Valorization of requesón Cheese Whey through Synthesis of Galactooligosaccharides with Fungal and Recombinant β-Galactosidases

Requesón is a traditional Mexican whey cheese produced by protein precipitation from the whey remaining after cheese manufacturing. This process generates a byproduct called requesón whey (RW), which retains significant lactose content and represents a considerable organic waste stream. This study evaluated the potential of RW as a lactose source for synthesizing galactooligosaccharides (GOS), valuable prebiotic ingredients. Two commercial enzymes were assessed: β-galactosidase from Aspergillus oryzae (β-gal-Ao) and a recombinant hyperthermophilic glycosidase (GLY 001–02). Response surface methodology optimized transgalactosylation conditions, achieving maximum GOS yields of 44.35% for β-gal-Ao (52.56 °C, 22.73% lactose) and 61.56% for GLY 001–02 (84 °C, 23% lactose). Both enzymes successfully produced GOS yields exceeding 40% when using RW as the lactose source, demonstrating its viability as an alternative raw material. These findings underscore the biotechnological potential of RW for functional food ingredients production while supporting circular economy principles through waste stream valorization.