Extraction and characterization of pectic substances from pulp of grape berries

Pectic substances were successively extracted from the alcohol-insoluble residue (AIR) of the pulp of grape berries, by water (WSP), oxalate (OXP), hot dilute HCl (HP) and cold dilute NaOH (OHP). Pectins (WSP, OXP, HP) were purified by ion-exchange chromatography (DEAE-Sephacel) or precipitation with cupric ions (OHP). Total pectic substances represent 20·8% (w/w) of the AIR, WSP and HP being the main components (6% and 12% of the AIR, respectively). An alternative to oxalate extraction of the water-insoluble pectin was extraction with NaCl solutions of increasing concentrations, which had released small amounts of pectins. Each of the fractions contained mainly galacturonic acid, arabinose and galactose, lower amounts of rhamnose and xylose, and minor amounts of glucose and mannose. Ion-exchange chromatography was performed on DEAE-Sephacel, M_w distribution was checked by gel permeation on Sepharose CL-2B, and M_v was determined as well as degrees of methylesterification (DM), acetylation (DA), and protein content.     

Enzymic degradation of apple pectins

Purified apple pectins extracted under mild conditions were degraded by purified pectin lyase (EC 4.2.2.10) and pectate lyase (EC 4.2.2.2). The degraded pectins were fractionated by gel permeation chromatography and the degree of esterification and the sugar composition determined for each fraction. More than 90% of the uronic acid residues can be isolated as homogalacturonan chains. The neutral sugar residues can be detected in the column eluates as high molecular weight fragments. Models of the apple pectin molecules are presented. In the models the neutral sugars are present as side chains, arranged in blocks (in so-called ‘hairy regions’). The galacturonate residues in the hairy regions are esterified with methanol.     

Comparison of the structural features of apple and citrus pectic substances

Pectic substances were extracted from Alcohol Insoluble Solids from lemon peel (albedo) and fractionated by ion exchange chromatography and gelfiltration. The pectin molecules contained rhamnose, arabinose, galactose, glucose and galacturonic acid residues; xylose residues were almost absent. Degradation with purified pectolytic enzymes and subsequent gelfiltration of the resulting pectin fragments showed that the neutral sugar side chains were present in ‘hairy regions’ (blocks of neutral sugar side chains). The distribution of the methoxyl groups was studied by HPLC analysis of enzyme-degraded pectins. Some influence of native pectinesterase on the distribution of the methoxyl groups was found. The results are compared with those of similarly extracted and purified apple pectic substances.     

Methods for obtaining neutral and acid oligosaccharides from flax pectins

Esterified acid soluble pectins from flax (Linun usitatissimum L.) were degraded either with HCl or pectin lyase. Centrifugation and 2-propanol precipitation led to the isolation of two low molecular weight polygalacturonates after acid hydrolysis of pectins. However, after pectin lyase digestion and purification by size-exclusion HPLC, ¹H NMR analyses indicated that acetylated hairy regions, large methylated and acetylated oligogalacturonides together with small unsubstituted oligogalacturonides were produced. Thus, in a few steps, a panel of substituted neutral and acidic oligosaccharides was produced from a raw plant material. Such oligosaccharides could be useful for further fractionations such as chemical saponification and enzymatic removal of neutral sugar chains from the hairy regions. The procedures used for pectin extraction, for degradation, and for the purification of fragments seem appropriate for large-scale production of biologically active oligosaccharides from flax.Revisions requested 24 September 2004; Revisions received 4 November 2004     

Extraction and purification of pectins from Alcohol Insoluble Solids from ripe and unripe apples

Pectins are extracted from Alcohol Insoluble Solids of ripe and unripe apples and fractionated by ion exchange chromatography and gelfiltration. In the extracts mainly pectins with neutral sugar contents of 0·15, 0·24 and 0·53 mol neutral sugar residues/mole galacturonate residues are present. The pectin molecules contain rhamnose, arabinose, xylose, galactose, glucose and galacturonic acid residues. No mannose could be detected. The neutral sugar composition of the glycans bound to the galacturonan was found to be constant, except for the relative amount of galactose. During ripening the neutral sugar composition of the extractable pectin does not change.     

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.     

Structural features of pectic polysaccharides from the skin of Opuntia ficus-indica prickly pear fruits

After removal of the mucilage with water at room temperature, pectic polysaccharides were solubilized from Opuntia ficus-indica fruit skin, by sequential extraction with water at 60 degrees C (WSP) and EDTA solution at 60 degrees C (CSP). Polysaccharides with neutral sugar content of 0.48 and 0.36 mol/mol galacturonic acid residue were obtained, respectively, in the WSP and CSP extracts. These pectic polysaccharides were de-esterified and fractionated by anion-exchange chromatography, yielding for each extract five fractions, which were thereafter purified by size-exclusion chromatography. Two of these purified fractions were characterized by sugar analysis combined with methylation and reduction-methylation analysis. The study was then supported by (1)H and (13)C NMR spectroscopy. The results showed that the water-soluble fraction WSP3 and the EDTA soluble fraction CSP3, consisted of a disaccharide repeating unit -->2)-alpha-l-Rhap-(1-->4)-alpha-d-GalpA-(1--> backbone, with side chains attached to O-4 of the rhamnosyl residues. The side chains contained highly branched alpha-(1-->5)-linked arabinan and short linear beta-(1-->4)-linked galactan.     

Heterologous expression of a Penicillium purpurogenum pectin lyase in Pichia pastoris and its characterization

Lignocellulose is the major component of plant cell walls and it represents a great source of renewable organic matter. One of lignocellulose constituents is pectin. Pectin is composed of two basic structures: a ‘smooth’ region and a ‘hairy’ region. The ‘smooth’ region (homogalacturonan) is a linear polymer of galacturonic acid residues with α-(1→4) linkages, substituted by methyl and acetyl residues. The ‘hairy’ region is more complex, containing xylogalacturonan and rhamnogalacturonans I and II. Among the enzymes which degrade pectin (pectinases) is pectin lyase (E.C. 4.2.2.10). This enzyme acts on highly esterified homogalacturonan, catalysing the cleavage of α-(1→4) glycosidic bonds between methoxylated residues of galacturonic acid by means of β-elimination, with the formation of 4,5-unsaturated products. In this work, the gene and cDNA of a pectin lyase from Penicillium purpurogenum have been sequenced, and the cDNA has been expressed in Pichia pastoris. The gene is 1334 pb long, has three introns and codes for a protein of 376 amino acid residues. The recombinant enzyme was purified to homogeneity and characterized. Pectin lyase has a molecular mass of 45 kDa as determined by SDS-PAGE. It is active on highly esterified pectin, and decreases 40 % the viscosity of pectin with a degree of esterification ≥85 %. The enzyme showed no activity on polygalacturonic acid and pectin from citrus fruit 8 % esterified. The optimum pH and temperature for the recombinant enzyme are 6.0 and 50 °C, respectively, and it is stable up to 50 °C when exposed for 3 h. A purified pectin lyase may be useful in biotechnological applications such as the food industry where the liberation of toxic methanol in pectin degradation should be avoided.     

香蕉果实成熟软化过程中细胞壁物质的变化

系统研究了香蕉果实软化过程中细胞壁物质―醇不溶性固形物(AIS)以及3种不同性质的果胶物质:水溶性果胶(WSP)、酸溶性果胶(HP)和碱溶性果胶(OHP)含量的变化。结果表明:随果实的成熟软化,AIS的含量不断降低,且在呼吸跃变时急剧降低;WSP的含量不断增加,HP和OHP的含量不断减少,且均表现出在早期变化量少,在果实硬度迅速降低时变化明显。该研究进一步证明细胞壁物质的变化是导致香蕉果实软化的主要原因。     

Extraction, purification and chemical characterisation of xylogalacturonans from pea hulls

Pea hulls contained 925 mg/g sugar including 659 mg/g cellulosic glucose and 90 mg/g uronic acid. They were de-esterified by NaOH (pH>13 at 4°C, 2 h) and treated with HCl (0.1 mol/l, 80°C, 24 h). The HCl-soluble fraction represented 95 mg/g initial pea hulls. It was rich in galacturonic acid (259 mg/g), xylose (93 mg/g) and rhamnose (91 mg/g), which co-eluted in anion-exchange chromatography. The HCl-soluble fraction was degraded by a rhamnogalacturonan-hydrolase and the reaction products were fractionated by size-exclusion chromatography. Two fractions, representing together 18 mg/g initial pea hulls, were composed almost exclusively of galacturonic acid and xylose and could be defined as xylogalacturonans. The first fraction exhibited a high molar mass, a molar ratio Xyl/GalA of 1 and contained almost 5% of rhamnose. The molar mass of the second fraction was much lower and the molar ratio Xyl/GalA was 0.6. Methylation analysis showed the presence in both fractions of a (1→4) galacturonan backbone highly substituted on O-3 either by terminal xylosyl residues or by short side-chains of (1→2) linked xylosyl residues.     

Structure of citrus pectins and viscometric study of their solution properties

Citrus pectins with degrees of methylation between 30 and 72% were carefully characterized in order to determine their charge density and molecular weight distribution, the content in galacturonic acid and in neutral sugars, the degree of methylation and acetylation. Using enzymic degradation it has been found that pectin molecules consist mainly of long homogalacturonan regions with some regions of neutral sugars as side chains attached on rhamnose residues. The viscometric behaviour of the different samples indicates that 0.1 M NaCl, at 25 degrees C, is a good solvent of sodium pectinates. From the evolution of the Huggins parameter, it appears that pectins with 50% of methylated galacturonic groups exhibit a maximum flexibility. A Mark-Houwink exponent of 0.8 has been found in good agreement with theoretical predictions for flexible polymers in a good solvent.     

Characterization of non-esterified galacturonic acid sequences in pectin with endopolygalacturonase

A method was developed that enabled the study of non-esterified galacturonic acid sequences (so-called blocks) in pectin. Endopolygalacturonase of Kluyveromyces fragilis was used to extensively degrade pectin, and the composition of the galacturonic acid molecules produced was determined with high-performance anion-exchange chromatography at pH 5. With this technique, the amount of non-esterified mono-, di-, and trigalacturonic acid released was determined. In addition, the relative amounts of methyl-esterified oligomers--up to 10 galacturonic acid residues could be observed. By comparing the percentages of non-esterified mono-, di-, and trigalacturonic acids released, pectins with large enzyme-degradable blocks could be distinguished from pectins with small enzyme-degradable blocks. High percentages of mono- and digalacturonic acid were found for pectins containing small non-esterified blocks. The total area of all peaks corresponding to methyl-esterified oligomers was found to be indicative for the distribution of these blocks. The higher the ratio of the methyl- to non-esterified peak areas, the more closely associated blocks are present. Randomly esterified pectins, with degrees of methyl esterification of 50 and higher, contained smaller, more clustered blocks than commercial extracted pectins of comparable degrees of esterification. The approach developed enables a very detailed study of the methyl-ester distribution of pectin to be carried out and is a very important addition in the study of the functional behavior of this complex polymer.     

Rapid quantification of O-acetyl and O-methyl residues in pectin extracts

A rapid method for the determination of the degrees of methylation (DM) and acetylation (DA) of pectins was developed. The polymer substitution degree as determined after saponification at 80 degrees C with NaOD during 1H NMR analysis. Under alkaline conditions, the cleavage of O-acetyl and O-methyl linkages allows the detection and the integration of the H-4 signal from galacturonic acid residues in the newly unesterified pectins. So, after a 10-min NMR recording, sodium acetate and sodium methanolate can be easily quantified relative to the clearly identified H-4 signal in galacturonic acid residues. Protons signals from pectin neutral sugars do not interfere with H-4. During the analysis, a limited (<3%) methanol evaporation leading to a weak reduced signal from the methanolate protons was observed. The proposed method allows in few minutes an accurate simultaneous quantification of DM and DA from few mg of pectin extracts, without the need of external standards.     

Simultaneous Measurement of the Galacturonate and Neutral Sugar Contents of Pectic Substances by an Enzymic-HPLC Method

An enzymic-HPLC method was successfully applied to the simultaneous analysis of the galacturonate and neutral sugar contents of pectic substances. A mixture of seven neutral sugars that are present in oridnary pectins was eluted as one peak on a Shodex SUGAR SH1821 column, using 0.001 N sulfuric acid as the eluent; the peak was completely separated from that of galacturonate. No difference in the peak-area ratios of individual neutral sugars to glycerol (internal standard) was found among the seven; the relationship between the peak response and concentration was strictly linear throughout the entire concentration range studied. Seventeen pectic samples, including pectins, pectates, and rhamnogalacturonan fragments, were completely prehydrolyzed by Driselase, an industrial enzyme product from Irpex lacteus, and then analyzed for their constituent sugar contents. The enzymic-HPLC method was simple, accurate, and particularly useful for routine pectin analyses.     

Sugar beet (Beta vulgaris) pectins are covalently cross-linked through diferulic bridges in the cell wall

Arabinan and galactan side chains of sugar beet pectins are esterified by ferulic acid residues that can undergo in vivo oxidative reactions to form dehydrodiferulates. After acid and enzymatic degradation of sugar beet cell walls and fractionation of the solubilized products by hydrophobic interaction chromatography, three dehydrodiferulate-rich fractions were isolated. The structural identification of the different compounds present in these fractions was performed by electrospray-ion trap-mass spectrometry (before and after (18)O labeling) and high-performance anion-exchange chromatography. Several compounds contained solely Ara (terminal or alpha-1-->5-linked-dimer) and dehydrodiferulate. The location of the dehydrodiferulate was assigned in some cases to the O-2 and in others to the O-5 of non-reducing Ara residues. One compound contained Gal (beta-1-->4-linked-dimer), Ara (alpha-1-->5-linked-dimer) and dehydrodiferulate. The location of the dehydrodiferulate was unambiguously assigned to the O-2 of the non-reducing Ara residue and O-6 of the non-reducing Gal residue. These results provide direct evidence that pectic arabinans and galactans are covalently cross-linked (intra- or inter-molecularly) through dehydrodiferulates in sugar beet cell walls. Molecular modeling was used to compute and structurally characterize the low energy conformations of the isolated compounds. Interestingly, the conformations of the dehydrodiferulate-bridged arabinan and galactan fragments selected from an energetic criterion, evidenced very nice agreement with the experimental occurrence of the dehydrodiferulated pectins. The present work combines for the first time intensive mass spectrometry data and molecular modeling to give structural relevance of a molecular cohesion between rhamnogalacturonan fragments.     

Studies on the Pectic Substances of Plant Cell Walls: III. DEGRADATION OF CARROT ROOT CELL WALLS BY ENDOPECTATE LYASE PURIFIED FROM ERWINIA AROIDEAE

Pectate lyase was isolated from the cell extract of Erwinia aroideae. The enzyme was further purified to a high degree by a procedure involving ammonium sulfate fractionation and chromatography on CM-Sephadex C-50 and on Sephadex G-200. The enzyme attacked its substrate in an endo fashion and was more active on the sodium salt of acid-insoluble polygalacturonate or pectic acid than it was on the methoxylated pectin. The enzyme had an optimum pH at 9.3, was stimulated by calcium ions, and was completely inhibited by ethylenediaminetetraacetic acid. In addition, the reaction products showed an absorption maximum between 230 and 235 nm and reacted with thiobarbituric acid. These results indicate that the purified enzyme is an endopectate lyase. The endopectate lyase also had the ability to solubilize effectively the pectic fraction from the cell walls of carrot (Daucus carota) root tissue. The enzyme released 30.5% of the wall as soluble products and also liberated all of the galacturonic acid present in the walls. The total neutral sugars released by the enzyme were 10.6% of the walls, which corresponded to 71.5% of noncellulosic neutral sugars. The soluble products were separated into five fractions by DEAE-Sephadex A-50 column chromatography. Based on the analysis of sugar composition of each fraction, the pectic fraction of carrot cell wall is presented.     

Polysaccharides from the callus tissue of the rowan tree Sorbus aucuparia L. stem

Polysaccharide fractions SAcI and SAcII were isolated from callus tissues of rowan tree stems. The SAcI fraction was shown to contain compounds belonging to the arabinogalactan II group. The SAcII fraction, called sorban, comprised pectic polysaccharides composing the protopectin complex of the cell wall callus. The SAcII fraction was found to contain a large amount of galacturonic acid residues and a set of neutral sugars characteristic of rhamnogalacturonan I. The composition and properties allowed a suggestion that the sorban backbone is mainly formed by 1,4-α-D-galactopyranosyluronic acid residues, while the neutral sugars are represented by 1,4-linked glucopyranose and xylopyranose residues, 1,5-linked arabinofuranose, 1,6-linked galactopyranose and mannofuranose residues as well as terminal glucopyranose and xylopyranose residues. The callus growth was shown to be associated with nearly a constant content of galacturonic acid and neutral sugar residues in sorban (fraction SAcII).     

Sugar constituents of fucose-containing polysaccharides from various Japanese brown algae

Sugar constituents of the fucose-containing polysaccharides (FCPs) from 21 species of brown algae were analyzed. FCPs were extracted with hot water (100 °C, 4 h), separated by precipitation with 20% (v:v) ethanol in the presence of 0.05 M MgCl₂ to remove contaminating soluble alginate, and purified by DEAE-Sephadex column chromatography. The samples were hydrolyzed with HCI, and neutral sugar and uronic acid were separated by anion exchange chromatography. Their amounts were determined by gas-liquid chromatography. The neutral sugars in the FCPs from Ishige okamurae, Laminaria ochotensis, Myelophycus simplex, Padina arborescens and Sargassum thunbergii all contained arabinose, fucose, galactose, glucose, mannose, rhamnose and xylose residues. The FCPs from Ishige okamurae, Padina arborescens, Sargassum hemiphyllum, S. patents and S. sagamianum contained the four uronic acids, galacturonic acid, glucuronic acid, guluronic acid and mannuronic acid.     

Comparative study of the cell wall composition of broccoli, carrot, and tomato: structural characterization of the extractable pectins and hemicelluloses

This study delivers a comparison of the pectic and hemicellulosic cell wall polysaccharides between the commonly used vegetables broccoli (stem and florets separately), carrot, and tomato. Alcohol-insoluble residues were prepared from the plant sources and sequentially extracted with water, cyclohexane-trans-1,2-diamine tetra-acetic acid, sodium carbonate, and potassium hydroxide solutions, to obtain individual fractions, each containing polysaccharides bound to the cell wall in a specific manner. Structural characterization of the polysaccharide fractions was conducted using colorimetric and chromatographic approaches. Sugar ratios were defined to ameliorate data interpretation. These ratios allowed gaining information concerning polysaccharide structure from sugar composition data. Structural analysis of broccoli revealed organ-specific characteristics: the pectin degree of methoxylation (DM) of stem and florets differed, the sugar composition data inferred differences in polymeric composition. On the other hand, the molar mass (MM) distribution profiles of the polysaccharide fractions were virtually identical for both organs. Carrot root displayed a different MM distribution for the polysaccharides solubilized by potassium hydroxide compared to broccoli and tomato, possibly due to the high contribution of branched pectins to this otherwise hemicellulose-enriched fraction. Tomato fruit showed the pectins with the broadest range in DM, the highest MM, the greatest overall linearity and the lowest extent of branching of rhamnogalacturonan I, pointing to particularly long, linear pectins in tomato compared with the other vegetable organs studied, suggesting possible implications toward functional behavior.     

L-Altruronic acid formed by epimerization of D-galacturonic acid methyl esters during saponification of citrus pectin

While searching for oligosaccharides containing rhamnose residues in the endopolygalacturonase (EPG) digest of saponified citrus pectin, we found several oligomers containing, in addition to galacturonic acid, a sugar previously unreported in pectin. The 1- and 2-D 1H NMR spectra of the oligosaccharides were consistent with the sugar being a uronic acid with its 2- and 3-hydroxyls being axial and 4-hydroxyl being equatorial. MALDI-TOF mass spectrometry indicated that the oligomers consisted solely of uronic acids. Reduction of the uronic acids in the oligosaccharides converted them to galactose and altrose. The altrose was found to be the L enantiomer by comparison of its trimethylsilyl (-)-2-butyl glycosides to those of authentic D-altrose and a racemic mixture. The sugar was not found in oligosaccharides prepared from EPG digestion of citrus pectin deesterified with pectin methylesterase rather than saponification. Thus, it appears that during saponification, a small proportion of the methylesterified galacturonic acid residues in pectins is epimerized at C-5 leading to formation of L-altruronic acid residues.