Food Technology. Vol. 38, Num. 5. May 1984. pp. 67-96.

Symposium: Gelation in Food Protein Systems

Quantitative Analysis of Gelation in Egg Protein Systems

Authors Rizvi and Baker are, respectively, Associate Professor, Dept. of Food Science, and Professor and Chairmen, Dept. of Poultry and Avian Sciences, Cornell University Ithaca, NY 14853. Author Gossett formerly with Cornell, is a Scientist with The Pillsbury Co. 311 Second St. S E., Minneapolis, MN 55414
Based on a paper presented during the symposium, "Gelation in Food Protein Systems," at the 43rd Annual Meeting of the Institute of Food Technologists, New Orleans. La., June 19-22, 1984.

An important function of proteins in food systems is gelation. This phenomenon involves the formation of a three-dimensional matrix mainly through inter-protein hydrogen bonding and allows the immobilization of water within the gel structure. The coagulation or gelation of proteins—in particular, the irreversible, heat-induced coagulation or gelation of egg proteins—often controls the success of certain conked food products. It is of interest to the food scientist to be able to quantitatively monitor the gelation process to better predict end-product characteristics, as well as understand the mechanism of network formation. A better understanding of the gelation process will permit manipulation of variables to obtain a gel of desired textural characteristics and functional properties.

Although the coagulation, i.e., denaturation of proteins can be brought about by heat, high pressure, salts, acids, alkalies, alcohols, or denaturing agents such as urea (Mirsky and Pauling 1936), this article will concentrate on the quantitative analysis of heat-induced coagulation or gelation of egg-albumen proteins (Table 1). Factors affecting the kinetic parameters of gelation and the rheological properties of the gel will be discussed, as well as methods used to measure gelation.

Table 1-PHYSICAL AND CHEMICAL PROPERTIES of egg albumen proteins^a

Protein	% in albumen	pI	Molecular weight	Intrinsic viscosity (dL/g)	Characteristics
Ovalbumin	54	45-4.6	45,000	0.043b	Phosphoglycoprotein; denatures easily; has four sulfhydryls
Ovotransferin	12-13	6.1-6.6	76,000-80,000	0084c	Glycoprotein, complexes iron and other metals
Ovomucoid	11	3.9-4.3	28.000	0.055d	Glycoprotein, trypsin inhibitor
Ovomucin	3.5	4.5-5.0	110,000	2.10e	Glycoprotein, fibrous, viscous
Lysozyme	3.4-3.5	10.7	14,300-14,600	0.027c	Spherical protein; four disulfide bonds; lytic action
Ovoinhibitor	1.5	5.1-5.2	44,000-49,000	-	Inhibits trypsin and chymotrypsin
Ovoglycoprotein	1.0	3.9	24,000.24,400	-	Glycoprotein
Ovoflavoprotein	0.8	4.0-4.1	32.000-35,000	-	Binds riboflavin
Ovomacroglobulin	0.5	4.5-4.7	760,000-900,000	0.0651d	Glycoprotein
Avidin	0.5	9.5-10	55,000-68,300	-	Binds biotin

^a Adapted from Osuga and Feeney (1977) and Powrie (1977); data refer to albumen from chicken eggs
^b From Yang (1961)
^c From Kuntz and Kauzmann (1974)
^d From Vadehra and Nath (1973); ovomucoid pH 3.8-4.6; ovomacroglobulin: pH 6-9
^e From Fasman (1976)

Terminology

A discussion of gelation necessitates defining some commonly used terms associated with the phenomenon. At first glance, some of these terms, may appear to overlap in meaning. However, each has its particular role in this discussion.

• Denaturation is (1) the process in which a protein or polypeptide is transformed from an ordered to a disordered state without rupture of covalent bonds (Scheraga, 1961), or (2) any process, except chemical modification, not involving rupture of peptide bonds which causes a change in the three-dimensional structure of a protein from its "native" in-vivo form (Haschemeyer end Haschemeyer, 1973). Denaturation involves protein-solvent interactions and leads to changes in physical properties, such as loss of solubility of the protein. Sometimes unfolding of the protein structure is considered part of denaturation.
• Aggregation is a general term referring to protein-protein Interactions, with formation of complexes of higher molecular weights Hermansson 1979). Aggregation is usually governed by a balance between attractive and repulsive forces. Attractive forces can involve hydrogen bonds, covalent bonds such as disulfide linkages, and hydrophobic associations, whereas repulsive forces can involve coloumbic forces which are affected by the net charge of the protein molecule or the ionic strength of the solution (Egelandsdal, 1980).
• Coagulation is the random aggregation of already denatured protein molecules (Hermansson, 1979), in which polymer-polymer interactions are favored over polymer-solvent reactions (Schmidt, 1981). The coagulum is often turbid, and the formation of the coagulum is usually thermally irreversible (Shimada and Matsushita, 1981). A coagulum may settle out of solution.
• Gelation. is an orderly aggregation of proteins, which may or may not be denatured, forming a three-dimensional network (Hermansson 1979). Polymer-polymer and polymer-solvent interactions, as well as attractive and repulsive forces, are balanced such that a well-ordered matrix can be formed (Schmidt, 1981). The gel may be turbid or translucent; in the latter case, the gel may be thermoreversible (Shimada and Matsushita, 1981). The term gelation is also used in another context with respect to egg-yolk proteins. The phenomenon of egg-yolk gelation refers to the formation of an irreversibly gelled product upon freezing of the yolk (Cotterill, 1977). However, in this article, the term gelation will refer to the former definition only.

Theory of Gelation

Attempts to describe the mechanism and theory of gel network formation are numerous. The classic explanation of heat induced aggregation of protein molecules is the following two-step process (Ferry, 1948):

Native protein -> denatured protein (long chains) -> aggregated protein (associated network)

The first step is considered a denaturation process and the second step an aggregation process. Comparison of the rate of the denaturation step vs that of the aggregation step helps determine gel characteristics. For example, Ferry (1948) suggested that for a given rate of denaturation the rate of aggregation will be slow if the attractive forces between the denatured protein chain are small. The resulting gel will be a finer network of protein chains, will be less opaque, and will exhibit less syneresis than one with a faster rate of aggregation. A coarser network of protein chains yields an opaque gel with large interstices capable of holding solvent which is easily expressed from the matrix. Hermansson (1979) suggested that conditions favoring denaturation, such as high or low pH, have the opposite effect on aggregation of globular proteins, possibly due to the fact that at high net charge, protein-solvent interactions such as denaturation are favored, rather than protein-protein interactions such as aggregation. A gel network with a certain degree of order can be attained if the aggregation step occurs more slowly than the denaturation step. thus giving the denatured protein molecule, time to orient themselves before aggregation; this is lower in opacity and higher in elasticity then one where aggregation is not suppressed. Schmidt (1981) suggested that if aggregation occurs simultaneously with denaturation, an opaque. less elastic gel results.

Since the kinetics of the denaturation step relative to the aggregation step appear to he important in determining the type of gel produced, it is useful to review some kinetic terms that aid in describing the gelation process:

l. Reaction Rate Constant. The first is the reaction rate constant k (min ^-1), which is obtained from the first-order relationship (Land, 1975):

        dc

    -   --  = kc

        dt

where c=concentration and -dc/dt=the rate at which concentration decreases. Integrating between limits et at time l=U and c et time t gives:

A plot of ln c vs t gives a line of slope -k.

The rate constant may be temperature dependent, end this dependence of reaction rate constant on temperature can best be described by the Arrhenius equation:

    k=S exp (-Ea/RT)

where s=frequency factor (min ^-1), e.=activation energy (cal/mole). r=gas constant (1.987 cal/mole.°K), and t=absolute temperature (°K). A plot of In k vs 1/T gives e straight line of slope -E_a/R.

2. Z Value. Another useful kinetic quantity in describing the temperature dependency of the reaction rate is the Z value, which a defined as the necessary rise in heating temperature (°C) needed for a 10-fold increase in the reaction rate (Dagerskod, 1977). If some parameter of the gelation process is measured, say heating time to reach a certain gel strength, then the Z_c value is the temperature rise needed to increase the heating time or reaction rate 10-fold, This value is useful in calculating the cook value, which is the time required at temperature T for a sample with a certain Z_c value to receive a heat treatment equivalent to 1 min at 100°C (Dagerskod, 1977). To quantitate gelation, it in useful to coagulate samples to equal cook values to ensure equivalent heat treatment

Factors Affecting Gelation

Many researchers have tried to identify the factors which effect aggregation of proteins, since altering the rate of aggregation relative to the rate of denaturation appears to affect gel characteristics. The following are some of the factors which have been studied:

• Electrostatic Charge is one of the must commonly investigated factors. The pH as well as the ionic strength of the protein environment can alter the charge distribution among the amino acid side chains and can either decrease or increase the protein-protein interaction,. Nakamura et al. (1978) concluded that the main factor contributing to the heat-induced aggregation of ovalbumin (pI 4.5-4.6) is the degree of electrostatic repulsion among the denatured protein molecules. When the heat-denatured protein concentration is high (>0.5%), the aggregate size decreases as the pH increases from 5.8 to 10.0; this is due to increased repulsive forces among the protein molecules at the alkaline pH levels. Conversely, decreasing the pH or adding cations decreases the negative charge end accelerates aggregate formation, as does increasing the ionic strength.

  Shimada end Matsushita (1980a; 1981) found that the turbidity of a 4.5% protein solution (3.6% ovalbumin, 0.9% conalbumin) heated for 15 min at 80°C decreases as the pH increases from 8 to 11, after which no turbidity is observed. Hardness of the gel is a maximum at pH 8.5 and decreases on either side of this pH. The turbidity increases upon addition of cations (Ca⁺⁺ >> Li⁺ > Na⁺ Cs⁺) to a 2.8% albumin solution heated for 30 min at 80°C. Anions increase the turbidity in the alkaline pH regions (SO₄⁼ > Cl^- > Br^- > I- > SCN^-). Salts shift the critical pH for coagulant formation to alkaline pH levels as ionic strength increases, perhaps by decreasing the amount of water bound to the albumin and making it less soluble (Bull and Breese, 1970) or by decreasing water protein interactions and increasing protein-protein interactions.

  Holme (1963) observed that the rate of aggregation of heated l.8% ovalbumin solutions is higher at pH 5.5 and 8.5 then at pH 7.0. However, at pH 5.5 the development of turbidity is rapid, with actual precipitation of the protein and slight increases in the levorotation and intrinsic viscosity of the supernatant, while at pH 8.5 there is slight turbidity and marked increases in levorotation and viscosity of the clarified solution.

  Hegg et al. (1979) found from differential scanning calorimetry measurements that the denaturation temperature for a 4.4% ovalbumin solution increases from 62°C at pH 3 to 79°C at pH 6-10 but decreases to 77°C al pH 11. The aggregation temperature, defined as the temperature at which 10% of the protein has aggregated, was found to be 65-70°C at pH 4.0-5.5. The fact that aggregation only occurs around the isoelectric point implies that aggregation behavior is determined by the number of net charges on the ovalbumin molecule. When 1% NaCl is added to the ovalbumin, aggregation occurs at conditions ranging from 53°C at pH 3 to over 85°C at pH 11. The relationship between denaturation temperature T_D and aggregation temperature T_A determines the quality of the thermal aggregates; if T_A is 6 -7°C lower than T_D, precipitates of over 13% dry matter result; if T_A is 4-6°C lower than T_D, gel-like precipitates of 9-13% dry matter appear: if T_D-T_A is 2-4°C, opaque gels of 5-9 % dry matter form; and if T_D-T_A is 1-2°C or negative, transparent gels of less than 5% dry matter are found. Unlike Ferry (1948), Hegg et al. (1979) implied that aggregation takes place prior to denaturation, at all pH levels except pH 11, but that T_D-T_A decreases as pH is increased. At pH 10.5, denaturation and aggregation are predicted to occur simultaneously. Hegg and Lofqvist (1974) hypothesized that at extreme pH values or upon addition of anionic detergents the greater number of charged groups of the same sign repel each other and cause the protein to expend. The expanded molecules also repel each ether and thus hinder aggregation.

  When liquid egg white is heated at 58°C for 5, 15, 30, and 60 min, the optical density decreases as the pH increases from 6.0 to 10.0 (Seideman at al., 1963); this agrees with analogous work using dilute albumin solutions. Egg white containing 50% sucrose requires a higher temperature for coagulation, which is also pH dependent.

• Protein Concentration is also a factor affecting aggregation. Nakamura et al. (1978) found that almost all of the protein aggregates, regardless of concentration, when ovalbumin is heated at 80°C. About 80% of the protein (pH 6.2) heated at 75°C for 5 min aggregates, regardless of albumin concentration; but at 70°C, a concentration of at least 1% is required for aggregation. A higher protein concentration is probably needed to allow a closer association of molecules for aggregate formation at the lower temperatures.

  Shimada and Matsushita (1980a; b) observed that as albumin concentration increases from 0.09%, to 12.5%, turbidity of the solutions heated at 80°C for 15 min is observed over a wider pH range. The pH required for equal turbidity increases as the protein concentration increases on the alkaline side of the isoelectric point from pH 6 to 11, and decreases on the acidic side from pH 4.5 to 3.

• The Formation of Disulfide Bonds and the exposure of hydrophobic amino acid residues are thought to be involved in the first step of coagulation (Shimada and Matsushita, 1980a; b). Proteins with higher percentages of hydrophobic amino acids are classified as coagulation-type proteins and concentration dependent, while proteins with a lower percentage are gelation-type proteins and concentration independent. Further heating causes egg albumin to polymerize by intermolecular sulfhydryl-disulfide, exchange, forming a network (Buttkus, 1974; Shimada and Matsushita, 1980b). However, Hegg (1982) has indicated that many globular proteins with differing sulfhydryl convents can form heat induced gels and that no correlation between disulfide or sulfhydryl .content and gel-forming ability can be found.
• The Composition of the albumin mixture also affects the aggregation of the proteins. Denaturation temperatures of conalbumin, globulins, ovalbumin, and lysozyme are 57.3, 72.0, 71..5, and 81.5°C, respectively, while ovomucin and ovomucoid do not coagulate (Johnson and Zabik. 1981). The gel strength of the lysozyme gel is the highest, followed by that of globulin. Conalbumin gels exhibit the most drainage. In binary mixtures of albumin proteins, aggregation occurs near the denaturation temperature of the least-heat-stable protein. The lysozyme-globulin gel is the firmest, while the ovomucoid-ovalbumin gel is the least firm. Ovomucoid combinations exhibit high drainage.

Measurement of Heat-Induced Gelation

Many methods for measuring gelation involve testing individual samples after discrete time intervals of heating until coagulation or gelation is completed. Some of the methods which have been used are discussed below:

• Gel Strength. A common method is to measure gel strength or firmness of the gel after various heating times. The rigidity of heat-induced 8.2% ovalbumin gels, as in measured by the initial slope of a force-distance curve given by a compression test on the Instron Universal Testing Machine, varies with the pH of the solution, with maximum gel strength exhibited an either side of the isoelectric point (Egelandsdal, 1980). Using the Instron force applied to a probe when the surface yield point of a gel is reached as an indicator of gel strength, Dunkerley and Hayes (1980) showed that the gel strength of egg white at pH 7.0 is at a maximum of 2.3 Newtons (N) at 85°C; 1.3 N at pH 7 and 90°C; and 8.5 N at pH 9.3 end 92-95°C. Beveridge et el. (1980) measured firmness of albumen coagulum as the maximum force required to shear a sample in a shear compression cell; they found that coagulant shearing force increases by approximately 0.1 kg of force/min of heating (0-60 min) and with temperature (77-90°C). The increase in firmness is not a linear function of temperature; the higher temperatures produce gels considerably firmer then those at lower temperatures. Hickson et al. (1982) used an annular back-extrusion device attached to an Instron machine to measure the penetration force, viscosity index, and apparent elasticity of egg albumen gels heated at 80°C. They found that the penetration force and viscosity index increase at the rate of 0.0625 N/min and 8.75 Poise/min, respectively, up to 80 min of heating, after which decreases are observed. Elasticity increased from 0 to 30 N/cm² after 120 min of heating. Shimada ad Matsushita (1980,) determined hardness of albumin gels from profile analysis of the first chew as measured on a Texturometer using a "visco-type" plunger and a 24-mm-diameter cup; they found that coagulum begins to form 3 min after heating at 80°C and that hardness reaches 300 kg of force after 12 min.

  Using a capillary extrusion apparatus fitted to an Instron machine, Gossett (1983) and Gossett et al. (1983a) measured the apparent viscosity and yield force of albumen gels at various pH levels and heating temperatures; they found that gel strength increases with increasing pH and with increasing heating temperature (Table 2).

• Solubility. The change in solubility of a heated protein system has also been monitored as an indicator of the coagulation or gelation process. Upon heating at 80°C, the turbidity, measured as absorbance at 600 nm, of a 4.5% solution of egg albumin was shown to increase rapidly within the first minute, then slowly (Shimada and Matsushita, 1980a). Seideman et al. (1963) found that the optical density of egg white at 550 nm increases with time of heating (58°C, 0-60 min) and increases more rapidly with decreasing pH (8.0-10.5).
• Gravimetric Analysis. Gravimetric analysis of the coagulum or the supernatant after centrifugation has been another method of following the coagulation process. Recovering or separating the precipitate or coagulum from the solution can be difficult because the precipitates can vary from rapidly sedimenting flocculates to nonsedimenting, sol-like opaque aggregates (Hegg et al., 1978). To follow thermal precipitation of protein solutions, Hegg et al (1978; 1979) removed volumes of solution at various time or temperature intervals of heating, centrifuged the solution, and calculated the percentage of aggregated protein as the decrease in absorbance of the supernatants at 380 mm. For slower-sedimenting plateaus, absorbance at 340 nm (opalescence) of the supernatants was measured (Hegg et al., 1978).The weight of the aggregate can also be monitored and the dry matter content DM calculated by the following equation (Hegg et al., 1979):

  
      %DM=C0V0a/Wa
  
  

  where, C₀ =initial protein concentration, V₀ =initial solution volume, a= percent aggregation and W_a = weight of the aggregate. Nakamura et a1. (1978) have measured the amount of aggregated proteins as the difference between the soluble protein concentration before and after heat treatment and centrifugation.

• Formation of Disulfide Linkages. Chemical properties such as the formation of disulfide linkages can be determined as coagulation proceeds. Shimada and Matsushita (1980a) measured the number of free sulfhydryl groups with 5,5'-dithiobios(2-nitrobenzoic acid) (DTNB) on solutions of dissolved albumin coagulums formed by heating 4.5% solutions at 80°C; the number of moles of SH groups/10⁵ g of protein decreased from 5 to 3.5 after 1 min of heating and reached a plateau of 3 after 5 min.

  

  Fig. I -Typical Plots of electrobalance force vs time of heating for egg albumen (PH 9.0) at various temperatures of hearing. Plots shown terminate at or shortly after maximum force values.

• Electrophoresis. The disappearance or appearance of different polyacrylamide gel electrophoretic (PAGE) bands of proteins with different heating time and temperatures has been observed. Matsuda et al. (1981a) found that the intensity of bands for ovalbumin (at pH 7 and 9), ovotransferrin (pH 7), globulins Al, A2, and G3A (pH 7 and 9), and flavoprotein (pH 7 and 9) decreases with heating time (0-60 min) but for ovotransferrin (pH 9) and ovomucoid (pH 7) remains constant. At pH 7, bands of ovotransferrin disappear at around 60°C; globulin G3A, 65°C; ovalbumin, 80°C; globulins A1 and A2, 84°C; and flavoprotein, 85°C; ovomucoid does not disappear. Shimada and Matsushita (1980a) used sodium dodecyl sulfate PAGE to show that the relative amount of monomeric ovalbumin decreases rapidly after 5 min of heating and continues to slowly decrease up to 15 min, with the amounts of polymerized proteins increasing with heating time.
• Structural Changes. The denaturation step of the gelation process has been studied by observing structural changes of egg protein solutions upon heating. The reduced viscosity of a 1.8% ovalbumin solution increases with temperature of heating above 65°C and with time of heating (Holme, 1963). Removal of the aggregate by centrifugation yields supernatants with reduced viscosities and levorotation similar to those of the native, unheated solutions; this indicates that the intermediate denatured species of ovalbumin is rapidly converted to aggregates upon heating, since only aggregated and native molecules are present in a headed solution. Matsuda et al. (1981b) used circular dichroism at 222 nm and ultraviolet absorption at 287 nm to study heat induced structural changes and unfolding in ovomucoid, a very heat-resistant egg protein. Denaturation of egg white has also been studied using differential scanning calorimetry (Donavan et al., 1975); egg white (at pH 7) heated at 10°C/min exhibits two endotherms-one at 65°C (conalbumin) and the other at 84°C (ovalbumin).

New Method Developed

The above methods for measuring heat-induced coagulation do have certain limitations, one of which is the need to make single-point measurements on many samples at different time intervals throughout the process to obtain a continuous view of the phenomenon. These samples must be [cooled?] instantly; otherwise the coagulation process con[tinues?] another limitation of some gel-strength or gravimetric measurements is that the sample is destroyed during the measurement. Yet another limitation is that for some turbidity measurements, once the sample becomes opaque, no further change is registered by the spectrophotometer despite continuing changes in gel characteristics. Furthermore, a gel can be clear end still exhibit increasing gel strength with time.

With these limitations in mind, we devised a new method to quantitate, the gelation process (Gossett et al., (1983b). This nondestructive technique requires only one sample to observe the total process. It involves continuous monitoring of the force exerted by the gel as coagulation takes place and can be used to obtain kinetic data on the heat-induced gelation of egg albumen.

The technique uses a recording electrobalance from which a wire probe is suspended into a jacketed cylinder containing the albumen sample. The cylinder is connected to a water bath maintained at the appropriate temperature, and, as coagulation occurs with time, the force exerted by the gel on the probe as the matrix forms is recorded on e linear recorder.

Typical electrobalance force-vs-time curves (Fig. 1) for albumen heated at temperatures from 60 to 95°C show that the force increases until a maximum force is observed. For some treatments, however, there are decreases in force after the maximum force is attained; this is thought to be due to expansion of the gel once the gel coagulates—the expansion may have raised the probe and caused a decrease in observed force.

^a Quadruplicate determinations. From Gossett )1983), Gossett et al. (1983b)
^b Time at which first step "ends" and second step "starts"
^defg Means in the same column within a set that are followed by different superscripts are significantly different (P < 0.05)

Fig. 2-PLOT OF ln FORCE VS TIME showing rate constants k₁ and k₂ and the time t where the first step "stops" and the second step "starts." From Gossett at al. (1983b)

Rate constants for albumen (at pH 9) heated at 60-9.5°C and for albumen (pH 7.0-11.0) heated at 80°C are shown in Table 3. First-order kinetics and the existence of two reactions during the heat-coagulation of the albumen, as su[illegible] by the broken-line curve in Figure 2, yielded rate [constants] k₁ and k₂ and were obtained by best r₂ fit If k₁ is interpreted to be the rate constant for the denaturation process and k₂ the rate constant for the aggregation process, then the data suggest that the denaturation end aggregation rates both increase with increasing heating temperatures. The time at which the first step "ends" and the second step "starts" is found to decrease with increasing heating temperature; this suggests that at higher temperatures, denaturation occurs faster and the onset of aggregation is expedited. With increase in pH, rate constants for denaturation increase but those for aggregation decrease; the results agree with those of Nakamura et al. (1978) and Hermansson (1979), who suggested that aggregation is decelerated at higher pH levels.

Fig 3-ARRHENIUS PLOTS for rate constants k₁ (r=0.94) and k₂ r=0.931) From Gossett et al (1983b)

Activation energies for the rate constants were calculated using the Arrhenius relationship; Figure 3 shows E_a to be 8.7 kcal/mole for the denaturation step and 14.4 kcal/mole for the aggregation step (Dwek and Navon, 1972, estimated E_a for the denaturation of egg albumen to be approximately 24 kcal/mole). In addition, a Z_c value of 41.7°C was calculated by plotting the ln of the heating time required to reach a certain force value vs the heating temperatures.

Although this method offers the advantage of using just one a ample to continuously observe the gelation process, there are presently some limitations to the technique. The true source and physical meaning of the measured force are not yet understood and should be a topic of future research. Change. in the geometry of the gelling apparatus need to be explored to differentiate between the contribution of gelation and that of thermal expansion to the force. Lastly, optimization of probe design for various gel types would be desirable.

The gelation of food proteins is important to textural and rheological characteristics of a food product. Future, research should be directed toward finding quick, reliable methods that quantitate the gelation phenomenon so that food scientists can better manipulate the process to obtain desirable, high-quality foods.

References

Beveridge, T., Arntfield, S., Ko, S., and Chung, J.K.L. 1980. Firmness of heat-induced albumen coagulum. Poultry Sci. 59: 1229.

Bull, H.B. and Breese, K. 1970. Water and solute binding by proteins. I. Electrolytes. Arch. Biochem. Biophys. 137: 299.

Buttkus, H. 1974. On the nature of the chemical and physical bonds which contribute to some structural properties of protein foods: A hypothesis. J. Food Sci. 39: 484.

Cotterill, O.J. 1977. Freezing egg products. In "Egg Science and Technology," W.J. Stadelman and O.J. Cotterill, eds., 2nd ed., p. 145. AVI Pub. Co., Westport, CT.

Dagerskod, M. 1977. Time-temperature relationships in industrial cooking and frying. In "Physical, Chemical and Biological Changes in Food Caused by Thermal Processing," T. Hoyem and O. Kvale, eds., p. 77. Applied Science Publishers, Ltd., London.

Donovan, J.W., Mapes, C.J., Davis, J.G., and Garibaldi, J.A. 1975. A differential scanning calorimetric study of the stability of egg white to heat denaturation. J. Sci. Food Agric. 26: 73.

Dunkerley, J.A. and Hayes, J.F. 1980. Characterization of whey protein gels using a temperature gradient block. New Zealand J. Dairy Sci. Technol. 15:191.

Dwek, R.A. and Navon, G. 1972. On boiling an egg. Nature 240: 491.

Egelandsdal, B. 1980. Heat-induced gelling in solutions of ovalbumin. J. Food Sci. 45: 570.

Fasman, G.D. 1976. "Handbook of Biochemistry and Molecular Biology. Vol. II. Proteins," 3rd ed. CRC Press, Cleveland, OH.

Ferry, J.D. 1948. Protein gels. Adv. Prot. Chem. 4: 2.

Gossett, P.W. 1983. Some physical and rheological properties of raw and coagulated pH-adjusted or succinylated egg albumen with respect to water retention properties. Ph.D. thesis, Cornell Univ., Ithaca, NY.

Gossett, P.W. Rizvi, S.S.H., and Baker, R.C. 1983a. Selected rheological properties of pH-adjusted or succinylated egg albumen. J. Food Sci. 48: 1395.

Gossett, P.W., Rizvi, S.S.H., and Baker, R.C. 1983b. A new method to quantitate the coagulation process. J Food Sci. 48: 1400.

Haschemeyer, RH. and Haschemeyer, A.E.V. 1973. "Proteins," p. 353. John Wiley and Sons, NY.

Hegg, P.O. 1982. Conditions for the formation of heat induced gels of some globular food proteins. J. Food Sci. 47: 1241.

Hegg, P.O. and Lofqvist, B. 1974. The protective effect of small amounts of anionic detergents on the thermal aggregation of crude ovalbumin. J. Food Sci. 39: 1231.

Hegg. P.O., Martens, H., and Lofqvist, B. 1978. The protective effect of sodium dodecylsulphate on the thermal precipitation of conalbumin. A study on thermal aggregation and denaturation. J. Sci. Food Agric. 29: 245.

Hegg, P.O., Martens, H., and Lofqvist, B. 1979. Effects of pH and neutral salts on the formation and quality of thermal aggregates of ovalbumin. A study on thermal aggregation and denaturation. J. Sci. Food Agric. 30: 981.

Hermansson, A-M. 1979. Aggregation and denaturation involved in gel formation. In "Functionality and Protein Structure," A. Pour-El, ed., ACS Symp. Series 92, p. 81. Am. Chem. Soc., Washington, DC.

Hickson, D.W., Dill, C.W., Morgan, R.G., Sweat, V.E., Suter, D.A., and Carpenter, Z.L. 1982. Rheological properties of two heat-induced protein gels. J. Food Sci. 47: 783.

Holme, J. 1963. The thermal denaturation and aggregation of ovalbumin. J. Phys. Chem. 67: 782.

Johnson, TM. and Zabik, M.E. 1981. Gelation properties of albumen proteins, singly and in combination. Poultry Sci. 60: 2071.

Kuntz, ID. and Kauzmann, W. 1974. Hydration of proteins and polypeptides. Adv. Prot. Chem. 28: 239.

Lund, D.B. 1975, Heat processing. in "Principles of Food Science. Part II. Physical Principles of Food Preservation," O.R. Fennema, ed., p. 28. Marcel Dekker, Inc., NY.

Matsuda, T., Watanabe, K., and Sato, V. 1981a. Heat-induced aggregation of egg white proteins as studied by vertical flat-sheet polyacrylamide gel electrophoresis. J. Food Sci. 46: 1829.

Matsuda, T., Watanabe, K., and Sate, V. 1981b. Independent thermal unfolding of ovomucoid domains, Biochem. Biophys. Acta 669: 109.

Matsuda, T., Watanabe, K., and Sate, V. 1981c. Temperature-induced structural changes in chicken egg white ovomucoid. Agric. Biol. Chew. 45:1609.

Mirsky, A.E. and Pauling, L. 1936. On the structure of native, denatured, and coagulated proteins. Proc. Natl. Acad. Sci. 22: 439.

Nakamura, R., Sugiyama, H., and Sato, Y. 1978. Factors contributing to heat-induced aggregation of ovalbumin. Agric. Biol. Chem. 42: 819.

Osuga, D.T. and Feeney, RE. 1977. Egg proteins. In "Food Proteins," J.R. Whitaker and S.R. Tannenbaum, eds., p. 209. AVI Pub. Co., Inc., Westport, CT.

Powrie, W.D. 1977. Chemistry of eggs and egg products. In "Egg Science and Technology," W.J. Stadelman, and O.J. Cotterill, eds., 2nd ed., p. 65. AVI Pub. Co., Inc., Westport, CT.

Scheraga, HA. 1961. "Protein Structure," p. 81. Academic Press, New York.

Schmidt, RH. 1981. Gelation and coagulation. In "Protein Functionality in Foods," J.P. Cherry, ed., ACS Symp. Series 147, P. 131. Am. Chem. Soc., Washington, D.C.

Seideman, WE., Cotterill, O.J., and Funk, E.M. 1963. Factors affecting heat coagulation of egg white. Poultry Sci. 42: 406.

Shimada, K. and Matsushita, S. 1980a Thermal coagulation of egg albumin. J. Agric. Food Chem. 28: 409.

Shimada, K. and Matsushita, S. 1980b. Relationship between thermocoagulation of proteins and amino acid compositions. J. Agric. Food Chem. 28:413.

Shimada, K. and Matsushita, S. 1981. Effects of salts and denaturants on thermocoagulation of proteins. J. Agric. Food. Chem. 29: 15.

Vadehra, D.V. and Nath, KR., 1973. Eggs as a source of protein. CRC Crit. Rev. Food Technol., 4:193.

Yang, J.T. 1961. The viscosity of macromolecules in relation to molecular conformation. Adv, Prot. Chem. 16: 323.