**2.1 Key points in obtaining a high quality VOO**

Processing of healthy olives:

When the common olive fly (*Bactrocera oleae*) attacks olives (from the beginning of summer to the start of harvesting), damage occurs as a result of larval growth: oils from damaged fruits show changes in both volatile and phenolic compounds that influence negatively the sensory properties and oxidative stability of the product, especially during oil storage (polar phenols have a fundamental role as antioxidants during storage). The bad taste due to these changes caused by the olive fly is well known as a *grubby defect* (Angerosa et al., 1992; Gómez-Caravaca et al. 2008).

In order to obtain a high quality olive oil, it is necessary to process olives that are not overripe. The use of fruits that have partially degraded tissues cause an increase in enzymatic and microrganism activities and oxidative reactions; therefore the produced oil probably will be characterized by an higher free acidity and perceivable sensory defects. When olives are accumulated in piles for many days, the high temperature and humidity inside the mass promotes proliferation of bacteria, yeasts and moulds, producing undesirable fermentation and degradation that give rise to specific volatile molecules responsible for unpleasant odours (i.e. winey, fusty and mouldy).

Winey, the typical pungent sensory note perceptible in oils produced by olives stored in piles or in jute sacks for several days, arises from alcoholic fermentation: *Lactobacillus* and *Acetobacter* have been detected in olives inducing fermentative processes. The main microorganism found in olives depends on the length of storage: at the beginning the enterobacteriaceae genera *Aerobacter* and *Escherichia* prevail, while *Pseudomonas*, *Clostridium* and *Serratia* are predominant after longer periods of time. The activity of these microorganisms results in the presence of low concentrations of biosynthetic volatiles and large amounts of compounds such as the branched alcohols due to degradation of amino acids that lead to the typical undesirable sensory note known as fusty (Angerosa, 2002; Morales et al 2005). The most abundant deuteromycetes found in olives stored at high humidity are several species of the genus *Aspergillus* together with ascomycetes *Penicillium*; these organisms oxidize free fatty acids producing mainly methyl ketones, in contrast to yeasts of the genera *Candida*, *Saccharomyces* and *Pichia* which are able to reduce carbonylic compounds. Enzymes from these microorganisms interfere with the LOX pathway to produce volatile C8 molecules characterized by very low odour thresholds, and reduce some C6 compounds. This volatile profile is responsible for the musty defect of EVOO.

Selection of the most suitable milling conditions

The phenolic content is greatly influenced by this technological step. In general, the use of the more violent crushing systems (i. e. with hammers instead of blades) causes an increase

aspects that determine the level of enzymes in fruit (Angerosa et al., 1999) that are involved in synthesis of volatile molecules (LOX pathway) and phenol compounds (biosynthetic

Even if enzymatic activity depends on the stage of ripeness (Morales et al., 1996; Aparicio & Morales 1998) agronomic (fertilization, irrigation) and climatic (temperature and rainfall)

When the common olive fly (*Bactrocera oleae*) attacks olives (from the beginning of summer to the start of harvesting), damage occurs as a result of larval growth: oils from damaged fruits show changes in both volatile and phenolic compounds that influence negatively the sensory properties and oxidative stability of the product, especially during oil storage (polar phenols have a fundamental role as antioxidants during storage). The bad taste due to these changes caused by the olive fly is well known as a *grubby defect* (Angerosa et al., 1992;

In order to obtain a high quality olive oil, it is necessary to process olives that are not overripe. The use of fruits that have partially degraded tissues cause an increase in enzymatic and microrganism activities and oxidative reactions; therefore the produced oil probably will be characterized by an higher free acidity and perceivable sensory defects. When olives are accumulated in piles for many days, the high temperature and humidity inside the mass promotes proliferation of bacteria, yeasts and moulds, producing undesirable fermentation and degradation that give rise to specific volatile molecules

Winey, the typical pungent sensory note perceptible in oils produced by olives stored in piles or in jute sacks for several days, arises from alcoholic fermentation: *Lactobacillus* and *Acetobacter* have been detected in olives inducing fermentative processes. The main microorganism found in olives depends on the length of storage: at the beginning the enterobacteriaceae genera *Aerobacter* and *Escherichia* prevail, while *Pseudomonas*, *Clostridium* and *Serratia* are predominant after longer periods of time. The activity of these microorganisms results in the presence of low concentrations of biosynthetic volatiles and large amounts of compounds such as the branched alcohols due to degradation of amino acids that lead to the typical undesirable sensory note known as fusty (Angerosa, 2002; Morales et al 2005). The most abundant deuteromycetes found in olives stored at high humidity are several species of the genus *Aspergillus* together with ascomycetes *Penicillium*; these organisms oxidize free fatty acids producing mainly methyl ketones, in contrast to yeasts of the genera *Candida*, *Saccharomyces* and *Pichia* which are able to reduce carbonylic compounds. Enzymes from these microorganisms interfere with the LOX pathway to produce volatile C8 molecules characterized by very low odour thresholds, and reduce some

C6 compounds. This volatile profile is responsible for the musty defect of EVOO.

The phenolic content is greatly influenced by this technological step. In general, the use of the more violent crushing systems (i. e. with hammers instead of blades) causes an increase

Selection of the most suitable milling conditions

responsible for unpleasant odours (i.e. winey, fusty and mouldy).

pathways via PPO and -glucosidase) present in VOOs.

**2.1 Key points in obtaining a high quality VOO** 

conditions also play an important role.

Processing of healthy olives:

Gómez-Caravaca et al. 2008).

in extraction of phenolic compounds due to more intense tissue breaking; therefore, a more vigorous milling system should be used to process olive varieties that are naturally low in phenolic compounds, and permit enrichment of bitter and pungency intensities. The use of more violent milling systems also produces a significant increase in olive paste temperature and a corresponding decrease of the activity of enzymes that play a key role in the production of volatile compounds responsible for fruity and other green notes (Salas & Sanchez, 1999; Servili et al., 2002).

Concerning the malaxation phase, which consists in a slow kneading of the olive paste, the time-temperature pair should be carefully controlled to obtain a high quality EVOO. The lipoxygenase pathway is triggered by milling of olives and is active during malaxation. The volatile compounds produced are incorporated into the oil phase to confer its characteristic aroma. Specifically, a temperature above 28°C for more than 45 min should be avoided; in fact, these conditions can lead to the deactivation of enzymes that produce both positive volatile compounds and oxidize the phenolic compounds causing changes in oil flavour (Salas & Sanchez 1999; Kalua et al., 2007). The reduced concentration of oxygen in paste, obtained by replacing air with nitrogen in the headspace of malaxer during processing, can inhibit these enzymes and minimize the oxidative degradation of phenolic compounds during processing (Servili et al., 1999; Servili et al., 2003). Malaxation under erroneous conditions is responsible for the unpleasant flavor known as a "*heated defect*" due to the formation of specific volatile compounds (Angerosa et al., 2004).

The application of different oil separation systems

One of the main disadvantages of discontinuous mill systems is the possible fermentation and/or degradation phenomena of residues of pulp and vegetation waters on filtering diaphragms; these reactions give rise to a defect termed "*pressing mats*", but also promote winey and fusty defects (Angerosa et al., 2004). It is well known that among continuous systems, discontinuous mill systems with a three-phase decanter need lukewarm water to dilute olive paste in contrast to a two-phase decanter, which has two exits producing oil and pomace and separates the oil phase from the olive paste This latter system has advantages in terms of water reduction and major transfer of phenols from the olive paste to the oil, with a consequent increase in oxidative stability, bitterness and pungency.

The amount of water added determines the dilution of the aqueous phase and lowers the concentration of phenolic substances that are more soluble in vegetable waste water. Consequently, a large amount of antioxidants is lost with the wastewater during processing. In addition to phenolic compounds, some volatile compounds accumulate more in oil from a dual-phase decanter than in oils extracted with three-phase decanters. Therefore, the use of a two-phase decanter promotes greater accumulation of volatile and phenolic compounds that are not lost in the additional water as in a three phase decanter. The higher concentrations of these compounds are related to the high intensities of bitter, pungent, green fruity, freshly cut lawn, almond and tomato perceptions (Angerosa et al., 2000; Angerosa et al., 2004; Kalua et al., 2007).

Storage of oil under suitable conditions

In unfiltered oil, the low amounts of sugars or proteins that remain for extended times in oil can be fermented or degraded by specific anaerobic microrganisms of the *Clostridium* genus,

Sensory Analysis of Virgin Olive Oil 115

experts of IOOC. In addition, the official method (IOOC/T.20/Doc.5/rev.1 and IOOC/T.20/Doc.14/rev.2) includes precise recording of the correct tasting temperature, as well as the dimensions and colour of the tasting glass and characteristics of the test room.

The panel leader is the person responsible for selecting, training and monitoring tasters to ascertain their level of aptitude according to (IOOC/T.20/Doc.14/rev.2). The number of candidates is generally greater than that needed in order to select people that have a grater sensitivity and discrimination capability. Screening criteria of candidates are founded on sensory capacity, but also on some personal characteristics of candidates. Given this, the panel leader will personally interview a large number of candidates to become familiar with their personality and understand habits, hobbies, and interest in the food field. He uses this information to screen candidates and rejects those who show little interest, are not readily

The determination of the detection threshold of the group of candidates for characteristic attributes is necessary because the "threshold concentration" is a point of reference common to a "normal group" and may be used to form homogeneous panels on the basis of

A selection of tasters is made by the intensity rating method, as described by Gutiérrez Rosales (Gutiérrez Rosales et al., 1984). A series of 12 samples is prepared by diluting a VOO characterized by a very high intensity of a given attribute in an odourless and tasteless medium (refined oil or paraffin). The panel leader sends out the candidate, removes one of the 12 tasting glasses from the series, and places the remaining together; the candidate is called back in the room and is asked to correctly replace the testing glass withdrawn from the series by comparing the intensity of this last with that of the others. The test is carried out for fusty, rancid, winey and bitter attributes to verify the discriminating capacity of the

The stage training of assessors is necessary to familiarize tasters with the specific sensory methodology, to heighten individual skill in recognizing, identifying and quantifying the sensory attributes and to improve sensitivity and retention with regards to the various attributes considered, so that the end result is precise and consistent. In addition, they learn

The maintenance of the panel is made through continuous training over all duration of life of the same panel, the check of the sensory acuity of tasters, and exercises that allow the

Every year, all panels must assess a number of reference samples in order to verify the reliability of the results obtained and to harmonize the perception criteria; they must also update the Member State on their activity and on composition changes of their group.

A method for the organoleptic evaluation of olive oils was introduced in the Regulation (EEC) No 2568/91, Annex XII, that is inspired by the COl/T.20/Doc. no.15, published in 1987. In the profile sheet of EEC Reg. 2568/91, a number of positive attributes and defects

available or who are incapable of expressing themselves clearly.

olfactory-gustatory sensitivity.

to use a profile sheet.

candidate on the entire scale of intensities.

measurement of the panel performance.

**3.1 Evolution of sensory methodology: From old to new** 

were evaluated, giving each a score from 0 to 5 (Figure 1).

producing volatile compounds responsible for an unpleasant muddy odour by butyric fermentation. The filtration of newly-produced oil can avoid this phenomenon. It is known (Fregapane et al., 2006; Mendez & Falque, 2007; Lozano-Sanchez et al., 2010) that EVOO has a low amount of water, and for this reason it can be considered as a water-in-oil emulsion (Koidis et al., 2008)

The orientation of phenolic compounds in the oil-water interface and the active surface of water droplets can protect against the oxidation of oil. According to some researchers (Tsimidou et al., 2004; Gómez-Caravaca et al., 2007), the stability of unfiltered samples is significantly higher than that of the corresponding filtered oils. This coincides with a higher total phenolic content in unfiltered oils due to a greater amount of emulsified water. On the other hand, higher water levels are expected to favour enzymatic catalysis, including lipase, lipoxygenase and polyphenol oxidase activities. Thus, a more rapid oxidation of unfiltered oil is expected. Some authors (Montedoro et al., 1993) observed that hydrolytic processes occurr in parallel with oxidation during long term storage.

Lipid oxidation is an inevitable process that begins immediately after oil extraction and leads to a deterioration that becomes increasingly problematic during oil storage. The presence of a rancid defect, typical off-flavour for the fatty matrices, can be avoided or substantially slowed. The most advanced oxidation stages are characterized by the complete disappearance of compounds arising from the LOX cascade and by very high concentrations of saturated and unsaturated aldehydes together with unsaturated hydrocarbons, furans and ketones that contribute mainly to the rancid defect because of their low odour thresholds (Guth & Grosch, 1990; Morales et al., 1997; Bendini et al., 2009). To avoid the rancid perception, it is fundamental to control factors that promote lipid oxidation. These include a decrease in the availability of oxygen, the protection of the oil from light and storage at a temperature of 12-14°C. Before bottling, it is advisable to maintain the oil in stainless steel tanks under an inert gas such as nitrogen equipped with devices that periodically eliminate sediments from the bottom of the tank.
