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' , -, ~ 1 a in: .3 “‘- J) ‘ 1““ .1 mil 55-3 $333 1..“ w—— This is to certify that the thesis entitled EGG ALBUMEN PROTEINS INTERACTIONS IN SELECTED FOOD SYSTEMS presented by TEIKO MURATA JOHNSON- has been accepted towards fulfillment of the requirements for PhD dggreein Food. Science 'rprof r Date September 26, 1980 0-7639 I WME Hugs: 25¢ per day per 1%. RETURNUG LIBRARY MTERIALS: Place in book return to remove charge from circulation records EGG ALBUMEN PROTEINS INTERACTIONS IN SELECTED FOOD SYSTEMS BY Teiko Murata Johnson A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Food Science and Human Nutrition 1980 é/ 67/5. ¢/’ ABSTRACT EGG ALBUMEN PROTEINS INTERACTIONS IN SELECTED FOOD SYSTEMS BY Teiko Murata Johnson The present study was aimed at the investigation of a1- bumen proteins functional properties and specific protein- protein interactions in two food systems. Foamability was evaluated in an angel food cake system and coagulability in a custard model system. Ovomucin, lysozyme, globulins, ovo- mucoid, conalbumin, and ovalbumin were isolated from egg white. They were further tested singly and in combinations in these food systems. Various combinations of three levels of each protein according to a mixtures experimental design constituted the treatments for the cake system. The effects of the proteins interactions in this system were analyzed through response surface methodology to define protein levels for optimization of cake parameters. Ultrastructural examina- tions of selected foams in transmission and scanning elec- tron microscopes and of coagulums in a scanning electron microsc0pe were performed. Foamability studies of the individual protein solutions showed that globulins solution had good foaming properties Teiko Murata Johnson and produced a cake with high volume and excellent textural characteristics. Ovalbumin solution did not exhibit good air incorporation ability and produce a coarse textured cake of relatively large volume. Conalbumin, lysozyme, ovomucin, and ovomucoid had little or no foaming capacity. In interaction studies it was found that ovomucin, lyso- zyme, and globulins were the proteins primarily involved in foaminess and volume of angel food cakes . Association of ovomucin with globulins significantly favored foam forma- tion. However, cake volume correlated negatively with foami- ness. Lysozyme depressed foaming capacity by complexing with ovomucin. This resulted in considerable cake volume improve- ment. Surface response analysis of foaming index and cake volume defined ovomucin, lysozyme, and globulins levels range for production of a target cake. These were 0.2-1.0%, 0.0- l.8%, and 12.2-14.8% for ovomucin, lysozyme, and globulins, respectively. Studies of gelation properties of the albumen proteins showed lysozyme, globulins and combinations of lysozyme with globulins produced the firmest gels. Ovomucin and ovomucoid showed no heat gelation abilities. The following order of heat stability of the proteins was found: conalbumin < oval- bumin < globulins < lysozyme. Denaturation transition Teiko Murata Johnson temperatures of these proteins were 57.3, 71.5, 72.0, and 81.5°C, respectively. It was observed that the primary effects of protein-protein interactions in this food system were a denaturing action exerted by the least heat stable proteins over the more stable ones. Transmission electron microsc0pic examinations of foams revealed the existence of a layer of partially cross-linked polypeptides at the film surface and scanning electron micro- sc0py studies showed that ovomucin concentrated at the film surface preventing the formation of a cohesive membranous layer. The depressive effect of ovomucin on cake volume seemed to conform with formation of less stable foams and lack of heat coagulative properties of the protein film. Lysozyme, by complexing with ovomucin, altered the viscous nature of this protein. Scanning electron microscopic investigations of coagu- lums showed a characteristic association of the polypeptides in grape-like clusters of variable sizes. Globulins poly- peptides aggregated in membrane-like arrangements and exhibited excellent binding abilities. Smaller cluster sizes seemed to correlate with firmer gels. To Craig, for his presence. ACKNOWLEDGMENTS The author wishes to express her gratitude to Dr. Mary Ellen Zabik for her continuous guidance and valuable sugges- tions throughout this entire study. Appreciation is extended to Dr. Karen K. Baker, Dr. Maurice E. Bennink, Dr. Lawrence E. Dawson, and Dr. Pericles Markakis for the advice and encouragement given as members of the guidance committee. Special thanks are expressed to Dr. Gary R. Hooper for the guidance given in the initial work, and to my fellow graduate students for their encouragement and interest in the project. Gratitude is extended to American Egg Board for the financial support throughout the graduate program. Lastly, I would like to thank my parents for their constant support throughout this graduate program, and to Craig, all I can say is: "Thank you." iii TABLE OF List of Tables . List of Figures . Introduction . . Review of Literature Egg Composition Albumen Proteins Ovalbumin Conalbumin Ovomucoid Lysozyme and Globulins Ovomucin Other Proteins Protein Structure Albumen Proteins Protein Denaturation Protein Bonding Functional PrOperties of Viscosity Surfactant Properties Foaming Properties Gelation (Coagulation) Experimental Procedure Protein Fractionation Source of Eggs Preparation Egg Albumen Globulins Ovalbumin Conalbumin Lysozyme Ovomucoid iv CONTENTS Proteins PAGE Vii Ovomucin . . Freeze-Drying . Removal of Iron from Conalbumin Assessment of Purity-Purification ElectrOphoresis Purification Chemical Analyses . Moisture Analysis Total Nitrogen Sulfhydryl Groups Total Sulfhydryl- -Disu1fide Groups Elemental Analyses Experimental Design Angel Food Cake System Protein Solution Preparation Cake Preparation Volume Determination Tenderness Determination Compressibility Determination Foaming Index Determination Texture . Custard Model System Gel Strength Determination Percentage Drainage Electron Microscopy Transmission Electron Microscopy Scanning Electron Microscopy Statistical Analyses of the Data Results and Discussion . Protein Fractionation . Angel Food Cake System' Protein Interactions Foaming Index Volume Tenderness and Compressibili Sulfhydryl Groups Response Surface Analyses Custard Model System Electron Microsc0py Examination of Foams . Examination of Gels Summary and Conclusions Y e o o o ff 0 o o o 160 Suggestions for Further Research References . . Appendices . . A. Experimental Design B. C. Observed and Predicted Values Comparison of Time-Temperature Relationships of Albumen Proteins with Their Double Combinations PAGE 165 166 178 178 180 187 LIST OF TABLES Table Page 1 Composition of Albumen, Yolk, and Whole Egg. 5 2 Physicochemical Characteristics of Egg-White Proteins (Powrie, 1977). . . . . . 6 3 Reactive Groups of Proteins (Olcott and Fraenkel-Conrat, 1947). . . . . . 12 4 Types of Chemical Bonds in Proteins (Wehrli and Pomeranz, 1969). . . . . . . 13 5 Amino Acid Composition of Egg Albumen Proteins. . . . . . . . . . 15 6 General Classes of Functional Pr0perties of Proteins Important in Food Applications (Kinsella, 1976). . . . . . . . 2M 7 Mineral Composition of Dried Egg-White and Several Isolated Proteins. . . . . 56 8 Proximate Protein Composition of Egg Albumen and Level Range Values of Each Protein . 58 9 Treatment Levels Combinations of 6 Proteins Used for the Preparation of Angel Food Cakes. . . . . . . . . . 60 10 Treatment Levels Combinations of 6 Proteins Used for the Preparation of Custard Type Gels. . . . . . . . . . . 63 11 Hot Plate Control Settings Used to Prepare the Custard Type Gels. . . . . . 72 12 Average Moisture, Nitrogen, and Protein Contents of Egg Albumen. . . . . . 81 13 Yields and Recovery of Protein Fractions Isolated from Egg Albumen. . . . . 81 Vii Table Page 14 Composition and Relative Purity of the Iso- lated Albumen Protein Fractions. . . . 8H 15 Sulfhydryl and Disulfide Contents of Albumen Proteins. . . . . . . . . . 86 16 Physico-Chemical Characteristics of Albumen Proteins Solutions and Angel Food Cake Parameters. . . . . . . . . 87 17 Significant Simple Correlation Coefficients Among Protein Mixtures Components, Physical Characteristics, and Cake Parameters. . . 94 18 Effect of Whipping on the Sulfhydryl Content of Albumen Protein Solutions. . . . . 107 19 Mean Square Values and F-Ratios Significance of Viscosity, Surface Tension, and Foaming Index of Albumen Proteins Mixtures. . . 109 20 Mean Square Values and F-Ratios Significance of Volume, Tenderness, and Compressibility of Angel Food Cakes. . . . . . . 109 21 Partial Regression Coefficients (b.), Stan- dard Errors (sb), Partial Correlation Co- efficients, and T-Test Significance for Viscosity of Protein Mixtures. . . . . 110 22 Partial Regression Coefficients (b.), Stan- dard Errors (3 ), Partial Correlation Co- efficients, an T-Test Significance for Foaming Index of Protein Mixtures. . . . 111 23 Partial Regression Coefficients (b.), Stan- dard Errors (3 ), Partial Correlation Co- Efficients, an T-Test Significance for Cake Volume. . . . . . . . . 112 24 Partial Regression Coefficient (bi), Stan- dard Errors (sb), Partial Correlation Co- efficients, and T-Test Significance for Cake Tenderness. . . . . . . . 113 25 Prediction Equations for Significant Phys- ical Parameters of Protein Mixtures, Stan- dard Error of the Estimate (8), and Co- Efficients of Multiple Determination (R2). . 116 viii Table 26 27 28 29 30 31 32 33 Prediction Equations for Significant Cake Standard Error of the Estimate (S), and Coefficients of Multiple Deter— Parameters, mination (R2). Coagulation Temperature Ranges of Albumen Pro- teins Solutions and T-Statistic. Firmness of Coagulums Prepared with Albumen Proteins Solutions and T-Statistic. Percentage Drainage of Gels Made With Albumen Proteins Solutions and T-Statistic. Effect of Heating on the Sulfhydryl Content and pH of Albumen Proteins Solutions. Physico-Chemical Characteristics of Gels Made from Egg Albumen Proteins Mixtures. Observed and Predicted Values of Protein Mix- tures Physical Parameters. Observed and Predicted Values of Angel Food Cake Parameters. Page 117 l2u 125 126 138 1M2 181 18u Figure LIST OF FIGURES Page Schematic diagram of bonds within and between polypeptide chains. (a) electrostratic in- teraction; (b) hydrogen bonding; (c) hydro- phobic interaction; (d) dipole-dipole inter- action; (e) disulfide bond . . . . . . 13 Flow diagram of the isolation of egg albumen proteins . . . . . . . . . . 36 Electrophoretogram of egg white proteins a) SDS PAGE. 1. Ovomucin; 2. Globulins; 3. Conalbumin; 4. Ovomucoid; 5. Ovalbumin; 6. Lysozyme . . . . . . . . . 83 b) PAGE. 1. Conalbumin; 2. Ovomucoid; 3. Globulins; 4. Ovalbumin; 5. Lysozyme; 6. Ovomucin . . . . . . . . . 83 Angel food cakes prepared with various albumen protein solutions. Protein concentration 10.4%, pH 5.7, ionic strength 0.20 . . . . . 92 Effect of ovomucin on the foaming ability of pro- tein solutions containing various levels of lyso- zyme and globulins. Protein concentration = 10.4%, pH = 5.7, ionic strength = 0.20. Ovomucoid = 11.5%, conalbumin‘= 13.5%, ovalbumin = 53.5 to 72.0% .ll= H. . . . . . . . . . 97 Effect of lysozyme on the foaming capacity of protein solutions containing various levels of globulins and ovomucin. Protein concentration = 11.5%, conalbumin = 13.5%, ovalbulin = 53.5 to72.0%.N=ll. . . . . . . . . 98 Effect of globulins on the foaming capacity of protein solutions containing various levels of lysozyme and ovomucin. Protein concentration = 10.4%, pH = 5.7, ionic strength = 0.20. Ovo- mucoid = 11.5%, conalbumin = 13.5%, ovalbumin = 53.5 to 72.0%. N =1L . . . . . . . 100 Figure Page 8 Effect of globulins on volume of angel food cakes prepared with protein solutions con- taining varying levels of lysozyme and ovo- mucin. Protein concentration = 10.4%, pH = 5.7, ionic strength = 0.20. Ovomucoid = 11.5%, conalbumin = 13.5%, ovalbumin = 53.5 to72.0%,N=u, . . . . . . . .102 9 Effect of ovomucin on volume of angel food cakes prepared with protein solutions con- taining varying levels of globulins and lysozyme. Protein concentration = 10.4%, pH = 5.7, ionic strength = 0.20. Ovomucoid = 11.5%, conalbumin = 13.5%, ovalbumin = 53.5% to 72.0% .DJ= A. . . . . . . 103 10 Effect of lysozyme on volume of angel food cakes prepared with protein solutions con- taining varying levels of globulins and ovomucin. Protein concentration = 10.4%, pH = 5.7, ionic strength = 0.20. Ovo- mucoid = 11.5%, conalbumin = 13.5%, oval- bumin = 53.5 to 72.0%. N==H. . . . . . 104 11 Protein mixture foaming index response sur- faces as a function of lysozyme and ovomucin. Other protein levels: globulins 6.50-14.95%, ovomucoid 12%, conalbumin 14%, and ovalbumin 59% a) Contour Surface . . . . . . 118 b) Perspective Response Surface . . . 118 12 Angel food cake volume response surfaces as a function of lysozyme and ovomucin. Other pro- tein levels: globulins 6.50-14.95%, ovo— mucoid 12%, conalbumin 14%, and ovalbumin 59% . a) Contour Surface . . . . . . 119 b) Perspective Response Surface . . . 119 13 Foaming index and volume contour plots overlay for designation of levels of ovomucin and lysozyme in angel food cake system . . . 121 14 Time-temperature curves of several albumen pro- tein solutions heated at a rate of 0.74 C/min. Protein concentration = 1.27%, ionic strength = 0.275, pH = 8.0 I O 0 O O O O O 122 xi Figure 15 16 17 18 19 20 21 22 A comparison of changes in coagulation temp- erature, gel firmness, and percentage drainage of lysozyme (LYS) coagulum with the addition of globulins (GLOB), conalbumin (CON), and ovalbumin (OVB) . . . . . . . . A comparison of changes in coagulation temp- erature, gel firmness, and percentage drainage of globulins coagulum with the addition of lysozyme (LYS), ovomucoid (OVD), conalbumin (CON), and ovalbumin (OVB) . . . . . A comparison of changes in coagulation temp- erature, gel firmness, and percentage drainage of ovalbumin (OVB) coagulum with the addition of lysozyme (LYS), globulins (GLOB), ovomucoid (OVD), and conalbumin (CON) . . . . . A comparison of changes in coagulation temp- erature, gel firmness, and percentage drainage of conalbumin (CON) coagulum with the addition of lysozyme (LYS), globulins (GLOB), ovomucoid (OVD), and ovalbumin (OVB) . . . . . Comparison of time—temperature curves of solu- tions with different protein composition. Heat- ing rate = 0.74°C/min, protein concentration = 1.27%, pH = 8.0, ionic strength = 0.275 . . Comparison of time-temperature curves of var- ious solutions with different protein compo- sition. Heating rate = 0.74 C/min, protein concentration = 1.27%, pH = 8.0, ionic strength = 0.275 . . . . . . . . . . Transmission electron micrographs of albumen protein foams a) Lysozyme . b) Globulins . c) Conalbumin d) Ovalbumin . e) Control . Scanning electron micrographs of albumen protein foams a) Lysozyme . . . . . . . . b) Globulins . . . . . . . . c) Conalbumin . . . . . . . Page 133 13M 135 136 lUO lUl 1M6 1&6 1&6 1&6 1H6 1H8 1H8 1&8 Figure Page d) Ovalbumin . . . . . . . . 148 e) Control . . . . . . . . 1H8 23 Scanning electron micrographs of albumen protein foams a) Lysozyme . . . . . . . . 1M9 b) Globulins . . . . . . . 1&9 c) Conalbumin . . . . . . . 1M9 d) Ovalbumin . . . . . . 1M9 e) Control . . . . . . . . 1H9 24 Scanning electron micrographs of albumen protein foams a) Lysozyme . . . . . 150 b) Globulins . . . . . 150 c) Conalbumin . . . . . . . 150 d) Ovalbumin . . . . . . . . 150 e) Control . . . . . . . . 150 25 Scanning electron micrographs of foams differing in lysozyme content. Other proteins levels: ovomucin 2.5%, globulins 3%, ovomucoid 5%, conalbumin 6%, ovalbumin 77.5-83.5% a) and b) 0% lysozyme . . . . . 152 c) and d) 6% lysozyme . . . . . 152 26 Scanning electron micrographs of albumen protein gels a) Lysozyme . . . . . . . . 155 b) Globulins . . . . . . . 155 c) Conalbumin . . . . . . 155 d) Ovalbumin . . . . . . . . 155 e) Control . . . . . . . . 155 27 Scanning electron micrographs of albumen protein gels 8) Lysozyme . . . . . . . 156 b) Globulins . . . . . . . . 156 c) Conalbumin . . . . . . . 156 d) Ovalbumin . . . . . . . 156 e) Control . . . . . . . . 156 xiii Figure Page 28 Scanning electron micrographs of gels prepared with,protein.mixtures. a) and b) Lysozyme-Globulins . . 158 c) and d) Ovomucin-Lysozyme- -Globulins- Conalbumin-Ovalbumin . . . 158 29 Changes in time-temperature curve of lysozyme solution with the addition of globulins (Glob), ovomucoid (Ovd), conalbumin (Con), and oval- bumin (OVb). Heating rate = 0.74°C/min, protein concentration = 1.27%, pH = 8.0, ionic strength = 0.275 . . . . . . . . 188 30 Changes in time-temperature curve of globulins solution with the addition of lysozyme (Lys), ovomucoid (Ovd), conalbumin (Con), and oval- bumin (Ovb). Heating rate = 0.74°C/min, protein concentration = 1.27%, ionic strength = 0.275 . . . . . . . . . . 189 31 Changes in time-temperature curve of conal— bumin solution with the addition of lysozyme (Lys), globulins (Glob), ovomucoid (Ovd), and ovalbumin (Ovb). Conditions are the same as those in Fig. 29 . . . . . . . 190 32 Changes in time-temperature curve of oval- bumin solution with the addition of lysozyme (Lys), globulins (Glob), ovomucoid (Ovd), and conalbumin (Con). Heating rate = 0. 74 oC/- min, protein concentration = 1. 27%, pH = 8. 0, ionic strength = 0.275 . . . . . . 191 xiv INTRODUCTION Increased interest in improving the egg processing technology has led to an extensive research concerning egg proteins functionality. Of the processes employed by the egg industry, drying, particularly spray-drying, has been known to adversely affect the whipping ability of dehydrated albumen and whole egg' (MacDonnell et al., 1950: Bergquist and Stewart, 1952; Joslin and Proctor, 1954). It also accelerates the flavor deteriorative changes of yolk- containing products (Bergquist, 1977). The incorporation of sugar or salts to liquid yolk is required to prevent changes in viscosity upon freezing (Cotterill, 1977). The microbiological problem of egg products was brought under control with the application of heat, such as pasteuriza- tion or other heat treatments. However, accompanying functional changes have required the use of whipping aids (Miller and Winter, 1950; Clinger et al., 1950; Forsythe, 1964). The changes observed in dried egg white are apparent- ly caused by alteration of one or more of the proteins. A combination of drying per se and physical treatment may be involved in promoting these changes (MacDonnell et al., 1950). Heat could also have an effect provided a temperature high enough is reached in the product during drying. The degree of alteration in functional properties is generally measured through performance tests in food pro- ducts. In studies on the proteins primarily responsible for foaming in an angel food cake, globulins, including lysozyme, were rated as excellent foamers while ovomucin was classed as a foam stabilizer (MacDonnel et al., 1955). By conducting physical tests it was concluded that globulins, ovomucin, and conalbumin have high foaming power, whereas ovalbumin, lysozyme, and ovomucoid do not foam as easily (Nakamura, 1963). In studies of coagulation ability of egg products several factors such as salts, sugar, acid, alkali, and temperature have been listed as the major parameters in- fluencing the heat induced gelling of eggs (Baldwin, 1977). The different heat sensitivity of albumen proteins (Cunning- ham and Lineweaver, 1965) suggests some proteins may play major roles in defining the gelling ability of egg white. While several workers have studied the functional aspects of the proteins in egg white by conducting simple performance and physical tests (Nakamura, 1964; Naka- mura and Sato, 1964; Snider and Cotterill, 1972; Cunning- ham, 1976; Egelandsdal, 1980), these studies could yield misleading information as to how the proteins would behave in the more complex food systems. The effects of interactive forces among ingredients, particularly protein-protein interactions, on foaming and coagulation abilities need to be evaluated in relation to performance in food systems. The findings associated with such studies may lead to the development of improved egg products or lead to new applications. Protein func- tionality data of extrapolative value for predicting per- formances of novel proteins in food systems could reduce production costs (Kinsella, 1976). Furthermore, such data may serve in comparative studies of proteins func- tions and molecular conformation. Therefore, in view of these considerations, the present study focused on the determination of possible interactions among the albumen proteins, as well as the characterization of functional properties of each protein. Two food systems with constant protein content were used: an angel food cake to investigate foamability and a custard model system to study coagulability. Six a1- bumen proteins were isolated and their foaming and gelling capacities were characterized at a physicochemical and ultrastructural level. The second objective of this study was to develop a statistically planned design from mixtures experiments to simplify and reduce the number of treatments. Multi- ple regression analysis was used to derive prediction equations for the analysis of response surfaces and op- timization of cake parameters. REVIEW OF LITERATURE Literature pertaining to egg albumen proteins' charac- teristics and functional properties are summarized in this review. Fundamentals of protein structure and function- ality in general are also reviewed to provide a better understanding of protein's functions in food systems. Egg Composition Eggs are a complex system of proteins and lipids dispersed in an aqueous phase with many other constituents. Whole eggs are composed of 8-11% shell, 56-61% albumen, and 27-32% yolk. The chemical composition of albumen, yolk and whole egg is displayed in Table 1 (Powrie, 1977). The major constituent of the total solids in albumen is proteins, whereas yolk solids are mostly proteins and lipids. Carbohydrates are present in the free form and in combination with proteins. In albumen, 44% of the carbo- hydrates are present in the free form and 56% are found in glycoproteins, while, in yolk, 70% are in the free form and 30% are present as protein-bound carbohydrates. Albumen Proteins Egg white has been reported to contain as many as 40 u Table 1. Composition of Albumen, Yolk, and Whole Egg (Powrie, 1977). Comgggent Protein Lipid Carbohydrate Ash ‘% Albumen 9.7-10.6 0.03 0.4-0.9 0.5-0.6 Yolk 15.7-16.6 31.8-35.5 0.2-1.0 1.1 Whole Egg 12.8-13.4 10.5-11.8 0.3-1.0 0.8-1.0 different proteins (Vadehra and Nath, 1973), most of which have not been isolated. The physicochemical characteris- tics of the major albumen proteins are summarized in Table 2. Ovalbumin Ovalbumin is a phosphoglycoprotein composed of three forms, A1, A2 and A3, which differ in their phosphate content. A1 has two phosphate groups per molecule; A2 has one; and A3 has none. The relative proportion of these components is approximately 85:12:3. The protein is easily denatured by exposure to new surfaces, action of various denaturating agents, and heat, but is resistant to pasteurization conditions (Powrie, 1977). During storage of eggs ovalbumin is converted to a more heat stable form, S-ovalbumin, possibly through rearrangement of the molecule to a more thermodynamically stable form (Smith and Back, 1965). .ammmHv mEMHHHHR can GGO3IO «abbqav xomm 6cm nuwfimln «Avmmav mosmmm can mmsmOIm .cfluofin meaflm m m m.m ooo.mm mo.o anefi>4 .mmmmmuoum Hmum>mm muflnfincH m m ~.m ooo.ve H.o soufinflrcflo>o m m h.v:m.v ooo.oom m.o sedan loco.omh IonouomEo>o m m m.m ooo.vm mm.o sflmu Ioumoomamo>o ..sw>mHmonfiu mpcwm m m H.v ooo.mm m.o :«muonmo>mHm .GOwumsausHmm .ImEms msuw> mufinfln new .cflmuoumoHMHm m m wo.m|m.v m m.a causeo>o a a m.m m no.4 ceasnoao mo ooo.mv N m m m.m uooo.om mo.¢ ceasnoHo o .HHM3 HHQO mflumuomn mmmsg v o a.oH oom.ea m.m mssuomsq .qflmdmu» mufinfircH m o m.qum.m ooo.m~ Ha chooseo>o . HwHQOHUflEIflUGw .mcoe Hmums mocfim omH o almo.ovm.m ooo.om ma cassnamcoo .cwmuoumoo usamormmonm 51Hc~ v m.¢ ooo.mq em cflesnam>o w.cmsan< mmsouw mmsouw usages mowumflnmuomumnu ovflmasmfla ahuphsmasm Hm unasooaoz “MVHMMMWM samuoum .Ahhma .mflu3omv mswmuoum mufinz:mmm mo moflumflumuomumnu HMOHEmnooowmhnm .m wanna Conalbumin Conalbumin, also known as ovotransferrin, is able to bind metal ions forming protein-metal complexes resistant to denaturation by heat, pressure, proteolytic enzymes, and denaturating agents (Fraenkel-Conrat and Fenney, 1950; Azari and Feeney, 1958, 1961). The protein complexes with two moles of metal ions per molecule with the relative binding stabilities: Fe3+ > Cr3+, Cu2+ > Mn2+, C02+, Cd 2n2+ > N12+ (Tan and Woodworth, 1969), and also with A13+ 2+> (Cunningham and Lineweaver, 1965). The iron binding ability of the protein imparts antimicrobial activity to egg white. In the metal free form, the protein is extremely susceptible to heat denaturation. Ovomucoid Ovomucoid is a glycoprotein of very high resistance to heat denaturation in acidic solutions and it shows a remarkable inhibitory activity against trypsin (Rhodes et al., 1960). The protein has an unusually high carbohydrate content (20-25%) which is present as three oligosaccha- ride units, each attached to the polypeptide through a glycosidic bond to asparagine (Powrie, 1977). Lysozyme and Globulins Lysozyme was identified as the protein in egg white with a lytic action on micrococcus cells by Fleming (1922). This enzyme cleaves the 8 - (1,4) - glycosidic linkages between N-acetylneuraminic acid and N-acetylglucosamine in bacterial cell walls (Osuga and Feeney, 1977). Lysozyme has been reported to be more resistant to heat inactiva- tion in phosphate buffer than in egg white (Cunningham and Lineweaver, 1965). This basic protein binds with several other egg white proteins possibly through electro- static interactions. Hawthorne (1950) demonstrated that addition of lysozyme to ovomucin induced drastic changes in the jelly-like protein to a stringy, compact mass. Other investigators have reported the interaction of lysozyme with conalbumin (Ehrenpreis and Warner, 1956) and with ovalbumin (Nakai and Kason, 1974). Longsworth et a1. (1940) noted two other globulins fractions, G2 and G3, distinct from lysozyme. Feeney et a1. (1963) isolated G2 globulins and partially characteriz- ed this protein fraction. They found negligible amounts of sulfhydryl and nucleic acids. Baker (1968) reported that G2 and G3 globulins have isoelectric points of 5.5 and 4.8, respectively. Ovomucin Ovomucin imparts the gel-like character to thick egg white. This extremely large polydispersed glycoprotein is filamentous and fiber-like in nature (Robinson and Monsey, 1975). Sugihara et a1. (1955) reported the virus anti- hemagglutinin activity of ovomucin and also pointed out the chalaziferous layers of eggs are formed by twisted ovomucin fibers. Ovomucin appears to be involved in the natural thin- ning of thick egg white which occurs during aging of eggs, but contradictory hypotheses have been proposed. Hawthorne (1950) suggested that the interaction of ovomucin with lysozyme causes thinning. On the other hand, Cotterill and Winter (l955),and Rhodes and Feeney (1957) proposed that the dissociation, rather than its formation, was responsible for thinning. Others have claimed that reduc- tion or hydrolysis of disulfide bonds in ovomucin (Mac Donnell et al., 1951; Tomimatsu and Donovan, 1972) and loss of the O-glycosidically linked carbohydrate units from the protein (Kato et al., 1979) were associated with deterioration of the gel structure. Variable molecular weights ranging from 7.6 x 106 to as high as 240 x 106 daltons have been reported for the protein (Lanni et al., 1949: Robinson and Monsey, 1975: Tomimatsu and Donovan, 1972). Other Proteins Ovomacroglobulin has been identified as component 18 by Lush (1961). Later, Miller and Feeney (1966) isolated and characterized this protein. They observed that the 10 protein contains low levels of sulfhydryl and sialic acid groups. Flavoprotein is the protein in egg white combined with riboflavin (Rhodes et al., 1958, 1959). The protein has from seven to eight phosphate moieties, but no sulf- hydryl groups. Avidin has been identified as the protein which binds biotin (Eakin et al., 1941). Other minor albumen proteins include a ficin and papain inhibitor (Possum and Whitaker, 1968), ovoglycoprotein (Ketterer, 1962), and ovoinhibitor (Matsushima, 1958). Protein Structure Study of a food protein at the molecular level can be aimed at understanding its behavior in food systems. The native structure of a protein is directed by its amino acid sequence and the surrounding microenvironment (water and cofactors such as metal ions). The sequence of amino acids will govern the folding of the main polypeptide chain whereas water will force the arrangement of the apolar side chains into the interior of the protein. Ultimately, this extremely complex three-dimensional configuration dic- tates the protein prOperties including functionality in food systems. The reactive groups in specific amino acid residues which may be involved in the various types of bonds that ll maintain the protein structure are displayed in Table 3. Many of these amino acid residues also participate in associations with water molecules. Table 4 displays the types of bonds present in proteins and the heat of formation (energy) involved in these interactions, whereas Fig. 1 illustrates the manner by which these bonds stabilize the protein molecule. Whitaker (1977) has summarized the various structural levels which are present in proteins. The primary sequence of the protein is formed by condensation between the carboxyl group of one amino acid with the amino group of another amino acid. This covalent peptide bond has a heat of formation of about 100 Kcal/mol which is broken only upon hydrolysis with strong acids or bases and heat, or with enzymes. The secondary structure consists of a-helices, B-folds, and B-turns. The helical regions are stabilized by multi- ple hydrogen bonds, an average of 3.6 bonds per turn, with a heat of formation of about 1-5 Kcal/mol. Water competing for binding can easily disrupt a hydrogen bond. The B- pleated sheets are primarily stabilized by hydrogen bonds, but hydrophobic and electrostatic bonds between the side chains of the amino acid residues also contribute to the structure maintenance. These B-regions are formed through inter or intrachain interactions between chain segments brought close together by B-turns. The tertiary structure is maintained by a combination Table 3. Reactive Groups of Proteins (Olcott and Fraen- kel-Conrat, 1947). Group Structure Origin Amino -NH2 Lysine, peptide chain end Guanidyl -NH|('2NH2 Arginine NH Imidazole -C?==CF Histidine N NH \\ / CH Amide -COONH2 Glutamine, Asparagine Aliphatic -OH Serine, Threonine hydroxyl Indole -fi 0 Tryptophan HC\ NH Thimethyl -SCH3 Methionine Disulfide -S-S- Cystine Phenol -<;::>>OH Tyrosine Sulfhydryl -SH Cysteine Carboxyl -COOH Glutamic acid, Aspartic Acid, peptide end chain l3 Table 4. Types of Chemical Bonds in Proteins (Wehrli and Pomeranz, 1969). Bond Mechanism (Kggififigl) Covalent 2 atoms bound by a common 30-100 electron pair Ionic Attraction between oppo- 10-100 (Electrostatic) site charges Hydrogen Affinity of hydrogen for 2-5 electronegative atom (i.e. oxygen) Van der Waals Long range interaction be- up to 0.5 tween nonpolar groups IIH . Q - E '11 .- \ i" I No) ' \ (c) l —J \ \(b) I \ °...\ '- \\o \\ — =0 ._‘: __ ...._i ___1 ______ :Cil-I:3 lCl-l3:(c) l he) I l cu let: on Lfifl /‘_"_2_l ._3J I .2.- Fig. 1. From Anfinson (I959) Schematic diagram of bonds within and between polypeptide chains. (a) electrostatic interaction: (b) hydrogen bonding: (c) hydrophobic interaction; (d) di- pole-dipole interaction; (e) disulfide bond. 14 of covalent, electrostatic, hydrogen, and hydrophobic bonds and by Van der Waals attractive forces. Of these bonds, the covalent disulfide bond is the strongest with an energy of 50 Kcal/mol. The quaternary structure of the protein is formed by the association of two or more polypeptide chains. These are held together by the same types of bonds which stabi- lize the tertiary structure. Albumen Proteins The conformation of the majority of the proteins present in egg white has not been elucidated. However, several studies have documented the amino acid composition of a number of them. The amino acid composition of ovo- mucin, lysozyme, ovomucoid, conalbumin and ovalbumin, reported by several investigators, is summarized in Table 5. Ovalbumin is a monomeric and nearly spherical globular protein (Osuga and Feeney, 1977). Approximately 50% of the amino acid residues in ovalbumin are hydrOphobic, and the protein contains four sulfhydryl groups and one disul- fide (Table 5). The thiol groups have variable reactivity towards several chemicals. Nitroprusside, ferricyanide and porphyridin do not react with native ovalbumin (Mirsky and Anson, 1936: Greenstein, 1938), whereas iodoacetate, iodoacetamide, p-chloromercuribenzoate (Anson, 1940:Nhr$vfl1 15 .oowuasmwo H .mssucsrudsm q - u ..ohms. Hsamumruom can Haamumnuom . o .Ammmav nEMfiaawz 0cm :cm3 I p .AHhmHv .Amwmuv .Hm no uw>mo I o .Hm um mmHHon - n ..Nsmdc ammcoz can comcnnom u a on a.m~ ~.k ~ m.so mcfisoua OCHEH ms H.ma m.~ N k.ss manganrumz no H.m~ m.oH m ~.cm .mflmrvwcsumso unmasm on s.mv m.HH on ~.om ocsuom ma G.om m.m~ 5 «.ms mancomure H>xoup>= m ~.~H us- m m.o~ caraouasue om m.m~ H.m m «.Hm mcflcasufiscora a m.- n.m m m.¢~ mcsxousa UHHMEOH< mm p.4m m.oa m ~.mm «chasm; «a o.m~ a.~ m m.~m mcaosodomH on m.cm «.4H m e.~m cansm> vm c.4m m.c~ NH v.~m wcncmac as °.wm 5.4H NH m.nm manosso onuormsaa an m.mm o.o as v.om mananmua m m.mn «.4 H m.m~ wcnonumsm oN m.vm m.~s m o.ov mafia»; venom om q.sn m.m~ m m.mm choc onemuaso an c.~m H.om Hm n.4m onoa ofiuummma oneno< lacs lacs .Hos .6mofl e um \mosowmmuv AHOE\mmspwnmuv \mospwmouv \noscfinmuv \nmaoev on“ Gaussnaw>o ccflquHmcmuuo>o ocuooseo>o awexuonmq acaoseo>o “Ed .mcaououm smfisnad man no :ofiuwnomeoo pact ocfie< .m manna l6 and Neuberger, 1972 ; MacDonnell et al. , 1951) will react with some, but not all, of the sulfhydryl groups. This masked nature of the protein also restricts the reactivity of other groups such as phenolic hydroxyl (Crammer and Neuberger, 1943), lysine residue (Steven and Tristram, 1958), and carboxyl groups (Atassi and Rosenthal, 1969). Upon denaturation, the phenolic hydroxyl in tyrosine residues ionize (Crammer and Neuberger, 1943). Fothergill and Fothergill (1970) reported that the C-terminal sequence of ovalbumin is Cys-Val-Ser-Pro. These authors also pointed out that one end of the disulfide was located in this C-terminal peptide. A single polypeptide chain forms conalbumin (Bezkoro- vainy et al., 1968; Greene and Feeney, 1968). The primary sequence of conalbumin has not been completed, but indica- tions do not support the presence of two identical half structures (Bezkorovainy and Zschocke, 1974). Williams (1975A, 1975B) isolated a 35,000 daltons C-terminal frac- tion which contained all of the carbohydrate present in the protein. The amino acid composition of conalbumin (Table 5) indicates the presence of approximately 15 disulfide bonds and about 55% of reactive residues. These include 159 potentially negatively charged groups, about 65 amino groups, 22 phenolic groups, and 35 guanidine groups. Ovomucoid has a single polypeptide chain with helical regions (Ikeda et al., 1968) and eight disulfide groups 17 in the chain. Kato et a1. (1974) reported on the presence of three similar domains in ovomucoid. Two fragments of molecular weights of 15,000 and 10,000 daltons were iso- lated from CNBr-treated ovomucoid (Beeley, 1972). Andrews (1965) suggested that ovomucoid is probably an expanded molecule, rather than compact. Donovan (1967) also con- cluded from hydrodynamic parameters that ovomucoid is neither compact nor highly asymmetric, but is highly hy- drated due to the presence of carbohydrate moieties. Approximately 60% of the amino acid residues in ovo- mucoid are potentially reactive (Table 5). Of these resi- dues, about 44% are acidic. Ovomucin is an extremely large protein probably com- posed of extended glycoprotein chains held together by disulfide links (Gottschalk et al., 1972). The protein appears to be in a flexible, extended-B or random-coil conformation containing little or no a-helix (Donovan et al., 1970). Tomimatsu and Donovan (1972) indicated that ovomucin is composed of short chains cross-linked in a branching arrangement and retains a random-coil conforma- tion upon alkaline hydrolysis. Robinson and Monsey (1975) observed that ovomucin is an aggregate (molecular weight 720,000) composed of chains of globular units, each with an approximate molecular weight of 103,000 daltons. Ovomucin has a relatively high content of disulfide groups. About 180 residues are acidic, 96 basic and 260 are apolar (hydrophobic) residues (Table 5). 18 Lysozyme is the only protein in egg white with a completely elucidated conformation. The amino acid sequence of this protein was characterized by Canfield (1963) and its conformation was determined with x-ray analysis by Blake et a1. (1966A). The molecule has an ellipsoidal shape with a marked cleft on one side, and dimensions of about 45 x 30 x 30 A. The main chain has a relatively small proportion of a-helix, three runs, each about 10 residues long, and long stretches with irregular conforma- tion. Several of these stretches have an extended 8- pleated sheet arrangement. The charged side chains, acidic and basic, and the terminal groups are distributed over the surface of the molecule. The remaining polar chains also appear to be on the surface whereas the majority of the non-polar (hydrophobic) chains are in the interior of the molecule. A number of hydrophobic groups are located on the surface of the cleft where the active site of the enzyme is situated. Lysozyme appears to exist as a dimer between pH 5 to 9 (Sophianopoulos and VanHolde, 1964). The contact between the molecules does not obstruct the access to the active site (Blake et al., 1966B). The proteins classed as globulins are poorly charac- terized. The fraction identified as 62 globulins has 0.8% tryptophan, 3.35% tyrosine, and traces of sulfhydryl and nucleic acids (Feeney et al., 1963). Ovomacroglobulin has, in 105 g of protein, about 19 143 acidic residues, 80 basic amino acids, 11.5 half cystine and approximately 235 hydrophobic groups (Miller and Feeney, 1966). Donovan et a1. (1969) reported that the protein was nearly spherical and had little a-helix. Protein Denaturation Protein denaturation is a familiar phenomenon though it means different things to different researchers. In all interpretations the most accepted concept is that of any modification involving the secondary, tertiary or quater- nary structure, without the rupture of any primary covalent bonds. Native proteins can show subtle or major changes in conformation during denaturation, usually through disul- fide bond scission (Kinsella, 1976). These changes increase the protein interfacial area and often result in a slight increase of bound water. However, the effects of protein unfolding (denaturation) followed by protein aggregation, result in an overall reduced solubility (Fen- nema, 1977). The extent and rate of denaturation may vary with protein sources and causative factors Such as heating, freezing, radiation, extreme dilution, sonication, and ex- posure to air-water or oil-water interfaces (Kinsella, 1976). Several denaturating chemical agents act by dis- rupting specific secondary forces which maintain the pro- tein structure. Urea, guanidine hydrochloride and some 2O surfactants break hydrophobic interactions (Gordon and Warren, 1968). Nonetheless, some anionic detergents may stabilize proteins against denaturation (Hegg and Lofquist, 1974). Perutz (1974) suggested that denaturation by acid or alkali involves the combined effects of total surface change of the protein and the ionization of specific groups on particular amino acids. These ionized groups attract water molecules which, in turn, disrupt hydrophobic associa- tions causing unfolding. The partial or total denaturation of protein mole- cules is obviously an important parameter of significant effects on protein functionality. Solubility is a critical factor in the functional behavior of proteins in food proteins. However, denaturation is required in the prepara- tion of many foods and in texturization to expose masked reactive groups of the protein for aggregation or a certain degree of association to occur. Protein Bonding The importance of the major types of bonds in main- taining the three-dimensional configuration of proteins was discussed in the Protein Structure section. The same types of bonds are also involved in the many phenomena or processes that characterize the behavior of proteins in food systems. Of these bonds, the disulfide (S-S) linkage 21 has the strongest stabilizing effect in protein conformation and is relatively common in many proteins. The sulfhydryl and disulfide are potentially the most reactive groups in proteins, though their availability may be hindered by the characteristic conformation of the protein. Nonetheless, a great deal of evidence indicates the sulfhydryl-disulfide interchange phenomena play a major role in proteins functionality. They are involved in aggregation of polypeptide chains to form a coagulum (Jensen et al., 1950), in the polymerization of actin and in the ATP-ase activity of myosin (Poglazov, 1966), in the pre- and post-rigor reactions important in muscle tenderi- zation (Gawronski et al., 1967), and in the formation of the gluten structure of bread dough (Wehrli and Pomeranz, 1969). One of the first indications of the participation of sulfhydryl groups in aggregation of denatured proteins stems from observations on the nature of coagulums formed by heated bovine plasma albumin (Jensen et al., 1950). Later, a chain-type mechanism for the exchange reaction under protein denaturation conditions was proposed by Jensen (1959). According to this investigator, the sul- hydryl group initiates a chain reaction with the disulfide groups as follows: RSSR + R's" = RSSR' + Rs" RSSR' + RHS' .~.___ R'SSR" + RS- 22 An important consideration in the proposed mechanism is that once a few sulfhydryl groups participate in the reaction, the polymerization or aggregation process may ensue without any further decrease in measurable sulfhy- dryl groups since, for every disulfide linkage formed, another mercaptide group is generated (Buttkus, 1974). Evidences of other types of chemical reactions in protein aggregation has been clearly shown in studies of gelatin, a protein with no sulfhydryl or disulfide groups (Harrington and Rao, 1970). Hydrogen bonds are primarily responsible for the gel structure and some hydrophobic and electrostatic interactions may be involved in cross-bonding. The gel structure can be easily disrupted by heating or by addition of 2M KCl solutions at pH 5 to 7. In extensively denatured proteins several types of bonds may occur. Buttkus (1974), in studies of aggregates of egg white and myosins, indicated that a combination of disulfide, hydrophobic, hydrogen and other types of bonds may be involved in protein-protein interactions. Functional Properties of Proteins Functional properties of proteins are defined as physicochemical properties that influence some performance aspect, especially in aqueous dispersion, affecting the characteristics of the product favorably (Hermansson and Akesson, 1975). 23 There are several functional characteristics or physicochemical properties desired in protein-containing products. Some of the broad categories are summarized in Table 6. These properties, singly or in combination, contribute to structural, foaming, binding, emulsifying, thickening, and gelling qualities in foods as well as to color, odor, flavor, and mouth-feel. The functional pro- perties discussed in this review are specific properties associated with foamability and coagulability. Viscosity Proteins absorb water and may swell in this process, thereby imparting thickening and an increase in viscosity of dispersions, slurries, or pastes (Kinsella, 1976).. Highly soluble, non-swelling proteins have low viscosity, whereas soluble proteins with swelling capacity exert high viscosities. This property is dependent on molecular size, shape and charge of the protein and presence of prosthetic groups, carbohydrates in particular (Kato and Sato, 1972). Surfactant Properties The lowering of water surface tension by proteins is primarily caused by absorption and orientation of the mole- cules at the surface (Peter and Bell, 1930) and this func- tion reflects the protein composition and conformation 24 Table 6. General Classes of Functional Properties of Proteins Important in Food Applications. (Kinsella, 1976). General Property Specific Functional Term Organoleptic Color, flavor, odor, texture, mouth-feel, etc. Hydration Solubility, dispersibility, water absorption, swelling, gelling, water holding capacity, synerisis, viscosity, etc. Surface Emulsification, foaming, lipid binding, flavor binding, stabili- zation, etc. Structural Elasticity, cohesion, chewiness, etc. Textural Aggregation, gelation, fiber formation, extrudability, dough formation, etc. Other Compatibility with additives, enzymatic, inertness, modifica- tion properties. (Adam, 1941). The tendency is to have the hydrophobic groups arranged on the surface of the liquid whereas the hydrOphylic groups are extended into the aqueous phase. Reduction of surface tension facilitates deformation of the liquid phase and, therefore, formation of new surfaces. Foaming Properties The ability of air incorporation exhibited by protein solutions depends on the formation of ully extended, semi-rigid, and coherent protein f'lm absorbed on the \ “I 25 air-solution interface (Thuman et al., 1949). Low surface tension appears to be necessary for readiness of foaming or for initiation of a foam, whereas high viscosity im- parts foam stability characteristics (Peter and Bell, 1930). These concepts have been accepted and further specula- tions on the mechanisms of foam formation essentially de- scribe the same phenomena with minor variations. In a description of food foams, Kinsella (1976) stated that food foams are dispersed air droplets in a liquid containing a surfactant. The surfactant lowers the surface tension of the liquid and facilitates deformation of the liquid. Proteins with excellent foaming power have the ability to surround air droplets and undergo a certain degree of denaturation followed by limited intermolecular attractions. Such protein-protein interactions enhance the cohesive nature of the film capsule, thereby imparting stability and elasticity to the membrane. Factors that influence foaming prOperties of proteins are all related to the effects they have on the inherent physicochemical characteristics of the protein. These factors include protein source, method of preparation, solubility, concentration, pH, temperature, salts, sugars, and lipids (Kinsella, 1976). The overall net charge on the molecule is affected by pH and it has been suggested that when there is a minimum net charge on the protein complex, which occurs at the isoelectric point, and at very high and low pH values 26 (Thuman et al., 1949), the rate of spreading (unfolding) of the protein molecule at the air-water interface is maxi- mum (Adam, 1941). In the alkaline range, cations improve spreading and on the acid side of the isoelectric point, anions are effective. Di and tri valent ions with least hydration capacity are the most effective and assistance to spreading may be due to salt formation between the ions in solution and the protein in the film. The effect of salts on foaming is highly dependent on concentration. Low levels may enhance solubility whereas salting out possibly occurs at higher concentrations. Therefore, varying results have been observed on whippabil- ity of soy protein (Watts, 1937; Eldridge et al., 1963), fish protein concentrate (Hermansson et al., 1972), egg foams (Sechler et al., 1959), and wheat protein (McDonald and Pence, 1961). Excessive whipping of protein solutions produces numerous smaller bubbles resulting in more unstable foams. This effect has been demonstrated by MacDonnell et a1. (1955) who reported that reduced angel food cake volumes were obtained when these cakes were prepared with over- whipped egg white. Decrease in bubble elasticity resulting from excessive insolubilization of proteins at the air- albumen interface was pointed out as a possible causative factor by these authors. Several proteins such as fish protein concentrate and soy isolates (Groninger and Miller, 1975: Eldridge et al., 27 1963), whey protein concentrates (DeVilbiss et al., 1974), sunflower isolate (Lin et al., 1974), and oilseed proteins (Lawhorn et al., 1972) exhibit the ability to incorporate air. Therefore, these proteins have potential food uses in aerated products. Gelation (Coagulation) Protein gels are composed of three-dimensional matrices of partially associated polypeptides with water held in the interstices (Kinsella, 1976). Ferri (1948) described gelation as.a two-stage process initiated by heat denaturation of the protein molecules into unfolded polypeptides and then association of the polypeptides form- ing the gel matrix. The interactions may involve hydrogen bonds, disulfide bonds, hydrophobic associations, or a combination of these (Catsimpoolas and Meyer, 1970). Typical protein gels are coagulated.egg white, soybean curd (tofu), casein curd (cheese), and gelatin jelly. Studies on gelation properties indicate most other proteins have the capacity to form gels upon heat treatment. These include soy proteins (Saio et al., 1974), fish protein (Hermansson and Akesson, 1974), leaf protein (Lu and Kinsel- 1a, 1972), sunflower, fababean, and field pea proteins (Fleming et al., 1975). Protein concentration, pH, salts, sugar, lipids, and temperature all affect firmness and characteristics of protein gels (Catsimpoolas and Meyer, 28 1970; Saio et al., 1974; Fleming et al., 1975; Lu and Kinsella, 1972). Albumen Proteins Functionality Studies concerning characterization of individual albumen proteins functionality reflect the possibility of determining which proteins are altered during processing. MacDonnell et a1. (1950) have demonstrated that shear stress forces caused by homogenization, and not pressure, are the factors responsible for damaging the whipping ability of egg white during spray drying. The high degree of surface formation, such as is found during atomization, has also been indicated as a causative factor (Bergquist and Stewart, 1952), as well as heat treatment (Clinger et al., 1951; Cunningham and Lineweaver, 1965: Hill et al., 1965: Garibaldi et al., 1968). Apparently, one of the proteins associated with the detrimental effects of physical treatments on whippability 'is ovomucin. Forsythe and Bergquist (1951) studied the ovomucin fraction in blended and homogenized egg white and observed that the decrease in whipping ability and in volume of angel food cake prepared with homogenized egg white appeared to be related to a decrease in ovomucin fibers length. In studies with pasteurized egg white, Garibaldi et a1. (1968) indicated that the heat induced formation of 29 ovomucin-lysozyme complex followed by denaturat1on and aggregation was associated with the longer whipping of the pasteurized stabilized egg white. Disappearance of ovo- macroglobulin was also observed with the treatment. Addition of whipping aids to pasteurized egg white and to spray-dried albumen are required to restore the foaming ability of these products. The anionic surface- active agents, such as sodium lauryl sulfate, are effective for dried albumen while cationic and nonionic agents do not improve foaming. On the other hand, sodium lauryl sulfate has limited effectiveness for pasteurized liquid and frozen products. For these products an organic ester, triethyl citrate, is commonly used, and bile salts and fatty acids salts have been reported to be effective as well (Bergquist, 1977). Mechanisms by which these whipping aids function are not known, but their variable effective- ness suggest the alterations caused by the different pro- cesses, may involve other proteins or other underlying effects. Foaminess Of the several studies conducted on foaming properties of individual albumen proteins, the work of MacDonnell et a1. (1955) is the only reported investigation utilizing a food system for testing. They isolated globulins, lyso- zyme, and ovomucin from egg white and through successive 30 tests in angel food cake, determined that the whipping ability of egg white depended on globulins and lysozyme, while the foam stability was associated with ovomucin. They also pointed out ovalbumin, easily coagulated with heat, as the protein responsible for supporting the structure of angel food cakes. Nakamura (1963) studied the spread surface mono- layer characteristics of several albumen proteins and ob- served that ovomucin, globulins, and conalbumin, which were easily spread (and denatured to a certain extent), have high foaming power, whereas lysozyme, ovomucoid, and ovalbumin resist spreading, and, therefore, exhibit low foaming characteristics. Later, Nakamura (1964) showed that changing ovalbumin conformation through denaturation with heat, alkali or acid, improved foaming power as much as three times over that of the native form. He also observed the increase in surface life time of the denatured protein monolayer. In investigations of egg albumen under repeated whip- ping conditions, Cunningham (1976) reported the retention of ovomucin, globulins, lysozyme, and to a lesser extent, of conalbumin in the foam of beaten egg white. Nakamura and Sato (1964), using a similar approach, observed the depen- dence of egg-white foam stability on the highly viscous ovomucin (B) fraction. Mechanisms of foam formation have been proposed. Proteins with good foaming ability are easily surface 31 'denatured and form a well developed network through peptide linkages. These linkages appear to be dependent on the presence of a high ratio of non-polar/polar side chains in the protein (Nakamura, 1963). Meyer and Potter (1975), in a study of whipped albumen with sodium hexametaphosphate and triethyl citrate plus trisodium citrate, pointed out the correlation between stable foams and denaturation of proteins at the air-albumen interface. They further suggested sodium hexametaphosphate and trisodium citrate increase foam stability possibly through cross-linking of the proteins and triethyl citrate improves foamability through ovalbumin denaturation. The effect of chemical modification of egg white on functional performance has been investigated. Egg white altered with 3, 3-dimethy1 glutaric anhydride exhibited no heat coagulation ability, but foaming capacity was not altered appreciably (Gandhi et al., 1968). Grunden et a1. (1974) treated egg white with several proteolytic enzymes and noted a considerable increase in foaming capacity with the treatments, but an adverse effect on foam stability. Gelation Although the gelation properties of isolated albumen proteins from the stand point of food applications have not been characterized, the effect of temperature on several proteins has been well documented. Most of these reported 32 studies have been undertaken for evaluation of factors influencing their biological activities. MacDonnell et a1. (1953) studied the virus antihemag- glutinin activity of ovomucin and noted the extreme stability of the protein to heating at 70 and 100°C for 30 and 60 minutes, respectively, and at pH values ranging from two to eleven. Ovomucoid is remarkably stable to heating at 100°C for 30 min., in acidic solution (Fredericq and Deustch, 1949). The antitryptic activity of this protein is de- stroyed with a heat treatment of 80°C for 30 min. at pH 9, but not at pH 7 and below (Lineweaver and Murray, 1947). Lysozyme has been shown to withstand heating at 100°C in acidic solution with little loss of lytic activity (Smolelis and Hartsell, 1952). However, on the alkaline side, the protein loses enzymatic activity or becomes insoluble (coagulates), with rates depending on pH and temperature (Sandow, 1926; Beychock and Warner, 1959). Conalbumin is very heat sensitive and denatures on heating at 61°C for 3-4 min. at pH 5.5-5.6, although not at pH 7.0 (Kline et al., 1953). Azari and Feeney (1958) showed that the iron-complex form was much more stable to heating at 64°C than the metal-free protein, and no precipitation occurred even after one hour of heating. Several studies have reported on the effect of heat on ovalbumin. Chick and Martin (1911, 1912) indicated 33 that the increase in rate of heat denaturation of ovalbumin was extremely high with relatively small increments in temperature. Lewis (1926) showed that ovalbumin is most stable to heating between pH 6.5 and 7.0 and found minimum denaturation even after 30 min. at 65°C. The gelation ability of denatured ovalbumin with urea was characterized by McKenzie et a1. (1963). They noted that with faster rate of unfolding and higher concentration of polypeptides, a finer gel network was formed. At pH 3 in 7M urea there was a rapid unfolding of the polypep- tide, but aggregation did not ensue. Cunningham and Lineweaver (1965), in studies of stability of egg white to pasteurization conditions, in- dicated that globulins G2 and G3 are fairly heat stable at 60°C in egg white adjusted to pH 6. Recently, Egelandsdal (1980) reported on the charac- teristics of ovalbumin gels obtained at various pH and ionic strength values. At low ionic strengths maximum gel rigidities occurred on each side of the isoelectric point of the protein. Increasing the ionic strength shifted the maxima away from the isoelectric point. The firmest gel was formed on the acid side which also correlated with a high net charge on the molecule. At very high net charges, on both alkaline and acid sides, rigidity was minimum. The author also suggested that ovalbumin gelling was primarily the effect of electrostatic attractions. EXPERIMENTAL PROCEDURE In order to investigate the foaming and gelling abilities of several egg albumen proteins and mixtures thereof, the experimental procedure was divided into five sections. The first section consisted of a selective fractionation of the egg white into six major proteins which constituted the variables in this study. The second sequence consisted of statistically planning the experimental design to minimize the number of observations for assessing the functional prOperties of the variables of interest. The third and fourth series described the testing of the proteins in an angel food cake to measure foamability and a model depicting custard gels for measure- ment of gelling properties, respectively. Lastly, selected samples were prepared for examination in transmission and scanning electron microscopes. Protein Fractionation All chemicals used in this section were ACS reagent grade. Proteins used as standards were ovalbumin (lot no. 105C-8022), lysozyme (lot no. 75C-8483), conalbumin (lot no. 46C-8125), globulins (lot no. 95C-8115), and ovomu- coid (lot no. 66C-8120), all obtained through Sigma 34 35 Chemical Company (St. Louis, Missouri). Source of Eggs Approximately one day old eggs from Single Comb White Leghorn hens, strain H & N, were purchased at a local farm. The hens were, on an average, 11 to 12 months old and were fed a ration containing a commercial pre- mix, corn and soy meals, and calcium salts. Eggs were brought back to the laboratory and separation of the white from the yolk proceeded immediately. Egg Albumen Preparation Eggs were thoroughly washed under cold running tap water, the shell broken, and the white separated from the yolk. Any chalaziferous material present in the whites was removed with a pair of tweezers. The whites were weighed and gently homogenized in a Osterizer blender, model 857-05JX at the slowest speed and lowest settings available for 30 to 45 seconds. The blender jar was filled two-thirds of its maximum capacity and a commercial food wrapping film was carefully layered on the surface of the whites and around the jar walls to eliminate air filled spaces and prevent incorporation of air during blending. Following homogenization, the albumen was treated as shown in Figure 2 to isolate the various proteins. Because of the lengthy procedure, and the considerably large quantity .msfimuonm sossnao mom mo cowuoHOmw on» mo Emummwp 30am .m .mflm >.¢ mm .unmum: unmasm as ooa\vommxvmzc endow .m as sassnamcoo 36 can» Imusumm «OmNAvmzv mom @ .k.v mm swaonam>o cog» :munumm eOmNAemzv mom @ .m.m so h.¢ mm .ucmumsuomsm He oOH\4Om~Aemzc eaaom .m as cassnamsoo soap Imusuom «Ommaemzv wom @ .b.v mm sflEanm>o sow» Imuoumm vomNAvmzv wom w .m.m mm msHHsQoHo Uofilo um mxoo3 N o .Huoz wm .m.m mm maaasnomwlllll 1||(1(|||||(|(||\|||||||wesNomsq muwnslmmo oouwsmmofiom »\\\\\\\\\\\\ :IlIIlIIIIIIIIIIIIIO coausflom Hos “a .Hmuo3 mmESHo> vumoufin3 mEDHo> a sfiosfio>o osoumoo .m.m mm .msouoomudua ofioooao>o 37 of proteins required, several batches of whites were pre- pared to solve the bulk handling problems and minimize the deterioration of samples. Referring to Figure 2, a single batch was divided into two equal portions for the preparation of globulins and lysozyme. The supernatants obtained from the preparations were subsequently treated to prepare the other proteins. Separate egg white batches were used for isolation of ovomucoid and ovomucin. Globulins This protein fraction was isolated using a modifi- cation of the procedure described by MacDonnel et al.(1955). To approximately 1500.0 9 of homogenized albumen an equal volume of a saturated ammonium sulfate solution was added and with constant stirring the pH of the resulting solution was adjusted to 8.5 with a 1N potassium hydroxide solution. After stirring proceeded for an additional 30 minutes, the solution was centrifuged in a Damon/IEC B-20A centrifuge, model 3444 at 10,000 rpm (15,000XG) for 5 minutes. The temperature of the centrifuge chamber was kept at 4°C throughout centrifugation. Following separation, the supernatant was saved for ovalbumin preparation, the precipitate was removed from the centrifuge bottles and redissolved in 600 m1 of deionized water. To this solution an equal volume of saturated ammonium sulfate solution was added and the pH readjusted to 8.5. After being kept at 2-4°C overnight, the precipitate was removed by 38 centrifugation as previously described. This procedure was repeated four times to ensure the removal of the other proteins. The supernatants from the first and second washings were stored at 2-4°C for further treatment to ob- tain conalbumin. After the final wash the protein was redissolved in 400 ml of deionized water and poured into a dialysis membrane of 3.6 cm of diameter, and a molecular‘ weight cut-off of 12,000 to 15,000. The ends were securely closed by tight knots and the tubing was placed in a large stainless steel bucket filled with deionized water. The dialysis set up was kept at 2-4°C and daily changes of the water was performed to accelerate the removal of the ammonium sulfate salt. The presence of the salt ions was monitored with barium chloride. To a small volume of the dialysis water a few drops of a barium chloride solution were added and the formation of an insoluble barium sulfate precipitate was taken as a qualitative measure of the presence of the salt ions. Dialysis con- tinued until the water was free from sulfate ions. At the end of this period the protein solution was again centrifuged to remove a large precipitate usually formed during dialysis. The clear supernatant containing the soluble globulins fraction was transferred to several nine inch. round aluminum pie pans and kept frozen at -23°C until further treatment. 39 Ovalbumin The procedure used for the preparation of ovalbumin was a modification of the method used by Sorensen and Hoyrup (1915-17). The supernatant obtained from the globulins preparation was adjusted to pH 4.7 with the gradual addition of 1N sulfuric acid solution and constant stirring. The resulting creamy white mixture was kept un- disturbed at 2-4°C overnight. The precipitated protein was then removed by centrifugation using the same con- ditions as described for globulins preparation. The supernatant was put aside for further treatment to obtain conalbumin and the precipitate was dissolved in 800 m1 of deionized water. An equal quantity of saturated ammo- nium sulfate solution was added, the mixture stirred, and kept for at least 15 hours at 2-4°C. Following this period, centrifugation was again performed, and the precipitated mass was redissolved in 800 m1 of deionized water. Wash- ings of the precipitated protein were repeated three additional times and then the protein fraction obtained was dissolved in 500 ml of deionized water and dialysed as previously described. The supernatants from the first two washings were saved for conalbumin preparation. The salt free protein solution was equally divided into round aluminum pie con- tainers, and stored at -23°C for further treatment. 4O Conalbumin This protein was prepared as the iron-free complex following the procedure outlined by Warner and Weber (1951) with several modifications. The supernatants ob- tained from the ovalbumin fractionation were combined and 10 grams of solid ammonium sulfate salt per 100 ml of supernatant were added. Following complete dissolution with constant stirring, the mixture was allowed to stay at room temperature for two hours, and then centrifuged at 10,000 rpm (15,000XG) for 5 minutes and at 4°C. The supernatants from globulins washing were adjusted to pH 4.7 with 1N sulfuric acid solution and then conalbumin was salted out with the addition of solid ammonium sulfate (10 grams/100 m1 supernatant). This mixture was left un- disturbed for two hours at room temperature followed by centrifugation at the end of this period. The precipi- tates obtained from both batches were combined, dissolved in 600 ml of deionized water and dialysed at 2-4°C against frequent changes of deionized water. After the majority of the salt ions were removed, usually after three days, the solution was centrifuged to separate a small precipitate formed during dialysis. The clear solution was then adjusted to pH 6.0 with 1N potassium hydroxide solution and made 0.02 M sodium chloride with a 1M sodium chloride solution. With constant monitoring of the solution pH, a 50% ethanol-0.02 M sodium chloride solution was gradually added to a final concentration of 41 20% ethanol-0.02 M sodium chloride. This solution was then kept at 2-4°C for two to five days to effect the crystallization of the protein. After a fairly large precipitate was formed, the mixture was centrifuged at 12,000 rpm (20,000XG) for eight minutes and at a tempera- ture of -10°C. The mixture was kept below 0°C at all times during centrifugation to prevent redissolution of the pro- tein and maximize its recovery. The supernatant was dis- carded and the material obtained was dissolved in 500 ml of deionized water. This solution was again made 20% ethanol-0.02M sodium chloride, pH 6.0, and the entire pro- cedure was performed 4 additional times to remove other proteins. After the final wash the protein solution was dialysed at 2-4°C against deionized water until the dialys- ate was free from chloride ions. The presence of these ions was detected with a silver nitrate solution. Following dialysis, the yellowish clear protein solu- tion was transferred to round aluminum pie pans and stored at -23°C. Lysozyme Lysozyme was obtained utilizing the crystallization method of Alderton and Fevold (1946). To approximately 1500.0 grams of homogenized egg white was added solid sodium chloride to a final concentration of 5%. After complete dissolution of the salt, the pH was adjusted to 9.5 with a 1N potassium hydroxide solution. A very small 42 amount of crystalline lysozyme was added without stirring, and the prepared albumen was left at 2-4°C for at least two weeks to effect the crystallization of the protein. During this period the pH was checked from time to time and readjusted to 9.5 when necessary. The crystalline material formed was then removed by centrifugation at 8,000 rpm (9,SOOXG) for five minutes and at a temperature of 4°C. The supernatant was further used for the preparation of globulins, ovalbumin, and conalbumin, as previously describ- ed, while the crystalline mass was dissolved in 500 ml of 0.1 M acetate buffer, pH 4.3. The insoluble material in the solution was removed by centrifugation at the conditions described above. The clear supernatant was again made 5% with respect to sodium chloride, the pH adjusted to 9.5, and placed in the refrigerator. Subsequent crystalli- zations usually occurred after 3-4 days at 2-4°C and were repeated three times. After the last crystalliza- tion, the material obtained was dissolved in 400 ml of the acetate buffer and dialysed against deionized water. The dialysis membrane used was a spectrapor membrane tubing with a diameter of 7.64 cm and a molecular weight cut off of 6,000-8,000. After removal of the salt ions, the protein solution was placed in round aluminum pie con- tainers and kept at -23°C until further treatment. Ovomucoid The procedure utilized for the fractionation of 43 ovomucoid was a slight modified method outlined by Lineweaver and Murray (1947). To portions of homogenized whites ranging from 1000.0 to 1500.0 grams were added an equal volume of a trichloroacetic acid-acetone solution or until a pH of 3.5 was attained. The trichloroacetic acid-acetone solution consisted of a mixture of lvolume of 0.5 M trichloroacetic acid and 2 volumes of acetone. The thick creamy white mass was centrifuged at 8,000 rpm (9,500 XG) for five minutes at 4°C, and the precipitate discarded. To the greenish-yellow clear supernatant were added approximately 2.5 volumes of acetone. The mixture was left undisturbed for approximately 15 minutes or until the precipitate settled. The clear upper solution was removed by suction with a glass tubing connected to a water pump. Three additional acetone washings were done, the residual acetone removed by centrifugation, and the precipitate dissolved in 500 ml of deionized water. The protein solution was adjusted to pH 4.5 with a 1N potas- sium hydroxide solution, and dialysed, using the same procedure as described for globulins, for approximately eight days. The protein solution was then placed in aluminum pie containers and kept frozen at -23°C. Ovomucin The procedure adopted for obtaining ovomucin has been described by Robinson and Monsey (1971). Since 44 drying or freezing adversely affect the solubility of this protein, fractionation was performed just prior to its use. A small-scale separation was done to test the adequacy of the purification procedure. The protein obtained in this preparation was stored at -23°C for further testing. To approximately 1000.0 grams of homogenized whites were added four volumes of deionized water and the resulting mix- ture was stirred for 15-20 minutes. Following this period the precipitate was removed by centrifugation at 13,000 rpm (21,000)(G) for five minutes at 4°C. The supernatant was discarded, the precipitated material dispersed in 2000 ml of deionized water, and centrifuged again. The protein mass was redispersed in 2000.0 ml of a 2% potassium chloride solution, the mixture stirred for 30 minutes and then centrifuged. Washings with the potassium chloride solution were repeated several times until the wash solution contained no measurable amount of proteins. The presence of these proteins was detected by measuring the absorbance of the wash solution on a Beckman spectrophotometer, model DB-G, at 290 nm. After the contaminating proteins were removed from the amorphous ovomucin mass, approximately 2000.0 m1 of deionized water were added, the mixture stirred for about 30 minutes and then centrifuged. After this stage it was very dif- ficult to separate the translucent ovomucin mass by centri- fugation; therefore the wash water was removed by filtering the mixture through 4 layers of cheese cloth. This procedure was re- peated until the wash water, monitored with a silver nitrate 45 solution, was free from chloride ions. The pH of the preparation was checked, adjusted to 8.0 with 1N potassium hydroxide, and moisture was determined to estimate the protein concentration. For preparing the cake containing only ovomucin, the excess moisture was removed under vacuum with frequent changes of phosphorus pentoxide at 2-4 °C 0 Freeze-Drying The frozen protein preparations were covered with perforated wrapping films and subsequently freeze-dried for 3-4 days in a Virtis Unitrap II freeze-drier with a capacity of eight liters, and equipped with a clear drum of dimensions 43.2 cm high x 30.5 cm diameter (17 x 12 in). The samples were dried at a system.pressure of 4 - 6 x 10.2 Torr and trays temperature of approximately 40-50°C. The dried materials were weighed and reduced to a fine powder by blending in a Osterizer blender, model 857-05 Jx, set at high speed, except for the glo- bulins fraction which was broken into small pieces. The proteins were placed in tightly covered glass jars and stored at -23°C. Removal of Iron from Conalbumin Small amounts of iron present in the conalbumin preparation were removed by treatment with a resin as 46 described by Warner and Weber (1951). Dowex-l, l x 8 - 50, chloride form, was recycled by suspending in a 10% sodium chloride, following by stirring for 15 minutes. The excess chloride ions were removed by several washes with deionized water. About 4 g of protein were dissolved in 200 ml of a 0.01 M potassium citrate buffer, pH 4.7, and approximately 10 g of the recycled resin were added to the resulting solution. After stirring for 30 minutes the mixture was filtered through filter paper no. 4, followed by three washes of the resin with approximately 10 m1 of citrate buffer. The filtrate was again treated with fresh resin two additional times and then dialysed and freeze-dried as previously described. The dried pro- tein mass was reduced to a fine powder and stored in securely closed glass jars at -23°C. Assessment of Purity-Purification Each individual batch of prepared protein was tested for purity by a disc-type polyacrylamide gel electro- phoresis (PAGE) and sodium dodecyl sulfate-polyacryla- mide gel electrophoresis (SDS-PAGE). If impurities were estimated to exceed 10%, the proteins were purified by employing the same washing or crystallization procedure used for their isolation. Relatively pure batches (over 90%) of the same protein were combined to form a common uniform lot for the functionality tests. Purifi- cation by gel filtration of small quantities of proteins 47 containing between 5-10% of contaminating proteins were performed for testing of the individual proteins func- tional properties. Electrophoresis. PAGE was performed using a modified pro- cedure described by Ornstein (1964), and Davis (1964), and the gels for SDS-PAGE were prepared according to the method outlined by Laemmli (1970) except that spacer gel was not used in both preparations. The electrophores- is apparatus was a Buchler Polyanalist, model 3-1750, connected to a Beckman Duostat power supply, model RD. Glass tubes of dimensions 10.0 cm long x 0.5 cm I.D. x 0.8 cm O.D. were carefully cleaned by immersing in a chromerge solution for 24 hours, followed by rinsing with cohi tap water and distilled water. A final rinse with a photoflo solution (1:200) was done, the tubes drained and allowed to dry. For PAGE the stock gel consisted of a 7.5% poly- acrylamide (cyanogum 41) solution in a 0.38 M tris-hydro- chloric acid buffer, pH 8.91, the running buffer a 0.055 M tris-0.767 M glycine buffer, pH 8.3, and the sample buffer a 0.062 M tris-hydrochloric acid buffer, pH 6.7. The stock gel and sample buffer were kept refrigerated between uses, and the chamber buffer was freshly prepared. To 28 ml of stock gel were added 35 ul of N, N, N’, N'-tetra methylethylenediamine (TEMED) and 130 ul of a 5% ammonium persulfate solution, freshly made. The solution 48 was swirled to mix the chemicals and immediately poured with a syringe filling the prepared tubes to about 1.5 cm from the top. Water was then carefully layered on top of the gel solution to exclude oxygen and allow for polymerization to take place (usually completed after 20 to 30 minutes). The samples were prepared by dissolving approximately 1 mg of protein in 1 ml of the sample buffer contining 0.2 m1 of a saturated sucrose solution and 0.01 ml of a bromophenol blue solution. Approximately 20 to 50 ug of each protein were layered on top of the gels, and the upper and lower reservoirs were filled with the chamber buffer. Electrophoresis was run at l mA/tube for 1 hour and then at 2 mA/tube until the tracking dye was at the bottom of the tube. The gels were removed from the tubes by squeezing water between the gel and the tube wall with a microsyringe connected to a tap water supply. Gels were stained with a 1% amido black, 50% methanol, 10% acetic acid solution for five minutes and destained by diffusion in a 7% acetic acid solution. For SDS-PAGE, stock solutions consisted of a stock gel prepared with a 37.5:l:150 mixture of acrylamide, N,N’-methylenebisacrylamide (Bis) and water, on a weight basis, a 1.5M tris-hydrochloric acid buffer, pH 8.8, a 0.2M ethylenedinitrilo tetraacetic acid disodium salt (EDTA) solution and a 10% sodium dodecyl sulfate (SDS) solution. The first three solutions were kept refri- gerated while the SDS solution was stored at room 49 temperature. The sample buffer was a 1% sodium dodecyl sul- fate, 0.05 M tris-hydrochloric acid, pH 6.72, 0.05% mer- captoethanol, 0.001% bromophenol blue, containing 10 ml of a saturated sucrose solution. This buffer was stored in a plastic bottle in the freezer. For the gel preparation, to 10 ml of the stock gel were added 3 ml of the tris-HCl buffer, 0.3 ml each of the SDS and EDTA solutions, 0.015 ml of TEMED, 11.3 ml of water and 0.6 m1 of a 5% ammonium persulfate solution, freshly prepared. The solution was immediately poured with a syringe, filling the tubes to 1.5 cm from the top. Oxygen was excluded by layering n-butanol on top of the gel solution and polymerization was allowed to take place. After the gel had hardened the butanol was removed by several rinses with distilled water and then with the chamber buffer. This buffer con- sisted of a 0.05 M tris - 0.38 M glycine solution, pH 8.41, and containing 10 ml each of 10% SDS and 0.2 M EDTA solutions. Samples were prepared by dissolving from 0.9 to 1.3 mg of each protein in 5 to 10 m1 of the sample buffer and incubating the resulting solution in a 40°C water bath for one hour. From 1 to 6 ug of the cooled samples were layered on top of the gels, the upper and lower reservoirs filled with the running buffer and electrophoresis was run at l mA/tube for 3-4 hours or un- til the tracking dye reached the bottom of the gel. The gels were removed using the previously described technique, and stained overnight with a 0.03% coomassie brilliant 50 blue (R-250), 50% methanol, 7% acetic acid solution. The gels were destained by diffusion with frequent changes of a 5% methanol - 7.5% acetic acid solution. The purity of each final common lot (all batches of a single protein combined) was determined quantita- tively with the SDS-PAGE by measuring the absorbance of the protein bands in a densitometer at 550 nm. The instru- ment consisted of a Beckman DU spectrophotometer, model 2400, coupled to a Gilford gel scanner, model 2520, and a Hewlett Packard integrator, model 33805. Gels were scanned at a rate of 2 cm/min while the chart was set to a speed of l cm/min. Purification. Conalbumin, ovomucoid, and globulins were purified by gel filtration with Sephadex G-75-120. The resin was equilibrated with a 0.1M sodium acetate - 0.1M sodium chloride buffer, pH 8.0, for three days at 2-4°C and then poured into a glass column of dimensions 3.5 cm I.D. x 140 cm long, equipped with a glass stopcock. The column was allowed to stabilize for a week by constantly running the acetate buffer through it. At this point the resin had settled to a constant height of 112 cm. About 1.5 g of protein were dissolved in 5 ml of the acetate buffer containing 1 ml of a concentrated blue dextran solution (5 mg/ml) and 2 ml of a saturated sucrose solution. The prepared sample was carefully layered on the surface of the resin through the buffer, the buffer 51 tank was connected to the top of the column, and then separation was performed at a flow rate of 0.5 - 0.7 ml/min. Soon after blue dextran was eluted from the column,collec- tion started with a GM chromatography automatic collector. Fraction size collected was 8 ml/tube and the protein con- centration was monitored at 280 nm on a Beckman DB-G spectrophotometer. Chemical Analyses Duplicate determinations were performed for all chemical tests. Moisture Analysis Moisture content determination was carried out in a Hotpack vacuum oven, model 633, at 100°C and a vacuum of 660-711 Torr (26-28 in. Hg) following the official AOAC method 1007 (1975) except that approximately 2.0 g of liquid sample were used. Samples were dried to a constant weight and the dried weight was expressed as the percen- tage of total solids. Total Nitrogen A micro-Kjeldahl method described by McKenzie (1970) was used to determine total nitrogen. Approximately 30 mg of dry sample were digested with 2.5 m1 of concentrated 52 sulfuric acid, 1.5 g of potassium sulfate and 0.5 m1 of a 0.46 M mercuric sulfate in 2M sulfuric acid solution. Following digestion the samples were cooled, diluted with about 20 ml of ammonia-free water, and transferred to a micro-Kjeldahl distillation apparatus. To the digest were added 10 ml of a 12.5 M sodium hydroxide - 0.13 M sodium thiosulfate solution and collection of the steam distillate started soon after immersing the condenser tip into 5 m1 of a boric acid indicator solution placed in a 50 ml beaker. This solution was prepared by combining 20 g of boric acid and 6.67 mg of methylene blue, dissolved in 800 m1 of water, with 13.3 mg of methyl red dissolved in 10 m1 of ethyl alcohol, and completing the volume to one liter. After the distillate-indicator solution reached the 40 m1 mark, the beaker was lowered from the condenser tip and distillation proceeded until about 5 ml more of distillate were collected. The tip was rinsed with a few ml of distilled water, the solution quantitatively trans- ferred to a 125 ml erlenmeyer flask and titrated with a 0.02N hydrochloric acid solution to a grey-lilac end point. Recoveries of nitrogen were determined with d1 - try- ptophan dried over phosphorus pentoxide for three days. Blanks were run along with the samples and the percentage of nitrogen was calculated with the formula: (sample-b1ank)m1 HCl x normality(HC1) x 14.007 x 100 %N = mg sample 53 After adjusting the values for the percentage re- covery the protein content was calculated by multiplying the corrected nitrogen values by the factor 6.25. Sulfhydryl Groups A modification of the method described by Ellman (1959) was used to determine "free" sulfhydryl groups. About 3 to 5 mg of sample were dissolved in 1 ml of distilled water, then 4 m1 of a 0.01 M sodium phosphate buffer, pH 8.0 - 1% sodium lauryl sulfate - 0.4% EDTA solution were added. The samples were boiled for 30 minutes, allowed to cool, and then 0.2 ml of a 0.01M 5, 5' dithiobis - 2-nitrobenzoic acid (DTNB) solution in 0.1M sodium phosphate buffer, pH 7.0, was added. The color was allowed to develop for one hour and then the absorbance of the solution was measured at 412 nm in a Beckman DB-G spectrophotometer. Blank determinations were run parallel to the tests and concentration of the sulfyhdryl groups was calculated using a extinction coefficient of 13,600 with the formula: (sample-blank)absorbance x final volume moles SH/g protein = 13,600 x mg sample The procedure described above was used for all samples except that for the gel samples a few modifications were introduced. The samples were dissolved in 5 m1 of the buffer solution containing 2% SDS. The concentration of 54 this chemical was raised to prevent reassociation of the polypeptide chains upon cooling. Solutions that were still cloudy were filtered through Whatman filter paper no. 4 before absorbance measurement. Total Sulfhydryl - Disulfide Groups The number of disulfide groups was determined by assessing the total sulfhydryl content according to the method of Cavallini et a1. (1966). To approximately 1.0 mg of sample were added 1 m1 of a 0.05 M sodium phosphate - 0.001 M EDTA buffer, pH 7.4, 0.5 m1 of l-octanol, and 1 ml of a freshly prepared 40:1:40 urea: sodium boro- hydride:water solution. The resulting solution was in- cubated in a water bath at 40°C for 30 minutes. Samples were cooled and 0.5 ml of a 1 M potassium phosphate - hydrochloric acid buffer, pH 2.7, was added drop wise to prevent excessive foaming. After five minutes,1 m1 of acetone was added and the tubes shaken to complete boro- hydride destruction. To this solution, 0.2 m1 of a 0.01 M DTNB solution in 0.1 M sodium phosphate buffer, pH 7.0, was added and color was allowed to develop for one hour. Before measuring the absorbance at 412 nm the solutions were diluted with 5-8 ml of deionized water, and filtered through filter paper no. 4 to remove l-octanol. Blank tests were run with the samples and the total sulfhydryl content, expressed as moles of SH/g of protein, was calculated using the formula for the "free" sulfhydryl determination. For 55 proteins of known molecular wieght, the number of sulf- hydryl groups was calculated with the equation: M.W. x A x V N = where MW = Molecular Weight 12’000 x m A = Absorbance V = final Volume m = sample weight in mg The disulfide content was estimated from the difference of total sulfhydryl and "free" sulfhydryl contents. Elemental Analyses Trace elements were determined at the Ohio Agricul- tural Research and Development Center (Wooster, Ohio), using a inductivehr coupled plasma - optical emission spectro- scopy technique as described by Fassel and Kniseley (1974). Samples were prepared according to Kenworthy (1960). Chlorine and sulfur content were determined by a private food testing laboratory (Micro-Tech Laboratories, Inc., Skokie, IL) using a combustion and titration techni- que. For chlorine, titration was done with a silver perchlorate solution with dichlorofluorescein as indicator, and for sulfur the samples were titrated with a barium perchlorate solution using dimethylsulfonazo-III as indicator. The concentration of the minerals present in dried egg-white and the various isolated proteins is shown in Table 7. These values were used to calculate the mineral content of the protein mixtures to be used in the 56 .Hmusou ucwfimon>mo .manmuomuoo won I o meHOHMHOQMn—H SowBIOHUH—z I Q pom noummmmm Housuasownmd owno I m 0¢.HH 05.0 om.0 0m.0 mh.m 00.0 «0.0 homaom 0oz 0H.0 00.0 002 mm.o 0m.m ov.mH commodnu AS IS a H0.0v Ho.0v H0.0v H0.0v H0.0v Ho.0v H0.ov Esflumm H0.0v H0.0v Ho.0v H0.0v H0.0v H0.ov H00v Eswucouum Ho.ov Ho.0v H0.ov H0.0 H0.0v mH.o H0.0v EscHEst H0.0 H0.0 Ho.0 Ho.0 Ho.o mo.0 Ho.ov osfim H0.0v Ho.0v H0.ov Ho.0v H0.ov 00.0 Ho.ov Hmmmoo Ho.ov H0.0v Ho.ov H0.0v Ho.0 no.0 H0.0v sonom H0.o no.0 H0.0 no.0 Ho.ov m0.0 H0.0v couH H0.0v H0.ov Ho.ov Ho.0v H0.0v a0.0v Ho.ov ommsoocmz o0.0 NH.0 0H.o no.0 H0.0v HH.0 no.0 Eowmosmmz hh.o Hm.0 50.0 mm.o H0.ov NN.0 mH.H mononmmonm 00.0 0H.o oH.0 NH.0 0H.0 50.0 00.0 Eofioom om.0 00.0 o>.0 0H.0 no.0 «v.~ 0H.H Esfloaou 00.H 00.~ vv.q HH.H mh.a ~0.ma H0.HH Esfimmouom moommo m\mE sHEsn .Am>o sflfisnamsoo owoosEo>o msflHsnoHU mahuoqu swoseo>o oufln3Immm Hammad: mcflmuoum oouoaomH Hono>om one ouwnzImmm omega mo sofiufimomfioo anyone: .5 magma 57 functionality tests. Potassium, calcium, sodium, and chlorine were replaced to their original values (levels found in egg-white) with the addition of sodium chloride, calcium chloride, and potassium hydroxide, all in 1N solutions. Experimental Design The extreme vertices design for experiments with mixtures described by McLean and Anderson (1966) was used to determine the levels of the various proteins in the formulation of the protein solutions to be used in the angel food cake system. Selection of this design was based on its unique feature of maintaining the sum of the mixture components at a constant value (i.e., .t Xi = 1 or 100%) while allowing for variation of the compghint levels within specific ranges, or constraints, chosen by the experimenter. The concentration in albumen of the six proteins of interest are listed in Table 8 (Powrie, 1977). These values were adjusted by a factor to 100% to account for the other minor albumen proteins not used in the mix- tures. In the same table are listed the minimum and maximum levels within which each protein was allowed to vary. The maximum value was set at approximately 1.5 times the normal adjusted levels of each protein. The set of treatment combinations found (protein formulations) described a geometrical region with five dimensional faces 58 Table 8. Proximate Protein Composition of Egg Albumen and Level Range Values of Each Protein Protein 3:13:83 fighiiifd MinimfiingeMaximum % % % Ovomucin 1.50 1.65 0.00 2.50 Lysozyme 3.50 3.80 0.00 6.00 Globulins 8.00 8.80 '3.00 13.00 Ovomucoid 11.00 12.10 5.00 18.00 Conalbumin 13.00 14.30 6.00 21.00 Ovalbumin 54.00 59.30 30.00 89.00 a - Powrie, 1977. b - normal values adjusted to 100%. c - constraints placed on each protein. (K-l) with the formulations located at the vertices and centroids of this region. To reduce the number of observa- tions only the centroids of the third and fifth dimensional faces were used. The procedure to find the vertices were to first construct all treatment combinations (K.2K-l) with the minimum and maximum levels and leaving one factor's level in blank, e.g. x1, X2, --, X4, X5, X6. Next, the blanks were filled with admissible values for each protein (within the constraints) to make the sum of the levels equal to 100% (or unity). This procedure yielded a total 59 of 32 vertices. The centroids of the 3-dimensional face were found by averaging the four remaining factor levels of the vertices with any two constant factor levels. The 5-dimensiona1 face centroid was obtained by averaging the levels of all the treatments found. Table 9 displays the vertices and centroids of the design used to prepare the cakes, and in the appendix the steps and calculations involved in developing the design are shown in more detail. For testing each factor separately, additional treatments with 100% of each protein were performed, and a control cake was prepared with the adjusted levels of each protein listed in Table 8. For the model system representing a custard type gel each factor was allowed to vary from 0 to 100% and tested individually and in combination of two or five proteins. The control gel was prepared with the adjusted levels of the proteins. Table 10 lists the treatments used for preparing the gels. Angel Food Cake System The formulation and procedure used to prepare the cakes were adapted from Dam et a1. (1970). The quantities of the ingredients were reduced to 50% of their basic recipe and consisted of 30.0 g of protein solution, 30.5 of sugar, 11.0 g of cake flour, and 0.15 g of solid sodium chloride. Variable quantities of cream of tartar 6O 00.00 00.0 00.0 00.0 00.0 00.0 mm 00.0w 00.Hm 00.0H 00.0H 00.0 00.0 «m 00.00 00.0 00.0H 00.0H 00.0 00.~ mm 00.00 00.H~ 00.0 00.0H 00.0 00.0 mm 00.00 00.0 00.0 00.0H 00.0 00.0 am 00.00 00.H~ 00.0H 00.0 00.0 o0.m 0N 00.00 00.0 00.0H 00.0 00.0 00.0 0H 00.00 00.HN 00.0 00.0 00.0 00.N 0H 00.00 00.0 00.0 00.0 00.0 00.m 0H 00.~v 00.H~ 00.0H 00.0H 00.0 00.0 0H 00.00 00.0 00.0H 00.0H 00.0 00.0 0H 00.00 00.HN 00.0 00.0H 00.0 00.0 va 00.00 00.0 00.0 00.0H 00.0 00.0 0H 00.00 00.Hm 00.0H 00.0 00.0 00.0 NH 00.00 00.0 00.0H 00.0 00.0 00.0 Ha 00.00 00.H~ 00.0 00.0 00.0 00.0 0H 00.00 00.0 00.0 00.0 00.0 00.0 0 00.00 00.H~ 00.0H 00.0H 00.0 00.0 0 00.00 00.0 00.0H 00.0H 00.0 00.0 h 00.H0 00.Hm 00.0 00.0H 00.0 00.0 0 00.00 00.0 00.0 00.0H 00.0 00.0 0 00.00 00.Hm 00.0H 00.0 00.0 00.0 0 00.00 00.0 00.0H 00.0 00.0 00.0 m 00.Hh 00.HN 00.0 00.0 00.0 00.0 m 00.00 00.0 00.0 00.0 00.0 00.0 H moofiuum> w sfifisnao>o afiesnaosou owoosfio>o msmaonoHu mE>NOmmA swosfio>o HoQEsz camuoum ucmEummHB .moxmu ooom Homn< mo GOwuoummoum mow now owns mswououm xwm mo macauosflofioo mHo>mA usoEuomuB .0 manma 61 00.00 00.0H 00.HH 00.0 00.0 0~.H 00 00.00 00.0 00.HH 00.0 00.0 00.~ 00 00.00 00.H~ 00.0H 00.0 00.0 00.0 00 00.H> 00.0 o0.HH 00.0 00.0 00.0 00 00.00 00.0H 00.0H 00.0 00.0 00.~ vv 00.00 00.0H 00.0 00.0 00.0 00.~ 00 00.00 00.0H 00.0H 00.0 00.0 00.0 m0 00.0w 00.0H 00.0 00.0 00.0 00.0 H0 00.00 00.0H 00.HH 00.0H 00.0 00.~ 00 00.00 00.0H 00.HH 00.0 00.0 00.~ 00 00.00 00.0H 00.HH 00.0H 00.0 00.0 00 00.00 00.0H 00.HH 00.0 00.0 00.0 00 00.00 00.0H 00.HH 00.0 00.0 00.~ 00 00.00 00.0H 00.HH 00.0 00.0 00.0 00 00.H0 00.0H 00.HH 00.0 00.0 00.0 00 00.00 00.0H 00.HH 00.0 00.0 00.0 mm mofiouucou 00.00 00.Hm 00.0H 00.0H 00.0 00.~ «0 00.00 00.0 00.0H 00.00 00.0 00.N H0 00.~0 00.Hm 00.0 00.0H 00.0 00.m 00 00.00 00.0 00.0 00.0H 00.0 00.~ am 00.00 00.H~ 00.0H 00.0 00.0 00.0 on 00.00 00.0 00.0H 00.0 00.0 00.0 mm 00.~0 00.H~ 00.0 00.0 00.0 00.0 00 moofluuo> m cfladnam>o cassnaocou ofloooeo>o mcwasnoHU oE>Nommq afloofio>o Hmoasz :wmuoum vamEummue GODGHUGOU m magma 62 00.00 00.0H 00.HH 00.0 00.0 0N.H 00 0N.00 00.0 00.HH 00.0 00.0 0N.H 00 0~.00 00.HN 00.HH 00.0 00.0 0~.H 00 00.00 00.0 00.HH 00.0 00.0 0m.a 00 00.00 00.0H 00.0H 00.0 00.0 0N.H 00 0~.00 00.0H 00.0 00.0 00.0 0N.H v0 0~.00 00.0H 00.0H 00.0 00.0 00.H 00 0~.~h 00.0H 00.0 00.0 00.0 0~.H am 00.00 00.0H 00.HH 00.0H 00.0 0N.H H0 05.00 00.0H 00.HH 00.0 00.0 0N.H 00 00.00 00.0H 00.HH 00.0H 00.0 0~.H 00 mofiouusmu w GHESQHM>O swasnamsou ofloosEo>o moflflsnoHU mahuoqu swosao>o Honfioz samuoum usofiumoua omscaucoo a magma 63 00.00 00.0H 0H.~H 00.0 00.0 00.H Houusoo 00.0H 00.0H 00.0H 00.0H 00.0H 00.0H mm 00.00 00.00 00.0N 00.0w 00.00 00.0 mm 00.0w 00.00 00.00 00.0w 00.0 00.00 am 00.0w 00.0w 00.0w 00.0 00.0w 00.0w 0N 00.00 00.00 00.0 00.00 00.0w 00.00 0H 00.00 00.0 00.00 00.00 00.0w 00.0w 0H 00.0 00.00 00.0w 00.0m 00.00 00.00 ha 00.00 00.00 00.0 00.0 00.0 00.0 0H 00.00 00.0 00.00 00.0 00.0 00.0 0H 00.0 00.00 00.00 00.0 00.0 00.0 «H 00.00 00.0 00.0 00.00 00.0 00.0 0H 00.0 00.00 00.0 00.00 00.0 00.0 NH 00.0 00.0 00.00 00.00 00.0 00.0 Ha 00.00 00.0 00.0 00.0 00.00 00.0 0H 00.0 00.00 00.0 00.0 00.00 00.0 0 00.0 00.0 00.00 00.0 00.00 00.0 0 00.0 00.0 00.0 00.00 00.00 00.0 h 00.00H 00.0 00.0 00.0 00.0 00.0 0 00.0 00.00H 00.0 00.0 00.0 00.0 0 00.0 00.0 00.00H 00.0 00.0 00.0 0 00.0 00.0 00.0 00.00H 00.0 00.0 m 00.0 00.0 00.0 00.0 00.00H 00.0 m 00.0 00.0 00.0 00.0 00.0 00.00H H 0 sfladnao>o swasnamcou oaoosao>o mfiwwonoHo mewuomhq swooso>o :fiououm .ucoEuooHB .maoo wows onmumsu mo coflumummmnm man now pom: mcfiououm x00 00 mcoflumsflnaoo mam>on usoEuomne .0H magma 64 were added to the protein solution to adjust the pH to 5.7. The ionic strength of these solutions ranged from 0.204 to 0.253. One cake was prepared per treatment. Protein Solution Preparation A total of 3.65 g of protein was used to prepare the equivalent of 35.0 g of egg white (10.4% protein, liquid basis). Sodium chloride, calcium chloride, and potassium hydroxide, all in 1N solutions were added to replace the ions normally present in egg albumen. For preparing the solutions, certain precautions had to be taken to prevent precipitation or complexing of lysozyme with conalbumin and/or ovomucin. For mixtures containing no ovomucin, the insolubilization problem was solved by dissolving the weighed globulins, ovomucoid, conalbumin, ovalbumin, and lysozyme, one at a time, in 10 ml of deionized water containing the sodium chloride and calcium chloride solutions, as well as the solid sodium chloride. Lastly, additional water with the potassium hydroxide solution was added to complete the weight to 35.0 9. For solutions with the maximum level of ovomucin, 50-60% of the alkali and the salt solutions were added to the weighed ovomucin mass contained in a 50 m1 erlenmeyer flask. After thorough mixing, globulins, ovomucoid, conalbumin, ovalbumin, and solid sodium chloride were 65 added one at a time, and carefully dissolved. Finally, to this resulting solution was added the previously dissolved lysozyme in the remaining water and potassium hydroxide solution. For mixtures with medium ovomucin content, globu- lins, ovomucoid, conalbumin, ovalbumin, and lysozyme were dissolved in 10 m1 of deionized water with the salt solu- tions and solid sodium chloride. After complete mixing, the previously prepared ovomucin-alkali mixture was slowly poured into the protein solution and the weight completed to 35.0 g with additional water. Following preparation, the viscosity and surface tension of the protein solutions were determined. The viscosity was measured on a Nametre Direct Readout Vis- cometer, model 7.006, at a temperature of 25 t 0.01°C, maintained by a Neslab Exacal circulating water bath, model Ex 100. The surface tension determination was performed on a Fisher Surface Tensiometer, model 20. The container for the protein solution was a crystallizing dish of 4.5 cm I.D. and surface tension forces were measured with a platinum-iridium ring of 6.0 cm of circumference. After these determinations, the pH of the protein solutions was adjusted to 5.7 by the addition of solid potassium acid tartrate (cream of tartar), the flasks covered with food wrapping film, and stored overnight in the refrigerator. 66 Cake Preparation A Kitchen Aid mixer, model K5-A, connected to a Gralab Universal Timer, model 171, was used to prepare the cakes. After the protein solution reached room temperature, 30.0 g were weighed into the mixer bowl, and whipped at speed 10 (210 rpm) until an optimum foam stage for the particular so- lution was attained. At this point, a portion of the foam was placed in a crystallizing dish of dimensions 4 . 5 cm I.D. x 3. 5 cm height, leveled with a spatula, and weighed to the nearest 0.01 g for foam specific volume determination. The foam was placed back into the mixer bowl and rewhipped for two to five seconds. One third of the sugar was sprinkled over the foam and mixed by whipping at speed 6 (145 rpm) for four seconds. The re- maining two portions of sugar were incorporated into the foam in the same manner, after which the sides of the bowl were scraped. The flour-sugar mixture (one fourth of the sugar sifted with the flour twice) was then folded into the foam in four additions. Each portion was sifted over the meringue and gently folded into it using 10 strokes of a stainless-steel spatula. A 55 g portion of the batter was weighed into a tared, un- greased mini loaf pan of dimensions 9 x 5 cm at the base, 5.5 cm height, and 12 x 7.5 cm at the top. The cake was baked at 175°C (350°F) for 25 minutes in a National Reel type test baking oven. After baking the cake, it was allowed to cool, in an inverted position, on cooling racks. Before addition of sugar, a one level teaspoon of 67 foam was removed, placed in a vial, and immediately frozen in dry ice. The remaining unused portion of the protein solution was also placed in vials and stored at -23°C along with the foams. These samples were further freeze- dried for sulfhydryl determinations. Volume Determination The top of the cooled cake was dusted with flour and the volume determined by rape-seed displacement before removing the cake from the pan. The seeds were placed over the cake, filling the pan to the top. The surface was leveled with a spatula and the remaining seeds were poured into a collecting pan. The volume of these seeds was determined by pouring into a 100 m1 graduated cylinder from a constant height. Cake volume was expressed in cm3 and calculated from the difference between the total pan volume (determined with rape-seeds) and the volume of the seeds. After volume determination, the cakes were removed from the pans, wrapped in plastic film, and stored at -23°C until further determinations. Tenderness Determination The slices for tenderness testing were prepared while the cakes were still frozen to prevent collapse of 68 the fragile structure. Cakes were cut vertically through the center; one-half was wrapped again and stored at -23°C, and from the other half a 1.3 cm (0.5 in.) thick section was cut horizontally from the bottom. Each slice was wrapped in plastic film and allowed to defrost at room temperature. The slices were then unwrapped, weighed to the nearest 0.01 g and sheared with a multiple blade com- pression cell in an Allo-Kramer shear press, model SP12. Measurements were performed with either the 100 lb or 3,000 lb maximum load transducer connected to the Food Technology Corporation Texturecorder, model TR 3. The areas of the curves outlined were calculated by triangulation and tenderness was expressed as the work, in lb x cm, required to shear through 1 g of sample. Compressibility Determination The unused upper portion of the cake prepared for tenderness determination was shaped by cutting with a 5.2 cm I.D. circular mold, wrapped, and allowed to defrost. After the slices reached room temperature, the minimum and maximum heights of each slice were measured with a caliper, and then compressed to 0.8 cm in an Allo-Kramer shear press, model SP12, equipped with a Food Technology Inc. Texturecorder, model TR3. For most determinations, the 100 1b maximum load transducer was used at a recorder range of 100, and a few samples required the use of the 69 3000 lb maximum load transducer at recorder ranges of either 10 or 33. The areas of the curves obtained were measured with a Keuffel and Esser Compensating Polar Planimeter. The average height of the slices was used for the calculations and compressibility was expressed as the work, in lb x cm, required to compress 1 cm of the sample. Foaming Index Determination Foaming index values were calculated by dividing the foam specific volume, in cm3/g, by the whip time, in minutes, of each protein solution. This ratio compensated for the variations in specific gravity and whip time and allowed for an easier comparison among samples. These values also correlated directly with foamability, e.g., larger numbers represented better air incorporation. Texture Photographs were used to show the textural charac- teristics of selected samples. Custard Model System To prevent any possible interactions among milk, yolk, and albumen proteins, as well as visual interferences at the ultrastructural level, yolk and milk were eliminated 70 from the system. Additional egg white proteins were used to replace the yolk proteins and a salt solution with a similar ionic strength (0.165), substituted for milk. This solution was prepared according to the milk mineral com- position listed in the Michigan State University Nutrient Data Bank (1978). The salts used were calcium, sodium, magnesium, potassium, and ferric, all in the chloride form. Duplicate gels were prepared per treatment. The formula used to prepare the gels consisted of a 1:4 mixture of protein solution:sa1t solution, resulting in a 1.27% protein concentration in the final mixture. A total of 0.78 g of proteins was dissolved in deionized water with added sodium and calcium chloride solutions to reconstitute the equivalent of 6.0 g of whole egg (13% protein, liquid basis). To this mixture was added the salt solution replacing milk, and with constant stirring, the pH was adjusted to 8.0 with 1N potassium hydroxide solution. The resulting solution was transferred to a 50 ml beaker which had been previously sprayed with sili- cone to prevent sticking of the gel to the container walls. The beaker was covered with a double layer of (R) wrapping film, and placed in a pan of dimensions Saran 10 x 10 x 4.5 cm, filled with an 8% sodium chloride solu- tion. This salt solution was used as the bath medium to prevent excessive evaporation of the water during heating. The beaker was held in place by positioning it in between two thick wires running across the top and around the 71 sides of the pan, and a flat piece of porcelain was placed under the container to prevent direct contact with the hot pan. The heating system consisted of two Corning Hot Plate Stirrer units, model PC-351, each connected to a Staco Variable Autotransformer, type 2PF 1010, set to 80. The water bath set-up containing the sample, was placed on the previously heated hot plates and a uniform tempera- ture throughout the liquid was ensured by constant agitation with two magnetic stirrers. To monitor the temperature of the protein solution and the water bath, a thermocouple was securely positioned into the protein solution to a depth of 0.5 cm from the surface, and another into the water bath. Temperature recording immediately started using a Honeywell Brown Electronik Potentiometer, model 153 x 65 - P12H - II - III - 81 - A8, equipped with 12 thermocouple terminals. The terminals were connected in parallel into two sets with the same type of wire used for the thermocouples to shorten the elapsed time between each temperature recording to 15 second intervals. For attainment of similar heating rates of the different protein solutions, particularly during coagula- tion, the hot plate's controls were set as displayed in Table 11. The settings were changed at water bath temperatures of 65°C (intermediate setting) and 79-80°C (final setting). At these conditions, the heating rate during coagulation of the various protein solutions, was approximately 0.74°C/min. For the mixtures the control 72 settings varied accordingly with the proteins present. After the end-point temperature of the gel was attained, it was removed from the water bath, covered with a plastic film and stored at 2-4°C for further determina- tions. Gel Strength Determination— Gel strength was determined the day following gel preparation using an Instron Universal testing instrument, floor model TT-BM. An Instron tension load cell, model D30-36 was adapted for the measurements since a compression load cell of equivalent sensitivity was not available. Table 11. Hot Plate Control Settings Used to Prepare the Custard Type Gels. _Setting Initial Intermediate Final Protein Solution Ovomucin 6.5 no change high Lysozyme 6.2 no change 6.5 Globulins 6.0 no change 6.5 Ovomucoid 6.5 no change high Conalbumin 4.2 no change no change Ovalbumin 6.0 no change 6.5 Control 4.2 6.0 6.5 A probe was made with a 100 9 reference weight and a micro syringe glass plunger of 0.35 cm of diameter and 6.8 cm 73 long firmly attached to it. The instrument was set to zero, the probe hooked to the tension cell, and the record- er pen brought back to zero with the balance fine knob. For calibration, a 10 9 standard weight was added to the cell and calibrated to full scale (at instrument range 1) with the calibration knob. Upon removal of the standard weight, the pen returned to zero, after which it was moved up to full scale with the balance fine knob. This reversed polarity position was used as the reference point for measurement of gel firmness. At such position, application of a force upwards against the plunger would decrease the tension force on the cell and result in a curve with the peak maximum directed towards the normal zero. Force resistance curves of the gels plunged with the devised probe, were obtained at a cross-head speed of 5 cm/min. with the gear shift set to high. After reaching room temperature, the sample was placed on the platform attached above the cross-head and slowly raised towards the probe until 1.0 cm of the plunger penetrated the gel. At this point the cross-head was stopped and immediately lowered. Force curves were obtained from two opposite locations on the gel at a chart speed of 20 cm/min. The area of the curves was determined with a Keuffel & Esser compensating polar planimeter and gel strength was ex- pressed as the work, in g xcnm required to plunge through 1 cm of the sample. 74 Percentage Drainage Following gel strength determination, the coagulums were carefully removed from the beakers onto a pre-weighed dish set-up devised for collecting the drained liquid. This set-up consisted of a medium size weighing boat to which a copper wire screen with 10 cm of diameter and seven openings/cm was molded. A Whatman filter paper no. 4 of 11.0 cm of diameter was then placed over the screen. The dishes containing the gels were covered with stainless steel bowls and the liquid allowed to drain for one hour. After this period the total gel weight and the liquid weight were determined. The percentage drainage was calculated by dividing the liquid weight by the sample total weight and multiplying by 100. Small gel portions of approximately 1 cm on a side were frozen in dry ice before the liquid was drained and further freeze-dried for sulfhydryl determinations. pH Determination The protein coagulum and drained liquid obtained from the previous determination were combined, and the curd was broken into small pieces with a metal spatula until a slurry was formed. The pH of this mixture was measured on a Beckman Expandomatic pH meter, model 76A. 75 Electron Microscopy A transmission electron microscope (TEM) was used to examine foams and a scanning electron microscope (SEM) was used for both foams and gels examination. Transmission Electron Microscopy Selected freeze-dried foams were fixed with osmium tetroxide vapors. A pyrex dish filled with a 2% osmium tetroxide solution was placed into a medium size petri- dish, and the samples, contained in small plastic vial caps, were positioned around the fixative. The petri dish was then covered, carefully sealed with an adhesive tape, and left under the hood for 72 hours. After fixation, the samples were transferred to small capped vials (4.5 m1 capacity) and infiltrated for 24 hours with anhydrous, TEM grade acetone. An Epon-Araldite: 13a, epoxy resin mixture (1:1), prepared according to Hooper et a1. (1979), was used as the embedding medium. The resin mix was dissolved in acetone (1:2 mixture), and added to the sample vials to a final 1:5 resin:acetone concentration. The vials were capped, gently swirled and left standing for 24 hours. After this period, the vials were uncapped and placed under the hood for four hours. At the end of this period, additional resin-acetone mix was added to 2/3 of the vial capacity, the vials gently agitated to ensure complete mixing, and left uncapped under the hood 77 grids were rinsed with a 0.02 N sodium hydroxide solution and two additional distilled water rinses of one minute each. The prepared grids were examined in a Philips 201 TEM at an accelerating voltage of 60kV. Scanning Electron Microscopy Small pieces of freeze-dried samples were mounted on aluminum stubs and gold coated with a sputter coater for nine to twelve minutes. The prepared samples were examined in a Japan Electron Optics Limited SEM, model JSM 35C, at an accelerating voltage of lSkV. Statistical Analyses of the Data The data obtained from the cakes were treated by multiple regression analysis using a least squares pro- gram available at Michigan State University Computer Center. For most dependent variables examined, the possible sub- set models contained only linear terms and interaction terms with two independent variables whereas, for foam- ability, a few interaction terms with three independent variables were introduced. A technique described by Becker (1968) was also used to detect components with a linear blending behavior resulting in models with non- polynomial forms. The procedure involved transformation of all interaction terms to the form xin/Xi + Xj,.and 78 components exhibiting linear blending were then detected through their improved correlation coefficients significance. The "best" final subset of variables was found by eliminat- ing variables of poor significance to improve the standard error of the estimate (S), the coefficient of multiple determination (R2), and the sum of squares of the residuals, simultaneously. The prediction equations for volume and foaming index parameters obtained with the least squares method were used to estimate regions of optimum response through response surface analysis. The contour plots and per- spective response surfaces were obtained as a function of two independent variables by a Surface II Graphics System program developed by Sampson (1975), also available at Michigan State University Computer Center. Ovomucin and lysozyme were used as the independent variables and ovo- mucoid, conalbumin, and ovalbumin levels were fixed to their normal values. Globulins levels of the mixture treatments plotted were obtained by the difference of the sum of the other five protein levels from 1. Gel data were analyzed by the Student's t-statistics for comparison of two means with unknown, but equal variances, with the following formula (Gill, 1978): §1-§2 S/(l/Nl) + (l/N2) 79 where {szzl - (2X1)2] + [2X22 - (2X2)2] S = + N1 N2 (N1 + N2-2) RESULTS AND DISCUSSION Protein Fractionation Determination of moisture and nitrogen contents were performed on several batches of prepared albumen. Table 12 displays data from these evaluations and corresponding calculated protein concentration. The average percent protein value was used to obtain yields and recoveries of the isolated fractions as well as for preparation of protein solutions used in the angel food cake system. Table 13 lists yields and recoveries of the various proteins isolated by the described fractionation procedure. Except for conalbumin and globulins, recoveries were satisfactory, with values ranging from 66 to 76%. Conal- bumin was prepared as the iron-free protein complex. Therefore, owing to its higher solubility, only partial crystallization was achieved resulting in low recoveries. The low recoveries of globulins appeared to be associated with formation of a large precipitate during dialysis. This highly insoluble material contained very high molecular weight protein fractions, as estimated by electrophoresis, and some globulins. In addition, considerable quantities of conalbumin present in globulins preparations necessi- tated purification of the material at less than half 80 81 Table 12. Average Moisture, Nitrogen, and Protein Contents of Egg Albumen.a . b Protein Albumen . N1trogen . M01sture . . (Wet weight Batch (Dry Weight Ba51s) Basis) % % % 1 88.4 13.05 10.39 2 88.4 13.13 10.34 3 88.5 13.23 10.39 4 88.2 13.23 10.65 Average 88.3 13.16 10.44 aValues are average of two determinations. b Determined with the Kjeldahl method. Table 13. Yield and Recovery of Protein Fractions Isolated from Egg Albumen. Protein Unfractionated . b Fraction Albumena Yield Recovery %c g g % Ovomucin 1.5 1.6 1.1 73.4 Lysozyme 3.5 5.5 4.1 76.0 Globulins 8.0 12.5 2.6 20.5 Ovomucoid 11.0 17.2 11.3 66.0 Conalbumin 13.0 20.3 4.4 21.9 Ovalbumin 54.0 84.2 58.0 68.9 aAlbumen weight 1250 g, protein content 130 g. bAverage of all fractionations. CSource: Powrie (1977). dSource: Parkinson (1966). Include globulins G2 and G3. 82 saturated ammonium sulfate solution (approximately 40-45%). Under these conditions some globulins also remained in solution. The various protein preparations were tested for purity with PAGE and SDS-PAGE. Figure 3 A and B show SDS-PAGE and PAGE, respectively, of the preparations used for functionality tests. The degree of purity of all proteins was estimated to be over 90% visually and the relative intensity of the protein bands of SDS-PAGE gels was determined from densitometer tracings. Under the conditions used in SDS-PAGE globulins appeared to have the same relative mobility as that of ovalbumin (Figure 3A, gels 2 and 5). This suggested that both proteins have comparable molecular weights. The composition and relative purity of the protein fractions are summarized in Table 14. Although dye binding ability varies among proteins, in general densi- tometric quantifications were comparable to gels visual inspection. An exception was observed with globulins preparation which appeared to contain a relatively high amount of ovomacroglobulin, calculated to be about 24% (Table 14). This apparent high concentration may have resulted from the combined effects of higher dye binding ability of ovomacroglobulin and the compactness of the protein band (Figure 3A, gel 2). For evaluation of foaming properties of individual proteins, fractions with relative purity below 95% Fig. 3. Electrophoretogram of egg white proteins. a) SDS PAGE. '1. Ovomucin; 2. Globulins; 3. Conal- bumin; 4. Ovomucoid; 5. Ovalbumin; 6. Lysozyme. b) PAGE. 1. Conalbumin; 2. Ovomucoid; 3. Globulins: 4. Ovalbumin; 5. Lysozyme; 6. Ovomucin. 83 owns— :6 cou__ ’. ova GLOB— ovo_ , lYS._ 84 .mwswomup Hmumeouflmsmo Eonm wouoasoaoon .mwflmlmflm Cfl WQHHHHHDOE 0>flHMHOH SCH“ Umfififiwflwn—M . . swash 0 00 0 m IHm>o . . . cwfion h m 0 N0 0 m IHmcoo . . . 000055 0 0 v 00 0 m Io>o 0.H H.00 v.0 0.H v.a> 0.H mcHHsQOAw +0.00 weanomma 0.0 0.0 0.00 swoseo>o . w mswououm samuonm nonfinflnca :fiHsnon :Heon GHEDQ ofioose mafia: oshn c0095 Hague Io>mHm Io>o Iouomso>o IHo>O Iamsoo Io>o IQOHU Iowan Io>o :oauomuh unwouonm coasnad mom caououm .msofiuoonm swououm smfisnad ooUMHOmH 0:» mo amufiusm o>HpoHom poo msofluwmomfioo .va magma 85 (Table 14) were purified by gel filtration. For inter- action studies the protein preparations were used without purification. Sulfhydryl (SH) and disulfide (88) contents were determined on the isolated fractions and these values are listed in Table 15. All protein preparations contained some SH groups and had relatively high SS contents. Ovalbumin contained most of the SH groups, 3.73 SH resi- dues/mole, whereas less than one SH group was present in the other proteins. The absence of thiol groups in conalbumin, lysozyme, and ovomucoid (Powrie, 1977), and in globulins (Feeney et al., 1963) has been reported. Therefore, the measurable SH content in these proteins probably resulted from slight denaturation during frac- tionation. The disulfide content obtained for most proteins was well within literature values. Lysozyme contained 4 SS groups, ovomucoid 8, conalbumin 15, and ovalbumin 1.35, while reported values are 4 (Jolles et al., 1963), 8.15 (Davis et al., 1971), 14.55 (Wenn and Williams, 1968), and 1 (Smith and Back, 1970), respectively. Angel Food Cake System The physico-chemical characteristics of the protein solutions used for testing foaming properties and the control solution are illustrated in Table 16. The 86 Table 15. Sulfhydryl and Disulfide Contentsa of Albumen Proteins. Sulfhydryl Disulfide P t . molis7 moles/ mole/ moles/ r° em 10 g mole 104g mole protein protein protein protein Ovomucin 0.12 3.79 Lysozyme 0.12 0.20 2.74 4.00 Globulins 0.36 1.51 Ovomucoid 0.10 0.31 3.00 8.00 Conalbumin 0.10 0.09 1.85 15.00 Ovalbumin 0.73 3.73 0.30 1.35 aValues are average of two determinations. parameters of angel food cakes prepared with these solu- tions are also summarized in Table 16. Viscosities ranged from 2.77 cps x g/cm3 for the globulins solutions, to 1.53 cps x g/cm3 for lysozyme solution. Viscosity and surface tension of ovomucin solution were not determined owing to the very poor flowing pr0perties of the solution. However, it was evident that ovomucin had the highest vis- cosity of all proteins in solution. The control solution exhibited a viscosity of 2.02 cps x g/cm3, which was primarily imparted by ovomucin. The influence of ovomucin on viscosity appears to conform with its very high molecular size and carbohydrate content. With these characteristics, the protein is 87 .MGOflumGflpp—Hmnvww 03“.. HO GOMHMKVM 0H0 WGSHM> oEUvnQH " H03 VA 0 .mufiommmo moflEmom 0s mmuoosoo osaw>o .owswahouoa nozo n .0.0 H mm .00.0 on 00.0 n H «00.0H u sofiuwuucmosoo samuoumm 000.0 000.0 000 0H.0000.m 0H.0HN0.0 00.0 0.00 00.0 Houusoo 000.0 000.0 000 00.0000.0 NH.0H¢H.0 00.0 0.H0 N0.H caesoam>o 000.0 000.0 00H H0.0H~0.0 mo.0u00.0 00.0 0.00 00.H :«Eanmcou 000.0 000.0H 00 00.0000.0 mo.oww0.o 000.0 0.00 00.H ofloosao>o 000.0 000.0 000 00.0Hom.m 00.0w00.0 H0.v 0.00 00.0 mafiannoHo 000.0 0H0.HH 000 00.0H00.0 00.0HH0.0 ~H.0 0.00 00.H mfihuoqu 00H.N 000.0 00 vo.ow00.0 00.0000.0 000.0 002 ooz sfiosso>o oEO\xH03 000303 080 0.3800 003300.38 suna\m\mfio 50\mofino mEO\0Xmm0 300m newusaom muaaanammoumfioo 0000,3003. 05.50.» xoooH sounmsma wuamoomdw sawuonm . . . mcafimom mommusm . . . 00:00:00 Hmuoarmasm . .mumuwamnmm 0x00 UOOM mecm wan mGOHHSHOm mCHmHOHQ :mESQHm MO mOHumHHmuOMHmno HMOHEmSOIOOHmmnm .0H manna M 88 capable of absorbing considerably large quantities of water, swelling tremendously in the process. Likewise, the ability of ovomucoid to promote in- creases in viscosity may be related to the high quantity of carbohydrate prosthetic groups attached to the molecule. These groups represent about 20-25% of the protein weight (Davis et al., 1971). The underlying causes for the high viscosity of glo- bulins solution were not apparent. As evidenced from SDS-PAGE, globulins have relatively small molecular size ( m 45,000 daltons). The possibility that some carbo- hydrate moieties may be attached to the polypeptide chains and/or that the proteins may associate in solution forming larger complexes, could justify their effects on viscosity. The proteins exhibited varying degrees of surface ten- sion lowering effects. Ovalbumin was the least effective with a surface tension value of 51.8 dynes/cm whereas ovomucoid reduced the surface tension of water from about 72.0 dynes/cm to 39.0 dynes/cm. Lysozyme and conalbumin solutions surface tension values were quite similar, 42.0 dynes/cm and 42.4 dynes/cm, respectively. Globulins solution surface tension was 45. 4 dynes/cm whereas the control solution showed a surface tension of 46.7 dynes/cm. For four of the proteins, the foaming index of the solutions correlated positively with viscosity, whereas surface tension did not seem to affect foamability. Glo- bulins showed the highest foaming capacity (4.71 cm3/g/min.). 89 The other proteins had rather poor foaming ability with values of 0.59 cm3/g/min for ovalbumin, 0.24 cm3/g/min for conalbumin, and 0.12 cm3/g/min for lysozyme. The absence of air incorporation ability of ovomucin again resulted from the extremely high viscosity and lack of flowing properties of the solution. Ovomucoid also exhibited no foaming capacity although its viscosity and surface tension characteristics favored foam formation. A certain degree of denaturation is necessary for formation of a cohesive film, and Adam (1941) indicated that the spreading ability of a protein at the liquid surface depends on the protein conformation. Ovo- mucoid has extremely high resistance to heat denaturation (Fredericq and Deutsch, 1949) and apparently, the mechani- cal whipping action also did not alter the protein con- formation. This high resistance to denaturation may be related to the large number of disulfide linkages (8) stabilizing the protein structure. It was observed that the same type of correlation between disulfide groups and foaminess was present in the other proteins. Therefore, lysozyme, with four disulfide bonds present in the mole- cule, also showed very poor foaming power, whereas oval- bumin, with one disulfide group, exhibited slightly better air incorporation. Whipping reduced the number of sulfhydryl groups of most proteins. A significant reduction of about 20% was observed for lysozyme (P < 0.01), of 12% for globulins 90 (P < 0.05), and of 8% for ovalbumin (P < 0.05). Although a decrease in SH content of whipped conalbumin and control solutions was also observed, the differences were not significant. No apparent effects of whipping on the sulfhydryl content of ovomucin and ovomucoid were observed. The reduction of SH groups for the other proteins, however, suggested that inter and/or intramolecular sulfhydryl- disulfide interchange reactions were involved in foam formation. Great variability in volume of angel food cakes was obtained when the different porteins were used for foam preparation. Globulins produced the cake with the largest volume (330 cm3) followed by ovalbumin (308 cm3) and con- trol (272 cm3). V01umes of 157 cm3 for conalbumin and 107 cm3 for lysozyme cakes resulted from fewer air inclusions and instability of the foams, whereas volumes of 54 cm3 for ovomucoid, and 52 cm3 for ovomucin cakes reflected complete lack of aeration. Tenderness and compressibility measurements reflected the degree of aeration of the cakes. Tenderness of glo- bulins, ovalbumin, and control cakes varied little, but ovalbumin produced a slightly tougher cake, with tender- ness value of 4.535 work/g. Cakes prepared with the con- trol mixture and with globulins showed tenderness values of 4.047 and 4.275 work/g, respectively. Conalbumin, lysozyme, ovomucin, and ovomucoid cakes were considerably 91 less tender with tenderness values of 8.347, 11.915, 6.703, and 16.982 work/g, respectively. It was interesting to note that ovomucin cake was very moist and "mushy" and these characteristics apparently lowered the work required to shear the cake. Compressibility values of globulins and control cakes were quite similar, 0.769 and 0.750 work/g, respectively. The work required to compress the cakes prepared with ovalbumin, conalbumin, lysozyme, ovo- mucin, and ovomucoid were 0.567, 4.738, 6.465, 2.158 and 2.320 work/g, respectively. The somewhat lower compressi- bility values of ovomucin and ovomucoid cakes probably resulted from improper compression since these cakes had very low heights. Figure 4 illustrates the cakes prepared with the various albumen proteins. Cake prepared with ovalbumin showed very coarse texture with several large air cells and thick cell walls, whereas cake prepared with globu- lins had numerous smaller air cells with thin walls. The control cake appeared to combine the effects of adding the various proteins, particularly globulins and ovalbumin. Textural characteristics of the other cakes varied accordingly with the degree of aeration in these products. Protein Interactions The effect of variations in levels of six albumen pro- teins on viscosity, surface tension, and foaming index of Fig. 4. Angel Food Cakes Prepared with Various Albumen Protein Solutions. Protein Concentration 10.4%, pH 5.7, Ionic Strength 0.20. CONALBUMI‘ GLOBULINS CONTROL OVOMUCIN OVOMUCOID E ! H '0 LY 3 02 Y M E 93 protein solutions, and on volume, tenderness, and com- pressibility of angel food cakes were evaluated. Table 1? summarizes the significant simple correlations among pro- tein mixtures components, physical characteristics, and cake parameters. The observed and predicted values of protein mixtures physical parameters and angel food cake parameters are summarized in Tables 32 and 33 in the appendix B section. Ovomucin and combinations of ovomucin with the other five proteins correlated positively with viscosity at P < 0.0005. Globulins and interaction terms of globulins with lysozyme, ovomucoid, and ovalbumin effectively re- duced the surface tension of the solutions (P < 0.05). Several linear and interacting variables affected foaming index. Positive correlations were observed with ovomucin (P < 0.0005), globulins (P < 0.01), the inter- acting variables of ovomucin with globulins, ovomucoid, conalbumin, and ovalbumin (P < 0.05). Viscosity also correlated positively with foaminess at P < 0.0005. Lyso- zyme, and the interacting variables of lysozyme with globulins, ovomucoid, conalbumin, and ovalbumin correlated negatively (P < 0.05) with foaming index. Ovomucin, and interaction terms of ovomucin with globulins, ovomucoid, conalbumin, and ovalbumin correlated negatively with volume. Viscosity and foaming index also showed a negative correlation with volume. In con- trast, lysozyme, and interaction of lysozyme with globulins, 94 .moee.e v a nu usmoemesmem444 “S... v m an usmoemmsmem«a 126 v a 08 usmmmmosmmmg «««H0.0I 0EDHO> «v~.o «««00.0I xmwcH mafifimom flmNoo COfimgme mUMHHDm aa0m.o 00%H0.0I ««%mm.o huHmOOmH> go x 200 *0N.0I m>O x Q>O «.«Nm.0l ZOO x 920 «0N.o «0N.0I m>o x monw «NN.¢I «mm.o 200 x mOAQ 0%H0.0I «v0.0 a0N.0I Q>O x moqw «0N.0I «asom.o «£00.0I m>O x mag «t0m.o «st.OI 200 x qu «aom.o «aHm.0I Q>O x qu «0N.o *0N.0I «ewm.0I mono x mud %«HM.O ssmm.o «£000.0I «0000.0 «aa0m.o m>O x Z>O «0N.o «£000.0I «%«m¢.o %««00.0 200 x Z>O «000.0 avN.0 «aam0.0I «0000.0 %«%00.0 Q>O x Z>O «amm.o «vm.o *««00.0I «aavm.o «««00.0 monw x Z>O «0N.o «aamv.o mNA x Z>O «tom . c GflEDQHMNVO GHESQHmGOU *0N.0I CHOOSEO>O *0N.0I «000.0 000.0I mdaaflQOHw «vN.0I «at0v.o «100.0I OE>NOmMQ «smm.o *0N.o «aamm.ol «aamv.o %«%H0.o GAODEO>O xmosH soflmsma muwaflnfimmmumaoo mmmsumocme wasHo> 0008000 moomnsm muflmoomfi> .mumumfioumm 0x00 000 .moflumfiumuomumnu Hmowmhnm .musms IomEoo mousuxwz samuonm 00054 musmflOAMmooo sodumHmuuoo mameflm ucmowmflc0am .0H magma 95 ovomucoid, conalbumin, and ovalbumin positively affected volume. The work required to shear the cakes (tenderness determination) was positively affected by ovomucin, oval- bumin, ovomucin x lysozyme, ovomucin x globulins, ovomu- cin x ovomucoid,_and ovomucin x ovalbumin. Globulins, ovomucoid, globulins x ovalbumin, ovomucoid x ovalbumin, globulins x ovomucoid, and ovomucoid x conalbumin negatively correlated with work/g values (produced more tender cakes). Compressibility showed a positive correlation with ovomucin, ovomucin x globulins, ovomucin x ovomucoid, ovomucin x ovalbumin, and viscosity at P < 0.01, and with ovomucin x conalbumin, surface tension, and foaming index at P < 0.05. Lysozyme and lysozyme x ovalbumin (P < 0.05), and volume (P < 0.0005) correlated negatively with com- pressibility. Foaminngndex. The effects of the various proteins on foaming index appeared to be related to viscosity and surface tension, and to other underlying effects apparently associated with lysozyme levels. The highly significant positive correlations of ovomucin with viscosity and with foaming index suggested this protein facilitated foam formation primarily by increasing the solution viscosity. Globulins appeared to positively influence foaminess, in part, by lowering surface tension. Lysozyme and its interaction terms correlated 96 negatively with foaming index. The influence of this pro- tein on foamability may be associated with the formation of a ovomucin-lysozyme complex. Garibaldi et al. (1968) observed that anovomucin-lysozyme complex formation during heating was related to loss of foaming proPerties of egg white. Heat apparently accelerates complex formation reaction rates at normal levels of ovomucin and lysozyme. Kato et al. (1975) showed the dependence of lysozyme con- centration on ovomucin-lysozyme interaction. These authors pointed out aggregation increased in proportion to lysozyme concentration and reached saturation when the concentration of the protein was twice that of ovomucin. Variations in levels of ovomucin and lysozyme from low (0%) to high (1.5 times normal levels) clearly showed the effects of these proteins on foaminess. Figures 5 and 6 illustrate the correlations among ovomucin and ly- sozyme levels and foaming index. Ovomucin exponentially increased the foaming index of solutions which contained 0% lysozyme and various levels of globulins (Figure 5). In the presence of 6% lysozyme, the magnitude of foaming in- dex values was much lower. Moreover, all solutions showed similar trends, in that the normal level of ovomucin caused a slight reduction in foaminess while higher levels of ovomucin improved foaminess. The drastic depression of foaming capacity of solutions containing a high level of ovomucin (2.5%) with the addition of lysozyme is seen in Figure 6. In absence of ovomucin, 97 0—0 3% Globulins 30" D—CI 8% Globulins A H 13% Globulins ___ 0% Lysozyme z 24_ __ 6% Lysozyme I \ o a" a u - "3. x H a z a f 12.. I < a b ovomucin, % Fig. 5. Effect of ovomucin on the foaming ability of pro- tein solutions containing various levels of globulins and lysozyme. Protein concentration 10.4%, pH 5.7, ionic strength 0.20. Ovomucoid = 11.5%; conalbumin = 13.5%; ovalbumin = 53.5 to 72.0%. N==h. 98 0—0 3% Globulins 30.. H 8% Globulins H 13% Globulins — 2.5% Ovomucin --- 0‘ Ovomucin f 24- I \ a n‘ I a ‘ 18- x an a z a z :I 12- " D a I lYSOZYIE,% Fig. 6. Effect of lysozyme on the foaming capacity of protein solutions containing various levels of globulins and ovomucin. Protein concentration = 10.4%, pH = 5.7, ionic strength 5 0.20. Ovomucoid = 11.5%, conalbumin = 13.5%, ovalbumin = 53.5 to 72.0%. N==u. 99 lysozyme improved foaminess of the solutions. This basic positively charged protein at pH 5.7 may interact electro- statically with the acidic proteins (ovomucoid and oval- bumin) and possibly with globulins facilitating foam formation. The effect of globulins on foaming index is shown in Figure 7. Globulins increased foaming capacity of solu- tions with any level of lysozyme and ovomucin. Linearity was observed for most solutions. However, for solutions with 2.5% ovomucin and 0% lysozyme, a saturation point was reached at 8% globulins level. No significant correlation between ovomucoid, conal- bumin, and ovalbumin and foaming index was detected. This result suggested these proteins had little or no influence on foamability. VOlume. The influence of the various proteins on volume was associated with the effects they have on foaming capacity. The observable trend was an inverse correlation between volume and foaming index. Thus, ovomucin and its interactions, which positively correlated with foaming index, had high depressive effects on volume. Lysozyme and its double combinations, negatively correlated with foaming index, showed positive relationship with volume. Globulins and its interacting variables had no significant effects on volume. The relationship between globulins, ovomucin, and 100 36 0—0 0% Lysozyme 0—0 3% Lysozyme H 6% Lysozyme 30 — 2.5% Ovomucin ——- 0% Ovomucin C 0 I I 3 ur \ n I a x an El 18L I G. I I ‘ a u- n. O s 9 u w cLoBULIus,% Fig. 7. Effect of globulins on the foaming capacity of protein solutions containing various levels of lysozyme and ovomucin. Protein concentration = 10.4%, pH = 5.7, ionic strength = 0.20. Ovomucoid = 11.5%, conalbumin = 13.5%, ovalbumin = 53.5 to 72.0%. IJ= u. lOl lysozyme and volume is more readily seen in Figures 8, 9, and 10, respectively. Although globulins (Figure 8) did not significantly affect volume, it seemed that at normal levels (8%), better air incorporation resulted in slightly larger cakes for most treatments, whereas at higher levels (13%), a slight reduction in cake volume was observed. For the combination of 6% lysozyme with 2.5% ovomucin, a pro- gressive increase in the level of globulins consistently reduced volume. In general, with cakes that contained ovomucin (Figure 9) the trend was a decrease in volume with increasing levels of the protein, except that for treatments with 3% globulins and 6% lysozyme, higher volumes were obtained with the higher level of ovomucin. In contrast, for cakes with varying levels of lysozyme (Figure 10), considerable im- provement of volume occurred for treatments with 2.5% ovomucin and 3, 8, and 13% globulins. For cakes with 0% ovomucin and 3, and 13% globulins increasing levels of lysozyme caused a slight improvement followed by a reduction of volume, whereas for treatment with 8% globulins, the inverse occurred. As it was observed previously, the effects of glo- bulins, ovomucin, and lysozyme on cake volume were clearly related to foaming capacities. Previously, MacDonnel et a1. (1955) reported that smaller cakes were produced when prepared with egg white containing extra quantities of ovo- mucin. They implicated that excessive insolubilization of 102 350 300 O\ _u_ /' ‘_;;\ a ————— m“”~\=::———I:I ’/ \\\““A \\ A’ \o 0" 250 n A I 9 " D Ill I a _a D 6 an 200 0 0 0—0 0% Lysozyme H 3% Lysozyme A—A 6% Lysozyme 150 _. 2.55 Ovomucin —— 0% Ovomucin 0__J 41 1 L, 1 0 3 6 9 12 15 GtOBlILIIS X Fig. 8. Effect of globulins on volume of angel food cakes prepared with protein solutions containing varying levels of lysozyme and ovomucin. Protein concentration 10.4%, pH 5.7, ionic strength 0.20. Ovomucoid = 11.5%, conalbumin 13.5%, ovalbumin = 53.5 to 72.0%.I(=u, 103 votuu£,003 Globufins Lysozyme X O-O 3 0 . zoo- m a o . D-D I3 0 ._‘. 3 6 twat 8 6 "it I---I 13 6 Jr. 1 I 0 1 2 ovomucin, a: Fig. 9. Effect of ovomucin on volume of angel food cakes prepared with protein solutions containing varying levels of globulins and lysozyme. Protein concentration = 10.4%, pH = 5.7, ionic strength é 0.20. Ovomucoid = 11.5%, con- albumin = 13.5%, ovalbumin = 53.5 to 72.0%.13= “- 1014 mm mm 1. 250 a lull a I —l 2 A 200‘; 0—0 3% Globulins o—o 8% clown... A—A 13% Globulin. _ 2.5% Ovomucin 150_ __ 0% Ovomucin lYSOZYIE , X Fig. 10. Effect of lysozyme on volume of angel food cakes prepared with protein solutions containing varying levels of lysozyme and ovomucin. Protein concentration lO.4°, pH 5.7, ionic strength 0.20. Ovomucoid = 11.5%, conalbumin = 13.5%, ovalbumin = 53.5 to 72.0%. N= 11. 105 ovomucin at the bubble surface decreased film elasticity and prevented cake expansion during baking. In the current study it was observed that cakes pre- pared with 2.5% ovomucin and 0% lysozyme expanded normally during baking, but then collapsed at the last stage of baking. This suggested that reduced heat coagulative pro- perties of the film surrounding the air cells was primarily responsible for low cake volumes. In addition, ovomucin has been reported to lack heat coagulative properties (Cun- ningham and Lineweaver, 1965; MacDonnell et al., 1953). The "protective" effect of lysozyme on volume seemed to correlate with formation of ovomucin-lysozyme complex. Apparently, this complex either does not insolubilize excessively at the bubble surface or has normal heat coagu- lative properties. The evidence for the reductive effects of high levels of globulins on cake volume points out this protein fraction may also insolubilize (denature) exces- sively at the air-albumen interface. Tenderness and Compressibility. Determination of tenderness and compressibility may indicate the degree of aeration of baked products. In general, highly aerated products ex- hibit low tenderness (numerical value) and compressibility scores. Compressibility correlated positively with visco- sity and foaming index and negatively with volume, although there were no apparent correlations of these parameters with tenderness. 106 As expected, ovomucin and its interaction terms correlated positively with both tenderness and compressi- bility. This indicated that cakes prepared with these variables were more compact. Lysozyme's aerating effect correlated negatively with compressibility values. Globulins and ovomucoid appeared to produce more tender cakes. This effect was also observed for the follow- ing interaction terms: GLOB x OVD, GLOB x CON, OVD x CON, and DVD x OVB. Sulfhydryl Groups The sulfhydryl content of the various protein solutions before and after whipping are summarized in Table 18. The sulfhydryl content of the solutions varied accordingly with the concentration of ovalbumin since the majority or all sulfhydryl groups in egg white are present in this protein (Powrie, 1977). In general, whipping reduced the number of sulfhydryl groups. This effect suggested that formation of the protein layer at the surface of the film involved sulfhydryl-disul- fide interchange type reactions, with formation of disulfide bonds. Although some mixtures exhibited more pronounced reduc- tions in the number of sulfhydryl groups than others, the specific reasons for these effects were not apparent. Additionally, the fact that extensive sulfhydryl-disulfide 107 Table 18. Effect of whipping on the Sulfhydryl Content8 of Albumen Proteins Solutions. _ML Solution Nean+ roan Nean+ t- e tat 1 ".1 ‘:c OVN LYS GLOB OVD CON 0V3 Standard Deviation Standard Deviation uolee/losg sample L L L L L H 5.83:0.02 5.52:0.18 NS L L L L N a 4.61:0.04 4.73:0.09 -4.595' L L L a L N 5.41:0.22 5.3020.08 NS L L L a N N 3.97:0.08 3.78:0.16 N! L L a L H N 4.01:0.03 3.Blzo.l2 NS L L a a a L 3.76:0.09 3.69:0.08 NS L N L L L 8 4.79:0.05 4.68:0.06 NS L N L L a N-N 4.14:0.29 4.01:0.11 NS L H L B L N-N 4.12:0.04 4.02:0.00 4.003' L a L N a N-L 3.58:0.19 3.42:0.07 NS L N N L L a 5.28:0.24 4.4820.24 3.333. L N 3 L a N 3.80:0.09 3.63:0.18 NS L N N a L N 3.94:0.00 4.17:0.12 N8 L N N N N L 3.12:0.09 2.85:0.03 4.025. N L L L L a 5.53:0.32 5.11:0.06 NS 3 L L L N N-N 4.35:0.13 4.28:0.06 NS 3 L L N L a 4.38:0.07 4.00:0.34 NS 3 L L a a N 3.41:0.04 3.89:0.14 -4.662' a L a L L N 4.79:0.25 4.83:0.07 N3 3 L N L N N 3.80:0.00 3.99:0.17 NS 8 L B N L N 6.58:0.10 4.36:0.04 NB 3 L N N n L 4.00:0.01 3.31:0.02 63.639'** 3 N L L L N 4.58:0.20 4.7720.29 NS 3 a L L N N 3.91:0.05 3.53:0.24 NS 3 N L N L N-N £.31:0.08 3.73:0.16 4.585. N N L N 8 N-L 3.10:0.02 3.10:0.03 NS 8 N N L L N-N 5.18:0.05 4.95:0.13 N3 8 N a L a N-L 3.61:0.06 3.16:0.09 NS 8 N N N L N 3.72:0.25 3.65:0.04 NS N N a N N L 2.95:0.08 2.70:0.38 N8 L L N N N N-N 6.56:0.04 4.36:0.07 3.157' L a N N N N 4.01:0.06 4.0830.02 NS 3 L N N N N-N 4.10:0.05 3.8630.05 4.800. a N N N N N 4.2720.12 4.0920.0l NS L N L N N N-N 4.25:0.16 3.7Sto.06 4.138. L N N N N N 4.37:0.l7 3.94:0.23 NS N N L N N N-H 4.03:0.00 3.94:0.0‘ NS 8 N N N N N 3.81:0.12 3.79;0.00 NS L N N L N N 4.4420.02 4.43:0.18 NS L N N N N N 4.25:0.23 3.99:0.04 NS 3 N N L N N-a 4.56:0.14 6.38:0.l6 NS 8 N N N N N 3.76:0.19 3.£9z0.35 NS L N N N L N 4.96:0.14 4.70:0.10 NS L N N N a N 4.11:0.04 3.36:0.31 3.393’ a N N N L N 5.07:0.30 4.72:0.29 NS N L L N N N 5.01:0.22 4.36:0.09 3.867. N L a N N N 4.15:0.13 3.9120.08 N8 N N N N N N-L 4.33:0.08 4.22:0.07 NS N L N L N ‘ H 4.94:0.17 4.74:0.00 NS N L. N N N N 4.93:0.09 4.02:0.15 7.357'* N a N L N N-N 4.53:0.22 4.50:0.03 NS N N N a N N-L 3.48:0.02 3.51:0.12 NS N L N N L a 4.67:0.17 4.47:0.36 NS N L N N N N 4.34:0.03 4.55:0.08 o3.476' N B N N L N-N £.86:0.21 4.27:0.03 3.933. N N N N N N 4.72:0.07 4.16:0.l3 5.460. '10-: how I ovomucin, Lrs-lyeozyne, GLOB-globulins. OVD-ovomcoid. CON-conalbumin OVB- ovalbunin. L-low, N-normal. anhigh. Numerical yaluee for low. normal, and high levels are listed in Table 9. 1'Si.gnificant': at P < 0.05. "Significant at P < 0.01. "'Significant at P < 0.0005. cNS-not significant at P - 0.05. 108 interchange reactions may take place without a significant decrease in sulfhydryl groups (Jensen, 1959) make it more difficult to pinpoint which solutions favored the inter- change reaction. The few mixtures which showed an increase in sulfhydryl content may reflect more extensive denatura- tion of the proteins, but most likely resulted from varia- tions in experimental conditions. Response Surface Analyses The effect of variations in levels of six albumen proteins on specific functional properties was analyzed. Significant effects on viscosity and foaming index of pro- tein solutions (Table 19) and on volume and tenderness of angel food cakes (Table 20) were observed. Changes in surface tension (Table 19) and in compressibility (Table 20) were not significantly associated with variations in pro- tein levels. The partial regression coefficients and standard errors, and partial correlation coefficients of the varia- bles influencing viscosity and foaming index of protein solutions, and cake volume and tenderness are summarized in Tables 21 through 24. Variables positively affecting viscosity (Table 21) were ovomucin (P < 0.005) and ovalbumin (P < 0.0005). Conalbumin and the interacting variables of ovomucin with the other five proteins negatively affected viscosity at P < 0.05. 109 Table 19. Mean Square Values and F-ratio Significance of Viscosity, Surface Tension, and Foaming Index of Albumen Proteins Mixtures. Mean Squares Degrees of Source Viscosity Surface Foaming Freedom Tension Index Regression *** ** (about mean) 20 .20 2.00 136.85 Residuala 39 .01 2.01 8.92 Total 59 ***Significant at P < 0.0005. aError term. Table 20. Mean Square Values and F-Ratios Significance of Volume, Tenderness, and Compressibility of Angel Food Cakes. Dependent Degrees of Freedom Mean Square Value Variable Regression ResidualaTotal Regression Residuala Volume 19 40 59 2494.01*** 180.07 Tenderness 18 41 59 0.39** 0.12 Compressi- bility 20 39 59 0.07 0.05 **Significant at P < 0.001. ***Significant at P < 0.0005. aError Term. 110 .cwfinnam>onm>o can .cwEdnHmcoouzou .pfiooseo>ouo>o .mcflaonoamnmoqw .mENNOmhauqu .cwosEo>onz>onm «mooonbxv m an “snowmacmmm««* “mo.o v m um unmowmacmwmw «mm.o h>.m mm.oa m>o x zoo wo.o| mh.m HH.m m>o x o>o ha.o vm.oa om.Ha ZOO x o>o mo.ot N~.ma 5v.m m>o x moqo ma.o nv.wa m~.wa zoo x moqw ma.on mm.mH mo.mH Q>O x moqw oH.oI mo.hv on.om m>o x mug oo.o1 mm.mv v~.ma 200 x wwq ma.ol H5.mv mo.ov o>o x mug ma.ou mm.mv bh.ov mono x qu *mm.01 av.ahm om.¢ont m>o x z>o «mm.o: ma.o>~ em.>mm| 200 x z>o «mm.o| om.cnm bn.Hoh| o>o x z>o «mm.o| mm.>mm mh.mchl mono x z>o enm.oa mm.mm~ mm.mvhl mwn x z>o cowuomumucH «agom.o ma.o mm.H m>o «mm.ot mo.m vo.HH zoo HH.o mm.m Hm.e a>o HH.o mm.~H mo.m mono Ha.o mm.vv mo.Hm mun “mm.o mm.¢m~ mm.hoh z>o Madden ucmwowmmmoo Anm. 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The interaction terms OVN x OVB, and OVN x LYS x GLOB, and the additive terms of ovomucin with ovomucoid and conalbumin showed negative signi- ficant effects on foaming index. Cake volume (Table 23) showed significant partial positive correlation with ovomucin, globulins and ovalbumin. The interaction terms of OVN x OVB and of lysozyme with globulins, ovomucoid, conalbumin, and ovalbumin nega- tively correlated with volume. The additive (Becker's model) terms of ovomucin with globulins and ovomucoid significantly reduced cake volume. Significant variables affecting cake tenderness (Table 24) positively were conalbumin, ovalbumin, and the interacting OVN x LYS variable. The interaction terms OVD x CON, and CON x OVB negatively correlated with tender- ness. Lysozyme and conalbumin additive variables also significantly reduced tenderness. The effects of the various protein mixtures on vis- cosity, foaming index, cake volume, and tenderness when evaluated by multiple regression analysis, revealed complex interactions among the individual mixture components. The underlying effects of the individual components, however, cannot be analysed as such, and the overall mixture response should be considered for evaluating functional performance. The prediction equations of non-polynomial form for the 115 significant protein solutions physical parameters and cake parameters are illustrated in Tables 25 and 26, respectively. The prediction equations for foaming index and volume were used for drawing contour response surface and perspective response surface with variations in ovomucin, lysozyme, and globulins. Figures 11 and 12 illustrate the effects of ovomucin, lysozyme, and globulins, on the response sur- faces of foaming index and cake volume, respectively.' Nearly linear correlations between ovomucin and lyso- zyme levels with foaming index values of three and above were observed (Figure 11A). Increasing lysozyme concentra- tion progressively decreased foaming index. Minimum foaming index values were characterized by high levels of lysozyme and normal levels of ovomucin. The region of Optimum response was localized in the corner defined by low levels of lysozyme and high levels of ovomucin. Figure 11B illustrates the three-dimensional correla- tion among ovomucin, lysozyme, and foaming index. It is clearly seen that the optimum response was in the region of high levels of ovomucin and low levels of lysozyme. The minimum extreme was observed in the region of normal levels of ovomucin and high lysozyme content. The effectsof lysozyme and ovomucin on volumeene seen in Figure 12. Increasing ovomucin levels consistently decreased volume, whereas, with lysozyme, the inverse occurred. Maximum volume responses were observed in the neighborhood of the regions with low ovomucin levels and Table 25. 116 Prediction Equations for Significant*** Physical Parameters of Protein Mixtures, Standard Error of the Estimate (8), and Coefficients of Multiple Determination (R2). Parameter Equationa s R2 lfixmdng Y Index 5057.97X1+1238.19X2+99.71X3- 2.99 0.89 “93.09Xu-125.74X5-2.11X5- “3537.14X1X5-103l.31X2X3-1117.07X2Xu- -l3l4.68X2X5-l317.51X2X5-l39.14X3X5+ +100.73XuX5-126893.04X1X2X3-24077.62X1X2Xu- -296.27 XIX: +1102.82 XIX: '1179.36 XIXI‘ "' (X1+X2) (X1+X3) (X1+Xu) -2407.80 XIX: +81.33 XEX§ +158.84 X§X§ (X1+Xs) (Xu+Xs) (Xs+xs) Viscosity Y==7O7.52X1-+3l.08X2+9.08X3+ 0.11 0.89 +4.91Xu-11.04X5+1.55X5- -745.99X1X2-709.75X1X3-701.77X1Xu- -667.34X1X5-704.80X1X5-46.77X2X3- -40.08X2Xu-18.24X2X5-30.70X2X5- '18.02X3Xg+16.23X3X5-8.47X3X5+ +11.56XuX5-3.11XQX5+16.62X5X5 ***Significant at P < 0.0005. ax; = ovomucin, X2 = lysozyme, X3 = globulins, XI. = ovomucoid, X 5 = conalbumin, X5 = ovalbumin. Table 26. 117 Prediction Equations for Significant Cake Parameters, Standard Error of the Estimate (S), and Coefficients of Multiple Determination (R2). Parameter Equationa S R2 Volume*** Tenderness** Y 13806.63X1+16474.39X2-2223.09X3- 13.42 0.86 -1296.85Xu+542.80X5+256.85X5- -6099.54X1X5-8281.42X1X5-l7926.O7X2x3- —15941.07x2x.-17o36.42x2x5-16935.44x2x5+ +3654.34x3x.+2764.02x3x5+3164.59x3x5- -515.07X5X5+983.13 X1X2 -3840.27 XIX; - (X1+X2) (X1+X3) -9l42.23 XIX}: +2002.96 XQXE (X1+Xu) (Xu+Xs) -1531.04X1-ll.18X2-31.68X3- 0.34 0.59 -4.60Xu+46.53X5+3.56X5+ +1938.02X1X2+1592.56X1X3+1486.13X1Xu+ +1511.82X1X5+1594.45X1X5+29.74X2X5- -28.14X3X5-72.50XuX5-55.45X5X5+ +25.87'.X2X& "44.72 :XZXS '+38.85 2X3X§ -+ (X2+Xu) (X2+X5) (X3+X6) +13. 56 XIIX§ (Xu+X6) ***Significant at P < 0.0005. **Significant at P = 0.001. ax, = ovomucin, X2 = lysozyme, X3 = globulins, XI = ovo- mucoid, X5 = conalbumin, X5 = ovalbumin. Fig. 11. Protein Mixture Foaming Index Response Surfaces as a Function of Lysozyme and Ovomucin. Other Pro- tein Levels: Globulins 6.50-14.95%, Ovomucoid 12%, Conalbumin 14%, and Ovalbumin 59%. a) Contour Surface. b) Perspective Response Surface. OVOMUCIN , % I L I l '4 l I film) "0.0 1.0 2.0 3.0 4.0 5.0 6.0 FOAMING INDEX, 95 LYSOZYME I 7‘ Fig. 12. Angel Food Cake VOlume Response Surfaces as a Function of Lysozyme and Ovomucin. Other Protein Levels: Globulins 6.50—14.95%, Ovomucoid 12%, Conalbumin 14%, and Ovalbumin 59%. a) Contour Surface b) Perspective Response Surface. 119 l I 1 I T I l 8.0 3.0 5.0 610 4.0 1.0 .04 O 4 9W 5 Al x. z_oaao>o AH 1.0 S 0.% Lvsozvue,% R mag—40> 120 low and high lysozyme levels. For optimization of foaming index and volume simul- taneously Figures 11A and 12A were overlaid to designate the point or regions of maximum response (Figure 13). Since foaming index and volume correlated inversely, the regions of satisfactory response, that is, the region deli- neated by volume values ranging from 255 cm3 and above and foaming index values above 4 cm3/g/min., were considered. This region was localized near the corner with low levels of ovomucin (0.2-1.0%) and lysozyme (0.0-l.8%). Accord- ingly, globulins levels ranged from 12.2 to 14.8%. The normal levels of these proteins in egg-white are 1.5, 3.5, and 8% for ovomucin, lysozyme and globulins, respectively. Custard Model System The time-temperature relationships for gels prepared with lysozyme, globulins, conalbumin, and ovalbumin are illustrated in Figure 14. A rapid increase in temperature followed by a decrease and levelling off during heating was observed for all gels. The decrease in temperature marked the onset of coagulation and also showed that aggregation of the polypeptides proceeded with absorption of energy from the system (endothermic process). The coagulation temperature ranges, firmness, and percentage drainage of gels prepared with lysozyme, globulins, conalbumin, and ovalbumin, and t-statistics 121 i / /; /// I — —1 _ .— e— l _ L0 05 0.0 I.I.I./V// 00 L0 20 30 40 50 SJ lY!501!YIlE,55 0N §h§ K Fig. 13. Foaming index and volume contour plots overlay for designation of levels of ovomucin and lysozyme in angel food cake system. 122 IEIPEIAIURE,°C Lysozyme Globulins Conalbumin Ovalbumin L s u a n (a 55 Ills, MIN Fig.14. Time-temperature curves of several albumen protein solutions heated at a rate of 0.74°C/min. Protein concentra- tion = 1.27%, ionic strength = 0.275, pH = 8.0. 123 are shown in Tables 27 through 29. Conalbumin coagulation initiated at 57.3°C and aggregation ensued promoting a drop of 1.3°C i: temperature. Ovalbumin coagulation tem- perature ranged from 71.5 to 71.0°C. Aggregation of the polypeptides proceeded with a modest drop of 0.5°C in temperature. A temperature range of 72.0-69.9°C for globulins coagulation was observed with a drop of about 2°C in temperature. Lysozyme showed the highest thermal requirement for unfolding of the polypeptides, 81.5°C. Formation of the coagulum ensued with a drOp of 7°C in temperature. The initial coagulation temperatures were significantly different among the various gels except for ovalbumin and globulins gels. Ovomucin and ovomucoid showed no gelation properties under the conditions used for evaluations. The observed heat stability of these proteins is in close agreement with the earlier findings of MacDonnell et a1. (1953) and Lineweaver and Murray (1947) for ovomucin and ovomucoid, respectively. Significant differences among the various protein gel firmness were observed (Table 28). Lysozyme produced the firmest gel (43.98 g x cm) of all proteins, followed by globulins (14.26 g x cm), ovalbumin (5.68 g x cm) and conalbumin (3.61 g x cm). Percentage drainage measurements reflect the ability of gels to hold liquid in the interstices and this in turn is dependent on gel strength. As expected, lysozyme Table 27. 1214 Coagulation Temperature Ranges of Albumen Proteins Solutionsa and T-Statistic. Protein Temperature RangeC t-statisticd Solution Initial Drop Lyso- Glob- Conal- Oval- zyme ulins buInin bum1n C Ovomucin NCe Ovomucoid NCe Lysozyme 81.510.7 74.5i0.0 13.435** 43.379***20.000** Globulins 72.0:0.7 59.9:0.1 26.385** usf Conalbumin 57.310.4 56.0i0.0 -56.993*** Ovalbumin 71.5:0.0 71.0:0.0 LYS-OVD NCe LYS-GLOB 67.510.0 66.310.4 -28.000**-9.000** LYS-CON 58.3:0.4 56.810.4 -41.590*** st LYS-OVB 70.6:O.5 69.81-0.7 -l7.400** NSf GLOB-OVD 76.1i0.2 75.1i0.2 8.004“ GLOB-CON 57.8i0.0 57.0i0.0 -28.500** NSf GLOB-OVB 70.410.2 69.0:0.0 -3.153* -8.999** OVD-CON 58.810.4 S7.8i0.4 4.242* OVD-OVB 73.5iO.7 72.610.5 4.000* CON-OVB 58.1i0.2 57.110.2 3.130*-106.986*** aProtein concentration = 1. 27% , pH = 8 . 0 , ionic strength = O. 275 . Mixtures are l:lon a weight basis; LYS = lysozyme , OVD = ovo- mucoid, GLOB = globulins, CON = conalbumin, OVB = ovalbumin. °N=L d eNC = no coagulation. f *significant at PI<0.05. **Significant at PIov caesnam>o cam .Acoov cflfisnamcoo .Ap>ov pwoosso>o .Aquv mfihuomma mo cofiuwppm on» zuH3 Enasmmoo AQOHOV mcwasnon mo mandamup mmmusmoumm pom .mmeEHHm Hum .musumummfiou sofiumasmmoo ca momcmno mo COmHHmmEoo 4.6H.mflm a2. .89 a»: a: a: can a: MI».— n>= .39 a»: a: afiaa an; may; name use tau—a eye a“; use 3.5 nun-a a“; nun—u nan—a :39 O ..' o ..'.C O on on a a .I I. n 3 I o. S I u u u I n no on ..V. .1. H :3 H 00 M x .9 .3 X 0 o a u on 2 I 3 . Acouv GHEDQHMCOO «Em . Sou/0V cunoosfio>o . Anodwv mcflasnoam . Amway 93309: HO scapegoat on» sun; Esasmmoo Ant/0v cfifisnamuyo mo mmmcflmup owmucmouom was .mmmcfiufiw Hum .wusumummfiwu coauoasmmoo a“ momcmno mo somHummEoo d .56.?“ :3 a; 2:“ m: :3 :3 23¢ m: :3 a: 3.3 a: + + + + + + + + + + + + a; :9 =9 :9 =9 :9 :9 :9 =9 :9 a: n: :9 a; a; n.“ \O JO . .00 Cl PO I on fl \\ l N 1 an no I. a S I. I u I Ion H H u H I. o an \x N I. I B so H 00 M I on I a 00 3 VP nu m 50% I 1 ON 1 O— I Do 136 .An>ov :flEonHm>o 0cm .Ap>ov tacosao>o .AQOHOV mcflasnon .Ammqv meanom>a mo coaufipom on» saws Esasmmoo Acoov definnamcoo mo mmmcflmnp mmmucooumm pom .mmmsfiuflm How .musumummamu scandasmmoo ca momcmno mo cOmHuwmfioo d 4:”.mwm a: :2. 3.5 a: a; a: ammo m: =9 a»: 93¢ a: + + + + + + + + zen .39 :8 .39 :3 2...? =3 23 :3 sec :3 :3 =3 .89 :3 ... e ... a C. \ 0.. eueue \ \ "one”. \ I s .3.” ”HUN” I 8 I . ”NH” 9 eueuee 3 eeeee ql. e e e n a s I... v I. 3 I M H I S v u as" a a a a .. M I s n x I s - m "a I. 3 I o u 3 I E. I 8 I w _ 137 The decrease in lysozyme gel strength reflected the combined effects of variable extent of aggregation of the different proteins and dilution effects. Increases in percentage drainage paralleled formation of weaker gels. Similar trends were observed for the effects of double combinations in the other protein gels (Figures 16, 17, and 18). Ovomucoid raised the denaturation temperature of globulins, but gel strength decreased considerably. It appeared that ovomucoid did not participate in the aggre- gation phenomena leading to formation of a weaker gel, which also reflected in the poor liquid holding ability of the gel. I Conalbumin and its combinations consistently showed the lowest coagulation temperature. Lysozyme and globu- lins double combinations showed the highest gel strengths which indicated these proteins were actively involved in development of the coagulums. The effect of heating on the sulfhydryl (SH) content and pH of the various protein solutions are summarized in Table 30. In general, heating decreased the number of SH groups of the coagulums. Significant reductions in SH groups of globulins, ovalbumin, and GLOB x OVB gels were observed. In contrast, heating significantly increased SH content of lysozyme, conalbumin, and OVD x CON gels. The occurrence of extensive unfolding of polypeptides without formation of inter and/or intramolecular SS bonds does not seem a viable possibility for these gels. In 138 .mooo.o v m on unmoHHHomHmIII .Ho.o v m on HomoHoHoonII .mo.o v o no HomoHoHaon. mo.o u m um HGMUHMHcmfim no: u mzm p Nuzo ofloooeo>0Io>O .cflfisnam>0Im>o .cwesn IamcooIzoo .mcflasnonImoqo .oE>NOm>HImwA “manna unmwm3 m :0 HHH mum mousuxfiz WGSH M> UQHMEIHHWW n .mhm.o u sumcmuum Deco“ .o.m n ma Hmwuflcfl .mhm.a u GOHHMHucmocoo cwououmm oo.o H mm.h vmo.o H mmm.m pmom.m m>0Izoo mo.o H ~>.h aho.o H mao.m paov.m m>oIo>o ha.o H o>.> Haomm.mI «mo.o H mmH.H moo.o H omm.o _ ZOOIQ>O Ho.o H H>.h «mma.m hoa.o H vam.~ moa.o H moo.v m>OImoqw Ho.o H mm.> oma.o H Hmm.a Umoa.~ ZOUImoqw oo.o H om.» mmo.o H vav.a pmom.~ Q>OImoqo oo.o H mm.> vmo.o H hmo.~ paaa.m m>oIqu mo.o H om.o mHo.o H -m.H omom.o , zooImwu oo.o H mm.h mao.o H mmm.H paao.m moqumwA No.0 H mm.h «Hom.m mm~.o H mom.v moo.o H mom.m GHEsQHm>o 00.0 H «h.h «oom.vHI hmo.o H mmm.H omo.o H ovo.a cHEsQHmcoo oo.o H mm.n «Haaam.mha moo.o H hmo.m moo.o H va.m mcHHSQOHw oo.o H mm.h *th.vI mmo.o H mNH.H mmo.o H mmm.o mshuomaq mHmEmm m moa\m0HOE m E: ammo I Esasmmoo somwsaom m H o oHHmHHmum H ouomuooo HauomaoHsm noHouoHo m.mcoflusHom mcfimu Ionm cmesnafi mo no can ucoucoo Hmnphnmasm wsu so mcfiummm mo Hommmm .om manna 139 turn, the observed incomplete solubilization of the gels during the determinations may have caused higher absorbance readings, affecting the results. A significant decrease in pH from 8.00 to 7.58 (treatment total) occurred with heating. This indicated more acidic groups were exposed with the treatment. The time-temperature relationship of protein mixtures gels are illustrated in Figures 19 and 20. As can be seen in Figure 19, the mixture containing all proteins showed a slight decrease in temperature (0.5°C) after about 15 min. of heating followed by a fairly rapid increase in temperature. The mixture lacking ovomucin coagulated at two different temperatures at about 23 and 38 min. of heating. 'The other protein mixtures did not show an apparent drop in temperature during coagulation, but the slight decrease in rate of heat penetration, seen in the curves, marked the onset of coagulation. The mixture with no globulins showed a slight drop in temperature during coagulation (Figure 20). It can be seen that the mixture lacking conalbumin coagulated after about 25 min. of heating, whereas without ovalbumin, coagu- lation took place earlier. The characteristics of the gels prepared with the protein mixtures are summarized in Table 31. Mixtures con- taining ovomucin, lysozyme, and conalbumin simultaneously showed an initial coagulation temperature ranging from 55 to 59°C. Removal of conalbumin raised coagulation 1110 90 e 80.. / i e 70- . ., ,/./ o‘ m 60_ a = I .- v c a In ”P o. . a »’ u . .— 40_ H All proteins H No ovomucin -’ M No lysozyme 30- v—v No ovomucoid e /V 0 ll L I l L I 0 5 15 25 35 45 55 IIIIE,IIIII Fig. 19. Comparison of time-temperature curves of various solutions with different protein composition. Heating rate 0.74°C/min, protein concentration = 1.27%, pH = 8.0, ionic strength = 0.275. 1111 “1 H1 .' 70? e o 0‘ m m_ a a .— ‘ 3 so - L I In .— 40.. D—-D No globulins 30.. H No conalbumin M No ovalbumin )/ 0 [III I I I I 0 5 15 25 35 45 IIIIE,IIIII \ Fig.20. Comparison of time-temperature curves of solu- tions with different protein composition. Heating rate = 0.74°C/min., protein concentration = 1.27%, pH = 8.0, ionic strength = 0.275. 1&2 .cHOSEO>OIZO “wm.ma . m 09HM> Mumumsflflm HM .mo.o v m um “snowmwcmwm mum :EoHoo m cflnuw3 mm:am>« .wom .cwsonam>onmo .pwooseo>0Ioo .mcwasnonlqw .mEmNommalwq o no mousuxfie ca u m mo mmusuxfia :fl mcfimuoum msu mo mao>oao .mh~.o u numcmuum owned .o.m H mm HmHuHCfl .w>~.H n coaumuucmocoo cwououmn .m:0flumcHEHmump 03» mo mmmum>m mum mooam>m 8.0.6326 3mg“ ooéfimfie $63.3 8.3%.... mfiwmmnmwo 33:8 mmoéfioemé mg; 8.33;. 353.3 afioimd m.mmuo.mm mouoouoouooumquzo gaggle; 2m.~ 8.38; 243.8 39336 M.WMHM.MT menoonoouqoufi 236334 S~.~ 863mg .5636 3.334 c.2393 mouoonoofiouzo «36384 3&4 3.3mm; m.~...m.$ 2.335 «.mmumém mouoouoousfiuzo 23.3234 mmo.~ 3.33.“ 3.35.3 «3.33.3 93:93 mouooaqoquuzo «8.3.3.04 Sim 8.335 253.3 Hméfimeé 0679mm mouoounoucfiuzo mooéflmmmé can; 8.3.36 343.3 «3.3.2.3 93.6.3 oouoonqoifiuzo Manama m moa\moaofi m onm 0o Esadmmoo mcofiuoaom mm ommcamuo numcmnum oHohHWMWWmB mnouxaz camuoum ucoucooa “pagoda . Hmo cofimasmmoo o . . n.mmusuxwz mcwopoum cofisnad mom nuwz mom: maow mo mmowumflnouomumno Hmoafionoloowmhnm .Hm ®HQMB 143 temperature range to 65-75°C. Similar effect was observed with the mixture lacking lysozyme. Omitting ovomucin in- duced coagulation at two temperatures, 59.4-58.9°C, and 71.3-70.8°C. The control also displayed two coagulation temperature ranges, 61.5-62.5°C and 73.0-71.0°C. The first coagulation temperature range indicated conalbumin and possibly some other protein(s) denatured with partial aggregation, and coagulation completed at the second tem- perature range. The destabilizing effect of the least heat stable protein (conalbumin) on the other proteins in a fashion similar to that of the double Combination treatments was affirmed with the mixtures of five or six proteins. In the mixture lacking lysozyme, the observed higher coagula- tion temperature may have resulted from the extremely vis- cous nature of the solution. This characteristic appeared to have, in part, prevented intermolecular associations of conalbumin with the other proteins. Firmest gels were produced with the mixtures with no ovalbumin (12.17 g x cm), and no ovomucoid (10.48 g x cm). Control and gel with no ovomucin ranked second, with values of 6.34 and 6.73 g x cm, respectively. Gel with no globulins (5.97 g x cm) and gel with all proteins (5.54 g x cm) ranked third, followed by the gel with no lysozyme (4.36 g x cm) and the gel with no conalbumin (3.79 g x cm). In general, gels containing ovomucin in the five proteins combinations exhibited better liquid retention luu with percentage drainage values ranging from 0—62%. No liquid drained from gel lacking lysozyme. Evidently ovo- mucin retained most of the liquid in the swollen carbo- hydrate moieties. This suggeststhat,in presence of lyso- zyme,formation of ovomucin-lysozyme complex results in decreased ability of ovomucin's water absorption capacity. The gel with no ovomucin had the highest liquid loss (66%) followed by the gel containing all proteins (65%). The control gel lost about 61% liquid. The observable trend in the effect of heating on pH was a decrease except that for gels with no ovalbumin or conalbumin, an increase was noted. The significance or causes for these higher pH values were not apparent. The sulfhydryl content of the coagulums were con- sistently lower than that of the solutions. This supports the idea of SH-SS interchange reactions involvement in cross-linking of polypeptides. Electron Microsc0py Ultrastructural examinations of protein foams and gels may provide some further insight as to the visual nature of protein-protein interactions in these systems. Selected foams were examined in a transmission electron microscope (TEM) and in a scanning electron microsc0pe (SEM), whereas gels were examined in a scanning electron microscope. 145 Examination of Foams The mechanism of foam formation has been postulated by many investigators (Peter and Bell, 1930; Adam, 1941; Thuman et al., 1949). These authors concurred that forma- tion of a cohesive layer of insoluble protein at the air- liquid interface was involved in the process of air incor- poration. As can be seen in Figure 21, transmission electron micrographs of whipped protein solutions revealed the presence of a layer of high electron density at the film surface, sustaining foam theory. The character of the layers seemed roughly similar in lysozyme, globulins, conal- bumin, and ovalbumin foams, with formation of conglomer- ates of unfolded polypeptides being evident. The protein clusters noticeably formed a network of parallel filaments that were easily dislodged from the liquid phase (micro- graph B). The surface film in the control foam varied in nature and displayed a double layering phenomenon of the unfolded polypeptides. The overall thickness of the film was diminished and the surface appeared smooth, but formation of conglomerates was still noticeable. There was no visual discernment of which proteins actively participated in formation of the film. In all likelihood, ovomucin, lyso- zyme, and globulins were involved. In all films the liquid phase displayed a sandy Fig. 21. Transmission electron micrographs of albumen pro- tein foams. a) Lysozyme b) Globulins c) Conalbumin d) Ovalbumin e) Control 1146 0.1 um ,1 147 background with no apparent preferencial distribution of the proteins. Occasionally, small clusters of medium electron density were visible in the light matrix (indi- cated by arrows). Figures 22, 23, and 24 show scanning electron micro- graphs of the various protein foams at several magnifica- tions. The severe breakdown of the fragile protein foams during handling concealed visualization of air cells (Figure 22). However, partial discernment of air inclu- sions was possible in globulins and control foams. In all foams the surface film delineating the air cells was exten- sively ruptured revealing numerous membrances underneath it. There was no apparent formation of a thin membranous cohesive film in lysozyme foam when examined at a higher magnification (Figure 23, micrograph A). The film was rather rough textured and appeared quite rigid. In con- trast, the other protein films, although severely interrupt- ed, showed the formation of a continuous smooth sheet which appeared flexible in nature. Some variability in membrane thickness was also observable. Globulins (micrograph B), ovalbumin (micro- graph D), and control (micrograph E) films looked very thin, conalbumin (micrograph C) film seemed slightly thicker and less flexible, whereas lysozyme film was considerably thicker. At the highest magnification (Figure 24) all protein films looked "textured," exhibiting numerous small granules. Fig. 22. Scanning electron micrographs of albumen protein foams. a) Lysozyme b) Globulins c) Conalbumin d) Ovalbumin e) Control 148 Fig. 23. Scanning electron micrographs of albumen protein foams. a) Lysozyme b) Globulins c) Conalbumin d) Ovalbumin e) Control 1119 Fig. 24. Scanning electron micrographs of albumen protein foams. a) Lysozyme b) Globulins c) Conalbumin d) Ovalbumin e) Control 150 151 These were probably unfolded polypeptide clusters forming the surface layer. A few salt crystals were also seen in most micrographs. Scanning electron micrographs of whipped protein solu- tions differing in lysozyme content are displayed in Figure 25. Likewise, it was noted that breakdown of the foams occurred with manipulation. However, it was quite evident that the foam with high levels of ovomucin and no lysozyme was more severely disrupted (micrograph A). At higher mag- nification (micrograph B) it seemed that the membrane was less adhesive in nature, as well as thick and fluid, draping over the torn edges. Lysozyme appeared to strengthen the foam (micrograph C). There was less rupturing of the mem- braneous layer and at higher magnification (micrograph D) the film looked thin and somewhat flexible, but not amorphous. In previous testings it was determined that the protein solution with high ovomucin content and without lysozyme exhibited excellent foaming ability but produced smaller cakes. In contrast, with lysozyme foaming was drastically depressed whereas cake volume improved considerably. The detrimental effect of ovomucin on volume has been attributed to its excessive insolubilization at the air- albumen interface leading to formation of more stable foams but with less expansive characteristics (MacDonnell et al., 1955). Examination at the microscopic level confirmed that without lysozyme the viscous ovomucin fraction concentrated at the film surface. It appeared that ovomucin contribution Fig. 25. Scanning electron micrographs of foams differing in lysozyme content. Other proteins levels: ovomucin 2.5%, glob- ulins 3%, ovomucoid 5%, conalbumin 6%, ovalbumin 77.5-83.S%. a) and b) 0% lysozyme c) and d) 6% lysozyme 152 153 to foaminess was primarily related to its ability to increase the solution viscosity, and thus facilitated film formation. However, ovomucin could not maintain or produce a cohesive membranous layer, which indicated the protein itself is unable to participate in intermolecular associations. Hence, the overall effect of ovomucin on the process of foam forma- tion does not lend support to the observations of MacDonnell et al. (1955) since considerably more unstable films were formed. In addition, the fact that the cake prepared with the protein solution containing a high level of ovomucin and no lysozyme expanded normally and then collapsed during baking, suggested ovomucin also affected the heat coagulative process of the protein film. The poor foaming characteristics of solutions with lyso- zyme and ovomucin were consistent with the observations of Garibaldi et al. (1968) in studies of ovomucin-lysozyme interaction. These authors pointed out that the formation of the complex depressed egg-white foaminess. Kato et al. (1975) indicated the carbohydrate units of ovomucin were involved in electrostatic associations with lysozyme. These evidences suggest that the viscous nature of ovomucin is altered in the interaction phenomenon. The appearance of the foam prepared with the solution containing ovomucin and lysozyme confirmed this observation. 15“ Examination of Gels When heated, proteins denature and usually association of the unfolded polypeptides into a three-dimensional net- work ensues. The extent of unfolding is dependent on the physicochemical characteristics of the proteins and environ- mental factors that exert an influence in these character- istics. Figures 26 and 27 show scanning electron micrographs of selected albumen protein gels. At the lowest magnifica- tion (Figure 26) lysozyme, conalbumin, ovalbumin, and control gels were similar in appearance, displaying a disorganized aggregation of the polypeptide chains. Globulins gel was remarkably different and showed a definite orientation of the cross-linked polypeptides into multiple parallel mem- branes which were interconnected by numerous projections. The possible causes for the differences observed in gel strength and percentage drainage of the various gels were not visually apparent at this level of magnification. The slightly better ability of the globulins gel to hold water in the interstices over that of conalbumin and ovalbumin gels could have been the result of the different orientation of the polypeptides in the gel matrix. At the next level of magnification (Figure 27) the dis- organized arrangement of the polypeptide aggregates in lyso- zyme, conalbumin, ovalbumin, and control gels appeared grape- like clusters. Moreover, differences in cluster size were noticeable in the various gels. Lysozyme gel (micrograph A) Fig. gels, 26. a) b) c) d) e) Scanning electron micrographs of albumen protein Lysozyme Globulins Conalbumin Ovalbumin Control 155 Fig. 27. Scanning electron micrographs of albumen protein gels. a) Lysozyme b) Globulins c) Conalbumin d) Ovalbumin e) Control 156 157 was characterized by a finer gel network of considerable small aggregates. Conalbumin and ovalbumin gels were roughly similar and showed mostly large conglomerates. The control gel exhibited clusters of intermediate size range, whereas globulins gel had the smallest polypeptide aggregates very tightly associated in the membrane-like arrangement. The scanning electron micrographs of albumen protein mixture gels are shown in Figure 28. Micrographs A and B illustrate lysozyme-globulins combination gel at two levels of magnification. Globulins exhibited the same trend in the orientation of the unfolded polypeptides with occasional in- clusion of lysozyme clusters. At higher magnification it was clearly seen that lysozyme clusters were larger in size (compare with micrograph A, Figure 27). The pebbly appear- ance of some clusters resulted from the association of lyso- zyme and globulins polypeptides. The striking difference in size of the lysozyme aggre- gates appeared to be related to its premature denaturation in the presence of globulins. McKenzie et a1. (1963) pointed out that gelation may occur before a large number of poly- peptide chains are unfolded and when this happens a coarser gel network forms. Micrographs C and D display the gel prepared with 5 pro- teins (ovomucoid omitted). There were several protein strands which appeared to be ovomucin held by aggregates of polypep- tides (micrograph C). A few large clusters of denatured Fig. 28. Scanning electron micrographs of gels prepared with protein.mixtures. a) and b) Lysozyme-Globulins c) and d) Ovomucin-Lysozyme-Globulins-Conalbumin- Ovalbumin 158 159 proteins (indicated by arrows) were noticeable. At higher magnification (micrograph D) the disorganized arrangement of the polypeptides in the gel matrix was evident. It seemed as though globulins predominated in the gel. The coagulated protein appeared to be binding, and in some in- stances, envelOping the other protein aggregates. These were relatively small in size and the overall appearance of the gel matrix suggested a stronger structure. This obser- vation was consistent with gel strength determination. SUMMARY AND CONCLUS IONS The primary objective of this study was to determine the functional properties of various albumen proteins and specific protein-protein interactions in two food systems. Foamability was evaluated in an angel food cake system and coagulability in a custard model system. Ovomucin, lysozyme, globulins, ovomucoid, conalbumin, and ovalbumin were isola- ted from egg white. They were further tested individually and in combinations in these food systems. Combinations of three levels of each protein determined with a mixtures ex- perimental design were studied in the cake system. The effects of the protein-protein interactions in this system were analyzed through response surface methodology to designate protein levels for optimization of cake parameters. Textural characteristics of selected foams were studied in transmission and scanning electron microscopes, whereas selected coagulums were examined in a scanning electron microsc0pe. Evaluation of foaming properties of individual protein solutions revealed that globulins solution had very good air incorporation capacity and produced a large cake of excellent textural characteristics. Ovalbumin solution exhibited poor foaming ability and formed a foam after a 160 161 relatively long whip time. The cake had a large volume but was coarse textured with thick air cell walls. Conalbumin and lysozyme solutions showed very poor foamability, whereas ovomucin and ovomucoid had none. In interactions studies, inclusion of both ovomucin and globulins at levels of 0.00, 1.25, and 2.50%, and 0, 3, and 13%, respectively, significantly improved foaminess of the solutions. However, cake volume progressively decreased, showing a strong significant negative correlation with foam- ability. Addition of lysozyme to these mixtures at levels of 0, 3, and 6% significantly depressed foaming power of the solutions, resulting in considerable improvement of cake volume. Conalbumin, ovomucoid, and ovalbumin showed no significant influence on foamability and angel food cake volume. Tenderness and compressibility measurements reflected the degree of aeration of the products. More tender cakes were associated with larger cakes and low compressibility values. In determining the sulfhydryl content of foams it was found that whipping reduced the number of SH groups. This implied that formation of the cohesive film at the air- solution interface involved inter- and/or intramolecular sulfhydryl-disulfide interchange. Multiple regression analysis revealed the protein mix- tures were significantly affecting viscosity, foaming index, volume, and cake tenderness. Partial positive effects on 162 viscosity were associated with ovomucin and ovalbumin, whereas conalbumin and the interaction terms of ovomucin with the other 5 proteins reduced viscosity. Foaming index was partially positively affected by ovomucin, and interaction of ovomucin with globulins. Interactions of ovomucin with ovalbumin, lysozyme, ovomucoid, and conal- bumin had a negative effect on foaminess. Partial positive effects on cake volume were observed for ovomucin, lysozyme, and ovalbumin. Interactions of ovomucin with ovalbumin, globulins, and ovomucoid, as well as lysozyme with globulins, ovomucoid, conalbumin, and ovalbumin, significantly reduced cake volume. Cake tenderness was reduced by conalbumin, ovalbumin, and the interaction of ovomucin with lysozyme. Ovomucoid and conalbumin, conalbumin and lysozyme, as well as conalbumin and ovalbumin interaction variables produced more tender cakes. Surface response methodology was used to evaluate pro- tein solution foaming index and cake volume responses as a function of ovomucin, lysozyme, and globulins levels. It was found that a target angel food cake could be prepared with ovomucin, lysozyme, and globulins levels ranging from 0.2-1.0%, 0.0-1.8%, and 12.2-14.8%, respectively. Studies of gelation properties of the albumen proteins showed that conalbumin was the least heat stable protein with a denaturation temperature of 57.3°C. Globulins and oval- bumin ranked second with denaturation transition temperatures of 72.0 and 71.5°C, respectively. Lysozyme denatured at 163 81.5°C while ovomucin and ovomucoid showed no coagulation abilities. Lysozyme produced the strongest gel, followed by globulins, ovalbumin, and conalbumin. In the double combinations studies it was found that aggregation of polypeptides occurred near the denaturation transition temperature of the least heat stable protein. Therefore, in combinations of lysozyme, globulins, and oval- bumin with conalbumin, denaturation was apparent at 58.3, 57.8, and 58.1°C, respectively. Ovomucoid consistently increased the coagulation temperature ranges of globulins, conalbumin, and ovalbumin, and prevented coagulation of lysozyme. Gel strength varied according with the proteins present. The combinations of 5 or all proteins resulted in subtler changes of temperature during coagulation. The destabilizing effects of conalbumin on the other proteins were still apparent. The control mixture exhibited two distinct coagulation temperature ranges of 61.5-62.5°C and 73.0-71.o°c. Transmission electron microsc0py studies of foams con- firmed the presence of a layer of cross-linked polypeptides at the surface of the film enveloping an air inclusion. Scanning electron microscopic examination of a whipped so- lution with high levels of ovomucin and without lysozyme showed that the protein concentrated at the film surface and considerably decreased foam stability. The membrane appeared less cohesive in nature and draped fluidly over 164 the torn edges. Inclusion of lysozyme seemed to improve the overall foam appearance. The effects of lysozyme on foaminess in the presence of ovomucin appeared to be asso- ciated with formation of ovomucin-lysozyme complex. This complex seemed to alter the viscous nature of ovomucin. In the scanning electron microscopic investigations of selected coagulums it was found that lysozyme, conalbumin, and ovalbumin polypeptides aggregated in grape-like clusters of variable size. The control mixture gel also exhibited the same pattern. Globulins polypeptides appeared to tightly associate in membrane-like arrangements and showed excellent binding abilities. Smaller cluster sizes seemed to parallel gel firmness. The results of this study indicated that complex protein- protein interactions influenced the overall functional prop- erties of the various proteins. Readiness of foam formation was associated with the highly viscous ovomucin fraction, whereas stability was dependent on formation of a cohesive membraneous layer of partially cross-linked polypeptides at the film surface. Low cake volumes resulted from unstable foams and lack of heat coagulative properties of the membrane. Globulins alone could produce an excellent angel food cake. Lysozyme and globulins, and combinations of lysozyme with globulins, produced the firmest gels. Changes in denatura- tion temperatures were associated with the destabilizing action of more heat unstable proteins. SUGGESTIONS FOR FURTHER RESEARCH The observations and findings associated with the present investigation raised a multitude of questions relevant to protein functionality: 1. Studies of foaming properties of the various albumen proteins should be elaborated further under different envi- ronmental conditions. Observations of maximum foam stability attainment in the neighborhood of the isoelectric point of the protein and the variable behavior of proteins at the extremes of pH values have been reported. 2. Similarly, the effects of salts and pH on the coagu- lation abilities of the proteins would warrant further in- vestigation. 3. The contribution of hydrogen bonds, hydrophobic and electrostatic interactions to protein-protein interactions in association of polypeptides should be evaluated with specific chemical reagents. Urea, guanidine HCl and sodium dodecyl sulfate are known to disrupt hydrogen, hydrophobic and ionic bonds. 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Hence, leaving ovalbumin in blank: ovu LYS GLOB ovp CON ova Total 1 0.0 0 3 5 6 86.0 100 2 0.0 0 3 5 21 "“’ . 3 0.0 0 3 18 6 "“‘ : 4 0.0 0 3 18 21 ““' : 5 0.0 0 13 5 6 "“' : 6 0.0 0 13 5 21 ““' : 7 0.0 0 13 18 6 ""7' : 8 0.0 0 13 18 21 "“' : 9 0 0 6 3 5 6 ““' : 16 0.0 6 13 18 21 E 17 2.5 6 3 5 6 "“" : 32 2.5 6 13 18 21 39 5 100 178 179 This procedure was repeated leaving the other protein levels in blank, i.e., xlxzx,X.-x5, xlxzxa-x5,x5, xlxz-x.x5x5, xl-xax.x5x5, and -x2x3x.x5xs. However, since ovalbumin (X5) levels were set to 30 (low) and 89 (high) there were no possible combinations allowable at these levels. As it can be seen in the above calculations, the minimum and maximum possible levels for ovalbumin were 39.5 and 86.0, respectively. Therefore the other combinations were automatically excluded from the design. A total of 32 vertices were generated fol- lowing this procedure. The centroids of the 3-dimensional face were found by averaging the vertices treatment levels with any two constant protein levels. Therefore, vertices l to 8, with ovomucin and lysozyme showing equal levels, were averaged; vertices l to 4 with ovomucin and globulins displaying equal levels, and so on. The fifth dimensional face represented the average of all treatment levels (ver- tices and 3-dimensiona1 face centroids). APPENDIX B Observed and Predicted Values 180 181 Table 32. Observed and Predicteda Values of Protein Mixtures Physical Parameters. Treatmentb VisCOSity Surface TenSion Foaming Index Ob- Pre- Ob- Pre- Ob- Pre- OVN LYS GLOB OVD CON OVB served dicted served dicted served dicted cps x g/cm3 Dynes/cm cm3/g/min L L L L L H 1.76 1.73 50.90 48.71 0.99 0.85 L L L L H H 1.69 1.68 49.40 49.54 0.93 0.07 L L L H L H 1.86 1.82 48.30 49.06 0.67 0.00 L L L H H N 1.73 1.73 49.50 49.55 0.75 0.00 L L H L L H 1.65 1.79 49.50 48.21 2.10 0.29 L L H L H N 1.95 ‘1.86 49.60 49.20 1.46 0.75 L L H H L N 1.83 1.79 48.80 48.38 1.34 0.00 L L H H H L 1.72 1.83 48.40 48.90 1.61 0.00 L H L L L H 1.63 1.72 50.30 48.25 1.16 1.99 L H L L H N-H 1.64 1.64 48.80 48.74 1.06 0.75 L H L H L N-H 1.75 1.76 47.30 48.26 0.80 1.71 L H L H H N-L 1.73 1.64 48.80 48.34 1.19 0.34 L H H L L H 1.91 1.74 46.20 46.52 4.12 3.97 L H H L H N 1.71 1.77 47.50 47.12 3.22 3.84 L H H H L N 1.62 1.69 46.00 46.35 3.36 4.16 L H H H H L 1.69 1.69 47.80 46.36 3.40 3.47 H L L L L H 2.20 2.24 47.50 48.48 5.55 6.37 H L L L H N-H 2.26 2.27 49.30 48.79 6.05 7.36 H L L H L H 2.26 2.35 49.00 48.13 9.45 10.25 H L L H H N 2.46 2.34 48.60 48.31 10.67 11.22 H L H L L H 2.37 2.31 47.50 48.21 21.83 23.08 H L H L H N 2.48 2.46 49.90 48.83 27.43 25.26 H L H H L N 2.43 2.33 47.50 47.81 29.63 27.44 H L H H H L 2.28 2.44 47.10 48.27 29.42 29.23 H H L L L H 2.41 2.22 49.40 49.77 1.89 2.04 H H L L H N 2.12 2.21 49.80 49.82 1.84 2.55 H H L H L N-H 2.23 2.28 48.70 49.16 1.27 2.00 H H L H H N-L 2.12 2.23 48.20 49.06 1.23 2.27 H H H L L N-H 2.04 2.24 48.60 48.33 1.76 2.26 182 Table 32--Continued Viscosity Surface Tension Foaming Index b Treatment Ob- Pre- served dicted Ob- Pre- served dicted Ob- Pre- OVN LYS GLOB OVD CON OVB served dicted cps x g/cm3 Dynes/cm cm3/g/min H H H L H N-L 2.38 2.35 49.00 48.67 2.24 3.81 H H H H L N 2.21 2.21 47.90 47.70 3.48 2.68 H H H H H L 2.47 2.29 46.10 47.89 4.26 3.53 L L N N N N-H 1.91 1.84 49.70 49.60 1.37 2.07 L H N N N N 1.71 1.76 49.30 47.71 1.28 4.66 H L N N N N-H 2.35 2.40 49.90 48.68 29.26 17.42 H H N N N N 2.26 2.31 47.80 48.66 2.87 2.65 L N L N N N-H 1.66 1.77 48.00 48.37 0.88 1.72 L N H N N N 1.75 1.82 45.50 47.34 2.63 3.33 H N L N N N-H 2.48 2.32 49.60 48.57 4.49 1.17 H N H N N 2.34 2.38 48.50 47.94 9.15 10.50 L N N L N 1.84 1.79 46.20 48.00 0.99 1.33 L N N H N 1.77 1.79 46.50 48.08 2.31 1.29 H N N L N N-H 2.46 2.33 49.50 48.91 6.72 6.74 H N N H N N 2.36 2.35 50.30 48.52 6.32 9.24 L N N N L H 1.75 1.69 46.70 47.18 1.89 0.73 L N N N H N 1.74 1.67 48.20 47.90 1.91 0.50 H N N N L H 2.14 2.21 48.30 47.73 4.90 7.74 N L L N N H 1.95 1.99 47.60 48.21 2.82 2.95 N L H N N N 2.28 2.07 49.70 47.86 3.77 10.74 N H L N N N-H 1.93 1.93 46.80 47.91 1.15 0.00 N H H N N N-L 1.94 1.97 46.00 46.33 2.79 0.67 N L N L N H 1.86 2.01 44.60 48.24 3.89 5.32 N L N H N N 2.09 2.04 48.30 48.16 3.22 8.50 N H N L N N-H 1.96 1.95 46.00 47.74 1.85 0.00 N H N H N N-L 1.98 1.94 50.00 47.31 1.34 1.11 N L N N L H 1.86 1.90 46.00 47.42 3.58 5.09 N L N N H N 1.93 1.93 47.80 47.83 3.36 8.15 183 Table 32--Continued Treatmentb Viscosthy Surface Ten51on Foaming Index Ob- Pre- Ob- Pre- Ob- Pre- OVN LYS GLOB OVD CON OVB served dicted served dicted served dicted cps x g/cm3 Dynes/cm cmB/g/min N H N N L N-H 1.95 1.84 46.80 46.91 1.21 0.00 N L N N H N 1.85 1.94 48.90 47.28 3.18 1.75 Control 2.02 2.04 46.70 47.27 3.08 2.60 a . . Based on regre351on analySis. bOVN = ovomucin, LYS = lysozyme, GLOB = globulins, OVD = ovo- mucoid, CON = conalbumin, OVB = ovalbumin. L = low, N = normal, H = high. Numerical values for low, normal, and high levels are listed in Table 9. 18A Table 33. Observed and Predicteda Values of Angel Food Cake Parameters. Compress- Treatmentb Volume Tenderness ibility OVN LYS GLOB ovp CON OVB Ob- Pre' Ob- Pm" Ob- Pre- served dicted served dicted served dicted 31113232212335:mmmmmt‘t‘t‘t‘r‘t‘t‘t‘t‘bfif‘t‘t‘t‘t‘ mmmmfit‘t‘t‘t‘t‘t‘t‘mmmmmmmmt‘fit't‘t‘L-‘t‘t‘ bbt‘hmmrnmr'vbvmmmmvbvvmmman-‘L-‘r't' mmbbmmbbmznbbmmbhmmhvmmbbznznr-‘r; vzzmzmma: N-H N-H N-L N :1: t" Z Z I I {1:1-'22 mbmumhmrmhmbmhmt—‘mhmvmbmr'mb 331.422.1231 :1: 2 H L 294 271 274 270 295 290 302 273 294 284 300 275 297 294 296 281 217 240 206 191 251 232 217 208 312 311 296 286 C!!! 3 282 272 281 267 302 294 299 284 297 290 298 287 292 286 290 275 232 232 208 204 237 238 210 203 302 305 280 277 work/gc 3.444 3.311 3.195 3.663 3.353 3.428 3.135 3.356 3.141 3.133 3.439 3.751 3.302 3.075 3.203 3.184 3.643 3.412 3.412 3.248 3.992 3.723 3.292 3.120 2.938 3.004 3.226 3.082 2.946 3.112 2.689 2.641 3.500 3.631 4.531 3.881 3.373 3.383 3.030 3.203 3.468 3.428 3.661 3.934 3.077 2.994 2.845 2.975 4.004 4.203 3.385 3.936 4.650 4.146 3.581 3.433 C work/cm 0.763 0.821 0.794 0.903 0.783 0.707 0.570 0.647 0.848 0.715 0.932 0.779 0.598 0.656 0.523 0.547 0.829 0.674 0.963 0.782 0.667 0.758 0.640 0.707 0.533 0.586 0.645 0.664 0.597 0.724 0.600 0.604 0.955 0.926 1.300 1.048 0.841 0.976 1.174 0.949 0.850 0.938 0.776 1.036 1.101 1.043 0.905 0.959 0.542 0.573 0.632 0.718 0.801 0.821 0.581 0.798 185 Table 33-—Continued Compress- Trea ntb Vblume Tenderness ibility 0VN LYS GLOB OVD CON ova Ob- Pre' Ob" Pre' Ob- Pre- served dicted served dicted served dicted cm3 work/gC work/cmc H H H L L N—H 288 282 4.046 3.767 0.674 0.603 H H H L H N-H 290 286 3.668 3.729 0.936 0.711 H H H H L N 245 256 3.573 3.493 0.723 0.905 H H H H H L 250 249 2.928 2.886 0.721 0.803 L L N N N NBH 290 301 3.574 3.266 0.771 0.660 L H N N N N 295 309 2.761 3.033 0.668 0.636 H L N N N N-H 223 220 2.980 3.340 0.881 0.970 H H N N N N 288 280 3.886 3.575 0.546 0.737 L N L N N N-H 294 277 2.824 3.105 0.739 0.802 L N H N N N 287 285 2.707 2.851 0-716 0.727 H N L N N N-H 230 242 3.352 3.427 0-950 0.918 H N H N N 211 234 3.047 3.135 1.410 0.959 L N N L N H 282 279 2.959 3-062 0-739 0-959 L N N H N 280 276 2.890 2.928 0.899 0.907 H N N L N N-H 236 252 3.765 3-554 0-978 1.072 H N N H N N 208 225 2.958 3-046 1-207 1-181 L N N N L H 289 296 3.483 3-422 0-670 0-653 L N N N H N 290 287 3.968 3.544 0.745 0.662 H N N N L H 247 241 3.462 3.785 1.011 0.838 N L L N N H 243 231 3.419 3.492 0.892 0.857 N L H N N N 257 248 3.548 3.346 0.968 0.814 N H L N N N-H 257 278 3.728 3.611 0.674 0.722 N H H N N N-L 258 270 3.319 3.207 0.422 0.691 N L N L N H 238 242 3.969 3.654 0-995 1-051 N L N H N N 245 235 3.611 3.296 0.803 0.975 N H N L N N-H 265 274 3.602 3.478 0.679 0.826 N H N H N N-L 272 269 2.369 3.291 1.872 0.940 186 Table 33--Continued b VOlume Tenderness Cigiiiis- Treatment Y OVN LYS GLOB OVD CON OVB Ob" Pre' Ob" Pm" Ob- Pre- served dicted served dicted served dicted cm3 work/gC work/cmc N L N N L H 235 250 3.398 3.738 0.778 0.733 N L N N H N 251 245 4.238 3.979 0.712 0.753 N H N N L N‘H 270 283 3.482 4.149 0.732 0.591 N L L N H N 285 251 3.597 3.517 0.558 0.902 Control 272 250 4.047 3.513 0.705 0.899 aBased on regression analysis. bOVN = ovomucin, LYS mucoid, CON normal, H = high. CWork = lb x cm. = lysozyme, GLOB = conalbumin, OVB Numerical values for low, normal, high levels are listed in Table 9. ovalbumin. L globulins, OVD = ovo- low, N = and APPENDIX C Comparison of Time-Temperature Relationships of Albumen Proteins with Their Double Combinations 187 188 70 _ 9 ° \ u 60 - 3 a .- d : so _ a. I u .— 40 L, Lysozyme D—D Glob.l.ys v—v Ovd.l.ys 30 ' O—-O Con-l.ys H Ovb-l.ys 011 1 L l 1 rs 15 25 35 45 55 TIIE,|IIII Fig.29.Changes in time-temperature curve of lysozyme solu- tion with the addition of globulins (Glob), ovomucoid (Ovd) ‘, conalbumin (Con), and ovalbumin (Ovb). Heating rate = 0.74°C/ min., protein concentration = 1.27%, ionic strength = 0.275, pH = 8.0. 189 90 O N). N). .9 so- u a = .- ‘ 50... 8 III a I fill '- 40,. / H Globulins :" O—-O Lys.Glob " v—v 0.78.0165 30 A—A Con-Glob D—D Ovb.Glob 1V 0 ,1 a 1 l i 1 0 5 15 25 35 45 55 nu, ma Fig.30. Changes in time-temperature curve of globulins solu— tion with the addition of lysozyme (Lys), ovomucoid (Ovd), conalbumin (Con), and ovalbumin (Ovb). Heating rate = 0.74°C/min., protein concentration - 1.27%, pH = 8.0, ionic strength = 0.275. 190 a) 80. "I. u 0‘ In HI_ 8 = .— ‘ a m wu- a. I m .— 401. H Conalbumin O—O Lys-Con H Glob.Con 30" V—V Ovd.Con H Ovh.Con x5 I OWLI 1 1 l J 5 I5 25 35 45 55 TIIE,III Fig.3fln Changes in time-temperature curve of conalbumin solution with the addition of lysozyme (Lys), globulins (Glob), ovomucoid (Ovd), and ovalbumin (Ovb). Conditions are the same as those in Fig.29 . 191 90 not 70b u o ‘ 60.. u x = .— < a: so. a. I u .— 40.. H Ovalbumin O—O Lys.0vb D—D Glob.Ovb 30“ H Ovd.OVb H COH.OVb a? o %4 4 4 1 1 5 15 25 35 45 55 TIIE,III Fig.32. Changes in time-temperature curve of ovalbumin solu- tion with the addition of lysozyme (Lys), globulins (Glob), ovomucoid (Ovd), and conalbumin (Con). Conditions are the same as those in Fig.29 . HICHIGRN sms UNIV. 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