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A Method for Determining ß-Galactosidase Activity of Yogurt Cultures in Skim Milk^1,2,

Department of Food Science and Nutrition, University of Minnesota, St. Paul 55108

ABSTRACT

A method was developed for determining the specific activity of bacterial ß-galactosidase (EC 3.2.1.23) during growth of Streptococcus thermophilus and Lactobacillus bulgaricus in skim milk. Individual and mixed strain cultures of S. thermophilus (St 3642, St14485) and L. bulgaricus (Lb11842, Lb880) were examined for growth (OD at 600 nm and viable cell counts), acid production, and ß-galactosidase activity (expressed as a function of recoverable TCA-precipitable cellular protein). Cultures were inoculated into 10% skim milk (2% inoculum) and incubated at 40°C for 12 h. Aliquots were removed at 2-h intervals and diluted with ice cold EDTA, pH 12. The EDTA chelates calcium and solubilizes milk protein, allowing separation of the bacteria by centrifugation. Cells were then washed twice with 20 mM phosphate buffer and disrupted by sonication. Cell debris and intact cells were removed by centrifugation and the cell-free extract evaluated for ß-galactosidase activity using o-nitrophenyl-ß-D-galactopyranoside as substrate. Specific activities ranged from 0 to 6 units/mg protein. This simple and reproducible method is applicable for enzyme assays and measurement of cellular components where contamination by milk proteins is a potential problem.

¹ Published as Paper Number of the scientific series of the Minnesota Agricultural Experiment Station on research conducted under the Minnesota Experiment Station Project Numbers 18-016 and 18-017.

² This project was funded by the National Dairy Board. The funding was administered in cooperation with the Dairy Research Board.