CE-LIF-MS n profiling of oligosaccharides in human milk and feces of breast-fed babies.

Mixtures of the complex human milk oligosaccharides (HMOs) are difficult to analyze and gastrointestinal bioconversion products of HMOs may complicate analysis even more. Their analysis, therefore, requires the combination of a sensitive and high-resolution separation technique with a mass identification tool. This study introduces for the first time the hyphenation of CE with an electrospray mass spectrometer, capable to perform multiple MS analysis (ESI-MS(n)) for the separation and characterization of HMOs in breast milk and feces of breast-fed babies. LIF was used for on- and off-line detections. From the overall 47 peaks detected in off-line CE-LIF electropherograms, 21 peaks could be unambiguously and 11 peaks could be tentatively assigned. The detailed structural characterization of a novel lacto-N-neo-tetraose isomer and a novel lacto-N-fucopentaose isomer was established in baby feces and pointed to gastrointestinal hydrolysis of higher-Mw HMOs. CE-LIF-ESI-MS(n) presents, therefore, a useful tool which contributes to an advanced understanding on the fate of individual HMOs during their gastrointestinal passage.

Overall charge and local charge density of pectin determines the enthalpic and entropic contributions to complexation with ß-lactoglobulin.

The complex formation between ß-lactoglobulin and pectins of varying overall charge and local charge density were investigated. Isothermal titration calorimetry experiments were carried out to determine the enthalpic contribution to the complex formation at pH 4.25 and various ionic strengths. Complex formation was found to be an exothermic process for all conditions. Combination with previously published binding constants by Sperber et al. (Sperber, B. L. H. M.; Cohen Stuart, M. A.; Schols, H. A.; Voragen, A. G. J.; Norde, W. Biomacromolecules 2009, 10, 3246-3252) allows for the determination of the changes in the Gibbs energy and the change in entropy of the system upon complex formation between ß-lactoglobulin and pectin. The local charge density of pectin is found to determine the balance between enthalpic and entropic contributions. For a high local charge density pectin, the main contribution to the Gibbs energy is of an enthalpic nature, supported by a favorable entropy effect due to the release of small counterions. A pectin with a low local charge density has a more even distribution of the enthalpic and entropic part to the change of the Gibbs energy. The enthalpic part is reduced due to the lower charge density, while the relative increase of the entropic contribution is thought to be caused by a change in the location of the binding place for pectin on the ß-lactoglobulin molecule. The association of the hydrophobic methyl esters on pectin with an exposed hydrophobic region on ß-lg results in the release of water molecules from the hydrophobic region and surrounding the methyl esters of the pectin molecule. An increase in the ionic strength decreases the enthalpic contribution due to the shielding of electrostatic attraction in favor of the entropic contribution, supporting the idea that the release of water molecules from hydrophobic areas plays a part in the complex formation.

Antiproliferative and proapoptotic actions of okra pectin on B16F10 melanoma cells.

The proliferation and apoptosis of metastatic melanoma cells are often abnormal. We have evaluated the action of a pectic rhamnogalacturonan obtained by hot buffer extraction of okra pods (okra RG-I) on melanoma cell growth and survival in vitro. We added okra RG-I containing an almost pure RG-I carrying very short galactan side chains to 2D (on tissue culture polystyrene, tPS) and 3D (on poly(2-hydroxyethylmethacrylate), polyHEMA) cultures of highly metastatic B16F10 mouse melanoma cells. We then analyzed cell morphology, proliferation index, apoptosis, cell cycle progression and the expression of adhesion molecules. Immunostaining and western blotting were used to assay galectin-3 (Gal-3) protein.Incubation with okra RG-I altered the morphology of B16F10 cells and significantly reduced their proliferation on both tPS and polyHEMA. The cell cycle was arrested in G2/M, and apoptosis was induced, particularly in cells on polyHEMA. The expression of N-cadherin and alpha5 integrin subunit was reduced and that of the multifunctional carbohydrate-binding protein, Gal-3, at the cell membrane increased.These findings suggest that okra RG-I induces apoptosis in melanoma cells by interacting with Gal-3. As these interactions might open the way to new melanoma therapies, the next step will be to determine just how they occur.

Binding of beta-lactoglobulin to pectins varying in their overall and local charge density.

The formation of complexes between proteins and polysaccharides is of great importance for many food systems like foams, emulsions, acidified milk drinks, and so on. The complex formation between beta-lactoglobulin (beta-lg) and pectins with a well-defined physicochemical fine structure has been studied to elucidate the influence of overall charge and local charge density of pectin on the complex formation. Binding isotherms of beta-lg to pectin are constructed using fluorescence anisotropy, which is shown to be an excellent technique for this purpose, as it is fast and requires low sample volumes. From the binding isotherms the maximal adsorbed amount, binding constant (k(obs)) and the cooperativity of binding are obtained at different ionic strengths. The Hill model is used to fit the binding isotherms and is shown to be preferable over a Langmuir fit. At pH 4.25, k(obs) shows a maximum at an ionic strength of 10 mM when using a low methyl esterified pectin (LMP) due to the balance of attractive and repulsive electrostatic forces between beta-lg and pectin and beta-lg neighbors. For two high methyl esterified pectins, one with a blockwise distribution of methyl esters (HMP(B)) and one with a random distribution (HMP(R)), this ionic strength maximum is absent and k(obs) decreases with increasing ionic strength. k(obs) is found to be largest for LMP and HMP(B) and considerably lower for HMP(R). A positive cooperativity is observed for both LMP (above an ionic strength of 45 mM) and HMP(R) (above an ionic strength of 15 mM) but not for HMP(B). Positive cooperativity is thought to be caused by a rearrangement of the pectin helix structure caused by binding of beta-lg, thus creating new or binding sites with a higher affinity. To attain strong binding of beta-lg to pectin it is preferable to use a pectin with a blockwise distribution of methyl esters. When complex formation takes place in high ionic strength media an LMP gives the best results, while at low ionic strength a high methyl esterified pectin with blockwise distribution may give better results, due to reduced electrostatic repulsion between both pectin and beta-lg and beta-lg neighbors.

The chain length of lignan macromolecule from flaxseed hulls is determined by the incorporation of coumaric acid glucosides and ferulic acid glucosides.

Lignan macromolecule from flaxseed hulls is composed of secoisolariciresinol diglucoside (SDG) and herbacetin diglucoside (HDG) moieties ester-linked by 3-hydroxy-3-methylglutaric acid (HMGA), and of p-coumaric acid glucoside (CouAG) and ferulic acid glucoside (FeAG) moieties ester-linked directly to SDG. The linker molecule HMGA was found to account for 11% (w/w) of the lignan macromolecule. Based on the extinction coefficients and RP-HPLC data, it was determined that SDG contributes for 62.0% (w/w) to the lignan macromolecule, while CouAG, FeAG, and HDG contribute for 12.2, 9.0, and 5.7% (w/w), respectively. Analysis of fractions of lignan macromolecule showed that the higher the molecular mass, the higher the proportion of SDG was. An inverse relation between the molecular mass and the proportion (%) CouAG+FeAG was found. Together with the structural information of oligomers of lignan macromolecule obtained after partial saponification, it is hypothesized that the amount of CouAG+FeAG present during biosynthesis determines the chain length of lignan macromolecule. Furthermore, the chain length was estimated from a model describing lignan macromolecule based on structural and compositional data. The average chain length of the lignan macromolceule was calculated to be three SDG moieties with CouAG or FeAG at each of the terminal positions, with a variation between one and seven SDG moieties.

Hydroxycinnamic acids are ester-linked directly to glucosyl moieties within the lignan macromolecule from flaxseed hulls.

In flaxseed hulls, lignans are present in an oligomeric structure. Secoisolariciresinol diglucoside (SDG), ester-linked to hydroxy-methyl-glutaric acid (HMGA), forms the backbone of this lignan macromolecule. The hydroxycinnamic acids p-coumaric acid glucoside (CouAG) and ferulic acid glucoside (FeAG) are also part of the lignan macromolecule. However, their position and type of linkage are still unknown. The aim of this study was to investigate how CouAG and FeAG are linked within the lignan macromolecule from flaxseed hulls. Fragments of the lignan macromolecule were obtained by partial saponification. After isolation of the fragments by preparative RP-HPLC, several key structures were identified by MS and NMR. Within the lignan macromolecule, CouAG is attached to the C-6 position of a glucosyl moiety of SDG. FeA is linked to the C-2 position of a glucosyl moiety of SDG. FeAG is ester-linked within the lignan macromolecule with its carboxyl group, but it remains unclear whether FeAG links to the C-2 or C-6 position of SDG. Attachment of HMGA to the glucosyl moiety of CouAG or FeAG was not observed. The results clearly show that within the lignan macromolecule, the hydroxycinnamic acids are linked directly via an ester bond to the glucosyl moiety of SDG.

Bifidobacterium carbohydrases-their role in breakdown and synthesis of (potential) prebiotics.

There is an increasing interest to positively influence the human intestinal microbiota through the diet by the use of prebiotics and/or probiotics. It is anticipated that this will balance the microbial composition in the gastrointestinal tract in favor of health promoting genera such as Bifidobacterium and Lactobacillus. Carbohydrates like non-digestible oligosaccharides are potential prebiotics. To understand how these bacteria can grow on these carbon sources, knowledge of the carbohydrate-modifying enzymes is needed. Little is known about the carbohydrate-modifying enzymes of bifidobacteria. The genome sequence of Bifidobacterium adolescentis and Bifidobacterium longum biotype longum has been completed and it was observed that for B. longum biotype longum more than 8% of the annotated genes were involved in carbohydrate metabolism. In addition more sequence data of individual carbohydrases from other Bifidobacterium spp. became available. Besides the degradation of (potential) prebiotics by bifidobacterial glycoside hydrolases, we will focus in this review on the possibilities to produce new classes of non-digestible oligosaccharides by showing the presence and (transglycosylation) activity of the most important carbohydrate modifying enzymes in bifidobacteria. Approaches to use and improve carbohydrate-modifying enzymes in prebiotic design will be discussed.

The flavonoid herbacetin diglucoside as a constituent of the lignan macromolecule from flaxseed hulls.

Lignans in flaxseed are known to be part of a macromolecule in which they are connected through the linker-molecule hydroxy-methyl-glutaric acid (HMGA). In this study, the lignan macromolecule was extracted from flaxseed hulls and degraded to its monomeric constituents by complete saponification. Besides secoisolariciresinol diglucoside (SDG), the phenolic compounds p-coumaric acid glucoside (CouAG) and ferulic acid glucoside (FeAG) were isolated, which was expected based on indications from the literature. Also the flavonoid herbacetin diglucoside (HDG) was found. The presence of HDG was confirmed by NMR following preparative RP-HPLC purification. Also the presence of the three other constituents (CouAG, FeAG and SDG) was confirmed by NMR. To prove that HDG is a substructure of the lignan macromolecule, the macromolecule was fragmented by partial saponification. A fragment consisting of HDG and HMGA was indicated. This fragment was isolated by preparative RP-HPLC and its identity was confirmed by NMR. It is concluded that the flavonoid HDG is a substructure of the lignan macromolecule from flaxseed hulls and that it is incorporated in the macromolecule via the same linker-molecule as SDG.

Bilberry xyloglucan--novel building blocks containing beta-xylose within a complex structure.

Bilberries are known to have one of the most complex xyloglucan structures described in the plant kingdom until now. To characterise this structure, xyloglucans were enzymatically degraded and the oligosaccharides obtained were analysed. More than 20 different building blocks were found to make up the xyloglucan polymer. Bilberry xyloglucan was of XXXG-type, but some XXG-type oligomers were present, as well. The building blocks contain galactose-xylose (L) and fucose-galactose-xylose (F) side chains. In both side chains, the galactose units can be acetylated. In addition, beta-xylose-alpha-xylose (U) side chains were shown. This U chain was present in three building blocks described before (XUXG, XLUG and XUFG) and four novel blocks that have not been described in the literature previously: XUG, XUUG, XLUG and XXUG.

Aggregation of beta-lactoglobulin regulated by glucosylation.

A large number of proteins are glycosylated, either in vivo or as a result of industrial processing. Even though the effect of glycosylation on the aggregation of proteins has been studied extensively in the past, some reports show that the aggregation process is accelerated, whereas others found that the process is inhibited by glycosylation. This paper investigates the reasons behind these controversial results as well as the potential mechanism of the effect of glucosylation on aggregation using bovine beta-lactoglobulin as a model. Glucosylation was found to inhibit denaturant-induced aggregation, whereas heat-induced aggregation was accelerated. It was also found that the kinetic partitioning from an unfolded state was driven toward refolding for glucosylated protein, whereas aggregation was the preferred route for the nonglucosylated protein.

A new group of exo-acting family 28 glycoside hydrolases of Aspergillus niger that are involved in pectin degradation.

The fungus Aspergillus niger is an industrial producer of pectin-degrading enzymes. The recent solving of the genomic sequence of A. niger allowed an inventory of the entire genome of the fungus for potential carbohydrate-degrading enzymes. By applying bioinformatics tools, 12 new genes, putatively encoding family 28 glycoside hydrolases, were identified. Seven of the newly discovered genes form a new gene group, which we show to encode exoacting pectinolytic glycoside hydrolases. This group includes four exo-polygalacturonan hydrolases (PGAX, PGXA, PGXB and PGXC) and three putative exo-rhamnogalacturonan hydrolases (RGXA, RGXB and RGXC). Biochemical identification using polygalacturonic acid and xylogalacturonan as substrates demonstrated that indeed PGXB and PGXC act as exo-polygalacturonases, whereas PGXA acts as an exo-xylogalacturonan hydrolase. The expression levels of all 21 genes were assessed by microarray analysis. The results from the present study demonstrate that exo-acting glycoside hydrolases play a prominent role in pectin degradation.

Effect of pretreatment severity on xylan solubility and enzymatic breakdown of the remaining cellulose from wheat straw.

The effect of process conditions used for wheat straw pretreatments on the liquor- and residue-composition was studied. Hereto, the pretreatment conditions were expressed in a 'combined severity R(0)(')-factor'. The higher the combined severity factor (R(0)(')) the more xylan was released from the wheat straw, but the more xylan decomposed and furfural formation occurred. The percentage of residual xylan present after pretreatment appeared to be a good indicator concerning cellulose degradability or bio-ethanol production. Namely, cellulose degradation by using commercial enzymes was higher at higher severities corresponding to a lower amount of residual xylan. The xylan release and degradation was studied in more detail by using HPSEC and MALDI-TOF mass spectrometry. The more severe the treatment the more (acetylated) xylose oligomers with a DP lower than nine were analysed. The presence of (acetylated) xylans with a DP of 9-25 increased slightly from low to medium severity. The quantification of the DP-distribution of the (acetylated) xylans released proved to be a good tool to predict cellulose degradability.

Biochemical characterization of the major sorghum grain peroxidase.

The major cationic peroxidase in sorghum grain (SPC4) , which is ubiquitously present in all sorghum varieties was purified to apparent homogeneity, and found to be a highly basic protein (pI approximately 11). MS analysis showed that SPC4 consists of two glycoforms with molecular masses of 34,227 and 35,629 Da and it contains a type-b heme. Chemical deglycosylation allowed to estimate sugar contents of 3.0% and 6.7% (w/w) in glycoform I and II, respectively, and a mass of the apoprotein of 33,246 Da. High performance anion exchange chromatography allowed to determine the carbohydrate constituents of the polysaccharide chains. The N-terminal sequence of SPC4 is not blocked by pyroglutamate. MS analysis showed that six peptides, including the N-terminal sequence of SPC4 matched with the predicted tryptic peptides of gene indice TC102191 of sorghum chromosome 1, indicating that TC102191 codes for the N-terminal part of the sequence of SPC4, including a signal peptide of 31 amino acids. The N-terminal fragment of SPC4 (213 amino acids) has a high sequence identity with barley BP1 (85%), rice Prx23 (90%), wheat WSP1 (82%) and maize peroxidase (58%), indicative for a common ancestor. SPC4 is activated by calcium ions. Ca2+ binding increased the protein conformational stability by raising the melting temperature (Tm) from 67 to 82 degrees C. SPC4 catalyzed the oxidation of a wide range of aromatic substrates, being catalytically more efficient with hydroxycinnamates than with tyrosine derivatives. In spite of the conserved active sites, SPC4 differs from BP1 in being active with aromatic compounds above pH 5.

Importance of physical vs. chemical interactions in surface shear rheology.

The stability of adsorbed protein layers against deformation has in literature been attributed to the formation of a continuous gel-like network. This hypothesis is mostly based on measurements of the increase of the surface shear elasticity with time. For several proteins this increase has been attributed to the formation of intermolecular disulfide bridges between adsorbed proteins. However, according to an alternative model the shear elasticity results from the low mobility of the densely packed proteins. To contribute to this discussion, the actual role of disulfide bridges in interfacial layers is studied. Ovalbumin was thiolated with S-acetylmercaptosuccinic anhydride (S-AMSA), followed by removal of the acetylblock on the sulphur atom, resulting in respectively blocked (SX) and deblocked (SH) ovalbumin variants. This allows comparison of proteins with identical amino acid sequence and similar globular packing and charge distribution, but different chemical reactivity. The presence and reactivity of the introduced, deblocked sulfhydryl groups were confirmed using the sulfhydryl-disulfide exchange index (SEI). Despite the reactivity of the introduced sulfhydryl groups measured in solution, no increase in the surface shear elasticity could be detected with increasing reactivity. This indicates that physical rather than chemical interactions determine the surface shear behaviour. Further experiments were performed in bulk solution to study the conditions needed to induce covalent aggregate formation. From these studies it was found that mere concentration of proteins (to 200 mg/mL, equivalent to a surface concentration of around 2 mg/m(2)) is not sufficient to induce significant aggregation to form a continuous network. In view of these results, it was concluded that the adsorbed layer should not be considered a gelled network of aggregated material (in analogy with three-dimensional gels formed from heating protein solutions). Rather, it would appear that the adsorbed proteins form a highly packed system of proteins with net-repulsive interactions.

A comparison of liquid chromatography, capillary electrophoresis, and mass spectrometry methods to determine xyloglucan structures in black currants.

Different separation (HPAEC, RP-HPLC, CE) and identification (MALDI-TOF-MS, ESI-MS(n)) techniques were compared to analyse oligosaccharides obtained after incubation of xyloglucan with endo-glucanase. It was possible to analyse xyloglucan oligosaccharides with each technique. Several techniques, including off line (HPAEC-MALDI-TOF-MS) or online (CE-ESI-MS(n), RP-HPLC-ESI-MS(n)) connection provided complementary information on xyloglucan structure. Online CE-MS and RP-HPLC-MS are described for the first time in xyloglucan analysis. Advantages and disadvantages of the techniques for different purposes such as structural characterisation of oligosaccharides or oligosaccharide profiling are discussed. Black currant xyloglucans had a rather simple XXXG-type structure with galactose and fucose containing side chains.

Capillary electrophoresis fingerprinting, quantification and mass-identification of various 9-aminopyrene-1,4,6-trisulfonate-derivatized oligomers derived from plant polysaccharides.

Various plant polysaccharide derived mono- and oligosaccharides were derivatized with the fluorescent 9-aminopyrene-1,4,6-trisulfonate (APTS) and subjected to capillary electrophoresis (CE) in combination with laser induced fluorescence (LIF) detection. CE-LIF was suitable for mol-based quantification of various APTS-monosaccharides. CE-LIF of APTS-oligosaccharides showed high resolutions, while analysis times were at maximum 15 min. The coupling of CE to electrospray-iontrap mass spectrometery (MS) with online UV detection showed to be a powerful technique in the identification of APTS-oligosaccharides. For the first time, various APTS-xylo-oligosaccharides, having either no, O-acetyl, arabinosyl or xylosyl substitutions at varying positions, were identified by using CE-LIF and CE-MS(n).

Combined enzymatic and high-pressure processing affect cell wall polysaccharides in berries.

The effect of high-pressure processing (HPP) on cell wall polysaccharides in berries was investigated. HPP decreased the degree of methyl esterification (DM), probably by activation of pectin methyl esterase (PME), and improved the extractability of pectins. When commercial enzyme mixtures were added to mashed berries, a synergistic effect was observed between treatment with commercial enzymes and HPP. Compared to treatment at atmospheric pressure, pectic polysaccharides were degraded to a larger extent when HPP was used. In contrast, hemicelluloses were hardly affected by the added enzymes when HPP was included, although they were degraded during similar treatment at atmospheric pressure. Additionally, the activity of rhamnose-releasing enzymes present in minor quantities might be enhanced after HPP, resulting in a decrease of rhamnose in the polymeric cell wall material. These results exploring the effect of HPP at representative conditions clearly point out the potential of HPP for polysaccharide modification.

Do sulfhydryl groups affect aggregation and gelation properties of ovalbumin?

The aim of this work is to evaluate the impact of sulfhydryl groups on ovalbumin aggregation and gelation. Ovalbumin was chemically modified to add sulfhydryl groups in various degrees. The rate of aggregation was not affected by the introduction of sulfhydryl groups, and disulfide bond formation was preceded by physical interactions. Hence, disulfide interactions may not be the driving force for the aggregation of ovalbumin. Investigation of the aggregates and gels by electron microscopy and rheology suggested that a critical number of sulfhydryl groups can be introduced beyond which the microstructure of the aggregates transforms from fibrillar into amorphous. Rheological studies further suggested that covalent networks, once formed, do not have the possibility to rearrange, reducing the possibility to attain a stronger network. These results show that, even though aggregation of ovalbumin may be primarily driven by physical interactions, formed disulfide bonds are important to determine the resulting aggregate morphology and rheological properties.

Effects of protein composition and enzymatic activity on formation and properties of potato protein stabilized emulsions.

In the present study emulsions were made with various potato protein preparations, which varied in protease inhibitor and patatin content. These emulsions were characterized with respect to average droplet size, plateau surface excess, and the occurrence of droplet aggregation. Droplet aggregation occurred only with potato protein preparations that contained a substantial amount of protease inhibitors and could be prevented only at pH 3. The average droplet size of the emulsions made with potato proteins appeared to be related to the patatin content of the preparation used. Average droplet size was found to be dominated by the patatin-catalyzed lipolytic release of surface active fatty acids and monoglycerides from the tricaprylin oil phase during the emulsification process. Addition of monoglycerides and especially fatty acids, at concentrations representative of those during emulsification, was shown to cause a stronger and much faster decrease of the interfacial tension than that with protein alone and to result in a drastic decrease in droplet size. The patatin used was shown to have a lipolytic activity of 820 units/g with emulsified tricaprylin as the substrate. Because of the droplet aggregating properties of the protease inhibitors, the patatin-rich potato preparations seem to be the most promising for food emulsion applications over a broad pH range, provided the lipolytic activity can be diminished or circumvented.

The adsorption and unfolding kinetics determines the folding state of proteins at the air-water interface and thereby the equation of state.

Unfolding of proteins has often been mentioned as an important factor during the adsorption process at air-water interfaces and in the increase of surface pressure at later stages of the adsorption process. This work focuses on the question whether the folding state of the adsorbed protein depends on the rate of adsorption to the interface, which can be controlled by bulk concentration. Therefore, the adsorption of proteins with varying structural stabilities at several protein concentrations was studied using ellipsometry and surface tensiometry. For beta-lactoglobulin the adsorbed amount (Gamma) needed to reach a certain surface pressure (Pi) decreased with decreasing bulk concentration. Ovalbumin showed no such dependence. To verify whether this difference in behavior is caused by the difference in structural stability, similar experiments were performed with cytochrome c and a destabilized variant of this protein. Both proteins showed identical Pi-Gamma, and no dependence on bulk concentration. From this work it was concluded that unfolding will only take place if the kinetics of adsorption is similar or slower than the kinetics of unfolding. The latter depends on the activation energy of unfolding (which is in the order of 100-300 kJ/mol), rather than the free energy of unfolding (typically 10-50 kJ/mol).