CN114736851B

CN114736851B - Method for preparing vegetable-based fat-cultured meat

Info

Publication number: CN114736851B
Application number: CN202210105570.1A
Authority: CN
Inventors: 周光宏; 宋文娟; 丁世杰; 李惠侠; 唐长波; 刘裴裴
Original assignee: Nanjing Zhouzi Future Food Technology Co ltd; Nanjing Agricultural University
Current assignee: Nanjing Zhouzi Future Food Technology Co ltd; Nanjing Agricultural University
Priority date: 2022-01-28
Filing date: 2022-01-28
Publication date: 2024-04-12
Anticipated expiration: 2042-01-28
Also published as: CN114736851A

Abstract

In order to solve the defects of a conventional bracket for culturing lipid-forming stem cells and the defects of the existing bracket for culturing meat in aspects of flavor, nutrition, taste, juiciness and the like, the invention provides application of plant wiredrawing protein in serving as a bracket for culturing lipid-forming stem cells in vitro and a preparation method of plant-based fat-cultured meat, which specifically comprises the following steps of S1: pretreatment of a plant wiredrawing protein bracket: swelling plant wiredrawing protein with water, cutting into proper size, oven drying, sterilizing, and soaking plant wiredrawing protein bracket in growth medium under aseptic condition overnight; s2: adding the adipogenic stem cells into a growth culture medium, preparing a cell suspension, and then inoculating the cell suspension onto a plant wiredrawing protein bracket for proliferation culture; s3: and performing proliferation culture until the coverage rate of cells on the bracket reaches 80% -90%, performing induced differentiation culture until the cells are differentiated into fat cells, and finally preparing the plant-based fat culture meat with animal meat flavor.

Description

Method for preparing vegetable-based fat-cultured meat

Technical Field

The invention belongs to the technical field of stem cells and animal cell cultured meat, and particularly relates to a preparation method of plant-based fat cultured meat.

Background

Meat is rich in proteins and micronutrients and has been widely consumed by humans due to the ease of energy acquisition, high quality proteins, palatability and energy. With the world population growing and the abundance increasing, meat consumption increases year by year. Traditional meat production relies on the animal industry, not only with risks of animal disease, epidemics and antibiotic abuse, but also with negative effects on animal welfare, resources and environment, even threatens to public health, such as ischemic heart disease and colorectal cancer. Therefore, in order to achieve sustainable production of meat, development of a green and efficient meat production method to partially replace the traditional meat production is urgently needed.

The cultured meat, also called clean meat, is an in vitro synthesized or laboratory cultivated meat, which is produced in a bioreactor using tissue engineering techniques, intended to produce meat by cell culture. Unlike traditional meats, the cultured meats are expected to solve animal welfare, resource shortage and public health problems. A key goal of cell culturing meat is to replicate and ultimately improve the organoleptic and nutritional properties of traditional meat products. In 2013, research on cultured meat is rapidly developed internationally, and a series of important breakthroughs are achieved. However, most of the research on cell culture meat has been focused on producing muscle so far. Fat, although a small percentage of the total meat content, is a key determinant of flavor, texture, nutrition and visual appearance, all of which are related to consumer preferences and purchase intent. Thus, the impact of fat on meat flavor, nutrition, appearance and texture makes it an important area of research and development for cell culture meat.

In recent years, vegetable meats have become increasingly popular due to concerns about the sustainability of livestock production of meats. Many vegetable-derived proteins have been successfully used to produce vegetable protein meats, but mimicking the flavor and mouthfeel of animal fat remains a challenge. Therefore, the combination of the cell culture fat and the vegetable protein can obviously improve the sensory quality and the nutritional characteristics of vegetable meat, also meet the purchasing desire of consumers to a certain extent, and is beneficial to the sustainability of meat production. In addition, in vitro culture can control lipid profile to provide a fat supplement for meat processing for flavor, nutrition, mouthfeel, juiciness, to optimize human health and consumer perception related quality.

The use of in vitro cultured adipocytes as an improvement factor for vegetable protein meat allows for a relatively low number of cells to improve the quality of existing products and to reduce meat production in dependence of animal husbandry.

Large scale in vitro culture of adipose tissue requires the establishment of cell lines that differentiate directionally towards mature adipocytes. First, the cell line must have sufficient proliferative capacity to allow for initial isolation to large scale expansion. Second, cell lines must be able to accommodate low cost media, culture three-dimensional cell scaffolds, and not affect their proliferation and differentiation. Finally, the cell lines must be capable of efficient, food-safe differentiation into mature adipocytes or adipose tissue.

Currently, as conventional scaffolds for the culture of adipogenic stem cells, for example: animal-derived scaffolds such as collagen scaffolds, which are used for culturing meat in the original sense of reducing or avoiding the use of animal-derived materials, are contrary to the original sense of culturing meat; and plant-source scaffolds such as cellulose scaffolds, etc., have poor cell adhesion and poor culture effects. In addition, when the conventional bracket is used for inoculating cells, chemical reagents are needed to be crosslinked, most of crosslinking agents cannot be eaten due to safety problems, and the conventional bracket is high in price, so that popularization of the cultured meat is severely restricted. Therefore, there is a need to provide a culture scaffold and a culture method that can be applied to adipogenic stem cells.

Disclosure of Invention

In order to solve the technical problems in the prior art, the invention applies the plant wiredrawing protein to the preparation of the fat culture meat and provides a method for preparing the plant-based fat culture meat.

The technical scheme of the invention is as follows:

the first object of the present invention is to provide the use of a plant wire drawing protein, preferably peanut wire drawing protein, soybean wire drawing protein or wheat wire drawing protein, as an in vitro culture scaffold for lipid-forming stem cells.

Further, the application comprises the steps of:

S1: pretreatment of a plant wiredrawing protein bracket: rehydrating and sterilizing the plant wiredrawing protein, and soaking the plant wiredrawing protein in a growth medium under a sterile condition to obtain a plant wiredrawing protein bracket; preferably, the plant wiredrawing protein is firstly absorbed in pure water until the plant wiredrawing protein is fully expanded, cut into the required size and then sterilized;

s2: adding the adipogenic stem cells into a growth medium to prepare a cell suspension, and then inoculating the cell suspension onto a plant wiredrawing protein bracket for proliferation culture;

s3: and performing proliferation culture until the coverage rate of cells on the scaffold reaches 80-90%, and performing induced differentiation culture until the adipogenic stem cells are differentiated into mature adipocytes.

Further, in a particular embodiment, the plant wire drawing protein scaffold is cut into cubes 1.2cm long by 1.2cm wide by 1.2cm thick by 1mm thick in S1.

Further, the S1 sterilization mode is high-temperature sterilization, irradiation sterilization or 75% alcohol soaking sterilization; preferably, the sterilization mode is high-temperature sterilization; further preferably, the high-temperature sterilization is to sterilize the plant wiredrawing protein for 10 to 20 minutes at 120 to 130 ℃ after the drying, and further preferably, the high-temperature sterilization is to sterilize the plant wiredrawing protein for 15 minutes at 121 ℃ after the drying.

Further, the growth medium of S1 and S2 is a basal medium added with bFGF and 10% (volume ratio) fetal bovine serum with the concentration of 5ng/mL, and the basal medium is one of DMEM medium, MEM medium, DMEM/F12 medium and F10 medium.

Further, the concentration of the cell suspension of S2 was 5X 10 ⁴ ～5×10 ⁵ Individual cells/. Mu.L; preferably, S2 is as describedCell suspension concentration was 1X 10 ⁵ Individual cells/. Mu.L.

Further, the cell seeding density on the plant wire drawing protein scaffold in S2 is 2×10 ⁴ Individual cells/mm ³ ～3×10 ⁵ Individual cells/mm ³ The cell seeding density on the scaffold, preferably a plant wire drawing protein scaffold, is 1.4X10 ⁵ Individual cells/mm ³ And (3) a bracket.

Further, the induced differentiation culture method of S3 is as follows:

continuously culturing for 5 days by adopting a differentiation culture medium, wherein the differentiation culture medium is as follows: 10. Mu.g/mL insulin, 0.1mM 3-isobutyl-1-methylxanthine, 1. Mu.M dexamethasone, 0.1mM indomethacin, 1. Mu.M rosiglitazone and basal medium containing 10% fetal bovine serum;

the differentiation medium was then replaced with a maintenance differentiation medium for 2 days, which was: basal medium containing 10 μg/mL insulin and 10% fetal bovine serum;

finally, the maintenance medium is replaced by a basic medium of 10% fetal bovine serum, and the differentiation is continued for 3 days;

The basal medium is selected from one of DMEM medium, MEM medium, DMEM/F12 medium and F10 medium.

Further, the adipogenic stem cell is a stem cell capable of differentiating into a adipogenic lineage cell, preferably, the adipogenic stem cell is selected from adipose-derived mesenchymal stem cells, bone marrow mesenchymal stem cells, pluripotent stem cells or progenitor cells, embryonic stem cells, induced pluripotent stem cells, immortalized adipogenic stem cells.

A second object of the present invention is to provide a method for preparing a vegetable-based fat-cultured meat, comprising the steps of:

s3: and when the coverage rate of the proliferation culture cells on the scaffold reaches 80-90%, performing induced differentiation culture until the adipogenic stem cells are differentiated into mature adipocytes, wherein the plant wire drawing protein scaffold and the mature adipocytes adhered to the plant wire drawing protein scaffold are the plant-based fat culture meat.

Further, the concentration of the cell suspension of S2 was 5X 10 ⁴ ～5×10 ⁵ Individual cells/. Mu.L; preferably, the cell suspension of S2 has a concentration of 1X 10 ⁵ Individual cells/. Mu.L

Further, the cell seeding density on the plant wire drawing protein scaffold in S2 is 2×10 ⁴ Individual cells/mm ³ ～3×10 ⁵ Individual cells/mm ³ The cell seeding density on the scaffold, preferably plant wiredrawing protein scaffold, is 1×10 ⁵ Individual cells/mm ³ And (3) a bracket.

Further, the induced differentiation culture method of S3 is as follows:

A third object of the present invention is to provide a vegetable-based fat-cultured meat prepared by the aforementioned method.

The technical scheme of the invention has the beneficial effects that:

the invention combines the cell culture fat with the vegetable protein, and obviously improves the sensory quality and the nutritional characteristics of vegetable meat.

The application relates to a cell for preparing plant-based cultured meat. The method comprises the steps of inoculating the adipogenic stem cells to plant wire drawing protein scaffolds such as peanut wire drawing protein, wheat wire drawing protein scaffolds and soybean wire drawing protein scaffolds, and inducing the adipogenic stem cells to differentiate into mature adipocytes. Thereby the vegetable meat has the flavor, nutrition, taste and juiciness of animal fat, and the quality of vegetable meat is improved.

The invention discovers that the adipogenic stem cells can take the plant wiredrawing protein as a three-dimensional adipogenic bracket, better adhere and proliferate on the plant wiredrawing protein bracket and differentiate to mature adipocytes, and the prepared plant-based fat culture meat has a certain similarity with the traditional meat, is greatly different from the blank bracket in texture and volatile matter composition, and greatly improves the flavor and taste of the plant protein meat. In conclusion, the plant protein is inoculated with the adipogenic stem cells to induce the adipogenic stem cells to differentiate into mature fat cells, so that the quality of the vegetable meat can be improved.

Drawings

FIG. 1 is an illustration of adipose stem cell isolation and identification. Wherein, the pig subcutaneous adipose tissue collected by A. B is chopped pork subcutaneous adipose tissue. C is a type I collagenase digested cell suspension. D is a cell morphology diagram of the isolated adipose-derived stem cells cultured for 12h, 24h, 48h, 72 h. E is immunofluorescent staining of adipose stem cell specific surface marker proteins. F bright field plot of differentiation of adipose stem cells into mature adipocytes. G is oil red O staining.

FIG. 2 is a morphology of a plant protein scaffold. Wherein A is the form of plant protein after water swelling. B is a plant protein scaffold with the size of 0.6cm multiplied by 0.1 cm. C is the result of a plant protein bracket scanning electron microscope.

FIG. 3 adhesion of cells on plant wire-drawing protein scaffolds treated in different sterilization modes. Wherein A is 75% alcohol soaking for 1.5h sterilization treatment. B is a bracket after gamma-ray irradiation sterilization treatment. C is a plant wiredrawing protein bracket subjected to high-temperature high-pressure sterilization treatment.

FIG. 4 shows the cell adhesion of different plant protein scaffold materials and the absence of material pretreatment. Wherein A is the adhesion result of the soybean wiredrawing protein scaffold cells. Panel B shows the results of cell adhesion of soy protein scaffold pretreated with collagen. Panel C shows the results of cell adhesion of peanut wiredrawing protein scaffolds. Panel D shows the results of cell adhesion of peanut wiredrawing protein scaffolds pretreated with collagen. Panel E shows the results of cell adhesion of wheat wire drawing protein scaffolds. Panel F shows the results of cell adhesion of a collagen-pretreated wheat wire-drawing protein scaffold.

FIG. 5 shows growth of living cell tracer labelled cells on a peanut wiredrawing protein scaffold. A is a two-dimensional planar cultured adipose stem cells labeled with a living cell tracer. B is the cell state at day 3 when the adipose stem cells were seeded onto peanut wiredrawing protein scaffolds. C is the cellular status of the adipose-derived stem cells at day 7 when they were seeded onto peanut wiredrawing protein scaffolds.

FIG. 6 shows the results of observation of proliferation and growth states of adipose-derived stem cells on a peanut wiredrawing protein scaffold by a scanning electron microscope. Wherein, panel a is the scanning electron microscope results at days 3 and 7 after adipose stem cells were grafted into the scaffold. Panel B shows the survival of adipose-derived stem cells on a peanut wiredrawing protein scaffold under a high power microscope.

FIG. 7 shows the proliferation results of the DNA quantitative determination of adipose-derived stem cells on peanut wiredrawing protein scaffolds.

FIG. 8 results of oil red O staining of adipose-derived stem cells after induced differentiation on peanut wiredrawing protein scaffolds. Wherein, graph A is blank stent oil red O staining results. Panel B shows the results of oil red O staining induced by differentiation to day 10 after adipose stem cells were grafted onto the scaffolds.

FIG. 9 quantitative PCR results of expression of lipid-critical gene FABP4 after induced differentiation of adipose-derived stem cells on peanut wiredrawing protein scaffolds.

Fig. 10 shows the docking of adipose stem cells onto peanut wiredrawing protein scaffolds at different densities. Wherein, the inoculation density of each of the graphs A-C is 10 ⁷ Individual blocks (i.e. 2.8X10) ⁵ Individual cells/mm ³ )、5×10 ⁶ Individual blocks (i.e. 1.4X10) ⁵ Individual cells/mm ³ )、10 ⁶ Individual blocks (i.e. 2.8X10) ⁴ Individual cells/mm ³ )。

FIG. 11 immunofluorescent staining of perilipin (Plin 1) after induction of differentiation of adipose stem cells onto peanut wiredrawing protein scaffolds at different densities on day 10. Wherein, the inoculation density of each of the graphs A-C is 10 ⁷ Individual blocks (i.e. 2.8X10) ⁵ Individual cells/mm ³ )、5×10 ⁶ Individual blocks (i.e. 1.4X10) ⁵ Individual cells/mm ³ )、10 ⁶ Individual blocks (i.e. 2.8X10) ⁴ Individual cells/mm ³ )。

FIG. 12 results of oil red O staining after induction differentiation of adipose stem cells at different densities onto peanut wiredrawing protein scaffolds on day 10. Wherein, the inoculation density of each of the graphs A-C is 10 ⁷ Individual blocks (i.e. 2.8X10) ⁵ Individual cells/mm ³ )、5×10 ⁶ Individual blocks (i.e. 1.4X10) ⁵ Individual cells/mm ³ )、10 ⁶ Individual blocks (i.e. 2.8X10) ⁴ Individual cells/mm ³ )。

FIG. 13 induced differentiation of adipose stem cells at different densities onto peanut wiredrawing protein scaffoldsAfter day 10, adipogenic marker protein FABP4, plin1 expression results. Wherein, the inoculation density of the graph A is 10 respectively ⁷ Individual blocks (i.e. 2.8X10) ⁵ Individual cells/mm ³ )、5×10 ⁶ Individual blocks (i.e. 1.4X10) ⁵ Individual cells/mm ³ )、10 ⁶ Individual blocks (i.e. 2.8X10) ⁴ Individual cells/mm ³ ) FABP4 Western blotting results. Panel C inoculation densities of 10 respectively ⁷ Individual blocks (i.e. 2.8X10) ⁵ Individual cells/mm ³ )、5×10 ⁶ Individual blocks (i.e. 1.4X10) ⁵ Individual cells/mm ³ )、10 ⁶ Individual blocks (i.e. 2.8X10) ⁴ Individual cells/mm ³ ) FABP4 Western blot gray-scale analysis results. Panel B seed density of 10 respectively ⁷ Individual blocks (i.e. 2.8X10) ⁵ Individual cells/mm ³ )、5×10 ⁶ Individual blocks (i.e. 1.4X10) ⁵ Individual cells/mm ³ )、10 ⁶ Individual blocks (i.e. 2.8X10) ⁴ Individual cells/mm ³ ) Results of Plin1 western blotting. Panel D inoculation densities of 10 respectively ⁷ Individual blocks (i.e. 2.8X10) ⁵ Individual cells/mm ³ )、5×10 ⁶ Individual blocks (i.e. 1.4X10) ⁵ Individual cells/mm ³ )、10 ⁶ Individual blocks (i.e. 2.8X10) ⁴ Individual cells/mm ³ ) Results of the Plin1 western blot gray analysis.

FIG. 14 shows the results of expression of adipogenic marker gene FABP4 after induction of differentiation of adipose stem cells into peanut wiredrawing protein scaffolds at different densities on day 10.

Figure 15 product appearance of plant-based fat-cultured meat. Wherein A is the appearance of a blank bracket. B is the appearance of peanut wiredrawing protein cultured meat.

FIG. 16 Whole tissue nile red staining results of plant-based fat-cultured meat. Wherein A is the whole tissue nile red staining result of the blank peanut wiredrawing protein scaffold without inoculating cells. B is the nile red dyeing result of peanut wiredrawing protein cultured meat.

FIG. 17 shows the results of measurement of texture-related index of plant-based fat-cultured meat. Wherein A-F are the results of hardness, elasticity, chewiness, recovery, cohesiveness and tackiness of the vegetable-based fat-cultured meat, respectively.

FIG. 18 electronic nose measurement of flavor index of vegetable-based fat-cultured meat. PCA analyzes the volatile materials of four groups of materials.

FIG. 19 GC-IMS measurement of flavor index of vegetable-based fat culture meat. PCA analyzes the volatile materials of four groups of materials. Wherein 1 is pig muscle tissue, 2 is plant-based cell scaffold, 3 is pig subcutaneous fat tissue, and 4 is plant-based fat culture meat.

Detailed Description

The invention is further described in connection with specific embodiments, but the scope of the claims is not limited to these. The specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

The growth medium used in the proliferation stage of the adipose-derived stem cells on the plant-based protein porous scaffold is DMEM/F12 added with bFGF and fetal bovine serum with the concentration of 5ng/mL and 10% (volume ratio), and other aspects are consistent with the in-vitro culture method of the normal stem cells.

The following examples were conducted by inducing differentiation of adipose-derived stem cells into mature adipocytes: continuously culturing for 5 days by adopting a differentiation culture medium, wherein the differentiation culture medium is as follows: dexamethasone 1. Mu.M, 3-isobutyl-1-methylxanthine 0.1mM, insulin 10. Mu.g/mL, indomethacin 0.1mM, rosiglitazone 1. Mu.M in DMEM/F12 containing 10% foetal calf serum for 5 days; then the differentiation medium was replaced with maintenance medium, DMEM/F12 containing 10% fetal bovine serum with insulin of 10. Mu.g/mL for 2 days; finally, the maintenance medium was replaced with DMEM/F12 of 10% fetal bovine serum for 3 days.

The culture conditions used in the examples below were all CO ₂ Culturing at 37deg.C in incubator, CO ₂ The concentration of (C) was 5% (v/v).

The detection methods employed in the examples below are experimental methods, detection methods and preparation methods disclosed in the art unless otherwise specified.

Materials, reagents, instruments and the like used in the examples described below are commercially available unless otherwise specified.

Example 1 pig adipose mesenchymal stem cell isolation:

(1) Single cell isolation: the method comprises the steps of taking fresh slaughtered piglets, soaking the fresh slaughtered piglets in 75% (volume percentage) ethanol for one minute, taking neck subcutaneous adipose tissue under a sterile condition, and preserving the neck subcutaneous adipose tissue in a basic culture solution. Washing 3 times with PBS buffer containing high concentration of penicillin and streptomycin under aseptic condition, shearing off blood vessel and connective tissue visible in fatty tissue, and shearing fatty tissue into 1mm pieces ³ About small pieces, type I collagenase (0.1% by mass) and penicillin-streptomycin double anti-digestion solution (3% by volume) were added for digestion for 90min (37 ℃ C., shaking in a water bath) (FIGS. 1A-C).

(2) Neutralizing the digested solution with an equal volume of complete culture solution after digestion, filtering with 100 μm and 40 μm cell sieve, centrifuging at 1500rpm for 10min to remove suspended adipocytes and lipid droplets; the precipitation part is SVF (vascular matrix component, stroma vascular fraction), the supernatant is discarded, the erythrocyte lysate is added, the mixture is blown and beaten uniformly, the mixture is kept stand at room temperature for 10min, the mixture is centrifuged at 1000rpm for 5min, the supernatant is discarded, the serum-free culture solution is added, the mixture is blown and beaten uniformly, the mixture is centrifuged at 1000rpm for 5min, the supernatant is discarded, the complete culture medium is added, the mixture is blown and beaten uniformly, and the blood cell count plate is used for counting, and the mixture is counted according to the ratio of 5 multiplied by 10 ⁴ Density per mL cells were inoculated into unused 6-well plates and incubated at 37 ℃, 5% co2, saturated humidity; after 12 hours, the non-adherent cells, i.e., porcine adipose stem cells, were removed, and the adherent cells were observed by photographing at 24h, 48 h and 72h of culture (fig. 1D).

(3) Identification of adipose-derived stem cells: the adipose-derived stem cells cultured to the third day were fixed with 4% paraformaldehyde at room temperature for 15min, and washed with PBS 3 times for 3min each, and blotted with absorbent paper. 200. Mu.L of 0.5% Triton X-100 (PBS) was added, and the mixture was allowed to permeate at room temperature for 20min; the solution was washed 3 times with PBS for 3min each time and the water was sucked dry. 200. Mu.L of 1% BSA was added and blocked at room temperature for 1h; the blocking solution was blotted with blotter paper, 500 μl of the formulated primary antibodies CD29 and CD44 (1:100 primary antibody and 1% BSA by volume) were added, respectively, and placed into a wet box overnight at 4deg.C; washing with PBS for three times, each time for 5min, adding enough diluted fluorescent secondary antibody, and incubating for 1h at room temperature in dark place; nuclear dyeing: the PBS was washed three times, 5min each, and 10. Mu.L of DAPI cell nuclear dye with the cappings were added for imaging, and the results showed that the isolated cells contained a higher proportion of adipose stem cells (FIG. 1E).

(4) And (5) detecting the differentiation capacity of the two-dimensional cultured adipogenic cells. Accumulation of intracellular lipid droplets was examined under a bright field phase contrast microscope, and the results showed that intracellular lipid droplets could be accumulated by inducing differentiation to day 10 (fig. 1F). Two-dimensional induced differentiated cells were fixed with 4% paraformaldehyde at room temperature for 15min, washed 3 times with PBS for 3min each, and 500. Mu.L of the prepared oil red O dye was added. Incubation at room temperature for 15min, washing with PBS three times after incubation, sealing with glycerol gelatin, photographing, and the results showed that mature adipocytes were stained with red lipid droplets, showing that cells differentiated into mature adipocytes (fig. 1G).

Example 2 preparation of peanut wiredrawing protein scaffold and exploration of optimal Sterilization method

(1) Preparing a peanut wiredrawing protein bracket: the commercial peanut wiredrawing protein (Qingdao longevity food Co., ltd., filament type) is soaked in distilled water for 5min, and after the commercial peanut wiredrawing protein is fully absorbed and swelled, the result shows that the commercial peanut wiredrawing protein bracket is light yellow, has loose porous structure, is soft in wet state and has certain elasticity (figure 2A). The peanut wiredrawing protein scaffold was cut into cubes with the size of 0.6cm×0.6cm×0.1cm (fig. 2B), and the peanut wiredrawing protein scaffold was sterilized by different sterilization methods:

group 1: 15mL of 75% alcohol is added for soaking and sterilization for 1.5 hours, the alcohol is discarded, the washing is performed three times by 15mLPBS, and the mixture is soaked in 15mL of growth medium overnight;

group 2: drying the cut bracket in a 60 ℃ oven for 30min, sterilizing by gamma ray irradiation, and adding 15mL of growth medium for soaking overnight;

group 3: the cut scaffolds were dried in an oven at 60℃for 30min, sterilized at 121℃for 15min, and soaked overnight with 15mL of growth medium.

(2) Scanning electron microscope observation of peanut wiredrawing protein bracket: taking the peanut wiredrawing protein scaffold prepared in the embodiment 2 (1), adding the peanut wiredrawing protein scaffold into PBS containing 2.5% glutaraldehyde, fixing overnight, taking out, soaking in distilled water for 1 day, dehydrating by gradient ethanol, drying, spraying gold, observing the structure of the peanut protein scaffold under a scanning electron microscope, and observing the structure result of the scaffold by the scanning electron microscope shows that: peanut wiredrawing protein bracket is in a grid shape, and the pore sizes are different. (FIG. 2C).

(3) Adhesion capability of adipose-derived stem cells on peanut wiredrawing protein scaffolds: labeling cells with living cell tracer, namely diluting working solution with CM-Dil staining solution concentration of 1 mu M with basic culture medium DMEM/F12, adding 2mL of staining solution into cells to be labeled, incubating at 4 ℃ for 15min, discarding the staining solution, adding 4mL of PBS for three times, adding 2mL of pancreatin, incubating at 37 ℃ for 2min, adding 2mL of DMEM/F12 containing 10% FBS for neutralization digestion, collecting cell suspension and 15mL of centrifuge tube, centrifuging for 5min, discarding supernatant, re-suspending cell sediment with 1mL of growth medium, and recording living cell number with trypan blue staining.

Inoculating: centrifuging the labeled adipose-derived stem cell suspension at 330g for 5min, removing supernatant, adding 7.5 μl of growth medium to resuspend cells, wherein the concentration of the cell suspension is 1×10 ⁵ The growth medium is DMEM/F12 added with bFGF with concentration of 5ng/mL and containing 10% (volume ratio) of fetal bovine serum, the plant wire drawing protein bracket is soaked by the cell suspension, the cell suspension is connected to the peanut wire drawing protein bracket prepared in the example 2 (1), and the cell inoculation density on the plant wire drawing protein bracket is 2 multiplied by 10 ⁴ Individual cells/mm ³ 。

Proliferation culture: in order to avoid the premature addition of proliferation medium, the cells are flushed from the scaffold by the proliferation medium when not migrating into the interior of the wire drawing protein, preferably, after the scaffold which is inoculated with the cells is incubated for 1.5 hours at 37 ℃, a growth medium which is DMEM/F12 containing 10% (volume ratio) fetal bovine serum is added, and the proliferation culture is carried out for 6 days, and the growth medium is replaced every two days until the confluency coverage rate of the cells on the peanut wire drawing protein scaffold reaches 80-90%.

Three days later, the adhesion of cells on the peanut protein scaffold was observed by an inverted phase contrast fluorescence microscope, and the results showed that: after the three sterilization mode treated scaffolds are connected with cells, the cells can be well adhered to the peanut wiredrawing protein scaffold. In view of the safety of the product and the nutritional characteristics of the food, it is considered that the high-temperature sterilization is more suitable for the sterilization treatment of the plant-based protein scaffold as the fat-cultured meat. (FIGS. 3A-C)

Example 3 three-dimensional plant-based cell scaffold Material and Material pretreatment suitable for fat-cultured meat

(1) Adhesion of adipose-derived stem cells to three plant-based protein scaffolds: in example 2, it has been verified that peanut wire drawing protein scaffolds can provide cell adhesion sites for adipose stem cells, and this example further discusses whether wheat wire drawing protein and soybean wire drawing protein are equally suitable for use as cell scaffolds for fat cultured meat. The specific operation is as follows: according to the procedure of example 2 (3), the adipose-derived stem cells obtained in example 1 were labeled with a living cell tracer and then were put on a wheat wire-drawing protein scaffold (Qingdao longevity food Co., ltd., filament type) and a soybean wire-drawing protein scaffold (Qingdao longevity food Co., ltd., filament type), and after three days, the adhesion of the cells on the wheat protein scaffold and the soybean protein scaffold was observed by an inverted phase contrast fluorescence microscope, and the results showed that: the cells on the peanut, wheat and soybean wire drawing protein scaffolds were all adhered to the scaffolds by grafting adipose stem cells onto the scaffolds, respectively (fig. 4A and E).

(2) Pretreatment of a plant wiredrawing protein bracket: in example 2, cells were allowed to adhere to a peanut wiredrawing protein scaffold, but it was not determined whether the scaffold could promote adhesion of cells after being coated with collagen, so that the scaffold treated in example 2 (1) was prepared with 0.5mg/mL type i collagen (Corning), soaked for four hours to obtain a soybean wiredrawing protein scaffold pretreated with collagen, a peanut wiredrawing protein scaffold pretreated with collagen, a wheat wiredrawing protein scaffold pretreated with collagen, and the corresponding wiredrawing protein scaffold not pretreated with collagen was used as a control, and cells labeled with a living cell tracer in advance were inoculated to three scaffolds according to the labeling and inoculation method of example 2 (3), and adhesion of cells to scaffolds coated with or without collagen was observed and photographed, and the results showed that: the adipose-derived stem cells can be well adhered by using the collagen pretreatment scaffold and not using the collagen pretreatment scaffold. Therefore, the invention adopts the plant wiredrawing protein as a culture bracket of the adipogenic stem cells, and the adipogenic stem cells can be well adhered to the bracket without collagen pretreatment to assist cell adhesion before cell inoculation. (FIGS. 4A-E).

Example 4 proliferation Capacity of adipose Stem cells on peanut wiredrawing protein scaffolds

(1) Cell tracer staining characterizes the proliferation capacity of adipose stem cells on peanut wiredrawing protein scaffolds: cells were inoculated onto peanut wiredrawing protein scaffolds according to the labeled live cell tracer, seeding and proliferation culture method of example 2 (3), and the proliferation and migration ability of cells on scaffolds was observed with a fluorescent inverted microscope on days 3 and 7.

(2) The proliferation capacity of the adipose-derived stem cells on the peanut wiredrawing protein scaffold is characterized by a scanning electron microscope: adipose stem cells were grown at 2.8X10 ⁴ Individual/mm ³ The density of the cells was inoculated onto a peanut wiredrawing protein scaffold, and the coverage of the cells on the scaffold and the growth state of the cells on the scaffold were observed by a scanning electron microscope according to the scanning electron microscope sample treatment method of example 2 (2) when culturing to D3 and D7.

(3) Quantitative detection of proliferation of cells on scaffolds: adipose stem cells were grown at 2.8X10 ⁴ Individual/mm ³ And (3) inoculating the peanut wiredrawing protein bracket, adding 500 mu L of 2% NaOH (NaOH and ultrapure water in mass-volume ratio) lysate into the peanut wiredrawing protein bracket when culturing to D3 and D7, cracking for 1h at 95 ℃, centrifuging at 12000rpm for 10min, taking the supernatant, and measuring the DNA concentration by using a micro-spectrophotometer.

The results show that: the staining results of the cell tracer (figure 5) and the scanning electron microscope results (figures 6A-B) both prove that the coverage rate of the cells on the peanut wiredrawing protein bracket for D3 days and D7 days is increased along with the extension of the culture time, and the cells can be fully adhered and stretched under the scanning electron microscope high-power microscope, extend out of the pseudopodia and secrete a large amount of extracellular matrix. The DNA concentration measured by a micro-spectrophotometer shows that the DNA content of the cells on the peanut wiredrawing protein bracket is obviously increased along with the extension of the culture time compared with the DNA content on the 3 rd day, and the normal proliferation of the cells on the peanut wiredrawing protein bracket is proved (figure 7).

Example 5 fat Stem cells ability to adipogenic differentiate on peanut protein scaffolds

(1) Oil red O staining detects the adipogenic differentiation capacity of adipose stem cells on peanut wiredrawing protein scaffolds: the cell-inoculated peanut wiredrawing protein scaffold of the proliferation day 7 of the example 4 is taken for induction differentiation culture, the induction differentiation day is 10 days, and the induction differentiation culture method comprises the following steps: discarding the growth medium, and continuously culturing for five days by adopting a differentiation medium, wherein the differentiation medium is as follows: 10 mug/mL insulin, 0.1mM 3-isobutyl-1-methylxanthine, 1 mug dexamethasone, 0.1mM indomethacin, 1 mug rosiglitazone and a basal medium containing 10% fetal bovine serum, wherein the basal medium is DMEM/F12 medium.

The differentiation medium was then replaced with a maintenance differentiation medium for 2 days, which was: basal medium containing 10 μg/mL insulin and 10% fetal bovine serum, said basal medium DMEM/F12 medium.

Finally, the maintenance medium was replaced with DMEM/F12 of 10% fetal bovine serum for 3 days of differentiation.

After the culture is finished, the plant wiredrawing protein bracket and the fat cells adhered to the plant wiredrawing protein bracket are the plant-based fat culture meat. The obtained vegetable-based fat-cultured meat and a blank vegetable protein scaffold were fixed with 4% paraformaldehyde at room temperature for 15min, immersed in PBS for 3 times each for 3min, and after OCT embedding of the vegetable-based fat-cultured meat, the samples were cut into 7 μm thick slices using a frozen section scale, and 500. Mu.L of the prepared oil red O dye was added. Incubation is carried out for 15min at room temperature, PBS is used for washing three times after incubation is finished, glycerinum gelatin is used for sealing the tablets, and photographing is carried out. The results show that: induced differentiation of adipose stem cells into scaffolds to day 10 after scaffold access, oil red O staining stained neutral lipid droplets on scaffolds to red, indicating that adipose stem cells can differentiate into mature adipocytes on peanut protein scaffolds (fig. 8B), whereas empty scaffolds were not stained (fig. 8A).

(2) qPCR detects the adipogenic differentiation capacity of adipose stem cells on peanut protein scaffolds: collecting plant-based cell culture meat differentiated in the step (1) on days 0, 4, 8 and 12 respectively, extracting total RNA of the plant-based cell culture meat according to the specification of a total RNA extraction kit of a root organism, and measuring the total RNA concentration by a micro spectrophotometer; the RNA was then inverted to cDNA according to the instructions of the reverse transcription kit of Norpraise, and the expression of the marker gene FABP4 of mature adipocytes was determined according to the instructions of fluorescent quantitative PCR. The results show that: the expression level of the lipid-forming key gene FABP4 increases rapidly with the extension of the culture time. (FIG. 9).

Example 6 Effect of cell seeding Density on the adipogenic differentiation ability of adipose Stem cells on peanut protein scaffolds

The example 5 demonstrates that the incorporation of adipose stem cells into peanut wire-drawing protein scaffold adipose stem cells can differentiate into mature adipocytes on peanut wire-drawing protein scaffold, but the present invention also explores the optimal cell-incorporation density in order to investigate the effect of cell-seeding density on differentiation efficiency. Respectively is provided with 10 ⁶ Individual blocks (i.e. 2.8X10) ⁴ Individual cells/mm ³ )、5×10 ⁶ Individual blocks (i.e. 1.4X10) ⁵ Individual cells/mm ³ )、10 ⁷ Individual blocks (i.e. 2.8X10) ⁵ Individual cells/mm ³ ) Three density gradients plant-based fat cultures of different cell seeding densities were prepared according to the seeding and proliferation culture method of example 2, the cell induced differentiation method of example 5 (1) (fig. 10).

(1) Plin1 immunofluorescent staining to examine the adipogenic differentiation capacity of adipose stem cells on peanut protein scaffolds at different cell seeding densities: plant-based fat culture meat induced to differentiate to day 10 was sectioned as in example 5 (1), and immunofluorescent-stained for the marker protein perilipin 1 (Plin 1) of mature adipocytes as in example 1 (3), and the results showed that: at a cell seeding density of 5X 10 ⁶ Individual blocks (i.e. 1.4X10) ⁵ Individual cells/mm ³ ) When plin1 positive cells were the largest and 5×10 ⁶ Individual blocks (i.e. 1.4X10) ⁵ Individual cells/mm ³ ) The number of group cells DAPI stained cells was high (fig. 11).

(2) Oil red O staining detects the adipogenic differentiation capacity of adipose stem cells on peanut protein scaffolds at different cell seeding densities: fixing the plant-based fat culture meat with different cell inoculation densities induced to differentiate to 10 th day with 4% paraformaldehyde at room temperature for 15min, soaking and washing with PBS for 3 times and 3min each time, embedding the plant-based fat culture meat with OCT,samples were cut into 7 μm thick slices using a frozen section scale and 500 μl of the formulated oil red O dye was added. Incubation is carried out for 15min at room temperature, PBS is used for washing three times after incubation is finished, glycerinum gelatin is used for sealing the tablets, and photographing is carried out. Oil red O staining showed that three seed densities all had adipogenesis, 5X 10 ⁶ The fat production of each block group is more than 10 ⁶ Individual/block sum 10 ⁷ A group of blocks. (FIG. 12).

(3) Western blot testing the ability of different cell seeding densities to adipogenic differentiation of adipose stem cells on peanut protein scaffolds: will 10 ⁶ Individual/block, 5×10 ⁶ Individual/block, 10 ⁷ Three densities of each/block were inoculated, proliferated and differentiated to a plant-based cell culture meat on day 10, washed three times with pre-chilled PBS, blotted with water absorbing paper to dry the residual liquid, and placed on ice; 200. Mu.L of a protease inhibitor PMSF-containing protein lysate (10. Mu.L of PMSF was added to 1mL of lysate); cracking on ice for 30min; the tissue was crushed by the freeze crusher, centrifuged at 14 000r for 15min at 4℃and the supernatant was aspirated, and the protein concentration was determined according to the BCA protein concentration determination kit instructions. Western blot detects the expression of the marker proteins FABP4 and Plin1 of mature fat, and protein gray values are analyzed by using Image J, and the result shows that: 5X 10 ⁶ The expression level of the marker proteins FABP4 and Plin1 of mature adipocytes of each/block cell inoculum size is higher than 10 ⁶ Individual/block sum 10 ⁷ The amount of expression of individual/block (FIG. 13).

(4) qPCR assay of the ability of different cell seeding densities to adipogenic differentiation of adipose stem cells on peanut protein scaffolds: the expression of the adipogenic differentiation marker gene FABP4 was examined in the same manner as in example 5 (2), and the results showed that: expression of the adipogenic differentiation marker gene FABP4 at a cell seeding density of 5X 10 ⁶ Individual blocks (i.e. 1.4X10) ⁵ Individual cells/mm ³ ) The highest expression level (FIG. 14) indicates 1.4X10 ⁵ Individual cells/mm ³ May be the optimal cell seeding density.

EXAMPLE 7 preparation of plant-based cell culture meat

(1) Plant-based cell culture meat appearance: example 6 describes an optimal cell seeding density of 5X 10 ⁶ The cells differentiated into mature adipocytes at each/block are most efficient, and thus inoculatedThe inoculation and subsequent steps are carried out by the density, and the specific method is as follows:

s1: pretreatment of plant wiredrawing proteins: rehydrating peanut wiredrawing protein (Qingdao longevity food Co., ltd., filament type) by soaking the plant wiredrawing protein in distilled water for about 5-10min, expanding the volume of the bracket prepared according to the embodiment 2 (1) by 4 times after the plant wiredrawing protein is fully absorbed and expanded, namely preparing a cube with the length and width of 1.2cm multiplied by 0.1cm, drying the cut bracket in a 60 ℃ oven for 30min, sterilizing at 121 ℃ for 15min, and soaking the peanut wiredrawing protein bracket in a growth medium under the aseptic condition overnight;

S2: the pig adipose stem cells isolated in example 1 were added to a growth medium, and the proliferation medium had the following formulation: to DMEM/F12 containing 10% (volume ratio) of fetal bovine serum added with bFGF at a concentration of 5ng/mL, 2X 10 ⁷ The adipose-derived stem cells were prepared into cell suspensions (cell suspension volume: 200. Mu.L, cell suspension concentration: 1X 10) ⁵ Individual cells/. Mu.L), the plant wire drawing protein scaffold is soaked in the cell suspension, and the cells are directly inoculated on the plant wire drawing protein scaffold, so that the cell inoculation density on the cell scaffold is 1.4X10 ⁵ Individual cells/mm ³ 。37℃5％CO ₂ Culturing for 1.5h under the condition, adding a growth culture medium, performing proliferation culture for 7 days, and changing the growth culture medium every 2 days in the proliferation culture process;

s3: when proliferation culture is carried out until the day 7, the coverage rate of proliferation culture cells on a bracket reaches 80-90%, induced differentiation culture is carried out, the induced differentiation days are 10 days, and the induced differentiation culture method comprises the following steps: discarding the growth medium, adding a differentiation medium consisting of 10 mug/mL insulin, 0.1mM 3-isobutyl-1-methylxanthine, 1 mug dexamethasone, 0.1mM indomethacin, 1 mug rosiglitazone and DMEM/F12 medium containing 10% fetal bovine serum, and inducing differentiation for 5 days; the differentiation is induced and maintained by changing a differentiation medium on the 5 th day, wherein the differentiation medium is a DMEM/F12 medium containing 10 mu g/mL and 10% fetal bovine serum, and the differentiation is maintained for 2 days; day 7 of induced differentiation, DMEM/F12 medium containing 10% fbs was changed and differentiated for 3 days. The pig fat stem cells are differentiated into mature fat cells, and the peanut wiredrawing proteins and the mature fat cells adhered to the peanut wiredrawing proteins are the plant-based fat culture meat.

The product appearance of the plant-based fat-cultured meat is directly compared with that of blank peanut wiredrawing protein cultured by proliferation and differentiation culture medium under the same conditions, and the result shows that: the appearance of the prepared plant-based fat culture meat is obviously changed compared with that of a blank bracket, the pores on the blank bracket are obvious, and the bracket surface is drier and has no luster (figure 15A); however, the pores on the scaffold were hardly seen in the plant-based cell culture meat, and the surface of the plant-based cell culture meat was glossy due to the water retention of the cells (fig. 15B).

(2) Preparation of plant-based cell culture meat nile red whole tissue staining: fixing the plant-based cell culture meat prepared in the step (1) with 4% paraformaldehyde for 15min at room temperature, discarding the fixing solution, washing with PBS for three times, adding 500 mu L of nile red staining solution, incubating for 15min at 37 ℃, discarding the staining solution, washing with PBS for three times, and photographing with an inverted fluorescence microscope, wherein the result shows that: whole tissue nile red staining showed that the plant-based cell culture meat had a large accumulation of lipid droplets, proving that the plant-based fat culture meat was produced. (FIG. 16).

Example 8 plant based cell culture meat quality

(1) Texture characteristics of plant-based cell cultures: the texture characteristics of the plant-based cell culture meat prepared in example 7 were measured, and the probe type was selected as P50; the speeds before, during and after measurement are as follows: 1.0/2.0/10.0mm/s; compression degree: 75%; the interval time was 5s. The texture characteristics A-F of the plant-based plant-cultivated meat were measured for hardness, elasticity, chewing freshness, recovery, cohesiveness, and tackiness, respectively. The results show that: the texture characteristics of the plant-based fat culture meat were measured, and the hardness, chewiness, recovery, cohesiveness, and tackiness of the plant-based fat culture meat were extremely significantly reduced and the elasticity was significantly reduced as compared with the blank plant-based cell scaffold (fig. 17).

(2) Plant-based cell culture meat flavor index: the flavor of the plant-based cell culture meat was evaluated using an electronic nose device. Cutting the plant-based culture meat sample, adding into a headspace bottle, placing into a constant-temperature water bath at 50 ℃ for 30min, and enabling volatile flavor substances in the sample to fill the sample bottle and then starting detection. Before each measurement, the baseline was calibrated by running clean air in the instrument chamber until the pressure per probe was stabilized at 1 relative to conductivity. Before measuring the sample, the balanced air pressure needle and the sampling needle are simultaneously inserted into a sample bottle, and specific parameters are set as follows: the detection time is 120s, the cleaning time is 120s, the sample injection flow is 1L/min, the preparation time is 10s, and each sample is subjected to 3 groups of parallel tests, and the groups are as follows: the pig skin tissue comprises the following components of 1 pig skin adipose tissue, 2 plant-based cell culture meat, 3 plant-based cell scaffolds and 4 pig muscle tissue. The results show that: the flavor was measured by electronic nose and PCA analysis showed that the samples of the plant-based cell culture meat were closer to the subcutaneous adipose tissue of the pig than the blank plant-based scaffolds, indicating that the flavor components of the plant-based fat culture meat were closer to the subcutaneous adipose tissue of the pig, from the point of aggregation and dispersion of the four groups of samples (FIG. 18).

(3) Plant-based cell culture meat flavor index: volatile materials of the vegetable-based fat-cultured meat were analyzed by gas phase ion mobility spectrometry (GC-IMS). The grouping is as follows: 1 is pig muscle tissue, 2 is plant-based cell scaffold, 3 is pig subcutaneous adipose tissue, 4 is plant-based fat culture meat, and four groups of samples are cut and placed into a GC-IMS sample headspace bottle. Gas chromatography conditions: type of column: FS-SE-54-CB-0.5 15m ID:0.53mm; chromatographic column temperature: 60 ℃; analysis time: 20min; carrier gas: high-purity nitrogen (purity is more than or equal to 99.999%); carrier gas flow rate: 0-2min,2ml/min;2-10min,2ml/min-20ml/min;10-20min, 15-100 ml/min. Ion mobility spectrometry conditions: type of column: FS-SE-54-CB-0.5 15m ID:0.53mm; chromatographic column temperature: 60 ℃; analysis time: 20min; carrier gas: high-purity nitrogen (purity is more than or equal to 99.999%); carrier gas flow rate: 0-2min,2ml/min;2-10min,2ml/min-20ml/min;10-20min, 15-100 ml/min. Automatic headspace sample introduction conditions: sample injection volume: 500. Mu.L; incubation time: 20min; incubation temperature: 70 ℃; the heating mode is as follows: oscillating and heating; sample injection needle temperature: 85 ℃; incubation rotation speed: 500rpm. The results show that: GC-IMS measures the flavor substances of the vegetable-based fat cultured meat, and PCA analysis shows that the contribution rate of the first main component (PC 1) is 50%, the contribution rate of the second main component (PC 2) is 21%, and the contribution rate of the two main components is 71% in a cumulative way, so that the difference of volatile substance components among four groups of samples can be well reflected. From the point of view of aggregation and dispersion, it is quite visual that the plant-based cell culture meat sample is closer to the subcutaneous adipose tissue of the pig than the blank plant-based scaffold, indicating that the flavor component of the plant-based adipose culture meat is closer to the subcutaneous adipose tissue of the pig (fig. 19).

In the present specification, each embodiment is described in a progressive manner, and each embodiment is mainly described in a different point from other embodiments, and identical and similar parts between the embodiments are all enough to refer to each other. For the device disclosed in the embodiment, since it corresponds to the method disclosed in the embodiment, the description is relatively simple, and the relevant points refer to the description of the method section.

The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. Use of plant wire drawing protein as a scaffold for in vitro culture of adipogenic stem cells, characterized in that the use comprises the steps of:

s1: pretreatment of a plant wiredrawing protein bracket: sterilizing the plant wiredrawing protein, and soaking the plant wiredrawing protein in a growth medium under a sterile condition to obtain a plant wiredrawing protein bracket;

s3: performing proliferation culture until the coverage rate of cells on a bracket reaches 80-90%, and performing induced differentiation culture until the adipogenic stem cells are differentiated into mature adipocytes;

the adipogenic stem cells are stem cells capable of differentiating towards adipogenic lineage cells, the adipogenic stem cells being derived from fat;

s1, sterilizing at high temperature;

the growth medium is a basic medium added with bFGF with the concentration of 5ng/mL and 10% fetal bovine serum, and the basic medium is one of DMEM medium, MEM medium, DMEM/F12 medium and F10 medium;

the induced differentiation culture method of S3 is as follows:

the basal medium is selected from one of DMEM medium, MEM medium, DMEM/F12 medium and F10 medium;

the plant wiredrawing protein is peanut wiredrawing protein, soybean wiredrawing protein or wheat wiredrawing protein.

2. The use according to claim 1, wherein the high temperature sterilization is sterilization for 10-20 min at 120-130 ℃ after drying the plant wire drawing proteins.

3. The use according to claim 2, wherein the high temperature sterilization is a sterilization at 121 ℃ for 15min after drying the plant wire drawing proteins.

4. The use according to claim 1, wherein the concentration of the cell suspension of S2 is 5 x 10 ⁴ ~5×10 ⁵ Individual cells/. Mu.L.

5. According toThe use according to claim 4, wherein the concentration of the cell suspension of S2 is 1X 10 ⁵ Individual cells/. Mu.L.

6. The use according to claim 1, wherein the cell seeding density on the plant wire drawing protein scaffold in S2 is 2 x 10 ⁴ Individual cells/mm ³ ~3×10 ⁵ Individual cells/mm ³ 。

7. The use according to claim 6, wherein the cell seeding density on the plant wire drawing protein scaffold in S2 is 1.4X10 ⁵ Individual cells/mm ³ 。

8. A method for preparing vegetable-based fat-cultured meat, the method comprising the steps of:

s1: pretreatment of a plant wiredrawing protein bracket: rehydrating and sterilizing the plant wiredrawing protein, and soaking the plant wiredrawing protein in a growth medium under a sterile condition to obtain a plant wiredrawing protein bracket;

s2: adding the adipogenic stem cells into a growth medium to prepare a cell suspension, and directly inoculating the cell suspension onto a plant wiredrawing protein bracket for proliferation culture;

s3: when the coverage rate of proliferation culture cells on a bracket reaches 80-90%, performing induced differentiation culture until the adipogenic stem cells are differentiated into mature fat cells, wherein the plant wire drawing protein bracket and the mature fat cells adhered to the plant wire drawing protein bracket are the plant-based fat culture meat;

s1, sterilizing at high temperature;

The induced differentiation culture method of S3 is as follows:

the basic culture medium is selected from one of DMEM culture medium, MEM culture medium, DMEM/F12 culture medium and F10 culture medium

9. The method of claim 8, wherein the high temperature sterilization is performed by drying the plant wire drawing protein and then sterilizing at 120-130 ℃ for 10-20 min.

10. The method of claim 9, wherein the high temperature sterilization is a sterilization at 121 ℃ for 15min after drying the plant wire drawing proteins.

11. The method of claim 8, wherein the concentration of the cell suspension of S2 is 5 x 10 ⁴ ~5×10 ⁵ Individual cells/. Mu.L.

12. The method of claim 11, wherein the concentration of the cell suspension of S2 is 1X 10 ⁵ Individual cells/. Mu.L.

13. The method of claim 8, wherein the seeding density of cells on the plant wire drawing protein scaffold in S2 is 2 x 10 ⁴ Individual cells/mm ³ ~3×10 ⁵ Individual cells/mm ³ 。

14. The method of claim 13, wherein the cell seeding density on the plant wire drawing protein scaffold in S2 is 1.4 x 10 ⁵ Individual cells/mm ³ 。

15. The use according to claim 1 or the method according to claim 8, wherein in S1 the plant wire drawing proteins are first absorbed in pure water to a complete expansion, cut to the desired size and sterilized.

16. A vegetable-based fat-cultured meat produced by the method of claim 8.