Globular Protein Gels Research Articles

Influence of amyloid fibril length and ionic strength on WPI-based fiber-hydrogel composites: Microstructural, rheological and water holding properties

One of the possible advantages of protein fibers in composites is that they can interact with the continuous gel matrix as active fillers at neutral pH. This study aimed to investigate the impacts of short (SAF) and long amyloid fibrils (LAF) and ionic strength (0–150 mM NaCl) on the whey protein isolate (WPI)-based fiber-hydrogel composites. The water holding capacity of the composite gels more depended on the gel coarseness rather than their elasticity. Significant differences were observed between the rheological data of the SAF-WPI and LAF-WPI composites, and the maximum elastic modulus (G′) was achieved at 100 mM NaCl where the LAF-WPI composites outperformed the SAF-WPI ones in enhancing G′ and hardness. Nevertheless, the elasticity and hardness of the composites dramatically dropped at 150 mM NaCl due to the change in the gel microstructure from fine strand to particulate, according to CLSM and SEM images. Interestingly, the trend in average particle size changed at 150 mM NaCl for the SAF- and LAF-WPI soluble aggregates compared to the WPI soluble aggregates. The effects of amyloid fibril and NaCl concentration on the protein aggregates as the initial building blocks of the gel were confirmed by size exclusion chromatography. These results indicated that the properties of composite gel were related to the balance between aggregate creation and the development of cross-links between them during gelation. The findings of the current study could be exploited to predict the functional behavior of globular protein gels containing amyloid fibrils with different lengths at various NaCl concentrations.

Understanding the function of water during the gelation of globular proteins by temperature-dependent near infrared spectroscopy.

Water plays an indispensable role in the gelation of proteins, but its function still remains unclear. In this work, the variation of water species with the structural changes of globular proteins was investigated using temperature-dependent near infrared (NIR) spectroscopy. Ovalbumin (OVA) was used as a model protein, which forms a gel-like structure as the temperature increases through three phases, i.e., phase I (native), phase II (molten globule state), and phase III (gel state). The structural change and the content variation of different water species in the three phases of gelation were analyzed by two-dimensional correlation NIR spectroscopy and Gaussian fitting. A decrease in the water species with two hydrogen bonds (S2) was found and the change follows the same phases as OVA. In the first two phases, the change occurs after those of other water species but in the third phase, the change is faster than that of free water species. The result indicates that in the native and molten globule states, S2 is located in the hydration shell of OVA to maintain the stability of the protein structure, and then in the gel state, high temperature weakens the hydrogen bonding of S2 and leads to the destruction of the hydration shell, making OVA clusters form a gel structure.

Heat- and pH-induced BSA conformational changes, hydrogel formation and application as 3D cell scaffold

Aggregation and gelation of globular proteins can be an advantage to generate new forms of nanoscale biomaterials based on the fibrillar architecture. Here, we report results obtained by exploiting the proteins' natural tendency to self-organize in 3D network, for the production of new material based on BSA for medical application. In particular, at five different pH values the conformational and structural changes of the BSA during all the steps of the thermal aggregation and gelation have been analyzed by FTIR spectroscopy. The macroscopic mechanical properties of these hydrogels have been obtained by rheological measurements. The microscopic structure of the gels have been studied by AFM and SEM images to have a picture of their different spatial arrangement. Finally, the use of the BSA hydrogels as scaffold has been tested in two different cell cultures.

Water Holding as Determinant for the Elastically Stored Energy in Protein-Based Gels.

To evaluate the importance of the water holding capacity for the elastically stored energy of protein gels, a range of gels were created from proteins from different origin (plant: pea and soy proteins, and animal: whey, blood plasma, egg white proteins, and ovalbumin) varying in network morphology set by the protein concentration, pH, ionic strength, or the presence of specific ions. The results showed that the observed positive and linear relation between water holding (WH) and elastically stored energy (RE) is generic for globular protein gels studied. The slopes of this relation are comparable for all globular protein gels (except for soy protein gels) whereas the intercept is close to 0 for most of the systems except for ovalbumin and egg white gels. The slope and intercept obtained allows one to predict the impact of tuning WH, by gel morphology or network stiffness, on the mechanical deformation of the protein-based gel. Addition of charged polysaccharides to a protein system leads to a deviation from the linear relation between WH and RE and this deviation coincides with a change in phase behavior.

Microstructure and rheology of globular protein gels in the presence of gelatin

The microstructure and rheological response of globular protein gels (whey protein isolate (WPI) and soy protein isolate (SPI)) in the presence of gelatin (type A, type B and hydrolyzed type A) was investigated. Microstructural information was obtained using a combination of confocal laser scanning microscopy (CLSM) and spin echo small-angle neutron scattering (SESANS). Addition of gelatin led to a coarsening of the globular protein gel structure and to a reduction in storage modulus of globular protein gels. The presence of hydrolyzed gelatin on the other hand did not induce these changes in the globular protein gels. Results were obtained at conditions where proteins only interact via hard body interactions. For these conditions it was concluded that the ratio of molecular sizes is the most important determinant for the occurrence or absence of rheological and microstructural changes in globular protein gels prepared in the presence of gelatin.

Relation between the gel structure and the mobility of tracers in globular protein gels

Diffusion of fluorescent-labeled dextran with different molecular weights was investigated in β-lactoglobulin (β-lg) solutions and gels over a wide range of salt and protein concentrations at pH 7 by combining confocal laser scanning microscope (CLSM) with fluorescence recovery after photobleaching (FRAP). Effects of the protein concentration, the salt concentration and the tracer size were investigated in detail. Diffusion in turbid heterogeneous gels formed at 0.2M NaCl depended weakly on the probe size and the protein concentration and remained close to that in unheated solutions. A strong decrease of the diffusion coefficient with increasing tracer size and protein concentration was observed in more homogeneous gels formed at lower salt concentrations. Larger dextran chains were trapped in transparent gels formed at NaCl concentration below 0.1M. The present investigation complements an earlier study of tracer diffusion of larger spherical probes in β-lg gels using multi-particle tracking.

Fractal Structures of the Hydrogels Formed in Situ from Poly(N-isopropylacrylamide) Microgel Dispersions

Dispersions of poly(N-isopropylacrylamide) (PNIPAM) microgel thermally gel in the presence of inorganic salts. The in situ-formed hydrogels, with a network of soft particles, represent a new type of colloidal gels. Here, their fractal structures were determined by rheological measurements, using the models of both Shih et al. and Wu and Morbidelli. According to the definition of Shih et al., the colloidal PNIPAM gels fall into the strong-link regime. Yet the calculated fractal dimension of the floc backbone, x, yielded unrealistic negative values, suggesting this model is inapplicable for the present system. The Wu-Morbidelli model gives physically sounder results. According to this model, the strengths of the inter- and intrafloc links are comparable, and the in situ-formed gels are in the transition regime. The fractal dimension, d(f), of the hydrogel decreases from ∼2.5 to ∼1.8 when the heating temperature increases from 34 to 40 °C. The d(f) values suggest different aggregation mechanisms at different temperatures, that is, a reaction-limited one accompanied by rearrangement at low temperature, a typical reaction-limited one at the intermediate temperature, and a diffusion-limited one at high temperature. With increasing salt concentration, the d(f) of the hydrogel decreases from ∼2.1 to ∼1.7, suggesting the aggregation mechanism changes from reaction-limited to diffusion-limited. The effects of both temperature and salt concentration can be explained by the changes in the interactions among the microgel particles. The thermogellable PNIPAM microgel dispersions may serve as a model system for the study of heat-induced gelation of globular proteins.

Effects of Heating Rate and pH on Fracture and Water‐Holding Properties of Globular Protein Gels as Explained by Micro‐Phase Separation

The effect of heating rate and pH on fracture properties and held water (HW) of globular protein gels was investigated. The study was divided into 2 experiments. In the 1st experiment, whey protein isolate (WPI) and egg white protein (EWP) gels were formed at pH 4.5 and 7.0 using heating rates ranging from 0.1 to 35 °C/min and holding times at 80 °C up to 240 min. The 2nd experiment used one heating condition (80 °C for 60 min) and probed in detail the pH range of 4.5 to 7.0 for EWP gels. Fracture properties of gels were measured by torsional deformation and HW was measured as the amount of fluid retained after a mild centrifugation. Single or micro-phase separated conditions were determined by confocal laser scanning microscopy. The effect of heating rate on fracture properties and HW of globular protein gels can be explained by phase stability of the protein dispersion and total thermal input. Minimal difference in fracture properties and HW of EWP gels at pH 4.5 compared with pH 7.0 were observed while WPI gels were stronger and had higher HW at pH 7.0 as compared to 4.5. This was due to a mild degree of micro-phase separation of EWP gels across the pH range whereas WPI gels only showed an extreme micro-phase separation in a narrow pH range. In summary, gel formation and physical properties of globular protein gels can be explained by micro-phase separation. The effect of heating conditions on hardness and water-holding properties of protein gels is explained by the relative percentage of micro-phase separated proteins. Heating rates that are too rapid require additional holding time at the end-point temperature to allow for full network development. Increase in degree of micro-phase separation decreases the ability for protein gels to hold water.

How Micro‐Phase Separation Alters the Heating Rate Effects on Globular Protein Gelation

This study was conducted to determine how the combination of heating rate and pH can be used to alter viscoelastic properties and microstructure of egg white protein and whey protein isolate gels. Protein solutions (1% to 7% w/v protein, pH 3.0 to 8.5) were heated using a range of heating rates (0.2 to 60 °C/min) to achieve a final temperature of 80 °C. The gelation process and viscoelastic properties of formed gels were evaluated using small strain rheology. Single phase or micro-phase separated solution conditions were determined by confocal laser scanning microscopy. In the single phase region, gels prepared by the faster heating rates had the lowest rigidity at 80 °C; however, a common G' was achieved after holding for 4 h at 80 °C . On the other hand, under micro-phase separation conditions, faster heating rates allowed phase separated particles to be frozen in the network prior to precipitation. Thus, gels produced by slower heating rates had lower rigidities than gels produced by faster heating rates. The effect of heating rate appears to depend on if the solution is under single phase or micro-phase separated conditions. The effect of heating rate and/or time on protein gel firmness can be explained based on protein charge. When proteins have a high net negative charge and form soluble aggregates, there is no heating rate effect and gels with equal firmness will be formed if given enough time. In contrast, when electrostatic repulsion is low, there is a competition between protein precipitation and gel formation; thus, a faster heating rate produces a firmer gel.

Particle Diffusion in Globular Protein Gels in Relation to the Gel Structure

Globular protein gels with a variety of structures were prepared by heating β-lactoglobulin solutions at different concentrations and different ionic strengths. The structure was analyzed in terms of the pair correlation function of the protein concentration, and the volume fraction of the gels was determined. A strong coarsening of the gel structure was observed upon increasing the NaCl concentration between 0.1 and 0.25 M. The mean square displacement of spherical particles with diameters between 0.2 and 2 μm was determined in solutions and in gels by multiparticle tracking of confocal laser scanning microscopy images. Brownian diffusion or trapping of spheres with different sizes was observed, depending on the gel structure. In few cases the diffusion was anomalous. The relationship between gel structure and particle mobility is discussed.

Gelation properties of salt-extracted pea protein induced by heat treatment

Gelation is one of the most important properties of plant proteins. In this paper, a low denaturation salt extraction method was used to extract pea (Pisum sativum L.) protein isolate from commercial pea flour. The gelation properties of this isolate were examined and compared to commercial products. The pea protein isolate followed the three-step process of gelation that is generally accepted for heat-induced gelation of globular proteins. The minimum gelation concentration of salt-extracted pea protein isolate (PPIs) was 5.5% while that of commercial pea protein isolate (PPIc) was 14.5%. The gelling point was in the range of 82–86 °C for 14.5% PPIs, 0.3 M NaCl at natural pH (5.65). With increasing heating rate, the gelling point tended to increase. Higher heating and cooling rates resulted in decreased final G′ (storage modulus) and G″ (loss modulus) values, indicative of decreased gel strength. A higher protein concentration resulted in higher G′ and G″ values and it was found that there was a power law relationship between protein concentration and G′ and G″. Tan delta (δ) values decreased with increasing protein concentration and at concentrations of 5.5% and above, tan δ remained constant which means the critical concentration for gel formation was 5.5%. The values of G′ and G″ for PPIs were greater than those of PPIc, and tan δ of PPIs was smaller, indicative of a stronger gel network. DSC data showed that PPIc had undergone denaturation whereas PPIs had not (ΔH = 15.81 J/g protein). Although rheometer data showed that the final G′ value of commercial soy protein isolate (SPIc) was smaller than that of PPIs, the gel prepared with SPIc was visually stronger than that of PPIs. The rheological data obtained with small amplitude oscillatory testing was not consistent with the actual observations. Overall, the low degree of denaturation of the PPIs resulted in a stronger gel than that of PPIc making the PPIs a more attractive food ingredient.

Micro-phase separation explains the abrupt structural change of denatured globular protein gels on varying the ionic strength or the pH

Aqueous solutions of globular proteins gel after heat-induced denaturation. The structure of β-lactoglobulin gels was studied as a function of the pH (2–8) and the NaCl concentration (0–1 M) over a wide range of length scales (1 nm-100 µm) using a combination of scattering techniques and microscopy. The gel structure depended on the strength of the electrostatic interaction that was varied by changing the charge density of the proteins (pH) or the screening length (ionic strength). Homogeneous so-called finely stranded gels were formed at strong interaction, while more heterogeneous so-called particulate gels were formed at weak interaction. It is shown that phase separation of growing irreversibly bound protein aggregates explains the abrupt transition between the two structures for small changes of the ionic strength or the pH. Phase separation was limited to the formation of protein rich domains with a radius of several microns that associated into the particulate gels. The structure of homogeneous gels is shown to be determined by the amplitude of the (critical) concentration fluctuations of protein aggregates that were frozen-in by gelation. The characteristic length scales of the homogeneous gels varied between about 20 nm and 1 µm.

Quantitative analysis of confocal laser scanning microscopy images of heat-set globular protein gels

Gels of the globular protein β-lactoglobulin were made by heating solutions at pH 7 and different NaCl concentrations ( C s). The influence of the ionic strength on the gel structure was studied by confocal laser scanning microscopy (CLSM). For C s < 0.2 M the images were homogeneous, but at higher NaCl concentrations micro-phase separation was observed. The protein concentration in the two phases was determined from the images. It is shown how CLSM images can be quantitatively analysed in terms of the pair correlation function of the protein concentration fluctuations, yielding results that can be directly compared to those obtained from light scattering. The transition between so-called finely stranded and particulate gels is explained by a switch from net repulsive to net attractive interaction between growing protein aggregates.

The influence of electrostatic interaction on the structure and the shear modulus of heat-set globular protein gels.

Gels formed by the globular protein β-lactoglobulin after heat denaturation were studied using light scattering, turbidity and shear oscillation measurements. The structure of the gels was characterized in terms of the amplitude and the correlation length of concentration fluctuations. The strength of electrostatic interactions was varied by changing the pH in the absence of added salt or by changing the NaCl concentration at pH 7. A very strong increase of the heterogeneity of the gels was observed when decreasing the pH towards the isoelectric point or when increasing the salt concentration. The structural change was interpreted in terms of a decrease of the net repulsion between the growing aggregates leading to increased concentration fluctuations and finally microscopic phase separation. The elastic shear modulus increased with decreasing pH and showed a maximum as a function of the NaCl concentration. No direct correlation between the change in the structure and the elastic modulus was found.

Denaturation and aggregation processes in thermal gelation of whey proteins resolved by differential scanning calorimetry

Gelation of globular proteins on heating involves two separate stages. The first is partial unfolding (denaturation) of the native globular structure; the second is intermolecular aggregation. Denaturation involves dissociation of intramolecular bonds (non-covalent and, in some cases, disulfide) and is therefore an endothermic process. The aggregation step involves formation of new bonds between protein molecules, and would therefore be expected to give a differential scanning calorimetry (DSC) exotherm on heating, but numerous previous studies of the thermal gelation of whey proteins, carried out on conventional (fast scanning) DSC calorimeters (typical sample mass ∼15–50 mg), have shown only endothermic transitions. In the present work, however, we have observed the endothermic (denaturation) and exothermic (aggregation) processes in thermogelation of whey protein isolate (WPI) as separate transitions in DSC heating traces recorded on a Setaram microcalorimeter (sample mass ∼850 mg). Under conditions where aggregation occurs much more slowly than denaturation (low protein concentration; low ionic strength) the two transitions are well resolved, with the exotherm from aggregation following the endotherm from denaturation. The position of the exotherm, however, appears to be time-dependent rather than temperature-dependent. On reduction in heating rate, the apparent peak-maximum temperature of the aggregation exotherm decreases towards the (essentially constant) position of the denaturation endotherm, and, at sufficiently low scan rates, the exotherm becomes obscured by the more intense endotherm. Progressive displacement of the exotherm into the temperature range of the denaturation endotherm also occurs in response to changes that accelerate intermolecular aggregation and accompanying gelation (addition of salt; increasing protein concentration). The absence of a detectable exotherm in previous studies using conventional calorimeters is attributed to the much smaller sample mass than in the Setaram instrument, giving much faster heat transfer, which may cause the exothermic heat flow from the slow aggregation process to be swamped by the endothermic heat flow from the more rapid denaturation process.

Food Biophysics of Protein Gels: A Challenge of Nano and Macroscopic Proportions

Aggregation and gelation of proteins are key reactions used to generate food texture. Heat-induced gelation of globular proteins produces two general types of gels designated as fine-stranded and particulate. Fine-stranded gels are formed from denatured proteins that aggregate into curved, flexible strands (pH > pI) or rigid, linear fibrils (pH pI breakdown into large, inhomogeneous particles that have irregular shapes and do not form a cohesive mass or stick to the teeth during chewing. In contrast, particulate gels are formed from proteins with a lower degree of unfolding that aggregate into large particles. Particulate gels break down rapidly into a homogeneous distribution of small particles forming a cohesive mass that adheres to teeth during chewing. This review discusses the mechanisms related to the formation and breakdown of fine-stranded and particulate gels. Although there has been extensive research on gel formation, understanding gel breakdown based on mechanical (rheological and fracture properties) and sensory testing is limited. Further research is required to understand how the nanostructure of a gel network translates into the complex fracture pattern seen when evaluating the macroscopic property of food texture.

Strain hardening and fracture of heat-set fractal globular protein gels

Non-linear mechanical behavior at large shear deformation was been investigated for heat-set β-lactoglobulin gels at pH 7 and 0.1 M NaCl using both oscillatory shear and shear flow. These gels have a self-similar structure at length scales smaller than the correlation length of the gel with fractal dimension d f = 2 . Strain hardening is observed that can be well described using the model proposed by Gisler et al. [T.C. Gisler, R.C. Ball, D.A. Weitz, Phys. Rev. Let. 82 (1999) 1064] for fractal colloidal gels. The increase of the shear modulus normalized by the low strain value ( G 0 ) is independent of G 0 . For weak gels the elasticity increases up to a factor of ten, while for strong gels the increase is very small. At higher deformation irreversible fracture occurs, which leads eventually to macroscopic failure of the gel. For weak gels formed at low concentrations the deformation at failure is about 2, independent of the shear modulus. For strong gels fracture occurs at approximately constant stress ( 2 × 10 3 Pa ).

Influence of Cosolvent Systems on the Gelation Mechanism of Globular Protein: Thermodynamic, Kinetic, and Structural Aspects of Globular Protein Gelation.

How thermostability and gelation of globular protein are affected by cosolvent systems present in food systems is critical to understanding their functionality. The expression of these functional attributes depends on the molecular structure and thermal-mechanical history of the protein, as well as its chemical environment. To improve the design of processing protein-containing food systems, one must fully understand the thermodynamic, kinetic, and structural impact of cosolvent on globular protein gelation. This review focuses on the impact of weakly interacting neutral cosolvent systems (for example, sugars and polyols) on the gelation of globular proteins. The physicochemical mechanisms by which these cosolvent systems can modulate protein gelation are highlighted from a thermodynamic, kinetic, and structural point of view.

Influence of the Ionic Strength on the Structure of Heat-Set Globular Protein Gels at pH 7. Ovalbumin

The effect of the NaCl concentration (Cs) on the structure factor and the turbidity of heated ovalbumin solutions was investigated using cross-correlation dynamic light scattering. The heated systems are characterized by a correlation length, which depends on protein and salt concentration, beyond which they are homogeneous. At length scales below this correlation length, the systems have a self-similar, fractal, structure. For Cs ≥ 100 mM, very turbid systems are formed with correlation lengths that show strong concentration dependence, and the correlation length rapidly rises above 1 μm at protein concentrations above 4 g/L. For Cs ≤ 30 mM NaCl the systems remain transparent with relatively small correlation lengths (<20 nm). At 50 mM NaCl the correlation length first increases strongly with increasing protein concentration followed by a decrease above 8 g/L. At both 30 and 50 mM, an unexpected increase in the correlation length and turbidity is observed at high protein concentrations (above 40 and 30 g/L, respectively). From the present work we conclude that the structure of heated globular protein solutions and gels is generally determined by the interplay between the growth of the aggregates and the electrostatic interaction between the aggregates.

Fibrillar β-Lactoglobulin Gels: Part 2. Dynamic Mechanical Characterization of Heat-Set Systems

Oscillatory shear rheometry (mechanical spectroscopy) has been used to study the heat-set gelation of beta-lactoglobulin at pH 2. Modulus-concentration relationships were obtained by extrapolating cure data to infinite time. In terms of theory, these fail to provide a clear distinction between the fractal description of biopolymer gels and the classical random f-functional polycondensation branching theory (cascade) approach, though the latter is preferred. Critical exponents for the sol-gel transition, derived from these data, are also discussed. Where gel time-concentration results are concerned the fractal model makes no predictions, and the cascade approach in its simplest form must be rejected in favor of a more sophisticated version involving delivery of fibrils by nucleation and growth into the random aggregation process. Over the limited concentration range accessed experimentally, cure data for the different beta-lactoglobulin solutions, reduced to the universal form G'/G'inf versus t/tgel, superimposed well for samples heated both at 80 and 75 degrees C and for different batches of protein. Studies of the frequency responses of the fully cured gels confirm the validity of the gel description given to these materials, and a study of the temperature dependence of the frequency spectrum suggests a fall in the elastic component of the modulus as temperature decreases. This contrasts with what has been found for other heat-set globular protein gels such as those from serum albumin where the gel modulus increases at lower temperatures. The present results are in good agreement with more limited amounts of pH 2 beta-lactoglobulin data published earlier, though some differences arise through a previous neglect of measurement "dead time".