Ana S.P. Moreira

Evaluation of the effect of roasting on the structure of coffee galactomannans using model oligosaccharides.

The roasting process induces structural changes in coffee galactomannans. To know more about the reaction pathways that occur during the roasting of coffee, mannosyl and galactomannosyl oligosaccharides, having a degree of polymerization (DP) between 3 and 4, were used as models for galactomannans. These compounds were dry-heated under air atmosphere from room temperature to 200 °C, being maintained at 200 °C for different periods of time. The roasted materials were analyzed by mass spectrometry (ESI-MS, MALDI-MS, and ESI-MSn) and methylation analysis. In the MS spectra were identified several [M+Na]+ ions belonging to a series from a single hexose to 10 hexose residues ([Hex1-10+Na]+). The ions corresponding to their respective mono- and tridehydrated derivatives ([Hex2-10-H2O+Na]+ and [Hex2-10-3H2O+Na]+, respectively) were also identified. ESI-MSn as well as deuterium-labeling and alditol derivatization experiments showed that the tridehydrations occur at the reducing end of the oligosaccharides. The identification of (1→2)- and (1→6)-linked mannose residues and (1→4)-linked glucose residues by methylation analysis allowed the conclusion that transglycosylation and isomerization reactions occur during dry thermal processing.

Sequential microwave superheated water extraction of mannans from spent coffee grounds.

The feasibility of using sequential microwave superheated water extraction (MAE) for the recovery of mannans from spent coffee grounds (SCG) was studied. Due to the high contents of mannose still present in the SCG residue left after two consecutive MAE, the unextracted material was re-suspended in water and submitted to a third microwave irradiation (MAE3) at 200 °C for 3 min. With MAE3, mannose recovery achieved 48%, increasing to 56% by MAE4, and reaching a maximum of 69% with MAE5. Glycosidic-linkage analysis showed that in MAE3 mainly galactomannans were recovered, while debranched galactomannans were recovered with MAE4 and MAE5. With increasing the number of extractions, the average degree of polymerization of the mannans decreased, as observed by size-exclusion chromatography and by methylation analysis. Scanning electron microscopy images showed a decrease on cell walls thickness. After final MAE5, the remaining un-extracted insoluble material, representing 22% of the initial SCG, was composed mainly by cellulose (84%).

Roasting-induced changes in arabinotriose, a model of coffee arabinogalactan side chains

Thermal processing can promote reactions that change the structure of food constituents, often by unknown mechanisms, such as those occurring in arabinose residues of coffee arabinogalactan side chains. Aiming to know more about these modifications, the structurally related α-(1→5)-l-arabinotriose was roasted at 200°C and the products obtained were identified by ESI-MS and MALDI-MS and characterised by ESI-MS(n). Depolymerised and polymerised oligosaccharides with up to 16 residues and new types of linkages were formed. Also, products resulting from dehydration, oxidation, and cleavage of a carbon-carbon bond at the reducing end of the corresponding non-modified oligosaccharide were formed, probably promoting the release of formaldehyde, formic acid, glycolaldehyde, glyoxal, acetic acid, glycolic acid, glyceraldehyde, 2-hydroxypropanedialdehyde and lactic acid. As many of these compounds have been reported to occur in roasted coffee beans and/or brews, it can be suggested that the degradation of coffee arabinogalactan side chains can contribute to their formation upon roasting.

Differentiation of isomeric pentose disaccharides by electrospray ionization tandem mass spectrometry and discriminant analysis

RATIONALE The structural characterization of unknown oligosaccharides remains a big challenge since a large number of isomeric structures are possible even for disaccharides. In this work, electrospray ionization collision-induced dissociation tandem mass spectrometry (ESI-CID-MS/MS) was used for the differentiation of isomeric pentose disaccharides, α-(1 → 5)-L-arabinobiose (Ara(2)) and β-(1 → 4)-D-xylobiose (Xyl(2)). METHODS ESI-MS/MS spectra of [M + Li](+) and [M + Na](+) ions of Ara(2) and Xyl(2), as well as these precursor ions of (18)O-labelled disaccharides, were acquired using two mass spectrometers equipped with different analyzers: LIT (linear ion trap) and Q-TOF (quadrupole time-of-flight). RESULTS Product ions observed in MS/MS spectra arise from the cleavage at the nonreducing side of the glycosidic bond (Y(1)(+)) and from cross-ring cleavages (0,1)A(2)(+), (0,2)A(2)(+), and (0,3)A(2)(+) at the reducing residue. Statistically significant differences were observed between the relative abundance of specific product ions, when comparing both disaccharides. These differences allowed discriminant models to be built and to propose a criterion using the relative abundances of selected ions capable of discriminating between the isomers for both adduct ions and spectrometers. CONCLUSIONS Isomeric pentose disaccharides can be distinguished based on the fragmentation of both [M + Li](+) and [M + Na](+) ions and using different mass spectrometers. However, LIT instrument has a better discriminant power.

Lipidomics of Mesenchymal Stromal Cells: Understanding the Adaptation of Phospholipid Profile in Response to Pro-Inflammatory Cytokines

Mesenchymal stromal cells (MSCs) present anti‐inflammatory properties and are being used with great success as treatment for inflammatory and autoimmune diseases. In clinical applications MSCs are subjected to a strong pro‐inflammatory environment, essential to their immunosuppressive action. Despite the wide clinical use of these cells, how MSCs exert their effect remains unclear. Several lipids are known to be involved in cells signaling and modulation of cellular functions. The aim of this paper is to examine the variation in lipid profile of MSCs under pro‐inflammatory environment, induced by the presence of tumor necrosis factor alpha (TNF‐α) and interferon gamma (IFN‐γ), using the most modern lipidomic approach. Major changes in lipid molecular profile of phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), lysoPC (LPC), and sphingomyelin (SM) classes were found. No changes were observed in the phosphatidylinositol (PI) profile. The levels of PC species with shorter fatty acids (FAs), mainly C16:0, decreased under pro‐inflammatory stimuli. The level of PC(40:6) also decreased, which may be correlated with enhanced levels of LPC(18:0), which is known to be an anti‐inflammatory LPC, observed in MSCs subjected to TNF‐α and IFN‐γ. Simultaneously, the relative amounts of PC(36:1) and PC(38:4) increased. TNF‐α and IFN‐γ also enhanced the levels of PE(40:6) and decreased the levels of PE(O‐38:6). Higher expression of PS(36:1) and SM(34:0) along with a decrease in PS(38:6) levels were observed. These results indicate that lipid metabolism and signaling are modulated during MSCs activation, which suggests that lipids may be involved in MSCs functional and anti‐inflammatory activities. J. Cell. Physiol. 231: 1024–1032, 2016.

Mid-infrared (MIR) metabolic fingerprinting of amniotic fluid: a possible avenue for early diagnosis of prenatal disorders?

This work describes a mid-infrared (MIR) metabolic profiling study of 2nd trimester amniotic fluid in relation to selected prenatal disorders, with results focusing on fetal malformations (FM), preterm delivery (PTD) and premature rupture of membranes (PROM), the latter two conditions occurring later in pregnancy. Partial least squares-discriminant analysis (PLS-DA) models were obtained for FM and pre-PTD subject groups, supported by Monte Carlo Cross Validation (MCCV), and identified specific MIR profile changes. For pre-PROM subjects, minor changes were noted. MIR interpretation was assisted by intra- (MIR/MIR) and inter- (MIR/NMR) domain statistical correlation analysis, the results unveiling possible biomarker MIR signatures for FM and pre-PTD subjects. Biofluid MIR metabolic profiling holds enticing possibilities as a low cost, easy to use, rapid method and the results presented have shown its sensitivity to clinically diagnosed conditions such as FM, and to the pre-clinical stages of PTD. Specific improvement needs are discussed, namely regarding sample numbers and experimental reproducibility.

Structural analysis of dextrins and characterization of dextrin-based biomedical hydrogels.

The characterization of several commercial dextrins and the analysis of the potential of dextrin derived hydrogels for biomedical applications were performed in this work. The structural characterization of dextrins allowed the determination of the polymerization and branching degrees, which ranged from 6 to 17 glucose residues and 2 to 13%, respectively. Tackidex, a medical grade dextrin was choosen for further characterization. The combination of hydrogel with a dextrin nanogel and urinary bladder matrix was achieved without compromising the mechanical properties or microstructure. The encapsulation of cells, preserving its viability, confirms the biocompatibility of the injectable hydrogels, which have therefore great potential for biomedical applications.

Alteration in Phospholipidome Profile of Myoblast H9c2 Cell Line in a Model of Myocardium Starvation and Ischemia

Myocardium infarction is one of the most deathly cardiovascular diseases. It is characterized by myocardium ischemia as a result of nutrients depletion and hypoxia. The cell can respond to this injury by autophagy or apoptosis, which determines the evolution and possible recovery of the myocardium infarction. Lipids play an important role in cardiovascular disease. However reports stating lipidome variations in cardiovascular disease are scarce and the role that lipids play in this pathological condition is not completely understood. The aim of this work was to identify changes in lipid profile of a myoblast H9c2 cell line under starvation and ischemia, to better understand and recognize new biomarkers for myocardial infarction. Lipidomic profile was evaluated by HILIC‐LC‐MS and GC‐MS. Cardiac cells showed alterations in phosphatidylcholines PC (34:1) and PC (36:2), lysophosphatidylcholines lyso PC(16:0), lysoPC(18:1) and lysoPC(18:0), phosphatidylethanolamine PE (34:1), phosphatidylserine PS (36:1), phosphatidylinositol PI (36:2), PI (38:3) and PI (38:5), sphingomyelin SM (34:1) and cardiolipins CL(68:4), CL(72:5) and CL(74:7) in ischemia and/or starvation, in comparison with control. Specific differences observed only in starvation were decrease of SM (34:1) and FA (20:4), and increase of PS (36:1). Differences observed only in ischemia were decrease of PC (36:2), lyso PC (16:0) and FA (18:1) and simultaneous increase of FA (16:0), and FA (18:0). Interestingly, PC (34:1) increased in ischemia and decreased in starvation. In conclusion, our work suggests that lipids are potential markers for evaluation of cell fate, either cell death or recovery, which will be useful to improve diagnosis and prognostic of cardiovascular diseases. J. Cell. Physiol. 231: 2266–2274, 2016.

Oxidation of amylose and amylopectin by hydroxyl radicals assessed by electrospray ionisation mass spectrometry.

The hydroxyl radicals (HO) are one of the most reactive oxygen species (ROS) involved in the oxidative damage of biological molecules, including carbohydrates. During the industrial processing of food, ROS can be formed. In order to identify the structural changes induced in starch by oxidation, amylose, amylopectin, and maltotriose, an oligosaccharide structurally related to these polysaccharides, were subjected to oxidation with HO generated under Fenton reaction conditions (Fe(2+)/H2O2). The oxidised polysaccharides were hydrolysed by α-amylase and the obtained oligosaccharides were fractionated by ligand-exchange/size-exclusion chromatography. Both acidic and neutral α-amylase resistant oligosaccharides were characterized by mass spectrometry. In oxidised neutral products, new keto, hydroxyl, and hydroperoxy moieties, and oxidative ring scission were observed at the reducing end of the oligosaccharides. The acid sugar residues occurred at the reducing end and included gluconic and glucuronic acid derivatives, and acids formed by oxidative ring scission, namely, arabinonic, erythronic, glyceric and glycolic acids.

Chlorogenic acid–arabinose hybrid domains in coffee melanoidins: Evidences from a model system

Arabinose from arabinogalactan side chains was hypothesized as a possible binding site for chlorogenic acids in coffee melanoidins. To investigate this hypothesis, a mixture of 5-O-caffeoylquinic acid (5-CQA), the most abundant chlorogenic acid in green coffee beans, and (α1 → 5)-L-arabinotriose, structurally related to arabinogalactan side chains, was submitted to dry thermal treatments. The compounds formed during thermal processing were identified by electrospray ionization mass spectrometry (ESI-MS) and characterized by tandem MS (ESI-MS(n)). Compounds composed by one or two CQAs covalently linked with pentose (Pent) residues (1-12) were identified, along with compounds bearing a sugar moiety but composed exclusively by the quinic or caffeic acid moiety of CQAs. The presence of isomers was demonstrated by liquid chromatography online coupled to ESI-MS and ESI-MS(n). Pent1-2CQA were identified in coffee samples. These results give evidence for a diversity of chlorogenic acid-arabinose hybrids formed during roasting, opening new perspectives for their identification in melanoidin structures.