Synthesis of Oligosaccharides Derived from Lactulose (OsLu) Using Soluble and Immobilized Aspergillus oryzae β-Galactosidase

β-Galactosidase from Aspergillus oryzae offers a high yield for the synthesis of oligosaccharides derived from lactulose (OsLu) by transgalactosylation. Oligosaccharides with degree of polymerization (DP) ≥ 3 have shown to possess higher in vitro bifidogenic effect than di- and tetrasaccharides. Thus, in this work, an optimization of reaction conditions affecting the specific selectivity of A. oryzae β-galactosidase for synthesis of OsLu has been carried out to enhance OsLu with DP ≥ 3 production. Assays with β-galactosidase immobilized onto a glutaraldehyde–agarose support were also carried out with the aim of making the process cost-effective and industrially viable. Optimal conditions with both soluble and immobilized enzyme for the synthesis of OsLu with DP ≥ 3 were 50 °C, pH 6.5, 450 g/L of lactulose, and 8 U/mL of enzyme, reaching yields of ca. 50% (w/v) of total OsLu and ca. 20% (w/v) of OsLu with DP 3, being 6′-galactosyl-lactulose the major one, after a short reaction time. Selective formation of disaccharides, however, was favored at 60 °C, pH 4.5, 450 g/L of lactulose and 8 U/mL of enzyme. Immobilization increased the enzymatic stability to temperature changes and allowed to reuse the enzyme. We can conclude that the use, under determined optimal conditions, of the A. oryzae β-galactosidase immobilized on a support of glutaraldehyde–agarose constitutes an efficient and cost-effective alternative to the use of soluble β-galactosidases for the synthesis of prebiotic OsLu mixturesThis work has been supported by projects AGL2014-53445-R from Ministerio de Economía y Competitividad; ALIBIRD-CM S-2013/ABI-2728 from Comunidad Autónoma de Madrid and COOPB20099 from CSI

Detailed kinetic model describing new oligosaccharides synthesis using different β-galactosidases

The production of prebiotic galactooligosaccharides (GOS) from lactose has been widely 20 studied whereas the synthesis of new prebiotic oligosaccharides with improved properties as 21 those derived from lactulose is receiving an increasing interest. Understanding the mechanism 22 of enzymatic oligosaccharides synthesis from lactulose would help to improve the quality of 23 the products in a rational way as well as to increase the production efficiency by optimally 24 selecting the operating conditions. A detailed kinetic model describing the enzymatic 25 transgalactosylation reaction during lactulose hydrolysis is presented here for the first time. 26 The model was calibrated with the experimental data obtained in batch assays with two 27 different -galactosidases at various temperatures and concentrations of substrate. A complete 28 system identification loop, including model selection, robust estimation of the parameters by 29 means of a global optimization method and computation of confidence intervals was 30 performed. The kinetic model showed a good agreement between experimental data and 31 predictions for lactulose conversion and provided important insights into the mechanism of 32 formation of new oligosaccharides with potential prebiotic properties. 33Peer reviewe

Monomer and Linkage Type of Galacto-Oligosaccharides Affect Their Resistance to Ileal Digestion and Prebiotic Properties in Rats

A detailed study was performed to compare the in vivo ileal digestibility and modulatory effects in fecal microbiota of novel galacto-oligosaccharides (GOS) derived from lactulose [GOS-Lu; degree of polymerization (DP) $2, 14.0and commercial GOS derived from lactose (GOS-La; DP$3, 35.1% trisaccharides) in growing rats (5 wk old). Rats were fed either a control diet or diets containing 1% (wt:wt) of GOS-Lu or GOS-La for 14 d. Quantitative analysis of carbohydrates from dietary and ileal samples demonstrated that the trisaccharide fraction of GOS-Lu was significantly more resistant to gut digestion than that from GOS-La, as indicated by their ileal digestibility rates of 12.5 6 2.6% and 52.9 6 2.7%, respectively, whereas the disaccharide fraction of GOS-Lu was fully resistant to the extreme environment of the upper digestive tract. The low ileal digestibility of GOS-Lu was due to the great resistance of galactosyl-fructoses to mammalian digestive enzymes, highlighting the key role played by the monomer type and linkage involved in the oligosaccharide chain. The partial digestion of GOS-La trisaccharides showed that glycosidic linkages (1/6) and (1/2) between galactose and glucose monomers were significantly more resistant to in vivo gastrointestinal digestion than the linkage (1/4) between galactose units. The absence of GOS-La and GOS-Lu digestion-resistant oligosaccharides in fecal samples indicated that they were readily fermented within the large intestine, enabling both types of GOS to have a potential prebiotic function. Indeed, compared with controls, the GOS-Lu group had significantly more bifidobacteria in fecal samples after 14 d of treatment. The number of Eubacterium rectale also was greater in the GOS-Lu and GOS-La groups than in controls. These novel data support a direct relationship between patterns of resistance to digestion and prebiotic properties of GOS. J. Nutr. 142: 1232–1239, 2012.Peer reviewe

Evaluación in Vitro de las propiedades de fermentación de oligosacáridos derivados de la lactulosa

Trabajo presentado al II Workshop Probióticos, Prebióticos y Salud: Evidencia Científica, celebrado en Madrid del 16 al 17 de diciembre de 2010.La lactulosa es un disacárido constituido por una molécula de galactosa y otra de fructosa (ß-D-Gal (1¿4)-D-Fru) cuyos efectos beneficiosos sobre el intestino grueso se llevan estudiando desde hace 50 años. Es un carbohidrato resistente a las enzimas digestivas humanas llegando inalterado a los primeros tramos del colon donde es selectivamente metabolizado por las bifidobacterias y lactobacilos. Sin embargo, no alcanza las zonas más distales del intestino grueso donde la incidencia de determinadas patologías es mayor. Diferentes estudios sobre las propiedades de los carbohidratos prebióticos han demostrado que su estructura influye en la velocidad de fermentación de los microorganismos probióticos. Así, el objetivo de este trabajo ha sido evaluar in vitro las propiedades de fermentación de oligosacáridos derivados de la lactulosa (previamente sintetizados con ¿-galactosidasas, purificados y caracterizados). Los ensayos de fermentación (0, 5, 10 y 24 h) se llevaron a cabo utilizando muestras fecales de voluntarios adultos sanos inoculadas de una mezcla enriquecida de oligosacáridos derivados de la lactulosa (1%). Las poblaciones bacterianas se cuantificaron utilizando fluorescencia con hibridación in situ (FISH) con sondas de oligonucleótidos específicos para cada género de bacterias estudiado (Lactobacillus/Enterococcus; Bifidobacterium spp; Bacteroides/Prevotella; Clostridium; Atopobium; Eubacterium). Las mezclas de síntesis enriquecidas contenían oligosacáridos derivados de la lactulosa con un grado de polimerización (GP) ¿ 3 y enlaces ¿ (1¿6), ¿ (1¿4) y ¿ (1¿3). En general, durante todo el período de incubación considerado, se observó un incremento en las poblaciones de bifidobacterias y lactobacilos. Las mayores diferencias en las poblaciones bacterianas se obtuvieron a las 10 h de fermentación. Las fracciones enriquecidas en los oligosacáridos derivados de lactulosa presentaron un valor del índice prebiótico (medida cuantitativa de los cambios de la población bacteriana beneficiosa y de la indeseable) notablemente superior al de la lactulosa, indicando una mayor selectividad hacia los nuevos oligosacáridos por parte de las bacterias beneficiosas de la microflora intestinal.Este trabajo ha sido financiado por diferentes proyectos de investigación: AGL 2008-00941/ALI y CONSOLIDER Ingenio 2010 (FUN-C-FOOD): CSD 2007-00063 (MCINN); ALIBIRD-CM-P 2009/AGR-1469 (C.A.M.)Peer reviewe

Study of influential factors on oligosaccharide formation by fructosyltransferase activity during stachyose hydrolysis by pectinex ultra SP-L

The influence of reaction conditions for oligosaccharide synthesis from stachyose using a commercial enzymatic preparation from Aspergillus aculeatus (Pectinex Ultra SP-L) was studied. Oligosaccharides were analyzed by gas chromatography with flame ionization detection (GC-FID) and matrix-assisted laser desorption/ionization–time-of-flight–mass spectrometry (MALDI-TOF-MS). Galactosyl-melibiose (DP3) was synthesized as a result of fructosidase activity, whereas fructosyl-stachyose (DP5) and difructosyl-stachyose (DP6) were formed as a consequence of the fructosyltransferase activity of Pectinex Ultra SP-L. The optimal reaction conditions for the synthesis of penta- and hexasaccharides were 60 °C, pH 5.5, 600 mg/mL stachyose, and 34 U/mL enzyme. Reaction time played an important role in oligosaccharide mixture composition constituted by 20% DP5, 0.7% DP6, 55% stachyose, 21% galactosyl-melibiose, and 1% monosaccharides after 1 h and 16% DP5, 4% DP6, 27% stachyose, 44% galactosyl-melibiose, and 2% monosaccharides after 3 h. In conclusion, stachyose could be used as a substrate for the enzymatic synthesis of new oligosaccharides that may open new opportunities in the development of future prebiotics.This work has been financed under a R+D program of the Spanish Ministry of Science and Innovation Science, Projects AGL-2008-00941/ALI and Consolider Ingenio 2010 (FUN-CFOOD) CSD 2007-00063; a R+D program of the Comunidad de Madrid, Project ALIBIRD P2009/AGR-1469; and as a R+D program of the Comunidad de Castilla-La Mancha, POII10-0178-4685.Peer Reviewe