Improvement of Fish Freshness Determination Method by the Application of Amorphous Freeze-Dried Enzymes

Alkaline phosphatase (ALP), nucleoside phosphorylase (NP), and xanthine oxidase (XOD) were used in a calorimetric method for evaluation of fish freshness based on the K-i value. Two enzyme mixtures, NP-XOD and ALP-NP-XOD, were prepared with a color developing agent, and stabilities of the enzymes were improved by freeze-drying with glass-forming additives, i.e., sucrose and sucrose-gelatin. As a result, a linear relationship was obtained between the K-i values determined by the developed colorimetric method and a conventional high-performance liquid chromatography with a high correlation coefficient of 0.997. All enzyme samples containing the additive(s) were amorphous, and higher enzymes activities were maintained compared to those freeze-dried without an additive. Sucrose-gelatin/enzyme mixtures showed higher glass transition temperature; consequently, the enzymes were better stabilized than the sucrose/enzyme formulations. Using the sucrose-gelatin/enzyme mixture, K-i values of fish meat could be accurately determined even after 6-month storage of the dried enzymes at 40 degrees C.