Protease Activity and Degree of Hydrolysis

Protease activity was determined in dry seeds and germinated legume flours. Protein extracts were obtained by an incubation of 200 mg of flour in 1 ml of double distilled water at 25°C and 1,500 rpm of agitation speed for 60 min using a Thermomixer C orbital shaker (Eppendorf, Thermo Fisher Scientific, Waltham, MA, USA). After incubation, the samples were centrifuged at 14,000 g, 25°C for 10 min. The Fluorescent Protease Assay kit (Pierce, Thermo Fisher Scientific, Waltham, MA, USA) was used for evaluating total protease activity in protein extracts. Fluorescence was read in a microplate reader (Biotek Instruments, Winooski, VT, USA) at excitation and emission wavelengths of 485 and 538 nm, respectively. The increase of relative fluorescence units within 10 min of reaction was plotted as a function of trypsin (standard) concentration. Different concentrations of trypsin ranging from 0 to 1,500 ng/ml (in distilled water) were used to plot an external calibration curve. The results were expressed as ng of trypsin activity equivalents/mg of flour.

The degree of hydrolysis (DH) was monitored using the reported o-phthaldialdehyde (OPA) spectrophotometric assay in 96-well plates (Nielsen et al., 2001). Briefly, 50 mg of flour were dispersed in 50 ml of bidistilled water and centrifuged at 10,000 g for 5 min at 23°C. Supernatant (30 μl) was placed into each well and mixed in 225 μl of OPA reagent (Sigma-Aldrich, St. Louis, MO, USA). After incubation at 23°C for 2 min, the absorbance was measured at 340 nm using a Synergy HT microplate spectrophotometer (BioTek Instruments, Winooski, VT, USA). Serine was used as a standard at the concentration of 0.1 mg/ml of deionized water (0.9516 meqv/L). The DH was calculated based on the total number of peptide bonds per protein equivalents and the number of hydrolyzed bonds according to theoretical general values (Nielsen et al., 2001).

where h_tot was 8.0 and h was expressed as

Here, β was 0.40, α was 1.00, and Ser-NH₂ was determined as

where X was a 0.05-g sample and 0.05 was the sample volume in liter.