Front-face fluorescence spectroscopy study of globular proteins in emulsions: influence of droplet flocculation

Measurement of the intensity (I(MAX)) and/or wavelength (lambda(MAX)) of the maximum in the tryptophan (TRP) emission spectrum using front-face fluorescence spectroscopy (FFFS) can be used to provide information about the molecular environment of proteins in nondiluted emulsions. Many protein-stabilized emulsions in the food industry are flocculated, and therefore, we examined the influence of droplet flocculation on FFFS. Stock oil-in-water emulsions stabilized by bovine serum albumin were prepared by high-pressure valve homogenization (30 wt % n-hexadecane, 0.35 wt % BSA, pH 7). These emulsions were used to create model systems with different degrees of droplet flocculation, either by changing the pH, adding surfactant, or adding xanthan. Emulsions (21 wt % n-hexadecane, 0.22 wt % BSA) with different pH (5 and 7) and molar ratios of Tween 20 to BSA (R = 0-131) were prepared by dilution of the stock emulsion. As the surfactant concentration was increased, the protein was displaced from the droplet surfaces, which caused an increase in both I(MAX) and lambda(MAX), because of the change in TRP environment. The dependence of I(MAX) and lambda(MAX) on surfactant concentration followed a similar pattern in emulsions that were initially flocculated (pH 5) and nonflocculated (pH 7). Relatively small changes in FFFS emission spectra were observed in emulsions (21 wt % n-hexadecane, 0.22 wt % BSA, pH 7) with different levels of depletion flocculation induced by adding xanthan. These results suggested that droplet flocculation did not have a major impact on FFFS. This study shows that FFFS is a powerful technique for nondestructively providing information about the molecular environment of proteins in concentrated and flocculated protein-stabilized emulsions. Nevertheless, in general the suitability of the technique may also depend on protein type and the nature of the physicochemical matrix surrounding the proteins.