Sequential enzymatic hydrolysis of egg yolk proteins: Kinetics, functionality, and bioactivity of hydrolysates.

The lecithin-free egg yolk (LFEY) by-product was hydrolyzed using Alcalase (A) or Flavourzyme (F), or a sequential enzymatic A-F system, under different pH and Flavourzyme concentrations. The hydrolysates' molecular weight (Mw) profile was analyzed, and their functionality and bioactivity were assessed in terms of soluble protein, emulsifying activity index (EAI), emulsion stability index (ESI), and antioxidant and Angiotensin I-converting enzyme (ACEI) inhibitory activities. Hydrolysates were fractionated using 5 and 1 kDa ultrafiltration (UF) membranes. Alcalase efficiently hydrolyzed LFEY proteins under pH control, but its activity declined sharply without pH control. Flavourzyme could not hydrolyze egg yolk proteins, but hydrolyzed the peptides >12 kDa produced by Alcalase in the sequential enzymatic reaction. The hydrolysis of LFEY using sequential reaction increased by 6 % the degree of hydrolysis and the soluble protein by 9 % compared to the single enzyme reaction. It also increased the antioxidant properties and the ACE inhibitory activity by nearly two and three times that of the LFEY, respectively. UF fractions of hydrolysates having peptides with Mw between 1 and 5 kDa showed higher antioxidant properties and ACE inhibition activity than peptides <1 kDa. The sequential reaction demonstrated the cooperation of enzymes in hydrolyzing protein by-products.