Abstract
Salted egg white is a nutrient-rich by-product that is usually discarded due to its high salt content. It has strong potential for value-added transformation through enzymatic hydrolysis. This study aimed to generate peptide-rich hydrolysates with antioxidant potential from desalted salted egg white using single and combined protease systems. Hydrolysis was carried out using three enzymatic systems: MSD (a dual endo- and exo-protease), Thermoase GL (a thermostable endo-protease), and a combination of both. The hydrolysis process was monitored at 0, 1, 3, and 6 h. The hydrolysates were characterized in terms of degree of hydrolysis (DH), amino acid release, kinetic parameters, synergy analysis and antioxidant activity. MSD exhibited the highest hydrolytic efficiency, releasing up to 338.46 ± 0.05 mg/g d.b. of free amino acids, while GL showed limited activity under the test conditions. The combined enzymes system enhanced peptide release, showing a synergy factor of 1.87 at 3 h. Kinetic modeling (zero-, first-and second-order) suggested that GL enhanced MSD-mediated hydrolysis by increasing substrate accessibility. Antioxidant assays revealed that MSD alone had the highest activity, with 84.14% 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging at 3 h and 31.20 nmol Trolox equivalents (TE)/mL ferric reducing antioxidant power (FRAP) at 1 h. These values were higher than those from GL and mixed treatments. Principal component analysis (PCA) revealed that amino acids such as phenylalanine, leucine, methionine, and tyrosine were key markers for distinguishing MSD and mixed enzyme hydrolysates from GL samples based on enzyme type and hydrolysis time. Overall, the results highlight the effectiveness of MSD in producing antioxidant-rich peptides and demonstrate that appropriate enzyme selection and combinations can yield functional hydrolysates from underutilized food by-products.