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A study of protein surface hydrophobicity and structure-function analysis of proteins in spray dried… Cheng, Eugene S. Y. 2001

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A STUDY OF PROTEIN SURFACE HYDROPHOBICITY AND STRUCTURE - FUNCTION ANALYSIS OF PROTEINS IN SPRAY DRIED EGG ALBUMEN by EUGENE S . Y . CHENG B.Sc. in Agriculture (Hons.), University of British Columbia, 1998 A thesis submitted in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE in THE F A C U L T Y OF G R A D U A T E STUDIES Food Science We accept this thesis as conforming To the required standard THE UNIVERSITY OF BRITISH COLUMBIA February 2001 © Eugene S.Y. Cheng, 2001 UBC Special Collections - Thesis Authorisation Form http://www.library.ubc.ca/spcoll/thesauth.html In presenting t h i s thesis i n p a r t i a l f u l f i l m e n t of the requirements for an advanced degree at the Univ e r s i t y of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y a v a i l a b l e for reference and study. I further agree that permission for extensive copying of t h i s thesis for sc h o l a r l y purposes may be granted by the head of my department or by his or her representatives. I t i s understood that copying or p u b l i c a t i o n of t h i s thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission. Department The U n i v e r s i t y of B r i t i s h Columbia Vancouver, Canada 1 of 1 2/19/01 1:48 PM ABSTRACT In examining functional properties of food proteins, the ultimate goal of all researchers is to understand basic information relating functional properties to particular conformational or structural features of the protein. It is generally accepted that the molecular property of hydrophobicity plays an important role in the function of food proteins. In the first part of the thesis, surface hydrophobicity (S0) of 10 proteins, under varying conditions of pH (3.0, 7.0, and 9.0) and salt concentrations (0.01 and 1.0 M NaCl), measured using an uncharged fluorescent probe, P R O D A N (6-propionyl-2- (dimethylamino)naphthalene) was compared with S 0 determined with an anionic probe, ANS (l-anilinonaphthalene-8-sulfonate). S 0 values measured by both P R O D A N and ANS for each protein were statistically different (P < 0.05) as a function of pH and salt concentration. Overall, lowest S 0 values using PRODAN were found at pH 3.0, while S 0 values using ANS were generally higher at acidic than neutral or alkaline pH. These results demonstrate the need to consider charged probe-protein interactions when applying anionic fluorescent probes for surface hydrophobicity quantification. In the second part of the thesis, the structure-function relationships of spray dried egg albumen and its functional properties (e.g. gelation and foaming properties) were investigated. Protein structure was evaluated by measurement of surface hydrophobicity, net charge as zeta potential (ZP), sulfhydryl (SH) and disulfide (SS) groups, and by differential scanning calorimetry. Simple linear correlations were performed to better understand the structure-function relationship between structural parameters and functionality (gel strength and foam volume). In general, gel strength was positively correlated with S 0 determined by A N S , reactive SH groups, SS bonds and ZP (P < 0.001) and negatively correlated with total S H groups (P = ii 0.009). In general, foam volume was positively correlated with S 0 determined by PROD A N and ANS, reactive SH groups, and SS bonds (P < 0.05) and negatively correlated with total SH groups and ZP (P < 0.05). Significant correlations of structural properties to gel strength and foam volume were 2 2 observed by multiple regression analysis using a full quadratic model (R = 0.956 and R = 0.918, respectively; P < 0.001; n = 53). T A B L E O F C O N T E N T S A B S T R A C T T A B L E OF C O N T E N T S LIST OF T A B L E S LIST OF F I G U R E S A C K N O W L E D G E M E N T S LIST O F A B B R E V I A T I O N S C H A P T E R 1. I N T R O D U C T I O N C H A P T E R 2. L I T E R A T U R E R E V I E W 2.1. O V E R V I E W O F P R O T E I N S T R U C T U R E 2.2. P R O T E I N S U R F A C E H Y D R O P H O B I C I T Y 2.2.1. Definition of 'Hydrophobic' 2.2.2. Hydrophobicity Scales 2.2.3. Fluorescent Probes for Measuring Surface Hydrophobicity 2.3. P R O T E I N N E T C H A R G E 2.3.1. Measurement of Net Charge (Zeta Potential) 2.4. S U L F H Y D R Y L G R O U P S A N D D I S U L F I D E BONDS 2.4.1. Measurement of Sulfhydryl Groups 2.4.2. Measurement of Disulfide Bonds 2.5. EGG ALBUMEN 2.5.1. Composition of egg white 2.5.2. Ovalbumin 2.5.3. Ovotransferrin 2.5.4. Ovomucoid 2.5.5. Ovomucin 2.5.6. Lysozyme 2.5.7. Globulins 2.6. E G G DEHYDRATION 2.7. GELATION AND GEL PROPERTIES 2.7.1 Mechanism of Gelation 2.7.2. Gelation of Egg Albumen Proteins 2.8. SURFACE DEN ATI JRATION AND FOAMING PROPERTIES 2.8.1. Mechanism of Foam Formation 2.8.2. Foaming Properties of Egg Albumen Proteins 2.9. DIFFERENTIAL SCANNING CALORIMETRY 2.9.1. General Principle of Calorimetry 2.9.2. General Principles of Differential Scanning Calorimetry 2.9.3. DSC and Egg Albumen C H A P T E R 3. M A T E R I A L S A N D M E T H O D S 3.1 P R O T E I N S U R F A C E H Y D R O P H O B I C I T Y 3.1.1. Preparation of Probe Solution 3.1.1.1 ANS Stock Solution 3.1.1.2. P R O D A N Stock Solution 3.1.2. Buffer Preparation 3.1.3. Protein Samples and Preparation 3.1.4. Evaluation of PRODAN Method 3.1.5. Protein Surface Hydrophobicity (S0) Measurement 3.1.6. Effect of Solvent Polarity and pH on Fluorescence of P R O D A N 3.1.7. Calculation of Bigelow's Average Hydrophobicity Value (H(|) a v e ) 3.1.8. Statistical Analysis 3.2. S P R A Y - D R I E D E G G A L B U M E N A N A L Y S I S 3.2.1. Materials 3.2.2. Buffer Preparati on 3.2.3. SDS-PAGE Electrophoresis 3.2.4. Protein Surface Hydrophobicity (S0) Determination 3.2.5. Zeta Potential (ZP) Determination 3.2.6. SH group and SS bond Determination 3.2.6.1. Sulfhydryl Group Measurement 3.2.6.2. Total Sulfhydryl and Disulfide Group Measurement 3.2.7. Gel Strength Determination 3.2.7.1. Gel Preparation 3.2.7.2. Gel Strength Measurement 3.2.8. Foam Volume 3.2.9. Differential Scanning Calorimetry (DSC) 3.2.11. Statistical Analysis CHAPTER 4. RESULTS AND DISCUSSION 4.1. PROTEIN SURFACE HYDROPHOBICITY 4.1.1. Evaluation of P R O D A N Method 4.1.2. Surface versus Average Hydrophobicity of the Ten Proteins 4.1.3. Effect of pH and Salt Concentration on Surface Hydrophobicity 4.2. SPRAY DRIED E G G ALBUMEN ANALYSIS 4.2.1. SDS-PAGE Electrophoresis 4.2.2. Protein Surface Hydrophobicity 4.2.2.1. Surface Hydrophobicity using P R O D A N 4.2.2.2. Surface Hydrophobicity using ANS 4.2.3. Zeta Potential 4.2.4. Sulfhydryl and Disulfide Groups 4.2.4.1. Reactive and Total Sulfhydryl Groups 4.2.4.2. Total Sulfhydryl Groups and Disulfide Bonds 4.2.5. Gel Strength 4.2.6. Foam Volume 4.2.7. Differential Scanning Calorimetry 4.3. SIMPLE CORRELATIONS OF INDIVIDUAL PARAMETERS AND FUNCTIONALITY 100 4.3.1. Correlation of Protein Surface Hydrophobicity and Zeta Potential 100 4.3.2. Correlation of Gel Strength and Structural Parameters 101 4.3.2.1. Correlation of Gel Strength and Surface Hydrophobicity 101 4.3.2.2. Correlation of Gel Strength and Zeta Potential 101 4.3.2.3. Correlation of Gel Strength and Sulfhydryl Groups and Disulfide Bonds 102 4.3.3. Correlation of Foam Volume and Structural Parameters 103 4.3.3.1. Correlation of Foam Volume and Surface Hydrophobicity 103 4.3.3.2. Correlation of Foam Volume and Zeta Potential 104 4.3.3.3. Correlation of Foam Volume and Sulfhydryl Groups and Disulfide Bonds 104 4.4. MULTIPLE REGRESSION ANALYSIS 110 4.4.1. Gel Strength , 110 4.4.2. Foam Volume 111 CHAPTER 5. GENERAL CONCLUSIONS 112 5.1. PROTEIN SURFACE HYDROPHOBICITY 112 5.2. SPRAY DRIED E G G ALBUMEN ANALYSIS 112 5.3. RECOMMENDATIONS AND FUTURE WORK 114 REFERENCES 115 viii APPENDICES Appendix 1. Correlation (Pearson) of Bigelow's H^avt and S 0 values of the 10 proteins measured by ANS and PROD A N probes 124 Appendix 2. The relative fluorescence intensity (RFI) and pH values of ANS in citrate-phosphate buffer and citrate-phosphate:methanol buffer mixtures 125 Appendix 3. Multiple Regression Analysis of Gel Strength and Structural Parameters 126 Appendix 4. Multiple Regression Analysis of Foam Volume and Structural Parameters 128 ix LIST OF TABLES Table 1 - Major proteins in egg albumen, composition, size and properties 18 Table 2 - Hydrophobicity Values (S0) of Proteins using ANS Probe under different pH and salt concentrations compared to H<J) a v e 58 Table 3 - Hydrophobicity Values (S0) of Proteins using PROD A N Probe under different pH and salt concentrations compared to H(|) a V e 59 Table 4 - The relative fluorescence intensity (RFI) and pH values of PROD A N in citrate phosphate buffer, citrate phosphate: methanol buffer mixtures, methanol, acidified methanol and alkalinized methanol 66 Table 5 - Summary of Structural Parameters and Functionality Determined for Spray Dried Egg Albumen (Sample Codes 827-875) and Ovalbumin (OVA) 79 Table 6 - Differential Scanning Calorimetry of Spray Dried Egg Albumen 99 Table 7 - Correlation (Pearson) of Gel Strength and Foam Volume with Structural Parameters at pH 3.0 105 Table 8 - Correlation (Pearson) of Gel Strength and Foam Volume with Structural Parameters at pH 7.0 106 Table 9 - Correlation (Pearson) of Gel Strength and Foam Volume with Structural Parameters at pH 9.0 107 Table 10 - Correlation (Pearson) of Gel Strength and Foam Volume with Structural Parameters for combined data at pH 3.0, 7.0 and 9.0 108 X LIST OF FIGURES Figure 1 - Chemical structure of some fluorescent probes that may be used for determination of protein surface hydrophobicity: ANS, CPA, D P H and P R O D A N 9 Figure 2 - The net relative fluorescence intensity of BSA (0.0075 % (w/v)) and O V A (0.0250 % (w/v)) solutions with incremental addition of P R O D A N (8.8 x 10"5 M) stock solution from 0 to 10 uL 54 Figure 3 - Variation in fluorescence emission spectrum of P R O D A N (8.8 x 10"5 M) increasing from 1 ul to 7 ul with BSA protein concentration at 0.0075%, in aqueous citrate phosphate buffer (pH 7.0 and 0.01 M NaCl) 55 Figure 4 - Variation in fluorescence emission spectrum of P R O D A N (8.8 x 10"5 M) increasing from 1 ul to 7 ul with O V A protein concentration at 0.0234%, in aqueous citrate phosphate buffer (pH 7.0 and 0.01 M NaCl) 56 Figure 5 - Effect of pH and ionic strength on hydrophobicity values of proteins measured using ANS probe 61 Figure 6 - Effect of pH and ionic strength on hydrophobicity values of proteins measured using P R O D A N probe 62 Figure 7 - Reducing SDS P A G E (10-15 Gradient Phastgel) of Spray Dried Egg Albumen (Sample Codes 827-875) and Fresh Egg Albumen 68 Figure 8 - Non-Reducing SDS P A G E (10-15 Gradient Phastgel) of Spray Dried Egg Albumen (Sample Codes 827-875) and Fresh Egg Albumen 69 Figure 9 - Surface Hydrophobicity of Spray Dried Egg Albumen (Sample Codes 827-875) and Ovalbumin (OVA) at pH 3.0, 7.0, and 9.0 using P R O D A N 73 Figure 10 - Surface Hydrophobicity of Spray Dried Egg Albumen (Sample Codes 827-875) and Ovalbumin (OVA) at pH 3.0, 7.0, and 9.0 using ANS 74 Figure 11 - Zeta Potential of Spray Dried Egg Albumen (Sample Codes 827-875) and Ovalbumin (OVA) at pH 3.0, 7.0, and 9.0 . 7 7 Figure 12 - Comparison of Zeta Potential of Spray Dried Egg Albumen (Sample Codes 827-875) and Ovalbumin (OVA) 78 Figure 13 - Reactive and Total Sulfhydryl Groups of Spray Dried Egg Albumen (Sample Codes 827-875) and Ovalbumin (OVA) at pH 7.0 81 Figure 14 - Total Sulfhydryl and Disulfide Groups of Spray Dried Egg Albumen (Sample Codes 827-875) and Ovalbumin (OVA) at pH 7.0 85 Figure 15 - Disulfide Groups of Spray Dried Egg Albumen (Sample Codes 827-875) and Ovalbumin (OVA) at pH 7.0 86 Figure 16 -Gel Strength of Spray Dried Egg Albumen (Sample Codes 827-875) and Ovalbumin (OVA) at pH 3.0, 7.0 and 9.0 88 Figure 17 - Force Deformation Curves of Spray Dried Egg Albumen (Sample Codes 827-875) and Ovalbumin (OVA) Gels at pH 3.0, 7.0 and 9.0 89 Figure 18 - Foam Volume of Spray Dried Egg Albumen (Sample Codes 827-875) and Ovalbumin (OVA) at pH 3.0, 7.0 and 9.0 94 Figure 19 - Photographic Illustration of Small Compact and Large Coalesced Bubbles in Foam produced from Spray Dried Egg Albumen (Sample Coded 827) and Ovalbumin (OVA) at pH 7.0 95 Figure 20 - Differential Scanning Calorimetry Thermograms of Spray Dried Egg Albumen (Sample Codes 827-875) 98 A C K N O W L E D G E M E N T S I would like to express my thanks to Dr. Eunice C .Y. Li-Chan for her grateful guidance and advice throughout my studies. Her excellent supervision, constant encouragement, and financial support are greatly appreciated. I would like to thank the University of British Columbia for a University Graduate Fellowship during the course of this study and funding from the Natural Sciences and Engineering Research Council of Canada is gratefully acknowledged. I am thankful to Dr. B.J. Skura and Dr. C. Seaman for being the members of my research supervisory committee and their helpful advice and comments oh my research program and thesis are gratefully acknowledged. I would also like to thank Dr. J. Richards and Dr. K. Keiver for being members in my examining committee. I would like to thank Canadian Inovatech Inc. (Abbotsford, B C ) for donating the spray dried egg albumen samples for analysis and Dr. E. Charter for his expertise. I am appreciative to Dr. C.Y. Ma (Department of Botany) at the University of Hong Kong for allowing the analysis of my egg albumen samples on the T A Instruments Thermal Analysis - DCS standard cell. I would like to thank Karen Tang for performing the DSC analysis. I am also indebted to Mr. Sherman Yee, Ms. Val Skura, Ms. Angela Gerber, Ms. Joyce Tom and Ms. Jeannette Law for their technical support and assistance. Many thanks to my friends and colleagues of the Food, Nutrition and Health program, your friendship, support and advice are greatly cherished. Finally, I wish to thank my parents and my sisters for their understanding, immeasurable support, love and encouragement. LIST OF ABBREVIATIONS ANS l-anilinonaphthalene-8-sulfonate CD circular dichroism CPA cis-parinaric acid D P H diphenylhexatriene DSC differential scanning calorimetry EDTA ethylenediaminetetraacetic acid g . gram H(j) ave average hydrophobicity hr hour J Joules kDa kilodalton 1 litre M molar (moles per litre) mg milligram min minute ml millilitre m M millimolar mm millimeter mV millivolt ul microlitre nm nanometer NTB 2-nitro-5-thiobenzoate NTSB 2-nitro-5-thiosulfobenzoate °C degrees Celsius P R O D A N 6-propionyl-2-(dimethylamino)naphthalene RFI relative fluorescence intensity RSAP Raman spectral analysis package So surface hydrophobicity SDS sodium dodecyl sulfate sec second SH sulfhydryl SS disulfide T d denaturation temperature T, initial temperature V volt w/w weigh t/weight ZP zeta potential PROTEINS a-CAS alpha-casein oc-LAC alpha-lactalbumin p-CAS beta-casein P-LG beta-lactoglobulin B G bovine globulins BSA bovine serum albumin L F lactoferrin O V A ovalbumin PEP pepsin TRYP trypsin C H A P T E R 1. I N T R O D U C T I O N In addition to being nutritionally important, proteins impart a variety of physical characteristics to foods. These physical characteristics are generally referred to as functional properties. In a broader sense, the functional properties of proteins are those affecting their behavior during preparation, processing, storage, and use of various protein-containing products (Magdassi and Kamyshny, 1996). They include such characteristics as the ability to form stable gels, emulsions, and foams, and to provide viscosity, mouthfeel and flavour binding (Kinsella, 1982). Functionality is considered to be a higher order concept rather than a simple "property", because it includes only those features of ingredient behavior that are identifiable by sensory analysis panels as being relevant to food quality (Eads, 1994). Proteins exhibiting a range of important functional properties may be naturally present in foods or they may be added as structure-forming ingredients during the processing of manufactured products (Dickinson and McClements, 1996). The knowledge of factors influencing the functional properties of proteins and the ability to regulate these properties are of great importance in developing new processes in the food, biotechnological, pharmaceutical, and cosmetic industries. Since functional properties of proteins are intimately related to their molecular structure and interactions, it is important to understand the basic information relating functional properties to particular conformational or structural features of food proteins (Kinsella, 1982). The ultimate goal of all researchers in this area has been to relate precisely the macroscopic molecular properties of protein with manifestations of protein functionality in its utilization (Pour-El, 1979). The intrinsic physical, chemical and structural properties of proteins have great relevance to their functional properties. These include size, shape, amino acid composition and sequence, net charge and distribution pattern of charges, and hydrophobicity/hydrophilicity ratio. Secondary structure, tertiary and quaternary structure arrangements, inter- and intrapeptide cross-links (disulfide bonds), molecular rigidity/flexibility in response to changes in environmental conditions, and the nature and extent of interactions with other components are also important (Damodaran, 1996b). It is generally accepted that the molecular property of hydrophobicity, especially surface or effective hydrophobicity, plays an important role in the function of food proteins. Therefore, information on surface hydrophobicity is essential for understanding protein functionality (Nakai et al., 1996). Comparison of fluorescent probe methods using the uncharged aromatic probe PRODAN, as described by Alizadeh-Pasdar and Li-Chan (2000), and the commonly used anionic aromatic probe ANS, as described by Kato and Nakai (1980), for measuring surface hydrophobicity of different proteins under varying conditions of pH and salt concentrations allows for consideration of the effects of presence or absence of a permanent charge on the probe. The potential for electrostatic interaction contributions in the measurement of protein surface hydrophobicity is less of a factor with P R O D A N due to the absence of a permanent charge. Therefore, P R O D A N may be a more useful probe than ANS for the investigation of the effects of pH or ionic strength on protein surface hydrophobicity. Hence, in the first part of the thesis, protein surface hydrophobicity of ten proteins were studied at various pH and salt concentrations using 6-propionyl-2-(dimethylamino) naphthalene (PRODAN) and 1-anilinonaphthalene-8-sulfonate (ANS) fluorescent probes. A known advantage of fluorescent probes is their applicability to complex systems composed of several interacting proteins. Both P R O D A N and ANS probe methods were compared for measuring the protein surface hydrophobicity of a food system comprised of heterogeneous proteins. The heterogeneous protein system of spray dried egg albumen was investigated in this study and compared to ovalbumin. The results obtained by this method gave 2 an average surface hydrophobicity of all the protein components in the spray dried egg albumen system. In the second part of the thesis, the structure-function relationships between protein surface hydrophobicity, net charge, sulfhydryl and disulfide groups, differential scanning calorimetry of spray dried egg albumen and its functional properties (e.g. gelation and foaming properties) were investigated. The various functional properties of food proteins may be regarded as manifestations of two molecular aspects of proteins: the hydrodynamic properties and the surface-related properties. The hydrodynamic properties are predominantly related to the size, shape and molecular flexibility of the protein. In contrast, the surface-related properties are governed by the hydrophobic, hydrophilic, and steric characteristics of the protein surface that contacts other phases in a system. Functional properties such as gelation are predominantly manifestations of the hydrodynamic properties, and functional properties such as foaming are manifestations of the properties of the protein surface. The examination of the correlation of parameters of structural properties (i.e., surface hydrophobicity, net charge, sulfhydryl and disulfide groups, and differential scanning calorimetry (DSC)) of spray dried egg albumen to functional properties allows for the investigation of its structure-function relationship. Surface hydrophobicity of the food system was determined using P R O D A N and ANS. Net charge of the protein was expressed as zeta potential. Both sulfhydryl and disulfide groups were quantified using Ellman's reagent, 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) and 2-nitro-5-thiosulfobenzoate (NTSB), respectively. The functional properties under investigation were gelation (gel strength) and foaming properties (foam volume). 3 CHAPTER 2. LITERATURE REVIEW 2.1. OVERVIEW OF PROTEIN STRUCTURE Proteins are linear polymers made from the condensation of amino acids. The polypeptide chains of some proteins are covalently linked to other groups, such as lipids, phospholipids, carbohydrates or prosthetic groups. Twenty amino acids commonly occur in proteins, and it is convenient to classify them, according to their side groups, as polar (ionic or non-ionic), non-polar or amphipathic. The type, number and location of amino acids along the polypeptide chain determine the overall three-dimensional structure and molecular interactions of a protein (Dickinson and McClements, 1996). Protein structure is characterized as primary, secondary, tertiary or quaternary, according to the level of molecular organization (Dickinson and McClements, 1996). The primary structure refers to the sequence of amino acids along the polypeptide chain, and the position of any disulfide bonds. The arrangement of the amino acids in a folded protein is not completely disordered. There are typically various regions of local order, referred to as secondary structure. The most common examples of this type of structure are the a-helix, the p-sheet and the reverse turn. Generally, the three-dimensional structure of a protein is referred to as the tertiary structure. The major molecular factors involved in determining the tertiary structures of proteins are configurational entropy, hydrophobic interactions, hydrogen bonding, electrostatic interactions, van der Waals forces and disulfide bonds (Dickinson and McClements, 1996). Finally, some proteins in the native state consist of aggregates of different polypeptide chains bound together by covalent and/or non-covalent interactions. This type of organization of polypeptide sub-units is referred to as the quaternary structure. Any significant alteration in the arrangement of a polypeptide chain, without breaking it, is termed denaturation (Magdassi and Kamyshny, 1996). This process is usually, but not 4 necessarily, irreversible and often takes place when the protein is adsorbed at high-energy air/water and oil/water interfaces. Unfolding of adsorbed molecules allows the polypeptide chains to orient with most of the polar groups in the water phase and most of the nonpolar groups towards the air or oil phase (Magdassi and Kamyshny, 1996). Some of the molecular properties of proteins responsible for their surface activity are hydrophobicity, charge and features of structure (i.e. sulfhydryl groups and disulfide bonds). These parameters will be described briefly. 2.2. PROTEIN SURFACE HYDROPHOBICITY 2.2.1. Definition of 'Hydrophobic' The term 'lyophobic' is used to describe a solute that has little or no affinity for the solvent medium in which it is placed (Li-Chan, 1999). When water or an aqueous solution is the composition of the medium, 'hydrophobic' is used as a more specific descriptor. 'Hydrophobic' is derived from Greek meaning hydro: water and phobia: fear - 'water fearing'. Protein molecules and amino acid side chains, which are mainly hydrocarbons, have a lower energy when they are clustered together than when they are distributed through an aqueous solution. Because of their attraction for one another and their reluctance to mix with water they are called 'hydrophobic'. The forces that hold the nonpolar regions of the molecules together in aqueous solution are called hydrophobic interactions. The strength of these interactions is due to not only the tendency to reduce the entropically unfavourable contact between nonpolar groups with water but also the tendency to form enthalpically favourable noncovalent associations including those interactions broadly classified as Van der Waals forces, such as the net effect of attractive London interactions, repulsive electron cloud overlap and inducible dipole orientation and induction effects (Li-Chan and Nakai, 1991). 5 2.2.2. Hydrophobicity Scales A popular assessment of protein hydrophobicity is based on hydrophobicity scales. Using the hydrophobicity scales of the constituent amino acids, various approaches have been taken to calculate values for proteins: (1) those that are based on the solubility behavior in solvents of different polarity and (2) those that are calculated using crystallographic or other data showing the location of amino acid residues in the molecular structure, assuming that hydrophobic residues will locate in the interior of the molecule (Li-Chan, 1999). The former scales include those based on the free energy of transfer of the amino acid residues or their derivatives from water to an organic solvent or vapour, or the partition coefficients measured as a solubility ratio between water and a nonpolar immiscible organic solvent. The latter scales include those based on accessible or buried area of the residues, or on the location of amino acid residues in proteins assessed either in terms of the distance from the protein centre of mass and average orientation of the side chain or in terms of the average surroundings of residue types (Li-Chan, 1999). An equation has been formulated to calculate average hydrophobicity values of proteins based on Tanford's scale for free energies of transfer of amino acid side chains from an organic to an aqueous environment. The average hydrophobicity (H^ve) of a protein, when multiplied by the number of residues in the molecule equals the total hydrophobicity, which is a measure of the stabilization that a molecule could achieve if all of its "nonpolar" residues were buried (Bigelow,1967). It should be emphasized that H§ave is based on free energies of transfer of amino acid chains from an organic environment to an aqueous environment. Bigelow's average hydrophobicity values are calculated using only information on the amino acid composition of the protein. 6 The significant drawback to this method of hydrophobicity value determination of proteins is the lack of consideration of the effect of three-dimensional structure of proteins on the extent of exposure of the residues. Potential changes in protein structure as a function of the surrounding environmental conditions such as pH or salt concentration are not considered in the calculation of the average hydrophobicity value. Therefore it should not be expected that H(()ave and surface hydrophobicity (S0) would follow any predictable trend or correlate in any way. Surface hydrophobicity is defined in this study as the accessible hydrophobic regions of proteins which are measured by fluorescent probes. Scales that were formulated on the basis of location or buriedness of residues measured in different proteins attempt to address this problem. However, this approach is limited in universality of application due to the difficulties in extrapolation of available data about the behavior of residues to proteins. In the case of food systems, the problem is even more complex, due to the heterogeneity of proteins in the system. The calculation of an average protein hydrophobicity value for all the proteins present requires calculation of a hydrophobicity value for each protein as well as knowledge of how each value might change through possible interactions among the proteins. Furthermore, the calculated values fail to take into account the effects of processing on buriedness or surface exposure of residues. For these reasons, various methods of measuring parameters that may relate to the hydrophobicity of complex food proteins are usually favoured over calculation of average values or profiles based on the constituent amino acids (Li-Chan, 1999). 7 2.2.3. Fluorescent Probes for Measuring Surface Hydrophobicity It is generally accepted that the molecular property of hydrophobicity, especially surface or effective hydrophobicity, plays an important role in the function of food proteins. Therefore, information on surface hydrophobicity is essential for understanding protein functionality (Nakai et al., 1996). Despite the many protocols that have been reported to quantify protein surface hydrophobicity, no consensus has been reached on a standard method for its measurement. Methods using fluorescent probes have proved most popular due to their simplicity, speed, ability to predict functionality and use of small quantities of purified proteins for analysis. Another advantage is the applicability to complex systems composed of several interacting proteins, giving an average surface hydrophobicity of the protein mixture (Li-Chan, 1999). The quantum yield of fluorescence and wavelength of maximal emission of the fluorescent probe will ultimately depend on the polarity of its environment. Although the fluorescent probe method is unable to yield quantitative information on the distribution of individual side chains, it can be useful to monitor changes in the accessible hydrophobic sites in folded or denatured protein molecules as a function of different processing or environmental conditions. Figure 1 shows several fluorescent probes reported in the literature for determination of protein surface hydrophobicity. A popular probe used to measure protein surface hydrophobicity belongs to the anionic probes of the aromatic sulfonic acid class, 1-anilinonaphthalene-8-sulfonate (ANS), or its dimeric form (bis-ANS). Another group of anionic fluorescence probes is of the fatty acid analog type, including cis-parinaric acid (CPA) and frans-parinaric acid, which has been used as a probe for proteins and biological membranes in particular (Nakai et al., 1996). 8 Fig. 1 - Chemical structure of some fluorescent probes that may be used for determination of protein surface hydrophobicity. ANS = l-anilinonapthalene-8-sulfonic acid; CPA = cw-parinaric acid; DPH = diphenylhexatriene; PROD AN = 6-propionyl-2-(dimethylamino)naphthalene. The two anionic probes (ANS and CPA) are shown in the dissociated acid form. 9 The results obtained by using the anionic probes ANS and C P A have given researchers a difficult task of interpretation. The main concern is the potential contribution of electrostatic interactions binding to these anionic probes, which leads to possible ionic as well as hydrophobic contribution to the probe-protein interactions. This factor is likely to result in inaccurate estimation or quantification of protein surface hydrophobicity depending on the pH and the charge of the protein. The use of neutral or uncharged probes may circumvent this problem. Researchers have investigated a neutral lipophilic fluorescent probe, diphenylhexatriene (DPH), as a possible method for protein surface hydrophobicity determination which might avoid the problem of electrostatic interactions associated with the anionic probes ANS and CPA. The primary application of D P H has been to estimate membrane fluidity and the effects of various drugs or treatments on membrane structure (Nakai et al., 1996). Unfortunately, truly hydrophobic dyes such as D P H cannot be dissolved in aqueous medium, and have a tendency to form aggregates. To enable its use as a probe in aqueous systems such as protein solutions, the dye must first be completely dissolved in a water-miscible solvent such as dimethylsulfoxide, tetrahydrofuran or acetone (Slavik, 1994). Even so, there is always the danger that the dye is not evenly dispersed and it can form clusters which may appear as opalescence (Slavik, 1994). This was confirmed in the study of Chan (1995) who reported turbidity development in protein solutions or buffer blanks upon addition of the D P H probe, and an increase in fluorescence with decreasing concentration of D P H added to the protein solution. Another uncharged fluorescent probe for monitoring protein hydrophobicity is 6-propionyl-2-(dimethylamino) naphthalene (PRODAN). This probe has been used to determine microscopic polarity in biological systems such as lipid bilayers, membranes and protein interior (Bunker et al., 1993). It has also been used to probe the interaction of alcohols with 10 biological membranes (Rottenberg, 1992) and may be useful to show exposure of hydrophobic sites on enzymes or to detect aggregation of hydrophobic moieties (Bruins and Epand, 1995). Recently, Haskard and Li-Chan (1998) proposed a method using P R O D A N for the determination of food protein hydrophobicity. In that study, the influence of ionic interactions on the quantitation of protein surface hydrophobicity was assessed by comparing the binding of P R O D A N versus ANS to bovine serum albumin (BSA) and ovalbumin (OVA) at various ionic strengths. Haskard and Li-Chan (1998) reported that increasing ionic strength up to 1.0 M decreased S 0 values of B S A measured by ANS, increased S 0 of B S A measured by P R O D A N and of O V A measured by ANS, and had no significant effect on the S 0 of O V A measured by PRODAN. These results demonstrate the importance of considering charge effects when determining protein surface hydrophobicity. Further research work was suggested by Haskard and Li-Chan (1998) to evaluate the potential advantages of the electrically neutral fluorescence probe P R O D A N over anionic probes such as ANS and CPA by determining surface hydrophobicity index values for a wide spectrum of food proteins. Also, the nondissociable nature of P R O D A N may be of particular value in investigating the effects of changes in broad ranges of pH and ionic strength on protein surface hydrophobicity. According to Haskard and Li-Chan (1998), the aqueous stock solution of P R O D A N was prepared by dissolving P R O D A N in distilled deionized water. Although low, solubility of PRODAN in aqueous solution gives this probe an advantage over DPH. However, due to the low solubility (3.5mM) of P R O D A N in water [the P R O D A N has to be stirred overnight to reach such solubility, as stated by Weber and Farris (1979)], the protocol established by Haskard and Li-Chan (1998) yielded low fluorescence readings and measurements were difficult to reproduce due to batch-to-batch variability in the solubility of the P R O D A N aqueous stock solution (Alizadeh-Pasdar, unpublished data, 1998). 11 In response to a concern about the possible effect of anionic probes on the binding of proteins, Alizadeh-Pasdar and Li-Chan (2000) established a fluorescent probe method using an uncharged probe (PRODAN), prepared as a methanol stock solution, to compare the values of protein surface hydrophobicity measured by aliphatic (CPA) and aromatic (ANS) anionic probes. Surface hydrophobicities of three proteins (whey protein isolate, BSA and p-lactoglobulin) before and after heating (80 °C for 30 min) at various pH values (3.0, 5.0, 7.0, and 9.0) were measured using these three probes. ANS and CPA yielded opposing results to each other for the effects of pH and heating on protein hydrophobicity. Hydrophobicity was lower at pH 3.0 than at other pH values for all proteins measured by PRODAN, whereas the values measured by ANS and CPA at pH 3.0 were quite high compared to those at other pH values, suggesting the influence of electrostatic interactions on anionic probes-protein binding (Alizadeh-Pasdar and Li-Chan, 2000). Alizadeh-Pasdar and Li-Chan (2000) concluded that the results suggest that the presence or absence of a permanent charge as well as the aromatic and aliphatic nature of fluorescent probes can affect protein hydrophobicity values measured under various pH conditions. 2.3. PROTEIN NET CHARGE Protein molecules bear a net charge except at their isoelectric point, being negatively or positively charged at pH values significantly higher or lower than their isoelectric point, respectively. Both the overall net charge on the protein and the distribution of charges play important roles in determining its molecular properties. Experimentally, proteins have frequently been found to exhibit greater surface activity near the isoelectric point, because of minimization of electrostatic repulsion between the identically charged adsorbed molecules (Magdassi and Kamyshny, 1996). This behavior should be especially apparent in the case of an 12 uncharged surface. For adsorption at ionic surfaces the main factor is, probably, the net opposite charge of the protein molecule, which may contribute to the enthalpic part of the adsorption free energy. At the same time, a non-uniform distribution of ionic patches on the surface of a protein can lead to attractive electrostatic interactions between the patches and the surface even when the net charge of the protein is the same type as that of the surface (Magdassi and Kamyshny, 1996). Most of the charged groups are located at the periphery of the protein molecule, where they are accessible to the solvent. This is probably because the free energy change associated with moving a charged group from a high dielectric medium (water) to a low dielectric medium (the protein interior) is positive, i.e. unfavourable (Dickinson and McClements, 1996). Examinations of the three-dimensional structures of protein crystals have shown that the charged groups on the protein surface tend to be surrounded by oppositely charged groups. Experiments with protein molecules which have been modified to alter their overall charge have shown that some charged groups are crucial to the stability of folded proteins, that is when they are removed, the protein unfolds. In contrast, other charged groups make little contribution to protein stability; when such groups are removed, the protein retains the same structure (Dickinson and McClements, 1996). 2.3.1. Measurement of Net Charge (Zeta Potential) Zeta potential denotes the effective charge on the particle, expressed in millivolts (mV), measured via electrical transport. According to the manufacturer of the zeta potential meter (Laser Zee ™ Model 500, Pen Kem Inc., Croton-On-Hudson, N Y ) , an electric field is applied across an eletrophoresis cell containing the protein colloid. Negatively charged particles migrate to the anode at a transfer velocity directly dependent on their charge and the applied 13 electric field. The higher the charge on the particle, the greater its velocity at a given applied voltage. Traditionally, electrophoresis measurements have been reported in terms of "mobility", expressed in microns/second/volt/centimeter. However, zeta potential can be calculated directly in volts, using the Helmholtz-Smoluchowski formula. Because the dielectric constant and viscosity are temperature dependent, zeta potential must be measured at 20°C; otherwise a compensating correction must be applied. In the Laser Zee Meter this correction is supplied by a thermistor. The expression for zeta potential is: ZP = Kr) V/eE, where ZP = zeta potential (mV); K = constant; r\ = viscosity (poise); V = transfer velocity (cm/sec); e = dielectric constant; and E = field gradient (volts/cm). The Laser Zee Meter automatically solves the equation for aqueous systems at 20°C. 2.4. SULFHYDR YL GROUPS AND DISULFIDE BONDS Cysteine is an amino acid commonly found in globular food proteins. It has a thiol group (-SH), which is capable of forming disulfide bonds (-S-S-) with other thiol groups by an oxidation reaction (Magdassi and Kamyshny, 1996). Free thiol groups may also participate in thiol-disulfide interchange reactions with disulfide bonds. Proteins are therefore capable of forming both intramolecular and intermolecular disulfide bonds under the appropriate conditions. Intramolecular bonds form when a pair of cysteine residues is brought into close proximity by the folding of a protein molecule. Disulfide bonds of cystine play an important role in stabilizing the structure of proteins against unfolding, and in determining their functional properties (Kinsella, 1982). Their major contribution to protein stability is to reduce the number of conformations that the unfolded 14 protein can take up, and therefore to reduce the configurational entropy term favouring the disordered state. The propensity of a protein to unfold can therefore be altered by varying the number of intramolecular disulfide bonds which it contains. Increasing the number enhances stability of the native state, while decreasing the number reduces stability (Magdassi and Kamyshny, 1996). Sulfhydryl groups and disulfide bonds in proteins are usually located in the interior of the folded molecule and are unavailable for interaction, even under conditions which would normally favour thiol-disulfide interchange. This is probably because steric hindrance of the polypeptide chains excludes reactive agents, and because of the hydrophobicity of the protein interior (Magdassi and Kamyshny, 1996). Unfolding of the protein molecule exposes reactive sulfhydryl groups and allows thiol-disulfide interchanges to take place under the appropriate conditions. This explains the increase in sulfhydryl-disulfide interchange which occurs when heating proteins, when proteins are adsorbed at an interface, or after proteins are treated with proteolytic enzymes such as trypsin and chymotryspin (Magdassi and Kamyshny, 1996). Sulfhydryl-disulfide interchange also occurs between egg white proteins during heating (Mine et al., 1990). 2.4.1. Measurement of Sulfhydryl Groups Sulfhydryl groups of proteins have often been analyzed by using Ellman's reagent, 5, 5'-dithiobis(2-nitrobenzoic acid) (DTNB) (Ellman, 1958). This reagent reacts quantitatively with the SH groups and gives rise to a characteristic chromophoric product, 5-thiobis-(2-nitrobenzoic acid (TNB). To quantify the number of reactive S H groups in proteins, the assay is measured in buffer, without a denaturant. To quantify the total number of free SH groups in proteins, the reaction is generally run in a concentrated denaturant solution to 15 unfold the protein. The limitation of this method is that, some of the SH groups which are buried in the interior of the protein may not be exposed by some denaturants. 2.4.2. Measurement of Disulfide Bonds Many methods have been proposed to determine the disulfide bonds in proteins. Among them, the method reported by Thannhauser and colleagues (1984), which involves the use of the reagent 2-nitro-5-thiosulfobenzoate (NTSB), has significant advantages in terms of sensitivity and quantitative capability. The disulfide bonds of proteins are cleaved quantitatively by excess sodium sulfite at pH 9.5 and ambient temperature. Guanidine thiocyanate (2 M) is added to the protein solution as a denaturant to make the disulfide bonds accessible. The reaction with sulfite leads to thiosulfonate and a free sulfhydryl group: RS - SR ' + S032" ^ w RS - Sty + R 'S" The liberated thiol subsequently reacts with NTSB in the presence of excess sodium sulfite (Thannhauser et al., 1984): + R ' S w R 'S-S0 3 " + HOOC^ ^ ^ S - S 0 3 - HOOC^ ^ "S" The limitation of this method is that the chromophoric product formed in the reaction disappears rapidly in the presence of light. Thus, the assay should be performed in the dark for the quantification of disulfide bonds using the NTSB method (Damodaran, 1985). 16 2.5. EGG ALBUMEN 2.5.1. Composition of egg white Egg albumen, also known as egg white, accounts for most of an egg's liquid weight, about 67%. It contains more than half the egg's total protein, niacin, riboflavin, chloride, magnesium, potassium, sodium and sulfur. The albumen consists of 4 alternating layers of thick and thin consistencies. From the yolk outward, they are designated as the inner thick or chalaziferous white, the inner thin white, the outer thick white and the outer thin white. Egg white tends to thin out as an egg ages because its protein changes in character. The major constituents of egg white and some of their physicochemical and biologically or functionally important characteristics are summarized in Table 1. Protein is the major solids component of egg white, constituting approximately 9.7-10.6% (w/w). The carbohydrates, existing either in a free form or combined with protein, account for 0.5-0.6% of egg white. Glucose, at 0.5%, accounts for -98% of the total free carbohydrates. The amount of lipids (0.01%) in egg white is negligible compared with the amounts present in egg yolk. Egg albumen contains more than 15 proteins, and it may be regarded as a protein system consisting of ovomucin fibers in an aqueous solution of numerous globular proteins (Li-Chan and Nakai, 1989). 17 Table 1. Major proteins in egg albumen, composition, size and properties 1 . % of MW Surface Albumen (kDa) Tension T d Protein Protein Composition Pi (mNrn1) (°C) Ovalbumin 54.0 386 residues, 4-SH, 1-SS- 45 4.5-4.7 51.8 84.0 Ovotransferrin 13.0 705 residues, 9-SS- in C-domain 76.6-80 6.1-6.6 42.4 61.0 6-SS- in N-domain Ovomucoid 11.0 186 residues, 7-9 - S S - 27.3 4.1 39.0 77.0 Ovomucin 1.5-3.0 Not sequenced, a1:135 4.5-5.0 ND ND SS-groups, a1, a2 a2:210 and P-ovomucin 3:720 Lysozyme 3.4-3.5 129 residues, 4-SS- groups 14.3 10.7 42.0 75.0 Ovoglobulins 4.0-4.2 Not sequenced, G2:47 5.5 45.4 92.5 G2 and G3 2 genetic variants G3:50 1 Compiled from Powrie and Nakai (1986) and Hammershoj et al. (1999); pi _ isoelectric point; T d - denaturation temperature; ND - not determined 18 2.5.2. Ovalbumin Ovalbumin is a monomelic phosphoglycoprotein which constitutes over half of the egg white proteins by weight. Ovalbumin is by far one of the most well studied egg white proteins because of its ease of purification and crystallization in large quantities. It can be easily purified in large quantities by crystallization from an ammonium sulfate solution of egg white. After several recrystallizations, a highly pure ovalbumin with homogenous molecular weight by sedimentation or SDS-PAGE can be obtained; however, some reports have shown heterogeneity in such preparations, and this has been attributed to factors such as differences in phosphorylation and glycosylation, the presence of genetic variants, and contamination of S-ovalbumin (Doi and Kitabatake, 1997). Ovalbumin possesses a molecular weight of 44.5 kDa and an isoelectric point (pi) of 4.5-4.7. Generally, purified ovalbumin is a mixture made up of A l , A2 and A3 ovalbumins (Doi and Kitabatake, 1997) which contain two, one and no phosphate groups per molecule, respectively. According to Perlmann (1952) and Kitabatake et al. (1988), the approximate ratio of A l , A2 and A3 are 85:12:3 and the isoelectric points are 4.75, 4.89 and 4.94, respectively (Kitabatake et al.,1988). Ovalbumin is the only egg white protein to contain free sulfhydryl groups. It is reported to have four sulfhydryl groups and one disulfide bond (Powrie and Nakai, 1986). However, the conflicting reports on the precise number of thiol versus disulfide groups are probably due to sulfhydryl-disulfide interchange (Li-Chan and Nakai, 1989). The complete amino acid sequence of hen ovalbumin, which comprises 385 residues (Nisbet et al., 1981), and its crystal structure at 1.95 A resolution (Stein et al., 1991) have both been reported. 19 2.5.3. Ovotransferrin Ovotransferrin, also known as conalbumin, is identified as the bacteria inhibiting, iron binding protein from avian egg white. It constitutes 12% of the egg white proteins, and has a molecular weight of 77.7 kDa and a pi of approximately 6.1. Hen ovotransferrin contains 686 amino acid residues, has 15 disulfide bridges, and is glycosylated, containing a single glycan chain in the C-terminal domain, composed of mannose and N-acetylglucosamine residues (Abola et al., 1982). According to Mine (1995), X-ray diffraction studies (5 A resolution) revealed the structure of ovotransferrin to be bilobal. 2.5.4. Ovomucoid Ovomucoid, a glycoprotein, has a molecular weight of approximately 28.0 kDa and a pi of 4.1. It is best known for its trypsin inhibitory activity. Hen ovomucoid has nine disulfide bridges and no sulfhydryl groups. The molecule consists of three tandem domains, each homologous to pancreatic secretory trypsin inhibitor (Kazal type), and has a putative reactive site for the inhibition of serine proteases (Mine, 1995). Approximately 25% of the carbohydrate present in this glycoprotein, is joined to the polypeptide chain through asparaginyl residues (Yasmashita et al., 1984). The native conformation of ovomucoid is resistant to extremes of pH and temperature (i.e. heated to 100°C under acidic conditions for 60 minutes) and to exposure to urea at 80°C, and trichloracetic acid-acetone mixtures (Kilara and Harwalker, 1996). 20 2.5.5. Ovomucin Ovomucin, a structurally important sulfated glycoprotein with a highly viscous and gel-like nature, is found in egg white chalazaes, and comprises 1.5-3.5% of the total egg white solids (Mine, 1995). Many physicochemical investigations into the properties and structural importance of ovomucin in egg white have been generated; however, data have not always been consistent. Robinson and Monsey (1975) reported the presence of carbohydrate-rich ((3-ovomucin) and carbohydrate-poor (a-ovomucin) components in ovomucin. Two different types of ovomucin complexes in hen egg white were reported by Kato et al. (1977): an insoluble ovomucin complex formed from whole thick egg white and a soluble one formed from both thick and thin egg white. The molecular weight of the soluble ovomucin complex in mild non-denaturing and non-reducing conditions is -5000-8000 kDa. Carbohydrate content of ovomucin has been reported to be as high as 33%. Unfractionated ovomucin consists of 10-12% hexosamine, 15% hexose and 2.6-8% sialic acid (Mine, 1995). 2.5.6. Lysozyme Lysozyme (mucopeptide N-acetylmuramoylhydryolase; E C 3.2.1.17) is another egg white protein that is often studied. It has a molecular weight of 14.3 kDa and a pi of 10.7. Lysozyme consists of 129 amino acid residues and has four disulfide bridges. Lysozyme hydrolyzes the (3(1—> 4) linkages between N-acetylmuramic acid and N-acetylglucosamine in the proteoglycan of the bacterial cell. It has also been implicated in electrostatic interactions with ovomucin that may have considerable practical importance on the thinning of egg white subjected to storage (Mine, 1995). 21 2.5.7. Globulins The presence of three globulins, G l , G2 and G3, in egg white was reported several decades ago by Longsworth and colleagues (1940). The G l globulin was subsequently identified as lysozyme, and has been well characterized. In contrast, very little work has been published on the G2 and G3 globulins, which each comprise approximately 4% of the egg white proteins. Analyses of amino acid composition and carbohydrate composition (3.2-3.7% hexose, 2.4-2.5% hexosamine) revealed G2 and G3 to be very similar to each other; furthermore, the molecular weights of both globulins were roughly estimated to be 49.0 kDa by SDS-PAGE (Mine, 1995). 22 2.6. EGG DEHYDRA TION Dehydration is a successful way of preserving eggs, and advancements in the spray-drying of eggs have been developed over the past decade. Research has played a major role in solving problems which involve chemical, functional, and microbiological properties of dried egg products. Considerable improvements have been made in the production of dried eggs and they are now used extensively in bakery foods, bakery mixes, mayonnaise and salad dressings, confections, ice cream, pastas, and many convenience foods (Bergquist, 1995). Bergquist (1995) noted that dried egg products have the following advantages: 1. They can be stored at low cost under dry storage or refrigeration and require less space than shell or liquid eggs. 2. Transportation costs are less for dried than frozen or liquid eggs. 3. They are easy to handle in a sanitary manner. 4. They are not susceptible to bacterial growth when held in storage. 5. They permit a precise control over the amount of water used in a formulation. 6. They have good uniformity. 7. They have made possible the development of many new convenience foods. The objective when drying egg products is to obtain a finished product whose properties, when reconstituted, are close to those of the raw, uncooked liquid egg. Bergquist (1995) noted that the properties of dried eggs could be affected by the following treatments: 1) Drying perse. 2) Physical forces, such as pumping, homogenizing, and atomization. 3) Heating of the liquid egg prior to and during dehydration 4) Heating of the dried material. Most dried egg white products are obtained by spray drying. Factors that influence the quality and storage stability of dried eggs have been studied extensively. Moisture level, 23 storage temperature, particle size, acidity, carbohydrate additives, and gas packing have been considered individually and in combination as to their influence on the stability of dried eggs. While each of these factors can be utilized in some beneficial manner, none has proven as successful in producing a stable egg product as the removal of glucose from the liquid prior to drying (Sebring, 1995). Several methods of desugarization are employed in industry; they include spontaneous microbial fermentation, controlled bacterial fermentation, yeast fermentation and enzyme incubation. A detailed description of glucose removal practices currently used in the egg industry is not readily available because of their confidential nature. A few generalizations may be made concerning the overall procedures employed in desugarization. The removal of glucose from egg white is done almost entirely by the controlled bacterial fermentation process (Sebring, 1995) due to its functional and financial advantages compared to enzyme and yeast methods. The use of bacterial fermentation results in a high whipping egg white solids product with good flavour, good solubility, and high whipping qualities at a nominal cost in terms of labour and materials (Sebring, 1995). In general, the successful desugarization from egg whites requires thorough microbial control, good quality raw material, controlled fermentation conditions (temperature and pH), and the appropriate culture recommended for the particular procedure utilized. Pasteurization of dried egg albumen is performed by storage at elevated temperatures. This hotroom treatment has been shown to be an effective means of freeing egg-white solids of pathogens but details of the specific conditions used are proprietary information. However, the effect of moisture content and temperature of storage of dried albumen on viability of S. senftenberg, S. oranienburg, and S. pullorum and on solubility, specific gravity of meringue, and angel-cake making properties of the reconstituted product have been studied. Results 24 showed that albumen can be stored at elevated temperatures (50°, 60°, or 70°C) to eliminate Salmonella without significant impairment of functional properties (Cunningham, 1995). Spray drying is a conventional method for concentrating liquids or for making them into powder or flour, in the food industry. Many researchers have reported changes in functional properties of protein during such processes, however reports on the effects are contradictory. Whipping properties of dried egg products are important for angel food cake, sponge cake, meringues, and confections. Egg whites have the ability to incorporate air and form a relatively stable foam when beaten with a wire whip or other mechanical devices. Foamability or foaming power refers to the rate at which the surface tension of the air/water interface decreases. The reduction of foaming ability (foaming power) of egg protein by dehydration with spray drying was noted in some reports (Rolfes et al., 1955; Bergquist, 1964), while other reports suggest that satisfactory whipping properties were retained after spray drying (Joslin and Proctor, 1954; Carlen and Ayres, 1953) or after freeze drying (Rolfes et al., 1955) of egg albumen. This controversy might be due to variability in the food constituents contained in the samples, that is, solids content, protein concentration, fat and others. The effects of heat experienced by the food materials during drying and the heat history must also influence the functionality of the final product (Kitabatake et al., 1989). 25 2.7. GELATION AND GEL PROPERTIES 2.7.1 Mechanism of Gelation When Ferry (1948) first introduced the two-stage mechanism of gelation, he speculated on the importance of the relationship between denaturation and association. According to Ferry (1948), protein gel formation is considered as a two-stage process involving initial denaturation of macromolecules followed by aggregation: xPn - 4 xPd <-» (Pd)x where "n" denotes the native state and "d" the denatured state. The denatured molecules can orient themselves to a certain degree of order before aggregation. Specifically, partially unfolded globular protein molecules aggregate in linear associates when repulsion is large, and in random ones when repulsion is small, e.g., at the isoelectric point (Magdassi and Kamyshny, 1996). If the attractive forces between chains are low, denaturation should proceed quickly relative to chain association. This would result in an accumulation of free denatured proteins as an intermediate. Under these conditions, a fine gel network should form. A coarse opaque gel should be created when an increase in attractive forces leads to gelation prior to accumulation of many free chains. As attractive forces are further increased only a precipitate should form. Although formation of a protein network has been described as a two-stage process, the presence of soluble aggregates prior to network formation has been reported for a number of proteins (e.g. egg albumen and glycinin). Therefore, a three-step mechanism, proposed by Shimada and Matsushita (1980), involves protein denaturation followed by soluble aggregation and then interaction of the aggregates into a network. According to Shimada and Matsushita (1980), the thermocoagulation of proteins is formed by intermolecular interactions which produce a continuous, three-dimensional network exhibiting structural rigidity. First of all, protein denaturation occurs by heating and then protein-protein interactions take place with a 26 three-dimensional gel network resulting. The overall scheme of the coagulation is illustrated below: Coagulum 71 Sol ! -> Conversion -> Sol _ -> Heat Aggregate Sol i, a native protein solution is converted to Sol z, a denatured protein solution, and then set to coagulum or aggregate by heating. Conversion from Sol i to Sol 2 involves changes in the structure of the protein molecules and may be brought about by heat, mechanical means, salts, acid, alkali, and other reagents such as urea. Various factors such as protein concentration, molecular weight and heating time are some of the conditions for forming either coagulum or aggregate. In the first step of coagulation, the Sol 1 Sol 2 reaction most likely involves formation of disulfide bonds and exposure of hydrophobic amino acid residues (Shimada and Matsushita, 1980). Further support for the three-step mechanism has come from electron micrographs in which the strands of gel network from whey protein concentrate (Beveridge et al., 1983) and egg white (Woodward and Cotterill, 1986) were shown to contain spherical or bead-like structures. A balance between the attractive forces and the repulsive forces of proteins is necessary for formation of a stable gel. This balance is determined by the molecular properties of protein (structure, size of molecules, hydrophobicity, surface charge and distribution) as well as processing parameters (temperature, heating and cooling rate, pH, ionic strength, etc.). The main forces responsible for gel formation are hydrogen bonding, electrostatic and hydrophobic interactions, and disulfide cross-links (Morrissey et al., 1991). Cross-linking is very important for gel formation and stabilization, and together with the fluidity of the solvent inside the gel 27 network it imparts to the gel its elasticity and strength. Hydrophobic interactions are also important, especially at the initial stages of the gelation process (Morrissey et al., 1991). Electrostatic forces can provide attraction between oppositely charged amino acid residues of adjacent parts of a polypeptide chain as well as cause mutual repulsion between residues with the same charge (Magdassi and Kamyshny, 1996). The importance of the electrostatic interactions is reflected in the effects of pH and salts on gel properties. In many cases, the formation of gels with necessary characteristics can be achieved only when optimizing all these types of interactions (Magdassi and Kamyshny, 1996). 2.7.2. Gelation of Egg Albumen Proteins Egg white is a transparent viscous liquid, but after heating it changes into a turbid gel. The term " egg white" might be derived from this phenomenon. When eggs are cooked, egg proteins undergo denaturation, coagulation, and gelation. Isolated egg white is used as a food ingredient to enrich the nutritional quality as well as to improve the functional properties such as gel strength and water holding capacity of foods (Doi and Kitabatake, 1997). In general, coagulum-type gels are characterized by a three-dimensional network structure formed by protein aggregates. The aggregation of ovalbumin during heating results mainly from hydrophobically driven protein-protein interactions (Doi and Kitabatake, 1997). Hatta and colleagues (1986) concluded that the hydrogen bonding and disulfide bridging are apparently not involved in the heat-induced gels of a 5.0% ovalbumin solution in 20 m M NaCl kept at 80°C for 1 hr. This reported irrelevance of disulfide bridging and heat-induced aggregation of ovalbumin contradicts the findings of Shimada and Matsushita (1980). As previously mentioned, in the first step of coagulation, the Sol i Sol 2 reaction most likely involves the formation of disulfide bonds (Shimada and Matsushita, 1980) and with further 28 heating, egg albumen forms cross-linkages by sulfhydryl-disulfide exchange to produce a continuous three-dimensional network. The far-UV circular dichroism (CD) spectrum of dried ovalbumin (prepared by spray drying at 70°C) reveals no significant loss of secondary structure compared to that of the native ovalbumin. However, the same dried ovalbumin exhibits a greater binding capacity for hydrophobic fluorescent dyes, such as ANS and CPA, than the native ovalbumin (Kitabatake et al., 1989). Hence, the conformation of heat-denatured ovalbumin, caused by spray drying, at the secondary structure level is not very different from that of the native molecule, but hydrophobic areas that were buried in the native molecule become exposed after drying. The intermolecular interactions between heat-denatured ovalbumin molecules, which are still in a globular shape, are controlled by both the attractive hydrophobic and repulsive electrostatic interactions (Doi and Kitabatake, 1997). Denatured ovalbumin molecules form soluble linear aggregates when the electrostatic repulsion is relatively strong and the attractive hydrophobic interaction restricted. Soluble linear aggregates are formed when ovalbumin is heated at a pH (e.g. pH 3.5 or 7.5) far from the isoelectric point of ovalbumin (pi = 4.5) and /or at low ionic strength (0-70 m M NaCl). Under these conditions, ovalbumin molecules are charged, and therefore electrostatic repulsion between them is significant (Doi and Kitabatake, 1997). Cross-linked three-dimensional gel networks are formed when these soluble linear aggregates are at high protein concentration. When protein concentration is low, soluble linear aggregates do not form gel networks, but rather a viscous transparent sol. Doi and Kitabatake (1997) noted that when electrostatic repulsion is repressed either by adjusting the pH to close to the isoelectric point (pi = 4.5) and/or by increasing the ionic strength (0.1M NaCl), denatured protein molecules aggregate randomly resulting in a turbid gel or suspension, depending on the protein concentration. 29 According to Doi and Kitabatake (1997), at pH values far from the pi and at low ionic strength, linear aggregates are formed. With decreasing electrostatic repulsion at low ionic strength or at pH 7.0 (i.e. pH > pi), three-dimensional networks form a transparent gel. At high ionic strength or at pH values near the pi, proteins aggregate to form a turbid gel composed of random aggregates. At intermediate ionic strength or pH, both linear aggregates and random aggregates are formed. In this case, the linear aggregates form a cross-linked primary gel network and the random aggregates are interdispersed within this network. This mixed gel of linear and random aggregates has either a translucent or opaque appearance depending on the relative amounts of the linear and random aggregates. Among these gel types, the transparent and the opaque/translucent gels exhibit higher gel strength and water-holding capacity than the others. This has been shown to be the case for egg albumen gels. Many researchers have reported that the changes of sulfhydryl groups by heating are closely related to the formation of protein gel. During further heating, egg albumen was cross-linked by sulfhydryl-disulfide exchange and then a continuous, three-dimensional network was produced. Since hydrophobic interaction is favoured by a rise in temperature, this type of bonding may mainly form an associated structural network, a coagulum (Shimada and Matsushita, 1980). 30 2.8. SURFACE DENATURATION AND FOAMING PROPERTIES 2.8.1. Mechanism of Foam Formation In food products, where a gas is dispersed into a liquid, the foaming ability of the system is an important functional property. Foam creation is a dynamic process during which the surface of the protein film surrounding the gas bubbles is being compressed and expanded. The foam can be created by different methods, e.g. whipping, stirring, bubbling, sparging and shaking, varying with regard to the amount of gas available for incorporation (Hammershoj et al., 1999). The mechanism, as described by Nakamura and Doi (2000), by which foams are formed may be characterized as follows: (1) Protein molecules in bulk solutions contact the air/water interface, where they are adsorbed. (2) The protein molecules are denatured at the interface. (3) A monolayer or film of denatured protein molecules is formed at the interface. (4) The film traps air to form bubbles. (5) Continued adsorption of protein molecules around the bubbles enhances the coagulated regions of the film. (6) Protein films of adjacent bubbles come in contact to prevent flow of the liquid. The foaming properties of proteins are evaluated by their foamability (foaming power). Foamability is related quantitatively to the rate at which the surface tension of the air-water interface decreases. Thus, highly ordered globular protein molecules that do not readily denature at the surface show poor foamability (Doi and Kitabatake, 1997). Formation of a protein network can proceed by thiol and disulfide exchange, which is very rapid at neutral to alkaline pH (Creighton, 1993) and by oxidation of the sulfhydryl groups of cysteine residues into disulfide bonds. At high pH values the fast exchange of the thiol-groups and disulfide bonds can influence surface properties of the protein film, as cleavage of disulfide bonds increases the flexibility, opportunity of orientation, and unfolding of the 31 molecule in the interface (Hammershoj et al., 1999). Hammershoj and colleagues (1999) noted that this phenomenon has great relevance to ovalbumin, which is the only protein in egg albumen known to have free SH-groups. Such exchanges may play a role in stabilizing a protein film by polymerization and thereby influence the surface properties. 2.8.2. Foaming Properties of Egg Albumen Proteins Egg albumen is an important foaming ingredient for food applications. It is widely used in different food products as a foaming agent and is known to have excellent foaming properties. The dry matter of egg albumen contains approximately 80% protein. As the egg albumen consists of several proteins with a range of pi from 4.1 (ovomucoid) to 10.7 (lysozyme), it is important to understand the surface behavior of this complex protein mixture and the variation of functionality with pH. A range of net charges will exist at different pH values in egg albumen. Egg albumen contains 40 known proteins, with about half as minor components, but knowledge of the role of these minor proteins, in relation to foamability and foam stability and how the surface properties of egg albumen films are acting under different conditions, is limited. Surface denaturation of ovalbumin has been detected from an increase in the reactivity of the sulfhydryl groups. When an ovalbumin solution is whipped, the ovalbumin molecules are adsorbed at the air/water interface, rearrange themselves and change their conformation to orient their hydrophobic portion in the direction of the gas phase (Doi and Kitabatake, 1997). This change exposes the cysteinyl residues that are buried in the interior of the molecule and makes them susceptible to oxidation to form disulfide bridges with cysteinyl residues of neighbouring ovalbumin molecules at the air/water interface. Formation of aggregates and sulfhydryl-disulfide interchange reaction between ovalbumin molecules will take place at the interface. The aggregates formed at the air/water interface seem to produce a surface network, 32 and this may be responsible for the stability of ovalbumin foam (Doi and Kitabatake, 1997). However, the aggregates formed by disulfide bridges do not seem to be essential for stabilization of the foam. Thus, the stability of ovalbumin is mainly related to surface denaturation-induced aggregation and surface gelation and, to a lesser extent, to disulfide cross-linking between the denatured aggregated molecules. In other words, conformational changes at the air/water interface strengthen interaction between neighbouring molecules via non-covalent bonding as well as via disulfide bridges (Doi and Kitabatake, 1997). Foamabilities and foam stabilities of protein solutions differ depending on the choice of method for making foam. Therefore, a comparison of the data obtained by different authors is difficult to make (Kitabatake and Doi, 1982). Some representative results are described below. A number of studies of surface behaviour and properties of single protein systems have been performed, especially the foamability of ovalbumin. Kitabatake and Doi (1982) reported that the foamability of ovalbumin is relatively low while Nakamura (1964) reported that the foamability of ovalbumin increased under acid treatment, alkali treatment, or heat denaturation. Nakamura (1964) examined the foaming properties of egg white proteins. The proteins had decreasing foamabilities in the following order: ovomucin, ovoglobulin, ovotransferrin, ovalbumin, ovomucoid and lysozyme. The proteins which foamed well denatured easily at the surface and formed a well-developed network. Hydrophobic side chains of peptides were partly exposed on the surface of the in denatured proteins. The hydrophobic interactions and hydrogen bonds acted as binding forces to form a network. Intramolecular disulfide bonds gave conformational stability to lysozyme and ovomucoid. The restricted unfolding in these proteins limited surface denaturation, thereby resulting in low foamabilities (Nakamura, 1964). Hammershoj and colleagues (1999) examined the surface properties of egg albumen protein solutions at pH 4.8 (near pi of ovalbumin and ovomucin), 7.0 (fresh egg), 9.2 (aged egg) and 10.7 (pi of lysozyme) in relation to foamability, foam stability and bubble size 33 distribution of foam. The results showed that at pH 4.8, where proteins with both net positive and negative charge were present resulting in electrostatic attractions, the increase in surface and foaming properties were significant (Hammershoj et al., 1999). These results agreed with the review of Mine (1995), where electrostatic interactions were regarded as important for egg albumen's foaming properties. However, protein interactions by intermolecular cross-linking decreased the flexibility and foaming properties (Mine, 1995). The high foamability and the presence of small bubbles at pH 4.8 correlated well, as the decreasing foamability at higher pH is accompanied by an increase in mean bubble size, although for pH 7.0 larger bubbles dominated the picture, but the corresponding foamability was not poor. The significantly high effect on the foamability of egg albumen at pH 4.8 (Hammershoj et al., 1999) contradict with the results of Howell and Taylor (1995), who reported that egg albumen at pH 3.0-3.4 did not produce foamabilities differing from egg albumen at pH 7.0. The results of Hammershoj and colleagues (1999) showed that foamability and foam stability are favoured by different conditions of the egg albumen protein solution. Foam formation and drainage were favoured by the presence of different protein net charges and attractive forces, while foam volume stability was favoured by the same or zero net charge of proteins resulting in repulsive forces. In conclusion, foaming behaviour of an aqueous egg albumen solution at pH 4.8, with high overrun, small bubble size and slow drainage of liquid from the foam, can be related to the dynamic surface properties, which are a more rigid behaviour of the surface and a lower dynamic surface tension compared to pH 7.0-10.7 (Hammershoj et al., 1999). 34 2.9. DIFFERENTIAL SCANNING CALORIMETRY 2.9.1. General Principle of Calorimetry Thermal processing is one of the major operations in the food industry. It is therefore vitally important to understand the way in which heat can affect the various constituents present in food systems in order to minimize damage or to optimize processing conditions (Wright, 1986). Calorimetry involves the measurement of temperature or heat, more specifically, the determination of the temperature or the quantity of heat absorbed or given off when a definite amount of material undergoes a specific physicochemical change (Davis, 1994). Davis (1994) noted that chemical changes in foods are coupled with energy transformations: 1) Oxidation is characterized by the net amount of heat being released during a temperature rise in the sample. These are called exothermic reactions. 2) Hydrolysis is characterized by little or no heat evolution. Reactions are referred to as isothermic. 3) Reduction is characterized by a net amount of heat absorbed during a temperature rise. Reactions are endothermic (energy is taken up by the sample). 2.9.2. General Principles of Differential Scanning Calorimetry Differential scanning calorimetry (DSC) can provide much information on the behavior of all of the major food components, i.e. proteins, polysaccharides, lipids and water. Since the beginning of the 1970s, high sensitivity and precise microcalorimetry, and particularly DSC techniques, have been developed as powerful tools for the study of protein unfolding (Lefebvre and Relkin, 1996). The range of instrumentation available ensures that studies can be conducted on whole food systems as well as on dilute solutions of isolated components. The advantage that DSC 35 has over many other physical techniques is that few restrictions are placed on the physical nature of the sample under investigation. Thus, it is not usually necessary to extrapolate from model to real systems (Wright, 1986). DSC measures the differential temperature or heat flow to or from a sample versus a reference material, and this is displayed as a function of temperature or time. These techniques can differentiate between endothermic and exothermic reactions. The types of thermal transitions and what they can represent have been summarized by Davis (1994): 1) Endothermic curves usually relate to physical rather than chemical changes. a) A sharp endotherm is indicative of crystalline rearrangement, fusion, or solid state transitions for pure materials b) A broad transition is indicative of transitions that relate to dehydration, temperature dependent phase behaviour, or polymer melt. 2) Exothermic curves that relate to reactions without decomposition can be caused by a decrease in enthalpy of a phase or chemical system. a) Narrow exotherms can result from crystallization (ordering) of a metastable system, whether undercooled organic, inorganic, amorphous polymer or liquid, or annealing of stored energy resulting from mechanical stress. 3) Exothermic curves that relate to decomposition can be narrow or broad, depending on kinetic behaviour. 4) A change in heat capacity of a material at a glass transition can be seen simply as a small change in heat capacity with no well defined peak being produced. 36 DSC allows the determination of the thermodynamic functions of the unfolding transition directly from the heat capacity curve recorded. The heat capacity peak, characteristic of the transition, reflects the excess heat capacity arising from the enhanced enthalpy fluctuations that occur in the temperature range of the transition (Lefebvre and Relkin, 1996). Thermal denaturation is a highly cooperative phenomenon, which can be detected as an endothermic peak in the DSC thermograms, since the disruption of intramolecular hydrogen bonds is an endothermic reaction (Ma and Harwalker, 1990). The enthalpy (AH) is calculated from the area under the transition peak, and can provide an estimate of the thermal energy required to denature the protein. The temperature of denaturation (Td) can be estimated from the transition, generally as the peak temperature. The limitations of DSC is that it is a secondary technique in the sense that it only monitors changes in thermal properties which are associated with unspecified physical phenomena. 2.9.3. DSC and Egg Albumen The heat denaturation of egg albumen and its component proteins (ovalbumin, lysozyme, ovotransferrin and other proteins) was studied by DSC by Donovan and colleagues (1975). At a heating rate of 10°C/min, egg albumen at pH 7.0 showed two major endotherms, at 65°C and 84°C, produced by denaturation of ovotransferrin and ovalbumin, respectively. The ovotransferrin endotherm was increased to 70°C by raising the pH to 9.0. Donovan and colleagues (1975) concluded that within experimental error, the enthalpy of denaturation of egg albumen equals the sum of the enthalpies of denaturation of its component proteins (ovalbumin, lysozyme, ovotransferrin and other proteins), and is independent of pH over the pH range of 7.0 - 9.0. 37 Kato et al. (1990) showed that when 10 % protein samples were analyzed, spray dried egg albumen exhibited three endothermic peaks with a total enthalpy of 12.6 J/g. Purified ovalbumin showed a fairly sharp peak with a Td value of 77.7 °C and an enthalpy of 13.6 J/g. The thermogram of the globulin fraction showed three endothermic peaks with a total enthalpy of 11.9 J/g, whereas, ovotransferrin showed a major peak with Td value of 61.3 °C and enthalpy value of 12.8 J/g. It should be evident from an inspection of thermograms of egg albumen that the DSC technique is capable of quickly giving a large amount of information about the heat stabilities of the protein components. A detailed analysis of a thermogram gives quantitative information about the amounts of the various components present and the rates at which the components are heat denatured (Donovan et al., 1975). Like other techniques, DSC has its limitations. It gives no information about components present in small amounts. For example, the macroglobulins of egg albumen, which denature at approximately 60°C are not detected in DSC thermograms of egg albumen, and the trace component avidin, which denatures at 95°C is likewise not observed (Donovan et al., 1975). 38 C H A P T E R 3. Materials and Methods 3.1 PROTEIN SURFACE HYDROPHOBICITY 3.1.1. Preparation of Probe Solution 3.1.1.1 ANS Stock Solution The magnesium salt of ANS was prepared according to Weber and Young (1964). Approximately 24.8 mg of ANS (l-anilinonaphthalene-8-sulfonate) was dissolved in 10 ml 0.01M phosphate buffer at pH 7.0. The concentration of ANS stock solution was determined to be 2.0 x 10" 4M in methanol using A372 and e = 7.8 x 103 M ' W (Haugland 1996). The ANS stock solution was kept at room temperature and in the dark, in vials wrapped with aluminum foil. 3.1.1.2. PRODAN Stock Solution P R O D A N (Molecular Probes, Inc., Eugene, OR) was used without further purification as no major impurities were detected upon excitation at 280 nm. Bunker et al (1993) reported a fluorescent contaminant in samples of PRODAN from Molecular Probes Inc. (Eugene, OR), which was revealed by the appearance of an emission near 430 nm upon 280 nm excitation. If present, this contaminant can be extracted from a saturated water solution with n-hexane. Approximately 3.2 mg of PRODAN (6-propionyl-2-(dimethylamino) naphthalene) was dissolved in 10 ml of methanol. The concentration of the P R O D A N stock solution was determined to be 8.8 x 10"5 M in methanol using A361 and e = 18 x 10 3M"'cm" 1 (Haugland, 1996). The P R O D A N stock solution was divided into aliquots in vials and stored at -8 °C. The probe is light sensitive and the processes of dissolving and distributing were performed in the dark and the vials were covered with aluminum foil. 39 3.1.2. Buffer Preparation Citric acid-Na2HP04 (Mcllvaine) buffer solutions (pH 3.0 and 7.0 containing 0.01 M and 1.0 M NaCl) were prepared using analytical reagent grade chemicals and deionized distilled water according to Dawson et al. (1986). Citric acid-Na2HP04 solutions at.pH 9.0 containing 0.01 M and 1.0M NaCl were prepared by the addition of I N NaOH to pH 7.0 buffer solution. Sodium azide (0.02%) was added as an antimicrobial agent to all buffer solutions. Citric acid-Na2HP04 buffer solutions will be referred to as citrate phosphate buffer in this thesis. The conductivity of citrate phosphate pH 3.0 (0.01 M and 1.0 M NaCl), pH 7.0 (0.01 M and 1.0 M NaCl), and pH 9.0 (0.01 M and 1.0 M NaCl) solutions were measured as 5.2 ds/m and 52.0 ds/m, 17.0 ds/m and 56.0 ds/m, and 10.0 ds/m and 58.0 ds/m, respectively. Conductivity was measured using a Yellow Springs Instrument Conductivity Bridge, YSI Model 31 (Yellow Springs, Ohio). 3.1.3. Protein Samples and Preparation The 10 proteins were chosen for this study based on the wide range of protein surface hydrophobicity at pH 7.0 as reported by Li-Chan (1999). Bovine globulins G-8512 (BG), bovine serum albumin A-4503 (BSA), cc-casein C-7891 (cc-CAS), a-lactalbumin L-6010 (cc-L A C ) , (3-lactoglobulin L-0130 (|3-LG), ovalbumin A-5503 (OVA), pepsin P-7012 (PEP) and trypsin T-1005 (TRYP) were purchased from Sigma (St. Louis, MO), while fj-casein Cat No. 100321 (3-CAS) and lactoferrin Cat No. 151535 (LF) were purchased from ICN Biochemical (Cleveland, OH). The proteins were used without purification. Protein stock solutions in distilled deionized water (1.5 % w/v) were prepared by mixing the powdered protein in buffer with a glass rod. Protein stock solutions were diluted for hydrophobicity measurement in pH 3.0, 7.0 or 9.0 citrate-phosphate buffer containing low or high salt concentrations (0.01M or 1.0M NaCl). A l l protein concentrations were determined by A280 and using E 1 % i c m values reported by Fasman (1992) with the exception of B G . No reported E 1 % ] c m was found for B G hence the biuret protein determination method standardized with BSA was employed using Total Protein Reagent from Sigma Diagnostics (St. Louis, MO). Sodium azide (0.02%) was included in stock solutions to prevent microbial growth. 3.1.4. Evaluation of PRODAN Method Evaluation of the PRODAN method was performed to establish the volume of P R O D A N stock solution required to interact with accessible or surface hydrophobic sites of the protein. Bovine serum albumin A-4503 (BSA) and ovalbumin A-5503 (OVA) from Sigma (St.Louis, MO), were chosen as proteins for evaluation of the P R O D A N method. BSA and O V A were used because of their respective high and low hydrophobicity values as reported by Haskard and Li-Chan (1998). Protein stock solutions in distilled deionized water (1.5 % w/v) were diluted in pH 7.0 citrate-phosphate buffer with low salt concentration (0.01M NaCl) to 0.0075 % (w/v) and 0.0250 % (w/v) for BSA and O V A , respectively. Each protein solution (4 mL) was titrated with 1 ul increments of P R O D A N stock solution. The relative fluorescence intensity (RFI) at excitation and emission wavelengths of 365 and 465 nm, respectively, was measured at ambient temperature with a Shimadzu RF-540 (Shimadzu Corporation, Kyoto, Japan) spectrofluorophotometer using a 1-cm path length quartz cuvette (Starna Ltd, Romford, Essex). Slit widths of 5 nm were used. 41 Fluorescence emission scans were also recorded for both BSA and OVA at an excitation wavelength of 365 nm and emission wavelength range of 290-800 nm after each incremental addition of PRODAN into the protein solution. 3.7.5. Protein Surface Hydrophobicity (S0) Measurement Measurements of protein surface hydrophobicity using ANS were performed essentially according to the method of Kato and Nakai (1980). To successive samples containing 2 mL of diluted protein solutions were added 10 uL ANS stock solution. The protein solutions were diluted to six concentrations in a typical range of 0.000 to 0.0250 %, with the exception of BSA (range of 0.000 to 0.0075 %) due to the high fluorescence of BSA at high protein concentrations (0.0250 %). The mixture of protein solution and ANS was incubated at ambient temperature and in the dark for 15 minutes before measurement of relative fluorescence intensity. The protocol of Haskard and Li-Chan (1998) as modified by Alizadeh-Pasdar and Li-Chan (2000) was employed for the protein surface hydrophobicity measurements using PRODAN. To successive samples containing 2 mL of diluted protein solutions (prepared as described above for ANS determination) were added 5 uL PRODAN stock solution. The mixture of protein solution and PRODAN was incubated at ambient temperature and in the dark for 15 minutes before measurement of relative fluorescence intensity. Fluorescence emission scans of PRODAN were also recorded for all proteins at pH 3.0, 7.0 and 9.0, and 0.01 M NaCl salt concentration at an excitation wavelength of 365 nm and emission wavelength range of 290-800 nm. Relative fluorescence intensity (RFI) was measured at ambient temperature with a Shimadzu RF-540 (Shimadzu Corporation, Kyoto, Japan) spectrofluorophotometer using either 42 a 1-cm path length quartz cuvette (Starna Ltd, Romford, Essex) or disposable polystyrene fluorometer cuvette C-0918 (Sigma, St.Louis, MO); The RFI obtained from both quartz and disposable polystyrene cuvettes were compared and found to be essentially the same. Excitation and emission wavelengths of 390 and 470 nm, and 365 and 465 nm, for ANS and PRODAN, respectively, and slit widths of 5 nm were used. To eliminate the factor of day-to-day instrumental fluctuations in the RFI values, standardization was performed by measuring the RFI of 4 mL of methanol with 10 ul of PRODAN or 10 ul of ANS, and determining the correction factor to adjust the measured RFI to a standard value of 50 or 30, respectively. The RFI of each protein solution after addition of probe or buffer alone (no probe) was measured for each of the diluted protein solutions (six concentrations). The net RFI of each protein solution with probe was then calculated by subtracting the RFI attributed to protein in buffer and standardized using the correction factor as described. Protein surface hydrophobicity was expressed as the initial slope (S0) of the plot of net RFI versus protein concentration (%), calculated by linear regression analysis using Microsoft Excel™ (Microsoft Corporation, Roselle, IL). No time dependence of the fluorescence intensity was observed in the systems studied within 15 to 30 minutes after mixing. Surface hydrophobicity values were rountinely determined using at least duplicate analyses. S0 values are presented in tables and figures as the mean value of duplicates. Preliminary studies on BSA and OVA were done in quadruplicates and showed a standard deviation of range of ± 1 to 20. 3.1.6. Effect of Solvent Polarity and pH on Fluorescence of PRODAN Methanolic and aqueous solutions were prepared to monitor the effects of solvent polarity and pH on the fluorescence emission of PRODAN. The RFI of PRODAN was 43 Specifically, 10 uL of PRODAN stock solution was added to one of the following solvents: 4 mL of citrate-phosphate buffer (at pH 3.0, 7.0 or 9.0, with 0.01 M or 1.0 M NaCl); 4 mL of citrate-phosphate buffer (at pH 3.0, 7.0 or 9.0, with 0.01 M or 1.0 M NaCl) and 2 mL of methanol; 4 mL of methanol; 4 mL of methanol acidified with 1 drop of 1M HCI; 4rnL of methanol alkalinized with 1 drop of 1M NaOH. The RFI was measured at ambient temperature with a Shimadzu RF-540 (Shimadzu Corporation, Kyoto, Japan) spectrofluorophotometer using a 1-cm path length quartz cuvette (Starna Ltd, Romford, Essex). Excitation and emission wavelengths of 365 and 465 nm, and slit widths of 5 nm were used. 3.1.7. Calculation of Bigelow's Average Hydrophobicity Value (H<j>ave) The average hydrophobicity (H(|) a v e ) values calculated according to Bigelow's method were as reported by Li-Chan (1999), except for lactoferrin which was calculated in this study based on the amino acid sequence reported by Shimazaki et al. (1993). 3.1.8. Statistical Analysis Analysis of variance followed by Tukey's pairwise comparison test was conducted on the S 0 values within each protein as a function of pH and salt concentration, and between proteins under the same pH and salt concentration conditions, using the software package MtNITAB™ version 12 (Minitab, Inc., State College, PA). The significance level of 5% was used unless otherwise specified. 44 3.2. SPRA Y-DRIED EGG ALBUMEN ANALYSIS 3.2.1. Materials Spray dried egg albumen powders were provided by Canadian Inovatech Inc. (Abbotsford, BC). In total six egg albumen powder samples with known gel strengths were studied. The egg albumen powders were coded as 827, 853, 860, 861, 862, and 875, with reported gel strengths at pH 7.0 of 700, 610, 422, 550, 500, and 655, respectively (Charter, E., personal communication). Ovalbumin A-5503 (OVA) from Sigma (St.Louis, MO) was used as a reference. Liquid albumen from an egg purchased from a local supermarket was used as a standard in the SDS-PAGE electrophoresis. The liquid albumen was separated by hand from the yolk and then mixed to obtain a uniform mixture (i.e. homogenous mixture of thin and thick albumen). The liquid albumen was then diluted and the protein concentration was determined by the biuret protein determination method standardized with OVA using Total Protein Reagent from Sigma Diagnostics (St. Louis, MO). 3.2.2. Buffer Preparation Citric acid-Na2HP04 (pH 3.0 and 7.0) and Na2HP04-NaH2P04 (pH 7.0) buffer solutions were prepared using analytical reagent grade chemicals according to Dawson et al. (1986) and will be referred to as citrate phosphate and sodium phosphate buffers, respectively, in this thesis. Citrate phosphate at pH 9.0 was prepared by the addition of 1M NaOH to citrate phosphate at pH 7.0. Sodium phosphate at pH 3.0 and 9.0 were prepared by the addition of 1M HCI and 1M NaOH to sodium phosphate at pH 7.0, respectively. Sodium azide (0.02%) was added as an antimicrobial agent to all buffer solutions. 45 The conductivity of citrate phosphate pH 3.0 (0.01 M and 1.0 M NaCl), pH 7.0 (0.01 M and 1.0 M NaCl), and pH 9.0 (0.01 M and 1.0 M NaCl) solutions were measured as 5.2 ds/m and 52.0 ds/m, 17.0 ds/m and 56.0 ds/m, and 10.0 ds/m and 58.0 ds/m, respectively. The conductivity of sodium phosphate pH 3.0, pH 7.0, and pH 9.0 solutions were measured as 8.2 ds/m, 6.8 ds/m, and 9.0 ds/m, respectively. Conductivity was measured using a Yellow Springs Instrument Conductivity Bridge, YSI Model 31 (Yellow Springs, Ohio). 3.2.3. SDS-PAGE Electrophoresis Reducing and non-reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) using PhastGel™ gradient 10-15 gels and SDS buffer strips were carried out on a PhastSystem™ Separation and Control Unit according to the manufacturer's instructions (Pharmacia, LKB Biotechnology, Uppsala, Sweden). A total mixture of approximately 100 u.1 containing 80 u.1 of protein solution (2-4 mg/ml) prepared in 0.01 M sodium phosphate buffer, pH 7.0, 20 pi 10 % SDS, and 2 ul 1 % bromophenol blue marker dye and with or without the addition of 2 ul 2-mercaptoethanol for reducing and non-reducing conditions, respectively, was boiled for 15 min prior to application to gels. Mixtures were prepared in 1.7 ml disposable conical micro-centrifuge tubes with caps (Catalogue # 20170-355, VWR Scientific Products, West Chester, PA). Molecular weight standards (BSA, 66 kDa; OVA, 45 kDa; P-CAS, 23 kDa; P-LG, 18 kDa and ot-LAC, 14 kDa) and diluted liquid egg albumen were utilized as standards. The standards were prepared as described above for protein solutions depending on application for reducing or non-reducing SDS-PAGE. 46 Lane 1 was loaded with a molecular weight standard which consisted of BSA, O V A , cc-L A C , p-CAS and (3-LG (each at ~ 2 mg/ml); Lanes 2-7 were loaded with spray-dried egg albumen samples (~ 4 mg/ml); Lane 8 was loaded with diluted liquid egg albumen (~ 4 mg/ml). The protein concentration of each sample was determined by the biuret protein determination method using Total Protein Reagent from Sigma Diagnostics (St. Louis, MO). 3.2.4. Protein Surface Hydrophobicity (S0) Determination Refer to sections 3.1.1. and 3.1.5. for the preparation of A N S and P R O D A N stocks and protein surface hydrophobicity determination, respectively. 3.2.5. Zeta Potential (ZP) Determination The net charge of the proteins in solution was determined as zeta potential essentially according to Hayakawa and Nakai (1985). A mixture of 5 ml of 0.1% protein solution (in 0.01M sodium phosphate at pH 3.0, 7.0 and 9.0) and 0.15 ml of 3,3'-dimethylbiphenyl (Aldrich Chemical Co.) were emulsified with the Ultra Turrax homogenizer (Hansen & Co., West Germany) at 11, 000 rpm for 20 seconds. The emulsion was diluted with a 100-fold volume of buffer (0.5 ml emulsion into 50 ml buffer with low ionic strength - 0.01M sodium phosphate at pH 3.0, 7.0 and 9.0). The chamber of the zeta potential meter (Laser Zee ™ Model 500, Pen Kem Inc., Croton-On-Hudson, NY) was filled with 25 ml of the protein colloid emulsion using a syringe, following the manufacturer's recommendations, and then placed on the stage of the zeta meter. The observed zeta potential (in mV) was corrected for temperature according to the equation: ZP (corrected) = ZP (measured) x (1 - 0.02T), where T is the measured temperature at the time of ZP measurement. Analysis of each sample was conducted 5 times. 47 3.2.6. SH group and SS bond Determination 3.2.6.1. Sulfhydryl Group Measurement Sulfhydryl group determination was performed by using Ellman's reagent (5,5'-dithiobis(2-nitrobenzoic acid) or DTNB) according to Mine (1997). To 1 ml of the 1% protein solution (in 0.01M sodium phosphate at pH 7.0) was added 4 ml of 0.1 M Tris-glycine buffer (pH 8.0) containing 0.01 M EDTA (for reactive sulfhydryl groups) or the same buffer with 0.01 M EDTA containing 2 M guanidine thiocyanate (for total sulfhydryl groups). After incubation at 40 °C for 30 min, 125 p i of Ellman's reagent solution (20 mg in 5 ml of 0.1 M Tris-glycine buffer, pH 8.0) was added and then incubated at 25 °C for 10 min. The absorbance was read at 412 nm on a Shimadzu UV-Visible Spectrophotometer UV-160 (Shimadzu Corporation, Kyoto, Japan). The absorbance at 412 nm was recorded against a blank of 1 ml of water, 4 ml of Tris-glycine buffer and 125 ul of Ellman's reagent. The sulfhydryl residues were calculated as: umole SH/g = 73.53A4i2(D/C) [derived from the extinction coefficient DSOOMT'cm"1 (Beveridge et al., 1974)] where Amis the absorbance at 412 nm, C is the sample concentration in mg/ml (10 mg/ml), and D is a dilution factor, 5.125. Analysis of each sample was conducted in triplicate. The Ellman's reagent assay (Beveridge et al., 1974) requires a specific pH environment of 8.0 for the reaction to occur. Due to this limitation of the sulfhydryl group assay, the reactive and total sulfhydryl groups were determined only at pH 8.0 for both the spray dried egg albumen and OVA samples. 3.2.6.2. Total Sulfhydryl and Disulfide Group Measurement The total content of SH and SS groups was investigated using the method of Thannhauser et al. (1984). The NTSB (2-nitro-5-thiosulfobenzoate) stock solution was 48 synthesized as follows: 100 mg Ellman's reagent was dissolved in 10 ml of 1 M Na2S03 and the pH of the solution was adjusted to 7.5 to produce a bright red colour. While heating in a 38 °C water bath, the solution was bubbled with oxygen until a pale yellow colour appeared (- 2 hrs). The stock solution was stored in two aliquots of 5 ml at -18 °C for future use. The NTSB assay solution was prepared by diluting the stock solution 1 to 100 with freshly made solution containing 2 M guanidine thiocyanate, 50 m M glycine, 100 m M sodium sulfite and 3 m M EDTA (pH 9.5). To measure the disulfide bond content, 100 u,L of protein solution (15 mg/ml in 0.01 M sodium phosphate buffer at pH 7.0) was mixed with 1.5 ml of NTSB assay solution, and the mixture was incubated in the dark for 25 min at ambient temperature. The absorbance was read at 412 nm with a Shimadzu UV-Visible Spectrophotometer UV-160 (Shimadzu Corporation, Kyoto, Japan). The absorbance at 412 nm was recorded against a blank of 1.5 ml of NTSB assay solution mixed with 100 ul water. The total SH and SS residues were calculated as: umole SH/g = 71.94A 4 i 2 (D/C) [derived from the extinction coefficient 13900M"1cm"1 (Thannhauser et al., 1984)] where A 4 ] 2 i s the absorbance at 412 nm, C is the sample concentration in mg/ml (15 mg/ml), and D is a dilution factor, 16. Analysis of each sample was conducted in triplicate. Disulfide groups were determined from the subtraction of total S H groups (Mine, 1997) from total SH and SS groups (Thannhauser et al., 1984). The limitation of this assay is that each SS group only results in A m equivalent to one SH (not 2). The NTSB assay (Thannhauser et al., 1984) requires a specific pH environment of 8.0 for the reaction to occur. Due to this limitation, determination of the disulfide bonds were only performed at pH 8.0 for both the spray dried egg albumen and O V A samples. 49 3.2.7. Gel Strength Determination 3.2.7.1. Gel Preparation Egg albumen powder solutions (10 % (w/v)) were prepared in 0.01 M sodium phosphate at pH 3.0, 7.0 and 9.0. Sodium azide (0.02 %) was added to prevent microbial growth during experiments and storage. After adjusting the pH of the solution with 1 M NaOH or HC1, the exact protein concentration of the albumen solutions were determined by using the biuret protein determination method (Total Protein Reagent, Sigma Diagnostics, St. Louis, MO) standardized with ovalbumin. The albumen solutions were degassed, for approximately 1.5 hours in 200 ml beakers in a desiccator with a drawn vacuum, prior to being filled into plastic cylindrical molds (Industrial Plastics and Paints, Richmond, BC) with a diameter of 3.1 cm and length of 4 cm. The top and bottom ends of the molds were wrapped in Resinite packaging film (Borden Packaging and Industrial Products-Canada, Barrie, ON) and sealed with rubber bands to avoid leakage of albumen solution and penetration of water. The gels were formed by heating at 80°C for 30 min in a water bath, then immediately placed into an ice bath for 30 min. The gels were kept in the cold room at 4°C for 24 hrs and equilibrated to ambient temperature before measuring the gel strength. 3.2.7.2. Gel Strength Measurement The albumen gels formed at pH 3.0, 7.0 and 9.0 were prepared in triplicate, and two measurements were taken for each gel. The top and bottom of each gel was sliced a wired cheese cutter to obtain a smooth uniform edge. Then 10 mm was sliced from either end for analysis. Gel strength was expressed as the peak force (Newton) multiplied by the distance (mm) to the peak, obtained from the force deformation curves measured by compression with the TA.XT2 Texture Analyzer (Texture Technologies Corp., Scardale NY/Stable Microsystems, Gogalmin, Surrey, UK) using the following conditions: distance format, strain; 50 pre-test and post-test speed, 10.0 mm/sec; test speed, O.lmm/sec; strain, 50.0%; trigger type, auto; trigger force, 0.05 kg. The diameter of compression plate was 50 mm, and the compression was applied until 50% of the gel height was attained. 3.2.8. Foam Volume Foam volume was determined by using a modified procedure of Kato et al. (1983). Ascending nitrogen gas was introduced at a constant flow rate of 60 cm3/min for 15 sec, from the bottom of the 150 ml column (2.0 cm internal diameter) through a glass frit with G-4 glass fiber filter circles (Fisher Scientific, Pittsburgh, PA) at both sides, into 10 ml of 0.1% protein solution in 0.01M sodium phosphate solution at pH 3.0, 7.0, or 9.0. Foam volume was determined by measuring the height of the foam in the graduated column. Analysis was performed in triplicate for each test solution. 3.2.9. Differential Scanning Calorimetry (DSC) DSC was performed using a T A Instruments Thermal Analysis - DCS standard cell (Instrument 2920 M D S C V2.2A) at the University of Hong Kong (Department of Botany) according to the recommended methodology of Dr. C. Y . Ma (University of Hong Kong). About 1 mg of each egg albumen powder sample was accurately weighed onto an aluminum hermetic pan. 0.01 M sodium phosphate buffer was added to the sample to make a 10 % slurry. For each liquid sample, 10 ul of the sample was loaded on the pan. Each pan was sealed and loaded onto the DSC cell with an empty sealed aluminum pan as reference. The pan was heated from 35 °C to 130 °C in the DSC at a programmed rate of 10 °C/min. The thermograms were analyzed by the Universal Analysis program (Universal VI.9D) in terms of the initial 51 temperature of the appearance of the endothermic peak, the enthalpy of the endothermic peak, the denaturation temperature and the temperature range at half of the peak area. 3.2.11. Statistical Analysis Analysis of variance followed by Tukey's pairwise comparison test, Pearson's correlations test and multiple regression analysis were conducted on data collected for each parameter of structural properties, using the software package M I N I T A B ™ version 12 (Minitab, Inc., State College, PA). The significance level of 5 % was used unless otherwise specified. 52 CHAPTER 4. RESULTS AND DISCUSSION 4.1. PROTEIN SURFACE HYDROPHOBICITY 4.1.1. Evaluation of PRODAN Method Figure 2 shows the net RFI values of BSA and O V A solutions upon the incremental addition of 1 u,l aliquots of P R O D A N stock solution to 4 ml of the protein solutions at pH 7.0 and 0.01 M NaCl. The observed plateau of the RFI values confirms that the volume of P R O D A N stock solution (10 uL) added per volume of protein solution (4 ml) reported by Alizadeh-Pasdar and Li-Chan (2000) was in excess. Figures 3 and 4 show the fluorescence emission spectra of B S A and O V A solutions upon incremental addition of 1 ul aliquots of PRODAN stock solution to 4 ml of the protein solutions at pH 7.0 and 0.01 M NaCl, respectively. A maximum fluorescence spectrum was achieved after the addition of 5 and 6 ul of PRODAN for B S A and O V A solutions, respectively. This also confirms the excess of PRODAN reported by Alizadeh-Pasdar and L i -Chan (2000). 5 3 4 0 3 5 -3 0 -Li- 2 5 -en • 2 0 -CD C 1 5 -1 0 -5 -0 i o A o o o o o o 0 B S A A A A A A O V A i 1 1 1 1 i i r~ 0 1 2 3 4 5 6 7 8 V o l u m e of P R O D A N (jal) — i 1 9 1 0 Fig. 2 - The net relative fluorescence intensity of 4 ml of B S A (0.0075 % (w/v)) and O V A (0.0250 % (w/v)) solutions with incremental increases of P R O D A N (8.8 x 10"5 M) stock solution from 0 to 10 uL. Open diamonds - BSA; Solid triangles - O V A . Excitation wavelength was 365 nm and emission wavelength was 465 nm. 5 4 c CD CD O C CD O in CD o zs CD > CD OC 4 e. G I ' 0 . 0_ 0 . o_ 2 9 8 . e A = 4 0 0 . 6 : 6 0 Q . 0 Emission Wavelength (nm) Fig. 3 - Variation in fluorescence emission spectrum of P R O D A N (8.8 x 10" M) increasing from 1 ul to 7 u,l with BSA protein concentration at 0.0075%, in aqueous citrate phosphate buffer (pH 7.0 and 0.01 M NaCl). Excitation wavelength was 365nm and emission wavelength range was 290 - 800 nm. Slit widths were 5 nm. Numbers pointing to each curve indicates the volume of added PRODAN solution. 55 4 y . y_ co — CD CD o c CD o co CD o CD > CD CC £ y y . y = 4- y y « y : 6 y y . y Emission Wavelength (nm) Fig. 4 - Variation in fluorescence emission spectrum of P R O D A N (8.8 x 10"5 M) increasing from 1 uJ to 7 ui with O V A protein concentration at 0.0234%, in aqueous citrate phosphate buffer (pH 7.0 and 0.01 M NaCl). Excitation wavelength was 365nm and emission wavelength range was 290 - 800 nm. Slit widths were 5 nm. Numbers pointing to each curve indicates the volume of added P R O D A N solution. 56 4.1.2. Surface versus Average Hydrophobicity of the Ten Proteins Tables 2 and 3 show the S 0 values of the 10 proteins studied using ANS and PRODAN, respectively, compared to the average hydrophobicity (H<t>ave) values of the proteins calculated by the method of Bigelow (1967). In general, similar trends for S 0 values were observed for ANS and P R O D A N probes. For example, BSA and O V A were reported by Haskard and L i -Chan (1998) to have high and low surface hydrophobicity, respectively. These high and low S 0 values for BSA and O V A , respectively, were observed for ANS and P R O D A N probes for all pH (3.0, 7.0, 9.0) and salt concentrations (0.01 and 1.0 M NaCl). The trend of S 0 values for the ANS method observed in this study at pH 7.0 also correlated with values at pH 7.0 reported by Li-Chan (1999). The H<have of a protein, when multiplied by the number of residues in the molecule equals the total hydrophobicity, which is a measure of the stabilization that a molecule could achieve if all of its "non polar" residues were buried (Bigelow, 1967). There was a noticeable difference observed between the trends for H<have and S 0 values for the ten proteins. It should be emphasized that H<t>aVe is based on free energies of transfer of amino acid chains from an organic environment to an aqueous environment. Bigelow's average hydrophobicity values are calculated using only information on the amino acid composition of the protein. The significant drawback to this method of hydrophobicity value determination of proteins is the lack of consideration of the effect of three-dimensional structure of proteins on the extent of exposure of the residues. Potential changes in protein structure as a function of the surrounding environmental conditions such as pH or salt concentration are not considered in the calculation of the average hydrophobicity value. Therefore it should not be expected that H(t>ave and S 0 would follow any predictable trend or correlate in any way. 5 7 ) Table 2: Hydrophobicity Values (So) of Proteins using ANS probe under different pH and salt concentrations compared to He) a v e 1 PROTEIN H<t> ave1 S 0 pH 3.0 S 0 PH7.0 S 0 pH 9.0 0.01 M 1.0M 0.01 M 1.0M 0.01M 1.0M Bovine Globulins ND 2 759 ' 588 h 378 9 400 9 297 9 356 h Bovine Serum Albumin 1120 388* 500 9 356 ' 413 h 694 h 700 1 Casein, a 1200 84 d 209 e 41 d 64 d 43 d 93 d Casein, p 1320 590 h 350 ' 167 e 397' 164* 328 9 Lactalbumin, a 1150 450 9 1019 ' 16b 11 a 37° 27 c Lactoferrin 1095 597 1 603 1 17 b 14b 18b 21 b Lactoglobuiin, p 1230 43 c 42 c 24 c 36 c 79 e 124 f Ovalbumin 1110 110 e 57 d 5 a 9 a 9 a 120 b Pepsin 1063 5 a 8 a 17b 88 e 17 b 116 e Trypsin 940 18 b 16 b NA * NA* 7 a 5 a a i Values in the same column with different superscripts are significantly different at P < 0.05 * NA - Not applicable - no correlation (P > 0.05) was observed between protein concentration and relative fluorescence intensity 1 He)) ave" average hydrophobicity calculated according to Bigelow's method as reported by Li-Chan (1999), except for lactoferrin which was calculated in this study based on the amino acid sequence reported by Shimazaki et al. (1993) 2 ND - Not determined 58 Table 3: Hydrophobicity Values (So) of Proteins using PRODAN probe under different pH and salt concentrations compared to H<t> w e 1 PROTEIN H(t>ave1 S 0 pH 3.0 S 0 pH 7.0 S 0 pH 9.0 0.01 M 1.0M 0.01 M 1.0M 0.01 M 1.0M Bovine Globulins ND 2 59 6 110 ' 361 9 455 9 252 1 97 h Bovine Serum Albumin 1120 20 d 32 b 174' 163 ' 257 1 279 ' Casein, a 1200 7 b 31 b 58 d 48 b 67 d 84* Casein, p 1320 18 c d 44 c 40 c 131 e 90 9 91 9 Lactalbumin, a 1150 14c 47 d 38 c 20 a 72 e 47 d Lactoferrin 1095 8 b 60 6 31 b 17 a 4 4 b 23 b Lactoglobulin, p 1230 5 a b 6 a 108 e 105 c 140 h 110 1 Ovalbumin 1110 2 a 4 a 15a 18 a 10a 15 a Pepsin 1063 NA* NA* 27 b 123 d 81* 73 e Trypsin .940 NA* N A * NA* _ NA* 63 c 40° a'j Values in the same column with different superscripts are significantly different at P < 0.05 * NA - Not applicable - no correlation (P > 0.05) was observed between protein concentration and relative fluorescence intensity 1 H(l> ave" average hydrophobicity calculated according to Bigelow's method as reported by Li-Chan (1999), except for lactoferrin which was calculated in this study based on the amino acid sequence reported by Shimazaki et al. (1993) 2 ND - Not determined 59 Linear regression analyses (Appendix 1) indicated no significant correlation (P > 0.05) between Bigelow's H(j)ave and S 0 values measured by either probe, for any of the 6 combinations of pH and salt concentrations. Although the fluorescent probe approach provides some information about the non-polarity of the protein surface, it must be cautioned that the measured value may or may not reflect the "true" hydrophobicity of the protein surface (Damodaran, 1997). It has been shown that binding of fluorescent probes to proteins occurs only at well-defined hydrophobic cavities formed by groupings of non-polar residues on the protein surface (Damodaran, 1989). These cavities are not accessible to water but are accessible to non-polar ligands. When a protein molecule is folded, non-polar residues may be buried in the interior with polar residues at or near the surface of the molecules (Nakai, 1996). But still many non-polar side chains are in contact or exposed to water at the surface. Thus knowledge about the distribution of those side chains is critical for predicting effective or accessible surface activity for proteins. 4.1.3. Effect ofpH and Salt Concentration on Surface Hydrophobicity Effect of pH and salt concentration on hydrophobicity values of proteins measured using ANS and P R O D A N probe are shown in Figures 5 and 6, respectively. For the PRODAN method, in general, the lowest S 0 values were found at pH 3.0 for all the proteins studied, with the exception of L F and cc-LAC. For O V A , PEP, P-CAS and B G , the highest S 0 values were observed at pH 7.0, whereas for BSA, R-LG, cc-LAC, oc-CAS, T R Y P , and L F , the highest S Q values were observed at pH 9.0 using PRODAN. In contrast to P R O D A N , ANS S 0 values were generally higher at acidic pH compared to neutral/alkaline pH with the exception of BSA, P-LG PEP and TRYP. 60 ° 3500 -r 3000 ~ 2500 o 1500 g- 1000 | 500 0 pH 3.0 ° 3500 -r 3000 £ 2500 2 2000 £ 1500 g- 1000 S 500 -I >. 0 pH 3.0 £. 800 1 | 600 I £ 400 d | £ 200 * n • 1 pH 3.0 3500 3000 -I £ 2500 2 2000 ° 1500 -| o. 1000 | 500 I ° pH 3.0 % 3500 -r 3000 £ 2500 2 2000 £ 1500 g. 1000 | 500 >. 0 pH 3.0 BG c d fl b 1 mi pH 7.0 pH 9.0 BSA J Q e f pH 7.0 pH 9.0 cc-CAS pH 7.0 pH 9.0 p-CAS _ d e c Mi J i in pH 7.0 pH 9.0 Ct-LAC b a d c pH 7.0 pH 9.0 S> 3500 — 3000 £ 2500 2 2000 9 1500 a 1000 . I 500^  z g 1000 5 800 2 6 0 0 £ 400 o 200 H d e pH 3.0 pH 3.0 % 1000 £ 800 £ 600 £ 400 -I o 200 ?. 0 X g 1000 £ 80° 2 6 0 0 £ 400 o 200 pH 3.0 a b pH 3.0 ° 1000 n £ 800 2 600 1 400 §• 200 -I I 0 c b pH 3.0 LF b a b c pH 7.0 pH 9.0 P-LG pH7.0 pH 9.0 O V A a b b c pH 7.0 pH 9.0 PEP pH 7.0 pH 9.0 TRYP NA NA pH7.0 pH 9.0 Fig. 5 - Effect of pH and ionic strength on hydrophobicity values of proteins measured using ANS probe. Solid bars (0.01M NaCl); open bars (1.0 M NaCl). Refer to section 3.1.3. for an explanation of protein abbieviations.abc>d,e,f Values with different superscripts are significantly different at P < 0.05. NA (not applicable) - no significant correlation was observed between protein concentration and relative fluorescence intensity. 61 £- 800 a -wo -I S 200 I 0 pH 3.0 pH 7.0 pH 9.0 pH 7.0 pH 9.0 CC-CAS pH 3.0 pH 7.0 pH 9.0 P-CAS d d pH 3.0 pH 7.0 pH 9.0 pH 3.0 pH 7.0 pH 9.0 £ 800 _ 600 2 400 CL 2 200 — 8 1000 & 800 I 600 £ 400 S 200 — £ 800 Z 600 £ 400 g 200 I 0 5 1000 £ 800 £ 6 0 0 £ 400 f 200 I ° pH 3.0 pH 7.0 pH 9.0 a a a a NA NA P -LG b c mi Wn pH 3.0 pH 7.0 pH 9.0 O V A c d b c pH 3.0 pH 7.0 pH 9.0 PEP c b pH 3.0 pH 7.0 pH 9.0 TRYP NA NA NA NA pH 3.0 pH 7.0 pH 9.0 Fig. 6 - Effect of pH and ionic strength on hydrophobicity values of proteins measured using PRODAN probe. Solid bars (0.01M NaCl); open bars (1.0 M NaCl). Refer to section 3.1.3. for an explanation of protein abbreviations. a , b , c , d e , f Values with different superscripts are significantly different at P < 0.05. NA (not applicable) - no significant correlation was observed between protein concentration and relative fluorescence intensity. 62 The S 0 values are not reported for PEP and T R Y P at pH 3.0 using P R O D A N , or for TRYP at 7.0 using both PRODAN and ANS, due to the fact that there was no significant correlation observed between protein concentration and relative fluorescence intensity. Possible reasons for this phenomenon could be weak or insignificant binding of the probe to these proteins under the particular pH and salt conditions, due to low protein surface hydrophobicity or steric hindrance. The increase in S 0 values measured by ANS for B S A with higher salt concentrations for all pH conditions in this study contradicts the findings of Haskard and Li-Chan (1998), who found a decrease in ANS measured S 0 of B S A with increasing ionic strength at pH 7.0. An increase in the PRODAN measured S 0 of B S A with increasing ionic strength at pH 7.0 was reported by Haskard and Li-Chan (1998). The increase in S D values of B S A measured with P R O D A N were also observed in this study for B S A at pH 3.0 and 9.0, but not at pH 7.0. The S 0 value of O V A measured by ANS was enhanced by an increase in salt concentration at pH 7.0 and 9.0 but reduced at pH 3.0. Haskard and Li-Chan (1998) also reported an enhancement of the ANS measured S 0 of O V A with an increase in ionic strength at pH 7.0. The S 0 of O V A measured by P R O D A N was enhanced by an increase in salt concentration at all three pH conditions used in this study, although this increase was only significant at pH 7.0 and 9.0. Haskard and Li-Chan (1998) also reported that increasing the ionic strength at pH 7.0 resulted in a slight increase in the S 0 of O V A measured using PRODAN. The differences between the findings of this study and those of Haskard and Li-Chan. (1998) for BSA and O V A may be related to various factors, including the use of different buffers (citrate-phosphate versus phosphate) for protein dissolution, and the different preparation of the P R O D A N probe stock solution (in methanol versus water). 63 The trends observed for the effects of pH on the proteins B S A and J3-LG in this study are similar to those reported by Alizadeh-Pasdar and Li-Chan (2000), with the exception of some discrepancies observed for the S 0 values at pH 9.0. It was hypothesized that the S 0 values could be reflecting different protein surface hydrophobicity values as a function of the two pH 9.0 buffers used in these studies, namely citrate-phosphate buffer in this study, versus borate buffer in the study by Alizadeh-Pasdar and Li-Chan (2000). Additional S 0 measurements were therefore conducted to compare S 0 values of BSA and P-LG solutions prepared in pH 9.0 citrate-phosphate and borate buffers. Differences in S 0 values for 3 -LG were indeed noted in pH 9.0 citrate-phosphate buffer versus borate buffer using both ANS (227 versus 119 respectively) and P R O D A N (417 versus 863 respectively). On the other hand, no difference in S 0 was observed for B S A in pH 9.0 citrate-phosphate versus borate buffer for either ANS (2240 versus 2230, respectively) or P R O D A N (746 versus 703, respectively). These results suggest that for some proteins but not others, the protein surface hydrophobicity measured by these fluorescent probes may be affected not only by the buffer pH, but also by the buffer composition. The nondissociable nature of the PRODAN probe is an advantage in enabling investigation of changes in protein surface hydrophobicity over a broad range of pH (Alizadeh-Pasdar and Li-Chan, 2000). However, it must be noted that the emission of P R O D A N is sensitive not only to solvent polarity, but also may be affected by solvent acidity (Catalan et al, 1991) and proticity or aproticity due to a hydrogen bonding effect (Rottenburg, 1992). The maximum emission wavelength of P R O D A N was reported to show a red shift with increasing dielectric constant, but to be insensitive in phosphate buffer to either pH (2.75-8.0) or ionic strength (0.12-1.20) (Harianawala and Bogner, 1998). In contrast, ANS is essentially considered non-fluorescent in aqueous solutions in the absence of proteins, and the RFI values 64 were not significantly different for ANS in citrate-phosphate buffer and citrate-phosphate:methanol buffer mixtures at pH 3.0, 7.0 and 9.0 (Appendix 2). Table 4 shows the relative fluorescence intensity (RFI) values measured for PRODAN in (a) citrate-phosphate buffers at pH 3.0, 7.0 and 9.0, (b) mixtures of methanol with the pH 3.0, 7.0 and 9.0 buffers, and (c) methanol alone and after pH adjustment to acidic and alkaline pH values. As expected, RFI increased with decreasing polarity of the solvent, i.e. buffer < methanokbuffer (1:2) < methanol. Furthermore, the RFI values of P R O D A N in all cases was lower (P < 0.05) at acidic pH than at neutral or alkaline pH. These results suggest that the RFI readings for protein surface hydrophobicity may also be underestimated at acidic pH compared to neutral or alkaline pH. The dampening effect of acidic pH on the RFI values obtained with PRODAN probe should be considered when comparing S 0 values and interpreting the effects of pH on protein surface hydrophobicity. Nevertheless, the relative magnitude of the solvent acidity effect on P R O D A N emission shown in Table 4 is small in comparison to the changes observed in S 0 values of the proteins as a function of pH (Figure 6). In fact, in the case of lactoferrin, the highest S 0 value was observed in 1.0M NaCl at pH 3.0. These results suggest that the hydrophobicity or non-polarity of the protein binding site may have a more dominant effect than solvent acidity on the observed RFI values and ultimately the S 0 values measured by PRODAN. 65 1 ' Table 4. The relative fluorescence intensity (RFI) and pH values of P R O D A N in citrate-phosphate buffer, citrate-phosphate:methanol buffer mixtures, methanol, acidified methanol and alkalanized methanol Solvent RFI of P R O D A N Measured pH Citrate Phosphate Buffer pH3 .0 0.01 M NaCl 0.2 + 0.0 a 3.0 1.0M NaCl 0.2 +0.0 a 3.0 pH 7.0 0.01 M NaCl 1.7 + 0.0 b 7.0 1.0M NaCl 1.7 + 0.0 b 7.0 pH 9.0 0.01 M NaCl 2.6 +0.0 c 9.0 1.0M NaCl 2.3 + 0.0 d 9.0 Citrate Phosphate Buffer: Methanol Mixture (2:1, v:v) pH3 .0 0.01 M NaCl 6.8 + 0.1 a 3.6 1.0M NaCl 4.8 +0.1 b 3.6 pH 7.0 0.01 M NaCl 9.0 +0.4 c 7.8 1.0M NaCl 8.4 +0.3 c 7.8 pH 9.0 9 .6+0 .2 0 6 0.01 M NaCl 8.9 1.0M NaCl 10.0 +0.2 d 9.2 Methanol Unadjusted 50.2 + 1 .9 a 8.2 Acidified 23.8 +0.1 b 0.7 Alkalanized 54.8 + 0.6 c 10.7 1 RFI values are the mean of triplicate analyses + S D of 10 pi P R O D A N stock solution in 4 ml buffer or methanol, or 6 ml of buffenmethanol mixture (k e x = 365 nm, X e m = 465 nm). a.b,c, etc. \ / a | u e s j n the same solvent system with different superscripts are significantly different at P < 0.05. 4.2. SPRAY DRIED EGG ALBUMEN ANALYSIS 4.2.1. SDS-PAGE Electrophoresis SDS-PAGE electrophoresis was performed to assess if proteolytic hydrolysis of spray dried egg albumen (sample codes 827-875) occurred during cold storage at approximately 4°C for 6 months. Fresh egg albumen was used for comparison. Figures 7 and 8 show the reducing and non-reducing SDS P A G E gels, respectively, of spray dried egg albumen (sample codes 827-875) and fresh egg albumen. Based on the molecular weight, bands were tentatively identified as ovotransferrin, ovalbumin, ovomucoid and lysozyme. No peptide bond hydrolysis was observed in the electrophoretic analysis of spray dried egg albumen proteins. However, some high molecular weight or aggregated material remained undissociated in the stacking gel especially in the absence of 2-mercaptoethanol (non-reducing SDS-PAGE), see Figure 8, lanes 2 and 7.. 67 Lanes 1 2 3 4 5 6 7 8 Fig. 7 - Reducing SDS-PAGE (10-15 Gradient Phastgel) of Spray-Dried Egg Albumen (Sample Codes 827-875) and Fresh Egg Albumen. Lane 1 - Molecular Weight Marker (BSA, 66 kDa; O V A , 45 kDa; P - C A S , 23 kDa; P - L G , 18 kDa; and a - L A C , 14 kDa); Lane 2 - Sample 827; Lane 3 - Sample 853; Lane 4 - Sample 860; Lane 5 - Sample 861; Lane 6 - Sample 862, Lane 7 - Sample 875; and Lane 8 - Liquid Egg Albumen a, bl/2, c and d are probably ovotransferrin, ovalbumin, ovomucoid and lysozyme, respectively. 68 a b c d Lanes 1 2 3 4 5 6 7 8 Fig. 8 - Non-Reducing SDS-PAGE (10-15 Gradient Phastgel) of Spray-Dried Egg Albumen (Sample Codes 827-875) and Fresh Egg Albumen. Lane 1 - Molecular Weight Marker (BSA, 66 kDa; O V A , 45 kDa; R-CAS, 23 kDa; R-LG, 18 kDa; and a - L A C , 14 kDa); Lane 2 - Sample 827; Lane 3 - Sample 853; Lane 4 - Sample 860; Lane 5 - Sample 861; Lane 6 - Sample 862, Lane 7 - Sample 875; and Lane 8 - Liquid Egg Albumen a, b, c and d are probably ovotransferrin, ovalbumin, ovomucoid and lysozyme, respectively. 69 4.2.2. Protein Surface Hydrophobicity 4.2.2.1. Surface Hydrophobicity using PRODAN Figure 9 compares the surface hydrophobicity values determined by P R O D A N of the spray dried egg albumen (sample codes 827-875) and ovalbumin (OVA) samples at a given pH level (i.e. pH 3.0, 7.0 and 9.0) and at different pH levels for the given sample. From the analysis of variance (data not shown), it can be concluded that the pH effect, the sample effect and the interaction of pH and sample were all significant (P < 0.001). S 0 values determined by P R O D A N of spray dried egg albumen (sample coded 827-875) at pH 3.0 and 7.0 were all significantly higher than the corresponding S 0 values of O V A at pH 3.0 and 7.0. At pH 9.0, sample 860 and 862 were not significantly different from O V A , while the other albumen samples had significantly higher S 0 values. At pH 3.0, the S D values were in the following order: albumen samples 827, 853 > 862, 861 and 875 > 860 > O V A . Samples 827 and 853 have higher S 0 values, whereas sample 860 has a lower S 0 value. Samples 861, 862 and 875 possessed intermediate S 0 values but they were significantly different from samples 827, 853 or 860. At pH 7.0, the S 0 values were in the following order: albumen samples 827, 853 > 875 > 860, 861 and 862 > O V A . The surface hydrophobicity of samples 827 and 853 were significantly higher compared to the other spray dried egg albumen samples. The surface hydrophobicity of sample 860, 861 and 862 were not significantly different from each other. At pH 9.0, the surface hydrophobicity of sample 827, 853, 861 and 875 were significantly higher compared to surface hydrophobicity of sample 860, 862 and O V A . When using P R O D A N , in general, at pH 3.0, the S 0 values for all samples 827-875 and O V A were significantly lower when compared to the S 0 values determined at pH 7.0 and 9.0, with the exceptions of samples 860 and 862. Significant difference was not observed for S 0 values determined at pH 3.0 and 9.0 for sample 860, and at all three pH's for sample 862. The 70 highest S 0 values were generally noted at pH 7.0 for all samples, although for some samples, there was no significant difference between S 0 values at pH 7.0 and 9.0. 4.2.2.2. Surface Hydrophobicity using ANS When the S 0 values are compared across the three pHs studied (pH 3.0, 7.0 and 9.0), the significant trends of high and low surface hydrophobicity observed for ANS were reversed for S 0 values determined by PRODAN. Figure 10 compares the surface hydrophobicity values determined by ANS of the spray dried egg albumen (sample codes 827-875) and ovalbumin (OVA) samples at a given pH level (i.e. pH 3.0, 7.0 and 9.0) and at different pH levels for the given sample. From the analysis of variance (data not shown), it can be concluded that the pH effect, the sample effect and the interaction of pH and sample were all significant (P < 0.001). At pH 3.0, the S 0 values were in the following order: albumen samples 827, 853 > 861, 875 > 862 > 860 > O V A . The S 0 values for all spray dried egg albumen (sample coded 827-875) determined by ANS at pH 3.0 were significantly higher when compared to O V A . Samples 827 and 853 at pH 3.0 were significantly higher in surface hydrophobicity compared to the other spray dried egg albumen samples and O V A . However, sample 860 was significantly lower in surface hydrophobicity compared to the corresponding spray dried egg albumen samples at pH 3.0. At pH 7.0, the surface hydrophobicity of all the spray dried egg albumens (sample coded 827-875) were not significantly different. Also samples 860 and 862 were not significantly different from O V A at pH 7.0. At pH 9.0, the S 0 values were in the following order: albumen samples 827 > 853 > 861, 875 > 862 > 860 > O V A . At pH 9.0, the surface hydrophobicity of sample 827 was 71 significantly higher than samples 860 and 862, and only sample 860 was not significantly different from O V A . When the S 0 values are compared across the three pHs studied (pH 3.0, 7.0 and 9.0), the S 0 values determined by ANS show a significant trend of highest surface hydrophobicity at pH 3.0 and lowest surface hydrophobicity at pH 7.0 and 9.0 for all albumen samples 827-875 and O V A . Also, the S 0 values determined at pH 7.0 were not significantly different from those at pH 9.0 within each of the albumen samples 827-875 and O V A studied. 72 pH 3.0 T150 8 -ft oo to •— w 50 o I ° az 827 az HMBH by • H I • 853 860 aby abx 861 Sample Code 862 abz cy 875 OVA pH7.0 V150 8 -fioo 3 ° w _ 50 o » f 0 ax ax 827 853 cx cx 860 861 862 Sample Code bx dx 875 OVA pH 9.0 T150 n ?, .£100 o _ n - ° co _ 50 o - o ay ay bxy bx 827 853 860 861 862 Sample Code bx 875 OVA Fig. 9 - Surface Hydrophobicity of Spray Dried Egg Albumen (Sample Codes 827-875) and Ovalbumin (OVA) at pH 3.0, 7.0, and 9.0 using PRODAN. a, b, c, etc. S 0 values of samples at a given pH not sharing common superscripts are significantly different at P < 0 .05 . x ' y ' z S 0 values at different pH for a given sample not sharing common superscripts are significantly different at P < 0.05. Bars show the mean of duplicate analysis (n = 2). 73 pH3.0 T1000 — 800 co 2? o o 600 | 2 400 W Q. o 200 | 0 ax 827 853 dx bx cx ex 860 861 862 Sample 875 OVA pH 7.0 V1000 i ^ 800-C D e1 I 5 600 -3 ° 400 -2 200 -•a ay ay aby ay mmni aby ay by X 0 " 827 853 860 861 Sample 862 875 I OVA pH 9.0 T1000 -- 800 -C O & | s 6oo -| | 400 -o 200 - ay r~n aby cdy abcy I I I I I I I I I I bey abcy dy x 0 • 827 853 r 860 861 Sample 862 i 875 OVA Fig. 10 - Surface Hydrophobicity of Spray Dried Egg Albumen (Sample Codes 827-875) and Ovalbumin (OVA) at pH 3.0, 7.0, and 9.0 using ANS. a b c - e t c SG values of samples at a given pH not sharing common superscripts are significantly different at P < 0 .05. x ' y ' z ' S„ values at different pH for a given sample not sharing common superscripts are significantly different at P < 0.05. Bars show the mean of duplicate analysis (n = 2). 74 4.2.3. Zeta Potential Figure 11 shows the zeta potential of the spray dried egg albumen (sample codes 827-875) and ovalbumin (OVA) determined at pH 3.0, 7.0, and 9.0. Figure 12 shows the comparison of the zeta potential of the spray dried egg albumen (sample codes 827-875) and ovalbumin (OVA) determined at pH 3.0, 7.0, and 9.0. From the analysis of variance (data not shown), it can be concluded that the pH effect, the sample effect and the interaction of pH and sample were all significant (P < 0.001). The linear regression trendline was drawn through the 15 charge (in mV) points. The isoelectric points (pi) of the egg albumen (sample codes 827-875) and ovalbumin (OVA) solutions were 4.61, 4.73, 4.70,4.80, 4.85,4.89 and 4.39, respectively. The isoelectric points were determined as the pH corresponding to ZP = 0 mV, at the intersection of the regression line through the pH x-axis. The pi of O V A determined in this study (pi = 4.39) is slightly lower than the reported pi of 4.5-4.7 (Powrie and Nakai, 1986; and Hammershoj et al, 1999). Table 5 shows the mean zeta potential values (n = 5). In general, the charge at pH 3.0 was positive. However, as pH increased to 7.0 and 9.0, the charges became significantly more negative for all albumen samples (827-875) and O V A . At pH 3.0, the absolute ZP were in the following order: albumen samples O V A , 875 > 861, 862, 860 > 827 > 853. At pH 3.0, the zeta potential of sample 853 was significantly lower compared to the other samples. Samples 860, 861 and 862 possessed intermediate ZP and were not significantly different from each other. Samples 875 and O V A had significantly higher zeta potential compared to other samples. At pH 7.0, the absolute ZP were in the following order: albumen samples O V A > 875 > 827 > 861, 860, 862 > 853. At pH 7.0, the highest zeta potential was observed for sample O V A , whereas the lowest zeta potential was observed for sample 853. Samples 860, 861 and 862 possessed intermediate ZP and were not significantly different from each other. 75 At pH 9.0, the absolute ZP were in the following order: albumen sample O V A > 860 > 861, 862 > 853, 875 > 827. At pH 9.0, O V A possessed the greatest ZP, whereas among the spray dried egg albumen samples significantly higher zeta potential was observed for sample 860 and significantly lower zeta potential was observed for sample 827. At the three pH levels studied (pH 3.0, 7.0 and 9.0), the absolute values of zeta potential for all albumen samples and O V A were significantly higher at pH 9.0 followed by pH 7.0, and then pH 3.0. 76 827 CO 50.0 v ~ 0.0 % ^-50.0°!° N -100.0 y = -7.589x + 35.023 R2 = 0.9339 10.0 pH 853 15 5 0 0 n S 5- o.o £ ^-50 .0° l 0 N -100.0 y = -7.3317x + 34.673 R2 = 0.9988 10.0 pH 860 50.0 ^ - 5 0 . 0 ° l 0 I N -100.0 y = -9.7435X + 45.759 R2 = 0.9948 PH 10.0 862 50.0 ~ 0.0 £ - 5 0 . 0 ° | ° -100.0 -y =-8.5511x +41.244 R2 = 0.9978 10.0 pH 861 TH 50.0 -l .a 1 2 - 0.0 | £ - 5 0 . 0 ° J ° N -100.0 y = -8.8262x +42.386 R2 = 0.9946 PH 10.0 875 g 50.0 n c | l - 5 0 . 0 ° - | ° N -100.0 0.0 y = -9.2523x + 45.266 R2 = 0.9098 10.0 pH OVA y = -15.014x +65.839 R2 = 0.9892 PH Fig. 11 - Zeta Potential of Spray Dried Egg Albumen (Sample Codes 827-875) and Ovalbumin (OVA) at pH 3.0, 7.0, and 9.0. Each data point represents the mean of 5 replicate analyses (n = 5). 77 30.0 827 853 860 861 862 875 OVA Linear (827) Linear (853) Linear (860) Linear (861) Linear (862) Linear (875) Linear (OVA)| -70.0 0.0 5.0 10.0 Fig. 12 - Comparison of Zeta Potential of Spray Dried Egg Albumen (Sample Codes 827-875) and Ovalbumin (OVA). 78 Table 5. Summary of Structural Parameters and Functionality Determined for Spray Dried Egg Albumen (Sample Codes 827-875) and Ovalbumin (OVA) Sample Sample Codes 1 827 2 853 3 860 4 861 5 862 6 875 Ovalbumin OVA Gel Strength * 700 612 422 550 500 655 ND Gel Strength (Nmm) pH 3.0 pH 7.0 pH 9.0 85 68 a y 50 3 2 71 b x 55 b y 49 a y 36 d x 38 c x 38 " 65 b x 52bdxy 44 a y 49 c x 43cdx 42" 67 b x 59 * x 44 a y oe z 20 ov 38" Foam Volume (mL) pH 3.0 pH 7.0 pH 9.0 127ay 138" 116^ 113 ^  120bx 101 b z 97* 107cx 95 * 120 b x 97 d z 102by 101 c x 102dx g g b C X 126" 123 b x 106by 111 <* 118 b x 99 te Surface Hydrophobicity (PRODAN) pH 3.0 5632 pH7.0 105 " pH 9.0 81 a y 49 3 2 101 ** 78 a y by 53 c x 45bxy 41 ** 63 c x 68 " 42 -* 50 c x 36 b x 40 •te 8 2 bx 66 a y 11 ^  32 dx 42 bx Surface Hydrophobicity (% 1) (ANS) pH 3.0 821 " pH7.0 99ay pH9.0 108ay 830" 100ay 91 "by 457 d x 64*" 44^ 711 b x 85 a y 72 abcy 604 c x 66 a b y 53^ 675 b x 96 a y ygabcy 276 e x 25 b y 12* Zeta Potential (mV) pH 3.0 pH 7.0 pH 9.0 14.6 b c x -25 a y -28.7 3 2 12.6CX -16.4 a y -31.5bz 15.8bx -20.2 b y -43.4 16.6bx -21.4by -35.7" 15.9 b x -19.6by -35.1 c z 20.9" -29.5 * -31.3 ^  22.5" -44.4 " -73.8 6 2 Pi 4.61 4.73 4.70 4.80 4.85 4.89 4.39 Sulfhydryl Groups (nmole /g protein) Reactive SH 3.3a 3.4a Total SH 29.3d 50.8a Total SH + SS 47.8d 77.6b 1.7° 40.8 b44.2 d 2.1 d 37.0 ^  62.6° 1.9* 51.6 a52.3 d 2.3 b32.2cd 53.5 d0.5 644.8* 83.9" Disulfide Bonds 18.5 26.8 3.4 25.6 0.8 21.3 39.1 a.b,c,etc. V a | u e s j n t n e s a m e r o w n o t sharing common superscripts are significantly different at P < 0.05. x , y , z Values in the same column not sharing common superscripts are significantly different at P < 0.05. * Values (unknown units) reported by Canadian Inovatech Inc. 79 4.2.4. Sulfhydryl and Disulfide Groups 4.2.4.1. Reactive and Total Sulfhydryl Groups Table 5 shows the reactive and total sulfhydryl groups of the spray dried egg albumen (sample codes 827-875) and ovalbumin (OVA) determined at pH 7.0. From the analysis of variance (not shown), the sample effect was significant (P < 0.001). Figure 13 compares the reactive and total sulfhydryl groups of spray dried egg albumen (sample codes 827-875) and ovalbumin (OVA) determined at pH 7.0. The reactive SH were in the following order: albumen samples 827, 853 > 875 > 861, 862 > 860 > O V A . Samples 827 and 853 possessed significantly higher reactive S H (umole SH/g protein) compared to the other albumen samples, whereas O V A possessed significantly lower reactive SH (umole SH/g protein). Among the spray dried egg albumen samples, the observed reactive SH for samples 860 and 862 was significantly lower compared to the other albumen samples. However, samples 861 and 862 were not significantly different from each other. The total SH were in the following order: albumen samples 853, 862 > O V A > 860, 861 > 875, 827. Samples 827 and 875 contained lower total S H (umole SH/g protein) compared to the other albumen samples, whereas samples 853, 862 and O V A showed higher total SH (umole SH/g protein). The range of total sulfhydryl groups determined for spray dried egg albumen (sample codes 827-875) were 29.3 ± 0.3 to 51.6 ± 3.0 umole SH/g protein. These values are comparable to the literature value of the total sulfhydryl groups of spray dried egg white proteins of 45.7 ± 0.6 umole SH/g protein reported by Mine (1997). 80 60 50 g 40 CD o Q. CD w 30 o o E 20 10 H B CD be 827 853 860 861 862 Sample Code • Reactive SH • Total SH BC AD 875 OVA Fig. 13 - Reactive and Total Sulfhydryl Groups of Spray Dried Egg Albumen (Sample Codes 827-875) and Ovalbumin (OVA) at pH 7.0. a, b, c, etc. Reactive S H values not sharing common superscripts are significantly different at P < 0.05. A ' B ' c ' e t c Total SH values not sharing common superscripts are significantly different at P < 0.05. Bars show the mean of triplicate analyses (n = 3) with error bars representing standard deviation range of 0.0 to 0.2 umole S H / g protein and 0.3 to 3.0 u.mole S H / g protein for surface and total SH, respectively (too small to be seen). 81 The majority of sulfhydryl residues in egg white proteins exist in the interior of protein molecules and are exposed with heat denaturation (Mine, 1997). Since the egg albumen (sample codes 827-875) were spray dried there would be some expectation of denaturation of the proteins. The state of denaturation was observed indirectly from the greater number of reactive sulfhydryl groups in the spray dried egg albumen samples compared to O V A . The O V A was purchased from Sigma (St. Louis, MO) and used without further modifications (i.e. did not go through the same spray drying process as the albumen samples), hence the observed reactive sulfhydryl groups of O V A was significantly lower compared to the albumen samples 827-875. As previously mentioned, O V A is a single pure protein which has been reported to have theoretically 4 SH groups and 1 SS bond. In this study, the SH groups and SS bonds for O V A were calculated to be 2.02 mol S H / mol O V A and 1.76 mol SS / mol O V A , respectively. However, the reports on the precise number of thiol versus disulfide groups have been conflicting probably due to sulfhydryl-disulfide interchange (Li-Chan and Nakai, 1989). The theoretical number of 4 SH/mole of O V A will be different for egg albumen due to the complexity of the proteins of which it is composed. O V A is the only major protein in egg albumen to contain SH, hence would be appropriate to use as a comparison. It may be possible that spray drying caused the SS bonds to convert to SH from the other proteins in egg albumen, therefore the observed increase in the reactive SH groups. 4.2.4.2. Total Sulfhydryl Groups and Disulfide Bonds Table 5, Figures 14 and 15 show the total sulfhydryl groups, sulfhydryl groups and disulfide bonds, and disulfide bonds of the spray dried egg albumen (sample codes 827-875) and ovalbumin (OVA) determined at pH 7.0. 82 According to the method of Thannhauser et al. (1984), the protein or peptide containing disulfide bonds is added to a solution of 2-nitro-5-thio-sulfobenzoate (NTSB) solution at pH 9.5 and incubated for 25 min. Under these conditions the disulfide bonds in the proteins or peptides are first cleaved by the excess sodium sulfite. The liberated thiols subsequently react with NTSB. The 2-nitro-5-thiobenzoate (NTB) liberated in the medium has an extinction coefficient of 13,600 M ' W at 412 nm (Thannhauser et al, 1984). Since 1 mol of NTB is liberated from the cleavage of 1 mol of disulfide bond, the concentration of disulfide bonds can be quantitatively determined simply by measuring the absorbance of the solution at 412 nm. Although the method is highly suitable for quantification of disulfide bonds in proteins and peptides, Damodaran (1985) reported that the NTB liberated in this system is readily converted to a nonchromophoric derivative in the presence of room light. The nonchromophoric derivative is tentatively identified as a sulfo derivative of NTB, regenerated from the photochemical reaction between NTB and excess sulfite in the assay medium (Damodaran, 1985). Explanation for the differences in the total S H group or SS bond content among the albumen samples might be the oxidation of cysteine and cystine of the proteins during the pretreatment and spray drying procedures. Under alkaline conditions, cysteine and cystine follow the R-elimination reaction pathway to produce dehydroalanine residues. However, at acidic pH, oxidation of cysteine and cystine results in formation of several intermediate oxidation products., cysteine sulfenic acid, cysteine sulfinic acid and cysteic acid, and cystine mono- or disulfoxide and cystine mono- or disulfone, respectively (Damodaran, 1996a). Some of these derivatives are unstable and are not detectable by the assays employed. 83 The number of total available sulfhydryl groups and disulfide bonds have been reported to range from 79.9 to 84.5 umole/g of native egg white protein (Mine et al., 1990). In this study, the range of sulfhydryl groups and disulfide bonds was 44.2 to 77.6 umole/ g of protein for samples 827-875 and 83.9 umole/g of protein for O V A . These results indicate that most sulfhydryl residues in egg albumen protein existed in the interior of the protein molecules which formed S-S bonds by SH oxidation or SH/S-S interchange reaction upon exposure with heat denaturation. 84 100 -I 90 -80 -70 -^ 60 co CO c 50 03 X CO ®- 40 o E 30 -20 -10 -0 -wwww xvsxwvs .\\\\\\\\ .\\\\\\\\ .\N\\\\\\ ,\\\\\\\\ \ \ \ \ \ s \ \ , \ \ \ \ \ \ \ \ . \ \ \ \ \ \ \ \ , \ \ \ \ \ \ V * . \ \ \ \ \ \ \ \ . \ \ \ \ \ \ \ \ .WWWW >V\VS\S\\ .\XS\WNN .\\\s\s\\ . \ \ \ \ \ w , \ \ \ \v\ \ \ ,W\\V\\N NN\\N\V>I , \ \ \ \ \ \ \ \ \ \ \ W \ \ \ . \ \ \ \ \ \ \ \ . \ \ \ \ \ \ \ \ .\sw\w\ , \ \ \ \ \ \ \ \ .wwvsw .\\\\\\\\ ,\\\\\\\\ \NS\\\\\ wwww 827 853 860 861 Sample Code 862 875 O V A Fig. 14 - Total Sulfhydryl and Disulfide Groups of Spray Dried Egg Albumen (Sample Codes 827-875) and Ovalbumin (OVA) at pH 7.0. Samples with different letters are significantly different at P < 0.05. Bars show the mean of triplicate analyses (n = 3) with error bars representing standard deviation range of ± 1.0 to 3.0 umole SH and SS / g protein (too small to be seen). 85 50 -i 45 -40 H 827 853 860 861 862 875 OVA Sample Code Fig. 15 - Disulfide Groups of Spray Dried Egg Albumen (Sample Codes 827-875) and Ovalbumin (OVA) at pH 7.0. 86 4.2.5. Gel Strength Gelation properties of egg albumen are similar to that of ovalbumin and involve the formation of extensive regions of antiparallel P-sheet between protein molecules during gelation upon heating (Painter and Koenig, 1976). Ovalbumin is the most abundant protein in egg albumen and thus it dominates the gelling properties. However, that does not exclude effects from other proteins. Egg albumen proteins such as lysozyme and ovotransferrin will gel independently, and egg albumen contains other proteins that can alter the gelation of ovalbumin. Figure 16 and Table 5 show the gel strengths of the spray dried egg albumen (sample codes 827-875) and ovalbumin (OVA) determined at pH 3.0, 7.0, and 9.0. From the analysis of variance (not shown), it can be concluded that the pH effect, the sample effect and the interaction of pH and sample were all significant (P < 0.001). Figure 17 shows the force deformation curves of spray dried egg albumen (sample codes 827-875) and ovalbumin (OVA) gels formed at pH 3.0, 7.0, and 9.0. In general, high gel strengths were observed for all gels made at pH 3.0 with the exceptions of samples coded 860, 862 and O V A . Samples 860 and 862 were not significantly different at the three pH levels studied and O V A did not form a gel at pH 3.0. Ziegler and Foegeding (1990) noted that the hardness or gel strength of egg albumen gels was greater at acidic pH compared to alkaline pH (e.g. gel strength was greater at pH 5.0 than 6.0,7.0 and 8.0). Arntfield and Bernatsky (1993) reported that mixed protein systems could produce stronger, less structured networks than can pure proteins. Hence, egg albumen gels have greater strength than ovalbumin gels formed under the same conditions. This was observed for the egg albumen and ovalbumin gels formed at pH 3.0 and 7.0, but not at pH 9.0. 87 pH7.0 E 100 I so ^ £ 60 1 40 _ 20 2 n o u ay by bdxy cdx K ey 827 853 860 861 862 Sample 875 OVA Fig. 16 - Gel Strength of Spray Dried Egg Albumen (Sample Codes 827-875) and Ovalbumin (OVA) at pH 3.0, 7.0 and 9.0. a , b - c - e t c Gel strength values of samples at a given pH not sharing common superscripts are significantly different at P < 0 .05 . x ' y ' z Gel strength values at different pH for a given sample not sharing common superscripts are significantly different at P < 0.05. Bars show the mean of triplicate analyses (n = 3) with error bars representing standard deviation range of 2.0 to 8.0 Nmm. 88 827 2 3 4 Distance (mm) OVA 20 -| in c o 15 -I (Ne 10 -CD (J 5 -O U . 0 -875 20 i in c o 15 -I (Ne 10 -CD o 5 -o L i . 0 -2 3 4 Distance (mm) 2 3 4 Distance (mm) pH 3.0 pH 7.0 pH 9.0 Fig. 17 - Force Deformation Curves of Spray Dried Egg Albumen (Sample Codes 827-875) and Ovalbumin (OVA) Gels at pH 3.0, 7.0 and 9.0. No curve is shown for O V A at pH 3.0 due to ho gel formation. 89 The gel strengths determined in this study at pH 7.0 followed the same trends as the gel strengths measured by the company providing the samples, Canadian Inovatech Inc. (Abbotsford, BC). Although the gel strength values of samples were generally higher at pH 3.0 than at pH 7.0, the overall trend with respect to the samples was similar at these two pH values. In contrast, at pH 9.0, the gel strengths of samples (827-875) and O V A were not significantly different from each other. Relatively weak turbid and translucent gels were observed for O V A at pH 7.0 and 9.0, respectively. Gelation of O V A did not occur at pH 3.0, resulting in a viscous opaque liquid. Translucent egg albumen gels were formed at pH 3.0 and 9.0, whereas turbid egg albumen gels were formed at pH 7.0. Egelandsdal (1980) noted that the maximum in gel strength for ovalbumin occurred between one and two pH units from the isoelectric point (pi = 4.65). At pH values between the two maxima the gels were opaque, probably reflecting the fact that ovalbumin has a tendency to form large, compact aggregates when its net charge is low. This tendency is obviously much weaker at high and low pH values since the gels at pH values beyond the two maxima were highly transparent. At extreme pH values the heated protein solutions behaved more like highly viscous, liquids than firm gels. However, the findings in this study for O V A at pH 3.0 contradict the observations made by Egelandsdal (1980). At pH 3.0, within one and two pH units from the isoelectric point of ovalbumin, no gel was formed for O V A , whereas the albumen samples formed strong(est) gels at pH 3.0. These observations suggest that maximum gel strength occurs at pH values where O V A has only a limited capacity for aggregation. In general, aggregation is governed by a balance between attractive and repulsive forces. The repulsive forces between protein molecules are coloumbic forces which depend upon the net charge of the protein itself, and on the ionic strength of the solution employed. The attractive forces may involve hydrophobic 90 associations, multiple hydrogen bonds, disulfide bridges and local ionic attractions (Kinsella, 1982). Since egg albumen is comprised of heterogeneous proteins including ovalbumin, ovotransferrin, ovomucoid, and lysozyme, the heat aggregation of egg albumen could be facilitated by interactions among such heterogeneous proteins. The balance between the attractive and repulsive forces as a function of pH is also an important factor to consider. With too strong tendency towards aggregation the molecules interact to form dense aggregates, thereby excluding themselves from making any contribution to the building of large elongated network structures which give mechanical strength to the gels. On the other hand, too low tendency towards aggregation may result in too few inter-molecular interactions, and correspondingly low gel strength (Egelandsdal, 1980). The maximum in gel strength occurred at a higher net charge (ZP) on the acid than on the alkaline side of pi. Egelandsdal (1980) reported that the maximum gel strength on the acid side of pi occurs at a higher degree of nonspecific electrostatic repulsion between the chains than on the alkaline side of pH. It is well known that the gelling properties can be affected by the cross-linkages of the unfolded molecules containing hydrogen bonds, ionic and hydrophobic interactions (Kato et al., 1989). Also, the increase of cross-links with disulfide bonds may be critical for gel formation. A possible elucidation is that the exposed and reactive residues of the protein molecules, in addition to disulfide bonds, are situated in the appropriate positions to form a strong and stable gel matrix by the interactions between proteins during heating for gelation, while the reactive residues of the native molecules may remained buried in the interior of the protein molecule (Kato et al, 1989). Egelandsdal (1980) noted the possibility that the higher gel strengths of the acid gels are due to higher degree of inter-molecular disulfide bridges. This was also observed in the present study. The gel strengths of samples 860 and 862 were not significantly different 91 at the three pH levels studied and these two samples also contained the lowest number of disulfide bonds of all the samples (sample codes 827-875 and O V A ) analyzed and the lowest level of reactive SH of all the albumen samples. There has been some indication in the literature that heat-treated ovalbumin is more expanded at acidic pH values compared to O V A at alkaline pH values. The degree of unfolding of the chains caused by the heat treatment may therefore be an important parameter in determining gel rigidity. The relatively high net charge at the maximum on the acid side of pi supports this idea, since the possibilities for electrostatic repulsion between the fixed charges within each chain should be high in these acid gels (Egelandsdal, 1980). 4.2.6. Foam Volume Figure 18 and Table 5 shows the foam volume of the spray dried egg albumen (sample codes 827-875) and ovalbumin (OVA) determined at pH 3.0, 7.0, and 9.0. From the analysis of variance (not shown), it can be concluded that the pH effect, the sample effect and the interaction of pH and sample were all significant (P < 0.001). Figure 19 shows a photographic illustration of the typical foams observed for spray dried egg albumen (sample code 827) and O V A at pH 7.0. The photographs were taken 15 -30 sec after foam production. In general, the bubbles produced in the egg albumen foams were relatively small and compact as compared to the large and coalesced bubbles in the foams of O V A . At pH 3.0, the data for foam volume were in the following order: albumen samples 827, 875 > 861 > 853, 862 and O V A > 860. Samples 827 and 875 gave significantly higher foam volumes compared to those of other egg albumen and O V A samples, whereas, the lowest foam volumes were observed for samples 860 and 862. 9 2 At pH 7.0, the data for foam volume were in the following order: albumen sample 827 > 875, 853 and O V A > 860 > 862, 861. Sample 827 was significantly higher compared to the foam volumes of other egg albumen and O V A samples, followed by the foam volume of samples 853, 875 and O V A . The foam volume of samples 861 and 862 were significantly the lowest at pH 7.0. At pH 9.0, the data for foam volume were in the following order: albumen sample 827 > 875, 861 and 853 > O V A , 862 > 860. Samples 827 was significantly higher compared to the foam volumes of other egg albumen and O V A samples at pH 9.0, whereas the lowest foam volume was observed for sample 860, 862 and O V A . In general, there was significant difference observed for the foam volume for each egg albumen sample and O V A , with the exception of samples 860 and 862. For sample 860, there was no significant difference between the foam volume produced at pH 3.0 and 9.0. For sample 862, there was no significant difference between the foam volume produced at all pH levels (pH, 3.0, 7.0 and 9.0). 93 pH 3.0 150 i | 100 — o > 50 03 o 1 a x bx cx • •I pH 7.0 150 i E, | 100 -| o > 50 E CO £ o pH 9.0 150 § 100 -j — o > 50 E co £ o cx ax I cx —I —I 827 853 860 861 862 Sample Code 875 OVA ay bx cy d dx 111 by 827 853 860 861 862 Sample Code 11 875 OVA az bz cx bz 111 827 853 860 861 862 Sample Code 875 OVA Fig. 18 - Foam Volume of Spray Dried Egg Albumen (Sample Codes 827-875) and Ovalbumin (OVA) at pH 3.0, 7.0 and 9 . 0 . 3 - b c , e t c Foam volume values of samples at a given pH not sharing common superscripts are significantly different at P < 0 .05 . x ' y ' z Foam volume values at different pH for a given sample not sharing common superscripts are significantly different at P < 0.05. Bars show the mean of triplicate analyses (n = 3) with error bars representing standard deviation range of 1.0 to 3.0 (too small to be seen). 94 Fig. 19 - Photographic Illustration of Small Compact and Large Coalesced Bubbles in Foam produced from 0.1% protein solution of Spray Dried Egg Albumen (Sample Code 827) and Ovalbumin (OVA) at pH 7.0 with nitrogen flow rate of 60 cm 3/min for 15 sec. Photos were taken within 15-30 sec after foam production. 95 4.2.7. Differential Scanning Calorimetry Figure 20 shows a typical DSC endothermic thermogram of spray dried egg albumen (sample code 827). Table 6 shows the initial temperature of the appearance of the endothermic peak (Tj), the enthalpy of the endothermic peak, the denaturation temperature (Td) and the temperature range at half of the peak height analyzed from the DSC thermograms of spray dried egg albumen (sample codes 827-875). The initial temperature Tj of the appearance of the endothermic peak were in the following order: albumen sample 853, 860, 861 and 862 > 827, 875. The enthalpy of the endothermic peak were in the following order: albumen samples 860, 875 > 827, 853, 861 and 862. Kato et al. (1990) showed that when 10 % protein samples were analyzed, spray dried egg albumen exhibited three endothermic peaks with a total enthalpy of 12.6 J/g. However, in this study, the 10 % solutions of spray dried egg albumens (sample code 827-875) showed only one endothermic peak with an enthalpy range of 3.81-6.59 J/g. The single endothermic peak suggests that the egg albumens studied were denatured to a greater extent by the spray drying process compared to the reported three endothermic peaks of spray dried egg albumen studied by Kato et al (1990). The denaturation temperatures Td were in the following order: albumen samples 860, 861 > 853, 862 and 875 > 827. Samples 860 and 861 possessed the highest T d , but the values were not significantly different from samples 853, 862 and 875 which had intermediate T d . The T d of sample 827 was significantly lower than to the other albumen samples. The denaturation temperature of the spray dried egg albumens (sample code 827-875) ranged from 82.11 to 83.33°C. This T d range is slightly higher than the reported denaturation value of 77.0°C for spray dried egg albumen (Kato et al., 1990) and lower then the reported denaturation value of 84°C for ovalbumin (Donovan et al., 1975). 96 The highest temperature ranges at half of the peak height were observed for samples 827 and 875, whereas significantly lower temperature ranges at half of the peak height were observed for samples 827, 853, 860, 861 and 862. Egg albumen samples 827, 853, 860, 861 and 862 were not significantly different from each other. Sample: EA827A Size: 0.9700 rag Method: HGT Comment: 0.1M phosphate buffer pH 7.0 -7 3 D S C F i l e : D:\TA\DSC\0ATA\EA827.01 Operator: karen Run Oate: 31-Jan-00 13:08 -7.5 -7.6 -7.7 -7.8 -7.9 80 90 Temperature (°C) 100 no Universal V1 .90 TA Instruments Fig. 20 - A Typical Differential Scanning Calorimetry Thermograms of Spray Dried Egg Albumen (Sample Code 827) ON CD c CD E < D ) LU •a CO 1 CL CO o CD E o CO o a> c c c (0 o CO TJ c CD k_ CD b CD © n co 1/3 CO TJ O O 03 a. E co CO cn co CM 00 CM oo cd cn" o cn oS co cn o co cn co" co c\j CO J 3 CO .o J 3 CO n CO o O? CO co in CM CO co CO o CM 00 CM oo co oo CM 00 CNJ oo CM 00 •a H in CO cn CM co" CO "«** 00 co" c\i oo CM oo co oo CO oo ((82 CM oo CO CO CO CO "55 oo m cn CO >% C L co CD co in CO thal CM O •«t oo T— CO cn o in CO En CO iri iri CO n . O JO CO o in in *t CO CM in O r— 00 CO cn o •<* in ^t in r-» H 00 in m CO 00 •<* in CO CO iri in cn CO CM 00 co o i -m co oo oo CM in co co h-oo oo oo IT) o d V 0_ rt c CD i — CD i t TJ C CO CO *-.2 E c 'oo CD (0 00 ••—• C L o CO 1 CD QL 00 c o E E o o CD c CO - C 00 ' o c o o CD E CO to CD 00 CD CO > CD O c co co CD CL CL CO CD O CD ZD -4—• CO k_ CD CL E CD CD -*—< II H CO CD 00 >, CO c CO CD •*—» CO o "5. 3 T3 00 CO c o J Z CO CO c E •3 •= II CD _3 CO > CD my—* © E CO 'CD J Z II h-X c CO » co CD C L E Q C _o V-' CO k -CO c CD T J T3 H (0 CD C L O E C >-z <~ CO CL sz o CO 111 CD J Z •4—' O • D C 0 4.3. SIMPLE CORRELATIONS OF INDIVIDUAL PARAMETERS & FUNCTIONALITY 4.3.1. Correlation of Protein Surface Hydrophobicity and Zeta Potential Tables 7 to 10 show the correlations (Pearson) of protein surface hydrophobicity using PRODAN and ANS with the IZPI of spray dried egg albumen (sample codes 827-875) and O V A at pH 3.0, 7.0, 9.0 and all pH values combined. The S 0 determined at each pH (3.0, 7.0 and 9.0) by P R O D A N versus ANS were highly positively correlated (P < 0.001). But when S 0 values at all three pH values were combined in the correlation analyses, the S e of P R O D A N versus ANS correlated negatively (P < 0.001). The negative correlation observed when the S 0 determined at all three pH's were pooled could be explained by the relationship of P R O D A N and ANS versus IZPI. This relationship is probably due to the effect of pH and resulting charge on the interaction of the probe with the protein, leading to very high S 0 values at pH 3.0 measured by ANS but not by PRODAN. At pH 3.0, a lack of correlation was observed for both P R O D A N and ANS versus IZPI. This was also observed for S 0 determined by P R O D A N and ANS versus IZPI at pH 7.0. At pH 9.0, significantly negative correlation between both P R O D A N and ANS S 0 versus IZPI was observed. But when all pH's were combined in the correlation analyses of P R O D A N and ANS S 0 versus IZPI, no correlation between P R O D A N S 0 and IZPI was observed, whereas the correlation between ANS S 0 and IZPI was significantly negative. In general, the net charge effect did not have a significant correlation on the surface hydrophobicity determination at pH 3.0 and 7.0. However, significantly negative correlation was observed at pH 9.0 for the absolute values of net charge and S 0 determined by both P R O D A N and ANS probes. Furthermore, no correlation between net charge and surface hydrophobicity determined by P R O D A N was observed when the data were combined for all pH levels, whereas a significantly negative correlation was observed for the absolute values of net charge and surface hydrophobicity determined by ANS. This observation suggests that the 100. anionic ANS probe was influenced by the environmental charge, whereas the neutral uncharged PRODAN probe was not affected. 4.3.2. Correlations of Gel Strength and Structural Parameters Tables 7 to 10 show the correlations (Pearson) of gel strength and structural parameters of spray dried egg albumen (sample codes 827-875) and O V A at pH 3.0, 7.0, 9.0 and all pH's combined. 4.3.2.1. Correlation of Gel Strength and Surface Hydrophobicity It is well known that the gelling properties can be affected by the presence of hydrophobic side chains of the unfolded molecules. The surface hydrophobicity determined at the individual pH's (pH 3.0, 7.0 and 9.0) using PRODAN and ANS were all significantly correlated to gel strength, but when the data from all three pH levels were combined in the correlation analysis, there was no correlation observed between gel strength and PRODAN. However, correlation was observed between gel strength and ANS. The significantly positive correlation between gel strength and S 0 determined by ANS is probably due to the anionic nature of the ANS probe which possibly incorporates a charge effect as well as a binding affinity to surface hydrophobic regions of proteins. 4.3.2.2. Correlation of Gel Strength and Zeta Potential As previously mentioned, it has been reported that the maximum in gel strength occurs at a higher net charge (ZP) on the acid than on the alkaline side of pi. Also, the maximum gel strength on the acid side of pi occurs at a higher degree of nonspecific electrostatic repulsion between the chains than on the alkaline side of pH (Egelandsdal, 1980). The higher degree of nonspecific electrostatic repulsion observed on the acid side of pi could possibly explain the 101 lack of correlation between gel strength and IZPI determined at pH 3.0. A lack of correlation was also observed between gel strength and IZPI determined at pH 7.0, whereas significantly negative correlation was observed for pH 9.0. When the three pH's were combined for correlation analysis between gel strength and IZPI, the correlation was significantly negative. The significance of correlation between gel strength and IZPI indicates that the net charge of the protein solutions prior to gelation plays an important role in gel strength. The net charge effect on gel strength could possibly explain why there was a significant positive correlation observed for gel strength versus ANS but not for PRODAN. The anionic nature of the ANS probe is able to estimate surface hydrophobicity as well as interactions of electrostatic nature, hence the correlation of gel strength to IZPI and ANS was observed. The net charge effect on gel strength could also explain why there was no correlation observed for gel strength versus PRODAN. Since the neutral uncharged nature of the P R O D A N probe is able to estimate surface hydrophobicity only without the electrostatic effect, no correlation was observed for gel strength and P R O D A N over a wide pH range. 4.3.2.3. Correlation of Gel Strength and Sulfhydryl Groups and Disulfide Bonds It is possible that decreases in sulfhydryl content are also responsible for gelation (Hayakawa and Nakai, 1985). Involvement of sulfhydryl groups in gelation of egg white has been reported. Ovalbumin is one of the relatively few proteins containing both thiol groups (4 moles) and disulfide groups (1 mole) in the molecule. Ovalbumin can be polymerized by intermolecular sulfhydryl-disulfide exchange during heating. This intermolecular sulfhydryl-disulfide exchange enables ovalbumin to form simple linear aggregates because the protein contains one mole of readily available disulfide bond. Therefore, conversion of sulfhydryl groups to disulfide bonds as well as interchange of sulfhydryl-disulfide groups may be important for gelation (Hayakawa and Nakai, 1985). Heat-induced sulfhydryl-disulfide exchange in dried egg albumen proteins, studied by Mine (1997), may affect the aggregation behavior of the proteins. Mine (1997) noted that sulfhydryl groups in dried egg albumen proteins were exposed to the surface on the protein and sulfhydryl-disulfide exchange reactions were accelerated by heating. The importance of the sulfhydryl-disulfide interchange for the polymerization of egg albumen proteins could be observed in the significant correlation between gel strength and reactive SH at the individual pH level (pH 3.0, 7.0 and 9.0) and combined pH levels, and the significant correlation between gel strength and SS bonds at the individual pH level (pH 3.0, 7.0 and 9.0) and combined pH levels. 4.3.3. Correlation of Foam Volume and Structural Parameters Tables 7 to 10 show the correlations (Pearson) of foam volume and structural parameters of spray dried egg albumen (sample codes 827-875) and O V A at pH 3.0,7.0, 9.0 and all pH's combined. 4.3.3.1. Correlation of Foam Volume and Surface Hydrophobicity Heat denaturation, leading to increased surface hydrophobicity of proteins, usually enhances foaming properties. The surface hydrophobicity determined at the individual pH levels (pH 3.0, 7.0 and 9.0) using PRODAN and ANS were all significantly correlated to foam volume. But when the all three pH's were combined in the correlation analysis, the correlation observed between foam volume and PRODAN was more highly significant compared to the correlation observed between foam volume and ANS. 103 4.3.3.2. Correlation of Foam Volume and Zeta Potential The results of Hammershoj and colleagues (1999) concerning foamability showed that egg albumen protein solutions seem to favour foam formation when different protein net charges and attractive forces were present. In general, at pH 3.0 and 7.0 there was no correlation observed between foam volume and IZPI. However, negative correlation between foam volume and IZPI was observed at pH 9.0. Furthermore, when data from all three pH levels were combined in the correlation analysis of foam volume and IZPI, the relationship was negatively significant. 4.3.3.3. Correlation of Foam Volume and Sulfhydryl Groups and Disulfide Bonds Formation of a protein network can proceed by thiol and disulfide exchange, which is very rapid at neutral to alkaline pH (Creighton, 1993) and by oxidation of cysteine residues into disulfide bonds. This could be confirmed with a significant correlation of foam volume versus reactive SH and SS bonds. Correlation of foam volume with SS bonds at neutral or alkaline pHs were not as significant when compared to the acidic pH. However, the correlation of reactive SH was more significant for foam volume at neutral and alkaline pH values when compared to acidic pH values. 104 O CO CO 13 o IS > u CO . w IS (0 4-* O CO z < z < Q O DC OL cu E 3 E re O) c 155 00 CO i - o co o d d • a 00 o o 00 o d d cn o to o oo o d d o o r» O d d CO o CM O cn o d d 00 ,_ ~ in 9 d o oo CO o T CO 9 d JO CM co § 9 d "2 in 9 d < Q O C C C O z < CO "5 o o m T - co ~ CO 9 d o o o o cn o d d o o CM o co o d d CM o cn i -in o d d r~ o n o CO o d d X CO cu > u CO CU C C CO CO O CO in o d d O) CM CD O d d in at en o in o d d in oo CM oo CO -r-d d in CM oo co i - •* d d r~ CM co r-oo — d d CO CO + X CO « o CM co o CO o d d co co T - o co o d d 92 <o co —. 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CM o 9 d O) O CM o in o 9 d Si o> in o co O p d < Q O CC 0. co z < X CO o O o ••- co 9 d oo •<* co CM i - CM d d co o in o co o d d in o cn o in o d d r~ o co o co o d d CO > u a o CC o o O i -o o d d t in o in d d cn oo cn CM CM o d d o m o cn d d o Tj-co cn CM o d d CO CO + X CO To o o o cn o n . o o d O CM i -T- CO d d m o T- O w o d d *t o o d d o o co o in o d d o 0 3 . in O _; 92 co — ^ s . CO cn CM t co •>- CM o d CO CO CO is 9 d CO CO a N c CD o 3r a> o O c g jo <D O O m o d v 0-c cc o c g> •<o CD w CO (/> CD 3 CD j 3 CO > > 32 a. o a CO. 4.4. MULTIPLE REGRESSION ANALYSIS 4.4.1. Gel Strength Multiple regression analysis was carried out using a quadratic model to relate gel strength with the structural parameters of surface hydrophobicity determined by P R O D A N and ANS, total S H groups, reactive S H groups, total S H groups + SS bonds, SS bonds and IZPI. Appendix 3 shows the regression analysis printout for gel strength and the structural parameters. A highly significant regression equation was obtained to describe gel strength (R 2 = 0.956, P < 0.001, n = 53): Gel Strength = - 191 + 1.76 X! + 0.222 X 2 - 532 X 3 - 41.6 X 4 - 524 X 5 + 529 X 6 + 1.00 X 7 + 0.0076 X! 2 - 0.000066 X 2 2 + 7.23 X 3 2 - 3.10 X 4 2 - 7.3 X 5 2 + 7.3 X 6 2 - 0.0107 X 7 2 - 0.00183 X,X 2 - 13.4 X 1 X 3 - 0.095 X 1 X 4 + 13.4 X,X 5 - 13.4 X!X 6 + 0.0231 X,X 7 - 1.30 X 2 X 3 + 0.0698 X 2 X 4 + 1.30 X 2 X 5 - 1.30 X 2 X 6 - 0.00259 X 2 X 7 - 0.29 X 3 X 4 + 14.6 X 3 X 6 + 19.2 X 3 X 7 + 0.852 X 4 X 5 - 0.709 X 4 X 7 -19.2 X 5 X 7 + 19.2 X ^ , where X i = S 0 using PRODAN; X 2 = S 0 using ANS; X 3 = Total S H groups; X 4 = Reactive S H groups; X 5 = Total S H groups and SS bonds; X 6 = SS bonds; and X 7 = IZPI. The interaction terms of X 3 X 5 , X 4 X 6 , and X 5 X 6 were highly correlated with other X variables and were not included in the final regression equation. According to the multiple regression analysis of gel strength and structural parameters (Appendix 3), the interaction terms X 2 X 3 , X 2 X 5 , X2Xo, XaX 7 , X5X-7, and X<;X7 were the only significant (P < 0.05) predictors in the regression equation. The interaction terms X1X3, X 1 X 5 , and X 1 X 6 were significant at P = 0.079. 110 4.4.2. Foam Volume Multiple regression analysis was carried out using a quadratic model to relate foam volume with the structural parameters of surface hydrophobicity determined by P R O D A N and ANS, total SH groups, reactive SH groups, total SH groups + SS bonds, SS bonds and IZPI. Appendix 4 shows the regression analysis printout for foam volume and the structural parameters. A highly significant regression equation was obtained to describe foam volume (R 2 = 0.918, P < 0.001, n = 53): Foam Volume = 47 + 0.80 X, + 0.062 X 2 + 184 X 3 + 119 X 4 - 186 X 5 + 179 X 6 - 1.31 X 7 +0.0022 X, 2 - 0.000146 X 2 2 -2 .3 X 3 2 - 10.1 X 4 2 + 2.3 X 5 2 - 2.3 X 6 2 - 0.0088 X 7 2 - 0.00088 X,X 2 + 3.08 X 1 X 3 - 0.328 X 1 X 4 - 3.08 X,X 5 + 3.12 X , X 6 + 0.0176 X!X 7 - 0.004 X 2 X 3 - 0.0777 X 2 X 4 + 0.009 X 2 X 5 -0.001 X 2 X 6 + 0.00398 X 2 X 7 - 1.61 X 3 X 4 - 4.5 X 3 X 6 + 5.09 X 3 X 7 + 0.859 X 4 X 5 + 0.865 X4X7 - 5.18 X 5 X 7 + 5.10 X ^ , where X , = S 0 using P R O D A N ; X 2 = SG using ANS; X 3 = Total S H groups; X 4 = Reactive SH groups; X 5 = Total SH groups and SS bonds; - SS bonds; and X 7 = IZPI. The interaction terms of X 3 X 5 , X 4 X 0 , and X 5 X 6 were highly correlated with other X variables and were not included in the final regression equation. According to the multiple regression analysis of foam volume and structural parameters (Appendix 4), there were no significant (P > 0.05) predictors of the regression equation. I l l C H A P T E R 5. G E N E R A L C O N C L U S I O N S 5.7. PROTEIN SURFACE HYDROPHOBICITY Until a more theoretically acceptable, universal method is established, simple approaches that give a measure of the hydrophobic groups on the protein surface that are available for participation in functionality may be the methods of choice for monitoring protein hydrophobicity (Li-Chan 1999). The hydrophobicity values (S0) obtained by a spectroscopic method using two different fluorescent probes were investigated in this study. Even though P R O D A N and ANS are both aromatic hydrophobic probes, different trends were observed in the S 0 values obtained by P R O D A N and ANS at acidic and alkaline pH or in the presence of low and high concentrations of NaCl. These differences may be attributable to the absence of an ionizable group in PRODAN, in contrast to the negatively charged sulfonate group of ANS. By avoiding potential contribution of electrostatic probe-protein interactions due to its uncharged nature, P R O D A N may be a more useful probe than ANS for the investigation of the effect of pH or ionic strength on protein surface hydrophobicity. 5.2. SPRA Y DRIED EGG ALBUMEN ANALYSIS The manifestation of functional properties of food proteins is the result of a complex interplay of various intrinsic properties of proteins, such as hydrophobicity, charge, sulfhydryl groups and disulfide bonds, and extrinsic factors such as pH, ionic strength, and temperature. Quantitative understanding of the relative importance of the various intrinsic properties of proteins in the expression of a specific functional property is still elusive (Damodaran, 1994). The basic problem is that it is difficult to change each of these molecular properties individually and study its effect on a given functional property. Because of these difficulties much of the information in the literature on the structure-function relationship of food proteins is qualitative and descriptive in nature. A realistic approach to achieving a basic understanding 112 of the structure-function relationship would be a multivariate approach in which the combined effects of various molecular factors on a functional property are studied simultaneously (Damodaran, 1994). A main focus of this study, therefore, was to evaluate and compare the structure and functional properties, including protein surface hydrophobicity, charge, total and reactive sulfhydryl groups, disulfide bonds, differential scanning calorimetry analysis, gelation and foaming properties, of six spray dried egg albumen and ovalbumin. Surface hydrophobicity determined at the individual pH's (pH 3.0, 7.0 and 9.0) using P R O D A N and ANS were all significantly correlated with gel strength. When data from all three pH levels were combined in the correlation analysis, there was no correlation observed between gel strength and PRODAN. However, correlation was observed between gel strength and ANS. The higher degree of nonspecific electrostatic repulsion observed on both side of pi could possibly explain the lack of correlation between gel strength and IZPI determined at pH 3.0 and 7.0. However, at pH 9.0 negative correlation was observed. When the three pH's were combined for correlation analysis between gel strength and IZPI, the correlation was significantly negative. The importance of the sulfhydryl-disulfide interchange for the polymerization of egg albumen proteins could be observed in the significant correlation between gel strength and reactive SH at the individual pH level (pH 3.0, 7.0 and 9.0), the combined pH levels, and the significant correlation between gel strength and SS bonds at the individual pH level (pH 3.0, 7.0 and 9.0) and combined pH levels. The surface hydrophobicity determined at the individual pH levels (pH 3.0, 7.0 and 9.0) using P R O D A N and ANS were all significantly correlated to foam volume. But when all three pH's were combined in the correlation analysis, the correlation observed between foam volume 113 and PRODAN was more significant compared to the correlation observed between foam volume and ANS. In general, at pH 3.0 and 7.0 there was no correlation observed between foam volume and IZPI. However, negative correlation between foam volume and IZPI was observed at pH 9.0. The correlation of foam volume with SS bonds at neutral or alkaline pHs were not as significant when compared to the acidic pH. However, the correlation of reactive SH was more significant for foam volume at neutral and alkaline pH values when compared to acidic pH values. The denaturation temperature of the spray dried egg albumens (sample code 827-875) ranged from 82.11 to 83.33°C. Significant models were obtained to describe gel strength and foam volume as a function of the structural parameters using multiple regression analysis (R 2 = 0.956 and R 2 = 0.918, respectively; P < 0.001; n = 53). 5.3. RECOMMENDATIONS AND FUTURE RESEARCH In hindsight, along with studying the foam volumes of the spray dried egg albumens and O V A , other foaming parameters (i.e. bubble size of the foam and foam stability) could have been investigated to better understand the correlation between the foaming functionality with structural parameters. The reasoning is that two systems could produce that same foam volume but have major differences in bubble size of the foam and ultimately the stability of the foam. For future research, investigation of structural properties of the proteins could be performed using circular dichroism, vibrational spectroscopy, nuclear magnetic resonance spectroscopy and/or Raman spectroscopy. 114 R E F E R E N C E S Abola, J.E., Wood, M.K. , Chwen, A. , Abraham, D. and Pulsinelli, P.D. 1982. The Biochemistry and Physiology of Iron, P. Saltman and J. Hegenarer (Eds.), p. 27. Elsevier Applied Science, London, U K . Alizadeh-Pasdar, N . and Li-Chan, E.C.Y. 2000. Comparison of protein surface hydrophobicity measured at various pH values using 3 different fluorescent probes. J. Agric. Food Chem. 48: 328-334. Arntfield, S.D. and Bernatsky, A. 1993. Characteristics of heat-induced networks from mixtures of ovalbumin and lysozyme. J. Agric. Food Chem. 41: 2291-2295. Bergquist, D.H. 1995. Egg Dehydration. 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Correlation (Pearson) between Bigelow's H<t> a v e and S 0 values of the 10 proteins measured by A N S and P R O D A N probes Bigelow's H<|> S 0 ANS pH 3.0 0.01 M NaCl 0.398 a 0.289 b 1.0M NaCl 0.191 0.623 S 0 ANS pH 7.0 0.01 M NaCl 0.272 0.479 1.0M NaCl 0.460 0.213 S 0 ANS pH 9.0 0.01 M NaCl 0.134 0.730 1.0M NaCl 0.278 0.469 S 0 PRODAN pH 3.0 0.01 M NaCl 0.568 0.111 1.0M NaCl 0.410 0.273 S 0 PRODAN pH 7.0 0.01 M NaCl 0.323 0.396 1.0M NaCl 0.461 0.212 S 0 PRODAN pH 9.0 0.01 M NaCl 0.166 0.669 1.0M NaCl 0.201 0.604 a Correlation Coefficient b P-Value Appendix 2. The relative fluorescence intensity (RFI)1 and pH values of A N S in citrate-phosphate buffer and citrate-phosphate:methanol buffer mixtures Solvent RFI of P R O D A N Measured pH Citrate Phosphate Buffer pH 3.0 0.01 M NaCl 0.6 3.0 1.0M NaCl 0.6 3.0 pH7.0 0.01 M NaCl 0.6 7.0 1.0M NaCl 0.6 7.0 pH 9.0 0.01 M NaCl 0.6 9.0 1.0M NaCl 0.6 9.0 Citrate Phosphate Buffer: Methanol Mixture (2:1, v:v) pH 3.0 0.01 M NaCl 0.6 3.6 1.0M NaCl 0.6 3.6 pH7 .0 0.01 M NaCl 0.6 7.8 1.0M NaCl 0.6 7.8 pH9 .0 0.01 M NaCl 0.6 9.1 1.0M NaCl 0.6 9.0 1 RFI values are the mean of triplicate analyses of 10 uL A N S stock solution in 4 ml buffer (X e x = 390 nm, X e m = 470 nm). Standard deviation for RFI of A N S in citrate phosphate (pH 3.0, 7.0 and 9.0) was 0.0 for both salt concentrations. Appendix 3. Multiple Regression Analysis of Gel Strength and Structural Parameters * X3*X5 i s hig h l y c o r r e l a t e d with other X variables * X3*X5 has been removed from the equation * X4*X6 i s highly c o r r e l a t e d with other X variables * X4*X6 has been removed from the equation * X5*X6 i s highly c o r r e l a t e d with other X variables * X5*X6 has been removed from the equation The regression equation i s GEL = - 191 + 1.76 XI + 0.222 X2 + 532 X3 - 41.6 X4 - 524 X5 + 529 X6 + 1.00 X7 + 0.0076 Xl*2 -0.000066 X2*2 + 7.23 X3*2 - 3.10 X4*2 - 7.3 X5*2 + 7.3 X6*2 - 0.0107 X7*2 - 0.00183 X1*X2 - 13.4 X1*X3 - 0.095 X1*X4 + 13.4 X1*X5 - 13.4 X1*X6 + 0.0231 X1*X7 - 1.30 X2*X3 + 0.0698 X2*X4 + 1.3.0 X2*X5 - 1.30 X2*X6 - 0.00259 X2*X7 - 0.29 X3*X4 + 14.6 X3*X6 + 19.2 X3*X7 + 0.852 X4*X5 - 0.709 X4*X7 - 19.2 X5*X7 + 19.2 X6*X7 Predictor Coef StDev T p Constant -190 . 6 146.5 -1. 30 0. 207 XI 1.762 1.690 1. 04 0. 309 X2 0.2224 0.1512 1. 47 0. 156 X3 532 1027 " 0. 52 0. 610 X4 -41.63 53.82 -0. 77 0. 448 X5 -524 1028 -o 51 0. 616 X6 529 1028 0 51 0. 612 X7 1.002 2.581 0 39 0 702 Xl* 2 0 . 00761 0.01300 0 59 0 565 X2*2 -0 .0000658 0.0001406 -0 47 0 645 X3*2 7.229 9.962 0 73 0 476 X4*2 -3 .101 9.529 -0 33 0 748 X5*2 -7 .30 10.00 -0 73 0 473 X6*2 7.27 10.00 0 73 0 475 X7*2 -0.01066 0 03269 -0 33 0 748 X1*X2 -0.001834 0.002978 -0 62 0 545 X1*X3 -13.413 7 .256 -1 85 0 079 X1*X4 -0.0953 0.6902 -0 14 0 891 X1*X5 13 .390 7.255 1 85 0 079 X1*X6 -13.421 7.263 -1 85 0 079 X1*X7 0.02314 0.02443 0 95 0 354 X2*X3 -1.2997 0.5868 -2 22 0 038 X2*X4 0.06976 0.07477 0 .93 0 361 X2*X5 1.2970 0.5874 2 .21 0 .038 X2*X6 -1.3004 0.5873 -2 .21 0 .038 X2*X7 -0.002595 0.003822 -0 .68 0 .505 X3*X4 -0 .286 1.003 -0 .29 0 .778 X3*X6 14.55 19.99 0 .73 0 .475 X3*X7 19.159 7 .582 2 .53 0 .020 X4*X5 0.8525 0.6763 1 .26 0 .221 X4*X7 -0 .7088 0.6775 .-1 .05 0 .307 X5*X7 -19.157 7.. 597 -2 .52 0 . 020 X6*X7 19.199 7.591 2 .53 0 .020 S = 2.813 R-Sq = 98.3% R-Sq(adj) = 95.6% 126 Analysis of Variance Source DF SS MS F P Regression 32 9375.16 292.97 37.03 0.000 Residual Error 21 166.17 7.91 Total 53 9541.33 Source DF Seq SS XI 1 456. 15 X2 1 6903 . 20 X3 1 241. 44 X4 1 149. 73 X5 1 1. 67 X6 1 244 39 X7 1 0 84 Xl*2 1 0 34 X2*2 1 685 29 X3*2 1 39 49 X4*2 1 46 75 X5*2 1 70 11 X6*2 1 1 07 X7*2 1 34 40 X1*X2 1 60 03 X1*X3 1 19 28 X1*X4 1 3 87 X1*X5 1 45 94 X1*X6 1 1 83 X1*X7 1 0 67 X2*X3 1 139 29 X2*X4 1 18 .24 X2*X5 1 32 .06 X2*X6 1 27 .53 X2*X7 1 6 .44 X3*X4 1 12 .10 X3*X6 1 1 .03 X3*X7 1 7 .31 X4*X5 1 30 .18 X4*X7 1 2 .47 X5*X7 1 41 .41 X6*X7 1 50 .62 127 Appendix 4 . Multiple Regression Analysis of Foam Volume and Structural Parameters * X3*X5 i s h i g h l y c o r r e l a t e d with other X variables * X3*X5 has been removed from the equation * X4*X6 i s hig h l y c o r r e l a t e d with other X variables * X4*X6 has been removed from the equation * X5*X6 i s hig h l y c o r r e l a t e d with other X variables * X5*X6 has been removed from the equation The regression equation i s FOAM = 47 + 0.80 XI + 0.062 X2 + 184 X3 + 119 X4 - 186 X5 + 179 X6 - 1.31 X7 + 0.0022 Xl*2 -0.000146 X2*2 - 2.3 X3*2 - 10.1 X4*2 + 2.3 X5*2 - 2.3 X6*2 - 0.0088 X7*2 - 0.00088 X1*X2 + 3.08 X1*X3 - 0.328 X1*X4 - 3.08 X1*X5 +3.12 X1*X6 + 0.0176 X1*X7 - 0.004 X2*X3 - 0.0777 X2*X4 + 0.009 X2*X5 - 0.001 X2*X6 + 0.00398 X2*X7 - 1.61 X3*X4 - 4.5 X3*X6 + 5.09 X3*X7 + 0.859 X4*X5 + 0.865 X4*X7 - 5.18 X5*X7 + 5.10 X6*X7 Predictor Coef StDev T p Constant 47.0 189.7 0. 25 0. 807 XI 0.801 2.188 0. 37 0 . 718 X2 0.0615 0.1958 0. 31 0. 756 X3 184 1330 0. 14 0. 891 X4 118.94 69.68 1. 71 0 . 103 X5 -186 1332 -0. 14 0. 890 X6 179 1331 0 13 0. 894 X7 -1.308 3 .341 -0 39 0 699 Xl*2 0.00216 0.01683 0 13 0 899 X2*2 -0.0001462 0.0001821 -0 80 0 431 X3*2 -2.26 12 .90 -0 18 0 863 X4*2 -10.13 12.34 -0 82 0 421 X5*2 2.26 12.95 0 17 0 863 X6*2 -2.29 12.95 -0 18 0 861 X7*2 -0 . 00880 0.04233 -0 21 0 837 X1*X2 -0.000884 0.003856 -0 23 0 821 X1*X3 3 .077 9.395 0 33 0 747 X1*X4 -0.3279 0.8936 -0 37 0 717 X1*X5 -3 . 076 9.394 -0 33 0 747 X1*X6 3.120 9.405 0 33 0 743 X1*X7 0.01762 0.03163 0 56 0 583 X2*X3 -0.0042 0.7598 -0 01 0 996 X2*X4 -0.07774 0.09681 -0 80 0 431 X2*X5 0.0091 0.7606 0 .01 0 991 X2*X6 -0.0010 0.7604 -0 .00 0 999 X2*X7 0.003975 0.004948 0 .80 0 .431 X3*X4 -1.608 1.298 -1 .24 0 .229 X3*X6 -4.52 25.89 -0 .17 0 .863 X3*X7 5.090 9.818 0 .52 0 .610 X4*X5 0.8587 0.8757 0 .98 0 .338 X4*X7 0.8648 0.8772 0 .99 0 .335 X5*X7 -5.178 9.836 -0 .53 0 .604 X6*X7 5.102 9.829 0 .52 0 . 609 S = 3.642 R-Sq = 96.8% R-Sq(adj) = 91.8% 128 Analysis of Variance Source DF SS MS F P Regression 32 8310.74 259.71 19.58 0.000 Residual Error 21 278.60 13.27 Total 53 8589.33 Source DF Seq SS XI 1 2124 30 X2 • 1 3394 86 X3 1 843 73 X4 . 1 9 90 X5 1 265 61 X6 1 68 88 X7 1 14 02 Xl*2 1 253 51 X2*2 1 290 27 X3*2 1 34 82 X4*2 1 131 16 X5*2 1 3 91 X6*2 1 5 26 X7*2 1 287 15 X1*X2 1 8 61 X1*X3 1 117 38 X1*X4 1 15 00 X1*X5 1 100 58 X1*X6 1 0 01 X1*X7 1 9 03 X2*X3 1 0 .71 X2*X4 1 5 .92 X2*X5 1 224 .11 X2*X6 • 1 14 .48 X2*X7 1 0 . 00 X3*X4 1 0 .02 X3*X6 1 8 .72 X3*X7 1 7 .36 X4*X5 1 0 .01 X4*X7 1 0 .03 X5*X7 1 67 .79 X6*X7 1 3 .57 129 

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