Enzymatic Synthesis and Identification of Two Trisaccharides Produced from Lactulose by Transgalactosylation CRISTINA MARTÍNEZ-VILLALUENGA, † ALEJANDRA CARDELLE-COBAS, † AGUSTÍN OLANO, † NIEVES CORZO, † MAR VILLAMIEL,* ,† AND MARIA LUISA JIMENO ‡ Instituto de Fermentaciones Industriales, and Centro Química Orgánica Manuel Lora Tamayo, Consejo Superior de Investigaciones Científicas (CSIC), Juan de la Cierva, 3, Madrid 28006, Spain The enzymatic transgalactosylation during lactulose hydrolysis was studied using the -galactosidase from Kluyveromyces lactis and an initial lactulose concentration of 250 g/L. During hydrolysis of lactulose, the formation of two novel trisaccharides was followed by high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD). A maximum trisaccharide yield of 14.05% was observed at 91.9% of lactulose hydrolysis. The two novel trisaccharides obtained by transglycosylation of lactulose were isolated and fully characterized by an extensive nuclear magnetic resonance (NMR) study. Complete structure elucidation and full proton and carbon assignment were carried out using 1D ( 1 H, 13 C, and 1D TOCSY) and 2D (gCOSY, TOCSY, ROESY, gHSQC, and gHMBC) NMR experiments. The trisaccharides were shown to be lactulose-based structures; the main one has a Gal unit linked to C-6 of the galactose moiety, and the other one has a Gal unit linked to C-1 of the fructose moiety. Transglycosylation of lactulose allows for the obtention of galacto- oligosaccharides with new glycosidic structures and would open new routes to the synthesis of prebiotics. KEYWORDS: -Galactosidase; trisaccharides; lactulose; transglycosylation INTRODUCTION The importance of the colonic microflora in health and nutrition is well-known. The human large intestine plays an important role as a nutritional organ mainly because of the metabolic activities of the resident microbiota, which is made up of a very complex different bacterial species. A large number of diseases are strongly related to the composition of colonic microbiota (1) and Bifidobacterium spp., and lactobacilli, present in the colon, can have beneficial effects on constipation, diarrhea, the immune system, cancer, and absorption of minerals (2, 3). The effectiveness of food enrichment with prebiotics to enhance the growth of bifidobacteria and lactobacilli in the intestine has been clearly recognized, and these products are drawing increased interest from consumers. Some carbohydrates that have established their status as prebiotic food ingredients and are currently used in the European market are fructo-oligosaccharides (FOS) and galacto- oligosaccharides (GOS) (4). GOS are usually produced by trans- glycosylation during enzymatic hydrolysis of lactose (5), and FOS are obtained by partial hydrolysis of inulin or from sucrose by action of fructosyltransferases (6, 7). The formation of GOS during enzymatic hydrolysis of lactose is well-known, and transfer reactions of -galactosidases from several sources as well as the effects that different factors, such as temperature and pH (8–10), use of organic solvents (11, 12), immobilization on different matrices (13), etc., have on enzy- matic activity and the amount and nature of oligosaccharides formed have been extensively studied. During the studies of prebiotic properties of carbohydrates, it has been observed that chemical structures of oligosaccharides (the number or type of hexose moieties and the position and conformation of links between the hexoses) may affect the fermentation properties of probiotic microorganisms (14–17); therefore, there is a high current interest in the obtention of new prebiotic carbohydrates with improved prebiotic potential. Lactulose (4-O--D-galactopyranosyl-D-fructose) is a disac- charide currently manufactured by lactose isomerization in basic media. It has several pharmaceutical applications and is also used as a prebiotic ingredient in nutrition (18–20). Lactulose may be hydrolyzed by the action of microbial -galactosi- dases (21, 22), but the study of its use as a substrate for oligosaccharide synthesis has not been undertaken. Because lactulose is resistant to the action of human digestive enzymes, * To whom correspondence should be addressed. Telephone: +34- 91-562-29-00 Ext. 397. Fax: +34-91-564-48-53. E-mail: mvillamiel@ ifi.csic.es. † Instituto de Fermentaciones Industriales. ‡ Centro Química Orgánica Manuel Lora Tamayo. J. Agric. Food Chem. 2008, 56, 557–563 557 10.1021/jf0721343 CCC: $40.75  2008 American Chemical Society Published on Web 12/21/2007