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.     

Effect of Partial Gelatinization and Lipid Addition on α‐Amylolysis of Barley Starch Granules

The effect of partial gelatinization with and without lipid addition on the granular structure and on α‐amylolysis of large barley starch granules was studied. The extent of hydrolysis was monitored by measuring the amount of soluble carbohydrates and the amount of total and free amylose and lipids in the insoluble residue. Similarly to the α‐amylolysis of native large barley starch granules, lipid‐complexed amylose (LAM) appeared to be more resistant than free amylose and amylopectin. Partial gelatinization changed the hydrolysis pattern of large barley starch granules; the pinholes typical of α‐amylase‐treated large barley starch granules could not be seen. Lipid addition during partial gelatinization decreased the formation of soluble carbohydrates during α‐amylolysis. Also free amylose remained in the granule residues and mostly amylopectin hydrolyzed into soluble carbohydrates. These findings indicate that lysophospholipid (LPL) complexation with amylose occurred either during pretreatment or after hydrolysis, and free amylose was now part of otherwise complexed molecules instead of being separate molecules. Partial gelatinization caused the granules to swell somewhat less during heating 2% starch‐water suspensions up to 90°C, and lipid addition prevented the swelling completely. α‐Amylolysis changed the microstructure of heated suspensions. No typical twisting of the granules was seen, although the extent of swelling appeared to be similar to the reference starch. The granules with added LPL were partly fragmented after hydrolysis.     

Structural properties of hydrolyzed high-amylose rice starch by α-amylase from Bacillus licheniformis

High-amylose cereal starch has a great benefit on human health through its resistant starch (RS) content. Enzyme hydrolysis of native starch is very helpful in understanding the structure of starch granules and utilizing them. In this paper, native starch granules were isolated from a transgenic rice line (TRS) enriched with amylose and RS and hydrolyzed by α-amylase. Structural properties of hydrolyzed TRS starches were studied by X-ray powder diffraction, Fourier transform infrared, and differential scanning calorimetry. The A-type polymorph of TRS C-type starch was hydrolyzed faster than the B-type polymorph, but the crystallinity did not significantly change during enzyme hydrolysis. The degree of order in the external region of starch granule increased with increasing enzyme hydrolysis time. The amylose content decreased at first and then went back up during enzyme hydrolysis. The hydrolyzed starches exhibited increased onset and peak gelatinization temperatures and decreased gelatinization enthalpy on hydrolysis. These results suggested that the B-type polymorph and high amylose that formed the double helices and amylose-lipid complex increased the resistance to BAA hydrolysis. Furthermore, the spectrum results of RS from TRS native starch digested by pancreatic α-amylase and amyloglucosidase also supported the above conclusion.     

Granule structure and distribution of allomorphs in C-type high-amylose rice starch granule modified by antisense RNA inhibition of starch branching enzyme

C-type starch, which is a combination of both A-type and B-type crystal starch, is usually found in legumes and rhizomes. We have developed a high-amylose transgenic line of rice (TRS) by antisense RNA inhibition of starch branching enzymes. The starch in the endosperm of this TRS was identified as typical C-type crystalline starch, but its fine granular structure and allomorph distribution remained unclear. In this study, we conducted morphological and spectroscopic studies on this TRS starch during acid hydrolysis to determine the distribution of A- and B-type allomorphs. The morphology of starch granules after various durations of acid hydrolysis was compared by optical microscopy, scanning electron microscopy, and transmission electron microscopy. The results showed that amorphous regions were located at the center part of TRS starch subgranules. During acid hydrolysis, starch was degraded from the interior of the subgranule to the outer surface, while the peripheral part of the subgranules and the surrounding band of the starch granule were highly resistant to acid hydrolysis. The spectroscopic changes detected by X-ray powder diffraction, 13C cross-polarization magic-angle spinning NMR, and attenuated total reflectance Fourier transform infrared showed that the A-type allomorph was hydrolyzed more rapidly than the B-type, and that the X-ray diffraction profile gradually changed from a native C-type to a CB-type with increasing hydrolysis time. Our results showed that, in TRS starch, the A-type allomorph was located around the amorphous region, and was surrounded by the B-type allomorph located in the peripheral region of the subgranules and the surrounding band of the starch granule. Thus, the positions of A- and B-type allomorphs in the TRS C-type starch granule differ markedly from those in C-type legume and rhizome starch.     

Effect of Small and Large Wheat Starch Granules on Thermomechanical Behavior of Starch

The physicochemical properties of small‐ and large‐granule wheat starches were investigated to reveal whether gelatinization properties and rheological behavior differ between size classes of wheat starch. All samples contained 60% water (w/w, wb). The starch granule size and shape were examined by scanning electron microscopy in the separated A‐ and B‐type granule populations and in the whole wheat starch granule population. Differential scanning calorimetry (DSC) and electron spin resonance (ESR) analyses were performed in parallel with rheological measurements using dynamic mechanical thermal analysis (DMTA) to relate the viscoelastic changes to modifications in dynamic properties of aqueous solutions and structural disorganization of starch. The small (B‐type) granules had slightly higher gelatinization temperature and lower gelatinization enthalpy than did the large (A‐type) granules. Also, B‐type granules had higher enthalpy for the amylose‐lipid complex transition. Moreover, our results suggested that small granules have higher affinity for water at room temperature. It seems that there is a less ordered arrangement of the polysaccharide chains in the smaller granules when compared with the larger ones. These differences in functional properties of small and large granules suggested that the granule size distribution is an important parameter in the baking process.     

Physicochemical Properties of Zero Amylose Hull-less Barley Starch

Zero amylose starch isolated from hull-less barley (HB) showed a typical A-type diffraction pattern. The X-ray analysis suggested that granules of zero amylose (SB94794) and 5% amylose (CDC Candle) HB starches had lower crystallinity than did commercial waxy corn starch. Differential scanning calorimetry showed lower transition temperatures and endothermal enthalpies for the HB starches than for the waxy corn starch. The zero amylose HB starch showed a Brabender pasting curve similar to that of waxy corn starch, but with lower pasting and peak temperatures and a higher peak viscosity. Noteworthy characteristics of zero amylose HB starch were its low pasting temperature and high paste clarity and freezethaw stability, which make this starch useful for many food and industrial applications.     

Morphologies and Thermal Properties of Hydroxypropylated High‐Amylose Corn Starch

High‐amylose (80%) corn starch was modified by hydroxypropylation with different molar substitution (MS). The unique microstructure of high‐amylose starch keeps its granules intact after hydroxypropylation. However, the microstructures and thermal properties strongly depend on the MS of hydroxypropylation. With increasing MS, the granule size was increased, which is partly due to disrupted granule structure, particularly in the amorphous region. Unlike normal starch, the modified high‐amylose corn starch showed a narrow gelatinization range measured by differential scanning calorimetry (DSC), which can be explained by destruction of amylose‐lipid complex. Internal microstructures and morphologies of hydroxypropylated starch were investigated using confocal laser scanning microscopy and to further explore the mechanism of chemical reaction and phase transitions.     

Starch from Hull‐less Barley: Ultrastructure and Distribution of Granule‐Bound Proteins

Starch granules isolated from waxy, normal, and high‐amylose hullless barley grains were examined by transmission electron microscopy with cytochemical techniques. The micrographs showed two distinct regions of different sizes: 1) densely packed granule growth rings (which varied in size and number depending on the genotype), and 2) a loose filamentous network located in the central region of the granule. The granule ring width decreased with increasing amylose content. In all three genotypes, the growth rings closer to the granule surface were narrower in width than those within the granule interior. The waxy starch had wider intercrystalline amorphous growth rings, semicrystalline growth rings, and more open crystalline lamellae than normal and high‐amylose starches. Granule bound proteins (mainly integral proteins) were located in the central and peripheral (growth ring) regions of the granule.     

Morphological, structural, thermal, and rheological characteristics of starches separated from apples of different cultivars

The starches were separated from unripe apples of five cultivars (Criterion, Ruspippum, Red Spur, Skyline Supreme, and Granny Smith) and evaluated using scanning electron microscopy (SEM), gel permeation chromatography (GPC), X-ray diffraction, differential scanning calorimetry (DSC), and dynamic viscoelasticity. SEM showed the presence of round granules as well as granules that had been partially degraded, probably by amylases. The starch granules in different apple starches ranged between 4.1 and 12.0 mum. Debranching of starch with isoamylase and subsequent fractionation of debranched materials by GPC revealed the presence of an apparent amylose, an intermediate fraction (mixture of amylose and amylopectin), long side chains of amylopectin, and short side chains of amylopectin in the range of 28-35.2, 3.6-4.4, 20-21.3, and 39.9-47.1%, respectively. The swelling power of starches ranged between 14.4 and 21.3 g/g. X-ray diffraction of apple starches showed a mixture of A- and B-type patterns. All apple starches showed peak intensities lower than that observed for normal corn and potato starch, indicating the lower crystallinity. The transition temperatures (onset temperature, T(o); peak temperature, T(p); and conclusion temperature, T(c)) and enthalpy of gelatinization (deltaH(gel)) determined using DSC ranged between 54.7 and 56.2 degrees C, between 57.1 and 59.1 degrees C, between 60.2 and 63.5 degrees C, and between 3.3 and 4.2 J/g, respectively. The viscoelastic properties of starch from different cultivars measured during heating and cooling using a rheometer differed significantly. Red Spur and Criterion starches with larger granule size showed higher G' and G' ' values, whereas those containing smaller size and amylolytically degraded granules showed lower G' and G' '.     

Resistant Starch Content Is Related to Granule Size in Barley

Physical properties of resistant starch (RS) were examined in a range of barley genotypes to determine the contribution of starch and seed physical characteristics to the RS component. Thirty‐three barley genotypes were studied, which varied significantly in their RS, amylose, and starch contents and grain yield. From 33 genotypes, 13 exhibiting high RS were selected for detailed physicochemical analysis of starch. In high‐RS varieties, granule size and number were unimodal, compared with normal starches from a reference genotype, which showed a bimodal distribution. Principal component analysis (PCA) showed that a higher content of granules <15 µm was positively correlated with RS and amylose content, whereas the proportion of granules 15–45 µm was negatively correlated with the RS and amylose contents. Physical fractionation of starches by centrifugation into different population sizes demonstrated that size alone is not an accurate indicator of the population of A‐type and B‐type granules within a given genotype. PCA also showed that large 15–45 µm granules were positively correlated with seed thickness and that thousand grain weight was positively correlated with seed width. High‐RS and high‐amylose genotypes showed variation in overall yield and starch content, with some genotypes showing yield comparable to the reference genotype. Analysis of amylopectin chain length distribution showed that high amylose or RS content was not associated with a higher proportion of amylopectin long chains when compared with either waxy or reference (normal) barley genotypes. This study highlights useful markers for screening barley genotypes with favorable starch characteristics.     

Mechanism and enzymatic contribution to in vitro test method of digestion for maize starches differing in amylose content

To determine the rapidly digestible starch (RDS), slowly digestible starch (SDS), and resistant starch (RS) contents in a starch sample, the addition of amyloglucosidase is often used to convert hydrolyzates from α-amylase digestion to glucose. The objectives of this study were to investigate the exact role of amyloglucosidase in determining the digestibility of starch and to understand the mechanism of enzymatic actions on starch granules. Four maize starches differing in amylose content were examined: waxy maize (0.5% amylose), normal maize (≈27% amylose), and two high-amylose starches (≈57 and ≈71% amylose). Notably, without amyloglucosidase addition, the RS content increased from 4.3 to 74.3% for waxy maize starch, 29.7 to 76.5% for normal maize starch, 65.8 to 88.0% for starch with 57% amylose, and 68.2 to 90.4% for the starch with 71% amylose. In the method without α-amylase addition, less RS was produced than without added amyloglucosidase, except in maize at 71% amylose content. Scanning electron microscopy (SEM) revealed the digestive patterns of pinholes with α-amylase and burrowing with amyloglucosidase as well as the degree of digestion between samples. To understand the roles of amyloglucosidase and α-amylase in the in vitro test, multiple analytical techniques including gel permeation chromatography, SEM, synchrotron wide-angle X-ray diffraction, and small-angle X-ray scattering were used to determine the molecular and crystalline structure before and after digestion. Amyloglucosidase has a significant impact on the SDS and RS contents of granular maize starches.     

Barley grain constituents, starch composition, and structure affect starch in vitro enzymatic hydrolysis

The relationship between starch physical properties and enzymatic hydrolysis was determined using ten different hulless barley genotypes with variable carbohydrate composition. The ten barley genotypes included one normal starch (CDC McGwire), three increased amylose starches (SH99250, SH99073, and SB94893), and six waxy starches (CDC Alamo, CDC Fibar, CDC Candle, Waxy Betzes, CDC Rattan, and SB94912). Total starch concentration positively influenced thousand grain weight (TGW) (r(2) = 0.70, p < 0.05). Increase in grain protein concentration was not only related to total starch concentration (r(2) = -0.80, p < 0.01) but also affected enzymatic hydrolysis of pure starch (r(2) = -0.67, p < 0.01). However, an increase in amylopectin unit chain length between DP 12-18 (F-II) was detrimental to starch concentration (r(2) = 0.46, p < 0.01). Amylose concentration influenced granule size distribution with increased amylose genotypes showing highly reduced volume percentage of very small C-granules (<5 μm diameter) and significantly increased (r(2) = 0.83, p < 0.01) medium sized B granules (5-15 μm diameter). Amylose affected smaller (F-I) and larger (F-III) amylopectin chains in opposite ways. Increased amylose concentration positively influenced the F-III (DP 19-36) fraction of longer DP amylopectin chains (DP 19-36) which was associated with resistant starch (RS) in meal and pure starch samples. The rate of starch hydrolysis was high in pure starch samples as compared to meal samples. Enzymatic hydrolysis rate both in meal and pure starch samples followed the order waxy > normal > increased amylose. Rapidly digestible starch (RDS) increased with a decrease in amylose concentration. Atomic force microscopy (AFM) analysis revealed a higher polydispersity index of amylose in CDC McGwire and increased amylose genotypes which could contribute to their reduced enzymatic hydrolysis, compared to waxy starch genotypes. Increased β-glucan and dietary fiber concentration also reduced the enzymatic hydrolysis of meal samples. An average linkage cluster analysis dendrogram revealed that variation in amylose concentration significantly (p < 0.01) influenced resistant starch concentration in meal and pure starch samples. RS is also associated with B-type granules (5-15 μm) and the amylopectin F-III (19-36 DP) fraction. In conclusion, the results suggest that barley genotype SH99250 with less decrease in grain weight in comparison to that of other increased amylose genotypes (SH99073 and SH94893) could be a promising genotype to develop cultivars with increased amylose grain starch without compromising grain weight and yield.     

Morphological,Structural, and Thermal Properties of Starch Nanocrystals Affected by Different Botanic Origins

Six types of starch nanocrystals were prepared from corn, barley, potato, tapioca, chickpea, and mungbean starches with an acid hydrolysis method. The yields and morphological, structural, and thermal properties of starch nanocrystals were characterized. Starch nanocrystals had yields ranging from 8.8 to 35.7%, depending on botanical origin. During acid hydrolysis, amylose was effectively degraded, and no amylose was detected in any starch nanocrystal. Shape and size of native starch granules varied between starches, whereas there was no obvious difference in shape among different types of starch nanocrystals. The average particle size of starch nanocrystals was mainly related to crystalline type of native starches. Compared with their native starch counterparts, changes in crystalline diffraction patterns of starch nanocrystals depended on the original botanical source and crystalline structure. Degree of crystallinity, melting temperature, and enthalpy of starch nanocrystals increased, whereas their thermal decomposition temperature decreased. Of six produced starch nanocrystals, potato starch nanocrystal had the lowest yield, degree of crystallinity, and onset and melting temperatures, the largest particle size, and obvious changes in crystalline diffraction pattern.     

Determination of the molecular and structural characteristics of okenia, mango, and banana starches

Starches were isolated from nonconventional sources (banana, mango, and okenia) and their characteristics were examined using polarized light microscopy, X-ray diffraction pattern, Fourier transform infrared (FTIR) spectroscopy, and differential scanning calorimetry (DSC). Banana starch granules were of an ellipsoidal shape with size between approximately 8 and 20 microm; okenia had the smallest granule size, between approximately 2 and 5 microm. The three starches showed the Maltese cross, indicative of an intact granule structure. Okenia and mango starches had the A-type X-ray diffraction pattern, common to native cereal starches, whereas banana starch showed a mixture between A- and B-type pattern. Banana starch had the highest temperature (77.6 degrees C) and enthalpy (23.4 J/g) of gelatinization in excess water conditions; okenia had the lowest temperature (71.2 degrees C) and enthalpy (15 J/g), which may be related to the X-ray diffraction pattern and its small granule size. Both the okenia and mango starches had a higher molar mass and gyration radius than banana starch, which may be related to the differences determined in their crystalline structures.     

Resistant Starch Escaped from Ethanol Production: Evidence from Confocal Laser Scanning Microscopy of Distiller's Dried Grains with Solubles (DDGS)

The origin of resistant starch (RS) in distiller's dried grains with solubles (DDGS) of triticale, wheat, barley, and corn from dry‐grind ethanol production was studied. A considerable portion of starch (up to 18% in DDGS) escaped from either granular starch hydrolysis or conventional jet‐cooking and fermentation processes. Confocal laser scanning microscopy revealed that some starch granules were still encapsulated in cells of grain kernel or embedded in protein matrix after milling and were thus physically inaccessible to amylases (type RS1). The crystalline structures of native starch granules were not completely degraded by amylases, retaining the skeletal structures in residual starch during granular starch hydrolysis or leaving residue granules and fragments with layered structures after jet‐cooking followed by the liquefaction and saccharification process, indicating the presence of RS2. Moreover, retrograded starch molecules (mainly amylose) as RS3, complexes of starch with other nonfermentable components as RS4, and starch–lipid complexes as RS5 were also present in DDGS. In general, the RS that escaped from the granular starch hydrolysis process was mainly RS1 and RS2, whereas that from the jet‐cooking process contained all types of RS (RS1 to RS5). Thus, the starch conversion efficiency and ethanol yield could be potentially affected by the presence of various RS in DDGS.     

Uptake of Cu2+ by starch granules as affected by counterions

Potato and wheat starch granules were soaked in 1% aqueous solutions of copper(II) salts: acetate, chloride, and sulfate. Such treatment caused sorption of Cu(2+) ions at the granule surface and their penetration into the granule interior as was proven, for sectioned granules of investigated starch, by scanning electron microscopy combined with an X-ray microanalysis system (energy dispersive spectroscopy). Copper ions incorporated into the granules influenced the starch thermal stability. Uptake of Cu(2+) by potato, determined by flame atomic absorption spectrometry, was much higher than obtained for the wheat starch. Moreover, it was dependent on copper counteranions present in the solution. In all investigated granules, the most effective sorption occurred in the acetate solution. Starch dehydration or/and freezing and thawing, affecting the water-dependent inner structure of the granules, also influenced the amount of Cu(2+) taken from the solutions. Thus, compared to that in native starch, this value was considerably higher in Cu(CH 3COO)2, almost unchanged in CuSO4, and significantly lower in the case of CuCl2 solution. The influence of chloride and sulfate anions seemed to correlate with their water structure-making and structure-breaking ability, affecting the migration of Cu(2+) in the amorphous parts of the granules. However, high Cu uptake observed for acetate solution could be explained on the basis of acetate anion hydrolysis activating the polysaccharide matrix for cation binding. The obtained results provide new information about interactions of starch granules with salt solution and therefore support our understanding of starch properties.     

Location of Sites of Reaction Within Starch Granules

To observe granular reaction patterns within modified starch granules, starch derivatives were converted to thallium(I) salts and viewed by scanning electron microscopy compositional backscattered electron imaging. Observation of phosphorylated potato and sorghum starches and a hydroxypropyl analog of waxy maize starch revealed that granular patterns of reaction were influenced by both starch and reagent types. In waxy maize and sorghum starches, flow of reagent into the granule matrix occurred from channels (laterally) and cavities (from the inside outward). In potato starch granules, which do not possess channels, reagent diffused inward through exterior granule surfaces. Phosphoryl chloride (highly reactive) reacted to a large extent at granule surfaces, while the propylene oxide analog (less reactive) appeared to diffuse into the granule matrix prior to reacting.     

Physicochemical and Enzyme Characterization of Small and Large Starch Granules Isolated from Two Maize Cultivars

In this study, some morphological, physicochemical, and the initial characterization of the starch granule enzymes of blue and white maizes of small and large granules are described. Starch was isolated from blue and white maizes, and the small and large granules were separated. The efficiency of separation was higher in large granules than small of the blue maize starch. The amylose content was slightly greater (1.3%) in large granules than smaller in both starches studied. No differences in the average gelatinization temperature were found between large and small granules, but the enthalpy of gelatinization value was higher in the small granules. The electrophoretic pattern showed that the granule‐bound starch synthase (GBSSI) had higher expression in large than small granules and that explain the higher amylose content in the former granules. The differences showed in the starch biosynthesis enzymes in small and large granules might explain partially the physicochemical and functional properties of maize.     

A Study of Maize Endosperm Hardness in Relation to Amylose Content and Susceptibility to Damage

The relative amounts of amylose and amylopectin in maize starch were determined in samples representing hard and soft endosperm. Although differences were small, amylose content differed significantly (P < 0.001 and P < 0.05) between the two types of endosperm, with hard endosperm containing a higher percentage of amylose. Scanning electron microscopy was used to determine that the surface appearance of starch granules from hard and soft endosperm differed. Starch granules from soft endosperm had randomly distributed pores on their surfaces, which had a rough appearance. Few pores were observed on granules from hard endosperm. A fairly common occurrence with starch granules from soft endosperm was the development of wrinkles or fissures upon prolonged exposure to the beam of the electron microscope. Thus, a correlation existed between endosperm hardness, amylose content, and susceptibility to wrinkling and fissures. The granules of the soft endosperm of maize, presumably less mature than the granules of the hard endosperm, have a lower amylose content (20.5 ± 1.9% vs. 23.0 ± 1.0%), exhibit more surface pores, and are more susceptible to wrinkling in an electron beam, compared with granules of the hard endosperm. Results suggested that the composition and internal architecture of the starch granule differ depending on the hardness of the endosperm from which it was obtained.