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Physicochemical Aspects of Whey Protein Functionality1
Physicochemical Aspects of Whey Protein Functionality 1 MICHAEL E. MANGINO Ohio Agricultural Research and Development Center Department of Food Science and Nutrition The Ohio State University 2121 Fyffe Road Columbus 43210
strength of the network but do not form a dough as such. Fiber spinning and thermal extrusion are two more examples of processes that are not applied generally to whey proteins. Although in practice almost any protein can be extruded or spun, these processes are utilized almost exclusively with oil seed proteins. Many excellent reviews and books (5, 19, 20, 24, 25, 26, 28, 30) have been written on protein functionality, and these may be consuited for more complete discussion of the body of experimental literature. My paper will be limited to a discussion o f the nature o f forces involved and types o f interactions that must occur for" whey proteins to function in water binding, gelatin, emulsification, and foam formation.
ABSTRACT
Many of the desirable attributes of foods may be directly or indirectly related to the functionality of their protein components. The manner in which food proteins interact with other food components as well as with themselves determines their functionality. Forces in the maintenance o f protein structure are discussed as they relate to physical requirements necessary for water binding, gelation, emulsification, and foam formation. Factors that affect these functional properties are discussed as they relate to changes o f protein structure. The nature of interactions required for optimal functionality are related to conditions that alter protein structure in such a way as to encourage occurrence of these interactions.
PROTEIN STRUCTURE
INTRODUCTION
Functionality has been defined (31) as: " a n y property of a food or food ingredient, except its nutritional ones, that affects its utilization." F o r proteins then there must be a large number of functions and functional properties. Kinsella (19) suggested that properties in Table 1 are some of the most important ones to consider in discussing proteins. As far as functionality o f whey proteins is concerned, this entire list need not be considered. F o r example, dough formation generally refers to interactions of certain grain proteins that lead to formation of a network that can entrain gases. Whey proteins can affect the
Received August 22, 1983. 1Salaries and research support provided by State and Federal Funds appropriated t o t h e Ohio Agricultural Research and Development Center, The Ohio S t a t e University. Journal Article No. 128-83. 1984 J Dairy Sci 67:2711--2722
A protein manifests functionality b y interacting with other components within the food system. These interactions may involve solvent molecules, solute molecules, other protein molecules, or substances that are dispersed in the solvent such as oil or air. To describe forces in these interactions, it is essential that forces and energies in achievement and maintenance of native protein structure be described. Although complete discussion of the forces is b e y o n d the scope of this paper, some observations on the nature of protein structure will be useful. Proteins exist in the lowest kinetically attainable state of free energy (1). Whether this is the lowest state possible or just the one readily available still is being debated. It can be stated, however, that given a set of conditions, a protein molecule spontaneously will assume the conformation of lowest free energy. Protein structure is highly dependent upon the environment, and protein will assume different conformation as environmental conditions change (2, 17, 32, 37). Factors of importance include pH, temperature, dielectric
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TABLE 1. Functions of proteins in food (19). Flavor Binding Water binding Viscosity Gelation
Dough formation Fiber spinning Thermal extrusion Emulsification Formation
constant, ionic strength, and presence of other molecules including air, fat, denaturants, etc. One of the main ways that proteins lower their free energy involves removal of hydrophobic groups from the aqueous environment. This may provide the greatest single decrease of free energy of all types of binding within proteins (20). The strength of hydrophobic binding is, however, sensitive to changes of temperature and dielectric constant, and, thus, changes of these can have large influences on protein structure. Protein molecules may contain crosslinks of a noncovalent, salt bridges, or a covalent, disulfide bonds, nature. These crosslinks lower the conformational entropy of the molecule, which must be compensated for by a decrease of binding energy. The presence of crosslinks adds greatly to the stability of the native protein structure and makes the molecules resistant to unfolding or denaturation. For example, simple unfolding is inhibited sterically by crosslinking, because portions of the molecule that might be allowed normally to become exposed to the solvent are held in place by the crosslinks. Denaturation is also less likely because one of the driving forces of denaturation, an increase in conformational entropy, is reduced greatly (18). In a noncrosslinked protein, if unfolding to a random coil structure can be induced, there is a large gain in the number of conformations the molecule can assume. This gain in conformational entropy is a large driving force for maintenance of the denatured state when the denaturing agents are removed. In contrast, a highly crosslinked protein cannot assume the same degree of random conformations, and, thus, the increase in e n t r o p y is much less. This helps explain why molecules that contain large numbers of disulfide bonds are often resistant to denaturation. Journal of Dairy Science Vol. 67, No. 11, 1984
Structures attained by proteins are not rigid but are dynamic (17). There is rotational freedom about many o f the bonds within the protein molecule, and the entropy gain o f this freedom lowers the total free energy of the native structure. There are also portions of the protein structure that are stabilized by weak secondary forces, and these are often free to assume different conformations. These alternate conformations lead to structures of higher free energy and, thus, are not stable or long lived. A protein may be envisioned as a dynamic entity that constantly is sampling a variety of structures (17). These new structures are usually only slightly different from the native conformation and almost always lead to a situation where the free energy o f this system increases. The increase o f free energy causes the protein to refold spontaneously into the state of lowest free energy. Thus, the native structure of a protein is not the only structure it can assume but rather the one of lowest free energy and, hence, of greatest probability. Slight changes of environment can cause alternate structures to be of lowest free energy and, thus, lead to protein denaturation. F o r portions of ribonuclease molecules, the difference between a molecule that is in the native format .02% of the time and one that is completely native can be achieved by a gain of only .37 kcal/residue (2). Hence, small localized changes of energy can cause large changes of protein conformation. F o r a protein to exhibit functionality, it must interact with other components of the food system. These interactions often may require that the protein be free either to move throughout the system or to alter its structure in a way to allow interactions with other components. In some cases the simple presence of other molecules in the protein solution will allow interaction, but more commonly interactions require input of energy into the system to ensure adequate mixing. This energy may alter the physical nature of the molecules being mixed, e.g., decrease average fat globule size and also alter conformation of the protein molecule. In the following section of this paper, some specific types of functionality will be examined, and changes of protein structure required will be discussed.
SYMPOSIUM: ASSESSING FUNCTIONALITY OF WHEY PROTEINS WATER BINDING
Interactions of water with proteins are important both to the structure of the proteins and to their behavior in food systems. Water molecules tend to associate with themselves through a network of hydrogen bonds. When solute molecules are placed in water, these molecules will be soluble if water solute interactions have lower free energy than do the separate solute-solute and water-water interactions. The nature of these interactions is complex and has been the subject of many reviews. Although there is still much to be learned about interactions of water both with itself and with solute molecules, we can describe types of interactions that are important to protein structure and functionality. Proteins contain a n u m b e r of amino acids that have side groups that contain electrical charges at certain pH's. The ion-dipole interactions between water and these charged groups are fairly strong with energy of about 5 kcal/ mol. With model peptides, from four to seven water molecules can be associated with each residue of charged amino acid (21). Proteins also contain amino acids that have polar side chains but that do not have a charge. These polar molecules are dipoles, and, thus, water can interact with them through dipoledipole interactions. Because all molecules involved contain hydrogen as part of the dipoles, the special class of dipole-dipole interactions known as hydrogen bonds can occur. These are typically stronger than other dipole-dipole interactions and can have energies of from 2 to 6 kcal/mole (42). The nonionized polar amino acids in proteins typically have two water molecules strongly associated with them (21). Nonpolar amino acids are not soluble in water, and, thus, the interaction of water with these molecules is minimal. Sometimes, however, nonpolar groups are forced into water as a part of a specific protein structure (41). Intrusion of hydrophobic groups into the aqueous environment causes an ordering of water molecules in their vicinity and, thus, a decrease in entropy (38). It has been estimated that the removal of a hydrophobic group from contact with water yields a reduction of free energy of the protein of about 4 kcal/mol. This is a strong driving force for the removal of these groups
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from the aqueous environment. Any water associated with these groups is highly ordered and has been termed hydrophobic hydration water. Kinsella (20) has listed factors in Table 2 as being those that affect water binding by food proteins. As noted earlier, some amino acids bind more water than do others, and hence proteins that contain large amounts of the charged amino acids will tend to bind large amounts of water. A n u m b e r of schemes have been devised to predict water binding of proteins based on amino acid composition. These generally work fairly well and demonstrate the importance of amino acid composition on water binding. The next three factors in Table 2 relate to how amino acids are arranged in the final protein structure. If groups that are capable of forming hydrogen bonds are removed from contact with water and allowed to form hydrogen bonds with themselves, the amount of water b o u n d to the protein will be decreased. However, if the protein molecule is unfolded, these groups may make better contact with the aqueous phase, and water binding actually may increase with denaturation (10). Some proteins require the presence of trace amounts of electrolytes to be soluble. These ions interact with the charged groups on the protein surface and also with water present. This tends to increase the amount of water b o u n d to the protein. At high salt concentrations, salt and protein may compete for water present, and protein water binding and solubiity may be decreased. The extent of these effects depend to a large extent on the nature of the ions present. The size of the hydrated radius of the ion seems to
TABLE 2. Factors affecting water binding by food proteins (20). Amino acid composition Protein conformation Surface polarity/hydrophobicity Ionic concentration Ion species pH Temperature
Journal of Dairy Science Vol. 67, No. 11, 1984
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be of prime importance for determining the dehydration effect of concentrated salts. The effect of pH on water binding of proteins can be manifested in many ways. As the pH of a protein solution changes, so does the number of charged groups on the molecule. As noted, the number of charged groups is strongly related to the amount of water associated with a protein. Changes of pH also can alter conformation of the protein which may either expose or bury potential water binding sites. Any discussion of water binding must include a definition of what is meant by bound (12). Types of b o u n d water associated with proteins have been described by Chou and Morr (7) and are listed in Table 3. In some proteins, a molecule of water may be intimately associated with the final structure of the molecule (3). In these cases, the water serves as a bridge to hydrogen bond charged groups within the molecule. These molecules of structural water do not behave like bulk phase water and are unavailable for chemical reactions. Water molecules that are b o u n d through ion-dipole of dipole-dipole interactions are described as monolayer water, in which the molecules form a layer of tightly b o u n d water around the protein molecule. Hydrophobic groups exposed to the aqueous phase tend to cause an increase in the order of the water molecules near them. These ordered molecules form cage-like structures called clathrates. The nature of the water b o u n d near these hydrophobic groups is not defined well, but it does not behave as normal bulk water. All of these types of water interact strongly with the protein molecule and exhibit properties that are different from those of free water. One of the most notable characteristics of b o u n d water is that is does not freeze at normal temperatures and is termed unfreezable water. Many of the methods to measure the amount of water bound to proteins are based on this phenomenon. This is also the type of water that can be predicted from a knowledge of amino acid composition of proteins a~ mentioned. Capillary water refers to water that is associated with proteins but that freezes at normal temperatures and is free to act as a solvent for small molecules. This water behaves much like bulk phase water but is difficult to remove from the protein mass. The amount of Journal of Dairy Science Vol. 67, No. 11, 1984
TABLE 3. Types of water associated with proteins (7). Structural Monolayer Unfreezable Hydrophobic hydration Capillary Hydrodynamic hydration
this type of water associated with a protein is dependent upon the nature of measurement. A good physical description of the forces that bind this water to proteins is not available, but at least some of it seems to be entrained in a three-dimensional network of protein molecules. Incorporation of large amounts of water into a protein structure can lead to formation of a gel, the next property to be discussed. GELATION
Protein gels can be formed by addition of salts, action of enzymes, changes of pH or by application of heat. Whey protein gels are obtained by heating, so this discussion will be limited to mechanisms of heat induced protein gelation. Ferry (11) suggested that the process of gelation was a two-stage one involving an initial denaturation or unfolding of a protein molecule followed by subsequent aggregation. The steps in thermal gelation are summarized in Table 4. When a protein is heated, the bonds that maintain its secondary and tertiary structures are weakened, and, at some temperature, broken. This breaking of noncovalent bonds with its resulting alteration of protein structure is denaturation. In the early stages of thermal denaturation, most protein molecules begin to unfold (42). This unfolding often leads to a slight increase of the a m o u n t of water tightly
TABLE 4. Stages in heat-induced protein gelation. Protein unfolding Water binding Protein-protein interactions Water immobilization
SYMPOSIUM: ASSESSING FUNCTIONALITY OF WHEY PROTEINS bound to the protein (10). If protein-protein interactions lead to the formation of a threedimensional network capable of entraining water molecules, a gel can form. If the network is too weak, viscosity will increase; but fluid flow will be possible, and a true gel will not form. If, however, the proteinprotein interactions are too strong, the network will collapse, and water will be expelled from the structure. A balance between attractive forces necessary to form a network and repulsive forces necessary to prevent its collapse is required for gel formation. Consider a fibrous protein being held together by a series of ionic bonds. This just as easily could be a protein that is stabilized by hydrogen bonds such as the collagen triple helix or a globular protein the structure of which is stabilized by a variety of noncovalent and possibly some covalent bonds. In any case, water does not interact with the interior of the protein because there are too many bonds. If water were to attempt to interact at some point, not only would that bond have to be broken, but also a number of neighboring bonds would have to be weakened. This is not possible at room temperature, and the protein remains in the conformation of lowest free energy. Application of heat will weaken these bonds and allow water to interact with the charge groups. At some temperature, attractive forces will have been weakened enough to allow for extensive water-ion interactions. This causes unfolding of the molecule and increase of water binding. It a network can be imagined to extend into three dimensions, we could envision the existence of "pockets" of bulk phase water that would be associated with the network. This water would freeze at normal temperatures and would be freely available to participate in chemical reactions. Thus, protein-protein interactions are necessary for a network to form. Interactions could involve calcium or other ionic bridges, hydrophobic interactions, disulfide bonds, or others. As long as there are enough of them to form a network and there are enough repulsive forces to prevent the network's collapse, a gel can be formed. Many factors affect formation and properties of protein gets. Some of the more important of these are in Table 5.
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TABLE 5. Factors affecting gel formation. Temperature Protein concentration pH Salt concentration Calcium concentration Free-sulfhydryl concentration
Temperature can affect formation of protein gets in a variety of ways. Heat can affect both rate of denaturation and rate of protein-protein interaction. If rate of protein aggregation is rapid compared to rate of protein unfolding, the gel structue will be affected adversely (15). If rate or extent of aggregation is too great, a precipitate will form without appreciable water immobilization. A temperature must be selected for gel formation that balances rate of protein unfolding with that of aggregation. Often at the temperature selected, there is only minimal aggregation, and the majority of protein-protein interaCtions occur as the system is cooled. Protein concentration determines both likelihood of gel formation and also characteristics of the gel that forms. Below a certain concentration that varies according to the protein utilized, gelation will not occur. When protein is too low, a protein network is difficult to establish. Protein-protein interactions then tend to occur within molecules rather than between molecules, and a gel framework cannot be established. As protein content increases, the likelihood ofintermolecular crosslinks increases, and at some concentration gelation can occur. Further increases of protein content change the strength and texture of the gel resulting in firmer gels as more water is tightly bond to protein molecules (34). As discussed, pH can have a marked effect on structure of proteins and amount of water that can be b o u n d to proteins. The pH also can affect extent of protein-protein interaction it if is of an ionic nature. In other cases, maintenance of proper pH can prevent collapse of a gel network from charge repulsion. Denaturation of proteins is highly dependent on pH (35), and so rate of protein unfolding can be influenced by pH of heating. Proper pH adjustment may be necessary to achieve the Journal of Dairy Science Vol. 67, No. 11, 1984
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proper balance between rate of denaturation and aggregation as well as forces of attraction and repulsion between adjacent protein chains that are necessary to achieve a protein gel. Salts in general can affect the structure of protein molecules as well as the nature of protein-water interactions. These effects markedly influence both solubility of protein and their rate of thermal denaturation. As with heating and pH there is generally an optimal concentration of salt that favors gel formation. Calcium ion concentration can have effects on gelation beyond what would be predicted from changes of ionic strength or its hydrated diameter. Calcium ion concentration can affect both rate of whey protein denaturation and solubility of denatured protein molecules (33). Calcium also is an effective protein crosslinking agent in casein systems as well as mediating interaction between whey proteins and caseins. Kalab and Emmons (16) studied effects of calcium on the nature of milk protein gels, and Schmidt et al. (36) demonstrated significant interaction between calcium and concentration of free sulfhydryl groups in whey protein system. The nature of the calcium effect on properties of whey protein gels deserves further study. Schmidt and coworkers (36) demonstrated that free sulfhydryl groups have a significant effect on properties of whey protein gels. At low concentrations, gel strength increases as free sulfhydryl group increases. At some maximal concentration, which depends upon pH, ionic strength, and calcium content of the system, gel strength begins to decrease with further increases of free sulfhydryls. At low concentrations sulfhydryl groups will tend to ease unfolding of whey proteins that are stabilized by disulfide linkages. Upon heating, disulfide interchange may occur that would p r o m o t e formation of a crosslinked matrix. Beyond a certain concentration, free sulfhydryl groups probably inhibit formation of disulfide linkages and thus weaken gel structure. Thus, all of the factors in Table 5 exhibit their effects on gelation by altering either rate of protein unfolding or rate and extent of protein-protein interaction. In many cases, these agents affect both steps in the process. In almost all cases, effects o f reagents in formation of thermal protein gels can be explained by a Journal of Dairy Science Vol. 67, No. 11, 1984
knowledge of their effects on protein structure. EMULSIFICATION
When two immiscible liquids are mixed, the state of free energy is lowest when the area of contact between the two liquids is minimized. This happens when phase separation occurs. Any increase of interfacial area and, thus, of interfacial energy requires input of work. If work is applied and the two liquids are dispersed, the system again will attempt to keep its total energy at a minimum. One way to minimize interfacial area is for components of the dispersed phase to form spherical particles. Large spheres have smaller ratio of surface to volume than do small ones and, hence, a lower surface energy. Unless an energy barrier is present to prevent it, coalescence occurs and tends to decrease the interfacial area. If there are differences of density between phases, coalescence will be accelerated, and a two-phase system will be obtained more rapidly. The dispersed system can be stabilized against coalescence and phase separation if another component that is partially soluble in both phases is added. F o r a mixture of oil in water, phospholipids can function this way. The fatty acid tail portions of the phospholipid are insoluble in the aqueous phase but are fully soluble in oil. The phosphate ester head group of the molecules contains a charge and is only soluble in the aqueous phase. Thus, a portion of the molecule extends into the lipid phase while another portion remains in the aqueous phase. The like charges on the phospholipid head groups present an energy barrier to coalescence, and discrete particles are maintained. Such an emulsion formed by the mixture of oil, water, and an emulsifier is still at higher energy than the deemulsified sytem. Thus, the emulsion is thermodynamically unstable and, given enough time, will break. Formation of an emulsion requires input of energy equal to the new interfacial energy generated upon its formation. The size distribution of an emulsion is a function of method of homogenization, concentration of emulsifying agent, and rate at which the emulsifier can reach the surface of the dispersed phase. If a mixture of oil and water is passed
SYMPOSIUM: ASSESSING FUNCTIONALITY OF WHEY PROTEINS through a homogenizer valve at high pressure, the shear causes dispersion of the oil into small droplets within the aqueous phase. Wherever two droplets encounter, they will merge to form a larger droplet of lower interracial energy. It an emulsifying agent is present, the droplets will continue to merge until the surface of the droplets is sufficiently coated with emulsifier to prevent their coalescence. If the newly created interface could be coated instantly with emulsifier molecules, the emulsion would consist of particles having the same diameter as they did at the instant of homogenization. In the real word, the emulsifier requires finite time to diffuse to the interfacial area to present a barrier to coalescence. The longer the time required for diffusion to the interface, the larger the droplets. When a protein is utilized as the emulsifying agent, the mechanism of interaction becomes more complicated. To visualize how this occurs, it is useful first to consider the absorption of a protein molecule at a static water/oil interface. A protein can be envisioned as a roughly spherical molecule containing b o t h positively and negatively charged groups at its surface and having a number of h y d r o p h o b i c groups buried in its interior. As the protein approaches the interface, some charged groups will tend to be less hydrated. This is energetically unfavorable, and these groups will be repelled from the interfacial area. If these groups occur in an area of the protein that has some flexibility, the structure of the protein will be altered. Movement of charged groups will tend to open the protein structure and expose hydrophobic groups to the solvent. If this had occurred as a random fluctuation in the bulk phase of the solvent, these groups would assume the configuration of lowest free energy, and the hydrophobic groups would be returned to the protein interior. When the hydrophobic groups are exposed while the proteins is at an oil/water interface, the groups may find a state of lower energy if they enter the nonaqueous phase. At low protein concentrations, this involves only a minimal energy barrier and occurs readily (22). Any fluctuation of protein structure that allows for insertion of hydrophobic groups into the oil phase will be favored. This will continue to occur until random fluctuations in protein structure no longer can
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yield a conformation o f lower free energy. The amount of unfolding that occurs at such an interface will depend on how rigid the threedimensional protein structure is and on the number and location of hydrophobic groups in the molecule (14). A flexible, noncrosslinked protein will be able to unfold easier than will a highly structured and crosslinked one (29). If energy is applied to cause shear, the process will be accelerated. The shear can cause the protein to unfold, thus exposing its hydrophobic groups to the nonaqueous phase. It also can increase the interracial area between the two phases and allow more protein to come into contact with the nonaqueous phase. For a homogenizer that creates many small droplets that tend rapidly to coalesce, the protein is involved in two ways. In the first case, the rate of protein diffusion to the interfacial suface is important to the final droplet size (39). Anything that tends to slow diffusion of the protein will tend to increase average particle size. Coalescence of particles will cease when enough protein molecules have been absorbed to the surface. Once protein is absorbed to a fat globule, it will tend to unfold and cover more of the surface. The rate of this unfolding also can affect final distribution of droplet sizes. If unfolding is slow compared to coalescence and diffusion o f protein to the surface, then it will have little effect on droplet size. If, however, rate of protein unfolding is greater than either rate of coalescence or absorption of new protein molecules, factors that increase unfolding can decrease average size o f fat globules. In most food systems, proteins are not utilized alone b u t in conjunction with smaller emulsifier molecules. In these systems, globule size is determined b y homogenization conditions and by properties of the small emulsifier. Only after stable globules have formed will appreciable amounts of proteins be adsorbed at the surface. The presence o f emulsifiers will decrease the amount of protein that can be absorbed at the surface (6, 23), b u t sufficient protein need only be added to stabilize the emulsion towards long effects. Protein tends to increase density of fat globules towards a density closer to that of the continuous phase. This tends to decrease the tendency for b o u y a n t creaming. More importantly, however, proJournal of Dairy Science Vol. 67, No. 11, 1984
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SYMPOSIUM: ASSESSING FUNCTIONALITY OF WHEY PROTEINS reins tend to offer steric hindrance to creaming (40). Soluble proteins tend to form better emulsions than do insoluble ones (8). Factors that tend to unfold protein structures tend to facilitate formation of emulsion, whereas those that increase rigidity of interfacial films tend to increase emulsion stability. Factors that increase surface rigidity and steric hindrance are those that tend to increase emulsion stability the most. FOAMING
Formation of foam is analgous to formation of an emulsion. Forces involved when air is
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incorporated into the aqueous phase are similar to those in the mixing of oil and water. In a foam, water molecules surround air droplets. Air is essentially nonpolar, and ordering of water molecules adjacent to air cells occurs. This results in high surface tension and high surface energy. Emulsifiers work in an air-water mixture much as they do in an oil-water system to lower interfacial energy and to provide a kinetic barrier to bubble coalescence. A protein that is utilized to form a stable foam will require many of the same properties as are required to form an emulsion. The protein must be able to diffuse rapidly to the interface and unfold in such a manner as
[
Figure 1. A protein foam observed 0 (A), 3 (B), 6 (C), 16 (D), and 36 (E) min after whipping. All photographs are of the same field. X100. Journal of Dairy Science Vol. 67, No. 11, 1984
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to lower interfacial tension betwen air and water phases. Once a foam is formed, there are three main forces that can lead to its decay. Forces are in Table 6. A foam initially will have a number of small air cells separated by an interfacial layer and by areas of water. As the foam ages, gravitational forces will cause water to drain, and air cells will come closer together. A phenomenon called capillary drainage works further to thin walls separating the air cells. Even though extensive drainage occurs, there is still an interfacial layer between the air cells that should be capable of preventing air cell coalescence. Mechanical disturbances, such as vibration, can cause the cells to collide and rupture. When this occurs, the smaller cells will give their contents to the larger cells because the pressure is lower in the larger ones (4). Figure 1A shows a protein foam within 1 min of whipping. In this figure, a number of large and small air cells are apparent. In some cases, the lamella have ruptured, and small air cells can be seen merging with larger ones. Also evident are areas rich in bulk phase water as well as areas where air cells are coming closer to each other and capillaries are forming. In Figure 1B the same field is shown 3 rain later. The average size of air cells has increased, and much of the free water has been lost through gravitational drainage. The same field after an additional 3 rain is in Figure 1C. More gravitational drainage has occurred, and many distinct capillaries can be seen. As these capillaries form, they tend to become thinner because of capillary drainage. The amount of free water has become so small that gravitational drainage does not occur to as great an extent. If one calculates the pressure in the areas between the air cells, pressure is lowest in the region where three capillaries meet to form the socalled Plateau borders (4). The water tends to migrate from the high pressure areas to those of lower pressure causing further thinning of capillaries. Figure 1D shows the same field of the foam after an additional 10 rain. Almost all noncapillary water has been removed, and air cells that remain are fairly stable. Rupture occurs primarily from disturbances in the air cells due to vibration. Some evaporative loss of water also must be occurring that also can weaken the foam. Figure 1E shows the same foam 20 min later. Some stable small cells are Journal of Dairy Science Vol. 67, No. 11, 1984
TABLE 6. Factors that affect the stability of foams. Decrease stability
Increase stability
Gravitational drainage Capillary pressure drainage Mechnical disturbances
Surface viscosity Gibbs-Marangonieffect Electric double layers
seen as well as a number of large ones. Further losses of.structure are almost all due to mechanical disturbances. Although reasons for foam collapse have been discussed, this figure demonstrates that other forces must be present that stabilize the film from collapse for fairly long times. The main stabilizing forces are in Table 6. If protein at the air-water interface binds water tightly and is rigid, there will be increased surface viscosity that will tend to reduce rate of water drainage (27). If viscosity is high enough, all flow may stop, and the film will be stabilized. Thus, proteins that are rigid and have a high surface viscosity, like lysoenzyme, do not form high overrun foams; but foams once formed are stable (29). Modifications of a protein that tend to increase its viscosity at a surface will increase stability of foams formed by the protein (13). The Gibbs-Marangoni effect relates to the increase of surface tension in areas where drainage of water also has removed some of the interfacial material. Increase of surface tension, as well as increase of concentration of the emulsifier molecules in areas adjacent to the location of water drainage, will cause diffusion of emulsifier back to this location. Emulsifier molecules will carry water with them, and thickness of the film will be restored. This "self-healing" of films is important to their stability. Foams also may gain stability due to electrical properties of protein or emulsifier molecules. As the lamella thin and emulsifiers move closer to each other, they may either be attracted due to the Van der Waal's forces or be repelled due to charge repulsion. In films that drain to this stage, residual charge on the
SYMPOSIUM: ASSESSING FUNCTIONALITY OF WHEY PROTEINS emulsifier m o l e c u l e s can b e i m p o r t a n t t o film stability. T o o m u c h charge, h o w e v e r , s h o u l d b e avoided, as this c a n lead t o charge r e p u l s i o n d u r i n g f o r m a t i o n o f t h e film a n d t o i n c o m p l e t e coverage b y p r o t e i n m o l e c u l e s . Also, t h e r e is a n energy barrier to removal of charged groups from the aqueous phase and to their insertion i n t o air cells. M o s t studies h a v e s h o w n t h a t f o a m i n g is m a x i m a l at p H ' s n e a r t h e iselectric p o i n t o f soluble p r o t e i n s i n v o l v e d (9). A t t h e s e pH's, t h e n u m b e r o f charge t o b e r e m o v e d to f o r m t h e a q u e o u s p h a s e are m i n i m a l , a n d surface viscosity o f p r o t e i n s t h e n is m a x i m a l . T h u s , t o f u n c t i o n well in f o a m f o r m a t i o n , p r o t e i n m o l e c u l e s m u s t b e able t o u n f o l d at an air-water i n t e r f a c e a n d to s p r e a d rapidly t o cover t h e e n t i r e i n t e r r a c i a l area. T h e y t h e n m u s t possess e i t h e r e n o u g h surface viscosity o r charge or b o t h t o p r e v e n t drainage.
REFERENCES
1 Anfinsen, C. B. 1973. Principles that govern the folding of protein chains. Science 181:223. 2 Anfinsen, C. B., and H. A. Scheraga. 1975. Experimental and theoretical aspects of protein folding. Adv. Protein Chem. 29:205. 3 Berendsen, J. C. 1975. Specific interactions of water with biopolymers, in water. A comprehensive treatise. Vol. 5. Water in dispersed systems. F. Franks, ed. Plenum Publ., Co., London. 4 Bikerman, J. J. 1973. Foams. Springer-Verlag, Berlin. 5 Cherry, J. P. 1981. Protein functionality in foods. Am. Chem. Sot., Washington, DC. 6 Chmura, J. N. 1982. The effect of protein hydration on emulsion stability. Ph.D. Diss., The Ohio State Univ., Columbus. 7 Chou, D. H., and C. V. Morr. 1979. Protein-water interactions and functional properties. J. Am. Oil Chem. Soc. 56:53A. 8 Crenwelge, D. D., C. W. Dill, P. T. Tybor, and W. A. Landrnann. 1974. A comparison of the emulsification capacities of some protein concentrates. J. Food Sci. 39:175. 9 Devilbiss, E. D., V. H. Holsinger, L. P. Posati, and M. J. Pallansch. 1974. Properties of whey protein concentrate foams. Food Technol. 28:40. 10 Fennema, O. 1977. Water and protein hydration. Food proteins. J. R. Whitaker and S. R. Tannenbaum, ed. Avi Publ. Co., Inc:, Westport, CT. 11 Ferry, J. D. 1948. Protein gels. Adv. Protein Chem. 4:1. 12 Franks, F. 1983. Bound water: Fact and fiction. Cryo Lett. 4:73. 13 Graham, D. E., S. Levy, and M. C. Phillips. 1976. The conformation of proteins at interfaces and their role in stabilising foams. Foams. R. J. Akers, ed. Academic Press, London. 14 Graham, D. C., and M. C. Phillips. 1979. Proteins
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16 17 18 19 20
21 22 23
24
25 26
27
28
29 30 31
32 33 34
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at liquid interfaces. III. Moelcular structure of adsorbed films. J. Colloid Interface Sci. 70:427. Hermansson, A.-M. 1979. Aggregation and denaturation involved in gel formation. Functionality and protein structure. P. A. Poul-Ed, ed. Am. Chem. Soc., Washington, DC. Kalab, M., and D. B. Emmons. 1972. Heat-induced milk gels. V. Some chemical factors in influencing firmness. J. Dairy Sci. 55:1225. Karplus, M., andJ. A. McCammon. 1983. Dynamics of proteins: Elements and function. Am. Rev. Biochem. 53:263. Kauzmann, W. 1959. Some factors in the interpretation of protein denaturation. Adv. Prot. Chem. 14:1. Kinsella, J. E. 1976. Functional properties of proteins in foods: A survey. CRC Crit. Rev., Food Sci. Nutr. 7:219. Kinsella, J. E. 1982. Relationship between structure and functional properties of food proteins. Food Proteins. P. F. Fox and J. J. Cowdon, ed. Appl. Sci. Publ., London. Kuntz, I. D., Jr. 1971. Hydration of macromolecules. 3. Hydration of polypeptides. J. Am. Chem. Soc. 93:514. MacRitchie, F. 1978. Proteins at interfaces. Adv. Protein Chem. 32:283. McDermott, R. L. 1981. Protein-lipid interactions of reductively methylated 14C soy protein fractions in a model soy-based infant formula system. Ph.D. Diss.,Ohio State Univ., Columbus. Mort, C. V. 1979. Conformation and functionality of milk proteins. Funcionality and Protein Structure. A. Pour-E1, ed. Am. Chem. Soc., Washington, DC. Morr, C. V. 1979. Functionality of whey protein products. N.Z.J. Dairy Sci. Technol. 14:185. Morr, C. V. 1982. Functional properties of milk proteins and their uses as food ingredients. Developments in dairy chemistry. 1. P. F. Fox, ed. Appl. Sci. Publ., London. Nakamura, R., and Y. Sato. 1964. Studies on the foaming property of the chicken egg white. X. On the role of ovomucin (B) in the egg white foaminess. Agric. Biol. Chem. 28: 530. Nakai, S. 1983. Structure-function relationships of food proteins with an emphasis on the importance of protein hydrophobiciry. J. Agric. Food Chem. 31:676. Phillips, M. C. 1981. Protein conformation at liquid interfaces and its role in stabilizing emulsions and foams. Food Technol. 35:50. Pout-El, A. 1979. Functionality and protein structure. Am. Chem. Soc., Washington, DC. Pout-El, A. 1981. Protein functionality: Classification, definition and methodology. Protein functionality in foods. J. R. Cherry, ed., Am. Chem. Soc., Washington, DC. Privalov, P. L. 1979. Stability of proteins. Adv. Protein Chem. 33:167. Richert, S. H. 1975. Current milk protein manufacturing processes. J. Dairy Sci. 58:985. Schmidt, R. H. 1/981. Gelation and coagulation. Protein functionality in foods. J. P. Cherry, ed. Am. Chem. Soc., Washington, DC. Journal of Dairy Science Vol. 67, No. 11, 1984
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35 Schmidt, R. H., B. L. Illingworth, and E. M. Ahmed. 1978. Heat-induced gelation of peanut protein/whey protein blends. J. Food Sci. 43:613. 36 Schmidt, R. H., L. B. lllingworth, J. C. Deng, and A. J. Cornell. 1979. Multiple regression and response surface analysis of the effects of calcium chloride and cysteine on heat-induced whey protein gelation. J. Agric. Food Chem. 27: 529. 37 Tanford, C. 1961. Physical chemistry of macromolecules. Wiley, New York, NY. 38 Tanford, C. 1970. Protein denaturation, Part C. Theoretical models for the mechanism of denaturation. Adv. Protein Chem. 24:1. 39 Tornberg, E. 1979. The adsorption behavior
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proteins at an interface as related to their emulsifying properties. Functionality and protein structure. A. Pout-El, ed., Am. Chem. Soc., Washington, DC. 40 Vincent, B. 1974. The effect of adsorbed polymers on dispersion stability. Adv. Colloid Interface Sci. 4:193. 41 Wetlaufer, D. B. 1973. Protein structure and stability: Conventional wisdom and new perspectives. J. Food Sci. 38:740. 42 Whitaker, J. R. 1977. Denaturation and denaturation of proteins. Food proteins. J. R. Whitaker and S. R. Tannenbaum, ed. Avi Publ. Co., Inc., Westport, CT.
strength of the network but do not form a dough as such. Fiber spinning and thermal extrusion are two more examples of processes that are not applied generally to whey proteins. Although in practice almost any protein can be extruded or spun, these processes are utilized almost exclusively with oil seed proteins. Many excellent reviews and books (5, 19, 20, 24, 25, 26, 28, 30) have been written on protein functionality, and these may be consuited for more complete discussion of the body of experimental literature. My paper will be limited to a discussion o f the nature o f forces involved and types o f interactions that must occur for" whey proteins to function in water binding, gelatin, emulsification, and foam formation.
ABSTRACT
Many of the desirable attributes of foods may be directly or indirectly related to the functionality of their protein components. The manner in which food proteins interact with other food components as well as with themselves determines their functionality. Forces in the maintenance o f protein structure are discussed as they relate to physical requirements necessary for water binding, gelation, emulsification, and foam formation. Factors that affect these functional properties are discussed as they relate to changes o f protein structure. The nature of interactions required for optimal functionality are related to conditions that alter protein structure in such a way as to encourage occurrence of these interactions.
PROTEIN STRUCTURE
INTRODUCTION
Functionality has been defined (31) as: " a n y property of a food or food ingredient, except its nutritional ones, that affects its utilization." F o r proteins then there must be a large number of functions and functional properties. Kinsella (19) suggested that properties in Table 1 are some of the most important ones to consider in discussing proteins. As far as functionality o f whey proteins is concerned, this entire list need not be considered. F o r example, dough formation generally refers to interactions of certain grain proteins that lead to formation of a network that can entrain gases. Whey proteins can affect the
Received August 22, 1983. 1Salaries and research support provided by State and Federal Funds appropriated t o t h e Ohio Agricultural Research and Development Center, The Ohio S t a t e University. Journal Article No. 128-83. 1984 J Dairy Sci 67:2711--2722
A protein manifests functionality b y interacting with other components within the food system. These interactions may involve solvent molecules, solute molecules, other protein molecules, or substances that are dispersed in the solvent such as oil or air. To describe forces in these interactions, it is essential that forces and energies in achievement and maintenance of native protein structure be described. Although complete discussion of the forces is b e y o n d the scope of this paper, some observations on the nature of protein structure will be useful. Proteins exist in the lowest kinetically attainable state of free energy (1). Whether this is the lowest state possible or just the one readily available still is being debated. It can be stated, however, that given a set of conditions, a protein molecule spontaneously will assume the conformation of lowest free energy. Protein structure is highly dependent upon the environment, and protein will assume different conformation as environmental conditions change (2, 17, 32, 37). Factors of importance include pH, temperature, dielectric
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TABLE 1. Functions of proteins in food (19). Flavor Binding Water binding Viscosity Gelation
Dough formation Fiber spinning Thermal extrusion Emulsification Formation
constant, ionic strength, and presence of other molecules including air, fat, denaturants, etc. One of the main ways that proteins lower their free energy involves removal of hydrophobic groups from the aqueous environment. This may provide the greatest single decrease of free energy of all types of binding within proteins (20). The strength of hydrophobic binding is, however, sensitive to changes of temperature and dielectric constant, and, thus, changes of these can have large influences on protein structure. Protein molecules may contain crosslinks of a noncovalent, salt bridges, or a covalent, disulfide bonds, nature. These crosslinks lower the conformational entropy of the molecule, which must be compensated for by a decrease of binding energy. The presence of crosslinks adds greatly to the stability of the native protein structure and makes the molecules resistant to unfolding or denaturation. For example, simple unfolding is inhibited sterically by crosslinking, because portions of the molecule that might be allowed normally to become exposed to the solvent are held in place by the crosslinks. Denaturation is also less likely because one of the driving forces of denaturation, an increase in conformational entropy, is reduced greatly (18). In a noncrosslinked protein, if unfolding to a random coil structure can be induced, there is a large gain in the number of conformations the molecule can assume. This gain in conformational entropy is a large driving force for maintenance of the denatured state when the denaturing agents are removed. In contrast, a highly crosslinked protein cannot assume the same degree of random conformations, and, thus, the increase in e n t r o p y is much less. This helps explain why molecules that contain large numbers of disulfide bonds are often resistant to denaturation. Journal of Dairy Science Vol. 67, No. 11, 1984
Structures attained by proteins are not rigid but are dynamic (17). There is rotational freedom about many o f the bonds within the protein molecule, and the entropy gain o f this freedom lowers the total free energy of the native structure. There are also portions of the protein structure that are stabilized by weak secondary forces, and these are often free to assume different conformations. These alternate conformations lead to structures of higher free energy and, thus, are not stable or long lived. A protein may be envisioned as a dynamic entity that constantly is sampling a variety of structures (17). These new structures are usually only slightly different from the native conformation and almost always lead to a situation where the free energy o f this system increases. The increase o f free energy causes the protein to refold spontaneously into the state of lowest free energy. Thus, the native structure of a protein is not the only structure it can assume but rather the one of lowest free energy and, hence, of greatest probability. Slight changes of environment can cause alternate structures to be of lowest free energy and, thus, lead to protein denaturation. F o r portions of ribonuclease molecules, the difference between a molecule that is in the native format .02% of the time and one that is completely native can be achieved by a gain of only .37 kcal/residue (2). Hence, small localized changes of energy can cause large changes of protein conformation. F o r a protein to exhibit functionality, it must interact with other components of the food system. These interactions often may require that the protein be free either to move throughout the system or to alter its structure in a way to allow interactions with other components. In some cases the simple presence of other molecules in the protein solution will allow interaction, but more commonly interactions require input of energy into the system to ensure adequate mixing. This energy may alter the physical nature of the molecules being mixed, e.g., decrease average fat globule size and also alter conformation of the protein molecule. In the following section of this paper, some specific types of functionality will be examined, and changes of protein structure required will be discussed.
SYMPOSIUM: ASSESSING FUNCTIONALITY OF WHEY PROTEINS WATER BINDING
Interactions of water with proteins are important both to the structure of the proteins and to their behavior in food systems. Water molecules tend to associate with themselves through a network of hydrogen bonds. When solute molecules are placed in water, these molecules will be soluble if water solute interactions have lower free energy than do the separate solute-solute and water-water interactions. The nature of these interactions is complex and has been the subject of many reviews. Although there is still much to be learned about interactions of water both with itself and with solute molecules, we can describe types of interactions that are important to protein structure and functionality. Proteins contain a n u m b e r of amino acids that have side groups that contain electrical charges at certain pH's. The ion-dipole interactions between water and these charged groups are fairly strong with energy of about 5 kcal/ mol. With model peptides, from four to seven water molecules can be associated with each residue of charged amino acid (21). Proteins also contain amino acids that have polar side chains but that do not have a charge. These polar molecules are dipoles, and, thus, water can interact with them through dipoledipole interactions. Because all molecules involved contain hydrogen as part of the dipoles, the special class of dipole-dipole interactions known as hydrogen bonds can occur. These are typically stronger than other dipole-dipole interactions and can have energies of from 2 to 6 kcal/mole (42). The nonionized polar amino acids in proteins typically have two water molecules strongly associated with them (21). Nonpolar amino acids are not soluble in water, and, thus, the interaction of water with these molecules is minimal. Sometimes, however, nonpolar groups are forced into water as a part of a specific protein structure (41). Intrusion of hydrophobic groups into the aqueous environment causes an ordering of water molecules in their vicinity and, thus, a decrease in entropy (38). It has been estimated that the removal of a hydrophobic group from contact with water yields a reduction of free energy of the protein of about 4 kcal/mol. This is a strong driving force for the removal of these groups
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from the aqueous environment. Any water associated with these groups is highly ordered and has been termed hydrophobic hydration water. Kinsella (20) has listed factors in Table 2 as being those that affect water binding by food proteins. As noted earlier, some amino acids bind more water than do others, and hence proteins that contain large amounts of the charged amino acids will tend to bind large amounts of water. A n u m b e r of schemes have been devised to predict water binding of proteins based on amino acid composition. These generally work fairly well and demonstrate the importance of amino acid composition on water binding. The next three factors in Table 2 relate to how amino acids are arranged in the final protein structure. If groups that are capable of forming hydrogen bonds are removed from contact with water and allowed to form hydrogen bonds with themselves, the amount of water b o u n d to the protein will be decreased. However, if the protein molecule is unfolded, these groups may make better contact with the aqueous phase, and water binding actually may increase with denaturation (10). Some proteins require the presence of trace amounts of electrolytes to be soluble. These ions interact with the charged groups on the protein surface and also with water present. This tends to increase the amount of water b o u n d to the protein. At high salt concentrations, salt and protein may compete for water present, and protein water binding and solubiity may be decreased. The extent of these effects depend to a large extent on the nature of the ions present. The size of the hydrated radius of the ion seems to
TABLE 2. Factors affecting water binding by food proteins (20). Amino acid composition Protein conformation Surface polarity/hydrophobicity Ionic concentration Ion species pH Temperature
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be of prime importance for determining the dehydration effect of concentrated salts. The effect of pH on water binding of proteins can be manifested in many ways. As the pH of a protein solution changes, so does the number of charged groups on the molecule. As noted, the number of charged groups is strongly related to the amount of water associated with a protein. Changes of pH also can alter conformation of the protein which may either expose or bury potential water binding sites. Any discussion of water binding must include a definition of what is meant by bound (12). Types of b o u n d water associated with proteins have been described by Chou and Morr (7) and are listed in Table 3. In some proteins, a molecule of water may be intimately associated with the final structure of the molecule (3). In these cases, the water serves as a bridge to hydrogen bond charged groups within the molecule. These molecules of structural water do not behave like bulk phase water and are unavailable for chemical reactions. Water molecules that are b o u n d through ion-dipole of dipole-dipole interactions are described as monolayer water, in which the molecules form a layer of tightly b o u n d water around the protein molecule. Hydrophobic groups exposed to the aqueous phase tend to cause an increase in the order of the water molecules near them. These ordered molecules form cage-like structures called clathrates. The nature of the water b o u n d near these hydrophobic groups is not defined well, but it does not behave as normal bulk water. All of these types of water interact strongly with the protein molecule and exhibit properties that are different from those of free water. One of the most notable characteristics of b o u n d water is that is does not freeze at normal temperatures and is termed unfreezable water. Many of the methods to measure the amount of water bound to proteins are based on this phenomenon. This is also the type of water that can be predicted from a knowledge of amino acid composition of proteins a~ mentioned. Capillary water refers to water that is associated with proteins but that freezes at normal temperatures and is free to act as a solvent for small molecules. This water behaves much like bulk phase water but is difficult to remove from the protein mass. The amount of Journal of Dairy Science Vol. 67, No. 11, 1984
TABLE 3. Types of water associated with proteins (7). Structural Monolayer Unfreezable Hydrophobic hydration Capillary Hydrodynamic hydration
this type of water associated with a protein is dependent upon the nature of measurement. A good physical description of the forces that bind this water to proteins is not available, but at least some of it seems to be entrained in a three-dimensional network of protein molecules. Incorporation of large amounts of water into a protein structure can lead to formation of a gel, the next property to be discussed. GELATION
Protein gels can be formed by addition of salts, action of enzymes, changes of pH or by application of heat. Whey protein gels are obtained by heating, so this discussion will be limited to mechanisms of heat induced protein gelation. Ferry (11) suggested that the process of gelation was a two-stage one involving an initial denaturation or unfolding of a protein molecule followed by subsequent aggregation. The steps in thermal gelation are summarized in Table 4. When a protein is heated, the bonds that maintain its secondary and tertiary structures are weakened, and, at some temperature, broken. This breaking of noncovalent bonds with its resulting alteration of protein structure is denaturation. In the early stages of thermal denaturation, most protein molecules begin to unfold (42). This unfolding often leads to a slight increase of the a m o u n t of water tightly
TABLE 4. Stages in heat-induced protein gelation. Protein unfolding Water binding Protein-protein interactions Water immobilization
SYMPOSIUM: ASSESSING FUNCTIONALITY OF WHEY PROTEINS bound to the protein (10). If protein-protein interactions lead to the formation of a threedimensional network capable of entraining water molecules, a gel can form. If the network is too weak, viscosity will increase; but fluid flow will be possible, and a true gel will not form. If, however, the proteinprotein interactions are too strong, the network will collapse, and water will be expelled from the structure. A balance between attractive forces necessary to form a network and repulsive forces necessary to prevent its collapse is required for gel formation. Consider a fibrous protein being held together by a series of ionic bonds. This just as easily could be a protein that is stabilized by hydrogen bonds such as the collagen triple helix or a globular protein the structure of which is stabilized by a variety of noncovalent and possibly some covalent bonds. In any case, water does not interact with the interior of the protein because there are too many bonds. If water were to attempt to interact at some point, not only would that bond have to be broken, but also a number of neighboring bonds would have to be weakened. This is not possible at room temperature, and the protein remains in the conformation of lowest free energy. Application of heat will weaken these bonds and allow water to interact with the charge groups. At some temperature, attractive forces will have been weakened enough to allow for extensive water-ion interactions. This causes unfolding of the molecule and increase of water binding. It a network can be imagined to extend into three dimensions, we could envision the existence of "pockets" of bulk phase water that would be associated with the network. This water would freeze at normal temperatures and would be freely available to participate in chemical reactions. Thus, protein-protein interactions are necessary for a network to form. Interactions could involve calcium or other ionic bridges, hydrophobic interactions, disulfide bonds, or others. As long as there are enough of them to form a network and there are enough repulsive forces to prevent the network's collapse, a gel can be formed. Many factors affect formation and properties of protein gets. Some of the more important of these are in Table 5.
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TABLE 5. Factors affecting gel formation. Temperature Protein concentration pH Salt concentration Calcium concentration Free-sulfhydryl concentration
Temperature can affect formation of protein gets in a variety of ways. Heat can affect both rate of denaturation and rate of protein-protein interaction. If rate of protein aggregation is rapid compared to rate of protein unfolding, the gel structue will be affected adversely (15). If rate or extent of aggregation is too great, a precipitate will form without appreciable water immobilization. A temperature must be selected for gel formation that balances rate of protein unfolding with that of aggregation. Often at the temperature selected, there is only minimal aggregation, and the majority of protein-protein interaCtions occur as the system is cooled. Protein concentration determines both likelihood of gel formation and also characteristics of the gel that forms. Below a certain concentration that varies according to the protein utilized, gelation will not occur. When protein is too low, a protein network is difficult to establish. Protein-protein interactions then tend to occur within molecules rather than between molecules, and a gel framework cannot be established. As protein content increases, the likelihood ofintermolecular crosslinks increases, and at some concentration gelation can occur. Further increases of protein content change the strength and texture of the gel resulting in firmer gels as more water is tightly bond to protein molecules (34). As discussed, pH can have a marked effect on structure of proteins and amount of water that can be b o u n d to proteins. The pH also can affect extent of protein-protein interaction it if is of an ionic nature. In other cases, maintenance of proper pH can prevent collapse of a gel network from charge repulsion. Denaturation of proteins is highly dependent on pH (35), and so rate of protein unfolding can be influenced by pH of heating. Proper pH adjustment may be necessary to achieve the Journal of Dairy Science Vol. 67, No. 11, 1984
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proper balance between rate of denaturation and aggregation as well as forces of attraction and repulsion between adjacent protein chains that are necessary to achieve a protein gel. Salts in general can affect the structure of protein molecules as well as the nature of protein-water interactions. These effects markedly influence both solubility of protein and their rate of thermal denaturation. As with heating and pH there is generally an optimal concentration of salt that favors gel formation. Calcium ion concentration can have effects on gelation beyond what would be predicted from changes of ionic strength or its hydrated diameter. Calcium ion concentration can affect both rate of whey protein denaturation and solubility of denatured protein molecules (33). Calcium also is an effective protein crosslinking agent in casein systems as well as mediating interaction between whey proteins and caseins. Kalab and Emmons (16) studied effects of calcium on the nature of milk protein gels, and Schmidt et al. (36) demonstrated significant interaction between calcium and concentration of free sulfhydryl groups in whey protein system. The nature of the calcium effect on properties of whey protein gels deserves further study. Schmidt and coworkers (36) demonstrated that free sulfhydryl groups have a significant effect on properties of whey protein gels. At low concentrations, gel strength increases as free sulfhydryl group increases. At some maximal concentration, which depends upon pH, ionic strength, and calcium content of the system, gel strength begins to decrease with further increases of free sulfhydryls. At low concentrations sulfhydryl groups will tend to ease unfolding of whey proteins that are stabilized by disulfide linkages. Upon heating, disulfide interchange may occur that would p r o m o t e formation of a crosslinked matrix. Beyond a certain concentration, free sulfhydryl groups probably inhibit formation of disulfide linkages and thus weaken gel structure. Thus, all of the factors in Table 5 exhibit their effects on gelation by altering either rate of protein unfolding or rate and extent of protein-protein interaction. In many cases, these agents affect both steps in the process. In almost all cases, effects o f reagents in formation of thermal protein gels can be explained by a Journal of Dairy Science Vol. 67, No. 11, 1984
knowledge of their effects on protein structure. EMULSIFICATION
When two immiscible liquids are mixed, the state of free energy is lowest when the area of contact between the two liquids is minimized. This happens when phase separation occurs. Any increase of interfacial area and, thus, of interfacial energy requires input of work. If work is applied and the two liquids are dispersed, the system again will attempt to keep its total energy at a minimum. One way to minimize interfacial area is for components of the dispersed phase to form spherical particles. Large spheres have smaller ratio of surface to volume than do small ones and, hence, a lower surface energy. Unless an energy barrier is present to prevent it, coalescence occurs and tends to decrease the interfacial area. If there are differences of density between phases, coalescence will be accelerated, and a two-phase system will be obtained more rapidly. The dispersed system can be stabilized against coalescence and phase separation if another component that is partially soluble in both phases is added. F o r a mixture of oil in water, phospholipids can function this way. The fatty acid tail portions of the phospholipid are insoluble in the aqueous phase but are fully soluble in oil. The phosphate ester head group of the molecules contains a charge and is only soluble in the aqueous phase. Thus, a portion of the molecule extends into the lipid phase while another portion remains in the aqueous phase. The like charges on the phospholipid head groups present an energy barrier to coalescence, and discrete particles are maintained. Such an emulsion formed by the mixture of oil, water, and an emulsifier is still at higher energy than the deemulsified sytem. Thus, the emulsion is thermodynamically unstable and, given enough time, will break. Formation of an emulsion requires input of energy equal to the new interfacial energy generated upon its formation. The size distribution of an emulsion is a function of method of homogenization, concentration of emulsifying agent, and rate at which the emulsifier can reach the surface of the dispersed phase. If a mixture of oil and water is passed
SYMPOSIUM: ASSESSING FUNCTIONALITY OF WHEY PROTEINS through a homogenizer valve at high pressure, the shear causes dispersion of the oil into small droplets within the aqueous phase. Wherever two droplets encounter, they will merge to form a larger droplet of lower interracial energy. It an emulsifying agent is present, the droplets will continue to merge until the surface of the droplets is sufficiently coated with emulsifier to prevent their coalescence. If the newly created interface could be coated instantly with emulsifier molecules, the emulsion would consist of particles having the same diameter as they did at the instant of homogenization. In the real word, the emulsifier requires finite time to diffuse to the interfacial area to present a barrier to coalescence. The longer the time required for diffusion to the interface, the larger the droplets. When a protein is utilized as the emulsifying agent, the mechanism of interaction becomes more complicated. To visualize how this occurs, it is useful first to consider the absorption of a protein molecule at a static water/oil interface. A protein can be envisioned as a roughly spherical molecule containing b o t h positively and negatively charged groups at its surface and having a number of h y d r o p h o b i c groups buried in its interior. As the protein approaches the interface, some charged groups will tend to be less hydrated. This is energetically unfavorable, and these groups will be repelled from the interfacial area. If these groups occur in an area of the protein that has some flexibility, the structure of the protein will be altered. Movement of charged groups will tend to open the protein structure and expose hydrophobic groups to the solvent. If this had occurred as a random fluctuation in the bulk phase of the solvent, these groups would assume the configuration of lowest free energy, and the hydrophobic groups would be returned to the protein interior. When the hydrophobic groups are exposed while the proteins is at an oil/water interface, the groups may find a state of lower energy if they enter the nonaqueous phase. At low protein concentrations, this involves only a minimal energy barrier and occurs readily (22). Any fluctuation of protein structure that allows for insertion of hydrophobic groups into the oil phase will be favored. This will continue to occur until random fluctuations in protein structure no longer can
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yield a conformation o f lower free energy. The amount of unfolding that occurs at such an interface will depend on how rigid the threedimensional protein structure is and on the number and location of hydrophobic groups in the molecule (14). A flexible, noncrosslinked protein will be able to unfold easier than will a highly structured and crosslinked one (29). If energy is applied to cause shear, the process will be accelerated. The shear can cause the protein to unfold, thus exposing its hydrophobic groups to the nonaqueous phase. It also can increase the interracial area between the two phases and allow more protein to come into contact with the nonaqueous phase. For a homogenizer that creates many small droplets that tend rapidly to coalesce, the protein is involved in two ways. In the first case, the rate of protein diffusion to the interfacial suface is important to the final droplet size (39). Anything that tends to slow diffusion of the protein will tend to increase average particle size. Coalescence of particles will cease when enough protein molecules have been absorbed to the surface. Once protein is absorbed to a fat globule, it will tend to unfold and cover more of the surface. The rate of this unfolding also can affect final distribution of droplet sizes. If unfolding is slow compared to coalescence and diffusion o f protein to the surface, then it will have little effect on droplet size. If, however, rate of protein unfolding is greater than either rate of coalescence or absorption of new protein molecules, factors that increase unfolding can decrease average size o f fat globules. In most food systems, proteins are not utilized alone b u t in conjunction with smaller emulsifier molecules. In these systems, globule size is determined b y homogenization conditions and by properties of the small emulsifier. Only after stable globules have formed will appreciable amounts of proteins be adsorbed at the surface. The presence o f emulsifiers will decrease the amount of protein that can be absorbed at the surface (6, 23), b u t sufficient protein need only be added to stabilize the emulsion towards long effects. Protein tends to increase density of fat globules towards a density closer to that of the continuous phase. This tends to decrease the tendency for b o u y a n t creaming. More importantly, however, proJournal of Dairy Science Vol. 67, No. 11, 1984
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SYMPOSIUM: ASSESSING FUNCTIONALITY OF WHEY PROTEINS reins tend to offer steric hindrance to creaming (40). Soluble proteins tend to form better emulsions than do insoluble ones (8). Factors that tend to unfold protein structures tend to facilitate formation of emulsion, whereas those that increase rigidity of interfacial films tend to increase emulsion stability. Factors that increase surface rigidity and steric hindrance are those that tend to increase emulsion stability the most. FOAMING
Formation of foam is analgous to formation of an emulsion. Forces involved when air is
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incorporated into the aqueous phase are similar to those in the mixing of oil and water. In a foam, water molecules surround air droplets. Air is essentially nonpolar, and ordering of water molecules adjacent to air cells occurs. This results in high surface tension and high surface energy. Emulsifiers work in an air-water mixture much as they do in an oil-water system to lower interfacial energy and to provide a kinetic barrier to bubble coalescence. A protein that is utilized to form a stable foam will require many of the same properties as are required to form an emulsion. The protein must be able to diffuse rapidly to the interface and unfold in such a manner as
[
Figure 1. A protein foam observed 0 (A), 3 (B), 6 (C), 16 (D), and 36 (E) min after whipping. All photographs are of the same field. X100. Journal of Dairy Science Vol. 67, No. 11, 1984
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to lower interfacial tension betwen air and water phases. Once a foam is formed, there are three main forces that can lead to its decay. Forces are in Table 6. A foam initially will have a number of small air cells separated by an interfacial layer and by areas of water. As the foam ages, gravitational forces will cause water to drain, and air cells will come closer together. A phenomenon called capillary drainage works further to thin walls separating the air cells. Even though extensive drainage occurs, there is still an interfacial layer between the air cells that should be capable of preventing air cell coalescence. Mechanical disturbances, such as vibration, can cause the cells to collide and rupture. When this occurs, the smaller cells will give their contents to the larger cells because the pressure is lower in the larger ones (4). Figure 1A shows a protein foam within 1 min of whipping. In this figure, a number of large and small air cells are apparent. In some cases, the lamella have ruptured, and small air cells can be seen merging with larger ones. Also evident are areas rich in bulk phase water as well as areas where air cells are coming closer to each other and capillaries are forming. In Figure 1B the same field is shown 3 rain later. The average size of air cells has increased, and much of the free water has been lost through gravitational drainage. The same field after an additional 3 rain is in Figure 1C. More gravitational drainage has occurred, and many distinct capillaries can be seen. As these capillaries form, they tend to become thinner because of capillary drainage. The amount of free water has become so small that gravitational drainage does not occur to as great an extent. If one calculates the pressure in the areas between the air cells, pressure is lowest in the region where three capillaries meet to form the socalled Plateau borders (4). The water tends to migrate from the high pressure areas to those of lower pressure causing further thinning of capillaries. Figure 1D shows the same field of the foam after an additional 10 rain. Almost all noncapillary water has been removed, and air cells that remain are fairly stable. Rupture occurs primarily from disturbances in the air cells due to vibration. Some evaporative loss of water also must be occurring that also can weaken the foam. Figure 1E shows the same foam 20 min later. Some stable small cells are Journal of Dairy Science Vol. 67, No. 11, 1984
TABLE 6. Factors that affect the stability of foams. Decrease stability
Increase stability
Gravitational drainage Capillary pressure drainage Mechnical disturbances
Surface viscosity Gibbs-Marangonieffect Electric double layers
seen as well as a number of large ones. Further losses of.structure are almost all due to mechanical disturbances. Although reasons for foam collapse have been discussed, this figure demonstrates that other forces must be present that stabilize the film from collapse for fairly long times. The main stabilizing forces are in Table 6. If protein at the air-water interface binds water tightly and is rigid, there will be increased surface viscosity that will tend to reduce rate of water drainage (27). If viscosity is high enough, all flow may stop, and the film will be stabilized. Thus, proteins that are rigid and have a high surface viscosity, like lysoenzyme, do not form high overrun foams; but foams once formed are stable (29). Modifications of a protein that tend to increase its viscosity at a surface will increase stability of foams formed by the protein (13). The Gibbs-Marangoni effect relates to the increase of surface tension in areas where drainage of water also has removed some of the interfacial material. Increase of surface tension, as well as increase of concentration of the emulsifier molecules in areas adjacent to the location of water drainage, will cause diffusion of emulsifier back to this location. Emulsifier molecules will carry water with them, and thickness of the film will be restored. This "self-healing" of films is important to their stability. Foams also may gain stability due to electrical properties of protein or emulsifier molecules. As the lamella thin and emulsifiers move closer to each other, they may either be attracted due to the Van der Waal's forces or be repelled due to charge repulsion. In films that drain to this stage, residual charge on the
SYMPOSIUM: ASSESSING FUNCTIONALITY OF WHEY PROTEINS emulsifier m o l e c u l e s can b e i m p o r t a n t t o film stability. T o o m u c h charge, h o w e v e r , s h o u l d b e avoided, as this c a n lead t o charge r e p u l s i o n d u r i n g f o r m a t i o n o f t h e film a n d t o i n c o m p l e t e coverage b y p r o t e i n m o l e c u l e s . Also, t h e r e is a n energy barrier to removal of charged groups from the aqueous phase and to their insertion i n t o air cells. M o s t studies h a v e s h o w n t h a t f o a m i n g is m a x i m a l at p H ' s n e a r t h e iselectric p o i n t o f soluble p r o t e i n s i n v o l v e d (9). A t t h e s e pH's, t h e n u m b e r o f charge t o b e r e m o v e d to f o r m t h e a q u e o u s p h a s e are m i n i m a l , a n d surface viscosity o f p r o t e i n s t h e n is m a x i m a l . T h u s , t o f u n c t i o n well in f o a m f o r m a t i o n , p r o t e i n m o l e c u l e s m u s t b e able t o u n f o l d at an air-water i n t e r f a c e a n d to s p r e a d rapidly t o cover t h e e n t i r e i n t e r r a c i a l area. T h e y t h e n m u s t possess e i t h e r e n o u g h surface viscosity o r charge or b o t h t o p r e v e n t drainage.
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