Olive Cultivation Italy

THE OLIVE CULTIVATION,
OLIVE GERMPLASM
TABLE OLIVE
AND OLIVE
OIL INDUSTRY IN ITALY
Edited by Innocenzo Muzzalupo

OLIVE GERMPLASM
THE OLIVE CULTIVATION,
TABLE OLIVE AND OLIVE
OIL INDUSTRY IN ITALY


Olive Germplasm
The Olive Cultivation, Table Olive and Olive Oil Industry in Italy
http://dx.doi.org/10.5772/3314

Contributors
Catherine Marie Breton, Peter Warnock, Andr Jean Bervill, Adriana Chiappetta, Innocenzo
Muzzalupo, Caterina Briccoli Bati, Elena Santilli, Ilaria Guagliardi, Pietro Toscano, Adolfo Rosati,
Silvia Caporali, Andrea Paoletti, Nino Iannotta, Stefano Scalercio, Giuliana Albanese, Maria
Saponari, Francesco Faggioli, Rosario Muleo, Michele Morgante, Riccardo Velasco, Andrea
Cavallini, Gaetano Perrotta, Luciana Baldoni, Maurizio Servili, Agnese Taticchi, Sonia Esposto,
Beatrice Sordini, Stefania Urbani, Pietro Toscano, Francesco Montemurro, Amalia Piscopo,
Marco Poiana, Massimiliano Pellegrino, Enzo Perri, Giovanni Sindona, Domenico Taverna, Enzo
Perri, Cinzia Benincasa, Domenico Britti, Daniela Impellizzeri, Antonio Procopio, Salvatore
Cuzzocrea, Flora Valeria Romeo, Barbara Lanza

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Olive Germplasm The Olive Cultivation, Table Olive and Olive Oil Industry in Italy,
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Contents

Preface IX
Section 1 Olive Germplasm 1
Chapter 1 Origin and History of the Olive 3
Catherine Marie Breton, Peter Warnock and Andr Jean Bervill
Chapter 2 Botanical Description 23
Adriana Chiappetta and Innocenzo Muzzalupo
Chapter 3 Cultivation Techniques 39
Caterina Briccoli Bati, Elena Santilli,
Ilaria Guagliardi and Pietro Toscano
Chapter 4 Floral Biology:
Implications for Fruit Characteristics and Yield 71
Adolfo Rosati, Silvia Caporali and Andrea Paoletti
Chapter 5 Susceptibility of Cultivars to Biotic Stresses 81
Nino Iannotta and Stefano Scalercio
Chapter 6 Phytosanitary Certification 107
Giuliana Albanese, Maria Saponari and Francesco Faggioli
Chapter 7 Olive Tree Genomic 133
Rosario Muleo, Michele Morgante, Riccardo Velasco,
Andrea Cavallini, Gaetano Perrotta and Luciana Baldoni
Section 2 Virgin Olive Oil 149
Chapter 8 Technological Aspects of Olive Oil Production 151
Maurizio Servili, Agnese Taticchi, Sonia Esposto,
Beatrice Sordini and Stefania Urbani
Chapter 9 Olive Mill By-Products Management 173
Pietro Toscano and Francesco Montemurro
VI Contents

Chapter 10 Packaging and Storage of Olive Oil 201
Amalia Piscopo and Marco Poiana
Chapter 11 Sensory Analysis of Virgin Olive Oils 223
Innocenzo Muzzalupo, Massimiliano Pellegrino and Enzo Perri
Chapter 12 Modern Methodologies to Assess the Olive Oil Quality 239
Giovanni Sindona and Domenico Taverna
Chapter 13 Olive Oil Traceability 265
Enzo Perri, Cinzia Benincasa and Innocenzo Muzzalupo
Chapter 14 Oleuropein an Olive Oil Compound in Acute and Chronic
Inflammation Models: Facts and Perspectives 287
Domenico Britti, Daniela Impellizzeri,
Antonio Procopio and Salvatore Cuzzocrea
Section 3 Table Olive 319
Chapter 15 Microbiological Aspects of Table Olives 321
Flora Valeria Romeo
Chapter 16 Nutritional and Sensory Quality of Table Olives 343
Barbara Lanza

Preface

Among cultivated plants, the olive (Olea europaea L.) is the sixth most important oil
crop in the world, presently spreading from the Mediterranean region of origin to new
production areas, due to the beneficial nutritional properties of olive oil and to its high
economic value. Olive oil has favourable nutritional properties, and consequently, its
consumption, which was traditionally restricted to the Mediterranean area (77% of the
world production area), is increasing worldwide (mainly the United States, Canada,
Australia and Japan). Some olive varieties are cultivated specifically for table
consumption, but the majority are used for oil extraction. Based on estimates by the
FAO 2010, Plant Production and Protection Division Olive Germplasm, the worlds
olive germplasm contains more than 2,629 different varieties, with many local varieties
and ecotypes contributing to this wealth.
Olive growing in Italy is very important, but there is still a high degree of confusion
regarding the genetic identity of olive cultivars. The problem of characterizing the
olive tree germplasm is complicated not only by the wealth of its genetic patrimony,
but also by the absence of reference standards and a well defined system of
nomenclature that is free from homonymy and synonymy. Only recently, some Italian
research projects (i.e. COLLEZIONI; CERTOLIO; GERMOLI; OLEA; OLVIVA; RGV-
FAO and RIOM projects) have raised the issue of the standard reference variety are
trying to achieve a standard certificate for each variety present in different Italian
regions. The extent of this diversity has important implications for both the adaptation
of varieties to their local environment and for the optimization of the agronomical
performance of these varieties under a given set of environmental conditions. For
example, every initiative promoting olive cultivation should consider the potential
repercussions of such action on any local olive varieties. Every region should preserve
its own plant material in order to safeguard both the adaptation and productivity of
the species and the unique characteristics of the regions olive oil. However, the study
of intra-varietal polymorphisms is important since they may have traits that, although
not considered important in the past, might be important to meet the challenges of
modern olive growing (i.e. resistance to low temperatures, salinity tolerance, etc.).
The systematic collection of Italian olive varieties for deposit into specific catalogue
fields began in Italy in the 1980s. A similar international collection was started in 1997
by the Consiglio per la Ricerca e la sperimentazione in Agricoltura centro di ricerca
X Preface

per lOLIvicoltura e lindustria olearia (Agricultural Research Council - Agricultural
Research Council - Olive Growing and Oil Industry Research Centre, CRA-OLI) in
Italy. Collection entailed the following steps: a survey of the territory, individuation,
basic characterization, and introduction into the germplasm field. To date, roughly 500
varieties have been introduced into the CRA-OLI collection, and this list has been
published (web site http://apps3.fao.org/wiews/olive/olcoll2.jsp).
A useful olive germplasm collection also requires an organizational system devoid of
homonymy, synonymy and mislabeling so that a reliable classification of all varieties
can be achieved without unnecessary confusion. Recent research has focused on using
morphological, bio-agronomical and molecular markers to characterize and identify
olive varieties. The identification of varieties by using molecular markers is a crucial
aim of modern horticulture, because such a technique would greatly facilitate breeding
programmers and germplasm collection management.
The book "Olive Germplasm" was born from the idea of gathering and evaluating
research results obtained on the growing of olive trees in the CRA-OLI collection field.
In fact, after more than 20 years from the planting of the olive trees in the collection
field, a quantity of validated data was obtained which was considered to be sufficient
for the realization of this work which contains the results of innovative research on its
main products such as olive oil and table olives.
The book is divided into two parts: I) the olive cultivation, table olive and olive oil industry
in Italy and II) Italian catalogue of olive varieties.
This book highlights the importance of studying the distribution, characterization and
valorization of genetic diversity for better exploitation of olive resources and for the
design of breeding programs. In addition, it reports some important aspects about the
current state of the art in the chemistry, analysis and quality assessment of table olives
and of olive oil and its minor constituents, extraction of olive oil from the fruits, water
treatment, and innovative approaches for the production of olive oil based products. It
also discusses bioavailability, pharmacological and other properties of bioactive
ingredients in the light of new evidence on the composition of olive oil and table
olives. Furthermore, it addresses some aspects related to biotechnology and other
technologies to retain optimum levels of such bioactive ingredients in the various olive
oil forms and to protect the environment from olive mill waste products.
The purpose of this book is to provide a glimpse into the table olive and olive oil
industry by presenting the thoughts of some of the scientists who are engaged in the
development of new tools and ideas used to improve the quality of products, often
from very different perspectives.
I hope and trust that the information in this report will be used as the basis for policy
and technical decisions to strengthen national efforts to conserve and utilize the
treasures incorporated in the worlds plant genetic resources to address the urgent
problems faced by agriculture today and tomorrow.
Preface XI

I would like to express my deepest gratitude to all the authors who contributed to this
book by sharing their valuable works with us. A special thanks to the reviewers who
have used their valuable time in the improvement of the chapters. Finally, thanks to
the publishing house that provided me with great professionalism in the realization of
the book.

Innocenzo Muzzalupo
Agricultural Research Council -
Olive Growing and Oil Industry Research Centre, Rende (CS),
Italy
Section 1

Olive Germplasm

Chapter 1
Origin and History of the Olive
Catherine Marie Breton, Peter Warnock and Andr Jean Bervill
Additional information is available at the end of the chapter
1. Introduction
To begin, the methodology followed to reconstruct the origin and history of the olive is
presented. The genetic structure (density of alleles across the geographic distribution of
individuals) based on allele frequencies of the present oleaster tree in the Mediterranean
Basin computed with different methods of comparison with the genetic structure of
cultivars grouped based on their geographic and genetic origins infers several possible
scenarios for the transition from the oleaster to the olive. To screen among the scenarios
requires solid dating in oleaster presence, diffusion and physical remains (from oleaster and
cultivar trees) from different sites. Consequently, reconstructing the origin of the olive is
based upon data from diverse disciplines and integrating them appears fruitful. Genetic
data show that an event (such as bottleneck, migration, differentiation, adaptation) has
occurred, but it cannot be dated. Thus it requires crossing genetic data with data gathered
from different biological disciplines to make a strong case for this history. We examine
successively:
1. The present distribution of the olive and its counterpart the wild olive,
2. Archaeological records of wood charcoals and artifact remains, ethno-botanical
methods, pollen databases, and chemical methods for oil traces;
3. Molecular data obtained through 1995 to the present from Olea europaea and including
relationships between varieties. We examined successively the evolution of methods to
analyze data, the data sets examined through Bayesian methods, and the relationships
between the oleaster and the olive in order to propose a wide scenario.
This article does not attempt to review all relevant literature on the history or background of
the research, but rather focuses on the history of the olive tree and infers some shortcuts in
the references of the work published. We apologize and readers can refer to recent general
publications that fill the gap here [1, 2, 3, 4].

Olive Germplasm The Olive Cultivation, Table Olive and Olive Oil Industry in Italy 4
2. Dogma on the olive tree history
Domestication is based upon conscious behavior by humans over several centuries aimed at
selecting among a species natural diversity those individuals that satisfied human
requirements [5] such as in yield (seeds or other organs), composition (sugar, starch, fats,
), and to harvest and maintain the cultivation of said species (seed attachment to spike or
capitulum, dormancy of seeds, ).
However, human practices have had effects on the plant genome other than those intended
through the conscious behavior of human domestication, effects which are highly
documented for many crop species, especially cereals [6] barley, maize [7] Maize, [8]
sunflower [9, 10], as examples.
For wheat and grains which were weeds in wheat fields, the main domestication center has
been the Near-East in the Fertile-Crescent documented by [11, 12, 13]. Botanists have
therefore inferred that the olive tree may have followed a similar history. The presence of
Olea cuspidata in the Mountains of Iran suggested a relationship between this species and
the olive (Figure 1). Initial molecular results have eliminated this hypothesis and definitively
anchored the origin of the olive in the wild olive only [14, 15]. Relationships between the
subspecies of Olea europaea are not addressed in this chapter.
Present populations of wild olive (called oleaster) have questioned researchers on their
origins. In the East of the Mitterrand they were considered natural. The famous botanist
Pelletier has written, the motherland of the wild olive tree is Anatolia, where numerous
wild olive trees make up dense groves, and De Candolle opines that olive was originated
from Asia Minor and spread from Syria over to Greece via Anatolia [16]. The Flora of the
Mediterranean basin is split into eastern and western halves by a line between the Cyrenaica
basin of Libya and the Adriatic sea [17, 18, 19, 20]. Effectively, Botanists believe that the
oleaster was native to the eastern or oriental half, and once domesticated the olive was
transported by humans in the western half, where it thrives as feral form. Thus, all oleaster
trees in the western Mediterranean would be feral trees [21].
This supposition has been widely accepted on plenty of open commercial internet sites
selling olive products. We believe that this assertion is false and will demonstrate that idea
through the results given from various teams working on the olive (Italy, Spain, France,
Portugal, Morocco, Tunisia, ).
Using the first molecular markers, namely isozymes, [22] has shown genetic differences
between the wild and the crop in the west, but the results did not support an exclusive crop
origin in the East, although in many internet sites, such as wikipedia, there is quotation of
[23] Wikipedia to sustain the theory.
Readers may therefore encounter confusing theories dealing with the olive history. A
number of commercial sites favor the olives origin as belonging to the commercial sites
country, but this has no scientific support. According to some commercial sites, the
motherland of the olive is the island of Crete, according to others Southern Caucasia, Iran,

Origin and History of the Olive 5
the Atlas Mountains in North Africa, Lower Egypt, the Sudan or even Ethiopia. Further, The
wild-olive is a tree of the maquis shrubland, itself in part the result of the long presence of
mankind. [24].
In the west of the Mediterranean basin they were considered today, as a result of natural
hybridization and the very ancient domestication and extensive cultivation of the olive
throughout the Mediterranean Basin, wild-looking feral forms of olive, called oleasters",
constitute a complex of populations, potentially ranging from feral forms to the wild-olive.
We believe that feral forms cannot be called oleaster due to their origin from the
domesticated olive.
3. State of the art on the olive tree
The olive tree contributes in shaping landscapes and has deep importance in the agro-
economy, including the industrial economies based upon its by-products. However, the
wild olive thrives in most of the domesticated olives cultivation area and the wild olives
contribution to landscapes is far from neglected even though its contribution to agro-
economy is weak. One faces some confusion on their respective identification [25].
The olive tree is now used for oil and canned-fruit production, with minor use of the wood
for handcrafts. The leaves are used in medicine as herb tea, due to mainly their high
phenolic compound content as oleuropein and hydroxytyrosol, which are beneficial in
nutrition and medicine (see chapter 15, this book). What was the first use of the olive tree
that justified initial care to wild trees? This remains a question and we suggest some tracks.
First, the use of the wood and oil as a fuel, since the wood burns green and further the oil
produces little smoke, an great advantage in caves in comparison to using animal fats for
light and to warm [26]. The spread of olive oil has been documented in the Bronze Age by
the features and artifacts (stones, pottery) and later by the containers (aryballos and
alabasters) of the perfume industry which used olive oil as a perfume base [27].
All European civilizations have tree symbols: Ash tree (Scandinavian), Sycomore (Egyptian),
Plane tree (Sparta, Greece), Oak tree (for the Gauls, Druids, to harvest mistletoe), Pinus
(Japanese), (For the Buddha, India) and for Adam and Eve, . The olive tree is markedly
present in all religions (Christian, Judaism, Islam) symbolizing peace, aging, longevity,
rejuvenating, authority, and plenty of legends and stories are anchored in its history in
Mediterranean cultures [28]. However, a primary feature of the olive is that olive oil may
also be sacred and has many religious associations. Chrism (consecrated or anointing oil) is
made of olive oil, usually includes balsam, and spices. There are many legends on the
origins of the olive tree, and all started with the myth of a spontaneous (Athena) or a foreign
origin, as Arbequina cv. (Museum Borges Blanca, Catalonia, Spain). Chrism is used for
Blessed Sacrament, unction (anointing) oil for baptism, confirmations, Eucharist or
communion, marriage, for doing penance, ordination of priests, and extreme unction or the
last rites. If olive oil did exist during the Bronze Age, its exact purpose is not well known
[27].


Figure 1. Europaea subsp. cuspidata x O. e. subsp. europaea grown in INRA nursery (Near
Montpellier, France) See [33]. Andr Bervill.
The area where the wild olive thrives is restricted in comparison to the area where the olive
is presently grown. Indeed, one of the consequences of the 7,000 to 8,000 years of olive
domestication was to spread the cultivated olive out of the biological area of the wild olive,
whereas the wild olive did not spread (Figure 3). Indeed, the history of the domesticated
olive is tightly linked to mankind and their trend to colonize all the environments, even
harsh ones, in order to avoid conflicts with other populations.
It could appear simple to recognize an olive tree [29, 30, 31, 32]. However, out of orchards
and in the ecological area of the wild olive, it is not so easy due to many tricks that could
lead to confusion with a wild olive. Now, we have abandoned the idea to make the
differences rational for all criteria retained (morphological, phenological, molecular) a
continuous variation between the two forms is recorded. Consequently, we have to keep in
mind that all a priori discrimination between the two forms is questionable and that their
confusion does not bias the results exposed here.
Other chapters develop the taxonomy of Olea that naturally thrives over all continents
except the Americas, and the species Olea europaea L. that spreads over Asia, Africa and
Europa and is used for its fruit in the Mediterranean basin, but is used for wood charcoal in
the mountains of India and Africa [33, 34].

From a scientific point of view the olive is an orphan species, that means there is no model
plant surrounding the genus Olea. Although several thousand DNA sequences are deposed
in databases, little is known from the Olea genome, which remains to be sequenced.

Figure 2. First plane: landscape of abandonned Medieval olive groves surrounded by stone walls
returned to natural appearance (Near Montpeyroux South of France). Catherine Breton.
4. The present distribution of the olive and its counter partner the wild
olive
At present the oleaster is native in the following regions, and we can consider that since the
last ice age the distribution has not changed, due to agricultural development the oleaster
has disappeared in the agro-ecosystem, but it has not declined in the natural ecosystem
(Figure 2). It is not an endangered species [30]. The wild olive tree thrives along the
Mediterranean coasts. It is genuine in Spain, continental France and Corsica, continental
Italy, Sardinia and Sicily islands, Greece and Turkey with Cyprus Island, and in all the east
and south Mediterranean countries (Jordan, Lebanon, Syria, Israel, Egypt (Sinai) and Libya,
it is present in plant formations. In Tunisia, Algeria and Morocco (Moulay-Idriss, Cascade
dOuzou, Morocco; Ichkheul, Tunisia) once other tree species have been eliminated it may
thrive as dense populations but is not a colonizing species.

Its current dispersal depends upon the wild olive trees that survived after the last ice age in
refugee populations. However, its spread during the middle-Pleniglacial (Late Pleistocene)
before the ice age is based upon wood charcoal and pollen sequences [17, 29, 35, 36] and it
was present both in the Levant and in Spain. Based on this evidence, the oleaster is also
genuine in the west. During the Holocene it is noticeable that it spread quickly and became
abundant or dominant [35]. From a botanical point of view, there is no difference between
the oleaster in the east and in the west.
Moreover, [35] analyzed botanical data to clearly identify the oleasters associated with
other thermophilous trees (Ceratonia, Lentiscus, Phillyrea, Rhamnus) in the Mediterranean
climate zone in comparison to those of the Atlantic formation (Pinus, Betula ), which enabled
them to define zones where the Olea has probably thrive.
However, the olive tree expanded widely outside the oleasters limits and the famous
French writer Georges [37] Duhamel has said L o lolivier renonce, finit la
Mditerrane or There the olive has given up, the Mediterranean finishes, that means the
olive tree is an excellent indicator of the Mediterranean climate. There is little knowledge on
the spread of the olive, it was probably slow following the human colonization of harsh
territories by populations seeking shelter to escape wars, and they were patient to adapt
their cultivar set to the harsh environments. The spread of the olive follows the trade and
settlement patterns of the Phoenicians from the Levant westward to North Africa and
Spain especially. Olive oil as both a trade good and utilitarian household item would have
been a premier crop for any colonizer. The present diversity of the olive - probably around
2,000 cultivars - is a witness of this permanent fight between peoples and Nature [38]. The
distribution of the olive tree around the Mediterranean basin goes in latitude northern and
southern [39,40] and in elevation higher than the distribution of the oleaster tree (max 500m
in Spain [31] (Figure 2).
The olive tree was introduced into the New World in South America by the Spanish
(explorers and monks) at the beginning of the 1500s (Colombia, Peru, but later on the west
coast of the USA). The common perception is that historic olive trees in California are
dominated by the Mission cultivar originally introduced by Spanish missionaries to the
present day Caribbean and central Mexico in the early 1500s [41, 42]. Thomas Jefferson
wrote to James Ronaldson on January 13, 1813, it is now twenty-five years since I sent them
(southern planters) two shipments of about 500 plants of the olive tree of Aix (Aix-en-
Provence, France), the finest olive trees in the world. [43]. Olive seeds are believed to have
been brought to California in 1769 to grow into trees hardy to 12 degrees Fahrenheit. Those
olive trees were cultivated in the Franciscan Spanish monasteries. It was the Spanish who
spread the olive to America. Catholic missionaries spread the olive to Mexico and later to
California, as well as to South America. The late Earnest Mortensen of the Texas
Agricultural Experiment Station brought olive trees to the Winter Garden area in the 1930s.
It was introduced in South Africa after the Boer colonization and there it coexists with the
subspecies cuspidata. In Australia the olive has been introduced by 1812 [21] and later
cultivars were introduced in China, Japan, Argentina and Chili and in all countries with a

Mediterranean climate. When introduced as cuttings the cultivars were maintained, but
when introduced as seeds unreferenced cultivars were obtained.
5. Archaeological records: wood charcoals, pollen sequences and artifact
remains
The Oleaster
Due to oleaster wood being used as biofuel during the prehistoric age, abundant evidence
exists to assess its presence from the pleniglacial to the Middle Holocene (for review see
[35]). These authors stated that these kinds of remains constituted safe indicators for the
presence of Olea, although endocarps and pollen grains may have accumulated due to
human and wind transport them over undetermined distances.
The first record of olive wood is by [44] who found a fireplace dating to around 790,000
years ago containing (wild) olive wood charcoals. Wood-charcoal analyses carried out at
prehistoric sites would reflect the local flora and therefore the frequency of Olea wood
indicates its presence. Wood charcoals may be due to natural fires or from fireplaces in
prehistoric sites depending on the sites and other remains in the site.
As reported by [35] the oldest site where Olea europaea thrived in Klissouva cave 1 (Southern
Greece) which is dated to 61,140 55,230 Cal. Yr. BP. At Higueral de Valleja Olea europaea
has been dated to 42,630 41,390 Cal. Yr. BP. Olea europaea has been present on both sides of
the Mediterranean Basin, but obviously, such sites are scarcely distributed and do not allow
us to draw an accurate map for the presence of Olea europaea.
Pollen sequences that contain Oleaceae family pollen may include pollen from Phillyrea,
Jasmine (Jasminum fruticans), and Mediterranean ash tree (Fraxinus angustifolia) which
thrives along rivers. The pollen is frequently transported long distances and accumulation
sites (ponds, swamps, peat-lands) are often far from forests where the oleaster thrives. Thus
some bias in pollen data may exist. However, pollen sequences are accurate for dating sites.
However, some of the oldest remains have been dated about to around one million years
(wood charcoals from Israel [44] and leaf fragments from Tuff conglomerate [45]. Tuff does
not give accurate aging of the site. These remains cannot attest to the presence of the actual
oleaster, they belong to an Olea europaea, but the sub-species cannot be given. Moreover,
such remains have been conserved due to exceptional favorable conditions and are too
scarce to infer any model of distribution from them.
The Olive
When the oleaster first was tamed and received care marks the beginning of the
domestication process. [29, 46] have shown that the wood charcoal kept traces of pruning
practices because of specific vessel architecture and shape, as early as 7500 BP in the
Portuguese Extremadura. Their results push back by 1,500 years the preceding estimation of
olive domestication given in the eastern Mediterranean region.

Recently, Terrals team has revealed that wood charcoal could also reveals traces of
watering in the Middle Ages [40]. If the reasons people were pruning the oleaster are
unknown, the consequences of pruning probably appeared to these peoples by more regular
blossoming over years and more fruits. Today olive cultivars display a wide variability in
response to pruning methods that raises questions on the origin of the diversity [15, 47, 48,
49] at the Neolithic C site of Atlit-Yam on the Levantine coast (dated to 7100-6300 yrs. BP,
uncalibrated C14) found underwater wells constructed of alternating layers of tree branches
and stones, stone-installations, some lined with undressed stones and others dug into the
clay sediment. Some of the crushing installations contained thousands of crushed olive-
stones and waste resulting from the extraction of olive oil. So far this is the oldest known
evidence for olive oil extraction.
Remains that enabled us to trace the olive tree are more and more numerous from the
Mesolithic to the Historical periods. The most informative remains are olive endocarps
(stones) that are frequently found in fireplaces (they are charred or carbonized). Terrals
team has developed morphometric methods that appeared efficient in analyzing such
remains [50]. The main features that result from their analyses are based on the fact that the
morphometry of the endocarps has change during the domestication process. Using modern
and ancient reference samples they screened and pointed out domesticated remains and
unraveled some cultivar relationships. If many stones together in an archaeological site can
reveal some transition phase between the wild to the domesticated olive (many broken
stones together probably represent oil processing), numerous remains are single or a few
stones and consequently these methods are limited on such samples. The accumulation of a
few stones probably represents eating olives. However, secondary usage of olive pressing
wastes may limit finding traces of olive oil production based on olive remains alone [51].
Pottery types absorb indications of the type of fat they have stored. Pottery types devoted to
olive oil as containers for perfumes are aryballos and alabasters, which are widely present
throughout the Mediterranean basin due to their diffusion by Greek and Roman cultures
[52]. Ceramic chronologies are strict and factories are well recorded, providing a large
corpus of data on exchange and trade during the historic periods. Documentation indicates
that people used several plant oils (at least flax, saffron, safflower, castor oil and poppy),
however, it is possible to differentiate plant oils from animal fats and to identify plant oils
by the fatty acid composition obtained from pottery remains [53, 54, 55, 56].
Remains are concrete and their preservation is of importance for future diagnostic methods.
The materials from sites studied by all the authors will probably tell more in the future.
Furthermore, archaeology continues to uncover new sites and materials and this is likely to
continue especially for the southern and eastern parts of the Mediterranean coasts.
In conclusion, the archaeological materials have enabled researchers from different
disciplines to anchor the wild and cultivated olive in regions where they naturally thrived
and colonized, respectively. Moreover, biologists and archaeologists have defined the basic
elicit statistical differences between the wild and the cultivated olive for key historical

periods. All this information lays the foundation required to set up genetic models derived
from the present genetic diversity recorded in the oleaster and in olive cultivars.
6. Molecular data enabling genetic inference for subsp. europaea in
Mediterranean basin
All molecular data based on several types of methods (isozymes, RAPD, RFLP, ISSR and
SSR) have been obtained since 1995 from Olea europaea [see 1-4 for review]. Obviously, we
neglected reports that established relationships between cultivars unless they are
informative in reconstituting the olives history. Molecular data should be based on samples
of wild trees and of cultivars representative of the present genetic diversity. If molecular
data are bias in the sampling the conclusion will be probably biased. There is no a priori
rational criterion for sampling oleaster and olive cultivar trees due to the ignorance of their
origin. Several hundreds of publications have reported data based on different types of
molecular markers on various samples of wild trees and cultivars (depending on the
country, the easy sampling of wild trees (from the local region or covering several regions)
and with different methods to analyze data.
Evolution of methods to get and to analyze molecular data
Evolution of methods is permanent due to progresses in their development. We will not
reiterate all methods of studying the history of the olive since the last ice age through
domestication, but will try to enable non geneticists to follow our reasoning. The progress in
developing molecular markers over the last twenty years has made some techniques lapsed,
although they have released plenty of information [4, 57].
Whatever the techniques used to visualize the genetic diversity, the main feature is to
aggregate data from the three DNA supports in the olive tree: the mitochondrial DNA (mt, ),
the Chloroplast DNA (cp, [58] and the nuclear DNA (nu-, [15, 59, 60, 61]. The information
brought by the three compartments is not proportional to the length of the DNA, but by the
mode of inheritance and by their mode of evolution. The mt-DNA is maternally inherited as
the cp-DNA [62], and evolves by recombination and by mutation and deletion, respectively.
These DNA pools are constituted from several copies of the same molecule (they are
haploid) and define haplotype. The nuclear DNA is made of two halves from each parent.
If the two alleles at one locus are discernible they are said to be codominant, and if only one
is discernible due to the other one being absent then the discernible allele is dominant over
the hinted allele. If the same dominant allele is found in two different individuals they are
said to be similar, whereas if the same two alleles are found in two different individuals
they are said to be identical.
Population genetic methods based on similarities (established for dominant alleles)
compares at each locus two heterogeneous groups, one homogenous (the double recessive)
and one heterogeneous (the double dominant and the heterozygous). All methods used to
structure the genetic diversity are based on the allelic frequencies that are firm with co-

dominant markers but are estimated with dominant markers, i.e., Correspondence (FCA)
and Hierarchic analyses (leading to dendrograms). Bayesian methods for nuclear DNA (nu-
DNA) appeared in 2000 to analyze the data sets and they enable to constitute clusters (based
on inferences of allele frequencies) and to check in each individual under examination the
proportion of the genome that is coming from the different groups made by the software
[63]. These clusters under some hypotheses may correspond to ancestor origins. However,
Bayesian method can mix data from nu-, and cp- or mt-DNA. All the methods have
contributed in eliciting the olives origin and they have opened the way to use more
adequate methods [64]. Obviously, old data sets could be treated with new methods to get
new information.
By the year 2000, after several completed projects (European projects and country projects),
the molecular diversity in the wild form appeared deeply structured, that means the
geographic distribution of the molecular markers in the wild tree was not homogenous [1,
24, 65, 66]. The genetic structure (estimated by the Fst) was stronger with mt- and cp-DNA
markers than with the nu-DNA. Moreover, the mt- and cp-DNA distribution in the eastern
and the western halves of the Mediterranean Sea appeared strongly structured. Even if
sampling problems for all the studies had biased their data, the trend from the whole data
supports that clines for allele frequencies do exist in the wild olive diversity. The clines
could be due to different causes and as for other tree species the spread of the wild olive at
the end of the last ice age may explain its present distribution.
Data sets examined
All data on the olive and oleaster can be analyzed as i) botanical samples to look for a key to
differentiate them ;ii) similarity records to try to differentiate the two forms from a statistical
point of view, and iii) genetic relationships using Bayesian methods.
The botanical differentiation between the oleaster and the olive, although with numerous
attempts, does not reveal key traits neither morphologic nor molecular [31, 47, 67].
Similarity records have been shown more effective in differentiating between the two forms.
Clear cut separation has occurred though some trees remained not clustered, which may
indicate that there are hybrid forms between trees from the two groups or that all trees did
not share all the traits recorded either morphological or molecular. Low frequency markers
should be eliminated in such analyses as they may weigh too much and may distort the
results. These methods lead to references of phylogenetic relationships between different
levels of taxa, even though, they are not accurate for distant taxa (species, genera), but they
have shed light on variety relationships for the olive [47, 64, 68, 69].
[57] Breton has examined a wide sampling of about 1900 trees including wild olives (950
from 55 sites), old trees (50) with undetermined status, and cultivars (about 900, either
abandoned, feral forms or from collections) sampled in most places around the
Mediterranean basin (Figure 3). Using 16 nu-DNA loci (Single Sequence Repeat or SSR) and
3 cp-DNA loci (single base repeats), the comprehensive data set was examined using

structure software [63] by different a priori packages made of oleaster trees, cultivars,
oleaster and feral trees. All clusters revealed by this study were checked by other
aggregation methods (FCA, Dendrograms) to verify their consistency. However, these
methods cannot ensure the biological existence of such groups. [70] have examined about
250 cultivars and two oleaster populations with AFLP markers (there are mostly dominant
markers) from the central Mediterranean with similar methods. [71] has examined 171 wild
trees with 8 SSR from the north-western Mediterranean [30] has studied about 32 cultivars
and 70 oleaster trees from Tunisia, with morphological and molecular methods.

Figure 3. Anti Atlas Morocco elevation 1525masl. Catherine Breton
7. Relationships between the oleaster and the olive
[70] Baldoni et al. concluded that most cultivars have been introduced into Central Italy
regions from the outside and that Umbrian cultivars have originated by selection from local
oleaster trees.[71] Belaj et al. concluded that the genetic structure (=density of alleles across
the geographic distribution of individuals) is not strong enough to positively establish
relationships between true oleaster trees and cultivated varieties. The impact of these
studies has probably been limited due to the limited sampling of the wild forms. [30]
Hannachi et al. (2009) has revealed that the cultivar sets can be split into those of local

origins and those introduced from the Near-East and western regions, making Tunisia in
central Mediterranean a key-place for olive and oleaster diversity. [57] Breton concluded
that the oleaster populations were structured in at least eleven ancestral populations, which
colonized the Mediterranean basin after the last ice age, following mostly the sea-coasts.
Based on coincidence of the sampling area and the clusters, some geographic zones for the
refugee populations have been suggested: 1) Four in the East (Turkey, Cyprus (2) and
Israel+ Lebanon), 2) four in the central Mediterranean (including North of Africa, and main
islands Sicily, Sardinia, and Corsica) and 3) three in the western Mediterranean (Continental
Spain (2) and South France). The zones were well defined in the East and wide in the West,
probably due to limited sampling in Spain and continental Italy.
Crossing models based on historic methods and genetic based-hypotheses
[57] Breton applied the same methods to the cultivar set. Although other methods could not
split the cultivars into different groups based on biological criteria [47], nine clusters were
clearly defined. Furthermore, domestication centers have been revealed by crossing the
oleaster and cultivar clusters. Nine domestication centers appeared. The main features from
all these results are that the genetics suggests clusters and relationships that remain obscure
without data to confirm them. Coincidences between the pre-domestication evidence in
Portuguese Extremadura [29] and one refugee zone in central Spain [57] strengthen each
other. Carrin et al. points out that the accurate records of all archaeological sites where the
oleaster or the olive was found sustains some other sites in the west (Spain, see fig. 3 in [35])
and in the east (Cyprus).
Using the same methods (Wide sampling, Bayesian clustering, FCA and dendrograms) [64]
have shown that by admixture analyses for some olive cultivars it is feasible to attribute
different origins in the glacial refugees and furthermore the proportion of each origin is
quantitatively computed by the Structure software. The application of this method to
cultivars will enable us to have a clear view of the cultivars origins. Data supports that in a
western country most cultivars have been introduced from the eastern Mediterranean, but
that some cultivars have their origin in local oleaster [72] Ozkaya. [30] Hannachi et al. used
this method to reveal three olive origins in Tunisia from the north of Africa (Maghreb), the
Near-East, and the west (Spain).
Archaeological remains could release more information by studying the DNA for other
species. The method has been applied successfully to olive stones [73]. Many olive remains
could be analyzed, but the method is still risky. Today, all these data converge to sustain
that in each region the present olive cultivar diversity is either or both the result of ancient
introductions from the Near-East and/or from other area (North of Africa, Cyprus, Turkey),
local selection from oleaster trees, and from crosses between oleaster and ancient cultivar
trees. More details on the history of the oleaster tree could be obtained at local levels and
with reference to sampling the whole Mediterranean. Dialogue between researchers from
the different fields will be required.

All these data converge to sustain that in each region the present olive cultivar diversity is
either or both the result of ancient introductions from the Near-East and/or from other area
(North of Africa, Cyprus, Turkey), local selection from oleaster trees and from crosses
between oleaster and ancient cultivar trees. However, the self-incompatibility system in the
wild olive and the olive is still not yet known, leaving the selection pressures that occurred
along the domestication processes unknown, which are required to gather enough S-alleles
in a region to enable fruit set. [74] Breton & Bervill have recently deciphered S-allele pair
wise combinations for a few varieties, and it appeared which varieties may combine
efficiently, at least in silico, but it remains to experimentally check coincidence in blossoming
and other compatibility levels, which may affect development of pulp and embryo. The
model developed infers which genotypes may coexist to ensure correct fruit set, even
though self-compatibility appears inherent to most varieties.

Figure 4. Abandonned olive trees along the Mediterraean coast (North of Catalogna, Spain)
Catherine Breton

8. Conclusion
The origin of the olive tree displays singularities in comparison with other tree species. As
well detailed by [35] the thermophilous requirements of the oleaster has constrained its
diffusion. The domestication process has spread out the crop into harsh environments (in
northern latitude, deserts, higher altitude) creating plenty of cultivars. About ten
domestication centers may be at the origin of this diversity for adaptation to these
environments. Recent findings in olive S-allele relationships have not been taken into
account here to show the olives history. The mode of reproduction of the species has
probably played a major role enabling self-progenies and thus narrow local adaptation, thus
explaining logically the huge diversification encountered in this species.
Author details
Catherine Marie Breton
Present address: CNRS ISE-M UMR 5554, Montpellier, France
Address: INRA , TGU AGAP, Equipe DAVEM, Montpellier, France
Peter Warnock
Missouri Valley College, USA
Andr Jean Bervill
INRA, UMR DIAPC, Montpellier, France
Acknowledgement
This work was supported by the ANR project PATERMED (2011-2014) coordinated by
Stphane Angls (UMR LADYSS) in the frame of the call SYSTERA.
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Chapter 2
Botanical Description
Adriana Chiappetta and Innocenzo Muzzalupo
1. Introduction
The olive (Olea europaea L.) is an emblematic species that represents one of the most
important fruit trees in the Mediterranean basin (Loumou & Giourga, 2003). The
Mediterranean form, Olea europaea, subspecies europaea, which includes wild (Olea europaea
subsp. europaea var. sylvestris) and cultivated olives (Olea europaea subsp. europaea var.
europaea), is a diploid species (2n = 2x = 46) (Kumar et al., 2011).
The origin of the olive tree has been lost over time, coinciding and mingling with the
expansion of Mediterranean civilizations which for centuries governed the destiny of
mankind and left their imprint on Western culture. From the eastern of the Mediterranean
basin, olive trees spread west throughout the Mediterranean area and into Greece, Italy,
Spain, Portugal, and France. In 1560, the Spanish Conquistadors carried olive cuttings and
seeds to Peru. From there or independently, olive trees were found in Mexico at Jesuit
missions. The Franciscan padres carried olives and other fruits from San Blas, Mexico, into
California. Sent by Jose de Galvez, Father Junipero Serra established the San Diego de Alcala
Mission in 1769. Though oil production began there in the next decade, the first mention of
oil was written in the records of the San Diego de Alcala Mission in 1803 as described by
Father Lasuen (Winifred, 1967).
Currently, a renewed emphasis of the health benefits of monosaturated olive oil has lead to
a resurgence of olive oil production. The olive tree has been widely used for shade around
homes and as a street tree in cities. Its distribution is only limited by cold weather in winter,
indeed temperatures below 10 C are lethal (Denney et al., 1993). Most olive-growing areas
lie between latitudes 30 and 45 north and south of the equator, although in Australia some
of the recently established commercial olive orchards are nearer to the equator than to the
30 latitude and are producing a good yield; this may be because of their altitude or for
other geographic reasons.
Olive (Olea europaea L.) is the main cultivated species belonging to the monophyletic Oleaceae
family that includes 30 genera and 600 species (Cronquist, 1981), within the clade of Asterids,

Olive Germplasm The Olive Cultivation, Table Olive and Olive Oil Industry in Italy

24
in which the majority of nuclear and organellar genomic sequences are unknown. The Olea
genus comprises 30 species and has spread to Europe, Asia, Oceania and Africa (Bracci et al.,
2011).
The olive is a member of the Oleaceae, the family that contains the genera Fraxinus (ash),
Forsythia (golden bell), Forestiera (F. neomexicana, the California wild-olive), Ligustrum
(privet), and Syringa (lilac) as well as Olea (olive). Its primary genetic resources are
taxonomically classified in the Olea europaea complex in which six subspecies are recognized
(Green, 2002) (scheme 1).

Scheme 1. Taxonomic scheme of Olea europaea L.
The Olea europaea subsp. europaea include the wild form, also named sylvestris, and the
cultivated form, called Olea europaea subsp. europaea var. europaea. The olive tree is self-
incompatible. Out - crossing is mediated by the wind that transports pollen over long
distances, with cytoplasmic male - sterile cultivars being pollinated efficiently by
surrounding cultivars or even by sylvestris (Besnard et al., 2000). It is assumed that cultivars
originated from the wild Mediterranean olive and have been disseminated all around the
Mediterranean countries following human displacement. It is also presumed that crosses
Kingdom: Plantae
Phylum: Magnoliophyta
Class: Rosopsida
Order: Lamiales

Family: Oleaceae
Sub-family: Oleideae

Genus: Olea
Sub-genera: Paniculatae
Tetrapilus
Olea

Sections: Ligustroides

Olea
Sub-species: cuspidata
laperrinei
maroccana
cerasiformis
guanchica
europaea
varieties: sylvestris (wild olive)
europaea (cultivated olive)


25
between wild and cultivated forms could have led to new cultivars around Mediterranean
countries (Besnard et al., 2001).
Nowadays, there are more than 2000 cultivars in the Mediterranean basin that displays huge
diversity based on fruit morphology and pit size and morphology and several modern
cultivars display small pits such as the sylvestris, making the distinction criteria doubtful
(Bartolini et al., 1998; 2002; Ganino et al., 2006).
Until recent years, cultivar identification was based only on morphological and agronomic
traits. However, recognition of olive cultivars based on phenotypic characters appeared to
be problematic, especially in the early stages of tree development. Traditionally diversity
within and between olive tree cultivars was determined by assessing differences in the olive
tree, namely leaf shape and color, and olive fruits morphology. These measures have the
advantage of being readily available, do not require sophisticated equipment and are the
most direct measure of phenotype, thus they are accessible for immediate use, an important
attribute. However, these morphological and phenological markers have the disadvantage
of the small number of polymorphism detected and of being environmentally dependent
(Mohan et al., 1997; Tanksley & Orton, 1983). Besides that, some of the phenological
characteristics are only accessible for a limited period (e.g., olive fruits) or when the olive
tree achieves a mature stage, which may delay correct identification. Due to the high genetic
diversity level observed in olive germoplasm and the presence of homonym and synonym
cases, efficient and rapid discriminatory methods are urgently required.
2. Description of the Olea europaea sub-species
2.1. Olea cuspidate
Olea europaea subsp. cuspidata is a native from South Africa, from which it spread through the
Middle East, Pakistan, India to China. Subtropical dry forests of Olea europaea subsp.
cuspidata are found in the Himalayan subtropical broadleaf forests ecoregion.
In the nineteenth century it was introduced to the Australian territory for economic
purposes (Spennemann & Allen, 2000; Bass et al, 2006). Since 1960, cuspidata naturalized
populations have been found in the Hawaii Archipelago (Starr et al., 2003). The Olea europaea
subsp. cuspidata includes much - branched evergreen trees, which vary their size between 2
to 15 m in height. The leaves have an opposite, decussate arrangement, and are entire. Their
size is between 3 to 7 cm in length and 0.8 to 2.5 cm in width. The leave apex is acute with a
small hook or point, and the base is attenuate to cuneate. Leaf margins are entire and
recurved, the upper surface is grey-green and glossy, and the lower surface has a dense
covering of silvery, golden or brown scales. Venation is obvious on the upper surface and
obscure on the lower surface; the petiole is up to 10 mm long.
Fruit are borne in panicles or racemes 50 to 60 mm long. The calyx is four-lobed and is about
1 mm long. The corolla is greenish-white or cream; the tube is 1 to 2 mm long; lobes are
about 3 mm long and reflexed at the anthesis. The two stamens are fused near the top of the
corolla tube, with bilobed stigma.


26
The fruit is a drupe whose shape varies from globose to ellipsoid, it is 6 mm in diameter and
15 to 25 mm long. The drupe is fleshy, glaucous to a dull shine when ripe, and purple-black.
The tree usually flowers in spring. The wood is much - prized and durable and it is used for
fine furniture and turnery.

Figure 1. Phenotypic aspect of Olea europaea subsp. cuspidata trees


27
2.2. Olea laperrinei
The Olea europaea subsp. laperrinei is restricted to the massifs of central - southern Sahara and
eastern Sahel (Wickens, 1976; Quzel, 1978; Maley, 1980; Mdail et al., 2001; Green, 2002).
The Olea europaea subsp. laperrinei are present at high altitudes, from 1800 to 2800 m, on
volcanic or eruptive rocks, generally in cliffs and canyon banks . This taxon is adapted to
very dry conditions and in Hoggar, a highland region in southern Algeria, along the Tropic
of Cancer, it persists in areas reaching a mean rainfall of about 20 100 mm per year (Quzel,
1965).
The Olea europaea subsp. laperrinei trees reach a height of 1.5 - 3 m and their trunk is mainly
monocaulous. Leaves have a lanceolate - linear to linear aspect, 2.8 - 4 to 5 - 7 cm long and
0.3 0.5 to 1 - 1.5 cm wide. They are ashy-green above and whitish - silvery beneath in
colour and their apex is clearly mucronate. The median vein is flat or canaliculated and the
petiole is 0.2 - 0.4 cm in length. Flowers are 4 - 6mm in diameter, white, with bracteoles
present and well developed. Fruit are borne in panicles. Their shape is ovoid - globose, they
are 4 - 5 mm in diameter and 5 - 8 mm long. The pulp is purplish in colour (Medail et al.,
2001).
2.3. Olea maroccana
The Olea europaea subsp. maroccana is located in the South - west of Morocco, in the western
part of the High Atlas. Its area of distribution is mainly on the southern slope of the Ida ou
- Tanane massif and in the western Anti Atlas (Maire, 1933; Jahandiez & Maire, 1934). The
existence and the taxonomic position of this tree have long remained uncertain, but the
combination of several morphological traits is unique.
The Olea europaea subsp. maroccana tree is arborescent or shrubby in appearance, and
evergreen. The trunk is 4 - 5 m high and generally pluricaulous. Branches and ramifications
are erect, internodes of terminal ramifications are elongate, reaching 20 - 60 mm. The bark is
smooth, grey-brown. Leaves are lanceolate or lanceolate-acute aspect; they are 3 - 4 to 7 - 8
cm long and 0.4 0.6 to 1.2 - 1.3 cm wide. They are slightly contracted into an acuminate
reddish-brown apex and progressively contracted into a medium petiole 0.2 - 0.7 cm long.
Lateral veins are not visible or scarcely visible, the median vein is partly canaliculate on the
lower side. Leaf-blades have a revolute margin; glossy dark-green and very sparsely
scattered with tectorous and star-like hairs above; whitish and densely covered by flattened
tectorous hairs beneath.
Flowers are rather large, 4 6 mm in diameter, white - yellowish; inferior flowers are
pedunculate and 2 - 4 mm long with 2 bracteoles ovoid - elongate of 1 - 2 mm, inserted
either on the pedicel or beneath the calyx. The calyx is urceolate, erect, cylindrical - oval, 1
mm long. Fruit are borne in panicles or axillary and terminal racemes, elongate and
flexuous; lateral ramets are 50 - 70 mm long, ramified; secondary ramets are 10 - 30 mm
long; terminal ramets are reflected, and 60 - 120 mm long. Inflorescence bracts are lanceolate
- obtuse, 3 - 4 mm long. The fruit is a globose - ovoid drupe; it is 5 - 7 mm in diameter and 9 -


28
11 mm long, obtuse at the apex, green then red - brown when unripe, becoming purplish -
black. Drupe pulp is carmine and aqueous, sweet or slightly bitter taste (Medail et al., 2001).

Figure 2. Morphological aspect of Olea europaea subsp. maroccana fruits
2.4. Olea cerasiformis
The Olea europaea subsp. cerasiformis tree is arborescent or shrubby, and evergreen. The trunk
is 1 - 2.5 m high, generally pluricaulous, and is grey or whitish in colour. Leaves are
oblanceolate to linear in shape, rarely suborbicular; they are 3 - 4 to 8 - 10 cm long and 0.4
0.6 to 1.0 - 1.4 cm wide. The leaf apex is acuminate and the colour is greyish-green above
and paler beneath. They have a petiole 0.2 - 0.7 cm long. The main vein protrudes on the
abassial surface.
Flowers are 4 mm in diameter, white; bracteoles are generally present and well developed.
Fruit are borne in panicles. The fruit is an ellipsoid drupe; it is 9 - 12 mm in diameter and 12
- 22 mm long. Drupes are green then purplish - black; pulp with a bitter taste (Medail et al.,
2001).
2.5. Olea guanchica
Traditionally wild olive populations present in the Canary Islands are ascribed to the
species Olea europaea subsp. cerasiformis. A recent genetic study concluded that populations
of Madeira and the Canary Islands were genetically separate enough as to be separated into
distinct subspecies, therefore the Canarian wild pass was renamed Olea europaea subsp.
guanchica.
This subspecies is present throughout the islands forming part of transition forests or
thermophiles. In Gran Canaria it is very abundant and it can be easily found around the
north, forming clumps, but especially in the north-east. In the south of the island it is much
more local and rare.


29
The Olea europaea subsp. guanchica is a small tree evergreen that can reach 6 m in height or
more often it has a shrub appearance. Generally, the trunk is pluricaulous, grey or whitish.
Leaves are bright green, oblanceolate to narrowly elliptic, 2 - 3 to 7 - 8 cm long and 0.4 0.6
to 1.1 - 2.1 cm wide. The apex is mucronate to cuspidate and the colour is greyish-brown
above and paler beneath. Leaves have a petiole 0.2 - 0.5 cm long. The main vein partly
protrudes on the abassial surface.
Flowers are 4 - 5 mm in diameter, white; bracteoles not well individualized or missing. Fruit
are borne in panicles. The fruit is an ovoid - globose drupe; it is 9 - 12 mm in diameter and
12 - 22 mm in length; their colour is green then purplish - black; pulp with a bitter taste
(Medail et al., 2001).

Figure 3. Morphological aspect of Olea europaea subsp. guanchica leaves and trunk
2.5.1. Olea europaea subsp. europaea var. sylvestris (wild olive)
Olea europaea subsp. europaea var. sylvestris is a typical tree of the Mediterranean regions and
it contributed to the Mediterranean forest. In fact, it is considered by many authors as a
forest tree. With the olive being the most longeve plant crop species, numerous ultra-
millennial still living sylvestris trees are present over all the European Mediterranean
countries (Baldoni et al., 2006).
However, forest fires and extensive urbanization that characterize the Mediterranean coast
have endangered the sylvestris variety determining a decline of this genetic resource.


30
Olea europaea subsp. europaea var. sylvestris has not been recorded and evaluated and thus it is
not used as a genetic resource although it spread to areas without olives and it seems well
adapted to hard environments such as drought, cold, salt, poor soils, etc.
The Olea europaea subsp. europaea var. sylvestris tree is arborescent or shrubby. The plant is
long-lived, despite the difficulty in determining the real age, in many cases it can exceed
1000 years old. The trunk is often twisted and cable, and it can reach a considerable size, up
to 15 m in the monumental specimens (Baldoni et al., 2000). The branches are numerous,
they have thorns in young plants, and can have erect, intermediate or pendulous aspect. The
bark is gray ash - colored, more or less smooth in young trees, becoming rough in the
adult ones. The leaves are opposite, leathery, with smooth margin. The lamina is elliptic -
lanceolate in shape, the adaxial surphace is green and glabrous, the abaxial one has small
silver shield-shaped scales. Flowers are white, pedunculated, very numerous and grouped
in axillary racemes (inflorescence). The calix normally has four leaves ovoid, while the
corolla, white, is formed by four petals of 2 - 4 mm. There are two stamens per flower, the
stigma is bifid and the ovary has four niches. The fruit is an ovoid-globose drupe; it is 5 - 7
mm in diameter and 10 - 15 mm in length. Pulp is initially green then black - brown at
maturity. The endocarp is hard and woody with a single seed, rarely two (Camarda et al.,
1983).

Figure 4. Olea europaea subsp. europaea var. sylvestris trees: one of the oldest sylvestris genotypes located
in Sardinia


31

Figure 5. Olea europaea subsp. europaea var. sylvestris: higher magnification of the trunk of the plant
represented in fig. 4

Figure 6. Olea europaea subsp. europaea var. sylvestris fruits
2.5.2. Olea europaea subsp. europaea var. europaea (cultivated olive)
Commercial olive fruits are products of Olea europaea subsp. europaea var. europaea, and only
this sub-species of the Olea genus produces edible fruits.
The cultivated olive tree can reach heights ranging from just a few meters to 20 m. The wood
resists decay, and when the top of the tree is killed by mechanical damage or environmental
extremes, new growth arises from the root system. Whether propagated by seed or cuttings,
the root system is generally is shallow, spreading to 0.9 - 1.2 m even in deep soils. The above
- ground portion of the olive tree is recognizable by the dense assembly of limbs, short
internodes, and compact nature of the foliage. Light does not readily penetrate to the
interior of an olive tree unless the tree is well managed and pruned to open light channels
toward the foliage. If unpruned, olives develop multiple branches with cascading limbs. The


32
branches are able to carry large populations of fruit on terminal twigs, which are pendulous
and flexible - swaying.

Figure 7. Olea europaea subsp. europaea var. europaea: morphological aspect of a cultivated olive tree (A),
leaves (B); inflorescence (C); fruits (D); endocarp (E)
Olive leaves are thick, leathery, and oppositely arranged. Each leaf grows over a 2-year
period. Leaves have stomata on their abassial surfaces only. Stomata are nestled in peltate
trichomes that restrict water loss and make the olive relatively resistant to drought. Some
multicellular hairs are present on leaf surfaces. Olive leaves usually abscise in the spring
when they are 2 or 3 years old; however, as with other evergreens, leaves older than 3 years
are often present.
Flower buds are borne in the axil of each leaf. Usually the bud is formed on the current
seasons growth and begins visible growth the next season. Buds may remain dormant for
more than a year and then begin growth, forming viable inflorescences with flowers a
season later than expected. When each leaf axil maintains a developing inflorescence, there
are hundreds of flowers per twig. Each inflorescence contains 15 - 30 flowers, depending on
the cultivar.
Olives are polygamo - monoecious. The flowers are born axially along the shoot, arranged in
panicles. Perfect flowers, those with both pistillate and staminate parts, normally consist of a
small calyx, 4 petals, 2 stamens with a filament supporting a large pollen-bearing anther,
and a plum green pistil with a short thick style and a large stigma. Perfect flowers are borne
apically in an inflorescence, and within the typical triple-flower inflorescence the middle


33
flower is generally perfect. Imperfect flowers are staminate, with the pistil either lacking or
rudimentary. The flowers are borne on the inflorescence and are small, yellow-white, and
inconspicuous.
The perfect flower is evidenced by its large pistil, which nearly fills the space within the
floral tube. The pistil is green when immature and deep green when open at full bloom.
Staminate flower pistils are tiny, barely rising above the floral tube base. The style is small
and brown, greenish white, or white, and the stigma is large and plumose in a functioning
pistil.
Floral initiation occurs by November (Pinney & Polito 1990), after which, the flower parts
form in March. The inductive phase of flowering in the olive may occur as early as July
(about 6 weeks after full bloom), but initiation is not easily seen until 8 months later in
February. Complex microscopic and histochemical techniques reveal evidence of floral
initiation by November, but the process of developing all the flower parts starts in March.
Some olive cultivars, such as those grown in Crete, southern Greece, Egypt, Israel, and
Tunisia, bloom and fruit heavily with very little winter chilling; whereas those originating in
Italy, Spain, and California require substantial chilling for good fruiting.
At full bloom, flowers are delicately poised for pollination, when some 500,000 flowers are
present in a mature tree; a commercial crop of 7 metric tons/ha or more can be achieved
when 1 or 2% of these flowers remain as developing fruit. By 14 days after full bloom, most
of the flowers destined to abscise have done so. By that time, about 494,000 flowers have
abscised from a tree that started with 500,000 flowers (Rosati et al., 2010).
Cultivars vary, but most abscission occurs soon after full bloom and final fruit set nearly
always occurs within 6 weeks of full bloom. Further fruit abscission can result from pest
infestation and environmental extremes. When trees have an inflorescence at nearly every
leaf axil a commercial crop occurs with 1 to 2% fruit set; with a small population of
inflorescence, a commercial crop may require 10% fruit set.
Shot berries (parthenocarpic fruits) occur randomly and for reasons which have not been
clearly understood. When shot berries occur, they may be seen in clusters on each
inflorescence. Here, the inter-fruit competition for raw materials differs from that of normal
olive fruits. Shot berries mature much earlier than normal fruit and may be more prevalent
when conditions favor a second large crop in succession.
The olive fruit is a drupe, botanically similar to almond, apricot, cherry, nectarine, peach,
and plum fruits. The olive fruit consists of a carpel, and the wall of the ovary has both fleshy
and dry portions. The endocarp (pit) enlarges to full size and hardens by 6 weeks after full
bloom. At that time, the endosperm begins to solidify and embryo development takes place,
leading to embryo maturity by September. The mesocarp (flesh) and exocarp (skin) continue
their gradual growth. The fruits begin changing from the green color to yellow-white
(straw) and accumulate anthocyanin from the distal or base end. Fruit shape and size and
pit size and surface morphology vary greatly among cultivars (see the elaiographic cards
attached to chapter Description of varieties)


34
The mature seed is covered with a thin coat that covers the starch-filled endosperm. The
latter surrounds the tapering, flat leaf like cotyledons, short radicle (root), and plumule
(stem). Seed size and absolute shape vary greatly with cultivar.
The seed undergoes most of its development starting in July and ending in about
September. The fruit is horticulturally mature in October or November (in Italy) and if
harvested and stratified at that time, it will achieve the maximum of germination. However,
seeds are physiologically mature in January or February when its germination is greatly
reduced (Lagarda et al., 1983a).
Olea europaea L. subsp. europaea var. europaea is a species of great economic importance in the
whole Mediterranean basin.
In fact, the genetic patrimony of the Mediterranean basin olive trees is very rich and is
characterised by an abundance of varieties. Based on estimates by the FAO Plant Production
and Protection Division Olive Germplasm (FAO, 2010), the worlds olive germplasm
contains more than 2.629 different varieties, with many local varieties and ecotypes
contributing to this richness. It is likely that the number of cultivars is underestimated
because of inadequate information about minor local cultivars that are widespread in
different olive-growing areas.
Current scientific knowledge offers the possibility of introducing new assessment systems,
based not only on the varietal character phenology, usually adopted, but also on genetic
traits.
For seed production, the fruits should be harvested when ripe, but before they turn black.
This period extends from late September to mid-November, depending on the cultivar
(Largarda et al., 1983a,b). Pits are removed from the flesh of the fruit with macerators. Pits
can be stored in a dry place for years or planted directly, but germination is slow and
uneven. Pre-germination treatments are designed to overcome both seed coat (mechanical)
and embryo dormancy. Mechanical or chemical scarification is used to treat mechanical
dormancy. During the scarification phase, the endocarp can be cracked mechanically or
clipped at the radicle end, with care taken not to damage the embryo. Clipping just the
cotyledonary end of the endocarp does not improve germination. Good germination results
can be obtained using a seed cracking device before subsequent handling procedures
(Martin et al., 1986). Pits may be soaked in concentrated sulfuric acid to soften the endocarp.
Soaking time depends on the thickness of the endocarp; typical soaking times for
Manzanillo are between 24 and 30 hours. The acid bath is followed by 1 to 2 hours of rinsing
in water (Crisosto & Sutter, 1985).
The pits can be planted directly after the endocarp treatments at a depth about 2 to 3
times their diameter. Seeds planted outdoors in December do not germinate until the
following spring. Pits can also be planted in pots or seedbeds in a greenhouse maintained
at a range of temperature between 21 - 24 C. Germination takes up to 3 months
(Hartmann, 1949).


35
Germination is quicker and more uniform when treatments to overcome internal dormancy
are carried out in addition to scarification. The most successful of these treatments on a
commercial scale is stratification. Pits are scarified as described above and then soaked in
water at room temperature for 24 hours. The pits are mixed with moist sand or vermiculite
and then placed in the dark in a controlled environment. The temperature is kept at 15 C
for 30 days. Stratification is thought to reduce abscisic acid, an inhibitor of germination,
within the embryo or seed-coat. After stratification, pits can be planted outdoors if the
weather is suitable; severe weather can cause losses. Pits can be planted in a greenhouse at
21 to 27 C. Bottom heat is necessary. Germination should occur within 1 month.
Transplanting seedlings from the greenhouse to the nursery should include steps to harden
the seedlings, such as partial shade provided by a lath house. Adequate irrigation and
fertilization are recommended to ensure continued rapid growth.
Virtually all olive trees are produced from rooted cuttings. Seed handling difficulties, low
germination percentage, and slow initial seedling growth rate make seedling production
impractical.
3. Conclusion
Olea europaea L. represents one of the most important trees in the Mediterranean basin and
the oldest cultivated plant. Among cultivated plants, the olive is the sixth most important oil
crop in the world, presently spreading from the Mediterranean region of origin to new
production areas, due to the beneficial nutritional properties of olive oil and to its high
economic value.
The Mediterranean basin is the traditional area of olive cultivation and has 95% of the
olive orchards of the world. From the Mediterranean basin, olive cultivation is presently
expanding into areas of Australia, South and North America (Argentina, Chile, United
States), South Africa and even in exotic place, like Hawaii. Given its wide range of
distribution, it is becoming increasingly urgent to identify plants into different ranges of
distribution in the world to avoid cases of homonymy, synonymy and mislabeling so
that a reliable classification of all varieties can be achieved without unnecessary
confusion.
In this context, along with morphological characteristics the acquisition of additional
information on biochemical markers is essential. This aspect represents a fundamental and
indispensable step to preserve the main olive varieties and also to safeguard minor
genotypes, in order to avoid a loss of genetic diversity.
Recent research has focused on using morphological markers associated with molecular
ones to characterize and identify olive varieties (Ercisli et al., 2009; Muzzalupo et al., 2009).
The identification of varieties by using molecular markers is a crucial aim of modern
horticulture, because such a technique would greatly facilitate breeding programmes and
germplasm collection management.


36
Author details
Adriana Chiappetta
*

University of Calabria (UNICAL), Dep. of Ecology,Arcavacata di Rende (CS), Italy
Innocenzo Muzzalupo
Agricultural Research Council - Olive Growing and Oil Industry Research Centre, Rende (CS), Italy
4. Acknowledgement
The authors thank the CERTOLIO project and University of Calabria funds for financial
support.
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*
Corresponding Author


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Chapter 3
Cultivation Techniques
Caterina Briccoli Bati, Elena Santilli, Ilaria Guagliardi and Pietro Toscano
1. Introduction
In an agricultural context, olive growing is emerging as a dynamic and interesting topic. The
majority of Italian olive growing still requires organic renewal interventions such as farm
restructuring and tree planting, varietal conversion, mechanization, technical assistance for
the implementation of technological innovations, better organization and contractual weight
in product marketing. Therefore, the main objective in this area can be obtained with a more
economical management of olive orchards in order to achieve a high production per unit
area at lower costs and while respecting the environment. Therefore, current olive growing
must be based on two pillars such as the reduction of management costs and the use of
cultivation techniques with a low environmental impact. More and more attention is being
paid, by the EU, and also by Italy, to environmental sustainability, biodiversity and
compliance requirements in agriculture. Indeed, in recent years the EU has issued a set of
regulations aimed at environmental protection and enhancement of rural areas by
improving the competitiveness of the agricultural sector in order to obtain high-quality
products aimed at enhancing the peculiarities of the different territories of origin (PGI,
PDO) and protecting agricultural and natural resources. Quality must always be considered
a key resource for agriculture, which will enable farms to survive and compete in both
Italian and International markets. The cultivation techniques used in olive orchards are
directed to preserve and improve the physical and chemical characteristics of the soil (soil
preparation and tillage, irrigation, fertilization) and to enhance plant production (training,
pruning, fruiting, production and pesticide treatments). The knowledge of olive
morphology and biology is a prerequisite for the rationalization of cultivation techniques to
improve the quantity and quality of production.
Although the olive tree can be considered a hardy plant and is cultivated in marginal areas,
it requires specific cultivation techniques coordinated and integrated with each other in
order to exalt their productive potentialities. This is why the wise use of tools such as
pruning, irrigation, nutrition and soil management plays an important role in achieving a

greater vegetative and reproductive plant balance, and cost containment should be the main
goal that guides management decisions.
2. Soil
Soil is defined as the top layer of the earths crust. It is formed by mineral particles, organic
matter, water, air and living organisms. It is in fact an extremely complex, variable and
living medium, and represents a non-renewable resource which performs many vital
functions: food and other biomass production, storage, filtration and transformation of
many substances including water, carbon, nitrogen. As long as 100 years ago, Wollny (1898)
described the positive effect of soil structure on root growth, water availability, gas
transport in soils as well as the positive effects of soil structure on soil strength. He
mentioned that the mechanisms involved in the interaction between soil structure and plant
growth and yield needed to be investigated. Since then, the positive effects of a favorable
soil structure and the negative effects of, for example, soil compaction on crop growth
and/or yield have been repeatedly described (e.g. Blank, 1932-1939; Dexter, 1988; Hakansson
et al., 1988; Kay, 1990). The anthropogenic activities such as tillage, mineral fertilization,
waste disposal and industrial pollution, affect both chemical and physical natural soil
properties (Kabata-Pendias & Mukherjee, 2007).
Recent improvements and new methods in analytical chemistry and increasing areas of
environmental investigation have substantially added to our knowledge of agricultural soil
science. For example, the soil characteristics of an olive plantation are especially important
in terms of vulnerability to erosion and, to a lesser extent, to leaching of potentially
contaminating elements contained in fertilisers and pesticides. The root system of the olive
is concentrated in the top 50-70 cm of soil although it may send out roots to a depth of more
than one meter to satisfy its water needs. Therefore, the soil must have an optimal texture,
structure and composition to a depth of at least one meter. The management of a cropping
system requires periodic evaluation that includes systematic testing, with the aim of
determining the nutritional status of soils in order to assess the existence of any nutritional
deficiency, excess or imbalance and form a basis for planning the nutrient supply as well as
other practices (tillage, amendment, correction). The following is a brief description of the
main chemical and physical soil properties.
2.1. Soil texture, porosity and density
The textural class is the first parameter that defines soil properties, and is determined by the
relative percentage of the three major soil compounds: sand, silt and clay, defined by the
respective particle diameter size.
Clayey grounds are characterized by particles of a diameter of less than 0.002 mm, constituted
by flinty minerals with different capacities to inflate in the presence of water and to contract in
dry conditions, forming cracks which are typical of vertisols. Clayey soils show a low water
permeability and high plasticity, which can induce stagnation phenomena and root asphyxia
in wet conditions; while in the dry state it has notable tenacity and cohesion.

Cultivation Techniques 41
Silty soils have elementary particles of greater dimensions than the clayey ones and, unlike
these, they have greater difficulty in reaching a glomerular structure. The lower structural
stability of silty soils causes a low macroporosity and a great bulk density, that determine
conditions of low aeration, low permeability and water stagnation in the profile. This type of
soil is subject to loosening conditions with greater facility in comparison to clayey soils.
However, they have a greater tendency to pulverization in the dry state, and the formation
of mud in the wet state.
Sandy soils have particles of a diameter between 2 and 0.02 mm; they are characterized by
high permeability of rainwater, and fast mineralization of organic matter.
The Olive tree responds best to soil textures with balanced proportions of sand, silt and clay.
Soils that are primarily sandy do not have good nutrient or water-holding capacities, but
they do provide good aeration and olives do well, especially when water is available and the
crop is properly fertilized to satisfy its mineral requirements. The soil should not contain too
much clay to avoid limiting air circulation and to prevent soil management problems. The
soil particles should aggregate in granules or crumbs to make the soil porous; this is ensured
by sufficient quantities of organic matter and rational soil management to prevent
compacting and erosion. Soil is composed of solid particles (mineral and organic matter) of
different sizes, usually bound together into aggregates by organic matter, mineral oxides,
and charged clay particles. The number and size of pores vary considerably among soils
exhibiting different organic matter content, texture and structure and cultivation techniques
have a great effect on bulk density and porosity: any management practice that increases
organic matter will increase the granular structure of the soil, increase the pore space, and
decrease the bulk density (Gisotti, 1988; Giordano, 1999; Hao et al., 2008).
2.2. Organic Matter and nitrogen ratio
The Organic Matter (OM) is a complex mixture of organic compounds deriving from
metabolic wastes and decaying residuals of plants, animals and microorganisms, at different
stages of decomposition. The OM percentage directly influences the structure and chemical-
physic properties of soil in terms of water infiltration and retention, element absorption,
particle aggregations, Cation Exchange Capacity (Al
3+
, Fe
3+
, Ca
2+
, Mg
2+
, NH4
+
), buffering
power, over the nutrient source for the plant. The quantity and nature of OM is highly
dependent upon farming practices and climatic conditions and is found as both chemically
stable humus (or passive OM) and partially decomposed plants, microbes and animal
residues (or active OM).
Measures to increase the organic content are a very important part of good soil management
in Mediterranean regions, especially in order to reduce vulnerability to erosion (European
Soil Bureau, 1999). Practical measures are based on the incorporation of organic matter such
as farm-yard manure, cover crops, pruning and processing residues, and soil tillage.
Plant availability of organic N is dependent on OM breakdown, which is difficult to
estimate. The ratio of total organic carbon and total nitrogen (C/N) is the traditional guide to
the nature of the organic matter present in the soil.

The basic premise behind this ratio is that organic carbon is the primary source of energy for
soil microbes, but these also require nitrogen to multiply and utilise this energy. The
microbes utilise soil carbon via respiration, with the consequent loss of carbon dioxide from
the soil. As the active fraction of the OM is thus degraded, the C/N ratio drops until a steady
state (the passive fraction) is finally attained. Interpreting this ratio is complicated, as it also
depends on the nature of the OM. The passive fraction of the OM can have a C/N ratio that
is medium. Consequently, medium C/N ratio soils can have a wide variation in
mineralisable N status, and this is a limitation when considering the C/N ratio in isolation.
2.3. Cation Exchange Capacity (CEC), pH, electrical and hydraulic conductivity,
water content
Plant nutrients usually exist as ions which carry an electrostatic charge. This electrostatic
charge is a result of atomic substitution in the lattices of soil minerals and because of
hydrolysis reactions on the broken edges of the lattices and the surface of oxides,
hydroxides, hydrous oxides and organic matter (Hendershot et al., 2008a). These charges
attract counterions (exchangeable ions) and form the exchange complex. Ions can be bound
to the soil in varying degrees. At one extreme, they may be an integral part of the soil,
strongly bound to silica and essentially unavailable to growing plants. At the other extreme,
they may be fully soluble and not interact with the soil to any significant extent.
Exchangeable ions are between these two extremes , and are weakly bound to soil particles.
The bonds between soil particles and exchangeable ions are not permanent, and are
continually broken reformed, as the ions move within the water surrounding soil particles.
The bonding of these ions largely prevents their loss by leaching, but is not so strong that
plants cannot extract them from the soil. In fact, plant roots absorb exchangeable ions by
'swapping' them for hydrogen cations (H
+
).
The cation exchange capacity is often estimated by summing the major exchangeable cations
(K, Ca, Mg, and Na) using units of cmol kg
-1
, even if the common expression for CEC is in
terms of milliequivalents per 100 grams (meq/100g) of soil. The CEC of soil can range from
less than 5 to 35 meq/100g for agricultural type soils, and is related to clay and organic
matter content.
CEC is important for maintaining adequate quantities of plant available calcium,
magnesium, sodium and potassium in soils. For many crops the magnesium level should
ideally be twice as much as that of potassium. When magnesium is lower than potassium,
suppression of magnesium uptake can occur. Sodium is only of secondary importance in the
soil test as its uptake by plants is largely dependent on the plant species involved and the
potassium status of the soil, rather than the level of sodium extractable from the soil.
The Total Base Saturation is related to CEC, which represents the proportion of the soils
total capacity for cations that is actually occupied by these nutrients. It is calculated by
summing together the levels of calcium, magnesium, potassium and sodium found in the
soil and expressing this sum as a percentage of the CEC value.

Soil pH is one of the most common and important measurements in standard soil analyses
(Hendershot et al., 2008b). The pH value expresses degree of acidity or alkalinity of the soil.
It is important because it influences the chemical and physiological processes in the soil, and
the availability of nutrients. Availability changes differently with pH levels: aluminium,
copper, iron, manganese and zinc increase when the pH decreases; unlike magnesium that
decreases when the pH decreases (Belsito et al., 1988; AA.VV., 1989; Jones, 2003).
Electrical conductivity (EC) is the ability of a material to conduct an electrical current and is
commonly expressed in units of microSiemens per meter (S cm
-1
). It is used to estimate the
level of soluble salts. The measurement of EC in the soil water extracted from the field-water
content is theoretically the best measure of salinity as it indicates the actual salinity level
experienced by the plant root (Miller & Curtin, 2008). However, this measurement has not
been widely used because it varies as soil-water content changes over time and so it is not a
single-valued parameter. A soil is considered saline if the EC of the saturation extract
exceeds 4000 S cm
-1
at 25C. The soil EC varies depending on the amount of moisture held
by soil particles. Consequently, the EC correlates strongly to soil particle size and texture
and affects crop productivity.
Soil water analyses can be organized into two main groups: analysis of storage properties
and analysis of hydraulic properties. The water content of soil is part of the analysis of
storage properties which refer to the soils ability to absorb and hold water. Instead
hydraulic conductivity is a hydraulic property which refers to the soils ability to transmit or
conduct water . It is more difficult for plants to absorb nutrient elements at low soil moisture
levels, so nutrient element contents will be lower.
3. Soil management
In order for plants to live, two key functions can be attributed to soil: habitability and
nutrition. The function of habitability mainly depends on the physico-chemical
characteristics of the soil. The function of nutrition depends on the factors that make
nutrients bio-available to the plants, described above, determining the fertility of the soil as
productive attitude. Biological soil functions depend on the micro-organic pattern,
responsible for processes on the organic matter such as: mineralization, humification,
nitrification, nitrogen fixation, symbioses, and parasitism. The agricultural management
systems of soils, such as crop rotation, nutrient application, plant species, kind of tillage, and
use of pesticides may have a strong impact on the composition of the soil microbial
community. Maintenance of sustainable soil fertility depends greatly on the ability to
harness the benets of rhizosphere microorganisms such as arbuscular mycorrhizal fungi
(AMF), which form a symbiotic association with the roots of most plant families. Olive
plants are known to form arbuscular mycorrhiza (Roldn-Fajardo & Barea, 1985; Briccoli
Bati et al., 1992; Calvente et al., 2004), the most common mycorrhizal type involved in
normal cropping systems, being considered as a key component in environmentally friendly
agro-biotechnologies (Jeffries & Barea, 2001).
Mycorrhizae act as biofertilizers, bioregulators, and biocontrol agents (Lovato et al., 1996;
Von, 1997). Arbuscular mycorrhizal fungi allow the plant to absorb greater quantities of

water and nutrients, particularly those less mobile in the soil such as phosphorus. In
addition to phosphorus, other elements such as nitrogen, zinc, calcium and sulfur are
involved in the mechanisms of mobilization and uptake by mycorrhizal fungi.
Mycorrhizal symbiosis also acts as a bio-regulator, able to influence some physiological
processes, growth regulators and the development of the plant, to modify the morphology
of the roots, the roots/foliage ratio and sometimes branching foliage and flowering. These
soil fungi improve the agronomic fertility of the soil through the formation and stabilization
of particle aggregates, in particular in land lacking structure.
3.1. Tillage
Tillage consists of some mechanical operations, performed with different tools, which
modify soil structure, according to the management needs, that can be summarized as:
increase of the soil mass (active layer); increase in soil permeability, runoff and erosive
phenomena; accumulation of water reserves; reduction of evaporation due to interruption of
superficial capillarity; destruction or containment of weeds; burial of fertilizers, corrective,
amendants, and crops residual.
Usually, for olive groves a deeper autumnal tillage is carried out, to increase the water
reserve and to bury the phospho-potassic fertilizers; while during the spring-summer
period, some harrowing is performed to reduce evaporation and to eliminate weeds. Soil
tillage was classified according to the epoch and the type of performance, distinguishing
into preparatory practices, performed before the plantation, to constitute suitable conditions
to sustain the crop after the implant, and subsequent practices, performed during crop
culture.
Soil tillage can be performed with different tools classified into three main groups:
mouldboards, rippers and scramblers. Mouldboards cut and upset the soil; rippers cut the
profile producing clods but without modifying soil stratigraphy; and scramblers break up
and remix the worked layer.
The choice of the best tillage technique must be performed in order to: reduce costs, in terms
of working times and fuel needs; increase the timeliness of intervention; maintain a suitable
productive level of crops and soil fertility; contain erosive phenomena.
The soil water content strongly influences the choice of the epoch and the type of
intervention performance. Dependent on this, there are different physical soil states: i)
cohesive, when the soil is dry; in this state the soil does not stick to utensils, it has maximum
tenacity, it is crushproof and resistant to breakup; ii) plastic, characterized by a progressive
warping and stickiness, that increases with an increase in damp; iii) liquid, when the soil
behaves like a suspension. Tillage is difficult and harmful to the soil when the soil is sticky;
over this limit, the passing of machines provokes undoing of the structural aggregates. With
dry soils the work needs higher powers, and forms compact clods of varying dimensions,
according to the type of performance: plowing causes large clods, while with rotary hoeing
a notable pulverization and formation of small clods occurs.

A soil is considered as loosening when it has an optimal damp for the execution of tillage,
approximately corresponding to half of their field capacity, with a more or less ampler range
depending on the soil type and intervention (Bonciarelli, 1981; Giardini, 1986).
Plowing is the most known and commonly used form of soil tillage in agricultural practice
and is performed with three type of tools: the ploughshare plow and the disk plow, that
work by traction; and the rollover plow, that acts by the tractor power take-off (PTO).
The plow operates by cutting and overthrowing a slice of soil, with an angle varying with
the type of plough, the operating velocity and the operation goal: the complete overthrow of
slices is necessary in green manuring and in weed control; while vertical slices improve
airing and rainwater infiltration. Using a cylindrical bending breast a greater crumbling
action is achieved; while a helical breast favours the slice overthrow with smaller
production of thin soil. The speed of ploughing acts on both the slice overthrow and
crumbling: a fast ploughing enhances the inversion of layers and the pulverization of clods.
The ploughshare action can cause compaction of the deep soil, called tillage pan. Such a
drawback can be enhanced using tractors working "within furrow", and in conditions of
high damp. The tillage pan hinders the vertical movement of rainwater and the gaseous
exchanges in the soil and the growth of the roots.
The drawbacks of the ploughshare plow are: excessive clod level, that requires other
refining tillage, with further passages of machinery that stamp on the soil and degrade its
structure; formation of tillage pan; high requirement of traction power. Such negative
aspects can be mitigated using the disk plow, in which the ploughshare and the breast are
replaced by a spherical cap, free rotating on an axle angled in respect of the operating
direction. The disk limits attrition and needs of traction power. During rotation the cap lifts
a slice of soil that is then crumbled and remixed. In comparison to the classical plow it better
prepares the bed for seeding; it is proper for light ploughing in loose or medium textured
soil rich of skeleton and in groves.
There are other tillage techniques that can be carried out, using different types of machinery
which can be complementary or alternatives to ploughing. Among these the most common
are:
- Ripping technique is characterized by the vertical breakup of the soil without inversion
of the layers. The tools used in this type of tillage are constituted by a varying number
of anchors (from 1 to 5) that practice a different action on the soil depending on their
shape and interaxle. Some machineries are endowed with vibrating tools that enhance
shattering of the soil, and can be joined to rolls or harrows to finish up and to level the
surface in a single pass.
- Rotary Hoeing consists in the shattering and remixing of the soil performed by
machinery moved with a tractor PTO., with tools that work on a horizontal axle
(millers) or vertical axle (rototiller). Their drawback is the excessive shattering of soil,
that worsens its structure, with compaction, formation of superficial crust and tillage
pan, fast tool usury; while requiring high working power. They are unsuitable in heavy
and/or skeleton soils.

- Weeding is a tillage practice complementary to ploughing, carried out to reduce the
clod of the soil, and to bring up weed roots. This machinery is formed by bent rigid or
elastic rippers and with different types of feet.
- Harrowing is performed to refine clods, eliminate weeds, bury fertilizers and break the
superficial crust. The tools for this operation are of different shapes and dimensions,
according to soil types, soil conditions and the needs of soil refining.
All tillage up to 15 cm of depth is included in the minimum tillage group, , with the aim of
energy saving, preserving soil structure and timeliness of work. Among the different
operative options, various types of machinery are available that can perform tillage,
fertilization, seeding and chemical weeding of the soil in a single pass (Toscano, 1998).
3.2. Tillage: Soil characteristics and erosion
The usable kind of tillage depends on the soil texture. In clayey soils, the minimum tillage
can have positive effects on the containment of erosive phenomena, due to the residual crop
on the soil surface, and on compaction, in order to reduce passing of the machinery.
In silty soils all the tilling techniques that do not involve the inversion of layers favour soil
structuring, and the presence of residual crop, and avoids the destructuring caused by the
beating action of rain water.
In sandy soils, the choice of tillage techniques should exclude deep intervention, while all
the minimum tillage techniques generally guarantee best results.
The different handling of the soil can determine a different availability of the nutrient
elements, as well as a different biological activity. The techniques that do not involve the
inversion of layers, allow maintaining or increasing the organic matter in the soil. With regards
to the availability of the principal nutrient elements, the effects produced by the different
tilling techniques vary according to the different movement of each element in the soil.
Nitrogen results mostly available for plants in a worked soil, due to the high aeration of the
mass and for the velocity whereby the residual and organic fertilizers are degraded into
mineral elements which can be assimilated.
For the phosphorus, a low mobile element, there are strong differences in its stratification
along the profile according to the type of tillage. With a plow this element has the tendency
to distribute itself in a more homogeneous way in the soil in comparison to how it is
distributed in soil that has not been worked, or only cracked soil, in which phosphorus
remains in the most superficial layers.
The common good supply of natural Italian agricultural land, particularly clayey ones, leads
to a substantial independence of availability of potassium from the plowing technique
adopted.
All tilling techniques that improve soil permeability, and allow maintenance of a vegetable
coverage, are very useful in the control of erosive phenomena (Stein et al., 1986; Rasiah &

Kay, 1995; Raglione et al., 2000; Toscano, 2000; Toscano et al., 2004a). Erosion consists in the
removal of the most fertile soil layers by wind and/or rainwater action. The eroded amount
is proportional to the intensity of the rainfall, to the slope and type of the soil.
3.3. Soil grassing
To protect the soil profile, structure and edaphic biocenosis, it is useful to apply less
expensive cultivation techniques which have a lower environmental impact than traditional
tillage. The minimum tillage or controlled soil grassing, generally determine a great activity
of the soil biota, due to the greater presence of organic matter and the low trouble of soil.
It is possible to implement soil grassing, which can be either natural or artificial, and to
partially or wholly cover the orchard surface. The benefits of controlled grassing in olive
orchards are: improvement of the soil structure, increase of soil organic matter and water
absorption, reduction of runoff and erosion, improvement of carrying capacity and
reduction of compactness, enhancement of microbial activity and nutritional balance;
simplified management at lower operating costs. The possible competition of turf for water
and nutrients with regards to the olive plants (Pastor, 1989), can be prevented with proper
grass management, such as cutting, or additional fertilization (Toscano et al., 2004b).
The simpler type of soil grassing is permanent, whereby the soil is constantly covered by
spontaneous vegetation that is periodically mowed or shredded.
Alternatively, in dry summer conditions, temporary grassing can be adopted, eliminating
grass, when competition for water competition begins, with superficial tillage or using
contact herbicides; the coverage naturally reestablishes itself with the resumption of the
rainy season, retaining its beneficial effects up to the following spring.
For artificial grassing the choice of the essences is very important, these must have fast
growth following planting and to be resistant to pounding and to mulching. However, the
artificial grassing presents some economic and managerial limits such as the difficult choice
of the essences and the seeding costs.
The machinery for grass management consists in the rotary mower, and the shredder. They
are of great working capacity and have low power needs, compared to the tools that operate
on the soil; the shredder has the advantage that it grinds the mown grass, thus reducing
degradation times, and it can also be used for pruning residues. Both these machines can be
equipped with intercept rotary mower, which allows cutting of the grass along the row,
avoiding damage to the tree trunks. Long-time experiences of controlled grassing in
different non-irrigated olive orchard soils have confirmed the effectiveness of this technique
in improving soil properties, in the drainage system, in the control of soil erosion and on
olive tree productivity (Briccoli Bati et al., 2002; Toscano et al., 1999, 2006). On the contrary
other tests, comparing different application methods of grassing, have evidenced better
agronomic and productive results with green manure in summer, to avoid the increased
competition for soil water occurred by permanent grassing in this environment (Toscano,
2009). Therefore, the choice of the best soil management system must be made according to

the specific soils and crop characteristics. In olive groves the replacement of tillage with
other techniques is possible, according to water availability, in order to obtain the best
maintenance of soil fertility, a reduction of the erosion in slopes, a timeliness of intervention,
a reduction of the costs and, therefore, the attainment of greater incomes.
4. Nutrition
The olive tree is still often considered a rustic plant, having little nutritional requirements
and capable to survive even in rough environments, with minimal care and management.
The olive plant grows in most soil types as long as they are well drained. These plants, could
also vegetate in the absence of fertilization, but require suitable nutrition to express their
productive and qualitative potentialities.
In the traditional olive-grove plant nutrition is mainly based on systematic and massive
inputs of chemical nutrients distributed to the soil, not always correctly and often
unnecessarily (if not harmful) for plants and the environment (groundwater pollution). In
many cases olive tree fertilization is often empirically approached and farmers apply much
more fertiliser than the crop really needs (Tombesi et al., 1996).
The compilation of adequate fertilization programs, in terms of type, doses, epochs and
disposal of the nourishing elements, are not of simple generalization and depend on local
environmental and climatic factors, as well as on the effectiveness of the fertilizer
composition and its application method.
It is indispensable to carry out a prior analysis of soil chemistry and of the nutrient contents
of the plant by plant tissue analysis (usually leaves are used). These analyses will give
significant data on the status of both soil and plant, indicating the most useful typology and
doses of nutrients to apply in the fertilization plans.
Leaf analysis, is a reliable method for assessing the nutritional status of the crop (Bouat,
1968; Freeman et al., 1994). The content of the major nutrients in the leaves differs not only
according to the cultivars, the soil and climatic conditions of the cultivation area, the time of
sampling for the analysis but also in relation to the pruning and irrigation applied to olive
orchard (Briccoli Bati et al., 1995). Some research on a regional scale has defined certain
relationships between the time of leaf sampling, the foliar nutrient content and the quality of
production (Failla et al., 1997; Soyergin et al., 2000). In fact, it was found that the leaf
diagnostic at flowering is conclusive for the less mobile elements (Ca, Mg, Fe and Zn) while
during the winter rest foliar analysis better shows the nutritional potential of the soil for
nutrients with increased mobility as N, P and K (Failla et al., 1997). The level of global
nutrition, generalizing, expressed as percentage amount on the leaf dry matter for nitrogen,
phosphorus and potassium, results as 3,5% divided, respectively, in 2,1 - 0,35 - 1,05 with a
physiological relationship of 6:1:3.
In profitable olive growing, the nutritional needs also vary in relation to the phenological
phases, to the climate, to the cultivar, to the trees productive potentiality, and to the olive
orchard management, i.e. presence of soil grassing and irrigation. For these reasons,

fertilization planning cannot be approached as a standard procedure and many authors
report different evaluations about the nourishment needs of olive trees (Natali, 1993;
Petruccioli & Parlati, 1983).
Fertilisation systems include: chemical fertilisers (NPK applied beneath the tree canopy
projection, usually in the form of combined fertilisers), organic fertilisation (green and
animal manures, leaves, compost, manufactured organic fertilisers), and fertilisation
through watering systems and through foliage.
During the first three years of the olive plantation, when vegetative activity prevails on
fructification, it is important to stimulate, with fertilization, rapid canopy and root growth of
the tree to predispose the plants quickly to flowering and fruiting (Palese et al., 1997). In this
phase, Nitrogen is the essential element, while phospho-potassic fertilizers at this time are
less important, provided that during the preparatory work of the soil for planting, such
fertilizers were distributed over the entire surface and buried with deep tillage.
When the plant completed the first phase of growth (5
th
- 6
th
year) and during the entire life
of the orchard, the scope of fertilization is to induce and support the yield and,
simultaneously, also to ensure the renewal of fruiting shoots and roots.
In order to calculate the amount of nutrient supply to plants it is helpful to adopt the
returning criteria of nutrients removed with fruit harvesting, with pruned wood and
abscised leaves: for 100 kg of drupes produced the olive tree needs around 900 g nitrogen,
200 g of phosphorus and 1.000 g of potassium. In fertilization planning, such doses must be
triplicate, due to the losses leaching, volatilization, fixation, etc.
Traditionally nitrogen is supplied annually and divided in at least two doses. Most of the
quantity to be given (2/3) at the end of winter before flower bud differentiation and before
the growth of new lateral shoots, and the second during the flowering period (from the pre-
flowering stage till fruit set). Usually the recommended nitrogen application ranges between
500-1500g for bearing tree, according to canopy volume.
Throughout the life of the olive-grove, phosphorus and potassium supply must be repeated
every 5-6 years, with the doses defined by the results of soil and leaf analysis. These
fertilizers are usually supplied in autumn, and burying with shallow tillage, on alternate
inter-rows to limit damage to the roots, with doses of 200-400 units of potassium and 100-
200 of phosphorus per hectare integrated with suitable doses of organic matter (manure,
green manure or compost).
During the annual cycle, nitrogen absorption is more intense from the flowering up to the
pit hardening, while the contents of N and P decrease in the leaves up to the pit hardening
and at the same time they increase in the drupes. Subsequently, both in the leaves and fruits
nitrogen and phosphorus decrease after veraison. Instead, potassium, constantly decreases
in the leaves, while increasing in the fruits.
Biennial or triennial interventions for phosphorus and potassium, in the poor soils are
useful, applied after harvesting in concomitance with deep tillage for rainwater storage, in

old or dry-condition raised olive orchard; or at the end of winter, with lighter tillage in a
young and intensive olive grove.
4.1. Nutrients typology and effects
The use of the appropriate fertilizer at the right time increases the efficiency and reduces the
cost of fertilization, with a positive impact on the produced olive fruit and oil content.
Fertilization can be distinguished into organic and mineral. The first one has the purpose to
improve the physical characteristics of the soil, such as the structure, the porosity, the
permeability, the tackiness, the consistency, the water retention, and the pH. The second one
is destined to feed the plants.
Nitrogen is fundamental in plant growth, it participates in the formation of amino acids and
in the formation of proteins, therefore, it is crucial in the growing processes when the plant
is young. In adult trees nitrogen supports the formation of shoots, a necessary condition to
ensure constant productivity and positively influence flower formation, fruit setting and
fruit development, especially during the early stages, up to the pit hardening. Nitrogen
fertilization consistently increases the olive yield but only when leaf N is below the
sufficiency threshold (Hartmann,1958). It is usually applied to the soil using urea,
ammonium sulphate, or ammonium nitrate. Nitrogen can also be supplied with either
organic materials such as feathers or blood meal, compost, or a leguminous cover crop. Its
deficiency is manifested by decreased growth activity, leaf yellowing, high ovary abortion,
low yield and alternate bearing (Cimato et al., 1990; 1995).
Phosphorus also has a role in growth, being essential for cell division and the development
of the meristematic tissues, fruit set, fruit growth and maturation, and in lignification of the
shoots. Even if absorbed by the olive tree in a relatively high quantity, the effects of
phosphatic fertilization are nevertheless manifested with extreme slowness. The most used
phosphate fertilizers are soluble phosphates and in particular superphosphates with 35-45%
of phosphorus pentoxide, which is the form absorbed by the plant. A content of 50-100 ppm
of phosphorus pentoxide in the soil detected by soil analysis, can be considered to be
satisfactory. The symptoms of deficiency of this element, which is very rare, occur with a
reddish or purplish coloration of the green parts of the plant, and it causes metabolic
problems for growth and fructification, with delays in drupe maturation.
Potassium promotes the accumulation of carbon hydrates such as starch, an energetic
reserve for metabolic processes. This element, regulates the water consumption of the plant
through an increase in water retention in the tissues and it also controls transpiration.
Potassium is an enzymatic activator, it increases the plants resistance to thermal extremes
and to some fungal diseases, and it promotes oil accumulation in the fruits. This element is,
usually, applied to the soil during winter in order to gradually reach the rooting zone with
the rain. Regular potassium fertilization is necessary in order to maximize both yield and
quality, especially in heavy yield years.

After nitrogen, phosphorus and potassium, other very important nourishing elements are
magnesium and calcium. Magnesium is an essential component of chlorophyll and
generally it is not considered in fertilization plans because it is already contained in many
fertilizers. Occasionally, magnesium deficiency can be revealed in orchards growing on
sandy, neutral soil. Fertilization based on magnesium sulfate corrects this deficiency.
Calcium is vital to olive plant growth, because it is an essential constituent of cell walls and
contributes to the mechanical resistance of tissues, it also acts as an activator of some
enzymes. Deficiencies of calcium due to soil acidity, can be corrected with an adequate lime
supply as calcium carbonate.
Sulphur is present in plant amino-acids such as cystine, cysteine and methionine and is
located in the soil in the organic matter. Fertilizers containing sulphur as ammonium or
potassium sulphate, etc. are distributed against possible deficiencies of this element.
The most important microelements are iron, copper, zinc, manganese, molybdenum and
especially boron, all developing a specific and exclusive role as enzymatic activators in the
biochemical processes of the plants. These elements, present in small amounts in olive
tissues, have a very narrow range between a sufficiency and toxicity level. Leaf tissue
analyses provide excellent information in order to directly diagnose the toxicity or the lack
of these microelements.
Above all it is very important to know the boron content of the leaves because it plays a
major role in pollen growth, fruit set and plant productivity. Visible symptoms of boron
deficiency are manifested with leaves with apical chlorosis, followed by necrosis and leaf
drop. In the cases of a slight boron deficiency, the fertility of the flowers is reduced due to
increased ovary abortion (Perica et al., 2001). Boron deficiency is nevertheless removable
with extreme rapidity and effectiveness through leaf treatments during the pre-flowering
stages. Foliar applications have had statistically significant effects on the yield and leaf B
contents, therefore, the most economic dose was found to be 0.4% foliar application of
sodium tetraborate.
The organic matter in soil plays a central role in controlling the availability of N, P and K
and it can also act as a chelate, making certain micronutrients more available for the roots in
the form of complexes.
4.2. Fertilization techniques
Plant nutrition is physiologically dependent on the absorption of nourishing elements
through the roots; it is therefore necessary to ensure that in the active soil layer there is a
suitable endowment of available nourishing elements for the plants. Normally fertilizers are
spread on the soil. Nitrogenous fertilizers, nitric, ureic or ammoniacal, are used annually
according to requirements and the time of intervention, the first one being easily soluble
with a fast effect, while ureic and ammoniacal products have a longer acting time and
greater persistence. The nitrogen amount usually provided is of 1kg N equal to,
approximately, 5kg of ammonium sulfate, 3kg of ammonium nitrate, 4kg of calcium nitrate

or 2kg of urea. The principal provisions of phosphorus and potassium, due to their low
mobility in the soil, are provided before the plantation of the orchard establishment, further
applications are made every 4 to 5 years in the autumn on the ground.
Fertilizers can also be supplied by the foliage, and in olive trees this characteristic can be
effectively exploited in order to satisfy the needs of the plants in situations of particular
demands (lacks of microelements), or as integration of soil fertilization in the different
phenological phases. This technique is considered to be a valid support to increase the
nutrient levels and the crop yield, reducing competition among metabolic sinks (shoots,
inflorescences and fruits) and increasing the absorption of nutrients through the roots
(Cimato et al., 1990, 1991, 1994, 1995; Toscano et al., 2000; Toscano, 2008). It provide
nutrients quickly, uses low amounts of fertilizer, can be combined with pesticide
applications, is well suited to rain-fed olive trees or when ground fertilizations would be
useless due to a lack of soil humidity. The advantages of this technique are manifold: timely
intervention, nutrients are given at the moment of greatest necessity, and is effective in a
short time, and allows an integral use of the administered element. If only a foliage solution
is applied, several applications take place (Fernndez-Escobar, 1999; Ben Mimoun et al.,
2004). Some results demonstrate, however, that foliar fertilization cannot entirely replace
nutrition through the roots, even though it permits a reduction of the fertilizer required to
be applied to the soil (Fontanazza, 1988; Toscano et al, 2000).
Many authors have studied the efficiency of olive foliar nourishment and for specific
nutrients good results have been achieved using urea solution (Cimato et al, 1991; 1994).
Potassium is easily absorbed and distributed through leaf tissues (California Fertilizer
Association, 1998) and foliar application is helpful to satisfy plant requirement having a
high efficiency (Inglese et al., 2002). Phosphorous is given during summer fruit growth, in
order to be readily absorbed and translocated to the fruits for quality purposes, therefore its
application is better in the form of a foliar fertilizer.
In addition, recent studies have assessed the effectiveness of some commercial products on
different olive cultivars, which behave differently. A helpful example of this efficiency is
shown using NutriVant (NV) foliar fertilizers in addition to soil fertilization and showed
that better results are obtained on the Carolea, than on Nocellara del Belice cultivar. This
difference is more clear in the off year orchards, during which an increase in the
vegetative parameters and yield entity, in comparison to the control tests, was recorded.
Conversely, in the on year, the NV test had good results on both observed cultivars
(Toscano et al., 2002b; Toscano, 2005; Toscano & Godino, 2010).
In irrigated orchards it is possible to supply nutrients to the plant by watering systems
(fertigation) (Toscano et al., 2002a). The advantages of such practice consist in the easiness of
application and in the efficiency of fertilizers, being able to reduce the needs of fertilizers by
up to 30% in comparison to soil distribution. Fertigation implies a sensitive reduction of the
management costs both in terms of purchase, transport and distribution of fertilizers ,
enhancing their efficacy in order to grant a better nutritional level to the trees, to maximize
yield, oil production and profitability.

5. Irrigation
The efficient use of water resources in agriculture is extremely important in order to
improve the economical and environmental sustainability of agricultural activity.
Mediterranean regions of Italy are characterized by a high evaporative demand of the
atmosphere, water scarcity and increasing negative consequences of climate change. In Italy
the rainfall can vary annually from less than 400 to over 800 millimeters, and the lack of
precipitation that is often manifested during the summer, involves the use of irrigation
during dry periods to ensure the constant productivity of olive orchards.
In traditional olive cultivation areas, characterized by water scarcity, rainfall and
underground water resources are the only supplies for the olive tree water requirements.
Rainfed olive groves, therefore, are characterized by low plantation density which allows
the exploitation of an adequate soil volume by the root system, minimizing competition for
water among plants.
The olive, a sclerophyllous evergreen tree, is able to tolerate the low availability of water in
the soil by means of morphological and physiological adaptations acquired in response to
coping with drought stress (Connor & Fereres, 2005; Bacelar et al., 2007). Under semi-arid
conditions olive trees were able to restrict water loss by modulating stomatal closure at
different levels of soil moisture and evaporative demand and show a non-balanced
allocation of dry matter among the different plant organs, resulting in a reduction of the
vegetative growth and a significant decline in the productive performance (low yield and
alternate bearing behaviour) in favour of development of the root system. Indeed, olive tree
roots can extend and go deep into the soil to exploit a wider soil volume (Fernndez et al.,
1991; Dichio et al., 2002). Olive plants maintain a high rate of photosynthesis during long
drought stress periods. The high efficiency of the olive is also due to its ability to continue to
absorb carbon dioxide and to produce carbohydrates in water deficit conditions that
determine the complete stomatal closure and threaten the survival of other species
(Xiloyannis & Dichio, 2006). A higher photosynthetic rate under drought is a decisive factor
for better drought tolerance in olive cultivars (Bacelar et al., 2007). Generally, when water is
not restricting growth, plants invest a considerable fraction of photoassimilates in the
expansion of photosynthetic tissues, maximising light interception and, as a consequence,
growth (Dale, 1988). The capacity to withstand severe and prolonged drought periods,
however, is negatively associated with olive tree growth and productivity, owing to the
decrease of assimilates under water deficit conditions. Reductions in photosynthetic
performance under water stress have also been observed by several authors (Inglese et al.,
1999; Patumi et al., 1999; Tognetti et al., 2005; Bacelar et al., 2006; Lavee et al., 2007; Ben
Ahmed et al., 2009).
Much research shows the productivity benefits of irrigation. Irrigation is highly effective in
increasing yield and yield components such as fruit size, fruit number and oil content,
moreover, irrigation affects the pulp-to-pit ratio, phenology and time of fruit maturation
(Agabbio, 1978; Goldhamer et al., 1994; Michelakis et al., 1994; Inglese et al., 1996; Gmez-
Rico et al., 2006, 2007; Dag et al., 2008; Servili et al., 2007).

A proper soil water availability enhances vegetative growth, such as shoot length, allowing
the olive trees to produce a higher number of buds able to provide the opportune basis for
the next years production (Patumi et al., 2002; DAndria et al., 2004; Gucci et al., 2007;Ben-
Gal et al., 2008; ). Stress levels and water requirements are highly dependent on fruit load
and best irrigation management must account for biannual bearing effects. Although
biennial bearing is basically genetically determined, the degree to which it occurs is greatly
affected by environmental conditions, especially the weather and cultivation practices
(Pandolfi et al., 2000). Alternate fruit bearing occurs under both extensive and intensive
growing conditions (Pannelli et al., 1996; Lavee, 2006). With irrigation, olive production can
increase up to five times that of olive groves in dry arid climates, in the Italian climate on
average a double production must be expected (Bini et al., 1997). Obviously the scale of
production will depend on soil conditions, average rainfall, evapo-transpiration and
temperatures, cultivars, planting distances and other cultural practices (Nuzzo et al., 1997).
Proper management of irrigation, especially during the summer drought, keeps leaves in
activities promoting fruit growth and accumulation of reserves in the various plant organs
(Xiloyannis & Palese, 2001), in any case, table olives cannot be cultivated without irrigation.
5.1. Olive tree water needs
The unitary water consumption of the olive tree, namely the quantity of water that must be
transpired in order to synthesize a gram of dry substance or commercial product, have been
estimated to be 1 liter of water by 1 m
2
of leaf, daily transpired in August. Such indexes,
with opportune calculations, can be useful to help establish watering volumes. Best
indications are drawn by the compilation of water budgets that, from the comparison
among the entity of the rains and the losses of damp from the soil by evapo-transpiration,
allow the determining of the water deficit or excess in the different periods of the year.
The criteria to be adopted in watering planning must be based on respect of the water
requirement of the crop, and on the knowledge of the critical phases of the vegetative cycle
of the plants, over that of the quantity of available water for irrigation, for the evaluation of
the economic convenience of the intervention. As for all the other production factors, the
economic principle of marginal productivity is in force also for water.
Olive tree water requirements are variable and depend upon factors such as soil type,
climate, planting density, age of trees, cultural management (e.g. fertilizing, pruning) and
the method of irrigation.
In the olive tree there are nevertheless some critical periods during the annual cycle, during
which the plant mostly needs water. The first one extends from bud differentiation up to
flowering and therefore to the fruit set: in these phases a water deficit can create trouble in
flower development with a smaller number of flowers for inflorescence, increasing ovary
abortion, and a lower fruit set. Generally during this period in Italy there are no deficiencies
in soil water. Subsequently, at the second phase of fruit growth, corresponding to the pit
hardening period, olive trees are most resistant to water deficit (Goldhamer et al., 1994;
Moriana et al., 2007), on the contrary the third phase, when olive oil is accumulated, the
olive tree again seems to be sensitive to water stress (Lavee & Wodner, 1991)

The inolition process starts around the pit hardening phase and reaches a maximum before
ripening. The effects of irrigation on oil content are nevertheless quite controversial
depending on different experimental conditions
Some authors did not find any difference in oil content between irrigated and non-irrigated
trees (Michelakis et al., 1994; dAndria et al., 2004), while Inglese et al. (1999) reported a lower
oil content in the fruits of trees grown under high soil water deficit conditions. The literature
suggests that the fruit and oil yield response to irrigation is highly cultivar specific (Lavee et
al., 2007). Despite the increasing use of irrigation in olive groves, there is still a poor
understanding of the effect of irrigation deficit on the qualitative parameters of olive oil.
Increasing irrigation leads to fruits with a greater water content (lower oil percentage), and
irrigation has been found to decrease the polyphenol content (Patumi et al., 1999; Gmez-
Rico et al., 2006; Ben-Gal et al., 2008; Dag et al., 2008), which then changes the oil bitterness
and spicy tastes.
Several studies, which focused on the effect of irrigation on olive oil composition, report that
irrigation increases free fatty acids in oil (Dag et al., 2008), can affect the fatty acid
composition (Ranalli et al., 1997; Aparicio & Luna 2002; Servili et al., 2007) and the
accumulation of secondary metabolites, that are fundamental in improving the organoleptic
characteristics of the oil, is increased (Pannelli et al., 1996; Inglese et al., 1996).
For the calculation of the water needs in an olive-grove, some formulas are used that consider
climatic environmental data, such as the rains and the potential evapo-transpiration (ETP),
adopting different coefficients in relation to the spacing of trees, the age and shape of the
plants, and season. The water deficit, will be given by the difference between the water used
by the crop and the water availability in the soil: such a deficit will be therefore compensated
for with irrigation to optimize the productive potentialities of the plants.
The calculated seasonal watering volumes, will be nevertheless reduced considering the
threshold of convenience, in relation to the efficiency of the irrigation system, the cost and
availability of water and the value of the product. For the intensive olive growing in South
Italy, it increased from 1.500 up to 3.000 m3 hectare
-1
per year (Agabbio, 1978).
An evaluation of the water needs, such as the water consumed by the crop (evaporation and
transpiration), can rationalize the irrigation technique. The evaporation potential (ET0) must be
determined through the compilation of the soil hydrological balance and the search for an
empirical correlation between the potential evapo-transpiration and one of the climatic factors.
To satisfy the needs of an intensive olive-grove the results of different watering trials
pointed out that for the olive tree it is enough to supply 30-50% of the evaporated water.
The beginning of the irrigation season should take place when the soil is still wet (60-70% of
available water) to ensure the maintenance of adequate reserves even in deeper layers and at
points not covered by providers in order, however, to maintain roots present in those areas.
Irrigation can be realized in different ways and the choice of the optimal method should be
made according to each single olive-grove typology and environments. Sprinkling methods,

with giant irrigators or wings, have the advantage of adapting to any soil condition, the
facility of moving and transfer, and the timeliness of intervention, but generally with high
costs and low efficiency of water. On the contrary localized irrigation, that allows water
distribution evenly in sloping land, is a technique which offers the possibility to intervene in
certain biologically critical phases for the plants (flowering, fruit setting, pit hardening, etc.),
allowing a significant reduction, of about 25-30%, in the consumption of water. Furthermore
it allows a more uniform distribution of water over time, with shorter shifts and increases
the efficiency of irrigation up to 90%, avoiding losses due evapo-transpiration, runoff, etc.
With sprinkling the volumes are calculated for the whole surface; with the localized
irrigation on the wet surface equal to 10% with drip irrigation and to 25% with microjets.
Microjets enable irrigation of a rather large surface to meet the different needs of the olive
tree during its development, but also creates constraints for tillage and weeds removal by
mechanical means and increase water loss by evapo-transpiration.
In the center-northern olive-grove environments of Italy, natural water availability is often
such to allow satisfactory production, even without resorting to irrigation. In the South of
Italy, generally the annual average rainfall is rather low, with rains concentrated in the
autumn-winter season, that does not coincide with the needs of the plant, therefore it is
necessary to apply irrigation during summer.
The irrigation intervals depend, more than from the evaporative demand of the
environment, on the type of soil and therefore from the quantity of water that it can retain.
In the case of localized irrigation shifts are on average 2-4 days with about 30 m
3
ha
-1
of
water, the turns will progressively be shorter passing from slimy-sandy to sandy soil.
The most critical phases in which water stress should be avoided are at floraison, at fruit set, at
fruit growth and at inolition. An effective watering season could start, according to watering
water availability, at the end of flowering (May-June) and continuing until late September.
In recent years many studies have tried to apply deficit irrigation strategies to olive trees.
These are based on the observation of Chalmers et al. (1981), who reported for peach trees
that the maintenance of a slight plant water deficit can improve the partitioning of
carbohydrate to reproductive structures, such as fruit, thus controlling excessive vegetative
growth. The asynchronous growth of olive fruits and shoots reduces competition for
assimilates at critical stages, providing a sound basis for the application of irrigation deficit.
The controlled irrigation deficit is a water management method that does not completely
satisfy the trees water requirements during the growing season. It causes a temporary and
regulated water deficit in a specific phenological stage. When it is applied in the pit-
hardening period, the olive oil yield is not affected while the water use efficiency (WUE) is
improved. On the contrary when the controlled irrigation deficit is applied from fruit set to
harvest, the oil yield decreases but the WUE and certain olive oil quality parameters
improve. As the productive tree responses are not affected by moderate levels of water
stress, irrigation deficit strategies are recommended in arid and semi-arid areas to save the
scarce conventional water resources (Angelakis et al., 1999; Massoud et al., 2003).

Finally it is interesting to note that the olive is quite resistant to salinity. This plant tolerates
brackish water (up to a salty residue of 4 g/liter), and therefore can allow the realization of
irrigated olive-groves, valorizing waters which are not usable for other crops (Basta et al.,
2002; Perica et al., 2008).
6. Pruning
Pruning is a very expensive practice in olive grove management, reaching up to 40% of total
cultivation costs, but it is also essential for olive grove profitability. It is finalized to modify
the natural shape and structure of the trees, to reduce to the least one skeletal structures, to
balance the vegetative and productive activity, and to maximize fructification. To reach the
best results, pruning must be rationally managed, and based on the harvesting system. A
modern approach to this practice allows to form and maintain the tree structure at a
relatively low cost, reducing and simplifying pruning operations without negatively
affecting yield, oil quality, or orchard sustainability.
Strategies of minimum pruning can be developed at a farm level independently of the
type and size of the orchard. Managing the canopy according to the criteria of minimum
pruning is suitable both for traditional olive groves and modern, high-density orchards.
The growing habit of the cultivar, the natural tendency for high vegetative activity, the type
of buds and branches, and alternate bearing are all important biological features of the olive
tree that it is important to consider for pruning.
In practice, pruning is distinguished into a formation and a production pruning: formation
pruning has the purpose to give the selected form to the olive tree; while the production
pruning is finalized to preserve the form and the size of the canopy, to eliminate inefficient
or unproductive structures, to facilitate the functional positioning of fruiting shoots to
enhance harvest efficiency, to maintain the trees vegetative and productive balance. Olive
trees bear fruits on the previous years shoots, so to have fruit every year an adequate
vegetative growth must be achieved. Annual interventions should be faster, smaller and
easier cuts using small tools such as shears and saws.
The execution of pruning should avoid the accumulation of too much wood caused by an
excess of primary branches and an excessive overlapping of secondary branches. To
stimulate olive production pruning must be reduced to strictly necessary interventions,
leaving the most possible greater number of leaves. Periodically some return cuts made on
the branches return the plants to their assigned volume to maintain the volume and the
shape of olive trees.
Extraordinary kinds of pruning are practiced when it is necessary to restructure the canopy
in another form held to be more convenient. Pruning old trees requires drastic cuts to
rejuvenate or to restore the health of the plants so as to stimulate their growth and renew
fruit-bearing shoots and branches.
Pruning also contributes to reducing the occurrence of pest and disease. Dense canopies
encourage the presence of parasites due to high relative humidity, whilst well-aerated

canopies considerably decrease the attack of pest and disease such as the olive knot which
appears on branches and is otherwise very difficult to control.
6.1. Training system
Around the Mediterranean basin, a traditional area of olive tree cultivation, there are many
different training systems for olive trees, but now in modern orchards the most common
shapes are:
- The vase with several different variants is by far the most popular shape and the
most practical for hand or semi-mechanical harvesting. Usually the vase has a single
trunk varying in height from 50 cm to 120 cm, branching into some primary branches,
in adult plants generally three, equally spaced so as to intercept as much sunlight as
possible. These branches, tilted about 45-50 degrees, support the scaffolding of the tree.
The only pruning required is in the centre of the canopy to allow enough sunlight to come
through and removing cross branches leaving the greatest possible number of leaves on the
plants, because productivity and the oil quantity in the drupes is dependent on them.
- The single-trunk is constituted by a central axis of the tree that raises a series of
primary branches chosen amongst those that grow vigorously inserted in a spiral,
alternated by 50-60 cm between them. To achieve this form few thinning cuts are made
during the training phase. When the plants are in production the primary branches are
periodically pruned by cutting or heads back, or eliminating them in order to renew the
fruit-bearing surface. This shape is good for the intensive olive growing systems where
the canopy cone reduces distances between the plants and for the mechanical harvest
with shakers, but it is difficult to manage.
- The bush shape is the result of natural basal sprouting of the olive tree with numerous
branches and those that arise from the bottom. This system is suitable for intensive
cultivation models to be collected by hand or with tool facilitators it requires very little
pruning during the training phase, but it is unsuitable for mechanical harvests with
shakers due to lateral branches shooting from the proximal part of the trunk.
- The hedgerow is a training system in which trees grow freely, so that the canopy forms
a productive wall along the row, usually managed with mechanical pruners to maintain
the trees with an available volume.
Lately the training system suitable for olive orchards with over 1000 trees per hectare, is a
single axis, obtained by thinning out the side branches in the apical part and by also
removing those located below 0.5 m from soil during the first year of planting the to allow
the passage of the machine. Once full production has been reached, plants are pruned so as
to contain vegetation within 2.2 m in height and prevent the development of little branches
of a diameter greater than 3 cm. In these groves mechanical pruning alternating with
manual interventions is used to contain the development of foliage.
The training system is the result of the growing habit of the tree and pruning practices and it
must be chosen before the planting as a function of the harvesting method and mainly of the

area climate. Indeed, experimental tests of comparison between the vase and the single-
trunk conducted over several years in the experimental field of CRA-OIL, located in Mirto
Crosia (CS-Italy), showed the extreme difficulty in maintaining the last shape due to the
climatic characteristics which are strongly favorable to olive plant development.
6.2. Pruning scopes and effects
A first objective of pruning is to provide a shape and structure to the tree which guarantees
proper illumination of the canopy to enhance photosynthesis, good circulation of air,
avoidance of pest disease, and a better disposition of fruiting shoots to facilitate and
maximize the harvest.
At plantation, the first cuts are executed to plan the scaffold and the principal branches are
chosen according to the selected shape. In the following years, pruning will be limited to the
elimination of unfit shoots, favoring correct skeletal development. After the third-fourth
year, with the beginning of the yield, annual pruning will have to balance volumetric
growth with the vegetative-productive equilibrium of the trees.
Pruning intensity increases with the age of the tree. Pruning is light on young trees to allow
the shaping and to grow and build energy reserves.
As general guidelines, for adult trees, given the light need of this species to perform
photosynthesis, it is necessary to reduce the density of the foliage, allow sunlight to
penetrate into every part of the olive canopy and promote air circulation. All the suckers
around base of the trunk and branches that have already produced should be removed. It is
also important to keep the upper parts of the tree open to allow the lower parts to remain
productive.
In adult olive-groves, in full production, annual pruning allows better regulating of the
balance between vegetative and reproductive activities and so contributes to reduce
alternate bearing. This phenomenon is more dramatic the wider the pruning shift. The shift
of pruning cycles should be chosen based on factors such as the growth of branches, the
fruit-laden, the training system, soil fertility and climate and structural aspects of the farm.
The execution of pruning every 2 or more years allows a reduction of the cost of such
practice but favors the occurrence of alternate bearing.
Tests for several years showed that it is necessary to maintain a large leafy area of the plant,
and pruning of medium intensity in shifts of two - three years, depending on the cultivar,
gives the best results (Tombesi et al., 2002; Tombesi et al., 2007). These pruning cycles,
compared with annual pruning, allow an increase in the production efficiency of the plant,
and also a net saving of human work, since the execution times are not very different, and
the number of cuts per plant is almost similar.
According to whether the mechanical harvest are adopted, with shakers or mechanization, in
the first instance it is necessary to build the canopy towards erect forms, with shoots which are
relatively short and rigid, to favor the transmission of vibrations up to the drupes in the whole

volume of the canopy, while in the second case, the pruning will have the aim of bringing up
the external wall of the canopy longer and pending fruiting shoots, to facilitate harvesting with
pneumatic combs(De Simone & Tombesi, 2006; Tombesi et al., 2008).
Pruning must be carried out at the end of winter, before the restart of vegetative growth. It
must be avoided after the harvest, because it reduces the cold resistance of the plants and
does not allow wounds to heal, favoring diseases from fungi or other parasites.
Traditionally in late summer a second pruning is performed on adult plants to eliminate
suckers inside the plants, with special reference to the vase shape, where the formation of
shoots within the canopy becomes a rule.
In profitable olive orchards pruning mechanization is essential to reduce management costs
and regain timeliness in working, even though it penalizes the productive efficiency of trees.
To balance the economic needs of management there are several ways to carry out
mechanical pruning that should not be performed simultaneously on the entire plant. To
ensure a good production of fruiting branches the canopy that remains after pruning should
not excessively reduced. Mechanical pruning can be applied alternately in different years
and/or rows by carrying out topping and hedging, reserving annual hand pruning to
eliminate shoots and suckers.
Several experiments have been performed in different types of olive orchards in order to
study the feasibility of mechanical pruning (Giametta & Zimbalatti, 1997; Ferguson et al.,
1999; Ferguson et al., 2002; Pea et al., 2002; Tombesi et al, 2008; Dias et al., 2008; Farinelli et
al., 2009). In our experience, the technical-economical convenience of pruning
mechanization, also considering equipment integrated with pneumatic combs for olive
harvesting, was evaluated. The results showed the good effectiveness of the pruning
equipment in terms of cutting quality and working capacity (Pascuzzi et al., 2007; Toscano
2010a ). Other trials have been carried out to assess the performances and the effects of
mechanical pruning, that was performed both with toothed disks and scissor blades pruning
machines. Both resulted in great efficacy and work productivity; nevertheless, the first ones
are more efficient for woody vegetation, up to 10 cm diameter, while the second ones work
better on thin branches, even though it can cut woody branches up to 5 cm diameter
(unpublished data).
7. Harvesting
In olive grove management, the harvest is the other most expensive practice, together with
pruning. Harvesting systems, can be considered rational only when they can reconcile the
operation costs with the necessity to pick up the maximum yield in respect of the product
quality. In the past, the availability of low cost manpower with a manual harvest allowed
the satisfaction of these two demands, but the low availability and increase of the cost of
labor, have made such operations excessively onerous, and applicable only to table olives.
Manual harvest can be improved using hand-held pneumatic combs to detach the olives
from the plant that assuage the work, and give maximum flexibility in terms of harvesting
time, and increase the operators productivity, but it is time-consuming and costly.

Mechanical harvesting is executed with shaker, also equipped with a reverse umbrella as an
olive interceptor, that has considerable economic advantages compared with traditional
manual picking procedures(Tombesi & Tombesi, 2007). In this way a great reduction in
labour costs, harvesting timeliness and good performance, is achieved. Nevertheless it is
difficult to apply in the majority of traditional olive groves due to the presence of
malformed, voluminous plants, or those of an unsuitable cultivar.
In the new intensive olive groves, with trees optimized for cultivar and structure,
mechanical harvest is instead applicable with positive results, usually in a step, getting up to
80-90% of yield (Hartmann & Reed, 1975; Ferguson et al., 1999; Giametta & Pipitone, 2004;
Toscano & Casacchia, 2006). In these orchards with well pruned trees it is possible to harvest
up to 50 trees/hour with a suitable shaker and collecting system (Lavee, 2010).
Using shakers, the more efficient harvesting yard is constituted by 5 or 7 operators, of which
one operates the shaker, and the others the nets and the moving of olives, reaching a
productivity up to 0,4 tons/man by hour (Briccoli et al., 2006; Tombesi, 2006; Toscano, 2007).
In super intensive olive orchard, or intensive olive orchard with trees structured in a
productive wall, continuous harvesters , derived from grape pickers that work on both walls
of a row (Bellomo et al., 2003; Arrivo et al., 2006) can be used. Wall pickers, that work on a
single side of tree walls, also reach a working productivity up to 1 hectare by hour, with
yield percentages similar to the shakers (Toscano, 2010b).
7.1. Ripening physiology of drupes
The olive tree fruits (Olea europaea L.) are oval or round drupes, of variable weight
according to the cultivar, the yield, the nutritional and health state of the tree. The drupe is
constituted by the external exocarp (peel), by the fleshy mesocarp (pulp) and by the internal
stone (pit). Fruit development and ripening are a combination of biochemical and
physiological changes that occurred during maturity of fruit. The development process is
characterized by changes in size, weight, composition, color, flavor and physical proprieties
of the fruit (Connor & Fereres, 2005) and is critical for final yield and oil quality. Oil
accumulation, which occurs from pit hardening to harvesting may be early or late
depending on the variety, generally it starts in the northern hemisphere from the month of
August and continues up to November-December, subsequently the increase of oil content
in the drupes is apparent being due to water reduction.
The maturation of olives also varies depending on the crop load, environmental conditions,
which are subject to strong annual variations, soil moisture, and cultural practices. At
harvest, within a tree, not all the fruit are at the same ripening stage, in fact this parameter
also depends on the position of the fruits.
For the olives to be used for the oil extraction, the harvest must have been carried out at the
beginning of the ripening phase of fruits, at veraison, when the pulp changes its color from
green to purplish red. This stadium corresponds to the maximum oil yield per hectare,
since, even if subsequently a slight increase in the oil content of the drupes is had, the loss

for natural fruit drop undoes the advantage. In many instances the oil quality also
decreases. An early harvest allows the production a good oil, rich in antioxidants and
aromatic flavors, that confers resistance to oxidation, and a "fruited taste. Instead, oils
obtained from olives harvested at an advanced ripening stage are less intense, less bitter
with a lower percentage of mono-unsaturated and saturated fatty acids and a higher
percentage of poly-unsaturated fatty acids, that penalize its stability.
With regards to the harvesting method, oils with excellent quality can be produced with
both manual and mechanical harvesting, as long as the drupes are intact and healthy.
For table olives harvesting is carried out manually from the plant, to avoid damage to the
fruits that would consequently depreciate their market value. In some cases harvesting in
the olive-grove is done in different steps as a consequence of the ripening scale, or
performed at the same time and the sub-size fruits are sent to the crusher for oil extraction.
To facilitate the harvest it is necessary that the plants are of a contained dimension, and with
suitable forms that assemble the fruits on the outside of the canopy and on lean shoots.
In table cultivars for green fruit processing the harvest is done when the peel color changes
to light green, that corresponds to the beginning of pulp softening and the maximum
content of sugars, fundamental during tanning, for the fermentation process that follows
sweetening, with lye or brine. For the olives destined for tanning to black, the harvest must
be effected when the pulp is also colored, based on the physiological maturation of the
fruits.
The most important parameters to determine the stage of maturation of the drupes are the
fruit separation force and the development of natural fruit drop. Before the natural fall of
olives there is an attenuation of the force with which they are attached to branches and
shoots. These physiological changes do not occur simultaneously on all the drupes of the
same tree but occur with a certain scaling. Therefore, the decrease of the attachment strength
of the fruit and the drop of the first fruits are the most important indices of the final stage of
ripening. These indices are easily determined and able to predict with sufficient reliability
the time to start harvesting.
The optimal time of harvest can be, further, defined as one in which there is a high amount
of fruit on the plant capable of being detached by the machines in considerable percentages
and with a high content in good quality oil when ripening the pulp becomes less consistent
(Farinelli et al., 2006).
8. Conclusions
The information contained in this paper highlights that it is possible to achieve some
improvements in olive tree productivity (in terms of quantity and quality) and a reduction
in costs, spreading more rational agronomic practices. Increasing the olive groves income
can be achieved through updated cultivation techniques. All these must be coordinated and
integrated with each other to obtain a rapid formation of the tree production structures that
allow the maximum expression of their productive potential and provide a high level of

mechanization. Soil management, plant structure, fertilizer, irrigation, pruning and
mechanical harvesting must be chosen according to variety and environmental features. The
paper provides useful indications on the introducing of the cover crop to better soil
management in order to control erosion and maintain soil fertility.
Moreover, olive trees respond very strongly to irrigation and take advantage of very low
volume of water also with regulated irrigation deficit. With regards to olive pruning, this
cultural practice must be managed rationally based on the harvesting system and both these
techniques (pruning and harvesting) must be done mechanically to reduce the running costs
for better crop competitiveness.
Author details
Caterina Briccoli Bati, Elena Santilli and Pietro Toscano
Ilaria Guagliardi
National Research Council - Institute for Agricultural and Forest Systems in the Mediterranean,
(ISAFOM), Rende (CS), Italy
Acknowledgement
Financial support for this study was provided by the Italian Ministry of Agriculture, Food
and Forestry Policy through the project GERMOLI Salvaguardia e valorizzazione del
GERMoplasma OLIvicolo delle collezioni del CRA-OLI.
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507-517.
Chapter 4
Floral Biology: Implications for Fruit
Characteristics and Yield
1. Introduction
Floral biology has important practical implications, in addition to its scientific relevance,
given that flower characteristics and bloom affect fruit characteristics and yield. Yield
derives from fruit quality (e.g. weight) and quantity (i.e. number), which, in turns, depend
on flower quantity and quality: flowers must be suitable to become fruits, and then must be
pollinated and fertilized, and must set fruits, which must then grow. Not all flowers can do
all of this: some flowers, for instance, have aborted ovaries which are partially developed or
absent at bloom, depending on when the abortion occurred. Even when still present, these
aborted ovaries are not capable of becoming fruits. Normal pistils, may not be pollinated or
fertilized, but also fertilized ovaries may drop after some growth, resulting in fruit drop.
From 100 flowers, in olive, all the above phenomena result in one to few fruits (Hartmann,
1950). Because of this low fruit set, it is often believed that cultural practices aimed at
improving pollination, increasing fruit set or reducing ovary abortion or fruit drop, may
lead to increased olive yields.
In the following paragraphs we will analyze in detail some of these passages (i.e. ovary
abortion and fruit set) allowing fruit formation from the flower. We will conclude that, in
olive, yield is affected mostly by the nutritional status of the tree and the environmental
conditions affecting it, which determine the potential yield. To achieve this potential yield, the
plant uses a series of compensating mechanisms which make the yield rather independent of
flowering or any single subsequent passage (i.e. ovary abortion, fruit set, drop and size).
Firstly, however, we will begin by describing how fruit characteristics, particularly fruit size,
are mostly determined by the ovary characteristics at bloom. In the following paragraphs we
will see how these ovary characteristics affect ovary abortion and fruit set. Finally we will
discuss the apparent redundant flowering in olive, which produces many more flowers than
necessary for the yield it can sustain.


72

Figure 1. Flower size differs among olive cultivars: Nocellara del Belice, a large-fruited cultivar, on the
left, and Koroneiki, a small-fruited cultivar, on the right.
2. Ovary vs. fruit characteristics
Fruit size is important commercially and the study of factors affecting it are of great
scientific interest. Fruit size depends mostly on the size and number of fruit cells, though
intercellular space may be also important (Bertin et al., 2002; Corelli-Grappardelli, 2004). In
olive, fruit size differs greatly among cultivars (Barranco, 1999). Both the endocarp and
mesocarp contribute to final fruit size differences among cultivars (Hammami et al., 2011).
Fuit size differences across cultivars are mostly due to cell number, while cell size tends to
be similar (Rapoport et al., 2004), despite the fact that fruit growth, from the ovary to the
mature fruit, is due in greater part to cell expansion than to cell division (Rapoport et al.,
2004; Hammami et al., 2011). In fact, the mature fruit has about 8.5 times the cell number
compared to the ovary, while cell size is about 250 times greater (Rapoport et al., 2004). Fruit
weight is about 2000 (from 1000 to 4000) times greater than ovary weight at bloom (Rosati et

Floral Biology: Implications for Fruit Characteristics and Yield

73
al., 2009). However, from the ovary to the mature fruit, the mesocarp grows much more than
the endocarp (Rosati et al., 2012), probably due to its longer growth period (i.e. up to fruit
maturity) compared to the endocarp, which stops growing about eight weeks after bloom
(Hammami et al., 2011). However, both tissues grow in strict proportion to their initial cell
number in the ovary. The greater growth of the mesocarp is related to its initial (i.e. in the
ovary at bloom) greater number of cells of smaller size (Rosati et al., 2012). These results
agree with the hypothesis that cell number, rather than tissue mass, is related to the sink
strength of an organ (Ho, 1992) even though this might be not always the case (Marcelis,
1996), because cell number is only one factor determining sink strength (Gillaspy et al.,
1993).
Mature fruit size correlates with flower and, particularly, ovary size at bloom (Rosati et al.,
2009). This is true for both the mesocarp and the endocarp independently, though, as stated
above, the mesocarp grows proportionally more: the endocarp volume in the fruit is about
4000 times greater than its volume in the ovary, while the same ratio is about 800 for the
endocarp (Rosati et al., 2012).
Both the endocarp and mesocarp contribute to the ovary size differences among and within
cultivars and even the locular space is proportional to ovary size (Rosati et al., 2012).
Similarly, proportionality among the ovary and other flower parts was found both within
trees (Cuevas & Polito, 2004) and between cultivars (Rosati et al., 2009). Ovary size depends
mostly on cell number rather than size, both across and within cultivars (Rosati et al., 2011a).
This is true both for the mesocarp and the endocarp, suggesting that similar mechanisms
regulate cell division in different tissues. Cell size does differ among cultivar, but does not
correlate with ovary size. Cell size differs greatly among tissues with bigger but fewer cells
in the endocarp compared to the mesocarp, suggesting that cell growth patterns differ
among tissues (Rosati et al., 2011a). In fact, cell size in the ovary correlates with tissue
relative growth (i.e. from bloom to fruit maturity), both across tissues and cultivars,
suggesting that cell size at bloom reflects the stage of growth of a given tissue: the larger the
cells, the less growth remains to be performed (Rosati et al., 2012). This agrees with the
observation that fruit growth is mostly achieved by cell expansion as mentioned above. The
larger cell size of the endocarp at bloom suggests that this tissue is at a more advanced stage
of growth, compared with the mesocarp and thus, its remaining growth, relative to its size
at bloom, is less, at least in terms of cell expansion. This would also explain why the
endocarp stops growing earlier than the mesocarp. It is possible that the endocarp might be
in a more advanced stage of development at bloom, since it is inside this tissue that vital
functions (e.g. fertilisation) take place soon after bloom and thus the endocarp needs
relatively more differentiated cells. The mesocarp, on the contrary, does not perform
particular activities at bloom, hence, it does not need advanced cell differentiation in this
period.
In conclusion, genetic differences in fruit size, among olive cultivars, appear to arise from
differences in cell division patterns occurring in the ovary (and probably in the whole
flower) before bloom. Similar results have been found in other species where genes coding
for cell division before bloom have been found to be responsible for differences in final fruit


74
size. The final fruit size, aside from genetic control, is also related to environmental and
endogenous plant conditions that allow the genetic potential growth to be achieved to a
varying degree. Analysis the genomics, or of environmental factors affecting fruit size is
beyond the scope of this review.

Figure 2. Fruit size differing in olive cultivars: Nocellara del Belice, a large-fruited cultivar, on the right,
and Koroneiki, a small-fruited cultivar, on the left at the same date during the fruit growth cycle. The
difference was already present among the ovaries at bloom.
3. Ovary (or pistil) abortion
Ovary or pistil abortion indicates the presence of flowers with absent or only partly
formed and non-viable ovaries: that is, ovaries incapable of becoming fruits and destined
to drop. Flowers with aborted ovaries are often called staminate flowers since only the
male organs are complete and functional. Normal flowers with both male and female
organs complete and functional are called hermaphrodite flowers. Ovary abortion varies
greatly with year, cultivar, individual tree, branch and shoot, and even among and within
inflorescences (Morettini, 1939; Bottari, 1951; Badr & Hartmann, 1971; Fabbri et al., 2004;
Martin & Sibbett, 2005). Ovary abortion occurs early during flower development, (Pirotta
& De Pergola, 1913), mostly 30-40 days before bloom (Uriu, 1959; Cuevas et al., 1999; Reale
et al., 2006).
Ovary abortion in olive appears to results mostly from competition among flowers (and
ovaries, the future fruits) for resources, which are insufficient for all flowers to develop,
given the redundant flowering. The onset of this competition occurs very early and affects
both pistil abortion and fruit set, as we will see later (Hartmann, 1950; Uriu, 1959; Cuevas et
al., 1994; Perica et al., 2001; Levin & Lavee, 2005). Conditions that affect competition among


75
flowers/fruits or that decrease available resources, usually result in increased abortion and
decreased fruit set. Among such conditions are: N deficiency, foliar diseases and low leaf-to-
bud ratio, (Petri, 1920; Morettini, 1951; Uriu, 1953, 1959; Fernandez-Escobar et al., 2008);
water stress (Melis, 1923; Brooks, 1948; Garca Glvez, 2005); insufficient light (Bottari, 1951;
Dimassi et al., 1999); adverse climactic conditions and high yield in previous year (Rallo et
al., 1981; Rapoport & Rallo, 1991; Lavee, 1996; Cuevas et al., 1994); abundant flowering in
current year (Reale et al., 2006); and unfavorable inflorescence position in the canopy
(Cuevas & Polito, 2004; Seifi et al., 2008). Aborted flowers do not contain starch, suggesting a
link between nutrient availability and pistil abortion (Reale et al., 2009). Removing part of
the inflorescences reduces drastically the ovary abortion in olive (Seifi et al., 2008).
On the other hand, pistil abortion is known to be under genetic control, varying with the
cultivar (Campbell, 1911; Morettini, 1939, 1951; Magherini, 1971; Lavee, 1996; Lavee et al.,
2002). In a recent paper it has been demonstrated that even the genetic component can be
interpreted with the competition theory (Rosati et al., 2011b). In fact, as described above,
fruit size differences are related to ovary size differences among cultivars. Large-fruited
cultivars tend to have bigger flowers, not just ovaries (Rosati et al., 2009). This implies
greater energetic costs for the same number of flowers, and thus greater pistil abortion,
which in fact tends to be higher in larger-fruited cultivars (Morettini, 1939; Magherini, 1971;
Acebedo, 2000; Rosati et al., 2011b). In the previous paragraph we have seen that greater
ovary size in large-fruited cultivars results from greater cell number (not greater cell size)
and cell number appears to be a key factor in determining the sink strength of an organ
(Marcelis, 1996). Larger ovaries with more cells, therefore, might have a greater ability to
compete for resources than smaller ovaries, determining both greater abortion and lower
fruit set.
4. Fruit set
As mentioned above, fruit set is very low in olive (Hartmann, 1950). It is often believed that
positively affecting it (i.e. reducing it) would lead to greater yields. However, many studies
show that when flower numbers are artificially reduced, fruit set increases proportionally,
leading to similar numbers of set fruits (Suarez et al., 1984, Rallo & Fernandez-Escobar, 1985,
Lavee et al., 1996, 1999). This is interpreted as a tendency of the olive tree to set a fixed mass
of fruits, that is independent of flower number. This fixed mass is referred to as the fruiting
potential (Lavee et al., 1996), and depends on the genotype and environmental factors.
Hence, as soon as fruit set reaches such potential, based on tree reserves, the rest of the
flowers may drop.
Competition for resources among flowers in olive is reported in many studies (Suarez et al.,
1984; Cuevas et al., 1994; Lavee et al., 1999; Seifi et al., 2008; Rapoport & Rallo, 1991; Cuevas et
al., 1995). Rugini and Pannelli (1993) showed that fruit set increases when shoot
development is mechanically or chemically slowed down, further supporting the hypothesis
that competition drives fruit set. Given that resource availability affects fruit set, large-
fruited cultivars, having larger ovaries/flowers (Rosati et al., 2009), which need more
resources, set fewer fruits, though total fruit mass is similar (Rosati et al., 2010). This


76
suggests compensation between fruit number and size. The genetic effect on fruit set,
appears thus to result from different degrees of competition for resources among
ovary/flowers of different sizes. This would explain why small-fruited cultivars like
Arbequina and Arbosana tend to produce several fruits per inflorescence, while table-olive
(i.e. large-fruited) cultivars typically produce only one, but bigger fruit. Nor can it be argued
that the small size in small-fruited cultivars is the result of a higher fruit set and consequent
source limitations. In fact, aside from the fact that the fruit of these small-fruited cultivars is
smaller already at bloom (i.e. the ovary), well before fruit set is decided, thinning fruits to
reduce competition increases fruit size minimally, but does not result in fruit size
comparable to that of large-fruited cultivars (Rosati et al., 2010). This suggests that
differences in fruit size are mainly of genetic origin. Clearly, the ultimate fruit size depends
also on resource availability, and varies up to more than 100% within the same cultivar, but
among cultivars fruit size may vary much more than that (Barranco, 1999), up to 600%
(Rosati et al., 2009). Hence, adjusting fruit load across cultivars with extremely different fruit
size needs a wider compensation mechanism than just adjusting fruit size: this mechanism
appears to be the adjustment of fruit set.
5. Redundant flowering, andromonecy and fitness
As stated above, the olive produces redundant flowers relative to its yield potential and
fruit set is very low. We have seen how ovary abortion increases under conditions that
increase competition for resources. In fact, ovary abortion is a means to save resources,
balancing the number of ovaries to the resources available (Primack & Lloyd, 1980; Bertin,
1982; Stephenson & Bertin, 1983). Plants, like the olive, that abort part of the ovaries in
otherwise hermaphrodite flowers are called andromonoecious (about 4000 species are
estimated to be andrommonoecious). From an evolutionary point of view, andromonoecy is
considered an intermediate step towards dioecy, and allows saving of resources without
affecting the total number of flowers and thus the male function and fitness (Vallejo-Marn
& Rausher, 2007). Aborting the ovary does not affect pollen production in olive (Cuevas &
Polito, 2004). In this species, large-fruited cultivars have greater ovary abortion, but similar
number of flowers (Rosati et al., 2010, 2011b), leaving the male function probably unaltered
(although differences in pollen production per flower, among cultivars with different
fruit/ovary/flower size have not been studied). Leaving the male function (and fitness)
unaltered might explain why only the ovary is aborted instead of the whole flower, which
would save more resources, given that the flower is several times bigger than the ovary
alone (Rosati et al., 2009; Cuevas & Polito, 2004). It would also explain why the flower
production is so redundant compared to the fruiting potential. Producing pollen is less
expensive than producing fruits and seeds, while still increasing the plants fitness, making
it more convenient to produce more (male) flowers that fruits. Modeling work demonstrates
that fitness is maximized, under limiting resources, when many more flowers than fruits are
produced (Morgan, 1993). This author finds that the optimal flower/fruit ratio increases for
andromonoecious and monoecious species compared to dioecious ones, and increases even
more for wind pollinated species, like the olive.


77
To increase olive productivity, therefore, it might be possible to breed plants that invest less
in male fitness (reduced flowering), thus saving resources, which can be spend in setting
and maturing more fruits. This appears reasonable since the energetic cost of flowering is
not negligible in olive (Famiani et al., unpublished data).
6. Conclusions
We have seen how competition for available resources among developing flowers and fruits
plays a continuous role during the whole developmental cycle of the reproductive organs
(Lavee et al., 1999). Hence, pistil abortion first, and fruit set afterwards, appear to be part of a
single mechanism that adjusts maternal investments to available resources. Even the genetic
component affecting fruit size (i.e. cultivar differences) may be explained with the
competition theory, based on the related differences in flower and ovary size (which
correlates with fruit size), implying different energetic costs for the development of one
fruit. In the absence of catastrophic events that might impair production (e.g. lack of
pollination, severe drought or nutrient deficiency, etc.), the olive tree appears to produce
fruits according to its potential, independent of the amount of flowers produced. To achieve
this potential, the tree uses the different compensatory mechanisms available at any given
developmental stage (i.e. pistil abortion, fruit drop, fruit size). Hence, studies reporting
positive correlations between abundance of flowering (or pollen in the air) and yield
(Moriondo et al., 2001; Fornaciari et al., 2002, Galn et al., 2004), would imply not a causal
relationship but simply correlated phenomena: bloom and yield both represent an
expression of the tree yield potential. The redundant flowering in olive, may serve the
purpose of increasing the mail and the overall fitness, which is advantageous from an
evolutionary point of view, but wasteful from an agronomical perspective. For the olive
grower, selecting for reduced mail fitness (i.e. fewer flowers) to benefit the female fitness
(i.e. yield) might prove beneficial.
Author details
Agricultural Research Council - Olive Growing and Oil Industry Research Centre, Spoleto (PG),
Italy
Acknowledgment
7. References
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Chapter 5
Susceptibility of Cultivars to Biotic Stresses
1. Introduction
The Italian history of crop protection has been dominated by the use of agrochemical,
generally having a negative impact on the environment and residues in final products. The
approach based on the use of genotypes with low susceptibility to biotic stresses searching
for sources of resistances was largely uninvestigated for long time. Only recently researchers
focussed their studies on this subject. Studies devoted to this subject are very difficult
mainly for permanent crop such as the olive tree because time consuming and because it is
very hard to compare the behaviour of a large germplasm in the same pedoclimatic
condition. Furthermore, it is very hard to design tests of resistances under controlled
conditions because of the difficulty due to the hard tolerance of captivity of pests such as
olive fly and many others. In any case, recently research activities on this field has been
greatly improved and several research projects are supported by many institutions.
On the other hand, abiotic stresses has been sufficiently studied, mainly for those
concerning the cold tolerance. The CRA-OLI of Rende studied from fifteen years the
behaviour of several cultivars in respect to the main biotic stresses utilising two large
varietal collections planted in two experimental fields, the largest on the ionian coast of
Calabria (Mirto-Crosia, Cosenza) and the smallest near Rende (Cosenza). Results showed a
high behavioural diversity of observed cultivars showing different degree of susceptibility
to main pathogens and phytofagous of the olive tree. Some cultivars has been deeply
investigated searching for the mechanisms determining such behavioural differences.
2. Susceptibility to biotic stresses
Many studies reported in this paragraph have been carried out in two experimental olive
groves 20-years old located in Calabria, South Italy, where hundreds of cultivars coming
from around the world grow in the same pedoclimatic conditions and permitted us to
produce useful data for comparing susceptibility to biotic stresses.

2.1. Susceptibility to Bactrocera oleae
It is well known that cultivars have a differentiated susceptibility to olive fly infestations. In
this paragraph are chronologically arranged some of papers devoted to the comparison of
susceptibility of cultivars to the major insect pest of the olive. The cultivars Carboncella di
Pianacce, Gentile, Bardhi i Tirana, Kokermadh i Berat and Nociara showed an infestation
level significantly lower (less than 10%) than the cultivars Carolea, Cassanese, Cucco,
Giarraffa, Intosso, Kalinjot, Nocellara del Belice, Picholine and Santa Caterina (more than 20
%) during two years of observations (1997-1998) (Iannotta et al., 2001) (Table 1). Other
cultivars investigated by the same authors (Dritta di Moscufo, Leccino, Maiatica di
Ferrandina and Mixan) showed intermediate infestation levels. Among these cultivars has
been observed that cultivars showing a low infestation level had a higher percentage of
sterile oviposition stings.
Iannotta et al. (2001) also underlined that the cultivars having a high amount of oleuropein in the
pulp of drupes are those with the lowest level of infestation in the period considered optimal for
the harvesting (end of October-beginning of November) (Table 1). In fact, the cultivars
Carboncella di Pianacce, Gentile di Chieti, Bardhi i Tirana, Kokermadh i Berat and Nociara have
an amount of oleuropein higher of 30g/kg of fresh pulp, while the cultivars with higher
infestation level have an amount of oleuropein lower than 20g/kg of fresh pulp. From these data
emerge a correlation between olive fly infestation and oleuropein content of drupes (Fig. 1).
Cultivar
Sterile Oviposition
Stings
Infestation Oleuropein
(%) (%) (g/Kg f.p.)
Bhardi i Tirana 27.5 C 8.5 A 36.60
Carboncella di Pianacce 26.0 C 9.5 A 34.09
Carolea 13.0 AB 22.5 CDE 20.21
Cassanese 17.0 ABC 24.5 E 16.12
Cucco 23.5 ABC 26.5 E 19.11
Dritta di Moscufo 26.0 C 11.0 AB 18.51
Gentile di Chieti 26.0 C 9.5 A 31.37
Giarraffa 16.5 AB 23.5 DE 10.81
Intosso 18.0 ABC 31.0 E 24.04
Kalinjot 12.5 A 23.0 DE 9.29
Kokermadh i Berat 26.5 C 10.0 AB 31.18
Leccino 21.5 ABC 20.0 BCDE 29.01
Maiatica di Ferrandina 19.5 ABC 12.5 ABCD 27.88
Mixan 19.0 ABC 11.5 ABC 19.80
Nocellara del Belice 18.5 ABC 23.0 DE 16.47
Nociara 25.0 BC 9.5 A 32.73
Picholine 17.0 ABC 24.0 E 19.58
Santa Caterina 16.5 ABC 23.5 DE 16.83
Table 1. Percentages of olive fly infestation showed by olive cultivars growing in the same
pedoclimatic conditions. Letters indicate significant statistical differences (P<0.01; ANOVA test) (from
Iannotta et al., 2001, modified).

Susceptibility of Cultivars to Biotic Stresses 83

Figure 1. Correlation between oleuropein content of drupes and olive fly infestation
(from Iannotta et al., 2001, modified).
Studies carried out in Sicily (Iannotta et al., 2002) highlighted the low susceptibility to olive
fly infestations of the cv. Turdunazza antimosca compared to cvs. Tonda Iblea, Moresca and
Verdese in the same olive grove. In this case, the low suceptibility of the cultivar
Turdunazza antimosca seems to be related to the repellent action against the olive fly of that
cultivar.
Iannotta et al. (2006a) observed a very low active infestation (percentage of drupes with
living stages of the olive fly, such as eggs, larvae or pupae) rate along the ripening season
for cvs. Cellina di Nard and Cima di Mola, strongly reducing drupes damages and
avoiding any kind of pesticide use for the production of a high quality olive oil (Table 2).
The cv. Ogliarola del Vulture showed a low infestation level only until the end of October
and this lead to an anticipated harvesting for producing a high quality olive oil without
field pesticide applications. The cv. Leccino registered a high infestation only during the
first period of the olive ripening, while during October and November infestation was
lower than the 20% (Table 2), i.e. tollerable for obtaining high quality olive oil. Other
cultivars, mainly cv. Maurino, Moraiolo and Grossa di Spagna, showed a high
susceptibility to olive fly attacks, showing active infestation levels higher than the 20%.
For last cultivars the field applications of pesticides are needed. Percentages of sterile
oviposition stings were higher at the end of September for cultivars with low
susceptibility, when the content of oleuropein within drupes is the highest of the ripening
season. These results confirmed previous observations that demonstrated the role of
oleuropein for increasing the mortality of eggs and reducing the hatching rate of young
larvae (Iannotta et al., 2002).
These data underline that increasing studies on cultivar susceptibility could effectively
produce significant results under a perspective of olive growing sustainability
Iannotta et al. (2006b) obtained results concerning 16 Italian cultivars (Table 3), displaying
data concerning different development stages of the olive fly, active and total (active

infestation plus exit holes of adults) infestation levels, detected amounts of oleuropein and
cyanidine and weight of 100 drupes.
Cultivar
First
ripening period
(%)
Second
ripening period
(%)
Third
ripening period
(%)
Cellina di Nard 16,50 D 6,50 D 14,50 CD
Cima di Mola 22,00 BCD 15,50 CD 13,00 D
Coratina 49,00 ABC 79,50 A 51,50 ABCD
Dolce Agogia 41,00 ABCD 20,50 CD 23,00 BCD
Frantoio 39,50 ABCD 35,00 BCD 45,00 ABCD
Grossa di Spagna 21,50 CD 16,50 CD 74,50 AB
Leccino 44,50 ABCD 15,00 CD 17,00 CD
Maurino 26,00 BCD 38,50 BCD 77,50 A
Moraiolo 56,00 A 49,50 ABC 65,00 ABC
Ogliarola Barese 32,00 ABCD 41,00 BCD 38,50 ABCD
Ogliarola Vulture 25,00 BCD 16,50 CD 71,00 AB
Peranzana 51,00 AB 66,00 AB 70,50 AB
Pisciottana 24,00 BCD 43,50 BC 53,00 ABCD
Table 2. Active infestation trend. Letters indicate significant statistical differences (P<0.01; ANOVA
test), from Iannotta et al. (2006a), modified.
The same table shows that cvs. Ascolana tenera and Nostrana di Brisighella turn out to be
significatively the less infested cultivars, both for active and total infestation in all observed
ripening times. Cellina di Nard shows the lowest susceptibility to olive fly attacks (9.83%
of active infestation and 17.67% of total infestation). Also cvs. Nera di Cantinelle, Frantoio,
Tonda di Strongoli, Nolca, Cima di Melfi and Termite di Bitetto exhibit low susceptibility
(active infestation lower than 13%), while cvs. Dolce Agogia, Dolce di Rossano, Nostrale di
Fiano Romano, Ogliarola del Bradano, Ogliarola garganica and Ogliarola del Vulture
display intermediate susceptibility values. The active and total infestations, obtained as
mean values for any cultivars concerning the different ripening times, turn out to be
increasing during the season (Table 4).
The results obtained for 9 non-Italian cultivars show cvs. Gordal sevillana and Hojiblanca
with the lowest level of active infestation (<15%), cv. Konservolia the most infested and cvs.
Arbequina, Kalamata, Koroneiki, Lucques, Manzanilla and Picual register intermediate
infestation percentages (Table 5).
The results of Iannotta et al. (2006b) confirm a different behavior of olive cultivars
concerning their susceptibility to olive fly attack. Investigated genotypes in the area of
observation displayed a contained percentage of attack lower than 13% in the Italian cvs.
Cellina di Nard, Nera di Cantinelle, Frantoio, Tonda di Strongoli, Nolca, Cima di Melfi and
Termite di Bitetto and lower than 15% in non-Italian cvs. Gordal sevillana and Hojiblanca.

Active percentage within the limit of 15% is compatible with a high qualitative product
(olive oil) avoiding the use of pesticides. It allows the achievement of the fixed aims,
consisting in ecosystem and biocoenotic balances safeguard, which make economically
positive the ecocultivation (organic and integrated farming).

Weight
100
drupes
(g)
Eggs
Sterile
oviposition
stings
Larvae
(pupae)
Exit
holes
Infestation
Oleuropein
(ppm)
Cyanidine
(ppm)
Fertile
(Aborted)
Active
(total)
Ascolana tenera

676.1
725.3
842.7
19.0 (2.5)
10.0 (2.0)
13.5 (2.5)
10.0
8.0
7.5
7.5 (3.5)
12.0 (6.0)
24.5 (4.0)
5.5
15.0
13.0
31.0 (49.0)
30.0 (55.0)
42.0 (65.0)
7974.1
3595.4
1419.0
0.0
17.1
129.3
Cellina di Nard

125.7
131.1
143.3
3.5 (1.0)
5.5 (0.5)
2.0 (2.5)
4.5
5.0
3.5
0.0 (0.0)
2.5 (1.0)
12.5 (2.5)
0.0
0.0
6.5
3.5 (9.0)
9.0 (14.5)
17.0 (29.5)
8165.2
1482.1
0.0
79.0
1019.7
1690.0
Cima di Melfi

247.4
271.0
260.8
9.5 (1.0)
11.5 (5.0)
5.5 (4.0)
8.5
17.0
10.0
0.0 (2.0)
0.0 (0.0)
4.5 (3.5)
3.0
4.5
6.0
11.5 (24.0)
11.5 (38.0)
13.5 (33.5)
-
2469.8
0.0
-
17.4
9.9
Dolce Agogia

181.2
209.5
227.2
8.0 (2.5)
3.0 (0.5)
7.0 (1.0)
7.5
11.0
16.5
4.5 (0.0)
16.0 (2.5)
2.5 (7.5)
2.0
7.5
17.5
12.5 (24.5)
26.5 (45.5)
17.0 (52.0)
8919.3
4889.6
42.9
27.3
0.0
0.0
Dolce di Rossano

133.7
186.6
175.5
7.0 (2.0)
4.0 (0.5)
7.5 (2.0)
13.5
6.5
5.5
3.0 (2.0)
8.0 (0.5)
15.5 (4.5)
0.0
3.5
14.5
12.0 (27.5)
12.5 (23.0)
27.5 (49.5)
11860.3
681.7
149.2
0.0
96.7
503.1
Frantoio

157.6
175.3
221.4
6.5 (3.0)
6.0 (0.0)
5.0 (0.5)
9.0
9.5
3.5
0.0 (0.0)
3.0 (0.0)
7.5 (4.5)
0.0
1.0
2.0
6.5 (18.5)
9.0 (19.5)
17.0 (23.0)
7831.2
1292.0
561.4
12.7
0.0
0.0
Moraiolo

163.6
183.0
231.2
8.0 (2.5)
13.0 (4.0)
5.5 (0.5)
5.5
5.5
8.5
3.5 (0.0)
0.5 (3.0)
12.0 (4.5)
0.5
1.0
5.0
11.5 (20.0)
16.5 (27.0)
22.5 (36.5)
11810.7
5162.9
1771.3
0.0
33.9
337.6
Nera di Cantinelle

226.2
253.3
3.0 (0.0)
4.5 (0.0)
9.0
11.0
1.5 (0.0)
9.0 (0.0)
0.5
6.5
4.5 (14.0)
13.5 (31.0)
8727.3
666.9
10.5
56.6

Weight
100
drupes
(g)
Eggs
Sterile
oviposition
stings
Larvae
(pupae)
Exit
holes
Infestation
Oleuropein
(ppm)
Cyanidine
(ppm)
Fertile
(Aborted)
Active
(total)
270.9 4.0 (0.0) 6.5 7.5 (2.5) 16.5 14.0 (37.0) 39.1 210.1

Nolca

250.2
327.0
325.1
1.0 (0.0)
2.0 (2.0)
2.0 (2.0)
13.0
6.5
8.0
2.5 (0.5)
3.5 (6.0)
13.5 (5.0)
2.5
7.0
10.0
4.0 (19.5)
11.5 (27.0)
21.0 (41.0)
155.9
0.0
0.0
91.1
601.8
1120.3
Nostrale di Fiano Romano

255.5
262.4
342.3
10.5 (2.0)
7.0 (1.5)
7.5 (0.0)
10.5
8.0
16.0
3.5 (3.5)
6.0 (3.5)
3.0 (4.5)
12.0
8.5
15.5
17.5 (42.0)
19.0 (37.0)
15.0 (46.5)
3174.0
463.4
93.8
34.0
25.4
13.8
Nostrana di Brisighella

481.7
801.2
674.9
13.5 (2.5)
3.5 (0.5)
15.5 (2.0)
10.5
0.0
6.5
12.5 (8.0)
5.0 (6.5)
15.5 (8.0)
5.0
19.5
29.5
36.0 (54.0)
22.0 (42.0)
39.0 (77.0)
6444.0
5002.3
454.0
0.0
17.2
0.0
Ogliarola del Bradano

153.9
171.6
235.4
10.0 (1.0)
13.5 (3.5)
0.0 (0.0)
6.0
6.0
5.0
2.5 (1.0)
4.5 (2.0)
11.0 (5.0)
1.5
3.0
7.5
13.5 (22.0)
20.0 (32.5)
16.0 (28.5)
3830.2
2238.0
88.8
0.0
0.0
0.0
Ogliarola Garganica

261.2
238.2
250.6
8.5 (4.5)
8.5 (1.0)
2.5 (1.0)
16.5
13.5
4.0
1.0 (0.0)
1.5 (3.5)
10.0 (5.0)
1.0
5.0
13.5
9.5 (31.5)
13.5 (33.0)
20.5 (39.0)
5387.2
641.6
444.0
71.6
46.3
705.6
Ogliarola del Vulture

195.4
188.8
265.4
4.5 (2.0)
3.5 (1.0)
12.0 (3.5)
11.5
6.0
21.0
1.5 (0.0)
7.0 (2.5)
12.5 (3.0)
2.5
3.0
13.5
6.0 (22.0)
13.0 (23.0)
27.5 (65.5)
9082.3
6669.3
588.0
36.9
21.4
0.0
Termite di Bitetto

228.0
268.8
382.9
4.5 (0.0)
2.0 (0.0)
2.5 (1.0)
8.0
6.5
11.0
4.5 (0.0)
10.0 (0.0)
5.5 (5.5)
1.5
14.0
10.0
9.0 (18.5)
12.0 (32.5)
17.5 (39.5)
1654.4
345.6
229.4
34.0
513.5
1647.1
Tonda di Strongoli

389.7
448.7
453.3
7.5 (1.0)
8.5 (0.0)
2.5 (0.5)
7.0
11.0
6.5
1.0 (1.0)
3.0 (0.0)
7.0 (4.0)
2.0
1.0
10.5
9.5 (19.5)
11.5 (23.5)
13.5 (31.0)
8299.6
4153.3
557.1
0.0
0.0
15.5
Table 3. Detailed data obtained in the different theses and ripening times concerning olive fly
infestation and oleuropein and cyanidine drupe contents. For any cultivar, the observation was
performed in three different ripening times (from Iannotta et al., 2006b, modified).

Ripening time

Active
infestation
Tukey test Total
infestation
Tukey test

26 Sept. 2006 12.38 B 28.81 c C
26 Oct. 2006 15.69 B 35.88 b B
29 Nov. 2006 21.28 A 49.06 a A
Table 4. Data concerning the comparison among the different investigated ripening times referred to
Italian cultivars (from Iannotta et al., 2006b, modified). Letters indicate significant statistical differences
(Capital letters: P<0.01; small letters: P<0.05; ANOVA test).
Weight
drupes
(g)
Eggs Sterile
oviposition
stings
Larvae
(pupae)
Emergency
holes
Infestation
Oleuropein
(ppm)
Cyanidine
(ppm)
Fertile
(Aborted)
Active
(total)
Arbequina

149.6
195.1
191.0
11.5 (1.0)
6.0 (1.0)
5.5 (0.0)
9.5
4.5
5.0
2.5 (0.0)
9.5 (1.5)
7.5 (3.0)
1.0
2.5
14.0
14.0 (25.5)
18.0 (26.0)
16.0 (35.0)
508.9
101.8
1956.4
11.5
23.2
0.0
Gordal sevillana

506.9
803.6
922.3
10.0 (0.5)
10.5 (1.0)
4.5 (2.0)
12.0
7.5
9.5
0.0 (0.0)
4.0 (1.5)
10 (2.5)
0.0
4.0
7.0
10.0 (22.5)
16.0 (28.5)
17.0 (35.5)
3994.2
1764.8
35.4
17.6
77.7
140.7
Hojiblanca

219.6
290.8
322.6
2.0 (3.0)
3.5 (0.0)
4.5 (0.0)
11.5
8.0
5.0
1.5 (0.0)
15.0
(2.0)
15.0
(1.5)
0.5
5.0
18.0
3.5 (18.5)
20.5 (33.5)
21.0 (44.0)
5808.2
5424.5
-
0.0
187.5
-
Kalamata

197.9
298.3
301.0
11.5 (3.5)
5.0 (1.0)
11.0 (2.0)
14.0
0.0
1.0
2.0 (0.0)
6.5 (3.5)
11.5
(6.5)
1.0
2.5
3.0
13.5 (32.0)
15.0 (18.5)
29.0 (35.0)
10571.0
3644.6
240.7
0.0
22.0
0.0
Konservolia

322.9
465.3
508.4
13.5 (3.5)
8.5 (1.5)
12.0 (1.5)
8.5
10.5
7.5
2.5 (0.0)
5.5 (2.5)
16.0
(3.0)
2.5
9.0
3.5
16.0 (30.5)
16.5 (37.5)
31.0 (43.5)
5761.2
3203.6
234.2
187.5
122.0
36.9
Koroneiki

93.0
87.3
89.7
3.5 (0.0)
5.5 (0.5)
9.5 (1.5)
10.0
4.5
7.5
3.5 (0.0)
10.0
(2.5)
14.0
(5.0)
2.5
4.5
5.0
7.0 (19.5)
18.0 (27.5)
28.5 (42.5)
6987.9
2029.9
5774.4
0.0
0.0
0.0

Weight
drupes
(g)
Eggs Sterile
oviposition
stings
Larvae
(pupae)
Emergency
holes
Infestation
Oleuropein
(ppm)
Cyanidine
(ppm)
Fertile
(Aborted)
Active
(total)
Lucques

515.7
623.9
853.3
6.0 (1.5)
2.0 (0.0)
5.0 (0.0)
0.0
12.0
5.5
6.5 (3.0)
6.5 (5.5)
12.5
(3.0)
5.0
6.5
13.5
15.5 (22.0)
14.0 (32.5)
20.5 (39.5)
3557.7
2119.3
0.0
12.6
96.4
12.4
Manzanilla

376.3
353.2
423.8
11.5 (2.5)
5.0 (1.0)
5.5 (1.5)
5.5
9.0
55
1.5 (0.0)
7.5 (2.5)
18.0
(2.0)
3.0
6.0
8.5
13.0 (24.0)
15.0 (31.0)
25.5 (41.0)
10517.0
6028.2
519.1
85.6
742.6
186.1
Picual

364.5
435.1
507.1
8.0 (1.5)
6.5 (2.5)
4.5 (2.0)
14.0
1.0
3.5
3.5 (0.0)
10.0
(5.0)
17.5
(1.5)
0.0
6.0
7.0
11.5 (27.0)
21.5 (31.0)
23.5 (36.0)
8915.3
5663.4
2596.7
21.81
178.5
1695.6
Table 5. Detailed percentages obtained in the different theses and ripening times concerning B. oleae
infestation and oleuropein and cyanidine drupe contents. For any non-Italian cultivar, the observation
was performed in three different ripening times (from Iannotta et al., 2006b, modified).
The susceptibility to B. oleae of other ten cultivars has been investigated by Iannotta et al.
(2007a). Observations were carried out detecting the percentages of sterile oviposition
stings, active infestation (presence of pre-imago stages: eggs, larvae and pupae) and total
infestation (emergence holes, feeding tunnels and pre-imago stages) on olive fruit samples,
200 drupes per cultivar. Samples were collected in three different times during fruit ripening
(Table 6).

Weight
drupes (g)
Eggs
Sterile
stings
Larvae
(pupae)
Emergence
holes
Reinfested by

Fertile
(Aborted)
Larvae (pupae)
Bardhi Tirana

309.69
358.91
415.48
9.5 (2.0)
2.5 (0.5)
7.5 (1.0)
9.0
9.0
6.0
0.5 (0.0)
4.5 (0.0)
12.5 (1.0)
0.5
4.5
7.0
0.0 (0.0)
0.0 (0.0)
2.5 (0.0)
Carboncella di Pianacce

100.86
128.96
129.91
11.0 (0.5)
9.5 (0.5)
9.5 (1.5)
16.5
9.5
5.5
1.5 (0.0)
11.0 (5.5)
17.5 (3.5)
5.0
9.5
10.5
0.0 (0.0)
0.0 (0.0)
1.0 (1.0)
Carolea
321.34 11.0 (1.0) 11.0 8.5 (2.0) 3.5 0.0 (0.0)


Weight
drupes (g)
Eggs
Sterile
stings
Larvae
(pupae)
Emergence
holes
Reinfested by

Fertile
(Aborted)
Larvae (pupae)
390.70
463.46
4.0 (1.0)
1.0 (1.0)
1.0
5.5
22.0 (4.0)
18.0 (5.5)
11.0
21.5
0.0 (2.5)
9.0 (2.0)
Cassanese

228.09
285.48
312.36
5.0 (0.0)
8.5 (0.5)
7.0 (2.5)
5.5
3.0
1.0
2.5 (0.0)
13.5 (0.0)
19.0 (2.0)
0.5
7.0
17.0
0.0 (0.0)
0.5 (1.0)
7.5 (0.0)
Gentile di Chieti

511.35
495.03
524.06
14.0 (0.5)
10.0 (3.0)
6.5 (0.0)
19.0
5.5
7.0
4.0 (0.0)
12.5 (3.0)
14.5 (1.0)
5.0
9.5
12.0
0.0 (0.0)
2.5 (0.0)
4.0 (0.0)
Giarraffa

606.61
711.97
771.70
5.0 (0.5)
5.0 (1.0)
2.5 (0.0)
15.0
6.5
2.0
6.0 (0.0)
20.0 (4.0)
23.0 (6.0)
8.0
8.5
17.5
0.0 (0.0)
5.0 (0.5)
12.0 (0.0)
Nocellara del Belice

349.88
408.33
496.77
5.0 (1.0)
0.5 (1.0)
7.5 (1.5)
10.0
3.0
4.0
3.0 (0.5)
20.5 (5.0)
19.0 (4.0)
4.0
10.0
9.0
0.0 (0.0)
8.0 (2.5)
4.0 (1.5)
Nociara

163.15
196.58
211.31
12.0 (6.5)
5.5 (0.0)
7.5 (2.5)
18.5
5.5
4.5
5.0 (0.0)
20.0 (3.0)
16.0 (5.0)
2.5
10.0
11.0
0.0 (0.0)
4.0 (0.0)
5.5 (1.0)
Picholine

416.12
422.25
466.44
5.0 (0.5)
6.5 (0.5)
10.0 (2.0)
14.0
5.5
1.0
1.0 (1.5)
13.0 (1.5)
25.0 (5.0)
2.5
13.5
16.0
0.0 (0.0)
4.0 (0.0)
2.0 (0.0)
Tonda nera dolce

242.92
301.60
298.97
2.0 (0.5)
5.0 (2.5)
4.0 (0.5)
3.5
10.5
12.5
0.0 (0.0)
3.5 (0.0)
4.0 (0.0)
0.0
3.0
3.5
0.0 (0.0)
0.0 (0.0)
0.0 (0.0)
Table 6. Detailed percentages obtained in the different cultivars and ripening times concerning olive fly
infestation (from Iannotta et al., 2007a). Observations were performed in three different ripening times
(03rd October, 04th November and 5th December) for any investigated cultivar. Reported values are
referred to 100 drupes.
Drupes weight increased during the season, according to the physiological processes
involved in fruit maturation. Fertile eggs were more abundant than aborted ones, which did
not exhibit a trend related to ripening times. For many observed cultivars, sterile oviposition
stings were much more abundant in the first ripening time. Few pupae were registered
within drupes in respect to larvae. As expected, emergence holes increased during the

season showing a low value in the first ripening time. Very low value of reinfestation were
observed according to female egg laying behavior.
Infestation values due to preimago stages seem to be generally related to the investigated
ripening time rather than to the single investigated cultivar. More evident trends were
determined by the olive plant phenology, however some cultivars showed an interesting
and peculiar behavior in relation to attack levels of olive fly.
Results show that the less susceptible cultivars to B. oleae attacks are cvs. Tonda nera dolce
and Bardhi Tirana while cvs. Carolea, Carboncella di Pianacce, Gentile di Chieti, Giarraffa,
Nocellara del Belice, Nociara and Picholine displayed a considerable susceptibility (Table 6).
The low susceptibility observed for cvs. Tonda nera dolce and Bardhi Tirana could be
attributed to different causes. The high amount of oleuropein present in the drupes of
Bardhi Tirana, according to previous studies (Iannotta et al., 2001; 2002; 2006a), could be
involved in processes determining a low incidence of olive fly attack. It hasnt been
observed an high presence of the glycoside in cv. Tonda nera dolce while a considerable
amount of cyanidine in the drupes was registered. The high value of cyanidine in cv. Tonda
nera dolce could determine the observed low susceptibility. Probably, the dark color given
by anthocyanins, achieved ever since in the early ripening stages, could confuse female olive
flies in drupe recognition with a consequent decreased ovideposition.
The results obtained in the present research confirm those ones achieved in previous
investigations, proving the need to explore the existent olive germplasm to search genetic
resistance sources. It suggests the utility to achieve these results both to transfer directly to
farmers world and to emphasize ecosystem health and biodiversity conservation.
2.2. Susceptibility to Pseudomonas savastanoi
To keep under control the olive knot disease, the use of preventive measures turns out to be
essential. Among these ones, the use of less susceptible cultivars emerged to be promising.
Behavioural investigations showed a large variability in severities of olive disease caused by
Pseudomonas savastanoi (Smith). A study carried out in the spring of 2005 by performing a
large-scale investigations on the different responses to the pathogen of 262 Italian and 43
non-Italian cultivars, in the germplasm conservation field, where plants are cultivated under
the same environmental and growing conditions (Iannotta et al., 2006c). The response to
pathogen was evaluated by examining the symptomatology on the basis of the quantity of
tubercles present on branches, arranged in classes of infection. During this time, several
adverse meteorological events took place, including record minimum temperatures which
influence the onset of the disease. Results displayed a different behaviour of olive cultivars
to P. savastanoi in relation to their different susceptibility to the pathogen. Among observed
Italian cultivars, 61% showed an infections percentage ranging from 0 to 20%, 22.5%
ranging from 20 to 40%, 11.1% ranging from 40 to 60%, 5% ranging from 60 to 80% and 0.4%
ranging from 80 and 100%. Among investigated cultivars, 86 displayed no symptom of
disease (Abunara, Aitana, Ascolana dura, Ascolana semitenera, Aurina, Bianchera,

Cacaredda, Capolga, Caprina Casalanguida, Caprina vastese, Carbonchia, Carpinetana,
Cavalieri, Cellina Rotello, Colombina, Corneglia, Cornia, Corniola, Corniolo, Correggiolo,
Dolce Andria, Fosco, Gentile Larino, Gentile Colletorto, Giusta, Gragnaro, Gragnan,
Grappolo, Grossa Venafro, Grossale, I/77, Laurina, Lavagnina, Mantonica, Marina pugliese,
Marzio, Minna di vacca, Morfa, Morchiaio, Morellona Grecia, Morinello, Nasitana, Nebbio
Chieti, Nocellara etnea ovale, Ogliara, Ogliastro grande, Oliva grossa, Olivastra seggionese,
Olivastro Bucchinico, Olivo da olio, Olivo della Madonna, Orbetana, Ortice, Pampagliosa,
Pennulara, Piangente, Piantone Moiano, Pignola, Posola, Posolella, Precoce, Puntella,
Racioppella, Rastellina, Raza, Razzo, Remugnana, Resciola Venafro, Romanella molisana,
Rosciola coltodino, Rosciola Rotello, Rustica, Saligna, Sammartinara, Sammartinenga, San
Benedetto, San Francesco, Santa Maria, Sperone di gallo, Tombarello, Tonda Alife, Tonda
dolce, Tonda dolce Partanna, Tunnulidda, Vicio, Zinzifarica) and no cultivar with a
percentage of infection equivalent to 100% was observed, emphasizing the presence of
genetic resources in Italian germplasm for olive knot disease prevention. Among analysed
non-Italian cultivars, 41.9% showed a percentage of infection included between 0 and 20%,
with 5 cultivars showing no sign of attack (Bardhi, Chetani, Hojiblanca, Lucques,
Salonenque); 23.3% included between 20 and 40%; 4.7% included between 40 and 60%; 4.7%
between 60 and 80%; 23.3% included between 80 and 100%. Cultivars Drobnica, Koroneiki
and Vasilikada showed an infection equivalent to 100%. This different susceptibility
evaluated under the same agro-environmental conditions, confirms a different response to
the pathogen in relation to the ratio plant/parasite and appears tightly dependent from
cultivars tolerance to low temperatures.
2.3. Susceptibility to Spilocaea oleagina
The different susceptibility of olive cultivars to knot disease has been observed by several
authors. Iannotta and Monardo (2004) observed that both percentage of the number of
leaves infected and surface occupied on leaves by the symptoms of the disease are
significantly different in observed cultivars. These authors observed that out of 35 cultivars
studied cultivars Bardhi i Tirana, Carboncella di Pianacce, Cassanese, Dritta di Moscufo,
Gentile di Chieti, Kalinjot and Leccino did not show any kind of symptoms of the disease,
while cultivars Bosana, Carolea, Nocellara del Belice, Nera di Villacidro, Maiatica di
Ferrandina, Itrana and Tonda di Cagliari were clearly infected by Spilocaea oleagina. The
others observed cultivars showed intermediate values of the disease incidence (Table 7). The
same authors related the cultivar susceptibility to the oleuropein content of leaves showing
a direct relation between oleuropein content and cultivar susceptibility. In fact, low
susceptible cultivars are those with the highest oleuropein content in leaves. Iannotta &
Monardo (2004) affirmed that oleuropein could play an important role in determining the
development inhibition of the fungus within the leaves.
Results are similar to those obtained by Iannotta et al. (2001) concerning the relation among
oleuropein content of drupes and susceptibility to olive fly infestation, demonstrating that
studies devoted to the exploration of genetic variability of olive cultivars is a focal subject, to
date not sufficiently developped but of great importance.


Table 7. Percentage of infected leaves (FI%) and percentage of surface occupied by symptoms on
leaves surface (SI%) of 35 cultivars (from Iannotta and Monardo, 2004). Letters indicate significant
statistical differences (P<0.01; ANOVA test).
2.4. Susceptibility to Camarosporium dalmaticum
Iannotta et al. (2006d) found a different level of susceptibility among cultivars for fruit rot
disease. In particular, a low susceptibility was observed for cvs. Frantoio, Tonda di Strongoli
and Dolce di Rossano. On the contrary, cvs. Ascolana tenera and Nostrana di Brisighella
showed a high susceptibility (Table 8). Since the cultivars displaying the lowest
susceptibility to the fungus are the same which show the lowest susceptibility to olive fly
attacks, a direct correlation between could be hypotised. Also data concerning non-Italian
cultivars show a different behaviour. In fact, cvs. Arbequina, Hojiblanca and Picual are the

less infected and cv. Gordal sevillana the most affected by mycosis (Table 9). The study
proves the utility of further investigations in order to characterize the different cultivars
behaviour in relation to their parasites, so as to define their specific susceptibility.
Cultivar
C. dalmaticum
Infection LSD test
Ascolana tenera 12.00 A
Cellina di Nard 3.33 BC
Cima di Melfi 4.67 BC
Dolce Agogia 3.17 BC
Dolce di Rossano 1.67 BC
Frantoio 0.67 C
Moraiolo 4.00 BC
Nera di Cantinelle 3.17 BC
Nolca 4.33 BC
Nostrale di Fiano Romano 3.67 BC
Nostrana di Brisighella 11.17 A
Ogliarola del Bradano 4.50 BC
Ogliarola garganica 2.83 BC
Ogliarola del Vulture 6.00 B
Termite di Bitetto 3.67 BC
Tonda di Strongoli 1.50 BC
Table 8. Mean values concerning C. dalmaticum infection referred to each Italian cultivars. Letters
indicate significant statistical differences (P<0.01; ANOVA test). (from Iannotta et al., 2006d, modified).
Cultivar
C. dalmaticum
Infection LSD test
Arbequina 3.33 b
Gordal sevillana 7.50 a
Hojiblanca 3.83 b
Kalamata 5.00 ab
Konservolia 5.50 ab
Koroneiki 4.50 ab
Lucques 5.50 ab
Manzanilla 5.00 ab
Picual 3.00 b
Table 9. Mean values concerning C. dalmaticum infection referred to each non-Italian cultivars. Letters
indicate significant statistical differences (P<0.01; ANOVA test). (from Iannotta et al., 2006d, modified).
Trials performed by Iannotta et al. (2007a) in the collection field of the CRA OLI compared
susceptibility to Camarosporium dalmaticum of cultivars planted in the same environmental

and agronomic conditions (Fig. 2). The investigation has been performed in 2005 by
analyzing 10 cultivars, in the experimental olive plantation made up by a cultivar collection
consisting in 20-years old plants. Observations were carried out detecting on 200 drupes per
cultivar the infection level (%) of C. dalmaticum by direct observation of drupes. Samples
were collected in three different period during fruit ripening (03rd October, 04th November
and 5th December).

Figure 2. Location of CRA-OLI experimental field, where several cultivars are planted under the same
agronomical and climatic conditions, on the ionian coast of Calabria (Mirto-Crosia, Cosenza, Italy).
Table 10. Mean infected drupes concerning Camarosporium dalmaticum infection. Letters indicate
significant statistical differences (P<0.01; ANOVA test). (From Iannotta et al., 2007a, modified).

Results display a different susceptibility of investigated genotypes in relation to the fungal
infection, as indicated by significativity letters concerning analysis of variance (Table 10).
The lowest susceptibility in relation to fungal infection has been observed for cv. Tonda nera
dolce. Also cvs. Bardhi Tirana, Carboncella di Pianacce and Nociara showed a low
susceptibility in relation to the pathogenic fungus infection. The cv. Giarraffa turn out to be
the most susceptible cultivar while cvs. Carolea, Cassanese, Gentile di Chieti, Nocellara del
Belice and Picholine display an intermediate susceptibility value. Comparison among
investigated ripening times for pathogenic fungus emphasise an increase of infection
percentages through the season.
2.5. Resistance to Verticillium dahliae
Verticillium wilt, caused by the fungus Verticillium dahliae Kleb., is a vascular wilt with a
very large host range. Verticillium wilt is increasing in several Mediterranean countries, and
it is very difficult to reduce its incidence because is not easy to apply in the field chemical
compounds such as fosetyl-Al, directly inoculated within vascular system. Very little is
known about biochemical and molecular mechanisms of olive resistance to the Verticillium
wilt. Among investigated aspects, it seems to be very important the role of phenolic
methabolism (Baidez et al., 2007; Markakis et al., 2010). Vizzarri et al. (2011) investigated the
susceptibility of cvs. Arbequina, Arbosana, Frantoio, Ottobratica, SantAgostino and Urano
by inoculating them with different isolates of Verticillium dahliae. Plantlets of 18 months and
160 cm high, has been inoculated by applying a small square of agar with sporulating fungal
hyphae on wounded wood. After inoculation plantlets were observed for one year in a
greenhouse. The severity of the disese was evaluated by utilising 5 classes of the percentage
of damaged plant and the intensity of symptoms: 0%, healthy plant; 20%, plant with large
clorosis and moderate foliar symptoms; 50%, severe foliar symptoms and desiccation of
vegetative apex; 75%, desiccation of more than half of the plant; 100% dead plant, without
defoliation (Colella et al., 2008). Furthermore, Vizzarri et al. (2011) defined a molecular
protocol for studying the expression of gene involved in defense mechanisms of the olive.
They utilised plants of 14 months of the cv. Leccino, some artificially inoculated as described
above and some non inoculated as negative control. Results showed that genetic resistance
of tested cultivars, inoculated with different Verticillium isolates, is subordinated to the
virulence of the inoculated isolate (Table 11).

Table 11. Results of the genotypic susceptibility assay. Number of plants showing a given range of
symptoms. Cultivars and isolates used (10 replicates for each cultivar). (from Vizzarri et al., 2011).

The cv. Frantoio and Urano were the most resistant with no symptoms on tested plants,
while cvs. Ottobratica and SantAgostino were higly susceptible. Further studies are needed
for assessing the behaviour of cvs. Arbequina and Arbosana, largely utilised in
superintensive olive plantations, that showed a differentiated susceptibility depending on
the utilised isolate for inoculation.
Vizzarri et al. (2011) also tested a method for evaluating the expression of the genes (PAL
and CHS) involved in defense mechanisms of olive. The level of trascript of these genes
showed significant increments after plantlets wounding. Attaining the highest value 9 hours
after plantlets wounding. Afterward, the level of transcript of both genes decrease, more
evidently for the gene PAL after 24 hours, in any case maintaining high expression levels
until 30 days after wounding (Fig. 3).

Figure 3. Time course of the relative transcript level of PAL and CHS genes in the leaves of stem-
wounded olive plantlets (cv. Leccino), as compared to unwounded plantlets (from Vizzarri et al., 2011).
The use oh this method permit to verify that the response of plant to the injury is quite
rapid, more or less 9-12 hours. Vizzarri et al. (2011) hypothised that the evaluation of the
expression level of genes PAL e CHS for cultivars with different resistance could be
important for verifying the role of phenolic methabolism in olive resistance to pathogenes.
In fact, recent papers demonstrated that phenolic compounds are very important for
modulating resistance/susceptibility of olive cultivars to verticillium wilt. Phenolic response
to verticillium wilt is very different in resistant and susceptible cultivars. The resistant cv.
Koroneiki showed higher increasing of phenols than susceptible cv. Amfissis when
inoculated with verticillium wilt (Markakis et al., 2010). Genes PAL e CHS play an important
role in the biosynthesis of phenolic compounds, then a role in determing resistance of olive
cultivars to verticillium wilt could be hypothesised.

3. Factors affecting cultivar susceptibility
Morphological parameters such as dimensions and coulor are known to be important in
determining susceptibility of cultivars affecting female choice for laying eggs. Recently, a
great effort is done for determining biochemical factors involved in resistance/susceptibility
of olive cultivars to biotic stresses.
3.1. The role of phenolic compounds
Differences in the response of olive cultivars to olive fly infestations have been observed
(Gmusay et al., 1990; Iannotta et al., 1999, 2006a, 2007a, 2007b; Pereira et al., 2004; Basile et
al., 2006; Rizzo and Caleca, 2006; Daane and Johnson, 2010). Two phenolic compounds,
oleuropein and cyanidine, were assessed to determine their role in the reduced
susceptibility of certain olive cultivars to olive fly (Iannotta et al., 2006a, 2007a, 2007b). In
addition, a positive correlation has been observed between the drupe oleuropein content
and a low susceptibility of olive cultivars to olive fly damage such that when the drupe
oleuropein content is high, the olive fruit is less susceptible to attack (Iannotta et al., 2006a,
2007a, 2007b).
3.1.1. Oleuropein
Iannotta et al. (2001) investigated the huge olive showing a low susceptibility to olive fly
infestations of some cultivars due to the high content of oleuropein within drupes. That
cultivars became particularly interesting in respect to cultivars having a low oleuropein
content within drupes also when planted in the same environmental and agronomical
conditions. Although a correlation between high oleuropein content and low
susceptibility of olive cultivars to olive fly infestations is generally accepted, it is nopt
clear the mechanism of action of this compound. Some authors hypothesised a mechanism
of action against eggs and young larvae of olive fly explicated by oleuropein and their
methabolites within the tissue of drupes, causing a reduction of the preimaginal
population of this pest. Iannotta et al. (2001) evaluated the amount and the localisation of
oleuropein within drupes of ten cultivars selected among them known as low-susceptible
and high-susceptible ones. Furthermore, absolute oleuropein has been applied directly on
the oviposition sting in order to evaluate its efficacy to control egg hatchling and the
following larval development. Results confirm the different behaviour of tested cultivars
with cvs. Bardhi i Tirana, Carboncella di Pianacce, Gentile di Chieti and Nociara less
susceptible than cvs. Carolea, Nocellara del Belice, Giarraffa, Cucco, Picholine and
Cassanese (Table 12). Susceptibility of cultivars is correlated to the amount of oleuropein
within drupes. Furthermore, the amount of oleuropein is higher where female lays eggs.
The higher amount of this compound in the epicarp found for the low susceptible
cultivars seems to be related to genetic characteristics of cultivars more than to
phisiological response to olive fle attacks, as demonstrated by comparing the distribution
of oleuropein in healthy and infested drupes.

Cultivar
Active
infestation
(%)
Sterile
oviposition
stings
(%)
Oleuropein
(drupe)
(mg/g)
Oleuropein
(epicarp)
(mg/g)
Oleuropeina
(mesocarp)
(mg/g)
Bardhi i Tirana 8,5A 27,5C 29,60cd 32,89 26,48
Carboncella di
Pianacce
9,5A 26,0C 60,04b 70,54 49,55
Gentile di Chieti 9,6A 26,4C 38,82bc 37,69 39,96
Nociara 8,9A 25,2BC 91,91a 141,37 42,45
Carolea 22,7CDE 13,8AB 2,25cd 23,90 20,61
Nocellara del Belice 23,1DE 18,6ABC 40,52bc 48,93 32,12
Giarraffa 23,6DE 16,5AB 11,41d 13,05 9,78
Cucco 26,8E 23,5ABC 21,10cd 23,24 18,87
Picholine 24,1E 17,1ABC 18,80cd 17,36 20,25
Cassanese 27,4E 16,9ABC 14,30d 17,24 11,36
Table 12. Detailed percentages obtained in the different cultivars concerning olive fly infestation (from
Iannotta et al., 2001, modified). Reported values are referred to 100 drupes. Letters indicate significant
statistical differences (P<0.01; ANOVA test).
The use of oleuropein directly on oviposition stings confirm the role of control agent of this
compound. After ten days from the oleuropein application, within treated sample only the
31% of olive were infested, while within the untreated sample the infested olive were the
65%.
Phenolic composition and concentration are related to genetic features of a given olive
cultivar. These genetic features can be used as varietal markers and as indicators of fruit
maturation (Esti et al., 1998). Furthermore, a correlation between olive fruit size and
oleuropein content has been shown. Small-fruit cultivars are characterised by high
oleuropein content (Amiot et al. 1986). Phenolic compounds are important for the defence of
plants against pathogens and insect infestations (Haukioja et al., 1985; Hudgins et al., 2003).
The antimicrobial activity of phenolic compounds is well documented (Bisignano et al. 1999;
Rauha et al., 2000; Proestos et al., 2005; Pereira et al., 2006, 2007).
It has been shown that oleuropein and cyanidine contents are inversely related. During olive
fruit maturation the oleuropein content decreases rapidly (Limiroli et al., 1995) while
flavonoid content as cyanidine increases (Amiot et al., 1989). In detail, olive fruit maturation
consists of three phases: the growth, green maturation and black maturation (Amiot et al.,
1989). While in the growth phase an accumulation of oleuropein occurs, in the green
maturation phase it decreases. The black maturation phase is characterized by the
appearance of anthocyanins and by the progressive decrease of oleuropein levels (Amiot et
al., 1989). In Iannotta et al. (2006a) the mean content of both phenolic compounds appears
genetically determined. Similar results were observed by Iannotta et al. (2007a, 2007b)
confirming a different olive genotype behavior which depends on the genetically
determined content of phenolic compounds (Esti et al., 1998). Moreover, the oleuropein

content of drupes is not affected by B. oleae attacks, as it is not moved to the damage site. No
differences in the oleuropein content were observed between non-infested and infested
drupes belonging to the same cultivar (Iannotta et al., 2002).

Figure 4. Results of treatment test with oleuropein of oviposition stings (from Iannotta et al., 2001,
modified).
In previous studies, it has been established that the drupe oleuropein content is genetically
determined since it hasnt been observed a statically significative difference between
oleuropein content in non infested and infested drupes by B. oleae belonging to the same
genotype (Iannotta et al., 2001). Moreover, it has been proved that the differences in
oleuropein amounts are correlated to the different behavior of the cultivars in relation to
olive fly attacks (Iannotta et al., 2001, 2002, 2006b).
Oleuropein was first isolated from olive leaves (Panizzi et al., 1960) where it is present in
high levels (Le Tutour and Guedon, 1992). In addition, it occurs throughout the tree and in
any constituent part of the fruit (Servili et al., 1999). Oleuropein confers resistance to
diseases and to insect infestation of the olive tree (Soler-Rivas et al., 2000). The bactericidal
and bacteriostatic activities of oleuropein and its degradation products against many
pathogenic microorganisms have been investigated (Hirschman, 1972; Federici and Bongi,
1983) and its in vitro activity has been detected in relation to several bacteria, fungi, viruses,
and parasitic protozoans (Hirschman, 1972; Walter et al., 1973; Gourama and Bullerman,
1987; Tassou et al., 1991; Tranter et al., 1993; Tassou and Nychas, 1994, 1995). Oleuropein can
also interfere with the synthesis of virus amino acids, prevent viral shedding, budding or
assembly at the cell membrane, inhibit viral replication and, in the case of retroviruses,
neutralize the production of reverse transcriptase and proteases. Oleuropein is also able to
stimulate phagocytosis, as a response of the immune system against pathogenic
microorganisms (Hirschman, 1972). A strong chemotactile repulsion exerted by oleuropein
in the oviposition of olive fly eggs has been described (Soler-Rivas et al., 2000). Small
droplets of olive sap exuded just after oviposition prevent other females from ovipositing on

the same fruit (Girolami et al., 1981; Lo Scalzo et al., 1994). Oleuropein acts by inhibiting the
development of olive fly immature stages, especially eggs and first instar larvae during the
early ripening period (Iannotta et al., 2002). The higher concentration of oleuropein in the
epicarp than in the mesocarp may be due to the biological function of oleuropein in drupe
protection against pests (Soler-Rivas et al., 2000). In fact, the epicarp is the interface between
the outer environment and the inner olive fruit. Therefore, high levels of oleuropein in the
epicarp protect the fruit against olive fly ovideposition (Iannotta et al., 2002).
Moreover, the defence response of fruits damaged both by pathogens and mechanical
means, is mediated by -glucosidase; this enzyme hydrolyses the oleuropein, producing
highly reactive aldehyde molecules. Olive cultivars with different levels of enzyme activity
have differing degrees of susceptibility to the olive fly. This may be related to the ability of
the -glucosidase to produce highly reactive aldehyde molecules in damaged tissues. A
strong peroxidase activity is thereafter detected as a consequence of damage (Spadafora et
al., 2008). Results obtained by Iannotta et al. (2001) showed that five cultivars (Bardhi i
Tirana, Carboncella di Pianacce, Gentile di Chieti, Kokermadh i Berat, and Nociara) with
high levels of drupe oleuropein (31.18 36.60 g kg
-1
) had low levels of infestation (lower
than 10%). When oleuropein content decreases, a corresponding increase in the amount of
damage caused by olive flies occurs. In the same cultivars, Iannotta et al. (2001) found that
the percentage of sterile oviposition stings ranged from 25.0 to 27.5%. Similar results were
also observed for cultivars SantAgostino, Leccino, and partially Frantoio (Basile et al., 2006).
Sterile sting numbers and oleuropein content are inversely proportional to infestation
(Iannotta et al., 2001). The role of oleuropein in the inhibition of the development of olive fly
immature stages has been shown by performing a comparison between untreated olive
samples and samples treated with oleuropein belonging to the cv. Carolea. The cultivar
Carolea was chosen because it is susceptible to the olive fly. After ten days, infestation levels
were 31% and 65%, respectively, in the oleuropein-treated and non-treated samples
(Iannotta et al. 2002). The concentration of oleuropein is greater in the epicarp rather than in
the mesocarp during the entire ripening process, except in the case of cvs. Gentile di Chieti
and Picholine (Iannotta et al., 2002, 2007a). In these varieties, there is a slightly lower content
of oleuropein in the epicarp during the early ripening period. In another study no
correlation was observed between infestation and oleuropein content (Iannotta et al., 2006a).
In fact, olive fly infestation may be different on the same olive cultivar under different
environmental conditions (Fontanazza, 2000) inasmuch as the oleuropein content might be
affected by climatic trend (Iannotta et al., 2006a). In a study performed in 2005 in an
experimental field located on the Ionian coast of Calabria (Southern Italy), it was observed
that cv Cellina di Nard was the least infested by the olive fly in terms of total infestation
(17.67%). In contrast, cvs. Ascolana tenera and Nostrana di Brisighella were the most
damaged attaining percentages of total infestation at 56.33% and 57.67%, respectively
(Iannotta et al., 2006a). This difference is presumably related also to fruit size (Daane and
Johnson, 2010). In fact, Cellina di Nard has relatively small fruits compared to Ascolana
tenera and Nostrana di Brisighella. In addition, it has been shown that small-fruit cultivars

are characterised by high oleuropein content (Amiot et al., 1986), playing a synergic role in
determining low susceptibility.
Interestingly, it has also been observed that many cultivars characterized by low
susceptibility to olive fly attacks showed low susceptibility to the fungal pathogen Spilocaea
oleagina (Cast.) Hugh. and a negative correlation between oleuropein content in olive leaves
and fungal infection has been found (Iannotta and Monardo, 2004). In addition, a correlation
between B. oleae infestation and Camarosporium dalmaticum (Thm.) Zachos & Tzav.-Klon.
infection has been established (Iannotta et al., 2007d). Since the same cultivars showed low
levels of susceptibility to both parasites, it could be assumed that high levels of oleuropein
may play a role also in determining low cultivar susceptibility to fungal disease (Iannotta et
al., 2006c, 2007a).
3.1.2. Cyanidine
Cyanidine occurs in olive fruits (Servili et al., 1999) and an increase of cyanidine content at
the end of the maturation stages of the olive fruit, as a consequence of hydrolytic processes,
was found (Vinha et al., 2005). On the reasons of different genotype behavior concerning the
susceptibility to olive fly attacks, the direct influence of cyanidine in the drupes could be, in
effect, supposed. It is evident in cvs. Cellina di Nard, Nolca and Termite di Bitetto which
register high value of cyanidine, increasing during the season (Iannotta et al., 2006b). When
investigated genotypes are cultivated in the same pedoclimatic conditions and samples
obtained from them are collected in the same ripening times, it is possible attribute the
differences, concerning cyanidine amount, to a strong influence of the different investigated
genotypes genetic diversity. It has been observed that the completely pigmented drupes are
not very recognizable by B. oleae females determining considerable difficulties for their
ovideposition (Caleca, pers. comm.).
A role played by cyanidine in resistance to herbivores was additionally assessed (Harborne
and Williams, 1998). Significant differences were found among cultivars in relation to active
and total infestations and cyanidine content (Iannotta et al., 2006a). Cultivars Ascolana
tenera and Nostrana di Brisighella had the highest level of active infestation (34.33% and
32.33%, respectively) while cv. Cellina di Nard was the least infested (9.83%). In addition,
cvs. Frantoio, Gordal sevillana, Koroneiki, Nera di Cantinelle, Nolca, Ogliarola garganica,
and Tonda di Strongoli showed low levels of susceptibility to olive fly (lower than 15%).
Cultivars Cellina di Nard, Nolca, and Termite di Bitetto had higher levels of cyanidine than
other cultivars in the study and had low levels of infestation.
In a study undertaken in 2005 in an experimental field located on the Ionian coast of
Calabria (Southern Italy), Iannotta et al. (2007a) found the lowest susceptibility to olive fly
attack for cvs Tonda nera dolce and Bardhi i Tirana (6.67% and 13.50%, respectively). On the
contrary, cvs. Carolea, Cassanese, Carboncella di Pianacce, Gentile di Chieti, Giarraffa,
Nocellara del Belice, Nociara and Picholine were susceptible with a mean percentage of
active infestation ranging from 22.17 to 29.83%. The presence of cyanidine in the first

ripening period only for cv. Tonda nera dolce suggests a possible role in determining the
lowest level of active infestation observed.
Studies on cultivars Bardhi i Tirana, Carolea and Tonda nera dolce (Iannotta et al., 2007a)
corroborate results obtained in previous investigations. Cultivars Bardhi i Tirana and
Carolea were selected because they have low and high levels of susceptibility, respectively,
to olive fly whilst cultivar Tonda nera dolce was selected because it shows high levels of
cyanidine. Differences were found among the three cultivars in relation to active and total
infestations and oleuropein and cyanidine contents. The lowest percentages of active and
total infestations were observed on cv. Tonda nera dolce (8.62% and 20.12, respectively),
while cv. Carolea had the most damage (29.00% and 49.38%, respectively). Cultivar Bardhi i
Tirana showed intermediate values of active and total infestations. The low susceptibility
found for cv Bardhi i Tirana, although greater than the susceptibility showed by cv Tonda
nera dolce, might be due to the highest oleuropein content observed. The content of
cyanidine in cv. Tonda nera dolce might be responsible for the lowest susceptibility found.
Cyanidine probably acts by giving olive fruits a dark colour during the early ripening
stages. This may confuse female olive flies in drupe recognition resulting in a decrease in
oviposition. This hypothesis is supported by the evidence that in herbivorous diurnal
insects, visual cues may play an important role in the location of host plants and essential
resources, such as food, mating, and oviposition sites (Prokopy and Owens 1983). This
situation is very common for frugivorous Tephritid flies that feed and oviposit on fruits
(Katsoyannos, 1989; Fletcher and Prokopy, 1991; Diaz-Fleischer et al., 2000; Prokopy and
Papaj, 2000). A laboratory experiment investigating the effect of fruit colour on attracting
olive fruit fly females was carried out by Katsoyannos et al. (1985). In this study, females
were left to select for oviposition sites among hollow, hemispheric, ceresin wax domes of
different colours. Yellow and orange domes were preferred for oviposition compared to
domes of other colours. Red, blue, black, and white domes were the least preferred. Red,
blue, and black correspond to the colour of ripening olives reached during the maturation
stages. Fruit colour is genetically determined in some cultivars and is related to the content
of anthocyanins. Olive fly females prefer green olives for oviposition compared to red and
black olives (Cirio, 1971; Rizzo and Caleca, 2006).
The high content of phenolic compounds found in several cultivars is a resource in olive
germplasm. Field researches demonstrated that a high content of phenolic compounds is
related to low susceptibility to olive fly attacks and other parasites. Therefore, the planting of
genotypes containing high amounts of these compounds may greatly contribute to a
significant reduction of pesticides inputs. In addition, the presence of phenolic compounds in
olive fruits is also associated with various benefits for human health deriving from high
quality olive oil intake. Therefore, the conservation of olive intraspecific biodiversity
preserves sources of genetic resistance to various pests. To preserve olive germplasm
biodiversity in accordance with CAP directives and minimize pesticide use diversifying
agronomic practices are strongly related. Strategic B. oleae control is thus a priority for
safeguarding both environmental integrity and consumer health. The identification of genetic

resistance sources may represent an effective means for olive crop management. In fact, using
olive cultivars with low susceptibility to olive fly may represent an effective strategy for
organic and integrated pest management, eliminating or decreasing pesticides inputs.
Author details
Acknowledgement
We thanks Veronica Vizzarri, Tiziana Belfiore, Maria Elena Noce, Luigi Perri and all the
other colleagues involved in field and laboratory works and that allow us to write this
contribute. Financial support was provided by the Italian Ministry of Agriculture, Food and
Forestry Policy through the project GERMOLI Salvaguardia e valorizzazione del
GERMoplasma OLIvicolo delle collezioni del CRA-OLI
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Iannotta, N.; Belfiore, T.; Monardo, D.; Noce, M.E.; Scalercio, S. & Vizzarri, V. (2007b). Indagine
nel germoplasma dellolivo sul comportamento di numerosi genotipi in relazione alla loro
suscettibilit agli attacchi parassitari. Acta Biologica, Vol.83, pp. 215-220.
Katsoyannos, B.I. (1989). Field responses of Mediterranean fruit flies to spheres of different
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Katsoyannos, B.I.; Patsouras, G. & Vrekoussi, M. (1985). Effect of colour hue and brightness
of artificial oviposition substrates on the selection of oviposition sites of Dacus oleae.
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13C NMR Characterization of new Oleuropein Aglycones. J. Chem. Soc., Perkin Trans. 1,
Vol.1, pp. 1519-1523.
Lo Scalzo, R.; Scarpati, M.L.; Verzengnassi, B. & Vita, G. (1994). Olea europaea chemical
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Panizzi, L.; Scarpati, M.L. & Oriente, E.G. (1960). Structure of oleuropein, bitter glycoside
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Chapter 6
Phytosanitary Certification
Giuliana Albanese, Maria Saponari and Francesco Faggioli
1. Introduction
Olive plants are among the most ancient cultivated fruit trees. Over the centuries,
propagation occurred mainly vegetatively. The longevity of trees and the latency of most of
the virus infections allowed the dissemination through the propagative material of hidden
viruses, which were not detected until recently, when the advent of novel diagnostic tools
surprisingly revealed that virus infections are in fact widespread.
In the past, the selection of high value olive germplasm has been mainly based on the
agronomic and pomological traits of the plants and on the quality and yield of the olive-
derived product (oil). Specifically, investigation on the sanitary status of the selected ecotypes
were done mainly by visually inspections. This fact leaded to the propagation and spread of
systemic pathogens harbored either in a latent form or in the form of specific symptoms that
initially have been confused with the phenotypic expression of the plant (as in the case of
symptoms caused by infection of Strawberry latent ringspot virus (SLRSV) in the cultivar
'Raggiola')
In order to prevent the spread of dangerous pathogens (Verticillium dahliae, Pseudomonas
savastanoi pv. savastanoi) and viruses in particular, remedies rely mainly on preventive
measures such as the use of pathogen-tested propagative material. The main approach used
to obtain, propagate and commercialize plants free from harmful pathogens is through
phytosanitary selection and certification programs, which also encompass pomological
selection for trueness to type and superior quality traits. In order to obtain pathogen-free
material from infected trees, sanitation treatments such as heat therapy, meristem tip culture
and micrografting, although still limited for their application for virus elimination in olive
plants, can be applied.
A certification program is a procedure whereby single well-analysed candidate mother
plants (nuclear stock plants) are used as sources of propagation material with a process of
filiation. In this way, it is possible to provide growers with high quality (genetic and


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sanitary) material. The certification scheme in general, and phytosanitary in particular, can
be adopted either for worldwide spread varieties or for those locally distributed.
Each step of the propagation (descendent filiation) (Pre-basic, Basic and Certified material)
must comply with the requirements that are intended to produce and maintain the selected
material in the best growing conditions as specified by the enforced phytosanitary
regulations. In particular, the sanitary status must be assessed following the officially
recognized technical procedures, regarding the list of the target pathogens, type of sample,
period of sampling and protocol for testing.
Phytosanitary selection requires the use of appropriate diagnostics protocols for pathogen
detection. The difficulty in recognising and/or diagnosing virus-infected olive trees during
field surveys imposes the use of laboratory tests in order to assess the absence of the target
pathogens. Due to the lack of indicators for the biological assays and the unreliability of the
ELISA test in olive, the application of molecular diagnostic techniques for viruses, fungi and
bacteria detection became, in the recent past, critical for the assessment of the sanitary
status of a given selected ecotype. These sensitive and reliable methods are absolutely
necessary as they are at the basis of efficient and valid certification programs.
The increasing international demand for olive products, and therefore the expansion of olive
crops, is stimulating the exchange of olive germplasm in new areas of the world, prompting
for the adoption at European and International level of harmonised Certification Programs
that reduce risks of pathogen dissemination and ensure the commercialisation of high
quality propagative material and, consequently, guarantee high quality olive productions.
2. Systemic pathogens transmissible with the propagative material
To date, 8 virus-like diseases have been described, and fifteen different viruses (Tab. 1) and
five phytoplasmas have been identified in olive plants. The actual Italian olive certification
law (DM 20/11/2006) imposes the absence, in the propagation material, of some of the
abovementioned pathogens as well as the most dangerous fungus and the most widespread
bacterium in olive crops (Tab. 2). An appraisal of other ways of transmission than vegetative
propagation if known, the susceptible hosts, effects and diseases caused by these pathogens
and Countries where they have been detected in olive is reported below.
Arabis mosaic virus (ArMV) is a member of the genus Nepovirus, family Secoviridae (Sanfaon
et al., 2011). It is transmitted by the longidorid nematode Xiphinema diversicaudatum, but
there is no evidence of its transmission to olive plants by this vector. The main hosts of this
virus are strawberry, hop, Vitis spp., raspberry (Rubus idaeus), Rheum spp., Sambucus nigra,
sugarbeet, celery, gladiolus, horseradish and lettuce. The most common symptoms induced
by ArMV are leaf mottling and flecking, stunting and several forms of deformation
including enations. Because of the serious damages caused on some crops this virus is
inserted among the harmful organisms known to occur in the community and relevant for
the entire community in Directive 2000/29/EC and its absence must be determined on plant
material of Fragaria and Rubus. The symptoms vary depending on the host plant but also on


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the virus isolate, cultivar, season and year. Many plant species infected with ArMV,
including olive trees, do not show any symptoms (Martelli et al., 2002). The virus has been
reported in olive trees from Italy (Savino et al., 1979), Portugal (Martelli, 2011) Egypt, USA
(Saponari & Savino, 2003), Turkey (alayan et al., 2004), Syria (Alabdullah et al., 2005) and
Lebanon (Fadel et al., 2005).
Cherry leaf roll virus (CLRV) belongs to the family Secoviridae, genus Nepovirus, subgroup c.
Even if it is classified as a Nepovirus, its transmission by nematodes has not yet been
demonstrated to date, whereas it effectively occurs by pollen and, in some hosts, very
efficiently by seed too. In olive plants, its transmission by means of pollen has not been
demonstrated, but has been ascertained by seeds at the rate of 41% (Saponari et al., 2002).
CLRV infects many herbaceous, shrubs and woody plants of genera: Betula, Celtis, Cornus,
Fagus, Juglans, Ligustrum, Olea, Populus, Ulmus, Rubus, Sambucus and Rheum. The virus often
induces symptoms in ash, birch, cherry, elderberry and walnut including delayed leaf
development, chlorotic leaf streaks or spots, as well as dieback of branches or whole trees
but it is symptomless in olive trees (Savino & Gallitelli, 1981). Its presence in olive trees was
reported in Italy, Portugal, Spain (Martelli, 1999), then in Egypt, USA (Saponari & Savino,
2003), Turkey (alayan et al., 2004), Syria (Alabdullah et al., 2005), Lebanon (Fadel et al.,
2005) and recently in Croatia (Luigi et al., 2011), where it has been shown to have a negative
impact on olive fruit and virgin oil quality (Godena et al., 2012).
Strawberry latent ringspot virus (SLRSV) is an unassigned species in the Secoviridae family. It is
transmitted by the nematode X. diversicaudatum and by seed in several species (Cooper,
1986), but in olive plants, these kinds of means of transmission have not been demonstrated.
SLRSV infects strawberry and raspberry, mostly without symptoms but resulting in various
degrees of mottle and decline in some cultivars. The virus was isolated for the first time
from olive in cv. Corregiolo in Italy (Savino et al., 1979) and later in Portugal (Henriques et
al., 1992), Spain (Bertolini et al., 1998), Egypt, USA (Saponari & Savino, 2003), Turkey
(alayan et al., 2004), Lebanon (Fadel et al., 2005), Syria (Alabdullah et al., 2005), Croatia
(Bjelis et al., 2007), Tunisia (Martelli, 2011) and Albania (Luigi et al., 2009). Small, pear-
shaped, puckered fruits with deformed kernels (bumpy fruits), narrow and twisted leaves,
bushy growth and reduced crop were described in olive trees of cv. Ascolana tenera
affected by SLRSV (Marte et al., 1986). Similar symptoms were observed in cvs Negrinha
and Galega in Portugal, associated with a severe reduced rooting ability of the cuttings
(Henriques et al., 1992). Among 15 different olive cultivars reporting plants being affected
by SLRSV in Portugal, only some showed symptoms (Henriques et al., 1992) in agreement
with what was observed in Italy (Savino et al., 1979; Marte et al., 1986); no symptoms are
apparently associated with SLRSV infections in Spain (Bertolini et al., 1998). Very interesting
is the fact that previously, the Raggiola and Frantoio were considered different olive
varieties due to morphological and agronomical dissimilarities. A relatively recent study
showed that the two cultivars are genetically identical and that their differentiations are
due to the constant presence of SLRSV in Raggiola and the repeated SLRSV absence in
Frantoio (Fig. 1) (Ferreti et al., 2002). Rooting trials conducted to compare SLRSV-infected
Raggiola with virus-free Frantoio showed that the virus does not influence the rooting


110
rate of olive cuttings (Roschetti et al., 2009), contrary to what had been previously reported
in Portugal.

Figure 1. Phenotypic expression of olive cultivars Frantoio SLRSV-affected, healthy Frantoio and
Raggiola SLRSV-affected. See as morphological aspect of Frantoio SLRSV-affected and Raggiola
SLRSV-affected are identical (narrow leaves and small inflorescences)
Cucumber mosaic virus (CMV) belongs to the genus Cucumovirus, family Bromoviridae. It is one
of the most dangerous virus affecting vegetable plants (about 800 wild and cultivated plant
species are its hosts). CMV induces important vegetative and productive reductions (up to
100% in plants such as tomato and pepper). When this virus affects herbaceous plants, it is
transmitted very efficiently by 75 different aphid species and with varying efficiency by
seed. CMV infection in olive is symptomless and its transmission by aphid vectors to/from
olive has not yet been proven. It was isolated the first time from olive trees by Savino and
Gallitelli (1983) in Italy. This report was confirmed in Portugal by Rei et al. (1993) who
detected CMV alone, as well as together with SLRSV. CMV in olive trees was also found in
Spain (Bertolini et al., 1998), Turkey (alayan et al., 2004), Syria (Alabdullah et al., 2005),
Croatia (Bjelis et al., 2007), Tunisia (Martelli, 2011) and recently in California (Al Rwahnih et
al., 2011).
Olive latent virus 1 (OLV-1) is a member of the genus Necrovirus, family Tombusviridae. The
virus is one of the few viruses detected in olive trees that is transmitted by seed (at a rate of
82%) (Saponari et al., 2002). It was detected in olive trees in Italy (Gallitelli & Savino, 1985),


111
Jordan (Martelli et al., 1995), Portugal (Felix & Clara, 2002), Egypt, USA (Saponari & Savino,
2003), Lebanon (Fadel et al., 2005), Syria (Alabdullah et al., 2005), Tunisia (Martelli, 2011)
and Turkey (Serce et al., 2007). Several OLV-1 isolates have been obtained from
symptomless or weakened trees. Since this virus has also been isolated from citrus in Turkey
and Italy (Martelli et al., 1996) and from tulips in Japan (Kanematsu et al., 2001) it is
reasonable to assume that it, as well as other olive viruses, may also have a larger host
range.
Olive latent virus 2 (OLV-2) is the type species of the monotypic genus Oleavirus, family
Bromoviridae (Grieco & Martelli, 1997). OLV-2 was isolated by mechanical inoculation from
symptomless olive trees in Apulia, Southern Italy (Savino et al., 1984). It has subsequently
been identified in Lebanon (Fadel et al., 2005), Syria (Alabdullah et al., 2005), Croatia (Bjelis
et al., 2007) and Tunisia (Martelli, 2011) from symptomless olive cultivars. The host range of
OLV-2 was limited to olive trees until castor beans (Ricinus communis L.), showing yellowish
vein netting and systemic mottling on leaves, were reported in Greece to be infected with
this virus (Grieco et al., 2002).
Olive latent virus 3 (OLV-3) is classified as a tentative member of the genus Marafivirus,
family Tymoviridae. The virus is not mechanically transmitted. Search of possible vectors,
Euphyllura olivina and Saissetia oleae, was not successful even if OLV- 3 was detected by RT-
PCR in the psyllid. A survey conducted in the Mediterranean region showed the OLV-3
presence in Italy, Syria, Malta, Tunisia, Portugal, Turkey, Lebanon and Greece with an
infection rate average of 30% always in symptomless olive trees (Alabdullah et al., 2010).
Olive latent ringspot virus (OLRSV) is an approved species of the genus Nepovirus, family
Secoviridae. The virus is transmitted by mechanical inoculation and the existence of a natural
vector is unknown. OLRSV is latent in olive trees, but it causes some symptoms on
diagnostically susceptible hosts, such as apical necrosis on Chenopodium quinoa and C.
amaranticolor, and red-rimmed local lesions and malformation on tip leaves of Gomphrena
globosa. The virus was isolated from asymptomatic olive trees in Lazio, Central Italy (Savino
et al., 1983), in Portugal in 1990, in Syria (Alabdullah et al., 2005) and then in Tunisia
(Martelli, 2011).
Olive leaf yellowing associated virus (OLYaV) is an unassigned species in the family
Closteroviridae. Various studies have been published (Sabanadzovic et al., 1999; Essakhi et al.,
2006; Luigi et al., 2010) and are still in progress to define its taxonomic position. OLYaV
presence in psyllid E. olivina and unidentified mealybugs of genus Pseudococcus gave the
indication that transmission by these vectors could be possible (Sabanazdovic et al., 1999). The
olive leaf yellowing (OLY) disease was recorded for the first time in Italy on cv. Biancolilla
(Savino et al., 1996) and it is characterized by a bright leaf yellow discoloration (Fig. 2). A
survey conducted in Italy showed that, in old OLYaV-affected olive trees, leaf yellowing
symptom is frequently absent (Albanese et al., 2003). The OLY syndrome, consisting of poor
fruit set, bright yellow discoloration of the foliage, mottling, necrosis, extensive defoliation and
dieback, has been associated to other viruses such as Olive vein yellowing associated virus
(OVYaV) (Faggioli & Barba, 1995) and Olive yellow mottle and decline associated virus (OYMDaV)


112
(Savino et al., 1996), but their presence on olive trees was very rare. On the other hand, OLYaV
seems to be one of the most widespread olive viruses: in Italy it infects more than 60% of
southern Italy olive cultivars (Faggioli et al., 2005) and it has also been reported in high
percentages in Israel (Martelli, 2011), Egypt, USA (Saponari & Savino, 2003), Lebanon (Fadel et
al., 2005), Spain (Martelli, 2011), Syria (Alabdullah et al., 2005), Albania (Luigi et al., 2009),
Croatia (Bjelis et al., 2007), Tunisia (Martelli, 2011) and California (Al Rwahnih et al., 2011). A
study on the rooting and grafting capacity of OLYaV-infected Carolea and its respective
healthy controls showed that the virus does not influence the rate of rooting of the cuttings
and does not interfere with the grafting success rate; positive significant effects in grafting
ability were observed on infected material only during a temperature stress, probably due to
the reduced water need of infected shoots (Roschetti et al., 2009). Significant difference in
vegetative growth was observed between virus-free and OLYaV-infected young olive plants,
demonstrating negative OLYaV interference (Cutuli et al., 2011). To date, no other hosts have
been found for this virus.

Figure 2. Yellowing symptoms in an olive cultivar Carolea (a) which tested positive for OLYaV and
detail of yellow leaves of the same tree (b)
Olive semilatent virus (OSLV) is still unclassified. It was transmitted mechanically from olive
tree to Nicotiana benthamiana (Materazzi et al., 1996). The main symptom observed in Italy on
OSLV-affected olive trees was a very mild chlorotic vein clearing of the leaves, but there is not
enough evidence of the etiological involvement of this virus in the disease (Martelli, 1999).


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Tobacco mosaic virus (TMV) belongs to the genus Tobamovirus, family Virgaviridae. Mechanical
transmission to herbaceous indicator plants was possible, but not easily. It was isolated in
central Italy from olive trees showing vein banding, discolorations along the main veins,
severe defoliation and decline (Triolo et al., 1996). However, there is no conclusive evidence
that TMV is agent of these symptoms.
Tobacco necrosis virus (TNV). Viruses with properties similar to those of TNV were first
detected in symptomless olive trees by Flix & Clara (2002) in Portugal. One isolate was
studied further, revealing its identity as TNV-D species (Cardoso et al., 2004). However,
further genomic characterization of this isolate led to its classification as a new species in the
Necrovirus genus named Olive mild mosaic virus (OMMV) (Cardoso et al., 2005). To date, it is
not clear if TNV-D can be considered among the viruses isolated from olive trees, even if
recent data shows the presence of this species in olive trees (Cardoso and colleagues
deposited the complete genome sequence of a TNV-D isolate from olive trees in the Gene
Bank, accession number FJ666328). Virions are readily transmitted by mechanical
inoculation and naturally by the fungus Olpidium brassicacae. TNV has a wide host range
that includes monocotyledonous and dicotyledonous plants, which frequently cause
necrotic lesions on the roots and leaves.

Acronym Virus species Genus Geographical distribution
OLV 1 Olive latent virus 1 Necrovirus
Italy, Jordan, Portugal, Egypt, USA ,
Lebanon, Syria, Turkey, Tunisia
OLV 2 Olive latent virus 2 Oleavirus Italy, Syria, Croatia, Lebanon, Tunisia
OLV 3 Olive latent virus 3 Marafivirus
Greece, Italy, Lebanon Malta, Portugal,
Syria, Tunisia, Turkey
OLRSV Olive latent ringspot virus Nepovirus Italy, Portugal, Syria, Tunisia
OVYaV Olive vein yelloving associated virus Potexvirus Italy
OYMDaV Olive yellov mottling and decline associated virus Unclassified Italy
OLYaV Olive leaf yellowing associated virus
Closteroviridae,
unassigned
species
Albania, Croatia, Egypt, Italy, Israel,
Lebanon, Spain, Syria, Tunisia, USA
OSLV Olive semilatent virus Unclassified Italy
OMMV Olive mild mosaic virus Necrovirus Portugal
SLRSV Strawberry latent ringspot virus
Secoviridae,
unassigned
species
Croatia, Egypt, Italy, Lebanon,
Portugal, Spain, Syria, Tunisia, Turkey,
USA
CLRV Cherry leafroll virus Nepovirus
Croatia, Egypt, Italy, Lebanon,
Portugal, Spain, Syria, Tunisia, Turkey,
USA
ArMV Arabis mosaic virus Nepovirus
Egypt, Italy, Lebanon, Portugal, Syria,
Turkey, USA
CMV Cucumber mosaic virus Cucumovirus
Croatia, Italy, Portugal, Spain, Syria,
Tunisia, Turkey, USA
TMV Tobacco mosaic virus Tobamovirus Italy
TNV Tobacco necrosis virus Necrovirus Portugal
Table 1. Viruses identified in olive trees and their geographical distribution (Martelli, 2011; alayan et
al., 2009)


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Phytoplasmas constitute a monophyletic clade within the Mollicutes class. Their classification
has been possible through the use of restriction fragment length polymorphism (RFLP)
analysis and sequencing of the conserved 16S rRNA gene (Lee et al., 1998; Semller et al.,
1998). A variable range of symptoms in olive trees such as shoot proliferation, shortening of
internodes, witches-brooms, little leaves (Fig. 3a), leaf rolling and yellowing, leaf bronzing,
phyllody, flower abortion, hypertrophied inflorescences (Fig. 3b), fasciation, erect growth,
dwarfing, decline and die-back have been frequently associated with the presence of
phytoplasma in Spain, Italy and Iran (Ahangara et al., 2006; Bertaccini et al., 2002; Font et al.,
1998; Pasquini et al., 2000). Identification of phytoplasmas detected in olive plants showed
they were members of the 16S-IB (Aster yellow), 16S-IC (Clover phyllody), 16Sr-III (Peach X
disease), 16S-VA (Elm yellow) or 16S-XIIA (Stolbur) groups and subgroups. The failure to
detect phytoplasmas in many symptomatic olive trees leads to doubts on whether these type
of alterations could be associated with other causes (Barba, 1993; Camele et al., 1999).
Nevertheless, phytoplasmas detected in olive plants are agents causing very well known
and severe diseases in other hosts. These include aster yellow, clover phyllody, peach X
disease, elm yellow and stolbur in solanaceous plants, as well as grapevine yellow (= Bois
Noir). Even if their transmission by leaf-hopper vectors has been proven for some of them
(among various host plants but not yet in and from olive plants) their presence in olive
plants indicates a serious potential threat for other important crops.

Figure 3. Shortened internodes, witches-brooms and little leaves (a); hypertrophied inflorescences (b)
on olive trees affected by phytoplasmas
The fungus Verticillium dahliae is a soil-borne pathogen that attacks olive trees (as well as
over a hundred woody and herbaceous species), particularly when their roots are stressed. It
causes the most severe disease suffered by olive plants, named Verticillium wilt that induces
yellow leaves, defoliation (Fig. 4) and death due to the fungus attacking the plants vascular
system. Internally, a dark reddish brown streak on the wood occurs in most plants. This is


115
visible on branches when the bark peeled off. If the cross-section of infected branches or
trunks is examined, the brown woody coloration may appear as a ring. Although some
plants may die quickly, more commonly trees with only a few wilted branches during a
growing season become more severely infected the following year. After the first report of
Verticillium wilt in Italy, it has later been detected in Algeria, Arizona, California, Egypt,
France, Greece, Iran, Malta, Marocco, Syria, Spain and Turkey (Bubici & Cirulli, 2011).
Another species, V. albo-atrum, may occasionally cause the same disease in olive plants.

Figure 4. Yellow leaves, defoliation and wilt of olive caused by V. dahliae (photo by Antonio Ippolito)
The bacterium Pseudomonas savastanoi pv. savastanoi causes the most frequent disorder
occurring in olive plants known as olive knot disease. The disease manifests itself through
the growth of tubercles (Fig. 5) , which either appear individually, or in clusters on any part
of the plant, but most commonly on twigs, young branches and around wounds on the main
trunk. Knots can damage the stem structure and can deform the scaffold of the tree if
infection is severe during the early stages of the tree. This may become a serious problem in
nurseries that grow olive plantlets for marketing. P. savastanoi causes a similar disease in
other plants as oleander, ash, jasmine, Japanese privet, Forsythia spp., Phyllirea spp., Retama
sphaerocarpa, Rhamnus alathernus and myrtle (Surico & Marchi, 2011). This bacterial disease is
present in all areas of the world where olive plants are cultivated. This is due to the ability
of its causal agent to colonize the phylloplane of the tree.


116

Figure 5. Olive knots: rough galls and swellings on twigs and branches caused by P. savastanoi pv.
savastanoi

Harmful organisms
Sanitary status
Acronym
Virus-free
(VF)
Virus-tested
(VT)
VIRUSES:
Arabis mosaic ArMV X X
Cherry leafroll CLRV X X
Strawberry latent ringspot SLRSV X X
Cucumber mosaic CMV X
Olive latent 1 OLV-1 X X
Olive latent 2 OLV-2 X
Olive leaf yellowing associated OLYaV X X
Tobacco necrosis TNV X
PHYTOPLASMAS X X
FUNGI:
Verticillium dahliae X X
BACTERIA:
Pseudomonas savastanoi pv. savastanoi X X
NEMATODES:
Meloidogyne incognita X X
Meloidogyne javanica X X
Pratylenchus vulnus X X
Xiphinema diversicaudatum X X
X = the absence of this organism must be ascertained
Table 2. Pathogens and pests that must be absent in order to obtain the virusfree or virustested
sanitary status according to Italian olive certification law (DM 20/11/2006)


117
3. Strategies to control invasive olive pathogens and the importance of
phytosanitary certification
Pathogens associated with olive propagative material may be systemic (viruses and
phytoplasmas and probably P. savastanoi pv. savastanoi) or associated with the vascular
system (V. dahliae) and they are unlikely to be eliminated during the vegetative propagation
of an infected source. Accordingly, local and long-distance spread of these pathogens
through the movement of infected propagative material has caused a highly threatening
worldwide distribution of infectious diseases. The symptomless nature of several olive virus
infections may also contribute to the inadvertent propagation and distribution of infected
material.
To avoid disease and/or pathogen dissemination through vegetative propagation, possible
remedies include mainly preventive strategies based on the use and propagation of
healthy mother plants. In fact, in order to attain sanitary improvements of any crop, a
system of preventive, protective and often of sanitation measures has to be established and
implemented, encompassing a complex series of interventions currently referred to as
phytosanitary selection and certification.
In the framework of a phytosanitary and clonal improvement program, the main activities
include: (i) field surveys for the selection of olive trees with no apparent disease
symptoms and fulfilling the pomological traits of the cultivar; (ii) samples collection for
laboratory tests, both for pathogen detection and DNA marker analysis; (iii) molecular
tests (RT-PCR, dot blot hybridization and dsRNA analysis) for the detection of viruses
included in the certification program; (iv) genetic characterisation using SSR markers; (v)
sanitation by heat therapy, meristem tip culture and micrografting in case of no healthy
trees being detected for one or more cultivars; (vi) propagation of the candidate nuclear
stocks under conditions that ensure freedom from re-infections, usually in insect-proof
greenhouses.
Field surveys should be carried out in the main olive-growing areas for the specific cultivar
undergoing the clonal and sanitary selection program. Usually mature trees are selected (i.e.
25-year-old) based on visual inspection during spring and autumn. Samples for virus testing
consist of 10-15 cuttings collected from 1- to 2-year-old twigs or young leaves for DNA
extraction and SSR marker analysis.
Despite limited information being available on the application and effectiveness of
sanitation protocols on olive plants, in vivo and in vitro heat therapy, in vitro shoot tip culture
and micrografting have all been applied in attempts to regenerate OLYaV- and CLRV-free
material and some successful results have been obtained (Bottalico et al, 2002). For in vivo
heat therapy, plants can be grown at 38C for 3 to 12 months. During heat therapy, 2 to 2.5
cm long shoot tips are excised no earlier than three months from the beginning of the
treatment. After surface-sterilization in 0,05% mercuric hydrochloride for 10 min, the shoot
tips are placed in vitro in petri dishes on different media according to the cultivar [OM
(Rugini, 1984); MSM media (Leva et al., 1994)] and grown at 24C with a 16 h photoperiod.


118
Regenerated explants are subcultured 3 or 4 times every four weeks using the proliferation
medium reported by Rugini (1984), prior to transplanting in jiffy pots. For in vitro heat
therapy, 2 to 3 cm long in vitro plantlets are exposed for 1 to 3 weeks to 38C. After heat
treatment and subcultures, the surviving plantlets are transplanted in jiffy pots.
For shoot tip culture, apexes are excised from well-established in vitro cultures maintained
on OM or MSM media. Regenerated apexes are subcultered on the same media prior to
being transplanted in jiffy pots.
Olive seedlings for micrografting are recovered from seeds soaked for a few seconds in 70%
alcohol before the endocarp removal. Kernels are then soaked for 10 min in 0.05% mercuric
hydrochloride solution, rinsed three times in sterile water, and placed on wet sterilized
paper in petri dishes at 25 in the dark. After 2-3 months, the regenerated seedlings are cut,
leaving about 1.0 cm of the epicotyl. Shoot apexes, excised from in vitro grown plantlets, are
then grafted on the top of decapitated seedlings. After grafting, plants are cultured in
rooting medium (Rugini, 1984). Generally less than 70% of the grafts are successful, and
only 10% of the plants survive after transplantation in the soil.
Although phytoplasma diseases may be cured by treatment with certain classes of
antibiotics and by heat water therapy, such approaches have not been applied to olive plants
in order to obtain sanitation from these pathogens. This is because olive trees affected by
phytoplasmas are very rare, and during a phytosanitary selection, phytoplasma-free plants
can be easily found. Since their transmission by leaf-hopper vectors is ascertained for other
crops, growing nuclear stock plants in insect-proof greenhouses ensures also freedom of
infection from phytoplasmas.
The detection of V. dahliae-free plants must be carried out with great care. A visual diagnosis
is in fact insufficient in guaranteeing the absence of this fungus and have recommended the
use of new and sensitive diagnostic tools that are now available (see paragraph 4).
In order to prevent infections by P. savastanoi pv. savastanoi selected materials must be free
from symptoms of the disease, and before propagation material is harvested, mother plants
must be sprayed with a copper-based treatment to reduce risk of infections by the epiphytic
bacterial population.
The candidate nuclear-stock material obtained through the field selection and/or sanitation
treatments describe above can enter the certification program upon official approval (see
paragraph 5), and genetically and sanitary certified propagative material will be available to
growers.
Demand for olive products is constantly increasing in local and foreign markets, stimulating
the expansion of olive crops and encouraging the exchange of olive germplasm at an
international level. The activation of a selection and certification program is thus crucial to
guarantee the quality of the propagative material and reduce risks for pathogen
dissemination.


119
4. Identification of olive pathogens: updates on diagnostic tools
Sanitary certification programs require reliable and sensitive diagnostic tests in order to
allow for the identification of pathogen-free trees and the assessment of their overall plant
production processes. Due to the latency of several infections caused for example by viruses,
visual inspections are not reliable and laboratory tests must be performed to certify virus-
free or virus-tested materials. Biological tests and serological assays, widely used to detect
pathogens affecting other crops like stone fruits, grapes, pome fruits, result ineffective in
olive plants due to the absence of differential woody indicators for the bioassays and the
low viral titre and/or to the interference by some contaminants. All these factors have made
olive tree virus diagnosis very problematic. Luckily, in the last decade several molecular
approaches have been developed and improved to detect olive viruses, bacteria and fungi in
the propagating materials. Different molecular techniques such as RT-PCR in single/double
step or nested, PCR, real time PCR, dot blot hybridization and dsRNA analysis, have been
implemented in recent years and drastically improved sensitivity and specificity of olive-
infecting pathogens diagnosis. Recently, molecular technology has been successfully
applied for routine and large scale detection and could easily be transferred to those
Countries that intend to develop their olive crops through production, maintenance and
distribution of healthy (virus-free or virus-tested) planting material.
Concerning viruses, RT-PCR assay has proved to be the most rapid, sensitive and reliable
technique for detecting an RNA target in infected plants, and in recent years, different
protocols have been developed for olive viruses detection (Grieco et al., 2000; Bertolini et al.,
2001a, 2003; Pantaleo et al., 2001; Faggioli et al., 2002, 2005). Recently, a one step RT-PCR
protocol has been set up and validated in an inter-laboratory ring test (Loconsole et al., 2010)
for the diagnosis of the eight most important olive viruses. This should be a starting point
for anyone wishing to approach the sanitary selection of olive plants. New and improved
diagnostic techniques (e.g. Real Time RT-PCR, multiplex RT-PCR, polyprobe for molecular
hybridization) will be continuously developed as the knowledge on the genetics and biology
of olive-infecting pathogens advances.
Phytoplasma detection is now accomplished through nested-PCR on total DNA extracted
from olive plants using the protocol of Barba et al. (1998). Gene amplification is performed
using a direct PCR with primers P1/P7 (Deng & Hiruki, 1991; Schneider et al., 1995), followed
by a nested-PCR with primers R16F2/R2 (Lee et al., 1993). The use of this analysis allows to
determine whether plants are affected by phytoplasma, but does not give information about
the identity of the pathogen. Identification of phytoplasma can be achieved through restriction
fragment length polymorphism (RFLP) analysis, but it is not required for sanitary certification
since the certified olive material must be free from all phytoplasmas. In recent years, the
diagnostic technique has also been developed and improved for phytoplasmas. Real time PCR
protocols for the identification and group characterization of phytoplasmas are now available.
Whilst not yet applied to the diagnosis of olive phytoplasmas, these techniques have all the
necessary features for this purpose, and there is therefore potential for their use in the near
future (Christensen et al., 2004; Hodgetts et al., 2009).


120
Diagnosis of V. dahliae is preliminarily performed through an accurate search for foliar
symptoms and vascular browning. Foliar chlorosis and necrosis could be due to other
causes such as root rot diseases, whereas browning on cross section of stems was sometimes
not found. Conclusive detection is attempted by isolating the fungus on agar media from
olive tissues and possibly using PCR or nested PCR with V. dahliae specific primers (Nigro
et al., 2002; Mercado-Blanco et al., 2002) or by Real Time Scorpion PCR (Schena et al., 2004).
Identification of P. savastanoi pv. savastanoi is very easy when the typical knots are present
on plants. Nevertheless, its presence in latent and systemic form has been reported by
Penyalver et al., (2006). The presence of P. savastanoi pv. savastanoi both as epiphytic and
entophytic agent makes its control in the certified material absolutely compulsory;
moreover, for a more sensitive and reliable diagnosis, molecular techniques are needed.
Recently, molecular protocols of nested-PCR have been set up to obtain reliable diagnoses of
latent infections (Bertolini et al., 2001b). This is also possible (and has been done)
simultaneously with four other olive viruses (Bertolini et al., 2003).
5. Phytosanitary certification program: The Italian experience in the last
twenty years
The production of healthy, high-quality olive products depend to a large extent on the
quality of the plant material used for olive cultivation. In general, the production of
healthy plants for planting occurs through defined certification procedures by which a
particular cultivated selection, whose health status and trueness to type have been officially
attested, is propagated following specific requirements. In a typical certification scheme, the
certified material is descended by a defined number of propagation steps from individual
plants, found to be free from pathogens and pests, maintained and propagated under
rigorous conditions that exclude recontamination.
To this end, appropriate regulations are necessary to ensure the production, marketing and
movement of certified plant propagation material with high standards and free from
harmful pathogens (Annex I). Over the past twenty years, through globalisation and the
expansion of several crops in new areas, concerns were raised about new disease
emergencies transmitted by infected propagating material. In an attempt to limit the
potential impact of the spread of pests and pathogens through the movement of infected
plants, specific laws have been issued at regional, national and European levels.
The Council Directive 2008/90/EEC of 29 September 2008 (recast version of Directive
92/34/EEC) on the marketing of fruit plant (including olive) propagating material and fruit
plants intended for fruit production established a harmonized Community regime which
ensures that growers throughout the Community receive propagating material and fruit
plants which are healthy and of good quality. This applies to fruit plant propagating
material and fruit plants of genera and species listed in Directive Annex I, which may only
be marketed if they are either CAC (Conformitas Agraria Communitatis), Pre-basic, Basic or
Certified material.


121
To be classified as such, material must comply with the criteria of quality, plant health,
testing methods and procedures, propagation systems and varietal aspects and must have
been recognised following official inspections. In addition, propagating material or fruit
plants may, in most circumstances, only be marketed by accredited suppliers, whose
production methods and facilities meet the requirements of the Directive. Propagating
material and fruit plants from Countries outside of the European Union (EU) may only be
marketed within the Community if they offer the same guarantees as materials produced in
the Community complying with Council Directive 92/34/EEC. Each EU Member State
adopts its own enforcement and implementation policies using the EU Directive as a guide.
In Italy, it is compulsory that the production and marketing of olive propagating material
fulfils the requirements established by the Italian Decree amended on 14/04/1997 in
compliance with the aforementioned EU Directive. According to this law, assessment of true
to type and certification of sanitary status of olive propagating materials are compulsory
and plants are certified as CAC. In this kind of certification plants must be free from
Euzophera pinguis, Meloidogine spp., S. oleae, P. savastanoi pv. savastanoi, V. dahliae and all
known viruses (15 to date). However, several aspects concerning for example the
procedures for inspections and controls are not well defined, leading to misinterpretations
and heterogeneous application of the law in the different Italian regions by the regional
Phytosanitary Services. Besides this compulsory system, which relies on the propagation of
olive mother plants identified by the single nurseryman and found free from the target
pathogens, a voluntary certification system has been activated since 1987 at the national
level by the Ministry of Agriculture. During the last 10 years, the legislation has been
revised, and in late 2006 the revision process was completed with the publication of 2
decrees concerning the revised organisation of the system (DM 24/7/2003, DM 4/5/2006) and
4 decrees concerning the updated official technical operations for the certification of
pommes, stone fruit, olive and strawberry (DM 20/11/06).
The DM 20/11/06 provides detailed guidance on the production of olive trees and rootstocks.
Plant material produced according to this certification scheme is derived from nuclear-stock
plants (also identified as Primary source) officially recognised and registered in the database
of certified accessions by the Ministry of Agriculture. The material deriving from the first
multiplication of the nuclear-stock material enters in the certification process as Pre-basic
material. Once the nuclear-stock has been registered, the breeders or Institutions or Research
Centres responsible for its production and selection must keep the material under
conditions that minimise recontamination risks.
The main outlines of the Italian certification scheme (Tab. 3 and Fig. 6) are the following:
a. Registration of nuclear-stock material: Breeders or researchers that intend to introduce a
new accession for a specific variety in the certification system must provide detailed
information about the trueness to type and sanitary controls performed by filling the
official forms defined in the DM 20/11/06. The evaluation and eligibility of the
registration request is carried out by a technical certification committee authorised by
the Ministry of Agriculture.


122
b. Maintenance and propagation of Pre-basic material: Pre-basic olive material derives directly
from the propagation of the nuclear-stock; the Pre-basic plants must be maintained in
insect-proof green-houses (at the Conservation for Premultiplication Repository) with at
least two replications. The plants are grown in V. dahliae and X. diversicaudatum -free soil
mixture and periodically tested for viruses using molecular tools (10% of the plants
each year, starting from the 5
th
year). Molecular tests should be also performed if, after
visual inspection, plants show symptoms of V. dahliae or phytoplasmas. Cuttings and
seeds collected from the Pre-basic material is used to produce, in the same facilities, the
Basic plants for the establishment of the Premultiplication Repository.
c. Maintenance and propagation of Basic material: Basic olive planting material is the
propagation material that is obtained from Pre-basic material, maintained in open
field (Repository for the Premultiplication) in a variable replication number (2
minimum) depending on the importance of the cultivar. Premultiplication field plots
must be tested and found free from V. dahliae and X. diversicaudatum, and have a 20
meters non-cultivated border. Basic plants must be periodically inspected and tested
as defined for the Pre-basic material. Cuttings and seeds collected from the Basic
material are used to produce the certified mother plants for the establishment of the
Multiplication mother blocks.
d. Maintenance and propagation of Certified material: Certified mother plants obtained from
the propagation of the Basic material represent the source for nursery certified olive
plant production. Mother plants are grown in open fields in variable replication
numbers depending on the market of the specific cultivar. Plants are visually inspected
at least once a year, while each plant must undergo laboratory tests for virus detection
at least once within a 30-year period.
e. Certified nursery productions: Production takes place in authorized nurseries that join the
certification program. The nursery production must comply with the requirements
established by the DM 20/11/06 in terms of: (i) soil mixture (free from V. dahliae, X.
diversicaudatum, Meloidogyne incognita, M. javanica and Pratylenchus vulnus), (ii) location
of the certifiable olive blocks; (iii) maintenance of a farm business registry. The regional
phytosanitary service, following visual inspections and examination of the
documentation, is in charge of releasing the official certification (blue label) for every
single plant or seedling.
In order to facilitate the certification and the availability of certified material for new olive
cultivars or clones, the Premultiplication and the Multiplication blocks may be created
directly using planting material deriving from the first multiplication of the nuclear-stock.
In this way, the timeframe between the approval of a new accession in the system and the
availability of certified plants in the nurseries is effectively reduced.
This certification program has been supported until now mainly by public funds that cover
the costs for the management of the Conservation for the Premultiplication and
Premultiplication repositories; starting in 2012 the program should shift to a self-sustaining
system, in which taxes recovered on each released certification label will make up for the
costs of the repository management.


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Steps Plant category Facilities
Current active Repository
for olive certified material
in Italy
Controls and
certification
released by
Selection of Nuclear-
stock
Nuclear-stock
Screen-
houses
Several Research Centers
Regional
Phytosanitary
Services
Conservation for
Premultiplication
Pre-basic
Screen-
houses
- CRA-PAV Rome
- University of Bari
- Azienda Agricola
Sperimentale Improsta
Premultiplication Basic Open field
- CRA-PAV Rome
- CRSA Basile Caramia,
Bari
- Azienda sperimentale di
Santa Paolina, Follonica
- CAV Tebano
Multiplication Certified
Open field ConsorzioVivaisticoPugliese
Nurseries --

Table 3. Organization of the Certification program for olive propagating material
As aforementioned, the Italian voluntary certification program involves several woody crop
species. In most cases, the main reason prompting for the certification of such accessions is
the presence of pathogens that can cause detrimental effects on the affected plants (i.e.
quarantine pests for stone fruit or citrus). Contrastingly, in the case of olive plants, the main
aspect that promoted the adoption of this program has been the high level of genetic and
phenotypic variability within each cultivar, which could results in heterogeneous plants and
misidentification of such cultivars.
The certification scheme adopted in Italy ensures trueness-to-type and uniformity, since the
certified plants are obtained through subsequent clonal propagation steps from a single
registered accession.


124

Figure 6. Outline of the general certification steps and facilities


125
Annex I. List of the reference European Union (EU) Directives and Italian national regulations
Basic EU Directive
Council Directive 92/34/EEC of 28 April 1992 on the marketing of fruit plant propagating
material and fruit plants intended for fruit production
Council Directive 2008/90/EC of 29 September 2008 (Recast version of Directive
92/34/EEC) on the marketing of fruit plant propagating material and fruit plants
intended for fruit production
Implementing measures of Directive 92/34/EEC :
Commission Directive 93/48/EEC of 23 June 1993 setting out the schedule indicating the
conditions to be met by fruit plant propagating material and fruit plants intended for
fruit production, pursuant to Council Directive 92/34/EEC
Commission Directive 93/64/EEC of 5 July 1993 setting out the implementing measures
concerning the supervision and monitoring of suppliers and establishments pursuant to
Council Directive 92/34/EEC on the marketing of fruit plant propagating material and
fruit plants intended for fruit production
Commission Directive 93/79/EEC of 21 September 1993 setting out additional
implementing provisions for lists of varieties of fruit plant propagating material and
fruit plants, as kept by suppliers under Council Directive 92/34/EEC
Basic Italian Regulations
DM 14 aprile 1997 Recepimento delle direttive delle Commissione n. 93/48/CEE del 23
giugno 1993, n. 93/64/CEE del 5 luglio 1993 e n. 93/79/CEE del 21 settembre 1993,
relative alle norme tecniche sulla commercializzazione dei materiali di moltiplicazione
delle piante da frutto e delle piante da frutto destinate alla produzione di frutto
DM 24 luglio 2003 Organizzazione del servizio nazionale di certificazione volontaria del
materiale di propagazione vegetale delle piante da frutto
DM 4 maggio 2006 Disposizioni generali per la produzione di materiale di
moltiplicazione delle specie arbustive ed arboree da frutto, nonch delle specie erbacee
a moltiplicazione agamica
DM 20 novembre 2006 norme tecniche per la produzione di materiali di moltiplicazione
certificati di Agrumi, Fragola, Olivo, Pomoidee, Prunoidee (supplemento ordinario alla
Gazzetta Ufficiale n. 141 del 20 giugno 2007)
Decreto Legislativo 25 giugno 2010, n.124 Attuazione della direttiva 2008/90 relativa alla
commercializzazione dei materiali di moltiplicazione delle piante da frutto destinate
alla produzione di frutti (refusione) (pubblicato nella Gazzetta Ufficiale n. 180 del 4
agosto 2010)
6. Conclusion
In this chapter an overview on the olive graft-transmissible pathogens and on the latest
phytosanitary directives embodied by the EU and by the Italian Ministry of Agriculture is
given.


126
As remarked, olive has always been considered a very resistant species to diseases caused
by different pathogens; however, several pathogens, mainly systemic, can affect the trees
and, in some case, invalidate the production. Recent advances in plant pathology and
molecular biology, significantly contributed to the discover of new olive pathogens, to
characterize their genome, biology and epidemiology.
Italy has been amongst the first Countries to adopt an effective certification system for the
production of plant propagation material with high quality standards. After 10 years from
its promulgation the Italian Regulation has been revised with the support of a technical
committee, in order to improve the program and meet the quality standards amended in
the late 90 by the EU, which are mandatory for all member States. In 2006 a revised
national Regulation was issued (DM 20/11/2006), updating the list of pathogens that need
to be checked and implementing the protocols for their identification. In the last 5 years
several valuable virus-free and true-to-type primary sources, belonging to the most
widespread or local Italian varieties, have been registered, propagated through the
certification system, and made available to the growers. Although, the EU directives and
the Italian regulations concerning the production of olive propagation material have been
critically revised and implemented, it is necessary to continuously update the list of the
pathogens and the diagnostic protocols, including the latest tools for genetic and
phytosanitary assessment. It should be considered for example that some specific olive
viruses such as OLV-1, OLV-2 and OLRSV are rare, infections are symptomless on olive
plants, and there are no evidences about their threat to other crops. OLYaV is currently
included in the list of the harmful pathogen for the Italian phytosanitary regulation, but
even if OLYaV-infected trees are widespread, there are very few plants showing
symptoms of yellowing, and more importantly the association of this virus with the OLY
disease has not been clearly demonstrated. Regarding CMV and TNV, although these
viruses are polyphagous and very damaging to other crops, they in olive are rare (CMV)
or present only in a restricted geographical area (TNV- Portugal). On the basis of these
data, the list of the viruses to be included in the phytosanitary certification program could
be restricted to the following: SLRSV, CLRV (both are associated to manifest diseases
either in olive plants or in other crops), ArMV (one of the harmful pathogens for Fragaria,
Rubus and other crops) and perhaps TNV. Whereas, it is important to ensure that the
certified olive material is free from phytoplasmas, V. dahliae and P. savastanoi pv.
savastanoi.
Long distance movement of plant propagation material and the expansion of olive crops in
new areas impose the use of common and harmonized certification procedures which are
crucial to restrict the spread of harmful pathogens and pests.
Author details
Giuliana Albanese
Dipartimento di Gestione dei Sistemi Agrari e Forestali,
Universit degli Studi Mediterranea di Reggio Calabria, Reggio Calabria, Italy


127
Maria Saponari
Istituto di Virologia Vegetale del CNR Unita Organizzativa di Supporto di Bari, Bari, Italy
Francesco Faggioli
CRA-Centro di Ricerca per la Patologia Vegetale, Roma, Italy
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Luigi, M., Godena, S., ermi, E., Barba, M., Faggioli, F. (2011). Detection of viruses in olive
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associated with a new disease of olive in Central Italy, Plant Dis. 70: 171-172.
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like diseases and viruses of olive in Jordan, Phytopathol. Medit. 34: 133-136.
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Chapter 7
Olive Tree Genomic
Rosario Muleo, Michele Morgante, Riccardo Velasco,
Andrea Cavallini, Gaetano Perrotta and Luciana Baldoni
1. Introduction
The cultivation of olive trees dates back to ancient time. Mythology ascribes to the divine
will the domestication of this species; the goddess Athena taught the people of the city of
Athens, as a gift, the cultivation of the tree and the treatment of the drupe (Kakridis, 1986).
Despite the economic, cultural and ecological importance of olive groves in the
Mediterranean area, now extending to other regions, olive has been a poorly characterized
species at genetic and genomic level among other fruit tree crops. Therefore, still remains
unknown the inheritance of most genes controlling the agronomical performance and
quality traits, even though in the last thirty years a wide molecular survey has been
performed on the olive germplasm (Rugini et al., 2011). In the Mediterranean Basin, in fact,
is conserved the majority of a large number of olive cultivars estimated in more than 1,200
(Bartolini et al., 2004).
Olea europaea subsp. europaea is present in two forms, namely wild (Olea europaea subsp. europaea
var. sylvestris) and cultivated (Olea europaea subsp. europaea var. europaea); it is a diploid species
(2n = 2x = 46), and the genome size range between 2.90 pg/2C and 3.07 pg/2C, with 1C = 1,400-
1,500 Mbp (Loureiro et al., 2007). Crosses with other subspecies and with the wild plants are
possible and may produce fertile offsprings, providing access to an enormous pool of genetic
variability. Over the last two decades, new knowledge on olive genetics has been produced,
with the development of nuclear and plastidial molecular markers and linkage maps.
The long generation time of the species has severely restricted breeding strategies to clonal
or varietal selection and, in a very few cases, to inter-varietal crosses. Approaches of marker
assisted selection could speed up the cross breeding programs but QTL markers are not yet
available. The first linkage map of Olea europaea was constructed by de La Rosa and co-
workers (2003), through the use of dominant PCR markers, such as RAPDs and AFLPs, and
codominant marker as RFLPs and SSRs on a cross progeny between two highly
heterozygous cultivars. Other maps have been constructed by the use of RAPDs,


134
microsatellites and SCAR markers on a Frantoio x Kalamata progeny (Wu et al., 2004) and,
more recently, a new maps has been derived through SSR, AFLP, ISSR, RAPD and SCAR
marker, scored on a 140 F1 progeny from a Picholine Marocaine x Picholine du Languedoc
cultivars cross (El Aabidine et al., 2010). In any case, no QTLs of agronomical interest for
olive breeding have been detected.
The Italian project, OLEA, is an initiative, mainly supported by Italian Minister of Agricultural,
Food and Forestry Policies, dedicated toward the development genomic resources of olive,
and it aims to identify, isolate and determine the function of genes that are associated with
both vegetative and reproductive phenotype. Therefore, the knowledge of the genetic
structural basis is the first step to identify the relevant differences in the control of gene
expression of the same sets of genes that exist among different genotypes. The development of
new molecular tools through approaches of structural and functional genomics, together with
those from proteomics, metabolomics, mapping and genotyping, will allow to advance in
molecular breeding of olive, pull out under-exploited natural diversity that is present in the
Olea complex and in olive germplasm, dissect the molecular mechanisms underlying traits
related to high valued compounds and those involved in plant-environment interactions,
establish a platform for a rapid and cost-effective transfer of knowledge and technologies.
2. Genome sequencing and assembly
The olive genome is being sequenced using a combination of Next Generation Sequencing
(NGS) technologies and a combination of assembly approaches, using the cultivar Leccino as
the genotype to be sequenced. The Whole Genome Shotgun approach to assembling the
genome is being pursued using Illumina and 454 sequencing with a combination of long single
reads, paired end reads and mate pairs until a coverage of at least 40 genome equivalents is
reached. The assembly is being performed using Abyss and CLC assemblers. A BAC pooling
approach is being used to sequence random pools of 384 BACs using Illumina paired end reads.
A BAC coverage of approximately 3-4 genome equivalents is going to be sequenced, with each
BAC clone sequenced on average at a 50X coverage. The advantages of the BAC approach are of
two types: on one hand each BAC pool is much smaller in size than the total genome size,
reducing the assembly complexity, on the other hand within each BAC pool we should not face
the problem posed by sequence heterozygosity among maternal and paternal-derived genomes
that strongly affects WGS approaches. The advantage of the WGS approach is the much more
complete and homogeneous coverage of the entire genome. The two assemblies derived, the
WGS and the pooled BAC assembly, will therefore be combined using a proprietary algorithm
(GAM) to produce a consensus assembly. The consensus assembly will finally be anchored to
the genetic map through the use of high throughput genotyping technologies.
As of today we have produced all of the data needed for the Whole Genome Shotgun
component. We have produced approximately 90 Gbp of Illumina sequence data,
corresponding to a nominal coverage of 60X of the olive genome. The Illumina sequences were
obtained from two paired-end libraries with 500-600 bp inserts that were sequenced on the
Illumina Genome Analyser IIx producing 150 bp reads for a total coverage of 43X (65 Gbp) and

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135
from one paired-end library with 1000 bp inserts that was sequenced on the Illumina
HiSeq2000 system producing 100 bp reads for the remaining 17X coverage (25 Gbp). Finally
two mate-pair libraries with 3 Kbp inserts were constructed and sequenced on the HiSeq2000
to produce 100 bp reads and reach a coverage of 4 genome equivalents (6 Gbp).
We have produced approximately 18 Gbp of Roche-454 sequence data, corresponding to
12X coverage approximately. 12 Gbp were obtained as long single reads of which
approximately one third were 400 bp long reads (FLX TITANIUM technology) and two
thirds were 700 bp long reads (FLX XL PLUS technology). Additionally 6.2 Gbp of sequence
data were obtained as paired end reads from 3 libraries with 3 Kbp inserts (3.8 Gbp) and 10
libraries with 8 Kbp inserts (4.4 Gbp).
The 454 single reds and the Illumina paired-end reads are being used in a traditional WGS
assembly. The Illumina mate-pair and the 454 paired end sequenced, i.e. all those sequences
that have been obtained from inserts of larger size, will be utilised in order to scaffold into
larger assemblies the contigs obtained from the assembly of the reads from the shorter
inserts and try to overcome the assembly problems posed by the occurrence of repetitive
elements. Since many of the transposable elements in plant genomes are larger than 3 Kbp
the larger inserts are going to be of crucial importance.
We have performed a number of assemblies to test different strategies and to obtain a first
rough draft of the olive genome. We tested assemblies both using the Illumina data only, as
well as using Illumina and 454 data. All data sets have been initially filtered for low quality
sequences and for chloroplast DNA contamination and then subject to assembly using the
CLCBio assembler. When only the Illumina data were used (53X coverage after filtering), we
produced an assembly of total size of 1.1 Gbp and N50 size of 1.7 Kbp. The scaffolding using
the mate pair and paired end information on the same assembly using the SSPACE tool
increased the N50 size to 2.3 Kbp. The addition of an initial set of 454 data (3.5 genome
equivalents after filtering, single reads only) increased the total assembly size to 1.5 Gbp and
the N50 size of contigs and scaffolds to 2.8 and 3.7 Kbp, respectively. We expect that the
addition of the remaining 454 sequenced from the large insert libraries (3 and 8 Kbp inserts)
should greatly improve the assembly by increasing considerably the N50 size of the
scaffolds. However, due to the problems posed by the high levels of sequence
heterozygosity present in the olive genome of cultivar Leccino, we consider the sequencing
of the pools of BACs a necessary component of our strategy in order to obtain a satisfactory
assembly. The problems here are represented by the difficulties in obtaining BAC libraries
with large insert sizes (>100 Kbp) from cultivar Leccino. Should this not prove feasible we
will anyhow resort to using a fosmid library (40 Kbp inserts).
3. Analysis of the repetitive component of the genome
3.1. Assembly of olive repetitive sequences
Some of the biggest technical challenges in sequencing eukaryotic genomes are caused by
repetitive DNA (Alkan et al., 2011): that is, sequences that are similar or identical to
sequences elsewhere in the genome.


136
The first step in characterizing and sequencing large genomes has to be a genome survey,
from which important information about common repeat sequences can be obtained. NGS
data are particularly suitable to identify sequences present in many copies per genome, by
assembling reads according to their sequence.
The olive genome is largely uncharacterized, despite the growing importance of this tree as
oil crop. Concerning repeated sequences, the most characterized are tandem repeats
belonging to 4 families, isolated from genomic libraries and, in some instances, localized by
cytological hybridization on olive chromosomes (Katsiotis et al., 1998; Minelli et al., 2000;
Lorite et al., 2001; Contento et al., 2002). Also putative retrotransposon fragments have been
isolated and sequenced (Stergiou et al., 2002; Natali et al., 2007), but a comprehensive
picture of RE landscape in the olive genome is still lacking.
We have performed a deep analysis of the repetitive component of olive genome, using
NGS techniques (454 and Illumina). We have used around 25 million Illumina paired-end
reads of 75 nt, corresponding to 1.8 billion nt and a 1.3 x coverage, and around 8 million 454
single reads, with mean read length of 407 nt, corresponding to a total of 3.3 billion nt and a
2.3 x coverage.
This large amount of sequencing data cannot be sufficient for whole genome assembly, but
it enables representative sampling of elements present in a genome in multiple copies.
Moreover, the proportion of individual sequences in the reads reflects their genomic
abundance, thus providing a simple and reliable means for quantification of repetitive
elements (Macas et al., 2007).
In our experiments, we performed de novo repeat identification and reconstruction by direct
assembly of the reads. Due to the relatively low genome coverage of the sequencing, most of
the contigs that are obtained do not represent specific genomic loci; instead, they are
probably composed of reads derived from multiple copies of repetitive elements, thus
representing consensus sequences of genomic repeats (Novak et al., 2010). Even though the
exact form of this consensus does not necessarily occur in the genome, this representation of
repetitive elements has been shown to be sufficiently accurate to enable amplification of the
full length repetitive elements using PCR (Swaminathan et al., 2007).
We assembled Illumina and 454 sequence reads by overlapping DNA sequence fragments
using CLC-BIO and CAP3 as aligners. In spite of recent progresses, a major challenge
remains when reads map to multiple locations, i.e. with multi-reads. The occurrence of
multi-reads is strongly dependent on the read length: they are most common in the Illumina
sequence packages, and less common in 454 sequence packages, in which sequence length is
rapidly growing to lengths similar to those achieved by classical Sanger sequencing, though
at higher costs than Illumina.
The sequencing coverage affects heavily the possibility to recover repeated sequences.
Obviously, the larger is the coverage, the higher is the possibility that multi-reads are not
resolved and discarded. For example, it has been demonstrated, in pea, that a very low
coverage (0.008 ) of the genome allows to obtain repetitive sequences present with at least

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137
1000 copies (Macas et al., 2007). Hence, we decided to proceed to the final assembly of
Illumina reads after having splitted the sequence read datasets into subpackages of different
genome coverages.
In a first assembly, we assembled the complete pool of Illumina reads using CLC-BIO and
subsequently CAP3 assembler. In other experiments, the pool of Illumina reads was splitted
into 8, 16, 32, 250, or 500 subpackages and assembled separately (indicated as split 1, 2, 3, 4,
and 5, respectively); for each splitting, the resulting contigs were assembled on their turn
using CAP3 assembler obtaining 210,063 supercontigs.
All supercontigs were then mapped with all Illumina 75 nt long reads (Table 1). It can be
observed that major splittings allow to recover the most redundant supercontigs, that are
not found in the lower splittings, because of their too large coverage and, hence, the
occurrence of multi-reads. Due to the different redundancy observed in the different
subpackages, we decided to use all supercontigs in the final assembly.

Split Nr. of
sub-
packages
Subpackage
coverage
Nr. of
assembled
supercontigs
Mean
length
Mean nr. of
mapped
reads
Average
coverage
N50
0 0 1.309 x 44336 235.6 19.61 6.07 243
1 4 0.327 x 78983 200.5 19.96 6.01 201
2 8 0.163 x 50698 204.2 31.72 9.56 204
3 32 0.041 x 22749 252.3 68.58 14.35 265
4 244 0.005 x 14748 240.6 218.57 74.42 258
5 489 0.003 x 11819 223.6 212.77 67.99 239
Table 1. Characteristics of supercontig sets obtained by CLC Bio Workbench and CAP3 assembly after
different splitting of Illumina reads.
Concerning 454 sequence reads, we did not proceed to such a subdivision, estimating that
the superior length of reads compared to that of Illumina ones allowed to recover also
highly repeated sequences. In fact, in longer sequences, the occurrence of multi-reads is
naturally reduced.
All Illumina- and 454-derived supercontigs and contigs longer than 80 nt were masked
against an in-house made database of chloroplast and mitochondrial sequences using
RepeatMasker, and organellar sequences were removed. Then, a final assembly was
performed, using CAP3, among all datasets, i.e. six Illumina datasets (split 0-5) and one 454
dataset. The resulting whole genome dataset included 238,914 supercontigs, with mean
length of 667.9 nt and N50 = 1.331.


138
3.2. Estimation of copy number of assembled sequences
Assuming that Illumina sequence reads in our experiments are sampled without bias for
particular sequence types, mapping the whole genomic dataset with Illumina sequence
reads provides a method of estimating the copy number of any genomic sequence in the
dataset (Swaminathan et al., 2007).
Data in the literature and slot blot experiments previously performed in our lab (Giordani,
personal communications) allowed estimation of the copy number per haploid genome of 16
sequences. The 16 sequences with known redundancy were inserted in the whole genomic
database and used as reference for the estimation of copy number by mapping on them a
pool of around 270 million Illumina 75 nt reads (coverage 14.4 x). We adopted a
classification commonly used in biochemical experiments (Britten & Kohne, 1968) and
defined supercontigs as highly repeated (HR, redundancy > 10.000 copies per genome, 3,619
supercontigs), medium repeated (MR, redundancy ranging between 100 and 10,000 copies
per genome, 67,045 supercontigs) and unique (U, redundancy < 100 copies per genome,
168,250 supercontigs).
3.3. Olive genome composition
HR and MR supercontig datasets were annotated to produce the OLEAREP 1.0 database.
The annotation pipeline is reported in Figure 1.

Figure 1. The annotation pipeline for the production of OLEAREP database.
The distribution of sequence type in the HR and MR datasets and in the whole OLEAREP
1.0 database is reported in Table 2.
The average coverage of each HR and MR sequence was used to estimate the redundancy of
the various types of repeat classes. Concerning the whole olive genome, around 50%
appears to be made of highly repeated sequences. Of these, around 2/3 are tandem repeats
belonging to five major families and other minor families (Figure 2). Such extreme
redundancy of tandem repeats appears a peculiar feature of olive genome, not found in the

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other plant species whose genome has been sequenced. On the contrary, medium repeated
component is mainly composed of LTR-retrotransposons, while tandem repeats are much
less represented in this genome portion.

Sequence type
Nr. of sequences (%)
HR MR
DNA transposons 31 (0.86) 2,183 (3.26)
Retrotransposons LTR-Copia 134 (3.70) 7,569 (11.29)
LTR-Gypsy 258 (7.13) 8,066 (12.03)
Non-LTR 29 (0.80) 949 (1.42)
Tandem repeats 1,535 (42.42) 6,718 (10.02)
rDNA 29 (0.80) 555 (0.83)
Putative genes 46 (1.27) 2,729 (4.07)
Unknown repeats 317 (8.76) 1,795 (2.68)
No hits found 1,240 (34.26) 36,481 (54.41)
Total 3,619 67,045
Table 2. Functional percentage distribution of the supercontigs in OLEAREP 1.0.

Figure 2. HR (left) and MR (right) fraction composition.
4. Olive chloroplast genome
The chloroplast genome of the olive has an organisation and gene order that is conserved
among numerous Angiosperm species and do not contain any of the inversions, gene
duplications, insertions, inverted repeat expansions and gene/intron losses that have been
found in the chloroplast genomes of the genera Jasminum and Menodora, from the same
family as Olea (Mariotti et al., 2010). 40 polymorphisms have been identified in the plastome
sequence, poorly able to differentiate among olive cultivars.


140
5. miRNA
A first inventory of sRNAs in olive has been obtained from juvenile and adult shoots,
revealing that the 24-nt class dominates the sRNA transcriptome and atypically accumulates
to levels never seen in other plant species, suggesting an active role of heterochromatin
silencing in the maintenance and integrity of its large genome (Donaire et al., 2011). A total
of 18 known miRNA families were identified in the libraries.
6. Analysis of transcriptome
Despite its global importance, genomic sequence resources available for olive are still scarce,
though an increasing number of expressed gene functions are being described in the last few
years through limited NGS approaches. Recently, many EST sequences from large scale
transcriptomic analyses of different organs, such as fruits and leaves have been released
(Alagna et al., 2009; Galla et al., 2009; Ozgenturk et al., 2009).
While these studies have highlighted the utility of cDNA sequencing for candidate gene
discovery and gene function, a comprehensive description of genes expressed in Olea
europaea remains unavailable.
Over the past several years, the NGS technology has emerged as a cutting edge approach for
high-throughput sequence determination and this has dramatically improved the efficiency
and speed of gene discovery (Ansorge, 2009). Furthermore, NGS has also significantly
accelerated and improved the sensitivity of gene-expression profiling and, is expected to
boost collaborative and comparative genomics studies (Strickler et al., 2012).
In this study, we generated over one million sequence reads with 454 FLX technology
(Roche Diagnostics Corporation, Basel, Switzerland) and identified a number of gene
functions potentially involved in the expression of major traits that control productivity and
quality of olive and oil production.
The starting materials used to explore the olive transcriptome were flower and fruit samples
from five different genotypes. Flower tissues at different developmental stages were
sampled from Leccino, Dolce Agogia and Frantoio varieties. Two 454 sequencing libraries
were obtained from retro-transcribed pooled RNA samples, extracted from flower buds at
all stages of development until anthesis of Leccino and Dolce Agogia genotypes,
respectively. Furthermore, pooled flower samples of Leccino and Frantoio genotypes,
collected after anthesis, were used for synthesis of cDNAs and for the subsequent
preparation of two additional 454 sequencing libraries.
In order to gain information on genes expressed in the drupe, with particular regard to
those involved in response to pathogen infections, another set of four 454 sequencing
libraries was obtained from fruit samples (at about 17 weeks after flowering) of Ortice and
Ruveia genotypes collected before and after the infection caused by the olive fruit fly
(Bactrocera oleae).

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The eight 454 cDNA libraries, four from flower and four from fruit tissues, were sequenced
in two separate runs by using 454 GS FLX Titanium Sequencer (Roche Diagnostics
Corporation, Basel, Switzerland); each library was loaded on sector of a picotiter
sequencing plate.
We identified a total of more than 1 million sequence reads with an average length of 356
bp, corresponding to a little less of half billion bases, about 60% of them are from fruit and
40% from flower samples (Table 3).

Sample Reads Total Bases Length Average (bp)
Flower_1 113,134 42,568,083 376.55
Flower_2 146,576 54,807,276 374.23
Flower_3 67,797 21,939,320 323.92
Flower_4 137,824 48,459,361 351.82
Flower_total 465,331 167,774,040 360.55
Fruit_1 173,118 63,611,956 367.45
Fruit_2 197,782 73,464,937 371.44
Fruit_3 146,765 52,792,933 359.71
Fruit_4 177,846 61,220,898 344.23
Fruit_total 695,611 251,090,724 361.02
Olea_total 1,160,942 418,864,764 356.63
Table 3. Raw sequencing data
Assembling of adaptor-trimmed 454 sequence data was performed using GSAssembler
Software (Roche Diagnostics Corporation, Basel, Switzerland). To build a compilation of
gene structures and functions expressed in Olea, we first assembled row data from all the
eight libraries together (Table 4).

Contigs Singletons
Samples in Assembly Reads in
Assembly
% Number Length Average
(bp)
Number Length Average
(bp)
Flower + Fruit
samples
964.266 83,05 25.342 892 112.717 323
Table 4. Total assembling
More than 83% of raw sequences were included in the assembly with 112,717 remaining as
singletons. This produced a set of 25,342 contigs with an average length of 892 bp (Table 4).
As expected, when sequences from flower and fruit samples are assembled separately, the


142
number of EST sequences assembled in contigs are significantly lower; however the average
length of contigs and singletons remains similar (Table 5).

Contigs Singletons
Samples in
Assembly
Reads in
Assembly
% Number Length Average
(bp)
Number Length Average
(bp)
Flower 338.853 72,82 14.599 804 91.999 345
Fruit 570.878 82,01 15.058 884 72.662 333
Table 5. Flower and Fruit Assembling
To assess the representativeness and the overall quality of the assembling, three randomly
chosen gene sequences, among those already characterized in Olea, were used as a reference
to map contig and singleton sequences produced by the assembling (Figure 3).

Figure 3. Overview of assembling procedure
The fact that two out of three selected genes are 100% covered by the total assembly with a
single contig composed by a great number of ESTs, indicates the coverage of the assembly
is sufficient to characterize the full coding sequence of high-medium expressed transcripts.
Only FAD 6 shows partial coverage, especially in the fruit assembly where only three

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matching singleton sequences were found (Figure 3). This is most probably due to the sharp
decrease of FAD 6 transcript abundance in fruits sampled at late developing stages, from 15
to 20 weeks after flowering (Matteucci et al., 2011).
To predict gene functions, we used a BlastX-based annotation (E-value 1-e
-5
) of unigenes
comparing them to NCBI non-redundant (nr) database (http://www.ncbi.nlm.nih.gov/).
About 52% of the unigenes match to known functional genes; while the remaining 48% has
no function assigned (Figure 4).
The majority of the BlastX annotated unigenes matches most to Vitis vinifera, Populus
trichocarpa and Ricinus communis counterpart sequences, in decreasing order (Figure 4).

Figure 4. Overall profile of unigenes based on homology with GenBank sequences
We also mapped the GI identifiers (http://www.ncbi.nlm.nih.gov/) of the best BlastX hits to
UniprotKB protein database (http://www.uniprot.org/) in order to extract Gene Ontology
(GO, http://www.geneontology.org/). Approximately one-fourth of the unigene set was
assigned to GO terms. This allowed us to group unigenes in 14 sub-categories of biological
processes, 9 sub-categories of cellular components and 11 sub-categories of molecular
functions (Figure 5).


144

Figure 5. GO terms distribution in the cellular components, biological processes and molecular
functions vocabularies.
Metabolic process sub-category, consisting of more than 11,000 genes, is dominant in
biological process. While, binding and cell part subcategories, consisting of about 13,0000
and 8,000 genes, are dominant in molecular function and cellular component, respectively.
We also noticed an appreciable number of genes included in cellular process, catalytic
activity and organelle sub-categories (Figure 5). However, at this level of detail no dramatic
differences are evident between flower and fruit tissue transcriptomes.
Next generation RNA sequence from additional organs/tissues/genotypes of Olea europaea,
as well as, full comparative data analysis between and within the sequenced samples are
currently in progress. These studies will certainly provide valuable information about gene
functions that trigger key metabolic pathways for the expression of desired traits.
7. Conclusion
Genomic sequences for olive will enable researchers to explore the breadth of genetic
diversity present within the species and within the breeding germplasm using high
throughput methods of resequencing based on NGS technologies. This will give access to all
types of variations, namely Single Nucleotide Polymorphisms (SNPs), small
insertion/deletions and structural variants (large insertion/deletions). It will allow a better

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145
assessment of the relationships among the different accessions, of the geographical patterns
of distribution of genetic variation and of the genetic consequences of olive trees
domestication. It will finally form the basis for the development of novel molecular marker
assays.
They will also allow the analysis of global gene expression and specific gene expression of
olive tissues in diverse developmental stages and conditions. The identification and
characterization of expression of important genes involved in agronomic and productive
traits affecting fruit production and quality, biotic and abiotic stress resistance, important
development characters (e.g., juvenility, self-incompatibility, ovary abortion, chill response),
it may offer a significant amount of tools and open new opportunities for improvement
either through molecular breeding and/or genetic engineering.
Many researches will be focused on gene network activities, using olive microarray and/or
qPCR to address the expression patterns of genes, during plant and fruit development and
ripening of drupe fruits, fatty acid metabolism as well as phenylpropanoid metabolism.
Moreover, new generation of molecular markers will be developed, helpful to localize genes
involved in both monogenic and polygenic agronomic traits, to construct genetic fine-maps;
these markers will be also used for marker-assisted selection (MAS) to obtain elite genotypes
by allowing the analysis of cross progenies at earlier stages.
Author details
Rosario Muleo
University of Tuscia, Dept. DAFNE, Viterbo, Italy
Michele Morgante
IGA-Institute of Applied Genomics, Udine, Italy
Riccardo Velasco
Edmund Mach Foundation, IASMA, San Michele allAdige, Italy
Andrea Cavallini
Dept. Crop Species Biology, Pisa, Italy
Gaetano Perrotta
ENEA, Trisaia, Rotondella (MT), Italy
Luciana Baldoni
CNR- Institute of Plant Genetics, Perugia, Italy
Acknowledgement
This research was partially supported by Progetto Strategico MIPAF OLEA - Genomica e
Miglioramento genetico dellolivo, D.M. 27011/7643/10, and by the Province of Trento and
Edmund Mach Foundation. We thank the Roche Diagnostic Spa, Applied Science to support
the OLEA Italian Project.


146
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Section 2

Virgin Olive Oil

Chapter 8
Technological Aspects of Olive Oil Production
Maurizio Servili, Agnese Taticchi, Sonia Esposto,
Beatrice Sordini and Stefania Urbani
1. Introduction
Virgin olive oil (VOO) is obtained exclusively by mechanical extraction from the olive fruit
and can be consumed crude without any further physical-chemical treatments of refining.
Its sensory and health properties are intimately linked to its chemical characteristics, in
particular to several minor components, which are strongly affected by the operative
conditions of oil processing and can thus be considered as analytical markers of the quality
of oil processing.
VOO contains different classes of phenolic compounds, such as phenolic acids, phenolic
alcohols, hydroxy-isochromans, flavonoids, secoiridoids and lignans. The phenolic acids
together with phenyl-alcohols, hydroxy-isochromans and flavonoids are present in small
amounts in VOO (Montedoro et al., 1992). Secoiridoids which, in combination with lignans,
are the main hydrophilic phenols of VOO, include the dialdehydic form of decarboxymethyl
elenolic acid linked to 3,4-DHPEA or p-HPEA (3,4-DHPEA-EDA or p-HPEA-EDA) an isomer
of the oleuropein aglycon (3,4-DHPEA-EA) and the ligstroside aglycon (p-HPEA-EA). The
main lignans found in VOO are (+)-1-acetoxypinoresinol and (+)-1-pinoresinol (Brenes et al.,
2000; Owen et al., 2000). Both secoiridoids and lignans affect the quality of the sensory and
health properties of VOO (Servili et al., 2004a), determining bitter, pungent sensations.
Many compounds, mainly carbonyl compounds, alcohols, esters and hydrocarbons, have
been found in the volatile fraction of VOO.
The C6 and C5 compounds, especially C6 linear unsaturated and saturated aldehydes
alcohols and esters represent the most important fraction of volatile compounds found in
high quality VOOs.
The C6 and C5 compounds, produced from polyunsaturated fatty acids by the enzymatic
activities exerted by the lipoxygenase (LOX) pathway and their concentrations, depend on
the level and the activity of each enzyme involved in this LOX pathway.

The pathway (Figure 1) begins with the production of 9- and 13-hydroperoxides of linoleic
(LA) and linolenic (LnA) acids mediated by lipoxygenase (LOX). The subsequent cleavage of
13-hydroperoxides is catalysed by very specific hydroperoxide lyases (HPL) and leads to C6
aldehydes, of which the unsaturated aldehydes can isomerize from cis-3 to the more stable
trans-2 form. The mediation of alcohol dehydrogenase (ADH) reduces C6 aldehydes to
corresponding alcohols, which can produce esters due to the catalytic activity of alcohol
acetyl transferases (AAT).
Furthermore, an additional branch of the LOX pathway (Figure 1) is active when the
substrate is LnA. LOX would catalyse the formation of stabilized 1,3-pentene radicals, which
can either dimerize leading to C10 hydrocarbons (known as pentene dimers), or can react
with a hydroxy radical, producing C5 alcohols. The latter can be enzymatically oxidated to
corresponding C5 carbonyl compounds. These compounds are responsible for the most
important sensory notes of VOO flavour, such as the Green and floral notes (Angerosa et
al., 2001; Angerosa et al., 2004; Servili et al., 2009a).

Figure 1. Lipoxygenase pathways involved in the production of C6 and C5 volatile compounds
(Angerosa et al., 2004).
The new approach to VOO processing should include as its first objective the improvement
of the quality of the sensory and health properties of oil. Since the presence of VOO
hydrophilic phenols and volatile compounds is strictly related to the activities of various
endogenous enzymes of olive fruit, their concentration in the oil is highly affected by the
operative conditions of the mechanical oil extraction process. By taking into account the
optimization of volatile and phenolic composition of VOO as the main goal of the new
13-alkoxy radical
pentene radical
pentene dimers
hexyl acetate
hexan -1-ol
cis -3-hexen -1-ol cis-3-hexenyl acetate
LA
LnA
trans -2-hexen -1-ol
trans -2-hexenal 13-hydroperoxides
LOX
hexanal
cis-3-hexenal
HPL
2-pentenal
1-penten -3-one
2-penten -1-ol
1-penten -3-ol
ADH AAT
isomerase
ADH
ADH
AAT
Hydro Peroxide Lyase
Alcohol Dehydrogenase
13-alkoxy radical
pentene radical
pentene dimers
hexyl acetate
hexan -1-ol
cis -3-hexen -1-ol cis-3-hexenyl acetate
LA
LnA
trans -2-hexen -1-ol
trans -2-hexenal 13-hydroperoxides
LOX
hexanal
cis-3-hexenal
HPL
2-pentenal
1-penten -3-one
2-penten -1-ol
1-penten -3-ol
ADH AAT
isomerase
ADH
ADH
AAT
Hydro Peroxide Lyase
Alcohol Dehydrogenase

Technological Aspects of Olive Oil Production 153
approach to the mechanical oil extraction process, the conditions during crushing and
malaxation can be considered as the most critical points (Capella et al., 1997; Caponio et al.,
1999; Servili et al., 2002; Servili et al., 2004a; Angerosa et al., 2004; Servili et al., 2009a).
2. The structure and composition of the olive fruit
The first factor for high quality production in the VOO industry is the structure of the raw
material, thus the quality and sanitary status of the olive are very important.
The olive fruit is a drupe, the weight of which varies between 0.5g and 20g. The constituent
parts of the fruit, which include the skin, pulp and stone, represent respectively 1.5-3.5%, 70-
80% and 15-28% of the weight of the fresh drupe. The stone contains the seed with a weight
ranging from 2% to 4% of the whole fruit (VV. AA., 2003).
It is difficult to define the average composition of the olive fruit, due to its remarkable
biodiversity, which produces high compositional variability. Water (40-70%) and fat (6-25%)
are the main constituents of the fresh fruit.
The fruit contains water-soluble compounds, including simple sugars, organic acids,
nitrogenous substances, phenolic compounds and an insoluble fraction of colloidal nature.
Colloids of the drupe include the components of the cell wall or the middle lamella, such as
hemicelluloses, celluloses, pectins, enzymatic and structural proteins.
The most important reducing sugars found in olives are glucose, fructose and sucrose,
whereas citric acid, malic acid and oxalic acid are the main organic acids of the olive drupe
(VV. AA., 2003).
In the composition of the olive, the phenolic fraction, which includes the precursors of
natural antioxidants present in VOO, is of major importance (Amiot et al., 1986; Servili et al.,
1999a, 1999b, 1999c; Servili & Montedoro, 2002). Phenolic compounds present in very high
quantities of drupe (from 0.5 to 2.5% fresh weight) include oleuropein and
demethyloleuropein. These substances are found mainly in the peel and pulp, whereas the
seed contains nzhenide, not found in the pulp and which is not considered a precursor of
the phenolic compounds of VOO (Servili et al., 1999b). Lignans were found both in the pulp
and in the woody core, but the latter cannot be released in VOO during oil processing
(Brenes et al., 2000; Garcia et al., 2001; Servili et al., 2007).
The peel and the pulp together contain more than 90% of the total phenolic concentration of
the fruit, which varies significantly according to the cultivar and stage of ripening of the
olives (VV. AA., 2003; Servili et al., 2004a). The oil fraction is present in the pulp (16.5-23.5%
fresh weight) and in the seed (1-1 .5% fresh weight).
Oilseed cells typically contain cytoplasmic and vacuolar oil. The compartmentalization of
the oil in the olive pulp cells is, in this context, unusual when compared with that of
oilseeds. In fact, the pulp cells of a ripe olive oil contain almost exclusively vacuolar oil,
whereas the amount of cytoplasmic oil in the oilseed cells is remarkable.

Vacuolar oil in the olive pulp is the result of the mechanical process of oil extraction, which
explains why even the most remote civilizations in the Mediterranean area have been using
this fruit as a natural source of dietary fat.
3. New technological approaches towards the quality of virgin olive oil
3.1. Olive fruit storage
Storage of the olives before the mechanical oil extraction process is a critical point, which
can reduce the quality of VOO. The first adverse effects are seen in the decrease in oil
phenols and in the reduction of volatile compounds responsible for the flavour of VOO
(Angerosa et al., 1996; Kiritsakis, 1998a; Kiritsakis et al., 1998b; Angerosa et al., 1999; Servili
et al., 2004a; Servili et al., 2009a). The activation during olive storage of endogenous
polyphenoloxidase (PPO) and peroxidise (POD), which catalyze the degradation of the
phenols, can explain the loss of phenolic compounds. The reduction of volatile compounds
responsible for the oil flavour can be due to the inhibition of the LOX pathway.
Moulds, yeasts and bacterial contaminations, and their corresponding metabolisms, are the
underlying cause of the off-flavour biogenesis in VOO. In several operative conditions
involving long-term storage of the olive and high relative humidity, mould contamination
increases the free acidity due to the production of fungal lipase, and simultaneously forms
the characteristic sensory defect of "mould" (Angerosa et al., 1999, 2004). Several mould
species can also produce mycotoxins. Sugar fermentation produces the formation of acetic
acid and ethyl acetate, which are considered responsible for the vinegar off-flavour.
During storage, the fatty acid alkyl esters of ethanol and methanol develop. The formation
of these compounds is used as a marker to recognize low quality virgin olive oil. In fact,
these compounds, combined with other sensory and chemical parameters, are used to
classify virgin olive oil according to the internationals standards set by the International
Olive Council (I.O.C., 1996).
These observations lead to the conclusion that olive storage should be avoided. To maintain
the quality of VOO, the olives should be processed within twenty-four hours after
harvesting. A thin, 30-40 cm thick layer of olives should be stored in perforated boxes on
pallets, in order to minimize the fermentation processes, which underlie not only the
formation of sensory defects, but also water condensation on the surface of the olive skin,
which can promote the attack by moulds. Perforated boxes and pallets are also the most
suitable to transport the olives from the olive grove to the mill (Angerosa et al., 1996, 1999;
Servili et al., 2004a; Servili et al., 2009a).
Another aspect, which affects the storage premises, is related to the handling of the pallets
or boxes. This process must be done by avoiding the use of forklift trucks run by petrol
engines, which produce polycyclic aromatic hydrocarbons. These compounds can
contaminate the olives, and subsequently, the oil (Angerosa et al., 2004).

3.2. Leaf removal and washing
Leaf removal is always recommended, especially when harvesting is done mechanically.
The presence of leaves during the mechanical oil extraction process does not add any
positive characteristic to the oil but, on the contrary, can change its taste and aroma.
Olives are generally washed by continuous washing machines. (Di Giovacchino et al., 2002;
Perez et al., 2003). Olive washing has a more or less significant effect on the quality of VOO,
depending on the characteristics and sanitary state of the olives. Fresh olives, harvested at
the correct degree of maturity and properly transported and stored, showed no direct effect
in the quality of the oil due to washing.
The most critical aspect of the washing process concerns the purity of the water used during
this stage. The water should be changed frequently during processing to prevent the use of
washing water containing too many earthy particles, which can release the compounds
responsible for the Earthy sensory off-flavour into the oil (Angerosa et al., 2004).
3.3. Olive crushing
The main hydrophilic phenols of VOO, such as secoiridoid aglycons, develop during
crushing from the hydrolysis of oleuropein, demethyloleuropein and ligstroside,
catalysed by the endogenous -glucosidases (Servili et al., 2004a; Obied et al., 2008).The
impact of crushing in the VOO phenolic and volatile compounds can be related to the
differentiated distribution of the endogenous oxidoreductases and phenolic compounds
in the constituent parts of the olive fruit (pulp, stone and seed). As reported in previous
papers, the POD, in combination with the PPO, are the main endogenous oxidoreductases
responsible for phenolic oxidation during processing. POD occurs in high amounts in the
olive seed. The phenolic compounds, on the contrary, are largely concentrated in the pulp,
whereas the stone and seed contain only small quantities of these substances (Servili et al.,
2004a; 2007). As a result, the crushing methods, such as the olive stoning process or the
use of mild seed crushers, which enable degradation of the seed tissues to be reduced by
limiting the release of POD in the pastes, prevent the oxidation of hydrophilic phenols
during malaxation, thus improving their concentration in the VOO (Figure 2) (Servili et
al., 1999a, 2004a, 2007).
The operative conditions of crushing also affect the volatile composition of VOO (Table 1).
As previously mentioned, almost all volatile compounds are responsible for the flavour of
high quality VOOs when the olive pulp tissue is ruptured, thus the effectiveness of the
crushing plays an important role in their production.
The traditional olive crusher used for many centuries was the stone crusher. The stone
crusher consists of a basin formed by a plinth and a stainless steel edge with an opening for
the unloading of olive paste at the end of milling. Two or four granite wheels rotate and
revolve on a rough granite base at different distances from the centre of the tank. Rotation
speed is normally 12- 15 rpm.


Figure 2. Effect of different crushing methods on the phenolic composition of VOO in Cv. Frantoio
(mg/kg). The vertical lines are the mean value of three independent experiments standard deviation.
The popularity of the stone crusher extraction system using pressure gradually declined. In
comparison with continuous crushers, this apparatus shows significant limitations in terms
of olive oil quality. In particular, it reduces the phenolic concentration as the olive pastes are
in long, extensive contact with the air during processing. Contact with the air stimulates
PPO and POD, producing a high oxidation of phenolic compounds. Other weaknesses of the
stone crusher are its low working capacity, the high hourly machine footprint, and its low
ability to release the chlorophyll found in the olive skin, responsible for the green colour of
EVOO, into the oil. This aspect is particularly relevant when the stone crusher is combined
with a solid-liquid centrifugal separation. The crushing operation in oil extraction by
centrifugation is generally replaced by the use of continuous crushers.
Continuous crushers include the hammer crushers, which were historically the first to be
introduced as an alternative to stone crushers. These machines have some benefits that are
attributable to their high working capacity, low footprint, and low installation costs
compared to the stone crushers. At the same time, the hammer crushers show some
disadvantages, such as the strong emulsifier effect produced on olive paste during crushing,
a considerable increase in paste temperature and the high degradation of the seed tissues
which, as mentioned earlier, can encourage phenolic oxidation (Angerosa & Di Giacinto.,
1995; Servili et al., 1999a; Servili & Esposto 2004).
The new approach to olive crushing is based on the differentiated crushing of the
constituent parts of the fruit, such as the skin, pulp and seed. In other words, the
Cv. Frantoio
0
50
100
150
200
250
300
350
400
hammer crusher pre-crusher + blade crusher crusher with a low number of
rotations
stoned
m
g
/
k
g

Sum of the phenolic compounds Sum of the oleuropein derivatives
Sum of the lignans Sum of the ligstroside derivatives

degradation process of the olive tissues should be strong for the skin and pulp, in order to
facilitate the release of oil and pigments, whereas impact on the seed should be limited. This
reduces the transfer of POD found in the seed to the olive paste pulp, which can increase the
oxidation of phenolic compounds during malaxation. The new generation of mild seed
crushers, which include the blade crusher, teeth crusher and double stoker crusher, reduce
the phenolic degradation and simultaneously improve the concentration of volatile
compounds, especially of hexanal, trans-2-hexenal and C6 esters, with a consequent positive
increase of the intensity of cut grass and floral sensory notes (Table 1) (Angerosa et al.,
2004).

hammer crusher
pre-crusher +
blade crusher
crusher with a low
number of
rotations
stoned
Aldehydes
Pentanal 236.5 4 273.4 2.1 17.9 1 66.5 6.7
Hexanal 280 2.9 511.4 35.7 553.7 0.3 579.6 5.3
2-Hexenal (E ) 43600.6 327 44718.9 208 39811.6 587 52228.1 521
2,4-Hexadienal
(E,E)
19.4 0.1 42 3.5 341.6 14.4 88.9 5.4
Alcohols
1-Pentanol 167 5.2 94.5 4.7 23.3 0.7 62.6 1.7
2-Penten-1-ol (E) 166 11.3 91.4 5.1 52.4 3.5 104.0 2.1
1-Penten-3-ol 960.3 53.2 899 43.3 522 49.2 300.0 4.6
1-Hexanol 1788 57 2152 74 512 41 1501.0 18.4
3-Hexen-1-ol (Z) 88.4 22.2 103.6 10.1 49.2 2.3 77.0 8.0
3-Hexen-1-ol (E) 22.2 0.2 20.2 0.1 9.9 0.2 20.4 2.3
Table 1. Effect of different crushing methods on the volatile composition of VOO in Cv. Frantoio
(g/Kg).

Results are the mean value of three independent experiments standard deviation.
Several researches have shown that olive stoning during the mechanical extraction
process of VOO increases the phenolic concentration in VOO (Figure 2) (Angerosa et al.,
1999; Mulinacci et al., 2005; Lavelli & Bondesan, 2005; Amirante et al., 2006) and, at the
same time, modifies the composition of volatile compounds produced by the LOX
pathway, increasing the concentration of those volatile substances correlated to the
green sensory notes (Table 1) (Servili et al., 2007). These results are particularly
important, because they would appear to demonstrate that the enzymes involved in the
LOX pathway have a different activity in the pulp and in the seed of the olive (Table 1)
(Servili et al., 2007).
The stoning process, moreover, produces a significant reduction in industrial oil yields. This
problem is caused by stone elimination which, when present in the pastes, produces an
important draining activity, which increases the efficiency of the separation of the oil from
the olive paste during solid-liquid extraction.

It should be noted that the use of enzymatic preparations with depolymerizing activities,
degrading the colloidal structure of fruit might partly solve the problem of low yields due to
the extraction of pastes for stoning. So far, however, the European Union (EU regulation)
does not currently allow the addition of enzymatic preparations.
3.4. Malaxation process
The mixing and heating (25-35 C) of the olive pastes during malaxation causes the
breakdown of water-oil emulsion, allowing oil droplets to form larger droplets, which
separate easily from the aqueous phase during the solid-liquid and liquid-liquid separation
processes.
The operative conditions applied during malaxation of the olive pastes largely affect VOO
quality (Servili et al., 1994; Montedoro et al., 2002; Servili et al., 2004a; Inarejos-Garcia et al.,
2009). As in the case of the crushing process, malaxation can also produce significant
modifications in the minor components of VOO with particular emphasis on volatile and
phenolic compounds. The technological parameters of temperature and oxygen
management show the highest impact on the volatile and phenolic composition of VOO.
The problems concerning temperature management during malaxation have been widely
studied for over twenty years and a substantial negative relationship between the
processing temperature and the quality of the VOO has been shown (Garcia et al., 2001; Di
Giovacchino et al., 2002; Servili et al., 2003a; Servili et al., 2004a; Kalua et al., 2006). However
some aspects of the relationship between the operative conditions of malaxation and oil
quality must be better defined.
As previously reported, the most sensitive quality markers linked to the effect of processing
temperature are the phenols and volatile compounds with their sensory impact. The
literature on phenolic compounds clearly shows that the phenolic concentration of VOO
could be more or less drastically reduced in relation to the increase in the mixing
temperature. In particular, the derivatives of oleuropein, demethyloleuropein and
ligstroside are highly affected by the processing temperature, whereas lignans are less
affected (Servili et al., 2004a).
The optimal temperature of activity for PPO and POD may explain the loss of phenolic
compounds in the oil depending on the processing temperature. These enzymes catalyze the
oxidative degradation of phenolic compounds during the mixing process and show an
optimal temperature of activity at approximately 50 C and 45 C respectively for PPO and
POD.
As a result, the oxidation of phenols by PPO and POD within the range from 20 C to 35 C
would be progressively higher, depending on the operating temperature. This explains the
widely published data about the differences in phenolic concentration between oils obtained
at different temperatures of between 20 and 40 C (Snchez & Harwood, 2002; Angerosa et
al., 2001).

These results are, in any case, obtained by performing malaxation with the pastes under
continuous contact with air, as shown in the traditional mixer (Servili et al., 1998, 2003a).
However, when the process is performed in the new-generation malaxer, known as a
"covered malaxer", which can control contact of the olive pastes with oxygen during mixing,
the results obtained in terms of relationships between phenol concentrations in VOO and
the processing temperature are completely different.
During processing, the olive pastes release CO2 and the dissolved O2 is simultaneously
consumed by the oxidoreductase activities. As a result, the reduction of the O2 content
obtained in the covered malaxer inhibits the PPO and POD activities, improving the
concentration of hydrophilic phenols in the olive pastes and in the corresponding VOO
(Figure 3) (Servili et al., 2008).

Figure 3. Phenolic composition (mg/Kg) of VOOs obtained after malaxation in different initial
atmospheric compositions (Servili et al., 2008).
0 kpa saturated with N2; 30 kpa corresponding to the air composition. The vertical lines are the mean
values of three independent experiments, standard deviation is reported in brackets.
As a result, the oxidative reactions occurring in the pastes during malaxation can explain the
relationships between VOO phenolic concentration and malaxation temperatures (Servili et
al., 2004a; 2009a; 2009b). The O2 dissolved in the pastes during malaxation, activate POD
and PPO, which oxidize phenolic compounds according to the temperature and
consequently reduce their concentration in VOOs obtained by pastes malaxed at high
temperatures. The traditional malaxer, which contains a high amount of O2 dissolved in the
paste during the process due to contact with the air, represents a classical example of the
aforementioned relationship between high temperatures and VOO phenolic loss. Low
0
200
400
600
800
1000
1200
1400
O
2
0 Kpa O
2
30 Kpa O
2
50 Kpa O
2
100 Kpa O
2
0 Kpa O
2
30 Kpa O
2
50 Kpa O
2
100 Kpa
Cv. CORATINA Cv. OGLIAROLA
m
g
/
k
g

Sum of the ligstroside derivatives Sum of the lignans
0
200
400
600
800
1000
1200
1400
O
2
0 Kpa O
2
30 Kpa O
2
50 Kpa O
2
100 Kpa O
2
0 Kpa O
2
30 Kpa O
2
50 Kpa O
2
100 Kpa
Cv. CORATINA Cv. OGLIAROLA
m
g
/
k
g

Sum of the ligstroside derivatives Sum of the lignans

amounts of O2, on the contrary, inhibit the oxidative reactions of phenols during malaxation
and, in this case, their concentration in the VOO increases according to the temperatures,
because of a major release of phenols into the oil (Servili et al., 2008; 2009a; 2009b).
Thus, O2 control during malaxation can be considered a new technological parameter which,
in combination with the traditional ones (time and temperature of the process), can be used
to optimize the VOO phenolic and volatile concentrations (Servili et al., 2004a, 2008, 2009a).
In this regard, the time of exposure of the olive pastes to air contact (TEOPAC) was studied
as a process parameter to regulate the O2 availability in the paste and, as a result, the
amount of phenols in the VOO (Servili et al., 2003a, 2003b).
Furthermore, the natural increase of an inert gas of this respiration catabolite, such as CO2,
released during malaxation after the destruction of the olive cell, may be combined with the
use of nitrogen or argon to reduce the O2 contact with the olive pastes during malaxation
(Parenti et al. 2006a, 2006b; Servili et al., 2008).
The latter have to be carefully controlled according to the time of malaxation. Moreover,
special attention must be paid to the traditional malaxers which work continuously in an air
saturated atmosphere. In fact, in this case, the longer the time at the same temperature
conditions, the greater the loss of phenolic compounds in the oil (Servili et al., 1994; Di
Giovacchino et al., 2002; Servili et al., 2004a). On the other hand, there is not a direct link in
the confined malaxer between the time of malaxation and the loss of phenolic compounds.
However, malaxation times of over 35-40 minutes do not involve an extraction yield
increase and, therefore, even though a loss in the quality of the oil is not observed, longer
periods of malaxation are negative for a correct plant management.
Time and temperature of malaxation also affect the volatile profile and, therefore, the
sensory characteristics of the resulting EVOOs (Angerosa et al. 2004; Servili et al. 2009a,
2009b). A large part of the volatile compounds, which explain the flavour of VOO, is due to
the activity of the enzymes involved in the lipoxygenase pathway (Figure 1).
This group of enzymes promotes the formation of aldehydes, alcohols at C5 and C6
saturated and unsaturated and esters. The main effect of malaxation time is the increase
of C6 and C5 carbonyl compounds, especially of trans-2-hexenal, which represent an
important contribution to the flavour of olive oil due to their low odour threshold,
whereas high temperatures of malaxation promote a fall of esters and cis-3-hexen-1-ol
and an accumulation of hexan-1-ol and trans-2-hexen-1-ol, both considered by some
authors as producing a not entirely agreeable odour (Angerosa et al., 2004; Servili et al.,
2009a).
The enzymes involved in the LOX pathway such as lipoxygenase, hydroperoxidelyase,
alcoholdehydrogenase and alcohol acyltransferase show an optimal temperature between 15
and 25 C, whereas their activity decreases after 30 C. Therefore, the malaxing process
carried out at temperatures of over 35 C can produce a reduction in the volatile compounds
generation during malaxation.

From the aforementioned considerations, it is, therefore, clear that the mixing temperature
control at levels lower than the 28-30 C represents an advisable stage in the extraction
process in order to get high quality VOO (Angerosa et al., 2001; Angerosa et al., 2004; Servili
et al., 2009a). We must also take into account that even temperatures of paste below 22 C in
the new generation of confined malaxer lead to a decrease in the solubilisation of phenolic
compounds and chlorophylls.
Considering the relationships between the volatile composition and O2 concentration of
VOO during malaxation, the results reported in literature indicate that the O2 concentration
in the pastes seems to have no effect on LPO activity during the malaxation (Table 2) (Servili
et al., 2008).
Initial O2 partial pressure in the malaxer chamber headspace (kPa)
0
a
30
b
50 100
OGLIAROLA cv.
ALDEHYDES
2-Pentenal (E)
z
291.5 (31.8)ab 343.0 (31.1)a 247.5 (11.7)b 269.5 (13.5)b
Hexanal 939.5 (9.2)a 1546.0 (200.8)b 1011.5 (27.6)a 1499.5 (16.3)b
2-Hexenal (E) 43645.0 (912.2)a 39130.0 (1054.7)b 37315.0 (233.3)b 38170.0 (1258.7)b
ALCOHOLS
1-Pentanol 28.5 (2.1)a 128.0 (6.8)b 122.5 (3.5)b 158.0 (1.4)c
2-Penten-1-ol (E) 55.5 (3.5)a 63.0 (4.6)a 50.5 (9.2)ab 38.5 (6.4)b
1-Penten-3-ol 567.0 (17)a 871.0 (4.7)b 690.0 (1.4)c 809.5 (3.5)d
1-Hexanol 8357.0 (102.6)a 9699.0 (106.1)b 11660.0 (99)c 13675.0 (63.6)d
3-Hexen-1-ol (E) 35.0 (1.2)a 41.0 (3.5)a 47.5 (2.1)b 61.5 (2.1)c
3-Hexen-1-ol (Z) 286.5 (4.9)a 434.0 (20.6)b 341.0 (11.3)c 400.5 (7.8)d
2-Hexen-1-ol (E) 7662.5 (75.7)a 8616.0 (87.9)b 9355.0 (353.6)c 9780.0 (60.8)c

CORATINA cv.
ALDEHYDES
2-Pentenal (E) 548.5 (16.3)ab 509.7 (5.8)b 636.7 (17.9)c 613.0 (51.2)ac
Hexanal 1187.0 (9.9)a 1624.3 (30)bc 1532.1 (27.3)b 1744.0 (121.2)c
2-Hexenal (E) 51565.0 (827.3)a 52900.0 (565.7)ab 54340.5 (355.7)b 53920.0 (332.1)b
ALCOHOLS
1-Pentanol 40.0 (5.7)a 54.3 (5)b 39.4 (5)a 48.0 (3.2)ab
2-Penten-1-ol (E) 87.5 (0.7)a 67.0 (0.2)b 105.8 (5.7)c 105.0 (8.3)c
1-Penten-3-ol 890.0 (2.8)a 820.0 (1.2)b 1093.5 (33.7)c 1185.0 (91.2)c
1-Hexanol 2326.0 (49.5)a 3694.2 (2)b 1788.0 (57.2)c 2170.0 (123.1)a
3-Hexen-1-ol (E) 25.5 (0.7)ab 31.6 (3.8)a 20.0 (1.9)b 21.0 (1.9)b
3-Hexen-1-ol (Z) 561.0 (4.2)a 513.6 (9.6)b 486.3 (11.1)b 498.0 (31.2)b
2-Hexen-1-ol (E) 3654.5 (30.4)a 5905.0 (321)b 3350.1 (80.5)a 4185.0 (35.6)c
Table 2. Volatile composition (g/kg) of EVOOs obtained after malaxation in different initial
atmospheric compositions (Servili et al. 2008).
a
saturated with N2; b corresponding to the air composition.
z
Data are the mean values of three
independent experiments, standard deviation is reported in brackets. Values in each row with different
letters (a-d) are significantly different from one another at p < 0.01.

In conclusion, it can be considered that, as regards the process variables adopted during
malaxation, the temperature should be set within the range of 24-27 C, both for the
traditional and for the new malaxer (i.e., the confined malaxer), while process times greater
than 35-40 minutes may result in a large loss in terms of product quality (in particular in the
traditional malaxer) without producing significant positive effects on the oil extraction yield.
Special attention must be paid to the control and regulation of oxygen percentage in contact
with the olive paste during malaxation to optimize the phenolic content as well as the
flavour of VOO. This new operating parameter can be used to act on the phenolic
concentration of the pastes and, therefore, of the oils, excluding negative collateral effects on
the volatile compound content of the product (Servili et al., 2003a; Migliorini et al., 2006;
Servili et al., 2008). In fact, the traditional Italian cvs differ with respect to their content of
phenolic substances and, because of this difference, the phenolic concentration in oil must be
optimized; this optimization can be obtained by regulating their oxidative degradation level
during malaxation. Thus, malaxation should be carried out without oxygen for the cvs with
low phenolic concentration, whereas malaxation should be carried out with controlled
supplementation of oxygen for those cvs characterized by higher phenolic concentrations
(Figure 3, Table 2).
Moreover, it must be pointed out that no additional gases, such as nitrogen or argon, are
required inside the head space in the confined malaxer to avoid the presence of oxygen. In
fact, if the malaxer is filled with crushed paste during the process, the olive tissues of pastes
naturally release carbon dioxide (CO2) (Weichmann, 1987), whereas the limited amount of
oxygen they adsorb during the crushing process will be consumed rapidly by endogenous
enzyme activities. As a consequence, the malaxer head space will be naturally saturated by
an inert gas, such as carbon dioxide (Servili et al., 2003a; Parenti et al., 2006a, 2006b; Servili
et al., 2008).
3.4.1. Use of the technological co-adjuvants in malaxation
According to the UE 2568/1991 and 1989/2003 standards, co-adjuvants can be added during
malaxation to break down emulsions and at the same time to guarantee a high oil extraction
yield. In particular, the most frequently employed co-adjuvants are micronized talc and in
some countries, although not in Europe, enzyme preparations are used.
Such enzyme preparations act on the structural colloids of the cells of not only the pulp, but
also the skin and assist the activity of the endogenous enzymes (pectinases, cellulases and
hemicellulases) resulting in an increase in the olive oil yield and in its quality (Vierhuis et
al., 2001). In particular, it has been demonstrated that enzyme preparations tend to increase
the phenolic content in VOO (Table 3). However, these effects are found only when working
with olives characterized by an early ripening (Ranalli & Serraiocco, 1996; Ranalli & De
Mattia, 1997; Vierhuis et al., 2001; Montedoro et al., 2002). In fact, the use of enzymatic
preparations in olives when they have reached an advanced stage of ripening does not lead
to any qualitative or quantitative benefit. This is due to the fact that the structural colloids in

mature olives, on which the aforementioned enzymes should act, have already been
degraded by the endogenous enzymatic activity during the ripening of the fruit (Heredia et
al., 1993). Therefore, the addition of enzymatic preparations would be useless in this case.
However, the EU regulations 2568/1991 and 1989/2003 do not allow the addition of
enzymatic preparations, whereas they authorise the use of micronized talc as co-adjuvant.
The use of talc as a co-adjuvant has been proved important to increase oil extraction yield
without any interference to the quality of VOO. The amount of talc used ranges from 0.7 to
1.5% of the weight of the olives being milled after the first 10 minutes of the process. In fact,
its addition to difficult pastes improves the paste structure and reduces emulsions. This
product acts on the olive pastes by increasing the drainage effect and, therefore, improving
the efficiency of solid-liquid separation during centrifugation of the crushed pastes (Servili
et al., 2004b). The micronized talc can be added to the pastes during malaxation (0.7-1.5% of
the paste weight) after the first 10 minutes of the trial. Several studies carried out on Italian
cultivars show that the use of the micronized talc does not involve any negative effects on
oil quality and, in some cases, its use leads to a meaningful increase in the extraction yield
(Servili et al., 2004b).

crushed
paste
malaxed
paste
malaxed paste +
enzyme preparations
3,4-DHPEA
a
2.7 0.3
a
0.7 0.1
b
1.09 0.1
c

p-HPEA 2.3 0.4
a
1.2 0.1
b
1.02 0.1
b

3,4-DHPEA-EDA 515.0 23
ab
317.0 16
c
439 16
d

p-HPEA-EDA 24.8 1.9
a
25.8 1.4
ab
29.4 0.8
c

Lignans 32.5 1.4
a
24.2 0.8
b
28.5 0.9
c

3,4-DHPEA-EA 357.0 13
a
177.0 8.0
b
218 8.0
c

Table 3. Phenolic composition of virgin olive oil (mg/kg) with and without enzymatic treatment during
malaxation (Vierhuis et al., 2001).
a
The phenolic content is the mean value of three independent experiments (standard deviation). Values
in each row bearing the same superscripts are not significantly different from one another (P < 0.05).
3.5. Olive oil extraction systems
Different extraction technologies, such as pressure and centrifugation and selective filtration
(i.e. "surface tension" or "percolation") enabling the separation of oily must from the olive
paste can be used (Boskou, 1996; Di Giovacchino et al., 1994, 1995).
3.5.1. Pressure extraction system
Pressing is one of the oldest methods of oil extraction and has evolved considerably over the
centuries. In olive oil mills equipped with this system the press separation of the oil from the
paste is currently carried out using open hydraulic presses, whereas close cage presses have
almost disappeared not only due to high purchase prices, but also to their maintenance
costs. The previously malaxed paste is subsequently stratified on stacked filter mats, each

covered with approximately 0.5 inches (1.25cm) of paste and interposed with metal disks.
This operation is carried out mechanically thanks to a dispenser, which takes the paste from
the malaxer and stores it on the nylon and/or polypropylene filter mats. Both types of filter
mats have a central hole to allow the expressed oil and water (olive juice) to exit in both
directions. From a theoretical point of view, this system guarantees intrinsic oil quality.
However, its use presents a few problems, mainly due not only to its low working capacity
per hour, in which case the storage of olives lengthens, but also to the proper use of the filter
mats and to the types of materials used to build the equipment. The critical aspects of the
process regarding the use of the press, which impacts on the quality of the oil, are concerned
with both the proper management of the filter mats and the use of construction materials
made of stainless steel. As regards the filter mats, it is important to point out that they can
represent a source of contamination, due to fact that they may introduce fermentation and
an oxidation defect into the oil, causing sensorial defects (Angerosa et al., 2004). This effect
can arise both from the contamination with oils obtained from poor batches of olives and
from fermentation processes of the vegetation water and pomace fragments, which remain
in the filter mats, when they are kept in storage during the different processing stages. The
latter problem occurs particularly when the oil harvest is interrupted by bad climatic
conditions and it is impossible to work continuously. In order to minimize the risk of defects
developing in the VOO, it would be desirable: i) to work in a continuous cycle; ii) to change
the stacked filter mats frequently during the process and to clean them periodically using a
pressure washer; iii) to store the aforementioned filter mats at a low temperature (0 C-5 C)
to avoid fermentative processes during breaks in the oil processing.
As regards the materials used to construct the press, all the metallic parts which come into
contact with the product must be made of, or at least covered with stainless steel to avoid
the transfer of metals, especially those metals which can speed up the oil oxidation, to the oil
during the extraction process.
3.5.2. Extraction by centrifugation
The majority of VOO is currently extracted by centrifugation in Mediterranean countries.
The idea of exploiting direct centrifugation of the malaxed paste to extract the oil dates back
to the late nineteenth century, when the use of the first decanter applied to food industries
was widespread. In the olive oil sector, this idea determined technological innovations in
the VOO mechanical extraction process, which were opposed to the traditional press. The
first operating patents, including the patent by Corteggiani, date back to 1956, followed by
new companies producing olive oil machines in the early sixties. This machine, called a
decanter, consists of a drum containing a cylindrical and a conical part with a horizontal
axis, inside which an additional cylinder worm is placed, which acts as a screw conveyor.
The differential speed of the latter is slower than that of the outer drum in order to
discharge the solid part. In recent years, this extraction system has evolved considerably in
order to reduce the amount of water used during the process. In fact, the decanters can be
classified as follows:

1. Traditional three-phases decanters, featuring water addition ranging from 0.5 to 1
m
3
/ton.
2. Two-phases decanters, which can operate without the addition of water and do not
produce vegetation water as a by-product of the extraction oil process.
3. New three-phases decanters, working at low water consumption ranging from 0.2 to 0.3
m
3
/ton.
The traditional three-phases decanters, which allow the oil to be separated both from the
vegetation water and from the pomace, feature a humidity level of between 50% and 55%
and dilute the pastes produced to reduce their viscosity. In doing so, they facilitate
separation of the oil-vegetation water with a dilution ratio ranging from 1:0.5 to 1:1 (from 50
to 100 l of water for every 100 Kg of paste to be decanted). In addition to the enormous
amounts of vegetation water which have to be drained, this implies a decrease in the oil
quality, principally due to the washing away of the phenolic compounds of the product,
with massive decreases in this important antioxidant fraction (Ranalli & Angerosa, 1996;
Servili et al., 1999c, 1999d; Stefanoudakii et al., 1999; Di Giovacchino et al., 2001; Servili &
Esposto, 2004).
The evolution of this technology has lead to the production of two and three-phases
decanters with low water consumption. By using these new systems, the extracted oils
feature a higher phenolic concentration than those extracted by means of the traditional
centrifugation process, because the loss of these hydrophilic phenolic compounds in the
vegetation water is reduced (Table 4) (Servili et al., 1999d). In this context, it is important to
point out how these new extraction systems, which do not take into account the addition of
water, enable high quality VOOs to be obtained. The focus of this problem consists in
linking together the low processing temperatures with a reduced use of water dilution in the
pastes: by using centrifugation systems, these two process variables should allow high

Coratina cv. Ogliarola cv.
Phenolic
compounds
two phases three phases

two phases three phases
3,4 DHPEA 0.87 0.02 0.58 0.08
b
0.66 0.11
a
0.50 0.11
a

p-HPEA 3.74 0.07
a
2.34 0.08
b
3.30 0.10
a
4.22 0.10
b

Vanillic acid 0.41 0.01
a
0.19 0.01
b
0.26 0.01
a
0.14 0.05
b

Caffeic acid 0.16 0.01
a
0.12 0.02
b
0.09 0.01
a
0.21 0.03
b

3,4 DHPEA-EDA 522.2 13.5
a
427.2 13.8
b
30.09 1.03
a
18.53 0.68
b

p-HPEA-EDA 78.16 0.52
a
67.26 2.55
b
20.99 0.82
a
22.40 0.33
a

Lignans 38.41 0.10
a
35.62 1.11
b
48.00 3.40
a
46.72 5.78
a

3,4 DHPEA-EA 351.7 11.0
a
244.9 13.6
b
68.01 6.00
a
52.04 3.11
b

Total polyphenols 673 4
a
585 7
b
304 5
a
263 4
b

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

Table 4. Effect of water reduction during centrifugation on the phenolic composition of virgin olive oil
(mg/kg) (Servili et al., 2002).
Data are the mean values of three independent experiments standard deviation. Values in each row,
with cvs with different letters, are significantly different from one another (p < 0,01).

quality oils and machines featuring high yields to be obtained (Ranalli & Angerosa, 1996;
Servili et al., 1999d; Stefanoudakii et al., 1999; Di Giovacchino et al., 2001).
As regards the by-products obtained by centrifugation, it is important to recall here that,
whereas the aforementioned problem concerning the use of three-phases traditional systems
is represented by the enormous amounts of vegetation water to be drained (0.7-1.2 m
3
/ton),
the main problem, when using the two-phases systems, is not only a decrease in oil quality,
but also the high humidity level of the pomaces (50% for the traditional three-phases
decanters, 55-60% for the two-phases). This last aspect implies two disadvantages: i) where
to store by-products of the extraction process; ii) how to transport to the pomace oil factory
and their subsequent use for residual oil recovery by solvent extraction.
The pomaces produced by two-phases system are generally employed for the production of
compost or for spreading to improve agriculture soil, following a process similar to that
used for the vegetation water of olives. In this context, three-phases decanters with low
water consumption can represent an adequate alternative with respect to the two-phases
extraction system, because they allow a quality of oil to be obtained which is comparable to
that of oils obtained using the two-phases system, and pomaces with a reduced humidity
content, similar to those produced by the traditional three-phases systems. On the contrary,
they produce a certain amount of vegetation waters, which imply water draining
procedures which comply with the regulations of the law.
3.6. Separation of the oil from vegetation water
The liquid coming from the extraction system is called oily must, and consists of oil and
vegetation water, which is separated by using vertical centrifuges. The oily must also
contains solid particles and mucilage (0.5 to 1.0%). These substances are suspended if they
are very small, whereas they are easily separated from the liquid if they are of an
appreciable size (seed fragments and/or epicarp of olive fruit fragments). In particular,
separation from the liquid is carried out by using a sieve (1~2 mm mesh) placed at the top of
the olive oil storage tanks.
Disk stack centrifuges, suitable for separating solid impurities with a specific weight
ranging from 1.050 to 1.150, are used to separate the oil from the oily must. The basic
principles of centrifugation are well known. If a vigorous rotational motion is applied to the
oily must (oil, vegetation water and impurities), the lighter part (i.e. the oil) is collected close
to the axis of rotation, while the heavier part (i.e. the water) is collected further away.
Finally, the impurities are collected even further away.
Moreover, spillways enable the oil as well as the vegetation waters and impurities to be
recovered. A fraction of the impurities is deposited on the rotating drum, which must be
periodically cleaned, even though self-cleaning decanters are widely used. In fact, these
decanters work in a continuous cycle, providing a periodical and automatic discharge of the
sediment, which could compromise the centrifugation process. The decanters most

frequently employed in the oil mills consist of a series of perforated, truncated cone-shaped
disks, mounted on the hollow shaft-mounted drum in order to leave a free space of
approximately 1 mm between the disks.
Centrifugal force forces the oily must poured in from the top through the hollow drum shaft
upwards and it is divided into three different layers of oil, vegetation water and impurities,
according to their specific weight. The drum diameter of decanters used in the oil mills
ranges from 400 to 700 mm, with a rotational velocity ranging from 5000 to 12000 rpm. The
working capacity of these machines in terms of litres of oily must poured in per hour is very
high and it varies between 500 and 2000 l/h. The work carried out by the vertical centrifuges
is qualitatively satisfactory, even though there is often a loss of oil in the vegetation waters.
This loss cannot exceed 500 g/ton of processed olives if centrifugation process is to be
considered adequate.
3.7. Olive oil storage
During storage, the phenolic composition of EVOO is modified by the endogenous
enzymatic activities contained in the cloudy phase. These enzymes may reduce the
pungent and bitter sensory notes, the intensity of which is strictly linked to the
content of aglycon secoiridoids, and, at the same time, can produce olfactory and taste
defects. Oil filtration partially removes the water and enzymes from EVOOs, and enables
the EVOO phenolic content to stabilize during its storage. The filtration process of EVOO
is a procedure carried out in two steps: first, the suspended solids are removed, and
second, the elimination of humidity gives the oil a brilliant aspect. Normally, organic or
inorganic materials are used in conjunction with a variety of filtration equipment to
enhance or enable the separation of suspended solids and water-oil. The type of such
equipment, often called filter aids, depends on the final objective (Montedoro et al.,
2005).
The olive oil profile changes during its storage, due to the simultaneous, drastic reduction in
compounds from the LOX pathway and to the neo-formation of volatile compounds,
responsible for some common defects referred to as rancid, cucumber and muddy
sediment (Morales & Aparicio, 1997; Angerosa et al., 2004; Servili et al,. 2009a).
This runs parallel to the increase in saturated aldehydes nonanal, and above all hexanal in
the oxidation process, but it cannot be considered a useful marker of oxidation, since it is
also present in the aroma of high quality EVOOs (Angerosa et al., 2004; Servili et al.,
2009a).
Furthermore, the presence of sediment as a result of the decantation of unfiltered olive oil
during its storage can determine, under suitable temperature conditions, the production of
unpleasant compounds responsible for the typical muddy sediment defect due to the
fermentation which produces compounds, probably of the butyric type (Angerosa et al.,
2004; Servili et al., 2009a).

Author details
Maurizio Servili, Agnese Taticchi, Sonia Esposto, Beatrice Sordini and Stefania Urbani
University of Perugia, Faculty of Agriculture, Perugia, Italy
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Chapter 9
Olive Mill By-Products Management
Pietro Toscano and Francesco Montemurro
1. Introduction
Among the Mediterranean countries, Italy results to be the second after the Spain for the
olive and olive oil production (FAOSTAT, 2012), with around 1.2 million of olive grove
hectares, by 80% displaced in the Italian southern regions (ISTAT, 2010). In the olive oil
industry, the oil extraction is carried out in oil mills, which are classified in pressure mills,
and in continuous two or three phases way mills. In all milling typologies, only not
over the 20% of processed olives constitute the oil production, while the milling by-
products, wastewater and pomace, represents up to 120% of processed olives. These wastes
could constitute a problem for their sustainable disposal as well as a resource for soil C
stabilization and sequestration, energy generation or production of value-add compounds
for the food, pharmaceutical or cosmetic industries.
A great part of husks derived by pressure and three phases mills is still destined to the
industry for the extraction of residual oil through solvents (n-exane: CH3(CH2)4CH3). The
residual defatted pomace is used as fuel in cogenerative processes (heat and electric power
generation) or, as well as the two phases mill husks (fluid pomace) and olive mill
wastewaters, disposed on soil as amendments, both raw and after composting. In the
Mediterranean countries, where soils have frequently problems of organic matter lack, and
active desertification processes, the recycle of the olive wastes as amendments should be
better and more important to protect the environment being a valid alternative and a useful
solution to the problems both of sustainable utilization of byproducts and soil fertility
conservation.
In many experimental trials carried out in many sites, the utilization of olive industry
byproducts as organic amendments, raw or stabilized through the aerobic fermentation,
frequently showed good agronomic efficiency, in terms of fertility and chemical, physic and
microbiological characteristics of the soils as well as crops productivity; generally pointing
out some negative effects of fermentable organic matter and better findings using stabilized
wastes.

Althought in literature can be found numerous findings on the influence of raw and
composted organic materials on soil fertility and crops growth, only few published studies
are focused on influences of waste waters and pomace compost on the soil-plant system, for
a sustainable crops production.
In this chapter on report the state of the art of the by-products composting, and results of
some application of raw and composted olive industry by-products on soil and crops.
2. Olive mill wastes
The olive mill byproducts are classified, according to the different systems of olive oil
extraction, as: Olive mill Waste Waters (WW); Virgin Olive Pomace (OP), by pressure mills
(OPP), with around 30% moisture; by centrifugal "three phases" mills (OP3), with around
50% moisture; or by centrifugal "two phases" mills (OP2), with moisture more than 60%. The
Solid Defatted Pomace (SDP), is the byproduct of the pomace industry, after the extraction
with solvents of the residual oil from virgin pomace.
2.1. Waste waters characteristics and treatments
The waste waters constitute the liquid fraction of the olive mill byproducts and are
composed by the olives water content and by the waters used in the milling (olives and
equipments washing waters and the waters of dilution process in the three phases
centrifugal systems). As reported in Table 1, the different extraction systems produce, from
100 kg of processed olives, around 10 liters of waste waters for the two phases system,
around 55 liters for the pressure crushers, and more than 100 litres for the continuous three
phases system, that they reduce to only 35 litres in case of recycle of water.

Milling technology H2O
added (%)
Pomace
(kg/100 kg olive)
Pomace
moisture (%)
Waste waters
(kg/100 kg olive)
3 phase continuous 50 55-57 48-54 80-110
Pressure 0-10 30-35 25-30 56-58
2 phase 0-10 75-80 60-70 10
3 phase with water
recycle
0-20 56-60 50-52 33-35
Table 1. Mass balance in the olive oil extraction
The main characteristic of WW is the presence of organic compounds such as organic acids,
lipids, alcohols and polyphenols that turn it into phytotoxic materials, that might have
unfavourable impact on plants (Capasso et al., 1992; Kavdir & Killi, 2008). It also represents
a great environmental hazard if not properly managed. However, WW contain valuable
resources, such as a high organic matter concentration and some nutrients, especially
potassium, that could be usefully used to improve the physic-chemical and biological
properties and then soil fertility and productivity; representing a valid option to close the

Olive Mill By-Products Management 175
residue-resource cycle (Roig et al., 2006). In Table 2 are reported the averages of main
chemical WW characteristics, given by several Authors (Aktas et al., 2001; Filidei et al., 2003;
Moreno et al., 1987; Paredes et al., 1999; Piperidou et al., 2000; Saviozzi et al., 1991; Vlyssides
et al., 1996; Vlyssides et al., 2004 as cited in Roig et al., 2006).

Dry matter (%) 6.75
pH 4.84
EC (dS/m) 8.36
O.M. (g l
-1
) 55.80
TOC (g l
-1
) 37.00
TN (g l
-1
) 0.97
P2O5 (g l
-1
) 0.56
K2O (g l
-1
) 4.82
Na (g l
-1
) 0.25
Ca (g l
-1
) 0.35
Mg (mg l
-1
) 121.25
Fe (mg l
-1
) 81.70
Cu (mg l
-1
) 3.15
Mn (mg l
-1
) 6.13
Zn (mg l
-1
) 6.13
d (g cm
3

-1
) 1.04
Lipids (g l
-1
) 6.38
Poliphenols (g l
-1
) 4.98
Carbohydrates (g l
-1
) 7.16
COD (g l
-1
) 124.67
BOD5 (g l
-1
) 65.00
Table 2. Average composition of olive mill waste waters.
About the microbiological characterization, the results of different analyses performed on
different kind of WW, have individualized 130 species of lipolytic microorganisms (56
Fungi, 22 Yeasts, and 52 Bacteria), cellulolytic Bacteria and pectinolytic Fungi, while not are
resulting the nitrificants nor the actinomycetes (Pacifico, 1989; Ramos-Cormenzana, 1986).
For the waste waters treatment have been proposed both physic-chemical and biological
processes (Amirante & Di Rienzo, 1993; McNamara et al., 2008; Rozzi & Malpei, 1996; Vigo
et al., 1990; Vitolo et al., 1999).
Among the first ones, finalized to the volumes reduction, and to the mineralization of
organic compounds, they are:
- distillation, filtering or flocculation and disposal in the water bodies;
- evaporation and agronomic utilization of concentrated material;
- raw application on the soil;

- adoption of two phases mills, and management of the fluid pomaces.
The biological treatments of purification include both aerobic and anaerobic processes. The
aerobic biological treatments are based on the microbic degradative activity that transform the
decomposable organic matter in not pollutants mineral elements and humus-like substances.
The anaerobic processes are characterized by microbial pools that works in absence of
oxygen, converting the organic polluting substances in biogas (methane) and carbon dioxide
or in hydrogenated volatile substances (fatty acids and alcohols), (Filidei et al., 2003). Three
main physiological groups of microorganisms are involved: fermenting bacteria, organic
acid oxidizing bacteria, and methanogenic archaea. Microorganisms degrade organic matter
with a sequence of biochemical conversions to methane and carbon dioxide. Syntrophic
relationships between hydrogen producers (acetogens) and hydrogen scavengers
(homoacetogens, hydrogenotrophic methanogens, etc.) are critical to the process. A wide
variety of process applications for biomethanation of wastewaters, slurries, and solid waste
have been developed. They vary from the simple WW open-air storage to the treatment in
Completely Stirred Tank Reactor (CSTR) through co-digestion with others organic matrixes,
up to treatments in special digesters, as UASB reactors (Up-flow Anaerobic Sludge Blanket)
and with different process conditions (retention times, loading rates, temperatures, etc.) to
maximize the biogas production (Angelidaki et al., 2011).
Even if these treatments are able to demolish the WW polluting power, in the practice they are
often not economically sustainable. The seasonality of WW production and the characteristics
of biological toxicity, make difficult the management of their treatment in the purification
installations of the urban waters. Conversely, the relative low quantity produced by single
milling plants do not make economic WW purification in specific installations.
The WW open-air storage is the simplest and economic system of storage and treatment.
During the open-air storage by the action of aerobes and anaerobes microorganisms, it will
be partially purified and stabilized. However, the WW open-air storage require ample
surfaces away from inhabited centers, for the stink development, and exposes to risks of soil
and subsoil water contamination, if the basins are not correctly waterproofed (Catalano et
al., 1985).
In fact, these wastes can constitute a secondary raw materials, to be consider as a resource,
being of a vegetable origin, which have not undergone chemical manipulations nor received
additives, and therefore without pathogenic microorganisms and viruses, as well as pollutants
or toxic products. The optimal operative option results therefore to be the agronomic
utilization of the WW that, when correctly managed, allows to exploit its fertilizing
characteristics with low costs of management, reducing the risks of environmental pollution.
Many studies have been conducted on soil application both on olive orchard and
herbaceous crops, studying differing time and doses of WW application. The results showed
both the agronomic benefits and the limitations of raw WW disposal. When correctly
managed, the WW application on soil generally results increasing the organic matter,
phosphorus and potassium content; improving both physical and hydraulic soil properties

as well as the crop yield (Alianiello et al., 1998; Andrich et al., 1992; A.R.S.S.A., 2001; Ben
Rouina et al., 1999; Bonari et al., 1993; Catalano et al., 1985; Di Giovacchino & Seghetti, 1990;
Di Giovacchino et al., 2001; Levi-Minzi et al., 1992; Montemurro et al., 2007, 2011; Saviozzi et
al., 1991; Tamburino et al., 1999; Tomati & Galli, 1992).
Another option concerns WW composting through biological processes of aerobic
stabilization, on supporting matrixes with adequate physical and mechanical properties
(porosity, structure, texture), to allow the reactions of bio-oxidation in the solid phase. In
this way it is possible to obtain a not phytotoxic organic material, humus-like partially
transformed, that could be used in fertilization plans to restore or maintain soil fertility on
partial or total substitution of mineral fertilizers (Benitez et al., 1997; Filippi et al., 2002; Galli
et al., 1997; Paredes et al., 2002; Tomati et al., 1995; Vallini et al., 2001).
2.1.1. Waste Waters agronomical value
The WW contain appreciable amount of mineral elements and organic substances, and can
be considered as liquid amendments by vegetal origin. WW application to the soil could
realize the double purpose to allow a natural chemical and biological degradation and to
enrich the soil in organic matter and in mineral elements. This means that the agronomical
use of these waters represents one of the most effective systems to decrease their BOD5. For
this reason, the elevated values of WW BOD5 and COD that make extremely risky WW
disposal in surface or ground water bodies, would come to lose importance in the case of
soil distribution. The WW poliphenols, held substances of elevated polluting power in the
case of disposal in the surface and ground water bodies, do not represent a pollution factor
for the soil, being instead precursory in the synthesis of humic substances that represent the
most active fractions of soil organic matter with important role in soil organic fertility and
protection from synthetic organic contaminating, as pesticides and heavy metals.
For this, the most effective option appears to be the use of WW as liquid organic amendment
or fertigant, and the WW recycle in soil could be a valid alternative to other depurative
treatments. In fact, WW affect positively crop performances, of olive, grapevine and cereals,
when distributed in dates and doses selected through rigorous agronomical criteria.
On the contrary, an incorrect WW management, may cause temporary immobilisation of
soil mineral nitrogen and, consequently, crop yield reduction, due to the deficiency of N
uptake by the plants; also increasing environmental pollution risks. To avoid these effects,
the maximum amount of waste water that can be applied is restricted by the Italian law to
50 and 80 m
3
ha
-1
yr
-1
, for the pressure and continuous milling systems, respectively.
Phytotoxic effects can also be avoided applying simple and few expensive WW
pretreatment before the application to the soil, using biological or mineral catalysts, or
submitting them to suitable composting processes, mixed with other organic by-products.
The stabilization of WW through composting on effect with repeated saturation of
supporting materials with suitable physical-mechanics characteristics (porosity, structure,
texture) as straws, leaves, sawdust, rapiers, or pomace, to maintain a moisture level useful

to the bioxidative reactions (Benitez et al., 1997; Garcia-Gomez et al., 2003; Paredes et al.,
2000; Tomati et al., 1995; Vallini et al., 2001).
In this way, on allow on the WW the bioxidative reactions in solid phase, handling to a final
product, without phytotoxic effects, useful in the agricultural soils as organic amendment.
2.1.2. Waste Water effects on the herbaceous crops
The effects of the WW distribution on soils destined to herbaceous crops, vary according to
the WW composition, the quantity, the distribution dates in relationship to seeding or crop
phenologic stage, the rainfall after the WW supply.
The olive mill WW do not generally affect the productivity of the spring-summer crops,
when the shedding is effected with an adequate interval time before the seeding. It is also
possible to distribute the WW with crop in action, i.e. on autumn-winters cereals, in the full
growth phase, limiting the WW doses to not over 40 m
3
ha
-1
(Bonari et al., 1993; Di
Giovacchino & Seghetti, 1990; Di Giovacchino et al., 2001; Marsilio et al., 1990; Montemurro
et al., 2007, 2011a; Roig et al., 2006).
Negative effects on grass crops are due to the elevated values of electrical conductivity, that
can induce salinity damages, flocculation of soil clay fraction, the phytotoxicity of
poliphenols (Della Monica et al., 1978, 1979).
In the last years, because of environmental protection policy, new technologies that
minimize these risks were developed. In this framework, the raw olive waste water was
differently treated to improve the percentage of recycle of these materials. In particular,
different studies indicate that the application of mineral catalyser (MnOX) on WW reduces
the level of poliphenols and other pollutants. As a consequence, this treatment decreases
both phytotoxicity and temporary immobilization of soil mineral N, thus making the treated
WW able to sustain good levels of crops yield and products quality (Montemurro et al.,
2007; Vigo et al, 1990).
2.1.3. Waste Water effects on the olive crop
Many experiences have confirmed the possibility to distribute the WW on the olive grove
more than over 400 m
3
ha
-1
without significant variations of vital parameters as the
photosynthetic activity, transpiration, stomatic conductance, leaves carbohydrates and
chlorophylls content. Conversely, improvements of the productivity on treated plants was
frequently observed. A decrease of the vegetative development has been recorded only on
young plants raised in pots and treated with higher WW doses (Proietti et al., 1988).
The organic matter content in WW treated soil always results greater in comparison to the
untreated control, with greater nitrogen contents and more elevated C/N ratio.
In the soils treated with WW a greater presence of the total microflora was found (Fungi and
other microbial groups), as well as an increase of respiratory and enzymatic activities. These
findings showed the absence of toxicity of the WW towards the microorganisms, and the

improvement of soil fertility. Moreover, the findings of different researches indicate the
increase in organic matter and nutrient contents, an improvement in the aggregate stability
and, as a consequence, a better physical soil properties.
In order to the possible ground water pollution, other results have shown that also up to 500
m
3
ha
-1
of WW application do not represent a pollution danger of the surface water in the
clay soils. (Andrich et al., 1992; Ben Rouina et al., 1999; Briccoli-Bati & Lombardo, 1990;
Lombardo et al., 1995; Palliotti & Proietti, 1992).
2.2. Pomace characteristics and treatments
The olive pomace is composed of fruit matter (olive skins, flesh, seeds and stone fragments),
and of different amount of vegetation and process water which contains the water-soluble
constituents of the fruits, in order to the extraction system used.
The OPP and OP3 are usually destined to the pomace industry, for the extraction of residual
oil by solvents; and then used as fuel, also in cogenerative processes (Molinari & Bonf,
2005). These by-products could also be used as animal food, or in biodegradative processes to
produce ethanol (Ballesteros et al., 2002), or compost for agricultural utilization. Nevertheless
the olive pomace, being constituted by vegetable not fermented organic matter, does not
contain heavy metals, toxic pollutants or pathogens, and can be considered as a vegetable
amendment (Table 3), (Alburquerque, et al. 2004). Therefore, it can be used in the agricultural
soils without any treatment, as allowed by the current normative (Law 574/1996).

Parameters Range
Humidity % 55 - 75
pH (H2O) 4.8 6.5
EC (dS m
-1
) 0.9 4.7
Ash (g kg
-1
) 24 - 151
TOC (g kg
-1
) 495 - 539
C/N ratio 28 - 73
Total N (g kg
-1
) 7 - 18
P(g kg
-1
) 0.7 2.2
K(g kg
-1
) 7.7 29.7
Ca(g kg
-1
) 1.7 9.2
Mg(g kg
-1
) 0.7 3.8
Na(g kg
-1
) 0.5 1.6
Fe (mg kg
-1
) 78 - 1462
Cu(mg kg
-1
) 12 - 29
Mn(mg kg
-1
) 5 - 39
Zn(mg kg
-1
) 10 - 37
Table 3. Main characteristics of the olive pomace (dry weight)

2.2.1. Pomace agronomical value
On the contrary to the WW, no many experiences was carried out on the OP application,
because both OP3 and OPP represent a better incoming value when destined to the pomace
industry and energetic production. The results of OP application both in olive orchard and
herbaceous crop, was different, depending both from doses applied and crop. Against to a
remarkable improvement in chemical-physics characteristics of olive orchard soil up to two
years after the pomace supply, has no given significant differences in vegetative and
productive parameters of treated olive trees. In herbaceous crop a phytotoxic effect was
observed on plant growth with highest doses applied, showing that the fresh organic matter
distributed in soil will be degraded producing intermediate metabolites which are not
compatible with normal plant growth and nutrients availability, with in some cases an
increase in the fungal disease (Bing et al., 1994; Bonanomi et al., 2006; Brunetti et al., 2005; Di
Giovacchino et al., 2004; Diacono & Montemurro, 2010; Tejada & Gonzales, 2003, 2004).
In the practice, the benefits of olive pomaces recycling in soil occur only if were applied
according to best agronomical practices, as taking into account suitable time and specific
plan of fertilization, needs of the soil-plant system and the climatic conditions.
Nevertheless, the application of raw olive pomace involves a more difficult management of
these biomasses and, also for possible negative aspects, a better management solution is to
compost the pomace, as we can see later in this paper.
2.3. Olive mill wastes legislation
According to their nature, the extraction methods and the different phases of management,
the olive mill by products have to follow different laws concerning storage, treatment,
transport, application to the soil, or other destinations.
2.3.1. Waste waters legislation
The WW legislation is based on their two possible different destinations: agronomic or
landfill. In case of agronomic use without any preventive treatment, the reference is the Law
n. 574/1996. As established by the Decree of the Ministry of the environment, on the
"Individuation of non dangerous wastes submitted to the simplified recovery procedures to
the senses of the articles 31 and 33 of the Legislative Decree 05.02.1997 n. 22", the WW can
also be recovered through the "Production of fluid fertilizers, as by the Law n. 748 of
19.10.1984: "New norms for the fertilizers discipline".
If the WW cannot be used in agronomical purposes, it is necessary to apply preventive
treatment. The products, resulting from the purification process, can be schematized as
follows:
- for the liquid phase, the reference is the Law n. 319, 10.05.1976 (Merli law) with his
modifications and integrations, and the Legislative Decree n. 22/1997 (Ronchi Decree)
and his modifications and integrations, in which the limits of acceptability are

established also for the discharge in surface and ground water, on the soils, or in the
subsoils (L.D. 389 of 8.11.1997).
- for the solid phase, the reference, related both to the treatment and discharge phases, is
constituted by the Legislative Decree 27.01.1992, n. 99, "Realization of the directive
86/278/CEE regarding the environment protection, particularly of the soil, in the use of
the solid phase purification in agriculture", and the Law 19.01.1984, n. 748 with his
modifications and integrations "New norms for the discipline of the fertilizers." The art.
8, par. 1, letter d of the "Ronchi Decree" establishes that WW are excluded by
application, because disciplined by specific dispositions of law "the activities of
byproducts treatment that give origin to the fertilizers, selected in reference to the
typology and the mode of utilization as by Law 19.10.1984, n. 748 with his modifications
and integrations.
Another possible destination of the WW is the agronomic use after an a intermediary phase
for production of liquid fertilizers, conforming to the Law 748 19.10.1984, New norms for
the discipline of the fertilizers", according to the norms of the D.M. 05.02.1998 of the
Ministry of the environment. As a consequence, WW must be recovered according to the
point R3 of the Annex C of Legislative Decree n. 22/1997, article 6, paragraph 1, letter h,
("Recycle/recover of the organic substances not used as solvents, included the composting
operations and other biological transformations").
2.3.2. Pomace legislation
The OP storage is disciplined by the Legislative Decree n. 22/1997 with his modifications
and integrations, according to which it should be considered as not dangerous special
wastes that (ex D.M. 05.02.1998 of the Ministry of the environment) can be submitted to the
simplified recovery procedures according to the articles 31 and 33 of the Legislative Decree
n. 22/1997 with his modifications and integrations.
About the treatments, in case of distribution on the soil, also the OP2 can be considered as
OP ex Law n. 574/1996, for which is not provide any preventive treatment for the agronomic
use, because: "To the goals of the application of the present law, the OP2 coming from the
olives milling and constituted by the waters and the fibrous part of fruit and the core
fragments, can be used as amendments in derogates to the characteristics established by the
Law 19.10.1984, n. 748 and modifications". As D.M. 05.02.1998 of the Ministry of the
environment, (non dangerous wastes that can be submitted to the simplified recovery
procedures as the articles 31 and 33 of the Legislative Decree n. 22/1997 with his
modifications and integrations) the pomace could be submitted to processes of anaerobic
biostabilization, with production of biogas. This material could also be submitted to the
aerobic processes, for the compost production in conformity with the annex to the Law
19.10.1984, n. 748 with modifications.
Other destinations of the OP, with exclusion of the OP2 for elevated moisture, is the
conferment to the pomace industry for the extraction with solvents of the residual oil. The
defatted pomace that resulted as byproduct of this process, are used as fuel, or as organic
amendments after aerobic co-composting.

3. Biological treatments
With this definition, are indicated the processes and the activities on organic materials that,
degraded and transformed by various decomposers microorganisms, allow its stabilization
in terms of mineralization of the mostly degradable components, and the hygienization of
the biomass by pasteurization.
The biological treatment of byproducts can be realized with different technologies and
processes, referable to three following typologies:
- composting of quality, on selected biomasses, for the production of amendments of
high agronomic value, according to the parameters defined by the Law 748/84 on the
fertilizers;
- biostabilization of organic matrixes of low quality (as organic fractions from mechanical
separation of the undiversified wastes residues, sludges with heavy metals, etc.);
- anaerobic digestion for production of biogas, both on high quality organic matrixes for
a successive agronomic utilization, and on lower quality or contaminated organic
residuals. The byproduct (digestate) can be afterward used as fuel, aerobically
stabilized, or arranged as sludge according to the Legislative Decree 99/1992 on the
sludge usage in agriculture.
4. Composting
The composting is a controlled process, based on the control of the natural bioxidative
process on organic substances (Figure 1) transformed by aerobes microorganisms naturally
present in the environment and in the matrixes, in a stabilized and sanitized organic
substance, defined compost (Casacchia et al., 2011; Insam & De Bertoldi, 2007).
The composting is classified by the law (Legislative Decree 22/97) as a recovery operation of
byproducts, among the actions of The recycle and/or retrieve of the organic substances not
used as solvents; being an interesting alternative in recycling large amounts of a wide
range of the residues produced in Mediterranean areas, resulting in an organic fertilizer
suitable to agricultural purposes, in improvement of o. m. content of soils, and reduction of
fertilization costs.
The composting process is usually divided in two phases: Active Composting Time (ACT),
and Curing.
During the ACT, there is an intense develop of degradative processes on to more easily
fermentescible organic components. In this thermophylic phase the biomass reaches
temperatures up to 65 C, that also allow its hygienization. The ACT can develop, according
to the characteristics of the matrix, in piles, with or without overturn and with or without
forced airing, or in more complex systems (bioreactors), and varying from 21-28 days in the
first case or 14-16 days in the second one.
During the curing phase, the degradative phenomena will be completed, and the synthesis
of the humic-like substances occurred. This process, which has a duration of more than 45
days, need a smaller oxygen and drain of heat.

The main parameters of the composting process, that ensure an optimal development of
microorganism able to transform the organic matrices, are synthetically reported in Table 4.

pH Should be between 5.5 - 8, to assure the activity of useful microbes.
Humidity % Range variable from 50-60% in the first weeks (ACT); to go down to
30-35% to the end of process, depending on the biomass characteristics
and the processing typology.
Temperature Range to develop microorganism: 40-45 C (3 days over 55 C for the
Italian legislation, to kill pathogens)
C/N ratio Nutrient balance: between 25 and 35 to avoid nitrogen losses for
volatilization (< 25) or decrease speed of metabolic reactions (> 35)
Porosity To ensure aerobic process; depending on particle size.
The better air-filled pore space: 25-50%
Particle size Surface air for microbial growth
O2 concentration Assured by forced airing and/or overturns. Optimal range for
degradative process: 15-20%
Table 4. Controllable composting parameters.
During the composting, the organic substance evolves both quantitatively, reducing weight
and volume, and qualitatively, with deep modifications of its chemical and structural
characteristics. The parameters that defines the composted materials are:
- stability, that characterize a specific stage of decomposition or state of organic matter
during composting, which is related to the type of residual organic compounds and the
resultant biological activity in the material. The specific stage of organic matter
represents the exhaustion of degradative processes due to the biological metabolic
activity (low static and/or dynamic respiration index).
- maturity, is the degree of composting completeness and concerns improved qualities
resulting from ageing or curing of a composted matter. Usually the level of
maturity is detected by the phytotoxicity, verifiable with the homonym test.
- humification, or the endowment of humic-like molecules originated from reactions on
the less fermentescible organic components. (Boulter et al., 2000; De Nobili & Petrussi,
1988; Franz et al., 2005; IPLA, 1992; Zucconi et al., 1981).
In order to the duration of the fermentative process, two typologies of compost can be
obtained, based on their degree of maturation:
- the ready-to-use compost, of greater fertilizing efficiency, that is obtained in 3-4 months,
at ACT end, that can be used for vegetable crops, gardens and in full field, on preseed
and before the transplantation;
- the mature compost, more stabile and humified, that is obtained after 5-6 or more
months of curing; usable as amendment in full field, or as substrate in the greenhouse
nursery.


Figure 1. Composting process flow.
The composting of the agro industrial by-products and agricultural residual biomasses can
be carried out according to two typologies:
- composting of crop residuals and animal manure by farmers; in this case the farmer
that compost in situ produced biomasses, is not subject to any restriction, provided that
the compost is used in the same firm;
- composting of extra farm biomasses; this kind of activity is not considered as normal
agricultural practice, but solid wastes recycling. According to the prevailing
interpretation, the extra-farm solid wastes included in the compostable materials list of
the D.M. 05.02.1998, are subjected to the "simplified procedure", as from artts. 31 and 33
of the Legislative Decree 22/97 (Ronchi decree).
The on-farm composting does not require particular technologies, while the necessary
machineries (carver, shovel loader, wagon mixer) are often already present in farm.
The biomasses to be composted, mixed with opportune percentages of appropriate
structural and absorbent fractions (lignocelluloses bulking), are typically placed in pile on
paved stage, preferably covered, and periodically overturned up to stabilization.
The shape and the dimensions of the pile will depend from the quantity and the quality of
the material to be treated. The forms are of triangular, parabolic or trapezoidal section, of 1.5
- 1.8 m height, while width and length depending from the quantity of material or from the
available space, and also in base to the machinery used for the overturns (hauled or self
moving over-turner, shovel loader).
During the composting, must be constantly monitored temperature and moisture of the pile,
taking care of maintain the porosity, to allow the aeration and thermic dispersion of
biomass.
According to the moisture and granulometry of biomass, the overturns will have to take
place to intervals of 2-3 days, in case of high moisture or low percentage of structural

fraction (bulking); while greater intervals (up to 10-15 days) can be applied on more porous
or coarse matrixes, and in the curing phase of compost.
The composting practices contemplate several operative options, in relationship to the
different typologies of biomasses to be treated, and to the different managerial and
environmental situations: from the simplest systems of aerobic stabilization on natural
conditions, up to the industrial plants with the complete control of the process conditions
(Alfano et al., 2008; Amirante & Montel, 1999; Baeta-Hall et al., 2005; Calvet et al., 1985; De
Bertoldi et al., 1983; Diaz et al., 2007; Madejon et al., 1998; Montemurro et al., 2009; Tomati et
al., 1996; Veronesi & Zampighi, 1997).
Although the OP is characterized from inadequate physical characteristics, in particular the
OP2, that makes difficult the aerobic degradation, this by-product can be easily composted
by using complementary residues such as pruning residues or cereal straw as bulking
agents; also mixed with other agricultural wastes as animal manures or horticultural
residues. During the process, the potentially dangerous organic substances in the olive
wastes are degraded, allowing the safe and useful use of the obtained compost in
agricultural fields; and valorizing these byproducts as a resource.
4.1. Composting normative
The composting sector is disciplined by the two following normatives: the wastes
normative, that defines the conditions of production of the compost from wastes or selected
fractions, and the fertilizers one that establish the marketing conditions of the compost.
- Wastes Normative: Legislative Decree n. 22, 05.02.1997 with modifications, and
Technical Normative of realization of the Legislative Decree 22/97, and the D.M.
05.02.1998: Individuation of non-dangerous wastes submitted to the simplified retrieving
procedures to the senses of the artts. 31 and 33 of the Legislative Decree 22/97.
- Fertilizers Normative: Law 748/84 (Annex 1C) and modifications and integrations.
The Legislative Decree 22/97, which follows the CEE/91/156 wastes directive, classifies the
composting among the recovery operations of wastes (Annex "C", R3: Recycle/recovery of
the organic substances not used as solvents, included the composting operations and other
biological transformations).
With the Technical Normative, the D. M. 05.02.1998, have been defined the technical aspects
tied up to the compost production, even if limited to the activities of recovery in simplified
regime, with a reference to the Law 748/84 "Norms for the fertilizers discipline", that in the
enclosure 1C, as modified by the D.M. 27.03.1998, defines the marketing characteristics for
the compost, indicating the following four types of final products:
- Simple not composted vegetable amendment: not fermented product composed of
barks and/or other vegetable residues as pomace, chaffs, peels, with exclusion of algae
and other marine plants (it is a product that, for definition, cannot considered as raw
material for composts);

- Green composted amendment: obtained through a controlled process of transformation
and stabilization of organic wastes constituted by urban green residues, crops residuals,
other residues of vegetable origin, with exclusion of algae and other marine plants;
- Mixed composted amendment: obtained through a controlled process of transformation
and stabilization of organic residues that can be constituted by the organic selected
fraction of the urban wastes, from animal slurry, included the animal sewages, from
agro industrial residues, and from the wood and textile untreated throughput as well as
from the matrixes of the Green composted Amendment;
- Peaty composted amendment: obtained by mixture of peat with Green composted
amendment or Mixed composted amendment.
For the realization and the management of the composting installation can be followed:
- the simplified procedure (as artts. 31 and 33 of the Legislative Decree 22/97) when the
installation, the first and the final products are entirely conforming to the prescriptions
of the D.M. 05.02.1998, particularly for the selection of the organic materials.
Furthermore, the characteristics of the final products should be conforming to the
requisite in the enclosure 1C to the L. 748/84 (as modified by the D.M. 27.03.98). This
procedure lay down a preventive communication of start of the recovery activity
together with a technical explanatory document, in the respect of the discipline
contained in the DPR 203/88, for the releases in atmosphere, and of that reported in the
Legislative Decree 152/99, for landfill.
- the ordinary procedure (as artts. 27 and 28 of the Legislative Decree 22/97), that require
a specific authorization, released from the local administrations.
4.2. Composting procedures
The compost should to have an adequate degree of stabilization (maturity); and qualified
amendments and nutritional properties.
The quality of the compost is function of the following factors:
- the composition of the mixture (raw materials), as percentage among different
components;
- the management of the composting process, particularly the duration, the moisture and
the oxygenation of mixture;
- the final conditioning (refinement, addition of other products).
The composting of selected organic substances has to be considered as a recovery system for
the production of high quality organic amendments to use for the agricultural purpose able
to replace, even though partially, the manure in the extensive agriculture, the chemical
fertilizers in the intensive crops and the peat in nursery.
The ACT phase, in relationship to the characteristics of the treated mixture, can happen in
piles on open air, or confined in more complex systems (bioreactors) with forced airing, with
or without overturns. During this phase must be checked the temperature, moisture, pH
and oxygen. The duration of this phase should be enough to guarantee the attainment of an

adequate Respiration Index (I.R.) of biomass, to be test with the IPLA (Institute of Plants,
Wood and Environment) method (IPLA, 1992). This duration corresponds to about 21 days
of treatment, in the case of stabilization in overturned piles and about 14-16 days in the case
of bioreactors.
The curing phase is always carried out in piles and require a lower amount of oxygen and
overturn in comparison to the ACT phase.
The biomasses to be composted must be previously grinded and mixed to optimize the
characteristics of the matrix (C/N ratio, porosity, moisture, pH). A good starting matrix for
the production of a Quality Composted Amendment (ACQ), has to have a moisture among
45 and 65%, C/N among 20 and 30, with at least a 30% in weight of lignocelluloses material.
The ACQ can be commercialized to the senses of the Law 748/84, also in mix with other
fertilizers, according to the same law prescriptions, as reported in Table 5. This material
could be used in the agricultural activities or similar (maintenance of the public or private
green, biofilters, etc.).

Parameter Green Composted
Amendment
Mixed Composted
Amendment
Peaty Composted
Amendment
pH 6.0-8.5 6.0-8.5 -
Humidity (%) <50 <50 -
TOC (% d.m.) >30 >25 >30
Organic Nitrogen (%) >80 >80 >80
C/N ratio <50 <25 <50
Humic and fulvic acids (% d.m.) >2.5 >7 >7
Peat (%) - - >50
Cd (mg kg
-1
) <1.5 <1.5 <1.5
Cu (mg kg
-1
) <150 <150 <150
Hg (mg kg
-1
) <1.5 <1.5 <1.5
Ni (mg kg
-1
) <50 <50 <50
Pb (mg kg
-1
) <140 <140 <140
Zn (mg kg
-1
) <500 <500 <500
Cr VI (mg kg
-1
) <0.5 <0.5 <0.5
Inert (>10 mm) (%) none none none
Salmonelle (on 25g sample) none none none
Enterobacteriacee (CFU g
-1
) < 100 < 100 < 100
Fecal Streptococcus (MPN g
-1
) < 1000 < 1000 < 1000
Nematodes (on 50g sample) none none none
Trematodes (on 50g sample) none none none
Cestodes (on 50g sample) none none none
Table 5. Limits of acceptability for the Quality Composted Amendment (ACQ), conforming to Annex
1C of the Law 748/84, as modified by the D.M. of 27.03.1998.

The composting process can also carried out with low-tech approaches and extreme
operational simplification, excluding the forced airing and effecting the biomass overturns
using machineries already in endowment to the farm (choppers, shovel loader, wagon
mixers).
4.3. Technological systems: classification and description
The bioxidative processes are carried out in different operative systems, classified as
(Amirante & Montel, 1999; Clodoveo et al., 2000; Goldstein, 1980; Willson et al., 1980):
- intensive or extensive systems, according to both the technological complexity and the
energetic needs;
- closed or open systems, according to be confined or not by the open air;
- static or dynamic systems, according to whether the mass has moved;
- aired or not aired systems, in presence or absence of forced airing.
The intensive systems are destined to high fermentability biomasses. They are generally
closed, dynamic and aired, and works through a first active phase (ACT), with control of
process parameters, and a second phase (curing) with lower-level of technological
complexity, similar to the extensive systems. The processing times vary between 25-30 and
120 days (in average 90 days). The energetic needs are around 40-60 kwh ton
-1
for the
machinery power (overturners, ventilation systems, grinders, sieves). The need areas are
around 0.7-1.5 m
2
ton
-1
of annual biomass to process.
The extensive systems are dedicate to biomasses of low fermentability and are open, or also
confined, static and not aired (aired for diffusion and natural convection), and do not
differentiate the operative phases, but they adopt an only lower technological step. Typically
they consist in macropiles open air, without or very time deferred overturns. The process
times varying around 6 months up to over 1 year. The energetic needs are low (10-20 kwh ton
-
1
), for the grinding, the optional overturn with generic machines (mechanical shovels) and the
sifting. They need surfaces around 1.5 - 2 m
2
ton
-1
of annual biomass to process.
4.3.1. Systems closed" or "open
In the closed systems the process is carried out in confined spaces (bioreactors) or in covered
areas (sheds), for biomasses that require greater control of the fermentative parameters
(high moisture, stinking as well as selected for high quality compost). Conversely, the open
systems are destined to biomasses of low fermentability, high percentage (i.e. 70%) of
lignocelluloses bulking, or for curing phases of biomasses already submitted to ACT.
4.3.2. Systems static" and "dynamic
These systems differ for the periodic or continuous biomass moving (dynamic) or for
immobility (static). The base idea of the static systems is not to disturb the growth and the
action of the fungi and of the microbial population, maintaining a microecological
environment. This situation is important to create in the biomass good growth conditions
with the purpose to facilitate the stabilization processes. This systems can work with

matrixes well structured and homogeneous, with at least 40 - 50% in weight of
lignocelluloses bulking, to avoid the compaction of the mass, and moisture less than 65%.
In the dynamic systems can be processed very moisture matrixes (> 65%) and with less than
30% in weight of lignocelluloses bulking.
4.3.3. Systems aired" and "not aired
The forced airing of the biomass is an important factor of optimization of the process
particularly for the treatment of low consistence and higher fermentability biomasses. For
the optimization of the forced airing it is necessary to calculate and monitoring:
- the specific air flow, generally expressed in Normal cubic meters for hour and of weight
biomass unit (NCM h
-1
ton
-1
). Generally, the airing needs for the drain are higher also of
a dimensional order, in comparison to those of oxygen in stechiometric demand for the
mineralization. The airing systems are therefore sized and used for the control of the
temperature, while the biomass oxygenation is obtained as derived effect. This sizing
involves nevertheless an excessive evaporation and drying of the biomass, with early
interruption of biostabilizative processes. For this reason, in airing systems are
necessary appropriate systems of control and remoisten.
- the proportion between times of airing activation and turning off. The intermittent
ventilation of the biomass, besides the energetic saving, has the purpose to allow during
the periods of turning off the equalization of moisture and temperature in the different
zones of the biomass, while the airing in continuous can behave more sensitive
stratifications of them. The proportion between times of working and turning off is
established in order to the better thermal level needed for the biomass, being the
optimization of the temperatures another of the essential parameters to the efficiency of
composting process. To the same flow of air and of heating power of the biomass
(dependent from moisture and fermentability), greater periods of working airing
proportionally involve lesser thermometric levels of the biomass (Higgins, 1982).
The different processing systems, classified according to the installations or the operational
methodology, can be associate to one or more of following categories. Typically, among the
open systems are included: Aired static pile; Overturned pile; Short pile. While, among the
closed systems they are: Biocontainer; Dynamic trenches; Dynamic basins; Silos.
4.3.4. Aired static pile
The aired static pile constitute a system of relative technological simplicity. It was developed
in America as "Beltsville system ", with the purpose to have a simple system of the wastes
biomasses bioconversion to the farmers for agronomic reuse. In his different variations, it
shapes as an open, static and aired system (Willson et al., 1980). Typically it foresees the
disposition of the biomass in pile, with forced airing in aspiration below the pile and
dispatch of the exhausted air to a compost-made biofilter. On the pile surface a layer of
mature compost is placed, with not permeable and filtering actions. As static system, it
requires of a high percentage of structural lignocelluloses bulking, and of a relatively low
moisture of the starting matrix. It also requires a appropriate homogenization pre-

treatments of biomass. In some different systems the forced airing works by inflation, while
for the pile coverage are adopted cloths or semi-permeable membranes, to limit the losses of
moisture.
4.3.5. Overturned piles
In this system the biomass are disposed in piles of great dimensions (3-4 m of height; width
up to over 20 m). The piles are typically open, not aired and overturned with ample
intervals (weeks or months), with mechanical shovels or specific overturning machines in
the case of continuous systems. This system is adopted for matrixes to tall prevalence of
lignocelluloses material (green residues) or in the curing of biomasses after the ACT. The
piles can be managed in batch, or in continuous, with progressive translation of the biomass
during the overturns, toward the unloading section. This second solution eliminates the
unused spaces between piles, while is forcing to adopt a fixed overturns periodicity, that
would not correspond to the real needs of process.
4.3.6. Short pile
The short pile differ from the piles for their smaller dimensions (max 2.5 m height) and can
be or not endowed with the forced airing. The overturn is generally frequent (from few days
up to daily intervals) and are made with specific overturners. The short piles are planned as
closed system for the active phase of high fermentable matrixes; or as open where the
stinking impacts do not constitute trouble, or for low fermentable material, or for the curing
of end-fermented biomasses.
4.3.7. Biocontainer and biocell
Biocontainer and biocell are horizontal closed reactors, typically static and airing. The
biomass is placed in beds of the maximum height of around 3 meters. The biocontainer are
made with metallic or concrete, insulate, and have unitary volumes of the order of many
about ten cubic meters. These systems generally adopt the recycle of the air, and survey
systems to manage the parameters of process (moisture, oxygen percentage in the inside
atmosphere, temperature) and feed-back regulation of the air flows and the percentages of
air recycle.
4.3.8. Dynamic trenches
Conversely to the biocontainer, in this system the reactor is divided in trenches served by
one or more lines of forced airing, with modulation of the air flows in the different sections
correspondents to the different times of process. The trenches race binary for the translation
of self-moving overturners that also effect the progressive transfer of biomass (continuous
system). The trenches are typically used for the management of the active phases of
biomasses to elevated fermentability, in closed environments. As dynamic system, can
effectively process also biomasses with high moisture content.
4.3.9. Dynamic basins
In this installation typology, typically closed, aired and dynamic, the composting biomass is
placed in basins, and moved with special self-moving overturners. It is generally used for

the active phase of higher fermentability biomasses or also for whole process (ACT and
Curing, without discontinuity). This system result to be economically competitive for
processing over 100 tons day
-1
of biomasses.
4.3.10. Silos
The silos are vertical reactors, to one or multi step, closed and airing, with continuous or
discontinuous loading and unloading of the biomass. The system can be static (for batch
processes) or semi-dynamic (top loading of composting matrix and bottom unloading of
stabilized biomass).
5. Composting experiences and results
Several experimental trials were carried out using different olive mill typology wastes (two
phase mill fluid pomace; three phase mill pomace and waste water), mixed with others
agricultural or agroindustrial byproducts, with the aim to evaluate both the composting
process efficiency, and the amendment and nutritional efficiency of obtained composts on: i)
maintain or improving soil properties and soil fertility; ii) growth and yield performance of
herbaceous crops; iii) growth and yield performance of olive orchards (Albuquerque et al.,
2007; Alfano et al, 2008; Baeta-Hall et al., 2005; Boulter et al., 2000; Calvet et al., 1985;
Casacchia et al., 2012; Diacono & Montemurro, 2010; Hoitink et al., 1997; Montemurro et al.,
2004, 2006, 2010, 2011b; Tejada et al., 2006, 2009; Toscano et al., 2009 b).
5.1. Results on olive orchards
In some experimental trials, the assessment of the composting process in natural conditions
was studied on two different typology of matrices, respectively composed by olive mill
wastes and other crop by-products as structural biomass: i) wet pomace by two phases mill
with olive leaves and cereals straw; ii) pomace and waste water by three phases mill with
carved pruning residuals. All materials were placed on a beaten-soil platform, and remixed
to homogenize the matrices. The evolution of bioxidation was monitored controlling the
temperature and humidity of matrices, and assuring oxygenation and thermal drain by
weekly blending with mechanical shovel on tractor front loader. At the end of the
fermentation period, both biomass microbial pools and chemical parameters were analyzed
to evaluate the characteristics of obtained composts. Results of composting parameters trend
and microbiological analysis, indicate a correct way of aerobic process; and the compost
analyses confirmed that a mixed composted amendments was obtained (Law 748/1984).
To evaluate amendment and nutritional efficiency, the composts were spread at doses of
approx. 150 kg tree
-1
(60 tons ha
-1
) on 6 x 4 scaled olive orchard, and buried with a light disk
arrow tillage. At the following years harvesting time, was compared the differences
between treated and untreated soil characteristics, and yield responses of 15 treated vs. 15
untreated olive trees of a 15 years old Nocellara messinese cultivar for OP2 compost, and
20 years old Leccino cultivar for OP3 compost.

The amendment and nutritional efficiency of these kinds of composts was confirmed both
by soil organic matter increment (+ 38.6%; + 40.6% for two phase and three phase compost,
respectively), and trees productive responses at the following year harvesting time (+ 10.5%
and + 15.1% oil yield increase on respective treated trees).
Even if the condition of fermentative process (open air, natural conditions) was not optimal,
the obtained compost demonstrate that the aerobic fermentation of olive mill residuals can
run in a correct way also in natural conditions, producing an hygienized and partially
stabilized organic amendment, that can be better spread out at the optimal time, and
without the negative effects related to the raw olive mill wastes supply. At the used doses,
both composts increased soil fertility, improved water retention and the availability of
nutrients in amended soils, and improved plant productivity. The showed nutritional effect
of these composts would allow a reduction in the use of chemical fertilizers, in agreement
with the energetic and economic sustainability principles in the use of renewable resources.
In common situations where industrial installations are unfeasible, the natural composting
process of olive mill wastes can therefore be a interesting alternative to the raw pomace and
waste-water spreading on soils; and could represent a solution of sustainable disposal
problem, allowing to increase soil organic matter contents and to reduce the desertification
processes (Casacchia et al., 2012; Toscano et al., 2009b).
5.2. Results of olive mill waste water application on herbaceous crops
As mentioned before, the national law (Law 574/1996) allows the olive mill waste water
(WW) spreading on soils with appropriate characteristics for agronomical use with the
maximum amount of 80 m
3
ha
-1
yr
-1
for the centrifuge system and of 50 m
3
ha
-1
yr
-1
for the
pressure method.
In a research carried out in the semi-arid environment of Southern Italy, the application of
two WW rates without preliminary treatments was studied. To reach this objective, the
effects on cereal and leguminous crops yield, quality, N uptake and on soil characteristics
were recorded.
The results obtained indicate that the WW treatments (both doses) positively affected the
yield of ryegrass, while a significant yield increase was found at the highest level of WW in
proteic pea. The clover crop showed a species-specific sensitiveness, but the WW
applications increased the protein content compared to the untreated plots. The WW rates
also increased total organic content in the soil, in respect to the unfertilized control. At the
end of this three-year experiment the values of soil total extracted carbon and humified
organic carbon were higher compared to the initial ones. The values of soil available P and K
of the control treatment found at the end of experiment were almost the same than those
recorded at the beginning of the research confirming that the increases found in WW plots
were due to the WW applications.
A two-year experiment was also carried out in controlled environment (lysimeters) to study
the effects of applying untreated and treated WW as soil amendments on both rye-grass

growth and soil characteristics. The results of this research indicated that the untreated and
treated WW application increased growth parameters, indicating the possible use of WW as
an amendment to rye-grass. A significant increase of total, extracted and humified organic
carbon in soil, and humification parameters (degree and not humified organic carbon) were
found, whereas no accumulations of heavy metals in the soil were measured at the end of
the experiment. Furthermore, N content in WW was used by rye-grass for plant growth that
increases N uptake and consequently the dry matter accumulation (Montemurro et al.,
2007).
5.3. Results of pomace application on herbaceous crops
As already mentioned, the composting of olive pomaces could be recycled for agricultural
purposes. In addition, a specific test could be performed to assess the phytotoxicity for both
the raw pomace and the stabilized composts. The phytotoxicity is one of the most important
criteria for evaluating the agronomical potential of organic materials as olive pomace, and
can be measured by specific test. A method can be used to assess phytotoxicity of this
residues, by combining the measurements of seeds germination and roots elongation of
cress (Lepidium sativum L.). Different experiments indicate that the olive pomace composts
were not phytotoxic. According to other researches, it can be suggested that repeated
compost application might preserve the soil organic carbon content and supply
macronutrients to crop. Finally, among different solutions, the addition to olive pomace of
manure, as a nitrogen source, and pruning wastes, as bulking-agent, may generate organic
amendments suitable for the organic cultivation management of some herbaceous crops.
Several experiments on herbaceous crops indicate that the use of organic pomace fertilizer,
as a partial substitution of mineral fertilizers, reached the same yield of the highest mineral
fertilizer treatment, ensured also an increase of soil total organic carbon and other soil
properties. Furthermore, the application of these organic composted wastes also induce a
lower nitrogen mineral soil level at the end of the experimental trials, indicating the
possibility to reduce pollution risks. These findings were found in maize and barley in a
same experimental research (Montemurro et al., 2006).
5.4. Results on greenhouse olive nursery
On the market are available different organic (peats, manures, urban wastes, barks, sawdusts)
and inorganic (sand, pumice, clay, perlite, vermiculite) materials, usable in the preparation of
olive nursery substrates. All these materials have some advantages, i.e. sterilization process
not necessary, and commercial fertilizers can be added. Even though the peat is the more used
organic material in the preparation of the substrates, other different material have been tested
with the aim to replace it, include the olive industry byproducts.
In some experimental trials has been valued the compatibility and efficiency of composts,
obtained from different olive mill wastes, in the composition of growth substrates for olive
nursery, with the aim of mostly valorizing these biomasses as partial or total substitution of

peat. Variable proportions of peat and/or two different olive waste composts deriving from
the continuous two and three phases extraction systems, were added to a basis of river sand,
used as control, for a total of six treatments for each of the three cultivars under observation
(Carolea, Nocellara messinese and Tondina). The self-rooted plantlets were kept for 28
months in greenhouse, making periodic measurements of linear growth (cm) and number of
internodes. At the end of trial, dry weights of leaves, branches and roots were detected. The
results showed a greater nutritional efficiency of the substrates containing composts derived
from the three phases olive mill wastes, with highly significant differences both in the linear
growths and in the number of internodes; while the compost derived from two phases olive
mill wastes do not showed particular benefits, giving results similar to peat, and to sand
(control), presumably due to the higher C/N ratio of this compound (Santilli et al., 2012;
Toscano et al., 2009a).
5.5. General findings
In the experimental conditions of southern Italy, the findings of the studies on repeated
applications of WW indicate that it could be possible both to sustain herbaceous crops
performance and support soil fertility, valorizing these wastes as organic amendment and
reducing the risks of soil degradation, giving also a useful practice to reuse purposes as EU
provides.
Also the composting experiences of olive mill and olive orchard wastes carried out in
natural conditions, demonstrate as relatively easy feasible and noticeable effective the
production of stabilized organic amendment of elevated agronomic value to very contained
costs. These materials have their natural utilization to maintain soil organic fertility, as well
as substrate for olive nursery, in perspective as substitute of peat. According to the modern
principles of the energetic and economic sustainability of agro industrial processes, the olive
mill and olive orchard wastes composting has been confirmed as a valid alternative to the
raw waste water and pomace shedding on the soil. In particular, a higher efficiency was
found in stabilizing the organic matter, which works better in the restoration and in long-
term maintenance of soil fertility. Furthermore must be considered that the nutritional effect
of compost can allow to reduce the use of chemical fertilizers in fertilization plans, mostly
raising the value of this kind of agro industrial by-products management. So could be a
better solution also to fit EU prescriptions concerning wastes reuse.
Author details
Pietro Toscano
Agricultural Research Council, Olive Growing and Oil Industry Research Centre, Rende (CS),
Italy
Francesco Montemurro
Agricultural Research Council, Research Unit for the Study of Cropping Systems, Metaponto (MT),
Italy

Acknowledgement
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Chapter 10
Packaging and Storage of Olive Oil
1. Introduction
Storage is a very important step of any food, including olive oil. In fact, olive oil shelf life
can be influenced by different factors, from olive quality to processing technologies,
however, the selection of proper storage conditions, including packaging, can be of great
importance. The Mediterranean diet is recommended as food model for the prevention of
various chronic-degenerative pathologies (cardiac diseases, cancer etc.) and olive oil is
surely the cornerstone of this type of nutrition with fruits, vegetables, legumes and fish. Its
peculiar nutritional characteristics depend on the presence of antioxidant components and
monounsaturated fatty acids that are predominant on unsaturated ones, with positive
results in the increase of HDL (High-Density Lipoproteins) and reduction of LDL (Low-
Density Lipoproteins) oxidation.
The consumer expresses its judgment on olive oil quality considering only some sensory
characteristics, such as the more or less pungent taste, fruity and mild flavour and within this
context a wide range of preferences can be found, because the sensory quality may match
specific dishes, cultural aspects or simple dietary habits. Incorrect storage practices influence
the sensory quality of the oil, as rancidity and off-flavours may develop. The lipid oxidation, in
fact, is one of the factors of olive oil quality deterioration. The rate of oxidation depends on the
availability of oxygen, the presence of light and the temperature. In absence of light, the auto-
oxidation follows a free radical mechanism with the formation of hydroperoxides. These labile
compounds further decompose to produce a complex mixture of volatile compounds such as
aldehydes, ketones, hydrocarbons, alcohols, and esters responsible for the already mentioned
deterioration of olive oil flavour known as oxidative rancidity. Many phenolic compounds
of the olive oil contribute to its resistance to oxidative rancidity. After light exposition, photo-
oxidation occurs through the action of natural photosensitizers (i.e. chlorophyll), which react
with triplet oxygen to form the excited state singlet oxygen. It then gives a free radical from
unsaturated fatty acids, leading to the production of hydroperoxides and eventually to
carbonyl compounds, which result in the development of undesirable off flavours in oils.

Packaging can directly influence olive oil quality by protecting the product from both
oxygen and light. The shelf life of the oils exposed to intense artificial light and diffused
daylight is shorter than that of oils kept in the dark. Moreover, the storage temperature, the
use of nitrogen atmosphere and the reduction of the oxygen in the headspace volume can
appreciably control quality changes during storage time.
Materials which have been used for olive oil packaging include glass, metals (tin-coated
steel) and more recently plastics and plastics coated paperboard. Among plastics,
polyethylene terephthalate (PET) has captured a large portion of the olive oil retail market
due to its many advantages including clarity, chemical inertness, low oxygen permeability,
and excellent mechanical properties. Incorporation of pigments and/or UV blocking agents
or oxygen scavengers may improve plastics properties with regard to quality retention of
olive oil. Besides PET, polyethylene (PE) in the form of LDPE-coated paperboard/aluminium
foil laminates, i.e. brick-type cartons and bag-in-box pouches and polypropylene (PP) are
being used today for the packaging of vegetable oils including olive oil. In this chapter,
factors affecting olive oil quality during storage will be discussed and various packaging
solutions will be approached.
The aim is to give evidence the best practices to adequately manage this important unit
operation, in order to improve/maintain the quality characteristics of the olive oil.
2. Nutritional value of olive oil
Mediterranean food tradition is sustained by three basic essentials: wheat, olives and grapes.
Nevertheless, olive oil is the central element inherent to this diet, and its importance is due
to the increasing consumption around the world, because of its nutritional and sensory
properties. European Union (EU) is the leading producer of olive oil and within the EU, the
Mediterranean members are the biggest producers, in fact Mediterranean area accounts for
95% of world production and 85% of world consumption of olive oil (IOOC, 2007).
Several clinical data have shown that consumption of olive oil can provide heart health
benefits, such as favourable effects on cholesterol regulation and LDL cholesterol oxidation,
and that it exerts anti-inflammatory, antithrombotic and antihypertensive effects (Lairon,
2007; Perez-Jimenez et al., 2007).
Virgin olive oil is a genuine fruit juice obtained from olive drupes (Olea europaea L.), using
exclusively mechanical procedures, without further treatments or chemical additions. The
saponifiable fraction is more representative (about 98%) than the unsaponifiable one and it
comprises principally triacylglycerols, esters formed by glycerol and fatty acids, mainly
unsaturated acids whose the major is oleic acid (about 65-80%). The olive oil contains also a
relatively reduced level of polyunsaturated essential fatty acids (PUFA), linoleic and
linolenic acids (C18:2 -6, C18:3 -3). Such composition gives good resistance to chemical
and biological oxidation, in contrast with other edible oils in which polyunsaturated fatty
acids prevail on monounsaturated ones (Rastrelli et al., 2002). Thus, the importance of virgin
olive oil is related to its high levels of monounsaturated fatty acids but also to the presence

Packaging and Storage of Olive Oil 203
of minor components including aliphatic and triterpenic alcohols, sterols, hydrocarbons,
volatile compounds, and several antioxidants. In fact, the unsaponifiable fraction which
covers a small percentage (0,5-3%) plays a significant role on human health.
Among the antioxidant compounds, squalene, a triterpenoid hydrocarbon and precursor of
sterol biosynthesis, occurs in olive oil with a high concentration (up to 1%). Other active
constituents in the olive oil are represented by tocopherols -, -, -, -: the concentration
of tocopherol (Vitamin E), traditionally considered as the major antioxidant, covers
almost 88% of the total tocopherols. The tocopherol content of olive oil depends not only on
the presence of these compounds in olive fruit but also on several other factors, involved in
the transportation, storage and olive fruit processing. Normally in animal fluids and tissues,
vitamin E works in synergy with coenzyme Q (CoQ) to protect cells and tissues against
lipoperoxidation, and some authors detected CoQ9 and CoQ10 in olive oil (Pregnolato et al.,
1994; Psomiadou & Tsimidou, 1998). According to Viola (1997), the ratio of vitamin-E to
polyunsaturated fatty acids in olive oils is better than in other edible oils.
Phenolic compounds make an important contribute to the nutritional properties, sensory
characteristics and the shelf life of olive oil, because they improve the resistance to the
autoxidation. Olive fruit contains simple and complex phenolic compounds: those derived
from the hydrolysis of oleuropein contribute to the intensity of the bitterness of virgin olive
oil, and especially hydroxytyrosol, tyrosol, caffeic, coumaric and p-hydroxybenzoic acids
influence the sensory characteristics of olive oil (Kiritsakis, 1998). The concentration and
composition of phenolic compounds in virgin olive oil is strongly affected by many
agronomical and technological factors, such as olive cultivar (Tura et al., 2007), place of
cultivation (Vinha et al., 2005), climate, degree of maturation (Kalua et al., 2005; Sicari et al.,
2009; Giuffr et al., 2010), crop season (Gmez-Alonso et al., 2002), irrigation (Tovar et al.,
2001) and production process (Cinquanta et al., 1997; Ranalli et al., 2001).
Regarding other compounds in the olive oil, its colour is mainly related to the presence of
chlorophyll and pheophytin a (Psomiadou & Tsimidou, 2001).
Carotenoids are also responsible for the colour of olive oil: the major are -carotene and
lutein in virgin olive oil (Gandul-Rojas & Mnguez-Mosquera, 1996). The presence of these
constituents depends on several factors, such as cultivar, soil and climate, and fruit
maturation as well as applied conditions during olive fruit processing (Gallardo-Guerrero et
al., 2002).
3. Deterioration reaction in olive oil
The olive oil quality is strongly related to the physiological conditions of the fruit from
which it is extracted. Important chemical changes occur inside the drupe during ripening.
They are related to the synthesis of organic substances, especially triglycerides, and to other
enzymatic activities that may affect virgin olive oil quality. From olive oil extraction to
storage operations the most common variations, recognized generally with the term
rancidity, are divided into hydrolytic and oxidative rancidity.

The hydrolytic rancidity is a change due to the presence of water in the drupe and the
catalytic action of an enzyme, the lipase, often derived by microorganisms. The reaction
consists of a triglyceride hydrolysis to give glycerol and fatty acids, which result in an
increase of free acidity. The release of mono-, diglycerides and finally fatty acids is obtained
by the enzymatic hydrolysis.
Besides lipases, also peroxidises and lipoxygenases are numbered among the involved
enzymes and they are responsible to the more or less selective formation of hydroperoxides,
which will damage the native antioxidants and the polyphenoloxidases that reduce the
polyphenol content, in particular on the olive paste. The enzyme activity becomes slower
due to the inhibition effect of the polyphenol oxidation products. This variation regards all
olive oil process, in particular the olive oil extraction systems.
The oxidative rancidity or autoxidation is due to the reaction between the oxygen and
unsaturated fatty acids, free and esteryfied. It follows the characteristic trend of radicalic
reactions with an induction, a propagation and a termination phase. The induction period is
characterized by production of free radicals by unsaturated fatty acids or lipid peroxides (so
called hydroperoxides), which constitute the primary autoxidation products. The direct
attack of atmospheric oxygen on the unsaturated fatty acid chain is unfavoured from the
thermodynamic point of view, because the activation energy of the reaction is high (145-270
kJ mol
-1
). The direct attack could be attained by singlet oxygen which can be formed by a
photochemical reaction. After the first formation, hydroperoxides are degraded to give a
chain-reaction in the propagation phase: it is the principal oxidation step. Oil-quality
changes related to the production of oxidized by-products that alter the sensory and
nutritional characteristics of the oil include the production of carbonyl compounds, a
decrease of the -tocopherol concentration, and the generation of off-flavour compounds.
These secondary products responsible of rancid odour and flavour are represented by
saturated and unsaturated aldehydes, ketones, volatile alchools, hydrocarbons, cyclic
oxygenated compounds, etc. (Frankel, 2005; Morales et al., 1997). In the end of the
autoxidation, reactions lead mainly to the formation of polymers (dymers oxygenated and
not oxygenated according as involved reagents).
When vegetable oils are exposed to light, photo-oxidation occurs through the action of
natural photosensitizers (i.e. chlorophyll), which react with triplet oxygen to form the
excited state singlet oxygen. Singlet oxygen then forms free radicals from unsaturated fatty
acids leading to the production of hydroperoxides and eventually to carbonyl compounds
resulting to the development of undesirable off flavours in oils (Skibsted, 2000).
The reaction responsible of oxidative rancidity is so promoted by light, heat, metals traces
(Fe, Cu, Co, Ni, Mn). Substrates of these reactions are principally unsaturated free fatty acids
which in general oxidize faster respect to triglycerides and phospholipids. The oxidation
velocity is affected mainly by the unsaturation degree. The saturated fatty acids oxidize at a
temperature higher than 60 C, whereas the polyunsaturated fatty acids oxidize at lower
temperatures.

Other unsaturated substrates can be submitted to similar oxidation reactions, among
which some hydrocarbons in the oils, in particular squalene, vitamin A, carotenoids and
vitamin E (-tocopherol). Oils and vegetable fats are more or less rich in -tocopherol that
has a natural antioxidant activity delaying the lipid oxidation. Tocopherols are known to
act as antioxidants by donating a hydrogen atom to chain-propagating peroxyl radicals
(Kamal-Eldin & Appelqvist, 1996). The oxidation of vitamins A and E and carotenoids can
be also due to the peroxides action formed by unsaturated fatty acids in the secondary
oxidation. This process leads to loss of vitamin activity and colour, while the essential
fatty acids oxidation involves a decrease of nutritional value (Sciancalepore, 1998).
In autoxidation, virgin olive oil stability is correlated with the polar phenol content: a linear
relationship between the phenolic content and oxidative stability of extra virgin olive oil has
been in fact noted (Di Giovacchino et al., 1994). The main active phenols are the o-diphenol
hydroxytyrosol and its oleosidic forms (Tsimidou, 1998, Aparicio et al., 1999) but relation
between total phenol content and oil stability is not always validated. The olive oil stability
to oxidation decreases in fact during the storage time, but a proportional trend to the
phenols decrease is not observed. It is possible that an equilibrium attains (or tends to
achieve) between polyphenols and their oxidation products, already present or formed
successively to its protection versus fatty acids. This equilibrium opposes to the normal
antioxidant activity of polyphenols still intact which, in these conditions, do not exert the
same antioxidant activity (Lercker, 2004).
Apart from contributing to colour, carotenoids protect the oil from photo-oxidation by
quenching singlet oxygen and acting as light filters (Fakourelis et al., 1987).
Chlorophyll pigments are also well-known to act as photosensitizers during light exposure.
They are able to catalyze photoxidation with a velocity higher (up to 30000 times more).
Chlorophylls exert an antioxidant activity dependent on the derivative present, the lipids
substrate and storage temperature (Endo et al., 1985; Gutierrez-Rosales et al., 1992).
The squalene hydrocarbon has a slight antioxidant activity which is concentration
dependent (Psomiadou & Tsimidou, 1999)
3.1. Deterioration agents in olive oil: Oxygen, light and temperature
Among physical agents, the oxygen plays a fundamental role in the oil alteration: in contact
with air it loses many qualitative characteristics as colour, flavour, odour and vitamins.
As index of oxidative deterioration, the peroxide formation in olive oil stored in closed tins
is in fact generally insufficient to lead to development of the typical rancid odour, because of
the limited amount of oxygen in the headspace. The lipid oxidation substrates, as previously
observed, are the unsaturated molecules, while the unsaturation degree influences the
oxidation velocity. The high level of natural antioxidants, associated with an excellent fatty
acid composition, confers the olive oil high stability against oxidation, apart from the
recognized nutritional value.

The adoption of proper operations is also important in the preliminary phase of olive oil
storage, as for example the laminar filtration by using of cartridges closed in steel cylinders
that reduce the air exposition, or the filtration under nitrogen, in controlled atmosphere.
The oxygen plays an important role on the entire and complex interactions in the olive oil.
Also antioxidant molecules are strongly influenced by oxygen presence. A study of changes
in total polar phenols, -tocopherols, -carotene, lutein, chlorophyll and squalene contents
during autoxidation indicated that most interactions may be suppressed if oxygen
availability is limited (Psomiadou & Tsimidou, 2002).
Another principal harmful agent of olive oil is light, both direct and diffused and in general
the UV area in the light spectrum at high energy, from 290 to 400 nm is the most charged.
The electromagnetic radiations promote some chemical and biochemical reactions
responsible of qualitative degradation as the lipid oxidation in the presence of air. In the
absence of air, however, direct sunlight causes a decrease in peroxide and Kreis values of
the oil. In particular, in a study on several olive oil packaging typologies, Kiritsakis (1984)
revealed that the oil oxidation proceeded slowly in darkness, faster in diffused light and
even faster in direct sunlight.
Moreover several natural substances have the property to absorb much the incident light
and, for it, to originate alterations and/or undesirable variations. Pigments have this
characteristics and colour degradation is in fact an unavoidable and well known event:
many vitamins (as A and B2) are photosensitive.
The temperature controls many of reactions catalyzed by enzymes. For each increase or
diminution of it a variation of biological food activity is shown. At low temperatures the
kinetic energy of reagent molecules decreases, the drop of mobility and collisions necessary
for the formation of enzyme-substrate complex and their products occurs. Some studies
(Gambacorta et al., 2004; Kanavouras & Coutelieris, 2006; Pristouri et al., 2010)
demonstrated that high storage temperatures modify the olive oil quality, but less than the
light exposition.
4. Storage of olive oil
According to olive variety, extra virgin olive oil has different sensorial attributes, e.g.
fragrance, flavour, colour and nutrient composition parameters. Thus, its quality can be
considered from diverse points of view: normative, commercial, nutritional, therapeutic and
sensorial. An excellent olive oil is produced with good olive growing, harvesting, storage
and processing practices. This condition considers the preservability an olive oil qualitative
characteristic.
Some quality parameters of extra virgin olive oil (acidity, peroxide value and UV
absorbance) can vary depending on time and storage method, reaching undesirable values
at the end of the shelf life. Acidity increases with time, both in oils stored in the dark and
exposed to light (Gmez-Alonso et al., 2007). Nowadays, consumers are imposing an

increasing demand for a higher quality of extra virgin olive oil during the shelf life period.
This is due to the expectation for a safe food and also for a reduction to a minimum of
undesired changes in sensorial quality (Hrncirik & Fritsche, 2005).
The olive oil storage could be shorter as possible: its assumption could not exceed the
production year: the oil gains the excellence about 3-8 months after production, after that its
quality starts decreasing. This behaviour depends on the olive cultivar and on the alteration
of fatty fraction.
During storage the oil tends to loss the typical pigmentation and the aromatic characteristics
of bitter and spicy, showing too many transparency and brilliance. Immediately after oil
extraction, the flavour appears too strong and also characterized by an unpleasant aftertaste.
If the olive oil is stored properly, as in well-sealed packages, it can reach the second storage
year maintaining its sensory properties.
A good quality olive oil with a low acidity degree preserves its opacity for a long time. This
is a characteristic of some oils, as those extracted from first harvested olives not yet
completely ripe, which are very fruity and with low free acidity. The consumption of these
oils, also named new oils is recommended up to a few weeks after bottling. For many
consumers the cloudy olive oil represents a certainty of authenticity and it is also preferred
in particular when the oil is purchased directly at the oil mill. It is characterized by a certain
water amount (about 2-4 g/kg ) and residual solids as pectins, hemicelluloses, cellulose and
mucilages in form of emulsion containing active or latent enzymatic activities as
lipoxygenase, polyphenoloxidase and, principally, some esterases able to hydrolyze
oleuropein, di-methyl-oleuropein and ligstroside aglycon derivatives responsible of bitter
and sour flavour in virgin olive oils. The association between the natural and the
healthy/nutritional aspect does not correspond to a scientific assessment because the
presence of cloud induces the phenols degradation, which results in sensory changes.
Moreover fermentative processes develop on olive polysaccharides with transfer of
compounds which confer sludge and warmed defects to the extracted oil. Thus, the filtration
operation appears necessary, indeed is advisable before bottling and also before storage with
the scope to maintain a major shelf life, avoiding settling and frequent decanting operations,
which in turn induce a higher oxidation. The industrial filtration can be conducted by several
systems, which differ for used material: hydrophilic cotton or cellulose at high porosity are
better than fossil flour which reduces the antioxidant property because acts directly on o-
diphenols, decreasing the amount in the olive oil and so its shelf life (Ricci, 2007).
The oil storage is favoured by the antioxidants content that prevents the rancidity, but their
activity initially slows and then stops with successive formation of free radicals. This action
can be enhanced by improper storage practices: for example, when storage temperature is
not controlled, or when the oil is held in contact with the direct light and or at high
temperatures at consumers house, or when an improper sealing is applied after the first
container opening. Concerning this last aspect, the use of screw caps is more advisable than
metal pourers which expose the oil to the oxidative agents.

As the extra virgin olive oil extraction is conducted with controlled temperatures lower than
28 C, as the extra virgin olive oil storage requests the same attention. It has to be done with
control of temperature, which may range from 10 C to 18 C: the correct storage
temperature is 14-15 C. The high temperatures increase the chemical variation velocity and
the major oil fluidity. This last effect promotes the oxygen permeation. When storage
temperature decreases at 8-9 C, white deposits can appear in the oil, due to triglyceride
crystallization. The higher is the content of saturated glycerides, the higher is the freeze
process. The step from the oil crystallization to the solidification by lower temperatures (3-4
C) slightly increases the oil stability to the oxidation and gives substantial modifications in
the sensory profile. If the clear colour maintains beside the transfer of solidified oils to
warmer places, it possible that a margarinization process involved or mucilage could be
present.
Finally, it is important that storing and packaging facilities respect hygiene and healthiness
standards. Aromatic polycyclic hydrocarbons and benzene present in the atmosphere are
absorbed by the oils contained in not hermetically sealed packages (Ricci, 2007).
5. Packaging of olive oil
After olive pressing, oils are transferred in tanks and after pouring they are put into storage
containers where they attain their sensory characteristics. Containers are of different
materials and capacity ; they are made mainly with AISI 304 inox steel which is also used for
the containers intended for olive oil clarification.
The packaging in the selling container is the last unit operation: it is very important for
product stability, maintaining high quality level and also value added, if carried out
properly.
Reduction of oxygen in the packaging headspace and light exposure are key factors in
lowering lipid oxidation and off-flavour development, thus keeping quality of olive oil.
5.1. Olive oil packaging method
The Regulation 1019/2002/CE (Official Journal European Union, 2002) sanctioned the
prohibition of the consumer sale of the on tap olive oils. This effect promotes the consumer
protection in product quality and the valorisation of farm identity. In this sense olive farms
can provide packaging or ask for it to external companies. As for the other foods, the oil
storage and packaging mill area must be maintained clean, ventilated, illuminated and all
the unit operations should follow the hygienic-health rules according to Regulation
852/2004/CE (Official Journal European Union, 2004). As previously discussed, olive oil
must be stored at constant temperature, about 14-15 C, protected from light and air. When
oils are stored in big containers (inox steel is the best material), these have to be preferably
maintained quite full or better with the headspace filled with nitrogen..
For smaller quantities, the bottling with depression and the pre-bottling with inert gas are
nowadays diffused by using bottlers apparatus , which saturate the headspace with nitrogen

in bottle filling operation. In particular, bottles are hooked up by a special machine, which
invert them and blow inside liquid nitrogen, which becomes gas when comes in contact
with the environment, dilutes and moves out the oxygen present in the free volume of the
bottle.
Then, bottle straighten up and proceeds toward the filling operation during which nitrogen
stays inside because is heavier than air. As the bottle fills up and the oil level goes up, the
nitrogen is carried outside avoiding anyway the oxygen penetration. The velocity of
successive capping guarantees that only presence of nitrogen between cap and oil (Soressi,
2009).
Using of inert gases, as argon and nitrogen, can solve many problems and provide an
optimal product storage in several production steps, as pressing, kneading and, most of all,
storage and bottling. A study demonstrated in fact, that extra virgin olive oils collected from
the previous harvesting season and stored under nitrogen atmosphere could be packaged in
glass bottles without appreciable quality changes, as compared with seasonal extra virgin
olive oil packaged in similar bottles (Guil-Guerrero & Urda-Romacho, 2009). Nitrogen is the
most used gas to protect oil from the air contact, able to remove oxygen by container
headspace and pre-formed volatile contaminants. Bottling lines are very versatile for
packaging typologies and easily cleanable. A past research on Italian plant typologies
revealed that the prevalent packaging line is the semi-automatic (50%), followed by the
automatic (27%) and manual (23%) ones. The first and the third show a low specialization
process level and a limited bottling capacity.
Bottle capping can be carried out by means of cork or more frequently by metal caps. A
good quality cork has not to be porous and present fungal contaminants, however it is not
suggested as may cause early deterioration, due to its high oxygen permeability. Metal caps
are provided of a screw and a plastic soft liner that permit a perfect airtight acting as
dripper. Also a thermally retractable capsule can be present on cap to seal the packaging
and improve the product from the esthetical point of view. Just these last are the most used
ones (72%) respect to ones with a simple screw (15%) and cork (11%) (Ricci, 2007).
5.2. Olive oil packaging materials
In Italy and in the world the majority of consumers purchases bottled olive oil. Nowadays
attention of many actors is focused on olive oil packaging, in particular on container
design that often communicates the natural concept and territory link associated to the
contained olive oil. Due to the changed life-style, oil container manifests also a functional
property.
The nature of the packaging material has a notable influence on oil quality (Gutierrez et al.,
1988). In general, a packaging is chosen depending on several criteria as product stability,
environmental conditions to which food will be exposed during storage and distribution,
and the product nature. In particular, the basic factors that may alter the quality of packed
oils are:

- Dissolved oxygen in the oil, that is the oxygen that remains in the container free space
after it is sealed and the oxygen diffused through the walls
- Light, which passes through containers, activates the oxidation process
- Trace metals, in particular copper and iron
- Autocatalytic oxidation
- Temperature during storage
- Humidity during storage
- Dissolution of some substances from the container to the oil (Richardson, 1976).
The principal used materials for olive oil packaging are represented by glass, steel, tinplate
and, in Italy to a lesser degree, plastic and polycoupled ones.
5.2.1. Glass
Glass represents an ancient material used in container structure: first glass containers
derived by rocks date back to 3000 BC. It is formed principally by silica obtained by sand,
flint or quartz. Silica is fused at very high temperatures (about 1720 C) to form silicated
glass. In most cases, silica is mixed with variable proportions to several raw materials: e.g.
sodium and potassium carbonates (which act as stabilizer and protect the glass from the
water solubilisation), lead (which confers transparency and lightness) and aluminium
(which increases its hardness and lastingness). Glass is neither in a solid nor in a liquid state
but glassy. Its molecules are arranged in disarray but with a sufficient cohesion to give
mechanical stiffness. The structural basic unit is represented by the silica-oxygen
tetrahedron, in which a silica atom is surrounded by four oxygen atoms to form a
tetrahedron. Big groups tend to dispose with disorder (amorphous structure) giving the
glass fragile with the tendency to rupture if submitted to an excessive tension. This induces
effects on its thermal resistance that is function of container type, packaging faces and
exposed surface to tension (internal if warmed and external if cooled) (Robertson, 2009).
In Italy glass is the king container for the olive oil packaging. Glass bottles can have
different forms and capacities and their colour can vary largely from white to green and
more dark tints. In Figure 1 several size of glass bottles are presented to demonstrate the
large offer in the Italian olive oil market also of aromatized oils with spices or hot pepper.
From an Italian research about the most diffused containers, 64%of olive farms use the green
glass whereas only the 10% of the total chooses the transparent one (Ricci, 2007). The best
containers for the olive oil are made in opaque and dark glass: it is advisable to prefer very
dark ones because little light can pass through. Since the consumer appreciates also the light
glass because it is transparent and so shows the oil colour, then it is advisable to supply the
bottle with a paper case which can protect the product by light. Guil-Guerriero and Urdo
Romacho (2009) reported a shelf life study carried out on oils produced from Picual,
Hojiblanca and Arbequina cultivars packaged in dark and transparent glass bottles. These oils
showed an increase in some parameters; the variation of peroxide value was significant in
extra virgin olive oils stored in transparent glass. Similar results were observed by Del Caro
et al. (2006) and Vacca et al. (2006) on Bosana extra virgin olive oils. Several studies

conducted on olive oil shelf life attested the glass as the best material for the storage
(Pristouri et al., 2010), especially when oil was stored in the dark (Kanavouras et al., 2004),
and for its acidity and peroxide value, with respect to other packages (Rababah et al., 2011).
Finally, in a comparative study among glass, High Density Polyethylene (HDPE) and PET,
results clearly indicated the glass was the best in the following ranking Glass > HDPE > PET
(Ben Tekaya et al., 2007).

Figure 1. Several types of commercial glass bottles for olive oil.
5.2.2. Steel and tinplate
First uses of this material date back to 1700 in domestic environment. Steel is an iron/carbon
alloy: the higher the carbon percentage, the higher the rupture limit.
The tinplate is a sheet of soft steel, which is more workable because of its lower carbon
content, coated on both faces by tin oxides layers and on face destined to the contact with
food also by organic synthetic lacquers. Tinplate container is mainly of rectangular shape; its
side seam is welded and protected with a food-approved special varnish: the welding
method assures a secure side seam, avoiding the dissolution of lead into the product (Tsimis
& Karakasides, 2002).
The inox steel is an alloy containing 11% of chrome which reacts with oxygen originating an
autopassivation condition due to the chrome oxides presence (Piergiovanni & Limbo, 2010).
It is the best material for the preservation of big volumes of olive oil. Compared to glass, it
has the same water resistant properties (so protects the product from oxygen, humidity and
microrganisms), and major advantages, e.g. handless, cleanness, shock resistance and total
light protection.
A research of Grover (1982) demonstrated that the quality of oils decreases if packed in re-
used tinplate containers, whereas new containers did not alter oils during the one year
storage period. A more recent study confirmed that the storage of oils in stainless and dark
glass appears more adequate, with respect to other packaging materials, as clear PET and
clear glass (Dabbou et al., 2011).

Tin plate is used for the olive oil retail both in bigger volume size (3-5 L) and in smaller
bottles (1 L). A recent study of Rababah et al. (2011) on olive oil samples stored for 60
months at light in bottles of 2.5 L denoted higher sedimentation amount in tinplate than in
the other containers and a strong reduction of sensory attributes, probably due to the
reaction of other components in olive oil, e.g. phenol compounds, with tinplate that
negatively affected the descriptive attributes.
5.2.3. Plastic materials
Plastic materials are partially or totally synthetic organic substances. Their principal
components are represented by polymers constituted by carbon, hydrogen and, in some
cases, oxygen, nitrogen, chloride, silica and sulphur. They have a relatively recent history
but in the time gained a large number of applications for their versatility, low production
cost, low weight, their good performance and their thermoplastic nature which guarantees
the recyclability and consequent low environmental impact.
Plastic containers are formed by blow-moulding (extrusion blow-moulding or injection
blow-moulding). One of their disadvantages is the wall permeability to gases and vapours.
If transparent, they also transmit light. Migration of small molecular weight substances (e.g.
monomers, oligomers and additives) from plastic to food can also occur, thus affecting the
quality of food (Tsimis & Karakasides, 2002). Oil contained in bottles with high air
permeability (PE, PP, etc.) should be sold within 4 weeks, in contrast to polyvinyl chloride
(PVC) bottles, which can hold olive oil for 3 months without appreciable quality loss.
PVC is a popular packaging material for edible oils in many countries, mainly due to its
transparency, adaptability to all types of closure, total compatibility with existing packaging
lines, and suitable for personalized design features (Kanavouras et al., 2004). PVC was used
in food packaging without any doubts in 1970s. Nowadays for the dissolution of Vinyl
Chloride Monomers (VCM) in oil during the storage time and for issues such as the
environmental protection, the ample supply, plastic shaping, and its mechanical properties,
PET has been supplanting PVC in the edible-oil market.
PET bottles are produced by bioriented extrusion and their advantages are: low water and
oxygen permeability, high hardness and stiffness. PET is also more resistant to oil and fats, than
the other plastic materials and for these properties it fits well for olive oil packaging. The
disadvantages of this material are the processing condition (to control the humidity content),
costs and the migration of acetaldehyde from bottle to food (Dipalma, 1986). As previously
affirmed, plastic materials are, however, porous and thus permit the penetration of humidity
and gases. Pristouri et al. (2010) demonstrated that between PET and PP, PET provided a better
protection to olive oil than the other one due to its significantly lower oxygen transmission rate.
Moreover Kiritsakis and Dugan (1984) reported that peroxide values were higher for olive oil
packaged in plastic containers as compared to those packaged in glass bottles in the dark.
Kanavouras et al. (2006) revealed instead that plastic containers had a particularly stronger
protective role when oil was stored in the light, with respect to transparent glass. Besides in
Italy the glass bottle is the most used, in other countries the olive oil packaging in plastic

containers is developing. Contemporary trends in olive oil packaging include dark coloured
glass bottles and PET bottles which have incorporated oxygen scavengers. These are used in
order to prolong the shelf life of foods whose degradation kinetic depends on the partial
pressure of oxygen inside the container. In particular, Del Nobile et al. (2003) confirmed that
oxidation kinetics slower than that found with glass bottles can be obtained by bottling the olive
oil into materials containing an oxygen scavenger. However, the slowest decay kinetics were
obtained by bottling the oil in PET containers and by reducing the oxygen concentration prior to
bottling to 10% of the equilibrium value. Also Gambacorta et al. (2004) studied the effect of
several percentages of oxygen scavengers and of barrier resin included in PET on olive oil
quality, and indicated that the materials having higher oxygen barrier properties can slow
down the quality decay kinetics of extra virgin olive oil.
5.2.4. Policoupled material
Policoupled materials are mainly represented by a triple coupled material formed by Low
Density Polyethylene (LDPE)/paper/LDPE and a thin aluminum foil between further
polyethylene layers as barrier respectively to gas, light and liquids. It is a parallelepiped
with rectangular section, the most known version of which is the Tetra Brik
, also named
Tetra Prisma.
Tetra Prisma allows an ideal storage for distributors and consumers. For the packaging of
vegetable oils including olive oil is being used today in some Mediterranean countries, e.g.
Spain and Greece, in the form of brick-type cartons and bag-in-box pouches. It is a very
revolutionary packaging because guarantees a total and prolonged protection of olive oil
chemical and physical characteristics up to two years. This material permits also to update
the packaging aspect, with a more coloured, snappy and modern graphic design. Besides
preserving the quality, Tetra Prisma allows the producer a major differentiation in
packaging, logistic efficiency, lower probabilities of rupture during transfer and, not least, a
100% recyclable package. From the consumers point of view, the pack is indestructible, easy
to take and handle. The realized cap contributes to protect from oxygen and it is for a
prolonged use, with the aim to avoid the drip, the product spill after opening and to
facilitate the pouring, regulating the flow and cutting the drip. In Italy, the olive oil
packaged in Tetra Prisma was proposed in 2005 with opposite considerations (Soressi, 2007).
Studies have been conducted also on this material type to evaluate its performance as
container for oil. It is efficient in maintaining antioxidants in stored product for several
months, due to its efficient barrier to light and oxygen, unlike the transparent glass and
plastic materials, despite these last show a low gas permeability (Mendez & Falque, 2007).
5.3. Olive oil packaging shape and size
Glass is the preferred packaging material in Italy for olive oil, and the most used bottle
shape is the marasca type. It is chosen for three aspects: small price, the best shelf
management and the functionality. The most diffused bottle volume is 0.75 L, followed by
0.5 L; the use of other volumes is very marginal. A research developed in 2005 on 48

producers of biological oil in the South and the Centre of Italy indicated that the bottle
largely used is that of 0,75 L (about 85% of the total), followed by 0,50 L (about 58%) and 0,1
L (about 17%) ones (Paffarini, 2007). Low capacity packaging is gaining market approval
above all for exported products and for those directed to a medium-high consumer segment.
Packaging in 0,1 L bottles is also used for very valuable oil to evidence the product value.
Producers propose on market sophisticated packaging created properly to contain one or
more bottles, as those for tasting of 6 mL volume. For the other materials (steel, tinplate and
plastic) the most used volumes have been previously cited.
It is noteworthy, the recent distribution of 10 mL single-dose sachets, constituted by 100%
extra virgin olive oil or olive oil mixed with vinegar, that is illustrated in Figure 2 with
tinplate and PET containers. This type of packaging developed after the coming into effect
of Italian Law 81/2006 (Official Bulletin, 2006), which bans the use of of olive oil containers
without label in restaurants .
Moreover containers that can be used without common rules of hygiene and safety are also
prohibited. So, it is admitted to present bottles provided of label (although their filling up is
also allowed) and single-dose sachets formed by appropriate machines, which guarantee
more safety and traceability of their material and because they are unable to be reused in
fraudulent practice. The use of cruets is so banned because the origin of contained oil is
impossible to be easily traced.
With the increase of container typologies and the research of more refined packaging in
materials and graphic the supplementary cost merges into the final price that is, however, a
product quality indicator (Paffarini, 2007).

Figure 2. PET, tinplate and single-dose sachets as olive oil packaging

5.4. Olive oil labelling
The olive oil labelling has two principal functions: safety for consumer and aesthetic quality.
It has to follow legal requirements and so present several technical informations, some of
these facultative, other obligatory. For the graphic point of view, it results as presentation of
the olive oil in market shelves.
When consumer purchases olive oil, he is also more interested on symbolic contents
reported in label, as the original production territory and extraction process, with respect to
the sensory and chemical-physical quality of product. The oil packaging in fact satisfies
curiosity about origin and characteristics by means of label, back label or attached paper,
especially buying particular containers in the organized distribution. Thus, packaging
elements have gained a relevant role to communicate not only product characteristics but
also its history, playing on emotions that are much linked to biological oils, for example, of
which label can evocate proper quality of biological agriculture.
The new Regulation 182/2009/CE (Official Journal European Union, 2009) established that
labelling must obligatory indicate the extra-virgin and virgin olive oil origin. This rule does
not regard a protected geographical indication (PGI) or a protected designation of origin
(PDO) qualified olive oils because they are submitted to another specific normative, that is
the Regulation 510/2006/CE (Official Journal European Union, 2006).
Three typologies were defined by Regulation 182/2009/CE to indicate olive oil origin:
1. Oil obtained in the same member State where olives are harvested: in this case origin
can be recalled indicating State name followed by phrases as Produced in, Obtained
in ., and also 100% produced in . The member State name can be substituted by
a Communitarian reference. Then using of produced in Italy is allowed only if olives
are produced and milled in Italy for the 100%
2. Oil obtained in a member State by olives derived from other member States: in this case
the origin indication can be referred as (Extra) virgin olive oil obtained in ..by olives
harvested in . The member State name can be substituted by a Communitarian
reference If it is necessary indication of more States, they are cited in decreasing order
in relation to the brought quantity
3. Mix of Communitarian and/or not Communitarian oils: in this case origin indication
mode is one of the following:
a. Mix of Communitarian olive oils, or a reference to the Community
b. Mix of not Communitarian olive oils, or a reference to the not Communitarian origin
c. Mix of Communitarian and not Communitarian olive oils, or a reference to
Communitarian and not Communitarian origin.
Regarding the indication about sensory characteristics, the Regulation 182/2009/CE
sanctions that they must be reported only in labelling of extra-virgin and virgin olive oils,
besides confirm of the voluntariness. Moreover, adjectives related to a positive attribute for
the oil (e.g. intense, medium, light, linked to fruity, green, ripe, etc.) are admitted
exclusively if derived by an objective evaluation based on the International Olive Oil

Council method described in XII attachment of Reg. 2568/91/CE (Official Journal European
Union, 1991) and revisions. The Regulation 182/2009/CE is a sort of Italian victory that can
let acknowledge in a Communitarian regulation the possibility to indicate the production
area of olive oils.
The question of mixing of olive oil with other vegetable oils is also well defined in the new
Regulation that establishes that member States can ban in the territory the production of mix
for internal consumption. Instead, they cannot prohibit on the territory the sale of mixes
produced by other States and ban the mix production for the commercialization in another
member State or the exportation. This regulation stopped an age-old argument in which
Italian farms were penalized in particular in not Communitarian markets where did not
compete at par with other member States for offer of oil mixtures. This product type is in
fact requested particularly in USA and Asiatic markets.
In olive oil packaging labelling is distinguished in:
- Principal, normally rectangular, big, obligatory, that refers all terms requested by
regulations
- Back label, not ever present, smaller and collocated generally on the back of the bottle.
It is obligatory to indicate on the label:
Commercial and sale denomination of oil
Oil category according to Reg. 1019/02/CE art. 3 (Official Journal European Union,
2002). For extra virgin olive oil: Olive oil of superior category obtained directly by
olives and only by mechanical extraction
Production or packaging plant location
Packaging batch
Product volume (e.g. 0,75 L followed by symbol)
Preferred consume date (as consume preferably up to the end.month and year)
Storage conditions (keep in a dry environment away from light and heat)
Recommendation "after using not disperse into environment"
Other facultative indications as:
"First cold-pressing" and "cold-pressed" are reserved to virgin oils obtained by
extractions not above 27 C. To guarantee the truth of their declaration, olive farm
owners could indicate the installed techniques for each stock of processed olives.
The term extracted could be substituted by obtained or produced
Acidity could be reported only if accompanied by peroxides value, waxes content
and spectrophotometrical values ( K232, K270, Delta K ). Values can be followed
by the phrase maximum values at packaging"
Polyphenol content only for olive oil that contains almost 5 mg of hydroxytirosol
and its derivatives per 20 g of olive oil. This indication must be accompanied by
information to the consumer that the beneficial effect is obtained after a daily
consumption of 20 g of olive oil. This last parameter has been fixed by a recent
Regulation (Official Journal European Union, 2012) on allowed healthy information
on food products.

Anyone who reports acidity and other parameters in labelling must organize the necessary
evidence to prove the conformity of product to indicated limits.
6. Conclusions
Olive oil storage and packaging are final steps of the production process and are as
important as the other ones. Deterioration agents can decrease the quality of this important
food also during these unit operations, so a correct control and monitoring of some
indicators can be useful to the olive oil shelf life. Storage environment and its characteristics
(temperature most of all) contribute to it.
Packaging typologies also influence the stored olive oil properties with different results
depending on materials. During the time various containers were adopted and evaluated
with evident feedback for qualitative aspects (some material have been in fact banned for
healthy causes). Moreover, the traditional uses can give to different preferences (e.g. in Italy
is very difficult to substitute the glass apart from its ascertained value as packaging material
for olive oil).
Future developments are surely expected because of many attentions on this subject also by
scientific community and probably some materials will be improved and other container
types will be proposed in a more and more advanced food market.
Author details
Dipartimento di Biotecnologie per il Monitoraggio,
Agroalimentare e Ambientale (Bio.M.A.A.), Mediterranean University of Reggio Calabria, Italy
7. References
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virgin olive oil stability measured by Rancimat, Journal of Agricultural and Food
Chemistry Vol. 47: 4150-4155.
Ben Tekaya, I., Ben Tekaya Ben Amor, I.,.Belgaied, S., El Atrache, A. & Hassouna M. (2007)
tude du conditionnement de lhuile dolive dans les emballages en plastique. Science
des Aliments Vol. 27 (No.3): 214-233.
Cinquanta, L., Esti, M., & La Notte, E. (1997) Evolution of phenolic compounds in virgin
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27.
Chapter 11
Sensory Analysis of Virgin Olive Oils
Innocenzo Muzzalupo, Massimiliano Pellegrino and Enzo Perri
1. Introduction
The evaluation of the sensory properties and determination of the importance of these
properties in consumer product acceptance represent a major accomplishment in sensory
analysis.
Most of the sensory evaluation carried out by a trained panel involves measurement in two
main areas, difference testing and descriptive analysis. Sensory difference tests are
procedures used to determine whether judges can distinguish between two similar stimuli. In
terms of food, the two stimuli are two very similar food samples. These evaluations are used
to determine whether slight changes occur due to either product reformulation or the change
in technological processing. Difference tests are particularly well adapted to the assessment of
vegetable oils during their processing, being used to control refining efficiency.
Furthermore, they may be used to measure slight flavour variation determined by changes
in storage or packaging. There are many different methods for sensory difference testing.
They are described in detail in several texts (Amerine et al., 1965; Lawless and Heymann,
1998; Meilgaard et al., 1999).
Descriptive analysis, the other main area of sensory evaluation, precisely describes and
measures the sensory attributes of food. It follows that this can be done only with trained
judges. There are various methodologies that can be used. One procedure is the Quantitative
Descriptive Analysis (QDA) developed by the Tragon Corporation of Palo Alto, California
(Stone et al., 1974). Judges examine the food and list all the relevant sensory properties. They
then use standards to obtain agreement on the descriptive terms being used. By using a
scaling procedure, the judge estimates the intensity of each sensory attribute considered.
The raw data obtained are then evaluated through common statistic techniques.
There are several applications for descriptive analysis: product development and
assessment, comparison of the sensory properties of products which cannot be compared

simultaneously (e.g., fresh olive virgin oil from consecutive years), shelf-life studies,
correlation of instrumental and sensory properties, studies of the effects of technological
processing on the sensory characteristics of a product, quality assurance, certification of a
preset quality standard, etc.
Another branch of sensory analysis is represented by consumer science. Generally, people eat
and drink food because they like it. But, food intake is not completely driven by hedonic
motives. Other factors such as market segmentation, advertising, price, packaging, opinions
and beliefs play a role too. The sensory perception of sensory quality is an important factor in
motivating consumer choice. It derives from the intrinsic properties of the product, perceived
by the consumer at the moment of buying (colour, shape, aspect, etc) and subsequently from
direct individual experiences (odour, taste). The measurement of liking is necessary before a
product is launched onto the market, with substantial capital being needed. This can therefore
save investing in a product that may not be liked due to a deficiency of sensory qualities.
The most frequently used method to measure acceptability and preference is the 9-point
hedonic scale, developed in 1955 (Jones et al., 1955). The subjects that take part in the sensory
testing are not trained, and should be relatively naive to this kind of task.
2. Sensory analysis of olive oil
The European Commission (EC) upon issuing the following regulations:
Commission Regulation n. 2568/91 of 11 July 1991 (EC Regulation, 1991);
Commission Regulation n. 2472/1997 of 11 December 1997 (EC Regulation, 1997);
Commission Regulation n. 796/2002 of 06 May 2002 (EC Regulation, 2002);
Commission Regulation n. 1989/2003 of 06 November 2003 (EC Regulation, 2003);
Commission Regulation n. 1640/2008 of 04 July 2008 (EC Regulation, 2008);
Commission Regulation n. 61/11 of 24 January 2011 (EC Regulation, 2011);
set the objective of establishing and developing the criteria needed to evaluate the chemical
and sensory characteristics of oil and virgin olive oil as well as the opportune methodology.
The introduction of the Panel Test has led to an evolution in the concept of oil quality. The
necessary cognitions are set in order to carry out the sensory analysis of virgin olive oil. It
tries to standardise the behaviour and procedures of the tasters, who should take into
consideration not only the more general indications but also those specific for tasting olive
oil. These procedures have led to an evaluation sheet being drawn up by the International
Olive Oil Council (IOOC), leading to results being obtained from different Panels, in
different areas of the same country as well as different countries to be compared.
Since 1991, this methodology has been part of the regulations of the European Commission
for classifying oils and is described in detail in Appendix II of the EU Regulation n. 2568/91.
The method, Quantitative Descriptive Analysis (QDA), defines the main attributes of oil,
both positive as well as negative. Appearance as colour was not selected as a quality
parameter of virgin olive oil, as a specific dark-coloured glass is used. The official evaluation

Sensory Analysis of Virgin Olive Oils 225
sheet used within the European Union to establish the sensory profile of virgin olive oil is
shown below (Fig. 1). The values, expressed as centimetres, are statistically processed to
calculate the median of each positive and negative characteristic.

Figure 1. Sheet for sensory analysis

Sensory analysis is an essential technique to characterize food and investigate consumer
preferences. International cooperative studies, supported by the International Olive Oil
Council (IOOC) have provided a sensory codified methodology for virgin olive oils, known
as the COI Panel test. Such an approach is based on the judgments of a panel of technicians,
conducted by a panel leader, who has sufficient knowledge and skills to prepare sessions of
sensory analysis, motivate judgement, process data, interpret results and draft the report.
The panel generally consists of a group of 8 to 12 persons, selected and trained to identify
and measure the intensity of the different positive and negative sensations perceived.
Sensory assessment is carried out according to codified rules, in a specific tasting room,
using controlled conditions to minimize external influences, using a proper tasting glass and
adopting both a specific vocabulary and a profile sheet that includes positive and negative
sensory attributes. Collection of the results and statistical elaboration must be standardized.
The colour of virgin olive oil, which is not significantly related to its quality, may produce
expectations and interferences in the flavour perception phase. In order to eliminate any
prejudices that may affect the smelling and tasting phases, panel lists use a dark-coloured
(blue or amber-coloured) tasting glass.
The panel leader is the person responsible for selecting, training and monitoring tasters to
ascertain their level of aptitude according to (IOOC/T.20/Doc.15/rev.3). The number of
candidates is generally greater than that needed in order to select people that have a grater
sensitivity and discriminatory capability. Screening criteria of candidates are founded on
sensory capacity, but also on some personal characteristics of candidates. Given this, the
panel leader will personally interview a large number of candidates to become familiar with
their personality and understand habits, hobbies, and interest in the food field. He uses this
information to screen candidates and rejects those who show little interest, who are not
readily available or who are incapable of expressing themselves clearly.
The determination of the detection threshold of the group of candidates for characteristic
attributes is necessary because the threshold concentration is a point of reference common
to a normal group and may be used to form homogeneous panels on the basis of olfactory-
gustatory sensitivity.
A selection of tasters is made by the intensity rating method, as described by Gutirrez
Rosales (Gutirrez Rosales et al., 1984). A series of 12 samples is prepared by diluting a
virgin olive oil characterized by a very high intensity of a given attribute in an odourless
and tasteless medium (refined oil or paraffin). The panel leader sends out the candidate,
removes one of the 12 tasting glasses from the series, and places the remaining together; the
candidate is called back in the room and is asked to correctly replace the testing glass
withdrawn from the series by comparing the intensity of this last with that of the others. The
test is carried out for fusty, rancid, winey and bitter attributes to verify the discriminating
capacity of the candidate on the entire scale of intensities.
The stage training of technicians is necessary to familiarize tasters with the specific sensory
methodology, to heighten individual skill in recognizing, identifying and quantifying the
sensory attributes and to improve sensitivity and retention with regards to the various

attributes considered, so that the end result is precise and consistent. In addition, they learn
to use a profile sheet.
The maintenance of the panel is made through continuous training during the entire life of
the same panel, the check of the sensory acuity of tasters, and exercises that allow the
measurement of panel performance.
Every year, every panel must assess a number of reference samples in order to verify the
reliability of the results obtained and to harmonize the perception criteria; they must also
update the Member State on their activity and on composition changes of their group.
3. Test conditions
The test conditions for tasting are described in COI/T.20/Doc. No. 15/Rev. 3:
The oil sample for analysis shall be presented in standardised tasting glasses conforming to
the standard COI7T.20/Doc. No. 5 (Fig. 2).

Figure 2. Tasting glass
The glass shall contain 14-16 ml of oil, or between 12.8 and 14.6 g if the samples are to be
weighed, and shall be covered with a watch-glass. Each glass shall be marked with a code
made up of digits or a combination of letters and digits chosen at random.

Figure 3. Heating the samples when in the glass

The oil samples intended for tasting shall be kept in the glass at 28 C 2 C throughout the
test (Fig. 3).
This temperature has been chosen because it makes it easier to observe organoleptic
differences than at ambient temperature and because at lower temperature the aromatic
compounds peculiar to these oils volatilize poorly while higher temperatures lead to the
formation of volatile compounds peculiar to heated oils. The test room must be at a
temperature between 20 and 25 C.
The morning is the best time for tasting oils. It has been proved that there are optimum
perception periods with regards to taste and smell during the day. Meals are preceded by a
period in which olfactory-gustatory sensitivity increases, whereas afterwards this perception
decreases. However, this criterion should not be taken to the extreme where hunger may
distract the tasters, thus decreasing their discriminatory capacity; therefore, it is recommended
to hold the tasting session between 10:00 in the morning and 12:00 noon (Fig. 4).

Figure 4. Cabins of the test room
The following recommendations apply to the conduct of the tasters during their work.
When called by the panel leader to participate in an organoleptic test, tasters should be able
to attend at the time set beforehand and shall observe the following:
- they shall not smoke or drink coffee at least 30 minutes before the time set for the test;
- they must not have used any fragrance, cosmetic or soap whose smell could linger until
the time of the test;

- they shall fast at least one hour before the tasting is carried out;
- should they feel physically unwell, and in particular if their sense of smell or taste is
affected, or if they are under any psychological effect that prevents them from
concentrating on their work, the tasters shall refrain from tasting and shall inform the
panel leader accordingly;
- when they have complied with the above, the tasters shall take up their place in the
booth allotted to them in an orderly, quiet manner;
- they shall carefully read the instructions given on the profile sheet and shall not begin to
examine the sample until fully prepared for the task they have to perform (relaxed and
unhurried). If any doubts should arise, they should consult the panel leader in private;
- they must remain silent while performing their tasks;
- they must keep their mobile phone switched off at all times to avoid interfering with
the concentration and work of their colleagues.
4. Technique for tasting of virgin olive oil
The technique applied for tasting is the one described in annex XXI of (EEC) Reg. N.
2568/91:
the taster shall pick up the glass (Fig. 2), keeping it covered with the watch glass, and shall
bend it gently fully in this position so as to wet the inside as much as possible.
Once this stage is completed, he shall remove the watch-glass and smell the sample taking
even, slow deep breaths until he has formed a criterion on the oil under assessment.
Smelling shall not exceed 30 seconds. If no conclusion has been reached during this time, he
shall take a short rest before trying again (Fig. 5).

Figure 5. The olfactory test during

When the olfactory test has been performed, the taster shall then judge the flavour (overall
olfactory-gustatory-tactile sensation). To do so, he shall take a small sip of approximately 3
ml of oil. It is very important to distribute the oil throughout the whole of the mouth
cavity, from the front part of the mouth and tongue along the sides to the back part and to
the palate support, since it is a known fact that the perception of the four primary tastes,
sweet, salty, acid and bitter varies in intensity depending on the area of the tongue and
palate.
It should be stressed that it is essential for a sufficient amount of the oil to be spread very
slowly over the back of the tongue towards the throat while the taster concentrates on the
order in which the bitter and pungent stimuli appear; if this is not done, both of these
stimuli may escape notice in some oils or else the bitter stimulus may be obscured by the
pungent stimulus.
Taking short, successive breaths, drawing in air through the mouth, enables the taster not
only to spread the sample extensively over the whole of the mouth but also to perceive the
volatile aromatic components via the back of the nose.
Tactile sensation shall also be taken into consideration. Consequently, fluidity, stickiness
and sharpness or sting shall be noted down when detected, and if required for the test, their
intensity shall be quantified.
When organoleptically assessing a virgin olive oil, only one sample shall be evaluated in
each session to avoid the contrast effect that could be produced by immediately tasting
other samples.
As successive tasting produces fatigue or loss of sensitivity, it is important to use a product
that can eliminate the remains of the oil from the preceding tasting from the mouth.
The use of a small slice of green apple (about 15 g) is recommended which, after being
chewed, can be spat out into a spittoon. Then the mouth should be rinsed out with a little
water at ambient temperature. At least 15 minutes should lapse between the end of one
session and the start of the next.
The panel leader will collect the profile sheets completed by each taster and review the
intensities assigned to the different attributes. Should they find any anomaly, they shall
invite the taster to revise his or her profile sheet and, if necessary, to repeat the test.
The panel leader will enter the assessment data of each panel member in a computer
program like that appended to this method with a view to statistically calculating the results
of the analysis, based on their median.
5. Definition and classification of olive oil.
Virgin olive oil is the oil obtained from the fruit of the olive tree either by mechanical or
other physical means under conditions, particularly thermal conditions, that do not lead to

alterations in the oil, and which has not undergone any treatment other than washing,
decantation, centrifugation and filtration.
In Table 1, the various classes of olive oil as divided by European legislation are reported.
The subdivisions in different classes are based on the degree of acidity as well as other
analytical parameters and sensory evaluation indices.
1. Extra virgin olive oil: is the virgin olive oil which has a free acidity, expressed as oleic
acid, of not more than 0.8 gram per 100 grams, and the sensory characteristics with
median defects is 3.5 and the median of fruitiness greater than 0.
2. Virgin olive oil: is the virgin olive oil which has a free acidity, expressed as oleic acid, of
not more than 2 grams per 100 grams and the sensory characteristics with median
defects greater than 0, but less than or equal to 2.5 and the median of fruitiness greater
than 0.
3. Lampante olive oil: is the virgin olive oil which has a free acidity, expressed as oleic acid,
of more than 2 grams per 100 grams and the sensory characteristics with median defects
greater than 3.5 or if the median defects is less than or equal to 3.5 and the median of
fruitiness is 0. Such olive oil is intended for refining purposes.
4. Refined olive oil: is the olive oil obtained from lampante olive oils by refining methods
which do not lead to alterations in the initial glyceridic structure, which has a free
acidity, expressed as oleic acid, of less than or equal to 0.3 grams per 100 grams.
5. Blended olive oil: is the oil consisting of a blend of refined olive oil and virgin olive oil
which has a free acidity, expressed as oleic acid, of less than or equal to 1 grams per 100
grams. It can be used for human consumption.
6. Crude olive-pomace oil: is the oil obtained by treating olive pomace with solvents, to the
exclusion of oils obtained by re-esterification processes and of any mixture with oils of
other kinds. This oil is intended for refining with a view to its use in food for human
consumption.
7. Refined olive-pomace oil: is the oil obtained from crude olive-pomace oil by refining
methods which do not lead to alterations in the initial glyceridic structure, which
has a free acidity, expressed as oleic acid, of less than or equal to 0.3 grams per 100
grams.
8. Olive-pomace oil: is the oil comprising the blend of refined olive-pomace oil and virgin
olive oil which has a free acidity, expressed as oleic acid, of less than or equal to 1 gram
per 100 grams. It can be used for consumption, but in no case should this blend be
called olive oil.
One important consideration is that the first two oils are the best for human consumption
due to them being within the set parameters, when obtained directly from the olive press.
The third type of oil cannot be consumed until it has been rectified, which gives the fourth
type of oil. A small amount of extra-virgin olive oil or virgin olive oil is added to this
rectified oil and is known as Olive oil.


Table 1. Analytical and sensory properties of olive oils

6. Olive oil tasting attributes
Olive oil as judged by experts shows a multitude of either positive or negative
characteristics.
6.1. Positive attributes
Almond: light smell recalling that of fresh or dried almond.
Apple: a sensation recalling this fruit.
Artichoke: a smell recalling raw artichoke.
Astringent: a puckering sensation in the mouth created by tannin.
Bitter: this is a preferred characteristic taste of olive oils, if it is not too highly intense.
Fruity: range of smells (dependent on variety) characteristic of oil from healthy fresh
fruit, green or ripe, perceived directly and/or retro nasally. Fruitiness is qualified as
green if the range of smells is reminiscent of green grass. Fruitiness is qualified as ripe if
the range of smells is reminiscent of ripe fruit and is characteristic of oil from green and
ripe fruit.
Green grass: a sensation recalling that of freshly cut grass.
Hay: a smell recalling that of dried grass.
Spicy: a tactile sensation similar to that of a light chilli pepper, especially in the back of the
throat, which can force a cough.
6.2. Negative attributes
Brine: salty taste of oil made from brined olives.
Coarse: a tactile sensation in the mouth due to texture of oil.
Cucumber: off flavour from prolonged storage, particularly in tin.
Dreggiest: odour of warm lubricating oil and is caused by the poor or lack of the decanting
process.
Earthy: this term is used when oil has acquired a musty humid odour because it has been
pressed from unwashed, muddy olives.
Esparto: hemp-like smell acquired when olive paste has been spread on Esparto mats. Smells
may differ according to whether the mates are green or dried.
Hemp: caused by the use of filtering panels, which are not perfectly clean, and recalls
hemp.

Flat: oils which have lost their characteristic aroma and have neither taste nor smell.
Frozen: due to olives which have been exposed to freezing temperatures. When cooked, this
oil gives off very unpleasant odours.
Fusty: due to olives fermenting in piles while in storage waiting to be pressed.
Grubby: smell imparted by grubs of the olive fly. The smell is both rotten and putrid at the
same time.
Heated: prolonged heating during extraction processing.
Muddy: typical odour of oil that has been stored to long on its own sediment.
Musty: mouldy smell from olives being stored too long before pressing.
Metallic: oils processed or stored with extended contact to metal surfaces.
Rancid: old oils that have started oxidizing due to exposure to light or air.
Vegetable water: oils that have absorbed the unpleasant odours and flavours of the vegetable
water after pressing that they have remained in contact for too long.
Wine-vinegar: typical odour of wine or vinegar due to fermentation of olives.
7. Factors that affect the sensory quality of virgin olive oil
The evaluation of exactly how various factors affect the sensory quality of the final product
is essential in order to distinguish among different types of oil. The following factors all play
a role in producing high quality olive oil (Angerosa, 2002; Angerosa et al., 2004, Benincasa et
al., 2011).
Cultivar. The numerous varieties constitute an important element for the production of extra
virgin olive oils, characterised by different organoleptic characteristics.
Cultivation Techniques (irrigation, fertilization, treatment of plants, diseases etc). Among the
environmental factors that influence the quality of the olives and therefore the oil, both the
temperature and the amount of water available have an important role, with the first
affecting the acidic composition of the olives, while the latter the amount of phenolic
substances.
Maturation of the olive. An early harvest generally gives a more bitter and spicy oil due to the
high phenol content.
Harvest and Storage of the olive. The quality of the oil is highly conditioned by the state
of integrity of the olive. Traditional manual harvesting techniques avoid damaging
the fruit in comparison to mechanical methods. Storage of the olives in not very big
crates, avoids an excessive mass of olives that could either become crushed or

overheated, facilitating attacks from micro-organisms as well as oxidation and
fermentation.
De-leafing and washing of the olive. Before being processed, the olives must be cleaned of any
superfluous material, including leaves, and branches. These are all elements that can
negatively influence the quality of the oil.
Pressing. The olives are broken during the pressing phase with the skin and the pulp being
lacerated as well as the stone crushed. The press can be a traditional pan-mill one, either
in a discontinuous system or combined with an extraction system in order to carry out
continual centrifugation. These presses can either be hammers or disks. Metal presses have a
more violent pressing of the olive (above all hammered ones) as well as a greater laceration
of the skin, giving a higher extraction of the phenolic composites and therefore a more bitter
and spicy oil that lasts longer.
Kneading. Prolonged kneading and high temperatures could increase the activity of pectoltic
and proteolitic enzymes, negatively modifying the chemical-physical characteristics and
therefore the quality of the oil.
Extraction. The systems to separate the liquid from the solid can be divided into two
groups:
a. mechanical pressure on the paste through a series of operations that make the process
discontinuous,
b. by centrifugation that is continuous. The extraction process by pressure has the risk of
contaminating the oil due to wear and dirtying of the filters. The continual
centrifugation system guarantees greater hygiene and therefore gives oil with elevated
qualitative characteristics.
Centrifugation. The oily liquid contains a certain amount of water (called of vegetation)
that is eliminated by centrifuging the product. This operation allows the suspended solid
substances to also be eliminated. Water is often added in order to rid the oil of the watery
impurities. However, this reduces the phenolic substances content.
Clarification and filtration. The oil obtained from centrifugation still contains mucilage, water
and small pieces of the fruit. It is also turbid and opalescent. A clarification process is then
carried out in order to eliminate these substances that can favour hydrolysis and/or
oxidation. Traditional clarification methods include sedimentation, and have now been
substituted by filtration. Light filtration systems are preferable rather than more drastic
ones that can provoke a reduction in the anti-oxidants and subsequently a reduced shelf-life
with the possibility of turning rancid.
Conservation of the oil. In order to maintain both the chemical-physical and organoleptic
properties of the oil, the conservation conditions must be controlled. The main factors
affecting the conservation of oil are:

a. the temperature (12-15C),
b. light (oil should be stored in the dark otherwise the photo-oxidization process on the
polyunsaturated fatty acids can determine the rancid defect),
c. oxygen in the air (a series of oxidation reactions occur when the oil comes into contact
with the air, modifying the chemical composition and subsequently the colour, smell
and flavour). It is therefore good practice to store extra virgin olive oil in a sealed
environment.
8. Conclusion
Virgin olive oil is the only product that since 1991 has undergone sensory analysis
regulated by European norms. It can be noted that the methodology for the sensory
evaluation of virgin olive oil is well documented and standardized. Sensory analysis of
olive oil is the evaluation of an oils organoleptic attributes, which are appreciated through
the senses of smell and taste. Sensory analysis is an essential part of evaluating olive oil
quality and complements chemical analysis, both of which are requirements for
determining the quality of olive oil according to International Olive Council (IOC) and new
USDA standards.
The studies on olive oil have highlighted that the sensory qualities are affected by several
agronomic and climatic parameters as well as extraction process. Due to here being a
variation in the chemical composition of the oil, there is a possibility that these chemical
compounds could be considered markers to be used to differentiate the various factors that
affect overall quality.
The results of the quantitative descriptive analysis carried out by 9 members of staff of the
CRA-OLI on the virgin olive oils produced by single varieties cultivated in the collection
field of CRA-OLI are given in the elaiographic cards attached to Chapter Description of
varieties
Author details
Innocenzo Muzzalupo
*
, Massimiliano Pellegrino and Enzo Perri
Acknowledgement
The authors thank all tasters and the panel leader of the panel at the CRA-OLI. The author
thank the Projects: CERTOLIO, GERMOLI and OLIOPIU for financial support.

*

9. References
Amerine, M.A.; Pangborn, R.M. & Roessler, E.B. (1965). Principles of sensory evaluation of
food. Academic Press, New York, ISBN 0120561506.
Angerosa F. (2002). Influence of volatile compounds on virgin olive oil quality evaluated by
analytical approaches and sensor panels. European Journal of Lipid Science and Technology,
Vol. 104, pp. 639660, ISSN 1438-9312.
Angerosa, F.; Servili, M; Selvaggini, R.; Taticchi, A.; Esposto, S. & Montedoro, G.F. (2004).
Volatile compounds in virgin olive oil: occurance and their relationship with the
quality. Journal of Chromatography A, Vol. 1054, pp. 17-31, ISSN 0021-9673.
Benincasa, C.; Russo, A.; Romano, E.; Elsorady, M.E.; Perri, E. & Muzzalupo I. (2011).
Chemical and sensory analysis of some egyptian virgin olive oils. Journal Nutrition &
Food Sciences, Vol. 1, pp. 1-8, ISSN 0034-6659.
European Community, Commission Regulation (1991) 2568/91. On the characteristics of
olive oil and olive residue oil and on the relevant methods of analysis. Official Journal of
the European Communities, July 11, L248, 1-83.
European Community, Commission Regulation (1997) 2472/97. Amending Regulation
No 2568/91/EEC. Official Journal of the European Communities, December 11, L341, 25-
39.
European Community, Commission Regulation (2002) 796/2002. Amending Regulation No
2568/91/EEC. Official Journal of the European Communities, May 6, L128, 8-28.
European Community, Commission Regulation (2003) 1989/2003. Amending Regulation
No 2568/91/EEC. Official Journal of the European Communities, November 6, L295, 57-
77.
2568/91/EEC. Official Journal of the European Communities, July 4, L178, 11-16.
2568/91/EEC. Official Journal of the European Communities, January 24, L23, 1-14.
Gutirrez-Rosales, F.; Risco, M.A. & Gutirrez Gonzlez-Quijano, R. (1984). Seleccin de
catadores mediante el mtodo de clasificacin por intensidad. Grasas y Aceites, Vol.35,
pp. 310-314, ISSN 0017-3495.
International Olive Oil Council (2007). Glass for oil tasting. IOOC/T.20/Doc. 5/rev.1
International Olive Oil Council (2010). Sensory analysis of olive oil. Method for the
organoleptic assessment of virgin olive oil. IOOC/T.20/Doc. No 15/Rev. 3.
Jones, L.V.; Peryam, D.R. & Thurstone, L.L. (1955). Development of a scale for measuring
soldiers food preferences. Food Research, Vol. 20, pp. 512-520.
Lawless, H.T. & Heymann, H. (1998). Sensory evaluation of food: principle and practices.
Ed. Chapman & Hall. New York, ISBN 0-412-99441-0.
Meilgaard, M.; Civille, G.V. & Carr, B.T. (1999). Sensory evaluation techniques. 3rd ed., CRC
Press, Boca Raton, Florida, ISBN 0-8493-0276-5.

Stone, H. & Sidel, J.L. (2004). Sensory evaluation practise. 3
nd
ed., Academic Press, San
Diego, CA, ISBN 0-12-672690-6.
Chapter 12
Modern Methodologies to
Assess the Olive Oil Quality
1. Introduction
1.1. Food quality and safety assessment
Food quality and safety assessments are the main issues that modern food industries are
required to fulfill in their production chains, being these properties directly correlated to the
claims of a holistic nutrition representing the fundamentals of a healthy balanced diet. In the
same direction goes, very recently, the need of checking the origin of an aliment even this is
aspect is apparently not directly correlated to the main issues above presented.
All the steps of olive production chain require, as for any other food, a careful evaluation of
the identity and absolute amounts of micro and macro components at the molecular level.
Protect Denomination of Origin (P.D.O.), Protected Geographical Identification (I.G.P.) and
other acronyms associated to olive oil quality, are often based on the apparent evaluation of
macroscopic properties which hardly deals with a scientific approach to the problem, and
whose official protocols of analysis lack often of the required specificity. A case study might
be represented by the food safety incident worldwide known as the melamine milk scandal.
Hundreds of babies fed with milk diluted with water and fraudulently treated with
melamine to artificially increase the apparent protein content, developed kidney stones.
Melamine is a plasticizer of molecular formula C3H6N6 thus with a nitrogen content equal to
67% of its molecular weight. The international official protocols for protein assay are still
based on the two hundred years old Kjeldahl method, whereby the nitrogen content is
evaluated from the amount of the released ammonia. This was the fate of melamine too!
Exactly in the same period researchers from the University of Shangai have published a
mass spectrometric assay of melamine in milk based on the specificity of isotope dilution
method by means of a contemporary, reliable and affordable approach.
1

The assessment of quality and safety of all the steps of a given food chain needs to be based
on the exploitation of high-tech analytical methodologies. Mass spectrometry represents a

valuable tool widely applicable to the identification and assay of those quality markers
present in olive oil.
1.2. The principle of mass spectrometry
Mass spectrometry (MS) is an analytical technique based on the principle of gas phase ion
chemistry which allows the identification of analytes of any molecular weight and in any
physical state. The availability of (i) multifaceted sample preparation, (ii) high throughput
methods for on-line multistep mixture separation, (iii) multistep (MS/MS) ion analysis, (iv)
high resolution acquisition and (v) specific isotopic analysis, confer to MS the unique
feature of a methodology that can be directly applied to complex natural mixtures such as
olive oil.
1.2.1. Multifaceted sample preparation
Any sample preparation protocol for MS evaluation of specific properties of foods requires
versatile, reliable and affordable approaches which suit structure properties and physical
state of the examined analytes. The sample preparation methods may include extensive
chemical modification, solvent extraction and different concentration approaches as in the
case of triglyceride (TGA) analysis from the alkyl esters of the corresponding fatty acid
constituents. This approach classically transform low vapor pressure species to relatively
volatile molecules amenable to gas-chromatographic (GC) separation followed by electron
impact (EI) ionization and single quadrupole MS analysis. It should be mentioned, even this
aspect goes beyond the scope of the present review, that the interest in the direct
transformation of TGAs into alkyl fatty ester mixtures, including the methyl derivative
(FAME) has found quite recently an enormous interest in the obtainment of biofuels under
chemical and/or enzymatic catalysis.
2

Scheme 1. Deglycosylation of secoiridoids 1,2 leading to the dealdehydes 3, 4.

Modern Methodologies to Assess the Olive Oil Quality 241
When minor compounds or active principles present in olive oil need to be identified the
sample preparation usually requires simple extraction of the analytes followed usually by a
direct injection into a multistep mass spectrometer (MS/MS). A case study is represented by
the anti-inflammatory dialdehydes formed in olive oil by enzymatic degradation of
secoiridoids (scheme 1).
3
A simple check on the existence of these molecules in olive oil can be performed from the
crude methanol extracts by means of LC-MS/ MS (Figure 1).
4

Figure 1. LC-MS chromatogram of a methanol extract.
The structure of many of the analytes present in the figure 1 was evaluated by further
MS/MS experiments. The species at m/z 379, 414 and 428 were, than, associated to the
protonated dialdehyde 4 (R= hydroxytyrosol, Scheme 1) and to the ammoniated species of
the corresponding geminal diol and hemiacetal. The latter are expected reaction products of
the very reactive system represented by the dialdehydes 3 and 4, when exposed to water
and methanol interaction. The observed results could be considered as a remarkable case of
freezing fast equilibrating species and detect them on-line, separately; the formation,
however, of stable derivatives from a given analyte, present in a complex mixture such as
olive oil, represents a false positive which can be eliminated by selecting a proper
derivatization procedure as it will considered later.
5
The recently introduced MS ambient ionization methods
6
are applicable to direct evaluation
of complex food matrices such as olive oil. In this case too, especially in the assay of a
specific component, in situ derivatization may still be needed.
7

When the isotope dilution approach is considered either for quantitative investigation or for
a reliable identification of analytes in complex natural mixtures
8
, sample preparation
requires the addition of suitable labelled internal standards which should not undergo
intramolecular or intermolecular isotope scrambling.
1.2.2. High throughput methods for on-line multistep mixture separation
The exploitation of MS coupling with separation techniques was initially hampered by the
experimental parameters of a conventional ion source operated at high vacuum. The
availability of multistage mass spectrometers has favored direct injections protocols of even
complex mixtures, nevertheless, many applications require the on-line separation of each
analyte before being submitted to identification and assay. Modern protocols are based on the
combination of GC, HPLC, capillary electrophoresis, and other separation techniques directly
linked MS/MS instruments. Ionization techniques such as electrospray (ESI), atmospheric
pressure chemical ionization (APCI), matrix-assisted laser desorption ionization (MALDI), and
differently performing ambient ionization devices are nowadays mainly used in food analysis.
The role of GC, as well as HPLC in high-resolution separation of complex mixtures is
unquestionable. Similarly, mass spectrometry has attained an indisputable position in
analytical chemistry as a highly structure-specific technique that can provide structural
identity of a wide range of compounds. Both chromatography and mass spectrometry have,
however, their limitations as stand-alone operations. The separation power of any
chromatography system is, actually, finite since it will be nearly impossible to achieve
complete separation of all components of a complex mixture. The identification of a compound
from a chromatographic peak is less than reliable when it has to be indentified by comparing
its retention time with that of a standard reference. Therefore, the commonly used separation
techniques such as GC and LC cannot provide unequivocal identity of the analyte when used
with conventional detection systems. A compound-specific detector is thus an essential
adjunct to characterize unambiguously the components that elute from any separation system.
In this respect, mass spectrometry offers the unique advantages of high molecular specificity,
detection sensitivity, and dynamic range. Mass spectrometry, only, has the advantage to
provide confirmatory evidence of an analyte because of its ability to distinguish closely related
compounds on the basis of the molecular mass and structure-specific fragment ion information
formed by gas phase processes. The confidence in identification of a target compound,
however, diminishes when it is present in a mixture, such as olive oil. Because of the universal
nature of mass spectrometry detection, the data obtained might also contain signal due to
other components of the mixture. The coupling of a separation device with mass spectrometry
thus benefits mutually. The result is a powerful two-dimensional analysis approach, where the
high-resolution separation and the highly sensitive and structure-specific detection are both
realized simultaneously. Some of the benefits that accrue when a separation technique and
mass spectrometry are coupled can be summarized as follows:
The capabilities of the techniques are enhanced synergistically; both instruments may,
therefore be operated at subpar performance levels without compromising the data
outcome.

The high selectivity of mass spectrometry detection allows one to identify co-eluting
components.
The certainty of identification is enhanced further because, in addition to the structure-
specific mass spectral data, the chromatographic retention time is also known.
Multicomponent samples can be analyzed directly without prior laborious off-line
separation steps, resulting in a minimal sample loss and saving of time.
The sensitivity of analysis is improved because the sample enters the mass spectrometer
in the form of a narrow focused band.
Less sample is required than the amount required for off-line analysis by the two
techniques separately.
Because of the removal of interferences, the quality of mass spectral data is improved
and any mutual signal suppression is minimized.
Various chromatographic techniques have been employed for separation, preparative
isolation, purification and identification of such kind of compounds in food. At date,
comprehensive two-dimensional separations are a definite trend in chromatography.
Comprehensive two-dimensional gas chromatography (GCGC) emerged some 15 years
ago.
9
The latter technique allows for the separation of complex mixtures of volatile analytes
GCxGC delivers more GC information in a shorter time than do other methods. GCxGC, at
its roots, is a multidimensional separation technique in which the resolving power of two or
more different columns is applied to some or all of the components in a sample. As the
name implies, comprehensive separations apply all the available resolving power of both
columns to all the peaks in a sample. In GCxGC each peak transits the first column, is
trapped at the end of the first or the beginning of the second column and is released onto the
second column (Figure 2).

Figure 2. GCxGC device.

The major difference between GCxGC and heartcutting is that for GCxGC the trapping time
and second column separation speeds are fast enough so that many heartcuts can be
analysed in the second column during the course of a single first-column run. The heartcuts
can be taken rapidly enough that individual peaks from the first column are sliced across
several sequential secondary column runs, but going much faster than that has no
advantage. Achieving this level of performance requires a high-speed separation for the
secondary column and encourages ordinary speeds for the first column. Faster separations
on the first column can drive the second-column speed requirement higher than what is
practical. So, the main advantages of the technique are vastly-expanded separation space
and the ability to resolve hundreds or thousands of peaks; improved sensitivity; and more
obvious structure in retention patterns for peaks. The increase in sensitivity is due to the fact
that most modulators compress and focus peaks into narrow bands, improving the signal,
and at the same time separate coeluting materials from the analyte of interest, decreasing the
noise.
10
Comprehensive Two-dimensional Gas Chromatography (GCxGC).
11
Comprehensive
two-dimensional separation is achieved also by liquid chromatography; the roots of
comprehensive two-dimensional liquid chromatography (LCLC) are older. Two-
dimensional thin-layer chromatography can be considered a form of LCLC, dating the
technique back at least some 50 years. The contemporary version of LCLC was first
demonstrated by Erni and Frei.
12
The common way to perform LCLC involves a first-
dimension separation that is relatively slow, with a typical analysis time of 1 h or longer.
Fractions of the effluent from this first-dimension column are collected in a loop of a
switching valve. When this valve is switched, the fraction is injected onto a second column,
which provides a much faster separation, typically with an analysis time of a minute or less.

Figure 3. LCxLC, 2 dimensions chromatograms.

While one fraction is being analyzed on the second-dimension column, a new fraction is
being collected in another loop connected to the same valve. The result of a comprehensive
two-dimensional separation is a series of many (for example, 100) fast second-dimension
chromatograms (Figure 3).
1.2.3. Multistep (MS/MS) ion analysis
The concept of in-space tandem mass spectrometry is illustrated in Figure 4. It involves two
mass spectrometry devices; the first one (MS-1) performs the mass selection of a desired
target ion from a stream of ions produced in the ion source. This mass-selected ion
undergoes either unimolecular fragmentation (equation 1) or a chemical reaction in the
intermediate region.

Figure 4. Basic principle of tandem mass spectrometry.
The second MS system (MS-2) performs the mass analysis of the product ions that are
formed in the intermediate step.

1
2
3
p n
n
n
m m m
m m
m m

(1)
The first stage of MS/MS separates a mixture of ions into individual components thus
resolving a mixture of compounds, whereas the second stage acts as an identification system
for the mass-resolved ions. By convention, the mass-selected ion is called the precursor ion (mp
+
)
and its fragments are called product ions (e.g., m1
+
, m2
+
, m3
+
, etc. in equation 1). The mn species in
equation 1 are neutral losses. Because of the incontrovertible link between the precursor ion
and all of its product ions, the molecular specificity of MS/MS is garanteed. This unique
attribute of tandem mass spectrometry is a highly useful feature that plays a role in the
unequivocal identification of a target compound in real-world samples. Tandem mass
spectrometry is not restricted to two stages of mass analysis (i.e., MS/MS or MS
2
); it is also
possible to perform multistage MS (i.e., higherorder MS) experiments, abbreviated as MS
n
.
These experiments can determine the genealogical relation between a precursor and its ionic

products. For example, MS
3
indicates three stages of tandem mass spectrometry, which
involves mass selection of one of the products, say either m1
+
, m2
+
, or m3
+
, formed from the
precursor mp
+
of the first-stage MS (equation 1), and determination of the second-generation
products of that mass-selected ion. Multistage MS experiments are performed mostly with ion-
trapping instruments. A maximum of 12 MS/MS experiments has been envisioned with a
quadrupole ion trap. Beam-type instruments can also be used for MS
n
experiments but require
as many discrete mass analyzers as there are number of stages in the experiment, making it
difficult to perform more than four stages of MS/MS experiments. As an example, a three-
sector magnetic field instrument can perform up to MS
3
experiments. Practical applications of
tandem mass spectrometry require data to be acquired in the following four scan modes. A
pictorial representation of these scans and their symbolism is given in Figure 5.

Figure 5. Representation of four scan modes of tandem mass spectrometry. AD refers to ion activation
and dissociation, and the filled and open circles stand for fixed and scanning mass analyzers,
respectively.
The product-ion scan (the old, now-unaccepted term, still used by some, is daughter-ion scan)
is the most common mode of MS/MS operation. That spectrum is useful in the structure
elucidation of a specified analyte. Information obtained in this scan is similar to that derived
from a normal mass spectrum, except that the spectrum contains only those product ions

that are formed exclusively from a mass-selected precursor ion. To acquire this spectrum,
the first mass analyzer is set to transmit only the precursor ion chosen, and the second mass
spectrometer is scanned over a required m/z range. Another popular MS/MS scan is the
precursor-ion scan (the past, now-unaccepted term is parent-ion scan). It provides a spectrum
of all precursor ions that might fragment to a common, diagnostic product ion. The
spectrum is obtained by adjusting the second mass spectrometer to transmit a chosen
product ion (e.g., m1) and scanning the first mass analyzer over a certain m/z range to
transmit only those precursor ions that fragment to yield the chosen product ion. This scan
is useful for the identification of a closely related class of compounds in a mixture. A typical
example is the detection of Sudan azo dyes within a food. A major product of fragmentation
of azo dyes is the ion at m/z 156. The precursor-ion scan of this m/z value will detect the
presence of azo dyes selectively in a complex mixture (see Figure 6).

Figure 6. Parent ion scan MS/MS spectrum of the fragment m/z 156 present in the APCI spectra of 5
azodyes extracted from baked products and sampled by flow injection.
In a constant-neutral-loss scan, all precursors that undergo the loss of a specified common
neutral are monitored. To obtain this information, both mass analyzers are scanned
simultaneously, but with a mass offset that correlates with the mass of the specified neutral.
Similar to the precursor ion scan, this technique is also useful in the selective identification
of closely related class of compounds in a mixture. For example, the loss of 44 Da is a
common reaction of carboxylic acids. Through the constant neutral loss scan, the identity of
all carboxylic acids present in a complex mixture can be revealed. Similarly, by monitoring
the 98-Da neutral loss, the presence of phosphopeptides can be detected in a complex
mixture.
13
The fourth scan, selected-reaction monitoring (SRM), is useful in quantitative
measurements of analytes present in complex mixtures. Conceptually, this scan mode is
similar to the product-ionscan. However, instead of scanning the second mass spectrometer
in abroad mass range, the two mass analyzers are adjusted to monitor one or more chosen
precursorproduct pairs of the analyte. This operation is identical to the selected-ion
monitoring mode (SIM) of data acquisition. Monitoring more than one reaction is termed
multiple-reaction monitoring (MRM). All four scan modes can be implemented with magnetic
sector- and quadrupole-based true or hybrid tandem instruments. Time-of-flight (TOF) and
tandem-in time devices are also suitable for product scan experiments, but they are unable

to perform the other three scans. Further, within the multistep tandem mass spectrometry
analyses have to be considered also Ion Mobility (IM). Those mass spectrometers are hybrid
instruments that combine an IM separation system with conventional MS systems. An ion
mobility spectrometer (IMS) can also serve as a stand-alone ion detection system.
14
A IMS uses
gas-phase mobility rather than the m/q ratio as a criterion to separate ions.
15, 16
The mobility
of ions is measured under the influence of an electrical field gradient and cross flow of a
buffer gas, and depends on ions collision cross section and net charge. A typical IMS
consists of a reaction region and a much longer drift region; both regions contain a series of
uniformly spaced electrodes that are connected via a series of high resistors to provide a
uniform electric field strength. The two regions are separated by an electrical shutter. Buffer
gas is also circulated in the drift tube. The ions are generated in the reaction chamber and
are allowed to enter the drift region by opening the electrical shutter for a brief period.
Under the influence of an electrical field, ions drift into the drift tube, where they are
separated according to their size-to-charge ratio. The mobility of ions is a combined effect of
ion acceleration by the electric field and retardation by collisions with the buffer gas. At the
end of the drift region is placed an ion detector (e.g., a Faraday cup) for the detection of the
separated ions. For more accurate mass analysis, an IMS is coupled to a quadrupole or TOF
mass analyzer.
17, 18

Similar to LCMS systems, the IMS serves as a separation device and the quadrupole or TOF
mass analyzer as a detection device, but has the added advantage that separation times are in
milliseconds. ESI and MALDI ion sources have both been coupled to IMMS instrument.
19, 20

Latter consists of a MALDI source, an ion mobility cell, a CID cell, and a TOF mass analyzer.
Ions exit the drift tube when the axial field strength of the ion mobility cell is ramped up, and
enter the source region of a TOF instrument, where the ions are detected intact in the usual
manner. Alternatively, ions can be fragmented in the CID prior to their detection by the TOF
MS. The resolution of an IMS, usually very low (10 to 12) can be increased to 200 to 400 by
increasing the pressure of the buffer gas, connecting the ion source directly with the drift
tube, increasing the length of the drift tube, and increasing the electric field gradient of the
drift tube. Unlike LC/MS separation, an ion chromatogram can be obtained within 1 s.
1.2.4. High-Resolution Acquisition
High-resolution mass spectrometry (HRMS) strongly competes with the classical tandem MS,
in particular in the field of quantitative methods (e.g., pesticides, drugs, etc). High-res
analyses are commonly used for residues in food: the availability of new and more sensitive,
selective instrumentation contributed to the development and the acceptance of such
technology in the last few years. Many benefits are gained by using HRMS, including the
collection of full-scan spectra, which provides greater insights into the composition of a
sample. High-res instruments, including double focusing, FTICR, orbitrap mass
spectrometers, are able to measure the mass of an ion and its associated isotope peaks with
sufficient accuracy to allow the determination of its elemental composition. This is possible
because each element has a slightly different characteristic mass defect. The accurate mass
measurement shows the total mass defect and provides a means to determine its elemental

composition. In the last years, the number of studies reposting HRMS-based approaches for
food analysis applications had a significant increase. Even if laboratories still analyze
pesticides, drugs and other microcomponents by using LC-MS/MS and other older
approaches, HRMS instruments are used in both quantitative and confirmative works. High-
res instruments allow not only for high resolution, superior sensitivity, larger dynamic range
and improved speed. Generally, a high-resolution analysis provides more comprehensive
information, e.g. identifying coeluted and therefore potentially suppressing matrix
compounds. Selectivity of detection in complex matrices is only ensured when the HRMS
instrument can physically resolve the analyte mass from that of other coeluted isobaric matrix
compounds. HRMS permits selectivity in their elemental composition and therefore have a
different exact mass. Many reports in the literature described the use of a narrow mass
window to improve selectivity.
21-23
Other studies have addressed the issue of the mass
resolution required for trace analysis in food matrices.
24
Resolutions of 50,000 or more FWHM
were suggested for low levels of analytes in complex matrices such as animal feed. The same
authors concluded that a resolving power of 25,000 FWHM is sufficient for less complex
matrices. Their conclusion was based on experiments that involved analyzing matrix samples
spiked at low levels with various veterinary drugs, mycotoxins, and pesticides. Moreover, the
latest generation of Orbitrap instrumentation provides resolutions well above 100,000 FWHM
(Figure 7). Such instrument provides a clearly higher selectivity than QqQ instrumentation.

Figure 7. High resolution MS spectra registered by a Orbitrap instrument; the signal at m/z 320 has
been resolved at 100,000 FWHM (top) and at 10,000 FWHM (bottom). The higher resolution allows for a
clear separation of the analyte ion from an isobaric matrix compound. The lower resolution does not
resolve these two ions.

The emergence of higher and higher resolving HRMS instruments is a significant advantage
over QqQ technology. It is a fact that in the recent past every QqQ instrument generation
showed an impressive increase in detector sensitivity as compared with the previous
generation. This provided not only lower limits of detection, but also permitted the dilution
of extracts in order to reduce signal suppression effects. The other side of the coin is the fact
that detection selectivity remained virtually unchanged. This resulted in SRM traces which
show many peaks corresponding to compounds being eluted before, after, or even together
with the targeted analyte.
25
Further, most of the currently available literature for HRMS
multiresidue screening is based on measurements made with instruments capable of
providing resolutions between 10,000 and 20,000 FWHM.
26, 27
Basically, higher resolution
leads to superior results when detecting low concentrations of analytes in complex matrices.
However, depending on the technology used: TOF instruments, for instance, achieve higher
resolution by employing very long flight tubes. This might be a physical limitation. Orbitraps
provide a user-defined resolution. There is no relevant sensitivity drop when choosing a
higher resolution. However, the resolution obtained is proportional to the measurement time.
The resulting consequences are fewer data points per time unit. This is a limitation when
using UPLC equipment or when attempting polarity-switching experiments. Furthermore,
higher resolutions produce more data which have to be processed by the instrument and later
accessed from the hard disk when extracting mass traces from the raw data.
1.3. Identification and assay of major components of olive oil
Triacylglycerols (TAG) are the most important lipid component of human diet and represent
by far the main constituent of olive oil (96-99%), they differ in fatty acid composition as well
as in their sequence within the glyceride. It is now well established that the biochemistry of
lipids assumed with diet, hence those present in olive oil, is strictly related to the action of
the digesting enzymes, such a pancreatic lipase, which are able to discriminate between the
fatty acids etherifying glycerol at different sites (Figure 8).
28
R
2
O
O
R
1
O
O
R
3
O
O

Figure 8. Schematic of TG structure. R, fatty acid, with R1, R2, and R3 indicating the fatty acid at the sn-
1, -2, and -3 positions, respectively.

Therefore the classic analytical methods based on the identification and assay of methyl or
ethyl fatty acid esters obtained by direct transesterification of TGAs, still the official method
of analysis, lack by nutritional, hence quality, validation.
29
Chromatographic
30, 31
and
1
H
NMR methods
32-34
have been introduced for a detailed analysis of TGA in olive oils. In a
recent application of chromatographic methodologies combined with statistical analysis
35
an
effective discrimination of extra virgin olive oils was achieved by direct injection of the
samples without any preliminary chemical derivatization.

Atmospheric pressure chemical
ionization (APCI), linked to a triple quadrupole, mass spectrometer provided sensitivity to
detect over 50 compounds in the sample.

In figure 9 is reported the MS profile of a TGA
mixture present in a given olive oil sample.

Figure 9. HPLC-MS profile of diluted extra virgin olive oil. The three letter acronyms have the conventional
meaning, where O, M. Po, P, S, L, Ln, E. Ma, and Mo, correspond to asingle fatty acid respectively.
A peculiar application to the evaluation of TAG profiling
36
of Matrix assisted laser desorption
(MALDI) time-of-flight (TOF) mass spectrometry was introduced (Figure 10). The method was
applied to a peculiar type of olive oil produced in a desert, by means of a conventional 2,5-
dihydroxybenzoic acid (DHB) matrix, using un-treated samples. The fatty acid composition of
the analyzed foodstuff was in agreement with what could be anticipated by applying
conventional GC techniques, with the main difference, however, that the presence of oleic acid,
for instance, was evaluated through the identification of differently structured TAGs, such as
Trioleyl (31.53%) dioleoyl-palmitoyl (23.06%) and dioleoyl-linoleoyl (14.31%) glycerol.
Microbiological studies have recently demonstrated the occurrence in the suspended
fraction of freshly produced olive oil of a rich microflora mainly composed of yeasts
37

which, probably, induces the presence of lipase.
38
This fact can explain the many changes
observed in the triglycerides profiles during aging as a consequence of the lipase action
39


Figure 10. TAG spectrum obtained from MALDI-TOF/MS of an olive oils sample in positive ion mode.
and to the presence of several mono and di carboxylic acids in olive oil derived from many
types of biotransformation.
40-42
The products resulted from this modifications are new type
of triglyceride oligopolymers present in olive oil, as it was reported for Japanese wax.
43
The relative activity of lipase to catalyze hydrolysis and transesterification of triglycerides
was studied and confirmed either with model systems mimic olive oil, or directly with the
foodstuff.
44
It was monitored, therefore, by mass spectrometry, the ability of lipase to
catalyze the formation of new functionalized triglycerides from triglycerides and free mono
and di-acids. The oligomerization of triglycerides has been monitored on real olive oil
samples, and verified in solution under lipase catalysis, whose presence in olive oil was
already documented. The hydrolytic pathways taken under enzymatic treatment is balanced
by the formation of triglycerides oligomers that should not alter the nutritional value of the
aliment. As a general view of the lipase action on triglyceride models in the presence of a
carboxylic acid, Tripalmitoyl- (1) and trioleoyl (2)- glycerol were incubated with succinic
under lipase catalysis at 70C (Scheme 2). The choice of succinic acid was motivated by the
known presence of this dienoic acid in olive oil.

Scheme 2. Incubation of triglyceride mixture with succinic acid in the presence of lipase.

In Figure 11 is reported the partial full scan spectrum of the succinoyl derivative of of
dioleylglycerol observed as sodiated species. The MS peak might correspond to other
isomeric species, in the sense that the succinoyl moiety could be linked to different glycerol
sites, according to the mechanism of lipase action. Nevertheless, it is clear that after thermal
mild treatment, similar to the deodorizing protocol, the presence of lipase and of succinic
acid in olive oil can cause, besides TAG isomerization, the formation of side products which
could be useful as markers of the industrial procedure.

Formula. 1,2-dioleyl-3-succinoylglycerol (6). C82H146O12

Figure 11. ESI-MS full scan spectrum of compound 6 identified as [M+Na
+
] adduct.

The ideal objective of any extraction method is to extract the largest possible amount of
matrix constituents without altering their identity. to measure total fat various methods
have been approved by the regulatory agencies of most major countries.
45
Most of the older
methods involve solvent extraction and gravimetric mass measurement of the lipid residue.
Several apparatus have been developed since 1939 for automatic extraction (e.g., Soxhlet,
Soxhterm, Soxtec, Butt, etc).
46
For total fat determination ethyl ether (diethyl ether) is often
the solvent of choice as it is relatively non-polar and extracts mostly the non-polar lipids
(triacylglycerols, sterols, tocopherols, etc) while poorly extracting the polar lipids
(glycolipids and phospholipids).
47, 48
Methods have been developed to use supercritical fluid
extraction for total fat determination.
49

However, because of the diversity of the matrix content, methods are designed to efficiently
extract specific molecule classes. The extraction of total lipids in foods is often considered to
be a simple procedure; to extract total lipids (non-polar and polar) more polar solvents must
be used as extractants (e.g., hexan or petroleum ether, chloroform, methanol, isopropanol,
water). The most popular method for total lipid extraction is the Folch method.
50
Lipids are
extracted from a sample by using chloroform-methanol (2:1 by volume). Many
modifications to the Folch procedure have been published.
51
1.4. Identification and assay of minor component of olive oil
The quality of olive oil is often associated with the presence of microcomponents whose
healing effects have been proved in some special cases.
52, 53
Virgin olive oils contains also
phenolic substances responsible for their stability against oxidation.
54
Phenolic fraction
includes simple phenols, tyrosol, and hydroxytyrosol, derivatives of hydroxybenzoic and
hydroxycinnamic acids, aglycons of some glucosides, namely oleuropein, demethyloleuropein,
ligstroside, and verbascoside.
55, 56
Absolute analytical methods for mass spectrometric detection as well as for quantification of
such bio-active molecules, such as oleopentadialdehydes have been developed. As
previously mentioned, the presence of dialdehydes in olive oil can be easily ascertained by a
simple LC-MS approach (scheme and figure above). However false positive can be easily
formed during the ionization process due to extreme reactivity of the sampled molecules
with polar solvents such as water or methanol.
57
Such novel method provides an in situ
chemical derivatization of the whole set of molecules into stable alkyloxime derivates,
allowing the concomitant use of a stable isotope standard which improves both the
precision and the accuracy of the measurements, thus reducing the drawbacks may arising
from the calibration procedure, sample preparation, and matrix effects.
The method was applied to a set of different Italian virgin olive oils showing the presence of
5 and 6 in the range of 70166 and 23132 ppm, respectively. The same approach was
exploited in the assay of hydroxytyrosol (1) and tyrosol (2), the strong antioxidant present in
large amount in virgin olive oil, by LCMS/MS under MRM condition and isotope dilution
method, using d2-labelled internal standards, 3 and 4, obtained by simple synthetic
procedures (Chart 1). This active principles ranged from 10 to 47 and 5 to 25 ppm in
experimental and commercial virgin olive oil, respectively.
58


Scheme 3. Derivatization of tyrosol (Tyr) and hydroxytyrosol dialdehydes with unlabelled and labeled
methoxyamine.

Scheme 4. Labelled and unlabelled hydroxytyrosol and tyrosol.
A renowned antioxidant present in olive oil is the secoiridoid oleuropein (OLP). A number
of studies have recognized that a diet rich in olive oil, particularly unrefined oils, provides a
healthy prevention of artery wall thickening as a consequence of low-density lipoprotein
(LDL) oxidation process. This beneficial effect has been associated to the presence of oleic
and linoleic monounsaturated fatty acids and to the action of potent antioxidants such as
tocopherols and the polyphenols.
59
OLP, a secondary metabolite of terpenoid origin, is the
main iridoid of the phenolic pool
60
of Olea europaea, whose activity is likely associated to
its o-dihydroxybenzene (catechol) moiety. The same moiety is shared by hydroxytyrosol
which is formed by enzymatic degradation of the intact secoiridoid and exhibits similar

redox activity. Other phenolic compounds, such as tyrosol, caffeic acid, etc., account for the
radical scavenger effect of virgin olive oil;
61
however, attention has been paid to the actual
content of oleuropein in foodstuffs due to its therapeutic action.
62
An ESR study performed
on the pure molecule has demonstrated that OLP can likely be exhibited in particular
physiological conditions a pro-oxidant activity.
63

Figure 12. ESI (-) MSMS spectra of compound HTyr (A) and labelled HTyr (B,insert).

Figure 13. ESI tandem mass spectra of OLP commercial standard. Product ion spectra (A) from [M+H]
+

and (B) from [M+NH4]
+
precursors.

A comprehensive MS characterization of OLP, showed the advantage of sampling [M+NH4]
+

ammonium adduct either for the sensitivity of the analytical method and for the peculiar
gas-phase chemistry of the species as shown by low and high energy collision experiments.
It was shown that the loss of ammonia neutral and the consequent formation of the [M+H]
+

species was controlling the unimolecular dissociations (Figure 13).
64

The analytical data suggested, therefore, that the [M+NH4]
+
species of oleuropein might
correspond to a mixture of different structures and react, in the gas phase, through the
elimination of neutral ammonia to give a transient [M+H]
+
which possesses enough internal
energy to undergo further fragmentation. Oleuropein is extremely reactive in the experimental
conditions leading to the preparation of olive oil from drupes, mainly due to the action of
glycosidase and esterase enzymes. Therefore its assay in oils derived from different cultivars
and different procedures could be considered a marker of process and of good practise. The
validated process previous discussed was therefore applied to the monitoring of OLP in oils
formed from different cultivars at different ripening stage. (Figure 14).
65

Figure 14. Ripening-phase-dependent oleuropein content in virgin olive oils of Cassanese and Carolea
cultivars. Oleuropein assay in olive oil.
The importance of oleuropein has prompted the development of an absolute method for its
assay in olive oil, based on APCI-MS/MS and isotope dilution.
66

Scheme 5. R1= CH3- OLPd0; R1= CD3- OLPd3
100 % green
100 % superficial
pigmentation
100 % deep
pigmentation
mean
Cassanese
Carolea
0,000
0,050
0,100
0,150
0,200
0,250
0,300
0,350
0,400
0,450

The sensitivity and accuracy of the method allowed the discrimination of olive oil samples
with very similar content of OLP. In particular filtered and not-filtered products from
different southern Italian regions, located at latitude ranging between (38 57 N) and (42
28 N), were discriminated by the amount of OLP which was in the range of 0.0930.225 and
0.1160.344 ppm, respectively.
A similar approach was exploited in the characterization, at the molecular level, of olive oils
produced from the whole fruits or from stoned olives obtained from a pilot plant.
67
The
absolute amount of OLP was 34 times higher in stoned oils, total phenol content showed a
similar behaviour, whereas the amount of -tocopherols was higher in oils obtained by
conventional milling procedures. The total phenol content should be enhanced either when
more lipophilic aglycones are formed, after the removal of the hydrophilic sugar moiety,
and, likely, when oleocanthal
55
and its hydroxytyrosol homologue are obtained after
deglycosylation and demethylation at position 11 of oleuropein.
68
This effect, indirectly,
resembles the action of cell-wall-degrading enzymes added to the paste to improve the olive
oil quality.
69

Figure 15. ESI-MS (-) spectra taken directly from the solution containing OLP and the water extracts of
Carolea pits after 60 min. of incubation.
The main technological difference in the production of the sampled oils was represented by
the presence or absence of stones during the manufacturing procedure. It was therefore
checked, always by LC-MS/MS, the action of water extracts of crushed stones on pure
samples of OLP was therefore planned. The extracted ion chromatogram showed, after 60
min of incubation, that elenoic acid and its open chain isomers were formed whereas
hydroxytyrosol was not detected (Figure 15). This was considered an evidence of the
preponderant presence of glucosidases in the stone enzyme pool. The quality of olive oils in
relation to the identification and assay of the nutraceutical pool has been recently performed
by the new tools of ambient ionization mass spectrometry. A low temperature plasma (LTP)
ion source has allowed a rapid screening of free fatty acids, selected bioactive phenolic
compounds, volatiles and adulterants in virgin olive oils (Figure 16).
70


Figure 16. LTP-MS full scan spectrum of crude virgin olive oil in the positive (+) and negative
ionization mode (-).
The peculiarity of the method is also represented by the possibility of analyzing each
component present in the full scan spectrum with conventional MS/MS devices. Further,
another ion source related to the ambient mass spectrometry techniques, named paper
spray, has been used for olive oil rapid analysis. The latter, allowed for qualitative
determination of several analytes as well as for their assay adding a suitable labeled internal
standard. Currently, this technique is applyed also to the is situ derivatization of olive oil
(e.g., by methoxyamine), allowing the formation of mass tags and increasing the ionization
efficiency for such king of molecules (e.g., oleopentanedialdehydes) in less than one minute.

Figure 17. Variation of the distribution of the aroma markers with the harvesting time in the olive oils
produced from two different southern Italy cultivar.
The volatile fraction of olive oil is considered as a source of quality data either for its
organoleptic properties and for its correlation to genetic, agronomic and environmental

factors. Aroma components of products of plant origin are dependent on genetic,
agronomic, and environmental factors.
71
The identification and assay of the terminal species
of the lipoxygenase pathway which are present in the volatile fraction of olive oils, has
been performed by electron impact and/or chemical ionization mass spectrometry in a
GC/MS Ion Trap apparatus. The quantitative data for each compound were subjected to
principal component analysis to characterize the different cultivars of this work. PCA
methodology was applied to confirm the hypothesis that the five selected markers of
specific lipoxygenase oxidation could be used to differentiate the various cultivars (Figure
17).
72
The same method was applied to distinguish the origin of experimental oils produced
from drupes harvested in different areas of Italian Calabria region and of Tunisia. An easy
discrimination was achieved from each cluster of samples. Olive oils produced from
irrigated and nonirrigated farms in Tunisia were also clearly distinguishable.
73
Quality and
safety of oil is also associated to the presence of dangerous organic residues such as
phthalates (PAEs). These compounds tend to be distributed mostly in fatty foods and this
can cause the presence of remarkable amounts of PAEs in olive oil. Their determination in
fatty matrices represents,therefore, a very important goal for the consumers health and
confidence. A rapid method for the analysis of phthalates in olive oil by GC-MS/MS after a
GPC clean-up has been developed which exploits the capability of tandem mass
spectrometry (GC-MS/MS) for the unequivocal confirmation and accurate quantification of
PAEs at low limits of detection (LOD) levels in fatty matrices without the need for a liquid-
liquid extraction prior to GPC and for SPE clean-up following GPC.
74
The interest in the
identification of secondary metabolites by MS methods has been extended to different olive
tissues. An extensive investigation, by means of high-resolution tandem mass spectrometry
(MS/MS) has shown that some of these micro components are strongly cultivar dependent,
and probably ripening dependent, while others are widespread and present in all the
analyzed cultivars.
75
The new secoiridoid metabolites found in drupes reveal that the key
molecules produced by secondary metabolism of terpenes can be conjugated with
hydroxytyrosol, a secondary metabolite of phenol biosynthesis, through the formation of
differently structured glucosides. The origin of this new species could be related to transport
phenomena which can be different among the various tissues of a given plant. Moreover,
more stringent evidence of the biogenetic similarity of the members of different oleaceae
families is provided by the discovery of metabolites typical of ligustrum and fraxinus in
olive tissues. Secondary metabolites of Olea europaea leaves have been selected as markers
for the discrimination of cultivars and cultivation zones by multivariate analysis; moreover,
the statistical approach has been exploited to correlated the identity and relative amounts of
metabolites in leaves with the harvesting period. The mean values of the concentration of
each compound, detected by tandem mass spectrometry were inputted into PCA analysis.
The bidimensional plot (Figure 18) shows a shifting along PC1 going from MarchApril to
January which indicates the increase of concentration of compounds on the left of plot and
the decrease for methoxytyrosol glucoside and 2-methoxyhydroxytyrosol glucoside.
Moreover, position of samples of July at highest score values on PC2 means a decrease of
concentration of variables verbascoside and hydroxytyrosol glucoside in leaves harvested in
this period.
76


Figure 18. Biplot of principal component scores and loadings for leaves of Carolea cv harvested in
March, April, July and January (variables are indicated as m/z values).
Author details
University of Calabria, Dept. Chemistry, Arcavacata di Rende (CS), Italy
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Chapter 13
Olive Oil Traceability
Enzo Perri, Cinzia Benincasa and Innocenzo Muzzalupo
1. Introduction
The market for imported, premium priced foods has increased dramatically over recent
years, as consumers become ever more aware of products originating from around the
world. There are many food products that are of superior quality (taste, texture, fragrance
etc.) because of the locale in which they are cultivated. Environmental conditions, such as
local climate and soil characteristics, combine to yield crops that exhibit specific traits.
Clearly, higher quality products demands higher market prices, therefore unscrupulous
traders may attempt to increase profits by deliberately mislabelling foods, or by increasing
the volume of a good quality batch through adulteration with sub-standard produce.
In recent years, there has been increasing legislation to protect the rights of both the
consumer and honest producers. On 2009 the European Union (EU) Member States agreed
to require origin labelling for virgin and extra virgin olive oils (EC Regulation 182/2009) to
defend consumers need about true characteristics and origin. To enforce these laws, a
measure of the authenticity of samples must be made, most often in the form of proving the
presence/absence of adulterants, or verifying geographical or cultivar origin by comparison
with known and reliable samples.
The term traceability is often used in modern language, however its usage is applied for
different meanings. In fact, in certain cases it is used to describe the origins of the raw
materials, whilst in others to describe from which geographical region a foodstuff originates.
It is also used more generically to identify a particular producer or from where it has been
purchased. Many consider it as a guarantee of food safety and therefore obligatory for all
food products; others however as a guarantee of quality and therefore a voluntary value.
Also the applied legislation is highly complex, with lawmakers giving different functions to
traceability depending on the application and environment of the law. With the event of the
food crisis in the 90s it was evident that new guidelines were urgently required to simplify
and streamline the processes which could also be applied to food contamination. For this
reason, the new laws of the European Union were considered as an umbrella and termed

General Laws for food safety and was not applied only to food safety but also: to
introduce the concept of traceability where food companies (producers, manufacturers and
importers) need to guarantee to be able to demonstrate the traceability of every food, animal
feed and ingredient, showing the chain from producer to consumer; create the European
Food Safety Authority (EFSA) to unify the various commities and to make public the
scientific process and risks; to strenghthen the ealry warning system adopted by European
governments and the European Commission to enable rapid interventions in cases of food
safety in the human and animal food chains.
In this chapter, we will only consider the "traceability" as a process that allows us to track
"from downstream to upstream" the informations distributed along the olive-oil production
chain, from producer to consumer (from farm to fork), and to include a evaluation of
analytical methods useful in ascertaining what is claimed on the label, in according to the
Regulation (CE) 178/2002.
2. Traceability in regulation (EC) 178/2002
Since 1 January 2005, a rule has required that EU countries implement labelling and
identification procedures for products sold by farmers, producers and first importers to the
EU to enable and facilitate their traceability when they are put on the market. The main
purpose is to be able to initiate a withdrawal and/or recall procedure for products in the
event of a food crisis. The quality of traceability will enable targeted and precise
withdrawals. It will also limit the extent of recalls and ensure the removal of holds on
products that are not involved.
The law (EC Regulation No. 178/2002) defines traceability as: The ability to reconstruct and
follow a food, feed, a food-producing animal or substance intended to be, or to join a food or
feed, through all stages of production, processing and distribution (Article 3, paragraph 15).
Many use the terms tracking and traceability synonymously. In reality, these two terms
identify two inverse processes:
tracking identifies the location of a product from upstream to downstream in the chain
and at every stage of the journey;
traceability or tracing is the inverse process, which allow us to gather the information
previously issued. It is evident that the two processes are strongly related and based on
the same system.
Traceability does not refer to the production of a generic good. It makes each unit of
production physically identifiable, managing production processes that are determined by
"lots", and manage traceability means identifying each group of products and following the
path.
It is necessary to record information relating to inputs (products and companies), processing
(product lots, which lots and what end products) and outflows (products/companies
/clients). The key is to define the composition of a set of products that have undergone the
same process of transformation.

Olive Oil Traceability 267
Moreover, the amount of information that identifies a batch may vary and of course the
complexity of the whole system increases with what information the company chooses to
include in the identification of a lot.
Internal traceability, then, helps to express the internal procedures of each company to trace
the origin of materials used.
The label is the instrument through which information is transferred to consumers. Its easy
to understand why this essential requirement is effective.
Finally, the information given in the production lot should be able to trace all the links along
the chain, back to the first producer and/or supplier of the product or substance to whom it
belongs. Traceability chain is an inter-company process, resulting from internal processes of
every operator in the industry. These systems should be linked by efficient information
systems. It is not governed by a single person in the chain, the relations between the
operators allow the tracing of the chain.
3. Analytical methods for the traceability of extra virgin olive oil
The increase in the demand for high-quality olive oils has led to the appearance in the market
of olive oils elaborated with specific characteristics. They include oils of certain regions
possessing well-known characteristics, that is, olive oils with a denomination of origin, or
with specific olive variety composition, that is, coupage or monovarietal olive oils.
Monovarietal olive oils have certain specific characteristics related to the olive variety from
which they are elaborated (Montealegre et al., 2010). However, coupage olive oils are
obtained from several olive varieties to achieve a special flavor or aroma.
The appearance of denominations and protected indications of origin has promoted the
existence of oils labeled according to these criteria. Regulation 2081/92, to promote and
protect food products, created the systems known as:
Protected Designation of Origin (PDO);
Protected Geographical Indication (PGI);
Traditional Speciality Guaranteed (TSG).
An olive oil with a PDO denomination requires meeting precise definition of several
parameters such as cultivar, geographical origin, agronomic practice, production
technology, and organoleptic qualities (Gimnez et al., 2010), and all of these parameters
have to be investigated to study its traceability and to certify its quality. The introduction of
certifications of origin and quality for virgin olive oil as PDO makes necessary the
implementation of traceability procedures.
Any research dealing with olive oil traceability is focused on investigating the botanical or
geographical origin. In both cases, the selection of the markers (compounds with
discriminating power) to be studied is complicated because the composition of extra virgin
olive oils is the result of complex interactions among olive variety, environmental conditions,
fruit ripening, and oil extraction technology (Araghipour et al., 2006). The verification of the

cultivars employed to produce an olive oil sample may contribute to address the oil origin.
This fact may have commercial interest in the case of monovarietal olive oils or olive oils with
PDO because these high-quality olive oils may be adulterated by other oils of lower quality,
using anonymous or less costly cultivars (Sanz-Cortes et al., 2003).
Unfortunately, morphological traits have been difficult to evaluate, are affected by
subjective interpretations, and are severely influenced by the environment and plant
developmental stage (Japon-Lujan et al., 2006). Nowadays, several efforts have been focused
on the investigation of one or several compounds present in olive oils usable to differentiate
olive varieties (Sanz-Cortes et al., 2003).
Compositional markers (substances that take part of the composition of the olive oils)
include major and minor components. Major components such as sterols, phenolic
compounds, volatile compounds, pigments, hydrocarbons, tocopherols, fatty acids and
triglycerides may provide basic information on olive cultivars. Minor components can
provide more useful information and have been more widely used to differentiate the
botanical origin of olive oils (Howarth and Vlahov, 1996; Lanteri et al., 2002).
In recent years, there has been increasing legislation to ensure consumer confidence and to
protect the rights of both the consumer and honest producers.
It is of great importance the development of analitycal methods to verify the correspondence
between what is stated on the label and what is contemplated in the documents (EC
Regulation 182/2009 and Reg. 834/2007) in relation to the production of olives and extra
virgin olive from organic farming. Moreover, to enforce these laws, a measure of the
authenticity of samples must be made, most often in the form of proving the
presence/absence of adulterants, or verifying geographical or cultivar origin by comparison
with known and reliable samples. The latter method often includes the use of multivariate
statistical techniques such as principal components analysis, linear discriminant analysis,
canonical variance analysis and partial least squares regression to investigate sample data
(Gimnez et al., 2010).
3.1. Chromatographic techniques for the traceability of olive oil
In the last decade, gas chromatography (GC), high performance liquid chromatography
(HPLC), supercritical fluid chromatography (SFC), and capillary electrophoresis (CE) have
been widely used in the authentication analysis of olive oil. The fat, sterols, protein,
carbohydrate or other natural compound profiles (Aparicio-Ruiz, 2000; Benincasa et al.,
2003; Vichi et al., 2005; Lopez Ortiz et al., 2006; Temime et al., 2006; Canabate-Diaz et al.,
2007; Cavaliere et al., 2007; Haddada et al., 2007; Vichi et al., 2007; Lazzez et al., 2008) have
been used to provide species and geographical differentiation. In several cases, enantiomeric
composition has been utilised (Rossmann et al., 2000). These methods of discrimination rely
on samples of different species and/or different geographical origin having different
chemical compositions. This is not always the case, samples from the same location have
been found to contain different components and conversely samples from different regions
may display identical chemical composition.

Detectors usually used, in combination with chromatographic techniques (GC and HPLC),
may be more or less selective and sensitive, but lack information about the identity of
compounds. Therefore, the coupling of chromatographic techniques and mass spectrometry
(MS) overcomes this drawback (Cavaliere et al., 2007; Lazzez et al., 2008).
MS is a sensitive and selective detector, sometimes allowing preparation steps to be
avoided. GCMS is a robust technique, used routinely in many laboratories for food
analysis; for example for the determination of aroma compounds and pesticide analysis.
More recently, LC coupled to quadrupoles, magnetic sectors or time-of-flight (TOF)
detectors, has also had a great expansion into the field of food analysis.
The recently introduced spray methods (ESI) have fostered qualitative and quantitative
analysis of medium to high polar analytes by mass spectrometry. The designing of ion
source houses for ESI has also fostered the rediscovery of atmospheric pressure ionization
(API) methods such APCI where the chemical ionization (CI) is achieved at atmospheric
pressure. Both techniques produce soft ionization, but additional fragmentation can be
achieved by performing in-source collision induced dissociation (CID) in tandem or trap
instruments. MS/MS in space (tandem, sectors, quadrupoles, TOFs, etc.) and in time (traps)
provide additional and unique information on the structure of analytes. ESI is useful for
polar and ionic solutes ranging in molecular weight from 100 to 15010
3
dalton. APCI is
applicable to non-polar and medium polarity molecules with a molecular weight from 100
to 2000 dalton. Although the choice of the right interface, as well as the detection polarity
are based mostly on the compounds polarity and thermal stability, and the HPLC operating
conditions, many classes of compounds can give good response with both ionization
techniques. In certain circumstances both positive and negative ionization modes are
needed, while in most of the cases the choice of only one operation mode is enough. The
number of applications of HPLCAPI-MS to food analysis has rapidly increased in recent
years. ESI is much more widespread than APCI, but for both techniques the trend is towards
an increase in the number of applications (Sindona et al., 1999; 2000; Di Donna et al., 2001).
3.2. Stable isotope techniques for the traceability of olive oil
Stable isotope techniques enable differentiation of chemically identical substances through
alterations in their isotopic fingerprint, and have been used in authenticity studies for many
food products. The isotopic composition of light elements (lighter than calcium) in plant
material can vary depending on location but, the dominant factor is the influence of latitude
on the fractionation of the elements in groundwater.
Fractionation occurs during physical processes such as evaporation. Lighter isotopes
evaporate very slightly faster than their heavier counterparts, therefore in warmer regions
where the amount of evaporation is higher, the isotopes are fractionated to a greater degree.
The discrimination between isotopes in such physical processes is only significant for light
elements, with a high relative mass difference between the isotopes. Thus hydrogen ratios,
measured by site-specific natural isotope fractionation nuclear magnetic resonance (SNIF-
NMR), and carbon, nitrogen, oxygen and sulphur isotope ratios measured by isotope ratio
mass spectrometry (IRMS) have been applied to the authentication of foods.

The elemental composition of vegetation reflects (to a certain extent) that of the soil in which
it has grown (Kelly et al., 2002) which in turn will depend on the topography, geology and
soil characteristics. Therefore, no two countries will have identical soil maps, and the
concentration of elements in food product, can then be used to assign their geographical
origin. As with other methods of authentication, a database of samples of known origin
must be available, against which unknown samples can be compared. The most useful
elements for the assignment of origin are those that are not homeostatically controlled.
Elements such as K, Ca and Zn are actively absorbed by organisms and will therefore be
present in samples at similar levels, regardless of the environmental conditions experienced.
Elements that have no role in normal physiological processes, such as the rare earth
elements (REEs) and the heavy metals, are passively absorbed and the concentration of these
elements in an organism will strongly reflect the environmental levels to which the
organism has been exposed.
3.2.1. Inductively coupled plasma mass spectrometry technique
Several studies have used multi-element concentration profiles in the determination of food
authenticity, either alone or in combination with chromatographic or stable isotope ratio
data.

Elemental concentrations were determined by atomic absorption spectroscopy (AAS),
inductively coupled plasma - atomic emission spectroscopy (ICP-AES) or inductively
coupled plasma mass spectrometry (ICP-MS) (Dugo et al., 2004; Benincasa et al., 2007;
Cindric et al., 2007).
Several trace elements have variable natural isotopic abundance due to the decay of
radioactive isotopes. These include Li, B, Cu, Sr, Nd, Hf, Pb and U.

The composition and age
of the local rocks dictates the abundance of the radioactive precursors and their daughter
species. Elements are taken up into plants in the same isotopic proportions as they occur in
the soil and in precipitation.
Therefore isotope ratios in plant-derived food products depend on the geology of the region
in which the source crop was grown, and are different in produce of different geographical
origin. There have, however, been relatively few reports of the use of heavier stable isotope
ratio measurements for the authentication of foodstuffs. For many years thermal ionization
mass spectrometry (TIMS) was the only technique capable of performing isotope ratio
measurements with sufficient precision to allow geographical assignment of food products
based on trace element isotopic composition. Samples for TIMS analysis must be loaded in
the form of the pure element, meaning that extensive sample preparation is required if this
technique is to be applied.
ICP-MS is now a well established technique for isotopic trace element determinations.

ICP-
MS allows rapid analysis of a large range of sample types, requires minimal sample
preparation and due to the ionising power of the ICP, can be applied to a wider range of
elements than TIMS. The precision of isotope ratio analyses by ICP-MS has only recently
matched that achievable by TIMS, through the application of double focusing mass
analysers coupled to multi-collector detection arrays. The increasing availability of multi-

collector ICP-MS is likely to lead to wider application of heavier stable isotope ratio
measurements to the authentication of food products.
3.2.2.
13
C compound-specific isotope analysis
One of the most powerful techniques to be used in food authenticity studies is stable isotope
ratio mass spectrometry (SIRMS). SIRMS has found application in the authentication of a
wide range of foodstuffs, especially for the detection of added cane sugar in fruit juices
(Bricout and Koziet, 1987), wines (Dunbar and Schmidt, 1984), spirits (Parker et al., 1998),
honey (White et al., 1998) and to detect the adulteration of flavour compounds with
synthetic analogues (Culp and Noakes, 1990). The majority of the reported research has
focused on bulk or global stable carbon isotope ratios (
13
C/
12
C).
Approximately 98.89% of all carbon in nature is
12
C and 1.1% is
13
C. The amount of
13
C
present in a sample is expressed as a ratio to the amount of
12
C present, relative to an
international standard using the notation. This notation has the units per mil (). The
13
C
value of plants varies because of isotopic fractionation during physical, chemical and
biological processes.
Photosynthetic fixation of CO2 by plants takes place by three different routes, depending on
the nature of the plant. Most terrestrial plants photosynthesise using the Calvin or C3
pathway in which CO2 is fixed via the carbon cycle to from a three-carbon compound. Some
plants, mainly tropical grasses, such as maize and sugar cane use the Hatch-Slack or C4
pathway in which the initial "Hatch-Slack" step is via a dicarboxylic acid, a four carbon
compound. A third photosynthetic class of plants uses the CAM (Crassulacean acid
metabolism) pathway.
Typical CAM plants are succulents and are of minor importance in the oleochemical
industry. In the C3 pathway carbon dioxide is fixed via the carboxylation of ribulose-1,5-
diphosphate to form phosphoglyceric acid (a 3-carbon molecule). This enzyme catalysed
reaction discriminates against
13
C and so proceeds with a relatively large isotope effect
(OLeary, 1981). This means that less
13
CO2 is incorporated into C3 plants than into C4
plants. C3 plants have bulk
13
C values in the range -24 to -30 whereas C4 plants have
bulk
13
C values from -9 to 14.
Olive oil shows a
13
C values of 28.7.
The most widely used technique to assess the authenticity of edible oils is to measure the
fatty acid composition. However it is not always possible to detect adulteration by this
technique because of the natural variation in fatty acid profiles and because blends of oils
with fatty acid compositions similar to the authentic oil may be prepared relatively easily. A
more sophisticated approach is to determine the fatty acid composition at the 2 position of
the triacylglycerol, since this is known to differ from the overall fatty acid composition.
Woodbury et al., (1998) used this technique to obtain the compound-specific
13
C values of
the fatty acids specifically at the 2-position of the triacylglycerol.

Royer et al., (1999) examined 188 olive oils produced mainly in Greece during 1993 to 1996.
The concentration and
13
C value of individual fatty acids present in the olive oils were
determined by gas chromatography and GC-C-IRMS respectively. The results were
examined in terms of geographical, temporal, and botanical factors. French and Italian olive
oils were securely classified at the 99.9% confidence interval using the
13
C values of the
principal fatty acids palmitic (C16:0), oleic (C18:1) and linoleic (C18:2). Regional
classifications for the Greek olive oils were also achieved on the basis of differences in the
13
C abundance of oleic acid compared to linoleic acid and palmitic acid.
Glycerol is a primary metabolite in plants. It is nominally present in its ester form as
glycerolipids in fats and oils (Kiritsakis and Christie, 1999). Glycerol is bio-synthesised
relatively early in the lipidic metabolic pathway compared to fatty acids (Weber et al., 1997;
Harwood and Snchez, 1999).
Consequently, it may be expected that the isotopic distribution in glycerol is a better
indicator of the botanical and environmental influences on any given plant. A number of
compound specific IRMS studies on glycerol have been performed, some of which include
data derived from vegetable oils.
3.2.3.
18
O and
2
H pyrolysis CF-IRMS
The abundance of the stable isotopes oxygen-18 (
18
O) and deuterium (
2
H) are particularly
interesting isotopic probes for both botanical and geographical identification of a variety of
different food products. The primary source of all organic hydrogen and oxygen is the
hydrosphere. The meteoric water that has passed through the meteorological cycle of
evaporation, condensation and precipitation finally constitutes the groundwater and
exhibits a systematic geographical isotope variation. Decreasing temperatures cause a
progressive heavy-isotope depletion of the precipitation when the water vapour from
oceans in equatorial regions moves to higher latitudes and altitudes. Evaporation of water
from the oceans is a fractionating process that decreases the concentration of the heavy
isotopomers of water (
1
H
2
H
16
O,
1
H
1
H
18
O) in the clouds compared to the sea. As the clouds
move inland and gain altitude further evaporation, condensation and precipitation events
occur decreasing the concentration of deuterium and oxygen-18.
Consequently, the ground water reflects this isotopic gradient from the coast to inland areas.
For land plants, a further pre-assimilation affects the isotopic composition of the water
substrate. The hydrogen and oxygen present in plant material originates from the water
taken up by the roots. The water is transported through the plants xylem system. The
isotopic composition of the xylem water is the same as that of water taken in by the roots,
and the water is taken into the leaves without a change in isotopic composition. Evapo-
transpiration of water through the leaf enriches the remaining water in the heavier
isotopomers. Therefore, it is expected that growing regions with relatively low humidity,
where the rate of evaporation from the leaf is higher, result in plant materials with relatively
enriched
2
H and
18
O values.

Over the past 5 years there has been a marked increase in the use and application of
2
H and
18
O stable isotopes in many areas of food research. This has been facilitated by recent
developments in on-line gas preparation devices that proceed by high temperature pyrolysis
of organic products and the availability of commercial IRMS analysers capable of measuring
2
H/
1
H ratios in the presence of a helium carrier gas. These innovations have, to a large
extent, overcome the difficulties associated with offline gas preparation for DI-MS and
greatly increased the applicability of this measurement. It is now possible to routinely
measure
2
H and
18
O abundances in organic samples by Pyrolysis-Continuous Flow-Isotope
Ratio Mass Spectrometry (Py-CF-IRMS).
Therefore, measurements of stable isotope ratios of the light elements (H, C ,N ,O, S and
bioelements) and of the heavy element stronzium, in natural cycles, have provided
geographical fingerprints (Romann et al., 2000).
Preliminary investigations into the application of
18
O-pyrolysis continuous-flow IRMS to
obtain information about the geographical origin of olive oil samples has been conducted by
Angerosa et al. (1999). They measured the
13
C and
18
O values of whole olive oil, sterols and
aliphatic alcohol fractions from fruits of Olea europaea L. produced in Greece, Italy, Morocco,
Spain, Tunisia, and Turkey. The results permitted provincial classification of the oils.
However, there were some misclassifications observed for oil samples coming from
neighbouring countries with similar climates.
A secure geographical classification of an olive oil, in order to ensure that the consumer is
not defrauded and that the honest trader is not disadvantaged by having their PDO oils
misrepresented by inferior products, can be achieved by performing heavy isotope ratios
(e.g.
88
Sr/
86
Sr) and multi-element analysis.
3.2.4. Nuclear magnetic resonance spectroscopy
During the last ten years, nuclear magnetic resonance spectroscopy (NMR) (Del Coco et al.,
2012; Mannina & Segre, 2002), has played an ever-increasing role in the study olive oil
characterization and autentication. In particular, it has been shown that high-resolution
NMR together with statistical analysis constitutes a powerful tool for the geographical
characterization of olive oils on Mediterranean, national, regional and PDO scales. On this
regard, innovative techniques like NMR spectroscopy seem to be able to distinguish olive
oils on the basis of their geographical origin, whereas the conventional analyses suitable for
the determination of quality and genuineness seem not to be so appropriate for this type of
discrimination (Frankel, 2010; Guillen & Ruiz, 2001).
Important information on the fatty acid distribution on the glycerol moiety can be obtained
by
13
C NMR (Rezzi et al., 2005; Petrakis et al., 2008; Alonso-Salces et al., 2010b; Mannina et al.,
2010; Alonso-Salces et al., 2011b). Two groups of resonances are observed in the carbonyl
region of the
13
C NMR spectrum of an olive oil: one group is due to fatty chains in position
sn-1,3 of the glycerol moiety, the other one is due to fatty acids in position sn-2.

It must be noted that although gas chromatographic methods give the full composition of
fatty chains, no information is given about the fatty chains distribution on glycerol. Thus in
this case, gas chromatography and
13
C NMR methodologies must be considered
complementary and not alternative.
13
C NMR spectroscopy together with discriminant
analysis has been proposed by Mavromoustakos et al., to detect the presence of soybean oil,
cottonseed oil, corn oil and sunflower seed oil in virgin olive oils. Only double bonds signals
have been used for the analysis. Fatty acids composition and fatty chain positional
distribution on glycerol moiety determined by
13
C NMR spectroscopy allow, in combination
with the multivariate statistical procedure, the classification of olive oils according to their
variety (Marini et al., 2004, 2006).
31
P NMR spectroscopy has been employed for the detection and quantification of the minor
compounds mono- and diacylglycerols, polyphenols, sterols, phospholipids and for the
determination of free acidity and moisture (Petrakis et al., 2008). This analytical technique
requires the derivatization of the labile hydrogens of the hydroxyl and carboxyl groups of
the olive oil constituents using the phosphorous reagent 2-chloro-4,4,5,5- tetramethyl
dioxaphospholane and the use of
31
P chemical shifts of the phosphitylated compounds to
identify the labile centers. The main phospholipids found in olive oil were phosphatidic
acid, lyso-phosphatidic acid and phosphatidylinosotol, although very small amounts of
other phospholipids were detected as well. In this way, the content of free fatty acids has
been determined showing a good correlation with official titration method. Besides, using
the same derivatization method, sn-1,2, sn-1,3-diacylglycerides and monoacylglycerides
have been quantified and have showed a characteristic trend during olive oil storage.
The composition of diacylglycerols (sn-1,2- 1,3-diacylglycerols and total) determined by
31
P
NMR derivatization method has been used to characterize olive oils from different Greek
areas (Petrakis et al., 2008). Some preliminary correlations between the diacylglycerols
content and the geographical regions have been observed.
The geographical characterization of monovarietal virgin olive oils from three regions of
Southern Greece, namely Peloponnesus, Crete and Zakynthos, has been performed applying
both
1
H and
31
P NMR (Petrakis et al., 2008). The correct geographical prediction at the level
of three regions, based on discriminant analysis, has been rather high (87%). Phenolic
compounds and free acidity determined by
31
P NMR spectroscopy have enabled to classify
Koroneiki, Athinolia and Kolovi monovarietal Greek extra virgin olive oils using ANOVA
and discriminant analysis.
1
H NMR spectrum of an olive oil does not provide this information directly; however, it is
possible to have an indirect measurement of acidity using the measurable amount of
diglycerides and monoglycerides.
Another study carried out by using
1
H NMR involved the characterization of the phenolic
fraction of olive oil from three different areas of the Apulia region (Sacco et al., 2000).
Statistical evaluation has showed discrimination between Coast, Hinterland and North
samples.

Extra virgin olive oils sampled in three harvesting years and coming from different Italian
regions (Tuscany, Lazio, Lake of Garda) have been analyzed by
1
H methodology. The
statistical elaboration applied on the intensity of b-sitosterol, aldehydes and some other
volatile compounds has allowed the classification of the olive oils according to the
geographical regions.
1
H-NMR fingerprinting of olive oil is a valuable analytical tool for the traceability of virgin
olive oils from different points of view, i.e. food authentication and food quality. As
described before,
1
H and
13
C NMR techniques provide different information:
1
H NMR
spectrum allows the measurement of minor components of olive oils such as -sitosterol,
hexanal, 2-(E)-hexenal, formaldehyde, squalene, cycloartenol and linolenic acid; the
13
C
NMR spectrum detects major components such as glycerol tri-esters of olive oils, defining
also acyl composition and positional distribution on the glycerol moiety. The main
sensitivity concern about
1
H NMR analysis is the dynamic range, which makes the detection
of signals below the threshold imposed by the ADC (analogue to digital converter)
impossible. Therefore, in a standard extra virgin oil spectrum, trace component detection is
limited to signals more intense than 105 times the intensity of the fatty acid CH2 signals.
Weak signals above the dynamic range threshold are in any case affected by severe baseline
distortion. Selective pulses coupled to gradient spin-echo refocusing such as DPFGSE (69),
in NMR modern instruments, allow these limitations to be circumvented, paving the way
for the easy detection of minor components, especially those presenting resonances away
from other interfering resonances.
The power of these new sequences could be demonstrated by the detection of aldehydes,
carotenoids or other specific minor components whose resonances fall in a relatively free
region. It was performed a specific DPFGSE analysis on the aldehydic components of some
extra virgin olive oils, mostly responsible of the sensorial properties of extra virgin olive oil.
Comparison with standard
1
HNMR spectra show tremendous improvement of quantitative
and qualitative information with drastic instrumental time reduction.
Fingerprinting techniques such as NMR, NIR (Galtier et al., 2007; Mignani et al., 2011), MIR
(Reid et al., 2006), fluorescence (Kunz et al., 2011), FT-IR, FT-MIR and FT-Raman (Baeten et
al., 2005; Lopez-Diez et al., 2003; Yang et al., 2005) spectroscopies, have been used for the
determination of food authenticity (Reid et al., 2006).
These types of techniques are particularly attractive since they are non selective, require
little or no sample pre-treatment; use small amounts of organic solvents or reagents; and the
analysis takes only a few minutes per sample.
3.3. Molecular markers for the traceability of olive oil
The food crisis situation seen in last years and the controversy about genetically modified
organisms (GMO), with a sharp increase in basic food prices, highlights the extreme
susceptibility of the current agricultural and food model and the need for more strict food
quality control, which should include determination of the origin of the product and the raw

materials used in it. Thats why a well documented traceability system has become a
requirement for quality control in the food chain.
Almost 84% from the total olive oil production derives from the European Union, especially
from Spain, Italy and Greece. The olive oil is a main constituent of the Mediterranean diet.
However there has recently been an increase in olive oil consumption internationally, due to
greater availability and the current consideration of high nutritive and health benefits,
including a qualified health claim from Food and Drug Administration (FDA, USA).
Molecular markers allow the detection of DNA polymorphisms and enable to effectively
distinguish different varieties in an effective way, without any environmental influence.
Olive oil extraction is the process of extracting the oil present in the olive drupes for food
use. Olive oil extraction is the process of separating the oil from the other fruit contents. It is
possible to attain this separation by physical means alone, i.e. oil and water do not mix, so
they are relatively easy to separate. The modern method of olive oil extraction uses an
industrial decanter to separate all the phases by centrifugation. In this method the olives are
crushed to a fine paste. This can be done by a hammer crusher, disc crusher, depicting
machine or knife crusher. This paste is then malaxed for about 30 minutes in order to allow
the small olive droplets to agglomerate.
When we blend olive oils of the same category, but from different provenances, most
chemical analyses are of limited significance. Due to their high variability according to
environmental conditions, neither morphological characteristics of different groups, nor the
analyses of chemical composition of fatty acid and secondary metabolites can provide
reliable results for oil traceability (Ben Ayed et al., 2009; Papadia et al., 2011). For this reason,
genetic identity seems to be the most appropriate method for identifying the variety from
which the olive oil under study derives.
Molecular markers allow the detection of DNA polymorphisms and enable to effectively
distinguish different varieties in an effective way, without any environmental influence. In
fact, DNA in oil is not affected by the environment and is identical to the mother tree DNA
since the oil containing tissues are formed by diploid somatic cells of the tree (Muzzalupo et
al., 2007). However, depending on the molecular markers used correctly, extra alleles can be
detected in the oil that do not correspond to the mother tree allele but to the pollinator
alleles contained in the embryo, itself located inside the seed (Muzzalupo and Perri, 2002;
Ben Ayed et al., 2010).
The use of DNA based technology in the field of food authenticity is gaining increasing
attention. This technique makes use of molecular markers that mostly use polymerase chain
reaction (PCR) and are thus easy to genotype. Even in a complex matrix such as olive oil,
molecular marker techniques such as RAPDs (random amplified polymorphic DNA,
Pasqualone et al., 2001; Muzzalupo and Perri, 2002).), AFLPs (amplified fragment length
polymorphism, Pafundo et al., 2005; Montemurro et al., 2007) and SSRs (simple sequence
repeat, Muzzalupo et al., 2007; Breton et al., 2004; Bracci et al., 2011; Ben Ayed et al., 2009) are
very useful in the study of the traceability of olive oil. Single nucleotide polymorphism

(SNPs) markers have been recently developed in olive and utilized to study the genetic
diversity of olive trees (Reale et al., 2006). SNP detection can be delivered in a number of
ways, but the simultaneous detection of multiple SNPs from a single DNA sample is of
particular interest (Consolandi et al., 2008). PDO oils are typically not monovarietal, so a
method for quantifying the components of the mixture is essential if conformity with
certification depends on a prescribed proportion of varietal types. So far, application of Real-
Time as a tool for olive oil authentication has been explored by Gimnez et al., (2010). The
authors evidenced that Real-Time PCR is useful to quantify DNA extracted from oil, and
thus to assess the yields of different methods of extraction. But the size of amplicon, is
critical for the success of analysis. A possibility of utilising qRT-PCR to quantify varieties in
PDO oils rests on the use of taqMan probes designed on SNPs specific of varieties entering
in the oil composition (Marmiroli et al., 2009). Several sequences from noncoding spacer
region between psbA-trnH and partial coding region of matK of plastid genome provided a
good discrimination of pure olive oil and its admixture by other vegetable oils such as canola
and sunflower. The plastid based molecular DNA technology has a great potential to be
used for rapid detection of adulteration easily up to 5% in olive oil (Kumar et al., 2011).
The knowledge of genome nucleotide sequences also could be useful to identify new
sequence polymorphisms, which will be very useful in the development of many new
variety-specific molecular markers and in the implementation of more efficient protocols for
tracking and protect olive oil origin.
A recent publication reported by Papadia et al., 2011 a systematic effort to obtain genetic
characterization by SSR amplification, soil analyses, and 1H-NMR spectra, is carried out in
order to make a direct connection between the olive tree variety (genetic information) and
the NMR spectra (chemical information) of the extra virgin olive oil produced. The results
reported show that a multidisciplinary approach, through the application of multivariate
statistical analysis, could be used to set up a method for variety and/or geographic origin
certification, based on the construction of a suitable database. Further research will be
directed to the growth of an organic genetic/NMR/soil database, in order to improve the
prediction ability of the LDA, and furthermore to develop a way to correlate
1
H-NMR
spectra of commercial extra virgin olive oils with their geographical and genetic origin.
3.4. Electrochemical sensor analysis for the traceability of olive oil
Electronic noses (e-noses) and electronic tongues (e-tongues) for liquid analysis, based on
the organizational principles of biological sensory systems, developed rapidly during the
last decade. E-noses and e-tongues crudely mimic the human smell and taste sensors (gas
and liquid sensors) and their communication with the human brain. The human olfactory
system is by far the more complex and contains thousands of receptors that bind odor
molecules and can detect some odors at parts per trillion levels (Breer, 1997) and include
between 10 and 100 million receptors (Deisingh et al., 2004). Apparently some of the
receptors in the olfactory mucus can bind more than one odor molecule and in some cases
one odor molecule can bind more than one receptor. This results in a mind-boggling amount

of combinations that send unique signal patterns to the human brain. The brain then
interprets these signals and makes a judgment and/or classification to identify the substance
consumed, based in part, on previous experiences or neural network pattern recognition.
The electronic nose often consists of non-selective sensors that interact with volatile
molecules that result in a physical or chemical change that sends a signal to a computer
which makes a classification based on a calibration and training process leading to pattern
recognition. The non-selectivity of the sensors results in many possibilities for unique signal
combinations, patterns or fingerprints. The human tongue contains sensors, in the form of
10,000 taste buds of 50100 taste cells each (Deisingh et al., 2004) for sweet, sour, bitter, salty
and umami and is much less complicated than the human olfactory system. The e-tongue
then uses a range of sensors that respond to salts, acids, sugars, bitter compounds, etc. and
sends signals to a computer for interpretation. The interpretation of the complex data sets
from e-nose and e-tongue signals is accomplished by use of multivariate statistics. For non-
linear responses, artificial neural networks (NAA) can be used for modeling the data.
Biosensors are also being developed, but are not yet commercialized. In contrast to chemical
sensing materials, that are broad spectrum to generate characteristic response patterns, there
are biological systems. The problem with chemical sensors is that these systems are
extensive, require large sample sizes for analysis, have low sensitivity and poor specificity
compared with the human nose. The bioelectronic nose utilizes olfactory receptors as
sensing mechanisms and are cell or protein-based to mimic a mammalian olfactory system
(Lee and Park, 2010). Another type of sensing system is based on colorimetric sensor array
built in disposable chips (Suslick et al., 2010). These arrays are based on the chemical
interactions between the analyte and a chemical dye. They are being developed for volatile
and non volatile molecules (Zhang et al., 2006; Zhang and Suslick, 2007; Musto et al., 2009)
for applications by the food industry.
The advantage of the human sensory system is that the brain can receive signals from both
olfactory and tongue receptors and integrate both sets of data to form classifications and/or
judgments. The e-nose and e-tongue are not integrated since each has its own software
package, but the data from both instruments could be imported into another program and
integrated. The disadvantage of the human sensory system is that no two brains are alike (of
course from another point of view, this is a good thing), and the same brain may react
differently from one day to the next, depending on an individuals health, mood or
environment, making the data subjective. On the contrary, e-nose and e-tongue instruments
can be calibrated to be reliably consistent and can give objective data for important
functions like quality and safety control. These instruments can also test samples that are
unfit for human consumption. A disadvantage for the e-nose and e-tongue systems (as with
humans) is that they are also affected by the environment including temperature for both e-
nose and e-tongue and humidity for e-nose, which can cause sensor drift, although
calibration systems and built-in algorithms help compensate for this. There are more or at
least not less different types of sensing materials for e-tongue (liquid sensors) compared to
e-nose systems, and liquid sensors often possess higher selectivity and significantly lower
detection limits compared to the gas sensors (e-nose).

There are several reviews on the subject of e-nose and e-tongue technology, including
reviews on e-noses (Di Giacinto et al., 2010; Wilson and Baietto, 2009),
biomimetic/biotechnology e-nose and/or e-tongue sensing systems (Rudnitskaya and Legin,
2008; Ghasemi-Varnamkhasti et al., 2010)], applications for e-noses and e-tongues
(Scampicchio et al., 2008), neural networks for e-noses (Lu et al., 2000), pattern recognition
techniques (Berrueta et al., 2007); meat quality assessment by e-nose (Ghasemi-
Varnamkhasti et al., 2009) and computational methods for analysis of e-nose data (Jurs et al.,
2000). This review will concentrate on the recent literature on applications of e-noses and e-
tongues in the food industry.
4. Conclusions
Some varieties of olive oil are recognized as being of higher quality because they derive
from well-defined geographical areas, command better prices and generally are legally
protected. Indeed, the aim of Protected Designations of Origin (PDO), Protected
Geographical Indication (PGI) and Traditional Specialty Guaranteed (TSG) is to add value to
certain specific high quality products from a particular origin.
The development of accurate analytical fingerprinting methods for the authentication of
olive oils and for the certification of the geographical origin is an actual issue and an
important challenge. In fact, to protect the rights of both the consumer and honest producers
and, to enforce the laws, it is important to develop analytical methods to measure the
authenticity of the samples, to verify the geographical or cultivar origin and to provide the
presence/absence of adulterants.
Author details
Enzo Perri
*
, Cinzia Benincasa and Innocenzo Muzzalupo
Acknowledgement
Financial support for this study was provided by Italian Ministry of Agriculture, Food and
Forestry through the project GERMOLI Salvaguardia e valorizzazione del GERMoplasma
OLIvicolo delle collezioni del CRA-OLI
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Chapter 14
Oleuropein an Olive Oil Compound in
Acute and Chronic Inflammation Models:
Facts and Perspectives
Domenico Britti, Daniela Impellizzeri,
Antonio Procopio and Salvatore Cuzzocrea
1. Introduction
A large mass of research has been accumulating to provide evidence for the health benefits
of olive oil feeding and to scientifically support the widespread adoption of traditional
Mediterranean diet as a model of healthy eating (Menendez et al., 2007). This evidence has
been attributed to the fact that olive oil, the predominant source of fat in the Mediterranean
diet (Petroni et al., 1995), contains several minor non-nutrients chemicals such as - and -
tocopherols and -carotene, phytosterols, pigments, terpenic acids, flavonoids such as
luteolin and quercetin, squalene, and phenolic compounds, usually and incorrectly termed
polyphenols (Menendez et al., 2007; Visioli et al., 2002; Trichopoulou et al., 2003; Tripoli et al.,
2005; Servili et al., 2004). The main phenolic compounds in virgin olive oil are secoiridoid
derivatives of 2-(3,4-dihydroxyphenyl)ethanol (3,4-DHPEA) and 2-(4-hydroxyphenyl)-
ethanol (p-HPEA) that occur as either simple phenols or esterified with elenolic acid to form,
respectively, oleuropein and its derivative demethyloleuropein, and ligstroside, their
aglycones 3,4-DHPEA-EA and p-HPEA-EA (Figure 1) (Bendini et al., 2007; Surez et al.,
2009). The aglyconic form of oleuropein and ligstroside, 3,4-DHPEA-EA and p-HPEA-EA
respectively, were reported for the first time by Montedoro et al, who also assigned their
chemical structures, later confirmed by other (Montedoro et al., 1992; Angerosa et al., 1996;
Owen et al., 2000). 3,4-DHPEA-EA and p-HPEA-EA, associated to the intense sensory of
bitterness and pungency respectively attribute in VOO (Gutierrez-Rosales et al., 2003), is
endowed with numerous beneficial effects on human health (Servili et al., 2004; Bendini et
al., 2007; Esti et al., 1998; Della Ragione et al., 2000; Paiva-Martins et al., 2001; Fabiani et al.,
2002; Carasco-Pancorbo et al., 2005; Artajo et al., 2006; Fabiani et al., 2006; Fabiani et al., 2008;
Paiva-Martins et al., 2009).


Figure 1. Chemical structures.
In particular, the anti-inflammatory properties of olive oil phenolic compounds seem to
overlap with those attributed to non-steroidal anti-inflammatory drugs (Procopio A, et al.,
2009). The majority of phenolic compounds found in olive oil or table olives are derived
from the hydrolysis of oleuropein, the major phenolic constituent of the leaves and
unprocessed olive drupes of Olea europaea and responsible for the bitter taste of immature
and unprocessed olives. Concentrations of up to 9.0 mg/l of oleouropein and 5.6 mg/l of its
hydrolysis product hydroxytyrosol, have been detected in some preparations of olive oil
(Montedoro et al., 1992). Oleuropein, a glucoside with hydroxyaromatic functionality, has
recognized several pharmacological properties including antioxidant, anti-inflammatory,
anti-atherogenic, anti-cancer, antidiabetic, antimicrobial, and antiviral, and for these
reasons, it is commercially available as food supplement in Mediterranean countries (Miles
et al., 2005; Covas, 2008; Omar, 2010). A more efficient anti-inflammatory role of the
aglyconic 3,4-DHPEA-EA compared with the glycosidic form of oleuropein possibly derives
from the greater lipophilicity of the former, a property that should allow better cell
membrane incorporation and/or interaction with other lipids (Saija et al., 1998). We focused
on the antinflammatory properties of 3,4-DHPEA-EA, a hydrolysis product obtained from
Oleuropein by the action of -glucosidase on the parent glucoside, has been evaluated in a
mice model of acute inflammation (carrageenan-induced pleurisy) and in a mice model of
chronic inflammation (collagen-induced arthritis) (Impellizzeri et al., 2011a-b).
2. 3,4-DHPEA-EA and acute inflammation
Premise - The inflammatory reaction is characterized by an initial increase in blood flow to
the site of injury, enhanced vascular permeability, production of mediators such us
prostaglandins, leukotrienes, histamine, bradykinin, platelet-activating factor (PAF) and the
ordered and directional influx and selective accumulation of different effector cells from the
peripheral blood at the site of injury. Influx of antigen non-specific but highly destructive
cells (neutrophils) is one of the earliest stages of the inflammatory response. Carrageenan-
induced local inflammation is commonly used to evaluate anti-inflammatory effects of non-
R
HO
O
O
CHO
O
R= OH: 3,4-DHPEA-EA
R= H: p-HPEA-EA
MeOOC
R
HO
O
O
OGluc
O
R= OH: R'=Me; oleuropein
R=OH: R'=H; demethyloleuropeine
R= H: R'=Me; ligstroside
R'OOC
R
HO
OH
R= OH: 3,4-DHPEA
R= H: p-HPEA

Oleuropein an Olive Oil Compound in Acute and Chronic Inflammation Models: Facts and Perspectives 289
steroidal drugs (NSAIDs). Therefore, carrageenan-induced local inflammation (pleurisy) is a
useful model to assess the contribution of mediators involved in cellular alterations during
the inflammatory process. In particular, the initial phase of acute inflammation (0-1h) which
is not inhibited by NSAIDs such as indomethacin or aspirin, has been attributed to the
release of histamine, 5-hydroxytryptamine and bradykinin, followed by a late phase (1-6 h)
mainly sustained by prostaglandin release and attributed to the induction of inducible
cyclo-oxygenase (COX-2) in the tissue (Nantel et al., 1999). It appears that the onset of the
carrageenan-induced acute inflammation has been linked to neutrophil infiltration and the
production of neutrophil-derived free radicals, such as hydrogen peroxide, superoxide and
hydroxyl radical, as well as the release of other neutrophil-derived mediators. Free radicals
are produced in small amounts by normal cellular processes as part of the mitochondrial
electron transport chain and the microsomal cytochrome P-450 system. They are formed
during traumatic or hypoxic injuries as a consequence of insufficient oxygenation. Reactive
oxygen species (ROS) and reactive nitrogen species (RNS) can react with and subsequently
damage proteins, nucleic acids, lipids, and extracellular matrix proteins. During the
inflammatory response, ROS and RNS modulate phagocytosis, secretion, gene expression,
and apoptosis. Indeed, under pathological circumstances such as acute lung injury and
sepsis, excess production of neutrophil-derived ROS and RNS may influence neighbouring
endothelial or epithelial cells, contributing to the amplification of inflammatory tissue
injury (Fialkow et al., 2007). Furthermore, oxidative stress elicits the activation of the
redox-sensitive transcription factors such as nuclear factor-B (NF-B) and AP-1, that play
a central and crucial role in inducing the expression of inflammatory cytokines and
intercellular adhesion molecule (ICAM-1) (Chen et al., 2004) and the activation of the
redox-sensitive protein kinases such as the mitogen-activated protein kinase (MAPK)
superfamily (Li et al., 2002). Thus, the study model was designed to evaluate the effects of
3,4-DHPEA-EA in a mice model of acute inflammation (0.1 ml of saline containing 2% -
carrageenan was injected into the pleural cavity). In particular, we investigated the effects
of 3,4-DHPEA-EA on the lung injury associated with carrageenan induced pleurisy. In
order to gain a better insight into the mechanism of action of 3,4-DHPEA-EA, we have
also investigated the effects on: 1) lung damage (histology), 2) polymorphonuclear (PMN)
infiltration (myeloperoxidase [MPO] activity), 3) ICAM-1 and platelet-adhesion-molecule
(P-selectin) expression, 4) nitrotyrosine and poly-ADP-ribose (PAR) formation, 5) pro-
inflammatory cytokines production, tumor necrosis factor- (TNF-) and interleukin-1
(IL-1), 6) lipid peroxidation (malondialdehyde [MDA] levels), and 7) nitric oxide (NO)
synthesis (nitrite-nitrate concentration).
2.1. Materials and methods
Animals - Male CD mice, weight 20-25 g; Harlan Nossan, Milan, Italy, were used in these
studies. The animals were housed in a controlled environment and provided with standard
rodent chow and water. Animal care was in compliance with Italian regulations on the
protection of animals used for experimental and other scientific purposes (D.lgs 116/92) as
well as with EEC regulations (O.J. of E.C. L358/1 12/18/1986).

Carrageenan-induced pleurisy - Carrageenan-induced pleurisy was induced as previously
described (Cuzzocrea et al., 1999). Mice were anaesthetized with isoflurane and subjected to
a skin incision at the level of the left sixth intercostals space. The underlying muscle was
dissected and saline (0.1 ml) or saline containing 2% -carrageenan (0.1 ml) was injected into
the pleural cavity. The skin incision was closed with a suture and the animals were allowed
to recover. At 4 h after the injection of carrageenan, the animals were killed by inhalation of
CO2. The chest was carefully opened and the pleural cavity rinsed with 1 ml of saline
solution containing heparin (5 U ml
-1
) and indomethacin (10 g ml
-1
). The exudate and
washing solution were removed by aspiration and the total volume measured. Any exudate,
which was contaminated with blood, was discarded.
Experimental Design - Mice were randomly allocated into the following groups: (i) CAR +
saline group. Mice were subjected to carrageenan-induced pleurisy (N = 10); (ii) CAR + 3,4-
DHPEA-EA group (100 M/kg). Same as the CAR + saline group but 3,4-DHPEA-EA (100
M/kg, i.p.) were administered 30min after to carrageenan (N = 10); (iii) CAR + 3,4-DHPEA-
EA group (40 M/kg). Same as the CAR + saline group but 3,4-DHPEA-EA (40 M/kg, i.p.)
were administered 30min after to carrageenan (N = 10); (iv) Sham + saline group. Sham-
operated group in which identical surgical procedures to the CAR group was performed,
except that the saline was administered instead of carrageenan (N = 10); (v) Sham + 3,4-
DHPEA-EA group. Same as the Sham+saline group but 3,4-DHPEA-EA (100 M /kg, i.p.)
were administered 30min after to carrageenan (N = 10). The doses of 3,4-DHPEA-EA (40 and
100 M /kg, i.p.) used here were based on previous in vivo studies (Procopio A, et al., 2009).
Histological examination - Lung tissues samples were taken 4 h after injection of carrageenan.
Lung tissues samples were fixed for 1 week in 10 % (w/v) PBS-buffered formaldehyde
solution at room temperature, dehydrated using graded ethanol and embedded in Paraplast
(Sherwood Medical, Mahwah, NJ, USA). Sections were then deparaffinized with xylene,
stained with hematoxylin and eosin. All sections were studied using Axiovision Zeiss
(Milan, Italy) microscope.
Measurement of cytokines - TNF- and IL-1 levels were evaluated in the exudate 4 h after the
induction of pleurisy by carrageenan injection as previously described (Cuzzocrea et al.,
1999). The assay was carried out using a colorimetric commercial ELISA kit (Calbiochem-
Novabiochem Corporation, Milan, Italy).
Measurement of nitrite-nitrate concentration - Total nitrite in exudates, an indicator of NO
synthesis, was measured as previously described (Cuzzocrea et al., 2001). Briefly, the nitrate
in the sample was first reduced to nitrite by incubation with nitrate reductase (670 mU/ml)
and -nicotinamide adenine dinucleotide 3-phosphate (NADPH) (160 M) at room
temperature for 3 h. The total nitrite concentration in the samples was then measured using
the Griess reaction, by adding 100 l of Griess reagent (0.1% w/v) naphthylethylendiamide
dihydrochloride in H2O and 1% (w/v) sulphanilamide in 5% (v/v) concentrated H3PO4; vol.
1:1) to the 100 l sample. The optical density at 550 nm (OD550) was measured using ELISA
microplate reader (SLT-Lab Instr., Salzburg, Austria). Nitrite concentrations were calculated
by comparison with OD550 of standard solutions of sodium nitrite prepared in H2O.

Immunohistochemical localization of ICAM-1, P-selectin, nitrotyrosine and PAR - At the end of
the experiment, the tissues were fixed in 10% (w/v) PBS-buffered formaldehyde and 8 m
sections were prepared from paraffin embedded tissues. After deparaffinization,
endogenous peroxidase was quenched with 0.3% (v/v) hydrogen peroxide in 60% (v/v)
methanol for 30 min. The sections were permeablized with 0.1% (w/v) Triton X-100 in PBS
for 20 min. Non-specific adsorption was minimized by incubating the section in 2% (v/v)
normal goat serum in PBS for 20 min. Endogenous biotin or avidin binding sites were
blocked by sequential incubation for 15 min with biotin and avidin, respectively. Sections
were incubated overnight with anti-nitrotyrosine rabbit polyclonal antibody (Upstate, 1:500
in PBS, v/v), anti-PAR antibody (BioMol, 1:200 in PBS, v/v), anti-ICAM-1 antibody (Santa
Cruz Biotechnology, 1:500 in PBS, v/v) or with anti-P-selectin polyclonal antibody (Santa
Cruz Biotechnology, 1:500 in PBS, v/v). Sections were washed with PBS, and incubated with
secondary antibody. Specific labeling was detected with a biotin-conjugated goat anti-rabbit
IgG and avidin-biotin peroxidase complex (Vector Laboratories, DBA). In order to confirm
that the immunoreaction for the nitrotyrosine was specific, some sections were also
incubated with the primary antibody (anti-nitrotyrosine) in the presence of excess
nitrotyrosine (10 mM) to verify the binding specificity. To verify the binding specificity for
PAR, ICAM-1, P-selectin, some sections were also incubated with only the primary antibody
(no secondary) or with only the secondary antibody (no primary). In these situations no
positive staining was found in the sections indicating that the immunoreaction was positive
in all the experiments carried out.
MPO activity - MPO activity, an indicator of PMN accumulation, was determined as
previously described (Mullane et al., 1985). At the specified time following injection of
carrageenan, lung tissues were obtained and weighed, each piece homogenized in a solution
containing 0.5% (w/v) hexadecyltrimethyl-ammonium bromide dissolved in 10 mM
potassium phosphate buffer (pH 7) and centrifuged for 30 min at 20,000 x g at 4C. An
aliquot of the supernatant was then allowed to react with a solution of tetramethylbenzidine
(1.6 mM) and 0.1 mM H2O2. The rate of change in absorbance was measured
spectrophotometrically at 650 nm. MPO activity was defined as the quantity of enzyme
degrading 1 mol of peroxide min
-1
at 37C and was expressed in milliunits per gram
weight of wet tissue.
MDA measurement - MDA levels in the lung tissue were determined as an indicator of lipid
peroxidation as previously described (Ohkawa et al., 1979). Lung tissue collected at the
specified time, was homogenized in 1.15% (w/v) KCl solution. A 100 l aliquot of the
homogenate was added to a reaction mixture containing 200 l of 8.1% (w/v) SDS, 1.5 ml of
20% (v/v) acetic acid (pH 3.5), 1.5 ml of 0.8% (w/v) thiobarbituric acid and 700 l distilled
water. Samples were then boiled for 1 h at 95C and centrifuged at 3,000 x g for 10 min. The
absorbance of the supernatant was measured using spectrophotometry at 650 nm.
Materials - Unless otherwise stated, all compounds were obtained from Sigma-Aldrich
Company Ltd. (Poole, Dorset, U.K.). 3,4-DHPEA-EA was obtained from the controlled

hydrolysis of oleuropein extracted from olive leaves by means the patented method reported
by Procopio et al. (2009). All other chemicals were of the highest commercial grade available.
All stock solutions were prepared in non-pyrogenic saline (0.9% NaCl; Baxter, Italy, UK).
Statistical evaluation - All values in the figures and text are expressed as mean standard
error (s.e.m.) of the mean of n observations. For the in vivo studies n represents the number
of animals studied. In the experiments involving histology or immunohistochemistry, the
figures shown are representative of at least three experiments (histological or
immunohistochemistry coloration) performed on different experimental days on the tissue
sections collected from all the animals in each group. The results were analyzed by one-way
ANOVA followed by a Bonferroni post-hoc test for multiple comparisons. A p-value less
than 0.05 were considered significant and individual group means were then compared
with Student's unpaired t test. A p-value of less than 0.05 was considered significant.
2.2. Results
Effects of 3,4-DHPEA-EA on carrageenan-induced pleurisy When compared to lung sections
taken from saline-treated animals (sham group Fig. 2A, see histological score 2D)
histological examination of lung sections taken from mice treated with carrageenan revealed
significant tissue damage and edema (Fig. 2B, see histological score 2D) as well as
infiltration of neutrophils (PMNs) within the tissues (Fig. 2B). 3,4-DHPEA-EA (100 M/kg)
reduced the degree of lung injury (Fig. 2C, see histological score 2D). The pleural infiltration
with PMN appeared to correlate with an influx of leukocytes into the lung tissue, thus we
investigated the effect of 3,4-DHPEA-EA on neutrophil infiltration by measurement of MPO
activity. MPO activity was significantly elevated at 4 h after carrageenan administration in
vehicle-treated mice (Fig. 2E). Treatment with 3,4-DHPEA-EA significantly attenuated
neutrophil infiltration into the lung tissue (Fig. 2E).
Effects of 3,4-DHPEA-EA on the expression of adhesion molecules (ICAM-1, P-selectin) - Staining
of lung tissue sections obtained from saline-treated mice with anti-ICAM-1 antibody
showed a specific staining along bronchial epithelium demonstrating that ICAM-1 is
constitutively expressed (Fig. 3A). No positive staining for P-selectin was found in lung
tissue sections from saline-treated mice (Fig. 3D). At 4 h after carrageenan injection, the
ICAM-1 staining intensity increased in the vascular endothelium (Fig. 3B). Lung tissue
sections obtained from carrageenan-treated mice showed positive staining for P-selectin
localized in the vessels (Fig. 3E). No positive staining for ICAM-1 or P-selectin was observed
in the lungs of carrageenan-treated mice treated with 3,4-DHPEA-EA (Fig. 3C and 3F
respectively).
Effects of 3,4-DHPEA-EA on the release of pro-inflammatory cytokine and nitritenitrate
concentration - When compared to sham animals, injection of carrageenan resulted in an
increase in the levels of TNF- and IL-1 in the pleural exudates (Fig. 4A,B). The release
of TNF- and IL-1 was significantly attenuated by treatment with 3,4-DHPEA-EA (Fig.
4A,B).


Figure 2. Effect of 3,4-DHPEA-EA (Ole aglycone) on histological alterations of lung tissue 4 h after
carrageenan-induced injury and on PMN infiltration in the lung. Lung sections taken from carrageenan-
treated mice treated with vehicle demonstrated edema, tissue injury (B) as well as infiltration of the
tissue with neutrophils (B). Carrageenan-treated animals treated with 3,4-DHPEA-EA
(C) demonstrated reduced lung injury and neutrophil infiltration. Section from sham animals
demonstrating the normal architecture of the lung tissue (A). The histological score (D) was made by an
independent observer. MPO activity, index of PMN infiltration, was significantly elevated at 4 h after
carrageenan (CAR) administration in vehicle-treated mice (E), if compared with sham mice (E). 3,4-
DHPEA-EA significantly reduced MPO activity in the lung (E). The figure is representative of at least 3
experiments performed on different experimental days. Data are expressed as mean S.E.M. from
n = 10 mice for each group. **, P < 0.01 versus sham group.
##
, P < 0.01 versus carrageenan.
No significant increase of TNF- and IL-1 exudates levels was found in the sham animal
(Fig. 4A,B). NO levels were also significantly increased in the exudate obtained from mice
administered carrageenan (Fig. 4C). Treatment of mice with 3,4-DHPEA-EA significantly
reduced NO exudates levels (Fig. 4C). No significant increase of NO exudates levels was
found in the sham animal (Fig. 4C).
Effects of 3,4-DHPEA-EA on carrageenan-induced nitrotyrosine formation, lipid
peroxidation and poly-ADP-ribosyl polymerase (PARP) activation - Immunohistochemical
analysis of lung sections obtained from mice treated with carrageenan revealed positive
staining for nitrotyrosine (Fig. 5B). In contrast, no positive staining for nitrotyrosine was
found in the lungs of carrageenan-treated mice, which had been treated with 3,4-DHPEA-
EA (100 M/kg) (Fig. 5C). In addition, at 4 hours after carrageenan-induced pleurisy, MDA
levels were also measured in the lungs as an indicator of lipid peroxidation. As shown in
Figure 5D, MDA levels were significantly increased in the lungs of carrageenan-treated
mice. Lipid peroxidation was significantly attenuated by the intraperitoneal injection of
3,4-DHPEA-EA (Fig. 5D). At the same time point (4 h after carrageenan administration),

lung tissue sections were taken in order to determine the immunehistological staining for
poly ADP-ribosylated proteins (an indicator of PARP activation). A positive staining for
the PAR (Fig. 5F) was found primarily localized in the inflammatory cells present in the
lung tissue from carrageenan-treated mice. 3,4-DHPEA-EA treatment reduced the degree
of PARP activation (Fig. 5G). Please note that there was no staining for either
nitrotyrosine (Fig. 5A) or PAR (Fig. 5E) in lung tissues obtained from the sham group of
mice.

Figure 3. Effect of 3,4-DHPEA-EA (Ole aglycone) on the immunohistochemical localization of ICAM-1
and P-selectin in the lung after carrageenan injection. No positive staining for ICAM-1 was observed in
lung sections taken from sham mice (A). Lung sections taken from carrageenan-treated mice showed
intense positive staining for ICAM-1 along the vessels (B). The degree of positive staining for ICAM-1
was markedly reduced in lung sections obtained from mice treated with 3,4-DHPEA-EA (C). No
positive staining for P-selectin was observed in lung sections taken from sham mice (D). Lung sections
taken from carrageenan-treated mice treated with vehicle showed intense positive staining for P-
selectin along the vessels (E). The degree of positive staining for P-selectin was markedly reduced in
tissue sections obtained from mice treated with 3,4-DHPEA-EA (F). The figure is representative of at
least three experiments performed on different experimental days.


Figure 4. Effect of 3,4-DHPEA-EA (Ole aglycone) on carrageenan-induced pro-inflammatory cytokine
release and NO formation in the lung. TNF- and Il-1 levels were significantly elevated at 4 h after
carrageenan administration in vehicle-treated mice (A and B respectively), if compared with sham mice
(A and B respectively). 3,4-DHPEA-EA significantly reduced TNF- and Il-1 levels (A and B
respectively). Moreover nitrite and nitrate levels, stable NO metabolites, were significantly increased in
the pleural exudates at 4 h after carrageenan administration (C) if compared with sham mice (C). 3,4-
DHPEA-EA significantly reduced the carrageenan-induced elevation of nitrite and nitrate exudates
levels (C). Data are expressed as mean S.E.M. from n 10 mice for each group. **, P < 0.01 versus sham
group.
##


Figure 5. Effect of 3,4-DHPEA-EA (Ole aglycone) on carrageenan-induced nitrotyrosine formation, lipid
peroxidation and PARP activation in the lung. No staining for nitrotyrosine is present in lung section from
sham mice (A). Lung sections taken from carrageenan-treated mice treated with vehicle showed positive
staining for nitrotyrosine, localized mainly in inflammatory cells (B). There was a marked reduction in the
immunostaining for nitrotyrosine in the lungs of carrageenan-treated mice treated with 3,4-DHPEA-EA
(C). Malondialdehyde (MDA) levels, an index of lipid peroxidation, were significantly increased in lung
tissues 4 h after carrageenan administration (D), if compared with lung from sham mice (D). 3,4-DHPEA-
EA significantly reduced the carrageenan-induced elevation of MDA tissues levels (D). Lung sections
taken from carrageenan-treated mice showed positive staining for PAR (F). There was a marked reduction
in the immunostaining for PAR in the lungs of carrageenan-treated mice treated with 3,4-DHPEA-EA (G).
Lung section from sham mice showed no staining for PAR (E). The figure is representative of at least 3
experiments performed on different experimental days. Data are expressed as mean S.E.M. from n 10
mice for each group. **, P < 0.01 versus sham group.
##

2.3. Discussion
All of the above findings are in support of the view that 3,4-DHPEA-EA attenuates the degree
of acute inflammation in the mouse. What, then, is the mechanism by which ole reduces acute
inflammation? One consequence of increased oxidative stress is the activation and inactivation
of redox-sensitive proteins (Bowie & ONeill, 2000). Recent studies have observed that the
acute consumption of olive oil decreased the activation of NF- B system on mononuclear cells
from healthy men (Perez-Martinez et al., 2007) and that 3,4-DHPEA-EA, trans-resveratrol, and
hydroxytyrosol incubated with human umbilical vein endothelial cells inhibit LPS-triggered
NF- B and AP-1 activation (Carluccio et al., 2003). Moreover, various experimental evidence
have clearly suggested that NF- B plays a central role in the regulation of many genes
responsible for the generation of mediators or proteins in acute lung inflammation associated
with carrageenan administration (Cuzzocrea et al., 2006) such us TNF-, IL-1, nitric oxide
synthase inducible (iNOS) and COX-2. By inhibiting the activation of NF- B, the production
of joint destructive inflammatory mediators may be reduced as well. In this regard, Miles et al.,
demonstrated that ole glycoside significantly decreased the concentration of IL-1 in LPS-
stimulated human whole blood cultures. Therefore, this study also demonstrates that 3,4-
DHPEA-EA attenuates the TNF- and IL-1 production in the lung of carrageenan-treated
mice. In addition, recent studies also showed that the potential cardioprotective activity of
oleuropein in acute cardiotoxicity induced by doxorubicin treatment was determined in vivo
in rats (Andreadou et al., 2007) by inhibiting lipid peroxidation products, decreasing oxidative
stress and reducing iNOS in cardiomyocytes. We show here that NO levels, evaluated as
NO2/NO3 and MDA levels which is the products of lipid peroxidation, were increased at 4h
after carrageenan injection while 3,4-DHPEA-EA decreased the levels of NO and MDA. For
many years, much attention has been paid to the effects of NO in respiratory diseases 26 but
recently, the focus has been shifted toward RNS in general, and to peroxynitrite (ONOO
) in
particular (Sadeghi-Hashjin et al., 1998). To probe the pathological contributions of ONOO
to
acute lung injury we have used the appearance of nitrotyrosine staining in the inflamed tissue.
We have observed here that the immunoassaying of nitrotyrosine is reduced in the lung of
carrageenan-treated mice and treated with 3,4-DHPEA-EA. Therefore, the inhibition of
nitrotyrosine formation by oleuropein described in the present study is most likely attributed
to the strong antioxidant activity of ole. During inflammation initiation, circulating leukocytes
must first be able to adhere selectively and efficiently to vascular endothelium. This process is
facilitated by induction of vascular cell adhesion molecules on the inflamed endothelium, such
as vascular cell adhesion molecule VCAM-1, ICAM-1, E-selectin demonstrated that 3,4-
DHPEA-EA was a more potent inhibitor of adhesion molecule expression on cultured human
endothelial cells than was the glycoside(Carluccio et al., 2003). Furthermore, the absence of an
increased expression of the adhesion molecule in the lung from CAR mice treated with 3,4-
DHPEA-EA was correlated with the reduction of leukocyte infiltration as assessed by the
specific granulocyte enzyme MPO and with the attenuation of the lung tissue damage as
evaluated by histological examination. Several studies also showed that in the auricular edema
induced by either arachidonic acid (AA) or 12-O-tetradecanoylphorbol acetate (TPA), the
topical application of the olive oil compounds such as ole also produced an inhibition of the

MPO in the inflamed tissue (de la Puerta et al., 2000). Various studies have demonstrated that
PARP activation after single DNA strand breakage induced by ROS plays an important role in
the process of acute lung injury (Szabo et al., 1998). In this study we confirm the increase in
PAR formation in the lung tissues from carrageenan-treated mice as well as that 3,4-DHPEA-
EA treatment attenuates PARP activation. In this regard, several studies demonstrated that
hydroxytyrosol, a hydrolysis product of 3,4-DHPEA-EA, also exerts an inhibitory effect on
peroxynitrite-dependent DNA base modifications and tyrosine nitration (Deiana et al., 1999).
Similarly, Salvini et al. (2006) showed a 30% reduction of oxidative DNA damage in peripheral
blood lymphocytes during intervention on postmenopausal women with virgin olive oil
containing high amounts of phenols. In conclusion, concomitant with inflammation is the
generation of free radicals, which increase oxidation of proteins and lipids, resulting in signals
that trigger more inflammation. Taken together, the results of the present study enhance our
understanding of the role of ROS generation in the pathophysiology of carrageenan-induced
pleurisy implying that olive oil compounds such as 3,4-DHPEA-EA may be useful in the
therapy of acute inflammation.
3. 3,4-DHPEA-EA and chronic inflammation
Premise - Reactive oxygen species (ROS) are produced in cells by several physiological and
environmental stimulations, such as infections, ultraviolet radiation and pollutants, known
collectively as oxidants. Interestingly, ROS have also been considered as risk and enhancer
factors for autoimmune diseases (Filippin et al., 2008) as there is a significant relation
between the oxidative stress and such diseases (Filippin et al., 2008; Avalos et al., 2007).
Rheumatoid arthritis (RA) is an autoimmune disease characterized by the sequestration of
various leukocyte subpopulations within both the developing pannus and synovial space.
The chronic nature of this disease results in multiple joint inflammation with subsequent
destruction of joint cartilage and erosion of bone. While this disease has a worldwide
distribution, its pathogenesis is not clearly understood (Harris, 1990). Type II collagen-
induced arthritis (CIA) in the mouse has proven to be a useful model of RA, as it possesses
many of the cell and humoral immunity characteristics found in human RA (Holmdahl et al.,
1990). The pathogenesis of CIA is dependent upon the host's response to type II collagen
challenge and the subsequent generation of antibodies that recognizes collagen rich joint
tissue (Holmdahl et al., 1990). The chronic activities initiated by immune complexes trigger a
variety of cell-mediated and humoral events. Moreover, the recruitment and activation of
neutrophils, macrophages, and lymphocytes into joint tissues and the formation of the
pannus are hallmarks of the pathogenesis of both CIA and human RA. Recently, it has been
demonstrated that interleukin (IL)-8, macrophage inflammatory protein (MIP)-l, MIP-1,
and RANTES are differentially chemotactic for lymphocyte subsets (Taub et al., 1993).
Chemokines may play prominent roles in RA, as neutrophil and mononuclear cell
stimulation and activation are prevalent in this disease. Concomitant with inflammation is
the generation of ROS (Trichopoulou et al., 2003)which increase oxidation of proteins and
lipids, resulting in signals that trigger more inflammation. Thus, the study model was
designed to evaluate the effects of 3,4-DHPEA-EA in a mice model of chronic inflammation

(development of CIA in the mice). We have evaluated the following endpoints of the
inflammatory process: (1) clinical score; (2) body weight; (3) inducible oxide nitric synthase
(iNOS) and cyclooxygenase expression (COX-2); (4) nitrotyrosine formation and activation
of the nuclear enzyme poly (ADP-ribose) polymerase (PARP); (5) cytokine and chemokines
production; (6) neutrophil infiltration; (7) joint histopathology.
Animals - DBA/1J mice (9 weeks, Harlan Nossan, Italy) were used for these studies. The
animals were housed in a controlled environment and provided with standard rodent chow
and water. Animal care was in compliance with Italian regulations on protection of animals
used for experimental and other scientific purposes (Dlgs 116/92) as well as with the EEC
regulations (O.J. of E.C. L 358/1 12/18/1986).
Experimental Design - Mice were divided into the following four experimental groups: (i)
CIA-Control; mice were subjected to collagen-induced arthritis (as described below) and
administered 200 l of 10% ethanol solution (i.p., vehicle for 3,4-DHPEA-EA) every 24 h,
starting from day 25 (n = 20); (ii) CIA-3,4-DHPEA-EA; mice subjected to collagen-induced
arthritis (as described below) were administered 3,4-DHPEA-EA (40 M/kg, i.p.) every 24 h,
starting from day 25 (n = 20); (iii) Sham-Control; mice subjected to an intradermal injection
at the base of the tail of 100 l of 0.01 M acetic acid instead of the emulsion containing 100
g of CII, were treated with 200 l of 10% ethanol solution (i.p., vehicle for 3,4-DHPEA-EA),
every 24 h starting from day 25 (n = 20); (iv) Sham-3,4-DHPEA-EA; mice subjected to an
intradermal injection at the base of the tail of 100 l of 0.01 M acetic acid instead of the
emulsion containing 100 g of CII, were administered 3,4-DHPEA-EA (40 M/kg, i.p.),
every 24 h starting from day 25 (n = 20). The dose of 3,4-DHPEA-EA used here to reduce
joint injury was chosen based on a previous study (Procopio A, et al., 2009). Collagen-
induced arthritis (CIA) is induced in mice by two consecutive (interval 21 days) intradermal
injection of 100 l of the emulsion (containing 100 g of bovine type II collagen) (CII) and
complete Freund's adjuvant (CFA) at the base of the tail. Mice develop erosive hind paw
arthritis with macroscopic clinical evidence of CIA as peri-articular erythema and edema in
the hind paws. The incidence of CIA is 100% by day 27 in the CII challenged and the
severity of CIA progressed over a 35-day period with a reabsorption of bone. The
histopathology of CIA include erosion of the cartilage at the joint.
Induction of CIA - Bovine CII was dissolved in 0.01 M acetic acid at a concentration of 2
mg/ml by stirring overnight at 4C. Dissolved CII was frozen at -70C until use. Complete
Freund's adjuvant (CFA) was prepared by addition of Mycobacterium tuberculosis H37Ra
at a concentration of 5 mg/ml. Before injection, CII was emulsified with an equal volume of
CFA. CIA was induced as previously described (Szabo et al., 1998). On day 1, mice were
injected intradermally at the base of the tail with 100 l of the emulsion containing 100 g of
CII. On day 21, a second injection of CII in CFA was administered.
Clinical assessment of CIA - The development of arthritis in mice in all experimental groups was
evaluated daily starting from day 20 after the first intradermal injection by using a
macroscopic scoring system: 0 = no signs of arthritis; 1 = swelling and/or redness of the paw or
one digit; 2 = two joints involved; 3 = more than two joints involved; and 4 = severe arthritis of

the entire paw and digits (Szabo et al., 1998). Arthritic index for each mouse was calculated by
adding the four scores of individual paws. Clinical severity was also determined by
quantitating the change in the paw volume using plethysmometry (model 7140; Ugo Basile).
Histological examination - On day 35, animals were sacrificed while they were under
anesthesia (sodium pentobarbital, 45 mg/kg, i.p), and paws and knees were removed and
fixed in 10% formalin. The paws were then trimmed, placed in decalcifying solution for 24
h, embedded in paraffin, sectioned at 5 m, stained with hematoxylin/eosin and Masson's
trichrome stain and studied using light microscopy (Dialux 22 Leitz).
Immunohistochemical localization of nitrotyrosine, Poly ADP Ribose (PAR), iNOS, and COX-2 - On
day 35, the joints were trimmed, placed in decalcifying solution for 24 h and 8 m sections
were prepared from paraffin embedded tissues. After deparaffinization, endogenous
peroxidase was quenched with 0.3% H2O2 in 60% methanol for 30 min. The sections were
permeabilized with 0.1% Triton X-100 in PBS for 20 min. Non-specific adsorption was
minimized by incubating the section in 2% normal goat serum in phosphate buffered saline for
20 min. Endogenous biotin or avidin binding sites were blocked by sequential incubation for
15 min with avidin and biotin. Sections were incubated overnight with 1) anti-rabbit
polyclonal antibody directed at iNOS (1:1000 in PBS, v/v) (DBA, Milan, Italy) or 2) anti-COX-2
goat polyclonal antibody (1:500 in PBS, v/v) or 3) anti- nitrotyrosine rabbit polyclonal antibody
(1:1000 in PBS, v/v) or 4) with anti-PAR goat polyclonal antibody rat (1:500 in PBS, v/v) or 5).
Controls included buffer alone or non-specific purified rabbit IgG. Specific labeling was
detected with a biotin-conjugated goat anti-rabbit IgG (for nitrotyrosine and iNOS) or with a
biotin-conjugated goat anti-rabbit IgG (for PAR and COX-2) and avidin-biotin peroxidase
complex. In order to confirm that the immunoreaction for the nitrotyrosine was specific some
sections were also incubated with the primary antibody (anti-nitrotyrosine) in the presence of
excess nitrotyrosine (10 mM) to verify the binding specificity. To verify the binding specificity
for PAR, COX-2 and iNOS, some sections were also incubated with only the primary antibody
(no secondary) or with only the secondary antibody (no primary). In these situations, no
positive staining was found in the sections indicating that the immunoreaction was positive in
all the experiments carried out. Immunocytochemistry photographs (N=5) were assessed by
densitometry by using Optilab Graftek software on a Macintosh personal computer.
Measurement of cytokines - Tumor Necrosis Factor- (TNF-) levels were evaluated in the
plasma from CIA and sham mice as previously described (Cuzzocrea et al., 2006). The assay
was carried out using a colorimetric commercial ELISA kit (Calbiochem-Novabiochem Co.,
Milan, Italy) with a lower detection limit of 10 pg/ml.
Measurement of chemokines - Levels of chemokines MIP-1 and MIP-2 were measured in the
aqueous joint extracts. Briefly, joint tissues were prepared by first removing the skin and
separating the limb below the ankle joint. Joint tissues were homogenized on ice in 3 ml
lysis buffer (PBS containing: 2 mM PMSF, and 0,1 mg/ml [final concentration], each of
aprotinin, antipain, leupeptin, and pepstatin A) using Polytron (Brinkinarm Instr.,
Westbury, NY). The homogenized tissues were then centrifuged at 2,000 g for 10 min.
Supernatant were sterilized with a millipore filter (0.2 m) and stored at 80
o
C until

analyzed. The extracts usually contained 0.2-1.5 mg protein/ml, as measured by protein
assay kit (Pierce Chemical Co., Rockford, IL). The levels of MIP-1 and MIP-2 were
quantified using a modification of a double ligand method, as previously described
(Kasama et al., 1994). Briefly, flat-bottomed 96- well microtiter plates were coated with 50
l/well of rabbit anti-cytokine antibodies (1 g/ml in 0.6 mol/liter NaCl, 0.26 mol/liter H3BO4
and 0.08 N NaOH, pH 9.6) for 16 h at 4C, and then washed with PBS, pH 7.5, 0.05% Tween
20 (wash buffer). Nonspecific binding sites on microtiter plates were blocked with 2% BSA
in PBS and incubated for 90 min at 37C. Plates were rinsed four times with wash buffer,
and diluted aqueous joint samples (50 l) were added, followed by incubation for 1 h at
37C. After washing of plates, chromogen substrate was added. The plates were incubated
at room temperature to the desired extinction, after which the reaction was terminated with
50 l/well of 3 M H2SO4 solution. The plates were then read at 490 nm in an ELISA reader.
This ELISA method consistently had a sensitivity limit of ~30 pg/ml.
Myeloperoxidase (MPO) assay - Neutrophil infiltration to the inflamed joints was indirectly
quantitated using an MPO assay, as previously described for neutrophil elicitation (Mullane
et al., 1985). Tissue was prepared as described above and placed in a 50 mM phosphate
buffer (pH = 6.0) with 5% hexadecyltrimethyl ammonium bromide (Sigma Chemical Co.).
Joint tissues were homogenized, sonicated, and centrifuged at 12,000 g for 15 min at 4C.
Supernatants were assayed for MPO activity using a spectrophotometric reaction with O-
dianisidine hydrochloride (Sigma Chemical Co.) at 460 nm.
Materials - 3,4-DHPEA-EA was obtained from Merck Biosciences (Calbiochem, Beecham,
Nottingham, UK). Unless otherwise stated, other compounds were obtained from Sigma-
Aldrich Company (Milan, Italy). All chemicals were of the highest commercial grade
available. All stock solutions were prepared in nonpyrogenic saline (0.9% NaCl; Baxter
Healthcare Ltd., Thetford, Norfolk, U.K.) or 10% ethanol (Sigma-Aldrich).
Data analysis - All values in the figures and text are expressed as mean standard error
(s.e.m.) of the mean of n observations. For the in vivo studies n represents the number of
animals studied. In the experiments involving histology or immunohistochemistry, the
figures shown are representative of at least three experiments (histological or
immunohistochemistry coloration) performed on different experimental days on the tissue
sections collected from all the animals in each group. Data sets were examined by one- or
two-way analysis of variance, and individual group means were then compared with
Student's unpaired t test. For the arthritis studies, Mann-Whitney U test (two-tailed,
independent) was used to compare medians of the arthritic indices (Szabo et al., 1998). A p-
value of less than 0.05 was considered significant.
3.1. Results
Effect of 3,4-DHPEA-EA on joint injury during experimental arthritis - To imitate the clinical
scenario of RA, mice were subjected to CIA. CIA developed rapidly in mice immunized with
CII and clinical signs (periarticular erythema and edema) (Fig. 6B) of the disease first appeared
in hind paws between 24 and 26 days post-challenge (Fig. 6D) leading to a 100% incidence of

CIA at day 28 (Fig. 6D). Hind paw erythema and swelling increased in frequency and severity
in a time-dependent mode with maximum arthritis indices of approximately 10 observed
between day 29 to 35 post immunization (Fig. 6D) in CIA-control mice. 3,4-DHPEA-EA
treatment demonstrated a significant reduction of joint inflammation, as identified by a
significant reduction in the incidence of arthritis (Figure 6C). CIA-3,4-DHPEA-EA mice
showed a 40% reduction in the development of arthritis and a significantly lower arthritis
index compared to CIA- control mice (Fig. 6D). There was no macroscopic evidence of either
hind paw erythema or edema in the sham-control group mice (Fig. 6A and D). The data in
Figure 6E demonstrate a time-dependent increase in hind paw volume (each value represents
the mean of both hind paws). The CIA-3,4-DHPEA-EA mice showed a significant reduction of
paw edema formation when compared to CIA-control mice (Fig. 6E). No increase in hind paw
volume over time was observed in the sham-control mice (Fig. 6E). The rate and the absolute
gain in body weight were comparable in sham-control and CIA-control mice in the first week
(Fig. 6F). From day 25, the CII-challenged mice gained significantly less weight than the sham-
control mice, and this trend continued through to day 35. 3,4-DHPEA-EA treatment
determined a significant increase of the weight gain compared with the vehicle-treatment in
CIA-control mice (Fig. 6F). The histological evaluation (on day 35) of the joint from CIA-
control mice (Fig. 7B) revealed signs of severe arthritis, with inflammatory cell infiltration and
bone erosion. The histological alterations of joint were significantly reduced in 3,4-DHPEA-
EA-treated mice (Fig. 7C). Moreover Massons trichrome stain reveals decreased collagen (blue
stain) in bone and cartilage of arthritic joint due to bone erosion and cartilage degradation in
CIA-control mice (Fig. 7E). The alterations of joint were significantly reduced in 3,4-DHPEA-
EA-treated mice (Fig. 7F). There was no evidence of pathology in the sham-control mice (Fig.
7A and D). The histological score (Fig. 7G) was determined by an independent observer.
Effect of 3,4-DHPEA-EA on cytokines, chemokine expression and neutrophil infiltration - We initiated
studies to assess the effect of 3,4-DHPEA-EA on the expression of chemokines in the aqueous
joint extracts during the development of CIA. As shown in Fig. 8, A and B, the expression of
MIP-1 and MIP-2, measured by ELISA, was significantly increased in the joint 35 days after
CII immunization. MIP-1 and MIP-2 levels in CIA-3,4-DHPEA-EA mice on day 35 were
significantly reduced in a dose-dependent manner in comparison with those in vehicle treated
CIA-control mice. Assessment of neutrophil infiltration into the inflamed joint tissue was
performed by measuring the activity of MPO. It was significantly elevated 35 days after CII
immunization in vehicle-treated CIA-control mice (Fig. 8F), whereas in the CIA-3,4-DHPEA-
EA group, MPO activity was markedly reduced in a dose-dependent manner (Fig. 8F). To test
whether 3,4-DHPEA-EA modulates the inflammatory process through the regulation of
cytokine secretion, we analyzed the plasma levels of the proinflammatory cytokines TNF-,
IL-1, and IL-6. A substantial increase in TNF-, (Fig. 8C), IL-1 (Fig. 8D), and IL-6 (Fig. 8E)
production was found in CIA-control mice 35 days after CII immunization. Levels of TNF-
(Fig. 8C), IL-1 (Fig. 8D), and IL-6 (Fig. 8E) were significantly reduced in a dose-dependent
manner in CIA-3,4-DHPEA-EA mice in comparison to CIA-control mice.
Effect of 3,4-DHPEA-EA treatment on iNOS, COX-2, PGE2, nitrotyrosine, and PAR formation -
Immunohistochemical analysis of the tibiotarsal joint sections obtained from CIA-control

mice revealed positive staining for iNOS (Fig. 10, A and A1) and COX-2 (Fig. 9, A and A1),
which were primarily localized in inflammatory cells. In contrast, staining for iNOS (Fig.
10B) and COX-2 (Fig. 9B) was markedly reduced in the tibiotarsal joints of CIA-3,4-DHPEA-
EA (40 g/kg) mice. No staining for either iNOS or COX-2 was detected in the tibiotarsal
joints obtained from sham control mice (data not shown). Moreover, we also evaluated the

Figure 6. Effect of 3,4-DHPEA-EA (Ole aglycone) on the clinical expression of CIA and on body weight.
A, no clinical signs were observed in sham mice. CIA developed rapidly in mice immunized with CII
and clinical signs such as periarticular erythema and edema (B) were seen with a 100% incidence of CIA
at day 28 (D). E, hind paw erythema and swelling increased in frequency and severity in a time
dependent mode. CIA-3,4-DHPEA-EA mice demonstrated a significant reduction in the clinical signs of
CIA (C), leading to a decrease in the incidence of arthritis in a dose-dependent manner (D). Swelling of
hind paws (F) over time was measured at 2-day intervals. G, beginning on day 25, the CII-challenged
mice gained significantly less weight and this trend continued through day 35. CIA-3,4-DHPEA-EA
mice demonstrated a significant reduced incidence of weight loss (G) as well as less paw edema in a
dose dependent manner (F). The figure is representative of all the animals in each group. Values are
means S.E.M. of 20 animals for each group. **, P < 0.01 versus sham-control; , P < 0.01 versus CIA.


Figure 7. Morphological changes of CIA. Representative hematoxylin and eosin-stained section of joint
was examined by light microscopy. The histological evaluation (on day 35) of joint from CIA-control
mice (B and G) revealed signs of severe arthritis, with inflammatory cell infiltration and bone erosion.
The histological alterations of the joint were significantly reduced in the tissues from CIA-3,4-DHPEA-
EA (40 g/kg)-treated mice (C and G). Massons trichrome stain reveals decreased collagen in bone and
cartilage of arthritic joint due to bone erosion and cartilage degradation in CIA-control mice (E and G).
The alterations of joint were significantly reduced in 3,4-DHPEA-EA (40 g/kg)-treated mice (F and G).
There was no evidence of pathology in the sham-control mice (A, D, and G). The histological score (G)
was made by an independent observer. The figure is representative of at least three experiments
performed on different experimental days. Values are means S.E.M. of 20 animals for each group. **, P
< 0.01 versus sham-control; , P < 0.01 versus CIA.
levels of PGE2, the metabolite of COX-2, in the serum during the development of CIA. A
substantial increase in PGE2 production was found in CIA-control mice 35 days after CII
immunization (Fig. 9E). Levels of PGE-2 were significantly reduced in CIA-3,4-DHPEA-EA
mice in a dose dependent manner compared with those in CIA-control mice (Fig. 9E). The
release of free radicals and oxidant molecules during chronic inflammation has been
suggested to contribute significantly to the tissue injury (Cuzzocrea et al., 2001). On day 35,
positive staining for nitrotyrosine, a marker of nitrosative injury, was found in the tibiotarsal
joints of vehicle-treated CIA-control mice (Fig. 11, A and A1). 3,4-DHPEA-EA (40 g/kg)
treatment significantly reduced the formation of nitrotyrosine (Fig. 11B).
Immunohistochemical analysis of joint sections obtained from CII-challenged mice revealed

positive staining for PAR (Fig. 12, A and A1). In contrast, no positive PAR was found in the
tibiotarsal joints of CII-challenged mice treated with 3,4-DHPEA-EA (40 g/kg) (Fig. 12B).
There was no staining for either nitrotyrosine or PAR in the tibiotarsal joints obtained from
sham-control mice (data not shown).

Figure 8. Effect of 3,4-DHPEA-EA (Ole aglycone) treatment on cytokine and chemokine expression and
neutrophil infiltration. A substantial increase in the expression of MIP-1 (A), MIP-2 (B), MPO activity
(F), plasma TNF- (C), IL-1 (D), and IL-6 levels (E) was found in CIA-control mice 35 days after CII
immunization. CIA-3,4-DHPEA-EA mice demonstrated a significant reduction in the expression of
MIP-1 (A), MIP-2 (B), MPO activity (F), plasma TNF- (C), IL-1 (D), and IL-6 levels in a dose
dependent manner (E). Values are means S.E.M. of 20 animals for each group. **, P < 0.01 versus sham
control; , P < 0.01 versus CIA-control. ND, not detectable.


Figure 9. Effect of 3,4-DHPEA-EA (Ole aglycone) treatment on COX-2 immunostaining and on serum PGE2
levels. A marked increase in COX-2 (A and in particular A1) staining was evident in the paw 35 days after
initiation of CIA. There was a marked reduction in the immunostaining for COX-2 (B) in the paw of CIA-3,4-
DHPEA-EA (40 g/kg) mice. To verify the binding specificity for COX-2, some sections were also incubated
with only the secondary antibody (no primary antibody). No positive staining for COX-2 was found in the
sections indicating that the immunoreaction was positive (see negative control C). In addition, a marked
increase of PGE2 levels was found in the serum of CIA control mice 35 days after CII immunization (E). The
treatment with 3,4-DHPEA-EA also caused a significant reduction in a dose-dependent manner of the serum
levels of the metabolite of COX-2 (E). The figure is representative of at least three experiments performed on
different experimental days. Densitometry analysis of immunocytochemistry photographs (n = 5) for COX-2
from paw section was assessed (D). The assay was performed by using Optilab Graftek software on a
Macintosh personal computer (CPU G3-266). Data are expressed as a percentage of total tissue area. **, P <
0.01 versus sham control; , P < 0.01 versus CIA. ND, not detectable.


Figure 10. Effect of 3,4-DHPEA-EA (Ole aglycone) treatment on iNOS immunostaining. A marked
increase in iNOS (A and in particular A1) staining was evident in the paw 35 days after initiation of
CIA. There was a marked reduction in the immunostaining for iNOS (B) in the paw of CIA-3,4-DHPEA-
EA (40 g/kg) mice. To verify the binding specificity for iNOS, some sections were also incubated with
only the secondary antibody (no primary antibody). No positive staining for iNOS was found in the
sections, indicating that the immunoreaction was positive (see negative control C). The figure is
representative of at least three experiments performed on different experimental days. Densitometry
analysis of immunocytochemistry photographs (n = 5) for iNOS from paw section was assessed (D). The
assay was performed by using Optilab Graftek software on a Macintosh personal computer (CPU G3-
266). Data are expressed as a percentage of total tissue area. **, P < 0.01 versus sham-control; , P < 0.01
versus CIA. ND, not detectable.


Figure 11. Effect of 3,4-DHPEA-EA (Ole aglycone) treatment on nitrotyrosine immunostaining. A
marked increase in nitrotyrosine (A and in particular A1) staining was evident in the paw 35 days after
initiation of CIA. There was a marked reduction in the immunostaining for nitrotyrosine (B) in the paw
of CIA-3,4-DHPEA-EA (40 g/kg)-treated mice. To verify the binding specificity for nitrotyrosine, some
sections were also incubated with only the secondary antibody (no primary antibody). No positive
staining for nitrotyrosine was found in the sections, indicating that the immunoreaction was positive
(see negative control C). The figure is representative of at least three experiments performed on
different experimental days. Densitometry analysis of immunocytochemistry photographs (n = 5) for
nitrotyrosine from paw section was assessed (D). The assay was performed by using Optilab Graftek
software on a Macintosh personal computer (CPU G3-266). Data are expressed as a percentage of total
tissue area. **, P < 0.01 versus sham control; , P < 0.01 versus CIA. ND, not detectable.


Figure 12. Effect of 3,4-DHPEA-EA (Ole aglycone) treatment on PARP immunostaining. A marked
increase in PARP (A and in particular A1), staining was evident in the paw 35 days after initiation of
CIA. There was a marked reduction in the immunostaining for PARP (B) in the paw of CIA-3,4-
DHPEA-EA (40 g/kg)-treated mice. To verify the binding specificity for PARP, some sections were also
incubated with only the secondary antibody (no primary antibody). No positive staining for PARP was
found in the sections, indicating that the immunoreaction was positive (see negative control C). The
figure is representative of at least three experiments performed on different experimental days.
Densitometry analysis of immunocytochemistry photographs (n = 5) for PARP from paw section was
assessed (D). The assay was performed by using Optilab Graftek software on a Macintosh personal
computer (CPU G3-266). Data are expressed as a percentage of total tissue area. **, P <0.01 versus sham-
control; , P<0.01 versus CIA. ND, not detectable.

3,4-DHPEA-EA Inhibits the Progression of Established Arthritis - To confirm that 3,4-
DHPEA-EA exerts beneficial effects in the experimental model of collagen-induced arthritis,
we have also evaluated its effect in a therapeutic regimen of post-treatment (40 g/kg),
starting the treatment at day 28. CIA-3,4-DHPEA-EA post-treatment mice also showed a
reduction in the development of arthritis and a significantly lower arthritis score compared
with those in CIA-control mice as shown in Fig. 13A. 3,4-DHPEA-EA post-treatment also
significantly reduced paw edema formation (Fig. 13B). In addition, 3,4-DHPEA-EA post-
treated mice showed significantly reduced histological alterations of the tibiotarsal joint as
shown in the histological score (Fig. 13C) and increased body weight (Fig. 13D).

Figure 13. Effect of 3,4-DHPEA-EA (Ole aglycone) post-treatment on joint inflammation. Starting the
treatment at day 28, we have also demonstrated that 3,4-DHPEA-EA post-treatment (40 g/kg) caused a
significantly lower arthritis score (A) and a reduction of foot increase (B) compared with the CIA-
control. In addition, we have also shown a reduction in the histological damage (C) and increased body
weight (D) in 3,4-DHPEA-EA -treated mice. Data are expressed as a percentage of total tissue area. **,
P< 0.01 versus sham-control; , P< 0.01 versus CIA.
3.2. Discussion
Rheumatoid arthritis is an inflammatory disease characterized by chronic inflammation of
the synovial joints associated with proliferation of synovial cells and infiltration of activated
immunoinflammatory cells, including memory T cells, macrophages, and plasma cells,
leading to progressive destruction of cartilage and bone (Hitchon et al., 2004). Another
central feature of RA synovitis is the transformation of fibroblast-like synovial cells into
autonomously proliferating cells with a tissue-infiltrating nature, forming hyperplastic

tissue with the potential for bone erosion and cartilage degradation known as pannus
(Filippin et al., 2008). Therefore, it is necessary to establish and characterize experimental
animal models to assess cellular and molecular events that contribute to the pathogenesis of
joint inflammation. Of interest, CII-induced arthritis in the mouse has proven to be a useful
model, because it possesses many of the cellular and humoral immune events found in
human rheumatoid arthritis. Oxidative stress describes an imbalance between ROS
synthesis and antioxidants. Many studies have demonstrated a role of ROS in the
pathogenesis of inflammatory chronic arthropathies, such as rheumatoid arthritis (Filippin
et al., 2008). In this regard, we investigate here the effects of 3,4-DHPEA-EA, a hydrolysis
product of oleuropein, in a mouse model of CII-induced arthritis. Although T-cell and
antibody responses against CII are a crucial event for the initiation of CIA (Holmdahl et al.,
1989), it has been demonstrated that several cytokines also appear to direct cell-to-cell
communication in a cascade fashion during the progression of CIA such as IL-1 (Hom et al.,
1992), TNF- (Dong et al., 2010), and IL-6 (Ferraccioli et al., 2010). In addition, it has been
demonstrated that monocyte chemotactic protein-1, MIP-, MIP-1, and regulated on
activation normal T cell expressed and secreted are differentially chemotactic for
lymphocyte subsets and are expressed in tissue from the inflamed joints of patients with
rheumatoid arthritis (Koch et al., 1994). In this study, we have confirmed that the cytokines
(TNF-, IL-1, and IL-6) as well as the chemokines (MIP-1 and MIP-2) are expressed at
sites of inflamed joints and probably contribute in different capacities to the progression of
chronic joint inflammation. Several cytokines, including TNF- and IL-1, are known
initiators of the nuclear factor (NF- B) activation cascade (Filippin et al., 2008) and are
under its transcriptional control, constituting a positive feedback loop. Recent studies have
observed that the acute consumption of olive oil decreased the activation of the NF- B
system on mononuclear cells from healthy men (Perez-Martinez et al., 2007) and that 3,4-
DHPEA-EA, trans-resveratrol, and hydroxytyrosol incubated with human umbilical vein
endothelial cells inhibit lipopolysaccharide-triggered NF- B and activator protein-1
activation (Carluccio et al., 2003). Of interest, using 3,4-DHPEA-EA, we have demonstrated
an inhibition of the release of proinflammatory cytokines and chemokines and a reduction
of leukocyte infiltration measured by MPO activity. Several studies also showed that the
potential cardioprotective activity of oleuropein in acute cardiotoxicity induced by
doxorubicin treatment was determined in vivo in rats (Andreadou et al., 2007) by inhibiting
lipid peroxidation products, decreasing oxidative stress, and reducing iNOS in
cardiomyocytes and that the olive oil polyphenols are capable of down-regulating COX-2
expression in colonic cancer cells by a mechanism involving the early inhibition of p38
mitogen-activated protein kinase and downstream inhibition of the transcription factor
cAMP response element-binding protein (Corona et al., 2007). We show here that 3,4-
DHPEA-EA decreased iNOS and COX-2 expression by immunohistochemical staining and
also reduced the levels of the metabolite of COX-2, PGE2, in the serum of 3,4-DHPEA-EA
treated mice. Reactive nitrogen species, such as the peroxynitrite radical (ONOO
_
) generated
by the reaction between O2 . and nitric oxide, can also cause oxidative damage (Soneja et al.,
2005). The addition of ONOO
_
to body cells, tissues, and fluids leads to fast protonation,
which may result in the depletion of -SH groups and other antioxidants, oxidation and

nitration of lipids, DNA disruption, and nitration and deamination of DNA bases (Filippin
et al., 2008). In this report, an intense immunostaining of nitrotyrosine formation also
suggested that a structural alteration of joint had occurred, most probably due to the
formation of highly reactive nitrogen derivatives ROS produce strand breaks in DNA, which
triggers energy-consuming DNA repair mechanisms and activates the nuclear enzyme
poly(ADP-ribosyl) polymerase (PARP). There is various evidence that the activation of
PARP may also play an important role in inflammation (Genovese et al., 2005). Continuous
or excessive activation of PARP produces extended chains of ADP-ribose (PAR) on nuclear
proteins and results in a substantial depletion of intracellular NAD and subsequently,
ATP, leading to cellular dysfunction and, ultimately, cell death (Chiarugi, 2002). We
demonstrate here that 3,4-DHPEA-EA treatment reduced the activation of PARP with a
decrease in PAR expression in the joint during CIA. In this regard, several studies
demonstrated that hydroxytyrosol, a hydrolysis product of 3,4-DHPEA-EA, also exerts an
inhibitory effect on peroxynitrite-dependent DNA base modifications and tyrosine
nitration (Deiana et al., 1999). Likewise, Salvini et al. (2006) showed a 30% reduction of
oxidative DNA damage in peripheral blood lymphocytes during intervention in
postmenopausal women with virgin olive oil containing high amounts of phenols. Thus,
3,4-DHPEA-EA, given at the onset of the disease, reduced paw swelling, clinical score,
and the histological severity of the disease when injected after the onset of clinical
arthritis. Amelioration of joint disease was associated with near to full inhibition of
cytokines as well as inhibition of neutrophil infiltration, which is a key player in RA.
Therefore, 3,4-DHPEA-EA was also administered from day 28 after collagen
immunization, targeting this early initiation phase of CIA. Then, with treatment starting
at day 28, 3,4-DHPEA-EA post-treatment caused a significant reduction of inflamed joints
collected at day 35. In conclusion, RA is a complex chronic inflammatory disease
dependent on multiple interacting environmental and genetic factors, making it difficult
to understand its pathogenesis and thereby to find effective therapies. Taken together, the
results of the present study enhance our understanding of the role of ROS generation in
the pathophysiology of CII-induced arthritis, implying that olive oil compounds such as
3,4-DHPEA-EA may be useful in the therapy of inflammation.
4. Overall conclusion
Oxidative stress is described as an imbalance between ROS synthesis and antioxidant
system in the mammal body where the normal production of oxidants is counteracted by
several antioxidative mechanisms. Food constituents are the normal substrate for energy
generation but a hypercaloric diet may result in higher ROS, thus inducing oxidative stress.
Epidemiological studies have shown that populations consuming a predominantly olive oil-
based Mediterranean-style diet exhibit lower incidences of breast cancer and other chronic
diseases than those eating a northern European or North American diet. Habitual high
intakes of olive oil, especially virgin olive oil, provide a continuous supply of antioxidants,
which may reduce oxidative stress via inhibition of lipid peroxidation, a factor that is
currently linked to a host of diseases such as cancer, heart disease, and ageing.

Author details
Domenico Britti and Antonio Procopio
Dept. of Health Sciences, University of Catanzaro, Catanzaro, Italy
Daniela Impellizzeri and Salvatore Cuzzocrea
Dept. of Clinical and Experimental Medicine and Pharmacology, University of Messina,
Torre Biologica Policlinico Universitario, Messina, Italy
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and olive oil. Med Res Rev 2002; 22: 65-75.
Section 3

Table Olive

Chapter 15
Microbiological Aspects of Table Olives
Flora Valeria Romeo
1. Introduction
Table olives are the most important fermented vegetables because of their worldwide
economic importance. The three main techniques for table olive production used in Italy
concern 82% green olives, 16% black olives and 2% processed at the cherry ripened stage
(UNAPROL, 2008). There are three main trade preparations of table olives: Spanish-style
olives, Californian-style olives and naturally black or turning colour olives (Garrido-
Fernndez et al., 1997). The Spanish processing method includes treatment with sodium
hydroxide solution, washing, brining, fermentation and packaging. The Greek-style method
is milder and includes washing, natural fermentation in brine, air-oxidation for colour
improvement, and packing. The Californian method includes lye treatment, washing, iron-
salt treatment and air-oxidation, canning and heat treatment. This last method includes a
final sterilization, so it is usually considered as a more safe production. Besides these most
prominent preparations, there are many other traditional table olive elaboration recipes that
are less known in the international market (Panagou et al., 2003) but very relevant at local
market level, where they are frequently sold in in glass jars or plastic pouches.
In the South of Italy the main traditional process method for table olive production is the
natural olive process, according which untreated, generally green, olives are washed, put
into containers and then filled with freshly prepared brine. Both treated and natural (un-
treated) olives have to fermented, and in order to enhance their safety extent, the current
practice requires the reduction of pH to a value of 4.5 or below. The fruits are maintained in
the brine until they lose their natural bitterness, at least partially (Arroyo-Lpez et al.,
2008a), and where they undergo the fermentation process whose characteristics depend on
the cultivar and on the applied conditions. At the end of the process the olives acquired
typical characteristics of final products.
Olives contain a significant amount of oil, ranging from 12% to 30%, depending on the
considered cultivar. The fermentable carbohydrates of flesh olives generally ranges from 2%
to 6%, however, when the olives are washed or lye treated sugars are also lost along with

other soluble compounds. Olive fermentation is considered to be over when the sugars are
totally consumed by microorganisms. The months necessary for this process might change
depending on several factors, such as the variety and olive size, the salt concentration and
temperature (Cardoso et al., 2010). Nowadays there are neither physico-chemical nor
microbiological controls to objectively determine the end of fermentation and producers
decide, according to personal criteria, when olives are ready to eat (Hurtado et al., 2008).
Other important components of olives are polyphenols. The most important classes of
phenolic compounds in table olives are phenolic acids, phenolic alcohols, flavonoids, and
secoiridoids (Sousa et al., 2006). The olive phenols are nutritionally interesting due to their
antioxidant activities, moreover, these compounds are determinant in the shelf-life of olive
oil and sensory qualities of both oil and table olives. Some of these, such as oleuropein and
its hydrolysis derivatives, have antimicrobial activities against a wide variety of
microorganisms, including lactic acid bacteria (LAB). The inhibiting effect of many
polyphenols on LAB growth has been widely studied (Fleming et al., 1973; Ruiz-Barba et al.,
1993). Moreover, the increase of oleuropein content in the growth medium reduces the
activity of bacteria to hydrolyse this glycoside (Romeo & Poiana, 2007). Recently several
studies on antimicrobial activity of olive products have been carried out, namely with olive
leaves, olive fruits and their pure compounds, such as oleuropein, hydroxytyrosol and
aliphatic aldehydes (Sousa et al., 2006) and it has been found that ferulic acid exhibits
toxicity effects toward several microorganisms (Sayadi et al., 2000).
Compared to the relatively few microbial species employed in other fermented foods,
microorganisms evolved in vegetable fermentations are many and different. In olive fruits
the epiphytic microbial population consists of yeast, fungi, and both Gram positive and
Gram negative bacteria but throughout the fermentation process, Enterobacteriaceae, LAB
and yeasts are the most relevant microorganisms (Garrido-Fernndez et al., 1997).
It has been generally established that LAB are responsible for the fermentation of treated
olives. While LAB and yeasts compete for the fermentation of untreated olives, and in some
cases yeasts can be exclusively responsible for fermentation on untreated olives.
LAB usually isolated from fermentation brines of treated olives include both
heterofermentative and homofermentative species (Hutkins, 2006), and Lactobacillus
plantarum is considered essential to produce the lactic acid needed for preservation and
typical flavour. L. plantarum generally coexists with a yeast population until the end of the
fermentation process and during storage, although other microorganisms may be involved
depending on the applied parameters of the process. Organic acids, such as lactic acid, and
sodium chloride are primary preservatives for table olives. Olives show a water activity
greater than 0.85 and a final pH close to 4.6 or below, which represent the most important
hygienic limit to avoid microbiological risks for consumers.
The control of temperature during the fermentation steps often led to beneficial effects,
especially in those region where the fermentation temperature follows environmental
fluctuations. Unfortunately, in most companies the temperature control is not applicable
because it is an expensive procedure.

Microbiological Aspects of Table Olives 323
The control of salt, temperature, anaerobiosis (or low oxygen percentage) and process
hygiene is necessary for successful fermentation. Under appropriate conditions, most non-
lactic acid bacteria will grow slower than LAB that are less affected and that will grow and
rapidly produce acid compounds, mainly lactic acid. These acids, along with CO2 that may
also be produced, create an even more stringent environment for competitors. So the
competitors disappear while LAB overcome the lactic acid fermentation.
In the past, fermentation was always considered to be an economical means for temporary
preservation of different kind of foods. Nowadays, the consumption of fermented foods is
also promoted because of their health benefits, nutritional value, sensorial properties and
functionality. This last aspect has been studied for some foods, such as yogurt and
fermented milk (Lavermicocca et al., 2005), but it must be improved with regards to
vegetable fermentation. The challenge of the next years must be the enhancement of this
research field and the design of new functional foods.
2. Microorganisms associated to table olives
2.1. Role of yeasts
The positive role of yeasts in table olive fermentation has recently been reconsidered. Yeasts
are especially relevant in directly brined green and black natural olive fermentations, where
fruits are not treated with NaOH solutions. In these conditions, in the first fermentation step
the LAB growth is slow because of the presence of phenolic compounds in brine. Growth of
oxidative yeasts and molds may occur in brine surfaces if the tanks are open. To prevent this
growth, the air layer between the liquid and the top of tank must be reduced as much as
possible.
The main roles of yeasts in the processing of fermented olives, are associated with the
production of alcohols, ethyl acetate, acetaldehyde and organic acids, compounds that are
relevant for the development of taste and aroma and for the preservation of the typical
characteristics of this fermented food (Alves et al., 2012).
Some yeast species seem to improve the growth of LAB. Yeasts are able to synthesize
substances such as vitamins, amino acids and purines, or breakdown complex
carbohydrates, which are essential for the growth of Lactobacillus species that request a
nutritionally rich environment for optimal growth (Viljoen, 2006). However, in table olive
processing yeasts can also produce spoilage such as off-flavour production, clouding of
brines and softening of fruits (Arroyo-Lpez et al., 2008a).
Recently, molecular methods have been applied for the identification of yeast associated
with table olives. These techniques confer a higher degree of accuracy in the final
identification than classical biochemical methods. Deiana et al. (1992) observed that the
species found depended on the degree of maturation of the olive fruits. The genera Candida,
Pichia, Rhodotorula, Saccharomyces, Debaryomyces, Kluyveromyces, Kloeckera, Torulopsis,
Trichosporon and Cryptococcus were found by several authors (Arroyo-Lpez et al., 2008a;
Rodrguez-Gmez et al., 2010). While the main frequently isolated species, in both naturally

black and Spanish-style olive brines, are Candida boidinii, Candida diddensiae, Pichia anomala,
Pichia kluyveri, Pichia membranifaciens and Saccharomyces cerevisiae (Oliveira et al., 2004; Coton
et al., 2005; Arroyo-Lpez et al., 2006). Recently Rodrguez-Gmez and co-workers (2012)
drawn up a list of isolates representative of the yeasts of table olives, and the most suitable
yeasts to be used as starters, alone or in combination with LAB.
The interrelationships between Lactobacillus species and yeasts in table olives may also play
an essential role in product preservation. Several authors have recently focused their
attention on yeast biodiversity associated with the different types of olive processes with
particular regard to their enzymatic activities, in order to propose yeast as starters (Bautista-
Gallego et al., 2011).
2.2. The microbiological hazards in table olives
Fermented vegetable technology is based on lactic acid and alcoholic fermentations, that
convert sugars to different end-products, and the obtained food products take on new and
different characteristics (Hutkins, 2006). To enhance the quality of final product is one of the
main scientific and technological challenges for table olive production together with the
reduction of cost of harvesting, of spoilage occurrence, and of environmental pollution
(Brenes, 2004).
The olive-ecosystem is influenced by the indigenous microbial population, by the intrinsic
factors related to the olives (pH, aw, phenols, sugar content, etc.) and extrinsic factors
(temperature, oxygen and salt levels). The microbial population characterizing the first days
of fermentation seem to be always the same: Enterobacteriaceae, lactic cocci, Bacillaceae and
yeasts, whose evolution is strongly related to the pH value. In addition to these populations,
Nychas and co-workers (2002) also reported the presence of Pseudomonas spp. at the start of
fermentation, that decreased as other Gram-negative bacteria, within the first two weeks of
fermentation.
Among the three main commercial preparations of table olives, there are some processing
parameters affecting the fermentation process. The most important is the pH. The
fermentation process in Spanish-style olives (treated olives) begins at an alkaline pH, higher
than 910, because the fruits are previously treated with NaOH to hydrolyse the oleuropein
(Medina et al., 2010). In this case, the microbial population is mainly composed of
Enterobacteriaceae, lactic cocci and other epiphytic microorganisms which are able to drop the
pH value below 7.0, creating the optimal conditions for LAB growth. Regarding the other
two main preparations, Greek-style and California-style, the fermentation is influenced
since the first steps by the processing conditions. In this case, the microbial population starts
to grow at an acidic pH because organic acid (acetic, citric, lactic) are added to prevent the
growth of Gram-negative bacteria.
No official microbiological criteria for table olives are available. However, the Standards of
the Codex Alimentarius prescribes the minimum requirements related to hygiene for table
olives. The final product shall be free from microorganisms and parasites in amounts which

may represent a hazard to health and shall not contain any substance originating from
microorganisms in amounts which may represent a hazard to health (Pereira et al., 2008). To
reduce the risk of food-borne illness and spoilage phenomenon, good practices in
agriculture (GAP), hygiene (GHP) and manufacturing (GMP) must be applied.
Although heat treatments have some negative effects such as alterations in consistency and
colour (Romeo et al., 2009), the correct use of temperature during pasteurization and/or
sterilization is essential to ensure microbiological safety and stability, inactivating enzymes,
and lessens the oxidizing processes. If heat sterilization is applied to olives, the treatment
must be sufficient both in time and temperature, to destroy spores of Clostridium botulinum
(COI, 2004). While olives preserved by salt and acidification or natural fermentation, are
usually C. botulinum and its toxin free, only if the pH is constantly monitored and
maintained below 4.6. Clostridial bacteria are relatively common in the environment
because they are spore-forming. Spores of C. botulinum were detected both in pasteurized
and sterilized olives (Pereira et al., 2008) indicating a poor attention to the application of
sterilisation parameters. The occurrence of C. botulinum appears, however, to be rare. The
sulphite reducing Clostridium spores are indicators of remote faecal contamination. Their
presence in pasteurized olives is due to the occurrence of anaerobic fermentations or to the
resistance of spores to pasteurization. However, the spores should be destroyed by
sterilisation as its presence in a sterilised product indicates either inadequate heat treatment
or post-sterilisation contamination.
The occurrence of Listeria monocytogenes in green table olives has been assessed,
demonstrating that the product, despite its low pH and high salt concentration, can support
Listeria survival for which an appropriate heat treatment must be applied (Caggia et al.,
2004). Another hazard in table olives is Escherichia coli O157:H7, a pathogenic bacterium
responsible for hemorrhagic colitis and hemolytic uremic syndrome. Its presence may be
particularly associated to the Spanish-style method because the drop in the pH is slower
than in natural fermented olive brine. The death rate of E. coli could be affected by using
starter strain (Spyropoulou et al., 2001). More recently, the species Enterobacter cloacae, an
opportunistic pathogen for humans, has been recovered in spontaneously fermented table
olives (Bevilacqua et al., 2010a). The occurrence of Listeria, Salmonella, Escherichia coli,
Yersinia pathogen strains and others are extensively reported in the scientific report of the
European Food Safety Authority (EFSA) and European Centre for Disease Prevention and
Control (ECDC), issued on 21 February 2012.
Other than the pH value, a parameter which strongly influences the storage and quality of
table olives is NaCl concentration. Its level is important for achieving stability of the
products because it prevents spoilage and growth of pathogens. During recent years,
consumers have developed an attitude on low sodium intake principally because a diet rich
in sodium leads to higher blood pressure. So, several scientific studies (Arroyo-Lpez et al.,
2008b; Romeo et al., 2009; Bautista-Gallego et al., 2010; Bautista-Gallego et al., 2011; Panagou
et al., 2011) have focalized on the viability, application and consequences of replacement of
sodium with calcium or potassium in table olive fermentation. Apparently, NaCl may be
substituted in diverse proportions with KCl or CaCl2 without substantially altering the

usual fermentation profiles and producing good sensorial characteristics. In particular, a
mixture of NaCl, CaCl2 showed the ability to reduce both bacterial and yeast growth, while
KCl showed similar effect of NaCl. Moreover, using different mixed salts, Tsapatsaris and
Kotzekidou (2004) showed that the replacement of NaCl by KCl in Kalamon olives resulted
in a strong synergy between calcium lactate and calcium acetate with higher growth rates of
starter cultures of Lactobacillus plantarum and Debaryomyces hansenii.
The replacement of NaCl with other chlorides could be important in those productions
traditionally processed in a high salt concentration, such as Greek-style olives, because this
action could lower the NaCl concentration without reaching the lowest limits necessary to
obtain a safe product. Therefore, besides the pH decrease and the NaCl concentration,
several actions have been proposed in order to overcome all the fermentation problems:
pasteurization, addition of sugars (glucose and sucrose), extra salt addition and use of
starter cultures. Sugar supplements increase the pH drop rate reducing the dangerous early
stage and ensuring the safety of the final product (Chorianopoulos et al., 2005).
2.3. LAB starter cultures
At industrial level LAB play a positive role in the production of wines and beers, but
therefore they represent major spoilage organisms for such products (Bamforth, 2005). Table
olive processing is still based on empirical methods despite its growing economical value.
However, interest in developing starter cultures to be used in table olives is increasing. LAB
have long been employed in fermentation as a food preservation technique owing to their
progressive acidification of the fermenting brine with a consequent pH decrease (Marsilio et
al., 2005). In addition, the use of LAB could standardize olive fermentation and reduce the
use of highly polluting chemicals as NaOH (lye solution), contribute significantly to storage
preventing microbial spoilage, and improve flavour.

Figure 1. L. plantarum strain detected by fluorescence microscopy.

In table olive processing, starter cultures must have some properties such as good resistance
to the inhibitory effect of polyphenols, good survival against wild strains of related species,
rapid acid production, complete utilization of fermentable sugars, good tolerance against
high levels of salt and low pH, and a possibly inhibitory effect against undesirable
organisms. The latter effect is due to the production of bacteriocin, peptides that were found
to be active against a number of natural competitors of L. plantarum in the fermentation
brines and also against bacteria that can cause olive spoilage (Leal-Snchez et al., 2003). This
property is considered of importance in the development of new preservation technologies
of foods (Devlieghere el al., 2004).
Moreover, lactobacilli are important members of the healthy human microbiota and exert
several beneficial physiological effects, such as antimicrobial and antitumorigenic activities
(Nguyen et al., 2007; Bevilacqua et al., 2010b). The reduction of cholesterol by LAB has been
demonstrated in human, mouse, and pig studies (Nguyen et al., 2007). Nowadays, foods
fortified with health-promoting probiotic bacteria are mainly produced with milk
derivatives, so functional food industries are focusing on new non-dairy foods that can
contribute to a regular assumption of probiotics (Lavermicocca et al., 2005).
The species of the genus Lactobacillus are widely occurring in many natural environments
often playing important roles in fermentation processes and in the regulation of
relationships among species of complex ecosystems. In particular, L. plantarum and L.
pentosus are regarded as the main species leading this process (Table 1) often being used as a
starter in guided olive fermentation (Snchez et al., 2001; Leal-Snchez et al., 2003; Hurtado
et al., 2009). L. pentosus and L. plantarum are also the most frequently isolated species in table
olives; the other species used as inocula, with little exception, have always been studied in
conjunction with them (Hurtado et al., 2012).
However, a significant occurrence of Leuconostoc spp. on olive fruits and leaves was
highlighted in the study of Ercolini and co-workers (2006), suggesting that Lactobacillus spp.
may also originate from the environment or tools of production and not exclusively from the
olives. Lavermicocca and others (2005) used table olives as a vehicle for delivering probiotic
bacterial species, such as Lactobacillus rhamnosus, L. paracasei, Bifidobacterium longum and B.
bifidum, but these strains are not involved in spontaneous fermentation and so they are not
well adapted to the environmental conditions of table olives (Perricone et al., 2010).
Isolation from olive brines of Enterococcus strains has been reported by several authors, so
mixed starters of E. faecium and L. plantarum (Lavermicocca et al., 1998) or E. casseliflavus and
L. pentosus have been studied. The suggestion to inoculate E. casseliflavus, isolated from
fermenting olives, is due to its good tolerance to the initial high pH (in case of lye
treatment), without the drawback of transmissible antibiotic resistance shown by E. faecium
(de Castro et al., 2002).
The selection of starters is based on diverse criteria including homo- and hetero-
fermentative metabolism, acid production, salt tolerance, flavour development, temperature
range growth, oleuropein-splitting capability and bacteriocin production (Panagou et al.,

2008). Furthermore, the ability to grow at low temperatures must be considered essential in
cold regions, since heating brine is complex and expensive (Durn Quintana et al., 1999).

Genus Species Authors & year
Lactobacillus
plantarum
Randazzo et al., 2011
Ruiz-Barba et al., 2010
Perricone et al., 2010
Hurtado et al., 2010
Kumral et al., 2009
Sabatini et al., 2008
Panagou et al., 2008
Saravanos et al., 2008
Romeo & Poiana, 2007
Marsilio et al., 2005
Chorianopoulos et al., 2005
Lamzira et al., 2005
Caggia et al., 2004
Leal-Snchez et al., 2003
Snchez et al., 2001
pentosus
Aponte et al., 2012
Hurtado et al., 2010
Medina et al., 2009; 2008
Panagou et al., 2008
Peres et al., 2008
Servili et al., 2006
Caggia et al., 2004
De Castro et al., 2002
Snchez et al., 2001
casei
Randazzo et al., 2011
Caggia et al., 2004
paracasei
De Bellis et al., 2010
Saravanos et al., 2008
paraplantarum Romeo & Poiana, 2007
brevis
Kumral et al., 2009
coryniformis Aponte et al., 2012
Leuconostoc
cremoris Kumral et al., 2009
paramesenteroides Kumral et al., 2009
Pediococcus pentosaceus Ruiz-Barba et al., 2010
Enterococcus
faecium Ruiz-Barba et al., 2010
casseliflavus De Castro et al., 2002
Table 1. LAB tested as starter cultures in table olives (references are shown for the last twelve years)

3. Enumeration of microorganisms in fermented olives:
Methods of analysis
Each microorganism should grow and form a separate colony when the sample is plated in
a solid medium during plate count procedures. Unfortunately, some organisms may not be
capable to grow under the conditions used. Moreover, some chains of organisms could
appear as a single colony (Swanson et al., 2001).
Up to now, methods available for detection and identification of microbial population
involved in table olive fermentation have been very limited and generally culture-
dependent, not providing reliable information on the composition of the entire microbial
community (Randazzo et al., 2012). A culture-independent method, such as the
denaturing gradient gel electrophoresis (DGGE), has the potential to study microbial
population quickly and economically, avoiding the use of selective cultivation and
isolation of bacteria (Rantsiou et al., 2005). So, plate count techniques are still the major
but not the most representative method. Using aseptic techniques, brine samples may be
taken and used directly to prepare serial dilutions. In order to analyse the whole olive, for
example dry salted olives, the containers should be shaken and after mixing the olives, a
sample should be taken aseptically with a sterile spoon, weighed and transferred to a
sterile container such as plastic bag. After adding a sterile volume of quarter-strength
Ringers solution or 0.9% NaCl, the bag is then pummelled in a stomacher to prepare
dilutions. Each mL collected by this bag represents 1 g of olive sample. The decimal
dilutions are usually used for the calculation of results. Different ranges to obtain a
readable number of colonies on plates may be necessary depending on the
microorganism, procedures and initial contamination of olives. After pipetting the diluted
sample into the petri plate, about 15 mL of each liquefied medium is added (the
temperature must not exceed 45C) into the plate. The medium is mixed with inoculum by
carefully rotating. Several dilutions or replicate plates for each dilution should be
prepared for each sample, making sure that the sample is tested, at least, in triplicates.
After solidification, the petri plates should be inverted and placed in the incubator at
specific temperature shown in the next sections. After incubation, the plates should
contain between 25 and 250 colonies for the best count accuracy (Swanson et al., 2001). If
microbial changes during fermentation are to be followed, the sampling must start at time
zero, when the olives are salted or brined, and samples should be collected at regular
intervals up to the end of fermentation. The sampling intervals may be of 1-3 days during
the first month of fermentation, then these intervals may be extended up to 7 days. The
brine samples, such as olives, should be examined as soon as possible, but if it is
temporarily impossible, the samples must be refrigerated and analysed within 24 hours.
3.1. Lactic acid bacteria
The simply enumeration of LAB may be carried out on MRS agar. It is probably the most
commonly used medium for the cultivation of lactobacilli and other LAB (Schillinger and
Holzapfel, 2003). This medium must be supplemented with nystatin (50 mg/L) or

cycloheximide (100 mg/L) as inhibitors of eukaryotic organisms (to prevent yeasts and
molds growth) or with sodium azide (200 mg/L) as Gram-negative bacteria inhibitor. A
number of differential and selective media were created for the isolation and
characterization of certain groups of LAB. HHD medium is the most used for the differential
enumeration of homofermentative and heterofermentative LAB. This medium contains
fructose which is reduced to mannitol by heterofermentative but not by homofermentative
LAB. Differences in the colour of the colonies are based on differences in the amounts of
acids produced by these bacterial groups (Schillinger & Holzapfel, 2003). So in this medium,
homofermentative LAB are blue to green, while heterofermentative LAB colonies are white
(Fleming et al., 2001). M17 medium should be used for lactococci isolation, but isolation of
these LAB is more frequent when analysing dairy products rather than fermented
vegetables. All LAB isolation requires anaerobic conditions and incubation at 30-32C for 48-
72 hours, depending on the medium used.
3.2. Mesophilic aerobic bacteria
The mesophilic count, or standard plate count, is generally obtained on Plate Count agar
(PCA) which is a generic medium for aerobic microorganisms that grow at mesophilic
temperatures. Aerobic plate counts are poor indicators of safety in some products such as
those fermented which commonly show a high aerobic count. However, this count gives
information about the hygienic and sensorial quality, about the adherence to good
manufacturing practice and shelf life of the product (Morton, 2001).
Alternatively to PCA, similar generic media are available as Nutrient agar. The growth
conditions are 25-30C for 24-48 hours, aerobically.
3.3. Yeasts and molds
Yeasts and molds are widely diffused eukaryotic microorganisms because of their
adaptation to different environmental conditions. Yeasts and molds can cause various
degrees of food decomposition. Invasion and growth may occur on virtually any type of
food (Beuchat & Cousin, 2001). Their contamination of food can lead to product losses but,
in particular, the highest risk is due to the mycotoxin production by the molds. Several
yeasts genera may be important for the sensorial properties of fermented products and for
their interrelation with Lactobacillus bacteria (see section 2.1) in fermented olives. However,
film-forming yeasts as Debaryomyces, Candida, Pichia and Endomycopsis, are often associated
with pickled products and vegetable brines (Fleming et al., 2001), representing the cause of
olive defects and consequent product losses.
Available media for yeasts and molds count are several: Rose Bengal Chloramphenicol agar,
YM agar, Oxytetracycline Glucose Yeast Extract agar, Sabouraud agar and others. When not
included in the medium, the addition of 100 mg/L chloramphenicol is recommended to
inhibit the growth of bacteria. For simple enumeration of yeasts and molds, the plates
should be incubated aerobically at 25C for 48 hours.

3.4. Enterobacteriaceae and coliform bacteria
The Enterobacteriaceae family consists of Gram-negative, facultative anaerobic rods widely
distributed in the environment, but which are usually associated with intestinal infections.

Figure 2. E. coli strain detected by fluorescence microscopy.
Numerous studies have determined that Escherichia coli, coliforms, faecal coliforms and
Enterobacteriaceae are unreliable when used as index of pathogen contamination of food. So
National and International advisory committees invalidated the prediction of food safety
based on levels of Enterobacteriaceae, coliforms, faecal coliforms and E. coli (Kornacki &
Johnson, 2001). The most important application of Enterobacteriaceae and coliforms is their
enumeration to assess if pasteurization has been adequately performed for example in
pasteurized milk, because a proper pasteurization under appropriate conditions inactivates
E. coli cells present in raw material.
Total coliform bacteria belong to the Enterobacteriaceae family, and include E. coli as well as
various members of the genera Enterobacter, Klebsiella and Citrobacter. All ferment lactose
with gas and acid production in 48 hours at 35-37C. While faecal coliforms are coliforms
which can ferment lactose to acid and gas within 48 hours at 45C and they are so called
because are more closely associated with faecal pollution (Manafi, 2003). The term coliform
is based only on lactose hydrolysis and has no taxonomy validity. In order to perform the
best examination of olive samples, it is preferable enumerate both Enterobacteriaceae and
coliforms, because some lactose-negative bacteria, such as Salmonella, are pathogens.
The most probable number (MPN) is a method which indicates the most likely number of
microorganisms present in the analysed sample. This method, used for several years, has
now been replaced with techniques on agar media because the MPN is based on a statistical
approximation. So, in addition to Violet Red Bile agar or Violet Red Bile Glucose agar, other
differential media based on a chromatic response were developed.

Mac Conkey's MUG agar and Eosin Methylene Blue agar (EMB) are differential and
selective media, suitable to obtain at the same time the isolation of Salmonella, Shigella and
coliform bacteria, in particular E. coli. In the first medium, lactose-negative colonies are
colourless, lactose-positive colonies are red and often surrounded by a turbid zone due to
the precipitation of bile acids. E. coli can be identified by fluorescence in UV due to its -D-
glucoronidase production. In EMB agar, lactose-fermenters form colonies with dark-blue
centres (E. coli may also have a green metallic sheen) while the non-lactose fermenters form
completely colourless colonies.
3.5. Staphylococcus aureus
The foods most associated with staphylococcal poisoning are meat products, dairy products
and cream filled bakery products. In processed foods in which S. aureus is destroyed by
processing, its presence usually indicates post-processing contamination from human skin,
mouth, nose or food handlers (Lancette & Bennett, 2003). Due to the high salt tolerance of S.
aureus, it can grow in table olives even though the low pH and the olive phenols may
represent natural inhibitors (Tassou & Nychas, 1994). However, it may be isolated and
enumerated in table olives for the same above mentioned reason (as contamination index).
A variety of coagulase-positive and coagulase-negative staphylococci are able to produce
enterotoxins, but S. aureus still plays a predominant role in staphylococcal food poisoning.
Baird-Parker agar and Rabbit Plasma Fibrinogen agar are the media recommended by the
International Organisation for Standardisation (ISO). Moreover, Baird-Parker agar is also
used in the official AOAC method in the United States (Zangerl &Asperger, 2003). Tellurite
reduction, egg yolk reaction and a high level of sodium chloride are the most applied
selective chemicals added in media for S. aureus isolation and enumeration.

Figure 3. S. aureus colonies on Mannitol Salt Agar.
S. aureus colonies in Baird-Parker agar are black, with an opaque zone around the colony
because of the egg yolk reaction. Another medium frequently used is the Mannitol Salt agar,

which contains mannitol. Coagulase-positive staphylococci grow and produce acid from
mannitol showing a yellow colony and halo in a red medium. For all these media, the
chemical inhibitors usually used are not completely selective, so additional diagnostic tests
may be necessary to identify S. aureus colonies. Microscopic examination, catalase test and
coagulase test may be rapidly executed to identify S. aureus from isolates. Common MPN
procedures may be used also for enumeration of S. aureus. In most cases, the methodologies
need a liquid enrichment procedure to detect low numbers of staphylococci (<100 UFC/g).
Culture conditions are usually 37C for 48 hours aerobically.

Figure 4. S. aureus strain detected by fluorescence microscopy.
3.6. Sulphite reducing clostridia
Clostridia are a widespread heterogeneous group of bacteria showing metabolic and
nutritional differences. They easily contaminate foods because they produce resistant spores
which can survive under mild processing conditions. The group of sulphite reducing
clostridia may be used as marker of raw material quality and hygiene of manufacturing
practices. Of particular concern for public health are C. botulinum, which forms a deadly
toxin in foods, and C. perfringens which causes enteritis when present in high numbers.
Other species or strains are also known to be toxinogenic or neurotoxinogenic (Bredius &
Ree, 2003). C. perfringens is the agent of a food poisoning usually associated with
consumption of cooked meats or poultry products. Sporulating cells of C. perfringens
produce a heat-labile enterotoxin which appears to be released in vivo in the intestine
(Labbe, 2001).
Most isolation media include sulphite and an appropriate iron salt. Sulphite is reduced to
sulphide by the clostridial enzyme sulphite reductase; the sulphide will then precipitate as a
black deposit in the presence of iron salt. This causes a blackening of the liquid media and
clostridia will appear as black colonies. To obtain a higher selectivity for C. perfringens
isolation, the Oleandomycin Polymixin Sulphadiazine Perfringens (OPSP) agar medium was
performed. It contains selective antibiotics and utilises sodium metabisulphite and liver

extract as sources of H2S with ferric ammonium citrate as the indicator. In any case,
subsequent confirmation tests such as motility, reduction of nitrate, lactose fermentation,
gelatin liquefaction (Labbe, 2001) should be necessary. The plates are incubated
anaerobically at 37C for 18-48 hours.
3.7. Listeria monocytogenes
Listeria species are ubiquitous organisms widely distributed in the environment, especially
in plant matter and soil. The microorganism is established as an important foodborne
pathogen, which can grow at high salt concentration (up to 10% of NaCl) and at refrigerated
temperatures. A study carried out by the U.S. Food and Drug Administration, U.S.
Department of Agriculture (USDA, 2001), indicated that vegetables are able to support L.
monocytogenes growth and, after a few years, Caggia and co-workers (2004) demonstrated
that L. monocytogenes can survive and grow in green table olives. As indicated by the most
recent scientific report of the European Food Safety Authority (2012), the number of
listeriosis cases in humans in the EU slightly decreased, and 1,601 confirmed human cases
were reported in 2010.
The minimum infective dose of L. monocytogenes has not yet been established and many
authorities require that the organism must be absent in 25 g of product. This has led to the
development of methods following the sequence of pre-enrichment, selective enrichment
and diagnostic plating. The whole procedure may take about five days. Then, presumptive
positive results need to be confirmed adding further time to complete the examination
(Beumer & Curtis, 2003).

Figure 5. L. monocytogenes colonies on ALOA medium.
Most of the isolation media differentiate Listeria spp. by means of aesculin hydrolysis which,
in the presence of iron, forms a black phenolic compound. According to the USDA method

(McClain & Lee, 1988) 25 mL of brine sample are added to 225 mL of Listeria Enrichment
broth base for the resuscitation of stressed cells. After blending, the bag is incubated at 30C
and then, at intervals from 4 hours to 7 days, an aliquot is plated for enumeration in Listeria
Selective agar base and incubated at 37C for 24 h. Black colonies on selective agar base
medium were considered presumptive Listeria colonies.
The presumptive Listeria isolates are to be tested for sugar fermentation, tumbling motility,
hemolytic reaction and growth at different salt concentrations (Caggia et al., 2004).
Alternatively to this medium, ALOA agar contains the chromogenic compound for the
detection of -glucosidase, common to all Listeria, which appear as blue coloured colonies.
While L. monocytogenes, which possesses a specific phospholipase, hydrolyses the specific
substrate added to the medium producing an opaque halo around the colonies (Beumer &
Curtis, 2003). Another chromogenic medium used is the Rapid L. mono agar.
Where counts < 100 CFU/g are expected, it is necessary to use the Most Probable Number
(MPN) technique (USDA, 2001).
In order to rapidly perform additional confirmation tests, systems of strips are commercially
available for Listeria (API Listeria) as for other pathogens.
3.8. Propionibacteria
Propionibacteria are often associated with food spoilage. The zapateria spoilage, which
can occur in brined olives, is characterized by a malodorous fermentation due to propionic
acid produced by certain species of Propionibacterium (Jay, 2000), alone or together with
Clostridium spp.. The propionibacteria may be considered as marker of the end of shelf life of
table olives, because the growth of these bacteria cause an increase in pH values creating
conditions for the growth of spoilage or pathogen bacteria and the arising of off-odors
(Plastourgos & Vaughn, 1957).
Unfortunately, propionibacteria are difficult to isolate because they grow very slowly on
solid media. The complex medium usually used is not able to suppress competing
organisms. This medium is Sodium Lactate agar (1% typticase, 1% yeast extract, 1% sodium
lactate, 0.025% dipotassium phosphate, 1.5% agar) in which the propionibacteria growth
appears in 5-7 days at 32C under anaerobic or microaerophilic conditions (Richter &
Vedamuthu, 2001).
4. Conclusion
A successful olive treatment depends on different factors. The olive cultivars show different
fermentation behaviours when directly brined. In fact, besides the correct application of
manufacturing practices, the knowledge of the chemical and physical characteristics of olive
cultivar used and its attitude to treatments are decisive. A more complete knowledge of the
olive cultivars is necessary to bring fermentation to a successful condition and attain a good
final product. A correlation between each method and chemical-physical composition of

each cultivar is needed. Moreover, the presence of potential pathogens detected in all olive
treatments by several authors suggests the necessity of pH control, following good hygienic
practices throughout the process, and the necessity of a heat treatment of traditional
products which are often empirically performed. Control and verification systems should be
employed in order to guarantee a safe and hygienic product.
Author details
Flora Valeria Romeo
Acknowledgement
The author greatly thanks Emanuela Fornasari and Barbara Bonvini of the Fodder and Dairy
Productions Research Centre (CRA-FLC, Lodi) for helpfully providing the images of
bacteria.
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Chapter 16
Nutritional and Sensory Quality of Table Olives
Barbara Lanza
1. Introduction
Table olives are greatly consumed mainly by Mediterranean Sea area populations. The
italian cuisine, for example, offers many dishes, aperitifs and appetizer in which olives are
an essential ingredient: fish and meat cooked with olives, table olive-based condiments for
pasta and pizza, bread-dough mixed with green/black olives, bruschetta with olive paste, and
so on. The gastronomic uses of olives are widely and very well-known, but the same cannot
be said about its nutritional, nutraceutical and sensory properties. The various Italian olive
cultivars with remarkable aptitude for processing as table olives have allowed the
development of specific and diversified process technologies. Both chemical and biological
treatments perform the dual function of hydrolyzing the compounds responsible for the
bitter taste of olive fruit and stabilizing the end product to overcome the constraint of
seasonal production. All technological interventions have negative collateral effects that
must be minimized or at least kept under control to avoid compromising the quality of the
processed product to satisfy the expectation, of the consumer, directed towards natural or
minimally processed products, whose nutritional properties and health benefits remain
unaltered.
The most important production zones of table olives are located in the Mediterranean area
and their consumption is expanding, due to the increasing popularity of the Mediterranean
diet (Table 1). In Italy, during the last three years, the average consumption of table olives
was approximately 124.000 tonnes/year with a pro-capite assumption of 2.1 kg/year. The
Italian production covers only 48.1% of the consumers demand, the remaining part is
imported from Spain and Greece.
The Italian table olive-growing boasts millenary traditions and its history is an integral part
of Italian culture, since its first inhabitants, in prehistoric epoch, used the alimentary
resources offered by the primitive Mediterranean Macchia and began to domesticate the
wild olive tree. Among the over 500 varieties of Olea europaea on the national territory only a

fraction is specifically suitable for processing as table olives. Italy is rich in typical table olive
products, obtained by traditional methods, many of which have obtained or aspired to the
recognition of trademarks (PDO, PGI, TAP). The reference to the origin through the
Protected Designation of Origin and the Protected Geographical Indication gives to the
product added value and it is greatly appreciated by the consumer. The Italian PDO
recognized are only three, Nocellara del Belice (Sicilia), La Bella della Daunia (Puglia)
and Oliva Ascolana del Piceno (Marche and Abruzzo). The trademark Traditional
Agrifood Products-TAP" (Decree of Italian Ministry of Agriculture 18 July 2000; XII Revision
of 7 June 2012) indicate those food products whose methods of processing are consolidated
and have been used for a period not less than 25 years. Italy recognize many traditional
table olive products, thanks to the country tradition that has passed to the present unique
and singular processing technologies for table olives, reflecting the different realities that
characterize the different Italian regions (Table 2).

Table 1. Table olive consumption. Data are expressed as 1000 tonnes. Source: International Olive
Council (November 2011). * provisional; **previsional.

Nutritional and Sensory Quality of Table Olives 345
Region Traditional Agrifood Product (TAP) Cultivar
Sicilia oliva nebba, oliva nera passuluni Nocellara del Belice
Calabria
olive alla calce, olive in salamoia, olive nella giara,
olive nere infornate, olive schiacciate, olive sotto sale
Carolea
Tonda di Strongoli
Cassanese
Borgese
Basilicata olive nere secche, oliva da forno di Ferrandina Majatica
Puglia
oliva da mensa (mele di bitetto, ualie dolc), olive
cazzate o schiacciate, olive celline di Nard in concia
tradizionale - olive in concia (ciline alla capsa volie
alla capsa), olive in salamoia, olive verdi, Peranzana
da mensa di Torremaggiore
Termite di Bitetto
Ogliarola leccese
Bella di Cerignola
Cellina di Nard
Peranzana
Campania
olive pisciottane schiacciate sottolio, oliva caiazzara,
oliva vernacciola di Melizzano
Pisciottana
Caiazzana
Vernacciola
Lazio
olive da mensa bianche e nere (olive calce e cenere,
olive di Gaeta, oliva bianca di Itri, olive al fumo, olive
sottolio, olive spaccate e condite, olive in salamoia,
olive essiccate) , pestato di olive di Gaeta
Itrana
Carboncella
Abruzzo olive Intosso (olive ndosse, olive in salamoia) Intosso
Molise olive al naturale (live curvate, olie allacqua e sale)
Sperone di gallo
Olivone
Olivoncello
Leccino
Marche olive nere marinate (olive nere strinate), salsa di olive
Raggiola
Raggia
Piantone di Falerone
Leccino
Toscana olive in salamoia
Leccino
Frantoio
Liguria olivo taggiasca Taggiasca
Sardegna olive a scabecciu, olive verdi in salamoia
Tonda di Cagliari
Pizz'e Carroga
Table 2. TAP table olives (XII Revision 2012).
2. Preparations and styles
The numerous Italian olive cultivars with remarkable aptitude for processing as table olives
have allowed the development of specific and diversified process technologies. The Italian
table olive production is mainly concentrated in the Southern and Central regions of Italy
(Figure 1).


Figure 1. Regional distribution of Italian table olive production.
The main Italian table olive preparations are the following:
2.1. Treated olives
The treated olives, according to "Trade Standard Applying to Table Olives" (IOC, 2004)
are green olives, olives turning color or black olives that have undergone alkaline
treatment, then packed in brine in which they undergo complete or partial fermentation,
and preserved or not by the addition of acidifying agents.
2.1.1. Treated green olives in brine by Sevillan-style
This method is also known as Sevillan or Spanish-style and it is one of the most common
and oldest methods for Italian green table olives (Tavanti, 1819). To obtain the treated
green olives in brine, the fruits are debittered with NaOH aqueous solution ranging from
2.0% to 3.5%, mainly depending on the variety and ripeness of the olives. The alkaline
treatment performs the function of hydrolyzing the compound principally responsible for
the bitter taste (oleuropein). The lye solution completely covers the fruits and the olives
remain in this solution until the lye has penetrated between 2/3 and 3/4 of the distance

between the skin and the pit. To verify a correct lye treatment, olives are cut with a
particularly sharp blade such as razor blade or scalpel near the pit, checking the surface after
air exposition. After alkaline treatment, the olives are washed with potable water. The
sequence of washings is the following:
- the first one is a strong washing with potable water made using a shower lasting 15-20
minutes in order to eliminate the lye attached to the fruit surface; the olives washed are
then left in the final washing water
- after 2-3 hours a second faster and simpler washing takes place using a filling-
emptying procedure
- in the following 24-48 hours 3-4 other washings are carried out, using always a filling-
emptying procedure.
After water-washings to eliminate the residual lye, olives are covered with a sodium
chloride solution (brine) and left to develop a spontaneous lactic fermentation. Initial brine
concentrations are 8-10% NaCl but rapidly drop to 5% due to the high content of
interchangeable water in the olives. A spontaneous fermentation starts as soon as the olives
are placed in brine. After alkaline treatment, the pH of olive flesh reaches the value of 11.0-
13.0 down to the value of 8.0-9.0 after the repeated washings. In this broth culture, a
complex and variable microbiota grows. Reducing sugars and glucosides, the basic sources
of carbon needed in the development of lactobacilli and other microrganisms, pass from
olive flesh to the brine, where they are used by heterofermentative or homofermentative
microrganisms to produce lactic acid. In the first phase of fermentation, when Gram-
negative bacteria prevail, the pH ranges from 8.0-9.0 to about 6.0. This low pH promotes the
growth of lactic acid bacteria that are aciduric with optimal growth between pH 5.5 and 5.8.
At the end of lactic fermentation, the pH reaches values <4.0 and acidity increases, ensuring
thus the preservation of the product. The lactic fermentation ends with the exhaustion of
available carbohydrates (glucose from glucosides and reducing sugars). Several researches
have been carried out to evaluate the technological functionality of selected lactic acid
bacteria or enterococci in Spanish-style green olive processing. Enterococcus casseliflavus and
Lactobacillus pentosus (de Castro et al., 2002; Snchez et al., 2001) have been proposed as
starter cultures to accelerate lactic acid formation at pH 9 (immediately after washings). In
this case, obviously, the strain used as starter is not necessarily oleuropeinolytic because lye
has just demolished the bitter glucoside. The importance of this type of starter is to reduce
the lag phase and the risk of spoilage (Bevilacqua et al., 2010; Ruiz-Barba et al., 1994; Leal-
Sanchez et al., 2003). As a result of ill-treatment alkali set or poorly conducted, the tissues of
the fruit may also undergo profound changes in structural and nutritional constituents
(Marsilio & Lanza, 1995).
2.1.2. Sweet green olives by Castelvetrano-style
This is a production method used in Sicilia, almost exclusively in the Castelvetrano district
using the Nocellara del Belice cultivar. The product is mainly consumed in central and
southern Italy (Cappello & Poiana, 2005; Lanza & Poiana, 2011). The olives are hand-

harvested at the green stage of ripening and, once they arrive at the processing plant, they
are graded, since only fruits of more than 19 mm in diameter are used. The selected olives
are put into plastic vessels and covered with 2.5-3.5 B NaOH solution (1.7-2.4%),
depending on the fruit ripeness and size. These vessels have 220 L total capacity, and are
filled with around 140 kg of fruits. Eight hours after the lye treatment begins, 6-7 kg of
grinded salt is added to each container, and the olives are kept in this alkaline brine for 8-
10 days. Salting is performed by placing the grinded salt over the press-fruit of each barrel
(Figure 2).

Figure 2. Addition of grinded salt directly on the press-fruit at the surface of the barrel
(photograph by A. Cappello from Lanza & Poiana, 2011).
The sodium chloride in contact with the liquid is slowly dissolved and is distributed by
gravity in the whole volume of the container. The salt traditionally used in this type of
procedure is sea salt coming from the saltworks of Trapani. It is particularly rich in trace
elements such as K, Mg, Ca, Fe and I, unlike the rock salt composed almost exclusively of
NaCl. In this environment, the olives are softened quickly highlighting a deep green color.
When the barrel is opened, the lye/salt brine is drained and the olives are washed to remove
excess lye. A mild washing step, carried out before marketing, does not totally eliminate the
lye, whose soapy taste is appreciated by the consumers of these olives. Under ambient
storage conditions the Castelvetrano olives have a shelf-life of only a few months, especially
under hot conditions. Deteriorated olives lose colour and develop off-odours. As this olive
style is a seasonal product, long-term storage is not required. Future research, however, may
refine the process and include steps for long-term storage under refrigeration, with the
temperature maintained between 4 C and 7 C, or in packing solutions.

2.1.3. Green olives by lime-and-ash treatment
In some Italian regions (Puglia, Campania, Lazio and Calabria), olives are prepared with an
ancient and traditional method that includes a debittering phase obtained using a lime-ash
mixture, preparing these treated green olives by mixing CaO (lime) with olive wood ash and
adding water to a paste, leaving submerged the green olives for some hours at room
temperature. The ratio of lime and ash is different (1:4, 1:8, 1:10, etc) and depends on the
cultivar and shape of the fruit. The action of lime-ash is similar to that of NaOH. After
alkaline treatment, the olives are washed with potable water. The sequence of washings is
similar to the sequence previously described for the treated green olives by Sevillan-style. It
is possible to eat these olives right after the debittering treatment or after brining. In this
case, after the washing step, olives were placed in an initial brine solution of NaCl (8% w/v),
where a spontaneous fermentation takes place. NaCl concentration is carefully monitored
during this phase. The brine concentration decreases quickly as a consequence of the
osmotic phenomena between the brine and the fruits from 8% to about 5% in 48-72 h. For
this reason after 4-5 days from brining, grinded salt is added to the brine to restore the initial
concentration. After 1-2 months of fermentation olives are ready to eat.
2.2. Natural olives
The natural olives, according to the "Trade Standard Applying to Table Olives" (IOC,
2004) are green olives, olives turning color or black olives placed directly in brine in which
they undergo complete or partial fermentation, preserved or not by the addition of
acidifying agents. The most important industrial preparation for natural black olives takes
the name Greek-style because it is traditionally practiced in Greece utilizing Conservolea
cv. (Balatsouras, 1990).
2.2.1. Natural turning colour olives
Italy has a long tradition in producing natural turning colour olives. In this process the
olives are directly brined in 8-10% of sodium chloride. The brine stimulates the microbial
activity for fermentation and reduces the bitterness of the oleuropein. Fermentation of these
olives takes a long time because diffusion of soluble components through the epidermis, in
fruits not treated with alkali, is slow. A diverse microbiota grows in these brines, although
yeasts are the microorganisms always present throughout the process. Enterobacteriaceae can
be found during the first 7-15 days, but they disappear as the brine characteristics do not
support their growth. The presence of lactic acid bacteria depends on the salt concentration
and the polyphenol content of the variety used. The traditional brining is carried out under
anaerobic conditions. However, an aerobic method can be applied, using a central column in
the fermenter through which air is bubbled. This system changes the ratio between
fermentative and oxidative yeasts, and a final product with better quality is attained (Garcia
Garcia et al., 1985 ; Garrido Fernndez et al., 1997). The colour fades during the process, but it is
corrected by aerating the olives for two or three days; sometimes they are treated with 0.1%
ferrous gluconate or lactate to make them deep black. Lastly, the olives are selected and

packed in barrels or in varnished cans, which are filled with 8% fresh brine. They are popular
because of their slightly bitter taste and aroma. When the bitterness has been sufficiently
weakened the fruit can be marketed. To reduce the debittering phase, several researches have
evaluated the use of selected oleuropeinolytic lactic acid bacteria (LAB) as starter cultures in
Greek-style olive processing. Lactobacillus plantarum (Ciafardini et al., 1994; Marsilio et al., 1996;
Marsilio & Lanza, 1998; Marsilio et al., 2005; Panagou et al., 2008) and Lactobacillus pentosus
(Panagou et al., 2003; Panagou et al., 2008; Servili et al., 2006), in addition to developing a fast
growth and a good acidifying capacity even in the presence of high concentrations of salt
(brine), were able to grow in the presence of phenolic compounds (known for their
antimicrobial action) and, thanks to the marked oleuropeinolytic activity, degraded the
oleuropein in no-bitter compounds, reducing thus considerably the time of debittering.
2.2.2. Natural green and black olives by Itrana-style
The Itrana variety is grown mainly in Lazio region and affects the hilly area of Ausoni,
Lepini and Aurunci mountains. The production is concentrated in Latina district (Itri, Cori,
Rocca Massima and Sonnino). The olives destined to produce the famous Oliva nera di
Gaeta are harvested at the stage of full maturity in the months of February-March (very late
compared to most table olive cultivars). This method provides an initial step of immersion
in water for about 1 month to stimulate the growth of specific microflora that contributes to
the debittering of the fruits (Lanza, 2010). After 10-30 days salt is added to the liquid, in
quantities not exceeding 7 kg per 100 kg of fresh olives. After 4-6 months of storage in brine
the olive flesh shows a typical red-wine colour and acidic taste probably due to the
contribution of heterofermentative bacteria and yeasts. In recent years another type of
product named Oliva bianca di Itri has evolved. The processing system is basically the
same but the Itrana fruits are collected at the beginning of ripening in the months of
November-December, when they reach their final size and look green or turning-color, and
immersed immediately in water. After 6-8 months, the product results more bitter and acid
respect to Gaeta olives and kinesthetic characteristics (hardness, fibrousness and
crunchiness) are more pronounced.
2.2.3. Cracked/crushed olives
This type of processing is typical of the Calabria, Puglia and Campania regions. In Puglia
(Salento) the olives of Ogliarola salentina cv., harvested at the green stage of ripening, are
crushed with a wooden hammer (or a crushing machine) taking care not to break the stone,
then they are put in an earthenware pot with water for at least 2 weeks changing the water
at least twice a day. The olives suitable for this preparation must have a crunchy flesh with a
strong detachment of the flesh from the stone. The finished product is seasoned with garlic,
pepper, oregano and other spices (local name: volia cazzata). In Calabria (Crotonese) olives of
Carolea cv. are similarly prepared (local name: aliva maccata). In Campania (Cilento) the
olives of Pisciottana cv. are still prepared according to an ancient recipe that has very special
ingredients and an elaborate preparation process. The green olives are picked, washed with
a lot of water and left in a bowl, in which powdered lime and ashes are added; everything is

then covered with water. The bowl is left to rest for 48 hours, then everything is drained and
rinsed; the next step is to let the olives steep for 24 hours in a container with salt and bay
leaves. After that the olives are drained and laid out on wood or stone board and squashed
with a wood pin, until the seeds come away. Once squashed, the olives are gathered and
placed in a glass container, they are pressed with the hands and dressed with chili pepper and
other spices and condiments and covered with extra virgin olive oil. During the fermentation
of cracked green olives, the predominant microorganisms are yeasts while lactic acid bacteria
are not detected. The main role of yeasts in the processing of fermented olives, is associated
with the production of alcohols, ethyl acetate, acetaldehyde and organic acids, compounds
that are relevant for the development of taste and aroma and the preservation characteristics of
this fermented food (Alves et al., 2012; Arroyo-Lopez et al., 2008).
2.2.4. Scabecciu olives
The Scabecciu olives are a traditional preparation typical of Sardegna region. The ripe olives
of Tonda di Cagliari and Pizze Carroga varieties are engraved in three points, put in brine for
about 3 days, washed with water and blanched in vinegar-water and then dried in the sun.
Finally, they are fried with garlic and parsley and placed in oil. After about 1 month of
preparation they can be consumed as appetizer.
2.3. Oven-dried black olives by Ferrandina-style
In Basilicata region, olives are prepared with an ancient and traditional method, Ferrandina
method, which name derives from the small city Ferrandina, near Matera, where olive trees
of Olea europaea L. Majatica cv. are cultivated (Savastano, 1937; Di Gioia, 1959; Cucurachi et
al., 1971). The olives are hand-harvested at the black stage of ripening at the end of
November-early December. Initially the olives are blanched in water at 90C for 3 min to
make the skin more permeable and facilitate the osmotic processes. After blanching, the
olives are salted with grinded NaCl (10:1 w/w) for 3 days and finally they are placed in
single layer and oven-dried in an air-oven at 50 C for 36 hours until the humidity is around
8 % and Aw around 0.7. This product is ready to eat in 1 week. The first two steps of the
process (blanching and salting) contribute to fruit debittering; the second and the final steps
(salting and oven-drying) result in water loss for better preservation of the product. This
trade preparation is in accordance with the "Trade Standard Applying to Table Olives" (IOC,
2004) as dehydrated and/or shrivelled black olives, black olives that have undergone or
not mild alkaline treatment, preserved in brine or partially dehydrated in dry salt and/or by
heating or by any other technological process. Today, olives from cv. Majatica are almost
entirely destined to the production of oil. The production of oven-dried black olives by
Ferrandina-style is in drastic decline and manufacturers are few. The product is prevalently
exported in the USA and not all olives sold in the Italian market as oven-dried olives
originate from cv. Majatica.
Textural changes occurring in oven-dried olive -tissues during each processing step were
visualized by scanning electron microscope, texture analyzer, analysis of cell wall

polysaccharides, pectins and biophenols (Borzillo et al., 2000; Marsilio et al., 2000; Cardoso et
al., 2009; Piga et al., 2005). Recently, some authors (Lanza et al., 2012) evaluated chemical and
nutrient characteristics of Ferrandina oven-dried table olives with the aim to enhance the
value of this end product from a nutritional point of view.
2.4. Dry-salted black olives
Salt-dried olives are prepared by packing naturally black-ripe olives in alternating layers
with dry coarse salt (equivalent to 1020% w/w of the weight of olives) and spices (oregano,
orange peel, bay leaves, fennel, garlic, etc) in slatted containers that allow drainage of the
vegetable water drawn out by the salt. The resulting olives, or date olives, are shrivelled in
appearance and have a salty bitter-sweet taste (Panagou, 2006). Salt is also taken up by the
olive, which acts as a preservative. Processing time is around four to six weeks and the
olives are best eaten within three months of processing. Addition of olive oil enhances the
flavour of the olive; however, oxidation of the oil can give the olives a rancid taste. In all
Italian regions the most common cultivar that is prepared by salt-drying is Leccino cv. but
there are also some local variations:
2.4.1. Strinate olives
In Marche region (areas near Ascoli Piceno and Macerata) black olives from Leccino,
Raggiola, Raggia, Piantone di Falerone and Lea cvs. are stored in a jute bag after being
combined with coarse salt. The bag, tied with a string, is hang outdoors sheltered from the
rain and in a very cold place, taking care to mix the olives twice daily to prevent formation
of mold and to promote a better distribution of salt. Under the bag a basin is placed to
collect the vegetation water produced by the olives. The low winter temperatures favor the
loss of bitter taste and cause dehydration of the olives, which assume a shriveled
appearance. After 20-40 days, they are placed in glass containers for storage and with the
addition of all the ingredients already described above for the preparation of dry-salted
olives. The product thus obtained is called strinate olives from dialectal word that means
"dead of cold".
2.4.2 Passuluna olives
In Sicilia region (Palermo), the passuluna olives are natural over-ripe olives, left on the tree,
harvested in December-January, washed with hot water and leave to air-dry. Really, those
olives are shriveled and debittered as the result of attack of the fungus Camarosporium
dalmaticum, introduced in the drupe from dipteran Prolasioptera berlesiana, parasite of
Bactrocera oleae eggs.
2.5. Stuffed olives by Ascolana-style
The stuffed olives by Ascolana-style are prepared from treated green olives in brine from
Ascolana tenera cv. (PDO Oliva Ascolana del Piceno). The meat (beef 40-70%; pork 30-50%;

chicken/turkey max 10%) is triturated and browned with onion, carrot and celery in olive oil
and cooked on low heat with the addition of dry white wine and salt. When cooked, meat
and add-ingredients are shredded and combined with beaten egg, grated cheese and ground
nutmeg. The pre-pitted olives are filled with the mixture, are dipped in flour, then beaten
egg and finally in breadcrumbs. The final product is intended for frying.
3. Nutritional and nutraceutical characterization
Table olives are a complete food from a nutritional point of view (Cannata, 1939). It is a
drupe consisting primarily of water, fat, carbohydrates, protein, fiber, pectin, biophenols,
vitamins, organic acids and mineral elements. The quality of this product is linked to the
combined effect of various factors, such as the suitability of raw materials, the processing
technologies, the nutritional composition and, in no small measure, the sensory properties.
Olive fruit is a drupe, constituted by three distinct anatomical zones: epicarp (skin),
mesocarp (pulp) and endocarp (stone) containing the seed. All three influence the quality of
the end product (Garrido Fernndez et al., 1997; Montao et al., 2010). The epicarp and
mesocarp constitute the edible part of the olive fruit that is around 70-85 %. Information on
the nutritional composition is related to 100g of edible portion (e.p.) (Table 2).
The energy value of 100 g of e.p. of Italian olives is around 200-250 kilocalories with some
exceptions (455 kcal for Majatica olives and 164 kcal for Bella di Cerignola olives; Table 3).
This value, referred to a diet of 2000 kcal for an adult of average build with limited physical
activity, accounts for 10-12.5 % of total calories.

Table 3. Nutritional characteristics of some Italian table olives. n.d. = not detected; tr = traces.
The protein content is low (1.0-2.2 g; Table 3), but nutritional quality is high for the presence
of essential amino acids for adults, threonine, valine, leucine, isoleucine, phenylalanine and
lysine (Young, 1994), and for childrens, arginine, histidine and tyrosine (Imura & Okada,
Nutrients/
100g e.p.
Sevillan
green
olives
Intosso
Sevillan
green
olives
Bella di Cerignola
Castelvetrano
green
olives
Nocellara B.
Ferrandina
black
olives
Majatica
Natural
black
olives
Taggiasca
Natural
black
olives
Peranzana
Natural
black
olives
Itrana
Natural
green
olives
Itrana
Natural
black
olives
Cellina N.
Energy kcal 190 164 204 455 226 247 235 193 223
Proteins g 1.0 1.2 1.0 2.2 1.5 1.7 1.4 1.5 1.3
Carbohydrates g
Sugars g
2.8
tr
2.5
<0.6
3.6
0.4
n.d.
4.4
8.9
tr
5.8
0.6
6.5
0.3
5.0
0.6
7.2
1.7
Fats g 17.5 15.5 19.8 46.9 19.9 23.2 21.7 17.7 19.9
SFA g 2.7 2.1 3.9 6.3 3.7 4.1 2.7 2.8 4.4
MUFA g 13.6 12.5 13.9 36.7 15.2 17.0 17.7 14.0 14.5
PUFA g 1.2 0.9 2.0 4.0 0.9 2.1 1.3 0.9 1.0
Fiber g 2.6 4.8 3.8 3.4 2.6 4.1 4.0 3.6 4.8
Sodium g 1.3 1.1 0.9 0.9 1.8 1.4 1.5 1.2 1.5
Calcium mg 33.6 34.9 n.d. 168.1 92.7 83.1 28.9 21.9 58.7
Polyphenols mg 168 104 24 263 206 334 211 109 299

1998). Aspartic and glutamic acids are the most representative amino acids, however in
some preparations threonine, valine, leucine and arginine contents are >100mg (Table 4).
These results are confirmed by other authors for other cultivars and treatments (Lanza et al.,
2010; Lazovic et al., 1999; Lpez et al., 2007; Lpez-Lpez et al., 2010b; Montao et al., 2005).

Table 4. Amino acid pattern of some Italian table olives. tr = traces.
The carbohydrate content in the olive fruit is, by itself, lower than any other edible fruit
(Marsilio et al., 2001). However, table olives have even lower proportions of these
compounds since during the fermentation process or brine storage the microorganisms
present in brines consume sugars. Then table olives can be considered as practically free of
sugar products. Therefore, the calculation of total carbohydrates as difference, as must be
made for nutritional labeling in the US, may lead to an overestimation of the amounts of
these compounds in table olives, resulting in an error for consumers. However, unfermented
products such as Ferrandina olives, appreciable quantities (4.4g) of simple sugars are
present (Table 3).
Table olives are a good source of dietary fibre, which in addition, has a high digestibility
rate (Jimenez et al., 2000; Lpez-Lpez et al., 2007). In European Union countries (Reg. CE
1924/2006 and Reg. UE 116/2010) it is possible to write on the label the claim source of
fibre if the product contains at least 3g of fiber/100g of e.p. If the product contains at least
Amino acid
(mg/100g e.p.)
Treated green
olives
Intosso
Ferrandina
black
olives
Majatica
Natural black
olives
Peranzana
Aspartic acid 150 214 131
Threonine 70 129 80
Serine 80 124 74
Glutamic acid 150 226 128
Proline 50 tr tr
Glycine 70 115 65
Alanine 80 115 66
Valine 60 104 63
Isoleucine 100 86 50
Leucine 140 173 98
Phenylalanine + Tyrosine 100 168 117
Lysine 10 18 tr
Histidine 30 26 28
Arginine 70 120 68
Other amino acids tr tr tr

6g of fiber/ 100 g of e.p. (as it can be the case for some varieties) the claim "with high
content of fiber" could be used. Most preparations have a content of fibre 3g/100g of e.p.,
so they can be considered as a source of fibre (Table 3).
It is worth highlighting on table olives mineral content. Discrete calcium content was found
in some samples (168.1 mg for Majatica, 92.7 mg for Taggiasca and 83.1 mg for Peranzana;
Table 3). The contribution of this element, albeit insignificant, contributes along with other
foods to reach the daily requirement of calcium which is 800 mg. The high Na content of
some traditional preparations ( 1.5 g; Table 3), resulting by fermenting and packing brines,
is not in contrast with sodium RDV by Dietary Guidelines for Italians (LARN, 1996), that
consists of 2.27 g/day. The consumption of these table olives may be unadvisable only in
hypertensive pathologies. There are some studies (Bautista Gallego et al., 2011; Marsilio et
al., 2000; Panagou et al., 2011) on use of alternative chloride salt mixtures in substitution of
common salt (NaCl) to reduce the intake of sodium, with an exception of the dry-salted
olives (4-10g/100g of e.p.) (Figure 3).

Figure 3. NaCl crystals on olive skin. Bar = 15 m.
Table olives are also rich in natural antioxidants such as vitamins. They provide small
amounts of B group vitamins as well as liposoluble vitamins such as pro-vitamin A and
vitamin E, considered to have great antioxidant effects. The vitamin C content is low (<1
mg/kg of e.p.). Many green olive commercial presentations add, as antioxidant, ascorbic
acid, which becomes a part of their final ingredients and increases the vitamin C content of
the product. This compound may be progressively lost during shelf life, but depending on
the time elapsed from packing, such table olives may eventually represent an interesting
source of vitamin C.
All table olives analyzed (except Nocellara del Belice olives) are, despite treatments, still rich
in natural antioxidants such as polyphenols (Table 3) and their antioxidant capacity and
functional effects on human wellbeing is well ascertained (Baiano et al., 2009; Ben Othman et
al., 2008; Boskou et al., 2006). Table olives contain simple and complex phenolic compounds

(at least 30 different phenolic compounds) in amounts ranging between 100 and 350
mg/100g of e.p. (the same quantity of 1kg of extra virgin olive oil!). It was demonstrated that
the variability of those main phenolic compounds can be related to a combination of
agronomic and/or technological processes. The polyphenol content and composition
depends on several factors such as cultivar, stage of ripening, location and processing
(Blekas et al., 2002; Bianchi, 2003; Romero et al., 2004). Recent research has focused on a
phenolic compound of nutraceutical value, oleocanthal (dialdehydic form of deacetoxy-
ligstroside aglycon) as NSAI-like drugs (non-steroidal anti-inflammatory drugs), with
Ibuprofene-like activity (Beauchamp et al., 2005).
Organic acids (oxalic, succinic, malic, citric and lactic) are present in rather low percentage,
such as to give the olive pulp a total acidity between 4-10g/kg (expressed as citric acid) and a
pH between 3.8 and 5.0. The content of oxalic and malic acids decreases in the course of
maturation and the content of citric acid increases, while succinic acid seems to remain
constant. In addition, the ratio of citric and malic acids decreases in the course of maturation
to reach, at the moment of maximum oil accumulation (inolition), values close to 1 (Garrido
Fernndez et al., 1997).
Crude fat content was determined by extraction in a Soxhlet apparatus, according to the
procedures described previously for olives (Lanza et al., 2010a). The olive product with the
lowest fat content was that of Bella di Cerignola processed with the Sevillan method (15.5 g),
while the highest was that of Majatica oven-drying by Ferrandina-style (46.9 g). It is
noticeable the difference between the fat content of Itrana processed as Oliva Bianca di Itri
and Oliva Nera di Gaeta (17.7 and 21.7 g, respectively) is certainly due to the different
periods of harvesting and maturation.
To analyze the fat composition, the olive fruits were pitted and triturated with a grinder.
The olive paste was warmed up in a water bath at 282 C for 30 min and the oil was
extracted by centrifugation at 5000 rpm for 30 min. The resulting surnatant oil, preleased
with a Pasteur pipette, was filtered in the presence of Na-sulphate anhydrous and stored at
4 C in aluminum foil wrapped falcon tubes until analyses. This procedure simulates the
extraction of olive oil in olive mill (crushing, mixing and centrifugation) and was used to
prevent changes in the oil quality as much as possible (Lanza et al., 2012).
Table 5 shows the detailed fatty acid composition (relative percentage within the lipid
fraction) of oil extracted from for different table olives. Oleic acid is the predominant one
(63.4-80.7 %), palmitic acid was the second most abundant fatty acid (9.8-19.6 %), followed
by linoleic acid (4.7-13.6 %) and stearic acid (1.6-3.0 %), a pattern common to most reported
data (Lanza et al., 2010a; Sousa et al., 2011; Sakouhi et al., 2008; Borzillo et al., 2000; Lpez et
al., 2006; Lpez-Lpez et al., 2010a; nal & Nergiz, 2003; Issaoui et al., 2011). Recent research
showed that intestinal mucosal cells utilize dietary oleic acid as a substrate to produce the
lipid messenger oleoylethanolamide (OEA) that plays an important role in the regulation of
animal food intake and body weight in human physiological and pathophysiological
conditions (Schwartz et al., 2008).


Table 5. Fatty acid composition percentage of some Italian table olives. n.d. = not detected; tr = traces.
Monounsaturated fatty acids (MUFA) were the major group (66.8-82.1 %), saturated fatty
acids (SFA) represented less than 22.4 % and polyunsaturated fatty acids (PUFA) range 4.9-
14.2 %. The trans fatty acids had a very limited occurrence (less than 0.02%). The intake of o-
linolenic acid (C18:3 e3), precursor for the synthesis of long chain e3 fatty acids, is
appreciable but the ratio e6:e3 is still too far towards e6 and depends on the cultivar (6.7-
23.5; Table 5). Several sources of information suggest that human beings evolved on a diet
with a ratio of e6 to e3 essential fatty acids of approximately 1 whereas in Western diets the
ratio is 15-16.7. Western diets are deficient in omega-3 fatty acids, and have excessive
amounts of omega-6 fatty acids compared with the diet on which human beings evolved
and their genetic patterns were established (Simopoulos, 2008). The ratio of oleic to palmitic
acid in dietary fats has a regulatory influence on certain thrombogenic and fibrinolytic
markers during the postprandial state in healthy subjects (Pacheco et al., 2006). It has a
recommended ratio of at least 5. Also the polyunsaturated/saturated fatty acid (PUFA/SFA)
ratio is used to assess the nutritional quality of the lipid fraction in foods. Consumption of
saturated fatty acids has been associated with coronary heart disease (Serrano et al., 2005).
Consequently, nutritional guidelines have recommended that the PUFA/SFA ratio should be
above 0.40.5 (Wood et al., 2008). The mean values of the indexes (cis PUFA/SFA + TFA) and
(cis MUFA + cis PUFA/SFA + TFA), which were most commonly used to express nutritional
value of edible fats (Alonso et al. 2002; Anwar et al., 2006; Garrido-Fernandez, 2008),
presented very high values due to the absence of trans fatty acids. These results are in
Fatty acids
Sevillan
green
olives
Intosso
Sevillan
green
olives
Bella di
Cerignola
Ferrandina
black
olives
Majatica
Natural
black
olives
Moresca
Natural
black
olives
Peranzana
Natural
black
olives
Itrana
Natural
green
olives
Itrana
Natural
black
olives
Cellina N.
Myristic acid C14:0
n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d.
Palmitic acid C16:0
11.8 11.4 9.8 15.1 14.7 10.1 13.6 19.6
Palmitoleic acid C16:1
0.6 0.8 1.1 2.9 1.3 1.1 1.4 1.9
Heptadecanoic acid C17:0
0.1 0.1 n.d. 0.1 n.d. n.d. 0.1 n.d.
Heptadecenoic acid C17:1
0.3 0.1 0.1 0.3 0.1 0.1 0.1 n.d.
Stearic acid C18:0
2.9 2.2 3.0 1.6 1.7 1.7 1.8 2.2
Oleic acid C18:1
76.4 79.6 76.8 63.4 70.2 80.7 76.5 70.8
Linoleic acid C18:2 e6
6.3 4.7 8.0 13.6 10.7 5.2 5.5 4.7
Arachic acid C20:0
0.8 0,4 0.4 0.3 0.3 0.2 0.3 0.4
Linolenic acid C18:3 e3
0.5 0.7 0.5 0.6 0.7 0.6 0.5 0.2
Eicosenoic acid C20:1
0.3 0.2 0.3 0.2 0.2 0.2 0.3 0.2
Behenic acid C22:0
0.1 n.d. 0.1 0.1 0.1 0.1 0.1 0.1
Lignoceric acid C24:0
n.d. 0.1 n.d. n.d. n.d. n.d. 0.1 0.1
Trans fatty acids
0.02 tr 0.02 0.01 tr tr 0.01 tr

accordance, but even higher, with those found for alcaparra olives (0.3 and 5.0,
respectively; Sousa et al., 2011). Only for Cellina di Nard olives these indexes are lower for
alcaparra olives (Table 6).

Table 6. Fatty acid indexes.
As regard to sterol, fatty and triterpenic alcohol composition, there are some studies relating
to the changes during processing (Lpez- Lpez et al., 2008, 2009). The main phytosterols
and phytostanols found in Ferrandina table olives (Table 7) are |-sitosterol (59.1-89.6 %) and
A5-avenasterol (1.5-34.3%), followed by campesterol (1.8-4.2%), A5,24-stigmastadienol (0.4-
1.2%) and chlerosterol (1.0-1.5 %). The low content of |-sitosterol and high content of A5-
avenasterol in Majatica olives is typical for this cultivar (Rotundo & Marone, 2002), but |-
sitosterol (including A5,23-stigmastadienol, clerosterol, |-sitosterol, sitostanol, A5-
avenasterol and A5,24-stigmastadienol) is higher than 93%, the limit fixed for extra virgin
olive oil (EVOO) by the Commission Regulation (EEC) 2568/91 and its subsequent
modifications. Epidemiologic and experimental studies suggest that dietary phytosterols,
and in particular |-sitosterol, may offer protection from the most common cancers in
Western societies, such as colon, breast and prostate cancer and contribute to lower the
cardiovascular disease risk. (Awad & Fink, 2000; Woyengo et al., 2009).
Finally, table olives could be utilized as a vehicle for incorporating probiotic bacteria and
transporting bacterial cells into the human gastrointestinal tract. Food industries are now
focusing on new foods which are part of a normal diet and can contribute to a regular
assumption of probiotics (functional foods). The incorporation of health-promoting bacteria
into table olives would add functional features to their current nutritional properties.
The consumption of table olives, in combination with the consumption of olive oil, which
are basic components of the Mediterranean diet, provide a large amount of natural
compounds of nutraceutical value (polyphenols, phytosterols and fatty acids) with
antioxidant, anti-inflammatory or hormone-like properties.
Fatty acid index
Sevillan
green
olives
Intosso
Sevillan
green
olives
Bella di
Cerignola
Ferrandin
a black
olives
Majatica
Natural
black
olives
Moresca
Natural
black
olives
Peranzana
Natural
black
olives
Itrana
Natural
green
olives
Itrana
Natural
black
olives
Cellina N.
SFA 15.7 14.2 13.3 17.2 16.8 12.1 16.0 22.4
MUFA 77.6 80.7 78.3 66.8 71.8 82.1 78.3 72.9
PUFA 6.8 5.4 8.5 14.2 11.4 5.8 6.0 4.9
MUFA/SFA 4.9 5.7 5.9 3.9 4.3 6.8 4.9 3.3
PUFA/SFA 0.4 0.4 0.6 0.8 0.7 0.5 0.4 0.2
(cis MUFA + cis
PUFA/(SFA+TFA) 5.4 6.1 6.5 4.7 5.0 7.3 5.3 3.5
cis PUFA/(SFA+TFA) 0.4 0.4 0.6 0.8 0.7 0.5 0.4 0.2
Oleic/palmitic 6.5 7.0 7.8 4.2 4.8 8.0 5.6 3.6
e6/ e3 12.6 6.7 16.0 22.7 15.3 8.7 11.0 23.5


Table 7. Sterolic composition (%) of some Italian table olives. n.d. = not detected.
3.1. The nutritional label
A separate discussion is made about nutritional labelling which, while optional until 13
December 2016, could add value to our product. What information do we expect to find on a
nutrition label of a jar of olives? Currently the provision of a nutrition label is regulated by
the Regulation (EU) 1169/2011 of the European Parliament and of the Council on nutrition
labelling for foodstuffs as regards recommended daily allowances, energy conversion
factors and definitions. The information written on the label should be referred to 100 g of
product: in the case of whole olives reference should be made to 100 g of drained product
(therefore considering the stone even if not edible) or 100 g of edible portion (in this case
only the pulp of olives). For pitted olives and olive paste this problem does not arise. The
nutritional information may also be referred to as a portion or "serving size", based on the
amount of food consumed by a person. For table olives, a serving size could be formed by
about 10 medium-sized olives and expressed in grams (taking into account the weight of the
stone). It is also useful to relate the content of each nutrient with a daily reference value for a
diet of 2000 kcal of an average weight adult performing limited physical activity.
Percentages above 20% are considered significant, below 5% modest.
4. The IOC Method for the sensory analysis of table olives
Since 21 November 2008 the procedure for the classification of table olives based on
parameters of quality has become official: COI/OT/MO/Doc. No 1. Method for the sensory
Sterol
Sevillan
green
olives
Bella di
Cerignola
Castelvetrano
green
olives
Nocellara B.
Ferrandina
black
olives
Majatica
Natural
black
olives
Moresca
Natural
black
olives
Peranzana
Natural
black
olives
Itrana
Natural
green
olives
Itrana
Natural
black
olives
Cellina N.
Cholesterol 0.8 1.4 0.4 0.8 0.5 0.4 0.5 0.6
Brassicasterol 0.1 0.3 0.1 0.1 n.d. n.d. 0.1 n.d.
24-methylen cholesterol n.d. 0.2 0.5 n.d. 0.2 0.1 0.1 0.2
Campesterol 3.1 4.2 1.8 2.1 2.0 2.6 2.4 3.1
Campestanol n.d. 0.1 0.1 0.2 0.4 n.d. n.d. 0.2
Stigmasterol 1.2 1.8 0.5 1.0 0.7 0.4 0.7 0.9
A7-campesterol 0.7 0.2 0.1 0.6 0.3 0.8 0.6 0.3
A5,23-stigmastadienol n.d. n.d. 0.1 n.d. n.d. n.d. n.d. 0.1
Chlerosterol 1.5 1.4 1.0 1.5 1.2 1.0 1,0 1.0
|-sytosterol 89.6 84.5 59.1 80.2 77.4 87.8 88.7 85.4
Sitostanol 1.3 0.6 0.4 2.5 0.6 0.6 0.7 2.3
A5-avenasterol 1.5 4.7 34.3 8.5 15.9 5.6 4.3 5.3
A5,24-stigmastadienol 0.1 0.6 1.2 1.0 0.5 0.4 0.6 0.7
A7-stigmastenol 0.3 n.d. 0.3 0.7 0.2 0.2 0.3 0.1
A7-avenasterol 0.3 n.d. 0.1 0.8 0.2 0.2 0.2 0.1

analysis of table olives. On 25 November 2011 (Decision No DEC-18/99-V/2011) the
International Olive Council adopted the revised version of the method (COI/OT/MO No
1/Rev. 2). The method establishes the necessary criteria and procedures for the sensory
analysis of the odour, taste and texture of table olives and sets out the systematics for their
commercial classification. It is applicable solely to the fruit of the cultivated olive tree (Olea
europaea L.) which has been suitably treated or processed and which has been prepared for
trade or for final consumption as table olives in accordance with the trade standard
applying to table olives referenced COI/OT/NC No 1 (2004).
4.1. The panel
The sensory evaluation of table olives is done by a group of 8-10 expert tasters selected on
the basis of aptitude and led by a panel leader. This group constitutes the taste panel. The
tasters are chosen by means of a selection process implemented in accordance with an
international standard according to his/her sensitivity and discriminatory power with
regard to the organoleptic characteristics of table olives, who becomes skilled after suitable
training and whose performance is objectively evaluated on the basis of rules established
beforehand by the leader of the panel to which the taster belongs. The panel leader is the
person whose chief duties are to lead panel activities, including taster recruitment, selection,
training, skill building and monitoring. He/she designs and leads the sensory tests and
analyses and interprets the data and may be assisted by one or more panel technicians.
4.2. Main facilities and equipment of test room
Tasting booth, referred to standard COI/T.20/Doc. No 6/Rev. 1 Guide for the installation of a
test room or to ISO 8589:2007 General guidance for the design of test rooms;
Glasses, according to standard COI/T.20/Doc. No 5 Glass for oil tasting, covered with watch-
glasses;
Plastic or metal cocktail sticks, two-pronged forks, spoons or tongs;
Profile sheet on hard or soft copy. The line for each attribute must measure exactly 10 cm.
4.3. Sample presentation and tasting session
The sample of table olives for analysis shall be presented in standard tasting glasses (Figure
4). The glass shall contain as many olives as the bottom of the glass can hold when the olives
are placed side by side in a single layer. When brined table olives are undergoing analysis,
sufficient covering liquid shall be poured over the olives to cover them fully. When the
olives are above the 91/100 size-grade, the volume of sample contained in the glass shall in
no case be more than half the height of the glass (i.e. 30 mm). In the case of table olives
belonging to a size-grade below 91/100, the sample for testing in the glass shall comprise no
less than three olives. When brined table olives are undergoing analysis, the quantity of
covering liquid in the glass shall come up to at least three-quarters of the height of the
olives. The glass shall be covered with the attendant watch-glass.


Figure 4. Different distribution of olives and brine in the glass.
The samples of table olives intended for tasting shall be kept in the glasses at ambient
temperature, between 20 and 25 C, under white light (daylight). To avoid tasting fatigue
and the appearance of bias or contrast effects, each tasting session should entail the sensory
analysis of not more than three samples. Between each session the tasters should rinse out
their mouth fully and take a break of at least fifteen minutes. No more than three tasting
sessions should be conducted in any given day. It should be kept in mind that the morning,
before lunch, is the period when olfactory-gustatory sharpness is optimal (between 10 a.m.
and 12 noon).
4.4. Tasting session and use of the profile sheet by tasters
The tasters shall pick up the glass, keeping it covered with the watch-glass, and shall bend it
gently to help the sample aromas to be released and blended. After doing so, they shall

remove the watch-glass and smell the sample, taking slow deep breaths to evaluate the
direct olfactory sensations cited in the profile sheet. Smelling shall not last more than 20
seconds. If no conclusion has been reached during this time, the tasters shall take a short rest
before trying again. The tasters shall then assess the other sensations cited in the profile
sheet. To do so, they shall place one of the olives contained in the glass in their mouth; they
shall chew the olive after removing the stone, making sure to spread the chewed olive
throughout the whole of the mouth cavity. They shall concentrate on the order of
appearance of the salty, bitter and acid stimuli, the retronasal olfactory sensations and the
kinaesthetic sensations of hardness, crunchiness and fibrousness and shall assess the
intensity of each of these sensations by making the corresponding mark on the intensity
scale of the tasting sheet. Next they shall spit out the chewed olives, rinse out their mouth
with water and recommence the assessment of the sensations produced by each of the olives
contained in the glass. They shall enter the intensity with which they perceive each of the
attributes in the scales of the profile sheet provided. Tasters may refrain from placing the
olives in their mouth when they observe an extremely intense negative attribute. They shall
record this exceptional circumstance in the profile sheet. They must, however, mark the
intensity of the odorous attribute on the corresponding scale. They shall enter in the profile
sheet the intensity of each of the sensations perceived when smelling and chewing the
olives.
4.5. Attributes to be perceived
4.5.1. Negative attributes
Unpleasant sensations are caused by the production of substances responsible for off-
odours, which are not present in the fresh fruit or formed during well-performed processing
treatments. The term abnormal fermentation includes all those olfactory sensations
perceived directly or retronasally, reminiscent of the odour of decomposing organic matter,
cheese, butter, rotten eggs, muddy sediment, sewer, rotten leather, caused by the
development of contaminating microorganisms (butyric fermentation, putrid fermentation
and zapateria). If the tasters perceive any negative attributes other than abnormal
fermentations, they shall record them under the 'other defects' heading, using the following
terms: musty, rancid, cooking effect, soapy, metallic, earthy and winey/vinegary. Musty
sensation is an olfactory-gustatory sensation perceived directly or retronasally, characteristic
of olives attacked by moulds. Rancid sensation is an olfactory sensation perceived directly or
retronasally, characteristic of olives that have undergone a process of rancidity. Cooking effect
is an olfactory sensation perceived directly or retronasally, characteristic of olives that have
undergone excessive heating in terms of temperature and/or duration during pasteurisation
or sterilisation. This taste may be typical of some cooked preparations as oven-dried black
olives. Winey-vinegary is an olfactorygustatory sensation reminiscent of wine or vinegar.
Winey-vinegary taste should not be confused with the acid sensation. The defect winey-
vinegary is due to alcoholic fermentation by yeasts, while the feeling of acid defines the taste
associated with acids naturally present or produced during the lactic fermentation by homo

and hetero fermentative lactic acid bacteria. Both sensations may be more or less marked by
inappropriate use of acids used as acidity correctors (e.g., acetic or citric acid), but may also
be typical of some preparations involving the use of vinegar (e.g., Kalamata olives). Soapy is
an olfactory-gustatory sensation reminiscent of soap. This taste is found primarily in olives
treated with lye (Spanish and Castelvetrano systems) and not sufficiently rinsed with water
or consumed shortly after debittering. Metallic is an olfactory-gustatory sensation
reminiscent of metals. It is found primarily in olives darkened by oxidation with the
addition of iron salts such as gluconate or ferrous lactate, used as stabilizers of the color (eg.
Californian black olives), but can also be perceived of olives preserved in metal packs.
Earthy is an olfactory-gustatory sensation reminiscent of soil or dust. It is found in olives
that have been in contact with soil and dust for a long time, usually harvested after they fall
from the tree.
4.5.2. Descriptive gustatory attributes
The gustatory sensations involve distinct areas of tongue: the region affected by the
perception of salty taste is the lateral-anterior, the region affected by the perception of acid
taste is the posterior and the region affected by the perception of bitter taste is the basis of
tongue. The salty sensation is associated to the basic taste produced by aqueous solutions of
substances such as sodium chloride and depends on the concentration of fermentation or
conditioning brines. The bitter sensation is associated to the basic taste produced by dilute
aqueous solutions of substances such as quinine or caffeine and depends on the presence
of bitter substances, mainly polyphenols. It may therefore be more intense in preparations
in which the debittering, incomplete, is due to the action of microrganisms (natural
olives). The acid sensation is associated to the basic taste produced by dilute aqueous
solutions of most acid substances, such as tartaric or citric acids. It defines the taste
associated with acids naturally present in the olive flesh (e.g., tartaric acid, malic acid or
citric acid) or produced during the lactic fermentation by homo and heterofermentative
lactic acid bacteria (e.g., lactic acid or acetic acid), but may also depend on the
inappropriate use of acids as correctives of acidity (e.g., citric acid). High level of acid
sensation is also found in the olives whose preparation involves the addition of vinegar
(e.g., Kalamata olives).
4.5.3. Kinaesthetic sensations (texture)
With the term kinaesthetic sensations (from the greek kivqoq = movement and oiouqoq =
sense) are indicated the sensations deriving from the contact of the fruit with the mouth. We
could translate it as "musculoskeletal overall perception, in the oral cavity, of the mechanical
characteristics of the fruit. Texture is defined as the set of rheological (related to the flow
and deformation of matter) and structural (geometrical and surface) properties of a product
perceptible to the mechanical receptors, tactile receptors and in some cases the visual and
auditory receptors. The following attributes are assessed in table olives: hardness,

crunchiness and fibrousness. Hardness is defined as the mechanical properties of consistency
related to the force required to obtain the deformation or penetration of a product. It is
perceived through the compression of the product between the teeth (solid products) or
between the tongue and palate (semi-solid products); as regards the solid products such as
table olives, the force required to compress the product is evaluated first between the
incisors (Hardness 1), then between the molars (Hardness 2) and finally during mastication
(Hardness 3) (Kim et al., 2012). Crunchiness is the property related to the noise produced by
friction or fracture between two surfaces. In our case, it is related to the force required to
fracture a product with the teeth and is determined by compressing the fruit between the
molars. Fibrousness is a geometric textural attribute relating to the perception of the shape
and the orientation of particles in a product. Fibrousness refers to the elongated
conformation of the particles, oriented in the same direction. It is evaluated by perceiving
the fibres between the tongue and palate when chewing the olive. Hardness, crunchiness
and fibrousness may also be assessed by instrumental puncture, compression, stretching
and acoustic tests using Texture Analyzer. The texture of edible fruits and vegetables is
strongly influenced by the chemical, physical and structural properties of the plant cell walls
which surround every cell and, through adhesion to the adjacent ones, provide mechanical
strength and protection (Brett & Waldron, 1996). Such properties will be affected by the
stage of ripening (Mafra et al., 2001) and processing technologies (Lanza & Marsilio, 2001;
Tassou et al., 2007). Tissues in which cell-to-cell adhesion is very strong may only be
disrupted by breakage of the cell walls: such tissues are usually crunchy in texture. Tissues
in which cell-to-cell adhesion is very weak may be disrupted through cleavage along the
plane of the middle lamella: such tissues are usually soft in texture. The examination of the
fracture surface can indicate whether tissue failure occurs as a result of cell rupture or cell-
to-cell debonding. A simple olive fracturing method is described in Lanza et al. (2010a). In a
recent research (Lanza & Di Serio, 2011), information provided by sensory and other
instrumental measurements of tissue hardness were compared with that provided by
scanning electron microscopy. The different levels of hardness applied to olives by sensory
analysis are as follows: soft (low level), firm (moderate level) and hard (high level).
Fractured epicarp and mesocarp olive fruits in longitudinal view, corresponding to the three
levels of hardness, were examined by SEM (Figure 5). In hard olives, the thin-walled
parenchyma cells were uniform and tightly packed. Tissue fracture involved cell walls
breaking, both in epicarp and mesocarp and cell separation at the middle lamella level was
not observed. The fracture surface of firm olives consisted of two regions: (a) one with 400
500 m containing broken cell walls and some separated cells and (b) a second one
containing intact cell walls. The region with broken cell walls extended from the olive
epidermis as far as the eighth-ninth layer of mesocarp. In soft olives, the region of broken cell
walls was smaller (100200 m) and involved only the epicarp and the first layers of the
mesocarp (hypodermis and, sometimes, the following first layer). The other cells showed
rounded outlines and were divided along the middle lamella, showing cell separation.
Summarizing, in hard fruits, tissue fracture involves cell walls breaking and the thin-walled

parenchyma cells olives are uniform and tightly packed (cell rupture). In soft olives the cells
show rounded outlines and are divided along the middle lamella (cell separation). This is
due to an increase in cell separation which consists mainly in the dissolution of middle
lamella pectic polysaccharides.

Figure 5. The different levels of hardness (H). Bar = 300 m.
4.6. Elaboration of sensory data
The panel leaders shall collect the profile sheets completed by each of the tasters and shall
review the intensities recorded for each of the descriptors. If they find any anomaly, they
shall invite the taster concerned to revise the profile sheet and, if necessary, to repeat the
test. The panel leaders shall determine the intensities of the attributes listed in the profile
sheet by using a ruler to measure the segment running from the origin of the scale to the
mark made by the taster. When this mark lies between two notches on the ruler, they shall
assign the value lying closest to one of the notches. The segment shall be expressed to one
decimal place. The scale shall measure 10 cm long and the intensity shall range from 1 to 11.
The panel leaders shall apply the method for calculating the median and the confidence
intervals according to the method contained in Annex 1 (COI/OT/MO/n1/Rev.2 Annex 1
Method for calculating the median and the confidence intervals) and shall only take into account

those attributes with a robust coefficient of variation of 20% or less. The computer program
for carrying out the calculations is presented in Annex 3 (COI/OT/MO/n1/Rev.2 Annex 3
Sensory analysis of table olives computer program). When a defect is entered under the Other
defects heading by at least 50 percent of the panel tasters, the panel leaders shall carry out
the statistical calculation of this defect and shall arrive at the corresponding classification if
the coefficient of variation is 20% or less.
4.7. Classification according to the defect predominantly perceived (DPP)
For classification purposes, the panel leader shall solely take into account the median of the
defect predominantly perceived (DPP) i.e. perceived with the greatest intensity, that satisfies
the requirements specified in the preceding paragraph. According to the intensity of DPP,
the samples shall be classified in four categories:
Extra or Fancy: DPP 3
First, 1st, Choice or Select: 3 < DPP 4.5
Second, 2nd or Standard: 4.5 < DPP 7.0
Olives that may not be sold as table olives: DPP > 7.0
The organoleptic analysis of table olives deriving by the same variety (Itrana) but processed
at a different stage of ripening (Oliva bianca di Itri and Oliva nera di Gaeta) shows a
different sensory profile with regard to kinaesthetic properties (hardness, fibrousness and
crunchiness) and bitter sensation (Figure 6 a and b) (Lanza et al., 2010b). The organoleptic
analysis of table olives deriving by the same variety (Itrana) and processing (Oliva Bianca
di Itri) show a different sensory profile between undefected and defected samples.
Defected samples show a decrease in kinaesthetic properties (hardness, fibrousness and
crunchiness) and an increase in acid sensation (Figure 7 a and b). The median value of DPP
is less or equal to 3 and the olives remain of Extra or Fancy category.

Figure 6. Sensory profiles of Oliva bianca di Itri and Oliva nera di Gaeta.


Figure 7. Sensory profiles of undefected (a) and defected (b) olives from Oliva bianca di Itri.
Acknowledgement
Financial support for this study was provided by Italian Ministry of Agriculture, Food and
Forestry through the project GERMOLI Salvaguardia e valorizzazione del GERMoplasma
OLIvicolo delle collezioni del CRA-OLI.
Author details
Barbara Lanza
Agricultural Research Council - Olive Growing and Oil Industry Research Centre, Citt
SantAngelo (PE), Italy
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