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Morphologies and microstructures of cornstarches with different amylose–amylopectin ratios studied by confocal laser scanning microscope
Journal of Cereal Science 50 (2009) 241–247
Contents lists available at ScienceDirect
Journal of Cereal Science journal homepage: www.elsevier.com/locate/jcs
Morphologies and microstructures of cornstarches with different amylose–amylopectin ratios studied by confocal laser scanning microscope Pei Chen a, b, c, Long Yu a, c, *, George Simon b, **, Eustathios Petinakis c, Katherine Dean c, Ling Chen a a
Centre for Polymers from Renewable Resources, ERCPSP South China University of Technology, Guangzhou, PR China Department of Materials Engineering, Monash University, Melbourne, Vic 3169, Australia c CSIRO, Materials Science and Engineering, Clayton South, Melbourne, Vic 3169, Australia b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 11 March 2009 Received in revised form 1 June 2009 Accepted 2 June 2009
The morphologies and microstructures of cornstarches with different amylose–amylopectin ratios (waxy: 0/100; maize: 23/77; Gelose 50: 50/50; and Gelose 80: 80/20) were studied by a confocal laser scanning microscope (CLSM). The temperature-induced changes of the cornstarch granules in excess of water were also studied under CLSM. Acid hybridization of starch by HCl was used to enhance the difference between amorphous and crystalline ranges. It was found that the high-amylose starches (G50 and G80) were brighter than those of low-amylopectin starches (waxy and maize) under confocal laser light, and the average (decreasing) fluorescence intensity sequence of the granules was G80 > G50 > maize > waxy. Waxy and maize starches showed clear internal cavities and channels, whilst G50 and G80 had bright cores. Sharp growth ring structures can be clearly observed for low-amylose starches (waxy and maize) after acid hydrolysis. Gelatinization of all starches starts at the hilum and the adjacent of the channels, and spreads rapidly to the periphery. This work is the first time that three-dimensional images of partly gelatinized granules have been constructed and presented from different confocal images, which allows further exploration of the mechanisms of gelatinization. Ó 2009 Elsevier Ltd. All rights reserved.
Keywords: Starch Amylose Microstructure Confocal image Gelatinization
1. Introduction The starch granule is a heterogeneous material containing both amorphous and crystalline regions. In terms of their chemical structure, most native starches are a mixture of amylose (a linear structure of alpha-1,4-linked glucose units) and amylopectin (a highly branched structure of short alpha-1,4 chains linked by alpha-1,6 bonds) (Zobel, 1984, 1988). The physicochemical properties of starch and its suitability for various industrial applications depend on the proportion, composition, and structure of the amylose and amylopectin molecules, the amylose–amylopectin ratio, the length and pattern of the amylopectin branches, the substitution of the glucose monomers, and the size and modality of the granules (Boren et al., 2008). Determination of the molecular distribution of amylose and amylopectin molecules in the native starch granule, along with the analysis of starch granule internal and surface topography, is important in order to gain an understanding of the behavior of starch and starch-based products.
* Corresponding author. CSIRO, Materials Science and Engineering, Clayton South, Melbourne, Vic 3169, Australia. Tel.: þ61 3 9545 2797; fax: þ61 3 9544 1128. ** Corresponding author. Tel.: þ61 3 9905 4936; fax: þ61 3 9905 4934. E-mail addresses: [email protected] (L. Yu), [email protected] (G. Simon). 0733-5210/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.jcs.2009.06.001
Various native starches exhibit significant differences in granular appearance and microstructure (Chen et al., 2006; Gregorov et al., 2006), and research involving the imaging of the morphology of starch granules and the subsequent changes which take place as they undergo digestion has been carried out for a long time. With the improvement and digitisation of analytical and microscopic techniques, a wider range of microscopic techniques can be usefully applied to this field, giving rise to a large amount of new and structural information about starch. The relevant imaging technologies developed include scanning electron microscopy (SEM) (Chanzy et al., 1990; Evers et al., 1970; Yamaguchi et al., 1979), transmission electron microscopy (TEM) (Chanzy et al., 1990; Helbert and Chanzy, 1996; Oostergetel and Van Bruggen, 1989), atomic force microscopy (AFM) (Baldwin et al., 1998; Neethikajan et al., 2008), and confocal laser scanning microscopy (CLSM) (Boren et al., 2008; Chung and Lai, 2006; van de Velde et al., 2002), assisted by other equipments able to probe microstructure such as X-ray(Jenkins et al., 1993; Zobel and Illinois, 1988), small angle neutron scattering (Donald et al., 1997, 2001), and 13C NMR (Cheetham and Tao, 1998; Wang et al., 2008). Despite significant efforts to characterize and differentiate various aspects of starch microstructure, the nature of their granular microstructure has not yet been fully elucidated (Chen et al., 2006; Kim and Huber, 2008; Liu et al., 2006, 2007, 2009; Xie et al., 2009).
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Table 1 Materials used in the experimental work. Sample No.
Name
Ratio of amylose–amylopectin
Molecular weight (g/mol)a
Supplier
1 2 3 4
Waxy Maize G50 G80
0/100 23/77 50/50 80/20
20,787,000 13,000,000 5,115,000 673,000
Australia Australia Australia Australia
a
Molecular weight measured by GPC [provided by Penford (Australia)].
The development of novel microscopy techniques, in particular CLSM, has enabled the study of the morphology and internal structure of starch granules without the use of complicated or invasive sample preparation methods, thus increasing new possibilities (Boren et al., 2008; Chung and Lai, 2006; Huber and BeMiller, 2000; Mira et al., 2007; van de Velde, 2002). The key feature of confocal scanning laser microscopy is its ability to image a single focal plane of the sample. This enables researchers to visualize the internal structure of starch granules without the need for sectioning techniques, which can result in destruction of the microstructure of the sample (Durrenberger et al., 2001). Furthermore, CSLM imaging enables the visualization of a single starch granule in slices of less than 0.1 mm, whereas sectioning with a precision microtome system results in slices of 10 mm without the possibility to view several sections of the same granule. van de Velde et al. (2002) have studied granule characteristics by CSLM, as
well as the gelatinization behavior of starch granules from different botanical sources, and detected the cross-section of cornstarch granules. Blennow et al. (2003) studied the molecular deposition of transgenically modified starch in the potato starch granule using a combination of confocal laser scanning microscopy, light microscopy, and environmental SEM techniques. They found that starch extracted from tubers with low-amylose contents showed very weak 8-amino-1,3,6-pyrenetrisulfonic acid (APTS) fluorescence. The starch granules with low molecular weight amylopectin and/or high-amylose granules showed a relatively even distribution in fluorescene, whilst normal and low-amylose granules had an intense fluorescence in the hilum, indicating a high concentration of amylose in the center of these granules. In our previous work (Chen et al., 2006), the morphologies and microstructures of cornstarches with different amylose–amylopectin ratios (waxy: 0/100; normal maize: 23/77; Gelose 50: 50/50; and Gelose 80: 80/20) were studied by microscopy with normal and polarized light, SEM, and XRD. It was found that birefringence of the granules under polarized light were waxy > maize > G50 > G80, which correspond with trends in molecular weight and crystallinity. In this work, the cornstarches were investigated by CLSM to systematically determine internal morphologies and microstructures of a single type of starch with different amylose–amylopectin ratios. The temperature-induced changes of the cornstarch granules in excess of water were also studied under CLSM.
Fig. 1. CLSM optical sections of different starches with same PMT: (a) waxy, (b) maize, (c) G50, and (d) G80.
P. Chen et al. / Journal of Cereal Science 50 (2009) 241–247 Table 2 Observation results of different starches under CLSM. Sample
Fluorescence intensitya (arbitrary units)
Channel
Hilum
Growth ring/crack
Waxy Maize G50 G80
40.27 61.61 83.64 90.73
Many Some Rare Rare
Dark Main dark Highlight Highlight
Growth ring Growth ring Crack Crack
a
Parenthesis: Fluorescence intensity from five independent measurements.
2. Experimental
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suspension was then filtered with Whatman No. 1 filter paper, with the acid-hydrolyzed starch dried at room temperature overnight under an air stream. 2.2.2. Partly gelatinized starch A quantity of 10 mg (dry basis) native starch was dispersed with 1 ml distilled water in a glass vial and then the glass vials were placed in the water bath for 2 min. Waxy and normal maize starch were heated to 70 C; and G50 and G80 were heated to 80 C. After thermal treatment, the samples were immediately cooled with tap water, the slurry found to separate into two layers after standing for 30 min.
2.1. Materials Cornstarches with different amylose–amylopectin ratios were used in the experimental work as model materials. All the starches are commercially available and were kindly supplied by Penford P/L (Australia). Table 1 lists the starches and their amylose–amylopectin ratios and other properties. APTS, sodium cyanoborohydride, and HCl acid were chemically pure and purchased as received from Sigma. 2.2. Sample preparation 2.2.1. Acid hydrolysis A quantity of 5 g (dry basis) of native starch was hydrolyzed by suspending it in 100 ml of 4 mol/L HCl solution at room temperature (about 21 C) for 5 days. After hydrolysis, the insoluble residue was washed several times with distilled water to neutrality. The
2.2.3. Dying Native and partly gelatinized starch samples were prepared for CLCM as previously described (Blennow et al., 2003). Starch granules (10 mg) were dispersed in 15 ml of freshly made APTS solution (10 mM APTS dissolved in 15% acetic acid), and 15 ml of 1 M sodium cyanoborohydride was added. The reaction mixture was incubated at 30 C for 15–18 h, with the granules washed five times with 1 ml of distilled water and finally suspended in 20 ml of 1:1 (v/v) glycerol–water mixture. A drop of the mixture was then mounted on a glass plate for microscopy. 2.3. Confocal laser canning microscopy A CLSM equipped with an Ar–Hg laser. (TCS SP2, Leica Microsystems, Wetzlar) with a stand for fixed fluorescent cell samples was used for the detection of the fluorescene signal from
Fig. 2. CLSM optical sections of HCl-hydrolyzed different starches: (a) waxy, (b) maize, (c) G50, and (d) G80.
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dye-stained starch granules. The details of the Leica objective lens used were: 100 plan apo/1.40 oil UV. The excitation wavelength was 488 nm with 52 capacity, and the format of the image was 512 512. During image acquisition, each line was scanned four times and averaged to reduce noise. For each starch sample, a stack of horizontal optical sections was obtained, encompassing the whole starch granule in three dimensions. Measurements of the fluorescence intensity were made with Imaris 6.1.5 and the 3D-image analysis was performed using the software of the Leica TCS SP2. 3. Results and discussion There have been many reports over the years utilizing various methods to unravel the internal structures of starch. The key feature of CLSM is the ability to image a single focal plane in the sample. This enables researchers to visualize cross-sections of starch granules without the need for sectioning techniques which can result in destruction of the microstructure of the sample (Durrenberger et al., 2001). However, starch does not show autofluorescence. It is necessary to choose an effective fluorescent dye. APTS is one of the fluorescence dyes with a small molecular structure that has previously been used successfully for specific labeling of the reducing ends of starch fragments (O’Shea et al., 1998). The intense and stable fluorescence of APTS allows both surface and internal images of starch granules to be recorded at high resolution. Previous researches have used APTS to dye starches for CLSM observation (Blennow et al., 2003; Chung and Lai, 2006;
Glaring et al., 2006). In these studies, CLSM imaging with APTS as a probe for starch molecule distribution was used to study the internal structural features of starches, such as growth ring, channels, and cracks, as well as the general molecular distribution of amylose and amylopectin within the starch granule. Fig. 1 shows the CLSM optical sections of different cornstarches obtained at the same photomultiplier tube (PMT) after dying with APTS. It can be seen that most of the high-amylose starches (G50 and G80) showed an intensely stained center and were brighter than low-amylose starches (waxy and maize). It is well known that amylose has a lower molecular weight than amylopectin and contains a much higher molar ratio of reducing ends per anhydrous glucose residues, which results in a higher by-weight labeling of amylose (Blennow et al., 2003) and thus the granules with high-amylose content showed higher fluorescence. It can be seen from Table 2 that the fluorescence intensity sequence of the granules is G80 > G50 > maize > waxy, which corresponds to decreasing intensity with decreasing amylose content. The center section of CLSM also reveals the structures in the granule core, where it can be seen that the core of most of the waxy granules (low amylose) were dark, and that the dark core is connected with the channels in the granules. Some dark cores can also be found in maize starch. However, the cores of most of the G50 and G80 (high amylose) starch granules are bright, and the bright core in G80 is larger than that in G50, which indicates that high-amylose starch granules were more easily highlighted by fluorescent dye in the hilum. One of the possible explanations for this phenomenon is that amylose content at the hilum is higher for the high-amylose
Fig. 3. CLSM optical sections of different starches after particularly gelatinized: (a) waxy, (b) maize, (c) G50, and (d) G80.
P. Chen et al. / Journal of Cereal Science 50 (2009) 241–247
cornstarches G50 and G80, whilst another explanation is related to the different microstructures of the channel and hilum. The cavities in the granular core for waxy and maize starches have been clearly observed, but with different structures for G50 and G80, the higher density of hilum of G50 and G80 showing brighter cores. Further studies are warranted in this area due to the observation of such contradictory phenomena. It should also be noted that there is a dark line or channel in the center for G80, which separates the
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bright core into two parts. This channel could relate to the granular growth and will be studied in detail in future work. From the cross-sections of the starch granule, the internal channels in starch granules can also be seen and are visible as dark lines running from the border of the granule toward the hilum in waxy and maize. The channels in starch granules have been observed by SEM and CLSM previously (Huber and BeMiller, 2000; Kim and Huber, 2008), and it has been noticed that the channels are
Fig. 4. 3D images of native (left) and partially gelatinized starches (right).
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more frequently and clearly observed in waxy and maize starch (low amylose) than that in the G50 and G80 (high amylose). Such channels extend to the granule surface connected with cavities within the granules in waxy and maize starch, whilst the channel (if it is observed) is a ring along the bright core in G50 and G80. Recently Kim and Huber (2008) studied the channels within different wheat starch granules and found that A-type wheat starch has relatively larger channels than B-type wheat starch, which corresponds to our observations reported above since waxy and maize are A-type, while G50 and G80 are B-type starches. Acid hydrolysis can be used to remove the amorphous and less crystalline parts in the starch granule. The acid-etched granule resulted in better contrast between amorphous and semicrystalline layers within starch granules (Chung and Lai, 2006). The growth rings within high-amylopectin starches (waxy and maize) were clearly observed after acid hydrolysis, whilst sharp edge cracks from the hilum toward the border in the high-amylose starches (G50 and G80) were observed after such acid treatment (see Fig. 2). The growth rings showed spacings of about 300–500 nm in waxy starches, whilst there are slightly finer rings with widths of about 200–400 nm in maize material. Generally, the high-amylose starch (G50 and G80) granules have a more compact microstructure than that of low-amylose starches, which show more acid-resistant and thermally stable behavior (Chen et al., 2007; Liu et al., 2006). Moreover, the acid-hydrolyzed corn starches showed stronger fluorescence intensity than native starches, indicating that there are more ends available to react with APTS groups after hydrolysis. The temperature-induced changes of the cornstarch granules in excess of water were also studied under CLSM. Phase transitions of starch granular during thermal processing are complicated and unique, especially the gelatinization process (Chen et al., 2007; Liu et al., 2006; Yeh and Li, 1996; Yu et al., 2006). The well-accepted definition of gelatinization for starch involves destruction of its order or crystalline regions. The starch suspensions were heated to just above their gelatinization temperatures (waxy and maize 70 C; G50 and G80 80 C) for 2 min, in order to avoid whole granules being completely destroyed, followed by rapid cooling by quenching with water. Following this they were then dyed with APTS and visualized using CSLM (see Fig. 3). The heterogeneity and complexity of such a starch structure within granules directly affects the gelatinization of starches. It can be seen from Fig. 3 that all the gelatinization starts at the hilum or botanical center of the granules and spreads rapidly to the periphery. Gelatinization thus begins in the intercellular and leastorganized region. As soluble components (primarily amylose) leach out of the granules during the process, they form a solution, whilst the center and adjacent components of the channels begin to gelatinize, and subsequently the other parts of the granules. The central area of the granule around the hilum is believed to be the least-organized region, since both acid hydrolysis and gelatinization start from there, which is in agreement with the previous observations (Chung and Lai, 2006). Fig. 4 shows 3D images of native and partly gelatinized starch granules constructed from different sections of CLSM images. The 3D images of native starch granules are similar to those observed from SEM (Chen et al., 2006). To the best of our knowledge, this is first time the full image of partly gelatinized granules in water suspension has been observed since other techniques are not able to view it under these conditions. For example, SEM cannot be used to view a water suspension, whilst other optical microscope techniques are not able to ‘‘slice’’ the object and construct such a 3D image. Using confocal microscopy, it can be clearly seen that the granules of waxy and maize are broken through the cavity and channels when the granules swell, whilst the granules of G50 and G80 remained as granular after swelling. This technique clearly
shows promise to further explore the mechanisms of gelatinization, and we are currently carrying out this work. 4. Conclusion The morphology and microstructure of maize starches with different amylose–amylopectin ratios were systematically studied by confocal laser scanning microscopy (CLSM). Temperatureinduced changes of the cornstarch granules in excess water were also studied by this technique. The different microstructures of cornstarch granules with different amylose contents were evident, in particular the distribution of amylose and amylopectin materials and cavity, channel, and growth ring–crack locations within the starch granule. It can be seen that the high-amylose starches (G50 and G80) are brighter than low-amylose starches (waxy and normal maize), and the average fluorescence intensity sequence of the granules is G80 > G50 > maize > waxy. The acid-hydrolyzed starches show stronger fluorescence intensity than native starches due to the increased end groups as a result of chain scission being more amenable to dye attachment. Waxy and maize starches clearly show cavities and channels, whilst G50 and G80 present a bright core, indicative of dyed amylose. Waxy and maize starches show sharp growth ring structures after acid hydrolysis due to etching of the amorphous layer, whilst G50 and G80 granules showed different structures. The gelatinization starts at the hilum and adjacent to the channels of the granules and spreads rapidly to the periphery. Three-dimensional images of partly gelatinized granules composed of different sections of CLSM images were obtained, which were used to study the mechanisms of gelatinization. The granules of waxy and maize broke through the cavity and channels when the granules became swollen during gelatinization, while the granules of G50 and G80 remained as granular. Acknowledgements The authors from SCUT, China, would like to acknowledge the research funds NRDPHT (863) (2007AA10Z312, 2007AA100407), NFSC (50540420129), and GNSF (05200617). We acknowledge Monash Micro Imaging Laboratory for providing CLSM for this work. P. Chen would like to acknowledge the State Scholarship Fund provided by China Scholarship Council which supported her study in Australia. References Baldwin, P.M., Adler, J., Davies, M.C., Melia, C.D., 1998. High resolution imaging of starch granule surfaces by atomic force microscopy. Journal of Cereal Science 27, 255–265. Blennow, A., Hansen, M., Schulz, A., Jorgensen, K., Donald, A.M., Sanderson, J., 2003. The molecular deposition of transgenically modified starch in the starch granule as imaged by functional microscopy. Journal of Structural Biology 143, 229–241. Boren, M., Glaring, M.A., Ghebremedhin, H., Olsson, H., Blennow, A., Jansson, C., 2008. Molecular and physicochemical characterization of the high-amylose barley mutant Amo1. Journal of Cereal Science 47, 79–89. Chanzy, H., Vuong, R., Jesior, J.C., 1990. An electron diffraction study on whole granules of lintnerized potato starch. Starch/Sta¨rke 42, 377–379. Cheetham, N.W.H., Tao, L., 1998. Solid state NMR studies on the structural and conformational properties of natural maize starches. Carbohydrate Polymers 36, 285–292. Chen, P., Yu, L., Chen, L., Li, X., 2006. Morphology and microstructure of maize starches with different amylose/amylopectin content. Starch/Sta¨rke 58, 611–615. Chen, P., Yu, L., Kealy, T., Chen, L., Li, L., 2007. Phase transition of starch granules observed by microscope under shearless and shear conditions. Carbohydrate Polymers 68, 495–501. Chung, Y.-L., Lai, H.-M., 2006. Molecular and granular characteristics of corn starch modified by HCl–methanol at different temperatures. Carbohydrate Polymers 63, 527–534. Donald, A.M., Waigh, T.A., Jenkins, P.J., Gidley, M.J., Debet, M., Smith, A., 1997. Internal structure of starch granules revealed by scattering studies. In:
P. Chen et al. / Journal of Cereal Science 50 (2009) 241–247 Frazier, P.J., Richmond, P., Donald, A.M. (Eds.), Starch: Starch Structure and Functionality. The Royal Society of Chemistry, London, pp. 172–179. Donald, A.M., Kato, K.L., Perry, P.A., Waigh, T.A., 2001. Scattering studies of the internal structure of starch granules. Starch/Sta¨rke 53, 504–512. Durrenberger, M.B., Handschin, S., Conde-Petit, B., Escher, F., 2001. Visualization of food structure by confocal laser scanning microscopy (CLSM). LebensmittelWissenschaft und-Technologie 34, 11–17. Evers, A.D., McDermott, E.E., Albans, S., 1970. Scanning electron microscopy of wheat starch. II. Structure of granules modified by alpha-amylolysis – Preliminary report. Starch/Sta¨rke 22, 23–26. Glaring, M.A., Koch, C.B., Blennow, A., 2006. Genotype-specific spatial distribution of starch molecules in the starch granule: A combined CLSM and SEM approach. Biomacromolecules 7, 2310–2320. Gregorov, E., Pabst, W., Bohacenko, I., 2006. Characterization of different starch types for their application in ceramic processing. Journal of the European Ceramic Society 26, 1301–1309. Helbert, W., Chanzy, H., 1996. The ultrastructure of starch from ultrathin sectioning in melamine resin. Starch/Sta¨rke 48, 185–188. Huber, K.C., BeMiller, J.N., 2000. Channels of maize and sorghum starch granules. Carbohydrate Polymers 41, 269–276. Jenkins, P.J., Cameron, R.E., Donald, A.M., 1993. A universal feature in the structure of starch granules from different botanical sources. Starch/Sta¨rke 45, 417–420. Kim, H.-S., Huber, K.C., 2008. Channels within soft wheat starch A- and B-type granules. Journal of Cereal Science 48, 159–172. Liu, H., Yu, L., Xie, F., Chen, L., 2006. Gelatinization of cornstarch with different amylose/amylopectin content. Carbohydrate Polymers 65, 357–363. Liu, H., Yu, L., Chen, L., Li, L., 2007. Retrogradation of corn starch after thermal treatment at different temperatures. Carbohydrate Polymers 69, 756–762. Liu, P., Yu, L., Liu, H., Chen, L., Li, L., 2009. Glass transition temperature of starch studied by a high-speed DSC. Carbohydrate Polymers 77, 250–253. Mira, I., Kurtis Villwock, V., Persson, K., 2007. On the effect of surface active agents and their structure on the temperature-induced changes of normal and waxy
247
wheat starch in aqueous suspension. Part II: A confocal laser scanning microscopy study. Carbohydrate Polymers 68, 637–646. Neethikajan, S., Thomson, D.J., Jayas, D.S., White, N.D.G., 2008. Characterization of the surface morphology of durum wheat starch granules using atomic force microscopy. Microscopy Research and Technique 71, 125–132. O’Shea, M.G., Samuel, M.S., Konik, C.M., Morell, M.K., 1998. Fluorophore-assisted carbohydrate electrophoresis (FACE) of oligosaccharides: efficiency of labelling and high-resolution separation. Carbohydrate Research 307, 1–12. Oostergetel, G.T., Van Bruggen, E.F.J., 1989. On the origin of a low angle spacing in starch. Starch/Sta¨rke 41, 331–335. van de Velde, F., van Riel, J., Hans Tromp, R., 2002. Visualisation of starch granule morphologies using confocal scanning laser microscopy (CSLM). Journal of the Science of Food and Agriculture 82, 1528–1536. Wang, S., Yu, J., Yu, J., 2008. Conformation and location of amorphous and semicrystalline regions in C-type starch granules revealed by SEM, NMR and XRD. Food Chemistry 110, 39–46. Xie, F., Yu, L., Su, B., Liu, P., Wang, J., Liu, H., Chen, L., 2009. Rheological properties of starches with different amylose/amylopectin ratios. Journal of Cereal Science 49, 371–377. Yamaguchi, M., Kainuma, K., French, D., 1979. Electron microscopic observations of waxy maize starch. Journal of Ultrastructure Research 69, 249–261. Yeh, A.-I., Li, J.-Y., 1996. A continuous measurement of swelling of rice starch during heating. Journal of Cereal Science 23, 277–283. Yu, L., Kealy, T., Chen, P., 2006. Study of starch gelatinization in a flow field using simultaneous rheometric data collection and microscopic observation. International Polymer Processing XXI, 283–289. Zobel, H.F., 1984. Gelatinization of starch and mechanical properties of starch pastes. In: Whistler, R.L., BeMiller, J.N., Paschall, E.F. (Eds.), Starch: Chemistry and Technology, second ed. Academic Press, Orlando, pp. 285–298. Zobel, H.F., 1988. Molecules to granules: A comprehensive starch review. Starch/ Sta¨rke 40, 44–50. Zobel, H.F., Illinois, A., 1988. Starch crystal transformations and their industrial importance. Starch/Sta¨rke 40, 1–7.
Contents lists available at ScienceDirect
Journal of Cereal Science journal homepage: www.elsevier.com/locate/jcs
Morphologies and microstructures of cornstarches with different amylose–amylopectin ratios studied by confocal laser scanning microscope Pei Chen a, b, c, Long Yu a, c, *, George Simon b, **, Eustathios Petinakis c, Katherine Dean c, Ling Chen a a
Centre for Polymers from Renewable Resources, ERCPSP South China University of Technology, Guangzhou, PR China Department of Materials Engineering, Monash University, Melbourne, Vic 3169, Australia c CSIRO, Materials Science and Engineering, Clayton South, Melbourne, Vic 3169, Australia b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 11 March 2009 Received in revised form 1 June 2009 Accepted 2 June 2009
The morphologies and microstructures of cornstarches with different amylose–amylopectin ratios (waxy: 0/100; maize: 23/77; Gelose 50: 50/50; and Gelose 80: 80/20) were studied by a confocal laser scanning microscope (CLSM). The temperature-induced changes of the cornstarch granules in excess of water were also studied under CLSM. Acid hybridization of starch by HCl was used to enhance the difference between amorphous and crystalline ranges. It was found that the high-amylose starches (G50 and G80) were brighter than those of low-amylopectin starches (waxy and maize) under confocal laser light, and the average (decreasing) fluorescence intensity sequence of the granules was G80 > G50 > maize > waxy. Waxy and maize starches showed clear internal cavities and channels, whilst G50 and G80 had bright cores. Sharp growth ring structures can be clearly observed for low-amylose starches (waxy and maize) after acid hydrolysis. Gelatinization of all starches starts at the hilum and the adjacent of the channels, and spreads rapidly to the periphery. This work is the first time that three-dimensional images of partly gelatinized granules have been constructed and presented from different confocal images, which allows further exploration of the mechanisms of gelatinization. Ó 2009 Elsevier Ltd. All rights reserved.
Keywords: Starch Amylose Microstructure Confocal image Gelatinization
1. Introduction The starch granule is a heterogeneous material containing both amorphous and crystalline regions. In terms of their chemical structure, most native starches are a mixture of amylose (a linear structure of alpha-1,4-linked glucose units) and amylopectin (a highly branched structure of short alpha-1,4 chains linked by alpha-1,6 bonds) (Zobel, 1984, 1988). The physicochemical properties of starch and its suitability for various industrial applications depend on the proportion, composition, and structure of the amylose and amylopectin molecules, the amylose–amylopectin ratio, the length and pattern of the amylopectin branches, the substitution of the glucose monomers, and the size and modality of the granules (Boren et al., 2008). Determination of the molecular distribution of amylose and amylopectin molecules in the native starch granule, along with the analysis of starch granule internal and surface topography, is important in order to gain an understanding of the behavior of starch and starch-based products.
* Corresponding author. CSIRO, Materials Science and Engineering, Clayton South, Melbourne, Vic 3169, Australia. Tel.: þ61 3 9545 2797; fax: þ61 3 9544 1128. ** Corresponding author. Tel.: þ61 3 9905 4936; fax: þ61 3 9905 4934. E-mail addresses: [email protected] (L. Yu), [email protected] (G. Simon). 0733-5210/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.jcs.2009.06.001
Various native starches exhibit significant differences in granular appearance and microstructure (Chen et al., 2006; Gregorov et al., 2006), and research involving the imaging of the morphology of starch granules and the subsequent changes which take place as they undergo digestion has been carried out for a long time. With the improvement and digitisation of analytical and microscopic techniques, a wider range of microscopic techniques can be usefully applied to this field, giving rise to a large amount of new and structural information about starch. The relevant imaging technologies developed include scanning electron microscopy (SEM) (Chanzy et al., 1990; Evers et al., 1970; Yamaguchi et al., 1979), transmission electron microscopy (TEM) (Chanzy et al., 1990; Helbert and Chanzy, 1996; Oostergetel and Van Bruggen, 1989), atomic force microscopy (AFM) (Baldwin et al., 1998; Neethikajan et al., 2008), and confocal laser scanning microscopy (CLSM) (Boren et al., 2008; Chung and Lai, 2006; van de Velde et al., 2002), assisted by other equipments able to probe microstructure such as X-ray(Jenkins et al., 1993; Zobel and Illinois, 1988), small angle neutron scattering (Donald et al., 1997, 2001), and 13C NMR (Cheetham and Tao, 1998; Wang et al., 2008). Despite significant efforts to characterize and differentiate various aspects of starch microstructure, the nature of their granular microstructure has not yet been fully elucidated (Chen et al., 2006; Kim and Huber, 2008; Liu et al., 2006, 2007, 2009; Xie et al., 2009).
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Table 1 Materials used in the experimental work. Sample No.
Name
Ratio of amylose–amylopectin
Molecular weight (g/mol)a
Supplier
1 2 3 4
Waxy Maize G50 G80
0/100 23/77 50/50 80/20
20,787,000 13,000,000 5,115,000 673,000
Australia Australia Australia Australia
a
Molecular weight measured by GPC [provided by Penford (Australia)].
The development of novel microscopy techniques, in particular CLSM, has enabled the study of the morphology and internal structure of starch granules without the use of complicated or invasive sample preparation methods, thus increasing new possibilities (Boren et al., 2008; Chung and Lai, 2006; Huber and BeMiller, 2000; Mira et al., 2007; van de Velde, 2002). The key feature of confocal scanning laser microscopy is its ability to image a single focal plane of the sample. This enables researchers to visualize the internal structure of starch granules without the need for sectioning techniques, which can result in destruction of the microstructure of the sample (Durrenberger et al., 2001). Furthermore, CSLM imaging enables the visualization of a single starch granule in slices of less than 0.1 mm, whereas sectioning with a precision microtome system results in slices of 10 mm without the possibility to view several sections of the same granule. van de Velde et al. (2002) have studied granule characteristics by CSLM, as
well as the gelatinization behavior of starch granules from different botanical sources, and detected the cross-section of cornstarch granules. Blennow et al. (2003) studied the molecular deposition of transgenically modified starch in the potato starch granule using a combination of confocal laser scanning microscopy, light microscopy, and environmental SEM techniques. They found that starch extracted from tubers with low-amylose contents showed very weak 8-amino-1,3,6-pyrenetrisulfonic acid (APTS) fluorescence. The starch granules with low molecular weight amylopectin and/or high-amylose granules showed a relatively even distribution in fluorescene, whilst normal and low-amylose granules had an intense fluorescence in the hilum, indicating a high concentration of amylose in the center of these granules. In our previous work (Chen et al., 2006), the morphologies and microstructures of cornstarches with different amylose–amylopectin ratios (waxy: 0/100; normal maize: 23/77; Gelose 50: 50/50; and Gelose 80: 80/20) were studied by microscopy with normal and polarized light, SEM, and XRD. It was found that birefringence of the granules under polarized light were waxy > maize > G50 > G80, which correspond with trends in molecular weight and crystallinity. In this work, the cornstarches were investigated by CLSM to systematically determine internal morphologies and microstructures of a single type of starch with different amylose–amylopectin ratios. The temperature-induced changes of the cornstarch granules in excess of water were also studied under CLSM.
Fig. 1. CLSM optical sections of different starches with same PMT: (a) waxy, (b) maize, (c) G50, and (d) G80.
P. Chen et al. / Journal of Cereal Science 50 (2009) 241–247 Table 2 Observation results of different starches under CLSM. Sample
Fluorescence intensitya (arbitrary units)
Channel
Hilum
Growth ring/crack
Waxy Maize G50 G80
40.27 61.61 83.64 90.73
Many Some Rare Rare
Dark Main dark Highlight Highlight
Growth ring Growth ring Crack Crack
a
Parenthesis: Fluorescence intensity from five independent measurements.
2. Experimental
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suspension was then filtered with Whatman No. 1 filter paper, with the acid-hydrolyzed starch dried at room temperature overnight under an air stream. 2.2.2. Partly gelatinized starch A quantity of 10 mg (dry basis) native starch was dispersed with 1 ml distilled water in a glass vial and then the glass vials were placed in the water bath for 2 min. Waxy and normal maize starch were heated to 70 C; and G50 and G80 were heated to 80 C. After thermal treatment, the samples were immediately cooled with tap water, the slurry found to separate into two layers after standing for 30 min.
2.1. Materials Cornstarches with different amylose–amylopectin ratios were used in the experimental work as model materials. All the starches are commercially available and were kindly supplied by Penford P/L (Australia). Table 1 lists the starches and their amylose–amylopectin ratios and other properties. APTS, sodium cyanoborohydride, and HCl acid were chemically pure and purchased as received from Sigma. 2.2. Sample preparation 2.2.1. Acid hydrolysis A quantity of 5 g (dry basis) of native starch was hydrolyzed by suspending it in 100 ml of 4 mol/L HCl solution at room temperature (about 21 C) for 5 days. After hydrolysis, the insoluble residue was washed several times with distilled water to neutrality. The
2.2.3. Dying Native and partly gelatinized starch samples were prepared for CLCM as previously described (Blennow et al., 2003). Starch granules (10 mg) were dispersed in 15 ml of freshly made APTS solution (10 mM APTS dissolved in 15% acetic acid), and 15 ml of 1 M sodium cyanoborohydride was added. The reaction mixture was incubated at 30 C for 15–18 h, with the granules washed five times with 1 ml of distilled water and finally suspended in 20 ml of 1:1 (v/v) glycerol–water mixture. A drop of the mixture was then mounted on a glass plate for microscopy. 2.3. Confocal laser canning microscopy A CLSM equipped with an Ar–Hg laser. (TCS SP2, Leica Microsystems, Wetzlar) with a stand for fixed fluorescent cell samples was used for the detection of the fluorescene signal from
Fig. 2. CLSM optical sections of HCl-hydrolyzed different starches: (a) waxy, (b) maize, (c) G50, and (d) G80.
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dye-stained starch granules. The details of the Leica objective lens used were: 100 plan apo/1.40 oil UV. The excitation wavelength was 488 nm with 52 capacity, and the format of the image was 512 512. During image acquisition, each line was scanned four times and averaged to reduce noise. For each starch sample, a stack of horizontal optical sections was obtained, encompassing the whole starch granule in three dimensions. Measurements of the fluorescence intensity were made with Imaris 6.1.5 and the 3D-image analysis was performed using the software of the Leica TCS SP2. 3. Results and discussion There have been many reports over the years utilizing various methods to unravel the internal structures of starch. The key feature of CLSM is the ability to image a single focal plane in the sample. This enables researchers to visualize cross-sections of starch granules without the need for sectioning techniques which can result in destruction of the microstructure of the sample (Durrenberger et al., 2001). However, starch does not show autofluorescence. It is necessary to choose an effective fluorescent dye. APTS is one of the fluorescence dyes with a small molecular structure that has previously been used successfully for specific labeling of the reducing ends of starch fragments (O’Shea et al., 1998). The intense and stable fluorescence of APTS allows both surface and internal images of starch granules to be recorded at high resolution. Previous researches have used APTS to dye starches for CLSM observation (Blennow et al., 2003; Chung and Lai, 2006;
Glaring et al., 2006). In these studies, CLSM imaging with APTS as a probe for starch molecule distribution was used to study the internal structural features of starches, such as growth ring, channels, and cracks, as well as the general molecular distribution of amylose and amylopectin within the starch granule. Fig. 1 shows the CLSM optical sections of different cornstarches obtained at the same photomultiplier tube (PMT) after dying with APTS. It can be seen that most of the high-amylose starches (G50 and G80) showed an intensely stained center and were brighter than low-amylose starches (waxy and maize). It is well known that amylose has a lower molecular weight than amylopectin and contains a much higher molar ratio of reducing ends per anhydrous glucose residues, which results in a higher by-weight labeling of amylose (Blennow et al., 2003) and thus the granules with high-amylose content showed higher fluorescence. It can be seen from Table 2 that the fluorescence intensity sequence of the granules is G80 > G50 > maize > waxy, which corresponds to decreasing intensity with decreasing amylose content. The center section of CLSM also reveals the structures in the granule core, where it can be seen that the core of most of the waxy granules (low amylose) were dark, and that the dark core is connected with the channels in the granules. Some dark cores can also be found in maize starch. However, the cores of most of the G50 and G80 (high amylose) starch granules are bright, and the bright core in G80 is larger than that in G50, which indicates that high-amylose starch granules were more easily highlighted by fluorescent dye in the hilum. One of the possible explanations for this phenomenon is that amylose content at the hilum is higher for the high-amylose
Fig. 3. CLSM optical sections of different starches after particularly gelatinized: (a) waxy, (b) maize, (c) G50, and (d) G80.
P. Chen et al. / Journal of Cereal Science 50 (2009) 241–247
cornstarches G50 and G80, whilst another explanation is related to the different microstructures of the channel and hilum. The cavities in the granular core for waxy and maize starches have been clearly observed, but with different structures for G50 and G80, the higher density of hilum of G50 and G80 showing brighter cores. Further studies are warranted in this area due to the observation of such contradictory phenomena. It should also be noted that there is a dark line or channel in the center for G80, which separates the
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bright core into two parts. This channel could relate to the granular growth and will be studied in detail in future work. From the cross-sections of the starch granule, the internal channels in starch granules can also be seen and are visible as dark lines running from the border of the granule toward the hilum in waxy and maize. The channels in starch granules have been observed by SEM and CLSM previously (Huber and BeMiller, 2000; Kim and Huber, 2008), and it has been noticed that the channels are
Fig. 4. 3D images of native (left) and partially gelatinized starches (right).
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more frequently and clearly observed in waxy and maize starch (low amylose) than that in the G50 and G80 (high amylose). Such channels extend to the granule surface connected with cavities within the granules in waxy and maize starch, whilst the channel (if it is observed) is a ring along the bright core in G50 and G80. Recently Kim and Huber (2008) studied the channels within different wheat starch granules and found that A-type wheat starch has relatively larger channels than B-type wheat starch, which corresponds to our observations reported above since waxy and maize are A-type, while G50 and G80 are B-type starches. Acid hydrolysis can be used to remove the amorphous and less crystalline parts in the starch granule. The acid-etched granule resulted in better contrast between amorphous and semicrystalline layers within starch granules (Chung and Lai, 2006). The growth rings within high-amylopectin starches (waxy and maize) were clearly observed after acid hydrolysis, whilst sharp edge cracks from the hilum toward the border in the high-amylose starches (G50 and G80) were observed after such acid treatment (see Fig. 2). The growth rings showed spacings of about 300–500 nm in waxy starches, whilst there are slightly finer rings with widths of about 200–400 nm in maize material. Generally, the high-amylose starch (G50 and G80) granules have a more compact microstructure than that of low-amylose starches, which show more acid-resistant and thermally stable behavior (Chen et al., 2007; Liu et al., 2006). Moreover, the acid-hydrolyzed corn starches showed stronger fluorescence intensity than native starches, indicating that there are more ends available to react with APTS groups after hydrolysis. The temperature-induced changes of the cornstarch granules in excess of water were also studied under CLSM. Phase transitions of starch granular during thermal processing are complicated and unique, especially the gelatinization process (Chen et al., 2007; Liu et al., 2006; Yeh and Li, 1996; Yu et al., 2006). The well-accepted definition of gelatinization for starch involves destruction of its order or crystalline regions. The starch suspensions were heated to just above their gelatinization temperatures (waxy and maize 70 C; G50 and G80 80 C) for 2 min, in order to avoid whole granules being completely destroyed, followed by rapid cooling by quenching with water. Following this they were then dyed with APTS and visualized using CSLM (see Fig. 3). The heterogeneity and complexity of such a starch structure within granules directly affects the gelatinization of starches. It can be seen from Fig. 3 that all the gelatinization starts at the hilum or botanical center of the granules and spreads rapidly to the periphery. Gelatinization thus begins in the intercellular and leastorganized region. As soluble components (primarily amylose) leach out of the granules during the process, they form a solution, whilst the center and adjacent components of the channels begin to gelatinize, and subsequently the other parts of the granules. The central area of the granule around the hilum is believed to be the least-organized region, since both acid hydrolysis and gelatinization start from there, which is in agreement with the previous observations (Chung and Lai, 2006). Fig. 4 shows 3D images of native and partly gelatinized starch granules constructed from different sections of CLSM images. The 3D images of native starch granules are similar to those observed from SEM (Chen et al., 2006). To the best of our knowledge, this is first time the full image of partly gelatinized granules in water suspension has been observed since other techniques are not able to view it under these conditions. For example, SEM cannot be used to view a water suspension, whilst other optical microscope techniques are not able to ‘‘slice’’ the object and construct such a 3D image. Using confocal microscopy, it can be clearly seen that the granules of waxy and maize are broken through the cavity and channels when the granules swell, whilst the granules of G50 and G80 remained as granular after swelling. This technique clearly
shows promise to further explore the mechanisms of gelatinization, and we are currently carrying out this work. 4. Conclusion The morphology and microstructure of maize starches with different amylose–amylopectin ratios were systematically studied by confocal laser scanning microscopy (CLSM). Temperatureinduced changes of the cornstarch granules in excess water were also studied by this technique. The different microstructures of cornstarch granules with different amylose contents were evident, in particular the distribution of amylose and amylopectin materials and cavity, channel, and growth ring–crack locations within the starch granule. It can be seen that the high-amylose starches (G50 and G80) are brighter than low-amylose starches (waxy and normal maize), and the average fluorescence intensity sequence of the granules is G80 > G50 > maize > waxy. The acid-hydrolyzed starches show stronger fluorescence intensity than native starches due to the increased end groups as a result of chain scission being more amenable to dye attachment. Waxy and maize starches clearly show cavities and channels, whilst G50 and G80 present a bright core, indicative of dyed amylose. Waxy and maize starches show sharp growth ring structures after acid hydrolysis due to etching of the amorphous layer, whilst G50 and G80 granules showed different structures. The gelatinization starts at the hilum and adjacent to the channels of the granules and spreads rapidly to the periphery. Three-dimensional images of partly gelatinized granules composed of different sections of CLSM images were obtained, which were used to study the mechanisms of gelatinization. The granules of waxy and maize broke through the cavity and channels when the granules became swollen during gelatinization, while the granules of G50 and G80 remained as granular. Acknowledgements The authors from SCUT, China, would like to acknowledge the research funds NRDPHT (863) (2007AA10Z312, 2007AA100407), NFSC (50540420129), and GNSF (05200617). We acknowledge Monash Micro Imaging Laboratory for providing CLSM for this work. P. Chen would like to acknowledge the State Scholarship Fund provided by China Scholarship Council which supported her study in Australia. References Baldwin, P.M., Adler, J., Davies, M.C., Melia, C.D., 1998. High resolution imaging of starch granule surfaces by atomic force microscopy. Journal of Cereal Science 27, 255–265. Blennow, A., Hansen, M., Schulz, A., Jorgensen, K., Donald, A.M., Sanderson, J., 2003. The molecular deposition of transgenically modified starch in the starch granule as imaged by functional microscopy. Journal of Structural Biology 143, 229–241. Boren, M., Glaring, M.A., Ghebremedhin, H., Olsson, H., Blennow, A., Jansson, C., 2008. Molecular and physicochemical characterization of the high-amylose barley mutant Amo1. Journal of Cereal Science 47, 79–89. Chanzy, H., Vuong, R., Jesior, J.C., 1990. An electron diffraction study on whole granules of lintnerized potato starch. Starch/Sta¨rke 42, 377–379. Cheetham, N.W.H., Tao, L., 1998. Solid state NMR studies on the structural and conformational properties of natural maize starches. Carbohydrate Polymers 36, 285–292. Chen, P., Yu, L., Chen, L., Li, X., 2006. Morphology and microstructure of maize starches with different amylose/amylopectin content. Starch/Sta¨rke 58, 611–615. Chen, P., Yu, L., Kealy, T., Chen, L., Li, L., 2007. Phase transition of starch granules observed by microscope under shearless and shear conditions. Carbohydrate Polymers 68, 495–501. Chung, Y.-L., Lai, H.-M., 2006. Molecular and granular characteristics of corn starch modified by HCl–methanol at different temperatures. Carbohydrate Polymers 63, 527–534. Donald, A.M., Waigh, T.A., Jenkins, P.J., Gidley, M.J., Debet, M., Smith, A., 1997. Internal structure of starch granules revealed by scattering studies. In:
P. Chen et al. / Journal of Cereal Science 50 (2009) 241–247 Frazier, P.J., Richmond, P., Donald, A.M. (Eds.), Starch: Starch Structure and Functionality. The Royal Society of Chemistry, London, pp. 172–179. Donald, A.M., Kato, K.L., Perry, P.A., Waigh, T.A., 2001. Scattering studies of the internal structure of starch granules. Starch/Sta¨rke 53, 504–512. Durrenberger, M.B., Handschin, S., Conde-Petit, B., Escher, F., 2001. Visualization of food structure by confocal laser scanning microscopy (CLSM). LebensmittelWissenschaft und-Technologie 34, 11–17. Evers, A.D., McDermott, E.E., Albans, S., 1970. Scanning electron microscopy of wheat starch. II. Structure of granules modified by alpha-amylolysis – Preliminary report. Starch/Sta¨rke 22, 23–26. Glaring, M.A., Koch, C.B., Blennow, A., 2006. Genotype-specific spatial distribution of starch molecules in the starch granule: A combined CLSM and SEM approach. Biomacromolecules 7, 2310–2320. Gregorov, E., Pabst, W., Bohacenko, I., 2006. Characterization of different starch types for their application in ceramic processing. Journal of the European Ceramic Society 26, 1301–1309. Helbert, W., Chanzy, H., 1996. The ultrastructure of starch from ultrathin sectioning in melamine resin. Starch/Sta¨rke 48, 185–188. Huber, K.C., BeMiller, J.N., 2000. Channels of maize and sorghum starch granules. Carbohydrate Polymers 41, 269–276. Jenkins, P.J., Cameron, R.E., Donald, A.M., 1993. A universal feature in the structure of starch granules from different botanical sources. Starch/Sta¨rke 45, 417–420. Kim, H.-S., Huber, K.C., 2008. Channels within soft wheat starch A- and B-type granules. Journal of Cereal Science 48, 159–172. Liu, H., Yu, L., Xie, F., Chen, L., 2006. Gelatinization of cornstarch with different amylose/amylopectin content. Carbohydrate Polymers 65, 357–363. Liu, H., Yu, L., Chen, L., Li, L., 2007. Retrogradation of corn starch after thermal treatment at different temperatures. Carbohydrate Polymers 69, 756–762. Liu, P., Yu, L., Liu, H., Chen, L., Li, L., 2009. Glass transition temperature of starch studied by a high-speed DSC. Carbohydrate Polymers 77, 250–253. Mira, I., Kurtis Villwock, V., Persson, K., 2007. On the effect of surface active agents and their structure on the temperature-induced changes of normal and waxy
247
wheat starch in aqueous suspension. Part II: A confocal laser scanning microscopy study. Carbohydrate Polymers 68, 637–646. Neethikajan, S., Thomson, D.J., Jayas, D.S., White, N.D.G., 2008. Characterization of the surface morphology of durum wheat starch granules using atomic force microscopy. Microscopy Research and Technique 71, 125–132. O’Shea, M.G., Samuel, M.S., Konik, C.M., Morell, M.K., 1998. Fluorophore-assisted carbohydrate electrophoresis (FACE) of oligosaccharides: efficiency of labelling and high-resolution separation. Carbohydrate Research 307, 1–12. Oostergetel, G.T., Van Bruggen, E.F.J., 1989. On the origin of a low angle spacing in starch. Starch/Sta¨rke 41, 331–335. van de Velde, F., van Riel, J., Hans Tromp, R., 2002. Visualisation of starch granule morphologies using confocal scanning laser microscopy (CSLM). Journal of the Science of Food and Agriculture 82, 1528–1536. Wang, S., Yu, J., Yu, J., 2008. Conformation and location of amorphous and semicrystalline regions in C-type starch granules revealed by SEM, NMR and XRD. Food Chemistry 110, 39–46. Xie, F., Yu, L., Su, B., Liu, P., Wang, J., Liu, H., Chen, L., 2009. Rheological properties of starches with different amylose/amylopectin ratios. Journal of Cereal Science 49, 371–377. Yamaguchi, M., Kainuma, K., French, D., 1979. Electron microscopic observations of waxy maize starch. Journal of Ultrastructure Research 69, 249–261. Yeh, A.-I., Li, J.-Y., 1996. A continuous measurement of swelling of rice starch during heating. Journal of Cereal Science 23, 277–283. Yu, L., Kealy, T., Chen, P., 2006. Study of starch gelatinization in a flow field using simultaneous rheometric data collection and microscopic observation. International Polymer Processing XXI, 283–289. Zobel, H.F., 1984. Gelatinization of starch and mechanical properties of starch pastes. In: Whistler, R.L., BeMiller, J.N., Paschall, E.F. (Eds.), Starch: Chemistry and Technology, second ed. Academic Press, Orlando, pp. 285–298. Zobel, H.F., 1988. Molecules to granules: A comprehensive starch review. Starch/ Sta¨rke 40, 44–50. Zobel, H.F., Illinois, A., 1988. Starch crystal transformations and their industrial importance. Starch/Sta¨rke 40, 1–7.