Lateral Phase Separation in Adsorbed Binary Protein Films at the Air−Water Interface

Abstract

Lateral phase separation in two-dimensional mixed films of soy 11S/beta-casein, acidic subunits of soy 11 (AS11S)/beta-casein, and alpha-lactalbumin/beta-casein adsorbed at the air-water interface has been studied using an epifluorescence microscopy method. No distinct lateral phase separation was observed in the mixed protein films when they were examined after 24 h of adsorption from the bulk phase. However, when the soy 11S/beta-casein and AS11S/beta-casein films were aged at the air-water interface for 96 h, phase-separated regions of the constituent proteins were evident, indicating that the phase separation process was kinetically limited by a viscosity barrier against lateral diffusion. In these films, beta-casein always formed the continuous phase and the other globular protein the dispersed phase. The morphology of the dispersed patches was affected by the protein composition in the film. In contrast with soy 11S/beta-casein and AS11S/beta-casein films, no lateral phase separation was observed in the alpha-lactalbumin/beta-casein film at both low and high concentration ratios in the film. The results of these studies proved that proteins in adsorbed binary films exhibit limited miscibility, and the deviation of competitive adsorption behavior of proteins at the air-water interface from that predicted by the ideal Langmuir model (Razumovsky, L.; Damodaran, S. J. Agric. Food Chem. 2001, 49, 3080-3086) is in fact due to thermodynamic incompatibility of mixing of the proteins in the binary film. It is hypothesized that phase separation in adsorbed mixed protein films at the air-water and possibly oil-water interfaces of foams and emulsions might be a source of instability in these dispersed systems.

Stabilization of bubbles and foams

“…Mackie and Wilde [60 • ] have reviewed these surface structural aspects. In fact, the inhomogeneity of adsorbed layers is one important general factor that is increasingly recognized in the field of food colloids, due to further atomic force microscopy (AFM) measurements [60 • ] of films transferred from the A-W interface to solid substrates, fluorescence microscopy of labeled spread films [61,62] and in situ measurements of adsorbed and spread films via Brewster angle microscopy (BAM) [63,64]. Xu et al [65] recently used BAM to show how successive compressions and expansions in situ could generate film inhomogeneity on a macroscopic scale in adsorbed films of individual proteins (β-lactoglobulin and ovalbumin).…”

“…Proteins that have different adsorbed properties can also exhibit incompatibility at the interface [61,62,66]. The dynamics of protein-protein interfacial competition are significantly more complex, because the approach to the equilibrium adsorbed state of individual proteins is slow and the energy of desorption can increase considerably with prolonged adsorption and greater unfolding at the interface.…”

Importance of physical vs. chemical interactions in surface shear rheology

“…To test whether the introduced sulfhydryl groups are capable of covalent cross-links between proteins, solutions of each variant (in the same buffer as in the surface shear experiment) were concentrated to concentrations that are comparable to those encountered at the air -water interface [45]: 100 and 200 mg/mL. Then the samples were left to equilibrate for 22 h, identical to the surface shear experiments.…”

First-order phase transition during displacement of amphiphilic biomacromolecules from interfaces by surfactant molecules

“…As a result, it is observed that the mixed solution breaks up into two distinct phases, one rich in the associating molecules and the other in unmodified ones [27][28][29][30]. We also suspect that the possible surface phase separation reported for certain mixed protein films occurring at air-water interfaces [31][32][33][34][35] is also the result of a very similar mechanism.…”