Production of galactooligosaccharides and heterooligosaccharides with disrupted cell extracts and whole cells of lactic acid bacteria and bifidobacteria Clarissa Schwab a, * , Vivian Lee a , Kim I. Sørensen b , Michael G. Gänzle a a Department of Agricultural, Food and Nutritional Science, University of Alberta, 4-10 Ag/For Centre, Edmonton AB T6E 2P5, Canada b Chr Hansen A/S, Bøge Allé 10-12, DK-2970 Hørsholm, Denmark article info Article history: Received 15 December 2010 Received in revised form 19 April 2011 Accepted 19 April 2011 abstract This study employed b-galactosidases present in disrupted crude cell extracts (CCEs) and whole cells of lactic acid bacteria (LAB) and bifidobacteria for formation of galactooligosaccharides (GOSs) and heter- ooligosaccharides (HeOSs) from lactose and the acceptor carbohydrates mannose, fucose, N-acetylglu- cosamine (GlcNAc) and sialic acid. CCEs and whole cells successfully produced up to three HeOSs with mannose, fucose or GlcNAc in addition to GOS, but did not utilize sialic acid as acceptor. Synthesis of HeOS by a heterologously expressed b-galactosidase of Streptococcus thermophilus verified that HeOSs were indeed formed by b-galactosidases present in CCEs or whole cells. Heterologously expressed b-galactosidases of S. thermophilus and Lactobacillus plantarum hydrolysed the novel HeOSs and confirmed, for the first time, fucose as an acceptor carbohydrate. In summary, LAB CCEs and bifidobac- teria CCEs and whole cells are suitable sources of b-galactosidases that can be used to synthesize novel HeOSs with potentially expanded functionality in addition to GOSs. Ó 2011 Elsevier Ltd. All rights reserved. 1. Introduction Beta-galactosidases are glycoside hydrolases and are catego- rized within the glycoside hydrolase families (GH) 1, 2, 35 and 42. GH2 b-galactosidases catalyze the hydrolysis of lactose into glucose and galactose, and the transfer of the galactosyl-moiety to suitable acceptors (Aronson, 1952; Pazur, 1953). The rate of hydrolysis depends on the source of enzyme and the availability of water as an acceptor molecule; transgalactosylation is favoured over hydrolysis in presence of high substrate concentrations and low amounts of water (Boon, Janssen, & van’t Riet, 2000). In the presence of high concentrations of lactose, GH2 b-galactosidases produce galactooligosaccharides (GOSs) (Hsu, Lee, & Chou, 2007; Hung & Lee, 2002; Møller, Jørgensen, Hansen, Madsen, & Stougaard, 2001; Nguyen et al., 2006; Splechtna et al., 2006, 2007). GOSs are commercially employed in infant formula and have shown therapeutic properties reducing the adherence of entero- pathogenic Escherichia coli (Moro et al., 2002; Nakamura et al., 2009; Shoaf, Mulvey, Armstrong, & Hutkins, 2006; Ziegler et al., 2007). b-Galactosidases have been also used to produce heterooligosaccharides (HeOSs); e.g., lactulose was enzymatically synthesized by a Sulfolobus solfataricus b-galactosidase from lactose and fructose (Kim, Park, & Oh, 2006), whereas Kluyver- omyces lactis b-galactosidase galactosylated aromatic primary alcohols (Bridiau, Taboubi, Marzouki, Legoy, & Maugard, 2006). A wide range of sugar alcohols and mono- and disaccharides have been shown to act as an acceptor carbohydrate for a b-galactosi- dase of Enterobacter cloacae B5 (Lu, Xiao, Li, & Wang, 2009). The choice of acceptor carbohydrate and enzyme allows the formation of tailor-made HeOSs with potential application as food additives or therapeutics. Carbohydrate receptors containing galactosyl units are frequently involved in bacterial adhesion (Pieters, 2007). Human milk oligosaccharides, whose core molecule lactose is cross linked with fucose, N-acetylglucosamine (GlcNAc) or N-acetylneuramic acid (sialic acid), act as substrate for infant bifidobacteria and interact with binding receptors of intestinal pathogens thereby preventing attachment to the epithelium and reducing the inci- dence of childhood diarrhea (Bode, 2009; Martìn, Martìn-Sosa, & Hueso, 2002; Sela et al., 2008). Utilizing the transgalactosylation properties of b-galactosidases, it is possible to imitate carbohydrate receptors of bacterial adhesion or the terminal structures of human milk oligosaccharides. The aim of this study was to exploit the transfer reaction of b-galactosidases of strains of lactic acid bacteria (LAB) and bifido- bacteria to produce GOSs and HeOSs. Beta-galactosidases were obtained from bacterial disrupted crude cell extracts (CCEs) or * Corresponding author. Tel.: þ1 780 492 3634; fax: þ1 780 492 4265. E-mail address: cschwab@ualberta.ca (C. Schwab). Contents lists available at ScienceDirect International Dairy Journal journal homepage: www.elsevier.com/locate/idairyj 0958-6946/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.idairyj.2011.04.010 International Dairy Journal 21 (2011) 748e754