*3.2. Thermal Analysis*

The phase transitions of olive oils measured by DSC are affected by molecular composition changes [38,39]. Figure 1 shows the cooling (A, B, C, D) and heating (E, F, G, H) thermograms of the thirteen studied monovarietal extra virgin olive oils at t1, divided by region of origin. All the curves show common traits: two main transitions upon cooling (p1c, p2c), three main transitions upon heating (p1h, p2h, p3h); analogous thermograms have been already observed for extra virgin olive oils [38,39]. The thermal phenomena observed during cooling are basically influenced by the chemical composition of the samples [38]. In particular, the main exothermic event, peaked at lower temperatures (p1c, Figure 1) has been related to the crystallization of TAG rich in oleic acid. The shape of this transition always appeared as a symmetrical Gaussian curve; suggesting an ordered and cooperative event involving homogenous molecules. The second major exothermic event peak occurred at higher temperatures upon cooling, p2c, and had an asymmetrical shape, indicating the involvement of more heterogeneous molecules, previously identified as saturated triglycerides (TAG) fractions [39].

**Figure 1.** DSC thermograms of the thirteen olive oils (t1) divided by region of origin. **A**–**D**: cooling thermograms; **E**–**H**: heating thermograms. p1c, p2c: main thermal events on cooling; p1h, p2h, p3h: main thermal events on heating.

Intuitively, it might be presumed that the thermograms found upon heating would mirror the ones observed during cooling, in which the formed crystals melt. However, the heating thermograms are more complex. In detail, the first thermal event, p1h, is an exothermic transition, related to a solid-state transformation of the TAG crystals towards more stable forms [40]. p2h and p3h are two endothermic events related to the melting of other TAG polymorphic forms [40]. Bayés-García et al. [40] described the nature of these phenomena very well, by explaining how three main groups of TAGs: triunsaturated OOO and OOL, saturated-unsaturated-unsaturated POO, POL, and SOO, and saturatedsaturated-unsaturated PPO, were responsible for the polymorphic behavior observed.

Besides these similarities, each sample showed specific transition temperatures and peak amplitudes and shapes. In some cases, additional thermal events, often visible as shoulders of the main thermal events, both upon cooling and heating, were observed. These minor transitions have not been examined in this study.

Tables 3 and 4 report the thermal parameters extrapolated from the cooling and heating thermograms, respectively.

Looking at the cooling parameters (Table 3), significant differences between the samples were observed, also showing high correlations with the fatty acid composition. The range of cooling (Range\_C), calculated as the difference between Ton\_C and Toff\_C, at t1 varied from 32.5 ◦C of TOR, CM, and OR to 42.5 ◦C of COR. The larger is the cooling transition, the more heterogeneous are the molecules involved in the crystallization [41]. In general, the Apulian and Abruzzi samples had narrower ranges of transition, with lower Ton\_C and higher Toff\_C, than the Sardinian and Calabrian ones. Narrow cooling transition ranges and low Ton\_C have been previously associated with olive oils rich in oleic acid [39], able to perform cooperative crystallization phenomena at lower temperatures. In support of this hypothesis, negative correlations have been found between oleic acid content, Ton\_C, and Range\_C (*p* < 0.01; R = −0.326; −0.633). The cooling enthalpy (ΔH\_C) was also influenced by the oleic acid content. In particular, it was positively correlated with C18:1 (*p* < 0.01; R= 0.397) and negatively with C16:0, C18:2 and C:20 (*p* < 0.01, R= −0.270; −0.392; −0.238). The cooling enthalpy, calculated as the area under the cooling curve, is influenced by the number of molecules involved in the exothermic phenomenon [32]. In this study, at t1, it ranges from 67.5 J/g of TOR and DR, to 62 J/g of SIV. The temperature of the major crystallization peak (Tp1\_C) ranged from −42 ◦C of SIV and COR to −35 ◦C of BAM. This thermal event, previously associated with the crystallization of oleic rich TAGs [40], in this study, showed positive correlations with C18:1 (*p* < 0.01; R = 0.686) and negative with C16:0, C16:1, C18:2 (*p* < 0.01; R = −0.538; −347; 0.665). The minor exothermal event (p2\_c) peaked in a range of temperatures from −9.7 ◦C of OTT, to −18 ◦C of OR. Tp2\_C correlated positively with C16:0, C16:1, and C20:0 (*p* < 0.01; R = 0.287; 0.401; 0.238) and negatively with C18:1 (*p* < 0.05; R = −0.182) and C18:3 (*p* < 0.01; R = −0.270). These results confirm that p2\_c occurs at higher temperatures for oils richer in saturated fatty acids [40].




*Foods* **2021** , *10*, 1004

**Table 3.** *Cont*.

141

t1 t2 t3

−31.03 ± 0.28 dAB

−30.58 ± 0.09 dA

−31.47 ± 0.32 eB

GEN

14.10 ± 0.26 abA

13.53 ± 0.28 abAB

13.14 ± 0.26 abB

45.56 ± 0.27 bA

44.11 ± 0.36 bB

44.17 ± 0.54 bB

66.95 ± 2.05 deB

68.28 ± 0.68 bcdeAB

72.03 ± 2.38 abA

−19.19 ± 0.12 deA

−19.89 ± 0.35 efA

−19.28 ± 1.15 cdeA

−8.07 ± 0.26 iB

−8.28 ± 0.60 eB

−7.09 ± 0.10 eA

9.79 ± 0.22 abA

9.46 ± 0.12 abA

8.67 ± 0.07 bB


**Table 4.** *Cont*. significant differences between the means (*<sup>p</sup>* < 0.05). Different small letters in the same column, at the same harvesting time for the different cultivars, indicate significant differences

between the means (*<sup>p</sup>* < 0.05).

Fewer differences have been observed comparing the different harvesting times. The parameters that were more affected by olive ripening were Tp1\_C and Tp2\_C. In detail, Tp1\_C increased over time for GEN, CM, OR and decreased for SIV, SEM, and BAM; these trends may be related to the changes of C18:1 observed during ripening (Table 1). Interestingly, for samples not affected by the change in Tp1\_C over time, a change in Tp2\_C was instead observed. In particular, a decrease in this temperature was observed for TOR, DR, COR, OTT, OTTC, TDF, CIC; it can be related to a decrease in the saturated fatty acids and an increase in the unsaturated ones, with the exception of TDF, CIC for which the opposite trend was observed. Chiavaro et al. [39] observed a significant shift of Ton towards higher temperatures and enlargement of the temperature range as a consequence of ripening on the cooling curves of three monovarietal extra virgin Italian olive oils. These authors suggested an increase in the complexity of oil composition, due to TAG lysis and lipid oxidation. In this study only SIV, OR, and BAM demonstrated a broadening of the crystallization range; however, TDF even showed a narrowing of the transition.

Looking at the heating parameters (Table 4), at t1 significant differences have been observed between the samples. Ton\_H ranged from <sup>−</sup>26.5 ◦C of CM, OR and CIC, to <sup>−</sup><sup>37</sup> ◦C of TOR and DR. Toff\_H ranged from 14.5 ◦C of BAM to 10.5 ◦C of OR. From these results, it is visible that OR had the narrowest heating transition (Range\_H: 37 ◦C), while TOR, DR, and BAM showed a broader transition (Range\_H: 50 ◦C). Range\_H was negatively correlated with C18:0 and C20:0 (*p* < 0.05, R = −465, −0.651); it suggests that the presence of heterogeneous TAGs containing saturated fatty acids formed different polymorphic crystals during the cooling phase, which melt over a wider range of temperatures. The enthalpy of the heating transition ranged from 71.8 J/g of CIC to 64.64 of SIV. This parameter was positively correlated with C18:1 (*p* < 0.01, R = 0.484) and negatively with C16:0 and C18:2 (*p* < 0.01, R = 0.474, 0.431), as already reported in previous studies [18].

Tp1\_H ranged from −21.35 ◦C of SIV to −16 ◦C of TOR and CIC. Tp2\_H ranged from −8 ◦C of GEN to −4.74 ◦C of CIC, while Tp3\_H ranged from 6 ◦C of OR to 10.5 ◦C of BAM. Obtaining a clear correlation of this phenomena with the fatty acid composition is complicated by the kinetic nature of peak p1h and the polymorphisms that characterize p2h and p3h. However, the presence of additional characteristic melting phenomena in the region between peak 1 and peak 2, makes the DSC heating curves of extra virgin olive oil a unique fingerprint for this kind of sample [21].

Comparing the different ripening times, only the olive oil from the cultivar DR did not show any modification. Moreover, Ton\_H and ΔH\_H were almost stable for all the studied samples. The temperature ranges of the heating transition (Range\_H) showed a narrowing tendency for TOR, GEN, SIV, CM, and OTT, as a consequence of the Toff\_H shifting towards lower temperatures. Among the three thermal events observed during heating, the first one (p1\_h) was exothermic and shifted towards lower temperatures during ripening only for TOR and the three Sardinian cultivars (SIV, SEM, COR). It is not easy to find an explanation of this trend, as it is more related to kinetic phenomena. Tp2\_H shifted towards higher temperatures during ripening for TOR, GEN, CM, OR, while it moved to lower temperatures for OTT and OTTC. For these last two samples, it was more clear that this phenomenon could be due to a decrease in the saturated and polyunsaturated fatty acids and an increase in the monounsaturated ones. Tp3\_H was the most affected during ripening time; except DR and OR, this thermal event in all other samples shifted towards lower temperatures. These events may be related to the melting of the most saturated TAG polymorphic forms, which tend to decrease over time.

#### *3.3. Viscosity*

In this study, all the tested olive oils exhibited a linear relationship between shear stress and shear rate, as expected [18,42], allowing olive oil to be classified as a Newtonian fluid. The viscosity of the samples (Figure 2), calculated by Newton's law (Equation (1)), at t1 ranged from 65.97 mPa\*s of SIV to 69.83 mPa\*s of TOR, without significant differences between them. These values were in the same order of magnitude as that reported by other authors on virgin olive oils at 25 ◦C [18,42]. Comparing the viscosity values of the oils obtained from the same cultivar at different ripening times, few differences were measured: COR and OTTC showed, respectively, a decrease and increase in viscosity passing from t1 to t3.

**Figure 2.** Viscosity of 13 olive oils from minor Italian cultivars harvested at three different maturation stages. Data are expressed as the mean of three replicates ± standard deviations. Different capital letters, between the three harvesting times for the same cultivar, indicate significant differences (*p* < 0.05). Different small letters, at the same harvesting time for the different cultivars, indicate significant differences (*p* < 0.05).

Exploring possible correlations with fatty acids composition, a positive Pearson correlation was found between viscosity and oleic acid (*p* < 0.01; R = 0.276), while an inverse correlation was found with linoleic acid (*p* < 0.01; R = −0.333,). These findings have been already reported by other authors [42,43], as fatty acids with more double bonds, being loosely packed, and exhibiting a more fluid-like behavior.

#### *3.4. Chlorophyll Content*

Even though the color is not considered a quality attribute in olive oil quality assessment by panel experts [23], consumers use color as a parameter to evaluate olive oil quality and authenticity [44].

The green color of an extra virgin olive oil is due to the presence of chlorophyll; a photosynthetic pigment extracted from olives during milling. During olive ripening, due to catabolic enzymes, chlorophyll undergoes chemical modifications, involving a shift in color from brilliant green to black while going through several shades of purple/pink [25]. This phenomenon, called véraison, literally means a change of color, and is used as an indicator of the ripening stage. Olive farmers start harvesting the olives when they are in the middle of véraison, before full ripeness [13]. The change of color is due to chlorophyll loss and a concomitant increase in anthocyanin pigmentation [45].

It is assumed that the degree of ripening of the olive fruit, and consequently its chlorophyll content, will determine the amount of chlorophyll in the final oil [26].

Figure 3 shows the levels of chlorophyll found in the thirteen olive oils obtained from minor olive Italian cultivars, harvested in three different periods, shifted about two weeks from each other. At time 1 (t1), which represents the optimal olive ripening period, according to the farmers' experience, the chlorophyll levels ranged from 58.5 mg/kg of BAM, to 5.6 mg/kg of TDF. In general, at t1, the Apulian cultivars (CM, OR, BAM) and SEM, which is a Sardinian cultivar, showed the highest levels of chlorophyll. On the other hand, the Calabrian cultivars (OTT, OTTC, TDF, CIC) had the lowest level of chlorophyll. The amount of chlorophylls in olive oil depends on the olive cultivar, pedoclimatic conditions, and agronomic practices [46].

**Figure 3.** Chlorophyll content of 13 olive oils from minor Italian cultivars harvested at three different maturation stages. Data are expressed as the mean of three replicates ± standard deviations. Different capital letters, between the three harvesting times for the same cultivar, indicate significant differences (*p* < 0.05). Different small letters, at the same harvesting time for the different cultivars, indicate significant differences (*p* < 0.05).

Comparing the different olives' harvesting times, in most of the cases the amount of chlorophyll decreased over time. In particular, passing from t1 to t3, the highest chlorophyll loss was registered for CM, which undergoes an 85% loss. Similarly, Criado et al. [47] studied the pigment content in fruit from different olive varieties in six consecutive stages of ripeness. They found that the concentrations of chlorophyll decreased continuously in all the varieties during ripening.

In a few cases, the amount of chlorophyll remained rather constant between t1 and t3 (DR, SEM, OTTC).

#### *3.5. Color*

As previously reported, the color of olive oil is strictly connected with its pigment content. Confirming this hypothesis, significant correlations between chlorophyll content and colorimetric parameters (Table 5) have been found. In particular, negative Pearson correlations between chlorophylls and the chromatic parameters *L* (*p* < 0.01 R = −0.799) and *a*\* (*p* < 0.01 R = −0.637) were observed. The higher the chlorophyll content, the darker and greener the olive oil. Surprisingly, the correlation between chlorophylls and the chromatic parameters *b*\* was positive (*p* < 0.01 R = 0.668). The *b*\* parameter represents the yellow tones; it was previously related to the carotenoid content in olive oil [47]. We assume that, in this study, the method used for chlorophyll detection measured both chlorophylls *a* and *b*, known to generate intense blue-green and yellow-green shades, respectively [25]. Possibly, the amount of chlorophyll *b* may have influenced this result.


**Table 5.** Color parameters of 13 minor Italian olive oils harvested at three maturation stages 1.

1. Data are expressed as the mean ± standard deviation of three replicates. Different capital letters in the same column, between the three harvesting times for the same cultivar, indicate significant differences between the means (*p* < 0.05). Different small letters in the same column, at the same harvesting time for the different cultivars, indicate significant differences between the means (*p* < 0.05).

Looking at the differences between the cultivars at t1, the L values ranged from 46–47 of SEM and OR to 56 of DR, resulting in, respectively, the darkest and the lightest samples. Negative *a*\* values indicate the green color. At t1, OR, and BAM were the greenest samples, with values of −9. On the other hand, DR was the least green sample with values of −6. The color parameter *b*\* indicates yellow tones; a large variability of this parameter was

observed between the samples at t1. The values of *b*\* ranged from 25 of DR to 56 of COR, resulting in, respectively, olive oils that were less or more yellow.

Focusing on the differences between the harvesting times, for L the general tendency was to increase over time, in relation to the chlorophyll decrease. This phenomenon was especially visible passing from t1 to t2 for most of the cultivars. BAM underwent the highest lightening from t1 to t3 (14%), while DR and GEN did not show any significant change of L. The parameter *a*\* underwent a general increase from t1 to t3, indicating a progressive loss of greenness. TOR, DR, GEN, and the Apulian cultivar CM did not show significant differences of *a*\*. On the other hand, the highest loss of greenness was observed for the Calabrian cultivars already at t1. In particular, TDF suffered around a 52% loss of this value prolonging the harvesting time. A large variability between the cultivars was observed in the *b*\* value trend. A general decrease in the *b*\* value was observed for the Calabrian cultivars (OTT, OTTC, TDF, CIC), particularly for TDF with a 63.5% loss. On the other hand, the *b*\* value of the Apulian cultivars (CM, OR, BAM) increased during this time, and DR showed the highest increase in *b*\* from t1 to t3 (42%). Criado and co-workers [47] observed a decrease in L, an increase in *a*\*, and a decrease in *b*\* in two olive oil samples, in relation to the ripening stage of the olive fruit.
