Water availability affects extracellular hydrolytic enzyme production by Aspergillus flavus and Aspergillus parasiticus

Abstract

The objectives of this study were to examine the effect of different water activities (a_w; 0.99, 0.96 and 0.94) and time (up to 120 h) on quantitative and specific enzyme production during germination and initial growth of Aspergillus flavus and A. parasiticus strains at 25 °C. This is an important early indicator of potential for aflatoxin production under conducive conditions. Qualitative API ZYM generic enzyme strips were used to identify key hydrolytic enzymes produced. Subsequently, the temporal effects of a_w on the total/specific activity of the key 4-5 hydrolytic enzymes were determined using 4-nitrophenyl substrates in a 96-well microtitre plate assay. The main enzymes produced by germinating conidia of A. flavus were esterase, lipase, acid phosphatase, β-glucosidase and N-acetyl-β-D-glucosaminidase, while for A. parasiticus these were alkaline phosphatase, lipase, acid phosphatase and β-fucosidase for both total (µmol 4-nitrophenol/min/g) and specific activity (nmol 4-nitrophenol/min/µg protein). There were significant increases in the specific activity of all these enzymes of germinating spores of A. flavus (0-120 h) except for β-glucosidase which was maximum at 72 h. The total/specific activities of the enzymes produced by A. flavus were maximum at 0.99 a_w, with the exception of acid phosphatase and N-acetyl-β-D-glucosaminidase at 0.94 a_w. For A. parasiticus, maximum total activity occurred at 0.99 a_w for fucosidase activity, while specific activity was found to be higher at lower a_w levels. These enzymes are important in early colonisation of food matrices by these species and single factors (a_w, time) and two-way interactions were all statistically significant for the enzymes assayed for both species. These enzymes could be used as an early and rapid indicator of the activity of Aspergillus section flavi species and suggests that rapid infection may occur over a wide range of a_w conditions.