Biblioteca Digital

Monitoring the industrial production of galacto-oligosaccharides (GOS) requires a fast and accurate methodology able to quantify, in real time, the substrate level and the product yield. In this work, a simple, fast and inexpensive UV spectrophotometric method, together with partial least squares regression (PLS) and artificial neural networks (ANN), was applied to simultaneously estimate the products (GOS) and the substrate (lactose) concentrations in fermentation samples. The selected multiple models were trained and their prediction abilities evaluated by cross-validation and external validation being the results obtained compared with HPLC measurements. ANN models, generated from absorbance spectra data of the fermentation samples, gave, in general, the best performance being able to accurately and precisely predict lactose and total GOS levels, with standard error of prediction lower than 13 g kg-1 and coefficient of determination for the external validation set of 0.93–0.94, showing residual predictive deviations higher than five, whereas lower precision was obtained with the multiple model generated with PLS. The results obtained show that UV spectrophotometry allowed an accurate and non-destructive determination of sugars in fermentation samples and could be used as a fast alternative method for monitoring GOS production.

An endoxylanase from Streptomyces halstedii was stabilized by multipoint covalent immobilization on glyoxyl-agarose supports. The immobilized enzyme derivatives preserved 65% of the catalytic activity corresponding to the one of soluble enzyme that had been immobilized. These immobilized derivatives were 200 times more stable 200 times more stable than the one-point covalently immobilized derivative in experiments involving thermal inactivation at 60 °C. The activity and stability of the immobilized enzyme was higher at pH 5.0 than at pH 7.0. The optimal temperature for xylan hydrolysis was 10 °C higher for the stabilized derivative than for the non-stabilized derivative. On the other hand, the highest loading capacity of activated 10% agarose gels was 75 mg of enzyme per mL of support. To prevent diffusional limitations, low loaded derivatives (containing 0.2 mg of enzyme per mL of support) were used to study the hydrolysis of xylan at high concentration (close to 1% (w/v)). 80% of the reducing sugars were released after 3 h at 55 °C. After 80% of enzymatic hydrolysis, a mixture of small xylo-oligosaccharides was obtained (from xylobiose to xylohexose) with a high percentage of xylobiose and minimal amounts of xylose. The immobilized-stabilized derivatives were used for 10 reaction cycles with no loss of catalytic activity. © 2013 Elsevier Ltd. All rights reserved.

Currently, there is worldwide interest in the technological use of agro-industrial residues as a renewable source of food and biofuels. Lignocellulosic materials (LCMs) are a rich source of cellulose and hemicellulose. Hemicellulose is rich in xylan, a polysaccharide used to develop technology for producing alcohol, xylose, xylitol and xylo-oligosaccharides (XOSs). The XOSs are unusual oligosaccharides whose main constituent is xylose linked by β 1-4 bonds. The XOS applications described in this paper highlight that they are considered soluble dietary fibers that have prebiotic activity, favoring the improvement of bowel functions and immune function and having antimicrobial and other health benefits. These effects open a new perspective on potential applications for animal production and human consumption. The raw materials that are rich in hemicellulose include sugar cane bagasse, corncobs, rice husks, olive pits, barley straw, tobacco stalk, cotton stalk, sunflower stalk and wheat straw. The XOS-yielding treatments that have been studied include acid hydrolysis, alkaline hydrolysis, auto-hydrolysis and enzymatic hydrolysis, but the breaking of bonds present in these compounds is relatively difficult and costly, thus limiting the production of XOS. To obviate this limitation...

β-(1→3)-Glucanases were produced by Trichoderma harzianum Rifai PAMB-86 cultivated on botryosphaeran in a bench-fermenter and optimised by the response surface method. Maximal enzyme titres occurred at 5 days, initial pH 5.5 and aeration of 1.5vvm. β-(1→3)-The β-glucanolytic enzyme complex produced by T. harzianum Rifai PAMB- 86 was fractionated by gel filtration into 2 fractions (F-I, F-II), and employed to produce gluco-oligosaccharides from algal paramylon ((1→3)-β-D-glucan) and lichen pustulan ((1→6)-β-D-glucan). Both enzymes attacked paramylon to the extent of ~15-20% in 30 min releasing glucose and laminaribiose as major end-products, and laminarioligosaccharides of degree of polymerization (DP) ≥3. Only F-I degraded pustulan resulting in ~2% degradation at 30 min, with glucose, gentiobiose and gentio-oligosaccharides of DP ≥4 as major products. The difference in the nature of the hydrolysis products can be explained by the substrate specificities of each enzyme fraction, and the structural differences of the β-D-glucans attacked.

Galacto-oligosacarídeos (GOS) são prebióticos obtidos via transgalactosilação enzimática da lactose. Dentre os vários benefícios associados ao consumo de GOS destaca-se a capacidade de estimular o crescimento e atividade de bactérias benéficas no cólon. O objetivo deste estudo foi avaliar as propriedades prebióticas dos GOS sintetizados, a partir da lactose, por ß-galactosidase de Scopulariopsis sp. A digestibilidade e a fermentabilidade foram avaliadas in vitro, enquanto os efeitos prebióticos foram avaliados in vivo em um conjunto de experimentos com ratos Wistar. Os resultados observados in vitro demonstraram que os GOS produzidos neste estudo são indigeríveis, altamente fermentáveis e convertidos em ácidos graxos de cadeia curta (acetato, propionato e butirato). Estudos in vivo demonstraram que o consumo de diferentes doses de GOS por 42 dias não produziu efeitos tóxicos nos animais, evidenciado a partir de avaliações clínicas, exames hematológicos, bioquímicos, necroscópicos e histológicos. Os ratos suplementados com GOS apresentaram maior (p<0.05) população cecal de bifidobactérias (log10 10,05 ± 0,27 UFC/g) e lactobacilos (log10 8,92 ± 0,16 UFC/g). Para os ratos não suplementados com GOS estas proporções foram de log10 8...

Estudos sao realizados com oligossacarideos, probioticos e a combinação deles; sao varios os fatores que envolvem os beneficios que estes em combinacao podem agregar para a saude dos consumidores. Esta pesquisa teve como objetivo avaliar o comportamento simbiotico in vitro das linhagens probioticas Bifidobacterium animalis (Bb-12) e Lactobacillus acidophillus (LA-05), em substratos enriquecidos com oligossacarideos, ou seja, avaliar se esses potencializavam a capacidade probiotica das linhagens estudadas. Os oligossacarideos utilizados no estudo foram o galactooligossacarideo (GOS) sintetizado pela ?-galactosidase de Scopulariops sp., frutooligossacarideo (FOSOrafti) e extrato bruto de Yacon. Avaliou-se o efeito bifidogenico de diferentes concentracoes de GOS, FOS e Yacon utilizando as culturas probioticas e sua capacidade de acidificacao do meio. Foi avaliado o perfil hidrofobico e acido da membrana celular dos probioticos usando meio enriquecido com GOS, FOS e controle meio MRS (Man Rogosa e Sharpe) sem fonte de dextrose; pelo método MATH (aderencia microbiana de hidrocarbonetos). Realizou-se tambem a producao, extracao e quantificacao de exopolissacarideos (EPS) por Lactobacillus acidophillus (LA-05) em meios enriquecidos com FOS e GOS...

Monitoring the industrial production of galacto-oligosaccharides (GOS) requires a fast and accurate methodology able to quantify, in real time, the substrate level and the product yield. In this work, a simple, fast and inexpensive UV spectrophotometric method, together with partial least squares regression (PLS) and artificial neural networks (ANN), was applied to simultaneously estimate the products (GOS) and the substrate (lactose) concentrations in fermentation samples. The selected multiple models were trained and their prediction abilities evaluated by cross-validation and external validation being the results obtained compared with HPLC measurements. ANN models, generated from absorbance spectra data of the fermentation samples, gave, in general, the best performance being able to accurately and precisely predict lactose and total GOS levels, with standard error of prediction lower than 13 g kg 1 and coefficient of determination for the external validation set of 0.93–0.94, showing residual predictive deviations higher than five, whereas lower precision was obtained with the multiple model generated with PLS. The results obtained show that UV spectrophotometry allowed an accurate and non-destructive determination of sugars in fermentation samples and could be used as a fast alternative method for monitoring GOS production.; Agência da Inovação – Programa IDEIA (Potugal); Fundação para a Ciência e a Tecnologia (FCT) - Bolsa de doutouramento SFRH/BDE/15510/2004

The synthesis of galacto-oligosaccharides (GOS) by the action of Aspergillus oryzae β-galactosidase free and immobilized on magnetic polysiloxane-polyvinyl alcohol (mPOS-PVA) was studied. A maximum GOS concentration of 26% (w/v) of total sugars was achieved at near 55% lactose conversion from 50%, w/v lactose solution at pH 4.5 and 40 °C. Trisaccharides accounted for more than 81% of the total GOS produced. GOS formation was not considerably affected by pH and temperature. The concentrations of glucose and galactose encountered near maximum GOS concentration greatly inhibited the reactions and reduced GOS yield. GOS formation was not affected by enzyme immobilization in the mPOS-PVA matrix, indicating the absence of diffusional limitations in the enzyme carrier. Furthermore, this water insoluble magnetic derivative was reutilized 10-times and retained about 84% of the initial activity. In addition, the kinetic parameters for various initial lactose concentrations were determined and compared for the free and immobilized enzyme.; European Union Programme of High Level Scholarships for Latin America - Programme Alban (Scholarship No. E05D057787BR); Brazilian National Research Council (CNPq).

The objective of this study was to compare the in vitro fermentability of xylo-oligosaccharides (XOS) with different degrees of polymerisation (DP) by the intestinal digesta collected in three distinct intestinal sections of the porcine intestinal tract: ileum, caecum, and distal colon. The studied oligosaccharides included commercial short-chain XOS (DP 2e5), and medium-chain (DP 2e14) and long-chain (DP 2e25) XOS obtained by autohydrolysis of brewery’s spent grain (BSG), corn cobs (CC) and Eucalyptus globulus wood (EUC). The oligosaccharide and monosaccharide consumption, lactate and short-chain fatty acids concentrations were correlated with shifts on PCR titres of Bacteroides/ Prevotella, Bifidobacterium and Lactobacillus/Pediococcus populations, by using group- and genus-specific primers. All tested XOS were extensively fermented by the piglet ileal, caecal and colonic microbiota. The rate of consumption of medium- and longchain XOS was notably reduced in the fermentations by the ileal inoculum as compared to commercial XOS. EUC XOS, CC XOS and commercial XOS supported an enhancement of bifidobacteria and lactobacilli replication in a first stage of the fermentations. Apparently this stimulation was not selective, because Bacteroides/Prevotella replication increased in a second stage of the fermentations...

The present work proposes the production of prebiotic xylo-oligosaccharides (XOS) as high-value co-products of the Lignocellulose Feedstock Biorefinery concept, foreseeing potential applications on food, feed and nutraceutical industries. Autohydrolysis was used to selectively solubilise the hemicellulosic fraction of several xylan-rich, widely available, agricultural, agro-industrial and forestry by-products: corn cobs, brewery’s spent grain and Eucalyptus wood chips. The soluble hemicellulose-rich and the solid cellulose- and lignin-rich fractions were separated, and the crude XOS-rich hydrolysates were further purified by gel filtration chromatography. Selected fractions of purified XOS within the desired ranges of polymerization degree were characterised and their prebiotic potential was investigated in in vitro fermentations by bifidobacteria, lactobacilli and intestinal inocula. Parameters such as bacterial growth and XOS consumption were evaluated and compared with commercially available xylo-oligosaccharides. The differences observed were considered of relevance for the formulation of symbiotic preparations and the future design of targeted, tailor-made prebiotic xylo-oligosaccharides.

Mild fractionation/pretreatment processes are becoming the most preferred choices for biomass processing within the biorefinery framework. To further explore their advantages, new developments are needed, especially to increase the extent of the hydrolysis of poly- and oligosaccharides. A possible way forward is the use of solid acid catalysts that may overcome many current drawbacks of other common methods. In this Review, the advantages and limitations of the use of heterogeneous catalysis for the main groups of solid acid catalysts (zeolites, resins, carbon materials, clays, silicas, and other oxides) and their relation to the hydrolysis of model soluble disaccharides and soluble poly- and oligosaccharides are presented and discussed. Special attention is given to the hydrolysis of hemicelluloses and hemicellulose-derived saccharides into monosaccharides, the impact on process performance of potential catalyst poisons originating from biomass and biomass hydrolysates (e.g., proteins, mineral ions, etc.). The data clearly point out the need for studying hemicelluloses in natura rather than in model compound solutions that do not retain the relevant factors influencing process performance. Furthermore, the desirable traits that solid acid catalysts must possess for the efficient hemicellulose hydrolysis are also presented and discussed with regard to the design of new catalysts.

Agaro- and carra-oligosaccharides were produced by partial acid hydrolysis of commercial agarose and kappa-carrageenan. Di- and tetrasaccharides were purified by gel filtration chromatography and characterized by NMR (1D and 2D) spectroscopy and ESIMS. The following oligosaccharides were obtained: agarobiose, agarotetraose, kappa-carrabiose and kappa-carratetraose. Agarobiose and agarotetraose were used as standards to develop a high performance size exclusion chromatography (HPSEC) method which was utilized to study the hydrolysis rate of agarose and oligosaccharide production. Six hours of hydrolysis (0.1 M TFA, 65 ºC) produced mainly di- and tetrasaccharides. The methodology for oligosaccharide production and evaluation developed in the present work shows good potential for the production of bioactive oligosaccharides.

Human milk oligosaccharides (HMO) are discussed to play a crucial role in an infant’s development. Lewis blood group epitopes, in particular, seem to remarkably contribute to the beneficial effects of HMO. In this regard, large-scale functional human studies could provide evidence of the variety of results from in vitro investigations, although increasing the amount and complexity of sample and data handling. Therefore, reliable screening approaches are needed. To predict the oligosaccharide pattern in milk, the routine serological Lewis blood group typing of blood samples can be applied due to the close relationship between the biosynthesis of HMO and the Lewis antigens on erythrocytes. However, the actual HMO profile of the individual samples does not necessarily correspond to the serological determinations. This review demonstrates the capabilities of merging the traditional serological Lewis blood group typing with the additional information provided by the comprehensive elucidation of individual HMO patterns by means of state-of-the-art analytics. Deduced from the association of the suggested HMO biosynthesis with the Lewis blood group, the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry profiles of oligosaccharides in individual milk samples exemplify the advantages and the limitations of sample assignment to distinct groups.

During the decade of the 1990s and the first years of the current century, our group embarked on a project to study and synthesize human milk oligosaccharides. This report describes 2 unexpected collateral observations from that endeavor. The first observation was the detection and confirmation of 2 rare neutral human milk oligosaccharides profiles that were uncovered while assessing oligosaccharide content in hundreds of samples of human milk. One of these lacked fucosylated structures altogether, and the other lacked the oligosaccharide 3-fucosyllactose [Galβ1–4(Fucα1–3)Glc]. We used glycoconjugate probes to determine whether the unusual profiles were mirrored by fucosylation of milk glycoproteins. The results show that the lack of fucosylated oligosaccharides in these samples corresponds to the absence of equivalent fucosylated motifs in milk glycoproteins. The second finding was a shortened and distinct lactation process in transgenic rabbits expressing the human fucosyltransferase 1. During the first day of lactation, these animals expressed milk that contained both lactose and 2′-fucosylactose, but on the second day, the production of milk was severely diminished, and by the fourth day, no lactose was detected in their milk. Meanwhile...

Human milk and colostrum contain ∼12–13 g/L and ∼22–24 g/L of oligosaccharides, respectively. The chemical structures of >100 human milk oligosaccharides (HMO) have been characterized to date. We determined the concentrations of 10 neutral and 9 acidic colostrum HMO collected during the first 3 d of lactation by using reverse phase HPLC after derivatization with 2-aminopyridine or 1-methyl-3-phenyl-5-pyrazolon. The predominant oligosaccharides were Fuc(α1-2)Gal(β1-4Glc (2′-FL), Fuc(α1-2)Gal(β1-3)GlcNAc(β1-3)Gal(β1-4)Glc (LNFP I), Fuc(α1-2)Gal(β1-3)[Fuc(α1-4)]GlcNAc(β1-3)Gal(β1-4)Glc (LNDFH I), and Gal(β1-3)GlcNAc(β1-3)Gal(β1-4)Glc (LNT), the concentration of each of which was ∼1–3 g/L. Because these HMO, other than 2′-FL, all contain the Lacto-N-biose type I structure [Gal(β1-3)GlcNAc], we conclude that HMO containing the type I structure predominate over those containing the N-acetyllactosamine type II structure [Gal(β1-4)GlcNAc]. This appears to be a feature that is specific to humans, because the milk and colostrum of other species, including apes and monkeys, either contain only type II oligosaccharides or type II predominate over type I. It is possible that type I HMO may have importance as substrates for beneficial bifidobacteria in breast-fed infants. The biological importance of type I HMO predominance warrants further study...

A method to semiquantify urinary oligosaccharides from patients suffering from oligosaccharidurias is presented. 1-Phenyl-3-methyl-5-pyrazolone has been used to derivatize urinary oligosaccharides prior to analysis by electrospray ionization-tandem mass spectrometry (ESI-MS/MS). Disease-specific oligosaccharides were identified for several oligosaccharidurias, including GM1 gangliosidosis, GM2 gangliosidosis, sialic acid storage disease, sialidase/neuraminidase deficiency, galactosialidosis, I-cell disease, fucosidosis, Pompe and Gaucher diseases, and alpha-mannosidosis. The oligosaccharides were referenced against the internal standard, methyl lactose, to produce ratios for comparison with control samples. Elevations in specific urinary oligosaccharides were indicative of lysosomal disease and the defective catabolic enzyme. This method has been adapted to enable assay of large sample numbers and could readily be extended to other oligosaccharidurias and to monitor oligosaccharide levels in patients receiving treatment. It also has immediate potential for incorporation into a newborn screening program.

Complex oligosaccharides with newly formed (1,3)-β-glycosidic linkages were obtained in good to excellent yields when substituted or unsubstituted α-laminaribiosyl fluorides, acting as donors, were condensed onto mono- and disaccharide β-D-hexopyranoside acceptors by using a (1,3)-β-D-glycosynthase. These linear and branched (1,3)-β-linked oligosaccharides could prove to be important in a range of medical, pharmaceutical, and agricultural applications. Furthermore, the observation that the (1,3)-β-D-glucan glycosynthase accommodates (1,3)-, (1,4), - and (1,6)-β-oligosaccharides in its acceptor subsites suggests novel, yet unexpected physiological roles for the wild type (1,3)-β-D-glucan endohydrolase from higher plants.; Jon K. Fairweather, Maria Hrmova, Simon J. Rutten, Geoffrey B. Fincher, Hugues Driguez

This protocol describes a method to allow for the detection of specific oligosaccharide fragments in urine by tandem mass spectrometry. The detection of fragments with specific masses indicates the presence of one of a number of diseases where the deficiency of lysosomal enzymes involved in the degradation of the glyco- moieties of glycoproteins is present in the patient. This method describes the derivatization of oligosaccharides present in urine with phenyl-1-methylpyrazolone, which renders them hydrophobic, thus allowing desalting with Combi cleanup columns prior to injection. This method allows the detection of storage of oligosaccharides, which may indicate the presence of one of the infantile Pompe disease, α-mannosidosis, Gm1-gangliosidosis, Sandhoff disease, sialidosis, galactosialidosis, I-cell disease, and aspartylglucosaminuria.; Peter R. Clements

In der vorliegenden Arbeit wurden neue Oligosaccharidmimetika in Form von S-Glycopeptiden über eine kombinatorische SPOT-Synthese mit S-Glycopeptid-Bausteinen dargestellt. Dazu wurden 1-Thioglycopyranosen über eine Mitsunobureaktion mit Aminoalkoholen (Spacer) und Asparaginsäure (Peptidrückgrataminosäure) zu den S-Glycopeptid-Bausteinen verknüpft. Dadurch konnten die Zucker (Monosaccharide, Disaccharide) und der Spacer (n-Pentyl-, Aminosäure-, Aromat-Spacer) über die Knüpfung von enzymatisch stabilen Thioethern gut variiert werden. Eine weitere Variante über eine 1,3-dipolare Cycloaddition ermöglichte die Synthese von Bausteinen mit 1,2,3-Triazol-Spacern. Anschließend erfolgte die Synthese der S-Glycopeptide über quantitative Peptidkuplungsreaktionen über ein Beta-Asparaginsäurerückgrat einzelner S-Glycopeptid-Bausteine. Dafür wurde die Asparaginsäure der S-Glycopeptid-Bausteine mit Pentafluorphenol unter Zugabe von Hydroxy-1,2,3-benzotriazol-4-(3H)-on aktiviert und das S-Glycopeptid vom N- zum C-Terminus stufenweie über eine Fmoc-Strategie aufgebaut. Durch Automatisierung mit einem SPOT-Roboter wurden aus 4 S-Glycopeptid-Bausteinen 256 neue S-Glycopeptide auf einer Zellulosemembran (9 cm x 13 cm) synthetisiert mit denen anschließend ein Lectin-Screening durchgeführt wurde. Dazu waren die Lectine mit Peroxidase (POD) markiert und konnten über eine Farbreaktion mit 3-Amino-9-ethylcarbazol (AEC) auf der Membran sichtbar gemacht werden. Versuche mit dem Lectin Galentus nivalis (GNA) zeigten...

The occurrence of the alditol oligosaccharides in the Claviceps afriana honeydew is partly as a rational expression of the pathogen's selective nutritive metabolism of the sucrose supplied by the host plant. The experiments were carried out in laboratory and when 14C-D-sucrose, 14C-D-fructose or 14C-D-mannitol radiolabelled saccharides were incorporated into: a) sorghum plant infected by C. africana, b) whole and macereted micelia tissue and c) cell-free honeydew of C. africana, it was observed that the glucose moiety of sucrose was not involved in oligosaccharides formation. Glucose was used by the pathogen as nutritional source. Part of the unused fructose moiety was reduced to mannitol by the pathogen's enzymes which was also excreted into honeydew where reductase activity accepted 14C mannitol. The mannitol was linked with fructose in a 2-position synthesizing the disaccharide 1-beta-D-fructofuranosyl-D-mannitol and then the process was repeated by the mannitol moiety of the disaccharide to yield the trisaccharide 1,6-di-beta-D-fructofuranosyl-D-mannitol, which became dominant. The direct formation of alditol saccharides from monosaccharides in this way seems to be unique to C. africana, contrasting with the fructosyl transfer from sucrose to sucrose which is usual in others ergot parasites.