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Many β-galactosidase enzymes convert lactose into a mixture of galacto-oligosaccharides (GOS) when incubated under the right conditions. Recently, the composition of commercial Vivinal GOS produced by Bacillus circulans β-galactosidase was studied in much detail in another study by van Leeuwen et al. As a spin-off of this study, we used the developed analytical strategy for the evaluation of 6 anonymous commercial GOS products, in comparison with Vivinal GOS. These GOS products were first subjected to HPLC-SEC, calibrated HPAEC-PAD profiling (glucose units in relation to a malto-oligosaccharide ladder), and 1D (1)H NMR spectroscopy. For a more detailed analysis and support of the conclusions based on the initial analysis, the GOS products were separated into DP-pure subpools on Bio-Gel P-2 (MALDI-TOF-MS analysis), which were subjected to calibrated HPAEC-PAD profiling and (1)H NMR analysis. Unidentified peaks from different GOS products, not present in Vivinal GOS, were isolated for detailed structural characterization. In this way, the differences between the various GOS products in terms of DP distribution and type of glycosidic linkages were established. A total of 13 new GOS structures were characterized, adding structural-reporter-group signals and HPAEC-PAD based glucose unit G.U. values to the analytical toolbox. The newly characterized products enhance the quality of the database with GOS structures up to DP4. The combined data provide a firm basis for the rapid profiling of the GOS products of microbial β-galactosidase enzymes.

Vivinal (R) GOS is a galacto-oligosaccharide (GOS) product, prepared from lactose by incubation with Bacillus circulans beta-galactosidase (EC 3.2.1.23). This complex mixture of saccharides with degree of polymerization (DP) between 1 and 8 is generally applied in infant nutrition. Here, a detailed structural description of the commercial product up to the DP5 level is given. First, Vivinal (R) GOS was subjected to DP analysis using HPLC-SEC (Rezex RSO-01 oligosaccharide Ag+ column) and H-1 NMR analysis. Then, the product was fractionated on Bio-Gel P-2, and the obtained fractions were pooled according to DP, as indicated by MALDI-TOF-MS analysis. Finally, fractions of single DP, as well as their subfractions obtained by HPAEC-PAD on CarboPac PA-1, were analyzed by 1D/2D H-1/C-13 NMR spectroscopy and linkage analysis. In total, over 40 structures, providing a structural coverage of over 99% of the product, have been characterized. Detailed H-1 and C-13 NMR data, as well as G.U. values (glucose units; malto-oligosaccharide ladder) on CarboPac PA-1 of all oligosaccharides are included. (C) 2014 Elsevier Ltd. All rights reserved.

Some beta-galactosidases (EC 3.2.1.23) are capable of producing mixtures of linear and branched galacto-oligosaccharides (GOS) with various types of glycosidic linkages [degree of polymerization (DP) 2-8; mainly Gal(n)Glc] when incubated under specific conditions with lactose. These products are generally applied in infant formula. However, for most galacto-oligosaccharide products only major components (low DP) or linkage patterns have been described. To build up a library of H-1 and C-13 NMR data, a detailed NMR study on commercially available GOS di- and trisaccharides, and some larger GOS oligosaccharides was carried out. Based on the fully assigned H-1 and C-13 chemical shifts of these model compounds, a H-1 NMR structural-reporter-group concept was formulated to function as a tool in the structural analysis of single GOS components and GOS mixtures. (C) 2014 Elsevier Ltd. All rights reserved.