Search

CN-120944812-B - Block cell culture meat and preparation method thereof

CN120944812BCN 120944812 BCN120944812 BCN 120944812BCN-120944812-B

Abstract

The invention provides a massive cell culture meat and a preparation method thereof. According to the invention, the requirement of massive cell culture meat on large-scale preparation of seed cells is met through large-scale culture of suspension cells, and adhesion, proliferation and integration of the seed cells on an edible bracket material are realized through induction of suspension cell adhesion by a specific culture medium. The method can ensure that the adherence rate of cells and the scaffold material reaches 96.8% by aseptically putting the scaffold material in the stirred tank cell bioreactor and matching with a specific induction culture solution and a stirring process. The method adopts the suspension cells as seed cells and combines the corresponding process and culture medium, thereby effectively solving the difficult problems that the seed cells are difficult to prepare on a large scale and difficult to be integrated with the bracket materials effectively in the process of culturing the meat by the massive cells, and providing an expandable scheme for the industrialized production of the meat by the massive cell culture.

Inventors

  • BAO LIHUI
  • QU YAPING
  • ZHOU FENG
  • WANG WEIHUA

Assignees

  • 未来肉膳健康科技(威海)有限公司

Dates

Publication Date
20260512
Application Date
20250703

Claims (8)

  1. 1. A method for preparing a bulk cell culture meat, comprising the steps of: Inoculating the suspension cells into a No.1 culture medium for proliferation culture, and obtaining the cells after proliferation culture when the cell density reaches a peak value, wherein the suspension cells are chicken embryo fibroblast lines; centrifuging the cells after proliferation culture, collecting cell sediment, and re-suspending the cell sediment by using a No. 2 culture medium to obtain cell suspension; inoculating the cell suspension on a gelatin modified plant wire drawing protein bracket, and sequentially carrying out variable speed culture and constant speed culture after supplementing a No.2 culture medium to obtain massive cell culture meat; Wherein the formula of the culture medium No. 1 comprises a basic culture medium, 8-12% v/v of fetal bovine serum, 23-25 ug/mL of anti-cell-agglomerating agent and 0.8-1 mg/mL of poloxamer 188; The formula of the No. 2 culture medium comprises 8-12% v/v of basal culture medium, 95-100 ug/mL of astragalus polysaccharide, 2-2.5 mg/mL of inulin, 166.12-176.12 ug/mL of vitamin C, 0.2-0.5 ug/mL of selenium-enriched yeast and 0.0106-0.0116 ug/mL of insulin.
  2. 2. The method of claim 1, wherein the cell suspension density is 1E6-5E6 cells/mL.
  3. 3. The method for producing a bulk cell culture meat according to claim 1, wherein in the centrifugation, the rotation speed is 800 to 1000rpm for 5 to 8 minutes; And/or the basal medium comprises any one of DMEM medium, DMEM/F12 medium and MEM medium.
  4. 4. The method of preparing bulk cell culture meat according to claim 1, wherein the gelatin modified plant wire drawing protein scaffold is obtained by a preparation method comprising the steps of: Crosslinking the plant wiredrawing protein by using gelatin, and sterilizing to obtain the initial gelatin modified plant wiredrawing protein; soaking the initial gelatin modified plant wiredrawing protein in sterile water, fully absorbing water, and expanding to obtain the expanded gelatin modified plant wiredrawing protein; Soaking the expanded gelatin modified plant wiredrawing protein by using a No. 2 culture medium to obtain a gelatin modified plant wiredrawing protein bracket; Wherein the culture medium No.2 is the culture medium No.2 according to claim 1.
  5. 5. The method for producing a bulk cell culture meat according to claim 4, wherein the soaking treatment is performed at a temperature of 38 to 39 ℃ for 20 to 30 minutes; and/or the plant wire drawing protein is any one of soybean wire drawing protein, peanut wire drawing protein and wheat wire drawing protein.
  6. 6. The method for producing a bulk cell culture meat according to claim 1, wherein the amount of the suspension cells to be inoculated is 2E5-3E5 cells/mL.
  7. 7. The method for preparing a bulk cell culture meat according to claim 1, wherein the step of inoculating the suspension cells into a culture medium No.1 for proliferation culture, and obtaining the cells after proliferation culture when the cell density reaches a peak value, specifically comprises: Inoculating the suspension cells into a No. 1 culture medium, performing proliferation culture for 4-6 days under the conditions of 110-120 rpm,38-39 ℃ and pH 7.2-7.4,50% -60% dissolved oxygen, changing the No. 1 culture medium every 1-2 days during the proliferation culture, and obtaining the cells after proliferation culture when the cell density reaches the peak value.
  8. 8. The method for preparing the massive cell culture meat according to claim 1, wherein the cell suspension is subjected to constant-speed culture after the surface of the gelatin-modified plant wiredrawing protein scaffold is attached to the surface, so as to obtain the massive cell culture meat, and the method specifically comprises the following steps: After the cells are adhered to the surface of the gelatin-modified plant wiredrawing protein bracket, the cells are subjected to constant-speed culture for 7-10 days under the conditions of 90-110 rpm,38-39 ℃ and pH=7.2-7.4,50-60% dissolved oxygen, and a No.2 culture medium is replaced every 1-2 days during the constant-speed culture period, so that the massive cell culture meat is obtained.

Description

Block cell culture meat and preparation method thereof Technical Field The invention relates to the technical field of cell culture, in particular to a massive cell culture meat and a preparation method thereof. Background As the global population increases in protein demand, the traditional animal industry scale has shown a rapid trend, and the development of animal industry has led to a series of environmental problems and resource pressures. Cell culture meat is an environment-friendly and sustainable protein production mode by utilizing animal cells to produce proteins in a controlled environment. At present, the production mode of cell culture meat is mainly in the form of microparticle tissue of culture single cells or cell composite materials, the harvested cell meat is often loose meat emulsion without texture, the texture and texture of animal meat are difficult to reproduce in the production mode, and the cell meat is required to be mixed with plant-based components to be shaped to be processed into various meat substitutes. The prior culture technology is difficult to reproduce the complex structure of natural muscles, so that most of culture products are in the form of crushed meat, and the requirements of the market on the form and taste of blocky meat (such as steak and chicken breast meat) cannot be met. The blocky cell culture meat is prepared by inoculating cells in a bracket material, adhering and proliferating the cells in the bracket material and completing matrix remodeling (integration), and finally the blocky meat with animal meat texture and structure is formed, which represents the trend and direction of the next development of the cell culture meat, but the prior art for preparing the blocky cell culture meat has obvious technical bottlenecks (1) the large-scale preparation of seed cells is difficult. At present, the meat seed cells are cultured by using the block cells, the production of the block cells depends on plate culture and microcarrier culture, the plate culture has the defects of large occupied area, complex operation, high labor cost, difficult scale and the like, and in microcarrier culture, the separation and centrifugation process of the cells and microcarriers increases the problems of process complexity, quality consistency challenges and the like. (2) efficient integration of seed cells with scaffold materials is difficult. At present, a cell and material integration mode based on a tissue engineering construction strategy mainly comprises a static inoculation method and cell 3D printing. The static inoculation method is subject to the restriction of uneven cell distribution, difficult amplification and the like, and the cell 3D printing is subject to the problems of low efficiency, high equipment cost and the like. Therefore, the problem that seed cells are difficult to prepare on a large scale and difficult to effectively integrate with a bracket material in the process of culturing the massive cell cultured meat is a key for promoting the establishment of the massive cell cultured meat industrial production technology. Disclosure of Invention The invention provides a preparation method of a massive cell culture meat, which comprises the steps of controlling interaction and signal transduction among cells by adjusting culture medium components and optimizing culture process when proliferation of suspension cells reaches a density peak value, inducing cells to cling to a bracket material (a gelatin-modified plant wiredrawing protein bracket), and then carrying out proliferation and migration activities of the cells and secreting extracellular matrix (ECM), so as to culture the massive cell culture meat which is close to the structure and the texture of real meat. The method adopts the suspension cells as seed cells, is suitable for industrialized mass proliferation culture, can effectively improve the production efficiency and scale, meets the requirement of cell culture meat on mass preparation of the seed cells, realizes high-efficiency integration of the seed cells and a bracket material, has a cell adherence rate as high as 96.8%, effectively solves the problems that the seed cells are difficult to prepare on a large scale and difficult to integrate with the bracket material in the process of culturing the massive cell culture meat, and provides a new path for mass production of the massive cell culture meat. The invention also provides the massive cell culture meat, which is prepared by the preparation method of the massive cell culture meat. The inventor researches show that the massive cell culture meat can keep good morphological structure. The first aspect of the present invention provides a method for preparing bulk cell culture meat, comprising the steps of: Inoculating the suspension cells into a No. 1 culture medium for proliferation culture, and obtaining the cells after proliferation culture when the cell density reaches a peak value; centrifuging