Evaluation and modification of existing methods for the quantification of exopolysaccharides in milk-based media

Two EPS assay procedures were evaluated using a ropy culture (Lactobacillus delbrueckii subsp. bulgaricus NCFB 2483) grown in a milk-based medium. Reproducible EPS measurements were unachievable because the proteins and lactose in the growth medium interfered with the separation of EPS and phenol-sulphuric acid method. Accurate determination of EPS in a complex system requires an effective regime to separate EPS from non-EPS components in fermented broths. In the development of a new EPS assay procedure, the reliability of the assay was assessed by adding a known amount of dextran to a fresh growth medium. Each processing step was then evaluated based on dextran recovery. Key improvements made to the EPS assay procedures included the use of Flavourzyme for protein hydrolysis; optimizing ethanol concentration to prevent lactose crystallization yet allowing complete EPS precipitation; and a suitable centrifugation regime to minimize EPS loss. The improved EPS assay gave reproducible results (5% coefficient of variation) with greater accuracy.