Atzori, G.

Azzarello, E.

Mancuso, S

Marone, E.

Redwan, M.

Taiti, C.

English

Mancuso, S.

Florence Research

143Adv. Hort. Sci., 2018 32(1): 143-147 DOI: 10.13128/ahs-22715Comparative analysis of volatile com-pounds (potential aromatic ability) inthe fruit of 15 olive Italian cultivarsC. Taiti 1, M. Redwan 1, E. Marone 2 (*), G. Atzori 1, E. Azzarello 1, S.Mancuso 11 Dipartimento di Scienze delle Produzioni Agroalimentari e dell’Ambiente,Università degli studi di Firenze, Viale delle Idee, 30, 50019 SestoFiorentino (FI), Italy.2 Facoltà di Bioscienze e Tecnologie Agro-Alimentari e Ambientali,Università degli Studi di Teramo, Via R. Balzarini, 1, 64100 Teramo, Italy.Key words: olive fruits, PTR-ToF-MS, volatile organic compounds (VOCs).Abstract: Virgin olive oils (VOOs) are characterized by peculiar flavors appreci-ated by the consumers all over the world. Their organoleptic characteristicsdepend on the aromatic properties of the fruits of the different cultivars, whichwill originate the final products. VOCs spectra of fifteen certified Italian olivecultivars of the University of Florence Germplasm collection, chosen as theirdifferent geographical origin, diffusion, and product purpose, were acquiredusing a Proton Transfer Reaction Time-of-Flight Mass Spectrometer (PTR-ToF-MS). The VOCs analyses highlighted a great variability among the fifteen culti-vars, mostly due to compounds (C6 and C5) deriving from polyunsaturated fattyacids through the LOX pathway. The early identification in the olive fruit ofthese compounds which are considered among the major contributors to thepositive VOOs attributes, would be useful to produce high quality olive oils, andget useful information to individuate the best parents for the genetic improve-ment. 1. IntroductionVirgin olive oil is worldwide considered a “commodity”, and until theSecond World War, a strategic food (Fiorino et al., 2010). The complex fla-vor of virgin olive oil is mainly produced by volatile organic compounds(VOCs) whose formation is related to olive fruit cell destruction (Moraleset al., 1996; Angerosa, 2004). The total amount and types of VOCs emit-ted change during the processing steps of olive fruit in the olive mill(Morales et al., 1997). In particular, VOCs emission is linked to thedestruction of the cell structure of olive fruits which activates a specificchain of enzymatic reaction (LOX cascade) (Angerosa et al., 2004). The C6and C5 are the compounds which most affect the aroma of olive oil gen-(*) Corresponding author: emarone@unite.itCitation:TAITI C., REDWAN M., MARONE E., ATZORI G.,AZZARELLO E., MANCUSO S., 2018 - Comparativeanalysis of volatile compounds (potential aromat-ic ability) in the fruit of 15 olive Italian cultivars. -Adv. Hort. Sci., 32(1): 143-147Copyright:© 2018 Taiti C., Redwan M., Marone E., Atzori G.,Azzarello E., Mancuso S. This is an open access,peer reviewed article published by FirenzeUniversity Press(http://www.fupress.net/index.php/ahs/) anddistribuited under the terms of the CreativeCommons Attribution License, which permitsunrestricted use, distribution, and reproductionin any medium, provided the original author andsource are credited.Data Availability Statement:All relevant data are within the paper and itsSupporting Information files.Competing Interests:The authors declare no competing interests.Received for publication 20 February 2018Accepted for publication 6 March 2018AHSAdvances in Horticultural ScienceShort noteAdv. Hort. Sci., 2018 32(1): 143-147144erating the positive attributes such as fruity andherbaceous notes (Marone et al., 2017). These com-pounds are usually responsible for typical aromasand flavors and play a dominant role in determiningthe peculiar aroma and quality of olive oils (Zunin etal., 2005). The synthesis of C5 and C6 compounds islinked to the level of LipOXygenase (LOX) activity tobe catabolized through its pathway during the millprocess (Garçia-Vico et al., 2017). Different C6straight chains are produced by the action ofhydroperoxide lyase on polyunsaturated C18 fattyacids (linoleic C18:2 and linolenic C18:3). While, C5seem to be synthesized through another branch ofthe LOX pathway starting from 13-hydroperoxidesderived from linolenic acid (Garçia-Vico et al., 2017).Currently, the most innovative analytical tech-nique used to detect VOCs emitted by fruits, whichprovides a high resolution coupled to a rapid screen-ing power of samples and easy to handle withoutsample manipulation, is the Proton TransferReaction-Time of Flight-Mass Spectrometer (PTR-ToF-MS) (Mayr et al., 2002; Masi et al., 2015; Taiti etal., 2017 a).Currently, a little information is  available and atthe best of our knowledge one study was carried outto understand the VOCs profile emission from olivefruits related to two cultivars (Masi et al., 2015). Theaim of this study was to develop a quick, accurateand relatively simple evaluation method based onthe PTR-MS technique, to define the aromatic poten-tiality of each olive cultivar starting from  olive fruits.This method could potentially help to address thechoice of the cultivars in new plantations and toidentify parents for future crosses, aimed to improvesome organoleptic characteristics of the oils.2. Materials and MethodsPlant materialFruits of 15 certified Italian olive cultivars, clonallypropagated, different in their geographical origin,distribution and fruit’s use were obtained from plantsof Olive Germplasm collection deriving from theItalian and World olive Gene bank (Montepaldiexperimental farm (43° 40’ 39’’ North latitude, 11°08’ 46” East longitude, 210 m asl), University ofFlorence) (Table 1). The rainfed, bush trained, four-teen years old olive plants are ordinarily cultivated,grown on a slight slope of a sedimentarious gipsy-arenaceous soil; the annual rainfall average is 867mm, the average annual temperature results 13.3°C.Fruit samplingFor each cultivar, 1 kg of sound and healthy fruitswas collected on October 20th from different parts ofthe trees of each cultivar. The day after, 100 washedfruits from each cultivar were weighted (W) and thecolor index (CI) was determined (Uceda and Hermoso,1998). Subsequently, for each cultivar nine whole andcut samples of fruits (~10 g) were submitted to theVOCs analysis.Statistical analysesOne way analysis of variance (ANOVA) was per-Cultivar OriginProductuseFruit averageweight(g)Color Index (0-7)Ascolana tenera Marche Table 4.80 ± 0.51 2.2Bianchera Friuli Venezia Giulia Olive oil 2.68 ± 0.12 1.5Carolea Calabria Olive oil/Table 3.91 ± 1.17 1.2Coratina Apulia Olive oil 2.05 ± 0.60 1.5Fasolona Basilicata Olive oil/Table 3.80 ± 0.58 3.1Frantoio Tuscany Olive oil 1.96 ± 0.36 1.5Itrana Lazio Olive oil/Table 2.19 ± 0.94 1.4Leccino Tuscany Olive oil 2.02 ± 0.19 2.2Maiatica di Ferrandina Basilicata Olive oil/Table 2.75 ± 0.93 1.2Moraiolo Tuscany Olive oil 1.42 ± 0.28 2.1Nocellara del Belice Sicily Olive oil/Table 3.84 ± 0.76 1.8Palmarola Apulia Olive oil/Table 2.15 ± 0.72 3.1Sant’Agostino Apulia Table 4.36 ± 0.81 0.7Santa Caterina Tuscany Table 5.80 ± 0.48 0.4San Francesco Tuscany Table 2.68 ± 0.22 1.5Table 1 - List of the characteristics of the genetic materialTaiti et al. - Comparative VOCs analysis of 15 Italian olive cultivars145formed to compare the considered groups of chemi-cal compounds: C5, C6, other VOCs, and total VOCs.Separation of means was performed by the Fisher’sLSD test (p = 0.01). Computations were performed byStatgraphics Centurion XV v. 15.0.04.A Factor Analysis (FA) was applied to the spectraldata of 273 olive oil samples, considering as factorsthree grouping of compounds: total VOCs, C5, and C6,respectively. Computations were performed by XLSTAT2014.5.03.A Principal Component Analysis (PCA, unsuper-vised method) was applied to the whole spectraldata of 273 olive oil samples, submitted to a logarith-mic transformation and mean centering as pre-pro-cessing. Computations were performed by PLS-Toolbox v. 8.0.2 (Eigenvector Research Inc., WestEaglerock Drive, Wenatchee, WA) for MATLAB_R2015b (Mathworks Inc., Natick, MA, USA).Volatile compounds detectionThrough a PTR-ToF-8000 (IONICON Analytik,GmbH, Innsbruck, Austria) measurements were per-formed in a similar way to the one reported by Taitiet al. (2017 b). Each analyzed sample consisted of~10 g of olive fruits intact or cut into 4 parts.Subsequently the sample was inserted into a 3/4 Lglass container plugged with a cover in which twoTeflon tubes are inserted, connected respectively toa zero-air generator (Peak Scientific) and to the PTR-ToF-MS. The drift tube had the following ionizationconditions: pressure of 2.20 mbar, voltage 600V andtemperature 110°C. Mass spectrometric data werecollected over a mass range of m/z 21 to 210 and theacquisition time for each samples was 0.1 ns, for 120seconds. The instrument internal calibration wasbased on: m/z = 29.997 (NO+); m/z = 59.049 (C3H7O+)and m/z = 137.132 (C10H17+) and was performed off-line. The raw data were acquired with the TofDaqSoftware (Tofwerk AG, Switzerland) as cps (count persecond), and subsequently were converted in ppbvfollowing the formula described by Lindinger andJordan (1998) on the basis of the primary ion signal.3. Results and DiscussionThe whole intact olive fruits of the fifteen cultivarsshowed no detectable quantities of VOCs emission;in fact, the LipOXygenase pathway is activated whenthe fruit is damaged by the cut, producing a largequantity of volatile compounds, among which differ-ent C5 and C6 compounds, which are the mainvolatiles responsible for the positive aroma of oliveoil (Angerosa et al., 2004). Meanwhile a great vari-ability exists among the total VOCs emitted by thecut fruits of fifteen different cultivars (Table 2), rang-ing from 1061.8 ppbv in ‘Ascolana Tenera’ to 8767.5ppbv for ‘Carolea’. Therefore, the total emission from‘Carolea’ was significantly higher compared to‘Ascolana Tenera’. The cultivars can be arbitrarilydivided in three main groups according to their totalVOCs emission: (a) low VOCs emission, includingtogether with the cv. Ascolana tenera, Fasolona andCultivarC5 Compounds (ppbv)C6 Compounds (ppbv)Total VOCs(ppbv)VOCs emission(Arbitrary ranking)Ascolana tenera 8.01±0.61 A 180.73 ± 47.17 A 1061.77 ± 120.13 A lowBianchera 35.29±2.15 G 1143.77 ± 148.60 F 5101.34 ± 305.77 G highCarolea 53.90±7.83 I 2864.58 ± 543.60 I 8767.54 ± 1024.09 G highCoratina 12.02±1.15 D 261.36 ± 34.62 ABC 3177.13 ± 281.13 F mediumFasolona 6.64±0.44 AB 229.19 ± 35.91 AB 1845.41 ± 124.20 DE lowFrantoio 11.86±1.43 D 525.42 ± 96.22 DE 2076.17 ± 149.65 ABC mediumItrana 43.63±1.75 H 2497.20 ± 222.50 H 7877.17 ± 491.75 G highLeccino 9.07± 0.51 BC 264.10 ± 58.53 ABC 3380.28 ± 148.72 F mediumMaiatica di Ferrandina 24.49±3.21 F 1581.16 ± 262.15 G 5339.86 ± 472.56 EF highMoraiolo 17.41±1.52 E 533.93 ± 67.89 DE 2768.21 ± 150.04 BC mediumNocellara del Belice 16.77±1.15 E 544.07 ± 104.33 E 2986.40 ± 128.63 D mediumPalmarola 9.01±0.57 B 404.83 ± 64.22 CDE 3142.02 ± 218.24 DE mediumSant’Agostino 7.65±1.66 AB 148.79 ± 27.75 A 2309.40 ± 141.08 DE mediumSanta Caterina 8.73±1.61 B 402.58 ± 55.28 CDE 2121.11 ± 148.07 AB mediumSan Francesco 11.54±0.76 CD 368.17 ± 43.71 BCD 1934.07 ± 65.61 C lowTable 2 - Analysis of variance (ANOVA) for the C5, C6, and total VOCs related to the fifteen cultivarsDifferent upper case letters within a column indicate the difference by the LSD test at the 99% confidence leve (p= 0.01).Adv. Hort. Sci., 2018 32(1): 143-147146San Francesco; (b) medium VOCs emission, including‘Leccino’, ‘Coratina’, ‘Palmarora’, ‘Nocellara delBelice’, ‘Moraiolo’, ‘Sant’Agostino’, ‘Santa Caterina’,‘Frantoio’ and (c) high VOCs emission, including‘Carolea’, ‘Itrana’, ‘Maiatica di Ferrandina’,‘Bianchera’. Also C6 and C5 straight chain showed anample variability; in particular, C6 compounds rangedfrom 148.79 ppbv for the cv. Sant’Agostino to2864.58 ppbv for the cv. Carolea, with a fluctuationamong the different cultivar of about 1:20. As thehigh amount of developed C6 compounds it is note-worthy to highlight the cv. Carolea, Coratina, andItrana. On the other hand, the C5 compoundsshowed a minimum value of 6.64 ppbv (cv. Fasolona),and a maximum of 53.90 ppbv (cv. Carolea).A biplot from Factor Analysis (Fig. 1) simultane-ously represents the relationship between totalVOCs, C6, and C5 compounds, highlighting the rela-tive distances among the fifteen olive cultivars. Thefirst axis explains the 97.25% of the total variability inthe spectral data. The samples are clustered in twomain groups (Fig. 1), characterized by higher VOCsemission (right quadrants), and lower VOCs emission(left quadrants). Although the different totalamount, and despite the quantitatively differentresponses of the different cultivars, C5 and C6 com-pounds resulted directly related to the total VOCsemission (0.952 ppbv, and 0.964 ppbv, respectively),as a consequence of a more general phenomenon,the LipOXygenase (LOX) pathway, common to all theexamined cultivars. Therefore, by evaluating thetotal VOCs amount, it is possible to analyticallyextrapolate the contribution of C5 and C6 in the totalvolatile compounds emission. Moreover, by theFactor Analysis plot it can be noted that some culti-vars are characterized by higher C6 compoundsemission (e.g. ‘Itrana’ and ‘Maiatica di Ferrandina’),while other cultivars showed higher C5 emission (e.g.‘Bianchera’ and ‘Carolea’).Subsequently a PCA (Fig. 2) was performed on thewhole VOCs data set to (1) give a general overview ofthe cultivars ordination and (2) to detect if the culti-vars can be grouped basing on their geographical ori-gin and/or final product use. ANOVA results confirm(Table 2) that a high variability appears in the VOCsemission from the different cultivars, widely distrib-uted without any particular tendency to clustering,excluding any possible link of the different cultivarsdue to their geographical origin or to the final prod-uct use. The first two components justified 58.32%for PC1 and 9.43% for PC2 of the total variability,respectively, indicating that the greatest amount ofthe total variance is explained by the PC1. The mostimportant PC1 loadings resulted in eight m/z asreported in figure 2. In particular, m/z = 81.069[Tentatively Identified (T.I.) as Hexenal fragment]and m/z = 99.080 (T.I. as 2,3-Hexenal) directly deriv-ing from linolenic acid and responsible for the freshlycut grass odors, are previously reported as positiveattributes in oils by Marone et al. (2017) and Taitiand Marone (2017).Fig. 1 - Biplot from Factor analysis. Relationships among the fif-teen cultivars and the three groups of chemical com-pounds (C5, C6, total VOCs).Fig. 2 - Score plot from PCA related to the VOCs dataset (ppbv) for the fifteen cultivars; in the table the main PC1 loadings are reported.Taiti et al. - Comparative VOCs analysis of 15 Italian olive cultivars147The reported data confirmed the possibility topredict and quickly evaluate during all stages of thefruit ripening the presence of VOCs characterizing thepositive flavors of the olive fruits obtained fromdefined cultivars or new breeding.4. ConclusionsThe data show a great variability in both amountand type of VOCs emitted by olive fruits, simultane-ously collected in the same environment from fifteendifferent Italian cultivars. For example the total emis-sion by ‘Carolea’ was significantly higher compared to‘Ascolana Tenera’. On the other hand, C5 and C6 com-pounds which most affect the aroma adding positiveattributes to olive oil, were respectively 95% and 96%correlated to the total VOCs. Among the cultivars it ispossible to note a different relationship between C5and C6: the cv. Bianchera showed the highest C5/totalVOCs ratio, while the cv. Itrana, and Carolea the high-est C6/total VOCs ratio.The presence of these two groups of compounds(C5 and C6) confirms that the cut of the fruit triggersthe same phenomena that verifies at the moment ofthe olive processing, that is the activation of the LOXcascade, which gives rise to VOCs considered to bepositive attributes for the olive oil aroma.Further studies would be necessary to deepenand to develop the information that can beobtained from the use of the fruits to: (1) under-stand the behavior of the derived oils, and (2) towiden the genetic platform from which informationcan be acquired and (3) to start  verifying theresponses of fruits deriving from predeterminedcrosses. In conclusion, the possibility to early identi-fy these compounds at the fruit level can allow toindividuate the best conditions to produce high quali-ty olive oils, and get useful information to individuatethe best parents for the olive genetic improvement.AcknowledgementsThe Authors would like to thank the MontepaldiExperimental farm of University of Florence for pro-viding the samples used for these trials.ReferencesANGEROSA F., SERVILI M., SELVAGGINI R., TATICCHI A.,ESPOSTO S., MONTEDORO G.F., 2004 - Review: Volatilecompounds in virgin olive oil: occurrence and their rela-tionship with the quality. - J. Chromatogr. A, 1054(1-2):17-31.FIORINO P., MARONE E., OTTANELLI A., 2010 - Mechanicalharvesting, productivity and superintensive plantingsystems in olive groves. - Adv. Hort. 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Comparative analysis of volatile compounds (Potential aromatic ability) in the fruit of 15 olive italian cultivars

Virgin olive oils (VOOs) are characterized by peculiar flavors appreciated by the consumers all over the world. Their organoleptic characteristics depend on the aromatic properties of the fruits of the different cultivars, which will originate the final products. VOCs spectra of fifteen certified Italian olive cultivars of the University of Florence Germplasm collection, chosen as their different geographical origin, diffusion, and product purpose, were acquired using a Proton Transfer Reaction Time-of-Flight Mass Spectrometer (PTR-ToFMS). The VOCs analyses highlighted a great variability among the fifteen cultivars, mostly due to compounds (C6 and C5) deriving from polyunsaturated fatty acids through the LOX pathway. The early identification in the olive fruit of these compounds which are considered among the major contributors to the positive VOOs attributes, would be useful to produce high quality olive oils, and get useful information to individuate the best parents for the genetic improvement

Taiti C.

Redwan M.

Marone E.

Atzori G.

Azzarello E.

Mancuso S

Archivio della Ricerca - Università degli Studi di Teramo

Comparative analysis of volatile compounds (potential aromatic ability) in the fruit of 15 olive Italian cultivars

Schaffer, Laurie S.

Fordham University School of Law