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 Olive 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 olive oil (VOO) is typified by characteristic, pleasant sensory notes that differentiate it from other edible oils. Sensory taste, together with nutritional aspects, is the main reason for the increase in consumption of VOO in recent years. Sensory analysis is required by European Official Regulations for olive oil in order to classify the product in commercial categories. In this study, the relationship between sensory and chemical composition has been investigated. In particular, 16 VOO samples (15 from a single variety of olives), produced in Italy and Spain (eight from each country), were analyzed. Sensory attributes were valued by four panels (three officially recognized both by IOOC and National Ministry: two Italian and two Spanish) employing a total of 59 tasters. Volatile and phenolic compounds were related to olfactory and gustative notes, respectively. Volatile compounds were then separated, identified, and quantified, starting from oil samples, by solid phase microextraction and capillary gas chromatographic analysis using a mass spectrometry detector (SPME/cGC-MSD). Furthermore, the phenolic profile was examined by high-performance liquid chromatography with diode-array and mass spectrometry detectors (HPLC-DAD/ MSD). Correlations were found between the major volatile L. Cerretani (*) : A. Bendini Dipartimento di Scienze degli Alimenti, Università di Bologna, P.zza Goidanich 60, 47023 Cesena (FC), Italy e-mail:
[email protected] M. D. Salvador (*) : G. Fregapane Departamento de Tecnología de Alimentos, Universidad de Castilla-La Mancha, Avda Camilo José Cela 10, 13071 Ciudad Real, Spain e-mail:
[email protected]
compounds (sum of aldehydes C6) and orthonasal perception of olive fruity and retronasal odor of almond. Additional correlations with bitterness and pungency were observed for tyrosol and oleuropein aglycon, respectively. Keywords Virgin Olive Oil . Sensory Analysis . Panel Test . Volatile Compounds . Phenolic Compounds Abbreviations cGC Capillary gas chromatography DAD Diode-array detector EI Electron ionization HPLC High-performance liquid chromatography IOOC International Olive Oil Council LOX Lipoxygenase MSD Mass-spectrometer detector QDA Quantitative descriptive analysis SPME Solid phase microextraction VOO Virgin olive oil
Introduction In terms of composition, virgin olive oils (VOO) differ from the other edible oils in minor components (Angerosa et al. 2004; Carrasco-Pancorbo et al. 2005a; Bendini et al. 2007a) that determine sensorial stimuli (Andrewes et al. 2003; Gutierrez et al. 2003). For the merceological classification of virgin olive oils, the European Union Commission has provided 26 chemical-physical parameters and organoleptic evaluation (EEC Reg. 2568/91; EC Reg. 1989/03). Sensory analysis of VOO initiated from Collaborative International studies, which has been supported for many years by the International Olive Oil Council (IOOC)
Chem. Percept. (2008) 1:258–267
that developed the Quantitative descriptive analysis sensory methodology for virgin olive oils, known as IOOC-Panel test. This study was finally published in June 1987, and the method was adopted for sensory analysis of VOO (IOOC 1987). Sensory evaluation was introduced in the current, official regulations in 1991 (EEC Reg. 2568/91 Annex XII). This analysis is carried out by a fully trained panel composed of 8 to 12 assessors, and the regulation provides a specific sensory sheet for assigning the merceological category. Successively, in 2002, the European Union Commission adopted the IOOC methodology, which modifies and simplifies the score sheet, employing an unstructured 100-mm scale (EC Reg. 796/02). Finally, the 4th July 2008 other modifications suggested by IOOC were introduced (EC Reg. 640/08) concerning the sensory vocabulary and the conditions for the optional use, on labels, of certain terms and expressions relating to the organoleptic characteristics of virgin olive oil. Recently, the evolution of sensory analysis of VOO has led to the proposal of a method for oil and food pairing (Cerretani et al. 2007). Sensory attributes of VOO mainly depend on the content of minor components like phenolic and volatile compounds. Each single component can contribute to different sensory perceptions. It is well established that phenolic compounds are responsible for bitterness in VOO (Gutierrez et al. 1989, 1992, 2003; Siliani et al. 2006; Beltrán et al. 2007). Andrewes et al. (2003) isolated individual compounds, and for each attributed sensory characteristic, these authors found that carboxymethyl-ligstroside aglycon, also called oleocanthal (Beauchamp et al. 2005), is the major phenolic molecule responsible for the burning pungent sensation of VOO. Moreover, the phenolic content has been related to protection against important chronic and degenerative diseases (Menendez et al. 2007). Approximately 180 compounds belonging to several chemical classes such as aldehydes, ketones, ethers, hydrocarbons, alcohols, and esters have been separated from the volatile fractions of different quality VOO (Angerosa 2002; Angerosa et al. 2004). Typical flavors and off-flavor compounds that affect the volatile fraction of a VOO originate by different mechanisms: enzymatically by linoleic and linolenic fatty acids through the so-called lipoxygenase (LOX) pathway, by sugar fermentation or amino acid (leucine, isoleucine, and valine) conversion, or from enzymatic activities of molds or auto-oxidative processes (Morales and Tsimidou 2000; Angerosa 2002). Volatile molecules can be perceived in very small amounts (micrograms per kilogram). These compounds do not contribute to the global aroma of VOO with the same importance; in fact, their influence must be evaluated not only on the basis of their concentration but also on their sensory threshold values. The independent odors of different volatile compounds that
259
contribute to various VOO aromas have been investigated extensively by GC sniffing techniques (Morales et al. 1994). In a large number of investigations on VOO (Andrewes et al. 2003; Gutierrez et al. 1989, 1992, 2003; Siliani et al. 2006; Beltrán et al. 2007), sensory and chemical parameters have been correlated, but in all cases, sensory analysis has been carried out only by a single panel group or by panels from the same producer country. To assess correlations between sensory attributes and the minor components of VOO, mainly phenolic and volatile profiles, four official panels from Italy and Spain and a set of 16 VOO (15 monovarietal) Italian and Spanish samples were employed in order to verify the quality of the tasters’ scores and determine the variability of the sensory characteristics of VOO samples. Together, Italy and Spain contribute to more than 60% of worldwide olive oil production (IOOC 2007), and therefore, the capacity of the Official Taste Panels employed in this research work is guaranteed.
Materials and Methods Samples Table 1 shows the characteristics of the VOO samples employed. With the exception of one sample, all oils were produced from a single variety of olives. Samples came from different industrial olive mills covering the main producing areas in the two countries during two consecutive crop seasons: 2003–2004 and 2004–2005. Reagents and Standards The standards used for quantification of volatile and phenolic compounds (1-nonanol and 3,4-dihydroxyphenylacetic acid, respectively) were from Sigma-Aldrich (St. Louis, MO, USA). All solvents used were analytical or HPLC grade (Merck, Darmstadt, Germany). Sensory Analysis Sensory analysis was performed by four Official Taste Panels. The Regional Panel of Agenzia Servizi Settore Agroalimentare Marche—A.S.S.A.M. (Ancona, Italy) was composed by a large number of members (20 plus the panel leader) with an average age of 35 years; this panel was recognized by IOOC in the year 2000 and successively by the Italian Ministry in 2004. The Panel of Institute for Mediterranean Agriculture and Forest Systems—CNRISAFoM (Perugia, Italy) is composed of 12 tasters with an average age of 35 years and are in the final training 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 code
Olive cultivar
Country of production
Area of olive production
A B
Cerasuola Correggiolo
Italy Italy
C D
Dritta Frantoio
Italy Italy
E
Leccino
Italy
F
Italy
Italy
Sicilia, Palermo
H
Frantoio, Leccino, Moraiolo Nocellara del Belice Orfana
Sicilia, Palermo Emilia-Romagna, Rimini Abruzzo, Pescara Emilia-Romagna, Ravenna Emilia-Romagna, Forlì-Cesena Toscana
Italy
I J
Arbequina Arbosana
Spain Spain
K L
Empeltre Gordalilla
Spain Spain
M N
Spain Spain
O
Lechin Manzanilla cacereña Morisca
P
Pico limon
Spain
Emilia-Romagna, Ravenna Cataluña, LLeida Cataluña, Tarragona Aragón, Teruel Andalucía, Málaga Murcia Extremadura, Cáceres Extremadura, Badajoz Andalucía, Sevilla
G
Spain
Denominación de Origen Montes de Toledo—DOMT (Toledo, Spain) was recognized by IOOC in 2003, by the Spanish Ministry (MAPA) in 2004, and by ENAC (Entidad Nacional Acreditación) in 2006; this panel is composed of 15 assessors with an average age of 32 years. Finally, the Panel del Laboratorio Arbitral Agroalimentario (Madrid, Spain) was recognized by IOOC in 1989 and by MAPA in 1987; this has 12 members, with an average age of 56 years. All panels evaluated VOO following an incomplete randomized design. A maximum of six oils, randomly selected among the 16 samples, were analyzed in each panel sessions. Each sample was tested twice in two different sessions. Moreover, to reduce the number of tests, a balanced incomplete block design was applied according to Cochran and Cox (1957). For the present study, a standard profile sheet (Fig. 1), realized according to IOOC method T20 and modified by IBIMET-CNR (Rotondi et al. 2004; Cerretani et al. 2005), was used. This sensory ballot permitted to obtain a complete profile of the sensory properties of samples, and a total of 59 tasters were employed.
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 headspace were adsorbed on a solid phase microextraction (SPME) under magnetic stirring. The fiber in PDMS 50/30 μm (Supelco, Bellefonte, PA, USA) was exposed to the vapor phase for 30 min at 40 °C. Desorption of volatile compounds trapped in the SPME fiber was done directly in the gas chromatograph injector set at 220 °C in splitless mode using a splitless inlet liner of 0.75 mm i.d. for thermal desorption where it was left for 3 min. A GC CP 3800 coupled with a mass-spectrometer Varian 2000 was used, and a fused-silica capillary column CP-Sil 8 CB (60 m, 0.25 mm i.d., 0.25-μm film thickness; Chrompack) was employed. The column was operated with helium at a pressure of 12 psi and a flow rate of 1 mL min−1. The GC oven heating was started at 40 °C; this temperature was maintained for 10 min, then increased to 55 °C at a rate of 1 °C min−1, increased to 80 °C at a rate of 5 °C min−1, and finally increased to 220 °C at a rate of 18 °C min−1. The temperature of the transfer line was fixed at 180 °C. The mass spectrometer operated in the electron ionization mode at an ionization voltage of 70 eV in the mass range of 39–300 amu at a scan rate of 1 scan s−1 and a manifold temperature of 180 °C. The GC-MS operated through the software Saturn GC-MS version. Volatile compounds were identified by comparison of their mass spectra and retention times with those of 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 based on calibration curve of 1-nonanol (from 0.1 to 200 mg kg−1 of oil) as an external standard. Phenolic Compounds Analysis The phenolic fraction was extracted from VOO by a liquid/ liquid extraction method described by Pirisi et al. (2000). The dry extracts were redissolved in 0.5 mL of methanol/ water (50:50, vol/vol) and filtered through a 0.2-μm nylon filter (Whatman, Clinton, NJ, USA). Extracts were frozen and stored at −43 °C. HPLC analyses were performed using a HP 1100 Series instrument (Agilent Technologies, Palo Alto, CA, USA) equipped with a binary pump delivery system, degasser, autosampler, diode-array UV–VIS detector (DAD) and mass-spectrometer detector (MSD). All solvents were of HPLC grade and filtered through a 0.45-μm nylon filter disk (Lida Manufacturing, Kenosha, WI, USA) prior to use. All analyses were carried out at room temperature. The phenolic profile was assessed by HPLC-DAD/ESIMSD 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, USA) according to Rotondi et al. (2004). The injection volume was 10 μL. Phenolic compounds detected at 280 nm were quantified using a 3,4-dihydroxyphenylacetic acid standard calibration curve (r2 =0.999).
linear correlations, at p < 0.05, were evaluated using Statistica 6.0 (2001, StatSoft, Tulsa, OK, USA).
Statistical Analysis
Sensory Analysis
The results reported in this study are the averages of at least three repetitions (n=3), unless otherwise stated. Pearson’s
Sensory analysis was performed by four different fully trained analytical VOO taste panels from Italy and Spain
Results and Discussion
262
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 in the Nocellara del Belice Italian sample (sample G) to a score of 21 in the Empeltre Spanish variety (sample K). On the other hand, pleasant orthonasal flavor notes were evaluated by tasters in a similar way, but in a wider range, from 45 in Nocellara del Belice (sample G) to a minimum value of 5 (sample K) in Spanish Empeltre. However, the values of fruitiness by retronasal perception routes were similar for all samples, ranging from 30 to 48, with the exception of sample K, showing a comparable behavior as observed for O-olive fruity attributes.
Chem. Percept. (2008) 1:258–267
Fig. 3 Sensory profiles of Spanish samples (for sample codes see Table 1). Different perception routes: (O) orthonasal, (R) retronasal, (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; 16=P
In accordance with all panels, sample D (Frantoio variety, sensory score of 44) was the most and Arbequina (score of 10) the least bitter. The same samples (D and I), but in this case firstly I (48) followed by D (45) showed the highest intensity of pungency. Italian samples were generally 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
Fig. 2 Sensory profiles of Italian samples (for sample codes see Table 1). Different perception routes: (O) orthonasal, (R) retronasal, (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; 16=P
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 represent the 89% and 72% of whole profiles for Italian and Spanish VOO, respectively (Fig. 4). The C6 compounds originating from the LOX pathway are 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
A B C D E F G H I J K L M N O P
TOT
ALD C6
Z-3/E-2
ALC C6
EST C6
TERP10
TERP15
90.3±5.6a 77.0±4.5 82.8±2.0 74.5±3.2 87.0±2.9 84.3±2.7 109±0.4 106±3 77.2±0.2 95.5±3.8 54.1±1.3 69.4±0.2 104±2 85.0±0.0 121±0.2 87.6±1.0
18.2±0.8 54.6±2.7 49.9±2.6 50.7±3.1 69.0±2.7 59.0±2.3 57.6±0.6 63.5±2.2 20.9±0.3 10.9±0.5 7.93±0.56 8.36±0.13 45.8±1.9 32.2±1.0 6.85±0.03 17.6±0.8
2.19±0.06
