A fluorometric β-glucuronidase assay for analysis of bacterial growth in milk

Abstract

The growth of common mastitis-causing bacteria in milk was followed by a fluorometric technique based on the release of fluorescent 4-methylumbelliferone (4-MU) from 4-methylumbelliferyl-β-D-glucuronide by the β-glucuronidase of bacterial or milk origin. Three of four Escherichia coli strains, all four strains of Streptococcus uberis ( 4 4 ) and Streptococcus agalactiae ( 4 4 ) produced β-glucuronidase. Four Staphylococcus aureus strains ( 4 4 ) and one E. coli strain, though unable to produce the enzyme, activated the milk β-glucuronidase most probably by lowering the pH of bacterial cultures in milk for optimum activity of the indigenous enzyme. The β-glucuronidase of milk, Str. uberis and Str. agalactiae origin had similar optimum pH ranges (5.3–6.6) while E. coli β-glucuronidase was more active at neutral or slightly alkaline pH (6.8–7.7). The increase of β-glucuronidase activity in milk cultures of E. coli, Str. uberis, Str. agalactiae and S. aureus seemed to parallel the increase of colony forming units and were dependent on the inoculum size. The time to reach a predetermined enzyme threshold in E. coli-milk cultures showed excellent linear relationship with the inoculum size.