- Email: [email protected]
Influence of amylopectin structure on rheological and retrogradation properties of waxy rice starches
Journal of Cereal Science 56 (2012) 367e373
Contents lists available at SciVerse ScienceDirect
Journal of Cereal Science journal homepage: www.elsevier.com/locate/jcs
Influence of amylopectin structure on rheological and retrogradation properties of waxy rice starches Harinder Singh a, Jheng-Hua Lin b, Wei-Hsiang Huang a, Yung-Ho Chang a, * a b
Department of Food and Nutrition, Providence University, 200 Chung-Chi Road, Shalu, Taichung 43301, Taiwan Department of Hospitality Management, MingDao University, 369 Wen-Hwa Road, Peetow, Chunghua 52345, Taiwan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 14 December 2011 Received in revised form 9 April 2012 Accepted 14 April 2012
A set of 13 waxy rice genotypes prepared by chemical-induced mutation of rice variety TNG67 and 7 waxy rice varieties widely grown in Taiwan were compared for structural, rheological and retrogradation characteristics of starches. Wide differences in retrogradation enthalpy (DHret), gel firmness and storage modulus (G’ret) were observed for 2-week stored gels of 20 starches. Ratio of short-to-long amylopectin chains was significantly higher (p < 0.05) in starches from mutant genotypes than in commercial varieties. DHret and G’ret of starch pastes stored over 4 weeks showed stronger correlation with amylopectin chain-profile compared to those stored for 2 weeks. Amount of long amylopectin chains was correlated positively (p < 0.05) with DHret and gel firmness. Overall, ratio of short-to-long amylopectin chains affected almost all the rheological and retrogradation parameters. Results of this study can be useful to plant breeders and food industry for quality improvement and selection of waxy rice mutants for various applications. Ó 2012 Elsevier Ltd. All rights reserved.
Keywords: Waxy rice starch Rheological properties Retrogradation Molecular structure
1. Introduction Starch is the major constituent of the polished rice, and rice eating and cooking qualities are mainly influenced by the starch properties (Bao et al., 2004). Many traditional foods are made from rice in oriental countries, which include rice puffs, snacks, cakes and noodles (Wang and Wang, 2002). Amylose contents of 0e2, 5e12, 12e20, 20e25 and 25e33%, in rice, have been classified, respectively, as waxy, very low, low, intermediate and high amylose (Juliano, 1992). The difference in amylose content in rice varieties has been described by a single-nucleotide polymorphism in an allele of the waxy gene encoding the granule bound starch synthase enzyme (Ayres et al., 1997). The textural properties of rice flours are mainly contributed by starch due to its gelatinization, retrogradation and rheological behavior (Lin et al., 2011; Tan and Corke, 2002). Gelatinization of starches involve granule swelling, pasting, loss of birefringence and unraveling and melting of double helices. The branches of the amylopectin molecule have been reported to form double helices that are arranged in crystalline domains (Sarko and Wu, 1978). Amylopectin fraction in starches has been reported to be responsible for the long-term changes that occur in stored starch gels
* Corresponding author. Tel.: þ886 4 2632 8001x15302; fax: þ886 4 2653 0027. E-mail address: [email protected] (Y.-H. Chang). 0733-5210/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.jcs.2012.04.007
(Eliasson, 1985). Retrogradation involves re-association of amylopectin chains during storage of starch, and the value of enthalpy change during gelatinization of retrograded starch provides an estimate of the melting energy of recrystallized amylopectin (Karim et al., 2000). Understanding of the retrogradation of starches is very important for the potential application of any cultivar or its mutant genotype. Retrogradation of starch has been shown to decrease with increase in amylose contents for waxy and non-waxy starches (Sasaki et al., 2000). Vandeputte et al. (2003) investigated retrograding behavior of waxy and non-waxy rice starches and concluded that: (1) the short rice amylopectin chains (with degree of polymerization (DP) 6e9) do not form double helices to a great extent; and (2) amylose lowers the recrystallization tendency and perfection of amylopectin. Wang and Wang (2002) attributed the low retrogradation tendency in waxy rice starches to a larger amount of A chains and a shorter exterior chain length of amylopectin. Bao et al. (2004) in their study on gel textural traits of rice starches reasoned that gel firmness was influenced by amylose content and since amylose content is controlled by the Wx gene, gel firmness was mapped at the Wx locus. Waxy rice starches can be a good model for studying the influence of amylopectin fine structure on rheological and retrogradation characteristics of starches. There have been constant efforts for a long time to improve the quality of rice cultivars for the food industry using mutation. In the present study, rice variety
368
H. Singh et al. / Journal of Cereal Science 56 (2012) 367e373
TNG67 widely grown in Taiwan was mutated using the chemical sodium azide (NaN3) for producing 13 new waxy mutants, and the aim of the present study was to study the viability of the mutants for food industry utilization. Broadly speaking, the aim of the present work was to assess the genetic difference between 13 waxy rice mutants and 7 commercial waxy varieties through measurement of functional properties of their starches. 2. Materials and methods 2.1. Materials Seven waxy rice genotypes namely BW, ESW, TCSW1, TCW70, TKW1, TKW5 and TSW2 were widely planted varieties in Taiwan. The other 13 waxy rice accessions, WR1eWR13, were NaN3induced mutants of the variety named TNG67. All the 20 waxy rice genotypes were obtained from Agricultural Research Institute of Taiwan, Wufong, Taichung, Taiwan. The 7 named genotypes were planted in different areas of Taiwan, and the 13 mutants were grown and harvested at Wufong under the same planting conditions. 2.2. Methods 2.2.1. Isolation of starch The starch was isolated according to the alkaline-steeping method reported by Lin and Chang (2006) with some modifications. Rice grains (2 kg, dry basis) were steeped overnight in 5 L of NaOH solution (0.1%). The supernatant was decanted, and the kernels were washed with fresh NaOH solution (0.1%). After washing, the grains were milled with 10 L of 0.1% NaOH solution by a stone Wet-Mill (CL-010, Ladyship, Taipei, Taiwan). The slurry was diluted to 35 L, and poured into a glass container (30 cm in diameter and 60 cm in length). After standing for 30 min, the slurry separated into three layers. The top and bottom layers exhibited yellow color, which were impurity layers. The middle layer was a starch layer, which was siphoned out. The impurity layers were collected and the process of diluting, standing, and siphoning was repeated till the starch layer was clear. The starch layers were collected and centrifuged at 10,000g in a continuous phase centrifuge (T1A, Sharples, Warminster, PA, USA). The precipitate was suspended in distilled water and neutralized with 0.1% HCl. Then, the slurry was washed many times and centrifuged by distilled water until the absence of NaCl from the supernatant was detected using 1% AgNO3. The precipitate was again suspended in 95% ethanol and air-oven dried at 40 C, and the starch that passed through a 100-mesh sieve was stored. The isolated starch of all the genotypes showed negligible iodine affinity, indicating that the amylose content of starch was very low (<1.0%). 2.2.2. Chain-length distribution The chain-length distribution of starch after debranching by isoamylase was determined by high-performance size-exclusion chromatography (HPSEC). The starch solution was prepared by dissolving 75 mg (db) of starch with 15 mL, 90% dimethyl sulfoxide (DMSO) solution in a boiling water bath for 1 h with constant stirring. A continuous stirring for 24 h at room temperature was then followed. Starch was precipitated from an aliquot of DMSO solution (5 mL) by using excess absolute ethyl alcohol and centrifugation at 4000g for 10 min. The precipitated amorphous starch pellet was dissolved in deionized water (2.45 mL, 95 C) and stirred with a magnetic stirrer in a boiling water bath for 30 min. Starch solution prepared as above was cooled to room temperature, acetate buffer (0.05 mL, 1.0 M, pH 3.5) and isoamylase solutions
(10 mL, 5.9 U/ml) were added, and the mixture incubated in a shaker bath at 45 C for 24 h. The solution was neutralized with 0.1 M NaOH, and deionized with Amberlite IR-120-P and Amberlite IR-93 (Sigma, USA) ion exchanger. The solution was diluted to 5 mL, and heated in a boiling water bath for 10 min. Debranched starch solutions were then filtered using a 0.45-mm syringe filter. The filtrate was injected (100 mL) into the HPSEC system. The columns used were one G3000PWXL and two G2500PWXL (TSK-Gel, Tosoh, Japan) connected in series. A typical HPSEC profile of debranched starch showed bimodal distribution. The average molecular weight of the peaks (long-chain and short-chain of amylopectin) were calculated from the refractive index (RI) signal using a calibration curve constructed from a series of pullulan molecular weight standards ranging from 1.0 kDa to 46.0 kDa (Polymer Standards Service, USA). The ratio of percentage of short-to-long chains (S/L) was also calculated. 2.2.3. Rheological properties Starch suspension (25%, w/w) was prepared and analyzed on a dynamic rheometer (Carri-Med-CSL 100, TA Instruments Ltd., Surrey, England) with a 4-cm diameter parallel disc. The gap was set at 1 mm and a frequency of 1 Hz and deformation of 1% was used. The starch suspension was pre-heated at 45 C for 10 min, and an appropriate amount of starch suspension was transferred to a rheometer that was kept at 45 C. Drops of mineral oil were applied around the probe to prevent water evaporation. The temperature profile of the rheometer was set at 45 C, followed by heating at a rate of 1 C/min from 45 C to 90 C, holding at 90 C for 5 min, and cooling at a rate of 2 C/min from 90 C to 5 C. The parameters collected were TG’, G’max, tan dG’max , TG0max , G090 C , G05 C , tan d5 C , and G’BDr, which stand for temperature at the initial increase of G’, maximum value of G’ during heating, tan d at G’max, temperature at G’max, G’ at temperature rising to 90 C, G’ at temperature cooling to 5 C, tan d at 5 C, and breakdown ratio of G’ (G’BDr ¼ (G’max G090 C )/G’max 100%) during heating, respectively (Fig. 1A). 2.2.4. Retrogradation Starch was weighed in aluminum sample pans, mixed with distilled water (two times dry starch weight), and sealed. For hydration of starch the pans were stood at room temperature for 1 h, and heated from 30 C to 140 C at a rate of 10 C/min in a differential scanning calorimeter (DSC, model 2910, TA Instruments, Surrey, England). After gelatinization, the pans were cooled and stored at 5 C for 2 and 4 weeks. After storage, the pans were equilibrated for 1 h before rescanning in DSC from 10 C to 100 C at a rate of 10 C/min. The retrogradation enthalpy (DHret) was quantified (Fig. 1B). Retrogradation of starch was also followed using the dynamic rheometer. The starch suspension (25%, w/w) was fully gelatinized in boiling water with continuous shaking for 10 min. The gelatinized starch was transferred on to two sheets of glass (1 mm in thickness) and wrapped with plastic film to prevent water evaporation. The starch gel was cooled at 5 C and stored for 2 and 4 weeks. Before analysis, the gel was stood at room temperature for 1 h and cut to 4 cm in diameter. The gel was placed in the rheometer with preset temperature of 25 C. A parallel disc of 4-cm diameter was used and the gap was set at 1 mm. The frequency sweep experiments were performed from 0.1 Hz to 10 Hz at 1% deformation. G’ret (storage modulus where the subscript, ret, stands for retrograded gel) value was read at a frequency of 1 Hz (Fig. 1C). 2.2.5. Firmness The firmness of starch gels was measured by using a Texture Analyzer (TA XT2, TA Instruments, UK). Starch (5 g, d.b.) was
H. Singh et al. / Journal of Cereal Science 56 (2012) 367e373
369
Fig. 1. Typical profiles of dynamic rheology during (A) heating and cooling, (B) DSC retrogradation, (C) frequency sweep, and (D) textural analysis of waxy rice starch.
weighed into a glass bottle (internal diameter 30 mm) and adjusted by addition of water until the suspension concentration was 25% (w/w). The bottle cap was sealed and heated in a boiling water bath with continuous shaking for 10 min, and stored at 5 C for 2 and 4 weeks. Measurements were done at room temperature after standing the gels for half an hour. The starch gel was penetrated (two penetration cycles) at a speed of 1 mm/s using a standard probe having a diameter of 10 mm (P/10). The penetration strain was 50% of the gel height. The firmness was taken as peak height of curve of the first penetration cycle (Fig. 1D). 2.2.6. Statistical analysis Three replications of each parameter were done and the mean was reported. Analysis of variance, least significant difference and Pearson correlation were applied on the determined parameters using SAS software (SAS Institute, Cary, NC) to compare and to determine the interrelationships between various parameters. 3. Results and discussion 3.1. Chain-length distribution The HPSEC profile of debranched waxy starches showed two fractions. The first fraction (f1) of the HPSEC profile corresponds to long chains of amylopectin (B2 and longer chains) and the second fraction (f2) corresponds to short chains of amylopectin (A and B1 chains). The average chain lengths of amylopectin (CLw) of the 20 waxy rice starches ranged between 15.8 (WR11) and 18.7 (BW) (Table 1). Jane et al. (1999) reported that the CLw of cereal amylopectin ranged between 18.8 (waxy rice) and 23.5 (amylomaize VII). Length of long amylopectin chains (f1) varied between 51.3 (WR11)
and 58.5 (WR5), respectively, whereas short amylopectin chain length (f2) ranged between 13.5 (WR11) and 15.6 (BW, TKW1). On an average, the proportion of long and short chains of amylopectin by weight was 20.7 and 79.3% for the 20 waxy rice starches, respectively. Previous reports also confirm that waxy cereal starches consisted of about 70e80% of chains with DP 6e25 (Benmoussa et al., 2007; Jane et al., 1999; Shi and Seib, 1992). The average ratio of short-to-long chains (S/L) was found to be 3.9 for the 20 waxy rice starches. The range of S/L ratio for starches from the 13 mutants was 3.43 (WR6) to 4.95 (WR9) and for starches of the 7 named varieties was from 3.23 (TCW70) to 3.97 (TKW5), respectively. Hence it can be said that mutant starches have more short chains in proportion to long chains than starches of the named varieties (p < 0.05). 3.2. Rheological properties Rheological behavior of waxy rice starch gels is summarized in Table 2. Storage modulus (G’) increased first to a maximum value corresponding to completion of starch gelatinization and water absorption. Starches exhibited G’max in the range between 41 and 815 Pa. Mutant genotype WR6 recorded the lowest G’max at a temperature of 71.2 C and mutant genotype WR9 exhibited the highest G’max at a temperature of 62.0 C. Difference between temperatures at which G’ started increasing (TG’) and temperatures at which G’ reached a maximum ðTG0max Þ was within 4 C. Gelatinization of starches, including loss of birefringence, is usually complete within a temperature interval of 10 C for each type of starch (Salvador et al., 2006). The increase in G’ during the heating phase also corresponds to starch granules getting closely packed due to swelling (Eliasson, 1985).
370
H. Singh et al. / Journal of Cereal Science 56 (2012) 367e373
Table 1 Weight percentage and weight-average chain length (CLw) of fractions of isoamylase-debranched waxy rice starches from different genotypes. S/L ratioa
Genotype
Average CLw
f1 %
CLw
%
CLw
BW ESW TCSW1 TCW70 TKW1 TKW5 TSW2 WR1 WR2 WR3 WR4 WR5 WR6 WR7 WR8 WR9 WR10 WR11 WR12 WR13 LSDb
18.7 18.4 17.7 18.6 18.4 18.0 18.2 17.1 18.0 17.6 17.6 17.8 17.3 17.1 16.9 16.4 16.5 15.8 16.6 16.6 0.2
23.0 21.6 20.5 23.7 20.9 20.1 20.6 19.4 21.0 20.8 20.3 21.6 22.6 20.1 19.5 16.8 20.0 20.1 20.5 21.2 0.5
58.4 57.7 56.2 57.2 58.2 57.6 57.7 55.8 57.8 58.3 57.5 58.5 55.6 55.9 55.3 57.1 53.5 51.3 54.6 53.0 0.4
77.0 78.4 79.5 76.3 79.1 79.9 79.4 80.6 79.0 79.2 79.7 78.4 77.4 79.9 80.5 83.2 80.0 79.9 79.5 78.8 0.5
15.6 15.5 15.1 15.4 15.6 15.3 15.5 14.7 15.2 14.9 15.0 15.0 14.4 14.5 14.4 14.4 14.1 13.5 14.1 14.1 0.1
a b
f2
3.34 3.64 3.88 3.23 3.78 3.97 3.85 4.17 3.76 3.82 3.92 3.62 3.43 3.99 4.12 4.95 4.00 3.97 3.87 3.72 0.13
Ratio of f2% to f1%. Least significant difference, n ¼ 3.
Different G’ exhibited by waxy rice starches may be attributed to the difference in the breakdown of the amylopectin matrix inside the swollen starch granules (Keetels and van Vliet, 1994). G0 depends upon factors like granule rigidity and swollen granule phase volume. Lii et al. (1996) postulated that rheological behavior of gelatinized starch suspension could be influenced by inter-granular interactions, such as entanglement between surface molecules of adjacent granules and the granular properties. The loss factor, tan d represents the ratio of energy lost to energy stored during a cycle (Ferry, 1970). During prolonged heating, starch suspensions show a rise in G’ and reach a maximum and then decrease which may depend upon the swelling factor and breakdown resistance (Singh et al., 2003). Starch gels showed G’ and G’’ values at 90 C in the range from 17 Pa to 25 Pa and 10e12 Pa, respectively. Lii et al. (1995) reported a G’ value at 95 C of 39 Pa for rice starch (20%, w/w) with amylose content of 0.99%. The decrease in G’ upon heating may be attributed to the deformation/loss of granular structure due to melting of remaining crystallites in swollen granules (Eliasson, 1986). Keetels et al. (1996b) hypothesized and attributed the decrease in G’ on prolonged heating to loosen non-covalent bonds by disentanglement. (%) ¼ [(G’max G090 C )/ The breakdown ratio (G’BDr 0 G90 C 100%]), an indirect measure of the thermal stability and degree of disintegration of starch granules varied widely between 50.7% (WR6) and 97.4% (WR9). Keetels et al. (1996b) postulated that breakdown of the amylopectin matrix inside the swollen starch granules and disentanglement of branches during prolonged heating would cause the granules to become less rigid. G’BDr showed significant negative correlations with TG’ (r ¼ 0.665) and TG0max (r ¼ 0.810). Thus, it can be confirmed that breakdown ratio is inversely proportional to swelling extent of starch granules. The cooling phase, which can be related to the short-time retrogradation of starches, saw a rise in G’ and G’’. The increase in G’ and G’’ during cooling from 90 C to 5 C may be due to interactions and associations between amylopectin chains and partly due to chain stiffness and stiffness of remaining intact starch granules. Jane et al. (1999) reported that long amylopectin chains (DP > 50) accelerated retrogradation. A positive correlation between percent
proportion of amylopectin B chains (DP > 40) and difference in values of G’ during cooling from 95 C to 4 C was observed by Chung et al. (2008). However, no significant correlation between G’ and amylopectin chain-profile was found, yet both long and short chains may have been re-associated during this short time. It is also noteworthy that the G’ during heating was observed to be much greater than during cooling. The tan d of starches during cooling decreased to less than 1 ðtan d5 C Þ, indicating the formation of molecular interaction network of starch. 3.3. Retrogradation Miles et al. (1985) indicated that the short-term development of gel structure and crystallinity in starch gels was found to be dominated by gelation and crystallization within the amylose. While long-term increase in the modulus of starch gels was linked to crystallization of amylopectin. Storage time of 2 and 4 weeks was selected for observing retrogradation of starch, since all samples exhibited very low amylose content. Values of DHret of starch gels after storage for 2 and 4 weeks are summarized in Table 3. DHret for all starches increased with increasing storage duration. DHret for the 20 waxy starches varied from 0.9 J/g (WR12) to 6.4 J/g (BW) and 5.1 J/g (TKW5) to 10.1 J/g (BW) after 2 and 4 weeks of storage, respectively. Vandeputte et al. (2003) showed a range of DHret (amylopectin basis) values from 1.8 to 4.7 J/g for five waxy rice starch gels (with 1.9e3.4% amylose content) stored at 6 C for 4 weeks. Sasaki and Matsuki (1998) indicated that longer chains of amylopectin formed more stable crystallites and more regions of crystallinity during storage. Percentage retrogradation (%R) was observed to vary from 5.8% (WR12) to 41.6% (BW) for the first 2 weeks of storage and subsequently from 33.3% (TKW5) to 65.6% (BW) for the next 2 weeks. Rice amylopectins have been reported to display two stages of retrogradation behavior during early (<7 days) and late storage (>7 days) (Lai et al., 2000). The former was not influenced by
Table 2 Rheological propertiesa during heating and cooling of waxy rice starches from different genotypes. Genotype TG’ ( C)
G’max (Pa)
tan dG’max TG0max ( C)
G090 C (Pa)
G05 C (Pa)
tan d5 C G’BDr (%)
BW ESW TCSW1 TCW70 TKW1 TKW5 TSW2 WR1 WR2 WR3 WR4 WR5 WR6 WR7 WR8 WR9 WR10 WR11 WR12 WR13 LSDb
327 321 148 163 180 451 166 296 235 60 114 376 41 280 259 815 260 164 337 311 28
0.263 0.264 0.259 0.278 0.256 0.227 0.290 0.240 0.247 0.322 0.245 0.240 0.425 0.259 0.233 0.196 0.227 0.261 0.223 0.243 0.009
25 22 21 20 18 19 22 18 19 20 21 19 20 17 21 21 19 19 19 17 3
42 35 35 38 33 32 39 34 36 37 36 36 38 34 37 37 35 37 34 35 5
0.788 0.843 0.866 0.879 0.909 0.865 0.845 0.919 0.863 0.851 0.800 0.870 0.868 0.949 0.844 0.856 0.880 0.904 0.903 0.929 0.017
64.8 63.6 64.0 64.8 60.5 60.0 64.3 59.5 62.2 64.3 61.1 62.4 67.3 62.1 61.8 60.1 60.9 60.7 59.5 61.8 0.6
66.7 65.4 65.9 66.7 62.6 62.4 66.5 62.1 64.2 67.9 64.3 64.1 71.2 64.0 63.9 62.0 63.0 62.4 61.9 64.1 0.9
92.2 93.3 87.1 87.6 90.1 95.5 86.8 93.8 91.8 66.0 81.5 95.1 50.7 93.9 92.2 97.4 92.6 88.7 94.4 94.6 2.2
a TG’: temperature at the initial increasing of G’; G’max: maximum value of G’ during heating; tan dG’max : tan d at G’max; TG0max : temperature at G’max; G090 C : G’ at temperature rising to 90 C; G05 C : G’ at temperature cooling to 5 C; tan d5 C : tan d at 5 C; G’BDr: breakdown ratio of G’ (G’BDr ¼ (G’max G090 C )/G’max 100%) during heating. b Least significant difference, n ¼ 3.
H. Singh et al. / Journal of Cereal Science 56 (2012) 367e373
amylopectin chain-length distribution whereas, for late stage retrogradation, amylopectin retrogradation enthalpies showed positive correlation with the population of DP 15. The starch retrogradation has been postulated to be controlled by the nonequilibrium recrystallization behavior of amylopectin (Slade and Levine, 1986). The early stages in starch retrogradation have been reported to be dominated by the chain folded lamellar microcrystalline junction zones of amylose (DP w 15e50) and the later stages by amylopectin branched chains (DP > 12e16) (Shi and Seib, 1992; Slade and Levine, 1986). Sasaki and Matsuki (1998) indicated that longer chains of amylopectin formed more stable crystallites and more regions of crystallinity during storage. Thus, the difference between retrogradation values of 2 weeks and 4 weeks could have been influenced by amylopectin branched chains, and the effect of amylopectin short chains on recrystallization perfection during storage (Vandeputte et al., 2003). Table 3 also summarizes values of G’ret of starch gels measured at a frequency of 1 Hz after storage for 2 and 4 weeks. Due to storage of gels, G’ret can be related to retrogradation of starch gels as chains re-associate during the period. G’ret measured after 2 weeks of storage duration ranged between 111 Pa (TKW5, WR1) and 2080 Pa (BW). G’ret increased drastically for all starches after storage for 4 weeks and ranged from 2957 Pa (TKW5) to 37,600 Pa (BW). This indicates increased tendency of amylopectin chains to re-associate. Nonetheless, G’ret would also depend upon rigidity of starch granules. Keetels et al. (1996a) have given two important postulations about the process of recrystallization of amylopectin in concentrated starch gels: (1) formation of crystalline clusters along the glucan chains in the amylopectin molecule, resulting in stiffening of strands between entanglements, and (2) formation of cross-links between adjacent clusters. This observation of increase in G’ret is consistent with the observations of increase in DHret upon storage in the present study. Significant positive correlations between G’ret and DHret were also observed (r ¼ 0.596). 3.4. Firmness Firmness of waxy rice starch gels is presented in Table 4. Gels were observed to become harder as storage duration increased. These results are consistent with observations on increase in Table 3 Retrogradation enthalpy (ΔHret) and storage modulus (G’ret) of starch gels from different genotypes of waxy rice after storage at 5 C for 2 and 4 weeks. Genotype
BW ESW TCSW1 TCW70 TKW1 TKW5 TSW2 WR1 WR2 WR3 WR4 WR5 WR6 WR7 WR8 WR9 WR10 WR11 WR12 WR13 LSDa a
ΔHret (J/g)
G’ret (Pa)
371
retrogradation of starch gels in the present study. Firmness of 2and 4-week stored gels ranged from 1.44 N (WR12) to 9.20 N (BW) and 6.84 N (TKW5) to 27.36 N (ESW), respectively. The average values of firmness for 2- and 4-week stored gels were higher for starch from the named varieties than from the mutant genotypes. The mechanical properties of starch gels are affected by the volume fraction and rigidity of gelatinized starch granules, as well as the interactions between dispersed and continuous phases of the gel, rheological properties of the continuous phase (includes solubilized starch in water), and deformability of particles (Biliaderis, 1998). These factors, in turn, have been observed to show dependence on amylopectin structure (Yamin et al., 1999). 3.5. Correlation analysis Table 5 shows the relationship between various parameters analyzed according to Pearson correlation. Significantly (p < 0.05) positive correlations of TG’ with average CLw (r ¼ 0.481) and percentage proportion of long amylopectin chains (f1%) (r ¼ 0.609) were observed. A negative relationship between percentage of short amylopectin chains (f2%) (r ¼ 0.689) and S/L ratio (r ¼ 0.649) with TG’ was also observed. Thus, longer amylopectin chains can be said to result in waxy starch granules with higher gelatinization temperature. Positive correlations of G’max with f2% (r ¼ 0.566) and S/L ratio (r ¼ 0.625) were observed. Keetels et al. (1996a) postulated that swollen granule rigidity was partly contributed by double helices made by short chains. The negative relationship between tan d and average chain length (average CLw) (r ¼ 0.449) and long amylopectin chain length (f1CLw) (r ¼ 0.521) showed that long chains can be attributed to the formation of the gel network. In the present study, DHret for samples after storage for either 2 (DHret-2wk) or 4 (DHret-4wk) weeks showed positive correlations with f1% (r ¼ 0.600 for DHret-2wk and 0.662 for DHret-4wk, respectively) and negative dependence on f2% (r ¼ 0.488 for DHret-2wk and 0.605 for DHret-4wk, respectively). S/L ratio was also observed to have negative correlations with DHret-2wk (r ¼ 0.461) and DHret-4wk (r ¼ 0.569). These results of interrelationships show that long amylopectin chains can associate faster during storage, thus accelerating retrogradation. DHret-4wk was observed Table 4 Firmness (N) of starch gels from different genotypes of waxy rice after storage at 5 C for 2 and 4 weeks.
2 weeks
4 weeks
2 weeks
4 weeks
Genotype
2 weeks
4 weeks
6.4 5.3 2.5 2.5 1.6 1.4 4.1 2.6 1.4 2.8 2.0 2.9 3.2 1.5 1.6 1.8 2.2 1.8 0.9 5.1 0.2
10.1 9.3 7.9 8.7 6.1 5.1 9.3 6.8 6.1 7.6 6.1 6.8 8.1 6.0 7.3 5.8 7.3 6.1 6.2 7.2 0.4
2080 135 316 391 125 111 1639 111 846 978 338 610 614 530 500 250 248 391 123 594 68
37,600 26,350 21,310 19,750 8653 2957 17,930 8088 14,410 21,920 11,100 13,990 10,720 9062 9675 8909 8184 5262 4858 18,120 1693
BW ESW TCSW1 TCW70 TKW1 TKW5 TSW2 WR1 WR2 WR3 WR4 WR5 WR6 WR7 WR8 WR9 WR10 WR11 WR12 WR13 LSDa
9.20 3.27 6.00 5.45 2.73 2.10 8.24 3.59 6.53 4.97 3.81 4.33 6.28 4.18 5.33 3.82 3.63 4.50 1.44 5.88 1.08
18.64 27.36 24.94 23.42 8.33 6.84 15.01 10.04 14.53 15.94 11.62 11.41 15.69 10.61 12.44 9.65 9.31 10.99 9.98 16.80 2.28
Least significant difference, n ¼ 3.
a
Least significant difference, n ¼ 3.
372
H. Singh et al. / Journal of Cereal Science 56 (2012) 367e373
Table 5 Pearson correlations between molecular structure parameters and retrogradation and rheological behaviors of waxy rice starches. Parametersa
Average CLw
f1%
f1CLw
f2%
f2CLw
S/L ratio
TG’ G’max tan dG’max TG0max G090 C G05 C G’BDr tan d5 C DHret-2wk DHret-4wk G’ret-2wk G’ret-4wk Fret-2wk Fret-4wk
0.481* ec e e e e e 0.449* e 0.494* e 0.596* e 0.488*
0.609* e e 0.527* e e e e 0.600* 0.662* e 0.618* e 0.757**
e e e e e e e 0.521* e e e e e e
0.689** 0.566* 0.550* 0.631* e e e e 0.488* 0.605* e 0.562* 0.472* 0.600*
e e e e
0.649* 0.625* 0.537* 0.598* e e e e 0.461* 0.569* e 0.524* e 0.557*
0.447* e e 0.470* e e e 0.542* e e
*Significant at p < 0.05; **significant at p < 0.001. a DHret-2wk: retrogradation enthalpy of starch gel after 2 weeks of storage; DHret4wk: retrogradation enthalpy of starch gel after 4 weeks of storage; G’ret-2wk: G’ of starch gel after 2 weeks of storage; G’ret-4wk: G’ of starch gel after 4 weeks of storage; Fret-2wk: firmness of starch gel after 2 weeks of storage; F4w: Firmness of starch gel after 4 weeks of storage. Rest symbols have the same meaning as mentioned in text and other tables.
to have a positive relationship with average CLw (r ¼ 0.494). However, the relationship of average CLw with DHret-2wk was not observed. Vandeputte et al. (2003) did not find any significant relationship between amylopectin chain-profile, amylose and retrogradation enthalpies for waxy rice starches. These differences in the interrelationships between the present study and Vandeputte et al. (2003) may be due to different numbers of genotypes and starch concentrations used for retrogradation analysis. Results of Qi et al. (2003) on starches from six waxy rice genotypes (amylose content 0.2e1.5%), however, suggest that recrystallization tendency of starches during storage depends upon the proportion of short amylopectin chains and attributed the stability of crystalline structure formed during retrogradation in vitro to integrity of amylopectin molecules. This difference in results with the present study may be due to difference in amylose content and ratio of weight percentages of short-to-long amylopectin chains. The dependence of G’ret of starch gels after storage for 4 weeks on structural parameters was also observed in the present study. The pattern of interrelationship of G’ret of 4-week stored gel (G’ret4wk) with amylopectin chain-profile was the same as that for DHret. It is also noteworthy that G’ret of 2-week stored gels (G’ret–2wk), however, did not record any significant relationships with amylopectin chain-profiles, whereas DHret-2wk showed some significant relationships (Table 5). The different sensitivity in the analysis methods of retrogradation of gels by rheometer and DSC may be attributed to the difference in interrelationships observed. The interrelationships observed in the case of G’ret-4wk with average CLw (r ¼ 0.596) and f1% (r ¼ 0.618) may be attributed to the formation of entanglements and re-association between amylopectin chains, which impart stiffness. The gel firmness of 4-week stored gel (F4wk) showed a positive relationship with average CLw (r ¼ 0.488) and f1% (r ¼ 0.757). A negative correlation (r ¼ 0.557) between F4wk and S/L ratio was also observed. Gel firmness of 2-week stored gel (F2wk) also showed a negative relationship with f2% (r ¼ 0.472) and a positive relationship with f1% (r ¼ 0.267). These observations on interrelationship are in line with the relationship of DHret with other parameters although the preparation procedures of gels were different for DSC and texture analyzer. Significant (p < 0.05) positive interrelationships between F2wk (r ¼ 0.479) and F4wk (r ¼ 0.682) were found
with G’ret-4wk. F4wk showed positive relationship with DHret-2wk (r ¼ 0.468, p < 0.05) and DHret-4wk (r ¼ 0.574; p < 0.001), which was not, however, found for F2wk. F2wk and F4wk were also interrelated (r ¼ 0.479, p < 0.05), which is also a significant result. Thus it can be safely concluded that reordering duration (storage time) of amylopectin chains had a greater impact on F4wk than F2wk. Vandeputte et al. (2003) reported no difference in firmness of 2-day and 2-week stored waxy rice starch gels (1.9e3.4% absolute amylose content). As mentioned earlier, these authors observed retrogradation for waxy rice starch gels after 4 weeks when stored at 6 C. These authors also have shown an increase in firmness of normal rice starch (amylose content from 13.6 to 28.2%) gels with storage. Interestingly, these authors did not find any relationship of amylopectin unit length distribution with starch textural attributes for normal or waxy rice starches. Wang and Wang (2002) observed an increase in firmness of waxy rice starch gels (10%, w/w) from four cultivars over storage duration of 1 day and a slight increase thereafter for 7-day stored gel. The authors reported a positive correlation between amounts of amylopectin A and B1 chains and gel firmness, which is opposite to our present study. The difference in relationships observed from the above two studies mentioned may be due to a difference in proportions of short amylopectin chains, amylose content (negligible in present study) and concentration of starch gels and number of genotypes used. Thus, reassociation of long chains during retrogradation or storage contributes to rigidity of gels as observed from rheology and gel textural results. Mutation may change the distribution of amylopectin chains and, consequently, affects the formation of cross-links and double helices in retrograded starch gels. The amount of long amylopectin chains was an important determinant in accelerating the retrogradation of starch. 4. Conclusions Starches from mutant waxy genotypes showed widely different retrogradation, rheological and textural attributes than starches from named waxy rice varieties. Long amylopectin chains showed an accelerating effect on retrogradation of waxy rice starch gels. Reassociation of long chains during retrogradation or storage contributes to rigidity of gels as observed from rheology results. Mutation was observed to change the percentage of short and long chains, hence may have also affected the ability to form cross-links and double helices in retrograded starch gels. Mutant genotypes WR2, WR7 and WR9 showed lower retrogradation and higher gel firmness than some named commercial varieties (such as TKW1 and TKW5) and could be useful on rice processing. Acknowledgment We thank the Council of Agriculture (COA), Taiwan, for financial support. The supply of polished rice by Agricultural Research Institute of Taiwan is also highly acknowledged. References Ayres, N.M., McClung, A.M., Larkin, P.D., Bligh, H.F.J., Jones, C.A., Park, W.D., 1997. Micro satellites and a single-nucleotide polymorphism differentiate apparent amylose classes in an extended pedigree of US rice germ plasma. Theoretical and Applied Genetics 94, 773e781. Bao, J., Sun, M., Zhu, L., Corke, H., 2004. Analysis of quantitative trait loci for some starch properties of rice (Oryza sativa L.): thermal properties, gel texture and swelling volume. Journal of Cereal Science 39, 379e385. Benmoussa, M., Moldenhauer, K.A.K., Hamaker, B.R., 2007. Rice amylopectin fine structure variability affects starch digestion properties. Journal of Agriculture and Food Chemistry 55, 1475e1479. Biliaderis, C.G., 1998. Structures and phase transitions of starch polymers. In: Walter, R.H. (Ed.), Polysaccharide Association Structures in Foods. Marcel Dekker, New York, pp. 57e168.
H. Singh et al. / Journal of Cereal Science 56 (2012) 367e373 Chung, J.H., Han, J.-A., Yoo, B., Seib, P.A., Lim, S.-T., 2008. Effects of molecular size and chain profile of waxy cereal amylopectins on paste rheology during retrogradation. Carbohydrate Polymers 71, 365e371. Eliasson, A.C., 1985. Retrogradation of starch as measured by differential scanning calorimetry. In: Hill, R.D., Munck, I. (Eds.), New Approaches to Research on Cereal Carbohydrate. Elsevier, Amsterdam, pp. 93e98. Eliasson, A.C., 1986. Viscoelastic behaviour during the gelatinization of starch. II. Effects of emulsifiers. Journal of Texture Studies 17, 357e375. Ferry, J.D., 1970. Illustrations of viscoelastic behaviour of polymeric systems. In: Ferry, J.D. (Ed.), Viscoelastic Properties of Polymers. J. Wiley & Sons, New York, pp. 34e58. Jane, J., Chen, Y.Y., Lee, L.F., McPherson, A.E., Wong, K.S., Radosavljevic, M., 1999. Effects of amylopectin branch chain length and amylose content on the gelatinization and pasting properties of starch. Cereal Chemistry 76, 629e637. Juliano, B.O., 1992. Structure, chemistry, and function of the rice grain and its fractions. Cereal Foods World 37, 772e774. Karim, A.A., Norziah, M.H., Seow, C.C., 2000. Methods for the study of starch retrogradation. Food Chemistry 71, 9e36. Keetels, C.J.A.M., van Vliet, T., 1994. Gelation and retrogradation of concentrated starch gels. In: Phillips, G.O., Williams, P.A., Wedlock, D.J. (Eds.), Gums and Stabilizers for the Food Industry. IRL, New York, pp. 271e280. Keetels, C.J.A.M., Oostergete, G.T., van Vliet, T., 1996a. Recrystallization of amylopectin in concentrated starch gels. Carbohydrate Polymers 30, 61e64. Keetels, C.J.A.M., van Vliet, T., Walstra, P., 1996b. Gelation and retrogradation of concentrated starch systems: 1. Gelation. Food Hydrocolloids 10, 343e353. Lai, V.M.-F., Lu, S., Lii, C.-Y., 2000. Molecular characteristics influencing retrogradation kinetics of rice amylopectins. Cereal Chemistry 77, 272e278. Lii, C.Y., Shao, Y.Y., Tseng, K.H., 1995. Gelation mechanism and rheological properties of rice starch. Cereal Chemistry 72, 393e400. Lii, C.Y., Tsai, M.L., Tseng, K.H., 1996. Effect of amylose content on the rheological properties of rice starch. Cereal Chemistry 73, 415e420. Lin, J.H., Chang, Y.H., 2006. Molecular degradation rate of rice and corn starches during acid-methanol treatment and its relation to the molecular structure of starch. Journal of Agricultural and Food Chemistry 54, 5880e5886. Lin, J.H., Singh, H., Chang, Y.T., Chang, Y.H., 2011. Factor analysis of the functional properties of rice flours from mutant genotypes. Food Chemistry 126, 1108e1114.
373
Miles, M.J., Morris, V.J., Orford, P.D., Ring, S.G., 1985. The roles of amylose and amylopectin in the gelation and retrogradation of starch. Carbohydrate Research 135, 271e281. Qi, X., Tester, R.F., Snape, C.E., Ansell, R., 2003. Molecular basis of the gelatinisation and swelling characteristics of waxy rice starches grown in the same location during the same season. Journal of Cereal Science 37, 363e376. Salvador, A., Sanz, T., Fiszman, S.M., 2006. Dynamic rheological characteristics of wheat flour-water dough. Effect of adding NaCl, sucrose and yeast. Food Hydrocolloids 20, 780e786. Sarko, A., Wu, H.C.H., 1978. The crystal structures of A-, B-, and C-polymorphs of amylose and starch. Starch/Stärke 30, 73e78. Sasaki, T., Matsuki, J., 1998. Effect of wheat starch structure on swelling power. Cereal Chemistry 75, 525e529. Sasaki, T., Yasui, T., Matsuki, J., 2000. Effect of amylose content on gelatinization, retrogradation and pasting properties of starches from waxy and non-waxy wheat and their F1 seeds. Cereal Chemistry 77, 58e63. Shi, Y.-C., Seib, P.A., 1992. The structure of four waxy starches related to gelatinisation and retrogradation. Carbohydrate Research 227, 131e145. Singh, N., Singh, J., Kaur, L., Sodhi, N.S., Gill, B.S., 2003. Morphological, thermal and rheological properties of starches from different botanical sources. Food Chemistry 81, 219e231. Slade, L., Levine, H., 1986. Recent advances in starch retrogradation. In: Stivala, S.S., Crescenzi, V., Dea, I.C.M. (Eds.), Proceedings of the Conference of Recent Developments in Industrial Polysaccharides: The Impact of Biotechnology and Advanced Methodologies. The Stevens Institute of Technology Gordon and Breach Science, New York, pp. 387e430. Tan, Y., Corke, H., 2002. Factor analysis of physicochemical properties of 63 rice varieties. Journal of Science of Food and Agriculture 82, 745e752. Vandeputte, G.E., Vermeylen, R., Geeroms, J., Delcour, J.A., 2003. Rice starches. III. Structural aspects provide insight in amylopectin retrogradation properties and gel texture. Journal of Cereal Science 38, 61e68. Wang, Y.-J., Wang, L., 2002. Structures of four waxy rice starches in relation to thermal, pasting and textural properties. Cereal Chemistry 79, 252e256. Yamin, F.F., Lee, M., Pollak, L.M., White, P.J., 1999. Thermal properties of starch in corn variants isolated after chemical mutagenesis of inbred line B73. Cereal Chemistry 76, 175e181.
Contents lists available at SciVerse ScienceDirect
Journal of Cereal Science journal homepage: www.elsevier.com/locate/jcs
Influence of amylopectin structure on rheological and retrogradation properties of waxy rice starches Harinder Singh a, Jheng-Hua Lin b, Wei-Hsiang Huang a, Yung-Ho Chang a, * a b
Department of Food and Nutrition, Providence University, 200 Chung-Chi Road, Shalu, Taichung 43301, Taiwan Department of Hospitality Management, MingDao University, 369 Wen-Hwa Road, Peetow, Chunghua 52345, Taiwan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 14 December 2011 Received in revised form 9 April 2012 Accepted 14 April 2012
A set of 13 waxy rice genotypes prepared by chemical-induced mutation of rice variety TNG67 and 7 waxy rice varieties widely grown in Taiwan were compared for structural, rheological and retrogradation characteristics of starches. Wide differences in retrogradation enthalpy (DHret), gel firmness and storage modulus (G’ret) were observed for 2-week stored gels of 20 starches. Ratio of short-to-long amylopectin chains was significantly higher (p < 0.05) in starches from mutant genotypes than in commercial varieties. DHret and G’ret of starch pastes stored over 4 weeks showed stronger correlation with amylopectin chain-profile compared to those stored for 2 weeks. Amount of long amylopectin chains was correlated positively (p < 0.05) with DHret and gel firmness. Overall, ratio of short-to-long amylopectin chains affected almost all the rheological and retrogradation parameters. Results of this study can be useful to plant breeders and food industry for quality improvement and selection of waxy rice mutants for various applications. Ó 2012 Elsevier Ltd. All rights reserved.
Keywords: Waxy rice starch Rheological properties Retrogradation Molecular structure
1. Introduction Starch is the major constituent of the polished rice, and rice eating and cooking qualities are mainly influenced by the starch properties (Bao et al., 2004). Many traditional foods are made from rice in oriental countries, which include rice puffs, snacks, cakes and noodles (Wang and Wang, 2002). Amylose contents of 0e2, 5e12, 12e20, 20e25 and 25e33%, in rice, have been classified, respectively, as waxy, very low, low, intermediate and high amylose (Juliano, 1992). The difference in amylose content in rice varieties has been described by a single-nucleotide polymorphism in an allele of the waxy gene encoding the granule bound starch synthase enzyme (Ayres et al., 1997). The textural properties of rice flours are mainly contributed by starch due to its gelatinization, retrogradation and rheological behavior (Lin et al., 2011; Tan and Corke, 2002). Gelatinization of starches involve granule swelling, pasting, loss of birefringence and unraveling and melting of double helices. The branches of the amylopectin molecule have been reported to form double helices that are arranged in crystalline domains (Sarko and Wu, 1978). Amylopectin fraction in starches has been reported to be responsible for the long-term changes that occur in stored starch gels
* Corresponding author. Tel.: þ886 4 2632 8001x15302; fax: þ886 4 2653 0027. E-mail address: [email protected] (Y.-H. Chang). 0733-5210/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.jcs.2012.04.007
(Eliasson, 1985). Retrogradation involves re-association of amylopectin chains during storage of starch, and the value of enthalpy change during gelatinization of retrograded starch provides an estimate of the melting energy of recrystallized amylopectin (Karim et al., 2000). Understanding of the retrogradation of starches is very important for the potential application of any cultivar or its mutant genotype. Retrogradation of starch has been shown to decrease with increase in amylose contents for waxy and non-waxy starches (Sasaki et al., 2000). Vandeputte et al. (2003) investigated retrograding behavior of waxy and non-waxy rice starches and concluded that: (1) the short rice amylopectin chains (with degree of polymerization (DP) 6e9) do not form double helices to a great extent; and (2) amylose lowers the recrystallization tendency and perfection of amylopectin. Wang and Wang (2002) attributed the low retrogradation tendency in waxy rice starches to a larger amount of A chains and a shorter exterior chain length of amylopectin. Bao et al. (2004) in their study on gel textural traits of rice starches reasoned that gel firmness was influenced by amylose content and since amylose content is controlled by the Wx gene, gel firmness was mapped at the Wx locus. Waxy rice starches can be a good model for studying the influence of amylopectin fine structure on rheological and retrogradation characteristics of starches. There have been constant efforts for a long time to improve the quality of rice cultivars for the food industry using mutation. In the present study, rice variety
368
H. Singh et al. / Journal of Cereal Science 56 (2012) 367e373
TNG67 widely grown in Taiwan was mutated using the chemical sodium azide (NaN3) for producing 13 new waxy mutants, and the aim of the present study was to study the viability of the mutants for food industry utilization. Broadly speaking, the aim of the present work was to assess the genetic difference between 13 waxy rice mutants and 7 commercial waxy varieties through measurement of functional properties of their starches. 2. Materials and methods 2.1. Materials Seven waxy rice genotypes namely BW, ESW, TCSW1, TCW70, TKW1, TKW5 and TSW2 were widely planted varieties in Taiwan. The other 13 waxy rice accessions, WR1eWR13, were NaN3induced mutants of the variety named TNG67. All the 20 waxy rice genotypes were obtained from Agricultural Research Institute of Taiwan, Wufong, Taichung, Taiwan. The 7 named genotypes were planted in different areas of Taiwan, and the 13 mutants were grown and harvested at Wufong under the same planting conditions. 2.2. Methods 2.2.1. Isolation of starch The starch was isolated according to the alkaline-steeping method reported by Lin and Chang (2006) with some modifications. Rice grains (2 kg, dry basis) were steeped overnight in 5 L of NaOH solution (0.1%). The supernatant was decanted, and the kernels were washed with fresh NaOH solution (0.1%). After washing, the grains were milled with 10 L of 0.1% NaOH solution by a stone Wet-Mill (CL-010, Ladyship, Taipei, Taiwan). The slurry was diluted to 35 L, and poured into a glass container (30 cm in diameter and 60 cm in length). After standing for 30 min, the slurry separated into three layers. The top and bottom layers exhibited yellow color, which were impurity layers. The middle layer was a starch layer, which was siphoned out. The impurity layers were collected and the process of diluting, standing, and siphoning was repeated till the starch layer was clear. The starch layers were collected and centrifuged at 10,000g in a continuous phase centrifuge (T1A, Sharples, Warminster, PA, USA). The precipitate was suspended in distilled water and neutralized with 0.1% HCl. Then, the slurry was washed many times and centrifuged by distilled water until the absence of NaCl from the supernatant was detected using 1% AgNO3. The precipitate was again suspended in 95% ethanol and air-oven dried at 40 C, and the starch that passed through a 100-mesh sieve was stored. The isolated starch of all the genotypes showed negligible iodine affinity, indicating that the amylose content of starch was very low (<1.0%). 2.2.2. Chain-length distribution The chain-length distribution of starch after debranching by isoamylase was determined by high-performance size-exclusion chromatography (HPSEC). The starch solution was prepared by dissolving 75 mg (db) of starch with 15 mL, 90% dimethyl sulfoxide (DMSO) solution in a boiling water bath for 1 h with constant stirring. A continuous stirring for 24 h at room temperature was then followed. Starch was precipitated from an aliquot of DMSO solution (5 mL) by using excess absolute ethyl alcohol and centrifugation at 4000g for 10 min. The precipitated amorphous starch pellet was dissolved in deionized water (2.45 mL, 95 C) and stirred with a magnetic stirrer in a boiling water bath for 30 min. Starch solution prepared as above was cooled to room temperature, acetate buffer (0.05 mL, 1.0 M, pH 3.5) and isoamylase solutions
(10 mL, 5.9 U/ml) were added, and the mixture incubated in a shaker bath at 45 C for 24 h. The solution was neutralized with 0.1 M NaOH, and deionized with Amberlite IR-120-P and Amberlite IR-93 (Sigma, USA) ion exchanger. The solution was diluted to 5 mL, and heated in a boiling water bath for 10 min. Debranched starch solutions were then filtered using a 0.45-mm syringe filter. The filtrate was injected (100 mL) into the HPSEC system. The columns used were one G3000PWXL and two G2500PWXL (TSK-Gel, Tosoh, Japan) connected in series. A typical HPSEC profile of debranched starch showed bimodal distribution. The average molecular weight of the peaks (long-chain and short-chain of amylopectin) were calculated from the refractive index (RI) signal using a calibration curve constructed from a series of pullulan molecular weight standards ranging from 1.0 kDa to 46.0 kDa (Polymer Standards Service, USA). The ratio of percentage of short-to-long chains (S/L) was also calculated. 2.2.3. Rheological properties Starch suspension (25%, w/w) was prepared and analyzed on a dynamic rheometer (Carri-Med-CSL 100, TA Instruments Ltd., Surrey, England) with a 4-cm diameter parallel disc. The gap was set at 1 mm and a frequency of 1 Hz and deformation of 1% was used. The starch suspension was pre-heated at 45 C for 10 min, and an appropriate amount of starch suspension was transferred to a rheometer that was kept at 45 C. Drops of mineral oil were applied around the probe to prevent water evaporation. The temperature profile of the rheometer was set at 45 C, followed by heating at a rate of 1 C/min from 45 C to 90 C, holding at 90 C for 5 min, and cooling at a rate of 2 C/min from 90 C to 5 C. The parameters collected were TG’, G’max, tan dG’max , TG0max , G090 C , G05 C , tan d5 C , and G’BDr, which stand for temperature at the initial increase of G’, maximum value of G’ during heating, tan d at G’max, temperature at G’max, G’ at temperature rising to 90 C, G’ at temperature cooling to 5 C, tan d at 5 C, and breakdown ratio of G’ (G’BDr ¼ (G’max G090 C )/G’max 100%) during heating, respectively (Fig. 1A). 2.2.4. Retrogradation Starch was weighed in aluminum sample pans, mixed with distilled water (two times dry starch weight), and sealed. For hydration of starch the pans were stood at room temperature for 1 h, and heated from 30 C to 140 C at a rate of 10 C/min in a differential scanning calorimeter (DSC, model 2910, TA Instruments, Surrey, England). After gelatinization, the pans were cooled and stored at 5 C for 2 and 4 weeks. After storage, the pans were equilibrated for 1 h before rescanning in DSC from 10 C to 100 C at a rate of 10 C/min. The retrogradation enthalpy (DHret) was quantified (Fig. 1B). Retrogradation of starch was also followed using the dynamic rheometer. The starch suspension (25%, w/w) was fully gelatinized in boiling water with continuous shaking for 10 min. The gelatinized starch was transferred on to two sheets of glass (1 mm in thickness) and wrapped with plastic film to prevent water evaporation. The starch gel was cooled at 5 C and stored for 2 and 4 weeks. Before analysis, the gel was stood at room temperature for 1 h and cut to 4 cm in diameter. The gel was placed in the rheometer with preset temperature of 25 C. A parallel disc of 4-cm diameter was used and the gap was set at 1 mm. The frequency sweep experiments were performed from 0.1 Hz to 10 Hz at 1% deformation. G’ret (storage modulus where the subscript, ret, stands for retrograded gel) value was read at a frequency of 1 Hz (Fig. 1C). 2.2.5. Firmness The firmness of starch gels was measured by using a Texture Analyzer (TA XT2, TA Instruments, UK). Starch (5 g, d.b.) was
H. Singh et al. / Journal of Cereal Science 56 (2012) 367e373
369
Fig. 1. Typical profiles of dynamic rheology during (A) heating and cooling, (B) DSC retrogradation, (C) frequency sweep, and (D) textural analysis of waxy rice starch.
weighed into a glass bottle (internal diameter 30 mm) and adjusted by addition of water until the suspension concentration was 25% (w/w). The bottle cap was sealed and heated in a boiling water bath with continuous shaking for 10 min, and stored at 5 C for 2 and 4 weeks. Measurements were done at room temperature after standing the gels for half an hour. The starch gel was penetrated (two penetration cycles) at a speed of 1 mm/s using a standard probe having a diameter of 10 mm (P/10). The penetration strain was 50% of the gel height. The firmness was taken as peak height of curve of the first penetration cycle (Fig. 1D). 2.2.6. Statistical analysis Three replications of each parameter were done and the mean was reported. Analysis of variance, least significant difference and Pearson correlation were applied on the determined parameters using SAS software (SAS Institute, Cary, NC) to compare and to determine the interrelationships between various parameters. 3. Results and discussion 3.1. Chain-length distribution The HPSEC profile of debranched waxy starches showed two fractions. The first fraction (f1) of the HPSEC profile corresponds to long chains of amylopectin (B2 and longer chains) and the second fraction (f2) corresponds to short chains of amylopectin (A and B1 chains). The average chain lengths of amylopectin (CLw) of the 20 waxy rice starches ranged between 15.8 (WR11) and 18.7 (BW) (Table 1). Jane et al. (1999) reported that the CLw of cereal amylopectin ranged between 18.8 (waxy rice) and 23.5 (amylomaize VII). Length of long amylopectin chains (f1) varied between 51.3 (WR11)
and 58.5 (WR5), respectively, whereas short amylopectin chain length (f2) ranged between 13.5 (WR11) and 15.6 (BW, TKW1). On an average, the proportion of long and short chains of amylopectin by weight was 20.7 and 79.3% for the 20 waxy rice starches, respectively. Previous reports also confirm that waxy cereal starches consisted of about 70e80% of chains with DP 6e25 (Benmoussa et al., 2007; Jane et al., 1999; Shi and Seib, 1992). The average ratio of short-to-long chains (S/L) was found to be 3.9 for the 20 waxy rice starches. The range of S/L ratio for starches from the 13 mutants was 3.43 (WR6) to 4.95 (WR9) and for starches of the 7 named varieties was from 3.23 (TCW70) to 3.97 (TKW5), respectively. Hence it can be said that mutant starches have more short chains in proportion to long chains than starches of the named varieties (p < 0.05). 3.2. Rheological properties Rheological behavior of waxy rice starch gels is summarized in Table 2. Storage modulus (G’) increased first to a maximum value corresponding to completion of starch gelatinization and water absorption. Starches exhibited G’max in the range between 41 and 815 Pa. Mutant genotype WR6 recorded the lowest G’max at a temperature of 71.2 C and mutant genotype WR9 exhibited the highest G’max at a temperature of 62.0 C. Difference between temperatures at which G’ started increasing (TG’) and temperatures at which G’ reached a maximum ðTG0max Þ was within 4 C. Gelatinization of starches, including loss of birefringence, is usually complete within a temperature interval of 10 C for each type of starch (Salvador et al., 2006). The increase in G’ during the heating phase also corresponds to starch granules getting closely packed due to swelling (Eliasson, 1985).
370
H. Singh et al. / Journal of Cereal Science 56 (2012) 367e373
Table 1 Weight percentage and weight-average chain length (CLw) of fractions of isoamylase-debranched waxy rice starches from different genotypes. S/L ratioa
Genotype
Average CLw
f1 %
CLw
%
CLw
BW ESW TCSW1 TCW70 TKW1 TKW5 TSW2 WR1 WR2 WR3 WR4 WR5 WR6 WR7 WR8 WR9 WR10 WR11 WR12 WR13 LSDb
18.7 18.4 17.7 18.6 18.4 18.0 18.2 17.1 18.0 17.6 17.6 17.8 17.3 17.1 16.9 16.4 16.5 15.8 16.6 16.6 0.2
23.0 21.6 20.5 23.7 20.9 20.1 20.6 19.4 21.0 20.8 20.3 21.6 22.6 20.1 19.5 16.8 20.0 20.1 20.5 21.2 0.5
58.4 57.7 56.2 57.2 58.2 57.6 57.7 55.8 57.8 58.3 57.5 58.5 55.6 55.9 55.3 57.1 53.5 51.3 54.6 53.0 0.4
77.0 78.4 79.5 76.3 79.1 79.9 79.4 80.6 79.0 79.2 79.7 78.4 77.4 79.9 80.5 83.2 80.0 79.9 79.5 78.8 0.5
15.6 15.5 15.1 15.4 15.6 15.3 15.5 14.7 15.2 14.9 15.0 15.0 14.4 14.5 14.4 14.4 14.1 13.5 14.1 14.1 0.1
a b
f2
3.34 3.64 3.88 3.23 3.78 3.97 3.85 4.17 3.76 3.82 3.92 3.62 3.43 3.99 4.12 4.95 4.00 3.97 3.87 3.72 0.13
Ratio of f2% to f1%. Least significant difference, n ¼ 3.
Different G’ exhibited by waxy rice starches may be attributed to the difference in the breakdown of the amylopectin matrix inside the swollen starch granules (Keetels and van Vliet, 1994). G0 depends upon factors like granule rigidity and swollen granule phase volume. Lii et al. (1996) postulated that rheological behavior of gelatinized starch suspension could be influenced by inter-granular interactions, such as entanglement between surface molecules of adjacent granules and the granular properties. The loss factor, tan d represents the ratio of energy lost to energy stored during a cycle (Ferry, 1970). During prolonged heating, starch suspensions show a rise in G’ and reach a maximum and then decrease which may depend upon the swelling factor and breakdown resistance (Singh et al., 2003). Starch gels showed G’ and G’’ values at 90 C in the range from 17 Pa to 25 Pa and 10e12 Pa, respectively. Lii et al. (1995) reported a G’ value at 95 C of 39 Pa for rice starch (20%, w/w) with amylose content of 0.99%. The decrease in G’ upon heating may be attributed to the deformation/loss of granular structure due to melting of remaining crystallites in swollen granules (Eliasson, 1986). Keetels et al. (1996b) hypothesized and attributed the decrease in G’ on prolonged heating to loosen non-covalent bonds by disentanglement. (%) ¼ [(G’max G090 C )/ The breakdown ratio (G’BDr 0 G90 C 100%]), an indirect measure of the thermal stability and degree of disintegration of starch granules varied widely between 50.7% (WR6) and 97.4% (WR9). Keetels et al. (1996b) postulated that breakdown of the amylopectin matrix inside the swollen starch granules and disentanglement of branches during prolonged heating would cause the granules to become less rigid. G’BDr showed significant negative correlations with TG’ (r ¼ 0.665) and TG0max (r ¼ 0.810). Thus, it can be confirmed that breakdown ratio is inversely proportional to swelling extent of starch granules. The cooling phase, which can be related to the short-time retrogradation of starches, saw a rise in G’ and G’’. The increase in G’ and G’’ during cooling from 90 C to 5 C may be due to interactions and associations between amylopectin chains and partly due to chain stiffness and stiffness of remaining intact starch granules. Jane et al. (1999) reported that long amylopectin chains (DP > 50) accelerated retrogradation. A positive correlation between percent
proportion of amylopectin B chains (DP > 40) and difference in values of G’ during cooling from 95 C to 4 C was observed by Chung et al. (2008). However, no significant correlation between G’ and amylopectin chain-profile was found, yet both long and short chains may have been re-associated during this short time. It is also noteworthy that the G’ during heating was observed to be much greater than during cooling. The tan d of starches during cooling decreased to less than 1 ðtan d5 C Þ, indicating the formation of molecular interaction network of starch. 3.3. Retrogradation Miles et al. (1985) indicated that the short-term development of gel structure and crystallinity in starch gels was found to be dominated by gelation and crystallization within the amylose. While long-term increase in the modulus of starch gels was linked to crystallization of amylopectin. Storage time of 2 and 4 weeks was selected for observing retrogradation of starch, since all samples exhibited very low amylose content. Values of DHret of starch gels after storage for 2 and 4 weeks are summarized in Table 3. DHret for all starches increased with increasing storage duration. DHret for the 20 waxy starches varied from 0.9 J/g (WR12) to 6.4 J/g (BW) and 5.1 J/g (TKW5) to 10.1 J/g (BW) after 2 and 4 weeks of storage, respectively. Vandeputte et al. (2003) showed a range of DHret (amylopectin basis) values from 1.8 to 4.7 J/g for five waxy rice starch gels (with 1.9e3.4% amylose content) stored at 6 C for 4 weeks. Sasaki and Matsuki (1998) indicated that longer chains of amylopectin formed more stable crystallites and more regions of crystallinity during storage. Percentage retrogradation (%R) was observed to vary from 5.8% (WR12) to 41.6% (BW) for the first 2 weeks of storage and subsequently from 33.3% (TKW5) to 65.6% (BW) for the next 2 weeks. Rice amylopectins have been reported to display two stages of retrogradation behavior during early (<7 days) and late storage (>7 days) (Lai et al., 2000). The former was not influenced by
Table 2 Rheological propertiesa during heating and cooling of waxy rice starches from different genotypes. Genotype TG’ ( C)
G’max (Pa)
tan dG’max TG0max ( C)
G090 C (Pa)
G05 C (Pa)
tan d5 C G’BDr (%)
BW ESW TCSW1 TCW70 TKW1 TKW5 TSW2 WR1 WR2 WR3 WR4 WR5 WR6 WR7 WR8 WR9 WR10 WR11 WR12 WR13 LSDb
327 321 148 163 180 451 166 296 235 60 114 376 41 280 259 815 260 164 337 311 28
0.263 0.264 0.259 0.278 0.256 0.227 0.290 0.240 0.247 0.322 0.245 0.240 0.425 0.259 0.233 0.196 0.227 0.261 0.223 0.243 0.009
25 22 21 20 18 19 22 18 19 20 21 19 20 17 21 21 19 19 19 17 3
42 35 35 38 33 32 39 34 36 37 36 36 38 34 37 37 35 37 34 35 5
0.788 0.843 0.866 0.879 0.909 0.865 0.845 0.919 0.863 0.851 0.800 0.870 0.868 0.949 0.844 0.856 0.880 0.904 0.903 0.929 0.017
64.8 63.6 64.0 64.8 60.5 60.0 64.3 59.5 62.2 64.3 61.1 62.4 67.3 62.1 61.8 60.1 60.9 60.7 59.5 61.8 0.6
66.7 65.4 65.9 66.7 62.6 62.4 66.5 62.1 64.2 67.9 64.3 64.1 71.2 64.0 63.9 62.0 63.0 62.4 61.9 64.1 0.9
92.2 93.3 87.1 87.6 90.1 95.5 86.8 93.8 91.8 66.0 81.5 95.1 50.7 93.9 92.2 97.4 92.6 88.7 94.4 94.6 2.2
a TG’: temperature at the initial increasing of G’; G’max: maximum value of G’ during heating; tan dG’max : tan d at G’max; TG0max : temperature at G’max; G090 C : G’ at temperature rising to 90 C; G05 C : G’ at temperature cooling to 5 C; tan d5 C : tan d at 5 C; G’BDr: breakdown ratio of G’ (G’BDr ¼ (G’max G090 C )/G’max 100%) during heating. b Least significant difference, n ¼ 3.
H. Singh et al. / Journal of Cereal Science 56 (2012) 367e373
amylopectin chain-length distribution whereas, for late stage retrogradation, amylopectin retrogradation enthalpies showed positive correlation with the population of DP 15. The starch retrogradation has been postulated to be controlled by the nonequilibrium recrystallization behavior of amylopectin (Slade and Levine, 1986). The early stages in starch retrogradation have been reported to be dominated by the chain folded lamellar microcrystalline junction zones of amylose (DP w 15e50) and the later stages by amylopectin branched chains (DP > 12e16) (Shi and Seib, 1992; Slade and Levine, 1986). Sasaki and Matsuki (1998) indicated that longer chains of amylopectin formed more stable crystallites and more regions of crystallinity during storage. Thus, the difference between retrogradation values of 2 weeks and 4 weeks could have been influenced by amylopectin branched chains, and the effect of amylopectin short chains on recrystallization perfection during storage (Vandeputte et al., 2003). Table 3 also summarizes values of G’ret of starch gels measured at a frequency of 1 Hz after storage for 2 and 4 weeks. Due to storage of gels, G’ret can be related to retrogradation of starch gels as chains re-associate during the period. G’ret measured after 2 weeks of storage duration ranged between 111 Pa (TKW5, WR1) and 2080 Pa (BW). G’ret increased drastically for all starches after storage for 4 weeks and ranged from 2957 Pa (TKW5) to 37,600 Pa (BW). This indicates increased tendency of amylopectin chains to re-associate. Nonetheless, G’ret would also depend upon rigidity of starch granules. Keetels et al. (1996a) have given two important postulations about the process of recrystallization of amylopectin in concentrated starch gels: (1) formation of crystalline clusters along the glucan chains in the amylopectin molecule, resulting in stiffening of strands between entanglements, and (2) formation of cross-links between adjacent clusters. This observation of increase in G’ret is consistent with the observations of increase in DHret upon storage in the present study. Significant positive correlations between G’ret and DHret were also observed (r ¼ 0.596). 3.4. Firmness Firmness of waxy rice starch gels is presented in Table 4. Gels were observed to become harder as storage duration increased. These results are consistent with observations on increase in Table 3 Retrogradation enthalpy (ΔHret) and storage modulus (G’ret) of starch gels from different genotypes of waxy rice after storage at 5 C for 2 and 4 weeks. Genotype
BW ESW TCSW1 TCW70 TKW1 TKW5 TSW2 WR1 WR2 WR3 WR4 WR5 WR6 WR7 WR8 WR9 WR10 WR11 WR12 WR13 LSDa a
ΔHret (J/g)
G’ret (Pa)
371
retrogradation of starch gels in the present study. Firmness of 2and 4-week stored gels ranged from 1.44 N (WR12) to 9.20 N (BW) and 6.84 N (TKW5) to 27.36 N (ESW), respectively. The average values of firmness for 2- and 4-week stored gels were higher for starch from the named varieties than from the mutant genotypes. The mechanical properties of starch gels are affected by the volume fraction and rigidity of gelatinized starch granules, as well as the interactions between dispersed and continuous phases of the gel, rheological properties of the continuous phase (includes solubilized starch in water), and deformability of particles (Biliaderis, 1998). These factors, in turn, have been observed to show dependence on amylopectin structure (Yamin et al., 1999). 3.5. Correlation analysis Table 5 shows the relationship between various parameters analyzed according to Pearson correlation. Significantly (p < 0.05) positive correlations of TG’ with average CLw (r ¼ 0.481) and percentage proportion of long amylopectin chains (f1%) (r ¼ 0.609) were observed. A negative relationship between percentage of short amylopectin chains (f2%) (r ¼ 0.689) and S/L ratio (r ¼ 0.649) with TG’ was also observed. Thus, longer amylopectin chains can be said to result in waxy starch granules with higher gelatinization temperature. Positive correlations of G’max with f2% (r ¼ 0.566) and S/L ratio (r ¼ 0.625) were observed. Keetels et al. (1996a) postulated that swollen granule rigidity was partly contributed by double helices made by short chains. The negative relationship between tan d and average chain length (average CLw) (r ¼ 0.449) and long amylopectin chain length (f1CLw) (r ¼ 0.521) showed that long chains can be attributed to the formation of the gel network. In the present study, DHret for samples after storage for either 2 (DHret-2wk) or 4 (DHret-4wk) weeks showed positive correlations with f1% (r ¼ 0.600 for DHret-2wk and 0.662 for DHret-4wk, respectively) and negative dependence on f2% (r ¼ 0.488 for DHret-2wk and 0.605 for DHret-4wk, respectively). S/L ratio was also observed to have negative correlations with DHret-2wk (r ¼ 0.461) and DHret-4wk (r ¼ 0.569). These results of interrelationships show that long amylopectin chains can associate faster during storage, thus accelerating retrogradation. DHret-4wk was observed Table 4 Firmness (N) of starch gels from different genotypes of waxy rice after storage at 5 C for 2 and 4 weeks.
2 weeks
4 weeks
2 weeks
4 weeks
Genotype
2 weeks
4 weeks
6.4 5.3 2.5 2.5 1.6 1.4 4.1 2.6 1.4 2.8 2.0 2.9 3.2 1.5 1.6 1.8 2.2 1.8 0.9 5.1 0.2
10.1 9.3 7.9 8.7 6.1 5.1 9.3 6.8 6.1 7.6 6.1 6.8 8.1 6.0 7.3 5.8 7.3 6.1 6.2 7.2 0.4
2080 135 316 391 125 111 1639 111 846 978 338 610 614 530 500 250 248 391 123 594 68
37,600 26,350 21,310 19,750 8653 2957 17,930 8088 14,410 21,920 11,100 13,990 10,720 9062 9675 8909 8184 5262 4858 18,120 1693
BW ESW TCSW1 TCW70 TKW1 TKW5 TSW2 WR1 WR2 WR3 WR4 WR5 WR6 WR7 WR8 WR9 WR10 WR11 WR12 WR13 LSDa
9.20 3.27 6.00 5.45 2.73 2.10 8.24 3.59 6.53 4.97 3.81 4.33 6.28 4.18 5.33 3.82 3.63 4.50 1.44 5.88 1.08
18.64 27.36 24.94 23.42 8.33 6.84 15.01 10.04 14.53 15.94 11.62 11.41 15.69 10.61 12.44 9.65 9.31 10.99 9.98 16.80 2.28
Least significant difference, n ¼ 3.
a
Least significant difference, n ¼ 3.
372
H. Singh et al. / Journal of Cereal Science 56 (2012) 367e373
Table 5 Pearson correlations between molecular structure parameters and retrogradation and rheological behaviors of waxy rice starches. Parametersa
Average CLw
f1%
f1CLw
f2%
f2CLw
S/L ratio
TG’ G’max tan dG’max TG0max G090 C G05 C G’BDr tan d5 C DHret-2wk DHret-4wk G’ret-2wk G’ret-4wk Fret-2wk Fret-4wk
0.481* ec e e e e e 0.449* e 0.494* e 0.596* e 0.488*
0.609* e e 0.527* e e e e 0.600* 0.662* e 0.618* e 0.757**
e e e e e e e 0.521* e e e e e e
0.689** 0.566* 0.550* 0.631* e e e e 0.488* 0.605* e 0.562* 0.472* 0.600*
e e e e
0.649* 0.625* 0.537* 0.598* e e e e 0.461* 0.569* e 0.524* e 0.557*
0.447* e e 0.470* e e e 0.542* e e
*Significant at p < 0.05; **significant at p < 0.001. a DHret-2wk: retrogradation enthalpy of starch gel after 2 weeks of storage; DHret4wk: retrogradation enthalpy of starch gel after 4 weeks of storage; G’ret-2wk: G’ of starch gel after 2 weeks of storage; G’ret-4wk: G’ of starch gel after 4 weeks of storage; Fret-2wk: firmness of starch gel after 2 weeks of storage; F4w: Firmness of starch gel after 4 weeks of storage. Rest symbols have the same meaning as mentioned in text and other tables.
to have a positive relationship with average CLw (r ¼ 0.494). However, the relationship of average CLw with DHret-2wk was not observed. Vandeputte et al. (2003) did not find any significant relationship between amylopectin chain-profile, amylose and retrogradation enthalpies for waxy rice starches. These differences in the interrelationships between the present study and Vandeputte et al. (2003) may be due to different numbers of genotypes and starch concentrations used for retrogradation analysis. Results of Qi et al. (2003) on starches from six waxy rice genotypes (amylose content 0.2e1.5%), however, suggest that recrystallization tendency of starches during storage depends upon the proportion of short amylopectin chains and attributed the stability of crystalline structure formed during retrogradation in vitro to integrity of amylopectin molecules. This difference in results with the present study may be due to difference in amylose content and ratio of weight percentages of short-to-long amylopectin chains. The dependence of G’ret of starch gels after storage for 4 weeks on structural parameters was also observed in the present study. The pattern of interrelationship of G’ret of 4-week stored gel (G’ret4wk) with amylopectin chain-profile was the same as that for DHret. It is also noteworthy that G’ret of 2-week stored gels (G’ret–2wk), however, did not record any significant relationships with amylopectin chain-profiles, whereas DHret-2wk showed some significant relationships (Table 5). The different sensitivity in the analysis methods of retrogradation of gels by rheometer and DSC may be attributed to the difference in interrelationships observed. The interrelationships observed in the case of G’ret-4wk with average CLw (r ¼ 0.596) and f1% (r ¼ 0.618) may be attributed to the formation of entanglements and re-association between amylopectin chains, which impart stiffness. The gel firmness of 4-week stored gel (F4wk) showed a positive relationship with average CLw (r ¼ 0.488) and f1% (r ¼ 0.757). A negative correlation (r ¼ 0.557) between F4wk and S/L ratio was also observed. Gel firmness of 2-week stored gel (F2wk) also showed a negative relationship with f2% (r ¼ 0.472) and a positive relationship with f1% (r ¼ 0.267). These observations on interrelationship are in line with the relationship of DHret with other parameters although the preparation procedures of gels were different for DSC and texture analyzer. Significant (p < 0.05) positive interrelationships between F2wk (r ¼ 0.479) and F4wk (r ¼ 0.682) were found
with G’ret-4wk. F4wk showed positive relationship with DHret-2wk (r ¼ 0.468, p < 0.05) and DHret-4wk (r ¼ 0.574; p < 0.001), which was not, however, found for F2wk. F2wk and F4wk were also interrelated (r ¼ 0.479, p < 0.05), which is also a significant result. Thus it can be safely concluded that reordering duration (storage time) of amylopectin chains had a greater impact on F4wk than F2wk. Vandeputte et al. (2003) reported no difference in firmness of 2-day and 2-week stored waxy rice starch gels (1.9e3.4% absolute amylose content). As mentioned earlier, these authors observed retrogradation for waxy rice starch gels after 4 weeks when stored at 6 C. These authors also have shown an increase in firmness of normal rice starch (amylose content from 13.6 to 28.2%) gels with storage. Interestingly, these authors did not find any relationship of amylopectin unit length distribution with starch textural attributes for normal or waxy rice starches. Wang and Wang (2002) observed an increase in firmness of waxy rice starch gels (10%, w/w) from four cultivars over storage duration of 1 day and a slight increase thereafter for 7-day stored gel. The authors reported a positive correlation between amounts of amylopectin A and B1 chains and gel firmness, which is opposite to our present study. The difference in relationships observed from the above two studies mentioned may be due to a difference in proportions of short amylopectin chains, amylose content (negligible in present study) and concentration of starch gels and number of genotypes used. Thus, reassociation of long chains during retrogradation or storage contributes to rigidity of gels as observed from rheology and gel textural results. Mutation may change the distribution of amylopectin chains and, consequently, affects the formation of cross-links and double helices in retrograded starch gels. The amount of long amylopectin chains was an important determinant in accelerating the retrogradation of starch. 4. Conclusions Starches from mutant waxy genotypes showed widely different retrogradation, rheological and textural attributes than starches from named waxy rice varieties. Long amylopectin chains showed an accelerating effect on retrogradation of waxy rice starch gels. Reassociation of long chains during retrogradation or storage contributes to rigidity of gels as observed from rheology results. Mutation was observed to change the percentage of short and long chains, hence may have also affected the ability to form cross-links and double helices in retrograded starch gels. Mutant genotypes WR2, WR7 and WR9 showed lower retrogradation and higher gel firmness than some named commercial varieties (such as TKW1 and TKW5) and could be useful on rice processing. Acknowledgment We thank the Council of Agriculture (COA), Taiwan, for financial support. The supply of polished rice by Agricultural Research Institute of Taiwan is also highly acknowledged. References Ayres, N.M., McClung, A.M., Larkin, P.D., Bligh, H.F.J., Jones, C.A., Park, W.D., 1997. Micro satellites and a single-nucleotide polymorphism differentiate apparent amylose classes in an extended pedigree of US rice germ plasma. Theoretical and Applied Genetics 94, 773e781. Bao, J., Sun, M., Zhu, L., Corke, H., 2004. Analysis of quantitative trait loci for some starch properties of rice (Oryza sativa L.): thermal properties, gel texture and swelling volume. Journal of Cereal Science 39, 379e385. Benmoussa, M., Moldenhauer, K.A.K., Hamaker, B.R., 2007. Rice amylopectin fine structure variability affects starch digestion properties. Journal of Agriculture and Food Chemistry 55, 1475e1479. Biliaderis, C.G., 1998. Structures and phase transitions of starch polymers. In: Walter, R.H. (Ed.), Polysaccharide Association Structures in Foods. Marcel Dekker, New York, pp. 57e168.
H. Singh et al. / Journal of Cereal Science 56 (2012) 367e373 Chung, J.H., Han, J.-A., Yoo, B., Seib, P.A., Lim, S.-T., 2008. Effects of molecular size and chain profile of waxy cereal amylopectins on paste rheology during retrogradation. Carbohydrate Polymers 71, 365e371. Eliasson, A.C., 1985. Retrogradation of starch as measured by differential scanning calorimetry. In: Hill, R.D., Munck, I. (Eds.), New Approaches to Research on Cereal Carbohydrate. Elsevier, Amsterdam, pp. 93e98. Eliasson, A.C., 1986. Viscoelastic behaviour during the gelatinization of starch. II. Effects of emulsifiers. Journal of Texture Studies 17, 357e375. Ferry, J.D., 1970. Illustrations of viscoelastic behaviour of polymeric systems. In: Ferry, J.D. (Ed.), Viscoelastic Properties of Polymers. J. Wiley & Sons, New York, pp. 34e58. Jane, J., Chen, Y.Y., Lee, L.F., McPherson, A.E., Wong, K.S., Radosavljevic, M., 1999. Effects of amylopectin branch chain length and amylose content on the gelatinization and pasting properties of starch. Cereal Chemistry 76, 629e637. Juliano, B.O., 1992. Structure, chemistry, and function of the rice grain and its fractions. Cereal Foods World 37, 772e774. Karim, A.A., Norziah, M.H., Seow, C.C., 2000. Methods for the study of starch retrogradation. Food Chemistry 71, 9e36. Keetels, C.J.A.M., van Vliet, T., 1994. Gelation and retrogradation of concentrated starch gels. In: Phillips, G.O., Williams, P.A., Wedlock, D.J. (Eds.), Gums and Stabilizers for the Food Industry. IRL, New York, pp. 271e280. Keetels, C.J.A.M., Oostergete, G.T., van Vliet, T., 1996a. Recrystallization of amylopectin in concentrated starch gels. Carbohydrate Polymers 30, 61e64. Keetels, C.J.A.M., van Vliet, T., Walstra, P., 1996b. Gelation and retrogradation of concentrated starch systems: 1. Gelation. Food Hydrocolloids 10, 343e353. Lai, V.M.-F., Lu, S., Lii, C.-Y., 2000. Molecular characteristics influencing retrogradation kinetics of rice amylopectins. Cereal Chemistry 77, 272e278. Lii, C.Y., Shao, Y.Y., Tseng, K.H., 1995. Gelation mechanism and rheological properties of rice starch. Cereal Chemistry 72, 393e400. Lii, C.Y., Tsai, M.L., Tseng, K.H., 1996. Effect of amylose content on the rheological properties of rice starch. Cereal Chemistry 73, 415e420. Lin, J.H., Chang, Y.H., 2006. Molecular degradation rate of rice and corn starches during acid-methanol treatment and its relation to the molecular structure of starch. Journal of Agricultural and Food Chemistry 54, 5880e5886. Lin, J.H., Singh, H., Chang, Y.T., Chang, Y.H., 2011. Factor analysis of the functional properties of rice flours from mutant genotypes. Food Chemistry 126, 1108e1114.
373
Miles, M.J., Morris, V.J., Orford, P.D., Ring, S.G., 1985. The roles of amylose and amylopectin in the gelation and retrogradation of starch. Carbohydrate Research 135, 271e281. Qi, X., Tester, R.F., Snape, C.E., Ansell, R., 2003. Molecular basis of the gelatinisation and swelling characteristics of waxy rice starches grown in the same location during the same season. Journal of Cereal Science 37, 363e376. Salvador, A., Sanz, T., Fiszman, S.M., 2006. Dynamic rheological characteristics of wheat flour-water dough. Effect of adding NaCl, sucrose and yeast. Food Hydrocolloids 20, 780e786. Sarko, A., Wu, H.C.H., 1978. The crystal structures of A-, B-, and C-polymorphs of amylose and starch. Starch/Stärke 30, 73e78. Sasaki, T., Matsuki, J., 1998. Effect of wheat starch structure on swelling power. Cereal Chemistry 75, 525e529. Sasaki, T., Yasui, T., Matsuki, J., 2000. Effect of amylose content on gelatinization, retrogradation and pasting properties of starches from waxy and non-waxy wheat and their F1 seeds. Cereal Chemistry 77, 58e63. Shi, Y.-C., Seib, P.A., 1992. The structure of four waxy starches related to gelatinisation and retrogradation. Carbohydrate Research 227, 131e145. Singh, N., Singh, J., Kaur, L., Sodhi, N.S., Gill, B.S., 2003. Morphological, thermal and rheological properties of starches from different botanical sources. Food Chemistry 81, 219e231. Slade, L., Levine, H., 1986. Recent advances in starch retrogradation. In: Stivala, S.S., Crescenzi, V., Dea, I.C.M. (Eds.), Proceedings of the Conference of Recent Developments in Industrial Polysaccharides: The Impact of Biotechnology and Advanced Methodologies. The Stevens Institute of Technology Gordon and Breach Science, New York, pp. 387e430. Tan, Y., Corke, H., 2002. Factor analysis of physicochemical properties of 63 rice varieties. Journal of Science of Food and Agriculture 82, 745e752. Vandeputte, G.E., Vermeylen, R., Geeroms, J., Delcour, J.A., 2003. Rice starches. III. Structural aspects provide insight in amylopectin retrogradation properties and gel texture. Journal of Cereal Science 38, 61e68. Wang, Y.-J., Wang, L., 2002. Structures of four waxy rice starches in relation to thermal, pasting and textural properties. Cereal Chemistry 79, 252e256. Yamin, F.F., Lee, M., Pollak, L.M., White, P.J., 1999. Thermal properties of starch in corn variants isolated after chemical mutagenesis of inbred line B73. Cereal Chemistry 76, 175e181.