3

Factors Affecting Extra-Virgin Olive

Oil Composition

Paolo IngleseDipartimento di Colture Arboree, Universit�a degli Studi di Palermo,Viale delle Scienze, 90128, Palermo, Italy

Franco FamianiDipartimento di Scienze Agrarie e Ambientali, Universit�a degli Studi diPerugia, Borgo XX Giugno, 06121, Perugia, Italy

Fabio GalvanoDipartimento di Chimica Biologica, Chimica Medica e BiologiaMolecolare, Universit�a degli Studi di Catania, 95125, Catania, Italy

Maurizio Servili, Sonia Esposto and Stefania UrbaniDipartimento di Scienze Economico-estimative e degli Alimenti,Sezione di Tecnologie e Biotecnologie degli Alimenti, Universit�a degliStudi di Perugia, 06121, Perugia, Italy

ABBREVIATIONS

I. THE CONCEPT OF OLIVE OIL QUALITY

II. EVOO COMPOSITION AND NUTRITIONAL PROPERTIES

III. SOURCES OF VARIABILITY OF EVOO COMPOSITION AND PROPERTIES

IV. AGRONOMICAL AND ENVIRONMENTAL FACTORS AFFECTING EVOO

COMPOSITION AND QUALITY

A. Genotype

B. Growing Area and Seasonal Conditions

C. Tree Water Status

D. Productivity and Alternate Bearing

E. Orchard Management

1. Cultivation Method

2. Training System and Pruning

3. Fertilization and Soil Management

83

Horticultural Reviews, Volume 38 Edited by Jules JanickCopyright � 2011 Wiley-Blackwell.

4. Pest and Disease Control

F. Fruit Ripening and Harvest

1. Ripening

2. Harvest Time and Production Objectives

3. Harvesting Systems

V. TECHNOLOGICAL FACTORS AFFECTING EVOO COMPOSITION AND QUALITY

A. Olive Fruit Storage

B. Olive Fruit Crushing

C. Olive Paste Malaxation

D. EVOO Extraction Systems

E. EVOO Storage

VI. SUMMARY AND CONCLUSIONS

LITERATURE CITED

ABBREVIATIONS

CVD cardiovascular diseaseEVOO extra-virgin olive oilHDL high-density lipoproteinLDLA low-density lipoproteinLOX lypoxygenaseMUFA monounsaturated fatty acidVOO virgin olive oilPDO protected designation of originPOD protected origin of denominationPPO polyphenyloxidase

Uvapendet in vitibus, et oliva in arboribus. . . et necuva vinumest, nec olivaoleum, ante pressuram. [Grape droops in grapevine and olives in the olivetree. . . but neither grapes arewine, nor olives are oil, until they are pressed.]—Agostino Enarrationes in Psamos 83, 1, 22, 16–20

I. THE CONCEPT OF OLIVE OIL QUALITY

The oil of the fruit of the olive (Olea europaea L.) is the cornerstone of theMediterraneandiet (Keys 1980). This product represents a heritage at thevery center of the civilization, history, religion, and economy of allcountries surrounding the Mediterranean Sea, which Braudel (1986)referred to as the �sea of the olive trees.� In antiquity, the principal use of

84 P. INGLESE ET AL.

olive oil was for illumination. Although oil lamps are no longer used,olive oil is still important for nonfood uses, such as cosmetics and bodycare. However, as a result of the extraordinary nutraceutical propertiesand alimentary value of extra-virgin olive oil (Keys 1980; Bendiniet al. 2007), consumption and trading is increasing worldwide, evenin countries with relatively low production, such as Argentina,Australia, Chile, New Zealand, South Africa, and the United States.According to the international regulations and trade standards of theInternational Olive Council (COI/T.15/NC no. 3/Rev. 3, Nov. 2008),olive oil is the oil obtained solely from the fruit of the olive tree, to theexclusion of oils obtained using solvents or re-esterification processes,and of anymixture with oils of other kinds. Before pressing the olives toextract the oil, the Romans, upon a very slight pressure, obtained fromthe fruit a liquid of watery consistency dark in color, bitter to the taste,called amurca (amorgh by the Greeks), which was used as a manure aswell as for various purposes in the domestic and agricultural economy.Nowadays, among other designations, we can distinguish extra-virginolive oil and virgin olive oil. The extra-virgin olive oil (EVOO), which isa marketable class of oil extracted from the olive fruit using onlymechanical extraction process, can be consumed directly as crude oil,without any additional physical or chemical treatments, other thanwashing, decantation, centrifugation, and filtration. EVOO must berelatively low in free acidity (lower that 0.8% expressed in oleic acid),with low peroxide number (lower that 20meq O2/kg). Virgin olive oil(VOO) is a second marketable class of olive oil extracted using the samemechanical process but characterized by higher free acidity (between0.8% and 2.0%).

Up to now, the marketable quality of EVOO has not included para-meters that are important in determining the health and sensory char-acteristics of the oil. In fact, several markers, such as phenolic andtocopherols composition, are not considered to define the EVOO mar-ketable class. In the last few decades, many aspects related to EVOOorganoleptic properties and nutritional quality have been intenselyinvestigated, with the ultimate goal of distinguishing the product andincreasing its commercial value. EVOO composition and propertiesgreatly changes with the peculiar characteristics of the different geno-types (cultivar) and their interaction with environmental conditions,orchard management, and oil extraction technologies. As a result of thisdifferentiation of EVOO quality, the awareness and the preferences ofnew and traditional consumers are rapidly changing in relation to itsdifferent uses in cooking and gastronomy. At the present time, EVOO isperceived as a functional food rather than as a simple dressing for salads

3. FACTORS AFFECTING EXTRA-VIRGIN OLIVE OIL COMPOSITION 85

or cooking oil. Different reviews have been published on the flavor andvolatile components of olive oil (Kiritsakis 1998; Kalua et al., 2007) aswell as on its functional properties (Newmark 1997; Fito et al. 2007) andon the relation between olive oil extractionmethods and its final quality(Uceda et al. 2006). This chapter presents and discusses the sources ofvariability of olive oil composition and quality, such as genotype,environment, cultural, and technological factors, with particular em-phasis given to aspects that may play an important role in the furtherdevelopment of the olive oil industry.

II. EVOO COMPOSITION AND NUTRITIONAL PROPERTIES

EVOOis themain sourceof edible lipids andoneof the staple foodsof theMediterraneandiet. In comparison toother vegetable oils, EVOOmust beconsidered a unique dietary food due to its fatty acid composition, suchas the prevalent presence of the monounsaturated oleic acid (C18:1) andits hydrophilic compound content, such as phenolic alcohols and acids,flavonoids, lignans, and secoiridoids. Evidence from an increasingnumber of scientific studies attribute the health properties of EVOO toits peculiar fatty acid composition, which confers consistent beneficialeffects onhumanhealth, particularly in the prevention of cardiovasculardiseases (Keys 1980). The fatty acid fraction accounts for not less than98% of the oil components and is characterized by a relative low level ofpolyunsaturated fatty acids and a high level of monounsaturated fattyacids (MUFA). Triglycerides account for 98% to 99% of total fatty acidcomposition, with diglycerides accounting for 1% to 1.5% and mono-glycerides less than 1%. Almost 99% of the fatty acid fraction iscomposed of: saturated palmitic (C16:0; 7.5–20%) and stearic fatty acids(C18:0; 0.5–3.5%); monounsaturated palmitoleic (C16:1; 0.3–3.5%) andoleic fatty acids (C18:1; 56.0–85.0%); and polyunsaturated linoleic(C18:2; 3.5–20%) and linolenic fatty acids (C18:3; 0–1.5%) that are notsynthesized by humans (Montedoro et al. 2003). Total MUFA content ofEVOO is 70–80 g/100 g, a valuemuch higher than those of vegetable oils,such as canola (59 g/100 g), peanut (46 g/100 g), sunflower (32 g/100 g),corn (29 g/100 g), soybean (24 g/100 g), and safflower (14 g/100 g)(Nicklas et al. 2004).

In contrast to all other vegetable oils, are obtained from seeds, EVOO isobtained from a fruit (drupe). As in other fruits, olives contain a pool ofcompounds that although quantitatively ofminor importance (about 2%of EVOO�s weight) show important biological properties. Indeed, EVOOcontains more than 230 chemical compounds, including the triterpene

86 P. INGLESE ET AL.

hydrocarbon squalene, the phytosterol b-sitosterol, and a pool of anti-oxidant polyphenols such as tyrosol, hydroxytyrosol, secoiridoids, andlignans that importantly contribute to its distinctive flavor due to thetypical pungent and bitter taste properties but also to its chemicalstability in terms of both shelf life and resistance to lipid oxidationduring cooking (Servili et al. 2009a). Phenols of EVOObelong todifferentchemical classes, such as phenolic acids, phenolic alcohols, flavonoids,secoiridoids, and lignans. Secoiridoids, which include aglycon deriva-tives from oleuropein, demethyloleuropein, and ligstroside, found ex-clusively in the Oleaceae, are the most abundant phenolic antioxidantsof EVOO (Servili et al. 2004). The concentration of secoiridoides andlignans in EVOO is highly variable as it largely depends on agronomicfactors, fruit ripening stage at harvest, and oil extraction techniques.Eventually, the total phenols concentration of an EVOOmay range from20 to 900mgkg�1 (Montedoro e Garofolo 1984; Servili et al. 2007).The natural antioxidants of EVOO are lipophilic and hydrophilic

polyphenols and carotenes (Boskou 1996). Carotenes, which includelutein as the main component, violaxanthin, and b-carotene, can befound in small amounts in EVOO, with concentrations, expressed astotal carotenes, between 5 and 24mgkg�1 (Servili et al. 2004). Thelipophilic phenols including tocopherols and tocotrienols that do notoccur in EVOO can be found in other vegetable oils. In EVOO,more than90% of total tocopherols are represented by a-tocopherol, which showshigh variation according to soil and climatic conditions and agronomicfactors, such as area of origin, cultivar, and fruit ripening stage (Serviliet al. 2009b).

EVOO contains different classes of hydrophilic phenols (Table 3.1,Figs. 3.1, 3.2) (Boskou 1996; Shahidi 1996). Phenolic acids, representedby caffeic, vanillic, syringic, p-coumaric, o-coumaric, protocatechuic,sinapic, p-hydroxybenzoic, and gallic acid, were the first group ofphenols discovered in EVOO (Montedoro et al. 1992a; Tsimidouet al. 1996; Servili et al. 2004). Phenolic alcohols include (3,4-dihydrox-yphenyl) ethanol (3,4-DHPEA) and (p-hydroxyphenyl) ethanol (p-HPEA). Their concentration is generally low in fresh oils but increasesduring oil storage (Montedoro et al. 1992a) as a result of the hydrolysis ofEVOO secoiridoids, such as 3,4-DHPEA-EDA, p-HPEA-EDA, and 3,4-DHPEA-EA, which contain 3,4-DHPEA and p-HPEA in their molecularstructures (Fig. 3.1) (Brenes et al. 2001). Flavonoids, such as luteolinand apigenin, were also reported as phenolic components of EVOO(Rovellini et al. 1997). The lignans include (þ )-1-acetoxypinoresinoland (þ )-1-pinoresinol (Fig. 3.2) (Brenes et al. 2000; Owen et al. 2000).Technological parameters of oil extraction process have a marginal

3. FACTORS AFFECTING EXTRA-VIRGIN OLIVE OIL COMPOSITION 87

impact on their concentration (Servili et al. 2004). Other phenoliccompounds, including oleuropein glucoside (with a range of 5–60mgkg�1), were found in EVOO as minor components. Secoiridoids arethe main compounds of EVOO and include the dialdehydic form ofdecarboxymethyl elenolic acid linked to 3,4-DHPEA or p-HPEA(3,4-DHPEA-EDA or p-HPEA-EDA), an isomer of oleuropein aglycon(3,4-DHPEA-EA), and the ligstroside aglycon (p-HPEA-EA) (Montedoroet al. 1992a,b, 1993; Angerosa et al. 1996a; Owen et al. 2000) (Fig. 3.1).These substances, aglycon derivatives of secoiridoid glucosides con-tained in the olive fruit, originate during oil mechanical extractionprocess by the enzymatichydrolysis of oleuropein, demethyloleuropein,and ligstroside, catalyzed by the endogenous b-glucosidase (Montedoroet al. 2002; Gonz�alez-Pombo et al. 2008). High amount of phenols also

Table 3.1. Phenolic composition of an olive fruit. Source: Servili et al. 2004, 2009a.

Anthocyanins Phenolic alcoholsCyanidin-3-glucoside (3,4 Dihydroxiphenil) ethanol

(3,4-DHPEA)Cyanidin-3-rutinoside

(p-Hydroxyphenyl) ethanol (p-HPEA)Cyanidin-3-caffeyglucoside

Cyanidin-3-caffeylrutinoside

Delphinidin 3-rhamosylglucoside-7-xyloside Secoiridoids

Oleuropein

Flavonols Demethyloleuropein

Quercetin-3-rutinoside Ligstroside

N€uzhenide

FlavonesLuteolin-7-glucoside

Luteolin-5-glucoside LignansApigenin-7-glucoside (þ )-1-Acetoxypinoresinol

(þ )-Pinoresinol

Phenolic acidsClorogenic acid Hydroxycinnamic acid derivativesCaffeic acid Verbascoside

p-Hidroxybenzoic acid

Protocatechuic acid

Vanillic acid

Syringic acid

p-Cumaric acid

o-Cumaric acid

Ferulic acid

Sinapic acid

Benzoic acid

Cinnamic acid

Gallic acid

88 P. INGLESE ET AL.

can be found in olive fruit, mainly in the pulp, where the total phenolscan range between 1% and 3%of fresh pulpweight (Garrido et al. 1997).Oleuropein, demethyloleuropein, ligstroside, and n€uzhenide are themost abundant secoiridoid glucosides in the olive fruit (Gariboldi

OCOOCH3

O

OH

LIGSTROSIDE AGLYCON

( p-HPEA-EA)

OLEUROPEIN AGLYCON

(3,4-DHPEA-EA)

DIALDIALDEHYDIC FORM OF

DECARBOXYMETHYL

ELENOLIC ACID LINKED TO p-HPEA

( p-HPEA-EDA) = OLEOCHANTAL

DIALDIALDEHYDIC FORM OF DECARBOXYMETHYL

ENOLIC ACID LINKED TO 3,4 -HPEA

( 3,4 DHPEA-EDA)

( 3,4-DIHYDROXYPHENYL) ETHANOL

( 3,4-DHPEA)

(p-HYDROXYPHENYL) ETHANOL

(p-HPEA)

HO O3′ 3′

3′

2′ 2′7′ 7′

2′ 7′8′ 8′

8′

3 34 47 7

7

6

6′ 6′

6′6

9 95 5

5

1 1

1′

1′1′

1′

1

8 810 10

4′ 4′5′

3′

2′ 7′8′

34

7

6

6′

9

8

5

4′5′

5′

3′

2′ 7′8′

34

6′

6

91

1′

8

10

4′5′

4′5′

HOO

O

O

O

OH

3′

2′ 7′8′

6′

1′

4′5′

OH

HO

HO

OCOOCH3

O

O

OHO HOO

O

O

O

HO

HO

HO

Fig. 3.1. Chemical structures of secoiridoids derivatives and phenyl alcohols of EVOO.

Source: Servili et al. 2004, 2009.

O

O

H

O

OH

OCCH3

OHO

4″ 3″

6″ 1″

5″ 2″

4″ 3″

6″

1

2

5

4

6 8

1

2

5

4

6 8

1″

5″ 2″

1′ 2′

5′ 4′

6′ 3′

1′ 2′

5′ 4′

6′ 3′

CH3

O

O

O

H

O

OH

H

OHO

OH

CH

(+)-1-PINORESIONAL

3

CH3CH3

Fig. 3.2. Chemical structures of lignans found in olives and in EVOO. Source: Servili

et al. 2004, 2009.

3. FACTORS AFFECTING EXTRA-VIRGIN OLIVE OIL COMPOSITION 89

et al. 1986; Garrido et al. 1997; Servili et al. 1999b) (Table 3.1),which alsocontains, as main phenolic compounds, the verbascoside in two iso-meric forms (Ryan and Robards 1998; Servili et al. 1999a). Sensoryproperties of EVOO are largely affected by phenolic content, which isresponsible for its �bitter� and �pungent� flavor (Morales et al. 2000).

Volatile compounds are responsible for EVOO aroma and flavor. Morethan 180 volatile compounds have been described in EVOO (Angerosaet al. 2004). It has been demonstrated that the relationship between the�fruitiness� note in olive oil and the presence of aldehydes and C5–C6

saturated and unsaturated alcohols originate from the enzymes involvedin the lipoxygenase pathway during olive crushing and following ma-laxation (slowly mixing of the olive paste after crushing) (Moraleset al. 1999; Angerosa et al. 2004). The use of the statistical sensorywheelas an appropriate method to relate volatile compounds and sensory datawas clearly demonstrated, and the aroma notes of 32 virgin oil samplesfrom three Mediterranean countries corresponded well to olive oilvolatile compounds (Aparicio et al. 1996).

The volatile fraction of EVOO is mainly composed by carbonylcompounds, alcohols, esters, and hydrocarbons (Flath et al. 1973;Angerosa 2000). However, the typical aroma of an EVOO rises fromseveral volatile compounds, which are responsible for fragrances de-scribed by these attributes: �fruity,� �cut grass,� �tomato leaf,� �tomato,��artichoke,� �walnut husk,� �apple,� or other fruits. The C6 and C5 sub-stances, especially C6 linear unsaturated and saturated aldheydes andalcohols, represent themost important fractionof the volatile compoundsthat were associated to several EVOO sensory notes, such as �fruity,� �cutgrass,� and �tomato leaf� (Angerosa et al. 2004; Servili et al. 2009b).

From a quantitative point of view, C6 and C5 compounds (Vick andZimmermann 1987; Hatanaka 1993; Angerosa et al. 1998a; Aparicio andMorales 1998), in particular C6 linear unsaturated and saturated alde-hydes, are the most important volatile substances of high-qualityEVOOs, whereas other neo-formation volatile compounds, namelyC7–C11 monounsaturated aldehydes (Solinas et al. 1987, 1988), C6–C10

dienals (Aparicio et al. 2000), C5 branched aldehydes and alcohols(Angerosa et al. 1996b), and some C8 ketones (Angerosa et al. 1999a),reach high concentrations in the aroma of EVOOs affected by organo-leptic defects.

C6 and C5 compounds are produced from polyunsaturated fatty acidsby the lipoxygenase (LOX) pathway, and their concentrations depend onthe level and the activity of each enzyme involved (Montedoro andGarofolo, 1984; Angerosa and Di Giacinto 1995; Di Giovacchinoand Serraiocco 1995; Morales et al. 1996; Aparicio and Morales 1998;

90 P. INGLESE ET AL.

Angerosa et al. 1998a;Morales et al. 1999;Angerosa et al. 2001;Angerosaand Basti 2003; Angerosa et al. 2004).

The LOX pathway (Fig. 3.3) starts with the production of 9- and 13-hydroperoxides of linoleic (LA) and linolenic (LnA) acids mediated byLOX. Very specific hydroperoxide lyases (HPL) catalyze the subsequentcleavage of 13-hydroperoxides and lead to C6 aldehydes, whose unsat-urated onesQ1 can isomerize from Z-3 to the more stable E-2 form. Themediation of alcohol dehydrogenase (ADH) reduces C6 aldehydes tocorresponding alcohols, which can produce esters because of thecatalytic activity of alcohol acetyl transferases (AAT) (Vick andZimmermann 1987; Hatanaka 1993).

A collateral byway of the LOX pathway (Fig. 3.3) is active when thesubstrate is LnA. LOX would also catalyze the formation of stabilized1,3-pentene radicals. These compounds could dimerize leading to C10

hydrocarbons (known as pentene dimers) or couple with a hydroxyradical present in the medium producing C5 alcohols, which canbe enzymatically oxidated to corresponding C5 carbonyl compounds(Angerosa et al. 1998b).

The pathways involved in the EVOO aroma production are shown inFig. 3.4. (The size of arrows gives an idea of the importance of each path.)In high-quality EVOOs, the LOX pathway predominates, while in oils

LA

13-hydroperoxides

hexanal hexan -1-ol hexyl acetate

trans-2-hexen -1-ol

cis-3-hexenyl acetate

trans-2-hexenal

cis-3-hexen -1-ol

cis-3-hexenal

13-alkoxy radical

pentene radical

pentene dimers 2-penten -1-ol1-penten -3-ol

2-pentenal1-penten -3-one

LnA

LOX HPL

ADH AAT

AAT

ADH

ADH

Isomerase

Fig. 3.3. Lipoxygenase (LOX) pathways involved in the production of EVOO C6 and C5

volatile compounds. Source: Angerosa et al. 2004; Servili et al. 2009.

3. FACTORS AFFECTING EXTRA-VIRGIN OLIVE OIL COMPOSITION 91

with sensory defects, other pathways occur that result in disagreeablearomas (Solinas et al. 1987; Solinas et al. 1988; Angerosa et al. 1996b;Aparicio et al. 2000; Morales et al. 2000; Garc�ıa-Gonzales et al. 2002a,2000b;Morales et al., 2005). The volatile fractionQ2 represent an importantmarker for the genetic and geographical origin of EVOO (Angerosaet al. 2004; Servili et al. 2009b).

Clinical and epidemiological evidence of the health properties ofEVOO are constantly increasing. As a result, a plethora of reviews areavailable emphasizing the ability of macro- and micro-components ofEVOO to reduce the risk of a number of chronic degenerative maladies,such as cardiovascular diseases (CVD) (Covas 2008; Huang andSumpio 2008), atherosclerosis (Covas 2008), obesity and metabolicsyndrome (Soriguer et al. 2007; Babio et al. 2009), Parkinson�s disease(Perez-Jimenez et al. 2005), Alzheimer�s disease (Pasinetti and Eber-stein 2008), some types of cancer (Escrich et al. 2007; La Vecchia 2009),insulin sensitivity and diabetes (Schr€oder 2007; Tierney andRoche 2007), nonalcoholic fatty liver disease (Assy et al. 2009), andinflammatory diseases (Patrick and Uzick 2001; Sales et al. 2009).

The ability of EVOO to reduce the risk of CVD has to be linkedprimarily to a series of beneficial health effects on the atheroscleroticand thrombotic pathways, including lipid oxidation, hemostasis, plate-let aggregation, coagulation, and fibrinolysis. Both oleic acid and thepolyphenols seem to exert antiatherosclerotic effects jointly. Plasma

Virgin Olive Oil

LOX pathway

Volatile Compounds Fatty acid metabolism

AutoxidationSugar fermentation

Conversion of amino acids

Homolytic cleavage ofI3-hydroperoxides

Fig. 3.4. The enzymatic and chemical paths involved in the production of EVOO volatile

compounds. Source: Angerosa et al. 2004; Servili et al. 2009.

92 P. INGLESE ET AL.

lipoproteins are carriers of plasmatic cholesterol, and their ratio is afundamental factor in the onset and development of atherosclerosis andCVD. Due to their high susceptibility to oxidation, low-density lipopro-teins (LDL) are a well-acknowledged risk factor of CVD, as their oxida-tion is a crucial step in the progress of atherogenic process. In contrast,high-density lipoproteins (HDL) are a protective factor, due to theirability to remove cholesterol from arteries and carry it to the liver. Forthis reason, the HDL/LDL ratio is considered a reliable marker of CVDrisk. Oleic acid and, particularly, EVOO�s antioxidant polyphenols areefficacious in reducing the susceptibility of LDL to oxidation (Ciceraleet al. 2009). Besides, EVOO is able to reduce the circulating levels of LDLand to increase those of HDL. However, EVOO�s beneficial effects are notexclusively linked to the plasma lipoprotein balance but also to thereductionofplasma triglycerides and total cholesterol and to thepositivemodulation of endothelial and platelet function (Cicerale et al. 2009).

EVOO�s consumption also provides protection against the risk of sometypes of cancer. Recently, La Vecchia (2009) reviewed literature dataobtained from a series of case control studies conducted in Italy andcomprising over 20,000 cases affecting 20 cancer sites. The authorconcluded that olive oil consumption is associated with reductions inthe risk of breast and colorectal cancer as well as of upper digestive tractneoplasms (i.e., oral/pharyngeal and laryngeal neoplasms, and esoph-ageal cancer). Additional evidence arises from a number of laboratorystudies suggesting that minor components of EVOO have the ability tomitigate the initiation, promotion, and progression of the multistagecarcinogenesis process. Interestingly, the anticarcinogenic properties ofEVOO�s polyphenols are not related per se to itsQ3 antioxidant ability butrather to theirQ4 capacity to induce cell differentiation, inhibit cell cycleprogression, and exert antiproliferative effects (Eschrich et al. 2007;Corona et al. 2009).

Squalene is thefirstminor compoundof EVOO indicated as apotentialanticancer agent (Newmark 1997). Animal and in vitro studies putforward a protective mechanism likely due to the remarkable squaleneability to inhibit the activity of beta-hydroxy-beta-methylglutaryl-CoAreductase, leading to reduced farnesyl pyrophosphate availability forprenylation of the ras oncogene (Soutirodis 2003; Owen et al. 2004).However, it must be emphasized that clinical confirmations of theanticarcinogenic properties of squalene are still lacking (Sotiroudis andKyrtopoulos 2008).

Numerous cell culture studies highlighted the ability of EVOOto modulate the expression and activity of some oncogenes, playinga crucial role in the initiation and progress of tumorigenesis and

3. FACTORS AFFECTING EXTRA-VIRGIN OLIVE OIL COMPOSITION 93

metastasis. In this regard, the modulation of HER2, an oncogene dra-matically overexpressed in breast cancer cells, was suggested as theprotective mechanism induced by oleoeuroperin aglycone (Menendezet al. 2008). Besides the modulation of oncogenes, in human promye-locytic HL60 leukemia cells EVOO�s polyphenols appear able to reducecell growth and to promote apoptosis, thus inhibiting cell proliferation(Fabiani et al. 2006). Antiproliferative properties of hydroxytyrosol,through inhibition of ERK1/2 and cyclin D1, have been also reportedinhumancolonadenocarcinomacells (Coronaet al. 2009). It is a fact that,analogously to what was mentioned earlier for squalene, the EVOO�spolyphenols anticancer properties have been observed exclusively inlaboratory studies; thus clinical confirmation is needed. It is remarkablethat the anticancer properties of EVOO are not attributable solely topolyphenols. Indeed, as accurately described by Menendez andLupu (2006), the regulation of the amount and/or activity of diversetranscription factors is the underlying mechanism by which oleic acidcan interact with the human genome.

Mounting epidemiological evidence indicates a favorable effect ofEVOO on obesity, type II diabetes, and metabolic syndrome(Schr€oder 2007). De Ferranti and Mozaffarian (2008) efficaciously de-fined the vicious circle linking obesity, oxidative stress, inflammation,and metabolic disorders as the perfect storm. Hence, it is conceivablethat the putative protection provided by EVOO is due to its antioxidantand antinflammatory properties. Adipose tissue produces a number ofproinflammatory factors, such as tumor necrosis factor (TNF-a), inter-leukin (IL) 6, and IL-1, which have been demonstrated to play animportant role in the onset of the major obesity-related comorbidities.Vassiliou et al. (2009) reported that oleic acid can counteract the negativeeffects of inflammatory cytokines by reversing their inhibitory effect ininsulin production. In a human study, Jim�enez-Gomez et al. (2009)observed that consumption of an olive oil–based meal elicits low post-prandial expression of proinflammatory cytokine. Beauchamp et al.(2005) attributed to the EVOO�s dialdehydic form of deacetoxy-ligstro-side aglycone (also called oleocanthal) the ability to inhibit the cyclo-oxygenase enzymes COX-1 and COX-2, thus exerting a pharmacologicalanti-inflammatory action comparable to that of the structurally similardrug ibuprofen.Hydroxytyrosolwas also attributedof anti-inflammatoryproperties by reducing TNF-alpha and COX-2 (Zhang et al. 2009).

Like other fat sources, EVOO is an energy-dense food. However,epidemiological studies have demonstrated that EVOO consumption isnot associated with increased body weight (Schr€oder 2007). A physio-logical explanation of why EVOO consumption is less prone to promote

94 P. INGLESE ET AL.

weight gain could be that oleic acid is oxidized more easily thansaturated fatty acids. Moreover, administration of EVOO promotedpostprandial fat oxidation and diet-induced thermogenesis in abdom-inally obesewomen (Soares et al. 2004). Recent data froman interventiontrial revealed that increases in dietary palmitic acid decreased fatoxidation and daily energy expenditure, whereas oleic acid had theopposite effect (Kien et al. 2005). Also in this regard, polyphenols seemto contribute importantly. Indeed, additional evidence arises from lab-oratory study reporting that EVOO�s polyphenols upregulates in rats theexpression and the activity of uncoupling proteins (Rodr�ıguezet al., 2002; Oi-Kano et al. 2007, 2008), which are able to increase heatproduction in brown adipose tissue and muscles. EVOO compositionand nutritional properties have also been correlated with genotype andgeographical origin (Galvano et al. 2007; Mineo et al. 2007).

III. SOURCES OF VARIABILITY OF EVOO COMPOSITIONAND PROPERTIES

Vetustas oleo taedium adfert, no item ut vino, plurimumque aetatis annuoest. [Aging affects oil butmore thanwine and it can last for nomore than oneyear.]—Pliny the Elder, Naturalis Historiae XV, 7

Oil accumulation in olive fruits starts toward the end of the pit-hard-ening stage and becomes very rapid from 9 to 17 weeks after fruit set(Tombesi 1994). The basic pattern of oil accumulation is linear duringmost of the fruit development period and until peel color breakage;however, the oil accumulation pattern may change considerably underlimiting growing contitions, such as major water stress or highly com-petitive fruit growth (Lavee andWodner 1991, 2004; Tombesi 1994). Oilcontent and percent yield in the olive fruit is genetically determinedand depend on cultural and environmental conditions (Lavee andWodner 1991). The oil accumulation rate pattern also depends ongenotype (Lavee and Wodner 1991; Fiorino e Ottanelli 2004), seasonalenvironmental conditions and tree water status (Lavee and Wod-ner 1991), and sink-source relationships related to the amount of cropload (Barone et al. 1994; Lavee and Wodner 2004). At full black fruitripening stage, the relative oil content in themesocarp is not yield or fruitsize dependent, but differences may exist in the oil accumulation ratepattern (Lavee and Wodner 2004). The amount of oil produced by each

3. FACTORS AFFECTING EXTRA-VIRGIN OLIVE OIL COMPOSITION 95

fruit of the same cultivar is regulated by the size of themesocarp (Ingleseet al. 1996; Lavee and Wodner 2004). Differences in fruit size betweencultivars often have no relation to the relative yield and the absolute oilcontent in the fruit. During the fruit maturation process, a number ofphysical and biochemical changes occurwithin the pericarp. The rate ofthese changes varies with the cultivar and the growing conditions andinclude the change in peel color, fruit water content, and the profiles ofthe fatty acid fraction, phenols, tocopherols, chlorophylls, and volatilecompounds. All these changes account for large differences in thesensory profile as well as in the oxidative stability and nutritional valueof the oil (Baccouri et al. 2006).

Olive oil composition in fruits of any specific cultivar results from avery complex multivariate interaction between the genotypic potentialand the environmental, agronomic, and technological factors that char-acterize fruit growth and ripening as well as oil extraction and storage(Montedoro and Garofolo, 1984; Lavee and Wodner 1991). In terms ofrelative content, the individual components of the fatty acid fraction aswell as theminor components may range independently and dependingon factors that are not always interrelated. This creates a �field ofindetermination� (Fiorino and Nizzi Grifi 1991) that makes it difficultto find markers to consistently identify the geographical area of originand the genetic inheritance of any EVOO. However, the analytical andsensory profiles of most of the EVOOs produced by the most importantcultivars worldwide have been largely described, and an acceptablelevel of probability can be reached using combined information on fattyacid composition and minor compound, analyzed with specific statis-tical analysis (Mannina et al. 2003).

The range of variability of the individual fatty acid content in EVOOofdifferent genotypes appears similar to the variation induced, within agenotype, by factors such as fruit ripening stage at harvest (Fiorino andOttanelli 2004) or seasonal environmental conditions during fruitgrowth and ripening (Lombardo et al. 2008; Ripa et al. 2008). Use ofthe fatty acid fraction as a potential marker of the geographical andgenetic origin of olive oil has been proposed, whether in terms ofabsolute content of its individual components or considering therelative oleic/linoleic or oleic/palmiticþ linoleic ratios (Fiorino andAlessandri 1996; D�Imperio et al. 2007). Indeed, fruit characteristicsvarywith location and between years in the same orchard. Even the fruitpopulation in the olive tree is highly variable in terms of size, flesh/pitratio, ripening, and oil accumulation rate pattern, due to the prolongedbloom and fruit set period, within-tree different source-sink relation-ships and environmental (i.e., light availability) conditions (Lavee and

96 P. INGLESE ET AL.

Wodner 2004). However, the range of variability of pomological char-acteristics does not necessarily imply a similar range in oil relative andabsolute yield and composition (Lavee and Wodner 2004) since fruitgrowth and the oil accumulation rate patterns are only partially inter-related (Lavee and Wodner 1991). From this point of view, all factorsaffecting the genotypic fruit growth potential (size and flesh/pit ratio)may not play a similar role on oil composition. However, the fruitripening stage at harvest time is one of the main sources of variability ofthe composition of the EVOO fatty acid fraction and, even more, ofphenolic and volatile compounds. This means that all the agronomic(crop load, irrigation, pruning) and environmental factors (tempera-tures, soil water content) that influence the oil accumulation ratepattern and the nature of fruit ripening also account for the seasonaland geographical variability of the composition and properties of theEVOO of a specific genotype (Fiorino and Nizzi Grifi 1991; Lavee andWodner 1991, 2004; Gucci and Servili 2006; D�Imperio et al. 2007;Lombardo et al. 2008). Pliny the Elder, in his Naturalis Historiae morethan 20 centuries ago, distinguished the value and the quality of theolive oil according to fruit ripening stage at harvest as follows: oleum exalbis ulivis or oleum acerbum (oil from very clear unripe fruits), oleumviride (greenish oil), oleum maturum (mature oil), and oleum caducum(defective oil). The finest oil was considered the one obtained bypressing fruits before they were fully ripe. Cato, in his De agri cultura,stated: Quam acerbissimus olea oleum facies tam oleum optimum erit(the earlier the ripening stage of the olives the better the oil whichresults from them). Columella, in his De re rustica, recommends thegrower to harvest oleum viride, because of the considerable yield andthe high price.

IV. AGRONOMICAL AND ENVIRONMENTAL FACTORSAFFECTING EVOO COMPOSITION AND QUALITY

A. Genotype

The statement that EVOO quality is genotype dependent is a clear andwell-known concept that now appears self-evident. However, manyfactors may overlap and override genetic potential of a single cultivar.Furthermore,most of the time, commercial olive oil is basedonablendofdifferent cultivars. The great diffusion of Protected Designation ofOrigin (PDO) olive oils, based on Regulation EEC 2081/92 and 2082/92 in the EuropeanCommission countries, is based on the definition and

3. FACTORS AFFECTING EXTRA-VIRGIN OLIVE OIL COMPOSITION 97

regulation of cultivar- and environment-specific analytical and sensoryprofiles. There are more than 30 PDOs in Italy alone. Olive oil PDO isbased on the assumption that EVOO chemical and sensory profile isdependent on the interaction between the genetic potential of thecultivar and the growing conditions, which should result in a typicaland consistent phenotypic expression. Genotype clearly affects theevolution of the fatty acid, phenolic, and volatile fraction as well as oilcolor (Angerosa et al. 2004; Fiorino andOttanelli 2004; Servili et al. 2004;Lombardo et al. 2008) (Tables 3.2, 3.3; Fig. 3.5).

Olive cultivars differ in seasonal crop load, fruit size, flesh/pit ratio,and ripening pattern as well as in their adaptive response to water stressor high temperatures during fruit growth and ripening. The seasonal orenvironmental variability of the olive oil composition of a given cultivardepends on its phenotypic stability (Lavee and Wodner 1991; Salvadoret al. 2001; Sweeney et al. 2002; Fiorino e Ottanelli 2004; D�Imperioet al. 2007; Lombardo et al. 2008). Indeed, more than 1,200 cultivars arecultivated worldwide, but 3 cultivars cover 63% of Spanish production,24 cultivars account for 58% of the olive-cultivated area in Italy, 3cultivars cover more than 90% of the olive area in Greece and Portugal,and a single cultivar represents 97% of the cultivated area in Morocco(Inglese and Famiani 2008). Many cultivars have only a very localdiffusion (Inglese and Famiani 2008). Differences in the linoleic acidcontent up to 500% have been measured between cultivars or seedlingsin the same season or for oils of the same genotype coming fromdifferentgrowing sites,whiledifferences in theoleic content accountedup to42%(between environments) and 57% (between cultivars) (Fiorino andOttanelli 2004; Lombardo et al. 2008).

The fatty acids profile of an EVOO is genotype dependent in terms ofunsaturated/saturated fatty acids ratio or, among the unsaturated ones,in terms of the monounsatured/polyunsatured ratio (Cucurachi 1965;Gouveia 1997; Stefanoudakii et al. 1999a). Leon et al. (2004a) reportedwide ranges of variation for all the fatty acids on progenies deriving fromcrossing programs that were as large or even larger than the rangesreported from the evaluation of olive cultivar collections.

EVOO of cultivars with an early or late ripening pattern (�Moraiolo�and �Leccino�) are also different in terms of fatty acid composition andunsaturated/saturated fatty acids ratio (Servili et al. 1990). The appli-cation of statistical methods, such as the multivariate analysis of vari-ance (MANOVA), principal component analysis (PCA), and the lineardiscriminant analysis (LDA), has demonstrated the usefulness of fattyacids analysis to group monovarietal EVOO (Mannina et al. 2003;D�Imperio et al. 2007).

98 P. INGLESE ET AL.

Table

3.2.

Fattyacid

compositionofextra-virgin

oliveoilsobtainedfrom

severalolivecultivars

cultivatedin

theCatamarcaregion

(Argentina)andin

Italy.Source:Manninaetal.2001.

Fattyacid

composition(%

)

Cultivar

Origin

C16:0

C16:1

C17:0

C17:1

C18:0

C18:1

C18:2

C18:3

C20:0

C20:1

Arbequina

Argentina

20.66

3.69

0.04

0.20

1.53

53.39

18.72

1.16

0.29

0.22

Biancolilla

Argentina

16.31

1.81

0.11

0.19

1.80

70.47

7.34

1.12

0.37

0.31

Italy

11.61

0.52

0.12

0.20

2.23

74.10

9.81

0.69

0.39

0.31

Cerasu

ola

Argentina

13.75

0.51

0.05

0.07

1.87

70.98

10.84

1.12

0.37

0.41

Italy

9.86

0.22

0.02

0.03

2.54

76.83

9.34

0.51

0.36

0.34

Coratina

Argentina

16.29

0.67

0.05

0.08

1.77

71.50

7.99

1.27

0.37

0.35

Italy

12.36

0.51

0.08

0.05

2.1

75.43

7.94

0.72

0.31

0.33

I-77

Argentina

15.34

0.91

0.05

0.08

1.52

70.52

9.54

1.45

0.32

0.25

Italy

9.82

0.50

0.05

0.12

1.58

80.54

5.82

0.70

0.32

0.39

Frantoio

Argentina

17.19

1.65

0.01

0.09

1.63

63.55

14.03

1.23

0.28

0.31

Italy

12.34

1.01

0.01

0.02

1.65

75.77

8.04

0.55

0.29

0.29

Kalamata

Argentina

12.93

1.46

0.04

0.13

1.78

65.79

16.04

1.33

0.22

0.29

Italy

9.87

0.61

0.01

0.56

1.52

78.95

6.56

0.72

0.40

0.52

Leccino

Argentina

17.39

1.16

0.05

0.09

1.71

68.45

9.19

1.43

0.33

0.25

Italy

13.23

1.25

0.01

0.09

1.53

77.96

4.54

0.68

0.28

0.33

Peranzana

Argentina

18.16

1.79

0.02

0.07

2.21

62.57

13.08

1.37

0.36

0.32

Italy

12.27

0.80

0.07

0.11

1.86

76.45

7.21

0.58

0.33

0.28

99

The UV, VIS, and NIRQ5 absorption spectroscopy covering a200–1,700nm spectral range, associated with LDA and PCA showedsignificant correlations with individual fatty acid contents, such as theoleic, the palmitic, and the total content of palmitic þ stearic (Mignani

Table 3.3. Concentrations of phenolic compounds (mg/kg) in extra-virgin olive

oils of different Italian olive cultivars.z Data represent the mean� sd of 10 samples.

Source: Servili et al. 2004.

Phenolic

compoundCultivar

Coratina Moraiolo Frantoio Carolea Leccino

3,4-DHPEAy 1.96�0.30 2.08� 1.79 1.38� 1.42 2.70�2.03 7.94�1.10

p-HPEA 0.89�0.99 0.87� 0.65 0.82� 0.91 0.72�1.11 12.3�1.6

3,4-DHPEA-EDA 382.4� 138.2 340.0� 26.3 154.0� 26.1 268.0� 11.4 67.6�15.5

p-HPEA-EDA 193.2� 65.2 99.8� 61.2 89.8� 7.8 189.6� 89.7 12.5�6.2

3,4-DHPEA-EA 177.5� 92.6 157.1� 84.5 84.1� 103.0 134.5� 56.3 47.2�15.0

Total polyphenols 755.9� 153.1 599.9� 67.1 330.1� 27.3 595.5� 106.5 147.5� 22.5

zOliveswere harvested at the industrial ripening stage andmalaxed at 30�C for 60min and

extracted by pressure on lab scale.y The concentrations of hydrophilic phenols were evaluated by HPLC.

Cultivar

Bo

rgio

na

Co

rreg

gio

lo

Do

lce

Ag

og

ia

Fra

nto

io

Lec

cin

o

Mora

iolo

Neb

bia

Nost

rale

R.

Orb

etan

a

Pic

ciolo

Rai

a

Rai

o

San

Fel

ice

Ten

del

lon

e

Voci

o

Tota

l ch

loro

phy

ll c

onte

nt

(pp

m)

0

20

40

60

80

100

120

140

160

180

200

Fig. 3.5. Total chlorophyll content in extra-virgin olive oils of different cultivars, in

central Italy (Umbria region). Source: Pannelli et al. 2000.

100 P. INGLESE ET AL.

et al. 2006). DNA analysis of the oil also discriminated monovarietalEVOOs (Cresti et al. 1996). Research of appropriatemethods andmarkers(Lain et al. 2004; Marmiroli et al. 2004) is still ongoing. However, noattempts have been made to define the genotype origin of blendedEVOOs, on an analytical basis.

Due to its variability, also in terms of analytical methodology, thephenol fraction is largely ignored by PDO protocols, which usually donot indicateQ6 or indicate very low thresholds (100ppm), generally lessthan the genetic potential of most Italian cultivars (Table 3.3). Phenolcontent and composition vary greatly with genotype and among geno-types, with environmental conditions such as water shortage, and par-ticularly with fruit ripening stage at harvest and the extraction technol-ogies. Nevertheless, the phenolic fraction and the relation betweensecoiridoids and lignans have been proposed as potential markers of thegenetic origin of an EVOO (Servili et al. 2004). Indeed, the variability ofthe EVOO phenolic profile, among other factors, can be related to thegenetic potential (Briante et al. 2002) in terms of range of variation of totalor individual content of polyphenols (Table 3.3) (Lo Curto et al. 2001).

The sensory analysis made by a panel of experts, which is essential inthe evaluation of EVOO, is a powerful tool for the discrimination of thegenetic inheritance of monovarietal and blended EVOOs in terms ofaroma and taste. The flavor notes of �fruitness,� �green,� �herbaceous,��sweet,� �bitter,� �pungent,� as well as �apple,� �almond,� �artichoke,� or�tomato� may characterize different EVOOs, particularly in relation totheir use in gastronomy (Panneli and Alfei 2008). As a matter of fact, theapplication of the panel test for the analysis of the genetic origin ofEVOOS is very often associated with the promotion of nutritional andsensory value in oil marketing,

Cross-breeding programs have been carried out with oil quality beingconsideredoneof themost important objectives. Theseprogramsmade itpossible to obtain new interesting genotypes in terms of oil quality(Guerin et al. 2000; Bellini et al. 2002; Leon et al. 2004, 2008; Baccouriet al. 2007a;Manai et al. 2008; Ripa et al. 2008). Hybridization provided awider range of variability than the original parents in several cases (Leonet al. 2008).

Information on comparison of oil content and composition amongcommercial olive cultivarsOlea europaea var. sylvestrisMiller-Brot andO. cuspidata Wall. is also available (Hannachi et al. 2009). Olive culti-vars had a higher oil content than fruits of oleaster andO. cuspidata fromKenya, but O. cuspidata from Pakistan showed the highest oil content(Gulfraz et al. 2009; Hannachi et al. 2009). Oil from O. cuspidata fromKenya had a relatively low oleic acid content (Hannachi et al. 2009),

3. FACTORS AFFECTING EXTRA-VIRGIN OLIVE OIL COMPOSITION 101

whereas O. cuspidata oils from Pakistan had a fatty acid compositioncomparable to that of oils obtained from commercial olive cultivars,although with a high linolenic acid content (> 1%) (Gulfraz et al. 2009).The fatty acid composition of oils from oleasters is variable, and in somecases they gave oils with less saturated fatty acids and higher oleic acidcontents compared to those obtained from commercial cultivars, such as�Chetoui�, �Chemlali�, and �Gerboui� (Hannachi et al. 2009). Evaluationand selection of wild olives on the basis of fatty acid composition,chlorophylls, carotenoids, tocopherols, phenolic and volatile com-pounds show a wide variability in chemical and aroma characteristicsof oleaster virgin olive oils, with aromatic profiles distinctively differentfrom those of European and Tunisian commercial oils (Baccouriet al. 2007b, 2008a).

B. Growing Area and Seasonal Conditions

Lavee and Wodner 1991 have shown that the genetic control of oilaccumulation rate and pattern acts via cultivar-environment interac-tions. However, seasonal and environmental conditions also affect boththe fatty acid and the insaponifiable fraction of the EVOOs. If the olivesare healthy and processed soon after harvesting (within 24hr), environ-mental conditions donot appear to have any substantial influence on thefree acidity, peroxide number, and UVabsorbencies of the oil, which arenormally within the values that allow the classification of the oils asEVOOs (Pannelli et al. 1990a; Ripa et al. 2008).

The earliest investigations indicate that latitude and altitude modifythe relative proportions of unsaturated and saturated fatty acids(Frezzotti 1934). Higher contents of oleic acid and, consequently, anincrease of the unsaturated/saturated fatty acid ratio move from thewarmer areas in southern Italy to the cooler ones in northern Italy(latitude effects) or from the lower altitudes to the higher ones (altitudeeffects). This response has been observed analyzing oils collected fromthemain areas of olive cultivation in Italy (Vitagliano et al. 1961) or fromthe same cultivars (�Frantoio� and �Coratina�) cultivated in areas atdifferent latitudes (Lotti et al. 1982). Lombardo et al. (2008), withdifferent genotypes grown in the same orchard, and Ripa et al. (2008),with a high number of genotypes cultivated in three different areas inItaly (Basilicata, Calabria, andUmbria regions), determined the relation-ships between temperature and fatty acid composition and demonstrat-ed that season-dependent and site-dependent fluctuations in the fattyacid compositions were related to average degree-days accumulateduntil harvest (Ripa et al. 2008), or from pit hardening to harvesting

102 P. INGLESE ET AL.

(Lombardo et al. 2008). In warm seasons and areas, oils had lowercontents of oleic acid, which are associated with higher contents ofpalmitic and/or linoleic acids (Lombardo et al. 2008; Ripa et al. 2008).Linolenic acid can also be higher under warm conditions (Lombardoet al. 2008) (Table 3.4).A similar behavior can be seen inprevious studiesthat compared the fatty acid composition of oils obtained from the samecultivars cultivated in environments characterized by different temper-ature regimes, such as the hot region of Catamarca (Argentina), and Italy(Mannina et al. 2001), Tuscany (Italy), Saudi Arabia, and Australia(Fiorino 2005), or areas at different altitudes in Chania, Greece (Mousaet al. 1996), or inAndalusia, Spain (PazAguilera et al. 2005). In addition,in cultivar comparative studies carried out in an arid region in Tunisia,most oils showed a decrease in oleic acid percentage and an increase inpalmitic and linoleic acid percentages as compared to those from theiroriginal sites (Zarrouk et al. 2009). In Australia, oils originating fromsouthern cooler areas had significantly higher oleic acid and lowerpolyunsaturated and saturated fatty acids (Ganz et al. 2002). Sweeneyet al. (2002) showed higher levels of oleic acid in oils from southernlatitudes of Australia. However, it is not possible to correlate specificenvironmental thresholdswith consistent genotype behavior in terms ofEVOO fatty acid composition. The environmental effect on fatty acidcomposition changes, indeed, with the different genotype-environmentcombinations. As amatter of fact, EVOOs from the �Carolea� and �Canino�cultivars showed no significant site variations for their fatty acidcompositions (Montedoro et al. 2003), whereas comparisons of the fatty

Table 3.4. Annual variation of the main fatty acids in extra-virgin olive oils

extracted from 68 olive cultivars cultivated in southern Italy. The warmest and the

coldest years were 2003 and 2005, respectively. CV¼ variability coefficient.

Source: Lombardo et al. 2008.

C 16 : 0z C 16 : 1 C 18 : 0 C 18 : 1z C 18 : 2z C 18 : 3

Year Mean CV Mean CV Mean CV Mean CV Mean CV Mean CV

2001 15.8 16.6 1.5 41.9 1.3 27.5 68.4 7.5 11.6 32.4 0.69 26.2

2002 14.7 18.8 1.3 44.0 1.9 33.0 70.1 8.0 10.5 34.1 0.74 23.9

2003 15.4 13.2 1.8 43.0 1.8 32.0 67.9 7.7 11.3 33.2 0.87x 26.2

2004 13.4 14.4 1.5 39.8 1.7 31.0 72.0 6.1 9.7 35.3 0.71 23.1

2005 13.1 14.9 1.7 42.3 1.1 58.0 73.8 6.4 9.0 38.5 0.55x 24.0

General

mean

14.5 17.1y 1.6 43.7y 1.6 41.5y 70.4 7.9y 10.4 35.7y 0.71 30.1y

z All data in the columns are statistically different at P� 0.05.y The general coefficient of variation (CV) is calculated on the overall data.x In the column of C 18:3, only the data with x are statistically different for P� 0.03.

3. FACTORS AFFECTING EXTRA-VIRGIN OLIVE OIL COMPOSITION 103

acid compositions of oils obtained in the hot region of Catamarca,Argentina, with those of oils of the same cultivars produced in Italyshowed that the oils produced in Argentina had a lower content of oleicacid and higher contents of palmitic, linoleic, and linolenic acids.These variations were, however, cultivar dependent, with 5% to 8%reductions in the oleic acid content in the �Biancolilla�, �Cerasuola�, and�Coratina� cultivars, and 16% to 18% reductions in �Frantoio� and�Peranzana� (Mannina et al. 2001) (Table 3.2). Comparison of the fattyacid compositions of the oils of the �Frantoio� and �Coratina� in Tuscany(Italy), Saudi Arabia, and Australia showed larger variations in the oilsof �Frantoio� than �Coratina� (Fiorino 2005). The fatty acid compositionof �Koroneiki� EVOO did not change in relation to growing site (Greeceand Tunisia), whereas when �Sigoise� was cultivated in Tunisia, itprovided an oil with lower oleic acid and higher palmitic and linoleicacids than is normally obtained in Algeria (Mahjoub Haddada et al.2007; Zarrouk et al. 2009). In a relatively hot year, the oils of the mostnumerous group of cultivars showed a decrease in oleic acid that wasmainly compensated for by an increase in palmitic acid. The oils ofanother group of cultivars showed a decrease in oleic acid that wasmainly compensated for by an increase in polyunsatured fatty acids(linoleic and linolenic acids). The oils of a very few cultivars showed nosignificant variations in the monounsaturated fatty acid contents (pal-mitoleic and oleic), whereas there was an increase in saturated fattyacids and a decrease in polyunsaturated fatty acids (Lombardo et al.2008). Apparently, cultivars that originated in northern environmentshave higher phenotypic instability, in terms of fatty acid composition,than cultivars that originated in southern environments (Lombardoet al. 2008).

The variations in fatty acid composition induced by the environmentcan be so large as to affect the commercial suitability of the oil as definedby the lawsof theEuropeanUnion (Reg. ECNo702/2007) andof the tradestandards of the InternationalOliveCouncil (COI/T.15/NCno. 3/Rev. 3—Nov. 2008). For example, in the Catamarca region of Argentina, which ischaracterized by high temperatures during the development and ripen-ing of the olives, �Arbequina� produced an oil with 53.4% oleic acid and1.2% linolenic acid (Mannina et al. 2001); these percentages are, re-spectively, lower andhigher than those established by trade standards ofthe International Olive Council (IOC) and the rules of the EuropeanUnion. Moreover, oils of �Biancolilla, �Cerasuola�, �Coratina�, �I-7�7Q7 ,�Frantoio�, �Kalamata�, �Leccino�, and �Peranzana� had percentages oflinolenic acid greater than 1%, which is the maximum value allowedby the above-cited rules for all the categories of olive oils.

104 P. INGLESE ET AL.

High rainfall reducedEVOOpolyphenol content (Pannelli et al. 1994).The role of temperatures on polyphenols content is controversial, de-pending on genotype and environmental conditions. In a study carriedon in different Italian regions, the higher the degree-days accumulatedfrom fruit set to harvest, the lower was the amount of total polyphenols(Ripa et al., 2008), whereas the total amount of polyphenol content of�Casaliva�, but not �Leccino�, increased with the degree-days accumulat-ed from August to October in the cool area of northern Italy (Turaet al. 2008). Moreover, in some studies on the effects of altitude, whichis a factor that affects thermal regime, in some cases (Chania, Greece)polyphenols content decreased with altitude (Osman et al. 1994; Mousaet al. 1996), whereas in other cases, the effects of altitude was unclear(Paz Aguilera et al. 2005).

As far as the contents and composition of volatile compounds in oliveoils are concerned, the effects of rainfall and temperature were notunambiguous and depend on environment and interaction with geno-type. In a study conducted in central Italy, rainfall negatively correlatedwith hexanal and isobutyl-acetate contents and positively correlatedwith the other compounds in the head-space of the oil (Pannelliet al. 1994). In a northern and relatively rainy area of Italy, no significanteffects of temperature on total volatile compounds in oils of the cultivar�Leccino� were observed, whereas positive relationships were seenbetween the cumulated degree-days in the period August to October inoils of the cultivar �Casaliva� (Tura et al. 2008). Oils of �Biancolilla�,�Carpellese�, and �Racioppella�produced in awarmcoastal areawere lessfruity, bitter, pungent, and sweeter than those produced in the fresh hillyareas where these cultivars are traditionally grown (Di Vaio et al. 2006).Temperature and rainfall can also have indirect effects on oil quality, asthey can affect fruit ripening patterns. Earlier olive ripening occurs inyears with low rainfall and warm temperatures (Pannelli et al. 1996; DiVaio et al. 2006).

There is no clear information on the influence of soil type on EVOOcomposition, although some specific relationships between the quali-tative parameters of an oil and the soil characteristics have beenreported (Angerosa et al. 1996a; Ranalli et al. 1997). The oil of the�Moraiolo� obtained in a stony soil had a higher polyphenol content andoxidation stability than that obtained in a clay soil, but these effectswere mainly attributed to the lower water availability in the stony soils,with respect to the clay ones, rather than to the texture of the soil(Pannelli et al. 1990a; Servili et al. 1990). In Sardinia, soil differencesappeared not to have a strong influence on �Bosana� oil quality (Deiddaet al. 1994).

3. FACTORS AFFECTING EXTRA-VIRGIN OLIVE OIL COMPOSITION 105

Altitude affects the tocopherol content of the oils. At lower altitudes,higher amounts of tocopherols were observed in several studies (Osmanet al. 1994;Mousa et al. 1996; PazAguilera et al. 2005). Altitudewas alsoable to affect the chlorophyll content, oxidation stability and quantity ofhydrocarbons, triterpenic alcohols, and sterols of olive oils (Ferreiro andAparicio, 1992; Osman et al. 1994; Mousa et al. 1996; Paz Aguileraet al. 2005).

In the coldest areas in which olive trees are grown, the fruits, andconsequently the oils extracted from them, can be damaged by freezingtemperatures during the ripening period. In an oil extractedwithin 24hrof the occurrence of freezing temperatures, reductions were shown forthe chlorophyll, carotenoid, and total polyphenols contents and theoxidation stability and bitterness index (K 225), whereas no significantnegative effects were seen on free acidity, peroxide number, ultravioletabsorbency (K 270), or a-tocopherol (Morello et al. 2003). Oil fromfrosted olives also showed significant changes in the concentrations ofseveral phenolic compounds and reductions in the bitter and pungentnotes, with the consequent onset of sensory defects that prevent the oilfrom being marketable as EVOO (Morello et al. 2003). The variability ofthe effects of environmental factors, which sometimes is rather large,also arises from interaction effects with other factors, such as genotypeand ripening stage of the fruit (Tous and Romero 1994; Pannelliet al. 1996).

C. Tree Water Status

In the Mediterranean area, olive trees traditionally have been grownunder rainfed conditions and with limited water resources during thefruit developmental stages, since most of rains occur during the winterperiod. Complementary irrigation, distributed during critical stages offruit growth, particularly during mesocarp development and oil accu-mulation, increases fruit size, flesh-to-pit ratio, and oil yield per hectare,although thepercent of oil in an individual fruitmaydecline because of aproportionally larger increase in the water content in the fruit and aminor efficiency inoil physical extraction (Spiegel 1955;Vitagliano1969;Lavee et al. 1990; Lavee and Wodner 1991; Goldhamer et al., 1994;D�Andria et al. 2002; Berenguer et al. 2006). The nature of fruit ripeningand the rate pattern of oil accumulation are also significantly affected bywater availability (Vitagliano 1969; Lavee andWodner 1991). Indeed, thefruit is the organmost sensitive towater stress, and severewater shortage,lasting up to the third stage of the fruit development period, may resultin shriveling of the fruits, advanced and rapid ripening, early and

106 P. INGLESE ET AL.

pronounced preharvest fruit drop, and, eventually, a complete or tem-porary arrest of the oil metabolism, to such an extent that its finalaccumulationcouldbesharply reducedand themetabolismof individualcomponents may also be affected (Lavee 1986; Dettori et al. 1990; Dettoriand Russo 1993; Inglese et al. 1996; Gucci and Servili 2006). Underirrigated intensive growing conditions, oil accumulation is linear duringmost of the fruits� growing period. The degree of diversity of the oilaccumulation from linearity in a single cultivar could serve as a partialindexofwater stress andcultivar sensitivity. Thewater deficit that occursduring the first stage of fruit growth determines a significant reduction ofmesocarp cells dimension, which is only partially recovered during thesubsequent developmental stages, even if the tree is regularly watered(Rapoport et al. 2004). Complementary irrigations, however, even withlowvolumes, distributed during the cell extension in themesocarp resultin an increase of fresh and dry fruit weight, flesh percent, and oil content(Lavee et al. 1990; Lavee and Wodner 1991; Dettori and Russo 1993;Motilva et al. 2000; Gucci et al. 2004; Servili et al. 2007).

The effect of water availability on the relative growth of the endocarpand themesocarp is controversial, since it also depends on other factors,such as crop load (Barone et al. 1994; Inglese et al. 1999), irrigationstrategy, and fruit growth potential defined by the genotype (Laveeet al. 1990; Lavee and Wodner 1991, 2004; Inglese et al. 1996; Rapoportet al. 2004; Gucci and Servili 2006). The prolonged duration of thesecond stage of fruit growth could be related towater deficit (Lavee 1986;Inglese et al. 1996). The effects of water availability on EVOO compo-sition and quality have been investigated under a wide range of envi-ronmental conditions, with different genotypes andwater deficit levels.Water shortage affects fruit ripening pattern, and differences of EVOOcomposition might be both a direct effect of water stress on oil accumu-lation pattern and single component metabolism or on the frequency atharvest of populations of fruits highly differentiated in terms of ripeningstage. These differences increase with time because fruits from nonir-rigated trees mature earlier and faster than those on irrigated trees(Inglese et al. 1996). Nevertheless, it is generally agreed that the oilscoming from trees under a severe water deficit (25% restitution of actualevapotranspiration [Etc]), or fully irrigated (100% restitution of ETc)complies with the international merceologicalQ8 standards of high-qualityEVOOs, with differences that, in most cases, concern the phenolicfraction and the sensory parameters.

There is no evidence of significant effects of water shortage on oilacidity, peroxide number, and spectrophotometric indexes, while thefatty acid composition changes slightly with tree irrigation. However,

3. FACTORS AFFECTING EXTRA-VIRGIN OLIVE OIL COMPOSITION 107

fluctuations of oleic acid and stearic acid occur inconsistently amongyears, cultivars, and irrigation treatments (Inglese et al. 1996; Berengueret al. 2006; Gucci e Servili 2006; Gomez-Rico et al. 2007; Serviliet al. 2007). Berenguer et al. (2006) report a significant and consistentdecrease of stearic fatty acid from 15% ETc to 107% ETc, while palmi-toleic, linoleic, and linolenic fatty acid levels significantly increasedwith irrigation only in one year out of two. In any case, the range ofvariations of the fatty acid compositiondonot account for any significantchange of the physical and nutraceutical properties of the oil (Faciet al. 2002; Tovar et al. 2002). Treewater status, however, has a consistenteffect on the phenolic fraction and the volatile compounds of the oil, andon its sensory properties (Inglese et al. 1996; Gucci et al. 2004; Berengueret al. 2006; Gucci and Servili 2006; Servili et al. 2007).

Most studies report adecrease of total phenol andpolyphenols contentand oxidation stability as the amount of supplied water increases(Patumi et al. 2002; Berenguer et al. 2006; Gucci and Servili 2006;Gomez-Rico et al. 2007). The effect is clear at low (66% ETc) (D�Andriaet al. 2002) and high (25% ETc) water stress level (Motilva et al. 2002).Nevertheless, some studies report no effect of tree water status onphenolic composition and total content or an increase of polyphenoland total phenols, particularlyduring the early stages of fruit ripening, inirrigated trees compared to rainfed ones (Dettori and Russo 1993; Ingleseet al. 1996). Reasons for such differences may lie on the overlap of cropload, fruit ripening rate pattern, and water regime effects (Gucci andServili 2006). The reduction of polyphenol content in the oil of irrigatedtrees could be a consequence either of a greater dilution of hydrosolublecompunds during oil extraction or reduced activity of the enzymesresponsible for phenolic compound synthesis, such as L-phenylalanineammonya-lyase, whose activity is greater under water stress conditions(Tovar et al. 2002; Gomez-Rico 2007; Servili et al. 2007).

Total phenol content decreased with fruit ripening from 1,700 to 900mg/kg and from 1,080 to 650, respectively, for EVOO from rainfed orirrigated olive trees (Gomez-Rico et al. 2007). Treewater status affects thecomposition of the phenol fraction; a greater concentration of secoir-idoids andaglyconderivatives of oleuropein togetherwith a reduction oftyrosol andhydrossid tyrosol content has beenmeasured in nonirrigatedolive trees (Servili et al. 2007). Berenguer et al. (2006) report significantbut inconsistent through the years variation of total and individual sterolcontent in oil from 15% to 107% ETc treatments.

Tree water status has a marked effect on volatile compounds andEVOO sensory properties (Servili et al. 2007). Rainfed or poorly irrigatedolive trees produce oils characterized by marked notes of pungency and

108 P. INGLESE ET AL.

bitterness, which decrease with irrigation (Patumi et al. 1999; Tovaret al. 2002; Berenguer et al. 2004; Gucci et al. 2004; Gucci e Servili 2006;Servili et al. 2007). Fruitiness, herbaceous, and floral flavors may alsodecrease with increasing water availability, although variationsmay notbe always consistent (Berenguer et al. 2006; Servili et al. 2007). Thesevariations may be related to changes of the lypoxigenase pathway inrelations C6 saturated and unsaturated aldehydes, alcohols, and esters(Servili et al. 2007).

Very few studies have examined the effect of saline water on oilquality (Gucci and Tattini, 1997). Cresti et al. (1994) reported thatsalinity increased aliphatic and triterpenic alcohol content and theoleic-linolenic acid ratio while Royo et al. (2005) report a decrease ofthe aliphatic alcohols and palmitoleic acidwith salinity in �Arbequina�oils.

In Israel, 48 olive oils from the years 2002 to 2004 were compared andgraded; oils produced under high evaporation and saline irrigation didnot differ significantly from most other oils produced from rainfed andfreshwater-irrigated orchards (Dag et al. 2008a). No significant differ-ences were found between saline- and control-water-irrigated �Barnea�trees in terms of olive oil basic quality parameters, such as free fattyacids, peroxide value, and fatty acid profile. However, the saline treat-ments increased the levels of certain antioxidant components (poly-phenols and vitamin E) in the oil extracted from the olives as comparedwith the control (Wiesman et al. 2004).

Eventually, rainfed olive trees produce EVOOwith strong taste, with aclear pungent and bitter flavor, while oils from irrigated trees are morearomatic and sweet, with a sharp reduction of bitterness and pungency.Result obtainedon �Leccino� indicate that anappropriate useof irrigation(qualitative irrigation) may modulate EVOO sensory properties, takinginto account the different evolution of the fruit ripening pattern (Gucci eServili 2006).

D. Productivity and Alternate Bearing

The olive tree shows a typical alternate bearing behavior with a frequen-cy and intensity regulated by the genotype and the growing conditions.The olive treemay show �on� and �off� years on single brancheswithin atree, single trees within an orchard, single orchards within the samelocation or betweendifferent geographical area. The intense competitionfor assimilates andwater between fruits during the earliest stages of theirdevelopment is primarly responsible for the regulation of crop load(Lavee 1986). It has been reported (Vitagliano 1969; Zucconi et al. 1978;

3. FACTORS AFFECTING EXTRA-VIRGIN OLIVE OIL COMPOSITION 109

Shulman and Lavee 1979; Lavee 1986; Lavee and Wodner 1991, 2004)that not only final fruit size and flesh-pit ratio but also the nature of fruitgrowth and ripening and both rate pattern and final oil accumulation inthe mesocarp depend on crop load. In the case of table olive, fruitthinning becomes essential to regulate fruit growth andfinal commercialsize (Barone et al. 1994; Inglese et al. 1999; Lavee and Wodner 2004).

In trees with a light crop load, oil accumulation and ripening ratepattern are fastenedQ9 ; fruit ripening occurs earlier and is more concen-trated than in heavily cropping trees (Barone et al. 1994; Gucci 2006).Eventually, the oil content in the single fruit may be reduced by heavycrop loads, due to a consistent reduction of the mesocarp size and aslower oil accumulation rate than in lightly cropping trees (Baroneet al. 1994; Lavee and Wodner 2004; Gucci and Servili 2006). Theinfluence of crop load on oil quality is less clear, and it may occur onlyin caseof veryheavy reductionsof crop load (>50%),with effects on fattyacid composition and polyphenols content (Barone et al. 1994; Gucci eServili 2006) (Fig. 3.6). The effect of harvest date, hence the differentdegrees of fruit ripening at picking, is much stronger than the effect ofcrop load in determining the variability of oil composition (MaestroDur�an, 1990), and differences in oil compositon between trees withheavy or light crop loadsmay largely dependon thedifferent time course

Harvest date

9 Jan.13 Dec.29 Nov.14 Nov.30 Oct.

Poly

hen

ol

conte

nt

(ppm

)

100

150

200

250

300

Fig. 3.6. Changes during olive ripening of total polyphenol content of oils obtained from

fruit produced from treeswith full load (FL¼.), partially reduced load (70%FL¼~), and

halved load (50% FL¼&). Analysis of variance showed significant effects for, P < 0.01, of

date of harvesting, fruit load, and their interaction. Source: Barone et al. 1994.

110 P. INGLESE ET AL.

of fruit ripening and uniformity of fruit ripening stage of the pendingfruits. Considering the evolution of single oil components, fruits fromlightly cropping trees show a more rapid metabolism and accumulationrate, which eventually result in a higher total palmitic and linoleic acidand polyphenol content than in heavily cropping trees (Baroneet al. 1994). Crop load has no effect on peroxides and sensory parametersof EVOOs (Gucci e Servili 2006).

The effect of crop density and different source-sink relationships onfruit growth and ripening rate pattern can be measured even at a singlefruiting branchlet (Inglese et al. 1999). Thismeans that treemanagementin terms of training system, pruning, and fruit thinning may account forthe uniformity of fruit growth and ripening and, ultimately, for the finaloil accumulation and composition.

Optimum harvest time to maximize oil yield and quality changes,then, with tree crop load; in trees with a light crop load, it occurs earlyand lasts for a short period, resulting in rapid changes of the degree theripening stages of pending fruitsQ10 . The timing and the speed of harvest aretherefore crucial in trees with a light yield, while the picking season ofheavily loaded trees, with a slower fruit ripening pattern, is later andmore extended. Provided these differences of the fruit ripening patternare taken into account in determining the optimum harvest time, largedifferences in tree crop load have only a limited effect on EVOOcomposition (Barone et al. 1994).

E. Orchard Management

1. Cultivation Method. The organic farming of olive groves has spreadthrough all of themost important olive-growing areas andparticularly inItaly, where it covers about 10% of the total area for olives. Despite this,few studies have been carried out to determine the influence that organicfarming has on the characteristics of the oil produced.

In a one-year study on the cultivar �Picual�, Guti�errez et al. (1999)showed that the EVOO obtained with organic farming had lower freeacidity, peroxide number, and linoleic acid content and higher organ-oleptic score, oxidation stability, oleic acid, a-tocopherol, total polyphe-nol, ortho-diphenol, and D5-avenasterol contents. Perri et al. (2002) andNinfali et al. 2008, comparing the qualitative characteristics of EVOOs of�Coratina� and �Ogliarola Salentina�, and �Frantoio� and �Leccino� ob-tained by organic and integrated methods, found inconsistent resultswith a large season- and genotype- dependent variability. The sensoryanalyses showed only slight differences in a few aromatic notes. InSpain, the oil of the �Hojiblanca� obtained with integrated farming had

3. FACTORS AFFECTING EXTRA-VIRGIN OLIVE OIL COMPOSITION 111

higher levels of sterols and tocopherols than those from conventionalfarming while the free acidity, peroxide number, ultraviolet absorben-cies, acid composition, polyphenol content, and sensory traits were notaffected by the cultivation system (Cayuela et al. 2006). The inconstantresults obtained in the comparison of the organic, integrated, andconventional cultivation systems indicate that the effects of the culti-vation method on the quality of the oil is very dependent on theinteraction with the other factors that affect oil quality (genotype,environment, and season).

2. Training System and Pruning. Comparisons of different trainingsystems (monocone, Y-shape, and vase) in young trees of the cultivars�Frantoio�, �Leccino�, �Maurino�, �Moraiolo�, and �Nostrale di Rigali�showed no significant differences in terms of free acidity, peroxidenumber, total polyphenol and chlorophyll contents, fatty acid compo-sition, and oxidation stability (Preziosi et al. 1994; Palliotti et al. 1999).

At the end of the 20th century a new concept of olive orchard arose,based on high-density plantations (1,100–2,050 trees/ha), forminghedgerows, and the use of straddle harvesting machines to collect thefruit (superintensive or high-density hedgerow orchards). Until now,the cultivars most used in these groves are the low vigor and compactcultivars such as �Arbequina�, �Arbosana�, and �Koroneiki� and theirclones, along with new cultivars such as �Urano�, �FS-17�, and �Askal�.The evaluation of suitability of some traditional cultivars (�Picual�,�Leccino�, �Barnea�, �Souri�, �Picholine�, �Chemlali�, �Chetoui�, �PicholineMarocaine�) to this cultivation system is in progress (Dag et al. 2006;Godini et al. 2006; Larbi et al. 2006; Leon et al. 2006; Pastor et al. 2006;De la Rosa et al. 2007; Tous et al. 2007). There is no experimentalevidence of oil quality with this new cultivation system as compared tothe traditional growing systems. The oils obtained with high-densityhedgerow orchards had physicochemical characteristics (free acidity,peroxide number, ultraviolet absorbencies) within the ranges estab-lished for extra-virgin olive oil by UEQ11 (Reg. EC No 702/2007) and IOC(COI/T.15NC no 3/Rev. 3—Nov. 2008) (Berenguer et al. 2006; Allaloutet al. 2009). The main cultivars used for high-density hedgerow orch-ards showed differences in contents of fatty acids, pigments, phenoliccompounds, tocopherols, and oxidative stability (Larbi et al., 2006; Dela Rosa et al. 2007; Allalout et al. 2009). An interaction is suggestedbetween the main cultivars used for this cultivation system andthe environment in determining fatty acid composition and totalphenols amounts (Berenguer et al. 2006; De la Rosa et al. 2007; Allaloutet al. 2009).

112 P. INGLESE ET AL.

Fruit locationwithin the canopypositioncanbeconsidereda sourceofvariability for the composition of the oil, resulting from large differencesin light environments within the canopy. Oils of �Frantoio� and �Leccino�obtained from fruits that developed under optimal and low-light avail-ability showed no differences in terms of free acidity, peroxide number,and fatty acid composition, whereas those obtained under optimal lightavailability had higher total polyphenol and chlorophyll contents and,in the case of �Frantoio�, greater fruity, bitter, and spicy tastes (Proiettiet al. 2009). This supports the importance of a balanced pruning to allowlight diffusionwithin the canopy, even though no light thresholds for oilquality have been defined.

3. Fertilization and Soil Management. The little information availableon the effects of fertilization on the qualitative characteristics of oliveoils relates primarily to nitrogen and is in some cases contradictory.Uceda Ojeda (1985) reported a positive correlation between adminis-tration of nitrogen and the levels of oleic and stearic acids in the oil,while a lack of nitrogen resulted in an increase in the palmitic andlinoleic acid contents. Cimato et al. (1994) showed an increase in thetotal polyphenol and tocopherol contents in the oil obtained from�Frantoio� and �Moraiolo� as a result of foliar treatments with urea.These effects were explained as a result of the delay in the ripeningof the fruits on treated plants, caused by the greater vegetative activityinduced by the nitrogen. In a study carried out in Portugal on�Carrasquenha�, Simoes et al. (2002) suggested that high levels ofnitrogen fertilization had a negative influence on the content of satu-rated fatty acids of the oil. Fern�andez-Escobar et al. (2006), supplyingnitrogen to plants of �Picual� where the leaf nitrogen status was alwaysabove the limit for deficiency even in the nonfertilized control trees,showed that overfertilized trees produced oils with lower total poly-phenol content, oxidation stability, and compounds responsible for thebitter taste and higher contents in tocopherols, in particular a-tocoph-erol. No effects were found on the carotenoids and chlorophylls con-centrations and fatty acid composition. In the oil of �Manzanilla deSevilla�, with an increase in the supply of a complex fertilizer (4 : 1 : 3 ofN-P-K) through fertigationQ12 , Morales-Sillero et al. (2006) found dose-related reductions in total polyphenol content, oxidation stability, andmonounsaturated to polyunsaturated fatty acid ratio. Eventually, itseems that high availability/excess of nutrients, especially nitrogen,may result in a significant worsening of the oil quality.

Very little information is available on the influenceof soilmanagementonoil quality.Differences in the sensoryprofilewerenoticed for �Carolea�

3. FACTORS AFFECTING EXTRA-VIRGIN OLIVE OIL COMPOSITION 113

EVOOs, although there were no differences in the panel test scores(Briccoli Bati et al. 2002).

4. Pest and Disease Control. Attacks of olive fly (Bactrocera oleae) thatare not controlled can cause serious damage to oil production andquality. Quantitative damage is due to premature olive drop and lossof part of theolivepulpcausedby the larvae.Qualitativedamage isdue tothe galleries produced by the larvae (rupture of tissue and presence ofoxygen) where fungi (decay) and bacteria can also develop. The greatestamount of damage has been observed when there is a large number ofolives with mature larvae and galleries with exit holes (Zuninet al. 1992). Increasing intensity of infestation corresponds to a progres-sive worsening of oil quality, because of an increase in free acidity,peroxide number, and UV absorbencies and a decrease in oxidationstability and polyphenol content (Parlati et al. 1990; Perri et al. 1996;Gomez-Caravaca et al. 2008; Tamendjari et al. 2009). Fly attack results inthe loss of phenols, o-diphenols, and, in particular, some secoiridoidderivates (Gomez-Caravaca et al. 2008).When the percentage of infectedolives increases, phenols decreases and stability of resulting oils iscompromised, probably because of an increase of polyphenoloxidaseactivity caused by larval damages and by the presence of exit holes thatexpose the olive pulp to oxygen (Tamendjari et al. 2004). Attack of olivefly causes a reduction in the chlorophyll and carotenoid contents as thedegree of infestation increases (Tamendjari et al. 2004). The fatty acidcomposition is quite stable, but in case ofmassive attacks, levelsmight bealtered (Parlati et al. 1990; Zunin et al. 1992; Perri et al. 1996; Pereiraet al. 2004; Tamendjari et al. 2004). Fly attack causes a reduction of thetotal amount of volatile compounds, attributable mainly to a decreasedconcentration of trans-2-hexenal, and a quicker flattening of fruity,pungent, andbitter attributes of theoilduringolive ripening (Tamendjariet al. 2004, 2009); moreover, it can cause the onset of sensory defects,such as fusty, winy, and grubby sensation, especially in advanced stagesof olive ripening (Angerosa et al. 1992; Tamendjari et al. 2009). Thequicker reduction of the positive bitterness and pungent attributes arecaused by the losses in phenolic compounds under the effects of flyattack. Postharvest storage of olives causes aworsening of the qualitativeparameters of the oil. This epresents more problems in infected olivesthat have worse qualitative characteristics at harvest time and canpresent higher rates of worsening of qualitative parameters during thestorage periodQ13 (Kyriakidis and Dourou 2001). During oil storage, thesensory characteristics worsen more rapidly in oils extracted frominfected olives (Esposito et al. 2004). In some cases, olive fly attack

114 P. INGLESE ET AL.

caused a reduction of the total b-sitosterol content below the level legallyrequired for olive oil (Zunin et al. 1992).

Olive anthracnose caused by Colletotrichum spp. affects olive pro-duction and oil quality. Experiments have shown that as the oliveinfestation increases, there is aparallelworsening of oil quality extractedfrom attacked fruit. This disease causes an increase in peroxide numberand free acidity and a decrease in the oxidative-tocopherol contents and,in some cases, a stability and polyphenol are reduced.With infestationsof 15% to 20% and 40% to 45%, the oil quality was beyond the limits tobemarketed as EVOOor as VOOdue to too-high free acidity (> 0.8% and2%, respectively) (Iannotta et al. 1999; Mincione et al. 2004; Carvalhoet al. 2008). Fatty acid composition is little affected by anthracnosewhereas sometimes the changes induced in individual and total amountof sterol contents can cause problems in respectingQ14 the internationaltrade standards for olive oils (i.e., total b-sitosterol < 93% and totalsterols < 1,000mg/kg, the minimum values established by the tradestandards of IOC andUE rules, Reg. ECNo702/2007; Iannotta et al. 1999;Mincione et al. 2004). Anthracnose attack increases the content ofaldehydes, such as heptaldehyde, octyl aldehyde, and nonanal (Runcioet al. 2008). Colletotrichum spp. also affects the aliphatic and terpenicalcohols and wax contents (Mincione et al. 2004).

Olive rot caused by Camosporium dalmatica does not greatly affectfree acidity and peroxide number. Oils can be classified as EVOO evenwhen100%of the oliveswere attacked, but an increase in the percentageof olive infection corresponds to a decrease in total phenols and oxida-tive stability (Iannotta et al. 1999).

Because of the great negative effects of disease and insect predation onoil, these biotic stresses must be carefully controlled to obtain a high-quality EVOO, especially in environments or seasons that are favorableto themandwith sensitive cultivars. In case of late attacks by theolivefly,earlier fruit harvesting and rapidmilling are important to avoid or reduceits deleterious affect on oil quality, especially in organic olive orchards,where the control of olive fly could be more difficult.

F. Fruit Ripening and Harvest

1. Ripening. During fruit ripening, a number of changes take place bothin the fruit and in the oil. Olives show a reduction in the resistance todetachment, which results in falling of the more mature fruits (fruitdrop), and consistenceQ15 of the pulp and a progressive increase in thepigmentation of the skin (the epicarp) and, later, of the pulp (themesocarp), starting from the outside layers; moreover, they complete

3. FACTORS AFFECTING EXTRA-VIRGIN OLIVE OIL COMPOSITION 115

their growth and accumulate oil (Pannelli et al. 1990a; Famianiet al. 2002; Tombesi et al. 2009) (Table 3.5).

The quantity of olive oil per tree is based on number of fruits, fruitweight, oil content, and fruit drop (Zucconi et al. 1978; Famianiet al. 2002). Generally, total quantity of oil decreases when fruit dropexceeds about 5%-Q16 (Famiani et al. 2002; Tombesi et al. 2006).

During fruit ripening, oleic acid, and sometimes linoleic acid, tends toincrease, while stearic acid and especially palmitic acid tend to de-crease,with a consequent increase in the ratio of unsaturated to saturatedfatty acids (Servili et al. 1990; Fiorino and Nizzi Grifi 1991; Di Matteoet al. 1992; FiorinoandAlessandri 1996;Tombesi et al. 2009).Hoever, thefatty acid composition does not always show significant variations,particularly in the period in which the harvesting is effectively carriedout, which is shorter than the entureQ17 ripening period (Famianiet al. 2002). In some cases, a different pattern of fatty acid modificationswas observed. In �Cornicabra�, during fruit ripening, a reduction in oleicacid and an increase in stearic and linoleic acids contents occurs

Table 3.5. Changes in fruit characteristics and fruit drop during fruit ripening

determined in olives collected from an intensive olive grove in central Italy.

Source: Famiani et al. 2002.

Cultivar

Harvesttime

Dry weight

(g fruit�1)

Oil content

(g fruit�1)

Detachment

force (N)

Pigmentation

(0–4)zFruit

dropy (%)

FrantoioBegin-Nov. 1.062 ax 0.368 a 5.60 c 0.7 a 6.8 a

End-Nov. 1.148 b 0.468 b 3.98 b 1.0 b 11.1 b

Mid-Dec. 1.150 b 0.471 b 3.60 a 1.4 c 16.7 c

LeccinoBegin-Nov. 0.972 a 0.348 a 5.77 b 2.5 a 4.5 a

End-Nov. 1.041 ab 0.428 ab 4.41 ab 3.1 b 5.3 a

Mid-Dec. 1.058 b 0.445 b 3.82 a 3.5 c 11.6 b

MaurinoBegin-Nov 0.698 a 0.276 a 4.63 b 2.6 a 2.8 a

End-Nov. 0.729 ab 0.330 b 3.95 ab 2.8 b 4.8 b

Mid-Dec. 0.740 b 0.349 b 3.10 a 3.1 c 12.7 c

z 0¼ green; 1¼pigmentation on less than 50% of fruit surface; 2¼pigmentation on more

than 50% of fruit surface; 3¼pigmentation on 100%of fruit surface; 4¼pigmentation on

100% of fruit surface and on the pulp.y Expressed as percentage of total olive production.x For each parameter and cultivar, means followed by different letters are significantly

different at P� 0.05.

116 P. INGLESE ET AL.

(Salvador et al. 2001). Also, in Tunisia, recent studies of fruit ripeningshowed reductions in oleic acid and increases in linoleic acid, andpalmitic acid decreased in some cultivars and increased in others(Baccouri et al. 2008b; Sakouhi et al. 2008). These results might havearisen due to the occurrence of high temperatures during the ripeningperiod.

The peroxide number of oil obtained from healthy olives often de-creases through the ripening period. This is related to a reduction in thelipoxygenase enzymeactivity (Guti�errez et al., 1999; Salvador et al. 2001;Baccouri et al. 2008b).

Ripening also has large effects on the sensory and nutritional char-acteristics of olive oils, affecting the contents of volatile substances,phenolic compounds, and pigments (Angerosa et al. 2004; Serviliet al. 2004). The contents of volatile compounds is greatest in the initialphases of the fruit surface pigmentation, with particular reference to thecomponents involved in the lipoxygenase pathway, such as aldehydesand saturated and unsaturated C5 and C6 alcohols and, in particular,trans-2-hexenal (Solinas et al. 1987; Montedoro et al. 2003; Angerosaet al. 2004). These then decrease because of a lower activity of theenzymes involved in their synthesis, with the consequent reduction inthe intensity of the fruity aroma that can be smelled/tasted, in particularthe �green� sensory notes (Montedoro and Garofolo, 1984; Solinaset al. 1987; Angerosa and Basti 2001; Montedoro et al. 2003; Angerosaet al. 2004) (Table 3.6). In very mature fruits (with pigmentation thatincludes themesocarp), there is amarked inactivation of the endogenousenzyme activities of the lipoxygenase pathway,with a consequent strongdecrease in the volatile compounds in the oil obtained from these fruits(Servili et al. 1990; Angerosa et al. 2004).

The total content of the phenolic compounds increases during theearly stages of fruit maturation and then decreases more or less rapidlywith the intensification of the pigmentation of the epicarp and themesocarp, according to the cultivar ripening pattern (Table 3.6). Thelowest concentrations are seen in oils obtained from very mature olives(Servili et al. 1990; Brenes et al. 1999; Uceda et al. 1999; Salvadoret al. 2001; Gimeno et al. 2002; Montedoro et al. 2003; Panaroet al. 2003; Servili et al. 2004; Baccouri et al. 2008b). The hydrophilicphenolic compounds are exclusively present in virgin olive oil, and theyare of great importance: Aswell as being antioxidants, they contribute tothe sensory characteristics of the oil, as they are responsible for the bitterand spicy tastes. Indeed, the analytical index of �bitterness� (K225) that isconsidered in some studies also tends to decrease during ripening(Garc�ıa et al. 1996). During the development of the fruits, there are also

3. FACTORS AFFECTING EXTRA-VIRGIN OLIVE OIL COMPOSITION 117

Table

3.6.

Influenceoffruitripeningstage(expressedasfruitpigmentation)onfattyacid

andaromaticcompoundcompositions,

chlorophyllandtotalpolyphenolcontents

andoxidationstability(inductiontime—

Swifttest

withRancim

atapparatus)

oftheolive

oil.D

ata

representtheaveragevaluesof6cultivars

(�Frantoio�,�Leccino�,�SanFelice�,�Tendellone�,�N

ostrale

diRigali�and�M

oraiolo�)

cultivatedin

centralItaly.O/L

¼oleic/linoleic

ratio;U/S

unsaturated/saturatedfattyacidsratio.Source:Servilietal.1990.

Fattyacid

composition(%

)z

Fruitpigmentation

C16:0

C16:1

C18:0

C18:1

C18:2

C18:3

O/L

U/S

Green

14.09Cz

1.02a

2.27a

72.99A

8.28a

1.34B

8.90a

5.12A

50%

ofsu

rface

13.10B

1.08a

2.44a

74.47B

7.87a

1.05A

9.64a

5.45A

100%

ofsu

rface

12.11A

1.06a

2.42a

75.35BC

8.06a

0.99A

9.58a

5.92B

100%

ofsu

rface,

50%

ofpulp

11.63A

0.95a

2.49a

76.19C

7.78a

0.95A

9.97a

6.10B

100%

ofsu

rface,

100%

ofpulp

11.37A

0.95a

2.52a

76.31C

7.91a

0.94A

9.76a

6.22B

Aromaticcomposition(I.U

.n�103)

Total

chlorophyll

(ppm)

Total

polyphenols

(ppm)

Induction

time(h)

Alcohols

Esters

Aldehydes

Trans-2-hexenal

Green

303b

135a

985b

621b

57.5

C253ab

9.1

A

50%

ofsu

rface

352b

203a

831b

596b

39.4

BC

310b

12.5

AB

100%

ofsu

rface

291ab

260a

758b

589b

27.1

B276b

13.6

B

100%

ofsu

rface,

50%

ofpulp

131a

137a

653ab

484ab

20.8

AB

226a

12.0

AB

100%

ofsu

rface,

100%

ofpulp

109a

148a

390a

215a

11.8

A203a

7.6

A

zIn

eachcolumn,foreachparameter,meansfollowedbydifferentletters

are

significantlydifferentatP�0.05andP�0.01(capitalletters).

yI.U.¼

IntegrationUnit.

Q35

118

variations in the ratios between the individual phenols (Salvadoret al. 2001; Briante et al. 2002). As has been shown in fruits of�Arbequina�, �Morrut�, and �Farga�, the concentrations of the aglyconicderivatives of oleuropeine decrease with the beginning of pigmentation,and this coincides with an increase in the phenolic alcohols, such ashydroxytirosol and tirosol (Morello et al. 2005). The different equilib-rium between phenolic compounds toward the most simple formsappears to be connected with a greater activity of the glucosidases andof the esterases during the first stages of fruit ripening (Brianteet al. 2002).

Generally, tocopherols show a decrease in their content during rip-ening (Solinas 1990; Di Matteo et al. 1992; Garc�ıa et al. 1996; Gimenoet al. 2002). Nevertheless, some results showno changes in a-tocopherolor g-tocopherol content (Garc�ıa et al. 1996; Beltr�an et al. 2005; Sakouhiet al. 2008).

During ripening, there are variations in oxidation stability of the oilsthat are known to depend on the antioxidant content (phenolic com-pounds, tocopherols, carotenoids) and fatty acid composition. General-ly, there is an increase in the initial phases of ripening and then adecrease or, in some cases, a constant reduction (Servili et al. 1990;Garc�ıa et al. 1996; Panaro et al. 2003; Baccouri et al. 2008b) (Table 3.6).The content in pigments, such as chlorophylls and carotenoids, de-creases during ripening (Servili et al. 1990; Guiti�errez et al. 1999;Salvador et al. 2001; Gimeno et al. 2002; Montedoro et al. 2003; Beltr�anet al. 2005; Baccouri et al. 2008b) (Table 3.6).

As ripening proceeds, the oils go from a very green color with intensegreen fruity (grassy), bitter, and spicy flavors that are often not very wellbalanced, to green or green/yellow oils with these various flavors veryevident andwell balanced, to yellow/green to yellow oils that tend to beorganoleptically flat (very weak fruity, bitter, and spicy hints) with anoverall sweetness. In general, the score attributed by the panel test tendsto decrease in the latest stages of ripening, sometimes to a great extent(Garc�ıa et al. 1996; Salvador et al. 2001). However, it isworth consideringthat for the organoleptic characteristics, there is no unique reference ofquality, because the best typology is that which best satisfies the sectionof themarket being targeted and thegastronomicuseof the oil. In general,oil quality variations during fruit ripening correlatewith the level of fruitpigmentation, although with a certain genotypic variability (Serviliet al. 1990).

In very ripe olives, free acidity and oxidation level of the oil canincrease (Pannelli and Montedoro 1988; Pannelli et al. 1990a; Dugoet al. 2004). In fruit advanced in stage ofmaturation, the decrease in pulp

3. FACTORS AFFECTING EXTRA-VIRGIN OLIVE OIL COMPOSITION 119

texture can determine conditions such as marking and bruising duringharvesting, transport, and later conservation that allow the breaking upof the cellular compartments, putting the oil in contact with enzymesthat can cause endogenous oxidative or hydrolytic processes, particu-larly if the temperature is high. Damages caused by parasites (e.g.,Bactrocera oleae) or diseases (e.g., Colletotrichum spp.) that have neg-ative effects on the quality of the oil are alsomuchmore accentuatedwithan advanced maturation state of the fruits at harvest.

2. Harvest Time and Production Objectives. As time of harvest canhave very important effects on the quantity and quality of the finalproduct, its choice needs to be made according to production objectives(Famiani et al. 2005). By correctly modifying the harvesting period, it ispossible to produce various different types of product: �novel� early oilby early harvest to get to the market at the beginning of the oil season,with a product that is well characterized in terms of color (green) andintensity of green fruity, bitter, and spicy flavors; POD (Protected OriginDenomination) or PGI (Protected Geographic Denomination) oil byharvesting olives at the ripening level that allows one to obtain an oilwith qualitative characteristics that satisfy the production rules thatmust be respected for this kind of product; �typical� or �differentiated�oil by harvesting the olives when the composition of the oil obtainedallows the typical or differential characteristics to be accentuated (e.g.,particular organoleptic flavors and/or higher levels of antioxidant com-pounds that provide nutritional value to the oil); standard extra-virginoil, byharvesting the oliveswhen theyhave themaximumamounts of oiland, at the same time, theextractedoil satisfies the commercial standardsof that type of product; �sweet� oil, ideal for delicate dishes and oftenpreferredbyconsumerswhoarenotused tooliveoil, byharvestingolivesat a relatively late stage. It is important to note that the search for aparticular oil quality can result in a decrease in the quantity of productobtained.

Numerous studies have been carried out to determine the optimalperiod for mechanical harvesting in different environments in relationto the quantity and quality of the oil (Famiani et al. 1993, 2002; Panaroet al. 2003; Dugo et al. 2004; Tombesi et al. 2006, 2009). In most cases,the best results, in terms of oil quality, are obtained by harvesting thefruits when their pigmentation is limited to the epicarp (surfacepigmentation).

3. Harvesting Systems. Fruit damage needs to beminimized in harvest-ing systems as it can reduce oil quality if olives are not processed

120 P. INGLESE ET AL.

immediately. Mechanical beaters can result in some damage to the fruit,but trunk shakers do not generally cause damage. Hand-held harvestingmachines do not produce excessive damages if used correctly (Tombesiet al. 1996).

In central Italy (Umbria region), the use of hand-held machines andtrunk shakers hadnonegative consequence on the oil quality of �Leccino�and �Moraiolo� in terms of free acidity, peroxide number, ultravioletabsorbencies, fatty acid composition, oxidation stability, and contents inpolyphenols and chlorophylls (Pannelli et al. 1990b; Tombesiet al. 1996), while in Sicily, mechanical harvesting of �Biancolilla�,�Cerasuola�, �Nocellara of Belice�, and �Tonda Iblea� was associated withreduced free acidity and increased tocoferolQ18 content (Dugo et al. 2004). InIsrael, hand-held machine harvesting resulted in an increase of freeacidity and peroxide number and a reduction of polyphenol content ascompared to oils obtained frommanual harvest (Dag et al. 2008b). Olivesfrom irrigated trees appeared to have a higher sensitivity to mechanicalwounding thatwas associatedwith reducedoil quality (Dag et al. 2008b).

Trunk shakers did not completely remove all the olives on the trees,but the fruits that remained on the trees, being generally located in thelower and more shaded parts of the canopy, did not reduce qualitybecause the oil extracted from these fruits have lower total polyphenols(Pannelli et al. 1990b). Mechanical harvesting, particularly with trunkshakers, greatly improves harvesting productivity andmakes it possibleto concentrate the harvest in the period that is optimal for productionobjectives. Moreover, the greater productivity provided by mechanicalharvesting has positive effects on oil quality by providing sufficientproduct required by the processing system and avoiding or reducing theneed for storage.

High-density hedgeroworchards are harvestedusing straddle harvest-ers able to remove 90% of fruits, regardless of fruit size, position in thecanopy, and ripening level (Tous et al. 2007). This makes it possible toconcentrate harvesting in a very short time and, usually, has a positiveeffect on oil quality.

V. TECHNOLOGICAL FACTORS AFFECTING EVOO COMPOSITIONAND QUALITY

Since the occurrence of EVOO hydrophilic phenols and volatile com-pounds is directly related to the activities of various endogenous en-zymes of olive fruit, their concentration in the oil is strongly affected bythe extraction conditions. All the steps of EVOO mechanical extraction

3. FACTORS AFFECTING EXTRA-VIRGIN OLIVE OIL COMPOSITION 121

process may influence its volatile and phenolic composition, but thestorage condition of the fruit before the EVOO extraction and crushingand malaxation can be considered the most critical point (Capellaet al. 1997; Caponio et al. 1999; Angerosa et al. 2004; Serviliet al. 2004; Servili et al. 2002b; Servili et al. 2009a,b).

A. Olive Fruit Storage

The storage of olives in unsuitable conditions, in sacks or in piles, hasheavy negative repercussions on the sensory quality of the resultingoils. Microorganisms produce different metabolites that give rise todifferent sensory defects related to the decrease in concentrations ofcompounds from the LOX cascade (Angerosa et al. 1996a). Microor-ganism development is promoted by the temperature reached in the pileand humidity. Clostridia and Pseudomonas develop, producingbranched aldehydes, branched alcohols, and their corresponding acids(Solinas et al. 1987; Solinas et al. 1988; Angerosa et al. 1996b); con-centrations in a few days overreach the threshold levels for the per-ception of �fusty� defects. If temperatures are high, the growth of yeastscan produce considerable ethanol and ethyl acetate, leading to the onsetof the �winey� defect. The possible presence of Acetobacter is respon-sible for the �vinegary� defect because of the production of acetic acidAngerosa et al. 1996a).

If fruit storage lasts several days, mold may develop, generally Pen-icillium and Aspergillus (Marsilio and Spotti 1997), whose enzymesinterfere with those of the olive fruit involved in the LOX pathway(Kaminski et al., 1974; Wurzenberger and Grosch 1984; B€orjessonet al. 1993). Mold invasion not only can cause complete rotting of fruitsbut can reduce production of C6 and C8 compounds (Angerosaet al. 1999b). Storage temperatures of about 5�C in air considerablyreduce fungal growth, so olives could be stored for at least 30 days atthat temperature without great changes in the sensory quality of theresulting oil (Kiritsakis et al. 1998). Olive storage also has a strong effectin the phenolic degradation in the fruit beforemechanical extraction. Asconsequence, EVOO after olive storage shows lower amount of phenolsin comparison to fresh fruits (Angerosa et al. 2004; Servili et al. 2004,2009a).

B. Olive Fruit Crushing

Crushing and malaxation are critical points in the mechanical ex-traction process that affect phenolic and volatile compositions of

122 P. INGLESE ET AL.

EVOO. In fact, the main hydrophilic phenols of EVOO, such assecoiridoid aglycons, originate during this phase by the hydrolysisof oleuropein, demethyloleuropein, and ligstroside, and catalyzedQ19by endogenous a-glucosidases. During malaxation, the concentrationof secoiridoid aglycons and phenolic alcohols decreases in olivepastes and in the relative oils, with increasing temperature andprocess time.

The impact of crushing in EVOO phenolic and volatile compoundscan be related to the different distribution of endogenous oxidoreduc-tases and of phenolic compounds in the constitutive parts of the olivefruit (pulp, stone, and seed). The peroxidase (POD), in combinationwiththe polyphenoloxidase (PPO), is the main endogenous oxidoreductaseresponsible for the phenolic oxidation during the process, and it occursin high amounts in the olive seed. The phenolic compounds, on thecontrary, are largely concentrated in the pulp while stone and seedcontain little quantities of these substances (Servili et al. 2004, 2007).Consquently, the crushing methods that reduce seed tissue degradation,such as the stoning process or mild seed crushers, limit the release ofPOD in the pastes. This prevents the oxidation of hydrophilic phenolsduring malaxation and improves their concentration in the EVOO(Table 3.7) (Servili et al. 1999a, 2004, 2007).

Crushing operative conditions also affect the volatile composition ofEVOO (Table 3.8). Almost all volatile compounds responsible for theflavor of high-quality EVOOs arise at the moment of olive pulp tissuedisruption. Thus the effectiveness of crushing plays an important role intheir production. The use of a hammer mill crusher, or other crushersthat produce a more violent grinding of pulp tissues, causes an increaseof the olive paste temperature and a reduction of HPL activity. The use ofnew crushers, such as blade crushers, improves the concentration ofvolatile compounds, especially hexanal, trans-2-hexenal, and C6 esters,leading to a positive increase of the intensity of �cut grass� and �floral�notes (Table 3.8) (Angerosa et al. 2004).

Several researchers have shown that olive stoning during EVOOmechanical extraction process increases the phenolic concentration inEVOO (Angerosa et al. 1999a; Lavelli and Bondesan 2005; Mulinacciet al. 2005) and, at the same time, modifies the composition of volatilecompounds produced by the LOX pathway. This increases the concen-tration of volatile substances related to the �green� sensory notes(Table 3.7) (Servili et al. 2007). These results are particularly importantbecause they demonstrate that the enzymes involved in the LOX path-way have a different activity in the pulp and in the seed (Table 3.8)(Servili et al., 2007).

3. FACTORS AFFECTING EXTRA-VIRGIN OLIVE OIL COMPOSITION 123

C. Olive Paste Malaxation

The distribution of phenols between the oil and the water phase, asrelated to their liposolubility, is not the onlymechanism involved in thereduction of the EVOO phenolic concentration during malaxation. Theoxidative reactions catalyzed by endogenous oxidoreductases such asPPO and POD, which promote the phenolic oxidation in the pastesduring this step, strongly affect their concentration in the oily phase. Theenzyme inhibition obtained by the reduction of the O2 concentration inthe covered malaxers improves the concentration of hydrophilic phe-nols in the olive pastes and in the corresponding EVOO. In this context,the O2 control duringmalaxation can be considered a new technologicalparameter that, in combination with the traditional ones (time andtemperature of the process), can be used to optimize the EVOO phenolicand volatile concentrations (Tables 3.9 and 3.10) (Servili et al. 2004,

Table 3.7. Effect of different crushers and the stoning process on EVOOs phenolic

compositionz (mg/kg) of �Franotio� cultivar. Source: upubl. data.

Phenolic compound

Hammer

crusher

Pre-crusher

þ blade

crusher

Crusher with

low turns

number Stoned

I Ripening stage (pigmentation index 0.95)y

3,4 DHPEA 1.0� 0.1 0.5� 0.0 2.2�0.0 8.0� 0.2

p-HPEA 11.2�0.7 12.8� 0.4 16.0� 0.2 19.8� 0.3

3–4 DHPEA-EDA 71.8�1.9 78.8� 2.3 89.6� 1.6 98.8� 2.9

p-HPEA-EDA 54.3�0.8 58.7� 0.9 60.1� 0.8 55.4� 0.9

(þ )-1-acetoxypinoresinol 31.0�1.7 41.9� 1.4 39.4� 0.8 43.7� 1.2

(þ )-pynoresinol 9.8� 0.2 11.9� 0.2 13.8� 0.3 15.3� 0.1

3–4 DHPEA-EA 76.0�1.1 93.6� 2.1 98.5� 3.0 94.3� 3.1

Sum of the phenolic

compounds

255.1� 2.4 298.2�3.3 319.6� 3.5 335.3�4.4

II Ripening stage (pigmentation index 1.49)

3,4 DHPEA 3.4� 0.1 1.1� 0.0 1.1�0.3 4.2� 0.1

p-HPEA 8.4� 0.3 13.4� 0.7 13.8� 0.3 7.5� 0.3

3–4 DHPEA-EDA 35.7�1.2 40.8� 2.2 44.0� 1.9 51.6� 4.2

p-HPEA-EDA 32.2�1.5 39.2� 0.6 45.2� 0.9 40.5� 3.7

(þ )-1-acetoxypinoresinol 26.3�1.0 29.1� 0.4 31.4� 0.2 30.9� 0.9

(þ )-pynoresinol 6.6� 0.4 7.6� 0.2 8.7�0.6 8.3� 0.6

3–4 DHPEA-EA 54.8�1.9 54.6� 0.3 58.3� 1.1 65.8� 4.3

Sum of the phenolic

compounds

167.5� 2.8 185.7�2.5 202.4� 2.5 208.7�6.9

z Phenolic content is the mean value�SD of three independent experiments.y Pigmentation index was determined according to Pannelli et al. (1994).

124 P. INGLESE ET AL.

2008, 2009b). The time of exposure of olive pastes to the air contact(TEOPAC) was studied as process parameter to regulate the averagedconcentration of O2 in the paste and as consequence the phenolicamount in theEVOO (Servili et al. 2003a, 2003b). Thenatural productionof inert gas, such as CO2, due to the olive cell metabolism duringmalaxationmay be combinedwith the use of nitrogen or argon to reducethe O2 contact with the olive pastes during malaxation (Parentiet al. 2006a, 2006b; Servili et al. 2008).

The application of new technologies, such as the EVOO mechanicalextraction from destoned pastes, improves the oil phenolic concentra-tion, confirming the relationships between the control of oxidativereactions during extraction process of the EVOO and its phenoliccontent. Since the POD is highly concentrated in the olive seed, thestoning processQ20 , excluding this constitutive olive part before themalaxa-tion and partially removing the POD activity in the pastes can reduce theenzymatic degradation of the phenols in the oils during this phase, thusincreasing their concentration in the EVOO and its oxidative stability(Servili et al. 2007, 2009b).

The oxidative reactions occurred in the pastes during the malaxationexplains the relationships between EVOO phenolic concentration andmalaxing temperatures (Servili et al. 2004, 2009a,b). The O2 present inthe pastes during the malaxation activate POD and PPO that oxidizephenolic compounds according to temperature and, as a consequence,

Table 3.8. Effect of different crushers and the stoning process on the volatile

compositionz (mg/kg) of �Frantoio� EVOO. Source: unpubl. data.

Volatiles

Hammer

crusher

Pre-crusher

þ blade

crusher

Crusher with

low turns

number Stoned

AldehydesPentanal 236� 4 273� 2.1 18� 1 36� 2.7a

Hexanal 280� 2.9 511� 35.7 554� 0.3 748� 27.9b

2-Hexenal (E) 43600� 327 44719� 208 39812� 587 27866� 705.5b

2,4-Hexadienal (E,E) 19� 0.1 42� 3.5 342� 14.4 256� 12.1b

Alcohols1-Pentanol 167� 5.2 94� 4.7 23� 0.7 21� 1.7b

2-Penten-1-ol (E) 16611 91� 5.1 52� 3.5 13� 2.1b

1-Penten-3-ol 960� 53.2 899� 43.3 522� 49.2 114� 4.6b

1-Hexanol 1788�57 2152� 74 512� 41 405� 18.4b

3-Hexen-1-ol (Z) 88� 22.2 104� 10.1 49� 2.3 164� 8.0b

3-Hexen-1-ol (E) 22� 0.2 20� 0.1 10� 0.2 22� 2.3b

z Data are the mean values of three independent experiments,�SD.

3. FACTORS AFFECTING EXTRA-VIRGIN OLIVE OIL COMPOSITION 125

reduce their concentration in EVOOs obtained by pastesmalaxed at hightemperatures. The traditional malaxers, which contain high amounts ofO2 dissolved in the paste during the process due to the air contactrepresent a classical example of the relationship between high tempera-tures and EVOO phenolic loss. Low amounts of O2, on the contrary,inhibit the oxidative reaction of phenols during the malaxation; in thiscase, their concentration in the EVOO increaseswith high temperatures,because of a higher solubility of such substances in the EVOOs (Serviliet al. 2008, 2009a,b).

Interactions between polysaccharides and phenolic compounds inthe olive pastes may also be involved in the loss of such substancesduring the processQ21 . Polysaccharides may link the phenols, thus reduc-ing their release in the oil during crushing and malaxation. In thisregard, it has been shown that the use of technical enzymatic prepara-tions containing cell wall–degrading enzymes during the malaxationcan improve the EVOO phenolic concentration (Ranalli and De Mattia1997; Vierhus et al. 2001).

Table 3.9. Phenolic compositionz (mg/kg) of EVOOs obtained after malaxation in

different initial atmosphere compositions. Source: Servili et al. 2008.

Initial O2 partial pressure in the malaxer chamber

headspace (kPa)

Phenolics 0y Xx 50 100

Ogliarola3,4-DHPEA 1.0 a 0.8 b 0.6 c 0.8 d

p-HPEA 3.1 a 3.1 a 4.0 b 4.2 b

3,4-DHPEA-EDA 247.7 a 235.2 b 117.8 c 118.1 c

p-HPEA-EDA 126.4 a 118.6 b 86.3 c 85.4 c

(þ )-1-acetoxypinoresinol 21.0 a 25.4 b 22.3 ac 24.1 bc

(þ )-pinoresinol 6.8 a 7.6 b 7.0 a 7.1 a

3,4-DHPEA-EA 212.2 a 186.4 b 100.9 c 98.2 c

Coratina3,4-DHPEA 6.8 a 3.2 b 4.4 b 1.4 c

p-HPEA 10.0 a 5.9 bc 7.8 b 4.4 c

3,4-DHPEA-EDA 478.9 a 437.7 b 343.1 c 229.9 d

p-HPEA-EDA 144.2 a 135.3 b 126.2 c 125.1 c

(þ )-1-acetoxypinoresinol 30.8 a 25.8 b 29.2 ab 27.1 ab

(þ )-pinoresinol 8.1 ab 8.0 a 8.6 b 7.9 a

3,4-DHPEA-EA 475.6 a 361.9 b 339.2 b 170.6 c

z Data are means of three independent experiments, Values in each row having different

letters (a–d) are significantly different from one another at p < 0.0.y Saturated with N2.x Corresponding to the air composition.

126 P. INGLESE ET AL.

Furthermore, it has been observed that the enzymes involved in theLOX pathway remain active during the malaxation process since theconcentration of volatile compounds increases in the pastes (Espostoet al. 2008; Servili et al. 2009a). The analysis of the composition ofEVOOs produced from traditional and stoned olive pastes confirms thatthe amounts of C6 unsaturated aldehydes were higher in �stoned�EVOOs than in the �traditional� ones because of the presence of the

Table 3.10. Volatile composition (mg/kg) of extra-virgin olive oils obtained after

malaxation in different initial atmosphere compositions. Source: Servili et al. 2008.

Initial O2 partial pressure in the malaxer chamber

headspace (kPa)

Volitiles 0a 30b 50 100

OgliarolaAldehydes2-Pentenal (E)z 291.5 ab 343.0 a 247.5 b 269.5 b

Hexanal 939.5 a 1546.0 b 1011.5 a 1499.5 b

2-Hexenal (E) 43645.0 a 39130.0 b 37315.0 b 38170.0 b

Alcohols1-Pentanol 28.5 a 128.0 b 122.5 b 158.0 c

2-Penten-1-ol (E) 55.5 a 63.0 a 50.5 ab 38.5 b

1-Penten-3-ol 567.0 a 871.0 b 690.0 c 809.5 d

1-Hexanol 8357.0 a 9699.0 b 11660.0 c 13675.0 e

3-Hexen-1-ol (E) 35.0 a 41.0 a 47.5 b 61.5 c

3-Hexen-1-ol (Z) 286.5 a 434.0 b 341.0 c 400.5 d

2-Hexen-1-ol (E) 7662.5 a 8616.0 b 9355.0 c 9780.0 c

CoratinaAldehydes2-Pentenal (E) 548.5 ab 509.7 b 636.7 c 613.0 ac

Hexanal 1187.0 a 1624.3 bc 1532.1 b 1744.0 c

2-Hexenal (E) 51565.0 a 52900.0 ab 54340.5 b 53920.0 b

Alcohols1-Pentanol 40.0 a 54.3 b 39.4 a 48.0 ab

2-Penten-1-ol (E) 87.5 a 67.0 b 105.8 c 105.0 c

1-Penten-3-ol 890.0 a 820.0 b 1093.5 c 1185.0 c

1-Hexanol 2326.0 a 3694.2 b 1788.0 c 2170.0 a

3-Hexen-1-ol (E) 25.5 ab 31.6 a 20.0 b 21.0 b

3-Hexen-1-ol (Z) 561.0 a 513.6 b 486.3 b 498.0 b

2-Hexen-1-ol (E) 3654.5 a 5905.0 b 3350.1 a 4185.0 c

a Saturated with N2;b Ccorresponding to the air composition.z Data are the mean values of three independent experiments, standard deviation is

reported in brackets. Values in each row having different letters (a–d) are significantly

different from one another at p <0.0.

3. FACTORS AFFECTING EXTRA-VIRGIN OLIVE OIL COMPOSITION 127

seed responsible of the C6 alcohols increase during the traditionalprocess (Servili et al. 2007).

Time and temperature of malaxation affect the volatile profile andtherefore the sensory characteristics of the resulting EVOOs (Angerosaet al. 2004; Servili et al. 2009a,b). The increase of C6 and C5 carbonylcompounds, especially of hexanal, which, due to its low odor thresh-old, represents an important contributor to the olive oil flavor, is themain effect of the malaxation time, whereas high temperatures ofmalaxation promote a fall of esters and cis-3-hexen-1-ol and an accu-mulation of hexan-1-ol and trans-2-hexen-1-ol, both considered bysome authors as elicitingQ22 odor not completely agreeable (Angerosaet al. 2004; Servili et al. 2009b). In addition, high temperatures in themalaxation step make active the amino acid conversion pathway withproduction of considerable amounts of 2-methyl-butanal and 3-methyl-butanal, but without accumulation of corresponding alcohols corre-lated with the �fusty� defect. The sensory analysis of the relativeEVOOs highlights a weakening of typical �green� attributes with theprolonging of malaxation time and of all sensory notes with hightemperatures during the malaxation (Angerosa et al. 2004; Serviliet al. 2009b).

D. EVOO Extraction Systems

Extraction systems, such as pressure and centrifugation, play an impor-tant role in the EVOO phenolic and volatile composition. The dilutionwater added to the olive pastes during the centrifugation modifies thedistribution of hydrophilic phenols between oil and water, enhancingtheir loss through thewater phase. Several studies have been carried outto compare the traditional three-phase decanter with the new two-phasedecanter (Di Giovacchino et al. 1994; Montedoro 1996; Ranalli andAngerosa 1996; Stefanoudakii et al. 1999b; Servili et al. 2002b). Similarresults were obtained, using Spanish and Greek cultivars, by otherauthors (Di Giovacchino et al. 2001; Garcia et al. 2001). An increasedconcentrationofphenolswas alsoobserved inEVOOsextractedby three-phase decanters at low water addition as compared to the traditionalthree-phase centrifuges (Table 3.11) (Amirante et al. 2001). The increaseof the EVOO phenols concentration was observed according to thereduction of water addition used during the mechanical extractionprocess by three- and two-phase decanters (Montedoro 1996).Other results obtained using two typical Italian cultivars, such as�Coratina� and �Ogliarola�, provide evidence that higher concentrations

128 P. INGLESE ET AL.

of hydrophilic phenols in EVOOs were obtained using two-phase de-canters as compared to the traditional three-phase centrifuges (DeStefano et al. 1999; Servili et al. 2002a).

E. EVOO Storage

During storage, thephenolic composition of the EVOO ismodifiedby theendogenous enzymatic activities contained in the cloudy phase. Theseenzymes may reduce the �pungent� and �bitter� sensory notes, theintensity of which is strictly to the aglicon secoiridoids� content and,at the same time, can cause olfactive and taste defects. The oil filtration,partially removing water and enzymes from EVOOs, allows the stabili-zation of EVOOphenolic content during storage (Montedoro et al. 2005).The olive oil profile changes during storage because of the simultaneousdrastic reduction of compounds from LOX pathway and the neo-formationQ23 of volatile compounds responsible for some common defectsknown as �rancid,� �cucumber,� and �muddy sediment� (Morales andAparicio 1997, Angerosa et al. 2004; Servili et al. 2009a). Amongsaturated aldehydes, nonanal and above all hexanal increase in parallelto the oxidation process, but this last cannot be considered a usefulmarker of oxidation since it is also present in the aroma of high-qualityEVOOs (Angerosa et al. 2004; Servili et al. 2009b). Furthermore, thepresence of sediment consequent to unfiltered olive oil decantationduring its storage candetermine, under suitable temperature conditions,

Table 3.11. Effect of thewater reductionduring centrifugation onEVOOsphenolic

composition (mg/kg). Source: Servili et al. 2002.

Coratina Ogliarola

Two phases Three phases Two phases Three phases

3,4 DHPEA 0.9� 0.02a 0.6� 0.08b 0.7�0.11a 0.5� 0.11a

p-HPEA 3.7� 0.07a 2.3� 0.08b 3.3�0.10a 4.2� 0.10b

Vanillic acid 0.4� 0.01a 0.2� 0.01b 0.3�0.01a 0.1� 0.05b

Caffeic acid 0.2� 0.01a 0.1� 0.02b 0.1�0.01a 0.2� 0.03b

3,4 DHPEA-EDA 522.2�13.5a 427.2� 13.8b 30.1� 1.03a 18.5�0.68b

p-HPEA-EDA 78.2� 0.52a 67.3� 2.55b 21.0� 0.82a 22.4�0.33a

Lignans 38.4� 0.10a 35.6� 1.11b 48.0� 3.40a 46.7�5.78a

3,4 DHPEA-EA 351.7�11.0a 244.9� 13.6b 68.0� 6.00a 52.0�3.11b

Total polyphenols 673.0�4.0a 585.0� 7.0b 304.0� 5.0a 263.0� 4.0b

Induction time [h] 17.8� 0.1a 15.5� 0.2b 5.2�0.1a 4.6� 0.1b

Q1z Data are the mean values�SD of three independent experiments. Values in each row

within cvs. having different letters are significantly different fromone another at p < 0.01Q2 .

Q36Q37

3. FACTORS AFFECTING EXTRA-VIRGIN OLIVE OIL COMPOSITION 129

the production of unpleasant compounds responsible for the typical�muddy sediment� defect due to the fermentation that produces com-pounds, likely of the butyric kind (Angerosa et al. 2004; Servili et al.2009b).

VI. SUMMARY AND CONCLUSIONS

The composition of olive oil results from a multivariate interaction inwhich genotype-, environment-, and agronomic-dependent factors areinvolved. The genotypemay control genetic traits accounting for the ratepattern of fruit growth, oil accumulation in mesocarp cells, and fruitripening,while the genotype� environment interaction changes the rateof fruit growth, oil accumulation, and fruit ripening pattern. The latteraccounts for large changes in oil composition and sensory features. Theinfluence of genotype is linked to differences in the fruit growth andripeningpattern, although all those factors thatmayhave an influence onfruit size, flesh/pit ratio, and relative growth rate have a lower and moreerratic influence on the olive oil composition. The genotype is theprimary source of sensory differences. This has been proven for mostof the cultivars, giving themQ24 a specific role in gastronomy. The influenceon olive oil composition of environmental factors, such as temperaturesduring fruit growth and ripening or water availability, may also be afunction of changes in the fruit growth and ripening patterns and of theoil accumulation rate pattern. Some facts have been generally recog-nized, such as the changes of saturated versus unsaturated fatty acidsratio in relation to temperature and latitude or the progressive reductionof polyphenols content in the oil along with fruit ripening or with theincrease of water availability. Crop load influences the fruit ripeningrate pattern and the rate pattern of oil accumulation, and this mayaccount for differences in oil composition related to polyphenols andfatty acid content.

From a historical point of view, the recognition of EVOO geographicaland genetic originwas not amajor cultural and commercial issue of oliveoil production and trading, as it always has been for wine production.The multivarietal composition of the traditional groves, due to thebiology of flowering and pollination of the species, the highly differen-tiated orchard systems and fruit harvesting periods andmethods, aswellas the different oil extraction technologies and storage systems, and,eventually, the historical separation between the olive farmers and theoil mill industry, together made it very difficult to establish effectivepolicies endorsing the geographical and genetic origin of the EVOO as a

130 P. INGLESE ET AL.

cultural and commercial value recognized by consumers. At present,even at the legislative level, the EVOO origin has become a central issuein the marketing strategies, particularly for those Mediterranean coun-tries with a wide olive germplasm and rich gastronomy.

However, provenience-related differences of EVOO quality wereclaimed even by Pliny the Elder during the Roman age, and modernresearch activityhasmade it possible to appreciate the large variability ofEVOO composition and properties. Nevertheless, it is difficult to un-derstand the interrelation of the different sources of variation and toapply appropriate strategies for a consistent and clear EVOOproductionand identification. This is the greatest paradox of EVOO production:Typical cultivar-related sensory properties are largely unknown orunrecognizable by consumers and still are underutilized in marketingstrategies.More information is needed on the genotype and environmentinfluence on fruit ripening and EVOO components evolution in order toregulate the ripening process and standardize the characteristics thatmake any EVOO unique and typical for its geographical and geneticorigin.

On the basis of our understanding of the technological factors affectingEVOOquality,we suggest the following approach to improve this rapidlyexpanding industry:

1. Improve management of oil mechanical extraction processes forworking olive pastes with and without olive stones.

2. Improve the malaxation efficiency through technological coadiu-vantsQ25 (inert materials and enzymatic preparations) to increasecoalescence and, as a consequence, the recovery of oil andphenoliccompounds.

3. Apply technologies, in malaxation particularly, to improve theefficiency of thermal exchange.

4. Control and regulate oxygen percentage in contact with the olivepaste during the malaxation to optimize the phenolic content aswell as the flavor of EVOO.

The olive oil consumption has increased, worldwide, from 1990 to2008 by 70%,moving from 1,666Mt to 2875Mt. Spain, Italy, and Greeceaccount for 77% of the olive oil production and for 65% of its consump-tion. Per capita consumption ranges fromQ26 25 l year�1 in Greece to15 l year�1 in Spain and Italy, 5.5 l year�1 for the European Community,and 0.6 l year�1 for the United States. The United States is the fourthlargest market, and the oil consumption has increased, in the sameperiod, three times (88Mt in 1990 and 250Mt in 2008) (IOC 2008). This

3. FACTORS AFFECTING EXTRA-VIRGIN OLIVE OIL COMPOSITION 131

increase in oil use has been accompanied by a general decrease of itsprice at world level. This creates large problems for those countries suchas Italy,which have high labor costs, a very traditional industry based onsmall crop areas of single farmers, and thousands of artisan mills (5 tday�1). Spain and the New World producers are focusing on high-density orchards and large mills, which makes it possible to reducecosts. Different strategies in Spain and Italy are, then, related to thestructure of their olive oil industry. For instance, 66% of the olive farmsin Italy arededicated to self-consumption, and thevariability of cultivarsand environments explain the great number of PDO in Italy (37, whichmeans 43%ofPDOdedicated to olive in theEuropeanUnion).Hundredsof new brands appear in themarket every year, and consumers are likelyto expect a large opportunities to choose high-quality oils with differentlevels of price. It is hoped that the role of genotype (cultivar) andcultivar� environment interaction will be more and more appreciatedby consumers and that the fraudulentmarketing of inferior productswillbe eliminated.

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3. FACTORS AFFECTING EXTRA-VIRGIN OLIVE OIL COMPOSITION 147

Author Query1. use noun instead of �ones�?

2. is this word cut?

3. EVOO�s?

4. polyphenols?

5. spell these out?

6. do not indicate what?

7. is this correct?

8. is this correct word?

9. correct word?

10. clarify

11. spell out

12. correct word?

13. clarify

14. meaning OK?

15. correct word?

16. is minus symbol after % correct?

17. clarify word: entire?

18. correct word?

19. what is catalyzed?

20. is this phrase correct here?

21. which process?

22. correct word?

23. word OK?

24. the sensory differences?

25. correct word?

26. clarify all these numbers; is the one correct? Is the symbol in thesuperscript a minus or just a dash?

27. verify title

28. word OK?

29. clarify these numbers?

30. here and above: clarify which way accent belongs

31. update.

32. full source needed?

33. name of publisher?

34. is this correct? to what does Ed. refer?

35. where is this on table? I don�t see superscript y, just I.U.

36. here and throughout, should p be lowercase or capped? style varies

37. Earlier tables had space after number and before letters a and b.please verify. Also where is superscript z on the table?