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Lactulose synthesis from fructose and lactose in continuous stirred tank (CSTR) reactor operation with glyoxyl-agarose immobilized Aspergillus oryzae β-galactosidase is reported for the first time. The effect of operational variables: inlet concentrations of sugar substrates, temperature, feed substrate molar ratio, enzyme loading and feed flow rate was studied on reactor performance. Even though the variation of each one affected to a certain extent lactulose yield (Y Lactulose ), specific productivity (π Lactulose ) and selectivity of the reaction (lactulose/transgalactosylated oligosaccharides molar ratio) (S Lu/TOS ), the most significant effects were obtained by varying the inlet concentrations of sugar substrates and the feed substrate molar ratio. Maximum Y Lactulose of 0.54 g⋅g –1 was obtained at 50°C, pH 4.5, 50% w/w inlet concentrations of sugar substrates, feed flowrate of 12 mL⋅min –1 , fructose/lactose molar ratio of 8 and reactor enzyme load of 29.06 IU H ⋅mL –1 . At such conditions S Lu/TOS was 3.7, lactose conversion (X Lactose ) was 0.39 and total transgalactosylation yield was 0.762 g⋅g –1 , meaning that 76% of the reacted lactose corresponded to transgalactosylation and 24% to hydrolysis, which is a definite advantage of this mode of operation. Even though X Lactose in CSTR was lower than in other reported modes of operation for lactulose synthesis, transgalactosylation was more favored over hydrolysis which reduced the inhibitory effect of galactose on β-galactosidase.

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