Structural changes during heat-induced gelation of globular protein dispersions

Macroscopic and molecular structural changes during heat-induced gelation of beta-lactoglobulin, bovine serum albumin, ovalbumin, and alpha-lactalbumin aqueous dispersions were probed by the mechanical and CD spectroscopy, respectively. Aqueous solutions of the native globular proteins, except for alpha-lactalbumin, exhibited solid-like mechanical spectra-namely, the predominant storage modulus G' over the loss modulus G" in the entire frequency range examined (0.1-100 rad/s), suggesting that these protein solutions were highly structured even before gelation, possibly due to strong repulsions among protein molecules. Such solid-like structures were susceptible to nonlinearly large shear but recovered almost immediately at rest. During gelation by isothermal heating, major changes in the secondary structure of the globular proteins completed within a few minutes, while values of the modulus continued to develop for hours with maintaining values of tandelta (= G"/G') less than unity. As a result, a conventional criterion for mechanically defining the gelation point, such as a crossover between G' and G", was inapplicable to these globular protein systems. beta-Lactoglobulin gels that had passed the gelation point satisfied power laws (G' approximately G" approximately omega(n)) believed to be valid only at the gelation point, suggesting that fractal gel networks, similar to those of critical gels (i.e., gels at the gelation point), were formed.     

Single and multi-step carboxymethylation of water yam (Dioscorea alata) starch: synthesis and characterization

Water yam (Dioscorea alata) starch was carboxymethylated by single and multi-step processes for nine times successively. Optimal degree of substitution (DS) of 0.98 and reaction efficiency (RE) of 70.5% were obtained in the single-step carboxymethylation at 40 degrees C, 3 h in isopropanol-water reaction mixture ratio 1:0.16. The ratio of sodium hydroxide and sodium monochloroacetate moles to anhydroglucose unit moles for the optimal DS and RE were 1.62 and 1.39, respectively. The DS increased progressively as the steps of carboxymethylation increased from 2 to 9 and an optimal DS of 2.24 was obtained. Initial increases in carboxymethylation step increased the RE progressively up to 82.1% after the seventh carboxymethylation step but declined with further increases in the carboxymethylation step. The wide angle X-ray diffractometry and the DSC revealed remarkable changes after carboxymethylation because starch crystallinity reduced significantly. Thermogram of native starch showed a characteristic three-step decomposition with 13.16%, 61.54% and 24.79% weight losses progressively while carboxymethyl derivative showed four decomposition stages with 9.86%, 36.57%, 3.04% and 23.07% weight losses progressively. Studies revealed that carboxymethylation improved thermal stability of the native starch. IR spectrometry revealed that carboxymethyl starch showed new bands at nu=1600, 1426 and 1324 cm(-1). The broad band (13)C NMR spectra of the ultrasonically degraded carboxymethyl starch showed a peak at delta=180 ppm which was assigned to carbonyl carbon.     

Biosynthesis of hetero-polysaccharides by Acetobacter xylinum - Synthesis and characterization of metal-ion adsorptive properties of partially carboxymethylated cellulose

Biological reconstruction of water-soluble carboxymethylated cellulose (CMC; D.S. =0.47) has been achieved by culturing Acetobacter xylinum in medium containing CMC and -glucose to give a novel hetero-polysaccharide having a carboxymethyl function. The novel extracellular polysaccharide, carboxymethylated-bacterial cellulose (CM-BC), had an ion exchange ability with enhanced specific adsorption for lead and uranyl ions compared to the original CMC and bacterial cellulose. The contribution of the hydroxy group at C-2 was confirmed by applying carboxymethylated chitin, which possesses acetamido group at C-2 of the glucose residue, as the carbon source of the incubation.     

Synergistic interaction of xyloglucan and xanthan investigated by rheology, differential scanning calorimetry, and NMR

A new synergistic interaction between tamarind seed xyloglucan and xanthan was found and investigated by rheology, differential scanning calorimetry (DSC), and NMR. The effect of the acetyl and pyruvate groups in the side chain in xanthan on the synergistic interaction was also examined. The shear moduli G' and G' ' of the mixture solution of xyloglucan and native (or acetate-free) xanthan increased steeply at around 22 degrees C upon cooling. An exothermic DSC peak appeared at the same temperature. A drastic decrease in the of the acetyl and pyruvate groups of the xanthan side chain was observed from 1H NMR spectra only in the mixture at low temperatures (<25 degrees C). It was found that the pyruvate group is more restricted in the mixture solution compared with the acetyl group. The mixture of xyloglucan and pyruvate-free xanthan showed no synergistic interaction. We concluded that this synergistic interaction is caused by the intermolecular binding between xyloglucan and xanthan, and, in the heterotypic junction zones, the xanthan side chain becomes a new state that is different from both the coil and helix states.     

Network structure of curdlan in DMSO and mixture of DMSO and water

The viscoelastic property of curdlan solution in dimethyl sulfoxide (DMSO) was investigated. We discuss the difference in the viscoelastic properties of curdlan solution in DMSO and that in 0.1 N NaOH aqueous solution. The viscoelastic function for curdlan solution in 0.1 N NaOH aqueous solution showed the Newtonian flow at the concentration of curdlan as high as 10 wt %. On the other hand, the Newtonian flow was observed in the concentrations below 7 wt % for curdlan solution in DMSO, and the plateau region appeared beyond this concentration. It was revealed by small angle x-ray diffraction measurements that the difference in the mechanical property would be originated from the difference in the network structure. The overlapping concentration c* was calculated on the basis of the mean field theory, and disagreement between theoretical prediction and experimental result was shown. We clarified that the above disagreement can be explained by the polydispersity of the curdlan sample, assuming adequate distribution functions. The static structure of the gel prepared by adding water to curdlan solution in DMSO was investigated. It was clarified by the dynamic viscoelasticity measurement that the cross-linking density increases with increasing the water content.     

Fibrillar beta-lactoglobulin gels: Part 3. Dynamic mechanical characterization of solvent-induced systems

Oscillatory shear rheometry has been used to study the gelation of beta-lactoglobulin at ambient in 50% v/v trifluoroethanol (TFE)/pH 7 aqueous buffer and in 50% v/v ethanol (EtOH)/water at pH 2. In contrast to what was found on heating aqueous solutions at pH 2 (Part 2 of this series), a more expected "chemical gelation"-like profile was found with modulus components G' and G' ' crossing over as the gels formed and then with G' ' passing through a maximum. In addition, for the EtOH system, there was a significant modulus increase at long time, suggestive of a more complex two-step aggregation scheme. Modulus-concentration relationships were obtained for both systems by extrapolating cure data to infinite time. For the TFE gels, this data was accurately described by classical branching theory, although it could also be approximated by a constant power--law relationship. Only the latter described the modulus--concentration data for the gels in ethanol, but there were problems here of greater frequency dependence of the modulus values and much less certain extrapolation. Gel times for the TFE systems showed higher power laws in the concentration than could be explained by the branching theory in its simplest form being similar, in this respect, to the heat-set systems at pH 2. Such power laws were harder to establish for the EtOH gels as for these there was evidence of gel time divergence close to a critical concentration. Reduced G'/G'inf versus t/tgel data were difficult to interpret for the gels in ethanol, but for the TFE system they were consistent with previous results for the heat-set gels and approximated master curve superposition. The frequency and temperature dependences of the final gel moduli were also studied. In general, the networks induced by alcohols appeared more flexible than those obtained by heating.     

Gelation behaviour of konjac glucomannan with different molecular weights

The deacetylation and gelation of konjac glucomannan (KGM) following alkali addition was investigated by Fourier transform infrared, while the rheological properties of KGM with different molecular weights were studied by dynamic viscoelastic measurements in shear mode and penetration force tests. It was found that gelation occurred after significant deacetylation had taken place. Rheometrical studies revealed that KGM with different molecular weights exhibited different gelation characteristics in small amplitude oscillatory shear flow. For the samples of fractionated KGM with lower molecular weights, a decrease in both the storage shear modulus (G') and loss shear modulus (G") was observed during gelation at temperatures above 75 degrees C. It is suggested that the decrease results from the wall slip between sample and measuring geometry owing to a rapid gelation process with syneresis and/or disentanglement of molecular chains adsorbed on the surface of parallel plates from those located in the bulk. Penetration force tests were employed to confirm the occurrence of slippage and thereby no decreases in rigidity of samples were observed during gelation. For the native KGM samples decreases in G' and G" during gelation were not observed, and it is suggested that this is due to the effect of the higher molecular weight and increased solution viscosity of these samples on the gelation kinetics.     

Synergistic gel formation of xyloglucan/gellan mixtures as sudied by rheology, DSC, and circular dichroism

The gelation behavior of mixtures of tamarind seed xyloglucan (TSX) and sodium form gellan (Na-G) was investigated. The storage and loss shear moduli, G' and G', of the mixtures showed that a thermoreversible gel was obtained although each polysaccharide alone did not form a gel at experimental conditions. The viscoelastic behavior of the mixtures showed a gel formation of TSX and Na-G induced by synergistic interaction. This synergistic interaction was also revealed by differential scanning calorimetry (DSC) and circular dichroism. Although TSX alone did not show any peak in DSC curves, mixtures with only a small amount of Na-G, which by itself did not show any peak, showed a single peak. With increasing Na-G content, another peak began to appear at the same temperature at which a peak of Na-G alone appeared. Thermally induced changes in circular dichroism of the mixtures were different from those expected from the individual behavior of TSX and Na-G.     

Pine wood carboxymethylation under microwave radiation

The use of microwave radiation was proposed for the intensification of the pine wood carboxymethylation process performed without prior separation into individual components. To obtain the carboxymethylated pine wood in the form of potassium salt, the pine wood was treated with potassium hydroxide and monochloroacetic acid in 2-propanol media. The composition and properties of the resulting products were studied. Carboxymethylated cellulose was extracted from the carboxymethylated pine wood. It was found that the increase of the microwave radiation power (from 210 to 700 W) and of the duration of the first and second carboxymethylation stages (20–30 sec) increases the content of carboxymethyl groups (18.3–25.6%). The solubility of the potassium salts of the carboxymethylated pine wood (10–18%) was abnormally low as compared with that of the sodium salts at similar content of carboxymethyl groups. The viscosity of the aqueous solutions of the carboxymethylated pine wood and the carboxymethyl cellulose extracted from it was studied. It is shown that the samples obtained using the microwave radiation power of 210 W are characterized by abnormal flow curves.     

Rheological evidence of the gelation behavior of hyaluronan-gellan mixtures

It was found that solutions of calcium hyaluronate (CaHA) (0.1 to approximately 0.5 wt%) could form a gel by mixing with solutions of sodium type gellan (0.1 to approximately 0.5 wt%), although neither polymer by itself forms a gel at low concentrations (0.1 to approximately 0.5 wt% in this experiment). The rheological properties of CaHA-gellan mixtures were investigated by dynamic and steady shear measurements. Both storage shear modulus G' and loss shear modulus G' for CaHA-gellan mixtures increased with increasing time, and tended to an equilibrium value after 1 h. After reaching steady values of G' and G", the frequency dependence of G' and G' was observed. G' was always larger than G' in the accessible frequency range from 10(-2) to 10(2) rad/s. The effects of pH and calcium ions were examined. Gel formation of the mixtures was promoted by decreasing pH and adding from 0.01 to 0.1 M calcium ions, but excessive calcium ions weakened the gel.     

Rheology of synovial fluid

After a discussion of the role of synovial fluid as a joint lubricant, rheological measurements are described with both normal (healthy) synovial fluids and pathological ones. Shear stress and first normal stress difference are measured as a function of shear gradient to calculate the apparent shear viscosity eta 1 and the apparent normal viscosity psi 7 as well as an apparent shear modulus G'. It is found, that in case of diseased synoviae all rheological parameters deteriorate. Most significant changes are observed with the zero shear viscosity eta 0, the shear modulus G', and a characteristic time theta 1, which is the reciprocal of the critical shear rate Dc which determines the onset of shear thinning. The rheological deterioration of synovial fluids is explained in terms of solute structure, whereby a molecular mass of the backbone hyaluronic acid of at least 10(7) g.mol-1 is required for satisfactory function. A theory of the rheological performance of normal synovial fluid as well as its pathological deterioration is proposed.     

Gel formation and low-temperature intramolecular conformation transition of a triple-helical polysaccharide lentinan in water

The gelation behavior of the triple-helical polysaccharide lentinan fractions having different molecular weights in water at 25 degrees C were studied by using a rheometer. The analysis of concentration and molecular weight dependence of shear stress and shear viscosity showed that aqueous lentinan is a typical shear-thinning fluid, possessing potential as a viscosity control agent, and that a weak gel with entangled network structure formed. The dynamic oscillatory behavior of lentinan in the temperature range of 1-15 degrees C was also investigated by rheologic method. The storage modulus G' and complex viscosity eta* increased first with decreasing temperature, and underwent a maximum centered at 7-9 degrees C, and then decreased with further decreasing temperature. This abnormal phenomenon was ascribed to formation of rigid structure in the gel state, which was confirmed by the experimental results from micro-DSC. The micro-DSC curves showed that an endothermic peak appeared at 7-9 degrees C for lentinan in water upon heating, which was attributable to the intramolecular order-disorder structure transition similar to triple-helical polysaccharide schizophyllan. Namely, at lower temperature, the side glucose residues of lentinan (triplix II) formed a well-organized triple-helical structure (triplix I) through hydrogen-bonding with the surrounding water molecules. Moreover, this conformation transition was proved to be thermally reversible. (c) 2008 Wiley Periodicals, Inc. Biopolymers 89: 852-861, 2008.This article was originally published online as an accepted preprint. The "Published Online" date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com.     

Carboxymethylated cyclodextrins and their complexes with Pr(III) and Yb(III) as water‐soluble chiral NMR solvating agents for cationic compounds

Cyclodextrins that are indiscriminately carboxymethylated at the 2‐, 3‐, and 6‐positions are used as chiral NMR solvating agents for cationic substrates with phenyl, naphthyl, pyridyl, indoline, and indole rings. Enantiodifferentiation with the α‐, β‐, and γ‐cyclodextrin derivatives is compared. The carboxymethylated derivatives are almost always more effective as chiral NMR solvating agents for cationic substrates than native cyclodextrins. The most effective carboxymethylated cyclodextrin varies for different substrates, and at times even different resonances of the substrate. Addition of paramagnetic praseodymium(III) or ytterbium(III) to mixtures of the carboxymethylated cyclodextrin and substrate often causes enhancements in enantiomeric discrimination and facilitates measurements of enantiomeric purity. The lanthanide ion bonds to the carboxymethyl groups and causes perturbations in the chemical shifts in the NMR spectra of substrate molecules in the cyclodextrin cavity. Chirality, 2010. © 2009 Wiley‐Liss, Inc.     

Mechanical characterization of network formation during heat-induced gelation of whey protein dispersions

The formation of gel network structures during isothermal heating of whey protein aqueous dispersions was probed by mechanical spectroscopy. It was anticipated that the pathway of the sol-to-gel transition of whey protein dispersions is quite different from that of ordinary cross-linking polymers (e.g., percolation-type transition), since aqueous solutions of native whey proteins have been shown to be highly structured even before gelation, in our previous study. At 20 degrees C, aqueous dispersions of beta-lactoglobulin, the major whey protein, and those of whey protein isolate (WPI), a mixture of whey proteins, exhibited solid-like mechanical spectra, i.e., the predominant storage modulus G' over the loss modulus G", in a certain range of the frequency omega (1-100 rad/s), regardless of the presence or absence of added NaCl. The existence of the added salt was, however, a critical factor for determining transitions in mechanical spectra during gelation at 70 degrees C. beta-Lactoglobulin dispersions in 0.1 mol/dm(3) NaCl maintained the solid-like nature during the entire gelation process and, after passing through the gelation point, satisfied parallel power laws (G' approximately G" approximately omega(n)) that have been proposed for a critical gel (i.e., the gel at the gelation point) that possesses a self-similar or fractal network structure. In contrast, beta-lactoglobulin dispersions without added salt exhibited a transition from solid-like [G'(omega) > G"(omega)] to liquid-like [G'(omega) < G"(omega)] mechanical spectra before gelation, but no parallel power law behavior was recognized at the gelation point. During extended heating time (aging), beta-lactoglobulin gels with 0.1 mol/dm(3) NaCl showed deviations from the parallel power laws, while spectra of gels without added NaCl approached the parallel power laws, suggesting that post-gelation reactions also significantly affect gel network structures. A percolation-type sol-to-gel transition was found only for WPI dispersions without added salt.     

Synthesis and macrophage activation of lentinan-mimic branched amino polysaccharides: curdlans having N-Acetyl-d-glucosamine branches

N-Acetyl-d-glucosamine branches were incorporated at the C-6 position of curdlan, a linear β-1,3-d-glucan, and the resulting nonnatural branched polysaccharides were evaluated in terms of the immunomodulation activities in comparison with lentinan, a β-1,3-d-glucan having d-glucose branches at C-6. To incorporate the amino sugar branches, we conducted a series of regioselective protection-deprotections of curdlan involving triphenylmethylation at C-6, phenylcarbamoylation at C-2 and C-4, and detriphenylmethylation. Subsequent glycosylation with a d-glucosamine-derived oxazoline, followed by deprotection gave rise to the branched curdlans with various substitution degrees. The products exhibited remarkable solubility in both organic solvents and water. Their immunomodulation activities were determined using mouse macrophagelike cells, and the secretions of both the tumor necrosis factor and nitric oxide proved to be significantly higher than those with lentinan. These results conclude that the amino sugar/curdlan hybrid materials are promising as a new type of polysaccharide immunoadjuvants useful for cancer chemotherapy.     

Clinical assessment of rheumatic diseases using viscoelastic parameters for synovial fluid

For the first time it is clearly exhibited that synovial fluid (SF) is thixotropic. Although no hysteresis loops were observed for SF, not even at high shear rates, thixotropy may be exhibited by measuring the rate of recovery after extensive shearing. The rebuilding of the structure in a small-amplitude oscillatory state following the high-shear-rate state reveals the thixotropic behaviour. Five different viscoelastic parameters for various synovial fluids (SF) were obtained using oscillatory rheometry. It was also shown that for SF in the low frequency range, corresponding to a knee joint almost at rest, the shear loss modulus G" is greater than the shear storage modulus G', since the system is allowed to dissipate energy at rest. However, with movement, G' increases and eventually becomes greater than G" at a characteristic frequency above which the system has insufficient time to dissipate energy and hence responds as an elastic body. This functional behaviour, characteristic for normal SF, broke down in the SF of rheumatoid arthritis. It was also absent in the SF of knee joints with meniscus lesions and ligament defects.     

Flow properties of N-(carboxymethyl) chitosan aqueous systems in the sol and gel domains

The flow behaviour and the viscoelastic properties of N-(carboxymethyl) chitosan aqueous systems in the sol and gel domains have been investigated by means of dynamic, steady and transient shear techniques. For polymer concentrations Cp up to 1%, a typical response of moderately concentrated polymer solutions was observed under continuous and oscillatory shear conditions. No time-dependent properties were detected during transient shear experiments. On the other hand, for all the samples with Cp greater than 1%, the rheological properties were more similar to those of a weak gel system. The continuous shear flow behaviour was of the plastic type and the viscoelastic quantities G' and G" were parallel to each other and slightly dependent on the frequency of oscillation omega. Stress overshoots were observed during transient shear experiments, and the kinetics of the structural breakdown and build-up processes were found to be dependent both on the polymer concentration and the applied shear rate.     

Viscoelastic evaluation of topical creams containing microcrystalline cellulose/sodium carboxymethyl cellulose as stabilizer

The purpose of this study was to examine the viscoelastic properties of topical creams containing various concentrations of microcrystalline cellulose and sodium carboxymethyl cellulose (Avicel(R) CL-611) as a stabilizer. Avicel CL-611 was used at 4 different levels (1%, 2%, 4%, and 6% dispersion) to prepare topical creams, and hydrocortisone acetate was used as a model drug. The viscoelastic properties such as loss modulus (G"), storage modulus (G'), and loss tangent (tan delta) of these creams were measured using a TA Instruments AR 1000 Rheometer and compared to a commercially available formulation. Continuous flow test to determine the yield stress and thixotropic behavior, and dynamic mechanical tests for determining the linear viscosity time sweep data, were performed. Drug release from the various formulations was studied using an Enhancer TM Cell assembly. Formulations containing 1% and 2% Avicel CL-611 had relative viscosity, yield stress, and thixotropic values that were similar to those of the commercial formulation. The elastic modulus (G') of the 1% and 2% formulation was relatively high and did not cross the loss modulus (G"), indicating that the gels were strong. In the commercial formulation, G' increased after preshearing and broke down after 600 seconds. The strain sweep tests showed that for all formulations containing Avicel CL-611, the G' was above G" with a good distance between them. The gel strength and the predominance of G' can be ranked 6% > 4% > 2%. The strain profiles for the 1% and 2% formulations were similar to those of the commercial formulation. The delta values for the 1% and 2% formulations were similar, and the formulations containing 4% Avicel CL-611 had lower delta values, indicating greater elasticity. Drug release from the commercial preparation was fastest compared to the formulations prepared using Avicel CL-611, a correlation with the viscoelastic properties. It was found that viscoelastic data, especially the strain sweep profiles of products containing Avicel CL-611 1% and 2%, correlated with the commercial formulation. Rheological tests that measure the viscosity, yield stress, thixotropic behavior, other oscillatory parameters such as G' and G" are necessary tools in predicting performance of semisolids.     

Mechanical properties of actin filament networks depend on preparation, polymerization conditions, and storage of actin monomers.

This study investigates possible sources for the variance of more than two orders of magnitude in the published values for the shear moduli of purified actin filaments. Two types of forced oscillatory rheometers used in some of our previous work agree within a factor of three for identical samples. Polymers assembled in EGTA and Mg2+ from fresh, gel-filtered ATP-actin at 1 mg/ml typically have an elastic storage modulus (G') of approximately 1 Pa at a deformation frequency of 0.1-1 Hz. G' is slightly higher when actin is polymerized in KCl with Ca2+ and Mg2+. Gel filtration removes minor contaminants from actin but has little effect on G' for most preparations of actin from acetone powder. Storage of actin monomers without frequent changes of buffer containing fresh ATP and dithiothreitol can result in changes that increase the G' of filaments by more than a factor of 10. Frozen storage can preserve the properties of monomeric actin, but care is necessary to prevent protein denaturation or aggregation due to freezing or thawing.     

Laser photolysis of carboxymethylated chitin derivatives in aqueous solution. Part 1. Formation of hydrated electron and a long-lived radical

Laser photolysis experiments on carboxymethylated chitin derivatives, such as carboxymethyl chitin (CM-chitin) and carboxymethyl chitosan (CM-chitosan), in aqueous solution by a 248 nm excimer laser were carried out for the first time. The transient absorption spectra of photolyzed CM-chitin or CM-chitosan solutions revealed a strong band with the maximum at 720 nm, which was assigned to the hydrated electron (eaq-). In the presence of argon, the eaq- decays by reacting with CM-chitin or CM-chitosan, and the rate constants are (6.1 +/- 0.1) x 10(7) M(-1) s(-1) and (3.7 +/- 0.1) x 10(7) M(-1) s(-1), respectively. Long-lived radicals with relatively weak absorption intensity were detected in the near-UV region. The absorption band was not notably characteristic and showed only an increasing absorption toward shorter wavelengths. It is similar to the signal of *CM-chitin or *CM-chitosan macroradicals formed by the reaction of CM-chitin or CM-chitosan with an OH* radical. It was assigned to *CM-chitin- or *CM-chitosan- macroradicals formed by eaq- + CM-chitin or CM-chitosan reaction. CM-chitin aqueous solutions were further examined by pulse radiolysis in order to confirm the site of the long-lived radical.