Role of postadsorption conformation changes of β-lactoglobulin on its ability to stabilize oil droplets against flocculation during heating at neutral pH

The influence of heating on droplet aggregation in hydrocarbon oil-in-water emulsions stabilized by a globular protein was examined using laser diffraction. Different levels of salt (0 or 150 mM NaCl) were added to n-hexadecane oil-in-water emulsions stabilized by beta-lactoglobulin (beta-Lg, pH 7.0) either before or after isothermal heat treatment (30-95 degreesC for 20 min). At 0 mM NaCl, no aggregation was observed in any of the emulsions because of strong electrostatic repulsion between the droplets. At 150 mM NaCl, droplet flocculation occurred between 30 and 65 degreesC due to surface denaturation of beta-Lg after adsorption. Above 70 degreesC, droplet flocculation became less extensive when 150 mM NaCl was added to the emulsions after heating but more extensive when the- salt was added before heating, which was attributed to thermal denaturation of adsorbed beta-Lg. When the droplets are in close proximity during heating (150 mM NaCl), interactions between proteins adsorbed onto different droplets are favored, but when droplets are not in close proximity (0 mM NaCl), interactions between proteins adsorbed onto the same droplets are-favored. Addition of N-ethylmaleimide, a sulfhydryl blocking agent, to the emulsions immediately after homogenization prevented droplet aggregation due to surface or thermal denaturation, highlighting the importance of disulfide bond formation on droplet flocculation stability. The mean droplet size decreased when small molecule surfactant (1 wt % Tween 20) and reducing agent (1 wt % 2-mercaptoethanol) were added to the emulsions, which indicated that the droplets were flocculated rather than coalesced. Our data show that the magnitude of droplet-droplet interactions during thermal denaturation of adsorbed globular proteins has a pronounced influence on the heat stability of protein-Aabilized emulsions. This study has important implications for the formulation and production of protein stabilized oil-in-water emulsions.