One-Pot Enzymatic Bioconversion of Native Whey for the Simultaneous Production of Galacto-Oligosaccharides and Antioxidant Peptides

The integrated valorization of whey into multifunctional food ingredients is constrained by sequential processing routes and the need for purified lactose and protein fractions. The simultaneous enzymatic conversion of lactose and whey proteins in a single reactor remains underexplored despite the frequent co-formulation of galacto-oligosaccharides (GOS) and whey protein hydrolysates in functional foods. This study evaluated the feasibility of a one-pot enzymatic system using native whey as the sole substrate for the concurrent production of GOS and antioxidant peptide fractions. A batch process combining β-galactosidase from <i>Aspergillus oryzae</i> and Alcalase^® was assessed through a 3² factorial design, analyzing the effects of pH (4.5-6.5) and temperature (40-60 °C) on GOS yield and degree of protein hydrolysis. The system enabled simultaneous transgalactosylation and proteolysis under mildly acidic conditions without significant mutual enzyme inhibition. Multi-response optimization identified pH 6.0 and 59.5 °C as the optimal conditions, yielding 25.7 ± 0.2% GOSs and 10.5 ± 0.3% protein hydrolysis. The antioxidant capacity and emulsifying and foaming properties were strongly dependent on pH, temperature, and reaction time. The results demonstrate that native whey can be directly transformed into a multifunctional ingredient through a one-pot enzymatic strategy, offering a simplified valorization approach.