Fermentia (Hungary)

A simple thin-layer chromatography (TLC) method for continuous determination of metabolism of carbohydrate and production of polysaccharides in the course of bacterial fermentations is presented in this paper. Samples were taken from the supernatants of decanted fermentation broth and from the hydrolysates of both the supernatants and the fermentation broth. Hydrolysis of the exo-polysaccharides (EPSs) to the constituent monosaccharides was carried out by 10% v/v hydrochloric acid at 100°C. These samples were applied directly onto a precoated nano silica plate besides a series of mono-, oligo-, and polysaccharide reference solutions.The plates were developed in a twin-trough chamber previously saturated with a solution of chloroform-toluene-35% formic acid-methanol (10:2:2:7 v/v) for 1 h. The plates were placed in the empty trough of the chamber in order to be saturated and then transferred to the trough containing the solvent. After reaching a migration distance of 160 mm, the plates were dried out and placed in a chamber saturated with I2 vapor in order to detect starch-type polysaccharides.After drying, the plates were dipped into a dish containing a solution of 10% v/v concentrated sulfuric acid in n-propanol-toluene (1:1 v/v). The excess reagent was eliminated with filter papers. Visualization was achieved in an electric oven at 115°C for 10 min. Evaluation was accomplished by comparison of the RF values, and the intensities of the spots were evaluated instrumentally using a CAMAG TLC Scanner II. The presented method is suitable for monitoring the bacterial EPS production and determining the monosaccharide constituents.