cc_byReichenberger, KatrinLutz-Wahl, SabineKettner, LucasFischer, Lutz2025-08-202025-08-202025https://doi.org/10.1007/s12161-025-02781-3https://hohpublica.uni-hohenheim.de/handle/123456789/17949An extremely sensitive amylase activity assay was developed using the natural substrate starch and two ancillary enzymes: a glucose oxidase (GOD) and a peroxidase, to measure the residual activity of the α -amylase from Bacillus subtilis in white bread. Firstly, the concentrations of the assay components: electron acceptor DA-67 (50 μM), horseradish peroxidase (681 nkat mL −1 ), a GOD from Aspergillus niger (1550 nkat mL −1 ) and the natural substrate starch (0.01% (w/v)), were optimized to achieve high sensitivity. The linearity of the assay was then tested with both an endo- ( α -amylase from B. subtilis ) and exo-acting amylase (maltogenic amylase from Geobacillus stearothermophilus ), and the effect of the incubation time on the assay sensitivity was investigated and optimized. The optimized assay was capable of determining a minimal amylase activity of 0.33 pkat mL −1 for both amylases tested with an assay run time of 7.5 h. This new DA-67 amylase assay demonstrated 4.7- and 4.2-times higher sensitivity, respectively, compared to optimized versions of the commercial Ceralpha (determination of endo-amylase activities) and Betamyl3 (determination of exo-amylase activities) assays. The new DA-67 amylase assay was used to determine the residual activity of α -amylase from B. subtilis in white bread. A consistent residual activity of 2.26 ± 0.15% was reliably determined.engAmylase assayα-amylaseThermal stabilityResidual activityBread bakingEnzyme declarationFood enzyme660Article2025-07-18