C-type Starch Research Articles

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.

Formation of Semi-compound C-Type Starch Granule in High-Amylose Rice Developed by Antisense RNA Inhibition of Starch-Branching Enzyme

Cereal starch granules with high-amylose and resistant starch (RS) always show irregular morphology and special crystalline structure, but their formation during grain development is not yet clear. In our previous studies, we had generated a transgenic rice line (TRS) enriched with amylose and RS, which contained semi-compound starch showing a C-type crystalline structure. In this study, the formation of semi-compound C-type starch granule during TRS endosperm development was carefully investigated with light, scanning electron, and transmission electron microscopes and X-ray powder diffraction. The results showed that the TRS starch subgranules, each with a central hilum, were individually initiated in amyloplast and showed an A-type crystal at the early stage of starch granule development, which was similar to that in its wild type. However, with the endosperm development, the amylose content in TRS endosperm starch increased and the B-type starch crystal was deposited in the periphery of subgranules; then, the adjacent subgranules fused together and finally formed a continuous outer layer band surrounding the entire circumference of the starch granule. Accordingly, a mechanistic model for the formation of semi-compound C-type starch granules is proposed.

Characteristics of native and enzymatically hydrolyzed Zea mays L., Fritillaria ussuriensis Maxim. and Dioscorea opposita Thunb. starches

Comparative studies on glucoamylase hydrolysis of A-type Zea mays L., B-type F. ussuriensis Maxim., and C-type Dioscorea opposita Thunb. were carried out by scanning electron microscope (SEM), X-ray diffractometer (XRD), Fourier transform infrared (FT-IR) spectra and differential scanning calorimetry (DSC). Maize, Fritillaria, Dioscorea starches were hydrolyzed with glucoamylase for 2, 4, 8, 12 and 24 h, respectively. The SEM and XRD results revealed that A-type, B-type starch and C-type starch displayed different hydrolysis mechanisms. A-type starch was digested with enzyme penetrating into starch granules through natural pores on the surface and disrupted the interior of the starch granules. The glucoamylase worked by attacking the surface of B-type starch and forming cracks. When endo-corrosion occurred, the internal part of the granule was corroded through small cracks. However, the glucoamylase primarily attacked the interior of the C-type starch granules and then the exterior of starch granules. FT-IR confirmed that the amorphous regions in the starch granules are firstly hydrolyzed and could be hydrolyzed completely as long as the hydrolysis time is sufficient. The transition temperatures and enthalpy of gelatinization (Δ H gel) were determined using DSC. According to the gelatinization parameters, it could be further proved amorphous and crystalline structures were hydrolyzed.

C-Type Starch from High-Amylose Rice Resistant Starch Granules Modified by Antisense RNA Inhibition of Starch Branching Enzyme

High-amylose starch is a source of resistant starch (RS) which has a great benefit on human health. A transgenic rice line (TRS) enriched amylose and RS had been developed by antisense RNA inhibition of starch branching enzymes. In this study, the native starch granules were isolated from TRS grains as well as the wild type, and their crystalline type was carefully investigated before and after acid hydrolysis. In high-amylose TRS rice, the C-type starch, which might result from the combination of both A-type and B-type starch, was observed and subsequently confirmed by multiple physical techniques, including X-ray powder diffraction, solid-state nuclear magnetic resonance, and Fourier transform infrared. Moreover, the change of starch crystalline structure from C- to B-type during acid hydrolysis was also observed in this RS-rich rice. These data could add to our understanding of not only the polymorph structure of cereal starch but also why high-amylose starch is more resistant to digestion.

Physicochemical properties and development of wheat large and small starch granules during endosperm development

Wheat mature seeds have large, lenticular A-type starch granules, and small, spherical B-type and irregular C-type starch granules. During endosperm development, large amyloplasts came from proplastid, divided and increased in number through binary fission from 4 to 12 days after flowering (DAF). Large starch granules formed and developed in the large amyloplast. One large amyloplast had only one large starch granule. Small amyloplasts came from the protrusion of large amyloplast envelope, divided and increased in number through envelope protrusion after 12 DAF. B-type starch granules formed and developed in small amyloplast from 12 to 18 DAF, C-type starch granules formed and developed in small amyloplast after 18 DAF. Many B- and C-type starch granules might form and develop in one small amyloplast. The amyloplast envelopes were asynchronously degraded and starch granules released into cell matrix when amyloplasts were full of starch granules. Apparent amylose contents of large starch granules were higher than that of small starch granules, and increased with endosperm development. The swelling powers and crystallinity of large starch granule were lower than that of small starch granules, and decreased with endosperm development. Small starch granules displayed broader gelatinization temperature ranges than did large starch granules.

Starch granules size distribution in superior and inferior grains of wheat is related to enzyme activities and their gene expressions during grain filling

Mature wheat endosperm contains A-, B-, C-type starch granules, and each class has unique physiochemical properties which determine the quality of starch. The dynamics of the starch granule size distribution, activities of starch synthases and expression of starch synthase encoding genes were studied in superior and inferior grains during grain filling. Compared with inferior grains, superior grains showed higher grain weight, contents of starch, amylose and amylopectin. The formation of A-, B-, C-type starch granules initiated at 4, 8, 20 DAF, respectively, and was well consistent with the temporally change patterns of starch synthase activities and relative gene expression levels. For instance, activities of soluble and granule-bound starch synthases (designated SSS and GBSS) peaked at 20 and 24 DAF. Genes encoding isoforms of starch synthases expressed at different grain filling periods. In addition, SS I was generally expressed over the grain filling stage; the SS II and SS III were expressed over the early and mid grain filling stage, and the GBSS I was expressed during the mid to late grain filling stage. In addition, the time-course changes in activities of starch synthases and expression of starch synthase encoding genes explained well the dynamics of the starch granule size distribution.

The structure of C-type Rhizoma Dioscorea starch granule revealed by acid hydrolysis method

Scanning electron microscope (SEM), X-ray powder diffraction (XRD) and cross polarisation/magic-angle spinning (CP/MAS) 13C nuclear magic resonance (NMR) have been used for the structural characterisation of C-type starch granule during acid hydrolysis. SEM shows that the amorphous areas mainly locate the core part of C-type starch granules, while the crystalline areas mainly exist in the peripheral region of starch granules. XRD analysis reveals that the B-type polymorph present in the C-type starch granule are preferentially degraded or degraded faster than the A-type polymorph. NMR spectra confirm that the amorphous regions in the starch granules are firstly hydrolysed and could be hydrolysed completely as long as the hydrolysis time is sufficient. After 40 days of hydrolysis, the acid-modified starch shows typical A-type characteristics upon analysis of the XRD pattern or the 13C CP/MAS NMR spectra.

Study on the morphology, crystalline structure and thermal properties of yam starch acetates with different degrees of substitution

This study was carried out to understand and establish the changes in physicochemical properties of starch extracted from Chinese yam (Dioscorea opposita Thunb.) after acetylation. Yam starch acetates with different degrees of substitution (DS) were prepared by the reaction of yam starch with glacial acetic acid/acetic anhydride using sulfuric acid as the catalyst. Their formation was confirmed by the presence of the carbonyl signal around 1750 cm−1 in the Fourier transform infrared (FT-IR) spectra. The thermal behavior of the native starch and starch acetate were investigated using thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC). The results reveal that the starch acetates are more thermally stable than the native starch. The starch esters showed 50% weight loss at temperatures from 328°C to 372°C, while the native starch underwent 50% weight loss at 325°C. The glass transition temperature (Tg) of the starch decreased from 273°C to 226°c. The X-ray diffraction (XRD) patterns could be classified as typical of the C-type for yam starch. X-ray diffraction also showed the loss of the ordered C-type starch crystalline structure and the degree of crystallinity of starch decreased from 36.10% to 10.96% with the increasing DS. The scanning electron microscopy (SEM) suggested that the most of the starch granules disintegrated with many visible fragments with the increasing DS.

Effect of the Alkaline Treatment on the Ultrastructure of C-Type Starch Granules

The effect of alkaline treatment on the ultrastructure of C-type starch granules was investigated during the alkaline extraction of Araucaria angustifolia (pinhao) starch. The efficiency in protein removal was evaluated using intrinsic fluorescence and Kjeldahl's method. In parallel, morphological changes of starch granules were observed using scanning electron microscopy and atomic force microscopy. The starch crystallinity was monitored by wide-angle X-ray scattering and the lamellar structure was studied by small-angle X-ray scattering (SAXS). The paracrystalline model was employed to interpret the SAXS curves. It was found that the granular organization was significantly altered when alkaline solutions were used during the extraction. A partial degradation of B-type allomorph of starch and a significant compression of semicrystalline growth rings were observed.

The new insight on ultrastructure of C-type starch granules revealed by acid hydrolysis

C-type starch granule could be considered as the mixture of A- and B-polymorphs. The ultrastructure of C-type starch granules has not been elucidated detailedly by comparison with that of A- or B-type starch. To better understand the ultrastructure of C-type starch granules, Environment Scanning Electron Microscope (ESEM) and Field Emission Gun Transmission Electron Microscope (FEG-TEM) have been used to analyze the conformation and ultrastructure of C-type starch granule from Rhizoma Dioscorea during acid hydrolysis. SEM results showed that the amorphous areas were mainly located interior part of C-type starch granules whereas the crystalline regions were found mostly in the peripheral region of the granules. The grain size can be confirmed to be about 4.5–9 nm from the HR-TEM micrographs. The nanocrystals from acid-thinned starch displayed the typical face-centered cubic structure. This selected area electron diffraction patterns showed that individual C-type starch granule consisted of A- and B-type polymorphs.

The semi-crystalline growth rings of C-type pea starch granule revealed by SEM and HR-TEM during acid hydrolysis

Semi-crystalline growth rings and the crystalline lamella of C-type pea starch were studied by Scanning Electron Microscope (SEM) and Field Emission Gun Transmission Electron Microscope (FEG-TEM) during acid hydrolysis. The alternating growth rings (semi-crystalline and amorphous growth rings) are clearly observed under SEM. The thickness of semi-crystalline growth rings is variable and approximately 200 nm apart. SEM results also reveals that the amorphous areas mainly locate core part of C-type starch granules, while the crystalline areas mainly exist in the peripheral region of starch granules. The crystalline and the amorphous areas are hydrolyzed concomitantly, although slowly. The clear lattice fringes with different orientation corresponding to the different thickness of crystalline lamella were observable from high-resolution-TEM (HR-TEM) images. The crystalline lamella size can be confirmed to be about 20 nm with the length of 20 nm and the thickness of 20 nm. The clear lattice fringes should correspond to the crystalline phases in the semi-crystalline growth rings while the blurry regions correspond to the amorphous growth rings among the semi-crystalline growth rings.

Granular structure and allomorph position in C-type Chinese yam starch granule revealed by SEM, 13C CP/MAS NMR and XRD

The crystalline microstructure and polymorphism of C-type starch from Chinese yam were evaluated by scanning electron microscope (SEM), 13C cross-polarization magic-angle spinning NMR ( 13C CP/MAS NMR) and powder X-ray diffraction (XRD) technique and acid hydrolysis method. Morphological changes during acid hydrolysis showed that the amorphous or the less crystalline areas were essentially located at the center part of C-type starch granules whereas the semi-crystalline and amorphous growth rings were found mainly in the outer part of the granules. 13C CP/MAS NMR and XRD results revealed that B-type allomorph was hydrolyzed more rapidly than A-type one. The amorphous or less crystalline areas were predominantly composed of B-allomorph whereas the outer semi-crystalline and amorphous growth rings were mostly composed of A-type allomorph. The A- and B-type allomorph coexisted in the individual C-type starch granule. B-type allomorph basically existed at the center part of the granules which was surrounded by the A-type allomorph in the peripheral part of granules.

Conformation and location of amorphous and semi-crystalline regions in C-type starch granules revealed by SEM, NMR and XRD

The conformations and locations of amorphous and semi-crystalline regions in C-type starch granules from Chinese yam were evaluated by a combination of morphology and spectroscopy studies during acid hydrolysis. Scanning electron micrographs showed that amorphous or less crystalline areas were essentially located in the centre part of C-type starch granules, whereas the semi-crystalline and amorphous growth rings were found mainly in the outer part of the granules. 13C cross-polarization magic angle spinning NMR (13C CP/MAS NMR) showed that amorphous regions were hydrolyzed faster than the crystalline ones. In addition, B-type polymorphs were shown to be hydrolyzed more rapidly than A-types. Powder X-ray diffraction (XRD) also revealed that the B-polymorph was hydrolyzed more rapidly than the A-type. XRD showed that the amorphous or less crystalline areas were mainly located in the core of starch granules, while the amorphous growth rings are distributed toward the outside of the granules and alternatively arranged with semi-crystalline growth rings. The amorphous or less crystalline areas predominantly consisted of the B-polymorph whereas the outer semi-crystalline and amorphous growth rings were mostly composed of the A-polymorph.

Structure characterization of C-type starch granule by acid hydrolysis

The structural changes of C-type Dioscorea rhizoma starch are evaluated by scanning electron microscope (SEM), X-ray powder diffraction (XRD) and cross polarization/magic-angle spinning (CP/MAS) 13C nuclear magic resonance (NMR) during acid hydrolysis. SEM shows that the amorphous areas are mainly located in the core part of C-type starch granules or distribute alternately in the crystalline regions. XRD analysis reveals that the B-type polymorphs present in the C-type starch granules are preferentially degraded or degraded faster than A-type polymorphs. NMR spectra confirm that the amorphous regions in the starch granules are firstly hydrolyzed and could be hydrolyzed completely as long as the hydrolysis time is enough. After 40 days of hydrolysis, the acid-modified starch shows typical A-type characteristics whether for the X-ray diffraction pattern or for the 13C CP/MAS NMR spectra. The finding suggests that the B-type polymorphs consisting of the C-type starch granules are more unstable than the A-type polymorphs. This is not in agreement with the fact that B-type starches (potato starch) are more resistant to acid and enzyme than A-type cereal starches.

Granule structure of C-type Chinese Yam ( Dioscorea opposita Thunb var. Zhongbowen) starch by acid hydrolysis

Granule structure of C-type starch separated from Dioscorea opposita Thunb var. Zhongbowen (D. ZBW) was characterized by scanning electron microscope (SEM), X-ray powder diffractometer (XRD) and laser light scattering particle size analyzer (LLSPSA) in the process of acid hydrolysis. D. ZBW starch was treated with 2.2 mol/l hydrochloric acid for different lengths of time. The SEM results revealed that the amorphous regions of the starch located at the centre of the granules while the outer layer of the granules was mainly composed of the crystalline areas. The crystal type of the starch changed from typical C-type X-ray diffraction pattern to the representative A-type pattern in the process of acid hydrolysis by XRD analysis. The above results demonstrated that B-type polymorphs present in C-type starch granule was preferentially degraded at the first stage of hydrolysis followed by A-type polymorphs. The acid-thinned starch granules displayed a gradual reduction in the particle diameter followed the acid thinning.

Comparative studies on morphological and crystalline properties of B-type and C-type starches by acid hydrolysis

Comparative studies on acid hydrolysis of B-type Fritillaria starch and C-type Rhizoma Dioscorea and Radix Puerariae starches were carried out using a scanning electron microscope (SEM) and X-ray diffraction (XRD). Fritillaria, Rhizoma Dioscorea and Radix Puerariae starches were hydrolyzed with 2.2 mol/L at 35 °C for 2, 4, 8, 16 and 32 days, respectively. The SEM and XRD results revealed that B-type starch and C-type starch displayed different hydrolysis mechanisms. The acid corrosion started from the exterior surface of B-type starch granules followed by the interior core of starch granules. However, the hydrogen ion primarily attacked the interior of the C-type starch granules and then the exterior of starch granules. B-type starch granule started to crack at the hydrolysis period of 4 days while C-type starch granule was not cracked until the hydrolysis progressed up to 16 days. The crystalline type of B-type starch was not changed with increasing hydrolysis time. However, the crystalline type was gradually changed from C-type to A-type for the Rhizoma Dioscorea and Radix Puerariae starches with increase in the hydrolysis time. This result showed that the B-type polymorphs present in the C-type starch granule was preferentially hydrolyzed during the first stage of hydrolysis.

The effect of acid hydrolysis on morphological and crystalline properties of Rhizoma Dioscorea starch

Starch isolated from Rhizoma Dioscorea (Jichengerhao-JCEH) was subjected to acid hydrolysis for 2, 4, 8, 16 and 32days, respectively. The acid-thinned starch was evaluated for starch yield, morphological, crystalline and particle size distribution properties by scanning electron microscope (SEM), wide-angle X-ray powder diffractometer (WAXRD) and light scattering particle size analyzer (LSPSA). The starch yield reduced from 100% for unmodified starch to 25.5% for starch subjected to 32days of acid hydrolysis. The SEM results revealed that the hydrogen ion preferentially attacked the inner amorphous regions of the starch granules followed by the outer crystalline areas. That is why starch granules did not show any corrosions or fissures at the first stage of acid hydrolysis. The average particle size of starch decreased from 23.4 μm for unmodified starch to 19.9 μm for acid-thinned starch for 16days. Wide-angle X-ray diffraction patterns indicated that the crystalline type of starch changed from C-type to A-type after 16 days of acid hydrolysis. This result demonstrated that B-type starch present in C-type starch was firstly degraded in the process of acid hydrolysis. The degree of crystallinity of starches increased gradually following the acid thinning.

Structure of lintnerized starch is related to X-ray diffraction pattern and susceptibility to acid and enzyme hydrolysis of starch granules

Acid-resistant residues (lintnerized starches, Ls) were prepared from starches showing A-, B- and C- X-ray diffraction patterns. Ls retained the same X-ray crystalline type as their native counterparts with an improvement in diffraction intensity. Fluorophore-assisted capillary electrophoresis (FACE) study indicated that structural characteristics of Ls were associated with X-ray diffraction patterns. Double helices originated from linear chains with an approximate average degree of polymerisation (DP) 14, 16, and 15 would span the entire length of crystalline lamellae of A-, B-, and C-type starches, respectively. The proportion of singly branched materials (SB) with DP 25 protected in Ls was higher for A-type Ls (10–17%) than for B-type Ls (4–6%) and C-type Ls (8%). The structures of SB were similar in which branched chain (DP 13–15) was longer than main chain (DP 10–12). The structural characteristics of Ls are discussed in relation to acid and enzymatic degradations of starch granules.

Structural studies of starches with different water contents.

The proportion of double helices in starches from a series of pea [rb, rug4-b, rug3-a, and lam-c mutants, and the wild type (WT) parental line], potato and maize (normal and low amylose), and wheat (normal) lines, ranged from about 30-50% on a dry weight basis. In relatively dry starch powders, only about half of the double helices were in crystalline order, this proportion being higher for A-type than for B-type starches. Using starch from WT pea as an example, it was found that increasing water content results in an increase in total crystallinity. When the water content was raised to a level similar to that in excess water, the proportion of crystallinity was close to the proportion of double helices (DH). Measuring crystallinity in starches with a high water content is difficult using traditional methods such as x-ray diffraction. A method was developed, therefore, for determining starch structural characteristics in excess water by measuring the enthalpy of gelatinization transition in quasi-equilibrium differential scanning calorimetry (DSC) experiments. It is suggested that DH% = DeltaH(sp)/DeltaH(DH) x 100%, where DeltaH(sp) and DeltaH(DH) represent the specific enthalpies of gelatinisation transition, DeltaH(sp) being measured as J/g dry starch weight and DeltaH(DH) as J/g DH, in starch. Studies on potato and maize starches in excess water and in 0.6M KCl showed, respectively, that DeltaH(DH) was 36.3 and 35.6 J/g for B-type polymorphs and 33.0 and 35.0 J/g for A-type polymorphs. For C-type starches, such as those from pea, intermediate values of DeltaH(DH), related to the proportions A-/B-polymorphs, should be used. The type of crystallinity in starch can be determined by the shift in peak temperature for thermograms in excess water and in excess 0.6M KCl. For B-polymorphs this shift was found to be approximately 2-3 degrees C and for A-polymorphs approximately 7-12 degrees C. The ratio between ordered areas with both A- and B-polymorphs can be determined from the enthalpies of disruption of each area. These enthalpies can be obtained by deconvolution of bimodal thermograms produced by C-type starches in excess 0.6M KCl. This methodical approach can be applied to all starches that give a sharp gelatinisation thermogram in excess water. Using a range of methods, including DSC, it was found that starch granules from the mutant peas are constructed in a similar way to those from the WT, with B-polymorphs in the centre and A-polymorphs at the periphery of all granules. The proportion of A/B-polymorphs, however, differed between the mutants. It was found that in addition to increasing the total crystallinity, increasing the water content within the granules also resulted in an increase in the proportion of B-polymorphs.

In situ observation of pressure-induced gelation of starches studied with FTIR in the diamond anvil cell

Pressure-induced gelation of six different starch types has been followed by Fourier-transform infrared (FTIR) spectroscopy. The diamond anvil cell allowed us to follow changes in situ at high pressure. Changes in the infrared spectrum due to conformational modifications and solvation changes in the starch granule were analysed. The spectra of these starches showed a similar behaviour at increasing pressure. Bands in the region 900–1300 cm −1 increased in intensity and changed position at high pressure. A correlation was found between the stability and the structure of the different starch types. B-type starches tend to be more pressure resistant than A- and C-type starches. In situ observation of the gelation was also performed with a microscope. At the pressure at which gelation occurred, a swelling of the starch granules was observed.