Authors Kato, Ibrahim, and Kobayashi are with the Dept. of Agricultural Chemistry, Faculty of Agriculture, Yamaguchi Univ., Yamaguchi 753, Japan. Authors Watanabe and Honma are with OP. Corporation, Fuchu-shi, Tokyo 183, Japan.
The enthalpy of denaturation (H) and surface properties of proteins were related to elucidate the mechanisms of foaming and emulsifying properties by using various heated egg white proteins in the dry state. Foaming and emulsifying properties of all sample proteins were greatly increased with a decrease in the enthalpy of denaturation as determined by differential scanning calorimetry analysis. In plots, foaming and emulsifying properties correlated linearly with H values for various dry-heated egg white proteins. Thus, the enthalpy of denaturation of proteins seemed to be a significant structural factor governing surface functional properties.
AMPHIPHILIC PROTEINS are principally surface active agents (Kato and Nakai, 1980; Kinsella, 1981; Halling, 1981). Thus, awareness has increased on the role of surface hydrophobicity in the functional properties of proteins. Many studies have focused on the protein hydrophobicity as a structural factor affecting functional properties such as emulsification (Kato and Nakai, 1980; Shimizu et al., 1983) and foaming (Horiuchi and Fukushima, 1978; Townsend and Nakai, 1983). In addition to protein hydrophobicity, the surface functional properties are also related to molecular size (Kato et al., 1985b), conformational stability (Kato and Yutani, 1988), and net charge (Kato et al., 1987).
Aside from the structural factors, interfacial film formation is an essential event in formation of foam and emulsion. In foaming systems the formation of strong cohesive viscoelastic film is desirable for stable foaming. This may be a case of emulsion stability. Protein diffuses and adheres to the interface, the tertiary structure of polypeptide unfolds to a certain extent and spreads, because of the favorable thermodynamic situation at the interface. These dynamic events are influenced by the stability of the tertiary structure of proteins and the predominant environmental conditions. Recently, Kato and Yutani (1988) proved the importance of protein stability in surface functional properties using protein engineering techniques. The surface properties of natural and six mutant tryptophan synthase substituted at the same position, 49, were measured by surface tension, foaming and emulsifying properties and the surface properties were correlated with stabilities. Good correlations were observed between these surface properties and values of the Gibbs free energy of proteins unfolding in water. The parameter of protein stability used in that experiment was calculated from the folding-unfolding equilibrium state. On the other hand, the enthalpy of denaturation, reflecting protein stability, can be determined directly by calorimetry using differential scanning microcalorimetry. Thus, we were interested in investigating whether the enthalpy of denaturation is related to surface properties in order to correlate more closely the structural and functional properties of proteins.
We reported the improvement of functional properties of egg white proteins by dry-heating without loss of solubility or deteriorative effect on conformation. This phenomenon is suitable for studying the structure-function relation of proteins, because various conformational states can be obtained using the same protein. In our present study, various egg white proteins heated in the dry state, were used to estimate the influence of enthalpy of denaturation (H) on their functional properties.
EGG WHITE (DEW), spray-dried at 60-70°C after decarbohydrate treatment, was provided by Q.P. Corporation, Tokyo. Heat treatment of DEW was done as follow; 5g DEW were placed in a test tube, tightly sealed and incubated at 80°C for various periods of time (days) in the dry state (7.5% moisture). After heating to the given time, the tube was removed from the incubator and cooled to room temperature (25°C); subsequently surface functional properties were measured. The measurements were performed after passing the sample solutions through a filter paper to remove insoluble materials and then protein concentration was adjusted spectrophotometrically. However, we found the absorbance at 280 nm of the samples before and after filtration remained essentially the same.
Foaming properties of heated DEW in the dry state were determined by measuring the conductivity of foams produced when air at a constant flow rate of 90 cm2/min was introduced for 15 sec into 5 mL of 0.125% DEW protein concentration in 20 mM phosphate buffer, pH 7.4, in a vertical glass column (2.4x30 cm) with a glass filter at the bottom (Kato et al. ,1983). The conductivity of foams was measured by an electrode with a cell fixed inside the glass column 1 cm away from and 2.4 cm above the filter, connected to a conductivity meter (Kyoto Electrics Industry Co., Model CM- 07). Foaming power was defined as the maximum conductivity of the foams 15 sec after air was introduced. Foam stability was calculated from conductivity curves as time until the foam was not apparent.
Emulsifying properties of heated DEW in the dry state were determined by the conductivity method (Kato et al., 1985a). The emulsions were prepared as follow: 5 mL of corn oil and 15 mL of 0.185% DEW protein solution in 100 mM phosphate buffer, pH 7.4, were homogenized in Ultra Turax equipment (Hansen and Co., West Germany) at 12000 rpm for 1 min at 20°C. The emulsifying activity of each emulsion was calculated from the difference between the conductivity of protein solution and emulsion. The stability of each emulsion was calculated from the initial slope of the conductivity curve, as described previously (Kato et al., 1985a).
The thermal characteristics of egg white proteins heated in the dry state for various periods were examined by differential scanning calorimetry (DSC-100, Seiko, thermal analyzer, equipped with a DSC cell). In a typical experiment, 50 µL of about 10% protein solution was sealed in a preweighed hermetic aluminum pan and weighed. Another pan containing water with no protein was used as the reference. The pans were heated in the calorimeter at a linear rate of 1°C/min over the range of 30-120°C. The denaturation temperature (Td) and enthalpy of denaturation (H) were computed from the thermograms by the SSC-5000 analyzer (Seiko Electric Industry Co.).
All tests in this study were performed in duplicate, and the deviations were minor.
OUR PREVIOUS REPORT (Kato et al., 1990) showed the data of calorimetric analysis for DEW, globulin or albumin fractions and purified ovalbumin as a function of heating time in the dry state. The values of H dramatically decreased with an increase of heating time in the dry state for all protein samples. The rate of decrease in H was dependent on the protein. Reduction in the H was accompanied with a slight decrease in denaturation temperature (Td). This result suggested changes in molecular conformation, i.e., unfolding of the tertiary structures as reflected by the marked decrease in 'H and Td values. Accordingly, the approach was attempted relate improved surface properties with such thermally altered molecules.
Fig. 1--Foaming properties of egg white fractions as a function of heating time in the dry state. (A) foaming power; (B) foam stability. The determination was carried out for DEW (•), globulin fraction (0), albumin fraction (0) and purified ovalbumin (A).
Fig. 2--Emulsifying properties of egg white fractions as a function of heating time in the dry state. A) emulsifying activity; B) emulsion stability. Symbols as in Fig. 1.
The foaming properties of native and dry heated DEW, globulin or albumin fractions and purified ovalbumin are shown in Fig. 1. Increased heating time in the dry state caused a significant increase in both foaming power and foam stability of all samples. The greatest foaming power was obtained from heated DEW. The globulin fractions had a higher foaming power than the albumin fractions or purified ovalbumin. Increase in heating time in the dry state resulted in increased foam stability of all samples. Foam stability of DEW was much greater than that of other samples at any heating time including the non-heated sample. Foam stability of the native globulin fraction was higher than that of albumin fraction or purified ovalbumin. These observations suggested that occurrence of protein-protein interaction might be facilitated in DEW leading to the formation of strong foam film.
The emulsifying properties of dry heated DEW, globulin or albumin fractions and purified ovalbumin are shown in Fig. 2.
Fig. 3--Plots of foaming properties versus A H of heated egg white proteins in the dry state for various periods of time. Key:
(A) foaming power; (B) foam stability. Symbols as in Fig. 1.
With increased heating time in the dry state, marked improvement in both emulsifying activity and emulsion stability was observed for all samples. The highest emulsifying activity was obtained from DEW and the globulin fraction. The emulsion formed from purified ovalbumin was usually less active compared to those formed from other fractions. The emulsion stability of the globulin fraction was much higher than that of DEW, albumin fraction or purified ovalbumin at any heating time. The emulsion stability of DEW remained higher than that of albumin fraction. Emulsion stability of purified ovalbumin was less altered by dry heating than the other egg white fractions -
Fig. 4--Plots of emulsifying properties versus A H of heated egg white proteins in the dry state for various periods of time. Key: (A) emulsifying activity; (B) emulsion stability. Symbols as in Fig. 1.
Plots of foaming properties and enthalpy of molecular unfolding for dry heated DEW were constructed to study the contribution of thermodynamic stability of proteins to surface properties (Fig. 3). Good linear correlations were observed between decrease in H and increase in foaming power and foam stability for the protein constituents of each fraction of egg white. Therefore, these results indicated a clear relationship between enthalpy of unfolding values of dry heated DEW proteins and their functionality. The plots of H vs. emulsifying properties of dry heated DEW proteins are shown in Fig. 4. Similarly, linear correlation between H and emulsifying activity and emulsion stability were observed. Although linear correlation between H and emulsion stability of ovalbumin was observed, it was quite deviated from the regression line. This behavior of ovalbumin was presumed to be predominantly due to its amphiphilic nature since this structural element is well known to have a critical role in emulsion stability. Also it may be that multi-protein components of egg white are required to attain strong correlations between H and emulsifying properties of DEW as a function of heating time in the dry state. However, the decrease in enthalpy of denaturation of the proteins by dry heating appeared to be a major contributor to the significant improvement of foaming and emulsifying properties.
Our approach indicates that H is a determinant of foaming and emulsifying properties. Our previous studies revealed that heating of DEW in the dry state caused a substantial increase in its molecular flexibility (Kato et al., 1990) and surface hydrophobicity (Kato et al., 1989). In addition, circular dichroism spectra of dry heated DEW indicated very slight conformational changes. Our experimental results enable thermodynamic considerations for surface and functional properties proteins. The reduction of H of egg white proteins by heating may have enhanced the rate of diffusion of the thermally altered molecules to the interface, facilitating protein-protein association in formation of interfacial films. This would not be surprising, since Kato et al (1990) found definitive experimental proof of a strong relationship between enthalpy of denaturation and gel strength. High foaming and emulsifying properties of dry heated proteins reflect faster unfolding and greater intermolecular interaction at the interface. Proteins having high entropy due to reduction of AH and more flexible polypeptide chains should facilitate a greater degree of association by hydrophobic and electrostatic interactions. The high foam stability of dry heated proteins reflected greater molecular interaction to form more cohesive film. This film comprised extensive overlapping of coiled polypeptides, which allowed it to expand upon application of stress.
Our study is clearly broadening our concept of the relationship between thermodynamics and foam or emulsion forming ability of egg white proteins. Generally we can say that foams and emulsions from dry heated proteins were more stable than those from native proteins. Foaming and emulsifying properties were greater when the enthalpy of denaturation of these proteins was lowered by dry heating.
In conclusion, our observations indicate that the formation and the properties of interfacial films were mainly attributed to thermodynamic characteristics of the proteins. Further studies are needed to determine whether these relationships are applicable for other proteins of different food sources as well.
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Ms received 9/8/89; revised 12/2/89; accepted 3/6/90.