Abstract:[Objective]The effects of different extraction methods on the yield, purity, structural characteristics, and physicochemical properties of tortoise shell collagen were investigated.[Method]The ultrasound-assisted enzymatic method, acid method, and enzymatic method were used to extract collagen from tortoise shell powder.[Result]The yield and purity of tortoise shell collagen extracted by ultrasound-assisted enzymatic method were 2.29% and 85.65%, respectively, significantly higher than those extracted by acid method (0.78% and 73.15%) and enzymatic method (1.77% and 75.63%). Ultraviolet absorption spectroscopy, infrared absorption spectroscopy, circular dichroism spectroscopy, and electrophoresis profiles showed that collagens extracted from tortoise shell by three methods were all type I collagen and retained intact triple helix structures. X-ray diffraction patterns revealed that the molecular structure of tortoise shell collagen treated with ultrasound-assisted enzymatic method was more compact, with a higher degree of order. The particle sizes of tortoise shell collagens extracted by ultrasound-assisted enzymatic method and enzymatic method were both distributed around 340 nm, while tortoise shell collagen extracted by ultrasound-assisted enzymatic method exhibited a lower particle size distribution. The thermal denaturation temperatures (Td) of the tortoise shell collagens extracted by the three methods were all 44 ℃. Scanning electron microscopy results showed that the microstructure of tortoise shell collagen extracted by ultrasound-assisted enzymatic method was more uniform and regular.[Conclusion]The combination of ultrasound treatment and enzymatic methods provide a new perspective for the extraction of collagen from tortoise shell. Compared with the conventional acid method and enzymatic method, the ultrasound-assisted enzymatic extraction method shows improvements in the yield and purity of tortoise shell collagen, thus demonstrating a broad application prospect.