Separation and quantitative determination of carbohydrates in microbial submerged cultures using different planar chromatography techniques (HPTLC, AMD, OPLC)

Carbohydrates are the principal sources of nutrient and energy in large-scale submerged fermentationprocesses. A method for detection and quantification of sugar levels are very advantageous because they can be considered as key indicators in determining the yields and the productivity of the process. For this reason, the objective of this study is to develop and validate a simple and relatively economical analytical method for detecting complex sugar mixtures in fermentation broth based on High-Performance Thin-Layer Chromatography (HPTLC). HPTLC is a widely used, fast and accurate method of separating complex mixtures. The proposed method involved the chromatographic separations of xilo-, galacto-, fructo-oligosaccharides mixtures at different molecular weights, tri- and disaccharides (raffinose, sucrose, lactose), and the corresponding monosaccharides (xylose, fructose, galactose) on HPTLC plates, using different eluent mixtures and elution conditions. The documentation of plates was performed using TLC visualization device and the images of plates were processed using a digital processor. HPTLC methods development using instrumental techniques as OPLC (Over Pressure Liquid Chromatography) and AMD (Automated Multiple Development) has been also described, as to simultaneously monitor several samples in the same elution, with significant time and solvent savings. Four different carbohydrates complex mixtures were analyzed using HPTLC techniques, as to optimize the quality of the separation among components. The methods set up were then applied for quantitative determination of sugars. As a model of submerged fermentations, a strain of Bifidobacterium spp. was used, saccharolytic bacterium with probiotic activities in the human gut, able to anaerobically ferment complex sugar mixtures. Results could be easily extended to other fermentation processes.