Crystallite orientation maps of starch granules from polarized Raman spectroscopy and synchrotron X‐ray microdiffraction data
Authors: Galvis Leonardo , Bertinetto Carlo , Weinhausen Britta , Montesanti Nicole , Lancelon‐Pin Christine , Vuorinen Tapani , Burghammer Manfred , Putaux Jean‐Luc 
Published: December 2016

Polarized Raman spectroscopy (PRS) and synchrotron X‐ray microdiffraction were used to determine the local orientation of crystalline regions in giant starch granules extracted from bulbs of tulip and the orchid
Phajus grandifolius
. Starch granules can be described as distorted spherulites composed of concentric growth rings in which molecules are radially oriented.

As previously validated on starch specimens [Galvis et al.
J. Cereal Sci. 62
(2015), 73], the molecular orientation in the native granules was determined by measuring the anisotropic Raman response of certain chemical bonds at different polarization directions of the incident laser radiation. Wellner et al. [
Starch
‐
Stärke
63 (2011), 128] had shown that the response of the Raman band at 865 cm
−1
assigned to the stretching of the glycosidic bonds C–O–C and ring breathing of glucose units exhibited a high spatial variation that could not only be explained by variations in the degree of crystallinity but also by the local molecular orientation in ordered structures. First, we have evaluated the response of the band at 865 cm
−1
using model acicular “A‐type” single crystals prepared from a fraction of short‐chain amylose biosynthesized
in vitro
[Montesanti et al.,
Biomacromolecules 11
(2010), 3049]. The A‐amylose crystals oriented “in plane” showed a maximal intensity when the polarization of the laser was along the chain axis of the crystal,
i.e.,
parallel to the axis of the amylose double helices, and minimal when perpendicular. In addition, the Raman band at 1343 cm
−1
, assigned to C–O–H bending, showed only a small variation and was used as “internal standard” to calculate the intensity ratio of bands 865 / 1343. In parallel, hydrated single starch granules have been probed with 3–5 µm synchrotron X‐ray beams and a raster step of 5 µm, at the ID13 microfocus beamline of ESRF. The collected fiber microdiffraction patterns were analyzed to deduce the local average orientation of the crystallites and produce maps over the whole granules.

PRS orientation maps of tulip (Figure 1a) and
P. grandifolius
starch granules revealed regions with an isotropic response close to the eccentred hilum (origin of the growth) and others with a high anisotropic response at the distal end (Figure 1b) [Galvis et al., in preparation]. The orientation maps of
P. grandifolius
granules were compared to those previously determined from synchrotron X‐ray microdiffraction data [Chanzy et al.,
J. Struct. Biol. 154
(2006), 100] and those from tulip granules, to the data newly collected at ESRF (Figure 2a). Again, the diffraction patterns showed that the crystallite orientation was very high far from the hilum, in regions where the curvature of the growth rings is low (Figure 2b). Around the hilum, the crystallinity remained high and therefore, the lower orientation was likely due to the high curvature of the growth rings and the resulting 3D distribution of crystallites within the probed volume. The spatial resolution of the orientation maps is limited by the size/volume of the region over which the signal is collected and thus averaged, which, in particular, results in a lack of information along the incident laser (for PRS) or X‐ray beam (for microdiffraction). However, both techniques are complementary and provide unique pictures of the local molecular organization in single objects.