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Laccase was immobilized on a chitosan/polyvinyl alcohol/tetraethylorthosilicate electrospun film (ceCPTL) and colored with guaiacol to obtain a laccase time-temperature indicator (TTI) prototype. The activation energy ( Ea ) of coloration of the prototype was 50.89-33.62 kJ/mol when 8-25 μg/cm 2 laccase was immobilized on ceCPTL, and that of lactic acid bacteria (LAB) growth in milk was 73.32 kJ/mol. The Ea of coloration of the TTI prototype onto which 8-10 μg/cm 2 laccase was immobilized was in the required range for predicting LAB growth in milk. The coloration endpoint of the TTI prototype onto which 10 μg/cm 2 (0.01 U) laccase was immobilized could respond to the LAB count reaching 10 6 colony-forming units (CFU)/mL in milk during a static temperature response test, and the prediction error was discovered to be low. In dynamic temperature response experiments with intermittent temperature changes between 4 and 25 °C, the coloration rate of the laccase TTI prototype was consistent with LAB growth. The results of this study indicate that the laccase TTI prototype can be applied as a visual monitoring indicator to assist in evaluating milk quality in cold chains.

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Enzymatic Time-Temperature Indicator Prototype Developed by Immobilizing Laccase on Electrospun Fibers to Predict Lactic Acid Bacterial Growth in Milk during Storage