GC-FID determination of monosaccharides from apple and citrus pectin hydrolyzates produced by three commercial enzyme preparations

Resumen del póster presentado a la XV Reunión Científica Sociedad Española de Cromatografía y Técnicas Afines (SECyTA) y VII Reunión Nacional Sociedad Española de Espectrometría de Masas (SEEM), celebradas en castellón del 27 al 30 de octubre de 2015.Pectin is one of the most structurally complex families of polysaccharides in nature. This heterogeneous polymer is mainly composed of a linear chain of α-1, 4-D-galacturonic acid called homogalacturonan (HG) which is the backbone where are ramified chains rhamnogalacturonan I (RGI), (alternating sequences GalAc and α-(1, 2) linked L-rhamnosyl residues) and rhamnogalacturonan II (RGI) a most complex chain. The properties and applications of pectin are affected by several factors related to pectin structure, including composition, presence and distribution of side chains, degree of methyl-esterification and acetylation, molar mass, and the charge distribution along their backbone. Different chromatographic methods can be used to analyze monosaccharides such as HPAEC-PAD and GC (as their alditols and aldonic acids) or trimethylsilyl oximes (TMSO). Regardless of the methods used to determine the monosaccharide composition, pectins needs to be hydrolyzed in order to release its monomeric units. The aim of this work was to determine monosaccharide composition (neutral sugars and galacturonic acid) by GC-FID in commercial apple and citrus hydrolyzates obtained using three commercial enzymatic preparations. Solutions from apple and citrus pectins (10 mg/mL) were prepared in acetate buffer 0.05M pH 4.5. For enzymatic hydrolysis, three commercial enzymatic extracts, Viscozyme, Mannozyme and Gluconex (50 U/mL) were used. Reactions were performed at 50 ºC during 24 h. Sampling was at 0, 1, 3 and 24 h. Monosaccharides and galacturonic acid in reaction mixtures were analyzed as their trimethylsilyl oximes by GG-FID using a capillary column DB-5HT (15m x 0.32 x 0.10μm). GC profiles of carbohydrates from apple and citrus pectin enzymatic hydrolyzates showed the presence of arabinose, rhamnose, galactose, mannose, glucose and galacturonic acid. Enzymatic hydrolysis after 24h of apple pectin was almost complete using Viscozyme or Mannozyme while only a minor release of monosaccharide was observed in Gluconex hydrolyzates. In citrus pectins, the highest content of neutral sugars was detected in Viscozyme hydrolyzates followed by Mannozyme and Gluconex being of 731, 652 and 189 mg/g of pectin, respectively, measured after 24h of hydrolysis. These differences could be attributed to the low release of mannose and galactose. On the contrary, galacturonic acid was found at high levels in apple pectins reaching 726, 682 and 555 mg/g of pectins when, respectively, Viscozyme, Manozyme and Gluconex were used. These results highlight the influence of the commercial enzyme characteristics on the determination of monosaccharides from apple and citrus pectin.This work has been funded by MINECO of Spain: Project AGL2014-53445-R.Peer Reviewe