M.L. Sanz

Derivatization of carbohydrates for GC and GC–MS analyses

GC and GC-MS are excellent techniques for the analysis of carbohydrates; nevertheless the preparation of adequate derivatives is necessary. The different functional groups that can be found and the diversity of samples require specific methods. This review aims to collect the most important methodologies currently used, either published as new procedures or as new applications, for the analysis of carbohydrates. A high diversity of compounds with diverse functionalities has been selected: neutral carbohydrates (saccharides and polyalcohols), sugar acids, amino and iminosugars, polysaccharides, glycosides, glycoconjugates, anhydrosugars, difructose anhydrides and products resulting of Maillard reaction (osuloses, Amadori compounds). Chiral analysis has also been considered, describing the use of diastereomers and derivatives to be eluted on chiral stationary phases.

Effect of prebiotic carbohydrates on the growth and tolerance of Lactobacillus

Resistance to gastrointestinal conditions is a requirement for bacteria to be considered probiotics. In this work, we tested the resistance of six different Lactobacillus strains and the effect of carbon source to four different gastrointestinal conditions: presence of α-amylase, pancreatin, bile extract and low pH. Novel galactooligosaccharides synthesized from lactulose (GOS-Lu) as well as commercial galactooligosaccharides synthesized from lactose (GOS-La) and lactulose were used as carbon sources and compared with glucose. In general, all strains grew in all carbon sources, although after 24 h of fermentation the population of all Lactobacillus strains was higher for both types of GOS than for glucose and lactulose. No differences were found among GOS-Lu and GOS-La. α-amylase and pancreatin resistance was retained at all times for all strains. However, a dependence on carbon source and Lactobacillus strain was observed for bile extract and low pH resistance. High hydrophobicity was found for all strains with GOS-Lu when compared with other carbon sources. However, concentrations of lactic and acetic acids were higher in glucose and lactulose than GOS-Lu and GOS-La. These results show that the resistance to gastrointestinal conditions and hydrophobicity is directly related with the carbon source and Lactobacillus strains. In this sense, the use of prebiotics as GOS and lactulose could be an excellent alternative to monosaccharides to support growth of probiotic Lactobacillus strains and improve their survival through the gastrointestinal tract.

The RIM101 Pathway Contributes to Yeast Cell Wall Assembly and Its Function Becomes Essential in the Absence of Mitogen-Activated Protein Kinase Slt2p

ABSTRACT The Saccharomyces cerevisiae ynl294cΔ (rim21Δ) mutant was identified in our lab owing to its moderate resistance to calcofluor, although it also displayed all of the phenotypic traits associated with its function as the putative sensor (Rim21p) of the RIM101 pathway. rim21Δ also showed moderate hypersensitivity to sodium dodecyl sulfate, caffeine, and zymolyase, and the cell wall compensatory response in this mutant was very poor, as indicated by the almost complete absence of Slt2 phosphorylation and the modest increase in chitin synthesis after calcofluor treatment. However, the cell integrity pathway appeared functional after caffeine treatment or thermal stress. rim21Δ and rim101Δ mutant strains shared all of the cell-wall-associated phenotypes, which were reverted by the expression of Rim101-531p, the constitutively active form of this transcription factor. Therefore, the absence of a functional RIM101 pathway leads to cell wall defects. rim21Δ, as well as rim101Δ, was synthetic lethal with slt2Δ, a synthetic defect alleviated by osmotic stabilization of the media. The double mutants grown in osmotically stabilized media were extremely hypersensitive to zymolyase and showed thicker cell walls, with poorly defined mannoprotein layers. In contrast, rim21Δ rlm1Δ and rim101Δ rlm1Δ double mutants were fully viable. Taken together, these results show that the RIM101 pathway participates directly in cell wall assembly and that it acts in parallel with the protein kinase C pathway (PKC) in this process independently of the transcriptional effect of the compensatory response mediated by this route. In addition, these results provide new experimental evidence of the direct involvement of the PKC signal transduction pathway through the Sltp2 kinase in the construction of yeast cell walls.

Mass spectrometric characterization of glycated β-lactoglobulin peptides derived from galacto-oligosaccharides surviving the in vitro gastrointestinal digestion

A mass spectrometric study has been carried out to elucidate the structures of glycated peptides obtained after in vitro gastrointestinal digestion of bovine β-lactoglobulin (β-LG) glycated with prebiotic galacto-oligosaccharides (GOS). The digests of both native and glycated β-LG were analyzed by MALDI-MS, LC-ESI-MS, and LC-ESI-MS/MS. MALDI-MS profiles showed marked differences mainly related to the lower intensity of ions corresponding to the digest of glycated β-LG. Overall, 58 and 23 unglycated peptides covering 97% and 63% of the mature β-LG sequence could be identified in the digests of native and glycated samples, respectively. The LC-ESI-MS analyses corroborated the MALDI-MS results regarding the unglycated peptides but they also enabled an extensive investigation into the digest of glycated β-LG. Thus, a total of 19 peptides glycated with GOS from two to seven hexose units could be identified. The tandem mass spectra of glycated peptides were mostly characterized by two neutral losses of 1026/1056, 864/894, 702/732, 540/570, 378/408, and 216/246 u, corresponding to the formation of the furylium ion and its subsequent “CHOH” loss, indicative of the peptide glycation with hepta-, hexa-, penta-, tetra-, tri-, and disaccharides, respectively. Also, other minor ionic species containing the furylium ring linked to different galactose units could be also detected, showing the diversity of the fragmentation pattern of peptides glycated with larger size carbohydrates. Finally, the putative GOS glycation sites could be determined at the NH2-terminal Leu residue and at Lys residues located in positions 14, 47, 75, 77, 83, 91, 100, 135, and 138.

Characterization ofO-trimethylsilyl oximes of disaccharides by gas chromatography-mass spectrometry

SummaryTwenty-three disaccharides have been analysed by GC-MS as theirO-trimethylsilyl oximes. The relationships between their mass spectral fragmentation and their structural characteristics have been studied in order to use GC-MS information for qualitative purposes. The presence of some structural features, such as the 1–2 or 1–6 linkages, can be estimated from the relative intensity of fragment ions. The method has been applied to the determination of the components present in the disaccharide fraction obtained from lactose treatment with β-galactosidase fromK. fragilis.

Carbohydrate Composition of High-Fructose Corn Syrups (HFCS) Used for Bee Feeding: Effect on Honey Composition

In this study, the carbohydrate composition of high-fructose corn syrups (HFCS) from commercial manufacturers as well as from beekeepers was characterized by GC-MS. Sucrose syrups (SS) were also included in this work for comparison. Fructosyl-fructoses and some unknown carbohydrates, which could correspond to fructosyl-glucoses, have been detected in HFCS for the first time, whereas SS were mainly characterized by the high contents of sucrose. Hydroxymethylfurfural (HMF) content of samples supplied by beekeepers was much more variable; the mean level of HMF was 64.61 ppm (+/-16.92 ppm, 95% CI ranging from 26.91 to 102.31 ppm). Syrups were used to feed caged bees and the resulting honeys produced were analyzed in order to determine their influence in carbohydrate composition. Fructosyl-fructoses were mainly detected in honeys from bees fed with HFCS, but not from those honeys coming from free-flying bees or bees fed with SS.

Chitin synthase III requires Chs4p-dependent translocation of Chs3p into the plasma membrane

In Saccharomyces cerevisiae, Chs4p is required for chitin synthase III (CSIII) activity and hence for chitin synthesis. This protein is transported in vesicles in a polarized fashion independently of the other Chs proteins. Its association with membranes depends not only on prenylation, but also on its interaction with other proteins, mainly Chs3p, which is the catalytic subunit of CSIII and is able to properly direct Chs4p to the bud neck in the absence of prenylation. Chs4p is present in functionally limiting amounts and its overexpression increases Chs3p accumulation at the plasma membrane with a concomitant increase in chitin synthesis. In the absence of Chs4p, Chs3p is delivered to the plasma membrane but fails to accumulate there because it is rapidly endocytosed and accumulates in intracellular vesicles. A blockade of endocytosis stops Chs3p internalization, triggering a significant increase in chitin synthesis. This blockade is independent of Chs4p function, allowing the accumulation of Chs3p at the plasma membrane even in the chs4Δ mutant. However, the absence of Chs4p renders CSIII functionally inactive, independently of Chs3p accumulation at the plasma membrane. Chs4p thus promotes Chs3p translocation into the plasma membrane in a stable and active form. Proper CSIII turnover is maintained through the endocytic internalization of Chs3p.

Evaluation of different operation modes of high performance liquid chromatography for the analysis of complex mixtures of neutral oligosaccharides

Chromatographic methods based on different HPLC operation modes, reverse phase (RP), high performance anion exchange chromatography (HPAEC), graphitized carbon chromatography (GCC) and hydrophilic interaction liquid chromatography (HILIC), have been developed and compared for the analysis of complex mixtures of neutral oligosaccharides with functional properties. Whereas GCC gave the best chromatographic separation of isomeric oligosaccharides with the same molecular weight (R(s) values in the range 1.0-4.0 and 2.4-5.6 for tetra- and pentasaccharides, respectively), HILIC provided the best results for mixtures including oligosaccharides of different degrees of polymerization (R(s) values of maltooligosaccharides between 3.4 and 6.2). Validation of the HILIC LC-MS method proved its utility for the analysis of oligosaccharide mixtures with functional properties: relative standard deviations lower than 10%, LODs and LOQs in the range 12.7-130.2 ng mL(-1) and 39.3-402.2 ng mL(-1), respectively, and linearity up to 10-20 μg mL(-1). Quantitative data for fructooligosaccharides, gentiooligosaccharides and dextransucrase cellobiose acceptor oligosaccharides were obtained by using this method.

Control of chitin synthesis through Shc1p, a functional homologue of Chs4p specifically induced during sporulation

The Saccharomyces cerevisiae SHC1 gene encodes a protein with a high homology to Chs4p, a positive regulator of chitin synthase III (CSIII) during vegetative growth. SHC1 is not expressed during vegetative growth but is strongly induced during sporulation as a mid‐late gene. shc1/shc1 mutants do not show any defect in the total rate of sporulation and meiosis occurs normally. However, shc1/shc1 ascospores be‐come highly permeable to DAPI, much more sensitive to glusulase treatment, and have very low levels of chitosan in their cell walls. All these observations indicate that Shc1p is required for proper maturation of the ascospore through its participation in the synthesis of the chitosan layer. Lack of SHC1 during sporulation can be partially compensated by over‐expression of the CHS4 gene. During vegetative growth, SHC1 has no apparent function but, when ectopically overexpressed, it can substitute Chs4p as an activator of the CSIII activity; however, Shc1p fails to localize it properly, as Chs4p does. In conclusion, S. cerevisiae contains two functionally redundant genes in the control of CSIII activity: CHS4, whose function is restricted to vegetative growth because Chs4p is rapidly degraded during sporulation, and SHC1, whose function in cell wall ascospore assembly is transcriptionally restricted to the sporulation process.

Hydrophilic interaction liquid chromatography coupled to mass spectrometry for the characterization of prebiotic galactooligosaccharides.

Three different stationary phases (sulfoalkylbetaine zwitterionic, polyhydroxyethyl aspartamide and ethylene bridge hybrid (BEH) with trifunctionally bonded amide), operating at hydrophilic interaction liquid chromatographic (HILIC) mode, have been assayed and compared for the analysis of complex mixtures of galactooligosaccharides (GOS). Chromatographic methods have been optimized to obtain the best separation between two consecutive galactose containing standards and maltodextrins, measured on the basis of resolution. Influence of several factors such as chemical modifiers (formic acid, ammonium acetate and ammonium hydroxide), organic solvent and gradients of the mobile phases in the separation of oligosaccharides have been studied. The best results were achieved on the BEH amide stationary phase, using acetonitrile:water with 0.1% ammonium hydroxide as mobile phase, where the most of oligosaccharides were successfully resolved. Characteristic MS(2) fragmentation profiles of disaccharides containing galactose, glucose and/or fructose units with different linkages were evaluated and used for the characterization of di-, tri- and tetrasaccharides of three commercial prebiotic GOS mixtures (GOS-1, GOS-2 and GOS-3) by HILIC-MS(n). Similar qualitative and quantitative composition was observed for GOS-1 and GOS-3, whereas different linkages and abundances were detected for GOS-2. In general, (1→4) and (1→6) glycosidic linkages were the main structures found in GOS, although (1→2) and (1→3) linkages were also identified. Regarding molecular weight, up to pentasaccharides were detected in these samples, disaccharides being the most abundant carbohydrates.