Synthesis of galactooligosaccharides with four β-galactosidases: Structural comparison of the products by HPLC, ESI-MS and NMR

Galactooligosaccharides (GOS) are lactose-derived functional ingredients applied in food products and have great potential in health protection. The conversion of lactose to GOS commonly occurs using (3-galactosidases of mould, yeast and bacterial origin. The yield and structure of the resulting GOS depend on the enzyme used and the reaction conditions. This work focuses on the structural analysis of the products obtained with four commercial (3-galactosidases Maxilact LGI 5000 (ML), Maxilact A4 MG (MA), Saphera 2600 L (SA) and NOLA Fit 5500 (NL) to evaluate their efficiency and specificity. HPLC, ESI-MS and NMR spectroscopy were applied to characterise the GOS preparations. GOS were separated from the reaction mixture using activated charcoal treatment. HPLC analysis confirmed that most of the monosaccharides and a part of the lactose, but also some other disaccharides, probably allolactose and 6-galactobiose, were retained by charcoal. In all the products, ESIMS analysis detects oligosaccharides up to hexamers. NMR spectra confirmed the presence of GOS of various configurations and polymerisation degrees and evaluated the specificity of used enzymes. MA preferably forms 1,6- and 1,4-glycosidic bonds, and bacterial enzymes NL and SA also form 1,2- and 1,3- glycosidic bonds, while yeast enzyme ML cannot produce new 1,4-glycosidic bonds. The mould enzyme MA showed the highest transgalactosylation activity, forming longer GOS oligomers than the other enzymes.