Biosci. Biotechnol. Biochem., 70 (12), 2859–2867, 2006
Rheological Characteristics of Heat-Induced Custard Pudding Gels
with High Antioxidative Activity
Yuanxia S UN,y Shigeru H AYAKAWA, Huihong JIANG, Masahiro O GAWA, and Ken I ZUMORI
Department of Biochemistry and Food Science, Kagawa University, Ikenobe,
Miki, Kagawa 761-0795, Japan
Received May 9, 2006; Accepted August 1, 2006; Online Publication, December 7, 2006
[doi:10.1271/bbb.60256]
Custard pudding gels were prepared from fresh
whole egg, milk and sugar. The effects of D-psicose
(Psi), a non-calorie rare hexose, on the antioxidative
activity and rheological properties of the custard
pudding gels were investigated at different temperatures
for comparison with those of control sugars (sucrose,
Suc; D-fructose, Fru). The rheological behavior of the
heat-induced pudding gels was evaluated by using
breaking and creep tests. During the heat-induced gel
formation, custard pudding containing Psi (15%, wt/wt)
demonstrated a stronger breaking strength and higher
viscoelasticity than those containing Fru and Suc. The
thermodynamic parameters obtained from DSC indicated that the egg white (EW) proteins were made less
thermally stable when heated in the presence of Psi than
in the presence of Fru and Suc. These findings are
consistent with enhanced aggregation of the EW solution in the presence of Psi. Furthermore, the Psi
pudding gels possessed higher antioxidative activity
than the control sugar pudding gels by using an analysis
of the scavenging activity on DPPH radicals and the
ferric-reducing antioxidative power. These results suggest that Psi favored cross-linking of Psi-protein molecules through the Maillard reaction which increased the
formation of intermediate products to improve functionality. Custard pudding containing Psi could therefore be an effective functional sweet dessert with high
antioxidative activity and the outstanding gelling characteristics.
Key words:
D-psicose;
custard pudding; aggregation;
gelling property; antioxidative activity
Egg and milk proteins are widely used in the
production of a number of foodstuffs. These food
proteins (egg albumen, whey, casein, etc.) not only
add a high nutritional value to egg- and milk-containing
foods, but are also associated with several functional
properties for industrial applications, including gel
formation, foaming capacity and emulsifying ability.1,2)
Heat-induced gelation is one of these important funcy
tional properties which is considered to involve the
formation of a continuous and well-defined network,
assembled from protein molecules or aggregates, and
be surrounded by water. Therefore, protein gelling and
aggregation behavior is widely used to control the
structure, texture and stability of food colloids, and
further improve the consistency of a product.3–5)
The Maillard reaction (MR) of protein with sugar is a
nonenzymatic browning reaction that takes place during
the processing and storage of foods. The development
of a certain degree of browning as a consequence of
heating is often a desirable one for many food products;
this is the case in baking, cooking, roasting and frying.
Furthermore, the potential of the Maillard reaction
products (MRPs) to act as antioxidants, or to provide
other beneficial effects, has been noted in many
studies.6–8) During food processing and storage, natural
antioxidants are considerably degraded. On the other
hand, chemical reactions among food components lead
to the formation of secondary antioxidants like MRPs.9)
There is substantial evidence that antioxidative food
components suppress oxidative stress and play a
protective role against coronary heart diseases.10) In
addition, the reactive oxygen species can cause the
deterioration of foods during storage, consequently
decreasing the nutritional quality and safety of foods.
The antioxidative activity of MRPs could result in
retarding lipid autoxidation, and increase the stability
and shelf life of food products that are vulnerable to
oxidation reactions.11,12)
Rare sugars, which are defined as ‘‘monosaccharides
and their derivatives that are rare in nature,’’13) have
recently attracted a great deal of attention mainly
concerning their application. D-psicose (Psi), a rare
hexose, is expected to be approved for commercial use
because now there is large-scale production of Psi from
D-fructose (Fru) using an immobilized D-tagatose 3epimerase bio-reactor.14) This could provide an alternative to the presently dominant sweeteners due to its
lack of calories, and thus be useful in the food industry
as an aid to a large number of people suffering from
To whom correspondence should be addressed. Fax: +81-87-891-3021; E-mail: [email protected]
2860
Y. S UN et al.
15)
diabetes and obesity.
It has been reported that a
custard pudding dessert containing Psi as a substitute for
sucrose possessed remarkable antioxidative activity.16)
Hence, the addition of Psi could be a feasible strategy
to develop new functional foods for preventing some
diseases associated with oxidative stress.
An important consideration in designing new functional foodstuffs is the potential interplay of functionality between proteins and cosolvents like sugars. These
cosolvents can alter the conformation and interaction
between proteins and, therefore, their functional properties, through a variety of different physicochemical
mechanisms. Sucrose is a naturally occurring polyhydric
compound and can be found in a wide variety of food
products. It is recognized that the presence of sucrose
can alter the thermodynamic and functional properties
of food proteins, and then affect the texture, taste
and flavor of food products.17,18) Reducing sugars can
interact with a protein molecule and affect the physicochemical and functional properties of protein through
the MR.6,19) It has been indicated that the different
reducing sugars have characteristic reaction rates resulting in the formation of MRPs.20,21) The precise nature of
the interactions and their influence on protein functionality depend on the type and concentration of the
cosolvents present.22,23) Although the effect on proteins
of some mono- and disaccharides as well as the number
of polyalcohols is well documented in the literature,
little is known about the actual importance of this
behavior with respect to food protein interactions and
their functionality in the presence of rare sugars.
The objective in the present study was to obtain a
better quantitative understanding of the influence of rare
sugars on thermal denaturation and aggregation by using
a well-defined egg white (EW) protein system. We also
evaluated the impact of Psi on the antioxidative activity
and characteristics of heat-induced custard pudding gels
at different temperatures in comparison to Suc and Fru.
The component interactions in the rare sugar–protein
custard pudding composite system are important for
improving the functional properties of pudding products.
Materials and Methods
Materials. D-psicose (Psi) was obtained from Kagawa
Rare Sugar Research Center (Takamatsu, Japan). Potassium ferricyanide (K3 Fe(SCN)6 ) was purchased from
Wako Pure Chemical Industries (Osaka, Japan), and 1,1diphenyl-2-picrylhydrazyl (DPPH) was purchased from
Nacalai Tesque (Kyoto, Japan). Fru, Suc, milk (Shikoku
Rakuren Co., Japan) and egg were of commercial grade.
All other chemicals used were of analytical grade.
Preparation of heat-induced protein-sugar mixtures.
An amount of EW to achieve the final concentrations of
1, 3, 5, and 10 mg/ml was dispersed in distilled water.
The different sugars (Psi, Fru and Suc) at a concentration of 15% (wt/v) were added to the various protein
solutions. Pure water was added to the control sample
(Ct) as a substitute for Suc, so that the Ct and Suc
samples could be maintained at the same protein
concentration as determined by the Lowry method.24)
The different sample solutions were heated in a water
bath at 90 C for 30 min, after which the sample was
immediately cooled in an ice-water to stop further
denaturation.
Determination of turbidity. The turbidity of each EWsugar mixture after the heat treatment was determined
as the optical density (OD) at 500 nm with a UVspectrophotometer (Shimadzu Co., Tokyo, Japan) at
room temperature.
SDS–PAGE. The heat-treated samples were resolved
by 12.5% sodium dodecyl sulfate–polyacrylamide gel
electrophoresis (SDS–PAGE) and then stained with
Coomassie Brilliant Blue R-250 (CBB) according to the
method of Laemmli.25) Electrophoresis was done under
both reducing and non-reducing conditions.
Determination of the thermal denaturation. The
thermodynamic parameters of the EW protein in the
presence of different sugars were investigated by differential scanning calorimetry (DSC) (Micro-DSC VII,
Setaram, France). The sample solution of 500 mg (EW,
5% wt/wt; sugar, 15% wt/wt) and the same amount of
water as a reference were heated at 0.5 C/min from
25 C to 110 C and then cooled to 20 C in an N2
atmosphere. The data obtained from DSC were applied
to nonlinear fitting algorithms to calculate the thermodynamic parameters, thermal denaturation temperature
(Td ) and calorimetric enthalpy (H). The total energy
required for denaturing the EW proteins, the enthalpy
change (H), was measured by integrating the area
under the peaks. The DSC analysis was performed in
triplicate and the results are given as an average.
Preparation of the custard pudding gel samples. The
pudding gel samples were prepared from an egg solution
and milk at a wt/wt ratio of 1:2, and sugar (final
concentration of 15%, wt/wt). The fresh egg solution
was homogenized with a mixer (Nihonseiki Kaisha,
Tokyo, Japan) at 1000 rpm for 20 min and then filtered
through a sieve (150 mesh). Suc, Fru and Psi were each
dissolved in milk at 50 C. The egg solution and sugarmilk solution were mixed and then degassed for 30 min.
For the breaking (puncture) test, the sample solution
was heated in a water bath from 80 C to 95 C at 5 C
intervals for 30 min in a small Petri dish (30 mm
diameter, 10 mm depth) covered with a silicone plate
and a glass plate. In addition, an aliquot of the mixed
solution was poured into a beaker. The beaker was put
into a steel plate containing boiling water and heated at
170 C for 30 min in an electric bakery oven (Nichiwa
Electric Co., Hyogo, Japan). For the creep test, each
sample was poured into a cellulose casing (25.5 mm
Rheological and Antioxidative Properties of Custard Pudding Gels
diameter) and then heated in a water bath at 80 C for
30 min. After heating, the pudding gel was immediately
cooled in a ice water for 20 min and allowed to set at a
temperature of 6 1 C in a refrigerator for the breaking
and creep tests.
Determination of the rheological properties. The
rheological properties were measured by a RheonerIIcreep meter (RE 2-3305; Yamaden Co., Tokyo, Japan).
Two tests were performed: (i) A breaking test to measure the breaking strength of the gel. The gel prepared
in a small Petri dish was placed on the stage of the
rheometer, and the breaking strength was measured
under constant-speed compressive breaking by means of
a 0.5-cm-diameter rod plunger. The breaking strength
was calculated from the stress against strain curve by the
value of the first force peak. (ii) A creep test to study the
development of viscoelasticity over a period of 2 min
carried out under uniaxial compression at 5 mm/s by
means of a 4-cm-diameter plate plunger. The gel from
the cellulose casing was put into a plastic tube, carefully
cut into cylindrical blocks (25.5 mm diameter and
20 mm height), and removed from the casing. The creep
curves were analyzed by a six-element mechanical
model described by equation 1 based on the relationship
between stress and strain.26) The compliance data of EW
gel samples from creep experiments were analyzed by
eqs. 2 and 3:27)
2
X
"ðtÞ ¼ S=E0 þ
S=Ei ð1 eðt=i Þ Þ þ ðS=N Þt ð1Þ
2861
28)
measured at 517 nm. For all experiments, the 75%
ethanol instead of sample solution was used as a control.
The DPPH radical scavenging effect was calculated
as:
½ðOD517 control OD517 sample Þ=ðOD517 control Þ 100%
ð4Þ
Ferric-reducing antioxidative power. The reducing
power of CPE was determined according to the method
of Oyaizu.29) The CPE samples (1 ml) were mixed with a
sodium phosphate buffer (1 ml, 0.2 M, pH 6.6) and
potassium ferricyanide (1 ml, 1%). The mixed solution
was incubated at 50 C for 20 min. After trichloroacetic
acid (1 ml, 10%) was added, the mixed solution was
centrifuged at 3000 g for 5 min. The resulting supernatant (2.5 ml) was mixed with distilled water (2.5 ml)
and ferric chloride (0.5 ml, 0.1%), and the absorbance
was measured at 700 nm. The higher the absorbance of
the reaction mixture, the greater the reducing power.
Statistical analysis. Creep tests were replicated 3
times, and at least 6 gel cylinders were analyzed for each
replication. The breaking strength was measured 5 times
for each dish or beaker, and each sample was measured
at least for 6 dishes or beakers. All other experiments
were analyzed in triplicate, and were measured 3 times
for each replication. Mean values were plotted in all
figures, and the standard deviation and t-test were
calculated by using data for each replicate. A two-tailed
p value lower than 0.05 was considered to be significant.
i¼1
JðtÞ ¼ "ðtÞ=S
2
X
JðtÞ ¼ J0 þ
Ji ð1 eðt=i Þ Þ þ t=N
ð2Þ
Results
ð3Þ
Effect of sugar on the thermal aggregation of egg
white proteins
Turbidity
The EW protein solution containing sugar was heated
at 90 C for 30 min to evaluate the effect of different
sugars (Suc, Fru and Psi) on protein aggregation.
Depending on the protein concentration and sugar used,
clear solutions or turbid suspensions of the protein
aggregate were observed, and the change in turbidity
as an indicator of the aggregation was determined as
the optical density (OD) at 500 nm (Fig. 1). First, no
significant difference in turbidity was found between
EW alone and the Suc-EW solution. However, replacing
Suc by Fru or Psi, for which the sugar concentration was
kept constant at 15%, was able to increase the turbidity
of the EW solution within the concentration range of 1–
10 mg/ml. As expected, the aggregation of the EW
solution increased with increasing protein concentration,
but the degree of increase was markedly different
depending on the added sugar in the following order:
Psi > Fru > Suc. In particular, the addition of Psi to the
1 mg/ml EW solution caused a significant increase in
the turbidity compared to that of Fru and Suc, this being
followed by a slight decrease, and then increase again.
Although the Psi-EW solution was significantly different
i¼1
where "(t) = strain (dimensionless), S = stress (N/m2 ),
E0 = elastic modulus of a Hookean body (instantaneous
modulus; N/m2 ), Ei = elastic modulus of a Voigt body
(N/m2 ), i = retardation time (s), N = Newtonian
viscosity (Pa s), J(t) = creep compliance (m2 /N), J0 =
instantaneous compliance (m2 /N), Ji = retarded compliance (m2 /N), and t = time (s).
Preparation of the pudding gel extract. A sample of
the pudding gel (10 g) was ground into a cream state in a
mortar, suspended in ethanol (20 ml, 99.5%), and mixed
to form a fine smooth slurry with a concentration of
0.5 g/ml (pudding/ethanol). The ethanolic sample was
centrifuged at 3000 g for 10 min. The custard pudding
gel extract (CPE) was obtained from the resulting
supernatant, and diluted to various concentrations with
75% ethanol to analyze the antioxidative profile.
Scavenging activity of the DPPH free radical. Twoml CPE samples were each mixed with 0.5 ml of a 1 mM
DPPH radical solution in 99.5% ethanol. The mixture
was shaken vigorously and allowed to stand at room
temperature for 30 min, and then the absorbance was
2862
Y. S UN et al.
1.2
1.0
Turbidity (OD500nm)
-ME
+ME
Ct
St
Suc
Fru
0.8
1
2
3
4
5
6
7
8
Aggregates
kDa
Psi
120
91
63
47
0.6
0.4
Ovotrans ferrin
Ovalbumin
37
0.2
28
0.0
19
0
2
4
6
8
10
12
Protein concentration (mg/ml)
Fig. 1. Plots of Turbidity (OD at 500 nm) against EW Protein
Concentration.
An EW solution in the presence of 15% sugar was heated at 90 C
for 30 min at different protein concentrations. Data shown are mean
values of three complete sets of experiments (n ¼ 9).
in the aggregation pattern from the Suc- and Fru-EW
solutions, all Psi-EW solutions at the different protein
concentrations tested underwent aggregation to the
highest degree. In addition, visible flocculation was also
observed in the Psi-EW solution at the protein concentration of 1 mg/ml. The results indicate that the
aggregation occurred rapidly by the interaction of Psi
and the EW protein molecules in the suspension at
higher Psi/protein ratios, with further association of the
conjugates into substantial aggregated assemblies. These
results indicate that the heat-induced EW solutions
resulted in the formation of aggregates with a clearly
different degree of variation of sugar under the restricted
concentration range. The occurrence of a heat-induced
aggregate as a significant new group of precipitable
protein particles may increase the network connectivity
and water retention, and introduce rigid covalent bonds
in the gel structure.
SDS–PAGE
The electrophoretic patterns of the EW and sugar-EW
solutions (protein concentration of 1 mg/ml and sugar
concentration of 15%) heated at 90 C for 30 min were
analyzed by SDS–PAGE under reducing and nonreducing conditions. As shown in Fig. 2, the Ct sample
presented the characteristic EW protein electrophoretic
profile, which was essentially composed of ovotransferrin, ovalbumin and lysozyme. The Suc-EW sample was
found to contain similar levels of the main EW proteins
with the EW solution alone in both the presence and
absence of 2-mercaptethanol (2-ME), suggesting that
no effect of Suc on the aggregation degree of EW was
apparent under the heat-treated conditions used. This
result is consistent with the turbidity as already shown
(Fig. 1). In the absence of ME, the bands of the highmolecular-weight aggregate appearing on the top of
separating and stacking gel were found in all four
samples (Fig. 2, lanes 5, 6, 7 and 8). After reducing with
2-ME, these polymeric bands partially disappeared,
Lysozyme
9.4
Fig. 2. SDS–PAGE Patterns of Heated EW and Sugar-EW Solutions
with and without Reduction.
EW protein solutions (1 mg/ml) with different added sugars
(15%) were heated at 90 C for 30 min. St, molecular weight
markers; lanes from 1 to 4 with reduction; lanes from 5 to 8 without
reduction; 1 and 5, EW; 2 and 6, Suc-EW; 3 and 7, Fru-EW; 4 and 8,
Psi-EW.
while monomeric bands appeared (Fig. 2, lanes 1, 2, 3
and 4), implying that disulfide interaction had occurred
between these aggregates. However, the intensity of the
monomeric bands from the EW solutions containing two
reducing sugars, especially for Psi, was lower than that
of the bands from EW alone. The aggregates of the
Fru- and Psi-EW solutions probably appeared during the
heat treatment as a result of Maillard cross-linking and
hydrogen interaction, besides disulfide interaction. In
addition, no residual monomers and lower intensity of
the polymers appearing on the top of the separating gel
were found in the heat-treated Psi-EW suspensions by
SDS–PAGE (Fig. 2, lane 8). It is possible that the partial
aggregates did not penetrate the acrylamide gel, because
it consisted of fairly large polymers.
Effect of sugar on the thermal denaturation of egg
white proteins
The ability of proteins to aggregate and be incorporated into a gel network depends on the heat-denatured
state of the protein. Thus, the effect of three sugars on
the thermal denaturation of EW proteins was investigated by the DSC method. Typical thermograms of
EW protein in the absence and the presence of each
sugar are shown in Fig. 3. The thermogram of EW in an
aqueous solution shows two main transitions with peak
temperatures (Td ) at 63.56 C and 76.07 C, respectively,
corresponding to the denaturation of ovotransferrin
(constituting 12% of EW proteins) and ovalbumin
(constituting 54% of EW proteins), while the overlapping transitions in the intermediate temperature range
may be related to the transition of lysozyme and
ovomucoid, for example.30,31) The thermodynamic parameters obtained from DSC for the EW proteins are
shown in Table 1. The Td values of the two main
transitions when heated in the presence of a sugar were
all significantly higher (P < 0:05) than those observed
Rheological and Antioxidative Properties of Custard Pudding Gels
0.8
Fru
0.6
Suc
0.4
14
A
Psi
Suc
Breaking stress (×10 3, N/m2 )
Heat flow (arbitrary scale)
1.0
Ct
2863
Fru
11
Psi
8
5
0.2
2
75
0.0
60
70
80
90
100
Temperature (°C)
Table 1. Thermodynamic Parameters Obtained from DSC for EW in
the Presence of Three Sugars
Td ( C)
Sample
Peak 1
EW
Psi + EW
Fru + EW
Suc + EW
63:56 0:16a
64:10 0:04b
65:25 0:03c
65:43 0:01d
H (J/g)
Peak 2
76:07 0:02a
77:73 0:16b
78:67 0:02c
78:25 0:09d
Td , denaturation temperature ( C); H, total enthalpy change (J/g). Each
value for Td and H is presented as the mean standard deviation (n ¼ 3).
Means in the same column with different letters are significantly different
(p < 0:05).
in the absence of any sugar, suggesting that each sugar
conferred thermal stability to the EW proteins. Furthermore, the stabilizing influence of Fru and Suc was
greater for the EW proteins than that of Psi, increasing
the Td value in the order of Ct < Psi < Suc/Fru.
According to this result, the EW protein was made less
thermally stable when heated in the presence of Psi than
in the presence of Fru or Suc.
Effect of sugars on the rheological properties of the
pudding gel
Breaking test
To understand the gelling properties of a custard
pudding gel containing Psi, the breaking stress and
breaking strain of the Psi-pudding gel were analyzed
in the temperature range of 80–95 C for comparison
with the Suc- and Fru-pudding gels (Fig. 4). The
breaking stress and strain increased with the increasing
temperature of the heat-induced gel, indicating that all
pudding gel samples became more rigid and elastic with
increasing temperature. Furthermore, the addition of
different sugars significantly affected the breaking stress
and strain of the custard pudding gel. Of the three
sugars, Psi showed the greatest enhancement in the gel
breaking stress and breaking strain, increasing in the
order of Psi > Fru > Scu. The findings for the breaking
95
100
95
100
Suc
Fru
30
Psi
25
20
15
75
17:16 0:22a
21:62 0:79b
21:24 0:68b
17:36 0:21a
90
35
B
Fig. 3. DSC Thermogram of EW Protein in the Absence (Ct) and the
Presence of Different Sugars (Suc, Fru and Psi).
The heating rate was 0.5 C/min.
85
Temperature (°C)
Breaking strain (%)
50
80
80
85
90
Temperature (°C)
Fig. 4. Variation of Breaking Stress (A) and Breaking Strain (B) as a
Function of Temperature for Custard Pudding Gel Samples.
Data shown are mean values of three complete sets of experiments (n ¼ 30).
strength of the different sugar-custard pudding gel
samples were in good agreement with change in
aggregation degree of the sugar–protein solutions.
Measurement of the gel strength was also performed
on baked custard pudding products containing the same
egg/milk ratio with different sugars at 170 C for
30 min. Figure 5 shows changes in the breaking stress
and strain for baked pudding products with 15% of a
sugar. These changes also indicated that Psi was more
(P < 0:05) effective in enhancing the pudding gel
strength than Fru, which in turn was greater (P <
0:05) than Suc.
Creep test
The effect of a sugar on the viscoelastic properties of
the pudding gel samples was investigated by a creep
test. The creep compliance curves were determined
under different stresses (shown in Table 2) in order to
remain within a constant strain (12 3%). Upon analysis of the creep curves according to the six-element
mechanical model from eqs. 1–3, the parameter values
for instantaneous compliance (J0 ), elasticity (E0 ) and
viscosity (N ) of the pudding gel samples were obtained
when heated at 80 C for 30 min (Table 2). First, J0 of
the gel, the reciprocal of the elastic modulus, was
studied because it yields more quantitative information
concerning the molecular structure of a material.
According to Lynch and Mulvihill,32) J0 may be related
to the undisturbed protein network structure, a higher
2864
Y. S UN et al.
20
A
1.2
A
Suc
Fru
0.9
b
15
Reducing power
Breaking stress (*10 3, n/m2 )
c
a
10
Psi
0.6
0.3
5
0.0
75
0
Suc
Fru
40
c
b
a
20
10
90
95
100
95
100
100
B
30
85
Temperature (°C)
DPPH scavenging activity (%)
Breaking strain (%)
B
80
Psi
Suc
Fru
80
Psi
60
40
20
0
75
0
Suc
Fru
Psi
80
85
90
Temperature (°C)
Fig. 5. Comparison of Breaking Stress (A) and Breaking Strain (B)
of Baked Custard Pudding Gel Samples with 3 Sugars at a
Concentration of 15%.
Baked custard pudding samples were heated at 170 C for 30 min.
Data shown are mean values of three complete sets of experiments
(n ¼ 30).
Fig. 6. Variation of the Reducing Power (A) and Scavenging Activity
on DPPH Radical (B) as a Function of Temperature for Ethanolic
Pudding Extracts.
The pudding extract was obtained from the supernatant with a
concentration of 0.2 g/ml (pudding/ethanol). Data shown are mean
values of three complete sets of experiments (n ¼ 9).
Table 2. Comparison of Viscoelastic Parameters for Custard Pudding
Gel Samples
can induce the formation of MRPs with antioxidative
activity in different food systems.9) The temperature
and duration of heating have been studied by
Maillard,33) who reported that an increase in temperature
led to an increase in the reactivity between the sugar and
the amino group. Hence, it was also interesting to note
that the addition of a rare sugar affected the antioxidative activity of egg- and milk-based gel products with
variation in the temperature for food processing.
The production of antioxidative capacity in the
present study was investigated for custard pudding gels,
as a function of the temperature, by the scavenging
DPPH radical activity and ferric-reducing power methods. In a preliminary experiment, it was found that the
Psi-pudding gel formed at 95 C for 30 min had
significant concentration-dependent antioxidative activity at a low concentration in the presence of Psi, but at
a higher concentration of the pudding extract (> 0:3
g/ml), a plateau value for the DPPH radical scavenging
activity was obtained. Hence, the concentration of 0.2
g/ml for the pudding extracts was selected as a functional concentration and further used to evaluate the
effect of rare sugars on the antioxidative activity at
different temperatures.
Figure 6 shows the reducing power and radical
scavenging activity of the three pudding extracts at a
concentration of 0.2 g/ml. The results indicate that the
S(102 ) J0 (103 )
Suc-pudding
Fru-pudding
Psi-pudding
3.99
3.99
5.99
0.20a
0.20a
0.17b
E0 (103 )
N (106 )
5:38a 0:45 1:15a 0:04
5:55a 0:37 1:27a 0:09
6:52b 0:54 1:81b 0:06
S, stress (N/m2 ); J0 , instantaneous compliance (m2 /N); E0 , instantaneous
modulus (N/m2 ); N , Newtonian modulus (Pa s). Each value for E0 and N
is presented as the mean standard deviation (n ¼ 18). Means in the same
column with different letters are significantly different (p < 0:05).
gel rigidity exhibiting lower J0 . As shown in Table 2,
J0 of the Psi-pudding gel was significantly lower than
that of the Suc- and Fru-pudding gels, while J0 of the
Suc- and Fru-pudding gels was not significantly different (P > 0:05). Furthermore, it was observed that the E0
and N values appeared to be higher in the presence of
Psi, indicating that the Psi-pudding gel exhibited a
higher viscoelastic property. In addition, the temperature-dependent trend in viscoelasticity was similar to
that of the breaking strength for the pudding gels (data
not shown).
Effect of sugars on the antioxidative activity of the
pudding gel
It has been demonstrated that a thermal treatment
Rheological and Antioxidative Properties of Custard Pudding Gels
reducing power and radical scavenging activity of the
Suc-pudding extract hardly changed, while the activity
of the Fru- and Psi-pudding extracts was significantly
increased with increasing temperature. In particular, the
DPPH radical scavenging activity of the Psi-pudding
extract was markedly increased to 60:39 2:60% with
increasing temperature to 95 C. In the same way, the
Fru- and Suc-puddings showed a DPPH radical scavenging effect of 30:06 1:15% and 9:80 1:01%,
respectively. At such a temperature, the reducing power
values of the extracts obtained from the Psi-, Fru-, and
Suc-pudding samples were 0:99 0:09, 0:61 0:05 and
0:08 0:01, respectively. These results show that the
highest antioxidative activity was obtained from the Psipudding gel, its respective reducing power and radical
scavenging activity being approximately 12- and 6-fold,
higher than that of the Suc-pudding sample. The results
demonstrate that the antioxidative activity could be
significantly improved by adding a rare sugar in food
processing, this improvement being strongly dependent
on the heating temperature.
Discussion
Heat-induced gel formation results from an aggregation process which has a strong influence on the
structure and properties of the resulting gel.26) Hence,
understanding the aggregation of a heat-induced EW
solution in the presence of a rare sugar may be helpful in
explaining the thermally induced gelling behavior of a
rare sugar–protein mixture. Our results indicate that the
Psi-EW solution possessed a higher degree of aggregation and cross-linking than the Fru- and Suc-EW solutions. This phenomenon might be interpreted as follows:
first, Psi might reduce the water activity and make
water-protein interaction less effective. Secondly, Psi
might interact directly with the protein through hydrogen bonding and lead to a change in the protein surface
hydrophilicity, which could be correlated with enhanced
hydrophobic interaction. Thirdly, Psi might favor crosslinking in Psi-protein molecules through MR, further
increasing the aggregation of the sugar–protein/protein–
protein molecules. Our results indicate that the turbidity
of the heat-induced Psi-EW solution was significantly
increased with increasing Psi/protein ratio, and that
extensive flocculation occurred in such suspension. This
could be attributed to adequate Psi around the protein
molecules, which sequentially affected the hydrogen
bonds and inhibited interaction between water molecules
and the hydrophilic groups in the protein backbone,
promoting the formation of a coagulum as a result of the
increased hydrophobicity.
There was a significant change of Td with the different
sugars, which indicated that the different sugars had a
different effect on the interaction between the protein
surface and the surrounding aqueous phase.34) As shown
in Table 1, the EW proteins were made less thermally
stable when heated in the presence of Psi than in the
2865
presence of Fru and Suc; that is, the proteins in the
aqueous Psi solution might have been unfolded easily.
Consequently, the unfolding of protein upon denaturation might have initiated a cross-linking interaction with
Psi which further speeded up the unfolding process. On
the other hand, protein unfolding is an integral part of
the gelation process; it leads to the exposure of nonpolar amino acids that were originally located in the
interior of the globular molecule. These exposed nonpolar groups could cause a strong hydrophobic attraction
and the formation of disulfide bonds between the protein
molecules, leading to gelation under suitable solution
conditions.
It has been reported previously that the heat-induced
gel of Psi-EW had a significant improved gel strength,
this effect being strongly related to the behavior of the
network structure due to strengthening of the Maillard
cross-linking.35) The influence of reducing sugars and
sucrose on the texture of a thermally processed soy
protein isolate-glucono--lactone gel has also been
studied by Peng,36) in which Maillard cross-linking
was considered to contribute to the hardness of the gel.
Maillard cross-linking might increase the molecular
entanglement in the gel structure, prevent the rupture of
weak non-covalent interaction, and thus stabilize the gel
network. In the present study, the Psi-pudding gel was
characterized by a higher number of cross-links and
stronger interaction between individual molecules, producing greater gel strength values. This result further
demonstrates that the greater the number of cross-links
within a gel network, the higher the gel strength.37)
The effect of different sugars on the visual appearance
of the pudding gel samples was also observed. A
significant difference in appearance was found among
the three pudding gels. The Psi-pudding gel was glossier
with a rubbery texture as compared to the Suc-pudding
gel which was softer and more brittle. The Fru-pudding
gel in its visual appearance was somewhere between the
Psi- and Suc-pudding gels. Moreover, the gels containing Suc showed no color development with increasing
temperature, whereas the gels containing Fru and Psi
became slight browner, especially the Psi-pudding gels,
from heating above 90 C. In general, a rare sugar could
alter the protein gelation mechanism in a number of
ways. As a consequence, a rare sugar might alter both
the formation and the final physicochemical properties
(appearance, texture, and antioxidative capacity) of a
protein gel in a complex manner.
MR has been shown to produce antioxidative components. One of the first observations was reported by
Griffith and Johnson,38) who demonstrated that the
addition of 5% glucose to sugar cookies produced a
marked browning of the cookies and resulted in a
greater stability against oxidative rancidity. Since then,
reaction products from various amino compounds and
sugars have been studied with regard to antioxidative
properties and were found able to protect food against
lipid oxidation.12) Our previous study has also noted that
2866
Y. S UN et al.
a custard pudding dessert baked at 170 C containing
non-calorie Psi possessed remarkable antioxidative
activity.16) At the same time, the baked Psi-pudding
dessert maintained its high level of antioxidative
capacity over a long period of storage, demonstrating
a delay in onset of lipid autoxidation and an increase in
the dessert storage time. It was identified in the present
study that the Psi-pudding gel still had the stronger
antioxidative activities in scavenging the DPPH radical
and providing reducing power when heated at a temperature lower than 100 C, and these activities being
strongly dependent on the temperature in the range of
80–95 C. Epidemiological studies have shown that
the diet can play an important role in the prevention
of certain diseases such as cancer, vascular disorders,
hypertension, and obesity. The findings here demonstrate that a thermal treatment can induce the formation
of a compound with new antioxidative properties in
an egg- and milk-based pudding gel containing Psi,
suggesting that the addition of Psi as a substitute for Suc
could develop a low-calorie functional food.
The interaction between a protein and reducing sugar
takes place by MR during the processing and storage of
foods, and is important for improving the functional
properties. Many studies have focused on MRPs and
their functional properties, but it is still not clear why the
same type of sugars, such as aldohexoses and ketohexoses, which have differences only in the configuration
of one or two hydroxyl (OH) groups, exhibit such
remarkable variation in the formation of MRPs and the
resulting functional properties. In our previous study, it
was indicated that the configuration of the OH groups
around C-3 and C-4 might be very important for the
formation of MRPs in different aldohexose-OVA systems.22) Fru and Psi are epimers of ketohexose with
respect to C3, differing only in the configuration around
one C atom. However, the thermal denaturation,
aggregation, and functional properties in EW proteins
and pudding gels were significantly different in the
presence of two such sugars. The results also suggest
that the configuration around the carbon C3 of hexose
might play a role in the cross-linking and functionality
of a protein. In addition, the differences in MR could
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.22) It might be
speculated that Psi possessed a higher content of acyclic
form in the aqueous phase. On the other hand, it has
been indicated that Psi-protein adducts, which might
undergo less oxidative cleavage in the formation of a
Schiff base or Amadori rearrangement, were available
for further reaction, either with the unreacted protein
monomer or with other protein adducts.19) Subsequently,
the presence of Psi was able to enhance cross-linking of
the protein–protein/sugar–protein and develop an enhanced gelling property. However, the mechanism for
the reaction between a rare sugar and protein is still
unclear, requiring further systematic investigations.
Conclusion
The presence of a rare sugar significantly affected the
thermal denaturation and aggregation of EW proteins,
and the rheological properties and antioxidative activities of pudding gel samples. The Td values of the two
main EW proteins in the presence of Psi were lower than
those in the presence of Fru and Suc, inferring that Psi
conferred less thermal stability to the EW proteins.
Furthermore, the aggregation of EW proteins was found
to be substantially increased by heating with the addition
of Psi. In respect of the custard pudding gel samples, the
presence of Psi significantly enhanced the viscoelastic
properties and breaking strength. It was also recognized
that the Psi-pudding gel exhibited the greatest enhancement in DPPH radical scavenging activity and reducing
power among the three sugar-pudding gels, this increasing in the order of Psi- > Fru- > Suc-pudding. From
these results, the addition of Psi significantly improved
not only the rheological properties but also the antioxidative capacity of the custard pudding gels. These
results could improve the understanding of the thermal
behavior of rare sugar–protein complexes and their
influence on the functional properties of food products.
Acknowledgments
This research was supported by the ‘‘Intellectual
Cluster’’ of the Ministry of Education, Culture, Sports,
Science and Technology of Japan.
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