In-situ crosslinked hydrogel based on amidated pectin/oxidized chitosan as potential wound dressing for skin repairing.

Hydrogel can provide a favorable moisture environment for skin wound healing. In this study, a novel in-situ crosslinked injectable hydrogel was prepared using the water-soluble amidated pectin (AP) and oxidized chitosan (OC) through Schiff-base reaction without any chemical crosslinker. The influence of AP content on the properties of the hydrogel was systemically investigated. It showed that gelation time, pore structure, swelling capability and degradability of the hydrogel can be tuned by varying the content of amine and aldehyde groups from AP and OC. All the porous hydrogels with various AP contents (65%, 70%, and 80%) presented desirable gelation time, swelling property, high hemocompatibility and biocompatibility. Particularly, AP-OC-65 hydrogel presented superior swelling capability and better hemo- and bio-compatibility, owing to more residual amine sites in the hydrogel. Therefore, the injectable AP-OC-65 hydrogel has a greater potential for application to wound dressing or skin substitute.

Pectin homogalacturonan nanofilament expansion drives morphogenesis in plant epidermal cells.

The process by which plant cells expand and gain shape has presented a challenge for researchers. Current models propose that these processes are driven by turgor pressure acting on the cell wall. Using nanoimaging, we show that the cell wall contains pectin nanofilaments that possess an intrinsic expansion capacity. Additionally, we use growth models containing such structures to show that a complex plant cell shape can derive from chemically induced local and polarized expansion of the pectin nanofilaments without turgor-driven growth. Thus, the plant cell wall, outside of the cell itself, is an active participant in shaping plant cells. Extracellular matrix function may similarly guide cell shape in other kingdoms, including Animalia.

Cell Wall Composition and Ultrastructural Immunolocalization of Pectin and Arabinogalactan Protein during Olea europaea L. Fruit Abscission.

Cell wall modification is integral to many plant developmental processes where cells need to separate, such as abscission. However, changes in cell wall composition during natural fruit abscission are poorly understood. In olive (Olea europaea L.), some cultivars such as 'Picual' undergo massive natural fruit abscission after fruit ripening. This study investigates the differences in cell wall polysaccharide composition and the localization of pectins and arabinogalactan protein (AGP) in the abscission zone (AZ) during cell separation to understand fruit abscission control in 'Picual' olive. To this end, immunogold labeling employing a suite of monoclonal antibodies to cell wall components (JIM13, LM5, LM6, LM19 and LM20) was investigated in olive fruit AZ. Cell wall polysaccharide extraction revealed that the AZ cell separation is related to the de-esterification and degradation of pectic polysaccharides. Moreover, ultrastructural localization showed that both esterified and unesterified homogalacturonans (HGs) localize mainly in the AZ cell walls, including the middle lamella and tricellular junction zones. Our results indicate that unesterified HGs are likely to contribute to cell separation in the olive fruit AZ. Similarly, immunogold labeling demonstrated a decrease in both galactose-rich and arabinose-rich pectins in AZ cell walls during ripe fruit abscission. In addition, AGPs were localized in the cell wall, plasma membrane and cytoplasm of AZ cells with lower levels of AGPs during ripe fruit abscission. This detailed temporal profile of the cell wall polysaccharide composition, and the pectins and AGP immunolocalization in the olive fruit AZ, offers new insights into cell wall remodeling during ripe fruit abscission.

Determination of chemical structure of pea pectin by using pectinolytic enzymes.

The chemical structure of pea pectin was delineated using pectin-degrading enzymes and biochemical methods. The molecular weight of the pea pectin preparation was 488,000, with 50 % arabinose content, and neutral sugar side chains attached to approximately 60 % of the rhamnose residues in rhamnogalacturonan-I (RG-I). Arabinan, an RG-I side chain, was highly branched, and the main chain was comprised of α-1,5-l-arabinan. Galactose and galactooligosaccharides were attached to approximately 35 % of the rhamnose residues in RG-I. Long chain ß-1,4-galactan was also present. The xylose substitution rate in xylogalacturonan (XGA) was 63 %. The molar ratio of RG-I/homogalacturonan (HG)/XGA in the backbone of the pea pectin was approximately 3:3:4. When considering neutral sugar side chain content (arabinose, galactose, and xylose), the molar ratio of RG-I/HG/XGA regions in the pea pectin was 7:1:2. These data will help understand the properties of pea pectin.

Analysis of pectin biopolymer phase states using acoustic emissions.

Acoustic emissions are stress or elastic waves produced by a material under external load. Since acoustic emissions are generated from within and transmitted through the substance, the acoustic signature provides insights into the physical and mechanical properties of the material. In this report, we used a constant velocity probe with force and acoustic emission monitoring to investigate the properties of glass phase and gel phase pectin films. In the gel phase films, a constant velocity uniaxial load produced periodic premonitory acoustic emissions with coincident force variations (saw-tooth pattern). SEM images of the gel phase microarchitecture indicated the presence of slip planes. In contrast, the glass phase films demonstrated early acoustic emissions, but effectively no force or acoustic evidence of periodic or premonitory emissions. Microstructural imaging of the glass phase films indicated the presence of early microcracks as well as dense polymerization of the pectin (without evidence of slip planes). We conclude that the water content in the pectin films contributes to not only the physical properties of the films, but also the stick-slip motion observed with constant uniaxial load. Further, acoustic emissions provide a sensitive and practical measure of this mechanical behavior.

AFM analysis reveals polymorphism of purified flax rhamnogalacturonans I of distinct functional types.

Functionally distinct polymers organized on the basis of rhamnogalacturonan I (RG-I) backbone with more than a half of rhamnose residues substituted by the side chains containing mostly galactose were purified from flaxseed mucilage, the primary cell wall of young hypocotyls and tertiary cell walls of bast fibers and characterized by atomic force microscopy. Seed mucilage RG-I with short side chains and unusual O3 substitution showed loose coils or star-like conformations. Primary cell wall RG-I, which included polygalacturonan (PGA) fragments, represented micellar objects and rare long chains. Pure RG-I with long galactan side chains, which was isolated as nascent polysaccharide before its incorporation into the tertiary cell wall of bast fibers was observed as long unbranched objects. RG-I entrapped by cellulose microfibrils in tertiary cell wall was visualized as compact micellar complexes. All types of flax RGs-I tended to aggregate. Relationships between RG-I structure and morphology are discussed.

Resonant soft X-ray scattering reveals cellulose microfibril spacing in plant primary cell walls.

Cellulose microfibrils are crucial for many of the remarkable mechanical properties of primary cell walls. Nevertheless, many structural features of cellulose microfibril organization in cell walls are not yet fully described. Microscopy techniques provide direct visualization of cell wall organization, and quantification of some aspects of wall microstructure is possible through image processing. Complementary to microscopy techniques, scattering yields structural information in reciprocal space over large sample areas. Using the onion epidermal wall as a model system, we introduce resonant soft X-ray scattering (RSoXS) to directly quantify the average interfibril spacing. Tuning the X-ray energy to the calcium L-edge enhances the contrast between cellulose and pectin due to the localization of calcium ions to homogalacturonan in the pectin matrix. As a consequence, RSoXS profiles reveal an average center-to-center distance between cellulose microfibrils or microfibril bundles of about 20 nm.

Immunogold scanning electron microscopy can reveal the polysaccharide architecture of xylem cell walls.

Immunofluorescence microscopy (IFM) and immunogold transmission electron microscopy (TEM) are the two main techniques commonly used to detect polysaccharides in plant cell walls. Both are important in localizing cell wall polysaccharides, but both have major limitations, such as low resolution in IFM and restricted sample size for immunogold TEM. In this study, we have developed a robust technique that combines immunocytochemistry with scanning electron microscopy (SEM) to study cell wall polysaccharide architecture in xylem cells at high resolution over large areas of sample. Using multiple cell wall monoclonal antibodies (mAbs), this immunogold SEM technique reliably localized groups of hemicellulosic and pectic polysaccharides in the cell walls of five different xylem structures (vessel elements, fibers, axial and ray parenchyma cells, and tyloses). This demonstrates its important advantages over the other two methods for studying cell wall polysaccharide composition and distribution in these structures. In addition, it can show the three-dimensional distribution of a polysaccharide group in the vessel lateral wall and the polysaccharide components in the cell wall of developing tyloses. This technique, therefore, should be valuable for understanding the cell wall polysaccharide composition, architecture and functions of diverse cell types.

Pectin-non-starch nanofibers biocomposites as novel gastrointestinal-resistant prebiotics.

Incorporation of nanofibers of chitin (NC), lignocellulose (NLC) and bacterial cellulose (BNC) in pectin was studied to improve prebiotic activity and gastrointestinal resistance of the pectin-nanofibers biocomposites for protection of probiotics under simulated gastrointestinal conditions. The biocomposites were prepared using various compositions of pectin and nanofibers, which were designed using D-optimal mixture method. The incorporation of the nanofibers in pectin led to a slow degradation of the pectin-nanofibers biocomposites in contrast to their rapid swelling. AFM analysis indicated the homogenous distribution of interconnected nanofibers network structure in the pectin-nanofibers biocomposite. FTIR spectra demonstrated fabrication of the biocomposites based on the inter- and intra-molecular hydrogen bonding and ionic interaction of pectin-Ca2+. XRD patterns revealed the amorphous structures of the biocomposites as compared to the crystalline structures of the nanofibers. Among the compositions, the optimal compositions were as follows: 60% pectin+40% NC, 50% pectin+50% NLC and 60% pectin+40% BNC, where the prebiotic score, probiotic survival under simulated gastric and intestinal conditions were optimum. The optimal biocomposite pectin-NC exhibited the highest survival of the entrapped probiotic bacteria under simulated gastric (97.7%) and intestinal (95.8%) conditions when compared with the corresponding to free cells (76.2 and 73.4%).

The nanostructural characterization of strawberry pectins in pectate lyase or polygalacturonase silenced fruits elucidates their role in softening.

To ascertain the role of pectin disassembly in fruit softening, chelated- (CSP) and sodium carbonate-soluble (SSP) pectins from plants with a pectate lyase, FaplC, or a polygalacturonase, FaPG1, downregulated by antisense transformation were characterized at the nanostructural level. Fruits from transgenic plants were firmer than the control, although FaPG1 suppression had a greater effect on firmness. Size exclusion chromatography showed that the average molecular masses of both transgenic pectins were higher than that of the control. Atomic force microscopy analysis of pectins confirmed the higher degree of polymerization as result of pectinase silencing. The mean length values for CSP chains increased from 84 nm in the control to 95.5 and 101 nm, in antisense FaplC and antisense FaPG1 samples, respectively. Similarly, SSP polyuronides were longer in transgenic fruits (61, 67.5 and 71 nm, in the control, antisense FaplC and antisense FaPG1 samples, respectively). Transgenic pectins showed a more complex structure, with a higher percentage of branched chains than the control, especially in the case of FaPG1 silenced fruits. Supramolecular pectin aggregates, supposedly formed by homogalacturonan and rhamnogalacturonan I, were more frequently observed in antisense FaPG1 samples. The larger modifications in the nanostructure of pectins in FaPG1 silenced fruits when compared with antisense pectate lyase plants correlate with the higher impact of polygalacturonase silencing on reducing strawberry fruit softening.

Fruit softening and pectin disassembly: an overview of nanostructural pectin modifications assessed by atomic force microscopy.

BACKGROUND: One of the main factors that reduce fruit quality and lead to economically important losses is oversoftening. Textural changes during fruit ripening are mainly due to the dissolution of the middle lamella, the reduction of cell-to-cell adhesion and the weakening of parenchyma cell walls as a result of the action of cell wall modifying enzymes. Pectins, major components of fruit cell walls, are extensively modified during ripening. These changes include solubilization, depolymerization and the loss of neutral side chains. Recent evidence in strawberry and apple, fruits with a soft or crisp texture at ripening, suggests that pectin disassembly is a key factor in textural changes. In both these fruits, softening was reduced as result of antisense downregulation of polygalacturonase genes. Changes in pectic polymer size, composition and structure have traditionally been studied by conventional techniques, most of them relying on bulk analysis of a population of polysaccharides, and studies focusing on modifications at the nanostructural level are scarce. Atomic force microscopy (AFM) allows the study of individual polymers at high magnification and with minimal sample preparation; however, AFM has rarely been employed to analyse pectin disassembly during fruit ripening. SCOPE: In this review, the main features of the pectin disassembly process during fruit ripening are first discussed, and then the nanostructural characterization of fruit pectins by AFM and its relationship with texture and postharvest fruit shelf life is reviewed. In general, fruit pectins are visualized under AFM as linear chains, a few of which show long branches, and aggregates. Number- and weight-average values obtained from these images are in good agreement with chromatographic analyses. Most AFM studies indicate reductions in the length of individual pectin chains and the frequency of aggregates as the fruits ripen. Pectins extracted with sodium carbonate, supposedly located within the primary cell wall, are the most affected.

Formulation of a pectin gel that efficiently traps mycotoxin deoxynivalenol and reduces its bioavailability.

We aimed to develop a new food-processing approach using pectin to reduce gastrointestinal absorption of mycotoxins. When Ca(2+) is added to low-methoxyl pectin, a gel resembling an egg box-like structure forms that is able to trap certain molecules. We examined whether or not low-methoxyl amidated pectin (LMA) and low-methoxyl non-amidated pectin (LMNA) trapped the mycotoxin deoxynivalenol (DON) after being ingested. We first determined the trapping effects of LMA and LMNA on DON in vitro under conditions similar to those in the human stomach, with results showing that LMA gel trapped DON to a greater extent than the LMNA gel. We then performed in vivo experiments and demonstrated that the LMA gel containing DON reduced DON's absorption from the gastrointestinal tract. This new food-processing technique holds great promise for reducing the bioavailability of DON in contaminated food and may be useful in mitigating the effects of other mycotoxins.

The relation of apple texture with cell wall nanostructure studied using an atomic force microscope.

In this study, the relation of the nanostructure of cell walls with their texture was investigated for six different apple cultivars. Cell wall material (CWM) and cellulose microfibrils were imaged by atomic force microscope (AFM). The mean diameter of cellulose microfibrils for each cultivar was estimated based on the AFM height topographs obtained using the tapping mode of dried specimens. Additionally, crystallinity of cellulose microfibrils and pectin content was determined. Texture of apple cultivars was evaluated by sensory and instrumental analysis. Differences in cellulose diameter as determined from the AFM height topographs of the nanostructure of cell walls of the apple cultivars are found to relate to the degree of crystallinity and pectin content. Cultivars with thicker cellulose microfibrils also revealed crisper, harder and juicier texture, and greater acoustic emission. The data suggest that microfibril thickness affects the mechanical strength of cell walls which has consequences for sensory and instrumental texture.

Properties of cellulose/pectins composites: implication for structural and mechanical properties of cell wall.

The primary cell wall of dicotyledonous plants can be considered as a concentrated polymer assembly, containing in particular polysaccharides among which cellulose and pectins are known to be the major components. In order to understand and control the textural quality of plant-derived foods, it is highly important to elucidate the rheological and microstructural properties of these components, individually and in mixture, in order to define their implication for structural and mechanical properties of primary plant cell wall. In this study, the rheological and microstructural properties of model systems composed of sugar-beet microfibrillated cellulose and HM pectins from various sources, with varied degrees of methylation and containing different amounts of neutral sugar side chains, were investigated. The influence of the presence of calcium and/or sodium ions and the biopolymer concentrations on the properties of the mixed systems were also studied. The characterizations of the mixed system, considered as a simplified model of primary plant cell wall, showed that whatever the structural characteristics of the pectins, the ionic conditions of the medium and the biopolymer concentrations, the gelation of the composite was mainly controlled by cellulose. Thus, the cellulose network would be the principal component governing the mechanical properties of the cell walls. However, the neutral sugar side chains of the pectins seem to play a part in the interactions with cellulose, as shown by the interesting viscoelastic properties of cellulose/apple HM pectins systems. The rigidity of cellulose/pectins composite was strongly influenced by the structural characteristics of pectins. The particular properties of primary plant cell walls would thus result from the solid viscoelastic properties of cellulose, its interactions with pectins according to their structural characteristics (implication of the neutral sugar side chains and the specific potential calcic interactions) and of the distribution of the components in separate phases.

Networks of polysaccharides with hydrophilic and hydrophobic characteristics in the presence of co-solute.

The present investigation deals with the changing network morphology of agarose and high methoxy pectin when mixed with polydextrose as co-solute at concentrations varying up to high level of solids. Thermomechanical analysis and micro-imaging were performed using small deformation dynamic oscillation in shear, modulated differential scanning calorimetry and environment scanning electron microscopy. Fourier transform infrared spectroscopy and wide angle X-ray diffraction were practised to examine the nature of interactions between polymer and co-solute, and the extent of amorphicity of preparations. We observed a decline in the mechanical strength of aqueous agarose preparations upon addition of high levels of polydextrose, which should be attributed to reduced enthalpic content of the coil-to-helix transition of the polysaccharide network. Glass transition phenomena were observed at subzero temperatures in condensed preparations, hence further arguing for the formation of a lightly cross-linked agarose network with changing solvent quality. High levels of co-solute induce formation of weak pectin gels at elevated temperatures (even at 95°C), which with lowering temperature exhibit increasing strength. This results in the formation of rubbery pectin gels at ambient temperature, which upon controlled cooling to subzero temperatures convert to a clear glass earlier than the agarose counterparts.

Combined QCMD and AFM studies of lysozyme and poly-L-lysine-poly-galacturonic acid multilayers.

A quartz crystal microbalance with dissipation monitoring (QCMD) has been used to monitor the adsorption and structure of lysozyme monolayers and multilayers, and poly-L-lysine (PLL)-polygalacturonic acid (PGalA) multilayers at a solid-liquid interface using freshly-cleaved mica as a substrate. QCMD measurements were complemented with atomic force microscopy (AFM). AFM images revealed that lysozyme formed incomplete monolayers and provided a basis for calculation of the thickness of the protein film. Comparative studies of adsorption onto standard and mica-coated quartz crystals showed higher areal mass adsorption and a longer-time adsorption process for mica-coated quartz crystals. Simultaneous AFM images and QCMD data were obtained for lysozyme, linear PLL-PGalA and 7 nm PLL dendrimer-PGalA multilayers. The layer-by-layer deposited multilayer films exhibited viscoelastic properties and their growth followed a non-linear regime, associated with the PLL diffusion in and out of the film formation for linear PLL-PGalA films. For the PLL 7 nm dendrimer-PGalA films the AFM images revealed marked changes in surface roughness during layer by layer deposition: these changes influence the interpretation of the QCMD data and provide additional information on the growth and structure of the multilayers.

NaCl effect on the distribution of wall ingrowth polymers and arabinogalactan proteins in type A transfer cells of Medicago sativa Gabès leaves.

We studied the distribution of wall ingrowth (WI) polymers by probing thin sections of companion cells specialized as transfer cells in minor veins of Medicago sativa cv Gabès blade with affinity probes and antibodies specific to polysaccharides and glycoproteins. The wall polymers in the controls were similar in WIs and in the primary wall but differently distributed. The extent of labeling in these papillate WIs differed for JIM5 and JIM7 homogalacturonans but was in the same range for LM5 and LM6 rhamnogalacturonans and xyloglucans. These data show that WI enhancement probably requires arabinogalactan proteins (JIM8) mainly localized on the outer part of the primary wall and WIs. By comparison, NaCl-treated plants exhibited cell wall polysaccharide modifications indicating (1) an increase in unesterified homogalacturonans (JIM5), probably implicated in Na(+) binding and/or polysaccharide network interaction for limiting turgor variations in mesophyll cells; (2) enhancement of the xyloglucan network with an accumulation of fucosylated xyloglucans (CCRC-M1) known to increase the capacity of cellulose binding; and (3) specific recognition of JIM8 arabinogalactan proteins that could participate in both wall enlargement and cohesion by increasing the number of molecular interactions with the other polymers. In conclusion, the cell wall polysaccharide distribution in enlarged WIs might (1) participate in wall resistance to sequestration of Na(+), allowing a better control of hydric homeostasis in mesophyll cells to maintain metabolic activity in source leaves, and (2) maintain tolerance of M. sativa to NaCl.

A new view of pectin structure revealed by acid hydrolysis and atomic force microscopy.

Individual pectin polymers and complexes, isolated from the pericarp of unripe tomato (Lycopersicon esculentum var. Rutgers), were subjected to a mild acid hydrolysis and visualised and characterised by atomic force microscopy (AFM). The AFM images confirm earlier studies showing that individual pectic polysaccharides often possess long branches. The AFM data have been used to construct size and molecular weight distributions for the single molecules and complexes, from which the calculated number-average and weight-average molecular weights can then be compared directly with the published literature data on the rheology of bulk samples. Loss of the neutral sugars arabinose, galactose and rhamnose from the pectin samples does not significantly alter either the size or the branching density of the individual polymers, but is reflected in a breakdown of the complexes. Significant loss of galacturonic acid at long hydrolysis times was found to be accompanied by changes in the size and branching of the single polymers and further breakdown of the complexes. The results suggest that rhamnose, arabinose and galactose are not the major components of the individual polymers but are, instead, confined to the complexes. The polysaccharides represent a previously unrecognised branched homogalacturonan with a minimum mean size some three times larger than that previously reported. The complexes consist of homogalacturonans (HGs) held together by rhamnogalacturonan I (RG-I) regions. Comparison of the rate of depolymerisation of the homogalacturonans and complexes with the published data on changes in the intrinsic viscosity of bulk pectin samples, subjected to similar acid hydrolysis, suggests that the different rates of depolymerisation of RG-I and HG contribute separately to the observed changes in intrinsic viscosity during acid hydrolysis. Thus data obtained using a single molecule microscopy technique provides new insights into the behaviour in the bulk.

Metal ion effects on hydraulic conductivity of bacterial cellulose-pectin composites used as plant cell wall analogs.

Low concentrations of some trace metals markedly reduce root elongation rate and cause ruptures to root rhizodermal and outer cortical cells in the elongation zone. The interactions between the trace metals and plant components responsible for these effects are not well understood but may be linked to changes in water uptake, cell turgor and cell wall extensibility. An experiment was conducted to investigate the effects of Al, La, Cu, Gd, Sc and Ru on the saturated hydraulic conductivity of bacterial cellulose (BC)-pectin composites, used as plant cell wall analogs. Hydraulic conductivity was reduced to approximately 30% of the initial flow rate by 39 microM Al and 0.6 microM Cu, approximately 40% by 4.6 microM La, 3 microM Sc and 4.4 microM Ru and approximately 55% by 3.4 microM Gd. Scanning electron microscopy (SEM) revealed changes in the ultrastructure of the composites. The results suggest that trace metal binding decreases the hydraulic conductivity through changes in pectin porosity. The experiment illustrates the importance of metal interactions with pectin, and the implications of such an interaction in plant metal toxicity and in normal cell wall processes.

Microrheological investigations give insights into the microstructure and functionality of pectin gels.

Many of the functional attributes of pectin, whether in the plant cell wall or in engineered food materials, are linked to its gelling properties and in particular to its ability to assemble in the presence of calcium. Pectin's fine structure and local concentration relative to that of its cross-linking ion play a major role in determining resultant gel micro-structures, and consequently the mechanical and transport properties of pectin matrices. Recent studies have sought to probe the basic properties of such calcium-induced matrices, using a light scattering technique called diffusing wave spectroscopy (DWS). In addition to the low frequency mechanical behaviour, which provides information about the nature and density of cross-links, microrheological measurements carried out with DWS are able to determine the high frequency behaviour, which is closely linked to the response of the basic strands of the network. By using these microrheological measurements, two distinct regimes have been identified into which pectin gels appear to fall: one corresponding to the presence of semi-flexible networks, a generally accepted paradigm in biological gels, and another where flexible networks dominate. In order to explain the origin of these dramatically different networks, distinct assembly pathways have been proposed in which the relative importance of the free energy gained by association and the frictional barrier to polymeric re-arrangement during network formation can differ significantly. By manipulating the local environment in the plant cell wall it is possible that Nature makes full use of both of these network types for fulfilling different tasks; such as providing strain-hardening, maximizing local elastic properties or controlling macromolecular transport.