QSAR of 1,4-benzoxazin-3-one antimicrobials and their drug design perspectives.

Synthetic derivatives of 1,4-benzoxazin-3-ones have been shown to possess promising antimicrobial activity, whereas their natural counterparts were found lacking in this respect. In this work, quantitative structure-activity relationships (QSAR) of natural and synthetic 1,4-benzoxazin-3-ones as antimicrobials were established. Data published in literature were curated into an extensive dataset of 111 compounds. Descriptor selection was performed by a genetic algorithm. QSAR models revealed differences in requirements for activity against fungi, gram-positive and gram-negative bacteria. Shape, VolSurf, and H-bonding property descriptors were frequently picked in all models. The models obtained for gram-positive and gram-negative bacteria showed good predictive power (Q2Ext 0.88 and 0.85, respectively). Based on the models generated, an additional set of 1,4-benzoxazin-3-ones, for which no antimicrobial activity had been determined in literature, were evaluated in silico. Additionally, newly designed lead compounds with a 1,4-benzoxazin-3-one scaffold were generated in silico by varying the positions and combinations of substituents. Two of these were predicted to be up to 5 times more active than any of the compounds in the current dataset. The 1,4-benzoxazin-3-one scaffold was concluded to possess potential for the design of new antimicrobial compounds with potent antibacterial activity, a multitarget mode of action, and possibly reduced susceptibility to gram negatives' efflux pumps.

Quantification of the catalytic performance of C1-cellulose-specific lytic polysaccharide monooxygenases.

Lytic polysaccharide monooxygenases (LPMOs) have recently been shown to significantly enhance the degradation of recalcitrant polysaccharides and are of interest for the production of biochemicals and bioethanol from plant biomass. The copper-containing LPMOs utilize electrons, provided by reducing agents, to oxidatively cleave polysaccharides. Here, we report the development of a ß-glucosidase-assisted method to quantify the release of C1-oxidized gluco-oligosaccharides from cellulose by two C1-oxidizing LPMOs from Myceliophthora thermophila C1. Based on this quantification method, we demonstrate that the catalytic performance of both MtLPMOs is strongly dependent on pH and temperature. The obtained results indicate that the catalytic performance of LPMOs depends on the interaction of multiple factors, which are affected by both pH and temperature.

Laminaria digitata phlorotannins decrease protein degradation and methanogenesis during in vitro ruminal fermentation.

BACKGROUND: Phlorotannins (PhTs) are marine tannins consisting of phloroglucinol subunits connected via carbon-carbon and ether linkages. These have non-covalent protein binding properties and are, therefore, expected to be beneficial in protecting protein from hydrolysis during ruminal fermentation. In this study, the effectiveness of a methanolic PhTs extract from Laminaria digitata (10, 20, 40, 50, 75 and 100 g kg-1 tannin-free grass silage, with or without addition of polyethylene glycol (PEG), was investigated in vitro on protection of dietary protein and reduction of methane (CH4 ) in ruminal fluid. RESULTS: Addition of PhTs had linear (P < 0.0001) and quadratic (P = 0.0003) effects on gas and CH4 production, respectively. Optimal dosage of PhTs was 40 g kg-1 as at this point CH4 decreased (P < 0.0001) from 24.5 to 15.2 mL g-1 organic matter (OM), without affecting gas production (P = 0.3115) and total volatile fatty acids (P = 1.000). Ammonia trended (P = 0.0903) to decrease from 0.49 to 0.39 mmol g-1 OM, indicating protection of protein. Addition of PEG inhibited the effect of tannins at all dosage levels, and none of the fermentation parameters differed from the control. CONCLUSION: PhTs effectively protected protein from fermentation and reduced ruminal methanogenesis. © 2017 Society of Chemical Industry.

Quantification of Lignin and Its Structural Features in Plant Biomass Using 13C Lignin as Internal Standard for Pyrolysis-GC-SIM-MS.

Understanding the mechanisms underlying plant biomass recalcitrance at the molecular level can only be achieved by accurate analyses of both the content and structural features of the molecules involved. Current quantification of lignin is, however, majorly based on unspecific gravimetric analysis after sulfuric acid hydrolysis. Hence, our research aimed at specific lignin quantification with concurrent characterization of its structural features. Hereto, for the first time, a polymeric 13C lignin was used as internal standard (IS) for lignin quantification via analytical pyrolysis coupled to gas chromatography with mass-spectrometric detection in selected ion monitoring mode (py-GC-SIM-MS). In addition, relative response factors (RRFs) for the various pyrolysis products obtained were determined and applied. First, 12C and 13C lignin were isolated from nonlabeled and uniformly 13C labeled wheat straw, respectively, and characterized by heteronuclear single quantum coherence (HSQC), nuclear magnetic resonance (NMR), and py-GC/MS. The two lignin isolates were found to have identical structures. Second, 13C-IS based lignin quantification by py-GC-SIM-MS was validated in reconstituted biomass model systems with known contents of the 12C lignin analogue and was shown to be extremely accurate (>99.9%, R2 > 0.999) and precise (RSD < 1.5%). Third, 13C-IS based lignin quantification was applied to four common poaceous biomass sources (wheat straw, barley straw, corn stover, and sugar cane bagasse), and lignin contents were in good agreement with the total gravimetrically determined lignin contents. Our robust method proves to be a promising alternative for the high-throughput quantification of lignin in milled biomass samples directly and simultaneously provides a direct insight into the structural features of lignin.

Phlorotannin Composition of Laminaria digitata.

INTRODUCTION: Phlorotannins are complex mixtures of phloroglucinol oligomers connected via C-C (fucols) or C-O-C (phlorethols) linkages. Their uniformity in subunits and large molecular weight hamper their structural analysis. Despite its commercial relevance for alginate extraction, phlorotannins in Laminaria digitata have not been studied. OBJECTIVE: To obtain quantitative and structural information on phlorotannins in a methanolic extract from L. digitata. METHODOLOGY: The combined use of 13 C and 1 H NMR spectroscopy allowed characterisation of linkage types and extract purity. The purity determined was used to calibrate the responses obtained with the colorimetric 2,4-dimethoxybenzaldehyde (DMBA) and Folin-Ciocalteu (FC) assays. Using NP-flash chromatography, phlorotannin fractions separated on oligomer size were obtained and enabled structural and molecular weight characterisation using ESI-MS and MALDI-TOF-MS. RESULTS: The fucol-to-phlorethol linkage ratio was 1:26 and the extract was 60.1% pure, determined by NMR spectroscopy. For DMBA, the response of the extract was 12 times lower than that of phloroglucinol, whereas there was no difference for FC. By accounting for differences in response, the colorimetric assays were applicable for quantification using phloroglucinol as a standard. The phlorotannin content was around 4.5% DM. Fucol- and phlorethol-linkage types were annotated based on characteristic MSn fragmentations. Structural isomers of phlorotannins up to a degree of polymerisation of 18 (DP18) were annotated and identification of several isomers hinted at branched phloroglucinol oligomers. With MALDI-TOF-MS phlorotannins up to DP27 were annotated. CONCLUSION: By combining several analytical techniques, phlorotannins in L. digitata were quantified and characterised with respect to fucol-to-phlorethol linkage ratio, molecular weight (distribution), and occurrence of structural isomers. Copyright © 2017 John Wiley & Sons, Ltd.

Transgenic modification of potato pectic polysaccharides also affects type and level of cell wall xyloglucan.

BACKGROUND: Genes encoding pectic enzymes were introduced into wild-type potato Karnico. Cell wall materials were extracted from Karnico and transgenic lines expressing ß-galactosidase (ß-Gal-14) or rhamnogalacturonan lyase (RGL-18). Pectic polysaccharides from the ß-Gal-14 transgenic line exhibited rhamnogalacturonan-I structural elements with shorter galactan side chains, whereas the RGL-18 transgenic line had less rhamnogalacturonan-I structures than Karnico. Xyloglucan in primary cell walls interacts with pectin and other cell wall polysaccharides and controls cell growth. RESULTS: Xyloglucan extracts from transgenic lines had different levels of monosaccharides compared to wild-type. Most XXGG-type xyloglucans from Karnico and RGL-18 alkali-extractable extracts predominantly consisted of XXGG and XSGG building blocks. Karnico and RGL-18 4 mol L-1 extracts had small proportions of the XXXG-type xyloglucan, whereas ß-Gal-14 extracts also contained the XXXG-type xyloglucan. The peak ratios of XSGG/XXGG were 1.9, 2.4 and 1.1 for 4 mol L-1 extracts of Karnico, RGL-18 and ß-Gal-14 lines, respectively. CONCLUSION: After transgenic modification on pectin, the xyloglucan building blocks may have been changed. The ß-Gal-14 lines mostly present XXXG-type repeating units instead of the XXGG-type in 4 mol L-1 extracts. The ratio of XSGG/XXGG repeating units also changed, indicating that the transgenic modification of pectin altered xyloglucan structure during plant development. © 2016 Society of Chemical Industry.

Effects of Toasting Time on Digestive Hydrolysis of Soluble and Insoluble 00-Rapeseed Meal Proteins.

Thermal damage to proteins can reduce their nutritional value. The effects of toasting time on the kinetics of hydrolysis, the resulting molecular weight distribution of 00-rapeseed meal (RSM) and the soluble and insoluble protein fractions separated from the RSM were studied. Hydrolysis was performed with pancreatic proteases to represent in vitro protein digestibility. Increasing the toasting time of RSM linearly decreased the rate of protein hydrolysis of RSM and the insoluble protein fractions. The extent of hydrolysis was, on average, 44% higher for the insoluble compared with the soluble protein fraction. In contrast, the rate of protein hydrolysis of the soluble protein fraction was 3-9-fold higher than that of the insoluble protein fraction. The rate of hydrolysis of the insoluble protein fraction linearly decreased by more than 60% when comparing the untoasted to the 120 min toasted RSM. Increasing the toasting time elicited the formation of Maillard reaction products (furosine, Nε-carboxymethyl-lysine and Nε-carboxyethyl-lysine) and disulfide bonds in the insoluble protein fraction, which is proposed to explain the reduction in the hydrolysis rate of this fraction. Overall, longer toasting times increased the size of the peptides resulting after hydrolysis of the RSM and the insoluble protein fraction. The hydrolysis kinetics of the soluble and insoluble protein fractions and the proportion of soluble:insoluble proteins in the RSM explain the reduction in the rate of protein hydrolysis observed in the RSM with increasing toasting time.

Peroxidase induced oligo-tyrosine cross-links during polymerization of α-lactalbumin.

Horseradish peroxidase (HRP) induced cross-linking of proteins has been reported to proceed through formation of di-tyrosine cross-links. In the case of low molar mass phenolic substrates, the enzymatic oxidation is reported to lead to polymerization of the phenols. The aim of this work was to investigate if during oxidative cross-linking of proteins oligo-tyrosine cross-links are formed in addition to dityrosine. To this end, α-lactalbumin (α-LA) was cross-linked using horseradish peroxidase (HRP) and hydrogen peroxide (H2O2). The reaction products were acid hydrolysed, after which the cross-linked amino acids were investigated by LC-MS and MALDI-MS. To test the effect of the size of the substrate, the cross-linking reaction was also performed with L-tyrosine, N-acetyl L-tyrosinamide and angiotensin. These products were analyzed by LC-MS directly, as well as after acid hydrolysis. In the acid hydrolysates of all samples oligo-tyrosine (Yn, n=3-8) was found in addition to di-tyrosine (Y2). Two stages of cross-linking of α-LA were identified: a) 1-2 cross-links were formed per monomer until the monomers were converted into oligomers, and b) subsequent cross-linking of oligomers formed in the first stage to form nanoparticles containing 3-4 cross-links per monomer. The transition from first stage to the second stage coincided with the point where di-tyrosine started to decrease and more oligo-tyrosines were formed. In conclusion, extensive polymerization of α-LA using HRP via oligo-tyrosine cross-links is possible, as is the case for low molar mass tyrosine containing substrates.

Spontaneous, non-enzymatic breakdown of peptides during enzymatic protein hydrolysis.

It is expected that during the hydrolysis of proteins with specific enzymes only peptides are formed that result from hydrolysis of the specific cleavage sites (i.e. specific peptides). It is, however, quite common to find a-specific peptides (i.e. resulting from a-specific cleavage), which are often ignored, or explained by impurities in the enzyme preparation. In recent work in a whey protein isolate (WPI) hydrolysate obtained with the specific Bacillus licheniformis protease (BLP), 13 peptides of 77 identified were found to be the result of a-specific cleavage. These were formed after degradation of 6 specific peptides, after 5 different types of amino acids. The fact that other peptides were not hydrolyzed after these 5 amino acids suggests that the cleavages were not the result of a contamination with a different enzyme. In other systems, certain peptide sequences have been described to degrade chemically, under relatively mild conditions. This process is referred to as spontaneous cleavage. To test if the a-specific peptides observed in the WPI hydrolysis are the results of spontaneous cleavages, the parental peptides were synthesized. Surprisingly, 4 of the 5 synthesized peptides were indeed spontaneously cleaved under the mild conditions used in this study (i.e. 40°C and pH 8) showing that peptides are less stable than typically considered. The rate of cleavage on the a-specific bonds was found to be enhanced in the presence of BLP. This suggests that the formation of a-specific peptides is not due to side activity but rather an enhancement of intrinsic instability of the peptides.

A tandem mass spectrometry method based on selected ions detects low-abundance phenolics in black tea - theatridimensins as products of the oxidative cascade.

RATIONALE: Mixtures of phenolics are widespread in plant-derived food products, for instance black tea. Detailed compositional analysis of phenolics present is important for quality control. Characterization of low-abundance compounds often requires extensive purification; hence, the need for rapid screening methods to annotate compounds in complex mixtures without extensive sample preparation. Opportunities of ultra-high performance liquid chromatography/tandem mass spectrometry (UHPLC/MS) as tool in a rapid screening method are discussed for black tea analysis, with the two-step-oxidation product theatridimensin (T3D) as example. METHODS: Three MS screening methods were compared for their ability to tentatively annotate two-step-oxidation products in black teas without the need for prior fractionation: (i) full MS; (ii) tandem mass spectrometry (MS/MS) on selected ions; and (iii) selected reaction monitoring (SRM), in combination with post-analysis extracted ion chromatography. A model system of theaflavin (TF), epicatechin (EC) and tyrosinase was used to prepare the two-step-oxidation product T3D, consisting of three oligomerized catechin subunits. Commercial teas were screened for the occurrence of T3Ds. RESULTS: The MS(2) fragmentation pattern of T3D was compared with that of an isomeric catechin trimer from black tea, TFsEC. MS(2) signature fragments were found to distinguish the two isomers, i.e. m/z 617 for T3D and m/z 563 for TFsEC. The MS screening methods, MS/MS on selected ions and SRM, both enabled monitoring MS(2) data of compounds present in low abundance. The former provided the most complete MS(2) data set, which facilitated the discovery of another isomer, i.e. theaflavate A. T3Ds, TFsECs, and theaflavate A could be tentatively annotated in all tested tea samples. CONCLUSIONS: When exploring black tea for the occurrence of two-step-oxidation products, the use of MS/MS on selected ions combined with extracted ion chromatography proved to be the most suitable. The occurrence of T3Ds and T3Dgs in various black teas was shown for the first time and the 'oxidative cascade hypothesis' was extended with novel oxidation products. Copyright © 2016 John Wiley & Sons, Ltd.

Involvement of a Hydrophobic Pocket and Helix†11 in Determining the Modes of Action of Prenylated Flavonoids and Isoflavonoids in the Human Estrogen Receptor.

Six prenylated (iso)flavonoids were purified from a licorice root extract and subjected to competition experiments with six commercially available (iso)flavonoids. The agonistic and antagonistic activities of these compounds towards both hERα (human estrogen receptor alpha) and hERß were determined. Differences in the modes of action (agonist or antagonist) were observed for the various compounds tested. In general, each compound had the same mode of action towards both ERs. In silico modeling was performed in order to study the differences in estrogenicity observed between the compounds. It is suggested that prenyl chains fit into a hydrophobic pocket present in the hER, resulting in an increased agonistic activity. In addition, it was shown that an increase in length (≈1.7 Å) of pyran prenylated isoflavonoids resulted in an antagonistic mode of action. This might be caused by collision of the pyran ring with helix 11 in the ligand binding cavity of the hER.

Stability properties of surfactant-free thin films at different ionic strengths: measurements and modeling.

Foam lamellae are the smallest structural elements in foam. Such lamellae can experimentally be studied by analysis of thin liquid films in glass cells. These thin liquid films usually have to be stabilized against rupture by surface active substances, such as proteins or low molecular weight surfactants. However, horizontal thin liquid films of pure water with a radius of 100 µm also show remarkable stability when created in closed Sheludko cells. To understand thin film stability of surfactant-free films, the drainage behavior and rupture times of films of water and NaCl solutions were determined. The drainage was modeled with an extended Derjaguin-Landau-Verwey-Overbeek (DLVO) model, which combines DLVO and hydrophobic contributions. Good correspondence between experiment and theory is observed, when hydrophobic interactions are included, with fitted values for surface potential (ψ(0,water)) of -60 ± 5 mV, hydrophobic strength (B(hb,water)) of 0.22 ± 0.02 mJ/m(2), and a range of the hydrophobic interaction (λ(hb, water)) of 15 ± 1 nm in thin liquid films. In addition, Vrij's rupture criterion was successfully applied to model the stability regions and rupture times of the films. The films of pure water are stable over long time scales (hours) and drain to a final thickness >40 nm if the concentration of electrolytes is low (resistivity 18.2 MQ). With increasing amounts of ions (NaCl) the thin films drain to <40 nm thickness and the rupture stability of the films is reduced from hours to seconds.

Enzymatic cross-linking of α-lactalbumin to produce nanoparticles with increased foam stability.

Hard colloidal nanoparticles (e.g. partly hydrophobised silica), are known to make foams with very high foam-stability. Nanoparticles can also be produced from proteins by enzymatic cross-linking. Such protein based particles are more suitable for food applications, but it is not known if they provide Pickering foam stabilisation to the same extent as hard colloidal particles. α-Lactalbumin (α-LA) was cross-linked with either microbial transglutaminase (mTG) or horseradish peroxidase (HRP) to produce α-LA/mTG and α-LA/HRP nanoparticles. With both enzymes a range of nanoparticles were produced with hydrodynamic radii ranging from 20-100 nm. The adsorption of nanoparticles to the air-water interface was probed by increase in surface pressure (Π) with time. In the beginning of the Π versus time curves, there was a lag time of 10-200 s, for nanoparticles with Rh of 30-100 nm, respectively. A faster increase of Π with time was observed by increasing the ionic strength (I = 0-125 mM). The foam-ability of the nanoparticles was also found to increase with increasing ionic strength. At a fixed I, the foam-ability of the nanoparticles decreased with increasing size while their foam-stability increased. Foams produced by low-shear whipping were found to be 2 to 6 times more stable for nanoparticles than for monomeric α-LA (Rh≈ 2 nm). At an ionic strength of 125 mM ionic strength and protein concentration ≥ 10 g L(-1), the foam-stability of α-LA/mTG nanoparticles (Rh = 100 nm, ρapp = 21.6 kg m(-3)) was 2-4 times higher than α-LA/HRP nanoparticles (Rh = 90 nm, ρapp = 10.6 kg m(-3)). This indicated that foam-stablity of nanoparticles is determined not only by size but also by differences in mesoscale structure. So, indeed enzymatic cross-linking of proteins to make nanoparticles is moving a step towards particle like behavior e.g. slower adsorption and higher foam stability. However, the cross-link density should be further increased to obtain hard particle-like rigidity and foam-stability.

Introducing enzyme selectivity: a quantitative parameter to describe enzymatic protein hydrolysis.

Enzyme selectivity is introduced as a quantitative parameter to describe the rate at which individual cleavage sites in a protein substrate are hydrolyzed relative to other cleavage sites. Whey protein isolate was hydrolyzed by Bacillus licheniformis protease, which is highly specific for Glu and Asp residues. The molar concentration of all peptides (58) from ß-lactoglobulin formed during hydrolysis was determined from the UV214 signal. The quality of identification and quantification of the peptides were described by newly defined parameters: the peptide sequence coverage (on average 94 %) and the molar sequence coverage (on average 75 %). The selectivity was calculated from the rate of hydrolysis of each cleavage site, and showed differences of up to a factor of 5,000. The ability to quantitatively discriminate the enzyme preference towards individual cleavage sites is considered essential to the understanding of enzymatic protein hydrolysis.

Understanding carbohydrate structures fermented or resistant to fermentation in broilers fed rapeseed (Brassica napus) meal to evaluate the effect of acid treatment and enzyme addition.

Unprocessed and acid-extruded rapeseed meal (RSM) was fed to broiler chickens, with and without addition of commercial pectolytic enzymes. Nonstarch polysaccharide (NSP) fermentability and unfermented NSP structures from RSM were studied in the excreta in detail. From unprocessed RSM, 24% of the nonglucose polysaccharides could be fermented. Acid treatment did not have a significant effect, but enzyme addition did improve fermentability to 38%. Most likely, the significant increase in NSP fermentability can be ascribed to the addition of pectolytic enzymes, which decreased branchiness of the water-soluble arabinan. Mainly xyloglucan, (glucurono-)xylan, (branched) arabinan, and cellulose remained in the excreta. The proportion of unextractable carbohydrates increased in excreta from broilers fed acid-extruded RSM. Probably, acid extrusion resulted in a less accessible NSP matrix, also decreasing the accessibility for pectolytic enzymes added in the diet. During alkaline extraction of the excreta, 39 to 52% (wt/wt) of the insoluble carbohydrates was released as glucosyl- and uronyl-rich carbohydrates, probably originally present via ester linkages or hydrogen bonding within the cellulose-lignin network. These linkages are expected to hinder complete NSP fermentation and indicate that digestibility of RSM may benefit substantially from an alkaline treatment or addition of esterases.

Carbohydrate utilization and metabolism is highly differentiated in Agaricus bisporus.

BACKGROUND: Agaricus bisporus is commercially grown on compost, in which the available carbon sources consist mainly of plant-derived polysaccharides that are built out of various different constituent monosaccharides. The major constituent monosaccharides of these polysaccharides are glucose, xylose, and arabinose, while smaller amounts of galactose, glucuronic acid, rhamnose and mannose are also present. RESULTS: In this study, genes encoding putative enzymes from carbon metabolism were identified and their expression was studied in different growth stages of A. bisporus. We correlated the expression of genes encoding plant and fungal polysaccharide modifying enzymes identified in the A. bisporus genome to the soluble carbohydrates and the composition of mycelium grown compost, casing layer and fruiting bodies. CONCLUSIONS: The compost grown vegetative mycelium of A. bisporus consumes a wide variety of monosaccharides. However, in fruiting bodies only hexose catabolism occurs, and no accumulation of other sugars was observed. This suggests that only hexoses or their conversion products are transported from the vegetative mycelium to the fruiting body, while the other sugars likely provide energy for growth and maintenance of the vegetative mycelium. Clear correlations were found between expression of the genes and composition of carbohydrates. Genes encoding plant cell wall polysaccharide degrading enzymes were mainly expressed in compost-grown mycelium, and largely absent in fruiting bodies. In contrast, genes encoding fungal cell wall polysaccharide modifying enzymes were expressed in both fruiting bodies and vegetative mycelium, but different gene sets were expressed in these samples.

Protein concentration and protein-exposed hydrophobicity as dominant parameters determining the flocculation of protein-stabilized oil-in-water emulsions.

DLVO theory is often considered to be applicable to the description of flocculation of protein-stabilized oil-in-water emulsions. To test this, emulsions made with different globular proteins (ß-lactoglobulin, ovalbumin, patatin, and two variants of ovalbumin) were compared under different conditions (pH and electrolyte concentration). As expected, flocculation was observed under conditions in which the zeta potential is decreased (around the isoelectric point and at high ionic strength). However, the extent of flocculation at higher ionic strength (>50 mM NaCl) decreased with increasing protein-exposed hydrophobicity. A higher exposed hydrophobicity resulted in a higher zeta potential of the emulsion droplets and consequently increased stability against flocculation. Furthermore, the addition of excess protein strongly increased the stability against salt-induced flocculation, which is not described by DLVO theory. In the protein-poor regime, emulsions showed flocculation at high ionic strength (>100 mM NaCl), whereas emulsions were stable against flocculation if excess protein was present. This research shows that the exposed hydrophobicity of the proteins and the presence of excess protein affect the flocculation behavior.

Effect of glycation on the flocculation behavior of protein-stabilized oil-in-water emulsions.

Glycation of proteins by the Maillard reaction is often considered as a method to prevent flocculation of protein-stabilized oil-in-water emulsions. The effect has been suggested, but not proven, to be the result of steric stabilization, and to depend on the molecular mass of the carbohydrate moiety. To test this, the stabilities of emulsions of patatin glycated to the same extent with different mono- and oligosaccharides (xylose, glucose, maltotriose, and maltopentaose) were compared under different conditions (pH and electrolyte concentration). The emulsions with non-modified patatin flocculate under conditions in which the zeta potential is decreased (around the iso-electric point and at high ionic strength). The attachment of monosaccharides (i.e., glucose) did not affect the flocculation behavior. Attachment of maltotriose and maltopentaose (Mw > 500 Da), on the other hand, provided stability against flocculation at the iso-electric point. Since the zeta potential and the interfacial properties of the emulsion droplets are not affected by the attachment of the carbohydrate moieties, this is attributed to steric stabilization. Experimentally, a critical thickness of the adsorbed layer required for steric stabilization against flocculation was found to be 2.29-3.90 nm. The theoretical determination based on the DLVO interactions with an additional steric interaction coincides with the experimental data. Hence, it can be concluded that the differences in stability against pH-induced flocculation are caused by steric interactions.

Characterisation of 3-aminoquinoline-derivatised isomeric oligogalacturonic acid by travelling-wave ion mobility mass spectrometry.

RATIONALE: Mass spectrometry has become a useful technique for elucidating the chemical structures of oligosaccharides. The combined use of chromatography and mass spectrometry for the separation and identification of oligosaccharides has shown much progress in recent years. However, no powerful method has yet been developed to quickly identify isomeric oligosaccharides in complex mixtures. METHODS: A rapid travelling-wave ion mobility mass spectrometry (TWIMS-MS) method was developed for the identification of various isomeric oligogalacturonic acids in mixtures and determined their structures, using 3-aminoquinoline (3-AQ) as a labelling agent. RESULTS: TWIMS successfully distinguished isomeric oligogalacturonic acids of various degrees of polymerisation (DPs) and levels of methyl-esterification. After derivatisation by 3-AQ, isomeric oligosaccharides of galacturonic acid, with the DP ranging from 2 to 9 and the number of methyl esters ranging from 1 to 5, were identified by 3-AQ-TWIMS-MS. The isomeric oligosaccharides with varying sites of methyl ester substitution were identified by the post-fragmentation mode of TWIMS using 3-AQ labelling to obtain simplified mass spectra. CONCLUSIONS: Using the 3-AQ-TWIMS-MS method, the precise distribution of methyl esters within the pectin molecule and isomeric oligogalacturonic acids after enzyme degradation was determined. Simplified product ion mass spectra and precise analysis of the isomers were achieved by labelling 3-AQ at the reducing end of the oligosaccharides. Series of methyl-esterified galacturonic acid oligomers have predictable drift times, depending on the precise position of the methyl ester.

UHPLC/PDA-ESI/MS analysis of the main berry and leaf flavonol glycosides from different Carpathian Hippophaë rhamnoides L. varieties.

INTRODUCTION: Sea buckthorn (Hippophaë rhamnoides L.) is known to be rich in many bioactive compounds (such as vitamins, phenolics, carotenoids) important for human health and nutrition. Among the phenolics, berries and leaves contain a wide range of flavonols that are good quality and authenticity biomarkers. OBJECTIVE: To compare the composition of the main flavonols of Romanian sea buckthorn berry and leaf varieties and to identify the specific biomarkers that contribute to sample differentiation among varieties. MATERIAL AND METHODS: Six varieties of cultivated sea buckthorn (ssp. Carpatica) berries and leaves were analysed by UHPLC/PDA-ESI/MS. RESULTS: Berries and leaves contained mainly isorhamnetin (I) glycosides in different ratios. Whereas I-3-neohesperidoside, I-3-glucoside, I-3-rhamnosylglucoside, I-3-sophoroside-7-rhamnoside and free isorhamnetin were predominant for berries (out of 17 compounds identified), I-3-rhamnosylglucoside, I-3-neohesperidoside, I-3-glucoside, quercetin-3-pentoside, kaempferol-3-rutinoside, and quercetin-3-glucoside were predominant in leaves (out of 19 compounds identified). Berries contained, on average, 917 mg/100 g DW flavonol glycosides. Leaves had higher content of flavonol glycosides than berries, on average 1118 mg/100 g DW. The variation of the quantitative dataset analysed using principal component analysis accounted for 91% of the total variance in the case of berries and 73% in case of leaves, demonstrating a good discrimination among samples. CONCLUSION: Based on quantitative analysis, by principal component analysis, the flavonol derivatives can be considered as biomarkers to discriminate among varieties and to recognise specifically the berry versus leaf composition.