Food Hydrocolloids 30 (2013) 53e60

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

Functional properties of Maillard reaction products of rice protein hydrolysates

with mono-, oligo- and polysaccharides
Yue Li a, Fang Zhong a, *, Wei Ji a, Wallace Yokoyama b, Charles F. Shoemaker c, Song Zhu a, Wenshui Xia a
a

Key Laboratory of Food Colloids and Biotechnology, Ministry of Education, School of Food Science and Technology, Jiangnan University, 1800 Lihu Avenue, Wuxi 214122, PR China
Processed Food Research, Agricultural Research Service, USDA, Albany, CA 94710, USA
c
Department of Food Science and Technology, University of California, Davis, CA 95616, USA
b

a r t i c l e i n f o

a b s t r a c t

Article history:
Received 28 October 2011
Accepted 26 April 2012

The Maillard reaction was carried out under wet reaction conditions using rice protein product from
limited hydrolysis to conjugate with various mono-, oligo- and polysaccharides. The Maillard reaction
products (MRPs) prepared with rice protein hydrolysates at 5% degree (hydrolysed by Protease N) and
dextran T20 (20 min at 100
C) showed the greatest improvement in functionality. The solubility,
emulsication activity (EA), and emulsication stability (ES) of the MRPs increased by factors of 3.5, 5.3
and 7.3 times, respectively as compared to MRPs formed with native rice proteins and dextran T20.
Amino acid analysis indicated that lysine and arginine decreased signicantly in the MRPs. Formation of
MRPs reduced the surface hydrophobicity, which was in agreement with the change of solubility.
However, further decreasing of the surface hydrophobicity resulted in lower EA and ES during the late
stage of the reaction. Fluorescence analysis suggested that formation of late stage MRPs occurred after
20 min of the Maillard reaction. The molecular weight distributions showed that the functional properties of MRPs and the mechanisms of formation were affected by the peptide chain length.
2012 Elsevier Ltd. All rights reserved.

Keywords:
Rice protein
Maillard reaction
Enzymatic hydrolysis
Emulsifying properties
Solubility

1. Introduction
Rice is the source of half the daily per capita of the worlds
protein supply. Protein from rice is high in biological nutritive
value, hypoallergenic, and bland in taste. These properties make it
ideal for infants (Cristina & Cristina, 2008; Jariwalla, 2001; Kennedy
& Burlingame, 2003; Parrado et al., 2006). However, the use of rice
protein as an ingredient in food has been limited, largely due to its
poor functional properties as a result of its low solubility at neutral
pH (Gujrala & Rosell, 2004).
Besides rice there are many other sources of vegetable proteins
whose utilization would be increased by improvements of their
functional properties through food approved physical and chemical
modications. Some success has been achieved by the use of
proteolytic enzymes. Improved functionalities were observed
following controlled or limited proteolysis (Dinakar & Kilara, 1996).
The Maillard reaction has also been reported to be an effective
method to improve the functional properties of proteins (Dickinson,
2008; Hiller & Lorenzen, 2010; Miralles & Martnez-Rodrguez, 2007;
Niu, Jiang, Pan, & Zhai, 2011; Oliver, Melton, & Stanley, 2006; Wooster
& Augustin, 2006). The modied protein from soy or milk protein has
* Corresponding author. Tel.: 86 138 12536912; fax: 86 510 85197876.
E-mail address: yue_li1@yahoo.com.cn (F. Zhong).
0268-005X/$ e see front matter 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodhyd.2012.04.013

been extensively studied, and Maillard reaction products (MRP) were

shown to have improved solubility and emulsifying properties
(Einhorn-Stoll, Ulbrich, Sever, & Kunzek, 2005; ter Haar, Westphal,
Wierenga, Schols, & Gruppen, 2011; Tian, Chen, & Small, 2009). In
foods and other biological systems containing proteins with free
amino groups or amino acids and carbohydrates, this reaction occurs
spontaneous and is considered to be a promising approach for the
improvement of the functional properties of proteins for food uses
(Akhtar & Dickinson, 2003; Chevaliera, Chobert, Popineau, Nicolas, &
Haertl, 2001; Li et al., 2009; Wooster & Augustin, 2006). Reports
related to rice protein modication by hydrolysis or Maillard reaction
have been limited (Li et al., 2009; Paraman, Hettiarachchy, & Schaefer,
2007), although rice proteins are a reasonable substrate for the
Maillard reaction since it contain a moderately high ratio of lysine.
There are two approaches, which have been reported in Maillard
reaction. The rst method involves the heating of a dry dispersion
of a protein and saccharide system under controlled temperature
and relative humidity conditions. It requires a long reaction time up
to several days or weeks, which was not feasible for industrial
production. The second method is to carry out the reaction in an
aqueous solution or dispersion. It has the advantage of better
control and shorter reaction time.
In our previous study, rice protein hydrolysates were prepared
with several food grade proteases to different hydrolysis degrees.

54

Y. Li et al. / Food Hydrocolloids 30 (2013) 53e60

The hydrolysates exhibited improved solubility, but no signicant

improvement in emulsifying activity (Data were not shown). It was
also found that MRP from unhydrolysed alkaline extracted rice
protein and various polysaccharides had improved functional
properties but were still inferior to those more soluble proteins,
such as whey (Li et al., 2009).
The objective of this study was to investigate the benets of rice
protein modication with combined action of limited hydrolysis
and glycation under wet reaction conditions. The small to large
carbohydrates (glucose, lactose, maltodextrin DE20, and dextran
T20) were used for glycation, and the functional properties and
structure of MRPs were evaluated. Although further research will
be needed to elucidate precise mechanistic details, this study will
extend our knowledge of the functionality changes due to hydrolysis and Maillard reaction of hydrophobic plant proteins.
2. Materials and methods
2.1. Preparation of rice protein concentrates
Milled medium-grain rice (Nanjing 39 variety, Jiangsu Baobao
Group Co., Rudong, China) was washed with deionized water, and
then soaked in three times its weight of deionized water for 15 h. The
rice and water were blended into slurry, and the protein was
extracted with 0.1 M NaOH at 40
C (the ratio of slurry to NaOH
solution was 1:3, w:w). The slurry was continuously stirred for 2 h
and then centrifuged (6000g for 10 min). The supernatant was
decanted, and its pH was adjusted to 5.5 using 0.1 M HCl. It was left
undisturbed at 4
C to precipitate overnight. The supernatant was
carefully siphoned off from the precipitate. The precipitate was
washed 4 times with deionized water. The nal slurry was
neutralized to pH 7.0 with 0.1 M NaOH and lyophilized. The protein
content of the lyophilized substrate was determined by the Kjeldahl
method (The value of 5.95 was used as a protein conversion factor),
and the rice protein content was 92% (dry basis). The average
molecular weight of the rice protein was around 9.65  105 Da.
2.2. Preparation of rice protein hydrolysates
Rice protein was dispersed in 1% (w/w) water, and the suspension was adjusted to 50
C and the optimal pH recommended by the
enzyme manufacturer for each protease, and then incubated for
30 min with continuously stirring. The optimal pH of Protease N
(Amano Enzyme Inc., Nagoya, Japan), Alcalase (Novozyme, Beijing,
China) and AS1398 (Genencor, Wuxi, China) were 9.0, 8.0 and 8.0,
respectively. The enzyme-to-substrate ratio was 0.0024:1 (w/w).
The pH of the dispersion was kept constant by continuous addition
of 0.03 M NaOH solution to the reaction mixture. The mixture was
incubated for 10.3, 12.7, 15.4 and 19.4 h which corresponded to the
degree of hydrolysis values of 4, 5, 6 and 7%, respectively. The
enzyme reaction was stopped by heating the dispersion at 100
C
for 5min. The dispersion was then cooled and stored at 4
C prior to
analysis.
2.3. Determination of degree of hydrolysis (pH-stat assay)
The degree of hydrolysis (DH), dened as the percent ratio of the
number of peptide bonds broken (h) to the total number of bonds
per unit weight (htot) was calculated from the amount of base
added during the hydrolysis to maintain a constant pH:

groups, and the value of 1/a for rice protein was 1.01; MP was the
mass of protein in gram; htot was the hydrolysis equivalents in
meqv/g protein (i.e., the total number of peptide bonds in the
protein, 7.4 meqv/g rice protein) (Adler-Nissen, 1986, pp. 122e124).
All samples were analysed in triplicate.
2.4. Preparation of MRPs
The MRP were prepared by the reaction of the rice protein
hydrolysates with glucose, lactose, maltodextrin DE20, and dextran
T20 (Sinpharm Chemical Reagent Co. Ltd, Shanghai, China). The
molecular weight of glucose, lactose, maltodextrin DE20 and
dextran T20 are 180, 342, around 1000 (based on the rule
DE  DP 120), and 20,000 Da. Each saccharide (1%, w/w) was
mixed with 1% (w/w) rice protein hydrolysate prepared at different
DH as described above. The materials were thoroughly dispersed in
water by vigorous and continuous agitation for 1 h, and then
adjusted to pH 11.0 with 10 M NaOH.
Aliquots of 30 mL were transferred to each of six 50 mL screwcapped glass tubes, and capped to prevent evaporation during heating. The samples were incubated in a boiling water bath at 100
C, and
removed at 0, 5, 10, 20, 30 or 40 min, respectively. The removed tubes
were immediately placed in an ice bath. The resultants were dialysed
against deionised water for 24 h and then centrifuged at 10,000g for
10 min. The supernatant termed as MRPs was collected, and stored at
4
C prior to analysis. Samples prepared by the same procedure were
freeze-dried for other experiments.
2.5. Emulsifying properties measurement
The emulsifying properties of the MRPs were determined by the
method of Pearce and Kinsella (1978) with some modications. An
MRP sample was diluted with buffer (0.05 M sodium phosphate
buffer, pH 8.0) to a nal protein concentration of 0.4%, and 9 mL was
combined with 3 mL of soy oil and mixed with continuous agitation.
The coarse emulsion was then homogenized using an Ultra-Turrax
T25 homogeniser (IKA Werke GmbH & Co. KG, Staufen, Germany)
at 10,000 rpm for 1 min. A sample of the emulsion (50 mL) was taken
from the bottom of the test tube at 0 and 10 min and immediately
diluted with 5 mL of 0.1% sodium dodecyl sulphate (SDS) solution.
The absorbance of the diluted emulsion was then measured at
500 nm. The emulsifying activity (EA) was determined from the
absorbance measured immediately after the emulsion had formed
(0 min). The emulsion stability (ES) was calculated as follows.
ES A0  10/(A0  A10), where A0 is the absorbance of the sample
immediately after the emulsion had formed (0 min), and A10 is the
absorbance of the sample after 10 min.
2.6. Solubility measurement
10 mL of an MRP was diluted with 10 mL of various buffers
(0.05 M sodium phosphate buffer, pH 5.0, 6.5 and 8.0; 0.05 M
sodium hydrogen carbonate buffer, pH 9.5 and 11.0; 0.05 M citrate
buffer, pH 2.0 and 3.5). The pH of the solution was readjusted to the
buffer pH with 0.1 M HCl or NaOH. Samples were centrifuged for
20 min at 10,000g. Protein content in the supernatant was
determined by the Kjeldahl method. Solubility (NS) was expressed
as a percentage of protein in the supernatant to the total protein
content (supernatant and precipitate). The measurement was
carried out in triplicate for each sample.

DH% 100  h=htot B Nb 1=a 1=MP1=htot  100

2.7. Determination of the degree of substitution

where B was the base consumption in mL; Nb was the normality of

the base; a was the average degree of dissociation of the a-NH2

The degree of substitution (DS) of the rice peptides was determined from the analysis of the free amino groups in the rice proteins

Y. Li et al. / Food Hydrocolloids 30 (2013) 53e60

and MRPs according to Adler-Nissen (1979). 1 mL protein or MRP

sample (1.2% w/v) was dispersed in 5 mL 0.1% SDS to obtain a solution with 0.2% protein concentration. It was then mixed with 2.0 mL
of 0.21 M phosphate buffer (pH 8.2) and 1.0 mL of 0.1% 2,4,6trinitrobenzenesulfonic acid (TNBS) solution. The solution was
thoroughly mixed and placed in a temperature-controlled water
bath at 50
C for 1 h in the dark. The reaction was terminated with
the addition of 2.0 mL of 0.1 M sodium sulphite. The mixture was
cooled at room temperature for 30 min. The absorbance was
measured at 340 nm. The degree of substitution was determined as:

DS % A0  At =A0 100%;
where A0 was the absorbance of the sample before heating, and At
was the absorbance of the sample after heating for t min. All
samples were analysed in triplicate.
2.8. Colour measurement
For measurement of the extent of browning by colour development, MRPs were diluted in 0.1% SDS to a concentration of 0.2% of
protein. The extent of browning was recorded as the absorbance at
420 nm. The measurement was carried out in triplicate for each
sample.
2.9. Fluorescence analysis
MRPs were dissolved in 0.2 M borate buffer (pH 8.5), then
centrifuged at 10000g for 20 min. The uorescence intensity of
the supernatant was measured in triplicate with a uorescence
spectrometer (Hitachi 650-60, Tokyo, Japan). The excitation wavelength was 347 nm, and the emission spectrum was recorded from
380 to 480 nm with a slit width of 5 nm.
2.10. Hydrophobicity measurement
The hydrophobicities of the MRPs were determined according to
Kato and Nakai (1980). A freeze-dried MRP sample was dissolved in
0.01 M phosphate buffer (pH 8.0) at different concentrations (0.1,
0.2, 0.3, 0.4, 0.5, and 0.6% w/v). The sample was centrifuged at
10000g for 20 min. The concentration of the protein in the
supernatant was measured by the Folin method (Lowry,
Rosebrough, Farr, & Randall, 1951). An aliquot of 2 mL was mixed
with 40 mL of 1-anilinonaphthalene-8-sulfonic Acid (ANS) (8 mM)
at 25
C. The uorescence intensity was measured with a uorescence spectrometer (Hitachi 650-60). The excitation and emission
wavelengths were set at 394 and 473 nm, respectively. The
hydrophobicity index was dened as the slope of the uorescence
intensity as a function of the protein concentration. All samples
were analysed in triplicate.
2.11. Amino acid analysis
The amino acid composition of MRPs was determined after
hydrolysis of the sample with 6 M HCl at 110
C. Measurement of
the amino acid content was carried out by HPLC analysis on an
Agilent 1100 series HPLC system (Agilent Technologies, Santa
Clara, CA) with on-line pre-column derivation by o-phthaldialdehyde (OPA) and 9-uorenylmethyl chloroformate (FMOC-Cl, for
proline analysis). Analysis was performed on a Hypersil C18
column (4.6 mm  150 mm, 5 mm lm thickness). The UV detector
was set at a wavelength of 338 nm. The column temperature was
30
C. Gradient elution was used. The gradients were formed with
20 mM sodium acetate (A) and 20 mM sodium acetate:methanol:acetonitrile (1:2:2 V/V/V; solvent B). The elution prole was:

55

0e10 min, 50%B; 10e20 min, 50e100% B; 20e25min, 100e50% B.

The ow rate was 1.0 mL/min. All samples were analysed in
triplicate.
2.12. Molecular weight distribution
The inuence of conjugation on the molecular weight distribution was determined by high-performance gel permeation
chromatography using a TSK Gel G4000 PWXL column (Tosoh
Corporation, Tokyo, Japan). A 0.05 M phosphate buffer (pH 8.5) was
used as the mobile phase. The sample was centrifuged at 10000 g
for 10 min. The supernatant was ltered (0.2 mm Whatman lter),
and applied to the column at a ow rate of 1 mL/min at 20
C. The
eluate was monitored for UV absorbance at 280 nm. All samples
were analysed in triplicate.
2.13. Statistical analysis
The averages and Duncan t-test were performed by SPSS 13.0 for
Windows software (SPSS Institute Inc., Cary, NC).
3. Results and discussion
3.1. Functional properties of MRPs from dextran and rice peptides
with different DH
In our previous study, a wide range of DH was investigated
among the 3 proteases used in the hydrolysis treatment, and the
range between 4% and 7% was found to be the most effective in
balancing solubility and emulsifying functionality (data not show).
Solubility and emulsifying properties of the MRP formed with
limited hydrolysis of rice protein and dextran T20 are presented in
Table 1. The NS, EA and ES were signicantly improved by the
combined modication with protease hydrolysis and glycation as
compared to the native rice protein. Under the same glycation
reaction conditions with dextran T20, the MRPs from hydrolysates
of Protease N showed the highest functional values among the 3
proteases (Table 1). It was observed that within a protease treatment, the DS for an MRP decreased, and the pH increased as the DH
increased. The nal pH of an MRP was higher with increasing DH of
the reactant peptide. Since the Maillard reaction results in the
reduction of pH due to the loss of free eNH2 groups after glycation,
the higher the nal pH value, the lower the DS for the MRPs. Lysine
is a hydrophilic amino acid, and the reactive eNH2 groups are
typically located at the surface of the native rice protein due to the
existence of 4 hydrophobic eCH2e units. The eCH2e units tend to
locate themselves in the protein interior (Damodaran, Parkin, &
Fennema, 2008, pp, 226e227). After hydrolysis, our results suggested that the greater mobility of the peptide resulted in movement of lysine to a more hydrophobic environment that made it
difcult for the reactive eNH2 to conjugate with the saccharide,
that resulted in a lower Maillard reaction activity. This may also be
the reason for the lack of dramatic changes in solubility after glycation by rice hydrolysates with different DHs (Table 1). The
decreased glycation at high DH would offset the advantage of
increased hydrolysis and result in similar solubilities within the
MRPs.
3.2. Functional properties of MRPs from glucose saccharides and 5%
DH rice peptides
From the above study, the modication of rice protein functionality was conrmed via the combination of protease hydrolysis
and saccharide glycation. The highest increase of EA and ES among
MRPs was obtained with glycated DH 5% rice peptides from

56

Y. Li et al. / Food Hydrocolloids 30 (2013) 53e60

Table 1
Effect of protease and degree of hydrolysis (%) on the functional propertiesa of Maillard reaction products (MRPs) formed with rice peptides and dextran T20.b
Protease used

DH%

EA

No protease (rice protein)

Protease N

0
4%
5%
6%
7%
4%
5%
6%
7%
4%
5%
6%
7%

0.36
1.29
1.71
1.63
1.10
0.81
0.90
0.72
0.60
0.88
1.02
1.00
0.79

Alcalase

AS1398















0.04
0.02b
0.03d
0.05c
0.03a
0.03c
0.04d
0.03b
0.04a
0.02b
0.03c
0.02c
0.01a

ES

NS%

DS

Browning absorbance (420 nm)

pH

11.57
66.5 
78.0 
56.8 
41.1 
55.5 
58.0 
54.2 
52.7 
63.9 
64.9 
53.2 
50.3 

13.64
45.1 
49.0 
52.5 
47.9 
40.1 
44.1 
44.4 
43.1 
42.5 
43.2 
46.0 
45.0 

0
13.4
12.2
14.1
7.2
11.0
10.8
9.3
6.4
10.4
9.9
7.0
5.3

0.070
0.29 
0.26 
0.26 
0.21 
0.27 
0.26 
0.26 
0.21 
0.25 
0.24 
0.23 
0.23 

11.0
9.9
10.1
10.2
10.5
10.0
10.1
10.1
10.3
10.4
10.5
10.5
10.6

0.2c
0.1d
0.3b
0.2a
0.3c
0.1d
0.2b
0.1a
0.2c
0.3c
0.2b
0.3a

0.3a
0.2c
0.4d
0.1b
0.2a
0.3cd
0.2d
0.1b
0.4a
0.3ab
0.3c
0.2d














0.6c
0.6bc
0.4c
0.2a
0.3c
0.2c
0.1b
0.3a
0.4c
0.3c
0.2b
0.1a

0.03c
0.02b
0.02b
0.01a
0.02c
0.03b
0.04b
0.01a
0.02c
0.01b
0.03a
0.02a














0.04a
0.05b
0.04c
0.06d
0.05a
0.06ab
0.04b
0.03c
0.06a
0.04ab
0.03b
0.02c

Values are means of three replicate values. Different superscript letters within the same enzyme treatment represent a signicant difference (p < 0.05) with respect to DH%.
a
EA is emulsifying activity, ES is emulsifying stability, NS is solubility index, DS is degree of substitution, and pH is the nal pH at the end of the glycation reaction. More
information is given in the text.
b
The MRPs were formed from 1% dextran (20 K Mw) and 1% rice protein (or peptide) at 100
C for 30 min.

Protease N hydrolysis (Table 1). In order to determine the effects of

saccharide structure on functionality, various mono-, oligo- and
polysaccharides were reacted with rice peptides (DH 5%) from
Protease N hydrolysate.
The EA and ES was positively correlated with DS, glycation
degree, up to a maximum value, but further glycation tended to
result in decreased EA and ES (Fig. 1 and Fig. 2). Parker, Gunning, Ng,
and Robins (1995) proposed that the polysaccharide portion of the
conjugate would exhibit a yield value high enough to resist the

Fig. 1. Effects of saccharide and reaction time on emulsifying activity (a) and emulsifying stability (b) of MRPs. MD: Maltodextrin (DE20); DT20: Dextran (T20). The MRPs
were formed with 1% saccharide and 1% rice peptides hydrolysed by Protease N at 5%
DH. The points in the graphic are means  standard deviation of three separate
determinations.

buoyant forces favouring upward movement and creaming. But

further glycation may result in loss of the binding spot of the
protein for the O/W interface.
There was a trend towards higher DS of glucose-rice and
lactose-rice peptide systems at the latter stages of reaction than
that of the maltodextrin-rice and dextran-rice peptide systems
(Fig. 2). These results were in agreement with Li et al. (2009),
who reported a slower glycation rate among high molecular
weight saccharides and protein as compared to lower molecular
weight saccharides. In addition to molar concentration differences, the lower reactivity of the maltodextrin and dextran has
been reported to be related to steric hindrance effects (Kato,
2002). This study is the rst report that supports the same
trend of Maillard reactions between protein hydrolysates and
saccharides.
There was no correlation between the functional properties of
MRPs and the molecular weight of the reactant saccharides (Figs. 1
and 3). Although the NS increased with reaction time in all sample
sets, the NS for MRPs produced with glucose and dextran glycated
rice peptides at the same reaction time were higher (Fig. 3). Higher
EA and ES indices were also observed for theses two sets of MRPs
(Fig. 1). The results conrmed the importance of increased solubility to improve the functional properties of highly hydrophobic

Fig. 2. Effects of saccharide and reaction time on degree of substitution (DS) of MRPs.
MD: Maltodextrin (DE20); DT20: Dextran (T20). The MRPs were formed with 1%
saccharide and 1% rice peptides hydrolysed by Protease N at 5% DH. The points in the
graphic are means  standard deviation of three separate determinations.

Y. Li et al. / Food Hydrocolloids 30 (2013) 53e60

57

The development of colour is an important feature of advanced

stages of the Maillard reaction. The reaction between aldose sugars
and sugar degradation products and available amino groups leads
to the formation of brown compounds (Brands & Van Boekel, 2003).
The decrease in pH could also be attributed to the conjugation
reaction of amines with aldoses to form compounds of lower
basicity (Brands & Van Boekel, 2001). As shown in Fig. 4, the brown
colour increased and pH decreased in glucose and lactose reactions
and both were higher at latter stages of the reaction than those
with maltodextrin or dextran, which suggested that monosaccharides and disaccharides were more easily formed brown
compounds than the polysaccharides.
3.3. Analysis of the development of MRPs
Fig. 3. Effects of saccharide and reaction time on solubility index (NS) of MRPs. MD:
Maltodextrin (DE20); DT20: Dextran (T20). The MRPs were formed with 1% saccharide
and 1% rice peptides hydrolysed by Protease N at 5% DH. The points in the graphic are
means  standard deviation of three separate determinations.

plant proteins through Maillard reaction. But the exact mechanism

involved in solubility modication of protein through saccharide
glycation is unknown. It was also observed from Figs. 1and 3 that in
each saccharideepeptide product the highest values of EA and ES
did not correlate with solubility. This might be due to the need of an
optimal hydrophilic-lipophilic balance (HLB) in the MRPs for
interfacial adsorption.

Fig. 4. Effects of saccharide and reaction time on Browning absorbance (a) and pH (b)
of MRPs. MD: Maltodextrin (DE20); DT20: Dextran (T20). The MRPs were formed with
1% saccharide and 1% rice peptides hydrolysed by Protease N at 5% DH. The points in
the graphic are means  standard deviation of three separate determinations.

With regard to the overall functionality of MRPs, the dextran

glycated MRPs were found to be better than MRPs obtained with
other saccharides, and the best reaction time was 20 min at
100
C (Fig. 1). The NS, EA and ES of the MRPs was 2.5, 4.3 and 6.3
times higher than that of the MRPs formed with native rice
protein and dextran, and 0.5, 1.2 and 7.0 times higher than that of
the rice protein hydrolysate before glycation (Table 1, Figs. 1and
3). In order to understand the inuence of saccharide structure on
the development of MRPs at different stages of the reaction and
on the functional properties of MRPs, various analyses were
performed.
The rst step in the Maillard reaction is the condensation of the
carbonyl group of a reducing sugar and a nucleophilic amino
group, which could be tracked by the loss of the reacted amino
acid. Amino acid analysis of MRPs (Table 2) showed that Lys and
Arg decreased dramatically within the rst 20 min, which was in
agreement with the rapid increase of DS, NS and emulsifying
capacity during that time as shown in Figs. 1e3. The results
conrmed that the early stage of glycosylation of peptides correlated with the functionality improvement. The loss of Cys-s and
Proline were also found to be signicant during initial heating. The
Cys-s loss maybe attributed to the formation of cystine-derived
crosslinks. The formation of a de-hydroalanyl residue from
cystine, which is capable of binding with the -amino group of
lysine to form lysinoalanine has been reported under heating (Gu,
Kim, Hayat, & Zhang, 2009).
As a result of the attachment of a hydrophilic saccharide to the
surface of a hydrophobic protein or peptide, the hydrogen
bonding capacity of saccharides eOH group changes the surface
hydrophobicity, leading to an increased solubility and an
improved functionality. The surface hydrophobicity of rice protein
hydrolysate was much lower than that of native rice protein
(Fig. 5), but still high when compared to other proteins such as
whey protein (Alizadeh-Pasdar & Li-Chan, 2000). The glycation
with saccharide dramatically reduced the surface hydrophobicity,
especially at the rst 5 min, which was in agreement with the
change of solubility during the reaction (Fig. 3). The surface
hydrophobicity of MRPs was further decreased during the reaction from 5 to 40 min, and dropped to around 200, which may be
too low to establish a reasonable hydrophilic-hydrophobic
balance for adsorption at oilewater interface, and resulted in
lower EA and ES values as compared to the MRPs formed at
20 min (Fig. 1 and Fig. 5).
In the initial stage of the Maillard reaction, Amadori compounds
are formed, which are not uorescent, but in advanced Maillard
reaction stages these compounds can form cross-links with adjacent proteins or with other amino groups, giving rise to uorescent
polymeric aggregates or the so-called advanced glycation products
(Caldern, Hernndez, & Villanova, 2009). The uorescence of the
products can be used as a marker of the extent of the Maillard

58

Y. Li et al. / Food Hydrocolloids 30 (2013) 53e60

Table 2
Effect of reaction time on content of amino acid component of conjugates formed with rice protein hydrolysed by protease N at 5% DH (1%) and dextran T20 (1%) at 100
C.
Content of amino acid component (mg/g)

Asp
Glu
Ser
His
Gly
Thr
Arg
Ala
Tyr
Cys-s
Val
Met
Phe
Ile
Leu
Lys
Pro

0 min

5 min

10 min

20 min

30 min

40 min

8.57
19.13
4.78
2.15
4.73
4.33
6.46 0.22b
4.23
2.17
1.13 0.11c
5.33
1.43
5.32
3.5
8.2
4.42 0.16d
5.8 0.19e

8.61
19.06
4.44
2.1
4.13
4.15
5.85
4.41
3.2
0.37
5.44
1.59
5.31
3.79
7.52
2.78
4.47

8.18
17.17
4.43
1.77
3.71
3.82
4.74
4.37
3.94
0.24
4.28
1.53
5.06
2.98
7.24
2.41
3.67

7.73
17.01
4.78
1.94
3.65
3.96
4.49 0.16a
4.64
4.75
0.17 0.08a
4.77
1.66
5.39
3.33
7.4
2.07 0.13a
2.7 0.18b

7.75
17.12
4.67
1.93
3.62
3.91
4.62
4.73
5.01
0.14
4.7
1.66
5.41
3.27
7.46
1.99
2.39

7.68
16.98
4.6
1.81
3.61
3.84
4.61
4.66
5.08
0.14
4.77
1.69
5.41
3.32
7.47
1.98
2.62

0.23b

0.10b

0.15c
0.22d

0.20a

0.09ab

0.16b
0.16c

0.19a

0.09a

0.09a
0.12a

0.18a

0.09a

0.11a
0.13ab

Values are means of three replicate values. Different superscript letters within the same amino acid component represent a signicant difference (p < 0.05).

reaction. The strongest uorescence intensity of rice protein-glycan

MRPs was found at an emission of 422 nm with an excitation of
347 nm (Fig. 6), which was in the suggested range of Maillard
reaction uorescent products, lex 340e370 nm and
lem 420e440 nm (Bosch & Alegra, 2007). The results suggested
the formation of advanced MRPs within 40 min after incubation.
The uorescence intensity of rice protein-glycan MRPs increased
gradually at the rst 20 min and accelerated thereafter. When the
uorescence was related to functional properties of MRPs (Fig. 1), it
was found that polymeric aggregation at later stage of the Maillard
reaction did not benet the emulsifying capacity of hydrophobic
protein.
In order to further study aggregates formed in the latter-stage
MRPs, HPSEC was used to follow molecular size changes with UV
detection. Chromatographs of the products at increasing reaction
times (0, 5, 10, 20, 30 and 40 min) were recorded (Fig. 7). Since
the extinction coefcients vary with the changes in composition
of the absorbing species, it is not possible to accurately determine
the amounts of the UV absorbing species. The eluate was monitored at 280 nm of the UV detector for the rice protein product.
The simple mixture of rice protein hydrolysate and dextran

separated into two peaks at 6.5 and 9.8 min (Fig. 7a). The peak
molecular weights were 8.95  105 (6.5 min) and 6.04  103 Da
(9.8 min), respectively. Since glycation had not occurred, the high
molecular was thought to be aggregates of rice peptides at pH 8.5.
The peak of lower molecular weight represented soluble
peptides. As the reaction continued, the appearance of intermediate peaks at 7.689 min (Fig. 7b) was probably due to
the partially release of peptides from the aggregates. While the
formation of proteinedextran conjugation resulted in the
increase of the high molecular weight group, the proportion of
low molecular weight species necessarily decreased with the
time (Fig. 7d). The average molecular weight of the low molecular
fraction was constant until 30 min, when an ultra-low molecular
weight group (average Mw 1326 Da) appeared (Fig. 7e), and this
peaks area increased at 40 min (Fig. 7f). This new peak could be
evidence for the degradation of early stage MRPs, which were
compounds dissociated from glycated proteins, containing chromophores that had absorbance at 280 nm. Further studies are
needed to obtain more detailed information to understand the
mechanisms of formation and identication of the degradation
compounds.

Fig. 5. Hydrophobicity of rice protein and MRPs with reaction time. Concentrations of
initial reactants were 1% dextran T20 and 1% rice peptides hydrolysed by Protease N at
5% DH. 0 min referred to the mixture of dextran T20 and peptides before conjugation
reaction. The points in the graphic are means  standard deviation of three separate
determinations.

Fig. 6. Fluorescence analysis of rice protein and MRPs with reaction time. Concentrations of initial reactants were 1% dextran T20 and 1% rice peptides hydrolysed by
Protease N at 5% DH. 0 min referred to the mixture of dextran T20 and peptides before
conjugation reaction. The points in the graphic are means  standard deviation of
three separate determinations.

Y. Li et al. / Food Hydrocolloids 30 (2013) 53e60

59

Fig. 7. Molecular weight distribution of simple mixture of rice protein hydrolysate and dextran T20 (a), and MRPs formed by Maillard reaction at the reaction time of 5 min (b),
10 min (c), 20 min (d), 30 min (e), and 40 min (f).

4. Conclusion
The combination of limited hydrolysis and Maillard glycation is
shown to be an efcient and economical method of rice protein
modication. Optimal reaction conditions obtained in this research
which improved the EA, ES and NS were found to be: Protease N
hydrolysis of rice protein to a DH of 5%, followed by glycation with
dextran T20 (1%) in an aqueous dispersion (pH 11), and reacted at
100
C for 20 min. The NS, EA and ES of the resulted MRPs increased
by factors of 3.5, 5.3 and 7.3 times, respectively as compared to that
of the MRPs formed with native rice protein and dextran, and 1.5,
2.2 and 8.0 times, respectively as compared to that of the rice
protein hydrolysate before glycation. Analysis of MPRs at different
Maillard reaction stages revealed that increased DH of the rice
protein would result in lower reactivity of peptides with saccharides. The increases of emulsifying activity and solubility of rice
peptide MRPs were coincidental with the increase in DS in certain
range.
Acknowledgements
We gratefully acknowledge the nancial support of National
Nature Science Foundation of China (30901000 and 31171686). This

work was also supported by the Fundamental Research Funds for

the Central Universities JUSRP11015 and National Twelfth-Five Year
Research Program of China 2011BAD23B02.
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