2. MATERIALS AND METHODS
Samples of subcutaneous adipose tissue removed from the coccyx of
Iberian pigs were obtained from the Instituto Nacional de Investigación
y Tecnología Agraria y Alimentaria (INIA) (Zafra, Spain). Lipid
fractions were extracted and dehydrated by the LiBiFood research group
of the Universitat de Barcelona, following the procedure explained
elsewhere (Bayés-García et al., 2016). All lipid extracts were stored in
topaz vials with liquid nitrogen inert atmosphere in the freezer to
avoid oxidation. 80 samples (20 samples for each category) were
available. Before performing any analysis, samples were melted at 65°C
and homogenized by using a vortex.
Gas chromatography experiments were carried out to quantify the fatty
acid moieties. The equipment consisted of a Shimadzu QP2010 coupled to a
SGE BPX70 column (30 m x 0.25 mm, 0.25 μm). The sample was diluted in
n-hexane-dichloromethane (5:1) and carried with He. The injector
temperature was 260 °C with split (split ratio 1:20). The mass
spectroscopy coupled with the column worked with an ion injector
temperature of 200 °C and an m/z range from 50.00 to 650.00.
DSC experiments were conducted for all samples (making a total of 80).
They were carried out at atmospheric pressure using a PerkinElmer
Diamond. Samples (9.0 – 9.9 mg) were weighted into 50 μL aluminum pans,
and covers were sealed into place. The instrument was calibrated with
reference to the enthalpy and the melting points of indium (melting
temperature 156.6 °C; ΔH = 28.45 J/g) and decane (melting temperature
-29.7 °C; ΔH = 202.1 J/g) standards. An empty pan was used for
reference. Dry nitrogen was used as purge gas in the differential
scanning calorimetry (DSC) cell at 20 cm3/min.
Thermograms were analyzed using Pyris Software to obtain the enthalpy
(J/g, Integration of the DSC signals) and onset and end temperatures of
the transformation (°C, intersection of the baseline and the initial and
final tangents at the transformation). The thermal program used for all
samples consisted of cooling from 65 °C to -80 °C at 2 °C/min followed
by a subsequent heating process from -80 °C to 65 °C at a rate of 2
°C/min.
Three independent measurements were carried out when sufficient amount
of sample was available. Then, random uncertainty was estimated with
95% threshold of reliability using the Student’s method, which enables
estimating the mean of normally distributed population when the
population is small.
X-ray diffraction experiments, with both laboratory-scale and
synchrotron radiation source, were performed for selected samples of
each Iberian pig fat category. The X-ray diffraction results obtained by
both laboratory-scale and synchrotron radiation source became
equivalent. However, in this work we will show the synchrotron data for
a better clarity and resolution. Laboratory-scale powder X-ray
diffraction (lab-scale XRD) measurements were performed by using a
PANalytical X’Pert Pro MPD powder diffractometer equipped with a hybrid
Monochromator and an X’Celerator Detector. The equipment also included
an Oxford Cryostream Plus 220 V (temperature 50-500 K). This
diffractometer operates with Debye-Scherrer transmission. The sample was
introduced in 1 mm-diameter Lindermann glass capillary that was rotated
around its axis during the experiment to minimize preferential
orientation of the crystallites. The step size was 0.013 ° from 1.004 °
to 28 ° 2θ, and the measuring time was 2.5 minutes per pattern.
Synchrotron radiation X-ray diffraction (SR-XRD) experiments were
conducted at the beamline BL11-NCD-SWEET of the Alba Synchrotron
facility (Cerdanyola del Vallès, Spain) at 12.4 keV. The sample-detector
distance was 2.2 m. X-ray scattering data were collected on a Quantum
210r ADSC detector with a pixel size of 102.4x102.4 µm2 for small-angle
X-ray diffraction (SAXD) data and on a LX255-HS Rayonix detector with a
pixel size of 40x40 mm2 for the wide-angle X-ray
diffraction (WAXD) data. The exposure time was 20 s. The temperature of
the sample was controlled by a Linkam stage. SR-XRD patterns were
acquired while the sample was cooled from 65 to -80 °C and reheated to
65 °C at the same controlled rate. The sample was placed in an aluminum
sample cell with a Kaptom film window. The q-axis calibration was
obtained by measuring silver behenate for SAXD and
Cr2O5 for WAXD. The software PyFAI was
used to integrate the 2D WAXD into 1D data: the SAXD data were processed
with in-house software.