Quantitative Fluorometric Assay for Rapid Enzymatic Characterization of Bifidobacterium longum and Related Bifidobacteria

Abstract

The quantitative, semi-automated assay described here is an alternative characterization method allowing for highly sensitive and specific detection of bifidobacterial enzymes. Twenty strains of Bifidobacterium longum, including the type strain ATCC 15707, and type strains of 15 other Bifidobacterium species were enzymatically characterized using 20 4-methylumbelliferyl conjugated substrates. Enzyme activities were determined by directly measuring the intensity of fluorescence derived from 4-methylumbelliferone, a fluorescent metabolic by-product. For this method, a Titertek^® Fluoroskan II fluorometer was used. Enzymes included glycosidases, an esterase, phosphatase, sulphatase, and neuraminidase. B. longum showed strong activity (>1, 000 absolute fluorescence units, afu) for α-L-Arabinopyranosidase and α-L-Arabinofuranosidase, β-D-Fucosidase, α- and β-D-Galactosidase, α-D-Glucosidase, and α-D-Mannosidase. No activity (≤50 afu) was observed for β-D-Cellobiosidase, α- and β-L-Fucosidase, β-D-Glucuronidase, β-D-Mannosidase, Neuraminidase and Sulphatase. Enzymatic activity profiles in other bifidobacteria were different according to the species. This assay is simple and rapid (6hr). Special cultural requirements are unnecessary. Results are objective and quantitative. This assay may be a useful tool for bifidobacterial taxonomy.