Differential scanning calorimetry: A potential tool for discrimination of olive oil commercial categories

a n a l y t i c a c h i m i c a a c t a 6 2 5 ( 2 0 0 8 ) 215–226

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Differential scanning calorimetry: A potential tool for discrimination of olive oil commercial categories Emma Chiavaro a,∗ , Maria Teresa Rodriguez-Estrada b , Carlo Barnaba a , Elena Vittadini a , Lorenzo Cerretani c , Alessandra Bendini c a b c

Dipartimento di Ingegneria Industriale, Università degli Studi di Parma, viale Usberti 181/A, I-43100, Parma, Italy Dipartimento di Scienze degli Alimenti, Università di Bologna, viale Fanin 40, I-40127, Bologna, Italy Dipartimento di Scienze degli Alimenti, Università di Bologna, piazza Goidanich 60, I-47023, Cesena, Italy

a r t i c l e

i n f o

a b s t r a c t

Article history:

Differential scanning calorimetry thermograms of five commercial categories of olive oils

Received 13 May 2008

(extra virgin olive oil, olive oil, refined olive oil, olive-pomace oil and refined olive-pomace oil)

Received in revised form 2 July 2008

were performed in both cooling and heating regimes. Overlapping transitions were resolved

Accepted 17 July 2008

by deconvolution analysis and all thermal properties were related to major (triacylglycerols,

Published on line 29 July 2008

total fatty acids) and minor (diacylglycerols, lipid oxidation products) chemical components.

Keywords:

enthalpies were significantly lower in olive oils due to a more ordered crystal structure,

Extra virgin olive oil

which may be related to the higher triolein content. Pomace oils exhibited a significantly

Olive oil

higher crystallization onset temperature and a larger transition range, possibly associated

Olive-pomace oil

to the higher amount of diacylglycerols. Heating thermograms were more complex: all

Differential scanning calorimetry

oils exhibited complex exo- and endothermic transitions that could differentiate samples

Thermal properties

especially with respect to the highest temperature endotherm.

All oils showed two well distinguishable exothermic events upon cooling. Crystallization

Chemical composition

These preliminary results suggest that both cooling and heating thermograms obtained by means of differential scanning calorimetry may be useful for discriminating among olive oils of different commercial categories. © 2008 Elsevier B.V. All rights reserved.

1.

Introduction

Commercial categories of olive and olive-pomace oils have been initially classified in 1966 and were reclassified in 2001, by the European Community (EC) Council of Regulation in an attempt to avoid misleading of consumers and operators and to preserve the image of high-quality products as extra virgin and virgin olive oils [1]. Extra virgin olive oil (EvOo) (free acidity <0.8 g 100 g−1 ) is the highest quality product among olive oils as it is obtained from olive fruits using only mechanical processing steps or other

∗

Corresponding author. Tel.: +39 0521 905888; fax: +39 0521 905705. E-mail address: [email protected] (E. Chiavaro). 0003-2670/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.aca.2008.07.031

physical means under conditions that do not lead to oil alteration [1]. Refining treatment can be applied to virgin olive oil of low quality to obtain products classified as refined virgin olive oil (ROo) (free acidity <0.3 g 100 g−1 ). Some mixtures of oils of different commercial categories are also legally permitted and are commonly performed by producers, who assess several chemical parameters (i.e. free acidity, peroxide value, fatty acid and sterols composition, etc.) of the oils to get a product that meets compositional Legal Limits set by the EU [2]. In particular, olive oil (Oo) (free acidity <1.0 g 100 g−1 ) can be produced by blending virgin olive oil with ROo.

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Among olive-pomace oils, refined crude olive-pomace oil (RPo) (free acidity <0.3 g 100 g−1 ) is produced by refining crude olive-pomace oil previously obtained from pomace by means of solvent extraction, and olive-pomace oil (Po) (free acidity <1.0 g 100 g−1 ) is made by blending RPo and virgin olive oil. The EC Council of Regulation has recently redefined the physico-chemical characteristics of olive and olive-pomace oils in an attempt to harmonize them with the international standards set by the International Olive Oil Council and the Codex Alimentarius [2,3]. Major differences between EvOo, Oo and Po compositions are linked to minor compound content: for example only EvOo contains a significant presence of phenolic compounds. On the other hand, Po has higher amount of waxes (≤250 mg kg−1 for EvOo, ≤350 mg kg−1 for Oo and >350 mg kg−1 for Po from EC Reg. no. 702/2007) and total aliphatic alcohols (erythrodiol and uvaol) (≤4.5% for EvOo and Oo, >4.5% for Po from EC Reg. no. 702/2007) that were more efficiently extracted from fruit husk by organic solvents. The olive oil classification is generally carried out with physico-chemical and sensory methods, but wrong classifications can still occur. Chemical methods are most commonly applied for this scope, but they are known to be expensive, time-consuming, and to have high environmental impact. Sensory evaluation is a powerful tool, but it is necessary a continuous training of the panel to achieve reliable oil assessments; furthermore, the correlations between sensory attributes and chemical composition have not been fully clarified yet [4]. Availability of new/additional analytical techniques as supporting tools for currently used methods may be helpful to improve olive oil classification and market management. Differential scanning calorimetry (DSC) is an analytical technique that has been applied in oil and fat research for the characterization of oils from different vegetables sources, providing a reproducible method for their identification [5,6] as thermal properties were found to be related to the chemical composition in extra virgin olive oils [7]. Jiménez Márquez and Beltrán Maza [8] were able to differentiate monovarietal virgin olive oils based on temperature (onset and transition range) of crystallization and melting profiles, which were well correlated with oleic and linoleic acid contents. More recently, a good relation was reported between thermal properties and major (triacylglycerols and fatty acids) and minor components (free fatty acids, diacylglycerols, and primary and secondary oxidation products) of monovarietal extra virgin olive oils for both cooling and heating profiles that were deconvoluted into the constituent peaks and related to specific triacylglycerol (TAG) species [9,10]. DSC application upon cooling and heating also appeared very promising in discriminating among oil samples from olives of different cultivars and/or harvesting periods [9,10]. Little information is reported in literature about DSC characterization of olive oils of different commercial categories. Jiménez Márquez associated the melting thermograms of virgin, refined, and lampante olive oils to TAG composition [11]; melting enthalpy and/or transition temperature were found to be related to the different content of TAG fractions in these oils. Angiuli et al. reported that calorimetric techniques (DSC in particular) may be used to efficiently discriminate between commercial and guaranteed origin extra virgin olive oils [12],

as well as for the recognition of the physical and/or mechanical treatments (refining, deodorization, filtration, etc.) in virgin olive oil [13]. However, chemical composition of the oils was not considered in these studies [12,13]. The aim of this preliminary work was to verify the potential DSC application to discriminate among olive oils of different commercial categories, evaluating the relationship between thermal properties (obtained upon cooling and heating) and chemical composition (major and minor components). Deconvolution analysis was applied to better characterize the complex nature of the transitions.

2.

Experimental

2.1.

Sampling

All commercial olive oil samples were supplied by Coppini Arte Olearia (Parma, Italy) and stored in dark bottles without headspace at room temperature before analysis. The olives used for oil production were hand-picked in 2006 and belonged to two cultivars (Nocellara del Belice and Ogliarola Messinese) from Trapani (Sicily, Italy). Olives were processed by a continuous industrial plant with a working capacity of 1 ton h−1 equipped with a hammer crusher, a horizontal malaxator (at a temperature of 27 ◦ C), and a three-phase decanter. Both EvOo and raw pomace oil were obtained with the same production cycle. All other oils were obtained as follows: RPo by physical refining of raw pomace oil; Po by blending EvOo and RPo; ROo by physical refining of an inedible extra virgin olive oil obtained from late, hand-picked olives of the same cultivars; Oo by blending EvOo and ROo. One sample per each category was taken and, thereafter, analyzed.

2.2.

Reagents, solvents and standards

All solvents used were analytical or high-performance liquid chromatography (HPLC) grade (Merck, Darmstadt, Germany). Commercial standards of triacylglycerols (triolein (OOO), trilinolein (LLL)), diacylglycerols (dimyristin, dipalmitin, distearin, diolein), tridecanoic acid, tridecanoic acid methyl ester and dihydrocholesterol (94.8% purity), were purchased from Sigma–Aldrich (St. Louis, MO, USA). The standard mixture of fatty acid methyl esters (GLC 463) was supplied by Nu-Chek (Elysian, MN, USA). Silica solid-phase extraction (SPE) cartridges (500-mg stationary phase/3-mL Strata cartridges) were purchased from Phenomenex (Torrance, CA, USA).

2.3.

DSC

Samples of oil (8–10 mg) were weighed into aluminium pans, covers were sealed into place and analyzed with a DSC Q100 (TA Instruments, New Castle, DE, USA). Indium (melting temperature 156.6 ◦ C, Hf = 28.45 J g−1 ) and n-dodecane (melting temperature −9.65 ◦ C, Hf = 216.73 J g−1 ) were used to calibrate the instrument and an empty pan was used as reference. Oil samples were equilibrated at 30 ◦ C for 3 min and then cooled at −80 ◦ C at the rate of 2 ◦ C min−1 , equilibrated at −80 ◦ C for 3 min and then heated from −80 ◦ C to 30 ◦ C at 2 ◦ C min−1 . Dry nitrogen was purged in the DSC cell at 50 cm3 min−1 . Thermograms

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were analyzed with Universal Analysis Software (Version 3.9A, TA Instruments) to obtain enthalpy (H, J g−1 ), Ton (◦ C) and Toff (◦ C) of the transitions (intersection of baseline and tangent at the transition) and peak temperature (Tp , ◦ C). Range of the transitions was calculated as temperature difference between Ton and Toff . Five replicates were analyzed per sample. Overlapping transitions of the cooling and heating thermograms were deconvoluted into individual constituent peaks using a PeakFitTM software (Jandel Scientific, San Rafael, CA, USA). The following parameters were considered for each deconvoluted peak: onset (Ton ), offset (Toff ) and peak temperature (Tp ). Range temperature of each peak was calculated as temperature difference between Ton and Toff .

2.4. High-performance liquid chromatography (HPLC) determination of triacylglycerols (TAG) HPLC analysis were carried out using a HP 1100 Series instrument (Agilent Technologies, Palo Alto, CA, USA), coupled to a 5-␮m LunaTM C18 (Phenomenex) column (25 cm × 3.0 mm i.d.) and equipped with a binary pump delivery system, a degasser, an autosampler, a diode-array (DAD) and a mass spectrometry (MSD) detectors, as previously reported [14]. A C18 precolumn filter (Phenomenex,) was used. All solvents were filtered through a 0.45-␮m nylon filter disk (Lida Manufacturing Corp., Kenosha, WI, USA) prior to use. Samples were prepared by dissolving the oil at 3% in a mixture of 2-propanol/n-hexane/acetonitrile (2:1:2, v/v/v). The injection volume was 10 ␮L. All the analyses were carried out at room temperature. The gradient elution was performed by using 2-propanol and acetonitrile as mobile phases A and B, respectively. The linear gradient elution system was: from 0 to 40 min held at 70% A; from 40 to 51 min, decreased to 45% A; from 51 to 60 min, decreased to 0% A; from 60 to 65 min, increased to 100% A; from 65 to 70 min, decreased to 60% A; from 70 to 75 min, increased to 70% A, as post-run. The flow rate was 0.4 mL min−1 from 0 to 51 min, 0.5 mL min−1 from 51 to 55 min, 0.6 mL min−1 from 55 to 60 min and 0.4 mL min−1 until the end of the HPLC run. The effluent was monitored with both DAD and MSD. The wavelength was set at 205 nm. MS detection was performed by using an atmospheric pressure chemical ionization (APCI) interface in the positive mode, according to the following conditions: drying gas flow, 3.0 L min−1 ; nebulizer pressure, 60 psi; drying gas temperature, 350 ◦ C; vaporizer temperature, 450 ◦ C; capillary voltage, 3000 V; corona current, 4 ␮A; and fragmentor voltage, 70 V. These compounds were identified based on their UV–vis and mass spectra obtained by HPLC-APCI-MSD and literature data [15]. The following TAG were identified: trilinolein (LLL), dilinoleoyl-palmitoleoyl-glycerol (LLPo), oleoyl-linoleoyllinolenoyl-glycerol (OLLn), dilinoleoyl-oleoyl-glycerol (OLL), palmitoleoyl-oleoyl-linoleoyl-glycerol (OLPo), dilinoleoylpalmitoyl-glycerol (LLP), dioleoyl-linolenoyl-glycerol (OLnO), dioleoyl-linoleoyl-glycerol (OLO), palmitoyl-oleoyl-linoleoylglycerol (OLP), dioleoyl-palmitoleoyl-glycerol (OOPo), palmitoyl-palmitoleoyl-oleoyl-glycerol (POPo), triolein (OOO), stearoyl-oleoyl-linoleoyl-glycerol (SLO), dipalmitoyloleoyl-glycerol (POP), dioleoyl-stearoyl-glycerol (SOO) and palmitoyl-stearoyl-oleoyl-glycerol (SOP). The limit of quanti-

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tation (LOQ) was 0.01 g 100 g−1 of TAG. Three replicates were prepared and analyzed per sample.

2.5. Gas chromatographic (GC) determination of total diacylglycerols (DAG) and major 1,2-DAG and 1,3-DAG DAG were determined according to a modified version of the method suggested by Bonoli et al. [16], where dihydrocholesterol was used as internal standard. Seventy microliters of a solution of dihydrocholesterol (1 mg of dihydrocholesterol in 1 mL of n-hexane:isopropanol (4:1, v/v)) were added to 100 mg oil and dissolved in 500 ␮L of n-hexane before loading into SPE. The rest of the DAG purification by SPE elution was the same as reported by Bortolomeazzi et al. [17]. Identification of DAG was carried out by comparing the peak retention times and the GC traces with those of the DAG standards and chromatograms reported in literature [18], respectively. The following DAG were identified: 1-palmitoyl-2-oleoyl-sn-glycerol (1,2-PO), 1palmitoyl-2-linoleoyl-sn-glycerol (1,2-PL), 1,2-diolein (1,2-OO), 1-oleoyl-2-linoleoyl-sn-glycerol (1,2-OL) and 1,3-diolein (1,3OO). LOQ was 0.04 g 100 g−1 of DAG. Three replicates were analyzed per sample.

2.6.

GC determination of total fatty acids (FA)

FA were determined according to Cercaci et al. [19]. About 10 mg of the oil were methylated twice with 100 ␮L of diazomethane, taken to dryness under nitrogen stream, dissolved in 500 ␮L of n-hexane and then transmethylated with 20 ␮L of 2N KOH solution in methanol. The mixture was vigorously shaken with a vortex for 1 min, then added with a known amount of internal standard solution (50 ␮L of tridecanoic acid methyl ester), centrifuged at 395 × g for 3 min and injected into a GC (1 ␮L of the supernatant). The GC instrument was a Carlo Erba HRGC Fractovap 4160 (Carlo Erba, Milan, Italy), which was coupled to a CPSil88 fused silica capillary column (50 m × 0.25 mm i.d. × 0.20 ␮m film thickness) (Restek, Bellefonte, PA, USA) coated with 90% biscyanopropyl–10% phenylcyanopropyl-polysiloxane. Oven temperature was programmed from 160 to 230 ◦ C at a rate of 1.5 ◦ C min−1 ; the final temperature was kept for 10 min. The injector and detector temperatures were both set at 230 ◦ C. Helium was used as carrier gas at 1.25 mL min−1 . The split ratio was 1:20. Peak identification was carried out by comparing the peak retention times with those of the GLC 463 FAME standard mixture. The internal standard was used for the quantification of fatty acids. The GC response factor of each fatty acid was calculated by using the GLC 463 FAME standard mixture and the internal standard (C13:0). LOQ was 0.01 g 100 g−1 of fatty acids. Three replicates were analyzed per sample.

2.7. Determination of free acidity (FA), peroxide value (POV) and p-anisidine value (PAV) Evaluation of free fatty acid content (expressed as % oleic acid) and primary oxidation products (expressed as meq O2 kg−1 oil) were performed according to the official methods described in annex III of EEC Regulation 2568/91 [20]. PAV determination was performed according to the IUPAC standard method 2.504,

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by measuring absorbance at 350 nm [21]. Three replicates for each determination were analyzed per sample.

2.8.

Oxidative stability index (OSI) time

The evaluation of the OSI was performed by using an eightchannel oxidative stability instrument (Omnion, Decatur, IL, USA), according to Jebe et al. [22]. The conductibility was measured in polycarbonate tubes using twice distilled water. The air flow was set at 120 mL min−1 . The OSI were run at 110 ± 0.1 ◦ C. Results were expressed as induction time (h). Three replicates were analyzed per sample.

2.9.

Statistical analysis

Means and standard deviations were calculated with SPSS (Version 14.0, SPSS Inc., Chicago, IL, USA) statistical software. SPSS was used to perform one-way analysis of variance (ANOVA) and Tukey’s honest significant difference test (HSD) at a 95% confidence level (p < 0.05) to identify differences among samples.

3.

Results and discussion

3.1.

Chemical composition

Chemical composition and oxidation parameters of EvOo, Oo, ROo, Po and RPo, obtained as described in Section 2, are reported in Table 1. Sixteen TAG were identified in all samples; eight of them were separately quantified and the others were quantified as pairs (LLL + LLPo, OLL + OLPo, LLP + OLnO and OLP + OOPo). OLL + OLPo, OLO, OLP + OOPo, OOO and SLO accounted for more than 85% of the total TAG in all commercial categories, whereas SOP was present in the lowest percentage (Table 1). Comparing olive and pomace oils, LLL + LLPo and LLP + OLnO were more represented in the latter oils than in the former samples. On the contrary, OOO was significantly higher in EvOo, Oo and ROo than in pomace oils. TAG were also grouped according to the type of FA bonded to the glycerol structure and percentage data of disaturated triacylglycerols (DSTAG), monosaturated triacylglycerols (MSTAG) and triunsaturated triacylglycerols (TUTAG) were reported in Table 1. EvOo exhibited significantly higher percentage of DSTAG than all other commercial categories. Both olive-pomace oils showed significantly greater MSTAG values than the other samples. TUTAG were significantly higher in Oo as compared to the other oils. Both olive-pomace oils also exhibited significantly greater amounts of DAG than olive oils. The concentrations of single DAG (Table 1) also varied among the different commercial categories. DAG were mostly present as 1,2-DAG (mainly 1,2OO and 1,2-OL) in all samples. EvOo presented the maximum value of 1,2-OO, which arose from the lysis of triolein, the major TAG in this oil category. On the contrary, Po and RPo showed significantly higher contents of 1,2-PL and 1,2-PO than oils from olives. FA percentages fell within the range indicated by the Commission Regulation for all categories [3], but different profiles were exhibited by olive and olive-pomace oils (Table 1). In par-

ticular, both olive-pomace oils showed significantly greater linoleic and linolenic acid content as compared to all olive oil categories. On the contrary, oleic acid percentages were significantly higher in olive oils than in olive-pomace samples (Table 1). FA were grouped into different classes according to their unsaturation degree and reported also in Table 1. The content of polyunsaturated fatty acids (PUFA) was slightly higher in Po than in EvOo and this is probably ascribable to their more efficient recovery from seeds due to the application of solvent extraction for Po production. EvOo exhibited significantly higher percentages of saturated fatty acids (SFA) and monounsaturated fatty acids (MUFA) than the other oils, ˜ whereas RPo had the maximum content of PUFA. Continas et al. [23] observed higher percentage of PUFA (about 5%) for Po as compared to EvOo. Free acidity of the oils ranged from 0.1 to 0.4% (Table 1), being all below the limit set for each category by the EC Regulation [3]. EvOo, which is the only non-refined vegetable edible oil, exhibited significantly higher acidity value, as expected. The oxidative status of the oils was assessed by OSI, primary and secondary lipid oxidation products. In general, oils were apparently at different stages of the oxidation process and displayed a typical oxidative behavior, where primary oxidation products are converted into secondary oxidation products. Pomace oil and refined pomace oil showed significantly higher OSI time than the other oils. POV were significantly higher in all olive oil samples than pomace oils, which is in agreement with OSI times. In addition, POV of ROo and RPo were slightly above the regulation limit for these categories [3]. EvOo exhibited not only high POV but also elevated PAV values. It was recently observed that commercial EvOo can exhibit lower oxidative stability as compared to high-quality products characterized by a protected mark or obtained from a single olive cultivar; this fact may be ascribable to different freshness/aging degree of olive fruits [24]. Oo and ROo exhibited the lowest PAV values. On the contrary, Po showed lower POV values, but PAV was significantly higher than in the other oil samples. Po displayed a more advanced stage of oxidation than the other oils, which might be ascribable to a higher and faster conversion rate of primary into secondary oxidation products.

3.2.

Cooling thermograms and their deconvolution

Commercial categories of olive and olive-pomace oils were characterized by DSC during cooling. Thermal properties were obtained at first from the thermograms and they were related to the chemical composition of the samples. Thermograms were then deconvoluted into their constituent peaks to resolve overlapping transitions and to more precisely relate thermal events to chemical composition. DSC representative cooling thermograms of all samples were reported in Fig. 1. Cooling profiles were quite similar for all samples and exhibited two well distinguishable exothermic events. The major, lower temperature exotherm showed a symmetrical lineshape in all samples. This event peaked at about −38.5, −39.2 and −40.6 ◦ C for EvOo, Oo and ROo, respectively and it slightly shifted towards lower temperature in olive-pomace oils (∼−45.3 and −42.0 ◦ C for Po and RPo, respectively), probably due to the higher unsaturation degree

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Table 1 – TAG, DAG and FA compositions, free acidity, OSI time, POV and PAV of the different olive and olive-pomace oilsa EvOo TAG (% of total TAG) LLL + LLPo OLLn OLL + OLPo LLP + OLnO OLO OLP + OOPo POPo OOO SLO POP SOO SOP DSTAG MSTAG TUTAG Total DAG (g 100 g−1 lipids)

3.3 1.3 13.3 4.3 21.4 11.4 1.6 25.6 11.8 1.7 3.7 0.7 2.4 32.9 64.7

± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.2 B 0.2 A 0.5 B 0.5 B 0.2 B 0.2 C 0.2 B 0.5 A 0.3 A 0.1 A 0.2 A 0.1 A 0.2 A 0.6 BC 0.8 AB

1.8 ± 0.1 D

Oo 3.1 0.9 15.5 3.1 22.8 12.6 1.6 23.8 11.4 1.2 3.5 0.5 1.7 32.3 66.1

± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

ROo

0.1 C 0.1 C 0.1 A 0.1 C 0.4 A 0.3 B 0.1 B 0.3 B 0.1 A 0.0 BC 0.5 A 0.1 BC 0.2 C 0.6 C 0.7 A

2.8 ± 0.1 C

2.9 0.8 15.3 3.6 22.4 13.0 1.7 23.0 11.7 1.3 3.8 0.7 2.0 33.7 64.4

± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.1 C 0.1 C 0.3 A 0.4 BC 0.1 A 0.1 B 0.1 B 0.2 B 0.3 A 0.0 B 0.2 A 0.1 A 0.2 B 0.3 B 0.3 B

3.3 ± 0.1 B

Po 4.9 1.1 13.3 9.5 21.6 13.6 1.8 20.8 10.7 1.1 3.4 0.6 1.7 38.2 60.1

± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.2 A 0.0 B 0.1 B 0.2 A 0.2 B 0.2 A 0.2 A 0.5 C 0.4 B 0.1 C 0.3 A 0.1 AB 0.1 C 0.4 A 0.5 C

4.3 ± 0.3 A

RPo 4.7 0.9 11.0 9.1 21.7 12.4 1.5 21.9 11.4 1.0 3.9 0.4 1.5 38.3 60.2

± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.2 A 0.1 C 0.3 C 0.1 A 0.2 B 0.3 B 0.2 B 0.5 C 0.4 A 0.1 C 0.4 A 0.0 C 0.2 C 0.3 A 0.3 C

4.1 ± 0.1 A

DAG (% of total DAG) 1,2-PO 1,2-PL 1,2-OO 1,2-OL 1,3-OO

5.0 11.9 22.0 45.1 10.8

± ± ± ± ±

0.1 C 0.2 D 0.1 A 0.5 A 0.2 BC

4.7 12.1 18.7 46.6 11.3

± ± ± ± ±

0.2 D 0.2 CD 0.6 C 0.6 A 0.4 B

4.7 12.5 17.2 46.2 11.2

± ± ± ± ±

0.2 D 0.3 B 0.9 D 0.5 A 0.4 B

5.4 13.2 18.7 42.5 12.2

± ± ± ± ±

0.2 B 0.2 A 0.1 C 2.5 B 0.2 A

6.0 13.0 20.8 45.2 10.4

± ± ± ± ±

0.0 A 0.3 A 0.1 B 1.2 A 0.1 C

FA (g 100 g−1 lipids) Palmitic acid Palmitoleic acid Stearic acid Oleic acid Linoleic acid Linolenic acid SFA MUFA PUFA

11.8 0.8 3.1 74.1 6.9 0.7 15.8 77.6 7.6

± ± ± ± ± ± ± ± ±

0.2 A 0.0 D 0.0 B 0.1 B 0.1 C 0.0 C 0.2 A 0.2 A 0.1 C

10.4 1.0 3.0 75.2 8.3 0.8 14.2 76.8 9.1

± ± ± ± ± ± ± ± ±

0.0 BC 0.0 B 0.0 B 0.2 A 0.1 B 0.0 B 0.1 B 0.4 B 0.1 B

10.9 1.2 3.1 74.4 8.4 0.8 14.7 76.1 9.2

± ± ± ± ± ± ± ± ±

0.7 B 0.1 A 0.1 B 0.5 B 0.0 B 0.0 B 0.5 B 0.5 B 0.0 B

10.8 0.9 3.5 71.8 8.8 0.9 15.6 76.4 9.6

± ± ± ± ± ± ± ± ±

0.1 B 0.0 C 0.0 A 0.5 C 0.0 A 0.0 A 0.2 A 0.3 B 0.1 B

9.9 0.7 2.9 72.4 9.0 0.9 13.8 75.1 9.8

± ± ± ± ± ± ± ± ±

0.2 C 0.0 E 0.0 C 0.5 C 0.0 A 0.0 A 0.2 C 0.2 C 0.1 A

Free acidity (%) OSI (h) POV (meq O2 kg−1 ) PAV a

0.4 ± 0.0 A

0.1 ± 0.0 C

0.1 ± 0.0 C

0.2 ± 0.0 B

0.1 ± 0.0 C

13.8 ± 0.0 C 18.3 ± 0.5 A 5.0 ± 0.2 B

11.1 ± 0.1 D 14.2 ± 0.1 B 4.2 ± 0.1 D

10.7 ± 0.0 E 11.7 ± 0.2 C 4.2 ± 0.1 D

17.7 ± 0.1 B 5.3 ± 0.2 E 5.8 ± 0.0 A

19.1 ± 0.1 A 7.4 ± 0.0 D 4.6 ± 0.1 C

Data are expressed as mean ± standard deviation of three determinations. Same capital letters within each row do not significantly differ (p < 0.05).

of these oils (higher LLL, linoleic acid, PUFA content), as previously observed in other vegetable oil with similar composition [14]. The minor exothermic event peaked at about −15 ◦ C for all samples, except for Po, where it was shifted towards higher temperature (−12 ◦ C). Similar cooling profiles were previously reported for Oo [6,7] and EvOo [8,9], whereas, to the authors’ best knowledge, DSC cooling thermograms of ROo, Po and RPo have not been previously reported in literature. The characterizing thermal properties (enthalpy, Ton , Toff ; and transition range) were reported in Table 2 for all samples. Significant differences were found in the crystallization enthalpy of the different oils. Po and ROo had the lowest and the highest crystallization enthalpies, respectively, while intermediate values were found in the other oils. Oo and ROo lipids started to crystallize at significantly lower temperature than EvOo, Po and RPo, also developing the phase transition in a significant narrower temperature range than both olive-pomace oils (Table 2). Differences in major (e.g.

TAG, FA) and minor (e.g. DAG, free acidity, and lipid oxidation products) chemical components (Table 1) may have affected crystallization of the samples. A more ordered crystal structure may be hypothesized for olive oil samples (EvOo, Oo and ROo) due to a more uniform chemical composition (higher TUTAG, especially OOO content) as compared to olive-pomace oils that resulted in a narrower range of transition, as previously observed [9,14]. The lower crystallization enthalpy of EvOo with respect to the other olive oils, may have been influenced by lipid oxidation products (higher values of both POV and PAV) and free fatty acids (higher acidity values), as these molecules have been reported to be adsorbed into the crystal lattices of TAG, forming mixed crystals [25] that may have required lower enthalpy to undergo phase transition. EvOo exhibited significantly higher Ton with respect to Oo and ROo, possibly due to its higher DSTAG and SFA content, as more saturated vegetable oils were reported to crystallize at higher temperature [7]. Po and RPo (richer in MSTAG, linoleic

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a n a l y t i c a c h i m i c a a c t a 6 2 5 ( 2 0 0 8 ) 215–226

Table 2 – DSC data obtained from the cooling thermograms of the different olive and olive-pomace oilsa Sample EvOo Oo ROo Po RPo a

b

H (J g−1 ) 65.8 70.3 77.9 63.9 68.4

± ± ± ± ±

2.9 BC 0.9 B 2.3 A 1.9 C 0.3 BC

Ton (◦ C) −10.5 −12.2 −12.8 −7.6 −10.7

± ± ± ± ±

0.2 B 0.1 C 0.1 D 0.3 A 0.0 B

Toff (◦ C) −45.4 −48.0 −48.5 −55.1 −50.4

± ± ± ± ±

0.2 A 0.7 B 0.5 B 0.8 D 0.1 C

Rangeb (◦ C) 34.9 36.0 35.6 47.5 39.7

± ± ± ± ±

0.4 C 0.8 C 0.5 C 1.0 A 0.1 B

Data are expressed as mean ± standard deviation of five determinations. Same capital letters within each column are not significantly different (p < 0.05). Temperature difference between Ton and Toff .

acid and PUFA) exhibited Ton similar to those of EvOo, although lower crystallization onset temperatures were found in vegetable oils with high unsaturation degree [7,14]. It might be hypothesized that high level of DAG (mainly 1,2-PO and 1,2-PL) may have a role in the high crystallization onset temperature of the olive-pomace oils. DAG were found to influence (accelerating or delaying) TAG crystallization in vegetable oils (palm and coconut) and in fats [26,27] and to shift crystallization onset temperature toward higher temperature in EvOo samples with DAG contents comparable to those found in olive-pomace oils in this study [9]. DAG were also reported to be adsorbed into crystal lattices of TAG, hindering their crystallization process [25]. The elevated DAG content, together with the high content of lipid oxidized molecules (PAV, Table 1), may also explain the larger transition range exhibited by both Po and RPo. Cooling thermograms were deconvoluted into their constituent peaks, as shown in Fig. 2; the experimental data, fitted curves and constituent peaks were reported for EvOo (Fig. 2A), Oo (Fig. 2B), ROo (Fig. 2C), Po (Fig. 2D) and RPo (Fig. 2E). All thermograms were best fitted with three peaks (R2 ≥ 0.98), as previously reported in EvOo [9]. The peaks were numbered starting from the lowest to the highest temperature and named as peaks 1, 2 and 3 (Fig. 2). The predominant peak (peak 1) was an asymmetric double Gaussian function, while peaks 2 and 3 were asymmetric double sigmoid functions and exhibited a more complex, asymmetrical line

shape, probably indicating a more complex crystallization pattern. Peaks 2 and 3 also appeared more flattened in Po and RPo as compared to the other oils. Deconvoluted peaks were more extensively studied by means of percentage area of the total peak area (%), Ton, Tp, Toff and temperature transition range (Table 3). Peaks 1 and 2 were previously found to be statistically correlated with TUTAG and MSTAG amounts, respectively [9]. Areas (%) of peak 1 were significantly higher in olive oils than in olivepomace oils (Table 1), confirming earlier findings [6,9]; this result may be related to the higher TUTAG content of olive oils, especially OOO. Peak 1 had significantly lower Ton , Tp and Toff in Po and RPo, probably due to the higher unsaturation degree (higher LLL, linoleic acid, PUFA content) in olive-pomace oils as compared to the other oils. Peak 2 of Po and RPo showed significantly higher % areas than the other samples, which is possibly related to their significantly higher MSTAG content [11], and it developed over a larger range of transition, likely due to the presence of a larger variety of crystallizing species (e.g. DAG), as previously reported [9]. Peak 3 of Po showed significantly higher % area and developed over a larger range of transition than the other samples, possibly because of the presence of high amount of minor component (e.g. DAG and secondary oxidation products) in this oil. Thermal properties of this deconvoluted peak were previously reported to be more influenced by minor components as compared to peaks 1 and 2, since a clear correlation between percentage area and DSTAG content was not established [9].

3.3.

Fig. 1 – DSC cooling thermograms of the different olive and olive-pomace oils.

Heating thermograms and their deconvolution

Characteristic DSC heating thermograms of olive oils of different categories were summarized in Fig. 3. All samples exhibited multiple transitions as heated from −80 to 30 ◦ C. To authors’ best knowledge, DSC thermograms of Po and RPo upon heating have not been previously reported in literature. Some marked differences in the lineshapes of the transition can be noted. A first exothermic event, occurring in the −30 to 15 ◦ C temperature range, was distinguishable for EvOo, Oo and RPo. This low temperature exotherm was less evident in Po, as it was spread over a larger temperature range (from −40 to −15 ◦ C). Exothermic events were previously reported in vegetable oils and were attributed to the transition/rearrangement of TAG polymorphic crystals into more stable (and higher temperature melting) forms [7]. ROo showed

a n a l y t i c a c h i m i c a a c t a 6 2 5 ( 2 0 0 8 ) 215–226

221

Fig. 2 – Deconvoluted cooling thermograms of EvOo (A), Oo (B), ROo (C), Po (D) and RPo (E): experimental data (), fitted curve (dotted line) and the constituent peaks (peaks 1–3) are shown.

a small distinct endothermic event peaking at −21.5 ◦ C. The presence of a small endothermic event (at −15.1 ◦ C), not completely resolved by the major endothermic peak, was already observed in heating thermogram of ROo and generically attributed to the melting of lowest stability polymorphic forms of TAG (e.g. ␣) [13]. The complex endothermic events occurring at higher temperatures were attributed to the melting of crystallized lipids and were characterized by multiple overlapping contributions, as previously observed in vegetable [5,7] and olive oils [5,8,10].

Two predominant endotherms (A at lower and B at higher temperature in Fig. 3) were clearly distinguishable in all samples. The major event (A) peaked at about −3.5 ◦ C for EvOo and at lower temperatures in all other samples (∼−6.0 ◦ C for Oo, Po, RPo and −8.0 ◦ C for ROo). An additional endothermic event (at ∼−14 ◦ C) was observed in all samples, displaying itself as a shoulder in peak A. This shoulder event was probably more important in ROo, which gave rise to a broader lineshape and less sharp peak A. The smaller event (B) peaked at about 6.5 ◦ C for ROo, Po and RPo and at higher temperature for Oo and

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a n a l y t i c a c h i m i c a a c t a 6 2 5 ( 2 0 0 8 ) 215–226

Table 3 – Deconvolution parameters of cooling thermograms of the different olive and olive-pomace oilsa Peakb

Ton (◦ C)

Area (%)

Tp (◦ C)

Toff (◦ C)

Rangec (◦ C)

1 EvOo Oo ROo Po RPo

74.0 72.8 73.5 62.6 68.1

± ± ± ± ±

0.6 A 0.2 B 0.9 B 1.0 D 0.3 C

−32.5 −34.3 −34.8 −39.4 −36.0

± ± ± ± ±

0.1 A 0.2 B 0.3 B 0.4 D 0.1 C

−38.2 −38.9 −40.4 −45.3 −41.6

± ± ± ± ±

0.4 A 0.4 A 0.1 B 0.1 D 0.1 C

−44.6 −45.7 −44.7 −52.8 −48.5

± ± ± ± ±

0.4 A 0.1 B 0.1 A 0.5 D 0.1 C

12.1 11.4 9.9 13.5 12.4

± ± ± ± ±

0.5 AB 0.3 B 0.4 C 0.9 A 0.2 AB

EvOo Oo ROo Po RPo

13.0 15.1 14.4 21.0 20.9

± ± ± ± ±

0.4 C 0.5 B 0.6 BC 1.1 A 0.5 A

−19.1 −16.4 −19.4 −16.1 −16.4

± ± ± ± ±

0.5 B 0.1 A 0.4 B 0.5 A 0.5 A

−32.6 −34.7 −35.2 −38.3 −35.8

± ± ± ± ±

0.7 A 0.1 B 0.4 BC 0.3 D 0.2 C

−36.7 −37.6 −39.2 −44.0 −39.4

± ± ± ± ±

0.5 A 0.3 A 0.6 B 0.6 C 0.3 B

17.6 21.2 19.8 27.9 23.0

± ± ± ± ±

0.6 D 0.4 C 0.4 C 1.1 A 0.4 B

EvOo Oo ROo Po RPo

13.1 12.1 12.2 16.3 11.1

± ± ± ± ±

0.5 B 0.7 B 0.3 B 0.4 A 0.2 C

−10.2 −11.8 −12.7 −7.7 −10.7

± ± ± ± ±

0.1 B 0.2 C 0.3 D 0.1 A 0.2 B

−13.4 −14.8 −15.2 −12.7 −14.8

± ± ± ± ±

0.2 B 0.0 C 0.1 D 0.1 A 0.0 C

−27.9 −30.5 −31.3 −37.4 −29.9

± ± ± ± ±

0.4 A 0.2 BC 0.6 C 0.5 D 0.7 B

17.8 18.7 18.6 29.7 19.2

± ± ± ± ±

0.5 B 0.4 B 0.4 B 0.4 A 0.5 B

2

3

a

b c

Data are expressed as mean ± standard deviation of five determinations. Same capital letters within each column for single peak are not significantly different (p < 0.05). See Fig. 2 for peak number assignation. Temperature difference between Ton and Toff .

Table 4 – DSC data obtained from the heating thermograms of the different olive and olive-pomace oilsa Sample

H (J g−1 )

EvOo Oo ROo Po RPo

67.1 75.6 83.4 63.5 73.3

a

b

± ± ± ± ±

3.5 C 2.1 B 1.3 A 1.8 C 1.1 B

Ton (◦ C) −26.8 −27.1 −32.0 −38.6 −28.9

± ± ± ± ±

Toff (◦ C)

0.7 A 0.2 A 0.2 C 0.2 D 0.2 B

12.2 11.3 10.6 10.5 10.4

± ± ± ± ±

0.2 A 0.0 B 0.5 C 0.2 C 0.2 C

Rangeb (◦ C) 39.0 38.3 42.7 49.1 39.3

± ± ± ± ±

0.9 CD 0.2 D 0.3 B 0.2 A 0.2 CD

Data are expressed as mean ± standard deviation of five determinations. Same capital letters within each column are not significantly different (p < 0.05). Temperature difference between Ton and Toff .

Fig. 3 – DSC heating thermograms of the different olive and olive-pomace oils. Major and minor endothermic events were indicated with (A) and (B), respectively.

EvOo (∼7.5 and 8.3 ◦ C, respectively). This peak appeared to be sharper in EvOo and Rpo, whereas it was wider and more asymmetric in the other samples. Heating thermal properties were obtained from the onset of the exothermal to the offset of the endothermic events, as these two events were not completely resolved (Table 4). Overall enthalpies were significantly lower in EvOo and Po than in the other samples, because of a larger contribution of the exothermic event. Po had significantly lower Ton and a larger range of transition than all the other samples, as the exothermic event started at the lowest temperature. EvOo exhibited the highest Toff and this may be related to a significantly larger highly saturated lipid fraction that were found to melt at higher temperature than those more unsaturated (DSTAG, SFA, Table 1) [28]. Deconvolution analysis was also applied to heating thermograms, and the peaks identified were numbered, starting from the lowest to the highest temperature, as shown in Fig. 4. Five peaks were necessary to properly deconvolute EvOo, Po and RPo thermograms, six for Oo and seven for ROo. Deconvoluted peaks 0, 1 and 2 were asymmetric double sigmoid and the others asymmetric double Gaussian functions (R2 ≥ 0.98 in all cases).

a n a l y t i c a c h i m i c a a c t a 6 2 5 ( 2 0 0 8 ) 215–226

223

Fig. 4 – Deconvoluted heating thermograms of EvOo (A), Oo (B), ROo (C), Po (D) and RPo (E); experimental data (), fitted curve (dotted line) and the constituent peaks (peaks 0–6) are shown.

Peak 0 was exothermic, while the others were all endothermic events. The low temperature endothermic peak (peak 1, Ton = −24.5 ◦ C) was observed only in ROo, indicating the presence of low temperature melting species (e.g. ␣ polymorphic forms of TAG). Peaks 2, 3 and 4 constituted the major low-temperature endotherm (peak A, Fig. 3) in all samples, while the minor endotherm (higher temperature, peak B) involved only one peak (peak 5) in EvOo (Fig. 4A), Po (Fig. 4D) and RPo (Fig. 4E), and two peaks (5 and 6) in Oo (Fig. 4B) and ROo (Fig. 4C).

Peaks 2, 3 and 4 may be attributed to the melting of three crystalline polymorphic forms of the most unsaturated fractions of triacylglycerols (TUTAG and in particular OOO), as previously reported [12]. Similarly, peaks 5 and 6, when present, were associated to the melting of MSTAG [12], although the DSTAG contribution cannot be excluded. Single peak (peak 5) in EvOo, Po and RPo may indicate the presence of a simultaneous and highly cooperative phase transition of TAG, possible related to the significantly higher amount of MSTAG in Po and RPo and to a closer cooperation between

224

a n a l y t i c a c h i m i c a a c t a 6 2 5 ( 2 0 0 8 ) 215–226

Table 5 – Deconvolution parameters of heating thermograms of the different olive and olive-pomace oilsa Peakb

Ton (◦ C)

Tp (◦ C)

Toff (◦ C)

Rangec (◦ C)

0 EvOo Oo ROo Po RPo

−26.8 −27.5 −33.4 −38.6 −28.6

± ± ± ± ±

ROo

−24.5 ± 0.2

−22.6 ± 0.1

EvOo Oo ROo Po RPo

−12.7 −15.7 −15.6 −16.2 −16.3

± ± ± ± ±

0.3 A 0.7 B 0.2 B 0.5 C 0.3 C

−10.1 −12.4 −13.2 −11.6 −12.5

± ± ± ± ±

0.3 A 0.1 C 0.0 D 0.1 B 0.3 C

−7.0 −8.9 −9.8 −8.9 −8.9

± ± ± ± ±

0.2 A 0.9 B 0.2 C 0.1 B 0.1 B

5.8 6.8 6.8 6.3 7.2

± ± ± ± ±

0.5 B 1.5 B 0.6 B 0.5 B 0.3 A

EvOo Oo ROo Po RPo

−11.4 −17.6 −25.3 −19.2 −20.4

± ± ± ± ±

0.5 A 1.0 B 1.4 D 0.1 BC 0.7 C

−4.6 −6.4 −8.0 −6.1 −6.2

± ± ± ± ±

0.0 A 0.1 B 0.1 C 0.1 B 0.1 B

3.6 7.5 3.7 3.1 7.0

± ± ± ± ±

0.2 C 0.2 A 0.3 C 0.1 D 0.1 B

15.1 25.1 29.1 22.3 27.4

± ± ± ± ±

0.6 D 0.9 B 1.2 A 0.1 C 0.8 A

EvOo Oo ROo Po RPo

−5.1 −6.5 −7.7 −5.9 −7.0

± ± ± ± ±

0.8 AB 0.5 BC 0.3 C 0.8 AB 0.6 ABC

−0.2 −2.1 −3.9 −1.9 −2.3

± ± ± ± ±

0.0 A 0.2 BC 0.1 D 0.1 B 0.1 C

2.9 0.1 −1.2 3.2 −0.6

± ± ± ± ±

0.6 A 0.0 B 0.1 C 0.7 A 0.1 BC

8.1 6.6 6.5 9.1 6.4

± ± ± ± ±

1.4 A 0.5 B 0.4 B 1.0 A 0.6 B

EvOo Oo ROo Po RPo

−5.6 −0.5 −3.9 −0.8 −0.1

± ± ± ± ±

0.1 D 0.1 AB 0.1 C 0.2 B 0.0 A

8.0 5.0 4.0 6.4 6.4

± ± ± ± ±

0.0 A 0.1 C 0.1 D 0.1 B 0.1 B

13.7 9.9 8.7 10.5 11.3

± ± ± ± ±

0.2 A 0.2 D 0.1 E 0.2 C 0.1 B

19.4 10.4 12.6 11.2 11.5

± ± ± ± ±

0.1 A 0.2 D 0.2 B 0.2 C 0.1 C

Oo ROo

−0.8 ± 0.3 −1.6 ± 0.1

0.7 A 0.3 A 0.6 C 0.2 D 0.2 B

−18.5 −18.6 −30.6 −24.2 −21.1

± ± ± ± ±

0.3 A 0.3 A 0.7 D 1.3 C 0.2 B

−13.7 −14.8 −25.7 −18.8 −16.7

± ± ± ± ±

0.4 A 0.1 B 0.2 E 0.5 D 0.5 C

13.0 12.8 7.7 19.8 12.0

± ± ± ± ±

0.7 B 0.4 B 0.7 D 0.5 A 0.4 B

1 −20.0 ± 0.1

4.5 ± 0.2

2

3

4

5

6

a

b c

8.3 ± 0.1 7.4 ± 0.1

11.2 ± 0.4 10.2 ± 0.2

12.2 ± 0.4 11.8 ± 0.2

Data are expressed as mean ± standard deviation of five determinations. Same capital letters within each column for single peak are not significantly different (p < 0.05). See Fig. 2 for peak number assignation. Temperature difference between Ton and Toff .

MSTAG and DSTAG in EvOo, as the latter were significantly higher in this sample than in the other oils. Attribution of the deconvoluted peaks to a single polymorphic form was difficult, due to the lack of literature data about the polymorphic behavior of olive oil TAG and the use of tandem techniques to characterize polymorphism. Hagemann et al. [29] observed that OOO, as standard, could exhibit four polymorphic forms (␤ 3 , ␤ 2 , ␤ 1 and ␤), which melt at −12, −8, −5 and 5 ◦ C, respectively. The ␤ –␤ transformation was reported to be sterically hindered in oil and fats where the mix of different chain length TAG reduced the efficiency of interplanar packing [30]. Thus, several ␤ forms generally occurred before transformation in the ␤ form [30]. Further and more detailed information on TAG polymorphic forms in olive oils could be obtained by coupling DSC with X-ray diffraction, as these tandem techniques were already applied to the analysis of polymorphic crystal forms of other vegetable oils [31].

Deconvoluted peaks were further characterized by means of Ton , Tp and Toff peak temperatures, as well as temperature transition range (Table 5). Peak 0 of Po showed significantly lower Ton value developing in a larger range of transition. This is probably ascribable to the presence of DAG and oxidized lipids (e.g. 1,3-DAG and secondary oxidation products) that, being absorbed into TAG crystals [32], may have hindered TAG crystal transition/rearrangement into more stable polymorphic forms. Peak 2 showed significantly lower Ton in both olive-pomace oils, which exhibited higher PUFA amount (Table 1). On the contrary, the highest Ton temperature was observed in EvOo, which showed significantly higher SFA amount as compared to all other samples (Table 1). Peak 3 of EvOo showed significantly higher Ton developing in a narrower range with respect to all other samples, which may be related to the higher OOO content of EvOo. The main endothermic transition of melting thermograms was already associated to the most unsaturated

a n a l y t i c a c h i m i c a a c t a 6 2 5 ( 2 0 0 8 ) 215–226

TAG fractions, especially OOO, in olive oil samples [5]. Peak 4 of EvOo and Po showed higher Ton and Toff developing in a larger range of transition as compared to the other samples. Peak 5 showed significantly lower Ton and higher Tp and Toff values in EvOo, developing in a larger range of transition. The formation of mixed MSTAG/DSTAG crystals may have influenced thermal properties of this deconvoluted peak.

4.

Conclusions

The results of this preliminary investigation confirmed that both cooling and heating thermograms may be a useful tool to discriminate among olive oil categories, as the oils developed different crystallization and melting profiles. Application of deconvolution analysis to DSC thermograms can provide additional information for olive oil classification and for a better understanding of the relationship among chemical composition (major and minor components) and thermal properties. The evaluation of the cooling thermal properties appeared promising for discrimination between olive and olive-pomace oils. In particular, Ton and Toff of the crystallization showed significant differences among different commercial categories, while enthalpy could differentiate between olive (Oo and ROo) from olive-pomace oils (Po and RPo). The application of deconvolution analysis to cooling profiles also appeared promising as thermal properties of peak 1, which was ascribable to the crystallization of the larger lipid fraction, showed significant differences among commercial categories. All oils exhibited distinctive heating thermograms profiles. In particular, the minor endothermic event (B, Fig. 4) had different lineshape among oil samples. In addition, the differences observed in ROo heating profile may suggest the potential DSC application for the recognition of EvOo adulteration with mildly deacidified and/or deodorized olive oils (refined samples), which is nowadays widely applied and very difficult to detect. The differentiation among oils of different commercial categories on the basis of thermal properties upon heating was found to be more difficult due to the complexity of the thermograms. The findings reported in this work must be confirmed by the appliance of multivariate statistical analysis to a larger set of samples to select parameters able to discriminate among different commercial categories from both cooling and heating thermograms. Samples should be obtained from different production cycles also taking into account the incidence of different chemical composition ascribable to olive cultivar, geographical origin, harvesting period, agronomical practices, that can influence the crystallization and melting transition of the oils.

Acknowledgments The authors would like to thank Stefano Savioli and Mara Mandrioli (University of Bologna) for their technical support and assistance during sample analysis.

225

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