A process for producing non-monosaccharide and high-purity galactooligosaccharides (GOS) was proposed. GOS were produced from lactose by Penicillium expansum F3 beta-galactosidase immobilized in calcium alginate. The effects of pH, temperature and lactose concentration on GOS production were investigated. A purity of 28.7% (w/w) GOS was obtained from.380 g/L lactose solution at pH 5.4 and 50 degrees C. The immobilized enzyme was used for repeated GOS synthesis and showed good operational stability. Digestible sugars in the GOS could be depleted after fermentation with Saccharomyces cerevisiae L, or Kluyveromyces lactis L-3 entrapped in the calcium alginate. Purity greater than 37% with yields greater than 27% of non-monosaccharide GOS were maintained by S. cerevisine L-1 for 19 batches and purity greater than 97% with a yield greater than 20% of high-purity GOS was produced using K. lactis L-3 for two batches. (C) 2008 Elsevier Ltd. All rights reserved.