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

Presence of galactooligosaccharides and furosine in special dairy products designed for elderly people

An evaluation of the formation of prebiotic carbohydrates during lactose hydrolysis has been carried out in industrially elaborated dairy preparations designed for elderly people. Due to the hydrolysis of lactose, high levels of galactose and glucose were found together with galactooligosaccharides (GOS), mainly allolactose, 6-galactobiose and 6-galactosyl lactose. Total GOS content was between 7.1% and 13.4% of total carbohydrates, depending on lactose hydrolysis extent. In addition, the determination of furosine as indicator of lysine loss during the Maillard reaction (MR) has been also studied. The high content of monosaccharides promoted the progress of the MR during UHT processing, as reflected by the levels of furosine found in samples. After storage at 20 °C for 4 months the content of furosine increased by 74-90%. These results underline the importance of controlling lactose hydrolysis, and processing and storage conditions to preserve the quality and increase the bioactivity of dairy preparations designed for elderly people.This work has been supported by project POII10-0178-4685 (JCCM and ERDF), ALIBIRD-CM S2009/AGR-1469 (CAM), AGL2011-27884 (MINECO) and SENIFOOD (CENIT Programme, CAPSA and MINECO).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

Propuesta de una modificación de la técnica de apertura craneal en la autopsia médico legal, con fines estéticos

existen múltiples técnicas en patología forense para la realización de un autopsia. A lo largo de los años y con la evolución de la medicina y sus ciencias auxiliares, estas técnicas se han ido adaptando a las necesidades tanto del estudio del óbito com o a la necesidad de la reconstrucción lo menos traumática posible para que el fallecido pueda volver a ser visto por sus seres querido s. Presentamos una modificación de la técnica de apertura craneal que nos gusta denominar “La modificación de López Carrasco”, siendo la utilizada en el Servicio de Patología del Instituto de Medicina Legal de Cádiz por su calidad estética final.multiple techniques in forensic pathology for conducting an autopsy. Throughout the years and with the evolution of medicine and its auxiliary sciences, these techniques have been adapted to the needs of both the study of death and the need fo r reconstruction as painless as possible so that the deceased can return to be seen by their loved ones. We present a modification of the technique of cranial opening we like to call "The m odification of López Carrasco" being used i n the Pathology Department of the Inst itute of Legal Medicine of Cádi z for his final aesthetic quality

Multi-compartment and multi-host vector suite for recombinant protein expression and purification

Recombinant protein expression has become an invaluable tool in basic and applied research. The accumulated knowledge in this field allowed the expression of thousands of protein targets in a soluble, pure, and homogeneous state, essential for biochemical and structural analyses. A lot of progress has been achieved in the last decades, where challenging proteins were expressed in a soluble manner after evaluating different parameters such as host, strain, and fusion partner or promoter strength, among others. In this regard, we have previously developed a vector suite that allows the evaluation of different promoters and solubility enhancer-proteins, through an easy and efficient cloning strategy. Nonetheless, the proper expression of many targets remains elusive, requiring, for example, the addition of complex post-translation modifications and/or passage through specialized compartments. In order to overcome the limitations found when working with a single subcellular localization and a single host type, we herein expanded our previously developed vector suite to include the evaluation of recombinant protein expression in different cell compartments and cell hosts. In addition, these vectors also allow the assessment of alternative purification strategies for the improvement of target protein yields

A reliable gas capillary chromatographic determination of lactulose in dairy samples

A gas capillary chromatography of silylated carbohydrates on SPB-17 phase (50% diphenyl/50% dimethylsiloxane) method for the determination of lactulose has been developed. The method has been evaluated for precision and accuracy using phenyl-β-D-glucoside as internal standard with satisfactory results and, then, applied to 27 commercial milk samples (pasteurized, UHT, sterilized, powder, condensed and chocolate-based milks). Results showed that it was suitable for the determination of lactulose in milks subjected to heat treatments of different intensity, giving good chromatographic resolution, as well as precise and reproducible results. Thus, lactulose levels found in pasteurised milk (n = 6) ranged from 13.0 - 32.1 mg L-1, UHT milk (n = 10) ranged from 95.6-437.1 mg L-1, sterilized milk (n = 2) above 622.7 mg L-1 and reconstituted powder milk (n = 3) ranged from 24.2 to 48.8 mg L-1 were similar to those previously reported. In addition, this method provided a good separation between sucrose and lactulose/lactose peaks which allowed the suitable quantification of lactulose in samples such as condensed and chocolate-based milks which contain a high concentration of sucrose.This work was supported by the Comisión Interministerial de Ciencia y Tecnología (CICYT), project number AGL 2004-07227-C02-02.Peer Reviewe

Formation of galactose during heat treatment of milk and model systems

The formation of galactose, lactulose and epilactose during heating of milk was studied. The concentration of these carbohydrates increased with pH and the severity of heat treatment. The presence of proteins reduced the amount of lactulose formed whereas it increased that of epilactose and galactose. The galactose in heated milks was derived from lactose degradation. Under the conditions studied, degradation of the Amadori rearrangement product did not result in the formation of galactose. The interaction between proteins and galactose increased the thermal stability of the monosaccharide.Project Nª 158/88 Comisión Interministerial de Ciencia y Tecnología, CICYT (España).Peer reviewe

Effect of the protein content and dilution on the lactulose/furosine ratio in heat-treated milk

Formation of lactulose and furosine during heat treatment at 145°C for 30 s of milk samples with protein content ranged from 30.0 to 36.6 g/l was studied. No significant differences in the formation of lactulose were observed whereas the formation of furosine was found to increase with protein concentration, therefore the values of the lactulose to furosine ratio (Lu/Fu) were inversely related to the protein content. When raw milk was diluted 10% or 20% with water the Lu/Fu ratio in the heated samples increased with dilution.Peer Reviewe