2.2. Hydrolysis Procedure

Hydrolysates of whey and soy protein were produced using subtilisin (EC 3.4.21.62) and trypsin (EC 3.4.21.4) (Novozymes, Denmark) as enzymes. To evaluate the effect of degree of hydrolysis on the emulsifying properties, five hydrolysates of a different degree of hydrolysis (2%, 4%, 6%, 10% and 14%.) were produced with each enzyme. Hydrolysis was carried out in a jacketed stirred tank reactor that maintained a temperature at 50 °C during operation. When the protein solution (40 g/L) reached 50 °C, enzyme was added to the reactor at an enzyme/substrate ratio of 0.5%. The pH was maintained constant at pH 8 by adding NaOH 1M. The reaction was monitored by the pH-stat method [28], which relates in real time the consumption of base with the number of peptide bonds cleaved according to Equation (1):

where B (mL) is the amount of base consumed, N_b (eq/L) is the normality of the base, α is the average degree of dissociation of the α-NH₂ groups released during hydrolysis, m_p (g) is the mass of protein in the substrate and h_TOT (meq/g) is the number of equivalents of peptides bonds per gram of protein. After reaching the desired DH, the reaction was stopped by thermal deactivation of the enzyme at 90 °C for 5 min. Afterward, the samples were freeze-dried for further use.