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International Journal of Biological Macromolecules

27 (2000) 112
Review
Annealing of starch a review
Richard F. Tester *, Stephane 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 specic 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
www.elsevier.com/locate/ijbiomac
1. Background and denitions
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
(4055% 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 (T
g
) but
below the gelatinisation temperature. However, the
term heat moisture is often used to describe high tem-
perature treatments, like 100C (up to 16 h at 27%
moisture) [2]. Stute [3] has also discussed the difference
between annealing and heat moisture treatments, ac-
knowledging that for work conducted in the early part
of last Century, annealing and heat moisture were
used as synonymous terms. More recently Collado and
Corke [4] have helped to clarify the situation. They
state that annealing represents physical modication of
starch slurries in water at temperatures below gelatini-
sation whereas heat moisture treatment refers to the
exposure of starch to higher temperatures at very re-
stricted moisture content (1827%).
These authors propose that the terminology is stan-
dardised which has implications in terms of the
denition of gelatinisation. Hence, the following deni-
tions 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 thermody-
namic transition and has been well described by Bili-
aderis 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 com-
pletely glassy polymers, T
g
is relatively distinct, where
an inection (increase) in the specic volume and en-
thalpy as a function of temperature occurs and is
* Corresponding author. Tel.: +44-141-3318514; fax: +44-141-
3313208.
E-mail address: r.f.tester@gcal.ac.uk (R.F. Tester)
0141-8130/00/$ - see front matter 2000 Elsevier Science B.V. All rights reserved.
PII: S0141- 8130( 99) 00121- X
R.F. Tester, S.J.J. Debon / International Journal of Biological Macromolecules 27 (2000) 112 2
reected in discontinuity in the specic heat capacity
(C
p
). Because starch contains both amorphous and
crystalline material, the exact thermal event represent-
ing T
g
is difcult to detect. However, high sensitivity
differential scanning calorimetry (DSC) has allowed for
the measurement of T
g
in amorphous and native
starches with various levels of crystallinity [68]. Water
is a very effective plasticiser of amorphous starch (and
hence T
g
), where the ratio of starch to water is critical
with respect to the temperature at which T
g
occurs (Fig.
1).
1.2. Annealing
Annealing represents the physical reorganisation of
starch granules (or appropriate polysaccharide matrices
like amyloselipid complexes) when heated in water (or
appropriate plasticiser) at a temperature between T
g
and the onset of gelatinisation (T
o
) of the native starch
(or polymeric system). It is recognised that annealing
can be associated with partial gelatinisation. However,
these authors believe the denition should be applied
only where gelatinisation does not occur and hence T
o
Fig. 1. State diagram of the starchwater system. The experimental data for the glass transition (T
g
) are from amorphous starch [8] while the
theoretical T
g
is derived from the CouchmanKarasz equation [9]. The experimental data for the melting transition (T
m
) are from DSC
(T
conclusion
) of wheat starches at different moisture content [1013]. The theoretical T
m
is tted from the FloryHuggins equation [14].
R.F. Tester, S.J.J. Debon / International Journal of Biological Macromolecules 27 (2000) 112 3
Fig. 2. DSC thermograms of a commercial wheat starch (BDH 30265): (a) native; (b) after annealing in excess water (45C, 100 days).
must not be exceeded. In addition, according to this
denition, 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 (dened
below) as shown in Fig. 2.
The annealing process has important industrial impli-
cations. Starches may be deliberately annealed to im-
part novel processing characteristics. However, there
are few commercial processes where annealing may be
justied in terms of energy and time to generate
starches with higher gelatinisation temperatures es-
pecially 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 molecu-
lar 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 T
g
,
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 gran-
ules as the polymers (and granule interstices) hydrate.
The onset temperature (T
onset
or T
o
, typically 45C)
by DSC reects the initiation of this process, which is
followed by a peak (T
peak
or T
p
, typically 60C) and
conclusion (T
conclusion
or T
c
, typically 75C) temperature
(Fig. 2). After T
c
, all amylopectin double helices have
dissociated, although swollen granule structures will be
retained until more extensive temperature and shear
have been applied. Beyond 95C an amorphous gel is
formed. The temperature range T
c
T
o
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 con-
fused with retrogradation. However, annealing of
starch granules is a process that retains granular struc-
ture 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 [1517], and how interactions be-
tween starch and other groups (especially water and
solutes) modify the temperature driven transitions.
Readers are referred to these publications for more
detail.
R.F. Tester, S.J.J. Debon / International Journal of Biological Macromolecules 27 (2000) 112 4
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 T
g
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 con-
ducted deliberately by industry to impart novel charac-
teristics. 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 freezethaw stability and improvement of the
baking quality of potato starch [4].
2. Relationship between T
g
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 amor-
phous lamellae and annealing of double helices is repre-
sented 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 dissoci-
ation of the crystallites begins after T
g
of amorphous
regions, and at this temperature (T
o
), limited dissociation
of amylopectin double helices (most of which are in
crystallites) is associated with limited swelling of gran-
ules. Gelatinisation proceeds as the temperature is in-
creased, progressively uncoiling all the double helices and
converting crystalline material to amorphous material.
If water is sufciently low so as to restrict gelatinisa-
tion, 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 (6
1
) is
reduced to B0.45, the shoulder becomes distinct and
represents the only endotherm observed. In high mois-
ture food systems, starch granules are completely gela-
tinised 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, essen-
tially native granule form is apparent under the micro-
scope-although this starch has presumably been heat
moisture treated.
The T
g
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 T
g
without facilitating the gelatinisation pro-
cess [17]. If starches are heated at progressively higher
temperature above T
g
, 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 [19,2427].
Perhaps because of the difculty associated with mea-
suring T
g
, some authors claim that starches can be
annealed below T
g
[28,29]. However, this would mean
that structural reorganisations of the crystalline compo-
nent of starch granules occur independently of reorgan-
isation 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 gelatini-
sation endotherm of starch using DSC, although this is
not true of T
g
. Whilst T
g
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 [68]. Primarily this is because
it is both small and submerged into the thermogram
baseline. Model polymeric systems, for example polyan-
hydroglucose compounds [30] have, however, been useful
in developing an understanding of how a-glucan struc-
ture itself moderates T
g
. For example branched regions
(which are also found in the amorphous zones of starch)
seem to depress T
g
similarly to plasticisation by small
molecules [30].
3. Effects of starch-to-moisture ratio on T
g
and
annealing
The original work of Gough and Pybus [31], which was
the rst to describe how wheat starch could be annealed
by heating in excess water at 50C, showed that gelatini-
sation 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 [23,29,3246]. Some-
times, the annealing process is conducted as a
R.F. Tester, S.J.J. Debon / International Journal of Biological Macromolecules 27 (2000) 112 5
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]).
R.F. Tester, S.J.J. Debon / International Journal of Biological Macromolecules 27 (2000) 112 6
Fig. 4. Inuence of water content on the differential scanning calorimetry thermograms prole 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 starchwater/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 difcult to compare re-
sults between the different studies.
The effect of the starch-to-water ratio, temperature
and time on annealing of wheat starch has been investi-
gated in detail [23]. This study demonstrated how criti-
cal the interrelationship of these parameters is. The
annealing process could be initiated when the moisture
content exceeded 20% by weight, (because T
g
is around
room temperature when this moisture content is ex-
ceeded, 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
15C below T
o
by DSC, the effect was more marked the
closer the annealing temperature was set to (below) T
o
.
Similar studies have been conducted on starches of
different botanical origins [32,40] and demonstrate the
additional complication of species specic variation.
During annealing of starches, there are in essence two
thermally driven processes which are intimately related
and reect the moisture content of the system the
elevation of T
g
and gelatinisation temperatures (espe-
cially T
o
). Low moisture causes elevation of (the rela-
tively unplasticised) T
g
of starches [6,7,22,47] and model
polymeric systems [30,48,49] which, in the case of
starch, intimately reects the increase in gelatinisation
temperatures. Indeed, the elevation of T
g
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
T
g
of starch in food systems is very complex because
R.F. Tester, S.J.J. Debon / International Journal of Biological Macromolecules 27 (2000) 112 7
Fig. 4. (Continued)
of the raft of potential interactions. More details con-
cerning (general) glass transitions in model systems can
be found elsewhere [50]. Similarly, sub-T
g
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 [5458]
there tends to be a small effect on the ne 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 tem-
perature 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 be-
tween 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 specic varia-
tion in starch physico-chemical properties, there is a
profound environmental effect on gelatinisation charac-
teristics. This effect is, in the experience of these au-
thors, far greater than cultivar specic (and hence
genetic) variation. The implications are, that product
quality is not simply a cultivar specic trait but is
largely dependent on environmental conditions experi-
enced during starch deposition.
5. Molecular basis for annealing
It has been difcult to dene, at the molecular level,
what happens to the internal structure of starch gran-
ules when they are annealed. Some authors have dis-
cussed the molecular event in terms of increasing
granule stability [32], reorganising granule structure
[39,40] or lowering free energy [17]. These descriptive
R.F. Tester, S.J.J. Debon / International Journal of Biological Macromolecules 27 (2000) 112 8
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. Crys-
tallinity and crystalline perfection (optimisation of
crystalline order) have been discussed in detail in this
context [28,36,38,42,43,59]. Similarly, granular reorgan-
isations 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 be-
tween T
g
and T
o
), 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 T
g
is elevated. The
constancy of double helix content pre- and post-anneal-
ing 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 amy-
lose helical structures [62,63]. This different structural
model within amylomaize starch granules helps to ex-
plain 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 mea-
sure 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 conrmed 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 de-
crease 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 signicance 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 signicant
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 conse-
quence of ordering of previously amorphous regions.
Rather, the enhanced ordering of double helices, due to
improved registration (alignment), with associated in-
creased rigidity of amorphous regions, probably under-
lies the annealing process.
Unlike WAXS which quanties crystalline order
throughout starch granules, small angle X-ray scatter-
ing (SAXS) quanties 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 crys-
talline 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 gran-
ules together with techniques used for their quantica-
tion are presented in Fig. 5. More detailed discussion
regarding the application of this technique to under-
stand structural, gelatinisation and annealing mecha-
nisms 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
gures quoted by Jacobs et al.
Native cereal starch granules contain amyloselipid
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 signicance of
these complexes is uncertain, although they have a
signicant effect on starch functionality. Whilst in
model amyloselipid complex systems annealing can be
induced [69] because of the relative ease of mobility,
annealing between T
g
of the starch and T
o
of amy-
lopectin in wheat starch has little effect on this amy-
loselipid endotherm [34,35]. The effect of annealing at
these temperatures (e.g. 3550C) on amyloselipid
complexes is predictably very unlikely, because the
temperature is far too low. The peak transition temper-
ature of these complexes is of the order of 95115C.
At temperatures where complexes have been annealed
(for example 80C, [69]) the starch would be fully
gelatinised in unlimiting water. Probably T
g
for the
complexes under these conditions is quite close to this
R.F. Tester, S.J.J. Debon / International Journal of Biological Macromolecules 27 (2000) 112 9
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 T
g
for
amylopectin as a function of moisture content [23].
There is a relationship between the amount of starch
phosphorylation and elevation of gelatinisation temper-
atures as a consequence of annealing [70]. The shift in
T
p
(or DT
p
) 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 crys-
tallite 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 intro-
duced phosphate groups have similar effects to natu-
rally (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
R.F. Tester, S.J.J. Debon / International Journal of Biological Macromolecules 27 (2000) 112 10
microscopic work indicated that wheat starch granule
dimensions increase after annealing [31]. Clearly, how-
ever, small differences in size cannot be accurately
quantied 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 B-
to A-type transition for potato starches, this does not
happen during annealing [3]. Hence, the molecular re-
orientation is more subtle.
The effect of annealing on gelatinisation characteris-
tics is well established, particularly using DSC, where
there tends to be an increase in T
o
and T
p
, decrease in
the gelatinisation range (T
c
T
o
) and either constancy or
an increase in gelatinisation enthalpy
[3,23,28,31,32,34,3741,43,46,64,70,71,73]. The increase
in gelatinisation temperatures is associated with a de-
crease in swelling power [23,32,41,46,73], provided that
some granular structure is retained. This is reected in
a higher temperature onset of swelling and reduced
swollen volume (below circa 90C, provided that water
is not limiting). The effects of annealing on pasting
characteristics are complex. In some studies the consis-
tency (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 re-
ported 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 amor-
phous regions within annealed starch granules and a
more ordered registration of amylopectin double he-
lices. 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 difcult to unravel the effects on
pasting characteristics, because this system will be
strongly inuenced 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 solubilisa-
tion 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. Anneal-
ing reduces solubilisation of a-glucan during swelling
below 100C [32,41,46,73]. As leachate is primarily
(amorphous) lipid free amylose (FAM) according to the
denition of Morrison et al. [68], the amylose must be
more restricted from leaching out of the granules. Al-
though at a given temperature post-annealing, the gran-
ules 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 T
g
.
8. Chemical hydrolysis
Annealing tends to reduce the amount of acid hy-
drolysis 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 rst phase of
acid hydrolysis (07 days, representing amorphous ma-
terial hydrolysis) annealed starch was more extensively
hydrolysed than native starch. After 7 days, where
crystalline material is progressively hydrolysed, the ex-
tent of hydrolysis for the native and annealed starches
was essentially the same. The explanation for the en-
hanced hydrolysis of amorphous regions after anneal-
ing 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) con-
rms that it is double helices (which remain constant)
which are the primary contributor to the hydrolysis
prole during this phase. Amyloselipid complexes
may affect the pattern of acid hydrolysis in cereal
starches, which could also inuence 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
R.F. Tester, S.J.J. Debon / International Journal of Biological Macromolecules 27 (2000) 112 11
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 unjustied compari-
sons between different phases of hydrolysis (amorphous
and crystalline material) rather than necessarily starch
specic 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 signicance than the
actual plant source. It is also possible that the anneal-
ing process creates pores or ssures which alter the
pattern of amylase hydrolysis from surface to internal
erosion [74]. Hence, although amorphous and crys-
talline lamellae become more ordered, accessibility to
the amorphous regions by enzymes is facilitated.
Annealing can be conducted in the presence of amy-
lases to selectively hydrolyse amorphous regions and
the possibility of novel products with unique gelatinisa-
tion 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 amy-
lopectin 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 T
g
and gelatinisation tempera-
tures are increased, swelling is decreased and leaching is
restricted. The situation in high amylose starches is
different from waxy and normal starches where anneal-
ing 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 informa-
tion about this process. There are important industrial
implications of the annealing process with relevance to
starch extraction and food production. As more sophis-
ticated physical techniques are developed, the molecular
basis will be understood in far more detail.
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