Thermal Stability of r-Amylase from Aspergillus oryzae Entrapped in Polyacrylamide Gel PATCHARIN RAVIYAN, † JUMING TANG, § AND BARBARA A. RASCO* ,# College of Agriculture, Chiangmai University, Chiangmai, Thailand; Department of Biological Systems Engineering, Washington State University, Box 646120, Pullman, Washington 99164-6120; and Department of Food Science and Human Nutrition, Washington State University, Box 646376, Pullman, Washington 99164-6376 To determine the suitability as a time-temperature indicator for dielectric pasteurization processes, the thermal stability (50-75 °C) of Aspergillus oryzae R-amylase immobilized in polyacrylamide gel in phosphate buffer, mashed potatoes, and minced shrimp was examined. Changing the cross-linking agent concentration from 3.3 to 5.3% and adding 2% salt did not markedly affect the thermal stability of the immobilized R-amylase. Thermal inactivation was first order, and immobilization generally improved the thermal stability of R-amylase. z values of the immobilized system in test food systems were 10.2 °C (phosphate buffer), 8.45 °C (minced shrimp), and 7.78 °C (mashed potatoes). KEYWORDS: r-Amylase; inactivation kinetics; polyacrylamide gel; time-temperature indicator; pas- teurization INTRODUCTION Microbial testing is a reference method for monitoring process lethality with microbes having z values from 5 to 12 °C useful for validating pasteurization processes (1). Microbial testing is often a time-consuming, burdensome, and costly proposition for food processors (2, 3). This has prompted the development of enzyme-based time-temperature indicators (TTIs) to monitor process lethality. Ideally, the thermal stability of a TTI should be somewhat higher than that of the target pathogen under the same experimental conditions; in this way residual enzyme activity can be detected following heat treatment. R-Amylases (R-1,4-R-D-glucan glucanohydrolase, EC 3.2.1.1) are of par- ticular interest because these enzymes are inexpensive and commercially available, and the assay is fast, simple, and inexpensive. Inactivation of free R-amylase may is generally first order (1, 4-6). End-point assays of endogenous enzyme activity have been used to determine the adequacy of a cooking process but are poor candidates for the quantitative monitoring of thermal inactivation (7-14). Use of exogenous enzyme-based TTIs shows more promise, but there has been little research conducted in this area, particularly in food systems. Inactivation of horseradish peroxidase (15-17) or R-amylase (4, 5) im- mobilized on glass beads has been studied, but not in foods. Entrapped B. amyloliquefaciens R-amylase in silicone particles or stainless steel capsules (1) has been studied in limited food applications. For TTIs in pasteurization processes (18), specif- ically those involving microwave or radio frequency (RF) heating, a TTI with dielectric properties that match the food would be important so that uniform coupling of microwave or RF radiation can be assured. A TTI based upon Aspergillus oryzae R-amylase immobilized in polyacrylamide gel provides a number of advantages over current designs, including ease of preparation, physical durabil- ity, easy separation of the gel from foods, a fast, simple, and inexpensive assay, and the simplicity of adjusting the sensitivity of enzyme assay. The objectives of this study were (1) to determine the temperature optima and thermal stability of free and immobilized R-amylase entrapped in polyacrylamide gel and (2) to determine inactivation kinetic parameters for free and immobilized R-amyl- ase in buffer solution and two food systems, minced shrimp and mashed potatoes. MATERIALS AND METHODS Enzyme Assay. A. oryzae R-amylase (EC 3.2.1.1) (Sigma Aldrich Co., St. Louis, MO) with a specific activity of 39 units/mg of solid or 185 units/mg of protein (by biuret method) was used. The 5 DE maltrodextrin substrate [Maltrin M040, Grain Processing Corp. (Muscatine, IA)] was prepared as described by Strumeyer (19). To block reducing ends, 5 g of maltodextrin was dispersed into 20 mL of distilled water and heated for 20-30 s until completely dissolved. The suspension was then diluted to 90 mL with distilled water and cooled in an ice bath. Then 10 mL of cold sodium borohydride (0.15 g of NaBH 4/10 mL) was added and stirred using a magnetic stirrer for 2 min. The solution was stored under refrigeration overnight before use and is stable for several days. * Corresponding author [telephone (509) 335-1858; fax (509) 335-4815; e-mail rasco@mail.wsu.edu]. † Chiangmai University. § Department of Biological Systems Engineering, Washington State University. # Department of Food Science and Human Nutrition, Washington State University. 5462 J. Agric. Food Chem. 2003, 51, 5462-5466 10.1021/jf020906j CCC: $25.00 © 2003 American Chemical Society Published on Web 08/05/2003