Microemulsions and Emulsions in Foods - American Chemical Society

Chapter 15 Importance of Hydrophobicity of Proteins in Food Emulsions E. Li-Chan and S. Nakai

Downloaded by UNIV OF ROCHESTER on April 17, 2013 | http://pubs.acs.org Publication Date: December 26, 1991 | doi: 10.1021/bk-1991-0448.ch015

Department of Food Science, University of British Columbia, 6650 NW Marine Drive, Vancouver, British Columbia V6T 1W5, Canada

The amphiphilic nature of proteins is important for their function as emulsifiers. Of particular relevance is the hydrophobic nature at the molecular surface, in conjunction with steric effects or flexibility which allow exposure of previously buried groups during emulsification. Various methods have been proposed to quantitate surface hydrophobicity of food proteins for elucidation of their emulsifying properties, including the use of fluorescence probes to investigate dilute protein solutions. This chapter discusses application of Raman spectroscopy to study hydrophobic interactions of proteins in concentrated solutions or gelled states, as well as in interactions with lipids in emulsions. Quantitative structure-activity relationship (QSAR) techniques use molecular structure and physical property data to make predictions about activity and reactivity of compounds. Hydrophobicity, topological descriptors, electronic descriptors and steric effects are common structure/property descriptors used in QSAR in environmental studies and in pharmaceutical research (1,2). The importance of hydrophobic, steric and charge parameters also extends to QSAR for elucidating functionality of proteins in food systems, including emulsifying properties (3,4). In particular, the importance of the amphiphilic nature of proteins in their role as emulsifiers has been widely recognized. Many methods have been proposed to measure the hydrophobicity of proteins which may be important in their emulsifying functions. At the same time, many researchers have focussed on processes during diffusion, adsorption and rearrangment of protein molecules at surfaces or interfaces. In most cases, dilute protein solutions and model hydrocarbons or triglycerides have been used in these studies. In order to obtain information which can be truly useful in elucidating QSAR in food emulsions, more effort should be focussed on 0097-6156/91AM48-0193$06.00/0 © 1991 American Chemical Society In Microemulsions and Emulsions in Foods; El-Nokaly, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.


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the complex w o r l d of r e a l food systems r a t h e r than the model system (5,6). For example, what i s the c o n c e n t r a t i o n of p r o t e i n commonly o c c u r r i n g i n the food emulsion? Is t h e p r o c e s s of a d s o r p t i o n at the i n t e r f a c e more l i k e l y t o be d i f f u s i o n - or t u r b u l e n c e - c o n t r o l l e d ? Can complex animal and vegetable fats and oils be used to study protein-lipid interactions? In t h i s c h a p t e r , the importance o f h y d r o p h o b i c i n t e r a c t i o n s of proteins i n food emulsions i s d i s c u s s e d by c o n s i d e r i n g some of t h e s e issues. The r o l e o f s t e r i c e f f e c t s and molecular flexibility on exposure of hydrophobic groups at the surface of the protein m o l e c u l e s which can p a r t i c i p a t e i n e m u l s i f i c a t i o n i s emphasized. A review of some e m p i r i c a l methods c u r r e n t l y used t o measure p r o t e i n hydrophobicity i s followed by recent research i n our laboratory applying l a s e r Raman s p e c t r o s c o p y t o study h y d r o p h o b i c i n t e r a c t i o n s and s t e r i c e f f e c t s of p r o t e i n s i n s o l u t i o n , g e l s and e m u l s i o n s . Molecular

Structure, Hydrophobicity

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Emulsifying

Properties

The m o l e c u l a r p r o c e s s e s which occur during emulsification in food systems have been the s u b j e c t o f s e v e r a l r e c e n t reviews and books (eg. 7-10), and c o n s i s t of a number of s t a g e s t h a t i n c l u d e migration to the oil-water i n t e r f a c e , adsorption at the interface, conformational re-arrangement and formation of multiple layers. Desorption or reversibility of the adsorption process is also p o s s i b l e , e s p e c i a l l y i n the case of mixed e m u l s i f i e r s . For many y e a r s , i t was accepted that the solubility of a p r o t e i n was the primary determinant i n i t s e m u l s i f y i n g p r o p e r t i e s ( L I ) . However, i n the last decade, key r o l e s have been assigned t o b o t h backbone f l e x i b i l i t y and p r o p e r h y d r o p h o b i c - h y d r o p h i l i c b a l a n c e o f the p r o t e i n molecule. Solubility facilitates diffusion of molecules to the surface or i n t e r f a c e . However, whether or not t h i s i s a c r i t i c a l f a c t o r under the t u r b u l e n t c o n d i t i o n s of h i g h shear and energy input which e x i s t d u r i n g e m u l s i f i c a t i o n i s u n c l e a r . In any c a s e , once a t the i n t e r f a c e , the a b i l i t y of the p r o t e i n t o i n t e r a c t w i t h the o i l or w i t h o t h e r p r o t e i n m o l e c u l e s t o form an i n t e r f a c i a l l a y e r depends on its flexibility and accessibility of surface exposed groups f o r i n t e r a c t i o n s , e s p e c i a l l y through hydrophobic i n t e r a c t i o n s . Charge effects play an a d d i t i o n a l important role since electrostatic r e p u l s i o n s can h i n d e r i n t e r m o l e c u l a r i n t e r a c t i o n s at the i n t e r f a c e . B - c a s e i n from cow's m i l k i s an example o f a food protein which possesses b o t h h i g h f l e x i b i l i t y and a m p h i p h i l i c i t y and i s r e c o g n i z e d as a s u p e r i o r e m u l s i f i e r . However, many food p r o t e i n s are g l o b u l a r i n n a t u r e and are o f t e n l i m i t e d i n b o t h m o l e c u l a r f l e x i b i l i t y as w e l l as h y d r o p h o b i c groups which are exposed on the s u r f a c e of the protein and a v a i l a b l e to participate in i t s f u n c t i o n a l i t y . For example, lysozyme from hen egg white has a compact g l o b u l a r s t r u c t u r e which i s s t a b i l i z e d by f o u r disulfide c r o s s - l i n k s . Lysozyme e x h i b i t s poor emulsifying and foaming p r o p e r t i e s , and i s r e s i s t a n t t o i r r e v e r s i b l e d e n a t u r a t i o n by h e a t i n g up t o 75°C. The o f t e n - c i t e d work of Graham and Phillips (12-14), which compared the b e h a v i o r between JB-casein, lysozyme and b o v i n e serum albumin a t air-water and "oil"-water or toluene-water i n t e r f a c e s , i s an excellent demonstration of the e f f e c t s of molecular structure on their properties. However, as

In Microemulsions and Emulsions in Foods; El-Nokaly, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

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Hydrophobicity of Proteins

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suggested by Mangino ( 6 ) , a p p l i c a b i l i t y of t h e i r c o n c l u s i o n s t o food emulsions i s q u e s t i o n a b l e due t o the low protein concentrations at which the d i f f e r e n c e s i n b e h a v i o r were o b s e r v e d . Surface or Exposed Hydrophobicity. The concept of hydrophilelipophile balance or HLB i n e x p r e s s i n g the r e l a t i v e p r o p o r t i o n s of h y d r o p h o b i c ( o r l i p o p h i l i c or n o n p o l a r ) v e r s u s h y d r o p h i l i c ( o r p o l a r ) m o i e t i e s i n low m o l e c u l a r weight compounds has been a u s e f u l aid in the s e l e c t i o n of suitable surface a c t i v e agents f o r f o r m a t i o n of o i l - i n - w a t e r or w a t e r - i n - o i l e m u l s i o n s . In the c a s e o f proteins, a value analogous to HLB can be o b t a i n e d by summing up o r t a k i n g the average o f the h y d r o p h o b i c i t y v a l u e s o f the constituent amino a c i d residues of the p r o t e i n . The c a l c u l a t i o n of total or average h y d r o p h o b i c i t y by the method of B i g e l o w (15) i s an example of this. However, such v a l u e s do not c o n s i d e r the e f f e c t of p r o t e i n s t r u c t u r e on the e x t e n t o f exposure of residues. Although i t is generally agreed t h a t charged amino a c i d r e s i d u e s a r e l o c a t e d p r e f e r e n t i a l l y at the surface of globular proteins while nonpolar or h y d r o p h o b i c r e s i d u e s a r e b u r i e d i n the i n t e r i o r of the m o l e c u l e , a n a l y s i s o f the three-dimensional s t r u c t u r e of p r o t e i n s by t e c h n i q u e s such as X-ray c r y s t a l l o g r a p h y has i n d i c a t e d the p r e s e n c e of h y d r o p h o b i c p a t c h e s on the surface of proteins. It i s t h e r e f o r e l i k e l y t h a t t h e groups which can participate in protein f u n c t i o n a l i t y , such as e m u l s i f i c a t i o n , are those amino a c i d r e s i d u e s which are l o c a t e d on the s u r f a c e o f the n a t i v e p r o t e i n m o l e c u l e s or become exposed during p r o c e s s i n g , such as h e a t i n g or h o m o g e n i z a t i o n or w h i p p i n g . The observation t h a t p r o t e i n s can d e c r e a s e i n t e r f a c i a l t e n s i o n at an o i l - w a t e r i n t e r f a c e has been s u g g e s t e d t o be a f a c t o r i n their a b i l i t y t o a c t as e m u l s i f i e r s . Keshavarz and Nakai (16) f i r s t showed that interfacial tension values a t 0.2% p r o t e i n s o l u t i o n / c o r n o i l i n t e r f a c e s were n e g a t i v e l y c o r r e l a t e d w i t h h y d r o p h o b i c i t y o f p r o t e i n s determined by e i t h e r r e t e n t i o n volume on hydrophobic i n t e r a c t i o n chromatography or p a r t i t i o n c o e f f i c i e n t between phases o f d i f f e r i n g polarity. No c o r r e l a t i o n was obtained between these "effective" hydrophobicity values and average h y d r o p h o b i c i t y values of the p r o t e i n s c a l c u l a t e d by Bigelow's method. The c o r r e l a t i o n between emulsifying activity and "surface" h y d r o p h o b i c i t y of p r o t e i n s was subsequently demonstrated by Kato and Nakai ( 1 7 ) , who measured s u r f a c e h y d r o p h o b i c i t y by the i n c r e a s e i n f l u o r e s c e n c e i n t e n s i t y upon b i n d i n g of h y d r o p h o b i c probes t o the p r o t e i n s o l u t i o n s . Molecular flexibility. Based on t h e a d s o r p t i o n b e h a v i o r o f l i n e a r polymers at solid s u r f a c e s , i t has been proposed that proteins undergo c o n f o r m a t i o n a l rearrangement a t the i n t e r f a c e r e s u l t i n g i n segments r e f e r r e d to as t r a i n s , loops and tails. However, the v a l i d i t y of t h i s model has been c o n t r o v e r s i a l . In cases where a d s o r p t i o n at the i n t e r f a c e occurs from c o n c e n t r a t e d b u l k s o l u t i o n , i t has been suggested t h a t a m i x t u r e of native and denatured m o l e c u l e s e x i s t s , and the p h y s i c a l s t a t e o f the adsorbed f i l m may resemble that of a gel (IS). Change in the c o n f o r m a t i o n o f whey p r o t e i n s adsorbed on e m u l s i f i e d f a t g l o b u l e s was suggested by t h e i r g r e a t e r s u s c e p t i b i l i t y t o p r o t e a s e d i g e s t i o n than the whey p r o t e i n s i n aqueous s o l u t i o n (19). No change in the secondary s t r u c t u r e of lysozyme upon adsorption was o b s e r v e d by



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c i r c u l a r dichroism ( 2 0 ) , b u t i t was n o t c l e a r i f any t e r t i a r y structural changes had o c c u r r e d o r whether such changes were r e s t r i c t e d by t h e d i s u l f i d e c r o s s l i n k s s t a b i l i z i n g t h i s p r o t e i n . D e s p i t e t h e l a c k o f consensus on t h e a c t u a l s t r u c t u r a l changes which o c c u r upon and a f t e r a d s o r p t i o n , i t i s p r o b a b l y agreed t h a t a c e r t a i n degree o f f l e x i b i l i t y i s required t o promote surface or interfacial activity. Amphiphilic proteins may be encouraged t o adsorb a t an i n t e r f a c e by p a r t i a l d e n a t u r a t i o n such as t h a t brought about by m i l d conditions of heating. Kato e t a l . (21-23) r e p o r t e d t h a t heat t r e a t m e n t which was c o n t r o l l e d so as not t o produce coagulation resulted i n increased surface hydrophobicity of the heated p r o t e i n s , and t h a t t h e s e i n c r e a s e d h y d r o p h o b i c i t y v a l u e s were linearly r e l a t e d t o improved e m u l s i f y i n g p r o p e r t i e s . On t h e o t h e r hand, when i n t r a m o l e c u l a r c r o s s l i n k s were c h e m i c a l l y i n t r o d u c e d t o a protein such as b o v i n e serum albumin, s u r f a c e a c t i v e p r o p e r t i e s were d r a m a t i c a l l y impaired ( 2 4 ) . E f f e c t o f D i s u l f i d e Bond R e d u c t i o n . D i s u l f i d e bonds s t a b i l i z e t h e t e r t i a r y s t r u c t u r e o f many g l o b u l a r p r o t e i n s such as lysozyme, b o v i n e serum albumin and soy g l y c i n i n . Treatment o f t h e s e p r o t e i n s w i t h a r e d u c i n g agent such as d i t h i o t h r e i t o l (DTT) was r e p o r t e d t o improve their surface a c t i v e p r o p e r t i e s , i n c l u d i n g foaming and e m u l s i f y i n g p r o p e r t i e s , as w e l l as t o induce g e l a t i o n i n some c a s e s ( 2 5 - 2 9 ) . In t h e c a s e o f soy g l y c i n i n , r e d u c t i o n o f 13 o f t h e 20 disulfide bonds by treatment w i t h 5 mM DTT i n t h e p r e s e n c e o f 8 M urea, f o l l o w e d by b l o c k i n g o f s u l f h y d r y l groups w i t h iodoacetamide, resulted i n dramatic increases i n surface hydrophobicity apparently through cleavage of intermolecular bonds linking the subunits; reduction o f a l l 20 d i s u l f i d e bonds, b o t h i n t r a - and i n t e r m o l e c u l a r , was a c h i e v e d u s i n g 10 mM DTT. The m o l e c u l a r conformational changes which enhanced m o l e c u l a r h y d r o p h o b i c i t y and i n c r e a s e d v i s c o s i t y were suggested t o be r e s p o n s i b l e f o r the s i g n i f i c a n t improvement i n surface a c t i v e p r o p e r t i e s o f t h e reduced p r o t e i n s (25,26) . However, the h a r s h conditions during the reducing treatment as w e l l as possible changes i n the p r o t e i n surface character by use o f a b l o c k i n g agent make i t d i f f i c u l t t o c o n c l u d e t h a t t h e improvements i n s u r f a c e a c t i v e p r o p e r t i e s were due t o enhanced m o l e c u l a r flexibility a r i s i n g from r e d u c t i o n p e r s e . When lysozyme solutions ( 0 . 5 % i n 50 mM NaCl a t pH 7) were t r e a t e d u s i n g much m i l d e r c o n d i t i o n s , i . e . w i t h 0.7 mM DTT a t e i t h e r 4°C o r room temperature f o r up t o 24 h o u r s , o n l y s l i g h t i n c r e a s e was o b s e r v e d both i n the s u l f h y d r y l content and in surface h y d r o p h o b i c i t y determined by f l u o r e s c e n c e probes ( 2 9 ) . Nevertheless, a s i g n i f i c a n t i n c r e a s e i n t h e e m u l s i f y i n g a c t i v i t y index was observed in the DTT-treated lysozyme compared t o t h e c o n t r o l . I t was suggested t h a t t h e m e c h a n i c a l energy o f t h e e m u l s i f i c a t i o n process itself was sufficient t o e n a b l e u n f o l d i n g o f t h e p a r t i a l l y reduced lysozyme. DTT-treated lysozyme subjected to the emulsification process i n t h e absence o f any o i l e x h i b i t e d a s i g n i f i c a n t l y h i g h e r surface hydrophobicity value compared to similarly emulsified lysozyme s o l u t i o n . Upon h e a t i n g o f 0.5% s o l u t i o n s o f t h e D T T - t r e a t e d lysoyzme e i t h e r a t 80°C f o r 12 minutes o r a t 37°C f o r 24 h o u r s , up t o 3 o f t h e 4 d i s u l f i d e bonds o f lysozyme were r e d u c e d , and s i g n i f i c a n t increases i n surface hydrophobicity, emulsifying a c t i v i t y


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and stability as w e l l as i n t u r b i d i t y o f t h e s o l u t i o n s were n o t e d . These r e s u l t s demonstrate t h a t changes i n molecular flexibility allowing exposure o f h y d r o p h o b i c groups can be brought about by t h e c l e a v a g e o f d i s u l f i d e bonds u s i n g low c o n c e n t r a t i o n s of reducing agent combined with energy i n p u t i n t h e form o f t h e e m u l s i f i c a t i o n p r o c e s s i t s e l f o r by h e a t i n g . In o t h e r words, t h e p o t e n t i a l f o r hydrophobic i n t e r a c t i o n s a t t h e i n t e r f a c e i s t h e key f a c t o r , r a t h e r than h i g h l y h y d r o p h o b i c c h a r a c t e r i s t i c s o f t h e p r o t e i n m o l e c u l e s i n bulk s o l u t i o n as t h e l a t t e r can l e a d t o f o r m a t i o n o f a g g r e g a t e s and may i n f a c t h i n d e r a d s o r p t i o n a t t h e i n t e r f a c e . E f f e c t o f P r o t e i n C o n c e n t r a t i o n i n t h e Bulk S o l u t i o n . The e f f e c t s o f concentration of p r o t e i n molecules i n the bulk s o l u t i o n t o the interfacial p r o t e i n c o n c e n t r a t i o n and c h a r a c t e r i s t i c s o f a d s o r p t i o n and m u l t i l a y e r f o r m a t i o n have been t h e s u b j e c t o f much controversy. Under q u i e s c e n t conditions of adsorption and a t low b u l k p r o t e i n c o n c e n t r a t i o n , t h e adsorbed l a y e r has been proposed t o resemble a two-dimensional gas with unfolded molecules; at intermediate c o n c e n t r a t i o n s , t h e s u r f a c e l a y e r becomes compressed and resembles a condensed liquid f i l m , while at s t i l l higher concentrations, the adsorbed l a y e r may e x h i b i t v i s c o e l a s t i c o r s o l i d - l i k e p r o p e r t i e s (j>). However, a d s o r p t i o n under q u i e s c e n t c o n d i t i o n s can d i f f e r from that in an e m u l s i o n , and i t has been suggested t h a t t h e much h i g h e r s u r f a c e - t o - v o l u m e r a t i o i n t h e l a t t e r may r e s u l t i n t h e s i g n i f i c a n t depletion of protein from t h e b u l k s o l u t i o n , a t low i n i t i a l b u l k concentrations (30). Many s t u d i e s have indicated that emulsifying c a p a c i t y or a c t i v i t y e x p r e s s e d p e r u n i t weight o f p r o t e i n d e c r e a s e s as a f u n c t i o n of i n c r e a s i n g bulk concentration o f p r o t e i n , and d i f f e r e n c e s i n e m u l s i f y i n g p r o p e r t i e s between p r o t e i n s have been reported t o be negligible at high protein concentration. H a i l i n g (30) i n d i c a t e d that the d i f f e r e n c e s i n emulsifying properties exhibited with respect to p r o t e i n c o n c e n t r a t i o n are a c t u a l l y a r e f l e c t i o n of differing soluble p r o t e i n content. However, as i l l u s t r a t e d i n F i g u r e 1, r e c e n t work i n o u r l a b o r a t o r y ( L e e , 6. U n i v e r s i t y o f B r i t i s h Columbia, B.Sc. t h e s i s , 1989) has demonstrated that there are inherent differences between soluble proteins i n their emulsifying a c t i v i t y b e h a v i o r as a f u n c t i o n o f p r o t e i n c o n c e n t r a t i o n . Viscosity of the continuous phase has been c i t e d as a parameter a f f e c t i n g e m u l s i o n s t a b i l i t y and may be a l t e r e d by p r o t e i n c o n c e n t r a t i o n . However, t h e r e l a t i o n s h i p i s n o t a s i m p l e one; no s i g n i f i c a n t c o r r e l a t i o n c o u l d be found between the t u r b i d i t y and v i s c o s i t y o f emulsions o f v a r i o u s p r o t e i n s a t d i f f e r e n t i n i t i a l b u l k c o n c e n t r a t i o n ( F i g u r e s l a and l b ) . A l t h o u g h i n c r e a s e d v i s c o s i t y may impart greater transient droplet stability during the e m u l s i f i c a t i o n process and t h u s reduce re-coalescence, the v i s c o s i t y of the continuous phase has a small effect on f i n a l droplet s i z e , and i n f a c t h i g h l y v i s c o u s aqueous phases c o n t a i n i n g c e l l u l o s e d e r i v a t i v e s were observed t o y i e l d l a r g e r droplet sizes (5). Dickinson and S t a i n s b y (2) concluded that empirical s t u d i e s have not r e v e a l e d any g e n e r a l r u l e s about t h e r e l a t i o n s h i p between p r o t e i n c o n c e n t r a t i o n and emulsion s t a b i l i t y .



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Protein Concentration, % Figure 1. (b) Kinematic viscosity of various soluble proteins as a function of initial bulk solution


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Comparison o f C u r r e n t

Methods f o r P r o t e i n H y d r o p h o b i c i t y

Studies

Methods f o r q u a n t i t a t i v e e s t i m a t i o n of p r o t e i n h y d r o p h o b i c i t y values have been r e c e n t l y reviewed (3 and L i - C h a n , E. In Encyclopedia of Food Science and Technology; H u i , Y. H., Ed.; W i l e y - I n t e r s c i e n c e : New York, 1990; i n p r e s s ) . G e n e r a l l y they may be categorized into (1) various algorithms f o r c o m p u t a t i o n o f h y d r o p h o b i c i t y v a l u e s or p r o f i l e s , u s i n g h y d r o p h o b i c i t y s c a l e s o f the i n d i v i d u a l amino acids and data of the amino a c i d c o m p o s i t i o n or primary sequence o f the p r o t e i n ; (2) partition methods, i n c l u d i n g r e l a t i v e solubility in p o l a r and n o n p o l a r s o l v e n t s o r r e l a t i v e r e t e n t i o n b e h a v i o r on r e v e r s e phase or h y d r o p h o b i c i n t e r a c t i o n chromatography; (3) b i n d i n g methods, including the b i n d i n g o f a l i p h a t i c and a r o m a t i c h y d r o c a r b o n s , sodium dodecyl s u l f a t e , simple triglycerides and corn oil; and (4) spectroscopic methods, i n c l u d i n g i n t r i n s i c f l u o r e s c e n c e , d e r i v a t i v e s p e c t r o s c o p y and use o f f l u o r e s c e n c e p r o b e s . C o n s i d e r a b l e v a r i a t i o n s i n the v a l u e s o b t a i n e d f o r p r o t e i n s by different methods have been r e p o r t e d , and caution i s warranted whenever t h e method i n v o l v e s use o f n o n p o l a r o r g a n i c solvents which may alter the native s t r u c t u r e and thus the s u r f a c e h y d r o p h o b i c c h a r a c t e r i s t i c s o f the p r o t e i n . S p e c t r o s c o p i c methods a r e usually non-destructive and l e s s l i k e l y t o i n d u c e c o n f o r m a t i o n a l changes i n the protein molecule. However, methods such as ultraviolet absorbance, d e r i v a t i v e s p e c t r o p h o t o m e t r y , or i n t r i n s i c f l u o r e s c e n c e , u s u a l l y o n l y g i v e i n f o r m a t i o n about the a r o m a t i c chromophores o f the protein. Algorithms f o r q u a n t i t a t i o n of protein hydrophobicity, w h i l e u s e f u l f o r c e r t a i n p r o t e i n s , are limited in universality of a p p l i c a t i o n , due t o the need f o r c o n s i d e r a b l e i n f o r m a t i o n on sequence or s t r u c t u r e of the p r o t e i n , assumption o f homologous b e h a v i o u r o f r e s i d u e s o f the p r o t e i n under i n v e s t i g a t i o n t o those i n the database upon which the a l g o r i t h m s were f o r m u l a t e d , and i n a b i l i t y t o e v a l u a t e h y d r o p h o b i c i t y o f complex food systems containing several protein s p e c i e s , whose i n t e r a c t i o n s may change d u r i n g food p r o c e s s i n g . E x t r i n s i c f l u o r e s c e n c e probes have become w i d e l y used t o probe hydrophobic s i t e s of p r o t e i n s , probably due t o the rapidity and simplicity of the methodology i n v o l v e d . The most commonly used probes i n the study of food p r o t e i n s have been the aromatic hydrophobic probe, l - a n i l i n o n a p h t h a l e n e - 8 - s u l f o n i c a c i d or ANS, and the a l i p h a t i c hydrophobic probe, c i s - p a r i n a r i c acid or CPA. The l a t t e r may be p a r t i c u l a r l y advantageous as i t s s t r u c t u r e i s analogous to fatty acids and thus i t may probe f o r s i t e s on t h e p r o t e i n m o l e c u l e which can b i n d food l i p i d s . During t h i s past decade, many reports have appeared i n the literature on the use of these f l u o r e s c e n c e probes t o i n v e s t i g a t e h y d r o p h o b i c i n t e r a c t i o n s o f food p r o t e i n systems and how the s u r f a c e h y d r o p h o b i c groups may be changed by p r o c e s s i n g (3). However, d e s p i t e the widespread use and p o t e n t i a l fundamental i n f o r m a t i o n which can be o b t a i n e d , two l i m i t a t i o n s may r e s t r i c t t h e i n t e r p r e t a t i o n o f r e s u l t s o b t a i n e d by t h e s e f l u o r e s c e n c e probes : (1) Both ANS and CPA c o n t a i n a n i o n i c m o i e t i e s which may c a s t doubt on whether t h e i r probing effect i s solely for hydrophobic sites; the effect of rigidity of the environment r a t h e r than h y d r o p h o b i c i t y on f l u o r e s c e n c e o f ANS has a l s o been c a u t i o n e d ; (2) low p r o t e i n c o n c e n t r a t i o n s ( t y p i c a l l y 0.005-0.05%) are used f o r the





201

measurement o f f l u o r e s c e n c e o f t h e bound probe, which i s i n c o n t r a s t to the much higher concentrations which would typically be encountered i n r e a l food systems such as e m u l s i o n s . Thus o t h e r methods t o q u a n t i t a t e h y d r o p h o b i c i n t e r a c t i o n s are being sought t o supplement t h e i n f o r m a t i o n which has been a c q u i r e d through f l u o r e s c e n c e probes. These new methods s h o u l d be a p p l i c a b l e at h i g h e r p r o t e i n c o n c e n t r a t i o n s , and w i t h o u t u s i n g e x t r i n s i c probes which may p e r t u r b t h e n a t i v e p r o t e i n s t r u c t u r e . Two approaches currently b e i n g i n v e s t i g a t e d i n our l a b o r a t o r y a r e t h e use o f p r o t o n magnetic r e s o n a n c e t o determine e x t e n t o f exposure o f a l i p h a t i c and aromatic s i d e chains o f p r o t e i n s under d i f f e r e n t c o n d i t i o n s , and application o f Raman s p e c t r o s c o p y (31.) • The remainder of this chapter will present some o f t h e r e s u l t s which we have r e c e n t l y obtained using the l a t t e r technique. Raman

Spectroscopy

Raman s p e c t r o s c o p y i s a l i g h t s c a t t e r i n g technique which can g i v e detailed information on t h e v i b r a t i o n a l motions o f atoms i n molecules. In essence, t h e t e c h n i q u e based on Raman scattering measures shifts i n t h e wavelength o f t h e e x c i t i n g l a s e r beam which a r i s e from t h e i n e l a s t i c c o l l i s i o n s between t h e sample m o l e c u l e s and the photons o r p a r t i c l e s o f l i g h t composing t h e l a s e r beam. Because the i n t e n s i t y and f r e q u e n c y o f t h e m o l e c u l a r v i b r a t i o n s a r e s e n s i t i v e t o c h e m i c a l changes and t h e environment o f t h e atoms, t h e Raman spectrum can be used as a monitor o f m o l e c u l a r c h e m i s t r y . Table I shows t y p i c a l assignments o f amino a c i d r e s i d u e s i d e c h a i n v i b r a t i o n s which c o r r e s p o n d t o peaks i n different wavenumber shift regions (cm ) o f t h e p r o t e i n Raman spectrum. A d d i t i o n a l i n f o r m a t i o n can be o b t a i n e d from v i b r a t i o n s a r i s i n g from the peptide backbone; analysis o f t h e amide I (1640-1680 c m ) and amide I I I (1230-1300 cm" ) r e g i o n s i n p a r t i c u l a r can y i e l d u s e f u l data on t h e secondary s t r u c t u r e o f t h e p r o t e i n (32-34). S i n c e t h e e a r l y r e s e a r c h on p e p t i d e s and p r o t e i n s i n t h e 1960's, Raman s p e c t r o s c o p i c a p p l i c a t i o n s i n t h e study o f b i o l o g i c a l m o l e c u l e s including p r o t e i n s have been r a p i d l y expanding, as e x e m p l i f i e d i n some r e c e n t monographs (35-39). Some d i s t i n c t advantages i n the a p p l i c a t i o n o f Raman over o t h e r s p e c t r o s c o p i c methods a r e t h a t i t can be a p p l i e d t o aqueous s o l u t i o n s o f m o l e c u l e s as w e l l as nonaqueous l i q u i d s , and has been s u c c e s s f u l l y used t o study m o l e c u l e s i n f i b e r s , f i l m s , powders, g e l s and c r y s t a l l i n e s o l i d s o f b i o p o l y m e r s . Thus, i t has g r e a t p o t e n t i a l t o study changes i n t h e p r o t e i n m o l e c u l e i n the dry versus hydrated or solution state, i n solvents of d i f f e r i n g p o l a r i t y , o r a f t e r c o a g u l a t i o n o r g e l a t i o n , as w e l l as t o monitor interactions with o t h e r m o l e c u l e s such as l i p i d s and c a r b o h y d r a t e s . P a i n t e r (40) p r e s e n t e d an e x c e l l e n t overview o f some a p p l i c a t i o n s o f Raman s p e c t r o s c o p y t o t h e c h a r a c t e r i z a t i o n o f f o o d , and noted t h a t t h i s t e c h n i q u e has a c l e a r b u t as y e t u n r e a l i z e d p o t e n t i a l f o r c h a r a c t e r i z i n g t h e i n d i v i d u a l components o f food systems. The p i o n e e r i n g work o f P r o f e s s o r R i c h a r d C o l l i n s L o r d y i e l d e d the f i r s t i n t e r p r e t a b l e l a s e r Raman spectrum of a native protein, lysozyme i n aqueous s o l u t i o n . In a s e r i e s o f p a p e r s p u b l i s h e d i n t h e early 1970's, L o r d and h i s co-workers s t u d i e d t h e Raman spectrum o f native lysozyme as w e l l as t h e changes observed by extensive - 1

- 1

1



202


T a b l e I . Assignments o f t y p i c a l amino a c i d s i d e c h a i n v i b r a t i o n s i n Raman spectrum o f p r o t e i n s ( c o m p i l e d from r e f e r e n c e s 35-38).

cm"

1

vibration assignment/interpretation

2700-3300 1465 + 20 1450 + 20

CH s t r e t c h C H bend C H a n t i s y m m e t r i c bend

1399 1725-1700 1425 1650(asn),1615 1640,1600 1491

s e r , t h r OH (weak) a s p , g l u C00H, u n - i o n i z e d C=0 s t r e t c h a s p , g l u COOH, i o n i z e d C=0 s t r e t c h a s n / g l n amide lys NH h i s i m i d a z o l e (probe i o n i z a t i o n s t a t e )

2

3

(gin)

1605,1585,1207,1006,622 1600,1590,850,830 1582,1553,1363,1014,879 761,577,544 540 525 510 745-700 670-630

^ > aliphatic J side chains

+

3

phe t y r (850/830 i s environment s e n s i t i v e ) t r p (1360 and 880 cm" i n t e n s i t y a r e e s p e c i a l l y s e n s i t i v e t o environment) 1

trans-gauche-trans 1 gauche-gauche-trans > c y s t i n e SS gauche-gauche-gauche J t r a n s C-S (met, c y s , cys/2) gauche C-S (met, c y s , cys/2)





203

denaturation u s i n g h e a t , complete r e d u c t i o n o f d i s u l f i d e bonds o r w i t h L i B r o r SDS (41-43). Based on t h i s work, they concluded that the denatured p r o t e i n e x i s t s i n r a n d o m - c o i l c o n f o r m a t i o n s , which may d i f f e r depending on t h e v a r i o u s d e n a t u r i n g a g e n t s . R e c e n t l y , we have used Raman s p e c t r o s c o p y t o s t u d y changes i n lysozyme i n c o n c e n t r a t e d (10-20%) s o l u t i o n s a f t e r treatment by h e a t and/or m i l d DTT r e d u c t i o n . The objective o f t h i s work was t o i n v e s t i g a t e changes b r o u g h t about by m i l d r e d u c t i o n ( i n c o n t r a s t t o complete r e d u c t i o n and e x t e n s i v e denaturation r e p o r t e d by L o r d and c o - w o r k e r s ) , which were a b l e t o confer improved g e l l i n g , foaming and e m u l s i f y i n g p r o p e r t i e s on lysozyme, and to compare these changes w i t h those previously demonstrated a t lower c o n c e n t r a t i o n (29). In a d d i t i o n , a p p l i c a t i o n of Raman s p e c t r o s c o p y t o study i n t e r a c t i o n s o f p r o t e i n w i t h c o r n o i l i s a l s o demonsrated. Experimental. Raman s p e c t r a were r e c o r d e d on a JASCO model NR-1100 l a s e r Raman s p e c t r o p h o t o m e t e r w i t h e x c i t a t i o n from t h e 488 nm l i n e o f a S p e c t r a - P h y s i c s Model 168B argon i o n l a s e r . The s p e c t r a o f 10 o r 20% (w/w) s o l u t i o n s o f lysozyme (Sigma L6876) i n water (final pH 5.75) o r i n deuterium oxide (final apparent pD 5.85), c o r n o i l (commercial grade Mazola b r a n d ) , o r t h e i r emulsions i n hematocrit capillary tubes were measured a t ambient temperature under t h e f o l l o w i n g c o n d i t i o n s : l a s e r power 200 mW, s l i t h e i g h t 2 mm; spectral resolution of 5.0 cm" a t 19,000 cm" , sampling speed 120 cm~ /min w i t h d a t a taken every cm" , 6-10 scans p e r sample. F o r samples treated with DTT ( D L - d i t h i o t h r e i t o l , Sigma D0632) and h e a t i n g , s o l u t i o n s o f t h e samples were p l a c e d i n t h e c a p i l l a r y tubes prior to heat-induced gelation. Background correction, n o r m a l i z a t i o n , smoothing and d i f f e r e n c e spectrum computation of the recorded spectra were performed with t h e NR-1100 d a t a s t a t i o n . F u r t h e r d e t a i l s o f t h e p r o c e d u r e s a r e r e p o r t e d elsewhere ( L i - C h a n and Nakai, 1990; m a n u s c r i p t s i n p r e p a r a t i o n ) . 1

1

1

1

Raman S p e c t r a o f Lysozyme S o l u t i o n and Heat Induced G e l s . F i g u r e s 2 and 3 show t h e Raman s p e c t r a o f 20% lysozyme s o l u t i o n s and t h e g e l s formed by h e a t i n g a t 100°C f o r 5 minutes. The changes brought about by t h i s s h o r t heat treatment a t h i g h p r o t e i n c o n c e n t r a t i o n a r e g e n e r a l l y s i m i l a r t o t h o s e r e p o r t e d when 7% s o l u t i o n s were h e a t e d a t 100°C f o r 2 hours ( 4 2 ) . Some o f t h e more e v i d e n t d i f f e r e n c e s arising from heating include the following: (1) t h e relative intensity o f t h e 510 and 525 cm" peaks i n t h e d i s u l f i d e (SS) s t r e t c h i n g r e g i o n changes from 3:1 t o 1:1 r a t i o , which c o r r e s p o n d s t o a change from an a l l gauche t o a gauche-gauche-trans conformation, respectively; (2) d e c r e a s e i n i n t e n s i t y o f t h e peaks a t 760, 880 and 1360 cm" , which i n d i c a t e i n c r e a s e d exposure o f t r y p t o p h a n r e s i d u e s t o t h e aqueous environment; (3) s h i f t s i n t h e c e n t r e o f t h e amide I I I region from 1257 t o 1245 cm" , and i n t h e amide I r e g i o n from 1661 t o a d o u b l e t a t 1660 and 1674 cm" , which a r e r e l a t e d t o a decrease in (X-helical structure and i n c r e a s e s i n p - s h e e t and random c o i l structure; (4) i n c r e a s e i n t h e i n t e n s i t y o f t h e CH stretching vibration a t 2938 cm" relative t o t h e broad water l i n e a t 3230 cm" which s u g g e s t i n c r e a s e d exposure o f a l i p h a t i c side c h a i n s as well as d e c r e a s e d m o b i l i t y o f t h e water m o l e c u l e s a f t e r h e a t - i n d u c e d gelation. These changes c o r r e l a t e well with the increases in h y d r o p h o b i c i t y a f t e r h e a t i n g measured by methods such as f l u o r e s c e n c e 1

1

1

1

1

1


204



o o

o o

o o o

in o

o o

o o

Avavenumber, cm Figure 2. Raman spectra (400-1700 20% lysozyme (a) s o l u t i o n and (b) g e l 100°C f o r 5 minutes. (Spectra were n o r m a l i z e d t o the d e f o r m a t i o n mode a t 1455 cm" , a f t e r recommended i n r e f e r e n c e 4 2 ) . 1

cm shift formed a f t e r intensity baseline

region) heating

of at

o f t h e H-C-H c o r r e c t i o n , as



205



to

A wavenumber, c m

- 1

A v a v e n u m b e r , cm"

Figure 3. Raman s p e c t r a (2500-3350 cm shift region) of 20% lysozyme (a) s o l u t i o n and (b) g e l formed a f t e r heating at 100°C f o r 5 m i n u t e s . ( S p e c t r a were n o r m a l i z e d t o t h e i n t e n s i t y o f t h e C-H s t r e t c h i n g band a t 2940 c m " ) . 1


206



probes ( 2 9 ) , and w i t h t h e s u g g e s t i o n t h a t h e a t - i n d u c e d g e l a t i o n does not always r e s u l t i n complete u n f o l d i n g t o a random c o i l b u t i n f a c t , t h e r e i s o f t e n an i n c r e a s e i n 3-sheet structure ( 4 4 ) . Furthermore, although chemical analysis u s i n g Edman's r e a g e n t d i d n o t show any s i g n i f i c a n t changes i n s u l f h y d r y l group c o n t e n t a f t e r h e a t i n g , i t i s interesting t o note t h e c a p a b i l i t y o f Raman s p e c t r o s c o p y t o d e t e c t changes i n t h e d i s u l f i d e bond c o n f o r m a t i o n i n t h e g e l . E f f e c t o f DTT and/or H e a t i n g on Raman S p e c t r a o f Lysozyme. The Raman s p e c t r a o f unheated 10-2056 lysozyme s o l u t i o n s t r e a t e d w i t h 10 mM DTT d i d n o t appear t o d i f f e r from that o f lysozyme, which i s consistent w i t h t h e s m a l l changes i n b o t h s u l f h y d r y l c o n t e n t and h y d r o p h o b i c i t y measured by f l u o r e s c e n c e probes reported previously. Firm opaque white g e l s were formed when lysozyme s o l u t i o n s a t ^ 5 % concentration were heated at either low (37-40°C) or high (75-80°C) temperature i n t h e p r e s e n c e o f low c o n c e n t r a t i o n s o f DTT (29). The p h y s i c a l changes m a n i f e s t e d i n g e l formation were accompanied by marked changes i n t h e Raman s p e c t r a . F i g u r e 4 shows the Raman d i f f e r e n c e s p e c t r a o f d e u t e r a t e d lysozyme w i t h DTT and/or heat treatment. In the presence o f DTT, b o t h low and h i g h temperature t r e a t m e n t r e s u l t e d i n t h e exposure o f a r o m a t i c r e s i d u e s (decrease i n peak i n t e n s i t y a t 760, 1005-1010, 1200 and 1555 c m " ) , decrease in ol - h e l i c a l content (930-950 cm" ), i n c r e a s e in antiparallel p-sheet structure (980-990 c m " ) , changes i n the d i s u l f i d e s t r e t c h i n g c o n f o r m a t i o n s (505-530 cm" ) (35) and i n c r e a s e in p e p t i d e backbone v i b r a t i o n s (C-C and C-N a t 1050-1150 cm" ) (37). The changes were more marked after h e a t i n g a t 75°C, and additional changes a t t h e h i g h e r temperature were noted i n t h e H-C-H bending v i b r a t i o n s a t 1445 cm" and t h e t r y p t o p h a n y l s i d e chain v i b r a t i o n s a t 1330-1340 cm" . In general, the changes noted i n t h e Raman s p e c t r a o f c o n c e n t r a t e d s o l u t i o n s and g e l s c o n f i r m t h o s e p r e v i o u s l y o b s e r v e d f o r i n c r e a s e d s u r f a c e h y d r o p h o b i c i t y determined on d i l u t e solutions. However, a d d i t i o n a l i n f o r m a t i o n on changes i n secondary s t r u c t u r e and more d e t a i l e d a n a l y s e s o f t h e changes i n a r o m a t i c and a l i p h a t i c residues are possible. Furthermore, a d i s t i n c t i n f l u e n c e o f p r o t e i n concentration was observed f o r e f f e c t o f DTT and heat t r e a t m e n t on r e s u l t i n g s u l f h y d r y l (SH) c o n t e n t o f t h e p r o t e i n . Whereas 3.4 and 6.0 moles SH/mole p r o t e i n resulted after h e a t i n g 0.5% lysozyme s o l u t i o n s c o n t a i n i n g 0.7 mM DTT a t 37°C and 80°C, r e s p e c t i v e l y , only 0.9 and 0.5 mole SH/mole, r e s p e c t i v e l y , r e s u l t e d by h e a t i n g 10% lysozyme solutions i n the presence of 10 mM DTT. A peak corresponding t o f r e e s u l f h y d r y l (2570-2580 cm" ) {35) c o u l d n o t be r e a d i l y d e t e c t e d i n t h e Raman spectrum o f t h e l a t t e r samples, b u t was d e t e c t e d i n t h e spectrum o f 10% lysozyme heated i n t h e p r e s e n c e o f 100 mM DTT, i n which c a s e 6 moles SH/mole were determined by Ellman's reagent. 1

1

1

1

1

1

1

1

Raman S p e c t r a o f I n t e r a c t i o n s o f P r o t e i n w i t h Corn o i l . Figure 5 shows t h e Raman s p e c t r a of DTT-treated lysozyme, c o r n o i l and emulsions formed by e i t h e r v o r t e x i n g or sonication of protein s o l u t i o n w i t h o i l ( o i l phase volume o r 0 o f 0.25). The Raman s p e c t r a i n t h i s r e g i o n c o r r e s p o n d s t o CH v i b r a t i o n a l bands o f b o t h protein and o i l components, as shown i n T a b l e I I (adapted from 3 5 ) .


LI-CHAN AND NAKAI

207


A Intensity 0.00

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Figure 6. Raman d i f f e r e n c e s p e c t r a o f (a) v o r t e x e d lysozyme, (b) vortexed DTT-lysozyme, ( c ) s o n i c a t e d lysozyme and (d) sonicated DTT-lysozyme and c o r n o i l emulsions, p r e p a r e d as d e s c r i b e d i n the legend t o F i g u r e 5. D i f f e r e n c e spectra were computed on the JASC0 NR-1100 data s t a t i o n , u s i n g s t a n d a r d s p e c t r a l wavenumbers o f 2940 and 3015 cm" f o r s u b t r a c t i o n o f protein and o i l s p e c t r a , r e s p e c t i v e l y , from t h e emulsion spectra. 1




211

non-equivalence in environment of the CH residues which is accentuated in the solid state. The complex pattern seen in the difference spectrum of the sonicated emulsions may thus be a reflection of interactions which lead to immobilization of the fatty acid chains. An additional feature which can be noted in these difference spectra is the highly negative slope at wavenumber above 3000 cm corresponding to the water band, which may be a result of changes in its Raman scattering coefficient and may suggest an immobilization of water molecules in these emulsions. 2

-1


Conclusions Molecular flexibility and the potential for hydrophobic interactions of proteins with other protein or lipid molecules at the oil-water interface are key structural factors in the ability of proteins to act as emulsifiers. While fluorescence probes can be used to study hydrophobic interactions of proteins in dilute solutions, Raman spectroscopy may be applied to investigate the interactions of proteins in more concentrated solutions, gels and in emulsions. Literature Cited 1. Borman, S. Chemical & Engineering News 1990, 68(8), 20-23. 2. Hansch, C.; Clayton, J.M. J. Pharm. Sci. 1973, 62, 1-21. 3. Nakai, S.; Li-Chan, E. Hydrophobic Interactions in Food Systems; CRC: Boca Raton, Florida, 1988. 4. Nakai, S. J. Agric. Food Chem. 1983, 31, 676-683. 5. Darling, D.F.; Birkett, R.J. In Food Emulsions and Foams; Dickinson, E., Ed.; Royal Society of Chemistry: London, 1987; Chapter 1. 6. Mangino, M.E. In Food Proteins; Kinsella, J.E.; Soucie, W.G., Eds.; Amer. Oil Chem. Soc.: Champaign, I1, 1989; Chapter 9. 7. Dickinson, E.; Stainsby, G. Colloids in Food; Applied Science:London, 1982. 8. Dickinson, E.; Stainsby, G., Eds. Advances in Food Emulsions and Foods; Elsevier Applied Science: London and NY, 1988. 9. Dickinson, E., Ed. Food Emulsions and Foams; Royal Society of Chemistry:London, 1987. 10. Becher, P. Encyclopaedia of Emulsion Technology, Volumes 1 and 2; Marcel Dekker: New York; 1983. 11. Kinsella, J.E. Crit. Rev. Food Sci. Nutr. 1976, 7, 219-280. 12. Graham, D.E.; Phillips, M.C. In Foams; Akers, R.J., Ed.; Academic: New York; 1976, pp. 237-255. 13. Graham, D.E.; Phillips, M.C. J. Colloid Interfac. Sci. 1979, 70, 403-414; 415-426; 427-439. 14. Graham, D.E.; Phillips, M.C. J. Colloid Interfac. Sci. 1980, 76, 227-339; 240-249. 15. Bigelow, C.C. J. Theoret. Biol. 1967, 16, 187-211. 16. Keshavarz, E.; Nakai, S. Biochim. Biophys. Acta 1979, 576, 269-279. 17. Kato, A.; Nakai, S. Biochim. Biophys. Acta 1980, 624, 13-20. 18. Dickinson, E.; Murray, B.S.; Stainsby, G. In Advances in Food Emulsions and Foams; Dickinson, E.; Stainsby, G., Eds.; Elsevier Applied Science:London, 1988; Chapter 4. 19. Shimizu, M.; Kamiya, T.; Yamauchi, K. Agric.Biol. Chem. 1981, 45, 2491-2496.


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20.

Izmailova, V.N.; Yampol'skaya, B.P.; Lapina, G.P.; Sorokin, M.M. Colloid J. USSR 1982, 44, 195. Cited in Reference 18. 21. Kato, A.; Tsutsui, N.; Matsudomi, N.; Kobayashi, K.; Nakai, S. Agric. Biol. Chem. 1981, 45, 2755-2760. 22. Kato, A.; Osako, Y.; Matsudomi, N.; Kobayashi, K. Agric. Biol. Chem. 1983, 47, 33-37. 23. Kato, A.; Komatsu, K.; Fujimoto, K.; Kobayashi, K. J. Agric. Food Chem. 1985, 33, 931-934. 24. Kato, A.; Yamaoka, H.; Matsudomi, N.; Kobayashi, K. J. Agric.Food Chem. 1986, 34, 370-372. 25. Kim, S.H.; Kinsella, J.E. J. Agric. Food Chem. 1986, 34, 623-7. 26. Kim, S.H.; Kinsella, J.E. J. Food Sci. 1987, 52, 128-131. 27. Nakamura, R.; Mizutani, R.; Yano, M.; Hayakawa, S. J. Agric. Food Chem. 1988, 36, 729-732. 28. Haque, Z.; Kinsella, J.E. J. Food Sci. 1988, 53, 416-420. 29. Li-Chan, E.; Nakai, S. In Food Proteins; Kinsella, J.E.; Soucie, W.G., Eds.; Amer. Oil Chem. Soc.:Champaign, I1, 1989; Chapter 14. 30. Hailing, P.J. CRC Crit.Rev.Food Sci. Nutr. 1981, 15, 155-203. 31. Nakai, S.; Li-Chan, E. International Chemical Congress of Pacific Basin Societies; Honolulu, 1989; Abstract 162. 32. Przybycien, T.M.; Bailey, J.E. Biochim. Biophys. Acta 1989, 995, 231-245. 33. Byler, D.M.; Farrell, H.M. Jr.; Susi, H. J. Dairy Sci. 1988, 71, 2622-2629. 34. Berjot, M.; Marx, J.; Alix, J.P. J. Raman Spec. 1987, 18, 289-300. 35. Tu, A.T. In Advances in Spectroscopy Vol. 13; Clark, R.J.H.; Hester, R.E., Eds.; Wiley: New York; 1986; Chapter 2. 36. Carey, P.R. Biochemical Applications of Raman and Resonance Raman Spectroscopies; Academic Press: New York; 1982. 37. Alix, A.J.P.; Bernard, L.; Manfait, M., Eds. Spectroscopy of Biological Molecules; Wiley: New York; 1985. 38. Spiro, T.G., Ed. Biological Applications of Raman Spectroscopy Volume 1; Wiley: New York; 1987. 39. Bertoluzza, M.; Fagnano, C.; Monti, P. Spectroscopy of Biological Molecules. State-of-the-Art. Proc. 3rd Eur. Conf., Esculapio: Bologna; 1989. 40. Painter, P.C. In Food Analysis: Principles and Techniques; Volume 2; Gruenwedel, D.W.; Whitaker, J.R., Eds.; Marcel Dekker: New York; 1984; Chapter 11. 41. Lord, R.C.; Yu, N.-T. J. Mol. Biol. 1970, 50, 509-524. 42. Chen, M.C.; Lord, R.C.; Mendelsohn, R. Biochim. Biophys. Acta 1973, 328, 252-260. 43. Chen, M.C.; Lord, R.C.; Mendelsohn, R. J. Amer. Chem. Soc. 1974, 96, 3038-3042. 44. Clark, A.H.; Lee-Tuffnell, C.D. In Functional Properties of Food Macromolecules; Mitchell; J.R.; Ledward, D.A., Eds.; Elsevier Applied Science: New York, 1986; Chapter 5. 45. Verma, S.P.; Wallach, D.F.H. Biochim. Biophys. Acta 1977, 486, 217-227. 46. Verma, S.P.; Wallach, D.F.H. Biochem. Biophys. Res. Comm. 1977, 74, 473-479. RECEIVED August 16, 1990


Microemulsions and Emulsions in Foods - American Chemical Society

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