Review. Influence of volatile compounds on virgin olive oil quality evaluated by analytical approaches and sensor panels.

Transcription

1 Eur. J. Lipid Sci. Technol. 104 (2002) Franca Angerosa Istituto Sperimentale per la Elaiotecnica, Città S. Angelo (PE), Italy Influence of volatile compounds on virgin olive oil quality evaluated by analytical approaches and sensor panels Volatile compounds, retained by virgin olive oils during their extraction process, are responsible for the oil aroma. Approximately one hundred and eighty compounds, whose structure was assigned by means of gas chromatography-mass spectrometry, were found in virgin olive oil aromas. The analytical approaches to their determination are briefly discussed, considering the problems related to the volatile compound collection and to adsorbents used for their trapping. The sensory methodology for the evaluation of the organoleptic characteristics of the virgin olive oils are reported and typical flavours and off-flavours are described. Compounds responsible for flavours, including factors affecting the volatile fraction, and for off-flavours are carefully examined, also considering the causes that give rise to offflavours. Relationships between volatile compounds and sensory characteristics, found by various researchers, are reviewed. Keywords: Olive oil, volatile compounds, sensory attributes, relationships between volatiles and the sensory characteristics. 1 Introduction Virgin olive oils, being mechanically extracted from olive fruits (Olea europaea L.), retain volatile and non volatile compounds, which are mainly responsible for their typical flavour that makes them highly appreciated by consumers not only in the countries of the Mediterranean basin where the olive oil production is concentrated. The salutistic properties of olive oil such as its high nutritional power, excellent digestibility, high oxidative stability even when used for cooking, strong capacity of prevention of heart and vascular troubles [1] do not explain the reasons for the increased popularity of the olive oil also in countries where it was a relatively underused commodity completely. The large increase in demand for high quality olive oils is thought to be related to their peculiar organoleptic characteristics that play an important role in human nutrition. Volatile aromatic compounds and also some non volatile compounds, strongly affecting sensory receptors, can decisively influence the food acceptability, direct the preference of consumer and, in a word, determine the quality of life to a great extent. Correspondence: Franca Angerosa, Istituto Sperimentale per la Elaiotecnica, Contrada Fonte Umano, Città S.Angelo (PE), Italy. Phone: , Fax: ; elaiotec@unich.it The sensory attributes of the olive oil perceived by consumers arise from the stimulation of the gustative and olfactive receptors through a large number of volatile [2-4] and some non volatile compounds [5, 6], such as phenolic substances. The latter mainly elicite the tasting perception of bitterness [7]. In addition, they stimulate the free endings of the trigeminal nerve located in all the palate and also in the gustative buds giving rise to the chemesthetic perceptions of pungency astringency and metallic attribute [8, 9]. All the other sensations experienced during the virgin olive oil tasting are attributed to the stimulation of the olfactory epithelium by a large number of volatile compounds present in the oil aroma in very low amounts. Tab. 1. Characteristics shared by volatile compounds responsible for virgin olive oil aroma. low molecular weight (<300 Da) high volatility so that a suitable number of molecules can reach the olfactory epithelium as molecular dispersion, transported by the air streams due to inhalation and expiration sufficient hydrosolubility to diffuse into the mucus that covers the sensitive olfactory cells fair liposolubility to dissolve in membrane lipids contiguous to proteins of receptors chemical features to bond specific proteins Review 2002 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim /2002/ $ /0

2 640 Angerosa Eur. J. Lipid Sci. Technol. 104 (2002) Tab. 2. Volatile carbonyl compounds identified in virgin olive oil aroma by MS. Tab. 3. Volatile ester identified in virgin olive oil aroma by MS. Compound Ref Compound Ref Aldehydes acetaldehyde [10] propanal [11] butanal [11] pentanal [10, 11] hexanal [10, 17] heptanal [10, 11] octanal [10, 11] nonanal [10, 11] decanal [21] acrolein [19] 2-butenal [19] pentenal (cis-2?) [10, 11] trans-2-pentenal [10, 11] cis-2-hexenal [10, 11] trans-2-hexenal [10, 17] cis-3-hexenal [12, 20] heptenal (cis-2?) [10, 11] trans-2-heptenal [10, 11] trans-2-octenal [10, 11] cis-2-nonenal [21] trans-2-nonenal [10, 11] cis-3-nonenal [21] trans-2-decenal [10, 11] trans-2-undecenal [10, 11] 2-methylbutanal [10, 17] 3-methylbutanal [10, 13] 2-methylbut-2-enal [10, 14] 2,4 hexadienal [10, 12] 2,4 -heptadienal (isomer A) [10, 12] 2,4 heptadienal (isomer B) [10, 12] 2,4-nonadienal [10, 21] 2,4-decadienal (isomer A) [10, 20] 2,4-decadienal (isomer B) [10, 20] benzaldehyde [10, 11] phenylacetaldehyde [21] Ketones acetone [11] 2-butanone [14] 2-hexanone [10] 2-heptanone [12] 2-octanone [10, 12] 2-nonanone [10, 12] 3-pentanone [10, 17] 3-octanone [4, 10] 3-metylbutan-2-one [10] 6-methyl-5-hepten-2-one [14] 4-methylpentan-2-one [14] 1-penten-3-one [13, 14] 1-octen-3-one [18, 21] 4-methyl-3-penten-3-one [17] 2-methyl-2-hepten-6-one [10] acetophenone [10] Identified by GLC-HPLC. Esters butyl acetate [12] ethyl acetate [10, 17] ethyl phenylacetate [10] ethyl propionate [10] ethyl butyrate [10, 12] ethyl octanoate [10, 12] ethyl heptanoate [11, 12] ethyl nonanoate [11] ethyl decanoate [11, 12] ethyl palmitate [11] heptyl acetate [12] hexyl acetate [12, 17] methyl acetate [14] methyl butyrate [10] methyl pentanoate [10] methyl hexanoate [10, 11] methyl heptanoate [10, 11] methyl octanoate [10, 11] methyl nonanoate [14] methyl decanoate [14] methyl oleate [11] methyl linoleate [11] 1-octyl acetate [10] propyl propionate [10] butyl 2-methylbutyrate [12] ethyl 2-methylpropionate [10, 12] ethyl 2-methylbutyrate [10, 12, 20] ethyl 3-methylbutyrate [10] methyl 2-methylbutyrate [10] methyl 3-methylbutyrate [10] 3-methyl-2-butenyl acetate [14] 2-methyl-1-butyl acetate [10, 17] 3-methyl-1-butyl acetate [10, 17] 2-methyl-1butyl 2-methyl-propionate [10] 2-methylbutyl propanoate [14] 2-methyl-1-propyl acetate [10, 14] 2-methyl-1-propyl 2-methylpropionate [10] 1-propyl 2-methylpropionate [10] ethyl benzoate [10] methyl benzoate [12] methyl salicilate [11] cis-3-hexenyl acetate [10, 17] ethyl cyclohexanoate [20] 2 Composition of volatile fraction Approximately one hundred and eighty compounds belonging to several chemical classes were separated from the volatile fractions of different quality virgin olive oils. Even though they belong to several chemical classes, they share the characteristics described in Tab. 1.

3 Eur. J. Lipid Sci. Technol. 104 (2002) Volatile compounds and sensorial analysis 641 Tab. 4. Alcohols identified in virgin olive oil aroma by MS. Compound Ref Alcohols methanol [15] ethanol [10, 17] propan-1-ol [18] butan-1-ol [12] pentan-1-ol [12, 13] hexan-1-ol [10, 12] heptan-1-ol [10, 12] octan-1-ol [10, 12] nonan-1-ol [10, 11] decan-1-ol [12] pentan-3-ol [10] octan-3-ol [18] 2-penten-1-ol [13, 14] trans-2-hexen-1-ol [10, 12] cis-3-hexen-1-ol [10, 12] 4-hexen-1-ol [12] 1-penten-3-ol [10, 12] 1-octen-3-ol [18] methylpropan-1-ol [10, 13] 2-methylbutan-1-ol [10] 3-methylbutan-1-ol [10, 12, 20] 3-methyl-1-pentanol [12] 2-phenylethanol [10, 12, 20] The chemical structure of most of the volatile compounds was assigned by gas chromatography-mass spectrometry (GC-MS) [10-21]. Tabs. 2, 3, 4 and 5 summarise carbonyl compounds, alcohols, esters and hydrocarbons, respectively, having been identified so far in virgin olive oil aromas by different researchers. Tab. 6 shows a compilation of other oxygenates compounds and thiophene derivatives found to be present among the volatile compounds of virgin olive oil. The number of volatile compounds detected in the aroma of an olive oil depends on the methodology adopted for their determination and on the quality of the virgin olive oil. Olive oil, obtained from healthy and rightly ripe fruits of the tree Olea europaea L., harvested at the right ripeness, by proper technological extraction methodologies, shows a volatile fraction mainly formed by compounds which are common contributors to the aroma of many fruits and vegetables. They are produced enzymatically from polyunsaturated fatty acids through the so-called lipoxygenase (LOX) pathway [22, 23]. It is stated that in the aroma of these oils C 6 aldehydes, C 6 alcohols and their corresponding esters are the most abundant accumulation products. Moreover, reasonable amounts of C 5 carbonyl Tab. 5. Hydrocarbons identified in the virgin olive oil aroma by MS. Compound Ref Hydrocarbons n-octane [10, 11] n-nonane [12] n-decane [12] n-undecane [12] n-dodecane [12] tridecane [12] tetradecane [12] methyldecane [12] hexene [14, 17] octene [12] C 8 C 14 [12] C 11 H 18 [12] tridecene [12] limonene [12] α-copaene [12] α-murolene [12] α-farnesene [12] 1,3-hexadien-5-yne [14] 3,5-diethyl-1,5hexadiene (2 isomers) [15] 3-ethyl-1,5 octadiene (2 isomers) known as pentene dimers [15] 3,7-decadiene (3 isomers) known as pentene dimers [15] benzene [12] ethylbenzene [12, 17] diethylbenzene [12] trimethylbenzene [12] tetramethylbenzene [12] propylbenzene [12] isopropylbenzene [12] xilene [12] stirene [12] naftalene [11] ethylnaftalene [11] dimethylnaftalene [11] acenaftene [11] compounds, C 5 alcohols and pentene dimers contribute to the virgin olive oil aroma [15]. A greater number of volatile compounds is present in the aroma of the virgin olive oils of worse categories. In those oils the concentrations of C 6 and C 5 compounds are quite lower than those detected in high quality oils or those compounds are even completely absent. At the same time C 7 -C 11 monounsaturated aldehydes [19, 24], or C 6 - C 9 dienals [16], or C 5 branched aldehydes [25] or some C 8 ketones [18] become important contributors to the oil aroma, they are responsible for negative attributes (defects), such as rancid, winey-vinegary, fusty, muddy sediment, musty. Gas chromatographic profiles of a virgin

4 642 Angerosa Eur. J. Lipid Sci. Technol. 104 (2002) compounds of high quality olive oils depends closely on the levels of enzymes involved in the pathways and on their activity. Genetic characteristics fix the contents of the different enzymes and are therefore responsible for the qualitative composition of volatile compounds [26]. Instead the quantitative accumulation of the different volatile compounds is connected with the enzyme activities which, in oils of good quality, are related to the ripening degree of fruits [27, 28] and on the operative conditions used during the oil extraction [29-33]. Other courses concerning the degradation of raw material will be followed when fruits show unsanitary conditions or are unsuitably stored before their processing. Fig. 2 briefly summarises the main pathways involved in the volatile production and depicts which compounds originate from each of them. Nevertheless, it has to be remembered that in addition to the volatile compounds deriving from the mentioned pathways, also other compounds, especially aldehydes derived from autoxidation processes can contribute to the aroma of the olive oils. Fig. 1. Gas chromatographic profiles of both a good quality virgin olive oil (A) and an oil showing musty defect (B). Peaks: 1 = octane; 2 = acetone; 3 = 1-octene; 4 = ethyl acetate; 5 = methanol; 6 = 2-methyl butanal; 7 = 3-methyl butanal; 8 = ethanol; 9 = ethyl 3-methyl butanoate 10 = pentan-3-one; 11 = pentene dimer; 12 = methyl 2-methyl butanoate; 13 = pentene dimer; 14 = 2-methyl propyl acetate; 15 = methyl 3-methyl butanoate; 16 = 1-penten-3- one; 17 = propan-1-ol; 18 = ethyl 2-methyl butanoate; 19 = ethyl 3-methyl butanoate; 20 = pentene dimer; 21 = pentene dimer; 22 = pentene dimer + hexanal; 23 = 2- methyl propan-1-ol; 24 = 3-methyl butyl acetate; 25 = 2- pentenal; 26 = 1-penten-3-ol; 27 = limonene; 28 = 3- methyl butan-1-ol; 29 = trans-2-hexenal; 30 = unknown; 31 = hexan-2-ol; 32 = pentan-1-ol; 33 = octan-3-one; 34 = hexyl acetate; 35 = heptan-3-ol; 36 = cis-3-hexenyl acetate; 37 = cis-2-penten-1-ol; 38 = hexan-1-ol; 39 = cis-3- hexen-1-ol; 40 = trans-2-hexen-1-ol; 41 = 1-octen-3-ol; 42 = heptan-1-ol; 43 = acetic acid; 44 = 2-methyl propanoic acid; 45 = 3-methyl butanoic acid; i.s. = nonan-1-ol (internal standard). olive oil without defects and an oil showing musty defect are depicted in Fig Biogenesis of virgin olive oil volatile compounds Volatiles in the vegetable kingdom can be considered to be metabolites of intracellular biogenetic pathways. The qualitative and quantitative composition of the volatile Tab. 6. Miscellany of volatile compounds identified in virgin olive oil aroma by MS. Compound Ref Acids acetic acid [13, 20] 2-methyl propanoic acid [18] 3-methyl butanoic acid [18] propanoic acid [18] butanoic acid [16] hexanoic acid [16] heptanoic acid [16] Furane derivatives 2-propylfuran (two isomers) [11] 2-propyl dihydrofuran [11] 2-pentyl-3-methylfuran [11] ethylfuran [14] 3-(4-methyl-3-pentenyl)furan [14] Ethers methoxybenzene (anisole) [10, 11] 1,2-dimetoxybenzene (veratrole) [10] Thiophene derivatives [11] 2-isopropenylthiophene [11] 2-ethyl-5-hexylthiophene [11] 2,5-dithiophene [11] 2-ethyl-5-hexyldihydrothiophene [11] 2-octyl-5-methylthiophene [11] Oxygenates terpenes linalool [10] α-terpineol [10, 11] lavandulol [11] 1,8-cineole [10]

5 Eur. J. Lipid Sci. Technol. 104 (2002) Volatile compounds and sensorial analysis 643 Fig. 2. The main pathways involved in the production of the volatile compounds of virgin olive oil aromas. The different nuances of the aroma of virgin olive oils are related to the importance of the various pathways that contribute to their formation. The aroma of the oil will not be defective when the most active pathway is the LOX cascade. On the other hand, the aroma of the oil will be defective, if some of the volatile compounds derive from fermentations or amino acid conversion or from enzymatic activities of moulds or from oxidative processes. 4 Volatile compounds analysis: The analytical approach to their evaluation The volatile fraction of the virgin olive oils consists of many compounds differing in molecular weights and chemical nature. Their concentrations, except for trans-2- hexenal, are generally very low, they can vary widely and reach minimum levels of about a hundred ppb or less. The analytical approach to their evaluation must solve the problem of determining compounds at trace levels, avoid the formation of artefacts and achieve rapid and reliable methods for the identification and the quantitation of chemical compounds responsible for different aromas. Generally, several steps are requested for the quantitative determination and the following identification of volatiles by the methods developed: the separation of the volatile fraction, its possible concentration, fractionation into the individual components and, finally, their identification. The fractionation of the individual components is performed by high resolution gas chromatography (HRGC) and their identification by GC-MS. All the steps are very important [34, 35] and, therefore, they should be specified carefully and observed closely to obtain comparable data. The methods reported in the literature for the collection of volatiles can be divided into two groups: techniques with or without an enrichment step. They were reviewed carefully by Morales and Tsimidou in 2000 [9] and the reader is referred to these papers for deepening the argument. Tab. 7 shows the different techniques for the quantification of the volatile compounds, briefly underlines the main problems of each technique, and reports references about the applications to olive oils [2, 9, 10, 14, 17-21, 24-28, 30-33, 36-39, 41, 48-51]. The techniques without an enrichment step are not commonly used because the amount of the sample is in any case too small to achieve a good sensitivity. Furthermore, in general, high temperatures are requested which may favour the formation of degradation products. Moreover, applying the direct injection memory effects in the chromatograph represent a further disadvantage; a static headspace, on the other hand, can be considered effective only for highly volatile compounds [9]. Among the techniques with an enrichment step the most popular one is the extraction of volatiles by means of a dynamic headspace. The volatile compounds are trapped on a suitable adsorbent and then the quantitative amount

6 644 Angerosa Eur. J. Lipid Sci. Technol. 104 (2002) Tab. 7. Most common techniques for the quantification of olive oil aroma volatiles and their main problems. Technique Sensitivity Problems References (olive oil) Techniques not involving an enrichment step Direct injection poor possible degradation products [9, 36] Static head space poor concentrations very low, often under [17, 37-41] gas cromatographic detectability thresholds [9] Techniques involving an enrichment step Direct extraction with solvent impossible to be applied Simple distillation not applied Combined distillation- good significant and selective lacks of solute due [10] extraction to co-evaporation during the removal of considerable amounts of solvent and concentration of impurities of solvent [9] Dynamic head space very good temperature, sample size, absolute quantity [2, 14, 18, 19, 24-28, of gas used for the stripping, chemical-physical 30-33, 37-39, 48] characteristics of both substances to be extracted and material employed for the trapping, of the length and diameter of the trap [42-47] Supercritical fluid quite good selective against the oxygenate compounds [49, 50] extraction (SFE) with low and medium molecular weights and also against many organic apolar compounds with low molecular weight Solid phase microextraction quite good extraction of a lower number of compounds [51] (SPME) than in both static and dynamic head-space. The major differences concern volatile compounds with lower molecular weight. Stable isotope dilution assay very good synthesis of a number of deutered volatile [20, 21] (SIDA) compounds of each individual volatile compounds is determined by HRGC after thermic desorption or elution with a solvent. In that case, the amount of volatile compounds depends on temperature, sample size, absolute quantity of the gas used for the stripping, chemical-physical characteristics of both substances to be extracted and material employed for the trapping, length as well as the diameter of the trap [42-48]. A special comment needs to be made on the technique known as stable isotope dilution assay (SIDA), a method which allows to quantify the volatile compounds very accurately (Tab. 7). SIDA methodology involves the adding of the deuterated compounds of all odorants to be analysed as internal standards. Due to the structural similarity of analyte and corresponding deuterated compound, problems related to losses during the sample preparation, to reactivity and chromatographic behaviour of the various analytes are overcome splendidly. However, this method requires the preparation of a number of deuterated compounds and, even if it is very accurate, it is not commonly used for quantifying all volatile compounds of virgin olive oils. Rather they are used to identify compounds considered to be the most potent contributors to olive oil aroma [20, 21]. In Tab. 8 the characteristics of the different adsorbents used for trapping volatile compounds are shown and references concerning the main applications to olive oils are reported [2-4, 9, 12, 14, 18, 19, 24, 25, 27, 30-33, 41, 42, 52-54, 56-59]. 5 Volatile compounds analysis: The sensory approach to their evaluation Volatile compounds, being responsible for most sensory properties of virgin olive oils, play a significant role in the evaluation of the overall oil quality and in the generation of preferences among consumers. Several investigations were carried out to find relationships between volatile compounds and sensory perceptions, but the results are not comprehensive enough to describe all the sensations experienced during tasting

7 Eur. J. Lipid Sci. Technol. 104 (2002) Volatile compounds and sensorial analysis 645 Tab. 8. Characteristics of the adsorbents used for trapping volatile compounds. Adsorbent Sensitivity Problems References (olive oil) Poropak good low thermal stability and production of artefacts [52-54] [42] Chromosorb very good good adsorption capacity of medium and high boiling [41] point compounds, the high thermal stability, poor affinity against water, easy cleaning procedures and possible recycling [9, 52] Tenax very good good adsorption capacity of medium and high boiling [2, 4, 14, 27, 32,48, 56] point compounds, higher thermal stability than Chromosorb, poor affinity against water, easy cleaning procedures and possible recycling [9, 52] Charcoal very good very strong adsorbent power against all classes of [3,12,18,19, 24,25, chemical compounds, ability of adsorbing compounds 30-33, 57-59] with low molecular weights and good affinity against water [9, 52] Tab. 9. Recovery [%] of C 5 and C 6 compounds, respectively, from virgin olive oils [3, 15, 65]. The confidence limits for recoveries at a probability level of 95% were obtained by using the standard deviation values calculated from four independent experiments. C 5 Compound Recovery C 6 Compound Recovery [%] [%] Hydrocarbons pentene dimers (C 10 H 18 ) 48.0 ± 0.5 Carbonyl compounds Carbonyl compounds 2-pentenal 36.8 ± 0.8 hexanal 21.6 ± 0.6 trans-2-hexenal 25.0 ± penten-3-one 80.6 ± 3.0 Alcohols Alcohols cis-2-penten-1-ol 31.0 ± 0.6 hexan-1-ol 20.2 ± 0.1 trans-2-penten-1-ol 37.6 ± 0.5 cis-2-hexen-1-ol 17.0 ± penten-3-ol 64.2 ± 2.0 trans-2-hexen-1-ol 18.1 ± 0.2 cis-3-hexen-1-ol 23.3 ± 0.7 trans-3-hexen-1-ol 24.0 ± 3.0 Esters hexyl acetate 11.3 ± 0.5 cis-3-hexenyl acetate 11.9 ± 0.4 trans-2-hexenyl acetate 10.5 ± 0.3 completely [9]. It should be remembered that the volatile compounds present at higher concentrations are not always the main contributors to the oil aroma [59]. The reason being that the thresholds the minimum concentration of a given stimulus able to give rise to a sensory response for taste and smell sense organs depend more on chemical factors and the stereochemical structure of the molecules than on their concentrations [60, 61]. An effective help in estimating the importance of a flavour compound provides the aroma extract dilution analysis (AE- DA). AEDA is based upon the calculation of the ratio of a compound concentration to its flavour threshold, the latter being evaluated nasally and retronasally [62]. However, the chemical determination of the volatile compounds, all of which contribute to the olive oil aroma, and of non volatile phenolic compounds, which are mainly responsible for taste and trigeminal sensations, does not render an account of its flavour. That is so because aroma, taste and trigeminal sensations contribute to sensory perceptions as well as complex interactions between these stimuli [63, 64]. On the other hand, the recovery of volatile compounds from virgin olive oils during their quantitative

8 646 Angerosa Eur. J. Lipid Sci. Technol. 104 (2002) determination (Tab. 9) is related to the number of carbon atoms in the molecule and to the kind and position of the functional group [3, 65]. Therefore the sensory analysis is still the most effective tool to evaluate quality, strength and differences among the stimuli elicited by the virgin olive oil during tasting and to investigate consumers preferences. A common perplexity about the sensory evaluation of virgin olive oils is represented by the fact that each taster judges the organoleptic characteristics in a subjective way. This was true until the evaluation of the sensory attributes was made exclusively by one or a few very specialised persons, who were able to define the quality characteristics and the defects of oils. Their judgement was still subjective, since each person has his/her own sensory threshold for each stimulus and may have considerable problems in describing the personal olfactory perceptions. The latter are based on personal previous experiences, and refer to an individual scale for every sensory note. For these reasons quantitative evaluations by different tasters are not comparable. The quantitative descriptive analysis (QDA) overcomes (i) the problem of the different individual thresholds among persons since the final judgement is the mean of judgements provided by a group of persons selected to represent the totality of consumers and fully-trained to evaluate a given product organoleptically; (ii) the difficulty in defining the sensory sensations by developing a vocabulary that allows all tasters to describe the different perceptions experienced during the tasting with the same words; (iii) the use of not fixed scales by adopting a defined scale to which all tasters must refer. This approach allows to compare the scores of different tasters and different group of tasters. Collaborative International studies, supported for many years by the International Olive Oil Council (IOOC), developed the QDA sensory methodology for virgin olive oils, known as COI-Panel test. The latter defines an agreed-on specific vocabulary of sensory attributes, performs a uniform tasting technique and eliminates all troubles that can compromise the sensory trial [66]. A group of persons, from 8 to 12, is selected in a codified way and trained suitably to identify and measure the strength of the different positive and negative sensations elicited by their sense organs. They use a defined structured scale for measuring the intensities of the different attributes. Since tasters are considered as measure instruments, it is absolutely essential to remove or, in any case, to minimise all troubles that can compromise the sensory trial. With this aim possible mental or physical stress of the tasters must be attentively considered since they can modify taste perception [67]. The physical environmental conditions have been carefully regulated and the room for the sensory trials must be organised with a number of booths where the taster can sit separately and concentrate during the tasting. Shape and dimensions of the glass, sample volume, and oil temperature are precisely established. Samples, never in a high number to avoid the sense organ fatigue, are presented in an anonymous and random way. Tasters until August 2002 also rated the overall grading (Fig. 3) for the characteristics of the olive oil on a 9-point scale (9 for exceptional qualities, 1 for the worst one). The mean score was considered as a measure of the oil quality and identified its grade in relation to its Fig. 3. Profile sheet and grading table adopted by European Union regulations until August 2002.

9 Eur. J. Lipid Sci. Technol. 104 (2002) Volatile compounds and sensorial analysis 647 Tab. 10. Scores (overall grading) for each category of virgin olive oil according to the old EU regulation. Scores Categories 6.5 Extra-virgin olive oil < Virgin olive oil < Ordinary virgin olive oil < 3.5 Lampante virgin olive oil quality. Statistical procedures were applied to evaluate the data provided by assessors and produced results that, due to their significance levels, could be considered as reliable as those of other methods usually adopted in scientific fields. Since 1991 this methodology had been included in the regulations of the European Union [68] for the classification of various virgin olive oil categories (Tab. 10) and was extensively described in the Annex II of the EU regulation (Reg. CEE n.2568/91). However, a poor reproducibility of the overall grading scores [69] was observed among different panels. The differences in the evaluations of the different attributes among the assessors of the various panels were mainly related to troubles deriving from an ineffective training, due to the absence of standards which can be reproduced because of the poor stability of the oil over time. Moreover, they were attributable to dissimilar origins, culture, and food habits [70]. Therefore the International Olive Oil Council promoted the revision of the organoleptic evaluation of the virgin olive oil and a new methodology was developed. A new profile sheet was proposed (Fig. 4) that mainly considers the negative attributes (e.g., fusty, musty, muddy sediment, winey-vinegary, metallic and rancid notes) that are the most commonly detectable ones in virgin olive oils [71]. Possible other defects described in the specific vocabulary can be named by means of designation to others. The profile sheet indicates only fruity, bitter and pungent sensations among positive notes. An unstructured scale, 100 mm long, was chosen to overcome the problems deriving from the fact that the amplitude of all intervals of the old structured scale (Fig. 3) could not be considered equally by tasters and to the reluctance of tasters to use the extreme portions of the structured scales [72]. The intensity data, expressed as centimetres, are statistically processed to calculate the median of each negative and positive attribute. The median value of the defect perceived with the strongest intensity identifies the olive oil grade, whereas the median value of the fruity attribute identifies the extra virgin and virgin types (Tab. 11). The method has been included recently in EU regulation (Reg. n. 796/02) [73]. The reliability of panel assessors is measured by the robust coefficient of variation that should be 20% for the median of defects in extra virgin, virgin, and ordinary grades and 10% in lampante. For the fruity median it should be 10% in extra virgin and virgin categories. 6 Flavours: Factors affecting the composition of the olive oil volatile fraction The fragrant and unique aroma of virgin olive oils of good quality is usually described by perceptions attributable to 1) the fruity sensation, the sensation reminiscent of healthy fresh fruit collected at the optimum of the harvesting time; 2) the sensations reminiscent of leaves, freshly cut grass, green fruits such as apple, banana or vegetables such as artichoke or tomato etc., accompanied by more or less intense taste notes of bitterness and pungency. C 6 and C 5 aromatic volatile compounds are mainly responsible for the green perceptions of the fragrant and unique aroma of virgin olive oils [23, 57], whereas bitterness and pungency have to be mainly attributed to secoiridoid compounds [9, 74, 75]. The perceptions indicated under point 2) are known as green odour notes, and characterise the flavour of oils extracted from not completely ripe olives. They are regarded as freshness and liveliness characteristics of good quality virgin olive oils by consumers. Tab. 11. Median values of defects and fruity aroma, and their corresponding robust coefficients of variation, in relation to virgin olive oil categories according to the new EU regulation. Median of defects Robust coefficient Median of fruity Robust coefficient Olive oil of variation [%] aroma of variation [%] category 0 20 > 0 10 Extra virgin > > 0 10 Virgin > >0 Ordinary > > 0 Lampante

10 648 Angerosa Eur. J. Lipid Sci. Technol. 104 (2002) Fig. 4. Profile sheet actual adopted by EU regulation. Oils from unripe fruits are characterised by quite intense green perceptions and by very high strengths of bitter and pungent attributes. A suitable blending with oils weaker from a sensory point of view make them acceptable for direct consumption. Conversely oils obtained from ripe fruits are lightly aromatic because of a low accumulation of volatile compounds that confer a typically fresh and herbal flavour, due to a reduced activity of enzymes involved in the lipoxygenase pathway [29, 58, 74-78]. They are also characterised by weak intensities of bitter and pungent perceptions due to a decreasing amount of phenolic compounds during the ripening of fruits [29]. The whole of both fruity attribute and the green sensations describe the different nuances of the aroma of virgin olive oils. Incorporating leaves in concentrations of about 2-3% prior to crushing enhances the flavour of the oils from overripe fruits and improves their quality [79]. The aroma is not distorted and tasters perceive higher intensities of green fruity and bitter taste. The analysis of volatile compounds in fact [79] does not show newly formed substances (Fig. 5), but a noticeable increase in C 6 aldehydes and C 6 alcohols that, as known, are related to the green attributes. The production of C 6 and C 5 compounds through the enzymatic oxidation of linolenic and linoleic acids [22, 23, 57] is affected by the cultivar, the degree of ripeness of fruits and by their processing conditions. The cultivar plays an essential role as the amount of the enzymes involved in the pathway is genetically characteristic. Montedoro and his group [17] already highlighted only quantitative differences in the composition of volatile

11 Eur. J. Lipid Sci. Technol. 104 (2002) Volatile compounds and sensorial analysis 649 Fig. 5. Changes of the fruity intensity, of C 6 aldehydes and alcohols with respect to the percentage of leaves added to over-ripe fruits before olive crushing. Fig. 7. Green note profiles of oils from fruits of two Italian cultivars, Provenzale and Leccino, harvested at the same degree of ripeness. Fig. 8. Percentage of the main compounds produced by enzymatic oxidation of α-linolenic acid in oils from fruits of Italian, Spanish and Greek cultivars harvested at the same degree of ripeness. Fig. 6. Percentage of the C 6 aldehydes, alcohols and esters of oils obtained from fruits harvested at the same degree of ripeness and processed in the same operating conditions in relation to cultivars. fractions. Fig. 6 shows the influence of the cultivar on the percentage of C 6 aldehydes, alcohols and esters of oils obtained from fruits of different cultivars harvested at the same degree of ripeness and processed under the same operating conditions [3, 26]. Different sensory green profiles are due to the different concentration of these three C 6 fractions of volatile compounds. As an example Fig. 7 shows profiles of oils from Provenzale and Leccino fruits. Moreover, a recent research [26] proved that, under the same conditions, the amount of the C 6 compounds coming only from the α-linolenic acid is practically the same during two harvesting years. Further, their accumulation in the oil, expressed as percentage of their total amount, is different according to the cultivars regardless of the climatic variables and where olives are grown. That means that the cultivar is a determing factor in the formation of the oil aroma (Fig. 8). The amount of the different metabolites from the LOX cascade, the most important pathway for the formation of the olive oil aroma, changes in relation to the ripening degree and storage time of fruits and the operative conditions used during the oil extraction.

12 650 Angerosa Eur. J. Lipid Sci. Technol. 104 (2002) Fig. 9. Changes of trans-2-hexenal in oils extracted from fruits at different ripening degree, measured by means of the Jaèn index, which is based on the colour of skin and pulp of olive fruits. tant with repercussions on the activity of some enzymes involved in the LOX pathway [77, 78]. The comparison of the volatile profiles [30] of oils from the same fruits extracted with the same processing diagram except for the crushing stage, resulted in concentration changes of some volatile compounds. Oils extracted by means of a stone mill were found to contain a higher amount of total volatile compounds (Fig. 10) and especially of trans-2- hexenal, hexanal and cis-3-hexen-1-ol than corresponding samples obtained by using a metallic crusher. The sensory analysis applied to oils obtained by the different crushing methods underlines the perception of higher intensities of the green attributes in oils extracted with a stone mill than in those obtained by metallic crushers [30]. Montedoro et al. [17, 29] and Solinas et al. [80], examining oils from different Italian cultivars, and Olias et al. [12], examining oils from Spanish varieties, found that the concentration of compounds responsible for the aroma, which is very high in oils from green olives, increases during olive ripeness. The latter was measured by means of the Jaèn index [80], based on the evaluation of the colours of the olive skin and pulp. The amount of volatiles increases until a maximum value is reached when fruits change skin colour from yellow-green to purple. Beyond this stage of ripeness, all researchers agree that the amount of the volatile fraction decreases. This behaviour is mainly affected by the changes in the concentration of trans-2-hexenal. It follows the trend just described (Fig. 9) and has been attributed to the dry climate of the production areas of the fruits [28]. A steady decrease in the concentration of the volatile compounds from the unripe to the over-ripe stages, including trans-2-hexenal, was found by Aparicio and Morales. One exception was the oil from Coratina fruits that, during one of the two crop years of their investigation, showed an increase of the amount of trans-2-hexenal until a maximum concentration, in agreement with other researchers results. However, the final volatile concentration in virgin olive oils depends also on technological aspects and, in particular, on the effectiveness of the grinding of pulp tissues, the temperature reached by the olive paste during crushing, the time and the temperature of malaxation and the kind of system used for oil extraction. Metallic crushers cause the disruption of a greater number of cells containing oil than stone mills, but the temperature of the olive paste, because of the violence of the grinding, rises to about 10 C over the temperature of pastes obtained by using a stone mill [81, 82], concomi- Fig. 10. Influence of the crusher type on the total amount of volatile compounds of oils extracted under the same conditions, except for the crushing stage. But also the paste malaxation causes modifications of volatile compound compositions. The malaxation step, consisting of a low and continuous kneading of olive pastes, is essential to break up the oil/water emulsion and thus to promote the merger of the small oil droplets formed during crushing, in particular by means of metallic crushers, into large drops that can be easily separated through mechanical systems. Time and temperature of malaxation affect the concentration of the volatile compounds and therefore the sensory characteristics of the resulting oils. The malaxation time mainly promotes the accumulation of alcohols and of C 6 and C 5 carbonyl compounds, especially of hexanal, one of the most important contributors to the olive oil flavour because of its low odour threshold [83]. The increase in the concentration of hexan-1-ol and trans-2-hexen-1-ol, the considerable decrease in the concentration of C 6 esters and in cis-3-hexen-1-ol, accompanied by the production of very high amounts of 2-methyl butanal and 3-

13 Eur. J. Lipid Sci. Technol. 104 (2002) Volatile compounds and sensorial analysis 651 Fig. 11. Influence of malaxation time and temperature on the amount of C 6 aldehydes, alcohols and esters. methyl butanal through the activation of the amino acid conversion pathway, represent the major effects due to the high malaxation temperature [32, 84] (Fig. 11). A general weakening of the oil flavour is recorded by tasters, especially for walnut husk and tomato, when the malaxation temperature rises, whereas prolonged periods of malaxation cause the weakening of leaf, freshly cut grass, walnut husk, bitter and pungent sensory notes [33]. Also bitter and pungent notes show the same behaviour according to the reduction of the phenolic compounds during the malaxation step as observed by some researchers [85]. The studies on the influence of the malaxation step on the quality of resulting oils reached the same conclusions; a low temperature ( 25 C) and medium times (35-45 min) are the best extraction conditions to promote the formation of green volatile compounds responsible for desirable perceptions. They also help to avoid high concentrations of some compounds of which production is closely connected with the degradation phenomena of raw material and inversely related to the virgin olive oil sensory quality [32, 33]. The systems adopted for extracting oil have some repercussion on the volatile composition. A lower content of Tab. 12. Influence of the decanter type on the amount of some C 6 compounds (ppm) arising from the lipoxygenase pathway. Decanter type Compound [ppm] dual-phase three-phase 1-penten-3-one penten-3-ol cis-2-penten-1-ol trans-2-hexenal trans-2-hexen-1-ol cis-3-hexen-1-ol cis-3-hexenyl acetate hexan-1-ol volatile compounds was found in oils extracted by means of three-phases centrifugal decanters than in oils extracted by pressure systems. The reduction is especially evident for C 6 alcohols, hexan-1-ol and trans-2-hexen-1-ol, probably removed by the warm water which is used for the dilution of olive pastes in order to facilitate their centrifugation [31, 86]. The introduction of new models of decanters on the market, which are able to separate the oily phase from the malaxated pastes without requiring any addition of warm water, allowed the production of oils whose volatile composition is more similar to that of oils extracted by pressure. In particular, in addition to a greater amount of phenolic substances, oils from dual phases decanter show a greater accumulation of C 6 compounds arising from the LOX pathway [87] (Tab. 12). 7 Origins of off-flavours When oils are of poor quality the sensory basic characteristics are considerably modified. Green, bitter and pungent notes are absent or very weakly perceived by tasters who identify the presence of some unpleasant sensations in the flavour. Tab. 13 summarises the causes of the defects detectable in virgin olive oils. The data show that the olive preservation before processing is the most important cause for the more common defects fusty, winey-vinegary and musty attributes detectable in virgin olive oils. 7.1 Defects from unsuitable conditions of olive fruit preservation The olive fruit preservation, even if carried out in ideal conditions (low temperature and very thin layer of olives in fully-air rooms), causes a decrease in the concentrations of volatile compounds especially of trans-2-hexenal [88]. The amount of the all volatile compounds, expressed as ppm of nonan-1-ol, decreases by about 30-40% in oils obtained from olives preserved for 15 d in relation to that detected in oils from fresh fruits [88]. The decrease becomes more evident for longer preservation times. In parallel with the ob-

14 652 Angerosa Eur. J. Lipid Sci. Technol. 104 (2002) Tab. 13. Causes of main defects perceived in virgin olive oil and description of the resulting flavour. Cause Defect Flavour description Bad sanitary conditions grubby typical of oils obtained from olives which have suffered a Dacus of fruits oleae infestation Wrong harvesting ground picked typical of oils obtained from ground-picked olives, spontaneously procedure olives fallen from trees and remained on the ground for several days Time and conditions of fusty typical of oils obtained from olives stored in piles which suffered fruit storage degradative phenomena winey typical of oils obtained from olives stored in piles which suffered some fermentation musty typical of oils obtained from olives stored in piles which suffered the more or less considerable fungal invasion Unsuitable extraction earth typical of oils obtained from fruits collected with earth or bespattered technology with mud and processed without washing heated typical of oils obtained when too long times or too high temperatures are adopted in the malaxation step metallic typical of oils extracted with both new processing plants and/or used the first time during the crop year Unsuitable oil rancid typical of oils strongly oxidized storage conditions muddy sediment typical of oils stored for a long time on their sediment cucumber typical of oils stored for a long time during the hermetic bottling served reduced amount of the volatile fraction, the scores of evaluation of the oil aromas show only a weakening of the intensity of the different attributes. Beyond 15 d the oil does not keep the original quality, but tasters perceive some defects at threshold level (Tab. 14). However, as the harvesting is done in a few months, the preservation of fruits often cannot be performed in ideal conditions because of the poor size of the processing plants. The reduced areas reserved for olive storage in relation to the fruit amount oblige to put them into jute sacks or to pile them at room temperature. Tab. 14. Scores, total amount of volatile compounds expressed as ppm of nonan-1-ol, their % loss in oils obtained from fruits stocked for different times with respect to the volatile fraction of the oil coming from fresh fruits. Days Panel test Total Loss score of volatile of volatile compounds compounds [ppm] [%] The profile of aromatic volatile compounds is significantly modified during the olive preservation [25, 89, 90]. Compounds from the LOX pathway decrease significantly and rapidly in the oils from the fruits stored in piles for different times. The suitable temperature conditions, the high humidity, and the loss from the epicarp of its ability to act as an antimicrobic barrier, due to an accelerated autolysis of the organic material and the fruit rotting, promote the microbial colonisation of the olive tissues by all the micro-organisms present in the environment [25]. According to the temperature and the degree of humidity reached in the pile, one genus of epiphytic microflora can develop better than another, thus it is possible that the production of different metabolites is responsible for different defects. The preferential growth of yeasts gives rise to the formation of ethanol and ethyl acetate. As a consequence, the winey defect appears when their concentrations are higher than those corresponding to their sensory thresholds. The possible presence of Acetobacter is responsible for the vinegary defect because it promotes the production of acetic acid [25]. Generally, Enterobacteriaceae, Clostridia and Pseudomonas meet with the better conditions for their growth. The accumulation of their metabolites, represented by branched aldehydes, branched alcohols and their corresponding acids [91, 92], is promoted by the contact time and the suitable temperature. In a few days, they over-

15 Eur. J. Lipid Sci. Technol. 104 (2002) Volatile compounds and sensorial analysis 653 step the threshold concentrations for the perception of fusty defect. In particular a significant correlation (R = ) was found between the intensity of the fusty defect and 2-methyl butan-1-ol + 3-methyl butan-1-ol/1 + trans-2-hexenal. Hereby 1 is a mathematical artefact to avoid that the function shows values to infinity which do not have any physical meaning for trans-2-hexenal values near to zero [90]. When the storage time is prolonged for several days, the humidity and temperature conditions encourage the development of moulds, whose pectolytic activity accelerates the complete rotting of fruits. An investigation carried out to isolate and identify micro-organisms present on the olive skin proved that the most moulds belong to Penicillium and Aspergillus species [93]. The enzymes of moulds interfere with those of olive fruit in the LOX pathway [94]. So that there is, parallel to the growth of moulds, a steady decrease in C 6 compounds and, at the same time, an increase in C 8 compounds which are common metabolites of the LOX pathway of moulds. In addition, as expected [95, 96] in parallel to the fungal growth great amounts of propan-1-ol, 2-methyl propan-1-ol, 3-methyl butan-1-ol and their corresponding acids and esters are produced. The musty defect average intensity was positively related (R = ) with the percentage of 1-octen-3-ol in relation to the total amount of C 8 compounds [18] (Fig. 12). The storage temperature plays an essential role in determining how long the preservation time can last without giving rise to the appearance of off-odours. Kiritsakis and co-workers [97] suggest that a storage temperature of about 5 C in air reduces the fungal growth considerably, so that olives can be preserved for at least 30 days. The resulting oil, sensory tested, was found to be still of good quality. The same authors state that storage temperatures near 0 C are detrimental because of the destruction of the natural antioxidants of olive fruits due to chilling injury. Fig. 12. Relationship between musty defect intensity and percent of 1-octen-3-ol with respect to total amount of C 8 compounds. 7.2 Defects from the oil preservation Other more common defects of virgin olive oils, such as muddy sediment, rancid, cucumber, originate during oil preservation. The oxidation is an inevitable process that starts after the virgin olive oil has been extracted and leads to a deterioration that always becomes more serious during oil storage. Initially lipids are radically oxidised to hydroperoxides, which are odourless and tasteless [98] and do not account for sensory changes. However, they are susceptible to further oxidation or decomposition into products of secondary reactions, which, conversely, are responsible for typical unpleasant sensory characteristics, identified on their whole as rancid attribute. Decomposition occurs through a homolytic cleavage of the hydroperoxide group with production of various compounds, including aldehydes, ketones, acids, alcohols, hydrocarbons, lactones, furans and esters [99]. Light, temperature, metals, pigments, unsaturated fatty acid composition, quantity and kind of natural antioxidants, as well as the amount of sterols promote the free radical mechanism of the autoxidation process in a different way [9, 99]. During the oil preservation the original volatile composition, mainly formed by compounds deriving from the LOX pathway responsible for pleasant properties, changes. The concentrations of C 6 aldehydes, especially that of trans-2-hexenal, and C 6 alcohols undergo a drastic reduction, whereas those of new compounds, C 5 -C 11 saturated and unsaturated aldehydes, increase gradually [24, 100]. The most advanced oxidation stages are characterised by the complete disappearance of compounds arising from the LOX cascade and by very high concentrations of the mentioned aldehydes [24]. They contribute mainly to undesirable aromas, because of their low odour thresholds [101]. Other contributors are represented by unsaturated hydrocarbons, furans and ketones. Unsaturated aldehydes and ketones can be further oxidised producing new off-flavour compounds, whose presence accounts for the different nuances of the unpleasant aromas described by tasters as rancid, painty, fishy, etc. [102]. Snyder et al. [103] observed that the volatile fraction of oxidised oils consisted mainly of saturated carbonyl compounds. Hexanal is always present in the flavour of good quality oils since it is formed through the LOX cascade. But its amount, because of the strict specificity displayed by some enzymes involved in its production [77], is rather low as compared with that of trans-2-hexenal, whose formation is promoted. However, Morales and his group [104] could not consider the considerable increase in the concentration of hexanal in oxidised oils, especially in