Degrees Of Methyl Esterification Research Articles

Structural, rheological and antioxidant properties of pectins from Equisetum arvense L. and Equisetum sylvaticum L.

The pectins were isolated from sterile stems of E. arvense (EA, yield 5.9%) and E. sylvaticum (ES, yield 4.8%) (Equisetaceae) using ammonium oxalate extraction after preliminary treatment with dilute HCl (рH 4.0). The pectins possessed high molecular weight (Mw, 340–360 kDa), high GalA content (ca. 85%), low degrees of methyl-esterification (14–16%) and acetylation (3–8%). NMR analysis indicated extensive regions of partially methyl-etherified and 3-O-acetylated HG and minor regions of low branched RG in the fragment isolated after hydrolysis of pectin EA by pectinase. Pectin EA produced a higher viscosity solution, formed a stronger and more rigid ionotropic hydrogel than pectin ES. The pectins scavenged DPPH and hydroxyl radicals, but not the superoxide radical and hydrogen peroxide. Phenolic compounds (0.11 and 0.23%) associated with polysaccharide moieties were apparently responsible for the differences in the anti-DPPH scavenging activity of pectins EA and ES (63 and 49%). The findings suggested that pectin from E. arvense should be more perspective than pectin from E. sylvaticum on their use as components of wound healing remedies.

Integrative analysis of pectin methylesterase (PME) and PME inhibitors in tomato (Solanum lycopersicum): Identification, tissue-specific expression, and biochemical characterization

Although previous studies have demonstrated that the degree of demethylesterification of pectin polysaccharides is modulated during tomato fruit ripening, its involvement in vegetative organ development has been seldom investigated. As a first step in understanding the importance of pectin modification during vegetative stages, we used chemical, biochemical, and molecular approaches to analyze PMEs and PMEIs in tomato plants. We found that tomato cell walls isolated from vegetative tissues as well as the fruit contain substantial quantities of pectin, and different degrees of methylesterification were evident in different tissues. Our chemical study was further substantiated by immunolocalization analysis, which showed that selective removal of pectin-bound methyl groups is required for proper organ development and growth. In the tomato genome, there exists 79 PMEs and 48 PMEIs with temporally and spatially regulated expression. As a case study, we showed that two tomato PMEIs (SolycPMEI13 and SolycPMEI14) exhibited PMEI activities. This is the first report regarding the genome-wide identification and expression profiling of PME/PMEIs in tomato and the first chemical evidence of the differential degrees of pectin methylesterification in vegetative and reproductive tissues. Taken together, our findings provide an important tool to unravel the molecular and physiological functions of tomato PME and PMEI in further study.

Cell wall polysaccharides from Ponkan mandarin (Citrus reticulata Blanco cv. Ponkan) peel

Cell wall polysaccharides from ponkan peel were investigated with the aim of gain knowledge about their potential for different applications and the use of ponkan peel as raw material for pectin extraction. The plant material was defatted using MeOH:CHCl3, pretreated with DMSO and then subjected to sequential extractions with cold and hot water, ammonium oxalate, HCl, Na2CO3, 2 M and 4 M NaOH in order to obtain polysaccharides. The polysaccharide fractions were analyzed by chemical, chromatographic and spectroscopic methods Cold and hot water-soluble pectins contained higher amounts of GalA and higher degrees of methyl-esterification (DM) than ammonium oxalate and HCl fractions. Na2CO3 extraction provided non-esterified arabinose-rich pectins which formed gel in a dialysis step. NaOH solubilized hemicelluloses, composed mainly of xyloglucans, galactomannans and galactoglucomannans. The water-soluble fraction (WSP) was purified using α-amylase and amyloglucosidase and gave rise to the subfraction named α-WSP. The α-WSP was a pectin composed of HG and RG-I domains containing side chains of arabinans and short-chains of galactans, with low DM (39.4%) and Mw of 1.615 × 105 g/mol.

Degree of pectin methyl esterification in endosperm cell walls is involved in embryo bending in Arabidopsis thaliana

The endosperm is a transitory structure involved in proper embryo elongation. The cell walls of mature seed endosperm are generally composed of a uniform distribution of cellulose, unesterified homogalacturonans, and arabinans. Recent studies suggest that changes in cell wall properties during endosperm development could be related to embryo growth. The degree of methyl esterification of homogalacturonans may be involved in this endosperm tissue remodelling. The relevance of the degree of homogalacturonan methyl esterification during seed development was determined by immunohistochemical analyses using a panel of probes with specificity for homogalaturonans with different degrees of methyl esterification. Low-esterified and un-esterified homogalacturonans were abundant in endosperm cells during embryo bending and were also detected in mature embryos. BIDXII (BDX) could be involved in seed development, because bdx-1 mutants had misshapen embryos. The methyl esterification pattern described for WT seeds was different during bdx-1 seed development; un-esterified homogalacturonans were scarcely present in the cell walls of endosperm in bending embryos and mature seeds. Our results suggested that the degree of methyl esterification of homogalacturonans in the endosperm cell wall may be involved in proper embryo development.

Morphology of complexes formed between β‐lactoglobulin nanofibrils and pectins is influenced by the pH and structural characteristics of the pectins

For the optimal use of β-lactoglobulin nanofibrils as a raw material in biological composites an in-depth knowledge of their interactions with other constituents is necessary. To understand the effect of electrostatic interactions on the morphology of resulting complexes, β-lactoglobulin nanofibrils were allowed to interact with pectins in which the amount of available negative charge was controlled by selecting their degree of methylesterification. In this study, citrus pectins having different degrees of methylesterification (∼48, 67, 86, and 97%) were selected and interacted with nanofibrils at pH 2 and pH 3, where they possess a net positive charge. Electrostatic complexes formed between β-lactoglobulin nanofibrils and all pectin types, except for the sample having a degree of methylesterification of 97%. The morphology of these complexes, however, differed significantly with the degree of methylesterification of the pectin, its concentration, and the pH of the medium, revealing that distinct desired biological architectures can be attained relatively easily through manipulating the electrostatic interactions. Interestingly, the pectin with a degree of methylesterification of 86% was found to crosslink the β-lactoglobulin nanofibrils into ordered 'nanotapes'.

Pectin nanostructure influences pectin-cation interactions and in vitro-bioaccessibility of Ca2+, Zn2+, Fe2+ and Mg2+-ions in model systems

This study explores the effect of pectin structure on mineral bioaccessibility in model systems. Commercial citrus and sugar beet pectin (CP and SBP, respectively) were demethylesterified by the action of carrot pectin methylesterase and characterisation of the resulting samples revealed different degrees of methylesterification (DM). Additionally, SBP exhibited a higher protein content, degree of acetylation, degree of feruloylation, degree of branching and molar mass compared to CP. In vitro-simulated digestion (stomach and small intestine phases) of mineral-enriched pectin solutions showed that decreasing pectin DM results in a decrease in mineral-bioaccessibility due to pronounced cation-pectin interactions. For a given DM, SBP digests exhibited higher mineral-bioaccessibilities than CP digests possibly due to structural differences of pectin in these sources. Furthermore, increasing the mineral-enrichment prior to in vitro-digestion resulted in a higher mineral-bioaccessibility while increasing the pectin concentration decreased the mineral-bioaccessibility. Generally, Mg2+-ions were the most bioaccessible followed by Ca2+, Zn2+, then Fe2+-ions.

Evaluation of cation-facilitated pectin-gel properties: Cryo-SEM visualisation and rheological properties

To provide more insight into the cation facilitated pectin-gel structure-function relationship, the microstructure of pectin-gels was explored using cryo-scanning electron microscopy (Cryo-SEM) in combination with physiochemical and rheological analysis. Pectin with different degrees of methylesterification, DM, was obtained from commercial citrus pectin (CP) and sugar beet pectin (SBP) by action of carrot pectin methylesterase. Pectin characterisation confirmed that pectin properties other than DM were unaltered and pectin from the two sources exhibited different structures. Cryo-SEM visualisation of pectin-gels prepared with different cations revealed that the network density increased with decreasing DM and increasing pH, pectin and cation-concentration. At a given DM, the more linear and less acetylated pectin (CP) exhibited a more intertwined fibrous network compared to the highly branched and more acetylated pectin (SBP). Furthermore, gel-microstructure was influenced by type of cation with Zn2+, Fe2+ and Ca2+-pectin gels showing a more intertwined network compared to Mg2+-pectin gels. Finally, rheological properties of gels complementing Cryo-SEM observations showed that as the network density increased, the gel-stiffness (G′) increased.

The impact of lemon pectin characteristics on TLR activation and T84 intestinal epithelial cell barrier function

Sufficient dietary fibre intake reduces the risk of several diseases, but the mechanisms linking fibre structure and health effects remain unclear. To analyse the influence of the amount of methyl groups esterified to the backbone, lemon pectins of different degrees of methyl esterification (30, 56, and 74DM) were studied for immune receptor activating potential and epithelial barrier protection. In reporter assays, the pectins demonstrated TLR/MyD88 dependent activation of NF-κB/AP-1, which increased with increasing pectin DM. To analyse the importance of backbone structure, the pectins were enzymatically digested into oligomers, which abrogated TLR activating potential. The 30 and 74DM pectins induced strong protection of the epithelial barrier measured by T84 transepithelial electrical resistance (TEER). These results indicate that activation of immune cells by lemon pectins is TLR dependent, and the intact polymer backbone is indispensable for activation. In addition, DM is a determining factor in activating potential and epithelial barrier protective effects.

Purification and Characterization of a Unique Pectin Lyase from Aspergillus giganteus Able to Release Unsaturated Monogalacturonate during Pectin Degradation.

A pectin lyase, named PLIII, was purified to homogeneity from the culture filtrate of Aspergillus giganteus grown in submerged culture containing orange peel waste as carbon source. PLIII was able to digest apple pectin and citrus pectins with different degrees of methyl esterification. Interestingly, the PLIII activity was stimulated in the presence of some divalent cations including Pb2+ and was not significantly affected by Hg2+. Like other pectin lyases, PLIII is stimulated by but is not dependent on Ca2+. The main soluble product released during the degradation of pectic substances promoted by the PLIII is compatible with an unsaturated monogalacturonate. PLIII is a unique enzyme able to release unsaturated monogalacturonate as the only soluble product during the degradation of pectic substances; therefore, PLIII was classified as an exo-pectin lyase. To our knowledge, this is the first characterization of an exo-pectin lyase. The PLIII described in this work is potentially useful for ethanol production from pectin-rich biomass, besides other common applications for alkaline pectinases like preparation of textile fibers, coffee and tea fermentation, vegetable oil extraction, and the treatment of pulp in papermaking.

A distinct role of pectate lyases in the formation of feeding structures induced by cyst and root-knot nematodes.

Pectin in the primary plant cell wall is thought to be responsible for its porosity, charge density, and microfibril spacing and is the main component of the middle lamella. Plant-parasitic nematodes secrete cell wall-degrading enzymes that macerate the plant tissue, facilitating the penetration and migration within the roots. In sedentary endoparasitic nematodes, these enzymes are released only during the migration of infective juveniles through the root. Later, nematodes manipulate the expression of host plant genes, including various cell wall enzymes, in order to induce specific feeding sites. In this study, we investigated expression of two Arabidopsis pectate lyase-like genes (PLL), PLL18 (At3g27400) and PLL19 (At4g24780), together with pectic epitopes with different degrees of methylesterification in both syncytia induced by the cyst nematode Heterodera schachtii and giant cells induced by the root-knot nematode Meloidogyne incognita. We confirmed upregulation of PLL18 and PLL19 in both types of feeding sites with quantitative reverse-transcriptase polymerase chain reaction (RT-PCR) and in situ RT-PCR. Furthermore, the functional analysis of mutants demonstrated the important role of both PLL genes in the development and maintenance of syncytia but not giant cells. Our results show that both enzymes play distinct roles in different infected root tissues as well as during parasitism of different nematodes.

Chemical and antihyperglycemic activity changes of ginseng pectin induced by heat processing

Six pectic polysaccharides were obtained from white ginseng (GPW-1 and GPW-2), red ginseng (GPR-1 and GPR-2, steamed ginseng at 100°C) and steamed ginseng (GPS-1 and GPS-2, steamed ginseng at 120°C) by combination of water extraction, ion-exchange and gel permeation chromatographies. Based on the data from monosaccharide composition and 13C NMR analysis, GPW-1, GPR-1 and GPS-1 were identified as type-I rhamnogalacturonan (RG-I)-rich pectins, GPW-2, GPR-2 and GPS-2 were homogalacturonan (HG)-rich pectins with different degrees of methyl-esterification. Remarkably, GalA increased with the increase of processing temperatures in these six fractions, which might be caused by the transformation of esterified GalA into un-esterified form during heat processing. In vivo animal experiments showed that GPs exhibited significant antiohyperglycemic and antioxidant activities in alloxan-induced diabetic mice, and the effects increased with the processing temperature, with the most potent activity in GPS.

Molecular structural differences between low methoxy pectins induced by pectin methyl esterase II: Effects on texture, release and perception of aroma in gels of similar modulus of elasticity

Six low-methoxy pectins with different degrees of methylesterification and amidation, and molecular weights were used to prepare gels with similar moduli of elasticity by varying the concentrations of pectin and calcium phosphate. Five aroma compounds were added to the gels and their sensory textural properties, release and perception of aromas were investigated. Sensory firmness, springiness, adhesiveness, chewiness and cohesiveness differed according to the gel type, even though the moduli of elasticity were not significantly different (p<0.05). Release and perception of aromas also displayed significant difference according to the gel type (p<0.05). Low-methoxy amidated pectin exhibited the lowest release and perception for all the aroma compounds, while pectin-methylesterase-treated pectin gels exhibited relatively higher aroma release and perception. These results showed that the structural properties of pectins and gelling factors that increase the non-polar character of the gel matrices could decrease the release and perception of aromas in pectin gel systems.

Wound-induced pectin methylesterases enhance banana (Musa spp. AAA) susceptibility to Fusarium oxysporum f. sp. cubense

Recent studies suggest that plant pectin methylesterases (PMEs) are directly involved in plant defence besides their roles in plant development. However, the molecular mechanisms of PME action on pectins are not well understood. In order to understand how PMEs modify pectins during banana (Musa spp.)–Fusarium interaction, the expression and enzyme activities of PMEs in two banana cultivars, highly resistant or susceptible to Fusarium, were compared with each other. Furthermore, the spatial distribution of PMEs and their effect on pectin methylesterification of 10 individual homogalacturonan (HG) epitopes with different degrees of methylesterification (DMs) were also examined. The results showed that, before pathogen treatment, the resistant cultivar displayed higher PME activity than the susceptible cultivar, corresponding well to the lower level of pectin DM. A significant increase in PME expression and activity and a decrease in pectin DM were observed in the susceptible cultivar but not in the resistant cultivar when plants were wounded, which was necessary for successful infection. With the increase of PME in the wounded susceptible cultivar, the JIM5 antigen (low methyestrified HGs) increased. Forty-eight hours after pathogen infection, the PME activity and expression in the susceptible cultivar were higher than those in the resistant cultivar, while the DM was lower. In conclusion, the resistant and the susceptible cultivars differ significantly in their response to wounding. Increased PMEs and thereafter decreased DMs acompanied by increased low methylesterified HGs in the root vascular cylinder appear to play a key role in determination of banana susceptibility to Fusarium.

Anti-inflammatory activity of low and high methoxylated citrus pectins

The anti-inflammatory activity of the citrus pectins, CU701 and CU201, with low and high degrees of methylesterification (LM and HM), respectively, was studied after oral administration in mice using three models of inflammation: acetic acid-induced colitis, cytokine production by blood leukocytes in response to lipopolysaccharide (LPS) and endotoxin shock. Oral administration of either CU701 or CU201 24h before the infusion of acetic acid into the colon was found to prevent the progression of colitis. However, CU701 was found to decrease the adhesion of peritoneal granulocytes and their production of reactive oxygen species in colitic mice, whereas CU201 had no effect on granulocyte activity. Only CU701 was found to decrease tumour necrosis factor-α (TNF-α) release and to increase production of the anti-inflammatory cytokine interleukin-10 (IL-10) 24h after oral administration in mice. CU201 had no effect on cytokine production. CU701, but not CU201, was found to improve the survival rate in mice that were subjected to a lethal dose of LPS. The present study demonstrates that LM pectin would be a more efficient anti-inflammatory agent than HM pectin upon oral administration. LM pectin was found to inhibit both local and systemic inflammation, whereas HM pectin was shown to prevent intestinal inflammation.

Dispersions of Nanoclays of Different Shapes into Aqueous and Solid Biopolymeric Matrices. Extended Physicochemical Study

Dispersions of nanofillers into aqueous and solid biopolymeric matrices were studied from the physicochemical viewpoint. This work was carried out based on the idea that the combination of biopolymers, derived from renewable resources, and nanofiller, environmentally friendly, may form a new generation of nanomaterials with excellent and unique properties at low cost. To this purpose, two pectins with different degrees of methyl esterification and nanoclays like halloysite and laponite RD were selected. The thermodynamic and structural studies on the aqueous mixtures of pectin and nanoclay were able to discriminate the interactions, which control the adsorption of pectin onto the filler and the aggregation of both pectin and clay particles. The gained insights were useful to interpret the mesoscopic structure of the nanocomposites (prepared from the aqueous mixtures by means of the casting method) evidenced by SEM, thermal stability, tensile properties, and transparency investigations. The attained knowledge represents a basic point for designing new hybrid nanostructures in both the aqueous and the solid phase for specific purposes.

RHEOLOGICAL AND STRUCTURAL PROPERTIES OF HIGH‐METHOXYL ESTERIFIED, LOW‐METHOXYL ESTERIFIED AND LOW‐METHOXYL AMIDATED PECTIN GELS

ABSTRACT Pectins are polygalacturonic acids with varying degrees of methyl esterification and are widely used to create gels. The commercial pectins studied by the authors were high‐methoxyl esterified pectin, low‐methoxyl esterified pectin and low‐methoxyl amidated pectin. A puncture test was performed at 5 and 20C using a texture analyzer. The compression force was recorded as a function of displacement. The resulting force‐displacement measurements were converted to stress‐strain to calculate Young's modulus by assuming incompressibility of the materials in a linear viscoelastic region (LVR). A rheometer was used for the shear flow curve, frequency sweep, amplitude sweep and creep tests, at 5 and 20C. The effects of temperature on storage and loss moduli were obtained. Further to that, a differential scanning calorimeter was used to investigate the melting behavior of pectin gels. The shear elastic moduli of pectin gels in an LVR were compared with Young's modulus. PRACTICAL APPLICATIONSUnderstanding the rheological properties of high‐methoxyl esterified pectin (HM), low‐methoxyl esterified pectin (LM) and low‐methoxyl amidated pectin (LMA) pectin gels is useful in the food, pharmaceutical and cosmetic industries for the improvement of product quality. The results may be used to model the relationship between the microstructural and mechanical properties of HM, LM and LMA pectin gels.

Electrophoretic Behavior of Copolymeric Galacturonans Including Comments on the Information Content of the Intermolecular Charge Distribution

Capillary electrophoresis has been used to characterize samples of the copolymeric anionic polysaccharides homo- and rhamno-galacturonan (HG and RGI, respectively). In the case of HG, the ratios of the two component sugar residues, galacturonic acid and its methylesterified analogue, have been controlled chemically to produce samples comprised of varying degrees of methylesterification (DM). The mapping of the measured electrophoretic mobilities to the biopolymer charge density has been considered in some detail and for HG substrates with random intramolecular patterns of methylesterification it is shown that the experimentally extracted intermolecular DM distribution agrees well with the predictions of calculations based on the binomial theorem. This demonstrates that the intermolecular distribution of the methylesterification of pectin samples contains information on the intramolecular pattern by virtue of the fact that they are both manifestations of the same statistical process.

Total fractionation and characterization of the water-soluble polysaccharides isolated from Panax ginseng C. A. Meyer

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 × 10 3 to 1.1 × 10 5. Lymphocyte proliferation assays showed that both the neutral polysaccharides and acidic polysaccharides were potent B and T cell stimulators.

Degradation of forage chicory by ruminal fibrolytic bacteria.

Determine the susceptibility of forage chicory (Cichorium intybus L.) to degradation by ruminal fibrolytic bacteria and measure the effects on cell-wall pectic polysaccharides. Large segments of fresh forage chicory were degraded in vitro by Lachnospira multiparus and Fibrobacter succinogenes, but not by Ruminococcus flavefaciens or Butyrivibrio hungatei. Cell-wall pectins were degraded extensively (95%) and rapidly by L. multiparus with a simultaneous release of uronic acids and the pectin-derived neutral monosaccharides arabinose, galactose and rhamnose. Fibrobacter succinogenes also degraded cell-wall pectins extensively, but at a slower rate than L. multiparus. Immunofluorescence microscopy using monoclonal antibodies revealed that, after incubation, homogalacturonans with both low and high degrees of methyl esterification were almost completely lost from walls of all cell types and from the middle lamella between cells. Only two of the four ruminal bacteria with pectinolytic activity degraded fresh chicory leaves, and each showed a different pattern of pectin breakdown. Degradation was greatest for F. succinogenes which also had cellulolytic activity. The finding of extensive removal of pectic polysaccharides from the middle lamella and the consequent decrease in particle size may explain the decreased rumination and the increased intake observed in ruminants grazing forage chicory.

Composition, structure and solution properties of polysaccharides extracted from leaves of Mesembryanthenum crystallinum

Water and Chelator-soluble polymers were independently isolated from the alcoholic-insoluble substance (AIS) of Mesembryanthenum crystallinum leaves. After precipitation in ethanol and ultrafiltration (40 kD cut-off) of recovered solids dissolved in water, the yield relative of the so-call Lw and LCh polymers to the AIS dry matter was 2.5 ± 0.2 and 7 ± 0.5%. The galacturonic acid contents were 67 ± 3% and 63 ± 5%. The degrees of methylesterification of ca. 45 ± 3% and 50 ± 3%, showed that Lw and LCh belonged to the fairly methylesterified pectin class. From sugar analysis, LCh was shown to contain at least two types of pectic blocks, homogalacturonan (HG: 58%) and rhamnogalacturonan-I (RG-I: 34%). Their structure were deduced after saponification, polygalacturonase treatments, size exclusion chromatography (SEC) onto Sephacryl S-200 and then sugar composition of the collected fractions. The main polymers were methylesterified HG (32%), some of them being linked to non-methylesterified HG (10%), and to polygalacturonase-resistant HG (17%). In addition, there were (1) a highly soluble RG-I with long galactan side chains (RG-I–gal accounting for 9%) and (2) a RG-I with short arabino-galactan side chains (25%), named RG-I–ara/gal that was almost totally lost during SEC analysis, due to its low solubility at room temperature and in absence of chelators. SEC coupled with differential refractive index and light scattering showed that highly methylesterified HG chains exhibited aggregate structures in solution due to intermolecular hydrophobic interactions. These interactions formed hydrophobic clusters, which have been characterized by surface tension measurements and with a polarity probe, the Coomassie Brilliant Blue dye. After alkaline treatment of LCh, the self-assembly of HG disappeared.