Starch/Stärke 2011, 63, 395–405 DOI 10.1002/star.

201000150 395

REVIEW

Human a-amylase and starch digestion: An interesting

marriage
Peter J. Butterworth, Frederick J. Warren and Peter R. Ellis

King’s College London, School of Medicine, Diabetes and Nutritional Sciences Division, Biopolymers Group, London, UK

a-Amylase catalyses the first step in the digestion of starch, a main source of carbohydrate in Received: December 6, 2010
the human diet. Amylase present in human saliva was one of the first enzymes ever to be Accepted: January 5, 2011
recognised but many puzzles remain about the molecular mechanisms involved in amylolysis
of starch and even of the physiological role of the salivary amylase itself. Native starch
granules represent a formidable challenge for attack from an enzyme in solution. Moreover
the frequently reported differences in the susceptibility to amylolysis of starches from various
botanical species, plus the changes that occur in starch structure and properties during
domestic and commercial food processing means that studies of the enzymology of starch
digestion can be challenging. We review the molecular properties of mammalian a-amylase
including its genetics, and speculate on the role of salivary amylase in digestion of dietary
starch. Also considered are enzyme kinetic approaches that have been used in vitro studies of
amylase digestion of starches. Such studies can result in better understanding of reasons for
the differences in glycaemic responses of humans following ingestion of different starch
containing foods.

Keywords:
a-Amylase / Genetics / Kinetics / Salivary / Starch digestion

1 Introduction classes of spitting into test tubes containing starch

In 1831, Erhard Leuchs reported that starch is broken
down when mixed with human saliva and used the named
ptyalin to describe the agent in saliva that was responsible
for the chemical reaction. This seems to have been one of
the earliest accounts of an enzyme experiment and soon
after, in 1833, Payen and Persoz isolated an enzyme from
barley that broke down starch and named it diastase. Thus
enzymes now named amylases have a long history and
most readers will have memories from school biology
Correspondence: Dr. Peter J. Butterworth, King’s College
London, School of Medicine, Diabetes and Nutritional Sciences
Division, Biopolymers Group, Franklin Wilkins Building,
150 Stamford Street, London SE1 9NH.
E-mail: peter.butterworth@kcl.ac.uk
Fax: þ44-207-848-4170
Abbreviations: AM, amylose; AP, amylopectin; CE, catalytic
efficiency; RDS, rapidly digested starch; RS, resistant starch.
suspensions stained dark blue with iodine and observing
the disappearance of the colour as the starch is hydrolysed
in the presence of salivary amylase.
Despite this long history and apparent familiarity with
amylase properties, there are still many aspects of amy-
lase and starch biochemistry that continue to puzzle inves-
tigators including the full physiological significance of that
familiar amylase in saliva. Starch is normally the main
source of digestible carbohydrate in the human diet and
as such, is the major source of glucose that appears
at relatively high concentrations in the blood circulation
following intestinal digestion of a starch-containing meal.
The first stage in the metabolism of starch is catalysed by
a-amylase which progressively brings about hydrolysis of
the polysaccharide resulting in the production of maltose,
maltotriose and limit dextrins as the main products [1].
Considerable differences, however, can occur in the post-
prandial blood glucose and the corresponding insulin
response, to the ingestion of different foods containing
identical amounts of starch [2]. That such differences

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396 P. J. Butterworth et al.
occur is evidence of large variations in the rate and extent
of starch digestion in the gastrointestinal tract [3–6].
Attenuating the fluctuations in postprandial glycaemia
and insulinaemia is important in the prevention and treat-
ment of life-style associated diseases, notably diabetes
mellitus and cardiovascular disease, and also has implica-
tions for obesity management [7–10].
The search for full explanations for the differences in
the rates of digestion of starch-containing foods continues
to be a main focus for research, but inter-alia, the differ-
ences are mainly attributed to inherent dissimilarities in the
structure of starch granules of different botanical species
and from structural alterations subsequent to domestic and
commercial processing of foods [11].
Native starch is packaged in granules that are semi-
crystalline and which are very large compared with the size
of the a-amylase molecule. This contrasts with the vast
majority of intracellular metabolic enzymes where the
enzyme molecules are usually huge in size compared with
the metabolites that they act upon. The starch granule size
seemingly presents a very favourable target for attack by
amylase with many potential sites for binding of the
enzyme. In spite of this apparent binding advantage, the
complete breakdown of starch within an intact granule is a
fairly slow process. Crystalline areas tend to be unfavour-
able for enzyme attack and, in addition, the granules may
contain small but variable amounts of proteins and lipids
that can also hinder starch–amylase interaction. Most of
the starch consumed by humans will have been cooked
and/or subjected to various processes during food pro-
duction that disrupts the granules to a greater or lesser
extent, but raw starch is consumed in many animal feeds.
Processing that disrupts general granule integrity and
reduces the degree of crystallinity, increases the suscepti-
bility to amylase.
In recent years, considerable advances have been
made in knowledge of the structures of starch and mam-
malian a-amylases and much has also been learned about
the genes coding for a-amylase and their tissue expres-
sion. It seems timely, therefore, to review relevant aspects
of what is known about starch and the enzyme to see
whether certain uncertainties in the starch-amylase story
can now begin to be clarified and also perhaps point up
aspects of this subject that still need to be resolved by
further experimentation.
2 Human a-amylase genes
The genes for a-amylase have been cloned by several
groups and the article by Meiser and Ting [12] provides an
excellent review of the subject. Salivary glands and the
pancreas express the enzyme in abundance but amylase
expression can also be found in the jejunum and mammary
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Starch/Stärke 2011, 63, 395–405
gland [13]. In humans and other mammals, two distinct but
closely related genes code for amylase. AMY1, the sali-
vary gene, produces the enzyme present in saliva and
mammary gland and AMY2 produces the enzyme synthes-
ised in the pancreas that is secreted into the duodenum in
digestive juices [13]. The expression of AMY1 can also
occur in certain tumour tissues, e.g. lung. In humans both
AMY1 and AMY2 are located on the short arm of
chromosome 1 [14–16]. The coding regions of AMY1
and AMY2 are very similar but the 50 untranslated region
of AMY2 is shorter than the equivalent region in AMY1
[12]. The genes code for enzymes with MWs of about
56 000 and the amino acid sequences of the salivary
and pancreatic enzymes differ by only 3%. Both AMY1
and AMY2 are polymorphic and alloenzyme forms of
the salivary and pancreatic amylases have been detected
[12, 13]. The two pancreatic forms are exclusive to the
pancreas and are expressed at nearly equal levels in
the gland [12]. The secreted salivary amylase has the
N-terminal blocked by a pyroglutamic acid residue [17]
that may confer some resistance to proteolysis.
The organisation of the amylase gene cluster shown
below is based on published data [12]. The genes are
arranged in a head to tail orientation except for AMY1B
which is arranged in the reverse order. AMY1P is a pseu-
dogene that lacks the first three exons of AMY1 [13].
AMY2____AMY2A____AMY1A____AMY1B____AMYP1____AMY1C
The promoter regions upstream of all the amylase genes
contain a g-actin pseudogene and this finding has been
interpreted to indicate that all the amylase genes are
derived from a single ancestral gene [18]. In all except
AMY2B, the g-actin pseudogene is interrupted by a retro-
viral-like sequence and this element probably serves a
regulatory role in the expression of the salivary enzymes
[12]. Meisler and Ting [12] suggest that an ancestral mam-
malian pancreatic gene gave rise to an ancestral AMY2
gene which in turn, after g-actin and retroviral insertion,
gave rise to the present AMY1 and AMY2 forms.
A remarkable feature is that the copy number of the
amylase genes is variable [12, 19]. For the AMY1 (salivary)
gene, variations in copy number from 2 to 15 have been
detected from the screening of 50 individuals [20].
3 a-Amylase structures and properties
X-ray crystallography has been used to obtain 3D struc-
tures of the a-amylase enzymes from human pancreas
[21] and saliva [17] and from porcine pancreas [22]. The
structures of these enzymes are all very closely related.
Mammalian amylases are composed of three structural
domains, the largest of which (domain A) contains import-
ant active site residues that include two aspartate and one
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Starch/Stärke 2011, 63, 395–405
glutamate residue. Domain A also contains a bound Cl

ion. The activation of amylase by chloride has long been
known [23, 24]. Domain B borders the active site region
and contains a bound Ca2þ ion that is probably important in
maintaining protein conformation in the region of the active
site. Domain C at the N-terminal region of the molecule
appears unlikely to play a direct role in the catalytic mech-
anism. It is less firmly associated with domains A and B
[21, 25].
The catalytic event in amylases is an example of a
double displacement mechanism and carboxyl groups of
aspartate and glutamate residues function as acid/base
catalysts and as a nucleophilic reactant for the formation of
a covalent intermediate during the catalytic sequence. The
activation by a Cl
ion may come about by facilitating the
protonation of a catalytically important carboxyl group
through a charge relay-system [26–28].
a-Amylase is an endoenzyme that carries out multiple
attack on linear portions of AM and AP with maltose and
maltotriose as the principal short chain products [1, 29].
Only minor amounts of glucose are produced [1, 30].
Kinetic studies suggested that the active site of porcine
pancreatic enzyme can accommodate up to five glucose
residues, i.e. there are five sub-sites at which glucose
residues can become bound [1]. The existence of multiple
sites was confirmed by the production of 3D structures
from X-ray crystallography, although a report of the struc-
ture of pig pancreatic amylase complexed with the potent
carbohydrate inhibitor acarbose provided evidence of a
sixth site [22]. The glucosidic bond susceptible to attack
is that linking residues 3 and 4 (Fig. 1) and the catalytically
important aspartate and glutamate residues of the
active site are suitably located for attack on the susceptible
link.
Kinetic studies using malto-oligosccharides as sub-
strates [31] were used to determine the binding free energy
(DG) associated with each of the five sites. DG values
become more negative (from approx
5 to
16 kJ/mol),
i.e. binding becomes stronger, as the sub-sites 1–5 become
Figure 1. Subsites at the active centre of a-amylase.
Bond breakage occurs between residues 3 and 4.
Carboxyl groups of key aspartate and glutamate residues
function as acid-base and nucleophilic catalysts [22].
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397
filled with glucose residues in a substrate [30–32]. The
binding energy of site 3 is þ17.6 kJ/mol and this unfavour-
able condition probably arises from forced distortion of the
glucan ring on binding. The binding energies are similar to
those observed for the sites on lysozyme [33]. The total
free energy of binding accruing from occupancy of all the
subsites is of consequence when considering the action of
amylase on native and hydrothermally treated starch (see
below).
4 Starch structure and implications for
a-amylase action
Descriptions and discussion of starch structure and prop-
erties are covered only briefly in this review. The infor-
mation presented here is that which seems most pertinent
to understanding the role of amylase in starch digestion.
The literature contains many excellent general accounts of
starch structure both in its native state and of the changes
that occur in structure resulting from various methods of
processing and readers are referred to these for more
detailed information [34–41].
Native starch granules are semi-crystalline with amor-
phous regions largely composed of AM and the branch
points in AP molecules, and crystallites formed from longer
chains of AP that twist into H-bonded double helices.
Crystalline regions are of two polymorphic types, A and
B [42–45], that differ in the packing of the helices. The
helices in A are orthogonally arranged whereas there is
hexagonal packing in B starches [43, 44]. The number of
associated water molecules is greater in the B crystals [43,
44]. Cereal starches have A type crystallinity and tuber
starches such as potato possess the B polymorph,
whereas legume starches have a so-called C pattern of
crystallinity that is known to arise from the presence of both
A and B polymorphs in different regions of the granule [39].
AM is enriched towards the periphery of the granules and
this peripheral material has shorter chain lengths than AM
located more centrally within the granule [46]. Granules
from cereal starches, e.g. maize, wheat and barley, have
surface pores that open onto channels with diameters that
range in size from 5 to 400 nm [46].
Native starches with a high B polymorph content, e.g.
potato, are attacked slowly by amylase but can be digested
more rapidly after hydrothermal processing [47]. Native A
type starches tend to be more favourable substrates [47].
During domestic and commercial food processing, gran-
ules can be subjected to varying degrees of hydrothermal
treatments which cause granules to swell by absorption of
water and crystalline structures are disrupted with an
increase in amorphous material [41]. The increase of
amorphous material makes the starch more digestible
by a-amylase [11]. Continued heating in aqueous
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398 P. J. Butterworth et al.
conditions results in solubilisation and gelatinisation
of the starch. On cooling, the disrupted material can re-
crystallise into a form that is resistant to amylase action
[48, 49]. This material is known as retrograded starch.
In crystalline starch, the packing of the double helices
are stabilised by hydrogen bonding between glucan resi-
dues. For optimal amylase action, a run of glucan residues
needs to fill the five subsites. Each glucan residue H bonds
to various amino acid side chains that line the active
site. Intuitively therefore, it can be expected that portions
of polysaccharide chain that are intimately associated
in forming crystallites are unlikely to bind securely to
the active site. Estimations of the binding energies associ-
ated with the binding of a glucan residue into each of
the subsites (see above) reveal the importance of filling
of all sites for optimal catalytic action. It is likely however,
that crystalline regions contain a number of areas
where the crystals are imperfect to give ‘amorphous zones’
where the chains are capable of becoming H-bonded to
active site residues of amylase. There would seem to be
a high probability of damage to crystalline regions during
industrial processing such as milling. Also, it can be envis-
aged that if damaged areas are relatively few in number,
enzyme binding to these amorphous zones is unlikely
unless the concentration of amylase is high. Provided that
sufficient water molecules are available for the hydrolytic
reaction, amylase can catalyse chain breakage.
Introducing breaks in polysaccharide chains at these
amorphous foci could lead to further local destabilisation
of crystallinity and favour continued amylolysis. The rates
of reaction are still likely to be relatively slow however,
because after bond breakage, the enzyme has to shuttle
along the glycan chain so that the subsites that span the
residues where bond breakage occurs are newly occupied.
Alternatively, amylase can associate with another section
of chain. Before either of these actions is possible, the
product from the first step must be expelled from subsites 4
and 5 of the active site and diffuse away. The constraints
imposed by localised crystallinity and relatively low water
availability are likely to be reflected in a low rate of
hydrolysis.
5 Salivary amylase
An editorial comment that appeared in a 1987 issue of
Digestive Diseases and Sciences described how the role of
salivary amylase in the digestion of starch was controver-
sial because of the assumption that most of the digestion of
starch occurs in the small intestine in the presence of a
very active pancreatic enzyme [50]. Since that article
appeared, considerably more has been learned about
starch structure and the molecular biology and metabolo-
mics of amylases, including that from saliva, but a degree
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Starch/Stärke 2011, 63, 395–405
of uncertainty still remains with regard to the physiological
significance of salivary amylase.
A significant paper was published in 2007 [20] that
reported on the relationship between the copy number
of the human amylase gene and dietary patterns in a
number of human populations. The authors reported that
individuals from populations that consume diets with rela-
tively high starch content have a higher number of AMY1
gene copies than populations who consume less starch.
They also reported that amylase protein levels correlated
to a considerable degree, with the gene copy number but
the presence of some discrepancy between copy number
and protein expression is evidence of other factors that
control the expression of the AMY1 genes. Thus it was
concluded that the evolution of salivary amylase was
associated with increases in the quantity of starch within
the diet. Unfortunately, their estimates of amylase protein
level in saliva are so much higher than other data in the
literature that there are reasons for questioning the validity
of their findings (see below).
An extension of the study to include other primates
revealed that chimpanzees possess fewer copies of
AMY1 and Perry et al. [20] point out that salivary amylase
protein levels are much higher in humans than in chim-
panzees. The diet of chimpanzees probably contains less
starch and therefore the greater copy number of AMY1 in
humans may reflect a response to increased evolutionary
pressure and that salivary amylase brings survival advan-
tages. Presumably, early hominids would have consumed
considerable amounts of uncooked and minimally proc-
essed (i.e. milled) food that would have contained native
starch granules. Native starch is digested much more
slowly than hydrothermally treated material (see below)
and perhaps higher copy numbers of the genes were
required in order to produce the enzyme in quantity so
that the relatively non-reactive starch was broken down
sufficiently fast enough to contribute usefully to an individ-
ual’s energy requirement. Given therefore, that the
enzyme brings advantages to human survival, what is
its exact role?
Amylase activity from the AMY1 gene can be detected
in saliva and gastric aspirates of prematurely born infants
and the level increases with gestational age from 26 to
42 wk [51]. The expression of pancreatic amylase is min-
imal until weaning introduces non-milk feeds [50, 51] and
the pancreatic gene is probably under partial control by
corticosteroids and other hormones [52]. Comparing esti-
mates of salivary amylase activity quoted in the literature is
complicated by the fact that authors do not always define
their units for expressing catalytic activity and different
assay temperatures may have been used. Some authors
express the enzyme concentration in terms of mg/mL. If
activity has been measured in international units (IU)
per mL, i.e. the production of 1 mmol of product
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Starch/Stärke 2011, 63, 395–405
per min, comparison is easier but again, assay tempera-
tures of 20, 30 and 378C have all been popular choices.
The commercial supplier Sigma–Aldrich1 list their amy-
lase activities in terms of milligram maltose produced in
3 min at 208C. This corresponds closely to 1 IU [53]. If the
turnover number for human salivary amylase is similar to
that of porcine pancreatic enzyme (i.e. approximately 105/
min), a concentration of 1 mg/mL would represent approxi-
mately 20  103 IU/mL of saliva. Sigma1 market a puri-
fied preparation of human salivary amylase with a specific
activity of 1.5  103 per mg that accords well with the
estimate given above that is based on the properties of
porcine pancreatic enzyme. Using this assumption, the
comparison of AMY1gene copy number with amylase con-
tent of saliva (see [20] and above) indicates that saliva
would contain up to 2  104 IU/mL. Activities obtained by
direct measurements and reported in the literature give
values that range from 10 to 160 IU/mL, dependent on the
degree of stimulation of saliva flow and the method of
collection [54–57].
Thus the expected amylase activity of secreted saliva
based on measurement of protein [20] is far in excess of
the activities reported from direct measurements [54–57].
There is an obvious disparity therefore, between calcu-
lations based on enzyme assay and those based on
protein content. Mastication and physical characteristics
of food influence the secretion rate of saliva and its amy-
lase activity [58]. The total protein content of saliva has
been reported to range from 0.1 to 1.0 mg/mL [58] and
therefore the report of concentrations of up to 7 mg/mL of
amylase per mL saliva [20] must be in error. This is a
problem that needs resolving.
In vitro experiments have demonstrated that the sali-
vary enzyme can be protected by starch and oligosacchar-
ides against inactivation by the gastric acidic environment
[59, 60] and thus it can be assumed that it could reach the
duodenum and continue to act upon dietary starch. The
AMY1 gene is also expressed in the lactating mammary
gland of humans and many other mammals including cows
and secreted a-amylase is present in human and cows’
milk [61, 62]. Thus although amylase from saliva and from
milk can reach the duodenum in an active condition it begs
the question of what, prior to weaning and the introduction
of some starch to the diet, does it act upon when it gets
there. Perhaps the enzyme protein possesses important
functional properties that have yet to be recognised.
Human milk contains non-starch oligosaccharides that
are largely formed from galactose but these oligosacchar-
ides are not acted upon by a-amylase. The undigested
oligosaccharides can reach the colon and provide a source
of nutrient for developing colonies of bacteria [63]. From
examination of faecal samples from infants, it was found
that bacteria capable of metabolising lactose and plant
polysaccharides appear at a stage prior to the introduction
ß 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
399
of solids into the infant diet. The milk oligosaccharides
could therefore have played a priming role. It seems that
in some populations from undeveloped areas of the world,
nursing mothers chew solid food materials containing
starch, e.g. roots and tubers, and then pass the pre-mas-
ticated food to their infants to supplement the main supply
of nutrients which the infants receive in milk [64]. The
amylase present in the saliva of the mother and the infant
will begin to digest any starch present and this process will
continue when the food bolus reaches the duodenum,
particularly if the amylase activity is boosted further by a
contribution from that present in a recent feed of milk. In
more developed nations, milk supplements containing
starch are used frequently and if these are introduced into
feeds at an early stage, digestion of the starch will require
the action of salivary amylase until the pancreatic enzyme
becomes expressed in response to dietary and/or hormo-
nal signals [52].
Archeologists have detected starch granules in dental
calculus taken from human skeletal remains [65] and pre-
sumably, there are potentially beneficial effects of salivary
enzyme that can come from attacks on starch lodged
between the teeth and thus aid removal of the obstruction.
The oral bacterium Streptococcal gordonii that is associ-
ated with dental plaque formation can bind to salivary
amylase [66]. The bacterial-bound enzyme retains most
of its activity and is thus able to catalyse the hydrolysis of
starch in the mouth. S. gordonni can convert the maltose
released from starch to glucose and subsequently to lactic
acid. A localised concentration of lactic acid in the dental
plaque area can damage the enamel on neighbouring
teeth [66] and lead to dental caries in circumstances of
poor oral hygiene. The acquisition in S. gordonii of a
specific amylase binding mechanism seems to represent
a developmental response to a ready supply of nutrient
albeit at increasing the risk of tooth decay in the host. The
potential benefit to humans that could arise from amylase-
catalysed removal of starch lodged between the teeth
seems hardly to be realised, therefore.
Recently it has become apparent that genes for taste
receptors are expressed in stomach and other areas of the
gastrointestinal tract. Sweetness receptors in upper sections
of the tract could detect any maltose formed from starch by
salivary amylase and if coupled to the release of a number of
signalling peptides such as GLP-1 and PYY, influence CNS-
mediated control of gastric emptying, insulin secretion and
appetite [67]. In the case of maltose formed in the mouth,
there could also be a feed-forward effect in that after detec-
tion by receptors in the gastrointestinal tract, the signals
could be interpreted as a warning that a starch containing
meal has been consumed and so lead to increased secretion
of pancreatic amylase to digest the expected starch load.
Perhaps not unrelated to this property, is the finding that
salivary amylase decreases the perception, by mouth taste
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400 P. J. Butterworth et al.
sensors, of the saltiness of savoury liquids when the latter
are thickened with starch [68].
6 Application of enzyme kinetic models to
analyse amylase action on starch in vitro
A recently published review contains a full account of
various kinetic approaches that have been taken by
authors in studies of a-amylase catalysed breakdown of
starch [69]. The methods include digestibility measure-
ments made over relatively long time periods and fits to
the Michaelis–Menten equation in both its standard and
integrated forms. This present article will mainly concen-
trate on the usefulness of Michaelis–Menten kinetics
because this is an area which our laboratory has concen-
trated upon for several years to yield fruitful information
about starch–amylase interactions. Rather surprisingly, it
has been stated that the Michaelis–Menten model cannot
be applied to in vitro studies of a-amylase action because of
high enzyme activities, uncertainties in structural changes
occurring in the substrate (starch) and the presence of
product inhibition [70]. Obviously, differences of opinion
exist among workers in the amylase area. Part of the con-
troversy may arise because of a lack of familiarity with the
kind of information that can be gleaned from studies of initial
rates of catalysis as opposed to studies of starch digestion
made over relatively long reaction times with very high
concentrations of enzyme. These conditions of time and
amylase concentration are probably selected due to the
insensitivity of the commonly used reductometric methods
for the detection of reducing sugar products of amylolysis.
Digestion curves are popular with many investigators.
The curves are usually fitted to a 1st order rate equation
first proposed by Goni et al. [71]:
C ¼ C1 ð1
e
kt Þ (1)
where C is the concentration of digested starch at time t,
C1 the digested concentration at the end of the reaction
and k is a first order rate constant. Various botanical
starches generate different values for k that reflect their
relative susceptibilities to digestion. Values typically
reported for k are in the range of 10
5–10
3 min
1. C1
is somewhat misleadingly referred to as the equilibrium
concentration [71]. There is no measurable back reaction
under the conditions pertaining in these experiments how-
ever, and so reference to an equilibrium is not correct. More
realistically, C1 represents the concentration of starch
digested when the reaction has reached a stage where
no more product is formed, i.e. when all starch that can be
hydrolysed has been exhausted and only so-called RS
remains. Figure 2 shows good examples, taken from the
literature, of digestibility curves and fits to the 1st order
ß 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Starch/Stärke 2011, 63, 395–405
Figure 2. Digestibility curves for amylase acting on
Sorghum grains. Particles of different size were produced
by a controlled milling process. The experimental points are
fitted by a first-order kinetic equation (reproduced, with
permission, from Mahasuhkonthachat et al. [70]).
equation to determine values for the apparent 1st order
constant, k [70]. These authors have also shown that 1/k is
proportional to the surface area of the granule calculated
from estimates of the diameter of the granules that are
assumed to be spherical. They state that if the rate-
determining step is related to the diffusion of amylase onto
the surface of the granule particle, a diffusion coefficient
can be calculated from the equation
1 X2
¼ (2)
k 6D
where X2 is the granule specific surface area (m2/g) of a
spherical particle and D is the diffusion coefficient. The
values obtained for D turn out to be extremely small, i.e.
approximately 1  10
10 cm2/s. Thus it takes about 100 s
to diffuse through 1 mm2. The granule–amylase collision
rate in water is probably at least as high as 108 s
1 [72].
We have recently studied the rates of binding of amylase to
starch granules at 08C and determined apparent rate con-
stants for binding (Warren et al. manuscript in preparation).
Binding involves collision with the granule and then cap-
ture. If the number of suitable binding areas on the granule
surface is very low, then the expected capture rate will be
low relative to the collision rate. Direct binding studies
provide some information on capture rates. In fact we
found that the binding constant kon is about 106 M
1/s.
This value seems reasonable given that the collision rate is
likely to be about 108 s
1 [72] and the turnover number for
amylase is approximately 104 s
1 [47, 53]. Our results
indicate that the binding rate is sufficiently fast to allow
a conclusion that diffusion of the enzyme to the surface of
granules must be much faster than the estimates derived
from digestibility constants. Since the latter are obtained
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Starch/Stärke 2011, 63, 395–405
from complete digestion of susceptible starch over rela-
tively long times, the very small diffusion coefficients prob-
ably represent gradual penetration of amylase into the
granule as digestion proceeds. Dhital et al. [73] came
partly to this conclusion from studies of the effects of
particle size on digestibility rate constants.
In spite of demonstrations that digestibility proceeds by
a single first order reaction process, many authors employ
a terminology introduced by Englyst et al. [74] whereby
digestibility curves are divided into phases of rapidly
digested (RDS) and slowly digested starch (SDS). The
RDS fraction is calculated from estimates of the amount
of products released within the first 20 min of digestion and
SDS from measurements of product formation after
periods of up to 2 h [74]. The percentage of RDS material
is said to correlate well with the glycaemic response
measured in vivo following ingestion of starch.
Unfortunately the use of RDS and SDS promulgates
the false idea that granules contain fractions of starch that
are digested at different rates. If the digestion reaction can
be described by a single first order reaction with only one
observed rate constant k, there are no inherent differences
in the potential rate of reaction of any digestible starch
fraction. The measured rate of digestion will be greater at
earlier stages of reaction than in later periods because the
fall in available starch substrate concentration as reaction
proceeds, inevitably results in a slower rate. There is a need
to provide a straightforward description of the relative rates
of digestibility of starches from different plant varieties and
food sources, and a good way of expressing this for a
particular starch source might be to quote the time required
for digestion of 50% of available starch under agreed stand-
ard conditions of amylase concentration and temperature
etc. This is calculated from the value of ln2/k.
Digestibility curves can also be used to calculate
hydrolysis indices (HI) from determination of the area
under the curves (AUC) obtained by integration of the
1st order rate equation with the bounds of t0–t.
 
C1 t þ C1 e
kt
AUC ¼ (3)
k large and hence the measured Km value will also be large
Hydrolysis indice values are an alternative to GI values
for starch foods. GI is defined as the AUC of a postprandial
blood glucose response curve following consumption of a
test meal expressed as a percentage of the AUC measured
with a reference food, e.g. glucose or white wheat bread
[75]. GI determinations require estimations of blood
glucose levels at various time points after the meal
and not all laboratories possess suitable facilities.
Hence, HI measurements may offer an easier alternative.
GI measurements seem to be becoming less popular,
perhaps because of the laboratory difficulties already
mentioned and of high cost implications [76]. There is also
ß 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
401
recognition that sharp excursions (peaks) in blood glucose
concentrations occurring relatively early in postprandial
periods may provide valuable patient information for die-
ticians and clinicians [76].
The rate of reaction (v) during the early stages of
amylase reaction on starch is linear with time and can
be related to the initial starch concentration (S) by the
familiar Michaelis–Menten equation:
Vmax S
v¼ (4)
Km þ S
where Vmax is the maximum velocity and Km is the
Michaelis constant.
Vmax ¼ kcat E0 (5)
where kcat is the catalytic rate constant (also called
turnover number) and E0 is the total enzyme concentration.
Reliable estimates of the kinetic parameters can be
obtained if rate measurements are made within the early
stages of the reaction before the onset of complications
arising from a combination of product inhibition and sub-
strate exhaustion. Reaction rates should be determined
from measurements of product release with time and
checked for linearity. Digestibility studies are often per-
formed with very large concentrations of amylase that
commonly far exceed the concentrations of enzyme that
are present in duodenal fluid or saliva. Initial rate measure-
ments are better performed (in order to maintain linear
product release with time) using reaction mixtures contain-
ing enzyme concentrations that approximate those found
in vivo. From published data we estimate this to be approx
50–450 IU/mL and 10–160 IU/mL for pancreatic juice and
saliva, respectively [54, 77]. We routinely use approximately
100 IU/mL of pancreatic amylase for kinetic work.
For any starch sample, the Km value is the concen-
tration of available (digestible) starch (As) that will support
an initial rate of reaction of Vmax/2. Digestible starch is the
fraction that can be readily acted upon by a-amylase. If
the total starch concentration is S, the ratio S/As will be
because it is calculated from total starch concentrations
added to the reaction mixture. Any treatment of starch to
increase the amount of As, will cause a fall in the S/As ratio
and the measured Km value will reflect this [78]. The Km
measurements can provide a direct indication of the frac-
tion of starch that can be readily digested and thus monitor
changes in starch structure resulting from hydrothermal
treatments and to study structure activity relationships in
general. The proportion of digestible polysaccharide at a
given temperature Tx relative to that present at the lowest
measured temperature T0 can be obtained from the frac-
T T
tion [KmTx =Km0 ], where KmTx and Km0 are the experimental
Km values determined for starches pre-treated at tempera-
www.starch-journal.com
402 P. J. Butterworth et al.
Figure 3. Relationship between catalytic parameters
and gelatinisation enthalpies for various starches. (A) CE
(mM/min (mg/mL)S1 and DHgel (J/g). (B) Hydrolysis rate
(mM/min) and DHgel (reproduced, with permission, from
ref. [53]).
tures Tx and T0, respectively. For example, for potato starch
the Km25 =Km60 ratio is 32.5 [78].
The turnover number kcat, gives the number of mol-
ecules of product formed per molecule of enzyme in unit
time. The dimensions of kcat are time
1. For amylase action
on starch, the measured kcat value will reach the maximum
possible when access to susceptible glycosidic linkages is
not hindered by structural constraints, water molecules for
the hydrolytic reaction are abundant and the reaction prod-
ucts are freely expelled from the catalytic site. These
conditions are likely to be met when the enzyme acts upon
gelatinised starch and the value for kcat should be the same
for all starches. We have found this to be broadly true for a
number of different botanical starches where the value
ranges from (1.0 to 1.6)  105/min [47, 53].
The parameter represented by kcat/Km is called the
specificity constant [79] or the catalytic efficiency (CE).
When an enzyme can react with more than one substrate,
CE is a handy way of comparing the relative reactivity of
the different substrates. Hence it can be very useful for
ß 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Starch/Stärke 2011, 63, 395–405
comparing amylase reacting with starches from different
botanical sources or foods and/or starches that have been
pre-treated to mimic the effects on starch structure of food
processing, e.g. hydrothermal treatments [80]. The ratio
provides an extremely valuable guide to the effectiveness
of the important early stages of a-amylase action on
starch. For potato starch, CE increases 117-fold after
pre-treatment at 608C [78].
The various kinetic parameters can be compared
with the gelatinisation enthalpies of starch granules and
with granule particle size. Figure 3 shows data from a
recent study [54]. Both CE and initial hydrolysis rate are
linearly related with granule surface area. Km increases
with DH (i.e. increased order of a-glucan chains) but
CE and initial hydrolysis rate decrease with DH (Fig. 3).
These findings add weight to the assumption that kinetic
data can provide sensitive monitors for changes in starch
structure and substrate availability induced by physical
treatments [80].
7 Conclusions
Knowledge of the structures and properties of starch and
a-amylase has increased significantly in recent years.
Such information is helping our understanding, at the
molecular level, of the reasons for variations in the rates
at which starches from different botanical origins and/or
from different starch foods are digested by amylase. A
number of uncertainties still remain, however, particularly
with regard to the full physiological importance of the
products of amylase gene AMY1 and the evolutionary
significance of possession of multiple copies of amylase
genes. The choice of kinetic models for studying starch
amylolysis in vitro is also a subject of some controversy.
The authors thank the BBSRC, Danone Research,
Paris, Unilever, Colworth and RHM Technology (Premier
Foods), High Wycombe for their various financial support
of the work of our group over several years.
The authors have declared no conflict of interest.
8 References
[1] Robyt, J. F., in: Fraser-Ried, B. O., Tatsuta, K., Thiem, J.,
Coté, G. L., et al. (Eds.), Glycoscience, Springer-Verlag,
Berlin, Heidelberg, Germany 2008, pp. 1437–1472.
[2] Crapo, P. A., Reaven, G., Olefsky, J., Postprandial plasma
glucose and insulin responses to different complex carbo-
hydrates. Diabetes 1977, 26, 1178–1183.
[3] Noah, L., Guillon, F., Bouchet, B., Buleon, A., et al., Digestion
of carbohydrate from white beans (Phaseolus viulgaris L.).
J. Nutr. 1998, 128, 977–985.
www.starch-journal.com
Starch/Stärke 2011, 63, 395–405
[4] Jenkins, D. J. A., Kendall, C. W. C., Glycemic index: Overview
of implications in health and disease. Am. J. Clin. Nutr. 2002,
76 (suppl), S266–S273.
[5] Ells, L. J., Seal, C. J., Kettlitz, B., Bal, W., Mathers, J. C.,
Postprandial glycaemic, lipaemic and haemostatic
responses to ingestion of rapidly and slowly digested
starches in healthy young women. Br. J. Nutr. 2005, 94,
948–955.
[6] Judd, P. A., Ellis, P. R., in: Souvamyanath, A. (Ed.), Traditional
Medicine for Modern Times, CRC Press, Taylor and Francis
Group, Florida, USA 2006, pp. 257–272.
[7] Jenkins, D. J. A., Kendall, C. W. C., Augustinson, L. S. A.,
Martini, M. C., et al., Effect of wheat bran on glycemic control
and risk factors for cardiovascular disease in type 2 diabetes.
Diab. Care 2002, 25, 1522–1528.
[8] Salmeron, J., Manson, J. E., Stampfer, M. J., Colditz, G. A., et
al., Dietary fiber, glycemic load and risk of NIDDM in women.
J. Am. Med. Assn. 1997, 277, 472–477.
[9] Frost, G., Leeds, A., Trew, G., Margava, R., Dornhorst, A.,
Insulin sensitivity in women at risk of coronary heart disease
and the effect of low glycaemic diet. Metabolism 1998, 47,
1245–1251.
[10] Mann, J., Dietary carbohydrate: Relationship to cardio-
vascular disease and disorders of carbohydrate
metabolism. Eur. J. Clin. Nutr. 2007, 61 (Suppl 1), S100–
S111.
[11] Gallant, D. J., Bouchet, B., Buleon, A., Perez, S., Physical
characteristics of starch granules and susceptibility to
enzymatic degradation. Eur. J. Clin. Nutr. 1992, 46 (Suppl
20), S3–S16.
[12] Meiser, M. H., Ting, C.-N., The remarkable history of the
human amylase genes. Crit. Rev. Oral Biol. Med. 1993, 4,
503–509.
[13] Groot, P. C., Bleeker, M. J., Pronk, J. C., Arwert, F., et al.,
The human a-amylase multigene family consists of haplo-
types with variable numbers of genes. Genomics 1989, 5,
29–42.
[14] Zabel, B. U., Naylor, S. D. L., Sakaguchi, A. Y., Bell, G. I.,
et al., High resolution chromosomal localization of human
genes for amylase, proopiomelanocortin, somatostatin and
a DNA fragment (D3S1) by in situ hybridization. Proc. Natl.
Acad. Sci. USA 1983, 80, 6932–6936.
[15] Muke, M., Lidgren, V., Martinville, B., Francke, U.,
Comparative analysis of mouse-human hybrids with
rearranged chromosomes 1 by in situ hydridization and
Southern blotting: High resolution of NRAS, NFGB and
AMY on human chromosome 1. Somatic Cell Mol. Genet.
1984, 10, 589–599.
[16] Tricoli, J. V., Shows, T. B., Regional assignment of human
amylase (AMY) to p22-p21 of chromosome 1. Somatic Cell
Mol. Genet. 1984, 10, 205–210.
[17] Ramasubbu, N., Paloth, V., Luo, Y., Brayer, G. D., et al.,
Structure of human salivary a-amylase at 1.6 Å resolution:
Implications for its role in the oral cavity. Acta Cryst. 1996, 52,
435–446.
[18] Samuelson, L. C., Wiebauer, K., Snow, C. M., Meisler, M. H.,
Retroviral and pseudogene insertion sites reveal the lineage
of human salivary and pancreatic a-amylase genes from a
single gene during primate evolution. Mol. Cell. Biol. 1990,
10, 2513–2520.
[19] Iofrate, A. J., Feuk, L., Rivera, M. N., Listewnik, M. I., et al.,
Detection of large scale variation in the human genome. Nat.
Genet. 2004, 36, 949–953.
ß 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
403
[20] Perry, G. W., Dominy, N. J., Claw, K. G., Lee, A. S., et al., Diet
and the evolution of human amylase gene copy number
variation. Nat. Genet. 2007, 39, 1256–1260.
[21] Brayer, G. D., Luo, Y., Withers, S. G., The structure of human
pancreatic a-amylase at 1.8 Å resolution and comparisons
with related enzymes. Protein Sci. 1995, 4, 1730–1742.
[22] Gilles, C., Astier, J.-P., Marchis-Mouren, G., Cambillau C., et
al., Crystal structure of pig pancreatic a-amylase isoenzyme
II, in complex with the carbohydrate inhibitor, acarbose. Eur.
J. Biochem. 1996, 238, 561–569.
[23] Levitski, A., Steer, M. L., The allosteric activation of
mammalian a-amylase by chloride. Eur. J. Biochem. 1974,
41, 171–180.
[24] Lifshitz, R., Levitski, A., Identity and properties of the chloride
effector binding site in hog pancreatic a-amylase.
Biochemistry 1976, 15, 1987–1993.
[25] MacGregor, E. A., Janacek, S., Svensson, B., Relationship of
sequence and structure to specificity in the a-amylase family
of enzymes. Biochim. Biophys. Acta 2001, 1546, 1–20.
[26] Agarjahi, N., Feller, G., Gerday, C., Haser, R., Structural basis
of a-amylase activation by chloride. Protein Sci. 2002, 11,
1435–1441.
[27] Qian, M., Haser, R., Buisson, G., Duee, E., Payan, F., The
active centre of a mammalian a-amylase. Structure of the
complex of a pancreatic a-amylase with a carbohydrate
inhibitor refined to 2.2 Å resolution. Biochemistry 1994,
33, 6284–6294.
[28] Feller, G., le Bussy, O., Houssier, C., Gerday, C., Structural
and functional aspects of chloride binding to Alteromonas
haloplanctis a-amylase. J. Biol. Chem. 1996, 271, 23836–
23841.
[29] Robyt, J. F., French, D., Multiple attack hypothesis of a-
amylase action: Action of porcine pancreatic, human salivary
and Aspergillus oryzae a-amylases. Arch. Biochem.
Biophys. 1967, 122, 8–16.
[30] Seigner, C., Proganov, E., Marchis-Mouren, G., The
determination of subsite binding energies of porcine pancre-
atic a-amylase by comparing hydrolytic activity towards
substrates. Biochim. Biophys. Acta 1987, 913, 200–209.
[31] Prodanov, E., Seigner, C., Marchis-Mouren, G., Subsite pro-
files of the active center of porcine pancreatic a-amylase.
Kinetic studies using maltooligosaccharides as substrates.
Biochem. Biophys. Res. Com. 1984, 122, 75–81.
[32] Seigner, C., Prodanov, E., Marchis-Mouren, G., On porcine
pancreatic a-amylase action: Kinetic evidence for the binding
of two maltooligosaccharide molecules (maltose, maltotriose
and o-nitrophenylmaltoside) by inhibition studies. Eur. J.
Biochem. 1985, 148, 161–168.
[33] Fersht, A., Enzyme Structure and Mechanism, 2nd Edn.,
W. H. Freeman and Company, New York, USA 1985,
pp. 435–436.
[34] Gallant, D. J., Bouchet, B., Baldwin, P. M., Microscopy of
starch: Evidence of new level of granule organisation.
Carbohydr. Polym. 1997, 32, 177–191.
[35] Buleon, A., Colonna, P., Planchot, V., Ball, S., Starch
granules: Structure and biosynthesis. Int. J. Biol.
Macromol. 1998, 23, 85–112.
[36] Wang, T. L., Bogracheva, T. Y., Hedley, C. L., Starch as simple
as A, B, C? J. Exp. Biol. 1998, 49, 481–502.
[37] Cooke, D. F., Gidley, M. J., Loss of crystalline and molecular
order during starch gelatinisation: Origin of the enthalpic
transition. Carbohydr. Res. 1992, 227, 103–112.
www.starch-journal.com
404 P. J. Butterworth et al.
[38] Donald, A. M., Waigh, T. A., Jenkins, P. J., Gidley, M. J., et al.,
in: Frazier, P. J., Donald, A. M., Richmond, P. (Eds.), Starch:
Structure and Functionality, Royal Society of Chemistry,
Cambridge, UK 1997.
[39] Bogracheva, T. Y., Morris, V. J., Ring, S. G., Hedley, C. L., The
granular structure of C-type pea starch and its role in gelati-
nisation. Biopolymers 1998, 45, 323–332.
[40] Svihus, B., Uhlen, A. K., Harstad, O. M., Effect of starch
granule structure, associated components and processing
on nutritive value of cereal starches. Animal Feed Sci.
Technol. 2005, 122, 303–320.
[41] Tester, R. F., Morrison, W. R., Swelling and gelatinisation of
cereal starches. I Effects of amylopectin, amylose and lipids.
Cereal Chem. 1990, 67, 551–557.
[42] Gidley, M. J., Bociek, S. M., Molecular organization in
starches: A carbon-13 CP/MAS NMR-study. J. Am. Chem.
Soc. 1985, 107, 7040–7044.
[43] Imberty, A., Perez, S., A revisit to the three dimensional
structure of B-type starch. Biopolymers 1988, 27, 1205–1221.
[44] Imberty, A., Chanzy, H., Perez, S., Buleon, A., Tran, V., The
double helical nature of the crystalline part of A starch. J.
Mol. Biol. 1988, 201, 365–378.
[45] Manners, D. J., Recent developments in our understanding of
amylopectin structure. Carbohydr. Polym. 1989, 11, 87–112.
[46] Perez, S., Baldwin, P., Gallant, D. J., in: BeMiller, J. N.,
Whistler, R. L. (Eds.), Starch: Chemistry and Technology,
3rd edn. Academic Press, London, UK, New York, USA 2009,
pp. 149–192.
[47] Slaughter, S. L., Ellis, P. R., Butterworth, P. J., An investigation
of the action of porcine pancreatic a-amylase on native and
gelatinised starches. Biochim. Biophys. Acta 2001, 1525,
29–36.
[48] Gidley, M. J., Cooke, D., Darke, A. H., Hoffman, P., et al.,
Molecular order and structure in enzyme resistant retro-
graded starch. Carbohydr. Polym. 1995, 28, 23–31.
[49] Hoover, R., Starch retrogradation. Food Rev. Int. 1995, 11,
331–346.
[50] Lebenthal, E., Role of salivary amylase in gastric and intes-
tinal digestion of starch. Dig. Dis. Sci. 1987, 32, 1155–1157.
[51] Hodge, C., Lebenthal, E., Lee, P. C., Topper, W., Amylase in
the saliva and in the gastric aspirates of premature infants: Its
potential role in glucose polymer hydrolysis. Pediatr. Res.
1983, 17, 998–1001.
[52] Swanson, K. C., Matthews, J. C., Matthews, A. D., Howell, J.
A., et al., Dietary carbohydrate source and energy intake
influence the expression of pancreatic a-amylase in lambs.
J. Nutr. 2000, 130, 2157–2165.
[53] Tahir, R., Ellis, P. R., Butterworth, P. J., The relation of physical
properties of native starch granules to the kinetics of amy-
lolysis catalysed by porcine pancreatic a-amylase.
Carbohydr. Polym. 2010, 81, 57–62.
[54] Froehlich, D. A., Pangborn, R. M., Whitaker, J. R., The effect
of oral stimulation on human parotid salivary flow rate and
amylase secretion. Physiol. Behav. 1987, 41, 209–217.
[55] Harthoorn, L. F., Salivary a-amylase: A measure associated
with satiety and subsequent food intake in humans. Int. Dairy
J. 2008, 18, 879–883.
[56] Beltzer, E. K., Fortunato, C. K., Guaderrama, M. M., Peckins,
M. K., Salivary flow and a-amylase: Collection technique,
duration and oral fluid type. Physiol. Behav. 2010, 101, 289–
296.
ß 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Starch/Stärke 2011, 63, 395–405
[57] Panchbhai, A. S., Degwekar, S. S., Bhowte, R. R., Estimation
of salivary glucose, salivary amylase, salivary total protein
and salivary flow rate in diabetics in India. J. Oral Sci. 2010,
52, 359–368.
[58] Mackie, D. A., Pangborn, R. M., Mastication and its influence
on human salivary flow and a-amylase secretion. Physiol.
Behav. 1990, 47, 593–595.
[59] Fried, M., Abramson, S., Meyer, J. H., Passage of salivary
amylase through the stomach in humans. Dig. Dis. Sci. 1987,
32, 1097–1103.
[60] Rosenblum, J. L., Irwin, C. L., Alpers, D. H., Starch and
glucose oligosaccharides protect salivary-type amylase
activity at acid pH. Am. J. Physiol. 1988, 254, G775–780.
[61] Lindberg, T., Skude, G., Amylase in milk. Pediatrics 1982, 70,
235–238.
[62] Fox, P. F., Kelly, A. L., Indigenous enzymes in milk: Overview
and historical perspectives. Int. Dairy J. 2006, 16, 500–516.
[63] Koenig, J. E., Spor, A., Scalfone, N., Frisker, A. D., et al.,
Succession of microbial consortia in the developing gut
microbiome. Proc. Natl. Acad. Sci. USA 2010, DOI:
10.1073/pnas.1000081107.
[64] Pelto, G. H., Zhang, Y., Habnicht, J.-P., Premastication: The
second arm of infant and young child feeding for health and
survival. Matern. Child Nutr. 2010, 6, 4–18.
[65] Hardy, K., Blakeney, T., Copeland, L., Kirkham, J., et al.,
Starch granules, dental calculus and new perspectives on
ancient diet. Archeol. Sci. 2009, 36, 248–255.
[66] Scannapieco, F. A., Bhandary, K., Ramasubbu, N., Levine,
M. J., Structural relationship between the enzymatic and
streptococcal binding sites of human salivary amylase.
Biochem. Biophys. Res. Comm. 1990, 173, 1109–1115.
[67] Little, T. J., Gupta, N., Maynard-Case, R., Thompson, D. G.,
McLaughlin, J. T., Sweetness and bitterness taste of meals
per se does not mediate gastric emptying in humans. Am. J.
Physiol. 2009, 297, R632–R639.
[68] Ferry, A.-L., Hort, J., Mitchell, J. R., Cook, D. J., et al.
Viscosity and flavour perception: Why is starch different
from hydrocolloids? Food Hydrocolloids 2006, 20, 855–862.
[69] Dona, A. C., Pages, G., Gilbert, R. G., Kuchel, P. W.,
Digestion of starch: In vivo and in vitro kinetic models
used to characterise oligosaccharide or glucose release.
Carbohydr. Polym. 2010, 80, 599–617.
[70] Mahasukhonthachat, K., Sopade, P. A., Gidley, M. J., Kinetics
of starch digestion in sorghum as affected by particle size. J.
Food Eng. 2010, 96, 18–28.
[71] Goni, I., Garcia-Alonso, A., Saura-Calixto, F., A starch
hydrolysis procedure to estimate glycemic index. Nutr.
Res. 1997, 17, 427–437.
[72] Fersht, A., Structure and Mechanism in Protein Science,
W. H. Freeman and Company, New York, USA 1999,
pp. 158–159.
[73] Dhital, S., Shrestha, A. K., Gidley, M. J., Relationship
between granule size and in vitro digestibility of maize
and potato starches. Carbohydr. Polym. 2010, 82, 480–
488.
[74] Englyst, H. N., Kingman, S. M., Cummings, J. H.,
Classification and measurement of nutritionally important
starch fractions. Eur. J. Clin. Nutr. 1992, 46, 33–50.
[75] Jenkins, D. J. A., Wolever, T. M. S., Taylor, R. H., Barker, H.,
et al., Index of foods. A physiological basis for carbohydrate
exchange. Am. J. Clin. Nutr. 1981, 34, 362–366.
www.starch-journal.com
Starch/Stärke 2011, 63, 395–405 405

[76] Seal, C. J., Daly, M. E., Thomas, L. C., Bal, W., et al., Ph.D Thesis, King’s College London, University of London
Postprandial carbohydrate metabolism in healthy subjects 2008.
and those with type 2 diabetes fed starches with slow and
[79] Fersht, A., Structure and Mechanism in Protein Science,
rapid hydrolysis rates determined in vitro. Br. J. Nutr. 2003,
W. H. Freeman and Company, New York, USA 1999,
90, 853–864.
pp. 110–111.
[77] Dahlqvist, A., Borgström, B., Digestion and absorption of
[80] Roder, N., Gerard, C., Verel, A., Bogracheva, T. Y., et al.,
disaccharides in man. Biochem. J. 1961, 81, 411–418.
Factors affecting the action of a-amylase on wheat starch:
[78] Egan, D., Biochemical mechanisms of starch digestion: Effects of water availability. An enzymic and structural study.
Enzyme kinetic studies of alpha-amylase action on starch. Food Chem. 2009, 113, 471–478.

ß 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.starch-journal.com