High Performance Liquid Chromatography Analysis of Oligosaccharides Formed During β-Galactosidase Action on Lactose

Abstract

High performance liquid chromatography was used to determine the formation of oligosaccharides during β-galactosidase action on lactose. Lactose solutions (5 and 20%) in .025 M potassium phosphate buffer (pH 6.60) were inoculated with β-galactosidase at 1 and 2 μmol orthonitrophenol units/ml from two different microbial sources and incubated at 4°C for 24 h and 37°C for 4 h. During hydrolysis, formation of oligosaccharides was followed with high performance liquid chromatography, using both resinbased (μ-Spherogel, Altex Scientific) and polar-bonded (Amino Spheri-5, Brownlee Labs) columns. Five oligosaccharides were detected during hydrolysis. However, only two of these occurred in significant concentrations. Maximum observed concentration was at .75 to 1.0 h in the 37°C incubation at 2 μmol orthonitrophenol units enzyme/ml substrate. The concentration of the two oligosaccharides decreased thereafter during hydrolysis. However, with 1 μmol enzyme used, one of the two major oligosaccharides showed no appreciable decrease in concentration after reaching its maximum at 45 min. At maximum concentration the oligosaccharides represented about 11.3% of the total lactose for 5% lactose solutions and 16.0% for 20% lactose. They decreased to 5.5% for 5% lactose and 10.8% for 20% lactose near the completion of hydrolysis. © 1985, American Dairy Science Association. All rights reserved.