Physicochemical studies of pectin/poly-L-lysine gelation

The effect of poly-L-lysine concentration and degree of polymerisation on the gelation of pectins differing in charge density and distribution was examined, through the determination of gel stiffness, swelling behaviour and the binding of poly-L-lysine to the gel network. Poly-L-lysine acts as a crosslinker of concentrated pectin solutions, with its effectiveness showing dependencies on pH and charge distribution on the pectin. Neutralisation of the anionic charge on the pectin with the polycationic peptide leads to gel opacity and eventually network collapse.     

Thermoreversible gelation of aqueous mixtures of pectin and chitosan. Rheology

The synergistic interaction between pectin and chitosan in aqueous acid solution and in the gel phase has been studied by oscillatory shear measurements. Mixtures of pectin and chitosan form thermoreversible gels over a broad composition range by lowering the temperature. The value of the gelation temperature depends on the composition of the mixture, with low values for mixtures with low pectin contents. For incipient gels, a power law can describe the frequency dependence of the complex viscosity, with power law exponents close to -1. The gel evolution of pectin-chitosan mixtures upon a temperature quench below the gel point has been studied. Evidence is provided for a relation between gelation and phase separation in the process of temperature-induced gelation of pectin-chitosan mixtures. A simple model is proposed to rationalize the gelation process in these systems.     

Influence of Interfacial Composition on in Vitro Digestibility of Emulsified Lipids: Potential Mechanism for Chitosan's Ability to Inhibit Fat Digestion

The objective of this study was to investigate the influence of interfacial composition and electrical charge on the in vitro digestion of emulsified fats by pancreatic lipase. An electrostatic layer-by-layer deposition technique was used to prepare corn oil-in-water emulsions (3 wt% oil) that contained droplets coated by (1) lecithin, (2) lecithin–chitosan, or (3) lecithin–chitosan–pectin. Pancreatic lipase (1.6 mg mL⁻¹) and/or bile extract (5.0 mg mL⁻¹) were added to each emulsion, and the particle charge, droplet aggregation, and free fatty acids released were measured. In the presence of bile extract, the amount of fatty acids released per unit amount of emulsion was much lower in the emulsions containing droplets coated by lecithin–chitosan (38 ± 16 μmol mL⁻¹) than those containing droplets coated by lecithin (250 ± 70 μmol mL⁻¹) or lecithin–chitosan–pectin (274 ± 80 μmol mL⁻¹). In addition, there was much more extensive droplet aggregation in the lecithin–chitosan emulsion than in the other two emulsions. We postulated that lipase activity was reduced in the lecithin–chitosan emulsion as a result of the formation of a relatively thick cationic layer around each droplet, as well as the formation of large flocs, which restricted the access of the pancreatic lipase to the lipids within the droplets. Our results also suggest that droplets initially coated by a lecithin–chitosan–pectin layer did not inhibit lipase activity, which may have been because the chitosan–pectin desorbed from the droplet surfaces thereby allowing the enzyme to reach the lipids; however, further work is needed to establish this. This information could be used to create food emulsions with low caloric level, or to optimize diets for individuals with lipid digestion problems.     

Effect of pectin charge density on formation of multilayer films with chitosan

The effect of pectin charge density on the formation of multilayer films with chitosan (PEC/CHI) is studied by means of electro-optics. Pectins of low (21%) and high (71%) degrees of esterification, which are inversely proportional to the pectin charge density, are used to form films on colloidal beta-FeOOH particles at pH 4.0 when the CHI is fully ionized. We find that, after deposition of the first 3-4 layers, the film thickness increases linearly with the number of adsorbed layers. However, the increase in the film thickness is larger when the film is terminated with CHI. Irregular increase of the film thickness is more marked for the PEC with higher density of charge. Oscillation in the electrical polarizability of the film-coated particles with the number of deposited layers is also registered in the PEC/CHI films. The charge balance of the multilayers, calculated from electrical polarizability of the film-coated particles, is positive, with larger excess of positive charge within the film constructed from CHI and less charged PEC. This is attributed to the ability of CHI to diffuse into the film at each deposition step. Despite the CHI diffusion, the film thickness increases linearly due to the dissolution of unstable PEC/CHI complexes from the film surface.     

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.     

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.     

Preparation and chemical and biological characterization of a pectin/chitosan polyelectrolyte complex scaffold for possible bone tissue engineering applications

In this work, porous scaffolds obtained from the freeze-drying of pectin/chitosan polyelectrolyte complexes were prepared and characterized by FTIR, SEM and weight loss studies. Additionally, the cytotoxicity of the prepared scaffolds was evaluated in vitro, using human osteoblast cells. The results obtained showed that cells adhered to scaffolds and proliferated. The study also confirmed that the degradation by-products of pectin/chitosan scaffold are noncytotoxic.     

Encapsulation of tannase for the hydrolysis of tea tannins

Tannase was encapsulated in alginate, chitosan, carrageenan or pectin gel matrices, and in the case of alginate, coated with high or low molecular weight chitosan to reduce enzyme release. Cross-linking with glutaraldehyde also improved enzyme retention. Active enzyme preparations were obtained, although carrageenan gels were unstable in tea. Tannase activity was evaluated by reduction in centrifugable (flocculated) tea solids, and a reduction in tea cream measured turbidimetrically after removal of flocculated solids. Tannin interactions with the polysaccharide gels increased the level of centrifugable solids (flocculent) in the tea. An optimum bead formulation consisted of an alginate core, coated with chitosan and cross-linked with glutaraldehyde. Both core and coating materials contained active enzyme. Beads were prepared in a single step procedure involving extrusion of alginate/tannase solution into a hardening bath containing tannase-loaded, chitosan solution. Tannase retained hydrolytic activity through three successive batch cycles, for a total period of 39h processing, and tea cream was visibly removed by treatment with the immobilized tannase. Activity remained stable during 1-month bead storage under refrigeration.     

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.     

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.     

Polydimethylsiloxane Films Modified with Chitosan/Pectin Multilayers as Scaffolds for Mesenchymal Stem Cells

Solid (smooth) and porous films of polydimethylsiloxane (PDMS) have been obtained; the effect of their structure on the adhesion of mesenchymal stem cells (MSCs) on their surface was found. It is shown that modification of these films with a (chitosan/pectin)₄ multilayer increased the efficiency of viable cell adhesion. A (3-aminopropyl)triethoxysilane–glutaraldehyde system was used to enhance the binding of the polysaccharide layer to the hydrophobic surface of PDMS. It was found that MSCs formed a monolayer culture of the fibroblast-like cells with high viability on porous PDMS modified with (chitosan/pectin)₄.     

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.     

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.     

Effect of oppositely charged polymer and dissolution medium on swelling, erosion, and drug release from chitosan matrices

The purpose of this research was to investigate the potential use of anionick-carrageenan and nonionic hydroxypropyl-methylcellulose (HPMC, K4) to improve the matrix integrity of directly compressed chitosan tablets containing naproxen sodium, an anionic drug. The influence of buffer pH and drug:polymer ratio on the water uptake, matrix erosion, and drug release were studied. The rapid release of naproxen sodium was seen from matrices containing 100% chitosan due to loss in the matrix cohesiveness; whereas, it was relatively slow for matrices containing optimum concentration ofk-carrageenan. In-situ interaction between oppositely charged moieties resulted in the formation of polyelectrolyte complexes with stoichiometric charge ratios of unity. Fourier transform in frared (FTIR) spectroscopy and powder x-ray diffraction (PXRD) data confirmed the importance of ionic bonds in polyelectrolyte complexation. The ionic interactions between polymers were absent in matrices containing HPMC and the integrity of tablets was improved owing to the presence of viscous gel barrier. The reasons for retarded release of naproxen sodium from the chitosan matrices at different pH include poor aqueous solubility of drug, the formation of a rate-limiting polymer gel barrier along the periphery of matrices, the interaction of naproxen sodium with protonated amino, groups of chitosan, and the interaction of ionized amino groups of chitosan with ionized sulfate groups ofk-carrageenan. Published: June 15, 2007     

Influence of chain length and polymer concentration on the gelation of (amidated) low-methoxyl pectin induced by calcium

The gelation of low-methoxyl pectin (LMP) induced by addition of Ca2+ was studied by measuring the storage modulus as a function of temperature during cooling. Samples with different molar masses were prepared by mechanical degradation. The effect of the molar mass and the pectin concentration on the gelation properties was investigated. The effect of partial amidation was studied by comparing LMP and partially amidated LMP with the same molar mass and degree of methylation. The results are compared to those from a model developed for Ca2+-induced pectin gelation, and good agreement is found except at low concentrations and low molar masses where the gels are weaker than predicted. At low concentrations intrachain bonding weakens the gel, while the presence of small pectin chains weakens the gel because it neutralizes binding sites on larger chains.     

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.     

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.     

Preparation and characterization of chitosan and trimethyl-chitosanmodified poly-(ε-caprolactone) nanoparticles as DNA carriers

The purpose of this research was to prepare poly-(ε-caprolactone) (PCL) particles by an emulsion-diffusion-evaporation method using a blend of poly-(vinyl alcohol) and chitosan derivatives as stabilizers. The chitosan derivatives used were chitosan hydrochloride and trimethyl chitosans (TMC) with varying degrees of quaternization. Particle characteristics-size, zeta potential, surface morphology, cytotoxicity, and transfection efficiency-were investigated. The developed method yields PCL nanoparticles in the size range of 250 to 300 nm with a positive surface charge (2.5 to 6.8 mV). The cytotoxicity was found to be moderate and virtually independent of the stabilizers' concentration with the exception of the highly quaternized TMC (degree of substitution 66%) being significantly more toxic. In immobilization experiments with gel electrophoresis, it could be shown that these cationic nanoparticles (NP) form stable complexes with DNA at a NP:DNA ratio of 3:1. These nanoplexes showed a significantly higher transfection efficiency on COS-1 cells than naked DNA. Published: August 10, 2005     

Influence of chitosan structure on the formation and stability of DNA-chitosan polyelectrolyte complexes

The interactions between DNA and chitosans varying in fractional content of acetylated units (FA), degree of polymerization (DP), and degree of ionization were investigated by several techniques, including an ethidium bromide (EtBr) fluorescence assay, gel retardation, atomic force microscopy, and dynamic and electrophoretic light scattering. The charge density of the chitosan and the number of charges per chain were found to be the dominating factors for the structure and stability of DNA-chitosan complexes. All high molecular weight chitosans condensed DNA into physically stable polyplexes; however, the properties of the complexes were strongly dependent on FA, and thereby the charge density of chitosan. By employing fully charged oligomers of constant charge density, it was shown that the complexation of DNA and stability of the polyplexes is governed by the number of cationic residues per chain. A minimum of 6-9 positive charges appeared necessary to provide interaction strength comparable to that of polycations. In contrast, further increase in the number of charges above 9 did not increase the apparent binding affinity as judged from the EtBr displacement assay. The chitosan oligomers exhibited a pH-dependent interaction with DNA, reflecting the number of ionized amino groups. The complexation of DNA and the stability of oligomer-based polyplexes became reduced above pH 7.4. Such pH-dependent dissociation of polyplexes around the physiological pH is highly relevant in gene delivery applications and might be one of the reasons for the high transfection activity of oligomer-based polyplexes observed.     

Effect of pectin on pectinases in autolysis of Botrytis cinerea

Pectic activity in autolyzed cultures of Botrytis cinerea in a medium with and without pectin was similar, but in the medium with pectin maximal activities occurred in younger cultures. The pectic activities found were polygalacturonase, polymethylgalacturonase, endo activity (pectin as substrate) and pectin lyase. The molecular weights of polygalacturonase, polymethylgalacturonase and endo activity (pectin as substrate) were 36000, 33000 and 30200 daltons respectively, and the molecular weight of pectin lyase was 18200 daltons. By gel electrophoresis four different pectic activities were detected, three in the top of the gel and one in the bottom. Two enzymes were characterized, the polygalacturonase activity (first band in the top) inhibited by Ca⁺⁺ and the pectin lyase activity (in the bottom) which was not inhibited by Ca⁺⁺. These enzymes are not induced by the presence of pectin in the medium during degradation of Botrytis cinerea.