3. RESULTS AND DISCUSSION
Figure 1 depicts the distribution of main fatty acids in lipid extracts
corresponding to the four Iberian pig categories: bellota(B ), recebo (R ), cebo de campo (CC )
and cebo (C ).
The results showed that the main fatty acid was monounsaturated oleic
acid (within the range 50-60%) for all categories, followed by
saturated palmitic (17-24%) and stearic (7-12%) acids, diunsaturated
linoleic acid (7-9%), monounsaturated palmitoleic acid (1-3%) and
saturated miristic acid (1-2%). These results were in agreement with
those reported by Diaz et al. (1996) and Gallardo et al. (2012) who
defined POO, OOO, POL and POS as main triacylglycerol components of
Iberian pig fat.
By comparing the lipid extracts compositions of the four categories, one
may define a clear tendency which is related to the
saturated/unsaturated nature of the three main fatty acids (oleic,
palmitic and stearic). The percentage of unsaturated oleic acid
decreased, whereas those of saturated palmitic and stearic increased in
the sequence bellota (B ) - recebo (R )- cebo de campo (CC ) - cebo (C ) as shown in
Figure 1b.
Although some differences in fatty acid compositions were detected,
these may not be determinant for a different crystallization and
polymorphic behavior for the four Iberian pig categories. Variations in
the polymorphic behavior may be mostly dictated by TAGs molecular
structures (and resulting interactions), in which fatty acids can occupy
different positions in the glycerol backbone.
The crystallization and polymorphic behavior of lipid extracts ofbellota, recebo, cebo de campo and cebo categories were
determined by DSC and XRD techniques when samples were cooled from 65°C
to -80°C at a rate of 2°C/min and subsequently heated in the same
temperature range. Although DSC experiments were carried out for the 80
samples (20 samples for each category), cooling and heating curves of
selected samples are shown in Figure 2. However, it is worth to mention
that DSC curves corresponding to the same category became highly
comparable.
All DSC thermograms consisted of four main thermal events: two exotermic
peaks present in the cooling curves, and two main endothermic peaks in
the heating curves. However, some differences were detected between the
four categories, such as the occurrence of additional DSC signals or
some shifting in temperatures at which phenomena occurred. In general
terms, one may note that initial and end temperatures of crystallization
in bellota and recebo samples (curves (1) and (2) in
Figure 2a) were significantly lower than those of cebo de campoand cebo samples (curves (3) and (4)). Regarding the DSC heating
curves, again bellota and recebo samples exhibited a
similar thermal profile, characterized by the presence of two initial
endothermic peaks with peak top temperatures between -20 and 0 °C, and
complex convoluted phenomena from 10 to 25 °C. By contrast, the DSC
heating curves corresponding to cebo de campo and cebosamples became less abrupt and continuous, as one may define just two
main sharp endothermic peaks, with peak top temperatures at around 5 and
30 °C (Figure 2b).
Then, due to the complexity of the DSC thermograms and the heterogeneity
of some of the thermal events, we defined two main temperatures as
indicators of the crystallization and melting behavior of the samples:
the initial crystallization temperature, and end melting temperature.
These temperatures may be also strongly related to the fatty acid
compositions of the lipid extracts. Figure 3 graphically shows onset
crystallization and end melting temperatures for all the analyzed
samples corresponding to each Iberian pig category (20 samples for each
category).
The results indicated an average initial crystallization temperature of
8.6°C (with standard deviation of 1.6) and 9.6°C (standard deviation of
2.9) for the bellota and recebo samples, respectively. By
contrast, cebo de campo and cebo samples exhibited higher
onset crystallization temperatures of 15.9°C (standard deviation = 1.6)
and 16.2°C (standard deviation = 3.1), respectively. Regarding the
melting behavior, again comparable temperatures were detected inbellota and recebo samples, with average end temperatures
of 28°C (standard deviation = 0.7) and 28.5°C (standard deviation =
1.2), respectively. As to cebo de campo and cebocategories, the corresponding average end melting temperature was 30.7
°C in both cases, with standard deviation of 0.9 and 1.0, respectively.
From the results described above, one may note that two clear groups can
be defined, in which crystallization and melting temperatures were
significantly comparable: bellota - recebo and cebo
de campo - cebo. As to standard deviation values, they were
lower for melting temperatures, as can be observed by lower dispersion
in temperature values in Figure 3. The different thermal behavior of the
samples was interpreted by analyzing the crystallization and polymorphic
behavior of selected samples for each category as will be described
further on.
Figure 4 shows SR-SAXD and SR-WAXD data obtained when a bellotasample was cooled from 65°C to -80°C at a rate of 2°C/min and reheated
to 65°C at the same rate.
During the cooling process, at a temperature of 6.3°C, the SR-SAXD
pattern revealed the occurrence of a triple chain length structure (3L)
peak at 3.5 nm. Simultaneously, in the SR-WAXD pattern, typical β’ peaks
at 0.41 and 0.38 nm were detected. Then, this crystallization process
corresponded to a β’-3L form and caused the first exothermic DSC peak
occurring at around 8°C (peak top temperature, see Figure 2a - curve
(1)). At -15.5°C, a double chain length structure peak (2L) at 4.3 nm
appeared in the SR-SAXD pattern accompanied by a SR-WAXD peak at 0.39
nm, which may correspond to a β’-2L form crystallization. This
crystallization process may be attributable to the exothermic peak with
peak top temperature at around -10°C (Figure 2a - curve (1)). On further
cooling, at -21.5°C, two extra SR-WAXD peaks occurred at 0.42 and 0.41
nm, probably due to the crystallization of an additional β’-2L
polymorphic form. This phenomenon may correspond to the last DSC peak at
-21°C observed when cooling. During the subsequent heating process, at
-2.5°C, the β’-2L form peaks at 0.42, 0.41 and 0.39 nm vanished, while
the intensity of the 2L peak at 4.3 nm considerably decreased. This
SR-SAXD peak completely disappeared at 5.4 °C. This changes may be due
to the melting process of the two β’-2L forms previously crystallized,
and they may correspond to the two broad endothermic DSC peaks with
maximum temperatures of -12 and 0°C. On further heating, at 17.4°C, the
SR-SAXD peak at 3.5 nm vanished, together with the SR-WAXD peaks at 0.41
and 0.38 nm, due to the melting of the β’-3L form. Soon after, new peaks
at 4.4 and 0.46 nm occurred, which may correspond to a newly-formed most
stable β-2L polymorph, probably through melt-mediation after the melting
of β’-2L forms. This most stable form finally melted at around 39.2°C
(see enlarged figure in Figure 4), temperature at which no peaks were
present in the SR-XRD patterns, that is in accordance with the
corresponding DSC thermograms. One may note the presence of a last and
very flat endothermic signal in the corresponding DSC thermogram (see
curve (1) in Figure 2b), which is related to the melting of this most
stable β-2L form.
The polymorphic behavior of recebo samples was highly similar to
that of bellota samples, as shown in Figure 5.
By cooling the molten recebo sample at a rate of 2°C/min, triple
chain length structure peak at 3.5 nm (SR-SAXD) and short spacing values
of 0.41 and 0.38 nm (SR-WAXD) occurred at 2.4°C, corresponding to the
crystallization of a β’-3L form. This event may correspond to the
exothermic DSC signal with peak top temperature of 5°C (see Figure 2a).
On further cooling, at -21.4°C, a 2L structure peak at 4.3 nm was
observed in the SR-SAXD pattern and, simultaneously, β’ WAXD peaks at
0.44, 0.43 and 0.39 nm were detected (β’-2L form crystallization).
Finally, at a lower temperature of -27.5°C, an additional peak at 0.42
nm appeared, which most probably corresponded to the crystallization of
another β’-2L form. These crystallization processes of β’-2L forms may
be attributable to the exothermic DSC events with maximum temperatures
of -12 and -25°C of the related cooling thermogram (see curve (2) in
Figure 2a). As to the heating step, when completely crystallizedrecebo sample was heated at 2°C/min, the first observable change
took place at 5.4°C, temperature at which a β’-2L form melted (vanishing
of the SR-WAXD peaks at 0.44, 0.43 and 0.39 nm and decrease of the
intensity of the SR-WAXD peak at 4.3 nm). Within the temperature range
from 11.4 to 17.3°C, all the SR-XRD peaks vanished, so that one may
deduce that β’-2L and β’-3L forms melted. Soon after, new SR-SAXD peak
at 4.4 nm appeared (see enlarged figure in Figure 5) and,
simultaneously, typical β SR-WAXD peak was observed at 0.46 nm. Then,
one may conclude that, similarly to the bellota sample case,
melt-mediated transformation occurred to obtain most stable β-2L form,
which finally melted at around 35.2°C (see very flat endothermic DSC
peak with peak top temperature of about 35 °C in curve (2) of Figure
2b). Recebo sample exhibited, therefore, a very similar
polymorphic behavior to that of bellota. However, crystallization
and melting temperatures were lower in recebo than inbellota in this specific case, which is in accordance with the
dispersion in crystallization and melting temperatures determined in the
characterized recebo samples (see Figure 3).
Different crystallization and polymorphic behavior was detected in thecebo de campo and cebo samples. Figure 6 shows the SR-XRD
data corresponding to cebo de campo , which allowed the
identification of the thermal phenomena observed by DSC (curve (3) in
Figure 2).
When cooled, an initial concurrent crystallization of β’-2L and β’-3L
forms was detected at 16.3 °C. These events were related to the DSC
exothermic peak with maximum temperature of 13 °C. These forms were
identified by long spacing values of 4.9 and 3.5 nm, respectively, and
short spacing values of 0.41 and 0.38 nm. On further cooling, at -11.5
°C, the SR-SAXD pattern showed the occurrence of an additional 2L peak
at 4.3 nm and, at -15.5 °C, the SR-WAXD pattern exhibited two new peaks
at 0.43 and 0.39 nm, which may refer to the formation of an additional
β’-2L form or two additional β’-2L forms, as will be justified through
the heating step. Regarding the DSC cooling thermogram, these phenomena
may correspond to the second main exothermic peak with peak top
temperature at around -10 °C. During the heating process, the first
observable change took place at 3.5 °C, when the 0.39 nm peak
disappeared, due to the melting of one of the newly-formed β’-2L forms
(on the DSC heating thermogram, this process was related to the
endothermic event with Tmax = 2 °C). At 25.3 °C, the 4.9
nm peak disappeared, at 27.3 °C, the peaks at 0.43, 0.41 and 0.38 nm
also vanished and, soon after, at 31.3 °C, the peak at 3.5 nm also
disappeared. These phenomena corresponded to the melting of the
remaining β’-2L forms and β’-3L form. Then, at 33.2 °C, the peak at 4.3
nm vanished and a new peak at 4.4 nm occurred in the SR-SAXD pattern,
together with a SR-WAXD peak at 0.46 nm, indicating the crystallization
of a most stable β-2L form, most probably through a melt-mediated
process. Finally, at a temperature of 43.2 °C, no SR-XRD peaks were
present, and the melting of most stable β-2L form may be related to the
flat endothermic DSC peak with maximum temperature of around 40 °C,
observable in curve (3) of Figure 2b.
A cebo sample was subjected to the same thermal treatment and the
SR-XRD pattern obtained are shown in Figure 7.
When cooled from 65 °C to -80 °C at a rate of 2 °C/min, the SR-SAXD
pattern exhibited, at 13.4 °C, the occurrence of an initial peak at 4.8
nm (2L) and, simultaneously, the SR-WAXD pattern showed the formation of
typical β’ form peaks at 0.41 and 0.38 nm (β’-2L form crystallization).
Soon after, at 11.3 °C, a SR-SAXD peak at 3.4 nm (3L) accompanied by
SR-WAXD peaks at 0.43 and 0.38 nm were detected, due to the
crystallization of a β’-3L polymorph. These two crystallization
processes (β’-2L and β’-3L forms) may be related to the first observable
exothermic DSC peak with peak top temperature of about 17 °C (curve (4)
in Figure 2a). Then, at -14.5 °C, an additional β’-2L form crystallized,
with long and short spacing values of 4.3 and 0.39 nm. This last event
may correspond to the DSC exothermic peak at Tmax = -9
°C. Finally, at -24.4 °C, the SR-WAXD pattern indicated the occurrence
of a β’ form peak at 0.42 nm (observed on the DSC cooling curve at
Tmax = -23 °C). Nevertheless, the chain length structure
of this newly formed polymorph could not be determined due to the
overlapping of the peaks. During the subsequent heating stage, the first
melting phenomenon corresponded to the lastly formed β’-2L form and it
was observed at 9.4 °C, temperature at which the SR-WAXD peak at 0.39 nm
vanished and the intensity of the SR-SAXD peak at 4.3 nm decreased
significantly. This event may be related to the first important
endothermic DSC signal with peak top temperature of around 4 °C.
According to the SR-XRD data, at 27.3 °C, the initially formed β’-2L
form and the β’ form with no definite long spacing value melted (peaks
at 4.8, 0.42, 0.41 and 0.38 nm). Later, at 29.3 °C, the peak at 3.5 nm
disappeared, which may be attributed to the melting of β’-3L form. These
melting processes were attributed to the second main endothermic signals
with maximum temperature of about 29 °C. Further on, and similarly to
other Iberian categories, at 35.2 °C, most stable β-2L form occurred,
with long and short spacing values of 4.4 nm and 0.46 nm, respectively.
Finally, this most stable form melted at 45.2 °C, temperature at which
no peaks were present. In the case of cebo sample, the last flat
endothermic DSC peak with maximum temperature of about 40 °C, which may
correspond to the melting of most stable β-2L form, becomes more
pronounced than for other categories.
The polymorphic behavior of the cebo de campo and cebosamples became highly similar, as will be discussed further on.
Moreover, both crystallization and melting temperatures became
comparable.
All samples of lipid extracts belonging to the different Iberian pig
categories of bellota , recebo , cebo de campo andcebo exhibited highly complex polymorphic behavior at the
experimental conditions examined. This complexity was detected in the
corresponding DSC cooling and heating thermograms, but also in the
SR-XRD patterns. As to the thermal behavior of the samples, the
occurrence of overlapped endothermic and exothermic DSC phenomena
predominated, often taking place continuously throughout all the thermal
treatment applied. The same happened with SR-XRD data, in which
concurrent crystallization of multiple polymorphic forms with similar
short-spacing values created significant difficulties in the
identification of polymorphic forms. However, SR-SAXD patterns
facilitated this task.
As a summary, Figure 8 depicts the polymorphic behavior determined for
the four Iberian pig categories.
SR-XRD data confirmed the tendencies observed by the DSC thermograms of
the samples (see Figures 2 and 3). The four Iberian pig categories could
be arranged in two main groups: that formed by the bellota andrecebo categories, and that constituted by the cebo de
campo and cebo categories. Bellota and recebosamples exhibited a simpler polymorphic behavior compared to that ofcebo de campo and cebo . When the two samples were cooled
from the molten state, three different polymorphic forms crystallized:
an initial β’-3L form followed by two different β’-2L forms. Then, when
heated, these forms melted, and some fraction of the liquid crystallized
to obtain most stable β-2L form, which finally melted at a temperature
of about 35-40 °C. By contrast, cebo de campo and cebosamples crystallized in a higher number of polymorphic forms. The first
characteristic event was the initial crystallization of a β’-2L form
with a long spacing value of 4.8 or 4.9 nm, which was not detected inbellota and recebo samples. This crystallization was
followed by the occurrence of a β’-3L form and at least two more β’-2L
forms. When samples were heated, and similarly to the bellota -recebo case, these polymorphic forms melted and some liquid
fraction re-crystallized to obtain most stable β-2L form through
melt-mediation. This most stable form melted at around 43-45 °C,
temperature significantly higher than that corresponding tobellota and recebo samples. Higher crystallization and
melting temperatures in cebo de campo and cebo samples
compared to bellota and recebo was another distinctive
characteristic previously confirmed by DSC data, as shown in Figures 2
and 3. Furthermore, in general bellota and recebocategories exhibited sharper and better-defined exothermic and
endothermic signals, whereas the phenomena showed by cebo de
campo and cebo categories became more continuous through all the
thermal program applied due to the occurrence of a higher number of
polymorphic forms which may be overlapping. The higher crystallization
and melting temperatures detected in cebo de campo andcebo categories may be related to a more saturated composition of
fatty acid moieties, as confirmed by chromatographic techniques (see
Figure 1).
By considering the characteristics that define each Iberian pig
category, regarding the feeding system during the fattening phase
(consumption of acorns and/or concentrated feed), grazing (that is,
doing physical exercise), etc., one may extract some conclusions about
which are the driving factors that determine the polymorphic behavior of
their lipid extracts. Figure 9 summarizes these distinctive
characteristics associated to the rearing system and grazing for the
four categories.
The results presented in this work demonstrated that bellota andrecebo samples exhibited essentially the same polymorphic
behavior, and the same occurred to cebo de campo and cebocategories. As shown in Figure 9, distinctive characteristics of bothbellota and recebo are the consumption of acorns and
grazing, which involves exercise. Grazing becomes another characteristic
activity of cebo de campo , similarly to the commercial feed
consumption, which is common in recebo , cebo de campo andcebo categories. Thus, one may conclude that the key factor which
determines the polymorphic behavior of Iberian pig lipid extracts is not
the physical exercise practiced by the pig, but the inclusion of acorns
in the feeding system. In this sense, the polymorphic behavior may be
used as a tool to discriminate among some of the Iberian pig categories.
In our previous work (Bayés-García et al. 2016) we concluded that both
DSC and XRD techniques could be used as identification tools to
discriminate between the two most differentiated categories:bellota and cebo categories. With the present work we
showed that it is also possible to discriminate among intermediate
categories of recebo and cebo de campo , as recebosamples exhibit an equivalent behavior to that of bellota ,
whereas cebo de campo becomes highly similar to cebo.Although further work is needed to analyze a higher number of samples of
all categories in order to confirm the results presented here, this work
becomes a good approach to show the potential of polymorphic tools to be
used as a fingerprint for the discrimination among different product
categories and, therefore, to combat food fraud.