Rheological study into the ageing process of high methoxyl pectin/sucrose aqueous gels

The ageing process of high methoxyl pectin (HMP)/sucrose gels was followed at different ageing temperatures by small amplitude oscillatory experiments. Dynamic mechanical measurements allowed the characterisation of the point at which the system undergoes the sol/gel transition. The HMP/sucrose system is extremely sensitive to temperature variation during ageing, especially in the lower temperature range. The viscoelastic behaviour through the gel point changes with the ageing temperature, probably due to variations in mobility of the pectin chains, and consequently, in the lifetime of junction zones. Weaker pectin networks are formed under thermal conditions unfavourable to the development of hydrophobic interactions. Gel time and elastic modulus have a complex dependence on temperature, which could be attributed to the different thermal behaviour of the intermolecular interactions that stabilise the nonpermanent cross links of these physical networks.     

Effect of sugar and sorbitol on the formation of low methoxyl pectin gels

Pectin gels were made with amidated low methoxyl pectin using sucrose, glucose, fructose and sorbitol as sweetening agents. The adsorption of water at controlled activity was measured by determining sorption isotherms and by differential scanning calorimetry. These results were correlated with the gel formation mechanism. ¹H NMR spectra were measured for sugar with and without Ca²⁺ or La³⁺ cations. Results demonstrated no correlation between water adsorption on sugars and gel rigidity. The effects of the different sugars appear to be associated with the competition between each sugar and the pectin for calcium cations.     

Action pattern of Valencia orange PME de-esterification of high methoxyl pectin and characterization of modified pectins

Valencia pectinmethylesterase (PME) fractions, B-PME, containing 36 and 13 kDa protein bands and U-PME, containing a 36 and 27 kDa protein bands, were used to de-esterify original pectin (O-Pec) from 73% degree of esterification (%DE) to 63% (B-Pec) and 61% DE (U-Pec), respectively. Most O-Pec eluted from ion exchange chromatography at low salt concentration and a smaller component eluted at higher ionic strength. B-Pec and U-Pec eluted as one broad peak at higher ionic strength. PME modification did not change molecular weight: O-pectin (134,000 g/mol), U-Pec (133,850 g/mol), and B-Pec (132,250 g/mol). The NMR signal of GG and GGG increased after modification, whereas the signal of EE and EEE decreased. The negative zeta-potential increased with pH for all pectins. U-PME and B-PME created differently modified pectins that vary in degree and length of multiple attacks and fraction of the pectin population that was modified.     

Advances in biomedical applications of pectin gels

Pectin, due to its simple and cytocompatible gelling mechanism, has been recently exploited for different biomedical applications including drug delivery, gene delivery, wound healing and tissue engineering. Recent studies involving pectin for the biomedical field are reviewed, with the aim to capture the state of art on current research about pectin gels for biomedical applications, moving outside the traditional fields of application such as the food industry or pharmaceutics. Pectin structure, sources and extraction procedures have been discussed focussing on the properties of the polysaccharide that can be tuned to optimize the gels for a desired application and possess a fundamental role in application of pectin in the biomedical field.     

Structure and properties of pectin gels in plant cell walls

Abstract This review deals with recent advances in the structural characterization of pectins and the gels which they form, in relation to auxin-induced extension growth, the ripening of fruit, and cellular recognition. Pectins are block polysaccharides. Heavily branched, largely methyl-esterified blocks alternate with unbranched blocks of varying degrees of esterification. The unbranched, non-esterified blocks can aggregate through calcium binding to form the junction zones that hold a gel together. The aggregates are of two, or possibly four, chains at low calcium levels, and larger with excess calcium. The fall in wall pH during auxin-induced growth activates glycanase enzymes. These may attack some components of the pectic fraction, as well as xyloglucans. Pectin-bound calcium ions may be displaced but this probably has little effect on gel strength. Pectins may be cross-linked by diferulate esters when growth stops. The softening of ripe fruit is due to loss of cohesion in the pectin gel. In apples this results from replacement of the pectins by more esterified forms. In many other fruits it results from depolymerization by polygalacturonases, assisted by pectinesterases, so that the remaining segments are too short for effective calcium binding. Pectins have a further role in the recognition reactions between plant cells and some of their bacterial and fungal pathogens.     

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.     

Mesoscopic structure of pectin in solution

Mesoscopic structure of pectin with different molecular characteristics was investigated by means of small angle X‐ray scattering (SAXS), electrokinetic measurements and data modelling. The influence of a broad range of pH (2–7) on chain conformation in the dilute and semi‐diluted regime was investigated. Scattering data and concomitant analysis revealed two length scales at all environmental conditions studied. pH showed greater influence at acidic values (pH 2.0) enhancing the globular component of the structure due to association of galacturonic acid residues. Double logarithmic scattering intensity plots revealed fractal dimensions of 1.9 ± 0.2 in the low‐q regime and 1.5 ± 0.2 in the high q‐region, irrespectively of the specific environment. Increase in branching of RG‐I regions of the polysaccharide chains enhanced the compact conformation irrespectively of the pH or concentration. The present work shows that radical changes in pectin conformation can be induced only under strongly acidic conditions a finding that has important consequences in tailoring the technological performance of these biopolymers.     

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.     

Microstructure and kinetic rheological behavior of amidated and nonamidated LM pectin gels

The microstructure, kinetics of gelation, and rheological properties have been investigated for gels of nonamidated pectin (C30), amidated pectin (G), and saponified pectin (sG) at different pH values, both with and without sucrose. The low-methoxyl (LM) pectin gels were characterized in the presence of Ca(2+) by oscillatory measurements and transmission electron microscopy (TEM). The appearance of the gel microstructure varied with the pH, the gel structure being sparse and aggregated at pH 3 but dense and somewhat entangled at pH 7. During gel formation of pectins G and C30 at pH 3 there was a rapid increase in G' initially followed by a small increase with time. At pH 7 G' increased very rapidly at first but then remained constant. The presence of sucrose influenced neither the kinetic behavior nor the microstructure of the gels but strongly increased the storage modulus. Pectins G and C30 showed large variations in G' at pH values 3, 4, 5, and 7 in the presence of sucrose, and the maximum in G' in the samples occurred at different pH values. Due to its high Ca(2+) sensitivity, pectin sG had a storage modulus that was about 50 times higher than that of its mother pectin G at pH 7.     

Correlation between sagging and shear elasticity in pectin, gelatin and polyacrylamide gels

The relationship between the height of gels determined by a sag test and their elastic shear modulus (G′) has been both investigated experimentally and simulated using a finite element analysis for the inhomogeneous deformation of gels due to gravity. It was assumed in the simulations that gels can be modeled as incompressible linear elastic materials. General relationships between the sag of gels and their elastic modulus were obtained from the simulations for slip and no-slip conditions. The relationships were tested experimentally on pectin, gelatin and polyacrylamide gels with a range of concentrations and rigidities. The good agreement between the predictions and the results shows that these gels can be modeled accurately as incompressible elastic materials. A standard 150° SAG pectin gel, which sags 23.5% in the SAG test, has G′ moduli of 429 and 379 Pa under slip and no-slip conditions, respectively.     

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.     

Combined rheological and ultrasonic study of alginate and pectin gels near the sol-gel transition

The sol-gel transition of biopolymer mixtures has been investigated by rheological and ultrasonic measurements. A scaling analysis of the data was performed for both types of measurements. A gel time was determined from rheology for the pure pectin samples, and the data could be fitted to a universal scaling form near the transition point. Its critical exponents are in good agreement with the predictions of scalar percolation theory. In addition, the ultrasonic signal of the pectin samples close to the transition was analyzed in terms of a high-frequency scaling approach for the attenuation and the velocity. For the alginate samples and the mixtures, for which the gel point cannot be determined reliably from rheology, the ultrasonic measurements were analyzed using the same scaling form as for the pectin sample, thus providing a method for estimating the gel point, even in the absence of rheological data.     

An investigation of pectin and pectic acid in dilute aqueous solution

Light-scattering measurements have been carried out on carefully clarified aqueous solutions of pectin and pectic acid. The strong dependence of the inverse excess scattering intensity on angle yields pectin chain dimensions that are inconsistent with the complete molecular dispersion of pectin molecules possessing the degrees of polymerization indicated by the measured osmotic molecular weights. Negligible or negative concertration dependences of the inverse scattering intensities coupled with the centrifugation, filtration, and gel permeation chromatography behavior of the samples likewise suggest nonequilibrium aggregation of the pectin molecules in solution. It is shown that the measured mean polymer chain dimensions can be rationalized in terms of the presence of a very small proportion of aggregated material.     

Influence of pectin fine structure on the mechanical properties of calcium-pectin and acid-pectin gels

The in vitro and in vivo functionality of the anionic plant polysaccharide pectin depends not only on the amount of ion-binding groups attached to the polymer but also on the distribution of such groups along the backbone. It has been proposed recently that information regarding this intramolecular distribution can be quantified by defining a degree of blockiness (DB or DB(abs)), and the usefulness of such measures in discriminating qualitatively between pectins originating from different sources has been demonstrated. Despite this, the value of these parameters in predicting the pseudoequilibrium elastic modulus of gels remains untested. This study seeks to address this problem through the sourcing and in-house modification of a variety of pectins in order to produce a library of distinct representative fine structures. These were subsequently characterized in terms of their relevant properties, including the determination of the proposed DB and DB(abs), and the formation of gels of these samples was monitored using small deformation mechanical spectroscopy. In addition to ionotropic calcium gels the effect of the fine structure on acid gelation was also studied.     

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.     

Microbial utilization and selectivity of pectin fractions with various structures

To evaluate the fermentation properties of oligosaccharides derived from pectins and their parent polysaccharides, a 5-ml-working-volume, pH- and temperature-controlled fermentor was tested. Six pectic oligosaccharides representing specific substructures found within pectins were prepared. These consisted of oligogalacturonides (average degrees of polymerization [DP] of 5 and 9), methylated oligogalacturonides (average DP of 5), oligorhamnogalacturonides (average DP of 10 as a disaccharide unit of galacturonic acid and rhamnose), oligogalactosides (average DP of 5), and oligoarabinosides (average DP of 6). The influence of these carbohydrates on the human fecal microbiota was evaluated. Use of neutral sugar fractions resulted in an increase in Bifidobacterium populations and gave higher organic acid yields. The Bacteroides-Prevotella group significantly increased on all oligosaccharides except oligogalacturonides with an average DP of 5. The most selective substrates for bifidobacteria were arabinan, galactan, oligoarabinosides, and oligogalactosides.     

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.     

An ethidium-acrylamide affinity medium for recovery of nucleic acids from free solution and from polyacrylamide and agarose gels

The simple preparation of an ethidium-bromide-based nucleic acid affinity medium is described. The medium is composed of an acrylamide matrix to which ethidium bromide is attached. Its use in preparative purification and fractionation of nucleic acids in solution and in electrophoretic elution of nucleic acids from gels is reported. Nucleic acids can be eluted from this medium with a buffered salt solution and concentrated by ethanol precipitation without persistent contamination with undesirable impurities.     

Functional characterization of the gels prepared with pectin methylesterase (PME)-treated pectins

High- and low-methoxyl pectins were treated with pectin methylesterase (PME) and the functional properties of the resulting pectin gels were characterized. The degree of esterification of high- and low-methoxyl pectins decreased from 74.5% to 6.3% and 40.0% to 6.5%, respectively while not changing their molecular weight. Also, the addition of glucono-delta-lactone (GDL) dramatically affected the gel strength and the pH reduction by the GDL led to the increased syneresis of the pectin gels, which was also observed in the PME-treated samples. When flavor compounds were incorporated into the pectin gels, the flavor release from the gels increased with decreasing the degree of esterification due to increased hydrophilic properties.