Journal of Cereal Science 34 (2001) 1–17 doi:10.1006/jcrs.2000.0402, available online at http://www.idealibrary.com on MINI REVIEW Aspects of the Physical Chemistry of Starch R. Parker and S. G. Ring∗ Institute of Food Research, Norwich Research Park, Colney Lane, Norwich, NR4 7UA, U.K. Received 20 November 2000 ABSTRACT Starch makes an important contribution to food structure and hence quality. Often there is a need to modify the behaviour of starch in food materials. One approach is to use knowledge of the physical chemistry of starch to modify component interactions and hence change behaviour. In this review we examine recent research on the phase behaviour and dynamics of starch relevant to this approach. 2001 Academic Press INTRODUCTION Many cereal products have been developed empirically over very many years. Often there is opportunity to develop new products to satisfy a market requirement, or a need to modify product performance for a particular application. Often this development will be largely empirical— diﬀerent ingredients will be mixed together or diﬀerent processes tried until the required material properties and consumer acceptability has been achieved. Any approach which ‘guides’ empiricism, or enables products to be ‘engineered’ for a particular application, is to be welcomed as development time will be reduced and improved products may result. The ability to ‘engineer’ a product is facilitated by prediction relating processing, to structure development, to material properties. Structure over a range of lengthscales, from the macroscopic to the molecular, has a potential impact on these properties. For example, the material properties of cereal foams are influenced by the density of the foam, whether it is closed or open cell, and the mechanical properties of the foam matrix. With a single cereal-based matrix of fixed composition it would be possible to prepared foamed structures with quite diﬀerent ∗ Corresponding author: E-mail [email protected] 0733–5210/01/040001+17 $35.00/0 behaviour1. The foam matrix will be multiphase, consisting of protein-rich and starch-rich domains, within which low molecular weight components, including water, will be distributed depending on their aﬃnity for the biopolymers. In this review we will focus on the molecular properties of one of those phases—the starch-rich phase. Our aim is to discuss research on the molecular properties of starch within the context of the physical chemistry of polymeric materials, more particularly synthetic polymers. This physical chemistry can help provide insight into how material properties may be altered through changing molecular interactions or composition. There are many excellent recent reviews on starch structure, biosynthesis and granule organisation so these aspects will only briefly be considered2,3. THE MACROMOLECULES Starch consists of two main polysaccharides, amylose and amylopectin. Both polysaccharides are based on chains of 1→4 linked -D-glucose [Figure 1(a)] but whereas amylose is essentially linear, amylopectin is highly branched containing on average one branch point which is 1→4→6 linked for every 20–25 straight chain residues. Linear regions of the amylose chain form a dark blue complex with polyiodide ions in aqueous solution at room temperature. This interaction is a basis 2001 Academic Press 2 R. Parker and S. G. Ring CH2OH O O Rotation angles (Φ , φ ) HO O CH2OH HO O (a) O H OH (b) A form a, b plane B form a, b plane 2.12 nm 1.17 nm 1.85 nm 1.85 nm (c) Figure 1 The glycosidic linkage of amylose showing the preferred chair conformations, and the possibility for conformational flexibility about the glycosidic linkage; (a) the 2dimensional cluster model of amylopectin with short chains arranged in clusters on longer chains, (b) the arrangement of double helices in the A and B crystalline forms of starch (c). for defining amylose as that starch polysaccharide which, under standardised conditions, binds 20% of its weight of iodine while under the same conditions amylopectin generally binds <1% w/w. This iodine binding allows a distinction to be made between amylose and amylopectin and permits the determination of the amylose content of native starch. Most starches contain between 20 and 25% w/w amylose although some waxy starches contain very little, if any, amylose (<1%) and other starches, such as amylomaize, which contain in the region of 65% amylose. Although a fraction of the amylose population is linear and is quantitatively hydrolysed by the exo-acting enzyme amylase, a fraction is lightly branched4–8. For example, purified wheat amyloses were hydrolysed from 79 to 85% by -amylase. Isoamylolysis allowed quantitation of a short chain fraction representing between 3·2 and 7·6% w/w of the amylose depending on the variety of wheat examined9. Typical molecular weights of extracted amylose are in the region of 105 to 106 g mol−1. In aqueous solution the amylose behaves as a somewhat stiﬀ flexible coil with a typical hydrodynamic radius of 7 to 22 nm2. The branching within amylopectin is not random3,10. Debranching amylopectin with a debranching enzyme such as isoamylase followed by size exclusion chromatography11, or high pressure anion exchange chromatography12 reveals an essentially bimodal population of chains with two main populations with peak DP of around 12–14 and >4511. The short chain fractions of many amylopectins also have shoulders of around DP 18–2013,14. The short chain fraction is the most abundant by weight and number. There is also the possibility that amylopectin can contain some longer chains, of suﬃcient uninterrupted length to give rise to significant iodine binding behaviour15,16. Classification of the constituent chains can also be based on how the chains are linked to the rest of the molecule. The C chain carries the sole reducing end of the molecule, the A chain is only linked to the rest of the molecule through its potential reducing end while the B chain is linked in this way and also carries one or more A chains. Typical estimates of the ratio of A : B chains is in the region of 1:1 to 1·5:110. The fine structure of amylopectin is dependent on botanical origin, with variations reported in the A : B chain ratio, the length and abundance of short and long chains, and the form of distribution of the constituent chains. The structure of diﬀerent amylopectins is generally characteristic of a particular species although small varietal diﬀerences are also observed9,11,17. Current models2 of amylopectin structure depict short linear chains, 10 to 20 units long, arranged in clusters on longer chains, with the longer chains spanning more than one cluster [Figure 1(b)]. Examination of the products of a limited -amylolysis of amylopectin reveals the Aspects of the physical chemistry of starch presence of branched clusters of short chains lending support to the model18,19. Typically, the cluster model is a two dimensional representation of a structure which must pack in the starch granule to account for the measured density of >1500 kg m−3, and the known crystallographic structure which imposes dimensional constraints on how the crystalline chains can be arranged. Amylopectin is one of the largest biopolymers known with typical molecular weights being in the region of 108 g mol−1 and a hydrodynamic radius of 21–75 nm. A consequence of the branching is that for its molecular weight the amylopectin molecule is relatively compact. A further consequence is that if the domains of amylopectin molecules interpenetrate the branching and clustering of chains will hinder their separation. GRANULE ORGANISATION Starch occurs naturally as water-insoluble granules whose form is characteristic of its botanical origin. When viewed under polarised light the granules are birefringent. As the radial refractive index is larger than the tangential refractive index a preferred radial distribution of chains is indicated2. The starch granule is also partially crystalline with crystallinities in the region of 30% being reported. A number of crystalline forms are known [Figure 1(c)], the A form20 which is found in most cereal starches, including wheat, consists of starch double helices packed into a monoclinic array. The B form21,22, which is found in some tuber starches and high amylose cereal starches, is a more highly hydrated and open structure, consisting of double helices packed in a hexagonal array. As waxy starches, containing only amylopectin, still have crystalline granules the participation of amylopectin chains within the crystalline domains is indicated. Acid etching of the granules, or lintnerisation as it is known, leads to a preferential erosion of the amorphous fraction. Examination of the product has shown that the length of chain participating in the crystalline domains is comparable to the short chain fraction of amylopectin hence the suggestion that it is the short chains of amylopectin which form the double helices23,24. Using high intensity X-ray sources it is now possible to map the crystallinity of diﬀerent regions of the starch granule25,26. This has given information on the orientation of crystalline domains within individual granules. For example, for potato 3 starch the B-type domains showed a preferred radial orientation while in wheat starch no such orientation was observed of the A-type crystallites. The approach has also been used to probe the distribution of A and B crystalline domains. For legume starch granules27 it was found that the A crystalline domains were concentrated in the granule periphery28. Other types of order can be probed by other techniques. Solid state NMR experiments have been used to complement Xray measurements29,30. The spectra of the A and the B forms of starch are characteristically diﬀerent, and diﬀerent to amorphous starch. X-ray and neutron scattering experiments31 on starch granules have revealed an intriguing peak in the scattering profile representing a periodicity in the range 8·9 to 10·9 nm32–35. Through analysis of the whole profile, in terms of a model of repeating amorphous and crystalline layers, it was found that, for a range of starches of diﬀerent botanical origin, within experimental error, this periodicity had a constant value of 9 nm. The constancy of this spacing is surprising given that the starches examined had diﬀerent crystalline forms and diﬀerent amylopectin structures. A recent interpretation of this proposed lamellar structure is that the amylopectin forms a side chain liquid crystal structure36–38. It is further proposed that the lamellae are tilted as in a proposed superhelical structure36,39. Another aspect of granule structure is the shells2 observed directly by light microscopy and by scanning electron microscopy of granules which have been etched by acid and enzyme treatment. These structures have a radial repeat length of between 120 and 400 nm and are considered to consist of repeating amorphous and semi-crystalline layers. Other structures40 observed on the surface of starch granules by atomic force and other microscopies are ‘blocklets’ of about 30 nm in diameter41. One interpretation is that these blocklets represent the amylopectin cluster, another is that the ‘blocklets’ are some surface contamination. If all this information is considered as a whole then clearly the starch granule is a very complex macromolecular assembly, and it is perhaps useful to distinguish between that which has received widespread acceptance and that which is still debated. Areas which remain a focus of discussion, largely because they represent recent research, are the blocklet concept and proposed models relating to the lamellar structures having a 9 nm spacing. Models of granule assembly should recognise the diversity of 4 R. Parker and S. G. Ring chemical structures which make up the granule, for example how is the amylose arranged, what is the significance of the variability of amylopectin structure, and the lengths of the constituent chains of amylopectin, to its organisation in the granule. GELATINISATION, MELTING AND DISSOLUTION Starch is usually processed by heating in the presence of water which disrupts the native crystalline structure, a phenomenon known as gelatinisation. In an excess of water (>90% w/w), above a characteristic temperature known as the gelatinisation temperature, the starch granule loses its native crystalline order and swells irreversibly to many times its original size. At the same time the starch polysaccharide amylose (if present) is preferentially solubilised. Substantial solubilisation of high molecular weight amylopectin is not observed although there are reports for some starches of the solubilisation of a low molecular weight amylopectin42. Although the gelatinisation temperature is relatively easy to determine experimentally it is important to bear in mind that the gelatinisation process occurs over a limited temperature range for a single granule and a somewhat wider temperature range for a population of granules. As heating is continued, past the gelatinisation temperature, the granule continues to swell. At temperatures less than 100 °C and in the absence of mechanical shear, the swollen granules, enriched in amylopectin, maintain their integrity. The increase in volume fraction of starch granules in the suspension leads to an increase in viscosity of the paste. If the temperature is such that the solubilised amylose is slow to aggregate, the viscosity of these pastes, in common with other particulate suspensions, is dominated by the volume fraction occupied by the starch granules43. At concentrations greater than about 6% w/w for cereal starches, the gelatinised granules fill the available volume producing a viscoelastic material, the properties of which are additionally influenced by the deformability of the swollen starch granule and hence the amylopectin concentration in the granule. The swelling of the granule on gelatinisation clearly has a major impact on the rheology of starch pastes. What molecular features aﬀect swelling? One approach for gaining insight into the swelling properties of starch might be to consider it as a network structure44. For neutral, chemically crosslinked, polymer networks the extent of equilibrium swelling observed is dependent on the extent of crosslinking and the aﬃnity of the network for the solvent45. The network will swell until the osmotic pressure generated by the network, as a result of its aﬃnity for the solvent, is balanced by the restorative stiﬀness of the network resisting swelling. Clearly, neutral amylopectins will largely have similar aﬃnities for water and it might be proposed that it is the eﬀective crosslinking of the entangled amylopectin network which might limit swelling. Changing the solvent characteristics, for example by increasing temperature, or by mixing with other compounds which have a high aﬃnity for the starch chain, would lead to increased swelling. For polyelectrolyte networks there is an additional contribution to the osmotic pressure driving swelling, coming from the mobile ions which are required to neutralise the charge on the polymer45. The pressure generated by this Donnan eﬀect is greatest at low ionic strength. Some amylopectins, for example potato amylopectin, are weakly phosphorylated and therefore behave as polyelectrolytes. Potato starch is also a high swelling starch whose swelling is reduced if ionic strength is increased. Clearly there is a polyelectrolyte eﬀect in operation and one potential way of modifying the swelling properties of these starches would be to alter their polyelectrolyte characteristics. While the consideration of the gelatinised granule as a network structure might be useful, it is important to bear in mind that the foregoing discussion is based on the equilibrium swelling of polymer networks i.e., there is a reversibility in swelling if the conditions are changed. The swelling of the starch granule after gelatinisation is profoundly non-equilibrium as illustrated by the observation that starches such as wheat starch which have been subjected to diﬀerent thermal histories, such as slow versus rapid heating, swell to diﬀerent extents and as a consequence the starch pastes have diﬀerent rheologies43,46. As the starch granule is a complex structure it is perhaps not surprising that the description of the processes occurring during gelatinisation are similarly complex. One approach has been to study the loss of various types of order with a range of techniques during the gelatinisation process. As the diﬀerent techniques probe diﬀerent aspects of the gelatinisation process, it should not be surprising that they do not exactly map onto each other. Ideally experiments should be carried out Aspects of the physical chemistry of starch during the gelatinisation process, often, for practical necessity, they are carried out on starch materials which have been quenched to room temperature, allowing a potential reordering of structure. One such study used solid state NMR, X-ray diﬀraction and diﬀerential scanning calorimetry47. Although these techniques probe diﬀerent aspects of the loss of order of the granule, it was found that the extent of loss of order as a function of temperature was broadly similar. This conclusion was consistent with a more recent study48 which followed the loss of crystallinity of maize starch, as a function of temperature, using a synchrotron source of X-rays. In this case, in addition to the disappearance of the native crystalline structure, it was also possible to monitor the crystallisation of starch as a lipid complex at higher temperatures. In addition to the loss of crystalline order, the loss of lamellar order has also been followed as a function of temperature32,38. In the initial stages of gelatinisation there is no marked shift in the peak in the scattering profile, and therefore no marked change in lamellar spacing. The feature does, however, become weaker. Finally, another study examined order loss by birefringence measurements, diﬀerential scanning calorimetry and X-ray diﬀraction. It was found that the loss of birefringence occurred over a narrower temperature range than the loss of crystalline order49,50. In conclusion, there is a broad consensus that, for a population of granules, all the forms of order (crystalline, lamellar, orientational) are lost or modified over a broadly similar temperature range. To further assess order loss as a function of temperature it is necessary to consider heterogeneity in structure over the population studied. For example, in pea starches which give a C-type diﬀraction pattern (a mixture of the A and B forms) it was found that the B form was present in the interior of the granule and the A form in the periphery27. On gelatinisation, birefringence is initially lost from the interior of the granule followed by the periphery. On examination of the loss in crystallinity it is found that the B-type crystallinity is lost first. A population of granules might be expected to show further heterogeneity. For example, in wheat starch the distribution in granule sizes is bimodal and the diﬀerent sizes might display somewhat diﬀerent gelatinisation behaviour. A further source of heterogeneity during gelatinisation occurs as a result of concentration gradients. As the population of starch granules gelatinises over a limited 5 range of temperature, those that gelatinise first have access to the available water. On gelatinisation the granules which swell reduce the amount of water available for the swelling of other granules51. At intermediate starch concentrations, of around 40–50% w/w, gradients in starch concentration are observed in a macroscopic sample. As reducing the water content elevates the temperature of ‘gelatinisation’, one eﬀect is to broaden the transition. Double peaks are observed at these concentrations in plots of order loss, as assessed by X-ray diﬀraction or diﬀerential scanning calorimetry, as a function of temperature52. This is at least in part due to the heterogeneous nature of the sample. As the physical descriptions of the gelatinisation process are becomingly increasingly sophisticated, it is worthwhile posing the question—how might the gelatinisation characteristics of a starch be changed through modifying aspects of granule organisation? To attempt to answer this question some physical insight is needed into the underlying physical processes. During gelatinisation crystalline order is lost. From comparison with synthetic polymer systems it can be proposed that the temperature dependence of this loss in crystallinity is dependent on the crystalline polymorph present, its degree of perfection, the length of chain involved in the crystalline unit and the diluent content (with increasing diluent content reducing the temperature of the transition)53. As an individual amylopectin molecule can participate in a number of crystalline domains or lamellae it is the local diluent content which is important. There are a number of ‘models’ of gelatinisation32 which give somewhat diﬀerence emphasis to the relative importance of these factors. For example, it has been proposed that the hydration of the amorphous regions of the granule is strongly temperature dependent, and that at a certain temperature there is a large change in the water content of these regions, with a result that the local diluent content is suﬃciently high that gelatinisation can occur. It has been suggested that this hydration is suﬃciently strong to overcome the forces holding the crystalline lattice together. The physical origin of this marked change in hydration has been attributed to exceeding a glass transition54 (see later), or simply that the aﬃnity of the amorphous regions of starch for water shows a strong temperature dependence. Estimates of the average water content of a hydrated starch granule are in the region of 27–33%51. For this average composition the glass transition occurs below ambient tem- 6 R. Parker and S. G. Ring Transition temperature (°C) 300 250 200 150 100 DP14 DP12 DP10 50 0 10 20 30 40 50 Water content (% w/w) 60 Figure 2 The eﬀect of water content on the melting and glass transitions of starch. Key: solid line, estimates of the melting temperature of A-type short-chain amylose crystals with degree of polymerisation (DP) 10, 12, 1461,63; Β, glass transition of pregelatinised starch55; •, melting transition of granular wheat starch64. peratures55. Furthermore, solid state NMR studies on granular starch have shown that the amorphous fraction is plasticised into a rubbery state at ambient temperatures38. Generally, water is a poor solvent for the starch polysaccharides and experiments which have measured the aﬃnity of the starch chain for water show a weak temperature dependence56. These observations suggest that the amorphous fraction of starch may not have a dominant role in controlling gelatinisation. One of the essential elements of the gelatinisation process is the loss of crystalline order52. To gain insight into the factors aﬀecting this loss of order, one approach is to examine the behaviour of highly crystalline materials, preferably single crystals of macroscopic size. Short linear chains of amylose crystallise readily and under appropriate conditions highly crystalline materials of the diﬀerent polymorphic forms of starch are obtained57–60. The eﬀects of diluent content, and polymorphic form, on the temperature dependence of dissolution have been examined61. It is also possible to examine the eﬀect of chain length on the process, with the expectation that the longer the length of chain involved in the crystalline unit the higher the melting temperature62—an expectation which was confirmed experimentally63. A schematic diagram of the expected dependence of the melting of A-type crystallites on water content is shown in Figure 2. Also included is an estimate of how the dissolution temperature varies with increasing chain length. The melting temperature of the low water content materials is experimentally inaccessible due to thermal degradation. In common with polymeric crystalline materials45,53, the addition of a diluent, in this case water, depresses the observed melting temperature, Tm. For an A-type starch crystallite formed from chains 12 units in length, the expected peak melting temperature at high water contents is about 75 °C. As a comparison, the predicted high molecular weight limit of Tm in excess water is 150 °C. With decreasing water content, the melting temperature rises, reaching a predicted 150 °C for chains 12 units in length at a water content of 16% w/w. The polymorphic form of starch also aﬀects the dissolution behaviour. At a fixed water content, the melting and dissolution of the B-form occurs at >20 °C lower temperature61. For the crystallites in wheat starch the gelatinisation/dissolution temperature in excess water is in the region of 60 °C rather than the 75 °C of the highly crystalline material, probably indicating an eﬀect of crystal perfection/size on the observed dissolution. The form of the increase with decreasing water content in the range 50 to 5% w/w water is remarkably similar64 (Figure 2), suggesting that dissolution experiments on very crystalline materials provide useful insight into the very much more complex phenomenon of gelatinisation. A further indication of usefulness is gained from experiments on smooth-seeded pea starches28 the granules of which contain a mixture of the A and B crystalline forms of starch. The prediction that the dissolution of the B domains should occur at a lower temperature than the A domains was confirmed by X-ray diﬀraction experiments on the gelatinisation process. Although the spherulites formed from the short amylosic chains are somewhat analogous to the starch granule, particularly for the B spherulites which are formed from the stacking of crystalline lamellae65, they diﬀer in at least one important respect. In the case of the spherulite, disruption of the crystalline lattice leads to a solubilisation of the amylosic chain. In the granular case, disruption of a crystalline domain will only lead to a partial solubilisation because the amylopectin molecule spans diﬀerent domains or lamellae. The initial partial solubilisation of a crystalline domain increases the local water content of the granule increases facilitating the subsequent dissolution of crystalline material. For native starches, the length of chain of the short chain fraction and the crystalline form of Aspects of the physical chemistry of starch the granule are associated11,66. It is found that starches which have a relatively short short-chain fraction e.g. wheat and maize starch, have an A crystalline form while starches which have a longer short-chain fraction, e.g. potato starch, have a B-crystalline form. In vitro experiments on short amylosic chains reveal the same type of behaviour with the shorter chains crystallising in the A form and longer chains in the B form57,59. Crystallisation at small undercoolings, below the equilibrium dissolution temperature of the crystallites, favours the production of A form while a large undercooling favours the production of the B form. This suggests that the B form is a kinetic product. The associations between amylopectin chain length and crystalline form suggest that these considerations remain relevant to granule assembly. Both crystal type, and chain length involved in the crystal, aﬀect dissolution. One strategy to ‘design’ a cereal starch (A-type crystalline form) with a reduced gelatinisation temperature, might be to reduce the chain length of the short chain fraction of amylopectin. If a higher gelatinisation temperature was required, then increasing the chain length of the short chain fraction might initially lead to a depression in gelatinisation temperature, as the crystalline form changed from A to B, followed by a subsequent increase as the chain length eﬀect started to dominate. INTERACTIONS WITH WATER AND LOW MOLECULAR WEIGHT SOLUTES To start to understand the role of water as a diluent, it is useful to examine predictive relationships describing the composition dependence of melting45,52. The classical description of the compositional dependence of polymer melting in the presence of a diluent is given by 1/Tm=1/Tm0+(R/Hu). (Vu/V1). [v1−v12] (1) where Tm0 is the melting temperature of the pure polymer, Vu and V1 are the molar volumes of polymer repeating unit and diluent, respectively, and v1 is the diluent volume fraction. Hu is the enthalpy of fusion per repeating unit and ; is the Flory-Huggins interaction parameter67 characterising the interaction energy per solvent molecule. This relationship predicts that the smaller the diluent size relative to that of the polymer repeating unit, in this case an anhydroglucose unit, 7 and the stronger the favourable interaction between the diluent and polymer, the stronger the depression in Tm. It is well known that the starch polysaccharides precipitate from aqueous solution at room temperature, indicating that water interacts weakly with the starch chain and at room temperature it can be considered a poor solvent. Physicochemical measurements on water sorption behaviour can provide more quantitative estimates of solvent quality. This requires equilibrium behaviour which can be achieved if the behaviour of oligosaccharides is examined63. For polysaccharides non-equilibrium eﬀects generally dominate68, particularly for compositions which are glassy at the temperature of measurement. The values of the parameter obtained for concentrated aqueous mixtures of maltooligomers were in the range 0·7 to 0·8 at room temperature63, confirming that water is a poor solvent under these conditions. To refine this approach more information is needed on the temperature and composition dependence of . The relatively small molecular size of water is predicted to lead to a strong depression in Tm. It has been found that the Flory-Huggins approach fits the available experimental data for starch crystallite dissolution reasonably well. The prediction that replacement of water by other larger water-soluble solutes that interact weakly with the starch chain, such as low molecular weight carbohydrates, would lead to an elevation of Tm is also confirmed experimentally69,70. BIOPOLYMER/BIOPOLYMER INTERACTIONS A further aspect of phase behaviour that should be considered is the miscibility of biopolymer mixtures. The immiscibility of concentrated solutions of chemically diﬀerent synthetic polymers is a well described phenomenon, and can result from either an unfavourable energetic interaction between the polymers45, or the solvent having very diﬀerent interactions with the diﬀerent polymers71. Biopolymers show the same general type of behaviour with aqueous mixtures of proteins and polysaccharides72, and diﬀerent polysaccharides73,74 exhibiting immiscibility. At equilibrium, macroscopic phase separation is observed with the formation of separate layers enriched in each of the biopolymers. This macroscopic phase separation can be arrested, for example by gelation of one of the components, and as a result useful 8 R. Parker and S. G. Ring textures can be produced. Even the two chemically similar starch biopolymers amylose and amylopectin, diﬀering primarily in their extent of branching, phase separate from concentrated solution73. This is an example of quite a small diﬀerence in molecular structure producing a potentially large eﬀect on the microstructure of the mixed biopolymeric material. The way that solvent distributes itself between ‘phases’ as a result of their relative aﬃnity for water can have an important impact on material properties. For example, if the same amounts of two glucan polymers amylose and dextran are mixed in concentrated solution, at a temperature where crystallisation is not observed on practical timescales, phase separation is observed with the formation of a dextran-rich and amylose-rich phases. The phase volume of the dextran phase is much larger than that of the amylose phase, i.e., water has a preferred interaction with the dextran. The material properties of this biphasic system will depend on the phase volumes and polymer concentrations in each phase. There will also be an eﬀect on stability. For example, consider a concentrated solution containing 40% w/w water with 30% w/w each of dextran and amylopectin at room temperature. If the water was distributed evenly then storage at room temperature for this composition would represent a quench of 80 °C for chains 12 units in length (Tm>100 °C), and the amylopectin in the amylopectin-rich phase would tend to crystallise. If the amylopectin phase was phase-concentrated, by the presence of dextran, then the resulting increase in amylopectin concentration would increase the driving force for crystallisation. GLASS TRANSITION BEHAVIOUR Included in the schematic of Figure 2 is another transition which has an important influence on material properties75,76—the glass transition which is characterised by the glass transition temperature Tg. Crystalline anhydrous -D-glucose melts at 150 °C, if this melt is cooled at a rate, which is rapid compared to the rate of crystallisation, then the viscosity of the liquid will progressively increase until at 7 °C the viscosity reaches about 1012 Pa s77. At these enormous viscosities the material behaves as a brittle solid. Glasses may be stable to crystallisation for many years, the enormous viscosity of the glass arresting crystal nucleation and growth. At the calorimetric Tg a sharp change in heat capacity is observed, indicative of a change from solid-like to liquid-like behaviour within the timescale of the calorimetric experiment. This provides an experimentally convenient method for the determination of Tg as the midpoint of the step change. The calorimetric approach is useful for the determination of the glass transition behaviour of simple amorphous biopolymer mixtures. As the degree of polymerisation of the glucan chain increases there is an increase in glass transition temperature, reaching 173 °C for maltohexaose78. Addition of water to the carbohydrates depresses the glass transition, as illustrated in Figure 2 which shows the composition dependence of the Tg of an starch/water mixture55. The Tg of the dry polymer is experimentally inaccessible for familiar reasons—thermal degradation intervenes. The addition of water has a strong plasticising eﬀect, causing a marked depression in Tg, until at 20% w/w water, Tg reaches room temperature. More recently a more detailed study on the glass transition behaviour of diﬀerent glucan polymers including amylose, amylopectin, pullulan and dextran has been reported79. While all the glucans behave in the same general way, in that water is a very eﬀective plasticising agent, there are subtle diﬀerences which lead to a glass transition range approaching 30 °C at a water content of 10% w/w. The branching of polymeric materials is thought to depress Tg as a result of an internal plasticisation from the short chain branches. Although water is an ubiquitous plasticiser of starchy materials, it is volatile, and small changes in its content can lead to large changes in mechanical behaviour. For this reason, the use of other, nonvolatile, plasticisers has been examined. Most studies have been carried out with low molecular weight hydroxy compounds such as glycerol80–83 or sorbitol84, often in combination with water. Mixing 29% w/w glycerol with amorphised barley starch containing a minimal quantity of water (1%) depressed the glass transition to 70 °C85. Glycerol is therefore a less eﬀective plasticiser than water. Initial additions of glycerol to starch resulted in a depression of a single glass transition. As water was added this glass transition was further depressed and an additional lower glass transition appeared, suggesting that the ‘ternary’ starch/ glycerol/water mixture phase separated. The lower Tg was close to the glass transition of pure glycerol. There are a number of relationships which describe the way that the Tg of mixed binary systems should vary as a function of composition, Aspects of the physical chemistry of starch Tgm=(w1Cp1Tg1+w2Cp2Tg2)/(w1Cp1+w2Cp2) (2) The glass transition temperature of the mixture, Tgm, is related to the glass transition temperatures of the individual components and the heat capacity increment at the glass transition, Cpi, where wi is the mass fraction of the i th component. Replacing water which has a relatively low Tg (−139 °C)88,89 with glycerol (Tg −80 °C)90 would lead to an elevation in the Tg of the mixture. Recently, experiments were reported where the development of the X-ray diﬀraction pattern of granular starches, on solvation in the presence of ethylene glycol and glycerol, was measured as a function of time91. It was found that the development of crystalline order took several hours at room temperature for ethylene glycol, and several days for glycerol. As the relative ability of the diluents to depress Tg would be in the order water > ethylene glycol > glycerol, the observations are consistent with the view that replacement of water by glycerol would elevate the Tg of the mixture, and as a consequence, at a fixed temperature slow the rate of development of crystalline order. At low water contents, the Tg of the starch/ water mixture is very sensitive to small changes in water content and it follows that some of the material properties are equally sensitive. At Tg the structural relaxation time is of the order of 100 s. This structural relaxation time is relevant to mechanical behaviour—how quickly a material should relax after a mechanical perturbation—and potentially transport properties within the material92. As a result there is interest in knowing not only Tg but also how the associated structural relaxation time will depend on temperature and composition. Fortunately for most amorphous organic materials—low molecular weight liquids, synthetic polymers, and biopolymers (including flexible proteins such as gluten and polysaccharides)—the expected dependence of structural relaxation time on temperature is broadly similar. An expression which has been shown to be widely applicable in describing this temperature dependence for synthetic polymers is the Williams-Landel-Ferry relationship93: log aT=−c1,0(T−T0)/(c2,0+T−T0) (3) where aT is the ratio of relaxation times at the 200 Temperature (°C) and these have found use in describing the behaviour of starch mixtures. One such is due to Couchman86,87 and is of the form 9 Rubbery region (beyond WLF region) 100 Loaf crust Glass 0 –100 0 10 τ , relaxation time centre 20 30 40 50 Water content (% w/w) 0.1 ns 1 ns 0.1 µ s 0.1 ms 100 s 60 Figure 3 Eﬀect of temperature and water content upon the relaxation time calculated from the glass transition curve55 and assuming WLF behaviour93. Water content of loaf crust and centre shown at 20 °C. temperature, T, and a reference temperature T0. If Tg becomes the reference temperature then, log aT=−c1g(T−Tg)/(c2g+T−Tg) (4) with values of the coeﬃcients, c1g and c2g, obtained from fitting data on a range of synthetic polymers, being 17·44 K−1 and 51·6 K, respectively. At the calorimetric glass transition the shear viscosity, , is of the order of 1012 Pa s and the shear stress relaxation time, , is of the order of 100 s, and is given by =/Gx, where Gx is the high frequency limit of the shear modulus which is 1010 Pa for many materials. The temperature and composition dependence of the relative relaxation time, aT, is shown schematically in Figure 3 for an amorphous, high molecular weight, biopolymer/water mixture and is based on the observed glass transition behaviour of starch/water mixtures. As the glass transition is approached, either through reducing the temperature or the water content, there is predicted to be a very marked change in relaxation behaviour. Structural relaxation and molecular mobility slows and, as a result, there is a very marked change in material properties for a relatively small change in either water content or temperature. Although the eﬀect is most marked as the glass transition is approached, and is particularly relevant to the behaviour of low moisture cereal products, or frozen products where crystallisation has freeze-concentrated the biopolymer, even for intermediate water content materials such as bread there is a very marked change in relaxation behaviour (i.e. orders of magnitude) for relatively small changes in water content. This 10 R. Parker and S. G. Ring change in molecular mobility will have an impact both on stability to crystallisation and local viscosity. The above discussion was concerned with a hypothetical biopolymer/water mixture. There is a need for more experimental data on the composition and temperature dependence of the relaxation behaviour of cereal biopolymers. When suﬃcient data have been gained there is potential for a ‘dynamic synthesis’ where the relaxation behaviour of cereal biopolymers over a wide temperature and composition range has been compared with the known behaviour of other polymeric materials. For a more detailed discussion on the use of equation 3 to describe the behaviour of synthetic polymers the reader is referred to the work of Ferry93. The use of this approach to predict temperature-dependent mechanical behaviour is useful. It could also be extended to make some prediction of how transport properties should vary with temperature but care is needed in the application of this approach. For example, there is an expectation that the increase in viscosity/structural relaxation time as an undercooled liquid approaches the glass transition would slow diﬀusion. While this is the case for a macroscopic particle embedded in a highly viscous matrix, for smaller molecules such as water there can be a marked uncoupling of its motion from the shear viscosity with diﬀusion being much more rapid than expected94–96. The examination of the dynamics of these mixtures reveals other relaxations as well as the main structural relaxation97,98. Their eﬀect on material properties of starchy materials remains to be established. STRUCTURAL RELAXATION If the structural relaxation is suﬃciently slow, it can lead to time dependent changes in the properties of glassy materials over practical timescales of hours to weeks99–107, and is potentially relevant to the observed ageing of low water content carbohydrate products108–110. The ‘equilibrium’ structure of an undercooled liquid is temperature dependent. For example it is expected that reducing temperature would increase the density of a material. As a liquid is undercooled towards the glass transition its viscosity increases as does the associated structural relaxation time. If an amorphous material is rapidly quenched into the glass state, the structural relaxation time may be so high that the amorphous structure, and resulting density, is eﬀectively ‘frozen’. The structure will gradually evolve and at very long times it will have a fully relaxed, ‘equilibrium’ structure. If it is cooled again, further structural relaxations and rearrangements within the undercooled liquid will occur until, given suﬃcient time, a new ‘equilibrium’ structure is obtained. The structure of the undercooled amorphous polymer liquid can therefore show a dependence on time and thermal history. This relaxation behaviour has been extensively studied, particularly for polymers and inorganic glasses. The temperature-dependent changes in bonding between molecules and their configuration are associated with changes in volume, enthalpy, heat capacity and material properties, including mechanical behaviour and diﬀusivity. The observed changes in mechanical behaviour of amorphous glasses with time is often described as embrittlement, as the material becomes stiﬀer and less compliant. At Tg there is a sharp change in behaviour as the liquid structure becomes arrested over the timescale of the cooling. If the cooling is slower, there is more time available for the structure to relax and the appearance of Tg will occur at a lower temperature. Liquid structure can be characterised on a temperature scale through the notion of a fictive temperature, Tf, the temperature at which a particular structure would be fully relaxed. Although the increase in density of a liquid with time is one way of probing this structural relaxation, its use is rather restricted. As densification also aﬀects the energetics of interaction between molecules, and the accessibility of liquid configurations, it can be probed in a calorimetric experiment where structural relaxation is observed as a peak in heat capacity preceding Tg or an overshoot at Tg. There is often a requirement to be able to predict the change in the material properties of a glassy product with time. Common questions might be ‘at what temperature do I need to store the product to minimise this timedependent change?’, ‘how might fluctuations in water content aﬀect the observed behaviour?’. Fortunately there are various phenomenological approaches for describing the observed time-dependent behaviour, a widely applied one, which has a useful predictive capability, is the ToolNarayanaswamy method104, which has been applied to polymeric systems. The dependence of structural relaxation on time, t, can be described by an empirical relaxation function, , of the form (5) (t)=exp[−(t/0) ] Aspects of the physical chemistry of starch (6) ∗ where A, x (0<xΖ1) and h are constants. h can be determined from the dependence of the calorimetric Tg on scanning rate. These relationships can be used to calculate the time dependence of Tf following a temperature step and from this the heat capacity change with temperature can be predicted. By appropriate selection of the constants A, x and the experimentally observed behaviour may be modelled. For a simple carbohydrate, such as maltose111, it was found that a single set of constants described the dependence of ageing on time and temperature and had a useful predictive utility. More recently it was found that the above approach was useful in describing the ageing of a plasticised starchy material. Figure 4(a) shows the predicted change in relaxation behaviour as a hypothetical starchy material is heated through the glass transition. On heating there is a marked decrease in relaxation time. The associated change in heat capacity is shown in Figure 4(b); a sharp change in heat capacity is observed in the vicinity of 315 K indicative of a glass transition, there is also an overshoot in heat capacity as a consequence of the structural relaxation. The way that the Tf evolves with time, on storage at 300 K is shown in Figure 4(c). At this temperature, although liquid structure slowly changes over the course of 100 h, it does not equilibrate over this timescale. The change in Tf gives an indication of how material properties might change with time on storage of this hypothetical product. If the eﬀects of variations in water content can be usefully incorporated in the model, then this approach could become more generally useful for describing the eﬀect of ageing on the material properties of low moisture cereal products. RETROGRADATION OF STARCH If a gelatinised starch/water mixture is cooled to room temperature there is a strong driving force favouring crystallisation. The processes which occur on cooling are generally known as retrogradation112–117. In the case of the high molecular weight linear polymer, amylose, the driving force 3 Relaxation time (10 s) ∗ (a) 300 200 100 0 300 310 320 Temperature (K) 330 340 1.2 (b) Normalised heat capacity 0=A exp [xh∗/RT+(1−x)h∗/RTf] 400 1.0 0.8 0.6 0.4 0.2 0.0 300 320 340 360 Temperature (K) 380 400 324 (c) Fictive temperature (K) and (0<Ζ1) is a measure of its non-exponentiality. 0 is a characteristic time which is dependent on both temperature, T, and, to an extent, liquid structure (characterised by Tf ) and has been successfully obtained using the expression 11 322 320 318 316 314 312 0.1 1 10 100 Time (h) Figure 4 Variation in structural relaxation time with temperature for a starch product (a) and its predicted eﬀect on the observed calorimetric behaviour (b) and the timedependent evolution of fictive temperature Tf (c). is strong, with an eﬀective quench of at least 120 °C if the material is held at room temperature. 12 R. Parker and S. G. Ring Such a strong quench is not conducive to the formation of very crystalline materials. In fact, if a concentrated aqueous amylose solution is cooled to room temperature there is a very rapid precipitation/phase separation process. The clear amylose solution rapidly becomes opaque indicating the formation of polymer aggregates. To produce the opacity the aggregates are at least the order of the wavelength of light in size. At the same time, for suﬃciently concentrated solutions (typically greater than 1–2% w/w), network formation is observed through the formation of an elastic gel. Network formation, as assessed by the development of stiﬀness, is generally complete within a few hours at room temperature. Electron microscopic examination of the network reveals coarse network strands which consist of assemblies of many individual polymer strands118. Subsequent to this precipitation, a slow crystallisation of the amylose is observed by X-ray diﬀraction over the course of a few days. This crystallisation does not have a marked eﬀect on material properties, at the end of this time, these materials are still poorly crystalline. One proposal for the structure of the initial aggregate is that it is formed from assemblies of double helices117 with a typical dimension, orthogonal to the helix axis, of from 10–20 nm119. These structures further aggregate to form a fractal structure. The aggregation process has also been likened to a phase separation in which polymerrich and polymer-deficient domains are formed and if the amylose is suﬃciently concentrated the polymer-rich domains will form an interconnected network114. The amylose gel is far from equilibrium and its structure will be very dependent on the condition of its preparation. The eﬀective quench for the formation of crystallites from short linear chains is much more modest. If concentrated amylopectin/water mixtures are quenched to room temperature or below, a slow crystallisation, as assessed by X-ray diﬀraction, of the amylopectin is observed. In this case the crystallisation is associated with the development of stiﬀness of the material and typically, at high water contents, takes days or weeks to approach a plateau value112,120. At the end of this time the extent of crystallinity of the amylopectin is comparable to that found in the native starch granule, i.e., in the region of 30%. The length of chain involved in the crystalline unit is relatively short. On examination of the behaviour of amylopectins from diﬀerent botanical sources it was observed that the longer the length and abundance of the short chain fraction of amylopectin the greater the tendency to retrograde and crystallise from aqueous solution120–122. Wheat amylopectin, which has a relatively short, short chain fraction, generally shows a reduced tendency to retrograde, even so it can lead to very significant timedependent changes in material properties. In a detailed examination of the retrogradation of maize amylopectins it was found that retrogradation was proportional to the amount of short chains having a DP of 16–30 and inversely proportional to the level of short chains with a DP of 6–11122. Treatment of starch with -amylase (an exo-acting enzyme) shortens the short chain fraction and reduces the rate of retrogradation123. The rate of retrogradation is expected to be at a maximum approximately mid way between the melting temperature of the crystallites, Tm and the glass transition temperature Tg. This reflects a balance between the eﬀect of temperature on the driving force favouring crystallisation, and the slowing of mobility as the glass transition is approached. Bell-shaped curves of extent of retrogradation versus temperature are observed supporting this suggestion124. For materials prepared by gelatinising starch and then cooling to room temperature the precipitation of amylose is a very rapid process. The crystallisation of amylopectin leads to an increase in stiﬀness of the gelatinised granule and a slow firming of the material. Amylopectin crystallisation and the associated firming can be abolished by reheating to 60 °C. If amylose in hot aqueous solution is cooled to room temperature, the amylose precipitates and retrogrades with the formation of a partially crystalline precipitate or gel. The precipitate needs to be heated to temperatures <120 °C to initiate dissolution. If the aqueous solution is saturated with 1-butanol prior to cooling, a precipitate is still formed which will dissolve on heating to 100 °C. This precipitate consists of amylose in a single helical form and can contain the 1-butanol as a complexed guest molecule. On the basis of X-ray diﬀraction experiments, the complex may be either crystalline or amorphous125. The amorphous material is formed on rapid cooling while slow cooling favours the formation of crystalline material. The important point is that the addition of a hydrophobic compound prevents the formation of the B-crystalline form—the single helical complex is the preferred product126–128. A further indication of this preference is the observation that Aspects of the physical chemistry of starch the amylose complex forms immediately after gelatinisation48. As the B form of amylose is thermally more stable this might again indicate the role of kinetics in influencing crystallisation. Depending on the hydrophobic compound added, (e.g., if it is charged) it is also possible to induce a conformational change to a helical form which does not readily crystallise or precipitate. From the foregoing it can be seen that the addition of hydrophobic surfactants to starch-rich products would modify the retrogradation behaviour through favouring the formation of single helical conformations. Depending on the conditions, this could be single helices in solution or an amorphous/crystalline complex. While the interaction with amylose is relatively strong, favouring the formation of crystalline materials, the interaction with the shorter chains of amylopectin would be expected to be weaker. It is known that after addition of surfactants to bread products, the crystalline single helical Vh form of amylose is observed and the development of the B form on ageing of the bread is retarded. CONCLUSIONS Although starch has long been studied from a polymeric perspective, recent research has added to this knowledge more particularly in terms of its phase behaviour and dynamics, and how they are modified by the presence of other compounds. 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