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Microstructure and rheological properties of mixtures of acid-deamidated rice protein and dextran
Journal of Cereal Science 51 (2010) 7–12
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Journal of Cereal Science journal homepage: www.elsevier.com/locate/jcs
Microstructure and rheological properties of mixtures of acid-deamidated rice protein and dextran Xianghong Li a, Yongle Liu a, Cuiping Yi a, *, Yunhui Cheng a, Sumei Zhou b, Yufei Hua c a
School of Chemistry and Biology Engineering, Changsha University of Science & Technology, 960 Wanjiali Road 2nd Section, Changsha 410114, Hunan Province, PR China Institute of Agro-Food Science and technology, The Chinese Academy of Agricultural Sciences, Beijing 100094, PR China c State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, Wuxi 214122, Jiangsu Province, PR China b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 25 March 2009 Received in revised form 13 August 2009 Accepted 28 August 2009
Microstructure and rheological properties of mixtures of acid-deamidated rice protein (ADRP) and dextran were studied. The microstructures of the ADRP/dextran mixtures were described using confocal laser scanning microscopy (CLSM), which revealed the effective association among ADRP and formation of a protein network-like structure in the mixture with higher protein concentration. Mechanical properties of the mixtures were observed by rheometer. The steady shear measurements showed a correlation with the CLSM results via a marked increase in the viscosity of the mixtures with protein association. Frequency sweeps further evidenced the build-up of the gelled network-like structure. The differences in fracture forces observed by textural measurements between mixture and single ADRP gel also suggested the difference in microstructures. The formation of network-like structure appeared to have occurred through a phase separation of ADRP and dextran. Ó 2009 Elsevier Ltd. All rights reserved.
Keywords: Rice protein Acid-deamidation Microstructure Rheology
1. Introduction Demand for relatively inexpensive sources of proteins that can be incorporated tin value-added food products is increasing. Much of the research involves various sources of plant proteins (Gorinstein et al., 2002; Rangel et al., 2003; Tomotake et al., 2002) that may help in increasing the nutritional value of food products at low cost. Rice is one of the most extensively cultivated cereals in the world, and is consumed as the staple food for more than half the world’s population and serves as the major source of energy and protein for large populations (Kato et al., 2000). Milled rice has an average protein concentration of 8-13% (Nakase et al., 1996) and these proteins are found to be one of the highest in nutritive value among cereal proteins for they are colorless, rich in essential amino acids, possess a bland taste, and are hypoallergenic and hypocholesterolemic (Chrastil, 1992), which suggests that rice protein should be considered when investigating the potential for adding value. This is especially so, considering the low commercial value of some rice products such as broken, chalky and debris rice or
Abbreviations: ADRP, acid-deamidated rice protein; CLSM, confocal laser scanning microscopy; SEC-HPLC, size-exclusion high-performance liquid chromatography. * Corresponding author. Tel./fax: þ86 731 85118969. E-mail address: [email protected] (C. Yi). 0733-5210/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.jcs.2009.08.006
by-product of rice starch. Their values could be significantly improved if the extracted rice protein’s functionality is suitable for food manufacture. Many previous studies on rice protein have been mainly focused on its isolation and characterization (Ellepola et al., 2006; Juliano, 1985; Van Der Borght et al., 2006), whereas studies on its application have been limited. Rice proteins do not meet some requirements necessary for processing stable food products because they actually exhibit low solubility in aqueous solution. This causes limited applications of rice proteins for various types of food, as solubility is the main characteristic of proteins selected for use in liquid foods and beverages, and is closely related to other functional properties of proteins such as foaming, emulsification, and gelling ability (Vojdani, 1996). Deamidation can improve the solubility and other functional properties of food proteins (Hamada, 1992). It has been shown that even small levels of deamidation could result in a significant improvement of protein functional properties (Matsudomi et al., 1985). Deamidation (acid treatments and enzymatic modifications) could improve the solubility of wheat gluten, since a high content of glutamine and asparagine residues may cause the aggregation of the protein molecules via hydrogen bonding (Matsudomi et al., 1985; Wu et al., 1976; Yong et al., 2006), just like the rice protein. Another reason for limited applications of rice proteins is that they cannot develop a viscoelastic network-like structure (Marco
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and Rosell, 2008), which is critical for obtaining an improvement in the quality of gel-based foods and bakery products such as gluten free products (Moore et al., 2004). Some food scientists made use of the interaction of polysaccharide and protein to obtain this kind of network-like structure, because the interaction plays an important role in macroscopic properties of food products such as flow, stability, texture and mouth feel (Ravindra et al., 2004); and a proper understanding and control of these interactions should enable design of products with desired structure and texture (Norton and Frith, 2001). The present study is part of research work aimed at altering the properties of rice proteins to make products similar to other protein based ones such as gluten and soy protein. Since rice proteins exhibit low solubility in aqueous solution and cannot develop a viscoelastic network-like structure, the present research aimed to design a biopolymer composite system using acid-deamidated rice protein and dextran for novel food applications. Dextran is a random coil in aqueous solutions and not able to form a gel. It has been extensively used in food products as a thickening agent (Schmitt et al., 1998). For the above purpose, a wide range of techniques were used, including zeta potential and size-exclusion high-performance liquid chromatography (SEC-HPLC) tests for molecular changes of single ADRP, confocal laser scanning microscopy (CLSM) for microscopic changes and rheological tests at small and large deformation for mechanical properties of mixtures.
disulfide bonds between the polypeptides (Hamada, 1996; Juliano, 1985). HCl is a kind of deamidation reagent. The functionalities (including solubility) of ADRP would be improved due to acid-deamidation and hydrolyzation of peptide bonds (Wu et al., 1976). We found that it provided a deamidation degree up to 49.76% under the above used deamidation conditions. The solubility of ADRP increased to 81.24% and the hydrolyzed degree was only 9.12%. 2.4. Preparation of mixtures
Debris rice, which was a by-product of polished rice, was supplied by Hunan Jinjian Cereal Industry Co., Ltd. The rice has a protein content of 7.8% (N 5.95, dry base, AOAC, 1995). All other reagents and chemicals were of analytical grade.
The ADRP were suspended in distilled water with a concentration of 20% (w/w) and stirred thoroughly. The suspensions were centrifuged at 10,000 g for 15 min and the supernatants were filtered through cellulose acetate membranes with pore size of 0.45 mm (Merck, Germany) to remove any insoluble particles. The solutions were characterized using a zeta potential analyzer and high-performance size-exclusion chromatography. The dextran with molecular weight of 2000 kDa was obtained from Sigma Co. (St Louis, MO, USA). The solutions were prepared as follows: the powders were suspended in distilled water and stirred for 2 h at room temperature. The pH was adjusted to 7.0, and the final solutions were filtered through cellulose acetate membranes with a pore size of 0.45 mm (Merck, Germany) to remove any insoluble particles. ADRP-dextran mixtures were prepared in cylindrical glass moulds containing 1–6%, w/w, protein and 1–3%, w/w, polysaccharide (with ionic strength of 0.5 mol/L) and stirred for 30 min in a controlled water bath at 25 0.1 C, then hermetically sealed and placed in a 25 0.1 C water bath for 24 h. NaN3 was added to avoid bacterial growth. A mixture was determined to be a gel when the mixture transformed into a solid. A spatula was used to test the mixture to detect the transition from fluid to solid (Zhang and Foegeding, 2003).
2.2. Defatting
2.5. Zeta potential and molecular weight distribution measurement
Rice flour was defatted with hexane (1:5, w/v) for 1 h at room temperature with continuous stirring. The flour was recovered using a Buchner funnel, air dried under a hood and passed through a 200 mesh sieve.
Zeta potentials of initial rice protein and ADRP were determined using a Zetasizer 2000 (Malvern Instruments, Southborough UK). The samples were diluted by a factor of 105 with distilled water and then injected into the apparatus. The averages of five measurements were reported as zeta potential. The molecular weight distributions of initial rice protein and ADRP were determined by SEC-HPLC. An Agilent liquid chromatography system equipped with a TSK gel2000 SWXL column (Tokyo, Japan) and an Agilent ultraviolet detector was used. The column was operated at a flow rate of 0.5 ml/min with 50 mmol/L phosphate buffer (pH 7.0), and the eluent was monitored at 220 nm. The sample was measured in triplicate and the representative example was selected for discussion. An Agilent Chemistry Station was used to analyze the peak area by integration. Due to the exclusion limit of the TSK gel2000 SWXL column, the molecular weight distribution of initial rice protein was separated with a Shodex protein KW-804 column (Shodex Separation and HPLC Group, Tokyo, Japan). The column, which has an exclusion limit of 106 Da, was operated at a flow rate of 0.5 ml/min with 3.5% SDS, since initial rice protein was not soluble in 50 mmol/L phosphate buffer (pH 7.0). The eluent was also monitored at 220 nm.
2. Materials and methods 2.1. Materials
2.3. Preparation of acid-deamidated rice protein (ADRP) Rice proteins were extracted from defatted rice flour using the procedure of Agboola et al. (2005) with minor modifications. Defatted rice flour was suspended in 0.05 mol/L NaOH with a liquid solid ratio of 5:1. After stirring for 1 h at room temperature, the suspension was centrifuged at 10,000 g for 30 min at 4 C to recover the supernatant. Rice protein was precipitated by adjusting pH to 4.1–4.8 with 2 mol/L HCl and centrifuged at 10,000 g for 30 min at 4 C and then freeze dried. Part of the protein sample was re-suspended in 0.38 mol/L HCl solution to deamidate at 88.5 C for 3.5 h. The deamidation degree was expressed as the ratio of the amount of released ammonia by acid deamination reaction to the total glutamine and asparagine residues of the protein. Amounts of ammonia released from deamidated glutamine and asparagine residues were determined using an ammonia test kit (Sigma). The number of total glutamine and asparagine residues was assessed by measuring the released ammonia when the proteins were treated with 3 mol/L HCl at 110 C for 3 h (Yi, 2006). Then the solution was dialyzed with distilled water at 4 C for 24 h, freeze dried and stored at 4 C. The major storage protein of rice is glutelin (66–78%). The insolubility of it is mainly due to hydrophobic, hydrogen and
2.6. Microscopic observations (CLSM) The proteins were stained by adding 25 ml of a 2% fluorescein isothiocyanate (FITC) to 100 ml protein solutions under magnetic stirring during 1 h and 30 min (Donato et al., 2005). Different mixtures with protein concentrations of 1%, 4.5% and 6%,
X. Li et al. / Journal of Cereal Science 51 (2010) 7–12
polysaccharide concentration of 3% and ionic strength of 0.5 mol/L were prepared as described above and poured between a concave slide and a coverslip, then hermetically sealed. Observations were performed on CLSM with a Leica TCS 4D confocal microscope (Leica Lasertechnik GmbH, Heidelberg, Germany) with a 100 oil-immersion objective lens. The excitation using an air-cooled Ar/Kr laser was performed at 485 nm and the emission was recorded between 500 and 540 nm. Observations in CLSM were verified by looking at various parts of the samples, and only repeatedly confirmed trends were reported and representative images shown. 2.7. Rheological measurements Rheological measurements were performed using an AR1000 controlled stress rheometer (TA Instruments, USA) equipped with a Peltier temperature controller and a flat-plane device (40 mm diameter, 0 angle). The large gap used in this geometry (1 mm) means that the possible interface effects are much reduced. The measurement temperature was 25 C and was controlled within 0.1 by a thermostat bath. The temperature was stabilized in less than 1 min. The steady shear viscosity was measured at shear rates from 102 to 102 s1. Single dextran (3%), single protein (6%, I ¼ 0.5 mol/L) and mixtures (with protein concentrations of 1%, 4.5% and 6%, polysaccharide concentration of 3% and ionic strength of 0.5 mol/L) were poured directly onto the measuring system of the rheometer. Strain sweep tests of the mixtures (with protein concentrations of 1%, 4.5% and 6%, polysaccharide concentration of 3% and ionic strength of 0.5 mol/L) were made in the range of 0.1–10% to determine the linear viscoelastic region at a frequency of 1 Hz and 25 0.1 C. That was determined to be below 1% and a target strain of 0.5% was thus employed in all subsequent experiments. Frequency sweep tests were made between 0.1 and 100 Hz at 0.5% strain and 25 0.1 C. All the experimental dynamic rheological data were obtained directly from the TA Rheology Advantage Data Analysis software. The deviation did not exceed 5% between duplicate runs, as the experiments were repeated.
9
carboxylation reaction significantly reduces intra-intermolecular hydrogen bonding (via the reduced number of NH2 groups) and increases the protein’s polyelectrolyte character which enhances electrostatic repulsion between protein molecules, thereby increasing the solubility of deamidated protein (Matsudomi et al., 1985). The molecular weight distributions of initial rice protein and ADRP are shown in Fig. 1. The SEC-HPLC profile of initial rice proteins (Fig. 1a) showed that the peaks with relative intensity of about 71.23% at 220 nm corresponded to Mw from 158,000 Da to above 106 Da, which were composed of HMW aggregates (glutelin, Mw > 65,000Da). A peak, corresponding to Mw of 19,800 Da with relative intensity of above 20%, was also detected. This part was composed of LMW protein materials comprising albumins, globulins and prolamins (Van Der Borght et al., 2006). The elution profile of ADRP (Fig. 1b) displayed two major elution peaks with total relative intensities of 91.58% at 220 nm, corresponding to Mw of 20,140 and 10,882 Da, respectively. Thus, the acid-deamidation of rice protein combined with a little hydrolyzation of peptide bond induced the dissociation of protein molecules, which also brought the increase of solubility. 3.2. CLSM observations and image analysis In present study, ADRP/dextran mixtures either developed into a stable solution, or a turbid bottom phase combined with a transparent upper phase, or a gel. CLSM images of ADRP/dextran systems (I ¼ 0.5 mol/L, protein concentration of 1%, 4.5% and 6%, respectively and polysaccharide concentration fixed at 3%) are presented in Fig. 2. The micrograph of the mixture consisting of 1% protein and 3% polysaccharide showed a homogeneous image (Fig. 2a). In the micrographs of mixtures consisting of 4.5%/6% protein and 3% polysaccharide (Fig. 2b and 2c), whatever the protein concentration
2.8. Textural measurements For large deformation measurements, the mixture (with protein concentration of 6%, polysaccharide concentration of 3% and ionic strength of 0.5 mol/L), which were transformed into a gel, and single ADRP gel (with protein concentration of 18.5% and ionic strength of 0.5 mol/L) were removed from the container and transferred to a Texture Analyzer (TA-XT2i, Stable Micro Systems, UK). A cylinder probe with diameter of 10 mm was chosen and the probe speed was set at 1 mm/s. The tests were performed at 25 C by compressing the samples until rupture. All samples were measured in triplicate. 3. Results and discussion 3.1. Characteristics of ADRP Wu et al. (1976) and Chan and Ma (1999) reported that relative molecular weight and surface potential related to the functionalities of acid-solubilized gluten and acid-deamidated soy protein, respectively. The characteristics were also strongly correlated to the mixing properties of protein and polysaccharide. Zeta potentials of rice protein and ADRP with different deamidation degree were measured. The zeta potential of rice protein was 29.5 2.5 mv, and that of ADRP increased to 43.8 1.6 mv as the deamidation degree increased to 49.76% and its solubility increased to 81.24%. Deamidation converts the amide group, from the glutamine and asparagine residue, to a carboxyl group. This
Fig. 1. (a) Molecular weight distribution of beginning rice protein. A Shodex protein KW-804 column was operated with 3.5% SDS; (b) Molecular weight distributions of acid-deaminated rice protein (ADRP). A TSKgel2000 SWXL column was operated with 50 mmol/L sodium phosphate buffer (pH 7.0).
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Fig. 2. CLSM images of ADRP and dextran mixtures at ionic strength of 0.5 mol/L. White color ¼ protein-rich area; black color ¼ polysaccharide-rich area. (a) 1%ADRP þ 3% dextran system; (b) 4.5%ADRP þ 3% dextran system; (c) 6%ADRP þ 3% dextran system.
All mixtures showed similar flow behavior where the viscosity decreased when shear rate increased. This behavior is defined as shear thinning and is produced when the stress disorganizes the arrangement of the macromolecules inside the matrix. The flow profiles of mixture with lower protein concentration (1%) and single polysaccharide or protein were very similar and a marked difference in viscosity was observed when the protein concentration increased to 4.5% and 6%. The steady shear measurements clearly showed a correlation with the CLSM results via a marked increase in the viscosity of the phase separated mixtures. 3.3.2. Strain sweeps Small amplitude oscillatory measurements afford the measurement of dynamic rheological functions without altering the internal network structure of materials tested and give more information about the microstructures than steady shear measurements. The strain sweep curves of the ADRP/dextran mixtures are shown in Fig. 4. It can be seen that for all the mixtures, elastic (G0 ) and viscous modulus (G00 ) remained virtually constant up to a relative strain of about 1%, after which they decreased with further increase in strain. Thus, the linear viscoelastic region was at strain values below 1% and frequency measurements were carried out at a strain of 0.5%. 3.3.3. Frequency sweeps Fig. 5 illustrates changes in G0 and G00 as a function of frequency for ADRP/dextran mixtures. The G0 and G00 curves for the mixture containing 1% protein and 3% dextran increased with frequency, Dextran
1.00E+02
ADRP 1%ADRP+3%Dextran 4.5%ADRP+3%Dextran
Viscosity (Pa.s)
of the mixtures, clear areas, corresponding to the fluorescence of FITC and thus revealing the presence of protein, were easily seen in the pictures. By contrast, dark areas corresponded to the localization of polysaccharide. It was thus seen that two components of both mixtures were distributed into two separated phases. The two micrographs also revealed the association of protein components. The degree of protein association appeared to depend on the concentration of protein. For the mixture consisting of 6% protein and 3% polysaccharide, protein seemed to connect into a networklike structure. At a macroscopic level, the system became a homogeneous gel at such lower protein concentration. Since dextran cannot form a gel and rice proteins cannot develop a viscoelastic network-like structure, the possible explanation is that the existence of dextran in the systems resulted in an increase in the amount of solvent that could be entrapped in the dextran phase, causing concentration of the protein phase and/or the solvent was redistributed between the phases as a result of the polymeric conformation changes that accompany gelation (Dickinson and McClements, 1995). Morris (1991) and Mounsey and O’Riordan (2008) believed that it’s a kind of exclusion effect, which was suggested to increase the effective concentration of each component, and produced the multi-textured gel. That’s a kind of ‘‘arrested’’ phase separation that behaved macroscopically as a stable highly viscous/gel-like mixture. In products such as salad dressing, this mechanism gives the product its characteristic consistency (De Kruif and Tuinier, 2001). We speculated that this kind of interaction could play an important role in macroscopic properties of food products such as flow, stability, texture and mouth feel, just like the research of Ravindra et al. (2004) which made use of the interaction between whey protein isolate and cross-linked waxy maize starch to produce such properties. The properties of the composite system strongly depend on the affinity of polysaccharide and protein to the solvent. Of course, parameters implied in the thermodynamics of phase separated systems such as protein configuration, polysaccharide molecular weight and concentration, as well as ionic strength and the type of salt of the medium, should be considered (Dickinson and McClements, 1995).
6%ADRP+3%Dextran
1.00E+01
1.00E+00
3.3. Rheological properties 3.3.1. Steady shear viscosity tests Fig. 3 shows viscosity as a function of shear rate for single ADRP (with protein concentration of 6% and I ¼ 0.5 mol/L), dextran (3%) and mixtures (I ¼ 0.5 mol/L) with protein concentration of 1%, 4.5% and 6% respectively and polysaccharide concentration fixed at 3%.
1.00E-01 1.00E-02
1.00E-01
1.00E+00
1.00E+01
1.00E+02
-1
Shear rate (s ) Fig. 3. Viscosity/shear rate profiles for single ADRP (6%, I ¼ 0.5 mol/L), dextran (3%) and ADRP/dextran mixtures.
X. Li et al. / Journal of Cereal Science 51 (2010) 7–12
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Table 1 Textural properties analysis of ADRP and ADRP/dextran mixture.
100
Sample a
ADRP ADRP/dextran
G',G''
10
Hardnessb(g)
Adhesivenessc(g s)
6.7 0.5 10.6 1.2
11.3 0.7 20.2 0.8
Average of three replications standard deviation. a Heating condition: heating in a Pyrex screwed tube at boiling water-bath. b Fracture ability: the biggest peak force that appeared in the first bite. c Adhesiveness: negative force area of the first bite.
1
0.1 0.1
1
10
Strain (%) Fig. 4. Strain sweep curves (1 Hz, 25 C) of ADRP/dextran mixtures (I ¼ 0.5 mol/L). -, : and represent 6%ADRP/dextran, 4.5%ADRP/dextran and 1%ADRP/dextran system, respectively. Solid and open symbols represent G0 and G00 .
and G0 was smaller than G00 all the time. Those of 4.5% the ADRP/ dextran system crossed over at a certain frequency, corresponding to the changes in the microstructure of the mixture. For the 6%ADRP/dextran system, G0 was larger than G00 over the entire range of frequency values and showed a small frequency dependence, indicating the formation of a typical gel (Musampa et al., 2007; Ould Eleya and Turgeon, 2000). Rheological measurements can therefore be interpreted in terms of a phase separation process. Both biopolymers had been concentrated in separated phases as a result of the phase separation process at a certain biopolymer concentration and a protein network-like structure was built up. We also found that ADRP, used as gelling agent in comminuted meat and other products, formed stable gels at a higher concentration range of 16.5–20% between the pH range 6.5 and 8.0. The fact that the ADRP/dextran mixtures are able to form stable gels at a lower concentration is very promising for their food application and the presence of phase separation may have contributed to this effect.
3.4. Large deformation textural properties of rice protein/dextran mixed gel The large deformation properties of gels are important for application in food products, because these are the properties that consumers observe during handling, slicing and eating of the product. Furthermore, mechanical properties provide information about network structure. Table 1 shows the texture properties of ADRP gel (with protein concentration of 18.5%, I ¼ 0.5 mol/L) and 6%ADRP/dextran mixture at the ionic strength of 0.5 mol/L. Although the protein 1.00E+02
G',G''
1.00E+01
1.00E+00
1.00E-01 1.00E-01
1.00E+00
1.00E+01
1.00E+02
Frequency (Hz) Fig. 5. Frequency sweep curves (0.5% strain, 25 C) of ADRP/dextran mixtures (with ionic strength of 0.5 mol/L). -, : and represent 6%ADRP/dextran, 4.5%ADRP/ dextran and 1%ADRP/dextran system, respectively. Solid and open symbols represent G0 and G00 .
concentration of the latter is much lower than the former, the gel hardness and adhesiveness were higher (calculated by Texture Expert Software, Stable Micro Systems, Godalming, Surrey UK). The differences in fracture forces between two samples as noticed in this experiment also suggested the difference in microstructures. In the former, rice proteins did not develop a viscoelastic network-like structure (Marco and Rosell, 2008). 4. Conclusion Microstructure and rheological properties of mixtures of ADRP and dextran were studied. Deamidation of rice protein induced an increase of zeta potential and decrease of molecular weight, which produced an increase of solubility. CLSM observations demonstrated an effective association between ADRP and formation of a network-like structure in the mixture with higher protein concentration. The steady shear measurements showed a correlation with the CLSM results via a marked increase in the viscosity of the phase separated mixtures, and frequency sweeps further evidenced the build-up of network-like structure. The differences in fracture forces between mixture and single ADRP gel also suggested the difference in microstructures. We speculated that the presence of redistribution of solvent between two phases and/or an exclusion effect of protein and polysaccharide may have contributed to the results. But the configuration and size of protein and many other properties also influence the phase behavior, so further studies are needed to focus on the phase separation mechanism of the ADRP and polysaccharide system. Acknowledgements This research project was funded by the Ministry of Science and Technology, PR China (Project No. 2006BAD05A10 and 2008AA100801) and Doctor Foundation of Changsha University of Science & Technology (Project No. 521), PR China. References Agboola, S., Ng, D., Mills, D., 2005. Characterization and functional properties of Australian rice protein isolates. Journal of Cereal Science 41, 283–290. Association of Official Analytical Chemists, 1995. AOAC method 990.03. In: Official Methods of Analysis, sixteenth ed. AOAC, Washington, DC. Chrastil, J., 1992. Correlations between the physicochemical and functional properties of rice. Journal of Agricultural and Food Chemistry 40, 1683–1686. Chan, W.M., Ma, C.Y., 1999. Acid modification of proteins from soymilk (okara). Food Research International 32, 119–127. De Kruif, C.G., Tuinier, R., 2001. Polysaccharide protein interactions. Food Hydrocolloids 15, 555–563. Dickinson, E., McClements, D.J., 1995. Protein-polysaccharide interactions. In: Dickinson, E., McClements, D.J. (Eds.), Advances in Food Colloids. Blackie Academic & Professional, London, pp. 81–101. Donato, L., Garnier, C., Novales, B., Doublier, J.L., 2005. Gelation of globular protein in presence of low methoxyl pectin: effect of Naþ and/or Ca2þ ions on rheology and microstructure of the systems. Food Hydrocolloids 19, 549–556. Ellepola, S.W., Choi, S.M., Phillips, D.L., Ma, C.Y., 2006. Raman spectroscopic study of rice globulin. Journal of Cereal Science 43, 85–93. Gorinstein, S., Pawelzlk, E., Licon, E.D., Haruenkit, R., Weisz, M., Trakhtenberg, S., 2002. Characterisation of pseudo cereal and cereal proteins by protein and
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Microstructure and rheological properties of mixtures of acid-deamidated rice protein and dextran Xianghong Li a, Yongle Liu a, Cuiping Yi a, *, Yunhui Cheng a, Sumei Zhou b, Yufei Hua c a
School of Chemistry and Biology Engineering, Changsha University of Science & Technology, 960 Wanjiali Road 2nd Section, Changsha 410114, Hunan Province, PR China Institute of Agro-Food Science and technology, The Chinese Academy of Agricultural Sciences, Beijing 100094, PR China c State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, Wuxi 214122, Jiangsu Province, PR China b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 25 March 2009 Received in revised form 13 August 2009 Accepted 28 August 2009
Microstructure and rheological properties of mixtures of acid-deamidated rice protein (ADRP) and dextran were studied. The microstructures of the ADRP/dextran mixtures were described using confocal laser scanning microscopy (CLSM), which revealed the effective association among ADRP and formation of a protein network-like structure in the mixture with higher protein concentration. Mechanical properties of the mixtures were observed by rheometer. The steady shear measurements showed a correlation with the CLSM results via a marked increase in the viscosity of the mixtures with protein association. Frequency sweeps further evidenced the build-up of the gelled network-like structure. The differences in fracture forces observed by textural measurements between mixture and single ADRP gel also suggested the difference in microstructures. The formation of network-like structure appeared to have occurred through a phase separation of ADRP and dextran. Ó 2009 Elsevier Ltd. All rights reserved.
Keywords: Rice protein Acid-deamidation Microstructure Rheology
1. Introduction Demand for relatively inexpensive sources of proteins that can be incorporated tin value-added food products is increasing. Much of the research involves various sources of plant proteins (Gorinstein et al., 2002; Rangel et al., 2003; Tomotake et al., 2002) that may help in increasing the nutritional value of food products at low cost. Rice is one of the most extensively cultivated cereals in the world, and is consumed as the staple food for more than half the world’s population and serves as the major source of energy and protein for large populations (Kato et al., 2000). Milled rice has an average protein concentration of 8-13% (Nakase et al., 1996) and these proteins are found to be one of the highest in nutritive value among cereal proteins for they are colorless, rich in essential amino acids, possess a bland taste, and are hypoallergenic and hypocholesterolemic (Chrastil, 1992), which suggests that rice protein should be considered when investigating the potential for adding value. This is especially so, considering the low commercial value of some rice products such as broken, chalky and debris rice or
Abbreviations: ADRP, acid-deamidated rice protein; CLSM, confocal laser scanning microscopy; SEC-HPLC, size-exclusion high-performance liquid chromatography. * Corresponding author. Tel./fax: þ86 731 85118969. E-mail address: [email protected] (C. Yi). 0733-5210/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.jcs.2009.08.006
by-product of rice starch. Their values could be significantly improved if the extracted rice protein’s functionality is suitable for food manufacture. Many previous studies on rice protein have been mainly focused on its isolation and characterization (Ellepola et al., 2006; Juliano, 1985; Van Der Borght et al., 2006), whereas studies on its application have been limited. Rice proteins do not meet some requirements necessary for processing stable food products because they actually exhibit low solubility in aqueous solution. This causes limited applications of rice proteins for various types of food, as solubility is the main characteristic of proteins selected for use in liquid foods and beverages, and is closely related to other functional properties of proteins such as foaming, emulsification, and gelling ability (Vojdani, 1996). Deamidation can improve the solubility and other functional properties of food proteins (Hamada, 1992). It has been shown that even small levels of deamidation could result in a significant improvement of protein functional properties (Matsudomi et al., 1985). Deamidation (acid treatments and enzymatic modifications) could improve the solubility of wheat gluten, since a high content of glutamine and asparagine residues may cause the aggregation of the protein molecules via hydrogen bonding (Matsudomi et al., 1985; Wu et al., 1976; Yong et al., 2006), just like the rice protein. Another reason for limited applications of rice proteins is that they cannot develop a viscoelastic network-like structure (Marco
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and Rosell, 2008), which is critical for obtaining an improvement in the quality of gel-based foods and bakery products such as gluten free products (Moore et al., 2004). Some food scientists made use of the interaction of polysaccharide and protein to obtain this kind of network-like structure, because the interaction plays an important role in macroscopic properties of food products such as flow, stability, texture and mouth feel (Ravindra et al., 2004); and a proper understanding and control of these interactions should enable design of products with desired structure and texture (Norton and Frith, 2001). The present study is part of research work aimed at altering the properties of rice proteins to make products similar to other protein based ones such as gluten and soy protein. Since rice proteins exhibit low solubility in aqueous solution and cannot develop a viscoelastic network-like structure, the present research aimed to design a biopolymer composite system using acid-deamidated rice protein and dextran for novel food applications. Dextran is a random coil in aqueous solutions and not able to form a gel. It has been extensively used in food products as a thickening agent (Schmitt et al., 1998). For the above purpose, a wide range of techniques were used, including zeta potential and size-exclusion high-performance liquid chromatography (SEC-HPLC) tests for molecular changes of single ADRP, confocal laser scanning microscopy (CLSM) for microscopic changes and rheological tests at small and large deformation for mechanical properties of mixtures.
disulfide bonds between the polypeptides (Hamada, 1996; Juliano, 1985). HCl is a kind of deamidation reagent. The functionalities (including solubility) of ADRP would be improved due to acid-deamidation and hydrolyzation of peptide bonds (Wu et al., 1976). We found that it provided a deamidation degree up to 49.76% under the above used deamidation conditions. The solubility of ADRP increased to 81.24% and the hydrolyzed degree was only 9.12%. 2.4. Preparation of mixtures
Debris rice, which was a by-product of polished rice, was supplied by Hunan Jinjian Cereal Industry Co., Ltd. The rice has a protein content of 7.8% (N 5.95, dry base, AOAC, 1995). All other reagents and chemicals were of analytical grade.
The ADRP were suspended in distilled water with a concentration of 20% (w/w) and stirred thoroughly. The suspensions were centrifuged at 10,000 g for 15 min and the supernatants were filtered through cellulose acetate membranes with pore size of 0.45 mm (Merck, Germany) to remove any insoluble particles. The solutions were characterized using a zeta potential analyzer and high-performance size-exclusion chromatography. The dextran with molecular weight of 2000 kDa was obtained from Sigma Co. (St Louis, MO, USA). The solutions were prepared as follows: the powders were suspended in distilled water and stirred for 2 h at room temperature. The pH was adjusted to 7.0, and the final solutions were filtered through cellulose acetate membranes with a pore size of 0.45 mm (Merck, Germany) to remove any insoluble particles. ADRP-dextran mixtures were prepared in cylindrical glass moulds containing 1–6%, w/w, protein and 1–3%, w/w, polysaccharide (with ionic strength of 0.5 mol/L) and stirred for 30 min in a controlled water bath at 25 0.1 C, then hermetically sealed and placed in a 25 0.1 C water bath for 24 h. NaN3 was added to avoid bacterial growth. A mixture was determined to be a gel when the mixture transformed into a solid. A spatula was used to test the mixture to detect the transition from fluid to solid (Zhang and Foegeding, 2003).
2.2. Defatting
2.5. Zeta potential and molecular weight distribution measurement
Rice flour was defatted with hexane (1:5, w/v) for 1 h at room temperature with continuous stirring. The flour was recovered using a Buchner funnel, air dried under a hood and passed through a 200 mesh sieve.
Zeta potentials of initial rice protein and ADRP were determined using a Zetasizer 2000 (Malvern Instruments, Southborough UK). The samples were diluted by a factor of 105 with distilled water and then injected into the apparatus. The averages of five measurements were reported as zeta potential. The molecular weight distributions of initial rice protein and ADRP were determined by SEC-HPLC. An Agilent liquid chromatography system equipped with a TSK gel2000 SWXL column (Tokyo, Japan) and an Agilent ultraviolet detector was used. The column was operated at a flow rate of 0.5 ml/min with 50 mmol/L phosphate buffer (pH 7.0), and the eluent was monitored at 220 nm. The sample was measured in triplicate and the representative example was selected for discussion. An Agilent Chemistry Station was used to analyze the peak area by integration. Due to the exclusion limit of the TSK gel2000 SWXL column, the molecular weight distribution of initial rice protein was separated with a Shodex protein KW-804 column (Shodex Separation and HPLC Group, Tokyo, Japan). The column, which has an exclusion limit of 106 Da, was operated at a flow rate of 0.5 ml/min with 3.5% SDS, since initial rice protein was not soluble in 50 mmol/L phosphate buffer (pH 7.0). The eluent was also monitored at 220 nm.
2. Materials and methods 2.1. Materials
2.3. Preparation of acid-deamidated rice protein (ADRP) Rice proteins were extracted from defatted rice flour using the procedure of Agboola et al. (2005) with minor modifications. Defatted rice flour was suspended in 0.05 mol/L NaOH with a liquid solid ratio of 5:1. After stirring for 1 h at room temperature, the suspension was centrifuged at 10,000 g for 30 min at 4 C to recover the supernatant. Rice protein was precipitated by adjusting pH to 4.1–4.8 with 2 mol/L HCl and centrifuged at 10,000 g for 30 min at 4 C and then freeze dried. Part of the protein sample was re-suspended in 0.38 mol/L HCl solution to deamidate at 88.5 C for 3.5 h. The deamidation degree was expressed as the ratio of the amount of released ammonia by acid deamination reaction to the total glutamine and asparagine residues of the protein. Amounts of ammonia released from deamidated glutamine and asparagine residues were determined using an ammonia test kit (Sigma). The number of total glutamine and asparagine residues was assessed by measuring the released ammonia when the proteins were treated with 3 mol/L HCl at 110 C for 3 h (Yi, 2006). Then the solution was dialyzed with distilled water at 4 C for 24 h, freeze dried and stored at 4 C. The major storage protein of rice is glutelin (66–78%). The insolubility of it is mainly due to hydrophobic, hydrogen and
2.6. Microscopic observations (CLSM) The proteins were stained by adding 25 ml of a 2% fluorescein isothiocyanate (FITC) to 100 ml protein solutions under magnetic stirring during 1 h and 30 min (Donato et al., 2005). Different mixtures with protein concentrations of 1%, 4.5% and 6%,
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polysaccharide concentration of 3% and ionic strength of 0.5 mol/L were prepared as described above and poured between a concave slide and a coverslip, then hermetically sealed. Observations were performed on CLSM with a Leica TCS 4D confocal microscope (Leica Lasertechnik GmbH, Heidelberg, Germany) with a 100 oil-immersion objective lens. The excitation using an air-cooled Ar/Kr laser was performed at 485 nm and the emission was recorded between 500 and 540 nm. Observations in CLSM were verified by looking at various parts of the samples, and only repeatedly confirmed trends were reported and representative images shown. 2.7. Rheological measurements Rheological measurements were performed using an AR1000 controlled stress rheometer (TA Instruments, USA) equipped with a Peltier temperature controller and a flat-plane device (40 mm diameter, 0 angle). The large gap used in this geometry (1 mm) means that the possible interface effects are much reduced. The measurement temperature was 25 C and was controlled within 0.1 by a thermostat bath. The temperature was stabilized in less than 1 min. The steady shear viscosity was measured at shear rates from 102 to 102 s1. Single dextran (3%), single protein (6%, I ¼ 0.5 mol/L) and mixtures (with protein concentrations of 1%, 4.5% and 6%, polysaccharide concentration of 3% and ionic strength of 0.5 mol/L) were poured directly onto the measuring system of the rheometer. Strain sweep tests of the mixtures (with protein concentrations of 1%, 4.5% and 6%, polysaccharide concentration of 3% and ionic strength of 0.5 mol/L) were made in the range of 0.1–10% to determine the linear viscoelastic region at a frequency of 1 Hz and 25 0.1 C. That was determined to be below 1% and a target strain of 0.5% was thus employed in all subsequent experiments. Frequency sweep tests were made between 0.1 and 100 Hz at 0.5% strain and 25 0.1 C. All the experimental dynamic rheological data were obtained directly from the TA Rheology Advantage Data Analysis software. The deviation did not exceed 5% between duplicate runs, as the experiments were repeated.
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carboxylation reaction significantly reduces intra-intermolecular hydrogen bonding (via the reduced number of NH2 groups) and increases the protein’s polyelectrolyte character which enhances electrostatic repulsion between protein molecules, thereby increasing the solubility of deamidated protein (Matsudomi et al., 1985). The molecular weight distributions of initial rice protein and ADRP are shown in Fig. 1. The SEC-HPLC profile of initial rice proteins (Fig. 1a) showed that the peaks with relative intensity of about 71.23% at 220 nm corresponded to Mw from 158,000 Da to above 106 Da, which were composed of HMW aggregates (glutelin, Mw > 65,000Da). A peak, corresponding to Mw of 19,800 Da with relative intensity of above 20%, was also detected. This part was composed of LMW protein materials comprising albumins, globulins and prolamins (Van Der Borght et al., 2006). The elution profile of ADRP (Fig. 1b) displayed two major elution peaks with total relative intensities of 91.58% at 220 nm, corresponding to Mw of 20,140 and 10,882 Da, respectively. Thus, the acid-deamidation of rice protein combined with a little hydrolyzation of peptide bond induced the dissociation of protein molecules, which also brought the increase of solubility. 3.2. CLSM observations and image analysis In present study, ADRP/dextran mixtures either developed into a stable solution, or a turbid bottom phase combined with a transparent upper phase, or a gel. CLSM images of ADRP/dextran systems (I ¼ 0.5 mol/L, protein concentration of 1%, 4.5% and 6%, respectively and polysaccharide concentration fixed at 3%) are presented in Fig. 2. The micrograph of the mixture consisting of 1% protein and 3% polysaccharide showed a homogeneous image (Fig. 2a). In the micrographs of mixtures consisting of 4.5%/6% protein and 3% polysaccharide (Fig. 2b and 2c), whatever the protein concentration
2.8. Textural measurements For large deformation measurements, the mixture (with protein concentration of 6%, polysaccharide concentration of 3% and ionic strength of 0.5 mol/L), which were transformed into a gel, and single ADRP gel (with protein concentration of 18.5% and ionic strength of 0.5 mol/L) were removed from the container and transferred to a Texture Analyzer (TA-XT2i, Stable Micro Systems, UK). A cylinder probe with diameter of 10 mm was chosen and the probe speed was set at 1 mm/s. The tests were performed at 25 C by compressing the samples until rupture. All samples were measured in triplicate. 3. Results and discussion 3.1. Characteristics of ADRP Wu et al. (1976) and Chan and Ma (1999) reported that relative molecular weight and surface potential related to the functionalities of acid-solubilized gluten and acid-deamidated soy protein, respectively. The characteristics were also strongly correlated to the mixing properties of protein and polysaccharide. Zeta potentials of rice protein and ADRP with different deamidation degree were measured. The zeta potential of rice protein was 29.5 2.5 mv, and that of ADRP increased to 43.8 1.6 mv as the deamidation degree increased to 49.76% and its solubility increased to 81.24%. Deamidation converts the amide group, from the glutamine and asparagine residue, to a carboxyl group. This
Fig. 1. (a) Molecular weight distribution of beginning rice protein. A Shodex protein KW-804 column was operated with 3.5% SDS; (b) Molecular weight distributions of acid-deaminated rice protein (ADRP). A TSKgel2000 SWXL column was operated with 50 mmol/L sodium phosphate buffer (pH 7.0).
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Fig. 2. CLSM images of ADRP and dextran mixtures at ionic strength of 0.5 mol/L. White color ¼ protein-rich area; black color ¼ polysaccharide-rich area. (a) 1%ADRP þ 3% dextran system; (b) 4.5%ADRP þ 3% dextran system; (c) 6%ADRP þ 3% dextran system.
All mixtures showed similar flow behavior where the viscosity decreased when shear rate increased. This behavior is defined as shear thinning and is produced when the stress disorganizes the arrangement of the macromolecules inside the matrix. The flow profiles of mixture with lower protein concentration (1%) and single polysaccharide or protein were very similar and a marked difference in viscosity was observed when the protein concentration increased to 4.5% and 6%. The steady shear measurements clearly showed a correlation with the CLSM results via a marked increase in the viscosity of the phase separated mixtures. 3.3.2. Strain sweeps Small amplitude oscillatory measurements afford the measurement of dynamic rheological functions without altering the internal network structure of materials tested and give more information about the microstructures than steady shear measurements. The strain sweep curves of the ADRP/dextran mixtures are shown in Fig. 4. It can be seen that for all the mixtures, elastic (G0 ) and viscous modulus (G00 ) remained virtually constant up to a relative strain of about 1%, after which they decreased with further increase in strain. Thus, the linear viscoelastic region was at strain values below 1% and frequency measurements were carried out at a strain of 0.5%. 3.3.3. Frequency sweeps Fig. 5 illustrates changes in G0 and G00 as a function of frequency for ADRP/dextran mixtures. The G0 and G00 curves for the mixture containing 1% protein and 3% dextran increased with frequency, Dextran
1.00E+02
ADRP 1%ADRP+3%Dextran 4.5%ADRP+3%Dextran
Viscosity (Pa.s)
of the mixtures, clear areas, corresponding to the fluorescence of FITC and thus revealing the presence of protein, were easily seen in the pictures. By contrast, dark areas corresponded to the localization of polysaccharide. It was thus seen that two components of both mixtures were distributed into two separated phases. The two micrographs also revealed the association of protein components. The degree of protein association appeared to depend on the concentration of protein. For the mixture consisting of 6% protein and 3% polysaccharide, protein seemed to connect into a networklike structure. At a macroscopic level, the system became a homogeneous gel at such lower protein concentration. Since dextran cannot form a gel and rice proteins cannot develop a viscoelastic network-like structure, the possible explanation is that the existence of dextran in the systems resulted in an increase in the amount of solvent that could be entrapped in the dextran phase, causing concentration of the protein phase and/or the solvent was redistributed between the phases as a result of the polymeric conformation changes that accompany gelation (Dickinson and McClements, 1995). Morris (1991) and Mounsey and O’Riordan (2008) believed that it’s a kind of exclusion effect, which was suggested to increase the effective concentration of each component, and produced the multi-textured gel. That’s a kind of ‘‘arrested’’ phase separation that behaved macroscopically as a stable highly viscous/gel-like mixture. In products such as salad dressing, this mechanism gives the product its characteristic consistency (De Kruif and Tuinier, 2001). We speculated that this kind of interaction could play an important role in macroscopic properties of food products such as flow, stability, texture and mouth feel, just like the research of Ravindra et al. (2004) which made use of the interaction between whey protein isolate and cross-linked waxy maize starch to produce such properties. The properties of the composite system strongly depend on the affinity of polysaccharide and protein to the solvent. Of course, parameters implied in the thermodynamics of phase separated systems such as protein configuration, polysaccharide molecular weight and concentration, as well as ionic strength and the type of salt of the medium, should be considered (Dickinson and McClements, 1995).
6%ADRP+3%Dextran
1.00E+01
1.00E+00
3.3. Rheological properties 3.3.1. Steady shear viscosity tests Fig. 3 shows viscosity as a function of shear rate for single ADRP (with protein concentration of 6% and I ¼ 0.5 mol/L), dextran (3%) and mixtures (I ¼ 0.5 mol/L) with protein concentration of 1%, 4.5% and 6% respectively and polysaccharide concentration fixed at 3%.
1.00E-01 1.00E-02
1.00E-01
1.00E+00
1.00E+01
1.00E+02
-1
Shear rate (s ) Fig. 3. Viscosity/shear rate profiles for single ADRP (6%, I ¼ 0.5 mol/L), dextran (3%) and ADRP/dextran mixtures.
X. Li et al. / Journal of Cereal Science 51 (2010) 7–12
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Table 1 Textural properties analysis of ADRP and ADRP/dextran mixture.
100
Sample a
ADRP ADRP/dextran
G',G''
10
Hardnessb(g)
Adhesivenessc(g s)
6.7 0.5 10.6 1.2
11.3 0.7 20.2 0.8
Average of three replications standard deviation. a Heating condition: heating in a Pyrex screwed tube at boiling water-bath. b Fracture ability: the biggest peak force that appeared in the first bite. c Adhesiveness: negative force area of the first bite.
1
0.1 0.1
1
10
Strain (%) Fig. 4. Strain sweep curves (1 Hz, 25 C) of ADRP/dextran mixtures (I ¼ 0.5 mol/L). -, : and represent 6%ADRP/dextran, 4.5%ADRP/dextran and 1%ADRP/dextran system, respectively. Solid and open symbols represent G0 and G00 .
and G0 was smaller than G00 all the time. Those of 4.5% the ADRP/ dextran system crossed over at a certain frequency, corresponding to the changes in the microstructure of the mixture. For the 6%ADRP/dextran system, G0 was larger than G00 over the entire range of frequency values and showed a small frequency dependence, indicating the formation of a typical gel (Musampa et al., 2007; Ould Eleya and Turgeon, 2000). Rheological measurements can therefore be interpreted in terms of a phase separation process. Both biopolymers had been concentrated in separated phases as a result of the phase separation process at a certain biopolymer concentration and a protein network-like structure was built up. We also found that ADRP, used as gelling agent in comminuted meat and other products, formed stable gels at a higher concentration range of 16.5–20% between the pH range 6.5 and 8.0. The fact that the ADRP/dextran mixtures are able to form stable gels at a lower concentration is very promising for their food application and the presence of phase separation may have contributed to this effect.
3.4. Large deformation textural properties of rice protein/dextran mixed gel The large deformation properties of gels are important for application in food products, because these are the properties that consumers observe during handling, slicing and eating of the product. Furthermore, mechanical properties provide information about network structure. Table 1 shows the texture properties of ADRP gel (with protein concentration of 18.5%, I ¼ 0.5 mol/L) and 6%ADRP/dextran mixture at the ionic strength of 0.5 mol/L. Although the protein 1.00E+02
G',G''
1.00E+01
1.00E+00
1.00E-01 1.00E-01
1.00E+00
1.00E+01
1.00E+02
Frequency (Hz) Fig. 5. Frequency sweep curves (0.5% strain, 25 C) of ADRP/dextran mixtures (with ionic strength of 0.5 mol/L). -, : and represent 6%ADRP/dextran, 4.5%ADRP/ dextran and 1%ADRP/dextran system, respectively. Solid and open symbols represent G0 and G00 .
concentration of the latter is much lower than the former, the gel hardness and adhesiveness were higher (calculated by Texture Expert Software, Stable Micro Systems, Godalming, Surrey UK). The differences in fracture forces between two samples as noticed in this experiment also suggested the difference in microstructures. In the former, rice proteins did not develop a viscoelastic network-like structure (Marco and Rosell, 2008). 4. Conclusion Microstructure and rheological properties of mixtures of ADRP and dextran were studied. Deamidation of rice protein induced an increase of zeta potential and decrease of molecular weight, which produced an increase of solubility. CLSM observations demonstrated an effective association between ADRP and formation of a network-like structure in the mixture with higher protein concentration. The steady shear measurements showed a correlation with the CLSM results via a marked increase in the viscosity of the phase separated mixtures, and frequency sweeps further evidenced the build-up of network-like structure. The differences in fracture forces between mixture and single ADRP gel also suggested the difference in microstructures. We speculated that the presence of redistribution of solvent between two phases and/or an exclusion effect of protein and polysaccharide may have contributed to the results. But the configuration and size of protein and many other properties also influence the phase behavior, so further studies are needed to focus on the phase separation mechanism of the ADRP and polysaccharide system. Acknowledgements This research project was funded by the Ministry of Science and Technology, PR China (Project No. 2006BAD05A10 and 2008AA100801) and Doctor Foundation of Changsha University of Science & Technology (Project No. 521), PR China. References Agboola, S., Ng, D., Mills, D., 2005. Characterization and functional properties of Australian rice protein isolates. Journal of Cereal Science 41, 283–290. Association of Official Analytical Chemists, 1995. AOAC method 990.03. In: Official Methods of Analysis, sixteenth ed. AOAC, Washington, DC. Chrastil, J., 1992. Correlations between the physicochemical and functional properties of rice. Journal of Agricultural and Food Chemistry 40, 1683–1686. Chan, W.M., Ma, C.Y., 1999. Acid modification of proteins from soymilk (okara). Food Research International 32, 119–127. De Kruif, C.G., Tuinier, R., 2001. Polysaccharide protein interactions. Food Hydrocolloids 15, 555–563. Dickinson, E., McClements, D.J., 1995. Protein-polysaccharide interactions. In: Dickinson, E., McClements, D.J. (Eds.), Advances in Food Colloids. Blackie Academic & Professional, London, pp. 81–101. Donato, L., Garnier, C., Novales, B., Doublier, J.L., 2005. Gelation of globular protein in presence of low methoxyl pectin: effect of Naþ and/or Ca2þ ions on rheology and microstructure of the systems. Food Hydrocolloids 19, 549–556. Ellepola, S.W., Choi, S.M., Phillips, D.L., Ma, C.Y., 2006. Raman spectroscopic study of rice globulin. Journal of Cereal Science 43, 85–93. Gorinstein, S., Pawelzlk, E., Licon, E.D., Haruenkit, R., Weisz, M., Trakhtenberg, S., 2002. Characterisation of pseudo cereal and cereal proteins by protein and
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