The production of prebiotic galacto-oligosaccharides (GOS) considers the enzymatic synthesis by lactose
transgalactosylation and the purification from the reacted mixture (raw GOS). Simultaneous synthesis
and purification of GOS (SSP) was conducted in a one-pot operation using b-galactosidase from Aspergillus
oryzae and Saccharomyces cerevisiae or Kluyveromyces marxianus cells, allowing the selective
removal of unwanted carbohydrates (monosaccharides and lactose). The reaction was carried out in a
stirred reactor at 40 C, pH 4.5 and 200 rpm, using lactose without additional supplements, evaluating
the effect of yeast genus (S. cerevisiae or K. marxianus), enzyme-substrate ratio (20e400 IUH/g lactose),
initial lactose concentration (20e50% w/w) and substrate mass-biomass ratio (0.05e0.4). The best GOS
yield was obtained with S. cerevisiae at 50 IUH/g lactose and 0.25 g cell biomass/g carbohydrates. These conditions
were used for scaling-up into a bioreactor (1000 g of 40% w/w lactose) operated under temperature and
pH control at an aeration rate of 5 vvm, obtaining a GOS yield of 40% at 24 h of reaction, which is
significantly higher than obtained in the conventional system of GOS synthesis. SSP may represent a
technological advantage in terms of productivity and yield of GOS production.