Journal of Cereal Science 40 (2004) 205–211 www.elsevier.com/locate/jnlabr/yjcrs
Effects of prolamin on the textural and pasting properties of rice flour and starch Graeme Baxtera,b, Christopher Blancharda,b, Jian Zhaoa,b,* a
School of Wine and Food Sciences, Charles Sturt University, Wagga Wagga NSW 2678, Australia b Farrer Centre, Charles Sturt University, Wagga Wagga NSW 2678, Australia Received 22 December 2003; revised 11 May 2004; accepted 9 July 2004
Abstract Prolamin is a major class of rice proteins but its influence on the physicochemical properties of rice is not clear. Rapid Visco Analyser (RVA) and TA-XT2 TPA textural analyses were performed on rice starch with the addition of prolamin extracted from three rice cultivars (Hitomebore, M103 and Amaroo), and on rice flour with the prolamin removed by propan-2-ol extraction. Addition of prolamin to rice starch was found to cause a significant (P!0.05) increase in RVA breakdown viscosity but significant (P!0.05) decreases in hardness, adhesiveness and gumminess of the starch gel. Similarly, when prolamin was removed from rice flour, exactly the opposite effect was observed. Addition of prolamin to rice starch also caused it to absorb water faster during cooking but the gelatinised starch absorbed less water compared with control samples without prolamin. q 2004 Elsevier Ltd. All rights reserved. Keywords: Prolamin; Rice; RVA pasting; Textural properties
1. Introduction The economic value of rice is strongly influenced by pasting and textural properties such as hardness and adhesiveness. Particular combinations of these traits can have an important influence on the end use of rice. For example, products such as sushi require soft, sticky rice while Indian style dishes are usually accompanied by rice with firmer, non-sticky characteristics (Barton, 1998; Bhattacharjee and Kulkarni, 2000). Despite their importance, it is not entirely clear what factors can affect these properties. Much of the research to date has focused on the role of starch with considerable success (Kokini et al., 1992; Vandeputte et al., 2003). However, Champagne et al. (1999) demonstrated that cultivars with similar starch content and composition could have rather different pasting and textural
Abbreviations: DTT 1, 4Dithio-DL-Threitol; RVA, Rapid viscoanalyser; TPA, Textural profile analysis. * Corresponding author. Tel.: C61-26932968; fax: C61-26932107. E-mail address: [email protected]
(J. Zhao). 0733-5210/$ - see front matter q 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.jcs.2004.07.004
properties, suggesting that components other than starch may contribute to pasting and textural traits. Recent studies suggest that protein can play a role in determining the pasting and textural properties of rice. Lim et al. (1999), for example, reported that reducing the protein content in rice flour increases its peak viscosity. This was confirmed by Tan and Corke (2002) who proposed that protein content is negatively correlated to peak viscosity and hot paste viscosity. Furthermore, Lyon et al. (2000) found that protein content was negatively correlated to adhesiveness of cooked rice. While these studies have suggested a link between total protein and physical properties of rice, it is not clear how individual protein fractions contribute to rice pasting and texture. Some preliminary studies have suggested that glutelin and the 60 kDa starch granule bound starch synthase protein are related to adhesiveness and other textural characteristics (Chrastil, 1992; Hamaker and Griffin, 1993). However, little is known about the effect of prolamin, the second most abundant class of rice endosperm protein (Evers et al., 1999; Takaiwa, 1998), on the physical properties of rice.
G. Baxter et al. / Journal of Cereal Science 40 (2004) 205–211
The objective of this study was to investigate the effect of prolamin removal and addition on the pasting and textural properties of rice flour and starch.
2. Experimental 2.1. Materials Three cultivars of rice (M103, Amaroo and Hitomebore) were used in this study. The varieties were grown at the Yanco Agricultural Institute, Yanco, NSW, Australia, during the 2001/2002 growing season. Rice grains with an average of 12% moisture were dehulled (THU35A Test Husker, Satake) and then milled (McGill No. 2 Mill) for 60 s. Broken grains were separated from whole grains by weight differentials and only whole grains were used for this study. Milled grains were ground to pass through a 0.12 mm screen (Retsch model, Zm100). Rice starch was obtained from Sigma Aldrich Pty Ltd (Product No. S 7260). 2.2. Defatting of rice flour Flour samples were defatted before prolamin extraction in the experiments involving the addition of prolamin to commercial rice starch in order to minimize lipid contamination in the protein extracts. Defatting was carried out by adding 14 volumes of a ethyl ether/methanol (1:1; v/v) mixture to rice flour samples, and the resultant slurries were mixed thoroughly, allowed to stand for 1 h and centrifuged at 10,000g for 10 min (Chrastil and Zarins, 1994). The defatting procedure was repeated a total of three times to maximise removal of the lipid components. 2.3. Optimisation of prolamin extraction To maximise prolamin extraction, trials were carried out using several different solvents and solvent/water mixtures: propan-2-ol (100%, 50%), ethanol (100%, 50%) and propan-2-ol (100%, 50%) with the addition of 1% DTT. Prolamin was extracted from rice flour using three volumes of the extraction solvent. The suspensions were mixed thoroughly, allowed to stand for 30 min, mixed thoroughly again and centrifuged at 10,000g for 10 min at 15 8C. The extraction process was repeated a total of four times and two different flour samples were used per solvent to serve as replicates. Protein concentrations of the extracts were determined after each extraction by the Lowry method using the Biorad DC protein assay kit (Catalogue No. 500-0111). Standard curves for the determination of prolamin concentration were created for each of the solvents using freeze-dried prolamin dissolved in the respective solvent.
2.4. Extraction of prolamin The above experiment established (details in Section 3) that 100% propan-2-ol was the most efficient solvent for the extraction of prolamin from rice flour. Therefore, in subsequent experiments, prolamin extraction was carried out using this solvent following the procedure outlined in Section 2.3. The extracts were dialysed against de-ionised water overnight at room temperature. Protein concentrations of the dialysed extracts were determined by the Lowry method as described in Section 2.3. To determine the purity of the extracted prolamin, a separate experiment was conducted where prolamin was extracted and dialysed exactly the same way as described above, and the extracts were freeze-dried. The freeze-dried extracts were weighed and the total nitrogen determined using a LECO FP-2000 analyser (Leco Corp., St Jeseph, MI, USA). Protein content (purity) of the solids was calculated by multiplying the percentage nitrogen found by the factor 5.95 (Chrastil, 1992). 2.5. RVA analysis Pasting properties were determined using a Rapid-Visco Analyser (Newport Scientific model 3D, Warriewood, Australia) following the AACC Approved Method 61–02 (2000), modified by extending the cooling time by 5 min and holding at 50 8C for 5 min to ensure that maximum peak viscosity was obtained. RVA analyses were performed on whole flour, whole flour with prolamin removed, reconstituted flour (described in Section 3.3) and Sigma rice starch with or without prolamin flour added. Reconstituted rice flour was prepared by first removing the prolamin from whole flour as described in Section 2.4. The prolamin extract was dialysed against de-ionised water overnight and then re-mixed with the flour residue from which it was extracted, and the mixture dried at 40 8C to constant weight. Each RVA canister contained 3 g of starch or flour and was made up to 28 g using de-ionised water or extracted protein in de-ionised water. Peak viscosity, hot paste viscosity, final viscosity, breakdown (peak viscosity–hot paste viscosity) and setback (final viscosity–peak viscosity) were recorded. Each analysis was performed at least twice. 2.6. Measurement of water absorption Water absorption of rice starch during cooking was measured using a variation of the method by Konik-Rose et al. (2001). Rice starch (100 mg) was accurately weighed into an 1.5 ml centrifuge tube. The sample was mixed well with 1 ml water or prolamin extract containing 3 mg prolamin and heated to 95 8C. Samples were removed at 3 min intervals from 0 to 18 min, cooled to room temperature and then centrifuged at 10,000g for 10 min. The pellets were resuspended in two volumes of methanol to remove unbound water. The starch–methanol mixtures were
G. Baxter et al. / Journal of Cereal Science 40 (2004) 205–211
Fig. 1. Concentrations of prolamin in sequential extracts as extracted with different solvents.
centrifuged at 10,000g for 10 min, the supernatant removed by suction, and the weight of the residue determined. Each analysis was repeated three times.
2.7. Textural analysis
The efficiancy of several different prolamin extraction media was tested to optimise prolamin extraction (Fig. 1). Propanol extracted more protein than ethanol at both concentrations (100 and 50%), while for both solvents, the extraction was more efficient at 100% than at 50%. The addition of DTT to propan-2-ol did not have a significant effect on protein extraction, however, a slight reduction in the amount of protein extracted in the solvent containing DTT was noted. Based on these results, 100% propan-2-ol without the addition of DTT was used for subsequent prolamin extractions. For this solvent, prolamin was essentially removed completely from the samples after the extraction was repeated three times (Fig. 1).
Textural properties of the rice flour gels formed after RVA analyses were determined with a TA XT2 textural analyser (Stable Microsystems, Surrey, Great Britain). Gels were sealed in the canisters with paraffin film to prevent moisture loss and were left overnight at 4 8C to allow even retrogradation. Analyses were made using a standard two-cycle program (TPA procedure) with a 10-mm cylindrical ebonite probe, which was programmed to move downwards for a distance of 48 mm at a speed of 2 mm/s. From the force-time curve obtained, textural parameters of hardness (height of the force peak on cycle 1, g), cohesiveness (ratio of the positive force areas under the first and second cycles), adhesiveness (negative force area of the first cycle, -gs) and gumminess (hardness!cohesiveness, g) were computed using the Texture Expert software supplied with the instrument. 2.8. Statistical analysis Data obtained were analysed by independent-samples t-tests or analysis of variance (ANOVA) following the procedure described by Miller and Miller (1993) using SPSS for WindowsTM version 11.0. For prolamin addition experiments, comparisons of sample means were made using ANOVA analysis. Differences of means were reported at the 5% significance level. For prolamin removal experiments, pairwise comparisons were made by t-tests between whole flour and prolamin depleted flour for each of the three cultivars. Differences of means were reported at the 5 or 1% significance level.
3.1. Optimisation of prolamin extraction
3.2. Prolamin extraction efficiency and purity To ascertain the purity of prolamin extracted from the flour of the three rice cultivars used in this investigation, Table 1 RVA pasting properties of starch with the addition of prolamina extracted from defatted flour from three rice cultivars Sample
Peak viscosity (RVU)
Starchb Hitomebore M103 Amaroo
239G0.7A 86G1.4A 233G2.4AB 113G0.7B 229G12.2AB 126G0.5C 219G4.7B 126G3.9C
Final viscosity (RVU)
280G1.7A 249G2.5B 232G3.0C 228G3.6C
41G1.0A 16G0.1B 3G2.8C 9G1.1C
Data are means of at least two analyses with standard deviation. Means in the same column with different uppercase letters were significantly different at the 5% level by ANOVA. a 100 mg of prolamin was added to 3 g rice starch. b Rice starch control.
G. Baxter et al. / Journal of Cereal Science 40 (2004) 205–211
the total solids in the prolamin extracts and the protein contents of the solids were determined. The average solids extracted from 10 g of flour of the cultivars Hitomebore, M103 and Amaroo were 0.143, 0.139 and 0.148 g,
Table 2 TPA textural properties of starch with the addition of prolamina extracted from defatted flour from three rice cultivars Sample
235.4G12.0A 0.63G7.8! 10K2A B 99.6G11.6 0.51G1.0! 10-4A C 30.2G10.3 0.45G2.8! 10K2A B 86.0G5.7 0.46G7.0! 10-3A
Gumminess (g) 88.0G9.9A 50.7G1.3B 41.6G9.5B 32.4G2.8B
Data are means of at least two analyses with standard deviation. Means in the same column with different uppercase letters were significantly different at the 5% level by ANOVA. a 100 mg of prolamin was added to 3 g starch. b Rice starch control.
respectively. The protein content of the solids was found to be 95.4, 94.8 and 95.7% for the cultivars Hitomebore, M103 and Amaroo, respectively. 3.3. Effect of prolamin addition on the pasting and textural properties of rice starch Addition of 100 mg prolamin to 3 g rice starch resulted in significant (P!0.01) changes in RVA pasting properties (Table 1). Irrespective of which rice variety was used as a source of prolaim, adding the protein to rice starch caused a significant (P!0.05) increase in RVA breakdown values and significant decreases (P! 0.05) in final viscosity and setback viscosity. While a general trend of decreasing peak viscosity with the addition of prolamin extract was also observed, the effect was only significant (P!0.05) for prolamin extracted from Amaroo. Table 3 Effect of prolamin removal on RVA pasting properties of flour from three rice cultivars Sample
Hitomebore Whole flour Prolamin removed M103 Whole flour Prolamin removed Amaroo Whole flour Prolamin removed
Fig. 2. Effect of prolamin concentration on RVA breakdown viscosity of rice starch (A) and the textural properties of hardness (B) and adhesiveness (C) of the starch gels.
Peak viscosity (RVU)
Final viscosity (RVU)
Data are means of at least two analyses with standard deviation. For each cultivar, means of whole flour and prolamin removed flour were compared by t-tests. * and ** indicate means were different at the 5 and 1% significance level, respectively.
G. Baxter et al. / Journal of Cereal Science 40 (2004) 205–211 Table 4 Effect of prolamin removal on the TPA textural properties of flour from three rice cultivars Sample Hitomebore Whole flour Prolamin removed M103 Whole flour Prolamin removed Amaroo Whole flour Prolamin removed
0.55G2.1x10K3 14.4G0.2 0.54G8.2x10-3 18.1G0.8**
0.64G5.0!10K3 10.6G0.6 0.64G3.8!10-3 14.4G0.7*
0.56G6.9!10K3 16.1G0.4 0.55G1.8!10-3 18.1G0.3*
Data are means of two analyses with standard deviation. For each cultivar, means of whole flour and prolamin removed flour were compared by t-test. * and ** indicate means were different at 5 and 1% significance level, respectively.
The effect of prolamin on RVA breakdown values was explored further by varying the concentrations of the protein fraction added to rice starch (Fig. 2). A positive linear trend was observed for samples with 0 to 45 mg of prolamin per gram of rice starch. Concentrations higher than 45 mg/g, however, did not cause any further increase in breakdown (Fig. 2A). Gels formed in the canisters after processing of rice starch samples by RVA were subjected to TPA analysis to investigate the effect of prolamin on rice gel texture. The addition of prolamin to starch produced significantly (P! 0.05) softer gels that had lower adhesiveness and gumminess values for all of the varieties tested (Table 2). Moreover, increasing the amount of prolamin added to rice starch caused linear decreases in hardness (r2Z0.99) and adhesiveness (r2Z0.93) of the rice starch gels (Fig. 2B and C). 3.4. Effect of prolamin removal on the pasting and textural properties of rice flour Removal of prolamin from rice flour resulted in a significant (P!0.01) decrease in breakdown viscosity for
all three varieties examined (Table 3). A significant (P!0.05) reduction in peak viscosity in Hitomebore and final viscosity in Hitomebore and M103 samples, respectively, was also observed. Removal of prolamin from rice flour resulted in significant (P!0.05) increases in hardness, adhesiveness and gumminess of the rice flour gels (Table 4). To determine whether the prolamin extraction and reconstitution procedures altered the pasting properties of rice, RVA viscosity curves of reconstituted rice flour were compared with those of whole flour for three rice varieties (Fig. 3). RVA profiles of the reconstituted flour were almost identical to those of the original native flour. 3.5. Effect of prolamin addition on water absorption of rice starch The amount of water absorbed by rice starch during cooking was followed at 3 min intervals for 18 min (Fig. 4). In the early stages of cooking, starch samples with added prolamin absorbed water more rapidly than the starch only samples. However, the amount of bound water in the samples containing prolamin decreased significantly (P!0.01) after three minutes with no significant change in the amount of water absorbed with further cooking. In comparison, water absorption by samples containing starch only was more gradual but the maximum amount of water absorbed was higher.
4. Discussion The present study suggests that prolamin concentration can significantly affect breakdown viscosity of rice pastes as well as hardness, adhesiveness and gumminess of rice flour gels. This is the first study that has demonstrated such effects of prolamin on rice pasting and textural properties. We have also shown that the procedure used for the extraction of prolamin from rice flour produced a protein that was pure enough to show that the changes in the pasting and textural properties of rice starch (Tables 1 and 2) following the addition of the extracted prolamin
Fig. 3. A comparison of RVA pasting profiles of original (—) and reconstituted (†) rice flour from three rice varieties. (A), Hitomebore; (B), M103; (C), Amaroo.
G. Baxter et al. / Journal of Cereal Science 40 (2004) 205–211
Fig. 4. Water absorption of rice starch (—) and with the addition of prolamin (- -) at a rate of 3 mg per gram of starch.
was most likely due to the protein and not to other rice components such as lipid or starch. Furthermore, the prolamin extraction and reconstitution process did not significantly alter the RVA profile of the rice flour samples (Fig. 3), suggesting that the properties of prolamin and other rice components had not been modified significantly, if at all, by the propan-2-ol extraction to cause a significant change in their pasting behaviour. This indicates that the observed changes in the pasting properties of rice flour (Table 3) following the removal of prolamin was due to the absence of the protein in the samples. There were, however, some inconsistencies between the results obtained from the samples with prolamin added to starch and those with prolamin removed from flour. This is not surprising as the former was a simple mixture of relatively pure starch and prolaminin whereas the latter was a much more complicated system consisting of starch, glutelin and other rice components. The presence of these components (glutelin, etc.) in the prolamin-depleted flour might have offset some of the effects caused by the removal of prolamin. It is generally accepted that the increase in viscosity that occurs during heating of starch suspension is mainly due to the swelling of the starch granules (with lesser contributions from the solubilisation of amylose and hydration of protein, if present) and breakdown of viscosity is caused by rupture of the swollen granules (Sandhya Rani and Bhattacharya, 1995). It is therefore likely that the observed increase in breakdown viscosity following the addition of prolamin to a starch-water mixture was due to an increased rate of starch granule rupturing during RVA processing. This may be caused by an increase in the rate of water absorption by starch granules, facilitated by the presence of the prolamin. This hypothesis was supported by the increase in the rate of water absorption in the early stages of cooking when prolamin was present (Fig. 4). Furthermore, the large decrease in the amount of bound water after cooking for longer than 3 min, observed in samples with the addition of prolamin, seemed to suggest that a greater proportion of
starch granules had ruptured during cooking, thus further supporting the hypothesis. The gel formed at the end of the RVA cooling cycle is essentially a three-dimensional network of intertwined amylose molecules incorporating dispersed swollen and ruptured starch granules. The decreased final viscosity of samples with prolamin added suggests that the threedimensional network is weakened by the presence of prolamin in the matrix. This hypothesis was supported by a reduction in both hardness and adhesiveness of rice gels containing prolamin (Table 4). Interestingly, Chrastil (1990) found that the adhesiveness of rice increases with the amount of glutelin it contains. The contrasting effects of prolamin and glutelin on adhesiveness may mean that the adhesiveness of rice is determined, at least in part, by the relative proportions of the two major protein fractions in rice. It may also mean that it might be possible to develop rice with desired levels of adhesiveness by breeding varieties with particular proportions of prolamin and glutelin.
Acknowledgements This project is funded by the Australian Research Council and Rice-growers co-operative. The authors wish to thank Tim Farrell and Rob Williams for the supply of the rice samples and Melissa Fitzgerald for the use of rice milling equipment.
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