Alkaline extractability of pectic arabinan and galactan and their mobility in sugar beet and potato cell walls

The organisation of sugar beet and potato cell walls was studied using alkaline extractions following a response surface methodology, simultaneously with solid-state ¹³C NMR spectroscopy. The influence of two extraction parameters: NaOH concentration (0.05, 0.275, 0.5 M) and temperature (40, 65, 90 °C) on the composition (neutral and acidic sugars) of the residues recovered was established. Treatments of increasing harshness progressively washed off non-cellulosic polysaccharides from the cell walls. Alkaline treatments applied to sugar beet cell wall material (SB-CWM) revealed the presence of diverse pectin populations. The existence of distinct pectin populations in potato cell wall material (P-CWM) was less outstanding. Solid-state ¹³C NMR applied to SB-CWM and P-CWM and residues after treatment by 0.275 M NaOH at 65 °C revealed two fractions of pectic arabinan and galactan side chains. One fraction was highly mobile, whereas the other one displayed restricted mobility.     

Pectin substances of the callus culture of <Emphasis Type="Italic">Silene vulgaris</Emphasis> (M.) G.

Pectin-protein fraction SVC was isolated from the callus culture of the bladder campion (Silene vulgaris). The main components in it were residues of D-galacturonic acid, galactose, arabinose, rhamnose, and protein. Using ion-exchange chromatography, ultrafiltration, and acid and enzymatic hydrolysis, it was shown that SVC contained a mixture of molecules of linear pectin, branched pectin polysaccharide, and pectin-protein polymer. A fragment of the linear chain of galacturonan amounted to more than half of the entire carbohydrate silenan chain. The branched area of the macromolecule is represented by rhamnogalacturonan I. The pectin-protein polymer consisted mainly of protein and weakly branched pectin fragments with molecular mass of more than 300 kDa.     

Pectin substances of the callus culture of Silene vulgaris (M.) G

Pectin-protein fraction SVC was isolated from the callus culture of the bladder campion (Silene vulgaris). The main components in it were residues of D-galacturonic acid, galactose, arabinose, rhamnose, and protein. Using ion-exchange chromatography, ultrafiltration, and acid and enzymatic hydrolysis, it was shown that SVC contained a mixture of molecules of linear pectin, branched pectin polysaccharide, and pectin-protein polymer. A fragment of the linear chain of galacturonan amounted to more than half of the entire carbohydrate silenan chain. The branched area of the macromolecule is represented by rhamnogalacturonan I. The pectin-protein polymer consisted mainly of protein and weakly branched pectin fragments with molecular mass of more than 300 kDa.     

Structural of a glucomannan from Lupinus varius seed

Polysaccharides extracted from seeds of Lupinus varius with hot ethanol 85% (polysaccharide FI) and 0.1 M sodium phosphate buffer, pH 7.5 (polysaccharide FII) were fractionated and purified by ion-exchange and gel-filtration chromatography. According to methylation and hydrolysis analysis, the main chains of FI and FII consisted of (1 → 4)-linked glucomannan; only traces of branched sugar residues were detected. This is the first report on the isolation of glucomannan from L. varius seeds.     

Structural characterization, degree of esterification and some gelling properties of Krueo Ma Noy (Cissampelos pareira) pectin

Pectins extracted from Krueo Ma Noy (Cissampelos pareira) leaves mainly consisted of galacturonic acid with trace amount of neutral sugars. The dominant structure of Krueo Ma Noy pectin was established as a 1,4-linked -D-galacturonan by a combination of carboxyl reduction and methylation analysis, and confirmed by FT-IR spectroscopy. The degree of esterification of Krueo Ma Noy pectins was 41.7 and 33.7% for crude and dialyzed pectins, respectively. Krueo Ma Noy pectin has an average molecular weight of 55 kDa, radius of gyration of 15.2 nm and intrinsic viscosity of 2.3 dl/g. Krueo Ma Noy pectin exhibited gelling properties in aqueous solutions at 0.5% (w/v) at 5 °C. Gels were formed at concentrations of 1.0% (w/v) and above even at room temperature. The gel strength, melting point, and melting enthalpy of Krueo Ma Noy pectin increased with polysaccharide concentration.     

Sugar Beet Pectin–Protein Complexes

The physical structure of pectin extracted from fresh sugar beet has been examined by atomic force microscopy (AFM). The images obtained reveal that these extracts contain a mixture of pectin polysaccharides and protein–pectin complexes. The AFM data demonstrate, for the first time, that these protein–pectin complexes consist of pectin molecules with protein attached to one end of the pectin chain.     

The ferulic acid esterases of Chrysosporium lucknowense C1: purification, characterization and their potential application in biorefinery

Three ferulic acid esterases from the filamentous fungus Chrysosporium lucknowense C1 were purified and characterized. The enzymes were most active at neutral pH and temperatures up to 45 °C. All enzymes released ferulic acid and p-coumaric acid from a soluble corn fibre fraction. Ferulic acid esterases FaeA1 and FaeA2 could also release complex dehydrodiferulic acids and dehydrotriferulic acids from corn fibre oligomers, but released only 20% of all ferulic acid present in sugar beet pectin oligomers. Ferulic acid esterase FaeB2 released almost no complex ferulic acid oligomers from corn fibre oligomers, but 60% of all ferulic acid from sugar beet pectin oligomers. The ferulic acid esterases were classified based on both, sequence similarity and their activities toward synthetic substrates. The type A ferulic acid esterases FaeA1 and FaeA2 are the first members of the phylogenetic subfamily 5 to be biochemically characterized. Type B ferulic acid esterase FaeB2 is a member of subfamily 6.     

Studies on Protopectinase-C Mode of Action: Analysis of the Chemical Structure of the Specific Substrate in Sugar Beet Protopectin and Characterization of the Enzyme Activity

Chemical structures of pectic substances degraded by protopectinase-C (PPase-C) were characterized to identify the releasing mechanism of pectin from sugar beet protopectin by the action of that enzyme. The substrate of PPase-C was a polysaccharide isolated from sugar beet pulp by extraction with NaOH and sequential digestions with rhamnogalacturonase (PPase-T), β-1,4-D-galactanase, and α-L-arabinofuranosidase. The structure of this polysaccharide was analyzed by gas-liquid chromatography (GLC), NMR analysis, and gas chromatography-mass spectrometry (GC-MS), and it was identified as α-1,5-L-arabinan. According to our results, arabinan chains seemed to be connected to rhamnogalacturonan through a chain of β-l,4-D-galactan. PPase-C hydrolyzed both linear α-1,5-L-arabinan and ramified L-arabinan in a random manner, producing L-arabinose. From these results, PPase-C could be classified as arabinan endo-1,5-α-L-arabinase [EC 3.2.1.99]. Moreover, PPase-C seemed to split the L-arabinan of the polysaccharides connecting the rhamnogalacturonan to the other constituents of the plant cell wall in sugar beet pulp, releasing water-soluble pectin.     

Global structures of high methoxyl pectin from solution and in gels

Images of high methoxyl orange pectin deposited from solution and high methoxyl sugar acid gels (HMSAG) were obtained by atomic force microscopy (AFM) in the tapping mode. For the first time, images of pectin deposited from water revealed that the transition from pectin networks to individual molecules or aggregates thereof occurred at concentrations between 6.5 and 13.1 microg/mL. At 6.5 microg/mL, shapes included rods, segmented rods, kinked rods, rings, branched molecules, and dense circular areas. At 13.1 microg/mL, all of these shapes were integrated into networks. These same structures were discernible in pectin high methoxyl sugar acid gels. Thus one might consider pectin networks in water at concentrations in excess of 10 microg/mL to be separate fluid precursors of networks in high methoxyl sugar acid gels. Examination of AFM images revealed that gels with "uniform" distribution of strands and pores between strands had higher gel strengths as measured by a penetrometer than gels in which strands were nonuniformly distributed and were separated by large and small spaces.     

A copolymer analysis approach to estimate the neutral sugar distribution of sugar beet pectin using size exclusion chromatography

Partially degraded sugar beet (Beta vulgaris) pectins were characterised in terms of galacturonic acid, neutral sugar and ferulic acids contents. It was shown that the total neutral sugar content is correlated with the ferulic acid content. One pectin (C) was further characterised by size exclusion chromatography coupled to refractive index and UV detectors (SEC-RI-UV). This gave the opportunity to estimate how the ferulic acid and neutral sugar contents changed with hydrodynamic radius. Pectin C was found to be heterogeneous in composition with neutral sugar-rich fractions of both high and low hydrodynamic radii. A neutral sugar-poor fraction was found at intermediate hydrodynamic radii.     

Dimeric calcium complexes of arabinan-rich pectic polysaccharides from Olea europaea L. cell walls

The study carried out in this work concerns the pectic polysaccharides of olive cell walls as present in olive pulp and that remained entrapped in the cellulosic residue after sequential extraction of the cell wall material (CWM) with imidazole, carbonate and KOH aqueous solutions. These polymers, obtained after neutralisation and dialysis of an aqueous suspension of the residue (sn-CR fraction), extracted with 4 M KOH, were arabinan-rich pectic polysaccharides. They accounted for 11–19% of the total pectic polysaccharides found in the olive pulp cell walls of fruits collected in two years and in three stages of ripening (green, cherry and black). The analysis by powder X-ray diffraction highlighted the existence, in all sn-CR fractions, of crystalline phases related with the presence of calcium-pectic polysaccharide complexes (CPPC) occurring in an amorphous carbohydrate network. The relative crystallinity of the CPPC varied linearly with the Ca²⁺/GalA molar ratio until a maximum of 0.57. Size-exclusion chromatography showed that sn-CR fractions possessed a bimodal molecular weight distribution. The lower molecular weight fraction of sn-CR (M_w = 70–135 kDa) was independent on the ripening stage of olive fruit, whereas the higher molecular weight fraction showed values of 1.1, 0.6–0.9 and 0.5–0.7 MDa, respectively, for green, cherry and black olives. Treatment of the sn-CR pectic polysaccharides with a 2 M imidazole solution disrupted the CPPC crystalline network showing the loss of low molecular weight galacturonan-rich material during dialysis (12–14 kDa cut off) and the decrease of molecular weight of the polymers to roughly half. These results allowed to infer the presence of oligogalacturonides held within cell walls by calcium ions and that the pectic polysaccharides of sn-CR fraction occurred in olive pulp cell walls as calcium bridged macrodimers.     

Structure and physiological activity of lemnan, Lemna minor L. pectin

A pectic polysaccharide, lemnan, was isolated from freshly collected duckweed Lemna minor L. Its sugar chain was shown to be mainly composed of the residues of D-galacturonic acid (64%), galactose, arabinose, xylose, and D-apiose, a branched chain sugar. The high content of D-apiose (25%) indicated that lemnan is an apiogalacturonan type pectin similar to zosteran, a pectic polysaccharide from a sea phanerogam of the Zosteraceae family. The results of partial acidic hydrolysis, pectinase digestion, and NMR studies of lemnan demonstrated that its macromolecule contains regions of the linear alpha-1,4-D-galacturonan and branched apiogalacturonan. The side chains of apiogalacturonan were found to be formed of single and 1,5-linked residues of D-apiofuranose attached to 2- and 3-positions of the D-galacturonic acid residues of the apiogalacturonan backbone. Lemnan was shown to exhibit an immunomodulatory effect by activating the system of phagocytosis.     

Evidence for in vitro binding of pectin side chains to cellulose

Pectins of varying structures were tested for their ability to interact with cellulose in comparison to the well-known adsorption of xyloglucan. Our results reveal that sugar beet (Beta vulgaris) and potato (Solanum tuberosum) pectins, which are rich in neutral sugar side chains, can bind in vitro to cellulose. The extent of binding varies with respect to the nature and structure of the side chains. Additionally, branched arabinans (Br-Arabinans) or debranched arabinans (Deb-Arabinans; isolated from sugar beet) and galactans (isolated from potato) were shown bind to cellulose microfibrils. The adsorption of Br-Arabinan and galactan was lower than that of Deb-Arabinan. The maximum adsorption affinity of Deb-Arabinan to cellulose was comparable to that of xyloglucan. The study of sugar beet and potato alkali-treated cell walls supports the hypothesis of pectin-cellulose interaction. Natural composites enriched in arabinans or galactans and cellulose were recovered. The binding of pectins to cellulose microfibrils may be of considerable significance in the modeling of primary cell walls of plants as well as in the process of cell wall assembly.     

Influence of different fibre sources on digestibility and nitrogen and energy balances in growing pigs

Abstract

The present study was undertaken to investigate how three different fibre sources, sugar beet pulp, soya bean hulls and pectin residue, constituting 15% of diets for growing pigs, influenced daily body gain, feed conversion, apparent faecal digestibility and nitrogen and energy balances. Eight castrated crossbreed pigs (30 – 80 kg live weight) were used in a replicated 4 × 4 Latin-square design with one control diet and three fibre containing diets. Daily body weight gain and feed conversion were not affected by the dietary treatments. The apparent faecal digestibility of organic matter (OM) and energy were significantly lower for the fibre diets (OM: 0.81 – 0.85; energy: 0.78 – 0.83) compared to the control diet (OM: 0.88; energy: 0.86). The apparent faecal digestibility of crude protein (CP) was lower for the fibre diets (0.71 – 0.78) compared to the control diet (0.83), although it was only significantly lower for the sugar beet pulp and pectin residue diets. The pectin residue diet, which contained the highest amount of dietary fibre, lignin and insoluble non-starch polysaccharides, had the lowest digestibility of OM, CP and energy. There was a tendency (p = 0.07) for a diet effect on retained nitrogen in proportion to digested nitrogen, where the sugar beet pulp and pectin residue diets had numerically the highest values. Heat production and retained energy in proportion to metabolizable energy intake were not affected by fibre inclusion. It was concluded that the inclusion of sugar beet pulp, soya bean hulls and pectin residue in diets for growing pigs decreased the apparent faecal digestibility and in the diets with sugar beet pulp and pectin residue higher utilization of digested nitrogen for retention compensated for the lower amount of digested nitrogen.     

Structure and thermal decomposition of methylamine borane

The crystal structure of methylamine borane has been determined and contains parallel chains of dihydrogen-bonded CH₃NH₂BH₃ molecules. Thermal decomposition takes place from the melt (ΔH_fusion = 8.5 kJ mol⁻¹) and begins with the formation of an ionic borohydride. Hydrogen is liberated in two stages, at ca. 100 and 190 °C, with the observed rates during the first stage (ΔH = −25 kJ mol⁻¹, E_a = 115 kJ mol⁻¹) strongly dependent on temperature and time. cis- and trans-N-trimethylcyclotriborazane are formed during the first stage and subsequently cross-link to yield a non-volatile solid. Before this cross-linking, the system exhibited a high degree of volatility, with weight losses in excess of 80% observed in TG experiments using flowing gas.     

Mapping sugar beet pectin acetylation pattern

Homogalacturonan-derived partly methylated and/or acetylated oligogalacturonates were recovered after enzymatic hydrolysis (endo-polygalacturonase+pectin methyl esterase+side-chain degrading enzymes) of sugar beet pectin followed by anion-exchange and size exclusion chromatography. Around 90% of the GalA and 75% of the acetyl groups present in the initial sugar beet pectin were recovered as homogalacturonan-derived oligogalacturonates, the remaining GalA and acetyl belonging to rhamnogalacturonic regions. Around 50% of the acetyl groups present in sugar beet homogalacturonans were recovered as partly methylated and/or acetylated oligogalacturonates of degree of polymerisation 5 whose structures were determined by electrospray ionization ion trap mass spectrometry (ESI-IT-MSn). 2-O-acetyl- and 3-O-acetyl-GalA were detected in roughly similar amounts but 2,3-di-O-acetylation was absent. Methyl-esterified GalA residues occurred mainly upstream 2-O-acetyl GalA. Oligogalacturonates containing GalA residues that are at once methyl- and acetyl-esterified were recovered in very limited amounts. A tentative mapping of the distribution of acetyl and methyl esters within sugar beet homogalacturonans is proposed. Unsubstituted GalA residues are likely to be present in limited amounts (approximately 10% of total GalA residues), due to the fact that methyl and acetyl groups are assumed to be most often not carried by the same residues.     

A new interpretation of eutectic behavior for distearoylphosphatidylcholine-cholesterol binary bilayer membrane

We investigated the thermotropic phase behavior of the distearoylphosphatidylcholine (DSPC)–cholesterol binary bilayer membrane as a function of the cholesterol composition (X_ch) by fluorescence spectroscopy using 6-propionyl-2-(dimethylamino)naphthalene (Prodan) and differential scanning calorimetry (DSC). The fluorescence spectra, each of which has a single maximum, showed that the wavelength at the maximum intensity (λ_max) changed depending on the bilayer state: ca. 440 nm for the lamellar gel (L_β′ or L_β) and the liquid ordered (L_o) phases, ca. 470 nm for the ripple gel (P_β′) phase and ca. 490 nm for the liquid crystalline (L) phase, respectively. The transition temperatures were determined from the temperature dependences of the λ_max and endothermic peaks of the DSC thermograms. Both measurements showed that the pretransition disappears around X_ch = 0.035. The constructed temperature–X_ch phase diagram indicated that the phase behavior of the binary bilayer membrane at X_ch ≤ 0.15 is similar to that of general liquid–solid equilibrium for a binary system where both components are completely miscible in the liquid phase and completely immiscible in the solid phase. It was also revealed that the diagram has two characteristic points: a congruent melting point at X_ch = 0.08 and a peritectic-like point at X_ch = 0.15. The hexagonal lattice model was used for the interpretation of the phase behavior of the binary bilayer membrane. These characteristic compositions well correspond to the bilayer states in each of which cholesterol molecules are regularly distributed in the hexagonal lattice in a different way. That is, each composition of 0.035, 0.08 and 0.15 is nearly equal to that for the binary bilayer membrane which is entirely occupied with units, each composed of a cholesterol and 30 surrounding DSPC molecules within the next-next-next nearest neighbor sites (Unit (1:30): L_β(1:30)), with units, each of a cholesterol and 12 surrounding DSPC molecules within the next nearest sites (Unit (1:12): L_β(1:12)) or with units, each of a cholesterol and 6 surrounding DSPC molecules at the nearest neighbor sites (Unit (1:6): L_β(1:6)), respectively. Therefore, the eutectic behavior observed in the phase diagram was fully explainable in terms of a kind of phase separation between two different types of regions with different types of regular distributions of cholesterol. Further, the L_o phase was found in the higher X_ch-region (X_ch > 0.15). No endothermic peak over the temperature range from 10 to 80 °C at X_ch = 0.50 suggested that the single L_o phase can exist at X_ch > 0.50.     

Effects of Hexametaphosphate on Soybean Pectic Polysaccharide Extraction

The effects of sodium hexametaphosphate concentration, incubation temperature, and pH on the extraction of polysaccharide from soybean okara (soybean curd waste) were examined. The results suggest that the soybean polysaccharides were almost completely extracted with 50 times their volume of a 2% hexametaphosphate solution at 100°C for 2h, avoiding protein contamination. The viscosity, molecular weight, and sugar composition of the polysaccharides were compared with those of the commercially available samples. No differences were observed in the molecular weight or neutral sugar composition. However, it was found that the galacturonate distribution in the molecules of the two polysaccharides was different. The viscosity of the polysaccharide solution was changed by adding small amount of salt.     

Effect of polymeric cosolutes on calcium pectinate gelation. Part 3. Gum arabic and overview

Addition of gum arabic (average M_r≈450 kDa; 0.5–2.0 wt%) to solutions of low methoxy pectin (DE 31; 2.0 wt%; pH≈2.9–3.0) with stoichiometric Ca²⁺ caused massive increases in G′ and G″ in the pre-gel state at 90 °C (attributed to segregative interactions promoting formation of calcium-mediated ‘egg-box’ junctions between pectin chains) but had little effect on the gels formed on cooling to 5 °C. This is in marked contrast to the behaviour of other polymeric cosolutes studied in the investigations reported in the two preceding papers, which caused large reductions in gel moduli (attributed to excessive association of calcium pectinate into large aggregated bundles); the difference is tentatively ascribed to strengthening of the calcium pectinate network by divalent counterions to the uronate residues in gum arabic. When the complication of cation exchange was eliminated by extensive dialysis of gum arabic against 100 mM Na⁺ and use of the final dialysate in preparation of mixtures with calcium pectinate, massive increases in G′ and G″ at high temperature were again observed, but with accompanying reductions in moduli at low temperature, which, at gum arabic concentrations above 1.0 wt%, arose from collapse of the developing calcium pectinate network during cooling. The tentative conclusion from this work, and from the two preceding papers, is that enthalpically unfavourable (segregative) interactions between low methoxy pectin and polymeric cosolutes can be relieved in two ways: (i) Ca²⁺-mediated self-association of pectin into compact ordered assemblies which occupy less of the total volume, and (ii) conformational rearrangement of the cosolute molecules to minimise segmental interactions with pectin; conformational rearrangement is inhibited by chain stiffness and by branching; thus polymeric cosolute molecules of limited flexibility are more effective in promoting self-association of pectin than more flexible molecules of comparable size, and branched molecules are more effective than linear chains of comparable stiffness.     

Inverse association of high-fat diet preference and anxiety-like behavior: a putative role for urocortin 2

The aim of this study was to investigate whether the preference for a palatable high-fat diet (HFD) is associated with response to novelty and with anxiety-like behavior in rats and whether such fat preference correlates with gene expression of hypothalamic neuropeptides related to feeding. We subjected male rats to two tests of exploration of novel environments: the multivariate concentric square field (MCSF) and the elevated plus maze (EPM). The rats were then exposed to a 5 - day test of preference for a palatable HFD versus reference diets. Messenger RNA (mRNA) levels of 21 neuropeptides were investigated by quantitative polymerase chain reaction. We found a strong positive correlation of HFD preference and open-arm activity in the EPM (% open-arm time, r _s = 0.629, df = 26, P   <   0.001). Thus, HFD preference was inversely associated with anxiety-like behavior. The same association was found for HFD preference and behavior in the MCSF (bridge entries, r _s = 0.399, df = 23, P  = 0.048). In addition, the HFD preference was positively correlated ( r _s = 0.433, df = 25, P  = 0.021) with hypothalamic mRNA levels of urocortin 2 (Ucn 2). Moreover, behavior in the EPM was significantly correlated with expression levels of the receptor for Ucn 2, the corticotropin-releasing factor receptor 2, in the hypothalamus ( r _s = 0.382, df = 33, P  = 0.022, pituitary ( r _s = 0.494, df = 31, P  = 0.004) and amygdala ( r _s = 0.381, df = 30, P  = 0.032). We conclude that preference for palatable HFD is inversely associated with anxiety and propose that Ucn 2 signaling may play a role in this association.