Water binding is an important functional property for sev- eral reasons: 1) most foods contain high amounts of water
and if their chemistry changes in a manner that would cause the formation of free water or drip loss, consumers would be displeased, 2) increasing the amount of water a product can hold effectively can increase the profitability of a given product, and 3) both product yield and sensory quality are highly dependent on the proper moisture con- tent of a finished food. Water-binding capacity is the amount of water that is bound or retained by a protein under highly defined conditions.
Water is usually be bound to the surface of a protein by hydrogen bonding, which is sometimes called dipole bonding. Hydrogen bonding results from water’s interac- tion with the R group of amino acids which are dipoles, such as serine and threonine, or ionic, such as lysine or aspartic acid. Water bound to the surface of proteins in this manner is called “monolayer” water and is very tightly associated with the protein. Other water associated with the protein or protein matrices can be trapped in cap- illary structures and pores. In food systems, water can also be held in cells and other structural networks. Water that is not associated with the monolayer on the protein sur- face is called free water and moves unhindered through- out the food system. Depending on the food or model system, water-binding capacity tests measure a mixture of the free and bound water present.
1. Factors Influencing Water Binding
For a given food system, the protein concentration, tem- perature, salt type and concentration, and degree of denat- uration impact on the water binding exhibited. In addition, small polar molecules, such as sugars or sugar alcohols, will enhance water binding by proteins in general. As the protein concentration increases, so does the water binding observed. Generally, water binding increases as tempera- ture increases. In some cases the proteins will form gels, which will enhance the binding of water by the system. Many types of whey protein and soy protein isolates will form gels when heated above 80°C. Enhanced binding in gels is due to water being bound by both hydrogen bonds and by being trapped in pores.
Ions also influence water binding. Sodium chloride binds to charged groups on protein surfaces and weakens intermolecular bonds. This is a positive effect in systems which utilize muscle fibers as part of the structural ele- ments of the food. Salt allows the muscle proteins to dis- tance themselves from others within the muscle fiber and thus increase the number of sites for water to bind. In var- ious muscle types, water binding may be increased 2–3 fold by the presence of salt (15).
The pH of a system markedly influences its ability to bind water. This is due to changes in the surface charges on a protein as the pH is altered. Water binding is lowest at the isoelectric point (pI) of a protein. As the pH is adjusted away from the isoelectric point the ionic charges
on the protein increase dramatically and water binding is enhanced. As the pH is lowered from the isoelectric point the protein assumes a net positive charge. Conversely, as the pH is increased, a net negative charge is seen.
2. Methods of Measurement
Water-binding model systems must be carefully defined if reproducible results are expected. Most of the methods are empirical and are usually designed for a specific product or application. At least 15 different methods have been developed to measure the water binding of various mus- cle/meat proteins (16). In general, two main types of tests exist. One is based on sorption—the adsorptions of water by a dry powder of protein. The other is sometimes called “expressible moisture” where a product is subjected to a force and the amount of moisture expelled is measured. The force is usually pressure or centrifugation. These types of tests must be carefully designed so that the actual internal structure of the gel or food is not destroyed when the pressure is applied. This would lead to an underesti- mation of the water binding for the system since water that normally would have been trapped in the matrix would be expressed (17).
D. EMULSIFICATION
An emulsion is a mixture of two immiscible liquids in which one is dispersed in the other in the form of droplets (18). It is common practice to call the liquid in the droplets the dispersed, internal, or discontinuous phase. The surrounding phase is called the external or continuous phase. Emulsions in which the dispersed phase is a lipid are called “oil in water” emulsions (o/w). Water in oil emulsions contain droplets of water dispersed in a lipid continuous phase. Egg yolk and milk are examples of nat- ural emulsions. Many manufactured foods are intentional emulsions including ice cream, salad dressings, chocolate, mayonnaise, cakes, frosting, butter, and spreads. Food emulsions are far more complex than a droplet of one phase suspended in another. Foods contain many other materials such as air, particulates or other dispersed solids, partially crystallized fat, and gels.
1. Principles of Emulsification
When a liquid is exposed to air, the surface between them is in a state of tension. This so-called “surface tension” is due to the attractive forces between molecules in the liq- uid that are enhanced by exposure to air. In lay terms, the molecules “bunch” together to decrease their exposure to the air surface. When two immiscible liquids, such as water and oil are in contact, the region of contact is called the interface, with the development of “interfacial ten- sion.” As the interfacial area increases, the stability of the mixture decreases. An example would be the creation of
temporary emulsion of oil in water by forming millions of small droplets of oil suspended in the water phase. One can imagine creating this mixture by blending several mil- liliters of oil into a cup of water in a high speed mixer. This type of mixture is called a “temporary emulsion,” for although there would initially be millions of oil droplets, they would rapidly coalesce into a separate oil layer which would form on top of the aqueous phase. Coalesced droplets have less surface area exposed to the water, and thus are more stable. Vinegar and oil salad dressing is a classic example of temporary emulsions.
To stabilize emulsions it is necessary to add molecules which decrease the interfacial tension between mixtures of lipids and water. Surfactants are molecules that contain both hydrophobic (non-polar) and hydrophylic (polar) regions in their structure. When added to a system that contains both lipids and water, surfactants rapidly migrate to the interfaces between the two phases. At the interface, the surfactants orient their polar region towards the aque- ous phase and their non-polar region towards the lipid phase. Since proteins contain amino acid residues that can be polar and non-polar they can be excellent surfactants in food systems. Figure 7.2 illustrates the coating of an oil or fat droplet with a protein molecule. Once the droplet or air bubble is coated, the interfacial tension between the two
phases is markedly lowered and the tendency to coalesce is greatly reduced. The simplistic illustration in Figure 7.2 shows a single molecule of protein unfolding on the oil surface. In a real emulsion, there are thousands of mole- cules involved on the surface of a single droplet. If a suffi- cient reduction in interfacial tension is achieved, the emulsion can be stable for long periods of time.
2. Factors Affecting Protein-Based Emulsions
Food emulsions are complex systems which are normally created by using large amounts of energy to form very small particles of one phase which become suspended in another. Higher energy inputs normally result in smaller droplets and enhanced activity of any surfactants that are present. To work well as a surfactant, a protein must be able to migrate to the interface, orient polar and non-polar side chains into the proper phases, and form a stable film around the droplet. In many cases proteins may partially unfold or denature during these activities. After unfolding and orientation towards the proper phase, proteins form multiple layers on the droplet surface due to intermolecu- lar ionic, hydrogen, and hydrophobic bonding between unfolded protein strands. The formation of superior emul- sions by proteins relates to their ability to form viscous yet flexible films around the surface of droplets (19).
Most food emulsions are oil in water. In forming o/w emulsions, the initial water solubility of the protein is very important. Therefore, factors such as protein concentra- tion, pH, salts, ionic strength, and temperature strongly affect the emulsification ability of proteins. Other factors that influence their ability to emulsify are related to the physicochemical properties of the protein, such as surface charge, surface hydrophobicity, molecular flexibility, ease of denaturation, and dissociation behavior of subunits. Table 7.3 lists the factors which can be important to the formation and stability of protein-based emulsions.
Since proteins vary in physicochemical properties, it is not unexpected that researchers have seen varying results when studying the effects of pH, salts, and tem- perature on the emulsification ability of proteins. Because proteins have unique primary, secondary, and quaternary structures, environmental effects such as pH must be determined for each individual protein. In general, pro- teins near their isoelectric point (pI) are poor emulsifiers, presumably due to poor solubility. As the pH is adjusted away from the pI, improvement in emulsification is gen- erally seen and is probably due to enhanced solubility. Very low or high pH may lead to poor emulsification even though the proteins might be very soluble. In this case, the proteins are so highly charged they do not interact to form films on the surface of the dispersed phase. If proteins are coated with an excess of charges they will not form cohe- sive, flexible films (19). Proteins with multiple subunits, such as the soybean storage protein legumin, may exhibit
Food Protein Functionality 7-7
Protein strand: P
P H H P H P
Oil or fat droplet
Aqueous environment
FIGURE 7.2 Formation of a protein stabilized emulsion. The protein is represented by the connected circles and squares, which are defined as follows:
Squares are hydrophobic (H) amino acid residues which orient towards the oil or fat phase.
Circles are hydrophylic (P) amino acid residues which orient towards the aqueous phase.
The continuous line represents the peptide bonds connecting the amino acids.
improved emulsification activity at pH values where the subunits are encouraged to dissociate (20).
Several researchers have shown that surface hydrophobicity can be highly related to the ability of pro- teins to act as emulsifiers (21). In general, proteins that are water soluble but which have large numbers of hydropho- bic groups on their surface tend to be superior emulsifiers. With increased surface hydrophobicity, a larger portion of the protein strand might be able to interact with the oil or fat droplet and form stronger protein films (22). Results have shown that proteins with high surface hydrophobici- ties, such as casein or bovine serum albumin (BSA), are superior emulsifiers compared to proteins with low sur- face hydrophobicities, such as collagen or gelatin (21).
3. Methods of Measurement
As with other model systems tests, the results of protein emulsification evaluations are often highly empirical. Comparison of results between laboratories is only possi- ble when strict testing conditions are maintained. Although the absolute numbers obtained in these tests may vary from lab to lab, the ranking of different proteins in terms of ability to emulsify or emulsion stability are often similar. The results seen in rapid tests may not give the same results seen in a true food system. Many food companies, however, cannot wait a year for results when they are evaluating proteins for use in products under development and subject to an accelerated development time table.
Emulsions are commonly tested for two parameters: stability and capacity. Emulsification capacity is normally measured as the maximum amount of oil a protein may emulsify. A classic test of emulsification capacity is that of Wang and Kinsella (23), where the maximum amount of oil that a protein can emulsify is determined by a titra- tion method. Oil is added to a protein solution in a blender or mixer until the emulsion fails, which is determined by a rapid decrease in viscosity. Advantages are that the pro- cedure is quick and simple. Disadvantages are that the results are highly dependent on the equipment used to generate the emulsions and the protein:lipid ratios encountered in the test are not commonly encountered in food systems (24). Another widely used technique is the Emulsification Activity Index (EAI) developed by Pearse and Kinsella (25). In this assay, the interfacial area created
in an emulsion is measured by a spectrophotometric assay. As the ability of a protein to emulsify increases, smaller and smaller emulsion droplets are formed, which can be measured by light scattering principles. Advantages of this method are that it is a rapid technique that does not rely on the use of an external force to break the emulsion. Disadvantages are: 1) emulsions must be prepared under very standardized conditions (e.g., sample container vol- ume, homogenizer or mixer manufacturer, speed or power settings) and 2) several studies have found poor correla- tions between EAI and emulsion stability (24).
Emulsion stability can be measured by a variety of methods. Choice of method depends on the type of insta- bility or emulsion breakdown observed in the actual food system where the protein may be used. Separation and clustering of fat or oil droplets on the top of an emulsion is called “creaming” and is a marked sign of emulsion breakdown. A standard test for creaming (26) involves the placement of an emulsion in a graduated cylinder which is held in an environmental chamber. The height of the boundary between the top of the creaming layer and the residual emulsion is measured as it decreases with time. Advantages of the test are that it can be set up rapidly and it can be easily used to determine how pH, ionic strength, and protein concentration impact emulsion stability. Disadvantages are that lengthy storage times under highly controlled conditions are required (24). A more rapid test is the Emulsion Volume Index where centrifugation in microhematocrit tubes is used to accelerate the forces which cause emulsion breakdown (27). Advantages are that analysis times are much shorter and other researchers have found correlations between EVI and longer term stability testing of actual products (24). If time is not essential, it is possible to use a long-term storage test to evaluate emulsion stability by measuring droplet size distribution and concentration in a hermetically sealed container (28). Although the test can duplicate actual stor- age conditions of products, it may take over a year to complete (24).
E. FOAMING
Emulsions and foams are similar types of food systems in that they contain two distinct phases. In foams, the liquid or solid continuous phase surrounds a dispersed gaseous phase which is usually air. Many times a protein which
TABLE 7.3
Factors Affecting Formation and Stability of Protein-Based Emulsions
Environmental factors Temperature, pH, salt concentration, salt type, ionic strength, other ions, other food components
Protein characteristics Solubility, ease of denaturation, surface hydrophobicity, surface charge, isoelectric point, flexibility, elasticity of protein film
Processing parameters Amount and rate of energy input (shear), oil or fat type, oxidative state of lipid, temperature
emulsifies well will also foam well. The first step in the formation of a foam is the migration of proteins to the interface between air bubbles and the aqueous phase. At the interface the protein will unfold and orient their non- polar regions toward the air phase. As proteins adsorb on the bubble surface, they begin to form layers of partially denatured proteins which encapsulate the air bubble and prevent the foam from collapsing (29). Foams may be pro- duced by mechanical agitation (whipping) or sparging, which is the injection of gas through very small orifices to produce bubbles. Most food foams are produced by whip- ping and include meringues, soufflés, whipped cream, non-dairy whipped toppings, angel food or sponge cakes, and ice cream. Yeast leavened breads are foams that are produced by the trapping of carbon dioxide bubbles by the gluten protein matrix.
1. Factors Affecting Foaming
As with other functional properties, solubility plays a crit- ical role in foaming. Good foaming proteins also exhibit one or more of the following molecular properties: 1) high rates of diffusion and adsorption at the interface, 2) abil- ity to unfold and denature at the interface, and 3) ability to form intermolecular associations with other molecules that result in the formation of cohesive films around the air bubble (1). The surface hydrophobicity of proteins cor- relates with its ability to form foams (21). Since it is important for a protein to unfold on the interface, the foaming ability of some proteins can be improved by a mild heat treatment or chemical modification which loosens the protein structure and allows it to unfold more rapidly. Enhanced film formation through increased inter- molecular bonding is also generally seen. Excessive denaturation will decrease foam formation and stability by decreasing initial protein solubility and causing protein films to form at the interface that are inflexible and stiff. This is a result of excessive intermolecular interaction between protein strands.
In regard to foaming ability, egg whites (albumen) form some of the highest quality foams due to the proper- ties of the constituent proteins, ovalbumin, globulins, and ovomucoid. During the foaming of egg whites, acids such as cream of tartar (potassium acid tartrate) are added after an initial whipping period. The acid lowers the pH and reduces the net charge on the protein which allows the protein strands to interact more strongly. It also facilitates the denaturation of proteins to increase the elasticity of protein films around the air bubbles. Overall, a more sta- ble film results. Addition of the tartrate before the initial foaming causes the proteins to unfold prematurely and interact before they reach the air bubble surface, which results in decreased foam volumes and stabilities (1).
In most cases, the foaming ability of proteins in inhib- ited by the presence of lipids. Contamination of egg white
with as little as 0.03% egg yolk completely inhibits foam- ing (30). It is theorized that lipid is absorbed at the air/water interface within the foam and causes the protein film to rupture. Conversely, high concentrations of satu- rated fat can stabilize foams. An example is whipped dairy cream in which cold coalesced fat droplets can sur- round protein-encapsulated air bubbles, resulting in a very stable foam.
The amount of energy used during foam formation impacts on foam stability. Energy inputs must be sufficient