Nanostar

Samples were mixed with deionized water to achieve ~60% of total moisture content and equilibrated at ambient temperature (~25 °C) for 24 h. A small-angle X-ray scattering diffractometer (NanoSTAR, Bruker AXS Inc., Madison, WI, USA), equipped with a Vantec 2000 detector (Bruker, Berlin, Germany) and pinhole collimation for point focus geometry, were used for sample lamellar structure analysis, as described elsewhere [19 (link)]. The average repeat distance (i.e., thickness of the semicrystalline lamellae) of the amorphous and crystalline lamellar of each sample was calculated as d = 2π/q, where d (nm) is the lamellar repeat distance and q (nm⁻¹) is the scattering vector, with q = (4π sinθ)/λ, where λ (nm) is the X-ray wavelength and 2θ is the scattering angle.

Each
3D-printed starch sample was dispersed into distilled water to form
a slurry (40% w/w) and slightly stirred with 56 s^–1 (367 G-force) at ambient conditions overnight. Next, it was centrifuged
(Eppendorf centrifuge 5417R, Hamburg, Germany) at 5000 G-force for
5 min, and the wet precipitate was collected. A Bruker small-angle
X-ray scattering (SAXS) instrument (NanoSTAR, Bruker AXS Inc., Billerica,
MA) was used to record the two-dimensional (2D) scattering pattern
of printed starches at 50 kV and a 30 W Cu Kα radiation wavelength
of 1.5418 Å. The SAXS instrument was equipped with a Vantec 2000
detector and pinhole collimation for point focus geometry. The one-dimensional
(1D) scattering curves were obtained in the range of 0.2 < q < 1.4 nm^–1 from the 2D scattering
patterns through the built-in software. The SAXS curves were further
analyzed with the help of the 1D linear correlation function L(r) (eq 3) Here, q is the scattering
vector, I(q) is the scattering intensity, r is the distance in real space, and the denominator is
the scattering invariant.

SAXS data were acquired
on a Bruker NanoStar instrument using 40.0 μM H-GbpA-FL or 32.0
μM D-GbpA-FL in 100 mM NaCl, 20 mM Tris–HCl pH 8.0, with
data acquisition times of 1 h per data set. Scattering intensities
were recorded as a function of the scattering vector q = (4π/λ)sinθ, where 2θ is the scattering
angle and λ is the wavelength (λ = 1.54 Å). Data
were collected in the q-range: 0.009 to 0.3 Å^–1.
The scattering intensities were corrected
for electronic noise, empty cell scattering, and detector sensitivity.
The scattering contribution from the buffer was subtracted, and intensities
were calibrated to absolute units with H₂O scattering as
standard, using the SUPERSAXS program package (CLP
Oliveira and JS Pedersen, unpublished; implementation explained in
ref (25 (link))).
Radii
of gyration and pair-distance distribution functions (from
inverse Fourier transform^{26 (link)}) were calculated
with PRIMUS(27 (link)) from the ATSAS(28 (link)) package. For both H-GbpA-FL
and D-GbpA-FL, 20 low-resolution models were calculated by ab initio shape determination using the DAMMIF(29 (link)) software. We built average models with DAMAVER(30 (link)) and refined them with DAMMIN.(31 (link)) All three programs
are from the ATSAS(28 (link)) package.
SAXS data are summarized in Table 2.

The SAXS experiments were performed using the NANOSTAR, Bruker installed at ANSTO. The obtained SAXS intensity was corrected for background, empty cell, buffer solution scattering, and transmittance.

The bulk samples were prepared from chloroform solution. After evaporating the solvent, the samples were annealed under vacuum at each temperature. The bulk morphologies of the BCPs were characterized by small-angle X-ray scattering (SAXS). The SAXS measurement was performed using a Bruker NanoSTAR with a 2D-PSPC detector.