Alpha-amylase/subtilisin inhibitor levels in Australian barleys

Abstract

An enzyme-linked immunosorbent assay (ELISA) was used to determine the bifunctional alpha-amylase/subtilisin inhibitor (BASI) content of barley grain from 11 cultivars grown in six diverse locations in Australia. The inhibitor ranged from 119 to 254 μg/g in 57 barley samples. Genotype had a significant (P<0·05) effect on BASI content but there was no effect due to environment. Total protein varied independently of BASI and was influenced by environment and genotype. BASI content was higher (P<0·05) in malting barley than in feed barley and was correlated positively (r=0·29;P<0·05) with alpha-amylase activity in corresponding malts. The ELISA used monoclonal and polyclonal antibodies raised against purified BASI. In immunoblot analysis the monoclonal antibody showed high specificity for the inhibitor in barley and also detected the inhibitor in wheat. Low levels of inhibitor (mean 3·2 μg/g) were found in 12 Australian wheat cultivars using the ELISA developed for barley. The assay had a linear working range of 5–50 ng/mL with a detection limit of 2 ng/mL. Reproducibility between assays was good (CV=4·9%) but mean recoveries were high, ranging from 116–129% when purified inhibitor was added to barley extracts. The ELISA may have useful applications in brewing research and barley breeding programmes.