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amylopectin content studied by differential scanning calorimetry
Journal o/Cereal Science 6 (1987) 147-158
The Ageing of Gels from Starches of Different Amylose/ Amylopectin Content Studied by Differential Scanning Calorimetry PETER L. RUSSELL Flour Milling and Baking Research Association, Chorleywood, Rickmansworth, Herts WD3 5SH, U.K. Received 6 October 1986
Differential scanning calorimetry (DSC) has been used to study the ageing of gels made from starches with different amylopectin contents (at 57% moisture content, vI 0'67). All the gels exhibited development of a •staling endotherm'S over a similar temperature range. The data were analysed by fitting an Avrami equation to each data set using the method of non-linear least squares. All the data sets were also analysed together employing different models, in which any or all of the Avrami parameters (limiting value A L , rate constant k or Avrami exponent n) were made identical for each starch. DSC results fitted best a model with n < I: however, the rankings of A L and k were little affected by different models. Rate constants were unrelated to amylopectin content; that for wheat was smallest. A L values were proportional to starch amylopectin content, identifying the S endotherm with reo ordering of the amylopectin. The results are consistent with the presence of amylopectin, amylose-lipid complexes and amylose in different domains within starch granules, as indicated by recent ultrastructural studies. Only weak coupling exists between phenomena occurring in different domains. DSC of ageing starch gels is sensitive to changes in the amylopectin domains: a comparison is made between DSC and some other physical techniques.
Introduction Because of its utility in the study of order ¢ disorder transitions, DSC has been applied to the study of both the disordering phenomena associated with starch gelatinisation 1 and the reordering during ageing of the subsequent gels H or the starch component of baked products, e.g. breadS-H. During ageing at 21°C, the development of an endotherm (the 'staling endotherm'S) was observed over a time scale of·~ 10 days 2-s. This time scale was similar to that for the development of crystallinity in bread under similar conditions 9 • For this and other reasons previously reviewed s, it was concluded that the process observed by DSC was related to redevelopment of crystallinity. Two features of starch behaviour during ageing are of particular interest: the identification of the species responsible for the S endotherm, and the exact relationship between the size of the S endotherm and crystallinity. Abbreviations used: DSC = differential scanning calorimetry; DTA = differential thermal analysis; GMS = glyceryl monostearate; CP/MAS 13C NMR = cross polarisation/magic angle spinning nuclear magnetic resonance.
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P. L. RUSSELL
Various lines of evidence indicate that the amylopectin component of starch is responsible for the crystallinity of native starch l . If ge1atinisation involved the destruction of amylopectin crystallites then the development of the S endotherm during ageing might reasonably be expected to be associated with the return of order within this fraction. Support for this has come from the observation that when separated amylopectin was subjected to a number of freeze-thaw cycles lO , or heated in water and then allowed to stand for 7 days at 21 °C ll , or 14 days at 25 °C l2 , an endotherm appeared centred at ~ 55 °e, a position similar to that of the S endotherm. However, to our knowledge no study has been made of the ageing of gels from whole starches of different amylopectin contents. The inferred association between the S endotherm and amylopectin, and between amylopectin content and crystallinity might be interpreted to mean that DSC provided a direct measure of crystallinity 2-8.ll.l2; it has recently become apparent that this is too simple an assumption. Thus, development of crystallinity (from X-ray diffraction measurements) with time for a 10 % wheat starch gel was shown to occur in a biphasic manner lB • One component, complete within ~ 1-2 days, was assigned to crystallisation in the amylose fraction of the starch. A second component with a time-scale of ~ 10 days (in agreement with previous work 9 ) was also observed: this was assigned to crystallisation in the amylopectin fraction lB • On subsequent heating to 95°C, only that part of the crystallinity due to amylopectin was thermally reversible and was lost, while some (due to amylose) remained lB. This indicates that identification of the crystallinity of starch gels during ageing solely with the amylopectin fraction is erroneous. The purpose of the work described in this paper was to use DSC to study the ageing of gels (at the same moisture content) from starches of differential botanical origin with different amylose/amylopectin contents. The starches chosen were amylomaize, wheat, potato and waxy maize, whose gelatinisation has been described in the accompanying paper. The aims were to study the kinetics of ageing of the gels, to establish whether or not S endotherm development occurred in each case, and whether or not any relationship existed between limiting S endotherm size and either amylose/amylopectin content, or gelatinisation endotherm size l •
Experimental Materials and methods not specifically referred to here have been described in the accompanying paperl .
Methods Preparation and characterisation of starch gels. Gels were made from all the starches at a water content of 57 % (100 x weight of water/total weight of dry starch + water), i.e. VI 0·67. Gels from wheat, waxy maize and potato were made by placing ~ 20 gof the starch-water mixture (containing 0'1 % w/w calcium propionate as a mould inhibitor) into a small polyethylene bag (105 x 165 mm), which was placed between two thin aluminium plates and sealed. The assembly was placed in a water bath at 65°C for 5 min, removed, and placed in a dry heating block at 97 °C for a further 5 min. Gels obtained by this two-step procedure did not show opaque regions, and were essentially homogeneous, Le. variation in moisture content throughout the gel slab was found to be 2% or less. Because the (G+Ml) and (M2) endotherms for amylomaize starch overlap! a higher temperature, one-step method was adopted for gel preparation, to ensure complete gela-
STARCH GEL AGEING KINETICS AND AMYLOPECTIN
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tinisation. Some of the starch-water mixture (~ 10 g) was placed between two circular sheets of MXT (transparent moisture-proof polyvinylidene chloride) film in a cylindrical aluminium cell (cavity internal diameter 5 em, depth 0·25 em), which was sealed. The closed cell was placed in an oven (130°C, 15 min), and allowed to cool to room temperature, to give a satisfactory gel. The pH was measured ofdispersions obtained by homogenising samples (- 0·5 g) of each gel with water (5 ml). A portion of each gel was also examined microscopically in either brightfield or polarisation modes. The use of DSC to study the ageing ofstarch gels at 57 % water. Samples (10 mg) of the gels (57 % water, vl 0'67) were weighed into Du Pont hermetic DSC pans which were sealed. Because even correctly prepared pans show some moisture loss on prolonged storage, they were stored over a piece of the gel (to inhibit this) in bottles that were maintained at a temperature of 21°C in a water bath. The samples were scanned at intervals using the Perkin-Elmer DSC-2 and the area of the 8 endotherm measured; zero time was taken as the time of removal from the oven. Endotherm onset (T~), peak (T,,) and termination (T~) temperatures have been previously defined l . Preliminary experiments had demonstrated that the progress curve was rapid at first; for this reason scans were made at frequent intervals (i.e. more or less continuously) for ~ 24 h after making the gels, and then much less frequently up to 65 days. Representative pans were reweighed after - 3 weeks storage which revealed a mean loss of weight of - 2'5 %. As in previous paperss-s, DSC data from each gel were first analysed separately by fitting an Avrami equation:
(1)
to each data set, where
Results and Discussion Characterisation of starch gels
As expected, X-ray diffraction measurements of all the gels (data not shown) showed that a B-pattern developed during ageing, irrespective of the pattern given by the native starch l , consistent with the conclusions of early workers l4 ,l5. The fact that the gels from different starches all exhibited a similar pattern implied that, regardless of the structure of the starches before gelatinisation, the same factors operated on all the gels during ageing and that comparison of gel-ageing kinetics by DSC should be meaningful. The pH values of the gels from waxy maize, potato, wheat and amylomaize starches were 6'12, 6·29, 5'74 and 5·69 respectively. Examination by DSC of the ageing of starch gels
Some representative thermograms during the course of ageing of all four of the starch gels were shown in Fig. 1. Gels from each starch exhibited the development of S endotherms with time: mean T p was 61'5 DC, and all the values for Tp were within a range of 6·4 dc. The similarity in temperature ranges (Table I) could be accounted for if the endotherm originated from a fraction common to each starch. This S endothenn temperature range was similar to those observed by previous workers during the ageing of wheat starch gels 4 (Tp 63'5 0c) or breads-so S endotherm enthalpy, after a given time, was much larger for waxy maize starch than for amylomaize starch, while wheat and
P. L. RUSSELL
150
(b)
(a)
~::: ~550h I
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I
I
I
I
(d)
~2'8h
~24h 10
30
50
70 90
~"oo,
10
110 130
30
50
70 90
110 130
Temperature (oG)
FIGURE 1. DSe thermograms for gels (containing 57 % water, VI 0,67) from (a) arnylomaize; (b) wheat; (c) waxy maize and (d) potato starches, at different times during ageing at 21°C. The numbers correspond to the ageing times (h) after gelatinisation. For clarity the curves are displaced relative to each other along the Y axis; the bar corresponds to 0·30 Jre g sample (including water). TABLE I. Thermal data from DSe scans of starch gels during ageing Staling endotherm S Temperatures ("q" Starch Waxy maize Potato Wheat Amylomaize
T'0
~
38'3 (0'5) 46·8 (0'4) 44·7 (0-2) 47-8 (0'6)
60·0 (0'3) 65·5 (0'4) 59·1
(0'5)
61·3 (0'6)
• For definition of parameters see text; figures in brackets are VI
0'67).
S.E.M.
r;n 75·1 (0'4) 85'5 (0'3) 72'7 (1'4) 71'5 (0-9) Starch-water 43: 57 (w/w, dry basis,
STARCH GEL AGEING KINETICS AND AMYLOPECTIN
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potato starches gave intermediate values (Fig. 1), suggesting that the amylopectin fraction is responsible for the S endotherm. Kinetics of ageing of starch gels DSC has been applied previously in the measurement of changes in the starch fraction of the crumb of bread during ageing and a non-linear least squares procedure has been used to model the experimental data by fitting an Avrami equation 6 - s. This equation has been applied to the crystallisation kinetics of high polymers: under these circumstances the value of n depends upon crystal shape and the time dependence of the nucleation process. It has been pointed out that direct visual observation of crystal morphology should be made before attempting to interpret n values in terms of crystal shape, etc. 6.16. For starch, the situation is potentially more complicated still than for some polymers, since starch granules themselves are not homogeneous, but possess a definite ultrastructure. For these reasons, as in other papers 6 - S , the Avrami equation has been used as a convenient means of fitting data, and of comparing the fits of different data sets with one another. In this context, it serves a purpose: no conclusions have been drawn regarding morphology. Figure 2 presents plots of S endotherm enthalpy vs. storage time at 21 DC for all four starches; it is clear that substantial differences exist between these different starches. The 7·5
(b)
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4·5
•
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•
•
['5~
Io
o
o
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1
• 3
400
800
1200
1600
o Time (h)
FIGURE 2. Plots of the enthalpy of the 'staling' endotherm, S vs. storage time at 21°C for gels (57 % water, VI 0'67) from (a) amylomaize; (b) wheat; (c) waxy maize and (d) potato starches. Curves were fitted as described in the text; numbers correspond to the appropriate model number in Table II.
u.
tv
TABLE II. Calculated kinetic parametersa from DSC measurements of gels made from different starches during ageing Limiting enthalpyb of staling endotherm, S A L (Jig gel) Model
1. Separate fits, free n, A L, k 2. Common n, free AL,k
3. Common n = 1 free A L, k
Rate constant" k x lOs (h- n )
Avrami exponent n
WM
POT
WH
AM
WM
POT
WH
AM
WM
POT
WH
AM
5·8
4·4
3-9
0·49
0·62
0·54
0·75
0·74
128
248
19
54
5·8
4·3
4·4
0'50
4
• •
127
208 (7'2) 86
29
75 (2'6) 24 (4'0)
5'5
4·2
3·7
0'48
[12-8] 90%
[9'8] 63%
[8'6] 88%
[1'1] I7%d
..
0·62 ]
(4'4)
46 (7'7)
(14'3)
(1)
6
(1)
• For definitions of parameters and a discussion of statistical significance, see text; figures in brackets are rate constants relative to those of wheat. Starch-water 43:57 (w/w, dry basis); divide by 0·43 to obtain Jig of dry starch, for example, figures in brackets [} for model 3. " Units of k depend on the value of the Avrami exponent, n; figures in parentheses () are rate constants relative to those of wheat. d % refer to (100 x A,jg dry starch/6H1/g dry starch) for model 3. b
:-c
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::0
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(/)
tTl t"" t""
STARCH GEL AGEING KINETICS AND AMYLOPECTIN
153.
choice of the equations giving the lines of best fit to these data was made in a way similar to that described previouslyS-8, and is outlined below. Firstly, the DSC data for all four gels were fitted separately by Avrami equations (l). A spread of values was obtained for all three unknowns, A L> k and n; in each case an Avrami exponent of less than I was found, varying from 0'54 for potato starch to 0·75 for wheat starch (modell, Table II). Fitted curves obtained by using this model are plotted in Fig. 2 (a)-(d), curve 1. Previous DSC results from the ageing of wheat starch gels were interpreted with n < 14, while for bread it has also been observed that the data were best fitted by a model with n < I S-8. Making the assumption that n was identical for all four gels, data analysis led to a value of n == 0,62, different rate constants and slightly different limiting values (model 2, Table II). This model was not a significantly worse fit than the original one of four separate Avrami equations so that, allowing for the reduction in the number of parameters employed, it was a better choice than model 1. To avoid confusing Fig. 2 with too many lines, no fitted curves are given for this model: they would be very similar to those for model I. Analysing the data with n constrained to be 1, free A L and k (model 3, Table II) gave a significantly worse fit than either of the other models. Fitted curves obtained by using this model are also plotted (Fig. 2(a)-(d), curve 3). From inspection of Fig. 2, both models I and 3 gave reasonable fits to the data. From these plots it is not obvious that the model with an Avrami exponent of < 1 gives a better fit. However, replotting using a larger scale (not shown), indicated that in general the data at early times ( < 24 h) were fitted better using models I or 2 than model 3. Despite the worse fit of model 3, the estimated parameters are given in Table II for comparison with literature data (see below). In fact the estimated limiting values for models 2 and 3 are very similar, and the rate constants, although different, because their dimension varies with n, have the same ranking; kWh < k am < k wm < k pot ' The conclusions described below are therefore essentially independent of the model chosen; for ease of comparison with literature data, the following discussion will assume the data of model 3 (n = I), unless otherwise stated. Inspection of the data (model 3, Table II, Figs. I and 2) indicates that substantial differences in limiting endotherm enthalpies (A LS) occur for gels from these four starches: the order of ALs is amylomaize < wheat - potato < waxy maize. This order parallels the amylopectin content of the starches, suggesting that the S endotherm is associated with the development of order in the amylopectin fraction. A plot of A L vs. amylopectin (100- % amylose) content for these starches gives a reasonably good straight line (Fig. 3), giving support to this interpretation. Interestingly, based on extrapolation of this plot, A L apparently becomes zero at an amylose content of -76% (amylopectin -24%), implying that starches containing higher proportions of amylose than this would not show the development of any S endotherm. Possible reasons for this behaviour are outlined in a succeeding section. Previous workers have studied the kinetics of ageing of wheat starch gels at 21°C by DTA 2 or DSC4, and have found rate constants (k x 10 3 ) of 11·1 and J 1·6 h-\ at moisture contents of 50 and 53 % respectively. The value obtained in this present paper, for a wheat starch gel of 57 % moisture content, was - 6·0 h -1. Although no measurements have been reported of the dependence of k on moisture content for wheat starch gels, it appears unlikely that the difference in water content was sufficient to account for such a sub-
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100
10
OL..-J.----'-----l_-'-----'-..........J.--":J..:::.......!----L--l
100
80
60
40
20
a
Amylopectin content of storch (%)
FIGURE 3. Plots of (1) rate constant for S endotherm development (k) and (2) limiting endotherm enthalpy (AI) during the ageing of starch gels (n = 1, model 3, Table II), vs. amylopectin content of starches. Open symbols are for rate constants, closed symbols are for ALs. 0, • = amylomaize; 0, • = wheat; /::", .. = waxy maize and \7, ,.. = potato starch.
stantial difference in k. Another factor that could have an influence is the pH of the gels l ; previous studies have not specified pH 2 ,4. The work described here differs from previous work in that data were taken over a much longer period than that employed by previous workers, and it is a possibility therefore that this may have affected the results. To check on this, the data for wheat starch were reanalysed with n = 1, but omitting data obtained after 250 h; under these circumstances (k x 103 ) was found to be 15·7 h- 1 • This figure is even higher than the literature data; the substantial change in the figure indicates the sensitivity of the Avrami equation to the number of data points included, particularly in the early and late stages of the process. Much more data has been included in this study than in previous work 2 ,4; furthermore, the same method of analysis has been applied to all the starches so that it should be possible to compare rate constants even if, as seems unlikely, their absolute magnitudes were in error. Striking differences exist between the rate constants for the different starches, those for amylomaize, waxy maize and potato being respectively ,...,4, 8 and 14 times that for wheat starch (see Table II, Fig. 3). This order apparently bears no relationship to the amylopectin content of the starch. Those starches with either no lipid (potato) or very little amylose (waxy maize) exhibit the largest rate constants, while wheat and amylomaize starches, with both amylose and lipid, exhibit much smaller rate constants. These results suggest that one factor contributing towards low rate constants is the presence of amylose-lipid complexes in the starch gels. In this respect it is interesting that we have recently observed a reduction in the rate of development of the S endotherm in bread containing glyceryl monostearate (GMS), compared with control (untreated) breadS. Another factor that might influence rate constants is the pH of the gels; in general the rate constants are larger, the higher the pH. No attempt has been made to compare gels at equal pH since to do so would have necessitated studying the effect of ionic strength on the process, because buffering would be necessary.
STARCH GEL AGEING KINETICS AND AMYLOPECTIN
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Starch gel ageing: the influence of separate domains
In general, the results described here, together with other data from the literature, suggest that the thermal and some other properties of starch granules after gelatinisation may be explained on the assumption that the granules behave as parcels, each containing a mixture of separate domains (e.g. amylopectin, amylose-lipid complexes, amylose) that are broadly independent of each other. This seems reasonable, given the mounting evidence from ultrastructural and other studies 1 , that the structure of the granule itself may be understood in terms of a compartmentalisation of the different components. Although various physical changes or reorganisations are envisaged as occurring within and between these fractions during gelatinisation, when gelatinisation is complete, domains still exist within which order can reappear. As an example, although after gelatinisation the amylopectin crystallites may be disrupted, the amylopectin molecules remain in situ, and, hence, can reorder. Even after gelatinisation, the granules still remain as discrete entities, albeit deformed. The evidence in favour of this 'separate domain' hypothesis is outlined below for each fraction in turn. The development of the S endotherm in starch gels appears, from the present results, to be associated with the amylopectin fraction of these starches. The close similarity in S endotherm position, and the fact that amylopectin separated from starch granules gives an endotherm in a very similar position 10-12, suggests also that in each case the developing structure is similar and that the S endotherm is associated with the crystal~ lisation of amylopectin 'domains'. The time-scale of several days for S endotherm development is similar to that observed for growth of the long-term component of crystallinity (amylopectin) in 10 % wheat starch gels 13 • Limiting S endotherm size in starch gels also appears to be dependent upon moisture content during ageing, not during gelatinisation 12. A reduction in the radii of gyration of spherically-shaped regions (assigned to extended amylopectin clusters) during ageing of gelatinised starch-water mixtures is also apparently associated with reorganisation of the amylopectin fraction!? Taken together, these observations suggest that as a result of ge1atinisation, the amylopectin domain is essentially decoupled from others, and the development of order in that domain then depends simply upon the amount of water present in the system. A second domain within the granules may consist of amylose-lipid complexes. Although the position of this fraction within starch granules has not yet been unambiguously identified, a possible location has been suggested, based on the results of small angle neutron scattering experiments 18 . These structures may be characterised by a number of physical techniques, including NMR and chiroptical methods 19 , and by thermal techniques where they lead to the M2 endotherm 19- 21 (see also the preceding paper 1 ). These X_ray 21 and thermaJl9-21 studies indicate that the ordered regions of amylose-lipid complexes are disrupted at a temperature different from that associated with gelatinisation (i.e. at 100-110 DC, see data for M2 endotherm in Table II, ref. 1) and show that the re-ordering of the material is rapid, i.e. that it occurs during cooling back to room temperature. The very different time scales for the restructing of amylopectin and amylose-lipid domains is itself evidence for the decoupling of these two separate fractions. The available evidence 1 suggests that although reorganisation (formation) of some amylose-lipid complexes may occur during gelatinisation, subsequent to this no further changes in M2 enthalpy occur during reheating!, or ageing ll •8 •
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Evidence for the presence and behaviour of the third domain i.e. amylose, also exists. Thus, the short term development of crystallinity has been assigned to the amylose fraction l3 • Contributions to the development of the shear modulus in dilute gels were identified both from amylose and amylopectin fractions with respectively short and long time scales. The faint birefringence observed in cooled, freshly-made amylomaize gels l may also reflect ordered regions of either amylose crystallites (or amylose-lipid complexes). Certainly this could not be due to ordered amylopectin regions, which have been shown to take much longer to re-order. Limited thermal evidence exists that may be relevant: thus we have observed an endotherm at ""' 160 ac in aged (""' I day, data not shown) amylomaize starch-water mixtures. Other workers have shown that amylosewater mixtures give an endotherm at a similar temperature lO . Despite the broad conclusion that independent domains exist to some extent in starch granules and in ageing gels, it also appears likely that a degree of co-operativity exists between these domains. For example, the observed reduction in the rate of development of the S endotherm due to addition of GMS to bread could be accounted for if some limited co-crystallisation of amylose and amylopectin occurred, so that the S endotherm included some contribution from the amylose fraction. Added OMS could complex with some of the amylose during gelatinisation, thus making it unavailable to participate in co-crystallisation during gel ageing. It is interesting that other workers have concluded that the mechanism by which GMS retards staling is not due to an alteration in moisture availability during starch retrogradation, as a result of surfactant addition 12 • Another observation indicating that the 'separate domain' hypothesis may be too simple is that although good linearity between limiting S endotherm enthalpy A L and starch amylopectin content exists (Fig. 3), this plot gives at} A L of zero at an (extrapolated) amylopectin content of ...... 24 %, not zero as might be expected. One interpretation is that'" 24 % of the amylopectin is distributed in the granules in a way different from the remainder. An alternative is that the reduction in A L with increasing amylose content results from a progressive disruption of amylopectin crystallites by interaction with material in other domains; until at 24 % amylopectin so much disruption of the amylopectin domains has occurred that their crystal forming capacity is destroyed. The information currently available does not allow us to distinguish between these possibilities. Starch gel ageing: relationship between DSC and other physical methods
A feature of interest in the results was the relationship between gelatinisation (G+ MI) and limiting S endotherm sizes (ALs) for these starches (Table II). Thus, for waxy maize and wheat starches, ALs were 90 and 88 % respectively of the original G+ Ml endotherms. In contrast, for potato starch A L represented only 63 %, and for amylomaize starch only 17 % of G endotherm size. The reason for this is not understood. However, potato starch has an unexpectedly high gelatinisation enthalpyl. In addition, based on cross polarisation/magic-angle spinning (CP/MAS) l3C NMR, potato starch possesses 50 % 'double-helix content' and only 25 % crystallinity (X-ray), while amylomaize starch had 38 % double-helix and again only 25 % crystallinity 22. The difference between the two measurements was taken as an indication of the amount of' short-range order';
STARCH GEL AGEING KINETICS AND AMYLOPECTIN
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i.e. that material that was present in the form of double helices, but that was not part of a crystalline array. This material with short-range order may have contributed to the (G + M I) endotherm, but not to the S endotherm. One possibility is that the short-range order originated in amorphous regions of the amylose and that on ageing after gelatinisation it became incorporated into amylose crystallites. It is noteworthy that both these starches possess B-type X-ray diffraction patterns\ unlike the other starches described in the present paper. It remains a possibility, therefore, that the excess doublehelix content is related to this. Whatever the reason, it is clear that X-ray diffraction and CPjMAS 13C NMR techniques do not measure the same fraction of native starches 22 • It is equally clear that DSC and X-ray methods are not sensitive to the same fractions of ageing starch gels 13 . Thus, DSC is evidently sensitive to the amylopectin fraction of the gels (and perhaps a small fraction of the amylose that co-crystallises with amylopectin domains) and not to the major proportion of the amylose. In contrast, X-ray diffraction gives a measure of crystallinity in both amylopectin and amylose combined. It follows that the assumption, either implicit or explicit in much previous work, that both X-ray diffraction and DSC measure overall starch crystallinity, is incorrect. A more appropriate model for starch is that, while X-ray diffraction measurements may measure total starch crystallinity, DSC (S endotherm) responds to the amylopectin fraction only. Application of the Avrami equation to the DSC results in effect applies it to the amylopectin fraction. This conclusion has testable implications, e.g. another DSC endotherm should also develop in ageing starch gels due to the reordering of the amylose fraction. One candidate is the endotherm at ~ 160°C observed in aged amylomaize gels or amylose. Because of the relatively high temperature of this endotherm, pressurised DSC would presumably be needed to explore this, to prevent the pans from leaking before the endotherm was reached. Conclusions
The behaviour of starch granules during gelatinisation and ageing can be accounted for in terms of a model involving separate domains of amylopectin, amylose-lipid complexes and amylose. During gelatinisation, some fractionation and interaction of these domains occurs. After gelatinisation, although some coupling occurs, the properties of the system are largely accounted for by the separate domains. The author thanks Dr T. Fearn for the statistical analyses, Dr 1. D. Schofield for useful discussions, Mr D. J. Nicholls for technical assistance and Mr S. Webb for assistance with artwork. The author is grateful to Mr C. R. W. Liley of Hatfield Polytechnic for X-ray diffraction measurements. This work forms part of a research project sponsored by the U.K. Ministry of Agriculture, Fisheries and Food to whom our thanks are due. The results of the research are the property of the Ministry of Agriculture Fisheries and Food and are © Crown Copyright 1987.
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References I. RusselJ, P. 1. J. Cereal Sci. 6 (1987) 133-145 2. McIver, R. G., Axford, D. W. E., Colwell, K. H. and Elton, G. A. H. J. Sci. Food Agric. 19 (1968) 560-563. 3. Colwell, K. H., Axford, D. W. E., Chamberlain, N. and Elton, G. A. H. J. Sci. Food Agric. 20 (1969) 550-555, 4. Longton, J. and LeGrys, G. A. Starch 33 (1981) 410-414. 5. Fearn, T, and Russell, P. 1. J. Sci. Food Agric. 33 (1982) 537-548. 6. Russell, P. 1. Starch 35 (1983) 277-281. 7. Russell, P. 1. J. Cereal Sci. 1 (1983) 285-296. 8. Russell, P. 1. J. Cereal Sci. 1 (1983) 297-303. 9. Wright, W. B. (Lecture presented to the Food Group of the Chemical Society, London. Summary), Chern. Ind. (London) 22 (1971) 568-570. 10. Von Eberstein, K. Hopcke, R., Konieczny-Janda, G. and Stute, R. Starch 32 (1980) 397-404. II. Eliasson, A.-C. in 'Proceedings of the Conference on New Approaches to Research on Cereal Carbohydrates', (R. D. Hill and 1. Munck, eds.) Carlsberg Laboratory, Copenhagen, June 24-29 (1984), Elsevier, Amsterdam 1985, pp 93-98. 12. Zeleznak, K. J. and Hoseney, R. C. Cereal Chern. 63 (1986) 407-41 I. 13. Miles, M. J., Morris, V. J., Orford, P. D. and Ring, S. G. Carbohydr. Res. 135 (1985) 271-281. 14. Katz, J. R. and Van Hallie, T. B. Z. Phys. Chern. A. 150 (1930) 90-99. 15. Hellman, N. N., Fairchild, B. and Senti, F. R. Cereal Chern. 31 (1954) 495-505. 16. Wunderlich, B. in 'Macromolecular Physics " Academic Press, New York (1976) p 139, 17, Yang, M., Grider, J., Gordon, J. and Davis, E. A. Food Microstruct. 4 (1985) 107-114. 18, Blanshard, J. M. V., Bates, D. R., Muhr, A. H. and Higgins, J. S. Carbohydr. Po/ym. 4 (1984) 427-442. 19. Bulpin, P. V., Welsh, E. J. and Morris, E. R. Starch 34 (1982) 335-339. 20. Donovan, J. W. and Mapes, C. J. Starch 32 (1980) 190-193. 21. Kugimiya, M., Donovan, J. W. and Wong, R. Y. Starch 32 (1980) 265-270. 22. Gidley, M. J. and Bociek, S. M. J. Am. Chern, Soc. 107 (1985) 7040-7044.
The Ageing of Gels from Starches of Different Amylose/ Amylopectin Content Studied by Differential Scanning Calorimetry PETER L. RUSSELL Flour Milling and Baking Research Association, Chorleywood, Rickmansworth, Herts WD3 5SH, U.K. Received 6 October 1986
Differential scanning calorimetry (DSC) has been used to study the ageing of gels made from starches with different amylopectin contents (at 57% moisture content, vI 0'67). All the gels exhibited development of a •staling endotherm'S over a similar temperature range. The data were analysed by fitting an Avrami equation to each data set using the method of non-linear least squares. All the data sets were also analysed together employing different models, in which any or all of the Avrami parameters (limiting value A L , rate constant k or Avrami exponent n) were made identical for each starch. DSC results fitted best a model with n < I: however, the rankings of A L and k were little affected by different models. Rate constants were unrelated to amylopectin content; that for wheat was smallest. A L values were proportional to starch amylopectin content, identifying the S endotherm with reo ordering of the amylopectin. The results are consistent with the presence of amylopectin, amylose-lipid complexes and amylose in different domains within starch granules, as indicated by recent ultrastructural studies. Only weak coupling exists between phenomena occurring in different domains. DSC of ageing starch gels is sensitive to changes in the amylopectin domains: a comparison is made between DSC and some other physical techniques.
Introduction Because of its utility in the study of order ¢ disorder transitions, DSC has been applied to the study of both the disordering phenomena associated with starch gelatinisation 1 and the reordering during ageing of the subsequent gels H or the starch component of baked products, e.g. breadS-H. During ageing at 21°C, the development of an endotherm (the 'staling endotherm'S) was observed over a time scale of·~ 10 days 2-s. This time scale was similar to that for the development of crystallinity in bread under similar conditions 9 • For this and other reasons previously reviewed s, it was concluded that the process observed by DSC was related to redevelopment of crystallinity. Two features of starch behaviour during ageing are of particular interest: the identification of the species responsible for the S endotherm, and the exact relationship between the size of the S endotherm and crystallinity. Abbreviations used: DSC = differential scanning calorimetry; DTA = differential thermal analysis; GMS = glyceryl monostearate; CP/MAS 13C NMR = cross polarisation/magic angle spinning nuclear magnetic resonance.
148
P. L. RUSSELL
Various lines of evidence indicate that the amylopectin component of starch is responsible for the crystallinity of native starch l . If ge1atinisation involved the destruction of amylopectin crystallites then the development of the S endotherm during ageing might reasonably be expected to be associated with the return of order within this fraction. Support for this has come from the observation that when separated amylopectin was subjected to a number of freeze-thaw cycles lO , or heated in water and then allowed to stand for 7 days at 21 °C ll , or 14 days at 25 °C l2 , an endotherm appeared centred at ~ 55 °e, a position similar to that of the S endotherm. However, to our knowledge no study has been made of the ageing of gels from whole starches of different amylopectin contents. The inferred association between the S endotherm and amylopectin, and between amylopectin content and crystallinity might be interpreted to mean that DSC provided a direct measure of crystallinity 2-8.ll.l2; it has recently become apparent that this is too simple an assumption. Thus, development of crystallinity (from X-ray diffraction measurements) with time for a 10 % wheat starch gel was shown to occur in a biphasic manner lB • One component, complete within ~ 1-2 days, was assigned to crystallisation in the amylose fraction of the starch. A second component with a time-scale of ~ 10 days (in agreement with previous work 9 ) was also observed: this was assigned to crystallisation in the amylopectin fraction lB • On subsequent heating to 95°C, only that part of the crystallinity due to amylopectin was thermally reversible and was lost, while some (due to amylose) remained lB. This indicates that identification of the crystallinity of starch gels during ageing solely with the amylopectin fraction is erroneous. The purpose of the work described in this paper was to use DSC to study the ageing of gels (at the same moisture content) from starches of differential botanical origin with different amylose/amylopectin contents. The starches chosen were amylomaize, wheat, potato and waxy maize, whose gelatinisation has been described in the accompanying paper. The aims were to study the kinetics of ageing of the gels, to establish whether or not S endotherm development occurred in each case, and whether or not any relationship existed between limiting S endotherm size and either amylose/amylopectin content, or gelatinisation endotherm size l •
Experimental Materials and methods not specifically referred to here have been described in the accompanying paperl .
Methods Preparation and characterisation of starch gels. Gels were made from all the starches at a water content of 57 % (100 x weight of water/total weight of dry starch + water), i.e. VI 0·67. Gels from wheat, waxy maize and potato were made by placing ~ 20 gof the starch-water mixture (containing 0'1 % w/w calcium propionate as a mould inhibitor) into a small polyethylene bag (105 x 165 mm), which was placed between two thin aluminium plates and sealed. The assembly was placed in a water bath at 65°C for 5 min, removed, and placed in a dry heating block at 97 °C for a further 5 min. Gels obtained by this two-step procedure did not show opaque regions, and were essentially homogeneous, Le. variation in moisture content throughout the gel slab was found to be 2% or less. Because the (G+Ml) and (M2) endotherms for amylomaize starch overlap! a higher temperature, one-step method was adopted for gel preparation, to ensure complete gela-
STARCH GEL AGEING KINETICS AND AMYLOPECTIN
149
tinisation. Some of the starch-water mixture (~ 10 g) was placed between two circular sheets of MXT (transparent moisture-proof polyvinylidene chloride) film in a cylindrical aluminium cell (cavity internal diameter 5 em, depth 0·25 em), which was sealed. The closed cell was placed in an oven (130°C, 15 min), and allowed to cool to room temperature, to give a satisfactory gel. The pH was measured ofdispersions obtained by homogenising samples (- 0·5 g) of each gel with water (5 ml). A portion of each gel was also examined microscopically in either brightfield or polarisation modes. The use of DSC to study the ageing ofstarch gels at 57 % water. Samples (10 mg) of the gels (57 % water, vl 0'67) were weighed into Du Pont hermetic DSC pans which were sealed. Because even correctly prepared pans show some moisture loss on prolonged storage, they were stored over a piece of the gel (to inhibit this) in bottles that were maintained at a temperature of 21°C in a water bath. The samples were scanned at intervals using the Perkin-Elmer DSC-2 and the area of the 8 endotherm measured; zero time was taken as the time of removal from the oven. Endotherm onset (T~), peak (T,,) and termination (T~) temperatures have been previously defined l . Preliminary experiments had demonstrated that the progress curve was rapid at first; for this reason scans were made at frequent intervals (i.e. more or less continuously) for ~ 24 h after making the gels, and then much less frequently up to 65 days. Representative pans were reweighed after - 3 weeks storage which revealed a mean loss of weight of - 2'5 %. As in previous paperss-s, DSC data from each gel were first analysed separately by fitting an Avrami equation:
(1)
to each data set, where
Results and Discussion Characterisation of starch gels
As expected, X-ray diffraction measurements of all the gels (data not shown) showed that a B-pattern developed during ageing, irrespective of the pattern given by the native starch l , consistent with the conclusions of early workers l4 ,l5. The fact that the gels from different starches all exhibited a similar pattern implied that, regardless of the structure of the starches before gelatinisation, the same factors operated on all the gels during ageing and that comparison of gel-ageing kinetics by DSC should be meaningful. The pH values of the gels from waxy maize, potato, wheat and amylomaize starches were 6'12, 6·29, 5'74 and 5·69 respectively. Examination by DSC of the ageing of starch gels
Some representative thermograms during the course of ageing of all four of the starch gels were shown in Fig. 1. Gels from each starch exhibited the development of S endotherms with time: mean T p was 61'5 DC, and all the values for Tp were within a range of 6·4 dc. The similarity in temperature ranges (Table I) could be accounted for if the endotherm originated from a fraction common to each starch. This S endothenn temperature range was similar to those observed by previous workers during the ageing of wheat starch gels 4 (Tp 63'5 0c) or breads-so S endotherm enthalpy, after a given time, was much larger for waxy maize starch than for amylomaize starch, while wheat and
P. L. RUSSELL
150
(b)
(a)
~::: ~550h I
\'
I
I
I
I
(d)
~2'8h
~24h 10
30
50
70 90
~"oo,
10
110 130
30
50
70 90
110 130
Temperature (oG)
FIGURE 1. DSe thermograms for gels (containing 57 % water, VI 0,67) from (a) arnylomaize; (b) wheat; (c) waxy maize and (d) potato starches, at different times during ageing at 21°C. The numbers correspond to the ageing times (h) after gelatinisation. For clarity the curves are displaced relative to each other along the Y axis; the bar corresponds to 0·30 Jre g sample (including water). TABLE I. Thermal data from DSe scans of starch gels during ageing Staling endotherm S Temperatures ("q" Starch Waxy maize Potato Wheat Amylomaize
T'0
~
38'3 (0'5) 46·8 (0'4) 44·7 (0-2) 47-8 (0'6)
60·0 (0'3) 65·5 (0'4) 59·1
(0'5)
61·3 (0'6)
• For definition of parameters see text; figures in brackets are VI
0'67).
S.E.M.
r;n 75·1 (0'4) 85'5 (0'3) 72'7 (1'4) 71'5 (0-9) Starch-water 43: 57 (w/w, dry basis,
STARCH GEL AGEING KINETICS AND AMYLOPECTIN
151
potato starches gave intermediate values (Fig. 1), suggesting that the amylopectin fraction is responsible for the S endotherm. Kinetics of ageing of starch gels DSC has been applied previously in the measurement of changes in the starch fraction of the crumb of bread during ageing and a non-linear least squares procedure has been used to model the experimental data by fitting an Avrami equation 6 - s. This equation has been applied to the crystallisation kinetics of high polymers: under these circumstances the value of n depends upon crystal shape and the time dependence of the nucleation process. It has been pointed out that direct visual observation of crystal morphology should be made before attempting to interpret n values in terms of crystal shape, etc. 6.16. For starch, the situation is potentially more complicated still than for some polymers, since starch granules themselves are not homogeneous, but possess a definite ultrastructure. For these reasons, as in other papers 6 - S , the Avrami equation has been used as a convenient means of fitting data, and of comparing the fits of different data sets with one another. In this context, it serves a purpose: no conclusions have been drawn regarding morphology. Figure 2 presents plots of S endotherm enthalpy vs. storage time at 21 DC for all four starches; it is clear that substantial differences exist between these different starches. The 7·5
(b)
(a)
6·0
• _-.===_=.=:::::;;......; I3
4·5 3·0
0. E 0
III
I
[·5
1 3 0,,0-0-
."
~.
i--'--'----'-....l..---..l....--JL-....L-.L---l
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"-
::2 >,
0-
"0
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7·5
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•
•~:=.• =====O=k
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4·5
•
3'0j
•
•
['5~
Io
o
o
•
1
• 3
400
800
1200
1600
o Time (h)
FIGURE 2. Plots of the enthalpy of the 'staling' endotherm, S vs. storage time at 21°C for gels (57 % water, VI 0'67) from (a) amylomaize; (b) wheat; (c) waxy maize and (d) potato starches. Curves were fitted as described in the text; numbers correspond to the appropriate model number in Table II.
u.
tv
TABLE II. Calculated kinetic parametersa from DSC measurements of gels made from different starches during ageing Limiting enthalpyb of staling endotherm, S A L (Jig gel) Model
1. Separate fits, free n, A L, k 2. Common n, free AL,k
3. Common n = 1 free A L, k
Rate constant" k x lOs (h- n )
Avrami exponent n
WM
POT
WH
AM
WM
POT
WH
AM
WM
POT
WH
AM
5·8
4·4
3-9
0·49
0·62
0·54
0·75
0·74
128
248
19
54
5·8
4·3
4·4
0'50
4
• •
127
208 (7'2) 86
29
75 (2'6) 24 (4'0)
5'5
4·2
3·7
0'48
[12-8] 90%
[9'8] 63%
[8'6] 88%
[1'1] I7%d
..
0·62 ]
(4'4)
46 (7'7)
(14'3)
(1)
6
(1)
• For definitions of parameters and a discussion of statistical significance, see text; figures in brackets are rate constants relative to those of wheat. Starch-water 43:57 (w/w, dry basis); divide by 0·43 to obtain Jig of dry starch, for example, figures in brackets [} for model 3. " Units of k depend on the value of the Avrami exponent, n; figures in parentheses () are rate constants relative to those of wheat. d % refer to (100 x A,jg dry starch/6H1/g dry starch) for model 3. b
:-c
r
::0
c: v.>
(/)
tTl t"" t""
STARCH GEL AGEING KINETICS AND AMYLOPECTIN
153.
choice of the equations giving the lines of best fit to these data was made in a way similar to that described previouslyS-8, and is outlined below. Firstly, the DSC data for all four gels were fitted separately by Avrami equations (l). A spread of values was obtained for all three unknowns, A L> k and n; in each case an Avrami exponent of less than I was found, varying from 0'54 for potato starch to 0·75 for wheat starch (modell, Table II). Fitted curves obtained by using this model are plotted in Fig. 2 (a)-(d), curve 1. Previous DSC results from the ageing of wheat starch gels were interpreted with n < 14, while for bread it has also been observed that the data were best fitted by a model with n < I S-8. Making the assumption that n was identical for all four gels, data analysis led to a value of n == 0,62, different rate constants and slightly different limiting values (model 2, Table II). This model was not a significantly worse fit than the original one of four separate Avrami equations so that, allowing for the reduction in the number of parameters employed, it was a better choice than model 1. To avoid confusing Fig. 2 with too many lines, no fitted curves are given for this model: they would be very similar to those for model I. Analysing the data with n constrained to be 1, free A L and k (model 3, Table II) gave a significantly worse fit than either of the other models. Fitted curves obtained by using this model are also plotted (Fig. 2(a)-(d), curve 3). From inspection of Fig. 2, both models I and 3 gave reasonable fits to the data. From these plots it is not obvious that the model with an Avrami exponent of < 1 gives a better fit. However, replotting using a larger scale (not shown), indicated that in general the data at early times ( < 24 h) were fitted better using models I or 2 than model 3. Despite the worse fit of model 3, the estimated parameters are given in Table II for comparison with literature data (see below). In fact the estimated limiting values for models 2 and 3 are very similar, and the rate constants, although different, because their dimension varies with n, have the same ranking; kWh < k am < k wm < k pot ' The conclusions described below are therefore essentially independent of the model chosen; for ease of comparison with literature data, the following discussion will assume the data of model 3 (n = I), unless otherwise stated. Inspection of the data (model 3, Table II, Figs. I and 2) indicates that substantial differences in limiting endotherm enthalpies (A LS) occur for gels from these four starches: the order of ALs is amylomaize < wheat - potato < waxy maize. This order parallels the amylopectin content of the starches, suggesting that the S endotherm is associated with the development of order in the amylopectin fraction. A plot of A L vs. amylopectin (100- % amylose) content for these starches gives a reasonably good straight line (Fig. 3), giving support to this interpretation. Interestingly, based on extrapolation of this plot, A L apparently becomes zero at an amylose content of -76% (amylopectin -24%), implying that starches containing higher proportions of amylose than this would not show the development of any S endotherm. Possible reasons for this behaviour are outlined in a succeeding section. Previous workers have studied the kinetics of ageing of wheat starch gels at 21°C by DTA 2 or DSC4, and have found rate constants (k x 10 3 ) of 11·1 and J 1·6 h-\ at moisture contents of 50 and 53 % respectively. The value obtained in this present paper, for a wheat starch gel of 57 % moisture content, was - 6·0 h -1. Although no measurements have been reported of the dependence of k on moisture content for wheat starch gels, it appears unlikely that the difference in water content was sufficient to account for such a sub-
P. L. RUSSELL
154
100
10
OL..-J.----'-----l_-'-----'-..........J.--":J..:::.......!----L--l
100
80
60
40
20
a
Amylopectin content of storch (%)
FIGURE 3. Plots of (1) rate constant for S endotherm development (k) and (2) limiting endotherm enthalpy (AI) during the ageing of starch gels (n = 1, model 3, Table II), vs. amylopectin content of starches. Open symbols are for rate constants, closed symbols are for ALs. 0, • = amylomaize; 0, • = wheat; /::", .. = waxy maize and \7, ,.. = potato starch.
stantial difference in k. Another factor that could have an influence is the pH of the gels l ; previous studies have not specified pH 2 ,4. The work described here differs from previous work in that data were taken over a much longer period than that employed by previous workers, and it is a possibility therefore that this may have affected the results. To check on this, the data for wheat starch were reanalysed with n = 1, but omitting data obtained after 250 h; under these circumstances (k x 103 ) was found to be 15·7 h- 1 • This figure is even higher than the literature data; the substantial change in the figure indicates the sensitivity of the Avrami equation to the number of data points included, particularly in the early and late stages of the process. Much more data has been included in this study than in previous work 2 ,4; furthermore, the same method of analysis has been applied to all the starches so that it should be possible to compare rate constants even if, as seems unlikely, their absolute magnitudes were in error. Striking differences exist between the rate constants for the different starches, those for amylomaize, waxy maize and potato being respectively ,...,4, 8 and 14 times that for wheat starch (see Table II, Fig. 3). This order apparently bears no relationship to the amylopectin content of the starch. Those starches with either no lipid (potato) or very little amylose (waxy maize) exhibit the largest rate constants, while wheat and amylomaize starches, with both amylose and lipid, exhibit much smaller rate constants. These results suggest that one factor contributing towards low rate constants is the presence of amylose-lipid complexes in the starch gels. In this respect it is interesting that we have recently observed a reduction in the rate of development of the S endotherm in bread containing glyceryl monostearate (GMS), compared with control (untreated) breadS. Another factor that might influence rate constants is the pH of the gels; in general the rate constants are larger, the higher the pH. No attempt has been made to compare gels at equal pH since to do so would have necessitated studying the effect of ionic strength on the process, because buffering would be necessary.
STARCH GEL AGEING KINETICS AND AMYLOPECTIN
155
Starch gel ageing: the influence of separate domains
In general, the results described here, together with other data from the literature, suggest that the thermal and some other properties of starch granules after gelatinisation may be explained on the assumption that the granules behave as parcels, each containing a mixture of separate domains (e.g. amylopectin, amylose-lipid complexes, amylose) that are broadly independent of each other. This seems reasonable, given the mounting evidence from ultrastructural and other studies 1 , that the structure of the granule itself may be understood in terms of a compartmentalisation of the different components. Although various physical changes or reorganisations are envisaged as occurring within and between these fractions during gelatinisation, when gelatinisation is complete, domains still exist within which order can reappear. As an example, although after gelatinisation the amylopectin crystallites may be disrupted, the amylopectin molecules remain in situ, and, hence, can reorder. Even after gelatinisation, the granules still remain as discrete entities, albeit deformed. The evidence in favour of this 'separate domain' hypothesis is outlined below for each fraction in turn. The development of the S endotherm in starch gels appears, from the present results, to be associated with the amylopectin fraction of these starches. The close similarity in S endotherm position, and the fact that amylopectin separated from starch granules gives an endotherm in a very similar position 10-12, suggests also that in each case the developing structure is similar and that the S endotherm is associated with the crystal~ lisation of amylopectin 'domains'. The time-scale of several days for S endotherm development is similar to that observed for growth of the long-term component of crystallinity (amylopectin) in 10 % wheat starch gels 13 • Limiting S endotherm size in starch gels also appears to be dependent upon moisture content during ageing, not during gelatinisation 12. A reduction in the radii of gyration of spherically-shaped regions (assigned to extended amylopectin clusters) during ageing of gelatinised starch-water mixtures is also apparently associated with reorganisation of the amylopectin fraction!? Taken together, these observations suggest that as a result of ge1atinisation, the amylopectin domain is essentially decoupled from others, and the development of order in that domain then depends simply upon the amount of water present in the system. A second domain within the granules may consist of amylose-lipid complexes. Although the position of this fraction within starch granules has not yet been unambiguously identified, a possible location has been suggested, based on the results of small angle neutron scattering experiments 18 . These structures may be characterised by a number of physical techniques, including NMR and chiroptical methods 19 , and by thermal techniques where they lead to the M2 endotherm 19- 21 (see also the preceding paper 1 ). These X_ray 21 and thermaJl9-21 studies indicate that the ordered regions of amylose-lipid complexes are disrupted at a temperature different from that associated with gelatinisation (i.e. at 100-110 DC, see data for M2 endotherm in Table II, ref. 1) and show that the re-ordering of the material is rapid, i.e. that it occurs during cooling back to room temperature. The very different time scales for the restructing of amylopectin and amylose-lipid domains is itself evidence for the decoupling of these two separate fractions. The available evidence 1 suggests that although reorganisation (formation) of some amylose-lipid complexes may occur during gelatinisation, subsequent to this no further changes in M2 enthalpy occur during reheating!, or ageing ll •8 •
156
P. L. RUSSELL
Evidence for the presence and behaviour of the third domain i.e. amylose, also exists. Thus, the short term development of crystallinity has been assigned to the amylose fraction l3 • Contributions to the development of the shear modulus in dilute gels were identified both from amylose and amylopectin fractions with respectively short and long time scales. The faint birefringence observed in cooled, freshly-made amylomaize gels l may also reflect ordered regions of either amylose crystallites (or amylose-lipid complexes). Certainly this could not be due to ordered amylopectin regions, which have been shown to take much longer to re-order. Limited thermal evidence exists that may be relevant: thus we have observed an endotherm at ""' 160 ac in aged (""' I day, data not shown) amylomaize starch-water mixtures. Other workers have shown that amylosewater mixtures give an endotherm at a similar temperature lO . Despite the broad conclusion that independent domains exist to some extent in starch granules and in ageing gels, it also appears likely that a degree of co-operativity exists between these domains. For example, the observed reduction in the rate of development of the S endotherm due to addition of GMS to bread could be accounted for if some limited co-crystallisation of amylose and amylopectin occurred, so that the S endotherm included some contribution from the amylose fraction. Added OMS could complex with some of the amylose during gelatinisation, thus making it unavailable to participate in co-crystallisation during gel ageing. It is interesting that other workers have concluded that the mechanism by which GMS retards staling is not due to an alteration in moisture availability during starch retrogradation, as a result of surfactant addition 12 • Another observation indicating that the 'separate domain' hypothesis may be too simple is that although good linearity between limiting S endotherm enthalpy A L and starch amylopectin content exists (Fig. 3), this plot gives at} A L of zero at an (extrapolated) amylopectin content of ...... 24 %, not zero as might be expected. One interpretation is that'" 24 % of the amylopectin is distributed in the granules in a way different from the remainder. An alternative is that the reduction in A L with increasing amylose content results from a progressive disruption of amylopectin crystallites by interaction with material in other domains; until at 24 % amylopectin so much disruption of the amylopectin domains has occurred that their crystal forming capacity is destroyed. The information currently available does not allow us to distinguish between these possibilities. Starch gel ageing: relationship between DSC and other physical methods
A feature of interest in the results was the relationship between gelatinisation (G+ MI) and limiting S endotherm sizes (ALs) for these starches (Table II). Thus, for waxy maize and wheat starches, ALs were 90 and 88 % respectively of the original G+ Ml endotherms. In contrast, for potato starch A L represented only 63 %, and for amylomaize starch only 17 % of G endotherm size. The reason for this is not understood. However, potato starch has an unexpectedly high gelatinisation enthalpyl. In addition, based on cross polarisation/magic-angle spinning (CP/MAS) l3C NMR, potato starch possesses 50 % 'double-helix content' and only 25 % crystallinity (X-ray), while amylomaize starch had 38 % double-helix and again only 25 % crystallinity 22. The difference between the two measurements was taken as an indication of the amount of' short-range order';
STARCH GEL AGEING KINETICS AND AMYLOPECTIN
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i.e. that material that was present in the form of double helices, but that was not part of a crystalline array. This material with short-range order may have contributed to the (G + M I) endotherm, but not to the S endotherm. One possibility is that the short-range order originated in amorphous regions of the amylose and that on ageing after gelatinisation it became incorporated into amylose crystallites. It is noteworthy that both these starches possess B-type X-ray diffraction patterns\ unlike the other starches described in the present paper. It remains a possibility, therefore, that the excess doublehelix content is related to this. Whatever the reason, it is clear that X-ray diffraction and CPjMAS 13C NMR techniques do not measure the same fraction of native starches 22 • It is equally clear that DSC and X-ray methods are not sensitive to the same fractions of ageing starch gels 13 . Thus, DSC is evidently sensitive to the amylopectin fraction of the gels (and perhaps a small fraction of the amylose that co-crystallises with amylopectin domains) and not to the major proportion of the amylose. In contrast, X-ray diffraction gives a measure of crystallinity in both amylopectin and amylose combined. It follows that the assumption, either implicit or explicit in much previous work, that both X-ray diffraction and DSC measure overall starch crystallinity, is incorrect. A more appropriate model for starch is that, while X-ray diffraction measurements may measure total starch crystallinity, DSC (S endotherm) responds to the amylopectin fraction only. Application of the Avrami equation to the DSC results in effect applies it to the amylopectin fraction. This conclusion has testable implications, e.g. another DSC endotherm should also develop in ageing starch gels due to the reordering of the amylose fraction. One candidate is the endotherm at ~ 160°C observed in aged amylomaize gels or amylose. Because of the relatively high temperature of this endotherm, pressurised DSC would presumably be needed to explore this, to prevent the pans from leaking before the endotherm was reached. Conclusions
The behaviour of starch granules during gelatinisation and ageing can be accounted for in terms of a model involving separate domains of amylopectin, amylose-lipid complexes and amylose. During gelatinisation, some fractionation and interaction of these domains occurs. After gelatinisation, although some coupling occurs, the properties of the system are largely accounted for by the separate domains. The author thanks Dr T. Fearn for the statistical analyses, Dr 1. D. Schofield for useful discussions, Mr D. J. Nicholls for technical assistance and Mr S. Webb for assistance with artwork. The author is grateful to Mr C. R. W. Liley of Hatfield Polytechnic for X-ray diffraction measurements. This work forms part of a research project sponsored by the U.K. Ministry of Agriculture, Fisheries and Food to whom our thanks are due. The results of the research are the property of the Ministry of Agriculture Fisheries and Food and are © Crown Copyright 1987.
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