Effects of Adsorbed Layer Composition and Structure on Interfacial Energy

Relative to small-molecule stabilizers and surfactants used in food systems, in a quanti-tative sense we know very little about how the molecular properties of a complex food polymer such as a globular protein influence its adsorption and eventual interfacial func-tion. Proteins constitute one of two classes of surface-active agents used in formulating and stabilizing industrial and colloidal food systems. Phospholipids (e.g., lecithins) and other nonprotein macromolecules are used as well, but only a few polysaccharides, such as modified cellulose derivatives or acetylated pectin, are considered sufficiently surface active for practical purposes [19]. Interfacial behavior is a cumulative property of a pro-tein, influenced by many factors. Among these are its size, shape, charge, and structural stability. Experimentally observed differences in interfacial behavior among different tein molecules have been particularly difficult to quantify in terms of these because pro-teins can vary substantially from one another in each category. Still, many experimental observations have been explained in terms of protein size, shape, charge, and tendency to unfold, as well as hydrophobicity of the interface itself [9]. Important findings in this regard are summarized in Table 2.3. The relevant interfacial dynamics contributing to changes in interfacial energy are strongly concentration dependent as well.

It is well accepted that a given protein can exist in multiple adsorbed conformational

“states” at an interface [7–9]. These states can be distinguished by differences in occupied area, binding strength, propensity to undergo exchange events with other proteins, and catalytic activity, or function. All of these features of adsorbed protein are interrelated, and can be time dependent. For example, decreases in surfactant-mediated elution of proteins from an adsorbed layer (an indirect measure of binding strength) are observed as protein–

surface contact time increases [20]. This time dependence is illustrated in Figure 2.5. As conformational change proceeds, the likelihood of desorption decreases. Another impor-tant observation is that protein adsorption is often a practically irreversible process, at least in the sense that it is often irreversible to dilution, or buffer elution. The adsorbed mass remains constant or decreases very little when the solution in contact with the interface is depleted of protein. However, although spontaneous desorption is not generally observed, adsorbed protein can undergo exchange reactions with similar or dissimilar protein mol-ecules, surfactants, or other sufficiently surface-active species adsorbing from solution [7–9].

Such exchange reactions are shown schematically in Figure 2.6. Adsorbed protein exchange rates are likely state dependent, being slower for more conformationally altered proteins.

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With reference to oil-in-water emulsions, qualitatively, the higher the “emulsifying activity” of a given protein or other stabilizer, the greater the amount of oil droplets “solu-bilized” during emulsion formation. After homogenization, dispersed oil droplets will rise and coalesce to form a floating layer. “Emulsion stability” is commonly measured in terms of the amount of oil separating from an emulsion during a certain period of time under certain conditions, reflecting the rate of this rise and coalescence. These properties can be measured by turbidimetric techniques or changes in suspension conductivity, for example [19]. In any event, higher emulsion activities and emulsion stabilities are achieved by molecules better able to reduce interfacial energy, as generally suggested by the guide-lines provided in Table 2.3.

Table 2.3 Factors that Affect Protein Adsorption

Factor Mechanism of Action Ref.

Size Small molecules have higher diffusivities than large molecules, but tighter binding is consistent with multiple noncovalent contacts

[20]

Flexibility An ability to bind to a relatively small unoccupied area is a

requirement for adsorption at a crowded interface [21]

Structural stability Protein unfolding exposes hydrophobic residues which can

associate with the surface [7,8,22]

Hydrophobicity Hydrophobic association is consistent with high binding strength in aqueous media

[20,23]

Electrostatic charge When electrostatic interactions predominate, the attractive force between a protein and an oppositely charged surface can be strong

[24–26]

low ionic strength: protein and surface charge contrast determines the electrostatic interaction

High ionic strength: reduces electrostatic effects between protein and surface

Time

Figure 2.5 Surface-induced conformational changes undergone by adsorbed protein, result-ing in multiple noncovalent bonds with the surface, and coverage of greater interfacial area/mol-ecule. (Reproduced with permission from J McGuire, CK Bower, MK Bothwell. In A Hubbard, Ed.

encyclopedia of surface and Colloid science. New York: Marcel Dekker, 2002, pp. 4382–4395.)

The molecular-level dynamics contributing to changes in interfacial energy with mixed protein–surfactant systems are strongly concentration-dependent, and difficult to predict.

These will be very briefly summarized by considering the change in interfacial energy at the air–water interface that might be produced by increasing surfactant concentration in a protein–surfactant mixture [27]. In general, at very low surfactant concentrations, the interfacial energy is the same as it would be for pure protein. Interfacial energy then decreases, due to surfactant occupation of “empty sites” at the interface, as well as forma-tion of surface active, surfactant–protein complexes. At higher surfactant concentraforma-tions, interfacial energy is seen to “plateau,” presumably because it is energetically favorable for surfactant to bind to protein at these concentrations. In this high concentration range, the critical micelle concentration (cmc) for pure surfactant preparation may be exceeded.

Interfacial energy then decreases again with increasing surfactant concentration, a result of complete displacement of protein from the interface by surfactant. After this decrease, further increases in surfactant concentration have no effect on interfacial energy, indicat-ing that the cmc of the protein–surfactant mixture has been met.

For pure liquids, surface energy decreases with increasing temperature and becomes zero at the critical point [28]. The surface tension of liquid water, for example, decreases from 75.6 to 58.9 mN/m as temperature is increased from 0 to 100°C, in a fairly linear man-ner [29]. Very little literature exists on the temperature dependence of properties related to solid surface energy. Dispersive forces exist in all types of matter and always give an attractive force between adjacent atoms or atomic groups, no matter how dissimilar their chemical natures may be. The forces depend on electrical properties of the volume ele-ments involved and the distance between them, and are independent of temperature [13]. Thus, while γld and γsd are expected to be temperature independent, Wa waterab

, would be expected to decrease with increasing temperature. Temperature effects on surface energet-ics of some solid materials which are commonly encountered in food processing have been reported by McGuire et al. [30].

For liquid foods, protein solutions, mixed protein–surfactant systems, and so on, the effect of temperature on surface properties is not predictable in any quantitative sense, and must usually be measured experimentally. In such cases, the solution chemistry and time- and concentration-dependent denaturation and aggregation phenomena near the interface will affect the observed interfacial energetics.

Time

Figure 2.6 Exchange reaction between a conformationally altered, adsorbed protein (white) and a dissimilar protein (gray) adsorbing from solution. (Reproduced with permission from J McGuire, CK Bower, MK Bothwell. In A Hubbard, Ed. encyclopedia of surface and Colloid science. New York:

Marcel Dekker, 2002, pp. 4382–4395.)

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2.3 MEASUREMENT TECHNIQUES