Abstract: Objective: To develop a time-temperature indicator (TTI) with amylase nanoflowers based on protein hybrid nanotechnology and enzyme reaction and to determine the activation energy for TTI and its suitability for different food types. Methods: Based on the interaction between amylase and copper ions, amylase was immobilized to form amylase@Cu nanoflowers. The amylase concentration for nanoflowers with the best shape was selected, and the biological activity and stability of amylase in the nanoflowers were tested. Six formulations of TTI were prepared by adding 10, 20, 30, 40, 50 mg and 60 mg of amylase@Cu nanoflowers and using 15 mL of 40 g/L soluble starch solution as the substrate and 4.5 mL of 1 g/L iodine solution as the indicator. At constant temperatures of 5, 15, 25 and 35 ℃, the discoloration process was observed, the kinetic parameters were measured, and the activation energy for the reaction was calculated. Results: When the amylase concentration was 0.5 mg/mL, the flower-like shape of amylase@Cu nanoflower was the most complete, the amylase activity increased by 3.42 times, and the storage stability was significantly improved. The six TTIs showed a change process from dark blue to colorless, and the activation energy values were 14.84, 21.00, 28.85, 33.03, 32.55 and 32.83 kJ/mol, respectively. Conclusion: The amylase nanoflower-based TTI was simple to operate and had good indication performance. According to the matching principle between TTIs and foods, this series of TTIs were suitable for monitoring food deterioration and spoilage caused by diffusion control, enzymatic reaction, fat oxidation or other reasons.

Key words: time-temperature indicator; amylase; nanoflowers; absorbance; activation energy