1. INTRODUCTION
The problem of food adulteration is by no means a contemporary phenomenon and is likely to be as old as the food processing and production systems themselves (Ellis, et al., 2012). Authentication of food products is of primary importance for consumers and industries at all levels of the production chain. The adulteration of food products normally becomes a means for fraudulent economic gain, as fraudulent actions are commonly applied to high-commercial-value products or to those which are produced in high tonnage. Food products, such as jam, fruit juice, meat, honey, milk, wine or vegetable oils are often adulterated with the addition of cheaper products, dilution processes or mislabeling (Cordella et al., 2002). Nevertheless, the detection of adulterations or the discrimination between different product categories become a very complicated issue when chemical compositions are similar.
Several methodologies have been used for foods authentication, such as spectroscopic techniques, methods based on mass spectrometry, chromatography or calorimetry. Characteristics of spectroscopic techniques are their rapidity and portability, and they can provide the fingerprint of a food product. On the other hand, calorimetric techniques, such as differential scanning calorimetry (DSC), are also rapid and highly sensible, and they can show the authenticity of a food product or the effect of an adulteration on the physicochemical properties of the sample (Chiavaro et al., 2008a and 2008b).
Among high-commercial-value products, traditional Spanish Iberian dry-cured ham is greatly appreciated for its particular and intense sensorial characteristics and associated with top quality gastronomy worldwide. Mainly produced in the southwest regions of Spain, Iberian pigs (pure breed or crossed with Duroc) are raised according to different systems, so that their dry-cured products are classified into different commercial categories. Four are the categories of Iberian pigs, namely bellota , recebo , cebo de campo andcebo. Although according to the current Spanish legislation (Royal Decree 4/2014), recebo category has been eliminated and, therefore, nowadays three are the existing categories (bellota, cebo de campo and cebo ), it is of primary importance to characterize all of them in order to find a robust method to authenticate the product and prevent fraudulent practices. Regarding the rearing systems which define each Iberian pig category, Table 1 summarizes their characteristics and requirements.
Bellota products, which are the most appreciated and those of highest commercial value, come from pigs with a final fattening period in oak forests (period called montanera ) during which they exclusively eat acorns, grass and other natural resources. Then, the minimum duration of this period is 60 days and the minimum weight gain of the pig must be of 46 kg. Recebo products are obtained from pigs that combine the consumption of acorn and grass with a supplementation of concentrated feeds (minimum duration of montanera: 60 days; minimum weight gain: 29 kg). By contrasts, cebo de campoand cebo products come from pigs fed with commercial feeds throughout their life. However, in the case of cebo de campo , pigs roam in pasture for at least 60 days prior to slaughter, although they keep eating mainly commercial feeds. The differences in animal nutrition in the final stage of the fattening period may determine the sensory quality of final products, due to different lipid deposition in adipose tissue and intramuscular fat (Tejeda et al., 2002).
Several methods have been used to discriminate among different Iberian dry-cured ham of different categories. Almost all the procedures are based on fat properties, such as melting and slip point, or fatty acid profiles. These methods rely on the fact that bellota usually contains higher amounts of oleic acid than other categories (Ruiz et al., 1998). By contrast, other methods include the determination of hydrocarbons (Narváez-Rivas et al., 2008) and volatile compounds profiles (Timón et al., 2001; Narváez-Rivas et al., 2011). Moreover, isotope analyses on the adipose tissue of the animals have also been applied to characterize and differentiate Iberian pork meat as a function of the diet of the animal (González-Martín et al., 1999). As to spectrometric techniques, near-infrared spectrometry (NIR) has also been applied with the same purpose (Arce et al., 2009), although this method could not perfectly discriminate among the different pig categories. Recently, more attention has been paid to the determination of the TAGs composition, as analyses become easier and faster, avoiding the use of saponification and the formation of methyl esters (Díaz et al., 1996; Gallardo et al., 2012). The TAGs composition has been also applied to elucidate the effect of genotype (Tejeda et al., 2002; Petrón et al., 2004; Viera-Alcaide et al., 2008). However, there are still some ambiguities in the results obtained by the methods used until now when high amounts of samples have been analyzed. Although the purpose of the new Spanish regulation consists of protecting the purity of Iberian pig, controlling the breeding and feeding and properly labelling the final product, there is still a lack of a normalized and robust method to discriminate among the different categories.
Some studies have focused on the characterization of physical properties of pork fat. Svenstrup et al. (2005) analyzed the influence of changing the cooling and reheating rates applied to pork fat. By using DSC and XRD techniques, they characterized samples of lard (pork dorsal fat) and leaf pork fat (surrounding the kidneys) in their raw state, their extracted fat fraction, and in liver pâté. The crystallization behavior observed permitted defining the textural properties obtained when pork fats were used in liver pâté. Campos et al. (2002) also applied different cooling rates to lard samples, but in order to observe the effects of such variations on the hardness of fat crystal networks. By contrast, Davenel et al. (1999) studied solid fat content variations of pig adipose tissues in relation to their lipid composition. As to spectroscopic techniques, Raman spectrometry has been applied toin situ analyze the crystalline states of fat in porcine adipose tissue (Motoyama et al., 2013). The purpose was to establish a tool for routine monitoring the physical conditions of meat carcasses in refrigerators. The same technique was also proposed to discriminate between pork and beef fat, by analyzing the polymorphic features of the characterized samples (Motoyama et al., 2010).
Our group has recently analyzed the polymorphic behavior of the two most differentiated categories of Iberian dry-cured ham (bellota andcebo ), and obtained very promising results (Bayés-García et al., 2016), as significant differences, related to the polymorphic behavior of the samples, were detected and permitted to clearly discriminate among the two categories. Crystallographic techniques, such as differential scanning calorimetry (DSC) and X-ray diffraction (XRD, with both laboratory-scale or synchrotron radiation source) have been widely used to study the polymorphism of lipid systems, in order to characterize their physical properties, such as melting, morphology, rheology and texture (Larsson et al., 2006; Bayés-García et al., 2020). Nevertheless, these techniques have been rarely used in food authentication.
In the present work we used DSC and XRD techniques to study the polymorphic behavior of the four Iberian pork categories, including intermediate recebo and cebo de campo to make a step further. Moreover, we used raw lipid samples, obtained before the curing of the ham, in order to define a more robust method, as curing becomes a very complex procedure in which it is not possible to control the variables which may modify the behavior of the samples. This study demonstrates that crystallographic tools may be promoted in the food authentication field to combat food fraud.