Biosci. Biotechnol. Biochem., 70 (3), 598–605, 2006
Antioxidant Effects of Maillard Reaction Products Obtained
from Ovalbumin and Different D-Aldohexoses
Yuanxia S UN,y Shigeru H AYAKAWA, Melin C HUAMANOCHAN,
Machiko F UJIMOTO, Aree I NNUN, and Ken I ZUMORI
Department of Biochemistry and Food Science, Kagawa University, Ikenobe, Miki, Kagawa 761-0795, Japan
Received July 15, 2005; Accepted November 19, 2005
Nonenzymatic glycation between ovalbumin (OVA)
and seven D-aldohexoses was carried out to study the
chemical and antioxidant characteristics of sugar–
protein complexes formed in the dry state at 55 C
and 65% relative humidity for 2 d through the Maillard
reaction (MR). The effects of Maillard reaction products
(MRPs) modified with different aldohexoses on radical
scavenging, lipid oxidation, and tetrazolium salt (XTT)
reducibility were investigated. The results showed that
the degree of browning and aggregation and the
tryptophan-related fluorescent intensity of glycated
proteins displayed a noticeable difference that depended
on the sugars used for modification. All the glycated
proteins exhibited higher antioxidant activity as compared to a heated control and native OVA, and the
antioxidant activity was well correlated with browning
development. Furthermore, the order of antioxidant activities for the seven complexes was as follows: altrose/
allose–OVAs > talose/galactose–OVAs > gulose–OVA >
mannose/glucose–OVAs. This implies that sugar–protein complexes with two sugars known as epimers about
C-2 showed a similar antioxidant capacity. From these
results, the configuration of a hydroxyl (OH) group
about position C-2 did not influence the advanced crosslinking reaction, but the configuration of OH groups
about C-3 and C-4 might be very important for
formation of MRPs and their antioxidant behaviors.
Key words:
antioxidant activity; ovalbumin;
hexose; Maillard reaction products
D-aldo-
The Maillard reaction (MR) or nonenzymatic glycation corresponds to a set of reactions occurring between
amines and carbonyl compounds, especially reducing
sugars. This reaction is thought to contribute to pathophysiological changes associated with diabetes and
atherosclerosis and be involved in the general aging
process in vivo,1) but it is a desirable consequence of
many industrial and domestic processes and is responsible for the attractive flavor and brown color of some
cooked foods. Moreover, MRPs have exhibited significant antioxidant properties and anticarcinogenic and
y
antimutagenic characteristics.2–5) The application of
food processing practices to derive MRPs can improve
the oxidative stability of food products,6) and preserve
food from oxidation and microorganism contamination.7) On the other hand, the functional properties of
MRPs have been reviewed with reference to their use as
potential new food ingredients that might play an
essential role in the prevention of cell aging, carcinogenesis, and cardiovascular disease.4) Therefore, sugar–
protein conjugates can be expected to have significant
potential for use in the food industry.
The International Society for Rare Sugars (ISRS) has
defined rare sugars as ‘‘monosaccharides and their
derivatives that rarely exist in nature’’. All eight possible
D-aldohexoses are shown in Fig. 1. D-Glucose (Glc), Dmannose (Man), and D-galactose (Gal) are naturallyoccurring saccharides, and the other five D-aldohexoses
are rare in nature and not utilized by living organisms.
Hence the latter five aldohexoses are referred to rare
sugars. Furthermore, bioproduction strategies for all rare
sugars are illustrated using ring-form structures given
CH=O
CH=O
|
|
HC-OH
HO-CH
|
|
HO-CH
HO-CH
|
|
HC-OH
HC-OH
|
|
HC-OH
HC-OH
|
|
CH2-OH
CH2-OH
D -Glucose
D -Mannose
CH=O
CH=O
|
|
HC-OH
HO-CH
|
|
HC-OH
HC-OH
|
|
HC-OH
HC-OH
|
|
HC-OH
HC-OH
|
|
CH2-OH
CH2-OH
D -Allose
D -Altrose
CH=O
CH=O
|
|
HC-OH
HO-CH
|
|
HC-OH
HC-OH
|
|
HO-CH
HO-CH
|
|
HC-OH
HC-OH
|
|
CH2-OH
CH2-OH
D -Gulose
D -Idose
CH=O
CH=O
|
|
HC-OH
HO-CH
|
|
HO-CH
HO-CH
|
|
HO-CH
HO-CH
|
|
HC-OH
HC-OH
|
|
CH2-OH
CH2-OH
D -Galactose
D -Talose
Fig. 1. The Stereochemical Relationships, Shown in Fischer Projection, among the D-Aldohexoses.
The configuration about C-2 distinguishes the members of each
pair shown with solid boxes.
To whom correspondence should be addressed. Fax: +81-87-891-3021; E-mail: [email protected]
Antioxidant Activity of Aldohexose–OVA Complexes
8,9)
the name Izumoring.
According to this ring, the
production of all hexoses from inexpensive monosaccharides such as Glc and D-fructose (Fru) has been
studied using microbial and enzymatic reactions. A
method of production of D-altrose (Alt) and D-allose
(All) from D-psicose (Psi) using immobilized L-rhamnose isomerase has been established,10) and also one for
mass production of Psi from Fru using an immobilized
D-tagatose (Tag) 3-epimerase bioreactor.11) Bioproduction of rare sugars should offer the possibility of making
all of the rare sugars available for research to discover
their unidentified physicochemical characteristics and
novel physiological functions.
Rare sugars have large potential for a wide range of
applications. Murata et al.12) reported that All has a
potent inhibitory effect on the production of reactive
oxygen species, and hence can be used for medical
purposes. Psi has been found to have beneficial health
properties. For example, it could be an alternative to
presently dominant sweeteners due to its lack of calories
and might be useful in the food industry as an aid to a
large number of people suffering from diabetes and
obesity.13) Moreover, proteins glycated with Psi and All
also exhibited a higher free radical-scavenging effect
and reducing power than proteins glycated with alimentary sugars.3,14) Hence the addition of rare sugars as
substitutes for alimentary sugars in foodstuffs might be a
feasible strategy to develop new functional food.
Maillard15) noted that the reactivities of sugars in the
MR decline in the order D-xylose, L-arabinose, hexoses,
and disaccharides, but he did not mention the differences
among the various hexoses. Yeboah et al.16) indicated
that big differences in MR behavior when heated in the
presence of a protein were observed between Glc and
Fru. These differences were mainly due to differences in
the reaction kinetics between aldose and ketose sugars.17) Much less information, however, is available on
the comparison of the chemical and functional properties of different aldohexose–protein complexes as a
consequence of MRP formation. Also, the relationship
between the chemical properties of glycated proteins
and their functionality has not yet been fully determined
in all aldohexose–protien conjugates. To control the MR
in foods and develop MRP functionality effectively, it is
a prerequisite to establish the chemical properties and to
elucidate the functional properties of MRPs. Egg-white
proteins are extensively utilized as food ingredients due
to their unique functional properties. Covalent attachment of sugar residues, using mild, nondenaturing heattreatment, has been reported to improve emulsifying
ability and gelling properties, and to enhance antioxidant
activity in egg-white proteins.3,18,19) Thus ovalbumin
(OVA) is used as a model protein to study chemical and
functional properties further.
The objective of the present study was to investigate
the chemical characteristics of all D-aldohexose–OVA
complexes, except for the glycated protein with D-idose
(Ido), by evaluating the formation of covalent aggre-
599
gates and browning substances, the extent of reaction
between carbonyl and amino groups, and tryptophan
(Trp)-related fluorescence intensity (FI). The antioxidant
activities of these complexes were investigated using
radical scavenging activities, inhibition of lipid oxidation, and tetrazolium salt (XTT) reducibility. Furthermore, the relationships between the chemical characteristics and antioxidant activities of different sugar–
protein complexes were investigated in order to understand the glycation derived from seven D-aldohexose
stereoisomers.
Materials and Methods
Materials. OVA was purified from fresh hen egg
white by a crystallization method using an ammonium
sulfate solution and recrystallyzation three times.20) All
and Alt were obtained from Kagawa Rare Sugar Cluster
(Takamatsu, Japan). D-talose (Tal) was obtained from
Extrasynthese (Genay Cedex, France). Gal and Man
were purchased from Nacalai Tesque, (Kyoto, Japan); Dgulose (Gul) from Sigma Chemical (St. Louis, MO); and
Glc from Wako Pure Chemical Industries (Osaka,
Japan). All other chemicals used in this study were of
analytical grade.
Preparation of Maillard reaction products. Glycation
was performed with seven aldohexoses (Glc, Man, Gul,
Gal, Tal, All, and Alt). In our preliminary experiment,
the results showed that the MR could be accelerated by
dissolving samples in carbonate buffer (pH 9.0). Hence
OVA and reducing sugar were dissolved in 10 mM
carbonate buffer (pH 9.0) at a protein concentration of
5% (w/v) and a sugar concentration of 0.4% (w/v)
(approximately a 1:1 ratio of sugar carbonyl to free
amino groups). OVA–sugar solutions were lyophilized.
The dried samples were incubated at 55 C and 65%
relative humidity (RH) for 2 d in an incubator (LHL-113,
Espect, Osaka, Japan). For these procedures, control
experiments were carried out with no added sugars.
Monitoring of Maillard reaction products. The brown
color of glycated OVA (10 mg/ml) was analyzed by
measuring the absorbance at 420 nm. Trp-related fluorescence (excitation at 280 nm) of samples (0.5 mg/ml)
was measured in a range from 300 nm to 400 nm using
an F-2500 spectrofluorometer (Hitachi, Tokyo), and was
expressed as relative FI.
Gel electrophoresis. The modified OVA samples were
resolved by 12.5% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE), followed by
staining with Coomassie Brilliant Blue R-250 (CBB),
according to the method of Laemmli.21) Electrophoresis
was done in the presence of 2-mercaptethanol (2-ME).
Determination of free amino groups. The quantity of
available amino groups was determined according to the
600
Y. S UN et al.
22)
OPA (o-Phthalaldehyde) method. To 50 ml aliquot of
samples, containing 3 mg/ml OVA, was added 1 ml of
OPA reagent. The absorbance was read at 340 nm after a
minimal delay of 2 min at room temperature. A
calibration curve was obtained using 0.25–2.00 mM
glycine as a standard. The protein content of all samples
in the present study was determined by the Lowry
method.23)
Assay of tetrazolium salt XTT reducibility. The XTT
assay was performed in a 96-well microtiter plate as
described by Ukeda et al.24) XTT solution was prepared
with 0.2 M potassium phosphate buffer (pH 7.0) containing menadione at the saturation level. Each well
contained 40 ml of sample (2% and 4%). Then 60 ml of
0.5 mM XTT solution was added into the well. After it
was mixed for 15 s, the difference in absorbance between
490 and 570 nm (as the reference) was read on a
microplate reader (Model 550; Bio-Rad, Tokyo) as the
absorbance at 0 min. Again, after 20 min incubation at
room temperature, the absorbance difference was recorded, and the increase in the absorbance difference was
calculated as the ability of the MRPs to reduce XTT.
Scavenging activity of ABTSþ radical cation. The
ABTS [2,20 -azino-bis(3-ethyl-benzthiazolin-6-sulfonic
acid)] radical cation method25) was used to evaluate
the free radical scavenging effect of MRPs. ABTSþ
was formed by adding potassium persulfate (K2 S2 O8 ) to
ABTS. ABTSþ stock solution was diluted with 10 mM
phosphate buffer (pH 7.4) to a final absorbance of 0:7 0:02 at 734 nm. The scavenging effects of MRPs were
determined from the percentage of decolorization at
734 nm after 2 min of reaction at room temperature. The
ABTSþ scavenging activity (%) was calculated as
follows:
½ðOD734 control OD734 sample Þ=ðOD734 control Þ 100%
ð1Þ
½ðOD517 control OD517 sample Þ=ðOD517 control Þ 100%
ð2Þ
The scavenging activity of a sample was expressed as
50% effective concentration (EC50 ) of sample having
50% of DPPH scavenging effect.
Antioxidant activity in SDS/linoleic acid-AAPH system. An aliquot (80 ml) of samples at a concentration of
10 mg/ml was added to the SDS miceller-linoleic acid
peroxidation system (6 ml) as described in Foti et al.27)
This miceller system contained 10 mM phosphate buffer
(pH 7.4), 0.1 M SDS, and 2.6 mM linoleic acid. The
radical reaction was started by adding 30 ml of 2%
AAPH [2,20 -Azobis(2-amidinopropane) dihydrochloride] after the addition of sample solution. The same
reaction mixture without a sample was used as a control.
The absorbance at 234 nm was measured before (OD0 )
and after (OD90 ) incubation for 90 min at 50 C,
and OD (OD90 OD0 ) was calculated. Antioxidant
activity against the SDS/linoleic acid-AAPH system
expressed by inhibition (%) of conjugated dienes
formation was represented as follows:
½ðOD234control OD234sample =OD234control Þ 100%
ð3Þ
Statistical analysis. Experimental results were obtained from three parallel measurements done at least in
triplicate. Mean values, standard deviation, and correlation coefficients (R2 ) were calculated using the data for
each replicate. Statistical analysis was performed by
one-way analysis of variance (ANOVA) with Tukey’s
test. Significant differences were graphically determined
through comparison of 95% confidence intervals.
Results
A dose-response curve of inhibition percentage against
different concentrations of trolox standard (0.5–2.0 mM)
was plotted. The antioxidant activity of samples was
expressed as trolox equivalent antioxidant capacity
(TEAC), which represented the concentration (mM) of
trolox, having the same activity as one mg of the sample.
The configurations of all the D-aldohexoses are
presented in Fig. 1, and eight D-aldohexoses were
classified into four groups depending on the epimers
about position C-2. In the present study, all the Daldohexoses, except Ido, were used to modify OVA, and
the seven sugar–OVA complexes in the chemical and
antioxidant properties were studied.
Scavenging activity of DPPH free radical. DPPH
[1,1-diphenyl-2-picrylhydrazyl] radical scavenging activity was determined according to the method of Yen
and Hsieh,26) with a slight modification. An aliquot of
MRPs (0.5 ml) was added to 2 ml of 0.125 mM DPPH
methanolic solution. The solution was then mixed
vigorously and allowed to stand at room temperature
in the dark for 30 min. The absorbance of mixtures was
measured at 517 nm. The control was prepared in the
same manner, except that distilled water was used
instead of the sample. DPPH scavenging activity (%)
was calculated as follows:
Chemical characteristics of D-aldohexose–OVA complexes
The MR is a series of complex reactions that occur
between reducing sugars and amino groups of protein,
leading to cross-linking and polymerization of the
protein, as well as the formation of brown and fluorescent compounds. Figure 2 shows the absorbance at
420 nm obtained for native, heated control (Ct), and
seven glycated OVA conjugates. No significant difference in absorbance between native and heated Ct
samples was found. As compared to nonglycated OVAs,
however, the absorbance values of all glycated samples
Antioxidant Activity of Aldohexose–OVA Complexes
0.25
Absorbance at 420 nm
d
d
d
100
Free amino groups (%)
d
0.20
c
0.15
b
b
0.10
a
a
0.05
a
a
80
60
b
40
b
b
c
b
b
Gal
Tal
All
Alt
c
20
0
0.00
Native
Ct
Glc
Man
Gul
Gal
Tal
All
Alt
Fig. 2. Comparison of Browning Intensity of Native, Heated Control
(Ct), and Glycated OVA with Seven D-Aldohexoses (Glc, Man, Gul,
Gal, Tal, All, and Alt) as Determined by Absorbance at 420 nm.
All results were determined at least in triplicate.
kDa
601
120
St
Ct
Glc
Man
Gul
Gal Tal
All
Alt
175
83
62
47.5
32.5
Fig. 3. SDS–PAGE Patterns of Heated Control (Ct) and Glycated
OVA Complexes with Seven D-Aldohexoses (Glc, Man, Gul, Gal,
Tal, All, and Alt) in the Presence of Mercaptoethanol.
were significantly higher (p < 0:05). Among the seven
aldohexose–OVA complexes, Glc– and Man–OVA
conjugates were found to have the lowest browning
intensity, and Gul–OVA displayed intermediate intensity. The four conjugates from the All–, Alt–, Gal–, and
Tal–OVA systems yielded the highest browning intensity.
As shown in Fig. 3, the effects of seven aldohexoes
on the cross-linking of sugar–protein complexes were
assessed by SDS–PAGE under the reducing condition.
The SDS–PAGE profile of heated Ct was similar to that
of native OVA (not shown). But a large amount of high
molecular weight aggregates retained in the resolving
gel were observed in all glycated samples, indicating
that the polymerization of OVA was due to the
incubation with reducing sugar. Furthermore, the density
of the broad bands from the high molecular weight
compounds that might correspond to heterogeneous
forms of OVA and sugar was remarkably different.
OVAs modified with All, Alt, Gal, or Tal exhibited a
greater extent of polymerization than those modified
with the other hexoses.
The reactivity of reducing sugars with respect to their
ability to utilize primary amino groups of OVA was
determined using the OPA method. Since no amino
Native
Ct
Glc
Man
Gul
Fig. 4. Comparison of Free Amino Groups of Native, Heated Control
(Ct), and Glycated OVA with Seven D-Aldohexoses (Glc, Man, Gul,
Gal, Tal, All, and Alt) as Determined by OPA Assay.
All results were determined at least in triplicate.
groups were modified on native OVA, all results are
reported as relative value to 100% of amino groups of
native sample. As shown in Fig. 4, the content of the
free amino groups in the heated Ct sample did not
change significantly as compared to native OVA. In
contrast, the content of free amino groups in the seven
glycated OVAs decreased dramatically and reached
approximately 30%–40% of amino groups of native
OVA. The terminal -amino group of OVA is acetylated, and therefore all free amino groups of OVA
originate from the "-amino groups of 20 Lys residues.
The average number of sugars bound per OVA molecule
by amino groups was about 12 for Glc, Gal, and Alt, 13
for Man and All, and 14 for the Gul and Tal molecules.
In our previous paper, it is indicated that the OVA
molecule was modified by 9.3 of Glc, 5.3 of Fru, and 4.5
of Psi molecules when they were incubated at a lower
RH (35%).28) All and Glc were also found to exhibit a
greater reactivity than Fru in undergoing condensation
reaction with amino groups of -lactalbumin.14) From
these findings, no remarkable difference in the available
amino groups in protein were found within ketoses or
aldoses but were found between ketose and aldose.
The fluorescence emission spectra of native, heated
Ct, and glycated OVAs were examined (Fig. 5). When
excited at 280 nm, native and heated Ct OVA exhibited
the same fluorescence emission maximum (max ) at
333.5 nm. In the case of sugar–OVA conjugates, the
max was at 337 nm for Glc– and Man–OVAs, at 339 nm
for Gul–OVA, and at 341 nm for the other glycated
OVAs modified with All, Alt, Gal, and Tal, revealing
that the glycated OVA complexes had slightly a red shift
of the max . The difference in max among complexes is
indicative of a different conformation around Trp
residues. It might be affected by the polarity of the
environment surrounding Trp residues due to the
modification by different reducing sugars. On the other
hand, the relative Trp-FI of all glycated OVAs was
remarkably lower than that of native and heated Ct
OVAs (Fig. 5). Particularly, the proteins modified with
All, Alt, Gal, and Tal displayed the lowest relative TrpFI among the seven complexes.
602
Y. S UN et al.
800
600
500
400
Glc
Man
300
200
Gal
Tal
All
Alt
Gul
100
ABTS radical scavenging activity (%)
Relative fluorescence intensity
A
Ct
Native
700
100
80
Native
Glc
Gul
Tal
Alt
Ct
Man
Gal
All
0.2
0.4
60
40
20
0
0
300
320
340
360
380
0.0
400
Wavelength (nm)
Antioxidant characteristics of D-aldohexose–OVA
complexes
First, the antioxidant properties of the different MRPs
was evaluated using assays of ABTS free radical
scavenging activity. In the scavenging activity of
ABTSþ , the stable free radical cation is generated by
direct oxidation of ABTS with K2 S2 O8 . The reduction of
ABTSþ was evaluated by monitoring the decrease in its
absorbance at 734 nm during the reaction with hydrogendonating antioxidant. Figure 6A shows a typical dependence of ABTSþ scavenging activity on the concentrations of native, heated Ct, and seven glycated proteins.
Interestingly, OVAs modified with a pair of epimers
about position C-2 (Glc and Man, Gal and Tal, and All
and Alt) exhibited similar concentration-dependent patterns. Among the seven complexes, ABTSþ scavenging
activity was highest in the case of All– and Alt–OVAs.
Furthermore, the antiradical effect of two such complexes increased rapidly in protein concentrations from
0.2 to 0.8 mg/ml, and then gradually reached the highest
level of about 100% at a concentration of 1 mg/ml.
To evaluate TEAC, a dose-response curve of radical
scavenging activity against different concentrations of
trolox standard (0.5–2.0 mM) was plotted (data not
shown), and the regression equation of standard curve
was expressed as y ¼ 499:92x þ 0:227 (R2 ¼ 0:999). At
the same time, the regression equations of concentration-response curves of samples in the range of 0.2–
0.8 mg/ml (plotted in Fig. 6A) were also calculated (not
shown). Based on these analyses, the TEAC values of
the seven complexes were evaluated (as shown in
Table 1). A TEAC with a higher value indicates a more
potent radical scavenging effect. As shown in Tukey’s
test results, all the MRPs significantly increased (p <
0:05) antiradical activity as compared to nonglycated
OVA, and the degree of increase depended on the
reducing sugar species used for modification. Among the
seven sugar–protein complexes, the TEAC values of
All/Alt–OVAs were the highest, their radical-scavenging effect being 2.5 times higher than that of non-
B
0.8
1.0
1.2
1.0
1.2
80
DPPH radical scavenging activity (%)
Fig. 5. Fluorescence Emission Spectra of Native, Heated Control
(Ct), and Glycated OVA Complexes with Seven D-Aldohexoses
(Glc, Man, Gul, Gal, Tal, All, and Alt).
0.6
Concentration (mg/ml)
Native
Glc
Gul
Tal
Alt
60
Ct
Man
Gal
All
40
20
0
0.0
0.2
0.4
0.6
0.8
Concentration (mg/ml)
Fig. 6. The Effects of MRPs Modified with Seven D-Aldohexoses
(Glc, Man, Gul, Gal, Tal, All, and Alt) on ABTS (A) and DPPH (B)
Radical Scavenging Activity.
All results were determined at least in triplicate.
Table 1. Antioxidant Properties of Native, Heated Control, and
Glycated OVAs with Seven D-Aldohexoses
Samples
TEAC
EC50 (mg/ml)
Inhibition of
conjugated dienes (%)
Native
Heated Ct
Glc–OVA
Man–OVA
Gul–OVA
Gal–OVA
Tal–OVA
All–OVA
Alt–OVA
0:088 0:003a
0:091 0:004a
0:119 0:005b
0:122 0:002b
0:141 0:009c
0:190 0:007d
0:195 0:009d
0:224 0:009e
0:228 0:010e
> 2a
> 2a
1.36b
1.32b
1.06c
0.86d
0.80d
0.69e
0.67e
38:60 1:91a
39:56 2:92a
42:64 1:93a
42:61 2:39a
45:71 1:89b
49:68 3:12c
50:92 2:68c
53:19 3:31c
52:77 3:16c
TEAC , trolox equivalent antioxidant capacity, expressed as mM trolox per
mg of sample.
EC50 , effective concentration of sample having 50% of DPPH radicalscavenging effect.
Each value is the mean SD of triplicate measurements. Values within a
column with different letters (a–e) differ significantly (p < 0:05).
glycated OVA. For other complexes, the order of TEAC
values was as follows: Gal/Tal–OVAs, Gul–OVA, and
Man/Glc–OVAs (the lowest). In addition, no significant
difference was found in the two proteins modified with a
pair of epimers about C-2.
The DPPH radical is a stable organic free radical
with an adsorption band at 515–528 nm. It loses this
adsorption when accepting an electron, which results in
Antioxidant Activity of Aldohexose–OVA Complexes
0.10
20 m g/ml
40 m g/ml
0.08
XTT Reducibility
0.06
0.04
0.02
A
lt
A
ll
Ta
l
G
al
G
ul
an
M
G
lc
Ct
iv
e
0.00
N
at
a visually noticeable discoloration from purple to
yellow. Figure 6B illustrates the DPPH scavenging
activity of the seven complexes derived from the MR at
various concentrations. All complexes showed a concentration-dependent manner in DPPH -scavenging activity. According to these concentration-dependent
curves, the EC50 values of the seven MRP complexes
were calculated, and they are shown in Table 1. The
EC50 values of the Glc–, Man–, Gul–, Gal–, Tal–, All–,
and Alt–OVA complexes were 1.36, 1.32, 1.06, 0.86,
0.80, 0.69, and 0.67 mg/ml respectively. The results
show that the DPPH activities were strongly inhibited
by All/Alt–OVAs, as indicated by their lowest EC50
values, suggesting that All/Alt–OVA conjugates were
the most effective scavenger for DPPH free radicals.
These results are in agreement with those obtained from
the ABTS assay. In the present study, the free sugars in
the glycated OVA samples were not removed. To test
the effect of free sugars in the glycated samples, the
antiradical activities of all seven sugar solutions in a
range of concentrations from 0.1 to 1.0 mg/ml were also
determined using the DPPH method. The results show
that no sugars exhibited any free-radical scavenging
activity in this concentration range (data not shown).
In the SDS/linoleic acid-AAPH system, the effects of
MRPs on the oxidation of linoleic acid were determined
spectrophotometrically at 234 nm and expressed by
inhibition of conjugated dienes formation (Table 1).
The results show that lipid oxidation rates were
significantly slowed when nonglycated and glycated
OVAs were added. No significant differences in the
inhibition of conjugated dienes formation were found
among native, Ct, and Glc/Man–OVAs, but the other
five glycated OVA complexes showed a significant
inhibition effect (p < 0:05) as compared to nonglycated
OVAs. Furthermore, the results of antioxidant activity
on lipid peroxidation, in decreasing order, were All/Alt/
Gal/Tal–OVAs > Gul–OVA > Glc/Man–OVAs native/Ct–OVAs. From these findings, although the
activity was not very strong, the glycated proteins had
a significant inhibition effect on linoleic acid peroxidation, except for Glc/Man–OVAs, as compared to nonglycated OVAs. It has been reported that MRPs exhibit
antioxidant activities in both model lipids and foods,
inhibit the oxidative degradation of natural organic
compounds, and improve the oxidative stability of food
products.29–31)
The ability of glycated OVAs with the seven
aldohexoses to reduce XTT was compared with nonglycated OVA at protein concentrations of 2% and 4%
(Fig. 7). Native and heated Ct OVA exhibited very weak
XTT reducibility, but all glycated OVAs showed a
potent XTT reducibility, especially at a high protein
concentration (4%). The results revealed that the sugar–
protein complexes, but not the protein itself, took part
principally in their reducing activity against XTT. Hence
MRPs are expected to have potential for use in the food
system as functional ingredients.
603
Fig. 7. Comparison of XTT Reducibility of Native, Heated Control
(Ct), and Glycated OVAs with Seven D-Aldohexoses (Glc, Man,
Gul, Gal, Tal, All, and Alt).
All results were determined at least in triplicate.
Discussion
In response to complex formation, the degree of
polymerization favored the All–, Alt–, Gal–, and Tal–
OVA complexes over the Glc–, Man–, and Gul–OVA
complexes (Fig. 3), and more browning development
was also detected in the former four complexes (Fig. 2).
These results indicate that the greater the protein
aggregation, the higher the browning extent. However,
in the case of the four glycated OVAs modified with All,
Alt, Gal, and Tal, the number of carbonyl groups in
reducing sugar bound on each OVA molecule was found
to be not remarkably different, compared to the Gul–,
Man– and Glc–OVA complexes. In addition, no relationship between the content of free amino groups and
browning intensity was observed (R2 ¼ 0:078). Thus the
results demonstrate that the formation of brown compounds did not directly depend on the Amadori reaction
at the initial stage of the MR, whereas it was caused by
the advanced covalently cross-linking reaction.
Figure 5 show a distinguishable decrease in the
relative Trp-FI of glycated OVAs as compared to
nonglycated OVAs. Hattori et al.32) also found that the
relative Trp-FI of -lactoglobulin-carboxymethyl dextran was lower than that of the native protein, and
indicated that this was to be attributed to a shielding
effect of the polysaccharide chain bound to protein. But
it was not clear whether the monosaccharide conjugated
to protein could cause a shielding effect. Hence the
blocking Lys residues were calculated from the free
amino group values (data not shown). It was found
that the relative Trp-FI did not correlate well with the
numbers of the blocking Lys residues per OVA
molecule in the seven aldohexose–OVA conjugates
(R2 ¼ 0:172). From the result shown above, OVAs
modified with All, Alt, Gal, and Tal showed much
higher extents of aggregation and browning. On the
contrary, the four such conjugates exhibited much
lower levels of relative Trp-FI. It is inferred that the
differences found in relative Trp-FI among the seven
sugar–OVA conjugates were not attributable to the
604
Y. S UN et al.
differences in the degree of blocking Lys residues but
rather to the other changes in protein conformation
arising from polymerization and the progress of the MR.
Antioxidant activity was assayed using four test
systems showing different sensitivities. The results
indicated that the glycated OVAs exhibited antioxidant
activity in all of the assay systems. According to the data
observed in the four assay systems, the relationships
were investigated between the antioxidant properties and
browning intensity as an indicator of the MRPs. The
ABTS and DPPH radical scavenging activities of MRPs
correlated well with the browning intensities (R2 ¼
0:921 and 0.947 respectively). Similar results were
found in the XTT reduced efficiency and inhibition of
linoleic acid peroxidation, showing correlation coefficients of R2 ¼ 0:951 and 0.952 with the browning
intensity of the seven complexes. Thus the high
correlation coefficients indicate that the browning pigment compounds might be the major antioxidant
components.
Many studies have focused on the MRPs and their
functional properties, but it is still not clear why the
same type of sugars, such as aldohexoses or ketohexoses, which have differences only in the configuration of
one or two OH groups, exhibit remarkable variation in
the formation of MRPs and their antioxidant activity.
Sugars that differ only by the configuration about one C
atom are known as epimers of one another. As shown in
Fig. 1, D-aldohexoses are divided into four pairs of
epimers according to configuration with respect to C-2.
In this study, it was shown that glycated proteins
modified with all two structural analogs which are
epimers about C-2, except Gul and Ido, displayed
similar antioxidant behaviors and chemical characteristics. However, the complexes derived from other
epimers about C-3 and C-4, for instance, Glc and All,
and Glc and Gal, exhibited significant variation in
antioxidant activities and chemical properties. These
results imply that the configuration of the OH group
about position C-2 did not influence the advanced crosslinking reaction, but that the configuration of OH groups
about C-3 and C-4 might be very important for
formation of MRPs. Thus a similar browning reaction
route might be formed in a pair of epimers about C-2 in
the presence of protein. On the other hand, the generally
accepted view is that the differences in the browning
reaction can be ascribed to the differences in open chain
form: the sugars with a higher concentration in the open
chain form brown faster and more intensely. It has been
indicated that the percentage of Gal and All in its acyclic
form is about 10 and 58 times higher than that of Glc at
neutral pH at room temperature respectively, which is
believed to be the reason for the higher reactivity of Gal
and All than Glc.17,33,34)
In our previous study, it was found that protein–All/
Psi complexes exhibited a stronger antioxidant activity,
as well as a greater aggregation and browning/fluorescence development than protein–Glc/Fru com-
plexes.3,14) Although the reasons were not clear, it has
been indicated that these chemical and antioxidant
properties of MRPs might be related to the rate of
conversion of Amadori rearrangement to chromo/fluorophoric adducts, while they are not related to the rate of
formation of the initial Schiff base adducts.35) MRPs are
a large group of compounds with a variety of structures
and a diversity of roles in foods. Consequently, how to
maximize their beneficial effects and minimize the
negative effects is an important subject for the food
industry. In this study, we found that all the MRPs
derived from glycated OVA with rare sugars, specifically All and Alt, exhibited stronger antioxidant activity
and a greater aggregation and browning development
than corresponding MRPs modified with Man and Glc.
These results should contribute to a better understanding
of MRPs and their antioxidant properties.
Conclusions
In the present study, all the MRPs significantly
increased (p < 0:05) antioxidant activity as compared
to nonglycated OVA, and the degree of increase
depended on the reducing sugar species used. Among
the seven aldohexose–protein complexes, All–, Alt–,
Gal–, and Tal–OVA conjugates exhibited higher browning development and underwent more conformational
changes than the Glc–, Man–, and Gul–OVA conjugates.
Furthermore, the MRPs, generated from Alt/All–OVA
systems, produced the highest antioxidant ability,
followed by Gal/Tal–OVAs, Gul–OVA, and Glc/
Man–OVAs. Significant differences in antioxidant activities were found between two sugar–OVA conjugates
modified with a pair of epimers about C-3 or C-4,
suggesting that the configuration of OH groups about
C-3 and C-4 might affect the formation of MRPs and
consequently cause variations in antioxidant behaviors.
The antioxidant properties of MRPs were well correlated
with their chemical characteristics, such as the formation
of browning and aggregation through the MR. The
remarkable differences in the MRPs and their antioxidant activities, as well as the strong relationships found
should offer possibilities to control chemical reactions
and utilize antioxidant activity in food with respect to
the MR.
Acknowledgments
The research was supported by the ‘‘Intellectual
Cluster’’ of the Ministry of Education, Culture, Sports,
Science and Technology of Japan.
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