Biomacromolecules 2003, 4, 1198-1202

1198

Platelet Nanocrystals Resulting from the Disruption of Waxy

Maize Starch Granules by Acid Hydrolysis
Jean-Luc Putaux,* Sonia Molina-Boisseau, Thomas Momaur, and Alain Dufresne
Centre de Recherches sur les Macromole cules Ve ge tales - CNRS, BP 53,
38041 Grenoble Cedex 9, France
Received February 6, 2003; Revised Manuscript Received June 2, 2003

Colloidal aqueous suspensions of starch nanocrystals were prepared by submitting native granules from
A-type amylopectin-rich waxy maize to a hydrochloric acid hydrolysis. The insoluble residue contains
polydisperse and more or less individualized platelet nanocrystals corresponding to the lamellae formed by
the association of amylopectin side branches into parallel arrays of double helices. After 2 weeks of hydrolysis,
5-7 nm thick lamellae still connected by R(1f6) linkages were seen edge-on using transmission electron
microscopy. As the hydrolysis progressed up to 6 weeks, more R(1f6) branching points located in the
inter-lamellar areas were severed and the platelets were thus observed in planar view. Despite a variety of
shapes, characteristic geometrical features of the nanocrystalsa were recognized, such as marked 60-65
acute angles and constituting parallelepipedal blocks with a length of 20-40 nm and a width of 15-30 nm.
X-ray and electron diffraction showed that these nanoplatelets retain the crystalline A-type of the parent
granules.
Introduction
Native starch occurs in the form of discrete and partially
crystalline granules mostly composed of two polysaccharides,
namely linear amylose and branched amylopectin, both
polymers consisting of R-linked D-glucosyl units.1-6 Starch
granules exhibit a so-called onion-like structure with more
or less concentric growth rings that are readily visible by
optical and electron microscopy.7-9 Small-angle neutron and
X-ray scattering experiments10-15 have revealed that the
granules exhibited a lamellar structure corresponding to the
stacking of nanometric subunits with a periodicity of 9-10
nm. It is now accepted that the partial crystallinity of native
starch granules is due to a clustered organization of amylopectin side chains.16 The radial orientation of the molecules
was demonstrated by electron diffraction carried out on thin
sections17,18 and, more recently, by microfocus synchrotron
X-ray diffraction experiments performed on native granules.15,19-21 Yamaguchi et al.22 as well as Oostergetel and
van Bruggen23,24 used transmission electron microscopy
(TEM) to observe fragments of granules from various starch
sources, obtained by wet-mashing and/or acid hydrolysis.
Negatively stained samples exhibit more or less organized
lamellar structures, mainly depending on the time of hydrolysis and the botanical origin of the granules. The results
obtained from the morphological and structural characterization of these smaller structural components were subsequently used to propose models describing possible grain
architectures. In particular, a model for potato starch was
proposed by Oostergetel and van Bruggen,24 then developed
by Waigh et al.,15,25,26 where amylopectin lamellae form a
complex superhelical architecture. Although the results of
* To whom correspondence should be addressed.
jean-luc.putaux@cermav.cnrs.fr.
Affiliated with the Joseph Fourier University of Grenoble.

E-mail:

the various studies converged to show that these lamellae

were made of parallel arrays of double-helices from the
amylopectin linear side chains, the main parameter provided
was the thickness of the crystalline lamella which may
slightly vary depending on the starch botanical source but
generally averages around 7 nm. However, only very few
authors have reported data about the lateral dimensions of
these lamellae. Hizukuri and Nikuni27 applied Scherrers
equation to selected peaks in X-ray diffraction spectra from
potato granules and described starch micelles as spheres
with a diameter of 14.5 nm. From TEM pictures of ultrathin
tangential sections of wheat starch granules, Kassenbeck
observed hexagonal networks of elongated units with a length
of 60 nm and a width of 30 nm.28 Expanding on the studies
of Kassenbeck,28,29 Yamaguchi et al.,22 and Oostergetel and
van Bruggen,23 we present here new morphological data on
the individual crystalline lamellae obtained by submitting
waxy maize starch granules to an extended-time hydrochloric
treatment called lintnerization.16 TEM images as well as
electron and X-ray diffraction data were used to characterize
the insoluble hydrolysis residues (referred to as lintners
in the following).
Experimental Section
Preparation of the Nanocrystals. A sample of amylopectin-rich (99%) waxy maize starch (Waxilys) was kindly
supplied by Roquette S.A. (Lestrem, France). According to
the lintnerization procedure described elsewhere,16,30,31 10
g of starch granules were dispersed in 200 mL of 2.2 N
hydrochloric acid, providing suspensions with 5 wt % of solid
product in water. The dispersions were stored at 36C. To
ensure a more homogeneous hydrolysis, they were stirred
for 1 min four times a day. At definite numbers of days,
aliquots were taken and diluted with an equal volume of

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Disruption of Waxy Maize Starch Granules

Biomacromolecules, Vol. 4, No. 5, 2003 1199

Figure 1. TEM micrographs of negatively stained waxy maize starch samples: (a)-(c) fragments of waxy maize starch granules after 2 weeks
of 2.2 N HCl hydrolysis at 36C. In a, a lamellar organization is clearly revealed with the platelets lying parallel to the incident electron beam.
In b and c, parallelepipedal platelets are seen lying flat on the carbon film. The arrow in b indicates a pyramidal stack of crystals. (d) Nearly
individual waxy maize starch nanocrystals obtained after 6 weeks of hydrolysis (scale bars: 50 nm).

water. The insoluble particles were washed by successive

centrifugations in distilled water to neutrality. The pellets
were finally redispersed in water and the suspensions kept
at 6C. The yields of hydrolysis were determined by calculating the ratio between the dry weight of insoluble residue
at a given time of hydrolysis and that of initial native starch
granules. Values were around 30 wt % after two weeks and
5 wt % after 6 weeks.
Transmission Electron Microscopy. Specimens for conventional TEM imaging were prepared. After a brief sonication, a drop of a dilute starch nanocrystals suspension was
deposited onto a glow discharged carbon-coated microscopy
grid. Prior to complete drying, a drop of 2% (w/v) uranyl
acetate negative stain was added. After 1 min, the liquid in
excess was blotted with filter paper and the remaining film
was allowed to dry. Unstained specimens for electron
diffraction purpose were also left to equilibrate overnight in
a 95% R.H. (relative humidity) atmosphere. Once positioned
into a Gatan 626 specimen holder, they were quench-frozen
in liquid nitrogen, transferred into the microscope, and
observed at low temperature (-180C). All specimens were
observed using a Philips CM200 Cryo TEM, operated at
80 kV for imaging and 200 kV for diffraction. Micrographs
were recorded on Kodak SO163 films.
X-ray Diffraction. Native waxy maize starch granules
were equilibrated overnight in a 95% R.H. atmosphere and
put into a glass capillary. Lintners obtained after a 6 week
hydrolysis of native granules were air-dried. After the liquid
in excess was evaporated, the solid residue was inserted into
a glass capillary and rehydrated overnight in a 95% R.H.

atmosphere. Both capillaries were sealed and X-rayed using

a Philips PW3830 generator operating at the Ni-filtered Cu
KR radiation wavelength ( ) 1.542 ). Powder diffraction
patterns were recorded on Fuji imaging plates read by a Fuji
BAS 1800 II Phospho-Imager.
Results and Discussion
After hydrolizing starch granules for 2 weeks, micrometersized fragments could be seen in the TEM samples. Some
200-500 nm wide compact fragments were also found.
Those were thin enough to be conveniently observed by TEM
after negative staining. Figure 1a-c shows different areas
of such fragments. In Figure 1a, the negative stain clearly
reveals a lamellar structure consisting of a stack of elongated
elements, with a width of 5-7 nm. These elements appear
as white objects protruding out of the contrasting layer of
stain. Such a lamellar organization was also reported by
Yamagushi et al. to describe fragments of wet-mashed waxy
maize starch granules22 and by Oostergetel and van Bruggen
for acid-hydrolyzed fragments of potato starch granules.23
Considering the cluster model describing the organization
of amylopectin molecules inside starch granules,1,16 the
objects visualized in Figure 1a likely correspond to an edgeon view of the lamellae formed by the association of
amylopectin side-branches into parallel arrays of double
helices. The thickness of the lamellae is consistent with the
results obtained by Robin et al.16 by gel chromatography on
lintnerized waxy maize starch: the lamellae would contain
linear chains of DP 15 and single-branched molecules of DP
25. The periodic organization is not as regular as that observ-

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Biomacromolecules, Vol. 4, No. 5, 2003

ed by Oostergetel et al. in images of negatively stained wetmashed fragments from native waxy maize granules.23 Thus,
the lamellar arrangement may have already been disrupted
to some extent, with the acid having hydrolyzed a certain
amount of R(1f6) bonds located in the amorphous region
between crystalline lamellae. The existence of intralamellar
R(1f6) branching points has been reported in the literature32
for waxy maize although it was shown that they were protected during acid hydrolysis.33
On the edges of some of the fragments obtained after 2
weeks of hydrolysis, polygonal objects with sharp 60-65
acute angles were also observed, either stacked in a pyramidal
fashion (such a stack is indicated by an arrow in Figure 1b)
or more randomly distributed (Figure 1c). If a sufficient
fraction of R(1f6) junctions located between crystalline
lamellae has been broken by the hydrolysis, we can assume
that the elongated units seen edge-on in Figure 1a can partially separate and consequently lie flat on the supporting
carbon film. The insoluble residue obtained after 6 weeks of
acid hydrolysis is shown in Figure 1d. Polygonal objects
similar to those present in Figure 1, parts b and c, can be seen
but they are now more frequent and fairly well individualized. As more remaining R(1f6) branching points located in
the interlamellar areas have been hydrolyzed, the crystalline
platelets could freely separate. The separation process is
schematized in Figure 2, parts b and c. However, it has to be
pointed out that fully individual nanocrystals could hardly
be seen. Although those shown in Figure 1d are fairly well
dispersed, we often observed aggregates of randomly flocculated platelets.
A specimen was prepared in frozen-hydrated conditions
from a 6 week hydrolysis dispersion and selected area electron
diffraction patterns were recorded on aggregates of flocculated
nanocrystals (Figure 3c). They were compared with the X-ray
diffraction powder patterns of native waxy maize granules
as well as hydrated lintners sealed in glass capillaries (Figure
3, parts a and b, respectively). The platelets clearly exhibit an
A-type pattern similar to that recorded from the parent
granules.
It is interesting to compare the overall shape of the starch
nanocrystals derived from the micrographs in Figure 1 with
the geometry of the (a,b) projection of the monoclinic unit
cell of the A-type amylose allomorph34,35 (Figure 2b). The
angle between the a and b axes is ) 123.5 35 which gives
a complementary acute angle of - ) 56.5. This value
is close to the acute angle measured from the platelets.
Considering the small size of the lamella and their very weak
contrast when observed at low magnification without negative staining, no single crystal diffraction pattern could be
successfully recorded from individual platelets either seen
edge-on or lying flat on the carbon film. Consequently, it
was not possible to confirm the results that van Breemen et
al., as well as Oostergetel and van Bruggen, obtained from
electron diffraction experiments on fragments of waxy
maize36 and potato granules23 respectively. In both reports,
it was stated that the platelet mean plane was inclined with
respect to the axis of the double helices. An angle of about
65 was proposed for potato starch.23 Waigh et al., fitting
small-angle X-ray scattering (SAXS) patterns, subsequently

Putaux et al.

Figure 2. Schematical drawing describing the ultrastructure, shape

and organization of the lamellae observed in Figure 1. (a) Projection
of the double helices in the (a,b) plane of the monoclinic A-type unit
cell.35 (b) Longitudinal and planar views of lamellar fragments as seen
in Figure 1, parts a and b. The longitudinal view shows the 9-10 nm
alternation of crystalline and amorphous lamellae. Circled above is
an enlarged view of a 5-7 nm thick crystalline lamella showing the
association of doubles helices (vertical rectangles) formed by the
amylopectin side chains. For clarity, the possibility that the lamellar
plane forms an angle with the helical axis15,24 was not taken into
account in the scheme. (c) Distribution of more individualized platelets
obtained after 6 weeks of hydrolysis and seen in planar view (cf.
Figure 1d).

proposed a value of about 72.14 No definite value was given

for waxy maize.36 Taking these results into account would
mean that the double helices in the lintners shown in Figure
1d are not parallel to the incident electron beam and that
the observation plane is not the (a,b) base plane. This tilting
effect may explain why the acute angle measured on several
platelets (60-65) is generally larger than that predicted from
the A amylose monoclinic unit cell (56.5).
The relative crystallinity of native waxy maize granules
has been estimated by various authors using X-ray diffraction. Values roughly vary from 30%37 to 50%.38 It is thus
surprising that only 5 wt % of the initial product was
retrieved after 6 weeks of HCl hydrolysis. What does this
5 wt % represent with respect to the parent granules?
Considering the 30 wt % yield estimated after 2 weeks, the
TEM data presented in our study preferentially illustrate the
behavior of the granule fraction that is the less susceptible

Disruption of Waxy Maize Starch Granules

Figure 3. X-ray powder diffraction patterns of (a) native waxy maize

starch granules and (b) lintners obtained after a 6 weeks hydrolysis.
(c) Low dose electron diffraction pattern recorded at low temperature
on a group of frozen-hydrated nanocrystals obtained after 6 weeks
of HCl hydrolysis. In all cases, the rings are typical of the A-type
allomorph.

to hydrolysis. We chose to focus on the morphological and

ultrastructural aspects of the degradation. For chromatographic data on the evolution of the soluble and insoluble
dextrins during waxy maize hydrolysis, the reader can refer
to the experiments reported by Robin et al.39 and Jakarody
and Hoover38 in HCl, as well as by Umeki et al.,32 Jackson
et al.,40 and Jane et al.33 in H2SO4.
The higher susceptibility to acid of amylopectin-rich waxy
maize compared to other cultivars was attibuted to the fact
that hydrolysis severed branched molecules more readily than
linear molecules. Moreoever, the two-stage profile of the
hydrolysis of waxy maize starch was explained by the fact
that acid attacked amorphous areas more rapidly than the
crystalline ones, less accessible to H3O+ ions.38,39 The
hydrolysis of the granules appears to be governed by two
factors: reactivity and accessibility. Because our starting
substrate was composed of whole native granules, the
accessibility of the compact crystalline areas to acid was
clearly reduced. However, platelets such as those shown in
Figure 1c could be detected as early as after a few days of
hydrolysis (not shown). This proves that the degradation of
the crystalline growth rings, although slow, and that of the
amorphous areas are concomitant. The disruption of the
lamellar structure starts as soon as the acid can diffuse in
the crystalline growth rings of the grain. It appears to be a
continuous process that was illustrated in the TEM images
of Figure 1. The lamellar distribution shown in Figure 1a is
not as regular as that observed by Oostergetel et al.23 in
fragments of wet-mashed native granules. Moreover, lamellae
can also be seen in planar view (Figure 1, parts b and c) as
a sufficient number of interlamellar R(1f6) linkages were
broken to allow the crystals to separate. However, it is likely
that the platelets that were unhinged in the earlier stages are
slowly degraded by the acid while new ones are separated
from compact fragments. Our results suggest that, in order
to account for the volumic decrease of the insoluble residue,
the nanoplatelets observed at a certain time represent the

Biomacromolecules, Vol. 4, No. 5, 2003 1201

more recently separated crystals and not necessarily the most

resistant ones. An initial fragmentation of the native granules
would certainly favor the accessibility of crystalline areas
to the acid. This would thus enhance the separation of
platelets and increase the yield. As the goal of our study
was the production of colloidal suspensions of starch lintners
and considering that the yield of hydrolysis was as low as 5
wt % after 6 weeks, we did not investigate what happened
after this time. It would be interesting to study the morphology and structure of the dextrins remaining after a few
additional weeks of hydrolysis.
Despite their dispersity in shape and size, the nanocrystals
share common morphological features (Figure 2), i.e., thin
parallelepipedal constituting blocks with marked angles. The
morphology of the blocks is in good agreement with the
current structural model of A-type amylose crystals.35 The
parallelepipedal shape of the idealized individual platelet
seems to be a homothetic view of the monoclinic unit cell
(Figure 2a). However, the lateral size of the crystallites
should be somehow related to the branching pattern of the
amylopectin molecules in native granules. Little is known
about the lateral extension of the lamellae described in the
cluster model. Several hypotheses can be made. The variation
in size of the crystallites may be related to the dispersity in
amylopectin molecular weight. The marked angular shape,
related to definite crystal planes, suggests that rather sharp
transitions exist between crystalline lamellae and less organized areas of amorphous amylopectin that may allow flexibility in the molecular architecture of the granule. Considering the mode of preparation of the specimens, it is not possible to know the region of the grain from which the observed
nanocrystals originate. Their shape and size may differ
depending on the location of the parent growth ring, and
they may be sensitive to the local curvature. Moreover, the
growth of contiguous molecules in a confined environment
may lead to a cross-crystallization phenomenon and result
in platelets that are laterally more extended. All of these
points are certainly worth investigating in further details.
Using tilt experiments and tomographic reconstruction
from TEM images of potato fragments, Oostergetel and van
Bruggen proposed that the crystalline domains were arranged
in a superhelical fashion.24 This model was later refined by
Waigh et al. on the basis of SAXS experiments15 and the
use of a chiral side-chain liquid-crystalline description of
starch granules.25,26 Our results do not confirm nor infirm
that the superhelical model applies to waxy maize starch.
Nevertheless, several remarks can be made. If the model is
valid, the platelets shown in Figure 1, parts c and d, would
correspond to the steps of the superhelical staircase
formed by the amylopectin molecules. However, the shape
of these steps is quite different from the piece of pieshaped lamellar motif proposed by Waigh et al.15 We got
no evidence that the earlier stage of hydrolysis led to the
lateral separation of superhelices followed by the destabilization and fragmentation of the helical structure into its
consituting steps. Indeed, we observed that several 2-weekold fragments seen lying flat on the film often contained
elongated and jagged elements (Figure 1, parts b and c).
These elements appear to be longer than the expected radius

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Biomacromolecules, Vol. 4, No. 5, 2003

of a superhelix. After a longer hydrolysis time, these elements would separate into smaller individual platelets. In
addition, although one may argue that the aggregates observed in our specimens may be a mixture of elements
originating from different areas of the starch granules, the
apparent polydispersity of the individual platelets in terms of
size and shape does not support the idea of a regular superhelical architecture.
Several facts allow us to rule out the possibility that the
observed platelets are artifacts arising from the recrystallization of soluble amylose molecules with a low DP. Metastable dilute aqueous amylose solutions can recrystallize into
different forms. Single crystals are only obtained when small
complexing molecules are added to the solution, with the
shape and crystal structure depending on the complexing
agent. A-type crystals are formed in the presence of acetone.35
Their shape is usually ogival, with a length of a few hundreds
nanometers, and the amylose double helices are oriented
parallel to the crystal long axis. Although sharing their crystal
type with waxy maize lintners, recrystallized A-type single
crystals exhibit a very different shape and chain orientation.
Hexagonal lamellar VH-type crystals made of parallel amylose single helices can be prepared from dilute aqueous
ethanol solutions.41 Electron diffraction patterns recorded
from our samples clearly showed that the lamellae retained
the crystal type of the native granules, namely, A. No V-type
pattern was recorded. When no complexing agent is added,
metastable aqueous amylose or amylopectin solutions retrograde at room temperature. In dilute solutions, the
molecules reorganize to form loose networks with vermicular
branches containing 10-nm-wide B-type semicrystalline
units.42 Such networks were anecdotally observed in our
preparations but their aspect could not be mistaken with that
of the platelet lintners.
Although the existence of starch lamellae had already been
reported elsewhere, it is the first time, to our knowledge,
that shapes and lateral dimensions are derived from the
observation of individual platelets in planar view. The starch
lintners shown in our study share some morphological
features with Laponite platelets (25 nm wide and 1 nm thick
synthetic clay discoids43), although their respective chemical
and crystal structures are very different. Being light and
biodegradable, starch nanocrystals may represent an interesting alternative as model platelet fillers in polymer matrixes.44,45 Work is in progress to determine with precision
the orientation of the c axis of the crystal with respect to the
mean plane of the lamellae in order to see if the superhelical
model developed by Oostergetel and van Bruggen24 and
Waigh et al.26 for potato starch is valid for waxy maize starch.
It would also be interesting to see if crystalline nanoplatelets
can be obtained by submitting starch granules from other
cultivars to an acid hydrolysis, in particular those containing
significant fractions of amylose.
Acknowledgment. The authors thank Dr. A. Buleon
(LPCM, INRA Nantes, France) for helpful discussions and
Dr. H. Chanzy (CERMAV) for continuous support and
critical readings of the manuscript, as well as Roquette S.A.
(Lestrem, France) for the gift of starch samples.

Putaux et al.

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