Purification and Initial Characterization of Ara h 7, a Peanut Allergen from the 2S Albumin Protein Family.

2S albumins are important peanut allergens. Within this protein family, Ara h 2 and Ara h 6 have been described in detail, but Ara h 7 has received little attention. We now describe the first purification of Ara h 7 and its characterization. Two Ara h 7 isoforms were purified from peanuts. Mass spectrometry revealed that both the isoforms have a post-translation cleavage, a hydroxyproline modification near the N-terminus, and four disulfide bonds. The secondary structure of both Ara h 7 isoforms is highly comparable to those of Ara h 2 and Ara h 6. Both Ara h 7 isoforms bind IgE, and Ara h 7 is capable of inhibiting the binding between Ara h 2 and IgE, suggesting at least partially cross-reactive IgE epitopes. Ara h 7 was found in all main market types of peanut, at comparable levels. This suggests that Ara h 7 is a relevant allergen from the peanut 2S albumin protein family.

Effect of heat treatment on the conformational stability of intact and cleaved forms ofthe peanut allergen Ara h 6 in relation to its IgE-binding potency.

This work reports on theeffect of heat treatment on the protein conformational stabilityof intact and post-translationallycleaved peanut allergen Ara h 6 in relation to IgE-binding. Intact and post-translationallycleaved Ara h 6 are structurally similar and theirstrong resistance to denaturant-inducedunfolding is comparable. Only upon exposure toautoclave conditions the twoforms of Ara h 6 demonstrated susceptibility toirreversible denaturationresulting in a significant decrease in IgE-binding potency. Thisreduction isfor the intact protein more pronounced than for than for the cleaved form. This isattributed to less conformational constrains of the cleaved form comparedtointact, as suggested by the 2-fold lower activation energy for unfoldingfound for the cleavedform. Overall, harsh conditionsare required to denature Ara h 6 and to significantly reduce its IgE-bindingpotency. The cleavedform possesses more resistance to such denaturation than the intactform.

Purification and Characterization of Naturally Occurring Post-Translationally Cleaved Ara h 6, an Allergen That Contributes Substantially to the Allergenic Potency of Peanut.

The 2S albumin Ara h 6 is one of the most important peanut allergens. A post-translationally cleaved Ara h 6 (pAra h 6) was purified from Virginia type peanuts, and the cleavage site was mapped using high-resolution mass spectrometry. Compared to intact Ara h 6, pAra h 6 lacks a 5-amino acid stretch, resembling amino acids 43-47 (UniProt accession number Q647G9) in the nonstructured loop. Consequently, pAra h 6 consists of two chains: an N-terminal chain of approximately 5 kDa and a C-terminal chain of approximately 9 kDa, held together by disulfide bonds. Intermediate post-translationally cleaved products, in which this stretch is cleaved yet still attached to one of the subunits, are also present. The secondary structure and immunoglobulin E (IgE) binding of pAra h 6 resembles that of intact Ara h 6, indicating that the loss of the nonstructured loop is not critical for maintaining the protein structure. Commercially available monoclonal and polyclonal immunoglobulin G (IgG) antibodies directed to Ara h 6 react with both intact Ara h 6 and pAra h 6, suggesting that the involved epitopes are not located in the area that is post-translationally cleaved. No differences between intact Ara h 6 and pAra h 6 in terms of IgE binding were found, suggesting that the area that is post-translationally cleaved is not involved in IgE epitopes either. For all main cultivars Runner, Virginia, Valencia, and Spanish, intact Ara h 6 and pAra h 6 occur in peanut at similar levels, indicating that pAra h 6 is a consistent and important contributor to the allergenic potency of peanut.

Conformational stability of digestion-resistant peptides of peanut conglutins reveals the molecular basis of their allergenicity.

Conglutins represent the major peanut allergens and are renowned for their resistance to gastro-intestinal digestion. Our aim was to characterize the digestion-resistant peptides (DRPs) of conglutins by biochemical and biophysical methods followed by a molecular dynamics simulation in order to better understand the molecular basis of food protein allergenicity. We have mapped proteolysis sites at the N- and C-termini and at a limited internal segment, while other potential proteolysis sites remained unaffected. Molecular dynamics simulation showed that proteolysis only occurred in the vibrant regions of the proteins. DRPs appeared to be conformationally stable as intact conglutins. Also, the overall secondary structure and IgE-binding potency of DRPs was comparable to that of intact conglutins. The stability of conglutins toward gastro-intestinal digestion, combined with the conformational stability of the resulting DRPs provide conditions for optimal exposure to the intestinal immune system, providing an explanation for the extraordinary allergenicity of peanut conglutins.

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.

Calcium Binding Restores Gel Formation of Succinylated Gelatin and Reduces Brittleness with Preservation of the Elastically Stored Energy.

To better tailor gelatins for textural characteristics in (food) gels, their interactions are destabilized by introduction of electrostatic repulsions and creation of affinity sites for calcium to "lock" intermolecular interactions. For that purpose gelatins with various degrees of succinylation are obtained. Extensive succinylation hampers helix formation and gel strength is slightly reduced. At high degrees of succinylation the helix propensity, gelling/melting temperatures, concomitant transition enthalpy, and gel strength become calcium-sensitive, and relatively low calcium concentrations largely restore these properties. Although succinylation has a major impact on the brittleness of the gels formed and the addition of calcium makes the material less brittle compared to nonmodified gelatin, the modification has no impact on the energy balance in the gel, where all energy applied is elastically stored in the material. This is explained by the unaffected stress relaxation by the network and high water-holding capacity related to the small mesh sizes in the gels.

Electrophoretic Behavior in Relation to the Structural Integrity of Codfish Parvalbumin upon Heat Treatment.

This work evaluates the impact of heat processing of parvalbumin, a major fish allergen, on the consequences for quantitative analysis of this protein embedded in different matrices during heating (either isolated, in an aqueous extract, or in whole fillets) to assess potential health risks. It is shown that oligomerization of parvalbumin does occur, but only upon heat treatment above 80 °C. This coincides with the ability of the isolated protein to refold up to this temperature in a fully reversible way, as demonstrated by circular dichroism analysis. In autoclaved samples a disintegration of the protein structure is observed. The situation becomes different when parvalbumin is embedded in a matrix with other constituents, as in fish extracts or whole fillets. The electrophoretic analysis of parvalbumin (SDS-PAGE and immunoblotting) is largely determined by complexation with other proteins resulting in insoluble materials caused by the partial unfolding of the parvalbumin at elevated temperatures. This effect is more strongly observed for cod fish extract, compared to whole cod fillets, as in the latter situation the integrity of the tissue hampers this interprotein complexation. Moreover, it is shown by ELISA analysis of heat-treated samples that using blotting procedures where disintegration of complexes may be promoted, restoring some of the IgG-binding propensity, may provide false outcomes. It was concluded that antibody binding to parvalbumin is dominated by the potential to form heat-induced complexes with other proteins. The possibly less-soluble or extractable character of these complexes may provide confusing information regarding potential health risks of fish and fish protein-containing food composites when such heat-treated samples are analyzed by immunochemical assays.

Modification of ovalbumin with fructooligosaccharides: consequences for network morphology and mechanical deformation responses.

The Maillardation of proteins has been used as a natural alternative to improve its functionality by covalent coupling of proteins with saccharides. However, the impact of Maillard reaction on the structural aspects of protein networks and, as a consequence, the mechanical breakdown properties of the gel networks has not been reported. The objective of this study was to evaluate how the attachment of linear oligo-sugar moieties onto ovalbumin affects its aggregation, network morphology, and consequently the mechanical deformation properties including the ability of the networks to elastically store energy in this material. To potentially alter the morphology of the network structure, ovalbumin was modified by conjugating some of its amino groups with fructooligosaccharide (FOS) moieties via the Maillard reaction. It was demonstrated that the attachment of FOS to ovalbumin does not affect the integrity of the secondary and tertiary structure as characterized using circular dichroism and tryptophan fluorescence. Differences in the network morphology were observed by scanning electron microscopy for FOS-modified ovalbumin variants. Upon increased modification, the microstructure of the gels had more and larger pores and had thinner strands than nonmodified variants. Evaluation of the large deformation properties of the gels demonstrated that FOS-modified gels were less strong and less brittle and showed lower stiffness than nonmodified variants. The recoverable energy (elastically stored energy) of gels reduced with an increase in the degree of modification. The results show that the attachment of FOS to ovalbumin alters the structural and mechanical (large) breakdown properties of the protein gels. The consequences of the alteration of the network structure and large deformation properties of FOS-modified ovalbumin offer opportunities to efficiently design food materials with desirable techno-functional applications.

Incorporation of mesoporous silica particles in gelatine gels: effect of particle type and surface modification on physical properties.

The aim of this work was to investigate the impact of mesoporous silica particles (MSPs) on the physicochemical properties of filled protein gels. We have studied the effect of the addition of different mesoporous silica particles, either bare or functionalized with amines or carboxylates, on the physical properties of gelatine gels (5% w/v). Textural properties of the filled gels were investigated by uniaxial compression, while optical properties were investigated by turbidity. The MSPs were characterized with the objective of correlating particle features with their impact on the corresponding filled-gel properties. The addition of MSPs (both with and without functionalization) increased the stiffness of the gelatine gels. Furthermore, functionalized MSPs showed a remarkable increase in the strength of the gels and a slight reduction in the brittleness of the gels, in contrast with nonfunctionalized MSPs which showed no effect on these two properties. The turbidity of the gels was also affected by the addition of all tested MSPs, showing that the particles that formed smaller aggregates resulted in a higher contribution to turbidity. MSPs are promising candidates for the development of functional food containing smart delivery systems, also being able to modulate the functionality of protein gels.

Fibril formation from pea protein and subsequent gel formation.

The objective of this study was to characterize fibrillar aggregates made using pea proteins, to assemble formed fibrils into protein-based gels, and to study the rheological behavior of these gels. Micrometer-long fibrillar aggregates were observed after pea protein solutions had been heated for 20 h at pH 2.0. Following heating of pea proteins, it was observed that all of the proteins were hydrolyzed into peptides and that 50% of these peptides were assembled into fibrils. Changes on a structural level in pea proteins were studied using circular dichroism, transmission electron microscopy, and particle size analysis. During the fibril assembly process, an increase in aggregate size was observed, which coincided with an increase in thioflavin T binding, indicating the presence of ß-sheet aggregates. Fibrils made using pea proteins were more branched and curly. Gel formation of preformed fibrils was induced by slow acidification from pH 7.0 to a final pH of around pH 5.0. The ability of pea protein-based fibrillar gels to fracture during an amplitude sweep was comparable to those of soy protein and whey protein-based fibrillar gels, although gels prepared from fibrils made using pea protein and soy protein were weaker than those of whey protein. The findings show that fibrils can be prepared from pea protein, which can be incorporated into protein-based fibrillar gels.

The ability to store energy in pea protein gels is set by network dimensions smaller than 50nm.

The objective of this study was to identify which length scales set the ability to elastically store energy in pea protein network structures. Various network structures were obtained from pea proteins by varying the pH and salt conditions during gel formation. The coarseness of the network structure was visualized by the use of confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM) and ranked from least coarse to most coarse networks. Least coarse networks were formed at a pH away from the isoelectric point (IEP) of pea proteins, and at a low ionic strength, whereas more coarse networks were formed at pH values close to the IEP and at a high ionic strength during gel formation. Mechanical deformation properties of the gels such as elastically stored (recoverable) energy, Young's moduli (stiffness of gels), fracture stress (gel strength), and fracture strain (brittleness of the gels) were measured by the use of a texture analyzer and correlated to the coarseness of the networks structure. The influence of coarseness on the ability of the networks to elastically store energy was observed for length scales below 50nm. The findings show that elastically stored energy of pea protein gels can be modulated via the creation of different network structures below 50nm length scales. The results from this study contribute to a better understanding of the dimensions that set the ability to elastically storage in pea protein gels. If the ability of pea proteins to store energy can be understood, products can be better tailored for consumers.

Modulation of the gelation efficiency of fibrillar and spherical aggregates by means of thiolation.

Fibrillar and spherical aggregates were prepared from whey protein isolate (WPI). These aggregates were thiolated to a substantial degree to observe any impact on functionality. Sulfur-containing groups were introduced on these aggregates which could be converted to thiol groups by deblocking. Changes on a molecular and microstructural level were studied using tryptophan fluorescence, transmission electron microscopy, and particle size analysis. The average size (nm) of spherical aggregates increased from 38 to 68 nm (blocked variant) and 106 nm (deblocked variant) after thiolation, whereas the structure of fibrillar aggregates was not affected. Subsequently, gels containing these different aggregates were prepared. Rheological measurements showed that thiolation decreased the gelation concentration and increased gel strength for both WPI fibrillar and spherical aggregates. This effect was more pronounced upon thiolation of preformed fibrillar aggregates. The findings suggest that thiolation at a protein aggregate level is a promising strategy to increase gelation efficiency.

The protein structure determines the sensitizing capacity of Brazil nut 2S albumin (Ber e1) in a rat food allergy model.

: It is not exactly known why certain food proteins are more likely to sensitize. One of the characteristics of most food allergens is that they are stable to the acidic and proteolytic conditions in the digestive tract. This property is thought to be a risk factor in allergic sensitization. The purpose of the present study was to investigate the contribution of the protein structure of 2S albumin (Ber e1), a major allergen from Brazil nut, on the sensitizing capacity in vivo using an oral Brown Norway rat food allergy model. Disulphide bridges of 2S albumin were reduced and alkylated resulting in loss of protein structure and an increased pepsin digestibility in vitro. Both native 2S albumin and reduced/alkylated 2S albumin were administered by daily gavage dosing (0.1 and 1 mg) to Brown Norway rats for 42 days. Intraperitoneal administration was used as a positive control. Sera were analysed by ELISA and passive cutaneous anaphylaxis. Oral exposure to native or reduced/alkylated 2S albumin resulted in specific IgG1 and IgG2a responses whereas only native 2S albumin induced specific IgE in this model, which was confirmed by passive cutaneous anaphylaxis. This study has shown that the disruption of the protein structure of Brazil nut 2S albumin decreased the sensitizing potential in a Brown Norway rat food allergy model, whereas the immunogenicity of 2S albumin remained preserved. This observation may open possibilities for developing immunotherapy for Brazil nut allergy.

Digestibility and IgE-binding of glycosylated codfish parvalbumin.

Food-processing conditions may alter the allergenicity of food proteins by different means. In this study, the effect of the glycosylation as a result of thermal treatment on the digestibility and IgE-binding of codfish parvalbumin is investigated. Native and glycosylated parvalbumins were digested with pepsin at various conditions relevant for the gastrointestinal tract. Intact proteins and peptides were analysed for apparent molecular weight and IgE-binding. Glycosylation did not substantially affect the digestion. Although the peptides resulting from digestion were relatively large (3 and 4 kDa), the IgE-binding was strongly diminished. However, the glycosylated parvalbumin had a strong propensity to form dimers and tetramers, and these multimers bound IgE intensely, suggesting stronger IgE-binding than monomeric parvalbumin. We conclude that glycosylation of codfish parvalbumin does not affect the digestibility of parvalbumin and that the peptides resulting from this digestion show low IgE-binding, regardless of glycosylation. Glycosylation of parvalbumin leads to the formation of higher order structures that are more potent IgE binders than native, monomeric parvalbumin. Therefore, food-processing conditions applied to fish allergen can potentially lead to increased allergenicity, even while the protein's digestibility is not affected by such processing.

Lubrication, adsorption, and rheology of aqueous polysaccharide solutions.

Aqueous lubrication is currently at the forefront of tribological research due to the desire to learn and potentially mimic how nature lubricates biotribological contacts. We focus here on understanding the lubrication properties of naturally occurring polysaccharides in aqueous solution using a combination of tribology, adsorption, and rheology. The polysaccharides include pectin, xanthan gum, gellan, and locus bean gum that are all widely used in food and nonfood applications. They form rheologically complex fluids in aqueous solution that are both shear thinning and elastic, and their normal stress differences at high shear rates are found to be characteristic of semiflexible/rigid molecules. Lubrication is studied using a ball-on-disk tribometer with hydrophobic elastomer surfaces, mimicking biotribological contacts, and the friction coefficient is measured as a function of speed across the boundary, mixed, and hydrodynamic lubrication regimes. The hydrodynamic regime, where the friction coefficient increases with increasing lubricant entrainment speed, is found to depend on the viscosity of the polysaccharide solutions at shear rates of around 10(4) s(-1). The boundary regime, which occurs at the lowest entrainment speeds, depends on the adsorption of polymer to the substrate. In this regime, the friction coefficient for a rough substrate (400 nm rms roughness) is dependent on the dry mass of polymer adsorbed to the surface (obtained from surface plasmon resonance), while for a smooth substrate (10 nm rms roughness) the friction coefficient is strongly dependent on the hydrated wet mass of adsorbed polymer (obtained from quartz crystal microbalance, QCM-D). The mixed regime is dependent on both the adsorbed film properties and lubricant's viscosity at high shear rates. In addition, the entrainment speed where the friction coefficient is a minimum, which corresponds to the transition between the hydrodynamic and mixed regime, correlates linearly with the ratio of the wet mass and viscosity at ∼10(4) s(-1) for the smooth surface. These findings are independent of the different polysaccharides used in the study and their different viscoelastic flow properties.

Controlling the aggregation propensity and thereby digestibility of allergens by Maillardation as illustrated for cod fish parvalbumin.

The aggregation propensity of heat-treated codfish parvalbumin is investigated upon Maillardation of the protein under food-relevant conditions. About twelve of the fourteen lysine residues had reacted with glucose under these conditions. It is shown using circular dichroism and fluorescence that extensive coupling of glucose moieties on the protein surface does not affect the secondary and tertiary structural fold nor severely impair calcium-binding under ambient conditions. The glucosylated protein has a 3 °C higher denaturation temperature, while the free energy change involved with denaturation is reduced by 5-10%. It is shown by establishing insight in the change in heat capacity involved in the denaturation process that glucosylation of the protein is likely to result in a lower exposed hydrophobicity in the unfolded state. Aggregation kinetics, as monitored using light scattering techniques, shows that this lower exposed hydrophobicity of the denatured state slows down the aggregation process, while the shape and size of formed aggregates are comparable to those formed by non-glucosylated protein. This lower aggregation propensity allows a significantly faster digestion of heat-treated materials by pepsin. This work demonstrates that (pre-)processing of ingredients containing potential allergens, like cod fish parvalbumin, using food-relevant conditions may provide an effective tool to minimize risks of provoked allergic responses during food consumption.

Purification of parvalbumin from carp: a protocol that avoids heat-treatment.

UNLABELLED: Parvalbumin from carp, a major allergen, was purified to homogeneity using ion exchange chromatography and size exclusion chromatography (estimated purity >95% to 98% based on SDS-PAGE and native PAGE) with a yield of 318 mg, and a number of basic biochemical characteristics were determined. The identity was confirmed by peptide-mass fingerprinting, and IgE-binding was demonstrated. The UV/Vis absorbance spectra were explained using the previously published amino acid sequences. Far UV-CD spectroscopy was used to confirm the folding character of parvalbumin. We conclude that parvalbumin from carp can be purified on a comparatively large (hundreds of milligrams) scale using a purification protocol that does not include denaturing steps. The purified protein resembles biochemical characteristics as were earlier published for carp parvalbumin, that is, a molecular weight of approximately 12 kDa, amino acid sequence identity and a secondary structure containing alpha-helices and beta-structures. The described method provides a yield sufficient to produce and characterize antibodies to construct immunochemical methods to detect parvalbumin in food, as well as for use as a standard calibrator for such assays. PRACTICAL APPLICATION: Parvalbumin is a major allergen from fish. Here, we have purified a comparatively large quantity from carp that can be used to develop antisera for use in an assay method to detect fish allergens.

Colloidal aspects of texture perception.

Recently, considerable attention has been given to the understanding of texture attributes that cannot directly be related to physical properties of food, such as creamy, crumbly and watery. The perception of these attributes is strongly related to the way the food is processed during food intake, mastication, swallowing of it and during the cleaning of the mouth after swallowing. Moreover, their perception is modulated by the interaction with other basic attributes, such as taste and aroma attributes (e.g. sourness and vanilla). To be able to link the composition and structure of food products to more complicated texture attributes, their initial physical/colloid chemical properties and the oral processing of these products must be well understood. Understanding of the processes in the mouth at colloidal length scales turned out to be essential to grasp the interplay between perception, oral physiology and food properties. In view of the huge differences in physical chemical properties between food products, it is practical to make a distinction between solid, semi-solid, and liquid food products. The latter ones are often liquid dispersions of emulsion droplets or particles in general. For liquid food products for instance flow behaviour and colloidal stability of dispersed particles play a main role in determining their textural properties. For most solid products stiffness and fracture behaviour in relation to water content are essential while for semi-solids a much larger range of mechanical properties will play a role. Examples of colloidal aspects of texture perception will be discussed for these three categories of products based on selected sensory attributes and/or relevant colloidal processes. For solid products some main factors determining crispness will be discussed. For crispiness of dry cellular solid products these are water content and the architecture of the product at mesoscopic length scales (20-1000 microm). In addition the distribution of water at mesoscopic length scales was found to be important. For semi-solid foods, sensory characteristics as spreadability, watery and crumbliness are primarily determined by food properties at mesoscopic length scales. Crumbliness is directly related to the formation of free running cracks that occur during eating of the product. Exudation of the continuous liquid phase of gels during compression gives rise to watery/juicy sensory attributes. For liquid food products, colloidal interactions of emulsion droplets, particles, proteins, and polysaccharides with saliva and oral surfaces were found to affect texture characteristics as creaminess, fattiness, roughness and astringency.

Physicochemical characterization of allergens: quantity, identity, purity, aggregation and conformation.

Allergens and allergoids can be characterized by means of physicochemical methods, resulting in a description of the protein on different structural levels. Several techniques are available and their suitability depends on the composition of the particular sample. Current European legislation on allergen products demands characterization of final products in particular focusing on identity, degree of modification (for allergoids) and stability of the composition. Structural parameters of allergens may be used to investigate the stability of an allergen product. The challenge is to identify and optimize techniques that allow determination of protein structure in the context of a formulated pharmaceutical product. As the majority of the products currently marketed are formulated with aluminium hydroxide or aluminium phosphate as a depot, most of the methods and techniques used for protein characterization in solution are not applicable. An additional hurdle is that allergen products are based on natural extracts, comprising a mixture of proteins, both allergens and non-allergens, sometimes in the presence of other uncharacterized components from the raw material. This paper describes which methods are suitable for the different stages of allergen product manufacturing, and how these relate to the current regulatory requirements. Some of the techniques are demonstrated using a model allergen, cod parvalbumin, and a chemically modified form thereof. We conclude that a variety of methods is available for characterization of proteins in solution, and that a limited number of techniques appear to be suitable for modified allergens (allergoids). Adaptation of existing methods, e.g. mass spectroscopy and infrared spectroscopy may be helpful to obtain protein parameters of allergens in a formulated allergen product. By choosing a combination of techniques, including those additional to physicochemical approaches, relevant parameters of allergens in formulated allergen products can be assessed in order to achieve a well-characterized pharmaceutical product.

Net charge affects morphology and visual properties of ovalbumin aggregates.

The effect of ovalbumin net charge on aggregate morphology and visual properties was investigated using chromatography, electrophoresis, electron microscopy, and turbidity measurements. A range of differently charged ovalbumin variants (net charge ranging from -1 to -26 at pH 7) was produced using chemical engineering. With increasing net charge, the degree of branching and flexibility of the aggregates decreased. The turbidity of the solutions reflected the aggregate morphology that was observed with transmission electron microscopy. Increasing the stiffness of the aggregates transformed the solutions from turbid to transparent. Artificially shielding the introduced net charge by introducing salt in the solution resulted in an aggregate morphology that was similar to that for low-net-charge variants. The morphology of heat-induced aggregates and the visual appearance of the solutions were significantly affected by net charge. We also found that the morphology of ovalbumin aggregates can be rapidly probed by high-throughput turbidity experiments.