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The Influence of Yolk on Egg White Lysozyme1,2,3
MECHANICAL GATHERING AND SHELL DAMAGE
3. Spoolated lifts as designed and used in the system studied, damaged eggs significantly. REFERENCES Forsythe, R. H., 1966. Modern marketing demands
1013
sound shells. Paper presented at Nebraska Egg Shell Quality Conference, March 23, 1966. Toleman, W. J., T. H. Coleman and L. E. Dawson 1964. Factors affecting the number of cracked eggs produced on Michigan farms. Research Report 17, Michigan State University Agr. Exp. Sta., East Lansing, Michigan.
The Influence of Yolk on Egg White Lysozyme1'2'3 F. E. CUNNINGHAM AND O. J. COTTERILL Dairy and Poultry Science Department, Kansas State University, Manhattan, Kansas 66502 and Food Science and Nutrition Department, University of Missouri-Columbia, Columbia, Missouri 6S201 (Received for publication October 10, 1970)
T
HE harmful effects of yolk and similar lipid material on the functional properties of egg white, long recognized, have been reviewed by several workers (Cotterill and Funk, 1963; Cunningham, 1963; Cunningham and Cotterill, 1964). The mechanism, however, whereby yolk is detrimental is not understood. Cotterill et al. (1963) showed that although the use of chemical additives inproved performance of yolk-contaminated egg white, additive actions were not solely associated with overcoming the adverse effects of the yolk. Cotterill and Funk (1963) found that lipase treatment improved yolk-contaminated white but did not determine if the improvement resulted from destruction of harmful yolk components or from products produced by hydrolysis (such as fatty acids). That pH is significant to functional performance, particularly for yolk-contaminated white, has 1 Contribution No. 801, Department of Dairy and Poultry Science, Kansas Agricultural Experiment Station, Manhattan, Kansas 66502. 2 Contribution from the Missouri Agricultural Experiment Station, Journal Series Number 6172. 3 Presented in part at the 59 th annual meeting of the Poultry Science Association, Knoxville, Tennessee, 1970.
been shown by Cotterill and Funk (1963) and Cunningham (1963). Cunningham and Cotterill (1964) found that centrifuging yolk-contaminated egg white improved its performance and that centrifuging at pH values around S.S caused maximum precipitation of yolk lipid and certain egg white proteins (presumably lysozyme-ovomucin). They suggested the possibility of complexing between yolk components and egg white ovomucin. Cotterill et al. (1965) found that the functional performance of yolk-contaminated egg white could be improved by heat treatment. They postulated that the mechanism for improvement in performance resulted from a heat-induced dissociation of a detrimental complex between some yolk component and certain egg white proteins. Lysozyme, a basic protein in egg white, is known to form electrostatic complexes with other substances. Its interaction with these substances has been demonstrated: thymus nucleate and yeast nucleate (Klotz and Walker, 1948), bovine plasma albumin (Steiner, 1953), ovomucin (Hawthorne, 1950; Cotterill and Winter, 1954), conalbumin (Ehrenpreis and Warner, 1956), ovalbumin (Forsythe and Foster, 1950; Nichol and Winzor, 1964; Cunningham
1014
F. E. CUNNINGHAM AND O. J. COTTERILL
and Lineweaver, 1967), and thyroxine or thyroglobulin (Litwack and Sears, 1965). This study concerns a reaction that occurs at room temperature (and over a wide range of pH) between lysozyme and egg yolk. MATERIALS AND METHODS
Egg white used in these experiments was obtained from fresh shell eggs, broken out in the laboratory, blended and frozen. Samples were thawed at room temperature as needed. Egg yolks, obtained from fresh shell eggs as needed, were hand separated from the white. While holding the yolk in a paper towel, the vitelline membrane was ruptured allowing the yolk to be collected in a beaker but retaining the membrane on the towel. Lysozyme was prepared from fresh, wellblended egg white by the isoelectric precipitation method of Alderton and Fevold (1946) and by separation from egg white utilizing ion-exchange chromatography with subsequent freeze drying of the fractions. Lysozyme activity was determined by the method of Parry et al. (1965). Samples of yolk-free and yolk-contaminated
egg white were analyzed by paper electrophoresis (Evans and Bandemer, 1956) and by column chromatography using the procedures of Mandeles (1960). RESULTS AND DISCUSSION
Samples of liquid egg white (pH 9.0) were intentionally contaminated with fresh, liquid yolk up to 1.0% (by weight) and well mixed in a Waring blendor. The presence of yolk apparently did not alter the electrophoretic behavior of the egg white proteins but did change the chromatographic profile. Subjecting 10-ml. portions of egg white to chromatography on DEAEcellulose produced the patterns shown in Figure 1. The continuous line shows the elution of yolk-free egg white, whereas the dashed line represents the sample contaminated with 0.1% yolk. The peaks in Figure 1 are identified as follows: A, B and C are lysozyme; peak D is conalbumin; peak E is ovomucoid; peaks G and H are ovalbumin; peak J is flavoprotein. Peaks F, I and K are not identified. The patterns obtained showed that the presence of yolk altered the behavior of several proteins, including lysozyme, ovomucoid and ovalbumin.
0.6-
0.4
0.2
SO
100
150 Fraction number
FIGURE 1. Chromatography of yolk-free (solid line) and yolk-contaminated (dotted line) egg albumin on DEAE-cellulose. Column size: 4 cm. diameter, 32 cm. length. Fraction size: 10 ml. Flow rate: 2 ml./min. Absorbance measured at 280 imi.
1015
LYSOZYME AND EGG WHITE
Peaks J and K were absent from the yolkcontaminated sample; also one of the lysozyme components was missing and peak A of lysozyme was greatly reduced. Lysozyme components (peaks A, B and C from yolk-free samples and peaks A and B from yolk-contaminated samples) collected from the ion-exchange column were freeze dried so that relative activities could be determined. The freeze dried fractions were dissolved in pH 6.2 phosphate buffer so that all solutions contained 0.3% protein and tested for lytic activity on Micrococcus lysodeikticus. Using Peak A from normal (yolk-free) egg white as a measuring standard (100%), it was found that peaks B and C collected from yolk-free egg white had higher specific activities than did peak A (Table 1). Peaks A and B collected from yolk-contaminated egg white had reduced activities (45 and 40%, respectively). The fate of the other lysozyme fraction in yolk-contaminated egg white is still unknown. In determining the concentration of yolk required to inactivate lysozyme in buffer at pH 6.2, the enzyme was crystallized from fresh, liquid egg white and redissolved in phosphate buffer. Various amounts (from 1 to 5%) of fresh, liquid yolk were added to solutions of 0.3% lysozyme and mixed well; 1 ml. of each mixture was transferred to 49 ml. of phosphate buffer to eliminate opaqueness. The relative activities are shown in Figure 2. In pH 6.2 buffer, the interaction between lysozyme and yolk at TABLE 1.—Relative activity of lysozymes* separated from egg white by chromatography Peak A
A B C
T, m i ' » Egg White
too 100 110 120
Yolk-Contaminated Egg White 55 60
' 0.3% lysozyme in pH 6.2 phosphate buffer.
100 80 >-
> 60 U < LU
40
>
N
O (/) 20 >-. O. 0
1
2 3 4 YOLK CONC.(%)
5
FIG. 2. Inactivation of egg white lysozyme (in pH 6.2 phosphate buffer) by various amounts of fresh liquid egg yolk.
the 5% level was sufficient to reduce lytic activity to less than 10%. Cunningham (1963) demonstrated that yolk, or some fraction thereof, was highly effective in reducing the foam capacity as well as amount of material insolubilized in the foam of egg white and suggested that the globulins were the egg white proteins most likely involved. MacDonnell et al. (1955) showed that the more rapid foamers in egg white were the globulins; therefore, it is possible that the harmful effects of yolk in liquid egg white are caused by the complexing between certain yolk lipids and lysozyme and other whipping proteins of egg white. Further work is needed to determine the yolk component(s) responsible for inactivating lysozyme and whether the reaction is reversible. SUMMARY
Proteins from yolk-free and yolk-contaminated egg white were separated by paper electrophoresis and ion-exchange chromatography. The presence of yolk did not
1016
F. E. CUNNINGHAM AND 0. J. COTTERILL
alter the electrophoretic patterns but did change the chromatographic profile. Three lysozyme components were apparent in yolk-free egg white; only two were found in yolk-contaminated samples. Lysozyme fractions were collected from chromatographic columns, freeze dried and tested for lytic activity. Peaks B and C obtained from yolk-free egg white had higher specific activities than peak A; peaks A and B from yolk-contaminated egg white had reduced activity (45 and 40%, respectively), using peak A from yolk-free egg white as a standard. Solutions of 0.3% lysozyme in pH 6.2 phosphate buffer were mixed with various levels of fresh yolk and tested for lytic activity. Under those conditions, 5% yolk reduced the activity to less than 10%. REFERENCES Alderton, G., and H. L. Fevold, 1945. Isolation of lysozyme from egg white. J. Biol. Chem. 157: 43-58. Cotterill, 0. J., and A. R. Winter, 1954. Egg white lysozyme. 3. The effect of pH on the lysozymeovomucin interaction. Poultry Sci. 34: 679686. Cotterill, 0. J., and E. M. Funk, 1963. Effect of pH and lipase treatment on yolk-contaminated egg white. Food Technol. 17: 1183-1188. Cotterill, O. J., F. E. Cunningham and E. M. Funk, 1963. Effect of chemical additives on yolk-contaminated liquid egg white. Poultry Sci. 42: 1049-1057. Cotterill, O. J., W. E. Seideman and E. M. Funk, 1965. Improving yolk-contaminated egg white by heat treatments. Poultry Sci. 44: 228-235. Cunningham, F. E., 1963. Factors effecting the insolubilization of egg white proteins. Ph.D. The-
sis. University of Missouri Library, Columbia, Missouri. Cunningham, F. E., and O. J. Cotterill, 1964. Effect of centrifuging yolk-contaminated liquid egg white on functional performance. Poultry Sci. 43: 283-291. Cunningham, F. E., and H. Lineweaver, 1967. Inactivation of lysozyme by native ovalbumin. Poultry Sci. 46: 1471-1477. Ehrenpreis, S., and R. C. Warner, 1956. The interaction of conalbumin and lysozyme. Arch. Biochem. Biophys. 61: 187-252. Evans, R. J., and S. L. Bandemer, 1956. Separation of egg white proteins by paper electrophoresis. Agri. Food Chem. 4 : 802-811. Forsythe, R. R., and J. F. Foster, 1950. Egg white proteins. I. Electrophoretic studies on whole white. J. Biol. Chem. 184: 377-383. Hawthorne, J. R., 1950. The action of egg white lysozyme on ovomucoid and ovomucin. Biochim. Biophys. Acta. 6: 28-35. Klotz, I. M„ and E. M. Walker, 1948. Complexes of lysozyme. Arch. Biochem. 18: 319-325. Litwack. G., and M. L. Sears, 1965. Precipitation of lysozyme with the thyroxine or thyroglobulin. J. Biol. Chem. 240: 674-678. MacDonnell, L. R., R. E. Feeney, H. L. Hanson, A. Campbell and T. F. Sugihara 1955. The functional properties of the egg white proteins. Food Technol. 9: 49-53. Mandeles, S., 1960. Use of DEAE-cellulose in the separation of proteins from egg white and other biological materials. J. Chromatog. 3 : 256-264. Nichol, L. W., and D. T. Winzor, 1964. The determination of equilibrium constants from rapidly reacting systems of the type A -f B ^ C. J. Phy. Chem. 68: 2455-2463. Parry, R. M. Jr., R. C. Chandan and K. M. Shahani, 1965. A rapid and sensitive assay of muramidase. Proc. Soc. Expl. Biol. Med. 19: 384386. Steiner, R. F., 1953. Reversible association processes of globular proteins. II. Electrostatic complexes of plasma albumin and lysozyme. Arch. Bioch. Biophys. 47: 56-75.
NEWS AND NOTES UNITED KINGDOM NOTES Dr. R. F. Gordon, Director of the Houghton Poultry Research Station, near Huntingdon, England, was honored with the Victory Medal of the Central Veterinary Society. The presentation was (Continued
made at the Society's Centenary Banquet held in the historic Guildhall of the City of London. The citation stated that the award was for services to the profession and pioneering work in poultry disease research. on page 1071)
3. Spoolated lifts as designed and used in the system studied, damaged eggs significantly. REFERENCES Forsythe, R. H., 1966. Modern marketing demands
1013
sound shells. Paper presented at Nebraska Egg Shell Quality Conference, March 23, 1966. Toleman, W. J., T. H. Coleman and L. E. Dawson 1964. Factors affecting the number of cracked eggs produced on Michigan farms. Research Report 17, Michigan State University Agr. Exp. Sta., East Lansing, Michigan.
The Influence of Yolk on Egg White Lysozyme1'2'3 F. E. CUNNINGHAM AND O. J. COTTERILL Dairy and Poultry Science Department, Kansas State University, Manhattan, Kansas 66502 and Food Science and Nutrition Department, University of Missouri-Columbia, Columbia, Missouri 6S201 (Received for publication October 10, 1970)
T
HE harmful effects of yolk and similar lipid material on the functional properties of egg white, long recognized, have been reviewed by several workers (Cotterill and Funk, 1963; Cunningham, 1963; Cunningham and Cotterill, 1964). The mechanism, however, whereby yolk is detrimental is not understood. Cotterill et al. (1963) showed that although the use of chemical additives inproved performance of yolk-contaminated egg white, additive actions were not solely associated with overcoming the adverse effects of the yolk. Cotterill and Funk (1963) found that lipase treatment improved yolk-contaminated white but did not determine if the improvement resulted from destruction of harmful yolk components or from products produced by hydrolysis (such as fatty acids). That pH is significant to functional performance, particularly for yolk-contaminated white, has 1 Contribution No. 801, Department of Dairy and Poultry Science, Kansas Agricultural Experiment Station, Manhattan, Kansas 66502. 2 Contribution from the Missouri Agricultural Experiment Station, Journal Series Number 6172. 3 Presented in part at the 59 th annual meeting of the Poultry Science Association, Knoxville, Tennessee, 1970.
been shown by Cotterill and Funk (1963) and Cunningham (1963). Cunningham and Cotterill (1964) found that centrifuging yolk-contaminated egg white improved its performance and that centrifuging at pH values around S.S caused maximum precipitation of yolk lipid and certain egg white proteins (presumably lysozyme-ovomucin). They suggested the possibility of complexing between yolk components and egg white ovomucin. Cotterill et al. (1965) found that the functional performance of yolk-contaminated egg white could be improved by heat treatment. They postulated that the mechanism for improvement in performance resulted from a heat-induced dissociation of a detrimental complex between some yolk component and certain egg white proteins. Lysozyme, a basic protein in egg white, is known to form electrostatic complexes with other substances. Its interaction with these substances has been demonstrated: thymus nucleate and yeast nucleate (Klotz and Walker, 1948), bovine plasma albumin (Steiner, 1953), ovomucin (Hawthorne, 1950; Cotterill and Winter, 1954), conalbumin (Ehrenpreis and Warner, 1956), ovalbumin (Forsythe and Foster, 1950; Nichol and Winzor, 1964; Cunningham
1014
F. E. CUNNINGHAM AND O. J. COTTERILL
and Lineweaver, 1967), and thyroxine or thyroglobulin (Litwack and Sears, 1965). This study concerns a reaction that occurs at room temperature (and over a wide range of pH) between lysozyme and egg yolk. MATERIALS AND METHODS
Egg white used in these experiments was obtained from fresh shell eggs, broken out in the laboratory, blended and frozen. Samples were thawed at room temperature as needed. Egg yolks, obtained from fresh shell eggs as needed, were hand separated from the white. While holding the yolk in a paper towel, the vitelline membrane was ruptured allowing the yolk to be collected in a beaker but retaining the membrane on the towel. Lysozyme was prepared from fresh, wellblended egg white by the isoelectric precipitation method of Alderton and Fevold (1946) and by separation from egg white utilizing ion-exchange chromatography with subsequent freeze drying of the fractions. Lysozyme activity was determined by the method of Parry et al. (1965). Samples of yolk-free and yolk-contaminated
egg white were analyzed by paper electrophoresis (Evans and Bandemer, 1956) and by column chromatography using the procedures of Mandeles (1960). RESULTS AND DISCUSSION
Samples of liquid egg white (pH 9.0) were intentionally contaminated with fresh, liquid yolk up to 1.0% (by weight) and well mixed in a Waring blendor. The presence of yolk apparently did not alter the electrophoretic behavior of the egg white proteins but did change the chromatographic profile. Subjecting 10-ml. portions of egg white to chromatography on DEAEcellulose produced the patterns shown in Figure 1. The continuous line shows the elution of yolk-free egg white, whereas the dashed line represents the sample contaminated with 0.1% yolk. The peaks in Figure 1 are identified as follows: A, B and C are lysozyme; peak D is conalbumin; peak E is ovomucoid; peaks G and H are ovalbumin; peak J is flavoprotein. Peaks F, I and K are not identified. The patterns obtained showed that the presence of yolk altered the behavior of several proteins, including lysozyme, ovomucoid and ovalbumin.
0.6-
0.4
0.2
SO
100
150 Fraction number
FIGURE 1. Chromatography of yolk-free (solid line) and yolk-contaminated (dotted line) egg albumin on DEAE-cellulose. Column size: 4 cm. diameter, 32 cm. length. Fraction size: 10 ml. Flow rate: 2 ml./min. Absorbance measured at 280 imi.
1015
LYSOZYME AND EGG WHITE
Peaks J and K were absent from the yolkcontaminated sample; also one of the lysozyme components was missing and peak A of lysozyme was greatly reduced. Lysozyme components (peaks A, B and C from yolk-free samples and peaks A and B from yolk-contaminated samples) collected from the ion-exchange column were freeze dried so that relative activities could be determined. The freeze dried fractions were dissolved in pH 6.2 phosphate buffer so that all solutions contained 0.3% protein and tested for lytic activity on Micrococcus lysodeikticus. Using Peak A from normal (yolk-free) egg white as a measuring standard (100%), it was found that peaks B and C collected from yolk-free egg white had higher specific activities than did peak A (Table 1). Peaks A and B collected from yolk-contaminated egg white had reduced activities (45 and 40%, respectively). The fate of the other lysozyme fraction in yolk-contaminated egg white is still unknown. In determining the concentration of yolk required to inactivate lysozyme in buffer at pH 6.2, the enzyme was crystallized from fresh, liquid egg white and redissolved in phosphate buffer. Various amounts (from 1 to 5%) of fresh, liquid yolk were added to solutions of 0.3% lysozyme and mixed well; 1 ml. of each mixture was transferred to 49 ml. of phosphate buffer to eliminate opaqueness. The relative activities are shown in Figure 2. In pH 6.2 buffer, the interaction between lysozyme and yolk at TABLE 1.—Relative activity of lysozymes* separated from egg white by chromatography Peak A
A B C
T, m i ' » Egg White
too 100 110 120
Yolk-Contaminated Egg White 55 60
' 0.3% lysozyme in pH 6.2 phosphate buffer.
100 80 >-
> 60 U < LU
40
>
N
O (/) 20 >-. O. 0
1
2 3 4 YOLK CONC.(%)
5
FIG. 2. Inactivation of egg white lysozyme (in pH 6.2 phosphate buffer) by various amounts of fresh liquid egg yolk.
the 5% level was sufficient to reduce lytic activity to less than 10%. Cunningham (1963) demonstrated that yolk, or some fraction thereof, was highly effective in reducing the foam capacity as well as amount of material insolubilized in the foam of egg white and suggested that the globulins were the egg white proteins most likely involved. MacDonnell et al. (1955) showed that the more rapid foamers in egg white were the globulins; therefore, it is possible that the harmful effects of yolk in liquid egg white are caused by the complexing between certain yolk lipids and lysozyme and other whipping proteins of egg white. Further work is needed to determine the yolk component(s) responsible for inactivating lysozyme and whether the reaction is reversible. SUMMARY
Proteins from yolk-free and yolk-contaminated egg white were separated by paper electrophoresis and ion-exchange chromatography. The presence of yolk did not
1016
F. E. CUNNINGHAM AND 0. J. COTTERILL
alter the electrophoretic patterns but did change the chromatographic profile. Three lysozyme components were apparent in yolk-free egg white; only two were found in yolk-contaminated samples. Lysozyme fractions were collected from chromatographic columns, freeze dried and tested for lytic activity. Peaks B and C obtained from yolk-free egg white had higher specific activities than peak A; peaks A and B from yolk-contaminated egg white had reduced activity (45 and 40%, respectively), using peak A from yolk-free egg white as a standard. Solutions of 0.3% lysozyme in pH 6.2 phosphate buffer were mixed with various levels of fresh yolk and tested for lytic activity. Under those conditions, 5% yolk reduced the activity to less than 10%. REFERENCES Alderton, G., and H. L. Fevold, 1945. Isolation of lysozyme from egg white. J. Biol. Chem. 157: 43-58. Cotterill, 0. J., and A. R. Winter, 1954. Egg white lysozyme. 3. The effect of pH on the lysozymeovomucin interaction. Poultry Sci. 34: 679686. Cotterill, 0. J., and E. M. Funk, 1963. Effect of pH and lipase treatment on yolk-contaminated egg white. Food Technol. 17: 1183-1188. Cotterill, O. J., F. E. Cunningham and E. M. Funk, 1963. Effect of chemical additives on yolk-contaminated liquid egg white. Poultry Sci. 42: 1049-1057. Cotterill, O. J., W. E. Seideman and E. M. Funk, 1965. Improving yolk-contaminated egg white by heat treatments. Poultry Sci. 44: 228-235. Cunningham, F. E., 1963. Factors effecting the insolubilization of egg white proteins. Ph.D. The-
sis. University of Missouri Library, Columbia, Missouri. Cunningham, F. E., and O. J. Cotterill, 1964. Effect of centrifuging yolk-contaminated liquid egg white on functional performance. Poultry Sci. 43: 283-291. Cunningham, F. E., and H. Lineweaver, 1967. Inactivation of lysozyme by native ovalbumin. Poultry Sci. 46: 1471-1477. Ehrenpreis, S., and R. C. Warner, 1956. The interaction of conalbumin and lysozyme. Arch. Biochem. Biophys. 61: 187-252. Evans, R. J., and S. L. Bandemer, 1956. Separation of egg white proteins by paper electrophoresis. Agri. Food Chem. 4 : 802-811. Forsythe, R. R., and J. F. Foster, 1950. Egg white proteins. I. Electrophoretic studies on whole white. J. Biol. Chem. 184: 377-383. Hawthorne, J. R., 1950. The action of egg white lysozyme on ovomucoid and ovomucin. Biochim. Biophys. Acta. 6: 28-35. Klotz, I. M„ and E. M. Walker, 1948. Complexes of lysozyme. Arch. Biochem. 18: 319-325. Litwack. G., and M. L. Sears, 1965. Precipitation of lysozyme with the thyroxine or thyroglobulin. J. Biol. Chem. 240: 674-678. MacDonnell, L. R., R. E. Feeney, H. L. Hanson, A. Campbell and T. F. Sugihara 1955. The functional properties of the egg white proteins. Food Technol. 9: 49-53. Mandeles, S., 1960. Use of DEAE-cellulose in the separation of proteins from egg white and other biological materials. J. Chromatog. 3 : 256-264. Nichol, L. W., and D. T. Winzor, 1964. The determination of equilibrium constants from rapidly reacting systems of the type A -f B ^ C. J. Phy. Chem. 68: 2455-2463. Parry, R. M. Jr., R. C. Chandan and K. M. Shahani, 1965. A rapid and sensitive assay of muramidase. Proc. Soc. Expl. Biol. Med. 19: 384386. Steiner, R. F., 1953. Reversible association processes of globular proteins. II. Electrostatic complexes of plasma albumin and lysozyme. Arch. Bioch. Biophys. 47: 56-75.
NEWS AND NOTES UNITED KINGDOM NOTES Dr. R. F. Gordon, Director of the Houghton Poultry Research Station, near Huntingdon, England, was honored with the Victory Medal of the Central Veterinary Society. The presentation was (Continued
made at the Society's Centenary Banquet held in the historic Guildhall of the City of London. The citation stated that the award was for services to the profession and pioneering work in poultry disease research. on page 1071)