Luis Cascão PereiraGraduate Studentco-advised by Harvey BlanchÉcole Polytechnique Fédérale de Lausanne,
Lausanne, Switzerland
Research Interests:
Dynamic Aspects of Enzyme Adsorption
Funding Source:
|
|
Research Summary:
The Mechanisms of Foam and Emulsion Stability
by Proteins
| Thin-film interactions between fluid interfaces with adsorbed protein are of pivotal importance for the stabilization of many foams and emulsions encountered in the dairy and pharmaceutical industries. We have developed using semiconductor manufacturing procedures a novel type of microfabricated film holder, which extends the thin-film balance (TFB) technique to the study of protein foam films. With this technique we can directly measure the forces responsible for stable film formation and investigate film drainage and coalescence. | 
Fig.1 - Detail of microfabricated film holder used for enzyme thin-film studies. |
Fig.2 Initial formation of a b-casein foam film. |
Direct force measurements are obtained for the first time for fresh b-casein foam films under varying conditions of protein concentration, solution ionic strength, and pH. The force law is expressed in terms of the disjoining pressure P(h) as a function of film thickness h, which is directly ammenable to the potential of mean force. At large separations b-casein films are stabilized by electrostatic disjoining forces that are low in magnitude (< 400 Pa) but long-ranged (< 80 nm). |
| Steric repulsive forces due to the excluded volume of the protein molecules stabilize b-casein films at short separations. These films are 18-nm thick, supporting the picture of a protein bilayer, and are stable over several orders of magnitude of the disjoining pressure. High concentrations favor the formation of thicker films, whereas addition of salt and pH close to the isoelectric point favor thinner films. At intermediate concentrations or at low electrolyte concentrations a first-order phase-transition between those two types of film is observed. In opposition, ovalbumin films are stable over a wide-range of separations under similar solution conditions and no phase transition is observed. Film history strongly influences thin-film stability and drainage. In aged films, proteins aggregate and prevent thinning. Aggregation is favored by the degree of aging at the interface, high concentrations, and solution pH. Aged films display solid-like behavior, are stable at a given separation but will rupture upon further thinning. The mechanism by which aging influences the phase transition is not yet clear and is under investigation. |
This work is being currently carried on in collaboration with Christian Johannson, visiting student from Lund, Sweeden.
Check out our work on the department's web page here!
This work has been presented at:
Tupy, M., Cascão-Pereira, L.G., Blanch, H.W., and Radke, C.J., "Protein Conformation at Fluid/Fluid Interfaces: Application to Food Technology," Mars Corporation Symposium on Properties of Colloids and Complex Fluids, Department of Nutrition, University of California, Davis, CA 95616, March 1, 1999.
Cascão-Pereira, L.G., Blanch, H.W., and Radke, C.J., "Disjoining Forces in Thin Liquid Films Stabilized by Proteins," paper 93k, 1998 Annual Meeting of American Institute of Chemical Engineers, Miami Beach, FL, November 15-20, 1998.
Cascão-Pereira, L.G., Blanch, H.W., and Radke, C.J., "Properties
of Thin Liquid Foam Films Stabilized by Proteins," paper LB4, Food
Emulsions and Foams Symposium, Seville, Spain, March 16-18, 1998.
Dynamic Aspects of Enzyme Adsorption and Deactivation at Interfaces
Protein adsorption studies in our laboratory at the oil/water interface using TIRFS indicate that the saturation coverage increases with bulk protein concentration. TIRFS and dynamic tensiometry washout experiments show that proteins are reversibly adsorbed at both oil/water and air/water interfaces at early times while progressively becoming fully irreversible for later times. Surface gel-formation is observed at longer times. Interfacial catalysis experiments with the hydroxynitrile lyase enzyme at the oil/water interface also show irreversibility at longer times and loss of catalytic activity depending on the degree of hydrophobicity of the organic phase. Adsorption tensiometry studies using the pendant-drop technique indicate three different time regimes: the first one characterized by loading of the interface, the second by conformational change to an irreversible state and the long-time third regime by gelation and additional exposure of tensio-active groups. No equilibrium state is ever observed.
A continuum kinetic model is developed to account for protein/enzyme adsorption dynamics, enzymatic activity, and interfacial tension activity at the oil/water and air/water interfaces. Proteins are initially reversibly adsorbed at the interface and in equilibrium with the bulk concentration near interfacial layers. Protein adsorption is area-dependent. Once at the interface, they can go through conformational rearrangement and become irreversibly adsorbed followed by exposure of tensio-active groups. Proteins irreversibly adsorbed are modeled as a population of alpha and beta states representing two extremes in conformational change. The alpha-state is of native size whereas the beta-state is a protein molecule that undergoes significant spreading at the interface. Spreading to the beta-state is area-dependent. Both states are enzymatically active for the case of an enzyme at a non-denaturing interface. For the case of a denaturing solvent the beta-state is inactive since the degree of conformational change represented by this state is of a destroyed active site. Multilayer growth is taken into account by considering a second-layer of protein molecules adsorbed underneath irreversibly bound molecules of the first layer. Gelation is the result of lateral interactions between irreversibly bound molecules once they reach sufficient coverage.