 I focus: individuation of the influence of olive ripening degree, irrigation regime and extraction technology on the quality of the EVOO produced.

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1

1. Summary

Olive oil, used for medical purposes since ancient times, nowadays represents an essential constituent of the Mediterranean diet. Epidemiological studies about its consumption, particularly related to extra virgin olive oil (EVOO) in Mediterranean countries, have showed important beneficial effects since it appears to be able to prevent many possible serious diseases and to modify immune and inflammatory responses (Alu’datt et al., 2017, Rigacci et al., 2016, Virruso et al., 2014, Sánchez-Fidalgo et al., 2013). Traditionally the health- protective effects of olive oil have been ascribed to its high monounsaturated fatty acid content (Bermudez et al., 2011, Escrich et al., 2011); although, nowadays, it is clear that many of the beneficial effects of olive oil intake are due to its antioxidants (bio-phenols, tocopherols, etc.) and its minor highly bioactive components.

In this context the research related to the present PhD thesis was developed “from olives to packaging, also looking at improve the nutraceutical value of oil obtained by refining process”. As described below, the aim of the research project was threefold:

 I focus: individuation of the influence of olive ripening degree, irrigation regime and extraction technology on the quality of the EVOO produced.

During the first part of the PhD research olive fruits from the same cultivar (Frantoio) with different ripening degrees and collected from the same grove in the same year were used.

Moreover, to evaluate the role played by irrigation on oil yield and phenol accumulation, a part of the olives was obtained from irrigated plants, whilst the other fraction derived from not irrigated olive trees. All the above-mentioned olive production lots were extracted with or without the addition of solid CO

2

during olive crushing. The results showed that olive oil yield and phenolic concentration increased with the ripening degree of the milled fruits.

Moreover, the olives harvested on the same date from irrigated plants produced more oil with more polyphenols than those coming from not irrigated trees. The addition of carbonic snow to fruits characterized by the same ripening index increased both the yield of oil and the concentration of total phenols dissolved in the olive oils.

 II focus: development of phenol-enriched olive oil with phenolic compounds extracted from wastewater produced by physical reﬁning.

Refining process is a characteristic phase in the olive oil production that allows to obtain

products with controlled quality and sensory characteristics, even starting from raw materials

that are not edible, but at the same time it involves the loss of many of the nutraceutical

elements present in them which, if recovered, could represent potential sources of phenols for

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2 the formulation of functional food. As widely reported in literature, the refining process eliminates most of the antioxidant compounds: polyphenols are eliminated by both the neutralization and the bleaching operations. The tocopherols are also stripped off during the deodorization process (Koidis et al., 2015). Considering that olive oil is one of the most exported excellence of Made in Italy, recently research tried solve this huge problem with a twofold aim: on the one hand, to improve the refining process reducing the loss of useful compounds like sterols, polyphenols and tocopherols, on the other hand, to recovery fine bioactive chemicals and/or production of added-value products. However, to the best of my knowledge, no data are available in the literature on the feasibility of the production of a phenol-enriched refined olive oil using its own phenolic compounds extracted from wastewater produced during physical refining. In this context this study had three main objectives: (i) to verify the effectiveness of a multi-step extraction process to recover the high-added-value phenolic compounds potentially contained in wastewater produced during the preliminary washing degumming step of physical refining of vegetal oils; (ii) to evaluate their potential application for the stabilization of olive oil obtained with refined olive oils; (iii) to evaluate their antioxidant activity in an in vitro model of endothelial cells. The results showed that wastewater collected during the physical refining process could be considered as a good source of bioactive compounds useful for a significant increase of nutraceutical value, as well as of the antioxidant capacity of olive oils. Thanks to a collaboration with the Department of Life Science, University of Siena, Italy, the antioxidant activity of high-added- value phenolic compounds recovered from wastewater produced by physical refining, has been evaluated in an in vitro model of endothelial cells. The results showed that the combination of hydroxytyrosol with tyrosol, when used at the recommended concentration from EVOO consumption, preserved cell functions from oxidative damage, rescuing their antioxidant properties.

 III focus: evaluation of the effects of different storage conditions on the shelf-life of EVOO, evaluating the maintenance of its chemical and sensory characteristics during time.

Over time olive oil chemical and sensory characteristics continuously change and proper

storage conditions can be the key factor to protect the quality, either nutritional, healthy and

merceological. Oxidation constitutes the major factor for quality deterioration of olive oil,

indeed it determines the loss of bioactive components, the degradation of lipidic fraction and

the consequent development of off-flavors due to the production of carbonyl and aldehyde

compounds. In particular the study of this aspect has been divided into two experimental

projects:

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3 a) evaluation of the effectiveness of different conditioner gases to develop a preservation method that can maintain the high quality of EVOO increasing its shelf life: the EVOO was stored in the dark at 12 °C in glass containers for about 9 months, and the effectiveness of N

₂

as head-space gas was compared to that of Ar and CO

₂

; the effects of conditioner gases were evaluated in comparison with air. The oil stored under air showed a marked decrease of quality after only two months of storage, while the use of conditioner gases in the headspace of the container during storage slowed oxidative degradation even if the use of nitrogen less protects the olive oil compared to the use of the other two gases (CO

₂

and Ar). Despite EVOOs stored under CO

₂

and Ar showed only slight differences as regards their chemical parameters, they appeared deeply modified for what concerns the sensorial characterization. In particular, CO

2

determined a negative organoleptic interference that would not support its use for EVOO long-term storage. Therefore, Ar treatment appears as the best solution alternative to nitrogen to preserve the quality of the EVOO over time.

b) evaluation of the effects of packaging and storage temperature on the shelf life of EVOO: in order to simulate conditions similar to the market storage, the two most commonly used packaging containers for olive oil, namely tinplate tin (TT) and greenish glass (GG), and the two storage temperatures of 6 ± 1 and 26 ± 1°C were chosen. Three replicates of EVOO were analyzed at the time of packaging (t0) and after 28 (t1), 78 (t2) and 125 (t3) days of storage at 6 and 26°C. Shelf-life of EVOO was differently affected by packaging and storage temperature, the latter being critical for the oxidative changes taking place in oil. In fact, the oil stored in GG at 6 °C mostly preserved positive attributes, whereas the one stored in TT at 26 °C showed an enhancement of oxidative processes leading to a significant presence of the rancid flavour. Moreover, the oil stored in GG at 6

°C maintained the highest bitterness intensity and did not show defects at the end of storage,

further suggesting that storage in GG at a low temperature (6 °C), could represent a

promising storage condition to slow-down the oil degradation during market storage.

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4

2. Introduction

2.1 Historical introduction

The olive tree has been present in the history from the beginning of civilization up the present day. Nowadays the olive plant (Olea europea, var. sativa) is very common all over the Mediterranean area, particularly in the central and western regions of Spain and Italy, in eastern Greece and Turkey and in southern Tunisia and Morocco. This is the result of a slow but continuous development of the various civilizations in the Mediterranean coasts and in the more inland areas of the Middle East.

It seems that the olive tree had its origin approximately 6,000–7,000 years ago in the region corresponding to ancient Persia and Mesopotamia (Polymerou-Kamilakis, 1996). All ancient civilizations located around the Mediterranean area and in the Middle East have left clear evidence that olive cultivation and oil production were activities that developed with the prosperity of peoples. At present, the most ancient known document in which the words olive and olive oil appeared for the first time is a tablet of clay discovered in the archaeological excavations of Ebla, near Aleppo (Syria), that dates back to the third millennium B.C. (about 2500 B.C.) (Blazquez 1997). Another ancient document is a stone, found at Susa (Iran) and dating back to the second millennium B.C., in which regulations regarding the commerce of agricultural products, including olive oil, are reported. The archaeological findings kept in the Haifa Museum and those of the Knossos Palace in Crete are other examples confirming the production of olive oil in ancient times. In ancient Egypt the olive plant was cultivated to obtain its oil used in religious ceremonies and as an ointment. The Phoenicians also cultivated olive trees (first millennium B.C.), but since they were also traders and navigators, they probably introduced the olive tree in colonies in many countries of the Mediterranean basin, such as in Libya, Tunisia (Carthage), Sicily, France and Spain.

Many references to the olive tree and its oil can be found in the Hebrew Scriptures and other

holy texts, (e.g. biblical reference, from 1000 B.C., concerning the return of the dove with a

small olive branch to confirm to the patriarch Noah the end of the Deluge and that the olive

trees cultivated near Mount Ararat in Turkey were no more covered by water). In Greece, the

olive tree and olive oil have existed since the fourteenth and thirteenth centuries B.C. The

importance of olive trees in ancient Greece is confirmed by the mythology: the olive tree was

presented as a gift of the gods of Olympus to humans (Polymerou-Kamilakis 1996). In

literature, references to olive trees can be found in the Homeric poems, in the Iliad and in the

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5 Odyssey, such as the bed of Odysseus built with the olive wood, or the use of olive oil to anoint the body of Odysseus (Schäfer-Schuchardt 1988).

In Italy, the olive tree has probably been known since the rise of the Etruscan civilization, but it is certain that it was disseminated by Greek colonizers who founded several cities in Southern Italy (Magna Grecia), especially in Calabria and Sicily. A recent study, carried out on finds kept at the Archaeological Museum of Syracuse, revealed the presence of olive oil traces dating back to over 4000 years ago (Tanasi et al., 2018). The Romans later developed this cultivation, which in turn increased their commerce with all the conquered lands that made up the Roman Empire. The Romans also contributed to technological developments in olive processing by speeding up the crushing operation with a millstone crusher, the trapetum (Figure 2.1), and improving the separation system thanks to the introduction of presses.

Figure 2.1: Example of trapetum, one of the first millstone crushers.

The screw press, used first by Greeks (fourth century B.C.) and then improved upon and spread by Romans, was, a major progress in olive processing (Schäfer- Schuchardt 1988). It’s interesting to note that even today, in some olive-growing countries of the Mediterranean basin, presses very similar to these are still used. With the fall of the Roman Empire, olive cultivation declined. Nevertheless, in the late Middle Ages, olive culture restarted both in Spain, under Arab dominion, and in Italy, mainly with the efforts of religious communities.

From the Middle Ages to the late 1800s, olive cultivation spread in the regions of central Italy

and more in the south, in Puglia region, where many old underground oil mills dug into the

rock remained. During the 1900s, several studies on percolation and centrifugation systems

were done in order to improve mechanical extraction. Thus, innovative systems were

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6 produced in 1951 with the Buendía patent for a percolation system, then they were built in Italy where they were known as Sinolea, and at the end of the 1960s the Centriolive plant, the first industrial decanter based on the continuous centrifugation of olive paste, was introduced.

The pressing system underwent continuous improvement with the aim of increasing both the yield and the quality of the finished product up to modern innovative systems.

2.2 The legal classification and denomination of olive oil

Figure 2.2 is a flow-chart of the classification and denomination of the various categories of olive oils as globally agreed (Council Regulation (EC) No 1234/2007 and following modification and integration)

Figure 1.2: Flow-chart of virgin and refined olive oils (Peri, 2014a) The six categories highlighted in green are suitable for human consumption.

The flow-chart starts with the olive milling process, whose products are “virgin” olive oils.

Two of them, extra virgin and virgin olive oil, are suitable for the consumption. The third

category, lampante, must be submitted to physical-chemical refining process to become edible

as refined olive oil. On the other hand, the pomace, which is the solid residue from the milling

process, still contains a small amount of olive oil that is impossible to extract by mechanical

means, but only with solvent; the row oil obtained from this extraction is refined with a

process that is very similar to the previous one. The refined oil obtained from pomace is

called refined olive-pomace oil. Refined olive oil and refined olive pomace oil can be mixed

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7 with extra virgin olive oil or virgin olive oil in various undefined portions to improve their flavor obtaining respectively “olive oil composed of refined and virgin olive oils” and “olive pomace oil”.

These categories of olive oil are recognized by European Community (EC Regulation No.

1019/2002, EC Regulation No 702/2007, EC Regulation No 1234/2007).

According to EC Regulation No 1234/2007 virgin olive oils are defined as:

“oils obtained from the fruit of the olive tree solely by mechanical or other physical means under conditions that not lead to alterations in the oil, which have not undergone any treatment other than washing, decantation, centrifugation or filtration, to the exclusion of oil obtained using solvents or adjuvant having a chemical or biochemical action, or by re- esterification process and any mixture of oil with other kinds”.

Quality standards of virgin olive oil can be divided into two groups: chemical and sensory standards.

The chemical standards are shown in Table 2.1.

Table 2.1: Chemical standards of extra virgin, virgin and lampante olive oil

CHEMICAL STANDARDS EXTRA VIRGIN VIRGIN LAMPANTE

Free acidity (%) ≤ 0,8 ≤ 2,0 > 2

Peroxide number (meqO₂/Kg) ≤ 20 ≤ 20 -

K₂₃₂ ≤ 2,50 ≤ 2,60 -

K270 ≤ 0,22 ≤ 0,25 -

∆K ≤ 0,01 ≤ 0,01 -

The sensory analysis as a method for the legal recognition and classification of olive oil is established by Commission Regulation (EEC) No 2568/91 f.m.i.

The sensory standards for classifying the various levels of quality of olive oil are reported in Table 2.2.

Table 2.2: Sensory standards of extra virgin, virgin and lampante olive oil

SENSORY STANDARDS EXTRA VIRGIN VIRGIN LAMPANTE

Median defects (Md) 0 ≤ 3,5 > 3,5

Median fruity (Mf) > 0 > 0 -

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8 2.3 Olive oil market

The International Olive Council (IOC) is the only intergovernmental organization in the world that brings together olive oil and table olive producing and consuming stakeholders. It was set up in Madrid, Spain, in 1959, under the auspices of the United Nations.

Among the several activities carried out by the Council, there is the monitoring of the global and european olive oil market. Once a year the IOC updates series of worldwide statistics on production, imports, exports and consumption.

2.3.1 Production

The worldwide production of olive oil in the 2016-2017 oil season was 2539000 tonnes, lower than the average of the previous five years (2921800 tonnes) (IOC, 2018).

2016-2017 is confirmed as a critical year for the production of olive oil. The crazy climate affected almost all the producing countries. The excess of humidity affected the olive trees during the bloom and fruit setting phases, followed by the infestation of Bactrocera oleae.

The world's largest producer of olive oil is Spain, followed by Italy and Greece; consequently,

at a continental level, the largest producer in the world is Europe. European production

amounts to 69,00 % (Figure 2.3); this data is slightly low if compared to the average of

previous years: in the period from 2004 to 2009, European production constituted 76% of the

worldwide total. This is due not so much to the reduction of European production, more to the

increase in production of the African and Asian continent. Indeed, among the main global

producers, there are countries overlooking the Mediterranean area, such as Turkey, Tunisia,

Morocco, that are increasing investments and production. Syria too has recently increased its

national production. The absolute novelty is China, which has started the production of olive

oil since 2014, reaching 5000 tonnes, as the USA.

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9 Figure 2.3: Production of olive oils of the 5 continents analyzed in percentage terms and

calculated on the basis of data concerning the 2016/2017 season.

In the European Union, Spain has strengthened its record by achieving a production corresponding to 74% of the European one (Figure 2.4)

Figure 2.4: Production of olive oils in EU countries analyzed in percentage terms and calculated as data concerning the 2016/2017 season.

Italy and Greece have significantly reduced their average production. Only Portugal seems to be on the opposite trend, in addition to Spain, with an average production increase from 45000 to 76600 tonnes.

69%

12%

15%

2% 1% 1%

Europe Africa Asia America Oceania Other

74%

11%

10%

4%

1%

Spain Greece Italy Portugal Other

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10 2.3.2 Export

The European Union is the first exporter all over the world (IOC, 2018) (Figure 2.5). The mass of olive oil exported (555800) has increased if compared to the previous period 2004- 2009 (361500).

Figure 2.5: Export of olive oils of the 5 continents analyzed in percentage terms and calculated as data concerning the 2016/2017 season.

Tunisia is in second place, followed by Turkey.

In the European Union, Spain has once again the main role: since 2009 it has almost doubled the volume of oil exported (from about 150000 to about 280000) (Figure 2.6).

Figure 2.6: Export of olive oils in EU countries analyzed in percentage terms and calculated as data concerning the 2016/2017 season.

73%

13%

7%

5%

1% 1%

51%

39%

7%

1% 2%

Spain Italy Portugal Greece Other

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11 Italy has recently lost the record in olive oil exportation and it is only in second place.

Portugal and Greece respectively follow the European classification.

2.3.3 Consumption

For historical and cultural reasons, the consumption of olive oil in the world has expanded significantly throughout the Mediterranean region, but it is significantly increasing in many other countries, thanks to its peculiar organoleptic and nutritional properties above all.

Although EU remains the world's largest consumer (Figure 7), its consumption has recently decreased.

Figure 2.7: Consumption of olive oils of the 5 continents analyzed in percentage terms and calculated as data concerning the 2016/2017 season.

Consumption has instead increased in other non-producing countries.

The most significant increase has been in Asia, particularly in Turkey, Japan, Syria and China, which in recent years has more than doubled its consumption (from 15,000 to around 40,000). The USA are the main consumers, followed by Brazil and Canada. The main consumer in Africa is Morocco followed by Algeria. In Tunisia there has been a slight decrease in consumption.

Europe is the only continent in which olive oil consumption has decreased, especially due to the main producing countries, while non-producing countries have a positive consumption trend. Italy, despite being the first European consumer (Figure 2.8), shows the biggest reduction, followed by Greece, and Spain. Among non-producing) countries, Germany and the United Kingdom have increased their consumption.

54%

9%

17%

16%

2% 2%

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12 Figure 2.8: Consumption of olive oils in EU countries analyzed in percentage terms and

calculated as data concerning the 2016/2017 season.

2.3.4 Import

Over the years, the quantities of olive oil imported in the world have increased: from about 640000 in the period 2004-2009 to about 800000 in the present day (IOC, 2018).

USA is the country that imports the most, followed by EU, Brazil, which recently doubled its imports, Japan, Canada and China. Thus, America is the most important country in terms of import volumes: it has a low production compared to a high consumption (Figure 2.9).

Figure 2.9: Import of olive oils of the 5 continents analyzed in percentage terms and calculated as data concerning the 2016/2017 season.

31%

7%

35%

5%

7%

4%

1%

4% 6% Spain

France Italy Portugal Greece Germany Belgium United King Other

21%

1%

17%

51%

3% 7%

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13 In EU, importation has decreased. The first country is Italy, followed by Spain and finally France and Belgium (Figure 2.10).

Figure 2.10: Import of olive oils in EU countries analyzed in percentage terms, calculated as data concerning the 2016/2017 season.

2.3.5 National characteristics: critical aspects and opportunities

Although the national production is lower than the Spanish one, Italy is at the center of the international market, because it has a solid tradition of bottling companies that work with national and imported oils, and then export to international countries, in particular the United States and Germany. The dependence on imports is very high, reaching over 100% at times in the past. Indeed, Italy is not self-sufficient even for its internal consumption. In order to investigate the critical aspects of production, it is necessary to highlight the extremely fragmented nature of Italian olive industry considering an average area of 1.30 ha. In recent years, the phenomenon of non-harvesting has also spread. This is due to the high production costs, which have to be attributed first of all to harvesting operations and then to the management of the olive grove, concerning pruning and defense. Prices recognized to the producer are not considered profitable. Hence the tendency to leave the olive groves.

A study of the International Olive Oil Council has compared olive oil production costs in the member countries, considering 7 different olive cultivation systems, from the traditional rainfed on steep slopes (rainfed orchards with a gradient > 20% and < 180 trees/ha) to the superintensive irrigated ones (irrigated orchards with > 800 trees/ha). The analysis of the data shows that the production costs in Italy, about 4 euro/kg of oil, are excessive if compared to

28%

61%

1% 6%

0% 3% 1%

Spain Italy Portugal France Germany Belgium Other

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14 countries like Morocco, Tunisia; Turkey; Greece and Spain where production costs range from 1.91 to 2.75 euro/kg (Figure 2.11).

Figure 2.11: Weighted average of oil production cost in several countries of the world.

It is reasonable to expect that the cultivation of the olive tree in North Africa and Turkey will extend in future years thanks to national capitals or foreign investments. The service institute for the Italian agricultural market (ISMEA) reports that the production cost of extra virgin olive oil varies from 3 to 8 euro/kg depending on the areas (ISMEA, 2018). These evidences are not encouraging for the future of olive oil in Italy, especially considering that in the Mediterranean area there are other countries that are able to produce with much more competitive costs and that are becoming increasingly important players in the international market. The major problem of national olive oil sector is the low profitability, considering that 49% of the value given to primary production hardly covers the costs, and that the milling and bottling companies have limited margins, too. Therefore, a matter of redistributing the value along the production chain would not be enough, but it is anyway necessary to increase the value to be distributed. For this reason, the constant promotional policies of large-scale retail trade represent a big limit.

Therefore, it is necessary to realize a set of strategies to enhance the national olive oil heritage

with interventions starting from primary production. What would be desirable is also a

conversion towards more intensive farming systems, if possible to mechanize, and

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15 interventions in the field of transformation and conservation of a product for which the recognized quality must be guaranteed and enhanced.

2.4 Nutritional and Health Benefits

The concept of the Mediterranean diet is now well known to the general public as well as health professionals. This diet is believed to be associated with numerous health beneﬁts that lead to increased longevity and a lower incidence of chronic diseases, including cardiovascular disease (CVD), cancer, and neurodegenerative conditions (Huang and Sumpio, 2008; Lairon, 2007; Pérez-Jiménez et al., 2007). Although the components of the diet vary somewhat between different cultures in the various Mediterranean countries, extra virgin olive oil is an important common factor. Extra virgin olive oil is becoming more important in daily diets due to its beneficial effects on human health. Epidemiological studies about consumption of extra virgin olive oil in Mediterranean countries, have showed important beneficial effects as antioxidant, anti-inflammatory, chemopreventive and anticancer (Bullo et al., 2011; Farr et al., 2012: Bulotta et al., 2014) and several studies have assigned to the extra virgin olive oil most of the beneficial effects on human health attributed to the Mediterranean diet (Sofi et al., 2013; Huang and Sumpio, 2008) (Table 2.3).

Approximately 85% of the fat content of the Mediterranean diet is provided by olive oil,

which contains mostly monounsaturated fatty acid (MUFA) in the form of oleic acid (Huang

and Sumpio 2008; Pérez-Jiménez et al., 2007). It has been known for many years that

replacing saturated fatty acid (SFA) in the diet with MUFA reduces the risk of heart disease,

and more recently a range of other health beneﬁts of MUFA consumption have been

discovered (Table 2.3).

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16 Table 2.3: Health beneﬁts of extra virgin olive oil

Beneﬁt Disease affected Active components References

Lower cardiovascular mortality Improved blood lipid proﬁle

Atherosclerosis, CVD MUFA, phenolic compounds

Covas et al., 2009;

Lairon, 2007

Reduced blood pressure Hypertension, CVD, particularly stroke

MUFAs, phenolic compounds

Covas, 2007; Pérez- Jiménez et al., 2007 Reduced inﬂammation Atherosclerosis, rheumatoid

arthritis, asthma

MUFAs, phenolic compounds, oleuropein, oleocanthal, α-

tocopherol, β-sitosterol, oleanolic acid

Covas et al. 2009;

Covas, 2007; Sales et al., 2009; Pérez- Jiménez et al., 2007;

Perona et al., 2006 Reduced oxidative

damage

Atherosclerosis, CVD, NAFLD (Non-alcoholic fatty liver disease) and NASH (Non- alcoholic steatohepatitis), cancer

MUFAs, phenolic compounds, α- tocopherol

Bester et al., 2010; Fito and de la Torre, 2007

Reduced hemostasis Thrombosis, CVD MUFAs, phenolic compounds

Huang and Sumpio, 2008; Covas, 2007;

López- Miranda et al., 2007; Pérez- Jiménez et al., 2007

Reduced risk of neurodegenerative diseases

Alzheimer’s disease, Parkinson’s disease

MUFAs, phenolic compounds

López-Miranda et al., 2010

Reduced cancer risk Breast, ovarian, colorectal, prostate, and upper aero- digestive tract cancers

MUFAs, squalene Berrino, 2016

Increased life span CVD, cancer,

neurodegenerative diseases

– Pérez-López et al., 2009; Pérez- Jiménez, 2005

Traditional olive oil health protective effects have been ascribed to its high monounsaturated

fatty acid content, although, nowadays, it is clear that many of the beneficial effects of olive

oil intake are due to its minor highly bioactive components, also because such compounds are

able to maintain their biological action when the oil is consumed in crude form. There are

more than 200 minor components in the unsaponifiable fraction of olive oil, which represent

about 2% of the total weight and include a number of heterogeneous compounds non-

chemically related to fatty acids (Bulotta et al., 2014). Particular attention has been focused

on the nutraceutical properties of those compounds provided with antioxidant activity. The

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17 most abundant antioxidants in olive oil are lipophilic and hydrophilic phenols (Table 2.4), which are physiologically produced in the plant to react against various pathogen attacks and/or insect injuries.

Table 2.4: The main phenolic compounds in olive oil (Bulotta et al., 2014)

Hydrophilic Lipophilic

Phenolic alchols Flavonoids Tocopherols

Hydroxytyrosol Apigenin α, β, γ, δ

Tyrosol Luteolin

Secoridoids Phenolic acids Tocotrienols

Oleuropein Gallic acid α, β, γ, δ

Ligstroside aglycon Vanillic acid

Lignans Benzoic acid

(+)-1-pinoresinol Cinnamic acid

(+)-1-acetoxypinoresinol Caffeic acid Coumaric acid

Phenolic compounds have shown anti-inflammatory, antioxidant, antimicrobial, anti- proliferative, antiarrhythmic, platelet antiaggregant and vasodilatory effects, as well as the ability to modulate important cellular signalling pathways (Covas et al., 2009; Covas 2007;

Pérez-Jiménez 2007; Perona et al., 2006; Sanchez-Fidalgo et al., 2013).

Phenolic compounds and their secondary metabolites also contribute to the long oil shelf-life and influence several organoleptic characteristics, including taste (e.g bitter, astringent and pungent) and color (Bulotta et al., 2014).

Such compounds are released from the olive fruit to olive oil during the extraction process. In

particular oleuropein is abundant in high amounts in unprocessed olive leaves and fruits,

while higher concentration of hydroxytyrosol may be found in the fruit and in olive oil, owing

to chemical and enzymatic reactions that in the plant occur during maturation of the fruit. In

addition, many agronomic factors, as cultivar, ripening index, geographic origin, olive trees

irrigation, as well as several extraction conditions, may influence their final concentration in

olive oil. It is therefore essential to optimize all process variables in order to increase the

concentration of bioactive compounds in the extracted oil. Furthermore, considering that they

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18 are sensitive to oxidative processes, it is important to identify the best storage conditions in order to preserve the bioactive compounds as much as possible during storage. Oleuropein and hydroxytyrosol represent the compound of major interest for their biological and pharmacological properties and they are the most investigated antioxidant natural compunds.

They have been studied as isolated compounds or as components of oil phenolic extracts, showing a wide variety of beneficial effect, mainly related to their antioxidants activity in many preclinical models of diseases (diabetes mellitus, cardiovascular diseases, neurodegenerative diseases and cancer) (Huang and Sumpio 2008; Bester et al., 2010; López- Miranda et al., 2007; Li et al., 2009; Pitt et al., 2009; Owen et al., 2000b)

2.4.1 Extra virgin olive oil claims

On the basis of generally accepted scientific data about the effect of the consumption of extra virgin olive oil on the human health, the European Food Safety Authority (EFSA) has approved a number of health claims for olive oil. The major claims are related to vitamin E, monounsaturated and polyunsaturated fatty acids and polyphenols (Table 2.5).

Table 2.5: Extra virgin olive oil claims, chemical compounds in object and its recommended dose.

Compound Recommended dose Claim (EFSA)

Vitamin E

at least 15% of 20 mg.

equal to 3 mg

Vitamin E contributes to the protection of cells from oxidative stress

Monounsaturated and polyunsaturated fatty

acids

Saturated fatty acids replacements with monounsaturated and polyunsaturated fatty acids in the extra virgin olive oil can maintain the normal blood LDL-cholesterol

concentrations

Polyphenols

5 mg of hydroxytyrosol per day

Olive oil polyphenols contribute to the

protection of blood lipids from oxidative stress

These claims can be reported on the extra virgin olive oil label according to Reg CE 1924/2006.

Considering the recommended doses to obtain the effect declared in claim and extravirgin

olive oil daily consumption in a balanced diet, it is clear that in order to exert a healthy action

the oil must have a vitamin E content of at least 150mg / kg and of polyphenols of at least 250

mg / kg.

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19 Unfortunately a minimum level of concentration of these functional components is not considered among the parameters indicated to classify an olive oil as extra virgin olive oil.

This lack is perhaps even more evident in the Disciplinary of Certified Oils, whose main purpose is to enhance the quality of the product. In some Disciplinary the minimum limit indicated for polyphenols and tocopherols is much lower than the effective concentration for health benefits. This could be due to the extreme variability of these components in quantitative terms, depending on several parameters: cultivars, pedo-climatic conditions, harvesting time, technological extraction and storage conditions.

2.5 Olive oil composition

The composition of olive oil is characterized by (Boskou et al., 2006):

- Triglycerides: 97-99% (mainly characterized by monounsatured fatty acid, followed by polyunsatured fatty acids and satured fatty acids)

- Minor components: 1-3% (mixture of polar, nonpolar and amphiphilic substances involved in the sensory and health promoting properties of extra-virgin olive oil) 2.5.1 Triglycerides (TG)

The fatty acid composition may vary depending on the sample, the area of production, the latitude, the climate, the variety and the stage of ripening of the fruit. Greek, Italian and Spanish olive oil is low in linoleic and palmitic acids and has a high percentage of oleic acid.

Tunisian olive oil is high in linoleic and palmitic acids and lower in oleic acid. On the basis of the analysis of samples from various countries, olive oils are classified into two types, one with a low content of linoleic-palmitic and high oleic acid and the other with a high content of linoleic-palmitic acid and low oleic content. The oleic acid is formed in the fruit and there is a strong antagonism between oleic acid and palmitic acid, palmitoleic acid and linoleic acid.

On average, the major fatty acids in olive oil are palmitic (C16: 0), palmitoleic (C16: 1), stearic (C18: 0), oleic (C18: 1), linoleic (C18: 2) and linolenic ( C18: 3) acid.

TG has a particular composition, with high amounts of triolein and other triglycerides

containing oleic acid. The fatty acid composition limits adopted in the latest editions of Codex

Alimentarius and the International Olive Oil Council are shown in Table 2.6.

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20 Table 2.6: Composition of fatty acids determined with gas chromatography according to Codex Alimentarius and to IOC

Fatty acids Codex Alimentarius International Olive Oil Council

Lauric C12:0 Not present Not specified

Myristic C14:0 <0.1 <0.05

Palmitic C16:0 7.5-20.0 7.5-20.0

Palmitoleic C16:1 0.3-3.5 0.3-3.5

Eptadecanoic C17:0 <0.5 ≤ 0.3

Eptadecenoic C17:1 <0.6 ≤ 0.3

Stearic C18:0 0.5-5.0 0.5-5.0

Oleic C18:1 55.0-83.0 55.0-83.0

Linoleic C18:2 3.5-21.0 3.5-21.0

Linolenic C18:3 ** ≤ 1.0

Arachidonic C20:0 0.8 ≤ 0.6

Eicosenoic C20:1 Not specified ≤ 0.4

Behenic C22:0 < 0.3 ≤ 0.2

Erucic C22:1 Not present Not present

Lignoceric C24:0 < 1.0 ≤ 0.2

In addition to the number of carbon atoms involved in their alkyl chain, fatty acids are also distinguished on the basis of the degree of unsaturation, i.e. the number of double bonds possibly present in the structure. In particular, in olive oil there is about 14% of saturated fatty acids (palmitic, stearic, myristic acids, etc.), 72% of monounsaturated (oleic and palmitoleic acids) while the remaining 14% is polyunsaturated (acidic linoleic and linolenic) (Barjol, 2013).

The saponifiable fraction promotes the absorption of fat-soluble vitamins, shows a plastic action in the structuring of cell membranes, provides the precursors that are necessary for the synthesis of prostaglandins, terminates free radicals and regulate positively the accumulation of cholesterol. The unsaturated fatty acids also have a very important role in nutrition: in particular linolenic and linoleic acids are fatty acids ω-3 and ω-6, essential for the growth and functionality of human tissues, which is not able to synthesize them and must therefore take them with the diet.

2.5.2 Minor components

1) Hydrocarbons: organic compounds that contain only carbon and hydrogen atoms. The

major hydrocarbon in olive oil is squalene: a triterpene hydrocarbon that shows

antioxidant activity by reacting with oxygen radicals and oxygen-reactive species,

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21 protecting the skin against UV rays. Squalene has also immune-stimulating properties and antineoplastic effects on colon, breast and prostate cancers. In olive oil, squalene achieves a concentration of 700 mg/100 g with a range variation comprised between 90 and 870 mg/100g.EVOO contains 200-700mg of squalene per 100g of oil (Beltran et al., 2016).

2) Tocopherols: fat-soluble alcohols. They can be found in variable quantities depending on biological and technological factors, with an average concentration for a virgin oil that is about 250 mg/kg. They are considered the most important natural antioxidant agents of lipid fractions, as they prevent lipid peroxidation by neutralizing free radicals and preferentially act at the level of cell membranes and plasma lipoproteins (Frankel, 2011).

In olive oil, there are the α-, β- and γ-tocopherol forms; the most frequent is α-tocopherol.

Tocopherols are vitamin E, obviously known for its antioxidant activity at the level of cell membranes and organelles, where this vitamin accumulates. (Condelli et al., 2015).

3) Aliphatic alcohols: in olive oil, they are esterified with fatty acids to produce the waxy components (docosanol, tetracosanol and esacosanol) that cover the surface of the fruit.

4) Diterpenic and triterpenic alcohols: they are biogenetic precursors of sterols and they are characterized by complex structures. They play a fundamental biological role since they hinder the absorption of cholesterol in the intestine. Among the diterpenic ones, we can find the cycloartenol and the methyl-cycloartenol. Among the triterpene, we find the uvaol and the erythrodiol, which are initially present in the epidermis of the fruit, so that their subsequent content in the oil produced depends on the extraction method adopted. In particular, pomace oil is rich in these components (Wang et al., 2017).

5) Triterpenic acids: the oleanolic, ursolic and maslinic acids, present on the skin of the drupe, stabilize its physical properties and protect it from attack by possible parasites.

6) Sterols: they appear in olive oil with a concentration varying between 0.2 ÷ 0.5% (on

average 1500 ppm) so that they constitute the fraction of the most representative

unsaponifiables after the hydrocarbons. They are compounds with high molecular weight,

with an alcoholic function whose synthesis has the acetylCoA as precursor and the

squalene as the most representative intermediate. They are partly free and partly esterified

with fatty acids. The major sterol in olive oil (> 90% of this fraction) is β-sitosterol, a

lower percentage means a fraudulent addition of other oil than olive oil (Lerma-Garcia et

al., 2010). From the nutritional point of view, sterols promote a reduction in the low-

density lipoprotein (LDL) content of blood vessel stenosis and finally of subsequent

thrombosis.

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22 7) Pigments: the color of virgin olive oil is due to the presence of particular pigments, such as carotenoids and chlorophylls. Carotenoids, whose color varies from yellow to red, are the precursors of vitamin A (pro-vitamin factors). The main carotenoids in olive oil are α- carotene, β-carotene, but there is also the γ-carotene. β -carotene is transformed into trans-retinol (vitamin A) in the intestinal mucosa. The amount of carotenoids in oil (~ 100 mg / 100 g oil) is influenced by several factors, such as the environment, the oil extraction system and the olive oil storage conditions. The typical greenish color of olive oil is due to the presence of chlorophylls, which are found associated with carotenoids. In the virgin olive oil there are the chlorophylls α and ß and some products of their decomposition, such as the phaeophytins. The chlorophyll content in olive oil is also influenced by the degree of ripeness achieved by the drupe (Mendoza et al., 2013): the oils obtained from not yet ripe olives may contain up to 50 mg of chlorophyll / kg of oil, as showed by the green color. The normal content of chlorophyll in young oils (1-2 months), on the other hand, varies from 1 to 10 mg of chlorophyll, whereas in the less young oils (7-8 months) it can also be completely absent. While carotenoids perform an antioxidant action by neutralizing the singlet oxygen, the chlorophylls have a pro- oxidizing action, catalyzing the production in the presence of light. It is therefore essential for the stability of the oil to reach a correct ratio between chlorophyll pigments and carotenoids. All the colored pigments in olive oils are easily altered by light and heat, especially in the presence of metals, oxygen and air.

8) Vitamins: in addition to vitamins E and A, in olive oil there are also other important fat- soluble vitamins, such as vitamin D (D2), which plays an important role in the metabolism of calcium fixation, and vitamin K, known for its antihemorrhagic action, whose concentration increases in the oils extracted in the presence of leaves.

9) Ubichinoni: they consist of a 2,3-dimethoxy-5methylbenzoquinone nucleus, with a lateral chain in position 6 formed by 6 to 10 isoprenic units; in olive oil, coenzyme Q10 can be found in concentrations between 0 ÷ 40 ppm depending on the harvesting period. From a biochemical point of view, ubiquinone plays a fundamental metabolic role as an electron transporter.

10) Phenolic substances: they take part in the determination of the antioxidant power and

they can be found in virgin olive oil in extremely variable quantities (50-550 ppm). They

accumulate in the lipid phase during the process of mechanical extraction of the oil from

the glucosidic polyphenols in olives (Termentzi et al., 2015), with a concentration

varying between 1 and 3% of the fresh weight of the pulp (Fernandez et al., 1997). The

phenolic units, because of their chemical structure, do not show a high solubility in oil,

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23 but they are found inside the micro-droplets of water dispersed in the lipid phase. The content in phenolic substances varies depending on many factors such as climate conditions, the cultivar, the degree of ripeness achieved by the drupes, their phytosanitary status, the extraction technique that is used, the oil storage condition. Phenolic substances contribute to the aromatic characteristics of olive oil (Clodoveo et al., 2015). In fact, they give the oil a bitter-pungent taste easily perceptible in the first months of life of this product. Over time this organoleptic characteristic tends to decrease. The phenolic compounds in virgin olive oil can be divided into five main classes:

- Phenethyl alcohol: it is characterized by a simple structure such as hydroxytyrosol (3,4- dihydroxyphenyl ethanol 3,4-DHPEA), known for its strong antioxidant activity, and tyrosol (p-hydroxyphenyl ethanol p-HPEA) (Cicerale et al., 2012). Their concentrations are generally limited in freshly extracted oil and increase during storage because secoridoids such as oleuropein and ligstroside progressively hydrolyze (Oueslati et al., 2018).

- Phenolic acids: they are derivatives of benzoic acid, cinnamic acid and phenyl acetic acid.

Caffeic, vanillic, syringic, p-coumaric, o-coumaric, protocatechuic, synaptic and p- hydroxybenzoic acid belong to this class of compounds and they are the first phenolic compounds to be found in virgin olive oil (Montedoro 1972; Vasquez Roncero., 1978).

These substances, when linked to other non-phenolic compounds such as elenoic acid, represent secoiridoid compounds, complex structures present in the olive, in virgin oil and in olive oil waste waters.

- Flavonoids: in olive oil, there are flavonols, such as luteolin-7-glucoside, rutin and apigenin, and also anthocyanins, such as cyanidin and delphinidin glucoside (Urbani, 2006).

- Secoiridoids: they can be found exclusively in plants belonging to the oleaceae family, and

therefore also in the European Olea unlike the more widespread phenyl-acids, phenyl-

alcohols and flavonoids. The secoiridoids of the olive oil are characterized by the

presence of elenolic acid and its derivatives (oleuropein, ligstroside, dimethyloleuropein,

verbascoside and nüzhenide (Urbani, 2006). Drupe and olive oil waste water are

characterized by high concentrations of the glycosidic form of these components and the

quantities of the aglycone form dissolved in the oil are considerably more limited. In the

oil, in fact, only the fraction that loses the glucose unit by hydrolysis (β- glucosidase) will

be transferred, because it is less polar and therefore more similar to the oil.

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24 - Lignans: they are phenolic substances with high antioxidant power and therefore anticarcinogenic activity like pinoresinol and 1-acetoxy-pinoresinol (Owen et al., 2000a).

2.6 Sensory quality of olive oil

The sensory properties of olive oil widely vary depending on several factors (Servili et al., 2004). Differences in genetic resources, environmental conditions, process speciﬁcations, and local know-how induce sensory differences among oils (Caporale et al., 2006).

Most of the attention on the sensory characteristics of olive oil is currently focused on how to evaluate whether a given oil is free of defects and how olive oil is qualiﬁed.

Indeed olive oil, to be defined as extra virgin olive oil, has not only to be characterized by specific chemical standards, but also to comply with precise sensory parameters. Official methods and standards are defined by the International Olive Council and signed by the EU legislation. IOC standards classify olive oil in categories such as extra virgin or virgin or

“lampante”. These standards are based on the evaluation of both “negative” and “positive”

attributes. Figure 2.12 shows the official technical sheet to evaluate negative and positive

attributes; the intensity of parameters is assigned on a 10 cm long scale from zero (no

perception) to 10 (extremely strong perception). Negative attributes are sensory defects and

cannot be present in EVOO, while positive sensory attributes are bitterness, pungency and

fruity notes and their intensity is important to determine extra virgin olive oil sensory

characteristics.

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25 INTENSITY OF PERCEPTION OF DEFECTS:

INTENSITY OF PERCEPTION OF POSITIVE ATTRIBUTES:

Figure 2.12: The scorecard for the official evaluation of the sensory characteristics of olive oil (Commission Regulation (EC) No 640/2008 of 4 July 2008 amending Regulation (EEC)

No. 2568/91 on sensory characteristics of olive oil)

According to the European regulation, oils should be classified according to the median of

defects and the median of “fruity” perception. The median of defects is defined as the median

of the defects perceived with the greatest intensity. The median of the defects and the median

of “fruity” are expressed to one decimal place, and the value of the robust variation

coefficient that defines them must be no greater than 20%. The following grading applies:

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26

SENSORY STANDARDS EXTRA VIRGIN VIRGIN LAMPANTE

Median defects (Md) 0 ≤ 3,5 > 3,5

Median fruity (Mf) > 0 > 0 -

Annex XXII of the Commission Regulation (EC) No 640/2008 of 4 July 2008 deals in detail with every aspect of the organoleptic analysis with particular reference to the method for evaluating extra-virgin olive oil, panel, panel head and tasters, tasting room, the use of the profile sheet, etc.

Moreover, in the Commission Regulation (EC) No 640/2008, there is also the glossary of terms for sensory defects and positive attributes. In particular, definitions of defects are:

 Fusty/muddy sediment: characteristic flavor of oil obtained from olives piled or stored in such conditions as to have undergone an advanced stage of anaerobic fermentation, or of oil which has been left in contact with the sediments that settles in the bottom of tanks and vats and which has also undergone a process of anaerobic fermentation.

 Musty-humid: characteristics flavor of oils obtained from fruit in which large numbers of fungi and yeasts have developed as a result of storage in humid conditions for several days

 Oily winey-vinegary acid-sour: characteristic flavor of certain oils reminiscent of wine or vinegar. This flavor is mainly due to aerobic fermentation of the olives or of the olive paste and leads to the formation of acetic acid, ethyl acetate and ethanol.

 Metallic: flavour reminiscent of metal, characteristic of oil that has been in prolonged contact with metallic surfaces during milling, malaxing, pressing or storage.

 Rancid: flavour of oils that have undergone an intense process of oxidation.

Definitions of positive attributes are:

 Fruity: range of smells (depending on cultivar, degree of maturity at harvest, and processing conditions) typical of oil from healthy fresh fruit, green or ripe, perceived directly as odor when smelling the oil and/or retronasally as flavor when tasting the oil in the mouth.

Fruitiness is qualified as green if the range of smells is reminiscent of green fruit and it is typical of oil from green fruit

Fruitiness is qualified as ripe if the range of smells is reminiscent of ripe fruit and it is typical of oil from green and ripe fruit.

 Bitter: characteristic primary taste of oil from green olives or olives turning color.

Bitterness is detected by the circumvallate papillae on the “V” region of the tongue

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27  Pungent: tingling sensation typical of oils produced at the beginning of the season, mainly from olives that are still green. It can be perceived throughout the mouth cavity, particularly in the throat.

The regulation allows the use of the following terms for fruitiness or bitterness or pungency on the package label:

 “Intense”: may be used when the median of parameter is greater than 6

 “medium”: may be used when the median of the parameter is between 3 and 6

 “light” may be used when the median of the parameter is less than 3

Bitterness and pungency are usually related to phenolic compounds in particular secoridoid derivatives such as the aldehydic form of oleuropein aglycone (Mateos et al., 2004; Siliani et al., 2006).

The use of Commission Regulation (EC) No 640/2008 is necessary for the classification of the olive oil product, but it is not appropriate if it is required to distinguish the olive oils according to the sensory characteristics, in order to enhance the wide panorama of positive parameters. Thus, in this case, the most widely used method is Descriptive Sensory Analysis (DSA). The aim of DSA is to describe the sensory profile of a product through the quantitative measurement of a series of appropriate descriptors; generally, for this reason, each panel develops the sensory language itself. In order to achieve the best reproducibility of sensory assessment, precise definition should be given for every sensory descriptor and reference standards should be used to select, train and compare the performance of sensory judges. Some of the most used descriptors are: grassy, tomato fruit, tomato leaf, apple, artichoke, almond, citrus, cut grass (Monteleone et al., 2012).

Both positive attributes and sensory defects in olive oil can be associated with volatile

compounds that are mainly produced by oxidation of fatty acids. It is generally agreed that

endogenous plant enzymes, through the lipoxygenase pathway, are responsible for the

positive aroma perception in olive oil, whereas chemical oxidation and exogenous enzymes,

usually from microbial activity, are associated with sensory defects (Kalua et al., 2007). The

aroma of olive oil is attributed to aldehydes, alcohols, esters, hydrocarbons, ketones, furans

and, probably, other unidentified volatile compounds. The volatile compounds that are mainly

responsible for the “green sensations”, typical of the freshly extracted olive oil, are the C6 and

the C5 compounds; trans-2-hexenal, cis-3-hexenal, cis-3-hexenol, cis-3-hexenyl acetate are

found in most virgin olive oils in Europe (Kalua et al., 2007).

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28 2.7 Olive oil processing

Olive oil quality depends on many factors related to olive tree cultivation, harvesting, extraction technology and storage (Servili et al., 2008; Cicerale et al., 2013; Zinnai et al., 2014, 2015, 2016). The good quality of olives is a decisive factor, but it is not the only one ensuring a good quality of the olive oil. It’s important that quality does not deteriorate during processing. Therefore wrong operations should be avoided.

2.7.1 Harvesting systems

The olive harvesting operation influences oil yield and quality as well as the cost of oil production. The harvesting operation includes olive detachment from the tree and collection of the detached olives. The first one can be carried out with different systems (Nasini et al., 2014):

1. Hand picking: it is the most expensive harvesting system due to high labor costs. It can be used with any type of tree training system and olive grove characteristics. The productivity of hand picking is 10-20 kg of olives per hour per operator. Hand picking is suitable for small-scale high-quality production.

2. Hand-held harvesting machines: there is a great variety of models and design with different performances. The productivity varies from 30-50 kg per hour per operator, but under optimal condition, a well-trained operator can harvest up to 150 kg per hour.

The best results are obtained with medium or high weight fruit, low resistance to detachment and low and well-pruned trees, so that the crown is easily accessible to the operating device of the harvesting machines, but tree height does not exceed 4 m. A hand-held harvesting machine consists of three parts: an operating device, a telescopic pole and a motor to provide the needed driving power. This technique may cause some damage to the trees (bruising the bark and leaf fall): bacterial and fungal growth may affect the damaged area of the bark, so a disinfectant treatment with copper products immediately after harvest is recommended. Mechanical damage to the fruit is generally low if the beating action is minimized. Anyway, it remains suitable for small-scale production.

3. Trunk shakers: olives are harvested by means of a vibrating grip head attached to the

trunk or, in case of very large trunks, to the main branches. The vibrating grip consists

of a jaw with a cushioned system to avoid damages to the bark of the trunk or

branches. Productivity may reach a value of 200-400 kg per hour per operator.

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29 Suitable for high-scale production in semi-intensive or intensive olive groves, it decreases its productivity when it must be attached to the branches.

4. Straddle harvesters: they are mechanical harvesters. In this case tree training and all of the concepts of cultivation are changed in order to adapt them to an already available harvesting machine. They are suitable to super high-density olive groves by simply increasing the number of shaker bars and in a single machine it combines both olive detachment and interception operations. Labour productivity may reach a value of 1000-1500 kg per hour per operator. It requires dwarf cultivars in the form of hedgerows with a planting density between 1500 and 2100 trees per hectare. The most suitable cultivars are Arbosana, Koroneiki and Arbequina. Two advantages compared to traditional systems are related to a very high yield or productivity and the standardization of oil quality. Two disadvantages compared to traditional system are related to the impossibility of enhancing the olive tree biodiversity and the high investment needed to implement the system, making it suitable only for large-scale companies.

2.7.2 Leaf removal and washing

Generally, olives are picked from the tree by hand or by mechanical devices. In some cases, olives are picked from the ground or nets are placed under the tree crown.

In any case, olives are contaminated with vegetal impurities, such as leaves or twigs, and with mineral impurities, like soil, dust and stone fragments. Separation of extraneous material is carried out by a leaf removal and washing machine. These operations are carried out by machinery equipped with a powerful aspirator, which removes leaves and sprigs, and a washing tank, with forced water circulation in which olives are washed (olive washing is generally carried out by recycling potable water). In addition, the machinery can be equipped with a special magnet to remove any iron objects that could be harmful to the metal crushers (Di Giovacchino, 2013).

2.7.3 Olive crushing

Olive crushing gives rise to the breakage of vegetable cells containing oil and helps to the oil

droplets to come out from the cells of olive flesh. Indeed, the aim of this phase is to reduce

olives to a homogeneous paste by breaking pits, skin and pulp cells and the vacuoles

containing tiny droplets of oil; the oil flowing freely from the vacuoles can then be separated

from the water and solid constituents. Olive crushing can increase the temperature of the olive

paste because a part of the kinetic energy of the crushers, which rotate either at a high speed

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30 (metallic crushers) or at a low speed (stone mill), is transformed into thermal energy for friction. The results reported in some papers (Di Giovacchino et al., 2002b) showed that the temperature of olive paste increased by 13-15 °C with respect to the ambient temperature, when a fixed-hammers metallic crusher was used, and by 4-5 °C, when a stone mill was used.

The different crushing methods may influence oil yields and quality (first of all they influence total phenol content of oils and affect the content of the volatile compounds of the head-space of virgin olive oil.

Table 2.7 shows the machines employed in olive oil production for the crushing step

(Amirante et al., 2010). Each machine has its advantages and disadvantages. The choice of

the crushing system depends on subjective circumstances.

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31 Table 2.7: Advantages and disadvantages of the machines employed in the crushing step of the extraction process for olive oil production (Amirante et al., 2010).