Characterisation of the extractable pectins and hemicelluloses of the cell wall of carrot

Pectic and hemicellulosic polysaccharides were successively extracted from an alcohol-insoluble residue (AIR) from carrot root by the actions of Pronase, hot dilute acid, cold dilute alkali, and concentrated alkali in yields corresponding to 12.6, 13.5, 21.7, and 6.7% of AIR, respectively. The first two products were fractionated further by ion-exchange chromatography. Carrot pectins contained 61.3–66.0% of galacturonic acid and 16.0–19.9% of neutral sugars, mainly galactose, arabinose, and rhamnose. Except for the alkali-soluble pectins, the degrees of methylation were high (62.9–67.1) and there was a significant degree of acetylation (7.2–13.5). Pectin fractions were homogeneous in gel-filtration chromatography with viscosity-average molecular weights varying between 36,200 and 56,500. Methylation analysis indicated the presence of arabinogalactans in the pectins extracted during the proteolysis, and fairly long chains of (1→4)-linked galactan with a branched arabinan in the two other pectic fractions. The hemicellulose fraction was mainly composed of (1→4)-linked glucan, (1→4)-linked mannan, (1→4)-linked xylan, and small but significant amounts of pectic polysaccharides. The possible association of cell-wall polymers is discussed.     

Cell wall changes during the expansion and senescence of carnation (Dianthus caryophyllus) petals

Senescence of carnation (Dianthus caryophyllus L. ev. White Sim) petals coincided with a decrease on a per flower basis in the yield of cell wall and ethanol-insoluble solids. The decrease in cell wall yield per flower was due largely to a loss of neutral sugars, primarily galactose (45%) and arabinose (23%). On a per flower basis, water-and chelator-soluble pectins increased throughout development, comprising in senescent petals 18 and 58%, respectively, of total pectin. Alkali-soluble pectins ranged from 35 to 45% of the total pectin and decreased during senescence. Gel chromatography of chelator- and alkali-soluble pectins revealed no change in molecular size and polygalacturonase activity was not detected. Large-molecular-size hemicelluloses decreased during development, an observation reminiscent of the changes affecting hemicelluloses during the ripening of a number of fruit types. Compositional analysis of the large hemicellulosic polymers revealed a decrease in xylose and galactose 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.     

Studies on the intermolecular distribution of industrial pectins by means of preparative size exclusion chromatography

Three industrial high methoxyl pectins have been fractionated by size exclusion chromatography (SEC) on a preparative scale and the chemical composition, viscosity and light scattering behaviour of the fractions have been investigated. Chemical analysis revealed that the composition varies greatly from one SEC fraction to another. In all three pectin samples, the fractions of low molecular size contain most of the free neutral polysaccharides as well as some free pectin ‘hairy regions’. In addition, the lemon pectin samples contain some pectin molecules of large size which are rich in neutral sugars. Phenolic and proteinaceous compounds coelute with neutral sugar-rich fractions. However, in the apple pectin, phenolics and proteins occur predominantly in the fractions of low molecular size. Lemon pectin molecules, especially that of the lemon A sample, are prone to aggregation in the presence of calcium cations. The aggregate fraction can be disrupted by shear forces, heating or the presence of a chelating agent. The formation of such calcium-pectinate aggregates seems to be due to the presence of some molecules with low degrees of methoxylation. Light scattering measurements also suggest that even very narrow SEC fractions remain highly heterogeneous on the basis of their molecular weight, thus indicating large differences in molecular conformation.     

Characterisation of pectin subunits released by an optimised combination of enzymes

Pectins from sugar beet, lime and apple were degraded by a rhamnogalacturonan hydrolase associated or not with pectin methylesterases and side chain degrading enzymes (galactanase and arabinanase). The composition of the enzymatic mixture was optimised by following the reaction by viscosimetric means. The reaction products were fractionated by ion exchange chromatography. Treatment with all the enzymes released four fractions: (1). 227-247 mg/g of initial pectins and corresponded to neutral sugars from the side chains; (2,3). represented together 184-220 mg/g of pectins and corresponded to rhamnogalacturonan; (4). 533-588 mg/g of pectins and corresponded to homogalacturonan. Lime pectins have the shortest rhamnogalacturonan regions. The molar masses of homogalacturonans were in the range of 16000-43400 g/mol according to the origin of pectins, corresponding to degrees of polymerisation of 85-250. The mode of action of the enzymes used is also discussed.     

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.     

Bibliography

Water-soluble polysaccharides isolated from the roots of Panax ginseng C. A. Meyer were completely fractionated into two neutral fractions (WGPN and WGPA-N) and six acidic fractions (WGPA-1-RG, WGPA-2-RG, WGPA-1-HG, WGPA-2-HG, WGPA-3-HG and WGPA-4-HG) by a combination of ethanol precipitation, ion-exchange and gel permeation chromatographies. The analytical results showed that WGPN was a starch-like glucan; WGPA-N was a mixture of starch-like glucan and arabinogalactan; WGPA-1-RG and WGPA-2-RG were composed of major neutral sugars and minor acidic sugars that belong to the type-I rhamnogalacturonan (RG-I)-rich pectins, while fractions WGPA-1-HG to WGPA-4-HG were mainly composed of galacturonic acid (GalA, 62.4–92.1%) and have been identified to be homogalacturonan (HG)-rich pectins with different degrees of methyl-esterification, ranging from 0% to 30%. High performance gel permeation chromatography (HPGPC) showed that the six acidic fractions were homogenous, with molecular weights approximately ranging from 3.5 × 10³ to 1.1 × 10⁵. Lymphocyte proliferation assays showed that both the neutral polysaccharides and acidic polysaccharides were potent B and T cell stimulators.     

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.     

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.     

Investigating the nature of branching in pectin by atomic force microscopy and carbohydrate analysis

Atomic force microscopy (AFM) has been used to investigate the nature of the long branches attached to pectin which were described in a previous report [Round, A. N.; MacDougall, A. J.; Ring, S. G.; Morris, V. J. Carbohydr. Res. 1997, 303, 251-253]. Analysis of the AFM images and comparison with neutral sugar and linkage analyses of the two pectin fractions suggest that the distribution and total amount of branches observed do not correspond with the pattern of neutral sugar distribution. It is thus postulated that the long chains consist of polygalacturonic acid, attached via an as yet undetermined linkage to the pectin backbone, with the neutral sugars present as short, undetected branches. This explanation would have important implications for the nature of 'in situ' pectin networks within plant cell walls and models of gelation in commercial extracted pectin, and the existence of significant branching will markedly influence the viscosity of extracted pectins.     

Characterization of hop pectins shows the presence of an arabinogalactan-protein

Hop pectins were extracted from spent hops using acid extraction conditions and were characterized chemically. The acid extraction of spent hops resulted in a yield of 2%, containing 59% of polysaccharides. The hop pectins under investigation had a relatively high molecular weight and an intrinsic viscosity comparable to that of commercially available apple and citrus pectins. The low degree of methyl esterification of these pectins implicates that they are mainly suitable for use in calcium gels. The degree of acetylation and the neutral sugar content were relatively high.

A high molecular weight fraction which contained arabinogalactan-proteins was shown to be present in the hop pectin extract after preparative size-exclusion chromatography. Additionally, a fraction with a lower molecular weight was present containing mainly homogalacturonans. The arabinogalactans in the high molecular weight population consisted of (1→3)- and (1→3,6)-linked galactans highly branched with arabinose and galactose side-chains. The protein part of the arabinogalactan-protein (13%) was found to be rich in cystein, threonin, serinin, alanin, and hydroxyprolin. The molecular weight distribution of the hop pectin after degradation with the enzymes endopolygalacturonase plus pectin methyl esterase suggested that the arabinogalactan-protein present in the hop pectin extract was linked to the pectin and that the arabinogalactan-protein itself had a fairly low molecular weight.     

Pectins in the fruits of Japanese quince (Chaenomeles japonica)

The pectin content, composition and physico-chemical properties were studied in the fruits of two genotypes of Japanese quince. On average, the fruits contained 11 g pectins/100 g dry fruit and 1.4 g pectins/100 g fresh fruit. A sequential extraction was used to isolate the pectins from the fruits. The cells from the flesh were examined using a confocal laser scan microscope, fresh and after each extraction step of the sequence. The dilute acid conditions were the most efficient for pectin extraction. Pectins extracted by water or potassium oxalate had higher (>600 ml/g) intrinsic viscosities than the pectins extracted by dilute acid (<400 ml/g). Anionic exchange chromatography was performed on the acid-extracted pectins. They were constituted of four populations, the first one being mainly composed of arabinans, the second one of homogalacturonans, the third one of rhamnogalacturonans. The composition of the last one varied with the genotype studied.     

Pectic polysaccharides of growing plant tissues

1. The polysaccharide compositions of the cell walls of sycamore cambium and sycamore callus tissue have been analysed and found to be directly comparable. 2. Electrophoretic analyses of the whole pectins prepared from actively growing callus and cambial tissue have shown that these preparations contain, in addition to the neutral and weakly acidic components present in apple fruit, a strongly acidic polygalacturonic acid component. 3. The weakly acidic component of all the pectins was directly comparable with that of the pectinic acid of apple fruit. 4. The components of the whole pectin of sycamore callus tissue have been partially purified and analysed. The neutral and weakly acidic components also found in apple fruit were isolated. 5. The pattern of the composition of the neutral sugars present in the pectins of actively growing tissues of cambium and callus has been compared with those present in apple-fruit pectinic acid. 6. The presence of rhamnose linked as galacturonosyl-(1-->2)-rhamnose has been found in sycamore whole pectin. 7. The difference in the pectins of callus, cambium and fruit appears not to be that of species difference but is more characteristic of the nature of the growth and growth conditions of the cells. This is discussed in relation to the problems of the control and mechanism of plant-cell growth and differentiation.     

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.     

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.     

Enzymic and chemical degradation and the fine structure of pectins from sugar-beet pulp

Pectins sequentially extracted from sugar-beet pulp with water (WSP), oxalate (OXP), hot acid (HP), and cold alkali (OHP) have been degraded variously by base-catalysed β-elimination, de-esterification, endopectin lyase, pectin methyl esterase, endopolygalacturonase, and endopectate lyase. The products were studied mainly by chromatography on Sephadex G-100. The pectins contain various amounts of degradation-resistant (hairy) fragments in which the molar ratios of neutral sugar residues to galacturonic acid residues were 4.8, 4.6, 3.8, and 3.7 for WSP, OXP, HP, and OHP, respectively. The molar ratios of rhamnose residues to galacturonic acid residues in these fragments were 0.15, 0.20, 0.38 and 0.35, respectively. The pectins also contained sequences of galacturonic acid residues with relatively little neutral sugar residues attached (smooth fragments). Methyl ester and acetyl groups were distributed fairly regularly along the smooth fragments. Evidence is presented for an association of oligogalacturonic acids with the hairy fragments under the conditions of gel chromatography. Feruloyl groups are located in the hairy fragments. Other phenolic compounds, associated with the purified pectins, appear not to be covalently linked.     

Production of polysaccharides by callus cultures of common duckweed

Callus lines of common duckweed produced acid arabinogalactan and pectin in an amount varying from 1 to 3% of dry weight. The arabinogalactan specimens from the cell lines studied displayed a similar monosaccharide composition. The duckweed callus lines whose arabinogalactans contained apiose residues (1–2%) were found. All pectin specimens had a similar qualitative monosaccharide composition but differed in the quantitative content of monosaccharide residues. The lines with high contents of galactose, arabinose, and apiose in pectin specimens were obtained. The total content of neutral monosaccharide residues in pectins varied from 26 to 50%.     

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.     

Composition and properties of pectin polysaccharides of St. John’s wort Hypericum Perforatum L.

Three fractions of acidic water-soluble polysaccharides (concentration of glucuronic acid 10?C65%) were obtained from the above-ground part of St. Johns wort Hypericum perforatum L. by serial extraction with water and 0.7% aqueous solution of ammonium oxalate. Enzymatic hydrolysis of these polysaccharides using endo-polygalacturonase indicates that their carbohydrate chains contain the units of galacturone formed by 1,4-??-linked residues of non-substituted D-galacturonic acid. The extracted polysaccharides have been purified by means of gel filtration. It has been shown that water-soluble polysaccharides obtained by extraction with water manly contain the residues of galactose, mannose, glucose, and arabinose (the concentration of glucuronic acid being 10?C27%) while the polysaccharide fraction extracted using 0.7% aqueous solution of ammonium oxalate is presented by pectin polysaccharides. Only the residues of galacturonic acid (55?C72%) have been identified among glucuronic acids in its composition using chromatography/mass spectrometry of trimethylsilyl derivatives. In addition, this fraction contains the residues of the neutral monosaccharides which are typical for pectins: arabinoses, galactoses, rhamnoses, and glucose; there are also minor concentrations of residues of xylose and mannose. IR spectra of pectin polysaccharides of St. John??s wort have absorption bands in the ranges 1740, 1640?C1620, 1236?C1200, and 1200?C1000 cm^?1 which are typical for pectins. It has been demonstrated that aqueous solutions of pectin polysaccharides of St. John??s wort (2 mg/mL) have pronounced antioxidant activity (44% of the activity of trolox taken for 100%).     

Interactions between apple (Malus x domestica Borkh.) polyphenols and cell walls modulate the extractability of polysaccharides

Apple polyphenol (procyanidin)–cell wall interactions were investigated and their impact on polysaccharide extractability were determined. Native and oxidised procyanidins with average degrees of polymerisation of 13 and 55 were incubated with cell walls. The effect of polyphenol oxidation was evaluated according to two designs: polyphenols were chemically oxidised either before or during interaction. The extent of procyanidin binding to cell walls was assessed by the weight increase of procyanidin–cell wall complexes as compared to weights of cell walls alone. Pectins and hemicelluloses were subsequently extracted from cell walls and from cell wall–procyanidin adducts using a chelating agent (ammonium oxalate), a pectin lyase treatment and NaOH.Weight increases of complexes ranged from 20% to 29%. Weight gains increased in the following order: native, pre-oxidised, simultaneously oxidised and bound procyanidins, these different fractions were, respectively, bound to cell walls. In presence of native procyanidins, oxalate extracted less pectins, and those pectins had lower degrees of methylation, as compared to cell walls alone. When cell walls were incubated with oxidised and oxidising procyanidins, even less pectins with lower degree of methylation were extracted. Major findings indicated that procyanidins mainly bound to pectins as compared to other cell wall compounds: (1) the procyanidin adsorption to cell walls limited the depolymerisation of pectins supposedly induced by pectin lyase. Thus less pectins were extracted but their degree of methylation increased, indicative of products of lysis of pectin lyase. (2) Hemicelluloses extracted using NaOH (4 M) were more abundant in pectins when oxidised or oxidising procyanidins were complexed rather than non complexed to cell walls.