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Bioscience, Biotechnology, and Biochemistry Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tbbb20
Development of formulae for estimating amylose content, amylopectin chain length distribution, and resistant starch content based on the iodine absorption curve of rice starch a
b
Sumiko Nakamura , Hikaru Satoh & Ken’ichi Ohtsubo a
a
Faculty of Agriculture, Niigata University, Niigata, Japan
b
Graduate School of Agriculture, Kyushu University, Fukuoka, Japan Published online: 11 Nov 2014.
To cite this article: Sumiko Nakamura, Hikaru Satoh & Ken’ichi Ohtsubo (2014): Development of formulae for estimating amylose content, amylopectin chain length distribution, and resistant starch content based on the iodine absorption curve of rice starch, Bioscience, Biotechnology, and Biochemistry, DOI: 10.1080/09168451.2014.978257 To link to this article: http://dx.doi.org/10.1080/09168451.2014.978257
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
Bioscience, Biotechnology, and Biochemistry, 2014
Development of formulae for estimating amylose content, amylopectin chain length distribution, and resistant starch content based on the iodine absorption curve of rice starch Sumiko Nakamura1, Hikaru Satoh2 and Ken’ichi Ohtsubo1,* 1
Faculty of Agriculture, Niigata University, Niigata, Japan; 2Graduate School of Agriculture, Kyushu University, Fukuoka, Japan
Received April 14, 2014; accepted September 30, 2014
Downloaded by [University of Chicago Library] at 12:31 19 November 2014
http://dx.doi.org/10.1080/09168451.2014.978257
Not only amylose but also amylopectin greatly affects the gelatinization properties of rice starch and the quality of cooked rice grains. We here characterized the starches of 32 rice cultivars and evaluated the relationship between their iodine absorption curve, apparent amylose content (AAC), pasting property, resistant starch (RS) content, and chain length distribution of amylopectin. We found that the iodine absorption curve differed among the various sample rice cultivars. Using the wavelength at which absorbance becomes maximum on iodine staining of starch (λmax), we propose a novel index, “new λmax” (AAC/(λmax of sample rice starches–λmax of glutinous rice starch)). We developed the novel estimation formulae for AAC, RS contents, and amylopectin fractions with the use of λmax and “new λmax.” These formulae would lead to the improved method for estimating starch properties using an easy and rapid iodine colorimetric method. Key words:
apparent amylose content; resistant starch; iodine absorption curve; amylopectin; rice
Starch is composed of essentially two kinds of α-glucans that have distinctive structure. Amylose is small, linear, and slightly branched molecules, whereas amylopectin is large and highly branched molecule. Particularly, amylose is one of the components of rice starch that greatly affects the quality and gelatinization properties of cooked rice.1) Low-amylose rice generally becomes soft and sticky after cooking, whereas highamylose rice becomes hard with fluffy separated grains.2) The group of high-amylose starches includes two types of rice starches with similar apparent amylose content (AAC) but different super-long chains (SLC) contents of amylopectin.3,4) The structure of amylopectin in the starch granule has been described using a cluster model.5–7) Nakamura et al. classified the
starches of 129 rice varieties cultivated in Asia into two types, L and S, based on the differences in the chain length of the amylopectin cluster.8) Rice starches contain 0–30% of amylose, and the content among varieties of rice varies with the ambient temperatures during development of the grain. The starches of rice grown at low temperature had a significantly higher amylose content than that of rice grown at high temperatures.9–13) Amylose is synthesized by starch granule-bound starch synthase encoded by the waxy gene. The fact that amylose content is usually higher in endosperm starch of indica rice than that of japonica rice has been explained by the presence of two types of waxy alleles, Wxa and Wx.b 14) Inouchi et al.9) and Hirano et al.15) showed a high positive correlation between the amounts of waxy (Wx) protein and the SLC contents of starch. A routine method for the quantification of amylose in rice improvement programs went through rapid development in the 1970s and early 1980s, resulting in the publication of two slightly different methods by International Organization for Standardization (ISO) and American Association of Cereal Chemists International (AACCI). Each different version of the iodine-binding method has led to revision of the protocol for the standard curve. The most widely used method for amylose determination is a colorimetric assay where iodine binds with amylose to produce a blue–purple color, which is measured spectrophotometrically at a single wavelength (620 nm).16) Amylopectin also has a color reaction with iodine, which interfering with the direct measurement of the color generated by the amylose–iodine complex. Dual-wavelength spectrophotometry obtains more accurate measurements for both amylose–iodine and amylopectin–iodine in mixture than the standard method of Juliano.17,18) Fukahori et al.19) showed that the amylose and amylopectin fractionated from different rice cultivars could be evaluated by the dual-wavelength spectrophotometry. By conducting a survey of the methods for measuring amylose in various rice quality evaluation
*Corresponding author. Email: [email protected] Abbreviations: AAC, apparent amylose content; CD, chain length distribution; RS, resistant starch; GI, glycemic index; SLC, super-long chains; SB, setback; BD, breakdown. © 2014 Japan Society for Bioscience, Biotechnology, and Agrochemistry
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laboratories, as well as testing the reproducibility between different laboratories, it was found that at least five different versions of the iodine method are in operation, with variability in the construction of the standard curve, standing time, and wavelength used for measurement.20) It was necessary to develop the method for measuring amylose and together standardize a method for quantifying actual values of amylose. Igarashi et al.21) used an automatic analyzer, developed to obtain an iodine absorption spectrum ranging from 400 to 900 nm setting 600 nm as a bordering wavelength. In this study, wavelength at which absorbance becomes maximum on iodine staining of starch (λmax) and absorbance at λmax (Aλmax) were used for estimating AAC (not used standard curve). Several researchers reported the development of high-resistant starch (RS) rice22,23) as well as high-amylose- and high-dietary-fiber rice,24) through physical and chemical mutations. The glycemic effect of foods depends on numerous factors, such as the structures of amylose and amylopectin structure.25,26) However, the cooked grains of ae mutant rice cultivars are too hard and non-sticky because ae mutant rice cultivars lack starch branching enzyme II b and the presence of SLC. They are promising in terms of their bio-functionality such as diabetes prevention.27–33) RS may be included within the term “fiber” on the nutrition labels used in some countries but not in others.34,35) Resistant starch is starch that escapes digestion in the small intestine and that may be fermented in the large intestine. Four main subtypes of resistant starch have been identified based on structure or source. There are several kinds of method of measurement for determining RS contents.36,37) RS differs depending on the different processing methods. It is necessary to develop the novel and simple method for measuring RS. Physicochemical properties of the starches were often evaluated as pasting characteristics using a Rapid Visco Analyzer (RVA). High-amylose rice cultivars are reported to have a higher final viscosity than lowamylose cultivars, where the final viscosity is related to the degree of starch retrogradation after cooling.3,38) The group of high-amylose starches had different SLC contents of amylopectin, different peak viscosities and setback (SB) values (we use the term “consistency” for “SB” in the this paper) according to the measurements using a RVA.39,40) RVA appears to be a useful tool for the rapid and simple evaluation of amylose contents or chain length distribution of amylopectin, while it was relatively expensive. Umemoto et al.41) showed that the fine structure of amylopectin in the japonica rice variety differed distinctly from that in the indica rice variety. Side-chain structure of amylopectin affects physicochemical properties of the starches, and several methods for determination of molar-based distribution of amylopectin have been reported. Among them, typical method is highperformance anion-exchange chromatography with pulsed amperometric detection（HPAEC-PAD）to analyze microstructure of amylopectin. Different from amylose, it is necessary to use the sophisticated ionchromatography to analyze the fine structure of amylopectin. HPAEC-PAD has been frequently used for the examination of the distribution for amylopectin.
We adopted a special detection system because starch does not show absorption in the region of ultraviolet wave length. As another way, analyzing method of molar molecular weight distribution of amylopectin labeling reducing-ends by fluorescent reagents was reported. Hanashiro et al.42) developed a method for determining the molar-based distribution of amylopectin using fluorescent 2-aminopyridine. The pre-column labeling enables online, direct measurement of a molarbased distribution of the molecules. Measurement of the structure of amylopectin cluster needs sophisticate technology or the expensive equipment. We here characterized the starch of various rice cultivars and evaluated the relationship between their iodine absorption curve and AACs, pasting properties, and resistant starch. The improvements, which we performed for analyzing the iodine colorimetric method, and the novel estimation formulae, which we developed in this paper against the amylose contents, resistant starch or a certain fractions of definite chain length amylopectin, would lead to an easy and low-cost spectroscopic method for starch characteristics.
Materials and methods Materials. Ae mutant rice (EM10, EM189, EM72, EM129, EM16, EM174, and EM145) and wx mutant rice (EM21), japonica-indica hybrid rice (Hoshiyutaka) and japonica rice (Kinmaze) were cultivated in an experimental field of Kyushu University in 2011. The ae mutant rice (Hokurikukona243go), high-quality premium japonica rice (Koshihikari), indica rice (Yumetoiro), and japonica-indica hybrid rice cultivars (Kareimai and Koshinokaori) were cultivated in an experimental field at the Hokuriku Research Center in the Central Agricultural Research Center, Joetsu in 2011. Glutinous rice (Koganemochi), japonica rice (Kamenoo), and high-amylose japonica rice (Ekkako) were cultivated at the Niigata Prefectural Agricultural Research Institute in 2011. Japonica rice cultivars (Kirara 397, Hinohikari, Hitomebore, and Akitakomachi), Japonica-indica hybrid rice cultivar (Jasmin rice), Tropical Japonica rice cultivar (Carnaroli), low-amylose japonica rice (Yumepirika), high-amylose japonica rice (Mizuhochikara), and glutinous rice cultivars (Hakuchomochi and Hiyokumochi) were purchased in a local market. Japonica-indica hybrid rice cultivars (Taiwan aromatic rice, Super hybrid rice, Kitsurin 88go) and japonica rice (Taiwan delicious rice) were kindly provided by Professor J. Cui of Tianjin Agricultural University, China. Preparation of polished white rice samples. Brown rice was polished using an experimental friction-type rice milling machine (Yamamotoseisakusyo, Co. Ltd., Tendoh, Japan) to obtain a milling yield (yield after polishing) of 90–91%. White rice flour was prepared using a cyclone mill (SFC-S1; Udy, Fort Collins, CO) with a screen of 1-mm-diameter pores. Preparation of starch granules. Starch granules were prepared from polished rice flour using the cold alkaline method.43,44)
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Estimation of Starch Properties of Rice by Colorimetric Analysis
Measurement of iodine absorption spectra. The iodine absorption spectrum of the rice starch (treated with alkaline solution) was measured using a Shimadzu UV-1800 spectrophotometer. The AAC of rice starch (treated with alkaline solution) was estimated using the iodine colorimetric method of Juliano.16,17) Potato amylose (type III; Sigma Chemical Co., St. Louis, MO) and waxy rice starch (fat and proteins removed from waxy rice) were used as standard amylose and standard amylopectin, respectively, for amylose determination. The iodine absorption spectrum was analyzed from 200 to 900 nm. The absorbance was measured at 620 nm (followed to Juliano’s method), λmax (peak wavelength on iodine staining of starch, which shows high correlation with the length of glucan chain; molecular size of amylose; and SLC), and absorbance at λmax, 400 nm, and B nm (wave length of first peak below 400 nm), is shown in Fig. 1. The area of F1 (from B nm to 400 nm), area of F2 (from 400 nm to λmax), and area of F3 (from λmax to 900 nm) were calculated. For distinguishing between AAC and SLC, we introduced a novel index, “new λmax,” which is calculated as follows: New kmax ¼ AAC=ðkmax of sample rice starches kmax of glutinous ricestarchÞ New λmax means “revised amylose content including the effect of amylopectin microstructure,” which includes the effect by the characteristics of amylopectin
3
microstructure by dividing AAC by (λmax of various rice starches‐λmax of glutinous starch). λmax of glutinous starch value was calculated from average of Koganemochi, Hakuchomochi, and Hiyokumochi. We defined New λmax of glutinous rice as zero because New λmax is “corrected amylose content including the effect by short-chain amylopectin.” Measurement of pasting properties of rice flours. The pasting properties of starch rice flours were measured using RVA (model Super 4; New-port Scientific Pty Ltd., Warriewood, Australia). A programmed heating and cooling cycle was followed, as described by Toyoshima et al.45) Measurement of RS. RS in the starch rice flour was measured according to the AOAC method using a RS assay kit (Megazyme, Wicklow, Ireland). Each sample (100 mg) was digested with pancreatin and amyloglucosidase at 37 °C for 6 h, and glucose was measured using a spectrophotometer at 510 nm. HPAEC-PAD of isoamylase-debranched materials of starch. The embryo and pericarp were removed from three de-hulled grains of average size. The endosperms were ground with a mortar and pestle, and 5 mg of the powder produced was suspended in 5 mL of methanol
(A)
(B)
(C)
(D)
Fig. 1. Analysis of the iodine absorption curve of starch. Notes: F1: Area from Bnm to 400nm (cm2), F2: Area from 400nm to λmax(cm2), F3: Area from λmax to 900nm (cm2). The iodine absorption spectrum was analyzed from 200 nm to 900 nm. The absorbance was measured at 620 nm, the maximum absorption wavelength (λmax), and absorbance at λmax, 400 nm and B nm is shown. The area of F1 (from B nm to 400 nm), area of F2 (from 400 nm to λmax), area of F3 (from λmax to 900 nm), and the new λmax index [the apparent amylose content of various rice starches / (the maximum absorption wavelength of various rice starches - the maximum absorption wavelength of glutinous starch)] were calculated. (A) Ae mutant rice; EM10, (B) Japonica rice; Koshihikari, (C) High- amylose rice; Koshinokaori, (D) Glutinous rice; Koganemochi.
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and boiled for 10 min. The homogenate was centrifuged at 2500 g for 5 min. The pelleted polyglucan was washed two times with 5 mL of 90% (v/v) methanol, suspended in 5 mL of distilled water, and boiled for 60 min. An aliquot (1.0 mL) of the sample of gelatinized polyglucan was added to 50 μL of 600 mM sodium acetate buffer (pH 4.4) with 10 μL of 2% (w/v) NaN3, and hydrolysis was achieved by the addition of 10 μL of isoamylase from Pseudomonas amyloderamosa (1400 units; Seikagaku Corp., Tokyo, Japan) incubating at 37 °C for 24 h. The aldehyde groups of the debranched glucans were reduced to alcohol groups by treatment with 0.5% (w/v) sodium borohydride under alkaline conditions for 20 h using the method of Nagamine et al.46) The preparation was dried in a vacuumo at room temperature and allowed to dissolve in 20 μL of 1 M NaOH for 60 min. Next, the solution was diluted with 180 μL of distilled water. An aliquot (25 μL) of the preparation was injected into a BioLC system (model DX-500; Dionex, Sunnyvale, CA) equipped with PAD and a Carbopac PA-1 column (4-mm i.d. × 25 cm). Size fractionation of α-1,4 glucans was performed using a linear gradient of sodium acetate (50–500 mM) in 0.1 M NaOH at a flow rate of 1 mL min−1. Statistical analyses. All results, including the significance of regression coefficients, were statistically analyzed using the t-test and one-way ANOVA, method of Tukey, and Excel Statistics (ver. 2006, Microsoft Corp., Tokyo, Japan).
Results and discussion Measurement of iodine absorption spectra The iodine absorption spectrum was analyzed from 200 to 900 nm (Fig. 1). The molecular structures of many starches, including amylose molecular sizes and amylopectin branch chain lengths, have been reported.47–49) The high molecular weight amyloses tend to have a longer wavelength of λmax.19) The λmax value of amylose was 598 nm with a molecular weight of approximately 70,000, and a high positive correlation was observed between absorbance at λmax and amylose content.21) Fig. 1 shows that the iodine absorption spectrum of ae mutant rice cultivar EM10; (A), japonica rice cultivar Koshihikari; (B), highamylose rice Koshinokaori; (C) (japonica-indica hybrid rice) and glutinous rice Koganemochi; (D). The λmax values of the ae mutant rice cultivars tend to be higher than the glutinous rice cultivars, whereas they are lower than the high-amylose rice cultivars, because ae mutant rice cultivars lack starch branching enzyme II b and contain SLC. Table 1 shows that the glutinous rice cultivars have very low λmax values, and indica rice, japonica-indica hybrid rice cultivars, and high-amylose japonica rice cultivar showed higher λmax values, especially Koshinokaori had the highest value. The λmax values of the japonica rice cultivars were intermediate. Table 1 shows that the absorbance at λmax of the ae mutant rice cultivars was higher than the japonica rice, japonica-indica hybrid rice, and the glutinous rice. In
case of similarly λmax value in various rice cultivars EM16 (582.5 nm), Hitomebore (583.0 nm), and Jasmin rice (583.0 nm), the absorbance at λmax of EM16 (0.59) was higher than Hitomebore (0.28) and Jasmin rice (0.28). Table 1 and Fig. 1 reveal that the ae mutant rice cultivar showed higher F2 values than japonica rice, japonica-indica hybrid rice, and the glutinous rice. The indica rice and Yumetoiro showed very high value, and Japonica-indica hybrid rice showed high values, and the japonica rice showed intermediate values. As shown in Table 1 and Fig. 1, the ae mutant rice cultivar had higher F3 values than the japonica rice, japonica-indica hybrid rice, and the glutinous rice. The F3 values of the glutinous rice cultivars were very low, whereas those of the ae mutant rice cultivars were very high. The F3 values of the indica rice, japonica-indica hybrid rice cultivars, and high-amylose japonica rice cultivars were also high, whereas those of the japonica rice cultivars were intermediate. As a result, F2 and F3 values had similar tendency to absorbance at λmax. Table 1 shows that the new λmax of the ae mutant rice cultivars was 2.23–2.99 times greater than those of the japonica rice cultivars, which were very low, particularly the low-amylose japonica rice Yumepirika. Koshihikari, Yumetoiro, and Hoshiyutaka also had high values. Furthermore, Koshinokaori, Carnaroli, Ekkako, and Mizuhochikara had intermediate values. As a result, in case of similar ACC (ACC contain a lot of amylose and a little SLC in amylopectin) in various rice cultivars, the difference of New λmax values is presumed to be related to SLC in amylopectin, as shown in the relationship between Hokurikukona243go (AAC; 21.6%, New λmax; 0.66) and Carnaroli (AAC; 22.0%, New λmax; 0.30), the relationship between Hoshiyutaka (AAC; 27.2%, New λmax; 0.44) and Koshinokaori (AAC; 25.8%, New λmax; 0.31), and the relationship between EM129 (AAC; 31.3%, New λmax; 0.64) and Yumetoiro (AAC; 30.7%, New λmax; 0.46). AAC of starch AAC has been used as a good parameter for estimating the cooking or eating qualities of rice grains and iodine colorimetric method at 620 nm for AAC measurement was developed by Juliano.16) In general, cereal starches are reported to have higher AAC than root and tuber starches, which tend to retrograde more rapidly after boiling, while cereal amyloses are smaller molecules than amyloses of other origins. Moreover, large molecular weight amyloses are readily gelatinized but not retrograded easily.50) Eating or cooking quality of rice is related to the amylose content and the fine structure of amylopectin.51) The AAC contents are higher than the actual amylose contents because of long-chain amylopectin binding with iodine.47) Table 1 shows that the AAC was 21.6–36.8 (%) in the ae mutant rice cultivars, that is, 1.05 times greater than that in the indica rice, Yumetoiro; 1.18 times greater than that in the japonica-indica hybrid rice, Hoshiyutaka; and 2.08 times greater than that in the japonica rice cultivar, Koshihikari. As a result, in case
Koganemochi Hakuchomochi Hiyokumochi
Yumepirika Koshihikari Hinohikari Hitomebore Akitakomachi Kirara397 Kinmaze Taiwan delicious rice Kamenoo Mizuhotikara Ekkako
Carnaroli
Hoshiyutaka Koshinokaori Kiturin88go Jasmin rice Taiwan aromatic rice Kareimai Super hybrid rice
Yumetoiro
B
C
D
E
F
0.71 2.12 0.00 1.41 0.71 0.71 0.00 0.71 0.00
0.00 0.71 0.00
2.83 0.00 0.71 1.41 2.12 0.71 0.71 4.95 2.83 3.54 0.71
2.12
0.00 3.54 0.00 1.41 2.83 2.83 0.00
0.00
566.50 555.50b 568.00a 569.00a 571.50a 582.50 574.00a 571.00a 523.00d
523.00d 522.50d 523.00d
585.00c 552.50b 583.50c 583.00c 585.50c 586.50c 587.50c 588.50c 578.00c 598.50e 581.50c
596.50e
585.00c 605.50e 599.00e 583.00c 594.00e 592.00e 578.00c
589.00e
SD
0.50a
0.45b 0.43a 0.32d 0.28d 0.33d 0.33d 0.29d
0.39b
0.27d 0.34d 0.32d 0.28d 0.29d 0.32d 0.34d 0.30d 0.31d 0.37d 0.33d
0.18c 0.18c 0.17c
0.59 0.47 0.60a 0.61a 0.57a 0.59a 0.54a 0.56a 0.20c
a
Aλmax
0.01
0.00 0.00 0.00 0.00 0.00 0.00 0.00
0.00
0.00 0.00 0.00 0.00 0.00 0.00 0.01 0.00 0.00 0.00 0.00
0.01 0.00 0.00
0.00 0.00 0.00 0.00 0.00 0.01 0.00 0.00 0.00
SD
1.70a
1.60b 1.77a 1.60b 1.54b 1.69a 1.59b 1.53b
1.70
1.48b 1.48b 1.53b 1.43b 1.48b 1.50b 1.57b 1.51b 1.47b 1.71b 1.51d
1.14d 1.13d 1.21d
1.76 1.58 1.75a 1.72a 1.74a 1.90c 1.85c 1.83c 1.21d
a
F1 ＋ F2 (cm2)
0.02
0.02 0.01 0.02 0.01 0.10 0.02 0.05
0.04
0.03 0.04 0.04 0.03 0.02 0.10 0.01 0.04 0.05 0.01 0.03
0.03 0.04 0.02
0.04 0.02 0.02 0.02 0.07 0.09 0.07 0.01 0.03
SD
0.64
0.62 0.80 0.78 0.83 0.80 0.76 0.80
0.79
0.76 0.70 0.84 0.67 0.70 0.76 1.12 0.75 0.68 0.79 0.75
0.75 0.72 0.81
0.63 0.71 0.69 0.66 0.58a 0.77 0.66 0.62 0.76
a
F1 (cm2)
0.02
0.02 0.02 0.02 0.01 0.09 0.02 0.04
0.03
0.03 0.02 0.03 0.03 0.03 0.08 0.00 0.04 0.05 0.01 0.02
0.02 0.02 0.03
0.04 0.02 0.02 0.03 0.04 0.05 0.05 0.01 0.02
SD
1.06
0.98 0.97 0.81 0.71 0.89 0.83 0.73
0.91
0.72 0.78 0.69 0.76 0.78 0.74 0.45 0.75 0.79 0.92 0.75
0.40 0.40 0.40
1.13 0.88a 1.06a 1.06a 1.16a 1.13a 1.19a 1.21a 0.45b
a
F2 (cm2)
Notes: A, Ae mutant rice; B, Glutinous rice; C, Japonica rice; D, Tropical japonica rice; E, Japonica-indica hybrid rice; F, Indica rice. Means with same letter are not significantly different (p
Bioscience, Biotechnology, and Biochemistry Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tbbb20
Development of formulae for estimating amylose content, amylopectin chain length distribution, and resistant starch content based on the iodine absorption curve of rice starch a
b
Sumiko Nakamura , Hikaru Satoh & Ken’ichi Ohtsubo a
a
Faculty of Agriculture, Niigata University, Niigata, Japan
b
Graduate School of Agriculture, Kyushu University, Fukuoka, Japan Published online: 11 Nov 2014.
To cite this article: Sumiko Nakamura, Hikaru Satoh & Ken’ichi Ohtsubo (2014): Development of formulae for estimating amylose content, amylopectin chain length distribution, and resistant starch content based on the iodine absorption curve of rice starch, Bioscience, Biotechnology, and Biochemistry, DOI: 10.1080/09168451.2014.978257 To link to this article: http://dx.doi.org/10.1080/09168451.2014.978257
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
Bioscience, Biotechnology, and Biochemistry, 2014
Development of formulae for estimating amylose content, amylopectin chain length distribution, and resistant starch content based on the iodine absorption curve of rice starch Sumiko Nakamura1, Hikaru Satoh2 and Ken’ichi Ohtsubo1,* 1
Faculty of Agriculture, Niigata University, Niigata, Japan; 2Graduate School of Agriculture, Kyushu University, Fukuoka, Japan
Received April 14, 2014; accepted September 30, 2014
Downloaded by [University of Chicago Library] at 12:31 19 November 2014
http://dx.doi.org/10.1080/09168451.2014.978257
Not only amylose but also amylopectin greatly affects the gelatinization properties of rice starch and the quality of cooked rice grains. We here characterized the starches of 32 rice cultivars and evaluated the relationship between their iodine absorption curve, apparent amylose content (AAC), pasting property, resistant starch (RS) content, and chain length distribution of amylopectin. We found that the iodine absorption curve differed among the various sample rice cultivars. Using the wavelength at which absorbance becomes maximum on iodine staining of starch (λmax), we propose a novel index, “new λmax” (AAC/(λmax of sample rice starches–λmax of glutinous rice starch)). We developed the novel estimation formulae for AAC, RS contents, and amylopectin fractions with the use of λmax and “new λmax.” These formulae would lead to the improved method for estimating starch properties using an easy and rapid iodine colorimetric method. Key words:
apparent amylose content; resistant starch; iodine absorption curve; amylopectin; rice
Starch is composed of essentially two kinds of α-glucans that have distinctive structure. Amylose is small, linear, and slightly branched molecules, whereas amylopectin is large and highly branched molecule. Particularly, amylose is one of the components of rice starch that greatly affects the quality and gelatinization properties of cooked rice.1) Low-amylose rice generally becomes soft and sticky after cooking, whereas highamylose rice becomes hard with fluffy separated grains.2) The group of high-amylose starches includes two types of rice starches with similar apparent amylose content (AAC) but different super-long chains (SLC) contents of amylopectin.3,4) The structure of amylopectin in the starch granule has been described using a cluster model.5–7) Nakamura et al. classified the
starches of 129 rice varieties cultivated in Asia into two types, L and S, based on the differences in the chain length of the amylopectin cluster.8) Rice starches contain 0–30% of amylose, and the content among varieties of rice varies with the ambient temperatures during development of the grain. The starches of rice grown at low temperature had a significantly higher amylose content than that of rice grown at high temperatures.9–13) Amylose is synthesized by starch granule-bound starch synthase encoded by the waxy gene. The fact that amylose content is usually higher in endosperm starch of indica rice than that of japonica rice has been explained by the presence of two types of waxy alleles, Wxa and Wx.b 14) Inouchi et al.9) and Hirano et al.15) showed a high positive correlation between the amounts of waxy (Wx) protein and the SLC contents of starch. A routine method for the quantification of amylose in rice improvement programs went through rapid development in the 1970s and early 1980s, resulting in the publication of two slightly different methods by International Organization for Standardization (ISO) and American Association of Cereal Chemists International (AACCI). Each different version of the iodine-binding method has led to revision of the protocol for the standard curve. The most widely used method for amylose determination is a colorimetric assay where iodine binds with amylose to produce a blue–purple color, which is measured spectrophotometrically at a single wavelength (620 nm).16) Amylopectin also has a color reaction with iodine, which interfering with the direct measurement of the color generated by the amylose–iodine complex. Dual-wavelength spectrophotometry obtains more accurate measurements for both amylose–iodine and amylopectin–iodine in mixture than the standard method of Juliano.17,18) Fukahori et al.19) showed that the amylose and amylopectin fractionated from different rice cultivars could be evaluated by the dual-wavelength spectrophotometry. By conducting a survey of the methods for measuring amylose in various rice quality evaluation
*Corresponding author. Email: [email protected] Abbreviations: AAC, apparent amylose content; CD, chain length distribution; RS, resistant starch; GI, glycemic index; SLC, super-long chains; SB, setback; BD, breakdown. © 2014 Japan Society for Bioscience, Biotechnology, and Agrochemistry
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laboratories, as well as testing the reproducibility between different laboratories, it was found that at least five different versions of the iodine method are in operation, with variability in the construction of the standard curve, standing time, and wavelength used for measurement.20) It was necessary to develop the method for measuring amylose and together standardize a method for quantifying actual values of amylose. Igarashi et al.21) used an automatic analyzer, developed to obtain an iodine absorption spectrum ranging from 400 to 900 nm setting 600 nm as a bordering wavelength. In this study, wavelength at which absorbance becomes maximum on iodine staining of starch (λmax) and absorbance at λmax (Aλmax) were used for estimating AAC (not used standard curve). Several researchers reported the development of high-resistant starch (RS) rice22,23) as well as high-amylose- and high-dietary-fiber rice,24) through physical and chemical mutations. The glycemic effect of foods depends on numerous factors, such as the structures of amylose and amylopectin structure.25,26) However, the cooked grains of ae mutant rice cultivars are too hard and non-sticky because ae mutant rice cultivars lack starch branching enzyme II b and the presence of SLC. They are promising in terms of their bio-functionality such as diabetes prevention.27–33) RS may be included within the term “fiber” on the nutrition labels used in some countries but not in others.34,35) Resistant starch is starch that escapes digestion in the small intestine and that may be fermented in the large intestine. Four main subtypes of resistant starch have been identified based on structure or source. There are several kinds of method of measurement for determining RS contents.36,37) RS differs depending on the different processing methods. It is necessary to develop the novel and simple method for measuring RS. Physicochemical properties of the starches were often evaluated as pasting characteristics using a Rapid Visco Analyzer (RVA). High-amylose rice cultivars are reported to have a higher final viscosity than lowamylose cultivars, where the final viscosity is related to the degree of starch retrogradation after cooling.3,38) The group of high-amylose starches had different SLC contents of amylopectin, different peak viscosities and setback (SB) values (we use the term “consistency” for “SB” in the this paper) according to the measurements using a RVA.39,40) RVA appears to be a useful tool for the rapid and simple evaluation of amylose contents or chain length distribution of amylopectin, while it was relatively expensive. Umemoto et al.41) showed that the fine structure of amylopectin in the japonica rice variety differed distinctly from that in the indica rice variety. Side-chain structure of amylopectin affects physicochemical properties of the starches, and several methods for determination of molar-based distribution of amylopectin have been reported. Among them, typical method is highperformance anion-exchange chromatography with pulsed amperometric detection（HPAEC-PAD）to analyze microstructure of amylopectin. Different from amylose, it is necessary to use the sophisticated ionchromatography to analyze the fine structure of amylopectin. HPAEC-PAD has been frequently used for the examination of the distribution for amylopectin.
We adopted a special detection system because starch does not show absorption in the region of ultraviolet wave length. As another way, analyzing method of molar molecular weight distribution of amylopectin labeling reducing-ends by fluorescent reagents was reported. Hanashiro et al.42) developed a method for determining the molar-based distribution of amylopectin using fluorescent 2-aminopyridine. The pre-column labeling enables online, direct measurement of a molarbased distribution of the molecules. Measurement of the structure of amylopectin cluster needs sophisticate technology or the expensive equipment. We here characterized the starch of various rice cultivars and evaluated the relationship between their iodine absorption curve and AACs, pasting properties, and resistant starch. The improvements, which we performed for analyzing the iodine colorimetric method, and the novel estimation formulae, which we developed in this paper against the amylose contents, resistant starch or a certain fractions of definite chain length amylopectin, would lead to an easy and low-cost spectroscopic method for starch characteristics.
Materials and methods Materials. Ae mutant rice (EM10, EM189, EM72, EM129, EM16, EM174, and EM145) and wx mutant rice (EM21), japonica-indica hybrid rice (Hoshiyutaka) and japonica rice (Kinmaze) were cultivated in an experimental field of Kyushu University in 2011. The ae mutant rice (Hokurikukona243go), high-quality premium japonica rice (Koshihikari), indica rice (Yumetoiro), and japonica-indica hybrid rice cultivars (Kareimai and Koshinokaori) were cultivated in an experimental field at the Hokuriku Research Center in the Central Agricultural Research Center, Joetsu in 2011. Glutinous rice (Koganemochi), japonica rice (Kamenoo), and high-amylose japonica rice (Ekkako) were cultivated at the Niigata Prefectural Agricultural Research Institute in 2011. Japonica rice cultivars (Kirara 397, Hinohikari, Hitomebore, and Akitakomachi), Japonica-indica hybrid rice cultivar (Jasmin rice), Tropical Japonica rice cultivar (Carnaroli), low-amylose japonica rice (Yumepirika), high-amylose japonica rice (Mizuhochikara), and glutinous rice cultivars (Hakuchomochi and Hiyokumochi) were purchased in a local market. Japonica-indica hybrid rice cultivars (Taiwan aromatic rice, Super hybrid rice, Kitsurin 88go) and japonica rice (Taiwan delicious rice) were kindly provided by Professor J. Cui of Tianjin Agricultural University, China. Preparation of polished white rice samples. Brown rice was polished using an experimental friction-type rice milling machine (Yamamotoseisakusyo, Co. Ltd., Tendoh, Japan) to obtain a milling yield (yield after polishing) of 90–91%. White rice flour was prepared using a cyclone mill (SFC-S1; Udy, Fort Collins, CO) with a screen of 1-mm-diameter pores. Preparation of starch granules. Starch granules were prepared from polished rice flour using the cold alkaline method.43,44)
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Estimation of Starch Properties of Rice by Colorimetric Analysis
Measurement of iodine absorption spectra. The iodine absorption spectrum of the rice starch (treated with alkaline solution) was measured using a Shimadzu UV-1800 spectrophotometer. The AAC of rice starch (treated with alkaline solution) was estimated using the iodine colorimetric method of Juliano.16,17) Potato amylose (type III; Sigma Chemical Co., St. Louis, MO) and waxy rice starch (fat and proteins removed from waxy rice) were used as standard amylose and standard amylopectin, respectively, for amylose determination. The iodine absorption spectrum was analyzed from 200 to 900 nm. The absorbance was measured at 620 nm (followed to Juliano’s method), λmax (peak wavelength on iodine staining of starch, which shows high correlation with the length of glucan chain; molecular size of amylose; and SLC), and absorbance at λmax, 400 nm, and B nm (wave length of first peak below 400 nm), is shown in Fig. 1. The area of F1 (from B nm to 400 nm), area of F2 (from 400 nm to λmax), and area of F3 (from λmax to 900 nm) were calculated. For distinguishing between AAC and SLC, we introduced a novel index, “new λmax,” which is calculated as follows: New kmax ¼ AAC=ðkmax of sample rice starches kmax of glutinous ricestarchÞ New λmax means “revised amylose content including the effect of amylopectin microstructure,” which includes the effect by the characteristics of amylopectin
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microstructure by dividing AAC by (λmax of various rice starches‐λmax of glutinous starch). λmax of glutinous starch value was calculated from average of Koganemochi, Hakuchomochi, and Hiyokumochi. We defined New λmax of glutinous rice as zero because New λmax is “corrected amylose content including the effect by short-chain amylopectin.” Measurement of pasting properties of rice flours. The pasting properties of starch rice flours were measured using RVA (model Super 4; New-port Scientific Pty Ltd., Warriewood, Australia). A programmed heating and cooling cycle was followed, as described by Toyoshima et al.45) Measurement of RS. RS in the starch rice flour was measured according to the AOAC method using a RS assay kit (Megazyme, Wicklow, Ireland). Each sample (100 mg) was digested with pancreatin and amyloglucosidase at 37 °C for 6 h, and glucose was measured using a spectrophotometer at 510 nm. HPAEC-PAD of isoamylase-debranched materials of starch. The embryo and pericarp were removed from three de-hulled grains of average size. The endosperms were ground with a mortar and pestle, and 5 mg of the powder produced was suspended in 5 mL of methanol
(A)
(B)
(C)
(D)
Fig. 1. Analysis of the iodine absorption curve of starch. Notes: F1: Area from Bnm to 400nm (cm2), F2: Area from 400nm to λmax(cm2), F3: Area from λmax to 900nm (cm2). The iodine absorption spectrum was analyzed from 200 nm to 900 nm. The absorbance was measured at 620 nm, the maximum absorption wavelength (λmax), and absorbance at λmax, 400 nm and B nm is shown. The area of F1 (from B nm to 400 nm), area of F2 (from 400 nm to λmax), area of F3 (from λmax to 900 nm), and the new λmax index [the apparent amylose content of various rice starches / (the maximum absorption wavelength of various rice starches - the maximum absorption wavelength of glutinous starch)] were calculated. (A) Ae mutant rice; EM10, (B) Japonica rice; Koshihikari, (C) High- amylose rice; Koshinokaori, (D) Glutinous rice; Koganemochi.
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and boiled for 10 min. The homogenate was centrifuged at 2500 g for 5 min. The pelleted polyglucan was washed two times with 5 mL of 90% (v/v) methanol, suspended in 5 mL of distilled water, and boiled for 60 min. An aliquot (1.0 mL) of the sample of gelatinized polyglucan was added to 50 μL of 600 mM sodium acetate buffer (pH 4.4) with 10 μL of 2% (w/v) NaN3, and hydrolysis was achieved by the addition of 10 μL of isoamylase from Pseudomonas amyloderamosa (1400 units; Seikagaku Corp., Tokyo, Japan) incubating at 37 °C for 24 h. The aldehyde groups of the debranched glucans were reduced to alcohol groups by treatment with 0.5% (w/v) sodium borohydride under alkaline conditions for 20 h using the method of Nagamine et al.46) The preparation was dried in a vacuumo at room temperature and allowed to dissolve in 20 μL of 1 M NaOH for 60 min. Next, the solution was diluted with 180 μL of distilled water. An aliquot (25 μL) of the preparation was injected into a BioLC system (model DX-500; Dionex, Sunnyvale, CA) equipped with PAD and a Carbopac PA-1 column (4-mm i.d. × 25 cm). Size fractionation of α-1,4 glucans was performed using a linear gradient of sodium acetate (50–500 mM) in 0.1 M NaOH at a flow rate of 1 mL min−1. Statistical analyses. All results, including the significance of regression coefficients, were statistically analyzed using the t-test and one-way ANOVA, method of Tukey, and Excel Statistics (ver. 2006, Microsoft Corp., Tokyo, Japan).
Results and discussion Measurement of iodine absorption spectra The iodine absorption spectrum was analyzed from 200 to 900 nm (Fig. 1). The molecular structures of many starches, including amylose molecular sizes and amylopectin branch chain lengths, have been reported.47–49) The high molecular weight amyloses tend to have a longer wavelength of λmax.19) The λmax value of amylose was 598 nm with a molecular weight of approximately 70,000, and a high positive correlation was observed between absorbance at λmax and amylose content.21) Fig. 1 shows that the iodine absorption spectrum of ae mutant rice cultivar EM10; (A), japonica rice cultivar Koshihikari; (B), highamylose rice Koshinokaori; (C) (japonica-indica hybrid rice) and glutinous rice Koganemochi; (D). The λmax values of the ae mutant rice cultivars tend to be higher than the glutinous rice cultivars, whereas they are lower than the high-amylose rice cultivars, because ae mutant rice cultivars lack starch branching enzyme II b and contain SLC. Table 1 shows that the glutinous rice cultivars have very low λmax values, and indica rice, japonica-indica hybrid rice cultivars, and high-amylose japonica rice cultivar showed higher λmax values, especially Koshinokaori had the highest value. The λmax values of the japonica rice cultivars were intermediate. Table 1 shows that the absorbance at λmax of the ae mutant rice cultivars was higher than the japonica rice, japonica-indica hybrid rice, and the glutinous rice. In
case of similarly λmax value in various rice cultivars EM16 (582.5 nm), Hitomebore (583.0 nm), and Jasmin rice (583.0 nm), the absorbance at λmax of EM16 (0.59) was higher than Hitomebore (0.28) and Jasmin rice (0.28). Table 1 and Fig. 1 reveal that the ae mutant rice cultivar showed higher F2 values than japonica rice, japonica-indica hybrid rice, and the glutinous rice. The indica rice and Yumetoiro showed very high value, and Japonica-indica hybrid rice showed high values, and the japonica rice showed intermediate values. As shown in Table 1 and Fig. 1, the ae mutant rice cultivar had higher F3 values than the japonica rice, japonica-indica hybrid rice, and the glutinous rice. The F3 values of the glutinous rice cultivars were very low, whereas those of the ae mutant rice cultivars were very high. The F3 values of the indica rice, japonica-indica hybrid rice cultivars, and high-amylose japonica rice cultivars were also high, whereas those of the japonica rice cultivars were intermediate. As a result, F2 and F3 values had similar tendency to absorbance at λmax. Table 1 shows that the new λmax of the ae mutant rice cultivars was 2.23–2.99 times greater than those of the japonica rice cultivars, which were very low, particularly the low-amylose japonica rice Yumepirika. Koshihikari, Yumetoiro, and Hoshiyutaka also had high values. Furthermore, Koshinokaori, Carnaroli, Ekkako, and Mizuhochikara had intermediate values. As a result, in case of similar ACC (ACC contain a lot of amylose and a little SLC in amylopectin) in various rice cultivars, the difference of New λmax values is presumed to be related to SLC in amylopectin, as shown in the relationship between Hokurikukona243go (AAC; 21.6%, New λmax; 0.66) and Carnaroli (AAC; 22.0%, New λmax; 0.30), the relationship between Hoshiyutaka (AAC; 27.2%, New λmax; 0.44) and Koshinokaori (AAC; 25.8%, New λmax; 0.31), and the relationship between EM129 (AAC; 31.3%, New λmax; 0.64) and Yumetoiro (AAC; 30.7%, New λmax; 0.46). AAC of starch AAC has been used as a good parameter for estimating the cooking or eating qualities of rice grains and iodine colorimetric method at 620 nm for AAC measurement was developed by Juliano.16) In general, cereal starches are reported to have higher AAC than root and tuber starches, which tend to retrograde more rapidly after boiling, while cereal amyloses are smaller molecules than amyloses of other origins. Moreover, large molecular weight amyloses are readily gelatinized but not retrograded easily.50) Eating or cooking quality of rice is related to the amylose content and the fine structure of amylopectin.51) The AAC contents are higher than the actual amylose contents because of long-chain amylopectin binding with iodine.47) Table 1 shows that the AAC was 21.6–36.8 (%) in the ae mutant rice cultivars, that is, 1.05 times greater than that in the indica rice, Yumetoiro; 1.18 times greater than that in the japonica-indica hybrid rice, Hoshiyutaka; and 2.08 times greater than that in the japonica rice cultivar, Koshihikari. As a result, in case
Koganemochi Hakuchomochi Hiyokumochi
Yumepirika Koshihikari Hinohikari Hitomebore Akitakomachi Kirara397 Kinmaze Taiwan delicious rice Kamenoo Mizuhotikara Ekkako
Carnaroli
Hoshiyutaka Koshinokaori Kiturin88go Jasmin rice Taiwan aromatic rice Kareimai Super hybrid rice
Yumetoiro
B
C
D
E
F
0.71 2.12 0.00 1.41 0.71 0.71 0.00 0.71 0.00
0.00 0.71 0.00
2.83 0.00 0.71 1.41 2.12 0.71 0.71 4.95 2.83 3.54 0.71
2.12
0.00 3.54 0.00 1.41 2.83 2.83 0.00
0.00
566.50 555.50b 568.00a 569.00a 571.50a 582.50 574.00a 571.00a 523.00d
523.00d 522.50d 523.00d
585.00c 552.50b 583.50c 583.00c 585.50c 586.50c 587.50c 588.50c 578.00c 598.50e 581.50c
596.50e
585.00c 605.50e 599.00e 583.00c 594.00e 592.00e 578.00c
589.00e
SD
0.50a
0.45b 0.43a 0.32d 0.28d 0.33d 0.33d 0.29d
0.39b
0.27d 0.34d 0.32d 0.28d 0.29d 0.32d 0.34d 0.30d 0.31d 0.37d 0.33d
0.18c 0.18c 0.17c
0.59 0.47 0.60a 0.61a 0.57a 0.59a 0.54a 0.56a 0.20c
a
Aλmax
0.01
0.00 0.00 0.00 0.00 0.00 0.00 0.00
0.00
0.00 0.00 0.00 0.00 0.00 0.00 0.01 0.00 0.00 0.00 0.00
0.01 0.00 0.00
0.00 0.00 0.00 0.00 0.00 0.01 0.00 0.00 0.00
SD
1.70a
1.60b 1.77a 1.60b 1.54b 1.69a 1.59b 1.53b
1.70
1.48b 1.48b 1.53b 1.43b 1.48b 1.50b 1.57b 1.51b 1.47b 1.71b 1.51d
1.14d 1.13d 1.21d
1.76 1.58 1.75a 1.72a 1.74a 1.90c 1.85c 1.83c 1.21d
a
F1 ＋ F2 (cm2)
0.02
0.02 0.01 0.02 0.01 0.10 0.02 0.05
0.04
0.03 0.04 0.04 0.03 0.02 0.10 0.01 0.04 0.05 0.01 0.03
0.03 0.04 0.02
0.04 0.02 0.02 0.02 0.07 0.09 0.07 0.01 0.03
SD
0.64
0.62 0.80 0.78 0.83 0.80 0.76 0.80
0.79
0.76 0.70 0.84 0.67 0.70 0.76 1.12 0.75 0.68 0.79 0.75
0.75 0.72 0.81
0.63 0.71 0.69 0.66 0.58a 0.77 0.66 0.62 0.76
a
F1 (cm2)
0.02
0.02 0.02 0.02 0.01 0.09 0.02 0.04
0.03
0.03 0.02 0.03 0.03 0.03 0.08 0.00 0.04 0.05 0.01 0.02
0.02 0.02 0.03
0.04 0.02 0.02 0.03 0.04 0.05 0.05 0.01 0.02
SD
1.06
0.98 0.97 0.81 0.71 0.89 0.83 0.73
0.91
0.72 0.78 0.69 0.76 0.78 0.74 0.45 0.75 0.79 0.92 0.75
0.40 0.40 0.40
1.13 0.88a 1.06a 1.06a 1.16a 1.13a 1.19a 1.21a 0.45b
a
F2 (cm2)
Notes: A, Ae mutant rice; B, Glutinous rice; C, Japonica rice; D, Tropical japonica rice; E, Japonica-indica hybrid rice; F, Indica rice. Means with same letter are not significantly different (p
