Enzymatic hydrolysis of chitosan into chitooligosaccharides: analysis of the reaction products by chromatographic techniques

Trabajo presentado en el ANQUE.ICCE.BIOTEC - Congress on Chemistry, Chemical Engineering and Biotechnology, celebrado en Madrid del 01 al 04 de julio de 2014.The control of the intestinal flora is one of the targets of the functional foods. A symbiotic equilibrium can be achieved by the intake of live microorganisms (probiotics) or by the so-called prebiotics (non-digested oligosaccharides). In the EU, the main prebiotics are inulin, fructooligosaccharides (FOS), galactooligosaccharides (GOS) and lactulose. Second generation prebiotics with improved activities, lower intestinal gas, major selectivity and/or stability are being investigated. Among them, chitooligosaccharides (COS) derived from chitin or chitosan exhibit interesting properties [1]. Chitin, which is formed by N-acetyl-D-glucosamine moieties with ß(1¿4) linkages, is the second most abundant natural polymer after cellulose. Chitosan is obtained by partial deacetylation of chitin and is a cationic polysaccharide composed of glucosamine and N-acetylglucosamine units. The low solubility of chitosan in neutral and alkaline solutions can be overcome by its partial hydrolysis into low-molecular-weight chitosan (LMWC) and COS, which renders molecules with higher aqueous solubility and improved biological properties [2]. We are currently investigating the synthesis of chitooligosaccharides from chitosan using glycosydic enzymes of the GH18 family (chitosanases). A colorimetric assay based on the DNS method was developed to screen chitosanase activity in different enzyme preparations. The sample with the highest activity was selected for further studies. The heterogeneity and low solubility of chitosan and related products makes difficult the analysis and identification of the reaction mixtures. In our work, the hydrolytic degradation of chitosan was analyzed in detail using various HPLC methodologies. First, the hydrolytic breakage of chitosan was visualized by size exclusion chromatography (SEC) with evaporative light-scattering detectors (ELSD). In order to analyze the composition of the resulting LMWC and COS, anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) was employed. The composition of the mixtures obtained under different experimental conditions will be addressed.Peer Reviewe