The heat-induced aggregation of bovine β-lactoglobulin, dispersed in water at neutral pH and in different concentrations (10, 30, or 50 g of dry matter/L), was studied at 65 °C, and the results are related to a kinetic model. Native PAGE and SDS−PAGE analysis under nonreducing and reducing conditions showed that on heating disulfide-linked aggregates were formed and that the average size of these aggregates increased with increasing initial β-lactoglobulin concentration. In the presence of the thiol-blocking agent N-ethylmaleimide (NEM), at a molar ratio of NEM/β-lactoglobulin monomer of 1, all thiol groups were blocked and no disulfide-linked aggregates were formed, although with native PAGE high molecular mass noncovalently linked aggregates were observed. The formation of these aggregates accelerated with increasing NEM concentration until a molar ratio of NEM/β-lactoglobulin monomer of 1 was reached. In separate experiments we studied the effect of pH (in the range pH 6.0−8.0) on the aggregation of β-lactoglobulin and related this to the pH dependent reactivity of the thiol group. Keywords: β-Lactoglobulin; thermal treatment; denaturation; aggregation; thiol reactivity
Five whey protein gels, with different gel hardnesses and waterholding capacities, were flavored with ethylbutyrate or diacetyl and evaluated by a 10-person panel to study the relation between the gel structure and the sensory perception, as well as the nosespace flavor concentration during eating. The sensory perception of the flavor compounds was measured by the time-intensity method, while simultaneously the nosespace flavor concentration was monitored by the MS-Nose. The nosespace flavor concentration was found to be independent of the gel hardness or waterholding capacity. However, significant changes in flavor intensity between the gels were perceived by the majority of the panelists, despite the fact that the panelists were instructed to focus only on flavor perception and to not take texture into account. From these observations it is concluded that the texture of gels determines perception of flavor intensity rather than the in-nose flavor concentration.
The effect of pH in the range 6.0-8.0 on the denaturation and aggregation of beta-lactoglobulin (beta-lg) was investigated. Results were interpreted in terms of the reaction scheme for the denaturation and aggregation of beta-lg proposed by Roefs and De Kruif (Eur. J. Biochem. 1994, 226, 883-889). The rate of conversion of native beta-lg increased strongly at higher pH values, whereas the molecular mass of the aggregates decreased strongly. In the pH range 6.4-8.0 aggregates were formed mainly by intermolecular disulfide bonds, but even at pH 6.0, thiol/disulfide exchange reactions were involved, although to a lesser extent. The time course of the exposure of the thiol group in native beta-lg upon heating and the subsequent disappearance of this group through the formation of disulfide-linked aggregates was investigated by reaction with 5,5'-dithiobis(2-nitrobenzoic acid) and varied strongly with pH. These observations could be used, in combination with the reaction steps of the reaction scheme, to describe qualitatively the strongly pH-dependent isothermal calorimetry curves, measured at 65 degrees C.
SummaryIn situ light scattering, where light scattered from a sample is measured directly while the sample is heated in the instrument, is presented as a simple and effective technique for studying the heat-induced aggregation of β-lactoglobulin. This technique was shown to be applicable not only to monitoring the initial aggregation steps, but to following the overall aggregation process with time. The experiments gave results similar to measurements carried out after a heat-quench treatment, but were more informative. From experiments on a standard NIZO β-lactoglobulin sample, a strongly desalted standard NIZO sample, different genetic variants of β-lactoglobulin and a mixture of these, we concluded that the standard NIZO sample was suitable for studying heat-induced aggregation. This sample has been investigated more extensively. Results with β-lactoglobulin (10–100 g/1) at 65 °C fitted a kinetic model for the denaturation and aggregation of β-lactoglobulin. This model, which held for β-lactoglobulin dissolved in water at near neutral pH and at 60–75 °C, recognizes an initiation, propagation and termination reaction, by analogy with polymer radical chemistry. It gave a quantitatively correct description of the dependence of the scattering intensity on the initial β-lactoglobulin concentration. Salt composition, pH and temperature strongly influenced the aggregation of β-lactoglobulin. Particle size increased with salt concentration in the range studied (up to 20 mM-NaCl and 1·0 mM-CaCl2). When the pH increased from 6°9 to 8·0 particle size was strongly reduced, whereas it strongly increased when pH was lowered to 6·2. Between 61·5 and 70 °C temperature did not affect particle size, whereas aggregation rate strongly increased. These effects could be incorporated in the kinetic model via the reaction constants of the reaction kinetic pathway.
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