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Effect of pH during the dry heating on the gelling properties of egg white proteins
Food Research International, Vol. 29, No. 2,
PII:
ELSEVIER
pp. 155-161, 1996
Copyright 0 1996 Published by Elsevier Science Ltd on behalf of the Canadian Institute of Food Science and Technology SO963-9969(96)00008-7 Printed in Great Britain 0963-9969/96 $15.00 + .OO
Effect of pH during the dry heating on the gelling properties of egg white proteins
Yoshinori Mine Department
of Food Science, University of Guelph, Guelph, Ontario, Canada NIG 2 WI
Gel strength and elasticity of dried egg white proteins greatly increased by heating in the dry state at alkaline pH region (under 9.5) for a short period of time (3-5 days) without losing water solubility. Circular dichroism and differential scanning calorimetry spectrum revealed that the increased degree of denaturation found during preheating was accelerated in alkaline pH region. The polymerization of the proteins was also enhanced by alkaline dry heating through sulfhydryl-disulfide interchange. Alkaline dry heating resulted in high molecular weight polymer of partially unfolded egg white proteins which, in turn, contribute in the formation of low molecular weight and narrow molecular distribution of the aggregate found during the subsequent heat for gelation. Thus, heating of dried egg white proteins in the dry state at alkaline pH (under 9.5) is an effective method to obtain firm and elastic gels. The degree of unfolding of the proteins upon dry heating may play a crucial role in the gelling process of the proteins. Copyright 0 1996 Published by Elsevier Science Ltd on behalf of the Canadian Institute of Food Science and Technology Keywords: egg white, gelation, dry heating, pH, aggregation.
mechanism and the structure-function relationship of food proteins. Kato et al. (1989, 1990a,b) reported that mild conformational changes and polymerization due to disulfide formation and/or sulfhydrykiisulfide (SH-SS) interchange in DEW proteins were attributable to the dry heating. The majority of sulfbydryl residues in egg white protein exist in the interior of protein molecules and are exposed with heat denaturation (Mine et al., 1990). Heat-induced changes of sulfhydryl levels in egg white proteins in solution are well characterized and SH-SS interchange is affected by pH (Beveridge & Arntfield, 1979). However, the process of protein interactions during dry heating and gelation processing of DEW proteins are not still well understood. The molecular unfolding of DEW proteins upon dry heating can be affected by pH, temperature and moisture content during dry heating. There are no available data concerning these effects of heating in dry state, despite the importance to industrial applications. Heat-induced gelation is a two-step process involving protein denaturation and
INTRODUCTION Dried egg white is generally heat treated at Y-65°C to reduce microbial number. The effect of high temperature storage of spray-dried egg white (DEW) on physical changes and thermal resistance of Salmonella organisms is well characterized (Slosberg et al., 1948; Cotterill et al., 1967; Baldwin ef al., 1967; McBee & Cotterill, 1971). These researchers studied the effect of low heating temperature (below 70°C) on DEW proteins. However, until the recent studies of Kato et al. (1989), there were no reports of the effects of heating egg white proteins in a dry state on their functional properties. These studies reported a significant improvement of functional properties, including foaming, emulsifying and gelling properties of DEW, on the protein heating in a dry state at 80°C for several days (7.5% moisture content). This method can be one of the most promising approaches for use in food processing. The mechanism of protein denaturation during dry heating is important in order to elucidate the gelation 155
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aggregation to form a cross-linked matrix. The mechanism of gelation of egg white proteins is considered to be similar to that of other globular protein. Monitoring the changes in protein structure of egg white protein upon dry heating and heat-induced gelation will help to further the understanding of the mechanisms involving gelation and texture formation by egg white proteins. The relationship between gel strength and the extent of aggregation of egg white proteins upon heat-induced gelation was studied using the low angle laser light scattering technique (Kato et al., 1990~). The average molecular weight of heat-induced DEW aggregates decreased greatly with increase of preheating time in the dry state and the gel strength greatly increased. A good correlation was observed between decrease in molecular weight and increase in gel strength of dry-heated DEW. However, Kato et al. only studied the heat-induced aggregation of dry-heated DEW proteins in solution. There is little information on the aggregation behavior of DEW proteins during dry heating. The purpose of this study was to clarify the effect of pH during dry heating on the functional and conformational changes of DEW proteins. In addition, the relationship between the aggregation behavior upon heat-induced and gelling properties of egg white proteins was examined.
MATERIALS
AND METHODS
Following treatment by a glucose oxidase catalase enzyme system, the egg white liquid was adjusted to pH 6.0, 6.5 and 7.3 with 1 N HCl or 1 N NaOH, respectively, before spray drying. Spray drying was done using a pilot plant dryer with an exhaust temperature of 65-70°C. The DEW with three pH levels before spray drying was stored at 75°C for various periods of time (days) in dry state (8.5% moisture content). Periodically, samples of DEW were taken out from the incubator and cooled at room temperature. These samples were used for testing the structural and functional properties after passing the sample solution through a filter paper to remove any insoluble materials. Preparation of DEW protein gels were carried out as follows: 10% protein solution of each sample in 20 mM phosphate buffer (pH 7.5) was put into 30 mm diameter polyvinylidene chloride tubes. The tubes were then sealed and immersed in a water bath at a temperature of 80°C for 40 min. After a given time, the tubes were taken from the hot bath and cooled to room temperature in an ice bath. The samples were kept at 4°C overnight before being subjected to testing. The quality of the gels was assessed by measuring their gel strength (g/cm) and deformation (cm). Every sample piece was prepared to have 30 mm height by cutting the original cylindrical shaped gel. Each gel piece was set on the detachable table of a Rheometer@
(Fudo Kogyo K. K., Tokyo) equipped with a spherical plunger (4 = 8 mm). Compression on the sample piece was executed with a table speed 60 mm/min. Jelly strength was obtained by deformation (g x cm). The measurement was repeated eight times on each sample gel and the averages were calculated. Measurement of sulfhydryl groups was performed by using Ellman’s reagent (Beveridge et al., 1974). A total of 4 ml of 0.1 M tris-glycine buffer, pH 8.0, containing 10 M urea and 0.01 M EDTA was added to 1 ml of the 1% protein solution. After incubation at 40°C for 30 min, 125 ~1 of 5,5’-dithiobis(2nitrobenzoic acid) solution (20 mg in 5 ml of 0.1 M tris-glycine buffer, pH 8.0) was added and then incubated at 25°C for 10 min. The color absorbance was read at 412 nm on a Hitachi U-2000 spectrophotometer. The total SH residues were calculated as follows: PM SH/g = 73.53 . A4,2 . D/C where A4i2 = the absorbance at 412 nm; C = the sample concentration in mg/ml; D = the dilution factor, 5.125 (Beveridge et al., 1974). Measurement of free amino groups was carried out as follows: 200 ~1 of DEW solution (0.2%) was added to 2.0 ml of 0.2 N sodium borate buffer, pH 9.2, and then 1.0 ml of TNBS reagent (equal volume of 0.1% trinitrobenzensulfonic acid and 0.126% sodium sulfide) was added. This mixture solution was allowed to stand at 40°C for 2 h covered with aluminum and subsequently cooled to room temperature before the absorbance was measured at 420 nm. The values of residual free amino groups of heat-treated samples were represented as the ratio to one of the nonheated DEW sample. The thermal characteristics of DEW proteins heated in the dry state for various pHs were examined using a differential scanning calorimetric (DSC) thermal analyzer (Seiko DSC-100, Tokyo, Japan) equipped with a DSC cell. A total of 50 ~1 of 8.0% protein solution in 20 mM phosphate buffer, pH 7.0, was sealed in a preweighted hermetic silver pan. Water was used as the reference. The pans were heated in the calorimeter at a linear rate Z”C/min over the range 30-120°C. The denaturation temperature (Td) and enthalpy of denaturation (AH) were computed from the thermograms by the SSC-5000 analyzer (Seiko Electric Industry Co., Tokyo, Japan). Circular dichroism (CD) analysis was carried out to assess the conformational changes of DEW heated in the dry state. DEW proteins were dissolved in 10 mM phosphate buffer, pH 7.2, and then filtered with a Millipore filter (pore size 0.22 pm) Each protein concentration was adjusted to 7.4 x 10m3 (%). CD spectra were measured on a JASCO J-720 spectropolarimeter using a 10 mm cell at 25°C in the far ultraviolet region (20&250 nm). The data was represented in terms of mean residue ellipticity (deg.cm*.dmol-‘).
EfSect of pH on egg white proteins
Heat-induced DEW protein aggregates were processed as follows: diluted DEW protein solution (0.1% protein in 10 mM phosphate buffer, pH 7.5) were passed through a membrane filter (0.5 pm, Shodex MX-25K) with subsequent adjustment of the protein concentration spectrophotometrically. A total of 3 ml of DEW solution was put into a test tube with a screw cap and the tube was immersed in a water bath at 80°C for 20 min and then cooled to room temperature immediately. Next, 200 ~1 of the sample was applied to a multiangle laser light scattering (MALLS) experiment. The MALLS experiments were conducted at room temperature on a DAWN DSP-F laser photometer (Wyatt technology, Santa Barbara, CA) using a 632.8 nm laser wavelength. The MALLS system was equipped with a high performance liquid chromatography system, consisting of Shodex KW-804 and KW-803 columns (Showa Denko K. K., 0.75 x 30 cm). A precision differential refractometer (Shodex RI) was used as a detector. As an elution buffer, 50 mM phosphate buffer, containing 0.15 M NaCl (pH 7.0) was used and flow rate was 1 ml/min. Scattering angle was 30-150” for each sample at room temperature. A specific refraction index increment (dn/dc) of 0.186 was obtained for a dialyzed DEW solution using a Wyatt Optilab differential refractometer, Model 903. Second virile coefficient of 1.94 x 10e4 was calculated from Zimm plot.
157
Ob Heating time (days) Fig. 1. Relationship between gel strength and heating time in the dry state of spray-dried egg white proteins at various pHs. (W) dry heated at pH 6.88 (control), (A) dry heated at pH 8.55 and (0) dry heated at pH 9.40.
RESULTS The pHs of DEW solution in water (10% protein), which were adjusted to pH 6.0, 6.5 and 7.3 before spray drying, were pH 6.88, 8.55 and 9.40, respectively. In general, liquid egg white is subjected to spray drying under pH 5.8-6.3 and the pH of DEW powder is 7.0-7.5. The increase in pH in DEW powder is due to the evaporation of soluble carbon dioxide in liquid egg white by spray drying. Thus, the DEW powders with different pHs were named as follows: DEW (pH 6.88-control), DEW (pH 8.55) and DEW (pH 9.40). The effect of pH during dry heating of DEW on the gelling properties was investigated (Figs 1 and 2). Figure 1 shows the relationship between heating time in the dry state at various pHs and the gel strength of DEW. The gel strength of DEW protein was greatly increased with increased heating time and resulted in an excellent firm gel structure. Interestingly, the final gel strength of all DEW samples after dry heating for 15 days showed almost the same values (900-950 g/cm). The increasing rate of gel strength in relation to heating time was larger in DEW (pH 9.40) than in DEW (pH 6.88-control). The relative strength of DEW (pH 9.40) indicated that it was about four times the value of nonheated DEW after 3 days. On the other hand, it took 7 and IO days to obtain the same gel strength in DEW (pH 8.55) and
0
5
10
15
Heating time (days) Fig. 2. Relationship between jelly strength and heating time in the dry state of spray-dried egg white proteins at various pHs. (m) dry heated at pH 6.88 (control), (A) dry heated at pH 8.55 and (0) dry heated at pH 9.40.
DEW (pH 6.88-control), respectively. The effect of heating in the dry state on the solubility of DEW proteins was very small (data are not shown). The relationship between heating time in the dry state at various pHs and the jelly strength of DEW protein gels are shown in Fig. 2. Heating in the dry state at pH 9.40 progressively increased the jelly strength within a short period of time (5 days) and resulted in excellent elastic gels of DEW proteins. The transparency of DEW (pH 9.40) gel was higher than those of DEW (pH 6.88-control) or DEW (pH 8.55) gels (data are not shown). Figures 3 and 4 show changes in total SH residues and free amino groups on the heat-treated DEW in the
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F *OM Heating time (days) Fig. 3. Changes of total sulfhydryl residues in spray-dried egg white proteins during the dry heating. (m) dry heated at pH 6.88 (control), (A) dry heated at pH 8.55 and (0) dry heated at pH 9.40.
210
230
250
Wavelength (nm) Fig. 5. Circular dichroism spectrum of nonheated and heated spray-dried egg white proteins in the dry state at various pHs. Key: ___ nonheated; . . . heated at pH 6.88 (control); - - heated at pH 8.55; - . - heated at pH 9.40 for 3 days at 75°C.
0
5
10
15
Heating time (days) Fig. 4. Changes in free amino groups of spray-dried egg white proteins during the dry heating. (m) dry heated at pH 6.88 (control), (A) dry heated at pH 8.55 and (0) dry heated at pH 9.40.
dry state for various periods at various pHs. The total SH residues of DEW proteins were gradually decreased with increasing heating time and pHs during heating in the dry state. This result suggests that SH-SS interchange reactions were accelerated by heating in the dry state. The SH-SS interchange reaction occurred more rapidly in the alkaline pHs than in neutral pHs. Little changes in the free amino group were observed at DEW (pH 6.88-control) and DEW (pH 8.55) upon heating, except at 15 days of heating time. On the other hand, the residual free amino group of DEW (pH 9.40) was decreased with increasing heating time. In total, 15% of total amino groups of the protein decreased by heating in the dry state for 5 days. Any browning reaction of each sample was not observed with heating time up to 10 days. However, a slight browning of the protein was
observed upon heating for 15 days. No differences in the electrophoretic patterns of DEW proteins with or without SDS were observed between native and dry-heated DEW proteins. No peptide bond hydrolysis of the proteins was detected. On the other hand, a dramatic change in color of DEW during dry heating was observed when the protein was heated at over pH 10.0 in the dry state. Solubility of this DEW (pH 10.0) was greatly decreased because of the aggregation of the proteins (data are not shown). The effect of heating in the dry state on the structure of DEW proteins at various pHs was studied by CD analysis. Figure 5 shows a typical CD spectra of nonheated and heat-treated samples for 3 days at 75°C in the dry state. No changes in CD spectrum were observed among the nonheated samples of each pH. As shown in Fig. 5, the change in CD spectrum of DEW (pH 6.88-control) protein was very mild. However, the heat treatment in the dry state of DEW at pH 8.55 or 9.40 affected their CD spectra, which was reflected as a decrease of a-helix content. The structural changes of DEW were further investigated by differential scanning calorjmetry (Fig. 6 and Table 1). The DEW proteins exhibited two major endothermic peaks which was tentatively identified as ovotransferrin (Td, 60.7”C) and ovalbumin (Td, 8O.l”Q respectively, with a total enthalpy of 14.0 J/g (Donovan et al., 1975). Changes in DSC peaks among the non-
Efect
ofpH
on egg white proteins
159
Table 1. Thermal characteristics and gel strength of spray-dried egg white (DEW) proteins under dry heating for 3 days at different pHs
a
Td (“C)
PH
(% b
Peak I
Peak 2
60.7 60.0 NDb ND
80.1 79.2 79.3 79.7
Controls pH 6.88 pH 8.55 pH 9.40
14.0 12.6 10.6 6.0 ____
Gel strength (g/cm)
123 297 481 728
“Nonheated DEW. ‘Peak not detectable.
Table 2. Laser light scattering characteristics of heat-induced spray-dried egg white aggregates -- ._
Samples”
5b
6b Heating
I
I
I
70
80
90
temperature
id0
(“C)
Fig. 6. Differential scanning calorimetric thermograms of spray-dried egg white proteins. Samples were heated in the dry state for 3 days at 75°C. Key: (a) nonheated; (b) heated at
pH 6.88 (control); (c) heated at pH 8.55; (d) heated at pH 9.40.
heated proteins
of each pH were not detected. When the
samples were heated in the dry state for 3 days at 75”C, there was a broadening of the peaks with a marked decrease in AH. Alkaline dry heating resulted in a further decrease of AH and broadening in the endothermic peaks, and their extent increased with increased pH of DEW proteins. However, no detectable decrease in Td value was observed upon heating in the dry state at various pHs. These data indicate the existence of intermediate forms different from the native form. Simply, the conformation of DEW protein molecule has shifted toward the partially unfolded state by dry heating. A good correlation was observed between the decrease in AH and the increase in gel strength of DEW proteins (Table 1). The molecular size and radius minimal square moments (RMS) of solution before heating and heatinduced soluble aggregates (progel) formed prior to gelation of DEW proteins were investigated by the MALLS system. The data are summarized in Table 2. The weight average molecular weight moments (Mw, g/ mol) and number average molecular weight moments (Mn, g/mol) of nonheated DEW proteins in the dry state were 54.8 kDa and 45.0 kDa, respectively. This value is close to the known value of ovalbumin, a major constituent of DEW proteins. This may indicate the general success of this measurement. The Mw and Mn of 0.1% DEW protein solution which was heated in dry state for 3 days increased with increasing pH during dry
Mw/Mn
RMS(W)~
(x!%)b
(x%4)
5.48
4.50
1.21
16.5
15.93 21.54 37.53
5.51 6.04 7.89
2.89 3.57 4.76
23.5 23.6 27.7
311.60
9.04
34.49
27.9
48.70 54.76 64.35
7.65 8.61 11.11
6.37 6.36 5.79
23.7 23.3 25.0
-___ Control Nonheated Dry heating at: pH 6.88 pH 8.55 pH 9.40 Heatede Nonheated Dry heating at: pH 6.88 pH 8.55 pH 9.40
“Samples were heated in dry state for 3 days at 75°C. bWeight-ave rag e molecular weight moments (g/mol). ‘Number-average molecular weight moments (g/mol). dRadius minimal square moments (nm). “0.1% protein in 50 mM phosphate buffer (pH 7.0) was heated at 80°C for 20 min.
heating. The relative Mw and Mn of DEW (pH 9.40) proteins showed about 7.0 and 1.7 times the value of nonheated DEW proteins after 3 days of dry heating. The Mw/Mn ratio of DEW (pH 9.40) increased about 4 times compared with the control. The RMS of nonheated DEW proteins was 16.5 nm and this value increased with dry heating and with increasing pH during dry heating based on the aggregation. These data indicate that DEW proteins were polymerized by dry heating. The polymerization of the proteins was accelerated by heating in the dry state in the higher pH region. The polydispersity of DEW protein aggregates was increased with increasing pH during the dry state. Interestingly, the heat-induced aggregation behavior of DEW proteins in solution between nonheated DEW and dry-heated DEW was quite different. The Mw of nonheated DEW was estimated to be 3116 kDa and the ratio of Mw/Mn was 34.5. The data indicate that the
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molecular weight distribution of the heat-induced aggregate of nonpreheated DEW was large and wide. On the contrary, the preheated DEW in the dry state showed small (400-650 kDa) and narrow molecular weight distribution of the heat-induced aggregates. No substantial differences of the RMS of heat-induced aggregates between nonheated DEW and dry heating DEW proteins was shown. These data show that dry heating led not only to a decrease in the molecular weight of DEW aggregates but also to the formation of an expanded structure of aggregates. This spread of aggregates resulted from the high molecular weight polymer of partially unfolded egg white proteins, increasing with increasing pH during dry heating.
DISCUSSION
A protein gel network is generally formed via noncovalent cross-linkages such as hydrophobic interactions, hydrogen bonds, or electrostatic interactions and less frequently by covalent interactions such as disulfide bonds (Clark, 1992). Dry heating of egg white powder is an effective method to reduce the microbial number, such as Salmonellae (McBee & Cotterill, 1971). This method has also been used on an empirical basis in Canada to improve the gel-forming properties of DEW since 1982 (Dr E. D. Murray, personal communication). It was reported that heating for 10 days in the dry state (8o”C, 7.5% moisture content) is proposed to gain the maximum functional properties of DEW (Kato et al., 1989). However, Kato et af. did not measure the pH of DEW upon dry heating. The present work revealed that the controlled heating at alkaline pHs (under 9.50) in the dry state is an effective method to improve the gelling properties of DEW proteins without losing solubility and browning reaction for a short period of time (3-5 days). These phenomena are important in elucidating the structure-function relationship of food proteins on a molecular basis. Mild conformational changes are caused in DEW proteins by the dry heating and these structures may play a critical role in the formation of strong and stable gels of dry-heated DEW. In addition protein-protein interactions due to disulfide formation and/or SH-SS interchange also occurred in DEW proteins upon dry heating. CD spectrum obtained in the present work suggested that the degree of conformational change of DEW proteins upon dry heating increased in the alkaline pH region without losing solubility. Molecular weight of the polymer of DEW (pH 9.40) was higher than that at neutral pHs and was due to disulfide formation and/or SH-SS interchange. An increase in pH in DEW during heating in the dry state resulted in an additional decrease of AH and more broadening in the endothermic peaks. It has been well documented that broadening of peaks indicates the existence of intermediate forms different from the
native form (Myers, 1990). Thus, partially unfolded conformations of DEW which occur upon dry heating may play an important role in the gelation process. It is particularly interesting that solubility of DEW proteins was not greatly affected even though the proteins were denatured and formed a polymer with molecular weight of 200-375 kDa upon dry heating. Protein solubility is often affected by temperature, pH and the presence of other solutes and salts. Solubility is related to the net free energy change from interaction of hydrophobic and hydrophillic residues on the protein surface with the surrounding solvent. No changes on the protein surface and no peptide bond hydrolysis of DEW proteins upon alkaline dry heating were detected by electrophoretic analysis. On the other hand, free amino groups of alkaline dry-heated proteins decreased with increasing heating time. It can be assumed that deamidation or isomerization of amino acid residues of DEW proteins also occurred during this process. However, it is difficult to conclude the exact chemical changes of the proteins from the present data. Further studies are required to determine the role of amino groups which might affect the protein solubility and gelling properties of DEW proteins upon alkaline dry heating. It was reported that the main factor contributing to the heat-induced aggregation of ovalbumin was the degree of electrostatic repulsion on the denatured protein molecules (Nakamura et al., 1978). Hatta et al. (1986) reported the importance of pH and ionic strength on the turbidity and hardness of heat-induced ovalbumin gels. It is suggested that the gel strength of DEW proteins could be determined by factors determining the state of aggregation. Alkaline dry heating egg white proteins results in the formation of low molecule weight soluble aggregates during the subsequent heat for gelation. In addition, the homogeneity of the molecular size in the soluble aggregate may be important in the formation of a firm gel matrix. The Mw/Mn ratios decreased with increasing pHs upon heating, due to initial aggregation caused by dry heating. These results basically support the concept obtained by Kato et al. (1990~). In addition, the present data suggest that the increased degree of denaturation of DEW proteins during dry heating and the homogeneity of the molecular size in soluble aggregates (under 1000 kDa) during the subsequent heat for gelation may be critical factors for the formation of an excellent gel network. The RMS data also predicted the existence of expanded structures or the formation of linear polymers (Mw < 1000 kDa) by preheated DEW proteins. In conclusion, it is proposed that dry heating of egg white proteins at alkaline pH (under 9.5) is an effective method to improve the gelling properties in a short period of time within 5 days without any loss of solubility and browning reaction. The formation of partially denatured proteins which have high solubility may be suitable form for improving the protein functionality.
E&t
of pH on egg white proteins
ACKNOWLEDGEMENT The
author
Producer’s
gratefully Marketing
161
Kato,
acknowledges Board
for
the financial
Ontario support
Egg of
this work.
REFERENCES Baldwin, R. E., Cotterill, 0. J., Thompson, M. M. & Myers, M. (1967). High temperature storage of spray dried egg white I. Whipping time and quality of angel cake. Poultr? Sci., 46, 1421-30. Beveridge, T. & Arnttield, S. (1979). Heat induced changes in sulphydryl levels in egg white. Can. Insf. Food Sci. Technol. J., 12, 173-6. Beveridge, T., Toma, S. J. & Nakai, S. (1974). Determination of SH- and SS-groups in some food proteins using Ellman’s reagent. J. Food Sci., 39, 49951. Clark. A. H. (1992). Gels and gelling. In Physical Chemistry q/ Food.r, ed. H. G. Schwartzberg & R. W. Hartel. Marcel Dekker, New York, pp. 2633305. Cotterill, 0. J., Baldwin, R. E. & Myers, M. (1967). High temperature storage of spray dried egg white 2. Electrophoretic mobility, conalbumin iron complexing, sulfhydryl activity, and evolution of volatile bases. Poultry Sci., 46, 1431-7. Donovan, J. W., Mapes, C. J., Davis, J. G. & Garibaldi, J. A. (1975). A differential scanning calorimetric study of the stability of egg white to heat denaturation. J. Sci. Food Agric., 26, 73-83. Hatta, H., Kitabatake, N. & Doi, E. (1986). Turbidity and hardness of a heat induced gel of hen egg ovalbumin. Agric. Biol. Chem., 50, 2083-9.
A., Ibrahim, H. R., Watanabe, H., Honma, K. & Kobayashi, K. (1989). New approach to improve the gelling and surface functional properties of dried egg white by heating in dry state. J. Agric. Food Chem., 37, 433-7. Kato, A., Ibrahim, H. R., Watanabe, H., Honma, K. & Kobayashi, K. (1990~). Structural and gelling properties of dry-heating egg white proteins. J. Agric. Food Chem., 38, 32-7. Kato, A., Ibrahim, H. R., Watanabe, H., Honma, K. & Kobayashi, K. (1990h). Enthalpy of denaturation and surface functional properties of heated egg white proteins in the dry state. J. Food Sci., 55, 128&3. Kato, A., Ibrahim, H. R., Takagi, T. & Kobayashi, K. (1990~). Excellent gelation of egg white preheated in the dry state is due to the decreasing degree of aggregation. J. Agric,. Food Chem., 38, 1868-72. McBee, L. E. & Cotterill, 0. J. (1971). High temperature storage of spray dried egg white 3. Thermal resistance of .salmonrllu organienburg. Poultr), Sci., 50, 452-8. Mine, Y., Noutomi, T. & Haga, N. (1990). Thermally induced changes in egg white proteins. J. Agric. Food Chem., 38, 212225. Myers, C. D. (I 990). Study of thermodynamics and kinetics of protein stability by thermal analysis. In Thermal Analysis qj” Foods, ed. V. R. Harwalkar & C.-Y. Ma. Elsevier Applied Science, New York, pp. 1650. Nakamura, R., Sugiyama, H. & Sato, Y. (1978). Factors contributing to the heat-induced aggregation of ovalbumin. Agric. Biol. Chem., 42, 819-24. Slosberg, H. M., Hanson, H. L., Stewart, G. F. & Lowe, B. (1948). Factor influencing the effects of heat transment on the leavening power of egg white. Poullry Sci., 27, 294-30 I.
(Received
I5 August
1995; accepted
6 November
1995)
PII:
ELSEVIER
pp. 155-161, 1996
Copyright 0 1996 Published by Elsevier Science Ltd on behalf of the Canadian Institute of Food Science and Technology SO963-9969(96)00008-7 Printed in Great Britain 0963-9969/96 $15.00 + .OO
Effect of pH during the dry heating on the gelling properties of egg white proteins
Yoshinori Mine Department
of Food Science, University of Guelph, Guelph, Ontario, Canada NIG 2 WI
Gel strength and elasticity of dried egg white proteins greatly increased by heating in the dry state at alkaline pH region (under 9.5) for a short period of time (3-5 days) without losing water solubility. Circular dichroism and differential scanning calorimetry spectrum revealed that the increased degree of denaturation found during preheating was accelerated in alkaline pH region. The polymerization of the proteins was also enhanced by alkaline dry heating through sulfhydryl-disulfide interchange. Alkaline dry heating resulted in high molecular weight polymer of partially unfolded egg white proteins which, in turn, contribute in the formation of low molecular weight and narrow molecular distribution of the aggregate found during the subsequent heat for gelation. Thus, heating of dried egg white proteins in the dry state at alkaline pH (under 9.5) is an effective method to obtain firm and elastic gels. The degree of unfolding of the proteins upon dry heating may play a crucial role in the gelling process of the proteins. Copyright 0 1996 Published by Elsevier Science Ltd on behalf of the Canadian Institute of Food Science and Technology Keywords: egg white, gelation, dry heating, pH, aggregation.
mechanism and the structure-function relationship of food proteins. Kato et al. (1989, 1990a,b) reported that mild conformational changes and polymerization due to disulfide formation and/or sulfhydrykiisulfide (SH-SS) interchange in DEW proteins were attributable to the dry heating. The majority of sulfbydryl residues in egg white protein exist in the interior of protein molecules and are exposed with heat denaturation (Mine et al., 1990). Heat-induced changes of sulfhydryl levels in egg white proteins in solution are well characterized and SH-SS interchange is affected by pH (Beveridge & Arntfield, 1979). However, the process of protein interactions during dry heating and gelation processing of DEW proteins are not still well understood. The molecular unfolding of DEW proteins upon dry heating can be affected by pH, temperature and moisture content during dry heating. There are no available data concerning these effects of heating in dry state, despite the importance to industrial applications. Heat-induced gelation is a two-step process involving protein denaturation and
INTRODUCTION Dried egg white is generally heat treated at Y-65°C to reduce microbial number. The effect of high temperature storage of spray-dried egg white (DEW) on physical changes and thermal resistance of Salmonella organisms is well characterized (Slosberg et al., 1948; Cotterill et al., 1967; Baldwin ef al., 1967; McBee & Cotterill, 1971). These researchers studied the effect of low heating temperature (below 70°C) on DEW proteins. However, until the recent studies of Kato et al. (1989), there were no reports of the effects of heating egg white proteins in a dry state on their functional properties. These studies reported a significant improvement of functional properties, including foaming, emulsifying and gelling properties of DEW, on the protein heating in a dry state at 80°C for several days (7.5% moisture content). This method can be one of the most promising approaches for use in food processing. The mechanism of protein denaturation during dry heating is important in order to elucidate the gelation 155
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aggregation to form a cross-linked matrix. The mechanism of gelation of egg white proteins is considered to be similar to that of other globular protein. Monitoring the changes in protein structure of egg white protein upon dry heating and heat-induced gelation will help to further the understanding of the mechanisms involving gelation and texture formation by egg white proteins. The relationship between gel strength and the extent of aggregation of egg white proteins upon heat-induced gelation was studied using the low angle laser light scattering technique (Kato et al., 1990~). The average molecular weight of heat-induced DEW aggregates decreased greatly with increase of preheating time in the dry state and the gel strength greatly increased. A good correlation was observed between decrease in molecular weight and increase in gel strength of dry-heated DEW. However, Kato et al. only studied the heat-induced aggregation of dry-heated DEW proteins in solution. There is little information on the aggregation behavior of DEW proteins during dry heating. The purpose of this study was to clarify the effect of pH during dry heating on the functional and conformational changes of DEW proteins. In addition, the relationship between the aggregation behavior upon heat-induced and gelling properties of egg white proteins was examined.
MATERIALS
AND METHODS
Following treatment by a glucose oxidase catalase enzyme system, the egg white liquid was adjusted to pH 6.0, 6.5 and 7.3 with 1 N HCl or 1 N NaOH, respectively, before spray drying. Spray drying was done using a pilot plant dryer with an exhaust temperature of 65-70°C. The DEW with three pH levels before spray drying was stored at 75°C for various periods of time (days) in dry state (8.5% moisture content). Periodically, samples of DEW were taken out from the incubator and cooled at room temperature. These samples were used for testing the structural and functional properties after passing the sample solution through a filter paper to remove any insoluble materials. Preparation of DEW protein gels were carried out as follows: 10% protein solution of each sample in 20 mM phosphate buffer (pH 7.5) was put into 30 mm diameter polyvinylidene chloride tubes. The tubes were then sealed and immersed in a water bath at a temperature of 80°C for 40 min. After a given time, the tubes were taken from the hot bath and cooled to room temperature in an ice bath. The samples were kept at 4°C overnight before being subjected to testing. The quality of the gels was assessed by measuring their gel strength (g/cm) and deformation (cm). Every sample piece was prepared to have 30 mm height by cutting the original cylindrical shaped gel. Each gel piece was set on the detachable table of a Rheometer@
(Fudo Kogyo K. K., Tokyo) equipped with a spherical plunger (4 = 8 mm). Compression on the sample piece was executed with a table speed 60 mm/min. Jelly strength was obtained by deformation (g x cm). The measurement was repeated eight times on each sample gel and the averages were calculated. Measurement of sulfhydryl groups was performed by using Ellman’s reagent (Beveridge et al., 1974). A total of 4 ml of 0.1 M tris-glycine buffer, pH 8.0, containing 10 M urea and 0.01 M EDTA was added to 1 ml of the 1% protein solution. After incubation at 40°C for 30 min, 125 ~1 of 5,5’-dithiobis(2nitrobenzoic acid) solution (20 mg in 5 ml of 0.1 M tris-glycine buffer, pH 8.0) was added and then incubated at 25°C for 10 min. The color absorbance was read at 412 nm on a Hitachi U-2000 spectrophotometer. The total SH residues were calculated as follows: PM SH/g = 73.53 . A4,2 . D/C where A4i2 = the absorbance at 412 nm; C = the sample concentration in mg/ml; D = the dilution factor, 5.125 (Beveridge et al., 1974). Measurement of free amino groups was carried out as follows: 200 ~1 of DEW solution (0.2%) was added to 2.0 ml of 0.2 N sodium borate buffer, pH 9.2, and then 1.0 ml of TNBS reagent (equal volume of 0.1% trinitrobenzensulfonic acid and 0.126% sodium sulfide) was added. This mixture solution was allowed to stand at 40°C for 2 h covered with aluminum and subsequently cooled to room temperature before the absorbance was measured at 420 nm. The values of residual free amino groups of heat-treated samples were represented as the ratio to one of the nonheated DEW sample. The thermal characteristics of DEW proteins heated in the dry state for various pHs were examined using a differential scanning calorimetric (DSC) thermal analyzer (Seiko DSC-100, Tokyo, Japan) equipped with a DSC cell. A total of 50 ~1 of 8.0% protein solution in 20 mM phosphate buffer, pH 7.0, was sealed in a preweighted hermetic silver pan. Water was used as the reference. The pans were heated in the calorimeter at a linear rate Z”C/min over the range 30-120°C. The denaturation temperature (Td) and enthalpy of denaturation (AH) were computed from the thermograms by the SSC-5000 analyzer (Seiko Electric Industry Co., Tokyo, Japan). Circular dichroism (CD) analysis was carried out to assess the conformational changes of DEW heated in the dry state. DEW proteins were dissolved in 10 mM phosphate buffer, pH 7.2, and then filtered with a Millipore filter (pore size 0.22 pm) Each protein concentration was adjusted to 7.4 x 10m3 (%). CD spectra were measured on a JASCO J-720 spectropolarimeter using a 10 mm cell at 25°C in the far ultraviolet region (20&250 nm). The data was represented in terms of mean residue ellipticity (deg.cm*.dmol-‘).
EfSect of pH on egg white proteins
Heat-induced DEW protein aggregates were processed as follows: diluted DEW protein solution (0.1% protein in 10 mM phosphate buffer, pH 7.5) were passed through a membrane filter (0.5 pm, Shodex MX-25K) with subsequent adjustment of the protein concentration spectrophotometrically. A total of 3 ml of DEW solution was put into a test tube with a screw cap and the tube was immersed in a water bath at 80°C for 20 min and then cooled to room temperature immediately. Next, 200 ~1 of the sample was applied to a multiangle laser light scattering (MALLS) experiment. The MALLS experiments were conducted at room temperature on a DAWN DSP-F laser photometer (Wyatt technology, Santa Barbara, CA) using a 632.8 nm laser wavelength. The MALLS system was equipped with a high performance liquid chromatography system, consisting of Shodex KW-804 and KW-803 columns (Showa Denko K. K., 0.75 x 30 cm). A precision differential refractometer (Shodex RI) was used as a detector. As an elution buffer, 50 mM phosphate buffer, containing 0.15 M NaCl (pH 7.0) was used and flow rate was 1 ml/min. Scattering angle was 30-150” for each sample at room temperature. A specific refraction index increment (dn/dc) of 0.186 was obtained for a dialyzed DEW solution using a Wyatt Optilab differential refractometer, Model 903. Second virile coefficient of 1.94 x 10e4 was calculated from Zimm plot.
157
Ob Heating time (days) Fig. 1. Relationship between gel strength and heating time in the dry state of spray-dried egg white proteins at various pHs. (W) dry heated at pH 6.88 (control), (A) dry heated at pH 8.55 and (0) dry heated at pH 9.40.
RESULTS The pHs of DEW solution in water (10% protein), which were adjusted to pH 6.0, 6.5 and 7.3 before spray drying, were pH 6.88, 8.55 and 9.40, respectively. In general, liquid egg white is subjected to spray drying under pH 5.8-6.3 and the pH of DEW powder is 7.0-7.5. The increase in pH in DEW powder is due to the evaporation of soluble carbon dioxide in liquid egg white by spray drying. Thus, the DEW powders with different pHs were named as follows: DEW (pH 6.88-control), DEW (pH 8.55) and DEW (pH 9.40). The effect of pH during dry heating of DEW on the gelling properties was investigated (Figs 1 and 2). Figure 1 shows the relationship between heating time in the dry state at various pHs and the gel strength of DEW. The gel strength of DEW protein was greatly increased with increased heating time and resulted in an excellent firm gel structure. Interestingly, the final gel strength of all DEW samples after dry heating for 15 days showed almost the same values (900-950 g/cm). The increasing rate of gel strength in relation to heating time was larger in DEW (pH 9.40) than in DEW (pH 6.88-control). The relative strength of DEW (pH 9.40) indicated that it was about four times the value of nonheated DEW after 3 days. On the other hand, it took 7 and IO days to obtain the same gel strength in DEW (pH 8.55) and
0
5
10
15
Heating time (days) Fig. 2. Relationship between jelly strength and heating time in the dry state of spray-dried egg white proteins at various pHs. (m) dry heated at pH 6.88 (control), (A) dry heated at pH 8.55 and (0) dry heated at pH 9.40.
DEW (pH 6.88-control), respectively. The effect of heating in the dry state on the solubility of DEW proteins was very small (data are not shown). The relationship between heating time in the dry state at various pHs and the jelly strength of DEW protein gels are shown in Fig. 2. Heating in the dry state at pH 9.40 progressively increased the jelly strength within a short period of time (5 days) and resulted in excellent elastic gels of DEW proteins. The transparency of DEW (pH 9.40) gel was higher than those of DEW (pH 6.88-control) or DEW (pH 8.55) gels (data are not shown). Figures 3 and 4 show changes in total SH residues and free amino groups on the heat-treated DEW in the
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158
F *OM Heating time (days) Fig. 3. Changes of total sulfhydryl residues in spray-dried egg white proteins during the dry heating. (m) dry heated at pH 6.88 (control), (A) dry heated at pH 8.55 and (0) dry heated at pH 9.40.
210
230
250
Wavelength (nm) Fig. 5. Circular dichroism spectrum of nonheated and heated spray-dried egg white proteins in the dry state at various pHs. Key: ___ nonheated; . . . heated at pH 6.88 (control); - - heated at pH 8.55; - . - heated at pH 9.40 for 3 days at 75°C.
0
5
10
15
Heating time (days) Fig. 4. Changes in free amino groups of spray-dried egg white proteins during the dry heating. (m) dry heated at pH 6.88 (control), (A) dry heated at pH 8.55 and (0) dry heated at pH 9.40.
dry state for various periods at various pHs. The total SH residues of DEW proteins were gradually decreased with increasing heating time and pHs during heating in the dry state. This result suggests that SH-SS interchange reactions were accelerated by heating in the dry state. The SH-SS interchange reaction occurred more rapidly in the alkaline pHs than in neutral pHs. Little changes in the free amino group were observed at DEW (pH 6.88-control) and DEW (pH 8.55) upon heating, except at 15 days of heating time. On the other hand, the residual free amino group of DEW (pH 9.40) was decreased with increasing heating time. In total, 15% of total amino groups of the protein decreased by heating in the dry state for 5 days. Any browning reaction of each sample was not observed with heating time up to 10 days. However, a slight browning of the protein was
observed upon heating for 15 days. No differences in the electrophoretic patterns of DEW proteins with or without SDS were observed between native and dry-heated DEW proteins. No peptide bond hydrolysis of the proteins was detected. On the other hand, a dramatic change in color of DEW during dry heating was observed when the protein was heated at over pH 10.0 in the dry state. Solubility of this DEW (pH 10.0) was greatly decreased because of the aggregation of the proteins (data are not shown). The effect of heating in the dry state on the structure of DEW proteins at various pHs was studied by CD analysis. Figure 5 shows a typical CD spectra of nonheated and heat-treated samples for 3 days at 75°C in the dry state. No changes in CD spectrum were observed among the nonheated samples of each pH. As shown in Fig. 5, the change in CD spectrum of DEW (pH 6.88-control) protein was very mild. However, the heat treatment in the dry state of DEW at pH 8.55 or 9.40 affected their CD spectra, which was reflected as a decrease of a-helix content. The structural changes of DEW were further investigated by differential scanning calorjmetry (Fig. 6 and Table 1). The DEW proteins exhibited two major endothermic peaks which was tentatively identified as ovotransferrin (Td, 60.7”C) and ovalbumin (Td, 8O.l”Q respectively, with a total enthalpy of 14.0 J/g (Donovan et al., 1975). Changes in DSC peaks among the non-
Efect
ofpH
on egg white proteins
159
Table 1. Thermal characteristics and gel strength of spray-dried egg white (DEW) proteins under dry heating for 3 days at different pHs
a
Td (“C)
PH
(% b
Peak I
Peak 2
60.7 60.0 NDb ND
80.1 79.2 79.3 79.7
Controls pH 6.88 pH 8.55 pH 9.40
14.0 12.6 10.6 6.0 ____
Gel strength (g/cm)
123 297 481 728
“Nonheated DEW. ‘Peak not detectable.
Table 2. Laser light scattering characteristics of heat-induced spray-dried egg white aggregates -- ._
Samples”
5b
6b Heating
I
I
I
70
80
90
temperature
id0
(“C)
Fig. 6. Differential scanning calorimetric thermograms of spray-dried egg white proteins. Samples were heated in the dry state for 3 days at 75°C. Key: (a) nonheated; (b) heated at
pH 6.88 (control); (c) heated at pH 8.55; (d) heated at pH 9.40.
heated proteins
of each pH were not detected. When the
samples were heated in the dry state for 3 days at 75”C, there was a broadening of the peaks with a marked decrease in AH. Alkaline dry heating resulted in a further decrease of AH and broadening in the endothermic peaks, and their extent increased with increased pH of DEW proteins. However, no detectable decrease in Td value was observed upon heating in the dry state at various pHs. These data indicate the existence of intermediate forms different from the native form. Simply, the conformation of DEW protein molecule has shifted toward the partially unfolded state by dry heating. A good correlation was observed between the decrease in AH and the increase in gel strength of DEW proteins (Table 1). The molecular size and radius minimal square moments (RMS) of solution before heating and heatinduced soluble aggregates (progel) formed prior to gelation of DEW proteins were investigated by the MALLS system. The data are summarized in Table 2. The weight average molecular weight moments (Mw, g/ mol) and number average molecular weight moments (Mn, g/mol) of nonheated DEW proteins in the dry state were 54.8 kDa and 45.0 kDa, respectively. This value is close to the known value of ovalbumin, a major constituent of DEW proteins. This may indicate the general success of this measurement. The Mw and Mn of 0.1% DEW protein solution which was heated in dry state for 3 days increased with increasing pH during dry
Mw/Mn
RMS(W)~
(x!%)b
(x%4)
5.48
4.50
1.21
16.5
15.93 21.54 37.53
5.51 6.04 7.89
2.89 3.57 4.76
23.5 23.6 27.7
311.60
9.04
34.49
27.9
48.70 54.76 64.35
7.65 8.61 11.11
6.37 6.36 5.79
23.7 23.3 25.0
-___ Control Nonheated Dry heating at: pH 6.88 pH 8.55 pH 9.40 Heatede Nonheated Dry heating at: pH 6.88 pH 8.55 pH 9.40
“Samples were heated in dry state for 3 days at 75°C. bWeight-ave rag e molecular weight moments (g/mol). ‘Number-average molecular weight moments (g/mol). dRadius minimal square moments (nm). “0.1% protein in 50 mM phosphate buffer (pH 7.0) was heated at 80°C for 20 min.
heating. The relative Mw and Mn of DEW (pH 9.40) proteins showed about 7.0 and 1.7 times the value of nonheated DEW proteins after 3 days of dry heating. The Mw/Mn ratio of DEW (pH 9.40) increased about 4 times compared with the control. The RMS of nonheated DEW proteins was 16.5 nm and this value increased with dry heating and with increasing pH during dry heating based on the aggregation. These data indicate that DEW proteins were polymerized by dry heating. The polymerization of the proteins was accelerated by heating in the dry state in the higher pH region. The polydispersity of DEW protein aggregates was increased with increasing pH during the dry state. Interestingly, the heat-induced aggregation behavior of DEW proteins in solution between nonheated DEW and dry-heated DEW was quite different. The Mw of nonheated DEW was estimated to be 3116 kDa and the ratio of Mw/Mn was 34.5. The data indicate that the
160
Y. Mine
molecular weight distribution of the heat-induced aggregate of nonpreheated DEW was large and wide. On the contrary, the preheated DEW in the dry state showed small (400-650 kDa) and narrow molecular weight distribution of the heat-induced aggregates. No substantial differences of the RMS of heat-induced aggregates between nonheated DEW and dry heating DEW proteins was shown. These data show that dry heating led not only to a decrease in the molecular weight of DEW aggregates but also to the formation of an expanded structure of aggregates. This spread of aggregates resulted from the high molecular weight polymer of partially unfolded egg white proteins, increasing with increasing pH during dry heating.
DISCUSSION
A protein gel network is generally formed via noncovalent cross-linkages such as hydrophobic interactions, hydrogen bonds, or electrostatic interactions and less frequently by covalent interactions such as disulfide bonds (Clark, 1992). Dry heating of egg white powder is an effective method to reduce the microbial number, such as Salmonellae (McBee & Cotterill, 1971). This method has also been used on an empirical basis in Canada to improve the gel-forming properties of DEW since 1982 (Dr E. D. Murray, personal communication). It was reported that heating for 10 days in the dry state (8o”C, 7.5% moisture content) is proposed to gain the maximum functional properties of DEW (Kato et al., 1989). However, Kato et af. did not measure the pH of DEW upon dry heating. The present work revealed that the controlled heating at alkaline pHs (under 9.50) in the dry state is an effective method to improve the gelling properties of DEW proteins without losing solubility and browning reaction for a short period of time (3-5 days). These phenomena are important in elucidating the structure-function relationship of food proteins on a molecular basis. Mild conformational changes are caused in DEW proteins by the dry heating and these structures may play a critical role in the formation of strong and stable gels of dry-heated DEW. In addition protein-protein interactions due to disulfide formation and/or SH-SS interchange also occurred in DEW proteins upon dry heating. CD spectrum obtained in the present work suggested that the degree of conformational change of DEW proteins upon dry heating increased in the alkaline pH region without losing solubility. Molecular weight of the polymer of DEW (pH 9.40) was higher than that at neutral pHs and was due to disulfide formation and/or SH-SS interchange. An increase in pH in DEW during heating in the dry state resulted in an additional decrease of AH and more broadening in the endothermic peaks. It has been well documented that broadening of peaks indicates the existence of intermediate forms different from the
native form (Myers, 1990). Thus, partially unfolded conformations of DEW which occur upon dry heating may play an important role in the gelation process. It is particularly interesting that solubility of DEW proteins was not greatly affected even though the proteins were denatured and formed a polymer with molecular weight of 200-375 kDa upon dry heating. Protein solubility is often affected by temperature, pH and the presence of other solutes and salts. Solubility is related to the net free energy change from interaction of hydrophobic and hydrophillic residues on the protein surface with the surrounding solvent. No changes on the protein surface and no peptide bond hydrolysis of DEW proteins upon alkaline dry heating were detected by electrophoretic analysis. On the other hand, free amino groups of alkaline dry-heated proteins decreased with increasing heating time. It can be assumed that deamidation or isomerization of amino acid residues of DEW proteins also occurred during this process. However, it is difficult to conclude the exact chemical changes of the proteins from the present data. Further studies are required to determine the role of amino groups which might affect the protein solubility and gelling properties of DEW proteins upon alkaline dry heating. It was reported that the main factor contributing to the heat-induced aggregation of ovalbumin was the degree of electrostatic repulsion on the denatured protein molecules (Nakamura et al., 1978). Hatta et al. (1986) reported the importance of pH and ionic strength on the turbidity and hardness of heat-induced ovalbumin gels. It is suggested that the gel strength of DEW proteins could be determined by factors determining the state of aggregation. Alkaline dry heating egg white proteins results in the formation of low molecule weight soluble aggregates during the subsequent heat for gelation. In addition, the homogeneity of the molecular size in the soluble aggregate may be important in the formation of a firm gel matrix. The Mw/Mn ratios decreased with increasing pHs upon heating, due to initial aggregation caused by dry heating. These results basically support the concept obtained by Kato et al. (1990~). In addition, the present data suggest that the increased degree of denaturation of DEW proteins during dry heating and the homogeneity of the molecular size in soluble aggregates (under 1000 kDa) during the subsequent heat for gelation may be critical factors for the formation of an excellent gel network. The RMS data also predicted the existence of expanded structures or the formation of linear polymers (Mw < 1000 kDa) by preheated DEW proteins. In conclusion, it is proposed that dry heating of egg white proteins at alkaline pH (under 9.5) is an effective method to improve the gelling properties in a short period of time within 5 days without any loss of solubility and browning reaction. The formation of partially denatured proteins which have high solubility may be suitable form for improving the protein functionality.
E&t
of pH on egg white proteins
ACKNOWLEDGEMENT The
author
Producer’s
gratefully Marketing
161
Kato,
acknowledges Board
for
the financial
Ontario support
Egg of
this work.
REFERENCES Baldwin, R. E., Cotterill, 0. J., Thompson, M. M. & Myers, M. (1967). High temperature storage of spray dried egg white I. Whipping time and quality of angel cake. Poultr? Sci., 46, 1421-30. Beveridge, T. & Arnttield, S. (1979). Heat induced changes in sulphydryl levels in egg white. Can. Insf. Food Sci. Technol. J., 12, 173-6. Beveridge, T., Toma, S. J. & Nakai, S. (1974). Determination of SH- and SS-groups in some food proteins using Ellman’s reagent. J. Food Sci., 39, 49951. Clark. A. H. (1992). Gels and gelling. In Physical Chemistry q/ Food.r, ed. H. G. Schwartzberg & R. W. Hartel. Marcel Dekker, New York, pp. 2633305. Cotterill, 0. J., Baldwin, R. E. & Myers, M. (1967). High temperature storage of spray dried egg white 2. Electrophoretic mobility, conalbumin iron complexing, sulfhydryl activity, and evolution of volatile bases. Poultry Sci., 46, 1431-7. Donovan, J. W., Mapes, C. J., Davis, J. G. & Garibaldi, J. A. (1975). A differential scanning calorimetric study of the stability of egg white to heat denaturation. J. Sci. Food Agric., 26, 73-83. Hatta, H., Kitabatake, N. & Doi, E. (1986). Turbidity and hardness of a heat induced gel of hen egg ovalbumin. Agric. Biol. Chem., 50, 2083-9.
A., Ibrahim, H. R., Watanabe, H., Honma, K. & Kobayashi, K. (1989). New approach to improve the gelling and surface functional properties of dried egg white by heating in dry state. J. Agric. Food Chem., 37, 433-7. Kato, A., Ibrahim, H. R., Watanabe, H., Honma, K. & Kobayashi, K. (1990~). Structural and gelling properties of dry-heating egg white proteins. J. Agric. Food Chem., 38, 32-7. Kato, A., Ibrahim, H. R., Watanabe, H., Honma, K. & Kobayashi, K. (1990h). Enthalpy of denaturation and surface functional properties of heated egg white proteins in the dry state. J. Food Sci., 55, 128&3. Kato, A., Ibrahim, H. R., Takagi, T. & Kobayashi, K. (1990~). Excellent gelation of egg white preheated in the dry state is due to the decreasing degree of aggregation. J. Agric,. Food Chem., 38, 1868-72. McBee, L. E. & Cotterill, 0. J. (1971). High temperature storage of spray dried egg white 3. Thermal resistance of .salmonrllu organienburg. Poultr), Sci., 50, 452-8. Mine, Y., Noutomi, T. & Haga, N. (1990). Thermally induced changes in egg white proteins. J. Agric. Food Chem., 38, 212225. Myers, C. D. (I 990). Study of thermodynamics and kinetics of protein stability by thermal analysis. In Thermal Analysis qj” Foods, ed. V. R. Harwalkar & C.-Y. Ma. Elsevier Applied Science, New York, pp. 1650. Nakamura, R., Sugiyama, H. & Sato, Y. (1978). Factors contributing to the heat-induced aggregation of ovalbumin. Agric. Biol. Chem., 42, 819-24. Slosberg, H. M., Hanson, H. L., Stewart, G. F. & Lowe, B. (1948). Factor influencing the effects of heat transment on the leavening power of egg white. Poullry Sci., 27, 294-30 I.
(Received
I5 August
1995; accepted
6 November
1995)