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

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-beta-D-glucuronide by the beta-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 beta-glucuronidase. Four Staphylococcus aureus strains (4/4) and one E. coli strain, though unable to produce the enzyme, activated the milk beta-glucuronidase most probably by lowering the pH of bacterial cultures in milk for optimum activity of the indigenous enzyme. The beta-glucuronidase of milk, Str. uberis and Str. agalactiae origin had similar optimum pH ranges (5.3-6.6) while E. coli beta-glucuronidase was more active at neutral or slightly alkaline pH (6.8-7.7). The increase of beta-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.