islet stem cells, mesenchymal stem cells, placental cells, bone marrow stromal cells, muscle side group cells, bone marrow-derived recycled cells, blood-derived mesenchymal precursor cells, bone marrow-derived side group cells, muscle precursor cells, circulating skeletal stem cells, neural progenitor cells, multipotent adult progenitor cells, mesoderm progenitor cells, spinal cord progenitor cells and spore-like cells and any combination thereof.

[0047] The technical solution of the present invention is further described below in conjunction with embodiments.

[0048] In the following examples, unless otherwise specified, the bottom of the directional freeze-drying mold used is a metal copper plate, and the upper layer is a fluorine-containing material PTFE.

[0049] The glutenin solution is obtained by dissolving gluten in acid and then removing the insoluble matter.

[0050] Unless otherwise specified, percentages are by mass.

[0051] Example 1

[0052] Preparation of bioscaffolds:

[0053] Solution configuration:

[0054] 1) Prepare 5% gluten powder with 1% food-grade glacial acetic acid, stir overnight at 1000 rpm; after stirring, centrifuge at 5000 rpm for 5 minutes. Take the supernatant, which is the glutenin solution;

[0055] 2) Prepare 2.5% shrimp shell chitosan (the chitosan source is a medium molecular weight chitosan provided by Sigma-Aldrich, and its deacetylation degree is >95%) with 1% food-grade glacial acetic acid;

[0056] 3) The glutenin solution and the chitosan solution are mixed at a mass ratio of 1:1 to form a uniform scaffold solution; and soybean dietary fiber is added to the above solution at a concentration of 0.1 mg / mL.

[0057] The biomimetic biological scaffold is prepared by directional freeze-drying using an ice crystal template method, wherein the preparation method is as follows:

[0058] 1) Pour the scaffold solution into a homemade mold, place it in a mixture of ethanol and liquid nitrogen at -80°C, and let it stand for 20 minutes to freeze the solution completely. After freeze-drying the water in a freeze dryer, an unfixed biological scaffold is obtained;

[0059] 2) After the sample is freeze-dried, fix it. The specific method is to heat crosslink, place the freeze-dried scaffold in 100℃ water and steam for 15 minutes; or in a steamer and baker, maintain 140℃, steam and bake for 10 minutes;

[0060] 3) After fixation is completed, use PBS to remove the acid and alkali and adjust to neutral.

[0061] Mechanical properties of bioscaffolds:

[0062] Texture analyzer is a commonly used instrument for texture measurement in food industry and scientific research. Using TA.XT Plus TextureAnalyser, the mechanical properties of the scaffold were tested in the full texture experiment (Texture Profile Abalysis, TPA, also known as the secondary chewing experiment) mode.

[0063] During the TPA test, a disc-shaped probe is used. The cross-sectional area of ​​the probe is larger than the sample area. The probe movement trajectory is as follows:

[0064] 1) After the probe's initial position is reset to zero, press it against the test sample at a rate of 1 mm / s;

[0065] 2) After contacting the sample surface, compress the sample at a certain distance (5mm) at the test rate; then return to the compression trigger point;

[0066] 3) After staying for a while, continue to compress downwards by the same distance; then return to the position before the probe test at the post-test rate. The mechanical properties of the bioscaffold can be obtained.

[0067] TPA test results Figure 1 As shown in the figure, it can be found that the directional freeze-dried scaffold has anisotropy. For example, the chewability of the scaffold, that is, the energy required to chew solid food into a swallowable state, is only 20% of that in the vertical direction along the freeze-drying direction. This anisotropy and biomimetic performance can provide a taste very close to natural muscle tissue.

[0068] Example 2

[0069] Preparation of bioscaffolds:

[0070] 1) Prepare 5% gluten powder with 1% food-grade glacial acetic acid, stir overnight at 1000 rpm; after stirring, centrifuge at 5000 rpm for 5 minutes. Take the supernatant, which is the glutenin solution;

[0071] 2) Prepare a 5% peanut protein solution with 0.5% food-grade hydrochloric acid, stir at 1000 rpm overnight to dissolve all the peanut protein, and obtain a peanut protein solution;

[0072] 3) using 1% food-grade glacial acetic acid to prepare 5% mushroom-derived chitosan (the chitosan source is food-grade mushroom chitosan with a viscosity of 100 cps) to obtain a chitosan solution;

[0073] 4) The glutenin solution, the peanut protein and the chitosan solution are mixed in a mass ratio of 1:1:1 to form a uniform scaffold solution;

[0074] 5) The biomimetic biological scaffold is prepared by freeze-drying using the ice crystal template method, wherein the preparation method is as follows: the scaffold solution is poured into a self-made mold. The scaffold is placed in a mixture of ethanol and liquid nitrogen at -80°C. The mixture is allowed to stand for 20 minutes to completely freeze the solution. After the water is freeze-dried in a freeze dryer, an unfixed biological scaffold is obtained;

[0075] 6) After the sample is freeze-dried, fix it. The specific method is to heat crosslink, place the freeze-dried scaffold in 100℃ water and steam for 15 minutes; or in a steamer and baker, maintain 140℃, steam and bake for 10 minutes;

[0076] 7) After fixation is completed, use PBS to remove the acid and alkali and adjust to neutral.

[0077] Color changes of bioscaffolds:

[0078] Natural meat products have typical hemoglobin. Therefore, the cell cultured meat industry also needs to provide more realistic colors. To this end, this embodiment uses a common optical camera to record the color changes of the bioscaffold after freeze-drying, thermal cross-linking fixation, and cell culture.

[0079] The results are as follows Figure 2 As shown in the figure, due to the typical Maillard reaction between glutenin and chitosan during the thermal cross-linking process, the color formed brown. After cell attachment, the prepared cell cultured meat showed a structure and color very close to natural meat.

[0080] Example 3

[0081] Preparation of bioscaffolds:

[0082] 1) Prepare 5% gluten powder with 1% food-grade glacial acetic acid, stir overnight at 1000 rpm; after stirring, centrifuge at 5000 rpm for 5 minutes. Take the supernatant, which is the glutenin solution;

[0083] 2) using 1% food-grade glacial acetic acid to prepare 5% mushroom-derived chitosan (the chitosan source is food-grade mushroom chitosan with a viscosity of 300 cps) to obtain a chitosan solution;

[0084] 3) The glutenin solution and the chitosan solution are mixed in a mass ratio of 1:1 to form a uniform scaffold solution;

[0085] 4) The biomimetic biological scaffold is prepared by freeze-drying using the ice crystal template method, wherein the preparation method is as follows: the scaffold solution is poured into a self-made mold. The scaffold is placed in a mixture of ethanol and liquid nitrogen at -80°C. The mixture is allowed to stand for 20 minutes to completely freeze the solution. After the water is freeze-dried in a freeze dryer, an unfixed biological scaffold is obtained;

[0086] 5) After the sample is freeze-dried, fix it. The specific method is to heat crosslink, place the freeze-dried scaffold in 100℃ water and steam for 15 minutes; or in a steamer and baker, maintain 140℃, steam and bake for 10 minutes;

[0087] 6) After fixation is completed, use PBS to remove the acid and alkali and adjust to neutral.

[0088] Cell culture:

[0089] 1) Mouse myoblast C2C12 cells with a cell number of no more than 6 generations were cultured in a high glucose medium / fetal bovine serum (DMEM / FBS) medium containing 10% FBS and 1% triple antibody. The cells were cultured in a 37°C, 5% CO2 incubator. After the cells reached 80% coverage in the culture dish, they were digested with trypsin and centrifuged, and resuspended to obtain a cell dispersion. After counting, the dispersed cells were 10 5cells / mL solution for later use.

[0090] 2) The biomimetic scaffold prepared in this example was sterilized by soaking in 75% medical alcohol. At the same time, the system was placed in a UV sterilizer for sterilization. Then it was washed with PBS 5 times in a biosafety cabinet, each time for 5 minutes. After removing the PBS, the cell suspension was inoculated on the bioscaffold. The cells were allowed to grow by adhering to the wall. The cells were cultured in a 37°C, 5% CO2 incubator.

[0091] 3) Cell characterization: After 5 days of cell culture, add an appropriate volume of Calcein AM / PI detection working solution. Usually, add 100μl to each well of a 96-well plate, 250μl to each well of a 24-well plate, 500μl to each well of a 12-well plate, and 1ml to each well of a 6-well plate. Incubate at 37°C in the dark for 30 minutes. After incubation, observe the staining effect under a fluorescence microscope (Calcein AM is green fluorescence, Ex / Em=494 / 517nm; PI is red fluorescence, Ex / Em=535 / 617nm).

[0092] 4) In order to track the adhesion and growth of cells over a longer period of time, the culture time was extended to 12 days and the above staining process was repeated.

[0093] Experimental results ( Figure 3 and Figure 4 ) showed that all cells showed green fluorescence, which indicated that the cell survival rate was more than 95%, and the cell adhesion density on the scaffold was high. The experimental results showed that the directional freeze-dried scaffold had good cell compatibility and could promote the adhesion and growth of cells. Compared with the morphology of cells on the 12th day, obvious oriented growth occurred, indicating that the bionic scaffold prepared by the present invention had a good promoting effect on the formation of muscle tissue.

[0094] The above is a further detailed description of the present invention, which should not be regarded as a limitation on the specific implementation of the present invention. For ordinary technicians in the technical field to which the present invention belongs, simple deduction or replacement without departing from the concept of the present invention is within the protection scope of the present invention.

Claims

1. An edible chitosan glutenin biomimetic oriented cell culture meat biological scaffold, the preparation method of which comprises the following steps: 1) dissolving glutenin and chitosan in an acetic acid solution and then fully mixing with water, adjusting the pH of the mixed solution to 9-10, to obtain a mixed protein solution, wherein the chitosan is selected from chitosan derived from shrimp shells and / or mushrooms, and the mass ratio of glutenin to chitosan is (3-8):(2-7); 2) transferring the mixed protein solution into a directional scaffold mold, placing the directional scaffold mold in a mixture of ethanol and liquid nitrogen for quick freezing, freezing the mixed protein solution within no more than 10 minutes, and freeze-drying after demolding to obtain an unfixed biological scaffold; 3) thermally cross-linking the unfixed biological scaffold at 90-150° C. to obtain a biomimetic oriented biological scaffold.

2. The cell culture meat biological scaffold according to claim 1, characterized in that: The viscosity of the chitosan ranges from 20 to 800 cps.

3. The cell culture meat biological scaffold according to claim 1, characterized in that: The toughness of the block-shaped biological scaffold per square centimeter is not less than 10000 g as measured by a texture analyzer TPA test.

4. A cell cultured meat obtained by culturing animal cells on a biological scaffold, characterized in that: The biological scaffold used for cell culture is as described in any one of claims 1 to 3, and has a bionic color ranging from light yellow to brown.

5. The cell cultured meat according to claim 4, characterized in that: The animal cells are selected from the group consisting of myoblasts, muscle satellite cells, fibroblasts, adipocytes, and preadipocytes.

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