Harmen H.J. de Jongh

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 β-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.

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.

UNLABELLEDnParvalbumin 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.nnnPRACTICAL APPLICATIONnParvalbumin 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.

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.

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.

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.