188.8.131.52. Protein Hydration and Water Solubility
Many of the functional properties of biopolymers in food emulsions are governed by their interactions with water (e.g., solubility, dispersibility, swelling, thickening, emulsification, foaming, and gelling) (Suggett 1975a,b; Damodaran 1996; Fennema 1996b). Biopolymer ingredients are usually added to the aqueous phase of food emulsions in a powdered form. The functional properties of many of these biopolymers are only exhibited when they are fully dissolved and evenly distributed throughout the aqueous phase. Consequently, the effective dissolution of the powdered biopolymer ingredient in an aqueous solution is an important part of emulsion preparation. This process involves a number of stages, including dispersion, wetting, swelling, and dissolution. The effectiveness and rate of dissolution depend on many factors, including the pH, ionic strength, temperature, and composition of the aqueous phase, as well as the application of shearing forces.
It is useful to examine the way in which successive water molecules bind to dried biopolymer molecules (Fennema 1996b). First, the charged groups are hydrated, then the polar groups, and finally the nonpolar groups. Charged groups bind more water molecules per group (3 to 7) than polar groups (2 to 3), which in turn bind more than nonpolar groups (~1). The water in a food containing biopolymers may exist in a number of different environments, including physically bound water, hydrodynamic water, capillary water, and free water (Fennema 1996b).
The water solubility of a biopolymer molecule depends on its compatibility with the aqueous solvent in which it is dispersed, which is governed by the relative magnitude of biopolymer-biopolymer, water-biopolymer, and water-water interactions. A biopolymer molecule has a low water solubility when the strength of the water-biopolymer interactions is significantly weaker than the average strength of the water-water and biopolymer-biopolymer interactions, because the molecules tend to associate with each other rather than with the solvent molecules (Section 2.6). An appreciation of the factors which determine the water solubility of a protein depends on an understanding of the various types of molecular interaction and entropy effects discussed in Section 4.6.2.
In general, the water solubility of biopolymer molecules is determined by a combination of van der Waals, electrostatic, hydrogen bonding, steric overlap, and hydrophobic interactions, as well as by the configurational entropy. In practice, the water solubility is usually dominated by only one or two of these interactions, the most common being the hydrophobic and electrostatic interactions (Damodaran 1996). It is therefore important for food scientists to identify the interactions which are most important for each type of biopolymer and to establish the influence of solvent conditions on these interactions.
Hydrophobic interactions promote aggregation and therefore lead to poor water solubility. They are particularly important when biopolymer molecules have high proportions of nonpolar groups on their surface. This accounts for the decreasing solubility of proteins with increasing surface hydrophobicity (Damodaran 1996) and for the fact that many globular proteins become insoluble in water when they are heated above a temperature where the protein molecule unfolds and exposes nonpolar groups (Damodaran 1996).
Electrostatic interactions play a major role in determining the water solubility of biopolymers that have charged groups and are important for all proteins and many polysaccharides (BeMiller and Whistler 1996, Damodaran 1996). Electrostatic interactions may be either attractive or repulsive, depending on the signs of the charges involved, and they may therefore either increase or decrease water solubility. A protein molecule is positively charged at pH values below its isoelectric point and negatively charged at pH values above it. As a consequence, there is an electrostatic repulsion between similarly charged molecules which prevents them from coming close enough together to aggregate and therefore enhances their water solubility (de Wit and van Kessel 1996). In addition, the molecules are also prevented from aggregating because the ionized groups are highly hydrated, which leads to a short-range hydration repulsion (Section 3.8). The water solubility of most proteins decreases dramatically at their isoelectric point (Franks 1991, Damodaran 1996). A protein molecule has no net charge at its isoelectric point, but it does have some groups that are positively charged and some that are negatively charged. Proteins are therefore particularly susceptible to aggregation at their isoelectric point because there is no electrostatic repulsion to prevent them from coming close together. In fact, there may even be an electrostatic attraction between the positive groups on one protein molecule and the negative groups on another, which promotes protein aggregation and insolubility. A number of proteins are highly soluble across the whole pH range, because the attractive forces between them are not sufficiently strong to overcome the thermal energy of the system and so the molecules remain dispersed (Damodaran 1996).
The water solubility of biopolymer molecules is highly dependent on the type and concentration of electrolyte ions in solution (Franks 1991). These ions can influence the water solubility of biopolymers in a number of different ways. At relatively low ionic strengths, electrolyte ions screen electrostatic interactions between molecules (Section 3.4.2). At the isoelectric point, one would expect screening to decrease the strength of the electrostatic attraction between protein molecules and therefore increase solubility. On the other hand, at pH values away from the isoelectric point, one would expect screening to decrease the electrostatic repulsion between charged biopolymers and therefore decrease their solubility. At intermediate ionic strengths, electrolyte ions may bind to the surface of biopolymers, thus increasing the short-range hydration repulsion interactions between them and thereby increasing their solubility (Israelachvili 1992). Alternatively, the electrolyte may alter the structural organization of the water molecules, which can either increase or decrease the magnitude of the hydrophobic attraction, depending on the nature of the ions involved (Israelachvili 1992, Damodaran 1996). At high ionic strengths, biopolymer molecules are often precipitated out of solution above a critical salt concentration, which is known as "salting out," because the majority of water molecules are strongly "bound" to the electrolyte ions and are therefore not available to hydrate the biopolymers (Damodaran 1996).
The temperature of an aqueous solution also plays an important role in determining the water solubility of biopolymers (Damodaran 1996). Altering the temperature of a solution changes the relative magnitude of the various kinds of molecular interaction and entropy effects mentioned in Section 4.6.2. This alters the balance between the forces favoring solubility and those favoring insolubility. An increase in temperature usually causes an increase in the strength of the hydrophobic attraction, a decrease in the strength of hydrogen bonds, a decrease in the magnitude of the hydration repulsion, an increase in the strength of any electrostatic interactions, and an increase in the configurational entropy. The effect of temperature on the solubility of a biopolymer therefore depends on the relative importance of the various types of interactions under a given set of experimental conditions.
The water solubility of a biopolymer can be characterized by a solubility index (SI), which is a measure of the percentage of biopolymer which is soluble in an aqueous solution under a specified set of solvent and environmental conditions (e.g., pH, ionic strength, temperature, and centrifugation speed) (Damodaran 1996). A known concentration of biopolymer is dispersed in solution, and then the solution is ultracentrifuged at a specific speed and time. The concentration of biopolymer remaining in solution is measured using an appropriate technique:
M initial where Mremaining and Minitial are the concentrations of protein in solution after and before centrifugation.
The solubility index is often a good indication of the degree of denaturation of a protein ingredient: the lower the solubility index, the greater the degree of denaturation.
Biopolymers that have significant amounts of both polar and nonpolar groups tend to be surface active (i.e., they are able to accumulate at oil-water or air-water interfaces (Dickinson 1992, Dalgleish 1996a, Damodaran 1996). The major driving force for adsorption is the hydrophobic effect. When the biopolymer is dispersed in an aqueous phase, some of the nonpolar groups are in contact with water, which is thermodynamically unfavorable, because of hydrophobic interactions (Section 4.4.3). When a biopolymer adsorbs to an interface, it can adopt a conformation where the nonpolar groups are located in the oil phase (away from the water) and the hydrophilic groups are located in the aqueous phase (in contact with the water). Adsorption also reduces the contact area between the oil and water molecules at the oil-water interface, which lowers the interfacial tension (Chapter 5). Both of these factors favor the adsorption of an amphiphilic molecule to an oil-water interface. The conformation that a biopolymer adopts at an interface and the physicochemical properties of the membrane formed depend on its molecular structure and interactions (Das and Kinsella 1990, Dickinson 1992, Dalgleish 1996a, Damodaran 1996). Flexible random-coil biopolymers adopt an arrangement where the predominantly nonpolar segments protrude into the oil phase, the predominantly polar segments protrude into the aqueous phase, and the neutral regions lie flat against the interface (Figure 4.17). The membranes formed by these types of molecules tend to be relatively open, thick, and of low viscoelasticity. Globular biopolymers (usually proteins) adsorb to an interface so that the predominantly nonpolar regions on the surface of the molecule face the oil phase, while the predominantly polar regions face the aqueous phase, and so they tend to have a definite orientation at an interface (Figure 4.18). Once they have adsorbed to an interface, biopolymers often undergo structural rearrangements so that they can maximize the number of contacts between nonpolar groups and oil. Random-coil biopolymers are relatively flexible molecules and can therefore rearrange their structures fairly rapidly, whereas globular biopolymers are more rigid molecules and therefore rearrange more slowly. The unfolding of a globular protein at an interface often exposes amino acids that were originally located in the hydrophobic interior of the molecule, which can lead to enhanced interactions with neighboring protein molecules through hydrophobic attraction or disulfide bond formation (Dickinson and Matsumura 1991, McClements et al. 1993d). Consequently, globular proteins tend to form relatively thin and compact membranes that have high viscoelasticities (Dickinson 1992).
FIGURE 4.17 The structure of the interfacial membrane depends on the molecular structure and interactions of the surface-active molecules.
Membranes formed by globular proteins therefore tend to be more resistant to rupture than those formed from random-coil proteins.
To be effective emulsifiers, biopolymers must rapidly adsorb to the surface of the emulsion droplets created during homogenization and then form a membrane that prevents the droplets from aggregating with one another (Chapter 6). Biopolymer membranes stabilize emulsion droplets against aggregation by a number of different mechanisms. All biopolymers provide short-range steric repulsive forces that are usually sufficiently strong to prevent droplets from getting close enough together to coalesce (Section 3.5). If the membrane is sufficiently thick, the steric repulsive forces can also prevent droplets from flocculating. Otherwise, it must be electrically charged so that it can prevent flocculation by electrostatic repulsion (Section 3.4). The properties of emulsions stabilized by charged biopolymers are particularly sensitive to pH and ionic strength. At pH values near the isoelectric point of proteins, or at high ionic strengths, the electrostatic repulsion between droplets may not be sufficiently large to prevent the droplets from aggregating.
A wide variety of proteins are used as emulsifiers in foods because they naturally have a high proportion of nonpolar groups and are therefore surface active (Charalambous and Doxstakis 1989, Dickinson 1992, Damodaran 1996). Most polysaccharides are predominantly hydrophilic and are therefore not particularly surface active (BeMiller and Whistler 1996). However, a small number of naturally occurring polysaccharides do have some hydrophobic character (e.g., gum arabic) or have been chemically modified to introduce nonpolar groups (e.g., some modified starches), and these biopolymers can be used as emulsifiers (BeMiller and Whistler 1996).
The second major role of biopolymers in food emulsions is to increase the viscosity of the aqueous phase (Mitchell and Ledward 1986). Viscosity enhancement modifies the texture and mouthfeel of food products ("thickening"), as well as reducing the rate at which particles sediment or cream ("stabilization"). Biopolymers used to increase the viscosity of aqueous solutions are usually highly hydrated and extended molecules or molecular aggregates. Their ability to increase the viscosity of a solution depends principally on their molecular weight, degree of branching, conformation, and flexibility (Launay et al. 1986, Rha and Pradipasena 1986).
The viscosity of a dilute solution of particles increases linearly as the concentration of the particles increases (Section 8.4):
where n is the viscosity of the particulate solution, no is the viscosity of the pure solvent, and ^ is the volume fraction of particles in solution. The ability of some biopolymers to greatly enhance the viscosity of aqueous solutions when used at very low concentrations is because their effective volume fraction is much greater than their actual volume fraction. A biopolymer rapidly rotates in solution because of its thermal energy, and so it sweeps out a spherical volume of water that has a diameter approximately equal to its end-to-end length (Figure 4.18). The volume of the biopolymer molecule is only a small fraction of the total volume of the sphere that is swept out, and so the effective volume fraction of a biopolymer is much greater than its actual volume fraction. The effectiveness of a biopolymer at increasing the viscosity increases as the volume fraction that it occupies within this sphere decreases. Thus, large highly extended linear biopolymers increase the viscosity more effectively than small compact branched biopolymers (Launay et al. 1986, Rha and Pradipasena 1986).
In a dilute biopolymer solution, the individual molecules (or molecular aggregates) do not interact with each other (Dickinson 1992, Lapasin and Pricl 1995). As the concentration of biopolymer increases above some critical value (c*), the viscosity of the solution increases rapidly because the spheres swept out by the biopolymers begin to interact with each other. This type of solution is known as a semidilute solution, because even though the molecules are interacting with one another, each individual biopolymer is still largely surrounded by solvent molecules. At still higher polymer concentrations, the molecules pack so closely together that they become entangled with each other and the system has more gel-like characteristics. Biopolymers which are used to thicken the aqueous phase of emulsions are often used in the semidilute concentration range (Dickinson 1992).
Solutions containing extended biopolymers often exhibit strong shear-thinning behavior (pseudoplasticity); that is, their apparent viscosity decreases with increasing shear stress (Lapasin and Pricl 1995). Shear thinning occurs because the biopolymer molecules become aligned with the shear field or because the weak physical interactions responsible for biopolymer-biopolymer interactions are disrupted. Some biopolymer solutions have a characteristic yield stress. If such a biopolymer solution experiences an applied stress which is below its yield stress, it acts like an elastic solid, but when it experiences a stress that exceeds the yield stress, it acts like a liquid (Section 8.2.3). The characteristic rheological behavior of biopolymer solutions plays an important role in determining their functional properties in food emulsions. For example, a salad dressing must be able to flow when it is poured from a container, but must maintain its shape under its own weight after it has been poured onto a salad. The amount and type of biopolymer used must therefore be carefully selected so that it provides a low viscosity when the salad dressing is poured (high applied stress) but a high viscosity when the salad dressing is allowed to sit under its own weight (low applied stress). The viscosity of biopolymer solutions is also related to the mouthfeel of a food product. Liquids that do not exhibit extensive shear-thinning behavior at the shear stresses experienced within the mouth are perceived as being "slimy." On the other hand, a certain amount of viscosity is needed to contribute to the "creaminess" of a product. The shear-thinning behavior of biopolymer solutions is also important for determining the stability of food emulsions to creaming. As an oil droplet moves through an aqueous phase, it only exerts a very small shear stress on the surrounding liquid. As a result of the shear-thinning behavior of the solution, it experiences a very high viscosity, which greatly slows down the rate at which it creams.*
Many biopolymer solutions also exhibit thixotropic behavior (their viscosity decreases with time when they are sheared at a constant rate), because the weak physical interactions that cause biopolymer molecules to aggregate are disrupted. Once the shearing stress is removed from the sample, the biopolymer molecules may be able to reform the weak physical bonds with their neighbors, and so the system regains its original structure and rheological properties. This type of system is said to be reversible, and the speed at which the structure is regained may be important for the practical application of a biopolymer in a food. If the bonds are unable to reform once they are disrupted or if they are only partially reformed, then the system is said to be irreversible or partially reversible, respectively.
A food manufacturer must therefore select an appropriate biopolymer or combination of biopolymers to produce a final product that has a desirable mouthfeel, stability, and texture. Both proteins and polysaccharides can be used as thickening agents, but polysaccharides are usually preferred because they tend to have higher molecular weights and be more extended so that they can be used at much lower concentrations.
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