New method for exopolysaccharide determination in culture broth using stirred ultrafiltration cells

A new method to remove simple carbohydrates from culture broth prior to the quantification of exopolysaccharides (EPS) was developed and validated for the EPS-producing strain. Lactobacillus rhamnosus RW-9595M. This method us es ultrafiltration (UF) in stirred cells followed by polysaccharide detecti on in the retentate by the phenol-sulfuric acid method. The UF method was c ompared with a conventional method based on ethanol extraction, dialysis, p rotein removal by trichloroacetic acid (TCA) and freeze-drying. EPS product ion during pH-controlled batch fermentations in basal minimum medium, whey permeate (WP), and whey permeate supplemented with yeast extract, minerals and Tween-80 (SWP) was determined by the new UF and conventional methods. E PS recovery by the new method ranged from 83% to 104% for EPS added in the concentration range 40-1,500 mg/l in 0.1 M NaCl solution or culture medium. The UF method was rapid (8 h), accurate and simple, and required only a sm all sample volume (1-5 ml). A very high maximum EPS production was measured in SWP by both the UF and conventional methods (1,718 and 1,755 mg/l).