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Mutants that have shorter amylopectin chains are promising materials for slow-hardening rice bread
Journal of Cereal Science 61 (2015) 105e110
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Mutants that have shorter amylopectin chains are promising materials for slow-hardening rice bread Noriaki Aoki a, b, Takayuki Umemoto c, Kazuyuki Okamoto d, Yasuhiro Suzuki e, Junichi Tanaka b, e, * a
National Agriculture and Food Research Organization, Headquarters 3-1-1 Kannondai, Tsukuba, Ibaraki 305-8666, Japan University of Tsukuba Graduate School of Life and Environmental Sciences, 2-1-18 Kannondai, Tsukuba, Ibaraki 305-8518, Japan NARO Hokkaido Agricultural Research Center, 1 Hitsujigaoka, Toyohiraku, Sapporo 062-8555, Japan d Ibaraki Agricultural Center Plant-Biotechnology Institute, 3402 Kamikunii, Mito, Ibaraki 311-4203, Japan e NARO Institute of Crop Science, 2-1-18 Kannondai, Tsukuba, Ibaraki 305-8518, Japan b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 26 May 2014 Received in revised form 20 August 2014 Accepted 22 September 2014 Available online 22 November 2014
Bread staling is a serious economic issue for the baking industry. Here, we found that shorter amylopectin chains caused by mutations play a role in maintaining the softer texture of rice bread. We used three rice cultivars that have a high proportion of short amylopectin chains in endosperm starch, two of which were starch branching enzyme I mutants, to make gluten-free and gluten-containing bread. Compression tests showed that the hardening rates for both types of bread made from these cultivars were markedly lower than those for control rice breads (gluten-free bread: 14%e39%, gluten-containing bread: 13%e27%), although there were no clear differences in the hardness values among the breads one day after baking. Sensory tests conducted two days after baking showed that gluten-free breads made from the three cultivars were softer than the control breads. Amylose contents, flour particle sizes, and damaged starch contents were similar among the flour samples, indicating that shorter amylopectin chains led to the slow-hardening of the rice bread. This finding can be applied not only to the breeding of rice cultivars for softer bread, but also to breeding of wheat and other cereals for bread. © 2014 Elsevier Ltd. All rights reserved.
Keywords: Amylopectin Rice Bread staling Starch branching enzyme I
1. Introduction Bread easily stales within a few days and its palatability reduces. Bread staling is not only a serious issue for bread quality to consumers but also causes huge economic losses to the baking and restaurant industries. Rice flour is used to make gluten-free bread, which is helpful for those who are allergic to wheat protein or have celiac disease (Cureton and Fasano, 2009); however, it stales faster than regular wheat bread (Ahlborn et al., 2005). Starch is thought to affect the staling of both gluten-free and regular wheat bread (Gray and Bemiller, 2003; Morgan et al., 1997). Therefore, one approach to solving the problem is to breed cultivars that produce breads that show less staling during preservation by changing the starch properties of such cultivars through mutations.
Abbreviations: DP, degree of polymerization; SbeI, starch branching enzyme I. * Corresponding author. NARO Institute of Crop Science, 2-1-18 Kannondai, Tsukuba, Ibaraki 305-8518, Japan. Tel.: þ81 29 838 8536. E-mail address: [email protected] (J. Tanaka). http://dx.doi.org/10.1016/j.jcs.2014.09.006 0733-5210/© 2014 Elsevier Ltd. All rights reserved.
Rice is suitable for investigating the relationship between starch and bread staling for two reasons. First, rice proteins do not form a gluten matrix like wheat proteins, hence the properties of starch directly affect bread qualities. Second, rice is a good material for genetic analyses, because it is a diploid self-pollinating species, that is, it is relatively easy to correct recessive alleles. Many rice mutants have been screened and characterized, including mutants with unique starch properties (Nakamura, 2002). Starch consists of two components, amylose and amylopectin. Amylose is essentially a linear molecule containing a-(1-4)-linked glucose units with few branches, whereas amylopectin is a branched molecule with linear chains of a-(1-4)-linked glucose units with a-(1-6)-linked branches (Hizukuri et al., 1989). Native starch is closely packed to form clusters of double helices, and starch granules comprise an amorphous region and a semicrystalline region (Donald, 2001). Starch is gelatinized by denaturing its structure under high temperature in the presence of water and the gelatinization temperature depends on amylopectin structure (Jane et al., 1999). When the gelatinized starch is cooled,
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starch is retrograded by partial recrystallization of the linear chains. This starch retrogradation is believed to play a major role in the staling process (Bosmans et al., 2013; Hug-Iten et al., 2003). Although both amylose and amylopectin undergo retrogradation, amylose is retrograded within hours, whereas amylopectin is retrograded for days (Miles et al., 1985; Ribotta and Le-Bail, 2007). Therefore, we hypothesized that amylopectin plays a major role in bread staling during long-term storage. Rice amylopectin is mainly classified into S-type and L-type (Nakamura, 2002) based on the absence or presence of Starch Synthase IIa activity (Umemoto et al., 2004). S-type amylopectin has a higher ratio of short chains than does L-type amylopectin. Moreover, the gelatinization temperature of starch with S-type amylopectin is lower than that of starch with L-type amylopectin. In our previous report, gluten-containing rice breads made from cultivars that have L-type amylopectin were more prone to hardening than bread made from cultivars that have S-type amylopectin (Aoki et al., 2012). Therefore, we hypothesized that softer bread (both gluten-containing and gluten-free) can be made by using cultivars that have shorter amylopectin chains than those in S-type amylopectin. Rice mutants of starch branching enzyme I (SbeI) have a high proportion of shorter amylopectin chains and a lower gelatinization temperature compared with cultivars that have regular Stype amylopectin (Okamoto et al., 2013a; Satoh et al., 2003). Here, we report that slow-hardening gluten-free and gluten-containing breads can be obtained by using mutants that have shorter amylopectin chains than those in S-type amylopectin.
2. Experimental 2.1. Materials Rice (Oryza sativa L.) cultivars, ‘Akita Sake 44’, ‘Hiderishirazu D’, and ‘Kurnai’ that are rich in shorter chains of amylopectin, were used. ‘Koshihikari’, which is the most popular cultivar in Japan, was used as a control (Table 1). ‘Hiderishirazu D’ and ‘Kurnai’ are natural loss-of-function mutants of SbeI. The mutations of ‘Hiderishirazu D’ and ‘Kurnai’ are considered to be of common origin, because no difference was detected between the DNA sequences of SbeI between ‘Hiderishirazu D’ and ‘Kurnai’ (Okamoto et al., 2013a). ‘Akita Sake 44’ is a gamma-ray-induced mutant with a low gelatinization temperature (Okamoto, unpublished result). Polished grains of rice were ground by using a jet mill (SPM-R290; Nishimura Machine Works, Osaka, Japan) after being soaked in a solution containing 0.3% trisodium citrate dihydrate and 0.05% pectinase (Pectinase G; Amano Enzyme, Nagoya, Japan) for 1 h at 40 C.
2.1.1. Genotyping of the wx and alk locus Total DNA was extracted by using diatomaceous earth and a spin filter (Tanaka and Ikeda, 2002). The waxy (wx) locus was genotyped by using the wx-allele-specific dCPAS marker (Yamanaka et al.,
2004). The alk locus, which encodes starch synthase IIa, was genotyped by using SNP markers (Hiratsuka et al., 2010). 2.2. Flour property measurements Flour particle sizes were measured with a laser diffraction particle size analyzer (LS 13 320; Beckman Coulter, Brea, USA). Damaged starch contents were measured according to the American Association of Cereal Chemists (AACC) method 76-31 (AACC, 2000b) with a starch damage assay kit (Megazyme International Ireland, Wicklow, Ireland). Apparent amylose contents were determined by using the iodine absorption method with an Auto Analyzer (BRAN þ LUEBBE, Norderstedt, Germany). The nitrogen content of flour was determined by using a Sumigraph NC-22F (Sumika Company Ltd., Osaka, Japan) and acetanilide as a standard; protein values were calculated by multiplying the nitrogen content by 5.95. Pasting properties were determined with a Rapid Visco Analyzer (RVA Model 3D; Newport Scientific, Warriewood, Australia) as previously described (Toyoshima et al., 1997). The temperature at the onset of the rise in viscosity was determined as the pasting temperature. The thermograms of the starch granules were recorded on a differential scanning calorimeter (DSC7; PerkinElmer Inc., Waltham, USA) with distilled water as the reference. Rice flour (10 mg) and distilled water (25 mL) were sealed in a platinum pan. Samples were heated from 10 C to 100 C at a rate of 10 C/min. The peak temperatures were determined as the gelatinization temperature. Water absorption of rice flour was determined as the amount of water needed to develop a standard dough of 500 Brabender units at the peak of the curve, as determined by using a Farinograph (Brabender Inc., Duisburg, Germany) according to the AACC method 54-21 (AACC, 2000a). A mixture of rice flour (40 g) and vital wheat gluten (10 g) was used (14% moisture basis). Amylopectin chain-length distribution was analyzed by using capillary electrophoresis as described by Fujita et al. (2001). Statistical evaluations were performed by using Tukey's test. 2.3. Bread making We made two types of bread, gluten-free bread and glutencontaining bread. Gluten-free bread was made by using 480 g of rice flour, 120 g of tapioca starch, 12.0 g of hydroxypropyl methylcellulose (Metolose; Shin-Etsu Chemical, Tokyo, Japan), 48.0 g of sugar, 24.0 g of skimmed milk, 12.0 g of salt, 42.0 g of shortening, 24.0 g of dry yeast, and 630 mL of water. Tapioca starch, which is often used in gluten-free bread-making (Schober, 2009), was added in an attempt to make bread larger (Kusunose et al., 1999; Sanchez et al., 2002). The dough was mixed in a Kanto mixer (HPi-20M; Kanto Mixer, Tokyo, Japan). Portions of dough (400 g) were placed in bread pans and proofed at 27 C and 80% humidity for 60 min. Breads were baked at 200 C for 20 min without steam. Gluten-containing bread was made by using 600 g of rice flour (14% moisture basis), 150 g of vital wheat gluten (14% moisture
Table 1 Properties of rice flour samples.
Koshihiakri Akita Sake 44 Hiderishirazu D Kurnai a
Average particle size (mm)a
Damaged starch content (%)b
32.9 30.3 31.3 34.9
1.9 2.4 2.7 3.0
± ± ± ±
0.1d 0.1c 0.1b 0.1a
Amylose content (%)b 18.0 20.2 20.7 17.6
± ± ± ±
0.2b 0.4a 0.2a 0.3b
Protein content (%)b 5.1 5.1 7.0 5.9
± ± ± ±
0.1c 0.2c 0.0a 0.1b
Pasting temperature ( C)b 68.8 66.4 65.7 65.2
± ± ± ±
0.8a 0.7a 0.7b 0.4b
Gelatinization temperature ( C)b 69.9 66.8 65.2 65.8
± ± ± ±
0.3a 0.1b 0.1c 0.4c
Water absorption (%) 77.4 77.1 77.1 79.3
Means of two replicates are shown. Means and standard deviations from three replicates are shown. Means ± standard deviations with the same letter are not significantly different from each other (p < 0.05, Tukey's test). b
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basis; Riken Vitamin, Tokyo, Japan), 52.5 g of sugar, 22.5 g of skimmed milk, 11.3 g of salt, 60 g of shortening, 11.3 g of dry yeast, and water according to the no-time method (Yamauchi et al., 2004). The amount of added water was determined on the basis of the water absorption value obtained as described earlier. A Kanto mixer was used to mix the dough and then 400-g portions were put in loaf pans and allowed to rise for about 1 h at 38 C and 80% humidity until the dough reached the top of the pan. The dough was then baked at 185 C for 28 min.
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3.2. Amylopectin structure Fig. 1 shows the differences in the amylopectin chains of the three mutant cultivars compared with those of ‘Koshihikari’. ‘Akita Sake 44’, ‘Hiderishirazu D’, and ‘Kurnai’ displayed a higher proportion of short-length chains and a lower proportion of middlelength chains compared with those of ‘Koshihikari’. The chainlength distributions of ‘Hiderishirazu D’ and ‘Kurnai’ were similar; however, ‘Akita Sake 44’ had a higher content of the DP 8e15 chains and a lower content of the DP 16e36 chains than ‘Hiderishirazu D’ and ‘Kurnai’.
2.4. Bread analysis The volume of the bread was measured by using a laser volume measurement unit (SELNAC-WinVM1100A/2100A; ASTEX, Tokyo, Japan). Specific loaf volume was calculated as volume per weight (mL/g). Statistical evaluations were performed by using Tukey's test. Bread hardness was evaluated with a compression test using a texture analyzer (TA.XTplus, Stable Micro Systems, Godalming, UK). A 1.4-cm-thick slice of bread was compressed to 25% of its thickness at a speed of 1.0 mm/s by using a plunger (3.6 cm diameter) with a flat surface. Multiple comparisons were conducted by using Dunnett's test with the values of ‘Koshihikari’ as the control. The hardening rates of the bread were calculated by means of linear regression analysis with hardness values of one, two, and three days after baking. The slope obtained from the analysis was used as the hardening rate. Statistical evaluations were performed by using Dunnett's test. The values of ‘Koshihikari’ were used as a reference. A sensory test was conducted two days after baking by using 1.4-cm sliced breads. The bread made from ‘Koshihikari’ served as the reference.
3. Results 3.1. Major constituents of the flour from rice cultivars Average particle sizes did not differ among the flour samples (Table 1). Damaged starch contents did not exceed 3% in the flour of any of the samples tested and were lowest in the flour from ‘Koshihikari’ and highest in that from ‘Kurnai’. The amylose content of ‘Kurnai’ was similar to that of ‘Koshihikari’, whereas that of ‘Akita Sake 44’ and ‘Hiderishirazu D’ was about 2% higher than that of ‘Koshihikari’. The genotypes of the Wx gene, which encodes granule-bound starch synthase I and genetically controls amylose contents, were the Wxb allele (japonica-type; lower type of amylose contents) in all four cultivars (data not shown). Genotyping of the alk gene showed that all four cultivars have non-functional alleles (‘Akita Sake 44’: haplotype 4; ‘Hiderishirazu D’ and ‘Kurnai’: haplotype 5 according to the classification of Hiratsuka et al. (2010)). The protein content of ‘Hiderishirazu D’ was slightly higher than that of the other cultivars. The gelatinization temperature of ‘Akita Sake 44’ was about 2 C lower than that of ‘Koshihikari’. The gelatinization temperatures of ‘Hiderishirazu D’ and ‘Kurnai’ were both about 3 C lower than that of ‘Koshihikari’. The pasting temperatures of ‘Akita Sake 44’, ‘Hiderishirazu D’, and ‘Kurnai’ were also lower than that of ‘Koshihikari’. The pasting temperature of ‘Akita Sake 44’ was higher than that of ‘Hiderishirazu D’ and ‘Kurnai’. The water absorption values of ‘Akita Sake 44’ and ‘Hiderishirazu D’ were similar to that of ‘Koshihikari’, but the water absorption value of ‘Kurnai’ was slightly higher than that of ‘Koshihikari’ (Table 1). The pasting properties of the bread dough showed no obvious differences among the cultivars (data not shown).
3.3. Bread shape The heights of the gluten-free breads made from ‘Akita Sake 44’, ‘Hiderishirazu D’, and ‘Kurnai’ were lower than that of ‘Koshihikari’ (Fig. 2A, Table 2). The specific loaf volumes of the gluten-free bread made from three mutant cultivars was lower than that of ‘Koshihikari’. The height of the gluten-containing bread made from ‘Akita Sake 44’ was similar to that of the bread made from ‘Koshihikari’, but the breads made from ‘Hiderishirazu D’ and ‘Kurnai’ were slightly shorter than the bread made from ‘Koshihikari’ (Fig. 2B, Table 2). The specific loaf volume of the gluten-containing bread made from ‘Akita Sake 44’ was higher than that of ‘Koshihikari’, whereas those of ‘Hiderishirazu D’ and ‘Kurnai’ were lower than that of ‘Koshihikari’.
3.4. Hardening rates of rice breads The hardening rates of the gluten-free breads made from ‘Akita Sake 44’, ‘Hiderishirazu D’, and ‘Kurnai’ were 39%, 22%, and 14% of that of ‘Koshihikari’, respectively. One day after baking, the hardness value of the gluten-free bread made from ‘Kurnai’ was slightly lower than that of the bread made from ‘Koshihikari’ (Fig. 3A) and there were no significant differences in hardness between bread made from ‘Akita Sake 44’ or ‘Hiderishirazu D’ compared with bread made from ‘Koshihikari’. However, significant differences were detected between the bread hardness of all three cultivars and that of ‘Koshihikari’ two and three days after baking. The hardening rates of the breads made from ‘Koshihikari’, ‘Akita Sake 44’,
Fig. 1. Differences in the chain-length distribution of isoamylase-debranched starches relative to ‘Koshihikari’.
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Fig. 2. Shapes of gluten-free bread and gluten-containing bread A: Gluten-free bread, B: Gluten-containing bread.
‘Hiderishirazu D’, and ‘Kurnai’ were 175.6, 68.9, 38.7, and 24.9 g/day respectively. The hardening rates of the gluten-containing breads made from ‘Akita Sake 44’, ‘Hiderishirazu D’, and ‘Kurnai’ were 27%, 16%, and 13% of that of ‘Koshihikari’, respectively. However, the bread hardness values of the gluten-containing breads made from ‘Akita Sake 44’, ‘Hiderishirazu D’, and ‘Kurnai’ were similar to those of breads made from ‘Koshihikari’ measured one day after baking (Fig. 3B). Significant differences were detected between the bread hardness values of the three cultivars and that of ‘Koshihikari’ at two and three days after baking. The hardening rates of the breads made from ‘Koshihikari’, ‘Akita Sake 44’, ‘Hiderishirazu D’, and ‘Kurnai’ were 53.9, 14.6, 8.7, and 7.2 g/day respectively.
Table 2 Loaf volumes and heights of rice breads. Gluten-free bread
Gluten-containing bread
Specific loaf Bread height Specific loaf Bread height volume (mL/g) (cm) volume (mL/g) (cm) Koshihikari Akita Sake 44 Hiderishirazu D Kurnai
4.3 3.9 3.6 3.8
± ± ± ±
0.1a 0.2b 0.1b 0.1b
12.6 11.6 10.1 10.5
± ± ± ±
0.4a 0.5a 0.1bc 0.1b
3.9 4.2 3.9 3.7
± ± ± ±
0.1b 0.1a 0.0b 0.0c
13.2 13.2 12.6 12.1
± ± ± ±
0.2a 0.1a 0.3b 0.1c
Means and standard deviations from three replicates are shown. Means ± standard deviations with the same letter are not significantly different from each other (p < 0.05, Tukey's test).
3.5. Sensory test of rice bread Fig. 4 shows a comparison of the softness scores of breads made from ‘Akita Sake 44’, ‘Hiderishirazu D’, and ‘Kurnai’ with the bread made from ‘Koshihikari’ serving as the standard. The ratios from the evaluators who selected positive values (softer than the bread made from ‘Koshihikari’) for the gluten-free bread made from ‘Akita Sake 44’, ‘Hiderishirazu D’, and ‘Kurnai’ were 74.1%, 66.7%, and 85.2%, respectively. The average scores of ‘Akita Sake 44’, ‘Hiderishirazu D’, and ‘Kurnai’ for the gluten-free breads were þ1.04, þ0.78 and þ1.56, respectively. The ratios from the evaluators who selected positive values for the gluten-containing bread made from ‘Akita Sake 44’, ‘Hiderishirazu D’, and ‘Kurnai’ were 37.5%, 59.4%, and 68.8%, respectively. The average sensory scores of ‘Akita Sake 44’, ‘Hiderishirazu D’, and ‘Kurnai’ for the gluten-containing breads were þ0.06, þ0.50, and þ1.13, respectively. Overall, the quality scores were diverse for both the glutencontaining and the gluten-free bread (data not shown).
4. Discussion In this study, we found that the amylopectin chains of ‘Akita Sake 44’, ‘Hiderishirazu D’, and ‘Kurnai’ clearly contained a high proportion of short chains compared with those of ‘Koshihikari’ (Fig. 1), although the other properties of the constituents were similar among the samples (Table 1). These results indicated that
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Fig. 3. Bread hardness A: Gluten-free bread, B: Gluten-containing bread * and ** indicate significant differences (p < 0.05) and (p < 0.01), respectively (Dunnett's test). The values of ‘Koshihikari’ were used as a reference.
these samples were suitable for investigating the relationship between amylopectin properties and bread qualities. A correlation between amylose content and the specific loaf volume of bread has been reported previously (Aoki et al., 2012; Lee et al., 2001). However, in this study, we found significant differences in the loaf volumes among samples (Fig. 2, Table 2), regardless of amylose content (Table 1). This result indicates that bread shape is affected by amylopectin structure. The differences in bread shape may be due to the differences in gelatinization temperature. Bubbles in the dough made from the amylopectin mutant cultivars may stabilize earlier due to the short time to gelatinize during baking. The difference in the bread made from ‘Hiderishirazu D’ and ‘Kurnai’ is, thus, probably due to the difference in amylose content. The compression test showed that the hardening rates of the breads made from ‘Akita Sake 44’, ‘Hiderishirazu D’, and ‘Kurnai’ were markedly lower than those of the control breads (Fig. 3). Sensory tests showed similar results (Fig. 4). There is significant positive correlation between amylose content and bread hardness (Aoki et al., 2012; Takahashi et al., 2009), but in the current study, the differences in amylose content among the samples were small (Table 1). Gluten-free bread contains tapioca starch, but its effect was also negligible, because the ratio between rice flour and tapioca starch was the same in all breads (4:1). These results indicate that it is the amylopectin structure, and not that the amylose content, that affects the slow-hardening of both gluten-
free and gluten-containing bread. The differences in hardening rates between ‘Akita Sake 44’ and ‘Hiderishirazu D’ or ‘Kurnai’ are also probably due to the difference in amylopectin structure (Fig. 1). Although further investigation will be necessary to determine which amylopectin chains are critical for the difference in bread hardness, the difference in gelatinization temperature may also contribute to the difference in bread hardness because gelatinization temperature is positively correlated with starch retrogradation (Vandeputte et al., 2003). The rice cultivars used in this study, ‘Akita Sake 44’, ‘Hiderishirazu D’, and ‘Kurnai’, are suitable for making gluten-free products because the hardening of the breads made from them was significantly slower than that for the control bread (Fig. 3). Nevertheless, there is room to improve these cultivars in order to introduce them to wider areas; ‘Hiderishirazu D’ and ‘Kurnai’ are upland cultivars and ‘Akita Sake 44’ is a cultivar for Japanese rice wine. A DNA marker that detects the mutant allele of SbeI is available (Okamoto et al., 2013a) and useful for selecting and breeding cultivars for slow-hardening rice bread. Okamoto et al. (2013b) reported that ‘Akita Sake 44’ possesses SBEI activity. The amylopectin chain length distribution of ‘Akita Sake 44’ is similar to that of rice endosperm mutants that lack starch phosphorylase activity (Satoh et al., 2008), indicating that ‘Akita Sake 44’ is a phosphorylase mutant. A DNA marker that detects the mutation of ‘Akita Sake 44’ could be generated by investigating the DNA
Fig. 4. Sensory evaluation of bread made from three cultivars with low gelatinization temperatures compared with bread made from ‘Koshihikari’ as a reference. A: Gluten-free bread, B: Gluten-containing bread Softness was scored from 3 (very hard) to 3 (very soft) with that of Koshihikari designated as 0.
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sequence of the starch phosphorylase gene. Such cultivars would be promising materials for producing slow-hardening rice bread. The ratio of short amylopectin chains in ‘Akita Sake 44’ was the highest among the samples (Fig. 1). However, the gelatinization temperature of ‘Akita Sake 44’ was higher than those of ‘Hiderishirazu D’ or ‘Kurnai’ (Table 1). Further investigations are necessary to unravel the cause of this discrepancy, but one possible cause is the difference in the proportion of the DP 8e15 amylopectin chains between ‘Akita Sake 44’ and ‘Hiderishirazu D’ or ‘Kunai’. About 3% of regular wheat bread cannot be sold because of staling (Zobel and Kulp, 1996) and economic losses due to bread staling are estimated to be one billion dollars every year (Berkowitz and Oleksyk, 1991). In this study, we found that shorter amylopectin chains caused by mutations play a role in maintaining the softer texture of rice bread (Figs. 3 and 4). In rice, single-locus recessive mutations of SbeI and the lack of starch phosphorylase activity result in shorter amylopectin chains (Okamoto et al., 2013a; Satoh et al., 2008). Unfortunately, mutants of wheat that lack SBEI activity have been produced but show no significant effects on the chain-length of amylopectin (Regina et al., 2004). If other wheat mutants that have shorter amylopectin chains, (similar to ‘Akita Sake 44’) can be developed, slow-hardening bread could be made by using such mutants. Our findings thus open new avenues for wheat breeding to produce slow-hardening bread, which could have a significant impact on consumers and the bread industry. Acknowledgments We thank T Fujimura at the University of Tsukuba for helpful comments on the manuscript. This study was supported by the Tojuro Iijima Memorial Foundation for the Promotion of Food Science and Technology. References American Association of Cereal Chemists (AACC), 2000a. Approved Methods of the AACC. Method 54-21, tenth ed. AACC, St Paul, MN. American Association of Cereal Chemists (AACC), 2000b. Approved Methods of the AACC. Method 76-13, tenth ed. AACC, St Paul, MN. Ahlborn, G.J., Pike, O.A., Hendrix, S.B., Hess, W.M., Huber, C.S., 2005. Sensory, mechanical, and microscopic evaluation of staling in low-protein and gluten-free breads. Cereal Chem. 82, 328e335. Aoki, N., Umemoto, T., Hamada, S., Suzuki, K., Suzuki, Y., 2012. The amylose content and amylopectin structure affect the shape and hardness of rice bread. J. Appl. Glycosci. 59, 75e82. Berkowitz, D., Oleksyk, L.E., 1991. Leavened Breads with Extended Shelf life. US Patent 5059432. Bosmans, G.M., Lagrain, B., Ooms, N., Fierens, E., Delcour, J.A., 2013. Biopolymer interactions, water dynamics, and bread crumb firming. J. Agric. Food Chem. 61, 4646e4654. Cureton, P., Fasano, A., 2009. The increasing incidence of coeliac disease and the range of gluten-free products in the marketplace. In: Gallagher, E. (Ed.), Glutenfree Food Science and Technology. Wiley-Blackwell Publishing, Oxford, pp. 1e16. Donald, A.M., 2001. Plasticization and self assembly in the starch granule. Cereal Chem. 78, 307e314. Fujita, N., Hasegawa, H., Taira, T., 2001. The isolation and characterization of a waxy mutant of diploid wheat (Triticum monococcum L.). Plant Sci. 160, 595e602. Gray, J.A., Bemiller, J.N., 2003. Bread staling: molecular basis and control. Compr. Rev. Food Sci. Food Saf. 2, 1e21. Hiratsuka, M., Umemoto, T., Aoki, N., Okamoto, K., 2010. Development of SNP markers of starch synthase IIa (alk) and haplotype distribution in rice core collections. Rice Genet. Newsl. 25, 80e82.
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The roles of amylose and amylopectin in the gelation and retrogradation of starch. Carbohydr. Res. 135, 271e281. Morgan, K.R., Gerrard, J., Every, D., Ross, M., Gilpin, M., 1997. Staling in starch breads: the effect of antistaling a-amylase. Starch-St€ arke 49, 54e59. Nakamura, Y., 2002. Towards a better understanding of the metabolic system for amylopectin biosynthesis in plants: rice endosperm as a model tissue. Plant Cell Physiol. 43, 718e725. Okamoto, K., Aoki, N., Fujii, H., Yanagihara, T., Nishi, A., Satoh, H., Umemoto, T., 2013a. Characterization and utilization of spontaneous deficiency in starch branching enzyme I of rice (Oryza sativa L.). J. Appl. Glycosci. 60, 53e60. Okamoto, K., Tabata, M., Kawamata, K., Aoki, N., Umemoto, T., 2013b. Characteristics about lower gelatinizated line selected from rice suited for sake brewing. Breed. Res. 15 (Suppl. 1), 79. Regina, A., Kosar-Hashemi, B., Li, Z., Rampling, L., Cmiel, M., Gianibelli, M.C., KonikRose, C., Larroque, O., Rahman, S., Morell, M.K., 2004. Multiple isoforms of starch branching enzyme-I in wheat: lack of the major SBE-I isoform does not alter starch phenotype. Funct. Plant Biol. 31, 591e601. Ribotta, P.D., Le Bail, A., 2007. Thermo-physical assessment of bread during staling. LWT e Food Sci. Technol. 40, 879e884. Sanchez, H.D., Osella, C.A., Torre, M.A., 2002. Optimization of gluten-free bread prepared from cornstarch, rice flour, and cassava starch. J. Food Sci. 67, 416e419. Satoh, H., Nishi, A., Yamashita, K., Takemoto, Y., Tanaka, Y., Hosaka, Y., Sakurai, A., Fujita, N., Nakamura, Y., 2003. Starch-branching enzyme I-deficient mutation specifically affects the structure and properties of starch in rice endosperm. Plant Physiol. 133, 1111e1121. Satoh, H., Shibahara, K., Tokunaga, T., Nishi, A., Tasaki, M., Hwang, S.-K., Okita, T.W., Kaneko, N., Fujita, N., Yoshida, M., Hosaka, Y., Sato, A., Utsumi, Y., Ohdan, T., Nakamura, Y., 2008. Mutation of the plastidial a-glucan phosphorylase gene in rice affects the synthesis and structure of starch in the endosperm. Plant Cell 20, 1833e1849. Schober, T.J., 2009. Manufacture of gluten-free specialty breads and confectionery products. In: Gallagher, E. (Ed.), Gluten-free Food Science and Technology. Wiley-Blackwell Publishing, Oxford, pp. 130e180. Takahashi, M., Homma, N., Morohashi, K., Nakamura, K., Suzuki, Y., 2009. Effect of rice cultivar characteristics on the rice flour bread quality (in Japanese). J. Jpn. Soc. Food Sci. Technol. 56, 394e402. Tanaka, J., Ikeda, S., 2002. Rapid and efficient DNA extraction method from various plant species using diatomaceous earth and a spin filter. Breed. Sci. 52, 151e155. Toyoshima, H., Okadome, H., Ohtsubo, K.i., Suto, M., Horisue, N., Inatsu, O., Narizuka, A., Aizaki, M., Okawa, T., Inouchi, N., Fuwa, H., 1997. Cooperative test on the small-scale rapid method for the gelatinization properties test of rice flours with a Rapid-Visco-Analyser (RVA) (in Japanese). J. Jpn. Soc. Food Sci. Technol. 44, 579e584. Umemoto, T., Aoki, N., Lin, H.X., Nakamura, Y., Inouchi, N., Sato, Y., Yano, M., Hirabayashi, H., Maruyama, S., 2004. Natural variation in rice starch synthase IIa affects enzyme and starch properties. Funct. Plant Biol. 31, 671e684. 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. J. Cereal Sci. 38, 61e68. Yamanaka, S., Nakamura, I., Watanabe, K., Sato, Y.-I., 2004. Identification of SNPs in the waxy gene among glutinous rice cultivars and their evolutionary significance during the domestication process of rice. Theor. Appl. Genet. 108, 1200e1204. Yamauchi, H., Noda, T., Matsuura-Endo, C., Takigawa, S., Saito, K., Oda, Y., Funatsuki, W., Iriki, N., Hashimoto, N., 2004. Bread-making quality of wheat/rice flour blends. Food Sci. Technol. Res. 10, 247e253. Zobel, H.F., Kulp, K., 1996. The staling mechanism. In: Hebeda, R.E., Zobel, H.F. (Eds.), Baked Goods Freshness: Technology, Evaluation and Inhibition of Staling. Marcel Dekker, New York, pp. 1e64.
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Journal of Cereal Science journal homepage: www.elsevier.com/locate/jcs
Mutants that have shorter amylopectin chains are promising materials for slow-hardening rice bread Noriaki Aoki a, b, Takayuki Umemoto c, Kazuyuki Okamoto d, Yasuhiro Suzuki e, Junichi Tanaka b, e, * a
National Agriculture and Food Research Organization, Headquarters 3-1-1 Kannondai, Tsukuba, Ibaraki 305-8666, Japan University of Tsukuba Graduate School of Life and Environmental Sciences, 2-1-18 Kannondai, Tsukuba, Ibaraki 305-8518, Japan NARO Hokkaido Agricultural Research Center, 1 Hitsujigaoka, Toyohiraku, Sapporo 062-8555, Japan d Ibaraki Agricultural Center Plant-Biotechnology Institute, 3402 Kamikunii, Mito, Ibaraki 311-4203, Japan e NARO Institute of Crop Science, 2-1-18 Kannondai, Tsukuba, Ibaraki 305-8518, Japan b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 26 May 2014 Received in revised form 20 August 2014 Accepted 22 September 2014 Available online 22 November 2014
Bread staling is a serious economic issue for the baking industry. Here, we found that shorter amylopectin chains caused by mutations play a role in maintaining the softer texture of rice bread. We used three rice cultivars that have a high proportion of short amylopectin chains in endosperm starch, two of which were starch branching enzyme I mutants, to make gluten-free and gluten-containing bread. Compression tests showed that the hardening rates for both types of bread made from these cultivars were markedly lower than those for control rice breads (gluten-free bread: 14%e39%, gluten-containing bread: 13%e27%), although there were no clear differences in the hardness values among the breads one day after baking. Sensory tests conducted two days after baking showed that gluten-free breads made from the three cultivars were softer than the control breads. Amylose contents, flour particle sizes, and damaged starch contents were similar among the flour samples, indicating that shorter amylopectin chains led to the slow-hardening of the rice bread. This finding can be applied not only to the breeding of rice cultivars for softer bread, but also to breeding of wheat and other cereals for bread. © 2014 Elsevier Ltd. All rights reserved.
Keywords: Amylopectin Rice Bread staling Starch branching enzyme I
1. Introduction Bread easily stales within a few days and its palatability reduces. Bread staling is not only a serious issue for bread quality to consumers but also causes huge economic losses to the baking and restaurant industries. Rice flour is used to make gluten-free bread, which is helpful for those who are allergic to wheat protein or have celiac disease (Cureton and Fasano, 2009); however, it stales faster than regular wheat bread (Ahlborn et al., 2005). Starch is thought to affect the staling of both gluten-free and regular wheat bread (Gray and Bemiller, 2003; Morgan et al., 1997). Therefore, one approach to solving the problem is to breed cultivars that produce breads that show less staling during preservation by changing the starch properties of such cultivars through mutations.
Abbreviations: DP, degree of polymerization; SbeI, starch branching enzyme I. * Corresponding author. NARO Institute of Crop Science, 2-1-18 Kannondai, Tsukuba, Ibaraki 305-8518, Japan. Tel.: þ81 29 838 8536. E-mail address: [email protected] (J. Tanaka). http://dx.doi.org/10.1016/j.jcs.2014.09.006 0733-5210/© 2014 Elsevier Ltd. All rights reserved.
Rice is suitable for investigating the relationship between starch and bread staling for two reasons. First, rice proteins do not form a gluten matrix like wheat proteins, hence the properties of starch directly affect bread qualities. Second, rice is a good material for genetic analyses, because it is a diploid self-pollinating species, that is, it is relatively easy to correct recessive alleles. Many rice mutants have been screened and characterized, including mutants with unique starch properties (Nakamura, 2002). Starch consists of two components, amylose and amylopectin. Amylose is essentially a linear molecule containing a-(1-4)-linked glucose units with few branches, whereas amylopectin is a branched molecule with linear chains of a-(1-4)-linked glucose units with a-(1-6)-linked branches (Hizukuri et al., 1989). Native starch is closely packed to form clusters of double helices, and starch granules comprise an amorphous region and a semicrystalline region (Donald, 2001). Starch is gelatinized by denaturing its structure under high temperature in the presence of water and the gelatinization temperature depends on amylopectin structure (Jane et al., 1999). When the gelatinized starch is cooled,
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starch is retrograded by partial recrystallization of the linear chains. This starch retrogradation is believed to play a major role in the staling process (Bosmans et al., 2013; Hug-Iten et al., 2003). Although both amylose and amylopectin undergo retrogradation, amylose is retrograded within hours, whereas amylopectin is retrograded for days (Miles et al., 1985; Ribotta and Le-Bail, 2007). Therefore, we hypothesized that amylopectin plays a major role in bread staling during long-term storage. Rice amylopectin is mainly classified into S-type and L-type (Nakamura, 2002) based on the absence or presence of Starch Synthase IIa activity (Umemoto et al., 2004). S-type amylopectin has a higher ratio of short chains than does L-type amylopectin. Moreover, the gelatinization temperature of starch with S-type amylopectin is lower than that of starch with L-type amylopectin. In our previous report, gluten-containing rice breads made from cultivars that have L-type amylopectin were more prone to hardening than bread made from cultivars that have S-type amylopectin (Aoki et al., 2012). Therefore, we hypothesized that softer bread (both gluten-containing and gluten-free) can be made by using cultivars that have shorter amylopectin chains than those in S-type amylopectin. Rice mutants of starch branching enzyme I (SbeI) have a high proportion of shorter amylopectin chains and a lower gelatinization temperature compared with cultivars that have regular Stype amylopectin (Okamoto et al., 2013a; Satoh et al., 2003). Here, we report that slow-hardening gluten-free and gluten-containing breads can be obtained by using mutants that have shorter amylopectin chains than those in S-type amylopectin.
2. Experimental 2.1. Materials Rice (Oryza sativa L.) cultivars, ‘Akita Sake 44’, ‘Hiderishirazu D’, and ‘Kurnai’ that are rich in shorter chains of amylopectin, were used. ‘Koshihikari’, which is the most popular cultivar in Japan, was used as a control (Table 1). ‘Hiderishirazu D’ and ‘Kurnai’ are natural loss-of-function mutants of SbeI. The mutations of ‘Hiderishirazu D’ and ‘Kurnai’ are considered to be of common origin, because no difference was detected between the DNA sequences of SbeI between ‘Hiderishirazu D’ and ‘Kurnai’ (Okamoto et al., 2013a). ‘Akita Sake 44’ is a gamma-ray-induced mutant with a low gelatinization temperature (Okamoto, unpublished result). Polished grains of rice were ground by using a jet mill (SPM-R290; Nishimura Machine Works, Osaka, Japan) after being soaked in a solution containing 0.3% trisodium citrate dihydrate and 0.05% pectinase (Pectinase G; Amano Enzyme, Nagoya, Japan) for 1 h at 40 C.
2.1.1. Genotyping of the wx and alk locus Total DNA was extracted by using diatomaceous earth and a spin filter (Tanaka and Ikeda, 2002). The waxy (wx) locus was genotyped by using the wx-allele-specific dCPAS marker (Yamanaka et al.,
2004). The alk locus, which encodes starch synthase IIa, was genotyped by using SNP markers (Hiratsuka et al., 2010). 2.2. Flour property measurements Flour particle sizes were measured with a laser diffraction particle size analyzer (LS 13 320; Beckman Coulter, Brea, USA). Damaged starch contents were measured according to the American Association of Cereal Chemists (AACC) method 76-31 (AACC, 2000b) with a starch damage assay kit (Megazyme International Ireland, Wicklow, Ireland). Apparent amylose contents were determined by using the iodine absorption method with an Auto Analyzer (BRAN þ LUEBBE, Norderstedt, Germany). The nitrogen content of flour was determined by using a Sumigraph NC-22F (Sumika Company Ltd., Osaka, Japan) and acetanilide as a standard; protein values were calculated by multiplying the nitrogen content by 5.95. Pasting properties were determined with a Rapid Visco Analyzer (RVA Model 3D; Newport Scientific, Warriewood, Australia) as previously described (Toyoshima et al., 1997). The temperature at the onset of the rise in viscosity was determined as the pasting temperature. The thermograms of the starch granules were recorded on a differential scanning calorimeter (DSC7; PerkinElmer Inc., Waltham, USA) with distilled water as the reference. Rice flour (10 mg) and distilled water (25 mL) were sealed in a platinum pan. Samples were heated from 10 C to 100 C at a rate of 10 C/min. The peak temperatures were determined as the gelatinization temperature. Water absorption of rice flour was determined as the amount of water needed to develop a standard dough of 500 Brabender units at the peak of the curve, as determined by using a Farinograph (Brabender Inc., Duisburg, Germany) according to the AACC method 54-21 (AACC, 2000a). A mixture of rice flour (40 g) and vital wheat gluten (10 g) was used (14% moisture basis). Amylopectin chain-length distribution was analyzed by using capillary electrophoresis as described by Fujita et al. (2001). Statistical evaluations were performed by using Tukey's test. 2.3. Bread making We made two types of bread, gluten-free bread and glutencontaining bread. Gluten-free bread was made by using 480 g of rice flour, 120 g of tapioca starch, 12.0 g of hydroxypropyl methylcellulose (Metolose; Shin-Etsu Chemical, Tokyo, Japan), 48.0 g of sugar, 24.0 g of skimmed milk, 12.0 g of salt, 42.0 g of shortening, 24.0 g of dry yeast, and 630 mL of water. Tapioca starch, which is often used in gluten-free bread-making (Schober, 2009), was added in an attempt to make bread larger (Kusunose et al., 1999; Sanchez et al., 2002). The dough was mixed in a Kanto mixer (HPi-20M; Kanto Mixer, Tokyo, Japan). Portions of dough (400 g) were placed in bread pans and proofed at 27 C and 80% humidity for 60 min. Breads were baked at 200 C for 20 min without steam. Gluten-containing bread was made by using 600 g of rice flour (14% moisture basis), 150 g of vital wheat gluten (14% moisture
Table 1 Properties of rice flour samples.
Koshihiakri Akita Sake 44 Hiderishirazu D Kurnai a
Average particle size (mm)a
Damaged starch content (%)b
32.9 30.3 31.3 34.9
1.9 2.4 2.7 3.0
± ± ± ±
0.1d 0.1c 0.1b 0.1a
Amylose content (%)b 18.0 20.2 20.7 17.6
± ± ± ±
0.2b 0.4a 0.2a 0.3b
Protein content (%)b 5.1 5.1 7.0 5.9
± ± ± ±
0.1c 0.2c 0.0a 0.1b
Pasting temperature ( C)b 68.8 66.4 65.7 65.2
± ± ± ±
0.8a 0.7a 0.7b 0.4b
Gelatinization temperature ( C)b 69.9 66.8 65.2 65.8
± ± ± ±
0.3a 0.1b 0.1c 0.4c
Water absorption (%) 77.4 77.1 77.1 79.3
Means of two replicates are shown. Means and standard deviations from three replicates are shown. Means ± standard deviations with the same letter are not significantly different from each other (p < 0.05, Tukey's test). b
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basis; Riken Vitamin, Tokyo, Japan), 52.5 g of sugar, 22.5 g of skimmed milk, 11.3 g of salt, 60 g of shortening, 11.3 g of dry yeast, and water according to the no-time method (Yamauchi et al., 2004). The amount of added water was determined on the basis of the water absorption value obtained as described earlier. A Kanto mixer was used to mix the dough and then 400-g portions were put in loaf pans and allowed to rise for about 1 h at 38 C and 80% humidity until the dough reached the top of the pan. The dough was then baked at 185 C for 28 min.
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3.2. Amylopectin structure Fig. 1 shows the differences in the amylopectin chains of the three mutant cultivars compared with those of ‘Koshihikari’. ‘Akita Sake 44’, ‘Hiderishirazu D’, and ‘Kurnai’ displayed a higher proportion of short-length chains and a lower proportion of middlelength chains compared with those of ‘Koshihikari’. The chainlength distributions of ‘Hiderishirazu D’ and ‘Kurnai’ were similar; however, ‘Akita Sake 44’ had a higher content of the DP 8e15 chains and a lower content of the DP 16e36 chains than ‘Hiderishirazu D’ and ‘Kurnai’.
2.4. Bread analysis The volume of the bread was measured by using a laser volume measurement unit (SELNAC-WinVM1100A/2100A; ASTEX, Tokyo, Japan). Specific loaf volume was calculated as volume per weight (mL/g). Statistical evaluations were performed by using Tukey's test. Bread hardness was evaluated with a compression test using a texture analyzer (TA.XTplus, Stable Micro Systems, Godalming, UK). A 1.4-cm-thick slice of bread was compressed to 25% of its thickness at a speed of 1.0 mm/s by using a plunger (3.6 cm diameter) with a flat surface. Multiple comparisons were conducted by using Dunnett's test with the values of ‘Koshihikari’ as the control. The hardening rates of the bread were calculated by means of linear regression analysis with hardness values of one, two, and three days after baking. The slope obtained from the analysis was used as the hardening rate. Statistical evaluations were performed by using Dunnett's test. The values of ‘Koshihikari’ were used as a reference. A sensory test was conducted two days after baking by using 1.4-cm sliced breads. The bread made from ‘Koshihikari’ served as the reference.
3. Results 3.1. Major constituents of the flour from rice cultivars Average particle sizes did not differ among the flour samples (Table 1). Damaged starch contents did not exceed 3% in the flour of any of the samples tested and were lowest in the flour from ‘Koshihikari’ and highest in that from ‘Kurnai’. The amylose content of ‘Kurnai’ was similar to that of ‘Koshihikari’, whereas that of ‘Akita Sake 44’ and ‘Hiderishirazu D’ was about 2% higher than that of ‘Koshihikari’. The genotypes of the Wx gene, which encodes granule-bound starch synthase I and genetically controls amylose contents, were the Wxb allele (japonica-type; lower type of amylose contents) in all four cultivars (data not shown). Genotyping of the alk gene showed that all four cultivars have non-functional alleles (‘Akita Sake 44’: haplotype 4; ‘Hiderishirazu D’ and ‘Kurnai’: haplotype 5 according to the classification of Hiratsuka et al. (2010)). The protein content of ‘Hiderishirazu D’ was slightly higher than that of the other cultivars. The gelatinization temperature of ‘Akita Sake 44’ was about 2 C lower than that of ‘Koshihikari’. The gelatinization temperatures of ‘Hiderishirazu D’ and ‘Kurnai’ were both about 3 C lower than that of ‘Koshihikari’. The pasting temperatures of ‘Akita Sake 44’, ‘Hiderishirazu D’, and ‘Kurnai’ were also lower than that of ‘Koshihikari’. The pasting temperature of ‘Akita Sake 44’ was higher than that of ‘Hiderishirazu D’ and ‘Kurnai’. The water absorption values of ‘Akita Sake 44’ and ‘Hiderishirazu D’ were similar to that of ‘Koshihikari’, but the water absorption value of ‘Kurnai’ was slightly higher than that of ‘Koshihikari’ (Table 1). The pasting properties of the bread dough showed no obvious differences among the cultivars (data not shown).
3.3. Bread shape The heights of the gluten-free breads made from ‘Akita Sake 44’, ‘Hiderishirazu D’, and ‘Kurnai’ were lower than that of ‘Koshihikari’ (Fig. 2A, Table 2). The specific loaf volumes of the gluten-free bread made from three mutant cultivars was lower than that of ‘Koshihikari’. The height of the gluten-containing bread made from ‘Akita Sake 44’ was similar to that of the bread made from ‘Koshihikari’, but the breads made from ‘Hiderishirazu D’ and ‘Kurnai’ were slightly shorter than the bread made from ‘Koshihikari’ (Fig. 2B, Table 2). The specific loaf volume of the gluten-containing bread made from ‘Akita Sake 44’ was higher than that of ‘Koshihikari’, whereas those of ‘Hiderishirazu D’ and ‘Kurnai’ were lower than that of ‘Koshihikari’.
3.4. Hardening rates of rice breads The hardening rates of the gluten-free breads made from ‘Akita Sake 44’, ‘Hiderishirazu D’, and ‘Kurnai’ were 39%, 22%, and 14% of that of ‘Koshihikari’, respectively. One day after baking, the hardness value of the gluten-free bread made from ‘Kurnai’ was slightly lower than that of the bread made from ‘Koshihikari’ (Fig. 3A) and there were no significant differences in hardness between bread made from ‘Akita Sake 44’ or ‘Hiderishirazu D’ compared with bread made from ‘Koshihikari’. However, significant differences were detected between the bread hardness of all three cultivars and that of ‘Koshihikari’ two and three days after baking. The hardening rates of the breads made from ‘Koshihikari’, ‘Akita Sake 44’,
Fig. 1. Differences in the chain-length distribution of isoamylase-debranched starches relative to ‘Koshihikari’.
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Fig. 2. Shapes of gluten-free bread and gluten-containing bread A: Gluten-free bread, B: Gluten-containing bread.
‘Hiderishirazu D’, and ‘Kurnai’ were 175.6, 68.9, 38.7, and 24.9 g/day respectively. The hardening rates of the gluten-containing breads made from ‘Akita Sake 44’, ‘Hiderishirazu D’, and ‘Kurnai’ were 27%, 16%, and 13% of that of ‘Koshihikari’, respectively. However, the bread hardness values of the gluten-containing breads made from ‘Akita Sake 44’, ‘Hiderishirazu D’, and ‘Kurnai’ were similar to those of breads made from ‘Koshihikari’ measured one day after baking (Fig. 3B). Significant differences were detected between the bread hardness values of the three cultivars and that of ‘Koshihikari’ at two and three days after baking. The hardening rates of the breads made from ‘Koshihikari’, ‘Akita Sake 44’, ‘Hiderishirazu D’, and ‘Kurnai’ were 53.9, 14.6, 8.7, and 7.2 g/day respectively.
Table 2 Loaf volumes and heights of rice breads. Gluten-free bread
Gluten-containing bread
Specific loaf Bread height Specific loaf Bread height volume (mL/g) (cm) volume (mL/g) (cm) Koshihikari Akita Sake 44 Hiderishirazu D Kurnai
4.3 3.9 3.6 3.8
± ± ± ±
0.1a 0.2b 0.1b 0.1b
12.6 11.6 10.1 10.5
± ± ± ±
0.4a 0.5a 0.1bc 0.1b
3.9 4.2 3.9 3.7
± ± ± ±
0.1b 0.1a 0.0b 0.0c
13.2 13.2 12.6 12.1
± ± ± ±
0.2a 0.1a 0.3b 0.1c
Means and standard deviations from three replicates are shown. Means ± standard deviations with the same letter are not significantly different from each other (p < 0.05, Tukey's test).
3.5. Sensory test of rice bread Fig. 4 shows a comparison of the softness scores of breads made from ‘Akita Sake 44’, ‘Hiderishirazu D’, and ‘Kurnai’ with the bread made from ‘Koshihikari’ serving as the standard. The ratios from the evaluators who selected positive values (softer than the bread made from ‘Koshihikari’) for the gluten-free bread made from ‘Akita Sake 44’, ‘Hiderishirazu D’, and ‘Kurnai’ were 74.1%, 66.7%, and 85.2%, respectively. The average scores of ‘Akita Sake 44’, ‘Hiderishirazu D’, and ‘Kurnai’ for the gluten-free breads were þ1.04, þ0.78 and þ1.56, respectively. The ratios from the evaluators who selected positive values for the gluten-containing bread made from ‘Akita Sake 44’, ‘Hiderishirazu D’, and ‘Kurnai’ were 37.5%, 59.4%, and 68.8%, respectively. The average sensory scores of ‘Akita Sake 44’, ‘Hiderishirazu D’, and ‘Kurnai’ for the gluten-containing breads were þ0.06, þ0.50, and þ1.13, respectively. Overall, the quality scores were diverse for both the glutencontaining and the gluten-free bread (data not shown).
4. Discussion In this study, we found that the amylopectin chains of ‘Akita Sake 44’, ‘Hiderishirazu D’, and ‘Kurnai’ clearly contained a high proportion of short chains compared with those of ‘Koshihikari’ (Fig. 1), although the other properties of the constituents were similar among the samples (Table 1). These results indicated that
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Fig. 3. Bread hardness A: Gluten-free bread, B: Gluten-containing bread * and ** indicate significant differences (p < 0.05) and (p < 0.01), respectively (Dunnett's test). The values of ‘Koshihikari’ were used as a reference.
these samples were suitable for investigating the relationship between amylopectin properties and bread qualities. A correlation between amylose content and the specific loaf volume of bread has been reported previously (Aoki et al., 2012; Lee et al., 2001). However, in this study, we found significant differences in the loaf volumes among samples (Fig. 2, Table 2), regardless of amylose content (Table 1). This result indicates that bread shape is affected by amylopectin structure. The differences in bread shape may be due to the differences in gelatinization temperature. Bubbles in the dough made from the amylopectin mutant cultivars may stabilize earlier due to the short time to gelatinize during baking. The difference in the bread made from ‘Hiderishirazu D’ and ‘Kurnai’ is, thus, probably due to the difference in amylose content. The compression test showed that the hardening rates of the breads made from ‘Akita Sake 44’, ‘Hiderishirazu D’, and ‘Kurnai’ were markedly lower than those of the control breads (Fig. 3). Sensory tests showed similar results (Fig. 4). There is significant positive correlation between amylose content and bread hardness (Aoki et al., 2012; Takahashi et al., 2009), but in the current study, the differences in amylose content among the samples were small (Table 1). Gluten-free bread contains tapioca starch, but its effect was also negligible, because the ratio between rice flour and tapioca starch was the same in all breads (4:1). These results indicate that it is the amylopectin structure, and not that the amylose content, that affects the slow-hardening of both gluten-
free and gluten-containing bread. The differences in hardening rates between ‘Akita Sake 44’ and ‘Hiderishirazu D’ or ‘Kurnai’ are also probably due to the difference in amylopectin structure (Fig. 1). Although further investigation will be necessary to determine which amylopectin chains are critical for the difference in bread hardness, the difference in gelatinization temperature may also contribute to the difference in bread hardness because gelatinization temperature is positively correlated with starch retrogradation (Vandeputte et al., 2003). The rice cultivars used in this study, ‘Akita Sake 44’, ‘Hiderishirazu D’, and ‘Kurnai’, are suitable for making gluten-free products because the hardening of the breads made from them was significantly slower than that for the control bread (Fig. 3). Nevertheless, there is room to improve these cultivars in order to introduce them to wider areas; ‘Hiderishirazu D’ and ‘Kurnai’ are upland cultivars and ‘Akita Sake 44’ is a cultivar for Japanese rice wine. A DNA marker that detects the mutant allele of SbeI is available (Okamoto et al., 2013a) and useful for selecting and breeding cultivars for slow-hardening rice bread. Okamoto et al. (2013b) reported that ‘Akita Sake 44’ possesses SBEI activity. The amylopectin chain length distribution of ‘Akita Sake 44’ is similar to that of rice endosperm mutants that lack starch phosphorylase activity (Satoh et al., 2008), indicating that ‘Akita Sake 44’ is a phosphorylase mutant. A DNA marker that detects the mutation of ‘Akita Sake 44’ could be generated by investigating the DNA
Fig. 4. Sensory evaluation of bread made from three cultivars with low gelatinization temperatures compared with bread made from ‘Koshihikari’ as a reference. A: Gluten-free bread, B: Gluten-containing bread Softness was scored from 3 (very hard) to 3 (very soft) with that of Koshihikari designated as 0.
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sequence of the starch phosphorylase gene. Such cultivars would be promising materials for producing slow-hardening rice bread. The ratio of short amylopectin chains in ‘Akita Sake 44’ was the highest among the samples (Fig. 1). However, the gelatinization temperature of ‘Akita Sake 44’ was higher than those of ‘Hiderishirazu D’ or ‘Kurnai’ (Table 1). Further investigations are necessary to unravel the cause of this discrepancy, but one possible cause is the difference in the proportion of the DP 8e15 amylopectin chains between ‘Akita Sake 44’ and ‘Hiderishirazu D’ or ‘Kunai’. About 3% of regular wheat bread cannot be sold because of staling (Zobel and Kulp, 1996) and economic losses due to bread staling are estimated to be one billion dollars every year (Berkowitz and Oleksyk, 1991). In this study, we found that shorter amylopectin chains caused by mutations play a role in maintaining the softer texture of rice bread (Figs. 3 and 4). In rice, single-locus recessive mutations of SbeI and the lack of starch phosphorylase activity result in shorter amylopectin chains (Okamoto et al., 2013a; Satoh et al., 2008). Unfortunately, mutants of wheat that lack SBEI activity have been produced but show no significant effects on the chain-length of amylopectin (Regina et al., 2004). If other wheat mutants that have shorter amylopectin chains, (similar to ‘Akita Sake 44’) can be developed, slow-hardening bread could be made by using such mutants. Our findings thus open new avenues for wheat breeding to produce slow-hardening bread, which could have a significant impact on consumers and the bread industry. Acknowledgments We thank T Fujimura at the University of Tsukuba for helpful comments on the manuscript. This study was supported by the Tojuro Iijima Memorial Foundation for the Promotion of Food Science and Technology. References American Association of Cereal Chemists (AACC), 2000a. Approved Methods of the AACC. Method 54-21, tenth ed. AACC, St Paul, MN. American Association of Cereal Chemists (AACC), 2000b. Approved Methods of the AACC. Method 76-13, tenth ed. AACC, St Paul, MN. Ahlborn, G.J., Pike, O.A., Hendrix, S.B., Hess, W.M., Huber, C.S., 2005. Sensory, mechanical, and microscopic evaluation of staling in low-protein and gluten-free breads. Cereal Chem. 82, 328e335. Aoki, N., Umemoto, T., Hamada, S., Suzuki, K., Suzuki, Y., 2012. The amylose content and amylopectin structure affect the shape and hardness of rice bread. J. Appl. Glycosci. 59, 75e82. Berkowitz, D., Oleksyk, L.E., 1991. Leavened Breads with Extended Shelf life. US Patent 5059432. Bosmans, G.M., Lagrain, B., Ooms, N., Fierens, E., Delcour, J.A., 2013. Biopolymer interactions, water dynamics, and bread crumb firming. J. Agric. Food Chem. 61, 4646e4654. Cureton, P., Fasano, A., 2009. The increasing incidence of coeliac disease and the range of gluten-free products in the marketplace. In: Gallagher, E. (Ed.), Glutenfree Food Science and Technology. Wiley-Blackwell Publishing, Oxford, pp. 1e16. Donald, A.M., 2001. Plasticization and self assembly in the starch granule. Cereal Chem. 78, 307e314. Fujita, N., Hasegawa, H., Taira, T., 2001. The isolation and characterization of a waxy mutant of diploid wheat (Triticum monococcum L.). Plant Sci. 160, 595e602. Gray, J.A., Bemiller, J.N., 2003. Bread staling: molecular basis and control. Compr. Rev. Food Sci. Food Saf. 2, 1e21. Hiratsuka, M., Umemoto, T., Aoki, N., Okamoto, K., 2010. Development of SNP markers of starch synthase IIa (alk) and haplotype distribution in rice core collections. Rice Genet. Newsl. 25, 80e82.
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