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P
a
g
e
1 o
36
J
ou
n
a
l o Ag i
c
ul
u
a
l
a
nd Food Ch
e
mi
s
y
1 A COMPREHENSIVE COLORIMETRIC STUDY OF ANTHOCYANIC
2 COPIGMENTATION IN MODEL SOLUTIONS. EFECTS OF pH AND MOLAR
3 RATIO
4
5 Belén Go
dillo, F
ancisco J. Rod
ígu
e
z-Pulido, M. Lui
sa
E
sc
ud
e
o-Gil
e
e
,
6 M. Loudes González-Mi
e , F
a
n
c
i
sc
o
J
. H
e e
di
a*
7 Food Colou & Quali y Lab., D
ep
. N
u
i i
on
& F
ood
Sci
en
c
e
. F
a
c
u
l
ad
de
8 Fa macia. Uni e sidad de Se ill
a
.
41012
-S
e
ill
a
, S
pa
i
n
.
9
10
11 Abb e ia ed
unning i le:
12 Colo and copigmen a ion o an
ho
cy
an
i
n
s
13
14
15 * Co esponding au ho
:
16 F ancisco J. He edia
17 Food Colou & Quali y Lab., D
ep
. N
u
i i
on
& F
ood
Sci
en
c
e
. F
a
c
u
l
ad
de
18 Fa macia. Uni e sidad de Se ill
a
.
41012
-S
e
ill
a
, S
pa
i
n
19 Tel.: +34 954556495 Fax: +
34
954557017
20 e-mail: he [email protected]
21
22
23
24
25
ACS P
a
a
gon Plu
s
En
i onm
e
n
1
J
ou
n
a
l o Ag i
c
ul
u
a
l
a
nd Food Ch
e
mi
s
y
P
a
g
e
2
o
36
26 Abs ac
27 New colo ime ic a iabl
e
s
ha
e
been
de
i
ned
i
n
he
un
i
o
m CIE
L
AB
28 colo space o assess he quan i
a
i
e
and
qua
li
a
i
e
c
o
l
o
c
hange
s i
ndu
c
ed
b
y
29 copigmen a ion, and hei incid
en
c
e
on
is
ua
l
pe
c
ep
i
on
. T
he
c
op
i
g
m
en
a
i
on
30 p ocess was assayed in mod
e
l s
o
l
u
i
on
s
be
w
een
M
a
l i
d
i
n
3
-
g
l
u
c
o
si
de
and
31 h ee phenolic compounds (ca
e
c
h
i
n
,
ep
ic
a
e
c
h
i
n
and
c
a
e
ic
a
ci
d
)
a
s
a
un
c i
on
32 o he pH and he pigmen /copi
g
m
en
m
o
l
a
a
i
o
. Al
ong
he
p
H
a
i
a
i
on
,
he
33 g ea es magni ude o copigm
en
a
i
on
w
a
s
ob
a
i
ned
a
p
H
3
.
0
,
be
i
ng
34 signi ican ly highe wi h epica ec
h
i
n
and
c
a
e
ic
a
ci
d
. A
h
i
gh
a
ci
d
ic
p
H,
he
m
a
i
n
35 con ibu ion o copigmen a ion
o
he
o
a
l c
o
l
o
w
a
s
qua
li
a
i
e
w
h
il
e
be
w
een
36 pH 2.0 and 4.0, he main c
o
l
o
im
e
ic c
on
i
bu
i
on
w
a
s
quan
i
a
i
e
. T
he
37 con ibu ion o epica echin and c
a
e
ic
a
ci
d
o
he
c
o
l
o
c
hange
s w
a
s m
o
e
38 ma ked o he quan i a i e cha
a
c
e
is ics. O
n
c
on
a
s ,
pa
ic
u
l
a
ly
a
h
i
ghe
p
H
39 alues, he quali a i e con ibu i
on
w
a
s m
o
e
im
po
an
i
n
c
a
e
c
h
i
n
c
op
i
g
m
en
ed
40 solu ions. Inc easing copigm
en
c
on
c
en
a
i
on
i
ndu
c
ed
pe
c
ep
i
b
l
e
c
o
l
o
41 changes a mola a ios highe
han
1
:
2
, c
on
sis i
ng
i
n
a
b
l
u
is
h
and
da
k
en
i
ng
42 e ec o he an hocyanin solu io
n
s. Am
ong
he
d
i
e
en
CIE
L
AB
a
i
bu
e
s,
hue
43 di e ences was he bes co el
a
ed
pa
a
m
e
e
wi
h
he
i
n
c
ea
s
e
o
c
op
i
g
m
en
44 concen a ion, p o ing he ele
an
c
e
o
h
is
ph
ysic
o
c
he
mic
a
l
pheno
m
enon
on
45 he quali a i e changes o an hocy
an
i
n
c
o
l
o
.
46
47 Keywo
ds: An hocyanin; colo ; c
op
i
g
m
en
a
i
on
; T is im
u
l
u
s C
o
l
o
im
e
y
ACS P
a
a
gon Plu
s
En
i onm
e
n
2
P
a
g
e
3 o
36
48
J
ou
n
a
l o Ag i
c
ul
u
a
l
a
nd Food Ch
e
mi
s
y
INTRODUCTION
49 An hocyanins a e he pigm
en
s
a
cc
oun
i
ng
o
he
b
illi
an
ed
,
pu
p
l
e
,
50 and blue colo s in many ui s,
ege
ab
l
e
s,
and
de
i
ed
ood
p
odu
c s s
u
c
h
a
s
51 ui s juices, jams, and ed wines (
1
).
52 I is well known ha h
e
s
ab
ili y
o
an
ho
cy
an
ic
p
i
g
m
en
s is
g
ea
ly
53 limi ed because hei esona in
g
s
u
c
u
e
c
on
e
s
he
m i
n
i
n
sic i
n
s
ab
ili y
54 agains se e al physical-chemical c
ond
i i
on
s. E
a
l
ua
i
on
o
he
a
c
o
s
a
e
c i
ng
55 he s abili y o an hocyanins in
d
ic
a
e
s
ha
p
H is
he
m
o
s im
po
an
e
x i
n
sic
56 ac o o an hocyanin deg ada i
on
. D
epend
i
ng
on
he
p
H
o
he
m
ed
i
u
m,
he
ed
57 colo ed la ylium ca ion coexis s
a
s
an
equ
ili
b
i
u
m mix
u
e
wi
h
o
he
o
ms
o
58 an hocyanins: he blue-pu ple q
u
i
nono
i
da
l
ba
s
e
s,
he
c
o
l
o
l
e
ss
he
mi
a
c
e
a
l B,
59 and he pale yellow chalcones. T
he
e
o
e
,
he
s
a
m
e
an
ho
cy
an
i
n
s
o
l
u
i
on
m
a
y
60 show di e en colo s (2).
61 Howe e , he chemical an
d
c
o
l
o
im
e
ic s
ab
ili y
o
an
ho
cy
an
i
n
s c
an
be
62 imp o ed by associa ions wi h o
he
s
ub
s
an
c
e
s. C
op
i
g
m
en
a
i
on
pheno
m
enon
,
63 among o he s, ep esen s one o
he
m
o
s c
o
m
p
l
e
x
and
e
ici
en
m
e
c
han
isms
o
64 an hocyanin ch omopho e s abiliz
a
i
on
i
n
na
u
e
and
ood
sys
e
ms (
3
). I
n
ood
65 science, his phenomenon is c
on
si
de
ed
a
e
l
e
an
i
n
e
a
c i
on
be
c
au
s
e
66 ob aining s able and a ac i e c
o
l
o
s is
a
m
a
j
o
o
c
u
s
o
qua
li y c
on
o
l
67 pu poses (4). Especially, in win
e
m
a
ki
ng
, is
a
ss
u
m
ed
i
p
l
a
ys
a
k
e
y
o
l
e
on
he
68 colo e olu ion and s abili y o h
e
y
oung
ed
wi
ne
s (
5
).
69 Copigmen a ion eac ions c
on
sis
o
non
-c
o
a
l
en
i
n
e
a
c i
on
s
be
w
een
70 an hocyanins among hemsel es (s
e
l -
a
ss
o
ci
a
i
on
)
o
wi
h
a
wi
de
a
i
e
y
o
71 colo less o ganic compounds
na
m
ed
c
op
i
g
m
en
s
o
c
op
i
g
m
en
a
i
on
c
o
a
c
o
s
ACS P
a
a
gon Plu
s
En
i onm
e
n
3

J
ou
n
a
l o Ag i
c
ul
u
a
l
a
nd Food Ch
e
mi
s
y
P
a
g
e
4
o
36
72 (basically o he phenolic comp
ound
s,
bu
a
ls
o
a
mi
no
a
ci
d
s, s
uga
s,
o
gan
ic
73 acids, polysaccha ides, e c.).
74 F om a molecula poin o i
e
w,
he
an
ho
cy
an
i
n
-c
op
i
g
m
en
s c
o
m
p
l
e
x
e
s
75 adop a ypical sandwich co
n
i
gu
a
i
on
(- s
a
cki
ng
) i
a
h
y
d
ophob
ic
76 in e ac ion. This s uc u al con o m
a
i
on
p
o
e
c s
he
ed
c
o
l
o
ed
l
a
yli
u
m c
a
i
on
77 agains he nucleophilic a ack o w
a
e
,
pe
o
xi
de
and
s
u
l
phu
d
i
o
xi
de
b
l
ea
c
h
i
ng
,
78 and pH changes educing he
o
m
a
i
on
o
he
o
he
c
o
l
o
l
e
ss s
pe
ci
e
s i
n
he
79 an hocyanin equilib ium (hemiac
e
a
l
and
c
ha
lc
one
) (
6
).
80 In addi ion, copigmen a i
on
no
on
ly c
on
e
s
g
ea
e
s
ab
ili y
o
81 an hocyanins, bu also induc
e
s c
o
l
o
a
i
a
i
on
s. T
h
is ki
nd
o
m
o
l
e
c
u
l
a
82 associa ion is esponsible o
he
y
p
ic
a
l c
hange
s i
n
he
s
pe
c
a
l
p
ope
i
e
s
o
83 he ch omopho e g oup, ha is,
an
i
n
c
ea
s
e
o
he
ab
s
o
p
i i y
and
equen
ly
a
84 shi o he isible λmax ow
a
d
g
ea
e
w
a
e
l
eng
h
s. C
on
s
equen
ly,
85 copigmen a ion p oduces bo h
quan
i
a
i
e
and
qua
li
a
i
e
c
o
l
o
c
hange
s
on
he
86 an hocyanins solu ions. In his se
n
s
e
,
he
m
ea
s
u
e
m
en
and
e
a
l
ua
i
on
o
he
s
e
87 colo ime ic changes is o g ea i
n
e
e
s i
n
ood
i
ndu
s y si
n
c
e
c
o
l
o
is
one
o
he
88 main senso y pa ame e o he
qua
li y
o
ood
s i
n
l
uen
ci
ng
c
u
s
o
m
e
s
e
l
e
c i
on
89 (7).
90 The con ibu ion o he c
op
i
g
m
en
a
i
on
pheno
m
enon
on
c
o
l
o
ha
s
been
91 widely s udied using spec op
ho
o
m
e
ic m
e
hod
s,
bo
h
i
n
m
ode
l
o
ood
92 sys ems which con ain indi idual
an
ho
cy
an
i
n
s
and
added
c
op
i
g
m
en
s;
o
i
n
ed
93 wines whe e an hocyanins na
u
a
lly c
o
-
o
cc
u
s wi
h
o
he
pheno
lic c
o
m
pound
s
94 (8-15). The e alua ion o he ch
ange
s i
n
he
VIS s
pe
c
u
m
o
an
ho
cy
an
i
n
s,
o
95 speci ically in he λmax (520 nm),
ha
s
pe
mi
ed
o
k
no
w
he
i
n
l
uen
c
e
o
96 nume ous ac o s on he e ec i
ene
ss
o
he
c
op
i
g
m
en
a
i
on
i
n
cl
ud
i
ng
he
ACS P
a
a
gon Plu
s
En
i onm
e
n
4
P
a
g
e
5 o
36
J
ou
n
a
l o Ag i
c
ul
u
a
l
a
nd Food Ch
e
mi
s
y
97 concen a ions o pigmen s
and
c
o
a
c
o
s,
he
i c
he
mic
a
l s
u
c
u
e
s,
he
98 co ac o /pigmen mola a io,
he
p
H
o
he
m
ed
i
u
m,
e
c. I
n
he
s
a
m
e
w
a
y,
99 se e al s udies in ed wines ha
e
s
ho
w
n
ha
he
m
agn
i
ude
o
c
op
i
g
m
en
a
i
on
100 and i s e olu ion du ing winem
a
ki
ng
is
e
x
e
m
e
ly
a
i
ab
l
e
a
cc
o
d
i
ng
o
101 i icul u al, ag onomical o oen
o
l
og
ic
a
l
p
a
c ic
e
s
a
cc
oun
i
ng
app
o
xim
a
e
ly
o
m
102 25 o 50% o he o al colo o yo
ung
ed
wi
ne
s (
5
,
16
-
20
).
103 In any case, al hough sp
e
c
a
l m
e
hod
s
ha
e
de
m
on
s
a
ed
o
be
a
a
li
d
,
104 simple and quick ool o quan i
a
i
e
e
s im
a
i
on
s, is
gene
a
lly
a
cc
ep
ed
ha
he
y
105 p o ide limi ed p ecision and
a
cc
u
a
cy
o
c
o
l
o
s
pe
ci ic
a
i
on
s. T
he
l
o
w
e
106 p ecision and accu acy achie
ab
l
e
c
an
be
e
x
p
l
a
i
n
be
c
au
s
e
an
adequa
e
107 desc ip ion o he colo a ia ions
equ
i
e
s i)
ha
s
pe
c
a
l
a
i
a
i
on
s c
on
si
de
ed
108 should be hose a ec ing he e
n
i
e
s
pe
c
a
l c
u
e
,
no
on
ly i s isi
b
l
e
λmáx,
and
109 ii) he use o a leas h ee colo im
e
ic
a
i
bu
e
s:
hue
, s
a
u
a
i
on
and
li
gh
ne
ss
110 (21). Mo eo e , he speci ic c
hange
s
a
he
λmáx
a
e
a
ls
o
o
en
i
n
e
p
e
ed
111 inco ec ly in colo ime ic e ms. I
ha
s
been
de
sc i
bed
ha
he
h
y
pe
c
h
o
mic
112 and ba hoc omic e ec s make
an
ho
cy
an
i
n
s s
o
l
u
i
on
s
appea
b
l
ue
and
wi
h
113 mo e in ense colo (3, 22-23). H
o
w
e
e
,
he
a
i
a
i
on
i
n
a
si
ng
l
e
w
a
e
l
eng
h
114 does no explain he comple e b
eha
i
o
o
he
c
o
l
o
due
o
h
is
pheno
m
enon
.
115 Fo hese easons, o ad
an
c
e
i
n
he
k
no
wl
edge
o
he
g
l
oba
l c
o
l
o
im
e
ic
116 ole o copigmen a ion is necess
a
y
o
c
on
si
de
bo
h
quan
i
a
i
e
and
qua
li
a
i
e
117 colo changes. In his con ex , T is im
u
l
u
s C
o
l
o
im
e
y, w
h
ic
h
is
ba
s
ed
on
118 ansmi ance alues o he whol
e
s
pe
c
a
,
ep
e
s
en
s
a
u
s
e
u
l m
e
hodo
ly
ha
119 widely imp o es he objec i e an
a
lysis
o
c
o
l
o
.
120 Thus, h ough T is imul
u
s C
o
l
o
im
e
y, i
n
h
is s
ud
y is
pe
o
m
ed
a
121 p ecise colo ime ic in e p e a i
on
o
he
c
op
i
g
m
en
a
i
on
pheno
m
enon
u
si
ng
he
ACS P
a
a
gon Plu
s
En
i onm
e
n
5
J
ou
n
a
l o Ag i
c
ul
u
a
l
a
nd Food Ch
e
mi
s
y
P
a
g
e
6
o
36
122 uni o m 1976-(L
*
a
*
b
*
) colo spac
e
(CIE
L
AB), w
h
ic
h
ha
s
been
e
c
o
mm
ended
b
y
123 he CIE (Commission In e na io
na
l
e
de
l
’
Écl
a
i
age
)
a
s
a
m
o
e
app
op
i
a
e
oo
l
124 o colo speci ica ion in mos i
ndu
s i
a
l
app
lic
a
i
on
s. F
o
h
is
pu
po
s
e
,
d
i
e
s
e
125 colo ime ic a iables based bo h
on
he
sc
a
l
a
(
L*
,
a*
,
b*
)
and
angu
l
a
(
L*
, C
*
ab,
126 hab) colo coo dina es has b
een
de
i
ned
o
a
ss
e
ss
he
quan
i
a
i
e
and
127 quali a i e colo implica ions ass
o
ci
a
ed
o
c
op
i
g
m
en
a
i
on
,
and
he
i i
n
ci
den
c
e
128 on isual pe cep ion. These c
o
l
o
im
e
ic
a
i
ab
l
e
s
ha
e
been
app
li
ed
and
129 compa ed wi h he mos used sim
p
li i
ed
m
e
hod
i
n
he
e
a
l
ua
i
on
o
he
130 copigmen a ion e ec s in model s
o
l
u
i
on
s.
131
132 MATERIALS AND METHODS
133
134 S anda ds and copigmen ed
m
od
e
l
s
s
olu ion
s
135 The pigmen mal idin 3-gl
u
c
o
si
de
(M
3
-
g
l) w
a
s is
o
l
a
ed
i
n
he
l
abo
a
o
y
136 om skins o Vi is ini e a ed
g
ape
s
o
T
e
m
p
an
ill
o
a
i
e
y. Ex
a
c i
on
w
a
s
137 made wi h acidic me hanol (me
hano
l: HCl
1
N;
95
:
5
/ )
and
he
e
x
a
c s w
e
e
138 pu i ica ed by semip essu e li
qu
i
d
c
h
o
m
a
og
aph
y
u
si
ng
a
e
e
s
ed
-
pha
s
e
139 column, as desc ibed by He
ed
i
a
, F
an
ci
a
-A ic
ha
, Ri
a
s-G
on
z
a
l
o
, Vic
a
i
o
&
140 San os-Buelga, 1998. The copigm
en
s (+)-c
a
e
c
h
i
n
(CAT)
and
(-) -
ep
ic
a
e
c
h
i
n
141 (EPI) and ca eic acid (CAF) w
e
e
pu
c
ha
s
ed
o
m Si
g
m
a
C
he
mic
a
l C
o
. (S .
142 Louis, MO).
143 All he model solu ions we
e
p
epa
ed
i
n
a
wi
ne
-lik
e
m
ed
i
u
m c
on
a
i
n
i
ng
5
144 g/L a a ic acid in 12% e hanol
and
i
on
ic s
eng
h
ad
j
u
s
ed
o
0
.
2
M
b
y
add
i i
ng
145 sodium chlo ide.
146 In o de o e alua e h
e
e
e
c
o
he
p
H
on
he
c
op
i
g
m
en
a
i
on
147 phenomenon, h ee copigmen e
d
s
o
l
u
i
on
s
o
m
a
l i
d
i
n
3
-
g
l:(+)-c
a
e
c
h
i
n
(MC),
ACS P
a
a
gon Plu
s
En
i onm
e
n
6
P
a
g
e
7 o
36
148
149
150
151
J
ou
n
a
l o Ag i
c
ul
u
a
l
a
nd Food Ch
e
mi
s
y
mal idin 3-gl:(-)-epica echin (ME),
and
m
a
l i
d
i
n
3g
l: c
a
e
ic
a
ci
d
(MF),
a
s w
e
ll
a
s
a e e ence solu ion (M 3-gl) w
e
e
p
epa
ed
i
n
wi
ne
-lik
e
m
ed
i
u
m
a
d
i
e
en
p
H
alues: 1.0, 2.0, 3.0, 4.0, and
5
.
0
. T
he
e
e
en
c
e
s
o
l
u
i
on
c
on
a
i
ned
200
m
g
/
L
(0.41 mM) o M 3-gl. Copigmen
ed
s
o
l
u
i
on
s c
on
a
i
ned
he
s
a
m
e
an
ho
cy
an
i
n
152 concen a ion and he co esp
ond
i
ng
c
op
i
g
m
en
u
si
ng
a
p
i
g
m
en
/c
op
i
g
m
en
153 mola a io o 1:5.
154 The e ec o he copigm
en
c
on
c
en
a
i
on
w
a
s
a
ls
o
a
ss
e
ss
ed
. Tw
o
155 copigmen ed solu ions o mal i
d
i
n
3
-
g
l:(+)-c
a
e
c
h
i
n
(MC)
and
m
a
l i
d
i
n
3
-
g
l:(-)-
156 epica echin (ME), and a e e enc
e
s
o
l
u
i
on
w
e
e
p
epa
ed
i
n
he
s
a
m
e
wi
ne
-lik
e
157 medium adjus ed o pH 3.60. C
op
i
g
m
en
ed
s
o
l
u
i
on
s c
on
a
i
ned
he
s
a
m
e
158 an hocyanin concen a ion (0.41mM)
and
he
c
o
e
s
pond
i
ng
c
op
i
g
m
en
s
o
g
i
e
159 he equi ed pigmen /copigmen m
o
l
a
a
i
o
:
1
:
1
,
1
:
2
,
1
:
5
,
and
1
:
7
.
160 All o he solu ions we e
p
epa
ed
i
n
i
p
lic
a
e
and
equ
ili
b
a
ed
o
ea
c
h
161 he equilib ium o 2 hou s, s o
ed
cl
o
s
ed
i
n
da
k
ne
ss
a
25
°C,
a
e
w
h
ic
h
he
i
162 abso p ion spec a we e eco de
d
.
163 Colo analysis
164 The abso p ion spec a (
380
-
770
n
m)
o
a
ll
he
m
ode
l s
o
l
u
i
on
s w
e
e
165 eco ded a cons an in e als (Δλ=
2
n
m) wi
h
a
H
e
wl
e
- P
a
ck
a
d
UV- is
166 HP8452 spec opho ome e (Pal
o
Al
o
, CA),
u
si
ng
2
mm
pa
h
l
eng
h
g
l
a
ss c
e
lls
167 and dis illed wa e as a e e enc
e
. T
he
CIE
L
AB
pa
a
m
e
e
s (
L*
,
a*
,
b*
, C
*
ab,
and
168 hab) we e de e mined by usin
g
he
C
o
m
aLab
s
o
w
a
e
(
24
),
o
ll
o
wi
ng
he
169 Commission In e na ional de
L’
Ecl
a
i
age’
s
e
c
o
mm
enda
i
on
s (
25
):
he
10
º
170 S anda d Obse e and he S an
da
d
Ill
u
mi
nan
D
65
.
171 The L
*
alue is he e ical
a
xis
and
de
i
ne
s
he
li
gh
ne
ss,
he
p
ope
y
172 acco ding o which each colo ca
n
be
c
on
si
de
ed
a
s
equ
i
a
l
en
o
a
m
e
m
be
o
ACS P
a
a
gon Plu
s
En
i onm
e
n
11

J
ou
n
a
l o Ag i
c
ul
u
a
l
a
nd Food Ch
e
mi
s
y
P
a
g
e
12
o
36
272 E0) / E0 ]
*
100. The esul s showe
d
ha
c
op
i
g
m
en
a
i
on
o
cc
u
ed
o
m
p
H
a
l
ue
s
273 close o 1 up o neu ali y o all
he
pheno
lics
u
s
ed
a
s c
op
i
g
m
en
s. H
o
w
e
e
,
274 he magni ude o he copigmen a i
on
and
i s c
o
l
o
im
e
ic
e
e
c w
a
s s
ong
ly
p
H-
275 dependen and in luenced by h
e
na
u
e
o
he
c
op
i
g
m
en
u
s
ed
. T
he
g
ea
e
s
276 magni ude o copigmen a ion was
ob
a
i
ned
a
p
H
3
.
0
,
be
i
ng
si
gn
i ic
an
ly
h
i
ghe
277 wi h (-)-epica echin and ca eic
a
ci
d
. T
he
y
bo
h
i
n
c
ea
s
ed
e
s
pe
c i
e
ly
he
o
a
l
278 colo o M 3-gl by 13.70% and
13
.
80%
, w
h
il
e
(+)-c
a
e
c
h
i
n
, w
h
ic
h
w
a
s
he
l
e
ss
279 e ec i e copigmen , only each
ed
an
i
n
c
ea
s
e
o
9
.
30%
. T
h
is
ag
ee
wi
h
o
he
280 s udies ha ha e shown ha am
ong
he
d
i
e
en
l
a
an
-
3
-
o
ls c
op
i
g
m
en
s,
he
281 mo e plana molecules as (-)-epic
a
e
c
h
i
n
,
o
wi
h
e
l
e
c
on
-
dono
s
ub
s i
uen
s
a
s
282 cinnamic acids can be e s
a
ck wi
h
an
ho
cy
an
i
n
s
e
s
u
l i
ng
i
n
h
i
ghe
283 copigmen a ion e ec (11,
28
-
30
). T
he
sli
gh
ly l
o
w
e
a
l
ue
s
o
he
284 copigmen a ion magni ude ob ai
ned
i
n
h
is s
ud
y
e
s
pe
c i
ng
o
ho
s
e
epo
ed
i
n
285 he li e a u e could be due bo h
o
he
l
o
w
e
p
i
g
m
en
/c
op
i
g
m
en
m
o
l
a
a
i
o
u
s
ed
286 and he chemical na u e o he
e
s
ed
c
op
i
g
m
en
s. I
ha
s
been
e
s
ab
lis
hed
ha
287 he di e ences in he numbe , h
e
siz
e
o
he
s
pa
i
a
l l
o
c
a
i
on
o
he
s
ub
s i
uen
s
288 make monome ic la an-3-ols as c
a
e
c
h
i
n
,
ep
ic
a
e
c
h
i
n
o
sim
p
l
e
pheno
lic
a
ci
d
s,
289 exhibi weake copigmen a ion
han
m
o
e
p
l
ana
pheno
lic c
o
m
pound
s
a
s
290 la onols (31).
291 As shown in Table 1, h
e
CIE
L
AB
d
i
e
en
c
e
s (E
*
ab, 
L*
, C
*
ab, 
h
ab)
292 be ween M 3-gl solu ions wi h
and
wi
hou
c
op
i
g
m
en
s w
e
e
a
ls
o
c
a
lc
u
l
a
ed
.
293 Along he pH a ia ion, he highes c
o
l
o
d
i
e
en
c
e
s (E
*
ab) w
e
e
p
odu
c
ed
o
m
294 pH 1.0 o pH 3.0, con i ming h
e
c
o
l
o
im
e
ic s
ab
iliz
a
i
on
o
he
l
a
yli
u
m i
on
a
295 lowe han a highe pH alues (Y
an
e
a
l.,
2011
). S
pe
ci ic
a
lly,
he
c
o
l
o
296 di e ences inc eased om 1.73
o
3
.
06
,
o
m
2
.
88
o
3
.
69
and
o
m
3
.
39
o
3
.
87
ACS P
a
a
gon Plu
s
En
i onm
e
n
12
P
a
g
e
13 o
36
297
298
299
300
301
302
303
304
305
306
307
308
309
310
311
312
313
314
315
316
317
318
319
320
321
J
ou
n
a
l o Ag i
c
ul
u
a
l
a
nd Food Ch
e
mi
s
y
CIELAB uni s, in M 3-gl solu ion c
op
i
g
m
en
ed
wi
h
(+)-c
a
e
c
h
i
n
, (-)-
ep
ic
a
e
c
h
i
n
and ca eic acid, espec i ely. Th
e
e
o
e
,
a
l
hough
l
a
an
-
3
-
o
ls
ha
s
been
u
s
ua
lly
conside ed poo e an hocyanin c
op
i
g
m
en
s,
a
m
o
s
a
ci
d
ic
p
H
a
l
ue
s,
he
c
o
l
o
changes induced we e always p
e
c
ep
i
b
l
e
b
y
he
hu
m
an
e
y
e
(
32
). H
o
w
e
e
,
a
pH 4.0 and 5.0, he e was no si
gn
i ic
an
d
i
e
en
c
e
be
w
een
he
c
o
l
o
e
e
c
induced by he la anols es ed,
and
a
ll
he
c
o
l
o
d
i
e
en
c
e
s c
a
lc
u
l
a
ed
w
e
e
smalle han 2 CIELAB uni s, ha is,
no
is
ua
lly
d
is i
ngu
is
hed
.
In gene al, ca eic acid
p
odu
c
ed
he
l
a
ge
s c
o
l
o
e
e
c s, w
h
ic
h
w
a
s
cohe en wi h he esul s ob ain
ed
abou
he
m
agn
i
ude
o
c
op
i
g
m
en
a
i
on
w
hen
i was assessed by T is imulus C
o
l
o
im
e
y. O
n
he
c
on
a
y,
a
s c
an
be
c
he
ck
ed
in Table 1, a some pH alues, w
h
il
e
c
o
l
o
c
hange
s w
e
e
de
e
c
ed
i
n
he
CIELAB colo space, no chan
ge
s
a
A520
n
m w
a
s
ob
s
e
ed
. T
h
is
d
i
e
gen
c
e
be ween bo h analy ical me hods
ha
s
been
p
e
i
ou
sly
epo
ed
b
y G
on
z
á
l
e
z-
Manzano (14) and con i ms ha
he
m
o
s sim
p
li i
ed
m
e
hod
s
no
a
lw
a
ys c
an
achie e a eliable e alua ion o h
e
c
op
i
g
m
en
a
i
on
p
o
c
e
ss.
Wi h espec o he indi id
ua
l c
o
l
o
a
i
bu
e
s,
ega
d
l
e
ss
o
p
H
a
l
ue
and
he copigmen used, copigmen
a
i
on
i
ndu
c
ed
si
gn
i ic
an
c
o
l
o
im
e
ic c
hange
s
consis ing in a dec ease o ligh
ne
ss (
L*
)
and
hue
(
h
ab),
a
s w
e
ll
a
s
an
i
n
c
ea
s
e
o ch oma (C
*
ab). The ligh ness
and
c
h
o
m
a
d
i
e
en
c
e
s (
L*
and
C
*
ab) w
e
e
mo e p onounced be ween pH
2
.
0
and
3
.
0
, w
h
il
e
he
l
a
ge
s c
hange
s
on
he
hue (hab) we e p oduced a hi
ghe
p
H
a
l
ue
s. T
h
is i
nd
i
ng
m
ean
s
ha
i
n
quan i a i ely e ms, ac oss he
d
i
e
en
p
H
a
l
ue
s s
ud
i
ed
,
he
o
i
g
i
na
l M
3
-
g
l
con ol solu ion change owa d d
a
k
e
and
m
o
e
i
n
en
s
e
c
o
l
o
due
s
pe
ci ic
a
lly
o
he copigmen a ion phenomen
on
. H
o
w
e
e
, i
n
qua
li
a
i
e
ly
e
ms,
due
o
he
no able in luence o he pH on
he
an
ho
cy
an
i
n
c
o
l
o
,
he
end
o
he
hue
ACS P
a
a
gon Plu
s
En
i onm
e
n
13
J
ou
n
a
l o Ag i
c
ul
u
a
l
a
nd Food Ch
e
mi
s
y
P
a
g
e
14
o
36
322 changes we e mo e a iable. F
o
m
p
H
1
.
0
o
4
.
0
, M
3
-
g
l c
on
o
l s
o
l
u
i
on
323 dec eased owa ds mo e bluish
hue
s w
h
il
e
de
c
ea
s
ed
o
w
a
d
s m
o
e
edd
is
h
a
324 pH 5.0, as can be obse ed i
n
Fi
gu
e
2
. T
he
a
c
ha
hue
s
d
i
e
en
c
e
s
a
e
325 almos indisce nible in he (a
*
b*)
d
i
ag
a
m
a
p
H
4
.
0
and
5
.
0
c
an
be
e
x
p
l
a
i
ned
326 based on he impo an deg a
da
i
on
o
he
c
o
l
o
ed
o
ms i
n
he
an
ho
cy
an
i
n
327 equilib ium a highe pH alu
e
s, w
h
ic
h
m
ade
M
3
-
g
l s
o
l
u
i
on
p
a
c ic
a
lly
328 ach oma ic.
329 Fo a mo e comp ehensi
e
ana
lysis
o
he
c
o
l
o
im
e
ic im
p
lic
a
i
on
o
330 copigmen a ion p ocess as a
un
c i
on
o
he
p
H,
he
e
l
a
i
e
c
on
i
bu
i
on
o
331 ligh ness (%L), ch oma (%C),
and
hue
(
%
H)
o
he
o
a
l c
o
l
o
d
i
e
en
c
e
o
332 each pigmen /copigmen solu i
on
w
e
e
a
ls
o
c
a
lc
u
l
a
ed
. As c
an
be
s
een
i
n
333 Figu e 3, he main con ibu ion
o
he
m
ea
s
u
ed
c
o
l
o
d
i
e
en
c
e
s ΔE
*
ab
a
p
H
334 1.0 was quali a i e, which was
e
i
den
c
ed
b
y
he
si
gn
i ic
an
h
i
ghe
c
on
i
bu
i
on
335 o hue %ΔH (80-88%) wi h es
pe
c
o
he
li
gh
ne
ss
%

L
o
c
h
o
m
a
%
C (
7
-
336 10% and 6-10%, espec i ely). T
he
s
e
e
s
u
l s
a
e
c
ohe
en
si
n
c
e
a
p
H 
2
,
he
337 an hocyanin exis s p ima ily in
he
o
m
o
he
ed
c
a
i
on
l
a
yli
u
m
and
he
338 amoun o he o he colo less s
pe
ci
e
s is
a
lm
o
s i
ne
xis
en
. T
he
e
o
e
,
a
h
is
p
H
339 alue, he copigmen ed complex
e
s c
an
i
ndu
c
e
ba
sic
a
lly c
o
l
o
a
i
a
i
on
bu
c
an
340 no p o ide impo an displaceme
n
o
he
an
ho
cy
n
i
n
equ
ili
b
i
u
m
o
w
a
d
he
ed
341 colo ed la ylium ca ion, which is
a
ls
o
e
l
e
c
ed
b
y
he
l
o
w
e
a
i
a
i
on
s
ob
a
i
ned
342 o E and E
*
ab. On he o he
hand
,
be
w
een
p
H
1
.
0
and
p
H
4
.
0
,
he
m
a
i
n
343 colo ime ic con ibu ion was
quan
i
a
i
e
;
be
i
ng
pa
ic
u
l
a
ly
he
c
h
o
m
a
344 modi ica ions %C mo e ma k
ed
han
li
gh
ne
ss
%

L
(
35
-
65%
and
22
-
38%
,
345 espec i ely). In his pH ange,
he
p
opo
i
on
o
he
o
he
c
o
l
o
l
e
ss s
pe
ci
e
s
346 inc ease coexis ing wi h he
ed
c
o
l
o
ed
l
a
yli
u
m c
a
i
on
i
n
s
o
l
u
i
on
, s
o
ACS P
a
a
gon Plu
s
En
i onm
e
n
14
P
a
g
e
15 o
36
347
348
349
350
351
352
353
354
355
356
357
358
359
360
361
362
J
ou
n
a
l o Ag i
c
ul
u
a
l
a
nd Food Ch
e
mi
s
y
copigmen a ion complexes can p
o
i
de
a
he
s
a
m
e
im
e
c
o
l
o
s
ab
iliz
a
i
on
and
a ia ion, which coincides wi h
he
g
ea
e
s c
hange
s
on
he
E
and
E
*
ab
a
l
ue
s.
F om pH 5.0 o neu ali y, he m
o
s
abundan
c
o
l
o
ed
s
pe
ci
e
s
p
e
s
en
is
he
quinoidal o m and hus M 3-gl i s
e
l
doe
s
no
c
on
e
m
u
c
h
c
o
l
o
o
a
s
o
l
u
i
on
,
so again he con ibu ion o he c
op
i
g
m
en
a
i
on
on
c
o
l
o
is m
a
i
n
ly
qua
li
a
i
e
,
ha is, signi ican ly due o hue ch
ange
s.
Mo eo e , he ela i e c
on
i
bu
i
on
o
li
gh
ne
ss (
%

L
), c
h
o
m
a
(
%
C),
and hue (%H) pe mi ed us o c
o
m
pa
e
he
c
o
l
o
im
e
ic
e
e
c i
ndu
c
ed
b
y
he
di e en la anols es ed. In g
ene
a
l
e
ms, (-)-
ep
ic
a
e
c
h
i
n
and
c
a
e
ic
a
ci
d
p oduced simila e ec s on he
o
a
l c
o
l
o
o
he
M
3
-
g
l
a
c
o
ss
o
he
p
H
changes, con ibu ing mo e ma k
ed
wi
h
quan
i
a
i
e
c
hange
s
han
qua
li
a
i
e
ones (%L + %C= 55-63%, an
d
%
ΔH=
45
-
37%
). O
n
he
c
on
a
y,
pa
ic
u
l
a
ly
a he highe pH alues, he quali
a
i
e
c
on
i
bu
i
on
w
a
s m
o
e
im
po
an
i
n
(+)-
ca echin copigmen ed solu ions. T
hu
s, i c
an
be
s
a
i
d
ha
he
c
op
i
g
m
en
a
i
on
o
(-)-epica echin and ca eic acid
p
o
i
de
no
ab
ly c
o
l
o
s
ab
iliz
a
i
on
and
a
i
a
i
on
,
while (+)-ca echin p o ide mo e i
n
en
s
e
c
o
l
o
a
i
a
i
on
han
s
ab
iliz
a
i
on
.
363
364 E ec o he mola
a io on copig
me
n
a
ion
365 The in luence o he pigm
en
/c
op
i
g
m
en
m
o
l
a
a
i
o
w
a
s
a
ls
o
a
ss
a
y
ed
a
366 pH 3.60. Fo his pu pose, w
o
c
op
i
g
m
en
ed
s
o
l
u
i
on
s
o
m
a
l i
d
i
n
3
-
g
l:(+)-
367 ca echin (MC) and mal idin 3-gl:(-)-
ep
ic
a
e
c
h
i
n
(ME) w
e
e
p
epa
ed
i
n
he
s
a
m
e
368 wine-like medium a he mola
a
i
o
s
1
:
1
,
1
:
2
,
1
:
5
,
and
1
:
7
.
369 The mean alues o he o
a
l c
o
l
o
(E)
o
he
o
i
g
i
na
l M
3
-
g
l s
o
l
u
i
on
and
370 each copigmen ed solu ion as a
un
c i
on
o
he
m
o
l
a
a
i
o
is
p
e
s
en
ed
i
n
T
ab
l
e
371 2. Fo bo h assays, he posi i e e
e
c
o
he
c
op
i
g
m
en
a
i
on
on
he
M
3
-
g
l
o
a
l

ACS P
a
a
gon Plu
s
En
i onm
e
n
15
J
ou
n
a
l o Ag i
c
ul
u
a
l
a
nd Food Ch
e
mi
s
y
P
a
g
e
16
o
36
372 colo was inc eased wi h inc
ea
si
ng
c
op
i
g
m
en
c
on
c
en
a
i
on
, w
h
ic
h
is i
n
373 ag eemen wi h p e ious s udi
e
s
epo
ed
i
n
b
i
b
li
og
aph
y
de
s
p
i
e
be
i
ng
l
o
w
e
374 he ange o he pigmen /copi
g
m
en
m
o
l
a
a
i
o
u
s
ed
(
33
). T
he
m
a
xim
u
m
375 displacemen o hyd a ion/dehy
d
a
i
on
equ
ili
b
i
u
m
o
w
a
d
s
he
c
o
l
o
ed
s
pe
ci
e
s
376 was achie ed using he highes m
o
l
a
a
i
o
(
1
:
7
) si
n
c
e
i c
au
s
ed
he
g
ea
e
s
377 o al colo inc eases ( om 49.26
o
57
.
32
and
57
.
90
CIE
L
AB
u
. i
n
MC
and
ME
378 solu ions, espec i ely).
379 Mo eo e , signi ican di e
en
c
e
s
on
he
L*
,
a*
,
and
b*
a
l
ue
s
a
m
ong
he
380 pu e M 3-gl solu ion and i s co
e
s
pond
i
ng
c
op
i
g
m
en
ed
w
e
e
ound
,
e
s
pe
ci
a
lly
381 a mola a ios highe han 1:2,
e
s
u
l i
ng
i
n
a
d
i
e
en
l
o
c
a
i
on
o
he
s
a
m
p
l
e
s i
n
382 he (a*b
*
) colo ime ic diag am (Fi
gu
e
4
). I
n
ab
s
en
c
e
o
an
y c
op
i
g
m
en
s,
he
383 o iginal an hocyanin colo ook p
o
si i
e
a*
and
b*
a
l
ue
s
and
appea
ed
l
o
c
a
ed
384 nea 0º, ha is, in a well de ined
edd
is
h
c
o
l
o
a
ea
(
L*
=
69
.
71
,
a*
=
38
.
84
,
and
385 b*= 0.39 CIELAB u.). Howe e , wi
h
he
i
n
c
ea
si
ng
add
i i
on
o
he
l
a
ano
l
386 copigmen s, he samples a
ppea
ed
l
o
c
a
ed
p
og
e
ssi
e
ly
a
e
o
he
387 coo dina es o igin and expe im
en
ed
a
e
m
a
k
ab
l
e
e
o
l
u
i
on
o
m
he
i s
388 owa d he ou h quad an , ha is,
o
m
he
edd
is
h
o
he
pu
p
l
e
o
ed
-
b
l
u
is
h
389 colo s egion. The beha io o
he
c
o
l
o
pa
a
m
e
e
s
a
s
a
un
c i
on
o
he
390 concen a ion was simila o b
o
h
he
c
op
i
g
m
en
u
s
ed
. A
he
h
i
ghe
s m
o
l
a
391 a io (1:7), copigmen ed samples
ea
c
hed
he
l
o
w
e
s
L*
and
b*
a
l
ue
s
bu
he
392 highes a
*
alues, and hus, hey
e
x
h
i
b
i
ed
he
da
k
e
s
and
m
o
s i i
d
b
l
u
is
h
393 colo (L* = 64.18 and 63.05, a* =
45
.
44
and
43
.
76
,
and
b*
= -
2
.
23
and
-
2
.
21
394 CIELAB u. o MC and ME solu i
on
s,
e
s
pe
c i
e
ly).
395 Rega ding he changes o
n
he
c
on
i
bu
i
on
o
he
c
op
i
g
m
en
a
i
on
o
he
396 o al an hocyanin colo , he adde
d
c
op
i
g
m
en
s c
au
s
ed
si
gn
i ic
an
c
on
c
en
a
i
on
-
ACS P
a
a
gon Plu
s
En
i onm
e
n
16
P
a
g
e
17 o
36
397
398
399
400
401
402
403
404
405
J
ou
n
a
l o Ag i
c
ul
u
a
l
a
nd Food Ch
e
mi
s
y
dependen inc eases on he ma
gn
i
ude
o
he
e
e
c ,
a
s c
an
be
s
een
i
n
Fi
gu
e
2
.
F om 1:1 o 1:7 mola a io, (+)-c
a
e
c
h
i
n
i
ndu
c
ed
an
i
n
c
ea
s
e
o
he
M
3
-
g
l
o
a
l
colo om 1.2% o 16.0%, whil
e
wi
h
(-)-
ep
ic
a
e
c
h
i
n
,
he
i
n
c
ea
s
e
w
a
s
o
m
3.9% o 17.9%. No wi hs andin
g
,
a
l
hough
he
pa
e
n
e
o
l
u
i
on
w
a
s simil
a
be ween bo h la anols copigm
en
s,
on
c
e
aga
i
n
,
he
ab
ili y
o
he
(-)-
ep
ic
a
e
c
h
i
n
o ac as M -3gl copigmen s was s
onge
han
o
(+)-c
a
e
c
h
i
n
si
n
c
e
si
gn
i ic
an
highes alues o copigmen a i
on
w
a
s
a
c
h
i
e
ed
o
m
o
s
o
he
m
o
l
a
a
i
o
s
es ed.
As was made in he p
e
i
ou
s s
e
c i
on
o
h
is s
ud
y,
he
dependen
c
e
406 be ween he ampli ude o he c
o
l
o
e
e
c i
ndu
c
ed
b
y c
op
i
g
m
en
a
i
on
w
a
s
407 e alua ed by means o he CIE
L
AB c
o
l
o
d
i
e
en
c
e
s (E
*
ab) (T
ab
l
e
2
). As
408 expec ed, he p og essi e o m
a
i
on
o
c
op
i
g
m
en
a
i
on
c
o
m
p
l
e
x
e
s w
a
s
409 con i med by a successi e i
n
c
ea
s
e
on
he
c
o
l
o
d
i
e
en
c
e
s
be
w
een
410 an hocyanins and la anols s
o
l
u
i
on
s w
hen
c
op
i
g
m
en
c
on
c
en
a
i
on
s w
e
e
411 inc easingly added. Fo all he c
on
c
en
a
i
on
l
e
e
l
e
s
ed
, (-)-
ep
ic
a
e
c
h
i
n
c
au
s
ed
412 always signi ican inc eases (p

0
.
05
)
on
he
c
o
l
o
d
i
e
en
c
e
s. I
n
c
on
a
s ,
413 highe inc eases on he mola
a
i
o
w
e
e
ne
c
e
ss
a
y
o
i
ndu
c
e
si
gn
i ic
a
i
e
414 changes wi h (+)-ca echin copigm
en
ed
s
o
l
u
i
on
s.
415 In any case, calcula ion
o
he
sim
p
l
e
eg
e
ssi
on
c
oe
ici
en
s
be
w
een
416 o al colo di e ences and he c
op
i
g
m
en
c
on
c
en
a
i
on
o
ea
c
h
c
op
i
g
m
en
ed
417 solu ion con i med ha he ela i
on
s
h
i
p
w
a
s si
gn
i ic
an
ly
h
i
gh
ega
d
i
ng
he
418 copigmen used (
*
alues an
ged
o
m +
0
.
95
o
+
0
.
98
un
i s,
p
<
0
.
05
). T
he
419 lowes colo di e ences we e
ound
be
w
een
pu
e
M
3
-
g
l s
o
l
u
i
on
and
he
420 copigmen ed ones a he lowes m
o
l
a
a
i
o
u
s
ed
(
1
:
1
),
a
ki
ng
a
l
ue
s
o
1
.
01
421 and 2.27 CIELAB u. wi h (+)-c
a
e
c
h
i
n
and
(-)-
ep
ic
a
e
c
h
i
n
,
e
s
pe
c i
e
ly.
ACS P
a
a
gon Plu
s
En
i onm
e
n
17
P
a
g
e
23 o
36
J
ou
n
a
l o Ag i
c
ul
u
a
l
a
nd Food Ch
e
mi
s
y
532 8. B ouilla d, R.; Mazza, G.; S
aad
, Z.; Al
b
e
c
h
-G
a
y, A.M.; C
he
mi
na
, A. T
he
533 copigmen a ion eac i
on
o
an
ho
cy
an
i
n
s: A mic
op
obe
o
he
534 s uc u al s udy o a
queou
s s
o
l
u
i
on
s. J
ou
na
l
o
he
Am
e
ic
an
535 Chemical Socie y. 1989,
111
,
2604
-
2610
.
536 9. Da ies, A.J.; Mazza, G. C
op
i
g
m
en
a
i
on
o
sim
p
l
e
and
a
cyl
a
ed
537 an hocyanins wi h colo l
e
ss
pheno
lic c
o
m
pound
s. J. A
g
ic. F
ood
C
he
m.
538 1993, 41, 716-720.
539 10. Ba anac, J.M.; Pe
ano
ic, N.A.; Dimi ic-M
a
k
o
ic, J.M.
540 Spec opho ome ic S
ud
y
o
A
n
ho
cy
an
C
op
i
g
m
en
a
i
on
R
ea
c i
on
s.
2
.
541 Mal in and he Nonglyc
o
si
d
iz
ed
Fl
a
one
Q
ue
c
e
i
n
. J. A
g
ic. F
ood
542 Chem. 1997, 45, 1694-1
697
.
543 11. Ei o, M.J.; Heinonen, M. A
n
ho
cy
an
i
n
C
o
l
o
B
eha
i
o
and
S
ab
ili y
du
i
ng
544 S o age:Çë E ec o I
n
e
m
o
l
e
c
u
l
a
C
op
i
g
m
en
a
i
on
. J. A
g
ic. F
ood
545 Chem. 2002, 50, 7461-7
466
.
546 12. Talco , S.T.; B enes, C.H.; Pi
e
s, D.M.; D
e
l P
o
z
o
-I
n
s
an
, D.
547 Phy ochemical S abili y
and
C
o
l
o
R
e
en
i
on
o
C
op
i
g
m
en
ed
and
548 P ocessed Muscadine G
ape
J
u
ic
e
. J. A
g
ic. F
ood
C
he
m.
2003
,
51
,
549 957-963.
550 13. G is, E.; Fe ei a, E.; Falc
ao
,
L
.; B
o
d
i
gnon
-
Lu
iz, M. I
n
l
uen
c
e
o
e
u
lic
551 acid on s abili y o an hocy
an
i
n
s
o
m C
abe
ne
S
au
i
gnon
g
ape
s i
n
a
552 model sys em and a y
ogu
sys
e
m. I
n
e
na
i
ona
l j
ou
na
l
o
ood
553 science and echnology.
2007
,
42
,
992
-
998
.
ACS P
a
a
gon Plu
s
En
i onm
e
n
23

J
ou
n
a
l o Ag i
c
ul
u
a
l
a
nd Food Ch
e
mi
s
y
P
a
g
e
24
o
36
554 14. González-Manzano, S.; D
ueña
s, M.; Ri
a
s-G
on
z
a
l
o
, J.C.; Esc i
bano
-
555 Bailón, M.T.; San os-B
ue
l
ga
, C. S
ud
i
e
s
on
he
c
op
i
g
m
en
a
i
on
556 be ween an hocyanins
and
l
a
an
-
3
-
o
ls
and
he
i i
n
l
uen
c
e
i
n
he
c
o
l
ou
557 exp ession o ed wine. F
ood
C
he
mis y.
2009
,
114
,
649
-
656
.
558 15. Lambe , S.G.; Asens o e , R.E.; Willi
a
ms
on
, N.M.; Il
and
, P.G.; J
one
s,
559 G.P. Copigmen a ion b
e
w
een
m
a
l i
d
i
n
-
3
-
g
l
u
c
o
si
de
and
s
o
m
e
wi
ne
560 cons i uen s and i s imp
o
an
c
e
o
c
o
l
ou
e
x
p
e
ssi
on
i
n
ed
wi
ne
. F
ood
561 Chemis y. 2011, 125,
106
-
115
.
562 16. He mosín-Gu ié ez, I. In l
uen
c
e
o
E
hano
l C
on
en
on
he
Ex
en
o
563 Copigmen a ion in a C
en
ci
be
l Y
oung
R
ed
Wi
ne
. J. A
g
ic. F
ood
C
he
m.
564 2003, 51, 4079-4083.
565 17. Gu ié ez, I.H.; Lo enzo, E.S.-P.; Es
p
i
no
s
a
, A.V. P
heno
lic c
o
m
po
si i
on
and
566 magni ude o copigme
n
a
i
on
i
n
y
oung
and
s
ho
ly
aged
ed
wi
ne
s
567 made om he cul i a s, C
abe
ne
S
au
i
gnon
, C
en
ci
be
l,
and
Sy
ah
.
568 Food Chemis y. 2005,
92
,
269
-
283
.
569 18. Da ias-Ma ín, J.; Ca illo-L
ópe
z, M.; Ec
ha
a
i-G
anado
, J.F.; í
a
z-R
o
m
e
o
,
570 C. The magni ude o co
p
i
g
m
en
a
i
on
i
n
he
c
o
l
ou
o
aged
ed
wi
ne
s
571 made in he Cana y Isl
and
s. E
u
opean
F
ood
R
e
s
ea
c
h
and
572 Technology. 2007, 224,
643
-
648
.
573 19. Guadalupe, Z.; Ayes a án, B. E
e
c
o
C
o
mm
e
ci
a
l M
annop
o
e
i
n
A
dd
i i
on
574 on Polysaccha ide, Poly
pheno
lic,
and
C
o
l
o
C
o
m
po
si i
on
i
n
R
ed
Wi
ne
s.
575 J. Ag ic. Food Chem. 20
08
,
56
,
9022
-
9029
.
ACS P
a
a
gon Plu
s
En
i onm
e
n
24
P
a
g
e
25 o
36
J
ou
n
a
l o Ag i
c
ul
u
a
l
a
nd Food Ch
e
mi
s
y
576 20. Go dillo, B.; López-In an e, M.I.; R
a
mí
e
z-P
e
¦
ü
e
z, P.; G
on
z
á
l
e
z-Mi
e
,
577 M.L.; He edia, F.J. In lue
n
c
e
o
P
e
e
m
en
a
i
e
C
o
l
d
M
a
c
e
a
i
on
on
he
578 Colo and An hocyanic C
op
i
g
m
en
a
i
on
o
O
gan
ic T
e
m
p
an
ill
o
Wi
ne
s
579 Elabo a ed in a Wa m Clim
a
e
. J. A
g
ic. F
ood
C
he
m.
2010
,
58
,
6797
-
580 6803.
581 21. Gonne , J.F. Colou e ec s
o
c
o
-
p
i
g
m
en
a
i
on
o
an
ho
cy
an
i
n
s
e
isi
ed
-
582 1. A colo ime ic de ini i
on
u
si
ng
he
CIE
L
AB sc
a
l
e
. F
ood
C
he
mis y.
583 1998, 63, 409-415.
584 22. Asen, S.; S ewa , R.N.; N
o
is, K.H. C
o
-
p
i
g
m
en
a
i
on
o
an
ho
cy
an
i
n
s i
n
585 plan issues and i s e
e
c
on
c
o
l
o
. P
h
y
o
c
he
mis y.
1972
,
11
,
1139
-
586 1144.
587 23. Dangles, O.; B ouilla d, R. A s
pe
c
o
sc
op
ic m
e
hod
ba
s
ed
on
he
588 an hocyanin copigmen a i
on
i
n
e
a
c i
on
and
app
li
ed
o
he
quan
i
a
i
e
589 s udy o molecula compl
e
x
e
s. J
ou
na
l
o
he
C
he
mic
a
l S
o
ci
e
y, P
e
ki
n
590 T ansac ions 2. 1992,
247
-
257
.
591 24. He edia, F.J.; Ál a ez, C.; G
on
z
á
l
e
z-Mi
e
, M.
L
.; & R
a
mí
e
z, A. C
o
m
aLab
,
592 análisis de colo . Regis
o
G
ene
a
l
de
l
a
P
op
i
edad
I
n
e
l
e
c
ua
l.
2004
.
593 25. CIE Technical Repo C
o
l
o
im
e
y; C
o
mmissi
on
I
n
e
na
i
ona
l
e
de
594 l'Eclai age Cen al Bu e
au
: Vi
enna
, A
u
s i
a
,
2004
.
595 26. Melgosa, M.; Hi a, E.; Poza, A.J.; Alm
an
, D.H.; B
e
n
s, R.S. S
up
a
h
e
s
ho
l
d
596 colo -di e ence ellipsoi
d
s
o
s
u
a
c
e
c
o
l
o
s. C
o
l. R
e
s. A
p
l.
1997
,
22
,
597 148-155.
ACS P
a
a
gon Plu
s
En
i onm
e
n
25
J
ou
n
a
l o Ag i
c
ul
u
a
l
a
nd Food Ch
e
mi
s
y
P
a
g
e
26
o
36
598 27. He edia, F.J.; F ancia-A ic
ha
, E.M.; Ri
a
s-G
on
z
a
l
o
, J.C.; Vic
a
i
o
, I.M.;
599 San os-Buelga, C. Ch
o
m
a
ic c
ha
a
c
e
iz
a
i
on
o
an
ho
cy
an
i
n
s
o
m
ed
600 g apes - I. pH e ec . Fo
od
C
he
mis y.
1998
,
63
,
491
-
498
.
601 28. Da ias-Ma ín, J.; Ma ín-L
u
is, B.; C
a
ill
o
-
Lópe
z, M.;
La
m
ue
l
a
-R
a
en
ó
¦s,
602 R.; Díaz-Rome o, C.; B
ou
l
on
, R. E
e
c
o
c
a
e
ic
a
ci
d
on
he
c
o
l
o
o
603 ed wine. J. Ag ic. Food C
he
m.
2002
,
50
,
2062
-
2067
.
604 29. Gómez-Míguez, M.; Gonz
a
l
e
z-M
an
z
ano
, S.; G
on
z
á
l
e
z-Mi
e
, M.
L
.; T
e
e
s
a
605 Esc ibano-Bailón, M.; H
e
ed
i
a
, F.J.; S
an
o
s-B
ue
l
ga
, C. I
n
l
uen
c
e
o
606 di e en phenolic copi
g
m
en
s
on
he
c
o
l
o
o
m
a
l i
d
i
n
3
-
g
l
u
c
o
si
de
. J.
607 Ag ic. Food Chem. 2006,
54
,
5422
-
5429
.
608 30. C uz, L.; B ás, N.F.; Teixei
a
, N.; M
a
eu
s, N.; R
a
m
o
s, M.J.; D
ang
l
e
s, O.;
609 De F ei as, V. Vinylca
e
c
h
i
n
d
im
e
s
a
e
m
u
c
h
be
e
c
op
i
g
m
en
s
o
610 an hocyanins han ca ec
h
i
n
d
im
e
p
o
cy
an
i
d
i
n
B
3
. J. A
g
ic. F
ood
C
he
m.
611 2010, 58, 3159-3166.
612 31. Kunsági-Ma é, S.; Szabó, K.; Nik
a
d
j
a
m, M.P.; K
o
ll
a
,
L
. D
e
e
mi
na
i
on
o
613 he he modynamic pa
a
m
e
e
s
o
he
c
o
m
p
l
e
x
o
m
a
i
on
be
w
een
614 mal idin-3-O-glucoside
and
po
ly
pheno
ls. C
op
i
g
m
en
a
i
on
e
e
c i
n
ed
615 wines. Jou nal o Biochemic
a
l
and
Bi
oph
ysic
a
l M
e
hod
s.
2006
,
69
,
113
-
616 119.
617 32. Ma ínez, J.A.; Melgosa, M.; P
é
e
z, M.M.; Hi
a
, E.; N
egue
ue
l
a
, A.I. N
o
e
.
618 Visual and ins umen al c
o
l
o
e
a
l
ua
i
on
i
n
ed
wi
ne
s. F
ood
Sci
en
c
e
619 and Technology In e na i
ona
l.
2001
,
7
,
439
-
444
.
ACS P
a
a
gon Plu
s
En
i onm
e
n
26
P
a
g
e
27 o
36
J
ou
n
a
l o Ag i
c
ul
u
a
l
a
nd Food Ch
e
mi
s
y
620 33. Be ké, B.; De F ei as, V. A c
o
l
o
im
e
ic s
ud
y
o
oen
i
n
c
op
i
g
m
en
ed
b
y
621 p ocyanidins. Jou nal o
he
Sci
en
c
e
o
F
ood
and
A
g
ic
u
l
u
e
.
2007
,
87
,
622 260-265.
623
624
ACS P
a
a
gon Plu
s
En
i onm
e
n
27
J
ou
n
a
l o Ag i
c
ul
u
a
l
a
nd Food Ch
e
mi
s
y
P
a
g
e
28
o
36
625 Table 1. Mean alues o he A
520
and he o al colo (E) ob ained o he pu e
626 an hocyanic solu ion and i s espec i e copigmen ed a each pH alue; as well as, he
627 CIELAB di e ences (E*
ab
, L*, C*
ab
, h
ab
) be ween M 3-gl solu ions wi h and
628 wi hou copigmen s.
629
Pigmen
pH
M
3-gl
Pigmen /Copigmen
MC
ME
MF
1
0
.
86
±
0
.
001
a
2
0.
73
±
0
.
002
a
A
520
3
0.
32
±
0
.
001
a
4
0.
10
±
0
.
003
a
5
0
.
06
±
0
.
001
a
1
59
.
51
±
0
.
98
a
2
54
.
37
±
1
.
67
a
E
3
2
9
.
13
±
0
.
98
a
4
9
.
47
±
1
.
67
a
5
5
.
12
±
0
.
60
a
1
-
2
-

E*
ab
3
-
4
-
5
-1
-
2
-

L*
3
-
4
-
5
-1
-
2
-

C
*ab
3
-
4
-
5
-1
-
2
-

h
ab
3
-
4
-
5
-
0
.
84
±
0
.
001
b
0
.
78
±
0
.
03
a
0
.
34
±
0
.
001
b
0
.
10
±
0
.
003
a
0
.
05
±
0
.
005
a
60
.
10
±
0
.
23
b
57
.
51
±
1
.
77
b
31
.
85
±
0
.
17
b
10
.
22
±
0
.
27
b
4
.
93
±
0
.
38
a
1
.
73
±
0
.
06
a
2
.
92
±
0
.
48
a
3
.
06
±
0
.
17
a
1
.
29
±
0
.
08
a
1
.
01
±
0
.
14
a
-
0
.
46
±
0
.
11
a
-
2
.
64
±
0
.
23
a
-
0
.
89
±
0
.
01
a
-
0
.
03
±
0
.
20
a
+
0
.
23
±
0
.
28
a
+
0
.
41
±
0
.
21
a
+
0
.
47
±
0
.
20
a
+
2
.
59
±
0
.
18
a
+
0
.
91
±
0
.
19
a
+
0
.
02
±
0
.
06
a
-
1
.
74
±
0
.
07
a
-
1
.
30
±
0
.
75
a
-
2
.
91
±
0
.
08
a
-
6
.
65
±
0
.
65
a
-
17
.
28
±
2
.
30
a
0
.
84
±
0
.
008
b
0
.
73
±
0
.
002
a
0
.
36
±
0
.
004
c
0
.
11
±
0
.
001
b
0
.
06
±
0
.
001
a
60
.
77
±
0
.
26
c
55
.
83
±
0
.
17
b
32
.
68
±
0
.
34
c
10
.
82
±
0
.
01
c
5
.
40
±
0
.
12
a
2
.
88
±
0
.
04
b
2
.
29
±
0
.
12
a
3
.
69
±
0
.
37
b
1
.
38
±
0
.
02
a
0
.
54
±
0
.
09
a
-
0
.
91
±
0
.
29
ab
-
1
.
16
±
0
.
02
a
-
1
.
97
±
0
.
20
b
-
0
.
77
±
0
.
21
b
-
0
.
29
±
0
.
10
a
+
0
.
95
±
0
.
15
b
+
1
.
03
±
0
.
19
a
+
2
.
95
±
0
.
27
a
+
1
.
09
±
0
.
01
a
+
0
.
05
±
0
.
01
a
-
2
.
74
±
0
.
20
b
-
1
.
93
±
0
.
07
a
-
2
.
12
±
0
.
29
b
-
2
.
75
±
0
.
84
b
-
5
.
56
±
0
.
92
b
0
.
83
±
0
.
004
b
0
.
77
±
0
.
03
a
0
.
37
±
0
.
003
c
0
.
12
±
0
.
00
5
b
0
.
05
±
0
.
002
a
60
.
79
±
0
.
16
c
57
.
88
±
1
.
4
2
b
32
.
79
±
0
.
2
0 c
11
.
06
±
0
.
38
c
5
.
52
±
0
.
47
a
3
.
39
±
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.
13
c
3
.
78
±
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.
06
a
3
.
87
±
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.
24
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.
59
±
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.
27
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.
71
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.
14
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-
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.
95
±
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.
10
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-
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.
27
±
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.
8
2
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.
14
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.
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17
b
+
2
.
98
±
0
.
1
5
a
+
1
.
17
±
0
.
1
6
a
+
0
.
44
±
0
.
24
b
-
3
.
35
±
0
.
15
c -
1
.
99
±
0
.
5
3
a
-
2
.
61
±
0
.
35
ab
-
2
.
20
±
0
.
1
7
b
-
6
.
41
±
3
.
07
b

ACS P
a
a
gon Plu
s
En
i onm
e
n
28
P
a
g
e
29 o
36
630
631
632
633
634
J
ou
n
a
l o Ag i
c
ul
u
a
l
a
nd Food Ch
e
mi
s
y
Table 2. Mean alues o he A
520
and he o al colo (E) ob ained o he pu e
an hocyanic solu ion and i s espec i e copigmen ed a each mola a io; as well as, he
CIELAB di e ences (E*
ab
, L*, C*
ab
, h
ab
) be ween M 3-gl solu ions wi h and
wi hou copigmen s.
Colo ime ic
pa ame e s
A
520
E
0
0
.
66
±
0
.
00
1
a
49
.
26
±
0
.
45
a
1
0
.
68
±
0
.
001
a
49
.
84
±
0
.
12
a
Mola Ra io
2
0
.
77
±
0
.
008
b
52
.
64
±
0
.
58
b
5
0
.
77
±
0
.
008
b
54
.
40
±
0
.
50
b
7
0
.
84
±
0.004
c
57
.
3
2
±
0.90
c
MC
ME
E*
ab
-
L* -
C*
ab
-
h
ab
-
A
520
0
.
67
±
0
.
004
a
E
49
.
26
±
0
.
001
a
E*
ab
-
L*
-C*
ab
-
h
ab
1
.
01
±
0
.
33
a
-
0
.
16
±
0
.
54
a
+
0
.
60
±
0
.
26
a
-
1
.
21
±
0
.
60
a
0
.
72
±
0
.
01
b
51
.
21
±
0
.
10
b
2
.
27
±
0
.
20
a
-
1
.
77
±
0
.
23
a
+
1
.
08
±
0
.
30
a
-
1
.
02
±
0
.
54
a
4
.
89
±
0
.
37
b
-
4
.
51
±
0
.
33
b
+
0
.
65
±
1
.
06
a
-
2
.
56
±
0
.
99
a
0
.
76
±
0
.
004
c
52
.
51
±
0
.
28
c
4
.
53
±
0
.
91
b
-
3
.
99
±
1
.
12
b
+
0
.
90
±
0
.
95
a
-
2
.
39
±
0
.
83
ab
6
.
36
±
0
.
19
b
-
3
.
95
±
0
.
66
b
+
3
.
40
±
1
.
17
a
-
5
.
05
±
0
.
13
b
0
.
78
±
0
.
008
d
55
.
50
±
0
.
38
d
6
.
65
±
0
.
33
c
-
4
.
16
±
0
.
37
b
+
4
.
67
±
0
.
34
b
-
3
.
11
±
0
.
47
b
9
.
7
6
±
1.27
c
-
8
.
56
±
0.99
c
+
2
.
5
9
±
3.60
a
-
5
.
1
2
±
0.13
b
0
.
82
±
0.002
e
57
.
90
±
0.36
e
10
.
69
±
0.44
d
-
5
.
5
3
±
0.24
b
+
6
.
64
±
0.63
c
-
3
.
3
8
±
0.50
b
ACS P
a
a
gon Plu
s
En
i onm
e
n
29
J
ou
n
a
l o Ag i
c
ul
u
a
l
a
nd Food Ch
e
mi
s
y
P
a
g
e
30
o
36
635 FIGURE CAPTIONS
636 Figu e 1. Change in he loca io
n
o
he
pu
e
M
3
-
g
l
and
c
op
i
g
m
en
ed
s
o
l
u
i
on
s
637 (MC, ME, and MF) wi hin (a) he (
a*b*
)
d
i
ag
a
m
and
(
b
) li
gh
ne
ss
a
l
ue
s (
L*
)
a
s
638 a unc ion o he pH alue.
639
640 Figu e 2. (a) Changes on he m
agn
i
ude
o
c
op
i
g
m
en
a
i
on
o
he
M
3
-
g
l
641 solu ions copigmen ed wi h (+)-c
a
e
c
h
i
n
(MC), (-)-
ep
ic
a
e
c
h
i
n
(ME),
and
c
a
e
ic
642 acid (MF) as a unc ion o he pH,
and
(
b
) wi
h
(+)-c
a
e
c
h
i
n
(MC), (-)-
ep
ic
a
e
c
h
i
n
643 (ME) as a unc ion o he mola
a
i
o
,
e
a
l
ua
ed
b
y T is im
u
l
u
s C
o
l
o
im
e
y
a
s
644 he a io [(EC- E0) / E0 ]
*
100.
645
646 Figu e 3. Rela i e con ibu ion
o
li
gh
ne
ss (
%

L
), c
h
o
m
a
(
%
C),
and
hue
647 (%H) o he o al colo di e enc
e
o
ea
c
h
p
i
g
m
en
/c
op
i
g
m
en
a
s
a
un
c i
on
o
648 he pH alue.
649
650 Figu e 4. Change in he loca io
n
o
he
pu
e
M
3
-
g
l
and
c
op
i
g
m
en
ed
s
o
l
u
i
on
s
651 (MC, ME) wi hin (a) he (a
*
b
*
)
d
i
ag
a
m
and
(
b
) li
gh
ne
ss
a
l
ue
s (
L*
)
a
s
a
652 unc ion o he mola a io.
653
654 Figu e 5. CIELAB hue di e
en
c
e
s (
h
ab)
a
s
a
un
c i
on
o
m
o
l
a
a
i
o
o
655 co esponding o M 3-gl: CAT
and
M
3
-
g
l: EPI s
o
l
u
i
on
s.
656
657 Figu e 6. Rela i e con ibu ion
o
li
gh
ne
ss (
%

L
), c
h
o
m
a
(
%
C),
and
hue
658 (%H) o he o al colo di e enc
e
o
ea
c
h
p
i
g
m
en
/c
op
i
g
m
en
a
s
a
un
c i
on
o
659 he mola a io.
ACS P
a
a
gon Plu
s
En
i onm
e
n
30
ACS P
a
a
gon Plu
s
En
i onm
e
n
34


P
a
g
e
35 o
36
J
ou
n
a
l o Ag i
c
ul
u
a
l
a
nd Food Ch
e
mi
s
y
Figu e 5.
2
1
0
-1
M 3-gl
:
CAT
M 3-gl
:
EP
I

h
ab
=
0.1158
-1.8512*MR
+
0.1595*MR
2
(CAT)

h
ab
=
0.0229
-
1.1267*MR
+
0.0956*MR
2
(EPI)
-2
-3
-4
-5
-6 1
:
0
1
:
1
1
:
2
1
:
5
1
:
7
Mola Ra io (MR)
ACS P
a
a
gon Plu
s
En
i onm
e
n
35
Ea
J
ou
n
a
l o Ag i
c
ul
u
a
l
a
nd Food Ch
e
mi
s
y
P
a
g
e
36
o
36
Figu e 6.
a
) MC
% %C %L
100
75
50
25
0 1:1 1:2 1:5 1:7
Mola a io pigmen /copigmen
b
) ME
%

%

C
%

L
100
75
50
25
0 1
:
1
1
:
2
1
:
5
1
:
7
Mola
Ra
io
pigmen
/
copigmen
ACS P
a
a
gon Plu
s
En
i onm
e
n
36