18 Davis, B.A. and Osswald, T.A. (1996) 'investigations of Non-linear Effects of 27 Baptista, P.N., Oliveira, F.A.R., Caldas, S.M.
M. and Oliveira, l.C. (1996)'Effect
Polymer Flows During Mixing' in Society of Plastic Engineers Technical Paper,
pp. 54-58, Society of Plastic Engineers, Brookfield, CT, USA
19 Coulson, I.M., Richardson, J.F. and Peacock, D.J. (1979) Chemical
Engineering, Pergamon Press
20 Nienow, A.W., Edwards, M.F. and Hamby, N. (1985) Mixing in the Process
Industries, Butterworths
21 Metzner, A.B., Feehs, R.H., Ramos, H.L, Otto, R.E. and Tuthill, J.D. i1961 )
'Agitation of Viscous Newtonian and Non-Newtonian Fluids' in AIChEJ. 7,
3-9
22 Nienow, A.W., Wisdom, D.J., Solomon, J., Machon, V. and Vlcek, J. (1983)
'The Effect of Rheological Complexities on Power Consumption in an Aerated
Agitated Vessel' in Chem. Eng. Commun. 19, 273-293
23 Dyster, K.N., Koutsakos, E., Jaworski, Z. and Nienow, A.W. (1993)'An LDA
Study oi the Radial Discharge Velocities Generated by a Rushton Turbine:
Newtonian Fluids, Re ~ 5' in Trans. IChemE 71, 11-23
24 Merchuk, J.C. (1991 ) 'Shear Effects on Suspended Cells' in Adv. Biochem.
Eng./Biotechnol. 44, 63-95
25 Nienow, A.W., Langheinrich, C., Stevenson, N.C., Emery, A., Clayton, T.M.
and Slater, N.K.H. (1996)'Homogenization and Oxygen Transfer Rates in
Large Agitated and Sparged Animal Cell Bioreactors - Some Implications for
Growth and Production' in Cytotechnology 22, 87-94
26 Ramaswamy, H.S., Abdelrahim, K.A., Simpson, B.K. and Smith, J.P. (1995)
'Residence Time Distribution (RTD) in Aseptic Processing of Particulate Foods
- A Review' in Food Res. Int. 28, 291-310
Review
Starch constitutes a major component of foods and also a
raw material for use in the production of industrial products.
The constituent macromolecules of starch are packaged in
a highly ordered and compact manner, resulting in inert, in-
soluble granules. Food processing destroys starch structure,
thereby releasing the component molecules, which are subse-
quently made available for hydrolysis or serve a functional
role in the food system. The application of starch as a raw
material usually requires the prior disruption of the inert gran-
ule structure, which involves additional processing steps. The
hydrolysis of native granules has ramifications at all levels
of the food processing chain, from postharvest losses to nu-
tritional consequences of the ingested food. Technologists
have not been able to take advantage of, or control, this pro-
cess because the body of the information that is available on
starch granule structure and the behaviour of hydrolysing
enzymes is still incomplete. In this review, I have highlighted
some of the more recent advances in this field, with the
view to opening up the way for more efficient native starch
hydrolysis.
ChristopherG. Oates is at the Department of Biochemistry, National University
of Singapore, 10 Kent Ridge Crescent, 119260 Singapore (fax: +65-779-1453;
e-mail: bcha°tes@nus'edusg/°ates@p°pulusnet)'
Trends in Food Science & Technology November 1997 [Vol. 8]
of Product and ProcessVariables in the How of Spherical Particles in a
Carrier Fluid Through Straight Tubes' in J. Focxt Process. Preserv. 20,
467-486
28 Willert, C.E. and Gharib, M. (1991) 'Digital Particle Image Velocimetry' in
Exp. Fluids 10, 181-193
29 Adrian, R.J. (1991) 'Particle Imaging Techniques for Experimental Fluid
Mechanics' in Anrru. Rev. Fluid Mech. 23,261-304
30 Naqwi, A. and Ziema, M. (1992) 'Extended Phase Doppler Anemometer for
Sizing Particles Smaller Than 10 I~m' in J. Aerosol Sci. 23, 613-621
31 Sommeffield, M. and Qiu, H.H. (1995)'Particle Concentration Measurement
by Phase-Doppler Anemometry in Complex Dispersed Two-phase Flows' in
Exp. Fluids18, 187 198
32 Zhang, L. and Fryer, P.J. (1994) 'Food Sterilization by Electrical Heating-
Sensitivity to Process Parameters' in AIChE J. 40, 888-898
33 Guo, l., Xu, Z. and Yin, C. (1996) 'Ultralow Speed Measurement by Laser
Doppler Velocimetry' in Acta Optica Sinica 16, 252-256
34 Damp, S. (1995)'Use of the Modified Miniatu'e-LDA in High Spatial
Resolution Application' in International Congress on Instrumentation in
Aerospace Simulation Facilities, pp. 21.1-21.5, IEEE,Piscataway, NJ, USA
35 Chandrasekaran, M. and Karwe, M.V. 'Velocity Profiles in the Translational
and Nip Regions of a Reverse Flighted Screw Element' in AIChEJ. (in press)
36 Chandrasekaran, M. and Karwe, M.V. 'Measurement of Velocity Profiles in a
Twin-screw Extruder: Transitions Between For,yard and Reverse Elements' in
J. Reinf. Plast. Compos. (in press)
Towards an understanding
of starch granule structure
and hydrolysis
ChristopherG. Oates
Starch granules are well suited to their role of storage.
They are insoluble in water and densely packed, but still
accessible to the plant's metabolic enzymes. Starch
technologists who are interested in the hydrolysis of the
component macromolecules view starch as useful only
once the granule structure has been destroyed; however,
this requires additional processing steps and econol~ic
resources. Several arguments prevail for a closer exami-
nation of the effects of hydrolytic enzymes on na'Live
starch granules, because hydrolysis affects all level,; of
food processing and nutrition.
Many processing operations are dependent on native
starch hydrolysis and/or could be improved by adopt:ing
or optimizing this process. Some natural fermenta'don
processes, which are dependent on the breakdowrL of
raw starch granules (e.g. gari, a traditional foodstuff in
Copyright~1997,ElsevierScienceLtd.All rightsreserved.O924-2244/97/$17.00 375
PII: SO924-2244(97)01090-X
Table 1. Influence of the major starch fractions on the properties of maize
starch"
Source Amylosecontent(%) Properties
Waxy maize 0-1 Non-gelling, low-setback and clear
paste; paste that is resistant to
syneresis; elastic and stringy paste
Maize 27 Firm gel; opaque paste; short paste
texture
Amylomaize 50-70 Granule that is resistant to swelling;
rigid gel; opaque paste; high paste
temperature
~Data taken from Ref.49
West Africa), can be improved by increasing the levels
of endogenous amylases that degrade the raw starch in a
controlled and efficient manner. Industrial fermentation
processes could be operated more cost-effectively by
carrying out the direct hydrolysis of native starch gran-
ules without prior gelatinization. However, such pro-
cedures do not exist because with our current technol-
ogy they remain economically unattractive.
Variable amounts of starch in food avoid digestion in
the human intestine and pass into the colonL The classi-
fication of poorly digestible starch material for nu-
tritional purposes is based on the intrinsic factors affect-
ing starch digestibility 2. Slowly digestible starch will be
slowly but completely digested in the small intestine;
this can include starch that is physically 'protected'
from pancreatic amylases. Such protection occurs if
starch is contained within intact plant structures includ-
ing uncooked cereals; if swelling of the granules is in-
hibited by the rigidity of the cell walls, as in legumes; or
if starch is densely packed in foods such as spaghetti:.
Generally, the crystalline structure of the starch granules
in slowly digestible starch will have been destroyed dur-
ing processing, although the granules may still appear to
be intact. In contrast, resistant starch is capable of resist-
ing hydrolysis by pancreatic amylases and will therefore
pass through the small intestine. Three subclasses are
generally identified: physically inaccessible starch, for
example partly milled grains and seeds (RS0; resistant
starch granules, as found in raw potato and banana
(RS2); and starch in which the macromolecules have re-
associated following gelatinization, usually a result of
cooling, and which is known as retrograded starch (RS3).
Intact resistant (RS2) starch granules can be present in
some foods that are ingested raw (e.g. banana) or in
foods that still contain a proportion of starch granules in
their native form after cooking (e.g. biscuits). Feeds for
poultry and pigs contain a substantial proportion of
starchy material that is still in the granular state.
The ability to manipulate and understand the hydroly-
sis of starch granules offers several nutritional impli-
cations. Blood glucose and insulin concentrations can be
regulated by controlling granule hydrolysis; resistant gran-
ules are hydrolysed slowly, thereby providing a more
376
continuous supply of absorbable glucose at lower con-
centrations. Starch that has been partly hydrolysed by
pancreatic amylases enters the colon and undergoes fer-
mentation, or in rare cases appears in the faeces 3. Starch
that escapes digestion in the small intestine is available
for fermentative breakdown by the anaerobic flora of the
colon. Products of such fermentation, short-chain fatty
acids, are reputed to influence the colon epithelium, im-
parting protection against cancer of the large boweP.
Starch granule structure
Macromolecules
Starch is defined as a mix of two distinct
polysaccharide fractions - amylose and amylopectin;
both are composed of glucose but differ in size and
shape. Amylopectin, the larger (107-109Da) fraction, is
highly branched; 5% of its structure is c~(1----6) branch
links 4. Current models for amylopectin fine structure
suggest two populations of chains, A- and B-chains,
which are present in almost equal proportions. The
designation of the two chain groups is based on the
relative position of a chain in the macromolecule
structure: A-chains are unbranched and attached to the
molecule by a single linkage, whereas B-chains are
branched and connected to two or more other chains.
Each amylopectin molecule will also possess a single
C-cMin, which contains the sole reducing group. Size
distribution of the chains reveals a bimodal distribution
in which the fraction associated with the smaller chain
size [degree of polymerization (DP)~-15] is composed
of A-chains and small B-chains. The second, larger,
population of chains (DP~-45) is thought to comprise
long B-chains. The chains are assembled in a cluster
structure based on early models proposed by Nikuni and
French (reviewed by Lineback s) and later modified by
Robin e t al. ~ Amylose is the smaller of the two fractions
(10s-106Da; DP 500-5000) and possesses very few
branches, 9-20 per molecule, with chain lengths of
between 4 and >100 glucose units 7. Structurally, and, in
many applications, functionally, amylopectin is the
more important of the two fractions, because alone it is
sufficient to generate granules, as occurs in mutant
starches that are devoid of amylose. Plants can be bred
that produce starches with amylose to amylopectin
contents outside the 'normal' range; for example, maize
can be grown with an amylose content as high as 70%
(amylomaize) or as low as zero (waxy maize). Such
families of starches are useful for studying the roles of
amylose and amylopectin within the granule but also
have immense commercial value because they provide a
'natural' means of modifying and extending the range of
functionality of a specific starch type (Table 1).
Granules are semi-crystalline
Native starches are semi-crystalline structures. This
general concept, first proposed in the 1920s, has subse-
quently been extended to describe the levels of struc-
tural complexity that are now regarded as important to
granule structure. The first level is the 'cluster arrange-
ment' of the amylopectin branches. Thi,; arrangement
Trends in Food Science & Technology November 1997 [Vol. 81
describes a structure characterized by
alternating regions of ordered, tightly
packed, parallel glucan chains and Amorphous
less-ordered regions that are predomi-
nantly composed of branch points. A
unit of an amylopectin cluster can thus
be regarded as comprising an amor-
Semi-crystalline
growth ring
A•mor.p• background
Amorphous ........... ~,,~.,. . . . .
growth rin ~,4~,.ys!al.l.!n'ei!.a~me.[[a~ii:'
Amorphous lamella~-
(c)
:~ A-chaiTn

phous region containing most of the

tightly spaced branches (amorphous ~ ~ .~~ : "
lamella) and a thin crystalline region
(~5nm) containing the parallel glu-
cans (crystalline lamellay; the size of
each cluster is highly conserved at 9 nm
~--
!__.~[_~Clust
[Z~usier
(Fig. 1)9.
Native starch granules contain be- ~ - ~'~:',: ~. •

tween 15% and 45% of crystallite ma-

terial (the measurement of absolute
crystallinity has been reviewed by
Blanshard 1°) and yield X-ray diffrac- \
tion patterns that correspond to one of
two limiting polymorphs (A or B) or an
intermediate form (C) (Table 2). Crys- Fig. 1
tallinity occurs within the ordered ar- Schematic diagram of the structure of a starch granule. (a), Single granule comprising concentric rings,
rays of amylopectin and is created by each containing stacks of amorphous and crystalline lamellae; (b), amorphous and crystalline lamellae;
the intertwining of chains with a linear (c), chains of amylopectin arranged in a cluster structure. (Reproduced with permission from Jenkins
length >10 glucose units to form double
and DonaldL)
helices. Crystallization or double helix
formation can occur either between
adjacent branches in the same amylopectin branch cluster
Table 2. Starch structure and raw granule hydrolysis
or between adjacent clusters in three dimensions. For all
starches, the number of granular double helices is greater
Enzyme Crystal Amylose
than that calculated as being necessary to yield the con- Starch source hydrolysis a polymorph b content (%)c
tent of crystal material, suggesting differences in the
composition of double helical material m. Cereals
Models that have been proposed for the crystal poly-
morphs of starch are based on similar structural designs Rice 5 A 18
and variation derived from differences in water content Waxy maize 5 A 0
and the packing configuration of double helices u J2.
Corn 4 A 28
Double helices associate in pairs that are nested together
and stabilized by hydrogen and van der Waals' bonds to Wheat 5 A 26
give either the 'A' or ' B ' crystal structures, for which Barley 5 A 22
atomic models have been developed and extensively re-
viewed". The most recent model for cereal starches ('A' Tubers
pattern) describes a face-centred monoclinic unit cell
Potato 1 B 20
incorporating 12 residues located in two left-handed
chains that contain four water molecules between the Tapioca 4 C 18
helices u. Previous models have described an ortho- Sweet potato 3 C 26
rhombic unit cell, or a triclinic unit cell, thus demon-
strating the complexity of these structures L~.The struc- Other
ture of B-pattern starches is more clearly defined and
Sago 2 C 26
composed of a basic repeat unit in which chains are
packed in a hexagonal array. The unit cell has two left- Banana 1 B 16
handed, parallel-stranded, double helices that are ar-
rayed in parallel, forming a hexagonal unit cell. It con- %cale devisedfrom a review of the literaturebasedon a scorefor overall
tains 12 glucose residues and 36 water molecules susceptibilityto amylaseattack (e~-amylaseand glucoamylase).Ranking
based on a scoreof 1-5, where 5 representsgreatestsusceptibility(in
(27%). Half of the water is tightly bound to the double
most studies,>80% hydrolysis)and 1 representsthe lowest susceptibilily
helices and the other half is centred on a 6-fold screw
(in most studies,<20% hydrolysis)
axis that is parallel to the c-axis H. The different crystal hData taken from Ref. 17
structures account for some of the variation observed CDatatakenfrom Ref.50
between the different starch types.

Trends in Food Science & Technology November 1997 [Vol. 8] 377

 molecules and is usually less organized than the rest of
the granule ~°. The non-reducing ends of amylose and
amylopectin radiate out towards the surface of the gran-
ule 17, allowing the addition of further glucose residues
(b)
to extend the amylopectin chains. Further growth occurs
from the surface as fresh shells of amylopectin molecules
are added, radiating outwards.
9 nl:'l Crystalline blocklets are evident in partly hydrolysed
starch granules in the form of residues comprising
k • small, more-or-less spherical blocks 18,19that are tangen-
tially arranged and tightly packed. Packing is in tiny
crystalline sheets (-5 nm thick), which are stacked tan-
gentially to the granule surface. In the hard (relatively
enzyme-resistant) parts of the shells, the blocklets may
Fig. 2 be up to 100nm in diameter, whereas they are only
A superhelical model for the three-dimensional organizatior of 25 nm in the soft parts. The tiny blocks are larger in B-
starch. (a), Superhelical three-dimensional arrangement of a gran~le and C-type granules than in A-type granules. In B-type
section; (b), enlargement of a single turn of the superhelix, displaying granules, much larger blocklets (4-500nm) are super-
the double helices and channel running down through the cenlre.
imposed at the peripheral level to a depth of 10 Dm. It is
tempting to explain the anomaly of greater susceptibility
(Based on Oostergetel and van Bruggen~4,and reproduced ~ith
to enzyme and chemical erosion2° of A-type granules, as
permission from Ball et a12; Copyright © 1996, Cell Press.) compared with that of B-type granules, to the presence
of structurally incorporated protective shells around the
Amylopectin and granule structure less tightly packed B-type granules. The presence of a
Granules are inert, structurally stable entities, whose 'protective' outer layer has been suggested in the case of
degree of stability cannot be explained by crystallinity sago starch; the contents of the granules appear to have
alone - granules contain too little crystalline material. been emptied by extensive enzyme hydrobsis 2~(Fig. 4).
Further levels of structural complexity are invoked to
devise models that explain this apparent anomaly and to A m y l o s e - the mobile fraction
describe the manner in which amorphous and crystalline The interrelationship between amylose and amylopectin
domains are aligned. However, starch structure remains is not completely understood, although the amylose frac-
an enigma because, unfortunately, much of the infor- tion is assumed to exist in the granule as an entity that is
mation that is required to generate a complete model is largely separated from the amylopectin fiaction. Thus,
still lacking TM. The most promising model describes the amylose is able to leach out of the granule. Unfortunately,
structure of a potato starch granule, in which crystalline assigning amylose a precise location within the granule
domains form continuous networks of left-handed heli- is evidently not that easy. Amylose has been located in
ces ~4. Voids inside the superhelices are assumed to be bundles between amylopectin clusters 1° and randomly
empty and are roughly 8-nm wide; the interpenetrating interspersed among clusters in both the amorphous and
superhelices are assumed to form a skeleton on which crystalline regions 22,23. As a result of a series of cross-
the granule is developed (Fig. 2). Considering the rela- linking studies, amylose molecules are thought to occur
tively minor variation in size, shape and properties of in the granule as individual molecules, randomly inter-
starch granules, together with the fact that structural de- spersed among the amylopectin molecules and in close
signs tend to be conserved in nature, it is not unreason- proximity with one another, in both the crystalline and
able to assume that this model will prove to be similar amorphous regions. The location of amylose with respect
for other granular starches is. to the amorphous and/or crystalline regions is dependent
Other structural features that have been identified on the botanical source of the starch. In wheat starch,
following the controlled enzymatic hydrolysis of gran- amylose is mainly found in the amorphous region, but in
ules include growth rings and crystalline blocklets. potato starch it may be partly co-crystallized with amylo-
Internally, granule material is present in the form of pectin 1°. Size is an important criterion. Large amylose
concentric rings, known as growth rings (Fig. 3). These molecules that are present in the granule core are able to
structures are visible under a light microscope in large participate in double helices with amylopectin, whereas
granules (potato and wheat), but are seldom seen in smaller amylose molecules, present at the granule pe-
small-granule starch (barley and rice). The existence of riphery, are able to leach from the granule 24.
growth rings suggests that starch is deposited in a diur- Despite its limited role in crystal formation, amylose
nal rhythm, and that newly synthesized material is de- can influence the arrangement of the double helices in the
posited on the surface as the granule increases in size 4'16. unit cells by interfering with the packing density of the
A model, incorporating growth rings, for granule dew~l- amylopectin chains. The mechanism is not fully under-
opment has been derived in which the first growth layer is stood but is assumed to be the result of either helix for-
initiated at the centre (hilum) of the granule, which con- mation between amylose and amylopectin chains or
tains a large proportion of the reducing ends of starch amylose-induced disruption within the amorphous layer9.

378 Trends in Food Science & Technology November 1997 [Vol. 8]

Fig. 3
Scanning electron photomicrograph of a fractured sago starch
granule, showing concentric growth rings (scale bar = 10 pro).
(C. Oates, unpublished.)
The physical properties of starch granules are complex,
reflecting the composite nature of the different regions. A
model based on the work of Morgan et aI.15 proposes that
in moistened starch, crystalline amylopectin is hard and
brittle, but is interspersed by regions that are composed
of amorphous amylopectin, which is assumed to possess
greater intramolecular mobility and to be similar to a rub-
bery polymer. The rubbery regions in granules are thought
to act as shock absorbers, protecting the granules from
compressive forces 15. The susceptibility of granules to
mechanical disruption will, in part, be influenced by the
contribution of these rubbery regions to the overall prop-
erties of the material. Amylose, which is present in the
amorphous region, possesses less intramolecular mobility
and imparts a more 'solid-like' structure to such regions.
The contribution by Oostergetel and van Bruggen 14
has provided a major step in our understanding of
granule structure. Although this model does not account
for all of the observations, it does provide a promis-
ing foundation for the development of future models
to explain such phenomena as growth rings.
Granules have a surface
A unified concept of the granule surface remains an
enigma, for which, unfortunately, the amount of interest
far outweighs current knowledge. The early suggestion
of a smooth surface, possibly membranous in nature, has
given way to the concept of a discontinuous boundary.
Little information is available on either the molecular
composition or the arrangement at the starch granule
surface. The basis of current models was first described
by Lineback's 25 'hairy billiard ball' model. In this model,
the granule surface is not smooth but characterized by pro-
truding chains. A later modification, by Stark and Lynn26,
described a granule whose surface is characterized by the
ends of amylose chains and protruding amylopectin clus-
ters, which are thought to be the start of the next growth
layer. These immature amylopectin molecules could be the
source of the intermediate material that is often reported
as the 'third' starch fraction. Newly but incompletely
Trends in Food Science & Technology November 1997 [Vol. 8]
Fig. 4
Extensively hydrolysed (>80%) sago starch granules that have been
'emptied' of their contents, leaving behind a shell of highly resistant
material. (a), Scanning electron micrograph; (b), section of
hydrolysed granules, as observed under a normal light
(scale bars = 0.1 ram). (C. Oates, unpublished.)
synthesized islands of amylopectin are loosely attached to
the granule and provide specific points for amylase at-
tack, the intensity of which is tempered by smoothing of
the surface as the amylose molecules fill the gaps/spaces 27.
Notwithstanding the contribution made by amylose, 1:he
granule surface is relatively impenetrable to large mol-
ecules such as amylases owing to the tight packing of
the amylopectin chains.
The porosity of starch granules to water or small, sol-
uble molecules, as outlined by French 17, is based on an
assumption that amorphous regions penetrate throughout
the entire granule and that on hydration they reversibly
swell, forming a continuous gel phase. Molecules of dif-
ferent sizes, but not exceeding 1000Da, are admit~:ed
into the granule to varying extents, suggesting the pres-
ence of a distribution of passage sizes or textures. The
structure of such passages, in the gel phase, is assumed
to consist of spaces between parallel crystalline plates.
The structural attributes for granule porosity invoked
by French 17 account only for the passage of small mol-
ecules into the granule. The entry of hydrolysing en-
zymes and other large molecules into the interior of starch
granules is restricted and only possible through the pores
379
 or channels. Surface pores on granules of corn, sorghum
and millet are suggested to be openings to channels that
penetrate in a roughly radial direction through the gran-
ule z8-3°. Transmission electron microscopy of starch gran-
ule sections has shown the presence of openings in several
granules and measurements suggest they have a diameter
of 0.1-0.3 p,m, compared with 0.07-0.1 I~m for interior
channels. However, the presence of surface pores has
been challengedzg, because of the artefactual nature of
the techniques, including light and electron microscopy,
that were used to demonstrate their presence.
The presence of pores, especially if extensive, would
result in a macroporous structure whose available sur-
face area would be much greater than the boundary
surface area 31. This model would account for the over-
estimates of surface area that are regularly obtained by
gas absorption studies. Provision for surface openings
and interior channels is accommodated in the model of
Oostergetel and van Bruggen~4; a channel runs through
the centre of the superhelical structures.
Holes inside starch granules, located at the centre of the
'Maltese cross' (the characteristic dark cross seen on starch
granules when viewed under polarized light, and the point
at which the hilum is centred), are generally believed to
exist and have been seen in potato, wheat and rice starch
granules 29. It is not clear whether surface or interior holes
are formed naturally or are merely an artefact of drying
during processing or sample preparation. Conceivably,
pressures imposed on the granule by the removal of
water during drying might generate a hole at the weak-
est point (the hilum).
Hydrolysis of raw starch
Native granule hydrolysis is a biological event
The heating of a starch suspension above a critical tem-
perature results in a multistage process known as gelati-
nization. This process involves tangential swelling of the
amorphous regions of the granule, disruption of the radi-
ally ordered structure and eventually opening of the crys-
tal structures as the polymer chains become increasingly
hydrated. Gelatinization greatly enhances the chemical
reactivity of inert starch granules towards amylolytic en-
zymes; this has become the most acceptable method of
enhancing starch hydrolysis and is widely adopted in the
manufacture of starch syrups (glucose, fructose syrups,
etc.). However, starch macromolecules can be hydrolysed
even when they are packaged in their native form; this
occurs in plants and during the digestion of certain foods.
Attempts to hydrolyse native granules invariably result
in a slowly and often poorly hydrolysed product. This pro-
cess is relatively efficient in plants, but plant biochemists
are only just beginning to offer useful insight into the
roles of amylases in this process32'33. Phospholytic degra-
dation of starch is commonly invoked as the main hydro-
lytic route, based on the apparent absence of amylolytic
enzymes and the presence of phospholytic enzymes. How-
ever, recent reports suggest amylases may have a greater
involvement; it is possible that they may not have pre-
viously been extracted because they form very tight asso-
ciations with starch granules 32'34.
380
The hydrolysis of native granules is much slower than
that of gelatinized starch35. This is better understood after
examining the mechanism for glucosidic bond hydrolysis.
Enzyme-catalysed hydrolysis of 0~(1~ 4) glucosidic bonds
involves the enzyme-induced ring distortion of one of the
I>glucosyl residues from the 4Cl-chair conformation to a
'half chair' conformation36. Ring distortion decreases the
enthalpy of activation and increases the susceptibility of
the glucosyl residues to nucleophilic attack by functional
groups on the enzyme and by water. The ability of amyl-
ases to unwind the double-helix structure is driven by the
influence of ring distortion on neighbouring units. Chains
with restricted mobility, either complexed or crystallized,
will be hydrolysed less readily because their constitu-
ent glucose units are more firmly locked into a specific
configuration, but given time, amylases can disentangle
macromolecular chains. The action of glucoamylases and
[3-amylases differs from that of c~-amylases. The exo-
enzymes, B-amylase and glucoamylase, accommodate the
non-reducing end of a substrate chain in their active cen-
tre, the three-dimensional structure of which is 'pocket
like '37. These structures ensure that only a single, low
molecular weight product can be produced. Glycosidic
bond cleavage is at the bond located at the non-reducing
end of the chain and is associated with inversion of the
configuration at the newly generated hydroxyl group at-
tached to C-1.
The digestion of native starch proceeds in two stages:
hydrolysis is initially rapid but continues at a slower and
more constant rate 27, which is more rapidly attained with
a higher initial enzyme concentration. The efficiency
of enzymatic solubilization of granular starch depends
on the type of starch38; surprisingly, the more densely
packed A-pattern polymorph granules are often more
readily hydrolysed than the less densely packed B-
pattern granules 19 (Table 2). This suggests that other
structural features, not yet incorporated into the models,
might also influence the susceptibility of granules to en-
zyme hydrolysis, for example the presence of lipids.
Hydrolysis in a two-phase system
Enzymatic reaction with insoluble substrates such as
starch granules occurs via several steps: diffusion to the
solid surface, adsorption and finally catalysis. The sec-
ond step is assumed to be a prerequisite for subsequent
catalytic activity, requiring the enzyme to pass across
the boundary between the aqueous and solid phases in
order to attach to the granule. The porosity and accessi-
bility of the substrate surface essentially control the
number of adsorption sites on natiw ~, starch granules.
The removal of the assumed adsorption area from the
surface of model granules has been ..shown to prevent
catalytic activity39, but the initial interaction between the
amylases and starch granules may vary.
The adsorption of enzyme to the granule surface does
not necessarily imply the occurrence of subsequent cata-
lytic activity; moreover, the relation between the extent of
adsorption, hydrolysis and granule morphology is not con-
sistent38. Granules from normal and waxy maize2v, despite
looking morphologically similar and be,ing of comparable
Trends in Food Science& Technology November 1997 [Vol. 8]
size and shape, adsorb amylases to different extents. En-
zyme is adsorbed onto waxy maize starch granules with
higher efficiency than onto normal maize granules, but
the rate of solubilization of the two starches is similar.
As well as adsorbing onto the granule surface, amylases
must functionally bind the glucan chains via several glu-
cose units to their subsites 4°. The subsites are located in
the active centre of the enzyme and are capable of inter-
acting with one glucose residue in the substrate; 4-9 sub-
sites are found within an active centre. The binding energy
of each subsite is different and thus promotes productive
amylase-substrate complex formation. The position of the
active site within the active centre and the number of sub-
sites are characteristic for each type of amylase 41. The re-
quirement for glucan units may not be fulfilled because
glucan chains at the granule surface are locked into a con-
formation that is determined by the formation of double
helices and packing into the unit cells. This restriction
ensures that a maximum linear portion of only three glu-
cose units is available; this is insufficient for complete
subsite binding, and promotes product inhibition of the
amylases by blocking the subsite and thus preventing the
formation of a productive enzyme-substrate complex 39.
Within a starch preparation, the granules are not equally
susceptible to enzymatic degradation, which is possibly
a function of the manner in which amylases adsorb to a
granule. Despite the specific mode of attack, starch hy-
drolysis occurs granule by granule, with an attacked gran-
ule being completely hydrolysed 2~,42,43. Susceptibility to
amylase attack represents the ability of the enzyme to at-
tach itself to the granule surface. Granules of different ages
and sizes will vary in the amount of 'loose' material at their
surface. Some granules may have become damaged during
extraction, causing faults or dislocations in their surface
that allow the enzyme greater access to the free gluco-
sidic chains. Minor constituents also influence enzyme
adsorption and the hydrolysis of starch granules: surface
proteins and lipids can reduce surface accessibility by
blocking the adsorption sites and will influence enzyme
binding by smoothing the amylopectin surface branches.
The initial adsorption of amylases is aided by the
presence of specialized starch-granule-binding domains,
which have been identified in some amylases and glu-
coamylases 44,45. The binding site is separate from the
active-centre site and in the case of some amylases is
essential for granule hydrolysis 44.
Granules are hydrolysed differently
The type and extent of morphological change undergone
by starch granules during hydrolysis have been well docu-
mented ~7,19. In general, enzymes either erode the entire
granule surface or sections of it (exocorrosion) or digest
channels from selected points on the surface towards the
centre of the granule (endocorrosion). Initial hydrolysis in
the case of most starch granules is superficial and depend-
ent on the type of starch. Potato and the B-type starches
are slightly eroded by exocorrosion. Wheat, barley, rye
and triticular lenticular starch granules as well as starches
from some tropical tubers (cassava, sweet potato) have
specific susceptible zones that become pitted owing to
Trends in Food Science & Technology November 1997 [Vol. 8]
endocorrosion. Pits become enlarged and canals of endo-
corrosion sink into the interior, and numerous channels are
formed around the granules. Starch granules from nomlal
maize, taro, rice and yam are hydrolysed by the formation
of random and deeply pitted canals that enlarge through
the granules at each shell level. Following the initial at-
tack, in many types of starches, enzymes will hydrol~se
one or more paths to the centre of the granule with sub-
sequent enzyme attack proceeding outwards over a broader
front. It is clean" that during all stages, from initial attack
to later digestion, some regions of the granule are more
readily digested than others ~9,46.Those areas that are sus-
ceptible to enzyme attack are the less organized amorpheus
rings, whereas the crystalline lamellae offer resistance
to enzyme erosion 21,42. Pretreatment, by partial anneal-
ing in an excess of water at 55°C for 2 h, can influence
the relative susceptibility of the granule regions such
that, following initial attack, hydrolysis occurs via the
degradation of sequential layers of the granule interior zl.
Annealing has been shown to alter the crystal strttc-
ture of starches 47, although it does not change the mor-
phology of the granules. Granule swelling, amylcse
leaching and the gelatinization temperature range are all
decreased on annealing, whereas the gelatinization tem-
perature and enthalpy both generally increase. Changes
in the susceptibility of annealed starches to amylase
attack depend on the type of starch; oat starch becomes
more susceptible, whereas lentil, wheat and potato
starches become less prone to attack 47.
Variation in the pattern of enzyme attack is evide.nt
between starch types and within the same population of
granules, and is influenced by the type of amylase. The
activity of amylolytic enzymes for a specific starch is
also dependent., to some extent, on the source of the en-
zyme. Early reports suggest that pancreatic a-amylase is
the most effective towards native starch, followed in
order by barley, bacterial and fungal amylases (reported
in Kimura and Robyt38). The reason for the supposed
activity of pancreatic amylase for starch granules is rLot
known, but the recent discovery of a third substrate bind-
ing site 48 may offer part of the explanation. The third
binding site, discovered in porcine pancreatic amylase,
is not near either the active centre or the surface starch-
binding domain.
Hydrolysis patterns are controlled by the structure of
starch granules. Starches that are hydrolysed from within
require that the enzyme gain access to the granule inl:e-
rior. Assuming a superhelical structure and that amylase
entry into the granule must be via pores and channels, it
is logical that the nature of these structures will control
hydrolysis. The', size of c~- and [3-amylases is - 5 - 6 nm 15,
which is small ,enough for them to fit inside the voids in-
side the superhelix, allowing access to the contents of the
granules. However, the voids are also the only portion,
within the superhelices, that is suitable for amylose-lipid
complex formation. The function of the amylose cona-
plexes may be to plug the voids, thereby restricting en-
zyme access to the granule interior. Starches that are
predominantly hydrolysed by surface abrasion may be
assumed to contain some type of protective layer around
381
 their outer surface, such as blocklets formed by the highly
structured packing of the unit cells. Therefore, in order
to understand or manipulate native granule hydrolysis,
further information is required particularly on the role of
enzyme flexibility, the importance of different oL-amylase
domains, the exact location of the adsorption and cata-
lytic sites, and the amino acid residues involved in the
hydrolysis process.
Conclusions
Our understanding of starch granule structure has im-
proved in recent years, but still remains incomplete. Future
models will have to address structural features that as yet
have not been incorporated, including a more detailed de-
scription of glucan chain availability and granule surface
characteristics. An understanding of both granule structure
and amylase action should improve our ability to regulate
starch granule hydrolysis. Benefits will be derived at dif-
ferent levels of the processing and food utilization chain,
including direct improvements in the industrial utilizat:ion
of starch and the regulation of hydrolysis to prevent post-
harvest losses or to provide novel nutritional benefits.
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