Excellent Emulsifying Properties of ProteinâDextran Conjugates

Chapter 16 Excellent Emulsifying Properties of Protein—Dextran Conjugates Akio Kato and Kunihiko Kobayashi

Downloaded by UNIV OF ALABAMA on March 17, 2016 | http://pubs.acs.org Publication Date: December 26, 1991 | doi: 10.1021/bk-1991-0448.ch016

Department of Agricultural Chemistry, Yamaguchi University, Yamaguchi 753, Japan

Protein-dextran conjugates having excellent emulsifying properties have been developed by coupling proteins to CNBr-activated dextran or by linking proteins with dextran through naturally occurring Maillard reaction. The emulsifying properties of both protein-dextran conjugates were much higher than native proteins. Furthermore, these conjugates were superior to commercial emulsifiers from sucrose-fatty acid esters and polyglycerin esters, especially at high salt concentrations and at acidic pH. In addition, the emulsifying properties of protein-dextran conjugates were greatly enhanced by preheating at 100°C. Thus, it was suggested that protein-dextran conjugates may be useful as a macromolecular emulsifier for food or drug application where conditions of acidic or alkaline pH and heat-treatment are required. Proteins have unique surface properties due to their large molecular weight and their amphiphilic properties. Proteins are generally unstable to heating for the pasteurization, to shaking and homogenization for the preparation of emulsion. Furthermore, some proteins coagulate during emulsifying process as a result of surface denaturation. Thus, many studies on the chemical and enzymatic modification of proteins have attempted to improve the emulsifying properties of proteins. We found that a soluble protein-dextran conjugate prepared by coupling proteins to cyanogen-bromide activated dextran exhibited emulsifying properties which were superior to commercial emulsifiers (1). This result suggested that a covalently linked protein-polysaccharide conjugate could be used to make new functional biopolymers. Since protein-polysaccharide conjugates formed by covalent attachment enhanced the stability and solubility of proteins, this technique for conjugate formation could be utilized for medical and food applications, if the safety of the conjugates were ensured. For these applications, the use of chemical reagents to make conjugates should be avoided. Here, we describe a O097-6156/91/0448-O213$06.00/0 © 1991 American Chemical Society El-Nokaly and Cornell; Microemulsions and Emulsions in Foods ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

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safe p r o t e i n - p o l y s a c c h a r i d e conjugate prepared without using chemical r e a g e n t s . One o f t h e most p r o m i s i n g way t o c o n j u g a t e i s t h r o u g h n a t u r a l l y o c c u r r i n g M a i l l a r d r e a c t i o n . S i n c e d e x t r a n has o n l y one a c t i v e reducing-end p e r molecule, the formation o f p r o t e i n - d e x t r a n c o n j u g a t e s i s p o s s i b l e by t h e M a i l l a r d r e a c t i o n . As e x p e c t e d , p r o t e i n - d e x t r a n c o n j u g a t e s were c o v a l e n t l y a t t a c h e d by t h e M a i l l a r d reaction t h r o u g h t h e p r o t e i n amino groups and t h e r e d u c i n g - e n d i n d e x t r a n . T h i s work d e s c r i b e s t h e p r o p e r t i e s o f t h e s e p r o t e i n - d e x t r a n conjugates.


MATERIALS AND METHODS M a t e r i a l s . D e x t r a n ( m o l e c u l a r w e i g h t , 60,000-90,000) was from Wako Pure C h e m i c a l I n d u s t r i e s , and S e p h a c r y l S-300 was from Pharmacia LKB. Ovalbumin was i s o l a t e d and r e c r y s t a l l i z e d f i v e t i m e s from f r e s h egg w h i t e w i t h sodium s u l f a t e ( 2 ) . Lysozyme was p r e p a r e d from f r e s h egg w h i t e by a d i r e c t c r y s t a l l i z a t i o n method (3) and r e c r y s t a l l i z e d f i v e times. S u n S o f t SE-11 and Q-18S were s u p p l i e d from T a i y o Kagaku Co.(Japan). P r e p a r a t i o n o f o v a l b u m i n - d e x t r a n c o n j u g a t e by C N B r - a c t i v a t e d dextran. Ovalbumin-dextran c o n j u g a t e was p r e p a r e d by t h e method o f M a r s h a l l and R a b i n o w i t z ( 4 ) . To a s t i r r e d s o l u t i o n o f d e x t r a n (2.5g) i n water (250ml), a d j u s t e d t o pH 10.7 w i t h 500mM sodium h y d r o x i d e s o l u t i o n , cyanogen bromide (0.625g) was added, f o l l o w e d by a second a d d i t i o n o f cyanogen bromide (0.625g), 30 min l a t e r . The pH was m a i n t a i n e d a t 10.7 d u r i n g t h i s p r o c e s s by a d d i t i o n o f sodium h y d r o x i d e s o l u t i o n (500mM). T h i r t y minutes a f t e r t h e second a d d i t i o n o f cyanogen bromide, t h e pH was a d j u s t e d t o 9.0 by a d d i t i o n o f lOOmM h y d r o c h l o r i c a c i d s o l u t i o n . A f t e r d i a l y s i s a t 4 °C f o r 2 h a g a i n s t 4 l i t e r s o f sodium c a r b o n a t e s o l u t i o n , pH 9.0 ( p r e p a r e d by a d d i t i o n o f 1.0 M sodium c a r b o n a t e s o l u t i o n t o d i s t i l l i z e d water u n t i l t h e pH r e a c h e d 9.0), ovalbumin (0.50g) was added. The pH was m a i n t a i n e d a t 9.0 d u r i n g a d d i t i o n o f ovalbumin, by a d d i t i o n o f sodium c a r b o n a t e s o l u t i o n (200mM). C o u p l i n g o f ovalbumin t o cyanogen b r o m i d e - a c t i v a t e d d e x t r a n was then a l l o w e d t o p r o c e e d d u r i n g 12 h a t 4°C. A f t e r t h i s p e r i o d , t h e s o l u t i o n was d i a l y z e d f o r 2 h a t room temperature a g a i n s t 4 l i t e r s o f sodium c a r b o n a t e s o l u t i o n ( p r e p a r e d as above), t h e n 20 ml o f g l y c i n e s o l u t i o n (lOOmg/ml) was added. A f t e r s t a n d i n g f o r an a d d i t i o n a l 12 h a t 4°C, t h e p r o d u c t was l y o p h i l i z e d . P r e p a r a t i o n o f o v a l b u m i n - d e x t r a n c o n j u g a t e by M a i l l a r d r e a c t i o n . F r e e z e - d r i e d o v a l b u m i n - d e x t r a n m i x t u r e s i n t h e weight r a t i o o f 1:5 were s t o r e d a t 60 °C and 65 % r e l a t i v e h u m i d i t y f o r 3 weeks ( 5 ) . To f u r t h e r p u r i f y ovalbumin-dextran conjugate, g e l f i l t r a t i o n o f the c o n j u g a t e was p e r f o r m e d on a column (70 x 3 cm) o f S e p h a c r y l S-300. E l u t i o n was c a r r i e d o u t w i t h 50 mM a c e t a t e b u f f e r , pH 5.0, c o n t a i n i n g 10 mM sodium c h l o r i d e , and 3.0 ml f r a c t i o n s were c o l l e c t e d . The c o n j u g a t e peak ( f r a c t i o n number 26 t o 36) was combined t o g e t h e r , d i a l y z e d a g a i n s t d e i o n i z e d water and l y o p h i l i z e d . Determination o f the m o l e c u l a r weight o f ovalbumin-dextran c o n j u g a t e . 0.1% o v a l b u m i n - d e x t r a n c o n j u g a t e s o l u t i o n i n 100 mM sodium phosphate b u f f e r (pH 7.0) c o n t a i n i n g 0.1 % SDS was a p p l i e d t o a h i g h performance g e l chromatography system, employing a TSK g e l G3000SW g e l column(Toyo Soda Co., 0.75 x 60 cm) a t a f l o w r a t e o f 0.3 ml/min. E f f l u e n t from t h e column was m o n i t o r e d w i t h a low-angle l a s e r l i g h t s c a t t e r i n g photometer (LS-8,Toyo Soda Co.) and t h e n w i t h a p r e c i s i o n

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


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d i f f e r e n t i a l r e f r a c t o m e t e r ( R I - 8 , T o y o Soda C o . ) . The m o l e c u l a r w e i g h t o f o v a l b u m i n - d e x t r a n c o n j u g a t e was e s t i m a t e d from the r a t i o o f t o t a l a r e a i n the peak o f a low-angle l a s e r l i g h t s c a t t e r i n g photometer(LS) t o t h a t o f a r e f r a c t o m e t e r (RI) by the method o f T a k a g i and H i z u k u r i (6). Measurement o f e m u l s i f y i n g p r o p e r t i e s . Emulsifying properties were measured by the method o f P e a r c e and K i n s e l l a ( 7 ) . To p r e p a r e e m u l s i o n s , 1.0 ml o f c o r n o i l and 3 ml o f p r o t e i n s o l u t i o n i n 0.1 M phosphate b u f f e r , pH 7.4, were shaken t o g e t h e r and homogenized i n an U l t r a T u r r a x (Hansen & Co., West Germany) a t 12,000 rpm f o r l m i n a t 20 °C. 50 u l o f e m u l s i o n was taken from the bottom o f the c o n t a i n e r a f t e r d i f f e r e n t times and d i l u t e d w i t h 5ml o f 0.1 % sodium d o d e c y l sulfate solution. The absorbance o f d i l u t e d e m u l s i o n was then determined a t 500nra. The e m u l s i f y i n g a c t i v i t y was d e t e r m i n e d from the absorbance measured immediately a f t e r e m u l s i o n f o r m a t i o n . The e m u l s i o n s t a b i l i t y was e s t i m a t e d by m e a s u r i n g the h a l f - t i m e o f the t u r b i d i t y of emulsion. SDS-slab p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s . SDS-slab p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s was c a r r i e d out by the method o f Laemmli(8) u s i n g 10 % a c r y l a m i d e s e p a r a t i n g g e l , and 3 % s t a c k i n g g e l c o n t a i n i n g 0.1 % SDS. P r o t e i n samples ( 2 0 u l , 0.1 % ) , were p r e p a r e d i n T r i s - g l y c i n e b u f f e r , pH 8.8, c o n t a i n i n g 1 % SDS and 1 % m e r c a p t o e t h a n o l . E l e c t r o p h o r e s i s were c a r r i e d o u t a t c o n s t a n t c u r r e n t o f 10 mA f o r 5 h u s i n g e l e c t r o p h o r e t i c b u f f e r o f T r i s - g l y c i n e c o n t a i n i n g 0.1 % SDS. The g e l s h e e t s were s t a i n e d f o r p r o t e i n s and c a r b o h y d r a t e s w i t h Coomassie b l u e G-250 and F u c h s i n , r e s p e c t i v e l y . Measurement o f lysozyme a c t i v i t y . Both l y s i s and h y d r o l y s i s were measured u s i n g M i c r o c o c c u s l y s o d e i k t i c u s and g l y c o l c h i t i n as s u b s t r a t e s , r e s p e c t i v e l y . L y t i c a c t i v i t y was e s t i m a t e d from the d e c r e a s e i n the t u r b i d i t y o f M i c r o c o c c u s l y s o d e i k t i c u s c e l l s u s p e n s i o n and the h y d r o l y s i s a c t i v i t y was c a l c u l a t e d from the i n c r e a s e i n the r e d u c i n g group o f h y d r o l y z e d N - a c e t y l g l u c o s a m i n e (9) . D e t e r m i n a t i o n o f amino groups i n p r o t e i n - d e x t r a n c o n j u g a t e . The c o n t e n t o f f r e e amino groups i n the c o n j u g a t e was d e t e r m i n e d by the method o f Haynes e t a l . ( 1 0 ) u s i n g a s p e c i f i c r e a g e n t f o r amino groups, t r i n i t r o b e n z e n e s u l f o n a t e . RESULT & DISCUSSION A scheme f o r the p r e p a r a t i o n and the b i n d i n g mode o f p r o t e i n d e x t r a n c o n j u g a t e s i s shown i n F i g . l . When p r o t e i n i s c o u p l e d w i t h C N B r - a c t i v a t e d d e x t r a n , p o l y m e r i z e d networks a r e formed between f u n c t i o n a l amino groups i n p r o t e i n and numerous a c t i v a t e d hydroxy1 groups c o n t a i n e d i n d e x t r a n m o l e c u l e . On the o t h e r hand, when powdered p r o t e i n - d e x t r a n m i x t u r e i s s t o r e d a t 60 °C and a t 65-79 % r e l a t i v e h u m i d i t y , c o n d i t i o n s o p t i m a l f o r the M a i l l a r d r e a c t i o n , o n l y one o r two moles o f d e x t r a n l i n k p e r moles o f p r o t e i n . Because t h e r e i s o n l y one r e d u c i n g - e n d group p e r d e x t r a n m o l e c u l e , d e x t r a n and p r o t e i n r e a c t w i t h o u t the f o r m a t i o n o f a network s t r u c t u r e . E v i d e n c e f o r t h i s h y p o t h e s i s was o b t a i n e d from m e a s u r i n g the m o l e c u l a r w e i g h t o f o v a l b u m i n - d e x t r a n c o n j u g a t e s . The m o l e c u l a r w e i g h t o f c o n j u g a t e s p r e p a r e d from C N B r - a c t i v a t e d d e x t r a n were 250,000-2,000,000, whereas t h o s e p r e p a r e d from the M a i l l a r d r e a c t i o n were 130,000 - 230,000 (Table I ) .



F i g u r e 1. Scheme f o r t h e b i n d i n g r e a c t i o n ( l e f t ) and t h e b i n d i n g mode ( r i g h t ) o f o v a l b u m i n - d e x t r a n c o n j u g a t e s t h r o u g h CNBra c t i v a t e d d e x t r a n (A) and M a i l l a r d r e a c t i o n ( B ) . D o t t e d a r e a s d e s i g n a t e p r o t e i n m o l e c u l e s whereas t h e branched s o l i d c i r c l e s designate dextran molecule.


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Protdn-Dextran Conjugates


16. KATO AND KOBAYASHI


217

218

MICROEMULSIONS AND EMULSIONS IN FOODS Table I.

M o l e c u l a r Weight D i s t r i b u t i o n o f Ovalbumin-Dextran C o n j u g a t e s E s t i m a t e d by Low-Angle L a s e r L i g h t S c a t t e r i n g Technique

Ovalbumin-dextran conjugates


CNBr-activated dextran Maillard reaction

M o l e c u l a r weight distribution 250,000 - 2,000,000 130,000 230,000

S D S - p o l y a c r y l a m i d e g e l e l e c t r o p h o r e t i c p a t t e r n s l e d t o the same c o n c l u s i o n ( F i g . 2 ) . The e l e c t r o p h o r e t i c p a t t e r n o f t h e ovalbumind e x t r a n c o n j u g a t e p r e p a r e d by the M a i l l a r d r e a c t i o n shows a s i n g l e band f o r p r o t e i n and c a r b o h y d r a t e s t a i n s n e a r the boundary between s t a c k i n g and s e p a r a t i n g g e l s , whereas the o v a l b u m i n - d e x t r a n c o n j u g a t e p r e p a r e d from C N B r - a c t i v a t e d d e x t r a n showed a b r o a d spectrum o f h i g h m o l e c u l a r weight bands i n the s t a c k i n g g e l , i n d i c a t i n g a p o l y d i s p e r s e d high molecular d i s t r i b u t i o n . As shown i n F i g . 3 - 5 , the e m u l s i f y i n g p r o p e r t i e s o f p r o t e i n d e x t r a n c o n j u g a t e p r e p a r e d from C N B r - a c t i v a t e d d e x t r a n were compared w i t h t h o s e o f commercial e m u l s i f i e r s . E m u l s i o n t u r b i d i t y ( o r d i n a t e ) i s p l o t t e d a g a i n s t the s t a n d i n g time a f t e r e m u l s i o n f o r m a t i o n ( a b s c i s s a ) . The commercial e m u l s i f i e r s were S u n S o f t SE-11 and Q-18S. S u n s o f t SE11 i s a s u c r o s e - f a t t y a c i d e s t e r whereas Q-18S i s a p o l y g l y c e r i n ester. E m u l s i f y i n g p r o p e r t i e s were compared i n two e m u l s i o n systems b o t h o f which had e q u a l volumes o f water and o i l ( F i g . 3 ) . In one system the e m u l s i f i e r was added t o the water phase whereas i n the o t h e r system i t was added t o the o i l phase. The o v a l b u m i n - d e x t r a n c o n j u g a t e was comparable t o the commercial e m u l s i f i e r s and was found s u i t a b l e f o r b o t h e m u l s i o n system. Apparently, ovalbumin-dextran c o n j u g a t e s can be used f o r e i t h e r o i l i n water o r water i n o i l e m u l s i o n s . In F i g . 4 i s shown the e f f e c t o f s a l t on the e m u l s i f y i n g p r o p e r t y o f the o i l i n water e m u l s i o n . 10 % NaCl was c o n t a i n e d i n the water phase. The r e l a t i v e e m u l s i f y i n g a c t i v i t y , which was d e t e r m i n e d from the e m u l s i o n t u r b i d i t y i m m e d i a t e l y a f t e r e m u l s i o n f o r m a t i o n , o f o v a l b u m i n - d e x t r a n c o n j u g a t e , SE-11 and Q-18S were 0.734, 0.130 and 0.077, r e s p e c t i v e l y . Comparison o f the commercial e m u l s i f i e r s w i t h the o v a l b u m i n - d e x t r a n c o n j u g a t e r e v e a l e d t h a t the c o n j u g a t e had b e t t e r e m u l s i f y i n g p r o p e r t i e s i n the p r e s e n c e o f 10 % N a C l . In F i g . 5 i s shown the e f f e c t o f a c i d on the e m u l s i f y i n g p r o p e r t y o f the o i l i n water e m u l s i o n . The pH o f the water phase was lowered t o 2.3 w i t h 1 % c i t r i c a c i d . In the p r e s e n c e o f a c i d i c s a l t , the o v a l b u m i n - d e x t r a n c o n j u g a t e was s t a b l e . The v a l u e o f r e l a t i v e e m u l s i f y i n g a c t i v i t y o f o v a l b u m i n - d e x t r a n c o n j u g a t e , SE-11 and Q-18S were 0.630, 0.150 and 0.235, r e s p e c t i v e l y . S t a b l e e m u l s i f y i n g p r o p e r t i e s i n the a c i d i c pH r e g i o n a r e i m p o r t a n t f o r i n d u s t r i a l a p p l i c a t i o n , i n t h a t the e m u l s i f y i n g p r o p e r t i e s o f most commercial e m u l s i f i e r s a r e g r e a t l y r e d u c e d i n the low pH r e g i o n . In F i g 6-8, the e m u l s i f y i n g p r o p e r t i e s o f o v a l b u m i n - d e x t r a n c o n j u g a t e p r e p a r e d by b o t h the M a i l l a r d r e a c t i o n and C N B r - a c t i v a t e d d e x t r a n a r e compared. In F i g . 6 a r e compared the e m u l s i f y i n g p r o p e r t i e s o f v a r i o u s o v a l b u m i n - d e x t r a n c o n j u g a t e s i n 0.1 M phosphate b u f f e r , pH 7.4. The e m u l s i f y i n g a c t i v i t y ( t h e t u r b i d i t y a t 0 time) and e m u l s i o n s t a b i l i t y ( h a l f - l i f e o f e m u l s i o n t u r b i d i t y ) o f ovalbumin-



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F i g u r e 2. S D S - P o l y a c r y l a m i d e g e l e l e c t r o p h o r e t i c p a t t e r n s o f o v a l b u m i n - d e x t r a n c o n j u g a t e s , l a n e 1, ovalbumin s t o r e d a t 60 °C f o r 3 weeks; l a n e 2,ovalbumin-dextran c o n j u g a t e o b t a i n e d by d r y h e a t i n g a t 60°C f o r 3 weeks; l a n e 3, o v a l b u m i n - d e x t r a n c o n j u g a t e p u r i f i e d from l a n e 2; l a n e 4,ovalbumin-dextran c o n j u g a t e p r e p a r e d by C N B r - a c t i v a t e d d e x t r a n . ( R e p r o d u c e d w i t h p e r m i s s i o n f r o m R e f . 5. C o p y r i g h t 1990 J a p a n S o c i e t y f o r B i o s c i e n c e , Biotechnology, and Agrochemistry.)



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TIME, min F i g u r e 3. E m u l s i f y i n g p r o p e r t i e s o f o v a l b u m i n - d e x t r a n c o n j u g a t e and commercial e m u l s i f i e r i n t h e e m u l s i o n systems where e m u l s i f i e r were added t o water phase (A) and o i l phase ( B ) . • , 0.1% o v a l b u m i n - d e x t r a n c o n j u g a t e ; • , 0.1% S u n S o f t SE-11; A , 0.1% S u n s o f t Q-18S. (Reproduced f r o m R e f . 1. C o p y r i g h t 1 9 8 8 A m e r i c a n Chemical Society.)



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TIME, min F i g u r e 4. E f f e c t o f s a l t on t h e e m u l s i f y i n g p r o p e r t i e s o f o v a l b u m i n - d e x t r a n c o n j u g a t e and commercial e m u l s i f i e r s i n o i l i n water emulsion(O/W = 1/3), w i t h 10 % NaCl c o n t a i n e d i n t h e water p h a s e . # , 0.1% o v a l b u m i n - d e x t r a n c o n j u g a t e ; • , 0.1% S u n S o f t SE-11; A , 0.1% S u n S o f t Q-18S. (Reproduced from R e f . Chemical S o c i e t y . )

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1

1—i II

TIME, min F i g u r e 5. E f f e c t o f a c i d on t h e e m u l s i f y i n g p r o p e r t i e s o f o v a l b u m i n - d e x t r a n c o n j u g a t e and commercial e m u l s i f i e r s i n o i l i n water e m u l s i o n (0/W = 1/3) w i t h 1 % c i t r i c acid contained i n the water phase, pH 2.3. # , 0.1% o v a l b u m i n - d e x t r a n c o n j u g a t e ; • , 0.1% S u n S o f t SE-11; A , 0.1% S u n s o f t Q-18S. (Reproduced f r o m R e f . 1. C o p y r i g h t Chemical Society.)

1988

American





V . . a 1 ? t 1 2

y

. y 9

TIME, min

t J II

F i g u r e 6. E m u l s i f y i n g p r o p e r t i e s o f o v a l b u m i n - d e x t r a n conjugate o b t a i n e d by d r y - h e a t i n g a t 60 °C f o r 3 weeks. • , f a c t i o n e l u t e d i n v o i d volume on a g e l f i l t r a t i o n w i t h S e p h a c r y l S-300; • , fraction e l u t e d l a t e r v o i d volume on a S e p h a c r y l S-300; A , o v a l b u m i n - d e x t r a n c o n j u g a t e p r e p a r e d by C N B r - a c t i v a t e d d e x t r a n . • , m i x t u r e o f ovalbumin w i t h d e x t r a n ( c o n t r o l ) . ( R e p r o d u c e d w i t h p e r m i s s i o n f r o m R e f . 5. C o p y r i g h t 1990 J a p a n S o c i e t y f o r B i o s c i e n c e , B i o t e c h n o l o g y , and Agrochemistry.)



224


Q1 « « i • 2

I 5

L_— 10

TIME, min F i g u r e 7. E f f e c t s o f v a r i o u s pH on t h e e m u l s i f y i n g p r o p e r t i e s o f o v a l b u m i n - d e x t r a n c o n j u g a t e o b t a i n e d by d r y - h e a t i n g a t 60 ° C f o r 3 weeks. • , pH 7.4 (1/15 M phosphate b u f f e r ) ; • , pH 10 (1/15 M c a r b o n a t e b u f f e r ) ; • , pH 3 (1/15 M c i t r a t e b u f f e r ) . ( R e p r o d u c e d w i t h p e r m i s s i o n f r o m R e f . 5. C o p y r i g h t 1990 J a p a n S o c i e t y f o r B i o s c i e n c e , Biotechnology, and A g r o c h e m i s t r y . )



16.


KATO AND KOBAYASHI

0

I 0

i

i 2

i

i 5

225

i 10

TIME , min F i g u r e 8. E f f e c t s o f h e a t i n g sample on the e m u l s i f y i n g p r o p e r t i e s o f o v a l b u m i n - d e x t r a n c o n j u g a t e o b t a i n e d by d r y - h e a t i n g a t 60 ° C f o r 3 weeks. • , unheated o v a l b u m i n - d e x t r a n c o n j u g a t e ; 0, o v a l b u m i n - d e x t r a n c o n j u g a t e h e a t e d a t 100 ° C . ( R e p r o d u c e d w i t h p e r m i s s i o n f r o m R e f . 5. Copyright 1990 J a p a n S o c i e t y f o r B i o s c i e n c e , Biotechnology, and Agrochemistry.)




226

d e x t r a n c o n j u g a t e s were h i g h e r than t h o s e o f n a t i v e ovalbumin. The e m u l s i o n s t a b i l i t y o f the o v a l b u m i n - d e x t r a n c o n j u g a t e o b t a i n e d by M a i l l a r d r e a c t i o n was h i g h e r than t h a t o f the c o n j u g a t e p r e p a r e d from C N B r - a c t i v a t e d d e x t r a n . A s l i g h t d i f f e r e n c e was o b s e r v e d between crude and p u r i f i e d c o n j u g a t e s p r e p a r e d by the M a i l l a r d r e a c t i o n . In F i g . 7 i s shown the e f f e c t o f v a r i o u s pH on the e m u l s i f y i n g p r o p e r t i e s of t h e o v a l b u m i n - d e x t r a n c o n j u g a t e p r e p a r e d by the M a i l l a r d r e a c t i o n . The h i g h e m u l s i f y i n g p r o p e r t i e s o f o v a l b u m i n - d e x t r a n c o n j u g a t e were m a i n t a i n e d as low as pH 3, and were f u r t h e r improved a t pH 10. In F i g . 8 i s shown the e f f e c t o f h e a t i n g the sample on the e m u l s i f y i n g p r o p e r t i e s o f t h e o v a l b u m i n - d e x t r a n c o n j u g a t e p r e p a r e d by M a i l l a r d r e a c t i o n . The e m u l s i f y i n g p r o p e r t i e s o f o v a l b u m i n - d e x t r a n c o n j u g a t e were g r e a t l y i n c r e a s e d by p r e h e a t i n g the c o n j u g a t e a t 100 °C i n t h a t no i n s o l u b l e m a t t e r was o b s e r v e d . Thus, a n o t h e r advantage o f t h e o v a l b u m i n - d e x t r a n c o n j u g a t e i s i t s h e a t r e s i s t a n c e which e n a b l e s i t to be p a s t e u r i z e d . We have shown t h a t o v a l b u m i n - d e x t r a n c o n j u g a t e s p r e p a r e d by the M a i l l a r d r e a c t i o n and by C N B r - a c t i v a t e d d e x t r a n had e x c e l l e n t e m u l s i f y i n g p r o p e r t i e s . Because o f the p r e p a r a t i o n w i t h o u t c h e m i c a l r e a g e n t s , the c o n j u g a t e formed by the M a i l l a r d r e a c t i o n i s more s u i t a b l e f o r i n d u s t r i a l a p p l i c a t i o n s t h a n the c o n j u g a t e formed from C N B r - a c t i v a t e d d e x t r a n . The c o n j u g a t e p r e p a r e d w i t h o u t c h e m i c a l s can be used as an e m u l s i f i e r and as a p r o t e i n f o o d a d d i t i v e w i t h h e a t stability. Another p r o t e i n - d e x t r a n conjugate with e x c e l l e n t f u n c t i o n a l p r o p e r t i e s i s t h a t o f lysozyme and d e x t r a n which were n a t u r a l l y l i n k e d t h r o u g h the M a i l l a r d r e a c t i o n . The d a t a i n F i g . 9 r e v e a l e d the p r o g r e s s i v e l y s o z y m e - d e x t r a n c o n j u g a t i o n d u r i n g the c o u r s e o f a M a i l l a r d r e a c t i o n o v e r a p e r i o d o f 3 weeks. As shown i n F i g . 1 0 , e m u l s i f y i n g p r o p e r t i e s o f l y s o z y m e - d e x t r a n a r e g r e a t l y i n c r e a s e d as M a i l l a r d r e a c t i o n proceeds. Lysozyme a c t i v i t y was c o n s i d e r a b l y r e s t o r e d i n l y s o z y m e - d e x t r a n c o n j u g a t e ( T a b l e I I ) and the h e a t s t a b i l i t y was g r e a t l y enhanced by c o n j u g a t e f o r m a t i o n (Table I I I ) . Table I I .

Enzymatic A c t i v i t y o f N a t i v e Lysozyme and Lysozyme-Dextran C o n j u g a t e P r e p a r e d by M a i l l a r d Reaction

Lysozyme

Substrate Glycol chitin M. l y s o d e i k t i c u s 100 % 100 % 32 % 80 %

Native Conjugate

Table I I I .

Heat S t a b i l i t y o f Lysozyme and Lysozyme-Dextran Conjugate P r e p a r e d by M a i l l a r d R e a c t i o n

Lysozyme Native Conjugate

Half l i f e

(min)

15 40

H a l f l i f e i s r e p r e s e n t e d as the time when l y t i c a c t i v i t y lowered t o 50 % on h e a t i n g a t 100 °C.



16.

KATO AND KOBAYASHI


FRACTION NUMBER (3ml/tube) F i g u r e 9. E l u t i o n p a t t e r n s i n 50 mM a c e t a t e b u f f e r (pH 5) on a S e p h a c r y l S-300 column o f lysozyme-dextran m i x t u r e s i n c u b a t e d a t 60°C f o r 0-3 weeks under 78 % r e l a t i v e h u m i d i t y . • • , absorbance a t 280 nm ( f o r p r o t e i n ) ; o — o , absorbance a t 470 nm t o f o l l o w the c o l o r development by p h e n o l - s u l f a t e method ( f o r carbohydrate).


in


228


TIME, min F i g u r e 10. E m u l s i f y i n g p r o p e r t i e s o f l y s o z y m e - d e x t r a n c o n j u g a t e p u r i f i e d by g e l f i l t r a t i o n on S e p h a c r y l S-300. © , 0 week; O , i n c u b a t e d a t 60 °C f o r 1 week; • , i n c u b a t e d a t 60°C f o r 2 weeks; 3 , i n c u b a t e d a t 60 °C f o r 3 weeks.




229

The binding mode was studied in detail- Two free amino groups were found to be reduced in lysozyme-dextran conjugate(Table IV). This suggests that two moles of dextran attach to one mole of lysozyme. This binding mode is supported from the data in the binding ratio of dextran to lysozyme, as shown in Table V. Table IV. Contents of Free Amino Group in Lysozyme-Dextran Conjugate Prepared by Maillard Reaction


Lysozyme Native Conjugate

Numbers of free amino groups per mole 7.0 4.8

Table V. Binding Ratio of Dextran to Lysozyme in Conjugate Prepared by Maillard Reaction

Weight ratio Molar ratio

Lysozyme

Dextran

1 1

11.3 2.1

Thus limited binding of dextran to lysozyme may result from the steric hindrance because of macromolecular interaction between them. This is also the case of ovalbumin-dextran conjugate (5). Lysozymedextran conjugate without the use of chemicals can be potentially used for food additive possessing bifunctional properties, either emulsifier or antimicrobial reagent. As mentioned above, the protein-dextran conjugates can be useful for the industrial application as a new functional emulsifier that is soluble, macromolecular, heat-stable, and stable in acid or in high salt concentration. Finally, it may be possible to make other functional protein food additives by selecting appropriate combinations of proteins with polysaccharides using the reactions described here. LITERATURE CITED 1. Kato,A.; Murata,K.; Kobayashi,K. J. Agric. Food Chem. 1988,36, 421-425. 2. Kekwick,R.A.; Cannan,R.K. Biochem. J. 1936, 30, 277-280. 3. Alderton,G.; Fevold,H.L. J. Biol. Chem. 1946, 164, 1-5. 4. Marshall,J.J.;Rabinowitz,M.L. J. Biol. Chem. 1976, 251, 1081-1087. 5. Kato,A.; Sasaki,Y.; Furuta,R.; Kobayashi,K. Agric. Biol. Chem. 1990, 54, 107-112. 6. Takagi,T. Hizukuri,S. J. Biochem. 1984, 95, 1459-1467. 7. Pearce,K.N.;Kinsella,J.E. J. Agric. Food Chem. 1978, 26, 716-723. 8. Laemmli,U.K. Nature 1970, 227, 680-685. 9. Imoto,T.; Yagishita,K. Agric. Biol. Chem. 1971, 35, 1154-1156. 10. Haynes,R.; Osuga,D.T.; Feeney,R.E. Biochemistry 1967, 6, 541-548. RECEIVED August 16, 1990


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