Abstract: Constructing an edible extracellular matrix (ECM) scaffold with good thermal stability, self-assembly properties and biocompatibility is crucial for manufacturing structured cell cultured meat products. In this study, carboxymethyl chitosan (CMCS) was introduced into the bovine bone collagen (BBC) system. Using ultraviolet (UV) absorption, infrared (IR) and fluorescence spectroscopy, it was found that the interaction between BBC and CMCS was enhanced with the increase in CMCS concentration, without affecting the triple-helical structure. The results of differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA) showed that the introduction of CMCS reinforced the thermal stability of BBC. Turbidity test, scanning electron microscopy (SEM) and transmission electron microscopy (TEM) confirmed an increase in the degree of fibrillogenesis and aggregation behavior and changes in self-assembly rate; a looser and twisted three-dimensional structure with a larger fibril diameter and a wider diameter distribution was observed after the introduction of CMCS. However, the introduction of CMCS did not significantly affect the formation and length of D-periodicity (a characteristic alternating light/dark transverse stripe structure formed during the self-assembly process of collagen fibers) in BBC, and there was no significant difference in the cell compatibility of the system before and after the introduction of CMCS. The BBC-CMCS interaction might be dominated more by electrostatic forces than covalent interactions and hydrogen bonds with the increase in CMCS concentration. These results indicated that the introduction of CMCS improved the thermal stability and self-assembly properties of BBC without affecting its biocompatibility or triple-helical integrity. This study provides a reference for the development of excellent edible collagen-based ECM as a biomimetic scaffold for cultured meat and for the deep processing and high-value utilization of livestock and poultry bone by-products.

Key words: cell cultured meat; extracellular matrix; bovine bone collagen; carboxymethyl chitosan; microstructure; thermal stability; self-assembly