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.