ACE-inhibitory activity is measured by fluorescence using the method of Sentandreu & Toldrá (2006) with some modifications (Coscueta, Campos, Osório, Nerli, & Pintado, 2019). A total of 40 µL of ultrapure water or ACE working solution are added to each microtiter-plate well, then adjusted to 80 µL by adding ultrapure water to blank, control or samples. For direct or 1/2 diluted samples a sample blank is also made. The enzyme reaction is started by adding 160 µL of substrate solution and the mixture is incubated at 37 °C. Serial dilutions are made of each sample, usually 1/1 to 1/32. The fluorescence generated is measured at 30 min using a Multidetection plate reader (Synergy H1, Vermont, USA). The assay is performed in a black 96-well microplate (Nunc, Denmark). Excitation and emission wavelengths are 350 and 420 nm, respectively.
Note: Generally the most concentrated dilutions are the ones that can fluoresce as the sample itself, so in the example sample blanks (SPLB) are used only for the two most concentrated dilutions. In the case of highly fluorescent samples, SPLB should be used for all dilutions.