Pectin-chitosan interactions and gel formation

The effect of chitosan concentration on the gelation of pectins differing in charge density and distribution was examined, through the determination of gel stiffness and the binding of chitosan to the gel network. Chitosan acts as a crosslinker of concentrated pectin solutions, with its effectiveness showing a dependency on charge on the pectin. The networks produced are clear even under conditions of charge neutralisation.     

Deposition of pectin/poly-L-lysine multilayers with pectins of varying degrees of esterification

The effect of pectin esterification on the assembly of multilayers consisting of poly-L-lysine (PLL) and pectin was studied using surface plasmon resonance (SPR), Fourier transform infrared-attenuated total reflection spectroscopy (FTIR-ATR), and a quartz crystal microbalance with dissipation monitoring (QCMD). With each layer deposited, there was a progressive increase in mass. The net charge of the multilayers was positive and increased with increasing degree of esterification of the pectin. Multilayer fabrication involved a limited fractionation of the pectin preparations, with the more highly esterified pectins having a weaker affinity for PLL. The multilayers were relatively hydrated structures with estimates of solids content in the range 10-32% w/w. The more highly esterified pectins had a tendency to form more hydrated structures, which showed a strong deswelling when PLL was added to a freshly deposited pectin layer.     

The structure of high-methoxyl sugar acid gels of citrus pectin as determined by AFM

Images of native high-methoxyl sugar acid gels (HMSAGs) were obtained by atomic force microscopy (AFM) in the Tapping Mode^™. Electronic thinning of the pectin strands to one-pixel wide allowed the pectin network to be viewed in the absence of variable strand widths related to preferentially solvated sugar. Thinned images revealed that HMSAGs of pectin comprise a partially cross-linked network, in that many of the cross-linking moieties are attached at only one end. Based on their structural similarities, aggregated pectin in water appears to be a fluid precursor of a HMSAG of pectin. Furthermore, examination of AFM images revealed that gels with ‘uniform’ distribution of strands and pores between strands had higher gel strengths than gels in which strands were non-uniformly distributed and were separated by large and small spaces.     

Complexation of ferric oxide particles with pectins of ordered and random distribution of charged units

Complexation between ferric oxide particles and pectins with degree of methylation 50% but having ordered or random arrangement of free carboxyl groups is investigated by electric light scattering and electrophoresis. The influence of charge distribution in pectin chain on the electrical properties of oppositely charged oxide particles and stability of their suspensions is examined as a function of pectin concentration. Although the difference in charge density of pectin samples is ~5%, we found small but measurable difference in the behavior of both oxide/pectin complexes. This is attributed to condensation of counterions near the chains of pectin with ordered distribution of charges, leading to a decrease in the effective charge density and to a corresponding decrease in the contour length of the adsorbing pectin chains. Two parameters are sensitive to the conformation of the adsorbed chains in suspensions, stabilized by pectin adsorption (at particle charge reversal). The electro-optical effect is higher for the complex with less charged pectin, which is explained with larger amount of chains, adsorbed in more coiled conformation than the chains of pectin with random distribution of free carboxyl groups. The addition of small amounts of CaCl(2) has no significant influence on the thickness of the layer from the less charged pectin, in agreement with a more compact conformation of the chains in this adsorbed layer. In contrast, the thickness of the layer from pectin with random distribution of charged groups decreases with increasing concentration of CaCl(2), indicating a more loose structure of this layer.     

Microstructure and rheological behavior of pure and mixed pectin gels

The microstructure and the rheological properties of pure HM (high methoxyl) and LM (low methoxyl) pectin gels and of mixed HM/LM pectin gels have been investigated. Gel formation of either the HM or LM pectin, or both, was initiated in the mixed gels by varying the sucrose and Ca(2+) content. The microstructure was characterized by transmission electron microscopy, light microscopy, and confocal laser scanning microscopy. HM and LM pectin gels showed aggregated networks with large pores around 500 nm and network strands of similar character. Small differences could be found, such as a more inhomogeneous LM pectin network with shorter and more branched strands of flexible appearance. LM pectin also formed a weak gel in 60% sucrose in the absence of calcium. A highly inhomogeneous mixed gel structure was formed in the presence of 60% sucrose and Ca(2+) ions, which showed large synergistic effects in rheological properties. Its formation was explained by the behavior of the corresponding pure gels. In the presence of 60% sucrose alone, a homogeneous, fine-stranded mixed network was formed, which showed weak synergistic effects. It is suggested that LM pectin interacts with HM pectin during gel formation, thereby hindering secondary aggregation leading to the aggregated networks observed for the pure gels.     

Pectin microgels and their subunit structure.

High-performance size exclusion chromatography revealed that alkaline-extracted peach fruit pectin dissolved in 0.05 M NaNO3 comprised a hierarchy of at least four aggregated, macromolecular-sized species. Each of the three largest species was found to be comparable in length to the three smallest subunits of an interconnecting gel network visualized by transmission electron microscopy of pectin, rapidly dried from solution. The interconnecting subunits of the gel network were rods or segmented rods and the integrated network formed a circular gel about 1 micron in diameter. Shadowed samples prepared from 5 and 50 mM NaCl or aqueous glycerol solutions produced images of partially dissociated subunits.     

Effects of calcium, pH, and blockiness on kinetic rheological behavior and microstructure of HM pectin gels

The kinetic behavior during gel formation and the microstructure of 0.75% high methoxyl (HM) pectin gels in 60% sucrose have been investigated by oscillatory measurements and transmission electron microscopy for three comparable citrus pectin samples differing in their degree of blockiness (DB). Ca2+ addition at pH 3.0 resulted in faster gel formation and a lower storage modulus after 3 h for gels of the blockwise pectin A. For gels of the randomly esterified pectin B, the Ca2+ addition resulted in faster gel formation and a higher storage modulus at pH 3.0. At pH 3.5, both pectins A and B were reinforced by the addition of Ca2+. In the absence of Ca2+, the shortest gelation time was obtained for the sample with the highest DB. Microstructural characterization of the gel network, 4 and 20 h after gel preparation, showed no visible changes on a nanometer scale. The microstructure of pectins A and B without Ca2+ was similar, whereas the presence of Ca2+ in pectin A resulted in an inhomogeneous structure.     

Overall charge and local charge density of pectin determines the enthalpic and entropic contributions to complexation with β-lactoglobulin

The complex formation between β-lactoglobulin and pectins of varying overall charge and local charge density were investigated. Isothermal titration calorimetry experiments were carried out to determine the enthalpic contribution to the complex formation at pH 4.25 and various ionic strengths. Complex formation was found to be an exothermic process for all conditions. Combination with previously published binding constants by Sperber et al. (Sperber, B. L. H. M.; Cohen Stuart, M. A.; Schols, H. A.; Voragen, A. G. J.; Norde, W. Biomacromolecules 2009, 10, 3246-3252) allows for the determination of the changes in the Gibbs energy and the change in entropy of the system upon complex formation between β-lactoglobulin and pectin. The local charge density of pectin is found to determine the balance between enthalpic and entropic contributions. For a high local charge density pectin, the main contribution to the Gibbs energy is of an enthalpic nature, supported by a favorable entropy effect due to the release of small counterions. A pectin with a low local charge density has a more even distribution of the enthalpic and entropic part to the change of the Gibbs energy. The enthalpic part is reduced due to the lower charge density, while the relative increase of the entropic contribution is thought to be caused by a change in the location of the binding place for pectin on the β-lactoglobulin molecule. The association of the hydrophobic methyl esters on pectin with an exposed hydrophobic region on β-lg results in the release of water molecules from the hydrophobic region and surrounding the methyl esters of the pectin molecule. An increase in the ionic strength decreases the enthalpic contribution due to the shielding of electrostatic attraction in favor of the entropic contribution, supporting the idea that the release of water molecules from hydrophobic areas plays a part in the complex formation.     

The effect of peptide-pectin interactions on the gelation behaviour of a plant cell wall pectin

The effect of basic peptides on the gelation of a pectin from the cell wall of tomato was examined through the determination of gel stiffness, and swelling behaviour of the gel in water. Poly-L-lysine, poly-L-arginine, and a synthetic peptide, designed to mimic a sequence of basic amino acids found in a plant cell wall extensin, act as crosslinking agents. Circular dichroism studies on the interaction of synthetic extensin peptides with sodium polygalacturonate demonstrated that a conformational change was induced as a result of their complexation. In addition to their effect as crosslinking agents, the polycationic peptides reduced the swelling of the pectin network in water.     

Rheological behaviour and microstructure of microfibrillated cellulose suspensions/low-methoxyl pectin mixed systems. Effect of calcium ions

In this study, the influence of the presence of low-methoxyl pectin (LM pectin) on the rheological and microstructural properties of microfibrillated cellulose suspensions was elucidated in order to create new structures with new and interesting textures. For that purpose, the rheological properties of the cellulose/LM pectin mixtures in variable proportions were compared with those of the individual biopolymers. The influence of the presence of calcium and/or sodium ions on the properties of the mixed systems was studied. The microstructure of the resulting system was studied by transmission electron microscopy and confocal laser scanning microscopy. It was found that, in the presence of LM pectin, a synergistic effect was observed when calcium ions were also present, leading to increased rheological properties of the composites. Indeed, addition of calcium to the mixtures induced LM pectin gelation, which was favoured in the presence of sodium, the pectin network contributing to the formation of a stronger cellulose/LM pectin composite gel. The presence of LM pectin alone in the microfibrillated cellulose suspensions does not significantly modify the viscoelastic and microstructural properties of microfibrillated cellulose suspensions. Whether calcium was added to the mixtures or not in water, the viscoelastic properties of the mixtures are mainly controlled by cellulose. The same behaviour was observed for the mixtures in NaCl without added calcium. Contrary to this observation, it was noticed that in presence of both sodium and calcium ions, the viscoelastic properties of the mixtures are largely governed by LM pectin. On the other hand, it was showed that the flow behaviour of microfibrillated cellulose suspensions is modified in the presence of LM pectin with an increase in thixotropic character shear-thinning behaviour, which was more pronounced in the presence of NaCl. It was also shown, from TEM observations, that an interpenetrating network formed in cellulose/LM pectin composites gel in the presence of calcium ions. In the same way, the CLSM observations allowed the separate localization of cellulose and LM pectin within the composite systems to be highlighted. The results obtained suggests that it is possible to thus create new structures with new interesting textures, by mixing microfibrillated cellulose suspensions and LM pectin in suitable proportions in the simultaneous presence of both sodium and calcium ions.     

Nanostructure of native pectin sugar acid gels visualized by atomic force microscopy

Height and phase shift images of high methoxyl sugar acid gels (HMSAG) of pectin were obtained by atomic force microscopy in the tapping mode. Images revealed that pores in these gels were fluid and flattened out when measured as a function of time. These images revealed for the first time the structure of adsorbed sugar on pectin in the hydrated native gels and how the pectin framework is organized within these gels. Segmentation of images revealed that the underlying pectin framework contained combinations of rods, segmented rods, and kinked rods connected end to end and laterally. The open network of strands was similar to pectin aggregates from 5 mM NaCl solution imaged earlier by electron microscopy (Fishman et al., Arch. Biochem. Biophys. 1992, 294, 253). Area measurements revealed that the ratio of bound sugar to pectin was in excess of 100 to 1 (w/w). Furthermore, images indicated relatively small differences in the organization of native commercial citrus pectin, orange albedo pectin, and lime albedo pectin gels at optimal pH as determined in this study. The findings are consistent with earlier gel strength measurements of these gels. In addition, values of gel strength were consistent with values of molar mass and viscosity of the constituent pectins in that they increased in the same order. Finally, we demonstrated the advantage of simultaneous visualization of height and phase shift images for observing and quantitating the nanostructure of relatively soft gels which are fully hydrated with a buffer.     

Analysis of methylated and unmethylated polygalacturonic acid structure by polysaccharide analysis using carbohydrate gel electrophoresis

The electrophoretic migration in polyacrylamide gels of oligogalacturonic acids (OGAs) derivatized by a fluorophore (2-aminoacridone) was studied. We found conditions such that OGAs can be separated up to a degree of polymerization (DP) of 40. The migration was dependent on degree of methylation and DP, because the OGA mobility relies on the charge of the galacturonic acid residues. Since both methylated and unmethylated oligosaccharides can be resolved, polysaccharide analysis using carbohydrate gel electrophoresis (PACE) is a powerful method for studying the fingerprint of pectin hydrolysis. It can be used to characterize endopolygalacturonase (Endo-PG) tolerance of methylation. Furthermore, using an Endo-PG that can distinguish low and highly methylated pectin, PACE can be used to investigate the blockwise or nonblockwise distribution of methylation of polygalacturonic acid. We show that the method can be applied to crude cell wall preparations of Arabidopsis inflorescence stems. Using chemical deesterification before or after Endo-PG digestion, we show that in the Arabidopsis cell wall, the pectins have both nonesterified and highly esterified regions.     

Strength of conjugate binding to plasmid DNA affects degradation rate and expression level in vivo

In vitro assays have demonstrated the capability of poly-L-lysine to protect plasmid DNA from serum nucleases and cellular lysates. Our purpose was to evaluate the stability and potency of poly-L-lysine-DNA polyplexes after intravenous injection into mice. Polyplexes consisted of 32P-radiolabeled plasmid DNA complexed with poly-L-lysine at specified charge ratios. Variations in conjugate hydrophobicity and levels of modification with polyethylene glycol were investigated. Our results show that, in contrast to in vitro studies, the systemically administered polyplexes exhibited marked DNA degradation in the vascular compartment within 5 min. Substitution of poly-L-lysine epsilon-amino sites with polyethylene glycol or hydrocarbon chains resulted in faster degradation even when complexed at higher charge (+/-) ratios. Use of excess cationic charge in the polyplexes (+/- 2.5) diminished degradation rates only slightly. An analysis was made of the strength of the poly-L-lysine:DNA interaction by competition with poly-aspartic acid. Polyplexes with the strongest binding between conjugate and DNA in the competition assay were also the most stable in blood. However, tighter binding was not enough to fully protect the polyplex in vivo and polyplex DNA was substantially degraded within 10 min. Increased polyplex stability did not correlate with improved in vivo transfection efficiency.     

Pectin-lipid assembly at the air-water interface: effect of the pectin charge distribution

Pectins are anionic polysaccharides that are sensitive to cations, a property that is widely used in food science. The interactions of a cationic lipid film (dimethyldioctadecylammonium bromide, DODAB) with a set of pectins bearing the same charge, which was either distributed randomly or pseudorandomly or blockwise, are investigated. The combination of Brewster angle microscopy BAM and infrared reflection-absorption spectroscopy IRRAS at the air-water interface reveals that pectin strongly binds to the cationic lipid film in forming a stacked layer underneath the lipid film. The detailed vibrational study of this stable mixed film indicates furthermore that pectin induces a disorder in the internal structure of the cationic film. The strong binding induces a splitting of the carboxylate stretching mode of pectin that is pressure and charge distribution dependent. The occurrence of an intermediate plateau below the collapse of the mixed film originates probably from a change in conformation of the pectin structure underneath the film.     

Multiple forms of pectin trans-eliminase from Verticillium dahliae Klebahn -- cotton wilt agent

From the culture liquid filtrate of Verticillium dahliae--cotton wilt agent--pectin trans-eliminase (EC) was isolated. The enzyme was isolated and examined, using ultrafiltration, gel filtration, ion exchange chromatography, isoelectrofocusing, and electrophoresis. The fungus was found capable to produce several forms of pectin trans-eliminase that differed in their molecular weight, charge, synthesis and release regulation, substrate action (position of bonding breakdowns in the pectin polymer molecule). Pectin trans-eliminase activity was also detected in cell walls of the fungal mycelium. Possible origin of multiple forms of the enzyme is discussed.     

Gelation of high methoxy pectin in the presence of pectin methylesterases and calcium

High methoxy pectin was submitted to various amounts of a fungal pectin methylesterase (PME) from Aspergillus aculeatus and of a plant PME from orange in the presence of calcium. The systems were characterized by rheological means during the gelation process. By the way of in situ demethoxylation with low amount of orange PME, it was possible to gel pectin from the beginning of the reaction although its high degree of methylation around 70. To understand this unusual properties, the behaviour of the two enzymes was investigated in pectic gels and in solution through the analysis of content and distribution of the remaining methyl esters. In the gel, the degree of methylation decreased slowly with orange PME and rapidly with Aspergillus PME. The degree of methylation and degree of blockiness after treatment with each PME in solution or in gels were slightly different. Possible explanations for this are evolving visco-elastic properties, including gel formation or influence of calcium on the enzyme–substrate complex.     

Heat-induced gelation of bovine serum albumin/low-methoxyl pectin systems and the effect of calcium ions

Influence of low-methoxyl pectin (LM pectin) and calcium ions (3 mM) on mechanical behavior and microstructure of bovine serum albumin (BSA) gels (pH 6.8, in 0.1 M NaCl) was evaluated. Protein and LM pectin concentrations were fixed at 2, 4, and 8 wt % and 0.21, 0.43, and 0.85 wt %, respectively. Rheological measurements and confocal laser scanning microscopy coupled with texture image analysis by use of the co-occurrence method were performed. Heat treatment of BSA/LM pectin mixtures induced protein gelation and a phase separation process between the two biopolymers, which was kinetically trapped. Calcium ions induced pectin gelation and modified BSA gel properties. Depending on biopolymer concentrations, a balance between pectin and/or protein gel contribution on final gel strength exists. The microstructures of the mixed systems in the presence of calcium can be interpreted as interpenetrated structures. Texture image analysis allowed one to classify more precisely the different microstructures observed in relation with mechanical properties.     

Interaction of HydSL hydrogenase from the purple sulfur bacterium Thiocapsa roseopersicina BBS with methyl viologen and positively charged polypeptides

The effect of polypeptides having different charge on the activity of Thiocapsa roseopersicina HydSL hydrogenase was studied. Strong inhibition was shown for poly-L-lysine bearing positive charge. The inhibition was reversible and competitive to methyl viologen, an electron acceptor, in the reaction of hydrogen oxidation catalyzed by the hydrogenase. Peptides carrying less positive charge had weaker inhibiting effect, while neutral and negatively charged peptides did not inhibit the hydrogenase. Molecular docking of poly-L-lysine to T. roseopersicina hydrogenase showed strong affinity of this polypeptide to the acceptor-binding site of the enzyme. The calculated binding constant is close to the experimentally measured value (K _i = 2.1 μM).     

Poly-L-lysine/heparin stimulates angiogenesis in chick embryo chorioallantoic membrane.

The effects of heparin on angiogenesis are controversial, with some studies claiming stimulatory and other studies claiming inhibitory effects. Since heparin in human plasma is complexed with basic peptides and proteins, we studied the angiogenic effect of complexes resulting by mixing poly-L-lysine (a basic heparin-binding polypeptide) and heparin. Angiogenesis was investigate by chorioallantoic membrane assay. In specimens treated with PBS (negative control), or poly-L-lysine, no significant vascular reaction was detectable. Heparin induced only moderate angiogenic response. However, neutral complexes purified from a mixture of poly-L-lysine and heparin (20/1, w/w) induced a very strong angiogenic response. These results demonstrate that the angiogenic effect of heparin was associated with neutralization of electric charge when the polysaccharide was complexed with a basic peptide.     

Purification and Characterization of an Endopolygalacturonase Produced by Sclerotinia Sclerotiorum

An endopolygalacturonase (endo-PG), was purified from the culture medium of a local isolate of Sclerotinia sclerotiorum with ammonium sulphate precipitation, cation exchange chromatography and gel filtration. The purified endo-PG had a molecular mass of approximately 18 kDa estimated by gel filtration. The isoelectric point was determined by isoelectric focusing to be approximately 8, suggesting that PG II possesses a net positive charge at physiological pHs. The pH optimum for the enzyme was at pH 4.5. The endo-PG showed essentially the same affinity for pectin and polygalacturonic acid as substrates. This revised version was published online in July 2006 with corrections to the Cover Date.