Poultry Science 1976. Vol. 55. pp. 738-743
Contribution No. 898, Kansas Agricultural Experiment Station, Kansas State University, Manhattan, Kansas 66506.
(Received for publication July 16. 1975)
This study was designed to provide a better understanding of egg white foams and the proteins involved by characterizing the drainage from foam. Samples of liquid egg white were whipped at high speed to a medium peak. After the foam had drained for an hour, drainages were collected and whipped again. That procedure was repeated until a 5th drainage had been collected. Portions of liquid drained from the 1st, 2nd, 3rd, 4th. and 5th whip sere saved and later were analyzed.
The foam became less stable with each successive whip. Drainage pH increased from. 9.0 to 9.3. Both angel cake volume and texture decreased with each drainage. Cakes prepared from the 4th or 5th drainage had very poor volumes and texture.
Viscosity of each drip was lower than that of the previous sample. Lysozyme activity decreased linearly, indicating that approximately the same amount of lysozyme remained in each foam.
The data showed that most major protein components in egg white are altered by repeated whipping. The ovalbumins were not greatly changed. but portions of ovomucin, conalbumin, lysozyme and globulins A1, and A2. were retained in the foam of beaten egg white
Egg white was subjected to a series of physical treatments by Forsythe and Bergquist (1951) to determine their effect on ovomucin structure, apparent ovomucin content, and functional performance. It has long been assumed that ovomucin plays an important role in egg white foam, but the data of Forsythe and Bergquist (1951) indicated that slightly less than half of the ovomucin was retained in egg white foam. Egg white drained from foams could perform satisfactorily provided the initial foam was not too stable. They concluded that the presence of ovomucin, either in native or modified form, was not sufficient to insure satisfactory formation of egg white foam.
Bergquist and Stewart (1952) founds that beating rate of foam drainage was not greatly affected unless the foams had been whipped to high specific volume (low density).
Nakamura and Sato (1964) reported that foam stability of each foam drain was less than that of the original egg white, as was the amount of coagulated proteins formed from the drain. They reported that most ovomucin was coagulated at the first whipping and that very little ovomucin returned to the foam-drainage. Their electrophoretic patterns of foam drainage indicated fewer globulins and less ovomucoid than in the original egg white, so they concluded that ovomucin played a major role in foam stability but had no part in foam formation. The other proteins acted as foam producers instead of foam stabilizers.
MacDonnell et al. (1955) investigated the role of individual egg white proteins in foam formation and in angel cake preparation. They also examined the liquid that drained from egg white foam. They found that viscosity and ovomucin nitrogen decreased most in the first whip, but that each additional whip caused a definite decrease in stability and increase in whip time. They further concluded that ovalbumin was not selectively removed from solution during the foaming process.
Because of its unique properties and the ability of its protein system to form a stable foam rapidly, egg white has found many uses in the food industry. A clearer understanding of the chemical and physical properties of the foam might lead to new applications. This study, an elaboration upon the work of MacDonnell et al. (1955). was designed to examine the characteristics of the drainage from egg white foams.
Egg white used in these experiments was Prepared as described by Cunningham et al. t 1960) and Cotterill et al. (1963).
Procedures used in determining pH, viscosity, foam drainage, absorbance and angel cake volumes were described in previous publications (Cunningham and Cotterill. 1963; Cotterill and Funk, 1963). Solids and total protein were determined by A.O.A.C. (1970) methods. Samples of blended egg white (100 ml.) were whipped in a Hobart K-4-B mixer at high speed to a medium peak. After the foam had drained for 60 min., drainage was collected and re-whipped. The procedure was repeated until the 5th drainage was collected.
Lysozyme Determination. Lysozyme activity- in foam drainage was determined by the method of Parry et al. (1965), as modified by Galyean et al. (1972).
Electrophoresis Procedure. Egg white proteins were separated according to the method of Davies (1964). Polyacrylamide gels were made with tris glycine buffer of pH 9.5. The tubes were prepared according to the instructions of the manufacturer with slight modifications. Stacking gel (0.5 ml. of large pore gel) was layered over 0.75 ml. of separating gel (small pore gel) of 7% acrylamide. Bromphenol blue was used as the tracking dye in the upper buffer. Samples of 0.1 ml. containing 200 ìg. were applied on top of the stacking gel. A current of 1 ma./tube was applied initially for 30 min., then raised to 3 ma./tube and continued for an additional 45 min. At the end of the run the gels were removed carefully from the tubes and the protein bands were fixed in 20% trichloracetic acid for 30 min.
Fixed gels were rinsed with distilled water and then stained for two hours in coomassie blue (0.25%). Excess dye was removed by placing the gels in 7% acetic acid solution 12 hours, and storing the gels in 1% acetic acid solution.
Chromatography Procedure. Egg white proteins were separated by column chromatography using the procedures of Mandeles (1960). The DEAE-cellulose was purchased from Schleicher and Schuell Co.. Keene, N.H. Column size was 4 x 34 cm. Fraction size was 10 ml., flow rate was 2 ml./min. and absorbance was measured at 280 nm.
Conalbumin Determination. Conalbumin concentration was determined by chromogenic iron-binding capacity measured at 470 nm. as outlined by Fraenkel-Conrat and Feeney (1950). Warner and Weber (1951) and Azari and Feeney (1958. 1961).
Ovomucin Analysis. The ovomucin content of experimental samples was determined by the method of Balls and Hoover (1940), modified by Forsythe and Bergquist (1951).
Fig. 1. Whip time and volume of drainage from egg white after repeated whipping.
Table 1. Selected characteristics of drainage from egg white foams after repeated whippings
|Drainage||PH||Solids (%)||mg. N/ml..||Volume (cc.)||Texture*|
* 5=normal texture. 1=extremely poor texture.
Whip Time and Drainage Volume. All egg white samples were whipped at 25°C. Successive drainages required longer beating times (Figure 1) to reach the medium peak stage. Also, the foam was less stable with each successive whip as indicated by volume of foam drainage. At least 100 ml. of drainage from each whip was collected and frozen for analysis later. Baldwin (1973) stated that the stage or extent of beating is the major factor; influencing the characteristics of egg foam. That is why in this study each foam was whipped to a medium peak, just as one would. in preparing an angel cake
Characteristics of Foam Drainage. Selected characteristics of drainage from egg white foams after repeated whipping are shown in Table 1. The pH of the drainage increased from 9.0 before whipping to 9.3 after the 5th whip. Solids content and mg. N/ml. varied considerably with no significant difference between whips. Both angel cake and texture decreased with each successive whip. Drainage from the 4th and 5th whip produced cakes with very poor volume and texture
Fig. 2. Relative viscosity of drainage from egg white after repeated whipping.
Viscosity. The viscosity of each drip was lower than that of the previous egg white sample (Fig. 2). Relative viscosity dropped from about 3.0 for the control to about 1.5 after the 5th whip.
Fig. 3. Loss of lysozyme activity in drainage from egg white foam after repeated whipping.
Lysozyme Activity. Lysozyme activity in original. unwhipped egg white (100%) was compared with activity in drainages from repeated whips. Loss of lysozyme activity in successive foam drainages is shown in Figure 3. The decrease of enzymatic activity was nearly linear, indicating that approximately the same amount of lysozyme was left behind in each foam. Only about 30% of the original activity remained after the 5th whip.
Fig. 4. Electrophoretic separation of proteins in control egg white (C) and in drainage from the 5th whip (D).
Conalbumin Content. The amount of conalbumin remaining in drip from successive foams is shown in Table 7. As indicated by iron-binding capacity, conalbumin drains from the foam to a greater extent than does lysozyme. The amount of conalbumin was nearly unaltered through three whippings.
Table 2.-Loss of conalbumin in drainage of egg white foams as indicated by chromogenic iron-binding
|Number of whips||Conalbumin loss (%)*|
* Estimated by iron-binding capacity measured at 470 nm.
Table 3.-Quantity of precipitate centrifuged from drainage of egg white foams. Drainages adjusted to pH 5.5
|Number of whips||Precipitate (cc.)*|
* Average of 3 measurements.
Ovomucin Content. An indication of the amount of ovomucin retained in egg white foam is shown in Table 3. Precipitate was only 0.9 cc. after the first whip, which agrees with the report of MacDonnell et al. (1955) that ovomucin decreases most in the first drip. However, even after the 5th whip, a small amount of ovomucin could still be separated from the drainage.
Electrophoretic Analysis. Changes in the electrophoretic pattern of control egg white (C) and drainage from the 5th whip (D) are shown in Figure 4. Crystalline ovalbumin and conalbumin were used as reference standards. Using the regions and numerical denotations of egg white components as assigned by Lush (1961). it can be seen that A1 component 14 in region III, was absent in the drainage. Conalbumin appeared to be only slightly, diminished which would agree with my iron-binding tests. Also, globulin A2, component 12 in region III. was diminished in the foam drainage. Regions II, which includes the ovomucoid and unidentified proteins, and region I, which includes the ovalbumins, did not appear to be altered
Fig. 5. Chromatographic separation of proteins in (A) normal egg white and (B) drainage from the fourth whip. Column of DEAE-cellulose: 4 cm. diameter, 34 cm. length. Fraction size: 10 ml. Flow rate: 2 ml./min. Absorbance measured at 280 nm.
Chromatographic Analysis. The difference in column chromatographic patterns between control egg white (A) and drainage front the 4th whip (B) is shown in Figure 5. The lysozymes (fractions 10-55) in the drainage were reduced considerably by repeated whipping which confirmed the enzymatic test (Fig. 3). Considerable lysozyme was retained in egg white foam. Conalbumin (fractions 75-120) was only slightly altered, which generally agrees with the chromogenic data in Table 2.
Globulins A1, and A2 (fractions 125 and 150) were absent in foam drainage. which confirmed the electrophoretic analysis (Fig. 4). The ovalbumins, as well as the remaining components including flavo-protein (fraction 10) were somewhat altered chromatographically
These data show that most major protein components in egg white are changed by repeated whipping. The ovalbumins are not greatly altered but indications are that ovomucin, lysozyme, globulins A1 and A2 and, to a lesser extent, conalbumin are retained in the foam of beaten egg white.
These data generally agree with those of Forsythe and Bergquist (1951) in that ovomucin was denatured in the foam interface to a lesser degree than is assumed by most workers. Half of the ovomucin returned in the drainage after the 3rd whip and, even after 5 whips, about a third of the ovomucin returned to the drainage. Admittedly, chose data could be altered by over-beating, in which case more ovomucin would be retained in the foam.
For normal egg white foams (such as used to prepare angel cakes) many other protein components are involved in the satisfactory formation of a voluminous, stable foam.
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