Chapter 3

Crystalline Structure in Starch

Denis Lourdin, Jean-Luc Putaux, Gabrielle Potocki-Véronèse,

Chloé Chevigny, Agnès Rolland-Sabaté, and Alain Buléon

Abstract Many reviews have been published on the crystalline structure of the
starch granule, addressing aspects such as birefringence, crystallinity, and structural
models for A- and B-type starches. After a synthetic presentation of the general
knowledge on this topic, the present review focuses on a critical description of
the main techniques used to investigate the starch crystalline structure, some new
data regarding crystalline lamellae, and the most recent models established for
the 3D structure of crystalline domains in the granules. Structural and phase
transitions occurring during hydrothermal treatments of starch are briefly presented
as an introduction to a more detailed description of local order and orientation in
amorphous starch materials. Recent results on the structure of amylose complexes
which form by heating in the presence of guest molecules are discussed as well.
Finally, results regarding the in vitro enzymatic synthesis of amylose, which self-
associates into gels or particles, and in vitro enzymatic extension of glycogen
external chains are described. They are evaluated as biomimetic systems for a
better understanding of the mechanisms involved in starch crystallization during
biosynthesis as well as in the different processes used for starch modification.

Keywords Self-association • Crystallinity • Polymorphism • Crystal structure •

Single/double helix • Amylose complexing • Local order • Amorphous • Enzy-
matic synthesis • Amylosucrase

D. Lourdin () • C. Chevigny • A. Rolland-Sabaté • A. Buléon

INRA-UR 1268 Biopolymères Interactions, Assemblages, Rue de la Géraudière, BP 71627
F-44316 Nantes cedex 3, France
e-mail: [email protected]
J.-L. Putaux
Centre de Recherches sur les Macromolécules Végétales (CERMAV), Université Grenoble Alpes,
F-38000 Grenoble, France
CNRS, CERMAV, F-38000 Grenoble, France
G. Potocki-Véronèse
Institut National des Sciences Appliquées, Université de Toulouse, Toulouse, France
Institut National Polytechnique, Université Paul Sabatier, Toulouse, France
Ingénierie des Systèmes Biologiques et des Procédés, Toulouse, France
UMR5504, UMR792 Ingénierie des Systèmes Biologiques et des Procédés, Centre National de la
Recherche Scientifique, Institut National de la Recherche Agronomique, Toulouse, France

© Springer Japan 2015 61

Y. Nakamura (ed.), Starch, DOI 10.1007/978-4-431-55495-0_3
62 D. Lourdin et al.

3.1 The Crystalline Structure of Native Starch

Starch is biosynthesized as semicrystalline granules whose shape and size depend

on the botanical origin (Fig. 3.1a, c). The starch granule presents a hierarchical
and multiscale organization with structural length scales ranging from the tenth of
nanometer for the glucose monomer up to a few micrometers for the granule size.
Intermediate structural levels include the crystalline and amorphous lamellae repeat
distance (9–10 nm), the blocklets (30–200 nm), the growth rings (200–600 nm), and
other supramolecular arrangements like superhelices (for reviews, see French 1984;
Buléon et al. 1998a, b; Tang et al. 2006; Pérez and Bertoft 2010).

Fig. 3.1 Potato starch granules observed by scanning electron microscopy (a), polarized light
optical microscopy (b), scanning confocal light microscopy (c). Scale bars: 20 m (From Buléon
et al. 2007 – images courtesy D. Dupeyre, CERMAV). (d, e) Synchrotron X-ray microfocus
mapping of a smooth pea starch granules (From Buléon et al. 1998a, b)
3 Crystalline Structure in Starch 63

3.1.1 About Chain Orientation in the Starch Granule

When starch granules are observed under polarized light, a characteristic Maltese
cross (centered at the hilum) can be seen (Fig. 3.1b), which has led to consider
the granules as distorted spherocrystals. The sign of birefringence is positive with
respect to the spherocrystal radius (French 1984) which indicates that, theoretically,
the average orientation of the polymer chains is radial. The intensity of birefringence
strongly depends on the shape and orientation of the granules with respect to the
light beam. Therefore, for nonspherical granules, it is more accurate to say that the
orientation is perpendicular to the growth rings and to the granule surface (French
1984). This was also confirmed by solid-state light scattering of starch granules
(Borch et al. 1972).
Further results on chain orientation in potato and wheat starch granules were
obtained by Chanzy et al. (1990) and Helbert and Chanzy (1996), using selected
area electron diffraction over 1 m2 on thin sections of partially hydrolyzed
starch granules. However, in both studies, the diffractograms were not very well
resolved because of the large amount of inelastic electron scattering inherent to this
technique.
Significant improvement was achieved using synchrotron microfocus X-ray
diffraction with a 1–2 m beam. Buléon et al. (1997) (Fig. 3.1d, e) and Waigh
et al. (1997) showed that the orientation of polymer chains in potato starch was
very high at the periphery of the granule and perpendicular to its surface. A lower
degree of orientation was found in the internal regions and at the hilum, but the
interpretation was more complex since in the center of the granule, the beam
averages over regions containing helices pointing forward, backward, and sideways.
No specific orientation at a 1–2 m scale was found for wheat starch granules, either
at the periphery or in the center (Buléon et al. 1997), which means that the radial
orientation in such granules is weak and limited to smaller domains. Nevertheless,
these pioneering works also showed that it was possible to record highly oriented
diffraction diagrams, very close to those stemming from recrystallized amylose
fibers used to solve the three-dimensional structure of B-type amylose (Buléon et al.
1997). These results show that the corresponding models can be transposed to the
crystalline domains in the starch granule.
More recently, many synchrotron microdiffraction experiments confirmed the
radial chain orientation in starch granules from various botanical origins (Chanzy
et al. 2006). The most recent advances of synchrotrons and the development of
the ID13 microfocus beamline at ESRF (Grenoble, France) (Riekel 2000) have
significantly improved the microdiffraction method which has now been applied
to a number of specimens to study specific properties of individual granules, for
instance, the structural changes induced by microdrop hydration (Lemke et al.
2004), high pressure (Gebhardt et al. 2007), or radiation damage propagation
(Riekel et al. 2010). X-ray probes with a diameter as small as 0.5 m are currently
available, with the brilliance of the synchrotron, which allows to probe the molecular
organization in individual concentric growth rings in the starch granule.
64 D. Lourdin et al.

3.1.2 About Crystallinity

The inner architecture of native starch granules is characterized by “growth rings”

(Fig. 3.2a, b) that correspond to concentric semicrystalline 120–400 nm-thick shells
separated by amorphous regions (French 1984; Buléon et al. 1998a, b; Donald
et al. 2001). The crystalline shells consist of a regular alternation of amorphous
and crystalline lamellae with a repeat distance of 9–10 nm (French 1984; Cameron
and Donald 1993; Jenkins et al. 1993; Cardoso and Westfahl 2010). Parallel arrays
of double helices made of amylopectin short branches form crystalline lamellae
(Fig. 3.2c, d). Their 5–7 nm thickness presents some variation depending on the
botanical origin. Native starch granules exhibit two main allomorphic types that can
be identified using X-ray scattering (Fig. 3.2g) or solid-state 13 C NMR spectroscopy.
The A type mainly occurs in cereal starches and the B type in tubers and amylose-
rich starches (Buléon et al. 1984; Zobel 1988). New morphological data on the
individual crystalline lamellae of waxy maize starch granules have been reported

Fig. 3.2 Schematic representation of the starch granule ultrastructure from growth rings to
amylopectin: (a) ultrathin section of a waxy maize starch granule (staining with uranyl acetate
and lead citrate (TEM image courtesy I. Paintrand, CERMAV); (b) alternation of amorphous and
semicrystalline growth rings; (c) clustered model of amylopectin; (d) detail of one cluster showing
the formation of double helices from the short branches of amylopectin; (e) nanocrystals observed
after 6 weeks of acid hydrolysis (TEM image after negative staining courtesy of H. Angellier,
CERMAV – scale bar: 50 nm); (f) simplified drawing of the crystalline platelets and proposed
molecular model in relation to the monoclinic unit cell of the A allomorph; (g) WAXS profiles of
A- and B-type starch (From Buléon et al. 2007)
3 Crystalline Structure in Starch 65

(Putaux et al. 2003). The mild hydrochloric acid hydrolysis of granules from A-
type amylopectin-rich waxy maize leads to an insoluble residue consisting of
polydisperse platelet nanocrystals that retain the crystalline A type of the parent
granules. They form parallelepipedal blocks with a length of 20–40 nm and a width
of 15–30 nm, with characteristic geometrical features such as 60–65ı acute angles
(Fig. 3.2e, f). Their 5–7 nm thickness is consistent with the presence of double
helices made of single strands having a degree of polymerization (DP) between
15 and 18. Such crystalline lamellae could not be as clearly individualized from
B-type starch, although lacy networks of acid-resistant units were obtained from
low-amylose Dianella potato starch (Wikman et al. 2014).
The crystallinity of native starch granules varies from 15 to 45 %, depending on
the starch origin, its hydration level, and the characterization method (Buléon et al.
1987; Paris et al. 1999; Zobel 1988; Gernat et al. 1993; Lemke et al. 2004; Lopez-
Rubio et al. 2008). The B-starch crystallinity depends more strongly on hydration
and increases with water content up to 30–33 % H2 O (dry basis), while dry potato
starch does not show any clearly visible diffraction peak (Buléon et al. 1987, 1998a,
b; Cleven et al. 1978). Wide-angle X-ray scattering (WAXS), solid-state 13 C NMR
(CP-MAS), differential scanning calorimetry (DSC), and, to a lesser extent, Fourier-
transform infrared spectroscopy (FTIR) are the most commonly used techniques
to determine the degree of crystallinity. They are based on different physical
phenomena which makes the comparison of the collected data very tricky, especially
for starch. Indeed, contrary to thermoplastic samples for which the melting enthalpy
is directly correlated with the crystallinity index, starch melting enthalpy cannot be
used to determine crystallinity since numerous and various processes are involved
during melting, including plasticization, swelling in water, competition between
melting and dissolution in water, etc. Therefore, the usual relevant interpretation
concerns the residual melting enthalpy which varies as the starch structure is
disrupted but cannot be quantitatively correlated with the crystallinity. Moreover
in case of amylose-lipid complexes, the melting of “isolated” or “amorphous”
complexes is very close to that of crystalline ones since the intramolecular hydrogen
bonds within the single amylose helix are much stronger than intermolecular H
bonds in the crystal (Biliaderis 1992).
When using FTIR, the 1,065870 cm1 spectral region is assigned to C-O-C, C-
C, and C-H stretching modes, and some peaks have been attributed to ordered (995,
1,047 cm1 ) or amorphous (1,022 cm1 ) starch (Bernazzani et al. 2000; Capron
et al. 2007; Wilson et al. 1987; van Soest et al. 1995). According to Fang and
coworkers (2002), the bands located at 1,023 and 1,083 cm1 could be assigned
to the C-O bond stretching in the C-O-C group of the anhydroglucose rings. The
peak at 1,023 cm1 has been shown to be typical of amorphous starch, while that at
1,049 cm1 increases with increasing crystallinity. However, even when using well-
defined crystalline samples, it is difficult to conclude if the FTIR signal is sensitive
to short-range order or crystallinity. More recently, the technique was successfully
used to determine local order in shape memory starch-based materials (Véchambre
et al. 2010).
66 D. Lourdin et al.

WAXS and solid-state NMR have proved to be the more reliable techniques to
determine the crystallinity and the allomorphic types of starch products. WAXS
signals directly stem from the crystalline regions and long-range order. Numerous
methods are available to determine the crystallinity from WAXS data. Starch crys-
tallinity was initially calculated by Sterling (1960) and Nara et al. (1978) following
the methods developed by Hermans and Weidinger (1948) and Wakelin et al. (1959)
for cellulose. They are based on the two-phase concept which assumes that relatively
perfect crystalline domains (crystallites) are interspersed with amorphous regions.
Solid-state NMR has proved to be a powerful tool to characterize some degree of
molecular order such as helicity in the structure of starchy substrates (Gidley and
Bociek 1985; Veregin et al. 1986; Horii et al. 1987; Morgan et al. 1995). Gidley and
Bociek (1985) demonstrated that C1 and C4 glycosidic sites were more sensitive to
conformational changes than the C2, C3, and C5 carbons since C1 and C4 showed
higher chemical shift dispersions under various conformations of the glycosidic
linkage in ’(1,4) glucans. These statements have been confirmed by Veregin et al.
(1987) and Gidley and Bociek (1988) who showed correlations between the C1
and C4 chemical shifts of ’(1,4) glucans and their torsion angles  and . The
multiplicity (stemming from crystallographic constraints in the crystalline regions)
and the chemical shifts assigned to C1 atoms have been correlated to the glycosidic
conformation and polymorphism of starch (Hewitt et al. 1986; Veregin et al. 1986;
Horii et al. 1987; Paris et al. 1999) and other polysaccharides (Jarvis 1994; Isogai
et al. 1989). The multiplicity of the C1 resonance is determined by space group
equivalent symmetry classes (Veregin et al. 1986; Horii et al. 1987), and for starch,
the C1 resonance appears as a triplet for A type and a doublet for B type. Strictly
integrating these resonances, directly linked to the crystalline domains, leads to
crystallinity values close to those determined by WAXS (Paris et al. 1999).
The influence of hydration on the ordering of starch chains and decrease of the
spectral bandwidth was also studied (Tanner et al. 1987; Horii et al. 1987; Paris et al.
1999). The NMR data have been used to identify, and sometimes quantify, the crys-
talline order of native, hydrolyzed, or gelatinized starches (Willenbucher et al. 1992;
Paris et al. 1999), but there is still some debate about the exact level of structure
reached with this technique, i.e., the degree of helicity (molecular order) or the frac-
tion of helices packed with respect to crystalline order (Gidley and Bociek, 1985)
and the quantitative character of the measurements. While diffraction methods are
the main techniques used to quantify crystallinity, shorter-range subcrystalline order
can be probed by solid-state NMR spectroscopy which is sensitive to structure at
the sub-nanometer level. Starch spectra are interpreted in terms of a combination
of amorphous and helical conformations, irrespective of whether helices are present
within crystallites or not. Thus, Lopez-Rubio and coworkers (2008) have recently
proposed to jointly use WAXS and 13 C CP-MAS NMR spectroscopy to determine
crystallinity, the amounts of single and double helices (Fig. 3.3), amorphous single
and double helical components being determined from NMR spectra as described
by Tan et al. (2007). In this work, the crystallinity was determined by fitting
experimental WAXS curves with theoretical discrete diffraction peaks determined
from the 3D structure of A or B starch and an amorphous background.
3 Crystalline Structure in Starch 67

Fig. 3.3 Combined use of WAXS and 13 C CP-MAS NMR for the determination of helical order
and crystallinity: (a) fit of a A-type WAXS profile using an amorphous background and discrete
diffraction peaks calculated from the A-type structure (From Lopez-Rubio et al. 2008); (b) solid-
state 13 C NMR spectra of A- and B-type recrystallized amylose (From Paris et al. 1999); (c)
decomposition of the 13 C NMR spectrum into contributions from the amorphous and ordered
phases in potato starch by subtraction at 84 ppm and (d) deconvolution of the ordered subspectrum
for potato starch (From Lopez-Rubio et al. 2008)

3.1.3 The Three-Dimensional Models of Crystalline Domains

in Starch

The low crystallinity and complex ultrastructure of the starch granule do not
allow direct determination of the three-dimensional shape and distribution of the
crystalline domains. Mild acid hydrolysis has widely been used to investigate the
structure and properties of starch crystallites by preferential degradation of the
amorphous regions (Buléon et al. 1987, 1998a, b). The extent of this preferential
erosion depends on the density difference between the crystalline and the amorphous
regions (Vermeylen et al. 2004). As previously mentioned, A-type nanocrystals
obtained by extended mild hydrolysis of waxy maize starch granules correspond to
the crystalline lamellae present in the native granule (Putaux et al. 2003) (Fig. 3.2).
However, electron diffraction data could not be collected from individual platelets,
because of their small lateral size and thickness.
68 D. Lourdin et al.

Contrary to amylopectin which constitutes the backbone of the semicrystalline

structure of starch granules, amylose can easily be crystallized from solution by
cooling or addition of a precipitant. This property has been used to prepare model
crystalline substrates whose diffraction data and thermal properties were analyzed
to propose three-dimensional models of starch crystallites and to understand the
phase transitions involved in various applications and the processing of starch
(Buléon et al. 1987; Whittam et al. 1990). The resulting morphology (aggregates,
precipitates, gels, single crystals, spherulites, etc. – Fig. 3.4) and allomorphic
type (A, B) depend on factors such as solvent, molar mass, branching degree,
concentration, or temperature (Buléon et al. 1984, 2007; Pfannemüller 1987; Gidley
and Bulpin 1987). A general rule is that long chains and low recrystallization
temperatures favor the B type, whereas high concentrations, high temperatures, and
short chains are known to induce A-type crystallization (Buléon et al. 2007). This
behavior has some similarities with the crystallization of starch during biosynthesis,

Fig. 3.4 Recrystallized amylose: (a) retrograded gel network from diluted long chains, (b) A-
type crystals prepared from a narrow fraction of amylose biosynthesized in vitro (DPn D 17.4 –
from Montesanti et al. 2010), (c) A-type spherocrystals and (d) corresponding electron diffraction
pattern recorded from a ultrathin section of a spherocrystal (From Helbert et al. 1993)
3 Crystalline Structure in Starch 69

since it is well known that (i) B-type starches amylopectin short chains are longer
than in A type (Hizukuri 1985); (ii) A type is mostly present in cereal grains, which
grow in warmer and dryer conditions than tubers starches; and (iii) an increase of
temperature during the plant growth favors A type (Gérard et al. 2000).
Most molecular models proposed to describe the structure of the crystalline
domains in starch granules were built using WAXS data collected from recrystal-
lized fibers (Wu and Sarko 1978a, b; Imberty and Pérez 1988) or lamellar crystals
(Imberty et al. 1988). The structural models established from A and B amylose
crystals were transposed to crystalline regions of native starch, which exhibit similar
but much less resolved diffraction diagrams. WAXS fiber diagrams recorded from
native potato and pea starch granules using synchrotron microbeam X-ray mapping
confirmed that these A- and B-type models could also be applied to describe native
starch crystallites (Waigh et al. 1997; Buléon et al. 1998a, b). In these A and
B structures, amylose exhibits a sixfold left-handed double helical conformation
with pitches of 2.08 and 2.13 nm, respectively (Imberty et al. 1988; Imberty et al.
1988, Takahashi et al. 2004). In the A structure, these double helices are packed
with the B2 space group in a monoclinic unit cell (a D 2.124 nm, b D 1.172 nm,
c D 1.069 nm,  D123.5ı) with four water molecules per unit cell (Imberty et al.
1988). In the B structure, double helices are packed with the P61 space group
in a hexagonal unit cell (a D b D 1.85 nm, c D 1.04 nm,  D120ı ) with 36 water
molecules per unit cell. The symmetry of the double helices differs in A and B
structures, since the repeated unit is a maltotriosyl unit in the A form and a maltosyl
unit in the B form, which is in agreement with the triplet and the doublet observed
by solid-state NMR for the C1 peak for the A form and the B form, respectively
(Fig. 3.3).
5–15 m-long needlelike A-amylose single crystals have recently been prepared
from dilute (0.05 % w/v) solutions of a short-chain amylose fraction (DPn D 17:4)
synthesized in vitro by amylosucrase from sucrose (see Sect. 3.3). Their size and
perfection allowed to collect WAXS datasets from single crystals, up to a resolution
of 0.151 nm, using a microbeam of synchrotron radiation (Fig. 3.5a, b) (Popov
et al. 2006, 2009). A total of 57 independent reflections were used to refine the unit
cell and confirm the space group proposed by Imberty et al. (1988). However, the
high resolution of the diffraction data, which was unique for a crystalline polymer,
allowed to resolve important new fine details. Pockets of two intracrystalline
water molecules were distributed between the double helices, resulting in helical
distortions (Fig. 3.5c). In addition, a tight network of hydrogen bonds involving
the primary and secondary hydroxyl groups of the glucosyl moieties stabilized the
structure (Fig. 3.5d). The refinement of the new structure indicated a “parallel-
down” organization of the amylose molecules within the unit cell as opposed to
the previous “parallel-up” model (Imberty et al. 1988). This new feature indicates
that within the crystals, the nonreducing ends of the amylose molecules are oriented
toward the c-axis direction of the unit cell. The description of this geometry
is important to correlate the crystallography of the A-type granules with their
ultrastructure and mode of biosynthesis.
70 D. Lourdin et al.

Fig. 3.5 (a) SEM image of A-amylose single crystals prepared by crystallizing amylose biosyn-
thesized in vitro; (b) one crystal glued to a borosilicate glass capillary tip; (c, d) projections on the
(a, b) and (a, c) planes of the structure determined by crystallographic analysis of the synchrotron
X-ray diffraction data collected from such single crystals (From Popov et al. 2009)

The general shape of the starch nanocrystals previously mentioned and obtained
by mild acid hydrolysis of waxy maize starch granules can also be described by
comparison to the unit cell of the A allomorph. The base-plane projection of the
nanoplatelets would be homothetic to the (a, b) plane of the monoclinic unit cell
(Fig. 3.2f). The experimental acute angles measured from the nanocrystals (60–
65ı ) are close to the 56.6ı acute angle complementary to the ” angle of the unit cell
(Fig. 3.2f) (Putaux et al. 2003; Pérez and Bertoft 2010).
3 Crystalline Structure in Starch 71

3.2 Phase Transitions and Crystalline Structure of Starch

In most uses of starch, the granule is disrupted, in particular during hydrothermal

treatments like extrusion, cooking, high-pressure treatments, etc. Many reviews
have been published on hydrothermal treatments of starch and related phenomena
such as gelatinization, melting, gelation, retrogradation, structural transition, and
amylose complexing (Buléon and Colonna 2007; Colonna and Buléon 2010). Once
the granule has been disrupted, the structure and properties of the resulting material
are governed by the way amylose and amylopectin rearrange during cooling, which
strongly depends on temperature, shear, and water content. Starch glassy materials
are obtained by melting of starch in processing conditions yielding a final water
content below the glass transition domain. On the contrary, as soon as a sufficient
amount of water is present, amylose and amylopectin recrystallize mostly into
B type. This starch retrogradation is the basis of many food uses of starch as
gelling agent. During hydrothermal treatments, amylose can form semicrystalline
single helical inclusion complexes with small molecules (like lipids, alcohols, flavor
compounds, and various small hydrophobic molecules). The helical conformation,
crystalline packing, and intra- or inter-helical inclusion depend on the complexing
molecules, and these parameters govern the thermal stability of the complex and
the way the guest molecule is released. Starch melting/retrogradation and starch
complexing by lipids and alcohols are well documented (see, e.g., Colonna and
Buléon 2010). The following section focuses, on one hand, on local order and
orientation in amorphous starch, as a function of the thermomechanical history, and,
on the other hand, on the most recent structures determined for amylose complexes.

3.2.1 Local Order and Orientation in Amorphous

and Semicrystalline Starch Materials

Amorphous starch materials present no long-range ordering (i.e., crystallinity),

but locally keep some organization and orientation which depend on the process
used for the starch structure disruption and the thermomechanical history of
the material. This local order can be studied by techniques such as solid-state
NMR for short-range order or two-dimensional WAXS and synchrotron radiation
(SR) infrared microspectroscopy for local orientation. Paris and coworkers, in
the early 2000s, used 13 C CP-MAS solid-state NMR for the characterization of
the different local-range orderings present in amorphous/semicrystalline starch,
amylose, or amylopectin, prepared by different techniques like casting, freeze-
drying, or 2-propanol precipitation from solution (Paris et al. 2001). All samples
were amorphous except reprecipitated ones. A major part of the project consisted
in providing a reliable spectral decomposition of the C1 resonance spectrum, which
revealed the existence of four to five main types of ’(1,4) linkages which were
quantified (Paris et al. 2001). Thus, accepting the classical correlation (Gidley
72 D. Lourdin et al.

and Bociek 1988) between the isotropic chemical shift and the conformational
angles (jˆjCj‰j), a specific ’(1,4) conformation was associated to each type.
The conformations used for the decomposition were extracted from a refined
literature analysis on NMR of ’-glucans and transposed to the determination of
local order within the amorphous samples studied. The bands stemming from the
spectral decomposition have the following chemical shift: A (103.4103.2 ppm), B
(102.9 ppm), C (101.4100.4 ppm), D (98.697.1 ppm), and E (94.594.4 ppm).
As an example, the C1 part of the NMR spectra of casted amylose and amylopectin
reprecipitated in 2-propanol, and their corresponding decomposition into four and
five components, respectively, is presented in Fig. 3.6c, d. Full spectra are shown in
Fig.3.6a, b for reference. A, C, D, and E peaks are present for all types of starchy
materials, while the B peak is only present in 2-propanol-reprecipitated samples.
The A peak is the most intense (around 50 % of total area) for all samples, except
for 2-propanol-reprecipitated samples. Its chemical shift (103.3 ppm) is similar to
the classical range of V-type spectra (Horii et al. 1987; Gidley and Bociek 1988)
and single sixfold helical conformation. The B peak, which is only observed for
2-propanol-reprecipitated samples presenting a WAXS diagram characteristic of a
mixture of amylose V2-propanol complex and B type, was attributed to VH -type single
helices, since at that time, amylose conformation present in amylose-2-propanol
complexes was assumed to be a single sixfold helix similar to that constituting the
VH structure. Considering the new structure recently established for the V2-propanol
crystalline complex (Nishiyama et al. 2010), this B line is more probably associated
to a sevenfold single helix. The remaining conformations (C, D, E) are the most
sample sensitive and subject to more variation in chemical shift. The C peak
appears in the range of double helical conformation and could be associated to
paracrystalline bundles stemming from uncompleted melting of starch structure or
very local retrogradation. The D peak has been observed in ’-cyclodextrins with
twisted glycosidic linkage (Gidley and Bociek 1988) and tentatively correlated to
’(1,6) linkages by Morgan et al. (1995). The chemical shift and C/D difference
observed between linear (amylose) and branched (amylopectin or starch) samples
suggest that D conformations are more sensitive to the presence of ’(1,6) and could
be closer to branching points than the C ones. E line, by far the weakest (2–8 %
depending on the sample), is related to energetically unfavorable conformations
such as constrained linkages.
Each type of amorphous starchy material has a specific signature with different
proportions of each conformation, bringing out their thermomechanical history.
Thus, the different local conformations present in amorphous amylose, amylopectin,
or starch are directly related to the preparation conditions. For example, for
casted samples twisted and/or constrained conformations are less present than
in freeze-dried corresponding samples since chains, heated in the presence of
water, may relax before complete removal of water. On contrary, during freeze-
drying the conformations are frozen before removal of water, and more constrained
conformations are created. More generally, E conformations are favored by more
drastic methods of preparation. Rehydration and plasticization by water lead to a
general decrease in line width and therefore to a more homogeneous distribution of
3 Crystalline Structure in Starch 73

Fig. 3.6 13 C CP-MAS NMR spectra of (a) amorphous amylose (obtained by hot casting) and (b)
2-propanol precipitated amylose. Enlarged C1 resonance for 2-propanol-precipitated (c) and casted
(d) amylose, with corresponding deconvolution of the C1 region (From Paris et al. 2001)
74 D. Lourdin et al.

conformation and show a higher sensitivity of more constrained conformations (D,

E lines) and freeze-dried samples. Recently, a similar spectral decomposition was
successfully performed on cross-linked starch (Thérien-Aubin et al. 2007).
The presence of a local organization in amorphous starch induces very important
changes on the properties and phase transition of starch materials. An example
is given by recent works showing very efficient shape memory properties of
extruded starch materials (Véchambre et al. 2010). Shape memory corresponds to
the ability for materials to recover their original shape after being deformed into
a temporary shape. In the case of “usual” thermoplastic shape memory materials,
the effect is attributed to the presence of two types of domain: one flexible and
one rigid. The rigid domains determine the initial shape, while the more flexible
domains become oriented in the temporary shape. This orientation remains stable
in “constrained” materials, below the glass transition temperature Tg . However, at
a temperature above Tg , these domains relax, which results in the recovery of the
initial macroscopic shape of the unconstrained sample.
The orientation and physical cross-links and their relation with thermome-
chanical history have been studied in amorphous extruded starch materials. Such
materials are completely transparent when observed using light microscopy, but
with polarized light microscopy, constrained samples show typical and clearly
visible birefringence fringes, contrary to unconstrained samples which do not
present any specific birefringence. This shows that the constrained samples are
anisotropic due to residual stress but that anisotropy disappears after shape recovery,
due to the relaxation of residual stress. Despite strong birefringence under polarized
light, no crystal melting is evidenced by DSC, and WAXS diagrams present no
clear diffraction peaks but only a broad amorphous scattering band. The scattering
band maximum corresponds to a repeat distance of about 0.5 nm. This is normally
considered to arise from the van der Waals (VDW) contact of nonbonded atoms
(VDW spacings) (Miller et al. 1984).
WAXS was used to determine the orientation within such constrained samples,
usually extruded rods or thermomolded barrels, after uniaxial deformation (Vécham-
bre et al., 2010). The WAXS diagrams were recorded in transmission mode using a
two-dimensional detector from specimens lying with their long axis lying parallel
to the vertical axis of the detector (Fig. 3.7a). Constrained samples exhibit periodic
scattering intensity changes on the maximum of the broad amorphous scattering
band, with the azimuthal angle (Fig. 3.7b). The orientation was determined by
azimuthal integration between 0.680 and 0.386 nm corresponding to the amorphous
scattering band. The presence of two maxima at 90 and 270ı shows that orientation
is parallel to the axis of deformation of the sample (Fig. 3.7c). It has been shown
that orientation increases with the level of deformation. Such orientation is stable as
long as moisture content keeps the sample in the glassy state (below Tg ). No periodic
scattered intensity was observed for the relaxed samples as for unconstrained
samples.
Similar measurements revealed that the orientation decreases when the tem-
perature of deformation increases. This decrease in orientation with increasing
deformation temperature was attributed to the greater ability of chains to rapidly
 3 Crystalline Structure in Starch 75

a b

3 13 17 23 30

2θ
Amorphous
Halo
Beam Ø500 μm λ:0.15406 nm Azimutal angle (Φ)
c
Td=Tg+40°C
0.02
Dr=80%
isotropic
Dr=60%
Orientation Intensity (u.a)

0.01
Dr=40%
Dr=0%
0
anisotropic

-0.01

-0.02
0 90 180 270 360
Azimutal angle (Φ°)

Fig. 3.7 Orientation diagrams from WAXS spectra: (a) WAXS setup used and positioning of the
specimen, (b) azimuthal scan of the amorphous broad scattering from the transversal sample, (c)
evolution of orientation as a function of the deformation rate

reorganize at higher temperatures between deformation temperature and Tg at the

end of the deformation process (Véchambre et al. 2011). The recovery stress is a
collateral effect of shape memory which happens when the sample is stimulated
and maintained in its initial shape. It has been shown that the maximum recovery
stress increases almost linearly with increasing orientation. This means that the
driving force for the starch shape memory is the behavior of oriented amorphous
segments that act as “entropic springs” above Tg . From these results, it is expected
that amylose, to which the linear structure induces a better ability to be oriented than
amylopectin, gives the higher recovery stress.
IR spectroscopy was used to look at the local orientation in constrained samples
and at the chemical groups potentially involved. The intensity of the two peaks at
1,023 and 1,049 cm1 , in the C-C, C-O, and C-H stretching and C-O-H bending
energy range, has been widely used to determine the amount of ordered and
amorphous starch, respectively (Smits et al. 1998; van Soest et al. 1995). The band
at 1,153 cm1 was assigned by Bernazzani and coworkers (2000) to vibrations
in the environment of ordered single helices. Finally, the band at 987 cm1 is
very sensitive to hydration and can shift from 987 to 1,003 cm1 depending on
the water content. It was attributed to intramolecular hydrogen bonding of the
hydroxyl group at C-6. It was also shown to develop with molecular ordering
76 D. Lourdin et al.

(Sevenou et al. 2002; Capron et al. 2007). Synchrotron IR microspectroscopy, which

yields higher signal-to-noise ratio, smaller recording time, and easier dichroism
experiments (the synchrotron beam being naturally polarized), was applied to con-
strained and unconstrained samples (Véchambre et al., 2010). With this technique,
spectra were collected on a surface of 12 m  12 m, and a mapping of samples
of about 100 m2 was performed with the synchrotron IR beam parallel (PAR) or
perpendicular (PER) to the axis of deformation of the sample (Fig. 3.8a). Small but
significant differences, demonstrated by a statistical analysis (PCA), were observed
between PAR and PER spectra contrary to unconstrained samples, which confirms
the presence of a specific orientation in the constrained samples. The PCA analysis
was also used to identify the main FTIR absorbance bands associate to PAR and
PER spectra (Fig. 3.8b). From band attribution presented previously, molecular
orientation in deformed sample is clearly related to amorphous domains. On the
contrary, all the bands assigned to higher order and/or crystallinity are linked to
spectra recorded in PER mode. It shows that local orientation is more present in

Fig. 3.8 Synchrotron infrared microspectroscopy of shape memory starch-based materials: (a)
orientation of the specimen for dichroism experiments, (b) first principal component loading vector
determined by the PCA and associated infrared absorptions
3 Crystalline Structure in Starch 77

amorphous domains and is limited by ordered domains. The presence of bands

linked to hydrogen bonds and ’(1,4) linkage could evidence the presence of local
order under the form of helical fragments which may be stabilized by intramolecular
H bonds and play as springs during the recovery process.

3.2.2 Starch Interactions with Small Molecules and Amylose

Complexing

Amylose shows the unique feature to form complexes with a large variety of
molecules. When heated in the presence of starch and water, monoacyl lipids and
emulsifiers as well as smaller ligands such as alcohols or flavor compounds are able
to induce the formation of left-handed amylose single helices. “V amylose” is the
generic term used to describe amyloses co-crystallized with such compounds. The
resulting helical conformation and crystalline packing depend on the nature of the
ligands and conditions for the formation of the complex. In particular, the specific
interaction of amylose with lipids or aroma compounds has a strong impact on
food quality (Heinemann et al. 2005; Conde-Petit et al. 2006), while the interaction
between amylose and some plasticizers impacts the mechanical properties of starch-
based materials. A foremost example is the use of fatty acids and monoglycerides as
anti-staling agents in bread and biscuits. The incorporation of such additives in the
dough induces a slower crystallization (retrogradation) of the amylopectin fraction
and therefore retards the staling of bread (Morrison et al. 1993a). Experimental
evidence that such complexes might be present in native starches in the amorphous
state was given by Morrison et al. (1993b). In that case, amylose chains are involved
in either isolated single helices or involved in crystallites too small to be resolved
by WAXS.
The crystalline structure of amylose complexes was widely studied by crystal-
lizing amylose from aqueous solutions in the presence of a large variety of small
organic and inorganic molecules. Amylose forms inclusion compounds with distinct
WAXS signatures that depend on the complexing agent (Takeo and Kuge 1969;
Tomasik and Schilling 1998a, b; Putseys et al. 2010). Several generic families of V-
amylose have been described (Buléon et al. 2007; Putaux et al. 2011a). Although the
knowledge of the molecular structure is important to locate the guest molecules and
understand how they are entrapped within the crystal lattice, only a small number of
structures have been resolved by crystallographic approaches, and several models
are still hypothetical. Besides many studies carried out on polycrystalline powders
or oriented fibers (Zobel et al. 1967), electron diffraction of lamellar single crystals,
in particular when performed in frozen-solvated conditions (Booy et al. 1979),
combined with molecular modeling, played a crucial role in the determination of
a number of structures (Yamashita et al. 1973; Helbert 1994; Brisson et al. 1991;
Nishiyama et al. 2010).
78 D. Lourdin et al.

The so-called VH type, obtained when amylose is crystallized in the presence

of fatty acids (Godet et al. 1993) and some alcohols (Brisson et al. 1991), is well
documented. The lamellar single crystals exhibit a characteristic hexagonal shape,
and the unit cell has been described by the hexagonal packing of left-handed sixfold
single helices (Brisson et al. 1991). In the case of complexes formed with lipids,
the aliphatic segment of the guest molecules has been shown to be located inside
the helical cavity, while the polar head remained outside (Godet et al. 1993). Other
sixfold helical systems have also been proposed to describe complexes formed in
the presence of n-butanol (Rundle and Edwards 1943; Helbert and Chanzy 1994),
dimethyl sulfoxide. (French and Zobel 1967), and glycerol (Hulleman et al. 1996).
Bear suggested that bulkier guest molecules should be accommodated in a larger
helix with more residues per turn (Bear 1944). By comparing the volume of the unit
cells of the complexes formed with tert-butanol and n-butanol, Zaslow obtained a
ratio of about 7:6 in favor of the existence of a sevenfold helix for the Vtert-butanol
complex (Zaslow 1963), a conclusion supported by Yamashita and Hirai (1966).
Later on, based on the electron diffraction data of V2-propanol single crystals, Buléon
et al. (1990) proposed an alternate model: the orthorhombic unit cell would contain
sixfold helices separated by a layer of guest molecules. Solving this dilemma
was important as it had been observed that a large number of complexing agents
(thymol, geraniol, menthone, etc.) promoted the formation of crystals exhibiting
similar diffraction patterns (Helbert 1994; Nuessli et al. 2003). Recently, Nishiyama
et al. (2010) refined the structure of V2-propanol crystals using electron diffraction
data combined with conformational and packing energy analyses. They proposed
an original unit cell based on sevenfold V-amylose helices whose packing was
described using two regularly alternating motifs: one is nearly tetragonal and holds
a cavity filled with 2-propanol and water molecules, whereas the other corresponds
to four close-packed helices with only water molecules located in the inter-helical
space (Fig. 3.9a–c).
An eightfold amylose single helix has been proposed to occur in the presence
of three complexing molecules: 1-naphthol (Yamashita and Monobe 1971; Helbert
1994), quinoline (Helbert 1994), and salicylic acid (Oguchi et al. 1998). The helices
would be organized in a tetragonal unit cell. So far, the strongest experimental
evidence of an eightfold helix has been provided by Cardoso et al. (2007) who
published a high-resolution transmission electron microscopy (TEM) image of the
crystal lattice viewed along the helical axis and showing a repeating ring motif of 8
subunits (Fig. 3.9d-f).
Sequential washing of powdered complexes with ethanol allowed to probe intra-
and inter-helical inclusions. Such approach was recently applied to the different
crystalline types of amylose alcohol complexes and to aromas like linalool and men-
thone (Rondeau-Mouro et al. 2004; Biais et al. 2006). High-resolution magic angle
spinning (HR-MAS) NMR spectra were also used to compare the chemical shifts of
free and bound aroma molecules and allowed to propose hydrogen bonding schemes
in amylose complexes. Moreover, free aroma was shown to be completely removed
by ethanol washing. Using CP-MAS NMR and X-ray scattering experiments, it
was demonstrated that the V2-propanol type was retained for linalool whatever the
3 Crystalline Structure in Starch 79

Fig. 3.9 (a, d) TEM images of amylose V2-propanol and V1-naphthol lamellar single crystals, respec-
tively; (b, e) corresponding low temperature base-plane electron diffraction patterns recorded
from frozen-wet crystals; (c) axial projection of the molecular model of the amylose V2-propanol
complex. For clarity, the hydrogen atoms have been omitted. The 2-propanol guest molecules are
located inside and between the sevenfold amylose single helices (From Nishiyama et al. 2010).
(f) Averaged high-resolution TEM lattice image of the crystal structure of amylose V1-naphthol .
The projection of the network of eightfold amylose single helices has been superimposed (From
Cardoso et al. 2007)

treatment used. On the contrary, it shifts toward VH type for menthone after ethanol
washing before the desorption step, reflecting the disappearance of inter-helical
associations between menthone and amylose. The stability of the complex prepared
with linalool shows that this ligand is more strongly linked to amylose helices.
The discrepancies observed in the chemical shifts attributed to carbons C1 and C4
in CP-MAS NMR spectra of V2-propanol and VH forms were attributed either to a
deformation of the single helix (with possible inclusion of the ligand inside) or to
the presence of the ligand between helices (only water molecules are present in the
VH form). As described above, it is now more probably related to the sixfold and
sevenfold nature of amylose helices in VH and V2-propanol complexes, respectively.
The formation of amylose complexes during in vitro enzymatic synthesis of
amylose by phosphorylase in the presence of fatty acids or polymers like polyethers,
polylactic acid, polycarbonate, or chemically modified cellulose has also been
reported. This direct wrapping of amylose chains around the complexing molecule
has been described as “vine-twining polymerization” (Kadokawa et al. 2002) or
“parallel enzymatic polymerization system” (Kaneko et al. 2008).
80 D. Lourdin et al.

3.3 In Vitro Enzymatic Synthesis of Starch Building Blocks

and Biomimetic Systems

Since the complete separation of amylose and amylopectin from native starch is
highly difficult, in vitro enzymatic synthesis of linear or branched ’(1,4)-linked
glucans has been significantly investigated during the last years, in order to mimic
starch biosynthesis and, in particular, to better understand how linear chains self-
associate.

3.3.1 In Vitro Synthesis of Amylose

Three classes of enzymes (glycosyltransferases, glycoside phosphorylases,

and transglycosylases) are naturally able to polymerize ’(1,4)-linked residues
(Fig. 3.10). However, the high-yield in vitro production of amylose based on
the use of Leloir glycosyltransferases (GTs) like granule-bound starch synthases
(GBSS) cannot be envisaged as these enzymes use an activated sugar nucleotide
donor (ADP-glucose) that is too expensive and are not active when they are
not embedded in the native starch granule (Ball and Morell 2003). Thus, only
glycoside phosphorylases and transglycosylases have been extensively used for in
vitro amylose synthesis.
Bacterial, animal, or plant ’-glucan phosphorylases (GPs), which are classified
in the GT35 family because of their structural similarity with real GTs, naturally
catalyze the breakdown of an ’(1,4) glucosidic linkage from amylose or glycogen
through a retaining mechanism, with concomitant phosphate glycosylation, to

Fig. 3.10 The various enzymatic routes of ’(1,4)-glycan synthesis

3 Crystalline Structure in Starch 81

yield a ’-D-glucose-1-phosphate (G-1-P) and a shorter ’-glucan chain. These

enzymes also perform reverse phosphorolysis to form a glycosidic bond between
the glycosyl unit originating from the glycosyl phosphate, which acts as the sugar
donor, and a carbohydrate acceptor. In this so-called synthetic reaction, the smallest
glycosyl acceptor, or primer, is maltotetraose. Phosphorylase-catalyzed enzymatic
polymerization is the only method used for production of amylose with low
dispersity (M w =M n < 1:2) and with a weight-average molar mass that can be easily
controlled by varying the G-1-P/primer ratio (Kadokawa 2012). However, G-1-P is
too expensive for industrial synthesis of amylose, even if two-step amylose synthesis
processes were developed, combining actions of phosphorylases to generate G-1-P
from sucrose or cellobiose (Ohdan et al. 2006).
Bacterial amylosucrases are thus a highly attractive alternative to mimic in vitro
amylose polymerization and to get knowledge on chain self-association during
starch synthesis or processing. Indeed, they are the only known enzymes that
catalyze, without any primer, the synthesis of an ’(1,4)-linked glucan from sucrose,
a cheap agroresource used as glycosyl donor, with the concomitant release of
fructose. These transglycosylases, classified in family 13 of glycoside hydrolases
(André et al. 2010), use a retaining non-processive mechanism to produce an
amylose-like polymer of which the average chain length, dispersity, morphology,
crystallinity, and chain length involved in crystals can simply be modulated by
varying initial sucrose concentration and reaction time (Potocki-Véronèse et al.
2005).
The longest chains (DPw D 58, M w =M n D 3:0) produced by Neisseria
polysaccharea amylosucrase (NpAS) from 100 mM sucrose entangle into net-
works similar to those observed by TEM for amylose gels (Fig. 3.11a). These
networks contain clusters of semicrystalline 10–15 nm elementary units, formed
by association of molecules into parallel double helices, linked by amorphous
sections containing loosely organized chains. A synchrotron SAXS study of the
amylose conformation during synthesis in such conditions revealed that at an
early stage of polymerization, amylose consists of a mixture of wormlike chains
and double helical cylindrical structures (Roblin et al. 2013). In a second stage,
individual double helices pack into clusters before crystallizing and precipitating.
All the dimensions determined for wormlike chains and cylindrical conformations
at different times of synthesis were in very good agreement with structural features
usually observed on gels of amylose extracted from starch. Furthermore, DSC and
WAXS analyses revealed that these short chains arrange into independent B-type
crystalline domains, as did the samples produced from 600 mM (DPw D 35,
M w =M n D 2:3) (Fig. 3.11b). Nevertheless, in these last conditions, the chains
suddenly precipitate as polycrystalline aggregates, when the limit chain length
and concentration are reached during synthesis (Potocki-Véronèse et al. 2005).
Remarkably, this behavior is generally also encountered for plant amylose chains,
the longest ones yielding B-type networks upon retrogradation at temperatures
between 4 and 30 ı C, while shorter chains form polycrystalline precipitates (Buléon
et al. 1984).
82 D. Lourdin et al.

Fig. 3.11 In situ crystallization of amylose during enzymatic synthesis by amylosucrase from
sucrose: (a) gel network (100 mM sucrose), (b) axialitic particles (600 mM sucrose) (From
Potocki-Véronèse et al. 2005). (c) WAXS profiles showing the structural transition of the axialitic
particles from B to A type after a heat-moisture treatment

In contrast, the crystallinity of the particles formed from the chains synthesized
in vitro from 600 mM sucrose is exceptionally high (94 %) considering that they
result from a self-aggregation process during enzymatic synthesis, without any
optimization of the crystallization conditions (Potocki-Véronèse et al. 2005). The
chain organization inside the particles is not known with precision. The analysis of
the polarized optical micrographs suggests there is an axial symmetry, hence the
term “axialites” used to describe the particles. Such a structure, if it is confirmed,
has never been reported before for amylose, but may correspond to the axialitic
organization of synthetic polymer chains (Encyclopedia of Polymer Science 1987).
In addition, by simply heating these amylose axialites for 5 min at 90 ı C, the
crystallinity increases further by 30 %, without any change in their external shape.
They should thus now be considered as a new standard for the determination of the
relative crystallinity of starchy products (Potocki-Véronèse et al. 2005). This ability
3 Crystalline Structure in Starch 83

of short chains to self-associate into a highly organized supramolecular structure

is helpful for understanding the crystallization stage during starch biosynthesis.
Besides amylose gelation, the behavior of these particles during hydrothermal
treatments like annealing in water excess or B- to A-type transition during heat
moisture treatment (Fig. 3.11c), both without any change in external morphology,
is also very similar to what happens with hydrothermal treatments of starch.
The exceptional crystallinity of these particles and the simplicity of the synthesis
process were exploited to obtain 13 C-labeled amylose from 600 mM 13 C-sucrose
and to characterize the conformation of B-type amylose by high-resolution solid-
state NMR (Rondeau-Mouro et al. 2006). The assignment of the complete B-type
amylose spectrum and correlations between 13 C-13 C distances and the atomic
positions in the three-dimensional model obtained by low-resolution electron and
X-ray diffraction of crystalline B-type fibers (Imberty and Pérez 1988) were
established.
The synthesis and fractionation conditions of maltooligosaccharides and short
amylose chains produced by NpAS were also optimized in order to prepare fractions
with a very low dispersity (1:003 < M w =M n < 1:01). Several fractions, with a
DPw ranging from 10 to 40, were crystallized from dilute solutions in the presence
of acetone vapors, resulting in needlelike A-type single crystals. The influence
of the molecular parameters of the narrow fractions of amylose chains on the
crystallization behavior and on the crystal morphology was investigated (Putaux
et al. 2011b). Indeed, a DPw of 15–19 and a low dispersity were necessary to form
crystals with a 5–10 m length, such as those shown in Fig. 3.4b (Montesanti et al.
2010). As previously mentioned in this article, the data collected by synchrotron
X-ray microdiffraction from such large model single crystals (Fig. 3.5a) were
successfully analyzed using classical crystallography methods, resulting in a revised
structure of A amylose (Popov et al. 2009).

3.3.2 Enzymatic Modification of Branched

˛(1,4)˛(1,6)Glucans

If many advances were done these last years regarding amylose chain self-
association, one cannot exclude the key role played by ’(1,6) branching points
of amylopectin in the elaboration of the starch granule ultrastructure. A large
panel of branched starchy macromolecules was synthesized in vitro either by
using NpAS, GP, or amylomaltase, alone or in combination with branching and/or
debranching enzymes to mimic the activities required for starch biosynthesis.
NpAS was also used to modify the structure of native glycogen by elongating
the external chains of the macromolecule in the presence of sucrose. In fact,
glycogen is particularly efficiently glucosylated by amylosucrase, the extension
rate being governed by the relative concentration of sucrose and glycogen (Putaux
et al. 2006). Using different sucrose/glycogen mass ratios, original glycodendrimers
84 D. Lourdin et al.

consisting of a glycogen core and a swollen amylose corona were formed within a
few hours (Fig. 3.12a, c). Upon aging or drying, the amylose chains intertwined,
resulting in the shrinking of the corona by the formation of crystallites and in a
decrease of the particle diameter (Putaux et al. 2006). The resulting morphology
depends on the initial sucrose/glycogen mass ratio. For a high ratio (around
340), the synthesized chains are long and reorganize in the form of a random
organization of crystallites by a retrogradation process (Fig. 3.12b). For a low
ratio (close to 1), small spikelike protrusions are seen on the surface (Fig. 3.12c).
The number of glucosyl units added to glycogen external chains was estimated
to be around 15. Consequently, the short chains intertwined in double helices
can be expected to lie perpendicularly to the particle surface (Fig. 3.12d), like
it is the case for the short branches of amylopectin in native starch granules.
Such systems can thus be used to develop a biomimetic approach and reproduce
in vitro the mechanisms involved in the biosynthesis of amylopectin and the
structuring process of starch granules in plants. It shows that there is a close
relationship between the length of the biosynthesized chains and the formation
of a semicrystalline lamellar organization. Long chains may not result in the
formation of lamellae, unless they are used by other biosynthetic enzymes. The

Fig. 3.12 Dendritic particles prepared by extension of glycogen external chains by amylosucrase
in the presence of sucrose: (a, c) cryo-TEM images of the particles obtained with initial
sucrose/glycogen weight ratios of 342 and 1, respectively; (b, d) schematic representation of the
growth and subsequent crystallization of the corona of neosynthesized linear glucan chains (LGC)
around the initial glycogen particle (IGP) (From Putaux et al. 2006)
3 Crystalline Structure in Starch 85

influence of a debranching activity may also be studied by adding isoamylase to

check the potential role of debranching enzymes during biosynthesis in amylopectin
formation/crystallization.
Hyperbranched alpha-glucans have been recently produced by jointly using
amylosucrase and a branching enzyme (Grimaud et al. 2013) or phosphorylase
and a branching enzyme (Ciric and Loos 2013). This is another approach of
amylopectin biosynthesis, but without the use of debranching enzyme, the branching
rate remains higher than that of amylopectin and the average chain length too low to
properly mimic the typical arborescent branching pattern observed in amylopectin
(Rolland-Sabaté et al. 2014). Thus, strictly, to date, amylopectin has never been
biosynthesized in vitro or at least mimicked, whatever the enzymatic cocktails used.
Two methods have been reported, described as “SP-GP-BE” (based on the use of
sucrose phosphorylase (SP), ’-glucan phosphorylase (GP), and branching enzyme
(BE)) with sucrose and maltotetraose as substrates (Ohdan et al. 2006; Kajiura
et al. 2011) and “IAM-BE-AM,” in which the branched linkages of starch were
first hydrolyzed by an isoamylase (IAM) to produce short amylose chains that were
assembled into spherical particles by a branching enzyme (BE) and an amylomaltase
(AM) (Kajiura et al. 2008). Indeed, both methods resulted in the formation of
glycogen-like molecules, rather than in a macromolecular architecture typical of
amylopectin.

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