High Degree Of Methylesterification Research Articles

Isolation and structure elucidation of pectic polysaccharide from rose hip fruits (Rosa canina L.)

A pectic polysaccharide from rose hip (RH) fruits has been obtained by extraction with 1% aqueous citric acid. It was found that the polysaccharide fraction mainly consisted of galacturonic acid (45.5%) next to galactose (5.5%) and arabinose (4.7%). RH pectin is having a relatively high degree of methylesterification (62%) and acetylation (10%) and consists of different molecular weight populations in the range of 10–100kDa. Enzymatic fingerprinting was performed using a combination of pectin lyase (PL) and endo-polygalacturonase. Detailed information about the structure and level of galacturonic acid oligomers released was obtained using LC-HILIC-MS/ELSD and HPAEC. Predominantly, unsaturated and methyl-esterified oligomers (DP 3–5) were released indicating that high proportions of methylesterified ‘PL degradable’ areas were present within the pectin. The data revealed that homogalacturonan is the main building block of the extracted pectin and consists of long methylesterified/acetylated GalA sequences interspersed with small blocks of non-methyl-esterified GalA units.

Microscopic evidence for Ca2+ mediated pectin–pectin interactions in carrot-based suspensions

This study explored the use of fluorescently labeled pectin to obtain evidence for Ca2+ mediated pectin–pectin interactions in situ. Specifically, carrots were either blanched at low temperature (LTB) or blanched at high temperature (HTB) to activate or inactivate endogenous pectin methylesterase, respectively. Consequently, pectin in tissue particles of LTB and HTB carrots exhibited low degree of methylesterification (DM) and high DM, respectively. Pectin present in the LTB carrot serum exhibited a lower DM, was more branched, and showed a higher molar mass compared to HTB carrot serum pectin. Ca2+ mediated pectin–pectin interactions were influenced by serum pectin molecular structure, increased with increasing pH and Ca2+ concentration, and decreasing DM. Presence of more linear pectin in the serum created a competition, leading to less intense interactions between labeled pectin and pectin at tissue particle surfaces. Generally, the most intense Ca2+ mediated pectin–pectin interactions were observed for pectin of LTB carrot particles.

The impact of rhamnogalacturonan-I side chain monosaccharides on the rheological properties of citrus pectin

Citrus peel is the preferred raw material for pectin production, mainly due to its high pectin content but also due to the gelling capabilities. Especially the high galacturonic acid content and the high degree of methyl esterification make it an attractive starting material for further modification. Until now the presence of rhamnogalacturonan-I (RG-I) in commercial pectin has received less attention due to its relatively low abundance. Using a gentle industrial extraction process we discovered a slightly higher composition in neutral sugars of RG-I. In particular, the arabinose concentration in mildly extracted pectin was higher compared to conventionally extracted pectin. The use of enzymes specific for either arabinan or galactan allowed assessing the implications of varying neutral sugar composition on the gelling capabilities of pectin. A comparison between untreated and debranched pectin revealed a significant decrease in both gelling temperature (up to 20 °C) and gel strength (up to 38%), following the specific reduction in neutral sugars concentration. Coil-overlap and entanglement effects were analysed to further understand the impact of RG-I side chains on polymer rheology.

Pectin characterisation in vegetable waste streams: A starting point for waste valorisation in the food industry

Vegetable waste streams, which are a major disposal issue in food industry, present a promising source of compounds which may be valorised because of their favourable functional features. In this context, the potential of five vegetable waste streams (rejected carrots, carrot steam peels, green beans cutting waste, leek cutting waste and celeriac steam peels) as a source for the extraction of pectin with interesting structural, and hence functional, properties was evaluated. Specifically, cell-wall components were extracted from the waste streams as alcohol-insoluble residue and subsequently fractionated into different (pectin) fractions based on their solubility. The pectic polysaccharides were characterised in terms of GalA content, neutral sugar content, linearity/branching, degree of methyl-esterification (DM), molar-mass distribution and the presence of bound protein. Pectin characterisation revealed considerable differences between the pectic polymers present in the investigated waste streams. For example, pectin in carrot steam peels showed a low DM, whereas the cutting waste of leek contained pectin with a very high DM. Furthermore, the level of protein bound to pectin was generally highest in carrot-derived waste streams. Depending on the intended pectin functionality, a deliberate choice for one of these vegetable waste streams as a source for pectin extraction can be made.

FT-IR spectroscopy, a reliable method for routine analysis of the degree of methylesterification of pectin in different fruit- and vegetable-based matrices

The use of Fourier transform infrared (FT-IR) spectroscopy as a method for routine analysis of the degree of methylesterification (DM) of pectin was validated. The relationship between the ratio of the intensity of the peak at around 1740cm−1 (due to ester carbonyl group stretching) to the sum of the intensities of the peaks at around 1740 and 1630–1600cm−1 (due to carboxylate group stretching) and the DM of pectin in model and real systems was investigated. In model systems of low to medium DM with low added protein (⩽20%), accurate DM determinations were obtained without spectra deconvolution whilst for medium to high DM pectin with high added protein (⩾30%), peak deconvolution was vital. In real systems, good DM determinations were obtained without peak deconvolution except for broccoli-derived samples. Considering that broccoli is a protein-rich vegetable, better determinations of the DM were obtained using deconvoluted FT-IR spectra.

Effect of Molecular Weight Reduction, Acetylation and Esterification on the Emulsification Properties of Citrus Pectin

Citrus pectin was chemically and thermally modified in order to increase the hydrophobic character of the molecule and its adsorptivity to the oil–water interface. The degree of acetylation and methylesterification was increased and the molecular weight was reduced. The emulsion formation and stabilization properties of these modified pectins were evaluated by surface and interfacial tension measurements and emulsification experiments. For the production of emulsions, a high pressure homogenizer was used. The viscosity ratio between oil and emulsion phase was adjusted by varying the amount of added sucrose. Pectins with a higher degree of methylesterification (DE) decrease the interfacial tension significantly compared to the unmodified pectin. Pectins with increased degree of acetylation (DAc), however, show higher interfacial tension values. In emulsification experiments, pectins with a reduced molecular weight do neither significantly reduce droplet sizes nor improve emulsion stability. Pectins with increased DE or DAc reduce the Sauter mean diameter d3,2 of emulsions significantly and, in case of an DE increase, also show excellent long term stability. Their performance is also superior to sugar beet pectin under comparable experimental conditions.

Rhamnogalacturonan II structure shows variation in the side chains monosaccharide composition and methylation status within and across different plant species

A paradigm regarding rhamnogalacturonansII (RGII) is their strictly conserved structure within a given plant. We developed and employed a fast structural characterization method based on chromatography and mass spectrometry, allowing analysis of RGII side chains from microgram amounts of cell wall. We found that RGII structures are much more diverse than so far described. In chainA of wild-type plants, up to 45% of the l-fucose is substituted by l-galactose, a state that is seemingly uncorrelated with RGII dimerization capacity. This led us to completely reinvestigate RGII structures of the Arabidopsis thaliana fucose-deficient mutant mur1, which provided insights into RGII chainA biosynthesis, and suggested that chainA truncation, rather than l-fucose to l-galactose substitution, is responsible for the mur1 dwarf phenotype. Mass spectrometry data for chainA coupled with NMR analysis revealed a high degree of methyl esterification of its glucuronic acid, providing a plausible explanation for the puzzling RGII antibody recognition. The β-galacturonic acid of chainA exhibits up to two methyl etherifications in an organ-specific manner. Combined with variation in the length of side chainB, this gives rise to a family of RGII structures instead of the unique structure described up to now. These findings pave the way for studies on the physiological roles of modulation of RGII composition.

Butternut and beetroot pectins: Characterization and functional properties

The physicochemical characteristics and functional properties of butternut (Cucumis moschata Duch. ex Poiret) and beetroot (Beta vulgaris L. var. conditiva) pectins obtained by enzymatic extraction from by-products of vegetable processing have been evaluated. The molecular mass distribution was determined using Gel Permeation Chromatography using light scattering, refractive index and UV detectors and the samples were found to be highly heterogeneous and polydisperse. Mw values of 136,000 and 1,309,000 g/mol were determined for butternut and beetroot pectins respectively. Butternut pectin had a high degree of methyl esterification. In the presence of high concentrations of sugar and at low pH, this pectin did not form gels but instead produced viscous solutions. Solutions showed pseudoplastic flow behaviour with a shear thinning index of 0.68 as determined from the Power law model. Beetroot pectin had a low degree of methyl esterification and formed gels with addition of Ca2+ at concentrations of 10 mg/g pectin or higher. The maximum value of the storage modulus was obtained at a Ca2+/GalA ratio of 0.25. The thermal stability of gels suggested that hydrogen bond interactions prevailed in the absence of Ca2+, whereas electrostatic junction zones increasingly developed between pectin chains as the calcium concentration increased. Aqueous solutions of butternut and beetroot pectins significantly reduced surface tension and both samples were able to form stable oil-in-water emulsions. It was found that protein and/or polyphenol – rich fractions present in the pectins adsorbed at the oil–water interface and were responsible for the emulsification properties.

Direct measurements of interfacial interactions between pectin and κ-casein and implications for the stabilisation of calcium-free casein micelle mimics

Using Surface Plasmon Resonance (SPR) it has been shown that the fine structure of the anionic polysaccharide pectin strongly influences its interfacial interaction with a κ-casein layer coated onto a gold surface (via a dextran linker) in the pH range 3.5–6.8, with the highest SPR signal being observed for pectin with the lowest charge density tested (a degree of methylesterification (DM) around 90%). Furthermore, the Brownian motions of κ-casein coated polystyrene beads (used to provide calcium-free ‘model casein micelles’) were studied in pectin solutions using Diffusing Wave Spectroscopy (DWS) and microscopy, and were compared with measurements made on naked beads. At every pH value studied (with the exception of 3.5), bridging of the protein-covered probe particles was observed for pectins of both DM 28 and DM 78. However, no aggregated complexes were found in these model casein micelle systems when pectin of an unusually high DM was used (90%). It was hypothesised that having a limited number of binding regions of spatially limited extent maximises the number of chains binding to the protein layer (as found with the SPR measurement), encourages the formation of loops and trains, and additionally limits the potential for destabilisation via bridging.

Diffusing wave spectroscopy investigations of acid milk gels containing pectin

The influence of the polysaccharide pectin on the gelation of acidified milk is studied in concentrated, undiluted, quiescent systems, primarily using diffusing wave spectroscopy. For pectins with a low degree of methylesterification (DM), interactions with milk-serum calcium yielded precipitated polysaccharide aggregates, even without acidification, that subsequently did not interact with casein micelles. However, high DM fine structures do not interact significantly with serum-calcium and absorb onto casein micelles as the pH is reduced below 5. A limited surface coverage of high DM pectin facilitates efficient bridging which enhances the rate of micelle aggregation and subsequent gelation and produces a clear signature in the shape of the measured MSD. The work highlights the fact that the behaviour of pectin in milk systems depends not only on the interaction of different polymeric fine structures with casein micelles, but also to a large extent on the interactions with calcium.

Deficiency of adenosine kinase activity affects the degree of pectin methyl-esterification in cell walls of Arabidopsis thaliana

Pectin methyl-esterification is catalysed by S-adenosyl-L: -methionine (SAM)-dependent methyltransferases. As deficiency in adenosine kinase (ADK; EC 2.7.1.20) activity impairs SAM recycling and utilization, we investigated the relationship between ADK-deficiency and the degree of pectin methyl-esterification in cell walls of Arabidopsis thaliana. The distribution patterns of epitopes associated with methyl-esterified homogalacturonan in leaves and hypocotyls of wild-type (WT) and ADK-deficient plants were examined using immunolocalization and biochemical techniques. JIM5 and LM7 epitopes, characteristic of low esterified pectins, were more irregularly distributed along the cell wall in ADK-deficient plants than in WT cell walls. In addition, epitopes recognized by JIM7, characteristic of pectins with a higher degree of methyl-esterification, were less abundant in ADK-deficient leaves and hypocotyls. Since de-esterified pectins have enhanced adhesion properties, we propose that the higher abundance and the altered distribution of low methyl-esterified pectin in ADK-deficient cell walls lead to the leaf shape abnormalities observed in these plants.

Immunocytochemical characterization of the cell walls of bean cell suspensions during habituation and dehabituation to dichlobenil

The effects of the cellulose inhibitor dichlobenil on the cell wall composition and structure during the habituation/dehabituation process of suspension‐cultured bean cells were assessed. A range of techniques were used including cell wall fractionation, sugar analysis, immunofluorescence and fluorochrome labelling of resin‐embedded sections, and immunodot assays (IDAs) of cell wall fractions. The cell walls from bean cell suspensions with initial levels of habituation to dichlobenil had decreased levels of cellulose, but this effect lessened with increasing numbers of subcultures. All cell walls analysed showed calcofluor‐stained appositions. However, in habituated and dehabituated cells, appositions were not recognized by an anticallose antibody. This finding suggested the accumulation of an extracellular polysaccharide different to callose, probably a 1,4‐β‐glucan in these cell lines. Appositions in habituated cells also contained homogalacturonan (HG) with a high degree of methyl esterification (DE), rhamnogalacturonan (RG) and xyloglucan. Habituated cell walls were also enriched in pectins, particularly HG, with a low DE, and RG. The levels of extensin epitope that colocalized with RG in habituated cells also diminished with the increasing number of subcultures. Habituated cells also liberated less extensin into the medium. In habituated cells, a decrease in the cell wall arabinogalactan protein (AGP) labelling was observed both in cell walls and in the culture medium. The increase in the number of subcultures in 0.3 µM dichlobenil was accompanied by an increment in some pectic epitopes (JIM5 and LM5) and a decrease in other pectic and in protein epitopes (JIM7, PAM1, LM6, LM2 and MAC207), indicating a re‐structuring of cell walls throughout the habituation procedure. Dehabituated cells showed an overall composition similar to that of non‐habituated cells, with exception of an increase in glucose in hemicellulosic fractions tightly bound to cellulose. However, these cells also showed reduced levels of extensin and AGP labelling. These differences could be related to the high tolerance to dichlobenil observed in dehabituated cells.

Pectate lyase C from Bacillus subtilis: a novel endo-cleaving enzyme with activity on highly methylated pectin

The gene yvpA from Bacillus subtilis was cloned and expressed in Escherichia coli. It encoded a pectate lyase of 221 amino acids that was denominated PelC. The heterologously expressed enzyme was purified by His-tag affinity chromatography and characterized. PelC depolymerized polygalacturonate and pectins of methyl esterification degree from 22 % to 89 %, exhibiting maximum activity on 22 % esterified citrus pectin. It showed an absolute Ca2+ requirement and the optimum temperature and pH were 65 degrees C and pH 10, respectively. The deduced amino acid sequence of PelC showed 53 % identity to pectate lyase PelA from Paenibacillus barcinonensis, which was also characterized. Similarly to PelC, purified PelA showed activity on polygalacturonate and pectins with a high degree of methyl esterification. The two enzymes cleaved pectic polymers to a mixture of oligogalacturonates, indicating an endo mode of action. Analysis of activity on trigalacturonate showed that PelC cleaved it to galacturonic acid and unsaturated digalacturonate, whereas PelA did not show activity on this substrate. PelC and PelA showed high homology to a few recently identified pectate lyases of family 3 and form with them a cluster of small-sized pectate lyases from non-pathogenic micro-organisms.

Polysaccharide Constituents of Coffee‐Bean Mucilage

ABSTRACT:Alcohol‐insoluble residues (AIRs) were isolated from hand‐dissected and commercial mucilages of depulped coffee beans. Both AIRs had similar polysaccharide composition: pectic substances (about 30%), cellulose (about 8%), and neutral noncellulosic polysaccharides (about 18%). Crude pectins were extracted from AIRs (dry‐matter yield: about 23% to 35%) with dilute nitric acid (pH 1.5, 90 °C). Both pectins contained about 60% uronic acids with high degree of methyl esterification (about 62%) and moderate degree of acetylation (about 5%). Their molecular weights were low (about12 to 29 kDa). They did not gel in the presence of sucrose at acidic pH.

THE DISTRIBUTION OF ACIDIC AND ESTERIFIED PECTIN IN CAMBIUM, DEVELOPING XYLEM AND MATURE XYLEM OF PINUS SYLVESTRIS

Homogalacturonans with low and high degree of methyl esterification have been immuno-localised in the cambium, differentiating xylem and mature xylem of Pinus sylvestris, by monoclonal antibodies JIM5 and JIM7. In the unlignified cambial tissue the antibodies revealed a similar distribution for acidic and esterified pectin in the compound middle lamella, ray cell walls and pit membranes. In the lignified xylem tissue, pectin was also found in the compound middle lamella, although dominantly in the methylesterified form. Lignification seemed to coincide with a decrease in the presence of acidic pectin in the compound middle lamella. Both antibodies indicated labelling in pit membranes and ray cell walls in partially and fully lignified wood fibres.