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

Spectrophotometric tool for the determination of the total carboxylate content in proteins; Molar extinction coefficient of the enol ester from Woodward's reagent K reacted with protein carboxylates

A number of relevant properties of Woodward's reagent K have been determined, such as the stability of the reactant and the optimal reaction conditions of the reactant with protein carboxylates. A Woodward's reagent K stock solution was stable at 4 C for prolonged time, whereas upon storage at 22 C, almost 20% of the reactive compound was lost within 1 week. The pH-dependency of the spontaneous degradation reaction of Woodward's reagent K was studied and was shown to be base-mediated. A molar extinction coefficient of 3150 M-1 cm-1 at 269 nm for the enol ester resulting from the reaction between Woodward's reagent K and the protein carboxylates was established using the conditions laid out in this work. This value was validated using a variety of proteins that were modified by Woodward's reagent K. In addition, upon methylation of the carboxylates of a single protein, ovalbumin in this case, the degree of modification could be determined accurately and was confirmed by cation exchange chromatography elution profiles

Assessing the Extent of Protein Intermolecular Interactions at Air-Water Interfaces Using Spectroscopic Techniques

There is an ongoing debate about whether a protein surface film at an air-water interface can be regarded as a gelled layer. There is literature reporting that such films show macroscopic fracture behavior and a rheology comparable to three-dimensional protein bulk-networks. Ifthis is the case, a complete description of the formation of adsorbed layers should include a transition from single, freely moving proteins to a gelled layer. This report presents studies using spectroscopic techniques, such as infrared, fluorescence and neutron spectroscopy, or ellipsometry, to derive molecular insight in situ to substantiate the intermolecular networking in surface films of chicken egg ovalbumin. It is concluded that protein films, generated by equilibrium adsorption from the bulk, behave as a densely packed colloidal repulsive particle system, where the proteins still have a significant rotational mobility, have a predominantly retained globular fold, and show distinct (lateral) diffusion. Applied stresses on the surface film (by compressions of the interface) may result in protein denaturation and aggregation. This process renders a surface film from a colloidal particle into that of a gelled system

Differential clearance kinetics of adhered layer constituents from the oral cavity as modulator for afterfeel of dressings: ATR FT-IR measurements of localized oral coatings

By analyzing swabs from different locations in the mouth (front and middle part of the tongue, cheek and palate) of four subjects after oral processing of dressings, the composition of the retained material was evaluated in time using infrared spectroscopy in an attempt to substantiate the physical origin of differences in afterfeel sensations indicated by a trained panel for these dressings. Three dressings were prepared, each containing 40% oil, 10¿14% protein and either starch, xanthan or a mixture of these two polysaccharides as thickener. There was no differential retention of fat/oil between the dressings, oral location or subject. Protein and carbohydrates followed that same clearance kinetics, with two exceptions: (1) xanthan/protein from the middle part of the tongue; and (2) starch/protein from the cheek and palate that both cleared only very slowly (10¿30 min). The relation of these findings with afterfeel and saliva action is discusse

Proteins at air-water interfaces studied using external reflection circular dichroism

In this report we describe the first attempts to record external reflection circular dichroism (ERCD) spectra of beta -lactoglobulin solutions. It is shown that the accumulated proteins at and near the air–water interface can be detected using ERCD and that the signals obtained contain information on the conformational properties and concentration of the proteins residing at the interface. The local protein concentration and its conformation are in full agreement with previous observations using external reflection infrared spectroscopy. The ERCD signals are dominated by linear dichroism (LD) due to non-ideal behavior of the instrumental optics, but can be explained for using the theoretical description of chiral reflection. This allows the analysis of ERCD spectra of protein solutions. The measured ERCD signals are described accurately in the region between 190 and 220 nm, but poor resemblance is obtained at higher wavelengths. We are however confident that improvement of experimental conditions and theoretical description will allow that in the near future, external reflection circular dichroism (CD) can be a valuable tool that complements the application of external reflection infrared spectroscopy to study interfacial system

The presence of heat-stable conformers of ovalbumin affects properties of thermally formed aggregates

The aim of this work was to study the effect of the formation of more heat-stable conformers of chicken egg ovalbumin during incubation at basic pH (9.9) and elevated temperature (55°C) on the protein aggregation properties at neutral pH. Native ovalbumin (N-OVA) is converted on the hours time-scale into more heat-stable forms denoted I- (intermediate) and S-OVA, that have denaturation temperatures 4.8 and 8.4°C, respectively, higher than that of N-OVA. The conversions most likely proceed via I-OVA, but direct conversion of N-OVA into S-OVA with slower kinetics can not be excluded. It is demonstrated that both I- and S-OVA have similar denaturation characteristics to N-OVA, except that higher temperatures are required for denaturation. The presence of even small contributions of I-OVA does, however, reduce the Stokes radius of the aggregates formed upon heat treatment of the material at 90°C about 2-fold. This affects the gel network formation considerably. Since many (commercial) preparations of ovalbumin contain varying contributions of the more heat-stable forms mentioned, proper characterization or standardization of the isolation procedure of the material is essential to control or predict the industrial application of this protein

Modification of beta-lactoglobulin by oligofructose: Impact on protein adsorption at the air-water interface

Maillard products of -lactoglobulin (Lg) and fructose oligosaccharide (FOS) were obtained in different degrees of modification depending on incubation time and pH. By use of a variety of biochemical and spectroscopic tools, it was demonstrated that the modification at limited degrees does not significantly affect the secondary, tertiary, and quaternary structure of Lg. The consequence of the modification on the thermodynamics of the protein was studied using differential scanning calorimetry, circular dichroism, and by monitoring the fluorescence intensity of protein samples with different concentrations of guanidine-HCl. The modification leads to lowering of the denaturation temperature by 5 C and a reduction of the free energy of stabilization of about 30%. Ellipsometry and drop tensiometry demonstrated that upon adsorption to air-water interfaces in equilibrium modified Lg exerts a lower surface pressure than native Lg (16 versus 22 mN/m). Moreover, the surface elastic modulus increased with increasing surface pressure but reached significantly smaller values in the case of FOS-Lg. Compared to native Lg, modification of the protein with oligofructose moieties results in higher surface loads and thicker surface layers. The consequences of these altered surface rheological properties are discussed in view of the functional behavior in technological applications