Chem. Percept. (2008) 1:258–267 DOI 10.1007/s12078-008-9031-3

Relationship Between Sensory Evaluation Performed by Italian and Spanish Official Panels and Volatile and Phenolic Profiles of Virgin Oils

Lorenzo Cerretani & Maria Desamparados Salvador & Alessandra Bendini & Giuseppe Fregapane

Received: 20 May 2008 /Accepted: 30 September 2008 /Published online: 24 October 2008 # 2008 Springer Science + Business Media, LLC

Abstract Virgin (VOO) is typified by character- compounds (sum of aldehydes C6) and orthonasal percep- istic, pleasant sensory notes that differentiate it from other tion of olive fruity and retronasal odor of almond. edible oils. Sensory taste, together with nutritional aspects, Additional correlations with bitterness and pungency were is the main reason for the increase in consumption of VOO observed for tyrosol and oleuropein aglycon, respectively. in recent years. Sensory analysis is required by European Official Regulations for olive oil in order to classify the Keywords Virgin Olive Oil . Sensory Analysis . Panel Test . product in commercial categories. In this study, the Volatile Compounds . Phenolic Compounds relationship between sensory and chemical composition has been investigated. In particular, 16 VOO samples (15 Abbreviations from a single variety of ), produced in Italy and cGC Capillary gas chromatography (eight from each country), were analyzed. Sensory attrib- DAD Diode-array detector utes were valued by four panels (three officially recognized EI Electron ionization both by IOOC and National Ministry: two Italian and two HPLC High-performance liquid chromatography Spanish) employing a total of 59 tasters. Volatile and IOOC International Olive Oil Council phenolic compounds were related to olfactory and gustative LOX Lipoxygenase notes, respectively. Volatile compounds were then separat- MSD Mass-spectrometer detector ed, identified, and quantified, starting from oil samples, by QDA Quantitative descriptive analysis solid phase microextraction and capillary gas chromato- SPME Solid phase microextraction graphic analysis using a mass spectrometry detector VOO Virgin olive oil (SPME/cGC-MSD). Furthermore, the phenolic profile was examined by high-performance liquid chromatography with diode-array and mass spectrometry detectors (HPLC-DAD/ Introduction MSD). Correlations were found between the major volatile In terms of composition, virgin olive oils (VOO) differ L. Cerretani (*) : A. Bendini from the other edible oils in minor components (Angerosa Dipartimento di Scienze degli Alimenti, Università di Bologna, et al. 2004; Carrasco-Pancorbo et al. 2005a; Bendini et al. P.zza Goidanich 60, 2007a) that determine sensorial stimuli (Andrewes et al. 47023 Cesena (FC), Italy 2003; Gutierrez et al. 2003). For the merceological e-mail: [email protected] classification of virgin olive oils, the European Union M. D. Salvador (*) : G. Fregapane Commission has provided 26 chemical-physical parameters Departamento de Tecnología de Alimentos, and organoleptic evaluation (EEC Reg. 2568/91; EC Reg. Universidad de Castilla-La Mancha, 1989/03). Sensory analysis of VOO initiated from Collab- Avda Camilo José Cela 10, 13071 Ciudad Real, Spain orative International studies, which has been supported for e-mail: [email protected] many years by the International Olive Oil Council (IOOC) Chem. Percept. (2008) 1:258–267 259 that developed the Quantitative descriptive analysis sensory contribute to various VOO aromas have been investigated methodology for virgin olive oils, known as IOOC-Panel extensively by GC sniffing techniques (Morales et al. 1994). test. This study was finally published in June 1987, and the In a large number of investigations on VOO (Andrewes method was adopted for sensory analysis of VOO (IOOC et al. 2003; Gutierrez et al. 1989, 1992, 2003; Siliani et al. 1987). Sensory evaluation was introduced in the current, 2006; Beltrán et al. 2007), sensory and chemical parameters official regulations in 1991 (EEC Reg. 2568/91 Annex have been correlated, but in all cases, sensory analysis has XII). This analysis is carried out by a fully trained panel been carried out only by a single panel group or by panels composed of 8 to 12 assessors, and the regulation provides from the same producer country. a specific sensory sheet for assigning the merceological To assess correlations between sensory attributes and the category. Successively, in 2002, the European Union minor components of VOO, mainly phenolic and volatile Commission adopted the IOOC methodology, which profiles, four official panels from Italy and Spain and a set modifies and simplifies the score sheet, employing an of 16 VOO (15 monovarietal) Italian and Spanish samples unstructured 100-mm scale (EC Reg. 796/02). Finally, the were employed in order to verify the quality of the tasters’ 4th July 2008 other modifications suggested by IOOC were scores and determine the variability of the sensory introduced (EC Reg. 640/08) concerning the sensory characteristics of VOO samples. Together, Italy and Spain vocabulary and the conditions for the optional use, on contribute to more than 60% of worldwide olive oil labels, of certain terms and expressions relating to the production (IOOC 2007), and therefore, the capacity of organoleptic characteristics of virgin olive oil. Recently, the Official Taste Panels employed in this research work is the evolution of sensory analysis of VOO has led to the guaranteed. proposal of a method for oil and food pairing (Cerretani et al. 2007). Sensory attributes of VOO mainly depend on the content of Materials and Methods minor components like phenolic and volatile compounds. Each single component can contribute to different sensory Samples perceptions. It is well established that phenolic compounds are responsible for bitterness in VOO (Gutierrez et al. 1989, 1992, Table 1 shows the characteristics of the VOO samples 2003; Siliani et al. 2006; Beltrán et al. 2007). Andrewes et al. employed. With the exception of one sample, all oils were (2003) isolated individual compounds, and for each attribut- produced from a single variety of olives. Samples came ed sensory characteristic, these authors found that carbox- from different industrial olive mills covering the main ymethyl-ligstroside aglycon, also called oleocanthal producing areas in the two countries during two consecu- (Beauchamp et al. 2005), is the major phenolic molecule tive crop seasons: 2003–2004 and 2004–2005. responsible for the burning pungent sensation of VOO. Moreover, the phenolic content has been related to protection Reagents and Standards against important chronic and degenerative diseases (Menendez et al. 2007). The standards used for quantification of volatile and Approximately 180 compounds belonging to several phenolic compounds (1-nonanol and 3,4-dihydroxyphenyl- chemical classes such as aldehydes, ketones, ethers, hydro- acetic acid, respectively) were from Sigma-Aldrich (St. carbons, alcohols, and esters have been separated from the Louis, MO, USA). All solvents used were analytical or volatile fractions of different quality VOO (Angerosa 2002; HPLC grade (Merck, Darmstadt, Germany). Angerosa et al. 2004). Typical flavors and off-flavor compounds that affect the volatile fraction of a VOO Sensory Analysis originate by different mechanisms: enzymatically by linoleic and linolenic fatty acids through the so-called lipoxygenase Sensory analysis was performed by four Official Taste (LOX) pathway, by sugar fermentation or amino acid Panels. The Regional Panel of Agenzia Servizi Settore (leucine, isoleucine, and valine) conversion, or from enzy- Agroalimentare Marche—A.S.S.A.M. (Ancona, Italy) was matic activities of molds or auto-oxidative processes composed by a large number of members (20 plus the panel (Morales and Tsimidou 2000;Angerosa2002). Volatile leader) with an average age of 35 years; this panel was molecules can be perceived in very small amounts (micro- recognized by IOOC in the year 2000 and successively by grams per kilogram). These compounds do not contribute to the Italian Ministry in 2004. The Panel of Institute for the global aroma of VOO with the same importance; in fact, Mediterranean Agriculture and Forest Systems—CNR- their influence must be evaluated not only on the basis of ISAFoM (Perugia, Italy) is composed of 12 tasters with their concentration but also on their sensory threshold values. an average age of 35 years and are in the final training The independent odors of different volatile compounds that process. The Panel of Fundación Consejo Regulador de la 260 Chem. Percept. (2008) 1:258–267

Table 1 Virgin olive oil samples Volatile Compounds Analysis Sample Olive Country of Area of olive code cultivar production production VOO (4 g) was put into a 10-mL vial and heated to 40 °C. After 2 min of equilibrium time, volatile compounds from the A Cerasuola Italy Sicilia, Palermo headspace were adsorbed on a solid phase microextraction B Correggiolo Italy Emilia-Romagna, (SPME) under magnetic stirring. The fiber in PDMS 50/30 μm Rimini (Supelco, Bellefonte, PA, USA) was exposed to the vapor C Dritta Italy Abruzzo, Pescara D Frantoio Italy Emilia-Romagna, phase for 30 min at 40 °C. Desorption of volatile compounds Ravenna trapped in the SPME fiber was done directly in the gas E Italy Emilia-Romagna, chromatograph injector set at 220 °C in splitless mode using a Forlì-Cesena splitless inlet liner of 0.75 mm i.d. for thermal desorption F Frantoio, Italy Toscana where it was left for 3 min. A GC CP 3800 coupled with a Leccino, mass-spectrometer Varian 2000 was used, and a fused-silica Moraiolo capillary column CP-Sil 8 CB (60 m, 0.25 mm i.d., 0.25-μm G Nocellara Italy Sicilia, Palermo film thickness; Chrompack) was employed. The column was del Belice H Orfana Italy Emilia-Romagna, operated with helium at a pressure of 12 psi and a flow rate of −1 Ravenna 1mLmin . The GC oven heating was started at 40 °C; this I Spain Cataluña, LLeida temperature was maintained for 10 min, then increased to − J Spain Cataluña, 55°Catarateof1°Cmin 1, increased to 80 °C at a rate of Tarragona 5°Cmin−1, and finally increased to 220 °C at a rate of 18 °C K Empeltre Spain Aragón, Teruel min−1. The temperature of the transfer line was fixed at L Gordalilla Spain Andalucía, 180 °C. The mass spectrometer operated in the electron Málaga M Lechin Spain Murcia ionization mode at an ionization voltage of 70 eV in the mass – −1 N Manzanilla Spain Extremadura, range of 39 300 amu at a scan rate of 1 scan s and a cacereña Cáceres manifold temperature of 180 °C. The GC-MS operated O Morisca Spain Extremadura, through the software Saturn GC-MS version. Badajoz Volatile compounds were identified by comparison of P Pico limon Spain Andalucía, their mass spectra and retention times with those of Sevilla authentic reference compounds. When standards were not available, identification of the volatile compounds was obtained by comparing their mass spectral data with those of the NIST/Wiley library. Quantification was obtained Denominación de Origen Montes de Toledo—DOMT based on calibration curve of 1-nonanol (from 0.1 to (Toledo, Spain) was recognized by IOOC in 2003, by the 200 mg kg−1 of oil) as an external standard. Spanish Ministry (MAPA) in 2004, and by ENAC (Entidad Nacional Acreditación) in 2006; this panel is composed of Phenolic Compounds Analysis 15 assessors with an average age of 32 years. Finally, the Panel del Laboratorio Arbitral Agroalimentario (Madrid, The phenolic fraction was extracted from VOO by a liquid/ Spain) was recognized by IOOC in 1989 and by MAPA in liquid extraction method described by Pirisi et al. (2000). 1987; this has 12 members, with an average age of 56 years. The dry extracts were redissolved in 0.5 mL of methanol/ All panels evaluated VOO following an incomplete water (50:50, vol/vol) and filtered through a 0.2-μm nylon randomized design. A maximum of six oils, randomly filter (Whatman, Clinton, NJ, USA). Extracts were frozen selected among the 16 samples, were analyzed in each and stored at −43 °C. panel sessions. Each sample was tested twice in two HPLC analyses were performed using a HP 1100 Series different sessions. Moreover, to reduce the number of tests, instrument (Agilent Technologies, Palo Alto, CA, USA) a balanced incomplete block design was applied according equipped with a binary pump delivery system, degasser, to Cochran and Cox (1957). autosampler, diode-array UV–VIS detector (DAD) and For the present study, a standard profile sheet (Fig. 1), mass-spectrometer detector (MSD). All solvents were of realized according to IOOC method T20 and modified by HPLC grade and filtered through a 0.45-μm nylon filter IBIMET-CNR (Rotondi et al. 2004; Cerretani et al. 2005), disk (Lida Manufacturing, Kenosha, WI, USA) prior to use. was used. This sensory ballot permitted to obtain a All analyses were carried out at room temperature. complete profile of the sensory properties of samples, and The phenolic profile was assessed by HPLC-DAD/ESI- a total of 59 tasters were employed. MSD equipped with a reverse phase C18 Luna™ column Chem. Percept. (2008) 1:258–267 261

Fig. 1 Sensory ballot for virgin olive oil evaluation (modified by IBIMET-CNR in Rotondi et al. 2004 and Cerretani et al. 2005)

(5 μm, 25 cm×3.00 mm i.d.; Phenomenex, Torrence, CA, linear correlations, at p<0.05, were evaluated using USA) according to Rotondi et al. (2004). The injection Statistica 6.0 (2001, StatSoft, Tulsa, OK, USA). volume was 10 μL. Phenolic compounds detected at 280 nm were quantified using a 3,4-dihydroxyphenylacetic acid standard calibration curve (r2=0.999). Results and Discussion

Statistical Analysis Sensory Analysis

The results reported in this study are the averages of at least Sensory analysis was performed by four different fully three repetitions (n=3), unless otherwise stated. Pearson’s trained analytical VOO taste panels from Italy and Spain 262 Chem. Percept. (2008) 1:258–267 using a modified profile sheet (Fig. 1) in order to provide a wider range of olfactory and gustative sensory attributes. The assessors evaluated the main positive attributes present in VOO as fruity, bitter, and pungent. Moreover, they assessed pleasant flavors related with different perception routes, orthonasal (O), retronasal (R), and gustative (G) like green and ripe notes from olive and others fruits. A total of eight attributes were used in the final sheet to provide a complete profile for each sample tasted. The median values for each VOO evaluated in two different sessions of four panels were used as final input for statistical analysis. The sensory scores of the Italian (Fig. 2) and Spanish (Fig. 3) samples are shown. The corresponding graphs highlight the large variability in sensory characteristics of the sample pool. In fact, monovarietal oils coming from a large geographical area produce a large variability of sensory and chemical responses (Cerretani et al. 2006). Figures 2 and 3 show the orthonasal perception of olive fruity (O), which varied widely from a sensory score of 57 Fig. 3 Sensory profiles of Spanish samples (for sample codes see Table 1). Different perception routes: (O) orthonasal, (R) retronasal, in the Italian sample (sample G) to a (G) gustative. For abbreviations see Table 1: 1=A; 2=B; 3=C; 4=D; score of 21 in the Empeltre Spanish variety (sample K). On 5=E; 6=F; 7=G; 8=H; 9=I; 10=J; 11=K; 12=L; 13=M; 14=N; 15=O; the other hand, pleasant orthonasal flavor notes were 16=P evaluated by tasters in a similar way, but in a wider range, from 45 in Nocellara del Belice (sample G) to a minimum In accordance with all panels, sample D (Frantoio value of 5 (sample K) in Spanish Empeltre. variety, sensory score of 44) was the most and Arbequina However, the values of fruitiness by retronasal percep- (score of 10) the least bitter. The same samples (D and I), tion routes were similar for all samples, ranging from 30 to but in this case firstly I (48) followed by D (45) showed the 48, with the exception of sample K, showing a comparable highest intensity of pungency. Italian samples were gener- behavior as observed for O-olive fruity attributes. ally evaluated by tasters as more bitter than Spanish ones (as mean value: 28 for the firsts and 25 for the others); nevertheless, Spanish varieties presented a wider range of intensities for this positive attribute (Fig. 3). The green and pleasant flavor notes in VOO tasted by a retronasal route were mainly present in Italian varieties, especially in sample A (34), but also in the Spanish Gordalilla variety. Retronasal ripe pleasant flavor notes in VOO were mainly perceived in Leccino (E) and Arbequina (I). All panels agreed on to judge defective two VOO (samples J and K); in particular, the assessors perceived a clear rancidity in Arbosana, whereas Empeltre oil was both winey/fusty and rancid.

Volatile Profiles vs. Olfactory Perceptions

Table 2 shows the volatile compounds identified and quantified in VOO, expressed as the mean value and its standard deviation grouped in major classes of compounds,

such as C6 structure aldehydes, alcohols and esters, or terpenes with 10 or 15 carbon atoms. These volatiles Fig. 2 Sensory profiles of Italian samples (for sample codes see represent the 89% and 72% of whole profiles for Italian and Table 1). Different perception routes: (O) orthonasal, (R) retronasal, Spanish VOO, respectively (Fig. 4). (G) gustative. For abbreviations see Table 1: 1=A; 2=B; 3=C; 4=D; 5=E; 6=F; 7=G; 8=H; 9=I; 10=J; 11=K; 12=L; 13=M; 14=N; 15=O; The C6 compounds originating from the LOX pathway are 16=P generally the volatiles that are more responsible for the Chem. Percept. (2008) 1:258–267 263

Table 2 Volatile compounds identified and quantified in VOO grouped in major classes of compounds values expressed as mean value and standard deviation

TOT ALD C6 Z-3/E-2 ALC C6 EST C6 TERP10 TERP15

A 90.3±5.6a 18.2±0.8 2.19±0.06 24.8±1.8 2.46±0.22 22.6±1.8 14.0±0.8 B 77.0±4.5 54.6±2.7 <0.04 2.18±0.14 1.36±0.27 11.3±0.8 2.22±0.20 C 82.8±2.0 49.9±2.6 <0.04 1.21±0.25 0.92±0.15 13.9±0.6 1.23±0.08 D 74.5±3.2 50.7±3.1 <0.04 1.65±0.17 0.48±0.08 12.0±0.5 1.01±0.07 E 87.0±2.9 69.0±2.7 <0.04 1.22±0.16 0.21±0.05 7.86±0.35 0.26±0.01 F 84.3±2.7 59.0±2.3 <0.04 0.80±0.12 0.28±0.03 13.1±0.4 1.89±0.02 G 109±0.4 57.6±0.6 2.58±0.10 15.7±0.6 1.93±0.05 14.8±0.2 9.99±0.72 H 106±3 63.5±2.2 <0.04 3.85±0.42 1.00±0.02 10.8±0.6 13.3±0.6 I 77.2±0.2 20.9±0.3 <0.04 2.94±0.06 19.3±0.3 17.2±0.1 4.63±0.09 J 95.5±3.8 10.9±0.5 nd 2.86±0.22 46.8±1.4 6.51±0.26 9.82±0.73 K 54.1±1.3 7.93±0.56 nd 0.57±0.01 13.8±0.2 4.54±0.03 2.89±0.11 L 69.4±0.2 8.36±0.13 0.28±0.01 1.34±0.11 2.57±0.08 21.1±0.5 8.45±0.62 M 104±2 45.8±1.9 nd 0.33±0.03 5.79±0.07 22.8±0.7 10.8±0.3 N 85.0±0.0 32.2±1.0 nd 0.89±0.04 13.4±0.5 8.85±0.90 16.4±0.6 O 121±0.2 6.85±0.03 0.64±0.03 6.12±0.12 54.7±0.2 9.04±0.12 7.31±0.18 P 87.6±1.0 17.6±0.8 0.58±0.04 1.63±0.09 6.49±0.21 10.7±0.3 17.7±0.8

Contents of different classes of volatile compounds in oil samples expressed as milligrams of 1-nonanol per kilogram of oil. (For sample names, see Table 1) TOT sum of volatile compounds, ALD C6 sum of hexanal, Z-3-hexenal and E-2-hexenal, ALC C6 sum of 1-hexanol, Z-3-hexenol, and E-2- hexenol, EST C6 sum of hexyl acetate and Z-3-hexenyl acetate, TERP10 sum of 3,3,5-trimethyl-1,4-hexadiene, 3,5-dimethyl-1,6-heptadiene, 3- ethyl-1,5-octadiene, 2,6-dimethyl-1,5-heptadiene, limonene, and β-ocimene, TERP15 sum of α-copaene, β-sesquiphellandrene, α-farnesene, α- muurolene, and δ-cadinene, nd not detected a Mean value±standard deviation

difference in green fruity notes, whereas the contribution of 7mgkg−1 in Morisca (O). In a recent study (Bendini et al. terpenoid hydrocarbons to flavor is still unclear. However, 2007b) it was observed that the group of C6 aldehydes was some authors (Bortolomeazzi et al. 2001; Vichi et al. 2003; the most abundant in oils with a medium–high intensity of Zunin et al. 2005) have proposed to use these compounds as orthonasal perception of fruitiness that gradually decreased markers to discriminate geographical areas of VOO origin. in samples with a medium–low intensity, up to a near

In general, for Italian samples, C6 aldehydes represented complete loss for samples that did not contain C6 the most important group with a medium content of aldehydes. According to the observations in the present 43.3 mg kg−1 of oil with respect to 15.5 mg kg−1 in study, sample K showed one of the lowest values for −1 Spanish oils, ranging from 69 mg kg in Leccino (E) to aldehyde C6 and was judged defective in fruitiness.

Fig. 4 Percentage distribution ALD C6 ALC C6 EST C6 TERP10 TERP15 of volatile classes in VOO sam- 100% ples. For abbreviations see Table 1: 1=A; 2=B; 3=C; 4=D; 5=E; 6=F; 7=G; 8=H; 9=I; 10=J; 80% 11=K; 12=L; 13=M; 14=N; 15=O; 16=P

60%

40%

20%

0% 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 264 Chem. Percept. (2008) 1:258–267

Table 3 Correlations (for p<0.05) between sensory and chemical evaluation

Sensory sheet attributes Volatile and phenolic compounds positively related Volatile and phenolic compounds negatively related

Olive fruitiness (O) Z-2-Penten-1-ol; 3,5-dimethyl-1,6-heptadiene; 3-Methyl-1-butanol; 2-methyl-1-butanol;

sum of aldehydes C6 2,4-dimethylheptane; hexyl acetate; nonanal; decanal; Z-2-decenal Pleasant flavors (O) Z-2-Penten-1-ol (grassya); Z-3-hexenal (green tomatoa); 3-Methyl-1-butanol (vinegary–fustya); 2,4- 2-methyl-6-methyl-1,5-heptadiene (grassya) dimethylheptane (cookeda); decanal (vinegarya) Olive fruitiness (R) Z-2-Penten-1-ol 3-Methyl-1-butanol; 2-methyl-1-butanol; 2,4-dimethylheptane; acetic acid hexyl ester; nonanal; decanal Grassy (R) Z-2-Penten-1-ol 3-Methyl-1-butanol; 2-methyl-1-butanol; hexyl acetate; decanal Green pleasant flavors (R) Z-2-Penten-1-ol (fresh almonda); Z-3-hexen-1-ol (tomatoa); Hexyl acetate; decanal; Z-2-decanal 2-methyl-6-methyl-1,5-heptadiene (artichokea); Z-3-hexenal/E-2-hexenal (tomato–artichokea)

Ripe pleasant flavors (R) Sum of aldehydes C6 (almond) – Bitterness (G) Tyrosol, oleuropein aglycon – Pungency (G) Oleuropein aglycon – Defect (O, R, G) 3-Methyl-1-butanol (vinegary–fustya); 2-methyl-1-butanol – (vinegary–fustya); heptanal (vinegarya)

The compounds and sensory attributes with a significant correlation of Pearson (for p<0.05) with respect to Italian and Spanish panels. Different perception routes: (O) orthonasal, (R) retronasal, (G) gustative a In the sensory sheet (Fig. 1), the taster can specify the relative attribute or defect in bracket the descriptor more correlated

The most representative compound of C6 aldehydes was Hexanal, ranging in concentration from 1.0 to 3.6 mg E-2-hexenal, which counted for 60% of total volatile kg−1, was the aldehyde that had the most similar concen- compounds in Italian and 21.6% in Spanish samples. tration among all samples. The Sicilian varieties Cerasuola

Table 3 reports the correlations between sensory and and Nocellara del Belice had very high amounts of C6 chemical profiles. In particular, it can be observed that C6 alcohols, and, in particular, the most abundant molecule aldehydes were correlated with O-olive fruitiness and with was Z-3-hexenol. With regards to C6 esters, this class of the ripe pleasant flavors of retronasally perceived almond. volatiles prevailed in Spanish samples, especially for Orthonasal pleasant flavor of green tomato was correlated Arbosana (J) and Morisca (O; mean values for 22.1% vs. with the Z-3-hexenal content. 1.2%). It is interesting to note that among Italian samples, However, as reported by Angerosa (2002), it is the amount of Z-3-hexenyl acetate was predominant in important to specify that each volatile compound, in Sicilian oils (samples A and G, Cerasuola and Nocellara addition to being present at different concentrations, varieties). It is likely that the high concentrations of Z-3- possesses a specific odor threshold. Based on this hexenal, Z-3-hexenol, and Z-3-hexenyl acetate are due to property, individual compounds can contribute differently the presence of specific enzymes, which would explain the to the global aroma of VOO. For example, Z-3-hexenal pleasant notes of grassy and tomato in these monovarietal appears to contribute more to green odor than E-2-hexenal oils described by assessors. because of its lower odor threshold (Guth and Grosch The total concentration of sesquiterpenes, molecular

1993). Regarding this aspect, the Z-3-hexenal/E-2-hexenal formula C15H24 (TERP15 in Table 2) ranged from 0.3 to ratioisworthyofcomment(Table2). In fact, Cerasuola 17.7 mg kg−1 (the highest values were found in P, N, A, and and Nocellara del Belice (samples A and G), both Sicilian H samples). Alpha-farnesene, a tetraunsaturated acyclic monocultivars, had higher ratios (2.2 and 2.6, respective- sesquiterpene, was present in the largest amounts with ly) and were judged by panels as richer in intensity in respect to the other sesquiterpenes (α-copaene, α-muur- olive fruitiness and green pleasant flavors (high level of olene, β-sesquiphellandrene, and δ-cadinene) in almost all olive fruitiness and pleasant flavors by orthonasal via and of the oils analyzed, followed by α-copaene. According to also for green pleasant flavor by a retronasal route). previous reports (Bortolomeazzi et al. 2001; Vichi et al. Moreover, as seen in Table 3, there was a correlation 2003; Zunin et al. 2005), terpenoid hydrocarbons show between the ratio Z-3-hexenal/E-2-hexenal and the retro- large differences that depend on both the olive variety and nasal green attributes of tomato and artichoke. geographical origin. Chem. Percept. (2008) 1:258–267 265

The data shown in Table 4, related to odor activity values calculated for the major compounds responsible for green pleasure notes and the most common defects in VOO, demonstrate a substantial difference among the 16 samples evaluated. Z-3-hexenal was present in higher amounts in Italian samples and particularly in those originating from -2-pentenol 0.300. Odor

Z Sicily (samples A and G). This compound has, as previously reported (Angerosa 2002; Kalua et al. 2007), a lower odor threshold (0.003 mg kg−1)betweenthe

compounds linked to green pleasure constituents of green aroma, also in a pure form (Burdock

5 2005). In this investigation, the significant presence of this

million of 1-nonanol, and its odor threshold compound in VOO (3,760 and 13,135 OAV values, respec- and C rancid defects: heptanal 0.500, nonanal 0.150, 6 tively), characterized with tomato odor notes, has been shown. Among alcohols, Z-3-pentenol and 1-hexanol were the most -3-hexenyl acetate 0.750, Z important due their lower sensory threshold, whereas E-2- hexenol did not contribute to whole aroma. Z-2-Penten-1-ol, practically absent in almost all Spanish oils, was found in low levels (highest mean value 0.77 mg kg−1)inItalian samples. However, for this latter compound, the OAV values ). Odor thresholds of C 1 were higher than 1.0 only in three Italian samples (A, C, and G, Cerasuola, Nocellara Sicilian, and Dritta), which were positively related with both olive fruity and green pleasant flavors perceived as ortho- and retronasal. Concerning esters, -3-6.000, hexyl acetate 1.040, Z both hexyl acetate and Z-3-hexenyl acetate have been found to be linked to some sweet and ripe pleasant flavor notes more intensively perceived in Spanish oils, particularly in Arbequina (sample I). As reported by Burdock (2005), as pure compounds,

-2-hexenol 5.000, 3-methyl-1-butanol and 2-methyl-1-butanol possess an E unpleasant pungent odor and alcoholic undertones, re- spectively. According to several authors (Procida et al. 2005; Morales et al. 2005) who found these compounds in real oils and in training samples provided by IOOC characterized by clear winey and fusty defective odors, the assessors perceived these defects in sample K, which had the highest concentrations of these two branched alcohols (Table 3). Higher contents of aldehydes like heptanal, nonanal, and decanal, originating from oxidative processes, was detected -3-hexenal 0.003, 1-hexanol 0.400, Z in some samples (Angerosa et al. 2004; Kalua et al. 2007).

). One to 16 Italian and Spanish oil samples (for sample names, see Table The four panels were in agreement on the presence of a light rancid defect only for samples J and K. 2007

Phenolic Profile vs. Gustative Perceptions -2-hexenal 0.400, E The phenolic profile of oil samples (Table 5) was similar in

ABCDEFGHIJKLMNOP terms of phenolic compounds identified to other oils analyzed with the same analytical method and sample storage conditions (Bendini et al. 2007b). In these samples (Table 5), low values for secoiridoid such as oleuropein and Odor activity values (OAV) of volatile profiles ligstroside aglycon were observed. As shown in previous studies, these compounds give rise to derivative forms -2-Hexenal-2-Hexenol 12.9 127 <1 120 <1 121 <1 162 <1 141 <1 38.2 <1 144 47.9 <1 23.8 <1 13.2 <1 11.8 <1 105 <1 73.9 <1 8.70 <1 26.2 <1 <1 <1 -3-Hexenal-3-Hexenol 3,764-3-Hexenyl acetate-2-Pentenol 230 3.1 2.6 167 <1 <1 1.0 151 <1 <1 <1 316 <1 <1 1.3 265 <1 <1 <1 13,135 <1 <1 750 <1 88 <1 2.0 2.3 <1 <1 2.6 1.3 <1 <1 <1 5.3 440 <1 <1 2.2 26.5 <1 1.2 <1 345 <1 1.3 <1 739 <1 <1 1.0 2,031 <1 1.8 <1 3.2 <1 <1 <1 <1 <1 <1 -Hexanol 13.2 2.5 1.3 1.7 1.5 <1 6.9 3.8 5.3 2.2 1.2 1.3 <1 1.8 3.2 <1 thresholds of volatiles linkeddecanal to 0.650 fusty and winey defects: 3-methyl-butanol 0.100, 2-methyl-butanol 0.480. Odor thresholds of volatiles linked to perceptions: hexanal 0.075, is in parts per meter (by Kalua et al. Table 4 OAVof different volatile molecules for all samples; the OAV were calculated by ratio between concentration of volatile compound express as parts per HexanalE Z 1 E Z Hexyl acetate 22.8Z Z 39.73-Methyl-butanol2-Methyl-butanolHeptanal <1 20.7Nonanal <1Decanal 23.0 <1 <1 5.2 42.2 <1 <1 <1 10.4 <1 23.0 <1 <1 <1 <1during 8.3 2.8 39.0 <1 1 <1 <1 17.4 storage 48.5 2.1 <1 <1 <1 1.9 8.9 19.9 (Di <1Lecce <1 18.0 1.2 <1 7.4 <1 <1 35.6 et <1 <1 8.1al. <1 <12006 31.0 <1 4.2 <1). 23.3 2.4 <1 47.9 The 4.2 19.4 <1 highest 3.9 21.3 2.4 <1 3.1 34.2 <1 1.6 15.3 values 1.4 <1 7.9 38.6 <1 1.4 7.7 of 13.3 <1 1.2 58.6 12.0 <1 1.2 <1 25.0 <1 12.2 <1 <1 26.7 <1 3.2 4.1 29.3 <1 2.8 <1 2.4 5.7 31.3 <1 <1 <1 5.5 <1 42.7 1.5 <1 5.2 1.4 10.0 266 Chem. Percept. (2008) 1:258–267

Table 5 Phenolic compound content in samples expressed as milligram of 3,4-dihydroxyphenyl acetic acid per kilogram of oil

Samples Hyty Tyr DMOA OA1 Pin AcPin+DLA OA2 LA TP

A 1.72 1.66 2.59 nd 9.79 10.8 2.93 2.44 31.9 B 0.276 0.367 3.13 1.23 4.88 29.9 5.40 7.21 52.4 C 6.91 7.55 46.5 1.64 10.2 23.9 5.82 7.79 110 D 15.5 30.0 33.3 1.52 9.95 20.4 6.68 2.62 120 E 1.29 0.808 52.3 1.28 10.1 16.3 5.52 4.85 92.5 F 0.677 3.18 15.9 1.16 10.5 23.9 4.36 8.13 67.8 G 0.267 0.934 1.99 0.428 14.1 19.0 2.94 9.47 49.1 H nd 0.195 3.11 0.775 13.6 17.2 1.34 14.5 50.7 I 1.35 2.13 2.61 nd 5.61 32.8 2.61 6.45 53.6 J 5.77 0.538 2.70 0.535 5.18 18.3 1.30 2.82 37.2 K 13.3 4.28 30.3 nd 3.69 19.4 9.39 1.31 81.7 L 2.87 2.08 31.7 nd 4.62 8.33 21.1 5.47 76.2 M 2.93 0.212 10.4 1.15 6.55 5.27 4.73 6.66 37.9 N 4.59 4.52 17.4 2.60 7.16 14.5 7.03 5.73 63.5 O 3.31 2.33 39.3 10.2 8.05 11.5 14.1 4.37 93.2 P 2.63 1.42 26.3 1.35 4.97 14.2 8.78 4.19 63.8 Mean 3.96 3.89 20.0 1.49 8.06 17.9 6.50 5.87 67.6

Hyty hydroxytyrosol, Tyr tyrosol, DMOA decarboxymethyl-oleuropein aglycon, OA1 aldehydic form of oleuropein aglycon, Pin (+)-pinoresinol, AcPin (+)-1-acetoxypinoresinol, DLA decarboxymethyl-ligstroside aglycon, OA2 oleuropein aglycon, LA ligstroside aglycon, TP total phenols phenolic compounds were seen for samples D and C. In behavior is probably due to the low variability of the these latter samples, the most abundant compound was the phenolic profile on the samples considered in this study. decarboxymethyl-oleuropein aglycon that, as previously demonstrated (Carrasco-Pancorbo et al. 2005b), has good Acknowledgments The authors would like to acknowledge the antioxidant activity, which extends the freshness of VOO. enormous help and willingness of the four taste panel leaders Barbara However, other samples were characterized by the presence Alfei (Ancona), Stefano Terenziani (Perugia), Juan Ramón Izquierdo of acetoxypinoresinol and decarboxymethyl-ligstroside (Madrid), Sergio Gómez-Alonso (Toledo), and especially all the aglycon. These molecules do not have high antioxidant assessors that contributed to this experimental research. activity (Carrasco-Pancorbo et al. 2005b), and this charac- teristic is linked to a lower degree of freshness of the oils. Correlations between phenolic compounds (known as References the most responsible for taste perceptions) and gustative attributes demonstrated low positive values between bitter- Andrewes P, Busch J-L-H-C, De Joode T, Groenewegen A, Alexandre ness and tyrosol and oleuropein aglycon and between H (2003) Sensory properties of virgin olive oil polyphenols: identification of deacetoxy-ligstroside aglycon as a key contrib- pungent and oleuropein aglycon. utor to pungency. J Agric Food Chem. 51:1415–1420 Angerosa A (2002) Influence of volatile compounds on virgin olive oil quality evaluated by analytical approaches and sensor panels. Eur J Lipid Sci Technol 104:639–660 Conclusions Angerosa F, Servili M, Selvaggini R, Taticchi A, Esposto S, Montedoro G-F (2004) Volatile compounds in virgin olive oil: The present study examined the sensory characteristics in occurrence and their relationship with the quality. J Chromatogr A 1054:17–31 16 VOO evaluated by four official panels, employing a Beauchamp G-K, Keast R-S-J, Morel D, Lin J-M, Pika J, Han Q, total of 59 tasters, from two major olive-producing Lee C-H, Smith A-B, Breslin P-A-S (2005) Phytochemistry— countries (Spain and Italy). ibuprofen-like activity in extra-virgin olive oil. Nature The relationships between the chemical composition of 437:45–46 Beltrán G, Ruano MT, Jiménez A, Uceda M, Aguilera MP (2007) VOO and sensory characteristics were explored. Evaluation of virgin olive oil bitterness by total phenol content The volatile composition evidences several correlations analysis. Eur J Lipid Sci Technol 109:193–197 with olfactory attributes perceived by sensory analysis: The Bendini A, Cerretani L, Carrasco-Pancorbo A, Gómez-Caravaca AM, Segura-Carretero A, Fernández-Gutiérrez A, Lercker G (2007a) sum of aldehydes C6 was correlated with orthonasal Phenolic molecules in virgin olive oils: a survey of their sensory perception of olive fruity and retronasal odor of almond. properties, health effects, antioxidant activity and analytical Finally, data about phenolic fraction and bitterness and methods. An overview of the last decade. Molecules 12:1679– pungent perceptions showed only slight correlations; this 1719 Chem. Percept. (2008) 1:258–267 267

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