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High Performance Liquid Chromatography Analysis of Oligosaccharides Formed During ß-Galactosidase Action on Lactose¹

Department of Animal Sciences and Industry, Kansas State University, Manhattan 66506

ABSTRACT

High performance liquid chromatography was used to determine the formation of oligosaccharides during ß-galactosidase action on lactose. Lactose solutions (5 and 20%) in .025 M potassium phosphate buffer (pH 6.60) were inoculated with ß-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.

¹ Contribution No. 84-492-J, Department of Animal Sciences and Industry, Kansas Agricultural Experiment Station, Kansas State University, Manhattan 66506.

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