2.2. Enzymatic Hydrolysis of Whey Protein

Enzymatic hydrolysis of whey protein (WP) was carried out in an automatic titrator 718 Stat Titrino (Metrohm AG, Herisau, Switzerland) to a degree of hydrolysis 10% (DH 10) with alcalase. For this purpose, a solution containing 36 g of protein was prepared with distilled water to a final volume of 0.9 L. The process conditions were set to 50 °C and the pH to 8, and the enzyme-substrate ratio was fixed to 0.55 (w/w). The degree of hydrolysis was estimated with the pH-stat-method, as described by Camacho et al. [18]. The hydrolysis reaction took ca. 1.5 h, and the enzyme was deactivated at 100 °C for 5 min. The whey protein hydrolysate (WPH) solution was stored at −20 °C until further use.