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Functional Properties of Maillard Reaction Products of Rice Protein Hydrolysates With Mono-, Oligo - and Polysaccharides
Functional Properties of Maillard Reaction Products of Rice Protein Hydrolysates With Mono-, Oligo - and Polysaccharides
Functional Properties of Maillard Reaction Products of Rice Protein Hydrolysates With Mono-, Oligo - and Polysaccharides
Food Hydrocolloids
journal homepage: www.elsevier.com/locate/foodhyd
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
54
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.
The degree of substitution (DS) of the rice peptides was determined from the analysis of the free amino groups in the rice proteins
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
56
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
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
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.
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.
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.
57
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.
58
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).
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.
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
60
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