Characterization and application of a crude bacterial protease to produce antioxidant hydrolysates from whey protein.

sp. CL14 crude protease was partially characterized and applied to obtain antioxidant whey protein isolate (WPI) hydrolysates. Optimal activity occurred at pH 9.0 and 60 °C. Ca, Mg, and Mn (5 mM) enhanced activity (12-26%), whereas Co, Cu, Fe, and Zn inhibited it (50-94%). At 1% (v/v), Tween 20 and Triton X-100 enhanced activities (21-27%), β-mercaptoethanol decreased it (15%), and dimethyl sulfoxide (DMSO) had no effect. Sodium dodecyl sulfate (SDS; 0.1%, w/v) increased activity by 36%. Complete inhibition by phenylmethylsulfonyl fluoride (PMSF), and 85% inhibition by ethylenediaminotetraacetic acid, indicates its serine protease character and the importance of cations for activity/stability. With 5 mM Ca, protease was optimally active at 65 °C and completely stable after 20 min at 40-55 °C. Crude protease preferentially hydrolyzed WPI and soy protein, followed by casein. WPI hydrolysis was then performed (55 °C, pH 9.0, 5 mM Ca) for 0-180 min. Contents of trichloroacetic acid (TCA)-soluble proteins in WPI hydrolysates (HWPI) increased from 29% (0 min) to 50-52% (60-180 min), accompanied by enhanced radical scavenging activity (14%, 0 min; ∼34%, 60-180 min) and Fe-chelating ability (56%, 0 min; ∼74%, 45-180 min). CL14 protease might represent an alternative biocatalyst to obtain antioxidant hydrolysates from WPI and, potentially, from other food proteins.