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The effects of heat treatment on emulsifying properties of beta-lactoglobulin were studied in order to compare them with previous studies on foaming properties. Both of them are closely linked to the structural changes on the protein. Aliquots from 5.5% (w/v) beta-lactoglobulin solution in 20 mM phosphate buffer pH 6.8 were heated at 85 ºC for different time periods, from 1 to 15 min. Protein solubilities were measured for unheated and heated beta-lactoglobulin samples. Protein-stabilized O/W emulsions were prepared with these samples and corn oil. Droplet size distribution in the emulsions and emulsifying activity index were determined for each system, as parameters of the emulsifying ability of the protein. Emulsion stability was estimated from three different methods: backscattering, determination of the remaining protein concentration after creaming and monitoring the oiling off process. With the assayed methodology, heat treatment of beta-lactoglobulin led to different effects on foaming and emulsifying properties of the protein, depending on the time of heating. For shorter times of heating, both foamability and foam stability improved, while emulsifying properties diminished. After 10 min of heating at 85 °C, both foaming and emulsifying properties diminished. Formation time scales, as well as size of the sedimentable aggregates and their steric effect on the interfacial film, play an important role in explaining these differences between foaming and emulsifying properties. Fil: Moro, Andrea. Universidad Nacional de Rosario. Facultad de Cs.bioquimicas y Farmaceuticas. Departamento de Quimica y Fisica. Area Fisicoquimica; Argentina Fil: Báez, Germán David. Universidad Nacional de Rosario. Facultad de Cs.bioquimicas y Farmaceuticas. Departamento de Quimica y Fisica. Area Fisicoquimica; Argentina Fil: Ballerini, Griselda A.. Universidad Nacional de Rosario. Facultad de Cs.bioquimicas y Farmaceuticas. Departamento de Quimica y Fisica. Area Fisicoquimica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Instituto de Química Rosario; Argentina. Universidad Tecnológica Nacional. Facultad Regional Rosario; Argentina Fil: Busti, Pablo Andres. Universidad Nacional de Rosario. Facultad de Cs.bioquimicas y Farmaceuticas. Departamento de Quimica y Fisica. Area Fisicoquimica; Argentina Fil: Delorenzi, Nestor Jorge. Universidad Nacional de Rosario. Facultad de Cs.bioquimicas y Farmaceuticas. Departamento de Quimica y Fisica. Area Fisicoquimica; Argentina

In spite of numerous physical and biochemical studies on thermal denaturation of bovine β-lactoglobulin (β-LG), the definition of the N- and U-states, and the extent of reversibility assigned to the process are not clearly determined. The application of two-state model and dissociation coupled unfolding model to interpret the thermal unfolding profiles obtained by different authors using UV-difference absorbance at 293 nm, was analyzed and discussed. We conclude that both models fail to describe correctly the β-LG thermal unfolding process in the conditions assayed in these previous works (at protein concentrations below 4 mg mL−1, at low ionic strength and near neutral pH). The probable causes of this failure are: (1) below 55 °C, β-LG solutions are a mixture of monomers and dimers, (2) between 55 and 90 °C, β-LG heating induces irreversible changes in the monomeric protein structure and promotes the formation of aggregates of low molecular weight, and (3) above 90 °C, β-LG aggregates of high molecular weight are formed irreversibly. In this work, the presence of irreversible changes in β-LG heating was demonstrated by quenching the intrinsic fluorescence of the protein by added acrylamide.

The degree of exposure of tryptophanyl residues (Trp) in beta-lactoglobulin (β-LG) molecules can be evaluated by following the external quenching of the intrinsic protein fluorescence by added acrylamide. Based on this technique, we propose a method to monitor β-LG equilibrium denaturation profile by urea. The results were analyzed by the dissociation coupled unfolding (DCU) model. This model takes into account the impact of dimerization on β-LG stability. The values of free energy change for denaturing β-LG (ΔG°DCU) obtained in this work were 63.3 (±0.5) kJ mol−1 at pH 6.8 and 73.4 (±2.3) kJ mol−1 at pH 2.5. These results are in good agreement with previous results reported by other authors, that monitored the denaturation process by ultraviolet difference spectrophotometry. In addition, the protein dependence of denaturation equilibrium profiles by urea followed by fluorescence polarization measurements suggests that β-LG denatures by 3-state DCU process at both pH 6.8 and pH 2.5, with the dissociation of dimers preceding the unfolding of the monomers.