Comparison of Amylose Determination Methods and the Development of a Dual Wavelength Iodine Binding Technique

It has long been recognized that limitations exist in the analytical methodology for amylose determination. This study was conducted to evaluate various amylose determination methods. Purified amylose and amylopectin fractions were obtained from corn, rice, wheat, and potato and then mixed in proportion to make 10, 20, 30, 50, and 80% amylose content starch samples for each source. These samples, considered amylose standards, were analyzed using differential scanning calorimetry (DSC), high-performance size-exclusion chromatography (HPSEC), and iodine binding procedures to generate standard curves for each of the methods. A single DSC standard equation for cereal starches was developed. The standard curve of potato starch was significantly different. Amylose standard curves prepared using the iodine binding method were also similar for the cereal starches, but different for potato starch. An iodine binding procedure using wavelengths at 620 nm and 510 nm increased the precision of the method. When HPSEC was used to determine % amylose, calculations based on dividing the injected starch mass by amylose peak mass, rather than calculations based on the apparent amylose/amylopectin ratio, decreased the inaccuracies associated with sample dispersion and made the generation of a cereal amylose standard curve possible. Amylose contents of pure starch, starch mixtures from different sources with different amylose ranges, and tortillas were measured using DSC, HPSEC, iodine binding, and the Megazyme amylose/amylopectin kit. All the methods were reproducible (±3.0%). Amylose contents measured by these methods were significantly different (P < 0.05). Amylose measurements using iodine binding, DSC, and Megazyme procedures were highly correlated (correlation coefficient >0.95). DSC and traditional iodine binding procedures likely overestimated true amylose contents as residual butanol in the amylose standards caused interference. The modified two-wavelength iodine binding procedure seemed to be the most precise and generally applicable method. Each amylose determination method has its benefits and limitations.     

Effects of Amylopectin Branch Chain Length and Amylose Content on the Gelatinization and Pasting Properties of Starch

Structures and properties of starches isolated from different botanical sources were investigated. Apparent and absolute amylose contents of starches were determined by measuring the iodine affinity of defatted whole starch and of fractionated and purified amylopectin. Branch chain-length distributions of amylopectins were analyzed quantitatively using a high-performance anion-exchange chromatography system equipped with a postcolumn enzyme reactor and a pulsed amperometric detector. Thermal and pasting properties were measured using differential scanning calorimetry and a rapid viscoanalyzer, respectively. Absolute amylose contents of most of the starches studied were lower than their apparent amylose contents. This difference correlated with the number of very long branch chains of amylopectin. Studies of amylopectin structures showed that each starch had a distinct branch chain-length distribution profile. Average degrees of polymerization (dp) of amylopectin branch chain length ranged from 18.8 for waxy rice to 30.7 for high-amylose maize VII. Compared with X-ray A-type starches, B-type starches had longer chains. A shoulder of dp 18–21 (chain length of 6.3–7.4 nm) was found in many starches; the chain length of 6.3–7.4 nm was in the proximity of the length of the amylopectin crystalline region. Starches with short average amylopectin branch chain lengths (e.g., waxy rice and sweet rice starch), with large proportions of short branch chains (dp 11–16) relative to the shoulder of dp 18–21 (e.g., wheat and barley starch), and with high starch phosphate monoester content (e.g., potato starch) displayed low gelatinization temperatures. Amylose contents and amylopectin branch chain-length distributions predominantly affected the pasting properties of starch.     

Structure-retrogradation relationship of rice starch in purified starches and cooked rice grains: a statistical investigation

Amylose content and amylopectin chain length distribution, the two most commonly used structural parameters of starch, have significant effects on starch retrogradation. In the present work, starches were separated and purified from 18 rice cultivars. The amylopectin was purified from each starch. Amylopectin chain length distribution was analyzed by high-performance size-exclusion chromatography after debranched using isoamylase. The blue value was used to measure the amylose content before and after the defatting of starch. The amount of amylose associated with lipid was calculated. Pulsed nuclear magnetic resonance was used to follow the retrogradation of starch both in cooked rice grains and in the purified form. The Avrami equation was employed to describe the retrogradation kinetics of rice starch. To look into the relationship between the starch structure and retrogradation behavior, the structural parameters were correlated with retrogradation kinetics parameters using both Pearson and partial correlations. The results indicated the following: first, the retrogradation behavior of rice starch remains similar in both the purified form and cooked rice grains; second, the peak value of amylopectin short-chain length has a significant positive relationship with the amylopectin crystallization rate constant k; third, the amylose content after defatting has a significant positive relationship with the parameter k and a negative relationship with the Avrami exponent n; and fourth, the amount of amylose associated with lipid has a negative relationship with the parameter k.     

Rice Quality by Spectroscopic Analysis: Precision of Three Spectral Regions

Three types of spectroscopy were used to examine rice quality: near infrared (NIR), Raman, and proton nuclear magnetic resonance (¹H NMR). Samples from 96 rice cultivars were tested. Protein, amylose, transparency, alkali spreading values, whiteness, and degree of milling were measured by standard techniques and the values were regressed against NIR and Raman spectra data. The NMR spectra were used for a qualitative or semiquantitative assessment of the amylose/amylopectin ratio by determining the 1–4 to 1–6 ratio for glucans. Protein can be measured by almost any instrument in any configuration because of the strong relationship between the spectral response and the precision of the reference method. Amylose has an equally strong relationship to the vibrational spectra, but its determination by any reference method is far less precise, resulting in a 10× increase in the standard error of cross‐validation (SECv) or standard error of performance (SEP) with R ² values equal to that of the protein measurement.     

Structural Characteristics,Properties, and In Vitro Digestibility of Rice

Using rice samples derived from normal rice cultivars and endosperm starch mutant, we investigated key factors contributing to the enzyme digestibility of steamed rice grains. The chemical composition of polished rice grains, structural features of endosperm starch, and enzyme digestibility of steamed rice grains were examined. The protein content of polished rice grains was 4.6–9.1%, amylose content was 4–27%, the DP_n of purified amylose was 900–1,600, the amylopectin short/long chain ratio was 1.2–5.9, and the enzyme digestibilities of steamed polished rice grains were 0.9–12.6 °Brix. Amylose content and RVA parameters (viscosity, breakdown, and setback) correlated significantly with enzyme digestibility of steamed rice grains. Multiple regression formulas were constructed to predict digestibility of steamed rice grain as a function of the molecular characteristics of the starch. When both amylose content and the short/long chain amylopectin ratio were used as predictor variables, they accounted for >80% of the observed variance in digestibility of steamed rice grains. Multiple regression revealed that the more digestible rice samples had starch with a lower amylose content and more short‐chain amylopectin. Reassociation of amylose‐lipid complex and recrystallization of amylopectin in the stored steamed rice grains was monitored by differential scanning calorimetry (DSC), and the observed retrogradation properties were related to the structural characteristics of starch and to the enzyme digestibility of steamed rice grains.     

Characterization of Starch in Aegilops Species

Starch was extracted and cleaned from 99 accessions of 20 species of Aegilops and also from 200 accessions of hexaploid wheat. Amylose content was determined by iodine staining and absorbance at 535 and 620 nm. Particle‐size distribution was determined by laser scattering. The amylose content of the Aegilops accessions did not exceed the extremes found in domesticated wheat. Aegilops species, on the whole, had a lower content of small particles than the hexaploid wheats. There was no correlation between amylose content and particle‐size distribution. Some species of Aegilops may be useful sources of low‐starch B‐type granules for hexaploid wheat, if the trait can be transferred, but they are unlikely to contribute to further variation in amylose content.     

A Better Understanding of Factors That Affect the Hardness and Stickiness of Long‐Grain Rice

Eight U.S. long‐grain rice cultivars were studied for chemical compositions, physicochemical properties, and leaching characteristics in relation to hardness and stickiness of rice flour paste and cooked rice. There were differences in the chemical composition of rice kernels among the eight rice cultivars, including crude protein (6.6–9.3%), crude fat (0.18–0.51%), and apparent amylose content by iodine colorimetry (19.6–27.0%). Differences were also observed in gelatinization temperatures and enthalpies, pasting temperatures and viscosities, leached/insoluble amylose, soluble solids, and hardness and stickiness of rice flour pastes and cooked rice kernels. The quantity and molecular size distribution of the leached starch molecules varied greatly among the samples. Protein and crude lipid contents negatively correlated with hardness of rice flour paste and cooked rice, but positively correlated with stickiness. Apparent amylose content correlated with gel properties but not cooked rice texture, whereas the ratio of A and short B chains to long B chains of amylopectin correlated significantly with cooked rice texture.     

Properties of Starches from Several Low‐Amylose Rice Cultivars

The starch properties of five low‐amylose rice cultivars, Yawarakomachi, Soft 158, Hanabusa, Aya, and Snow Pearl, were compared with those of two normal amylose rice cultivars, Nipponbare and Hinohikari. There were no large differences in the distributions of the amylopectin chain length determined by high‐performance anion‐exchange chromatography, and the starch gelatinization properties determined by differential scanning calorimetry, between normal and low‐amylose rice cultivars. Results obtained using rapid viscosity analysis indicated that low‐amylose rice starches had lower peak viscosity, breakdown, and setback values than normal amylose rice starches. Starch granules from low‐amylose rice cultivars had a higher susceptibility to glucoamylase than those from normal amylose rice cultivars. The results of this study showed some differences between normal and low‐amylose rice starches in pasting properties and enzymatic digestibility.     

氮肥施用量对甘薯产量和品质性状的影响

【目的】氮是影响作物产量和品质性状的重要因素,合理施氮是提高作物产量和改善品质的主要途径。研究施氮量对甘薯块根产量形成、营养品质和淀粉糊化特性的影响,对于明确江苏徐淮地区甘薯种植的适宜施氮量具有重要意义。【方法】本文选用淀粉型甘薯品种徐薯22和兼用型甘薯品种徐薯28为试验材料,设置5个施氮水平0、60、120、180、240 kg/hm2。调查了甘薯不同生育期的叶面积指数、光合势、干物质积累和产量构成要素,采用分光光度法测定了块根主要营养品质指标,利用Tech-master型RVA快速测定淀粉谱粘滞特性,分析明确施氮量对块根主要品质指标和淀粉糊化特征值的影响。【结果】施氮N 60 kg/hm2增加了各生育期甘薯的叶面积,提高了甘薯的光合势,显著增加了块根产量(徐薯28增产16.38%,徐薯22增产9.31%),过量施氮(N 240 kg/hm2)则产量降低。施氮并未显著降低块根中的淀粉和直链淀粉含量,但明显增加了可溶性糖和蔗糖含量。施氮显著提高最高粘度、最低粘度、最终粘度和消减值。相关分析显示,直链淀粉含量与最高粘度值和崩解值呈极显著或显著负相关(相关系数分别为-0.90**和-0.71*);直链淀粉含量与消减值呈显著正相关(相关系数为0.73*)。【结论】综合考虑甘薯的产量和品质,在本实验条件下甘薯较为适宜的氮肥用量约为60 kg/hm2。     

Fine Structures of Starches from Long‐Grain Rice Cultivars with Different Functionality

The structural features of rice starch that may contribute to differences in the functionality of three long‐grain rice cultivars were studied. Dried rough rice samples of cultivars Cypress, Drew, and Wells were analyzed for milling quality, grain physical attributes, and starch structures and physicochemical properties. Drew was lower in head rice yield and translucency and higher in percentage of chalky grains compared with Cypress and Wells. Apparent amylose content (21.3–23.1%), crude protein (8.3–8.6%), and crude fat (0.48–0.64%) of milled rice flours were comparable, but pasting properties of rice flours as measured by viscoamylography, as well as starch iodine affinity and thermal properties determined by differential scanning calorimetry were different for the three cultivars. Drew had higher peak, hot paste, and breakdown viscosities, and gelatinization temperature and enthalpy. Molecular size distribution of starch fractions determined by high‐performance size‐exclusion chromatography showed that the three samples were similar in amylose content (AM) (20.0–21.8%) but differed in amylopectin (AP) (64.7–68.3%) and intermediate material (IM) (10.9–13.5%). Drew had highest AP and lowest IM contents, whereas Cypress had the lowest AP and highest IM contents. High‐performance anion‐exchange chromatography of isoamylase‐debranched starch indicated that the AP of Drew was lower in A and B1 chains but higher in B2, B3, and longer chains.     

Physicochemical Properties and In Vitro Starch Digestibility of Cooked Rice from Commercially Available Cultivars in Canada

The influence of amylose content, cooking, and storage on starch structure, thermal behaviors, pasting properties, and rapidly digestible starch (RDS), slowly digestible starch (SDS), and resistant starch (RS) in different commercial rice cultivars was investigated. Long grain rice with high‐amylose content had a higher gelatinization temperature and a lower gelatinization enthalpy than the other rice cultivars with intermediate amylose content (Arborio and Calrose) and waxy type (glutinous). The intensity ratio of 1047/1022 cm^–1 determined by Fourier Transform Infrared (FT‐IR), which indicated the ordered structure in starch granules, was the highest in glutinous and the lowest in long grain. Results from Rapid ViscoAnalyser (RVA) showed that the rice cultivar with higher amylose content had lower peak viscosity and breakdown, but higher pasting temperature, setback, and final viscosity. The RDS content was 28.1, 38.6, 41.5, and 57.5% in long grain, Arborio, Calrose, and glutinous rice, respectively, which was inversely related to amylose content. However, the SDS and RS contents were positively correlated with amylose content. During storage of cooked rice, long grain showed a continuous increase in pasting viscosity, while glutinous exhibited the sharp cold‐water swelling peak. The retrogradation rate was greater in rice cultivars with high amylose content. The ratio of 1047/1022 cm^–1 was substantially decreased by cooking and then increased during storage of cooked rice due to the crystalline structure, newly formed by retrogradation. Storage of cooked rice decreased RDS content and increased SDS content in all rice cultivars. However, no increase in RS content during storage was observed. The enthalpy for retrogradation and the intensity ratio 1047/1022 cm^–1 during storage were correlated negatively with RDS and positively with SDS (P ≤ 0.01).     

Structural and Retrogradation Properties of Rice Endosperm Starch Affect Enzyme Digestibility of Steamed Milled-Rice Grains Used in Sake Production

Structural and physicochemical characteristics of endosperm starch from milled rice grains of seven Japanese cultivars used in sake production were examined. Amylose content was 15.2–20.2%, number-average degree of polymerization (DP_n) of amylose was 900–1,400, and the ratio of short-to-long chain amylopectin was 2.7–3.5, respectively. The degree of retrogradation of purified starch stored for seven days at 4°C after gelatinization was 20–31%. The degree of retrogradation correlated negatively with the ratio of short-to-long chain amylopectin. The effect of holding time after steaming on enzyme digestibility and starch retrogradation of steamed rice grains was investigated. The longer the holding time after steaming, the greater the extent of retrogradation, and the less the degree of enzymatic digestibility. The decreased rate of enzyme digestibility correlated with amylopectin chain length distribution. Samples with short-chain amylopectin exhibited a slow decrease in enzyme digestibility. It was determined that the structure and retrogradation properties of endosperm starch in Japanese rice cultivars affect the decreasing rate of enzyme digestibility of the steamed, milled rice grains.     

Morphological Changes of Granules of Different Starches by Surface Gelatinization with Calcium Chloride

Native starch granules of 11 selected cultivars (potato, waxy potato, sweet potato, normal maize, high‐amylose maize, waxy maize, wheat, normal barley, high‐amylose barley, waxy barley, and rice) were treated with a calcium chloride solution (4M) for surface gelatinization. The surface‐gelatinized starch granules were investigated using light microscopy and scanning electron microscopy (SEM) and differential scanning calorimetry (DSC). In general, those starches with larger granule sizes required longer treatment time to complete the gelatinization. The salt solution treatment of starch was monitored by light microscopy and stopped when the outer layer of the granule was gelatinized. The surface gelatinized starch granules were studied using scanning electron microscopy. On the basis of the gelatinization pattern from calcium chloride treatments, the starches could be divided into three groups: 1) starches with evenly gelatinized granule surface, such as normal potato, waxy potato, sweet potato, maize, and high‐amylose maize; 2) starches with salt gelatinization concentrated on specific sites of the granule (i.e., equatorial groove), such as wheat, barley, and high‐amylose barley; and 3) starches that, after surface gelatinization, can no longer be separated to individual granules for SEM studies, such as waxy barley, waxy maize, and normal rice. The morphology of the surface gelatinized starch resembled that of enzyme‐hydrolyzed starch granules.     

Structures and physicochemical properties of six wild rice starches

Starches from six wild rice cultivars were studied for their chemical structures and physicochemical properties and compared with a long-grain rice starch. The six wild rice starches were similar in morphological appearance, X-ray diffraction patterns, swelling power, and water solubility index but different in amylose content, beta-amylolysis limit, branch chain length distribution, thermal properties, and pasting properties. The structure of the wild rice amylopectins was close to that of waxy rice amylopectin with more branching and a larger proportion of short branch chains of degree of polymerization 6-12 as compared with that of amylopectin from rice starch with a similar amylose content. The differences in branch chain length distribution of amylopectin and amylose content were assumed to contribute to the differences in physicochemical properties among the six wild rice starches as well as to the differences between the wild rice starches and the rice starch.     

Effects of Cultivars,Transplanting Patterns,Environment, and Their Interactions on Grain Quality of Japonica Rice

With the increasing scarcity of rural labor, the rice transplanting pattern is encountering a shift from artificial transplanting (AT) to mechanical transplanting (MT) in numerous rice‐growing districts of China. The shift of transplanting patterns combined with altered growing environment during the grain‐filling stage in different years presumably affects rice quality. Nevertheless, related information is currently limited. This study investigated the effects of cultivars, transplanting patterns, environment, and their interactions on appearance, milling, eating, and nutritional qualities of four japonica rice varieties. The significant interactive effects of cultivars, environment, and transplanting patterns on almost all rice quality parameters (except Thr, Met, and Ile) were observed. Cultivars and environment were the main factors influencing rice appearance and milling and eating qualities. Cultivar was the primary factor affecting rice nutritional quality. Among all treatments, environment showed the strongest effect on percentage of chalky kernel, milled rice yield, peak viscosity, breakdown, setback, consistence, amylose, Glu, Tyr, and Met contents. However, Leu and Phe contents were unaffected by environment but only by cultivars and transplanting patterns. In addition to amylose and protein, Glu and Met contents were also involved in determining rice eating quality. Amino acid contents (except Cys, Tyr, and Met) were significantly negatively correlated with head rice yield, showing the function of amino acids in controlling rice milling quality. Percentage of chalky kernel as well as protein and almost all amino acid contents were significantly negatively correlated with the difference of maximum and minimum temperature (DMMT) and positively correlated with relative humidity (RH), whereas head rice yield did it reversely. Amylose content and setback were significantly negatively related to daily maximum temperature (DMAT), daily minimum temperature (DMIT), daily average temperature (DAT), and effective temperature accumulation (ETA). However, peak viscosity, breakdown, and consistence had contrary performances. According to these results, we can infer that DMMT and RH are important environmental factors affecting rice appearance, milling qualities, and nutritional qualities and that DMAT, DMIT, DAT, and ETA are key environmental factors influencing rice eating quality.     

Genotype and Environmental Influences on Pasting Properties of Rice Flour

The pasting behavior of flour from several Australian rice (Oryza sativa L.) cultivars, differing in amylose content and grown in three different locations and three seasons, were determined using the Rapid Visco Analyser. Genotype, growth season, and growth location all affected the pasting behavior of rice flour. The amylose content of the same cultivar was significantly higher in the coolest growing season, resulting in RVA traces with lower peak viscosity and higher setback than samples with lower amylose content. When the same cultivar of rice was grown in different locations in the same season, there were no significant differences in the total starch, protein, lipid, and amylose content of the flour, but there were significant differences in the pasting behavior. This indicates that environmental as well as genetic factors influence the pasting behavior of rice flour. Flour from parboiled and quick‐cooking rice did not paste and had low viscosities compared with unprocessed rice. Results from this study showed that the pasting behavior of rice flour was related to genotype and was influenced by environmental factors that brought about subtle changes in the grains that were not picked up by chemical analyses.     

Use of Mixolab in Predicting Rice Quality

Mixolab is a new instrument with capability to measure starch pasting properties on actual dough. It characterizes dough rheological behavior using a dual constraints of mixing and temperature. Rice samples (183) collected from 15 provinces across China were tested to determine the possibility of using Mixolab in predicting rice quality. Mixolab measurements, torque (Nm) at different mixing and heating stages (C1 to C5) were compared with rice quality characteristics (gelatinization temperature and consistency, amylose and protein contents), Rapid Visco‐Analyser (RVA) parameters and sensory assessments scores of cooked rice. Our results showed that Mixolab parameters were good indicators of amylose and protein content and quality suggested by significant correlations among Mixolab parameters, and between Mixolab and RVA measurements. Based on a subsample of 30 rice cultivars, correlation coefficients between the Mixolab parameter C4 and sensory assessment characteristics of palatability and total sensory score was negatively significant (P < 0.05). C_b (C3 – C4) was also significantly correlated with flavor (P < 0.05). The rice samples that gave high palatability and total sensory scores had low C4 values and low amylose contents. The cooked rice with high flavor had high values of C_b and GT but low protein content. It is possible to determine physicochemical properties of rice flour and sensory characteristics of cooked rice using Mixolab parameters.