Food Chemistry 117 (2009) 69–74

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Food Chemistry

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Functional properties of the Maillard reaction products of rice protein with sugar

Yue Li a, Fang Lu a, Changrong Luo b, Zhengxing Chen a, Jian Mao a, Charles Shoemaker c, Fang Zhong a,*

a The State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, Wuxi 214122, PR Chinab Hua Bao Food Flavour and Fragrance (Shanghai) Co. Ltd., Shanghai 201821, PR Chinac Department of Food Science and Technology, University of California, Davis, CA 95616, USA

a r t i c l e i n f o a b s t r a c t

Article history:Received 5 December 2008Received in revised form 23 February 2009Accepted 19 March 2009

Keywords:Rice proteinMaillard reactionEmulsifying propertiesProtein solubility

0308-8146/$ - see front matter � 2009 Elsevier Ltd. Adoi:10.1016/j.foodchem.2009.03.078

* Corresponding author. Tel.: +86 510 85912123; faE-mail address: fzhong@jiangnan.edu.cn (F. Zhong

The glycation of rice proteins with reducing sugars was investigated in an attempt to improve their sol-ubility and functionality. Rice proteins isolated from a Chinese milled medium-grain rice were glycatedwith glucose, lactose, maltodextrin, or dextran in 2% aqueous dispersions. The sugar that provided themost improvement of the solubility, emulsification activity (EA) and emulsification stability (ES) of theMaillard reaction products was glucose. The optimum reaction conditions were at pH 11, 100 �C, andreaction time 15 min, which increased the solubility, EA and ES of rice protein from 20%, 0.46 and 11.1to 92%, 0.64 and 18.2, respectively. Extending the reaction time beyond 15 min continued the develop-ment of latter-stage Maillard browning products without improvements in the functional properties ofthe Maillard reaction products. SEC–HPLC analysis with light scattering detection showed a decrease ofthe weight-averaged molecular weight from 500 to 100 K during the initial 15 min.

� 2009 Elsevier Ltd. All rights reserved.

1. Introduction

Rice protein has been considered to have a high biological valueand low hypoallergenic characteristics. However, the use of riceprotein as an ingredient in foods has been limited, due to its poorfunctional properties, such as solubility. Whole rice grains containdifferent types of proteins which have been isolated and character-ised. Over 80% of rice proteins are glutelins, which have low solu-bility at neutral pH. With low solubility the presence of otherfunctional properties, such as foaming, gelling and emulsifying,are also limited (Agboola, Ng, & Mills, 2005).

Numerous attempts have been made to improve the functionalproperties of proteins through physical, chemical, enzymatic andgenetic modifications (Matheis & Whitaker, 1984; Matsudomi,Sasaki, Kato, & Kibayashi, 1985). A chemical method to improvethe functional properties of proteins has been the reaction of pro-teins with reducing sugars via the Maillard reaction (Wooster &Augustin, 2006). The modified proteins have often showed bettersolubility and improved emulsifying properties (Oliver, Melton, &Stanley, 2006). This reaction is natural and spontaneous and doesnot require the addition of extraneous chemicals. Thus it has beenconsidered a promising approach to the improvement of the func-tional properties of proteins for food uses.

Different proteins, such as ovalbumin, lysozyme and soy pro-teins, have been conjugated with various sugars, in order to im-

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prove their solubility, emulsifying, and heat stability properties(Chevalier, Chobert, Popineau, Nicolas, & Haetle, 2001; Miralles,Martinez-Rodriguez, Santiago, van de Laemaat, & Heras, 2007).The improvement of the functionality of rice proteins by the Mail-lard reaction has not been reported. However, rice proteins containa moderately high ratio of lysine, which has a free amino groupthat is liable to undergo Maillard reaction.

There are two approaches, which have been used to carry outthe Maillard reaction (Guan, Qiu, Liu, Hua, & Ma, 2006). The firstmethod involves the heating of a dry dispersion of a protein andsaccharide under controlled temperature and relative humidityconditions. A disadvantage of this method is that some of the prod-ucts are insoluble aggregates with poor surface wetting properties;a second method, which can overcome some of these problems, isto carry out the reaction in an aqueous solution or dispersion (Jing& Kitts, 2002; Lertittikul, Benjakul, & Tanaka, 2007). The lattermethod has the advantage of better control and shorter reactionstimes. Its disadvantages include the removal of the water and buf-fer when recovering the products.

In our initial studies, rice protein was reacted with sugars bydry heating. Initial results showed that the modified rice proteinsexhibited slightly improved emulsifying activity, but there wasno significant improvement in solubility. The goal of this studywas to improve the solubility and emulsifying properties of riceproteins by glycation under wet reaction conditions. The influenceof pH, type of sugar, the weight ratio of protein:sugar, reactiontemperature and reaction time were studied. The degree of substi-tution and browning were also studied. The use of high pHs waschosen to solubilise the rice proteins and to enhance the rate of

70 Y. Li et al. / Food Chemistry 117 (2009) 69–74

Maillard reaction, with the hypothesis that improvements in solu-bility and emulsifying properties would be made.

2. Materials and methods

2.1. Preparation of rice protein concentrate

The common method for the separation of rice protein from thestarch of milled rice grains is by alkali solubilisation of the proteinsfollowed by acid precipitation (Connor, Saunders, & Kohler, 1977;Yamamoto, Sumie, & Toshio, 1973). In this study, milled med-ium-grain rice (Nan Jing 39 from Jiangsu Baobao Group Co., Ru-dong, China) was washed with deionised water, and then soakedin three times its weight of deionised water for 15 h. The riceand water were then placed in a food blender and blended into aslurry. The protein was extracted from the slurry with 0.1 M NaOHat 40 �C (the ratio of slurry to NaOH solution was 1:3 w:w). Theslurry was continuously stirred for 2 h and then centrifuged(6000g for 10 min). The supernatant was removed, and its pHwas adjusted to 5.5 using 0.1 M HCl. It was left undisturbed for acold precipitation overnight (4 �C). The supernatant was carefullysiphoned off from the precipitate. The precipitate was washed 4times with deionised water. The final slurry was neutralised to apH 7.0 with 0.1 M NaOH and lyophilised. The protein content ofthe lyophilised substrate was determined by the Kjeldahl method(The value of 5.95 was used as a protein conversion factor), andthe obtained rice protein content was 92% (dry weight).

2.2. Preparation of Maillard reaction products (MRPs) from riceprotein–sugar model systems

Rice protein (2.4 g) was dispersed in 200 ml deionised waterand the pH was adjusted to 12 with 10 M NaOH. The protein wasthoroughly dispersed by vigorous and continuous agitation for1 h. Glucose (2.4 g) was added to the dispersion. The pH of disper-sion was readjusted to pH 11 with 0.1 M HCl. Aliquots of 15 mlwere transferred to each of twelve 50 ml screw-capped glasstubes; they were capped to prevent evaporation during heating.The samples were heated in a water bath at 100 �C. Two sampletubes were removed during heating at the following times: 0, 5,10, 15, 20, and 30 min. When removed the tubes were immediatelyplaced in an ice-bath and then stored at 4 �C prior to analysis. Twoadditional samples were removed after 15 min of heating andfreeze–dried and used for further experiments. Samples with otherpH values, reaction temperatures, and ratios of protein to reducingsugar were prepared by the same procedure.

2.3. Solubility measurement of MRPs

Ten millilitres of an MRP sample was diluted with 10 ml of var-ious buffers (0.05 M sodium phosphate buffer, pH 5, 6.5 and 8;0.05 M sodium hydrogen carbonate buffer, pH 9.5 and 11; 0.05 Mcitrate buffer, pH 3.5 and 0.05 M KCl–acetic acid buffer, pH 2).The pH of the solution was readjusted to the buffer pH with0.1 M HCl or NaOH. Samples were centrifuged for 20 min at10,000g. Protein content in the supernatant was determined bythe Kjeldahl method. Solubility was expressed as a percentage ofprotein in the supernatant to the total protein content.

2.4. Measurement of hydrophobicity

The hydrophobicity of the MRPs was determined according tothe ANS method. A freeze–dried MRP sample was dissolved in0.01 M phosphate buffer (pH 8) at different concentrations. Thesample was centrifuged at 7000 rpm for 20 min. The concentration

of the protein in the supernatant was measured by the Folin meth-od. An aliquot of 2 ml was mixed with 40 ll of 1-anilinonaphtha-lene-8-sulfonic Acid (ANS) (8 mM) at 25 �C. The fluorescenceintensity was measured with a fluorimeter. The excitation andemission wavelengths were set at 394 and 473 nm, respectively.The fluorescence intensity was measured as a function of proteinconcentration. The slope of the function was expressed as thehydrophobicity index.

2.5. Emulsifying activity and stability measurement of MRPs

The emulsifying properties of the samples were determined bythe method of Pearce and Kinsella (1978) with some modifications.An MRP sample was diluted with buffer (0.05 M sodium phosphatebuffer, pH 8) to a final protein concentration of 0.4% for the analysisof emulsifying properties. To form an emulsion, 15 ml of a 0.4%sample solution and 5 ml of soy oil were mixed together, with con-tinuous agitation. The crude emulsion was homogenised using anUltra-Turrax T25 homogeniser (IKA Werke GmbH & Co. KG, Stau-fen, Germany) at 10,000 rpm for 1 min. A sample of the emulsion(50 ll) was taken from the bottom of the test tube at 0, 3, 5, and10 min and immediately diluted with 5 ml of 0.1% sodium dodecylsulphate solution. The absorbance of the diluted emulsion wasthen determined at 500 nm. The emulsifying activity (EA) wasdetermined from the absorbance measured immediately after theemulsion had formed (0 min). The emulsion stability (ES) wasdetermined as the half-life of the initial turbidity of the emulsion(Pearce & Kinsella, 1978; Tang, Hettiarachchy, Horax, & Eswara-nandam, 2003).

2.6. Determination of the degree of substitution of the MRPs

The degree of substitution (DS) of the rice proteins was deter-mined from the analysis of the free amino groups in the rice pro-teins and MRPs. The level of free amino groups in the riceprotein–sugar MRPs was determined with the TNBS method (Ad-ler-Nissen, 1979). A protein sample (1.2% w/v) was dispersed in0.1% SDS to obtain a solution with 0.2% protein concentration. Itwas then mixed with 2.0 ml of 0.21 M phosphate buffer, pH 8.2and 1.0 ml of 0.1% 2,4,6-trinitrobenzenesulfonic acid (TNBS) solu-tion. The solution was thoroughly mixed and placed in a tempera-ture-controlled water bath at 50 �C for 1 h in the dark. The reactionwas terminated with the addition of 2.0 ml of 0.1 M sodium sulph-ite. The mixture was cooled at room temperature for 30 min. Theabsorbance was measured at 340 nm.The degree of substitutionwas determined as:

DS ð%Þ ¼ ððAo � AtÞ=AoÞ � 100%;

where Ao was the absorbance of the sample before heating, and At

was the absorbance of the sample after heating for t min.

2.7. Colour measurement of MRPs

For measurement of the extent of browning, MRPs were dilutedin 0.1% SDS to a concentration of 0.2% of protein. The extent ofbrowning was recorded as a measure of the absorbance at 420 nm.

2.8. Fluorescence analysis of MRPs

MRPs were dissolved in 0.2 M borate buffer (pH 8.5), thencentrifuged at 7000 rpm for 20 min. The fluorescence intensity ofthe supernatant was measured with a fluorescence spectrometer(Hitachi 650-60). The excitation wavelength was 347 nm, and aspectrum was recorded from 380 to 480 nm with a slit width of5 nm.

Y. Li et al. / Food Chemistry 117 (2009) 69–74 71

2.9. Amino acid analysis of MRPs

Measurement of the amino acid content of the MRPs was car-ried out by HPLC analysis on an Agilent 1100 series HPLC system(Agilent Technologies, Santa Clara, CA) with on-line pre-columnderivation by o-phthaldialdehyde (OPA) and 9-fluorenylmethylchloroformate (FMOC-Cl, for proline analysis). Analysis was per-formed on a Hypersil ODS C18 column (4.6 mm � 150 mm, 5 lmfilm thickness). The UV detector was set at a wavelength of338 nm. The column temperature was 30 �C. Gradient elutionwas used. The gradients were formed by 20 mM sodium acetate(A) and 20 mM sodium acetate:methanol:acetonitrile (1:2:2 V/V/V; solvent B). The elution system was:–10 min, 50% B; 10–20 min, 50–100% B; 20–25 min, 100–50% B. The flow rate was1.0 ml/min.

2.10. Size exclusion chromatography – multi-angle laser lightscattering analysis of MRPs

The high performance size-exclusion chromatography/multi-angle laser light scattering (HPSEC/MALLS) system consisted ofan HP1050 series pump, an HP1050 autoinjector (Hewlett Packard,Valley Forge, PA), a MALLS detector (Dawn DSP-F, Wyatt Technol-ogies, Santa Barbara, CA), a differential refractometer detector(ERC-7512, ERMA Incorporated, Tokyo, Japan) and a UV detector(Shimadzu, Kyoto, Japan). For the SEC analysis, two PL aquagel-OH linear columns (Polymer Laboratories, Amherst, MA) were con-nected in series, and an aqueous solution of 0.1 N NaNO3 was usedas the mobile phase at a flow rate of 0.6 ml/min. The column tem-perature was maintained at 50 �C with a column heater (Bio-Rad,Hercules, CA).

3. Results and discussion

Many cereal proteins lack functionality, largely due to theirinsolubility. The covalent bonding of polysaccharides or smallerreducing sugars with proteins via the Maillard reaction can be auseful way of increasing their solubility and functionality. Forother proteins this has been shown to be the case (Oliver et al.,2006). However, the establishment of optimum reaction conditionsfor preparing useful MRPs is needed for the synthesis of more func-tional rice proteins. An important functional property of MRPs istheir emulsifying properties. The emulsifying properties of MRPsdepend on the degree of the glycation of the protein. The emulsify-ing properties have been reported as being enhanced during theearly stages of conjugation, and at longer times, they gradually de-creased. To produce an effective emulsifying agent there should bea good balance between the degrees of hydrophobic and hydro-philic properties. In addition, the loss of emulsifying power is pre-sumably caused by the degradation of initial MRPs, which has beensuggested by the appearance of low molecular weight productsfound by electrophoresis gel analysis at longer reaction times(Miralles et al., 2007).

Table 1Effect of saccharide type on Maillard browning reaction products (MRPs) formed with rice

Saccharide Degree of substitution (%) Solubility (%) Emulsifying ac

No saccharide 0a 20.0a 0.496a

Glucose 28.0b 90.0e 0.620b,c

Lactose 26.8b 83.2d 0.618c,d

Maltodextrin 17.7c 73.0 c 0.589b

Dextrin 12.2d 60.1b 0.585b

* Concentrations of initial reactants were 1% saccharide and 1% rice protein. Values arecolumn represent a significant difference (p < 0.05).

3.1. Influence of varieties of sugars

The effect of the type of saccharide (glucose, lactose, maltodex-trin, or dextran) on the functionality of MRPs formed by glycationof rice proteins (weight ratio of protein:sugar of 1:1) at pH 11 wasinvestigated at a reaction temperature of 100 �C (Table 1). The DSand solubility of the MRPs increased with a decrease in size ofthe reactant saccharide. Several investigations have shown thatthe reaction mechanisms of monosaccharides and disaccharidesdiffer and that reaction products obtained from monosaccharidesare different from those obtained from disaccharides (Kato, Matsu-da, Kato, & Nakamura, 1988). There has been reported a linear cor-relation between the DS and the saccharide size (Nacka, Chobert,Burova, Leonil, & Haertle, 1998).

There were effects on other properties of the MRPs as well.There was little difference observed between MRPs formed withglucose or lactose as compared to larger differences between theMRPs formed with maltodextrins or dextrans. Lactose showed agreater effect than glucose on the emulsion activity (EA), and glu-cose had a greater affect on the emulsion stability (ES). Lactose hada greater extent of browning, and lowering of pH, but both saccha-rides had greater effects on these properties than the maltodextrinand dextran. The lower reactivity of the maltodextrin and dextranhave been reported to be related to steric hindrance effects (Kato,2002).

The effect of saccharide type on DS, pH, and extent of browningwas measured at intervals over a 30 min heating period. The DS in-creased during the first 15 min of heating and remained ratherconstant upon continued heating (Fig. 1). The rice protein–glucoseMRPs exhibited the highest DS. Similar trends for the solubility, EA,and ES were also observed (not shown). The extent of browningand acidity also increased during the first 15 min of heating (Huiy-ing, Tao, Dongxiang, & Fang, 2007), and continued to increase dur-ing the latter 15 min of the 30 min period (Fig. 2). The reduction ofpH occurring during the Maillard reaction could be partially due tothe degradation of MRPs into acids, such as formic and acetic acids(Brands & Van Boekel, 2002). On the other hand, the decrease in pHcould also be attributed to the reaction of amines to form com-pounds of lower basicity (Van Boekel & Brands, 1998).

The extent of browning of the protein–lactose MRPs was greaterthan the MRPs formed with other sugars, and the pH of the pro-tein–lactose MRPs decreased more than other MRPs. Brands andVan Boekel (2001) reported that compared to monosaccharide sys-tems, about four times more formic acid was formed with disac-charide-casein MRPs. The pH of all MRPs decreased markedlyfrom their initial values within the first 15 min. Thereafter, thereduction of pH values was less during the last 15 min. It has beenreported that pH has an effect on both the reaction rate and thereaction mechanism (Brands & Van Boekel, 2002).

From these results, it appeared that useful functional propertiesfor the MRPs were optimum when formed with glucose at 15 min,and they did not appear to increase with longer reaction times. Thebest solubility of the MRPs (90%) was obtained with glucose. How-

protein after 15 min at 100 �C*.

tivity Emulsifying stability Browning absorbance (420 nm) pH

10.3a 0.07a 11e

18.0d 0.247d 8.7b

16.5c 0.268e 8.3a

13.6b 0.223c 9.3c

13.6b 0.128b 10.6d

means of three replicate values and different superscript letters within the same

0

5

10

15

20

25

30

35

0 5 10 15 20 25 30 35

GlucoseLactoseMaltodextrinDextran

DS

(%

)

Time (min)

Fig. 1. The effect of saccharide on the degree of substitution (DS) of the free aminogroups on rice proteins was measured over 30 min at 90 �C and an initial pH of 11.

7

8

9

10

11

12

13

14

15

0

0.1

0.2

0.3

0.4

0.5

0 10 20 30

Glucose

Lactose

Maltodextrin

Dextran

Glucose

Lactose

Maltdextrin

Dextran

pH

A(420

nm)

Time (min)

Fig. 2. The effect of saccharide on the pH and extent of browning of the reaction ofsaccharide with rice proteins was measured over 30 min. The open symbolsrepresent the pH values, and the closed symbols represent absorption (A) at420 nm.

72 Y. Li et al. / Food Chemistry 117 (2009) 69–74

ever, an increase in the extent of browning and reduction of pHcontinued throughout the 30 min reaction period.

3.2. Study of the effect of reaction conditions on the formation of MRPs

For rice proteins, pH may have several roles in the formation ofMRPs. High pH enhances rice protein solubility and increases therate of glycation. Rice proteins were glycated with glucose (weightratio 1:1) at pH values 10 and 11 at 100 �C. The DS for the MRPs atpH values of 10 and 11 increased rapidly during the first 10 minand reached values of 10% and 34%, respectively. With continuedheating after 10 min, the DS values increased more slowly, to reachvalues of 12% and 37%, respectively, after 30 min.

When the rice proteins were reacted with glucose at 100 �Cwith different weight ratios (WR) of protein:glucose (2:1, 1:1 and

1:2) with the same total weight (2%) for 30 min, differences amongthe functional properties of the MRPs were observed. Even thoughthe DS values for the WR 1:2 and 2:1 showed the same overalltrend as WR 1:1 over the reaction period (Fig. 1), the DS values at15 min were 26, 28, and 21 for the WRs of 1:2, 1:1, and 2:1, respec-tively. The ranking of the final DS values at 30 min was the same asthe 15 min ranking order. The trends of the pHs of the reactionmixtures and development of brown colour (420 nm) of the WRs1:2 and 2:1 were also similar to the WR 1:1 (Fig. 2). The pH valuesat 15 min were 8.0, 8.7 and 9.1 for the WR of 1:2, 1:1, and 2:1,respectively. Likewise, the absorbances at 420 nm were 0.33,0.25, and 0.22 for WR 1:2, 1:1, and 2:1, respectively. Thus for afixed total concentration (2% w/w) of glucose and protein, theWR of 1:1 gave the highest DS. The reduction of pH and develop-ment of brown colour increased with an increase in WR of glucoseunder these conditions.

The effect of three reaction temperatures (80, 90, and 100 �C) onthe solubility of the MRPs formed with WR 1:1 with the abovereaction conditions was measured. After 15 min, MRPs were iso-lated from the reaction mixtures with WR of 1:1 and their solubil-ity was measured. The solubilities of the MRPs were 15%, 86% and90% at 80, 90, and 100 �C, respectively.

With a WR of 1:1 (2% w/w), and initial pH of 11, the effect ofreaction times between 0 and 30 min was evaluated at 100 �C (Ta-ble 2). Both DS and degree of solubility of the MRPs showed rapidincreases during the first 10 min and became constant after15 min. The degree of hydrophobicity decreased with the increasein DS and solubility, as would be expected. The EA and ES values in-creased during the first 15 min and then showed a slight decreasein their values during the latter 15 min. A maximum in the EA andES values at 15 min may suggest that the best balance between thehydrophobic and hydrophilic balance had been obtained at thattime. As compared to native rice proteins, the amounts of the ami-no acids S-Cys, Lys, Pro and Arg decreased by 61.1%, 48.3%, 28.7%and 10.9% in the MRPs, which were formed after 15 min, respec-tively. Lys is commonly believed to be the most reactive amino acidin terms of glycation with reducing sugars. Arg (Yen, Tsai, & Lii,1992), S-Cys (Billaud, Maraschin, & Nicolas, 2004) and Pro (Davi-dek, Clety, Devaud, Robert, & Blank, 2003) were also found to belost in the Maillard reaction with proteins. The decrease of S-Cyscontent also reduces the chance of protein aggregation through –S–S bonds, which could help to increase the solubility of the MRPsin water.

The objective of this work was to improve the functional prop-erties of rice proteins by the formation of MRPs. The results suggestthat the reaction conditions which favoured the formation of use-ful MRPs with limited amounts of latter-stage products were WR1:1, pH 11, 100 �C for 15 min. These MRPs had optimum emulsify-ing activity and solubility. In order to confirm that reaction timesof longer than 15 min did produce latter-stage MRPs, severalexperiments were made to detect evidence for their production.

3.3. Analysis of latter-stage MRPs

The Maillard reaction is associated with the development offluorescent compounds formed prior to the generation of brownpigments (Morales, Romero, & Jimenez-Perez, 1996; Morales &Van Boekel, 1997), which may be precursors of brown pigments.The presence of fluorescent compounds was analysed during thecourse of the reaction, in order to find possible markers of thedevelopment of latter-stage MRPs. The development of fluorescentcompounds was found to increase with longer heating times of therice protein and glucose (Fig. 3). It appeared that after 10 min ofheating at 100 �C there was a large increase in the concentrationof fluorescent compounds. Even as these compounds may havebeen formed prior to the generation of brown pigments, their con-

Table 2Effect of reaction time on properties of MRPs formed with rice proteins (1%) and glucose (1%) at 100 �C.

Reaction time(min)

Degree of substitution(%)

Solubility(%)

Hydrophobicity Emulsifyingactivity

Emulsifyingstability

Browning absorbance(420 nm)

pH

MRPs 0 – 10.9a 2491f 0.461a 11.1a 0.08a 11.0e

5 20.0a 18.6b 1747e 0.539b 11.4a 0.16b 10.2d

10 29.6b 85.6c 1564d 0.631c,d 14.0b 0.21c 9.3c

15 28.2b 90.3d 1416c 0.642d 18.2d 0.25d 8.7b

20 28.8b 90.0d 1288b 0.599c 15.4c 0.26d,e 8.5b

30 27.7b 90.0d 1143a 0.563b,c 14.4b,c 0.28e 8.2a

Values are means of three replicate values and different superscript letters within the same column represent a significant different (p < 0.05).

0.1

0.2

0.3

0.4

0.5

0.6

0

2 105

4 105

6 105

0 5 1 0 1 5 2 0 2 5 30

Mass of Low Mw (mg)

Mass of High Mw (mg)Mw of High Mw

Mas

s (

arb

itra

ry u

nit

s)

Mw

(Da)

Time (min)

Fig. 4. Glucose MRP products analysed by HPLC size-exclusion analysis. Thecollection samples were divided into two fractions. The high molecular fraction wasin the range of 1000–10 kDa. The low Mw range is that below 10 kDa. The recoveredmass of each fraction is in arbitrary units. Mw is the weight average of themolecular weight of each fraction.

Y. Li et al. / Food Chemistry 117 (2009) 69–74 73

centrations continued to increase during the latter 15 min, as theEA and ES values were decreasing and the solubility of the MRPsshowed no significant change. This suggested that at longer heat-ing times the rate of production of latter-stage MRPs did increase,which was also supported by the increase in brown colour (Table2).

In order to further study the latter-stage MRPs, HPSEC-MALLSwas used with UV detection at 280 nm and multi-angle laser lightscattering. With HPSEC-MALLS the MRPs were separated by molec-ular weight. With a laser light scattering detector the weight-aver-aged molecular weights (Mw) of the larger region were measured.Chromatographs of the products from each reaction time (0, 5, 10,15, 20 and 30 min) were recorded; each was divided into two frac-tions, the first fraction had a low Mw cutoff of 10,000 Daltons andthe second was the fraction, which had Mws of less than 10,000(Fig. 4). For each fraction, the integration of absorbencies at280 nm was calculated. For the high Mw fraction, the recoveredmass increased during the first 15 min and remained relativelyconstant during the latter 15 min of heating. This trend was similarto the increase in solubility of the MRPs (Table 2). The Mw of thelarger molecular weight fraction decreased from 500 to 80 kDaduring the first 15 min and then remained relatively constant dur-ing the latter 15 min. This suggested that initially the rice proteinsmay have been aggregated. During the initial 15 min of the reac-tion, the degree of aggregation decreased with the formation ofMRPs. The final Mws of these fractions for the heating times after10 min of heating were between 100 and 80 kDa. For the lower

0

50

100

150

200

250

350 400 450 500

Protein0 Min5 Min10 Min15 Min20 Min30 min

Fluo

resc

ence

inte

nsity

Emission wavelength (nm)

Fig. 3. The effect of reaction time on the fluorescence spectra of glucose MRPsmeasured with an excitation frequency of 347 nm. The MRPs were formed at100 �C.

Mw fraction, there was little change in the recovered mass duringthe first 10 min and its Mw was also constant at 40 kDa. At latertimes the concentrations of the low Mw species absorbing at280 nm increased continuously and the final Mw was 20 kDa.These products may have been latter-stage MRPs, which werecompounds that were dissociated from glycated proteins, and con-tained chromophores absorbing at 280 nm.

4. Conclusion

The Maillard reaction can be successfully used as a couplingmethod for rice protein and glucose to form MRPs with improvedfunctional properties. Using methods to detect latter-stage MRPs,optimum reaction conditions were found which provided im-proved functional properties of rice proteins with reduced levelsof latter-stage MRPs. At a reaction time of 15 min the MRP hadthe best emulsifying capacity and solubility. The conditions forobtaining these MRPs were a 2% (w/w) protein:glucose (WR 1:1)aqueous dispersion with an initial pH of 11 which was heated for15 min at 100 �C. At these conditions, the functional properties(the emulsifying activity, emulsifying stability and solubility ofthe MRPs) increased by factors of 1.29, 1.77 and 8.30 times, respec-tively. However, there was a loss of 48% of the original lysine con-tent of the rice proteins. Longer reaction times did not improve theyield or functional properties and there was evidence that at longertimes other products were formed.

74 Y. Li et al. / Food Chemistry 117 (2009) 69–74

Acknowledgements

This work was financially supported by National 863 Program2006AA10Z327, Nature Science Foundation of Jiangsu Province,China (BK 2007502), NSFC 30871744, 111 project-B07029 andPCSIRT0627.

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