Key Technology Research Of Cell Cultured Meat Production By Gellan Gum-Gelatin-Based 3D Edible Scaffold

Cell cultured meat is a new meat production technology to alleviate the shortage of protein resources and has advantages in terms of food safety issues and achieving precise regulation of nutrition.3D scaffolds can provide mechanical support for cells to be distributed in three dimensions on the scaffold,thus proliferating and differentiating to form complete muscle tissue,which has great potential for application in the field of cell cultured meat.Gellan gum is an anionic polysaccharide that gels spontaneously at room temperature and becomes a thermally irreversible gel when crosslinked with cations.Gelatin is a hydrolysis product of collagen that retains its natural cell binding sequence;however,gelatin can only gel at temperatures below physiological,limiting its application in cell culture.In this study,a 3D edible scaffold for cell cultured meat was prepared using gellan gum and gelatin as substrates.Using chicken skeletal muscle satellite cells as a cell source for animal tissue culture,3D culture of cells was performed on a suitable scaffold for cell culture meat production.Details and the results of the study are as follows:(1)The effects of gelatin concentration and calcium ion crosslinking on the swelling ratio,degradation ratio and microstructure of the scaffolds were investigated.The scaffolds showed high water absorption,high water holding and low degradation ratio,with interconnected porous lamellar stacking structures,which were suitable for cell culture.In addition,fourier transform infrared spectroscopy results showed the existence of hydrogen bonding and electrostatic interactions between gellan gum and gelatin,demonstrating the successful crosslinking of gellan gum with calcium ions.(2)Cell adhesion ability and biocompatibility of different scaffolds were investigated using C2C12 mouse myogenic cells.The results of live/dead cell staining and cytoskeleton staining showed that the prepared scaffolds had higher biocompatibility and cell adhesion ability when the gelatin concentration was 0.5%(w/v),the gellan gum to gelatin ratio was 2:3,and the calcium chloride concentration was 0.180 M.The scaffolds prepared under these conditions had a swelling ratio of 2278.57±133.63% and a degradation ratio of 28.7±4.1%after 2 weeks,and C2C12 cells were able to proliferate and fuse and differentiate to form myotubes on the scaffolds.(3)The proliferation and differentiation characteristics of chicken skeletal muscle satellite cells under 2D culture conditions and on scaffolds were investigated.When chicken skeletal muscle satellite cells were seeded on the screened optimal scaffolds,chicken skeletal muscle satellite cells showed gradually increasing metabolic activity during the proliferative phase with elevated DNA content and were able to perform normal physiological activities and enter the cell cycle.The results of immunofluorescence staining,western blot,and RT-q PCR showed that chicken skeletal muscle satellite cells formed a complete cytoskeleton during the differentiation phase,cells fused with each other and formed myotube-associated proteins,and scanning electron microscopy observed the formation of a large number of parallel-arranged myofibers on the scaffold.(4)After culturing the cells,the scaffolds became compact in terms of appearance and their dry weights increased significantly.The results of colony analysis showed that the scaffolds were sterile before and after culturing the cells.The textural properties of the scaffolds were significantly improved after culturing the cells and had a similar texture to chicken leg meat,which can be used for the research and production of cell cultured meat.