Starch characteristics and their influences on in vitro and pig prececal starch digestion

The main objective of this research was to study the characteristics of starch granules and their influences on in vitro and pig prececal starch digestion of corn, dehulled barley, wheat, and potato. Scanning electron microscopy was used to study the starch endosperm structure in the parent material as well as in vitro starch digestion. The results showed that corn starch granules were polyhedral, with a diameter ranging from 2 to 10 μm, whereas those of dehulled barley and wheat were spherical, with a diameter ranging from 5 to 20 μm. Potato had the largest starch granules among starch sources reported herein, with oval spheres of 10-50 μm in diameter. In vitro starch hydrolysis showed that starch granules of corn degraded faster than the starch of dehulled barley and wheat, with the potato starch being degraded the slowest. The in vivo digestibility trial using ileal-cannulated pigs confirmed the starch degradation of grains. The in vitro (x, %) and in vivo (y, %) digestibility were highly correlated [y = 6.5304x - 538.48 (R(2) = 0.9924)]. On the basis of the results, in vitro starch hydrolysis might be useful in predicting in vivo prececal starch digestibility. The digestion kinetic characteristics of different starch sources might be employed to evaluate the starch digestive rate at the pig ileum.     

Associations of Starch Gel Hardness,Granule Size,Waxy Allelic Expression,Thermal Pasting,Milling Quality,and Kernel Texture of 12 Soft Wheat Cultivars

Starches were isolated from 12 soft wheat (Triticum aestivum L.) cultivars and were characterized for waxy (Wx) allelic expression, thermal pasting characteristics, and starch granule size. Gels were produced from the thermally degraded starches and were evaluated using large deformation rheological measurements. Data were compared with cultivar kernel texture, milling characteristics, starch chemical analyses, and flour pasting characteristics. Larger flour yields were produced from cultivars that had larger starch granules. Flour yield also was correlated with lower amylose content and greater starch content. Harder starch gels were correlated with higher levels of amylose content and softer kernel texture. The cultivar Fillmore, which had a partial waxy mutation at the B locus, produced the highest peak pasting viscosity and the lowest gel hardness. Softer textured wheats had greater lipid‐complexed amylose and starch phosphorus contents and had less total starch content. Among these wheats of the soft market class, softer textured wheats had larger starch granules and harder textured wheats had smaller starch granules. In part, this may explain why soft wheats vary in texture. The smaller granules have larger surface area available for noncovalent bonding with the endosperm protein matrix and they also may pack more efficiently, producing harder endosperm.     

Starch Hydrolyzing Enzymes for Retarding the Staling of Rice Bread

Previous attempts have been made to obtain gluten‐free bread of acceptable quality for bread specific volume and crumb texture. Rice bread is a good alternative to celiac patients, but it has a very rapid staling during storage. Rice starch is more prone to retrograde during storage than wheat starch, and the special hydrophobic nature of the rice proteins requires specific enzymes to be used in the rice bread process. To retard rice bread staling, two different starch hydrolyzing enzymes (α‐amylase of intermediate thermostability and cyclodextrin glycoxyl transferase [CGTase]) have been tested and their effect on fresh bread quality and staling during storage has been evaluated. The addition of α‐amylase improved bread specific volume and crumb firmness but very sticky textures were obtained. The addition of CGTase produced even higher specific volume and similar crumb firmness with better texture. Both enzymes decreased the ability of amylopectin to retrograde during storage. The firming kinetic was lowered by the α‐amylase but not the limiting firmness, while the rice crumb from CGTase firmed quickly with a very short range of firmness increase. Results revealed that the starch hydrolysis brought about by the α‐amylase was not sufficient to retard staling. CGTase was considered a better antistaling agent because of its starch hydrolyzing and cyclizing activity.     

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.     

Effects of Heat-Moisture Treatment and Lipids on Gelatinization and Retrogradation of Maize and Potato Starches

Effects of heat-moisture treatment (HMT) and lipids on the structure and gelatinization of maize and potato starches were studied, and the retrogradation process of 20% HMT starch gels was also investigated. Maize starch was physically modified by HMT or by defatting. Potato starch was physically modified by HMT or by adding monoglycerides. The X-ray pattern of the HMT maize starch was assigned to a combination of A and V patterns, which indicated that HMT formed crystallized amylose complexes and recrystallized amylose in maize starch granules. However, the X-ray pattern of defatted maize starch did not change for HMT, so the lipids originally existing in starch granules were important to the formation of new crystallites during this treatment. Differential scanning calorimetry (DSC) results suggested that weaker structures in amylopectin crystallites were more susceptible to degradation after HMT, while crystallized amylose complexes developed thermal stability after treatment. The amylose contents increased with increasing degree of HMT, which suggested that the newly created amylose arose from exterior linear chains of amylopectin degraded by the treatment. Investigation of retrogradation process showed that HMT significantly promoted retrogradation of starch gels, especially the initiation of recrystallization.     

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.     

Improved bread-baking process using Saccharomyces cerevisiae displayed with engineered cyclodextrin glucanotransferase

A bread-baking process was developed using a potential novel enzyme, cyclodextrin glucanotransferase[3-18] (CGTase[3-18]), that had previously been engineered to have enhanced hydrolyzing activity with little cyclodextrin (CD) formation activity toward starch. CGTase[3-18] was primarily manipulated to be displayed on the cell surface of Saccharomyces cerevisiae. S. cerevisiae carrying pdeltaCGT integrated into the chromosome exhibited starch-hydrolyzing activity at the same optimal pH and temperature as the free enzyme. Volumes of the bread loaves and rice cakes prepared using S. cerevisiae/pdeltaCGT increased by 20% and 45%, respectively, with no detectable CD. Retrogradation rates of the bread and rice cakes decreased significantly during storage. In comparison to the wild type, S. cerevisiae/pdeltaCGT showed improved viability during four freeze-thaw cycles. The results indicated that CGTase[3-18] displayed on the surface of yeast hydrolyzed starch to glucose and maltose that can be used more efficiently for yeast fermentation. Therefore, display of an antistaling enzyme on the cell surface of yeast has potential for enhancing the baking process.     

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.     

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' '.     

Plantain and banana starches: granule structural characteristics explain the differences in their starch degradation patterns

Different banana cultivars were used to investigate the influences of starch granule structure and hydrolases on degradation. The highest degrees of starch degradation were observed in dessert bananas during ripening. Scanning electron microscopy images revealed smooth granule surface in the green stage in all cultivars, except for Mysore. The small and round granules were preferentially degraded in all of the cultivars. Terra demonstrated a higher degree of crystallinity and a short amylopectin chain length distribution, resulting in high starch content in the ripe stage. Amylose content and the crystallinity index were more strongly correlated than the distribution of amylopectin branch chain lengths in banana starches. α- and β-amylase activities were found in both forms, soluble in the pulp and associated with the starch granule. Starch-phosphorylase was not found in Mysore. On the basis of the profile of α-amylase in vitro digestion and the structural characteristics, it could be concluded that the starch of plantains has an arrangement of granules more resistant to enzymes than the starch of dessert bananas.     

Study of Three-Dimensional Structure of Wheat Starch Granules Stained with Remazolbrilliant Blue Dye and Extracted with Aqueous Sodium Dodecyl Sulfate and Mercaptoethanol Solution

Wheat starch granules were obtained from soft wheat flour by acetic acid fractionation (pH 3.5), and the starch was stained by reaction with Remazolbrilliant blue (RBB) dye. RBB-stained starch was extracted with 1% sodium dodecyl sulfate (SDS) and 1% 2-mercaptoethanol (ME) for 14.5 hr at room temperature. This extraction step was repeated five times (extracts 1–5). SDS-ME extracts were subjected to size-exclusion column chromatography, and comparisons of their profiles for specific absorbance at 650 nm (A₆₅₀) and carbohydrates were made. After high molecular weight (HMW) carbohydrates on the starch granule surface were extracted, HMW carbohydrates inside the granule appeared to be extracted. Finally, low molecular weight (LMW) carbohydrates near the granule surface were extracted. Phase-contrast light microscopy of the treated starch granules showed that all granules became transparent. Two different interior structures were observed. Scanning electron microscopy indicated that the granule was split into two parts at the equatorial groove. The interior of the granule showed two different areas: a central hole area and the surrounding stratified area. Extraction beyond five times with the same solvent dissolved the weak part of the granule structure and left two types of skeletal structures. The appearance of the skeletal structure of the granule surface was different from the appearance of interior structures.     

Why do gelatinized starch granules not dissolve completely? Roles for amylose, protein, and lipid in granule "ghost" integrity

After gelatinization in water, starch granules persist in swollen hydrated forms known as ghosts. Three potential mechanisms for ghost formation are tested. Proteins and lipids on the granule surface are found to be a determinant of ghost robustness, but not ghost formation. Proteins inside pre-made maize or wheat starch ghosts are degraded extensively by proteases without any apparent change in ghost properties, making an internal protein cross-linking mechanism unlikely. Waxy maize mutants with a range of amylose contents have ghost integrities that correlate with (low) apparent amylose levels. It is hypothesized that ghost formation is due to cross-linking of polysaccharide chains within swollen granules, most likely involving double helices formed from polymer chains that become free to move following heat-induced granule swelling. The size and robustness of granule ghosts is proposed to be determined by the relative rates of swelling and cross-linking, modulated by surface non-polysaccharide components.     

Distribution of Granule Channels,Protein, and Phospholipid in Triticale and Corn Starches as Revealed by Confocal Laser Scanning Microscopy

The morphology and microstructure of starch granules from two cultivars of triticale and from normal corn were characterized using scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM). Compared to numerous pores distributed randomly on the surfaces of corn starch granules, markedly fewer pores were observed on the surfaces of starch granules isolated from Pronghorn triticale, and even fewer on the surfaces of starch granules isolated from Ultima triticale. CLSM with fluorescence staining revealed that starch‐associated protein was predominately distributed on the granule surface and in the internal channels of both triticale and corn starches. However, after triticale starch was treated with SDS or SO₂, the radially oriented, protein‐filled internal channels of the granules were observed more frequently and extended to the central region of granules. Phospholipid was located mainly on the granule surface but also in channels and throughout granules in triticale starches, whereas in corn starch granules, it was mainly in the channels. The amount of protein and phospholipid in chemically and protease‐treated starches varied with starch source and treatment conditions. In treated triticale starches, the nitrogen content was positively correlated with the phosphorus content, indicating a close association between protein and phospholipid within starch granules. Starch‐associated protein and phospholipid may play an important role in maintaining the structural stability of both the granule surface and the internal channels.     

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.     

Effects of Varying Weight Ratios of Large and Small Wheat Starch Granules on Experimental Straight‐Dough Bread

One commercial bread wheat flour with medium strength (11.3% protein content, 14% mb) was fractionated into starch, gluten, and water solubles by hand‐washing. The starch fraction was separated further into large and small granules by repeated sedimentation. Large (10–40 μm diameter) and small (1–15 μm diameter) starch fractions were examined. Flour fractions were reconstituted to original levels in the flour using composites of varying weight percentages of starch granules: 0% small granules (100% large granules), 30, 60, and 100% (0% large granules). A modified straight‐dough method was used in an experimental baking test. Crumb grain and texture were significantly affected. The bread made from the reconstituted flour with 30% small granules and 70% large granules starch had the highest crumb grain score (4.0, subjective method), the highest peak fineness value (1,029), and the second‐highest elongation ratio (1.55). Inferior crumb grain scores and low fineness and elongation ratios were observed in breads made from flours with starch fractions with 100% small granules or 100% large granules. As the proportion of small granules increased in the reconstituted flour, it yielded bread with softer texture that was better maintained than the bread made from the reconstituted reference flour during storage.     

Separation and Characterization of A- and B-Type Starch Granules in Wheat Endosperm

Mature wheat (Triticum aestivum L.) endosperm contains two types of starch granules: large A-type and small B-type. Two methods, microsieving or centrifugal sedimentation through aqueous solutions of sucrose, maltose, or Percoll were used to separate A- and B-type starch granules. Microsieving could not completely separate the two types of starch granules, while centrifuging through maltose and sucrose solutions gave a homogenous population for B-type starch granules only. Centrifuging through two Percoll solutions (70 and 100%, v/v) produced purified populations of both the A- and B-type starch granules. Analysis of starch granule size distribution in the purified A- and B-type granule populations and in the whole-starch granule population obtained directly from wheat endosperm confirmed that the purified A- and B-type starch granule populations represented their counterparts in mature wheat endosperm. Centrifugations through two Percoll solutions were used to purify A- and B-type starch granule populations from six wheat cultivars. The amylose concentrations and gelatinization properties of these populations were analyzed. All of the A-type starch granules contained higher amylose concentrations and had higher gelatinization enthalpies than did B-type starch granules. Although A- and B-type starch granules started to gelatinize at a similar temperature, B-type starch granules had higher gelatinization peak and completion temperatures than did A-type starch granules     

Effects of Wheat Starch Granule Size Distribution on Qualities of Chinese Steamed Bread and Raw White Noodles

Starch is a crucial component determining the processing quality of wheat‐based products such as Chinese steamed bread (CSB) and raw white noodles (RWN). Flour from wheat cultivar Zhongmai 175 was used for fractionation into starch, gluten, and water solubles by hand washing. The starch fraction was successfully separated into large (>10 μm diameter) and small starch granules (<10 μm diameter) by repeated sedimentation. Flour fractions were reconstituted to original levels in the flour by using constant gluten and water solubles and varying the weight ratio of large and small starch granules. As the proportion of small granules increased in the reconstituted flours, farinograph water absorption increased, and amylose content, pasting peak viscosity, trough, and final viscosity decreased. Starch granule size distribution significantly affected processing quality of CSB and RWN. Superior crumb structure score (12.0) was observed in CSB made from reconstituted flour with 35% small starch granules. CSB made from reconstituted flours with 30 and 35% small starch granules exhibited the highest total scores, with values of 85.4 and 83.3, respectively. Significant improvements in color, viscoelasticity, and smoothness of RWN were obtained with an increase in small starch granule content, and reconstituted flours with 30–40% small starch granules produced RWN with moderate firmness.     

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.     

Extrusion of Cross‐Linked Hydroxypropylated Corn Starches II. Morphological and Molecular Characterization

A series of cross‐linked hydroxypropylated corn starches were extruded with a Leistritz micro‐18 co‐rotating extruder. Extrusion process variables including moisture (30, 35, and 40%), barrel temperature (60, 80, and 100°C), and screw design (low, medium, and high shear) were investigated. Scanning electron microscopy (SEM) of extruded starches showed a gel phase with distorted granules and granule fragments after extrusion at 60°C. After extrusion at 100°C only a gel phase was observed with no granular structures remaining. High performance size exclusion chromatography (HPSEC) equipped with multiangle laser light‐scattering (MALLS) and refractive index (RI) detectors showed extruded starches degraded to different extents, depending on extrusion conditions. The average molecular weight of the amylopectin of unextruded native corn starch was 7.7 × 10⁸. Extrusion at 30% moisture, 100°C, and high shear reduced the molecular weight of amylopectin to 1.0 × 10⁸. Hydroxypropylated normal corn starch extruded at identical conditions showed greater decreases in amylopectin molecular weight. With the addition of cross‐linking, the amylopectin fractions of the extruded starches were less degraded than those of their native and hydroxypropylated corn starch counterparts. Similarly, increasing moisture content during extrusion lowered amylopectin degradation in the extruded starches. Increasing temperature during extrusion of cross‐linked hydroxypropylated starches at high moisture content (e.g., 40%) lowered amylopectin molecular weights of the extruded starches, whereas increasing extrusion temperature at low moisture content (30%) resulted in less degraded molecules. This difference was attributed to the higher glass transition temperatures of the cross‐linked starches.