Effects of monovalent cations (Na+ and K+) on galacto-oligosaccharides production during lactose hydrolysis by kluyveromyces lactis β-galactosidase

The influence of cations, Na+ and K+ on the oligosaccharides produced in the process of transglycosylation during the hydrolysis of lactose using β-galactosidase from Kluyveromyces (K.) lactis has been investigated. The enzyme exhibited higher hydrolytic activity in the presence of K+ than in that of Na+ regardless of the anion present (acetate or phosphate), thus 96% of lactose was hydrolyzed after 3h of reaction. Formation of di- and tri-saccharides also proceeded faster in presence of K+ than of Na+, however, the highest levels were reached in presence of Na+. Transgalactosylation was favoured at high concentrations of sodium buffer and, on the contrary, hydrolysis of both lactose and oligosaccharides was favoured at low salt concentrations. After 48h of reaction in 1M sodium acetate buffer a yield of 39.5% of galactooligosaccharides (GOS) was achieved (disaccharides 18.4% and trisaccharides 21.1%). The study of influence of Mg 2+ revealed that a concentration of 1 mM was necessary to obtain a 90% hydrolysis of lactose and a 16% of disaccharides formation after 8h of reaction; however, a maximum yield of trisaccharides of 19% was attained using the highest concentration of Mg 2+ (2 mM). These results could be applied to improve the GOS formation by β-galactosidase from K. lactis.This work has been financed under R + D program of the Spanish Ministry of Innovation and Science, projects AGL2008/00941/ALI and Consolider Ingenio 2010: FUNC-C-FOOD CSD2007-00063; and an R + D program of the Comunidad de Madrid, project ALIBIRD- CM-P 2009/AGR-1469.Peer Reviewe

Obtención de pectooligosacáridos derivados de pectinas de cítricos y de manzana utilizando extractos enzimáticos comerciales

Póster presentado a las II Jornadas de Estudiantes de Máster en Química Agrícola y Nuevos Alimentos, celebradas en la Facultad de Ciencias de la Universidad Autónoma de Madrid del 30 de septiembre al 2 de octubre de 2015.Uno de los problemas de la industria transformadora de vegetales es la gestión y tratamiento los subproductos generados. Los subproductos pueden ser aprovechados para la obtención de ingredientes funcionales tales como las pectinas. Estos polisacáridos estructurales, presentes en la pared celular, son heteropolímeros constituidos por una cadena de ácido galacturónico (AGal) (homogalacturonano, HG), interrumpida por regiones ramificadas formadas por azúcares neutros, Ramnogalacturonano I y II (RG-I y RG-II). A partir de las pectinas pueden obtenerse pectooligosacáridos, POS, que pueden presentar propiedades funcionales que van a depender, en gran medida de sus características estructurales, es decir, grado de polimerización, grado de esterificación y composición en monosacáridos. Los POS pueden obtenerse por métodos físicos, químicos o enzimáticos, siendo estos últimos los mas utilizados.La presente investigación se ha realizado con financiación de los proyectos AGL2014-53445, ALIBIRD-CM S-2013/ABI-2728 e I-COOPB20099.Peer Reviewe

Carbohidratos prebióticos derivados de la lactosa

El término prebiótico, definido como ingrediente alimentario no digerible que afecta beneficiosamente al huésped mediante la estimulación del crecimiento y/o la actividad de un número limitado de bacterias en el colón, fue introducido por Gibson y Roberfroid en 1995, sin embargo, hace más de cincuenta años que es conocido el efecto beneficioso de algunos carbohidratos sobre la flora intestinal. Los prebióticos se caracterizan por no ser digeridos por los jugos digestivos y llegar intactos al colon donde son fermentados selectivamente por la microflora colónica considerada beneficiosa (bifidobacterias y lactobacilos) en detrimento de las no deseables (bacteroides, clostridia, E coli) (Roberfroid, 2003; García Peris y Velasco Gimeno, 2007).Este trabajo ha sido financiado por diferentes proyectos de investigación: CYTED N° XI.24; ALIBIRD S-0505/AGR/000153 de la Comunidad de Madrid y CONSOLIDER Ingenio 2010 (FUN-C-FOOD) CSD 2007-00063 del Ministerio de Educación y Ciencia.Peer Reviewe

Quantification of prebiotics in commercial infant formulas

Since breastfeeding is not always possible, infant formulas (IFs) are supplemented with prebiotic oligosaccharides, such as galactooligosaccharides (GOS) and/or fructooligosaccharides (FOS) to exert similar effects to those of the breast milk. Nowadays, a great number of infant formulas enriched with prebiotics are disposal in the market, however there are scarce data about their composition. In this study, the combined use of two chromatographic methods (GC-FID and HPLC-RID) for the quantification of carbohydrates present in commercial infant formulas have been used. According to the results obtained by GC-FID for products containing prebiotics, the content of FOS, GOS and GOS/FOS was in the ranges of 1.6-5.0, 1.7-3.2, and 0.08-0.25/2.3-3.8 g/100 g of product, respectively. HPLC-RID analysis allowed quantification of maltodextrins with degree of polymerization (DP) up to 19. The methodology proposed here may be used for routine quality control of infant formula and other food ingredients containing prebiotics.This work has been supported by projects AGL2011-27884 and AGL2014-58205-REDC from Ministerio de Economía y Competitividad; ALIBIRD-CM S-2013/ABI-272 (Comunidad de Madrid).Peer Reviewe

Isolation of bovine β-lactoglobulin from complexes with chitosan

A simple, economical and non-toxic method is described for the solubilization of undenatured β-lactoglobulin (β-lg) from complexes with chitosan. The effect of pH (8-10), ionic strength (0.08-0.3 m) and volume ratio between sodium acetate solutions and whey on the dissociation of β-lg-chitosan complexes was evaluated. Following a single extraction step with the addition of 10 mL of 0.1 m sodium acetate solution at pH 9 to the β-lg-chitosan complexes obtained from 1 mL of cheese-whey, a recovery of 90% of β-lg with a protein purity of 95% was achieved, suggesting that electrostatic interactions play a key role in the complexation of β-lg with chitosan. The presence of free chitosan in solution was ruled out according to gas chromatography with flame-ionization detector analysis after acid hydrolysis. NMR spectroscopy showed that the recovered β-lg after further dialysis had structural features very similar to the native protein.This work was supported by the Comisión Interministerial de Ciencia y Tecnología (CICYT), Project Nos. AGL2004-07227-C02-02 and AGL2004-03322.Peer Reviewe

Selective linkage detection of O-sialoglycan isomers by negative electrospray ionization ion trap tandem mass spectrometry

9 páginas, 6 figuras, 2 esquemas.-- El pdf del artículo es la versión de autor.Sialylated O-linked oligosaccharides are involved in many biological processes, such as cell-cell interactions, cell-substance adhesion, and virus-host interactions. These activities depend on their structure, which is frequently determined by tandem mass spectrometry. However, these spectra are frequently analyzer-dependent, which makes it difficult to develop widely applicable analytical methods. In order to deepen the origin of this behavior, two couples of isomers of sialylated O-linked oligosaccharides, NeuAcα2-3Galβ1-3GalNAc-ol/Galβ1-3(NeuAcα2-6)GalNAc-ol and NeuGcα2-3Galβ1-3GalNAc-ol/Galβ1-3(NeuGcα2-6)GalNAc-ol, were analyzed by liquid chromatography/negative electrospray ionization ion trap tandem mass spectrometry (LC/ESI(−)-MSn) using both an ion trap and a triple quadrupole mass spectrometer. Results clearly showed that while ions obtained in the triple quadrupole instrument fitted very well with the standard fragmentation routes, in the ion trap several intense ions could not be explained by these rules, specially a fragment at m/z 597. Furthermore, this ion was observed in the mass spectrum of those isomers that sialic acid binds to GalNAc by an α2-6 linkage. From the MS3 spectrum of this ion an unexpected structure was deduced, and it led to propose alternative fragmentation pathways. Molecular mechanics calculations suggested that the found atypical route could be promoted by a hydrogen bond located only in α2-6-linked oligosaccharides. It has also been demonstrated that this process follows a slow kinetic, explaining why it cannot be observed using an ion beam-type mass analyzer. In conclusion, ion traps seem to be more appropriate than triple quadrupoles to develop a reliable analytical method to distinguish between isomeric O-linked glycans.Financial support was obtained from projects ANALISYC S-0505/ AGR/000312 from the Comunidad de Madrid, Consolider Ingenio 2010 (FUN-C-FOOD); CSD 2007-00063 from the Ministerio de Educación y Ciencia; and PIF-SIALOBIOTIC 200870F010-1 and -2 from CSIC.Peer reviewe

Intestinal anti-inflammatory effects of artichoke pectin and modified pectin fractions in the dextran sulfate sodium model of mice colitis. Artificial neural network modelling of inflammatory markers

Anti-inflammatory properties of artichoke pectin and modified fractions (arabinose- and galactose-free) used at two doses (40 and 80 mg kg−1) in mice with colitis induced by dextran sulfate sodium have been investigated. Expression of pro-inflammatory markers TNF-α and ICAM-I decreased in groups of mice treated with original and arabinose-free artichoke pectin while IL-1β and IL-6 liberation was reduced only in mice groups treated with original artichoke pectin. A decrease in iNOS and TLR-4 expression was observed for most treatments. Intestinal barrier gene expression was also determined. MUC-1 and Occludin increased in groups treated with original artichoke pectin while MUC-3 expression also increased in arabinose-free pectin treatment. Galactose elimination led to a loss of pectin bioactivity. Characteristic expression profiles were established for each treatment through artificial neural networks showing high accuracy rates (≥90%). These results highlight the potential amelioration of inflammatory bowel disease on mice model colitis through artichoke pectin administration.This work has been funded by MICINN of Spain, Projects AGL2014-53445-R and AGL2017-84614-C2-1-R. Carlos Sabater thanks his FPU Predoc contract from Spanish MECD (FPU14/ 03619)

Intestinal anti-inflammatory effects of artichoke pectin and modified pectin fractions in the dextran sulfate sodium model of mice colitis. Artificial neural network modelling of inflammatory markers

Anti-inflammatory properties of artichoke pectin and modified fractions (arabinose- and galactose-free) used at two doses (40 and 80 mg kg−1) in mice with colitis induced by dextran sulfate sodium have been investigated. Expression of pro-inflammatory markers TNF-α and ICAM-I decreased in groups of mice treated with original and arabinose-free artichoke pectin while IL-1β and IL-6 liberation was reduced only in mice groups treated with original artichoke pectin. A decrease in iNOS and TLR-4 expression was observed for most treatments. Intestinal barrier gene expression was also determined. MUC-1 and Occludin increased in groups treated with original artichoke pectin while MUC-3 expression also increased in arabinose-free pectin treatment. Galactose elimination led to a loss of pectin bioactivity. Characteristic expression profiles were established for each treatment through artificial neural networks showing high accuracy rates (≥90%). These results highlight the potential amelioration of inflammatory bowel disease on mice model colitis through artichoke pectin administration.This work has been funded by MICINN of Spain, Projects AGL2014-53445-R and AGL2017-84614-C2-1-R. Carlos Sabater thanks his FPU Predoc contract from Spanish MECD (FPU14/ 03619)