Food Hydrocolloids 38 (2014) 110e118

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Food Hydrocolloids
journal homepage: www.elsevier.com/locate/foodhyd

Effect of Maillard reaction conditions on the degree of glycation

and functional properties of whey protein isolate e Maltodextrin
conjugates
M.S. Martinez-Alvarenga, E.Y. Martinez-Rodriguez, L.E. Garcia-Amezquita, G.I. Olivas,
P.B. Zamudio-Flores, C.H. Acosta-Muniz, D.R. Sepulveda*
Centro de Investigacion en Alimentacion y Desarrollo, Av. Rio Conchos S/N, Parque Industrial, Cd. Cuauhtemoc, Chihuahua 31570, Mexico

a r t i c l e i n f o

a b s t r a c t

Article history:
Received 20 September 2013
Accepted 6 November 2013

The Maillard reaction has been used as a natural alternative to improve protein functionality by covalent
coupling with saccharides. However, if reaction conditions are not properly selected, glycation can lead to
a loss in functional properties. The objective of our research was to study the effect of temperature, time,
water activity and reactants molar ratio on the degree of glycation and color development in whey
protein isolate conjugated with maltodextrins. Three different levels of glycation (low, medium and high)
were selected to investigate functional properties. The extent of glycation was assessed by quantifying
the loss of amino groups using the o-phthaldialdehyde technique. Sodium dodecyl sulfatepolyacrylamide gel electrophoresis was used to evaluate the molecular weight of the glycoproteins.
Color changes were determined using a Minolta colorimeter and calculating the browning index.
Functional properties evaluated were solubility, rheological behavior, foam overrun and foam stability.
Temperature and water activity were the most inuential factors determining the degree of glycation
and color change. The correlation coefcient between blocked amino groups and color was of 0.743.
Whey protein isolate exhibited lower solubility at pH 5 and conjugates at pH 4. Consistency index and
foaming properties improved according to the level of glycation achieved.
2013 Elsevier Ltd. All rights reserved.

Keywords:
Maillard reaction
Whey protein isolate
Maltodextrin
Functional properties

1. Introduction
Milk proteins consist mainly of caseins and whey proteins, 80%
and 20% respectively (Walstra, Wouters, & Geurts, 2006, chap. 21).
Whey proteins are widely used as raw materials in the food industry due to their nutritional value and functional properties.
Modern processing techniques have allowed the separation and
fractionation of whey proteins to create whey protein powders
with different protein contents (Tunick, 2008). Whey protein
isolate (WPI) contains at least 90% protein, with negligible amounts
of fat, sodium, and lactose and is an excellent source of essential
amino acids. Despite the good functional properties of proteins
from different sources, increasing consumer demand for functional
foods has prompted food scientists to improve protein
functionality.
The Maillard reaction (MR) is a complex series of chemical reactions that occur naturally between the amino group of an amino

* Corresponding author. Tel.: 52 625 5812920.

E-mail address: dsepulveda@ciad.mx (D.R. Sepulveda).
0268-005X/$ e see front matter 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.foodhyd.2013.11.006

acid, peptide or protein and the carbonyl group of a reducing sugar

or an end-product of lipid peroxidation. It is classied in 3 stages
(Hodge, 1953): initial, intermediate, and nal. There is now extensive scientic evidence proving that protein functionality can be
signicantly improved by covalent coupling with saccharides
through the MR without the use of any chemical reagents (Akhtar &
Dickinson, 2007; Chevalier, Chobert, Popineau, Nicolas, & Haertl,
2001; Liu et al., 2012; Nakamura, Kato, & Kobayashi, 1992; Oliver,
Melton, & Stanley, 2006a; Sun, Hayakawa, & Izumori, 2004; Zeng,
Zhang, Guan, & Sun, 2011). This strategy has been regarded by
many scientists as the safest and most promising alternative for the
food industry (Kato, 2002; Mine & Yang, 2010; Oliver, Melton, &
Stanley, 2006b). Conjugation is based on the Amadori rearrangement of the MR (Shepherd, Robertson, & Ofman, 2000). Different
factors such as temperature, time, relative humidity (RH), pH, and
reactants molar ratio affect the rate and extent of the MR, hence,
the nature of the products formed and their functional properties
(Pan & Melton, 2007; Scaman, Nakai, & Aminlari, 2006). The majority of research studies report glycation of proteins with saccharides at high temperatures or for an extended period of time which
could lead to browning, odor formation, and irreversible loss of the

M.S. Martinez-Alvarenga et al. / Food Hydrocolloids 38 (2014) 110e118

protein structural integrity (Akhtar & Dickinson, 2007; Broersen,

Voragen, Hamer, & Jongh, 2004; Hiller & Lorenzen, 2010; Jiang &
Brodkorb, 2012; Li et al., 2009; Lillard, Clare, & Daubert, 2009;
Mesa, Silvn, Olza, Gil, & Castillo, 2008). On the other hand, there
is scarce information on the effect of mild MR conditions on the
degree of glycation and functional properties of proteins with
different levels of glycation produced by this method. This is a
relevant study topic since it has been reported that under such
conditions (60  C), the Amadori product formed in the initial
stage is rather stable (Malec, Pereyra Gonzales, Naranjo, & Vigo,
2002). The present study was therefore aimed at evaluating the
effect of temperature, time, relative humidity (RH), and reactants
molar ratio on the degree of glycation between WPI and maltodextrin (MD) in order to obtain mixtures with different levels of
glycation which were then used to evaluate their functional
properties.
2. Materials and methods
2.1. Materials
Whey protein isolate (WPI) (92.7% protein content), BiPRO Lot
no. JE 214-9-420, from Davisco Foods International (Eden Prairie,
MN, USA) was used in the preparation of protein solutions and
emulsions. It was not denatured, lactose-free, and comprised
mainly of b-lactoglobulin and a-lactalbumin, including as well
small amounts of bovine serum albumin, immunoglobulins, glycomacropeptide, lactoferrin, lactoperoxidase and proteose peptones. Maltodextrin with an average molecular weight of 1672 Da,
o-phthaldialdehyde (OPA, 79760), L-lysine, sodium bromide,
ammonium sulfate, sodium tetraborate, sodium dodecyl sulfate
(SDS), ethanol, and b-mercaptoethanol were purchased from
SigmaeAldrich Co. (Toluca, Edo de Mex, Mexico). All chemicals
used in this study were of analytical grade.
2.2. Description of the study
The present investigation was divided in two parts. Initially, the
effect of temperature (50, 60  C), time (24, 48 h), RH (50, 80%), and
amino/carbonyl ratio (1:1, 1:2) on the degree of glycation between
WPI and MD was evaluated employing a 24 full factorial design,
measuring the percentage of blocked lysine in each treatment as
response variable with the OPA method. These conditions have
shown to cause minimal changes to the protein structure
(Chevalier, Chobert, Dalgalarrondo, Choiset, & Haertl, 2002;
Gauthier, Bouhallab, & Renault, 2001; Morgan, Leonil, Moll, &
Bouhallab, 1999; Morgan et al., 1999; Wooster & Augustin,
2007a). Color was also evaluated as an indirect measurement of
the different stages of the MR. In the second part of the study, 3
glycoproteins (GP) with different levels of glycation (low, medium,
and high) were selected. The solubility, foaming properties, and
viscosity of the conjugates were evaluated and compared with their
respective controls with the aim of knowing how many saccharide
molecules need to be attached per molecule of protein in order to
obtain improved functionality.

treated separately under the same conditions as previously

described. Lyophilized mixtures of the biopolymers without any
treatments (Mixture 1:1, and Mixture 1:2) were evaluated as well.
2.3.1. Determination of degree of glycation
The degree of glycation was dened as the average number of
lysine residues conjugated with MD moieties (Wooster &
Augustin, 2007a) and was determined using the OPA colorimetric assay based on the method of Church, Swaisgood, Porter,
and Catignani (1983). The OPA solution was prepared by dissolving 200 mg of OPA in 5 mL of pure ethanol and mixing this solution with 125 mL of 0.1 M sodium tetraborate buffer (pH 9.75),
0.5 mL of b-mercaptoethanol and 12.5 mL of 10% (w/v) SDS solution. Afterwards, the solution was diluted to a nal volume of
250 mL with milli-Q water. The assay consisted of briey mixing
by inversion in a quartz cuvette 3 mL of OPA solution with 50 mL of
sample solution (1 mg/mL) and measuring the absorbance at
340 nm after 1 min of incubation at room temperature. A standard
curve was constructed using L-lysine by preparing a stock solution
(1 mg/mL) and then diluting to concentrations ranging from
0.02 mg/mL up to 0.1 mg/mL with milli-Q water. The moisture
content of each conjugate was taken into account when calculating the percentage of blocked lysine (Equation (1))

Available amino groups

X
Lmw *2
1
Pmw

(1)

where x is the value obtained in the equation of the standard curve,

Lmw, the Lysine molecular weight, and Pmw, the protein molecular
weight.
2.3.2. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE)
Polyacrylamide gel electrophoresis was performed under
denaturing conditions following the method of Laemmli (1970), in
a Bio-Rad Mini-Protean III apparatus, using a stacking gel of 10% and
a running gel of 4% (w/v). Samples of 20 mg of protein were mixed
(1:1) and denatured in 2X sample buffer (62.5 mM TriseHCl, pH
6.8; 25% glycerol; 2% SDS; 0.01% Bromophenol Blue; 10% b-mercaptoethanol) at 95  C for 3 min. Electrophoresis was carried out in
tris-glycine-SDS buffer (pH 8.35) at room temperature for 30 min at
80 V, and for 2 h at 100 V. Subsequently, a xing solution (40%
methanol, 10% acetic acid, and 50% distilled water) was applied to
gels, which were manually shaken for 15 min. Gels were rinsed
with distilled water and stained with a Coomassie brilliant blue
(CBB G-250) solution for 30 min. Finally, staining solution was
removed with an acetic acid solution (10%) thrice within 24 h.
2.3.3. Color development
Color changes were determined using a Konica Minolta Chroma
Meter CR-300 series (Konica Minolta Sensing, Inc., USA) with
diffuse illumination/0 to obtain the CIE L* a* b* values and then
calculate the browning index (Equation (3)). The instrument was
calibrated with a standard white tile (Y 88.2, x 0.309, y 0.316)
before measurements.

2.3. Preparation of Maillard conjugates

Appropriate amounts of WPI and MD at 1:1 (30 g of protein and
45 g of polysaccharide) and 1:2 (18.75 g of protein and 56.25 g of
polysaccharide) amino:carbonyl ratio were dissolved in Milli-Q
water and stirred for approximately 1 h at room temperature.
Mixtures were then lyophilized, ground and the resulting powders
were kept at 50 and 60  C in desiccators previously equilibrated at
50 and 80% RH for 24 and 48 h. As controls, WPI and MD were

111

BI

a 1:75L
5:645L a  3:012b
100x  0:31
0:172

(2)

(3)

where L, a, and b are the values obtained in the colorimeter, BI is the

browning index, and x is the value obtained in equation (2).

112

M.S. Martinez-Alvarenga et al. / Food Hydrocolloids 38 (2014) 110e118

2.4. Evaluation of functional properties

2.4.1. Solubility
Solubility was determined following the method of JimnezCastao, Lpez-Fandio, Olano, and Villamiel (2005). Lyophilized
samples were dispersed in distilled water and the pH of sample
solutions (3% w/v) was adjusted from 3e7 using HCl or NaOH at 0.1
or 1 M. After 30 min of stirring at room temperature, samples were
centrifuged (Eppendorf 5804-R, USA) for 15 min at 4  C and 3500  g.
Solubility was expressed as the percentage of the initial WPI concentration, determined by measuring the absorbance at 280 nm of
the supernatants using a standard curve of WPI (0.12e0.6 mg/mL).
2.4.2. Foaming properties
Foam overrun and stability were determined following the method
of Phillips et al. (1990) with modications. Lyophilized samples hydration was carried out by dissolving each sample in distilled water (5%
w/v protein equivalent, pH 7) with a magnetic stirrer for 1 h at room
temperature. Subsequently, 100 mL of sample solution was weighed
and foam was formed by whipping for 20 min in an Oster 2700 Stand
Mixer 480 W Mixmaster (Sunbeam Products, USA). The mixer was
turned on and set at speed 11 for the beaters and 2 for the bowl.
2.4.2.1. Foam overrun. Overrun measurements began immediately
after the whipping stopped. Foam was carefully scooped out with a
rubber spatula and placed in 2 tared weighing boats (100 mL) avoiding
air pockets. The excess foam was scrapped off the top with a metal
spatula. The mean value was used to calculate overrun following
equation (4).

fc

evaluated by analysis of variance to determine the inuence of

factors on the variables studied, as well as interactions between
them. Comparison of means was evaluated by least signicant
difference test with a condence level of 95%. Each experiment was
performed in triplicate.
3. Results and discussion
3.1. Determination of degree of glycation
The percentage of blocked amino groups in proteinmaltodextrin mixtures subjected to the experimental conditions
studied is shown in Table 1. The OPA assay proved to be accurate in
monitoring the availability of amino groups, since the experimental
value obtained for WPI in this study (15.38 amino groups per
protein molecule) is very similar to the value of 15.25 reported by
Wooster and Augustin (2007a). Measurements conducted on control mixtures (mixtures not subjected to treatments) showed a
negligible percentage of blocked amino groups, having 15.07 and
15.16 amino groups per protein molecule for Mixture 1:1 and
Mixture 1:2, respectively. WPI controls (WPI samples subjected to
reaction conditions but in absence of maltodextrin) showed as well
a minimum percentage of blocked amino groups, having a mean
value of 15.16 amino groups per protein molecule. Therefore, the
reduction of the available amino groups observed in the conjugates,
as shown in Table 1, can be safely attributed to the binding of MD
molecules to WPI molecules. Under the reaction conditions
employed in this study, multiple degrees of glycation were obtained
among treatments, with a minimum of w2.5 MD attached per WPI

weight of 100 mL of sample solution  weight of 100 mL of foam

*100
weight of 100 mL of foam

where fc is the foaming capability.

2.4.2.2. Foam stability. Foam stability was measured by monitoring
drainage. A plastic bowl with a 0.6 cm hole in the bottom 5.1 cm
from the center containing foam (40.4 g) was placed in a ring stand
at a 30 angle above a tared weighing boat on an analytical balance
pan. The drained liquid was collected in the tared container on the
balance pan and the time to attain half of the initial foam weight
was used as an index of foam stability.
2.4.3. Viscosity measurements
Flow curves were constructed to determine ow behavior constants using an AR 1500ex rheometer (TA Instruments, USA)
equipped with parallel plates geometry (60 mm diameter). All tests
were performed with 1.5 mL of sample solution (5% w/v) at a
controlled temperature of 20  C. The gap between the plates was
adjusted to 300 mm. Shear stress (s) was measured as a function of
increasing shear rate g_ from 10 to 1000 s1. Flow curves were
tted using the Herschel - Bulkley model (Equation (5)), where
consistent coefcient (k), yield stress (so), and ow index (n),
constant tting parameters, were obtained.

s so kg_ n

(5)

2.5. Statistical analyses

Experimental data were analyzed using the SAS statistical
package version 9.00 (SAS Institute Inc., USA). The results were

(4)

molecule at 50  C, 50% RH, 24 h, and 1:1 to a maximum of w8 MD

attached per WPI molecule at 60  C, 80% RH, 48 h, and 1:2. In
general terms, the percentage of blocked amino groups increased
signicantly as the temperature and RH increased, being this effect
more pronounced in the rst 24 h of the reaction (up to w43%
compared to a maximum of w13% between 24 h and 48 h).
3.1.1. Effect of reaction conditions on the degree of glycation
All factors evaluated had a statistically signicant effect
(p < 0.0001) on the degree of glycation, being from the most to the
least inuential: temperature > RH > time > molar ratio, representing 48, 34, 13 and 3% of the observed variation respectively.
There was also a signicant effect of the interactions between
temperature and relative humidity (P < 0.05), and time and relative
humidity (P < 0.001).
3.1.1.1. Effect of temperature. A rise of 10  C (50e60  C) increased
the percentage of glycation from w26% to w40%. This increase can
be attributed not only to an increase in reactivity between the
carbonyl group and amino group, but also to a greater unfolding of
the protein structure, thereby exhibiting a greater amount of
reactive functional groups (lysine residues) which is favored with
increasing temperature. Broersen et al. (2004) reported that an
increase of 15  C (45e60  C) resulted in a 56% increase of blocked
amino groups in conjugates formed between b-lactoglobulin and
glucose. Similarly, an increase of 10  C (40e50  C) showed an increase of 62% of blocked amino groups in conjugates created between b-lactoglobulin and tagatose (considering only the 15 lysine

M.S. Martinez-Alvarenga et al. / Food Hydrocolloids 38 (2014) 110e118

113

Table 1
Degree of glycation and browning index obtained at different reaction conditions.
Temperature ( C)

Relative
Humidity (%)

Time (h)

Molar Ratio
(Amino:Carbonyl)

Available Amino Groups

(Residues per mole WPI)

Blocked Amino
Groups (%  SD)a

60
60
60
60
50
60
60
50
60
60
50
50
50
50
50
50

80
80
80
80
80
50
50
80
50
50
80
80
50
50
50
50

48
48
24
24
48
48
48
48
24
24
24
24
48
48
24
24

1:2
1:1
1:2
1:1
1:2
1:2
1:1
1:1
1:2
1:1
1:2
1:1
1:2
1:1
1:2
1:1

7.42
8.33
8.79
9.13
9.16
9.45
9.88
10.00
10.28
10.83
11.10
11.49
11.75
12.34
12.73
13.03

51.75
45.82
42.87
40.61
40.47
38.54
35.75
35.00
33.15
29.55
27.84
25.28
23.57
19.74
17.20
15.28


















1.75
2.49
1.66
1.79
0.96
1.69
2.10
1.29
1.43
1.03
1.61
2.14
1.05
1.30
1.47
2.00

a
b
c
c,d
c,d
d
e
e
e
f
f,g
g,f
g,f
i
i,j
j

Browning Index
30.87
26.57
17.59
16.20
12.43
9.37
11.84
14.58
6.30
7.48
9.85
7.97
5.65
6.20
4.65
5.18


















2.3
0.9
0.2
0.8
1.6
0.6
0.3
0.1
0.3
0.3
0.7
0.3
0.1
0.1
0.3
0.2

a
b
c
d
f
g
f
e
i,j
h,i
g
h,i
j,k
i,j
k
j,k

a
Each value is an average of three samples  standard deviation. The means followed by different online letters in the same column are signicantly different (p  0.05) by
Tukeys test.

residues and the N-terminal) (Corzo-Martnez, Moreno, Olano, &

Villamiel, 2008). In conjugates formed between lysozyme and
dextran, there was a difference of 20% in glycation between 40  C
and 60  C (Scaman et al., 2006). Zhu, Damodaran, and Lucey (2008)
found little Schiff base formation in WPIedextran conjugates at
temperatures between 40 and 50 C. However, a signicant increase was observed at temperatures between 50 and 60  C. The
differences observed in these studies are understandable and expected due to the structure and reactivity of the different carbohydrates used.
3.1.1.2. Effect of relative humidity. An increase in the degree of
glycation was observed when increasing the RH from 50 to 80%. Our
results agree with those reported by Labuza and Baisier (1992)
where the reaction rate increases gradually with increasing aw
from 0.3 to 0.8 and to those of Pan and Melton (2007) who noted
that maximum reaction rates occurred at intermediate RHs. At aw
values below 0.3, the decrease in reaction rate is attributed to
diffusional limitations between the reactants and at aw values
above 0.8, the decrease is related to dilution effects and to inhibition by water (Buera & Karel, 1993). Desrosiers and Savoie (1991)
observed a higher percentage of blocked amino groups in whey
proteins in the presence of lactose at aw values of 0.3, 0.5 and 0.7
than at 0.97. Similarly, Malec et al. (2002) reported the greatest
amount of blocked amino groups at low and intermediate aw values
rather than high aw values (0.85 and 0.98).
3.1.1.3. Effect of time. Different research groups have reported incubation times of up to 3 weeks at mild temperatures in order to
achieve glycation between globular proteins and polysaccharides
arguing that their folded structure hides many reactive groups
which become accessible as time goes by (Kato, 2002; Nakamura
et al., 1992). Other research groups report shorter times, from 1.5
to 14 days (Dunlap & Ct, 2005; Einhorn-Stoll, Ulbrich, Sever, &
Kunzek, 2005; Miralles, Martnez-Rodrguez, Santiago, Lagemaat, &
Heras, 2007; Wooster & Augustin, 2007b). The values in Table 1
show that a signicant percentage of blocked amino groups were
achieved in only 24 h of incubation. Furthermore, glycation was
more pronounced in the rst 24 h, reaching values of up to w43%
compared to an average of 7.5% between 24 and 48 h. Similar trends
have been reported for lysozyme, ovalbumin, sodium caseinate and
the 11S fraction of soy protein (Achouri, Boye, Yaylayan, & Yeboah,
2005; Aoki et al., 1999; Morris, Sims, Robertson, & Furneaux, 2004;
Sun et al., 2004; Yeboah, Alli, Yaylayan, Konishi, & Stefanowicz,

2000). Reaction rates observed during the rst and second 24h periods (Fig. 1) were signicantly different among them,
showing a strong effect of reaction temperature and relative humidity during the rst 24 h and a highly reduced reaction rate
during the second 24-h period, when the effect of temperature and
relative humidity was no longer relevant, having an average reaction rate value of only 0.046 blocked residues per mole per hour.
It is interesting to note that the lowest levels of blocked amino
groups obtained in this study (15e19% blocked amino groups in
samples subjected to 50  C and 50% RH) roughly coincide with the
number of accessible amino groups in a-lactalbumin alone. According to Jimnez-Castao, Villamiel, and Lpez-Fandio (2007),
80e90% of lysine residues in the three dimensional structure of alactalbumin are readily accessible for interaction with other molecules. This number of free residues would correspond to 18.4%e
20.7% of the total number of amino residues in WPI, considering the
occurrence of b-lactoglobulin and a-lactoglobulin in a mole ratio of
approximately 2.7 to 1, having 16 and 13 free amino groups,
respectively (Wooster & Augustin, 2007a). This observation suggests that glycation during the rst 24 h may have proceeded
mainly with a-lactalbumin amino residues, requiring of more time
and more stringent conditions in order to be able to proceed with
b-lactoglobulin amino residues. This hypothesis is further supported by the results reported by Nacka, Chobert, Burova, Leonil,
and Haertl (1998) who found that glycation of a-lactalbumin
was faster and more pronounced compared to that of b-lactoglobulin in the presence of mono- and disaccharides.
3.1.1.4. Effect of reactants molar ratio. Generally, an excess of
carbonyl groups in the molar ratio of reactants promotes the
development of the MR. In this study, an increase of 1:1 to 1:2
(amino/carbonyl ratio) increased glycation by w3.5%. An increase in
the molar ratio of 1:3 to 1:15 in conjugates formed between bovine
serum albumin and galactomannan resulted in the binding of 2.5e
6.7 molecules of polysaccharide to protein. Wooster and Augustin
(2007b) evaluated the degree of glycation of conjugates created
between b-lactoglobulin and MD according to the following molar
ratios: 1:0.09, 1:0.17, 1:0.25, 1:0.33, 1:0.66, and 1:1.7 obtaining 10.4,
12.9, 16, 25.5, 35 and 47% of blocked amino groups, respectively.
3.1.2. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE)
SDS-PAGE under denaturing conditions was employed to evaluate the molecular weight of the produced GPs. In Fig. 2, GPs are

114

M.S. Martinez-Alvarenga et al. / Food Hydrocolloids 38 (2014) 110e118

Reaction Rate (Blocked residues per mole per hour)

0.3

First 24 h
0.25

Second 24 h
0.2

0.15

0.1

0.05

0
60C/80%

60C/50%

50C/80%

50C/50%

Reaction Conditions (Temperature / Relative Humidity)

Fig. 1. Reaction rates observed during the rst and second 24-h periods in which the samples were subjected to the studied experimental conditions.

arranged according to the OPA results, from the one with the
highest percentage of blocked amino groups to the one with the
lowest level, and also according to the employed treatment temperature. The lanes from 2 to 9 correspond to GPs obtained at 60  C,
and lanes from 10 to 17 to those obtained at 50  C. Controls (mixtures not treated and WPI subjected to treatments in absence of
MD) are not included in the shown gel because no signicant
reduction of free amino groups was observed during OPA analyses
and because their SDS-PAGE results did not signicantly differ from
those corresponding to untreated WPI. Two intense bands are
shown in lane 1, which are characteristic of WPI and belong to alactalbumin (lower band) and b-lactoglobulin. The WPI protein
bands tend to disappear, and new compounds with higher molecular weight start to form as the degree of glycation increase, which
seems to indicate that all the WPI proteins were glycosylated.
However, when comparison was made between the OPA results
(available amino groups) and the assumption of the formation of
highly glycosylated molecules from all the a-lactalbumin and blactoglobulin proteins, it would be expected a lower protein glycation. This demonstrates that OPA method shows an average of
available amino groups per molecule and does not distinguish between glycosylated and non-glycosylated molecules (Wooster &
Augustin, 2007a).

3.1.3. Color development

Color development of the GP, as an indirect measurement of the
development of the MR, is also shown in Table 1. The browning
index has shown to be a good indicator of the changes in the color
of the MR products (Matiacevich & Buera, 2006; Pagliarini, Peri, &
Abba, 1990). The browning index in WPI increased from 4.49
(sample without any treatment) to a mean value of all treated WPI
controls of 5.14  0.29. On the other hand non-treated MD showed
a browning index of 2.35, lower than WPI. An average browning
index of 3.85  0.57 of all treated MD controls was obtained, which
indicated a possible caramelization of the MD. Caramelization was
caused by the thermal dehydration of MD and could be suppressed
by dialysis (Shepherd et al., 2000). On the other hand, treated
mixtures WPI-MD clearly developed color changes due to MR. The
lowest browning index (4.65) was found in GP obtained at 50  C,
50% HR, 24 h, 1:2; and the highest (30.87) at 60  C, 80% HR, 48 h,
1:2, with a predominant yellow color (Fig. 3), which was probably
due to the MR step reached under those conditions or to the above
mentioned caramelization effect. Several studies report color
development when dairy proteins are conjugated with MD, suggesting that the presence of low molecular weight sugars in the MD
(up to 25% of sugars with molecular weight lower than 1200 Da)
may be responsible for the observed color changes (Akhtar &

Fig. 2. SDS-PAGE of WPI and GP. Lane 1) WPI, 2) GP (60-80-48-1:2), 3) GP (60-80-48-1:1), 4) GP (60-80-24-1:2), 5) GP (60-80-24-1:1), 6) GP (60-50-48-1:2), 7) GP (60-50-48-1:1),
8) GP (60-50-24-1:2), 9) GP (60-50-24-1:1), 10) GP (50-80-48-1:2), 11) GP (50-80-48-1:1), 12) GP (50-80-24-1:2), 13) GP (50-80-24-1:1), 14) GP (50-50-48-1:2), 15) GP (50-50-481:1), 16) GP (50-50-24-1:2), 17) GP (50-50-24-1:1). WPI means whey protein isolate and GP means glycoprotein. Numbers in parentheses correspond to the reaction conditions:
temperature ( C), relative humidity (%), time (h), and molar ratio, respectively.

M.S. Martinez-Alvarenga et al. / Food Hydrocolloids 38 (2014) 110e118

115

Fig. 3. Color development in samples treated under different experimental conditions. WPI means whey protein isolate and GP means glycoprotein. Numbers in parentheses
correspond to the reaction conditions: temperature ( C), relative humidity (%), time (h), and molar ratio, respectively. (For interpretation of the references to color in this gure
legend, the reader is referred to the web version of this article.)

3.1.3.1. Effect of reaction conditions on color development. The factors that showed a statistically signicant effect (p < 0.0001) on
color development were relative humidity, temperature and time,
from the most to the least inuential, representing 46, 26, and 13%,
respectively. Molar ratio did not showed a signicant effect
(p 0.8325). Medrano, Abirached, Panizzolo, Moyna, and An
(2009) reported that molar ratio had a signicant effect on the
color development of GP obtained from b-lactoglobulin and
glucose, but no effect was observed on GP made from b-lactoglobulin and lactose, which indicated that the carbohydrate reactivity is a determinant factor. In a monosaccharide e single amino

14
Available amino Groups

Dickinson, 2007; Morris et al., 2004; ORegan & Mulvihill, 2009;

Shepherd et al., 2000). A positive lineal correlation (0.743) between available amino groups and color development was found
(Fig. 4).

y = -0.1839x + 12.572
R = 0.7432

13
12
11

10
9
8
7

6
0.0

10.0

20.0
Browning Index

30.0

40.0

Fig. 4. Lineal correlation between available amino groups (Residues per WPI mole)
and Browning index (obtained from equation (3)) of glycoprotein samples.

116

M.S. Martinez-Alvarenga et al. / Food Hydrocolloids 38 (2014) 110e118

acid system, the molar ratio showed a signicant effect on the color
development at pH values higher than 8 (Renn & Sathe, 1997). At
RH of 50%, the browning index average was 7.08, whereas at 80% an
average of 17.01 was reached. These results concur with those obtained by Pan and Melton (2007), where the maximum color
attained in GP made from sodium caseinate and lactose occurred at
80% of RH. Results shown in Table 1 suggest that as the temperature
increased, the development of color also increased, producing an
average difference of w7.5 units between 50 and 60  C treatments.
Likewise, an average difference of w5.2 units was found between
24 h and 48 h treatments.
The browning index increased as the temperature and time rose,
getting the highest browning index value at 60  C for 48 h. Color
was approximately 3 times higher at 60  C and 80% RH, than at
60  C and 50% RH. Similarly, a considerable increase was observed
as time and RH increased. Similar results were reported for GP
prepared from whey proteins and polysaccharides, and also for
protein/monosaccharide and amino acid/monosaccharide systems
(Bosch, Alegra, Farre, & Clemente, 2007; Einhorn-Stoll et al., 2005;
Rozycki, Buera, Piagentini, Costa, & Pauletti, 2010).
3.2. Functional properties
3.2.1. Solubility
Solubility of WPI, controls, and GPs was assessed as a function of
pH (Fig. 5). Untreated WPI was almost completely soluble at all pH
conditions evaluated in this study, showing a minimum solubility
value of 85% at its isoelectric point. Solubility of control treated at
60  C, 80% RH for 48 h (WPI treated under glycosylation conditions
without MD) decreased considerably to a minimum solubility value
of 27% at pH 5. A decrease in solubility of b-lactoglobulin from 30 to
40% was reported by Jimnez-Castao et al. (2005) when protein
was heated at 60  C, 44% HR for 4 days in a concentration of 1 mg/
mL. The low solubility of WPI controls is probably due to a higher
exposition of hydrophobic amino acid residuals to water owing the
spatial conformation of proteins. The GP sample with the lowest
glycosylation level (GP, 50-50-24-1:1) resulted in a solubility
comparable to that of the WPI sample, with an increase of 3% at its
isoelectric point, possibly due to the addition of hydroxyl groups
provided by MD. GP samples with high and moderate glycosylation
levels showed a change in the minimum solubility value from pH 5
100

to pH 4e4.5, due to an increment of the negative net charge of GP,

moving the isoelectric point to a more acidic region (Fenaille,
Morgan, Parisod, Tabet, & Guy, 2003). Other research groups have
reported a greater reduction in solubility (up to 70%) in GP prepared
with b-lactoglobulin and ribose or arabinose at pH 4 (Chevalier
et al., 2001; Nacka et al., 1998). In general terms, GP showed better solubility than that of WPI samples equally treated (WPI controls). Furthermore, a change in the isoelectric point was observed
in GP with moderate and high glycosylation levels.
3.2.2. Foaming properties
All the assessed samples were able to foam by mechanical
stirring (Fig. 6). WPI exhibited a similar foaming capability than
that of control mixtures (Mixture 1:1 and Mixture 1:2) and WPI
treated at 50  C, 80% RH for 48 h. WPI treated at 50  C, 50% RH for
24 h, showed a higher foaming capability than the previously
mentioned samples; whereas WPI treated at 60  C, 80% RH for 48 h
presented the lowest foaming capability. Typically, a thermal
treatment enhances the foaming capability of proteins (Phillips,
Schulman, & Kinsella, 1990); nevertheless, the foaming capability
reduction of the WPI sample (60  C, 80% RH, 48 h) could be due to
its low solubility. GP samples showed a higher foaming capability
than WPI controls, being the highest glycosylated GP the one which
exhibited the best foaming capability.
Control mixtures (Mixture 1:1 and Mixture 1:2 without treatment) showed a signicant improvement in the foam stability of
WPI, being superior when mixtures with high polysaccharide
content were employed (Fig. 7). Foam stability showed a similar
behavior than foaming capability. WPI sample (50  C, 50%, 24 h)
showed a higher foam stability than that of untreated WPI, WPI
(50  C, 80% RH, 48 h), and WPI (60  C, 80% RH, 48 h). Moreover, GPs
exhibited a higher foam stability than that of untreated WPI, being
the highest glycosylated GP the one which showed superior
stability.
Foaming properties of WPI became considerably better when
glycosylated, and subjected to a heat treatment for 24 h at 50  C and
50% RH. The best foaming properties were achieved in GP with w8
MD attached per WPI molecule.
3.2.3. Viscosity
Flow curves were tted using the HerscheleBulkley model
(Equation (5)). Although all studied samples showed a ow index

1200

70
60

GP 50-50-24-1:1
GP 50-80-48-1:1
GP 60-80-48-1:2
WPI
WPI 50-50-24
WPI 50-80-48
WPI 60-80-48
4

5
pH

Fig. 5. Solubility of WPI controls and GPs in water at different pH values. Samples
nomenclature corresponds to temperature ( C) e relative humidity (%) e time (h) e
molar ratio, respectively. Results are expressed as the percentage of the initial WPI
solubility.

WPI 60-80-48

10

200
WPI50-80-48

20

400

WPI 50-50-24

30

600

Mixture 1:2

40

800

Mixture 1:1

50

1000

WPI

Solubility (%)

80

GP 50-50-241:1
GP 50-80-481:1
GP 60-80-481:2

Foaming Capability (%)

90

Fig. 6. Foaming capability of whey protein isolate (WPI), untreated mixtures of WPI
and maltodextrins, treated WPI controls, and selected glycoproteins (GP) produced
under different conditions (temperature ( C) e relative humidity (%) e time (h) e
molar ratio). Results are expressed as a percentage of the sample solution volume
before stirring.

M.S. Martinez-Alvarenga et al. / Food Hydrocolloids 38 (2014) 110e118

80
60
40
20

WPI 60-80-48

WPI50-80-48

WPI 50-50-24

Mixture 1:2

Mixture 1:1

WPI

GP 50-50-241:1
GP 50-80-481:1
GP 60-80-481:2

Foam Stability (min)

100

Fig. 7. Foam stability of whey protein isolate (WPI), untreated mixtures of WPI and
maltodextrins, treated WPI controls, and glycoproteins (GP) produced under different
conditions (temperature ( C) e relative humidity (%) e time (h) e molar ratio). Results
are expressed as the time to attain half of the initial foam weight obtained after
stirring.

value (n) of 1, the presence of yield stress (so) in all of them turned
the classication of their behavior into non-Newtonian (Bingham
plastic). Table 2 shows the consistency coefcient (k) and yield
stress (so) observed in the different studied solutions. Consistency
coefcients in general increased as the glycosylation level of the
samples increased, indicating that glycosylation tends to cause a
thickening effect on solutions, probably due to an increased molecular size (hydrodynamic volume). Similar results were published
by Corzo-Martnez et al. (2009). It is noted that only the consistency
coefcient of GP samples with medium and highest glycosylation
levels (GP, 50-80-48-1:1, and GP, 60-80-48-1:2, respectively) were
statistically signicantly different than their own controls. On the
contrary, yield stress tended to decrease with the increase in the
degree of glycosylation, implying that less stress is necessary to
start ow, probably suggesting a lower level of interaction between
proteins as the glycosylation level increases. The yield stress of WPI
controls was not statistically different among them.

4. Conclusions
Glycoproteins with w2.5ew8 maltodextrins attached per WPI
molecule were successfully produced in this study. GPs obtained at
Table 2
Consistency coefcient and yield stress of studied solutions (5% w/v).
Treatment
b

WPI
MD
Mixture 1:1
Mixture 1:2
WPI 50-50-24
WPI 50-80-48
WPI 60-80-48
GP 50-50-24-1:1
GP 50-80-48-1:1
GP 60-80-48-1:2

so (Pa s)a

k (Pa s)a
0.00115
0.00108
0.00134
0.00141
0.00121
0.00113
0.00136
0.00141
0.00154
0.00300












0.00009
0.00006
0.00008
0.00020
0.00001
0.00001
0.00001
0.00003
0.00003
0.00004

c
c
b,c
b,c
b,c
c
b,c
b,c
b
a

0.11913
0.04609
0.13311
0.14733
0.05888
0.05688
0.06228
0.23288
0.12696
0.12608












0.01495
0.00038
0.00288
0.01294
0.00001
0.00001
0.00001
0.00003
0.00003
0.00004

b
c
b
b
c
c
c
a
b
b

a
k and so are the HerscheleBulkley model parameters: consistent coefcient and
yield stress, respectively.
b
Each value is an average of three samples  standard deviation. The means
followed by different online letters in the same column are signicantly different
(p  0.05) by Tukeys test. WPI means whey protein isolate, MD means maltodextrin,
and GP means glycoprotein. Numbers of GP correspond to the reaction conditions:
temperature ( C), relative humidity (%), time (h), and molar ratio, respectively.

117

50  C, 50 RH, 24 h, 1:2 showed the lowest browning index, while

the highest BI, with a predominant yellow color, was observed in
molecules obtained at 60  C, 80 RH, 48 h, 1:2. A positive correlation
of 0.743 between the percentage of blocked amino groups and the
color development was found. The temperature and water activity
were the most inuent factors on the degree of glycation achieved,
representing 48% and 34% of the effect on the degree of glycation
and 26% and 46% on the effect on color development, respectively.
Changes in the molecular weight of the GPs were observed
employing gel electrophoresis as glycosylation percentage
increased, according to OPA assays. The lowest solubility of WPI in
water shifted from its standard isoelectric point (wpH 5) to pH 4e
4.5 when a high degree of glycosylation was achieved. The consistency coefcient of the studied GPs aqueous solutions increased
as the degree of glycation rose. The foaming properties of GP were
improved compared to those of the native protein, where highly
glycosylated GP showed the best foaming properties. This study
demonstrates that the Maillard reaction is a viable alternative to
improve protein functionality. Results from this study suggest that
the functional properties of glycoproteins may depend not only on
their degree of glycation but also on the employed production
method, making relevant for future research the study of the
functional properties of glycoproteins with equivalent degree of
glycation but produced by different methods.
Acknowledgments
The authors thank Angel Esparza and Arturo Ramos for their
technical assistance. This work was supported by the Mexican
National Council of Science and Technology (CONACYT) and
Chihuahua State Government, through FOMIX project number
CHIH-2009-C02-126256.
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