**4. Discussion**

From the results of the GC-IT-MS and UPLC-DAD analysis of volatile compounds and phenols, respectively, it was clear that each of the investigated monovarietal EVOOs was characterized by a unique volatile and phenol profile. Since the samples were collected from various producers and were relatively heterogeneous in terms of geographical microlocations, growing conditions, harvest date, olive processing technology, and EVOO finalization and storage parameters, it could be assumed, with a high degree of certainty, that the effects of all of these factors were random, and that varietal origin was the main source of the observed differences. In fact, varietal origin was previously found to have a greater impact on volatile composition than various environmental factors [28]. However, the geographical origin possibly had an effect, which was impossible to evaluate separately from the effect of variety considering the varieties studied were specific for their regions. Istrian EVOOs, mostly those made of native Buža and Rosinjola, as well as those of the international variety Leccino, were characterized by higher concentrations of many LOX volatiles in relation to Dalmatian Oblica and Lastovka EVOO, including the most odoriferous ones, such as hexenals and 1-penten-3-one (Table 1). It is probable that this was directly reflected on the differences in their sensory profiles, since Istrian EVOOs had higher intensities of the majority of positive odor attributes, especially those

of *green grass*/*leaves* and *chicory*/*rocket* (Figure 3, Table S3). Lower OAV values of the most potent odorants, (*Z*)-3-hexenal, 1-penten-3-one, and (*E*)-2-hexenal found in the Dalmatian, especially Lastovka EVOO, corroborated this assumption (Table 2). Since Istria is a region characterized by lower average temperatures than Dalmatia (Table S1), these results basically corroborated what was previously found in the majority of such studies that the temperature of environment is negatively correlated with the concentrations of LOX-derived volatile aroma compounds and the resulting EVOO positive sensory attributes [48,49].

Although without statistical significance in some cases, Buža EVOO excelled with the highest concentrations of the majority of positive LOX volatiles (Table 1), as well as with the highest intensities of positive odor sensory attributes (Figure 3, Table S3), which were probably in a causal relationship. It is worth emphasizing the highest cumulative odor activity value (OAV) of (*Z*)-3-hexenal and 1-penten-3-one, the two most powerful known odorants in EVOO with very low odor perception thresholds of 0.0017 and 0.00073 mg/kg, respectively [22,46] (Table 2), which certainly exhibited key roles. A large proportion of LOX volatiles among those extracted by the PLSDA as the most significant VIP compounds discriminating Buža from the other EVOOs (Figure 2) corroborated the assumption that this variety is characterized by strong lipoxygenase and hydroperoxide lyase activities in the LOX pathway.

Generally, the most similar to Buža in terms of high concentrations of LOX volatiles (Table 1) and their OAVs (Table 2), as well as high intensities of positive odor attributes (Figure 3, Table S3), was Rosinjola EVOO. When it came down to the discriminating VIP compounds, those extracted by PLSDA were mostly benzenoids (Figure 2). Many benzenoids which were found in relatively high concentration in Rosinjola EVOO, including methyl benzoate, acetophenone, and methyl salicylate, were previously reported to be important almond odorants [50], however their impact in olive oil has not been investigated yet. As well, *almond* note was not especially accentuated in Rosinjola EVOO (Figure 3, Table S3).

Istarska bjelica had lower concentrations of many important LOX volatiles (Table 1). Since it is a late ripening variety [51] it is possible that it was characterized by a slightly weaker LOX enzymatic load with respect to Buža and Rosinjola EVOO. As it is known that phenols may act as LOX enzymatic activity inhibitors [52], the possibility that the high concentrations of phenols found in this monovarietal EVOO (Table 3) acted in this way during milling and malaxation should not be excluded. However, the concentrations and OAVs of some other major LOX odorants, such as (*E*)-2-hexenal and 1-penten-3-one, were relatively high, suggesting a notable activity of (*Z*)-3:(*E*)-2-enal isomerase which catalyzes the conversion of (*Z*)-3- to (*E*)-2-hexenal, as well as relatively high activity of the enzymes or availability of the substrates involved in the synthesis of C5 compounds via 13-alkoxy radicals in this side-branch of the LOX pathway. The concentrations and OAVs of these volatiles were not lower that those found in Buža and Rosinjola EVOOs (Tables 1 and 2), so it is probable that (*E*)-2-hexenal and 1-penten-3-one were the key odorants in the formation of I. bjelica aroma and were the most responsible for the high intensity of several positive sensory attributes observed in this EVOO (Figure 3, Table S3). The most typical VIP chemical markers distinguishing I. bjelica EVOO were mostly non-LOX volatiles, namely middle-chain fatty acids and C6 alcohol acetates (Figure 2). Judging on the determined OAV values (Table 2), their sensory relevance was probably minor to medium. High concentrations of C6 alcohol acetates (Table 1) implied a possible high alcohol acyl transferase activity in olives and olive paste of this variety [53].

As stated previously, Oblica was characterized by a slightly lower contribution of the LOX volatiles and, consequently, lower intensities of particular positive odor attributes with respect to Istrian EVOOs, but was still superior to Lastovka EVOO (Table 1, Figure 3, Table S3). It was possibly mostly due to lower 1-penten-3-one and (*E*)-2-hexenal concentrations and OAVs, since the level of (*Z*)-3-hexenal was relatively high (Tables 1 and 2). As well, it is possible that a part of the *fruity* and *green* aroma originated from hexanal, found in higher concentration with respect to the Istrian EVOOs (Tables 1 and 2). *Green banana* odor sensory attribute which was found to be typical for Oblica EVOO (Figure 3, Table S3) could

have, at least partly, originated from the volatiles often associated with this nuance. (*Z*)-3-hexen-1-ol was certainly a candidate for this role [45], since its concentration was the highest in this EVOO (Table 1) and at the same time above the corresponding odor detection threshold (Table 2). For other LOX volatiles with the odor commonly described as banana-like, such as hexanol, hexenyl acetates and penten-1-ols [45,54], no significant differences between varieties were found. As well, their levels in Oblica were not among the highest among the investigated EVOOs (Table 1), implying their impact in the formation of *green banana* nuance was probably not crucial. The same applies for other minor volatiles commonly reported as carriers of banana odor, such as isoamyl and other acetates. The VIP compounds responsible for the differentiation of Oblica EVOO (Figure 2), which pertained to several chemical families, could have not been meaningfully related to the occurrence of *green banana* odor.

Lastovka EVOO was characterized by the most distinguishable volatile profile among the investigated monovarietal EVOOs. It contained the lowest concentrations of the majority of LOX volatiles (Table 1), including the most potent odorants with the highest OAVs (Table 2), which was certainly a direct cause of the lowest intensities of the majority of positive odor attributes perceived in this EVOO (Figure 3, Table S3). On the other hand, it was found to have high amounts of hexanal, particular C6 alcohols, and hexyl acetate, compounds often accounted among the carriers of *green* odor which derive from the enzymatic degradation of linoleic acid but also oxidation [10,28,55]. Lastovka EVOO contained the highest concentrations of particular monoterpenes and sesquiterpenes, whose sensory contribution is generally described by descriptors such as *citrus*, *camphor*, *eucalyptus*, *roses*, etc., as well as *wood*. Although sensory relevance of terpenes in olive oil is currently still unknown and it is certainly limited by the lipid matrix in which these lipophilic molecules are highly soluble, the possibility of their contribution to the specific *wood* odor perceived in Lastovka EVOO during sensory analysis (Figure 3, Table S3) should not be excluded. Several terpenes were extracted by PLSDA as among the most discriminative compounds for this variety (Figure 2). Particular sesquiterpenes, on the other hand, such as (+)-cycloisosativene, α-copaene, α-muurolene, δ-cadinene, and several unidentified ones were found in the lowest concentrations in Lastovka EVOO (Table 1). Terpenes were previously found to have large potential to differentiate EVOO according to variety [16,56], which was basically confirmed in this study. Other compounds found to be characteristic for Lastovka EVOO, such as particular saturated short-chain aldehydes, ketones, alcohols, and acids, could also have had a sensory impact with their *malty*, *pungent*, *rancid*, and *sweaty* nuances (Table 2).

One of probably the most important characteristics found typical for Leccino EVOO was the ratio between the important (*E*)-2- and (*Z*)-3-C6 forms, which was generally the highest and discriminated well this EVOO from the majority of the other studied EVOOs (Tables 1 and 2). The highest concentration of (*E*)-2-hexenal and the lowest concentration of (*Z*)-3-hexenal, (*Z*)-3-hexen-1-ol, as well as the low concentration of (*Z*)-3-hexenyl acetate in Leccino EVOO were likely the result of high (*Z*)-3:(*E*)-2-enal isomerase activity in Leccino olives, i.e., olive paste during milling and malaxation steps [57]. Considering that the estimated contribution of (*E*)-2-hexenal to the aroma of EVOOs was generally lower than that of (*Z*)-3-hexenal (Table 2), it is possible that one of the consequences of the observed differences was a slightly lower intensity of particular odor sensory attributes, such as *green*/*grass leaves* and *chicory*/*rocket*, observed in Leccino with respect to the EVOOs from the other, native Istrian varieties Buža, I. bjelica and Rosinjola (Figure 3, Table S3). Other interesting features of Leccino EVOO included lower concentrations of particular sesquiterpenes and furanoids. In fact, many sesquiterpenes were among those with the highest VIP scores extracted by PLSDA, but with a negative sign (Figure 2).

Phenols, especially secoiridoids, are responsible for the characteristic EVOO bitterness and pungency, but the specific sensory contribution of each individual major secoiridoid has not been precisely elucidated up to date. Nevertheless, there is solid evidence that *p*-HPEA-EDA is a key contributor to pungency, while the pungency of other, monoaldehydic ligstroside aglycons is weaker, although still strong [24]. Ligstroside aglycons were found to generally be less bitter than pungent, which was especially the case for *p*-HPEA-EDA. In the same study [24] it was found that the majority of

oleuropein aglycons, including 3,4-DHPEA-EDA, was described as both bitter and pungent, with some of them exhibiting rather strong bitterness. The lowest intensity of bitterness observed in Oblica and Leccino (Figure 2, Table S3) could be tentatively linked to the lowest oleuropein aglycon I concentrations found in these EVOO (Table 3). The intensity of pungency did not quite correlate with the average *p*-HPEA-EDA concentrations (Figure 3, Table 3, Table S3). In contrast to I. bjelica, for which a positive correlation was observed, Rosinjola EVOO was characterized as intensively pungent according to the official method [1] (intensity >6) despite containing relatively low concentration of this secoiridoid. The highest average concentration of oleuropein aglycon II in Rosinjola EVOO (Table 3) could have possibly compensated for this deficiency. The pungency of I. bjelica possibly partly originated also from the highest concentrations of all the three monoaldehydic ligstroside aglycons found in this EVOO (Table 3). Especially interesting was the highest ratio of bitterness to pungency found in Lastovka EVOO (Figure 3, Table S3). Roughly, Lastovka EVOO contained among the highest concentrations of oleuropein aglycones and among the lowest concentrations of ligstroside aglycones, which could have had such an impact. This EVOO had the highest concentration of *p*-coumaric acid which, although relatively low, possibly contributed to the bitterness observed. It is worth mentioning that the UPLC chromatograms of Lastovka EVOO contained several unidentified peaks in addition to those observed in the other monovarietal EVOOs (data not shown), which possibly originated from the compounds with sensory relevance. The so-called *sweetness* in most cases coincided with the lower amounts of total phenols, which was as expected (Figure 3, Table 3). Again, several features turned out to be specific for Leccino EVOO (Table 3), the most important being the highest concentration of 3,4-DHPEA-EDA, which implied variety-dependent differences with respect to the availability of precursors and enzymatic activity between Leccino and Croatian native olive varieties.

#### **5. Conclusions**

The use of GC-IT-MS and UPLC-DAD proved to be a powerful combination for studying the inter-varietal diversity of typical volatile and phenolic profiles of Croatian EVOOs, respectively. Each of the investigated monovarietal EVOO displayed unique volatile aroma and phenol composition. The qualitative and quantitative chromatographic data was useful for tentative elucidation of some of the perceived sensory attributes including the variety-typical ones, which though has to be taken with caution due to the extreme complexity of the established chemical profiles with the majority of volatiles with still unknown sensory relevance. Many potential varietal markers were extracted by uniand multivariate statistical analysis despite high intra-varietal heterogeneity. It was demonstrated that volatiles and phenols from all the investigated chemical classes can be useful for this purpose. Many of the volatile compounds which turned out to have a notable discrimination power were (tentatively) identified for the first time in EVOO, or were generally neglected in previous studies, especially from sensorial point of view. In fact, only in a few cases were the major LOX compounds, studied most extensively among the volatiles up to date, sufficient for a robust varietal differentiation in this work. This indicates a large potential of the untargeted fingerprinting approach for EVOO characterization, differentiation, and authentication studies.

The number of the extracted robust varietal markers among the investigated chemical compounds largely exceeded the number of typical sensory attributes useful to differentiate monovarietal EVOOs. It is reasonable to conclude that the approach which comprises GC-IT-MS and UPLC-DAD analytical techniques may provide additional objective information about varietal origin which successfully complement those obtained by sensory analysis. Probably the best example for this is the case of Rosinjola EVOO which was relatively similar and hardly distinguishable from that of Buža variety based solely on the sensory analysis, but was characterized by many exclusive chemical markers among benzenoid and furanoid volatiles which discriminated this EVOO rather successfully.

The results obtained in this study could certainly be useful for improving the quality management and control in the production of Croatian monovarietal/PDO EVOO. These findings could contribute to strengthening their PDO identities and position on the market, and could be especially useful for discriminating EVOOs of Croatian native varieties from the world famous Leccino variety.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2304-8158/8/11/565/s1, Table S1: Climate parameters in the Istria and Dalmatia regions of Croatia in 2015, Table S2: Standardized coefficients of the variables selected for the differentiation of monovarietal Buža, Istarska bjelica, Rosinjola, Oblica, Lastovka, and Leccino extra virgin olive oils on the first three discriminant functions obtained by stepwise linear discriminant analysis, and the percentage of correct classification at each step, Table S3: The intensities and scores of the sensory attributes perceived in monovarietal extra virgin olive oils produced from Buža, Istarska bjelica, Rosinjola, Oblica, Lastovka, and Leccino varieties in Croatia.

**Author Contributions:** Conceptualization, I.L.; Methodology, I.L.; Formal analysis, I.L., M.L., M.Ž., M.K., S.G., K.B.B.; Resources, I.L., M.L., K.B.B.; Data curation, I.L., M.L., K.B.B.; Writing—original draft preparation, I.L.; Writing—review and editing, I.L., M.L., K.B.B.; Supervision, I.L.; Project administration, I.L.; Funding acquisition, I.L.

**Funding:** This research was funded by Croatian Science Foundation grant number UIP-2014-09-1194.

**Acknowledgments:** The authors would like to thank olive oil producers from Istria and Dalmatia (Croatia, EU) for donating the samples of extra virgin olive oils, and Ivana Horvat M.Sc. for technical assistance.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.
