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Annealing of starch — a review
International Journal of Biological Macromolecules 27 (2000) 1 – 12 www.elsevier.com/locate/ijbiomac
Review
Annealing of starch — a review Richard F. Tester *, Ste´phane J.J. Debon Food Research Laboratories, School of Biological and Biomedical Sciences, Glasgow Caledonian Uni6ersity, Glasgow G4 0BA, UK Received 22 July 1999; accepted 1 December 1999
Abstract Annealing processes, involving specific heating protocols, have been used by man for centuries to impart desirable properties to materials — especially metals and particularly tools and weapons. The terminology has also been applied to biopolymers such as starches, where the effects of the processing have been known for decades although the molecular basis has not been at all well understood. Because of the marked effect the annealing process has on starch functionality and consequently industrial applications, it is critical that the underlying molecular events are understood. This review is an attempt to clarify the process of starch annealing with an emphasis on data generated in the authors’ laboratory. © 2000 Elsevier Science B.V. All rights reserved. Keywords: Annealing process; Heat–moisture treatment; Starch
1. Background and definitions The annealing process, when related to starches, has been variously described. Both annealing and heat– moisture treatments are related processes, where the starch to moisture ratio, temperature and heating time are critical parameters to control. Jacobs and Delcour [1] have discussed the difference between annealing and heat –moisture treatment of starch. They state that treatments in excess (\60% w/w) or at intermediate (40 – 55% w/w) water contents represent annealing while treatments below 35% (w/w) can be described as heat– moisture treatment. Also, they state that both processes occur at above the glass transition temperature (Tg) but below the gelatinisation temperature. However, the term heat–moisture is often used to describe high temperature treatments, like 100°C (up to 16 h at 27% moisture) [2]. Stute [3] has also discussed the difference between annealing and heat – moisture treatments, acknowledging that for work conducted in the early part of last Century, annealing and heat – moisture were used as synonymous terms. More recently Collado and * Corresponding author. Tel.: +44-141-3318514; fax: + 44-1413313208. E-mail address: [email protected] (R.F. Tester)
Corke [4] have helped to clarify the situation. They state that annealing represents ‘physical modification of starch slurries in water at temperatures below gelatinisation’ whereas heat–moisture treatment ‘refers to the exposure of starch to higher temperatures at very restricted moisture content (18–27%)’. These authors propose that the terminology is standardised — which has implications in terms of the definition of gelatinisation. Hence, the following definitions are proposed with respect to starch (and related polymeric systems).
1.1. Glass transition temperatures Glass transition temperatures are very important parameters that affect polymeric physical properties. The transition is similar to a second-order thermodynamic transition and has been well described by Biliaderis et al. [5]. The term describes the temperature induced transition of an amorphous glassy polymer system to a progressively more rubbery state when it is heated (usually in the presence of a solvent/plasticiser, when applied to polysaccharides). In the case of completely glassy polymers, Tg is relatively distinct, where an inflection (increase) in the specific volume and enthalpy as a function of temperature occurs and is
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reflected in discontinuity in the specific heat capacity (Cp). Because starch contains both amorphous and crystalline material, the exact thermal event representing Tg is difficult to detect. However, high sensitivity differential scanning calorimetry (DSC) has allowed for the measurement of Tg in amorphous and native starches with various levels of crystallinity [6 – 8]. Water is a very effective plasticiser of amorphous starch (and hence Tg), where the ratio of starch to water is critical with respect to the temperature at which Tg occurs (Fig. 1).
1.2. Annealing Annealing represents the physical reorganisation of starch granules (or appropriate polysaccharide matrices like amylose–lipid complexes) when heated in water (or appropriate plasticiser) at a temperature between Tg and the onset of gelatinisation (To) of the native starch (or polymeric system). It is recognised that annealing can be associated with partial gelatinisation. However, these authors believe the definition should be applied only where gelatinisation does not occur and hence To
Fig. 1. State diagram of the starch–water system. The experimental data for the glass transition (Tg) are from amorphous starch [8] while the theoretical Tg is derived from the Couchman–Karasz equation [9]. The experimental data for the melting transition (Tm) are from DSC (Tconclusion) of wheat starches at different moisture content [10 – 13]. The theoretical Tm is fitted from the Flory – Huggins equation [14].
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Fig. 2. DSC thermograms of a commercial wheat starch (BDH 30265): (a) native; (b) after annealing in excess water (45°C, 100 days).
must not be exceeded. In addition, according to this definition, the enthalpy of gelatinisation post-annealing cannot be less than for the native starch. Annealing leads to elevation of starch gelatinisation temperatures and sharpening of the gelatinisation range (defined below) as shown in Fig. 2. The annealing process has important industrial implications. Starches may be deliberately annealed to impart novel processing characteristics. However, there are few commercial processes where annealing may be justified in terms of energy and time to generate starches with higher gelatinisation temperatures — especially when many inexpensive chemical processes can be employed, over a short time frame, to selectively modify starch characteristics. Often annealing is achieved unintentionally. One example is the wet milling of maize when used to extract starch.
1.3. Gelatinisation Gelatinisation is a term used to describe the molecular events associated with heating starch in water. Starch is converted from a semi-crystalline, relatively indigestible form to (eventually) an amorphous (readily digestible) form. The gelatinisation process (in excess water) is believed by these authors to involve primary hydration of amorphous regions around and above Tg, with an associated glassy-rubbery transition. This in turn facilitates molecular mobility in the amorphous regions (with reversible swelling) which then provokes
an irreversible molecular transition. This irreversible step involves dissociation of double helices (most of which are in crystalline regions) and expansion of granules as the polymers (and granule interstices) hydrate. The onset temperature (Tonset or To, typically 45°C) by DSC reflects the initiation of this process, which is followed by a peak (Tpeak or Tp, typically 60°C) and conclusion (Tconclusion or Tc, typically 75°C) temperature (Fig. 2). After Tc, all amylopectin double helices have dissociated, although swollen granule structures will be retained until more extensive temperature and shear have been applied. Beyond 95°C an amorphous gel is formed. The temperature range Tc –To represents the gelatinisation period. After gelatinisation, a-glucan chains re-form double helices if the conditions are desirable. This process — retrogradation — occurs when, for example, bread stales. Sometimes, annealing type processes are confused with retrogradation. However, annealing of starch granules is a process that retains granular structure and original order. Retrogradation occurs as amorphous a-glucan chains form double helices and, perhaps eventually, align themselves in crystallites. Blanshard has discussed gelatinisation type processes in depth elsewhere [15–17], and how interactions between starch and other groups (especially water and solutes) modify the temperature driven transitions. Readers are referred to these publications for more detail.
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1.4. Heat–moisture Heat–moisture treatments represent the control of molecular mobility at high temperatures by limiting the amount of water and hence gelatinisation. In common with annealing, physical reorganisation is manifested. The low levels of water in the system lead to an elevation of Tg — the trigger for polymeric reorganisation — as discussed below. Hence, high temperatures are required to cause physical reorganisation within granules. Heat–moisture treatments of starches may be conducted deliberately by industry to impart novel characteristics. One example is the pre-treatment of starches for infant foods. Other examples of industrial applications of the process include processing of potato starch to replace maize starch in times of shortage, creation of excellent freeze–thaw stability and improvement of the baking quality of potato starch [4].
2. Relationship between Tg on both annealing and gelatinisation We view the gelatinisation process as a co-operative event between amorphous and crystalline regions in starches. In unlimited water, amorphous regions imbibe water as the starch granules are progressively heated. Perhaps the relative large amorphous growth ring type regions are the primary amorphous regions to hydrate, followed by amorphous lamellae ‘sandwiched’ between the crystalline lamellae. The plasticisation of the amorphous lamellae and annealing of double helices is represented in Fig. 3. The absorption of water into amorphous regions is certainly possible, as for example, potato starch can reversibly absorb up to 0.53 g water/g dry starch before the irreversible steps within the gelatinisation process are exceeded [17]. The water induces a transition of the amorphous regions from a rigid glassy state to a mobile rubbery state which in turn facilitates the hydration and dissociation of double helices in crystallites. The dissociation of the crystallites begins after Tg of amorphous regions, and at this temperature (To), limited dissociation of amylopectin double helices (most of which are in crystallites) is associated with limited swelling of granules. Gelatinisation proceeds as the temperature is increased, progressively uncoiling all the double helices and converting crystalline material to amorphous material. If water is sufficiently low so as to restrict gelatinisation, the primary gelatinisation endotherm (G) develops a high temperature trailing shoulder (M) [19,20], as shown in Fig. 4. As the volume fraction of water (61) is reduced to B 0.45, the shoulder becomes distinct and represents the only endotherm observed. In high moisture food systems, starch granules are completely gelatinised and often no granule form is discernible (e.g.
custard). Drier food products have often been processed under high moisture conditions and equally, little granule form is apparent (e.g. wafer biscuits). Where water is limiting, however, like fat rich shortbread biscuits, essentially native granule form is apparent under the microscope-although this starch has presumably been heat moisture treated. The Tg must be reached or exceeded for annealing to occur. Many authors accept that this is a prerequisite of the annealing process [17,22,23]. Indeed, the annealing process has been discussed in terms of the process itself improving Tg without facilitating the gelatinisation process [17]. If starches are heated at progressively higher temperature above Tg, they do eventually completely gelatinise, having gone through an early phase involving enhanced mobility of amorphous regions. It is logical that this phase is comparable to the phase that initiates and forms part of the annealing process [19,24–27]. Perhaps because of the difficulty associated with measuring Tg, some authors claim that starches can be annealed below Tg [28,29]. However, this would mean that structural reorganisations of the crystalline component of starch granules occur independently of reorganisation of the amorphous phase. This is an almost impossible situation to imagine in view of the relatively impenetrable nature of these regions by water molecules, with no associated passage through (and associated reorganisation of) amorphous regions. It is relatively straightforward to measure the gelatinisation endotherm of starch using DSC, although this is not true of Tg. Whilst Tg has reportedly been determined prior to gelatinisation [24], it is in fact very hard to detect and quantify [5,22] — unless high sensitivity DSC is used at low moisture contents [6–8]. Primarily this is because it is both small and submerged into the thermogram baseline. Model polymeric systems, for example polyanhydroglucose compounds [30] have, however, been useful in developing an understanding of how a-glucan structure itself moderates Tg. For example branched regions (which are also found in the amorphous zones of starch) seem to depress Tg similarly to plasticisation by small molecules [30].
3. Effects of starch-to-moisture ratio on Tg and annealing The original work of Gough and Pybus [31], which was the first to describe how wheat starch could be annealed by heating in excess water at 50°C, showed that gelatinisation temperatures could be increased while the gelatinisation range could be sharpened. Many studies have been conducted on the effects of annealing on different starches, with different starch-to-moisture ratios and different storage times [23,29,32–46]. Sometimes, the annealing process is conducted as a
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Fig. 3. Pictorial representation of the effect of hydration and subsequent annealing on the semi-crystalline lamellae (amylopectin double helices are represented as rectangles): (a) dry starch with glassy amorphous regions; (b) hydrated annealed starch with rubbery amorphous regions (adapted from [18]).
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Fig. 4. Influence of water content on the differential scanning calorimetry thermograms profile of potato starch: (a) the onset ( ) and conclusion () gelatinisation temperatures (adapted from [21]); (b) the corresponding DSC thermograms (adapted from [19]).
single event (single-step) whilst at other times it is conducted as two starch – water/temperature/time events (double-step) or even many individual steps (multi-step) as discussed elsewhere [1]. This double or multi-step approach is often used to promote annealing without gelatinisation, and the double step process potentially produces higher gelatinisation temperatures than the single step process [1]. The lack of standardisation of annealing conditions makes it difficult to compare results between the different studies. The effect of the starch-to-water ratio, temperature and time on annealing of wheat starch has been investigated in detail [23]. This study demonstrated how critical the interrelationship of these parameters is. The annealing process could be initiated when the moisture content exceeded 20% by weight, (because Tg is around room temperature when this moisture content is exceeded, Fig. 1) but was restricted (in terms of its effect on increasing gelatinisation temperatures) unless it ex-
ceeded 60%. Although annealing could be initiated at 15°C below To by DSC, the effect was more marked the closer the annealing temperature was set to (below) To. Similar studies have been conducted on starches of different botanical origins [32,40] and demonstrate the additional complication of species specific variation. During annealing of starches, there are in essence two thermally driven processes which are intimately related and reflect the moisture content of the system — the elevation of Tg and gelatinisation temperatures (especially To). Low moisture causes elevation of (the relatively unplasticised) Tg of starches [6,7,22,47] and model polymeric systems [30,48,49] which, in the case of starch, intimately reflects the increase in gelatinisation temperatures. Indeed, the elevation of Tg implies a more glassy state and hence reorganisation of amorphous regions. This is associated with improved order of crystalline regions (below). The situation with respect to Tg of starch in food systems is very complex because
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Fig. 4. (Continued)
of the raft of potential interactions. More details concerning (general) glass transitions in model systems can be found elsewhere [50]. Similarly, sub-Tg transitions of starches (which probably represent enthalpy relaxation [51]) have been described by other authors [51,52].
4. Environmental considerations The effect of environmental temperature on starch synthesis and properties has been the subject of much recent research. Apart from direct growth temperature effects on the activity of enzymes involved in starch biosynthesis (which are discussed in some detail in a recent publication by these authors [53]), there is a distinct effect on starch physico-chemical properties. In general, for mature cereal and tuber starches [54–58] there tends to be a small effect on the fine structure (chain length distribution) of amylose or amylopectin. Granule size decreases as growth temperature increases, while amylose content remains approximately the same. In the case of lipid (lysophospholipids and free fatty acids) in cereal starches (only), there is a distinct temperature dependent increase. Growth temperature also causes a distinct increase in gelatinisation temperatures
of starches (which can also be modelled using potato microtubers [53]), and parallels have been drawn between laboratory based annealing processes (in vitro annealing) and environmentally driven reorganisations (in vivo ‘annealing’) of starch granule architecture [23,53,57,58]. Hence, apart from species and cultivar specific variation in starch physico-chemical properties, there is a profound environmental effect on gelatinisation characteristics. This effect is, in the experience of these authors, far greater than cultivar specific (and hence genetic) variation. The implications are, that product quality is not simply a cultivar specific trait — but is largely dependent on environmental conditions experienced during starch deposition.
5. Molecular basis for annealing It has been difficult to define, at the molecular level, what happens to the internal structure of starch granules when they are annealed. Some authors have discussed the molecular event in terms of increasing granule stability [32], reorganising granule structure [39,40] or lowering free energy [17]. These descriptive
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terms do not, however, give readers a clear molecular picture associated with the reorganisations involved within granules when they are annealed. A number of authors have discussed annealing with more emphasis on the crystalline and amorphous domains. Crystallinity and crystalline ‘perfection’ (optimisation of crystalline order) have been discussed in detail in this context [28,36,38,42,43,59]. Similarly, granular reorganisations have been discussed in terms of rigidity [33] and realignments and partial melting [45,60]. Others recognise the importance of interactions between, and mobility of, amorphous and crystalline regions [3,61] and the constituent amylose and amylopectin molecules [37]. Tester et al. [23] (working on wheat starches), have discussed annealing in the context of hydration and swelling of amorphous regions (temperature range between Tg and To), which facilitates ordering of double helices in crystalline regions. This ordering of double helices could be associated with minor optimisation of double helix length, although no additional double helical material is formed [23]. The amorphous material post-annealing probably becomes more ‘glassy’ (more rigid and less mobile) whereupon Tg is elevated. The constancy of double helix content pre- and post-annealing has also been shown by Jacobs [1] for a range of starches (pea, potato and wheat). With respect to the effects of annealing on the double helix content of starches, the situation in amylomaize is far more complex than for waxy or normal starches. In amylomaize starch there is evidence from NMR that amylose also forms some double helices and that upon annealing there is partitioning of amylopectin and amylose helical structures [62,63]. This different structural model within amylomaize starch granules helps to explain the characteristic gelatinisation characteristics of these starches (below). Whilst NMR can be used to quantify the number of double helices within starch granules, it does not measure crystallinity per se. For this purpose, wide angle X-ray scattering (WAXS) may be employed. Early work on annealing of wheat starch indicated that there was little detectable effect on the X-ray diffraction pattern [31]. This has been confirmed for potato starch [3]. However, other workers have reported a small increase in intensity of the diffraction pattern (but with little or no effect on d-line spacings) for wheat, oat, lentil [32,64] and barley starches [46] but with a decrease in intensity for potato starch [32]. It is very useful to link together both NMR and WAXS data for starches, as they measure different levels of order. One could, for example, have non-crystalline double helices (outside crystalline domains) within starch granules that give a strong NMR signal but not a WAXS diffraction pattern. The relative significance of these techniques for determining starch structure and order
during annealing and gelatinisation may be supported by the use of scanning electron microscopy [65], where dimensions of amorphous and crystalline lamellae may be estimated. Overall, the NMR and WAXS data support the same general picture that annealing causes no significant increase in crystalline material formed within starch granules by either of two possible mechanisms: (i) formation of double helices (which need not necessarily be associated with existing crystalline domains) or; (ii) major increase in amount of crystallinity as a consequence of ordering of previously amorphous regions. Rather, the enhanced ordering of double helices, due to improved registration (alignment), with associated increased rigidity of amorphous regions, probably underlies the annealing process. Unlike WAXS which quantifies crystalline order throughout starch granules, small angle X-ray scattering (SAXS) quantifies differences (periodicity) at the level of amorphous-crystalline lamellae radiating from the hilum to the periphery of starch granules. Using this technique, Jacobs et al. [36] showed that (for wheat and potato starches) the repeat distances of the crystalline and amorphous lamellae remain unchanged (10.5 nm in wheat and 9.9 nm in potato), although there was an increase in peak intensity. A pictorial representation of the length scales within starch granules together with techniques used for their quantification are presented in Fig. 5. More detailed discussion regarding the application of this technique to understand structural, gelatinisation and annealing mechanisms of starches can be found elsewhere [65]. Those authors, however, reported that the lamellar repeat distance for wheat starch is smaller (8.85 nm) than the figures quoted by Jacobs et al. Native cereal starch granules contain amylose–lipid complexes, as shown by NMR [67,68]. The lipid (lysophospholipid or free fatty acid) is immobilised within the a-glucan helices and the corresponding lack of mobility of methylene groups can be determined using this technique. The biochemical significance of these complexes is uncertain, although they have a significant effect on starch functionality. Whilst in model amylose–lipid complex systems annealing can be induced [69] because of the relative ease of mobility, annealing between Tg of the starch and To of amylopectin in wheat starch has little effect on this amylose–lipid endotherm [34,35]. The effect of annealing at these temperatures (e.g. 35–50°C) on amylose–lipid complexes is predictably very unlikely, because the temperature is far too low. The peak transition temperature of these complexes is of the order of 95–115°C. At temperatures where complexes have been annealed (for example 80°C, [69]) the starch would be fully gelatinised in unlimiting water. Probably Tg for the complexes under these conditions is quite close to this
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Fig. 5. Pictorial representation of the length scales within the starch granule together with techniques used to characterise the structural features (adapted from [66]).
temperature, although it is perhaps as mobile as Tg for amylopectin as a function of moisture content [23]. There is a relationship between the amount of starch phosphorylation and elevation of gelatinisation temperatures as a consequence of annealing [70]. The shift in Tp (or DTp) is greater for starches with low levels of phosphorylation. The authors [70] proposed that this is because the number of potential dislocations is smaller. In other words, phosphate moieties restrict double helix (and consequently crystallite) formation. The higher the phosphate content, the greater the interference. Because of the detrimental effect of phosphate groups on crystallite formation, however, the increase in enthalpy is largest for the high phosphate starches indicating that
steric hindrance is diminished as a consequence of the increased mobility during annealing. Chemically introduced phosphate groups have similar effects to naturally (during biosynthesis) inserted groups [71]. This should be viewed in the context of phosphate esters affecting the crystallinity of native starches [72], where the gelatinisation enthalpy is inversely related to the phosphate content.
6. Physical consequences of annealing According to some authors [32], annealing causes no effect on granule dimensions or shapes, although early
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microscopic work indicated that wheat starch granule dimensions increase after annealing [31]. Clearly, however, small differences in size cannot be accurately quantified using microscopy and care should be placed on reliance upon this data. The A-type diffraction pattern of wheat starch is retained after annealing [31], although the line intensity may increase as discussed above. Although heat moisture treatments causes a Bto A-type transition for potato starches, this does not happen during annealing [3]. Hence, the molecular reorientation is more subtle. The effect of annealing on gelatinisation characteristics is well established, particularly using DSC, where there tends to be an increase in To and Tp, decrease in the gelatinisation range (Tc – To) and either constancy or an increase in gelatinisation enthalpy [3,23,28,31,32,34,37 – 41,43,46,64,70,71,73]. The increase in gelatinisation temperatures is associated with a decrease in swelling power [23,32,41,46,73], provided that some granular structure is retained. This is reflected in a higher temperature onset of swelling and reduced swollen volume (below circa 90°C, provided that water is not limiting). The effects of annealing on pasting characteristics are complex. In some studies the consistency (viscosity) of annealed starches (wheat and potato) increases (with associated decrease of peak viscosity for potato starch) while for lentil and oat it tends to decrease [32]. Similar results have been reported for wheat and potato starch, with annealed pea and rice starches exhibiting increased viscosity [3,33,41]. Using the model proposed by these authors and co-workers [23], the physical properties discussed above can be explained on the basis of more glassy amorphous regions within annealed starch granules and a more ordered registration of amylopectin double helices. These molecular events restrict ease of hydration of the starch granules during gelatinisation and elevate gelatinisation temperatures. In parallel, these events restrict swelling. It is difficult to unravel the effects on pasting characteristics, because this system will be strongly influenced by granule size and polysaccharide solubilisation — more so than gelatinisation (by DSC) and swelling power determinations. This is probably why there is a lack of consistency in response to annealing for starches from different botanical origins.
7. Solubility The annealing process itself leads to little solubilisation of a-glucan [23]. This is important as it shows that improved order is a genuine molecular event rather than a consequence of leaching amorphous a-glucan and hence ‘concentrating’ crystalline material. Annealing reduces solubilisation of a-glucan during swelling below 100°C [32,41,46,73]. As leachate is primarily
(amorphous) lipid free amylose (FAM) according to the definition of Morrison et al. [68], the amylose must be more restricted from leaching out of the granules. Although at a given temperature post-annealing, the granules will swell less than un-annealed starches and this will be the primary restraint to leaching. This does, however, strengthen the view that there is molecular reorientation in the starch granule which makes the amorphous material more glassy with an elevated Tg.
8. Chemical hydrolysis Annealing tends to reduce the amount of acid hydrolysis of starch granules, although small granules sometimes exhibit little difference or even enhanced hydrolysis [32,34]. This discrepancy has to some extent been resolved by Tester et al. [23] who investigated acid hydrolysis for native and annealed wheat starch as a function of time. They found that for the first phase of acid hydrolysis (0–7 days, representing amorphous material hydrolysis) annealed starch was more extensively hydrolysed than native starch. After 7 days, where crystalline material is progressively hydrolysed, the extent of hydrolysis for the native and annealed starches was essentially the same. The explanation for the enhanced hydrolysis of amorphous regions after annealing is that the amorphous regions become more concentrated due to the enhanced glassy structure. On the other hand, the similarity in hydrolysis pattern during the crystalline hydrolysis phase (\ 7 days) confirms that it is double helices (which remain constant) which are the primary contributor to the hydrolysis profile during this phase. Amylose–lipid complexes may affect the pattern of acid hydrolysis in cereal starches, which could also influence enzymic hydrolysis (below).
9. Enzymatic hydrolysis Certain studies have indicated that annealed wheat, barley and sago starches are more easily hydrolysed by a-amylases than native starches [31,46,74]. This has, however, been contradicted by other research on wheat, lentil and potato starches [32,41], although small starch granules (oat) have been reported to be much more easily hydrolysed post-annealing [32]. However, the rate of a-amylase hydrolysis for different starches follows two distinct phases: an initial rapid then subsequent slow phase. Annealing alters the extent of hydrolysis of these different phases as a function of botanical origin [35]. During the second phase of hydrolysis, annealed wheat and pea starches are more resistant to a-amylase hydrolysis whilst the inverse is true for potato starch [35]. Annealed potato starch is less easily hydrolysed by
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amyloglucosidase than native starch [41]. In common with the statements above regarding acid hydrolysis patterns of starches, reported differences in a-amylase hydrolysis patterns may represent unjustified comparisons between different phases of hydrolysis (amorphous and crystalline material) rather than necessarily starch specific responses to enzyme hydrolysis post annealing. Although, the botanical origin of starch is important with respect to hydrolytic pattern, the surface area to volume ratio is probably of more significance than the actual plant source. It is also possible that the annealing process creates pores or fissures which alter the pattern of amylase hydrolysis from surface to internal erosion [74]. Hence, although amorphous and crystalline lamellae become more ordered, accessibility to the amorphous regions by enzymes is facilitated. Annealing can be conducted in the presence of amylases to selectively hydrolyse amorphous regions and the possibility of novel products with unique gelatinisation and swelling characteristics [75,76]. Both potential starch and glucose syrup products are possible using this general approach.
10. Overview Much data has been published on annealing of starches. Whilst the molecular basis is not absolutely certain, these authors believe that the physico-chemical data are consistent with enhanced registration of amylopectin double helices within crystalline zones (with perhaps some slight enhancement of helical length) and greater rigidity (more glassy) of amorphous regions. The consequence is that Tg and gelatinisation temperatures are increased, swelling is decreased and leaching is restricted. The situation in high amylose starches is different from waxy and normal starches where annealing facilitates partitioning of amylose and amylopectin double helices. Annealed starch provides a very useful model to investigate amylopectin crystallisation. Whilst a very simple technique, it provides a great deal of information about this process. There are important industrial implications of the annealing process with relevance to starch extraction and food production. As more sophisticated physical techniques are developed, the molecular basis will be understood in far more detail.
References [1] Jacobs H, Delcour JAJ. Agric Food Chem 1998;46:2895 – 905. [2] Franco CML, Ciacco CF, Tavares DQ. Starch/Sta¨rke 1995;47:223 – 8. [3] Stute R. Starch/Sta¨rke 1992;44:205–14. [4] Collado LS, Corke H. Food Chem 1999;65:339–46.
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[5] Biliaderis CG, Page CM, Maurice TJ, Juliano BOJ. Agric Food Chem 1986;34:6 – 14. [6] Zeleznak KJ, Hoseney RC. Cereal Chem 1987;64:121 –4. [7] Mizuno A, Mitsuiki M, Motoki M. J Agric Food Chem 1998;46:98 – 103. [8] Bencze´di D, Tomka I, Escher F. Macromolecules 1998;31:3055– 61. [9] Kalichevsky MT, Jaroszkiewicz EM, Blanshard JMV. Polymer 1993;34:346 – 58. [10] Eliasson A-C. Starch/Sta¨rke 1980;32:270 – 2. [11] Donovan JW, Lorenz K, Kulp K. Cereal Chem 1983;60:381–7. [12] Burt DJ, Russell PL. Starch/Sta¨rke 1983;35:354 – 60. [13] Svensson E, Eliasson A-C. Carbohydr Polym 1995;26:171–6. [14] Farhat IA, Blanshard JMV. Carbohydr Polym 1997;34:263–5. [15] Blanshard JMV. In: Blanshard JMV, Mitchell JR, editors. Polysaccharides in Foods. London: Butterworths, 1979:139–52. [16] Blanshard JMV. In: Blanshard JMV, Frazier PJ, Galliard T, editors. Chemistry and Physics of Baking. London: Royal Society of Chemistry, 1986:1 – 13. [17] Blanshard JMV. In: Galliard T, editor. Starch: Properties and Potential. Chichester: Wiley, 1987:16 – 54. [18] Waigh TA, Hopkinson I, Donald AM, Butler MF, Heidelbach F, Riekel C. Macromolecules 1997;30:3813 – 20. [19] Donovan JW. Biopolymers 1979;18:263 – 75. [20] Donovan JW, Mapes CJ. Starch/Sta¨rke 1980;32:190 – 3. [21] Evans ID, Haisman DR. Starch/Sta¨rke 1982;34:224 – 31. [22] Biliaderis CG. Food Technol 1992;6:98 – 109, 145. [23] Tester RF, Debon SJJ, Karkalas J. J Cereal Sci 1998;28:259–72. [24] Maurice TJ, Slade L, Sirett RR, Page CM. In: Simatos D, Multon JL, editors. Properties of Water in Foods. Dordrecht: Martinus Nijhoff, 1985:211 – 27. [25] Ong MH, Blanshard JMV. Food Sci Technol Today 1994;8:217 – 26. [26] Pravisani CI, Califano AN, Calvelo A. J Food Sci 1985;50:657– 60. [27] Biliaderis CG, Maurice TJ, Vose JR. J Food Sci 1980;45:1669– 74. [28] Yost DA, Hoseney RC. Starch/Sta¨rke 1986;38:289 – 92. [29] Seow CC, Vasanti-Nair CK. Carbohydr Res 1994;261:307–16. [30] Bizot H, Le Bail P, Leroux B, Davy J, Roger P, Buleon A. Carbohydr Polym 1997;32:33 – 50. [31] Gough BM, Pybus JN. Starch/Sta¨rke 1971;23:210 – 2. [32] Hoover R, Vasanthan T. J Food Biochem 1994;17:303–25. [33] Jacobs H, Eerlingen RC, Clauwaert W, Delcour JA. Cereal Chem 1995;72:480 – 7. [34] Jacobs H, Eerlingen RC, Rouseu N, Colonna P, Delcour JA. Carbohydr Res 1998;308:359 – 71. [35] Jacobs H, Eerlingen RC, Spaepen H, Grobet PJ, Delcour JA. Carbohydr Res 1998;305:193 – 207. [36] Jacobs H, Mischenko N, Koch MHJ, Eerlingen RC, Delcour JA, Reynaers H. Carbohydr Res 1998;306:1 – 10. [37] Knutson CA. Cereal Chem 1990;67:376 – 84. [38] Tester RF, Morrison WR. Cereal Chem 1990;67:558 – 63. [39] Krueger BR, Walker CE, Knutson CA, Inglett GE. Cereal Chem 1987;64:187 – 90. [40] Krueger BR, Knutson CA, Inglett GE, Walker CE. J Food Sci 1987;52:715 – 8. [41] Kuge T, Kitamura S. J Jpn Soc Starch Sci 1985;32:65–83. [42] Larsson I, Eliasson A-C. Starch/Sta¨rke 1991;43:227 – 31. [43] Parades-Lo´pez O, Herna´ndez-Lo´pez D. Starch/Sta¨rke 1991;43:57 – 61. [44] Kempf W. Starch/Sta¨rke 1955;7:161. [45] Marchant JL, Blanshard JMV. Starch/Sta¨rke 1980;32:223–6. [46] Lorenz K, Kulp K. Starch/Sta¨rke 1984;36:122 – 6. [47] Biliaderis CG. In: Levine H, Slade L, editors. Water Relationships in Food. New York: Plenum, 1991:251 – 73.
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[48] Kalichevisky MT, Jaroszkiewicz EM, Ablett S, Blanshard JMV, Lillford PJ. Carbohydr Polym 1992;18:77–88. [49] Vodovotz Y, Chinachoti P. J Agric Food Chem 1998;46:446 – 53. [50] Schenz TW. Food Hydrocolloids 1995;9:307–15. [51] Thiewes HJ, Steeneken PAM. Carbohydr Polym 1997;32:123 – 30. [52] Yuan RC, Thompson DB. Carbohydr Polym 1994;25:1– 6. [53] Debon SJJ, Tester RF, Millam S, Davies HV. J Sci Food Agric 1998;76:599 – 607. [54] Tester RF, South JB, Morrison WR, Ellis RP. J Cereal Sci 1991;13:113 – 27. [55] Tester RF, Morrison WR, Ellis RH, Piggott JR, Batts GR, Wheeler TR, Morison JIL, Hadley P, Ledward DA. J Cereal Sci 1995;22:63 – 71. [56] Shi Y-C, Seib PA, Bernardin JE. Cereal Chem 1994;71:369 – 83. [57] Tester RF. Int J Biol Macromol 1997;21:37–45. [58] Tester RF, Debon SJJ, Davies HV, Gidley MJ. J Sci Food Agric 1999; in press. [59] Shi Y-C, Seib PA, Lu SPW. In: Levine H, Slade L, editors. Water Relationships in Food. New York: Plenum, 1991:667 – 86. [60] Marchant JL, Blanshard JMV. Starch/Sta¨rke 1978;30:257 – 64. [61] Nazakawa F, Noguchi S, Takahashi J, Takada M. Agric Biol Chem 1984;48:2647 –53.
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[62] Shi Y-C, Capitani T, Trzasko P, Jeffcoat R. J Cereal Sci 1998;27:289 – 99. [63] Tester RF, Debon SJJ, Sommerville MD. Carbohydr Polym 1999; in press. [64] Lorenz K, Kulp K. Starch/Sta¨rke 1980;32:181 – 6. [65] Cameron RE, Donald AM. Polymer 1992;33:2628 – 36. [66] Gidley MJ. The 5th European Training Course on Carbohydrates. CRF: The Netherlands, 1998. [67] Morrison WR, Law RV, Snape CE. J Cereal Sci 1993;18:107–9. [68] Morrison WR, Tester RF, Snape CE, Law R, Gidley MJ. Cereal Chem 1993;70:385 – 91. [69] Karkalas J, Ma S, Morrison WR, Pethrick RA. Carbohydr Res 1995;268:233 – 47. [70] Muhrbeck P, Svensson E. Carbohydr Polym 1996;31:263–7. [71] Muhrbeck P, Wischmann B. Starch/Sta¨rke 1998;10:423–6. [72] Muhrbeck P, Eliasson A-C. J Sci Food Agric 1991;55:13–8. [73] Lorenz K, Kulp K. Starch/Sta¨rke 1978;30:333 – 6. [74] Wang WJ, Powell AD, Oates CG. Carbohydr Polym 1997;33:195 – 202. [75] Stoof G, Anger H, Schmeidl D, Bergthaller W. Starch/Sta¨rke 1997;49:225 – 31. [76] Stoof G, Anger H, Schmeidl D, Bergthaller W. Starch/Sta¨rke 1998;50:108 – 14.
Review
Annealing of starch — a review Richard F. Tester *, Ste´phane J.J. Debon Food Research Laboratories, School of Biological and Biomedical Sciences, Glasgow Caledonian Uni6ersity, Glasgow G4 0BA, UK Received 22 July 1999; accepted 1 December 1999
Abstract Annealing processes, involving specific heating protocols, have been used by man for centuries to impart desirable properties to materials — especially metals and particularly tools and weapons. The terminology has also been applied to biopolymers such as starches, where the effects of the processing have been known for decades although the molecular basis has not been at all well understood. Because of the marked effect the annealing process has on starch functionality and consequently industrial applications, it is critical that the underlying molecular events are understood. This review is an attempt to clarify the process of starch annealing with an emphasis on data generated in the authors’ laboratory. © 2000 Elsevier Science B.V. All rights reserved. Keywords: Annealing process; Heat–moisture treatment; Starch
1. Background and definitions The annealing process, when related to starches, has been variously described. Both annealing and heat– moisture treatments are related processes, where the starch to moisture ratio, temperature and heating time are critical parameters to control. Jacobs and Delcour [1] have discussed the difference between annealing and heat –moisture treatment of starch. They state that treatments in excess (\60% w/w) or at intermediate (40 – 55% w/w) water contents represent annealing while treatments below 35% (w/w) can be described as heat– moisture treatment. Also, they state that both processes occur at above the glass transition temperature (Tg) but below the gelatinisation temperature. However, the term heat–moisture is often used to describe high temperature treatments, like 100°C (up to 16 h at 27% moisture) [2]. Stute [3] has also discussed the difference between annealing and heat – moisture treatments, acknowledging that for work conducted in the early part of last Century, annealing and heat – moisture were used as synonymous terms. More recently Collado and * Corresponding author. Tel.: +44-141-3318514; fax: + 44-1413313208. E-mail address: [email protected] (R.F. Tester)
Corke [4] have helped to clarify the situation. They state that annealing represents ‘physical modification of starch slurries in water at temperatures below gelatinisation’ whereas heat–moisture treatment ‘refers to the exposure of starch to higher temperatures at very restricted moisture content (18–27%)’. These authors propose that the terminology is standardised — which has implications in terms of the definition of gelatinisation. Hence, the following definitions are proposed with respect to starch (and related polymeric systems).
1.1. Glass transition temperatures Glass transition temperatures are very important parameters that affect polymeric physical properties. The transition is similar to a second-order thermodynamic transition and has been well described by Biliaderis et al. [5]. The term describes the temperature induced transition of an amorphous glassy polymer system to a progressively more rubbery state when it is heated (usually in the presence of a solvent/plasticiser, when applied to polysaccharides). In the case of completely glassy polymers, Tg is relatively distinct, where an inflection (increase) in the specific volume and enthalpy as a function of temperature occurs and is
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reflected in discontinuity in the specific heat capacity (Cp). Because starch contains both amorphous and crystalline material, the exact thermal event representing Tg is difficult to detect. However, high sensitivity differential scanning calorimetry (DSC) has allowed for the measurement of Tg in amorphous and native starches with various levels of crystallinity [6 – 8]. Water is a very effective plasticiser of amorphous starch (and hence Tg), where the ratio of starch to water is critical with respect to the temperature at which Tg occurs (Fig. 1).
1.2. Annealing Annealing represents the physical reorganisation of starch granules (or appropriate polysaccharide matrices like amylose–lipid complexes) when heated in water (or appropriate plasticiser) at a temperature between Tg and the onset of gelatinisation (To) of the native starch (or polymeric system). It is recognised that annealing can be associated with partial gelatinisation. However, these authors believe the definition should be applied only where gelatinisation does not occur and hence To
Fig. 1. State diagram of the starch–water system. The experimental data for the glass transition (Tg) are from amorphous starch [8] while the theoretical Tg is derived from the Couchman–Karasz equation [9]. The experimental data for the melting transition (Tm) are from DSC (Tconclusion) of wheat starches at different moisture content [10 – 13]. The theoretical Tm is fitted from the Flory – Huggins equation [14].
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Fig. 2. DSC thermograms of a commercial wheat starch (BDH 30265): (a) native; (b) after annealing in excess water (45°C, 100 days).
must not be exceeded. In addition, according to this definition, the enthalpy of gelatinisation post-annealing cannot be less than for the native starch. Annealing leads to elevation of starch gelatinisation temperatures and sharpening of the gelatinisation range (defined below) as shown in Fig. 2. The annealing process has important industrial implications. Starches may be deliberately annealed to impart novel processing characteristics. However, there are few commercial processes where annealing may be justified in terms of energy and time to generate starches with higher gelatinisation temperatures — especially when many inexpensive chemical processes can be employed, over a short time frame, to selectively modify starch characteristics. Often annealing is achieved unintentionally. One example is the wet milling of maize when used to extract starch.
1.3. Gelatinisation Gelatinisation is a term used to describe the molecular events associated with heating starch in water. Starch is converted from a semi-crystalline, relatively indigestible form to (eventually) an amorphous (readily digestible) form. The gelatinisation process (in excess water) is believed by these authors to involve primary hydration of amorphous regions around and above Tg, with an associated glassy-rubbery transition. This in turn facilitates molecular mobility in the amorphous regions (with reversible swelling) which then provokes
an irreversible molecular transition. This irreversible step involves dissociation of double helices (most of which are in crystalline regions) and expansion of granules as the polymers (and granule interstices) hydrate. The onset temperature (Tonset or To, typically 45°C) by DSC reflects the initiation of this process, which is followed by a peak (Tpeak or Tp, typically 60°C) and conclusion (Tconclusion or Tc, typically 75°C) temperature (Fig. 2). After Tc, all amylopectin double helices have dissociated, although swollen granule structures will be retained until more extensive temperature and shear have been applied. Beyond 95°C an amorphous gel is formed. The temperature range Tc –To represents the gelatinisation period. After gelatinisation, a-glucan chains re-form double helices if the conditions are desirable. This process — retrogradation — occurs when, for example, bread stales. Sometimes, annealing type processes are confused with retrogradation. However, annealing of starch granules is a process that retains granular structure and original order. Retrogradation occurs as amorphous a-glucan chains form double helices and, perhaps eventually, align themselves in crystallites. Blanshard has discussed gelatinisation type processes in depth elsewhere [15–17], and how interactions between starch and other groups (especially water and solutes) modify the temperature driven transitions. Readers are referred to these publications for more detail.
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1.4. Heat–moisture Heat–moisture treatments represent the control of molecular mobility at high temperatures by limiting the amount of water and hence gelatinisation. In common with annealing, physical reorganisation is manifested. The low levels of water in the system lead to an elevation of Tg — the trigger for polymeric reorganisation — as discussed below. Hence, high temperatures are required to cause physical reorganisation within granules. Heat–moisture treatments of starches may be conducted deliberately by industry to impart novel characteristics. One example is the pre-treatment of starches for infant foods. Other examples of industrial applications of the process include processing of potato starch to replace maize starch in times of shortage, creation of excellent freeze–thaw stability and improvement of the baking quality of potato starch [4].
2. Relationship between Tg on both annealing and gelatinisation We view the gelatinisation process as a co-operative event between amorphous and crystalline regions in starches. In unlimited water, amorphous regions imbibe water as the starch granules are progressively heated. Perhaps the relative large amorphous growth ring type regions are the primary amorphous regions to hydrate, followed by amorphous lamellae ‘sandwiched’ between the crystalline lamellae. The plasticisation of the amorphous lamellae and annealing of double helices is represented in Fig. 3. The absorption of water into amorphous regions is certainly possible, as for example, potato starch can reversibly absorb up to 0.53 g water/g dry starch before the irreversible steps within the gelatinisation process are exceeded [17]. The water induces a transition of the amorphous regions from a rigid glassy state to a mobile rubbery state which in turn facilitates the hydration and dissociation of double helices in crystallites. The dissociation of the crystallites begins after Tg of amorphous regions, and at this temperature (To), limited dissociation of amylopectin double helices (most of which are in crystallites) is associated with limited swelling of granules. Gelatinisation proceeds as the temperature is increased, progressively uncoiling all the double helices and converting crystalline material to amorphous material. If water is sufficiently low so as to restrict gelatinisation, the primary gelatinisation endotherm (G) develops a high temperature trailing shoulder (M) [19,20], as shown in Fig. 4. As the volume fraction of water (61) is reduced to B 0.45, the shoulder becomes distinct and represents the only endotherm observed. In high moisture food systems, starch granules are completely gelatinised and often no granule form is discernible (e.g.
custard). Drier food products have often been processed under high moisture conditions and equally, little granule form is apparent (e.g. wafer biscuits). Where water is limiting, however, like fat rich shortbread biscuits, essentially native granule form is apparent under the microscope-although this starch has presumably been heat moisture treated. The Tg must be reached or exceeded for annealing to occur. Many authors accept that this is a prerequisite of the annealing process [17,22,23]. Indeed, the annealing process has been discussed in terms of the process itself improving Tg without facilitating the gelatinisation process [17]. If starches are heated at progressively higher temperature above Tg, they do eventually completely gelatinise, having gone through an early phase involving enhanced mobility of amorphous regions. It is logical that this phase is comparable to the phase that initiates and forms part of the annealing process [19,24–27]. Perhaps because of the difficulty associated with measuring Tg, some authors claim that starches can be annealed below Tg [28,29]. However, this would mean that structural reorganisations of the crystalline component of starch granules occur independently of reorganisation of the amorphous phase. This is an almost impossible situation to imagine in view of the relatively impenetrable nature of these regions by water molecules, with no associated passage through (and associated reorganisation of) amorphous regions. It is relatively straightforward to measure the gelatinisation endotherm of starch using DSC, although this is not true of Tg. Whilst Tg has reportedly been determined prior to gelatinisation [24], it is in fact very hard to detect and quantify [5,22] — unless high sensitivity DSC is used at low moisture contents [6–8]. Primarily this is because it is both small and submerged into the thermogram baseline. Model polymeric systems, for example polyanhydroglucose compounds [30] have, however, been useful in developing an understanding of how a-glucan structure itself moderates Tg. For example branched regions (which are also found in the amorphous zones of starch) seem to depress Tg similarly to plasticisation by small molecules [30].
3. Effects of starch-to-moisture ratio on Tg and annealing The original work of Gough and Pybus [31], which was the first to describe how wheat starch could be annealed by heating in excess water at 50°C, showed that gelatinisation temperatures could be increased while the gelatinisation range could be sharpened. Many studies have been conducted on the effects of annealing on different starches, with different starch-to-moisture ratios and different storage times [23,29,32–46]. Sometimes, the annealing process is conducted as a
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Fig. 3. Pictorial representation of the effect of hydration and subsequent annealing on the semi-crystalline lamellae (amylopectin double helices are represented as rectangles): (a) dry starch with glassy amorphous regions; (b) hydrated annealed starch with rubbery amorphous regions (adapted from [18]).
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Fig. 4. Influence of water content on the differential scanning calorimetry thermograms profile of potato starch: (a) the onset ( ) and conclusion () gelatinisation temperatures (adapted from [21]); (b) the corresponding DSC thermograms (adapted from [19]).
single event (single-step) whilst at other times it is conducted as two starch – water/temperature/time events (double-step) or even many individual steps (multi-step) as discussed elsewhere [1]. This double or multi-step approach is often used to promote annealing without gelatinisation, and the double step process potentially produces higher gelatinisation temperatures than the single step process [1]. The lack of standardisation of annealing conditions makes it difficult to compare results between the different studies. The effect of the starch-to-water ratio, temperature and time on annealing of wheat starch has been investigated in detail [23]. This study demonstrated how critical the interrelationship of these parameters is. The annealing process could be initiated when the moisture content exceeded 20% by weight, (because Tg is around room temperature when this moisture content is exceeded, Fig. 1) but was restricted (in terms of its effect on increasing gelatinisation temperatures) unless it ex-
ceeded 60%. Although annealing could be initiated at 15°C below To by DSC, the effect was more marked the closer the annealing temperature was set to (below) To. Similar studies have been conducted on starches of different botanical origins [32,40] and demonstrate the additional complication of species specific variation. During annealing of starches, there are in essence two thermally driven processes which are intimately related and reflect the moisture content of the system — the elevation of Tg and gelatinisation temperatures (especially To). Low moisture causes elevation of (the relatively unplasticised) Tg of starches [6,7,22,47] and model polymeric systems [30,48,49] which, in the case of starch, intimately reflects the increase in gelatinisation temperatures. Indeed, the elevation of Tg implies a more glassy state and hence reorganisation of amorphous regions. This is associated with improved order of crystalline regions (below). The situation with respect to Tg of starch in food systems is very complex because
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Fig. 4. (Continued)
of the raft of potential interactions. More details concerning (general) glass transitions in model systems can be found elsewhere [50]. Similarly, sub-Tg transitions of starches (which probably represent enthalpy relaxation [51]) have been described by other authors [51,52].
4. Environmental considerations The effect of environmental temperature on starch synthesis and properties has been the subject of much recent research. Apart from direct growth temperature effects on the activity of enzymes involved in starch biosynthesis (which are discussed in some detail in a recent publication by these authors [53]), there is a distinct effect on starch physico-chemical properties. In general, for mature cereal and tuber starches [54–58] there tends to be a small effect on the fine structure (chain length distribution) of amylose or amylopectin. Granule size decreases as growth temperature increases, while amylose content remains approximately the same. In the case of lipid (lysophospholipids and free fatty acids) in cereal starches (only), there is a distinct temperature dependent increase. Growth temperature also causes a distinct increase in gelatinisation temperatures
of starches (which can also be modelled using potato microtubers [53]), and parallels have been drawn between laboratory based annealing processes (in vitro annealing) and environmentally driven reorganisations (in vivo ‘annealing’) of starch granule architecture [23,53,57,58]. Hence, apart from species and cultivar specific variation in starch physico-chemical properties, there is a profound environmental effect on gelatinisation characteristics. This effect is, in the experience of these authors, far greater than cultivar specific (and hence genetic) variation. The implications are, that product quality is not simply a cultivar specific trait — but is largely dependent on environmental conditions experienced during starch deposition.
5. Molecular basis for annealing It has been difficult to define, at the molecular level, what happens to the internal structure of starch granules when they are annealed. Some authors have discussed the molecular event in terms of increasing granule stability [32], reorganising granule structure [39,40] or lowering free energy [17]. These descriptive
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terms do not, however, give readers a clear molecular picture associated with the reorganisations involved within granules when they are annealed. A number of authors have discussed annealing with more emphasis on the crystalline and amorphous domains. Crystallinity and crystalline ‘perfection’ (optimisation of crystalline order) have been discussed in detail in this context [28,36,38,42,43,59]. Similarly, granular reorganisations have been discussed in terms of rigidity [33] and realignments and partial melting [45,60]. Others recognise the importance of interactions between, and mobility of, amorphous and crystalline regions [3,61] and the constituent amylose and amylopectin molecules [37]. Tester et al. [23] (working on wheat starches), have discussed annealing in the context of hydration and swelling of amorphous regions (temperature range between Tg and To), which facilitates ordering of double helices in crystalline regions. This ordering of double helices could be associated with minor optimisation of double helix length, although no additional double helical material is formed [23]. The amorphous material post-annealing probably becomes more ‘glassy’ (more rigid and less mobile) whereupon Tg is elevated. The constancy of double helix content pre- and post-annealing has also been shown by Jacobs [1] for a range of starches (pea, potato and wheat). With respect to the effects of annealing on the double helix content of starches, the situation in amylomaize is far more complex than for waxy or normal starches. In amylomaize starch there is evidence from NMR that amylose also forms some double helices and that upon annealing there is partitioning of amylopectin and amylose helical structures [62,63]. This different structural model within amylomaize starch granules helps to explain the characteristic gelatinisation characteristics of these starches (below). Whilst NMR can be used to quantify the number of double helices within starch granules, it does not measure crystallinity per se. For this purpose, wide angle X-ray scattering (WAXS) may be employed. Early work on annealing of wheat starch indicated that there was little detectable effect on the X-ray diffraction pattern [31]. This has been confirmed for potato starch [3]. However, other workers have reported a small increase in intensity of the diffraction pattern (but with little or no effect on d-line spacings) for wheat, oat, lentil [32,64] and barley starches [46] but with a decrease in intensity for potato starch [32]. It is very useful to link together both NMR and WAXS data for starches, as they measure different levels of order. One could, for example, have non-crystalline double helices (outside crystalline domains) within starch granules that give a strong NMR signal but not a WAXS diffraction pattern. The relative significance of these techniques for determining starch structure and order
during annealing and gelatinisation may be supported by the use of scanning electron microscopy [65], where dimensions of amorphous and crystalline lamellae may be estimated. Overall, the NMR and WAXS data support the same general picture that annealing causes no significant increase in crystalline material formed within starch granules by either of two possible mechanisms: (i) formation of double helices (which need not necessarily be associated with existing crystalline domains) or; (ii) major increase in amount of crystallinity as a consequence of ordering of previously amorphous regions. Rather, the enhanced ordering of double helices, due to improved registration (alignment), with associated increased rigidity of amorphous regions, probably underlies the annealing process. Unlike WAXS which quantifies crystalline order throughout starch granules, small angle X-ray scattering (SAXS) quantifies differences (periodicity) at the level of amorphous-crystalline lamellae radiating from the hilum to the periphery of starch granules. Using this technique, Jacobs et al. [36] showed that (for wheat and potato starches) the repeat distances of the crystalline and amorphous lamellae remain unchanged (10.5 nm in wheat and 9.9 nm in potato), although there was an increase in peak intensity. A pictorial representation of the length scales within starch granules together with techniques used for their quantification are presented in Fig. 5. More detailed discussion regarding the application of this technique to understand structural, gelatinisation and annealing mechanisms of starches can be found elsewhere [65]. Those authors, however, reported that the lamellar repeat distance for wheat starch is smaller (8.85 nm) than the figures quoted by Jacobs et al. Native cereal starch granules contain amylose–lipid complexes, as shown by NMR [67,68]. The lipid (lysophospholipid or free fatty acid) is immobilised within the a-glucan helices and the corresponding lack of mobility of methylene groups can be determined using this technique. The biochemical significance of these complexes is uncertain, although they have a significant effect on starch functionality. Whilst in model amylose–lipid complex systems annealing can be induced [69] because of the relative ease of mobility, annealing between Tg of the starch and To of amylopectin in wheat starch has little effect on this amylose–lipid endotherm [34,35]. The effect of annealing at these temperatures (e.g. 35–50°C) on amylose–lipid complexes is predictably very unlikely, because the temperature is far too low. The peak transition temperature of these complexes is of the order of 95–115°C. At temperatures where complexes have been annealed (for example 80°C, [69]) the starch would be fully gelatinised in unlimiting water. Probably Tg for the complexes under these conditions is quite close to this
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Fig. 5. Pictorial representation of the length scales within the starch granule together with techniques used to characterise the structural features (adapted from [66]).
temperature, although it is perhaps as mobile as Tg for amylopectin as a function of moisture content [23]. There is a relationship between the amount of starch phosphorylation and elevation of gelatinisation temperatures as a consequence of annealing [70]. The shift in Tp (or DTp) is greater for starches with low levels of phosphorylation. The authors [70] proposed that this is because the number of potential dislocations is smaller. In other words, phosphate moieties restrict double helix (and consequently crystallite) formation. The higher the phosphate content, the greater the interference. Because of the detrimental effect of phosphate groups on crystallite formation, however, the increase in enthalpy is largest for the high phosphate starches indicating that
steric hindrance is diminished as a consequence of the increased mobility during annealing. Chemically introduced phosphate groups have similar effects to naturally (during biosynthesis) inserted groups [71]. This should be viewed in the context of phosphate esters affecting the crystallinity of native starches [72], where the gelatinisation enthalpy is inversely related to the phosphate content.
6. Physical consequences of annealing According to some authors [32], annealing causes no effect on granule dimensions or shapes, although early
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microscopic work indicated that wheat starch granule dimensions increase after annealing [31]. Clearly, however, small differences in size cannot be accurately quantified using microscopy and care should be placed on reliance upon this data. The A-type diffraction pattern of wheat starch is retained after annealing [31], although the line intensity may increase as discussed above. Although heat moisture treatments causes a Bto A-type transition for potato starches, this does not happen during annealing [3]. Hence, the molecular reorientation is more subtle. The effect of annealing on gelatinisation characteristics is well established, particularly using DSC, where there tends to be an increase in To and Tp, decrease in the gelatinisation range (Tc – To) and either constancy or an increase in gelatinisation enthalpy [3,23,28,31,32,34,37 – 41,43,46,64,70,71,73]. The increase in gelatinisation temperatures is associated with a decrease in swelling power [23,32,41,46,73], provided that some granular structure is retained. This is reflected in a higher temperature onset of swelling and reduced swollen volume (below circa 90°C, provided that water is not limiting). The effects of annealing on pasting characteristics are complex. In some studies the consistency (viscosity) of annealed starches (wheat and potato) increases (with associated decrease of peak viscosity for potato starch) while for lentil and oat it tends to decrease [32]. Similar results have been reported for wheat and potato starch, with annealed pea and rice starches exhibiting increased viscosity [3,33,41]. Using the model proposed by these authors and co-workers [23], the physical properties discussed above can be explained on the basis of more glassy amorphous regions within annealed starch granules and a more ordered registration of amylopectin double helices. These molecular events restrict ease of hydration of the starch granules during gelatinisation and elevate gelatinisation temperatures. In parallel, these events restrict swelling. It is difficult to unravel the effects on pasting characteristics, because this system will be strongly influenced by granule size and polysaccharide solubilisation — more so than gelatinisation (by DSC) and swelling power determinations. This is probably why there is a lack of consistency in response to annealing for starches from different botanical origins.
7. Solubility The annealing process itself leads to little solubilisation of a-glucan [23]. This is important as it shows that improved order is a genuine molecular event rather than a consequence of leaching amorphous a-glucan and hence ‘concentrating’ crystalline material. Annealing reduces solubilisation of a-glucan during swelling below 100°C [32,41,46,73]. As leachate is primarily
(amorphous) lipid free amylose (FAM) according to the definition of Morrison et al. [68], the amylose must be more restricted from leaching out of the granules. Although at a given temperature post-annealing, the granules will swell less than un-annealed starches and this will be the primary restraint to leaching. This does, however, strengthen the view that there is molecular reorientation in the starch granule which makes the amorphous material more glassy with an elevated Tg.
8. Chemical hydrolysis Annealing tends to reduce the amount of acid hydrolysis of starch granules, although small granules sometimes exhibit little difference or even enhanced hydrolysis [32,34]. This discrepancy has to some extent been resolved by Tester et al. [23] who investigated acid hydrolysis for native and annealed wheat starch as a function of time. They found that for the first phase of acid hydrolysis (0–7 days, representing amorphous material hydrolysis) annealed starch was more extensively hydrolysed than native starch. After 7 days, where crystalline material is progressively hydrolysed, the extent of hydrolysis for the native and annealed starches was essentially the same. The explanation for the enhanced hydrolysis of amorphous regions after annealing is that the amorphous regions become more concentrated due to the enhanced glassy structure. On the other hand, the similarity in hydrolysis pattern during the crystalline hydrolysis phase (\ 7 days) confirms that it is double helices (which remain constant) which are the primary contributor to the hydrolysis profile during this phase. Amylose–lipid complexes may affect the pattern of acid hydrolysis in cereal starches, which could also influence enzymic hydrolysis (below).
9. Enzymatic hydrolysis Certain studies have indicated that annealed wheat, barley and sago starches are more easily hydrolysed by a-amylases than native starches [31,46,74]. This has, however, been contradicted by other research on wheat, lentil and potato starches [32,41], although small starch granules (oat) have been reported to be much more easily hydrolysed post-annealing [32]. However, the rate of a-amylase hydrolysis for different starches follows two distinct phases: an initial rapid then subsequent slow phase. Annealing alters the extent of hydrolysis of these different phases as a function of botanical origin [35]. During the second phase of hydrolysis, annealed wheat and pea starches are more resistant to a-amylase hydrolysis whilst the inverse is true for potato starch [35]. Annealed potato starch is less easily hydrolysed by
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amyloglucosidase than native starch [41]. In common with the statements above regarding acid hydrolysis patterns of starches, reported differences in a-amylase hydrolysis patterns may represent unjustified comparisons between different phases of hydrolysis (amorphous and crystalline material) rather than necessarily starch specific responses to enzyme hydrolysis post annealing. Although, the botanical origin of starch is important with respect to hydrolytic pattern, the surface area to volume ratio is probably of more significance than the actual plant source. It is also possible that the annealing process creates pores or fissures which alter the pattern of amylase hydrolysis from surface to internal erosion [74]. Hence, although amorphous and crystalline lamellae become more ordered, accessibility to the amorphous regions by enzymes is facilitated. Annealing can be conducted in the presence of amylases to selectively hydrolyse amorphous regions and the possibility of novel products with unique gelatinisation and swelling characteristics [75,76]. Both potential starch and glucose syrup products are possible using this general approach.
10. Overview Much data has been published on annealing of starches. Whilst the molecular basis is not absolutely certain, these authors believe that the physico-chemical data are consistent with enhanced registration of amylopectin double helices within crystalline zones (with perhaps some slight enhancement of helical length) and greater rigidity (more glassy) of amorphous regions. The consequence is that Tg and gelatinisation temperatures are increased, swelling is decreased and leaching is restricted. The situation in high amylose starches is different from waxy and normal starches where annealing facilitates partitioning of amylose and amylopectin double helices. Annealed starch provides a very useful model to investigate amylopectin crystallisation. Whilst a very simple technique, it provides a great deal of information about this process. There are important industrial implications of the annealing process with relevance to starch extraction and food production. As more sophisticated physical techniques are developed, the molecular basis will be understood in far more detail.
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