Image courtesy of Dietmar Klement / iStockphoto

A string of glucose molecules: starch. It sounds simple, but it isn’t. Dominique Cornuéjols and Serge Pérez explore the intricacies of its structure – and show that the mystery is by no means solved. Take a handful of spaghetti and throw it into boiling water. The stiff sticks will quickly become softer and gently curve while swelling. We take this behaviour of noodles in hot water for granted, but what exactly is happening to the fine structure of spaghetti to result in such a dramatic metamorphosis?

The beginning of the answer is that pasta – like rice, potatoes or bread – contains a large amount of starch. But what is starch? Produced in plants by the photosynthesis of carbon dioxide, starch granules are made out of glucose polymers and serve as energy stores. Towards the end of the growing season, starch accumulates in twigs of trees, close to the buds. It is also found in fruits, seeds, rhizomes and tubers. Starch granules are very suitable for such long-term storage, because of their compactness, relative dryness and high stability.

However, this essential source of energy only became accessible to humans once they had tamed fire, because raw starch granules are so compact that they are hardly digestible. In order to increase its digestibility, starch needs to be cooked: it is only once it has been heated that it becomes water-soluble and edible.
The transformation of raw starch in hot water is called gelatinisation: the granules swell and burst, forming a paste. During cooling or prolonged storage, the starch paste often thickens due to a phenomenon called retrogradation. Gelatinisation and retrogradation, which correspond to structural modifications in the granules, affect the behaviour of starch-containing systems.

Consequently, starch is excellent for modifying the texture of many processed and home-cooked foods (for example, as flour or corn flour to thicken sauces), and has also been used for centuries for other purposes, including the manufacture of paper (sizing), glues or fabric stiffener. Today, new applications of starch are emerging, including low-calorie dietary fibres, biodegradable packaging materials, thin films and thermoplastic materials.

Science: one small step at a time

Starch, therefore, is widely used in industry – and has been for thousands of years. The scientific study of starch started in 1833 when the French chemist Anselme Payen identified starch as being composed of glucose units. However, even today, its biochemistry and detailed structure are not yet well understood. At a molecular level, we know that native starch (as it occurs naturally) is made of two distinct components, amylose and amylopectin, which can be isolated by fractionation and studied independently.

Figure 1: Basic structural motifs of amylose (a) and amylopectin (b). Click to enlarge image Image courtesy of Serge Pérez, ESRF

Both components contain polymer chains of glucose units, but the chains are linked differently. Amylose is mainly linear (with glucose units linked by (1-4) bonds (Figure 1a), whereas amylopectin has a highly branched, very dense structure, due to (1-6) linkage (Figure 1b). Amylopectin can contain up to a hundred thousand glucose residues and is the largest known biomacromolecule.

Figure 2: a) Raw starch granule observed by scanning electron microscopy b) The corresponding granule under polarised light Image courtesy of Serge Pérez, ESRF

Native starch granules vary enormously in shape and size (from 0.1 to 200 mm), but they all have a common characteristic: under the microscope and illuminated with polarised light, starch grains stained with iodine exhibit a distinctive ‘Maltese cross’ (shown in orange in Figure 2b), indicating the existence of some common internal ordering. When granules are heated in excess water (as when spaghetti is cooked), the polarisation cross begins to disappear, demonstrating that this molecular order is being disrupted. The physical properties of starch – its stability and phase transformations, for example from starch granules to gels, or from brittle, raw pasta to soft, cooked pasta – are directly linked to this molecular order. However, understanding the detailed structure of starch requires very advanced research tools and techniques, such as X-ray crystallography, electron microscopy, nuclear magnetic resonance and computer modelling. With the help of these tools, scientists are slowly beginning to build up a picture of how starch – something we are so familiar with – is constructed.

Starting from the nanoscale: double helices, lamellae and superhelices

X-ray diffraction investigations at the nanoscopic level indicate that: Starch is composed of thin lamellar domains (about 4.5 nm thick); Each lamella is made up of about 100 double-stranded helices, each consisting of about 20 glucose units (Figure 3); The double helices are very densely packed, with a high degree of regularity, like in a crystal (see glossary for all italicised terms). (For an explanation of how X-ray diffraction is used to analyse crystalline structures, see Cornuéjols, 2009.)

Figure 3: Double-helix structure in amylopectin Image courtesy of Serge Pérez, ESRF

These X-ray results corroborate biochemical studies of the amylopectin molecule which show that this huge molecule is organised in crystalline clusters of double helices (Figure 4a): the lamellae revealed in the X-ray diffraction research consist of the clusters of double helices demonstrated in the biochemical studies. In this model, the branch points (the (1-6) links) in the amylopectin molecules are located in the less organised (or more amorphous) regions between the clusters. Amylose is entangled with amylopectin (Figure 4b), but so far nobody knows exactly how. Additional X-ray studies, using the techniques of small-angle and wide-angle scattering (SAXS and WAXS) to analyse hydrated starch, show that the lamellae of double helices are probably organised in a helical superstructure, or superhelix (Figure 5). (For an explanation of SAXS, see Stanley, 2009.)

Figure 5: Possible model of a superhelix structure Image courtesy of Serge Pérez, ESRF

Down from the microscale: growth rings and blocklets

Figure 6: Growth rings consisting of alternate layers of amorphous and semi-crystalline regions. Click to enlarge image Image courtesy of Serge Pérez, ESRF

The lamellar and superhelix structures of amylopectin are only a small part of the whole picture, however. On a larger (microscopic) scale, it is known that starch granules are made up of alternating amorphous and semi-crystalline shells, between 100 and 800 nm thick. These structures are termed growth rings (Figure 6).

We know almost nothing about the amorphous parts of the growth rings. The more crystalline regions, however, can be studied by X-ray diffraction. Recently experiments using extremely focused X-ray beams at a synchrotron (see box) have demonstrated that within the semi-crystalline regions, the nanoscopic lamellae are parallel to the surface of the starch granule (Figure 7). This enabled scientists to make the link between the small (microscale, e.g. the starch granules) and the very, very small (nanoscale, e.g. lamellae) – a link that is difficult to obtain in such structural investigations. Another recent study, using atomic force microscopy to look at the starch granule’s surface, showed the presence of blocklets within the growth rings. These blocklets are more or less spherical and have a size of 20–100 nm. So far, however, nothing more is known about these blocklets.

Figure 7: a) A series of diffraction images taken at different places in the granule (‘mapping’ of the granule) b) Orientation of the lamellae vis-à-vis the surface of the granule. Click to enlarge image Image courtesy of Serge Pérez, ESRF

Taking all the studies together, we can be fairly sure about nanoscale structure (double helices forming lamellae) and the growth rings (alternating amorphous and semi-crystalline shells); however, the evidence for the intermediate structures (the superhelices and the blocklets) is less solid. Furthermore, it is still unclear how the superhelices, blocklets and growth rings relate to each other. Figure 8 summarises the different structural levels (glucose units, helices, lamellae, superhelices, blocklets and growth rings), from the molecular (10^-9 m) to the microscopic (10^-5 m) level.

As early as 1858, the Swiss botanist Carl von Nägeli had a brilliant intuition, stating that “The starch grain... opens the door to the establishment of a new discipline... the molecular mechanics of organised bodies.” He would no doubt be astonished that, more than 150 years later, we are still struggling to understand the complex architecture of starch granules.

References

Angellier-Coussy H, et al. (2009) The molecular structure of waxy maize starch nanocrystals. Carbohydrate Research 344: 1558-1566. doi: 10.1016/j.carres.2009.04.002

Buléon A, Véronèse G, Putaux JL (2007) Self-association and crystallization of amylose. Australian Journal of Chemistry 60: 706-718. doi: 10.1071/CH07168

Cornuéjols D (2009). Biological crystals: at the interface between physics, chemistry and biology. Science in School 11: 70-76. www.scienceinschool.org/2009/issue11/crystallography

Gallant DJ, Bouchet B, Baldwin PM (1997) Microscopy of starch: evidence of a new level of granule organization. Carbohydrate Polymers 32: 177-191. doi: 10.1016/S0144-8617(97)00008-8

Imberty A et al. (1988) The double-helical nature of the crystalline part of A-starch. Journal of Molecular Biology 201: 365-378. doi: 10.1016/0022-2836(88)90144-1

Stanley H (2009) Plasma balls: creating the 4th state of matter with microwaves. Science in School 12: 24-29. www.scienceinschool.org/2009/issue12/fireballs

Waigh TA et al. (2000) Side-chain liquid-crystalline model for starch. Starch 53: 450-460. doi: 10.1002/1521-379X(200012)52:12<450::AID-STAR450>3.0.CO;2-5

Web reference

w1 - To learn more about ESRF, see: www.esrf.eu

Resources

For an extensive review of starch, see ESRF scientists’ Serge Pérez and Anne Imberty’s ‘Starch: structure and morphology’ website: www.cermav.cnrs.fr/glyco3d/lessons/starch

Imberty A, Pérez S (1988) A revisit to the three-dimensional structure of B-type starch. Biopolymers 27: 1205-1221. doi: 10.1002/bip.360270803

Pérez S, Baldwin P, Gallant DJ (2009) Structural features of starch. In: Starch-Chemistry and Technology, 3rd edition. BeMiller J, Whistler R (eds.). pp149-192. New York, NY, USA: Academic Press. ISBN: 978-0127462752

Chanzy H, et al. (2006) Morphological and structural aspects of the giant starch granules from Phajus grandifolius. Journal of Structural Biology 154(1): 100-120. doi: 10.1016./j.jsb.2005.11.007

If you enjoyed this article, you might like to browse all the other Science in School articles about ESRF science: www.scienceinschool.org/esrf

Dominique Cornuéjols, a physicist by training, has worked at the ESRF since 1993 as the communication manager. She is particularly involved in the ESRF’s outreach and education programmes.

After receiving his doctorate in crystallography from the Université de Bordeaux, France, Serge Pérez spent years doing research in America, Canada and France. He took up his first directorate position at the Centre de Recherches AgroAlimentaires in Canada in 1987. As director of research at the French National Center for Scientific Research (Centre National de la Recherche Scientifique, CNRS) he moved to Grenoble in the 1990s, and 12 years later moved to ESRF as the research director. Since 2003, he has also been the co-director and then director of the Doctoral School of Chemistry and Life Sciences at the Université Joseph Fourier, in Grenoble.

Review

The ultra structure of starch is not often considered, but this article describes how the components of starch – amylose and amylopectin – form the complicated levels of structure within the polymer. The article could be used as extension work in a lesson on starch digestion or measurement. It could also be used as an example of the use of the technique of X-ray diffraction or polarising light microscopy. Posters could be made (or homework set) on the uses of starch in industry, with each group taking a different use. Methods for producing and / or assaying starch in industry could be investigated. Comprehension questions could be set; for example:

1. Starch is made up of 2 components that are:
• Amylase and amylopectin
• Amylose and amylopectin
• Glucose and amylase
• Glucose and amylose


2. Describe what gelatinisation is.


3. What are growth rings in starch?


4. What is the difference between amylose and amylase?


5. Convert the sizes of the different structural levels from 10-9 m into smaller units, e.g. micrometres or nanometres.


6. X-ray diffraction was used in the 1950s to determine the structure of a well known molecule. Which molecule?

Shelley Goodman, UK

---

Referee's recommendations: Biology, Chemistry, Biochemistry