Heat-set Gels Research Articles

Rheological study of the effect of Cassia javanica galactomannans on the heat-set gelation of a whey protein isolate at pH 7

The effect of Cassia gum galactomannan on the thermal gelation of a whey protein isolate (WPI) at 80 °C has been monitored by rheological dynamic measurements at fixed frequency and the mechanical spectra of the gels have been recorded at 80 °C and after quenching to 20 °C. The mechanical spectra were analysed quantitatively by fitting a Cole–Cole model to the storage and loss compliance versus frequency data. The concentration of WPI was 110 g/l and that of Cassia gum varied from 0 to 11 g/l. At low concentration, the presence of the galactomannan enhanced the aggregation rate and the strength of the protein gel but did not modify intrinsically the gelation process and the structure of the system. At higher galactomannan concentrations, the gels displayed substantially lower moduli values and the shape of the mechanical spectra was modified. This was tentatively interpreted as the consequence of a depletion-induced liquid–liquid phase separation occurring during the course of the aggregation process, responsible for the downturn observed on the loss modulus versus time curves monitored at 80 °C.

Thermally induced structural changes in glycinin, the 11S globulin of soya bean ( Glycine max)—an in situ spectroscopic study

The thermal denaturation behaviour of glycinin solutions has been studied in situ as a function of ionic strength using various spectroscopic methods . Changes in secondary structure occurred at temperatures above 60 °C, well before the onset of gelation. Even after heating to 95 °C, much of the native β-sheet structure of glycinin was retained, as indicated by the amide I peak maximum at 1635 cm −1 in the Fourier transformed infrared (FT-IR) spectrum. This was accompanied by an increase in the 1625 cm −1 band, indicative of the formation of intermolecular β-sheet associated with protein aggregation. Nuclear magnetic resonance (NMR) spectroscopy confirmed the presence of highly mobile regions in glycinin comprising predominantly of Gln and Glu residues, corresponding to mobile regions previously identified by crystallographic studies. There was also evidence of a hydrogen-bonded structure within this mobile region, which may correspond to an α-helical region from Pro 256 to (or just before) Pro 269 in proglycinin. This structure disappeared at 95 °C, when heat-set gel formation occurred, as indicated by a sudden broadening and weakening of the NMR signal. Otherwise the NMR spectrum changed little during heating, emphasising the remarkable thermal stability of glycinin. It is proposed that during heating the core β-barrel structure remains intact, but that the interface between the β-domains melts, revealing hydrophobic faces which may then form new structures in a gel-network. As Cys 45, which forms the disulfide with Cys 12 linking the acidic and basic polypeptides, is found in this interface, such a rearrangement of the individual β-domains could be accompanied by cleavage of this disulfide bond, as is observed experimentally. Such information contributes to our understanding the aggregative behaviour of proteins, and hence develops knowledge-based strategies for controlling and manipulating it.

Heat-induced flocculation of microparticulated whey proteins (MWP); consequences for mixed gels made of MWP and β-lactoglobulin

Flocculation/aggregation processes occurred when suspensions of microparticulated whey proteins (MWP) were heated (or sheared). Large flocs were observed above both a critical volume fraction ( Φ MWP>0.099) and a critical temperature ( T>60 °C). These large structures combined at high volume fraction to form a coarse network which developed a liquid-like viscoelastic response. The rheological and structural consequences of such instabilities on mixed heat-set gels made of MWP and β-lactoglobulin (BLG) were studied. In particular, cooperative effects at low MWP volume fraction were observed. In addition, steric hindrance effects or thermodynamic incompatibility could occur between the two components at high MWP volume fraction leading to micro-phase separation in the mixed system. The theory of van der Poel failed to describe the experimental results. This was attributed to the strongly flocculated state of the MWP system and to the chemical affinity of MWP for the BLG network. These views were supported by diffusing wave spectroscopy measurements and confocal scanning laser microscopy observations.

Origin of multiexponential T(2) relaxation in muscle myowater.

To obtain a further understanding of the nature of the multiexponential T(2) relaxation seen in muscle tissue water (myowater), relaxation measurements were carried out on whole, minced, and homogenized pork of three different qualities with regard to water-holding capacity (normal, red soft exudative, and dark firm dry). Whole, minced, and homogenized pork all resulted in multiexponential T(2) relaxation (three components) independently of the quality, even though microscopic studies on homogenized meat revealed considerable disruption of the macroscopic structure. This states that the relaxation behavior in meat cannot be explained by intra-/extracellular compartmentalization of the water as suggested in earlier studies. Subsequent studies of T(2) relaxation in either whole meat, where the structure integrity was changed by the introduction of dimethyl sulfoxide (membrane disruption) or urea (protein denaturation), or minced meat with added NaCl (inter-/intraprotein interactions) lead to the suggestion that in whole meat (i) the fastest relaxation component reflects water tightly associated with macromolecules, (ii) the intermediate relaxation component reflects water located within highly organized protein structures, for example, water in tertiary and/or quaternary protein structures and spatials with high myofibrillar protein densities including actin and myosin filament structures, and (iii) the slowest relaxation component reflects the extra-myofibrillar water containing the sarcoplasmatic protein fraction. Finally, relaxation patterns in heat-set gels of superprecipitated actomyosin and bovine serum albumin similar to that identified in whole meat support the proposed nature of T(2) relaxation in muscle myowater.

Heat-Set Bovine Serum Albumin−Sodium Dodecyl Sulfate Gels Studied by Fluorescence Probe Methods, NMR, and Light Scattering

In this work, concentrated protein−surfactant solutions and their corresponding heat-set gels were studied by fluorescence probe methods, NMR, and light scattering. Bovine serum albumin (BSA) was used as the protein, and sodium dodecyl sulfate (SDS) as the surfactant. Heating concentrated BSA solutions gives turbid gels. Heat-set BSA−SDS gels are transparent. From fluorescence measurements it was concluded that SDS forms micelle-like clusters on BSA, both in solution and in the corresponding heat-set gel. Aggregation numbers were found to be similar in solution and gel. Also, I1/I3 values in solution and gel were similar. 2H NMR relaxation measurements of specifically deuterated SDS at the α-carbon position next to the headgroup were performed, and the longitudinal relaxation rates R1 were found to be the same in solution and gel. High values for the transverse relaxation rate R2 (indicating slow motions of SDS bound to large aggregates) were obtained, and the largest R2 value was found for the gel. Dynam...

Heat-induced gelation of globular proteins: part 3. Molecular studies on low pH β-lactoglobulin gels

Heat-set gels and aggregates from β-lactoglobulin (β-Lg), one of the major globular proteins from milk, have been studied on a molecular distance scale using negative-staining transmission electron microscopy (TEM), wide-angle X-ray diffraction (WAXD), and Fourier transform infrared spectroscopy (FTIR). The microscopy showed long linear aggregates forming in solutions at pH 2 (and sometimes 2.5) after prolonged heating. While there appeared to be no differences in aggregates formed under these conditions in H 2O as compared with D 2O, at all other pH and pD values, and in the presence of added salt, much shorter linear aggregates were formed. These became slightly more extended the further the pH was removed from pI. Wide-angle X-ray diffraction (WAXD) showed a diffuse β-sheet halo at 2 θ=19° in patterns for both dried native and aggregated protein (irrespective of pH) with only a small change (sharpening) of this feature on heat treatment. Solution FTIR spectra, measured at pD=2, 2.5, 3, and 7, during heating, indicated shoulder development at 1612 cm −1 in the carbonyl-stretching Amide I region diagnostic of a modest increase in intermolecular β-sheet. In terms of the shoulder size, no distinctions could be made between acid and neutral aggregate structures. At all pHs, β-lactoglobulin showed only limited secondary and tertiary structural changes in aggregation, in contrast to previous studies of insulin aggregation, where highly ordered crystalline fibrils were indicated. The current work has implications both in structural studies of food biopolymers and in ongoing studies of pathological protein self-assembly in disease states, such as spongiform encephalopathies.

Unfolding and Intermolecular Association in Globular Proteins Adsorbed at Interfaces

The conformational transitions that occur on heating solutions of globular proteins, unfolding and aggregation, were compared with the analogous transitions undergone by proteins adsorbed at interfaces. Fourier transform infrared spectrometry in solution and in the attenuated total reflection geometry revealed, for the globular proteins hen egg lysozyme and bovine serum albumen, both qualitative and quantitative differences between the transitions as they occur in bulk and adsorbed at an interface. In the bulk, unfolding is a sharp transition, followed sequentially on further heating by the relatively sharp onset of the intermolecular association associated with heat set gelation. In contrast, for adsorbed proteins, we found that both processes occur simultaneously over a wide range of temperatures. Proteins were more structurally stable adsorbed at a relatively hydrophilic, solid surface than at a liquid, hydrophobic surface; in the latter case, onset temperatures for both unfolding and intermolecular as...

“Ordered” structure in solutions and gels of a globular protein as studied by small angle neutron scattering

Small-angle neutron scattering measurements were used for structural investigation of β-lactoglobulin solutions and heat-set gels in conditions of strong double layer repulsions. At pH 9 and low ionic strength, a correlation peak was observed on the scattering curves of the solutions whatever the protein concentration C used (in the range C = 0.02–0.10 g/mL). The wave vector value qmax corresponding to these maxima scaled as C0.25. This exponent value is in agreement with those reported in the literature for other globular proteins in the same concentration range. Increasing the ionic strength decreased the peak which vanished without changing position at 0.1M NaCl. This polyelectrolyte-like behaviour suggests a local structure in the protein solution due to double layer repulsions. In the case of heat-set aggregates and gels (0.02–0.13 g/mL) formed at pH 9 and low ionic strength, a peak in the scattering curves was also observed indicating that even after gelation a correlation is still present; qmax varied as C0.5. As in the case of the solutions, the correlation peak decreased with increasing ionic strength, and it vanished at 0.06M NaCl. The dilution of the aggregates in order to determine their intraparticle structure factor showed that the correlations were lost and that the aggregates displayed the same internal structure as the elementary subunit in the gels. At high ionic strength, fractal structures of the aggregates down to a length scale of about 40 A were observed with df = 1.3–1.75 ± 0.05, increasing with protein concentration. Subsequent dilution didn't change the fractal dimension of these structures. © 1996 John Wiley & Sons, Inc.

Structure and rheology of heat-set gels of globular proteins

The structure and the rheology of systems resulting from heating at 80°C isoelectric solutions of bovine serum albumin (BSA) in the concentration range 10–200mg/ml were studied. Small-angle neutron scattering measurements view the systems as being formed of large aggregates of micrometric size with a close packed arrangement of denatured protein molecules. No indication of a fractal structure stands out. The viscoelastic behaviour is linear up to about 5% strain, except in the BSA concentration range 30–90mg/ml where the linearity limit is below 1% strain. The viscoelastic response was analysed in the linear domain, or as close as possible to it, by combining the results of dynamic and creep recovery measurements. The dependence on concentration of the steady state viscosity, of the steady state compliance, and of the average retardation time shows a marked change around a concentration C0∼50mg/ml, corresponding probably to a percolation threshold.

Effect of Pressure/Heat Combinations on Blue Whiting (Micromesistius poutassou) Washed Mince: Thermal and Mechanical Properties

Washed ground muscle of blue whiting was comminuted at low ionic strength and low temperature, and the batter was subjected to different pressure/heat processings. The functionality induced in the combined processed gels was evaluated by water holding and several rheological and fracture properties and compared to that obtained for traditional heat-induced gel. Ultrastructure was viewed by scanning electron microscopy. Protein denaturation was assessed by differential scanning calorimetry (DSC), and the thermal behavior of pressurized gels revealed that pressure and temperature were interdependent. At nondenaturing temperatures, the higher the pressure, the larger was the protein unfolding effect. At denaturing temperatures, pressure prevented fish protein from subsequent thermal denaturation. DSC demonstrated that pressurized samples underwent both types of pressure effects at the same time. It transmitted a mixed character to the pressure-induced gels, which displayed a rheological behavior intermediate between cold-set and heat-set gels. Keywords: High pressure/thermal treatments; cold gelation; heat gelation; blue whiting (Micromesistius poutassou) washed mince; protein denaturation; mechanical properties; ultrastructure; DSC; TPA; SEM

Effects of Limited Proteolysis on Gelation and Gel Properties of Whey Protein Isolate

Whey protein isolate was hydrolyzed by three proteolytic enzymes, porcine trypsin, a Bacillus subtilis protease (Neutrase®), and a Bacillus licheniformis protease. The heat-induced gelation properties of the hydrolysates were investigated as a function of pH, whey protein concentration, and the degree of hydrolysis, which was determined by osmometry. At pH 5.2, all control solutions (not hydrolyzed) containing 3 to 12% whey protein formed gels on heating to 80°C, but, at pH 3.0 and 7.0, only the most concentrated solution gelled. After hydrolysis by the B. licheniformis protease, all whey protein solutions at pH 7.0 formed gels, and the strength of the 12% whey protein gel was 10 times higher than that of the corresponding control gel. Trypsin hydrolysis prevented gelation of the 12% whey protein solution at pH 3.0 and 7.0 and caused a pronounced weakening of the gels formed at pH 5.2. Hydrolysis by Neutrase® did not change the ability to form a gel, but the gels formed at pH 5.2 and 7.0 were weaker and, at pH 3, were slightly stronger than the control gels. Gelation properties of whey proteins, thus, can be manipulated by limited proteolysis. Of special interest is the possibility of making very strong heat-set gels in the neutral pH range.

Conformational changes in adsorbed proteins

Attenuated total reflection Fourier transform infrared (FTIR-ATR) spectroscopy was used to follow the adsorption of the globular protein lysozyme from aqueous (D 2 O) solution onto a silicon surface. The effect of changing temperature and/or pD on the structure of protein in the adsorbed layer is observed and compared with the effect of these changes on concentrated solutions. We show that the same spectral changes as those occurring in the heat-set gelation of concentrated solutions of globular proteins can also be observed in adsorbed proteins, but at lower temperatures. We suggest that this is due to the presence of the surface having the effect of promoting molecular structural rearrangements.

Spray‐Dried Soy Protein Isolate Solubility, Gelling Characteristics, and Extractable Protein as Affected by Antioxidants

ABSTRACTWith the addition of antioxidants, spray‐dried soy protein isolates (SPI) exhibited increased solubilities by 8% over the control in 0.1M NaCl. Increased solubilities corresponded to a 15% and 8% reduction in protein oxidation as determined by protein carbonyl contents. The greatest differences in solubility between a spray‐dried SPI with antioxidant (66% protein solubility) and the control (54% protein solubility), as well as the minimum protein solubility, occurred in 0.2M NaCl. Following the initial solubilization step in SPI processing, amount of extracted proteins was increased by 4.5% over the control with addition of antioxidants. Gel fracturability, hardness and adhesiveness for heat set gels of 12% SPI processed with added antioxidants were increased by 26.3%, 23.5% and 24.6%, respectively.

A comparative rheological study of heat and high pressure induced whey protein gels

The rheological properties of whey protein concentrate (WPC) gels induced by high pressure (4000 bar/30 min), were compared to those induced by heat (80 °C/30 min) at protein concentrations ranging from 110 up to 183 g/liter. Oscillation measurements at 1 Hz and 0.001 strain showed the highest storage and loss moduli for heat set gels, while creep experiments at a stress level of 40 Pa gave larger sample deformations for high pressure induced gels. Relaxation experiments performed at 17 and 33% deformation were characterized by a higher force decay as a function of time for the high pressure gels, while during compression the compression modulus was always higher in the case of heat set gels. Electron microscopy showed a higher level of cross links in the heat induced gels; high pressure generated a more porous network with a lower amount of intermolecular cross links.

Evaluation of Protease Inhibitors in Pacific Whiting Surimi

Bovine plasma powder, whey protein concentrate and would potato powder were compared with purified bovine serum albumin, chicken egg albumin and a-lactalbumin as protease inhibitors in Pacific whiting surimi. Spectrophotometric and sodium dodecyl sulfate gel electrophoretic analyses demonstrated that the extent of proteolysis in samples containing the purified albumins was, in all cases, equal to the control. The same test also showed that at equal concentrations, whole plasma powder was a more effective inhibitor of proteolysis than whey protein concentrate or potato powder. However, electrophortic gels and punch test scores indicated that at higher concentrations, whey protein concentrate and potato powder could substitute for plasma powder in directly heat set gels, but not in suwari gels.

Muscle protein gelation at low ionic strength

The process of muscle protein gelation was monitored by dynamic rheological techniques. Gels formed from beef myofibrillar proteins extracted at low ionic strengths ( Γ 2 = 0·02 m ) were characterized as a function of concentration and Γ 2 of the pregelled sol. Network development was studied in real time by measuring changes in the elastic ( G′) and viscous ( G″) components and tan δ as a function of increasing temperature (20–80°C). Rheological thermograms indicated that the apparent onset of heat-initiated gelation occurred at ∼50°C; this thermal event was unaffected significantly by protein concentration. The concentration dependence of G′ for the heat-set gel followed approximately the relationship G′ α C 2 (in this case, G′ α C 2·2) reported for myosin and other gelling protein species. At low Γ 2 (0·01 m), no indication of SS bond formation previously reported for myosin head-head association was seen, but increasing this parameter (0·11 and 0·31 m) led to a thermal event ( dG′ dT plot) possibly coinciding with such an interaction. The point of increase in G′ related to myosin tail-tail interactions was unaffected by ionic strength. However, increasing the ionic strength (0·31 and 0·61 m) did produce more elastic (increased G′) gels. Comparison with previously reported data for the gelation of pure myosin indicated that the presence of non-gelling regulatory species may influence the mechanisms of network development.

Thermally induced globular protein gels: peculiarities of formation, melting, and restoration of gels of different structure

The results of the calorimetric study of the globular protein heat-set gels performed by the authors during recent years have been generalized in the present paper. The conformational variations have been studied which took place after the denaturation in globular protein concentrated solutions with continuous heating in the temperature range 20–140°C. The effects of pH, the ionic strength of the solution and the protein concentration on the formation of heat-set gels and their melting have been studied in parallel with visual observations of the solution states under the same conditions. Four different types of globular protein gels have been discovered (e.g. lysozyme), and the ranges of their existence have been determined. The results obtained have shown that two types of gel can be formed within the pH range 1.7–9.0 at fixed ionic strength and protein concentration: clear meltable gel (type I) and a turbid one which cannot melt (type II). The heating thermograms of the type I gel demonstrate besides denaturation heat absorption an additional high temperature maximum, which is the calorimetric manifestation of cooperative transformation proceeding at the melting of this gel. The incipient stage of the melting gel formation is described at critical values of pH, ionic strength and protein concentratión. The adjustment of protein aggregation in solution with the help of pH, ionic strength and concentration has been shown to broaden considerably the pH range in which thermoreversible gel is formed and to provide such changes in the structure of the turbid gel which make it meltable (type III). This results in the appearance of a high temperature maximum in the thermograms of such a gel. The melting of such a gel induces cooperative stratification of the system into the collapsed gel (type IV) and water.

Differential scanning calorimetric study of heat-set gels of globular protein

A calorimetric study of concentrated solutions of globular protein lysozyme has been carried out in the temperature range 20–140°C. The effects of pH, ionic strength of the solution, and protein concentration on thermotropic gelation have been studied. Calorimetric studies were complemented by the visual control of solution state and properties in identical heating conditions. The results obtained have shown that two types of gel can be formed within the pH range 1.7–9.0: a clear meltable gel (type I) and a turbid one (type II), which does not melt. The thremograms of the type I gel heating demonstrate that, in addition to the denaturation heat absorption, there also exists a high temperature maximum (HTM), which is the calorimetric manifestation of cooperative transformation accompanying the melting of this gel. The thermodynamic melting parameters of heat-set lysozyme gel are obtained. The incipient stage of type I gel formation is observed at critical values of pH, ionic strength and protein concentration. It is shown that by adjusting the protein aggregation in solution by the use of these parameters, one can expand considerably the pH range in which heat-gel is formed, and provide such changes in the structure of branched gel hich bring up its melting. This results in the appearance of HTM in the thermograms of such a gel.

Correlation of the Rheological Behavior of Egg Albumen to Temperature, pH, and NaCl Concentration

Rheological parameters of viscosity, gel strength, and elasticity were determined on heat-set egg albumen gels over various treatment combinations ranging from 65-90°C, pH 6.4-9.6, and NaCl concentrations of 0.0-0.1M added NaCl. Maximum viscosity was measured at a treatment combination of 77.5°C, pH 8.00, and 0.1M NaCl. Elasticity and gel strength were highest in gels with a treatment combination of 85.2°C, pH 9.0, and 0.08M NaCl. Temperature had the greatest effect on all three rheological parameters. Gels heated above 80°C were of unusual character, exhibiting syneresis and shrinkage.

Infrared and laser-Raman spectroscopic studies of thermally-induced globular protein gels.

Infrared and laser-Raman spectroscopy have been used to follow secondary structure changes during the heat-set gelation of a number of aqueous (D2O) globular protein solutions. Measurements of the infrared Amide I' absorption band around 1650 cm-1, for BSA gels of varying clarity and texture, have shown that the very considerable variations in network structure underlying these materials are not reflected in obvious differences in secondary structure. In all cases aggregation is accompanied by development of beta-sheet of a kind common in fibrous protein systems, but for BSA at least this does not appear to vary significantly in amount from one gel type to another. Infrared studies of gels formed from other protein systems have confirmed this tendency for beta-sheet to develop during aggregation, and the tendency is further substantiated by laser-Raman evidence which provides the extra information that in most of the examples studied alpha-helix content simultaneously falls. From these, and other observations, some generalisations are made about the thermally-induced sol-to-gel transformations of globular proteins.