For Peer Review · 2019-04-19 · For Peer Review 3 1 Mayonnaise is an acidic oil-in-water emulsion...
Transcript of For Peer Review · 2019-04-19 · For Peer Review 3 1 Mayonnaise is an acidic oil-in-water emulsion...
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Egg white hydrolysate inhibits oxidation in mayonnaise and
a model system
Journal: Bioscience, Biotechnology, and Biochemistry
Manuscript ID BBB-160707.R2
Manuscript Type: Regular Paper
Date Submitted by the Author: 23-Jan-2017
Complete List of Authors: Kobayashi, Hideaki ; Kewpie Corporation, Institute of Technology R&D Div. Sasahara, Ryou ; Kewpie Corporation, Institute of Technology R&D Div. Yoda, Shoichi ; Kewpie Corporation, Institute of Technology R&D Div. Kotake-Nara, Eiichi; National Agriculture and Food Research Organization, National Food Research Institute
Keywords: chelate, egg white, hydrolysate, mayonnaise, oxidation
Subject Categories: Food & Nutrition Science
Classification of Research Fields:
V - 2) Chemistry and Biochemistry < V. Foods, V - 4) Processing, Preservation, and Safety < V. Foods
Bioscience, Biotechnology, and Biochemistry
This is an Accepted Manuscript of an article published by Taylor & Francis in Bioscience, Biotechnology, and Biochemistry on 16 Feb 2017, available online: http://www.tandfonline.com/10.1080/09168451.2017.1290519
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Ar t i c l e t y pe : R e g u l a r p a p e r 1
2
Runn i ng t i t l e : A n t i o x i d a n t a c t i o n o f e g g w h i t e h y d r o l y s a t e 3
4
Ti t l e : E g g w h i t e h y d r o l y s a t e i n h i b i t s o x i d a t i o n i n m a y o n n a i s e 5
a n d a m o d e l s y s t e m 6
7
Au thor s : H i d e a k i K o b a y a s h i1 , 3 , ∗
, Ry o u S a s a h a r a1, S h o i c h i Yo d a
1, 8
E i i c h i K o t a k e - N a r a2 , 3 , ∗
9
10
Af f i l i a t i o n : 11
1 I n s t i t u t e o f Te c h no l o g y R&D D i v. , K ewp i e Co r p o r a t i o n , 2 - 5 - 7 , 12
S en gawa - c ho , Ch o f u - s h i , To k y o , 1 8 2 - 00 02 , J a pa n 13
2Food R e s e a rc h I n s t i t u t e , Na t i o na l A g r i c u l t u re a nd Fo o d 14
Re s e a rc h O rga n i z a t i o n , 2 - 1 - 12 K annon da i , Ts u k ub a , I b a r a k i 15
3 0 5 -8 64 2 , J ap a n 16
3I n s t i t u t e o f B i omed i c a l S c i e n c e s , To ku s h ima Un i v e r s i t y 17
Gradua t e S c ho o l , 3 - 1 8 - 1 5 Ku ramo t o - c h o , To ku s h ima 7 70 - 85 03 , 18
J ap an 19
20
∗∗∗∗To whom c o r re spondenc e s h ou l d b e add re s s e d . 21
1Te l : + 8 1 - 3 - 5 3 8 4 - 7 7 5 8 ; F a x : + 8 1 - 3 - 5 3 8 4 - 7 8 6 0 ; E - m a i l : 22
h i d e a k i _ k o b a y a s h i @ k e w p i e . c o . j p , 2Te l : + 8 1 - 2 9 - 8 3 8 - 8 0 3 9 ; F a x : 23
+ 8 1 - 2 9 - 8 3 8 - 7 9 9 6 ; E - m a i l : e k o t a k e @ a f f r c . g o . j p . 24
25
Abb re v i a t i o n s : D M S O , d i m e t h y l s u l f o x i d e ; D P P H , 26
1 , 1 - d i p h e n y l - 2 - p i c r y l h y d r a z y l ; E W H , e g g w h i t e h y d r o l y s a t e ; 27
E D TA , e t h y l e n e d i a m i n e t e t r a a c e t i c a c i d ; P V, p e r o x i d e v a l u e ; T C A , 28
t r i ch l o r oa ce t i c a c i d . 29
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Abs t r a c t 1
T h e f l a v o r d e t e r i o r a t i o n o f m a y o n n a i s e i s i n d u c e d b y i r o n , 2
w h i c h i s r e l e a s e d f r o m e g g y o l k p h o s v i t i n u n d e r a c i d i c 3
c o n d i t i o n s a n d p r o m o t e s l i p i d o x i d a t i o n . To p r e v e n t o x i d a t i v e 4
d e t e r i o r a t i o n , n a t u r a l c o m p o n e n t s , r a t h e r t h a n s y n t h e t i c 5
c h e m i c a l s s u c h a s e t h y l e n e d i a m i n e t e t r a a c e t i c a c i d h a v e b e e n 6
r e q u i r e d b y c o n s u m e r s . I n t h e p r e s e n t s t u d y, w e e v a l u a t e d t h e 7
i n h i b i t o r y e f f e c t s o f t h r e e e g g w h i t e c o m p o n e n t s w i t h t h e s a m e 8
a m i n o a c i d c o m p o s i t i o n , n a m e l y e g g w h i t e p r o t e i n , h y d r o l y s a t e , 9
a n d t h e a m i n o a c i d m i x t u r e , o n l i p i d o x i d a t i o n i n m a y o n n a i s e a n d 10
a n a c i d i c e g g y o l k s o l u t i o n a s a m o d e l s y s t e m . We f o u n d t h a t t h e 11
h y d r o l y s a t e h a d t h e s t r o n g e s t i n h i b i t o r y e f f e c t o n l i p i d o x i d a t i o n 12
a m o n g t h e t h r e e c o m p o n e n t s . T h e m e c h a n i s m u n d e r l y i n g t h e 13
a n t i o x i d a n t e f f e c t w a s a s s o c i a t e d w i t h F e2 +
- c h e l a t i n g a c t i v i t y. 14
T h u s , e g g w h i t e h y d r o l y s a t e m a y h a v e t h e p o t e n t i a l a s n a t u r a l 15
i n h i b i t o r s o f l i p i d o x i d a t i o n i n m a y o n n a i s e . 16
17
18
Key wor d s : c h e l a t e , e g g w h i t e , h y d r o l y s a t e , m a y o n n a i s e , 19
o x i d a t i o n 20
21
22
23
24
25
26
27
28
29
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M a y o n n a i s e i s a n a c i d i c o i l - i n - w a t e r e m u l s i o n f o o d t h a t 1
c o n s i s t s o f v e g e t a b l e o i l , e g g s , a n d v i n e g a r. I t c o n t a i n s n u t r i e n t s 2
s u c h a s p o l y u n s a t u r a t e d f a t t y a c i d s a n d i r o n d e r i v e d f r o m e g g 3
y o l k p h o s v i t i n ,1 )
w h i c h c o n t a i n s m a n y p h o s p h o r y l a t e d s e r i n e 4
r e s i d u e s .2 )
I r o n , w h i c h i s r e l e a s e d f r o m e g g y o l k p h o s v i t i n 5
b e c a u s e o f t h e d e c r e a s e i n p H c a u s e d b y a c e t i c a c i d i n v i n e g a r, 6
i n d u c e s t h e o x i d a t i v e d e g r a d a t i o n o f m a y o n n a i s e .3 )
I n o t h e r 7
w o r d s , p h o s v i t i n h a s l i t t l e c h e l a t i n g a c t i v i t y i n m a y o n n a i s e . To 8
m a i n t a i n t h e c o m m e r c i a l v a l u e o f m a y o n n a i s e , c a l c i u m d i s o d i u m 9
e t h y l e n e d i a m i n e t e t r a a c e t i c a c i d ( E D TA ) h a s b e e n u s e d a s a f o o d 10
a d d i t i v e o v e r a l o n g p e r i o d i n m o s t c o u n t r i e s . E D TA i s a n 11
e c o n o m i c a l a g e n t t h a t i s h i g h l y e f f e c t i v e i n s t r o n g l y c h e l a t i n g 12
i r o n . H o w e v e r , E D TA i s a l s o a c h e m i c a l s y n t h e t i c p r o d u c t t h a t 13
t e n d s t o g i v e a n e g a t i v e i m a g e t o c o n s u m e r s . T h u s , a n t i o x i d a n t s 14
d e r i v e d f r o m a n a t u r a l p r o d u c t a r e r e q u i r e d a s f o l l o w s .4 - 8 )
15
P r e v i o u s l y, i t w a s e v a l u a t e d w h e t h e r n a t u r a l a n t i o x i d a n t s s u c h a s 16
p h y t i c a c i d ,4 )
g a l l i c a c i d ,5 )
t o c o p h e r o l ,6 )
a s c o r b i c a c i d ,7 )
a n d 17
p u r p l e c o r n h u s k e x t r a c t s8 )
i n h i b i t e d t h e o x i d a t i o n o f l i p i d s i n 18
m a y o n n a i s e . A m o n g t h e s e c o m p o n e n t s , o n l y p u r p l e c o r n h u s k 19
e x t r a c t s i n h i b i t e d l i p i d o x i d a t i o n i n m a y o n n a i s e , b u t t h e c o l o r o f 20
m a y o n n a i s e t u r n e d p u r p l e . We h a v e a c o n c e r n t h a t t h e p u r p l e 21
c o l o r w i l l n o t b e a c c e p t a b l e t o c o n s u m e r s . 22
I n a d d i t i o n t o t h e s e n a t u r a l a n t i o x i d a n t s , s o m e p r o t e i n s a n d 23
p e p t i d e s a r e a l s o k n o w n t o f u n c t i o n a s a n t i o x i d a n t s .9 )
E g g 24
a l b u m i n i n h i b i t e d t h e i r o n - c a t a l y z e d o x i d a t i o n o f e g g 25
p h o s p h a t i d y l c h o l i n e .1 0 )
F a t - f r e e e g g y o l k p r o t e i n e x e r t e d a n 26
a n t i o x i d a n t e f f e c t o n e t h a n o l / w a t e r o r o n c o o k i e s c o n t a i n i n g 27
l i n o l e i c a c i d . C o m p a r e d w i t h e g g - y o l k p r o t e i n , t h e h y d r o l y s a t e 28
e x h i b i t e d a s t r o n g e r a n t i o x i d a n t e f f e c t .1 1 )
B e c a u s e t h e c o l o r o f 29
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t h e s e e g g p r o t e i n s / h y d r o l y s a t e i s w h i t e , t h e e g g - d e r i v e d 1
c o m p o n e n t s m a y p r e v e n t t h e o x i d a t i o n o f l i p i d s i n f o o d s w i t h o u t 2
c h a n g i n g t h e c o l o r o f m a y o n n a i s e . 3
A s d e s c r i b e d a b o v e , a c e t i c a c i d c a u s e d t h e r e l e a s e o f i r o n , 4
s u b s e q u e n t l y l e a d i n g t o l i p i d o x i d a t i o n . I n a d d i t i o n , a c e t i c a c i d 5
i t s e l f m a y d i r e c t l y p a r t i c i p a t e i n l i p i d o x i d a t i o n i n m a y o n n a i s e 6
b e c a u s e a c e t i c a c i d w a s r e p o r t e d t o a c c e l e r a t e t h e o x i d a t i o n o f 7
s o y b e a n o i l .1 2 )
O n t h e o t h e r h a n d , s o m e o r g a n i c a c i d s h a v e a n 8
a n t i o x i d a t i v e e f f e c t ,1 2 - 1 4 )
a n d t h e y m a y i n h i b i t t h e l i p i d 9
o x i d a t i o n i n m a y o n n a i s e . 10
H e n c e , i n t h e p r e s e n t s t u d y, w e d e t e r m i n e d t h e i n h i b i t o r y 11
e f f e c t o f e g g w h i t e c o m p o n e n t s a n d o r g a n i c a c i d s , i n s t e a d o f 12
a c e t i c a c i d , o n l i p i d o x i d a t i o n u s i n g r e a l m a y o n n a i s e a n d a m o d e l 13
s y s t e m , a n d t h e a n t i o x i d a t i v e m e c h a n i s m s w e r e a l s o e v a l u a t e d . 14
15
Mate r i a l s a nd Me thods 16
Ma t e r i a l s . S h e l l e d e g g s w e r e p u r c h a s e d f r o m a 17
s u p e r m a r k e t i n To k y o . G l a c i a l a c e t i c a c i d o f f o o d a d d i t i v e g r a d e 18
w a s p u r c h a s e d f r o m T h e N i p p o n S y n t h e t i c C h e m i c a l I n d u s t r y C o . , 19
L t d . ( O s a k a , J a p a n ) . C a l c i u m d i s o d i u m E D TA w a s p u r c h a s e d 20
f r o m M a r u z e n C h e m i c a l s C o . , L t d . ( To k y o , J a p a n ) . Tr o l o x a n d 21
n e o c u p r o i n e w e r e p u r c h a s e d f r o m S i g m a - A l d r i c h ( S t L o u i s , M O , 22
U S A ) . E g g w h i t e p r o t e i n h y d r o l y s a t e E P - 1 ® i s a p r o d u c t o f 23
K e w p i e C o . ( To k y o , J a p a n ) . I t i s o b t a i n e d b y t r e a t i n g a l b u m i n 24
w i t h n e u t r a l p r o t e a s e o f As pe rg i l l u s o r i g i n . I t s a v e r a g e 25
m o l e c u l a r w e i g h t i s 11 0 0 . C i t r i c a c i d , 26
1 , 1 - d i p h e n y l - 2 - p i c r y l h y d r a z y l ( D P P H ) , a n d 27
3 - ( 2 - p y r i d y l ) - 5 , 6 - d i p h e n y l - 1 , 2 , 4 - t r i a z i n e - 4 ′ , 4 ″ - d i s u l f o n i c a c i d 28
m o n o s o d i u m s a l t h y d r a t e ( f e r r o z i n e ) w e r e p u r c h a s e d f r o m Wa k o 29
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P u r e C h e m i c a l I n d u s t r i e s , L t d . ( O s a k a , J a p a n ) . A m m o n i u m i r o n 1
( I I ) s u l f a t e h e x a h y d r a t e , F e C l 3 , 1 0 0 % t r i c h l o r o a c e t i c a c i d ( T C A ) , 2
a n d 5 - s u l f o s a l i c y l i c a c i d d i h y d r a t e w e r e p u r c h a s e d f r o m N a c a l a i 3
Te s q u e , I n c . , ( K y o t o , J a p a n ) . E D TA - 2 N a w a s p u r c h a s e d f r o m 4
D o j i n d o L a b o r a t o r i e s ( K u m a m o t o , J a p a n ) . A l l o t h e r c h e m i c a l s 5
w e r e o f a n a l y t i c a l r e a g e n t g r a d e . 6
7
The am i no a c i d c ompo s i t i o n s o f t h e e gg w h i t e p ro t e i n a n d 8
h yd ro l y s a t e , a n d p re p a r a t i o n o f t h e am in o a c i d m i x t u re . 9
E g g w h i t e w a s f r e e z e - d r i e d a n d t h e n p o u n d e d i n a m o r t a r. T h e 10
r e s u l t i n g c o m p o u n d w a s u s e d a s e g g w h i t e p r o t e i n . T h e a m i n o 11
a c i d c o m p o s i t i o n s o f e g g w h i t e p r o t e i n a n d E P - 1 w e r e m e a s u r e d 12
u s i n g a J L C / 5 0 0 V 2 a m i n o a c i d a n a l y z e r ( N i p p o n D e n s h i , To k y o , 13
J a p a n ) a s f o l l o w s . E g g w h i t e p r o t e i n a n d E P - 1 w e r e h y d r o l y z e d 14
b y H C l . C y s t e i n e w a s o x i d i z e d t o c y s t e i c a c i d u s i n g p e r f o r m i c 15
a c i d p r i o r t o h y d r o l y s i s .1 5 )
Tr y p t o p h a n c o n t e n t w a s d e t e r m i n e d 16
v i a b a r i u m h y d r o x i d e h y d r o l y s i s .1 6 )
T h e a m i n o a c i d c o n t e n t s o f 17
e g g w h i t e p r o t e i n a n d E P - 1 a r e s h o w n i n Ta b l e 1 . T h e f i n d i n g s 18
w e r e s i m i l a r b e t w e e n t h e t w o . 19
To p r e p a r e a n a m i n o a c i d m i x t u r e t h a t h a s t h e s a m e 20
f o r m u l a t i o n r a t i o a s E P - 1 , 1 8 a m i n o a c i d s w e r e p u r c h a s e d f r o m 21
K a n t o C h e m i c a l C o . , I n c . ( To k y o , J a p a n ) a n d w e r e t h e n 22
c o m p o u n d e d . 23
24
Pre p a r a t i o n o f a c i d i c e g g y o l k s o l u t i o n a s a mod e l s y s t em f o r 25
l i p i d o x i d a t i o n i n may o nn a i s e . T h e s o l i d c o n t e n t o f e g g 26
y o l k w a s a d j u s t e d t o 4 3 % v i a t h e a d d i t i o n o f a s m a l l a m o u n t o f 27
e g g w h i t e , a c c o r d i n g t o t h e l i t e r a t u r e .1 7 )
A f t e r t h e e g g y o l k 28
m i x t u r e ( 1 0 g ) w a s d i l u t e d w i t h d i s t i l l e d w a t e r ( 1 3 0 g ) , t h e 29
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d i l u t e d e g g y o l k s o l u t i o n w a s h e a t e d a t 6 0 °C f o r 3 m i n . F u r t h e r , 1
t h e e g g y o l k s o l u t i o n w a s a d j u s t e d t o p H 4 . 0 u s i n g o r g a n i c a c i d s 2
s u c h a s g l a c i a l a c e t i c a c i d . T h e f i n a l s o l i d c o n t e n t o f e g g y o l k i n 3
t h e a c i d i c e g g y o l k s o l u t i o n w a s 1 % . E g g w h i t e c o m p o n e n t s a s 4
c a n d i d a t e a n t i o x i d a n t s w e r e d i s p e r s e d w i t h a v o r t e x m i x e r 5
( m o d e l T M - 1 5 1 ; A G C Te c h n o G l a s s C o . , L t d . S h i z u o k a , J a p a n ) i n 6
t h e d i s t i l l e d w a t e r. T h e s e c o m p o n e n t s w e r e a d d e d t o t h e a c i d i c 7
e g g y o l k s o l u t i o n , a n d t h e n t h e a n t i o x i d a n t a c t i o n w a s e v a l u a t e d 8
a s f o l l o w s . 9
T h e a c i d i c e g g y o l k s o l u t i o n ( 1 0 m L ) i n a 1 5 - m L g l a s s t u b e o f 1 0
11 m m i n n e r d i a m e t e r w i t h a s c r e w c a p w a s i n c u b a t e d a t 5 5 °C i n 11
t h e d a r k . L i p i d o x i d a t i o n w a s e v a l u a t e d b y m e a s u r i n g t h e 12
f l u o r e s c e n c e i n t e n s i t y a c c o r d i n g t o p r e v i o u s r e p o r t s .1 8 - 2 0 )
I n 13
b r i e f , e t h e r / e t h a n o l ( 1 : 3 , v / v ) w a s a d d e d t o a n a l i q u o t o f t h e 14
o x i d i z e d r e a c t i o n m i x t u r e a n d t h e n c e n t r i f u g e d a t 1 , 2 0 0 g f o r 5 15
m i n a t r o o m t e m p e r a t u r e . T h e u p p e r p h a s e w a s m e a s u r e d a t 16
e x c i t a t i o n a n d e m i s s i o n w a v e l e n g t h s o f 3 6 0 a n d 4 4 0 n m , 17
r e s p e c t i v e l y, u s i n g a H i t a c h i F - 2 0 0 0 s p e c t r o p h o t o f l u o r o m e t e r. 18
T h e f l u o r e s c e n c e i n t e n s i t y w a s e x p r e s s e d a s a v a l u e r e l a t i v e t o 19
t h e s t a n d a r d v a l u e f o r 1 µg / m L q u i n i n e s u l f a t e / 0 . 1 N s u l f u r i c a c i d 20
s e t a s 1 0 0 . 21
22
GC-MS an a l y s i s . Vo l a t i l e c o m p o u n d s p r o d u c e d b y l i p i d 23
o x i d a t i o n f r o m t h e a c i d i c e g g y o l k s o l u t i o n w e r e a n a l y z e d b y 24
G C - M S a c c o r d i n g t o a p r e v i o u s r e p o r t .2 1 )
I n b r i e f , t h e a c i d i c e g g 25
y o l k s o l u t i o n ( 3 m L ) w a s i n c u b a t e d i n a s e a l e d g l a s s t u b e a t 4 0 °C 26
f o r 5 m i n . Vo l a t i l e c o m p o u n d s w e r e a d s o r b e d v i a h e a d s p a c e s o l i d 27
p h a s e m i c r o e x t r a c t i o n u s i n g a 28
p o l y d i m e t h y l s i l o x a n e / C a r b o x e n / d i v i n y l b e n z e n e ( S i g m a - A l d r i c h ) 29
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f i b e r a t 4 0 °C f o r 2 0 m i n a n d t h e n a n a l y z e d b y G C u s i n g a n 1
A g i l e n t 6 8 9 0 g a s c h r o m a t o g r a p h c o u p l e d t o a 5 9 7 3 m a s s 2
s p e c t r o m e t e r ( A g i l e n t Te c h n o l o g i e s , P a l o A l t o , C A ) w i t h 3
e l e c t r o n i m p a c t i o n i z a t i o n a t 7 0 e V. M a s s u n i t s w e r e m o n i t o r e d 4
f r o m 2 9 t o 2 9 0 m / z . S e p a r a t i o n w a s p e r f o r m e d o n a S o l - G e l - Wa x 5
c a p i l l a r y c o l u m n ( 3 0 m × 0 . 2 5 m m i . d . , 0 . 2 5 µm f i l m t h i c k n e s s ; 6
S G E A n a l y t i c a l S c i e n c e , Vi c t o r i a , A u s t r a l i a ) c a r r y i n g a c o n s t a n t 7
f l o w o f h e l i u m ( 1 . 0 m L / m i n ) . T h e i n l e t t e m p e r a t u r e w a s s e t a t 8
2 5 0 °C . T h e c o l u m n o v e n t e m p e r a t u r e p r o g r a m c o n s i s t e d o f a n 9
i n i t i a l c o n d i t i o n o f 3 5 °C h e l d f o r 5 m i n , f o l l o w e d b y a n i n c r e a s e 10
t o 1 2 0 °C a t a r a t e o f 5 °C / m i n a n d t h e n 2 2 0 °C a t a r a t e o f 11
1 5 °C / m i n , f o l l o w e d b y a f i n a l h o l d t i m e o f 6 m i n . T h e m a s s 12
s p e c t r a o f p e a k s w e r e i d e n t i f i e d b y c o m p a r i s o n w i t h t h e N I S T 13
d a t a b a s e m a s s s p e c t r a l l i b r a r y. 14
15
Pre p a r a t i o n o f ma yon na i s e . M a t e r i a l s f o r m a y o n n a i s e 16
w e r e c o m p o u n d e d u s i n g t h e f o r m u l a t i o n s h o w n i n Ta b l e 2 a n d 17
t h e n p a s s e d t h r o u g h t h e c o l l o i d m i l l o f a K e w p i e p i l o t p l a n t 18
( K e w p i e C o . ) a t r o o m t e m p e r a t u r e . T h e r e b y, m a y o n n a i s e 19
f o r m u l a t i o n s h a v i n g a n a v e r a g e p a r t i c l e d i a m e t e r o f 2− 5 µ m w e r e 20
o b t a i n e d . T h e p a r t i c l e d i a m e t e r w a s d e t e r m i n e d u s i n g a l a s e r 21
d i f f r a c t i o n p a r t i c l e s i z e a n a l y z e r ( m o d e l S A L D - 2 0 0 V ; S h i m a d z u , 22
K y o t o , J a p a n ) . T h e m a y o n n a i s e f o r m u l a t i o n s ( 2 0 0 g ) w e r e 23
e n c l o s e d i n s q u a r e b a g s ( 1 4 0 m m w i d e × 1 7 0 m m d e e p ) o f t h i n 24
p l a s t i c m a d e o f n y l o n 1 5 µm / l i n e a r l o w - d e n s i t y p o l y e t h y l e n e 6 0 25
µm . 26
27
L ip i d o x i d a t i o n i n ma y o nn a i s e a n d t a s t e s e n s o r y e v a l u a t i o n . 28
M a y o n n a i s e f o r m u l a t i o n s w e r e a u t o x i d i z e d a t 5 5 °C f o r 7 d a y s i n 29
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t h e d a r k t o m e a s u r e t h e p e r o x i d e v a l u e ( P V ) . A f t e r o x i d i z a t i o n , 1
l i p i d s i n t h e m a y o n n a i s e w e r e e x t r a c t e d u s i n g t h e B l i g h a n d D y e r 2
m e t h o d .2 2 )
P V w a s d e t e r m i n e d a c c o r d i n g t o t h e J a p a n O i l 3
C h e m i s t s S o c i e t y ( J O C S ) o f f i c i a l m e t h o d 2 . 5 . 2 . 2 - 2 0 1 3 . 4
I n a d d i t i o n t o t h e a u t o x i d a t i o n , m a y o n n a i s e f o r m u l a t i o n s w e r e 5
a l s o p h o t o - o x i d i z e d a t 2 5 °C f o r 7 d a y s u n d e r a f l u o r e s c e n t l a m p 6
a t 5 0 0− 6 0 0 l x f o r s e n s o r y t e s t i n g . Ta s t e , a s e v a l u a t e d b y 7
e i g h t - t r a i n e d p a n e l i s t s , w a s s c o r e d f r o m 0 t o 7 , w i t h h i g h e r 8
n u m b e r s i n d i c a t i n g b e t t e r t a s t e , o n t h e b a s i s o f a s c o r e o f 7 a s 9
t h e s t a n d a r d t a s t e o f m a y o n n a i s e s t o r e d a t 4 °C f o r 7 d a y s i n t h e 10
d a r k . 11
12
DPPH r ad i c a l - s c a v e ng i n g a c t i v i t y . E g g w h i t e p r o t e i n 13
w a s d i s p e r s e d i n 1 % ( w / v ) t a u r o c h o l a t e a n d t h e n s o n i c a t e d w i t h a 14
p r o b e - t y p e h o m o g e n i z e r ( U l t r a S , V P - 5 S ; Ta i t e c , S a i t a m a ) . E g g 15
w h i t e p r o t e i n h y d r o l y s a t e ( E P - 1 ) a n d t h e a m i n o a c i d m i x t u r e 16
w e r e d i s s o l v e d i n 6 N H C l / D M S O ( 0 . 0 1 6 : 1 , v / v ) a n d 6 N 17
H C l / D M S O ( 0 . 0 5 6 : 1 , v / v ) , r e s p e c t i v e l y. T h e t h r e e r e s u l t i n g 18
s o l u t i o n s w e r e v i s u a l l y c l e a r. T h e c o n c e n t r a t i o n o f t h e s e s a m p l e s 19
w a s 2 0 m g / m L , a n d t h e n e g a t i v e c o n t r o l s a n d d i l u t i o n s w e r e 20
p r e p a r e d u s i n g t h e c o r r e s p o n d i n g v e h i c l e s . 21
T h e s c a v e n g i n g a c t i v i t y o f t h e s e s a m p l e s o n D P P H r a d i c a l s 22
w a s e v a l u a t e d u s i n g a m o d i f i c a t i o n o f a m e t h o d d e s c r i b e d 23
p r e v i o u s l y.2 3 )
T h e s o l u t i o n s ( 2 0 µL ) o f e g g w h i t e p r o t e i n , e g g 24
w h i t e p r o t e i n h y d r o l y s a t e , a n d t h e a m i n o a c i d m i x t u r e w e r e 25
i n c u b a t e d w i t h D P P H w o r k i n g s o l u t i o n c o n s i s t i n g o f 9 0 µL o f 26
1 0 0 m M D P P H - e t h a n o l a n d 9 0 µL o f 1 0 0 m M a c e t a t e b u f f e r ( p H 27
4 . 0 ) i n e a c h w e l l o f a 9 6 - w e l l p l a t e f o r 3 0 m i n a t r o o m 28
t e m p e r a t u r e . T h e s u p e r n a t a n t s ( 1 0 0 µL ) w e r e c e n t r i f u g e d a t 29
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1 , 6 0 0 g f o r 1 0 m i n a t 4 °C , t r a n s f e r r e d t o a n e w 9 6 - w e l l p l a t e , a n d 1
t h e n d i l u t e d 2 - f o l d w i t h d i s t i l l e d w a t e r. T h e D P P H c o n t e n t w a s 2
a n a l y z e d a t 5 1 0 n m u s i n g a m i c r o p l a t e r e a d e r ( Te c a n G r o u p , 3
M ä n n e d o r f , S w i t z e r l a n d ) . Tr o l o x - e t h a n o l w a s u s e d a s a p o s i t i v e 4
c o n t r o l . 5
6
Fe2 +
- c h e l a t i n g a c t i v i t y. F e2 +
- c h e l a t i n g a c t i v i t y w a s 7
e v a l u a t e d u s i n g a m o d i f i c a t i o n o f a p r e v i o u s l y d e s c r i b e d 8
m e t h o d .2 4 , 2 5 )
T h e s o l u t i o n s ( 7 5 µL ) o f e g g w h i t e p r o t e i n , e g g 9
w h i t e p r o t e i n h y d r o l y s a t e , t h e a m i n o a c i d m i x t u r e , a n d n e g a t i v e 10
c o n t r o l s a n d d i l u t i o n s a s d e s c r i b e d p r e v i o u s l y w e r e m i x e d w i t h 11
75 µL o f ammonium i r on ( I I ) s u l f a t e he xahydr a te a t 1 0 0 µM 12
a n d 6 0 0 µL o f 1 0 0 m M a c e t a t e b u f f e r ( p H 4 . 0 ) . A f t e r i n c u b a t i o n 13
a t 5 5 °C f o r 3 0 m i n , 7 . 5 µL o f 1 0 0 % T C A w e r e a d d e d t o 1 5 0 µL o f 14
t h e r e a c t i o n m i x t u r e a n d t h e n c e n t r i f u g e d a t 9 , 6 0 0 g f o r 1 0 m i n a t 15
4 °C . T h e s u p e r n a t a n t s ( 1 0 0 µL ) w e r e i n c u b a t e d w i t h 8 0 µL o f 16
1 0 % ( w / v ) a m m o n i u m a c e t a t e a n d 2 0 µL o f t h e f e r r o u s i r on c o l o r 17
i n d i c a t o r c o n s i s t i n g o f 6 . 1 m M f e r r o z i ne and 1 4 . 4 m M 18
n e o c u p r o i n e , w h i c h c o u l d b e d i s s o l v e d b y t h e a d d i t i o n o f s e v e r a l 19
d r o p s o f 6 N H C l , i n 9 6 - w e l l p l a t e s f o r 5 m i n a t r o o m t e m p e r a t u r e , 20
a n d t h e n a n a l y z e d a t 5 6 0 n m u s i n g t h e m i c r o p l a t e r e a d e r. 21
E D TA - 2 N a w a s u s e d a s a p o s i t i v e c o n t r o l . Ve h i c l e a l o n e w a s 22
u s e d a s t h e n e g a t i v e c o n t r o l f o r e a c h s a m p l e . 23
D u r i n g t h e i n c u b a t i o n , t h e r e w a s a c o n c e r n r e g a r d i n g 24
r e d u c t i o n s o f F e2 +
c o n t e n t b y a u t o x i d a t i o n . C o n c o m i t a n t w i t h t h e 25
m e a s u r e m e n t o f c h e l a t i n g a c t i v i t y, t h e o x i d a t i v e s t a b i l i t y o f F e2 +
26
i n e a c h n e g a t i v e c o n t r o l s o l u t i o n w a s e v a l u a t e d . I n a d d i t i o n , d u e 27
t o t h e p o s s i b l e c o n v e r s i o n o f F e2 +
t o F e3 +
c a u s e d b y t h e e g g 28
w h i t e c o m p o n e n t s , s u p e r n a t a n t s ( 1 0 0 µL ) o f s a m p l e s w e r e a l s o 29
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m i x e d w i t h 1 0 0 µL o f 5 - s u l f o s a l i c y l i c a c i d d i h y d r a t e a t 2 0 0 µM 1
i n 9 6 - w e l l p l a t e s f o r 1 0 m i n a t r o o m t e m p e r a t u r e , f o l l o w e d b y 2
a n a l y s i s a t 5 1 0 n m u s i n g a m i c r o p l a t e r e a d e r v i a a m o d i f i c a t i o n 3
o f a p r e v i o u s l y d e s c r i b e d m e t h o d f o r e v a l u a t i n g F e3 +
.2 6 )
B e c a u s e 4
F e3 +
d i d n o t c a u s e a c o l o r c h a n g e i n t h e r e a c t i o n u s i n g t h e 5
f e r r o u s i r o n c o l o r i n d i c a t o r, t h e p r o d u c t i o n o f F e3 +
i n d u c e d b y 6
t h e c o m p o n e n t s m i g h t c r e a t e t h e m i s t a k e n i m p r e s s i o n o f 7
e f f e c t i v e F e2 +
- c h e l a t i n g a c t i v i t y. 8
9
S t a t i s t i c a l a n a l y s i s . T h e d a t a w e r e a n a l y z e d u s i n g 10
o n e - w a y A N O VA w i t h D u n n e t t t e s t o r w i t h Tu k e y−K r a m e r t e s t . 11
P - v a l u e s < 0 . 0 5 w e r e c o n s i d e r e d s i g n i f i c a n t . 12
13
Re su l t s 14
L ip i d o x i d a t i o n i n t h e a c i d i c e g g y o l k s o l u t i o n 15
We m e a s u r e d t h e f l u o r e s c e n c e o f s u b s t a n c e s p r o d u c e d b y t h e 16
o x i d a t i o n o f a c i d i c e g g y o l k s o l u t i o n a s a m a y o n n a i s e m o d e l . T h e 17
f l u o r e s c e n t s u b s t a n c e s a r e t h o u g h t t o b e p r o d u c t s o f a l d e h y d e 18
g r o u p s i n v o l a t i l e c o m p o u n d s a n d a m i n o g r o u p s a s d e s c r i b e d 19
b e l o w. 20
F i g . 1 A s h o w s a G C c h r o m a t o g r a m o f t h e v o l a t i l e c o m p o u n d s 21
f o r m e d b y o x i d a t i o n i n t h e a c i d i c e g g y o l k s o l u t i o n a t 5 5 °C f o r 22
7 2 h . T h e t h r e e m a i n p e a k s w e r e a s s i g n e d b y c o m p a r i n g M S 23
s p e c t r a w i t h t h o s e o f t h e d a t a b a s e l i b r a r y a s f o l l o w s : p e a k a , 24
h e x a n a l ( F i g . 1 B ) ; p e a k b , 2 - p e n t y l f u r a n ( F i g . 1 B ) ; a n d p e a k c , 25
a c e t i c a c i d ( d a t a n o t s h o w n ) . 26
We p l o t t e d t h e f l u o r e s c e n c e i n t e n s i t y o f s u b s t a n c e s a g a i n s t G C 27
r e s p o n s e s o f t h e f i r s t t w o c o m p o u n d s p r o d u c e d b y t h e o x i d a t i o n 28
o f a c i d i c e g g y o l k s o l u t i o n a t 5 5 °C f o r 7 2 h u n d e r v a r i a b l e 29
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c o n d i t i o n s ( p H 4 . 0 o r 7 . 0 w i t h E D TA a t 0 o r 2 5 µM ) . P o s i t i v e 1
c o r r e l a t i o n s w e r e f o u n d b e t w e e n t h e f l u o r e s c e n c e i n t e n s i t y a n d 2
t h e f o r m a t i o n o f h e x a n a l ( R2 = 0 . 9 7 5 7 ) o r 2 - p e n t y l f u r a n ( R
2 = 3
0 . 7 3 1 8 ) ( F i g . 1 C ) . I n p a r t i c u l a r , t h e f l u o r e s c e n c e i n t e n s i t y 4
e x h i b i t e d a s t r o n g c o r r e l a t i o n a l r e l a t i o n s h i p w i t h t h e h e x a n a l 5
f o r m a t i o n . 6
7
The i n h i b i t o r y e f f e c t s o f e gg w h i t e p ro t e i n / h y d ro l y s a t e / am i n o 8
a c i d m i x t u re o n l i p i d o x i d a t i o n i n a c i d i c e g g y o l k s o l u t i o n a s a 9
may onna i s e mode l 10
We e v a l u a t e d t h e a n t i o x i d a t i v e e f f e c t o f e g g w h i t e c o m p o n e n t s 11
u s i n g a c i d i c e g g y o l k s o l u t i o n a s a m a y o n n a i s e m o d e l . 12
T h e f l u o r e s c e n c e i n t e n s i t y i n a c i d i c e g g y o l k s o l u t i o n w a s 13
i n c r e a s e d d u r i n g i n c u b a t i o n i n a t i m e - d e p e n d e n t m a n n e r ( F i g . 2 A , 14
C o n t r o l ) . E D TA ( 0 . 0 0 4 7 % ) , p o s i t i v e c o n t r o l , s i g n i f i c a n t l y 15
d e c r e a s e d t h e i n t e n s i t y a t 8 8 h ( o n e - w a y A N O VA , p<0 . 0 5 ) ( F i g . 16
2 A ) . E g g w h i t e p r o t e i n , t h e h y d r o l y s a t e , a n d t h e a m i n o a c i d 17
m i x t u r e a l s o t r e n d e d t o d e c r e a s e t h e f l u o r e s c e n c e i n t e n s i t y i n a 18
c o n c e n t r a t i o n - d e p e n d e n t m a n n e r o v e r t h e r a n g e o f 19
0 . 0 1 2 5 %−0 . 1 % ( F i g s 2 B - E ) . 20
A s s h o w n i n F i g s 2 B - E , e g g w h i t e h y d r o l y s a t e a n d t h e a m i n o 21
a c i d m i x t u r e m o r e s t r o n g l y d e c r e a s e d t h e f l u o r e s c e n c e i n t e n s i t y 22
t h a n e g g w h i t e p r o t e i n ( T h e v a l u e s a t 8 8 h , p< 0 . 0 5 ) . A m o n g t h e 23
t h r e e e g g w h i t e c o m p o n e n t s , t h e h y d r o l y s a t e a t 0 . 1 % m o s t 24
e f f e c t i v e l y d e c r e a s e d t h e i n t e n s i t y ( T h e v a l u e s a t 8 8 h , p<0 . 0 5 ) 25
( F i g . 2 E ) . 26
27
DPPH r ad i c a l - s c a v e ng i n g a c t i v i t y o f e gg wh i t e 28
p ro t e i n / h y d ro l y s a t e / am i no a c i d m i x t u re 29
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A s d e s c r i b e d a b o v e , e g g w h i t e c o m p o n e n t s s h o w e d t h e 1
a n t i o x i d a t i v e e f f e c t i n a c i d i c e g g y o l k s o l u t i o n a s a m a y o n n a i s e 2
m o d e l . B e c a u s e t h e l i p i d o x i d a t i o n i s f u r t h e r a c c e l e r a t e d b y 3
r a d i c a l s p r o d u c e d f r o m l i p i d p e r o x i d e ,2 7 )
r a d i c a l - s c a v e n g i n g 4
a c t i v i t y i s t h o u g h t a s a p o s s i b l e m e c h a n i s m f o r t h e a n t i o x i d a t i v e 5
e f f e c t . D P P H w a s a p p r o p r i a t e l y u s e d f o r e v a l u a t i n g t h e a c t i v i t y 6
a s d e s c r i b e d b e l o w. E D TA s h o w e d n o e f f e c t a t a l l o n t h e D P P H 7
r a d i c a l - s c a v e n g i n g a c t i v i t y ( d a t a n o t s h o w n ) . Tr o l o x i s 8
c o m m o n l y u s e d a s a p o s i t i v e c o n t r o l . A s s h o w n i n F i g . 3 A , t h e 9
r e m a i n i n g l e v e l s o f D P P H r a d i c a l s ( % ) ( Y ) w e r e p l o t t e d a g a i n s t 10
t h e Tr o l o x c o n c e n t r a t i o n s ( m M ) ( X ) , r e v e a l i n g a n e g a t i v e 11
c o r r e l a t i o n u n d e r t h e e x p e r i m e n t a l c o n d i t i o n u s e d ( R2 = 0 . 9 9 0 7 ) . 12
E g g w h i t e p r o t e i n , t h e h y d r o l y s a t e , a n d t h e a m i n o a c i d m i x t u r e 13
r e d u c e d D P P H r a d i c a l l e v e l s i n a c o n c e n t r a t i o n - d e p e n d e n t 14
m a n n e r. T h e a m i n o a c i d m i x t u r e h a d t h e s t r o n g e s t e f f e c t o n 15
D P P H r a d i c a l a m o n g t h e t h r e e c o m p o n e n t s a t 2 . 0 % ( F i g . 3 B ) . 16
W h e n t h e D P P H r a d i c a l - s c a v e n g i n g a c t i v i t y o f t h e s e c o m p o n e n t s 17
a t 2 . 0 % w a s e v a l u a t e d b a s e d o n t h a t o f Tr o l o x u s i n g t h e f o r m u l a 18
Y = 1 6 6 . 6 6 X , t h e v a l u e s w e r e 0 . 1 5 5 ± 0 . 0 11 m M f o r p r o t e i n , 19
0 . 1 2 6 ± 0 . 0 0 4 m M f o r t h e h y d r o l y s a t e , a n d 0 . 2 9 3 ± 0 . 0 2 6 m M f o r 20
t h e a m i n o a c i d m i x t u r e . 21
22
Fe2 +
- c h e l a t i n g a c t i v i t y o f e g g w h i t e p ro t e i n / h y d ro l y s a t e / am i no 23
a c i d m i x t u re 24
A s i r o n r e l e a s e c a u s e d l i p i d o x i d a t i o n , t h e i r o n c h e l a t i n g 25
e f f e c t w o u l d b e o n e o f t h e p o s s i b l e m e c h a n i s m s o f t h e 26
a n t i o x i d a t i v e e f f e c t . A n e x t r e m e l y l o w c o n c e n t r a t i o n ( 5 0 µM , 27
0 . 0 1 8 6 m g / m L ) o f E D TA - 2 N a a s a p o s i t i v e c o n t r o l c h e l a t e d 28
n e a r l y a l l o f t h e F e2 +
c o n t e n t ( F i g . 4 ) . A m o n g t h e t h r e e e g g w h i t e 29
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c o m p o n e n t s , t h e h y d r o l y s a t e e x h i b i t e d t h e g r e a t e s t 1
F e2 +
- c h e l a t i n g a c t i v i t y a t 1 . 0 % ( F i g . 4 ) , w i t h a p p r o x i m a t e l y 5 0 % 2
o f F e2 +
c h e l a t e d a f t e r i n c u b a t i o n f o r 3 0 m i n . E g g w h i t e p r o t e i n 3
a n d t h e a m i n o a c i d m i x t u r e h a d l i t t l e t o n o F e2 +
- c h e l a t i n g 4
a c t i v i t y. 5
N o o x i d a t i o n o f F e2 +
t o F e3 +
w a s o b s e r v e d d u r i n g i n c u b a t i o n 6
f o r 3 0 m i n ( d a t a n o t s h o w n ) . 7
8
The i n h i b i t o r y e f f e c t o f t h e hy d ro l y s a t e o n l i p i d o x i d a t i o n i n 9
may onna i s e 10
We e v a l u a t e d t h e a n t i o x i d a t i v e e f f e c t o f e g g w h i t e c o m p o n e n t s 11
o n r e a l m a y o n n a i s e . A f t e r i n c u b a t i o n f o r 7 d a y s i n t h e d a r k a t 12
4 °C , t h e P V i n m a y o n n a i s e w a s a p p r o x i m a t e l y z e r o ( d a t a n o t 13
s h o w n ) , w h e r e a s t h e v a l u e s a t 5 5 °C r e a c h e d a p p r o x i m a t e l y 6 ( F i g . 14
5 , C o n t r o l ) . E D TA s i g n i f i c a n t l y d e c r e a s e d t h e P V. T h e 15
h y d r o l y s a t e ( E W H ) a l s o s i g n i f i c a n t l y d e c r e a s e d t h e v a l u e o v e r 16
t h e r a n g e o f 0 . 0 9 %−0 . 9 % ( F i g . 5 ) . 17
F i g . 6 s h o w s t h e r e s u l t s o f t h e s e n s o r y t a s t e e v a l u a t i o n o f 18
m a y o n n a i s e a f t e r i n c u b a t i o n f o r 7 d a y s . U n d e r t h e c o n d i t i o n o f 19
2 5 °C w i t h l i g h t a t 5 0 0− 6 0 0 l x ( C o n t r o l ) , t h e a v e r a g e v a l u e o f 20
t a s t e s c o r e w a s l o w ( 2 . 5 ) . E D TA s i g n i f i c a n t l y i n h i b i t e d t h e 21
d e t e r i o r a t i o n o f m a y o n n a i s e , w i t h t h e a v e r a g e s c o r e i n c r e a s i n g t o 22
5 . 5 . T h e h y d r o l y s a t e a t 0 . 0 9 % a n d 0 . 4 5 % a l s o s i g n i f i c a n t l y 23
i n h i b i t e d t h e d e t e r i o r a t i o n ( a v e r a g e s c o r e s , 4 . 2 5 a n d 5 . 0 , 24
r e s p e c t i v e l y ) , b u t n o e f f e c t w a s o b s e r v e d a t a h i g h e r 25
c o n c e n t r a t i o n ( 0 . 9 % ) . 26
D u r i n g t h e e v a l u a t i n g p e r i o d , t i m e - d e p e n d e n t v i s c o s i t y 27
c h a n g e s w e r e n o t s i g n i f i c a n t l y d i f f e r e n t a m o n g t h e m a y o n n a i s e 28
s a m p l e s ( d a t a n o t s h o w n ) . Vi s i b l e o i l l a y e r s e p a r a t i o n w a s a l s o 29
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n o t o b s e r v e d i n a n y s a m p l e s ( d a t a n o t s h o w n ) . 1
2
The e f f e c t o f v a r i o u s a c i d s o n l i p i d o x i d a t i o n i n a c i d i c e gg 3
y o l k s o l u t i o n 4
I n g e n e r a l , m a y o n n a i s e i s a d j u s t e d t o a n a c i d i c c o n d i t i o n u s i n g 5
v i n e g a r c o n t a i n i n g a c e t i c a c i d . H o w e v e r , a s d e s c r i b e d a b o v e , 6
a c e t i c a c i d i t s e l f m a y a c c e l e r a t e t h e l i p i d o x i d a t i o n . We 7
e v a l u a t e d t h e e f f e c t o f v a r i o u s a c i d s ( s i x o r g a n i c a c i d s a n d t w o 8
i n o r g a n i c a c i d s : h y d r o c h l o r i c a c i d a n d p h o s p h o r i c a c i d ) o n l i p i d 9
o x i d a t i o n i n t h e e g g y o l k s o l u t i o n a n d t h e n c o m p a r e d t h e m w i t h 10
a c e t i c a c i d . E a c h e g g y o l k s o l u t i o n w a s a d j u s t e d t o p H 4 . 0 w i t h 11
e i g h t c o r r e s p o n d i n g a c i d s . A m o n g t h e o r g a n i c a c i d s t e s t e d , c i t r i c 12
a c i d a n d t a r t a r i c a c i d s i g n i f i c a n t l y r e d u c e d t h e f l u o r e s c e n c e 13
i n t e n s i t y a f t e r i n c u b a t i o n f o r b o t h 4 0 h a n d 8 8 h c o m p a r e d w i t h 14
t h e e f f e c t o f a c e t i c a c i d ( F i g . 7 ) . 15
F i g . 8 p r e s e n t s t h e F e2 +
- c h e l a t i n g a c t i v i t y o f c i t r i c a c i d a t p H 16
4 . 0 . C i t r i c a c i d s i g n i f i c a n t l y r e d u c e d t h e a m o u n t s o f F e2 +
. A t 17
1 0 0 0 µM , t h e a m o u n t o f F e2 +
w a s r e d u c e d t o a p p r o x i m a t e l y 6 0 % 18
o f t h e i n i t i a l l e v e l . C i t r i c a c i d a t t h e s a m e c o n c e n t r a t i o n s s h o w e d 19
n o e f f e c t a t a l l o n t h e D P P H r a d i c a l s c a v e n g i n g a c t i v i t y ( d a t a n o t 20
s h o w n ) . 21
22
Di s c u s s i o n 23
24
I n t h e p r e s e n t s t u d y, w e e v a l u a t e d t h e e f f e c t o f e g g 25
w h i t e - d e r i v e d c o m p o n e n t s s u c h a s e g g w h i t e p r o t e i n , t h e 26
h y d r o l y s a t e , a n d t h e a m i n o a c i d m i x t u r e o n l i p i d o x i d a t i o n i n 27
m a y o n n a i s e . 28
We c o n s t r u c t e d a m a y o n n a i s e m o d e l u s i n g a c i d i c e g g y o l k 29
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s o l u t i o n ( p H 4 . 0 ) a n d t h e n a s s e s s e d t h e a n t i o x i d a n t a c t i o n o f e g g 1
w h i t e - d e r i v e d c o m p o n e n t s . T h e f o r m a t i o n o f f l u o r e s c e n t l i p i d 2
p e r o x i d a t i o n p r o d u c t s w a s m e a s u r e d a s a n i n d e x o f l i p i d 3
o x i d a t i o n i n t h e a c i d i c e g g y o l k s o l u t i o n . T h e f l u o r e s c e n t 4
p r o d u c t s a r e k n o w n t o b e p r o d u c e d f r o m a S c h i f f b a s e o f a n 5
a m i n o g r o u p a n d a n a l d e h y d e g r o u p .2 0 )
S o m e v o l a t i l e c o m p o u n d s 6
w e r e p r o d u c e d d u r i n g t h e o x i d a t i o n o f a c i d i c e g g y o l k s o l u t i o n . 7
T h e a m o u n t s o f h e x a n a l ( c a p r o a l d e h y d e ) a n d 2 - p e n t y l f u r a n 8
c o m p o u n d s a m o n g t h e v o l a t i l e c o m p o u n d s w e r e c o r r e l a t e d w i t h 9
a n 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 ( F i g . 1 C ) . H e x a n a l w a s 10
r e p o r t e d t o f o r m f l u o r e s c e n t p r o d u c t s w i t h l y s i n e .2 8 )
A n o t h e r 11
v o l a t i l e c o m p o u n d , 2 - p e n t y l f u r a n , w a s r e p o r t e d a s o n e o f t h e 12
o x i d a t i v e d e g r a d a t i o n p r o d u c t s p r o d u c e d f r o m l i n o l e i c a c i d ,2 9 )
a 13
m a j o r p o l y u n s a t u r a t e d f a t t y a c i d i n e g g y o l k , a n d a s a p o t e n t i a l 14
m a r k e r o f l i p i d p e r o x i d a t i o n . H o w e v e r , i t d o e s n o t h a v e a n 15
a l d e h y d e g r o u p w i t h i n i t s m o l e c u l e . T h e p r o d u c t i o n o f 16
2 - p e n t y l f u r a n w o u l d b e i n a p p e a r a n c e c o r r e l a t e d w i t h a n i n c r e a s e 17
i n f l u o r e s c e n t p r o d u c t s . T h u s , i n t h e p r e s e n t s t u d y, h e x a n a l 18
p r o d u c t i o n w o u l d i n v o l v e t h e p r o d u c t i o n o f f l u o r e s c e n t p r o d u c t s . 19
S o m e p r o t e i n s a n d a m i n o a c i d s a r e k n o w n a s n a t u r a l 20
a n t i o x i d a t i v e c o m p o n e n t s w i t h i r o n - c h e l a t i n g a n d / o r 21
r a d i c a l - s c a v e n g i n g a c t i v i t y. A m i n o a c i d s3 0 )
s u c h a s h i s t i d i n e , 22
l y s i n e , a n d c y s t e i n e a n d p r o t e i n s s u c h a s e g g a l b u m i n ,3 1 )
s o y 23
p r o t e i n ,1 0 )
c a s e i n ,3 2 )
a n d g e l a t i n3 3 )
i n h i b i t e d t h e o x i d a t i o n o f 24
l i n o l e i c a c i d i n v a r i o u s m o d e l s y s t e m s . I n a d d i t i o n , t h e 25
h y d r o l y s a t e / p e p t i d e s o f s o m e p r o t e i n s w e r e m o r e e f f e c t i v e i n 26
i n h i b i t i n g l i p i d o x i d a t i o n t h a n t h e i r p a r e n t a l p r o t e i n s . A f t e r t h e 27
m i l k w a s t r e a t e d w i t h t r y p s i n , t h e o x i d a t i o n o f m i l k f a t w a s 28
i n h i b i t e d c o m p a r e d w i t h b e f o r e t h e t r e a t m e n t , s u g g e s t i n g t h a t 29
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c a s e i n h y d r o l y s a t e e x e r t e d s t r o n g e r a n t i o x i d a t i v e e f f e c t s .3 4 )
S o y 1
p r o t e i n h y d r o l y s a t e m o r e e f f e c t i v e l y i n h i b i t e d t h e o x i d a t i o n o f 2
l i n o l e i c a c i d t h a n s o y p r o t e i n .3 5 )
E g g y o l k p r o t e i n h y d r o l y s a t e 3
d i s p l a y e d s t r o n g e r a n t i o x i d a t i v e e f f e c t s o n t h e o x i d a t i o n o f 4
l i n o l e i c a c i d i n c o o k i e s t h a n e g g y o l k p r o t e i n a n d a m i n o a c i d s .1 1 )
5
I n a g r e e m e n t w i t h t h e s e s t u d i e s , w e f o u n d t h a t e g g w h i t e p r o t e i n 6
h y d r o l y s a t e m o s t s t r o n g l y i n h i b i t e d l i p i d o x i d a t i o n ( F i g . 2 E ) . 7
T h e a n t i o x i d a t i v e e f f e c t o f t h e h y d r o l y s a t e w a s r e p o r t e d t o b e 8
d e p e n d e n t o n t h e v a r i e t y o f e n z y m e s u s e d t o c l e a v e p e p t i d e 9
b o n d s , t h e r e b y i n d i c a t i n g t h a t t h e a n t i o x i d a t i v e e f f e c t s o f 10
h y d r o l y s a t e / p e p t i d e s w o u l d b e a f f e c t e d b y t h e a m i n o a c i d 11
r e s i d u e s a n d t h e t e r m i n a l s i t e s .3 6 ) 12
To i n v e s t i g a t e t h e m e c h a n i s m u n d e r l y i n g t h e i n h i b i t o r y e f f e c t 13
o f e g g w h i t e h y d r o l y s a t e o n t h e o x i d a t i o n o f a c i d i c e g g y o l k 14
s o l u t i o n , w e e v a l u a t e d t h e f o l l o w i n g t w o p o i n t s : 15
r a d i c a l - s c a v e n g i n g a n d F e2 +
- c h e l a t i n g a c t i v i t y. I n t h e p r e s e n t 16
s t u d y, a m o n g t h e t h r e e e g g w h i t e c o m p o n e n t s , a m i n o a c i d 17
m i x t u r e d i s p l a y e d t h e s t r o n g e s t D P P H r a d i c a l - s c a v e n g i n g 18
a c t i v i t y ( F i g . 3 B ) . A m i n o a c i d s s u c h a s h i s t i d i n e ,3 7 , 3 8 )
a n d 19
c y s t e i n e3 9 )
w e r e p r e v i o u s l y r e p o r t e d t o h a v e D P P H 20
r a d i c a l - s c a v e n g i n g a c t i v i t y. T h e a m i n o a c i d m i x t u r e u s e d i n t h e 21
p r e s e n t s t u d y c o n t a i n e d t h e s e a m i n o a c i d s , a n d t h u s t h e y a l s o 22
w o u l d e x e r t t h e s t r o n g e s t e f f e c t u n d e r a c i d i c c o n d i t i o n s . A s 23
d e s c r i b e d p r e v i o u s l y, s o m e s t u d i e s1 1 , 3 4 , 3 5 , 4 0 )
r e p o r t e d t h a t 24
h y d r o l y s a t e e x h i b i t e d s t r o n g e r i n h i b i t o r y e f f e c t s t h a n t h e 25
p a r e n t a l p r o t e i n s o n l i p i d o x i d a t i o n a n d t h a t t h e D P P H 26
r a d i c a l - s c a v e n g i n g a c t i v i t y o f t h e h y d r o l y s a t e w a s o n e o f t h e 27
m e c h a n i s m s r e s p o n s i b l e f o r t h e s t r o n g e r a n t i o x i d a t i v e e f f e c t . 28
E g g w h i t e h y d r o l y s a t e a l s o d i s p l a y e d D P P H r a d i c a l - s c a v e n g i n g 29
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a c t i v i t y. H o w e v e r, t h e e f f e c t t e n d e d t o b e w e a k e r t h a n t h a t o f 1
p r o t e i n , a l t h o u g h t h e r e w a s n o s t a t i s t i c a l l y d i f f e r e n c e b e t w e e n 2
t h e t w o . T h e r e s u l t s s u g g e s t e d t h a t t h e m a i n i n h i b i t o r y e f f e c t o f 3
t h e h y d r o l y s a t e o n l i p i d o x i d a t i o n i n t h e a c i d i c e g g y o l k s o l u t i o n 4
w a s n o t c a u s e d b y r a d i c a l - s c a v e n g i n g a c t i v i t y. 5
We e v a l u a t e d F e2 +
- c h e l a t i n g a c t i v i t y a s a n o t h e r p o s s i b l e 6
m e c h a n i s m f o r t h e a n t i o x i d a t i v e a c t i o n o f e g g w h i t e h y d r o l y s a t e . 7
T h e F e2 +
- c h e l a t i n g a c t i v i t y t e n d e d t o b e i n t h e o r d e r o f 8
h y d r o l y s a t e > > a m i n o a c i d m i x t u r e s = p r o t e i n , s u g g e s t i n g t h a t 9
t h e F e2 +
- c h e l a t i n g a c t i v i t y e x p l a i n e d t h e a n t i o x i d a t i v e e f f e c t o f 10
t h e h y d r o l y s a t e ( F i g . 4 ) . T h u s , t h e r e s u l t s s u g g e s t e d t h a t t h e 11
i n h i b i t o r y e f f e c t o f t h e h y d r o l y s a t e o n l i p i d o x i d a t i o n i n t h e 12
a c i d i c e g g y o l k s o l u t i o n c o u l d b e m a i n l y d u e t o i t s 13
F e2 +
- c h e l a t i n g a c t i v i t y. D u r i n g t h e e v a l u a t i o n o f F e2 +
- c h e l a t i n g 14
a c t i v i t y, F e2 +
w a s e x t r e m e l y s t a b l e u n d e r a n a c i d i c c o n d i t i o n i n 15
t h e c u r r e n t s t u d y, i n a g r e e m e n t w i t h p r e v i o u s r e p o r t s .4 1 )
T h u s , 16
t h e a u t o x i d a t i o n o f F e2 +
d i d n o t i n t e r f e r e w i t h t h e m e a s u r e m e n t 17
o f F e2 +
- c h e l a t i n g a c t i v i t y. 18
T h e e n z y m a t i c h y d r o l y s a t e o f e g g w h i t e a l b u m i n w a s 19
p r e v i o u s l y r e p o r t e d t o i n h i b i t t h e o x i d a t i o n o f l i n o l e i c a c i d i n 20
e t h a n o l / p h o s p h a t e b u f f e r4 2 )
a n d c o r n o i l e m u l s i o n .2 5 )
T h e 21
h y d r o l y s a t e d i s p l a y e d s t r o n g F e2 +
- c h e l a t i n g a c t i v i t y, a n d t h i s 22
a c t i v i t y w a s b e l i e v e d t o e x p l a i n t h e a n t i o x i d a t i v e e f f e c t , 23
a g r e e i n g w i t h o u r r e s u l t s . S o m e p e p t i d e s i n t h e h y d r o l y s a t e h a v e 24
b e e n c o n s i d e r e d t o h a v e a n t i o x i d a t i v e e f f e c t s . A l a - H i s - Ly s w a s 25
p r e v i o u s l y i d e n t i f i e d a s a c a n d i d a t e s u b s t a n c e r e s p o n s i b l e f o r 26
t h e a n t i o x i d a t i v e a c t i o n o f p r o t e i n h y d r o l y s a t e .4 2 )
I n t h e p r e s e n t 27
s t u d y, t h e a n t i o x i d a t i v e a n d c h e l a t i n g a c t i v i t i e s o f p e p t i d e s i n 28
e g g w h i t e h y d r o l y s a t e w e r e n o t c l a r i f i e d . T h u s , a d e t a i l e d 29
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d e t e r m i n a t i o n o f t h e a m i n o a c i d r e s i d u e i n v o l v e d i n t h e s e e f f e c t s 1
r e q u i r e s f u r t h e r s t u d y. 2
We c o n f i r m e d t h e i n h i b i t o r y e f f e c t o f e g g w h i t e h y d r o l y s a t e o n 3
l i p i d o x i d a t i o n i n r e a l m a y o n n a i s e b y d e t e r m i n i n g t h e P V. T h e 4
h y d r o l y s a t e s i g n i f i c a n t l y i n h i b i t e d l i p i d o x i d a t i o n i n a 5
c o n c e n t r a t i o n - d e p e n d e n t m a n n e r. H y d r o l y s a t e a t 0 . 4 5 a n d 0 . 9 % 6
e x e r t e d a s i m i l a r e f f e c t a s 0 . 0 1 % E D TA , s u g g e s t i n g t h a t t h e 7
h y d r o l y s a t e h a v e t h e p o t e n t i a l t o i n h i b i t l i p i d o x i d a t i o n a s a n 8
a l t e r n a t i v e o f E D TA ( F i g . 5 ) . I n a d d i t i o n t o t h e i r e f f e c t o n 9
o x i d a t i v e s t a b i l i t y, t h e r a t i n g i n t h e s e n s o r y e v a l u a t i o n o f 10
m a y o n n a i s e w a s a l s o h i g h e s t f o r t h e h y d r o l y s a t e a t 0 . 4 5 % a s w e l l 11
a s E D TA a t 0 . 0 1 % ( F i g . 6 ) . A l t h o u g h t h e r a t i n g f o r h y d r o l y s a t e a t 12
0 . 9 % w a s l o w e r t h a n t h a t a t 0 . 4 5 % , o f f - f l a v o r s s u c h a s o x i d i z e d 13
f l a v o r s w e r e n o t o b s e r v e d i n m a y o n n a i s e . 14
I n m a y o n n a i s e c o n t a i n i n g e g g w h i t e h y d r o l y s a t e a t 0 . 9 % , t h e 15
p a n e l i s t s s e n s e d a b i t t e r t a s t e , w h i c h w a s s i g n i f i c a n t l y d i f f e r e n t 16
f r o m t h e s t a n d a r d t a s t e . I n f a c t , t h e b i t t e r t a s t e o f e g g w h i t e 17
h y d r o l y s a t e h a s b e e n p r e v i o u s l y r e p o r t e d .4 3 )
T h e s e r e s u l t s 18
s u g g e s t t h a t a t t h e h i g h e r c o n c e n t r a t i o n , t h e t a s t e o f t h e 19
h y d r o l y s a t e t h e m s e l v e s w a s r e s p o n s i b l e f o r t h e l o w e r r a t i n g i n 20
t h e s e n s o r y e v a l u a t i o n . O n t h e b a s i s o f t h e s e r e s u l t s , a 21
h y d r o l y s a t e c o n c e n t r a t i o n o f 0 . 4 5 % w o u l d b e n e c e s s a r y t o o b t a i n 22
t h e s a m e i n h i b i t o r y e f f e c t a s 0 . 0 1 % E D TA . T h e c o n c e n t r a t i o n o f 23
t h e h y d r o l y s a t e d i d n o t a f f e c t t h e f l a v o r, t a s t e , c o l o r t o n e , o r 24
p h y s i c a l p r o p e r t i e s o f m a y o n n a i s e . T h u s , t h e e g g w h i t e 25
h y d r o l y s a t e w o u l d b e u s e f u l c o m p o n e n t s a s n a t u r a l a n t i o x i d a n t s 26
i n i n h i b i t i n g d e t e r i o r a t i o n o f m a y o n n a i s e 27
A l t h o u g h a c e t i c a c i d i s g e n e r a l l y u s e d t o m a k e m a y o n n a i s e , i t 28
h a s p r o - o x i d a t i v e a c t i v i t y.1 2 )
T h e u s e o f o t h e r o r g a n i c a c i d s m a y 29
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f u r t h e r e n h a n c e r e s i s t a n c e t o l i p i d o x i d a t i o n i n m a y o n n a i s e , a s 1
o r g a n i c a c i d s s u c h a s c i t r i c a c i d h a v e b e e n r e p o r t e d t o h a v e 2
a n t i o x i d a t i v e a n d / o r i r o n c h e l a t i n g a c t i v i t y.1 3 , 1 4 )
I n t h e p r e s e n t 3
s t u d y, w e e v a l u a t e d t h e a n t i o x i d a n t a c t i o n o f e i g h t a c i d s , n a m e l y 4
s i x o r g a n i c a c i d s , h y d r o c h l o r i c a c i d , a n d p h o s p h o r i c a c i d . 5
A m o n g t h e a c i d s t e s t e d , c i t r i c a c i d e f f e c t i v e l y i n h i b i t e d l i p i d 6
o x i d a t i o n i n t h e a c i d i c e g g y o l k s o l u t i o n ( F i g . 7 ) . C i t r i c a c i d 7
a l s o d i s p l a y e d F e2 +
- c h e l a t i n g a c t i v i t y u n d e r t h e a c i d i c c o n d i t i o n , 8
a l t h o u g h t h e e f f e c t w a s w e a k e r t h a n t h a t o f E D TA ( F i g . 8 ) . T h e s e 9
r e s u l t s s u g g e s t e d t h a t t h e e f f e c t o f c i t r i c a c i d o n l i p i d o x i d a t i o n 10
i n t h e a c i d i c e g g y o l k s o l u t i o n w a s c a u s e d b y t h e c h e l a t i n g 11
a c t i v i t y. F r o m t h e p o i n t o f v i e w o f m i c r o o r g a n i s m p r o p a g a t i o n , 12
i n t h e p r o c e s s i n g o f m a y o n n a i s e , t h e w h o l e a c e t i c a c i d c a n n o t b e 13
r e p l a c e d t o c i t r i c a c i d b e c a u s e t h e a n t i m i c r o b i a l a c t i o n o f c i t r i c 14
a c i d w a s l o w e r t h a n t h a t o f a c e t i c a c i d .4 4 )
H o w e v e r, a c e t i c a c i d 15
m a y b e p a r t l y r e p l a c e d b y c i t r i c a c i d a s f o l l o w s . I t m a y b e 16
p o s s i b l e t o u s e t h e l e m o n f r u i t j u i c e , i n c l u d i n g t h e c i t r i c a c i d , 17
f o r t h e p r o d u c t i o n o f m a y o n n a i s e . T h e o x i d a t i v e s t a b i l i t y o f 18
l i p i d s i n r e a l m a y o n n a i s e u s i n g c i t r i c a c i d t o g e t h e r w i t h a c e t i c 19
a c i d d e s e r v e s f u r t h e r s t u d y. 20
I n c o n c l u s i o n , t o e s t i m a t e t h e i n h i b i t o r y e f f e c t o f e g g w h i t e 21
c o m p o n e n t s o n l i p i d o x i d a t i o n i n m a y o n n a i s e , w e u s e d a c i d i c e g g 22
y o l k s o l u t i o n a s a s i m p l e m o d e l s y s t e m . O x i d a t i o n w a s m e a s u r e d 23
u s i n g f l u o r e s c e n c e i n t e n s i t y, w h i c h w a s c o r r e l a t e d w i t h 24
i n c r e a s i n g h e x a n a l f o r m a t i o n d u r i n g t h e i n c u b a t i o n . A m o n g t h e 25
e g g w h i t e c o m p o n e n t s t e s t e d , h y d r o l y s a t e d i s p l a y e d t h e s t r o n g e s t 26
i n h i b i t i n g e f f e c t . T h e a n t i o x i d a n t a c t i v i t y o f t h e e g g w h i t e 27
h y d r o l y s a t e c o u l d b e m a i n l y d u e t o i t s F e2 +
- c h e l a t i n g a c t i v i t y. 28
T h e h y d r o l y s a t e a l s o i n h i b i t e d l i p i d o x i d a t i o n b a s e d o n 29
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m e a s u r e m e n t s o f t h e P V i n r e a l m a y o n n a i s e a n d s i g n i f i c a n t l y 1
s u p p r e s s e d t h e a p p e a r a n c e o f o f f - f l a v o r i n s e n s o r y t a s t e 2
e x p e r i m e n t s . T h e p r e s e n t s t u d y i n d i c a t e s t h a t t h e e g g w h i t e 3
h y d r o l y s a t e i s a n a t u r a l p r o d u c t w i t h t h e p o t e n t i a l t o e f f e c t i v e l y 4
i n h i b i t t h e d e g r a d a t i o n o f m a y o n n a i s e . 5
6
Au thor s c o n t r i bu t i o n s 7
8
H . K . d e s i g n e d t h e s t u d y. H . K . a n d E . K . - N . w r o t e t h e 9
m a n u s c r i p t . R . S . a n d S . Y. p e r f o r m e d l i p i d o x i d a t i o n i n 10
m a y o n n a i s e a n d t a s t e s e n s o r y e v a l u a t i o n a n d a n a l y z e d t h e d a t a . 11
E . K. - N . p e r f o r m e d t h e e x p e r i m e n t s o f D P P H a n d i r o n c h e l a t e a n d 12
a n a l y z e d t h e d a t a . A l l a u t h o r s c o n t r i b u t e d t o t h e c r i t i c a l r e v i s i o n 13
o f t h e m a n u s c r i p t . 14
15
Acknow l e dgemen t s 16
17
We t h a n k t h e s t a f f o f I n s t i t u t e o f Te c h n o l o g y, K e w p i e C o . , 18
M i n e o H a s e g a w a a n d M a r i Ya m a d a f o r a d v i c e r e g a r d i n g t h e 19
a p p l i c a t i o n o f e g g w h i t e h y d r o l y s a t e i n m a y o n n a i s e , S a t o s h i 20
Te r a o k a f o r t h e p r e p a r a t i o n o f m a y o n n a i s e , a n d S h i r o O g i h a r a f o r 21
t h e P V m e a s u r e m e n t s . We a l s o w o u l d l i k e t o t h a n k E n a g o 22
( w w w. e n a g o . j p ) f o r t h e E n g l i s h l a n g u a g e r e v i e w. 23
24
Di s c l o sure s t a t emen t 25
26
T h e a u t h o r s d e c l a r e n o c o n f l i c t s o f i n t e r e s t . 27
28
29
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Re f e re nc e s 1
[ 1 ] C o t t e r i l l O J , M a r i o n W W, N a b e r E C . A n u t r i e n t r e - e v a l u a t i o n 2
o f s h e l l e g g s . P o u l t S c i . 1 9 7 7 ; 5 6 : 1 9 2 7 – 1 9 3 4 . 3
[ 2 ] S a m a r a w e e r a H , Zh a n g W G , Le e E J , e t a l . E g g y o l k p h o s v i t i n 4
a n d f u n c t i o n a l p h o s p h o p e p t i d e s - r e v i e w. J F o o d S c i . 5
2 0 11 ; 7 6 : R 1 4 3 – 1 5 0 . 6
[ 3 ] J a c o b s e n C , Ti m m M , M e y e r A S . O x i d a t i o n i n f i s h o i l 7
e n r i c h e d m a y o n n a i s e : a s c o r b i c a c i d a n d l o w p H i n c r e a s e 8
o x i d a t i v e d e t e r i o r a t i o n . J A g r i c F o o d C h e m . 2 0 0 1 ; 9
4 9 ( 8 ) : 3 9 4 7 - 5 6 . 10
[ 4 ] N i e l s e n N S , P e t e r s e n A , M e y e r A S . Ti m m - H e i n r i c h M , 11
J a c o b s e n C . E f f e c t s o f l a c t o f e r r i n , p h y t i c a c i d , a n d E D TA o n 12
o x i d a t i o n i n t w o f o o d e m u l s i o n s e n r i c h e d w i t h l o n g - c h a i n 13
p o l y u n s a t u r a t e d f a t t y a c i d s . J A g r i c F o o d C h e m . 2 0 0 4 ; 5 2 : 14
7 6 9 0 – 7 6 9 9 . 15
[ 5 ] J a c o b s e n C , H a r t v i g s e n K , T h o m s e n M K, e t a l . L i p i d 16
o x i d a t i o n i n f i s h o i l e n r i c h e d m a y o n n a i s e : c a l c i u m d i s o d i u m 17
e t h y l e n e d i a m i n e t e t r a a c e t a t e , b u t n o t g a l l i c a c i d , s t r o n g l y 18
i n h i b i t e d o x i d a t i v e d e t e r i o r a t i o n . J A g r i c F o o d C h e m . 2 0 0 1 ; 19
4 9 : 1 0 0 9 – 1 0 1 9 . 20
[ 6 ] J a c o b s e n C , H a r t v i g s e n , K , L u n d P, e t a l . O x i d a t i o n i n 21
f i s h - o i l - e n r i c h e d m a y o n n a i s e 2 . A s s e s s m e n t o f t h e e f f i c a c y 22
o f d i f f e r e n t t o c o p h e r o l a n t i o x i d a n t s y s t e m s b y d i s c r i m i n a n t 23
p a r t i a l l e a s t s q u a r e s r e g r e s s i o n a n a l y s i s . E u r F o o d R e s 24
Te c h n o . 2 0 0 0 ; 2 1 0 : 2 4 2 – 2 5 7 . 25
[ 7 ] J a c o b s e n C , A d l e r - N i s s e n J , M e y e r A S . E f f e c t o f a s c o r b i c 26
a c i d o n i r o n r e l e a s e f r o m t h e e m u l s i f i e r i n t e r f a c e a n d o n t h e 27
o x i d a t i v e f l a v o r d e t e r i o r a t i o n i n f i s h o i l e n r i c h e d m a y o n n a i s e . 28
J A g r i c F o o d C h e m . 1 9 9 9 ; 4 7 : 4 9 1 7 – 4 9 2 6 . 29
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[ 8 ] L i C Y, K i m H W, L i H , e t a l . A n t i o x i d a t i v e e f f e c t o f p u r p l e 1
c o r n e x t r a c t s d u r i n g s t o r a g e o f m a y o n n a i s e . F o o d C h e m . 2
2 0 1 4 ; 1 5 2 : 5 9 2 – 5 9 6 . 3
[ 9 ] E l i a s R J , K e l l e r b y S S , D e c k e r E A . A n t i o x i d a n t a c t i v i t y o f 4
p r o t e i n s a n d p e p t i d e s . C r i t R e v F o o d S c i N u t r. 5
2 0 0 8 ; 4 8 : 4 3 0 – 4 4 1 . 6
[ 1 0 ] N a r a E , M i y a s h i t a K , O t a T. O x i d a t i v e s t a b i l i t y o f l i p o s o m e s 7
p r e p a r e d f r o m s o y b e a n P C , c h i c k e n e g g P C , a n d s a l m o n e g g 8
P C . B i o s c i B i o t e c h n o l B i o c h e m . 1 9 9 7 ; 6 1 : 1 7 3 6 – 1 7 3 8 . 9
[ 11 ] S a k a n a k a S , Ta c h i b a n a Y, I s h i h a r a N , e t a l . A n t i o x i d a n t 10
a c t i v i t y o f e g g - y o l k p r o t e i n h y d r o l y s a t e i n a l i n o l e i c a c i d 11
o x i d a t i o n s y s t e m . F o o d C h e m . 2 0 0 4 ; 8 6 : 9 9 – 1 0 3 . 12
[ 1 2 ] Ka j i m o t o G , Ta k a s h i m a R , M u r a k a m i C . E f f e c t s o f 13
A n t i o x i d a n t o n t h e O x i d a t i v e D e t e r i o r a t i o n o f O i l s i n t h e 14
P r e s e n c e o f B a s i c a n d A c i d i c M a t e r i a l s . J J p n O i l C h e m S o c 15
( J a p a n e s e ) . 1 9 9 8 ; 4 7 : 5 9 1 – 5 9 7 . 16
[ 1 3 ] F u k u z a w a K, S o u m i K , I e m u r a M , e t a l . D y n a m i c s o f 17
x a n t h i n e o x i d a s e - a n d F e3 +
- A D P - d e p e n d e n t l i p i d 18
p e r o x i d a t i o n i n n e g a t i v e l y c h a r g e d p h o s p h o l i p i d v e s i c l e s . 19
A r c h B i o c h e m B i o p h y s . 1 9 9 5 ; 3 1 6 : 8 3 – 9 1 . 20
[ 1 4 ] M i w a S , N a k a m u r a M , O k u n o M , e t a l . P r o d u c t i o n o f s t a r c h 21
w i t h a n t i o x i d a t i v e a c t i v i t y b y b a k i n g s t a r c h w i t h o r g a n i c 22
a c i d s . B i o s c i B i o t e c h n o l B i o c h e m . 2 0 11 ; 7 5 : 1 6 4 9 – 1 6 5 3 . 23
[ 1 5 ] M o o r e S . O n t h e d e t e r m i n a t i o n o f c y s t i n e a s c y s t e i c a c i d . J 24
B i o l C h e m . 1 9 6 3 ; 2 3 8 : 2 3 5 – 2 3 7 . 25
[ 1 6 ] P o n N G , S c h n a c k e r z K D , B l a c k b u r n M N , e t a l . M o l e c u l a r 26
w e i g h t a n d a m i n o a c i d c o m p o s i t i o n o f f i v e - t i m e s - c r y s t a l l i z e d 27
p h o s p h o g l u c o s e i s o m e r a s e f r o m r a b b i t s k e l e t a l m u s c l e . 28
B i o c h e m i s t r y. 1 9 7 0 ; 9 : 1 5 0 6 – 1 5 1 4 . 29
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[ 1 7 ] S t r i x n e r T, K u l o z i k U . E g g p r o t e i n s . I n : P h i l l i p s G O , 1
Wi l l i a m s PA . H a n d b o o k o f F o o d P r o t e i n s : E g g p r o t e i n s . 2
C a m b r i d g e ( U K ) : Wo o d h e a d p u b l i s h i n g ; 2 0 11 . P. 1 5 0 – 2 0 9 . 3
[ 1 8 ] S h i m a s a k i H , S a t o J , H a r a I . F l u o r e s c e n t s p e c t r o p h o t o m e t r y 4
o f a u t o x i d i z e d e g g y o l k l i p o p r o t e i n s . J J p n O i l C h e m S o c 5
( J a p a n e s e ) . 1 9 7 5 ; 2 4 : 4 6 4 – 4 6 8 . 6
[ 1 9 ] H a r a I , S h i m a s a k i H , S a t o J . E f f e c t o f o x i d a t i o n o n c h e m i c a l 7
s p e c t r a l a n d i m m u n o c h e m i c a l p r o p e r t i e s o f e g g y o l k 8
l i p o p r o t e i n . L i p i d s . 1 9 7 3 ; 8 : 6 2 3 – 6 2 6 . 9
[ 2 0 ] S h i m a s a k i H . A s s a y o f f l u o r e s c e n t l i p i d p e r o x i d a t i o n 10
p r o d u c t s . M e t h E n z y m o l . 1 9 9 4 ; 2 3 3 : 3 3 8 – 3 4 6 . 11
[ 2 1 ] Ya n a g i s a w a T, Wa t a n u k i C , A r i i z u m i M , e t a l . S u p e r c h i l l i n g 12
e n h a n c e s p r e s e r v a t i o n o f t h e f r e s h n e s s o f s a l t e d e g g y o l k 13
d u r i n g l o n g - t e r m s t o r a g e . J F o o d S c i . 2 0 0 9 ; 7 4 : E 6 2 – E 6 9 . 14
[ 2 2 ] B l i g h E G , D y e r W J . A r a p i d m e t h o d o f t o t a l l i p i d e x t r a c t i o n 15
a n d p u r i f i c a t i o n . C a n J B i o c h e m P h y s i o l . 1 9 5 9 ; 3 7 : 9 11 – 9 1 7 . 16
[ 2 3 ] Ta n a k a M , N a k a g a w a M . A n t i o x i d a n t a c t i v i t y o f 17
t h i o c h o l e s t e r o l o n c o p p e r - i n d u c e d o x i d a t i o n o f l o w - d e n s i t y 18
l i p o p r o t e i n . L i p i d s . 1 9 9 5 ; 3 0 : 3 2 1 – 3 2 5 . 19
[ 2 4 ] C a r t e r P. S p e c t r o p h o t o m e t r i c d e t e r m i n a t i o n o f s e r u m i r o n a t 20
t h e s u b m i c r o g r a m l e v e l w i t h a n e w r e a g e n t ( f e r r o z i n e ) . A n a l 21
B i o c h e m . 1 9 7 1 ; 4 0 : 4 5 0 – 4 5 8 . 22
[ 2 5 ] A b e y r a t h n e E D , L e e H Y, J o C , e t a l . E n z y m a t i c h y d r o l y s i s 23
o f o v a l b u m i n a n d t h e f u n c t i o n a l p r o p e r t i e s o f t h e h y d r o l y s a t e . 24
P o u l t S c i . 2 0 1 4 ; 9 3 : 2 6 7 8 – 2 6 8 6 . 25
[ 2 6 ] S h a r a f E l - D i n M , S c h a u r R J , S c h a u e n s t e i n E . U p t a k e o f 26
f e r r o u s i r o n h i s t i d i n a t e , a p r o m o t e r o f l i p i d p e r o x i d a t i o n , b y 27
E h r l i c h a s c i t e s t u m o r c e l l s . B i o c h i m B i o p h y s A c t a . 28
1 9 8 8 ; 9 6 2 : 3 7 – 4 1 . 29
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[ 2 7 ] F r a n k e l E N . L i p i d o x i d a t i o n . P r o g L i p i d R e s . 1 9 8 0 ; 1 9 : 1 – 2 2 . 1
[ 2 8 ] S t a p e l f e l d t H , S k i b s t e d L H . K i n e t i c s o f f o r m a t i o n o f 2
f l u o r e s c e n t p r o d u c t s f r o m h e x a n a l a n d L - l y s i n e i n a 3
t w o - p h a s e s y s t e m . L i p i d s . 1 9 9 6 ; 3 1 : 11 2 5 – 11 3 2 . 4
[ 2 9 ] K r i s h n a m u r t h y R G , S m o u s e T H , M o o k h e r j e e B D , e t a l . 5
I d e n t i f i c a t i o n o f 2 - p e n t y l f u r a n i n f a t s a n d o i l s a n d i t s 6
r e l a t i o n s h i p t o t h e r e v e r s i o n f l a v o r o f s o y b e a n o i l . J F o o d S c i . 7
1 9 6 7 ; 3 2 : 3 7 2 – 3 7 4 . 8
[ 3 0 ] Ka r e l M , Ta n n e n b a u m S R , Wa l l a c e D H , e t a l . A u t o x i d a t i o n 9
o f M e t h y l L i n o l e a t e i n F r e e z e - D r i e d M o d e l S y s t e m s . I I I . 10
E f f e c t s o f A d d e d A m i n o A c i d s . J F o o d S c i . 1 9 6 6 ; 3 1 : 8 9 2 – 8 9 6 . 11
[ 3 1 ] G o t o M , S h i b a z a k i K . E f f e c t o f t h e o x i d a t i o n o f o i l s o n t h e 12
d e t e r i o r a t i o n o f f o o d s p a r t I I . E f f e c t s o f t h e f o o d c o m p o n e n t s 13
o n l i n o l e i c a c i d o x i d a t i o n . J J p n S o c F o o d S c i Te c h n o l 14
( J a p a n e s e ) . 1 9 7 1 ; 1 8 : 2 7 7 – 2 8 3 . 15
[ 3 2 ] R i v a l S G , B o e r i u C G , Wi c h e r s H J . C a s e i n s a n d c a s e i n 16
h y d r o l y s a t e . 2 . A n t i o x i d a t i v e p r o p e r t i e s a n d r e l e v a n c e t o 17
l i p o x y g e n a s e i n h i b i t i o n . J . A g r i c F o o d C h e m . 2 0 0 1 ; 4 9 : 2 9 5 –18
3 0 2 . 19
[ 3 3 ] Z i r l i n A , K a r e l M . O x i d a t i o n e f f e c t s i n a f r e e z e - d r i e d 20
g e l a t i n - m e t h y l l i n o l e a t e s y s t e m . J F o o d S c i . 1 9 6 9 ; 3 4 : 1 6 0 –21
1 6 5 . 22
[ 3 4 ] L i m i D , S h i p e W F. P r o p o s e d m e c h a n i s m f o r t h e a n t i o x y g e n i c 23
a c t i o n o f t r y p s i n i n m i l k . J D a i r y S c i . 1 9 7 2 ; 5 5 : 7 5 3 – 7 5 8 . 24
[ 3 5 ] Ya m a g u c h i N , Yo k o o Y, F u j i m a k i M . S t u d i e s o n 25
a n t i o x i d a t i v e a c t i v i t i e s o f a m i n o c o m p o u n d s o n f a t s a n d o i l s 26
p a r t I I I . A n t i o x i d a t i v e a c t i v i t i e s o f s o y b e a n p r o t e i n 27
h y d r o l y z a t e s a n d s y n e r g i s t i c e f f e c t o f h y d r o l y z a t e o n 28
t o c o p h e r o l , J J p n S o c F o o d S c i Te c h n o l ( J a p a n e s e ) . 29
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1 9 7 5 ; 2 2 : 4 3 1 – 4 3 5 . 1
[ 3 6 ] Ya m a g u c h i N , Yo k o o Y, F u j i m a k i M . S t u d i e s o n 2
A n t i o x i d a t i v e A c t i v i t i e s o f A m i n o C o m p o u n d s o n F a t s a n d 3
O i l s P a r t I I . A n t i o x i d a t i v e a c t i v i t i e s o f d i p e p t i d e s a n d t h e i r 4
s y n e r g i s t i c e f f e c t s o n t o c o p h e r o l . J J p n S o c F o o d S c i Te c h n o l 5
( J a p a n e s e ) . 1 9 7 5 ; 2 2 : 4 2 5 – 4 3 0 . 6
[ 3 7 ] Wu H C , S h i a u C Y, C h e n H M , e t a l . A n t i o x i d a n t a c t i v i t i e s o f 7
c a r n o s i n e , a n s e r i n e , s o m e f r e e a m i n o a c i d s a n d t h e i r 8
c o m b i n a t i o n . J F o o d D r u g A n a l . 2 0 0 3 ; 11 : 1 4 8 – 1 5 3 . 9
[ 3 8 ] G ü l ç i n I . C o m p a r i s o n o f i n v i t r o a n t i o x i d a n t a n d a n t i r a d i c a l 10
a c t i v i t i e s o f L - t y r o s i n e a n d L - D o p a . A m i n o A c i d s , 11
2 0 0 7 ; 3 2 : 4 3 1 – 4 3 8 . 12
[ 3 9 ] B l o i s M S . A n t i o x i d a n t d e t e r m i n a t i o n s b y t h e u s e o f a s t a b l e 13
f r e e r a d i c a l . N a t u r e , 1 9 5 8 ; 1 8 1 : 11 9 9 – 1 2 0 0 . 14
[ 4 0 ] N o h D O , S u h H J . P r e p a r a t i o n o f e g g w h i t e l i q u i d 15
h y d r o l y s a t e ( E L H ) a n d i t s r a d i c a l - s c a v e n g i n g a c t i v i t y. P r e v 16
N u t r F o o d S c i . 2 0 1 5 ; 2 0 : 1 8 3 – 1 8 9 . 17
[ 4 1 ] L a m b e t h D O , E r i c s o n G R , Yo r e k M A , e t a l . I m p l i c a t i o n s f o r 18
i n v i t r o s t u d i e s o f t h e a u t o x i d a t i o n o f f e r r o u s i o n a n d t h e 19
i r o n - c a t a l y z e d a u t o x i d a t i o n o f d i t h i o t h r e i t o l . B i o c h i m 20
B i o p h y s A c t a . 1 9 8 2 ; 7 1 9 : 5 0 1 – 5 0 8 . 21
[ 4 2 ] Ts u g e N , E i k a w a Y, N o m u r a Y, e t a l . A n t i o x i d a t i v e a c t i v i t y 22
o f p e p t i d e s p r e p a r e d b y e n z y m a t i c h y d r o l y s i s o f e g g - w h i t e 23
a l b u m i n . J A g r i c C h e m S o c J p n ( J a p a n e s e ) . 1 9 9 1 ; 6 5 : 1 6 3 5 –24
1 6 4 1 . 25
[ 4 3 ] C i g i ć B , Z e l e n i k - B l a t n i k M . P r e p a r a t i o n a n d 26
c h a r a c t e r i z a t i o n o f c h i c k e n e g g w h i t e h y d r o l y s a t e . A c t a C h i m 27
S l o v. 2 0 0 4 ; 5 1 : 1 7 7 – 1 8 8 . 28
[ 4 4 ] Ya m a m o t o Y, H i g a s h i K , Yo s h i i H . I n h i b i t o r y a c t i v i t y o f 29
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o r g a n i c a c i d s o n f o o d s p o i l a g e b a c t e r i a . J J p n S o c F o o d S c i 1
Te c h n o l ( J a p a n e s e ) . 1 9 8 4 ; 3 1 : 5 2 5 – 5 3 0 . 2
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F i gure l e g e nd s 1
2
F i g . 1 Vo l a t i l e c ompounds a nd f l uo re s c e n t p roduc t s i n t h e 3
o x i da t i o n o f a c i d i c e gg yo l k s o l u t i o n a s a mode l s y s t em o f 4
mayonna i s e . 5
( A ) G C p r o f i l e i n a c i d i c e g g y o l k s o l u t i o n a f t e r i n c u b a t i o n 6
a t 5 5 °C f o r 7 2 h . P e a k a , h e x a n a l ; p e a k b , 2 - p e n t y l f u r a n ; p e a k c , 7
a c e t i c a c i d . ( B ) M a s s s p e c t r a o f p e a k s a a n d b . ( C ) R e l a t i o n s h i p 8
b e t w e e n f l u o r e s c e n c e i n t e n s i t y a t E x . 3 6 0 n m / E m . 4 4 0 n m i n e g g 9
y o l k s o l u t i o n a n d G C - M S r e s p o n s e s o f v o l a t i l e c o m p o u n d s 10
( h e x a n a l a n d 2 - p e n t y l f u r a n ) a f t e r i n c u b a t i o n a t 5 5 °C f o r 7 2 h 11
u n d e r v a r i a b l e p H ( 4 . 0 o r 7 . 0 ) a n d e t h y l e n e d i a m i n e t e t r a a c e t i c 12
a c i d c o n c e n t r a t i o n s ( 0 o r 2 5 µM ) . T h e d a t a a r e p r e s e n t e d a s t h e 13
m e a n ± S D ( n = 3 ) . 14
15
F i g . 2 I nh i b i t o r y e f f e c t s o f t h re e e gg wh i t e c ompon en t s o n 16
l i p i d o x i da t i o n i n a c i d i c e g g y o l k s o l u t i o n . 17
A c i d i c e g g y o l k s o l u t i o n s w e r e i n c u b a t e d a t 5 5 °C f o r t h e 18
i n d i c a t e d t i m e s , a n d t h e f l u o r e s c e n c e i n t e n s i t y a t E x . 3 6 0 n m / E m . 19
4 4 0 n m w a s m e a s u r e d . ( A ) N e g a t i v e c o n t r o l ( n o c o m p o n e n t ) , 20
o p e n c i r c l e s ; p o s i t i v e c o n t r o l ( e t h y l e n e d i a m i n e t e t r a a c e t i c a c i d a t 21
0 . 0 0 4 7 % ) , o p e n t r i a n g l e s . T h e d a t a a r e p r e s e n t e d a s t h e m e a n ± 22
S D ( n = 3 ) . T h e a s t e r i s k f o r t h e v a l u e s a t 8 8 h i n d i c a t e s 23
s i g n i f i c a n t d i f f e r e n c e s ( o n e - w a y A N O VA , p < 0 . 0 5 ) . ( B – E ) E g g 24
w h i t e p r o t e i n , f i l l e d c i r c l e s ; h y d r o l y s a t e , f i l l e d s q u a r e s ; a m i n o 25
a c i d m i x t u r e , f i l l e d t r i a n g l e s . T h e s e c o m p o n e n t s w e r e a d d e d t o 26
a c i d i c e g g y o l k s o l u t i o n a t c o n c e n t r a t i o n s o f 0 . 0 1 2 5 % ( B ) , 27
0 . 0 2 5 % ( C ) , 0 . 0 5 % ( D ) , a n d 0 . 1 % ( E ) . T h e d a t a a r e p r e s e n t e d a s 28
t h e m e a n ± S D ( n = 3 ) . T h e a s t e r i s k s f o r t h e v a l u e s a t 8 8 h 29
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i n d i c a t e s i g n i f i c a n t d i f f e r e n c e s ( o n e - w a y A N O VA w i t h 1
Tu k e y−K r a m e r t e s t , p < 0 . 0 5 ) . 2
3
F i g . 3 DPPH r a d i c a l - s c a v e ng i n g a c t i v i t y und e r a c i d i c 4
c ond i t i o n s . 5
D P P H r a d i c a l s c a v e n g i n g a c t i v i t y. D P P H / e t h a n o l w a s 6
m i x e d w i t h t r o l o x a s a p o s i t i v e c o n t r o l o r e g g w h i t e 7
c o m p o n e n t s / a c e t a t e b u f f e r ( p H 4 . 0 ) a n d t h e n i n c u b a t e d a t r o o m 8
t e m p e r a t u r e f o r 3 0 m i n . D P P H w a s m e a s u r e d a t 5 1 0 n m . ( A ) 9
Tr o l o x . ( B ) E g g w h i t e p r o t e i n , f i l l e d c i r c l e s ; h y d r o l y s a t e , f i l l e d 10
s q u a r e s ; a m i n o a c i d m i x t u r e , f i l l e d t r i a n g l e s . T h e r e m a i n i n g 11
D P P H r a d i c a l c o n t e n t w a s e x p r e s s e d a s t h e p e r c e n t a g e o f t h e 12
v a l u e o f t h e c o n t r o l . T h e d a t a a r e p r e s e n t e d a s t h e m e a n ± S D o f 13
e i g h t w e l l s . T h e a s t e r i s k s f o r t h e v a l u e s a t a c o n c e n t r a t i o n o f 14
2 . 0 % i n d i c a t e s i g n i f i c a n t d i f f e r e n c e s ( o n e - w a y A N O VA w i t h 15
Tu k e y−K r a m e r t e s t , p < 0 . 0 5 ) . 16
17
F i g . 4 F e2 +
- c he l a t i n g a c t i v i t y o f t h re e e g g wh i t e c ompone n t s 18
unde r a c i d i c c o nd i t i o n s . 19
F e2 +
- c h e l a t i n g a c t i v i t y. F e2 +
w a s m i x e d w i t h e g g w h i t e 20
c o m p o n e n t s / a c e t a t e b u f f e r ( p H 4 . 0 ) a n d t h e n i n c u b a t e d a t 5 5 °C 21
f o r 3 0 m i n , a f t e r w h i c h t h e f e r r o u s i r on c o n t e n t w a s m e a s u r e d 22
v i a f e r r o z i n e c o l o r i m e t r y a t 5 6 0 n m . E t h y l e n e d i a m i n e t e t r a a c e t i c 23
a c i d ( E D TA ) a t 2 5 µM ( 0 . 0 0 0 9 % ) a n d 5 0 µM ( 0 . 0 0 1 9 % ) a s a 24
p o s i t i v e c o n t r o l , o p e n c i r c l e . E g g w h i t e p r o t e i n , f i l l e d c i r c l e s ; 25
h y d r o l y s a t e , f i l l e d s q u a r e s ; a m i n o a c i d m i x t u r e , f i l l e d t r i a n g l e s . 26
T h e r e m a i n i n g F e2 +
a m o u n t s w e r e e x p r e s s e d a s a p e r c e n t a g e o f 27
t h e c o n t r o l . T h e d a t a a r e p r e s e n t a s t h e m e a n ± S D o f f o u r w e l l s . 28
T h e a s t e r i s k s f o r t h e v a l u e s a t a c o n c e n t r a t i o n o f 1 . 0 % i n d i c a t e 29
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29
s i g n i f i c a n t d i f f e r e n c e s ( o n e - w a y A N O VA w i t h Tu k e y−K r a m e r 1
t e s t , p < 0 . 0 5 ) . 2
3
F i g . 5 I nh i b i t o r y e f f e c t o f e g g wh i t e hydro l y s a t e o n l i p i d 4
o x i da t i o n i n mayo nna i s e . 5
M a y o n n a i s e w a s p r e p a r e d a c c o r d i n g t o t h e f o r m u l a s h o w n 6
i n Ta b l e 2 . I t w a s i n c u b a t e d a t 5 5 °C f o r 7 d a y s i n t h e d a r k . T h e 7
e f f e c t o f e g g w h i t e h y d r o l y s a t e ( E W H ) a t t h e i n d i c a t e d 8
c o n c e n t r a t i o n s w a s e v a l u a t e d b y m e a s u r i n g t h e p e r o x i d e v a l u e 9
( P V ) . E t h y l e n e d i a m i n e t e t r a a c e t i c a c i d ( E D TA ) a t 0 . 0 1 % w a s u s e d 10
a s a p o s i t i v e c o n t r o l . T h e d a t a a r e p r e s e n t e d a s t h e m e a n ± S D ( n 11
= 3 ) . T h e a s t e r i s k s i n d i c a t e s i g n i f i c a n t d i f f e r e n c e s f r o m c o n t r o l 12
( o n e - w a y A N O VA w i t h D u n n e t t t e s t , p < 0 . 0 5 ) . 13
14
F i g . 6 I nh i b i t o r y e f f e c t o f e g g wh i t e hydro l y s a t e o n 15
de t e r i o r a t i o n o f mayonna i s e by l i p i d o x i da t i o n . 16
M a y o n n a i s e w a s i n c u b a t e d a t 2 5 °C f o r 7 d a y s u n d e r l i g h t . 17
T h e e f f e c t o f e g g w h i t e h y d r o l y s a t e ( E W H ) a t t h e i n d i c a t e d 18
c o n c e n t r a t i o n s w a s e v a l u a t e d b y e i g h t - t r a i n e d s e n s o r y t a s t e 19
p a n e l s . E D TA a t 0 . 0 1 % w a s u s e d a s a p o s i t i v e c o n t r o l . E a c h 20
a v e r a g e s c o r e w a s e x p r e s s e d a t a l o w e r s i d e . T h e a s t e r i s k s 21
i n d i c a t e s i g n i f i c a n t d i f f e r e n c e s f r o m c o n t r o l ( o n e - w a y A N O VA 22
w i t h D u n n e t t t e s t , p < 0 . 0 5 ) . 23
24
F i g . 7 I nh i b i t o r y e f f e c t s o f v a r i o u s a c i d s o n l i p i d o x i da t i o n 25
i n t h e a c i d i c e g g yo l k s o l u t i o n . 26
T h e p H o f e g g y o l k s o l u t i o n s w a s a d j u s t e d t o 4 . 0 w i t h e i g h t 27
a c i d s , a n d f o l l o w e d b y i n c u b a t i o n a t 5 5 °C f o r 4 0 h o r 8 8 h . T h e 28
f l u o r e s c e n c e i n t e n s i t y a t E x . 3 6 0 n m / E m . 4 4 0 n m w a s m e a s u r e d . 29
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30
T h e d a t a a r e p r e s e n t e d a s t h e m e a n ± S D ( n = 3 ) . T h e a s t e r i s k s 1
i n d i c a t e s i g n i f i c a n t d i f f e r e n c e s f r o m t h e v a l u e s o f a c e t i c a c i d 2
( o n e - w a y A N O VA w i t h D u n n e t t t e s t , p < 0 . 0 5 ) . 3
4
F i g . 8 F e2 +
- c he l a t i n g a c t i v i t y o f c i t r i c a c i d unde r a c i d i c 5
c ond i t i o n s . 6
C i t r i c a c i d w a s u s e d a t a c o n c e n t r a t i o n o f 2 5 0− 1 0 0 0 µM 7
( 0 . 0 0 5 %− 0 . 0 2 % ) . F e2 +
w a s m i x e d w i t h c i t r i c a c i d / a c e t a t e b u f f e r 8
( p H 4 . 0 ) a n d t h e n i n c u b a t e d a t 5 5 °C f o r 3 0 m i n , a n d t h e f e r r o u s 9
i r o n c o n t e n t s w e r e m e a s u r e d v i a f e r r o z i n e c o l o r i m e t r y a t 5 6 0 n m . 10
T h e d a t a a r e p r e s e n t e d a s t h e m e a n ± S D o f f o u r w e l l s . T h e 11
a s t e r i s k s i n d i c a t e s i g n i f i c a n t d i f f e r e n c e s f r o m c o n t r o l ( o n e - w a y 12
A N O VA w i t h D u n n e t t t e s t , p < 0 . 0 5 ) . 13
14
Graph i c a l a b s t r a c t s 15
P r o p o s e d m e c h a n i s m o f l i p i d o x i d a t i o n i n m a y o n n a i s e a n d 16
a n t i o x i d a t i v e a c t i v i t y o f e g g w h i t e p r o t e i n h y d r o l y s a t e . 17
“ - P < ” , p h o s p h a t e g r o u p . 18
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Table 1 Amino acid composition of egg white protein and hydrolysate
Amino acid Protein Hydrolysate
(EP-1) ∗3
Threonine 44 46
Tyrosine 37 39
Phenylalanine 59 60
Cysteine 27 26
Methionine 39 39
Valine 68 70
Isoleucine 52 53
Leucine 82 84
Lysine 72 73
Tryptophan 16 15
Histidine 23 27
Aspartic acid ∗1 99 92
Serine 64 68
Glutamic acid ∗2 121 123
Proline 34 29
Glycine 34 33
Alanine 70 66
Arginine 59 57
Total
(mg/g protein)
1000
1000
∗1 Total of asparagine and aspartic acid.
∗2 Total of glutamine and glutamic acid.
∗3 The hydrolysate is a commercial product. The amino acid mixture was compounded in
the same formulation ratio.
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Table 2 Formulation of mayonnaise
Control
EDTA・
Ca・2Na
Egg white hydrolysate
0.09% 0.45% 0.9%
Vegetable oil ∗1 7.5 7.5 7.5 7.5 7.5
10% salted egg yolk 1.0 1.0 1.0 1.0 1.0
Vinegar ∗2 0.7 0.7 0.7 0.7 0.7
Water 0.720 0.719 0.711 0.675 0.630
Salt 0.05 0.05 0.05 0.05 0.05
Sodium glutamate 0.03 0.03 0.03 0.03 0.03
Mustard 0.006 0.006 0.006 0.006 0.006
Egg-white hydrolysate - - 0.009 0.045 0.090
EDTA・Ca・2Na - 0.001 - - -
Total (kg) 10.006 10.006 10.006 10.006 10.006
∗1Canola oil/soybean oil (1:1).
∗2Acetic acid 9%.
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For Peer ReviewF
luo
resce
nce inte
nsity
0
GC-MS response (10,000)
Figure 1
H. Kobayashi et al.
(A)
(B)
(C)
Ab
un
da
nce
Ab
un
da
nce
m/z
50 50 100 100 150
Hexanal 2-Pentylfuran
m/z
29
44 56
100 41
72
53 138
81
y = 0.0922x
R² = 0.7318
y = 0.3344x
R² = 0.9757
Ab
un
da
nce
a
b
c
4 8 12 16 20 Retention time (min)
39 82
67 95 109
0.5
1.0
2.5
2.0
0 10 20 30
1.5
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For Peer Review
Figure 2
H. Kobayashi et al.
Time (h) 0 20 40 60 80 100
Time (h) 0 20 40 60 80 100
0
2
1
3
4
5
Flu
ore
sce
nce inte
nsity
Time (h)
0 20 40 60 80 100 0
2
1
3
4
5 0.0125%
(B)
Control
(A)
0.025%
(C)
0.05%
(D)
0.1%
(E)
Time (h)
0 20 40 60 80 100
0
2
1
3
4
5
Time (h) 0 20 40 60 80 100
Flu
ore
sce
nce inte
nsity
Flu
ore
sce
nce inte
nsity
Flu
ore
sce
nce inte
nsity
Flu
ore
sce
nce inte
nsity
a
0
2
1
3
4
5
0
2
1
3
4
5
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For Peer Review
Figure 3
H. Kobayashi et al.
0 0.2 0.4 0.6
100
80
60
40
20
0
y = -166.66x + 99.132
R² = 0.9907
Concentration (mM)
(A)
0 0.5 1.0 1.5 2.0
Concentrations (%)
100
80
60
40
20
Am
ou
nts
of D
PP
H
rad
ical re
ma
inin
g (
%)
Am
ou
nts
of D
PP
H
rad
ical re
ma
inin
g (
%)
(B)
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Figure 4
H. Kobayashi et al.
Concentrations (%)
Re
ma
inin
g F
e2+
(%
)
0
20
40
60
80
100
0 0.25 0.5 0.75 1.0
EDTA
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For Peer ReviewC
on
trol
PV
(me
q/k
g)
8
6
4
2
0
ED
TA
(0.0
1%
)
EW
H(0
.09%
)
EW
H(0
.45%
)
EW
H(0
.90%
)
Figure 5
H. Kobayashi et al.
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↑Figure 6H. Kobayashi et al.
Con
trol
Sen
sory
scor
e8
6
4
2
0
ED
TA(0
.01%
)
EWH
(0.0
9%)
EWH
(0.4
5%)
EWH
(0.9
0%)
∗ ∗ ∗2.5 5.5 4.25 5.0 3.75
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For Peer ReviewF
luo
resce
nce inte
nsity
0
8
6
4
2
0
8
6
4
2
Ph
osp
ho
ric a
cid
Ace
tic a
cid
Citric a
cid
La
ctic a
cid
Glu
co
nic
acid
Ma
lic a
cid
Ta
rtaric a
cid
Hyd
roch
loric a
cid
88 h
40 h
Figure 7
H. Kobayashi et al.
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Figure 8
H. Kobayashi et al.
Concentrations (M)
Rem
ain
ing F
e2+
(%
)
0 250 500 750 1000 0
20
40
60
80
100
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egg yolk
phosvitin
neutral pH acidic pH
Graphical abstract
H. Kobayashi et al.
vinegar
reductants
Fe3+
Fe3+
Fe2+
Fe2+
lipid oxidation O2
Fe3+ -P P-
Fe3+ -P P-
-P P-
-P P-
Fe3+ -P P- Fe3+ -P P-
aldehyde
alcohol
ketone
volatile compounds
hydroperoxide
EDTA
(synthesis
component)
off flavor
egg white
hydrolysate
(natural
component)
chelate
effect
phosvitin
egg white
Page 41 of 41 Bioscience, Biotechnology, and Biochemistry