Streamlined alpha-amylase assays for wheat preharvest sprouting and late maturity alpha-amylase detection

Late maturity alpha-amylase (LMA) and preharvest sprouting (PHS) lead to elevated alpha-amylase in wheat (Triticum aestivum L.) grain. Risk of poor end-product quality due to elevated alpha-amylase is detected in the wheat industry using the Hagberg-Perten falling number (FN) method. In breeding programs, selection for PHS and LMA tolerance requires higher throughput methods requiring a smaller sample size than the 7 g required for the FN method. Specifically, LMA can only be screened only using detection of alpha-amylase activity or protein after cold treatment of individual wheat spikes at a specific stage of grain development resulting in very small samples (<= 1 g). This study developed and evaluated a high throughput 96-well method for the Phadebas alpha-amylase enzyme assay for small wheat grain samples and compared this method to FN and the Megazyme Alpha-Amylase SD (Sprout Damage) Assay Kit performed on the automated Awareness Technology ChemWell-T Analyzer. In parallel, the efficacy of low-cost small-scale milling methods was evaluated relative to traditional larger scale mills. The Phadebas enzyme activity was highly reproducible and showed a strong correlation to the SD enzyme assay and FN method regardless of which mill was used to process the grain. The SD assay offers simpler standardization and calculation of enzyme activity, whereas the Phadebas assay offers higher sensitivity and lower expense. Both the 96-well Phadebas and automated Megazyme SD assays are suitable for alpha-amylase detection from small samples, and the use of low-cost coffee grinders to process small samples did not significantly impact assay performance.