Permeabilized probiotic Lactobacillus plantarum was used as a source of β-galactosidase for the synthesis of galactooligosaccharides (GOS) from lactose. β-galactosidase activity was highest when galactose (1,724 Miller Units) was used as a carbon source compared to lactose, sucrose or glucose at 37 °C, 18 h. Permeabilized cells had the highest transgalactosylation activity resulting in 34 % (w/w) GOS synthesis from 40 % (w/v) lactose at 50 °C over 12 h. HPLC revealed that the GOS were composed of 13 % disaccharides (non-lactose), 17 % trisaccharides and 4 % tetrasaccharides that were further confirmed by ESI–MS.