Comparison of droplet Flocculation in hexadecane oil-in-water emulsions stabilized by β-lactoglobulin at pH 3 and 7

The influence of surface and thermal denaturation of adsorbed beta-lactoglobulin (beta-Lg) on the flocculation of hydrocarbon oil droplets was measured at pH 3 and compared with that at pH 7. Oil-in-water emulsions (5 wt % n-hexadecane, 0.5 wt % beta-Lg, pH 3.0) were prepared that contained different levels of salt (0-150 mM NaCl) added immediately after homogenization. The mean particle diameter (d(43)) and particle size distribution of diluted emulsions were measured by laser diffraction when they were either (i) stored at 30degreesC for 48 h or (ii) subjected to different thermal treatments (30-95 degreesC for 20 min). In the absence of salt, little droplet flocculation was observed at pH 3 or 7 because of the strong electrostatic repulsion between the droplets. In the presence of 150 mM NaCl, a progressive increase in mean particle size with time was observed in pH 7 emulsions during storage at 30degreesC, but no significant change in mean particle diameter with time (d(43) similar to 1.4 +/- 0.2 mum) was observed in the pH 3 emulsions. Droplet aggregation became more extensive in pH 7 emulsions containing salt (added before thermal processing) when they were heated above 70degreesC, which was attributed to thermal denaturation of adsorbed beta-Lg leading to interdroplet disulfide bond formation. In contrast, the mean particle size decreased and the creaming stability improved when pH 3 emulsions were heated above 70degreesC. These results suggest that the droplets in the pH 3 emulsions were weakly flocculated at temperatures below the thermal denaturation temperature of beta-Lg (T < 70degreesC) but that floes did not form so readily above this temperature, which was attributed to a reduction in droplet surface hydrophobicity due to protein conformational changes. The most likely explanation for the difference in behavior of the emulsions is that disulfide bond formation occurs much more readily at pH 7 than at pH 3.