Removal of lactose in crude galacto-oligosaccharides by β-galactosidase from Kluyveromyces lactis

In order to remove the residual lactose in crude galacto-oligosaccharides (GOS), different commercial soluble β-galactosidases from Kluyveromyces lactis (Lactozym Pure 6500L, Maxilact L2000, Lactase NL and Biolactasa-NL) and reaction conditions (temperature, total carbohydrate concentration and enzyme:substrate mass ratio) were evaluated. To select the best biocatalyst, the hydrolytic activity on o-NPG and thermal stability of all enzymes were evaluated in the absence and presence of three cations (Co²⁺, Mg²⁺, Mn²⁺) at different concentrations. The enzyme source, cation and cation concentration were selected to obtain the highest hydrolytic activity and thermal stability. Then lactose hydrolysis of raw GOS was assessed varying the temperature (30 °C–45 °C), total carbohydrate concentration (10%-50%) and enzyme:substrate mass ratio (50 IU g⁻¹–400 IU g⁻¹) and considering the lactose percentage decrease as response parameter (D_L). Lactase NL was selected as the best enzyme, with a hydrolytic activity of 286 IU mg⁻¹ and a half-life of 9 h at 35 °C in the presence of 1 mM Mn²⁺. The best reaction conditions for lactose hydrolysis employing the selected enzyme were 35 °C, 50% initial carbohydrate concentration and 135 IU g⁻¹. At such conditions of lactose hydrolysis, 70% reduction of lactose in raw GOS was obtained, with an increase of 48% in monosaccharides and of 30% in GOS. This pre-hydrolytic step is a key aspect for the subsequent purification of GOS by nanofiltration or selected bioconversion, in which monosaccharides can be removed efficiently producing GOS of high purity.