Optimized Cross-Linked Enzyme Aggregates For Lactose Conversion in Whey

Whey permeate, a dairy industry co-product rich in lactose, represents an underutilized resource for the production of value-added ingredients. Free enzymes can catalyze its conversion, but suffer from limited stability and reusability, restricting industrial application. Enzyme immobilization provides a strategy to overcome these limitations. In this study, cross-linked enzyme aggregates (CLEA) of β-galactosidase and glucose isomerase were developed, optimized, and characterized as carrier-free biocatalysts for the transformation of lactose into a sweetener blend of glucose, galactose, and fructose. β-galactosidase and glucose isomerase CLEAs were successfully formed, yielding CLEAs densities of 30 mg and 10 mg of wet CLEA/mL, respectively, and demonstrating recyclability with activity retention after seven cycles. Applied in a batch process to whey permeate (40 mM lactose), the CLEAs enabled complete hydrolysis of lactose within 15 min at 60°C and progressive isomerization of glucose into fructose, reaching a fructose concentration of 8 mM after 7 h. These findings highlight the potential of CLEAs as efficient, recyclable biocatalysts for lactose up cycling, contributing to the sustainable valorization of dairy co-products into blended sweeteners and other value-added ingredients. PRACTICAL APPLICATIONS: This research presents an alternative approach for the valorization of whey permeate into value-added ingredients to be used directly as sweeteners or further transformed into rare sugars. Additionally, the study explores the variables influencing enzyme immobilization and describes the optimization of CLEA for use in whey permeate valorization, offering a reusable alternative to free enzymes.