Gel-forming structures and stages of red algal galactans of different sulfation levels

Sol-gel transition processes of algal galactans were studied using cryofixation method in combination with freeze-drying and scanning electron microscopy (SEM) techniques. The structures formed in successive stages of gelling process upon cooling were rapidly frozen at defined temperature points and viewed by SEM. It was established that in the case of both types of gelling galactans investigated, a fine honeycomb-like network exists for a wide range of solution temperatures. The formation and structure of this network depends on the structural type, gelling stage, and concentration of the galactan in solution. The honeycomb suprastructures exist also in carrageenan and agarose sols (at temperatures considerably exceeding the gelling temperatures). An additional helical network formed showed different behaviour in the case of carrageenan and agar-type polysaccharides. In the gel-formation process, tightening of the network takes place in both types of galactan gels; the honeycomb structures persist in carrageenan (furcellaran) but not in agarose gels.     

Gelatin vs polysaccharide in mixture with sugar

The structural behavior of a well-characterized gelatin sample has been revisited to investigate the morphology of its network in the presence of sugar. This was then contrasted with the corresponding properties of the gelling polysaccharides agarose, kappa-carrageenan, and deacylated gellan. Small deformation dynamic oscillation, differential scanning calorimetry in plain and modulated mode, visual observations, and transmission electron microscopy were used to identify the structural characteristics of the biopolymers from the rubbery plateau through the transition region to the glassy state. In contrast to the collapse of the polysaccharide gels at intermediate levels of co-solute, gelatin forms reinforced networks. The drop in polysaccharide network strength is accompanied by a decline in the enthalpy of the coil-to-helix transition, whereas the transition enthalpy is more pronounced in gelatin gels in accordance with their strengthening. Tangible evidence of the molecular transformations was obtained using microscopy, with polysaccharides disaggregating and dissolving in the saturated sugar environment. Gelatin, on the other hand, is visualized in an aggregated form thus producing a phase-separated topology with sugar.     

Peptides and proteins in a confined environment: NMR spectra at natural isotopic abundance.

Confinement of proteins and peptides in a small inert space mimics the natural environment of the cell, allowing structural studies in conditions that stabilize folded conformations. We have previously shown that confinement in polyacrylamide gels (PAGs) is sufficient to induce a change in the viscosity of the aqueous solution without changing the composition and temperature of the solvent. The main limitation of a PAG to run NMR experiments in a confined environment is the need for labelling the peptides. Here we report the use of the agarose gel to run the NMR spectra of proteins and peptides. We show that agarose gels are completely transparent in NMR experiments, relieving the need for labelling. Although it is necessary to expose biomolecules to fairly high temperatures during sample preparation, we believe that this is not generally an obstacle to the study of peptides, and found that the method is also compatible with temperature-resistant proteins. The mesh of agarose gels is too wide for direct effects of confinement on the stability of proteins but confinement can be easily exploited to interact the proteins with other reagents, including crowding macromolecules that can eventually lead to fold stabilization. The use of these gels is ideally suited for low-temperature studies; we show that a very flexible peptide at subzero temperatures is stabilized into a well-folded conformation.     

Effect of co-solute and gelation temperature on milk protein and gum tragacanth interaction in acidified gels

The aim of this study was to investigate the role of process conditions and system composition on the acid-induced gelation of a mixture of milk protein and gum tragacanth. This was studied by determining the effects of co-solute (lactose) addition (3, 5 and 7%) and gelation temperature (25, 37 and 45°C) on the mixture's rheological properties and microstructure using a combination of techniques including small-deformation rheology and scanning electron microscopy. The presence of lactose played an important role in the microstructure formation of gels but did not change most rheological properties. The microstructure of gels formed in the presence of lactose was coarser and more particulate, but less interconnected; this can be explained by lactose's role in improving protein aggregation. Gels prepared at a lower temperature had a high structure strength, as indicated by their high storage modulus, τ(f) and G(f) values. Low gelation temperature also caused a more branched and homogenous microstructure.     

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.     

Interpenetrating network formation in agarose--kappa-carrageenan gel composites

Thermal, mechanical, turbidity, and microscope evidence is provided which strongly suggests molecular interpenetrating network (IPN) formation by mixtures of the seaweed polysaccharides agarose and kappa-carrageenan. Over a range of ionic strength, and potassium content, there is no evidence for synergistic coupling of the networks, and simple phase separation (demixing) can definitely be ruled out. At low ionic strength, where the agarose gels first, differential scanning calorimetry evidence shows some influence of the carrageenan on the agarose ordering enthalpy, particularly at higher polymer concentrations. As the potassium level is increased, however, and the order of gelling is reversed, this effect disappears. Cure behavior for the systems at high ionic strength can be described as a simple summation of the pure component contributions. At low ionic strength, on the other hand, the modulus behavior is more complex, suggesting either a modification, in the mixture, of the kappa-carrageenan gelling parameters or a more complex modulus additivity rule.     

Segregative phase separation in agarose/whey protein systems induced by sequence-dependent trapping and change in pH

The structural properties and morphology of mixed gels made of aqueous preparations of agarose and whey protein were modified by changing thermal treatment and pH. The conformationally dissimilar polymers phase separated and this process was followed by small-deformation dynamic oscillation in shear, differential scanning calorimetry and environmental scanning electron microscopy. Experimental protocol encourages formation of a range of two-phase systems from continuous agarose matrices perforated by liquid-like whey protein inclusions to phase inverted preparations where a soft protein matrix suspends hard agarose-filler particles. These distinct morphologies have widely different mechanical moduli, which were followed by adapting a theoretical analysis (isostress-isostrain and Lewis-Nielsen blending laws) from the literature in synthetic block polymers and polyblends. Based on this framework of thought, reasonable predictions of the elastic moduli in the composite gels were made that led to patterns of solvent partition between the two polymeric networks. It was shown that proteins, in mixture with polysaccharide, exhibit favorable relative affinity (P-factor) for water molecules at a pH above their isoelectric point. This is an unexpected outcome that adds to the central finding of a single P value for the distribution of solvent between the continuous matrix and discontinuous inclusions of binary gels. It was thus proposed that phase continuity and solvent distribution in agarose/whey protein systems are under kinetic control that can be heavily governed by pH changes in the aqueous environment.     

Unexpected high pressure effects on the structural properties of condensed whey protein systems

We show that application of high hydrostatic pressure (600 MPa for 15 min) on condensed whey protein (WP) systems (e.g., 80% w/w solids content) results in unexpected structure–function behavior when compared with conventional thermal treatment. Unraveling the relaxation properties in first‐order thermodynamic transitions, the manifestation of glass transition phenomena and the preservation of native conformation in condensed preparations were recorded using small‐deformation dynamic oscillation in shear, modulated differential scanning calorimetry, and infrared spectroscopy. Informed temperature application results in the formation of continuous networks at the denaturation temperature, which undergo vitrification at subzero temperatures. In contrast, high‐pressure‐treated WPs resist physicochemical denaturation, hence preserving the native conformation of secondary and tertiary structures. This was rationalized on the basis of a critical concentration threshold where transfer of water molecules to nonpolar residues in the protein interior is minimized because of low moisture content and restricted molecular mobility. The physical state and morphology of these high‐solid preparations were further examined by the combined framework of reduced variables and Williams, Landel, and Ferry equation/free volume theory. Theoretical treatment of experimental observations unveils the dynamic range of the mechanical manifestation of the glass transition region in samples subjected to heat or pressure. In addition to preserving native conformation, WPs subjected to high pressure form glassy systems at parity with the structural functionality of the thermally treated counterparts. © 2012 Wiley Periodicals, Inc. Biopolymers 97:963–973, 2012.     

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.     

Steady-state and pulsed NMR studies of gelation in aqueous agarose

Steady-state and pulsed NMR techniques have been used to investigate molecular motion in sols and gels of agarose. In passing through the sol–gel transition, the molecular mobility of water molecules is reduced only by a small amount, whereas motion of the polymer chains is greatly attenuated. The results are discused in terms of the network theory of gelation, with references to the role of water in the process and the nature of the “junction zones” between polymer chains. T₂ and line-width measurements are dominated by exchange broadening. The effects of exchange rate and differences in relaxation time between the exchanging sites are discussed. The temperature hysteresis behavior of agarose gels has been investigated and the effects of “ageing” correlated with changes in nuclear relaxation times. The synergistic increase in gel strength obtained on adding locust bean gum (LBG) to agarose has been investigated. The results indicate that LBG does not form double-helix junctions and may decrease rates of gelation by steric effects. At high agarose concentration, the LBG remains mainly in solution in interstitial water, but at low agarose concentration, it is suggested that the LBG can link gel aggregates together into a self-supporting structure, producing a synergistic increase in gel strength. Comparisons have been made between the nature of the agarose–LBG interaction and agarose–cellulose interactions in biological systems.     

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.     

Gelation of xyloglucan by addition of epigallocatechin gallate as studied by rheology and differential scanning calorimetry

Interaction of tamarind seed xyloglucan (TSX) and epigallocatechin gallate (EGCG) was investigated. TSX alone showed the rheological behaviors of dilute and semidilute solution types in the temperature range from 10 to 50 degrees C and the concentration range from 1 to 10%. Addition of a small amount of EGCG changed the rheological properties of TSX solutions to induce a thermoreversible gelation. The sol-gel transition was detected as a crossover of the storage and loss shear moduli at a certain temperature in thermal scanning rheological measurements and an endo- and exo- thermic peaks in curves obtained by differential scanning calorimetry on heating and cooling. High storage modulus of the gels at all experimental frequencies also indicated the formation of a network structure. Increase in the gel strength and the enthalpy of the transition with increasing EGCG concentration at fixed TSX concentration suggested that EGCG was directly involved in the network formation through association with TSX. The TSX gel was obtained by addition of appropriate amount of EGCG. Addition of an excessive amount of EGCG induced precipitation.     

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.     

Structural properties of pectin-gelatin gels. Part II: effect of sucrose/glucose syrup

Small deformation dynamic oscillation and bright field microscopy were used to examine the structural properties of single and mixed high methoxy pectin and gelatin systems in the presence of sucrose/glucose syrup blends. Co-solute concentrated (≥78%) systems of the polysaccharide form rubbery structures which are readily transformed into glassy consistencies according to the time-temperature superposition principle. Increasing amounts of co-solute in the gelatin samples induce changes in viscoelasticity from that of conventional hydrogels to mechanical traces that cover much of the plateau region and the beginning of the glass transition area. Furthermore, manipulation of the protein/ sugar ratio can result in strong crystalline matrices, or viscoelastic solutions where the co-solute forms the continuous phase and the gelatin inclusions can undertake a conformational transition. The properties of the single components were used to rationalise the phase behaviour of their mixtures. Upon triggering the gelation of pectin, mixtures can be made where either gelatin or both components form a continuous phase. Results are discussed in the light of evidence obtained from the ethylene glycol work in Part I.     

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.     

Membrane permeability parameters for freezing of stallion sperm as determined by Fourier transform infrared spectroscopy

Cellular membranes are one of the primary sites of injury during freezing and thawing for cryopreservation of cells. Fourier transform infrared spectroscopy (FTIR) was used to monitor membrane phase behavior and ice formation during freezing of stallion sperm. At high subzero ice nucleation temperatures which result in cellular dehydration, membranes undergo a profound transition to a highly ordered gel phase. By contrast, low subzero nucleation temperatures, that are likely to result in intracellular ice formation, leave membrane lipids in a relatively hydrated fluid state. The extent of freezing-induced membrane dehydration was found to be dependent on the ice nucleation temperature, and showed Arrhenius behavior. The presence of glycerol did not prevent the freezing-induced membrane phase transition, but membrane dehydration occurred more gradual and over a wider temperature range. We describe a method to determine membrane hydraulic permeability parameters (E_Lp, Lpg) at subzero temperatures from membrane phase behavior data. In order to do this, it was assumed that the measured freezing-induced shift in wavenumber position of the symmetric CH₂ stretching band arising from the lipid acyl chains is proportional to cellular dehydration. Membrane permeability parameters were also determined by analyzing the H₂O-bending and -libration combination band, which yielded higher values for both E_Lp and Lpg as compared to lipid band analysis. These differences likely reflect differences between transport of free and membrane-bound water. FTIR allows for direct assessment of membrane properties at subzero temperatures in intact cells. The derived biophysical membrane parameters are dependent on intrinsic cell properties as well as freezing extender composition.     

Global structures of high methoxyl pectin from solution and in gels

Images of high methoxyl orange pectin deposited from solution and high methoxyl sugar acid gels (HMSAG) were obtained by atomic force microscopy (AFM) in the tapping mode. For the first time, images of pectin deposited from water revealed that the transition from pectin networks to individual molecules or aggregates thereof occurred at concentrations between 6.5 and 13.1 microg/mL. At 6.5 microg/mL, shapes included rods, segmented rods, kinked rods, rings, branched molecules, and dense circular areas. At 13.1 microg/mL, all of these shapes were integrated into networks. These same structures were discernible in pectin high methoxyl sugar acid gels. Thus one might consider pectin networks in water at concentrations in excess of 10 microg/mL to be separate fluid precursors of networks in high methoxyl sugar acid gels. Examination of AFM images revealed that gels with "uniform" distribution of strands and pores between strands had higher gel strengths as measured by a penetrometer than gels in which strands were nonuniformly distributed and were separated by large and small spaces.     

Definition of the rheological glass transition temperature in association with the concept of iso-free-volume

Small deformation dynamic oscillation was used to develop an index of physical significance for the rationalisation of the mechanical properties of high co-solute/biopolymer systems during vitrification. The index is based on the combined framework of Williams–Landel–Ferry equation with the free volume theory and is called the ‘rheological glass transition temperature, T_g’ thus differentiating it from the empirical calorimetric T_g used in thermal analysis. The rheological T_g is located at the conjunction of two distinct molecular processes, namely: free-volume effects in the glass transition region and the predictions of the reaction-rate theory in the glassy state. The method of reduced variables was used to shift the mechanical spectra of shear moduli to composite curves. The temperature dependence of shift factors for all materials was identical provided that they were normalised at suitably different reference temperatures, which reflect iso-free-volume states. The treatment makes free volume the overriding parameter governing the mechanical relaxation times during vitrification of high co-solute/biopolymer systems regardless of physicochemical characteristics. We believe that potential applications resulting from this fundamental work are numerous for the food and pharmaceutical industries.     

Rheological Study of the Gelation Process of Agarose-Based Solutions

The gelation of agarose is investigated by rheological methods and electron microscopy, as well as the thickening properties of xanthan. The gelling and thickening agents have been investigated in pure water to compare the results with theoretical models. The gelation of agarose was shown to follow two steps upon cooling, which could be addressed to the formation of helices and their aggregation. In addition to the rheology, transmission electron micrographs of freeze-dried samples have been taken to underline the date by corresponding structures at different stages of the gelling process. The xanthan molecules, which have been approximated by rigid highly charged rodlike molecules, undergo a jamming transition at a critical concentration. This concentration shows a strong dependence on the length of the molecules, which supports the high thickening effect of xanthan. When both, agarose and xanthan are mixed, the gel structure becomes very different. The gelling process is now determined by one step only. It is proposed that the jamming xanthan molecules prevent the formation of the aggregates of the agarose gel. The gels themselves appear then less elastic, and should yield a better mouth feeling.     

Stereocontrolled Synthesis of an Octasaccharide Part of I-Active Glycolipids

The effects of glycerin and ethylene glycol on the elastic modulus and DSC thermograms of agarose and kappa-carrageenan gels were examined to clarify the relation between structure and properties. The elastic modulus of these gels as a function of the concentration of polyols increased up to a certain concentration and then decreased with increasing concentration of polyols. These polyols shifted the melting temperature of the gel to higher temperatures in kappa-carrageenan gels but to lower temperatures in agarose gels. The temperature dependence of elastic modulus was changed in opposite directions in agarose and kappa-carrageenan gels by the addition of polyols, and this is discussed on the basis of model consisting of junction zones which are connected by Langevin chains. It was suggested that the mean distance between junction zones became shorter in the presence of a small amount of polyols.