Application of laccase-inorganic nanoflowers based time-temperature integrator to real-time monitor the freshness of pasteurized milk

A time-temperature integrator (TTI) based on laccase@Cu₃(PO4)₂ nanoflowers (laccase@NFs) was created to monitor the freshness of pasteurized milk during storage. To address the challenges of easy inactivation, poor stability, and high use-cost of laccase in the application of TTI, laccase@NFs were synthesized by ultrasonic-assisted biomineralization. The laccase@NFs-based TTI was created through the enzymatic reaction between laccase and 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid ammonium salt), and the effects of laccase@NFs addition amount on the reaction rate, discoloration lifetime and activation energy (Ea) were discussed. Furthermore, the spoilage pattern and kinetic properties of pasteurized milk were explored based on titratable acidity. The measurement of the Ea was determined as 49.98 kJ/mol, and an investigation was conducted to assess the suitability of the TTI with pasteurized milk under both constant and variable temperature conditions. This research aims to contribute valuable insights into the application of enzymatic TTI in monitoring the shelf life of pasteurized milk.