Collaborative Study of Detecting Method for Foreign Amylase in Honey using Native Polyacrylamide Gel Electrophoresis-Activity Staining

The Codex Standard for Honey adopts diastase activity as an indicator of honey freshness and as a measure to detect overheating during the manufacturing process. However, in some cases a foreign amylase is added to honey to increase its diastase activity. In our previous study, we developed a simple and sensitive method for screening foreign amylases using native polyacrylamide gel electrophoresis followed by activity staining. In this study, to confirm the effectiveness of this method, a collaborative study involving 12 laboratories was conducted in accordance with the AOAC guidelines for qualitative analysis, targeting amylases derived from the Aspergillus and Geobacillus, which were frequently detected in Japan. From the probability of detection (POD) curve confirmed in the preliminary test, three concentrations (low, medium, and high) calculated as diastase numbers were set, and eight samples of each concentration (total of 24 samples) were distributed in a blind manner and evaluated. The results showed that while the POD at the middle level was higher than expected, the method demonstrated sufficient performance to determine the presence or absence of foreign amylase.