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Lipophilization of beta-lactoglobulin : effect on hydrophobicity, surface functional properties, digestibility… Akita, Emmanuel E. 1988

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L I P O P H I L I Z A T I O N O F B E T A - L A C T O G L O B U L I N - E F F E C T O N H Y D R O P H O B I C 1 T Y , S U R F A C E F U N C T I O N A L P R O P E R T I E S , D I G E S T I B I L I T Y A N D A L L E R G E N I C I T Y B y Emmanuel E . A k i t a B . Sc. (Biochemistry and Food Science) University of Ghana A T H E S I S S U B M I T T E D I N P A R T I A L F U L F I L L M E N T O F T H E R E Q U I R E M E N T S F O R T H E D E G R E E O F M A S T E R O F S C I E N C E in T H E F A C U L T Y O F G R A D U A T E S T U D I E S D E P A R T M E N T O F F O O D S C I E N C E We accept this thesis as conforming to the required standard T H E U N I V E R S I T Y O F BR I T I S H C O L U M B I A August 1988 © Emmanuel E . A k i t a , 1988 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of The University of British Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date DE-6G/81) A b s t r a c t In this research, beta-lactoglobulin was chemically modified by attaching different levels of stearic acid to the protein. The effect of this modification on hydrophobic!ty, emulsi-fying and foam properties, digestibility and allergenicity of the protein was investigated. It was found that the effect of fatty acid attachment or lipophilization depended on the amount of fatty acids attached to the protein. Incorporation of the hydrophobic ligands led to increased hydrophobic interactions, resulting in a decreasing solubility with extent of incorporation. Furthermore, the surface hydrophobicity measurements showed that the two fluorescence probes 8-anilinonaphthalene-l-sulfona.te ( A N S ) and cis-parinaric acid ( C P A ) used for the surface hydrophobicity. measurements were not equivalent This may support the. observation by earlier workers that A N S measures aromatic hydrophobicity and C P A aliphatic hydrophobicity. • The studies on surface functional properties i.e. emulsifying and foaming properties, indicated that there was some improvement in these functional properties at low and medium levels of incorporation which decreased as the extent of fatty acid attachment further increased. The improvement, of these functional properties could be attributed to improved amphiphilicity of the proteins at these levels of incorporation. This research also showed that both high solubility and high A N S surface hydrophobicity is needed for the best emulsifying properties.. In vitro digestibility studies showed a decrease in digestibility of the modified proteins with increased lipophilization. From the passive cutaneous anaphylaxis experiments, it was found that the level of fatty acid attachment to the protein had a significant effect on its ability to elicit IgE 11 antibodies. Increased ability to elicit IgE antibodies was observed at a low level of fatty acid. When a medium level of fatty acid was attached the ability to elicit antibodies was reduced and almost completely destroyed when a higher level of fatty acid was incorporated. The above observations could be explained by the fact that the low level incorporation of fatty acid led to changes in the protein structure which exposed more allergenic sites. The almost complete destruction of the allergenicity could be attributed to denaturation of the protein which reduced or destroyed available allergenic sites. The antigenicity or binding of the modified proteins to the IgG antibodies raised against the native protein was studied by both direct and competitive enzyme linked immunosorbent assay. It was found that at low and medium levels of incorporation, the proteins demonstrated increased binding ability compared to the native protein. This was attributed to the increased exposure of antigenic sites on the protein with fatty acid incorporation. However, the protein with high level of incorporated fatty acid showed decreased binding ability. in T a b l e o f C o n t e n t s A b s t r a c t i i L i s t o f t a b l e s v i i L i s t o f f i g u r e s v i i i A c k n o w l e d g e m e n t x 1 I N T R O D U C T I O N 1 2 L I T E R A T U R E R E V I E W 5 2.1 Bet a - lactog lobu l in 5 2.2 Hydrophobicity of proteins 6 2.2.1 Hydrophobic interactions 6 2.2.2 Determination of protein hydrophobicity 6 2.2.3 Alterat ion of hydrophobicity to change functionality of proteins . 8 2.3 Protein emulsions 11 2.3.1 Formation and stabilization of emulsions 11 2.3.2 Relationship between emulsifying properties and hydrophobicity . 12 2.3.3 Determination of emulsifying properties 13 2.4 Protein foams 15 2.4.1 Formation and stabilization of protein foams . 15 2.4.2 Relationship between foaming properties a.nd hydrophobicity . . . 15 2.4.3 Determination of protein foam properties 16 iv 2.5 In vitro protein digestibility measurements 18 2.6 Allergenicity of proteins 20 2.6.1 Food protein allergy 20 2.6.2 Suppression of protein allergenicity 20 2.6.3 Antigenicity of /3-lactoglobulin 21 2.6.4 Determination of protein allergenicity 23 3 M A T E R I A L S A N D M E T H O D S 25 3.1 Materials 25 3.2 Methods 26 3.2.1 Esterification of stearic acid with N-hydroxysuccinimide 26 3.2.2 Stearic acyl-/31g preparation 26 3.2.3 Determination of degree of incorporation 27 • 3.2.4 Protein solubility 28 3.2.5 Foaming properties 28 3.2.6 Emulsifying properties 28 3.2.7 Protein hydrophobicity 29 3.2.8 Circular dichroism 30 3.2.9 Digestibility studies 30 3.2.10 Determination of amino groups 32 3.2.11 Z e t a potential 32 3.2.12 SDS-Polyacrylamide gel electrophoresis 32 3.2.13 Immunochemical studies 33 4 R E S U L T S A N D D I S C L J S S I O N 37 4.1 Lipophil ization of /3-lactoglobulin 37 4.2 Solubility 42 v 4.3 Hydrophobici ty and conformational changes 44 4.4 Net charge 48 4.5 Emulsifying properties 51 4.6 Foaming properties 59 4.7 In vitro enzyme digestion 65 4.7.1 Initial rate of digestion 65 4.7.2 Extent of digestion ' 75 4.8 Immunochemical studies 77 4.8.1 Passive cutaneous anaphylaxis 77 4.8.2 Direct and competitive EL1SA 81 4.9 General discussion 84 5 C O N C L U S I O N S 8 8 B i b l i o g r a p h y 91 vi L i s t o f T a b l e s 4.1 Effect of l ipophil ization on net charge of native and modified proteins at p H 7.0 50 4.2 Effect of l ipophil ization of f3\g on relative ini t ial rate of hydrolysis by a-chymotrypsin 67 4.3 Effect of l ipophilization of /31g on relative ini t ial rate of hydrolysis by pepsin 70 4.4 Effect of l ipophilization of (31g on relative init ial rate of hydrolysis by pepsin 71 4.5 Effect of l ipophilization of /?lg on relative ini t ia l rate of hydrolysis by pancreatin 73 4.6 Release of essential amino acids by an in vitro digestion with pepsin followed by pancreatin of na.tive and modified /51g 76 4.7 P C A titre in rats from sera of mice immunized with native and modified proteins and challenged by their corresponding proteins 78 4.8 P C A titre in rats from sera of mice immunized with native and modified proteins and challenged by the native protein 80 vn L i s t o f F i g u r e s 4.1 Amount of fatty acids attached as a function of the mole ratio of 18-OSU to lysine residues 39 4.2 Available amino groups as a function of the mole ratio of 18-OSU to lysine residues. . 40 4.3 S D S - P A G E Electrophoresis of native and modified proteins 41 4.4 Effect of incorporation of fatty acids on the solubility of /3-lactoglobulin. 43 4.5 Effect of incorporation of fatty acids on C P A hydrophobicity of the proteins. 45 4.6 Effect of incorporation of fatty acids on A N S hydrophobicity of the proteins. 46 4.7 Circular dichroism spectra of native-/31g, control-/3Ig and 0.3-/31g 49 4.8 Relationship between emulsifying activity index and degree of incorpora-tion of fatty acids 52 4.9 Relationship between emulsifying activity index and C P A hydrophobicity. 53 4.10 Relationship between emulsifying activity Index and A N S hydrophobicity. 54 4.11 Relationship between emulsion stability index and degree of incorporation of fatty acids 56 4.12 Relationship between solubility, A N S hydrophobicity and emulsifying ac-t ivi ty index 57 4.13 Relationship between solubility, A N S hydrophobicity and emulsion stabil-ity index 58 4.14 Relationship between foam capacity and degree of incorporation of fatty acids 60 4.15 Plot of foam serum drainage volume as a function of time 61 v m 4.16 Plot of reciprocal of foam serum drainage volume against the reciprocal of drainage time 62 4.17 Relationship between foam stability index and degree of incorporation of fatty acids , 64 4.18 Relationship between foam stability index and A N S hydrophobicity. . . . 66 4.19 Relationship between pancreatic in i t ia l rate of hydrolysis and a-chymotryptic in i t ia l rate of hydrolysis 74 4.20 Immunochemical reactivity of modified proteins with IgG antibodies to native protein, measured by E L I S A 82 4.21 Immunochemical reactivity of modified proteins with IgG antibodies to native protein, measured by competitive E L I S A 83 ix A c k n o w l e d g e m e n t I wish to express my sincere gratitude to my supervisor Dr. Shuryo Nakai of the de-partment of Food Science for his invaluable advice and guidance throughout my M S c program. Dr Nakai's hardwork and achievements in the field of Food Science was a source of great encouragement. I would also like to thank the other members of my re-search committee, Dr . W . D . Powrie, Dr . B . J . Skura, of the department of Food Science and Dr . R . C. Fitzsimmons of the department of A n i m a l Science for their assistance and helpful suggestions on my research program and thesis. The assistance of Dr. D . D . Ki t t s , Mrs . L . K w a n , Dr . E . L i - C h a n , Mrs . V . Skura and M r . S. Yee is gratefully acknowledged. Financial support in the form of a Canadian Commonwealth Scholarship throughout my program is greatly appreciated. This thesis is dedicated to the members of my family, especially my late grandmother. She taught me how to think like a scientist. x C h a p t e r 1 I N T R O D U C T I O N Whey proteins have excellent nutritional quality. The undenatured whey proteins have good emulsification capacity and whipping ability and form good gels when subjected to heat treatment under proper conditions (Marshal l , 1981). Unfortunately processing into concentrates adversely affect some of these functionalities especially when harsh processing conditions are used. For whey proteins to compete with other protein sources, both conventional and un-conventional, there is a need for more research to develop new markets. One possibility is the increased uti l ization of the individual proteins. Beta-Lactoglobulin (/ilg) was chosen for this research because it is the major whey protein and a well characterized globular protein. For increased utilization of /31g two conditions should be met. 1. Improvement of its functionality. 2. Reduction of the allergenicity of the protein. Chemical modification was the method of choice. The reason is that as well as being used directly to improve the functional properties it could be used as a powerful tool to study the protein structure-function relationship. It is believed that better understanding of this relationship will help predict the functionality of food proteins by measuring simple physicochemical properties. Earlier work implicated hydrophilic interactions between the proteins and aqueous phase as the most important factor to explain protein functionality. However several 1 Chapter 1. INTRODUCTION 2 contradictory reports have been made indicating that emulsifying properties and solubil-ity are not always closely correlated.(Aoki et al . , 1980; McWatt ters and Holmes, 1979; Wang and Kinsel la , 1976). Better correlations have been obtained when hydrophobic pa-rameters were used in conjunction with solubility (Voutsinas and Nakai , 1983; L i - C h a n et al., 1984). Current hypotheses suggest that protein hydrophobicity is also important in explaining surface functionality, namely foam and emulsification properties (Keshavarz and Nakai , 1979; Ka to and Nakai, 1980; Nakai, 1983; Townsend and Nakai , 1983; Shimizu et al., 1983). Proteins are surface active agents, due to their amphiphilic nature. They facilitate foam and emulsion formation by aligning themselves at the air-water and oil-water in-terfaces respectively, thus lowering the interfacial tension. One method to improve the surfactant properties of proteins is attachment of hydrophobic groups to impart greater amphiphilicity to the protein. A survey of the literature showed that a number of methods have been used to attach hydrophobic groups or change the hydrophobicity of proteins and these include, 1. At taching fatty acids (Haque et al., 1982). 2. Reductive alkylation (Galembeck et al., 1977; Sen et al., 1981). 3. Enzymat ic or chemical reactions to bind hydrophobic amino acids (Watanabe et al . , 1981). 4. Treatment with surfactants (Nakai et al . , 1980). 5. Alcoho l modification (Aoki et al., 1981). 6. Deamidation of gluten (Matsudomi et al., 1982). Chapter 1. INTRODUCTION 3 A l l the above workers reported improvement in either emulsifying or foaming properties upon modification. It is believed that there is an optimum protein hydrophobicity for a given functionality instead of a simple linear relation between them (Nakai, 1983). Consequently, addition of a proper degree of hydrophobicity can give rise to adequate amphiphilicity, thereby facilitating proper orientation at the interface. In this research, fatty acid attachment was selected to modify the hydrophobicity of /31g. The reasons for this selection were two fold. 1. Fatty acids are naturally occurring substances in the body. 2. It has been reported that fatty acid attachment reduced the allergenicity of oval-bumin (Segawa et al . , 1981). The change in hydrophobicity expected could be due to the introduction of the l ipophil ic groups per se, or the attachment could lead to conformational changes in the protein thereby exposing the hydrophobic core. There is therefore a need to monitor conformational changes in the protein. The effect the modification will have on the digestibility of the protein is important especially if it is to be used in human food systems. A number of workers have reported decreased digestibility of chemically modified proteins (Galembeck et al., 1977 and Shetty and Kinsel la , 1982). However, Waniska and Kinsella (1984) reported increased proteolysis of /3\g after glycosylation. Digestibility studies should therefore be conducted. W i t h the above considerations in mind, the specific objectives of this research were, 1. Modification of the hydrophobicity of (3lg through covalent attachment of fatty acids by a base catalyzed ester exchange ( Haque et al., 1982). Chapter 1. INTRODUCTION 4 2. Investigation of the effect of this incorporation on the conformation and surface functional properties of the protein. 3. Evaluation of the digestibility (in vitro ) of the modified proteins. 4. Investigation of the allergenicity of the protein. 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 B e t a - l a c t o g l o b u l i n Beta-lactoglobulin (/31g) is the predominant whey protein in ruminant milk. It is a glob-ular protein with a primary structure of 162 amino acid residues and molecular weight of 18,400. The primary structure has been reported by Whitney et al. (1976). It has a uni-form distribution of nonpolar, polar and charged residues. The /31g monomer has one free sulfhydryl group and two disulfide bonds. Thus, under appropriate conditions /31g readily participates in sulfhydryl-disulfide interchange reactions and this affects characteristics such as solubility. /3-lactoglobulin folds intramolecularly, burying most of the hydrophobic residues so that extensive self-association or interaction wi th other proteins does not occur. However it undergoes limited self association; at the p H of milk, a dimer is formed with a geometry resembling two impinging spheres (Swaisgood, 1985). The secondary structure of the monomeric form is believed to be a mixture of o-helical, /3-sheet and disordered chains. The structure of /31g is dependent on p H . Below p H 3.5 the dimer dissociates to a slightly expanded monomer, between p H 3.5 and 5.2 the dimer tetramerizes reversibly to give an octamer, and above p H 7.5 the dimer dissociates and undergoes a conformational change giving an expanded momomer. Irreversible denaturation occurs at higher pH's. /3-lactoglobulin exhibits heterogeneity resulting from genetic polymorphism (McKenzie , 1971). 5 Chapter 2. LITERATURE REVIEW 6 2.2 H y d r o p h o b i c i t y o f p r o t e i n s 2.2.1 H y d r o p h o b i c i n t e r a c t i o n s The hydrophobic properties of polymers have received increasing attention since the importance of hydrophobic interactions was first shown (Hofstee, 1973). In a recent comprehensive review of hydrophobic interactions in food systems, Nakai and L i - C h a n (1988) pointed out that there are two schools of thought for interpreting hydrophobic interactions. One explains hydrophobic interactions as entropic forces (Tanford, 1980) while the other regards it as a special case of van der Waals attraction when the solute molecules are placed in solvents (Van Oss et al. , 1986). 2.2.2 D e t e r m i n a t i o n o f p r o t e i n h y d r o p h o b i c i t y A number of methods have been used to quantify the hydrophobicity of protein. Earlier methods were mainly concerned with the total hydrophobicity of the protein. Waugh (1954) arbitrarily defined valine, leucine, isoleucine, proline, phenylalanine, tryptophan, and tyrosine as nonpolar residues, and calculated the nonpolar side chain frequencies for a series of proteins as the fraction of non polar residues in these proteins. It has been suggested that 100% ethanol can serve as a model for the inside of a protein molecule (Tanford, 1962). Consequently, Tanford (1962) established a hydrophobic scale by comparing the solubility of free amino acids in water and ethanol and calculating the free energy change on transferring one mole of the amino acid from an aqueous solution to an alcoholic solution. He substracted the transfer free energy for glycine to obtain the side chain hydrophobicity for a particular amino acid. He evaluated the total hydrophobicity of a protein by summing up the hydrophobicities of all its amino acid residues. Since then, for various reasons, many workers have suggested new scales for the hydrophobicity of amino acid residues. Some are based on empirical inspection of Chapter 2. LITERATURE REVIEW 7 protein structures, whilst others are based on experiments performed on isolated amino acids. Bigelow (1967) used the hydrophobicity scale for side chains of amino acid residues suggested by Tanford (1962) and calculated the average hydrophobicity by dividing the total hydrophobicity with the number of residues. Although some of the nonpolar residues of proteins could be found on the surface of the proteins (Lee and Richards, 1971), the nonpolar residues are mainly buried in the interior of the native protein. Consequently, the average hydrophobicity is not a good representation of the available hydrophobicity which is more likely of great biological and technological significance. According to Melander and Horvath (1977), the hydrophobicity of a molecule is essen-tial ly a surface property. They estimated the hydrophobic surface of proteins by normal-izing the nonpolar surface areas they calculated from salting-out data with respect to the molecular weight. They termed this parameter 'Relative Surface Hydrophobici ty ' . They indicated that the advantage of their approach in which the protein molecules themselves act as hydrophobic probes in the salting out process, offers a natural hydrophobicity scale. Other methods have been employed by many workers to estimate the surface hy-drophobicities of proteins. Keshavarz and Nakai (1979) applied hydrophobic chromatog-raphy and hydrophobic partition (also used by Shanbhag and Axelsson, 1975 ) to de-termine the 'Effective' hydrophobicty of nine proteins. Hydrophobic chromatography, however, is time consuming and the hydrophobic partition is limited by the low solubil-ity of some proteins in the solvent phase system used. To develop simple and rapid methods for measurements of surface hydrophobicity, fluorescence probes have been used. cis-Parinaric acid, ( C P A ) a natural polyene fatty acid that can readily simulate natural lipid-protein interactions (Nakai and Powrie, 1981) was first introduced as a probe of membrane structure and protein-lipid interactions by Chapter 2. LITERATURE REVIEW 8 Sklar et al . (1977). The method was later modified by Ka to and Nakai (1980), who used the ini t ia l slope of fluorescence intensity versus protein concentration as an index of effective hydrophobicity of proteins. l-Anilinonaphthalene-8-sulfonate ( A N S ) has also been used quite extensively. (Duke et al . , 1966; Clarke and Nakai, 1972; A o k i et al., 1981; Hayakawa and Nakai, 1985) However, Penzer (1972) and Ka to and Nakai (1980) have criticized the use of A N S as a probe partly on the grounds that there is no convincing evidence that the site of attachment is hydrophobic. However, these two probes are both dissociable under certain conditions, thus electro-static effects between the probes and protein molecule may be included in the hydrophobic measurements (Tsutsui et al . , 1986). A t acidic p H , the data for hydrophobicity become unreliable as the quantum yields of undissociated and dissociated forms of the probe are different. Recently, a simple fluorescence method for fat binding capacity as a index of protein hydrophobicity of proteins was described by Tsutsui et al. (1986). They used an undissociated probe, 1,6-diphenyl hexatriene ( D P H ) , as a fluorescent marker of fat bound to proteins. In addition to the fluorescence probes other hydrophobic probes have been used. They include binding of proteins with hydrocarbons (Mohammadzadeh-K et al., 1969; Mangino et al., 1985), triglycerides (Smith et al. , 1983) and sodium dodecyl sulfate (SDS) (Kato et al. , 1984). The drawback of these methods is that they are both tedious and time consuming and some of them may need gas liquid chromatography. 2.2.3 A l t e r a t i on of hydrophobici ty to change functionali ty of proteins Alteration of hydrophobicity of proteins could be achieved by two main ways. One approach involves derivatization of one or more of the many functional groups or amino acid side chains, especially the positively charged amino groups or the negatively charged Chapter 2. LITERATURE REVIEW 9 carboxyl groups. The change may result in a modified isoelectric point and conformation with exposure of the more hydrophobic groups buried within the protein. This in turn influences the overall functional characteristics of the protein. The other method used is the attachment of hydrophobic, groups to the protein. Summarized below are a few examples of how hydrophobicity has been altered by a number of workers. Nakai et al . (1980) treated a number of plant proteins wi th SDS and linoleic acid. They attributed the increased surface hydrophobicity to either the exposure of hydropho-bic sites on the protein due to the interaction between the surfactants and the protein and/or the hydrophobic ligands of the surfactants. They found good correlation between the surface hydrophobicity and the emulsifying activity index of the proteins. Aoki et al. (1981) working with soy proteins found that the emulsion stability of alcohol modified soy protein increased almost linearly with the increase in surface hydrophobic.it}' under slightly acidic conditions. Watanabe et al. (1981) covalently incorporated l ipophilic leucine a lkyl esters into gelatin by treatment with papain in their preparation of proteinaceous surfactants. They found that the products resulting from incorporation of C 4 to C 6 alkyl esters of leucine showed greater whippabili ty whereas the incorporation of C : 0 to C 1 2 a lkyl esters gave products having a higher ability to stabilize an oil/water type emulsion. Sen et al. (1981) studied the effect of reductive alkylation of caseins using methyl-, isopropyl-, butyl- , cyclopentyl-, cyclohexyl- and benzyl- groups. They found that the native conformation of casein was altered to an extent dependent upon the size of the hydrophobic alkyl groups, generally the larger the group the greater the deviation of the ultraviolet absorption spectrum from the native protein. W i t h the exception of butyl-casein they found marked increase in emulsifying activity. Matsudomi et al. (1982) suggested that the improvement in functional properties by Chapter 2. LITERATURE REVIEW 10 deamidation of gluten is due to the marked increase in surface hydrophobicity. They ex-plained that induction of amphiphilic nature due to an increase in surface hydrophobicity caused a decrease in free energy at the surface of the deamidated gluten and ensured good surface properties. Matarel la and Richardson (1983) attached ethyl residues to (5\g through esterification. Although the emulsifying activity was slightly lower than the native protein, the stability of the emulsion prepared with the ethyl esterified protein was significantly greater than emulsion prepared with the native protein. They found over 4 0 % of the ethyl esterified protein adsorbed to the interface of the oil and water, almost four times more than the native protein. This could perhaps explain the improved emulsion stability. Haque and K i t o (1983b) reported that covalent incorporation of palmitoyl residues into o: s l-casein dramatically improved the ability to form and stabilize emulsions. Foam activity increased unt i l the moles of palmitoyl residues attached per mole of protein reached six. They attributed the improved functionality to the improved amphipathic nature of the protein. Chapter 2. LITERATURE REVIEW 11 2.3 P r o t e i n e m u l s i o n s 2.3.1 F o r m a t i o n a n d s t a b i l i z a t i o n o f e m u l s i o n s To form an emulsion, a protein should have the ability to diffuse to the water-oil interface, unfold and orient in such away that the hydrophobic groups associate with the oil phase while hydrophilic. groups associate with the water phase (Schmidt et al., 1984). This leads to reduction of interfacial tension. The amount of unfolding that occurs at such an interface is said to depend on how rigid the three dimensional structure of the protein is, and on the number and location of the hydrophobic groups in the molecule (Graham and Phil ips, 1979). As would be expected, flexible proteins wi l l be able to unfold more easily than highly structured and crosslinked ones (Phil l ips, 1981). There is controversy in the literature as to whether proteins are completely unfolded at the water-oil interface. Mi tche l (1986) holds the view that proteins are not completely unfolded at the water-oil interface. He argued that if proteins were completely unfolded one might expect the interfacial tension to correlate with overall hydrophobicity rather than surface hydrophobicity. His view is corroborated by the work of Keshavarz and Nakai (1979) who found good correlation between interfacial tension and surface or ef-fective hydrophobicity measured by both hydrophobic chromatography and hydrophobic parti t ion methods. However, no correlation was found between the effective hydrophobic-ity determined by hydrophobic chromatography and Bigelow's average hydrophobicity. Emulsions are thermodynamically unstable because of the positive free energy caused by interfacial tension (Kinsella, 1979). According to Karel (1973), the major way to stabilize emulsions is to provide an energy barrier which prevents coalescence. The fac-tors that increase emulsion stability include increased viscosity of the continuous phase, Chapter 2. LITERATURE REVIEW 12 electrostatic repulsion betweeen charged groups located in the oil-water interface and for-mation of hydration layers outside the oil droplets because of water-orienting hydrophilic groups present at the surface. In addition factors that increase rigidity of interfacial films tend to increase emulsion stability (Mangino, 1984). 2.3.2 R e l a t i o n s h i p b e t w e e n e m u l s i f y i n g p r o p e r t i e s a n d h y d r o p h o b i c i t y The relationship between emulsifying properties and surface hydrophobicity has been investigated by a number of workers. Kato and Nakai (1980) found good correlation between surface hydrophobicity determined using a fluorescent probe and emulsifying activity and stability indices. Voutsinas and Nakai (1983) found that emulsifying prop-erties could be more accurately predicted if both hydrophobicity and solubility were used together. L i - C h a n et al . (1984) studied salt extracts of meat samples and demonstrated that the surface hydrophobicity was more important for predicting the emulsifying prop-erties of samples with high solubility. According to A o k i et al . (1981), the emulsifying properties of proteins ultimately depend on the suitable hydrophile and lipophile balance and do not necessarily increase as the proteins become more lipophilic. Furthermore, the distribution of the lipophilic and hydrophilic groups is important for surface activity of proteins. Morr (1976) explained that the lower emulsifying and foaming properties of whey protein concentrates compared to casein may be due to unfavourable balance between exposed hydrophilic and hydrophobic groups brought about by either an unfavourable amino acid sequence or the more compact globular conformation of native whey pro-teins compared to caseins. Casein subunits are known to contain strongly segregated hydrophilic and hydrophobic regions which impart surfactant-like properties to them. The whey protein components possess a more uniform sequence of hydrophilic and hy-drophobic groups, thus making them less surface active in nature. Chapter 2. LITERATURE REVIEW 13 2.3.3 D e t e r m i n a t i o n o f e m u l s i f y i n g p r o p e r t i e s A number of methods have been used to measure the ability of proteins to form emul-sions. Emulsifying capacity ( E C ) is the volume of oil (milliliters) that can be emulsified by a unit weight of protein before phase inversion occurs under specified conditions. E C is a property of the emulsion system, the equipment, and the conditions used. Standard-ization is difficult. Pearce and Kinsel la (1978) also pointed out the possible errors in measurement of E C when very viscous emulsions are formed. Emulsifying activity ( E A ) is measured by determining the particle size distribution of the dispersed phase (oil) by microscopy, Coulter counting or spectroturbidimetry (Wal-stra et al . , 1969). Pearce and Kinsel la (1978) proposed an emulsifying activity index (EAI) which is based on the interfacial area (calculated via spectroturbidimetry) per unit of protein. E A I is a function of oil volume fraction, protein concentration and type of equipment used to prepare the emulsion. The ability of proteins to stabilize emulsions has been investigated by measuring emulsion stability (ES) and emulsion stability index (ESI) . ES is usually measured in terms of the amount of cream and/or oil that separates in a given time at a stated temperature and gravitational field (Acton and Saffle, 1970). According to Stainsby (1986) acceleration of the test by raising the temperature wil l change the mode of break down and should therefore be discouraged. ESI is measured by monitoring emulsion breakdown from the decrease in absorbance with time resulting from the irreversible reduction in interfacial area (brought about by the processes of coalescence and oiling off). To facilitate the breakdown of emulsion, Pearce and Kinsel la (1978) quantified the decrease in absorbance with time of heating in boiling water. For the same reason mentioned above, Stainsby (1986) criticized this Chapter 2. LITERATURE REVIEW 14 approach and suggested that the heating should not be recommended even when the change in turbidity is a simple first order reaction. According to Pearce and Kinsella (1978) these two indices ( E A I and ESI) are easily measured, and from theoretical and practical considerations, seem more likely to be related to practical performance of products than E C , E A and E S . Chapter 2. LITERATURE REVIEW 15 2.4 P r o t e i n f o a m s 2.4.1 F o r m a t i o n a n d s t a b i l i z a t i o n o f p r o t e i n f o a m s Formation of foam is analogous to formation of an emulsion. The forces involved when air is incorporated into the aqueous phase are similar to those in mixing oil and water. Consequently, the protein must be able to diffuse rapidly to the interface and unfold in such a manner to lower the interfacial tension between the air and water phases. After the foam is formed there are three main forces that can lead to its decay, namely, gravitation drainage, capillary pressure drainage and mechanical disturbance. On the other hand, foam may be stabilized by factors which promote surface viscosity, Gibbs-Marangoni effect, and electric double layers. (Mangino, 1984). The Gibbs-Marangoni effect relates to the increase of surface tension in areas where drainage of water has removed some of the interfacial material. Increased surface tension and concentration of the emulsifier molecules in areas adjacent, to the location of water drainage will cause diffusion of emulsifier back to this location. Emulsifier molecules wil l carry water with them and thickness of the film wil l be restored. This 'self healing' of films is important to their, stability. In addition, the physical adsorption of solid surface materia] e.g. denatured proteins, plays a role in the stability of the foam. 2.4.2 R e l a t i o n s h i p b e t w e e n f o a m i n g p r o p e r t i e s a n d h y d r o p h o b i c i t y Attempts have been made to correlate surface hydrophobicity and foaming properties. Townsend and Nakai (1983) found no significant correlation between foam capacity and surface hydrophobicity measured fluorometricaily using C P A . It was found that the Bigelow average hydrophobicity significantly (p< 0.01) correlated with the foaming ca-pacity. They also heated 1.0% protein solutions at 100 °C for 10 min in the presence of 1.5% SDS prior to fluorometric measurements, a treatment which caused unfolding Chapter 2. LITERATURE REVIEW 16 of the protein. Significant correlation was obtained between foam capacity and surface hydrophobicity of unfolded proteins as measured with the fluorescent probe C P A . The correlation between overall hydrophobicity and foam capacity may suggest more extensive uncoiling of the protein molecule at the air-water interface than oil-water inter-face (Nakai, 1983). According to Nakai (1983), this may relate to the fact that tension at the air-water interface (73 dynes/cm) is far greater than that at the oil-water interface (13-19 dynes/cm). The suggestion that there is more uncoiling at the air-water interface than the oi l - water interface does not appear to be in agreement wi th the surface pressure-area isotherms obtained by Graham and Phi l l ips (1979) especially for globular proteins. According to them globular proteins are more unfolded at the water-oil interface because solvation by oil reduces the van der Waals cohesion between apolar side chains. Kato et al. (1983) found no correlation between surface hydrophobicity and foam sta-bility but a curvilinear relationship between this parameter and foaming power. Mitchel (1986) suggested that the lack of dependence of foam properties and surface hydropho-bicity found by Townsend and Nakai (1983) may be due to the small number of proteins examined. 2.4.3 D e t e r m i n a t i o n o f p r o t e i n f o a m p r o p e r t i e s A number of methods have been used to generate foams for study. A l l these methods are empirical. Hai l ing (1981) wrote an excellent review on this subject. Bubbl ing of gas through a porous sparger has been the most popular foam formation method in basic studies on foams, its main advantage being that it gives more reproducible and uniform bubble sizes and allows easy monitoring of the progress of formation (Hailing, 1981). Foam can also be produced by whipping or beating an aqueous protein solution in the presence of the bulk gas phase. This method is preferred by some workers as it is the most standard means of gas introduction in most aerated products. The method, Chapter 2. LITERATURE REVIEW 17 according to Hai l ing (1981) is not popular for basic, research, since the formation and the history of a single bubble are not well defined. Shaking is yet another method. The rate at which gas bubbles are introduced into the protein solution is said to be dependent on the frequency and amplitude of shaking, volume and shape of container and the volume and flow properties of the l iquid, making it difficult for the method to be standardized. A fourth procedure for the formation of foam, mentioned by Cheftel et al. (1985), involves sudden release of pressure from a previously pressurized solution. Foam stability is often measured as the volume of liquid drained from the foam in a given time or as the time required for a given foam to collapse. However these macroscopic processes do not correspond directly with the microscopic events of lamellae drainage and rupture. Measurement of foam volume could be complicated by noil uniform collapse of foam which causes large voids within the foam. Hail ing (1981) has recommended the procedures of Clark and Blackman (1948) and M i t a et al. (1978) for the measurement of foam stability. The former determined specific surface area from photographs as a function of time. The latter used photographs to determine mean bubble volume and used the reciprocal of this, the number of bubbles per unit volume as a function of time, as a measure of rate of coalescence. One major criticism of the above methods is that the rate of coalescence measured of the side of the foam may not truly represent the decay of the whole foam. However, this measurement provides more fundamental information on the processes occuring in the foam which is correspondingly easier to relate to physicochemical properties. Chapter 2. LITERATURE REVIEW 18 2.5 In vitro p r o t e i n d i g e s t i b i l i t y m e a s u r e m e n t s Anima l bioassays are considered the ultimate methods for evaluating the nutritive value of proteins. However, these assays are expensive and time consuming. Consequently, in vitro enzymatic hydrolysis methods have been developed to measure the digestibility and availability of proteins, the major factors that determine the nutritive quality of food proteins. The first step is obviously enzymatic hydrolysis. Bo th one-step and two-step processes as well as single and multienzyme systems have been employed in the enzymatic digestion. For example pepsin (Sheffner et al . , 1956), trypsin (Maga. et al . , 1973) and a combination of trypsin, chymotrypsin and peptidases (Hsu et a l . , 1977) have been used in the one - step processes. Peptic predigestion followed by pancreatin (Mauron et al., 1955) or trypsin (Saunders et al., 1973) have been used in the two - step processes. In most cases the reaction was carried out at 37°C. Many methods have been used to evaluate the digestibility and the availability of pro-tein constituents of the digestion products. Sheffner et al. (1956) combined the pattern of essential amino acids released during the in vitro pepsin digestion with amino acid pattern of the remainder of the protein to describe an integrated index, the pepsin-digest-residue ( P D R ) amino acid index. Dividing the P D R index wi th digestibility coefficient of the respective proteins was found to yield values that accurately predicted the biological value of the proteins studied. Akeson and Stahman (1964), after digestion of the proteins, precipitated the undi-gested protein and peptides with picric acid, and the amino acids were determined with an automated amino acid analyzer. Stahman and Woldegiorgis (1975) used sulfosalycylic acid instead of picric acid as a precipitant since it allows recovery of tryptophan during the subsequent amino acid analysis. Chapter 2. LITERATURE REVIEW 19 Trichloroacetic acid has been used by some workers to precipitate undigested proteins and peptides. This is followed by amino acid analysis, determination of amino groups by various methods or spectrophotometric measurement at A 2so-Horn et al . (1953) measured microbiologically the individual amino acids made avail-able by pepsin, trypsin and hog mucosa. Their method had a good correlation with the biological values of cottonseed meal which was processed to different degrees. However, this method may not allow comparison of proteins from different sources. M a g a et al . (1973) pointed out that the init ial rates of hydrolysis by trypsin on some proteins were good indicators of protein digestibility. Hsu et al. (1977) monitored the p H drop on digestion of protein using a multienzyme technique. The principle of the method is that as the proteolytic enzymes attack and break the peptide bonds within the protein's pr imary structure, the freed ca.rboxyl groups that are formed immediately release H + which in turn lower p H of the protein suspension. They found good correlation between p H of the protein suspension after ten minutes digestion and in vivo apparent digestibility of rats. Enzymat ic hydrolysis may be inhibited by the hydrolysis products which in vivo are rapidly absorbed. Consequently, a number of methods have been introduced to deal with the problem. These include separation of the hydrolysis products by dialysis (Mauron et al., 1955) and filtration through a membrane under pressure (Prahl and Taufel, 1966). Ford and Salter (1966) effected continuous removal of reaction products from the in vitro digestion system by causing the enzyme-substrate mixture to pass through a calibrated column of Sephadex G-25. Recently, Savoie and Gauthier (1986) deviced a 'dialysis cell ' which enables continuous elimination of digested products by dialysis. Chapter 2. LITERATURE REVIEW 20 2.6 A l l e r g e n i c i t y o f p r o t e i n s 2.6.1 F o o d p r o t e i n a l l e r g y Antigenicity is the. property of discrete surface structures for specific binding of anti-bodies and T cell receptors. Allergens are those antigens that can induce an immediate hypersensitive reaction in the airways, gastrointestinal tract, skin or mucous membrane of man following interaction with cell bound specific immunoglobulin E (IgE) molecule (Baldo and Wrigley, 1984). In an allergic reaction, IgE molecules bind to mast cells v ia the Fc receptor sites on the IgE heavy chains and to complementary allergen via the anti-body combining sites (Ishizaka et al . , 1970). Br idging of adjacent IgE molecules triggers cell degranulation and release of chemical mediators of sensitivity such as histamine and chemostatic factors (Mossman et al., 1974; Siraganian et al., 1975) 2.6.2 S u p p r e s s i o n o f p r o t e i n a l l e r g e n i c i t y Several attempts have been made to suppress IgE antibody response by administration of chemically modified antigens. These include the use of urea denatured protein (Ishizaka et al. , 1975; Takatsu and Ishizaka, 1975), allergens conjugated to polyethylene glycol (Lee and Sehon, 1978; Matsushima et al. , 1980) and conjugates of protein and a synthetic copolymer of D-glutamic acid and D-lysine (Liu et al., 1979). Segawa et al . (1981) prepared antigens (ovalbumin) which were covalently attached with fatty acids of different carbon numbers and tested their effect on the IgE antibody response against native antigen. They found that such modified antigens had no im-munogenicity by themselves and were able to suppress both primary and secondary IgE antibody formation in mice upon injection before and after the primary immunization with the modified antigen. Chapter 2. LITERATURE REVIEW 21 2.6.3 A n t i g e n i c i t y o f / 3 - l a c t o g l o b u l i n /3-lactoglobulin is recognized as one of the major allergenic components in bovine milk (Col l in -Wi l l i am, 1962; Goldman et al. , 1963). Goldman et al . (1963) found that out of 37 milk allergic patients challenged orally with /31g, 23 of them showed symptoms. Col l in -W i l l i a m (1962) and Goldman et al. (1963) reported that in atopic children hypersensitive to milk, /31g caused positive reactions in skin test. Haddad et al . (1979) also detected specific IgE antibodies against /31g in patients. In order to decrease or eliminate the allergenicity of this allergen, the antigenicity of /31g should be understood. Consequently, attempts have been made by a number of workers to study the antigenic structures of /?lg. Two types of antigenic sites are known in globular proteins. One type is the continuous site, which is composed of residues continuously linked by peptide bonds, and the other is the discontinuous site which consists spatially adjacent surface residues that are generally distant in sequence positions (Atassi and Smith, 1978). According to Otani et al. (1985), several approaches have been used to locate these antigenic sites. These include the effect of chemical modification of specific residues on the antigen, isolation of peptide fragments with antigenic activities from their parent molecules by enzymatic and chemical degradation and by direct synthesis of immuno-chemically active peptides. Some of these methods have been employed in the study of antigenicity of /31g. Kur isaki et al. (1982) studied the effect of enzymatic and chemical fragmentation and of chemical modification of sulfhydryl group, amino groups, arginine and tryptophan residues. They determined the antigenic reactivity of these /31g fragments and derivatives as their ability to neutralize specific mouse IgE antibodies assayed by passive cutaneous anaphilaxis ( P C A ) in rat. From the results they suggested that one sulfhydryl group, Chapter 2. LITERATURE REVIEW 22 two arginine and two tryptophan residues and most amino groups were not part of the antigenic sites in /?lg, and that the antigenicity depended on the conformation maintained by the disulfide bridges. Otani et al. (1985) also examined the antigenic reactivity of anti /31g serum in rabbit with /51g modified with chemical reagents by immunodiffusion, quantitative precipitin test and enzyme linked immunosorbent assay ( E L I S A ) . They also found that 1.1 of three arginine residues, two tryptophan residues and one sulfhydryl group were not part of the antigenic sites, but indicated a possibility of amino group, histidine residues and carboxyl groups playing important roles in the antigenicity of bovine /31g. Haddad et al. (1979) demonstrated that treatment of /?lg with pepsin or pepsin-trypsin, in vitro , resulted in the formation of a breakdown product with the ability to elicit the formation of antibodies. However, hydrolysates-from enzyme fragmentation of /51g by trypsin, chymotrypsin and pepsin had no antigenic reactivity (Kurisaki et al. , 1985). The difference in the results may be attributed to the fact that native {3lg was removed from the hydrolysate by gel filtration by the latter workers. Otani (1981) also observed no antigenic activity in peptides with molecular weights of 10,000 or less liberated from /?lg by the action of pepsin, trypsin and chymotrypsin. The susceptibility of the antigenic structure of /31g to the proteases mentioned above, and the observation that this was maintained by steric conformation suggest that the antigenic site of native /31g is of the discontinuous type (Otani and Hosono, 1987). Unfolded peptide chains are capable of eliciting antibody formation, and the anti-bodies to the unfolded proteins have been shown in some cases to be specific and not cross react with the respective native protein (Arnan and Maron , 1971). A t least four antigenic sites have been found in unfolded /?lg, which are associated with regions 41 -61, 62 - 107, 125 - 145 and 148 - 162 in the primary structure of /31g (Kurisaki et al., 1985; Otani and Hosono, 1987). This should be expected to be of the continuous type. Chapter 2. LITERATURE REVIEW 23 The effect of heat on the antigenic sites of (3\g has been studied. Ki lshaw et al. (1982) found that severe heat treatment, 121 °C for 20 minutes of diafiltered whey completely abolished the sensitizing capacity of (3lg. The IgE binding properties of heated and unheated milk preparations of milk proteins using sera, from allergic infants was examined (Baldo, 1984). According to Baldo (1984), a temperature of 80-100 °C for 15 minutes produced a marked drop in the IgE binding capacity of /31g and bovine serum albumin, but this response did not occur in al l infants. However, Otani et al. (1984) observed that j3\g in U H T processed milk retained stable antigenic sites after reductive carboxylation of intramolecular disulfide linkages in it. Thus the type and extent of heat treatment is important. 2.6.4 D e t e r m i n a t i o n o f p r o t e i n a l l e r g e n i c i t y A number of methods have been used to detect IgE, the antibody of great importance in allergic, reactions. In humans it can be detected by inoculation of the allergen into the skin of a sensitized person. Loca l swelling and erythema (wheal and flare reaction) occur within 5 to 10 minutes and subside in an hour or two. The sensitizing antibody can also be transferred passively in the P - K test, so called in recognition of Prausnitz and Kiistner, who discovered the reaction. In this test serum from an allergic individual is inoculated intracutaneously into a normal person, followed in 24 to 48 hours wi th intracutaneous injection of the allergen at the same site. A typical wheal and flare reaction occurs. According to Garvey et al. (1977), the current method of choice for detecting IgE antibodies in human serum is radioimmunoassay, the so called R A S T test, an acronym for radioallergosorbent test. However, in the study of allergenicity of proteins, one cannot always use humans. Hence a number of alternate approaches have been used. Coombs et al. (1978) initiated anaphylaxis in sensitized guinea pigs. Their major contribution is that they initiated the Chapter 2. LITERATURE REVIEW 24 sensitization orally. The advantage of this approach is that the oral route more closely reflects the intestinal route of sensitization to milk proteins. However, the large amount of proteins needed for sensitization could be a disadvantage under certain conditions. Passive cutaneous anaphylaxis ( P C A ) has been used by many workers to measure the allergenicity of proteins. P C A is a useful immunological tool for detecting as little as 0.1 /ug antibody protein. In P C A , the anaphylactic reaction is visualized as a local reaction rather than a systematic action (active anaphylaxis). According to Garvey et al. (1977) the components of the two reactions are the same. The advantages of P C A over active anaphylaxis include the high sensitivity of the method and the absence of painful death associated with active anaphylaxis. In the P C A reaction, the sample to be tested is injected intracutaneously into the skin of a suitable animal. After a latent period which allows fixation of antibody to the skin, specific allergen mixed with a nontoxic dye (Evans blue) is injected intravenously. Presence of antibodies wil l lead-to antigen-antibody complex formation in the skin with a release of vasoactive substances as histamine and serotonin, which increase the perme-ability of the capillaries at these sites. Blu ing of the skin occurs at the site of reaction due to extravasation of the dye into the surrounding tissue. In addition to IgE antibodies, it is known that homologous IgGi and some heterolo-gous IgG2 are able to sensitize the skin of some species (Ovary, 1964; Prouvost-Danon et al. , 1967). It has also been shown that mouse antibodies can sensitize rat mast cells. Ovary et al . (1975) proposed the use of mouse antibodies in the rat for P C A since mouse IgE and not IgGi selectively sensitized the rat skin. 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 M a t e r i a l s Beta-lactoglobulin from cow's milk (L-6879), pepsin (P-7012), a-chymotrypsin (C-4129), pancreatin (P-1750), stearic acid (S-4751), N,N'dicyclohexyl carbodiimide (D-3128), D i -ethanolamine (D-8885), tetrahydrofuran (T-5267), glycine (G-7126), antimouse IgG (whole molecule) alkaline phosphatase conjugate developed in goat (A-5153) and p-phenyl phos-phate disodium were obtained from Sigma Chemicals (St. Louis, M O ) . N-Hydroxysuccinimide and 3,3'-dimethyl biphenyl were obtained from Aldr ich Chemical Company (Milwaukee, W L ) . Acrylamide, boron trifluoride methanol complex (14% B F 3 ) and N,N'-methylene-bis-acrylamide were purchased from B . D . H . Chemicals (Toronto, O N ) . Insulin syringes (1 cc); single use, with Micro-Fine III needle (28 gauge, l / 2 i n needle) was purchased from Canlab Laboratories (Vancouver, B . O ) . Corn oil was from Fisher Scientific Com-pany (Fair Lawn, N J ) . Trinitrobenzenesulfonic acid and l-anilino-8-naphthalenesulfonate ( A N S ) were obtained from Eastman Kodak (Rochester, N Y ) . cis-Parinaric acid ( C P A ) was purchased from Molecular Probes (Junction City, O R ) . A l l other reagents used were of research grade. 25 Chapter 3. MATERIALS AND METHODS 26 3.2 M e t h o d s 3.2.1 E s t e r i f i c a t i o n o f s t e a r i c a c i d w i t h N - h y d r o x y s u c c i n i m i d e N-hydroxysuccinimide ester of stearic acid (18-OSU) was prepared as described by Lapi -dot et al. (1967) with some modifications. Stearic acid ( l g , 3.5mM) was added to a solution of N-hydroxysuccinimide (0.4g, 3.5mM) in tetrahydrofuran(15mL). A n equimo-lar solution of dicyclohexylcarbodimide (0.7g, 3.5mM) in tetrahydrofuran (2 m L ) was added and the reaction mixture left overnight at room temperature. The dicyclohexy-lurea formed was removed by filtration, and the filtrate was concentrated under pressure (by means of rotary evaporator) to yield white crystals. The product was recrystallized from absolute ethanol. The crystals were thoroughly dried under vacuum at 60°C and stored desiccated at 4°C. Pur i ty of the prepared sample was checked by thin layer chromatography ( T L C ) on a pre-coated silica gel plate (Polygram^ Sil G . , Macherey-Nagel & Co. Duren, W . Germany) using two solvent systems: 1. Chloroform and 2. Petroleum ether (bp 40-60 ° C ) - d i e t h y l ether in a ratio of 8:2. 3.2.2 S t e a r i c acyl-/31g p r e p a r a t i o n The procedure employed by Haque and K i t o (1983a) was used with modification. The reaction temperature was set at 35°C and the concentration of (3lg in the reaction medium fixed at 0.5%. The experiments were carried out at 18-OSU to lysine ratios of 0.0, 0.05, 0.10, 0.25, 0.50, 0.75, 1.0, and 1.25 and the solvent system used was 0.05M T r i s - H C l (pH 8.0) and tetrahydrofuran in a ratio of 1:1. Chapter 3. MATERIALS AND METHODS 27 Finely powdered fatty acy l -OSU was dissolved in tetrahydrofuran and added to pro-tein solution which had been solubilized in the buffer. The reaction was run for 10 hr and stopped by adjusting the p H to 7.0 . The modified protein was dialyzed for 96 hr at 4°C in 2% N a C l solution at pH 7 with two daily changes of buffer. The modified proteins were then freeze dried and stored at 4°C. For the various experiments, stock solutions (approximately 0.5%) of the protein were made and stored frozen during the experiments. Protein content was determined by a micro Kjeldahl method of Concon and Soltess (1973). After digestion, the nitrogen content was determined on a Technicon Auto Analyzer II system . Protein content was calculated from nitrogen content using the conversion factor of 6.38. 3.2.3 D e t e r m i n a t i o n o f d e g r e e o f i n c o r p o r a t i o n About 20 n ig of sample (4 m L ) was hydrolyzed with 6 N HC1 for 24 hr at 110°C. The hydrol}'sa.te was extracted three times with the same volume of water saturated ethyl ether. Methyla t ion was done with boron trifluoride-methanol reagent as described by Morrison and Smith (1964). The extracted fatty acid was evaporated under nitrogen in a centrifuge tube wi th a screw cap. 2 m L of 14% B F 3 - methanol reagent, was added and the tube was closed with the screw cap. The tube was then heated for 5 min in boiling water and cooled. The ester was extracted by adding two volumes of pent an e and one volume of water, mixed on a vortex mixer (Thermolyne maxi m i x ™ , Thermodyne Corp. Dubuque, 10) for 1 min and centrifuged at 3000 x g for 5 min at 20 °C. The organic, layer was quantitatively removed for fatty acid analysis. Determination of fatty acid was done by gas-liquid chromatography ( G L C ) . A M T 220 gas chromatograph (Microtek Instruments Corp. Aus t in , T X ) was used within a programmed temperature range of 140-190°C at the maximum rate setting. The injection and detector temperatures were 230 and 220 °C respectively. Palmitic, acid was used as Chapter 3. MATERIALS AND METHODS 28 an internal standard. Presence of free fatty acid in the modified protein was determined by extraction of the same volume of sample without hydrolysis. The difference between unhydrolyzed and hydrolyzed samples was taken as the amount of fatty acid attached to the protein. 3.2.4 Pro te in solubil i ty Aliquots of the protein samples (0.5%) were centrifuged at 27,000 x g for 30 min at 5 °C. Protein content in the supernatant was measured as described above. The protein solubility was calculated as 100 X (protein content of supernatant) Solubility = : — — (protein content of uncentrifugea sample) Determinations were done in duplicate. 3.2.5 Foaming properties Foaming capacity (F .C. ) was determined using the column aeration apparatus described by Townsend and Nakai (1983). A 100 x 2 cm glass column fitted with sintered glass disc, (pore size 40-60 am) at the bot tom was used. A i r was sparged into 5 m L of 0.1 % solution of protein at a flow rate of 40 m L / m i n for 1 min. The volume of foam produced was indicated as an index of the foam capacity of the protein. Measurements were done in triplicate. Foam stability, i.e. drainage, was taken as the time taken in minutes for the foam to collapse to half its maximum volume. 3.2.6 Emuls i fy ing properties Emulsifying activity index ( E A I ) was determined by the turbidimetric method of Pearce and Kinsel la (1978) as modified by L i - C h a n et al . (1985). Emulsions were prepared using 3 m L of 0.5% protein solution and 1 mL of corn oil , homogenized at 1750 rpm for 1 Chapter 3. MATERIALS AND METHODS 29 min in a Sorvall Omnimixer with micro-attachment assembly (Ivan Sorvall Inc. Norwalk, C N ) . Aliquots (0.01 m L ) of emulsion was taken from the bottom of the of the vessel after one min and diluted 100 times with 0.01M phosphate buffer (pH 7.0) and absorbance was measured at 500nm wi th a Gary 210 Spectrophotometer (Varian Associates Palo Al to , C A ) . E A I was calculated as (2.303) (2) ( A 5 0 0 x d i lu t ion) / (c) (<j>) (10,000) in units of m 2 / g , where <j> is the oil volume fraction of the emulsion and c is weight of the protein in g / m L of aqueous phase before emulsion formation. Emulsion stability index (ESI, min) of the emulsions prepared for E A I determina-tion was defined as the time in minutes for A 5 0 o to decrease to one-half at zero time. Measurements were done in duplicate. 3.2.7 P r o t e i n h y d r o p h o b i c i t y Protein surface hydrophobicity was determined using two hydrophobic, fluorescence probes, l-anilino-8-naphthalenesulfonate (ANS) and cis-parinaric acid ( C P A ) . Measurements were performed according to the method of Ka to and Nakai (1980) with slight modification. Experiment was carried out in the absence of SDS. Protein solutions were diluted to 0.001 - 0.02% protein using 0.01M phosphate buffer, p H 7.0. 50^L of A N S (S.OmM in 0.1M phosphate buffer, p H 7.0) or C P A (3.6mM in absolute ethanol containing butylated hydroxytoluene) was added to ten m L of diluted protein. Fluorescence intensity (FI) was measured with an Aminco-Bowman Spectrofluorophotometer (American Instrument Co. Inc. Silver Spring, M D ) at excitation wavelengths of 355nm and 380nm, and emission wavelengths of 415nm and 475nm for C P A and A N S respectively. The ini t ia l slope (S 0 ) of the F I versus protein concentration (%) plot was calculated by linear regression analysis with a Sharp Pocket Computer ( P C 1401, Sharp Corp. Japan) and used as an index of the protein hydrophobicity. Measurements were done in duplicate. Chapter 3. MATERIALS AND METHODS 30 3.2.8 C i r c u l a r d i c h r o i s m Circular dichroism was measured on a Jasco J-500 A spectropolarimeter (Japan Spec-troscopic. Co. L t d . Japan) at the far ultra-violet region (190-250nm) with a 0.1cm pathlength cell at 20 °C under a constant nitrogen flush. The polarimeter was calibrated with 0.05% (w/v) androsterone in dioxane as specified by the manufacturers. The spec-tral bandwidth was 1 nm, scanning rate was 1 n m / c m , the sensitivity was 2 m ° / c m and the time constant was 4 s. The protein was diluted in 0.01M sodium phosphate buffer p H 7.0 to 0.02%) and analysis was carried out within 1 hr of sample preparation. Each protein solution was measured a minimum of three times. Baseline correction was done; the baseline spectrum was obtained by running the buffer under identical conditions as the samples. The data was expressed as mean residue ellipticities in deg. c n r . / d m o l using a mean residue weight of 112 (Adler et al . , 1973) as established from amino acid composition of /31g. 3.2.9 D i g e s t i b i l i t y s t u d i e s a - c h y m o t r y p s i n Protease digestion was carried out with a-chymotrypsin using the method described by Ka to et al. (1985) with slight modification. 0.1% a-chymotrypsin solution was added to 0.1% protein sample both dissolved in 0 .1M Tris buffer, p H 8.0, and the reaction was carried out at 37 °C with protein to enzyme ratio of 16:1. At predetermined times (0, 1, 2 , 3, 4 , 5, 10, 15, 30 and 60 min). aliquots (2 m L ) of the digest were taken and added to the same volume of 20% trichloroacetic acid. This treatment precipitated out the native protein and the precipitate was removed by filtration. The amount of small peptides and amino acids in the filtrate was estimated by reading absorbance at 280nm. The release of small peptides and amino acids increased almost linearly with time for the first 5 min. Chapter 3. MATERIALS AND METHODS 31 The init ial rate of hydrolysis was taken as the slope of absorbance at 280nm for the first 5 min calculated by linear regression analysis with a Sharp Pocket Computer ( P C 1401, Sharp Corp. Japan). P e p s i n - p a n c r e a t i n The method of Akeson and Stahman (1964) as modified by Stahman and Woldegiorgis (1975) was followed with modification. 20 rag of protein was digested in 3 mL pepsin solution (dilute HC1 - p H 2.0) at an enzyme to protein ratio of 1:25 for 3 hr at 37 °C. Aliquots of solution (0.1 m L ) were taken for amino group determination at set intervals (0, 2, 4, 6, 8, 10, 30, 60 and 180 min). The init ial linear slope of amino group concentration versus time (for the first 8 min) was calculated by linear regression analysis with a Sharp Pocket Computer ( P C 1401) and taken as the in i t ia l rate of hydrolysis. After neutralization wi th 1 M N a O H to p H 8.0 digestion was continued for 24 hr at 37 °C by adding 0.1% pancreatin in 0 .1M phosphate, pH 8.0 to give an enzyme to protein ratio of 1:20 and 0.25 m L of 50 ppm merthiolate solution. Again , aliquots of solution (0.1 m L ) were taken for amino group determination as the same time intervals as above and initial rate of hydrolysis was determined as before. The reaction was stopped by adding 80% sulfosalicylic acid to give a final concentra-tion of 7% and the solution adjusted to pH 2.2. After centrifugation at 12000 x g for 10 min at 20 °C, the supernatant was used for amino acid analysis. Appropriate enzyme controls were run. The determinations were done in duplicate. A m i n o acid analysis was done on a Beckman System 6300 High Performance Amino A c i d Analyzer wi th a prepacked Beckman System 6300 High Performance column. Chapter 3. MATERIALS AND METHODS 32 3.2.10 Dete rmina t ion of amino groups Amino groups were determined using the trinitrobenzene sulfonic acid ( T N B S ) method described by Kwan et al. (1983). Protein solution (0.1 m L ) was mixed with 2.0 mL of 0.2M sodium borate buffer pH 9.2, containing 2% SDS. The pancreatic digest sample was heated at 80 °C for 5 min to ensure destruction of enzyme. 1.0 m L of 4.0 m M T N B S was added and incubated for 30 min at room temperature. 1.0 m L of 2.0 M N a H 2 P 0 4 containing 18mM N a 2 S 0 3 was added and absorbance was read at 420nm. The. determinations were done in duplicate. 3.2.11 Zeta potential Net charge was measured as the zeta potential (ZP) in m V with a particle microelec-trophoresis apparatus (Pen Kem, Laser Zee Model 501, Bedford Hil ls , N Y ) as described by Hayakawa and Nakai (1985). Protein emulsions were prepared by homogenizing the mixture 0.1% protein sample and 3,3'-dimethyl biphenyl (100:3) using an Ultra-Turax (Janke &: Kunke l , Staufen, West Germany) at 12,000rpm for 20 sec. The resulting emul-sions were diluted 100 times with 0.01 M N a phosphate buffer p H 7.0 with a specific conductance of 1.3 m mhos/cm. The measurements were done in triplicate. 3.2.12 SDS-Polyac ry lamide gel electrophoresis The method of Laemmli (1970) was used wi th modification. A slab type vertical gel system, the At to SJ 1060 S D H electrophoresis unit (Atto Co., Tokyo, Japan.) was used and the polyacrylamide gel electrophoresis was run in the presence of 0.2% SDS. The gel was made up of a separating gel and a stacking gel with polyacrylamide concentrations of 10 and 3% respectively. The ratio of acrylamide to N,N'-methylene-bis-acrylamide was 25. Polymerization was catalyzed with 0.02% ammonium persulfate. Chapter 3. MATERIALS AND METHODS 33 2 - 4 mg of protein was heated in 5% SDS in boiling water for 5 min , followed by addition of 200 mg of glycerol and 50 uh of 0.05% bromophenol blue tracking dye solution. 50 /zL of sample was applied. The electrode buffer used was Tris-glycine buffer (containing 3 g Tris, 14.4 g glycine and 1 g SDS in one liter, p H 8.3). Electrophoresis was performed at room temperature at a constant voltage (90 volts). After electrophoresis staining was done in 0.25% Coomasie Blue R-250 dye for 1.5 hr followed by destaining in a mixture of 10% acetic, acid and 7.5% methanol. 3.2.13 I m m u n o c h e m i c a l s t u d i e s A n i m a l s B a l b / c male mice and Sprague Dawley male rats were purchased through University of Br i t i sh Columbia's Animal Care Centre. Mice were used at the age of eight - ten weeks whilst rats were used at the age of 18 - 20 weeks. Animals were fed a commercial diet (5001 Pur ina Lab Chow diet, Ralston Pur ina Co. , St. Louis, M O ) in pellet form. Animals were randomly selected and housed in groups of five in plastic, cages under conditions of controlled temperature (20 - 23 °C) and lighting (alternating 12-hr periods of light and dark.) A n t i g e n s To study the effect of l ipophilization, (3\g modified to different levels were selected. The five antigens used for the immunization were native /31g, control /31g, 0.3-/?lg, 4.0-/31g and 13.0-/?lg (containing 0.0, 0.0, 0.3, 4.0 and 13.0 moles of fatty acids per mole of (3lg). Chapter 3. MATERIALS AND METHODS 34 I m m u n i z a t i o n Groups of five balb/c mice were immunized intraperitoneally with 10 ug of antigen ad-sorbed on 1 mg of aluminum hydroxide gel which was prepared according to the method described by Levine and Vaz (1970). The antigen on aluminum hydroxide was prepared by mixing 100 //.g/mL of antigen in P B S with an equal volume of 10 m g / m L aluminum hydroxide gel. Injections were performed with 200 ph of antigen-adjuvant suspension. Boosters were given on days 30 and 66. A n t i s e r a Bleedings from immunized mice were performed on days 15, 28, 50, 64 and 80 post primary challenge. Mice were bled from the orbital sinus using pasteur pipettes. About 0.5 mL of blood were collected from each mouse and blood from each group was pooled. Blood to serve as a control was also collected from a group of five mice which were not immunized. Antiserum was prepared from the collected blood as described by Garvey (1977). Freshly drawn blood was allowed to stand,for 2 hr at room temperature for clot formation and stored in cold room (4 °C) for 24 hr to allow clot contraction. Serum was collected and centrifuged at 12,000 x g for 30 min at 4 °C. The antiserum thus prepared was stored at -20 °C unti l use. P a s s i v e c u t a n e o u s a n a p h y l a x i s ( P C A ) The IgE antibody specific for native and modified /3\g in mouse serum was assayed by heterologous passive cutaneous anaphylaxis reaction in rat skin as described by Ovary et al. (1975) with slight modification. Two rats were sensitized for each antigen by intradermal injections of doubling dilutions of 200/iL of antiserum. The P C A reaction was elicited 4 hr later by intravenous (via the penile vein) injection of 1 mg of modified Chapter 3. MATERIALS AND METHODS 35 or native /31g in 1 m L of 2% Evans blue dye. P C A reactions were evaluated 45 min after challenge. The titre was expressed as the reciprocal of the highest dilution of the antiserum with positive reaction (bluing of injection site), and the negative reaction given at five fold dilution of the antiserum was regarded as no response. E n z y m e l i n k e d i m m u n o s o r b e n t a s s a y ( E L I S A ) Enzyme linked immunosorbent assay was performed using the method of Watabe et al. (1983) with slight modification. A polystyrene microtitre plate (96 wells, Dynatech Laboratories Inc. Chantil ly, V A ) was used as the solid support. Wells were coated with 100 • /nL of 0.01% antigen (native or modified (3lg) in 0.01 M phosphate buffer p H 7.0 , and incubated for 4 hr. Coated plates were washed three times with PBS-Tween (0.05% Tween 20, 200/ iL/well) . Plates were incubated with the appropriate dilutions of native (3\g antiserum (prepared from blood collected on day 50) for 3 hr at room temperature. The plates were then washed again three times with PBS-Tween and 100 uh of goat antimouse IgG coupled to alkaline phosphatase (1:500 in PBS-Tween) was added to each well. After 2hr incubation, plates were washed again followed by addition of 50 uh freshly prepared substrate solution (0.1% p-phenyl phosphate disodium in diethanolamine). The reaction was stopped by addition of 50 uh 2.5 N N a O H . For each plate, controls for non specific binding of anti /3lg antibodies and enzyme-labeled antibodies were prepared. To get the background level of antibodies a control experiment was done with antiserum collected at day 50 from unimmunized mice kept under the same conditions of experiments. This was used for background correction. C o m p e t i t i v e e n z y m e l i n k e d i m m u n o s o r b e n t a s s a y Antiserum (prepared from blood collected on day 50 and diluted 1000 times) containing anti-native /31g antibodies was incubated in test tubes with increasing concentrations of Chapter 3. MATERIALS AND METHODS 36 native and modified (3lg for 2 hr at 37 °C. 100 /xL of this mixture were then added to microplate wells (which had already been incubated for 3 hr wi th 100 JJLL native (3lg and washed) and allowed to react for 2 hr (Breton et al . , 1988). Detection of anti-native /3\g antibodies by addition of enzyme labeled antibodies and color forming reagent was as described earlier. C h a p t e r 4 R E S U L T S A N D D I S C U S S I O N 4.1 L i p o p h i l i z a t i o n o f / 3 - l a c t o g l o b u l i n The term lipophil izat ion has been used in a broad sense to describe the increase in hydrophobicity of proteins by modification (Aoki et al., 1981). However, it was used in a much narrower sense by Haque and Ki to (1983a) to describe the attachment of fatty acid to casein. The term has been used in this research to describe attachment of fatty acid to /3-lactoglobulin. In the init ial experiments, the procedure of Haque and K i t o (1983a) for lipophiliza-tion of asi-casein was used. It involved preparing a succinimide ester of stearic acid as described elsewhere. The ester (dissolved in ethanol) was reacted wi th /31g (dissolved in phosphate buffer) at 30 °C using different 18-OSU to lysine ratios. The pH was main-tained at 9.00 and the solvent mixture was 3.5:6.5 (phosphate buffer:ethanol). It was observed from the control experiment that the conditions of the experiment denatured the protein as demonstrated by the precipitation of the protein. The denaturation could have been caused by the solvent system and/or the p H of the reaction. Various solvent mixtures were tried but all gave negative results. Haque and Ki to (1983a) postulated that the reaction between the succinimide ester of fatty acid and protein is a base catalyzed ester exchange. It follows that the reaction is favoured at high p H . However, it has been reported that as the p H increase above 6.5, /31g undergo conformational transition and show increased dissociation. At higher pH's it 37 Chapter 4. RESULTS AND DISCUSSION 38 may aggregate and/or be denatured irreversibly (McKenzie , 1971). A t lower pH's there was little or no binding of fatty acid. As a compromise, p H of 8.0 was therefore used and the reaction time was extended ten fold, from 1 hr to 10 hr. This condition caused no loss of solubility in the control experiment. Under the conditions of the experiment, the incorporation (i.e. moles of stearic acid covalently bound per mole of (3lg) at 18:0-OSU to lysine mole ratios of 0.0 (control), 0.05, 0.1, 0.25, 0.5, 0.75, 1.0 and 1.25 were 0.0, 0.3, 1.0, 3.0, 4.0, 4.7, 9.0 and 13.1 respectively (Figure 4.1). 0.0 refers to the control protein. The difference between the control and native protein (/?lg) is that although no fatty acid was attached to both proteins, the control protein was taken through all the steps involved in attachment of fatty acid without adding fatty acid to the medium. It means that this protein exhibits the effect of reaction conditions. For easy reference 0.3 and 1.0, were referred to as less incorporated, 3.0, 4.0 and 4.7, medium and 9.0 and 13.1 highly incorporated J3lg. The attachment of the fatty acid is between the carboxyl group of the fatty acid and the amino groups of the protein, thus fatty acid attachment should result in decrease in available amino groups. Indeed this was the case in this work (Figure 4.2). The very good correlation obtained between available amino groups and the amount of fatty acid attached to the protein (r = -0.990, n = 9, p < 0.001), is an indication of the reliability of the data obtained on the amount of fatty acid attached to the protein. It should be noted that the values of moles of fatty acid attached per protein reported here are average values. This is because for a given modified sample, the fatty acid may not be attached to the same degree to all the protein molecules, thus giving a heterogenous population of modified proteins. Hence, even for the highest incorporated protein, unmodified protein would be present. Figure 4.3 shows electrophoretic. patterns of the modified proteins. C h a p t e r 4. RESULTS AND DISCUSSION 39 S 14 B i g 0.00 0.05 0.10 0.25 0.50 0.75 1.00 1.25 M O L E R A T I O O F 18-OSU / L Y S I N E R E S I D U E S Figure 4.1: A m o u n t o f f a t t y a c i d s a t t a c h e d as a f u n c t i o n o f t h e m o l e r a t i o o f 1 8 - O S U t o l y s i n e r e s i d u e s . Chapter 4. RESULTS AND DISCUSSION 40 B i g 0.00 0.05 0.10 0.25 0.50 0.75 1.00 1.25 M O L E RATIO OF 18-OSU / LYS INE RESIDUES Figure 4.2: A v a i l a b l e a m i n o g r o u p s as a f u n c t i o n o f t h e m o l e r a t i o o f 1 8 - O S U t o l y s i n e r e s i d u e s . Chapter 4. RESULTS AND DISCUSSION 41 1 2 3 4 5 6 7 8 9 10 11 12 Figure 4.3: S D S - P A G E E l e c t r o p h o r e s i s o f n a t i v e a n d m o d i f i e d p r o t e i n s . E l e c t r o p h o r e s i s w a s d o n e i n t h e a b s e n c e o f p - m e r c a p t o e t h a n o l . L i p o p h i l i z a t i o n l e d t o t h e f o r m a t i o n o f p o l y m e r s e s p e c i a l l y a t h i g h e r l e v e l s o f f a t t y a c i d i n c o r p o r a t i o n . • P r o t e i n s i n l a n e s 1, 7, 12 - n a t i v e p i g , 2 - c o n t r o l (3\g, 3 - 0 . 3 - d l g , 4 - 1.0-/31g, 5 - 3.0-/31g, 6 - 4.0-/31g, 8 - 4.7-/31g, 9 - 9.0- 3\g a n d 10 , 11 - 13.1-/?]g. Chapter 4. RESULTS AND DISCUSSION 42 In order to detect the presence of polymers the S D S - P A G E electrophoresis was performed without /3-mercaptoethanol which breaks the disulfide linkage. Lipophil izat ion as could be seen led to the formation of some polymers of /51g, especially when medium and high levels of fatty acid were attached to the protein. 4.2 S o l u b i l i t y Protein denaturation is a complex process. It is therefore not suprising that denaturation is defined differently by many workers. Cheftel et al. (1985) defined protein denaturation as any change in conformation (secondary, tertiary or quarternary) not accompanied by rupture of the peptide bonds involved in the primary structure. It may start with unfold-ing of the protein which may finally lead to precipitation resulting in loss in solubility, frequently used as index of denaturation. The decrease in solubility is an indication that l ipophilization caused denaturation of the protein (Figure 4.4). Although a good correlation was found between solubility and amount of fatty acid attached (r = -0.949, n = 9, p < 0.001), the decrease in solubility-appear to occur in stages. No solubility loss was observed when 0.3 moles of fatty acid was attached, followed by a very small loss in solubility when one to three moles were incorporated. Attachment of an additional one mole of fatty acid caused a sharp drop of solubility wi th another gradual decrease when up to nine moles of fatty acid were attached. A drastic loss of solubility was again observed when 13 moles of fatty acid were attached to a mole protein. The reduced solubility observed is primarily due to increased hydrophobic interactions with the incorporation of fatty acids, which caused precipitation of the proteins. The fact that hydrophobic interaction is important in the loss of solubility is supported by the observation that the precipitate became soluble in the presence of 2% SDS. Chapter 4. RESULTS AND DISCUSSION 43 M O L E OF STEARIC AC ID / M O L E OF PROTEIN Figure 4.4: Ef fect , o f i n c o r p o r a t i o n o f f a t t y a c i d s o n t h e s o l u b i l i t y o f / 3 - l a c t o g l o b u l i n . Chapter 4. RESULTS AND DISCUSSION 44 4.3 H y d r o p h o b i c i t y a n d c o n f o r m a t i o n a l c h a n g e s Lipophil izat ion as should be expected modified the surface hydrophobicity (S 0 ) of the protein. The fact that the changes in surface hydrophobicity with incorporation, as measured by two fluorescent probes, namely C P A and A N S , were different (Figures 4.5 and 4.6 respectively) may suggest that A N S and C P A measure two different types of hydrophobicities. Hayakawa and Nakai (1985) classified the hydrophobicity of proteins into aliphatic hydrophobicity due to aliphatic amino acid residues and aromatic, hydrophobicity due to aromatic amino acid residues. According to them C P A is more useful for determining the aliphatic hydrophobicity and A N S for determining aromatic hydrophobicity. The changes observed in surface hydrophobicity for the modified proteins using the two fluorescent probes also appear to occur in stages. This is, primarily, a reflection of the solubility of the modified proteins. Figure 4.5 shows the general relationship between C P A S 0 and incorporation of fatty acid. It should be noted that the C P A is measuring surface hydrophobicity due to both the aliphatic hydrocarbon chain (fatty acid) and aliphatic amino acids. The solvent system and/or the reaction conditions reduced the C P A S 0 of the native protein. There was then a small increase of C P A hydrophobicity when up to three moles of fatty acid was attached to the protein, followed by a sharp increase when one more mole of fatty acid was added. There was again a small general increase up to when nine moles were attached. After this attachment of more fatty acid led to a drop in C P A S 0 . On the otherhand, the solvent system and/or the reaction condition increased the A N S S 0 . A general increase in hydrophobicity was observed when up to three moles Chapter 4. RESULTS AND DISCUSSION 45 6 M O L E RATIO OF 18-OSU / LYS INE RESIDUES Figure 4.5: E f f e c t o f i n c o r p o r a t i o n o f f a t t y a c i d s o n C P A h y d r o p h o b i c i t y o f t h e p r o t e i n s . • V a l u e s on t o p o f b a r s r e f e r t o t h e a m o u n t , o f f a t t y a c i d a t t a c h e d t o t h e p r o t e i n . • C P A h y d r o p h o b i c i t y v a l u e s a r e t h e a v e r a g e s o f d u p l i c a t e d e t e r m i n a t i o n s . Chapter 4. RESULTS AND DISCUSSION 46 300 -r g 280 -< 260 -240 -MOLE RATIO OF 18-OSU / LYS INE RESIDUES F i gu re 4.6: E f f e c t o f i n c o r p o r a t i o n o f f a t t y a c i d s o n A N S h y d r o p h o b i c i t y o f t h e p r o t e i n s . • V a l u e s o n t o p o f b a r s r e f e r t o t h e a m o u n t o f f a t t y a c i d a t t a c h e d t o t h e p r o t e i n . • A N S h y d r o p h o b i c i t y v a l u e s a r e t h e a v e r a g e s o f d u p l i c a t e d e t e r m i n a t i o n s . Chapter 4. RESULTS AND DISCUSSION 47 were attached wi th the optimum at 0.3 moles. Attachment of one additional fatty acid resulted in a decrease of the A N S S 0- A gradual decrease was observed up to when nine moles were attached. Further attachment of fatty acid led to a sharp decrease. It is known that protein structure to a large extent depends on the environment and wil l assume different conformations as the environmental conditions change (Mangino, 1984). Mozhaev and Mart inek (1984) pointed out that although aromatic amino acids are more hydrophobic in nature, they are not normally buried inside the molecule due to their bulky structure. However, the less hydrophobic aliphatic amino acids find their way inside the protein as they are smaller in size and possess greater elasticity. It appears that the alkaline p H and the presence of organic solvent led to the re-arrangement of the protein. The fact that the C P A S 0 decreased whilst there was an increase in the A N S S 0 implies that more aromatic amino acids were exposed with the rearrangement, whilst the aliphatic amino acids may have been hidden. The significance of this observation is that by measuring the distribution of both aliphatic and aromatic hydrophobicity possible conformational changes could be monitored. When up to 0.3 moles of fatty acid were attached the rearrangement of the protein continued with subsequent increase in A N S and C P A hydrophobicity. According to Puigserver et al . (1979a), when hydrophobic groups are attached to the side chain of lysyl residues they tend to move into the hydrophobic interior of these proteins. This is expected to put a constraint on the molecule hence the rearrangement. As more fatty acids were incorporated and the protein became more unfolded, the increased hydrophobic interactions may promote polymerization (Figure 4.3) with sub-sequent precipitation of the protein. The resulting decreased solubility (Figure 4.4) may explain the lowering of the A N S hydrophobicity. The increased aliphatic hydrophobicity may be partly due to the presence of hydrophobic fatty acid ligands. The severe loss of solubility may account for the decrease in C P A hydrophobicity when 13 moles of fatty Chapter 4. RESULTS AND DISCUSSION 48 acid were incorporated. Circular dichroism ( C D . ) measurements were done on the three soluble proteins used in this study i.e.native, control and 0.3-/51g (modified protein with 0.3 moles of fatty acid attached) to study possible conformational changes. The C D . spectrum of native Beta-lactoglobulin (Figure 4.7) was in agreement with published spectra (Towend et al., 1967; Su and Jirgensons, 1977). The circular dichroism spectra were found to be the same although the surface hydrophobicites were different. This means that the secondary structures of the three proteins were similar. Shimizu et al. (1985) also found that although the surface hydrophobicity of Big significantly changed upon lowering of p H , no significant difference in the secondary structure of Big was observed. 4.4 N e t c h a r g e The results of net charge determination as measured by particle microelectrophoresis is shown in Table 4.1. Lipophil izat ion led to increase in net negative charges after the depression of net. negative charge in the control. For proteins, it is the charged groups on the outside of the molecule that contribute to the net charge of the protein at any particular p H . Hence, if the protein is uncoiled a different charge structure could be expected. Depression of the negative charges may be a further confirmation that the hydrophobic solvent led to conformational changes or rearrangement, of the protein molecule which exposed the hydrophobic core and caused some of the charged groups which are normally found on the outside to be buried. The new structure may have been stabilized by non-covalent forces, hence in the absence of the organic solvent (after dialysis) the structure Chapter 4. RESULTS AND DISCUSSION "~i 1 1 1 r -190 210 230 WAVELENGTH (nm) F igu re 4.7: C i r c u l a r d i c h r o i s m s p e c t r a o f na t i v e - / ? l g (•), c o n t r o l - / ? l g (+) a n d 0.3-/31g (o). Chapter 4. RESULTS AND DISCUSSION 50 Table 4.1: Effect of l ipophil izat ion on net charge of native and modified proteins at p H 7.0. Net Charge Protein Zeta Potential* (mV) (3lg (native) -53.39 ± 0.38 0.0 (control) -45.93 ± 0.43 0.3-/51g -45.99 ± 0.26 1.0-/?lg -46.04 ± 0.47 3.0-/31g -46.11 ± 0.09 4.0-/31g -47.03 ± 0.42 4.7-/31g -50.98 ± 0.56 9.0-/31g -53.34 ± 0.53 13.1-/?lg -59.39 ± 0.37 • f Each data point is the average of three determinations. Chapter 4. RESULTS AND DISCUSSION 51 was maintained. The increase of net negative charge with l ipophilization provides further proof of covalent attachment since the positively charged e-amino groups of lysine were used in the amide linkage with the carboxyl groups of fatty acid. 4.5 E m u l s i f y i n g p r o p e r t i e s The ability of the modified proteins to form emulsion was measured by determining the E A I . The emulsifying activity of /3-lactoglobulin was improved with fatty acid attach-ment. Good emulsifying activities were obtained at very low incorporation of fatty acids and decreased with the extent of incorporation (Figure 4.8). Morr (1976) pointed out that whey proteins possess a more uniform sequence of hydrophobic and hydrophilic groups thus making them less surfactant like in nature. Incorporation of fatty acid might have imparted a more surfactant-like property to /31g and this could account for the improvement in emulsifying properties. The highest E A I was obtained at 0.3 moles of fatty acid/mole of protein. This level of incorporation gave the highest A N S S c with 100% solubility. This reflects improved amphiphilicity of the protein without loss of solubility. The improved amphiphilicity facilitates the aligning of the protein at the oil-water interface, hence improving the ability of the protein to form emulsions. Nakai et al. (1980) also found improved emulsifying activity by noncovalent binding of linoleate to rapeseed, soy and sunflower proteins. Kato and Nakai (1980) found a significant correlation (p < 0.01) between E A I and the hydrophobicity of a number of proteins determined fluorometrically using C P A . However in this work no correlation was found between C P A hydrophobicity and E A I (Figure 4.9). Instead a significant correlation (r = 0.794, n = 9, p < 0.02) was obtained between E A I and A N S S Q (Figure 4.10). Chapter 4. RESULTS AND DISCUSSION 52 M O L E RATIO OF 18-OSU / LYS INE RESIDUES F i gu r e 4.8: R e l a t i o n s h i p b e t w e e n e m u l s i f y i n g a c t i v i t y i n d e x a n d d e g r e e o f i n c o r p o r a t i o n o f f a t t y a c i d s . • V a l u e s o n t o p o f b a r s r e f e r t o t h e a m o u n t o f f a t t y a c i d a t t a c h e d t o t h e p r o t e i n . • E m u l s i f y i n g a c t i v i t y i n d e x v a l u e s a r e t h e a v e r a g e s o f d u p l i c a t e d e t e r m i -n a t i o n s . Chapter 4. RESULTS AND DISCUSSION 5.5 < CL, U 5 A H 4.5 V 4H OH S3 _ _ d 2 g 6 3 < fT ( 5 2.5 -2 -1.5 50 70 90 110 130 150 EMULSIFYING ACTIVITY INDEX (m2/g) F i gu re 4.9: R e l a t i o n s h i p b e t w e e n e m u l s i f y i n g a c t i v i t y i n d e x a n d C P A h y d r o p h o b i c i t y Chapter 4. RESULTS AND DISCUSSION 54 F i gu re 4.10: R e l a t i o n s h i p b e t w e e n e m u l s i f y i n g a c t i v i t y i n d e x a n d A N S h y d r o p h o b i c i t y . Chapter 4. RESULTS AND DISCUSSION 55 Emulsion stability was also affected by l ipophil ization (Figure 4.11). The control showed a slight decrease in emulsion stability compared to the native protein. Although a very good emulsifying activity was obtained for 0.3-/31g the stability of the emulsion was not the best. The best emulsion stabilities were found for the samples in which three and four moles of fatty acid were attached. Higher fatty acid incorporation led to decreased emulsion stabilities. Similar observations have been made by other workers. Matarel la and Richardson (1983), attached ethyl residues to Big through esterifica-tion. Al though the emulsifying activity was slightly lower than the native protein, they reported that stability of the emulsion prepared with the ethyl ester esterified was sig-nificantly greater than emulsion prepared by the native protein. They found over 40% of the ethyl esterified protein adsorbed.to the interface of the oil and water, almost four times more than the native protein and this could perhaps explain the improved emulsion stability. Haque and K i t o (1983b) reported that covalent incorporation of palmitoyl residues into ctsi- casein dramatically improved the ability to form and stabilize emulsions. They found that generally the less incorporated protein showed a better emulsification activity than the highly incorporated samples. However the highly mcorpoprated samples showed excellent stability, contrarily to the results of this work. They attributed the improved functionality of improved amphipathic nature of the protein. L i - C h a n et al. (1984) showed that in general, both solubility and hydrophobicity were important, in describing emulsifying properties. This was confirmed in this study. 3-dimensional surface plots were generated to visualize the relationship between A N S hydrophobicity, solubility and emulsifying properties. It is evident from Figures 4.12 and 4.13. that the best emulsifying properties were favoured by either high A N S hydropho-bicity or solubility or a combination of both. Chapter 4. RESULTS AND DISCUSSION 56 150 -• - 140 -MOLE RATIO OF 18-OSU / LYS INE RESIDUES F i gu re 4.11: R e l a t i o n s h i p b e t w e e n e m u l s i o n s t a b i l i t y i n d e x a n d d e g r e e o f i n c o r p o r a t i o n o f f a t t y a c i d s . • V a l u e s o n t o p o f b a r s r e f e r t o t h e a m o u n t o f f a t t y a c i d a t t a c h e d t o t h e p r o t e i n . • E m u l s i o n s t a b i l i t y i n d e x v a l u e s a r e t h e a v e r a g e s o f d u p l i c a t e d e t e r m i n a -t i o n s . Chapter 4. RESULTS AND DISCUSSION 57 Figure 4.12: Relat ionship between solubil i ty. A N S hydrophobici ty and emulsifying act iv i ty index. Scale: Hydrophob ic i ty 0 - 200, solubil i ty 0 - 100% and emulsion act ivi ty index 0 - 160 m 2 / g . The 3-dimensional plot was rotated and t i l ted for the best view of the surface. The rotat ion about the z-axis measured clockwise from the positive x-axis was 225° and the angle of t i l t from the X Y plane was 30° ( A t 90° the observer is directly above the plot) . C h a p t e r 4. RESULTS AND DISCUSSION 58 Figure 4.13: Rela t ionship between solubil i ty, A N S hydrophobici ty and emulsion stabili ty index. Scale: Hydrophob ic i ty 0 - 200, solubil i ty 0 - 100% and emulsion s tabi l i ty index 0 - 130 min .The 3-dimensional plot was rotated and t i l ted for the best view of the surface. The rotation about the z-axis measured clockwise from the positive x-axis was 225° and the angle of t i l t from the X Y plane was 30° (At 90° the observer is directly above the plot) . Chapter 4. RESULTS AND DISCUSSION 59 4.6 F o a m i n g p r o p e r t i e s The ability of the modified proteins to form foam was investigated using the bubbling technique (Figure 4.14). There was a small but significant increase in foam capacity for the modified proteins, especially for the low and medium levels of of incorporation of fatty acid. However, 13.1-/31g which gave a very poor foam capacity. The improvement, observed is mainly due to the solvent and p H effect, considering the value of the control. On the otherhand Haque and K i t o (1983b) found that covalent. incorporation of palmitoyl residues into a s i -casein improved the foam activity considerably. According to them foam activity increased until the moles of palmitoyl residues attached per mole of protein reached six, then it started to decrease with further incorporation. Watanabe et al . (1981) covalently incorporated lipophilic leucine alkyl esters into gelatin by treatment with papain in their preparation of proteinaceous surfactants. They also found that products resulting from incorporation of C 4 - C 6 a lkyl esters of leucine showed greater whippability. Foam stability was monitored by measuring the drainage half life of the foam. A c -cording to Hai l ing (1981), drainage half life is a better indicator of foam stability than absolute drainage time. This is because the extent and rate of liquid drainage from film lamellae which are important in determining the stability of foams are taken into account, in calculating the drainage half life. Although a number of models have been proposed, no satisfactory mathematical model has yet been developed to describe the drainage half life. Figure 4.15 was a typical curve of liquid drainage obtained in this study. As could be seen the amount of serum collected increased in a. hyperbolic manner toward a maximum volume. Curve fitting was done to obtain the parameter Fs referred to as foam stability index defined as time it takes, in minutes, for half of the maximum volume to drain. Chapter 4. RESULTS AND DISCUSSION 60 H i—< U < o 0.00 0.00 0.30 1.00 3.01 3.96 4.74 9.03 13.07 Big 0.00 0.05 0.10 0.25 0.50 0.75 1.00 MOLE RATIO OF 18-OSU / LYSINE RESIDUES 1.25 F igu re 4.14: R e l a t i o n s h i p b e t w e e n f o a m c a p a c i t y a n d d e g r e e o f i n c o r p o r a t i o n o f f a t t y -a c i d s . • V a l u e s o n t o p o f b a r s r e f e r t o t h e a m o u n t o f f a t t y a c i d a t t a c h e d t o t h e p r o t e i n . F o a m c a p a c i t y v a l u e s a r e t h e a v e r a g e s o f t r i p l i c a t e d e t e r m i n a t i o n s . Chapter 4. RESULTS AND DISCUSSION 61 2.4 Max V o l f 1/2 Max Vol. OFs 20~ 40~ 60 DRAINAGE TIME (MIR ) F i gu re 4.15: P l o t o f f o a m s e r u m d r a i n a g e v o l u m e as a f u n c t i o n o f t i m e . CAa.pf.er 4. RESULTS AND DISCUSSION Figure 4.16: P l o t o f r e c i p r o c a l o f f o a m s e r u m d r a i n a g e v o l u m e a g a i n s t t h e r e c i p r o c a l d r a i n a g e t i m e . Chapter 4. RESULTS AND DISCUSSION 63 This was done by plotting the reciprocals of volume of serum and time of drainage. Figure 4.16 show a typical curve fitted graph. 1 _ 1 Fs V V max V maxT where, V = drainage volume V m a x = maximum drainage volume T = drainage time. „ slope Fs = intercept W i t h the exception of 13.1-/31g the other 8 proteins gave significant goodness of fit. Foam formed by 13.1-/31g collapsed almost immediately after formation and was considered zero. Figure 4.17 show the relationship between foam stability index and degree of fatty acid incorporation. The maximum foam stability was observed when one to four moles of fatty acid were attached per mole of the protein and this decreased at higher incorporation. The 13.1-/?lg exhibited no stability. In contrast, Haque and Ki to (1983b) found increased foam stability with an increased incorporation. The stability of foam observed in this wOrk may be partly due to stabilization by particles of lipophilized protein present in the medium. According to Bikerman (1973), some particles can stabilize foam by forming a physical barrier to coalescence. This phenomenon is associated with small particles which have medium wettability and are found in the air-water interface. Attachment of medium levels of fatty acid provided adequate amount of precipitated proteins which could stabilize foam as indicated above. In addition the higher negative net charge of these proteins compared to the control and 0.3-/31g incorporated protein may help to stabilize the foam by electrostatic repulsion. Townsend and Nakai (1983) found significant correlation between foam capacity and C P A hydrophobicity when the proteins were unfolded by heating in boiling water in the presence of 1.5% SDS. However, hydrophobicity measured without unfolding showed no Chapter 4. RESULTS AND DISCUSSION 64 3.5 X W Q < O 3 -2.5 2 -1.5 -0.5 -0.00 0.30 0.00 V, 1.00 3.01 w 3.96 4.74 9.03 Big 0.00 0.05 0.10 0.25 0.50 0.75 1.00 MOLE RATIO OF 18-OSU / LYSINE RESIDUES 13.07 — 1 — 1.25 Figure 4.17: R e l a t i o n s h i p b e t w e e n f o a m s t a b i l i t y i n d e x a n d d e g r e e o f i n c o r p o r a t i o n o f f a t t y a c i d s . • V a l u e s o n t o p o f b a r s r e f e r t o t h e a m o u n t o f f a t t y a c i d a t t a c h e d t o t h e p r o t e i n . • F o a m s t a b i l i t y i n d e x v a l u e s a r e t h e a v e r a g e s o f t r i p l i c a t e d e t e r m i n a t i o n s . C h a p t e r 4. RESULTS AND DISCUSSION 65 correlation wi th foam capacity. In this work, a significant correlation (r = 0.735, n = 9, p < 0.05) was observed between Foam Stability and A N S hydrophobicity. (Figure 4.18). 4.7 In vitro e n z y m e d i g e s t i o n The choice of proteolytic enzyme is a very important factor in in vitro digestibility studies. This is because the nature of the enzyme which is related to their specific action on protein wi l l influence the composition of digested products. Use a of single pure enzyme gives the opportunity to study the availability of specific amino acids involved in the digestion. The interpretation of results is also much easier. However, Marable and Sanzone (1981) pointed out that enzyme assays should be carried out as much as possible with mixtures of crude enzymes corresponding to those found in the gut instead of a purified enzyme. It is obvious that both approaches have their advantages and disadvantages. In this work, the in vitro digestibility of modified /31g was investigated using the two approaches. 1. Digestion with a-chymotrypsin 2. Predigestion with pepsin followed by digestion with pancreatin. 4.7.1 I n i t i a l r a t e o f d i g e s t i o n a - c h y m o t r y p s i n The apparent digestibility of lipophilized derivatives of /31g was estimated by init ial rate of hydrolysis using a-chymotrypsin. Table 4.2 show the relative ini t ia l rates (relative to the native /31g) of hydrolysis of the modified proteins by a-chymotrypsin. These were taken as indices of rate of digestion. As could be seen the init ial rate of digestion decreased pier 4. RESULTS AND DISCUSSION F i gu re 4.18: R e l a t i o n s h i p b e t w e e n f o a m s t a b i l i t y i n d e x a n d A N S h y d r o p h o b i c i t y . Chapter 4. RESULTS AND DISCUSSION 67 Table 4.2: Effect, of lipophilization of /51g on relative initial rate of hydrolysis by a-chymotrypsin. a-chymotrypsin Digestion Protein Rel . Init. Rate of Hydrolysis* (3]g (native) 1.0 0.0 (control) 1.01 0.3-/31g 0.93 1.0-/31g 0.71 3.0-/31g 0.49 4.0-/31g 0.31 4.7-/?lg 0.24 9.0-^lg 0.20 13.1-/51g 0.19 • f Values are reported relative terminations. to the native /31g and are averages of duplicate de-Chapter 4. RESULTS AND DISCUSSION 68 wi th increased incorporation of fatty acid. Similar results have been obtained by reductive alkylation of protein amino groups (Galembeck et al., 1979; Lee et al., 1979), and covalent attachment of amino acids to casein (Puigserver et al . , 1979a). Lee et al. (1979) explained the decrease in the ini t ial rate of hydrolysis in terms of steric hindrance of the added residues, product inhibition and/or formation of non-productive enzyme complexes. Matoba et al. (1980) attributed the decrease in the digestibility of their acetylated casein to the low solubility which was caused by aggregation. On the otherhand, Matoba and Doi (1979) reported a higher ini t ial rate of hydrolysis of succinylated casein by a-chymotrypsin. According to them the electrostatic repulsion of the succinyl residues enhanced the unordered conformation of casein, exposed some of the buried aromatic amino acid residues, and thereby increased the rate of hydrolysis. Waniska and Kinsel la (1984) also found that the rate of hydrolysis of maltosyl derivatives of Big by a-chymotrypsin were higher than the unmodified Big. They attributed this to the expansion of the tertiary structure of the maltosyl derivatives. A similar trend was observed with glucosaminyl derivatives of Big. The reduction of digestibility of the lipophilized Big as measured by the initial rate of hydrolysis could be attributed to one or two or a combination of these factors, namely steric hindrance, substrate or product inhibition and decreased solubility. The extent to which the above factors affect the digestibility of the modified proteins cannot be ascer-tained, since it was not investigated. However, it is believed that solubility and steric hindrance may not be very influencial in the low fatty acid incorporated derivatives but may play more important role in the highly incorporated proteins. On the otherhand substrate and/or product inhibition could be operative even in the low lipophilized pro-teins. Chapter 4. RESULTS AND DISCUSSION 69 P e p s i n - p a n c r e a t i n The relative ini t ial rates of hydrolysis of lipophilized pTg derivatives by pepsin is summa-rized in Table 4.3. These results also demonstrate that rate of hydrolysis was decreased by l ipophil izat ion. However, closer examination of the data indicates differences in the effect of the two enzymes. The init ial rates of hydrolysis of native and control proteins for chymotryptic digestion are similar (Table 4.2). However, the native protein gave a much lower in i t ia l rate compared to the control on peptic digestion (Table 4.3). This difference is even made more dramatic when the values of the modified proteins are related to the native protein (Table 4.4). This shows that when up to four moles of fatty acid were attached to /31g the relative ini t ia l rates were higher than that of the native protein. It is worth noticing that the A N S hydrophobicity values of modified proteins when up to four moles of fatty a.cid were attached were higher compared to the native (Figure 4.6). The increase in digestibility of low lipophilized proteins (relative to the native protein) with pepsin compared with a-chymotrypsin may be attributed to the broad specificity of pepsin. Pepsin exhibits specificities for peptide bonds C-terminal to aromatic residues, leucine, methionine, and glutamic acid (Tang, 1963), whereas a-chymotrypsin hydrolytic activity is essentially directed toward aromatic residues and to a much lesser extent leucine and methionine (Neil et al . , 1966). It may be argued that the differences observed between pepsin and a-chymotrypsin is rather an effect of p H since peptic digestion was done at p H 2.0 and chymotryptic digestion at pH 8.0. If this were the case chymotryptic digestion should have given higher values since 6-lg is more expanded at the alkaline pH's. However, on the contrary, peptic digestion gave higher values. Chapter 4. RESULTS AND DISCUSSION 70 Table 4.3: Effect of l ipophilization of f3\g on relative ini t ial rate of hydrolysis by pepsin. Pepsin Dij jestion Protein Rel . Init. Rate of Hydrolysis* 0.0 (control) 1.00 f3\g (native) 0.22 0.3-/31g 0.68 1.0-/31g 0.62 3.0-/?lg 0.52 4.0-/31g 0.34 4.7-/51g 0.11 9.0-/51g 0.10 13-l-/31g 0.08 • f Values are reported relative to the control /31g and are averages of duplicate determinations. Chapter 4. RESULTS AND DISCUSSION Table 4.4: Effect of lipophilization of Big on relative initial rate of hydrolysis by pepsin. Pepsin Digestion Protein Rel . Init. Rate of Hydrolysis^ Big (native) 1.0 0.0 (control) 4.56 0.3-/31g 3.56 1.0-/31g 2.81 3.0-/31g 2.37 4.0-f3lg - 1.59 4.7-Big 0.51 9-0-Blg 0.46 13.1-Blg 0.37 • f Values are reported relative to the native Big and are averages of duplicate terminations. Chapter 4. RESULTS AND DISCUSSION 72 Imoto et al. (1976) pointed out that the amount of proteins in the denatured state in the native-denatured transition could be detected by the protease digestion method, although it was impossible to detect by routine optical methods. Proteolytic enzymes were also used by Ueno and Harrington (1984) as probes to detect the slight structural changes that would be difficult or impossible to monitor by other physical methods. The high ini t ia l rate of hydrolysis obtained with pepsin for the control, low and medium incorporated proteins compared to the native protein suggests that these pro-teins have more expanded or unfolded structure. As already discussed circular dichroism studies showed no changes in the secondary structure with modification. This means that the conformational changes indicated by this protease digestion and changes in surface hydrophobicity affected the tertiary and quarternary structures. This type of confor-mational changes may involve only a few of the surface residues and the term 'subtle conformational change' has been used to describe it (Lumry and Bil tonen, 1969). The initial rate of hydrolysis with pancreatin was also monitored. It is interesting to note that the result obtained by digestion with pancreatin is similar to that of ct-chymotrypsin (Table 4.5) and also shows a decrease in the rate of digestion with degree of l ipophil ization. It should be noted that the protein solution became clear almost immediately upon addition of pancreatin to the turbid high incorporated protein solu-tions. A nonlinear relationship was found between the digestibilities by the two enzymes (Figure 4.19). This relationship is best described by the regression equation: LN Pancreatic Digestion = 1.12 -f 0.59 x LN Chymotryptic Digestion. (r = 0.95, n = 9, p < 0.001). This could be explained by the fact that the effect of l ipophilization on digestibility of the proteins decreased more rapidly with a-chymotrypsin than wi th pancreatin. Chapter 4. RESULTS AND DISCUSSION 73 Table 4.5: Effect of l ipophilization of pTg on relative in i t ia l rate of hydrolysis by pancreatin. Pancreatin Protein Rel . Init. Rate of Hydrolysis^ Big (native) 1.0 0.0 (control) 1.17 0.3-Blg 0.94 1.0-plg 0.93 3.0-/31g 0.90 4.0-/31g 0.-71 4.7-/31g 0.46 9.0-/31g 0.39 13.1-/?lg 0.38 • f Values are reported relative to the native Big and are averages of duplicate de-terminations. Chapter 4. RESULTS AND DISCUSSION 74 Figure 4.19: R e l a t i o n s h i p b e t w e e n p a n c r e a t i c i n i t i a l r a t e o f h y d r o l y s i s a n d a - c h y m o t r y p t i c i n i t i a l r a t e o f h y d r o l y s i s . Chapter 4. RESULTS AND DISCUSSION 75 For example, it could be observed from Table 4.5 that when up to three moles of fatty acid were attached, pancreatic digestion was only slightly reduced, i.e. 90% of the value of the native protein. On the other hand a- chymotryptic digestion gave only 49% of the value of the native protein (Figure 4.2) at the same level of incorporation. The difference could be attributed to the fact that pancreatin is a mixture of enzymes which are not affected to the same extent by lipophilization. According to Hsu (1977), a single enzyme system that attacks at specific peptide bond tends to underestimate digestibility of proteins. A mult i enzyme system on the other hand, among other things could reduce the effect caused by a specific enzyme inhibitor and thus give a better approximation of protein digestibility. It is therefore conceivable that digestibility of lipophilized /3-lactoglobulin in vivo w i l l be different from the results obtained in these in vitro assays in this work, since many more enzymes are involved in the in vivo digestion process. 4.7.2 E x t e n t o f d i g e s t i o n The digestibility of the lipophilized derivatives of /31g was also investigated by extent of hydrolysis determined as the amino acids released after 3 hr of digestion with pepsin followed by 24 hr digestion with pancreatin. Table 4.6 shows the release of essential amino acids. It could be seen that l ipophilization decreased the release of essential amino acids, once again confirming the negative effect of lipophilization. Chapter 4. RESULTS AND DISCUSSION 76 Table 4.6: Release of essential amino acids by an in vitro digestion with pepsin followed by pancreatin of native and modified Big. Essential Nat ive and VIodified Proteins Amino Acids 8lg 0.0 0.3 1.0 3.0 4.0 4.7 9.0 13.1 T H R + S E R 81.6+ 90.7 71.3 57.0 57.9 43.8 31.2 25.7 29.2 V A L 63.8 65.0 77.6 59.8 74.4 55.1 28.7 27.6 26.2 M E T 72.9 124.7 132.6 141.9 100.2 72.8 37.3 33.4 27.8 L E U 26.8 30.2 40.9 45.5 47.1 39.7 23.1 23.1 21.8 I L E 282.1 311.5 287.1 269.6 273.9 199.7 101.1 99.4 89.6 P H E 106.6 94.5 85.6 82.1 73.7 57.1 " 42.4 25.1 15.8 HIS 61.7 57.9 51.9 48.4 47.0 30.9 27.8 19.4 17.3 L Y S 271.8 308.7 241.1 238.4 174.7 91.9 31.0 19.4 17.3 Mean 120.9 135.4 124.3 117.8 106.1 73.9 40.3 35.0 32.4 • f Values are expressed as /xmoles of each amino acid in 20 mg of protein and are averages of duplicate determinations. Chapter 4. RESULTS AND DISCUSSION 77 The above method is subject to criticism. This is because digestion in vitro gen-erally involves the accumulation of products which in vivo are rapidly absorbed and which may progressively inhibit the digestion reaction (Ford and Salter, 1966). They also pointed out that in the presence of an accumulation of their products, trypsin and a-chymotrypsin may function increasingly as trans-peptidases. This could have been rectified by separating the products of digestion by gel filtration or dialysis. However, in spite of the shortcoming of the above method, Akeson and Stahmann (1964) found a good corelation between their in vitro method and in vivo assay, therefore this method has been continually a method of choice by many workers. The determination of ini t ial rate of hydrolysis as an index of hydrolysis used in this study was another way to circumvent this problem. 4.8 I m m u n o c h e m i c a l s t u d i e s 4 .8 . 1 P a s s i v e c u t a n e o u s a n a p h y l a x i s Passive cutaneous anaphylaxis was used to study the allergenicity or the ability of mod-ified and native /31g to elicit IgE antibodies. Table 4.7 is a summary of the P C A titre values obtained when the rats were challenged with the various antigens after intradermal injection with antibodies raised in mice against those antigens. The result show a similar trend when P C A reaction was done 20 days after the first boosting and 14 days after the second boosting. The highest titre was obtained with the 0.3-/31g. Two possible explanations could be advanced to explain the observation, the first being that the mice used for immunization may be high responders compared to the other mice. The probability of this taking place is very low since genetically homozygous mice (balb/c strain) were used, and the five mice making a group were selected randomly and their blood pooled for the analysis. Chapter 4. RESULTS AND DISCUSSION 78 Table 4.7: P C A titre in rats from sera of mice immunized with native and modified proteins and challenged by their corresponding proteins. Allergen P C A Titre Values* Days of bleeding Day 50 Day 80 f3\g (native) 40* 80* 0.0 (control) 40 80 0.3- /% 80 160 4.0-/31g 40 40 13.0-/31g no response* no response • f P C A titre values are reciprocals of highest dilutions to give positive reaction in at least one of the two rats used. • * no response — No response at the 1:5 dilution. Chapter 4. RESULTS AND DISCUSSION 79 A second, and more plausible explanation is that unfolding or rearrangement of the protein as a result of modification might have resulted in exposure of more antigenic sites on the protein. This may be supported by the fact that this highest P C A titre corre-sponded to the highest A N S hydrophobicity which is believed to be an index of surface hydrophobicity due to aromatic, amino acids. According to G i l l (1972), the presence of aromatic amino acids enhances immunogenicity of proteins and polypeptides and there is a particular amount necessary for optimal enhancement. The medium level fatty acid incorporated /31g, (4.0-Blg) showed a decreased ability to elicit IgE antibodies compared to the native and control proteins. 13.1-/?lg did not induce detectable anti 8lg IgE antibodies. The second boosting of the mice also failed to elicit detectable IgE antibodies. The elimination of the ability to elicit. IgE antibodies could be attributable to destruction of antigenic sites due to denaturation of the protein with modification as indicated by the decreased solubility of the protein. The ability of IgE antibodies raised against native and modified proteins to react with the native dig was also studied. Table 4.8 shows the P C A values when IgE elicited by the modified antigens were challenged wi th the native Big. In this case the response to 0.3-Blg was the same as for the control and native Big. The heterogenous nature of 0.3-/31g means that antibodies are raised against both the unreacted and modified proteins. The lower P C A titre obtained when 0.3-/31g was challenged with the native 81 g suggests that the antibodies specific, to the modified protein may not react or react, poorly with the native protein. Bixler and Atassi (1985) also observed that antibodies to native lyzozyme do not cross react with S-carboxylated derivatives. The small positive response observed with 13.1-pTg on challenge wi th the native Big could also be attributed to the heterogeneity of the protein. This may represent the reaction of IgE antibodies produced against the unmodified population of 13.1-/?lg. Chapter 4. RESULTS AND DISCUSSION 80 Table 4.8: P C A titre in rats from sera of mice immunized with native and modified proteins and challenged by the native protein. Allergen P C A Titre Values* Days of bleeding Day 50 Day 80 [3lg (native) 40* 80* 0.0 (control) 40 80 0.3-/31g 40 80 4.0-/31g 20 40 13.1-/31g 5 10 • f P C A titre values are reciprocals of highest dilutions to give positive reaction in at least one of the two rats used. Chapter 4. RESULTS AND DISCUSSION 81 The important conclusions from the P C A reaction could be summarized as follows: 1. Incorporation of low levels of fatty acid which caused rearrangement of the /?lg molecule might have led to exposure of more antigenic sites, culminating in an increased ability to elicit IgE antibodies. 2. Incorporation of medium level of fatty acid led to reduction of ability of (3lg to elicit IgE antibodies. 3. Incorporation of high level of fatty acid caused denaturation of the protein which almost destroyed the ability of the protein to elicit IgE antibodies. Segawa et al . (1981) reported a similar finding. Coupling of ovalbumin of hens wi th fatty acid produced modified antigens, which were unable to react, wi th mouse an-tiserum against native ovalbumin and incapable of eliciting primary and secondary anti ovalbumin IgE antibody response in balb/c mice. 4.8.2 D i r e c t a n d c o m p e t i t i v e E L I S A The antigenicity or ability of modified proteins to react with antibodies raised against the native [3\g was investigated by E L I S A (Figure 4.20). A higher antibody binding was obtained with 0.3-/51g compared with the native f3lg whilst the high fatty acid incorporated protein (13.1-/31g) exhibited a low antibody binding ability. The difference observed in direct E L I S A for the modified and native protein could be a reflection of the differences in their ability to bind to the microplates rather than true differences in the binding activity. In addition, the proteins may undergo different, levels of denaturation when adsorbed to the solid surface which can lead to loss of native epitopes in some and appearance of new epitopes normally hidden in others (Friquet et al., 1984; Soria et al., 1985). Chapter 4. RESULTS AND DISCUSSION 82 1.2 ANTISERUM DILUTIONS (1 /mL) F igure 4.20: Immunochemical reactivity of modified proteins wi th IgG antibodies to native protein, measured by E L I S A . • Each data point is an average of t r ipl icate determinations. • 1. Con t ro l 2. Nat ive 3. 0.3-/31g 4. 4.0-/51g 5. 13.1-^]g. Chapter 4. RESULTS AND DISCUSSION 83 1.1 1 0 H 1 1 1 r -0 200 400 ANTIGEN CONCENTRATION (ug/mL) F i gu r e 4.21: I m m u n o c h e m i c a l r e a c t i v i t y o f m o d i f i e d p r o t e i n s w i t h I g G a n t i b o d i e s t o n a t i v e p r o t e i n , m e a s u r e d b y c o m p e t i t i v e E L I S A . • E a c h d a t a p o i n t is a n a v e r a g e o f t r i p l i c a t e d e t e r m i n a t i o n s . • 1. N a t i v e 2. C o n t r o l , 3. 0.3-/31g 4. 4.0-/31g 5. 13.1-/31g. C h a p t e r 4. RESULTS AND DISCUSSION 84 To avoid this problem, the binding properties of native and modified proteins against the antibodies to the native protein were also investigated by competive E L I S A (Figure 4.21). The results show that the lowest inhibition was obtained by the 13.1-/31g, hence it gave the highest absorbance values. This observation was in agreement with the result obtained with direct E L I S A that 13.1-/31g showed a re-duced antibody binding. From the competitive E L I S A it could be concluded that the control, 0.3-/?lg and 4.0-/31g bound more antibodies than the native protein. This is reasonable since these modified proteins were more unfolded than the na-tive as already discussed. This may lead to exposure of more antigenic sites. Very recently, Breton et al . (1988) also reported that heat denatured ovalbumin was more antigenic than the native form using both direct and competitive E L I S A . 4.9 G e n e r a l d i s c u s s i o n Haque et al. (1982) attached palmitoyl residues to hydrophilic acidic soybean glycinin and reported an improvement in emulsification activity, foam activity and foam stability. In a subsequent study they attached palmitoyl residues to the more hydrophobic a,,i-casein and surprisingly, reported a very good improvement of the surface functional properties of the protein (Haque and K i t o , 1983b). In both instances they attributed the enhanced functionality to improved amphiphilicity of the proteins. Their results seem to suggest that even for relatively hydrophobic proteins the amphiphilic balance could be improved by attaching a hydrophobic ligand. In this work, improvement of functionality was observed with incorporation of stearic, acid to relatively hydrophobic globular dig, but it was not as dramatic, as that reported by Ki to ' s group. The improvement was observed when low and Chapter 4. RESULTS AND DISCUSSION 85 medium levels of fatty acid were attached. . The difference observed in the effect of l ipophil izat ion on /31g and a s l -casein, both of which are relatively hydrophobic, may be attributed in part to the differences in their structural organization. The E A I and ESI experiments showed that incorporation of low and medium levels of fatty acids improved emulsion properties which were diminished when higher levels of fatty acid were attached. It should be noted that these emulsions were made using oil to water ratio of 1:3. According to Haque and K i t o (1983b), their less incorporated proteins (when up to six moles of fatty acid were attached) showed higher emulsification activity up to an oil to water ratio of two but only the highly incorporated proteins showed emulsification activity at a ratio of three. Therefore, there is a strong possibility that better emulsion properties would have been obtained at higher oil to water ratio for the highly incorporated proteins. Hail ing (1981) pointed out that proteins are generally poor stabilizers of wa-ter /o i l (W/0) emulsions. This may be attributable to the predominantly hy-drophilic nature of most proteins, causing the bulk of the adsorbed protein molecule to reside on the water side of the interface. The medium and high fatty acid in-corporated (3\g should be expected to partly solve this problem, thus making them better candidates for water/oil emulsions. Further work is needed to study the effectiveness of stearoyl proteins in such emulsion systems. Jakobsson et al. (1982) suggested that the relatively slow hydrolysis of ot-lactalbumin and /51g may contribute to their being more allergenic than more easily digestible casein. The implication is that for the low and medium incorporated /31g, the decreased digestibility and increased hydrophobic nature of the protein should facilitate its passage across the membrane, and hence, make it even more allergenic. Segawa et al. (1981) reported that preadministration of the modified antigens, Chapter 4. RESULTS AND DISCUSSION 86 especially palmitoyl-ovalbumin, suppressed both primary and secondary anti oval-bumin IgE antibody response without affecting IgG antibody production. They attributed this suppressive effect to the priming of suppressor T cells by the hy-drophobically modified proteins. If this happens to be true for /31g, the decreased hydrolysis and increased hydrophobicity wil l have a rather positive effect. The more hydrophobic nature and the reduced hydrolysis of the stearoyl proteins should fa-cilitate their entry into the blood system. This may initiate the production of suppressor T cells, which should lead to suppression of allergenicity. This raises a number of interesting possibilities which need to be investigated later on. Puigserver et al. (1979b) found in in vivo experiments that covalent attach-ment of amino acid to casein did not significantly alter plasma amino acid pattern or protein efficiency values, although in vitro digestion of the modified protein was significantly decreased. They reported that proteolytic enzymes of either gastric or pancreatic secretions were unable to hydrolyze the isopeptide bond. However, isolated cells of kidneys, liver and intestine, as well as tissue homogenates were quite efficient in hydrolyzing those isopeptide bonds. The modified hydrophobic nature of some of these small peptides may facilitate their passage into the blood from the digestive tract to the above mentioned organs, where they should be hy-drolyzed. Consequently, the decrease in in vitro digestion following lipophilization may not necessarily mean that the biological value of the modified protein would be decreased. Further research involving animal studies is therefore needed. It is clear from this work that lipophilization decreased the allergenicity of the protein at the expense of other properties of the protein. This emphasizes the need for a comprehensive study of all related properties of the protein including nutrit ional and safety studies in chemical modification investigations. Chapter 4. RESULTS AND DISCUSSION 87 Although allergenicity was decreased or almost destroyed by lipophilization, its application in food use may not be facile. This is because of the great concern of Regulatory Agencies over the safety of such products. Some of the concerns expressed include harmful by-product formation and residual traces of the modify-ing reagents that may remain after completion of derivatization which may present toxicological problems. This does not mean that research involving altering protein properties through chemical modification is a wasteful venture. On the contrary, such investigations are very useful. A basic understanding of the role that structure has in determining functionality and other properties of the protein, wil l be required before modifica-tion schemes can be designed intentionally to tailor properties of whey proteins to fill specific food applications (Kester and Richardson, 1984). In addition such chemical modification studies m a y reveal less drastic and 'safer' methods including enzymatic, derivatization. C h a p t e r 5 C O N C L U S I O N S A number of conclusions may be drawn from this research. They include the following. (a) Different levels of fatty acid were covalently attached to 0\g. This led to changes in surface hydrophobicity and conformation, resulting in the denatu-ration of the protein. (b) This work also confirmed findings by other workers that, the C P A and A N S fluorescence probes do not measure the same surface hydrophobicities. There-fore, it is suggested that in order to obtain a. better picture of the surface hydrophobicities, both A N S and C P A hydrophobicities should be evaluated. (c) The effect of l ipophilization on the various properties of the protein was found to depend on the degree of fatty acid incorporation. Surface functional prop-erties, i.e. emulsifying and foaming properties, were generally found to be improved by attaching low and medium levels of fatty acid to the protein. This was found to reach an optimum. The improvement observed was at-tributed to improved amphiphilicity of these proteins. Further incorporation reduced these surface functional properties. It. was also observed that both high solubility and high A N S surface hydrophobicity were needed for the best, functionality. 88 Chapter 5. CONCLUSIONS 89 (d) Digestibil i ty of the protein, determined in vitro , decreased with extent of l ipophil izat ion. (e) Low level fatty acid incorporation increased the ability of B i g to elicit IgE an-tibodies production as measured by passive cutaneous anaphylaxis. However, there was a substantial decrease at the medium level of fatty acid incorpo-ration whilst the ability to elicit IgE antibodies was almost destroyed at the high level of fatty acid incorporation. Both direct and competitive E L I S A showed that the low and medium levels of fatty acid incorporation increased IgG antibody binding activity which decreased when higher levels of fatty acid were attached. The above observations could be explained by the fact that at-tachment of low and medium levels of fatty acid led to changes in the protein structure which exposed more antigenic or allergenic, sites. The diminished antigenicity and the almost complete destruction of the allergenicity could be attributable to the denaturation of the protein which decreased or destroyed available antigenic sites. In conclusion, it should also be observed that this research raised a number of unanswered questions which should be investigated later on. They include: • The investigation of the effectiveness of the modified proteins in formation and stabilization of water in oil type emulsions; • An ima l studies to evaluate the digestibility of these modified proteins; • Ant ibody suppression studies to determine whether the modified proteins wi l l cause the suppression of antibody production, especially IgE antibod-ies, elicited by the native protein when the modified proteins are administered prior or together with the native protein; Chapter 5. CONCLUSIONS • Incorporation of fatty acid to /3-lactoglobulin through enzymatic means. B i b l i o g r a p h y [l] Ac ton , J . C. and Same, R. L . 1970. Stability of water-in-oil emulsions. 1. Effects of surface tension, level of oi l , viscosity and type of meat protein. J . Food Sci. 35:852. [2] Adler , A . J . , Greenfield, N . J . and Fasman, G . D . 1973. In Methods in Enzy-m.ology V o l . 27. Eds. Hirs , C . H . W . and Timasheff, S. N . p. 675. Academic Press, New York. [3] Arnan , R. and Maron, E . 1971. A n immunological approach to the structural relationship between hen egg-white lysozyme and bovine a-lactalbumin. J . M o l . B i o l . 61:225. [4] Akeson, W . R. and Stahman, M . A . 1964. A pepsin pancreatin digest index of protein quality evaluation. J . Nutr . 83:257. [5] A o k i , H . , Taneyama, O. and Inami, M . 1980. Emulsifying properties of soy protein: Characteristics of 7S and 11S proteins. J . Food Sci 45:534 [6] A o k i , H . , Taneyama, O. and Orimo, N . 1981. Effect of l ipophilization of soy protein on its emulsion properties. J . Food Sci 46:1192. [7] Atassi , M . Z. and Smith, J . A . 1978. Immunochemistry 15:609. cited by Otani et al. (1985). [8] Baldo, B . A . 1984. M i l k allergies. The Aus. J . Dairy Tech.. 39:120. [9] Baldo, B . A . and Wrigley, C. W . 1984. Allergies to cereals. In Advances in Cereal Science and Technology. Vol. 4- E D . Pomeranz, Y . p. 289. Amer. 91 Bibliography 92 Assoc. Cereal Chem. Inc.. St. Paul , Minnesota. [10] Bigelow, C . C. 1967. On the average hydrophobicity of proteins and the re-lation between it and protein structure. J . Theoret. B io l . 16:187. [11] Bikerman, J . J . 1973. Foams . Springer - Verlag. New York, N Y . [12] Bixler , G . S. and Atassi , M . Z. 1985. T cell recognition of proteins. Conclu-sions from the localization of the full T-cell recognition profile of two native proteins. Biotechnology 3:47. [13] Breton, C , Phan Than , L . and Paraf, A . 1988. Immunochemical properties of native and denatured ovalbumin. J . Food Sci . 53:222. [14] Cheftel, J . C , Cuq , J - L . and Lorient, D . 1985. Amino acids, peptides and proteins. In Food Chemistry, 2nd Ed. E d . Fennema, O. R. p. 245. Marcel Dekker. Inc.. New York. [15] Clark, N . O. and Blackman, M . 1948. The degree of dispersion of the gas phase in foam. Trans._ Faraday Soc. 44:1 [16] Clarke, R. F. L. and Nakai, S. 1972. Fluorescent studies of ^.-casein with 8-anilinonaphthalene-l-sulphonate. Biochim. Biophys. A c t a 257:61. [17] Co l l in -Wi l l i am, C. 1962. Cow's milk allergy in infants and children. Int. Arch. Allergy 20:38. [18] Concon, J . M . and Soltess, D. 1973. Rapid micro kjeldahl digestion of cereal grains and other biological materials. A n a l . Biochem. 53:35. [19] Coombs, R . R. A . , Devey, M . and Anderson, K . J . 1978. Refractoroness to anaphylactic shock after continuous feeding of cow's milk to guinea pigs. Cl in . Exp . Immunol. 32:263. Bibliography 93 [20] Duke, J . A . , M c K a y , R. and Botts. L . 1966. Corformational change accom-panying modification of myosin ATPase . Biochim. Biophys. A c t a 126:600. [21] Ford, J . E . and Salter, D . N . 1966. Analysis of enzymatically digested food protein by sephadex-gel filtration. Br i t . J . Nutr. 20:843. [22] Friquet, B . , Djavadi-Ohaniance, L . and Goldberg, M . E . 1984. Some mono-clonal antibodies raised with a native protein bind preferentially to the de-natured antigen. M o l . Immunol. 21:673. [23] Galembeck, F . , Ryan, D . S., Whitaker, J . K . and Feeney, R . E . 1977. Re-action of proteins with formaldehyde in the presence and absence of sodium borohydride. J . Agric Food Chem. 25:238. [24] Garvey, J.S.,Cremer N . E . , and Sussdorf, D . H . 1977. Methods in Immunology. 3rd Ed. E d . , W . A . Benjamin, Inc., Reading. [25] G i l l , T . J . 1972. III. The chemistry of antigens and its influence on immuno-genicity. In Immunogenicity. E d . Borek, F . North- Holland, Amsterdam. [26] Goldman, A . S., Sellars, W . A . , Halpern, S. R. and Anderson Jr, D . W . 1963. M i l k allergy. II. Skin testing of allergic and normal children with purified milk proteins. Pediatrics 32:572. [27] Graham, D . C. and Phil l ips, M . C. 1979. Proteins at interfaces. III. Molecular structure of adsorbed films. J . Colloid Interface Sci. 70:427. [28] Haddad, Z. H . , K a l r a , V . and Verma, S. 1979. IgE-antibodies to peptic and peptic-tryptic digests of /3-lactoglobulin. Significance in food hypersensitivity. A n n . Allergy 42:368. [29] Hai l ing, P. J . 1981. Protein-stabilized foam and emulsions. C R C Cri t . Rev. Food Sci. Nutr . 15:155. Bibliography 94 [30] Haque, Z. and K i t o , M . 1983a. Lipophil izat ion of a s l -casein . 1. Covalent attachment of palmitoyl residue. J . Agric Food Chem. 31:1225. [31] Haque, Z. and K i t o , M . 1983b. Lipophil izat ion of a s l -case in . 2. Conforma-tional and functional effects. J . Agr ic Food Chem. 31:1231. [32] Haque, Z . , Matoba, T.and K i t o , M . 1982. Incorporation of fatty acid into food proteimpalmitoyl soybean glycinin. J . Agric Food Chem. 30:481. [33] Hayakawa, S. and Nakai, S. 1985. Relationship of hydrophobicity and net charge to the solubility of milk and soy protreins. J . Food Sci . 50:486 [34] Hofstee, B . H . 1973. Hydrophobic affinity chromatography of proteins. Ana l . Biochem. 52:430. [35] Horn, M . J . , Jones, D. B . and B l u m , A . E . 1953. Sources of error in microbio-logical determinations of amino acids on acid hydrolysates. I. Effect of humin on amino acid values. J . B io l . Chem. 203:907. [36] Hsu, H . W . , Vavak, D . L . , Satterlee, L . D . and Mil ler , G . A . 1977. A mul-tienzyme technique for estimating protein digestibility. J . Food Sci. 42:1269. [37] Imoto, T . , Fukuda, K . and Yagishita, K . 1976. A study of native-denatured (N ^ D) transition in lysozyme. J . Biochem. 80:1313. [38] Ishizaka, K . , Ishizaka, T. and Lee, E . H . 1970. Biological function of the Fc. fragments of E myeloma protein. Immunochemistry 7:687. [39] Ishizaka, K . , Okudaira, H . and K i n g , T. P. 1975. Immunogenic properties of modified antigen E . II. Abi l i ty of urea-denatured antigen and a-polypeptide chain to prime T cells specific for antigen E . J . Immun. 114:110. Bibliography 95 [40] Jakobsson, 1., Lindberg, T. and Benediktsson, B . 1982. In vitro digestion of cow's mi lk proteins by duodenal juice from infants with various gastrointesti-nal disorders. J . Pediatr Gastroenterol. Nutr . 1:183. [41] Kare l , M . 1973. Protein interactions in biosystems: protein-lipid interactions. J . Food Sci. 38:756. [42] Kato , A . , Komatsu, K . , Fujimoto, K . and Kobayashi, K . 1985. Relationship between surface functional properties and flexibility of proteins detected by the protease susceptibility. J . Agr ic . Food Chem. 33:931. [43] Kato , A . , Matsuda, T., Matsudomi, N . and Kobayashi, K . 1984. Deter-mination of protein hydrophobicity using a sodium dodecylsulfate- binding method. J . Agr ic . Food Chem. 32:284. [44] Kato , A . and Nakai , S. 1980. Hydrophobicity determined by a fluorescence probe method and its correlation with surface properties of proteins. Biochim. Biophys Ac ta 624:13. [45] Kato , A . , Osako, Y . , Matsudomi , N . and Kobayashi, K . 1983. Changes in the emulsifying and foaming properties of proteins during heat denaturation. Agric . B i o l . Chem. 47:33. [46] Keshavarz, E . and Nakai, S. 1979. The relationship between hydrophobicity and interfacial tension of proteins. Biochim. Biophys. A c t a 576:269. [47] Kester, J . J . and Richardson, T . 1984. Modification of whey proteins to im-prove functionality. J . Dairy Sci . 67:2757. [48] Ki lshaw, P. J . , Heppel, L . M . and Ford, J . E . 1982. Effect of heat treatment, of cow's milk and whey on the nutritional quality and antigenic properties. Arch . Dis . Childhood 57:842. Bibliography 96 [49] Kinsel la , J . E . 1979. Functional properties of soy proteins. J . Amer. Oi l Chem. Soc. 56:242. [50] Kur i sak i , J . , Nakamura, S., Kaminogawa, S., Yamauchi, K . , Watanabe, S., Hot ta , K . and Hattori , M . 1985. Antigenicity of modified (3- lactoglobulin examined by three different assays. Agr ic . B io l . Chem. 49:1733. [51] Kur i sak i , J . , Nakamura, S., Kaminogawa, S. and Yamauchi, K . 1982. The antigenic properties of /3-lactoglobulin examined with mouse IgE antibody. Agr ic . B io l . Chem. 46:2069. [52] K w a n , K . K . H . , Nakai, S. and Skura, B . J . 1983. Comparison of four methods for determining protease activity in milk . J . Food Sci. 48:1418. [53] Laemmli , U . K . 1970. Cleavage of structural proteins during the assembly of head of bacteriophage T 4 . Nature 222:680. [54] Lapidot , Y . , Rappoport, S. and Wolman, Y . 1967. Use of n-hydroxysuccinimide in the synthesis of n-acylamino acids. J . L i p i d Res. 8:142. [55] Lee, B . and Richards, F . M . 1971. The interpretation of protein struc-tures est imation of static accessibility. J . M o l . B i o l . 55:379. [56] Lee, W . Y . and Sehon, A . H . 1978. Suppression of reaginic antibodies with modified allergens. I. Reduction in allergenicity of protein allergens by con-jugation to polyethylene glycol. Int. A r c h . Allergy A p p l . Immun. 56:159. [57] Lee, H . S., Sen, L . O , Clifford, A . J . , Whitaker, J . R. and Feeney, R. E . 1979. Preparation and nutrit ional properties of caseins covalently modified wi th sugars. Reductive alkylation of lysines with glucose, fructose, or lactose. J . Agric . Food Chem. 27:1094. Bibliography 97 [58] Levine, B . B . and Vaz, N . M . 1970. Effect of combination of inbred strain, antigen, and antigen dose on immune responsiveness and reaginic production in the mouse. A potential mouse model for immune aspects of human atopic allergy. Int. Archs. Allergy A p p l . Iramun. 39:156. [59] L i - C h a n , E . , Nakai, S. and Wood, D . F . 1984. Hydrophobicity and solubility of meat proteins and their relationship to emulsifying properties. J . Food Sci. 49:345. [60] L i - C h a n , E . , Nakai, S. and Wood , D. F . 1985. Relationship between func-tional (fat binding, emulsifying) and physicochemical properties of muscle proteins. Effect of heating, freezing, pH and species. J . Food Sci. 50:1034. [61] L i u , F . T . , Bogowitz, C . A . , Bargatze, R. F . , Zinnerker, M . , Ka tz , L . R. and K a t z , D . H . 1979. Immunologic tolerance to allergenic, protein deter-minantsrproperties of tolerance induced in mice treated with conjugates of protein and a synthetic copolymer of D-glutamic acid and D-lysine ( D - G L ) . J . Immun. 123:2709. [62] Lumry, R. and Bil tonen, R.. 1969. Thermodynamic and kinetic aspects of pro-tein conformation in relation to physiological function. In Biological Macro-molecules. Vol. 2. Eds. Timasheff, S. N . and Fasman, G . D . p. 417. Marcel Dekker, New York. [63] Maga, J . A . , Lorenz, K . and Onayemi, O. 1973. Digestive acceptibility of proteins as measured by init ial rate of in vitro proteolysis. J . Food Sci. 38:173. [64] Mangino, M . E . 1984. Physicochemical aspects of whey protein functionality. J . Dairy Sci. 67:2711. Bibliography 98 [65] Mangino, M . E . , Fritsch, D . A . , Liao, S. Y . , Fayerman, A . M . and Harper, W . J . 1985. The binding of n-alkanes as a predictor of whey protein functionality. New Zealand J . Dairy Sci. Technol. 20:103. [66] Marable, N . L . and Sanzone, G . 1981. In vitro assays of protein quality assays ut i l iz ing enzymatic, hydrolyses. Discussion. In Protein Quality in Humans: Assessment and in vitro Estimation. Eds. Bodwell , C. E . , Adkins, J . S. and Hopkins, D . T. p. 275. A V I Pub. Co. Westport, C T . [67] Marshal l , K . R. 1981. Industrial isolation of milk proteins: whey proteins. In Developments in dairy chemistry. E d . Fox, P. F . p. 339. A p p l . Sci. Pub . Lond. U K . [68] Matarel la , N . L. and Richardson, T. 1983. Physicochemical and functional properties of positively-charged derivatives of bovine /3-lactoglobulin. J . Agric Food Chem. 31:972. [69] Matoba , T. and Doi , E . 1979. In vitro digestibility of succinylated proteins by pepsin and pancreatic proteases. J . Food Sci. 44:537. [70] Matoba , T. , Do i , E . and Yonezawa, D . 1980. Digestibility of acetylated and succinylated proteins by pepsin-pancreatin and some intracellular peptidases. Agric . B io l . Chem. 44:2323. [71] Matsudomi, N . , Ka to , A . and Kobayashi, K . 1982. Conformation and surface properties of deamidated gluten. Agric . B i o l . Chem. 46:1583 [72] Matsushima, A . , Nishimura, H . , Ashihara, Y . , Yokota, Y . and Inada, Y . 1980. Modification of E. coli asparaginase with 2,4-bis(0- methoxypolyethy-lene glycol)-6-chloro-S-triazine (activated P E G 2 ) : disappearance of binding ability towards anti-serum and retention of enzymatic activity. Chem. Lett . 7:773. Bibliography 99 [73] Mauron, J . 1973. The analysis of food proteins, amino acid composition and nutritive value. In Proteins in Human Nutrition. Eds. Porter, J . W . G . and R o l l , B . A . p. 139. Academic Press. London. [74] Mauron, J . , Mo t tu , F . , Bujard, E . and Egl i , R. H . 1955. The availability of lysine, methionine and tryptophan in condensed milk and milk powder. In vitro digestion studies. Arch . Biochem. Biophys. 59:433. [75] McKenz ie , H . A . 1971. Milk Proteins. Academic Press. New York, N Y . [76] McWatters , K . H . and Holmes, M . R . 1979. Influence of moist heat on sol-ubility and emulsification properties of soy and peanut flours. J . Food Sci. [77] Melander, W . and Horvath. C. 1977. Salt effects on hydrophobic, interactions in precipitation and chromatography of proteins; an interpretation of the lyotropic series. Arch. Biochem. Biophys. 183:200. [78] M i t a , T . , Ishida, E . and Matsumoto, H . 1978. Physicochemical studies on wheat protein foams. II. Relationship between bubble size and stability of foams prepared with gluten and gluten components. J . Col loid Interface Sci. [79] Mitchel l , J . R. 1986. Foaming and emulsifying properties of proteins. In De-velopments in Food Proteins - 4- E d . Hudson, B . J . F . p. 291. Elsevier A p p l . Sci. Pub. , New York. [80] Mohammadzadeh-K, A . , Feeney, R. E . and Smith, L . M . 1969. Hydrophobic binding of hydrocarbons by proteins 1.. Relationship of hydrocarbon struc-ture. Biochim. Bioplrys. A c t a 194:246. [81] Mor r , C. V . 1976. Whey protein concentrates: an update. Food Technol. 30(3):18. 44:774. 64:143. Bibliography 100 [82] Morrison, W . R . and Smith, L . M . 1964. Preparation of fatty acid methyl esters and dimethylacetals from lipids wi th boron fluoride-methanol. J . L ip id Res. 5:600. [83] Mossman, H . , Meyer-Delius, M . , Votisch, U . , Kickhofen, B . and Hammer, D. K . 1974. Experimental studies on the bridging hypothesis of anaphylaxis. Heptenic determinants required to elicit immediate-type reactions in calf skin by separate or concurrent sensitization wi th reagins of different specificity. J . Exp . Med . 140:1468. [84] Mozhaev, V . V . and Martinek, K . 1984. Structure- stability relationships in proteins: new approaches to stabilizing enzymes. Enzyme Microb . Technol. 6:50. [85] Nakai, S. 1983. Structure-function relationship of food proteins with an em-phasis on the importance of protein hydrophobicity. J . Agric . Food Chem. 31:676 [86] Nakai, S., Ho, L . , Helbig, N . and Tung, M . A . 1980. Relationship between hydrophobicity and emulsifying properties of some plant proteins. Can . Inst. Food Sci. Technol. J . 13:23. [87] Nakai, S., Ho, L . , Tung, M . A . and Qumn, J . R. 1980. Solubilization of rapeseed, soy and sunflower protein isolates by surfactant and proteinase treatments. Can. Inst. Food Sci. Technol. J . 13:14. [88] Nakai, S. and L i - C h a n , E . 1988. Hydrophobic Interactions in Food System,s. C R C Press, Inc. Boca Raton, Florida. [89] Nakai, S. and Powrie, W . D . 1981. Modification of proteins for functional and nutritional improvements. In Proceedings of International Symposium, on Cereal: A Renewable Resource. Aug . 11 - 14. Copenhagen, Denmark. Bibliography 101 [90] Nei l , G . G . , Niemann, C. and Hein G . E . 1966. Structural specificity of a-chymotrypsinrpolypeptide substrates. Nature 210:903. [91] Otani , H . 1981. Antigenicities of /3-lactoglobulin treated with proteolytic en-zymes. Jpn. J . Zootech. Sci. 52:47. [92] Otani , H . and Hosono, A . 1987. Antigenic reactive regions of s- carboxymethy-lated /3-lactoglobulin. Agric. B io l . Chem. 51:531. [93] Otani , H . , M o r i t a , S. and Tokita, F . 1984. Jpn. J . Zootech. Sci. 55:287. Cited by Otani and Hosono (1987). [94] Otani , H . , Uchio, T. and Toki ta , F . 1985. Antigenic reactivities of chemically modified /3-lactoglobulin with antiserum to bovine /3-lactoglobulin. Agric . B io l . Chem. 49:2531. [95] Ovary, Z., Bloch , K . J . and Benacerraf, B . 1964. Identification of rabbit, . monkey, and dog antibodies with P C A activity for guinea pigs. Pro. Soc. Exp . Bio l . M e d . 116:840. [96] Ovary, Z., Caiazza, S. S. and Ko j ima , S. 1975. P C A reactions with mouse antibodies in mice and rats. Int. Archs. Allergy A p p l . Immun. 48:16. [97] Pearce, K . W . and Kinsel la , J . E . 1978. Emulsifying properties of proteins: evaluation of a turbidimetric technique. J . Agric. Food Chem. 26:716. [98] Penzer, G . R. 1972. l-Anilinonaphthalene-8-sulfonate. The dependence of emission spectra on molecular conformation studied by fluorescence and proton-magnetic resonance. Eur . J . Biochem. 25:218. [99] Phil l ips , M . C. 1981. Protein conformation at l iquid interfaces and its role in stabilizing emulsions and foams. Food Technol. 35:50. Bibliography 102 [100] Prahl , L . and Taufel, K . 1966. Z. Lebenmittekmters. u. -Forsch. 133:73. Cited by Mauron (1973). [101] Prouvost-Danon, A . , Peixito, J . M . and Javierre, M . Q. 1967. Reagin- like antibody mediated passive anaphylactic reaction in mouse peritoneal mast cell in vitro . Life. Sci. 6:1793. [102] Puigserver, A . J . , Sen, L . C , Gonzales-Flores, E . , Feeney, R. E . and Whitaker, J . R. 1979a. Covalent attachment of amino acids to casein. 1. Chemical mod-ification and rates of in vitro enzymatic hydrolysis of derivatives. J . Agric. Food Chem. 27:1098. [103] Puigserver, A . J . , Sen, L . C , Clifford, A . J . , Feeney, R. E . and Whitaker, J . R. 1979b. Covalent attachment of amino acids to casein. 2. Bioavailability of methionine and n-acetylmethionine covalently linked to casein. J . Agric. Food Chem. 27:1286. [104] Saunders, R . M . , Connor, M . A . , Booth, A . N . , Bickoff, E . M . and Kohler, G . O. 1973. Measurement of digestibility of alfalfa protein concentrates by in vivo and in vitro methods. J . Nut r . 103:530. [105] Savoie, L . and Gauthier, S. F . 1986. Dialysis cell for the m vitro measurement of protein digestibility. J . Food Sci. 51:494. [106] Schmidt, R . H . , Packard, V . S. and Morris, H . A . 1984. Effect of processing on whey protein functionality. J . Dairy Sci. 67:2723. [107] Segawa, H . , Borges, M . S., Yokoda, Y . , Matsushima, A . , Inada, Y . and Tada, T . 1981. Suppression of IgE antibody response by fatty acid-modified antigen. Int. Arch . Allergy A p p l . Immun. 66:189. [108] Sen, L . C , Lee, H . C , Feeney, R. E . and Whitaker, J . R . 1981. In vitro digestibility and functional properties of chemically modified caseins. J . Agric Bibliography 103 Food Chem. 29:348. [109] Shanbhag, V . P. and Axelsson, C. G . 1975. Hydrophobic interaction deter-mined by partition in aqueous two-phase systems. Eur . J . Biochem. 60:17. [110] Sheffner, A . L . , Eckfeldt, G . A . and Spector, H . 1956. The pepsin- digest-residue ( P D R ) amino acid index of net protein ut i l izat ion. J . Nutr . 60:105. [ i l l ] Shetty, J . K . and Kinsel la , J . E . 1982. Isolation of yeast protein with reduced nucleic acid level using reversible acylating reagents: some properties of the isolated protein. J . Agric Food Chem. 30:1166. [112] Shimizu, M . , Saito, M . and Yamauchi, K . 1985. Emulsifying and structural properties of /3-lactoglobulin at different pHs. Agric . B i o l . Chem. 49:189. [113] Shimizu, M . , Takahashi, T . , Kaminogawa, S. and Yamauchi K . J . 1983. A d -sorption onto an oil surface and emulsifying properties of bovine a s l -casein, in relation to its molecular structure. J . Agr ic . Food Chem. 31:1214 [114] Siraganian, R. P., Hook, W . A . and Levine, B . B . 1975. Specific in vitro histamine release from basophils by bivalent haptens. Evidence for activation by simple bridging of membrane-bound antibody. Immunochemistry 12:149. [115] Sklar, L . A . , Hudson, B . S., Peterson, M . and Diamond, J . 1977. Conjugated polyene fatty acids as fluorescence probes: spectroscopic characterization. Biochem. 16:813. [116] Smith, L . M . , Fantozzi, P. and Creveling, K . 1983. Study of triglycerides-protein interaction using a microemulsion-filtration method. J . Amer. O i l Chem. Soc. 60:960. [117] Soria, J . , Soria, O , Mirshahi , M . , Boucheix, O , Aurengo, A . , Perrot, J . Y . , Bernadou, A . , Samama, M . and Rosenfeld. C. 1985. Conformational change Bibliography 104 in fibrinogen induced by adsorption to a surface. J . Colloid Interface Sci. 107:204. [118] Stahman, M . A . and Woldegiorgis, G . 1975. Enzymatic, methods for the pro-tein quality determination. In Protein Nutritional Quality of Foods and, Feeds. E d . Friedman, M . p. 211. Marcel Dekker, Inc. new York. [119] Stainsby, G . 1986. Foaming and emulsification. In Functional Properties of Food. Macromolecules. Eds. Mitchel l , J . R. and Ledward, D . A . p. 315. Else-vier A p p l . Sci. Pub. , New York. [120] Su, Y - Y . T . and Jirgensons, B . 1977. Further studies on detergent-induced conformational transistions in proteins: circular dichroism of ovalbbumin, bacterial a-amylase, papain and /3-lactoglobulin at various p H values. Arch. Biochem. Biophys. 181:137 [121] Swaisgood, H . E . 1985. Characteristics of edible fluids of animal origimmilk. In Food Chemistry, 2nd Ed. E d . Fennema, O. R. p. 791. Marcel Dekker. Inc. New York. [122] Takatsu. K . and Ishizaka, K . 1975. Reaginic antibody formation in the mouse. V I . Suppression of IgE and IgG antibody responses to ovalbumin following the administration of high dose urea-denatured antigen. Cel l . Immunol. 20:276. [123] Tanford, C. 1962. Contribution of hydrophobic interactions to the stability of the globular conformation of proteins. J . A m . Chem. Soc. 84:4240. [124] Tanford, C. 1980. The hydrophobic Effect: Formation of Micelles and Biolog-ical Membranes, 2nd ed. John Wiley & sons. New York, N Y . [125] Tang, J . 1963. Specificity of pepsin and its dependence on a possible 'hy-drophobic binding site'. Nature 199:1094. Bibliography 105 [126] Townend, R. , Herskovits, T . T. and Timasheff, S. N . 1967. The circular dichroism of variants of /3-lactoglobulin. J . B i o l . Chem. 242:4538. [127] Townsend, A - A . and Nakai , S. 1983. Relationship between protein hydropho-bicity and foaming characteristics of food proteins. J . Food Sci. 48:588. [128] Tsutsui, T . , L i - C h a n , E . and Nakai, S. 1986. A simple fluorometric method for fat binding capacity as an index of hydrophobicity of proteins. J . Food Sci. 51:1268. [129] Ueno, H . and Harrington, F . 1984. A n enzyme-probe method to detect struc-tural changes in the myosin rod. J . M o l . B io l . 173:35. [130] Van Oss, C . J . , Good, R. J . and Chaudbury, M . K . 1986. The role of van der Waals forces and hydrogen bonds in 'hydrophobic interactions' between biopolymers and low energy surfaces. J . Colloid Interf. Sci. 111:378. [131] Voutsinas, L . P. and Nakai, S. 1983. A simple turbidrimetric method for determining the fat binding capacity of proteins. J . Agric . Food Chem. 31:58. [132] Walstra, P., Ourwijn, H . and deGraaf, J . J . 1969. Neth. M i l k . Dairy J. 23:12. Cited by Waniska et al. (1981). [133] Wang, J . C . and Kinsella, J . E . 1976. Functional properties of alfalfa leaf protein: foaming. J . Food Sci 41:498. [134] Waniska, R . D. and Kinsella, J . E . 1984. Enzyme hydrolysis of maltosyl and glucosamyl derivatives of/3-lactoglobulin. J . Agr ic . Food Chem. 32:1042. [135] Waniska, R . D . , Shetty, J . K . and Kinsella, J . E . 1981. Protein-stabilized emulsions: effects of modification on the emulsifying activity of bovine serum albumin in a model system. J . Agric. Food Chem. 29:826. Bibliography 106 [136] Watabe, S., D inh , T . N . , Ochiai , Y . and Hashimoto, K . 1983. Immunochemi-cal specificity of myosin light chains from mackerel ordinary and dark muscles. J . Biochem. 94:1409. [137] Watanabe, M . , Toyokawa, H . , Shimada, A . and A r a i , S. 1981. Proteinaceous surfactants produced from gelatin by enzymatic modification evaluation for their functionality. J . Food Sci. 46:1467 [138] Waugh, D . F . 1954, Protein-protein interactions. A d v . Protein Chem. 9:325. [139] Whitney, R. M c L . , Brunner, J . R. , Ebner, K . E . , Farrell , H . M . , Josephson, R. V . , Mor r , C. V . and Swaigood, H . E . 1976. Nomenclature of the proteins of cow's milk:revision. J . Dairy Sci. 59:785. 

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