Dielectric properties determine the response

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1 USE OF DIELECTRIC PROPERTIES TO DETECT WHEY PROTEIN DENATURATION C. Bircan, S.A. Barringer and M.E. Mangino Denaturation of whey proteins can be detected by the dielectric properties. The dielectric properties of 20% whey protein with 0, 5 or 15% sugar, 2% salt, at ph 4 and isolated whey proteins were measured from room temperature to 100 C at MHz. The temperature at which a decrease in the dielectric loss factor, or increase in the dielectric constant, occurred was compared to the temperature of denaturation as determined by DSC and found to match. The change in the dielectric properties is likely caused by binding of water and/or ions that occurs during protein denaturation. For most of the samples, the dielectric loss factor showed the protein denaturation, but when ions were present due to salt or low ph, the dielectric constant showed the denaturation. Key Words: dielectric properties, protein, denaturation, whey ABOUT THE AUTHORS: Cavit Bircan, Sheryl A. Barringer and Michael E. Mangino are affiliated with The Ohio State University, Department of Food Science and Technolgy, Columbus, OH. Dielectric properties determine the response of a material to an electromagnetic field, such as occurs in a microwave oven. The inability of the molecules to instantaneously align with the applied electromagnetic field leads to dissipation of electromagnetic energy. The dielectric properties refer to a complex number consisting of a real portion (the dielectric constant or κ') and an imaginary portion (the dielectric loss factor or κ"). The dielectric constant is an indication of the polarizability of the molecules and their ability to store electric energy. The dielectric loss factor is related to the energy absorption and dissipation of electromagnetic energy from the field [Mudgett, 1985]. The dielectric constant is decreased by the presence of ions, which bind water and decrease its mobility [Mudgett, 1985]. In contrast, the dielectric loss factor is increased by the presence of ions. The dielectric loss factor measures the mobility of both the water and ions, but the conductive migration of the ions is much larger than the dipole motion. Therefore, as the mobility of the water and ions changes, the dielectric properties will change. The effect of salt addition on the dielectric constant is small, while the effect on the dielectric loss factor is large, therefore the dielectric loss factor is more sensitive to mobility changes. In food samples, water and salt are the two major ingredients which influence dielectric properties. Other food components usually have a minor influence on the dielectric properties. The electromagnetic field frequency and sample temperature affect dielectric properties as well. Water and salt content, frequency and temperature are commonly included in predictive equations, such as those of Sun et al. [1995] and Calay et al. [1995]. The physical state of the food is not included in these equations, though for some International Microwave Power Institute 179

2 foods it will influence the mobility of water and salt, which in turn will affect the dielectric properties. It has been reported that changes in physical state affect dielectric properties. The dielectric constant and loss factor change after the heating of gluten-starch mixtures [Umbach et al., 1992]. The dielectric loss of whey protein solutions increases during heating [Barringer et al., 1995]. The dielectric properties change as the electromagnetic field is oriented perpendicular or parallel to meat fibers [Bengtsson et al., 1963]. When starch gelatinizes the dielectric loss factor increases but the dielectric constant shows no change [Miller et al., 1991]. Therefore, protein denaturation, which either causes binding or release of water and salts, should cause a detectable change in the dielectric properties. This study was undertaken to determine whether or not the change is large enough to be important to predictive equations, as we have found it is for egg proteins (unpublished). There are several methods for detecting protein denaturation, but the most common is differential scanning calorimetry (DSC). The change in dielectric properties would provide an alternative method to DSC for detecting protein denaturation. Whey protein is used as an additive in many foods and has the advantage of being easily mixed with other ingredients for modeling purposes. Since the most common protein-containing food, muscle, contains approximately 20% protein, solutions of 20% whey protein were used. Sugar, acid and salt increase the denaturation temperature of protein by stabilizing the protein structure. Therefore these additives were used to change the denaturation temperature. Our objective was to determine whether protein denaturation in whey could be detected with the dielectric properties. Materials and Methods Twenty percent whey protein solutions were prepared from 80% whey protein concentrate (Land-O-Lakes, St. Paul, MN). 25 g of whey protein concentrate was gradually added to 75 g of water and stirred with a stirring bar for 30 min. The solution contained 0.2% sodium chloride as determined by Dichromat. A 0.2% sodium chloride solution was tested to determine the dielectric properties of the solutions without protein present. Whey protein solutions were also prepared with the addition of 5 or 15% sucrose, or 2% sodium chloride. The ph of the whey protein solution was reduced from 6.25 to 4.00 with 0.1 N HCl for the low ph samples. Individual whey protein solutions: β-lactoglobulin (10%), α-lactalbumin (20%) and bovine serum albumin (10%), were prepared by the same procedure used for the whey protein solutions. All individual proteins were purchased from Sigma. An open ended coaxial probe and network analyzer were used to measure dielectric properties (85070B and 8752C, Hewlett-Packard Company, Denver, CO). The probe was mounted with the ground flange facing up. An o-ring sealed the probe into a hole in the bottom of a 2.5-cm diameter stainless steel sample holder jacketed and attached to a silicon oil temperature bath. The probe and the cable were fixed so they could not be moved during sample measurement. A calibration was done using a short, air and water, before each set of experiments then checked to insure the calibration was stable. The dielectric properties were automatically calculated from the phase and amplitude of the reflected signal by the computer. The dielectric properties of the solutions were measured at frequencies that ranged from 300 to 2450 MHz from room temperature to 100 C. Samples were then cooled and reheated without removing the sample from the container to determine the dielectric properties of the denatured protein gel. All the measurements were repeated at least three times and were reproducible in a range ±6%. The change in the dielectric properties was reported as a range in Table 1, 180 Journal of Microwave Power & Electromagnetic Energy Vol. 36, No. 3, 2001

3 except where the change was large enough to determine the inflection point. Hermetic aluminum pans were used for sample measurement for DSC. The pan and lid were weighed and approximately 12 mg of the sample was placed into the pan with a syringe. Pans were sealed with the sample encapsulating press using crumpling kit # (TA Instruments, Inc., New Castle, DE). A reference of approximately 7 mg distilled water was prepared in a second pan. The pans were weighed to determine the exact weight. Tweezers were used to place the sample pan and the reference into the DSC cell (DSC 2920, TA Instruments, Inc., New Castle, DE). The heating rate was 10 C per min and the temperature was scanned from 20 to 95 C. The peak endotherm temperatures are reported. Results and Discussion Whey Protein Solution on dilute salt solutions. In a 0.2% salt solution containing no protein, the curve also decreased to 65 C then increased (Figure 1). TABLE 1: The dielectric properties and DSC indicate denaturation in the same temperature range. Sample Denaturation, C Dielectrics DSC 20% whey solution % whey + 5% sugar % whey + 15% sugar % whey + 2% salt % whey at ph % β-lactoglobulin % α-lactalbumin % Bovine serum albumin The dielectric loss factor of the whey protein solution decreased from room temperature to 65 C, increased from 65 to 75 C, decreased between C then increased beyond 80 C (Figure 1). The initial decrease to 65 C and increase to 75 C in the dielectric loss factor occurs merely because of the effect of temperature FIGURE 1: The dielectric loss factor of 20% whey protein solution with 0 and 15% sucrose, and of 0.2% sodium chloride without protein, 2450 MHz. International Microwave Power Institute 181

4 We propose that the decrease observed in the dielectric loss factor of the whey protein starting at 75 C is caused by the denaturation of the protein. This decrease is not caused by the temperature effect since the 0.2% salt solution, containing no protein, smoothly increased through this temperature range (Figure 1). When the whey protein solution was reheated after the protein was completely denatured, the decrease between C was not present (Figure 2); the dielectric loss factor of the reheat increased smoothly from 65 to 105 C. This is in keeping with the decrease being due to denaturation, an irreversible phase change. Further, protein denaturation is shown to occur at 78 C by DSC (Figure 3). The decrease in the dielectric loss factor occurred in the same temperature range as the peak of heat absorption as determined by DSC. The dielectric loss factor decreases due to water and ion binding by the protein during denaturation. Denaturation increases the water holding capacity of proteins [Walstra et al., 1984] causing the whey protein to bind water and ions when it denatures [Mangino, 1984]. When water and ions are bound, the dielectric loss factor decreases [Mudgett, 1985], causing the observed decrease in Figure 1. Miller et al. [1991] also used the dielectric loss factor to detect starch gelatinization. These results suggest that protein denaturation can be detected using the dielectric loss factor, however the change may not be large enough to warrant incorporation in predictive equations, based on calculations in Barringer et al. [1995]. Conversely, the dielectric constant of the whey protein solution showed no change during protein denaturation (Figure 4). Miller et al. [1991] also saw no changes in the dielectric constant during starch gelatinization. It is probable that the dielectric loss factor is more sensitive to small changes in water and ion mobility than the dielectric constant. The results to this point suggest that changes that occur during protein denaturation can be detected by measuring the dielectric loss factor of the whey solution. To show that the changes in the dielectric properties and those detected by DSC did not occur at the same temperature by coincidence, a series of experiments were done under conditions known to affect the temperature of protein denaturation. Adding sugar, salt and lowering the ph to 4 increases the conformational stability of the whey proteins and increases the temperature of protein denaturation. FIGURE 2: The dielectric loss factor of 20 % whey protein during reheating, 2450 MHz 182 Journal of Microwave Power & Electromagnetic Energy Vol. 36, No. 3, 2001

5 20% whey solution 20% whey with 5% sugar 78.6 C 20% whey with 15% sugar 79.3 C 20% whey with 2% salt 82.4 C 20% whey at ph C 10% β-lactoglobulin 85.5 C 20% α-lactalbumin 78.8 C 10% bovine serum albumin 75.0 C 87.6 C Temperature ( C) FIGURE 3: DSC of whey protein solutions. International Microwave Power Institute 183

6 FIGURE 4: The dielectric constant of 20% whey protein with 0 and 2% sodium chloride, 300 MHz. Whey Protein Solution With 5 and 15% Sucrose The dielectric loss factor versus temperature curve for whey protein plus 5 or 15% sucrose had the same shape as the whey protein without sugar added except for the temperature at which the loss factor began to decrease due to denaturation (Figure 1). The curves for 0 and 5% sucrose were similar so only 0 and 15% are shown. The presence of sugars increases the amount of water bound to the protein. This makes protein unfolding more difficult, because the energy difference between an exposed and unexposed hydrophobic group is greater in sugar water than in plain water [Back et al., 1979]. The denaturation temperature measured by DSC increased from 78.6 to 79.3 for 5% sugar and 82.4 C for 15% sugar. The decrease in the dielectric loss factor occurred in the same range, between for 0 and 5% sugar and C for 15% sugar (Table 1). The dielectric constant again showed no change. Whey Protein With 2% Sodium Chloride Solution When salt is added, the dielectric loss factor does not show a change at the temperature of protein denaturation. The ionic loss component from the salt becomes so large that changes in the dipole loss from water binding are no longer detectable. However, with the addition of ions the dielectric constant becomes sensitive enough to detect the denaturation of the protein. Lowering the frequency increased the magnitude of the change in the dielectric constant thus the results at 300 MHz are reported (Figure 4). The inflection point of the transition does not change with frequency, however the magnitude of the transition does change. 184 Journal of Microwave Power & Electromagnetic Energy Vol. 36, No. 3, 2001

7 Addition of salt induces crosslinking between proteins, increasing the denaturation temperature [Boye et al., 1995; Itoh et al., 1976]. Whey protein with 2% salt denatured at 81.2 C as measured by DSC (Table 1). The dielectric constant at 300 MHz increased with an inflection point at 83.8 (Figure 4). When a large number of ions are added, the increase occurs over a large enough range to allow the inflection point to be determined. The dielectric constant increases when ions are removed from solution [Mudgett, 1985]. The whey protein binds the ions during denaturation [Mangino, 1984]. The removal of some of the ions from solution by the protein would increase the dielectric constant as seen in Figure 4. No change in the dielectric constant was seen in whey protein solution without salt (Figure 1) because that solution contains only 0.2% salt. Such as small amount of salt binding would not noticeably affect the dielectric constant. Whey Protein Solution at ph 4 The effects of ph and salt on the dielectric properties are similar. The additional hydrogen ions affected the dielectric loss factor to such a large extent that small changes caused by whey protein denaturation were no longer detectable, therefore the dielectric constant was used. Reducing the ph from 6.25 to 4.00 increases the thermal stability of whey protein, increasing the denaturation temperature. The denaturation temperature of the whey protein increased from 78.3 at ph 6.25 to 85.5 C at ph 4.00, as measured by DSC (Table 1). The dielectric constant at ph 4.00 increased between C. Individual Whey Proteins Whey proteins contain 50% β-lactoglobulin, 19% α-lactalbumin and 5% bovine serum albumin. The individual whey proteins β-lactoglobulin, α-lactalbumin and bovine serum albumin were measured. β-lactoglobulin denatured at 78.8 C as determined by DSC (Table 1). The dielectric loss factor decreased between C. The shape and denaturation temperature of the curve was similar to that for the 20% whey protein solutions. β-lactoglobulin is the major protein in whey protein and therefore is responsible for most of the changes observed in the whey protein solutions. The shape of the curve for α-lactalbumin and bovine serum albumin were also similar to whey protein except the decrease in the dielectric loss factor occurred at different temperatures. The denaturation temperature of α-lactalbumin was 75.0 C using DSC and C by the dielectric loss factor. The denaturation temperature of bovine serum albumin was 87.6 C using DSC and C by the dielectric loss factor. For bovine serum albumin, the dielectric constant also showed an increase in the dielectric constant at the same temperature. This was the only protein which showed denaturation in both the dielectric constant and loss factor, which may indicate that the protein binds more water than the other proteins. Conclusions Whey protein denaturation affects the dielectric properties. The dielectric loss factor can be used to determine protein denaturation in solutions with a dielectric loss factor near that of water. For solutions with a high dielectric loss factor due to added ions, the dielectric constant can be used. Since this method detects changes in water and ion mobility associated with denaturation, instead of the change in heat capacity, it will give different information than can be found with DSC. Further refinements such as reducing sample size and temperature intervals could be made to increase the sensitivity. References Back, F. J., Oakenfull, D., Smith, B. M International Microwave Power Institute 185

8 Increased thermal stability of proteins in the presence of sugars and polyols. Biochemistry. 18: Barringer, S. A., Fleischmann, A. M., Davis, E. A., Gordon, J The dielectric properties of whey protein as indicators of change in polymer mobility. Food Hydrocolloids. 9(4): Boye, I.J, Alli, I., Ismail, A.A., Gibbs, F. B., Konishi, Y Factors affecting molecular characteristic of whey protein gelation. Int. Dairy Sci. 5: Bengtsson, E. N., Melin, J., Remi, K., Soderlind, S Measurements of the dielectric properties of frozen and defrosted meat and fish in the frequency range MHz. J. Sci. Food Agric. 14: Calay, R.K, Newborough, M., Probert, D., Calay, P.S Predictive equations for the dielectric properties of foods. Int. J. Food Sci. Technol. 29: Itoh, T., Wada, Y., Nakanishi, T.I Differential thermal analysis of milk proteins. Agric. Biol. Chem. 40: Mangino, M.E Pysico-chemical aspects of whey protein functionality. J. Dairy Sci. 67: Miller, L.A, Gordon, J., Davis, E.A Dielectric and thermal transition properties of chemically modified starches during heating. Cereal Chem. 68(5): Mudgett, R.E Dielectric Properties of Food. In: Decareau RV. editor. Microwave in the Food Processing Industry. Academic Press, Inc., New York. p Sun, E., Datta, A., Lobo, S Composition-based prediction of dielectric properties of foods. Int. Microwave Power Institute. 30(4): Umbach, S.L., Davis, E.A., Gordon, J., Callaghan, P.T Water self-diffusion coefficients and dielectric properties determined for starch-gluten-water mixtures heated by microwave and by conventional methods. Cereal Chem. 69(6): Walstra, P. and Jenness, R Dairy Chemistry and Physics. John Wiley and Sons, New York. 186 Journal of Microwave Power & Electromagnetic Energy Vol. 36, No. 3, 2001