STARCH STRUCTURE/FUNCTION RELATIONSHIPS: ACHIEVEMENTS AND CHALLENGES

M.J. Gidley

Unilever Research Colworth, Colworth House, Sharnbrook, Bedford, MK44 1LQ, UK

1 INTRODUCTION

The importance of starch, both biologically and technologically, is well known, as is its central role in the human diet. Many aspects of starch structure can be measured or detected by one or more chemical, physical, spectroscopic or microscopic methods. Functional performance of starches in technological applications can usually be assessed with appropriate end-use mimic tests, and underlying material properties can be measured using the arsenal of techniques available to the physical scientist. However, despite major progress in many aspects of starch structure / function relationships, there is still a significant gap in our ability to predict the functional properties of starch from a knowledge of structure. This is particularly relevant in the post-genomic era where it is, or soon will be, possible to select or modify plant sources of starch at the genetic level. There will therefore be a growing need to link structural features more precisely and predictably to defined functional performance. This review seeks to identify some of the key successes in describing and quantifying starch structure and function, as well as highlighting some of those areas where information or mechanistic understanding is lacking. Given the broad nature of the area, the choice of topics is necessarily subjective and will not include much in the way of introductory material.

2 LEVELS OF STRUCTURE IN STARCHES

Despite the textbook description as a 'simple' polymer of glucose, starch is one of Nature's most complex materials. There are two fundamental factors behind this complexity. One is the existence of characteristic structures over a wide range of distance scales, and the second is the heterogeneity of structure at all of these different distance scales both within a single granule as well as the natural variation inherent in populations of granules.

At each level of structure, there have been significant recent advances in methodology and information. Progress in tackling the heterogeneity problem has been slower reflecting the greater challenges involved.

2.1 Molecular Structure

Methods for probing the molecular structure of starch polymers are well advanced. A combination of chemical, enzymic and NMR methods are available for describing the branching pattern and branch length profile of polymers. When coupled with prior chromatographic fractionation, a high information content is obtained. It is now accepted that there is a continuum of starch polymer structures from purely linear to extensively branched, and that for some starches there is a difficulty in describing constituent polymers as purely amylose (lightly branched, molecular weight 104 - 106 Da) or amylopectin (heavily branched, molecular weight 106 - 108 Da). Although branching levels and branch lengths can be readily determined, it is more difficult to define the pattern of branching points. It is well established that branch points have a tendency to cluster, but it is more difficult to determine the local molecular architecture around branch points. NMR methods may help in this area in the future.

Molecular size is relatively easily determined for low or intermediate molecular weights, but sample preparation and handling requirements may make it difficult to identify true molecular weights for some large amylopectins. This is because above 108 Da, molecules may be sufficiently large that either they are mechanically unstable in solution or they are more colloidal than molecular in dimensions and consequently cannot be chromatographed or measured accurately by light scattering.

It is now clear that it cannot be assumed that molecular structure features are distributed evenly throughout granules. This can be shown by differential iodine staining or by sequential solubilisation from the outside of granules under defined chemical regimes. It is not known whether this distributional heterogeneity has any impact on functional properties.

2.2 Helices and Crystalline Order

There are significant stretches of contiguous linear glucan in all starches. This leads to the potential to form the repetitive glycosidic conformations characteristic of helices. A range of single- and double-stranded helices have been identified and characterised from native and treated starches. Starch chains usually show a high proportion of local glycosidic conformations characteristic of V-type structures that can be stabilised by complexation with iodine, fatty acids, monoglycerides etc. Endogenous starch granule lipid is present in such V-type complexes. Double helical structures are found for part of the amylopectin component within granules, and are formed from both amylose and amylopectin after gelatinisation. A minimum length of ten glucose residues is required for double helix formation, although chains as short as six residues can co-crystallise. It seems clear that the outer branch length of amylopectin is the major determinant of which double helical polymorph is found in native starch granules. Relatively short branches lead to A-type with longer branches giving B-type order. A- and B-type polymorphs have very similar individual helical structures but differ in packing arrangements. In vitro studies suggest that the A-type polymorph is the kinetic product and B-type the thermodynamic product of amylopectin (branch) crystallisation.

From comparative 13C NMR and X-ray diffraction measurements of double helical content and crystallinity extent respectively, it is found that typically about 40-50% be weight of starch chains are present as double helices in granules with approximately half of these helical chains present in crystallites large and perfect enough to diffract X-rays.

A significant recent advance has been the application of microfocused synchrotron X-ray beams to identify the relative extent and orientation of crystalline elements at defined positions within individual granules. For larger granules such as from potato, a detailed picture of crystalline ultrastructure can be obtained. No such description of non-crystalline double helix (or single helix complex) location or orientation is possible yet.

2.3 Granule Ultrastructure and Morphology

A well-known characteristic of granular starch is the appearance of a 'Maltese cross' birefringence pattern under polarised light. This indicates a radial distribution of sub-micron elements within granules, and may be related to a structural motif with a repeating distance of ca. 9nm observed by small angle scattering. This is ascribed to the regular repeating distance between adjacent clusters of branch points in the structure of amylopectin. Microfocus X-ray experiments show that these repeating distances have a radial arrangement as is inferred for the longer distance scale (hundreds of nms) evident from light birefringence.

Little is known about the micron scale architecture of granule segments, although it is tempting to infer the presence of 'super-helices' from electron microraphs of granule shavings and from detailed analysis of scattering and diffraction data. A super-helical framework would provide an appropriate mechanism by which granule development could occur during biosynthesis, but remains unproven at this stage.

It can be inferred from very different swelling and leaching properties, that the local environments of amylose and amylopectin differ significantly between granules from different botanical sources. In particular, the ability of a fraction of amylose to be leached from many starches around their gelatinisation temperature shows that the forces that retain amylose in hydrated granules are overcome at 60-80°C. This rules out amylose/amylose double helices which are not expected to dissociate until above 100°C. Despite the fact that amylose double helices form more readily than amylopectin double helices from solutions, it is the latter that predominate in native granules. Nature has therefore found a way of kinetically trapping amylose as individual / separated molecules within granules. How this is achieved over the long time periods involved in granule biosynthesis is an intriguing question.

The size, shape and surface characteristics of starches are relatively well conserved for a given biological source, yet essentially nothing is known about the biological or physicochemical factors that control these features. The surfaces of extracted starches frequently contain components (particularly proteins and lipids) characteristic of the matrix from which they were extracted e.g. cereal endosperms.

3 FUNCTIONALITY

From the above, it can be seen that starch structure has a series of characteristic length scales which all need to be taken into account when trying to derive mechanistic links to functional performance. To exemplify the challenge, consider a typical piece of e.g. food with ca. 2cm dimensions as an example of a functional application. If the length scale of an individual glucose residue (0.5nm) is compared with a person (1-2m), then 1-2cm is comparable to a global distance scale. This is illustrated below, including intermediate structural elements.

It is apparent from this analogy that attempts to describe cm ('global') scale functionality based on nm ('person') scale structures is a major challenge. This is true for all types of materials, but is magnified for starches due to (a) the presence of characteristic features not only at the polymer length scale but also at the granule length scale, and (b) biological variation (heterogeneity) at every length scale.

In the context of this review, functionality is primarily concerned with rheological and structuring properties obtained after cooking. The approach will be to identify which of the various properties associated with starch are primarily due to molecular level effects, which are mostly derived from granule level behaviour, and which are a complex mixture of both. Over-simplification will be used to emphasise this distinction.

3.1 Molecular Level Functionality

3.1.1 Gelatinisation Temperature. As a first approximation, the length of double helices is proposed to be a determinant of gelatinisation temperature. Amylose double helices in e.g. gels probably occur over a length scale of 40-80 residues and melt at approx. 150°C. Typical amylopectin-based double helices occur over 15-20 residues and melt at 60-80°C. Longer branch lengths found in so-called high amylose starches are proposed to be responsible for gelatinisation temperatures of 80-110°C. Lowest observed gelatinisation temperatures are around 50°C, probably corresponding to double helical lengths around the minimum of 10 residues. Presumably shorter branch lengths (as found e.g. in glycogens) will not lead to stable granules. It seems unlikely that gelatinisation temperatures much lower than 50°C will be achieved / found for native starches.

3.1.2 Solubilised Starch. When sufficient energy has been applied to a starch system to completely erase all supra-molecular order, then the expectation is that the constituent polymers will behave as any other polymeric system. In several respects this is the case. One example is in phase separation behaviour. Despite the chemical similarity of amylose and amylopectin, mixtures of the two in solution show evidence of phase separation both with themselves and added polymers. A second example is in the behaviour of depolymerised starches as modulators of freezing (at high moisture) and solidification or glass-like (at low moisture) properties. In the absence of retrogradation, there is nothing unexpected in these properties of starch polymers based on the general physico-chemical principles of polymer behaviour.

3.1.3 Retrogradation. Retrogradation is a word invented and defined by the starch community. In essence, however, the underlying mechanisms are analogous to those found for many other helix-forming polysaccharides. This stems from the central role of double helix formation in either amylose or amylopectin retrogradation behaviour. For long amylose chains, gelation and related network properties are a direct result of multiple helix formation creating a meshwork of cross-links between chains in an exactly analogous mechanism to e.g. gelatin or agar. For amylopectin, the analogies are fewer due to the unusual clustering of relatively short branches. Nevertheless the factors affecting double helix formation in amylopectin and underlying e.g. bread staling are in general predictable based on the mechanism involved.

Complexity increases when mixtures of amylose and amylopectin are retrograding. Relevant questions here include the kinetics of phase separation compared with those for double helix formation and the possibility of helices linking amylose and amylopectin molecules. These factors are likely to be in a delicate balance, resulting in a richness of potential properties but also a difficulty in predicting the outcome. The effects of detailed amylose and amylopectin structure on mixtures of the two have not yet been put into a mechanistic framework.

3.2 Granule Level Functionality

3.2.l Swelling. Following the molecular level melting induced by heating granules, a swelling process ensues. When observed microscopically, it is apparent that individual granules in any population go through the structural disorganisation phase of gelatinisation (typically monitored by optical birefringence) over a narrow temperature range followed by a characteristic swelling behaviour. Attempts to describe the gelatinisation of a collection of granules as a single process are therefore inappropriate. Although swelling behaviour is characteristic for botanical origin, there is no coherent explanation for observed differences. One factor which is certainly important for cereal starches such as maize and wheat is the amount and location of non-polysaccharide components. This is most graphically demonstrated by the consequences of removing surface lipids and proteins with sodium dodecyl sulphate (SDS) extraction. Whilst the swelling properties of starches such as waxy maize and tapioca (which naturally exhibit rapid and extensive swelling) are unaffected, wheat and maize starch swelling is dramatically altered. Following SDS extraction, these starches show similarly rapid and extensive swelling as waxy maize and tapioca. This emphasises that in assessing the structural basis for starch functional properties, minor components as well as the major polysaccharides need to be taken into account.