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.     

α-Amylolysis of Large Barley Starch Granules

The α-amylolysis of large (volume average 16 μm) barley starch granules was studied by measuring the amount of carbohydrates solubilizing during hydrolysis, and the changes in morphology and molecular structure of the granule residues by scanning electron microscopy, particlesize analysis, size-exclusion chromatography, X-ray diffraction, and differential scanning calorimetry. X-ray diffraction showed that, in the earlier stages of α-amylolysis, both amorphous and crystalline parts of the granules were equally solubilized. More extensive hydrolysis caused a gradual decrease in A-type crystallinity and degradation of the granular structure. Scanning electron microscopy revealed that hydrolysis proceeded through pinholes, and pitted and partially hollow granule residues were formed. The lipid-complexed amylose was less susceptible to α-amylolysis than free amylose and amylopectin. Lipid-complexed amylose started leaching out of the granule residues only after half of the starch had solubilized due to the α-amylase treatment. Even though scanning electron microscopy indicated that there were intact granules left throughout the hydrolysis, the results obtained suggested that α-amylolysis of large barley starch granules proceeded rather evenly among the granules.     

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.     

Molecular Structure and Organization of Starch Granules from Developing Wheat Endosperm

Wheat starches isolated from seeds harvested between 7 and 49 days after anthesis (DAA) were fractionated into large (>8 μm) and small (<8 μm) granules and studied for starch structure and architecture. Starch granules at 7 DAA possessed unimodal size distribution, whereas it was bimodal at later maturity stages. The apparent amylose fraction of starch granules at early maturity (7 and 14 DAA) consisted of intermediate‐type materials, whereas starch at later maturity stages (28 and 49 DAA) contained branched amylose. Wide‐angle X‐ray scattering (WAXS) revealed a well‐developed polymorphic structure already at 7 DAA. Although the presence of a small proportion of B‐type crystallites mixed with A‐type crystallites was observed in the X‐ray diffractogram of starches at early maturation (7 and 14 DAA), it was masked by the A‐type crystallites at later maturity stages. However, the large granules had a higher proportion of B‐type crystallites and lower relative crystallinity (RC) than their small‐granule counterpart. The iodine absorption properties of the starch granules demonstrated different levels of mobility of the starch polymers at different stages of maturity and the mobility of more glucan polymers in the large granule population compared with the small granules at the same maturity stage. Iodine did not change the characteristic A‐type crystalline pattern of starch, but it increased RC. Changes in peak width at half height based on WAXS data further suggested the possible interaction of iodine with amylopectin intercluster chain segments and branch chains in formation of inclusion complexes.     

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.     

碾轧时间和频率对玉米淀粉机械力化学效应的影响

为了研究碾轧对玉米淀粉机械力化学效应的影响,该研究以玉米淀粉为原料,采用扫描电镜、偏光显微镜、激光共聚焦显微镜、X-射线衍射、傅立叶变换红外光谱仪、差示扫描量热仪、快速黏度分析仪等手段来研究碾轧处理时间和转速对样品的表面形貌、粒度分布、结晶结构、糊化特性和热特性等结构和性质的影响。结果表明,在频率为20 Hz条件下,碾轧处理3~9 h时,碾轧对淀粉结晶结构破坏作用较弱,主要是对颗粒的无定型区产生了破坏作用,破坏了无定型区的双螺旋结构,中央腔变大,孔道模糊。碾轧处理3~6 h时,淀粉颗粒形状发生不规则变化,粒径也发生了相应的变化,热焓值下降,而结晶度下降不显著。在碾轧处理9 h时,球状凸起变大,水溶指数、膨胀度、透光率、峰值黏度和热焓值都有所减小,而淀粉乳稳定性增强。碾轧处理12~24 h时,淀粉颗粒表面球状凸起变的不明显,淀粉颗粒结晶区内部双螺旋结构破坏,孔道增多变粗,粒径增大,热焓值下降。总之,频率为20 Hz时的碾轧处理对淀粉颗粒的无定形区、结晶区产生不同程度的机械力化学作用,导致玉米淀粉颗粒内部依次发生了受力、聚集和团聚效应。而频率为30 Hz时,由于剪切力更强,碾轧处理对淀粉结构和性质的影响更为显著。     

Restriction of Starch Granule Swelling by Iodine During Heating

Differences in the degree of starch granule swelling and granule morphology are shown as a function of iodine concentration during heating. The observations reveal a restriction and delay in wheat and corn starch swelling in presence of iodine (0.02%) and a lack of swelling at higher iodine concentration (0.2%). The presence of iodine during heating did not influence waxy corn starch granule swelling, even at the higher concentration. A delay in the increase in paste viscosity during heating was observed, and gel formation was precluded after cooling at the higher iodine concentration. Waxy corn starch pastes formed a weak gel even at the higher iodine concentration. Spectrophotometric analyses showed that polymers leach into the solution when heated in the presence of 0.02% iodine, while no leaching was observed at 0.2% iodine concentration. Furthermore, the length and the amount of polymers leaching from normal corn were different from that observed for wheat starch. Thermal analyses reveal a shift in the onset of gelatinization temperature and an increase in the enthalpy in the amylose‐lipid region of the endotherm. While the iodine‐polymer complex did not appear to exhibit an endotherm during heating, the granules exhibited a diffused polarized cross, suggesting the presence of an ordered complex.     

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.     

Structural Changes of High-Amylose Rice Starch Residues following in Vitro and in Vivo Digestion

High-amylose cereal starch has a great benefit on human health through its resistant starch content. In this paper, starches were isolated from mature grains of high-amylose transgenic rice line (TRS) and its wild-type rice cultivar Te-qing (TQ) and digested in vitro and in vivo. The structural changes of digestive starch residues were characterized using DSC, XRD, (13)C CP/MAS NMR, and ATR-FTIR. TQ starch was very susceptible to digestion; its residues following in vitro and in vivo digestion showed similar structural characteristics with TQ control starch, which suggested that both amorphous and crystalline structures were simultaneously digested. Both amorphous and the long-range order structures were also simultaneously hydrolyzed in TRS starch, but the short-range order (double helix) structure in the external region of TRS starch granule increased with increasing digestion time. The A-type polymorph of TRS C-type starch was hydrolyzed more rapidly than the B-type polymorph. These results suggested that B-type crystallinity and short-range order structure in the external region of starch granule made TRS starch resistant to digestion.     

电子束辐照预处理对辛烯基琥珀酸改性芋艿淀粉性质的影响研究

为探究不同剂量电子束辐照预处理对辛烯基琥珀酸改性芋艿淀粉颗粒结构和功能特征的影响,本研究以奉化芋艿淀粉为材料,对其进行电子束辐照预处理,分析处理前后改性淀粉的颗粒结构、热力学性质、溶胀特性、消化性和乳化性。结果表明,电子束辐照预处理显著改变了辛烯基琥珀酸改性芋艿淀粉酯的理化与功能特性。随着辐照剂量从2 kGy逐步增加至50 kGy,辛烯基琥珀酸改性淀粉(OSTS)颗粒屑状点逐渐增多、表面形成的皱痕和突起加深,并出现颗粒破裂;快消化淀粉(RDS)含量和水溶性指数不断增加,结晶度、缓慢消化淀粉(SDS)、糊化峰值温度(T_p)不断下降;直链淀粉含量和溶胀力呈现先增加后减少的趋势,糊化焓、抗性淀粉、乳化稳定性和FTIR光谱中1 047 cm^-1/1 022 cm^-1 峰强度呈现先降低后升高的趋势。本研究为采用电子束辐照预处理技术改善辛烯基琥珀酸改性淀粉的功能特性提供了一定的理论依据。     

Association of Starch Granule Proteins with Starch Ghosts and Remnants Revealed by Confocal Laser Scanning Microscopy

Starch suspensions (0.25%) were gelatinized to 70 and 100°C, and starch ghosts (defined as gelatinized starch granule envelopes after the majority of internal starch polymers have been released) and remnants were collected by centrifugation and washed with water. Protein was revealed in isolated gelatinized normal starch ghosts using confocal laser scanning microscopy and a protein‐specific dye that fluoresces only after reaction with primary amines in protein. This technique eliminates background interference from residual dye. Observation of fluorescent‐labeled protein in the starch ghosts at different optical depths of field revealed that protein was concentrated in the envelopes of swollen, gelatinized potato, maize, and wheat starch ghosts. Only traces of protein were found in gelatinized starch granule remnants of waxy maize and amylose‐free potato starches after they were heated to 100°C, indicating that the proteins observed in gelatinized normal maize starch were largely granule‐bound starch synthase (GBSS). Moreover, fragility of the gelatinized waxy and amylose‐free starch granule remnants might be caused in part by the lack of GBSS. Gel electrophoresis of proteins in starch ghosts confirmed that GBSS in potato and maize was tightly associated with the starch ghosts. The study provides a structural explanation for a role of granule‐associated proteins in maintaining the integrity of starch ghosts and remnant structures, and their consequent effect on paste rheology.     

Dual modification of starch via partial enzymatic hydrolysis in the granular state and subsequent hydroxypropylation

The effect of enzymatic pretreatment on the degree of corn and mung bean starch derivatization by propylene oxide was investigated. The starch was enzymatically treated in the granular state with a mixture of fungal alpha-amylase and glucoamylase at 35 degrees C for 16 h and then chemically modified to produce enzyme-hydrolyzed-hydroxypropyl (HP) starch. Partial enzyme hydrolysis of starch in the granular state appeared to enhance the subsequent hydroxypropylation, as judged from the significant increase in the molar substitution. A variable degree of granule modification was obtained after enzyme hydrolysis, and one of the determinants of the modification degree appeared to be the presence of natural pores in the granules. Enzyme-hydrolyzed-HP starch exhibited significantly different functional properties compared to hydroxypropyl starch prepared from untreated (native) starch. It is evident that the dual modification of starch using this approach provides a range of functional properties that can be customized for specific applications.     

Resistance to α‐Amylase Digestion in Four Native High‐Amylose Maize Starches

Native and processed high‐amylose maize starch (HAMS) is an important source of resistant starch (RS). The objectives of this work were to use an in vitro procedure to estimate the RS content of native granules from a series of ae‐containing HAMS genotypes, and to examine the nature of the α‐amylase resistant starch (ARS). By the method of Englyst et al (1992), RS for ae V, ae VII, ae su2, and ae du were estimated to be 66.0, 69.5, 69.5, and 40.6%, respectively. By transmission electron microscopy, most of the residual granules from ae V, ae VII, and ae su2 showed little evidence of digestion. Partially digested granules had a radial digestion pattern in the interior and an enzyme‐resistant layer near the surface. Size and chain‐length profile of constituents of ARS were similar to those of the native HAMS (unlike type 3 RS), consistent with complete hydrolysis in susceptible granule regions. Between crossed polarizers, many iodine‐stained native and residual HAMS granules had blue centers and pink exteriors, which may be due to a difference in orientation of the amylose‐iodine complexes in the exterior. Four granule color types were observed for ae du, differing in enzyme resistance. The high‐enzyme resistance of native HAMS granules may result from altered granule organization, which appears to vary among and within granules from ae‐containing genotypes.     

Physicochemical,Molecular, and Digestion Characteristics of Annealed and Heat–Moisture Treated Starches Under Acidic,Neutral, or Alkaline pH

Rice, maize, and potato starches were incubated under acidic (2), neutral (7) or alkaline (11) pH conditions, and combined with annealing (ANN) or heat–moisture treatment (HMT), with the aim to evaluate their changes of physicochemical, digestion, and molecular characteristics. The applied treatments produced changes in all starches, showing void zones in the granules, which were more evident in ANN samples. The HMT starches promoted the formation of granular conglomerates that still showed birefringence. Overall, the evaluated conditions promoted changes in granule architecture (revealed by differences in gelatinization enthalpy) and crystallinity, for which an extensive degradation of their characteristics diffraction patterns occurred. These changes were more evident when incubation under acidic conditions was employed. Through principal component analysis, we found that the structural changes in starch granules have a direct influence on slowly digestible starch, resistant starch, and predicted glycemic index values, and this is the result of a higher proportion of organized crystallites, obtained from the acid hydrolysis process.     

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.     

Visualization of Channels and Cavities of Corn and Sorghum Starch Granules

Evidence is presented that corn (maize) and sorghum starch granules have channels that connect a central cavity to the external environment. A mutant sorghum starch with numerous, large surface pores was treated with a methanolic solution of merbromin and sectioned. Light, fluorescence, and compositional backscattering electron microscopy revealed channels connecting an internal cavity to the external surface in most granules. Cavities and channels could also be seen in whole corn and sorghum starch granules treated with merbromin in methanol and viewed by fluorescence microscopy. Treatment of sorghum starch granules with an aqueous solution of merbromin revealed that the molecule penetrated the granule matrix under even slightly swelling conditions. Light microscopy showed cavities in unstained, whole, dry corn and sorghum starch granules mounted in immersion oil.     

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.     

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.     

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.     

均质压力对玉米淀粉机械力化学效应的影响

为了研究均质压力对玉米淀粉微观结构及理化性质的影响,该文以玉米淀粉为原料,通过X-射线衍射(X-ray diffraction,XRD)、扫描电镜(scanning electron microscope,SEM)、快速黏性分析仪(rapid visco analyser,RVA)、偏光显微镜(polarizing microscope,PLM)、激光共聚焦显微镜(confocal laser scanning microscopy,CLSM)等手段研究不同压力(20、60、100、140 MPa)下淀粉结构及性质变化,并探究其相互关系,揭示均质压力对淀粉颗粒机械力化学效应。结果表明:均质压力处理对玉米淀粉结构及性质产生显著影响。经20~140 MPa处理后,与原淀粉相比,中央腔及孔道结构模糊,粒径、糊化黏度减小,结晶度下降,水溶指数和透光率呈上升趋势。20~100 MPa范围内,随均质压力增大,淀粉颗粒形貌逐渐破坏,球状凸起结构增加,100 MPa处理时中心球体最为明显,且与60 MPa相比,结晶度变大,膨胀度显著下降。当140 MPa处理时,颗粒内部球状凸起、碎片及孔洞结构显著减少,偏光十字破坏,糊化焓降低。可见不同均质压力对淀粉颗粒的无定形区、亚结晶区和结晶区产生不同程度的机械力化学作用,导致淀粉颗粒内部依次发生了聚集和团聚效应。该结果为研究淀粉化学活性及生产高性能变性淀粉提供理论支撑。