A new sugar nucleotide cycling (SNC) process was established in a one-pot three enzyme-coupled reaction using disaccharide trehalose. Trehalose synthase from Pyrococcus horikoshii could be applied to the SNC process for the synthesis of functional alpha-galactosyl oligosaccharides, alpha-galactose (Gal) epitopes and globotriose, using the effective regeneration of UDP-Gal. The alpha-Gal epitopes and globotriose were found to attach to the cell-surface of enteropathogenic Escherichia coil 0127 (EPEC) which were bound to human Caco-2 cells. These alpha-galactosyl oligosaccharides were able to prohibit the attachment of EPEC, which could have resulted in colonization and disease. The alpha-Gal epitope III with a lactulose acceptor showed the most inhibitory activity of anti-adhesion. The results suggest that the alpha-galactosyl oligosaccharides may be alternative anti-adhesion molecules that overcome antibiotic resistance. (C) 2013 Elsevier Ltd. All rights reserved.