Wheat Grain Biomechanics Data

Material and methods

<i>2.1
</i><i>Wheat grain
tempering treatment</i>

Prior to caring out biomechanical tests on
wheat grains a tempering phase was conducted to accurately mimic the industry
standard process of mellowing prior to milling. The cultivars selected for
optimising biomechanical characterisation techniques were the ‘soft’ biscuit
wheat, Cambrena, with samples taken from two provenances (1725: Posieux FR,
8212: Neuhausen SH, both in Switzerland), alongside Runal, a ‘hard’ inland top
wheat from three provenances (2852: Courtételle JU; 3052: Zollikofen BE; 4533:
Riedholz SO, all three in Switzerland).

A 2.5 g subsample of wheat grains was
processed in a manual burr grinder for 2 mins. The processed subsample was
then transferred to a Mettler Toledo HB43-S and its moisture content was
measured. The moisture content of the remaining subsamples was adjusted to 16%
(w/w) via the addition of further deionised water. 20 g of wheat grains were
shaken vigorously for 3 mins at 20ºC with the calculated addition of water
in a sealed Petri dish. Subsequently grains were incubated for a specified
tempering time, as indicated.

<i>2.2
</i><i>Shear testing</i>

A shear testing
regime was developed to mirror the type of forces that wheat grains undergo
during the milling process. This method determines the force and energy that is
required to shear the starchy endosperm (until break). This novel procedure
allows for very precise measurements of some of the key forces involved in the
milling processes.

Cambrena 1725 and 8212 grains were measured after 0, 1, and 6 h
tempering times. Runal 2852, 3052 and 4533 gain samples were measured after 0, 1,
6, 12, 18 and 24 h to represent typical tempering times for these
cultivars (n ≥ 26).

Runal 4533 grains were selected for further
testing to explore the effects of various pre-treatments on the shear force
required to shear the starchy endosperm. After 6 h tempering Runal 4533
grains were divided between 8 groups and subjected to a series of different
conditions (n = 12):

Control (tempering at 20ºC with distilled
water to adjust 16% grain moisture)

Heat (35ºC)

10 µM GA (gibberellin
A₄₊₇, Duchefa, in 0.005% (w/v) DMSO)

Microwave (20 g wheat material was
microwaved 1 h after the tempering at low power (520 W) for 20 s
(open lid). Temperature was checked during the experiment – 54°C)

Pressure (1 h, 30 bar for 30 min)

Vibration (Vortex-Genie® 2, shaking
motion: orbital, Orbit: 4 mm, Amplitude: 2 mm, Max. speed:
2700/min, for 32 s)

Vacuum (vacuum desiccator VWR 467-0102
DIN, negative pressure: -72±6.8 MPa)

8. Vacuum followed by the tempering step
(vacuum desiccator VWR 467-0102 DIN, negative pressure: -70.1±100 MPa)

The shear tests were conducted with a
custom-made testing machine with a load cell range 0-20 N. The application
of custom built sample holders for shear tests was implemented. Cylindrical
samples one millimetre in diameter were punched out of the tempered wheat grain
endosperm (hollow punch, Ø 1.0 mm, www.locheisen.com). The cylinders were
placed in custom made specimen holders (1.2 mm hole in the plexiglas
sample holder) and sheared. The measuring tip was lowered at a speed of 1.4 mm/min
to the sample at a constant speed, while force and displacement were recorded.
All testing was conducted at a constant 20°C.

The application of this shear testing method allows for the
calculation of several biomechanical indices. Firstly, the max force recorded
prior to the breaking point of the endosperm was calculated. Secondly the shear
strength of the material was calculated, as the cross sectional area of the
punched specimen is constant and known (π/4 mm²). The physical
work that was done in separation of the respective layers and the energy needed
was calculated by integrating the area enclosed by the force as the energy
dissipation. Finally, the force displacement curves were interpreted and
classified as to the breaking behaviour (failure type). We split this biological
material into three discrete categories of breaking behaviour a) a “brittle”
failure; force increases until a sudden complete failure takes place b) a
“composite” failure; a step-by step failure, creating a typical “zig-zag”
pattern and finally c) a “continuous” failure; typified by a continuous
decrease in force without sudden drops or steps. Example curves for all three
types of breaking behaviour can be found in the supplementary materials
appendix A. This method is so precise that individual layers of the bran can be
sheared independently.

<b> </b>

2.3
<i>Puncture force
testing</i>

In order to test the force required to puncture the
individual wheat grain layers a method was developed for the separation of
these layers using Runal 4533 to test this method. To prepare the samples the brush
and germ of the wheat grain was cut away and the remaining section of the grain
was soaked in deionised water for 12 h. After soaking, bran layers were
manually cleaned from starch and then the bran was separated into three experimental
layers by sliding a preparation needle between them. The isolated layers were the
aleurone (A), the intermediate layer (I) and the epicarp (E) (Figs. 1,5,6,
n ≥ 11).

The separated layers were flattened and left to dry on
microscope slides under cover slips for 10 h. Samples were assigned to different
treatment groups, an untreated control, two hormone treatments, and an enzyme
mixture. Following the addition of these treatments, samples were incubated for
22 h at 32ºC in darkness. Controls were incubated in deionised water (1 ml).
The two hormone treatments consisted of a treatment of gibberellin A₄₊₇
(GA) and one of <i>cis</i>-S(+)-abscisic
acid (ABA, Duchefa) which were used at final concentrations of 10 µM. The enzyme cocktail
consisted of equal volumes of Cellulase - Sigma, C-2605 (1000 U/ml);
Pectinase - Sigma P-2736; Pectinesterase - Sigma P-0764; Viscozyme L - Sigma
V2010 Xylanase - Sigma X-2753 (2500 U/g) used at concentrations of 1%,
0.1% and 0.01% (v/v) respectively. Water content of all samples was
equilibrated above a saturated NaCl / KCl mixture at 20ºC.

A second experiment
was conducted on the single layers in order to assess the influence of water
content on the force required to puncture the separated layers. Two moisture
content treatments were then created, one with a higher water content (approximately
16% grain moisture) and one at with a lower water content (approximately 5%
grain moisture). This was achieved by placing samples above a saturated salt
solution for 10 h. A high moisture content was created using a saturated
NaCl / KCl mixture at 20ºC and a low moisture content was created
using saturated LiCl solution at 10ºC.

The actual puncture force measurements of the samples were
conducted with a custom made testing machine with a load cell range 0-1 N.
The layer was placed in a custom made magnetic sample holder and a 0.5 mm
diameter probe (hemisphere shaped tip) was lowered at a speed of
0.7 mm/min onto the layer while force and displacement were recorded simultaneously.