Production of lactose-free galacto-oligosaccharide mixtures: comparison of two cellobiose dehydrogenases for the selective oxidation of lactose to lactobionic acid Thomas Maischberger, a,b Thu-Ha Nguyen, a,b Prakit Sukyai, b,c Roman Kittl, b Sergio Riva, d Roland Ludwig a,b and Dietmar Haltrich b, * a Research Centre Applied Biocatalysis, Petersgasse 10, 8010 Graz, Austria b Division of Food Biotechnology, Department of Food Sciences and Technology, University of Natural Resources and Applied Life Sciences (BOKU), Muthgasse 18, 1190 Wien, Austria c Suphanburi Campus Project, Kasetsart University, 50 Phahonyothin Road, Bangkok 10900, Thailand d Istituto di Chimica del Riconoscimento Molecolare, C.N.R., Via Mario Bianco 9, 20131 Milano, Italy Received 21 September 2007; received in revised form 24 January 2008; accepted 28 January 2008 Available online 6 February 2008 Presented at Eurocarb 14th Lu ¨beck, Germany, September 2007 Abstract—Galacto-oligosaccharides, complex mixtures of various sugars, are produced by transgalactosylation from lactose using b-galactosidase and are of great interest for food and feed applications because of their prebiotic properties. Most galacto-oligosac- charide preparations currently available in the market contain a significant amount of monosaccharides and lactose. The mixture of galacto-oligosaccharides (GalOS) in this study produced from lactose using recombinant b-galactosidase from Lactobacillus reuteri contains 48% monosaccharides, 26.5% lactose and 25.5% GalOS. To remove efficiently both monosaccharides and lactose from this GalOS mixture containing significant amounts of prebiotic non-lactose disaccharides, a biocatalytic approach coupled with subse- quent chromatographic steps was used. Lactose was first oxidised to lactobionic acid using fungal cellobiose dehydrogenases, and then lactobionic acid and monosaccharides were removed by ion-exchange and size-exclusion chromatography. Two different cel- lobiose dehydrogenases (CDH), originating from Sclerotium rolfsii and Myriococcum thermophilum, were compared with respect to their applicability for this process. CDH from S. rolfsii showed higher specificity for the substrate lactose, and only few other com- ponents of the GalOS mixture were oxidised during prolonged incubation. Since these sugars were only converted once lactose oxi- dation was almost complete, careful control of the CDH-catalysed reaction will significantly reduce the undesired oxidation, and hence subsequent removal, of any GalOS components. Removal of ions and monosaccharides by the chromatographic steps gave an essentially pure GalOS product, containing less than 0.3% lactose and monosaccharides, in a yield of 60.3%. Ó 2008 Elsevier Ltd. All rights reserved. Keywords: b-Galactosidase; Transgalactosylation; Cellobiose dehydrogenase; Galacto-oligosaccharides; Prebiotics; Lactobionic acid; Laccase 1. Introduction During the last years, galacto-oligosaccharides (GalOS) were reported to be beneficial for human health, and they are now recognised as prebiotics. 1,2 GalOS, non- digestible oligosaccharides, are not hydrolysed or ab- sorbed in the upper intestinal tract, they pass on to the colon where they are then fermented selectively by ben- eficial intestinal bacteria, which implicate a balanced 0008-6215/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.carres.2008.01.040 Abbreviations: ABTS, 2,2 0 -azinobis-(3-ethylbenzthiazoline-6-sulfonic acid); CDH, cellobiose dehydrogenase; DCIP, 2,6-dichloro-indophe- nol; GalOS, galacto-oligosaccharides; oNPG, o-nitrophenyl-b-D-galac- topyranoside; oNP, o-nitrophenol; TLC, thin-layer chromatography; CE, capillary electrophoresis; HPAEC-PAD, high-performance anion- exchange chromatography with pulsed amperometric detection * Corresponding author. Fax: +43 1 36006 6521; e-mail: dietmar.haltrich@boku.ac.at Available online at www.sciencedirect.com Carbohydrate Research 343 (2008) 2140–2147