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SIXTH FRAMEWORK PROGRAMME PRIORITY 5

Food Quality and Safety Priority, Call 4-C SPECIFIC SUPPORT ACTION

MAC-Oils

Mapping And Comparing Oils

THE SCIENTIFIC HANDBOOK www.mac-oils.eu

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Copyright notice

Except where otherwise stated, downloading and reproduction, for personal use or for further non-commercial or commercial dissemination, of documents or information available on the MAC-Oils website, MAC-Oils outputs (Scientific Handbook, Consumer’s Guide, SME Work Paper available as hardcopy and/or CD rom) are authorized to the condition that due acknowledgement is given as follows:

• to the appropriate copyright holder: European Communities, MAC-Oils Consortium

The permission granted above does not extend to any textual or artistic material (drawings, photos, audio, video, etc.) on this media which is identified as being the copyright of a third-party. In these circumstances, authorization to reproduce such material must be obtained from the appropriate copyright holders.

All logos and trademarks are excluded from the above-mentioned general permission.

Translations of documents or information into languages other than the language editions officially published on the MAC-Oils media are subject to the conclusion of a free of charge license agreement. For this purpose, relevant requests, as well as any other copyright queries if in doubt regarding the re-use of MAC-Oils documents or information, shall be addressed in writing to:

Dr. Gian Luigi Russo Istituto di Scienze dell'Alimentazione Consiglio Nazionale delle Ricerche Institute of Food Sciences National Research Council via Roma 64 83100 Avellino Italy Phone: +39 0825 299431 / 331 Fax: +39 0825 781585 e-mail: glrusso@isa.cnr.it

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Preface MAC-Oils (Mapping And Comparing Oils) is an European project granted by the Commission under the “Food Quality and Safety” frame in FP6. The main goal of this Specific Support Action consisted in the individual and comparative assessment of safety, quality, environmental impact aspects of the eight target oils (olive, peanut, sunflower, corn, argan, soybean, rice bran and rapeseed oils) and their respective production and manufacturing methods. Most of these edible oils have been selected since they are commonly used by European consumers or produced in European Countries, while others, considered to be “exotic”, even though their origins are well-known, have become recently available on the European marketplace. The three main outputs of MAC-Oils project have been: i. a Scientific Handbook addressed to researchers working in the field of edible oils; ii. a leaflet to SMEs and oil producers; iii. a Consumer’s guide.

Here, the MAC-Oils Consortium presents the Scientific Handbook consisting in a final, comparative analysis of the agronomical, industrial, physical-chemical, organoleptic and healthy properties of the edible oils investigated during the project. The Handbook critically reviews existing data and know-how, at European and extra-European level, collected during the eight thematic workshops organized during the project. The Chapters of the Handbook resemble the five main topics of interest investigated for each oil: 1. oil seeds cultivation; 2. extraction, conservation and packaging methods; 3. chemical and physical-chemical properties of oils; 4. organoleptic properties and acceptability by the European consumer; 5. risks/benefits of oil consumption for human health.

A special thank goes to the MAC-Oils colleagues who poured an incredible energy in coordinating the preparation of this Handbook: Arnon Dag for Chapter 1; Marileusa Chiarello, Zohar Kerem and Anne Rossignol-Castera for Chapter 2; Virginia Carbone, Amalia Carelli and Rosaria Cozzolino for Chapter 3; Mario Paolo Pellicano for Chapter 4; Rosalba Giacco for Chapter 5.

This Handbook will be spread among researchers and educational institutions in European, Mediterranean and Latin American Countries. It is also available, free of charge, on the MAC-Oils website at www.mac-oils.eu, and on CD format upon request.

We hope that this Handbook may stimulate discussion and exchange of know-how among readers and generate new ideas and collaboration among scientists working in the field of edible oils.

Gian Luigi Russo Project Coordinator

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This work has been made possible with the support of

European Commission 6th Framework Programme

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Contents CHAPTER 1 Cultivation: problems and perspectives 1 Abstract 21.1. Introduction 3

1.1.1 Botanical classification and description 31.1.2 Area of origin 41.1.3 Domestication 51.1.4 Source of oil 71.1.5 Traditional cultivation and folklore 7

1.2. Current situation of Growing in the World 111.2.1 Countries 111.2.2 Exportation/ importation 13

1.3. Environmental Requirements 151.3.1 Temperature 151.3.2 Soil and water needs 17

1.4. Cultural Practices 201.4.1 Tillage 201.4.2 Planting Date 201.4.3 Planting depth and row width 211.4.4 Fertilization 221.4.5 Water management 241.4.6 Harvesting 261.4.7 Training and pruning methods 29

1.5. Other Product of the Crop 291.6. Major Pests and Pathogens 321.7. Cultivars 34

1.7.1 Traditional 341.7.2 ‘New’- conventional 381.7.3 ‘New’- G.M. crops 39

1.8. Specific Cultivation Practice Affecting Oil Characteristics 411.8.1 Environment 431.8.2 Harvest time 431.8.3 Cultivars 441.8.4 Irrigation-water quality 451.8.5 Fertilization 46

1.9. Concluding remarks 47References 48  

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CHAPTER 2 Extraction, refining, conservation and packaging methods of edible oils 57 Abstract 582.1. Extraction and Preservation of Virgin Oils 59

2.1.1 Olive oil 592.1.2 Argan oil 63

2.2. Refined Oils: Preparation and Extraction 652.2.1 Preparing operations 662.2.2 Oil extraction 78

2.3. Refined Oils: Chemical and Physical Refining 912.3.1 Chemical refining: the case of sunflower oil 952.3.2 Particularities for other edible oils 1012.3.3 Physical Refining 1062.3.4. Specifications of refined oils 108

2.4 Packaging and Storing Oils 1082.4.1 Molecular oxygen and the oxidation of oils 1082.4.2 Oxygen scavengers and packaging 1092.4.3 Penetration of light and oxidation of oils 1102.4.4 Permeation, migration and absorption 1102.4.5 Influence of packaging on the oxidation kinetic of virgin olive oil 1112.4.6 Which package should be selected for edible vegetable oils? 1132.4.7. Conclusion 113

References 114 CHAPTER 3 Assessment of chemical and physical-chemical properties of edible oils 127 Abstract 1283.1. Comparison of Chemical and Physical-Chemical Properties 129

3.1.1. Introduction 1293.1.2. Chemical and physical parameters 1323.1.3 Major compounds 1333.1.4 Minor saponifiable compounds 1423.1.5 Unsaponifiable matter 1453.1.6. Other minor compounds 159

3.2. Instrumental Methods Used for Oil Analyses 1613.2.1 Classical methodologies 1623.2.2 New trends and recent developments in oil analysis 169

3.3 Concluding Remarks 179References 180 

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CHAPTER 4 Sensory properties and consumers acceptability of oils 199 Abstract 2004.1. Sensory Analysis 201

4.1.1 Sensory analysis of olive oil 2024.1.2 Factors that affect the sensory quality of virgin olive oil 2054.1.3 Sensory analysis of seed oils 206

4.2. Sensory Characterization of Oils 2094.2.1. Sensory properties of extra-virgin olive oils: agronomic and technological

aspects 2094.2.2. Technological aspects 2124.2.3. Sensory properties of seed oils 2134.2.4. Comparison of sensory properties: soybean, peanut, rapeseed/canola,

sunflower and extra virgin olive oils 2174.3.Relationship between Virgin Olive Oil Compounds and Sensory Attributes 218

4.3.1 Contribution of different compounds to the oil sensory quality 2184.3.2. Innovative instrumental characterisation of the flavours of oils: oral processing

effects 2274.4 Consumers Acceptability of Edible Oils 230

4.4.1. Factors that affect the acceptability of virgin olive oil 2304.4.2. Factors that affect the acceptability of seed oils. 2314.4.3. Acceptability of argan oil. 2334.4.4. Acceptability of rice bran oil 234

4.5. Concluding Remarks 236References 237 CHAPTER 5 Risk and benefits of edible oil consumption on human health 245 Abstract 2465.1. Introduction 2485.2. Effects of Fatty Acids Contained in Edible Oils on Human Health 248

5.2.1 Cardiovascular diseases 2495.2.2 Cancer 2615.2.3 Nutritional Recommendations and new prospective 262

5.3. Effects of Minor Components in Edible Oils on Human Health 2635.3.1 Tocopherols 2635.3.2 Phytosterols 2655.3.3 Phenols 2695.3.4 Combinatory prevention - synergistic effect 272

5.4. Concluding Remarks 274References 275

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CHAPTER 1

Cultivation: problems and perspectives

Authors Contributors DAG, A.1 DE SOUZA, P.F.A.3

BEN-DAVID, E.A.1 BERTIOLI, D.J.3

FIUME, P.2 TURLEY, D.4

PERRI, E.2 HUXTABLE, H.4

AREKIN, E.4

LIDDLE, N.4

GARGOURI, K.4

ZIPORI, I.1

MUZZALUPO, I.2

CHARROUF, Z.6

GUILLAUME, D.7

1 The Agricultural Research Organization, Gilat Research Center, M.P. Negev, 85280 (Israel) 2 CRA-OLI Consiglio per la Ricerca e la sperimentazione in Agricoltura – Centro di ricerca per l’olivicoltura e

l’industria olearia, Rende (CS) (Italy) 3 Universidade Católica de Brasília, 70790-150 Brasília (Brazil), 4 Central Science Laboratory, York, YO41 1LZ (United Kingdom) 5 Institut de l'Olivier, (Tunisia) 6 Faculté des Sciences Université Mohammed V- Agdal, FS-UMV-Agdal, 1014 Rabat (Morocco) 7 Institut de Chimie Moléculaire de Reims, UMR 6229, 51100 Reims (France)

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Abstract

Oil producing plants have been used in religious ceremonies, for personal use and adornment, and for flavouring throughout history by populations in Africa, Asia, Europe and the Americas. Nowdays there is a world-wide pressure by consumers to use perceived natural compounds in edible and personal products. It is vital that producers should be able to service this growing demand efficiently, economically and above all reliably. This publication adresses the need for a detailed informatin on the major oil producing crops namely Argan (Argania spinosa), corn (Zea mays), olive (Olea europaea), peanut (Arachis hypogaea), rapeseed (Brassica napus), rice (Oryza sativa), soybean (Glycine max) and sunflower (Helianthus annuus). For each of which a brief history of the use and economic data is given, and enviromental requirements, cultivation, and harvesting described. Results of current research and recommendations for improved agronomic practices, together with methods of adding value to the crop are also discussed.

The oil crops species belong to various botanical groups. The argan and the olive are species of evergreen perennial trees while the other oil crops are annual herbaceous plants. Some of the oil crops have been traditionally cultivated for centuries, while others are a product of a conventional breeding program or genetic modification (GM). Variety selection, environmental conditions, irrigation and fertilization all exert substantial influence on the oil quality parameters. In the past five years, about 56 percent of oilseeds produced and 80 percent of oilseeds traded worldwide have been soybeans. By country, the United States has the largest producer of soybean, peanut and corn. The Russian Federation is a major sunflower producing country while India, China and Canada are the largest producers of rapeseed. The olive oil production is concentrated in the Mediterranean countries Spain, Italy, Greece, Syrian, Turkey, Morocco and Algeria. The Argan oil production is exclusive to Morocco. In terms of exportation (1000 $), USA dominate maize oil exportation (246,163); Spain is the prime exporter of virgin olive oil (2,300,510); Argentina dominates peanut and soybean oils exportation (66,468 and 2,789,292 respectively) while Thailand, Canada and Ukraine are the prime exporters of oils from rice bran, rapeseed and sunflower respectively (2,696,248; 1,095,566 and 175,953 respectively).

The Mediterranean region provides optimal conditions for the olive growth. The argan tree is extremely well adapted to drought and environmentally conditions of southwestern Morocco. On the other hand, peanut, sunflower and the corn are native to the Americas. Although rice species are native to South Asia and certain parts of Africa, centuries of trade and exportation have made it commonplace in many cultures. Similarly, the soybeans are also native to east Asia and will grow well in climates with hot summers in other continents. The oil crops are prone to attacks by a large number of pests. Consequently the oil quality may deteriorate. Moreover, pesticides used by the famers may leave residues in the oil. In addition to their use in the manufacture of oil and food (with the exclusion of the non-edible argan and rapeseed), oil crops are being used in the manufacture of a variety of industrial and/or cosmetic products. Other olive products include table olives, olive paste (tapenade), skin care products, olive oil soap and even bio-gas production; peanuts have a variety of industrial end uses. Paint, varnish, lubricating oil, leather dressings, furniture polish, insecticides, and nitroglycerin are made from peanut oil; Sunflowers may be used to extract toxic ingredients from soil, such as lead, arsenic and uranium; Soybeans are the primary ingredient in many processed foods, including dairy product; substitutes Infant formulas based on soy are used by lactose-intolerant babies and for babies that are allergic to cow milk proteins; rapeseed oil is used in the manufacture of biodiesel for powering motor vehicles while the argan wood is being used in a more traditional manner as an energy source for heating.

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1.1. Introduction 1.1.1 Botanical classification and description Oil producing crops can be broadly classified into two groups, fruit trees and field crops. The olive and argan trees are amongst the former while the latter include the corn, peanuts, rapeseed, rice bran, soybean, and sunflower (Table 1.1). The genus Helianthus, to which the sunflower belongs, contains about sixty-seven species and nineteen subspecies, chiefly natives of North America; many are indigenous to the Rocky Mountains, others to tropical America, and a few species are found in Peru and Chile (Heiser, 1978). The most important species are H. annuus L., H. mollis L., H. argophyllus L., H. debilis L. and H. tuberosus L. The corn could be subdivided in 7 groups, indicated in the past as subspecies or botanical varieties: Zea mays indentata (dent corn), Zea mays indurata (flint corn), Zea mays amilacea (flour or soft corn), Zea mays saccharata (sweet corn), Zea mays everta (pop corn), Zea mays ceratina (waxy corn), Zea mays tunicata (pod corn). The dent corn type is the most cultivated in the world, followed by the flour corn which is preferred for human nutrition and for poultry. All the other types of a modest importance are used in alimentation industry (pop corn) or the chemical industry or ornamental plants (Eagles and Lothrop, 1994; Iltis, 2000; Orr et al., 2002; Wilkes, 1979). Peanut or groundnut (Arachis hypogaea L.) is an annual herbaceous plant, member of the family Papilionaceae native to South America, Mexico and Central America (Stephens, 1994). The soya bean (Glycine max) is a species of legume native to East Asia (Lackey, 1977). It is a papilionoid legume (family Fabaceae, subfamily Faboideae). The subtribe to which soybean belongs consists of 16 genera, none of which, except for soybean (Glycine) and kudzu (Pueraria), are commonly known outside of botanical science. Rapeseed cultivars are derived from the Brassica genus and the Cruciferae family (Musil, 1950). In this family, numerous species have been interbred to form a number of sub-species. The common commercial name rapeseed or oilseed rape widely used in Europe includes seeds of oilseed turnip rape (B. campestris1 synonymous with B. rapa), oilseed swede rape (B. napus) and mustards (B. juncea, B. nigra, B. hirta synonymous with Sinapis alba). Table 1.1. Botanical classification and description of major oil producing crops

Argan Corn Olive Peanut Rapeseed Rice Soybean Sunflower

Kingdom Plantae Plantae Plantae Plantae Plantae Plantae Plantae Plantae

Division Angiospermae Angiospermae Magnoliophyta Magnoliophyta Magnoliophyta Magnoliophyta Magnoliophyta Angiospermae

Class Magnoliopsida Liliopsida Magnoliopsida Magnoliopsida Magnoliopsida Liliopsida Magnoliopsida Magnoliopsida

Order Ebenales Cyperales Lamiales Fabales Brassicales Poales Fabales Asterales

Family Sapotaceae Poaceae Oleaceae Fabaceae Cruciferae Poaceae Fabaceae Asteraceae

Genus Argania Zea Olea Arachis Brassica Oryza Faboideae Helianthus

Species A. spinosa Skeels Zea mays O. europaea Arachis

hypogaea

B. nigra, B. oleracea B. rapa

O. glaberrima O. sativa Glycine max H. annuus.

Domesticated rice comprises two species of food crops in the Poaceae ("true grass") family, Oryza sativa and Oryza glaberrima. These plants are native to tropical and subtropical

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southern Asia and southeastern Africa (IRRI Knowledge Bank, 2006). The term "wild rice" can refer to the wild species of Oryza, but conventionally refers to species of the related genus Zizania, both wild and domesticated. Oryza sativa is a perennial, cultivated as an annual of the Poaceae (Gramineae) family. The Olive (Olea europaea) is a species of tree in the Oleaceae family, probably native to coastal areas of the Mediterranean region, from Lebanon and the maritime parts of Asia Minor and northern Iran at the south end of the Caspian Sea. The sapotaceae family, to which the argan (Argania spinosa) tree belongs, is a large family that includes almost 60 tropical genus. Surprizingly, Argania spinosa is the only species of the family sapotaceae remaining in the subtropical zone (Loumou and Giourga, 2003). 1.1.2 Area of origin The argan tree is almost exclusively endemically growing in South Morocco (Morton and Voss, 1987) in a region that extends South East, from Essaouira to the Souss plain. In addition, smaller and isolated forests grow near Benissnassen and Oued Grou, close of Rabat. A tiny argan forest can also be found in Tindouf (Algeria) but because of its reduced size, it can hardly be compared to the Moroccan forest. In contrast to the well defined origin of argan tree, the origin of the olive tree is still unknown. It is said to have appeared in prehistoric times, before humankind, and to have originated in southern Asia Minor where there are now abundant forests of wild olive trees. There are two cultivated species of rice. O. sativa, the Asian rice, is grown worldwide. O. glaberrima, the African rice, is grown on a limited scale in West Africa (Chang, 1976). The genus Oryza to which cultivated rice belongs, probably originated at least 130 million years ago and spread as a wild grass in Gondwanaland the super continent that eventually broke up and drifted apart to become Asia, Africa, the Americas, Australia and Antarctica (Chang, 1976). The origins of the soybean plant are obscure, but many botanists believe it to have derived from glycine ussuriensis, a legume native to central China (Anonymous, 2008). Sunflower (Helianthus annuus) is one of the few crop species that originated in West America. Peanut is believed to originate from Bolivia and North West Argentina. The origin of maize still continues to intrigue botanists after years of study and debate. However, archaeology and the language references show the presence of the maize in America already from the prehistoric age (Wilkes, 1979; Galinat, 1992; Eagles and Lothrop, 1994). Amongst the major rapeseed species, B. napus and B. Rapa are endemic to North America and Europe while B. juncea and B. rapa are mainly grown in India and the far East (Table 1.2; Fig. 1.1). Table 1.2. Area of origin of major oil producing crops

Argan Corn Olive Peanut Rapeseed Rice Soybean Sunflower

Area of origin

Southwestern Morocco

South America (Mexico?)

Mediterranean Basin

Bolivia; North West Argentina

Europe; North America; Asia

Asia; West Africa Central China

West America

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Figure 1.1. a. Argan (Argania spinosa); b. Corn (Zea mays); c. Olive (Olea europaea); d. Peanut (Arachis hypogaea L.); e. Rapeseed (Brassica napus); f. Rice (Oryza Sativa); g. Soybean (Glycine max); h. Sunflower (Helianthus annuus) 1.1.3 Domestication Among the oil producing fruit trees, the argan tree as long been considered as very difficult to grow. Early attempts, and failures, to cultivate it in various arid parts of the world have reinforced this idea (Morton and Voss, 1987). In addition the argan grove dwellers have long considered the presence of argan trees as natural and have not thought about it preservation or domestication. Consequently researchers in Morocco have decided to tackle the problem of the multiplication of the argan tree some years ago. Cultivation of the olive

a b c

d e f

g h

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tree on the other hand considerably developed along the Iranian plateau and the Mediterranean coasts of Syria and Israel, spreading from there to the island of Cyprus and towards Anatolia or from the island of Crete towards Egypt (Besnard and Bervillé, 2000). Probably in the 16th century BC the Phoenicians started disseminating the olive throughout the Greek isles, later introducing it to the Greek mainland between the 14th and 12th centuries BC where its cultivation increased and gained great importance in the 4th century BC (when Solon issued decrees regulating olive planting). Some historians, however, claim that archaeological discoveries prove that wild olive trees existed before civilization in Crete and that their cultivation began during the Paleolithic and Neolithic periods, i.e. between 3500 and 5000 BC. From Crete the trees spread to Egypt (2000 BC), and thereafter to the islands, Asia Minor, Israel and mainland Greece (1800 BC). In more modern times, the olive tree has continued to spread outside the Mediterranean and today is farmed in places as far removed from its origins as southern Africa, Australia, Japan and China. Many important field crops were brought into cultivation by the native Americans over 3000 years ago among them are the sunflower, corn, and peanut. The sunflower was much revered by the Aztecs, and in their temples of the sun as was evident the early Spanish conquerors findings of numerous representations of the sunflower wrought in pure gold in the Aztecs temples. During the 18th Century, the use of sunflower oil became very popular in Europe, particularly with members of the Russian Orthodox Church because sunflower oil was one of the few oils that was not prohibited during Lent. Before 1966, sunflower acreage in the USA was devoted primarily to non-oilseed varieties (Putt, 1978). Increased commercial interest in the sunflower increased as a consequent of the discovery of the male-sterile and restorer gene system that made hybrids feasible. Now cultivated around the world for food, oil and fuel it is one of the most familiar plants on the earth. Peanuts on the other hand were used by the Incas when burying their dead to give them food to take along to the hereafter. The domesticated peanut has two sets of chromosomes from two different species. The wild ancestors of the peanut were thought to be A. duranensis and A. ipaensis, a view recently confirmed by direct comparison of the peanut's chromosomes with those of several putative ancestors. This domestication might have taken place in Argentina or Bolivia, where the wildest strains grow today. These crops were probably first introduced to Europe through Spain, and spread through Europe as a curiosity. Then from Europe it diffuses in Africa and Asia and the pacific islands. Peanuts (originally from South America) were introduced to the United States from Africa having been carried on the slave ships. Peanut consumption increased enormously as an effect of the civil war in 1860, since both the Northern and the Southern army considered the peanut as a valuable food product (Robinson, 1984, and Pendleton, 1977). Domestication of rice, wild rice and soyabean begun in Asia. Wild rice domestication probably started about 9000 years ago. Development of annuals cultivation at different elevations in East India, Northern Southeast Asia and West China was enhanced by alternating periods of drought and variations in temperature during Neothermal age about 10 000–15 000 years ago (Whyte, 1972). The domestication of the soya bean began in China, at least 3.000 years ago, when farmers in the eastern half of northern China started planting the black or brown seeds of a wild recumbent vine. Cultivation of the soya bean spread very slowly from China to Korea, Japan, and Southeast Asia. Soya beans first appeared in Japan in the eighth century AD but did not appear in Europe until almost 1,000 years later (Anonymous. 2008b).

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1.1.4 Source of oil In olives the oil phase is contained in the pulp (mesocarp) and also in the stone (endocarp and kernal) of the drupe, therefore olive oil is generally extracted from the whole fruit. It is also possible to extract the oil phase just from the pulp of the drupe in order to obtain olive oils with very high qualitative characteristics. The argan oil similarly originates from the fruit. However, each fruit has to be cracked open to remove the kernels prior to the oil extraction. Peanut, rapeseed and sunflower oils are extracted from the seeds whilst the corn oil is produced from the kernel’s germ and endosperm. The soybean hulls needs to be removed because they absorb oil and give a lower yield (Table 1.3).

Table 1.3. Source of oil

Argan Corn Olive Peanut Rapeseed Rice Soybean Sunflower

Source of oil Seed kernels kernel Fruit flesh Groundnut Rapeseed Bran Soybean Seeds

1.1.5 Traditional cultivation and folklore Probably the first cultivation of the olive tree worldwide took place in Greece, and more specific in Crete (Besnard and Bervillé, 2000; Hesse et al., 2000; Loumou and Giourga, 2003; Zohary and Piegal-Roy, 1975). This happened about 3500 BC in the Early Minoan times. After 2000 BC the cultivation of the olive tree in Crete was very intense and systematic playing the most important role on the island's economy. From Crete started the first export of the olive oil not only in mainland Greece but in Northern Africa and Asia Minor as well. The olive tree was a symbol in ancient Greece and the olive oil was used not only for its valuable nutritional quality but also for medical purposes. Between the 7th and 3rd centuries BC ancient philosophers, physicians and historians undertook its botanical classifications and referred to the curative properties of olive oil. This knowledge is being "rediscovered" today as modern scientists research and find news why the Mediterranean diet is so healthy. The symbolic meaning of the olive tree as well as the exceptional value of the olive oil is visible in overall sectors of the ancient Greece's life. A number of facts show to us the relationship between the olive tree and its product with some social activities. The leafy branches of the olive tree, olive leaf as a symbol of abundance, glory and peace, were used to crown the victors of friendly games and bloody war. As emblems of benediction and purification, they were also ritually offered to deities and powerful figures: some were even found in Tutankhamen's tomb. Olive oil has long been considered sacred; it was used to anoint kings and athletes in ancient Greece. It was burnt in the sacred lamps of temples as well as being the "eternal flame" of the original Olympic Games. Victors in these games were crowned with its leaves. Today it is still used in many religious ceremonies. According to Greek mythology the Olive tree, her gift to the people of Attica, won Athena the patronage of the city of Athens over. The use of oil is found in many religions and cultures. During baptism in the Christian church, olive oil is used for anointment. At the Chrism mass olive oil blessed by the bishop, "chrism", is used in the ceremony. Like the grape, the Christian missionaries brought the olive tree with them to California for food but also for ceremonial use. Olive oil was used to anoint the early kings of the Greeks and Jews. Olive oil has also been used to anoint the dead in

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many cultures. In the past several hundred years the olive has spread to North and South America, Japan, New Zealand and Australia. Sunflower (Helianthus annuus L.) is one of the few crop species that originated in West America. Sunflower was brought into cultivation over three thousand years ago by Native Americans, who selected the largest seeds to plant and thus produced a much larger seed than the wild type (Erichsen-Brown, 1979; Densmore, 1974). The American Indians used sunflower as a foodstuff before the cultivation of corn; they ate the seeds raw, roasted, boiled, made them into gravy, gruel and breads. In addition to eating the seeds they produced oil from them, although the wild plants may have been preferred to the cultivated ones for oil production. Various tribes ascribed various medical and magical powers to the plant and it played a role in ceremony in some tribes as well; they uses it to treat everything from warts and snake bites to heatstroke and coughs. Dye was extracted from hulls and petals, while face paint was made from dried petals mixed with pollen. Dried stalks were utilized for building material. They also use it as a hunting calendar: when sunflower was tall and in bloom, the buffalo were fat and the meat good (Erichsen-Brown, 1979; Densmore, 1974). In Peru, this flower was much revered by the Aztecs, and in their temples of the Sun, the priestesses were crowned with Sunflowers and carried them in their hands. The early Spanish conquerors found in these temples numerous representations of the Sunflower wrought in pure gold. The first Europeans observed sunflower cultivated in many places from southern Canada to Mexico. Sunflower was probably first introduced to Europe through Spain, and spread through Europe as a curiosity. Early English and French explorers, finding sunflower in common use by the American Indians, also introduced it to their respective lands. It spread along the trade routes to Italy, Egypt, Afghanistan, India, China, and Russia. Introduced into Europe in the sixteenth century, it was the Russians who first cultivated it on a large scale; it was not used as an edible crop again until it reached Russia. In Russia, the Holy Orthodox Church forbade the use of many foods, including many rich in oil, during Lent and Advent. The Russians eagerly accepted the sunflower as an oil source that could be eaten without breaking the laws of the church. It also was grown as an ensilage crop for animal fodder in the late 1800s and early 1900s. Selection for high oil in Russia began in 1860 and was largely responsible for increasing oil content from 28% to almost 50%. Sunflower developed as a premier oilseed crop in Russia and has found wide acceptance throughout Europe. The high-oil lines from Russia were reintroduced into the U.S. after World War II, which rekindled interest in the crop. Before 1966, sunflower acreage in the USA was devoted primarily to non-oilseed varieties (Putt, 1978). However, it was the discovery of the male-sterile and restorer gene system that made hybrids feasible and increased commercial interest in the crop. Now cultivated around the world for food, oil and fuel it is one of the most familiar plants on the earth. To pin-point exactly when mankind first realised that the rice plant was a food source and began its cultivation is impossible. Many historians believe that rice was grown as far back as 5000 years BC. According to the Encyclopedia Brittanica (Encyclopaedia Britannica, 2008) the origin of rice culture has been traced to India in about 3000 BC. Rice culture gradually spread westward and was introduced to southern Europe in medieval times. Wild rice appeared in the Belan and Ganges valley regions of northern India as early as 4530 BC and 5440 BC respectively. Agricultural activity during the second millennium BC included rice cultivation in the Kashmir and mature Harrappan -Pakistan regions. (Anonymous, 2000) Mixed farming was the basis of Indus valley economy. Farmers planted their crops in integrated fields. Rice, grown on the west coast, was cultivated in the Indus valley (Kahn, 2005). However, the first recorded mention of rice originates from China in 2800 BC. The Chinese emperor, Shen Nung, realised the importance of rice to his people and to honour the grain he established annual rice ceremonies to be held at sowing time, with the emperor

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scattering the first seeds. Most likely, similar ceremonies took place throughout china with local dignitaries deputising for the emperor. Nowadays, the Chinese celebrate rice by specifically dedicating one of the days in the New Year festivities to it. Although we cannot identify China, India or Thailand as being the home of the rice plant we can be more certain of how rice was introduced to Europe and the Americas. For that we have to thank the traveller, whether explorer, soldier, merchant or pilgrim, who took with them the seeds of the crops that grew in their home or foreign lands. Not all seeds could be transplanted successfully, however. Great Britain has never been able to cultivate rice due to its adverse climatic conditions. The rice plant requires immense quantities of rainfall in its early days, followed by a long and uninterrupted season of hot dry weather. For this reason, farmers must find ways to either flood the fields or drain the water from them at crucial periods. In the West, parts of America and certain regions of Europe, such as Italy and Spain, are able to provide the correct climate thereby giving rise to a thriving rice industry. Some historians believe that rice travelled to America in 1694 in a British ship bound for Madagascar. Some years later, the British unfortunately blotted their copybook in relation to the rice industry they had probably initiated. During the American Revolution, they occupied the Charleston area and sent home the entire quantity of harvested rice, failing to leave any seed for the following year's crop. The American rice industry survived this set-back and cultivation continued, thanks to President Thomas Jefferson, who broke an Italian law by smuggling rice seed out of Italy during a diplomatic mission in the late 18th Century. The rice industry then transplanted itself from the Carolinas to the southern states surrounding the Mississippi basin. Rice is fundamentally important to various cultures that it is often directly associated with prosperity and much folklore and legend surrounds the grain. In many cultures and societies, rice is integrated directly into religious belief. In Japan rice enjoys the patronage of its own god, Inari, and in Indonesia its own goddess, the Dewie Srie. Rice is also linked to fertility and for this reason the custom of throwing rice at newly wedded couples exists. In India, rice is always the first food offered by a new bride to her husband, to ensure fertility in the marriage, and children are given rice as their first solid food. And, according to Louisiana folklore, the test of a true Cajun is whether he can calculate the precise quantity of gravy needed to accompany a crop of rice growing in a field. How easy to see that from its early beginnings to the present day, rice continues to play an integral role in sustaining both the world's appetites and cultural traditions. Hundreds of years ago, Asians and Europeans used rapeseed oil in lamps. Species from the genus Brassica were cultivated in ancient Rome and also in Gallia (Collumella and Palladius in Fussel, 1955). Seeds of these species were found in old German graves and Swiss constructions from the Bronze age (Witmack, 1904, Neuweiller, 1905, Schiemann, 1932). Term „Olisatum“ in Carl the Greate´s instructions related to German “Oelsaat” was very probably the name for Brassica species with oleaginous seeds. As time progressed, people employed it as a cooking oil and added it to foods. Its use was limited until the development of steam power, when machinists found rapeseed oil clung to water or steam-washed metal surfaces better than other lubricants. World War I saw high demand for the oil as a lubricant for the rapidly increasing number of steam engines in naval and merchant ships. When the war blocked European and Asian sources of rapeseed oil, a critical shortage developed and Canada began to expand its limited rapeseed production. After the war, demand declined sharply and farmers began to look for other uses for the plant and its products. Edible rapeseed oil extracts were first put on the market in 1956–1957, but these suffered from several unacceptable characteristics. Rapeseed oil had a distinctive taste and a disagreeable greenish colour due to the presence of chlorophyll. It also contained a high concentration of erucic acid. Experiments on animals have pointed to the possibility that erucic acid, consumed in large quantities, may cause heart damage, though Indian researchers have published

10

findings that call into question these conclusions and the implication that the consumption of mustard or rapeseed oil is dangerous. Feed meal from the rapeseed plant was not particularly appealing to livestock, due to high levels of sharp-tasting compounds called glucosinolates. Plant breeders in Canada, where rapeseed had been grown (mainly in Saskatchewan) since 1936, worked to improve the quality of the plant. In 1968 Dr Baldur Stefansson of the University of Manitoba used selective breeding to develop a variety of rapeseed low in erucic acid. In 1974 another variety was produced low in both erucic acid and glucosinolates; it was named Canola, from Canadian oil, low acid. A variety developed in 1998 is considered to be the most disease- and drought-resistant variety of Canola to date. This and other recent varieties have been produced by gene splicing techniques. Many pre-Columbian cultures, such as the Moche, depicted peanuts in their art (Berrin et al., 1997). Evidence demonstrates that peanuts were domesticated in prehistoric times in Peru. Archaeologists have thus far dated the oldest specimens to about 7,600 years before the present (Dillehay, 2006). Cultivation spread as far as Mesoamerica where the Spanish conquistadors found the tlalcacahuatl (Nahuatl = "cacao", whence Mexican Spanish, cacahuate and French, cacahuète) being offered for sale in the marketplace of Tenochtitlan (Mexico City). The plant was later spread worldwide by European traders. Cultivation in the English colonies of North America was popularized by African Americans, who brought the Kikongo word "nguba", which in the United States became "goober". When Africans were brought to North America as slaves, peanuts came with them. Slaves planted peanuts throughout the southern United States (the word goober comes from the Congo name for peanuts - nguba). In the 1700's, peanuts, then called groundnuts or ground peas, were studied by botanists and regarded as an excellent food for pigs. Records show that peanuts were grown commercially in South Carolina around 1800 and used for oil, food and a substitute for cocoa. However, until 1900 peanuts were not extensively grown, partially because they were regarded as food for the poor, and because growing and harvesting were slow and difficult until labor-saving equipment was invented around the turn of the century. Around 1900, equipment was invented for planting, cultivating, harvesting and picking peanuts from the plants, and for shelling and cleaning the kernels. With these mechanical aids, peanuts rapidly came into demand for oil, roasted and salted nuts, peanut butter and candy. Peanut production rose rapidly during and after World Wars I and II as a result of the peanut's popularity with Allied forces, and as a result of the post-war baby boom. Soybeans originate from China. In 2853 BC, Emperor Sheng-Nung of China named five sacred plants: soybeans, rice, wheat, barley, and millet (Anonymous, 2008c). Soybean plants were domesticated between 17th and 11th century BC in the eastern half of China where they were cultivated into a food crop. From about the first century AC to the Age of Discovery (15-16th century), soybeans were introduced into several countries such as Japan, Indonesia, the Philippines, Vietnam, Thailand, Malaysia, Burma, Nepal and India. The spread of the soybean was due to the establishment of sea and land trade routes. The earliest Japanese reference to the soybean is in the classic Kojiki (Records of Ancient Matters) which was completed in 712 AC. The first soybeans arrived in America in the early 1800's as ballast aboard a ship! It wasn't until 1879 that a few brave farmers began to plant soybeans as forage for their livestock. The plants flourished in the hot, humid summer weather characteristic of the north-eastern North Carolina. Around 1900 the US Department of Agriculture was conducting tests on soybeans and encouraging farmers to plant them as animal feed. In 1904, the famous American chemist, G. W. Carver discovered that soybeans are a valuable source of protein and oil. He encouraged farmers to rotate their crops with soybeans. To the surprise of farmers, this produced a better crop. In 1929 Morse spent two years researching soybeans in China, where he gathered more that 10,000 soybean varieties. It wasn't until the 1940's that farming of soybeans really took off in America. Although soybeans are native to southeast

11

Asia, 55 percent of production is in the United States. The US produced 75 million metric tons of soybeans in 2000 of which more than one-third was exported. Other leading producers of soybeans are Argentina, Brazil, China and India. Much of the US production is either fed to animals or exported, though US consumption of soy by people has been increasing. Brazil is expected to become the world's biggest soybean exporter in 2004, displacing the United States from the top seat. The Argania Spinosa is a small-leaved spiny tree that has been in existence for millions of years (Morton and Voss, 1987). It is a relic of the Tertiary Age. For centuries the Berber women have been extracting oil from its fruits. The argan trees grow almost exclusively in the southwest region of Morocco. It was first reported by the explorer Leo Africanus in 1510. An early specimen was taken to Amsterdam and then cultivated by Lady Beaufort at Badminton c1711. Now only 860,000 hectares remain in S.W. Morocco and these are declining at a rate of 50,000h per year. Measures are being put in place to protect this rare and endangered species and in 1999 the argan was listed as a UNESCO Biosphere Heritage. But it is the amazing sight of acrobatic goats browsing in the canopies of the trees that demonstrates the most extraordinary way this crop is harvested. The goatherd watches punctiliously and when he thinks the tree has been nibbled enough will whistle to his animals who then obediently leap from the branches. In the evening the flock will follow him home to the fold where these ruminants, only able to digest the flesh from the drupe, will quietly regurgitate and spit out the stone before settling down to chew the cud. In the morning the women gather every nut from the ground. Using only the ancestral method of “hammer and anvil” the Berber women crack each individual nut between two stones and remove the two or three “almonds” inside. Only those that are to made into arganti, the edible oil, are spread out on a clay plate and lightly toasted over a charcoal fire. Those to be used for cosmetics are left raw (Table 1.4).

Table 1.4. Traditional cultivation of oil crops by Country

Argan Corn Olive Peanut Rapeseed Rice Soybean Sunflower

Countries South-Western Morocco

North and South America; Asia; Europe; South Africa

Mediterranean countries

108 Countries; developing countries excluding Europe

Mainly India, China and Canada

All Continents

USA and Asia

Russian Federation, Ukraine, Argentina, China, France, United States of America, eastern Europe and South Africa

1.2. Current Situation of Growing in the World 1.2.1 Countries and production The world of oilseeds, meals, and oils has been evolving, both by variety and country. By variety, soybeans have dominated the production, consumption, and trade of oilseeds and meals. In the past five years, about 56 percent of oilseeds produced and 80 percent of oilseeds

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traded worldwide have been soybeans. Rapeseed and sunflower seed are other important varieties in the oilseed and meal markets (Mattson et al., 2004). By country, the United States has been the largest player in the complex, mainly due to its dominant position in soybeans and soybean meals. However, this dominance of the United States has been challenged. Specifically, the growth of Brazil and Argentina in the production of soybeans and soybean meal has eroded the market share of the United States in recent years. In the oil market, with the exception of traditional competition from Canadian rapeseed oil, Malaysia and Indonesia have been aggressively marketing their palm oil, especially in Asia, resulting in intense competition for the soybean oil exported from the United States. The two major exporting countries, Malaysia and Indonesia, rely on the international market for their palm oil industry, so it is expected that the competition will continue. Major sunflower producing countries are the Russian Federation, Ukraine, Argentina, China, France, United States of America, eastern Europe and South Africa. These seven countries or areas of the world produce about 86% of the world's production of both oilseed and non-oilseed sunflower. In the period 2001-2005 the Russian federation produced in average about 17.8 % of the world's production followed by Ukraine (13.9%) and Argentina (13.7%). In the last few years China has risen to fourth place, with 1710 thousands of tonnes produced on average over the last five years. As for soybean, according to FAO data of 2005, total land area under soybean in the world was 95.2 million ha and total production was 212.6 million tonnes. The three major soybean- producing countries were USA (29 million ha), Brazil (23 million ha), and Argentina (14 million ha). In terms of total production, USA produced 83 million tones, followed by Brazil (51 million tonnes) and Argentina (38 million tons). China is the world's fourth-largest producer of soybeans. The major Chinese soybean growing regions are in the northeast part of China. Yet, rapid growth of China's economy has spurred food consumption, turning the country into the world's leading soybean importer. Changes in China's agricultural and trade policies have greatly influenced world oilseed markets. China's WTO accession will further reduce import tariffs and quantitative restrictions to its oilseed market. The major Indian soybean growing region is in the central state of Madhya Pradesh. Indian production of soybeans and other traditionally grown oilseeds—such as peanuts, rapeseed, and cottonseed—has increased in the last decade, although yields are among the world's poorest. India imposes prohibitive barriers on oilseed imports, so its domestic crushing is limited to the oilseeds that can be produced within the country. Domestically produced oilseeds are highly valued for the vegetable oil, and India is now among the world's largest vegetable oil importers. India is a smaller (but growing) consumer of soybean meal, and exports its surplus to other Asian countries. With respect to Africa, soybean was grown on an average of 1.16 million hectares with an average production of 1.26 million tonnes in 2005. African countries with the largest area of production were Nigeria (601 000 ha), South Africa (150 000 ha), Uganda (144 000 ha), Malawi (68 000 ha), and Zimbabwe (61 000 ha). Other countries with sizeable areas are Rwanda (42 160 ha), DRC (30 000 ha), and Zambia (15 000 ha). Soybean is also grown in small scale in more than 10 other African countries. Maize is widely cultivated throughout the world, and a greater weight of maize is produced each year than any other grain. While the United States produces almost half of the world's harvest, other top producing countries are as widespread as China, Brazil, France, Indonesia, India and South Africa (Mattson, 2004). Worldwide production was over 600 million tonnes in 2003 — just slightly more than rice or wheat. In 2004, close to 33 million hectares of maize were planted worldwide, with a production value of more than $23 billion. The major producers/exporters of peanuts are the United States, Argentina, Sudan, Senegal, and Brazil. These five countries account for 71% of total world exports. In recent years, the United States has been the leading exporter of peanuts. India, China and Canada have the largest land area under rapeseed production and are the largest producers of rapeseed on a

13

tonnage basis despite the fact that their yields are much lower (by up to 50%) than yields of some of the largest rapeseed producing countries in Europe. At least 114 countries grow rice and more than 50 have an annual production of 100,000 tons or more. Asian farmers produce about 90% of the total, with two countries, China and India, growing more than half the total crop. For most rice-producing countries where annual production exceeds 1 million ton, rice is the staple food. In Bangladesh, Cambodia, Indonesia, Laos, Myanmar, Thailand, and Vietnam, rice provides 50-80% of the total calories consumed. The olive oil production is concentrated in the Mediterranean countries Spain, Italy, Greece, Syrian, Turkey, Morocco and Algeria. These seven countries alone account for 90 % of the world production. Since the mid ‘90s, Spain has consistently been the largest producer of olive oil, with a share of world production in volume in the most recent years varying between 46 (in 00/01) and 32 (in 04/05) per cent.

In 04/05 Italy and Greece accounted for 28 and 13 per cent of world

production, respectively, the EU-25 for 76 per cent; the main non-EU producers were, in the order, Syria (7 per cent), Tunisia (5), Turkey (5) and Morocco (3). Non-EU, non-Mediterranean countries accounted for 1 per cent only of world production (Mattson, 2004). 1.2.2 Exportation/importation Oilseeds are among the most important agricultural commodities worldwide. Oilseed meal and edible oil can be obtained from the crushing of oilseeds. Although in some Asian countries, as much as 40 percent of soybean consumption is in making soybean related products such as Tofu and milk, most demand for oilseeds still comes from the demand for meal and oil. In turn, demand for meal originates from the protein feed industry. The major types of oilseeds produced throughout the world include soybeans, rapeseed, cottonseed, and peanuts. However, soybeans account for over half of all oilseed production in the world, and about 85 percent of the world’s supply of soybeans is crushed to produce soybean meal and soybean oil. Processed soybeans are the largest source of protein feed and vegetable oil in the world. Although the United States is the leading producer and exporter of soybeans, but production in Brazil and Argentina has been increasing rapidly in recent years. USDA estimates for 2003/04, in fact, show that Brazilian soybean exports will surpass U.S. exports for the first time. Meal production and exports are dominated by soybean meal, followed by rapeseed meal. The United States is the leading producer and consumer of soybean meal and oil, while Argentina and Brazil are the top exporters. China is the leading producer of rapeseed, followed by Canada, India, Germany, and France. The leading exporter of sunflower seeds has changed over time. The United States was the leading exporter in the late 1970s and early 1980s. U.S. exports peaked at 1.8 million metric tons in 1979/80, but as production declined in the mid 1980s, exports dropped even more substantially. From 1998/99 to 2002/03, U.S. exports averaged 209 thousand metric tons, making the United States the fifth largest exporter. At the same time that U.S. exports decreased, exports from France rose dramatically. Sunflower seed production in France increased significantly in the 1980s, and French exports increased from 14 thousand metric tons in 1977/78 to a height of 1.5 million metric tons in 1987/88. French exports averaged 1.2 million metric tons per year from 1986/87 to 1991/92, but have since declined, averaging 452 thousand metric tons from 1998/99 to 2002/03. Exports from Russia and Ukraine increased considerably in the early 1990s, and throughout much of the mid and late 1990s, these two countries were the leading exporters of sunflower seed. However, exports dropped in 2001 and 2002. Among the remaining countries, Argentina is also a major exporter. The Netherlands is the leading

14

importer of sunflower seed, followed by Spain, Turkey, Germany, Portugal, and Italy. Imports by most of these countries have declined somewhat since 1999. The world trade market for peanuts may be considered a residual market, in the sense that only a small proportion of the world production is devoted to exports and imports, and most of the production is domestically utilized (Revoredo and Stanley, 2002). Thus, the average share of world peanut production exported since the 1970s has been about 5 percent. Despite its approximately constant share, the total volume of exports has been growing since the late 1980s, although at modest rates, increasing from an average for the 1976-80 period of 1.1 million metric tons to 1.5 million metric tons during 1996-2000. Although with some variability, since the late 1980s, the three major exporters of peanuts (Argentina, China and the United States) have comprised about 60 percent of the total world trade. In addition, it is important to note that while the composition of the main six exporters has changed over time, since the early 1990s (besides the three major exporters) India, Netherlands (a re-exporter), and Vietnam have been part of this group. It should also be noted that while during the 1970s African countries were among the major exporters, as the peanut trade shifted from a crush for oil to a market of peanuts for food purposes, their presence in the export market tended to decline in absolute and relative terms. In addition, their position was affected by the presence of aflatoxin in their production, which impeded them in competing in the edible peanut market and by the substitution of peanut oil by other oilseed oils namely soybean oil (see Carley et al., 1995). Olive oil is consumed worldwide, but it is mainly cultivated in the Mediterranean area. The trade of olives therefore plays an important role. Approximately 40 % of the total olive oil production with an equivalent value of more than 3 billion Dollars are imported/exported (Carlo et al., 2004; Giovanni et al., 2005). The Community is the reference importer and exporter on the world olive oil market, and is traditionally a net exporter. The countries of Euro-Med area as a whole represent the leading exporters (with 1,988 million of current dollars on average in 2003/04) and importer (with 2,257 million current dollars on average in 2003/04) on the olive oil world market. Spain and Italy are not only the main world producers of olive oil, but the largest exporters as well (with 1,292,392 thousand current dollars on average in 2003/04); the third largest exporter is Tunisia (328,659 thousand current dollars), which is the fifth largest producer.

These three countries alone accounted in 2004 for 89 per

cent of olive oil exports in value. The following countries are Greece (213,637 thousand current dollars), Turkey (147,504 thousand current dollars), Portugal (75,028 thousand current dollars) and Syria (31,020 thousand current dollars). The other countries show an export market share lower than 1%. Olive oil imports are less concentrated by country than exports. Italy, the second largest exporter of olive oil, is at the same time the largest importer, with 40 per cent of world imports in value in 2004; the other main importing countries are the US (15 per cent of world imports), France (6), Spain (6), UK (4), Germany (4), Portugal (4), Japan (3) and Australia (2) (Table 1.5). FAO’s estimate of world rice trade in 2007 has been revised downwards by around 175 000 tonnes since last September to 29.9 million tonnes, in milled rice equivalent (FAO, 2007). This would represent a 2.4 percent increase from the 29.2 million tonnes traded in 2006. The FAO forecast of rice trade in 2008 has been also lowered to 30.3 million tonnes, which would be 1 percent larger than in 2007. In Asia, imports to Bangladesh, China, Iraq, DPR Korea, Nepal and Turkey are forecast to rise, while they may fall in Indonesia, Islamic Republic of Iran, the Philippines and Sri Lanka. Shipments to African countries are forecast to rebound in 2008, sustained by larger deliveries to Cote d’Ivoire and Nigeria, while those to Latin America and the Caribbean may fall somewhat, given expectations of smaller purchases by Brazil and Colombia. As for exports, Argentina, Brazil, China, Guyana, Myanmar, Pakistan, the United States, Uruguay and Viet Nam are expected to be in a position to sell

15

more, as opposed to Egypt and India, where government restrictions may depress sales. Deliveries from Thailand, the leading exporter, may also fall, as supply availability from public inventories dwindled. Table 1.5. Major crops exporting and importing countries (1000 $) (FAOSTAT FAO Statistics Division 2006)

OILS Corn Olive (virgin) Groundnut Rapeseed Rice bran Soybean Sunflower

Export

USA Spain Argentina Canada Thailand Argentina Ukraine 246163 2300510 66468 756512 13427 2789292 922360 China Italy Senegal Germany S. Korea Brazil Argentina 76633 2169922 53227 368277 13 1228638 699233

Belgium Tunisia Belgium France Belgium USA Russia 55944 937854 27876 339282 0 360186 385315 Tunisia Greece China Netherlands Brazil Netherlands Netherlands 48395 486087 15464 289904 0 352013 285154 Oman Turkey France Belgium India Germany France 32729 238876 14937 207003 0 199596 266471

Import

Tunisia Italy USA Germany Japan China Iraq 88543 2311361 53529 997690 14621 802239 270000 Saudia USA France USA S. Korea India France 86155 981174 45110 438332 2609 685292 264622 Turkey Spain Italy Netherlands Thailand Iran Germany 85574 493292 42952 418185 211 390628 240878 UAE France Belgium Italy Belgium Morocco Turkey 57207 476190 35393 238228 0 216446 238430 Greece Portugal Germany France Brazil Venezuela Netherlands 29663 274139 18908 163247 0 193307 219312

Hard trade data on argan oil is difficult to obtain, for several reasons. Argan oil is a relatively new international product and the suppliers are rural Moroccans, not large corporations who are able to keep records. 1.3. Environment Requirements 1.3.1 Temperature Canola is adapted to wide environmental conditions, but particularly to the cool extremes of the temperate zones. Minimum temperatures for growth have been reported to be

16

near 0°C. The crop will germinate and emerge with soil temperatures at 5°C but the optimum is 10°C. Winter annual varieties are grown where adequate snow covers or mild winters are common. Planting date has a dramatic effect on survival however. Soybeans need a slightly higher minimum soil temperature of 12.8 to 15.6°C (55 to 60°F) to germinate (Berglund et al., 2007) Soybean germination rates increase at warmer temperatures. However, soybean plants can withstand temperatures as low as -2.8°C (27°F) for a short period of time, while corn experiences tissue damage at -2°C (28.4°F). Temperature is the major limiting factor for peanut yield since a minimum of 3,000 growing degree-days (with a base of 10°C) is required for proper growth and development (Grau et al., 1994). The elevate polymorphism provides to the maize Zea mays species a high adapting ability to the environment conditions. The optimal temperature is between 24 and 30°C and in function to the growing vegetative phase. The plant manifest during the growing cycle increasing requirements at the beginning then decreasing requirements. The optimal temperature values are: 22-23°C for the stem extension phase, 24-25°C for the flowering phase and 23-24°C for the grain filling phase. Temperatures over 32-33°C are harmful, under 10°C the development of the plant is blocked. The maize is a plant with high light requirements, in term of photoperiod can be defined as a short day specie (Crop and soil management, 2007/2008). Rice is usually grown with a minimum of 12 hr day and high intensity lighting. Day temperatures of 28-32°C are standard, with night temperatures about 3 degrees lower. Temperature may be critical to optimize growth for specific cultivars or wild species, and this needs to be determined for each variety or species, depending on its zone of adaptation (Horie, 1987). Temperature is the major limiting factor for peanut yield since a minimum of 3,000 growing degree-days (with a base of 10°C) is required for proper growth and development (Robinson, 1984). A peanut crop will not reach optimum maturity for a marketable yield to justify commercial production in areas with fewer heat units during the growing season. Little if any growth and development occur at temperatures below 13°C (Emery et al., 1969) and 30°C is reported to be optimal (Ketring, 1984). Sunflower is grown in many regions of the world from Argentina to Canada and from central Africa to Russian Federation. It is tolerant to both high and low temperatures. Sunflower seeds will germinate at 4°C, but temperatures of at least 8°C to 13°C are required for satisfactory germination. Periods of soil temperature below 10°C cause germination delay and extend the period of susceptibility to herbicide injury and to seedling diseases, such as downy mildew. Seedlings in the cotyledon stage can survive temperatures down to –5°C. At later phenological stages, freezing temperatures may injure the crop. Temperatures less than –2 °C are required to kill maturing sunflower plants. Optimum temperatures for growth are 21 to 26 °C, but a wider range of temperatures (18 to 33 °C) show little effect on productivity. Extremely high temperatures have been shown to lower oil percentage, seed fill and germination ability. Sunflower is often classified as insensitive to daylength, and photoperiod seems to be unimportant in choosing a planting date or production area in the temperate regions, where temperature is the more limiting factor on length of the growing season. Sunflower will adapt to grow in a wide range of soil types (Harris et al.,1978; Baldini and Vannozzi, 1996; ARC - LNR, NDA, 1998). Olive growing area is strictly associated with the Mediterranean climate. This climate is characterized by a rainy and mild winter and a hot and dry summer. Spring and the autumns are not well discernible. The olive-tree does not tolerate frost. Indeed, the tree is very sensitive to low temperatures, even during the period of vegetative rest. Beginning of vegetative growth need a range of temperature between 10 and 12 °C. Inflorescences

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development requires minimum of 15 °C, bloom would take place at 18 - 19°C and fertilization occurs between 21 and 22 °C and above. Olive winter chilling requirements are estimated to approximately 400 hours below 9°C during low vegetative activity periods. In experiments with the cultivars grown in Mediterranean area, optimum flowering occurred when the temperature fluctuated daily between 15.5 to 19 °C. Trees held at a constant temperature of 13 °C also bloomed profusely but had poor pistillate flower formation. If temperatures did not rise above 7.5 °C or fall below 15.5 °C, trees did not bloom. At 13 °C, both chilling and warmth are sufficient for flowering but not for complete flower development. In contrast to flower buds, vegetative buds of olive seem to have little if any dormancy, growing whenever the temperatures are much above 21 °C. In addition to winter chilling, inflorescence formation requires leaves on the fruiting shoots. Therefore, it is important to prevent defoliation. The occasional occurrence of hot, dry winds during the blooming period has been associated with reduced fruit set. Winds or heat increase the amount of natural abscission (Crisosto and Suffer, 1985; Lagarda et al, 1983; Pinney and Polito, 1990). The argan tree is particularly resistant to the dry and arid conditions. It can grow on poor and shallow soils and tolerate temperatures ranging between 3 and 50°C and grows on sun-facing slopes at altitudes up to 1500 meters. 1.3.2 Soil and water needs The water needs of the maize crop are very variable and depend on the productive level and water efficiency. Average water consumption of maize fluctuates between 4000 and 6000 m3 ha-1. The maize cultivation is impossible with less than 150 mm of rainfall (Crop and soil management, 2007/8). Peanuts require five months of warm weather, and an annual rainfall of 500 to 1000 mm or the equivalent in irrigation water. The most critical time to apply irrigation water is during the flowering period. Soil for peanut production should be light textured with good drainage, and moderately low amounts of organic matter. Such soil is preferred since it is usually loose and friable, permitting easier penetration of roots and pegs, better percolation of rainfall, and easier harvesting. Well-drained soils provide proper aeration for the roots and nitrifying bacteria that are necessary for proper mineral nutrition of the plant. Organic matter should be maintained at a level of 1 to 2% to improve water-holding capacity of the soil and supply plant nutrients. Peanut grows best in slightly acidic soils with a pH of 6.0 to 6.5, but a range of 5.5 to 7.0 is acceptable. Saline soils are not suitable since peanut has a very low salt tolerance (Weiss, 1983). Canola does best on medium textured, well drained soils. The crop is tolerant of a soil pH as low as 5.5 and saline conditions. Because of its tolerance to salinity, canola has been used as the first crop on newly drained dikes in the Netherlands. Canola requires approximately 16 to 18 inches (406 – 457 mm) of water through its growing season, with 8 to 8.3 inches (203 -211 mm) used by annual varieties in July near flower and pod fill. The demand of the sunflower crop for major soil nutrients is not as great as corn or wheat (Philbrook, 1986; Canola production handbook, 1989). Good soil drainage is required for sunflower production because it is sensitive to waterlogging, similar to other field crops. Sunflower is very sensitive to wind damage during the seedling stage and for this reason, cultivation on light-textured soils in situations susceptible to wind erosion, should be avoided. Sunflower is not sensitive to most soil pH

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values but should not be planted in soils with a pH lower than 4.6. It grows best on well-drained, high water-holding capacity soils with a near neutral pH (pH 6.5-7.5). However, the crop is grown commercially on soils ranging in pH from 5.7 to over 8.0 (Baldini and Vannozzi, 1996). Soybean is a hardy plant and well adapted to a variety of soils and soil conditions. Ideal soil for optimum soybean production is a loose, well-drained loam. Many fields have tight, high clay soil that becomes waterlogged when it rains. When the soil dries out, a hard crust surface may form which is a barrier to emerging seedlings. These high clay soils are low in humus and may have imbalance in mineral nutrients. Also, these soils may have few beneficial soil organisms (bacteria, fungi, algae, protozoa, earthworms and others). High clay soils may be amended with peat moss, sphagnum, organic mulch to increase the humus content. Sand may be added to loosen and aerate the soil and allow better drainage. The advantages of loose, well-aerated soil include: movement of air to roots and nitrogen-fixing root nodules; increased water-holding capacity with adequate drainage; reduced erosion; reduced weed populations: maintenance of steady and balanced nutrients to roots and balance pH: increased potential to protect roots from harmful nematodes, insects pests, and pathogens. Rice is a semi-aquatic plant, which has a high demand for water, particularly over the reproductive stage from panicle initiation to early grain development. Most field crops usually grow best when at least 50% of the usable soil moisture is available to the plant. With rice, this figure is closer to 75% especially during the reproductive phase. Continuous ponding of water in the rice fields is one way of meeting these moisture demands which also reduces the risk of plant stress and yield loss. Ponding also helps suppress weed growth, improves the efficiency of use of nitrogen and, in some environments, helps protect the crop from fluctuations in temperatures. Factors that determine the total water requirements of a crop are: evapotranspiration, permeability of the soil, drainage, the length of the growing season, and the levelness of the soil surface. Water is used by a rice crop through evaporation from the soil or water surface and by transpiration through the leaves. In the early stages of crop growth, most water is used through evaporation. However, when the crop develops a full canopy cover, transpiration accounts for most of the water used. The combined use, which is called evapotranspiration (ET), accounts for up to 80% of the water used by the rice crop. The total evapotranspiration of a rice crop is between 800 and 1200mm of water. This quantity depends on seasonal conditions such as temperature, humidity, wind and sunlight hours as well as the length of the growing period. In direct seeding situations which include nurseries, fields often need to be drained during the establishment stage to improve both the rate and number of plants established. While a growing rice crop can withstand total inundation for short periods, major yield losses will occur if lodged crops are flooded during the grain ripening stage and water cannot be removed because of poor drainage. In dry periods water may need to be re-circulated from one field to another to help save the crop. When pests such as the golden apple snail attack crops, the only recourse is to drain the whole field rapidly (Rotgers, et al.,; International Rice Research Institute, 2008) The olive tree is a typical dryland-farming crop, which uses natural rainfall as its only source of water. Over 90% of the olives grown worldwide are grown without supplemental irrigation; They are commonly grown without irrigation in areas with an annual rainfall of 400 to 600 mm but are even found in areas with about 200 mm rainfall. For high yields, 600 to 800 mm are required. The crop coefficient (kc) relating maximum evapotranspiration (ETm) to reference evapotranspiration (ET) is between 0.4 and 0.6. The olive tree overcomes irregularity in water supply that characterizes the Mediterranean climate (more than 70% of the total rainfall is received during the autumn and winter periods, which correspond to the rest period of the olive tree) by getting from deep soil layers the little of moisture they can stock and by exploiting a great volume of soil. However, under these conditions the olive

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production will be always uncertain and often marginal (weak production and marked alternate-bearing). However, olive trees respond well, in growth and yield, when irrigation is applied regularly. Many studies have been carried out on the response of olive to irrigation (Moriana and Orgaz 2003; Fernandez et al.,2006; Lavee et al., 2007). As a general rule, when the total amount of rainfall is 250–300 mm/year, the trees will survive, but hardly any production can be expected. As the amount of water increases, production increases as well. In most cases, the best response in terms of yield was achieved when the total amount of water applied to the trees in the orchard (rainfall included) was between 45% and 65% of the potential evapotranspiration (ETP). A cultivation coefficient of 0.5 is recommended when no further information is available. When the olives are widely spaced or small in size, and the ground surface covered by the crop is less than 45%, a reduction factor should be applied, ranging from 0.7 for 25% coverage to 0.9 for 40% coverage. In the Mediterranean basin, winter is the rainy season (October through April), accompanied by a dry, hot summer. With winter rain of about 500 mm, irrigation is applied during and after stone hardening. Under conditions of little winter rain, irrigation is applied during bud differentiation (early spring), prior to flowering (early summer) and during yield formation and particularly during stone hardening. Irrigation is also applied at (a) two to three weeks before flowering; (b) when the fruit reaches one third its full size; and (c) when the fruit reaches almost full size. For oil production, irrigation supply must be discontinued early enough to give a dry period during ripening. This will have little effect on the oil content but will reduce the water content of the fruit. Irrigation is applied by different surface methods, but when limited water is available, localized irrigation is preferred. When regular application of irrigation water is not possible, relief irrigation will result in improvement of crop response (Lavee et al., 1990). Relief irrigation should be applied during the critical periods of the crop. In most cases, flowering and fruit-setting takes place when there is sufficient water in the soil from winter rains, so water stress during this period is rare. The next critical stage is pit hardening, which occurs in mid-summer, at a time when there may already be a shortage of water, especially if the amount of rainfall during the winter was low. Relief irrigation at this stage will reduce fruit drop. Oil formation and accumulation processes are most intense in autumn. Oil yield will benefit if water stress during this period is prevented. Olives are considered to be capable of tolerating salinity well, although there are differences, depending on the variety. The use of marginal water for olive irrigation is becoming more and more widespread, especially in regions where fresh water prices are high and supply is limited. When using marginal water for olive irrigation, the irrigation regime should accommodate olive production and not hamper oil quality or cause soil properties to deteriorate. The use of saline water (EC = 4–5 dS/m) requires a leaching fraction to be applied regularly so that salts do not accumulate in the root zone. This leads to use of larger amounts of irrigation water. When the sodium adsorption ratio (SAR) of the water is high, measures must be taken to prevent soil-structure deterioration. Another important consideration when using marginal water is to select cultivars that are more resistant to salinity. The olive tree is able to adapt its development to very different soil conditions tolerating a wide margin of soil pH, but the neutral, slightly alkaline values to alkaline ones, comprised between pH 7.0 and 8.5, assure its best development conditions. The argan tree is particularly resistant to the dry and arid conditions of this region. It can grow on poor and shallow soils and tolerate temperatures ranging between 3 and 50°C and grows on sun-facing slopes at altitudes up to 1500 meters. The argan tree is also particularly well adapted to take advantage of the frequent fog covering this part of Morocco and a large part of argan tree needs in water is provided by atmospheric moisture.

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1.4. Cultural Practices Oil crops can be broadly classified as either annual (e.g. rice, soybean, rapeseed, sunflower and corn) or perennial (e.g., olive and argan). Annual crops are planted and harvested every year while perennials are planted once and harvested over a number of years. These differences affect cultural practices such as tillage, planting date, planting depth and row width, fertilization, water management, and harvesting. 1.4.1 Tillage Modification of the soil conditions by tillage affects growth, development and economic yield of crops. Tillage influences both root and shoot growth in pre and postestablishment phases. Tillage is a cultivation system of choice for many of the oil producing crops. Many different tillage systems can be used effectively for sunflower production. Conventional systems of seedbed preparation consist of moldboard plowing or chisel plowing to invert residue and several secondary field operations. For rice production, tillage may be done in fall or may be postponed until spring under wet conditions, but generally begins as early as possible. This tillage is carried out in 20 to 25 cm depth with waldboard plough. Then the levees are erected, their size is 30 to 40 cm height and 40 to 50 cm length. After then, livelong is done and a shallow tillage is conducted with a disk harrow or a field cultivator. Before flooding the field, the fertilizer and pre-emergence herbicide applications are done and incorporated into the soil with a spike-tooth harrow (Sürek). Tillage is also the most frequently used cultivation system in olive orchards. It can be carried out by a variety of tools but the most common one is the tine-spring cultivator, used before winter and in spring to improve water infiltration and to remove small weeds. Disking is used in spring to remove large weeds and finally, harrowing is carried out in summer to fill in cracks and prepare the surface for harvest. However, a no-tillage policy, where the soil surface remains covered by plant material and the weeds are controlled by herbicides and by cutting, gives very good results; soil erosion is reduced by the plant material, which prevents rain drops from impacting the bare soil (Castro and Pastor, 1991; Civantos, 1996). 1.4.2 Planting date Planting date management and variety selection are extremely important steps in terms of setting the crop off to a good start, achieving earliness, and establishing a strong yield potential. Sunflower is often classified as insensitive to day length, and photoperiod seems to be unimportant in choosing a planting date or production area in the temperate regions, where temperature is the more limiting factor on length of the growing season Sunflower will adapt to grow in a wide range of soil types. Sunflower can be planted at a wide range of dates, as most cultivars are earlier in maturity than the length of growing season in most areas. In northern regions, highest yields and oil percentages are obtained by planting early - as soon after the spring-sown small grain crops as possible. A later planting date tends to increase the

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proportion of linoleic acid in sunflower, especially at southern locations. Damage of sunflower heads by insect larvae may be increased by early planting (Unger and Thompson, 1982). Canola can be seeded in either the fall or the spring depending on the type of variety. Fall dates need to be timed to achieve about 6 true leaves and good root reserves before a killing frost (Philbrook , 1986). Planting between August 15 and September 1 should accomplish this in most areas of Wisconsin and Minnesota. Spring planting should begin as early as soil is dry and weather permits. Like spring small grains, spring canola generally yields the best with early planting. At Arlington, WI canola seeded the last week of April averaged 1325 lb/a compared to 1150 lb/a when seeded three weeks later on May 20 (Canola Production Handbook 1989). The highest yields of soybeans are obtained from early plantings, generally the first 10 days of May. Later plantings are likely to incur significant reductions in yield. However, yield depends on several other factors, too. Soybeans need a minimum soil temperature of 13 to 16o

C to germinate. Germination rates increase at warmer temperatures. When planting is delayed, fewer days are required for the plant to reach maturity. A one-month delay in planting results in a 9-day delay at maturity. Delayed planting can reduce the vegetative growth period. This results in shorter plants with lower pods. Late planting also reduces the number of pods per plant because of the shorter flowering period. In addition, planting date has some effect on the duration of the pod-filling period. Vegetative growth of late plantings can be improved by selecting taller varieties and planting in narrow rows. Seeding rates should be increased by 10% (Ablett, 1980-81). For optimal growth of peanut planting in early June was originally favored in Minnesota due to the warm temperature. However, planting in early May gave higher yields, larger seeds, and higher shelling percentage. Peanut planted in early May required 9 more days to emerge and had a slower development than a crop planted in June. However, the planting in early May flowered earlier which allowed more pods to reach maturity before frost (Robinson, 1984). Corn can be normally planted safely 10 to 14 days before the average date of the last killing frost. Ideally, soil temperatures in the seed zone should be 10 °C or above, and the 5-day extended weather forecast should indicate continued warm, or warmer, conditions (Alexander, 1988. 1.4.3 Planting depth and row width A planting depth of 1 to 3.5 in. allows sunflower seeds to reach available moisture and gives satisfactory stands. Row spacings of 30 in. are most common. Plant population has a strong effect on seed size, head size, and percent oil. A medium to high population produces higher oil percentage than does low populations. Canola is usually seeded with the small seed attachment of a grain drill to a depth of 1/2 to 1 inch. Rows should be spaced 7 inches or less. Research has shown highest yields with 3-inch row spacings. Canola should be seeded at 4-5 lb/a if drilled and 7-8 lb/a if broadcast depending on seed size and soil texture. Soybean seeds should be planted deep enough to meet the moisture and temperature requirements for germination. Due to the high water demand for germination, planting of 1 cm (approximately 1/2 in.) into moisture but not deeper than 7.5 cm (3 in.) is recommended. Row widths of 18 cm (7 in.) are recommended in short-season areas.

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Peanut planted in narrow 18-inch row spacing appears to result in greater yields, yet the row spacing used for planting will largely depend on the type of planting and harvesting equipment available. The highest peanut yields were produced in 18 in. rows planted with 105,000 seeds per acre while highest yields from 30 in. row spacing resulted from only 70,000 seeds per acre. Seventy-thousand seeds per acre produced highest yields on drylands and where row spacing had no effect. Seed should be planted 1 to 2 in. deep since at greater depths, slower and poorer emergence results. Corn sowing density can vary from 6 to 8 plants per square meters; usually the high density is adopted with early hybrids while the low density is used with medium or late varieties. With regard to sowing depth in dry conditions, it is better to plant up to 5 cm to get the seed to moisture. The sowing density of corn can vary from 6 to 8 plants per square meters; usually the high density is adopted with early hybrids while the low density is used with medium or late varieties. Rice seeds must be placed close to the soil surface. When dry seeding into heavier clay soils, place seeds within 10 to 15 mm of the surface. When wet seeding, seeds should not sink below the puddled surface. Rice can be direct seeded or transplanted. Direct seeded crops tend to mature faster than transplanted crops but have more competition from weeds. Direct seeded crops can be established using dry seed or pre-germinated seed and seedlings. They are broadcast by hand or planted by machine. The target number of plants to be established ranges from 100 to 150 plants per m2. To meet this target, seeding rates vary between 80 and 250 kg per ha. Transplanting of rice seedlings into puddled fields is widely practiced in Asia, primarily as a means of weed control. Transplanting requires less seed but much more labor, and the crop takes longer to mature due to transplanting shock. While the majority of rice fields in Asia are manually transplanted, China, Japan, South Korea and other countries also use mechanical transplanters. Local varieties are transplanted 40 to 80 days after establishment; improved varieties are transplanted within 20 days after establishment. Machine transplanted seedlings are transplanted 15 days after establishment. Seedlings are normally hand transplanted 20 cm apart, but this distance may be increased or decreased depending on soil fertility and water supply. The range is normally 15 to 30 cm (Rotgers et al.). Correct planting depth of olive trees should vary from 10 to 20 cm in heavy soil and 25 to 40 cm in light soil. Planting density for olives in rain-fed orchards is usually around 100 trees/ha. Under arid conditions, where rainfall is around 200 mm/year or less, a density of less than 20 trees/ha may be reached. With increasing rainfall or under supplemental irrigation, planting density may be increased and reach 300–400 trees/ha. In some cases, especially under controlled water supply with supplemental irrigation, trees are planted at a high density of 480 trees/ha (e.g. 3×7 m), initially reaching high yields. After some years, when competition for light becomes a limiting factor, the trees are spaced into 6×7 m (240 trees/ha) by removing every other tree (Fontanazza, 1984). The argan tree is not grown in plantations although its possible role in reforestation in Spain has been studied (Montoya, 1984). 1.4.4 Fertilization For sunflower, nitrogen recommendations of approximately 18 lb N/acre after fallow or legume sod, 60 lb N/acre after small grain or soybean and 80 to 100 lb N/acre after corn are common. On higher organic matter soils, amounts should be lowered. Nitrogen can be

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supplied from mineral or non-mineral sources (manures, legumes, compost). Row placement of P and K may be important in sunflower for maximizing efficiency of fertilizer use, as it is with many species. Recommendations for applications of P and K should be made from soil tests and the yield goal for each field. Recommendations range from 40 to 70 lbs P2O5 and -60 to 140 lbs K2O /acre for soils testing very low in P or K, depending on soil yield potential. These recommendations decrease as soil test P and/or K increase. Response to P is not expected if soil P exceeds 30 lb/acre nor to K if the K test is greater than 300 lb/acre (Merrien, 1986; Vrebalov, 1974). As for canola, some states recommend that P and K should be applied on the basis of soil test recommendations for winter wheat. For Wisconsin and Minnesota this means that when soil tests are in the medium range about 20-30 lbs of P2O5 and 20 lbs of K2O should be applied per acre. At lower soil tests these rates should be increased. Because canola is sensitive to direct seed contact with fertilizer, applications should be banded at least 2 inches to the side and below the seed or broadcast. Canola responds well to nitrogen fertilizer, with optimum yields occurring around 80-100 lbs N/acre. For spring canola, it should be broadcast and incorporated at seeding time along with the P and K. For winter canola, nitrogen may be best applied as a split application using starter nitrogen application of about 10-20 lbs/acre, followed by the remainder in the spring prior to regrowth. Canola is a heavy user of sulfur. Soils most likely to respond to S additions are light colored, sandy soils (Berglund et al., 2007). Soybean is a legume and which normally provides itself with adequate nitrogen through a symbiotic relationship with N-fixing bacteria of the species Bradyrhizobium japonicum. For this reason nitrogen fertilizers are not usually required for soybeans. If the soil is very low in potassium, a suggestion for an overall fertilizer source is potassium sulfate. Fertilizer formulations with chloride should be avoided because the chloride ion can injure soil microbes as well as soybeans themselves if present in high amounts. Fertilizer should not be placed in contact with soybean seeds due to the sensitivity to fertilizer salts. There is no yield advantage to this practice. The fertilizer may be broadcast and plowed down or worked into the soil either in the fall or spring (Orlovious, 2003). Peanut responds well to residual soil fertility from previous crops in the rotation, but usually has a low response to fertilizer in soils with medium to high fertility levels. When nutrients are needed (low or very low soil test levels) broadcast applications are recommended especially of potash due to the low salt tolerance of peanut. Rates should be similar to those used for soybean. Since it is a legume, peanut can biologically fix its own nitrogen. The adequacy of farm soils for fertility for peanut should be checked with soil tests. Optimum pH levels of 6.0 to 6.5 will usually result in adequate calcium being present, however on lighter soils especially where long term applications of potash have been made, Ca may be limiting pod formation. Soil test Ca should be above 600 to 800 ppm. Although plant analysis may be useful for micronutrient levels, it does not detect Ca shortages in storage organs such as peanut pods. The severe calcium and micronutrient deficiencies that occur in the major peanut production areas are not likely here. Nitrogen fertilizer or seed inoculation with the proper Rhizobium strain is needed for a crop on irrigated sandy soil. One-hundred-fifty pounds of nitrogen per acre were required to equal the yield produced with seed inoculation alone. Optimal nitrogen fertilization for corn is reported to be in a range of from 170 kg to 300 kg per hectare, depending on plant population and previous crop. Multiple applications of nitrogen through irrigation systems are also effective. These applications should be timed so that some nitrogen is applied by the sixth week after planting, and most of the nitrogen requirement is applied by the tenth week after planting. Manure can provide part or all of the phosphate and potash requirements for corn production. Recommended credits are 1.35 kg of P2O5/ton and 3.6 kg of K2O/ton of solid dairy manure (Lemon, 1996; Jordan, 1999; Shiedow,

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1989). For rice, the optimum levels of nitrogen, phosphorous and potassium are about 4 grams per seedling tray, respectively for semi-adult seedling. In case of nitrogen fertilizer, 2 gram of nitrogen fertilizer is applied respectively as basal and top-dressing at third leaf stage. Phosphorous and potash are applied as basal. Nitrogen is used at somewhat higher rates than phosphorous and potash in paddy field, and some differences in fertilizer requirements of rice varietal groups are also recognized. Split application of nitrogen is needed, with 50% as basal, 20% and 30% as top-dressing during the period of tillering and panicle formation, respectively (Rotgers et al.). Nitrogen fertilization of olive trees can be done by applying nitrogen to the soil in the irrigation water or it can be applied to the leaves (foliar application). In non-irrigated, traditional orchards, soil application of nitrogen should be associated with rainfall and therefore one nitrogen dressing in winter is common practice. An application of 50–100 kg N/ha.year is recommended for mature trees. In irrigated orchards, a dressing of 200 kg N/ha.year is recommended. One third of this amount has to be applied in winter, one third in summer and one third in autumn. In orchards where proportional fertilization is possible, especially in drip-irrigated orchards, nitrogen can be supplied with the irrigation water continuously, according to the critical uptake stages of the plant. Phosphorus deficiency is rare in olive orchards, perhaps due to the fact that the olive root system is very mycorrhizal, enabling the plant to extract all the phosphorus it needs from the soil. Although potassium plays an important role in the oil-production process, few trials showed a clear response to potassium fertilization, mostly in cases where potassium levels were extremely low. In irrigated orchards a dressing of 300 kg K/ha is recommended, preferably with the irrigation water. The use of compound fertilizers is also possible in irrigated orchards. In this case the N:K ratio should be 2:3 and P application will be based on leaf analysis (Baldini 1992; Hanoch et al., 2007; Hartman et al., 1986). 1.4.5 Water management Sunflower is not considered highly drought tolerant, but often produces satisfactory results when other crops are damaged during drought. Its extensively branched, deep taproot, penetrating to 2 m, aids the plant during water stress. A critical time for water stress is the period 20 days before and 20 days after flowering. If stress is likely during this period, irrigation will increase yield, oil percentage and test weight, but decrease protein percentage. Sunflower seasonal water use averages about 500 mm. Sunflower yield is most sensitive to moisture stress during the flowering period and least sensitive during the vegetative period from emergence to early bud. A 20% reduction of irrigation water application from plant emergence to the beginning of reproductive stage resulted in only a 5% reduction in yield, but the same reduction in irrigation water application during the reproductive period (bud to ray-petal appearance) resulted in a 50% yield reduction. Irrigations during the critical bud to ray-petal appearance period should be scheduled to maintain a low soil moisture stress condition (35 to 40% depletion). Irrigation should be avoided from beginning to end of flowering because of the susceptibility of the sunflower plant to head rot sclerotinia, so it’s useful to irrigate just before flowering in the bud stages. Soil moisture depletion can again approach 70% during late seed-fill and beyond with little or no depression in yield. Management of applied irrigation water requires the combination of periodic soil moisture measurement with a method of irrigation scheduling (Ennen, 1979; Robinson, 1985).

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Maize is an efficient user of water in terms of total dry matter production and among cereals it is potentially the highest yielding grain crop. For maximum production a medium maturity grain crop requires between 500 and 800 mm of water depending on climate. Frequency and depth of irrigation and rain has a pronounced effect on grain yield. Maize appears relatively tolerant to water deficits during the vegetative and ripening periods. Greatest decrease in grain yields is caused by water deficits during the flowering period including tasselling and silking and pollination, due mainly to a reduction in grain number per cob. Severe water deficits during the flowering period, particularly at the time of silking and pollination, may result in little or no grain yield due to silk drying. Water deficits during the yield formation period may lead to reduced yield due to a reduction in grain size. To obtain a good stand and rapid root development, the root zone should, where feasible, be wetted at or soon after sowing. Under conditions of marginal rainfall and limited irrigation water supply, the number of possible irrigation applications may vary between 2 and 5. As regards to irrigation methods of maize, both surface and sprinkler can be used. It is advisable to use the sprinkler in clay soils and when there isn’t enough land levelling (Crop and soil management, 2007/2008). A soybean crop will produce approximately 2 bu/ac for every inch of water it uses through the season. Yields in the 40 to 50 bu/ac range require 20 to 25 inches of available soil moisture during the growing season. The irrigation water needed will vary depending on the beginning soil moisture and the rainfall received during the growing season. An irrigation system needs to be capable of providing 10 to 15 inches of water during the season to assure an acceptable yield. Lack of early and late season irrigation is often responsible for a soybean crop not reaching its irrigated yield potential. Surface and sprinkler irrigation methods are used on soybeans. Each method has different characteristics that could make it the best for a particular situation. No one method can be labelled as the best – each has its place (Anonymous, 2008). Rice is a semi-aquatic plant, which has a high demand for water, particularly over the reproductive stage from panicle initiation to early grain development. Most field crops usually grow best when at least 50% of the usable soil moisture is available to the plant. With rice, this figure is closer to 75% especially during the reproductive phase. Continuous ponding of water in the rice fields is one way of meeting these moisture demands which also reduces the risk of plant stress and yield loss. Ponding also helps suppress weed growth, improves the efficiency of use of nitrogen and, in some environments, helps protect the crop from fluctuations in temperatures. Factors that determine the total water requirements of a crop are: evapotranspiration, permeability of the soil, drainage, the length of the growing season, and the levelness of the soil surface. Drainage of water from the field can be very important during the time of crop establishment, high rainfall events and during attacks by crop pests. In most level fields drains around the periphery of the field will be sufficient to drain off excess water in a timely manner. In large fields and some nurseries, small internal drains running from the center of the field to the extremities of the field may also be needed. The longer the crop growth period the higher will be the water requirement. A general rule is that a rice crop will need approximately 10mm of water per day. Therefore a crop that matures in 100 days will require approximately 1000mm of water while a crop that matures in 150 days will require 50% more. In areas that are affected by deep water or surface flooding, later maturing crops may be necessary so that the crop is sufficiently developed and tall enough to withstand the higher levels of water. Dry land preparation uses less water than wet land preparation, but this is not always possible in systems that rely on animal power for plowing. Land preparation systems that use animals or require free water on the soil surface before tillage can commence require up to 20% more water to grow the rice crop. Between 100mm and 300mm of water is required to saturate and weaken the soil so that animals are able plow it. When preparing the

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land wet, as in puddling, the field should initially be worked dry and water added just before the puddling operation (Rotgers et al.). The olive tree is a typical dryland-farming crop, which uses natural rainfall as its only source of water. Over 90% of the olives grown worldwide are grown without supplemental irrigation; however, olive trees respond well, in growth and yield, when irrigation is applied regularly. As a general rule, when the total amount of rainfall is 250–300 mm/year, the trees will survive, but hardly any production can be expected. As the amount of water increases, production increases as well. In most cases, the best response was achieved when the total amount applied to the trees in the orchard (rainfall included) was between 45% and 65% of the potential evapotranspiration (ETP). When regular application of irrigation water is not possible, relief irrigation will result in improvement of crop response. Relief irrigation should be applied during the critical periods of the crop. Oil formation and accumulation processes are most intense in autumn. Oil yield will benefit if water stress during this period is prevented. Olives are considered to be capable of tolerating salinity well, although there are differences, depending on the variety. The use of marginal water for olive irrigation is becoming more and more widespread, especially in regions where fresh water prices are high and supply is limited (Moriana and Orgaz 2003, Fernandez et al., 2006, Lavee et al., 2007). 1.4.6 Harvesting The sunflower plant is physiologically mature when the back of the head has turned from green to yellow and the bracts are turning brown, about 30 to 45 days after bloom, and seed moisture is about 35 percent; so sunflowers are generally mature long before they are dry enough for combining. Harvesting should commence as soon as 80 % of the sunflower heads are brown in order to minimise losses caused by birds, lodging and shattering; the fleshy sunflower head takes a long time to dry and so desiccants can be applied after physiological maturity to speed the dry-down process. These chemical compounds accelerate the drying process killing the green tissue on the plant like a frost. In wet areas, often growers are tempted to apply desiccants too early because dry-down of the seed is not as rapid as the dry down of the plant; application of a desiccant before the plant reaches physiological maturity will reduce yield and lower oil percentage. Seed shattering loss during harvest and loss from birds may be reduced by harvesting sunflower at moisture contents as high as 25 percent. Sunflower seed from the combine is then dried in a grain dryer to a safe storage level. Seeds should be below 12% moisture for temporary storage and below 10% for long term storage. Seed of up to 15% moisture content is satisfactory for temporary storage in freezing weather, but spoilage is likely after a few days of warm weather. Harvesting can be achieved using combines suitable for threshing small grains with appropriate adaptation; a variety of header attachments are available operating on a head stripper principle. The attachments are designed to gather only the sunflower heads and eliminate as much stalk as possible. Major components of this attachment are catch pan, deflector, and small reel. Long catch pans extend ahead of the cutter bar to catch the seed as it shatters. The deflector mounted above the catch pans pushes the stalk forward until only the heads remain above the cutter bar. As the heads move below the deflector, the stems contact the cutter bar and are cut just below the head. A small reel, mounted directly behind the deflector, pushes the heads into the combine feeder (Baldini and Vannozzi 1996; ARC – LNR, NDA, 1998). Grain corn harvesting can begin when the kernel moisture reaches 30%. Most producers aim to harvest grain corn between 20 and 27% moisture. If the moisture content is

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too high, many of the kernels will rupture before breaking away from the cob and longitudinal cob breakage may increase. If the moisture content is too low, field losses due to lodging and dropped ears will be increased and more kernels will be damaged when the cylinder bar strikes them. Artificial drying of grain corn is the common way to prepare the crop for storage. Corn is shelled when kernel moisture content is below 30% but to store it safely, the moisture must be reduced to 14-15%. Grain quality is the factor that can be most easily controlled by the dryer operator. Corn must be stored in a manner that will preserve its quality regardless of whether it is kept for a livestock feed or for sale to industrial users. Corn can be sold immediately after harvest and drying, but storage of the corn for later marketing can be advantageous (Crop and soil management). The optimum time for peanut harvesting is when most pods have a veined surface, seed coats are colored, and 75% of pods show darkening on the inner surface of the hull. However, in many peanut cultivated areas the crop does not reach this stage, so immature pods are removed in the threshing, drying, and cleaning operations. Harvesting usually starts with clipping or coultering. Rotary mowers remove up to half of the top growth when plant growth is too great for efficient harvesting. Varieties with prostrate growth may overlap between rows and a coulter makes the vertical cut between rows. The next operation frequently uses a digger-shaker-windrower. Dig deep enough to prevent cutting pegs. Windrow-inverting attachments orient plants as they leave the shaker so pods are primarily on the top of windrows to permit more air circulation and exposure to sunlight for a shorter drying time. Windrowed peanut may be combined-harvested wet (35 to 50% moisture), semidry (18 to 25%), or dry (8 to 10%). These peanuts may reach the semidry condition (seeds rattle in pods) 1 to 3 days after digging. Drying in the windrow to a moisture level of 8 to 10% requires 5 to 10 days of good drying weather. However, peanut remaining in windrows for several days is more susceptible to weather damage than when freshly dug. Combining wet (green) or preferably semidry peanut, followed by artificial drying, may result in better quality nuts (White and Roy, 1982; Shiedow et al., 1989; Robinson, 1984; Lemon, 1996). Timely harvest of canola is critical to prevent shattering. When pods first begin to yellow, the crop needs to be checked on a 3 to 4 day schedule. Harvest maturity can only be determined by observing the color of the seed. In canola that stands well, 30 to 40% of the seed on the main stem needs to be brownish-red in color prior to swathing. This corresponds to about 30 to 35% seed moisture. Canola does have a tendency to lodge, particularly with over-fertilization of susceptible varieties. In severely lodged canola, swathing should be done when 40 to 50% of the seed in exposed pods has turned color. Shattering can account for significant crop losses, therefore harvesting must not be delayed. Canola should be cut high on the stem and lightly pushed into the stubble with a windrower to prevent blowing. The crop is combined when it has dried to near 10% moisture. Direct combining with the use of a desiccant is possible in canola that is standing well, but determining application time is difficult and field losses are higher. The cylinder speed should be set at 450-1000 RPM and the cylinder concave clearance at 3/16 to 1/2 inch. Losses should be evaluated for further refinement of these adjustments. Canola that is to be stored for six months or more must be dried to near 8% moisture (Philbrook, 1986; Canola production handbook). Harvesting rice consists of four basic operations: cutting the mature panicles and straw above-ground, moving the cut crop to the threshing location, separating the paddy grain from the rest of the cut crop, and removing immature, unfilled and non-grain materials. Rice harvesting can be done manually using sickles and knives, or mechanically with the use of threshers or combine harvesters. In the firs way, the farmers cut the crop by hand with a sickle or reaper machine and then they leave it in the field to dry under the sun for a few days. After that, the sheaves of rice are carried to the threshing area. Threshing is done with combine or

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thresher. In the second way, rice is directly harvested with combine and the crop is dried to a storable moisture of 13 to 14 percent. For drying, mechanical dryers are used. The manual system is the most common method for rice harvest in Asia. Rice crop is cut by simple hand tools including sickles for cutting 15-25 cm above ground level, and simple hand-held knives to cut just below the panicle. In mechanized countries the mechanical system for cutting is the most widespread: the cutting is done by a reaper mounted on the front of a tractor. Most reapers lay the crop in a windrow, which allows for easy pick up of the harvested crop. A reaper with a cutting-width of 1.5 m can operate at a rate of 2 to 4 ha per day. For proper operation of reapers, fields need to be levelled. The common method for threshing by hand is separating the grain from the panicle by impact: this is done by hand beating, treading, or by holding the crop against a rotating drum with spikes or rasp bars. Hand beating methods are normally used for threshing of rice that easily shatters. Given the high labor requirements of manual threshing, the mechanized threshing by use of small stationary machine threshers is increasing. Stationary threshing is generally done in the field, or near or at the field side. In the mechanized countries the use of combine harvesters for paddy rice is the most widespread. Combine harvesters “combine” several operations into one: cutting, feeding into threshing mechanism, threshing, cleaning, and discharge of grain into a bulk wagon or directly into a bags. Drying is the most critical operation after harvesting a rice crop. Delays in drying, incomplete drying or ineffective drying will reduce grain quality and result in losses. Drying and storage are related processes and can sometimes be combined in piece of equipment (International Rice Research Institute, 2008; Sürek, 1982). In olives, determination of the optimal harvesting time is of crucial importance (Gutierrez et al., 1999; Gomez-Rico et al., 2006; and Caponio et al., 2001). Ripening time is affected by two major factors: the olive variety and the yield load on the tree. Under high yields, ripening is postponed and vice versa. It is common practice in many olive orchards to harvest when about 50% of the fruit have darkened in color. Oil accumulation in the fruit reaches its maximum when most fruit on the tree have fully ripened, i.e., are completely dark externally and the dark color has penetrated into the flesh. However, postponing the harvest until this stage may result in increased fruit drop and may negatively affect yield the following year. In addition, there may be logistical restrictions with large-scale olive orchards that make it impossible to harvest the whole orchard at the optimal time. Therefore, in most cases, the first trees are harvested a little too early, before oil content in the fruit is at its maximum and the last ones are picked a little too late, when natural fruit drop is already significant. One option to reduce the intensity of this problem is to select the cultivars that are planted such that harvesting time does not overlap too much. Olive harvesting methods can be classified into two groups: manual and mechanical. With manual picking, olives can be picked from the trees by hand or knocked down with poles. Hand picking is the least harmful method, both to the tree and to the fruit. This method involves stripping the olives from the tree by hand, by "milking" the branches into bags or into nets spread on the ground. This method involves intensive manual labor and is therefore costly. It is used only for very high quality table olives but hardly ever used for oil olives unless labor is very cheap. Knocking the fruit down with poles is very common in old, traditional orchards. This method damages the trees, as it breaks and knocks off twigs and damages buds, perhaps negatively affecting yield the following year. With this method, fruit skins are also damaged more, compared to hand-picking or mechanized methods, and oxidation processes are accelerated, which may negatively affect oil quality. In some cases the fruit are not picked, but collected from the ground instead, either mechanically or by hand. In most cases olive oil quality is lower when this method is used. The most commonly used method for mechanized olive harvesting is trunk shaking. To accommodate trunk shakers, the trees must be trained in advance into a single-trunk shape and the tree canopy must be maintained by pruning at a certain height.

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With this method, the shaker shakes the tree at a given frequency and the fruit drop onto nets spread on the ground. The fruit are collected either manually or by a built-in collection system. This method is much more efficient than hand picking and less costly; less damage is caused to branches and buds but sometimes the bark of the trunk is severely damaged, especially when trees are well watered. In super-intensive olive orchards, over-row harvesters are used. The initial investment in harvesting machinery is high but harvest costs are low and harvest capacity is very high. Very little manual labor is involved and harvesting at the proper stage of fruit ripening is much easier to accomplish. Harvesting of the argan tree takes place during September. Three main methods are used to harvest argan fruit, two of which involve the use of animals. Grazing goats sometimes eat the fallen fruits, spitting out the seeds which are then gathered up by hand. Camels are also fed the fruits, the seeds are indigestible and pass through the animal to be excreted. These seeds are then gathered from the dung. An alternative harvesting method is simply to shake the tree causing the fruits to fall to the ground for manual collection. The fruits are sun dried until they reach approximately 50% of their original weight. The fruit is hit with a stone and the outer dry pulp separates cleanly from the inner nut (Morton and Voss, 1987). 1.4.7 Training and pruning methods In intensive olive orchards the purpose of training is to achieve shapes that will take maximum advantage of the environment, particularly solar radiation, as early as possible. Formation of a single trunk is essential for mechanical harvesting by trunk shakers. The ideal tree shape proposed for intensive orchards is achieved from a single-trunk nursery-grown plant. These plants are truncated at a height of 80–120 cm and a hollow-vase shape is formed, usually with minimum manual interference. After the trees are fully formed, minimum pruning is required, especially in orchards with a good supply of water. The main objective of pruning at this stage is to keep the trunk and main branches shaded, to obtain a high leaf:wood ratio and to ensure good light penetration into the canopy. Canopy volume should be kept at a size that the planting pattern and the method of harvest permit. Pruning is carried out after harvest and before blooming and its intensity is usually related to the expected bearing potential of the trees the following year. After an "on" year, heavier pruning is recommended to improve light penetration and encourage new shoot growth. After an "off" year, lighter pruning is recommended to avoid removing potential fruit yielding branches. Tree height must be controlled regularly to facilitate harvesting and to avoid mutual shading between neighboring trees (Gucci and Cantini, 2000). 1.5. Other Products of the Crop The peanut groundnuts are grown mainly for the pods, but sometimes this crop is planted for hay, silage and pasture; this type of cultivation usually occurs on poorer soils. Whole cured plants can be fed with advantage to dairy and beef cattle; large cattle herds in parts of south America are allowed to graze on perennial wild groundnut plants. Moreover, when groundnuts are harvested, the aerial portions become available in large quantities for stock feed. These have proved to be an excellent feed and are also exceptionally palatable. It

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is common to find groundnuts fields planted for "Hogging off", which is the practice of allowing swine to feed on groundnuts; it represents a very cheap method of fattening pigs. Often this practice is used also for cleaning up waste groundnuts after harvesting. Moreover, by-products of the processing of peanuts for human consumption as skin and hulls can have a further utilization; the skin, about 3% of the seed weight, and hulls, 20-30% of the weight of the whole pod, can be used in ruminant feeds even if in small quantities, for the reason that the high content of crude fibre interfere with protein digestion; the hulls are often used as poultry litter (Woodroof, 1983). Peanuts also have a variety of industrial end uses. Paint, varnish, lubricating oil, leather dressings, furniture polish, insecticides, and nitroglycerin are made from peanut oil. Soap is made from saponified oil, and many cosmetics contain peanut oil and its derivatives. The protein portion of the oil is used in the manufacture of some textile fibers. Sunflower oil for cooking is being also used as biodiesel, or a vegetable-oil based fuel used for running many vehicles, including farming equipment (Bona and Vamerali, 1997; Carruthers and Smith, 1999; Hall and Overend, 1987). The by-product of biodesel is glycerin, which can be used in the manufacture of soap or hundreds of other products. Sunflower oils could be directly used without any treatments for heating purposes in domestic plants. In Europe sunflower bio-diesel production has increased substantially since 1992; the total European production in 2000 was ten times the production levels of 1992. For the human consumption the sunflower kernels, after dehulling, can be sold as confectionery nuts. Roasted in the same manner as coffee, they make an agreeable drink, and the seeds have been used in Portugal and Russia to make a wholesome and nutritious bread. One of the most beneficial uses of sunflowers is in the removal of toxic waste from the environment. Utilizing an emerging technology called rhizofiltration, hydroponically grown plants are grown floating over water. Possessing extensive root systems, they are able to reach deep into sources of polluted water and extract large amounts of toxic metals, including uranium (Nehnevajova et al., 2005). Such a process has been utilized in the former Soviet Union to decontaminate water polluted as a result of the 1986 accident at the Chernobyl nuclear power plant. The roots of floating rafts of sunflowers were able to extract 95% of the radioactivity in the water caused by that accident. Sunflower seed can be used as poultry and cattle food; sunflower seeds have a high feeding value; being so rich in oil, they are too stimulating to use alone and should only be used in combination with other feeding stuffs. As regards for medicinal action and uses should be stressed that sunflower oil helps to reduce the serum cholesterol levels; sunflower oil has high level of linoleic acid which is required for the cell membrane structure, cholesterol transportation in the blood and for prolonged blood clotting. The seeds have diuretic and expectorant properties and have been employed with success in the treatment of bronchial, laryngeal and pulmonary affections, coughs and colds, also in whooping cough. A tincture of the flowers and leaves has been recommended in combination with balsamics in the treatment of bronchiectasis (Deschauer, 1945). The rice plant seeds are first milled using a rice huller to remove the chaff (the outer husks of the grain). At this point in the process the product is called brown rice. This process may be continued, removing the germ and the rest of the husk, called the bran at this point, creating white rice. Where brown rice contains all of the ingredients of a healthy meal, tea rice, with the nutrients of rice germ and rice brain, is not a standard in counties for commercial offerings. White rice may be also buffed with glucose or talc powder (often called polished rice, though this term may also refer to white rice in general), parboiled, or processed into flour. The white rice may also be enriched by adding nutrients, especially those lost during the milling process. Rice bran is a valuable commodity in Asia and it is used for many daily needs and, particularly, to produce a lot of different kinds of whole foods. The raw rice may be ground into flour for many uses, including making many kinds of beverages such as

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amazake, horchata, rice milk, and sake. Rice flour is generally safe for people on a gluten-free diet because it does not contain the globulin fraction of proteins that is toxic for celiac people. The processed rice seeds are usually boiled or steamed to make them edible, after which they may be fried in oil or butter, or beaten in a tub to make mochi. Although rice is a good source of protein and a staple food in many parts of the world, it is not a complete protein. In fact, it does not contain all of the essential amino acids in sufficient amounts for good health, and should be combined with other sources of protein, like meat or soybeans (Jianguo et al., 2003). Rice, like other cereal grains, can be puffed (or popped). This process takes advantage of the grains' water content and typically involves heating grain pellets in a special chamber. Further puffing is sometimes accomplished by processing pre-puffed pellets in a low-pressure chamber. The ideal gas law means that either lowering the local pressure or raising the water temperature results in an increase in volume prior to water evaporation, resulting in a puffy texture. Bulk raw rice density is about 0.9 g/cm³. It decreases more than tenfold when puffed.

Table 1.6. Olive by products and their reuse (adapted from: Dally, Mullinger 2002; Yaman et al., 2000; Tekin and Dagic, 2000; Cliffe and Patumasawad, 2001).

The useful resources of olive crop are not only olive oil and table olives. Olive tree's wood, very hard and really beautiful, has been traditionally used for making furniture. Moreover, the olive-wood parquet floors are beginning to be used, due to their outstanding resistance and hardness. Apart from these traditional uses, very interesting results are being obtained from the use of subproducts from the olive oil production process, such as the use of olive pits to obtain animal food, active coal, etc., the cogeneration of electrical energy from

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the “orujillo” (olive pit and pulp waste), the use of waste water as fertiliser and anticryptogamic, extraction of vitamin E and polyphenols, etc. Olive kernels are useful for the production of seed oil and kernel wood. Pomace - There is still a small amount of oil in this cake so it can be further processed to obtain refined oil, used for heating, as animal feed supplement or returned to the olive trees as mulch (to prevent soil erosion or enrich the soil). Exhausted Pomace - When the refined oil is extracted the leftover fibrous material is primary lignin and cellulose. It can be composted, burned, used for heating, for animal feed supplement or returned to the olive trees as mulch. The two most important subproducts produced during olive oil extraction process are the waste water and the pomace. The actual uses of olives oil residues are shown in the table below, where it is explained which are the advantages and disadvantages of each use. As for the argan, its oil is mixed with almonds and honey to make an almond butter known locally as "amalou". Mixed with wheat germ and honey it makes a breakfast gruel locally called "sematar" (Mellado) (Table 1.6). 1.6. Major Pests and Pathogens Most crop diseases produce failed harvests rather than killing the plants outright. They do so by drastically reducing crop quality and quantity. Fungi present the biggest threat to crops. Crop diseases are caused by fungi, viruses and bacteria. These plant pathogens are transmitted by wind, water, or vectors. Because they depend heavily on environmental factors (e.g., temperature, humidity, rainfall, sunlight),the introduction of a pathogen does not necessarily result in widespread infection. There are three primary transmission modes of crop diseases. • Airborne (Fungal Diseases) - Fungi produce dry spores, which are dispersed on the wind

and can travel great distances. After a fungus has infected an area, it is very difficult to eliminate all of the spores. Although fungicides are helpful, fungi can persist in other hosts, allowing the disease to continue infecting plants for a long time.

• Vectors (Viruses and Bacteria) - Insects such as aphids are often virus carriers. When an aphid feeds on a leaf, it pierces cell walls and transmits the virus. Although viruses can be extremely damaging to crops, their ability to spread is limited by insect movement. Crop viruses are currently untreatable. Virus control depends on insect control and the use of virus-resistant crop strains. Insects also can transmit bacteria.

• Waterborne (Bacteria) - Bacteria require moisture for transmission. Although they cannot be transmitted on the wind, they can travel via wind-driven rain. Splashing rainwater can spread bacteria among individual plants, and irrigation runoff can spread bacteria over entire fields. Although bacteria can cause serious plant diseases, they generally cannot spread over vast areas.

Vertebrates, molluscs, nematodes and arthropod pests other than insects can damage crops directly. Infestations of particular insects can prompt export restrictions. The Mediterranean fruit fly, or “Medfly,” lays its eggs on many types of fruit on which the larvae later feed. If the Medfly became established in the United States, the USDA estimates that it would cost $1.5 billion per year in lost production and export restrictions. Pest management include natural control, biological control, control by disrupting reproduction, hormones, pheromones, indirect control measures, resistant varieties, direct methods, selectivity of control measures, pheonological control, integrated pest control, pest monitoring and

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simulation modelling and legislative measures and pesticides. Major pests and pathogens in oil field crops are shown in Table 1.7. Major corn diseases can be grouped into four categories: leaf blights, stalk rots, ear rots, and viral diseases (Rane and Ruhl, 2002). A number of leaf-blight diseases occur on corn. The most common are gray leaf spot, Stewart's bacterial leaf blight, and northern corn leaf blight. These diseases can be found in almost any field, depending on the year and susceptibility of the hybrid planted. Some leaf-blight diseases are most often found associated with continuous corn, especially in reduced-tillage, continuous corn fields. These are anthracnose, gray leaf spot, eyespot, and northern leaf spot. Stalk rots are the most important and common diseases of corn. Annual losses are estimated at 5 to 10 percent. There are several stalk-rot diseases, but Gibberella stalk rot and anthracnose stalk rot currently are the most prevalent. Both are fungal diseases that result in premature ripening, chaffy ears, and lodging of plants before harvest. White mold (Sclerotinia stem rot) can be a serious disease in rapeseed after flowering in seasons with cool, moist growing conditions (Weiss et al., 2006). Many insects may infest rapeseed at various stages of its growth. Probably the greatest problem is caused by the flea beetle, a shiny black beetle about 10 to 15 mm long which attacks rapeseed particularly at emergence, although it can be a problem later as well. Hot, sunny weather promotes feeding damage. Diamondback moth larvae can be a problem in dry years. The most serious diseases of sunflower are caused by fungi (Doll and Wedberg, 1980). The major diseases include rust, downy mildew, verticillium wilt, sclerotinia stalk and head rot, phoma black stem and leaf spot. The severity of these disease effects on total crop yield might be ranked: 1) sclerotinia, 2) verticillium, 3) rust (recently more severe), 4) phoma, and 5) downy mildew. Resistance to rust, downy mildew, and verticillium wilt has been incorporated into improved sunflower germplasm. Insect pests have become major potential yield-reducing factors in sunflower production in the northern Midwest of the USA. Insects specific to sunflower that feed on the heads include the larvae of three moths; sunflower moth, banded sunflower moth and sunflower bud moth. Largely diffused in the soybean field are the brown stem rot, stem canker and sudden death syndrome. These diseases are increasingly most likely to occur because of changes in tillage practices. Yield losses range from a few bushels to significant portions of the field being killed (especially for sudden death syndrome). Major rice diseases include Rice Ragged Stunt, Sheath Blight and Tungro. Rice blast, caused by the fungus Magnaporthe grisea, is the most significant disease affecting rice cultivation. Sheath blight damage can range from partial infection of the lower leaves with little effect on grain development to premature plant death. On some varieties, the panicle can be attacked during hot, humid weather. Both yield and grain quality may be reduced when the disease prevents the flow of water and nutrients to the grain. Grain may then develop only partially or not at all. Poorly developed grains usually break up during milling thus reducing quality. Sheath blight is more prevalent during periods of warm moist weather, and in thick, lush stands because of the high humidity which develops in the canopy. The pathogen thrives when the canopy humidity is above 95% and temperatures are hot (80-90oF). Rice Blast (Pyricularia oryzae) Blast symptoms can occur on leaves, leaf sheaths, nodes and panicles. The most serious damage occurs when the fungus attacks nodes just below the head. The stems often break at the diseased node. Several cultural practices are important to reduce risks from rice blast. These tactics include: reducing fungal overwintering sites through incorporation of the rice stubble soon after harvest to promote early decomposition; grow rice in open fields free of tree lines, particularly on east and south sides; grow rice in fields where flood levels are easily maintained (Beck and Smith, 2000). Disease problems may appear with continued peanut production. Early and late leaf spot are two of the most important foliar diseases observed. These diseases are caused by

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different organisms but are controlled by the same practices and fungicides. Crop rotations two or three years out of peanuts reduces the survival of leaf spot spores in the soil and makes chemical disease control more effective and profitable. Deep plowing reduces leaf spot inoculum. An important disease is produced by the fungus Aspergillus flavus; it cause extensive economic losses either by destroying the plant or by contaminating peanut kernels with the aflatoxins. This fungus grows when the temperature is between 30° and 35° C and when the moisture content is above 9% in the kernels or above 15% in the oilcake. The attack of this fungus can be prevented, with minimum damage to the hull or kernel, by careful harvesting and by quick drying and storage in low humidity. Thrips are cited as an important insect pest in peanut production. Thrips feed in terminal leaf clusters of young leaflets and cause dwarfing and malformation of leaves. Injury usually occurs during the first month after plant emergence (Lee et al., 1992; Melouk and Shokes, 1995; Smith et al., 1998). The olive moth is very widespread in many Mediterranean olive-growing countries (López-Villalta, 1999; Tzanakakis, 2006). The larvae fed on flower parts. Second-generation larvae penetrate young fruit. Depending on the seriousness of the injury, the fruitlets may or may not drop off. In some cases, massive fruit drop may cause severe crop loss. Jasmine moth is found in tropical and subtropical areas on all five continents. The damage in nurseries and young orchards can be considerable, affecting up to 90% of the leaf area. When fruit are attacked (August – September), heavy yield loss can occur. Pyralid moth is most prevalent in Tunisia, Morocco and Spain. The larval galleries damage the plant vessels and block circulation. Leopard moth is an insect that attacks numerous species of shrub from more than thirty botanical families. Found all over the world, it is a serious pest of olives only in Eastern Mediterranean regions; e.g., Lebanon, Syria, Jordan, Israel. Olive fly is the most widespread and best-known olive pest. Typically found in Mediterranean countries, it is also prevalent in other regions, including the United States. The olive fly is responsible for economic loss caused by direct damage (due to fruit drop and fruit weight loss) and by indirect damage (due to the poor quality of oil produced by infested orchards). Olive leaf spot is the most common olive disease, found in almost all olive-growing countries. Lesions produced by this disease cause leaves to fall off, resulting in lower production, loss of axillary buds and retarded tree development. Fruit stems and the fruit itself can also be affected. If the fruit become infected, ripening slows and low quantity and quality oil is produced. When relating oil quality to pests and diseases, two aspects must be considered: the first is the direct effect of the pest or pathogen on the extracted oil; the second is the possibility that chemical residue might be found in the extracted oil if the pest- and disease-controlling chemicals are used incorrectly (López-Villalta, 1999; Tzanakakis, 2006; Casacchia et al., 2009). 1.7. Cultivars 1.7.1 Traditional cultivars Botanically cultivated peanut can be classified into two subspecies which mainly differ in their branching pattern: subspecies hypogaea with alternate branching and subspecies fastigiata with sequential branching.

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Each subspecies is again divided into two botanical varieties; subsp. hypogaea into var. hypogaea (Virginia) and var. hirsuta; and subsp. into var. fastigiata (Valencia), var. vulgaris (Spanish), var. peruviana, and var. aequatoriana. Thousands of peanut cultivars are grown, with four major groups being the most popular: Spanish, Runner, Virginia, and Valencia; in trade, the bold-seeded types are referred to as Virginia, the small seeded as Spanish. Runner types are commonly used for peanuts butter production. Virginia types are directed to in-shell market or to snacks market. Spanish types are used mainly in confectionery industry but also for oil extraction for the higher oil content; Valencia types with three or four small seeds for pods and with sweet taste are produced for snacks market. The variety selected will depend largely on soil type and length of growing season (Knauft and Gorbet, 1989; Stephens, 1994; Whitty et al., 1999). Rapeseed cultivars are derived from the Brassica genus and the Cruciferae family. In this family, numerous species have been interbred to form a number of sub-species. The common commercial name rapeseed or oilseed rape widely used in Europe includes seeds of oilseed turnip rape (B. campestris synonymous with B. rapa), oilseed swede rape (B. napus) and mustards (B. juncea, B. nigra, B. hirta synonymous with Sinapis alba). There are three basic species; B. nigra, B. oleracea and B. rapa, which through hybridisation, produced the new species B. carinata, B. juncea and B. napus. The term Canola is also widely used in world literature and trade and refers to the marketing name developed by the Canadians for oilseed rape cultivars which are both low in glucosinolates and the fatty acid erucic acid, the so-called double-low (or “OO”) cultivars. All cultivars used for food products are of the canola type. The various species, sub-species and types of brassica have seeds that differ in size, seed coat colour and oil content (Berglund et al., 2007; Hardman, 1986; Philbrook1986; Canola production handbook, 1989). The genus Helianthus, to which the sunflower belongs, contains about sixty-seven species and nineteen subspecies. The majority of the species are perennial with only about a dozen annual species. The most important species are H. annuus L., H. mollis L., H. argophyllus L., H. debilis L. and H. tuberosus L. Sunflower (H. annuus L.), largely cultivated as oilseed crop, is an annual herbaceous plant, member of the Asteracae family with a typical composite flower (Robinson, 1973; Carter, 1978; Oplinger, 1978). The dent corn (Zea mays indentata) type is the most cultivated in the world, followed by the flour corn (Zea mays amilacea) which is preferred for human nutrition and for poultry. All the other types of a modest importance are used in alimentation industry (pop corn) or the chemical industry or ornamental plants (Alexaander, 1988; Crop and soil management, 2007/08). The soybean has a large number of cultivars. However, it is known that the progenitor of the modern soybean was a vine-like plant that grew prone on the ground. The genetic variation within the same species is enormous. There are thousands of varieties of soya including 'red', 'black' and 'green'’. The most popular variety is the 'yellow' soybean (Glycine max), which belongs to the family of the papilionaceous. The varieties can also be classified in geographical areas: Indian, Japanese, Manchurian and Chinese. The Manchurian and Japanese have high fat content, whereas the Chinese and Indian are characterized by high protein content. The genus Glycine Wild is divided into two subgenera (species), Glycine and Soja. The subgenus Soja(Moench) includes the cultivated Soybean, G. max (L.) Merrill, and the wild soybean, G. soja Sieb.& Zucc. Both species are annual. The soybean grows only under cultivation while G. soja grows wild in China, Japan, Korea, Taiwan and Russia. Glycine soja is the wild ancestor of the soybean: the wild progenitor. At present, the subgenus Glycine consists of at least 16 wild perennial species: for example, Glycine canescens, and G. tomentella Hayata found in Australia and Papua New Guinea (Anonymous, 1995; Fehr, 1987).

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Human selection and adaptation to diverse environments has resulted in numerous rice cultivars. It is estimated that about 120 000 varieties of rice exist in the world. After the establishment of International Rice Research Institute in 1960, rice varietal improvement was intensified and high yielding varieties were developed (Khush, 1987; Garris et al., 2004). While most breeding of rice is carried out for crop quality and productivity, there are varieties selected for other reasons. Cultivars exist that are adapted to deep flooding, and these are generally called 'floating rice' (IRRI rice knowledge bank, 2008). The largest collection of rice cultivars is at the International Rice Research. With over 100,000 rice accessions (IRRI rice knowledge bank, 2008) held in the International Rice Genebank Rice cultivar. s are often classified by their grain shapes and texture. For example, Thai Jasmine rice is long-grain and relatively less sticky, as long-grain rice contains less amylopectin. than short-grain cultivars. Chinese restaurants usually serve long-grain as plain unseasoned steamed rice. Japanese mochi rice and Chinese sticky rice are short-grain. Chinese people use sticky rice which is properly known as "glutinous rice" (note: glutinous refer to the glue-like characteristic of rice; does not refer to "gluten") to make zongzi. The Japanese table rice is a sticky, short-grain rice. Japanese sake. Indian rice cultivars include long-grained and aromatic Basmati. (grown in the North), long and medium-grained Patna rice and short-grained Sona Masoori (also spelled Sona Masuri. In South India the most prized cultivar is 'ponni' which is primarily grown in the delta regions of Kaveri River. Kaveri Within the olive species, more than 2000 cultivars have been already described for the Mediterranean area as a whole (Bartolini et al., 1998). In Italy alone at least three hundred cultivars have been enumerated, but only a few are grown to a large extent. The main Italian cultivars are 'Leccino', 'Frantoio' and 'Carolea'. A lot of these "cultivars" are difficult to classify because of the many different names given to the same plant in different regions and countries. Some particularly important cultivars of olive include:

• 'Frantoio' and 'Leccino'. These cultivars are the principal participants in Italian olive oils from Tuscany. Leccino has a mild sweet flavour while Frantoio is fruity with a stronger aftertaste. Due to their highly valued flavour, these cultivars have been migrated and are now grown in other countries.

• 'Arbequina' is a small, brown olive grown in Catalonia, Spain. • ‘Picual’ most important cultivar in Spain • 'Empeltre' is a medium sized, black olive grown in Spain. They are used both as a

table olive and to produce a high quality olive oil. • 'Kalamata' is a large, black olive, named after the city of Kalamata, Greece, used as a

table olive. These olives are of a smooth and meatlike taste. • 'Koroneiki' originates from the southern Peloponese, around Kalamata and Mani in

Greece. This small olive, though difficult to cultivate, has a high oil yield and produces olive oil of exceptional quality.

• 'Picholine' originated in the south of France. It is green, medium size, and elongated. Their flavour is mild and nutty.

• 'Lucques' originated in the south of France (Aude département). They are green, of a large size, and elongated. The stone has an arcuated shape. Their flavour is mild and nutty.

• 'Souri' originated in Lebanon and is widespread in Syria, Lebanon, and Israel. It has a high oil yield and exceptionally aromatic flavour.

• 'Barnea' is a modern cultivar bred in Israel and to produce a generous crop. The oil has a strong flavour with a hint of green leaf. Barnea is widely grown in Israel and in the southern hemisphere, particularly in Australia and New Zealand.

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• 'Maalot' is another modern, disease-resistant, Eastern Mediterranean cultivar derived from the North African 'Chemlali' cultivar. The olive is medium sized, round, has a fruity flavour and is used almost exclusively for oil production.

• 'Mission' originated on the California Missions and is now grown throughout the state. They are black and generally used for table consumption.

1.7.2 ‘New’ - conventional Traditional plant breeders improve the quality and yield of crops by crossing plants with desired traits to create a new, hopefully improved, hybrid strain. Consequently, the availability and performance of superior hybrids essentially have made the practical use of open-pollinated corn obsolete. Open pollinated corns often suffer from low yields and poor standability. Currently, much interest is being generated with new high-oil corn hybrids marketed. High-oil corn is a variety of corn that has been genetically selected to contain a greater germ portion in the corn kernel. Current high-oil corns will typically contain about 6.3 % (Alexander, 1988). Commercial high-oil corn hybrids have not been widely used because their yield potential is lower than that of normal hybrids. Recently, however, an alternative system for producing high-oil corn has been developed. This system of producing high-oil corn was developed by DuPont and is known as the Topcross system (Thomison and Geyer, 1998 and 2001). A TopCross Blend® is a mechanical mixture of two types of corn seed. One type, representing 90 to 95 percent of the seed in a bag, is a hybrid that is designated as the "Grain Parent." The normal male fertile version is called the "Grain Parent Check." The second type, representing all remaining seed, is a special "Pollinator". The Grain Parent is a male sterile version of an elite hybrid that may already be in commercial production. The Pollinator is a special line, available from a seed company, that sheds pollen within a TopCross Blend production field. The pollen shed from these Pollinator plants contain special genes that cause a kernel to produce a much larger than average germ or embryo (commonly called xenia effect). Since most of the oil and protein is in the germ, the oil, and thus the energy level, and protein quality of the grain produced by fertilization with these pollinators is enhanced. High oil corn also contains more protein, lysine, and metabolizable energy than normal corn; and less starch than normal corn. High oil corn have extra value because energy is a key feed requirement, and energy density has become important with modern livestock genetics allowing high growth rate animals. main corn hybrids available on the market are AgriGold A6415TC7, Beck 5405TC, Beck X5727TC, Callahan TC7616D, Callahan TC7761D, DeKalb DK595TC, DeKalb CR8691, Golden Harvest H-2515HOC, Golden Harvest H-2581HOC, LG Seeds 2583TC, LG Seeds 2604TC, Pfister SK2652-19, Pfister SK3049-19, Pioneer 34K79, Select Seed 4321, Select Seed 4897. With respect to sunflower, the development of a cytoplasmic male-sterile and restorer system for has enabled seed companies to produce high-quality hybrid seed (Robbelen et al., 1989). Most of these outyield open-pollinated varieties and are higher in percent oil. Performance of varieties tested over several environments is the best basis for selecting sunflower hybrids. The choice should consider yield, oil percentage, maturity, seed size (for non-oilseed markets), and disease resistance. Performance results from the Upper Midwest are usually available annually from North Dakota State University, University of Minnesota, and South Dakota State University. Low Erucic Acid Rape (LEAR), double low, and Canola refer to varieties that have been developed through conventional plant breeding and have been selected to produce oil that has reduced levels of the anti-nutritional factors; i.e. erucic acid

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and glucosinolates. Glucosinolates are responsible for the pungent odour and biting taste associated with brassicas. High Erucic Acid Rape (HEAR) is unsuitable for use in culinary markets because of its high erucic acid content and is only used for industrial purposes. Oil from HEAR contains 40 - 50% erucic acid. HEAR has applications in the lubricant, mastics, release agents and agrochemical markets. Oil produced from high oleic (HOLL) varieties contains increased quantities of oleic acid and a reduced quantity of linolenic acid compared with standard rapeseed oil. HOLL varieties have been developed through conventional plant breeding techniques. These varieties are of particular interest to food frying industries as they are approximately twice as stable as regular rapeseed oil and three times as stable as sunflower oil. Double low rapeseed varieties represent the typical rape oil accounting for the majority of food and fuel uses. These cultivars have reduced contents of both erucic acid and glucosinolates. Double low varieties have been developed since the 1970’s through traditional plant breeding methods and can be either winter or spring-sown varieties. 'Double low' or canola oil is high in monounsaturated fatty acids such as oleic acid, it is also a rich source of the essential fatty acids linoleic acid and alpha-linolenic acid that are necessary for growth and maintenance of cell membranes. The presence of vitamin E and phytosterols add to the nutritive value of the oil (see other MACOILS reports on oilseed rape). 1.7.3 GM crops A genetic engineering procedure where a gene from one, sometimes unrelated, organism is transferred to another can give a plant a new property, like resistance to insect or virus attack and tolerance to herbicides. Thus introducing new genes, using the tools of genetic engineering, is a targeted approach for improving plant characteristics (Pechan and de Vries, 2005). The use of these genes, or transgenes as they are sometimes called, should be a more predictable approach than traditional plant breeding where thousands of unknown genes are exchanged. Unlike traditional plant breeding however, the approach is new, can involve gene transfer that does not occur naturally and has not been tested for long-term side effects on the plant. Genetic engineering of crops is a new addition to traditional plant breeding. It is part of our quest to grow more and better food for our growing population. The total area under genetically modified (GM) crop cultivation has been steadily expanding with, however, a marked slowdown in the last few years. Soybean, corn and cotton are the most extensively cultivated GM crops (Table 1.8). Soybean represents over 60% of the total GM crop area. These crops were developed to allow better management of weeds and pests. In Europe, however, it is not currently possible to buy GM fresh produce. Even in North America, most fruit and vegetables are not yet available as GM products. Europe grows less than 0.5% of the world’s GM crops, mainly because of the de facto moratorium imposed on GM crops in Europe until the year 2003 and the refusal of the European consumers to buy GM products. Currently grown GM crops mainly benefit the farmer and not the consumer. Future uses of GM crops may bring more direct benefits, such as improved taste and quality, to the consumer. All food, whether GM and non-GM, might have certain risks of containing allergic or toxic compounds and all novel foods should be tested for possible allergic and toxic effects on living organisms. Some environmental groups are also concerned about the effect of GM crops on the environment. Most of all they are worried about the unknown effects these crops may have on our environment and the difficulty of controlling GM crops once released into the environment. The discussions about GM crops and food are indeed complex and include issues of choice, globalisation and alternative ways to grow crops.

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Europe is going ahead with comprehensive labelling and tracing of GM crops and their products. However, even if there were such standards, there is currently no mechanism to implement and enforce these standards on the international market. Soybeans are one of the "biotech food" crops that have been genetically modified, and GM soybeans are being used in an increasing number of products. In 1995 Monsanto introduced Roundup Ready (RR) soybeans that have had a copy of a gene from the bacterium, Agrobacterium sp. strain CP4, inserted into its genome by means of a gene gun, that allows the transgenic plant to survive being sprayed by this non-selective herbicide, Roundup. Glyphosate, the active ingredient in Roundup, kills conventional soybeans. RR soybeans allow a farmer to spray widely the herbicide Roundup and so to reduce tillage or even to sow the seed directly into an unplowed field, known as no-till farming or conservation tillage. No-till agriculture has many advantages, greatly reducing soil erosion and creating better wildlife habitat (Anonymous, 2008a) it also saves fossil fuels and sequesters CO2, a greenhouse effect gas. (Brookes and Barfoot, 2005). Similarly, the the main objectives of groundnut improvement are discovery, characterization and development of resistances or tolerances to single or multiple stresses in addition to the production of high oil yielding cultivars. The wild Arachis species are reservoir of genes for high levels of resistance to various stresses; for this reason genetic resources programs EMBRAPA, Brazil and of ICRISAT (International Crops Research Institute for the Semi-Arid Tropics) maintains a lot of groundnut accessions of wild Arachis species from 89 countries (www.icrisat.org). National genetic programs have done big efforts including in improved germplasm, multiple genetic resistance for rust, late and early leaf spots, aflatoxin, rosette, and peanut bud necrosis virus. Novel techniques such as genetic transformation, molecular markers added selection, and gene transfer from alien sources are yet to make an impact on groundnut research. Nowadays a large number of improved varieties are being tested on-farm in several countries. Right now, no large scale production of genetically modified rice is taking place. Although a GM rice cultivar (LL62) has been approved in the US, farmers have not yet begun using it (www.gmo-compass.org). An approval application for the food and feed use of LL62 rice has been submitted to the EU. It is still undergoing safety evaluations. This GM rice cultivar was genetically engineered to be resistant to an herbicide. This should make weeds easier to control than previously possible. Controlling weeds and pests are the main reasons why 80 percent of the world's rice fields are flooded. Rice was not originally an aquatic plant. Rather, it was adapted to flooded conditions by breeding. Other genetic engineering projects focus on altering rice's nutritional value. Golden Rice is the most well-known example of this. Rice is known for having too little iron and vitamin A. In regions where rice is eaten almost exclusively, vitamin A deficiency is widespread. Insufficient vitamin A leads to vision problems, and in some cases, blindness. Researchers in Zurich and in Freiburg, Germany, with funding from international foundations and enterprises, succeeded in creating a rice cultivar offering beta-carotein, a metabolic precursor to vitamin A. Owing to its yellow color, it was called Golden Rice. Golden Rice also possesses increased iron content. In 2004, Golden Rice underwent its first field tests and is to be available from 2011. Golden Rice will be provided free of cost to small-scale farmers in developing countries. Projects are underway in Japan for developing rice cultivars that are less of a problem for people with rice allergies. In order to do this, researchers are trying to repress the activity of a gene that leads to the formation of an important allergen (AS-Albumin). As of yet, researchers have not been able to completely eliminate all traces of albumin. Although many field trials with genetically modified rapeseed have been conducted in Europe, it is not yet being grown commercially (www.gmo-compass.org). Several lines of GM rapeseed have been approved for production and use as food and feed. GM rapeseed has been grown in Canada since 1996. In 2007, GM rapeseed was grown on 5.1 million hectares,

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which made up approximately 87 percent of Canada's rapeseed crop. GM rapeseed is grown to a lesser degree in the US and in certain states in Australia. All of the GM rapeseed grown throughout the world is herbicide resistant, which enables a more efficient and effective approach to weed control. Genetically modified rapeseed cultivars could soon be available that have changes in the composition of their oil. The types of fatty acids found in an oil are what determine its physical and nutritional properties.

• Genetically modified rapeseed could be developed with a higher content of long-chain fatty acids. Fatty acids with longer side chains remain solid at higher temperatures. This means that margarine could be produced with fewer processing steps.

• Certain applications require a higher composition of mid-chain fatty acids. For several years, a GM rapeseed cultivar was grown in the United States that contained a gene needed for the production of lauric acid. This mid-chain fatty acid, usually produced from coconut milk, is used as a raw material for the production of detergent additives. GM rapeseed enriched with lauric acid can also be used for producing fat-based coatings in food processing. Apparently, this GM rapeseed cultivar did not meet its expectations. It is no longer being grown.

Maize is the only GM crop that is grown commercially in the EU (www.gmo-compass.org). For the most part, maize is used for feeding livestock and as raw material for the starch industry. Starch, however, forms the basis of many foods and food additives. Genetically modified maize was grown for the first time in the US and Canada in 1997. Since then, GM maize production has expanded to more than 35 million hectares worldwide. Now, about 80 per cent of the maize produced in the US is genetically modified. Many countries in North and South America, Africa, and Asia grow GM maize. Two traits are expressed by today’s GM maize cultivars: insect resistance and herbicide tolerance. More and more, cultivars are being grown that express both of these traits simultaneously (stacked genes). 1.8. Specific Cultivation Practice Affecting Oil Characteristics Variety selection, environmental conditions, irrigation and fertilization all exert substantial influence on the oil quality parameters. These can be broadly separated into four principal groups including: environmental (soil, latitude, climatic), agronomic (irrigation, fertilization), cultivation (harvesting method, ripeness) and technological factors (storage, extraction procedure).

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1.8.1 Environment The fatty acid composition of rapeseed varies between varieties and changes according to environmental conditions (Kamel-Eldine & Appelquist, 1996). As seed matures, temperature can affect oleic and linoleic acid contents (see above) whereas the effect of nitrogen applications is less important and depends on the applications date (Steer & Seiler, 1990). A higher linoleic/oleic acid ratio (higher degree of unsaturation) is achieved within rape grown in temperature regions than in warmer regions. The increase in temperature can also promote the biosynthesis of tocopherols. Sunflower oil content and composition has been shown to be influenced by temperature. This phenomenon was studied on plants grown under field conditions. High temperature during the development of the seed was associated with a reduction in total oil yield. However, under field conditions this effect was variable owing to confounding with other environmental factors such as moisture stress, which also influence the yield of oil through their effects on growth and development of seed. Elevated temperatures, and in particularly high night temperatures, cause a marked reduction in the percentage of linoleic acid and higher oleic acid percentage, apparently due to the effect of temperature on the activity of the desaturase enzymes which are responsible for the conversion of oleic to linoleic acid (Harris et al., 1978). Infact it has been observed that oil from northern regions tends to be higher in linoleic acid and has a higher ratio of polyunsaturated to saturated fatty acids than oil produced in southern latitudes (Putnam et al., 1990). Fatty acid composition is affected by plant genotype and by environmental conditions with a major influence of temperature. The largest variation in oleic acid percentage was observed in the traditional hybrid and the lowest in the high oleic hybrids (Izquierdo, 2002). However, night temperature variation during fruit filling did not affect crop development and yield (Izquierdo, 2002). Even though temperature treatments can increase the oleic acid content of traditional hybrids, indicating that sowing date and location are important factors in determining oil quality; hybrid selection remain the most important agronomic practice affecting oil quality and composition. Other experiments on the dynamics of fruit growth and oil quality of sunflower exposed to high temperature (>35°C) during grain filling have shown that there is a direct detrimental effect of brief periods of heat stress on grain and embryo growth, oil deposition patterns and oil quality in sunflower (Rondanini, 2003). Salvador et al., (2003) showed that certain cultivation areas in Spain produced higher quality olive oil that was reflected by superior sterols composition. Mousa et al., (1996) demonstrated that olive oils from high-altitude orchards (800 m above sea level) were higher in their peroxide value and lower in the their total polyphenol content compared with low-altitude orchards (100 m above sea level). Cimato et al., (2003), comparing oil from 'Frantoio' olives grown in different soils in Italy, showed that soil environments affected polyphenol and tocopherol contents. Their findings were supported by Ranalli et al., (1999). 1.8.2 Harvest time Premature swathing of rapeseed may induce reductions in both seed yield and quality and therefore it is important that harvesting operations are undertaken at the appropriate optimum time. The effect of seeding dates and harvesting times on seed yield and quality was investigated by Vera., et al (2007) on a number of Canola varieties. Results identified that seed yield, weight, protein content (oil-free meal basis) and oil content tended to increase with

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seed development and delayed swathing time. A higher seed yield was more likely to be achieved with early seeding in good growing conditions, resulting in heavier mature seeds with high oil content. Seed oil composition was also seen to change during seed development with the proportion of oleic (C18:1) and linolenic (C18:3) acids increasing, whilst myristic (C14:0), palmitic (C16:0), palmitoleic (C16:1), stearic (C18:0), linoleic (C18:2) and arachidic (C20:0) acids decreased. Seeding dates and harvesting time had no effect on the levels of the long chain fatty acids eicosenoic (C20:1) and erucic (C22:1) acids although as seeds matured, the overall amount of fatty acids synthesised increased. Swathing was considered advantageous over direct combining in preventing weather induced shattering, which increases in windy conditions and heavy rain events. Olive oil compounds (phenols, tocopherols and pigments) have been reported to decrease in quantity and oxidation stability and shelf-life were reduced as ripenining proceded (Gutierrez et al., (1999), Gomez-Rico et al., (2006), Tawalbeh et al., (2006), Caponio et al., (2001) and Lassez et al., (2008)). Based on their findings, Caponio et al., (2001) recommended delaying harvesting of olive cultivars that usually yield bitter to pungent oils, but anticipating harvesting of olives that produce sweet-tasting oils. Delaying harvesting of olive cultivars has been also associated with a decrease in the saturated fatty acid content and MUFA/PUFA ratio and increased linoleic acid content (Beltran et al., 2004; Gutierrez et al., 1999; Simões et al., 2002). Ranalli et al., (1998) used the point of minimum fruit respiration rate during the ripening process as an index to obtain maximal oil quality. With respect to peanuts, dry basis chemical composition has not been reported to be influenced by is not affected by harvest date except for carbohydrates content which decrease slightly as digging is delayed. Peanut oil content increase and carbohydrates decrease as peanuts mature. Total unsaturated fatty acids content increase with maturity (Kim and Hung, 1991). Results from another has suggested that as seeds progressed from intermediate through nearly-mature to mature stages, palmitic and linoleic acids (%) decreased while oleic acid increased (Hinds and Singh, 1994). 1.8.3 Cultivars The most important factor affecting olive oil characteristics seems to be the choice of cultivar. As reported in numerous studies (e.g., Tura et al., 2007; Tous et al., 2005; Failla et al., 2002; Caravita 2007), different cultivars have different oil characteristics. A large survey of olive germplasm collected in Catalonia, Spain revealed variations in fatty acid composition (oleic-acid content varied from 61% with 'Blanqueta' to 78% with 'Picual'), in saturated-to-unsaturated fatty acid ratios (from 4.3 with 'Argudell' to 8.4 with 'I-55'), in MUFA/PUFA ratios (from 3.3 with 'Blanqueta' to 17.3 with 'Picual'), and in polyphenol content (from 127 ppm with 'Farga' to 888 ppm with 'Villalonga'). Similarly, 'Picual' tested in germplasm collection in Cordova, Spain gave superior results, while extremely high polyphenol content was obtained from the 'Chetoui' variety (1,240 ppm) (Tous et al., 2005). In general, palmitic-palmitoleic and palmitic-linoleic acid content ratios are strongly correlated in different olive genotypes. This group is negatively correlated with oleic acid content (Leon et al., 2004; Tous et al., 2005). New breeding programs are considering the sensorial, physicochemical and nutritional value of oil as positive criteria for selection of new cultivars (Ranalli et al., 2006). In particular, oil quality, in terms of fatty acid composition and content in phenolic compounds, was analysed for many new genotypes previously selected in an Italian breeding programme and cultivated in three different locations of central and southern Italy (Ripa et al.,

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2008). The availability of data from many genotypes cultivated in all three locations allowed quantitative analyses of the genetic and environmental effects on the oil quality traits studied. The acidic composition varied greatly both with genotype and with environment and so did the concentration in phenols, though the effect of genotype on phenols was not significant (P=0.17). The fatty acid composition, particularly the oleic/(palmitic+linoleic) ratio, appeared predominantly under genetic control, with heritability of 0.58 while the environmental effect explained 0.31 of the total variance. Phenolic compounds, instead, had lower heritability (0.29) and was more affected by the environment, which explained 0.50 of the total variance. Other breeding programs aim to 'correct' inferior oil characteristics; as was done in Tunisia to improve low oleic acid content in the local 'Chemlali' variety (Manai et al., 2007). The development of a cytoplasmic male-sterile and restorer system for sunflower has enabled seed companies to produce high-quality hybrid seed. Most of these outyield open-pollinated varieties and are higher in percent oil (Murphy D.J., 1994.). Recent studies highlight the main differences between seed oils produced from conventionally cultivated crops and organically cultivated ones and processed using mild extraction procedures. No significant trends were found in the oil samples for triacylglycerols and fatty acid composition, but remarkable differences were found in the composition of minor components and in the main chemical and analytical quality properties (Velasco et al., 2002). Trans fatty acid were found only in the conventional oils. Tocopherols are the most important compounds having antioxidant activity in sunflower seeds. Moreover, should be stressed that for alpha-, beta- and total tocopherol content, the effect of the genotype was larger than that of the environment, whereas the latter had a greater effect on gamma-tocopherol content (Velasco et al., 2002). While normal dent corn contains 3.5 to 4.5 percent oil, commercial high-oil types contain as much as 1.5 times this amount. The major difference between high oil corn and normal corn production is the need to follow management practices, from planting through storage, that will preserve grain identity in the system, i.e. prevent reduction in grain oil content. Certain high-yield management practices recommended for use in normal corn production become especially important in high oil corn production. Growers must use recommended agronomic practices, including the maintenance of good soil fertility and good control of weeds and other pests, to minimize the variability in performance between the two genetically different seed types contained in TC Blend seed corn. 1.8.4 Irrigation-water quality Oil extracted from the fruit of non-irrigated olive trees (as compared to irrigated trees) was reported to be higher in quality, showing lower acidity, higher phenol content, better stability and richer, fuller taste (Ben Gal et al., 2007; Gomez-Rico et al., 2006; Castro et al., 2006; Farinelli et al., 2006). Water stress may possibly be requisite to promotion of phenol production. Water stress has previously been demonstrated to increase the activity of certain enzymes, including L- phenylalanine ammonia-lyase, responsible for synthesis of phenolic compounds (Patumi et al., 1999; Tovar et al., 2002b). No effect of irrigation level was found on fatty acid composition (Patumi et al.,1999; 2002) in Italy and in Spain (Tovar et al., 2002a), while in Tunisia a reduction in oleic acid content was reported as water deficits increased (Gharb et al., 2006). The increased free fatty acid content found in irrigated olives could be a function of increased fruit sensitivity to pressure-induced mechanical injury, due to higher water content and thinner cuticle layer (Patumi et al., 2002). Mechanical injury incurred during harvest accelerates the enzymatic processes that are detrimental to oil quality.

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This phenomenon increases with increased delay between harvest and oil extraction. Organoleptic testing also indicated that non-irrigated olives resulted in higher quality oil. A possible explanation is that when water content of olives is greater, as it is in the fruit of irrigated trees, hydrophilic components of taste and smell in oil are removed to a greater extent during oil extraction and processing. The organoleptic differences corresponded to differences in polyphenol content of the oil; some taste parameters are polyphenol compounds themselves. It would seem, therefore, that a decrease in polyphenol content would result in a reduction in the richness of the taste of the oil. 1.8.5 Fertilization The effect of excess fertilizer application has been studied for several vegetable oils. Increased unsaturation and unsaturated-to-saturated fatty-acid ratios due to higher N concentrations have been found in a number of oils, including cottonseed, sesame, rapeseed and linseed. Increased unsaturation was also found as a consequence of raising K concentrations in linseed, sunflower and sesame oils. Rapeseed varieties are considered to be nitrogen-demanding plants with seed production increasing during maturation as total nitrogen applications increase (Sebei et al., 2004). As seed weight increases oil content is seen to decrease with increase of non-oil, proteinaceous fractions in seed. Palmitic and oleic acid contents also decrease in conventional double low cultivars as seed weight increases (Appelquist L. 1967, cited in Sebei K., et al, 2004). Tocopherol contents increase considerably during crop maturity and are affected by both state of crop maturity and nitrogen application rate. Total tocopherol content varies significantly between species. Research has shown that sunflower responds to N, P and K. Nitrogen is usually the most common limiting factor for yield. Nitrogen fertilizer tends to reduce oil percentage of the seed, change the amino acid balance, and increase leaf area of the plant. Yield increases from N fertilizer rates up to 175 lb/acre have been observed, but rates considerably lower than this are usually recommended. More yield increases are reported as a result of applications of P than from K in Europe and North America. Fernández-Escobar et al., found that N overfertilization in 'Picual' olive trees induces a decrease in total polyphenol content and consequently, reduces oxidative stability of the oil. Morales-Sillero et al., tested the response of 'Manzanilla de Sevilla' olives to increased fertigation levels (4N-1P-3K) and found lower polyphenol contents, K225 (bitterness), and oxidative stability in oils made from trees receiving greater doses of fertilizer. The MUFA content, of oleic acid in particular, decreased with increasing amounts of applied fertilizers, while PUFAs, specifically linoleic acid, increased. SimÕes et al., reported that a high level of N and K supplied to the soil around tree trunks in a 'Carrasqueña' orchard causes a significant decrease in saturated fatty acid (SAFA) content and an increase in both unsaturated-to-saturated and polyunsaturated-to-saturated fatty-acid ratios. In contrast, no differences in oil quality were found with foliar applications of N and K to 'Carolea' olive trees. Dag et al., (2008) found that greater P and N levels significantly influenced the composition of oil from fruit of the ‘Barnea’ cultivar, while K levels have only a minor effect. In addition the levels of PUFAs increased compared to the those of the MUFAs. Specifically, the level of the MUFA C18:1, polyphenol content and peroxide values decreased while the level of the omega 3 PUFA C18:3 increased in response to higher doses of N and P. The increased levels of PUFA suggest, on the one hand, a decrease in oil stability but on the other, increased nutritional benefits.

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1.9. Concluding remarks Agricultural practices have a major influence on the quality of oil produced from the various oil crops. It is therefore pertinent to provide the consumer with readily accessible information concerning the crop production practices. Furthermore, quality management systems such as ISO and euroGAP require from the farmer a detailed documentation of the production process. Extensive implementation of these standards by the growers is requires as well as making these data readily available to the consumer. There are large gaps in term of production optimization between the various oil crops cultivation practices that range from the naturally growing argan trees, through large scale traditional cultivation of olive, to the large scale industrial scale cultivation of canola and soybean. Thus, greater attention should be given to the traditionally cultivated oil producing crops. On the other hand, the recent tendency to return to sustainable and organic agriculture that relies on traditional cultivation practices cannot be overlooked. It is therefore important to address these issues by investigating the influence of various agro-technical practices on the oil quality of the oil producing crops. In this sense, the information contained in this chapter is just the tip of the iceberg.

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CHAPTER 2

Extraction, Refining, Conservation and Packaging

Methods of Edible Oils

Authors Contributors CHARROUF, Z.1 GHARB, S.1,6

CHIARELLO, M.D.2 GUILLAUME, D.7

DI STASIO, M.5 MATTHAÜS, B.8

KEREM, Z.3 MOR, W.3

PAGES, X.4

PINELI, L. de L. de O.2

ROSSIGNOL-CASTERA, A.4

VOLPE, M.G.5

1 Faculté des Sciences Université Mohammed V- Agdal, FS-UMV-Agdal, 1014 Rabat (Morocco) 2 Universidade Católica de Brasília (UCB), 70790-150 Brasília (Brazil) 3 Institute of Biochemistry, Food Science and Nutrition, The Robert H. Smith Faculty of Agriculture, Food and

Environment, The Hebrew University of Jerusalem (Israel) 4 French Institute for Fats and Oils (ITERG), 33600 PESSAC (France) 5 Instituto di Scienze dell’Alimentazione (ISA-CNR), 83100, Avellino (Italy) 6 Laboratoire contrôle qualité LESIEUR – CRISTAL, 20300 Roches Noires – Casablanca (Morocco) 7 Université de Reims Champagne-Ardenne, 51100 Reims (France) 8 Max Rubner-Institute,Federal Research Institute for Nutrition and Food Department for Lipid Research, D-

48147 Münster (Germany)

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Abstract The present chapter has the objective of presenting the methods of edible oil processing and packaging. The chapter is divided into four parts. In the first part, olive and argan virgin oils processings are described. To get high quality virgin oils, the process includes the selection of the fruits, the conditions of their transport and storage, the choice of the steps of the mechanical processing : milling, traditionnal cold pressing, decantation or centrifugation, filtration, and finally the control of the storage and packaging conditions of the final virgin oils. In the second part, are described the extraction conditions of the crude oil from seeds : maize, soyabean, sunflower, peanut and rapeseed seeds. The preparation steps include cleaning, drying, dehulling, milling and/or cooking operations. The crude oil is then obtained by mechanical ways (screw press extraction, expeller) and by hexane extraction including the operations of oil and cake desolventizing. The third part describes the physical and chemical refining processes that can give edible refined oils with good quality, safety, stability and organoleptic specifications. The traditional refining of crude oils include degumming, neutralization, washing, drying, dewaxing, bleaching and deodorization steps. New refining technologies, for instance with membrane processing, can give some economical, environmental, nutritional or sensorial advantages. The last part of the chapter approaches the aspects of packaging and storage of oils considering their stability to oxidation under different parameters, in particular light and heat. The choice of the material for packaging, in particular its permeability to air, is very important to get a correct shell life of the edible vegetable oils.

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2.1. Extraction and Peservation of Virgin Oils 2.1.1 Olive oil The craft of extracting precious oil from olive fruits has been honed in the Mediterranean region over thousands of years, and techniques have been passed down from generation to generation. The process is truly a regional art, i.e. the method used in Greece is different from the one used in Spain or in Morocco, and each individual grower may have his unique way of tending the trees and producing the olive oil. However, there is no particular secret to the technique in making olive oil, but rather an intense commitment to quality through great attention to detail: Starting with selecting a high quality fruit; protecting it with a good harvesting, transport, storage, and finally correct and careful processing, and ending with conservative oil storage techniques. In other words: Start with a good fruit, and as quickly as possible - crush it, separate the oil from the fruit-water and solids, then store it in a cool, dark container that excludes oxygen. Then you have captured the essence of fruity olive oil that reflects the characteristics of the variety, region and harvesting season. Some consumers pay more attention to the origin, and some prefer the sensorial qualities. Some use aromatic oil in their dishes, while others prefer delicate oil. All can be of extravirgin quality, and are the result, at least in part, of the production and storage practices. Storage of the olive fruit Olive oil quality is directly related to the physiological conditions of the olives from which the oil is extracted. Olive processing in the major producing countries (such as Spain, Italy, and Greece) is often not well synchronized with crop harvests (Garcia and Streif, 1991; Gutierrez, Perdiguero, Garcia, and Castellano, 1992). Olives are often piled into large heaps and stored at ambient temperature for up to several weeks prior to processing for oil extraction (Garcia, Gutierrez, Castellano, Perdiguero, and Albi, 1996), and this is when the greatest deterioration takes place (Olias and Garcia, 1997). Pressure within the olive pile during storage may cause secretion of fluid from the olives thereby providing an optimum medium for the growth of fungi and bacteria (Olias and Garcia, 1997). Under these conditions, anaerobiosis can occur in the inner part of the pile while aerobic losses occur in the outer part (Garcia and Streif, 1991). Furthermore, heat production from respiratory activity may accelerate the deterioration of the fruit (Garcia and Streif, 1991) and eventually cause the breakdown of cell structure (Gutierrez, Perdiguero, Garcia et al., 1992). Oil extracted from these damaged olives can be high in acidity and low in stability (Garcia, Gutierrez, Castellano et al., 1996) and can develop a great amount of volatile acids (acetic or butyric) that cause a characteristic musty smell (Olias and Garcia, 1997). In a few days, the physical and chemical structure of the olives is altered and the oil extracted from them has a very poor quality. This type of oil must be refined before consumption. (Gutierrez, Perdiguero, Garcia et al., 1992). Numerous methods to store fruits have been suggested to manage this important problem (Kader, 1986; Petruccioli and Parlati, 1987; Kader, Nanos, and Kerbel, 1989, 1990; Castellano, Garcia, Morilla, Perdiguero, and Gutierrez, 1993; Garcia, 1993a, 1993b; Garcia et al., 1994; Perez-Camino, Garcia, and Castellano, 1992; Garcia, Gutierrez, Barrera, and Albi, 1996; Pereira, Casal, Bento, and Oliveira, 2002; Koprivnjak, Conte, and Totis, 2002; Clodoveo et al., 2006).

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Washing and leaf removal Preliminary washing aims to remove foreign material that could damage machinery or contaminate the oil. With the increasing awareness to the presence of pollutants, more mills add washing to their common practice. Washing may lead to reduction in polyphenol content, lower bitterness rating, lower "Piquant" rating, and a less fruity flavor and loss of oil stability. Wash water is often dirty and can flavors into the oil. Milling Olive fruit is made up of roughly 1/3 solid material, 1/3 water, and 1/3 oil. Crushing, the first step of oil production aims to produce a fine, homogenous paste with easily extracted oil droplets. The olives may be crushed by a hammer, disc or knife crusher, and a depitting (de stoner) machine has also been introduced recently. The latter is a new procedure that includes ‘stone’ (seed or ‘pit’) removal before the olive oil extraction process (Amirante et al., 2001). Small quantities of leaves are not detrimental to the oil and sometimes leaves are added to produce a chlorophyll (green) color and flavor in the oil. Branches and wood material are however very detrimental to olive oil flavor producing a woody taste. Different crushers (Angerosa and Di Giacinto, 1995; Angerosa and Solinas, 1990) and different malaxation practices (Lanzani et al, 1990; Montedoro and Garofolo, 1984; Montedoro, 1992; Solinas et al, 1978) modify phenolics and volatiles contents and composition, partially due to the activation of enzymes of the lipoxygenase (LOX) pathway (Morales et al, 1994). Volatiles, with C6 and C5 compounds forming the major volatile fraction, form the various nuances of the positive attributes of olive oil (Angerosa et al., 1998a; Angerosa et al, 1998b; Angerosa et al, 2000; Hatanaka, 1993; Vick and Zimmerman, 1987; Salch et al., 1995). The enzymes levels are genetically and physiologically determined, whereas the technological operations dramatically affect their activities. The modern form of the oldest crushing system, stone crushers, consists of a stone base and upright millstones enclosed in a metal basin, often with scrapers and paddles to guide the fruit under the stones and to circulate and expel the paste. The slow movement of the stone crushers does not heat the paste and results in less emulsification so the oil is easier to extract without as much mixing (malaxation); the major disadvantages of this method are the bulky machinery and its slowness, its high cost, and its inability to be continuously operated. The stones are also more difficult to clean, and the slow milling time can increase oxygen exposure and paste fermentation. Stone mills, because of their inefficiency, are replaced by metallic mills in most large operations. The hammer mill, which may represent here metallic mills, generally consists of a metal body that rotates at high speed, hurling the olives against a metal grate. The major advantage of metal crushers is their speed and continuous operation, which translate into high output, compact size, and low cost. Their major disadvantage is the type of paste produced: The oil is more emulsified, requiring a longer mixing period to achieve a good oil extraction and the speed of metal crushing can produce elevated temperatures and possible metal contamination. Both lead to reduced oil quality. Metallic mills are rather easy to clean and fast, allowing for the deployment of a continuous flow system. Oil produced from a hammer mills is generally greener due to an extensive grinding of the skins. A new continuous processing line made up a de-stoner (instead of the usual metal crusher) allows the separation of the kernel, without an agressive crushing of the fruit, concurs the reduction of the mechanical actions and reduces the heating responsible of the degradation and oxidation phenomena (Amirante et al, 1987; Amirante et al., 2002). However, it results in a lower oil yield. Oils obtained from de-stoned pastes have higher polyphenol and C5 and C6 volatile compounds contents.

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Mixing of the olive paste (malaxation) Malaxation prepares the paste for separation of the oil from the pomace. This step is particularly important if the paste was produced in a metallic mill. The mixing process optimizes the amount of oil extracted through the formation of larger oil droplets and a reduction of the oil-water emulsion. The paste is slowly mixed, bringing small droplets of oil in contact with each other to form larger droplets to improve the extractability of the oil (Montedoro et al., 1992). The temperature of the paste during malaxation is very important (26°C to 30°C, which is still cold to the touch) to improve the viscosity of the oil and improve extractability. Temperatures above 30°C can cause problems such as loss of fruit flavors, increases in bitterness, increases in astringency, and unfavorable increase in deleterious enzymatic activities (Olias et al., 1993; Salas and Sanchez, 1998a,b,c). The LOX pathways become active at the olive crushing, and the concentrations of C6 and C5 volatile metabolites change in relation to malaxation time (Angerosa et al., 1998b; Salas and Sanchez, 1999a,b). Low temperatures are always recommended for the malaxation step, whereas the choice of the optimal malaxation time should be made so that a satisfactory yield and a good quality of oil can be achieved. Times ranging between 30 and 45 min, are common to satisfy these requirements. Temperature is mainly responsible for: (i) the sensory flattening of oils, (ii) very considerable losses of secoiridoid compounds, (iii) the marked decrease of concentration of C6 esters, very important contributors of delicate green perceptions, and of cis-3-hexen-1-ol which gives pleasant real green sensations, (iv) the increase of hexan-1-ol and trans-2-hexen-1-ol considered elicitors of less attractive perceptions (Aparicio et al., 1994; Bedukian, 1971; Aparicio et al., 1996); (v) the production of very high contents of 2-methyl butanal and 3-methyl butanal through the activation of the amino acid conversion pathway. Oil Extraction from the paste The next step is extracting the oil from the paste and fruit water (water of vegetation). The oil can be extracted by pressing, centrifugation, percolation, or through combinations of the different methods. Pressing is the oldest method of oil extraction. The method involves applying pressure to stacked filter mats, smeared with paste, that alternate with metal disks; a central spike allows the expressed oil and water (olive juice) to exit. The machinery, is cumbersome, the process requires more labor than other extraction methods, the cycle is not continuous, and the filter mats can easily become contaminated. Oil from presses falls into both extremes: producing the best oils when properly operated because they tend to have greater flavor and higher polyphenol content; and imducing defects, through fermentation of mats. This process is the opposite of the press system since no pressure is applied to the paste. It operates on the principle that in a paste containing solid particles, water and oil the oil alone will adhere to metal. The machine has stainless steel blades that dip into the paste, the adhering oil then drips off the blades into a separate container while the solids and water are left behind. The lack of pressure produces light oil with unique quality and value. Historically, olive paste, or olive juice containing both water and oil was allowed to sit in containers until the oil, with a lower specific gravity, rose to the top naturally. The oil was then decanted away from the remaining water and solid material. This natural separation takes considerable time and the contact of oil with enzymes, breakdown products and fermenting fruit water produced defective oils. Modern decanters are large horizontal centrifuges that separate the oil from the solids and water in the same process as in a decantation tank, just faster. The savings in time increases the efficiency of the system, but also decreases the time the oil is in contact with the fermenting fruit water.

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The regulation of the differential velocity screw/bowl allows a better performance of the decanter at low dilutions of the olive paste; it is possible to obtain high efficiency at a low dilution of the olive paste achieving a higher minor compounds content and the more advanced system of regulation leads to better results according to the variations of the ratio between liquid and solid phases, thus optimizing operational performances in relation to the rheological features of olive pastes (Catalano et al, 2003). The 3-Phase system decanter separates the paste into a relatively dry solid, fruit-water, and oil. A minimum quantity of water is added to separate the solid material better and to retain water-soluble polyphenols as much as possible. Preferably, the solid should have an oil content of no more than 6-7% and 50% moisture while the vegetation water should not contain over 0.3% oil and-8% solids. 2-Phase system decanters were introduced in the early 1990's. They function under the same principle as 3-Phase decanters except that the solid and fruit-water exit together. No water needs to be added to the 2-Phase system. The 2-Phase system has some advantages, i.e., good retention of polyphenols because no water is added and less loss of oil if the system is operated properly. One problem with the 2-Phase system is a greater potential to lose oil when the olives are low in moisture because there is a thinner interface between the two phases during centrifugation. Water can be added to the paste just prior to entering the 2-Phase decanter if the moisture content of the olives falls below 42%. Talc (a water absorbing neutral compound) can be added to the paste early in the season, if the olives have an excessive moisture content, to help extract a greater quantity of oil with no negative effect on quality. The 2-phase system produces the greatest weight of solid waste because it has the highest moisture content. It also produces the least amount of wastewater with the lowest Biological Oxygen Demand (BOD). The polyphenol content of the oil is lowest in the 3-phase system because of the addition of water . Vertical centrifuges spin at two times the velocity of a decanter and provide four times the separation force for the solid, water, and oil phases. They provide an additional separation of the three phases to further remove solid particles and water from the oil. Fresh warm water is added to "clean" the oil, creating a greater interface area between the phases. Many processors use two centrifuges, one for the "wet" oil from the decanter and a second one to separate the oil from the wastewater of the first centrifuge. Added water is only 2-4°C warmer than the water/oil mixture to be separated. Filtration The oil extracted from the olive paste is a turbid and opalescent must and contains impurities that can compromise its quality since they facilitate hydrolysis, fermentation and rancidity. Although filtration of this oil removes these otherwise damaging substances, it can also cause small changes in the oil. Bottino et al., (2004) proposed a membrane cross-flow filtration as a different and innovative filtration process for extra virgin olive oil. The preliminary results show that the removal of damaging substances through membrane filtration can be achieved in a single step, without the addition of filter aids (therefore reducing oil loss). Membrane filtration also does not alter the chemical composition of the oil.

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2.1.2 Argan oil Argan tree (Argania spinosa (L.) Skeels) is only endemic in Southwestern Morocco. The vast aera covered by argan trees is named the argan forest or argan grove and it goes along the Atlantic Ocean from Essaouira (northern limit) to Guelmim (Southern limit). On the world stage, the argan forest constitutes a unique ecosystem that was recognized as a biosphere reserve by the UNESCO in 1998. The argan forest is currently covering slightly more than 800,000 ha, but its extension was twice as large at the end of the nineteenth century (Chaussod, 2005). Protection of the argan grove is an urgent necessity today. Modernization of argan oil production, the only valuable product able to bring more income to the Berber population (the traditional argan forest dwellers) is an absolute necessity, that will lead to production of large quantities of oil and ascertain its quality. Argan oil exists as a cosmetic or edible grade oil. Both types of oil have traditional uses (Charrouf, 1999). Extraction For years, argan oil has been prepared exclusively by Berber women following an ancestral multistep process (Charrouf, 2002). From May to August, fallen ripe fruits are collected through the argan forest. The fruits are covered by a latex-rich peel, collected manually, and sun-dried for a few days. Only then, their dried peel can be manually removed, resulting in “argan nuts”. Argan nuts are then broken between two stones and the white kernels (one to three for each argan nut) are collected. 100 kg of fruits will produce 60 kg of argan nuts, from which only 6.5 kg of kernels. To prepare edible argan oil, the kernels have to be gently roasted in clay plates for a few minutes. The amount of time depends on each woman who evaluates it from the color of the kernels but an average of 1h15 to 1h45 hour is necessary to roast 6.5 kg of kernels. Roasting time strongly influences the final oil taste and a burning taste resulting from overheating of the material has to be avoided. To get a mild fire, the argan shells obtained during the stone breaking step are used. Next, the roasted kernels are crushed using a millstone. This simple and frequently home-made mill is composed of two stones: a bedstone and a cone-shaped rotating piece largely pierced in its center and in which the kernels are introduced. The brownish viscous liquid is then mixed with water and the dough is hand-malaxed for several minutes, slowly getting solid and releasing an emulsion from which argan oil is finally decanted. In summary, for a single person, from 100 kg of dried-fruits, 2 to 2.5L of oil can be obtained after 58 hours of work. The solid residue remaining after the maximum quantity of oil has been collected may still contain up to 25% of oil. Its taste is very bitter, but it has high energy content. This residue also contains a good amount of proteins, and sugar-containing derivatives so it is traditionally used to feed goats and cows. Obviously the traditional method is very slow and mainly suitable to satisfy family needs. The variable roasting time leads to oils with varing organoleptic properties (Charrouf, 2006). Furthermore, argan fruits are sometimes voluntarily given to goats and argan nuts can then be collected in goat stools, leading to potentially microbiological and quality problems (Hilali, 2005). All steps are inconsistent with the production of high quality argan oil, thus, improved methods have been designed and applied in women cooperatives. Improvements have recently been made to the argan oil preparation process to ascertain argan oil quality (Charrouf, 2002). For this, goat-depulped fruits can not be used and roasting by fire as well as water for extraction have been eliminated from the process. Depulping and pressing steps have also been modernized. Consequently, the temptation of collecting depulped fruits is strongly reduced. Optimizing the roasting step has allowed the

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production of an oil of repeatable taste. Finally, to replace the hand-press step, mechanical screw presses are used. Kernels are pressed at room temperature and no water is necessary. Multiple-head presses allow now the production of large quantities of high quality argan oil and the limiting factor, in oil production, is now the disposability of sufficient quantities of kernels. Conversely to most edible oils, edible argan oil, as well as virgin olive oil, is not refined. Because argan oil is not refined, its content in unsaponifiable matters (mainly polyphenols, tocopherols, and phytosterols) is high. These latter compounds are currently recognized for their contribution to the properties of edible argan oil. Large cosmetic laboratories that include argan oil in their products refine (by physical methods) argan oil prior to use it. This step is necessary to get limpid oil having reproducible color and odor characteristics. Effect of processing on the quality of Argan oil Sensory quality and storage stability are two important parameters for evaluating the quality of the product. Indeed, the oil will have success on the market only if its quality meets the demands and requirements of the consumer. Research shows that production conditions have a strong influence on the oxidative stability and the sensory characteristics of argan oil. Roasting of the seeds strongly improves the oxidative stability of argan oil (Fig. 2.1). Several parameters including a better extractability of antioxidant active compounds from the seeds or the formation of such compounds (e.g. Maillard reaction products) during the roasting process could be responsible for this effect (Eichner, 1980; Lingnert and Eriksson, 1981). At high storage temperature (60°C ) the effect of the roasting step on the oxidative stability is much more pronounced than during storage at 20°C. The effect of processing is much stronger on the sensory evaluation of the argan oil than on its oxidative stability (Fig. 2.2). Again roasting of the kernels led to an improvement of the oil quality. While oil from unroasted seeds had a nutty taste, oil prepared from roasted seeds was characterized by the appearance of nutty and roasty attributes. In oils from unroasted seeds the sensory sensations changed very fast within 4 weeks of storage and attributes like fusty as well as Roquefort cheese appeared resulting in oil inedible for humans. During storage the attribute nutty disappeared within 16 weeks and a taste for Roquefort cheese developed. The attribute roasty was perceivable during 20 weeks of storage, but resulting from the taste for Roquefort cheese the oil had to be assessed as inedible after 12 weeks of storage. No change of the sensory characteristics was found for oils from roasted kernels obtained by mechanical extraction. Most dramatically was the effect of seeds obtained from goat digestion on the sensory evaluation. Oil obtained from this raw material was characterized by a typical smell and taste for Roquefort cheese, which becomes obviously directly after pressing. During storage the intensity of aroma compounds responsible for this taste and smell increases resulting in oils with a very strong taste and smell for Roquefort cheese. Since the attribute Roquefort cheese can be correlated with the use of improper raw material (seeds obtained from goat digestion) or a decrease in quality during storage the appearance of this attribute in the oil is a clear indication for improper oil quality.

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2.2. Refined Oils: Preparation and Extraction The crude fats and oils from oilseeds can vary from pleasant-smelling products that contain few impurities to quite offensive-smelling, high impure materials. Processing techniques allow us to make them more useful to the food industry. Advances in lipid processing technology during the past century have resulted in dramatic increases in the consumption of edible fats and oils. Innovations such as deodorization, hydrogenation, fractionation and interesterification, along with improvement in other processes, have allowed the production of products that can satisfy demanding functional and nutritional requirements (O’Brien, 2004). It is important to the consumers to know the consequences of each unit operation on nutritional, safety and sensory quality of the final product, so they can choose properly which oil is better for their expectations. Fats and oils extraction and processing consists of a series of unit processing in which both physical and chemical changes are made to raw materials. Product quality and process efficiencies improvement, reduction of capital expenses and solution or reduction of environmental problems can motivate extraction and processing changes by oil industry.

0

2

4

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unroasted,mech. extracted

roasted,mech. extracted

roasted,trad. extracted

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Pero

xide

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e[m

eqO

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0 4 8 12 16Storage time at 20°C [weeks]

20

unroasted,mech. extracted

Pero

xide

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e[m

eqO

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0 5 7 14 21Storage time at 60°C [days]

28 35

0

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25

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Figure 2.1. Peroxide value of argan oil during storage at 20°C (Z. Charrouf, Faculté des Sciences Université Mohammed V- Agdal, Morocco)

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nutty roasty fusty Roquefort cheese

unroasted,mech. extracted

roasted,mech. extracted

roasted,trad. extracted

roasted, goats,trad. extracted

0 20124 8 16 0 20124 8 16 0 20124 8 16 0 20124 8 16 weeks

Inte

nsity

(0 =

not

dete

ctab

le; 5

= s

trong

dete

ctab

le)

0

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3

Figure 2.2. Sensory evaluation of argan oil during storage at 20°C (Z. Charrouf, Faculté des Sciences Université Mohammed V- Agdal, Morocco)

2.2.1 Preparing operations Oilseed processing starts just after harvest, when handling operations such as drying, cleaning and preparing steps (conditioning, cracking, dehulling, flaking, expanding) are conducted in order to optimize quality and yields in the extraction and refining. As oilseeds present different botanical structures and oil contents, different preparing operations are required to improve both extraction and refining steps. The operations before oil extraction are very important to the oil final quality. In fact, the seeds must be properly stored and prepared for extraction, to maintain high quality in the final product. Oils in the presence of water deteriorate rapidly, forming free fatty acids and rancid off-flavours (Casten and Snyder, 2001). Corn and corn oil Corn (Zea mays L.) is a plant of the family Poacea. Due to archaeological evidences it is believed that corn proceeded from Mexico, and America was the first continent to cultivate it, being subsequently introduced in Europe, Africa and Asia, becoming an essential food for the survival of the ancient civilizations and an ingredient base of several industrialized products of the modern civilization (Hui, 1996). The corn seed (kernel) is composed of three main parts: the outer layers (hull or bran, and tip-cap), which account for about 6% of corn kernel weight and is destined mainly to cattle feed, the germ, containing most of the oil and from which crude corn oil and the oil cake are obtained, and the endosperm, composed of gluten and starch. Industrial separation of the useful components of corn takes advantage of the biology of the corn kernel. The oil is in the germ, while the starch and gluten are found in the endosperm (Olson and Warren, 2000). From an average bushel of corn weighing 25 kilograms (kg) (56 pounds [lb]), approximately 14 kg (32 lb) of starch is produced, about 6.6 kg (14.5 lb) of feed and feed products, about 0.9 kg (2 lb) of oil, and the remainder is water (EPA, 1994)

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Corn germ obtained by wet milling contains 48-52% oil, whereas that obtained by dry milling containing 25-30% oil. In contrast, soybean contains 17-20% oil by weight; rapeseed (canola) contains about 42% oil; cottonseed (29%); sunflower meals (32%) (Maza, 2001). Corn oil is highly digestible and provides energy and essential fatty acids (Dupont et al., 1990). It is also known by its excellent oxidative stability in various applications, including frying. That high stability is due to the presence of natural antioxidants like tocopherols, tocotrienols, pherulic acids and ubiquinones (coenzyme Q), to its fatty acids composition, to the position of polyunsaturated in the middle of triacylglycerols and to the absence of chlorophyll (Souza, 2004; Moretto and Fett, 1998; Leibovitz and Ruckestein, 1983; Hui, 1996). Other components are phospholipids, glycolipids and unsaponified fraction which is composed mainly by tocopherols, tocotrienols, esterols and carotenoids, accounting for less than 3% of the oil (Gunstone et al., 1994). Desirable flavour and the oxidative stability of corn oil during use in combination with its lack of precipitation under refrigeration have contributed to its market demand. All but a minor fraction (<5%) is used in feed; the largest single use is bottled oil, followed by margarine and industrial snack-frying operations (Orthoefer et al., 2003). There are two primary types of milling: “dry milling” which produces flour and meal primarily and “wet milling” which produces high fructose corn syrup, oil, starch, some animal feed products, and ethanol primarily. Although the quality of corn oil is good, the yield of oil is not as high as in some other crops (Okoruwa and Kling, 1996). In general, at large scale the wet milling process is more used than dry milling. The wet milling apparently allows an efficient removal of aflatoxins and impurities from corn (Mejía, 2005). For dry milling, shelled corn arrives at the industrial plant and is accepted through quality check procedures. The corn is cleaned of foreign materials. Once clean, the moisture content of corn is raised to about 20%. The corn germs are then removed for oil extraction and the remaining corn is ground and sieved into many fractions which vary in particle size and composition. The primary products of dry milling are flour, corn meal and grits. Additional products include corn bran and feed mixtures. These products are used in brewing, foods, building products (binders), fermentations (pharmaceuticals and fuel), and animal feeds (EPA, 2001). In general, corn quality attributes such as high test weight, high percentage of horny endosperm, low broken corn and foreign matter and low breakage susceptibility are desirable for dry milling. Corn types with a high proportion of horny endosperm are hard. Hardness of corn kernels, an important intrinsic characteristic, affects power requirements for grinding, dust formation, kernel and bulk density and yield of dry milled products, especially grits. Dry milling requires kernels that are free of detectable mycotoxins. Kernel size and shape are also important. Too large proportions of small kernels, such as those from the tip of the ear, are objectionable. Hard kernels may require regrinding to produce smooth flours. Another factor to consider is storability. Soft kernels are susceptible to insects, ear rots, and possibly aflatoxin (Okoruwa and Kling, 1996). In wet milling, the protein and starch are separated. The starch is used in packaged foods. It is also hydrolysed to produce dextrose, corn syrup solids or glucose for use in food production. Fibre and protein by-products of wet milling are used as animal feed. The germ is used as raw material for corn oil production by mechanical pressing or by extraction methods. The basic steps for wet milling as described by the Corn Refiners Association (2006a) and Minnesota Corn Processors (1999) include steeping, germ separation, fine grinding, starch separation, fermentation, and syrup conversion. Corn processed by wet milling is typically separated into 5 basic components: starch, germ, gluten, fibre and steep liquor (Blanchard, 1992). By-products of wet milling along with corn germ and most corn gluten are used for animal feed products. A very small amount of corn gluten is subjected to acid hydrolysis

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resulting in amino acids and/or short peptide units called hydrolyzed vegetable protein (EPA, 2001). Shelled corn is delivered to the wet milling plant primarily by rail and truck and is unloaded into a receiving pit. Normally number 2 grade corn is purchased, based on U.S. Department of Agriculture standards (Corn Refiners Association, 2006b; USDA, 1975). The corn is then elevated to temporary storage bins and scale hoppers for weighing and sampling. The corn then passes through mechanical cleaners designed to remove unwanted material, such as pieces of cobs, sticks, and husks, as well as meal and stones. The cleaners agitate the kernels over a series of perforated metal sheets through which the smaller foreign materials drop. A blast of air blows away chaff and dust, and electromagnets remove bits of metal. It then is conveyed to storage silos, holding up to 350,000 bushels, until ready to go to the refinery. Coming out of storage bins, the corn is given a second cleaning before going into "steep" tanks (EPA, 1994). Steeping takes place in stainless steel steep tanks which hold from 70.5 to 458 cubic meters (m3) (2,000 to 13,000 bushels [bu]) of corn, operated in continuous-batch process. Corn is stored in these tanks for approximately 30 to 40 hours in 50° C soaking water which contains approximately 0.1% sulphur dioxide. Sulphur dioxide prevents excessive bacterial growth, separates starch and insoluble protein by disrupting the protein matrix through cleaving protein disulphide cross-links and allows dissolved sugars to be converted into lactic acid, which is helpful in maintaining pH near 4.0 (Weigel et al., 1997). During the incubation period, kernel moisture levels increase to between 15 and 45%, which also results in an increase in kernel size (up to 2X). The objective is softening the kernel for milling, helping break down the protein holding the starch particles, and removing certain soluble constituents as the corn is stored in the mildly acidic steep water, the gluten bonds within the corn loosen, which allows for starch release. Corn that has steeped for the desired length of time is discharged from its tank for further processing, and the tank is filled with fresh corn. New steeping liquid is added, along with recycled water from other mill operations, to the tank with the "oldest" corn (in steep time). The drained water used in steeping from the kernels is concentrated in multiple effect evaporators to yield concentrated steep water. This protein rich extract may be used as a nutrient for micro-organisms in the production of enzymes, antibiotics and other fermentation products. The major portion, however, is combined with fibre and gluten in the production of animal feed ingredients (Corn Refiners Association, 2006b). The steeped corn passes through degerminating mills, which tear the kernel apart to free the germ and about half of the starch and gluten (EPA, 1994) and to loosen the hull. Water is added to the attrition mills and a thick slurry of macerated kernels and whole germ results. Germ contains 25-50% of oil, so it is lighter than the endosperm and hull and can be separated from the slurry by centrifugation, using cyclone separators. They spin the low density corn germ out and pump the germ onto screens where the germ is washed repeatedly to eliminate residual starch, dewatered, and dried; the oil extracted; and the germ cake sold as corn oil meal or as part of corn gluten feed. Washings from the germ are piped to the starch centrifuges (Corn Refiners Association, 2006a,b). The corn and water slurry are moved from the germ separator into an impact or attrition-impact mill to release the starch and gluten from the fibre in the kernel. The suspension of starch, gluten and fibre flows over fixed concave screens which catch fibre but allow the starch and gluten to pass through. The fibre is then collected, slurred and screened again to reclaim any residual starch or protein, then piped to the feed house to be used as a major component of animal feed (EPA, 2001). The product stream contains starch, gluten, and soluble organic materials. The lower density gluten is separated from the starch by centrifugation, generally in two stages. The

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gluten fraction passes to a concentrator, and then it is filtered and dried. Final corn gluten is applied as feed product. The centrifuge underflow containing the starch passes to starch washing filters to remove any residual gluten and soluble particles. At this point, the starch has only approximately one to two percent of protein remaining. The starch is diluted 8 to 14 times, rediluted and washed again in hydroclones to remove the last trace of protein and produce high quality starch (usually > 99.5% pure) (EPA, 2001). The pure starch slurry is now directed into one of three basic finishing operations, namely, ordinary dry starch, modified starches, and corn syrup and sugar (EPA, 1994). Quality factors such as low percentages of broken or damaged kernels and foreign matter, and high test weight, are desired for maximum starch and oil yields in the wet milling process. If oil is an important end product, the corn should have a large germ and high oil content. For starch production, a smaller germ that is easily separated from the endosperm is preferred. Starch, amylose, fat and protein contents as well as water absorptivity of grains are other quality parameters of importance in corn wet milling (Okoruwa and Kling, 1996) Peanuts and peanut oil The cultivated peanut or groundnut (Arachis hypogaea L.), originated in South America and is now grown throughout the tropical and warm temperate regions of the world. The peanut, grown primarily for human consumption, has several uses as whole seeds or is processed to make peanut oil, butter and other products. The seed contains 25 to 32% protein (average of 25% digestible protein) and 42 to 52% oil (Putnam et al., 1991). Peanut is one of the most vulnerable crops to Aspergillus flavus contamination (Dorner and Cole, 1997). This fungus produces aflatoxins, which are highly carcinogenic mycotoxins (Kaaya et al., 2006). Presence of aflatoxin is a concern mainly in peanut production areas with warm climates, so the two most important operations in handling peanut after harvest are cleaning and drying to safe moisture content (5 to 10%). Pods should be kept dry and protected against infestation from insects or rodents, as well as from loss of natural colour and flavour, and prevention of the development of off-flavours and rancidity. Artificial drying of wet or semidry peanut should start immediately after combining to prevent mould growth and aflatoxin formation. Safe storage of peanut requires an atmosphere with low relative humidity (60 to 70%) (Putnam et al., 1991). The process of preparing for extraction, which is fairly well standardized, consists of four principal operations: cracking, dehulling/hull removal, conditioning, and flaking. Peanuts are conveyed from the process bins to the mill by means of belts or mass flow conveyors and bucket elevators. In the mill, the peanuts may be aspirated again, weighed, cleaned of tramp metal by magnets, and fed into corrugated cracking rolls. The cracking rolls "crack" each peanut to facilitate separation of the hulls from the oil-rich nuts. The cracked nuts are passed through an aspirator to remove the hulls, which are processed separately. The cracked nuts and residual nut chips are then conveyed to the conditioning area, where they are either put into a rotary steam tube device or into a stacked cooker and heated to "condition" them (cooked). Conditioning is necessary to permit the flaking of the chips and to prevent their being broken into smaller particles. Finally, the heated, cracked nuts are conveyed and fed to a continuous screw presses or expellers where they are prepressed and approximately 50 percent of the oil is removed. The prepressed peanut cake is then conveyed to extraction where the balance of the oil is removed by hexane extraction (Woodroof, 1983).

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Rapeseed and rapeseed oil Rapeseed (Cruciferae family) is one of the most important oilseeds in the world, ranking fourth with respect to production after soybean, palm, and cottonseed (FAO/WHO/UNU, 2002). The oil from the seed has gained an excellent reputation for its nutritional qualities in the human diet and is the most important component of the seed in terms of market value. Its nutritive value is excellent due to its unsaturated fatty acids (Vuorela et al., 2003). The meal is an important source of protein in animal feeding. Rapeseed refers to more than one plant species and is often used to denote the seeds derived from oil yielding members of Brassica family, including some mustard seeds grown for edible or industrial oil (Salunkhe et al., 1992; Bengtsson et al., 1972). Brassica napus and Brassica campestris are the two most important widely grown species with summer types grown in North America and a mixture of summer and winter types grown in Europe (Salunkhe et al., 1992). In the 1970s, Canadian plant breeders developed a new rapeseed cultivar that yields oil low in erucic acid (Ackman, 1983), a brand new concept, registered in 1978 as Canola - for “Canadian Oil - Low acid” - by the Western Canadian Oilseed Crushers’ Association. The name rapeseed applies to this “old” rapeseed, i.e. HEAR (High erucic acid rapeseed) or LEAR (low erucic acid rapeseed), known as “0”-variety, whilst the term canola is reserved for the “00”variety, i.e. cultivars containing less than 2% erucic acid in the oil and less than 3.9 mg/g of thioglucosinolates in the extracted meal. There is also a “triple O” variety of canola, known as candle, with a low fibre content which can reduce the need for decortication –(cellulose is mostly found in hulls), and which is a feasible and industrially proven, but very expensive process (De Smet, 2004). In 1988, The U.S. Food and Drug Administration (FDA) agreed that LEAR oil (less than 2% erucic acid) could be identified as canola oil (O’Brien, 2004). Canola oil is considered premium grade owing to its favourable fatty acid profile and tendency to remain clear at reduced temperatures. The latter property is an economic advantage as canola oil has not required winterization, unlike other vegetable oils such as sunflower oil. However, most canola crushers solvent-extract the seed and the hull together for operational efficiency. As the seed hulls contain waxes, they can solidify in retail bottled oil, which requires prior dewaxing (O’Brien, 2004). Canola oil is low in saturates and high in monounsaturated. Distribution of linoleic and linolenic acids is important for flavour stability. They are found primarily in sn-2 position, similar to high erucic rapeseed oil. This gives better resistance to oxidation than other oils with similar linoleic and linolenic fatty acids content. An important concern refers to the oil’s content of chlorophyll and sulphur compounds. If hydrogenation is applied a higher catalyst concentration may be necessary due to the presence of residual sulphur compounds, which can poison the hydrogenation catalyst (Mag, 1983). The formation of large β crystals limits the level of canola oil that can be utilised in margarine or shortenings formulations. In this case, the addition of β’ crystal formers can prevent sandy or grainy consistency (O’Brien, 2004). The first plant ever to process edible rapeseed oil started operation in 1956, in Canada (De Smet, 2004). The processing methods used to extract the oil from the seed to produce a high quality raw oil for further processing and a high quality protein meal as an animal feed have been developed over the years. These methods are continually being improved (Mag, 1990). As seeds has about 37-56.5% of oil content (DeClercq, 2006; Zhao et al., 2005), pre-pressing followed by solvent extraction is currently used for best yields. Alternatively, cold-pressing can be performed. Cold-pressed rapeseed oil has been introduced to the market relatively recently, and therefore data on it are rather limited. Recent advances in extraction are presented in literature, such as enzymatic extraction (Zhang et al., 2007 a,b)), and microwave treatment, followed by cold-pressing (Valentová et al., 2002).

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The main problem in the extraction of rapeseed oil is to achieve a high yield of oil while maintaining high quality of protein. Operational parameters should be optimized during preliminary operations, processing and isolation of oil in order to reach this target. The process includes pre-processing, extraction, utilization of by-products and refining. Rapeseed is normally harvested when the seed contains less than 10.5% moisture. If the moisture is higher, the product must be artificially dried to less than 10.5% immediately after harvest. If seed is dried at 85oC, it must be processed without delay to prevent loss of oil quality. The quality of oil which has been dried at temperatures high enough to destroy the seed viability deteriorates after several months’ storage of the seed (Salunkhe et al., 1992). Drying is also essential, as the acidity of the oil increases when seed moisture exceeds the limits of stability, i.e. approx. 10% moisture. The heat released also leads to some hydrolysis of the proteins, or of contamination by micro-organisms. If a long storage is desired, seed should contain less than 9.5% moisture. Before extraction, seed is graded according to a strict standard (Daun and Burch, 1984). Payment is based on grade. It is the first step to help ensure that a quality oil and meal are obtained. Matthäus and Brühl (2008) studied what factors are determinant in virgin rapeseed oil. The period after harvest until processing was considered crucial whereas extraction of the oilseed and purification has only a minor influence on the oil quality. The authors also stated that improper storage conditions result in increased metabolic processes in the seeds and an increase of the populations of micro-organisms and insects, which finally leads to the degradation of nutrients and the formation of unpleasant aroma compounds. The graded seed is cleaned to remove plant stems, straw, stalks, chaff, stones, loose hulls or other cereals and seeds from the bulk of the seed. Pre-cleaning is carried out using high-capacity perforated drums inside which special aspiration channels remove the undesirable by-products from the harvest (De Smet, 2004). Aspiration, indent cleaning, sieving, or some combinations of these are also used in the cleaning process. Dehulling of the seed is, at present, not a commercial process (Mag, 1990). Cracked or broken seeds contain high amounts of free fatty acids which decrease quality of the oil, therefore, they must be removed in this step. In order to produce a high quality oil and meal the seed entering a crushing plant must be well-matured and sound. After cleaning the moisture content is adjusted to about 8.5%. Pre-cooking is recommended before flaking, mostly in case there is a risk of having frozen or cold rapeseed. In many extraction plants, the cleaned seed is first heated to about 30-40ºC to prevent shattering. The seeds must then be flaked to break the walls of the oil cells making it possible for the oil to be pressed out and/or solvent extracted. The main points to watch are the steadiness of the feed to the unit, and the condition of the roll surfaces. Flaking rolls may be smooth or corrugated and may operate at 0 to 5% speed differential. The flaked seeds should be from 0.22 to 0.25mm for prepress solvent extraction (Salunkhe et al., 1992). Cooking reduces the viscosity of the oil, which can then flow out of the cells, and the cake, during pressing. Drying to 3.5-5% is done at the same time. Drying is necessary to make it possible for the press to apply sufficient pressure to the cake, (De Smet, 2004; Salunkhe et al., 1992). In this operation, the flaked seed is heated from 30oC to about 75-100ºC in cookers (Mag, 1990; Salunkhe et al., 1992). These may be either vertical tanks with agitated, steam-heated trays or, in more modern plants, horizontal, rotary kilns equipped with steam coils. This heating, also called cooking or conditioning, is important in order to: rupture remaining intact cells to release oil, coalesce small oil droplets to larger ones, coagulate protein for better diffusion during solvent extraction, adjust the moisture content of the seed prior to solvent extraction, increase oil fluidity, precipitate phosphatides and destroy moulds and bacteria. Another very important function of the cooking operation is to control enzymatic activity in the flaked seed, which depends on temperature and moisture content. Two enzyme systems

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are of importance: myrosinase or thioglucosidase enzymes, which hydrolyze glucosinolates; and lipases, which hydrolyze triglycerides and phosphatides. Myrosinase is active between 40 and 70oC. For optimum inactivation, the temperatures of the flakes must be rapidly increased to 80 to 100oC. The hydrolysis processes must be suppressed to produce quality oil and meal. Glucosinolate breakdown products impair the quality of the meal and the oil; triacylglycerol and, especially, phosphatide breakdown products make the oil more difficult to refine and whereas myrosinase breakdown product causes problems of hydrogenation. A small concentration of these products is usually unavoidable (Salunkhe et al., 1992; Mag, 1990). For pressing, the flakes go directly from the cooker to the press. For direct solvent extraction, the cooked flakes are crisped in an open screw conveyer to leave porous granules, and re-rolled on a smooth roller mill to a uniform flake thickness. Soybean oil Soy oil has found many food uses due to its excellent nutritional qualities, widespread availability, economic value and wide-use functionality. Soy oil is also an important ingredient for industrial products such as paints, plastics, lubricants and biofuels. Other by-products of soybean processing include soybean hulls - mainly used in animal feeds and as a source of soy fibre, and soy lecithin phospholipids - a nutritional supplement and functional emulsifying food ingredient (Johnson and Smith, 2004). Soybean oil is a very versatile oil, refined with a low loss. It is classified as a natural winter or salad oil. It has heat sensitive pigments that deodorize to a red colour much less than 1.0 Lovibond. It develops large, easily filtered crystals when partially hydrogenated and fractionated It has a tocopherol level of about 1300 ppm as crude and retains more than 500 ppm level required for oxidative stability. It has a high essential fatty-acid content linolenic fatty acid content (6-10%) which requests a careful handling and metal chelating. Another important chemical and physical property is the high content (58.7%) of triunsaturated triglycerides (O’ Brien, 2004). Soybean oil can be submitted to different processes, which result into different characteristics in the final product. General process steps consist of the following steps: oilseed handling operations, preparation of soybeans for extraction, extraction and oil refining (Regitano D’arce, 2006; EPA, 1995). Soybeans received at the facility by truck or rail are sampled and analyzed for moisture content, foreign matter, and damaged or green seeds. As soybeans are purchased by grade, it is necessary to draw representative samples for quality evaluation from each lot at the point of reception. Sometimes oil and protein content, free fatty acids and other quality factors are also determined (Islas-Rubio and Higuera-Ciapara, 2002; Berk, 1992). Moisture analysis is a very important variable in the receiving step. As beans storage conditions reflects directly upon the yield and the quality characteristics of the final product, some storage properties must be considered. Grains absorve oxygen and release carbonic gas, water and heat. Chemical reactions that occur during the beans respiratory activity are controlled by enzymes. A high moisture content leads to a higher biological activity and a rapid increase in the temperature, which elevates even more respiratory rates. The higher respiratory rate, the faster grain deterioration will be. Besides, there will be a higher heat generation and if it is not lost in the same velocity, it can produce such heating that the seeds can be carbonized or cause fire when seeds are stored with a high moisture, it is also probable to present free fat acids content increasing, oil browning, flavour and chemical changes. For example, a decrease in iodine value was observed during soybean storage (Moretto and Fett, 1998). Changes in relative humidity and temperature can promote enzymatic reactions, such as lipase hydrolysis and enzymatic or chemical oxidation (Regitano D’arce, 2006). Cooperatives usually establish moisture criteria for soybean payment. For example, Western Iowa Coop

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harvest policy (Western Iowa Coop., 2007) determines that beans greater than 15% moisture are subject to rejection. Wet beans are discounted 1.5% of the sales price for each 1/2% moisture over 13%. Another cooperative, from Brazil (Cooxupé, 2005) establishes that soybeans with moisture until 14% have no discount and seeds with 15.0% to 20% moisture are discounted 1,5%. After analyses, the beans are weighed, cleaned to remove large foreign matter and conveyed to large concrete silos or metal tanks for storage (EPA, 1995). Pre-cleaning is recommended to prevent deterioration and unjust use of the silo space (Moretto and Fett, 1998). The typical storage facility in soybean oil plants is the vertical cylindrical silo, provided that soybean seeds are easier to drain, compared to cottonseed, for example. Transportation to an empty cylinder body is done whenever ventilation and homogenization are necessary. According to EPA (1995) one of the advantages of the metal silos is the speed of erection to prepare soybeans for the solvent extraction process. Foreign matter reduces oil and protein yields, adversely affects quality and increases wear and damage to the processing equipment. Stems, pods, leaves, broken grain, dirt, small stones and extraneous seeds are typical components of the foreign material found in soybeans (O’ Brien, 2004). Typically, the seed cleaner consists of a two-deck vibrating screen or sieves (EPA, 1995; Berk, 1992). The upper screen retains the stones and other coarse materials but allows whole soybeans to fall through. The lower screen retains the soybeans but let finer particles such as sand to pass through. Light trash, free hull particles and dust are removed by aspiration and trapped in cyclones. Permanent or electromagnets are also used for the removal of tramp irons objects (O’ Brien, 2004). According to Islas-Rubio and Higuera-Ciapara (2002), since the beans may become re-contaminated with stray iron (loose nuts and bolts, nails etc.) as they pass through the machinery, magnetic cleaning is not a one-time operation but must be repeated several times along the line. It is therefore advisable to install magnetic separators at the entrance of each machine where the presence of metal particles may cause serious damage (cracking mills, flaking machines etc). The hulls may be combined later with hulls from the dehulling aspiration step. Hurburgh et al., (1996), reported aspiration cleaning soybean presenting from 0.5 to 4.0% of foreign material and from 3 to 22% of splits. The air velocities were 10m/s and 19 m/s. The high airflow rate removed 1.1% of whole soybeans compared with 0.4 percent at low airflow rate. At either airflow rate, the aspirator removed less saleable material and more non-grain material than previously reported for screen cleaning (Hurburgh et al., 1996). A quite driven drying with temperature control will prevent oxidative processes, thermal shocks must be avoided in the exit of the drier, because they can cause small trines that can make easy the access to mould mycelia in search of the beans nutrients (Regitano D’arce, 2006). If the soybeans are to be dehulled before extraction, they must be dried to a moisture content below 10% in order to facilitate separation of the hulls. Drying is usually accomplished in a vertical column, direct fired unit, although steam, and even solar energy have been used as a heat source for certain installations. Naturally, the hotter the drying temperature, the faster the drying operation will be. However, drying temperatures must be closely monitored as excessive temperatures will damage the seed. It has been found that temperatures in excess of 63(C will significantly increase the colour of both the meal and the oil, denature the protein, increase the nonhydratable phosphatide levels in the crude oil, and result in greater potential for grain dryer fires (Anderson, 2005). If the natural moisture content of the beans is 10% or less, or if dehulling is not practised, drying as a preparation step can be omitted (Islas-Rubio and Higuera-Ciapara, 2002). Tempering is the operation consisting of allowing the moisture content to equilibrate to loosen the hull, which improves dehulling (Johnson and Smith, 2004). After cooling, the dried soybeans are stored in bins for 2 to 5 days, in order to allow for moisture equilibration

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by diffusion. The tempering bins, which are usually outdoors silos of the vertical type, also serve as working bins (day bins), to secure uninterrupted feeding of the plant. As all the subsequent steps of processing are continuous, it is necessary to monitor the flow of soybeans from the working bins to the processing plant, in accordance with the planned processing capacity. This is done by means of automatic balances installed at the feed-end of the line (Islas-Rubio and Higuera-Ciapara, 2002) According to Regitano D’arce (2006), the oil industry must decide before processing, which kind of meal is desired to produce, with 44 or 50% of protein. In the first case, whole soybean is processed. In the second, soybean must be dehulled before processing. Soybean contains from 6 to 8% of hulls and they need to be separated because they occupy space in the extractor and are abrasive for the cooking and the roller mill equipments. Dehulling guarantees lower residual oil content in the meal. A correct relative humidity at storage for determined time after the harvest promotes partial separation of the hulls (Woerfel, 1995). Preparation of soybeans aims at breaching the oil bodies in the grains by means of thermal and mechanical treatments, making the oil readily extractable by the solvent, when it is the extraction method to be applied (Regitano D’arce, 2006; EPA, 1995). The unit operations typically involve scaling, cleaning, cracking, conditioning (or cooking), and flaking. Depending on the process and the oilseed in question, process drying, and dehulling (or decorticating) may be employed, as may be expanders and collect dryer/coolers. After the preparation process, the prepared flakes or collets are delivered to the extraction operation (Anderson, 2005). Soybeans are conveyed from the process bins to the preparation steps (mill) by means of belts or mass flow conveyors and bucket elevators. In the mill, the beans may be aspirated again, weighed, cleaned of tramp metal by magnets, and fed into corrugated cracking rolls (EPA, 1995). The purpose of this operation is to break the seeds into smaller particles in preparation for flaking. If the beans have been dried to 10% moisture and tempered as described above, cracking also loosens the hulls and permits their separation by aspiration. Cracking machines consist of pairs of counter-rotating, corrugated rolls. One roll in each pair rotates faster than the other, to provide the shearing effect necessary to break the seed. Roll diameters vary from 8 to 10 inches in diameter and are usually driven by a fixed speed gear motor at about 25 to 30 rpm. The rolls are cut with a round-bottom groove that simply serves to agitate the hopper above the roll (Boling, 2004). Roll length depends on the capacity. Two or three pairs of rolls are provided, mounted one on top of the other. A vibrating conveyor secures feeding of the mill at a uniform rate. The corrugations on the upper pair of rolls are coarser and deeper than those on the lower pairs (Islas-Rubio and Higuera-Ciapara, 2002). Uniformity and particle sizes are important aspects for quality and yields, to make dehulling easier and to prevent fines formation. The uniform reduction of the grain size to 1/6-1/8 of its natural size is done so that, besides obtaining complete cells exposition in the flaking, flakes of ideal thickness and lengths are gotten, which favours the perfect extraction. It is prevented trituration or the exaggerated reduction of the grain since the fines make it difficult the separation of the solvent and the oil from the humid meal. Trituration also hinders a good separation of the hull and cotyledon (Regitano D’arce, 2006). Berk (1992) describes that vibrating screen is provided at the exit from the mill. This is where the stream of broken particles is separated into hulls (removed by aspiration for further processing), oversize particles (returned to the cracking mill), meats of the correct size (sent to conditioning and flaking) and fines (usually mixed with the meats for conditioning). There have been several novel approaches applied to dehulling in the past few years, well described by Anderson (2005). Hot Dehulling is the ultimate system offered in areas where beans are processed directly from the field, since its use generally eliminates the process drying step traditionally identified with the storage function. It is considered to be suitable to obtain a high protein/low

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fibre meal. After traditional cleaning, the seed may be delivered directly to the crackers or may enter the hot dehulling operation. The basic principle shared by the three commonly used hot dehulling systems is to dry the bean from storage moisture to process moisture, dehull the seed while still hot, and deliver the conditioned cracks to the flakers without allowing the seed to subsequently cool. This not only saves the energy of one heating step, as much of the air is recycled reducing the energy required for the integrated facility, but reduces the fines generated compared with the traditional system where the grain is cracked cold. One system that has gained wide acceptance is the Escher-Wyss system, which uses a fluid-bed dryer-heater to perform the drying process. After the dryer-heater the grain is discharged to specially designed high-speed cracking rolls, where the seed is cracked while still hot, and then delivered to special high-shear impactors to separate the meats and hulls. The product is then delivered to aspirators, where the hulls are removed, and then to the conditioner, which allows the meats to cool slightly and to temper prior to flaking. Another process that has gained acceptance is the Buhler hot dehulling system, which uses a conditioning column with steam-heated elements to slowly bring the beans to 65°C. The beans are then subjected to a short treatment in a fluid-bed popper where the hull–meat bond is broken. The beans are then broken in half by impact splitters, the hulls removed in an aspirator, and the splits further cracked and sent to the flaking rolls. The Crown hot dehulling system uses a similar conditioning column followed by a jet dryer to crisp the soybean hull and free it from the meat. A proprietary Hulloosenator then splits the bean and rolls the hulls free where they are aspirated from the meats. The splits are then cracked to the final size for flaking and sent to the Crown Cascade Conditioner for additional aspiration with temperature and moisture adjustment. In addition to the obvious energy savings, these types of systems are reported to reduce residual oil content, improve extractability, and reduce refining loss. In all cases, the comments on drying temperatures presented during discussion of storage drying are valid with hot dehulling systems. Once separated, hulls can be added to the meal to adjust protein content, can be used in fibre production or food additives production or they can be burn (Bockisch, 1993; Fulmer, 1989). Studies with dairy and beef cattle demonstrated the effectiveness of soybean hulls as a highly digestible fibre substitute for dietary grain (Anderson et al., 1988; Nakamura and Owen, 1989). Next, the cracked beans and bean chips are conveyed to the conditioning area, where they are put either into a rotary steam tubed device or into a stacked cooker and are heated to "condition" them. The purpose of this operation is to increase the plasticity of the meats, in preparation for flaking. The conditioner is similar to the cooker described in connection with expellers. It can be a horizontal screw conveyor type heated reactor or a vertical stacked cooker. Heat can be provided by indirect steam or by direct steam injection, the latter being used to increase the moisture content when necessary. The meats are heated to 65-79oC for 20-60 minutes (Regitano D’arce, 2006; Erickson et al., 1980). However, studies have shown that phospholipase D, an enzyme that makes the phosphatides nonhydratable and more difficult to remove is highly active at 57 to 85°C, therefore this cooking temperature should be avoided (O’ Brien, 2004). At this point the plasticity of the meats is such that they can be flattened by pressure in the flakers, without breaking. Proper cooking can also result in the complete breakdown of the oil cells, coagulation of the proteins to facilitate the oil and the meal separation, insolubilisation of phospholipids, increased fluidity of the oil at higher temperatures, destruction of moulds and bacteria, inactivation of enzymes and drying to a proper moisture content (11%). The enzymes inactivated are lipase, which increases free fat acids content, lipoxygenase, which causes oxidative rancidity, myrosinase, which causes the formation of sulphur compounds, meal flavour and digestive problems, besides phospholipase D, whose activity is described above (O’ Brien, 2004). Soybean is not conditioned when it is intended to expand the flakes (Regitano D’arce, 2006; Bockisch, 1993).

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Flaking machines consist of a pair of horizontal counter-rotating smooth steel rolls at 60-68°C. Typical roll sizes are in the range of 60-80 cm. in diameter. The rolls are pressed one against the other by means of heavy springs or by controlled hydraulic systems. Conditioned soybean cotyledon particles are fed between the rolls and they are flattened as the rolls rotate one against the other. The roll-to-roll pressure can be regulated and it determines the average thickness of the flakes. The main purpose of flaking is to increase the contact surface between the oilseed tissues and the solvent, and to reduce the distance that the solvent and the extract will have to travel in the process of extraction. It is also believed that flaking disrupts the oilseed cells to some degree and thus makes the oil droplets more available for solvent extraction. Typical values for flake thickness are in the range of 0.2 to 0.35 millimetres (Islas-Rubio and Higuera-Ciapara, 2002; Berk, 1992). The thickness decreases extraction time (Anderson, 1990). It is said that there was a defect of lamination when the flakes leave with different thicknesses. An abnormal percentage of this effect will have repercussions on the extraction, i.e. flakes with 0.5 mm or more of thickness can determine very high values of residual oil in the meal. Irregular stresses along the cylinder, being concentrated in the centre, can be one of the causes of this problem (Regitano D’arce, 2006; Woerfel, 1995). Expansion is an alternative method developed in Brazil which aims at making meals more porous and more permeable to the solvent, resulting in increase of micella concentration. The expander is a low-shear extruder that heats, homogenizes and oilseeds into porous collets or pellets with a high bulk density. Expansion can increase the extractors’ capacity in 40% (Regitano D’arce, 2006). The basic principle of the expansion is humidifying the cracked grain, heating it with aid of the friction of the axle and the injection of steam, until the beans are expelled through a variable sized orifice at 155°C and 40 atm of pressure. The difference of pressure in and out the orifice results in expansion of the mass. This property of expansion is related to the characteristic of the soybean starch. The mass leaves the expander at 105 the 120°C. It is postulated that partial inactivation of the phospholipase occurs in the expander and, while the crude oil has a higher neutral oil loss, the quality of the degummed oil is higher. Expanders do have an impact on lecithin production as well, not only in terms of higher quantity but also with respect to the quality (Anderson, 2005). As advantages of expansion after flaking, O’ Brien (2004) reports that it is easier to extract oil from flakes, the solvent drains more completely, thus reducing the amount of solvent that must be evaporated from the meal, and the through-put capacity of the extractor is increased. Besides, advantages of expansion over flaking are the increase of density from 300 kg/m3 to 550 kg/m3 of flaking meat, the increase from 40 to 50 m2/m3 of the contact area solid-liquid in the extractor, the increase of percolation rate, the increase of extraction efficiency, the reduced solvent consume (5% at least), the reduced vapour consume in the desolventizer the reduced amount of extracted solids, helping oil filtration and the increased content of hydratable phosphatides, helping degumming. Expansion, like conditioning does not affect antinutritional factors of the soybean (Lusas et al., 1988). One of the greatest problems associated with physical refining of high-phosphorous oils (such as soybean or corn) is that nonhydratable phosphatides generally cannot be removed without extensive bleaching clays and acid treatments. Lurgi’s Alcon process is said to inactivate phospholipase D immediately after the flaking step, and provide an oil consistently acceptable for physical refining (Shoemaker, 1987). The flakes are humidified and heated in a conditioner, maintained at the desired moisture content and temperature for 15-20 minutes, then dried and cooled before being led to the extractor. According to Islas-Rubio and Higuera-Ciapara (2002), this is, essentially, an agglomeration process, whereby the flakes are fused into more compact, porous granules. As advantages of this step, it is claimed that: (a) the bulk density of the modified granules is by 50% higher than that of the original

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flakes (550 against 360 kg/m3), which results in a corresponding increase in extractor capacity, (b) the rate of percolation of micella or solvent through the granules is tripled, which results in improved extractor efficiency, (c) the solvent retention in the spent granules is about 25%, while conventional spent flakes may retain as much as 35% solvent. As a result, desolventizer capacity is increased, oil yield is improved and energy is saved, (d) the thermal treatment associated with the Alcon process inactivates these phospholipase and improves the efficiency and yield of the oil degumming process and (e) due to the thermal treatment mentioned above, meal toasting requirements are less severe. Sunflower and sunflower oil The purpose of oil recovery process is to obtain oil in high yield and purity and to produce co-products of maximum value. Oilseeds are processed by one of three types of process: (1) expeller or screw press extraction, (2) prepress solvent extraction and (3) expander solvent-extraction. However, some operations before oil extraction are very important to the oil final quality. In fact, the seeds must be properly stored and prepared for extraction, to maintain high quality in the final product. Oils in the presence of water deteriorate rapidly, forming free fatty acids and rancid off-flavours (Casten and Snyder, 2001). Sunflower originated probably in the Southwest United States to Mexico area (Salunke et al., 1992). The plant is a member of Compositae family. The seed of sunflower is more correctly an achene. The seed is compressed, flattish, oblong with top truncated and base four-sided. The colour of the seed is determined by the presence of pigments in the three seed coat layers. Sunflower seed consists of a pericarp or hull and kernel with two cotyledons and an embryo among the commonly cultivated oilseeds, sunflowers have a high proportion of hull (20-31%). According to Salunke et al., (1992) drying of seeds is the first step in sunflower seed processing. The moisture content of freshly harvested sunflower seeds are of about 20%, with a range from 6 to 35 % depending on the period of harvest, the climatic conditions and the variety of seeds. To ensure safe storage, seeds must be dried to less than 9% moisture to get a sufficient low water activity. The drying systems commonly used to sunflower seeds are: (1) in-storage-bin drying, (2) batch drying, (3) batch-in-bin drying and (4) continuous air flow drying. Incomplete drying tends to promote microbial activity and can lead to spoilage. The microbial activity increases the amount of soluble pigments and free fatty acids in the extracted oil. This step is not obligatory or could be only a partial dehulling. The seeds are passed through air separation chamber to remove foreign matter. The seeds are dehulled in a drum-shaped chamber which contains a revolving plate that impels seeds against the smooth inner of wall of the drum by centrifugal force. Another system for impact dehulling is a flow rotor. The hulls and kernels are separated by specific gravity in an air flow. If a total dehulling is performed, the kernels are then passed over to mechanical colour sorters and then go over a vibrating table for visual inspection and removal of unacceptable kernels. Kernel fragmentation occurs during dehulling process , particularly if the moisture content of seeds is bellow 10%. Complete dehulling before extraction reduces the transfer of pigments from hulls to flour and reduces fibre in the final product. Waxes are also reduced in the oil. Partially de-hulled sunflower seeds can be subjected to an hydrolytic treatment with cellulases during aqueous processing for oil and protein extraction. Sub-optimal extraction conditions (particle size and separation technology) were established in order to appreciate the potential improvement caused by the enzymatic treatment and to select the best operational conditions. The effects of three operational variables (extraction-treatment time, water/seeds

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ratio and enzyme/seed ratio) were studied on three objective functions (the extent of hydrolysis reaction, the oil extraction yield and the percent polyphenolics removal). After 2 h of enzymatic treatment-extraction a practical optimum in the range 7.5-8 g water g-1 seeds and 1.25-1.4 g enzyme 100 g-1 seeds could be defined. Under these conditions the oil extractability and the polyphenolics removal are improved by more than 30 and 80%, respectively (Sineiro et al, 1998). 2.2.2 Oil xtraction The purpose of oil recovery process is to obtain oil in high yield and purity and to produce co-products of maximum value. Oilseeds are processed by one of three types of process: (1) expeller or screw press extraction, (2) prepress solvent extraction and (3) expander solvent-extraction (O’Brien, 2004). Oil content in the seed and how oil is stored and related with other components in the seed are the principal criteria do choose among the different methods of extraction. Corn oil extraction If corn oil is obtained by dry milling once germ separation has been achieved by mechanical milling, the germ is ground and passed through a solvent extraction device. Solvent extraction is a standard method for recovering oil from oil seeds. However, corn germ from wet milling cannot be directly extracted with solvent after preparation by flaking, as is done for other oil seeds, because of its tendency to produce a substantial amount of fines which hinder solvent extraction (Maza, 2001). Oil obtained by wet milling is usually extracted by a combination of expelling in continuous screw presses and solvent extraction of the press cake (Corn Refiners Association, 2006a). Screw presses have the low extraction efficiency of about 50%. Although pressing has not been a major area of research in recent years, interest in pressing and especially cold pressing has recently been rekindled in the field of nutraceutical oils such as flaxseed and crambe (Moreau et al, 2006). After the expellers, the germ may be flaked and the remaining oil extracted using a solvent. The solvent extraction method requires stripping residual hexane from the oil and the soybean flakes. Solvent is driven off the spent germ meal by the desolventizer-toasters, dryers, and coolers (EPA, 1995). Maza (2001) describes many conventional processes for germ preparation. One of them consists of using dry germ as a starting material, which is rehydrated, cooked and full expelled, yielding an oil stream. Corn germ is expelled to 6-10% residual oil content. In order to enhance oil recovery economics, both germ preparation and germ expelling need to be conducted at high temperatures (250-275° F.). The majority of expelled oil is recirculated in hot and aerated state onto the expeller for external cooling of the barrel, while the quota withdrawn continually is processed hot. As a result, decomposition products leach out of the expeller cake along with the oil recovered. More decomposition products develop further through prolonged handling of hot oil. Another conventional process is partially similar to the previously described. Dry germ is submitted to rehydration, cooking and expelling to 20-30% residual oil content. This pre-expelling operation yields an oil stream. The expelled corn germ is again rehydrated, and then flaked. Pre-expelled cake is extracted with solvent to 3-4% residual oil content. This extraction operation yields another oil stream. Hot solvent extracts more degradation products out of the expeller cake. Consequently, the quality of crude oil (blend of pre-expelled and extracted oils) is inferior to full expelling.

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According to Maza (2001), a better process is to use extrusion as part of the pre-treatment process of corn germ, prior to the oil liberation step. Extrusion is an oil release technique which is based on the explosion of the oil seed matrix by controlled evaporation of cellular moisture. Extrusion has been very effective in improving the solvent extractability of many oleaginous plant materials and is well established in the preparation of soybean, rice bran, cottonseed, and pre-pressed canola, sunflower and other oilseeds. However, there are some problems in the extrusion of oleaginous plant materials having a high percentage of oil by weight, e.g., more than 30%. Williams (1990), in U.S. Pat. No. 4,901,635, addresses the problems of oil liberation occurring within the extruder as corn germ materials is processed through the extruder. The liberated oil forms pockets of free oil which squirt out of the dies and interrupt steady-state operation of the extruder. The squirting oil also results in the undesirable loss of oil, the principal product. An efficient process for recovering high quality corn oil from corn germ is proposed by Maza (2001). The process involves pre-treating corn germ by rehydration, conditioning, followed by flaking and extrusion. Water is provided up front in the process, prior to extrusion, reducing generation of fines and improving friction within the extruder, thereby allowing the material to move along and improving overall efficiency and streamlining of the process. The elevated moisture level in the extruder makes the corn germ less abrasive the material is forced out of the extruder through a restrictive device into an environment of lower pressure than within the extruder. As a result of the abrupt change of pressure, cellular water vaporizes instantly, rupturing the cells and releasing germ oil within the extruded meal. At given extrusion conditions, the fatty fines which may have been produced previously during flaking are agglomerated back with the extruded germ into a highly porous mass which retains the oil. The extruded material may be produced in either pellet or non-aggregated form. The short residence time in the extruder, as well as the relatively mild temperature and pressure conditions reduce any deleterious side effects on the corn germ being processed. The oil released from corn germ by this process is of high quality and high yield. The solvent extraction process consists of "washing" the oil from the germ with hexane solvent in a counter current extractor and then evaporating the solvent separately from both the solvent/oil mixture (micella) and the solvent-laden, defatted corn germ cake, which are removed separately from the extractor. The oil is desolventized by exposing the solvent/oil mixture to steam (contact and noncontact). The mixture oil/solvent is first pumped through heaters, then through film evaporators under vacuum and, finally, through a stripping column to remove the hexane. The hexane/water vapour mixture is removed from each oil desolventizing unit and condensed in a "separation" tank to separate the hexane from the water. Then the solvent is condensed, separated from the steam condensate, and reused. The desolventized oil crude corn oil is cooled and stored for further refining (EPA, 1995). The oil-depleted germ is freed from the solvent, toasted, ground and screened. The resulting corn germ meal is combined with fibre and gluten for animal feed (Corn Refiners Association, 2006b). Peanut oil extraction Peanut oil is obtained by one of three methods, including hydraulic, expeller and solvent extraction methods. The hydraulic method of extraction consists of pressing the shelled peanuts under 14,000 psi, while adding steam and heat. (Woodroof, 1983). Expeller pressing is the most popular method of peanut oil extraction. Screw presses use an electric motor to rotate a heavy iron shaft, which has flights, or worms built into it to push the seeds through a narrow opening. The pressure of forcing the seed mass through this slot releases part of the oil, which comes out through tiny slits in a metal barrel fitted around the

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rotating shaft. Expellers have a continuous flow of seed through the machine in contrast to the hydraulic system described above, which uses small, individual packages or batches of seed. To release as much oil as possible, the seeds must be dried to rather low moisture content and exposure to high temperature causes darkening of the oil. It also causes some scorching or overheating of the meal. In view of replace the traditional method of separation of groundnut oil slurry which is rather wasteful, unhygienic and labour intensive with a mechanized method, mechanical filtration by wide-angle conical screen centrifugal filter is necessary. A preliminary design analysis was made, using the data obtained from the physical and chemical properties of the groundnut oil and working upon the principle of continuous filtering centrifuges, a wide-angle conical screen centrifuge was designed. The design consists of a rotary conical screen as the main component of the machine driven by a variable speed motor via a v-belt and pulley power transmission system (Hassan et al, 2006). The widely-used second method is to extract the oil using a solvent, hexane, which dissolves the oil and strips it from the peanut flakes. The solvent extraction method requires stripping residual hexane from the oil and the peanut flakes. This method is frequently combined with the expeller methods (Woodroof, 1983). The extraction process consists of "washing" the oil from the peanut flakes with hexane solvent in a counter current extractor and then evaporating the solvent (i.e., desolventizing) separately from both the solvent/oil mixture and the solvent-laden, defatted flakes. The oil is desolventized by exposing the solvent/oil mixture to steam (contact and noncontact). Then the solvent is condensed, separated from the steam condensate, and reused. The desolventized oil, called "crude" peanut oil, is stored for further processing or load out. Specific steps in the solvent extraction and oil desolventizing processes are described below (Emission Factor Documentation for AP-42, 1995). Peanut flakes are conveyed into the extractor, where they are washed counter currently with various hexane/oil mixtures and, finally, with pure hexane. The initial oil content of the peanuts is approximately 18 percent to 20 percent by weight. After extraction, the defatted flakes contain approximately 0.5 percent to 2.0 percent oil by weight. Either a "deep bed" extractor or a "shallow bed" extractor is typically used in the extraction process. The deep bed extractor consists of pie-shaped mesh baskets that rotate around a shaft. The bed depth in this extractor varies from approximately 2 to 3 m. The shallow bed extractor, a more recent design than the deep bed extractor, conveys the peanut flakes horizontally over closely spaced "vee-bars" while washing them first with oil and hexane and then with pure hexane. Bed depth in the shallow bed extractor normally varies from approximately 0.5 to 1 m (Emission Factor Documentation for AP-42, 1995). The oil/hexane mixture is removed from the extractor separately from the defatted flakes. The mixture is first pumped through heaters, then through film evaporators under vacuum and, finally, through a stripping column to remove the hexane. The hexane/water vapour mixture is removed from each oil desolventizing unit and condensed in a "separation" tank to separate the hexane from the water. Once separated from the water, the hexane is reused in the extraction process. Crude oil is cooled and stored in tanks for further processing or load out (Emission Factor Documentation for AP-42, 1995). The flakes leaving the extractor contain up to 35 percent to 40 percent solvent and must be desolventized before use. Flakes are desolventized either through a "conventional" desolventizing process or a specialty ("flash") desolventizing process, depending on the end use of the material. Conventional desolventizing takes place in a desolventizer-toaster where both contact and noncontact steam are used to evaporate the hexane. The hexane is condensed, separated from the steam condensate, and reused. In addition, the contact steam "toasts" the flakes, making them more usable for animal feeds. The desolventized and toasted flakes then pass to a dryer, where excess moisture is removed by heat, and then finally to a

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cooler, where ambient air is used to reduce the temperature of the dried flakes. The desolventized, defatted flakes are then ground for use as peanut meal. In the specialty or "flash" process, solvent is removed from the defatted flakes either with superheated solvent (hexane) or in a vacuum with a small quantity of noncontact steam. Flakes desolventized in this manner are typically used for human foods (Emission Factor Documentation for AP-42, 1995). Rapeseed oil extraction Extraction of oil rapeseed can be performed by direct screw pressing, by direct solvent extraction or by pre-pressing solvent extraction. The latter is the most economical process. In the pressing and pre-pressing methods, the heat-conditioned flakes are passed into continuous screw-presses or expellers. The function of this equipment is to reduce the oil content of the seed to 4 to 12% mechanically. In the case of pre-pressing, a residual cake with 12 to 20% is released. For small capacities or special applications, full pressing may be applied. According to Martínez et al., (2008), screw-press performance depends on the method of preparing the raw material, which consists of the unit operations described before as cleaning, cracking, cooking, drying or moistening to optimal moisture content. The application of a thermal treatment before or during pressing generally improves oil recovery, but it may adversely influence the oil quality by increasing oxidative parameters. The seed moisture content at the time of pressing is another key process variable. It is known that moisture increases plasticity of seed materials, and contributes to press feeding owing to its effects as barrel lubricant. However, high moisture contents may result in poor oil recovery because of insufficient friction during pressing. Expellers consist of a rotating screw shaft in a cylindrical barrel. The barrel has flat steel bars mounted edgewise around the inside and spaced to allow oil to flow from between the bars while retaining the solid material within (Mag, 1990). Pressures of 1000 to 1400kg/cm2 force the oil from the flakes through the 0.25mm bar spacing. After pressing, the press cake is passed through a flaking mill to give flakes of 0.3 to 0.4mm thickness. Although this thickness does not give the most efficient extraction, the small size and fragility of rapeseed make this a necessary compromise (Salunkhe et al., 1992). Some discharge of very fine solids with the oil draining from the expeller is unavoidable. These fines are separated from the oil by gravity and filtration, and recycled to the conditioning stage (Mag, 1990). The press cake then goes to solvent extraction. In some plants, the press cake is subjected to mechanical extrusion to improve its solvent extraction properties. Extruders used for this purpose consist of a barrel with a rotating shaft fitted with flights. Steam is added and heating and mixing take place along the length of the barrel. Pressure is developed. The material is then discharged through the small openings of a die plate at the end of the barrel. The pressure release on discharge “expands” the extruded material, making it very porous. These small diameter, porous pieces of press cake (collets) have excellent solvent extraction properties. Capacity of solvent extraction equipment is significantly enhanced. Solvent extraction may take place in either percolation-type extractors where the solvent is allowed to percolate through the seed bed, or filtration-type extractors, where the solvent and seed mass are slurred and filtered (Salunkhe et al., 1992). Continuous counter current processes are recommended in order to remove higher amounts of oil with a minimum solvent. The ratio of solvent to meal is 1.1 to 1.3, and the concentration of oil in the final miscella is 10 to 50%. The meal in filtration-type extractors has 25-30% solvent. The main advantage of solvent extraction over the mechanical processes is that it improves the oil yield, since the residual oil content of the meal is around 1 per cent in industrial operation (De Smet, 2004). Although it is more advantageous for low oil content seeds, direct solvent extraction of

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rapeseeds can also be used in high capacity plants (Salunkhe et al., 1992). The meal and the miscella are desolventized to recover solvent-free meal and oil. The solvent-saturated meal is conveyed to a desolventizer, which is a vertical tank equipped with heated trays and agitators. Reduced pressure and sometimes live steam are used to evaporate the hexane and to dry the meal. The hexane and moisture vapours are condensed, the water and hexane are separated, and the hexane is reused. Several stages of heating and drying are applied to reduce the hexane content to negligible levels and moisture to 8-11 %. Some removal of glucosinolates and their breakdown products and some protein denaturation occur. The resulting meal has a dark colour, 1 to 2% of oil and low protein solubility. To achieve the best meal quality, the process must be well controlled with respect to temperature (110ºC max.) and time. Usually, the final treatment in desolventizing is a cooling stage. Cooled meal may be ground to a uniform particle size, or pelleted, ready for storage and marketing. Dehulling of the meal for non-ruminant feed may, in future, become commercial practice. Solvent is removed from miscella by distillation procedures involving heating with vent gases, direct steam-heating and the use of a “stripping column”. The hexane vapour from this operation is, also, condensed for reuse. A properly operated extraction plant loses no more than about 2-3 litres of hexane/mt of seed processed. The oil contains about 0.1% of residual solvent (Salunkhe et al., 1992; Mag, 1990). Oil extraction using supercritical fluids is an alternative method to replace or to complement conventional industrial process such as pressing and solvent extraction. The use of supercritical carbon dioxide (SC-CO2) in fats and oils processing as an extraction, fractionation, concentration and reaction solvent, has been investigated by several authors (Marrone et al., 1998; Oliveira et al., 2002; Santerre et al., 1994). Prior to SC-CO2 extraction, vegetable substrates are commonly subjected to mechanical pre-treatments aimed to improve the rate and/or yield of the extraction process. The pre-treatments may have multiple purposes, including releasing of solutes from cells, facilitating solvent flow through the packed bed, and increasing substrate load onto extraction vessels. One treatment frequently used for high-oil seeds is the pre-pressing tending to reduce the oil content (Eggers, 1996; Esquivel et al., 1999). In order to improve the quality of canola meal in terms of both composition and functionality, numerous processes have been studied, such as dehulling (Sosulski, 1981), heating (Eapen et al., 1968, solvent treatment (Bhatty and Solsulski, 1972), and protein isolation (Xu and Diosady, 2002; 1994). The products of these processes could be meals, protein concentrates or isolates, which are typically rich in protein, low in toxins, and have desirable functionalities for various food applications. However, so far none of these processes has been commercialized due to either poor quality or low yield. As a viable alternative to organic solvents, supercritical carbon dioxide (SC-CO2) has been investigated specifically for canola oil extraction (Dunford and Temelli, 1997; Temelli, 1992). Among many advantages of supercritical fluid extraction of canola is the elimination of the toasting step so that the proteins remain intact. Thus, the meal functionality could potentially be preserved. Sun et al., (2008) compared the chemical composition and functionality of canola meals defatted by SC-CO2 extraction without and with the addition of ethanol as a co-solvent to pressed meal and conventional meals extracted with hexane in the laboratory and industrial setting. The authors concluded that SC-CO2-extracted meal had lower glucosinolate and higher phosphorus contents than hexane extracted meal. The phenolic acid contents of hexane and SC-CO2-extracted meals were similar, but were higher than those of meals extracted with SC-CO2 + ethanol. The colour values of SC-CO2- and hexane-extracted meals were similar and both were brighter than commercial meals (pressed and toasted). The Nitrogen solubility index levels of SC-CO2- and hexane extracted meals were similar, but three times that of the commercial meal. Both hexane- and SC-CO2-extracted

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meals had high water holding capacity, oil absorption, emulsifying capacity, emulsion stability and overrun. Canola meal extracted with SC-CO2 was similar to hexane extracted meal in terms of both chemical composition and functionality, but was superior to commercial meals. The use of hexane for extracting rapeseed oil has recently become the focus of concerns with respect to its safety and environmental effects. (Zhang et al., 2007 a; b; Marlowe et al., 1991). An alternative process, aqueous (enzymatic) extraction, for extracting oil from many oil-bearing seeds/fruits has been attempted in the laboratory and/or at the pilot industrial scale level. Aqueous enzymatic extraction has emerged as a novel technology for coextraction of either oil or oil and protein from many oilseeds and oil-fruits (Domínguez et al., 1994; Rosenthal et al., 1996). In contrast to traditional processing the enzyme process is based on the use of water as solvent and cell wall degrading enzymes to facilitate an easy and mild fractionation of oil, protein and hulls. The oil is found inside plant cells, linked with proteins and a wide range of carbohydrates like starch, cellulose, hemi-cellulose and pectin. The cell content is surrounded by a rather thick wall which has to be opened so the protein and oil can be released. Thus, when opened by enzymatic degradation, down-stream processing makes fractionation of the components possible to a degree which cannot be reached when using the conventional technique like pressing and hexane extraction. The process usually consists of an aqueous (enzymatic) extraction of the comminuted materials, followed by a centrifugal separation of the slurry into oil, emulsion, and the aqueous and solid phases. Protein may be recovered in the aqueous or solid phase, depending on the conditions selected. Compared with the traditional technology, this process is mild and safe due to the complete avoidance of organic solvents (Zhang et al., 2007a; b). However, enzymatic extraction is told to be is 5 - 6 times more expensive than the conventional hexane process. Viability can be reached by a higher value and quality of sub-products. Fullbrook (1983) investigated aqueous hydrolysis of oilseeds followed by solvent extraction and also tried hydrolysis in the presence of a solvent to simultaneously extract the released oil. It was observed that yields could be considerably improved if hydrolysis of the finely ground flour of soybean and rapeseed was carried out in the presence of solvent. In the case of rapeseed, 50% more oil was thus obtained. Sosulski et al., (1988) evaluated the effect of different carbohydrases on the extraction time and yield of canola oil. The enzyme reaction was carried out on previously autoclaved and moisture adjusted canola flakes, and followed by drying and hexane extraction. The enzyme efficiency based on oil yield enhancement was: mixed activity enzyme > B-glucanase > pectinase > hemicellulase > cellulase. Enzymatic treatment before Soxhlet extraction for a given time gave 45% higher yields. Zuñiga et al., (2001) reported that a good quality canola oil was obtained through enzymatically-assisted process, which had 50% free fatty acids as compared to the control samples and the same peroxide value. However, Sosulski and Sosulski (1993) in a similar enzymatic-aided oil extraction process, recovered canola oil with a higher free fatty acids than a conventional extraction process. Zhang et al (2007b) developed downstream processes following aqueous enzymatic extraction and demulsification of rapeseed oil and protein hydrolysates to enhance the oil and protein yields as well as to purify the protein hydrolysates. As important conclusions, the authors verified that combination of the aqueous enzymatic extraction process with washing and demulsification steps, lead to yields of the total free oil and protein hydrolysates of 88–90% and 94–97%, respectively. The protein recovery was 66.7% and the protein content was enriched from 47.04 to 73.51% in the Crude Rapeseed Proteins, in which no glucosinolates and phytic acid were detected. In recent years, microwave-assisted extraction has attracted growing interest as it allows rapid extractions of solutes from plant material, with extraction efficiency comparable

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to that of the classical techniques. Indeed, microwaves interact selectively with the free water molecules present in the gland and vascular systems; this leads to localized heating, and the temperature increases rapidly near or above the boiling point of water. Thus, such systems undergo a dramatic expansion, with subsequent rupture of their walls, allowing the oil to flow towards free water (Benkaci-ali et al., 2006; Paré et al., 1994). Valentová et al., (2000) described the influence of microwave treatment on enhancing the rate of rapeseed oil extraction along with significantly positive effects on the usual quality parameters of crude extracted oil. Valentová et al., (2002) evaluated the quality of oil obtained by cold pressing of microwave-treated seeds compared to conventional heating of mechanically crushed seeds (flakes). Characteristics of crude oils obtained immediately and after 6 wk of seed storage after microwave heating are summarized. The yield of cold-pressed oil increased with increasing doses of irradiation up to about 40% (w/w) whereas the amount of oil obtained from the flakes was about 33% (w/w). The degree of lipid oxidation was significantly lower after treatment by doses up to 1.9 kW, with higher doses remaining approximately at the same level. The peroxide value for oils obtained after seed storage were much lower even for control seeds. Both findings were possibly due to the inactivation of oxidative enzymes, peroxidases, and lipoxygenases (Irfan and Pawelzik, 1999; Novótna et al., 1999) either by heat treatment (first pressing) or during storage (second pressing). Significantly increased oxidative stability of rapeseed oil after microwave treatment was also observed by Veldsink et al., (1999). Total phosphorus contents were higher in the oils from treated seeds, but significantly lower than in oil from flakes. It seems that microwave treatment damages membranes and thus higher amounts of phospholipids may be released into the crude oil. Rapeseed oil has traditionally been produced by mechanical pressing at high (>90 °C) temperatures followed by solvent extraction. Virgin rapeseed oil has become increasingly popular for the consumer because of the pleasant seed-like and nutty taste and smell. The oils are produced in small and medium-sized facilities by extraction of rapeseed using only cold pressing and purification by sedimentation or filtration. It is an art to produce high-quality virgin rapeseed oil that is accepted by the consumer because producers have no chance to improve the oil quality if the seed quality is bad (Matthäus and Brühl, 2008). There is a consensus about the importance of low temperature in cold pressing, but many different limits are presented in literature. In order to be labelled “cold pressed” in the United Kingdom, the oil temperature when exiting the screw press should be less than 50°C (Panfilis et al., 1998). Singh and Bargale (2000) reported exiting oil temperatures of less than or equal to 70°C. According to Kirk (2006), truly cold pressed oil (less than 32.2°C) has very special quality. Ferchau (2000) states that temperatures above 20oC have no additional benefit for oil quality and must be avoided. Mechanical screw presses typically recover 86 to 92% of the oil from oilseeds (Singh and Bargale, 2000). Adjusting pressing parameters can improve oil recovery; for example, increasing the internal pressure results in a decrease of the residual oil in the meal (Jacobsen and Backer, 1986). Oil recovery also can be enhanced by suitable pre-treatment of the oilseed, i.e., cracking, dehulling, conditioning, flaking, and cooking (Fils, 2000). Cold-pressed seed oils may retain more natural beneficial components of the seeds, including natural antioxidants, and are free of chemical contamination. This procedure involves neither heat nor chemical treatments, no solvents and no further processing other than filtering is applied. It is becoming a interesting substitute for conventional practices because of consumers’ desire for natural and safe food products (Yu et al., 2005). In the cold-pressed products, minor components affecting the colour, flavour and keeping qualities of the oil are thus preserved. These minor constituents can have either pro-oxidative (free fatty acids, hydroperoxides, chlorophylls, carotenoids) or antioxidative (tocopherols, phenols, phospholipids) effects. Cold-pressed rapeseed oil has been shown to be

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more susceptible to autoxidation than refined rapeseed oil (Pekkarinen et al., 1998). This has been attributed to the higher hydroperoxide and free fatty acid contents of the unrefined oil. On the other hand, refining processes destroy some of the tocopherols along with other phenolic compounds that protect the oil from autoxidation (Gutfinger, 1981; Salvador, 1991). Hellebrand et al., (1998) has found the presence of DNA from rapeseed in the cold pressed oil, indicating the possibility of detecting the use of GMO rapeseed as prime material in the processing. Soybean oil extraction Soybean oil can be extracted by mechanical or solvent method. The most popular method for recovering soy oil is solvent extraction. The yields in solvent extraction are approximately 18.5% of oil, 73.8% of soybean meal with 48% protein content, and 5.5% of hulls. For mechanical expelling, the recovering soy oil yields 12% of oil, 84.2% of high-energy meal, and 2.5% of hulls. The disadvantages of the mechanical method compared to the solvent extraction method are that (a) oil recovery is low. Even the most powerful presses cannot reduce the level of residual oil in the press-cake below 3 to 5%. In the case of soybeans, a 5% residual oil level in the cake represents an oil loss of about 25%. (b) The utility of a high-fat (or high-energy) meal is low. The commercial value of the meal is usually higher than the income from sales of the corresponding quantity of oil. The quality of the meal is therefore a factor of particular importance in the selection of a processing method for soybeans. The high content of residual oil, very sensitive to rancidity which leads to the rejection in the cattle feeding (Regitano D’Arce, 2006). The poor storage stability of the press-cake, due to its high oil content is very disadvantageous and the extreme temperatures prevailing in the expeller may impair the nutritive value of the meal protein, mainly by reducing the biological availability of the amino acid lysine. At any rate, expeller press-cake is not suitable for applications requiring a meal with high protein solubility (Islas-Rubio and Higuera-Ciapara, 2002; Berk, 1992). On the other side, screw pressing is thought to create Maillard browning reaction products that contribute natural antioxidants to the pressed oils (Warner and Gupta, 2005). Both concerns have limited the commercial use of the mechanical method. In some instances, soybeans are passed through extruders before being subjected to mechanical expelling. Soy oil obtained in this manner is even better because it has lower phosphatide content. Solvent extraction is normally applied in very large capacity plants that produce commodity soy oil. Small manufacturers that aim to produce specialty oils of high quality use the mechanical expelling process (Lence and Argawal, 2003). Continuous pressing by means of expellers (also known as screw presses) is a widely applied process for the extraction of oil from oilseeds and nuts. It replaces the historical method for the batch wise extraction of oil by mechanical or hydraulic pressing. The expeller consists of a screw, rotating inside a cylindrical cage (barrel). The material to be pressed is fed between the screw and the barrel and propelled by the rotating screw in a direction parallel to the axis. The configuration of the screw and its shaft is such that the material is progressively compressed as it moves on, towards the discharge end of the cylinder. The gradually increasing pressure releases the oil which flows out of the press through the slots provided on the periphery of the barrel, while the press-cake continues to move in the direction of the shaft, towards a discharge gate installed at the other extremity of the machine (Berk, 1992). The term expeller is the registered name of continuous screw presses patented by Anderson Expeller in 1903 (Regitano D’arce, 2006). Before entering the expeller, the oilseeds must be cleaned, dehulled (optional), flaked, cooked and dried. Flaking facilitates oil release in the press by decreasing the distance that the oil will have to travel to reach the particle surface. Cooking in the presence of moisture is essential for the denaturation of the

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proteins and, to some degree, for the coalescence of the oil bodies. Cooking plasticizes the flakes, renders them less brittle and thus reduces the extent of flake disintegration as a result of shear in the press. Extensive flake disintegration would reduce oil yield and produce a crude oil with a high content of fine solid particles. After cooking, excess moisture is removed in order to avoid the formation of muddy emulsions in the press. Cooking is usually achieved by mixing the flakes with live steam. Additional heat may be provided by indirect steam, while thoroughly mixing the mass. Johnson (2000) recommends flakes with 0.38 to 0.5mm and cooking at 115°C for 60 minutes. The advantages of mechanical extraction are: (a) the process is relatively simple and not capital-intensive. (b) While the smallest solvent extraction plant would have a processing capacity of 100-200 tons per day, expellers are available for much smaller capacities, from a few tons per day and up (Regitano D’arce, 2006; Berk, 1992), (c) solvent is not applied, which lowers processing and product costs, and (d) the product can be consumed without refining (Regitano D’arce, 2006; Masiero, 1995; Woerfel, 1995). Soybeans present an oil content as low as 20%. In this case, mechanical extraction becomes anti-economic and the process of extraction with solvent predominates. The extraction of oil from oilseeds by means of non-polar solvents is, basically, a process of solid-liquid extraction. The transfer of oil from the solid to the surrounding oil-solvent solution (micella) may be divided into three steps: (a) diffusion of the solvent into the solid, b dissolution of the oil droplets in the solvent, (c) of the oil from the solid particle to the surrounding liquid diffusion (Islas-Rubio and Higuera-Ciapara, 2002; Berk, 1992). Therefore, solvent extraction consists of leaching the oil out of the cake, flakes or collets with a solvent, usually hexane. This method is problematic for oilseeds with high content oil contents because the resulting flakes are easily disintegrated into fines during extraction process (O’ Brien, 2004). It is important that the grain preparation offers a porous structure that allows the ready absorption of the solvent on it sprinkled, the easy contact between the solvent and the oil for fast dissolution and fast drags of this oil by the solvent for out of the grain. In this form, little solvent or miscella are kept and lost in the matrix and all the oil is removed. Recently, it was concluded that solvent extraction is a set of operations that involves humidifying, diffusion, solution, osmosis and dialysis, among others, each one occurring to a variable and unknown speed (Regitano D’arce, 2006). The driving force in the diffusional processes is, obviously, the gradient of oil concentration in the direction of diffusion. Due to the relative inertness of the non-oil constituents of the oilseed, equilibrium is reached when the concentration of oil in the micella within the pores of the solid is equal to the concentration of oil in the free micella, outside the solid. Islas-Rubio and Higuera-Ciapara (2002) stated some practical conclusions from these considerations, such as: (a) since the rate-limiting process is diffusion, much can be gained by reducing the size of the solid particle. Yet, the raw material cannot be ground to a fine powder, because this would impair the flow of solvent around the particles and would make the separation of the micella from the spent solid extremely difficult. Instead, the oilseeds are rolled into thin flakes, as described in the previous paragraph, thus reducing one dimension to facilitate diffusion, without impairing too much the flow of solvent through the solid bed or contaminating the micella with an excessive quantity of fine solid particles. (b) The rate of extraction can be increased considerably by increasing the temperature in the extractor. Higher temperature means higher solubility of the oil, higher diffusion coefficients and lower micella viscosity. In fact, it is customary to heat the solvent and the intermediate micella to the highest temperature which would still provide an acceptable level of safety. Even tough, elevated temperatures reduce oil viscosity and enhances diffusion, the hexane vapour pressure limits the practical operating temperatures of the extractor and its contents to approximately 50 to 55°C (O’ Brien, 2004); (c) an open, porous structure of the solid material is desired, since such a structure facilitates diffusion and percolation; (d) although most of the resistance

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to mass transfer lies within the solid, the rate of extraction can be increased by agitation and free flow in the liquid phase around the solid particles. Too much agitation is to be avoided, so that extensive disintegration of the flakes is prevented; (e) the concentration gradient is the factor responsible for moving the oil out of the solid, it is important to keep this gradient high, at each point within the extractor. The principle of counter-current multistage extraction is recommended in this case for an economical solution. The process is divided to a number of contact stages. Each stage comprises means for mixing the solid and the solvent phases and for separating the two streams after extraction has been achieved. In going from one stage to the next, the flakes and the solvent move in opposite directions. Thus, flakes with the lowest oil content are contacted with the leanest solvent, resulting in high oil yield and high driving force throughout the extractor. Two different methods can be used to bring the solvent to intimate contact with the oilseed material: percolation and flooding. In the percolation method, the solvent trickles through a thick bed of flakes without filling the void space completely. A film of solvent flows rather rapidly over the surface of the solid particles and efficiently removes the oil which has diffused from the inside to the surface. This mode of contact is preferable whenever the resistance to diffusion inside the flake is relatively low, which means thin flakes with large surface area, open tissue structure. In the flooding mode the solid particles are totally immersed in a slowly moving, continuous phase of solvent. Immersion works better with materials offering a greater internal resistance to oil transfer, such as thick particles, dense tissue structure (Islas-Rubio and Higuera-Ciapara, 2002; Berk, 1992). A solvent universally adopted in the facilities is hexane. It is a derivative of petroleum refining and what reaches the industries is a mixture of n-paraffin fractions presenting not a specific boiling point, but a range of volatilization temperature (65 to 70°C), as expected, given that its variable composition. In spite of the presence of other components in the commercial hexane, they have little influence in its efficiency and form of use. Several solvents were employed before hexane, however the industrial practice led to its adoption because of some requirements, like being totally apolar and readily dissolving in oil, being immiscible in water, presenting low latent boiling heat, not attacking the pipings and the equipments with which it has contact (Regitano D’arce, 2006). While hexane is widely accepted as the most effective solvent used today, there are concerns about its inflammability, exposure, and environmental impacts (Anderson, 2005). Research has focused on various alternative solvents in the hopes of finding one with acceptable performance while providing greater safety. Alternative solvents that have received some attention include ethanol (Regitano D’arce, 2006) and isopropanol with ethanol (Hron and Koltun, 1984; Karnofsky, 1981; Rao and Arnold, 1958; Rao et al., 1955), supercritical carbon dioxide and other fluids (Boss, 2000). The unfavourable points to the application of these renewable solvents are the greatest energetic demand in the recuperation of the solvent, the demand of working in temperature nearer to that of boiling during the process of extraction, the necessity of rectification for recuperation of the solvent, which increases the process costs and the necessity of more careful refining given the greatest polarity of the solvent which extracts more undesirable compounds during crude oil production (Anderson, 2005). Supercritical technology uses as solvent the carbon dioxide (CO2). It is a process that has several advantages of the point of view of the environment and of the human health because it does not leave residues of toxic substances in the products or in the vegetable original matrix. After oil is extracted, the meal can be used in several applications like animal food, for example, because it has not residue (Meireles, 2005).The stages wrapped in the preparation of the seeds for the supercritical extraction with carbon dioxide do not differ from the conventional extraction with hexane. However, the stages of extraction and, principally, the stages of recuperation of the solvent of the oil and of the cake are significantly modified. Boss

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(2000) reported that extraction with carbon dioxide is better than conventional extraction when environmental impact and the quality of the product are considered. As advantages supercritical technology for oil processing the author cited (a) no inflammability, (b) gentle conditions of the critical point at 31ºC and 73.8bar, (c) chemical stability, (d) availability and low cost, since it can be obtained, for example, from fermentation, and (e) the omission of distillation and deodorization. The development of equipments for supercritical continuous extraction, for great amounts of seeds, is still necessary. It is also important to make industry aware about the damage caused in the nature by hexane emissions. The lack of more rigid laws with regard to the emission of pollutants, food solvent contamination and hazards of hexane related to occupational health and safety in the facilities, makes it difficult the introduction of a new method of extraction, since this hexane process is economically more advantageous than the others until the present moment. According to Islas-Rubio and Higuera-Ciapara (2002) and Berk (1992), solvent extractors are of three types: batch, semi-continuous and continuous. In batch processes, a certain quantity of flakes is contacted with a certain volume of fresh solvent. The micella is drained off, distilled and the solvent is recirculated through the extractor until the residual oil content in the batch of flakes is reduced to the desired level. Batch extractors as industrial units are now obsolete. Laboratory and pilot plant size extractors are still used for experimentation and instruction purposes. Semi-continuous systems consist of several batch extractors connected in series. The solvent or micella flows from one extractor to the next one in the series. The material in the first extractor is the most exhausted, since it has been treated with fresh solvent. After a while, the second extractor is made "head" of the series and connected to the fresh solvent line. The spent flakes are discharged from the first extractor, which is then filled with a batch of fresh flakes and is connected to the system as the "tail" unit, and so on. Semi-continuous systems of the type described above are seldom used for the solvent extraction of soybeans. In continuous extraction, both the oilseeds and the solvent are fed into the extractor continuously. The different available types are characterized by their geometrical configuration and the method by which solids and solvents are moved one in relation to the other, in counter-current fashion. The oil content in micella can reach 30% if counter current washing is well applied by a percolation system. Concentrations higher than 13% are rarely reached in immersion systems (Bockisch, 1993). Two streams leave the solvent extraction stage: an oil-rich fluid extract (full micella) and solvent-laden spent flakes. The next operations have the objective of removing and recovering the solvent from each one of the two streams (Berk et al., 1992). Full micella contains typically between 22 and 30% oil (Regitano D’arce, 2006; Berk, 1992). Thus, for every ton of crude oil some 2.5 tons of solvent must be removed by distillation. Most manufacturers of solvent extractors also offer micella distillation systems. This process consists of heating micella under vacuum (50 mmHg) in order to evaporate the solvent at a not too high temperature (until 105°C) which prevents oil alterations. Crude oil is recuperated and is pumped to a storage tank. Solvent vapours are condensed, separated from water in gravity tanks, heated and sent back to the extraction process. According to Berk (1992), the characteristics of a good micella distillation system are: good energy economy, minimal heat damage to the crude oil and its components, minimal solvent losses, efficient removal of the last traces of solvent from the oil and good operation safety. Generally, distillation takes place in a double effect evaporator and a high efficiency evaporator, which can be rising or falling film, followed by steam distillation, in order to remove solvent traces, under 100mmHg in the exit (Regitano D’arce, 2006; Masiero, 1995; Woerfel, 1995). In an efficient system, 75% of the energy for the distillation is provided by the heat loss of the discharge steam in the meal desolventization and toasting (Bockisch, 1993).

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The spent flakes carry with them about 35 to 40% solvent, 7 to 8% water and 0.5 to 1,0% oil (Regitano D’arce, 2006; EPA, 1995; Berk, 1992). The removal and recovery of this portion of the solvent is also one of the most critical operations in oil mill practice, since it determines, to a large extent, the quality of the meal and its derivatives. Flakes can be desolventized in traditional or flash desolventizing. The method used depends upon the end use of the flakes. Flakes that are flash desolventized are typically used for human foods, while conventionally desolventized flakes are used primarily in animal feeds (EPA, 1995). According to Berk (1992), in desolventizing-toasting (DT) applied in the production of soybean oil meal for animal feeding, the time-temperature-moisture profile of the process permits, in addition to solvent removal, a heat treatment sufficient to inactivate the undesirable enzymes and inhibitors and to improve the palatability of the meal to animals. The desolventized meal contains a high amount of urease activity (measured as a pH rise) that is detrimental for certain animal feeding purposes. Under conditions of heat, moisture, and retention time this enzyme is inactivated, and these variables provide the basis of control for the DT operation. After the desolventizing section, the solid-phase material passes through a series of steam-jacketed heating trays that provide the environment necessary for toasting (Anderson, 2005). Berk (1992) describes the most common type of desolventizer-toaster as a vertical cylindrical stack of compartments or "pans". Each compartment is fitted with stirrers or racks attached to a central vertical shaft. Spent flakes are fed at the top of the desolventizer-toaster. The pan floors are equipped with adjustable-speed rotating valve, to permit downward movement of the material, through the pans, at the desirable rate. Two methods of heating are used: direct steam heating and indirect steam heating. For heating with indirect steam, the pans are equipped with double bottoms acting as steam jackets. For direct steam heating, hot live steam is injected into the mass through spargers. The rotating stirrers spread the material and provide the necessary mixing action. Direct steam is used because direct contact between the solid material and condensing steam is a more efficient method of heating. Condensation of the steam adds moisture to the flakes. Besides, the added moisture facilitates the protein denaturation reactions leading to the inactivation of trypsin inhibitor. It is also believed that the toasting effect accomplished by the combined action of heat and moisture enhances the palatability of the meal to animals. Finally, the steam distillation effect is necessary in order to remove last traces of solvent from the meal. Desolventizing-toasting is the is not recommended for the production of white flakes, meal with minimum protein denaturation. Protein denaturation (expressed as the reduction in Nitrogen Solubility Index, NSI) by treatment with live steam is very rapid. According to Berk (1992), white flakes, which are the starting material for the production of soybean protein isolates, most concentrates and texturized products, must have a high NSI value. The best method of desolventizing for the production of white flakes is flash desolventizing. In this case, the flakes coming out from the extractor are fluidized in a stream of superheated solvent vapours. The superheat of the vapour provides the energy for the evaporation of solvent from the flakes. The turbulent nature of the flake-vapour flow permits extremely rapid heat and mass transfer. Protein denaturation is minimized, mainly because of the short heating time. A short stripping stage may be necessary to complete solvent removal and rapid cooling is a must for preventing undue reduction of NDI.

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Sunflower oil extraction Extraction of oil from sunflower seeds or kernel can be done using general equipments and operating conditions used for soybean or other oilseeds Seeds oil is obtained by one of three methods, including hydraulic, expeller and solvent extraction methods. The hydraulic method of extraction consists of pressing the shelled seeds under 14,000 psi, while adding steam and heat (Emission Factor Documentation For AP-42, 1995). A mathematical simulation of uniaxial compression of oilseeds for oil extraction was developed based upon combining Terzaghi's theory of consolidation for saturated soils with Darcy's law for unsaturated flow, while incorporating the time-varying nature of the coefficients of permeability and consolidation. The model was validated for extruded soy and for sunflower seeds. Material parameters were determined experimentally and predictions of oil recovery rates made for several levels of temperature, pressure and initial sample depth. Results indicated that while the model predicted the values of oil recovery for extruded soybean very well, the predictions were not satisfactory for sunflower seed samples. The higher error was attributed to material non-homogeneity and the presence of hulls in the sunflower seeds, which increased errors in measurement of the medium permeability function. The lack of experimental permeability data in the very early stages of pressing (t < 60 s) was an important source of error in general. The incorporation of varying material properties in the simulations resulted in substantially more accurate predictions of quantities and trends in oil recovery with time, when compared to using constant (averaged) values of material parameters (Bargale et al, 2000). Expeller pressing is the most popular method of seed oil extraction. Screw presses use an electric motor to rotate a heavy iron shaft, which has flights, or worms built into it to push the seeds through a narrow opening. The pressure of forcing the seed mass through this slot releases part of the oil, which comes out through tiny slits in a metal barrel fitted around the rotating shaft. Expellers have a continuous flow of seed through the machine in contrast to the hydraulic system described above, which uses small, individual packages or batches of seed. To release as much oil as possible, the seeds must be dried to rather low moisture content and exposure to high temperature causes darkening of the oil. It also causes some scorching or overheating of the meal. In view of replace the traditional method of separation of seed oil slurry which is rather wasteful, unhygienic and labour intensive with a mechanized method, mechanical filtration by wide-angle conical screen centrifugal filter is necessary. Kartika et al., (2006) investigates the screw configuration allowing oil extraction from sunflower seeds with a twin-screw extruder. Experiments were conducted using a co-rotating twin-screw extruder. Five screw profiles were examined to define the best performance (oil extraction yield, specific mechanical energy and oil quality) by studying the influence of operating conditions, barrel temperature, screw speed and feed rate. In all experiments tested, the extrusion process with different screw configurations and operating conditions produced oil of a good quality (Kartika et al., 2006) The widely-used second method is to extract the oil using a solvent, hexane, which dissolves the oil and strips it from the seed flakes. The solvent extraction method requires stripping residual hexane from the oil and the seed flakes. This method is frequently combined with the expeller methods.

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2.3. Refined Oils: Chemical and Physical Refining The aspect and the quality of the crude oils obtained by a mechanical extraction or by a solvent extraction depend directly on the process and mainly on the quality of the raw materials, oilseeds or fruits. Except if the raw material and consequently the crude oil, has a very high quality, a crude oil cannot be used directly for food or non food applications without some specific technological treatments. The objectives of these treatments is to get a better quality, a more acceptable aspect (limpidity), a lighter odor or color, a longer stability or a good safety with elimination of polluants. Effectively, a crude crude oil contains variable amounts of non triglyceridic natural components such as water, free fatty acids, phospholipids and other polar lipids (glycolipids) , unsaponifiable matter, waxes, pigments, solid impurities (fibers, cellulose) and oxidation products (peroxides, aldehydes, oxidised fatty acids). These components are not toxic but their presence in oils is undesirable for the stability or for the sensorial acceptability by the consumers. A crude oil can also contain some chemical and microbiological exogen polluants: pesticides, heavy toxic metals, hydrocarbons or mineral oils traces, aflatoxins, dioxins, hexane or other organic solvent traces, polycyclic aromatic hydrocarbons (PAH), bacteria or spores. The origin of theses polluants can be the environment during cultivation, the transport and the storage of the seeds, the process and the storage of the crude oils. All these components could be defined as “objectionable non triglyceridic constituents “. Their concentrations in crude oils and their effects are reported in Table 2.1. The treatments that can eliminate all the undesirable and toxic components in crude oils are the steps of the process called “refining”. There are two main industrial technologies for refining the oils: the chemical refining and the physical refining. In the both cases, the aim is to provide a “good quality” refined oil that can be used for food and non food applications nd for direct consumption. The “good quality” includes the respect of the sensorial quality (oil with light color, limpid, no odour, no taste), the stability (shell-life) and the safety of the oil.The final specifications of the refined edible oils are listed in Tables 2.2 and 2.3. According to Regitano D’arce (2006), the lowest the quality of crude oil, the highest the losses and wastes in refining process will be, wich lowers the industrial yields. Removal of the objectionable nontriglyceride constituents in the fat or oil with the least possible damage to the triglycerides and minimal loss of desirable constituents is the objective of refining (O’ Brien, 2004). The two major refining systems are chemical refining (Fig. 2.3) and physical refining (Fig. 2.4). In chemical refining, free fatty acids and most of the phosphatides and some of other objectionable components are removed during a chemical neutralization with caustic soda. In physical refining, free fatty acids are removed by distillation during deodorization at high temperature, under vacuum and with stripping steam. In this case, a pre-treatment of the crude oil must be done in order to remove phosphatides and other impurities prior to steam distillation. Physical refining cannot be applied to all fats and oils because a very high temperature is applied (240°C/ 260°C) and can promote degradation of sentisitive oils with high level of polyunsaturated fatty acids. During the refining, the losses of neutral oil, the contact with oxygen and metal contamination must be as low as possible. Besides, the expositions of the oil to high temperatures must be for short times and under vacuum, in order to prevent oxidation and other undesirable reactions.

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Table 2.1. Objectionable non triglyceridic components in crude vegetable oils (X. Pages, ITERG, French Institute for Fats and Oils, France)

Component Level in crude oils Origin Effects

Free Fatty Acids 0.3 - 5 % Natural compounds hydrolysis of triglycerids

Taste, smoke if heating, hydrolysis

Phosphatides (phospholipids) 0.2 - 2 % Natural components in

seeds

Cloudy aspect Deposit a residue in oil

Flavors Dark color if heating

Oxidation products Variable

x 10 mg/Kg Auto-oxidation of

unsaturated fatty acids

Undesiderable flavors Stability

Colour – Nutrition

Flavours < 0.1% Natural components of seeds auto-oxidation

Odorous components flavors

Waxes x 100 mg/kg Natural components of seeds Cloudy aspect

Pigments x 10 mg/kg Natural components of seeds

Colour Stability

Metals iron, copper x mg/kg Natural components

Technological pollution Oxidation catalysts

Stability Chemical polluants Heavy metals Pesticides PAHs Mycotoxines Dioxines

x 10 mg/ton x 10 μg/kg

Pollution during storage, transport, process Safety Toxicity

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Table 2.2. Specifications of refined oils (A. Rossignol-Castera, ITERG, French Institute for Fats and Oils, France)

Criterion and analytical method Codex Alimentarius value and value generally found

in edible refined oils

Sensorial parameters Panel test

Limpid at room temperature Neutral taste

Neutral flavor Color Lovibond NF ISO 15305 1.5 red / 15 yellow (5 p ¼ )

Content of water and volatiles NF ISO 662

< 0.2 g/100 g generally <0.05 g /100 g

Content of impurities or insoluble matter NF EN ISO 663

< 0.05 g/100g Generally < 0.01 g / 100 g

Oleic acidity or FFA level NF EN ISO 660

< 0.3 g /100 g Generally < 0.1 g / 100 g

Peroxide value NF ISO 3960

< 10 meq O2/kg Generally < 0.5 meq O2/kg just after

deodorization Trans Fatty acids NF EN ISO 5509/5508 < 1 % of total FA

Phosphorus content IUPAC 2.423

< 5 mg / kg Generally < 1 mg / kg

Soap traces < 10 mg / k Generally < 5 mg / kg

Waxes Cold test at 6°C Negative

Iron NF EN ISO 8294

< 1500 mg / kg Generally < 400 mg / kg

Copper NF EN ISO 8294

< 100 mg / kg Generally < 10 mg / kg

Stability test In conformity with shell life

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Table 2.3. Maximum limits for polluants in edible refined oils (A. Rossignol-Castera, ITERG, French Institute for Fats and Oils, France)

Criterion Codex Norm, or EC regulation or

recommendation ppb = μg/kg of oil

Lead < 100 ppb - Codex Alimentarius + EC 466/200

Arsenic < 100 ppb - Codex Alimentarius

Cadmium < 20 ppb

Mercury < 10 ppb

Hexane < 1 ppm - EC 97/60

Aflatoxin B1 < 5 ppb - French recommendation

Dioxins < 0.75 pg OMS-PCDD/F-TEQ/g EC 2375/2001

Organochloride and organophosphate pesticides Not detected or < 10 ppb - EC 396/2005

Benzo(a)pyren < 2 ppb - EC 208/2005

Heavy PAHs < 10 ppb - FEDIOL recommendation

Total PAHs < 25 ppb - FEDIOL recommendation

Pathogenic germs Coliforms < 1/g

E .coli < 1/g Salmonella 0/25 g

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2.3.1 Chemical refining: the case of sunflower oil The different steps of a chemical refining are listed in Fig. 2.3:

- Degumming : elimination of phospholipids - Neutralization : elimination of free fatty acids - Washings and drying : elimination of soaps and water - Bleaching : elimination of pigments - Dewaxing : elimination of waxes - Deodorization : elimination of volatiles

Degumming The chemical refining generally starts by a water degumming step or an acid conditioning step to can eliminate phospholipids from the crude oil. Crude oils contain phosphorous compounds called hydratable phospholipids, and small amounts of calcium and magnesium that complex with a portion of the phospholipids to form non-hydratable phospholipids (EPA, 1995). A degumming is crucial for physical refining but optional for chemical refining (O’ Brien, 2004). It consists of the treatment of crude oils with water, salt solutions, enzymes, caustic soda, or dilute acids such as phosphoric, citric or maleic to remove phosphatides, waxes, prooxidants and other impurities. The degumming process converts phosphatides to hydrated gums, wich are insoluble in oil and readly separated as a sludge by settling, filtering or centrifugal action. Degumming is necessary for lecithin production provided that hydrated gums are the raw materials for lecithin processing. This operation is also important to prevent impurities from settling out during transport and reduces neutral oil loss during chemical refining, given that phosphates act as emulsifiers in a caustic solution to increase the neutral oil entrained in the soapstock. This results in less impact upon the wastewater treatment systems Water degumming. Crude vegetable sunflower oils trend to deposit a residue during transportation and storage. This residue is due to the slow decantation of phospholipids. In order to remove these components, approximately 2% of water, by oil volume, is brought into contact with the crude oil by mechanical agitation in a mix tank. Complete hydratation requires approximately 30 minutes of agitation at 60/70 °C for batch processing. After hydratation, a centrifugation is applied to separate the gums (phospholipids + water) and the “degummed” oil phases. The gums can be vacuum dried for crude lecithin processing or added back to the meal; the process commonly used for soybean oil because of the high phospholipids level is also applied for sunflower oil despite to the lower gum quantities obtained, because of the non GMO source of these gums which offer new opportunities on the market.

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Figure 2.3. Flow diagram of the chemical/alkali refining process (M. Di Stasio, CNR-ISA, Institute of Food Sciences, Italy)

Acid conditioning. Acid degumming leads to a lower residual phosphorus content than water degumming and is therefore a good alternative if physical refining are to be the nest refining steps. The nonhydratable gums, consisting mainly of calcium and magnesium salts of phosphatidic acids and phosphatidyl ethanolamine is converted to hydratables forms in the presence of acid. A binding complex is formed with calcium and magnesium that can be removed with aqueous phase. Phosphoric and citric acids are the most used because they are food grade. The gums isolated with acid process is not suitable for standard lecithin because their phosphatide composition differs from those obtained with water degumming (higher phosphatidic acid) and they contain the degumming acid (O’ Brien, 2004). The treatment consists of food grade phosphoric or citric acid addition (about 0.1%) and mixing at 70/80°C before to go continuously to the second step of neutralization.

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Neutralization Bhosle and Subramaniam (2005) states that the removal of free fat acids from crude oil represents the most delicate and difficult stage in the refining cycle, since it determines the quality of the final product. Chemical, physical, and micella deacidification methods have been used industrially for neutralization of crude oils. The purpose of neutralization in the conventional chemical process is to remove nontriglyceride impurities, consisting principally of free fatty acids, along with substantial quantities of mucilaginous substances, phospholipids and colour pigments (Young et al., 1994). Neutralization is accomplished by the addition of an alkali to degummed oil, thereby precipitating the free fat acids as soap stock; the latter is then removed by mechanical separation from the neutral oil. Since the alkali most often used for neutralization is caustic soda (sodium hydroxide), the process is widely known as caustic deacidification. Oil sampling must be done before neutralization to measure phosphatides content, wich is desired to be lower than 0.3% (O’ Brien, 2004). If this level is exceeded, a blend with another oil is recommended to attain a maximum 1.0% phosphatide content. The steps applied in neutralization are: (a) conditioning, (b) neutralization, (c) washing and (d) drying.In conditioning, food grade phosphoric acid is added to the oil for 4 to 8 hours before refining. This step aims at precipitating phosphatidic materials and calcium and magnesium as insoluble salts, inactivating trace metals, reducing neutral oil losses and desestabilizing to improve the removal of chlorophyll in bleaching (O’ Brien, 2004). Then, emulsion is broken by mixing continuously the oil with a proportioned stream of dilute caustic soda. The gums are hydrolysed by the water in the caustic solution and become oil insoluble, the caustic and soft oil are mixed at 30 to 35°C in a dwell mixer with a 5 to 15-minute residence time if the method is long mix. Operation occurs at 80 to 90°C for 1 to 15 seconds if it is a short mix process, wich is recommended for oils with a higher free fat acids content (Regitano D’Arce, 2006). High oil temperatures during the caustic addition must be avoided because they can increase the neutral oil saponification and reduce the refined oil yield. Oil is heated until 74°C to break the emulsion and delivered to centrifuges for separation. After separation, oil is washed with hot softened water or recovered steam condensated proportioned into the oil in a rate of 10 to 20% of the oil flow. The fluids are high-speed mixed and are transported to a washwater centrifuge for separation. Washed oil is passed thurough nozzles into the evacuated section of a continuous vacuum dryer at 70cm Hg and 85°C. The final moisture must be bellow 0.1%. The dried oil is cooled to about 50°C before storage and nitrogen sparge or nitrogen blanket is applied to the surface in case of extended storage periods (O’ Brien, 2004). In the silica refining process the silica is used to remove soaps and some residual phospholipids from the neutral oil, thus replacing washing. This treatment before bleaching reduces the bleaching earth dosage.In chemical deacidification, there is considerable oil loss due to the hydrolysis of neutral oil by caustic. Besides, loss of oil also occurs in the form of occlusion in soapstock. The soapstock can hold as much as 50% of its weight of neutral oil, thereby reducing the overall yield of refined product. In spite of having several disadvantages, chemical deacidification is still commercially followed in many industries because of successful reduction of free fat acids up to the desired level irrespective of free fat acids content in crude oil. Chemical neutralization reduces the free fat acids to an acceptable level––down to 0.03%––depending on the characteristics of the vegetable oil (Hodgson, 1996). Usually the best results are obtained with relatively weak caustic solutions or lyes on low FFA oils and with stronger lyes on high FFA oils. A small excess of caustic solution is generally used in order to achieve low residual oleic acidity oil (<0.05%). Many refineries use an inline shear mixer to obtain the intimate contact time between caustic and oil followed by a delay period in the dwell mixer prior to centrifugation. In the same time caustic soda treatment trends to reduce primary oxidation products, pigments, metal traces and some of pesticides traces.

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Washings and drying After first separation of the soapstocks in a primary centrifuge, neutralized oil is washed with hot softened water from recovered steam condensate proportioned into the oil at the rate of 10 to 20 % of the oil flow. Softened water must be used to avoid the formation of insoluble soaps Residual soaps are readily washable and easily removed from the oil with generally a double washing. The water temperature should be about 85 to 90°C in order to get a good separation in the centrifuge. After this treatment, water-washed oil is dried with a vacuum dryer before bleaching. It is done by passing the oil through nozzles into the evacuated section of a continuous vacuum dryer that controls the moisture content of the oil below 0.1%. Bleaching The purpose of bleaching is not only to provide a lighter colored oil by removing pigments (carotenes, chlorophylls) by adsorption but also to purify the oil in preparation for further processing. After neutralization, washings and drying, it remains in oil traces of a number of undesirable impurities either in solution or as colloidal suspensions. The key parameters for the bleaching process are procedure, adsorbent type and quantity, temperature, time, moisture and filtration. The adsorbents used for bleaching can remove pigments and other impurities, such as soaps, trace metals, phospholipids, oxidation products, and polyaromatics (Mag, 1990). The removal of these impurities improves the sensory quality and the oxidative stability of the deodorized oil (De Greyt and Kellens, 2000). Activated adsorbents are hydrated aluminium silicates, commonly known as bleaching clays. They are purified and activated by a mineral acid treatment, resulting in the de-lamination of the structure, thus increasing clay specific surface and adsorption capacity (Rossi et al., 2003). Other adsorbents can be activated earths, activated carbon and amorphous silica. The three most used bleaching methods are batch atmospheric, batch vacuum and continuous vacuum (O’ Brien, 2004). In batch atmospheric bleaching, oil at 71°C is pumped into an open-top tank with steam coils or a steam jacket and a paddle agitator. Bleaching earth is fed from the top with agitation at the proportion of 0.15 to 3%. The temperature is raised until 110 °C and kept for a 15 to 20 minutes, when oil is pumped to a filter and recirculated to the tank until it is enough clear. For batch vacuum bleaching a small portion of refined oil at 71°C is added to an agitated slurry tank and bleaching earth is fed. The slurry is transferred to the 50 mmHg vaccum bleacher wich contains the balance of the oil batch. The bleaching vessel has steam coils or steam jackets, an agitator and a vacuum system. Temperature is raised to a maximum 85°C for 15 to 20 minutes, then the oil is cooled to 71°C, the vacuum is broken and the oil is filtered (Regitano D’arce, 2006). Finally, continuous vacuum method consists of feeding continuously the bleaching clay into a stream of oil at 71°C and spraying the mixture into a vaccum chamber to remove air and water from the clay and the oil during 7 minutes. Temperature is elevated in a heat exchanger and sprayed in a second chamber for bleaching. After 10 minutes, the oil is filtered and cooled to about 50°C before the vacuum is broken. Vacuum bleaching is preferred because it can use less clay, operates at lower temperatures, effects quicker moisture evacuation for less free fat acid development from hydrolysis and does not expose oil to oxidation at high temperatures (O’ Brien, 2004). According to Regitano D’arce (2006), the clays are good catalysers in the oxidation reaction, wich demands vacuum processing. Sunflower crude oil has a light yellow colour and is commonly treated with small quantity (0.5%) of activated bleaching clay. Activated carbon can also be used in mixture with bleaching earth to avoid any problems of filtration; carbon is effective in adsorbing certain impurities not affected by earths; for example some aromatic materials (PAHs). That is the case with Ukraine origin sunflower seeds which are often dried under bad operating

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conditions and could be polluted with PAHs. The spent bleaching earth removed from the bleached oil represents a substantial amount of waste material with an oil content from 25 to 60%. Some of the procedures for trying to recover this oil are cake steaming, hot water extraction, solvent extraction or water/lye extraction. Other ways of valorization are actually under experimentation such as biogas production. It is important to take into account the fact that the spent bleaching earth oxides rapidly when exposed to the air to develop a strong odour, and spontaneous combustion easily occurs. Dewaxing or winterization Some crude oils can contain waxes that can crystallise at room tempearature and give a cloudy aspect to the oil which is not suitable. Sunflower crude oils contain waxes, generally at levels of 0.02 to 0.35% but sometimes higher. Sunflower waxes are esters based on C20 to C22 fatty acids and C24 to C28 alcohols and melt at 70 to 80°C. The classical dewaxing process usually performed after bleaching and prior to deodorization consists of carefully cooling the oil to crystallize the waxes and then removing the waxes by filtration using a filter aid. The cooling is done slowly under controlled conditions: 8 hours at 8° C are usually needed for allowing a complete crystallization of sunflower waxes. Before filtration, the oil is carefully heated to 20°C in order to decrease the oil viscosity and facilate the separation. The filter cake rich in waxes gives similar problems as used bleaching clays and different ways of valorization have been studied recently. The use as raw material for manufacturing cleaning soaps or lubricant soaps, depending of the nature of the basic reactant used for saponification, has been patented by ITERG. Some of the procedures using to improve the economic impact of dewaxing include : a/ Simultaneous pre-dewaxing and degumming : the crude oil is cooled about 25°C and held at this temperature for 12 to 24 hours before water degumming. This process usually reduces the wax content to 200 to 400 ppm b/ Simultaneous dewaxing and chemical refining : the oil treated by phosphoric acid or citric acid and then neutralized with caustic soda is centrifuged using normal refining techniques. Before water washing, the oil is cooled to 8°C and held at this temperature for 4 to 5 hours under gentle agitation. Then 4 to 6% of water is added and the mixture is heated to 18 °C with agitation. During this mixing, the waxes crystals are wetted and suspended in the soapy water phase. This mixture is centrifuged to separate the water and oil phases. Usually a second water washing is required to complete removal of the waxes and soap traces from the oil. Deodorization The deodorization is the last step of a chemical refining. Deodorization is a vacuum- steam distillation process of the oil at an elevated temperature during which odoriferous volatile materials and residual FFAs are removed to obtain a bland and odourless oil. Sunflower oil as most vegetable oils retains characteristic undesirable flavours and odors and obtains others during processing. For example bleaching with activated bleaching clays imparts an earthy flavour and odour that can be described only as typical and certainly undesirable. The volatile or thermosensitive substances are FFAs, aldehydes, ketones, peroxides, alcohols, and other organic compounds. Additionally certain carotenoid pigments are destroyed, resulting in a heat-bleaching effect. Efficient removal of these substances depends upon their vapour pressure that is a function of the temperature with an increasing with the temperature. Deodorization is the last major processing step during which the flavour and odour and many of the stability qualities of an oil can be affected. Considerable care must given to the selection, operation and maintenance of the deodorizer equipment and the operating conditions. The four interrelated operating variables that influence deodorized oil

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quality are: vacuum, temperature, stripping steam rate and holding time at deodorization temperatures. Vacuum. If odoriferous compounds are to be distilled at as low temperature as possible, distillation must be carried out at low absolute pressure effected by the vacuum system. The boiling point of the fatty acids and the vapour pressure of the odoriferous materials decreases as the absolute pressure decreases. The required low absolute pressure, usually between 2 and 4 mbar, is commonly generated by vacuum systems consisting of a combination of steam jet ejectors, vapour condensers, and mechanical vacuum pumps. Special vacuum systems have been developed to reach lower pressures and operating costs and, at the same time, reduce emissions by a more efficient condensing of the volatiles. In the dry condensing systems, the sparge steam is condensed on surface condensers working alternatively. The remaining non-condensables are removed by mechanical pumps or by a vacuum ejector system. Temperature. Deodorization temperature must be high enough to make the vapour pressure of the volatile impurities in the oil conveniently high. The vapour pressure of the odoriferous materials increases rapidly as the temperature of the oil is increased. For example, the vapour pressure of palmitic fatty acid is 1,8 mm at 176.7°C, 7.4 mm at 204.4°C, 25 mm at 232.2°C, and 72 mm at 260°C. Assuming that the vapour pressure-temperature relationship for all the odoriferous materials is similar to that of palmitic acid, each 27.8°C deodorizer temperature increasing would triple the odoriferous material removal rate. Or, stated another way, it would take nine times as long to deodorize an oil at 176.7°C than at 232.2°C .Higher deodorizer temperatures definitely provide shorter deodorization times; however, excessive temperature results in the development of the polymerization, isomerisation to produce trans fatty acids, thermal cracking with formation of odoriferous and low boiling products, colour reversion, and distillation of tocopherols. Generally, trans formation during deodorization is negligible below 220°C, becomes significant between 220 and 240°C, and is nearly exponential above 240°C. Thermal degradation of the tocopherols becomes significant at deodorization temperatures above 260°C. It has been determinated that twice as many tocopherols and sterols are stripped out 275°C as at 240°C, and that pressure variations of 2 to 6 mbar had only a slight effect upon tocopherol/sterol stripping. Deodorizer operation at elevated temperatures can also promote thermal decomposition of some constituents naturally present in sunflower oils, such as pigments and some trace metal-prooxidant complexes. The carotenoid pigments can be decomposed and removed by deodorization beginning at 230°C; therefore, a compromise must be determined between time and temperature for deodorizing particular fats and oils. Optimum deodorizer operating temperatures vary from product to product. Chemically refined sunflower oils are easier to deodorize than physically refined oils due to lower FFA levels and more effective removal of polar components, oxidation products, and pigments. In general, deodorization temperatures will vary from 180 to 230°C. Stripping steam. The amount of stripping steam required is function of both the absolute operating pressure and the mixing efficiency of the equipment design. Typical stripping steam deodorization conditions for chemically refined oils are 5 to 15% of oil for batch systems and 0.5% to 2% for continuous and semi-continous deodorizer systems. Holding time. Stripping time for efficient deodorization has to be enough long to reduce the odoriferous components of the oils. Typically for sunflower oil, the holding time for continuous and semi-continous systems vary from 40 to 120 minutes.

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Deodorization systems. Deodorization equipment in current use to deodorize sunflower oil can be classified into three principal groups: batch, continuous, and semi-continuous. Nowadays continuous or semi-continuous systems are in use; continuous deodorizer provides uniform utility consumption. They can involve mainly tray or thin film deodorizers. Tray deodorisers are based on a series of steam agitated trays or compartments often stacked vertically in a cylindrical shell. The retention time per tray is usually 10 to 30 minutes. Thin-film deodorization has structured packing to create a maximum surface - to- volume ratio. The air flows over the packing and meets the sparging steam counter, currently for FFA stripping. Actually, all thermal heating fluids (THFs) became suspect carcinogenis materials. The European edible oil Community elected to phase out THF systems and replace them with hot water/steam heat transfer systems. After deodorization, the oil is cooled, normally first passing through a heat recovery economizer, and then through final cooling. During cooling, a small amount of chelating agent, such as citric acid, may be introduced into the oil, as may an antioxidant. The volatiles removed during the deodorizing process are condensed and usually recovered in a fatty material direct condenser, known as a vapor scrubber. 2.3.2 Particularities of other edible oils Peanut Oil The refining process of peanut oil is the same than sunflower oil except the dewaxing step. Peanut oil cannot be winterized because of the presence of high melting point fraction that solidifies at to 3°C (Krishmamurthy and White, 1996). Mould (Aspergillus flavous) can attack groundnut, leading to aflatoxin contamination, if the nuts are not dried sufficiently. Aflatoxin in peanuts is a serious problem. The peanuts can become infected either before or after harvest. Once they are infected, there is no way that the aflatoxin can be removed and the peanut becomes dangerous for consumption. If the peanut is free from the disease at harvest, correct drying to less than 10% moisture can prevent later infection. Aflatoxin is problematic in unrefined oil (Anon, 2001) or peanut oil prepared by small scale production (Diop et al,. 2000), since refining process is known to eliminate it (Ayres, 1983). Moldy peanuts that have been infected to the extent of 5500 ppb with aflatoxins yielded a peanut oil with 812 ppb, wich was reduced to 10 to 14 ppb after caustic refining and less than 1 ppb after bleaching (O’Brien, 2004). Corn Oil Crude corn oil, because of natural antioxidants it contains, undergoes little deterioration when stored for long periods, provided the temperature is kept well below 40ºC (102ºF) and moisture plus volatile matter level is below 0.4%. The refining conditions of crude corn oil are described in the doucment of the Corn Refiners Association, 2006. Soybean oil: exemple of membrane process O’Brien (2004) stated that soybean oil can be physically refined depending upon the treatment of the bean before and during the extraction. .The types of degumming O’Brien (2004) recommended for soybean oil are: (a) water degumming, (b) acid degumming, (c) enzymatic degumming. The presence of significant quantities of nonhydratable phosphatides usually indicates a poor-quality oil, for soybean oil from fresh good-quality beans about 90% of the phosphatides are normally hydratable. However, when seed is severely damaged, the

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hydratable phosphatide may be reduced by as much as 50% over time (Anderson, 2005; Rossel and Pritchar, 1991). Phospholipase A1, the preferred enzyme used for enzymatic degumming is produced by biological fermentation. The pH must be adjusted to about 4.5 and oil is kept at 40°C before adding the enzyme in the proportion of 200,000 units in 7.5 liters os water per ton of oil. After the reaction is finished, temperature is raised to 75°C for more efficient separation of the gums in a centrifuge. An enzymatic degumming trial of soybean oil was carried out by Yang et al., (2008) at a capacity of 400 tons/day by applying microbial phospholipase A1 from Thermomyces lanuginosus/Fusarium oxysporum. When the pH was kept in the range of 4.8–5.1, less than 10ppm of phosphorous content of the oil was obtained. The gum and oil were easily separated after centrifugation and the oil loss was minimal under the process conditions. The techniques of separation with membranes are presented as alternative of great interest to substitute the conventional degumming method in vegetable oil refining. This technology does not involve the use of water or acid solutions (diminishing the generation of effluent) and makes possible the reduction of losses of neutral oil and the associated energy cost to the process (Pagliero et al., 2003). According to Bhosle and Subramanian (2005), the membrane process, i.e. ultrafiltration, is a remarkably simple process offering many advantages over the conventional processes, such as low-energy consumption, ambient temperature operation, no addition of chemicals, and retention of nutrients as well as other desirable components. It has been estimated that a great potential for energy savings––to the tune of 15–22 trillion kJ/year––exists in replacing or supplementing conventional degumming, refining, and bleaching processes (Koseoglu and Engelgau, 1990). Ultrafiltered high phospholipids oils give rise to two products. The permeated, constituted of crude oil with low phospholipids content and hexane, and the retentate, constituted of oil high phospholipids content and hexane. In the following process, both are subjected to the evaporation of the solvent. Crude oil obtained in permeate contains from 3 to 7 ppm phospholipids, wich allows deodorization as next step. Degumming by ultrafiltration in ceramic membranes (0.01 μm) was evaluated by Soares et al., (2004), resulting in soybean oil with phosphorus content bellow 7 ppm, with good sensorial quality after deodorization. The ultrafiltered oils deodorized had shown more intense color, preserving carotenoids and 0.25% of free fatty acids. Soybean oil was rejected as a salad oil both at the retail level and by food processors until the flavor stability problem was remedied with partial hydrogenation to reduce the linolenic fatty acid content. However, hydrogenation also produced hard fraction in the soybean oil, wich crystallized at cool temperatures similar to cottonseed oil. Winterization was employed to separate the liquid and hard fractions, which elevated partially winterized soybean oil to the leading winterized salad oil product in United States (O’ Brien, 2004). Rapeseed oil Specific degumming. A number of acid degumming processes have been developed over the years such as the “superdegumming” and various other proprietary processes (Segers, 1982). Membrane degumming, countercurrent extraction with supercritical CO2, and ultrasonic degumming have also been reported (Young et al., 1994). The most recent development in degumming uses acid and aqueous sodium hydroxide, rather than acid and water, especially with lower quality oils. This represents an intermediate between acid-water degumming and alkali refining. Phosphatides as well as some of the other impurities are removed and, if sufficient alkali is applied to saponify the free fatty acids, fully refined oil is obtainable. Ohlson (1992) described some more recent processes, as total degumming processes. In top degumming, phosphatide/metal complexes are decomposed by a strong acid into

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insoluble metal salts and phosphatides in their acid form. The latter are converted into hydratable compounds by partial neutralization and can then be removed from the oil. It is essential that the acid is dispersed finely in the oil. After a certain contact time alkali is added and mixed into the acid-in-oil emulsion in such a way that soap formation is prevented. This is especially important for yield reasons. The oil is then degummed in a first centrifuge, yielding a gum phase with minimal oil content, and oil still containing residual gums. This oil is then passed to a second centrifugal separator, yielding gum-free oil and an oil-rich gum phase which is recycled. Centri-Ad, a new method for continuous adsorption of impurities, has been developed by Alfa-Laval. Centri-Ad utilizes low-density particles and centrifugal force. The particles form an annular bed of suspended solids that the liquid must pass through. The particles can be modified for different applications. The adsorption is counter-current, and used particles can be replaced during the operation. The main advantage is the potential to separate small amounts of substances, emulsified or dissolved, from large amounts of liquid on a continuous large scale. The centrifugal force exerts thousands of g on the particles. Potential applications for rapeseed oils are separation of sulfur or phosphorus components that would deactivate nickel catalysts and of colored components or substances affecting the flavor or odor. Enzymatic degumming. Enzymatic oil degumming is a suitable process for physical refining. It was first developed in the 1990s in initial industrial plant trials by the German Lurgi Company, as the “EnzyMax process” (Yang et al., 2006; Aalrust et al., 1992). In this process, enzymes change non-hydratable phospholipids into a hydratable form. The EnzyMax process consists of three important steps: adjusting the pH of the oil with buffer, carrying out the enzyme reaction in tanks, and separating gum/sludge from the oil. Every process step is important and must be controlled. Compared with a traditional degumming process, enzymatic degumming has many advantages. In addition to the reduction in the amounts of acid and base used and wastewater generated during the refining process, an enhancement in product yields and a reduction in operating costs can be observed (Klaus, 1998). Membrane degumming. A membrane process is remarkably simple and offers many advantages over conventional processes, namely, low energy consumption, ambient temperature operation, no addition of chemicals, and retention of all of the nutrients as well as other desirable components in the oil (Young et al., 1994). Pressure driven membrane processes are classified as reverse osmosis, nanofiltration, ultrafiltration, and microfiltration depending on the nature of particles or molecular sizes of solutes to be separated. Many researchers have used micelle-enhanced ultrafiltration to degum hexane–oil miscella (Segers, 198; Raman et al., 1994; Gupta, 1977). Keurentjes et al., (1991; 1992) attempted to remove free fatty acids with a combination of hydrophobic and hydrophilic membranes, and later by membrane extraction using 1,2-butanediol as an extractant. There are reports of removing free fatty acids from model vegetable oils and crude rice bran oil by alcohol extraction of free fatty acids followed by membrane separation (Subramanian et al., 2004; Raman et al., 1996). Studies were conducted by Subramanian et al., (1999) on surfactant-aided membrane degumming with soybean and rapeseed oils in a magnetically stirred flat membrane batch cell with different types of microfiltration membranes. The reduction of phospholipids in soybean oil was in the range of 85.8–92.8% during the membrane process. The phosphorus content of membrane permeates of soybean oil was in the range of 20–58 mg/kg. Crude rapeseed oil contained higher amount of non-hydratable phospholipids and hence resulted in lower reduction in phospholipids, in the range of 66.4–83.2%. Addition of hydratable phospholipids could improve the efficiency of degumming in the membrane process without using any electrolyte, resulting in improvement of quality as well as quantity of the phospholipids.

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Zenith process. The Zenith process was developed in Sweden in 1960 to enable better refining of rapeseed/canola oil. The stainless steel continuous process consists of three steps, two of which are semi-continuous to maintain the desired reaction times. In step one, the oil is treated with concentrated phosphoric acid to remove the non-fatty impurities that influence emulsions. The amount of phosphoric acid depends upon the oil quality but normally is about 0.2% by oil volume for rapeseed/canola oil. The reaction, performed under vacuum, requires 20 minutes. Such treatment with phosphoric acid is carried out at temperatures in the range of 35oC to 50oC. The acid sludge formed with the pigments, phosphatides, calcium, magnesium and other impurities is removed with a sludge separator. Water is introduced in the form of live steam to form liquid crystals of the remaining phosphatides at the interface between the water and the oil. Neutralization is performed in the second step by introducing the oil at 90oC in form of droplets to the bottom of a vessel almost filled with 0.35 N alkaline solution. The 1- to 2- mm diameter droplets rise by the difference in specific gravity and are collected in the upper conical part of the vessel, thus forming an oil layer with a typical analysis of 0.05% free fatty acid, 0.2 to 0.3% moisture and 100 ppm soap. Finally, in the last step, the neutralized oil is treated with citric acid to separate the trace quantities of soap for adsorption by the bleaching earth. The oil is dried and bleaching earth added before it is vacuum bleached for 30 minutes before filtering (O’Brien, 2004; Beharry et al., 1996). Membrane neutralization. The membrane process is a remarkably simple process offering many advantages over the conventional processes: namely, low-energy consumption, ambient temperature operation, no addition of chemicals, and retention of nutrients as well as other desirable components. Owing to the vast scope for energy savings as well as potential for improvement in oil quality, edible oil processing has become one of the prime areas for membrane applications. It has been estimated that a great potential for energy savings –– to the tune of 15–22 trillion kJ/year –– exists in replacing or supplementing conventional degumming, refining, and bleaching processes (Koseoglu and Engelgau, 1990). Conceptually, membranes could be used in almost all stages of oil production and purification (Cheryan, 1998). Ramam et al., (1994) listed some of the potential applications of membrane technology in vegetable oil processing. Many of them have been evaluated at the laboratory or pilot plant scale, nevertheless, except for gas separation for the production of nitrogen, there are few commercial membrane installations in the edible oil related industries, in spite of their vast potential and the considerable research efforts already put in. The pressure driven membrane processes are classified as reverse osmosis, nanofiltration, ultrafiltration and microfiltration depending on the nature of particle or on the molecular size of the solutes that are separated. Commercial membrane devices are available in four major types, namely plate and frame, tubular, spiral-wound and hollow fibre. Several researchers have attempted the deacidification of vegetable oils with and without solvents, by using porous as well as nonporous membranes. Despite these efforts, there has been no breakthrough in evolving a successful technology. The molecular weights of fatty acids are <300Da and that of triacylglycerols are >800Da. The ideal process would use a hydrophobic membrane with pores so precise that they could effectively separate the free fatty acids from the triacylglycerols (Raman et al., 1994). In the case of nonporous dense membranes, the selectivity for free fatty acids over triacylglycerols was completely lost upon dilution with hexane (Bhosle and Subramanian, 2005; Bhosle, 2002). Direct deacidification of model oil in acetone was observed when laboratory made, solvent stable, nanofiltration membranes, with either a PEBAX [poly(amide-b-ether) copolymer] or a cellulose-type top layer, were employed (Zwijnenberg et al., 1999). These membranes were stable in acetone, ethanol, 2-propanol and hexane. Fatty acids were retained less than triacylglycerols by these membranes,

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indicating the possibility of deacidification. Bhosle (2002) also observed that these hydrophilic nanofiltration membranes exhibited greater selectivity. For industrial adoption, however, the oil flux needs to be significantly improved. It appears that combination of solvent extraction and membrane separation seems to be technically feasible with the advent of solvent resistant membranes. Nevertheless, the introduction of another solvent in the process such as methanol/ethanol besides hexane used for extraction of oils/fats from oil-bearing material would not be a very attractive preposition as compared to direct membrane deacidification. Specific bleaching. Alkali refined oil, which still contains most of the chlorophylloid compounds present in the crude oil, requires bleaching. These compounds must be removed. They catalyze oil oxidation and give an undesirable green colour to the oil. According to Salunkhe et al., (1992), bleaching is the process in which finely divided clay (neutral or acid activated) is added to oil in order to improve the colour of the oil. It has become recognized that chlorophyll removal is the most important aspect of canola oil bleaching. Rapeseed oil contains significant amounts of chlorophyll and is usually more difficult to bleach than other oils. Oil from damaged or immature seed may be extremely difficult or impossible to bleach. Acid-activated clays are used. Their adsorptive properties are especially effective for the removal of these compounds. Other coloured compounds, some of the oxidation breakdown products and traces of iron are also removed in this process. The process itself is carried out under vacuum with the oil at about 100ºC. Many process versions are in use. About 5-30 minutes of contact time is given, while the oil/clay slurry is progressively dried to about 0.1% moisture content. This gives the best adsorption efficiency. As indicated earlier, about 1-3% clay (10% moisture content) may be required to achieve chlorophyll removal to <25 ppb. This level is innocuous in respect to oxidation and colour of the oil. Brimberg (1981, 1982) and Henderson (1993) have investigated aspects of chlorophyll adsorption important to the bleaching of rapeseed oil. Sepiolite bleaching. Sepiolite is a natural fibrous clay mineral with a hydrated magnesium silicate formula [(Si12)(Mg8)O30(OH)6(OH2)4.8H2O] (Brauner, 1956). It has a high decolorization capacity because of its high specific surface area, porosity, and surface activity, and its ability to form high and stable viscosities at low solids concentrations. The sorption ability of sepiolite is mainly ascribed to its high surface area (Alvarez, 1985). The efficiency of Turkish sepiolite in bleaching degummed rapeseed oil has been investigated by Sabah e Çelik (2005). Bleaching efficiency was more dependent on the ratio of sepiolite to oil than on operating parameters uch as contact time and temperature. An increase in the sepiolite dosage reduces the colour bodies of the rapeseed oil. Its effect on oxidation state, however, is complex and related to both primary and secondary oxidation products. The removal of impurities such as chlorophyll a, b-carotene, and phosphorus increases with increasing sepiolite dosage and reaches a maximum at 1.5% sepiolite addition and 100°C bleaching temperature (Sabah and Çelik, 2005). Dewaxing or winterization. Canola oil occasionally contains a small concentration of waxes (about 100 – 200 ppm). These compounds are related to triacylglycerols in composition, but are usually crystalline at room temperature, or lower. In some cases, it is desirable, therefore, to dewax the oil to avoid a hazy appearance. The process is carried out by chilling in a continuous heat exchanger to about 5ºC and metering about 0.1% of a filter aid into the chilled oil stream on the way to a filter. This reduces the wax content to <50 ppm, which no longer produces a visible haze. Hu et al., (1993) investigated the composition of turbidity refined canola oils by filtration at 4 and 20oC. Major components (thin-layer

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chromatography) at both temperatures were wax esters, hydrocarbons and triacylglycerols while free fatty acids and fatty alcohols were found in minor amounts at 4°C. Przybylski et al., (1993) examined the formation of sediment in bottled canola oil during storage at 2, 6 and 12°C over a 4-day period. Oils stored at 2°C showed the highest rate of sediment formation, followed by storage at 6°C. Removal of sediment from canola oil prior to storage by cold precipitation and filtration did not eliminate this phenomenon, which still developed rapidly at 2°C. Chemical composition and thermal properties of canola oil sediment were compared to sediment obtained from commercial winterization of this oil. The thermal properties of the purified winterization sediment (melting temperature, 74.9°C) closely resembled those of the sediment from bottled canola oil. Sediment from commercial winterization contained higher amounts of fatty acids and alcohols with more than 24 carbon atoms in the chain. Dewaxing/winterization is performed by chilling in a continuous heat exchanger to about 5ºC. The cooled oil is further filtered for removal of sediments. Deodorization. According to De Greyt (2004), chemical refining deodorization in Europe is carried out at 230 to 240oC with 2 to 3 mbar of absolute pressure for one hour. Henón et al., (1999) evaluated laboratory-scale treatments of canola oils similar to deodorization carried out by applying nitrogen or steam stripping at reduced pressure with temperatures ranging from 210 to 270°C for 2–65 h. Based on these experiments, a mathematical model was developed to describe the isomerization reaction steps occurring in linoleic and linolenic acids during deodorization. The calculated degrees of isomerization were independent of the composition of the oil but related to both time and temperature of deodorization. The degree of isomerization of linolenic acid was unaffected by the decrease of this acid content observed during the deodorization. Deodorization at about 220–230°C appears to be a critical limit beyond which the linolenic isomerization increases very strongly. The established model was considered a tool for manufacturers to reduce the total trans isomer content of refined oils, and was applied to produce a special selectively isomerized oil for a European nutritional project. The higher the temperature is, the lower will be the retention of tocopherols and sterols in the oil. It was also observed that for the same temperature, higher pressures increased the retention of tocopherols (De Greyt, 2004). 2.3.3 Physical refining Physical refining involves: (a) degumming to remove phosphatides, (b) bleaching and filtration to eliminate color pigments and (c) deodorization and physical removal of free fatty acids. In physical refining, acid-water degummed oil with a phosphorus content below 50 ppm is first subjected to a phosphoric acid pretreatment, as in the short-mix alkali refining process. It is then mixed with acid-activated bleaching clay in a standard bleaching process at 95-105ºC. The clay, along with precipitated phosphatides and adsorbed chlorophylloid and some carotenoid compounds, is then removed by filtration. This is the first and most important stage of physical refining. It delivers bleached oil ready for deodorizing. Except for the free fatty acids in the oil, all other minor constituents are reduced to the same concentrations as in alkali refining and in addition, chlorophyll is reduced to the concentration required of bleached oil, namely <25 ppb. Usually, 1-3% acid-activated bleaching clay is used, depending primarily on the concentration and type of chlorophylloid compounds present in the oil. The removal of the free fatty acids in the oil is achieved by steam distillation in a deodorizer. This simultaneously deodorizes the oil. The principal advantage for physical

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refining of sunflower oil is the reduction of plant pollution commonly caused by the acidulation of sopastocks produced with conventional caustic refining. A prerequisite for the successful process of physical refining is a very low content of phosphatides, i.e., a phosphorus content of less than 15 mg/kg, preferably less than 10 mg/kg. The process is more nearly ideal if the phosphorus content of the oil is less than 5 mg/kg (Narayana et al., 2002). Proper degumming is vital for the successful physical refining of vegetable oil. Inadequate degumming will directly influence the efficiency of refining and the quality of consumer-ready oils. However, degumming processes (water degumming, superdegumming, total degumming, ultrafiltration processes, acid treatment) cannot always achieve the low phosphorus contents required for physical refining, and they are not always optimally suited for all oil qualities. The refining loss, the cost of the equipment required, and the energy expenditure of these processes are also high (Gibon and Tirtiaux, 2000). Physical refining also requires oil of moderate chlorophyll and free fatty acid content, but it is then very economical. Little authoritative industrial experience with physical refining and the pretreatment of canola oil for physical refining has been published so far (Mag, 1990). For physical refining a temperature range of 230 to 250oC is currently used. These extreme conditions lead to the occurrence of important chemical reactions (Maza et al., 1992), such as the isomerization of polyunsaturated fatty acids (PUFA), that influence the final quality of vegetable oils. The main factor that controls the speed of the isomerization reaction is the deodorization temperature and the residence time (Ceriani and Meirelles, 2007; Devinat et al., 1980). Kinetic measurements concerning geometrical isomerization of linolenic acid showed that the reaction is a first-order one and that the isomerization constant varies with temperature according to Arrhenius’ law (Henón et al., 1999; Wolff, 1993; O’Keef et al., 1993).

Figure 2.4. Flow diagram of the physical refining process (source: M.Di Stasio, CNR-ISA, Institute of Food Sciences, Italy)

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2.3.4 Specifications of refined oils The specifications of refined oil are listed in Tables 2.2 and 2.3. The refined oil is stored until used or transported. Nitrogen gas may be placed in the headspaces of tanks or transport vessels to reduce oxygen contact with the oil, thus preventing degradation of the oil by oxidation (Emission Factor Documentation for AP-42, 1995). 2.4. Packaging and Storing Oils The quality of all edible oils is the sum of their hygienic, safety for consumption, organoleptic, and nutritional properties; this in-turn is determined by the plant source from which oil is produced, the nature of the production process, and last but not least, by the practices oils are stored and packed. Physical and chemical treatments may be applied to oils through processing, which enable the producer to stabilize the oil and preserve its quality characteristics over the course of its shelf life. However, being sensitive to oxidation, the properties of oils in storage and on the shelf change continuously as a function of exposure to the penetration of light and of oxygen, and the presence and levels of minor redox-active components. More changes in quality may and do occur when certain compounds are extracted, migrate, or exchanged between oils and their surrounding media: packaging material, stoppers, and in cases other food products packed together. The presence of oxygen in a package can trigger or accelerate oxidative reactions, and hence the role of packaging in preserving the quality of oils is immense. A good package should first provide the oil with barriers from the outside environment throughout the period of bulk storage at the warehouse and on the shelf, through transportation and finally at the consumer’s home. Indeed, a wide spectrum of retail oil packages can be found in the retail market, that differ by the type of packaging material: plastics of several qualities and properties, glass of various hues, and tinplate. Retail oil packages also differ by their size and shape, which define also the volume of headspace, and the size of the upper surface that is exposed to air prior to and after opening. Oxidative reactions result in adverse qualities such as off-odors, off-flavors, undesirable color changes, and reduced nutritional quality. Hence, oxidation of fatty acids is the most important and prominent deleterious process in oils. Many factors may contribute to the oxidation of stored oil, including storage conditions, i.e. temperature, the levels of polyunsaturated fatty acids and their profile, and the presence of prooxidants such as chlorophyll and heavy metals. The role of packaging in enabling or in blocking of the penetration of specific wavelengths of light and the levels and the availability of soluble and reactive oxygen is discussed. 2.4.1 Molecular oxygen and the oxidation of oils Autooxidation of unsaturated fatty acids during storage first leads the accumulation of hydroperoxides. Under favorable conditions, oxidation follows a free radical chemical process

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where the initially formed hydroperoxides may further decompose or even polymerize, resulting in a complex mixture of compounds that could be used to describe the oxidation level (Angelo, 1996). The generally accepted progression of these reactions is initiation, propagation and termination. These reactions increase in rate and intensity in the presence of light and heat. Molecular oxygen may reach the oil in several ways: Atmospheric oxygen may be entrained in the oil; oxygen can also be available in the headspace of the container and by permeation of the walls of the container. At packaging, the oxygen concentration is close to saturation, and hence the rate at which hydroperoxides are degraded is lower than the rate at which they are produced. As oxygen is consumed in the latter reactions, an oxygen concentration gradient is formed in the packed oil in a completely non permable package such as glass, tin and stainless steel, which in turn may stimulate permeation of external molecular oxygen through the wall of plastic containers. At the same time the rate at which hydroperoxides break degrade is increased. The above described phenomena lead to a decrease of concentrations of both local oxygen and hydroperoxides (Del Nobile et al, 2003). The most obvious marker of oxidation in oils and many food products is rancidity, which is also the main cause for loss of quality. Rancidity is the development of an off-flavor following oxidation of fatty acids and hydrolysis of oxidized intermediates to produce unacceptable flavor and aroma compounds. 2.4.2. Oxygen scavengers and packaging A common practice to enhance the shelf life and protect packged oil is de-aeration prior to filling into a package that has been sparged with an inert gas such as nitrogen. To overcome the residual levels of oxygen in a packaged oil and the propagation of oxidation reactions, and the potential later penetration of molecular oxygen into the Molecules that can be introduced into the packaging material and serve to remove oxygen (residual and/or entering), thereby retarding oxidative reactions, are generally referred to as oxygen scavengers. These may come in various forms: sachets in headspace, labels, or direct incorporation into package material and/or closures. Oxygen scavenging compounds are mostly agents that react with oxygen to reduce its concentration. Ferrous oxide is the most commonly used scavenger (Kerry et al, 2006). Others include ascorbic acid, sulfites, catechol, some nylons, photosensitive dyes, unsaturated hydrocarbons, ligands, and enzymes such as glucose oxidase. To prevent scavengers from acting prematurely, specialized mechanisms can trigger the scavenging reaction. For example, photosensitive dyes irradiated with ultraviolet light activate oxygen removal (Lopez-Rubio et al, 2004). Oxygen scavenging technologies have been successfully used in the meat industry (Kerry et al, 2006). In pressed oils, and specifically in olive oils, natural antioxidants that are present in the oil act as oxygen scavengers, and thus play an important role in conferring protection to oils. These natural antioxidants include tocopherols, which are found also in seed oils and polyphenols that are mostly found in cold pressed, non-filtered and non refined olive oil. Recently phytosterols were also suggested to protect oils from oxidation.

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2.4.3. Penetration of light and oxidation of oils The action of external energy sources such as light can also initiate the oxidation of oils and fats . Especially after bottling, the packaged oil may be exposed to different light sources: Lighting with fluorescent lamps (daylight type or warmlight type) at the dtore as well as at home, or natural sunlight. The regions of interest within the light spectrum are the ultraviolet (UV) with wavelengths up to 390 nm and the visible region of violet and blue in the wavelengths of 390 – 490 nm. It must be noted that a considerably higher portion of UV radiation can influence the product under natural daylight than under commercial lighting with fluorescent lamps. The reaction of the fatty acid moiety of a triglyceride is of the general type in which the fatty acid forms a free radical having been initiated by light quanta through hydrogen atom or electron abstraction. This can proceed in the absence of molecular oxygen. Virgin olive oil, and some other oils from vegetable sources contain chlorophyll pigments (8-24 mg/kg in refrain oil). Chlorophyll can act as a photosensitizer due to its ability to transfer energy from light to triplet oxygen, thus producing singlet oxygen, which then reacts with the unsaturated fatty acids. Studying the effect of visible light on the rate of oxidation of oils in a model system containing chlorophyll, Thron at el (2001) showed that the greatest quantum-dependent sensitivity was detected around 650 nm, the lowest around 500 nm. It was shown that the quanta absorbed by the absorption maximum of chlorophyll at 650 nm are most effective to photooxidation. Carotenoids in the oil serve as effective inhibitors of photo-oxidation by quenching singlet oxygen and triplet excited states of photosensitizers. The physical quenching mechanism of carotenoids is based on their low singlet energy state, which facilitates the acceptance of energy from singlet oxygen (Kiritsakis et al, 2002). Additionally, the antioxidant activity of carotenoids is related to a light-filtering effect due to the extended conjugation system (Fendler et al, 2007). These results point to role of both visible and UV light in accelerating oxidation of oil, and thus to the importance of using packging materials that completely block light penetration such as tin, stainless steel and colored bottles. Using other materials for packaging of oils, light filtering compounds must be used top protect from light induced oxidation. 2.4.4 Permeation, migration and absorption Interactions within a package system refer to the exchange of mass and energy between the packaged food, the package material and the external environment. Food-packaging interactions can be defined as interplay between food, packaging, and the environment, which produces an effect on the food, and/or the package (Hotchkiss, 1997).Mass transfer processes in packaging systems are normally referred to as permeation, migration and absorption. Permeation is the process resulting from two basic mechanisms: diffusion of molecules across the package wall, and absorption/desorption from/into the internal/external atmospheres. Migration is the release of compounds from the plastic packaging material into the product (Hernandez and Gavara, 1999). The migration of compounds from polymer packaging materials to foods was the first type of interaction to be investigated due to the concern that human health might be endangered by the leaching of residues from the polymerisation (e.g. monomers, oligomers, solvents), additives (e.g. plasticisers, colourants, UV-stabilisers, antioxidants) and printing inks. The fundamental driving force in the transfer of components through a package system is the tendency to

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equilibrate the chemical potential (Hernandez and Gavara, 1999). Mass transport through polymeric materials can be described as a multistep process. First, molecules collide with the polymer surface. Then they adsorb and dissolve into the polymer mass. In the polymer film, the molecules ‘hop’ or diffuse randomly as their own kinetic energy keeps them moving from vacancy to vacancy as the polymer chains move. The movement of the molecules depends on the availability of vacancies or ‘holes’ in the polymer film. These ‘holes’ are formed as large chain segments of the polymer slide over each other due to thermal agitation. The random diffusion yields a net movement from the side of the polymer film that is in contact with a high concentration or partial pressure of permeant to the side that is in contact with a low concentration of permeant. The last step involves desorption and evaporation of the molecules from the surface of the film on the downstream side (Singh and Heldman, 1993). Absorption involves the first two steps of this process, i.e. adsorption and diffusion, whereas permeation involves all three steps (Delassus, 1997).Later, absorption or scalping of components originally contained in the product by the packaging material attracted attention. Product components may penetrate the structure of the packaging material, causing loss of aroma, or changing barrier and/or mechanical properties, resulting in a reduced perception of quality. Concerning migration, though most of the plastic films were found to be almost inert towards food constituents, a small amount of monomeric and oligomeric constituents or additives used in their manufacture, to provide stability, plasticity and other desirable functional characteristics, are known to migrate into foods. It was shown that migration can be particularly extensive through direct contact with fatty food and at high temperature. 2.4.5 Influence of packaging on the oxidation kinetic of virgin olive oil Very interesting is also an approach with a two-dimensional mathematical model (Del Nobile et al., 2003) able to predict the time course of hydroperoxides and oxygen concentration profile inside bottled virgin olive oil during storage is presented. By simulating the behaviour of the bottled virgin olive oil it was possible to assess the influence of the bottles shape and size on the quality decay kinetics of virgin olive oil bottled in glass and plastic containers. In particular, five geometrically different containers were used to predict the storage behavior of bottled virgin olive oil. The obtained results show that the quality decay kinetics of bottled virgin olive oil greatly depends on container geometry. However, the extent to which the containers geometrical factors affect the quality decay kinetics depends on the material used to make the bottle, and on the initial value of the oxygen partial pressure in the bottle headspace. It has been proved that mathematical models able to predict the shelf life of packed foods are a valuable tool in designing packaging systems (Del Nobile, et al., 1997; Labuza and Contreras-Medellin, 1981; Tubert and Iglesias, 1985). In a previous paper, Del Nobile et al., (in press) presented a mathematical model able to predict the evolution of oxygen and hydroperoxide concentrations in virgin olive oil bottled in plastic and glass containers. The developed model was used to assess the effect of the following upon the quality decay kinetics of bottled olive oil: oxygen diffusivity and the thickness of the plastic container, the presence of an oxygen scavenger in the container wall and the concentration of oxygen in the oil prior to bottling. In particular, it was established that by increasing the barrier properties of the polymer used to manufacture the bottle it is possible to obtain a quality decay kinetic as slow as that obtained for olive oil bottled in glass containers. Oxidation kinetics slower than that found with glass bottles can be obtained by

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bottling olive oil in innovative materials containing an oxygen scavenger. However, the slowest decay kinetics were obtained by bottling the oil in polyethylene terephthalate (PET) containers and reducing the oxygen concentration prior to bottling to 10% of the equilibrium value. Even though the above model was advantageously used to demonstrate several advantageous aspects related to the design of plastic bottles for packaging of virgin olive oil, it has some limitations. In fact, it can not be used to predict the quality decay kinetics of small containers (i.e., only for hc=rb > 10) and/or to assess the influence of the bottle’s geometrical factors on the quality decay kinetics of the bottled oil. The above restrictions are a direct consequence of one of the hypotheses used to derive the model; i.e., oxygen diffusion takes place only in the radial direction (monodimensional model). In this work the mono-dimensional model was improved by taking into account also oxygen diffusion in the bottle’s axial direction (two-dimensional model). The new model was then used to assess the influence of some of the bottle’s geometrical factors on the quality decay kinetics of virgin olive oil bottled in glass and plastic containers.During the storage of bottled virgin olive oil hydroperoxides are formed through the oxidation of unsaturated fatty acids and consumed by hydroperoxide breakdown reactions. In the first stage of oxidation, when the oxygen concentration is close to saturation, the rate at which hydroperoxides are consumed is lower than the rate at which they are produced through the autooxidation of unsaturated fatty acids, leading to an increase in hydroperoxide concentration during storage. As the lipid oxidation reaction proceeds, oxygen is consumed to form hydroperoxides. This causes: (a) the formation of an oxygen concentration gradient in the bottled oil, which in turn brings about the permeation of external oxygen through the wall of the plastic container; (b) an increase in the rate at which hydroperoxides break down. As a result of the above phenomena, concentrations of both local oxygen and hydroperoxides decrease. Given the above scenario during oil storage, to properly describe the oxidation kinetics of bottled virgin olive oil a mathematical model was developed to predict the time course of oxygen and hydroperoxide concentrations in bottled oil during storage.By means of the two-dimensional mathematical model presented it was possible to assess the influence of some of the bottle geometrical factor (VHS=VOil and SBottle=VOil) on the quality decay kinetics of virgin olive oil bottled in polymer containers. The predictive ability of the mono- and two-dimensional models were also compared. In particular, it was observed that in the case of the bottle with a volumetric capacity of 1 l, the prediction of the mono-dimensional model is close to that of the more accurate two-dimensional model. This could be related to the fact that during the first stage of storage the oxidation reaction rate depends primarily on the oxygen dissolved into the oil prior to bottling. The results obtained in the case of PET bottles showed that when the ratio SBottle=VOil decreases, then the amount of oxygen permeating through the bottle wall decreases, thus slowing down the quality decay kinetics of the bottled oil. In the case of PET bottles with an oxygen scavenger uniformly dispersed in the bottle wall, the opposite is true. This is due to the fact that the oxygen scavenger consumes part of the oxygen dissolved in the oil prior to bottling. The ratio VHS=VOil also influences the evolution of the hydroperoxides during storage. These results suggested that, to control the oxidation kinetics during the storage of bottled oil, it may be useful to use well-designed plastic bottles, innovative plastic materials containing an oxygen scavenger and to perform the bottling operations under a nitrogen atmosphere (to reduce the oxygen pressure in the bottle headspace). These practices are often underestimated by the oil industry which defines empirically the period of shelf-life (date of recommended consumption) of bottled extra virgin olive oil, without carefully considering oil characteristics, packaging properties and the temperature conditions during product distribution.

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2.4.6 Which package should be selected for edible vegetable oils? Obviously, in the industrial world the manufacturer may not select a package, counting only on its quality. From an economic point of view the manufacturer will try to maximize the price for value ratio of the selected package: 1) the cost of the container: cost of the material (e.g. plastic, glass), place of manufacturing (in the bottling plant or buying a finish product), costs of transportation and storing and the production cost of using the chosen material (in some cases changing the container material may required a new bottling machine); 2) The type of oil, e.g. virgin olive oil, refined oil, seed oil, etc.; 3) The length of residence period in the container (shelf life), the level and type of preservatives added, if any, and the atmosphere within the container; 4) The consumer point of view: Ease of use, e.g. weight, ease of opening and plugging at home; and 5) Traceability of the packaging materials, and the potential to recycle. 2.4.7. Conclusions An understanding of the chemistry and processing of edible oils is critical to the supply of quality product to the world population. Packaging of oils must be considered as carefully as the processing, if acceptable quality is to be maintained. The challenge is to understand the manner in which the package can modify the quality of fats and oils and the directions and measures necessary to maintain quality. The container can only influence the accessibility of light, oxygen, heat and moisture to the product. Regulations for packaged foods including oils have a long history dating back to Roman times. Nowadays, great care is taken because of the potential toxicity of some of the monomers and other substances used to produce packaging materials. Many studies aimed to compare different materials suitable for oil packaging. The most important parameters, as mentioned before, are the permeability of package to oxygen and the permeability to light. In order to determine which of that parameter is more important most studies were performed in the presence and absence of light. It is commonly recognized that glass package is the best material for packaging of oil, due to its impermeability to gases.

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CHAPTER 3

Assessment of chemical and physical-chemical properties

of edible oils

Authors Contributors

CARBONE, V. 1 CHARROUF, Z.6

CARELLI, A.A.2 APARICIO-RUIZ, R.4

CARRIN, M.E.2 GHARSSELLAOUI, M.5

COZZOLINO, R.1 ROSSIGNOL-CASTERA, A.7

CREWS, C.3 SKOULIKA, R.8

APARICIO, R.4 ZOHAR, K.9

AYADI, M.5 MATTHÄUS, B.10

BAUMLER, E.R.2 CHIARELLO, M.D.11

PENCI, M.C.2 ZARROUK, M.12

GUILLAUME, D.13

CONSTENLA, D.2

GARCIA-GONZALEZ, D.L.4

HARHAR, H.6

GRATI-KAMOUN, N.5

BEN TEMIME, S.6

1 Istituto di Scienze dell’Alimentazione, CNR, 83100, Avellino (Italy)

2 Planta Piloto de Ingeniería Quimica (UNS-CONICET), 8000 Bahía Blanca (República Argentina) 3 Central Science Laboratory, York, YO41 1LZ (United Kingdom) 4 Instituto de la Grasa, CSIC, Sevilla. E-41012 (España) 5 Institut de l’Olivier, B.P: 1087, 3000 Sfax, (Tunisie) 6 Faculté des Sciences Université Mohammed V- Agdal, FS-UMV-Agdal, 1014 Rabat (Morocco) 7 French Institute for Fats and Oils, (ITERG), 33600 PESSAC, (France) 8 Food Industrial Research and Technological Development Company (ETAT SA), 117 43 Athens (Greece) 9 Institute of Biochemistry, Food Science and Nutrition, The Faculty of Agricultural, Food and Environmental

Quality Sciences, The Hebrew University of Jerusalem, Rehovot 76100, (Israel). 10 Federal Research Centre for Nutrition and Food, BFEL, 48147 Münster, (Germany) 11 Universidade Católica de Brasília, 70790-150 Brasília (Brazil) 12 Biotechnology Center, Borj-Cedria Technopark, 95, 2050, Hammam Lif (Tunisie) 13 Institut de Chimie Moléculaire de Reims, UMR 6229, 51100 Reims (France)

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Abstract The MAC-Oils project has been focused on five scientific Themes (A-E) related to the individual and comparative assessment of safety, quality, environmental impact aspects of eight target oils (argan, corn, olive, peanut, rapeseed, rice, soybean and sunflower) and their respective production and manufacturing methods. In particular, Chapter 3 reports the results of Theme A, whose purpose has been listing and comparing, in a critical way, data already present in literature concerning: the chemical composition of the oil, the physical-chemical parameters and the traditional and innovative analytical methodologies used for oils analyses. Regarding to the chemical composition, vegetable oils constituents can be divided into two categories: the saponifiable fraction (triglycerides, free fatty acids, phosphatides, other minor glyceridic compounds) and the unsaponifiable matter (sterols, triterpenic alcohols, tocopherols, pigments, carotenoids and derivatives, chlorophylls and derivatives, squalene, aliphatic compounds, phenolic compounds, other compounds). During the project it was possible to review some parameters used regularly to measure the chemical and physical properties of edible oils, which are: the iodine value, the saponification value, the refractive index and the density. During the MAC-Oils project instrumental methods used for oil analyses have been evaluated in relation to: investigate oil composition and detect adulterations. From the data reported in literature, it is possible to asses that, beside the analytical methodologies commonly used in vegetable oil analyses (UV-spectrophotometry, Gas-Chromatography, Nuclear Magnetic Resonance, Gas-Chromatography/Mass Spectrometry, High Performance Liquid Chromatography/Mass Spectrometry, High Performance Liquid Chromatography), today some innovative analytical tools, most of all Mass Spectrometric and Nuclear Magnetic Resonance methodologies, have been successfully applied for the analysis and characterization of vegetable oils.

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3.1. Comparison of Chemical and Physical-Chemical Properties 3.1.1 Introduction In the present Chapter a comparison is made of chemical and physical-chemical properties of eight edible oils (argan, corn, olive, peanut, rapeseed, rice, soybean and sunflower oils, see also Chapter 1). Argan oil. This oil has been known in Morocco for centuries. However, it only appeared on the European and developed country markets during 1998 and compared to other vegetable oils, it is a very “young oil”. Its worldwide marketing began only ten years ago after the development of its large-scale production in pure form in Moroccan rural cooperatives (Charrouf et al, 2002). Argan oil is prepared from fruits collected from wild trees growing exclusively in Morocco, and pressing the roasted kernels. At the present, growing argan trees is not a full-time farming activity and consequently neither industrial farming techniques nor large geographical changes can influence argan oil chemical composition. The combination of all these factors explains why argan oil chemical composition has not been studied in as much detail as other vegetable oils obtained from cultivated species. Its delicate hazelnut taste, combined with its high level of unsaturated fatty-acids, has allowed its swift commercial success and, nowadays, argan oil of standardized quality is marketed worldwide (Charrouf and Guillame, 2008). Corn Oil. Corn or maize, Zea mays, is native to both North and South America. Its oil is a by-product of the wet or dry milling of corn. The germ obtained with a wet degermination process contains about 50% of oil. In contrast, the germ obtained with a dry degermination process contains from 10 to 24% oil (O´Brien, 1998). Corn oil is one of a number of vegetable oils that are being promoted as more desirable for edible purposes because of their high omega 6 linoleic acid content and low saturated fatty-acid content. Olive Oil: The global consumption of olive oil has grown rapidly in recent decades. This enormous growth is due to several factors, amongst which are the public recognition in its health promoting potential as a part of the Mediterranean diet, and the global promotion campaigns initiated by the International Olive Council (IOC), (formerly the International Olive Oil Council). This has resulted in elevated prices for the quality product, e.g. VOO (Virgin Olive Oil) and EVOO (Extra-Virgin Olive Oil), but has also motivated fraud in many places and cases. As a part of its aggressive promotion, and to better control the authenticity of olive oil, the IOC has established in the EU (European Union) a set of standards and norms that define the quality and authenticity control of olive oil, the chemical and sensorial (organoleptic) characters that define degrees of quality, and the standard methods to assay oil. Nowadays, the IOC works in close cooperation with the International Organization for Standardization (ISO), the American Department of Agriculture (USDA), the Australian authorities, and will in the future collaborate with South Africa and South America. Purity criteria for olive oil have been fixed using compositional data for varietal oils, mainly from the European Community, and some others countries outside the Community. In the last decade, olive oil production has increased in countries from the Southern hemisphere (e.g. Argentina, Australia, and New Zealand) not traditionally associated with its production. Olive oil, as biological material, shows natural compositional changes between countries with

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different agro-climatic conditions from the Mediterranean region. In these countries, some genuine virgin olive oils have a number of compositional parameters outside the existing international normative. These deviations constitute a constant barrier for the international commerce of olive oil. For this reason, the countries involved have asked for amendments in the standards for olive oil. Peanut Oil. Botanically, the peanut, Arachis hypogaea, belongs to the same family of legumes as the soybean, but in composition it is more like other nuts than most beans or peas (O´Brien, 1998). Numerous peanut cultivars and wild species are found and have been collected in South America. The probable centres of origin of Arachis species and A. hypogaea were in the Gran Pantanal (Mato Grosso, Brazil) and on the eastern slopes of the Bolivian Andes. Peanuts are grown worldwide in the tropics and temperate zones primarily as an oilseed crop. Peanut seeds are rich in oil, naturally containing from 47 to 50% (Grosso, 1997). Rapeseed Oil. Rapeseed oil is today a major oil of commerce for both food and industrial uses. The main reasons for its commercial success are its very nutritional fatty acid profile and its wide range of cultivation. Rapeseed yields 40-60% of oil. Originally, rapeseed oil had unacceptably high levels of erucic acid (C22:1 ω9) and the meal contained high levels of undesirable glucosinolates. Erucic acid may constitute 30-50% of the total fatty acids in some Brassica varieties, such as in rapeseed (B. napus) and mustard seed (B. junca and B. nigra) oils. The permitted level of erucic acid has been established by Codex at 2% (Codex, 2005). Oilseed rape comprises the Brassica species B. napus and B. rapa which have been in cultivation for thousands of years. Low erucic acid and low glucosinolate cultivars of both of these species that are known as double zero or `00´ were introduced in Canada during the 1950s. The best known 00 cultivar is canola. It must contain less than 2% erucic acid in the oil fraction and less than 30 micromoles of any one or any mixture of 3-butenyl glucosinolate, 4-pentenyl glucosinolate, 2-hydroxy-3-butenyl glucosinolate, and 2-hydroxy-4-pentenyl glucosinolate per gram of air-dry, oil-free meal (Przybylski, 2008). Food-grade Brassica oils include oils with reduced saturated fatty acids (<7% total saturates), low linolenic acid (<3.5%), and with high oleic acid (over 75%) contents. Canola oil is obtained from B. napus and B. rapa cultivars which produce oils with very low levels of erucic acid, equivalent to the terms low-erucic acid rape or rapeseed (LEAR) and colza. Nowadays, the production of LEAR is very high in Europe, while the HEAR (high-erucic acid rapeseed) cultivars still predominate in India and China. Recently, the production and sale of cold-pressed oils have increased and data pertaining to the composition and quality of these oils is becoming available (Matthäus and Bruhl, 2003). Rice Oil. Domesticated rice comprises two species of food crops in the Poaceae (“true grass”) family, Oryza sativa and Oryza glaberrima. They are native to tropical and subtropical southern Asia and southeastern Africa, but now are cultivated between latitude 55° N in China and 36° S in Chile. The Asian cultivated rice (Oryza sativa) is grown all over the world, while the African cultivated rice (Oryza glaberrima) is grown in a small scale in West Africa. Rice bran, representing 6-8% of the rice grain, contains about 15-20% oil. Good-quality oil is obtained by solvent extraction immediately after milling. The oil has excellent oxidative stability due undoubtedly to its multicomponent system of natural antioxidants that includes esters of ferulic acid and high levels of tocopherols (Iqbal et al., 2005). It is also rich in unsaponifiable matter, which contains the micronutrients like vitamin E complexes, gamma oryzanol, phytosterols, polyphenols and squalene. Moreover, it has a very good balance in its

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fatty acid composition i.e., mono-unsaturates to poly-unsaturates/saturates ratio (Ghosh, 2007). Soybean Oil. Soybean (Glycine max) is one of the oldest crops cultivated by humans. It is native to eastern Asia. On a global scale, soybeans have by far the highest production rate with rapeseed and cottonseed following second and third. The majority of soybeans are grown in five countries: USA, Brazil, Australia, Argentina and China. Soybeans contain only roughly 21% of oil, this being a by-product of the production of soy protein for animal and human consumption. It is an excellent dietary source of essential linoleic acid and also tocopherols, which serve as sources of vitamin E and natural antioxidants. The oil has a tendency to oxidative flavour reversion, which is largely associated with the linolenic acid content (Padley et al., 1994). Sunflower Oil. Sunflower is native of North America. Nowadays, the cultivated sunflower (Helianthus annus L.) is the second largest world source of vegetable oil (O´Brien, 1998). The oil has excellent nutritional properties, is practically free of significant toxic compounds and has a high concentration of linoleic acid (Ceccarini et al., 2004). In the former USSR, seed quality and oil yields were substantially improved from 29% average yield in 1940 to a 46% oil yield in 1971 (O´Brien, 1998). Changes in fatty acid composition for certain oil markets, and changes in protein content and protein quality to enhance the value of the meal are also important breeding goals in some programmes (Fick and Miller, 1997). High-oleic sunflower oil, consisting of more than 700 g/kg oleic acid, became commercially available in the USSR in the late 1970s, and in 1985 it contained more than 800 g/kg oleic acid in the USA (Fick and Miller 1997). High-oleic oil has steadily gained in market acceptance, especially for food and industrial purposes where a high level of oxidative stability is required. Recently, new sunflower mutant lines were developed containing high levels of palmitic or stearic acids, almost exclusively at the sn-3 positions, and various concentrations of oleic and linoleic acids, being of great interest in those applications that require high thermal stability (Márquez-Ruiz et al., 1999). All plant oils are similar in many aspects, but a few minor differences have a significant effect on the characteristics of the individual oil. From the farmer’s point of view, the seed oil quality can hardly be altered through agronomical practices, other than choosing the best cultivars and grow the crop under the best conditions. Environmental conditions will dictate the oil content and chemical composition. Seed oil quality is generally very stable in the intact seed as long as the seed is free of admixture and stored under good conditions. For fruit oils, however, handling by the grower and processor will largely determine the quality. In the case of olive oil, bruising or damage to the fruit during harvest and storage will result in reduced quality in the oil. Olive growers therefore need to have a basic understanding of what oil quality is and how it is preserved. Similarly, the quality parameters of olive oil and their potential health promotion and sensorial values attract the modern consumer, who should also have a similar basic understanding of what oil quality is, to allow them to make the correct intelligent choices. In the case of argan oil, the time between fruit picking and kernel collection is an important factor to consider. Fruit picking is a seasonal activity whereas pressing is performed in the cooperatives all through the year; consequently, kernels can be pressed several months after the fruit collection. Mechanical fruit depulping is an important improvement in argan oil preparation, however it requires a sufficient level of fruit dryness. It has been found that a sun-drying period of two or three weeks leads to dried fruits that can be easily peeled by machine without modification of the oil chemical properties. It is likely that the shell protects the kernels from oxidative or enzymatic processes that would lead to oil degradation (Hilali et al., 2005).

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3.1.2. Chemical and physical parameters Parameters used regularly to measure the physical and chemical properties of edible oils are: the iodine value, the saponification value, the refractive index, and the density. The iodine value is a measure of the total number of double bonds present in fats and oils. High iodine-value oil contains a greater number of double bonds than low iodine-value oil and has usually a reduced oxidative stability. In addition, its melting point is lower than more saturated oils. The saponification value is a measure of the average chain length of all of the fatty acids present. The shorter the fatty acid, the higher the saponification value will be. The refraction index, density and viscosity depend on the temperature and on the fatty-acid composition of the oil. The refraction index increases with the unsaturation level of the oil. If the chain length is constant, the density increases with the unsaturation level while the viscosity decreases. If the unsaturation is constant, the density increases when the chain length decreases. In contrast, the viscosity increases with increasing molecular weight. If there is a secondary functional group (hydroxy, epoxy) in the fatty chain the viscosity increases strongly.

Table 3.1. Chemical and physical characteristics of the vegetable crude oil.

Oil Specific gravity

Iodine value Saponification

value Refractive

index

Olive at 25/25ºC

0.909-0.915a 80-88a 188-196a

at 25ºC

1.4677-

1.4705b

Soybeana at 25/25ºC

0.917-0.921 120-141 189-195

at 25ºC

1.470-1.476

Conventional at 20/20ºC

0.918-0.923 118-141 188-194

at 40ºC

1.461-1.468

High oleic at 25/20ºC

0.909-0.915 78-90 182-194

at 25ºC

1.467-1.471 Sunflowerc

Mid oleic at 20/20ºC

0.914-0.916 94-122 190-191

at 25ºC

1.461-1.471

Colza at 20/20ºC

0.910-0.920 94-120 168-181

at 40ºC

1.465-1.469 Rapeseedc

LEAR at 20/20ºC

0.914-0.920 105-126 182-193

at 40ºC

1.465-1.467

Rice branb at 25/25ºC

0.916-0.921 99-108 181-189

at 25ºC 1.470-1.473

Corna at 25/25ºC

0.915-0.920 103-128 187-193

at 25ºC 1.470-1.474

Peanutc at 20/20ºC

0.912-0.920 86-107 187-196

at 40ºC 1.460-1.465

Argan d at 20ºC

0.906-0.919 91-110 189.1-199.1

at 25ºC 1.463-1.472

(a)(Sonntag, 1985); (b)(Padley et al., 1994); (c)(CODEX, 2005); (d)(Hilali et al., 2005); (d) (SNIMA, 2003)

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Therefore, the values of these parameters are strongly dependent on the fatty acid composition of the oil and are mainly influenced by the climate and the variety. In addition to the agronomic conditions, the processing of the oil has a strong influence on the physical and chemical properties, because the processing influences the content and the composition of minor components in the oil. Table 3.1 summarizes the ranges of the main chemical and physical parameters reported in the bibliography for the eight vegetable oils. 3.1.3 Major compounds Fatty-acid composition The most important components in vegetable oils are the fatty acids, which generally comprise 93-99% of the oil, and provide the physical characteristics such as the density, frying and storage behavior and palatability. The fatty acids in plant edible oil usually have between 12 and 24 carbons. Nearly all of fatty acids of natural origin have an even number of carbons, e.g. 16, 18 or 20. However, olive oil contains a very small proportion of fatty acids with 17 carbon atoms. The proportion of each fatty acid strongly influences the characteristics and nutritive value of the oil. Fatty acids with double bonds are prone to oxidation, and their oxidation products, including peroxides, aldehydes and short acids, have considerable and noticeable sensorial effects. At the same time, linoleic and α-linolenic acid belong to the group of essential fatty acids. Humans are not able to biosynthesize these fatty acids, which are necessary for the formation of different hormone-like compounds, the so-called eicosanoids. These are made by introduction of further double bonds and elongation of the carbon chain. These eicosanoids control different processes in the body and they act as the body’s mediators and effectors in several metabolic processes (Ally and Horrobin, 1980; Brodt-Eppley and Myatt, 1998). Eicosanoids formed from linoleic acid produce blood coagulation, whereas eicosanoids formed from α-linolenic acid show the opposite effect. Therefore, the ratio between n-6/n-3 PUFAs in a diet has a remarkable effect on the tendency of blood to clot, which can result in an increasing risk for cardiovascular heart diseases. Additionally eicosanoids from linoleic acid are more potent than those synthesised from α-linolenic acid in mediating inflammation. The result is that consumption of linoleic acid increases inflammation, whereas α-linolenic acid in the diet reduces it. This is important for the treatment of inflammatory diseases like rheumatoid arthritis. The recommendations of the nutrition societies of Germany, Austria and Switzerland (D-A-CH recommendations for nutrition) have defined the optimal n-6/n-3 ratio as 4:1 to 5:1 (D-A-CH, 2000). The fatty acid composition of soybean oil is characterised by a high proportion of unsaturated fatty acids (about 81%) predominantly linoleic acid, which amounts to about 50% of the total fatty acids, and by a n-6/n-3 ratio even closer to the recommendations (see Table 3.2). The Western European diet has a ratio of more or less 10:1. Therefore, inclusion of soybean oil into the diet would shift the ratio towards the D-A-CH recommendations. On the other hand, it has to be taken into consideration that the fatty acid oxidation susceptibility increases with the unsaturation degree. In consequence, linoleic and linolenic acids are highly susceptible to oxidation, resulting in the favoured formation of oxidized LDL-cholesterol, which is responsible for the development of arteriosclerosis (Kratz et al., 2002). Additionally, oxidation products from polyunsaturated fatty acids play an important role in the aging processes and the development of tumours. Thus, nowadays it is recommended that the intake

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of linoleic acid is lowered and the intake of monounsaturated oleic acid is increased. From this point of view, a moderate consumption of soybean oil and a higher intake of those oils with high-oleic content (similar to olive oil) is recommended. The incorporation of high-oleic genes into seed-breeding lines (e.g. sunflower, peanut, rapeseed) can produce high-oleic varieties that consequently have extended shelf lives. With respect to erucic acid, feeding studies with laboratory rats suggested that this fatty acid may be undesirable from a nutritional point of view, because it causes fatty infiltration of heart muscle in experimental animals (de Wildt and Speijers, 1984; Hulan et al., 1976). These handicaps were removed after considerable modification and breeding produced low erucic acid oils, to the point where the C22:1 content is now usually below 1%. The permitted level of erucic acid has been established by Codex to 2% (Codex, 2005). A result of this intensive work has been the availability of rapeseed oils with a wide range of tailor-made compositions. Oils with only a trace of linolenic acid (C18:3) are considered more suitable as cooking and frying oils (e.g. argan, olive, corn, peanut, sunflower). The majority of olive oil fatty acid chains contain 16 or 18 carbon atoms, with oleic acid being the major component; while the main fatty acids in argan, corn, canola, rice and conventional peanut and sunflower oils are linoleic acid, C18:2 and oleic acid, C18:1. Canola oil has the lowest saturated fat content of commercial vegetable oils although forms of soya oils with similarly reduced saturated fat levels are available. The linoleic acid contents of canola/LEAR and argan oils are intermediate among the vegetable oils, lower than corn oil, soybean oil, rice oil and conventional sunflower oil, and higher than olive oil or conventional peanut oil, see Table 3.2. The profile of fatty acids and the composition of triacylglycerols also serve as initial markers to verify oil authenticity. Still, these markers may produce false results due to variations in the composition of oils that are produced from several cultivars, and under different climatic conditions. It has been shown that the fatty acid composition of vegetable oils varies depending on the genotype, maturity, climatic conditions, and growth location, and on interactions between these factors (Brown et al., 1975; Casini et al., 2003; Evrad et al., 2007; Grosso and Guzmán (1995); Grosso et al., 1996; Hinds, 1995, Holaday and Pearson, 1974; Kiritsiakis, 1998; Pryde, 1980; Rahmani, 2005; Richards et al., 2008; Sayed and Mohamend, 2002). Generally, lower temperatures during seed or fruit development are associated with a more unsaturated oil. Wider ranges of 43.7 to 93.5% for oleic acid and 1 to 30% for linoleic acid, mainly due to genetic and climatic conditions, have been found in olive oils (Kiritsiakis, 1998). Moreover, a few olive oils from new production areas show deviations from international normatives with respect to some fatty acid limits (low oleic-acid contents: < 55.0%, high values for linoleic acid: >20.0%, palmitic acid: >20.0%, linolenic acid: >1% and/or palmitoleic acid: >3.5%), (Ceci and Carelli, 2007). Regarding linolenic fatty acid, the Codex Alimentarius in its “Codex Standard for Olive Oils and Olive Pomace Oils” provides the following footnote for linolenic acid: “Pending the results of IOOC survey and further consideration by the Committee of Fats and Oils, national limits may remain in place” (Codex, 2003). Triacylglycerol composition The combination of the fatty acids in the triacylglycerol (TAG) molecule is a characteristic feature for a given vegetable oil. A slight variation in TAG composition occurs depending on the origin of the fruits or seed, the variety or hybrid, and the oil production process. In consequence, the profile of the TAG molecular species represents a key to understanding the physical characteristics of oil and it is a unique means of identification (Rezanka and Mares, 1991). For instance, the TAGs in all olive oils share many similarities,

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but are different from all other seed oils, for example, the presence of trilinolein can suggest adulteration with conventional sunflower, corn or soybean. Table 3.3 compares the main TAGs found in the vegetable oils that are the subjects of this study. The major TAGs in rice oils are mono-unsaturated (20.5%), diunsaturated ( 49.7%) and triunsaturated (29.8%) types. they have been identified by gas chromatography-mass spectrometry (GC-MS) as 1-palmitoyl (or oleoyl)-2-linoleoyl-3- oleoyl (or palmitoyl), 1-palmitoyl (or linoleoyl)- 2- linoleoyl-3-linoleoyl (or palmitoyl) and trioleyl types (Chakrabarty, 1990). While trilinoleate and mono-olein-dilinoleate predominate in corn oil TAGs, trioleate, monopalmitate-dioleate, mono-oleate-dilinoleate, and monolinoleate-dioleate are in preponderance in argan oil (Farines et al., 1984a; Maurin et al., 1992). Comparing conventional sunflower oil and high-oleic sunflower oil, it can be observed that trilinoleate dominates the TAGs of conventional sunflower oil, followed by mono-oleate-dilinoleate and monostearate-dilinoleate; while the TAGs composition of high-oleic sunflower oil is completely different. With more than 70% of trioleate the TAG structure of high-oleic sunflower oil appears very simple. Soybean fatty acid composition consists mainly of unsaturated fatty acids, with nearly all of the TAG molecules containing at least two unsaturated fatty acids, with di- and trisaturated glycerides being essentially absent (List et al., 1977; Pryde, 1980). Peanut and high-erucic rapeseed oils oils contain a high percentage (>15%) of TAGs with carbon number higher than 54 in comparison with other common vegetable oils (i.e. olive, soybean or sunflower oil). In peanut oil, TAGs from 56 to 60 carbon numbers mainly represent combinations of two C18 and one long-chain (C20-C24) fatty acid per TAG molecule (Mangaro et al., 1981). Erucic acid is found in the sn -1,3 position of rapeseed oil TAGs. In canola this is replaced by linoleic acid in that position, and gadoleic and erucic acids are found preferentially at the sn-3 position (Ohlson et al., 1975). In high erucic acid rapeseed oil (HEAR) at least 95% of the linoleic acid was present in the sn-2 position, whereas in canola oil only 54% was in that position (Jáky and Kurnik, 1981). In vegetable oils, long chain (C20:0-C24:0) and saturated fatty acids occur mostly in the 1- and 3-positions, while the octadecanoic (C18) fatty acids, especially linoleic and linolenic, are found in the 2-position. Dorschel (2002) found that most of the peanut TAGs are di- and tri-unsaturated, with long-chain saturated fatty acids (C20-C24) were confined almost exclusively to the sn-3-position, whereas the palmitic and stearic acids were more predominant in the sn-1 and sn-3 positions. Therefore, oleic acid was distributed almost evenly in the sn-1-, 2- or 3-position, while the linoleic acid was preferably found in the sn-2 position. Similarly, long chain (C20:0-C24:0) and saturated fatty acids occur mostly in the 1- and 3-positions of rapeseed TAGs, while the octadecanoic (C18) fatty acids (especially linoleic and linolenic) are found in the 2-position (Kallio and Currie, 1993; Ackman, 1983). Likewise, stereospecific analysis of argan oil has shown that palmitic and stearic acids, the two major saturated fatty acids, are more likely to be found at the glycerol extremities (sn-1 or sn-3) whereas linoleic acid is frequently encountered at the glycerol sn-2 position (Maurin et al., 1992). Finally, the stereospecific analysis of rice oil indicated that palmitic acid was found exclusively at 1- and 3- positions, while oleic acid occured in all three positions and linoleic acid predominantly occupied the 2-position (Chakrabarty, 1990).

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3.1.4 Minor saponifiable compounds Free-fatty acids The quantity of free-fatty acids (FFAs), usually referred as “the acid value”, is an important quality factor and has extensively been used as a traditional criterion for classifying olive oil into various commercial grades (Boskou, 2006; Rossell, 1986). The following maximum percentage limits have been established internationally for FFAs (as oleic acid): extra-virgin olive oil (0.8), virgin olive oil (2.0), ordinary virgin olive oil (3.3), refined olive oil (0.3), olive oil-a blend of refined olive oil and virgin olive oils- (1.0), refined olive pomace oil (0.3) and olive pomace oil (1.0). It is well known that the increase of free acidity is mainly due to enzyme activity caused by olive tissue damage (Boskou, 2006). When the fruit ripens or is affected by e.g. insects or microorganisms, or suffers environmental stresses (e.g. heat, draught, frost), TAGs are degraded and fatty acids are lost from by a process called “de-esterification”. The FFA content, also discriminates between extra virgin argan oil and virgin, ordinary virgin, and lampante argan oil. According to the Moroccan official norm (SNIMA, 2003), to be classified as an extra virgin argan oil, the acid value has to be lower than 0.8%. A value between 0.8 and 1.5% will apply to the respective virgin oil, between 1.5 and 2.5% to ordinary virgin argan oil, and above 2.5% to lampante argan oil. According to the Codex Alimentarius Commission, the limits of AV (acid value) recommended for human consumption are: 4 mg KOH/g oil for virgin oil and 0.6 mg KOH/g oil for refined oil (Codex, 2005). Crude peanut oil can have a FFA content as low as 0.3%, but most of the commercial oil is in the range 0.5-1.5% (Padley et al., 1994). However, crude rice oil tends to contain higher levels (2-6%) in comparison to other vegetable oils. High acidity is rather inherent in this oil, since rice bran contains an unusually active lipase (Sonntag, 1979). High AV has been found even in oils extracted promptly from bran freshly removed from the rice, and upon storage of the bran at 25 ºC, the acidity of the oil rose at the rate of 1% per hour (Sonntag, 1979). The FFA content of vegetable oils depends not only on the care exercised in handling the material prior to extraction of the oil, but also on other factors. In peanuts, high percentages of free fatty acids may indicate either poor handling, immaturity, mould growth or other ester hydrolysis activity (Sanders et al., 1992). The drying and storage of seeds constitute two fundamental steps in sunflower oil production, the effect of air temperature being greater than the effect of storage time on the oil quality. Seeds with high moisture content are susceptible to quality deterioration at high temperature because of the oil’s and phospholipids’ hydrolysis and an increase in acidity (Bax et al., 2004). FFA content depends on the extraction method. Brevedan et al., (2000) found that crude sunflower oils obtained by hexane extraction had a higher initial acidity than those obtained by pressing, showing the cold-pressed oils had the best initial quality, with relatively low hydrolytic and oxidative alteration. FFAs are also produced in any of the phases of production by hydrolytic reactions of the oil’s TAGs. The lower limit for FFA in refined oils is not due to the better quality of the raw materials, but to the complete removal of FFAs during neutralization in the refining process. It is known that FFAs can influence the organoleptic value of the oil and accelerate the oil's oxidation. During oil seed processing, experience has shown that oil flavour and odour removal correlate well with FFA reduction. The odour and flavour of an oil with a 0.1% FFA will be eliminated when the FFA is reduced to 0.01 to 0.03% (assuming a zero peroxide value) (O`Brien, 1998).

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Diacylglycerols Diacylglycerols (DAGs), usually termed as diglycerides, arise as intermediate products in triglycerides’ biosynthesis and/or are present owing to the acidic or enzymatic hydrolysis of TAGs (Ruiz-Gutiérrez and Pérez-Camino, 2000). In comparison to other vegetable oils, crude rice bran oil tends to contain higher amounts of DAGs (2-3%) and monoacylglycerols (MAGs, 1-2%) that are associated with enzymatic activity (Ghosh, 2007). Non-appreciable amounts of MAGs are found in olive oil (<0.2% of C18), high erucic rapeseed (<0.1% of C16), while they are not detected in corn, peanut, soybean and sunflower oils (Padley et al., 1994). DAGs with 34 and 36 carbon numbers are commonly present in peanut (C34: 0.5%, C36: 1.7%), corn (C34: 0.7%, C36: 2.1%), rapeseed (C34: 0.1%, C36: 0.7%), soybean (C34: 0.1%, C36: 0.9%), and sunflower oils (C34: 0.2%, C36: 1.8%), while olive oil also contains C32 (0.1%) (Padley et al., 1994). In rice oil, the amount of sn1,2 (2,3)-DAGs (1.9-2.1%) is slightly higher than sn-1,3-DAGs (1.6-1.8%) and is similar to that observed in peanut oil (Hemavathy and Prabhakar, 1987). In peanut oil, certain 1,2- and 1,3- isomers of DAGs have been reported. These are 1,2-PO; 1,2-PL; 1,3-PO; 1,3-PL; 1,2-OO; 1,2-OL; 1,3-OO; 1,3-OL and 1,3-LL (Frega et al., 1993). With reference to argan oil, DAGs can be found at proportions of 0.68 to 1.53% of oil, but their composition has not been studied in detail (Charrouf and Guillaume, 2008). DAGs are the major components of the polar fraction of fresh olive oils. They are found in virgin olive oils at low concentrations (1-2.8%) (Kiosseoglou and Kouzounas, 1993; Frega et al., 1993) while MAGs are present in much smaller quantities (less than 0.25%) (Kiosseoglou and Kouzounas, 1993; Paganuzzi, 1987). In the DAG fraction C-34 and C-36 prevail (Frega et al., 1993; Paganuzzi, 1987; Leone et al., 1988). Lampante and extracted olive oils have relatively high amounts of C-36 (Mariani and Fedeli, 1985). In the MAG fraction, glycerol oleate, glycerol linoleate, and glycerol palmitate are the major constituents. Other MAGs found are glyceryl stearate, glyceryl palmitoleate, glyceryl linolenate and glyceryl laurate (Paganuzzi, 1987). Knowledge of the amounts and composition of DAGs is of great importance for the evaluation of the quality of olive oil. In particular, the ratio of 1,3-DAGs/1,2-DAGs or the ratio of 1,2-DAGs to the total amount of DAGs [D= 1,2-DAGs/(1,2-DAGs +1,3-DAGs)] was found to be a useful index to assess the storage history of virgin olive oil (Pérez-Camino et al., 2001; Sacchi et al., 1991; Sacchi et al., 1997; Fronimaki et al., 2002). Pérez-Camino et al., (2001) reported the evolution of DAGs and the ratio of 1,3-DAGs/1,2-DAGs for Spanish virgin olive oils as a function of storage time, temperature, and free acidity. These investigators noted that the isomerization of 1,2-DAGs to 1,3-DAGs depended on these parameters, and in fact became faster with increasing temperature and free acidity. A few attempts have also been made to detect possible adulteration of virgin olive oil with deodorized and refined oils (Pérez-Camino et al., 2001; Serani et al., 2001) by DAG analysis. Phospholipids Phospholipids play an important role in the storage of TAGs in seed oils, participating in the formation of oil bodies in association with oleosin proteins. Oilseed phospholipids are also the major components of lecithins, which are by-products isolated during the refining of edible oils (Salas et al., 2006a). These lipids are composed of glycerol esterified with fatty acids and phosphoric acid. Phosphoric acid, in turn, is usually combined with a nitrogen or carbohydrate containing compound. Phospholipids (PLs) contribute to the smoothness, texture, and mouthfeel of foods and are undesirable in the oil since they are responsible for oil discoloration during deodorization and steam distillation, and losses of neutral lipids during neutralization (Cert et al., 2000). By other hand, the measurement of phospholipids is important in determining the stability and quality of vegetable oils. In fact, PLs not only affect the sensorial quality of the oil but also have an antioxidant effect (Carelli et al., 1997;

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Singleton and Stikeleather, 1995b). Their ability to bind to metals present in the oil may inhibit the catalytic activity of the metals, and their ability as radical scavengers is also well known. In addition, it has been reported that some of the phospholipids found in vegetable oils display important biological activity (Nomilos et al., 2002) and have been proved useful in the prevention of certain diseases like arteriosclerosis (Antonopoulou and Karantonis, 2002). Soybean oil is a major source for the production of vegetable lecithin for pharmaceutical and food purposes. The reasons for this are the ready availability of the raw material in bulk, (1.1-3.2% in the crude oil, Sonntag, 1985), and the extraordinary functionality of the product. The US Food Chemical Codex defines food-grade lecithin as obtained from soybeans and other plant sources. The major PLs in soybean oil are phophatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI) and phosphatidic acid (PA). Lecithin is a complex mixture of acetone-insoluble phosphatides combined with various amounts of other substances such as TAGs, fatty acids and carbohydrates. Crude soybean lecithin contains high amounts of neutral oil, which should be removed by separation of neutral and polar lipids with acetone. This is followed by alcohol separation of the deoiled lecithin, which provides two fractions, one enriched with PI (alcohol-insoluble) and the other with PC (alcohol-soluble). Phospholipids are the main non-TAGs or non-neutral oil components of sunflower oil but are still minor (about 1%) components (Dorrel and Vick, 1997). The lecithin composition of sunflower oil is comparable to that of soybean oil. The PL composition of oils from different sunflower mutants has been studied (Salas et al., 2006a) and the authors suggested that the mutant trait does not affect the synthesis of PLs because the relative polar lipid contents of standard and mutant seeds are similar. However, the acyl composition of PL molecular species appears to be influenced by the fatty acid composition (Salas et al., 2006b). Przybylski and Eskin, (1991) found that the total amount of PL in crude rapeseed oil was less than 2% of the total lipids, and that this fraction was highly unsaturated. PC contains the highest amount of unsaturated fatty acids, mostly oleic and linoleic. The other two phospholipids (PI and PE) are rich in palmitic, linoleic and linolenic acids (Smiles et al., 1988). LEAR varieties from winter rapeseed cultivars have similar phospholipid fatty acid profiles to canola oil (Sosulski et al., 1981). In comparision of other vegetable oils crude rice bran oil tends to contain high levels of non-TAG components, most of which have to be removed during the refining process. The PL content of this oil is between 1-2%, (Rajam et al., 2005; Ghosh, 2007). Experimental studies of PLs in olive oil are rather limited (Boskou, 2006). Freshly produced virgin olive oil may contain small amounts of PL (40-135 ppm). PC, PE, PI and phosphatidylserine (PS) were reported to be present in olive oil Pls (Alter and Gutfinger, 1982). Cherry and Kramer, (1989) reported the following PL composition for olive fruit: PC: 47.3-58.9%, PE: 5.3-8.0%, PI: 18.0-23.9%. Aged oils contained much smaller quantities. The level of PLs in corn oil is generally less than 1% (Sayed and Mohamed, 2002), while it is about 0.3-0.7% in peanut oil (Ayres, 1983; Jonnala et al., 2006). The PLs in peanuts are the major constituents of cell membranes, and they have a high degree of unsaturation. Changes in PL concentration may occur when peanuts are harvested prematurely, cured at a high temperature, and/or exposed to freezing temperatures (Singleton and Stikeleather, 1995b). They have a high degree of unsaturation, containing about 40 and 60% of C18:2/C18:2 and C18:2/C18:1, respectively in mature peanuts, (Singleton and Stikeleather, 1995a). Jonnala et al., (2006) found statistical differences among the phospholipid content and the composition of high-oleic peanut cultivars. However, these variations were within the range reported for traditional peanut varieties.

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The phospholipid composition of the seed oils described in this book are presented in Table 3.4. For some other vegetable oils including argan oil the presence of PLs has not been studied yet. In Table 3.5, the fatty acid composition of PLs from different oils is shown. The predominant fatty acids present in the total and individual PLs are oleic, linoleic and palmitic. In general, except for differences in linolenic acid composition, data for the major PLs and fatty acid are similar to those for soybean oil, (Cherry and Kramer, 1989). The relative PL concentration remaining in the oil after refining depends on the method of extraction and the type of degumming (Crapiste et al., 1998; Carelli et al., 2002a). The PL fraction recovered after the refining of oil has industrial aplication in food, cosmetical and pharmaceutical product manufacture because of the emulsifiying properties of PLs.(van Nieuwenhuyzen and Tomas, 2008). 3.1.5. Unsaponifiable matter Because the unsaponifiable matter contains a larger number of different compounds it is usually separated into several fractions or groups of constituents (e.g., sterols, alcohols, tocopherols, phenols, hydrocarbons, etc) for analysis. Table 3.6 shows the percentage of total unsaponifiable matter in the eight edible oils. The amount of unsaponifiable matter does not vary much between edible oils with exception of rice oil whose content is appreciably higher. In consequence, the total content of unsaponifiable matter is not of relevance in the authentication of edible oils and fats but the profile of the series of compounds included in this designation is. Table 3.4. Phospholipid distribution in different crude seed oils.

Oil PC (%) PE (%) PI (%) PA (%)

Soybeana-b 32.7-55.3 24.8-26.3 14.3-18.4 0.0-16.8

Sunflowerc 28.9-52.0 17.0-25.6 15.5-30.5 10.7-22.0

Canolad 15.1-31.2 17.8-18.8 10.3-19.7 21.6-48.9

Rice brane 35-38.4 27.2-29.0 21.0-23.2 7.2-9.6

Cornf 57.5-68.1 10.3-13.9 14.5-19.8 < 10

Peanutg-h 38.3-66.4 13.3-21.9 15.7-30.9 2.2-11.8 (a)(Padley et al., 1994), (b)(Wang et al., 1997), (c)(Carelli et al., 2002a), (d)(Przybybiski and Eskin, 1991), (e)(Hemavathy and Prabhakar, 1987), (f)(Harrabi et al., 2008), (g)(Jonnala et al., 2006a), (h)(Singleton and Stikeleather, 1995b). Abbreviations: PC: phosphatidylcholine, PE: phosphatidylethanolamine, PI: phosphatidylinositol, PA: phosphatidic acid.

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Table 3.5. Fatty acid composition of phospholipids from oilseeds.

Oil Palmitic acid

C16:0 Stearic acid

C18:0 Oleic acid

C18:1 Linoleic acid

C18:2 Linolenic acid

C18:3

Soybeana,b 11.7-17.4 4.0-4.0 9.8-17.7 54.0-63.3 4.0-6.8

Sunflowera 11.1-31.9 3.0-7.9 13.3-19.3 42.8-68.7 ---

Rapeseeda 18.3-27.7 0.6-1.1 22.3-23.1 38.047.9 7.4-9.4

Rice branc 18.1 4.0 42.8 33.6 1.5

Corna 17.7 1.8 25.3 54.2 1.0

Peanuta,b 12.9-33.9 2.6-2.8 30.9-47.1 22.7-35.6 --- (a)(Cherry and Kramer, 1989), (b)(Sonntang, 1985), (c)(Adhikari and Adhikari (Das Gupta), 1986), (---): no value or trace. Phenolic compounds Comparied to other oils, olive oil is rich in phenolic compounds, which comprise a large part of its non-glycerol fraction. They have been thoroughly investigated since they have a great impact on virgin olive oil quality and nutrition. They are responsible for providing sensory attributes (e.g. bitter, astringent, throat catching, pungent) (García-González et al., 2008). In addition, they are natural antioxidants that confer an effective defense system against free radical attacks. Some authors have estimated their contribution to olive-oil stability at around 29% (Aparicio et al, 1999). Although phenolic compounds are water-soluble, small quantities are present in olive oil, varying in virgin olive oils between 100 and 1000 mg/kg of oil. Their content depends on a number of factors like cultivar, origin of plantation, cultural practice, degree of fruit maturation, extraction procedure, storage of the olive oil, etc. (Abaza et al., 2005; Ben Temime et al., 2006a). The total phenol content is higher in virgin olive oil than in refined olive oil (Owen et al., 2000). The phenolic fraction of virgin olive oil is derived mainly from the hydrolysis products of oleuropein and ligstroside, aglycones and related compounds. It contains various classes of phenolic compounds such as phenolic acids, phenolic alcohols, flavonoids, hydroxy-isocromans, secoiridoids and lignans (Carrasco-Pancorbo et al., 2005). Phenolic acids were the first group of phenols observed as minor components in virgin olive oil. They have a basic benzoic acid or cinnamic acid structure and include caffeic, vanillic, syringic, p-coumaric, o-coumaric, protocatechic, sinapic and p-hydroxybenzoic acids (Montedoro et al., 1992). The main phenolic alcohols that occur in virgin olive oil are hydroxytyrosol and tyrosol. It is also possible to find in virgin olive oil hydroxytyrosol acetate and tyrosol acetate (Mateos et al., 2001; Abaza et al., 2005) and a glucosidic form of hydroxytyrosol. The prevalent phenols of virgin olive oil are, however, secoiridoids, that are characterized by the presence of either elenolic acid or its derivatives in their molecular structure. These compounds, e.g. oleuropein, demethyloleuropein and ligstroside, are derivatives of the secoiridoid glucosides of olive fruits. The most abundant secoiridoids of virgin olive oil are the dialdehydic form of oleuropein aglycon, the dialdehydic form of ligstroside aglycon, ligstroside aglycon and oleuropein aglycon (Montedoro et al., 1992). Several authors have reported that flavonoids such as luteolin and apigenin are also phenolic components of virgin olive oil. The flavanol, (+)- taxifolin has recently been found in Spanish virgin olive oil (Rovellini et al., 1997). Lignans are also prevalent phenolic compounds in virgin olive oil. Some authors have recently isolated and characterized 1-acetoxypinoresinol, pinoresinol and 1-hydroxypinoresinol as frequently occurring lignans in virgin olive oil (Owen et al., 2000).

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In addition, a new class of phenolic compounds, hydroxy-isocromans, was found in different samples of extra virgin olive oil, the presence of 1-phenyl-6,7-dihydroxy-isochroman and 1-(3’-methoxy-4’-hydroxy) phenyl-6,7,-dihydroxy-isochroman in particular, has been demonstrated.

With respect to argan oil, analytical studies have led to the identification of phenolic derivatives. These are caffeic acid, oleuropein, vanillic acid, tyrosol, ferulic acid, syringic acid, catechol, resorcinol, (-)epicatechin, and (+)catechin (Charrouf and Guillaume, 2007). Most of the phenolic compounds found in olive oil are not present in seed oils. Tuberoso et al., (2007) positively detected vanillic acid (2.8 mg/kg), ferulic acid (0.5 mg/kg) and trans-cinnamic acid (0.9 mg/kg) in corn oil (Tuberoso et al., 2007). Ferulic acid is generally present as sterol esters (Tileva et al., 2002). This component has special interest because of its antioxidant and photoprotective properties. It has been shown that a hexane extract from corn bran contains high levels of ferulate esters, similar in structure to “oryzanol” (Norton, 1994; Norton, 1995; Seitz, 1989), which is a cholesterol-lowering substance, found in rice bran and rice bran oil (Kahlon et al., 1992; Nicolosi et al., 1991). In corn oil ferulic acid has been detected as an ester with dihydro- β-sitosterol (Sonntag, 1979). The major phenolic compounds of crude rapeseed oil are 2,6-dimethoxy-4-vinylphenol (vinylsyringol), sinapine and sinapic acid. Vinylsyringol is formed by decarboxylation of sinapic acid at pressing temperatures. It gives stability to crude rapeseed oil but is removed by superdegumming or refining. The hydrophilic phenolics are removed progressively and completely during refining stages (Koski et al., 2003). Cold-pressed rapeseed oil has a high amount of syringic acid (6.8 mg/kg) and about 1.6 mg/kg of ferulic acid, while seeds from rape are described as rich in phenolic compounds, mainly sinapic acid (Amarowics et al., 2003). No phenolic compounds were detected in cold-pressed peanut, sunflower, or soybean oils (Tuberoso et al., 2007). However, the phenolic compound resveratrol (3,4,5-trihydroxystilbene) is usually found in fresh peanuts or in peanut products like peanut butter or roasted peanuts. In fresh peanuts, the resveratrol content varies from 0.01-1.79 mg/kg (Sobolev and Cole, 1999) up to 2.3 to 4.5 mg/kg (Wang et al., 2005). It is a potent antioxidant and exhibits a wide range of biological effects, including antiplatelet, anti-inflammatory, anticancer, antimutagenic and antifungal properties. Resveratrol reduces lipid peroxidation, oxidation and nitration of platelet and plasma proteins (Olas and Wachowicz, 2005). Phenolic compounds are among the substances eliminated during refining processes. Trials carried out with olive pomace oils at the industrial level confirmed the complete disappearance of polyphenols during refining process, since they were not detected in deodorized oils (García et al., 2006). As they are natural antioxidants, great interest has recently focused on the addition of polyphenols to refined oils. Tocopherols Tocopherols exist as α-, β-, γ-, and δ- isomers, the differences between them being the position of methyl groups on the phenol ring. A series of unsaturated analogues, the tocotrienols, are also present in some oils such as corn and rice. The series of tocopherols and tocotrienols are collectively called tocols. Tocopherols constitute an important group because of their biological and antioxidant properties. When relative tocopherol antioxidant properties were compared, the following order α> β ≈γ>δ was reported in vitro and the inverse order in vivo. Tocotrienols are less useful in inhibiting autoxidation than tocopherols (Elmadfa and Wagner, 1997). The tocol concentrations vary from oil to oil and the tocol profile and composition are often useful critera of purity or evidence of adulteration. The tocopherol pattern in seed oils has been reported to be influenced by environment, hybrids or genotype (Jonnala et al., 2006; Marwede et al., 2005; Nolasco et al., 2006; Richards et al., 2008) and by

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processing (Cmolik et al, 2000; Ferrari et al, 1996; Gogolewski et al, 2000; Tasan and Demirci, 2005). Table 3.6 shows the content of tocopherols in the vegetable oils studied. Argan oil is particularly rich in tocopherols whose concentration is between 600 to 900 mg/kg (Hilali et al, 2007; Rahmani, 2005) with the majority being γ-tocopherol. In contrast, α-tocopherol represents more than 90% of the total tocopherol content of olive and sunflower oils. The reported values of tocopherol content of virgin-olive oil include a wide range of values, from lower than 100 mg/kg up to higher than 300 mg/kg (Kiritsiakis, 1998, Boskou, 2006). However, even higher values (close to 500 mg/kg) have been detected in Argentinian virgin olive oils (Ceci and Carelli, 2007). Olive-pomace oils have a higher content of these compounds (200-600 mg/kg), highlighting the importance of the by-products of the olive oil extraction process as source of antioxidant compounds. In sunflower seed, the tocopherol concentration decreases when oil weight per seed increases (Nolasco et al., 2004). While the four-tocopherol isomers are present in soybean oil, the oil contains no appreciable amounts of tocotrienols. The tocopherol content consists mainly of γ-tocopherol, which amounts to about 60% of the total tocopherols, α-tocopherol which represents less than 10% of the total tocopherols and β-tocopherol which comes only to about 3%. It is clear from the tocopherol composition that soybean oil contains high amounts of the effective antioxidante γ-tocopherol, which stabilises the oil in the bottle, but the amount of the most biologically active α-tocopherol is comparable low. Corn oil contains tocopherols at a relatively high level, from 1100 to 1800 mg /kg, similar to soybean and rapeseed oils. The main forms are γ-tocopherol and α-tocopherol (Sayed and Mohamed, 2002). Of the tocotrienols, α-tocotrienol and γ-tocotrienol are most often reported in corn oil and canola. In canola oil the γ- isomer is normally present at twice the amount of the α- isomer. In contrast, crude peanut oil has only 30 to 40% of the soybean or rapeseed oil content of tocopherols. Ayres (1983) reported a considerable level of tocopherols in refined peanut oil, with a total tocopherol content of 530 mg/kg and most as α- and γ-tocopherol. Casini et al., (2005) reported total tocopherol levels between 199 and 816 mg/kg in crude Argentinian peanut oil over a four-harvest period and concluded that tocopherol content increases with higher precipitation and lower soil temperatures. Rice oil contains about 400 ppm of tocopherols, being majority the α- isomer, but also contains a significant quantity of tocotrienols (585-1296 mg/kg), being majoritary the alpha and gamma isomers, see Table 3.6. Sterols Sterols are important minor components of edible oils in terms of identification, authenticity and health effects. For good health, low levels of cholesterol are desirable with correspondingly high levels of other sterols. Sterols comprise an extensive series of compounds with more than 100 types reported in plant species. Sterols in plants, or phytosterols, exist in the form of free alcohols, fatty-acid esters, steryl glycosides and acylated steryl glycosides (Aparicio, 2000). They can be divided into three groups: (1) 4-desmethylsterols (the cholestane series), i.e. normal phytosterols; (2) 4-monomethylsterols (usually associated with the term methylsterols) and (3) 4,4´-dimethylsterols (the lanostane series, also known as triterpene alcohols). The three sterol groups differ in the conformation of carbon 4 in the steroid skeleton. The desmethylsterols have no methyl group at position 4, while the 4-monomethyl- and 4, 4´-dimethylsterols have one and two methyl groups there, respectively. For some edible oils (e.g.: corn, low-erucic rapeseed oil), the desmethylsterols can account for more than 50% of the unsaponifiable material, while 4-monomethylsterols and 4,4´-dimethylsterols together can constitute 10-30% (Wretensjö, 2004). Saturation of sterols at the 5-alpha position form compounds named stanols, including sitostanol and

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campestanol. In general terms, plant sterol and stanol consumption reduces plasma total and low density lipoprotein cholesterol (LDL-C) concentrations. The major 4-desmethylsterols identified in olive oil are cholesterol, 24-methylenecholesterol, campesterol, campestanol, stigmasterol, Δ7-campesterol, Δ5,23-stigmastadienol, clerosterol, β-sitosterol, sitostanol, Δ5-avenasterol, Δ5,24-stigmastadienol, Δ7-stigmastenol and Δ7-avenasterol, Table 3.7. However, some of them have not been quantified in virgin olive oils but appear in refined olive oils as a result of the refining process; this is the case of Δ5,23-stigmastadienol. Furthermore, Δ5,24-stigmastadienol increases in concentration during refining while the concentration of Δ5-avenasterol decreases (Morales and León-Camacho, 2000). These changes have been used to distinguish refined from virgin olive oils. Other Δ5-desmethylsterols - such as campesterol, stigmasterol and β-sitosterol - are affected by degradation (dehydration) in the refining process. The concentration of 4-desmethylsterols also varies with the cultivars so allowing their characterization (Aparicio, 1988). High campesterol (> 4%) was observed in olive oils grown under drought stress, during early stages of fruit ripening, or from new production zones (Ceci and Carelli, 2007). Finally, sterol esters are also present but as minor components relative to the free sterols. The sterol components of sterol esters are similar to those of the free sterols. Comparison of the sterol composition of free and esterified sterols has been proposed to determine the presence of hazelnut oil in olive oil even at very low percentages (Mariani et al., 2006). Argan oil does not contain a significant quantity of Δ5 sterols whereas these sterol are the major types found in most other vegetable oils. One hundred grams of argan oil can contain up to 320 mg of sterols. Four sterols have been identified as constituting the main part of the argan oil sterol fraction: stigmasta-8,22-diene-3-ol (3-6%), spinasterol (34-44%), dihydrospinasterol (schottenol) (44-49%), and stigmasta-7,24-diene-3-ol (4-7%) (Farines et al., 1981; Farines et al., 1984b). Interestingly, campesterol, a sterol common in most vegetable oils is almost lacking in argan oil sterol composition (<0.4%). Consequently, detection of the absence of campesterol has been proposed, in combination with other analyses such as fatty acid composition analysis and triglyceride composition analysis, as a method to ascertain argan oil authenticity (Hilali et al, 2007). Corn and rice oils have the highest levels of sterols (up to 2550 mg/100 g and 1800 mg/100 g, respectively) among the other edible vegetable oils studied here, being composed principally of β-sitosterol and campesterol. Sterol levels are lower in other oils - less than 500 mg/100 g in peanut, olive, argan, soybean and sunflower oil, and less than 1200 mg/100 g in rapeseed oil. Moreau et al., (2000) have determined the yields and made quantitative measurements of phytosterol classes in corn oils from aleurone, fibre (white), germ and pericarp, revealing that the germ oil contained phytosterols (β-sitosterol and campesterol) but not phytostanols. On the other hand, the aleurone and fibre oils contained high levels of phytostanols (sitostanol and campestanol) and some phytosterols. Peanuts and their products are good sources of phytosterols. The unsaponifiable fraction of peanut oil includes 0.15-0.90% sterol esters and 0.59-1.22% free sterols (Ayres, 1983). Unrefined peanut oil contains 200-300 mg/100 g of phytosterols. This value is higher than that of unrefined olive oil. Refining these oils results in a reduction of the phytosterol concentration. It has been reported that conventional refining does not significantly affect the percentage compositionof sterols, the relative proportions of the major sterols remain constant throughout the process (Wretensjö, 2004). Jonnala et al., (2006) found that a high oleic peanut line (Tamrun OL 01) had higher total phytosterol content (725 mg/100 g oil) than those of the parent lines (670 and 350

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mg/100g oil). β-Sitosterol was found to be the major phytosterol comprising 75-90% of the total phytosterols in all samples. No cholesterol was detected in those peanut lines. The total amount of sterols in rapeseed and canola oils ranges from about 0.5 to 1.0% by weight of the oil. The majority of the sterol fraction comprises desmethylsterols but minor amounts of some methyl- and dimethylsterols are also present (Itoh et al., 1976). As in most oils, β-sitosterol and campesterol dominate. Rapeseed and other brassicas uniquely contain the sterol brassicasterol (24-methyl-3,5,22-cholestatrienol) which can be used to detect the adulteration of more expensive oils with rapeseed oil (Strocchi, 1987; Ackman, 1990). Sitosterol and campesterol are equally distributed in the esterified and free sterol fractions of canola oil but twice the amount of brassicasterol is found in the free sterols than in the esterified ones. The sterol composition can be markedly affected by genetic modification. Brassicasterol, campesterol, and β-sitosterol levels were consistently lowered in one genotype, whereas increased brassicasterol content was observed in another variety (Abidi et al., 1999). The total sterol content of conventional sunflower oil ranges between 240 and 500 mg/100 g of oil (Padley et al., 1994; Codex, 2005; Delplanque, 2000) being a little wider in the case of the high-oleic line. Table 3.7 shows that the sterol compositions of conventional and high-oleic sunflower oil are similar, being mainly β-sitosterol (~60%), stigmasterol (~10%) and campesterol (~10%). Sunflower oil contains more free sterols (62.4%) than esterified sterols (37.6%) (Verleyen et al., 2002). Crude soybean oil can contain lower amounts of total sterols than sunflower oil, with the proportions of campesterol and stigmasterol being higher. The principal sterol in this oil is β-sitosterol, which is slightly reduced by refining, depending on the conditions. Methylsterols and triterpene alcohols The amount and composition of triterpene fractions vary markedly from one oil to another as is shown in Table 3.8. Rice oil has a very high level of triterpenic alcohols principally comprising cycloartenol and 24-methylene cycloartanol. Aparicio et al., (1994) studied 9 identified and 4 partially identified compounds (taraxerol, dammaradienol, β-amyrin, 28-nor-5α-olean-17-en-3β-ol, butyrospermol, α-amyrin, 24-methylene-lanostenol, 24-methylene-cycloartanol, cycloartenol, a Δ8-sterol, and three Δ7-sterols) in characterizing European virgin olive oils produced inside the by geographical origin and cultivar (Aparicio and Alonso, 1994; Aparicio et al., 1994; Luna et al., 2006). Finally, the presence of two pentacyclic triterpene dialcohols - erythrodiol and uvaol - has also been observed in the unsaponifiable matter of both olive-pomace oil and olive oil. These triterpene dialcohol compounds are used to detect the presence of olive-pomace oil in olive oil although the amount in olive oils varies depending on the cultivar (Albi et al., 1990). Triterpenes in peanut oil represent the 0.14% of the oil (Fedeli et al., 1966). Five triterpene alcohols have been identified in argan oil: tirucallol (27.9%), β-amyrin (27.3%), butyrospermol (18.1%), lupeol (7.1%), and 24-methylene cycloartanol (4.5%). Two methylsterols have also been identified, namely citrostadienol (3.9%), and cycloeucalenol (<5%) (Farines et al., 1984b). The 4-monomethylsterols are intermediate products of the biosynthesis of phytosterols. Table 3.9 comparises the contents of some of them in edible oils, showing that rice oil has the highest value. Aparicio and Alonso (1994) quantified 7 compounds (cycloeucalenol, citrostadienol, 24-dimethyl-24-cholestadienol, gramisterol, ethyllophenol, fridelanol, and obtusifoliol) for the characterisation of European olive oil by the SEXIA® Expert System.

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Pigments: carotenoids, chlorophylls and derivates Pigments are responsible for the colour of oil, one of the major attributes that affects consumer perception of quality. The most commonly encountered pigments in vegetable oils are chlorophylls and carotenoids. They play an important role in providing oxidative stability on account of their antioxidant properties in the dark and their pro-oxidant activity in the light. Our knowledge of the important roles that they play in health (Mayne et al., 1996) and food authenticity and traceability (Mínguez-Mosquera et al., 2000; Gandul-Rojas et al., 2000) is relatively recent. Today we know that they prevent cardiovascular diseases and degenerative eye pathologies, and that β-carotene and other carotenoids have anticancer activity. The quantity of pigments in vegetable oils is highly variable and depends on several factors. In the case of olive oils, the amount depends on olive cultivar, olive ripeness, olive oil processing system, storage conditions, and even the pedoclimatic characteristics of the farm. The presence of a specific pigment profile is a requirement of varietal virgin olive oil purity and the ratio between some pigments guarantees the authenticity of the product against fraudulent practices (Gandul-Rojas et al., 2000). Table 3.10 shows the main pigments quantified in Spanish and Italian monovarietal olive oils. Eight are carotenoids, usually quantified in virgin olive oils: neoxanthin, violaxanthin, anteraxanthin, lutein, β-carotene, monoesterified violaxanthin, esterified neoxanthin, and α-carotene (Mínguez-Mosquera et al., 1990). Five are derivatives of chlorophyll. Carotenoids exist in the form of a polyene chain which is sometimes terminated by benzene rings. The major carotenoids are β-carotene and lutein. Carotenoids that have been oxidized and subsequently contain hydroxyl groups on the ring, such as lutein and zeaxanthin, are classed as xanthophylls. They give oils their characteristic colour on account of their unsaturated conjugated double bond structure. One important factor for the quantification of individual carotenoids in olive oil is the ratio between minor carotenoids to lutein. This allows determination of the stage of ripeness of the olives processed for producing virgin olive oil. On the other hand, the ratio of lutein to β-carotene can be useful to differentiate monovarietal Spanish virgin olive oils, in which the ratio is between 1.3 and 5.1, from Italian and Greek single variety olive oils whose ratios are less than one. Carotenoids are responsible for argan oil's specific brown-orange colour. However, the exact composition of the carotenoid fraction that constitutes 37% of the unsaponifiable fraction has not been studied in detail yet. The xantophyll level can be up to 500 mg/kg (Rahmani, 2005), but only low levels of provitamin A and in trans-β-carotene havebeen reported (Collier and Lemaire, 1974). Kornsteiner et al., (2006) reported that the total quantity of carotenoids (α- and β-carotene, zeaxanthin, lutein, cryptoxanthin and lycopene) in peanut oil was low. Corn oil contains 200-400 mg/kg of carotenoids. In a study by Cabrini et al., (2001) sunflower oil contained slightly more β-carotene (3 mg/kg) than peanut (0.1 mg/kg), soybean (0.3 mg/kg) and corn (0.9 mg/kg) oils, and almost half that extra-virgin olive oil. The carotenes were mainly lutein and zeaxanthin, which together comprised just over 75% of the carotene fraction. Hazuka and Drozdowski (1987) reported that levels of β-carotene were lower in sunflower (0.1 mg/kg) and peanut than in rapeseed oil (2 mg/kg). Rade et al., (2004) also found that crude sunflower oil contained less than 3 mg/kg carotenes. Cabrini et al., (2001) also reported that, the related ubiquitinone antioxidants were present in a small number of unrefined sunflower oils (Cabrini et al., 2001).

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Sunflower oil contained low levels of reduced and oxidised ubiquinones (55 and 6 mg/L respectively) compared with soybean (about 200 and 70 mg/L) and corn oils (about 420 and 80 mg/L). It contained more reduced ubiquinones than extra virgin olive oil (about 10 mg/L) and lower levels of oxidised ubiquinones (about 110 mg/L). Crude canola oil contains carotenes at a level of about 100 mg/kg. They comprise 90% xanthophylls and 10% carotenes (Hazuka and Drozdowski, 1987). Lutein (3, 3´-dihydroxi-α-carotene) occurs as the main carotenoid in soybean (19.7 mg/kg) and rapeseed (39.5 mg/kg) oils. (Froehling et al., 1972). Crude rice bran oil contains low levels of β-carotene (11.5 mg/kg) as characterized on the basis of their UV-spectra (Kritchevsky et al., 2002; Stöggl et al., 2005). High levels of chlorophylls are associated with olive oil, with other vegetable oils containing much less. During oil storage chlorophyll A is degraded to uncoloured products: pheophytin A, and A’ and pyropheophytin A (PPP). The increment of PPP in virgin olive oils correlates with storage time (Gallardo-Guerrero et. al, 2005). In addition, thermal treatment at elevated temperatures augments significantly the PPP level, being an indicator of unpermitted thermal treatment of virgin olive oils or the presence of refined oil.

Table 3.10. Concentration ranges of individual pigments quantified in Spanish and Italian monovarietal olive oils.

Pigments (μg/kg)

Spain Italy

Carotenoids

Neoxanthin 70-400 310-790

Violaxanthin 10-150 260-770

Anteraxanthin 0-475 470-640

Mutatoxanthin 30-110 ---

Luteoxanthin 90-250 510-800

β-Cryptoxanthin 0.0-60 380-620

Lutein 1200-9800 2280-4490

β-Carotene 110-2400 8060-16270

Violaxanthin monoesterified 0-20 ---

Neoxanthin esterified 0-30 ---

Esterified xanthophylls 0-190 ---

Chlorophylls

Chlorophyll a 0-800 0-200

Chlorophyll b 0-1350 1030-1550

Pheophytin a 980-21700 19720-25040

Pheophytin b 0-840 150-2920

Pheophorbide a 0-565 ---

Total chlorophylls 400-25100 26100-31940

Compiled from: Aparicio, unpublished data; Criado et al., 2007; Gandul-Rojas and Mínguez-Mosquera, 1996; Giuffrida et al, 2007 (---): no value or trace.

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Chlorophyll and related pigments such as pheophytins are present in rapeseed oils (Endo et al., 1992). Changes in the composition and content of chlorophylls during rapeseed ripeness can have a direct impact on the processing and quality of this oil. The level of chlorophyll in the seed decreases as it matures, from over 1000 mg/kg to about 2 mg/kg. Also at maturity only chlorophyll a and b were present while all possible isomers/derivatives were observed at other stages of maturation. Rice bran oil usually shows a deep green colour due to its chlorophyll content. The pigments in the oil include carotenoids, chlorophyll and Maillard browning products (Kochhar 2002). Despite the facts that the chlorophyll content appears to be very high, and the bleaching of this oil is a challenging problem on account of heavy losses (Pagano et al., 2001), there are no published data on the pigment composition of rice bran oil. The extraction method and refining process affects the pigment content of vegetable oils, the pigment range reported being wide. Matthäus and Bruhl (2003) measured the chlorophyll content of 48 cold-pressed rapeseed oils in Germany. The level of chlorophyll in the oil increased with pressing intensity, with an average value of 43.6 mg/kg and a range of 0.3 to 175 mg/kg. The range was mostly between 20 and 60 mg/kg. In a survey of nine vegetable oils, retail cold-pressed sunflower oil contained about 2 mg/kg chlorophylls, less than any oil apart from peanut (Tuberoso et al., 2007). Levels of β-carotene were also lowest in sunflower (0.1mg/kg) and peanut than rapeseed oil (2mg/kg). However, Rade et al., (2004) found about 0.5 mg/kg chlorophylls in crude sunflower oil. Industrial crude oil obtained from pressing followed by solvent extraction contained the highest level of chlorophylls (0.63 mg/kg), considerably more than the values from the oil obtained in the laboratory (laboratory cold pressed: 0.02, laboratory hot-pressed 0.32 and laboratory extracted: 0.23) (Rade et al., 2004). Chlorophylls occurs in traces in soybean oil and about 5-35 mg/kg in canola and rapesseed oils (Przybylsky, 1993). The effect of processing on pigment content has also been studied (Usuki et al., 1984; Suzuki and Nishioka, 1993; Chapman et al., 1994; Rade et al., 2004; Kaynak et al., 2004). Chlorophylls A are almost removed during bleaching, while carotenoid levels are appreciable reduced during bleaching and deodorization. Heating oils causes the formation of products known as pyrochlorophylls and pyropheophytins that can act as markers of heat treatment of cold-pressed oils. Pheophytin a and pyropheophytin a are more absorptive than their b isomers when bleached with earth. Consequently, smaller amount of b isomers than a isomers are removed from the oil during bleaching. Stronger bleaching conditions and higher amounts of activated bleaching earth are needed to remove all chlorophyll derivatives (Suzuki and Nishioka, 1993). Waxes and fatty alcohols Wax esters occurring in vegetable oils are a group of compounds formed by esterification of high-molecular-mass alcohols with fatty acids. The length and structure of the alcoholic group is variable; thus, if the alcoholic groups are long chain aliphatic alcohols they result in aliphatic waxes. If the alcoholic groups are sterols, triterpene alcohols or methylsterols, the compounds are generally named terpenic waxes (Mariani et al., 1991; Gordon and Miller, 1997). During the oil extraction process, a fraction of these wax esters is transferred into the oil, depending on the oil extraction system (Cert et al, 2000). Aliphatic waxes have 36 to 60 carbon atoms (Hénon et al., 2001; Carelli et al., 2002b; Baümler et al., 2007). Waxes with chain lengths lower than C40 are denominated “soluble waxes”, those with lengths between C40 and C42 are called “partially-soluble waxes”, and those with more than C44 are named “crystallisable waxes”. The last fraction has to be removed from the oil, a high content of waxes in vegetable oils could produce turbidity after long term storage (Mariani et al., 1987). Wax content and profile differs between vegetable oils.

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The primary physiological function of waxes is to protect the seed of the environmental condition and avoid water stress. For sunflower seeds, their are mainly located on the hull surface in concentrations up to 3% in the lipid fraction depending on the hybrid and the origin of the seed (Cancalon, 1971; Morrison et al., 1984; Carelli et al., 2002b; Baümler et al., 2007). The hull contribution to the wax content in sunflower oil proved to be higher than 37%, reaching around 80% when only the C44-C48 fraction is considered (Carelli et al., 2002b). The oil wax content can be reduced appreciably by hexane-washing or by partial dehulling of the seed (Baümler et al., 2007). Wax extractability depends strongly on the extraction temperature (Brevedan et al., 2000). Pressed oil has a lower wax content than solvent-extracted oil (Mariani and Fedeli, 1989; Brevedan et al., 2000). Degumming and alkali neutralization has little effect on the total wax content, while bleaching and deodorization are usually responsible for higher losses (Mariani and Fedeli, 1989). Rice bran contains waxes that are extracted along with the TAGs during hexane-extraction. The recommended uses for these compounds include as a constituent of chocolate enrobing as a coating for lozenges, foodstuff, cosmetics and industrial products (Hamilton, 1995; Yoon and Rhee, 1982). The crude rice bran oil has a very high content of crystallizable waxes (up to 6700 mg/kg) with values of more than 85% for the C48-C54 waxes, with the soluble esters being absent or present at low levels (Hénon et al., 2001). The content of waxes varies among the various categories of olive oil. Hence, the profiles of these esters of fatty acids and fatty alcohols are of interest as indicators of both quality and purity. Waxes are found in comparatively larger amounts in refined and solvent extracted oil (olive-pomace oil) than in virgin olive oils (Kiritsakis, 1998). Thus, the sum of C40, C42, C44 and C46 aliphatic waxes is a parameter used to detect pomace-olive oil in virgin olive oil. This sum has been regulated as: ≤ 250 mg/kg for extra-virgin, virgin and ordinary virgin olive oils, and ≤ 350 mg/kg for other categories, (EEC, 1993). For soybean oil the crystallizable wax fraction is very similar to that of the winterized sunflower oil (Hénon et al., 2001), which is in the range of: 39-100 mg/kg (Carelli et al., 2002b). The crystallizable wax fraction of the peanut oil is similar to that of the soybean oil and contains only 50 mg/kg of C34-C42 soluble esters, compared to 153 mg/kg in refined soybean oil. Crude rapeseed and crude corn oil are also oils with low level of soluble esters, (Hénon et al., 2001). In the case of argan oil, because of its only recent commercial interest, the composition of some minor compound like waxes has not yet been reported. Canola oil is considered to be an excellent salad oil because of its clarity when is stored at low temperatures, but the industry has seen the occasional appearance of haze or sediment during storage in both refrigerator and store shelves (Xiaojun et al., 1993; Botha et al., 2000). Waxes are the major components present in the canola oil sediment, the carbon chain length of these waxes ranging from C36 to C56, with C36, C44, C46 and C48 being predominant (Xiaojun et al., 1993). Some studies have elucidated the chemical composition of waxes (Ramos-Ayerve and Rodriguez-Berbel, 1985; Mariani and Fedeli, 1989; Hénon et al., 2001; Sindhu Kanya et al., 2007). Sunflower oil wax esters are composed mainly of fatty acids in the range of 16-30 carbon atoms, especially C20 and C22, with fatty alcohols between 20 and 32 carbon atoms, principally C24 and C26 (Ramos-Ayerve and Rodriguez-Berbel, 1985; Mariani and Fedeli, 1989; Liu et al., 1996; Sindhu Kanya et al., 2007). Waxes with fewer than 42 carbon atoms were identified in sunflower oil by GC-mass selective detector as monounsaturated waxes, esters of long-chain saturated fatty acids, and a monounsaturated alcohol, mainly eicosenoic alcohol (Hénon et al., 2001).

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The waxes of crude rice oil consist of esters containing fatty acids with chain length of 22, 24 and 26 carbon atoms combined with fatty alcohols of 26, 28 and 30 carbon atoms, with triacontanol being the major alcohol constituent, (Hamilton, 1995). A series of long chain saturated fatty acids (C26-32) mainly esterified to long chain alcohols have been found in the sediment of canola oils and are assumed to be derived from seed coat waxes (Przybylski, 1993). In the case of peanuts, Kawanishi et al., (1991) analyzed the content of octacosanol in oil from seed coat and germ, and found them to contain 23.4 mg/kg and 9.2 mg/kg, respectively. Fatty alcohols are an important class of olive oil minor constituents because they can be used for detecting adulteration of olive oil with olive pomace oil (EEC, 1991). The main linear alcohols present in olive oil are docosanol, tetracosanol, hexacosanol and octacosanol (Tiscornia et al., 1982; Boskou, 2006). Odd carbon atom alcohols (tricosanol, pentacosanol, heptacosanol) may be found in trace amounts (Boskou, 2006). The total aliphatic alcohol content does not usually exceed 35 mg/100g oil. In extracted olive oil, the level of fatty alcohols may be more than ten times higher than in pressed oils (Boskou, 2006). Dry climatic conditions and high temperatures may cause a high aliphatic alcohol content as well as a high wax content. Hydrocarbons, squalene and stigmastadiene Squalene (2,6,10,15,19,23-hexamethyl-2,6,10,14,18,22-tetracosahexene) is widely distributed in nature with high proportions in olive oil compared to other vegetable oils. It is the major olive oil hydrocarbon making up more than 90% of the hydrocarbon fraction and ranging from 800 to 12000 mg/kg in quantity (Perrin, 1992; Cert et al., 2000). It is a key precursor in the biosynthesis of sterols and it is regarded as partially responsible for the beneficial effects of olive oil against certain cancers (Rao et al., 1998; Smith et al., 1998; Scolastici et al., 2004). The squalene content depends on olive cultivar (De Leonardis et al., 1998; Manzi et al., 1998), and oil extraction technology (Nergiz and Ünal, 1990), and it is dramatically reduced during the process of refining (Mariani et al., 1992; Lanzón et al., 1994). Sunflower oil may contain about 120-224 mg/kg squalene (Albi et al., 1997; Tuberoso et al., 2007), considerably less than olive oil, rice oil (up to 30000mg/kg, Chakrabarty, 1990), and argan oil (3100 mg/kg, Khallouki et al., 2003); but similar to rapeseed oil (10-440 mg/kg, Tuberoso et al., 2007); corn oil (130-340 mg/kg; Sayed & Mohamed, 2002, Tuberoso et al., 2007 ), soybean oil (0-143 mg/ kg, Tuberoso et al., 2007; Gutfinger and Letan, 1974), and peanut oil (270 mg/kg; O’Brien, 2004). The refining of vegetable oils with high β-sitosterol content produces considerable amounts of stigmasta-3,5-diene, a hydrocarbon derived from β-sitosterol by dehydration. Determination of stigmasta-3,5-diene can be used to establish whether an edible oil is crude or refined (Cert et al., 1994). The test is widely used for virgin olive oils where a limit of 0.10 mg/kg has been set. Another indicator of adulteration is the presence of campesta-3,5,22-triene (derived from the dehydration of brassicasterol) which reveals the presence of refined rapeseed oil. Other hydrocarbons reported to be present in vegetable oils are even and odd number paraffins, ranging from C11 to C30 (Fedeli, 1977), branched-chain hydrocarbons, iso-or anteiso, as well as aromatic and polycyclic hydrocarbons (Fedeli, 1977; Tiscornia et al., 1982). The proportion of C29 alkanes (unreported) in rapeseed was described as being higher than in olive oil and could be used as a monitor of adulteration (Webster et al., 2000). McGill et al., (1993) found a content of n-alkanes of 105 to 166 mg/kg in retail samples of refined sunflower oil, which was higher than any of 42 samples of 21 other vegetable and fish oils. The hard wax fraction isolated from rice oil contained C31(35.5%) and C29 (28.9%)

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hydrocarbons, and the soft wax fraction contained C21 (22.9%) (Chakrabarty, 1990). Other hydrocarbons (e.g. pentacosane to octacosane) have been used for characterizing monovarietal virgin olive oils and their geographical traceability (García-González et al., 2008). 3.1.6 Other minor compounds Volatiles Volatile compounds in oil are important in providing flavour to salad oils such as olive and cold-pressed rapeseed, but they can also be a source of taints. The measurement of volatile breakdown products of fatty acids, such as hexanal, can be used to determine oil quality. Analysis of volatiles can also show contamination with extraction solvents and volatile non-polar pollutants such as benzene and chlorinated hydrocarbons. The volatiles in argan oil have been qualitatively studied using GC-MS and GC-Olfatory analysis. Eighteen compounds have been identified: (Z)-non-3-enal, (E)-non-2-enal, (Z)-non-2-enal, dec-2-enal, (Z,Z)-deca-2,4-dienal, (E,E)-deca-2,4-dienal, vanillin, oct-1-en-3-one, acetophenone, p-methylacetophenone, (R)-δ-decalactone, 2-acetyltetrahydropyridine, acetylpyrazine, 2,5-dimethyl-3-ethylpyrazine, 2-methyl-3-furanthiol, 2-furfurylthiol, neoisomenthol, and 1-carvone. At least three additional components have not been identified (Charrouf et al., 2006) In olive oil volatiles are mainly produced by oxidation of fatty acids. The proportion of volatile organic compounds in olive oil is very small, less than 0.2%, but they are solely responsible for the aroma, and largely responsible for the taste. The volatile fraction of virgin olive oil consists of a complex mixture of more than one hundred compounds (Vichi et al., 2003). These are saturated and unsaturated aldehydes, alcohols, esters, hydrocarbons, terpenic hydrocarbons, ketones, furans and, probably, other yet unidentified volatile compounds. The major volatile compounds reported in virgin olive oils are C6 and the C5 compounds. Hexanal, trans-2-hexenal, hexan-1-ol and 3-methylbutan-1-ol are found in most virgin olive oils in Europe and in Tunisia (Angerosa, 2002; Aparicio et al., 1997; Ben Temime et al., 2006b; Haddada et al., 2007; Vichi et al., 2003). The volatile composition of olive oil depends on the levels and activity of the enzymes involved in the various metabolic pathways (Angerosa et al., 2004) which are genetically determined (Campeol et al., 2001). Corn oil under deep fat frying conditions generates monoaldehydic oxidation products such as alkanals, alk-2-enals, and alk-2,4-dienals. The formation of diethyl phthalate during the heating of corn oil has been reported, in addition to 45 different non-acidic compounds including hydrocarbons, alcohols, esters, lactones, aldehydes, ketones and aromatics (Sonntag, 1979). With respect to soybean oil, due to its high linolenic fatty-acid content, careful handling is mandatory to prevent oxidation and metal chelating that lead to avoid beany, painty, or green flavors (O´Brien, 1998). The pleasant nut-like flavor associated with peanuts remains in the oil on on extraction, rather than in the meal. The flavor is accentuated with oxidation but does not become offensive as quickly as some other vegetable oils (O’Brien, 2004). The most important volatile compounds found in peanut oil are 2,4-decadienal, hydrocarbons, menthol, nonanal and 2-nonenal (Caja et al., 2000), whereas hexanal and 1- methylpyrrole were the main volatiles found in raw peanut (Burroni et al., 1997). There are only very limited publications describing the volatiles of rapeseed oils. In a study of volatile formation on accelerated storage at 60 °C. Jelen et al., (2000) reported 55

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volatile compounds in rapeseed oil. The compounds pentanol, 3-nonen-2-one, 2,4-nonadienol, and four unidentified volatiles were present in refined but absent in cold-pressed oil. Limonene, 1-octanol and 2-nonanone were absent in refined but present in cold-pressed oil. The major aldehydes produced on accelerated storage were pentanal, hexanal, 2-hexenal, heptanal, 2-heptenal, octanal and nonanal. Katsuta et al., (2008) showed that when used for frying there were no significant differences in the quantity or range of volatiles released from rapeseed and high oleic sunflower. Bocci and Frega (1996) investigated the volatile components that may contribute to the formation of the flavor of the oil extracted from sunflower seeds. The main compounds found in the volatile fraction of cold-pressed sunflower oil analysed by gas chromatography/mass spectrometry (GC-MS) are terpenic hydrocarbons, being α-pinene the most abundant compound. About 150 compounds consisting of alkanes, aromatic hydrocarbons, pyrazines, quinolines, thiazoles, thiophenes, phenols and carboxylic acids have been identified in rice oil. These odor components are removed by normal dedorization processes (Chakrabarty, 1990). Metals Trace metals present in edible oils originate from two sources, they are taken up from the soil or atmospheric deposition especially from polluted sites, or they can be introduced during processing or storage. Most trace elements have detrimental effects. For safety control, certain toxic metallic polluants are analysed in edible oils, they include lead, arsenic, mercury and cadmium. Codex standard for named vegetable oils (Codex, 2005) gives a maximum admissible level of 0.1 mg/kg for lead and arsenic. Generally the measured levels in refined edible oils are very low, typically being 0.008-0.06 mg/kg for lead, less than 0.007 mg/kg for cadmium (Dugo et al., 2004), and less than 0.02 mg/kg for arsenic in sunflower (Dugo et al., 2004; Castera, 1992). Because iron and copper have pro-oxidant activity, the maximum recommended levels of iron are 1.5 mg/kg in refined oils and 5.0 mg/kg in virgin oils. With reference to copper, the maximum recommended levels are 0.1 mg/kg in refined oils and 0.4 mg/kg in virgin oils. Copper and zinc are considered micronutrients because they are essential for human nutrition at low doses exhibiting a wide range of biological functions such as components of enzymatic and redox systems (McLaughlin et al., 1999). Dugo et al., (2004) found the following ranges of zinc in commercial refined oils: peanut (0.05-0.5 mg/kg), sunflower (0.18-0.37 mg/kg), rice ( 0.13-0.26 mg/kg), soybean (0.03-0.04 mg/kg) and corn (0.03-0.07 mg/kg), concluding that peanut oil may be a good source of zinc. Among metals detected in olive oils are: Fe, Cu, Pb, Cd, Hg, As. The content of metals in olive oils should not exceed agreed levels, i.e. 0.05 to 1 mg/kg (Uzzan, 1996). Vitamin K Canola contains trans vitamin K1 (phylloquinone) at levels estimated as between 1 and 4 mg/kg oil (Zonta and Stancher, 1985; Ferland and Sadowski, 1992; Gao and Ackman, 1995; Shearer et al., 1996; Jakob and Elmadfa, 1996). These values are higher than those for most edible oils and probably reflect the high vitamin K1 content of brassica leaves. Olive oils contain moderate levels of phylloquinone (0.5- 0.8 mg/kg), but many other oils, such as sunflower and corn oils, contain less than 0.1 mg/kg. The levels are affected by production and processing methods (Zonta and Stancher, 1985; Gao and Ackman, 1995). Sulphur compounds Edible oils can also contain sulphur compounds. Soybean, rapeseed and sunflower oils contain sulphur at a level of 2-10 mg/kg. Levels of sulphur in crude and RBD (refined,

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bleached and deodorized) canola oils were measured at 25 mg/kg and 9.4 mg/kg, respectively (Wijesundera and Ackman, 1988; Cho-Ah-Ying and Deman, 1989). Sulphur inhibits hydrogenation catalysts and in organic form produces odorous taints. It occurs in rapeseed oil in the form of organic compounds derived from the decomposition of glucosinolates. Some sulphur components have antioxidant activity since they form stable complexes with hydroperoxy radicals. In addition, three related sulphur containing fatty acids have been reported in trace amounts (less than 0.01% of the fatty acid fraction) in canola (Wijesundera and Ackman, 1988). Their structure was of isomeric 9,12-; 8,11- and 7,10 epithiostearic acids, similar to that of furan fatty acids found in other oils but with a sulphur in the furan ring which was situated near the mid-point of the fatty acid chain. Glucosinolates Canola also contains glucosinolates, which are antinutritional factors as their degradation products have goitrogenic and toxic effects. They are also precursors of various taints. The degradation products include epithionitriles, nitriles, isothiocyanate, sulfinylnitrile, sulfinyl isothiocyanate, glucose, thioglucose and thioglucose dimer. Some traces of these can be detected in rapeseed oil (Shahidi et al., 1997). 3.2. Instrumental Methods Used for Edible Oil Analyses Several instrumental methods are nowadays used in the analysis of edible vegetable oil in order to establish oil identity, to assess quality status and to detect possible adulteration, in particular for olive oil. Classical chemical methods for identity assessment of vegetable oils include the evaluation of different indices such as iodine value, hydroxyl value, saponification value and refractive index. In addition, the composition of fatty acids, triglycerides, tocopherols and sterols provides useful information for oil identification. Many quality assessment methods are based on the measurement of oil oxidation or hydrolytic status. The oxidative process of oils is one of the main causes of the deterioration of the principal organoleptic and nutritional characteristics of foodstuffs. The oxidation process is very complex and can be summarized into two phases: in the first one fatty acids react with oxygen, leading to the production of odourless compounds such as peroxides; during the second phase, the peroxides degrade into many substances such as volatile aldehydes, responsible for the rancid odour and flavour, and in a non-volatile portion. The primary oxidation products are normally measured by means of the Peroxide Value test (PV) and the secondary products by means of the p-anisidine test that estimates the secondary oxidation products of unsaturated fatty acids, principally conjugated dienals and 2-alkenals. The oxidative status of the oil should be evaluated considering both its primary and secondary oxidation products. Indirect measures of the degree of oxidation are based on the measurement of colour or UV absorption. In the last forty years, the use of chromatographic techniques and mass spectrometry has provided a valuable tool for oil analysis, both for identity assessment and quality control. In particular, advances in instrumentation, improvement of on-line separation techniques and of data processing have contributed to a great expansion in the use of mass spectrometry in oil analysis.

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3.2.1. Classical methodologies UV-spectrophotometry UV spectroscopic methods can measure the percentages of conjugated diene, triene, tetraene and pentaene acids and also linoleic, linolenic, arachidonic and pentaenoic acids. A method based on this approach has been adopted by the American Oil Chemists' Society (AOCS Method Cd7–58, AOCS 1998). UV absorbance measurements are an index of the degree of oxidation and adulteration of a vegetable oil. Many authors reported a good correlation between the absorbance of olive oil at 232nm and the degree of oxidation (Montefredine and Luciano, 1968; Bartolomeo and Sergio 1969; Ninnis and Ninni, 1968; Jimenez and Gutierrez Conzalea-Quijiano, 1970). Moreover, spectrophotometric absorption in the ultraviolet region (K232 and K270) was used, together with other quality parameters such as acidity, peroxide values (PV), fatty acid composition, to monitor the quality of virgin olive oil (Psomiadou et al., 2003), to characterize extra-virgin olive oils of Appellation of Controlled Origin (ACO) (Rial and Falqué, 2003) and to define quality indices of five new olive cultivars (Baccouri et al., 2007). The effect of microwave heating on sunflower, peanut and soybean oils was studied by Hassanein et al., (2003) evaluating, together with other parameters, changes in colour absorbance at 420 nm. This value became higher with an increase in the heating period (Hassanein et al., 2003). Nuclear Magnetic Resonance (NMR) High resolution proton and carbon 13 nuclear magnetic resonance (1H and 13C NMR) are useful for a variety of analytical purposes in studies of oil chemistry. A careful observation of conveniently expanded 1H NMR spectral regions of different vegetable oils permits differentiation of oils of different composition. In particular, the most significant signals are those of methylic and methylenic protons, γ or further from carbonyl groups and β or further from double bonds. The first ones provide information on the proportions of linolenic, linoleic and saturated plus oleic acyl groups, while the second groups give information on the proportions of saturated, oleic, linoleic and linolenic acyl groups. Other relevant signals are those of allylic protons, which give information on the proportions of oleic, linoleic and linolenic acyl groups, and those of bis-allylic protons, which provide information on the proportions of linoleic and linolenic acyl groups. Due to the fact that vegetable oils are composed of triacylclycerols (TAG) with different acyl groups (see Table 3.11), using this approach Guillén and Ruiz (2003a) discriminated between 15 oils of different botanical origin. As an example, the observation of the methylic protons (-CH3) 1H NMR signals present in the spectra between 0.83 and 0.93 ppm of the 15 vegetable oils under study makes it possible to distinguish not only between oils of very different composition but also, in some cases, oils of similar composition (Guillén and Ruiz, 2003a). Guillén and Ruiz employed 1H NMR spectroscopy to determine the proportion of the different acyl groups in oils and fats (Guillén and Ruiz, 2003b), the unsaturation degree of a large group of vegetable oils (Guillén and Ruiz, 2003c) and to detect primary oxidation products, such as hydroperoxides and secondary oxidation products, like aldehydes (Guillén and Ruiz, 2001, Guillén and Ruiz, 2005). 1H-NMR spectroscopic and an FTIR spectroscopic methods were applied by Moya Moreno et al., to monitor the decomposition products formed in different edible oils (sunflower oil, virgin olive oil, corn oil, etc.) after heating at up to 300°C. They demonstrated that when the temperature reaches 150°C there is a formation of hydroperoxides, while a further temperature increase causes a second oxidation with the formation of new carbonyl

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compounds, mainly aldehydes. The results obtained showed that in all samples analysed, degradation became significant after heating at 200°C, a temperature which is often exceeded during normal cooking processes (Moya Moreno et al., 1999a). Minor oil components, such as saturated and unsaturated aldehydes, stigmasterol and β-sitosterol, can be analysed by 1H NMR when their signals do not overlap with those of the main components, their concentration is high enough to be detected, and high field equipment is used (Mannina et al., 1999; Segre and Mannina, 1997; Sacchi et al., 1996).

Table 3.11. Approximate typical proportions (%) of various acyl groups in 15 edible oilsa.

Edible oil Saturated groups

Oleic groups

Linoleic groups

Linolenic groups

Olive 14.5 75.5 7.5 1

Hazelnut 8.0 81.0 11.0 0

High-oleic sunflower 9.0 75.0 16.0 0

Peanut 17.5 41.0 35.5 0

Sesame 13.5 42.0 44.5 0

Corn 13.5 32.5 52.0 1

Sunflower 12.0 23.0 63.0 <0.5

Pumpkin seed 21.0 24.0 54.0 0.5

Safflower 9.0 12.0 78.0 0.5

Thistle 8.6 11.9 74.8 0

Rapeseed 6.0 63.0 20.0 9.0

Soybean 15.5 21.0 53.0 8.0

Wheat germ 18.0 20.0 52.0 10.0

Walnut 11.0 16.0 59.0 12.0

Linseed 10.0 18.0 14.0 58.0 a (Guillén et al., 2003)

The adulteration of high-value oils with oils of lesser value constitutes a problem of economic and commercial significance, particularly for olive oil, an expensive oil of recognized nutritional value. More important, the lower-value oils used for the adulteration have fatty acids profiles similar to olive oil. The detection of olive oil frauds by NMR is based on the qualitative and quantitative chemical information obtained from resonance data. For example, the determination of the proportions of linolenic acyl groups in an oil sample by 1H NMR can provide very valuable information. The addition of seed oil to olive oil could be presupposed when the proportion of linolenic acyl groups in the sample is higher than 0.9% (Mannina et al., 1999). Furthermore, Fauhl et al., investigated the possibility of detecting fraudulent mixtures of olive oil with sunflower oil or hazelnut oil by one-dimensional 1H NMR. These authors found that mixtures of hazelnut or sunflower oils with olive oil can be

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detected by this method if the proportion of hazelnut is higher than 25% and that of sunflower higher than 10% (Fauhl C. et al., 2000). Moreover, virgin oils from different olive varieties grown in the same environment and collected at the same ripening stage were discriminated on the basis of minor virgin olive components by 1H NMR (Sacchi et al., 1996). Similarly, Italian extra virgin olive oil samples obtained from different varieties were classified with respect to the region of origin (Sacchi et al., 1998). A classification according to geographical origin was also carried out in a study of Italian olive oils based on multivariate statistical analysis of the 1H NMR spectra of the oil phenolic fraction (Sacco et al., 2000). The first application of 13C NMR to the study of corn seeds and oil dates back to 1973 when Shoolery proposed this analytical approach to obtain high-resolution NMR spectra of different seed oils. Because 13C chemical shifts cover a range of nearly 200 ppm, the absorption lines of various chemically different 13C nuclei were very well resolved (Shoolery, 1973). In 1977, Rutar et al., applied this technique to corn and other seed oils to see whether 13C NMR could be used as a routine method for the characterization of oil composition. In particular they were interested in the ratio between the saturated and unsaturated fatty acids as well as in the relative concentration of linoleic acid. Fig. 3.1 reports the 13C high-resolution spectrum reported for corn oil. Peaks c and d are due to olefinic carbons with the linoleic acid that contributes to both the peaks and the oleic acid that contributes to peak c only. By measuring the intensity of these peaks it is possible to determine the ratio between the content of these two acids. The authors compared 13C high-resolution spectra registered for various different seed oils and concluded that high-resolution 13C NMR spectroscopy is a suitable method for determining the oil composition in single seeds (Rutar et al., 1977). High resolution 13C NMR spectroscopy is also able to provide valuable information about the acyl distribution and acyl positional distribution (1,3-acyl and 2-acyl) of TAG of different vegetable oils (Ng, 1985; Wollenberg, 1990). 13C NMR has been successfully used for olive oil cultivar classification (Brescia et al., 2003) and, in combination with multivariate analysis, has been successfully applied to differentiate olive oils by cultivar and geographical origin (Shaw et al., 1997). However, all the information collected from 1H-and 13C-NMR on major and minor compounds needs to be analysed with powerful mathematical tools due to the high complexity of NMR spectral data. On this basis Garcìa-Gonzàlez et al., designed an artificial neural network based on 1H and 13C-NMR data to detect the adulteration of olive oils with hazelnut oils at low percentages (Garcìa-Gonzàlez et al., 2004). Thin-layer chromatography (TLC) Thin–layer chromatography (TLC) is used for the preliminary isolation of lipid classes, principally for the separation of sterols from other non-saponifiables and it represents a major step in all official methods. It is also used to separate mono–, di– and triacylglycerols for fatty acid analysis (Sowa and Subbaiah, 2004). Gas-Chromatography (GC) and GC-MS (Mass Spectrometry) Gas chromatography with flame ionisation detection (GC-FID) has long been applied as a major technique for the determination of the major markers of oil composition, the fatty acids and sterols. Fatty acids are the major components of edible fats and oils and methods for their analysis are well established. They are found mainly as esters of glycerol as TAG or di– and monoacylglycerols, esters of sterols, and also as free acids. GC-FID methods have many advantages for the determination of fatty acids. Quantitative results can be obtained with an internal standard and without analytical standards for each

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acid. The time required for a chromatographic run is comparatively lengthy but sample preparation is minimal and brief, a large number of acids and isomers can be separated and quantified, and the methods have been very well validated and tested and have become accepted as standard procedures by official bodies.

Figure 3.1. 13C NMR high-resolution spectrum of corn oil (Rutar et al., 1977) [copyright granted].

For satisfactory GC performance, the fatty acids must be removed from the glycerol and converted to volatile derivatives such as fatty acid methyl esters (FAME). Several esterification methods, based on acid or base catalysis are available. They have been reviewed by Christie (1993), Kramer et al., (1997), and Rosenfeld (2002). The most common acid reagents are methanol solutions of sulphuric acid, hydrogen chloride, or boron trifluoride. Sodium methoxide is the preferred reagent for the basic process. Methanol/boron trifluoride is convenient to use and gives high yields of FAME (Craske, 1993), but adverse effects have been reported (Christie, 1993). Official procedures, such as Method 969.33 of the Association of Official Analytical Chemists (AOAC, 2006) and Method Ce 2–66 of the American Oil Chemists’ Society (AOCS, 2006) use a two-step process of transesterification with methanolic KOH followed by methylation with methanolic boron trifluoride. Nowadays, GC methods with high-quality capillary columns allow sensitive and reproducible fatty acid (FA) analysis. Statistical treatments applied to FAME data allow the differentiation of oils (Ollivier et al., 2006) and the study of the effect of different variables, such as cultivar, agro-climatic conditions, harvesting year, fruit-maturation stage, etc. (D´Imperio et al., 2006; Mannina et al., 2001; Matos et al., 2007) on the fatty acid profile of olive oil. The detection of the adulteration of olive oil samples has mainly focused on the comparison of their fatty acid composition (Gamazo-Vazquez et al., 2003; Christopoulou et al., 2004) and the chemometric analysis of their fatty acid content (Dourtoglou et al., 2003). The fatty acid composition of corn oil and other commercial vegetable oils (sesame, perilla, soybean, canola, rapeseed, olive and coconut oils) was studied by GC-FID and the data obtained were submitted to principal component analysis (PCA) and discriminant analysis (DA). PCA was used to characterize or classify the eight different oils that were clustered in distinct groups, while DA was employed to assign unknown samples into one of two groups. This approach could be very useful for detection of adulteration and quality control, (Lee et al., 1998). The fatty acid in the sn–2 position of triacylglycerols is often characteristic of individual oils and its identification is therefore of interest. The acid at position 2 can be identified by enzymatic cleavage with pancreatic lipase, followed by thin-layer chromatography (TLC) or

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high performance liquid chromatography (HPLC) isolation and GC-FID. This method has been adopted as an AOCS official procedure (AOCS 2006). Andrikopoulos (2002) reviewed the methods used for identification and quantification of triglyceride species in vegetable oils. GC offers high efficiency and high speed for the analysis of complex mixtures of acylglycerols with a broad range of relative molecular masses. The TAG are separated by carbon number on column with a phenylmethylsilicone stationary phases; each carbon number peak is split up giving a fine structure governed by the number of double bonds in order of increasing retention time (Geeraert, 1987). Chromatographic techniques for the TG analysis have been compared (Andrikopoulos et al., 2001; Carelli and Cert, 1993). The distribution of chromatographic peaks obtained by GC, together with the one obtained by HPLC-IR technique, permits, in some instances, the determination of individual TGs (Carelli and Cert, 1993). Moreover, GC allows the detection of TGs with very low or high carbon numbers without the interference with other TGs whose degree of unsaturation is different (Cert, 1995). GC has proved to be particularly suitable for the determination of minor constituents, such as sterols, triterpene and aliphatic alcohols, hydrocarbons and waxes, among others. Generally, a preliminary qualitative and quantitative isolation step from the TAG matrix is required. Many reports present in the literature describe the determination of the total amount of phenolic compounds in olive oils by spectrophotometric analysis and characterization of their phenolic patterns by GC (Gallina-Toschi et al., 2005, Carrasco-Pancorvo et al., 2005). GC is also used to study flavour and aroma of olive oils. Flavour and aroma are generated by a large number of volatile constituents that are present at low concentrations. These compounds include hydrocarbons, alcohols, aldehydes, ketones, esters and acids (Aparicio et al., 1996; Morales et al., 2005). Isolation methods, all using GC-FID, include direct injection, static headspace, dynamic headspace, high vacuum distillation and on-line LC-GC (Morales and Tsimidou, 2000). Recently, a simple and relatively fast method for the simultaneous determination of volatile and semi-volatile aromatic hydrocarbons in virgin olive oil was applied, based on headspace solid-phase microextraction (HS-SPME) (Vichi et al., 2005). A comparison of static headspace, HS-SPME, headspace sorption extraction and direct thermal desorption techniques has also been carried out (Cavalli et al., 2003). Kalogeropoulos et al., reported that the phenolic and terpenic compounds were extracted from virgin olive oil with methanol and a selective ion monitoring (SIM) GC-MS method was applied for detection of 28 target polyphenolic compounds and terpenic acids (Kalogeropoulos et al., 2007a; Kalogeropoulos et al., 2007b). Phenolic compounds in Sicilian olive oils were analyzed by GC–MS and GC–MS/MS. Numerous compounds were detected and 23 of them were identified by MS/MS experiments carried out on their molecular ions (Saitta et al., 2002). HS-SPME was used, in combination with GC-MS to isolate the volatile compounds formed during fatty acids peroxidation in some vegetable oils. This method was applied to the analysis of ten different vegetable oils with various degree of peroxidation (Jeleń et al., 2000). HS-SPME sampling and GC-MS and fast GC with FID detection analysis were applied to the investigation of fresh oils and oils subjected to storage at 60°C. The volatile compound profiles obtained were compared using multivariate analysis (MVA) for rapid differentiation of various plant oils and for monitoring changes in their storage (Mildner-Szkudlarz et al., 2003). High Performance Liquid Chromatography (HPLC) and HPLC-MS High Performance Liquid Chromatography (HPLC) has been used for the fractionation of lipid classes prior to GC but has not competed with GC-FID for the end

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determination of fatty acids on account of the ease of use of GC when analysing relatively small and volatile molecules such as FAMEs. Reverse Phase Liquid Chromatography directly coupled with gas-chromatography was used to identify volatile compounds present in different edible oils. The analytical method allowed the direct injection of the oil sample without any pre-treatment. The elution was monitored by UV detection, and the fraction of interest was transferred directly to the GC. Fig. 3.2 shows the gas chromatogram obtained after a selected fraction transfer from LC into GC (indicated in the figure), obtained in the LC pre-separation, of a peanut oil (Caja et al., 2000). HPLC has found more use in the analysis of triacylglycerols, tocopherols, and phenolic compounds. A drawback with HPLC is that triacylglycerols have absorbance maxima at low wavelengths, where the most commonly used solvents absorb, thus limiting the applicability of UV detection and the use of gradient elution. Cunha et al., reported a HPLC procedure for the determination of triacylglycerol profile in vegetable oils using a light scattering detector (ELSD). Quantification was based on the internal normalization method, assuming that the detector response was the same for all compounds (Cunha et al., 2006). Aranda et al., developed a method for the determination of triglyceride in virgin olive oil by employing a HPLC system equipped with a differential refractometer detector (Aranda et al., 2004), while Carelli et al., reported a method for the determination of triacylglycerol in olive oils by using a HPLC separations coupled with a refractive index detector (Carelli et al., 1993). At present, there is a great interest in the extraction of high-value compounds from natural products and vegetable oils and the residues from the oil refining process could represent important sources of high-value raw materials for chemical and nutraceutical industries. Supercritical fluid extraction (SFE) and fractionation (SFF) of vegetable oils using CO2 as solvent represents an interesting alternative to other separation processes, since CO2 is non-toxic, non-flammable and exhibits low cost, low operating temperatures and is easily removed from the sample. Hurtado-Benavides et al., have tested the efficiency of four different packed columns in countercurrent (CC) supercritical fluid extraction processes for extraction of high-value products from olive oil, such as vitamin E and sterols. The obtained extracts were analyzed using an HPLC equipped with photodiode array detector and detection of vitamin E and sterols was performed at 296 and 205 nm, respectively (Hurtado-Benavides et al., 2004). Cichelli et al., have described the HPLC analysis of chlorophylls, pheophytins and carotenoids in virgin olive oils. Pigment detection was performed with two detector systems, a fluorescence spectrometer and an UV–Vis spectrophotometer (Cichelli et al., 2004).

Luterotti et al., reported an HPLC method for selective, precise and simple profiling of carotenoids as well as for simultaneous ultrasensitive assaying of trans-β-carotene and cis-β-carotene(s) in virgin oils (Luterotti et al., 2002). Otles and Cagindi used a method for the determination of vitamin K1 content in olive oil. Oil samples were analyzed on a HPLC system equipped with a UV–Vis detector (Otles and Cagindi, 2007). An analytical method based on HPLC, with a specific injection system designed for the simultaneous concentration and separation of peanut phospholipids, was developed by Singleton and Stikeleather (1995a). Fig. 3.3 shows the HPLC chromatogram of phospholipids of peanuts oil obtained using this analytical method. The method was also used to analyse phospholipids of peanuts subjected to different post-harvest conditions. Fig. 3.4 shows the HPLC chromatograms of phospholipids of undamaged peanuts (Panel A), peanuts prematurely harvested (Panel B), and those cured at a high temperature (Panel C), or exposed to freezing temperatures (Panel D) (Singleton and Stikeleather, 1995b).

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Figure 3.2. LC preseparation obtained from the direct analysis of a peanut oil and GC separation resulting from on-line LC-GC transfer of the indicated fraction. (Caja et al., 2000) [with kind permission of Springer Science and Business Media]. A literature survey revealed that in the last years most investigators have preferred HPLC-MS techniques as the methods of choice for the analysis of different fat and oil systems (Careri et al., 2002). HPLC-MS procedures for the separation and identification of molecular species of triacylglycerols have been reviewed by Christie (1987), Nikolova–Damyanova (1997), and Nikolova–Damyanova and Momchilova (2001). Methods based on MS, particularly for the analysis of triacylglycerols, have been described by Byrdwell (2005), by Laakso (2002), Mottram (2005), and Rezanka and Sigler (2007). Interest in HPLC-MS for triacylglycerol analysis has grown considerably since the introduction of atmospheric pressure chemical ionization technique (APCI) which provides relatively simple mass spectra and some identification of positional isomers (Jakab et al., 2002a; 2002b). As an example, Fig. 3.5 shows a typical HPLC-APCI-MS profile of TAGs entracte from peanut oil sample (Jakab et al., 2002a). Rios et al., reported a qualitative method for the evaluation of phenolic compounds in virgin olive oil. The analyses of phenolic extracts were performed by a HPLC system coupled on-line with a MAT95’s magnetic sector mass spectrometer equipped with an APCI ionization interface. The APCI mass spectra were obtained in the positive-ion mode (Rios et al., 2005). Murkovic et al., described a method for the identification and quantitation of squalene and polyphenols in olive oil by HPLC-MS. Squalene and polyphenols were identified by comparing the HPLC retention time with an authentic substance and comparing the mass spectra of selected samples. For quantification, a standard addition method was used. For mass selective detection, the positively charged ions were analysed (Murkovic et al., 2004).

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Figure 3.3. Phospholipid profile of undamaged peanut oil, NL, neutral lipids; PA, phosphatidic acid; PG, phosphatidylglycerol; PE, phosphatidylethanolamine; PI, phosphatidylinositol; PC, phosphatidylcholine (Singleton and Stikeleather, 1995a) [with kind permission of Springer Science and Business Media].

3.2.2 New Trends and recent developments in oil analysis Edible oil characterisation is a very important task, which has been undertaken by different analytical methods. In this regard, mass spectrometric methodologies have recently been applied extensively to the analysis of vegetable oils, especially, electrospray ionization (ESI), which is a soft and wide-ranging ionization technique that is best applied to polar molecules, without the need of chemical derivatization or extraction from polar solutions. In this context, Catharino et al., (2005) have applied direct infusion ESI-MS in both negative and positive ion modes for vegetable oil classification. Fig. 3.6 presents the ESI-MS spectra, in the positive ion mode, of the methanol/water extracts of six (olive, soybean, corn, canola, sunflower and cottonseed) vegetable oils. In the range between 30-800 m/z, each oil gives rise to numerous diagnostic ions, which demonstrate that every ESI spectrum represents a fingerprint of that oil. This study also reports that the refined oils can also be differentiated using ESI-MS spectra obtained in the negative ion mode (ESI(-) MS). In particular, in the ESI(-) MS spectrum of olive oil, it can be noted that the water/methanol extraction permits the simultaneous observation of the phenolic acids, which are the prime importance for the biological antioxidant effect that olive oil is particularly famous for (Catharino et al., 2005). Kurata et al., (2005) have recently described a new, faster, easier method that enables the rapid discrimination of a number of fat and oil samples by ESI-MS over a short period of time. In particular, this study has demonstrated that 38 of the 42 types of saponified vegetable and animal oils analyzed showed that the fatty acid composition detected by ESI-MS analysis corresponded to those by GC-MS analysis. Comparisons of a base peak in the ESIMS spectra

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classified 28 types of the saponified vegetable oils into eight groups. The data reported in this study shows that the advantages of ESI-MS analysis compared with GC-MS. Oils can be analyzed within one minute by ESI-MS. Furthermore, the sample preparation for the ESI-MS analysis of fatty acids was reduced to two steps only: saponification and extraction, in contrast to the GC-MS analysis which requires three steps for sample preparation (saponification, extraction and esterification or transesterification) which is much faster than GC. However, GC-MS and GC-FID can discriminate between the isomers of unsaturated fatty acids, while ESI-MS analysis groups these compounds without discrimination of the isomers.

Figure 3.4. Effect of different postharvest treatments on the phospholipid profile of peanuts: (A) undamaged, (B) immature, (C) high-temperature cured, and (D) freeze-damaged; NL, neutral lipids; PA, phosphatidic acid; PG, phosphatidylglycerol; PE, phosphatidylethanolamine; PI, phosphatidylinositol; PC, phosphatidylcholine. (Singleton and Stikeleather, 1995b) [with kind permission of Springer Science and Business Media].

Figure 3.5. HPLC TAGs profile of a peanut oil sample. (Jakab et al., 2002a) [copyright granted].

Edible oils are complex mixtures containing a wide range of compounds. In particular, TAGs are considered to be good fingerprints for the detection of adulteration (Aparicio and Aparicio-Ruiz, 2000). HPLC-MS is the method of choice for the analysis of the TAG composition of oil samples and, due to the relatively simple mass spectra and the possibility of identifying the positional isomers, APCI is the most often used ionization method for the analysis of TAGs by HPLC-MS. The identification of TAG is based on the mass of the protonated molecular ion [M+H]+ and the diacylglycerol (DAG) [M-RCO2]+ fragment ions, while the positional isomers are identified from the relative intensities of the DAG fragments (Jakab et al., 2002a; Jakab et al., 2002b). In recent times it has been possible to characterize TAG in vegetable oils by silver-ion packed column supercritical fluid chromatography (SI-pSFC) with mass spectrometric detection using APCI and Coordination Ion Spray (CIS) with silver ions as ionization modes (Sandra et al., 2002). The SI-pSFC analysis of the triglycerides from a soybean oil sample is shown in Fig. 3.7A for UV detection at 210 nm and in Fig. 3.7B for the APCI-MS detection. For the MS analysis the sample is 100 times less concentrated, i.e. a 10% solution for UV and a 0.1% solution for MS, both in CHCl3. Solutes characterized by the same carbon number elute according to the number of unsaturated fatty acids. Fig. 3.8 shows some spectra taken

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from the analysis of soybean oil for TAGs present at low (POP at 33.2 min, SOO at 43.9 min), medium (SLO at 55.0 min) and high (OLL at 66.7 min) concentration. All spectra show a clear [M+H]+ ion. Spectra of highly unsaturated TAGs, e. g. OLL, have the [M+H]+ ion as base peak and fragment ions are not intense, while highly saturated TAGs show very intense fragment ions, but [M+H]+ ions of low intensity, e. g. POP. These disappear for fully saturated TAGs. In order to overcome this disadvantage, in this study Ag+-CIS-ESI-MS was evaluated for TAG analysis. For comparison, the four TAGs shown in Fig. 3.8, but extracted from sunflower oil, were analysed by Ag+-CIS-ESI-MS. The spectra are reported in Fig. 3.9. The main difference with APCI-MS is that, in CIS–ESI, the [M+Ag]+ ion shows the highest intensity irrespective of the degree of unsaturation. Elucidation of positional isomers was possible with CIS-ESI-MS. In conclusion, it is possible to say that APCI-MS and CIS-ESI-MS are complementary to each other because of the different separation mechanisms.

Figure 3.6. ESI (+) MS analysis of methanol/water extracts of six vegetable oils (Catharino et al., 2005) [copyright granted] Adulteration of vegetable oils is of concern for both commercial and health reasons. In this context, Wu et al., (2004) have demonstrated that ultrahigh mass resolving power (m/Δm50%> 350.000) and mass accuracy (<1 ppm) of Electrospray Ionization Fourier Transform Ion Cyclotron Resonance Mass Spectrometry (ESI FT-ICR MS) allows the detection of oil adulteration in distinguishing different oils by a fast and definite assignment of components having thousand of different elemental compositions within any of each of several chemical families present in the oils (fatty acids, DAG and TAG, tocopherols) (Wu et al., 2004). Fig. 3.10 shows the Negative–Ion ESI FT-ICR mass spectra of the three different oils studied: canola oil (top), olive oil (middle) and soybean oil (bottom). Fig. 3.11 presents the mass spectral segment from Fig. 3.10 containing four fatty acids (FAs) having the same number of carbons (18) but different degree of saturation. From this figure it is possible to see that it is possible to distinguish the three oils by the relative abundance (intensity patterns) of the FAs, which are diagnostic for each of the three oils. Wu et al., have also demonstrated that the tocopherol composition provides another way to distinguish different vegetable oils by

olive

soybean

corn

canola

sunflower

cottonseed

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using the relative abundance of β,γ-tocopherol in the three vegetable oils (Wu et al., 2004). Additionally it was demonstrated that the three oils can also be readily distinguished by the relative abundance patterns of their non-acidic compounds (such as TAGs and DAGs) analysed by Positive–Ion ESI FT-ICR MS (Wu et al., 2004).

Figure 3.7. SI-pSFC separation of soybean oil with UV at 210 nm (A) and APCI-MS (B). (Sandra et al., 2002) [copyright granted].

This study established that it is possible to detect adulteration of one vegetable (e.g. expensive) oil by addition of another oil (e.g. inexpensive) by both Negative and Positive–Ion ESI FT-ICR MS. To test that capability, olive oil was mixed with soybean oil in the weight ratios of 1:1; 2:1; 3:1; 4:1 and 5:1 and, Fig. 3.12 reports the fatty acid distribution for mixtures of olive oil and soybean oil. Proceeding from left to right in Fig. 3.12, it is clear that the presence of soybean oil may be determined from the fact that C18:3 FA (m/z 277), absent in pure olive oil, is immediately apparent even at a 5:1 olive soybean ratio, with increasing relative abundance on increasing proportion of soybean adulterant. Similar effects are seen for the DAGs and TAGs relative abundance, showing that the ratio C54:6/C54:5 is a sensitive indicator of the presence and relative proportion of soybean adulterant in olive oil, C54:6 being the most abundant TAG in soybean oil (Wu et al., 2004). An alternative to both APCI and ESI sources is the recently introduced technique of atmospheric pressure photoionization (APPI) (Syage et al., 2001). The ions present in a APPI MS spectrum are [M+H+], [M]+., [RiCO]+ and [RiCO-H2O]+, which, in particular, are absent in ESI ionization and, represent valuable complementary information. The APPI source coupled to quadrupole time-of-flight mass spectrometry (QqTOFMS) has been applied (Gomez-Ariza et al., 2006) to easily discriminate between different edible oils. This methodology has also been revealed an excellent approach for studies of olive oil adulteration (Gomez-Ariza et al., 2006). In recent years multistage tandem mass spectrometry (MSn) has given a great impulse to the analysis of lipids. Kalo et al (2006) have applied normal phase liquid chromatography-positive electrospray tandem mass spectrometry to a comprehensive analysis of low erucic acid rapeseed oil lipids (Kalo et al., 2006).

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Figure 3.8. Some representative APCI-MS spectra of the soybean oil analysis. (Sandra et al., 2002) [copyright granted].

Figure 3.9. Some representative Ag+-CIS-ESI-MS spectra of the sunflower oil analysis (Sandra et al., 2002) [copyright granted].

Figure 3.10. Broadband electrospray ionization FT-ICR negative-ion mass spectra of the acidic components of canola oil (top), olive oil (middle) and soybean oil (bottom). In each spectrum, peak heights are scaled relative to the highest-magnitude peak (Wu et al., 2004) [copyright granted].

Figure 3.11. Mass scale-expanded segment from Figure X7 showing relative abundances of various C18 fatty acids in three vegetable oils. The compositional differences readily distinguish soybean oil from canola or olive oils. (Wu et al., 2004) [copyright granted].

Fig. 3.13 shows a multistage tandem experiment related to the TAG molecule C54:3.

In particular, panel A of Fig. 3.13 reports the MS1 spectrum, in which, under the experimental conditions used, TAGs form only ammonium adducts without fragmentation. In MS2

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experiment, shown in panel B, [(M+NH4)–NH3]+ ions and abundant DAG ions [(M+NH4)–NH3–FA]+ with varying intensity are found. In MS3 spectrum, presented in panel C, the product ion [(M+NH4)–NH3]+, at m/z 886 Da, yielded [(M+NH4)–NH3–H2O]+ [(M+NH4)–NH3-FA]+ ions with medium and high abundance, respectively. Finally, panel D reports the MS3 experiment related to the product ion [(M+NH4)–NH3-FA]+ (604 Da),which showed abundant [Acyl]+, [Acyl+74]+ and medium abundant [(M+NH4)–NH3-FA-74]+ ions. These results clarify the stepwise fragmentation mechanism related to the multistage tandem mass spectra. In recent times, Matrix-Assisted Laser Desorption Ionization Time of Flight Mass Spectrometry (MALDI-TOF MS) have been shown to provide a rapid oil differentiation and classification by yielding detailed information about the molecular weight and fatty acid composition of TAGs in oils (Asbury et al., 1999; Ayorinde et al., 1999; Ayorinde, Eribo et al., 1999; Ayorinde, Keith et al., 1999; Ayorinde et al., 2000; Hlongwane et al., 2001; Robins et al.; 2003; Al-Saad et al., 2003; Lay et al., 2006). In fact, in all the paper reported in literature, it is evident that the resolution of the TOF experiment in the TAG region is sufficiently good so that the sodium adduct TAG ions and their main isotopic peaks can easily be differentiated.

The wide applicability of MALDI MS is due to the fact that it offers some advantages over other analytical methods. It is characterized by a fast and easy sample preparation and by the absence of the need for analyte purification, chemical modification or derivatization. However, the identification of the positional TAG isomers is only possible with APCI-MS (Christie, 1987; Byrdwell and Emken, 1995).

Figure 3.12. Fatty acid distributions for mixtures of olive oil and soybean oil. The presence and extent of soybean oil as an adulterant may be determined from these patterns (see text) (Wu et al., 2004) [copyright granted].

Figure 3.13. Mass spectra of trioleoylglycerol recorded in the flow injection mode. (A) Full scan mass spectrum of ammonium adduct. (B) Tandem mass spectrum (MS2) of ammonium adduct. (C) Mass spectrum of ion with m/z 886 produced in collision induced decay of ammonium adduct = MS3 (903 →886). (D) Mass spectrum of ion with m/z 604 produced in collision induced decay of ammonium adduct = MS3 (903 →604). (Kalo et al., 2006) [copyright granted].

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MALDI measurements are typically affected by intrinsic problems, i.e. complications arising from interference of matrix peaks in the low-mass region, and absence of shot-to-shot and sample-to-sample reproducibility as a result of lack of homogeneity in the matrix-sample crystals. Calvano et al., (Calvano et al., 2005) have demonstrated that these problems can be avoided by using laser desorption ionization time of flight mass spectrometry (LDI-TOF MS). This new approach has a simpler sample preparation, with no need to use a matrix with consequent absence of matrix interference peaks in the spectra, and potential improvements in shot-to-shot reproducibility due to the absence of the crystallization step resulting in a more homogenously deposited sample. The procedure was successfully applied to the determination of TAGs in whole oils, yielding TAG fingerprints very quickly (Calvano et al., 2005). The same authors have also shown that the present method can be successfully used to obtain a fast and easy identification of adulteration of an extra-virgin olive oil sample with sunflower oil. In fact, comparing the LDI-TOF mass spectra of a pure extra-virgin olive oil sample and sunflower oil sample, it is possible to notice the presence of same diagnostic ions, which can help in the detection of a percentage of up to 10% of sunflower oil in a sample of extra-virgin olive oil. Very recently, LDI-TOF MS has been employed for the first time to characterize olive and sunflower oils after thermally assisted oxidation in order to obtain information about their oxidation level (Calvano et al., 2007). Fig. 3.14 reports the expanded view of the high mass region of LDI-TOF mass spectra of a commercial extra-virgin olive oil sample (a), and the oil incubated at 200°C for 24 (b) and 48h (c). In spectra b and c, it is possible to note a progressive decrease in the TAG peak intensity and the simultaneous appearance of degradation/oxidation products. Taking for instance the peak at m/z 907.77, ions presenting a mass increase of 16, 32, 48 and 64 Da are present. This means that up to four oxygen atoms have been introduced into the original TAG, providing information about the extend of oxidation. The results obtained in this study have demonstrated the LDI-TOF MS ability to obtain TAG and oxidized TAG fingerprints. Recently, Apetrei et al., (2005) have set up a novel method able to discriminate oils of different origins and qualities using modified carbon paste electrodes (CPE), in which vegetable oils are employed as electroactive binder material. For this purpose, six oils of different qualities have been mixed with carbon powder to form the relative CPEs, and the electrochemical properties of these CPEs investigated by cyclic voltammetry (CV) and square wave voltammetry (SWV). The data obtained in this study show that all the plant oils analysed could be discriminated, taking advantage of the fact that the electrochemical signals obtained in these experiments depend on the nature and the composition of the antioxidant compounds found in the different oils. Furthermore, as the voltammograms are strongly influenced by the nature of the ions characterising the electrolytic solutions where the electrodes are immersed, the set of the responses of a particular oil towards a range of electrolytes could be used as a fingerprint of such oil. Recently, Tan and Man (1999) have developed a simple method based on Differential Scanning Calorimetry (DSC) for monitoring the oxidation of heated oils. In this study it was possible to observe that parameters such as total polar compounds (TPC), Free fatty acids (FFA), anisidine value (AnV), Peroxide value (PV), Totox value (TxV), increase with heating time, while the iodine value (IV) and ratio of linoleic acid/palmitic acid (C18:2/C16:0), DSC peak temperature and enthalpy decrease with increase in the time of heating. Further experiments on the crystallisation behaviour of polar and nonpolar fractions of heated oils gave supportive evidence to these experiments, allowing the conclusion that the DSC method can be a valuable alternative to existing approaches for assessing the quality of over-heated and frying oil samples (Tan and Man, 1999).

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Figure 3.14. Expanded view of the high mass region of LDI-TOF mass spectra of a commercial (a) and incubated at 200°C for 24 (b) and 48h (c) extra-virgin olive oil sample (Calvano et al., 2007) [with kind permission of Springer Science and Business Media].

Recently, innovative application of spectroscopic techniques to the analysis of vegetable oils have been reported in literature. Beaten et al., have been able to classify different edible oils using FT-Raman Spectroscopy (Beaten et al., 1998). Moya Moreno et al., (1999b), have employed Fourier Transform Infrared (FTIR) Spectroscopy to monitor thermal degradation in heated oils, studying the variation in lipids composition of some edible oils (sunflower, corn olive and seed oils) subjected to intense heat (80°C-300°C, 20-40 min). The method allowed the determination of the percentage of unsaturation and trans isomers at different temperatures and heating times. The results achieved by using FTIR demonstrate that in over-heated and frying oil samples there is a decrease in unsaturation and an increase in trans isomers and that these changes start after 150°C. At higher temperatures, of course, a more considerable variation is apparent. The peroxide value acquired for each sample by the iodometric method reveals that these modifications in composition coincide with the decomposition of hydroperoxides. Sikorska et al., (2005) have tested total luminescence spectroscopy (TLS) and synchronous scanning fluorescence spectroscopy regarding their ability to characterize and differentiate edible oils. As an example, Fig. 3.15 shows the contour map of total luminescence of a corn oil sample diluted in n-hexane. TLS, however, is very time consuming, because the acquisition of contour maps of sufficient resolution requires a large number of emission scans for each sample. Thus, for analytical purposes, it is better to use synchronous scanning fluorescence spectroscopy. In this method both excitation and emission characteristics are

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included into the spectrum by simultaneous scanning excitation and emission wavelength at a constant difference between them. The synchronous scan fluorescence spectrum of a corn oil sample diluted in n-hexane is reported in Fig. 3.16. The synchronous scan fluorescence spectra of all edible oil samples have a major band with a maximum at around 300 nm, but the spectral profiles of these spectra vary significantly between different oils samples. The spectral pattern, in fact, depends on excitation and emission profiles of fluorescent components, and thus is unique for each oil. For this reason, Sikorska et al., have proved that the synchronous fluorescence method can classify different classes of edible oils using a single scan. Furthermore, the method here discussed, analysing complex mixtures without separation, is extremely useful from a practical point of view. Fluorescence provides high sensitivity, simplicity and selectivity and may serve as a complement to other spectroscopic techniques used in edible oil analysis. One- or two-dimensional high-resolution nuclear magnetic resonance spectroscopic techniques and especially 13C NMR have been successfully employed in olive oil analysis. Anyway, quantitative 13C NMR spectroscopy at natural abundance, which requires spectra with a high signal-to-noise (S/N) ratio, faces experimental limitations related to a low natural abundance (1.1%) and to the inherent low sensitivity due to its small gyromagnetic ratio. Moreover, with this technique the time of signal-averaging is dramatically long. Recently, these experimental limitations, have been partly overcome in olive oil analysis by using the distortionless enhancement by polarization transfer (DEPT) pulse sequence (Bendal et al., 1982). DEPT pulse sequence, enhancing the sensitivity of carbon-13 nuclei, improves the signal-to-noise of 13C NMR spectra validating the quantitative measurements of the intensities of 13C resonance of triglycerides fatty acids of the whole olive oil spectrum.

Figure 3.15. Contour map of total luminescence of corn oil diluted in n-hexane. (Sikorska et al., 2005) [copyright granted].

Figure 3.16. Synchronous scan fluorescence spectrum of a corn oil sample diluted in n-hexane (Sikorska et al., 2005) [copyright granted].

13C NMR DEPT has been used a quantitative methodology for determining the adulteration of olive oil with soybean oil (Vlahov, 1997). Furthermore, it was successfully employed to check the difference/similarity of olive oil samples according to cultivar and geographical origin (Vlahov et al., 1999; Vlahov, 2005), in particular from areas labelled with the “Denomination of Protected Origin” (DOP) (Vlahov et al., 2001). Finally, considering the high potential of 13C NMR DEPT methodology, Vhahov (Vlahov, 2006) has lately demonstrated that the 13C-NMR DEPT, when applied to the measurement of spectra of olive oil samples of different grades, produced rigorously quantitative profiles of the TAG fraction of the oils which enables the discrimination of olive oils of the four grades, extra virgin olive oils, olive oils, olive pomace oils and lampante olive oils. Regarding to the application of proton nuclear magnetic resonance spectroscopy (1H NMR) to olive oil analysis, difficulties arise in relation to the information obtained from spectra of multicomponent mixture such as olive oil. In order to avoid these problems, in recent years the on-line coupling of liquid chromatography with NMR has been used in

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several instances (Lindon et al., 1996; Spraul, 2001). In particular Christophoridou et al (Christophoridou et al., 2005) have demonstrated that coupling HPLC with postcolumn solid-phase extraction to NMR (LC-SPE-NMR) the sensitivity of LC-NMR methodology improves and the S/N ratio is enhanced by up to a factor of 4. This study demonstrates that using LC-SPE-NMR, it is possible to unambiguously identify a large number of phenolic compounds and, more importantly, to detect new phenolic compounds not reported previously. Recently, phosphorus-31 NMR spectroscopy (31P NMR) has been employed in olive oil analysis (Christophoridou and Dais, 2006) supplementing 1H and 13C NMR spectroscopy, especially in cases where strong signal overlap and dynamic range problems in 1H NMR spectra and/or long relaxation times of 13C nuclei render the analysis of olive oil a difficult task (Fronimaki et al., 2002). Methods based on the derivatization of the labile hydrogen of functional groups, such as hydroxyl and carboxyl groups, with the phosphorus reagent 2-chloro-4,4,5,5-tetramethyldioxaphospholane, and the use of the 31P chemical shifts to identify the phosphorylated compounds (Spyros and Dais, 2000). An important advantage of 31P NMR method is the introduction of an internal standard of known amount (usually cyclohexanol) in the reaction mixture, which allows the determination of the absolute concentration of the phosphorylated product. Although the 31P NMR method is less simple and requires more time than conventional analytical techniques (GC and HPLC), it seems good for the quality control and authentication of extra-virgin olive oil (Spyros and Dais, 2007). A single run can rapidly detect all of the phosphorylated minor compounds present in the olive oil sample and provides signals, the intensities of which reflect the number of magnetically equivalent phosphorus nuclei. The assignment of the chemical shifts of the various functional groups is well documented, making this technique very appropriate for the screening of a large number of samples, and a valuable tool for the quality control and authentication of extra-virgin olive oil. Finally, in a very recent study (Dais et al., 2007) the amount of certain constituents (fatty acids, iodine value, diacylglycerols, phenolic compounds and free acidity) were determined by 1H NMR and 31P NMR spectroscopy and the data compared with that obtained by official and/or well-recognized methods of analysis showed very good agreement almost in all cases.

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3.3. Concluding Remarks • Extra-virgin olive oil can be considered the “gold standard” of vegetable oils, thanks to the high level of MUFA and to the presence of high levels of natural antioxidants components. • Extra-virgin olive oil is expensive. Therefore, MAC-Oils Project has focused the attention on other vegetable oils as a valid and cheaper alternative to extra-virgin olive oil. • Among them, Rapeseed oil, being particularly rich in MUFA, can be considered of special interest, in the same way as some vegetable oils obtained from new cultivars of Peanut, Sunflower and Soybean, characterized by a high oleic acid content. • During MAC-Oils project, different analytical methodologies used for oil analysis have been compared. In particular, beside classical techniques, today there are some innovative analytical tools, most of all MS and NMR methodologies, which have been shown to be very rapid and accurate to provide data regarding vegetable oil composition, genuineness and traceability, as well as to detect the presence of possible adulterations.

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CHAPTER 4

Sensory properties and consumers acceptability of oils

Authors Contributors

PELLICANO, M.P.1 MATTHÄUS, B.3

CAMMAROTA, G.1 LACOSTE, F.4

GRAZIANI, M.P.1 ZARROUK, M.5

APARICIO, R.2 BEN TEMINE, S.5

PINELI, L. de L. de O.6

ABD EL HADI, F.7

VAN RUTH, S.8

1 Istituto di Scienze dell’Alimentazione, National Research Council, Avellino (Italy) 2 Instituto de la Grasa, Consejo Superior de Investigaciones Científicas, Sevilla (Spain) 3 Federal Research Centre for Nutrition and Food - Institute for Lipid Research, Münster (Germany) 4 Institut des corps gras - Centre technique industriel, Pessac (France) 5 Centre de Biotechnologie de Borj-Cedria, Hammam Lif (Tunisia) 6 União Brasiliense de Educação e Cultura – Universidade Católica de Brasília, Brasília (Brazil) 7 Israeli Olive Oil Board (Israel) 8 RIKILT – Institute of Food Safety, Wageningen UR (the Netherlands)

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Abstract Chapter 4: “Sensory properties and consumers acceptability of oils” of MAC-Oils project includes four topics: introduction; sensory characterization of oils; relationship between oil compounds and sensory attributes; consumers acceptability of edible oils. In the first topic the tests commonly used by trained panels for the definition of sensory characteristics of foods as well as consumer tests to define theirs acceptability and preference are reported. Furthermore, the European Regulation that defines the chemical and sensory parameters that classify the different olive oils is reported. In this section the specific sensory methodology used to determine the sensory properties of oils as well as a list of positive and negative tasting attributes and theirs meaning are also briefly considered. The second topic includes the most important trained panel studies on the organoleptic properties of virgin olive oil in relation to agronomic and technological aspects as well as the studies on the sensory properties of seed oils. Comparison studies between virgin olive oil and seed oils have shown that the seed refined oils generally have a bland odour and flavour, in contrast to olive oils, which are rich in both. In the third topic the instrumental analysis of oils are reported in order to understand the relationship between the physicochemical composition of oils and their perceived flavour. The volatile compounds responsible for virgin olive oil flavour are about 180. The sensation of smell is not fully explained by the single substances, but it is possible to associate them, for example green odour with hexanal. The fourth topic focuses on consumer behaviour, i.e., the main factors that affect food choice, in particular intrinsic and extrinsic as well as social, environmental and communication factors. Fruity, bitter, pungency as well as brand name, typicality and geographic origin are important factors that influence acceptability and preference of oils.

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4.1. Sensory Analysis The evaluation of the sensory properties and determination of the importance of these properties to consumer product acceptance represent a major accomplishment in sensory analysis. Most of the sensory evaluation carried out by trained panel involves the 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. They can be distinguished on the basis of all or some of their sensory properties. 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 vegetale oils during their processing, being used to control the 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; Stone and Sidel, 1993; Lawless and Heymann, 1998; Meilgaard et al., 1999). Descriptive analysis, the other main area of sensory evaluation, describes and measures the sensory attributes of food precisely. 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 pre-set 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. Others 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/money being needed. This can therefore save investing in a product that could not liked due to a sensory qualities deficiency. The most frequently used method to measure acceptability and preference is the 9-point hedonic scale, developed since 1955 (Jones et al., 1955). The subjects that take part in the sensory testing are not trained, they should be relatively naive to this kind of task.

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4.1.1 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); 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 lead 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 lead 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 an oil, both positive as well as negative. Appearance as color was not selected as a quality parameter of virgin olive oil, a specific dark-colored glass is used. The official evaluation sheet used within the European Union to establish the sensory profile of virgin olive oil is shown below (Fig. 4.1). The values, expressed as centimetres, are statistically processed to calculate the median of each positive and negative characteristic.

Figure 4.1. Sensory profile sheet of virgin olive oil (See note below).

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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. Table 4.1. Analytical and sensory properties of olive oils (see note below)

In Table 4.1, 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. The following definitions are based on the classification of olive oil and olive-pomace oil reported in the Table above. 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 = 0 and the median of fruity 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 (See note below) and the median of fruity 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 2.5 (See note below) or if the median defects is less than or equal to 2,5 (See note below) and the median of fruity is 0. Such olive oil is intended for refining purpose. 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

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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”. The others are oils which have been chemically obtained from the pomace of olives. International legislation is different from European legislation and includes one more class as virgin olive oil as reported in the latest Trade Standard on Olive Oil by the IOOC (COI/T.15/NC no. 2/Rev. 6 of 5 June 1997): Ordinary virgin olive oil: is the virgin olive oil which has a free acidity, expressed as oleic acid, of not more than 3.3 grams per 100 grams and the sensory characteristics with median defects greater than 2.5 but less than or equal to 6.0 and fruity attribute greater than 0, or where median defects is greater than 0 but less than or equal to 6.0 and the median of fruity attribute equal to 0. Olive oil tasting attributes Olive oil as judged by experts shows a multitude of either positive or negative characteristics. 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 retronasally. 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. 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. Dreggish: 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.

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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. 4.1.2 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). 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 the 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, branches, earth. 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.

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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 including sedimentation, these 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 characteristics of the oil, the conservation conditions must be controlled. The main factors affecting the conservation of oil are: a) the temperature (12-15°C), b) light (oil should be stored in the dark otherwise the photo-oxidisation 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 still environment. 4.1.3 Sensory analysis of seed oils The sensory assessment of seed oils requires the setting-up of a motivated team used to distinguishing the different typical flavors and measuring their intensity. Assessors should not have any negative feelings towards the products they are testing. This implies a necessary training and selection of the assessors. The training will allow assessors to be obtained who are used to the products and will introduce them to developing a more accurate perception of the specific odors and flavors they are asked to detect. The selection and training of people involved in sensory analysis are standardized on an international level by the International Organization for Standardization (ISO). Identification of descriptors for establishing a sensory profile for refined oils The method was developed following the standardized method: ISO 11035- sensory analysis (La Coste, personal communication).

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It includes various stages. First stage: Selection of the refined oils which will enable the assessors to distinguish all the possible qualitative differences detectable in products for which the profile is drawn up for. Second stage: The assessors are asked to generate the maximum number of terms to describe all the sensations produced by these oils. Third stage: Selection of descriptors by multidimensional analysis PCA is used to either group together synonymous descriptors or eliminate descriptors which hardly contribute to showing the differences between the oils. Identification of the descriptors in order to establish a sensory profile for refined oil flavors resulted in a 6 terms descriptive list: butter, painty, fishy, rancid, fruity and grassy/beany. The intensity of each attribute is assessed on a continuous bipolar scale ranging from weak to strong (Fig.4.2).

Figure. 4.2. Profile sheet used for sensory profile of refined vegetable oils

The vocabulary necessary to explain these attributes to the assessors is the following. Fruity/seed: natural flavour of the cold oil pressed from the seed. Buttery: flavor of butter detected in oils at the beginning of their oxidation stage. Grassy/beany: flavour of slightly oxidized oil (flavour reversion). Fishy: flavor of fish detected in oxidized linolenic oils such as rapeseed or soybean oil. Painty: flavor of paint detected in oxidized linolenic oils such as rapeseed or soybean oil. Rancid: flavor detected in oxidized linoleic oils such as sunflower oil. For a correct use of this list, the panel must be trained to recognize each flavor by spiking tasteless oil with either chemical substances or other oils presenting the studied attribute. The panel is also trained to quantify the attributes on the intensity scale with the help of different dilutions of a sample presenting only one specific descriptor. A method was also standardized by the American Oil Chemists Society (AOCS) for the flavor panel evaluation of vegetable oils. Two different scoresheets are provided, one for

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oils with distinctive flavors and a second to evaluate the overall blandness of just refined vegetable oils. The quality level is scored according to a discrete scale. For example, a score of 8 for a fresh, unaged refined oil is indicative of an oil with good quality.

Figure. 4.3. Profile sheet used for sensory profile of virgin vegetable oils (except olive oil)

Identification of descriptors for establishing a sensory profile for virgin oils This research was developed in order to assess the quality of virgin sunflower oils (La Coste, personal communication). The list of attributes for virgin sunflower oils includes some of the descriptors from the virgin olive oil profile sheet, such as fusty, musty and rancid. It also includes specific attributes for virgin sunflower oils obtained with toasted seeds (Fig. 4.3). The vocabulary necessary to explain these attributes to the assessors is the following. Fruity/seed: natural flavor of the cold pressed oil from the seed. Grilled: flavor of toasted seeds. Burnt, smoked: flavor of over-toasted seeds. Grassy: flavor “grass” or “fermented grass” or flavor of slightly oxidized oil. Fusty: flavor of oils from stored seeds that have induced an anaerobic fermentation. Musty: flavor of oils from stored seeds in humid conditions fungi, yeasts. Rancid: flavor detected in oxidized linoleic oils such as sunflower oil. The European Communities, with the Commission Regulation No 640/2008 of 4 July 2008, has adopted the following modifications. In the Table 4.1, the organoleptic evaluation median defect (Md) value “2,5” is replaced by “3,5”. In addition, the profile sheet (Fig. 4.1) has been modified for the “Fruity” attribute. If a taster perceives the fruitiness to be of a green or ripe

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character, he or she must tick the corresponding box on the “new” profile sheet. 4.2. Sensory Characterization of Oils 4.2.1. Sensory properties of extra-virgin olive oils: agronomic and technological aspects Extra-virgin olive oil is a typical Mediterranean production whose typicality is strongly affected by the origin of its raw material and the manufacturing technology. In recent years, the European Union (EU) has recognised many protected extra virgin olive oils (protected designation of origin or protected geographic indication). These typical oils, which are mainly famous for their sensory properties, present a complex and specific qualitative profile including both intrinsic and extrinsic factors. A number of investigations were aimed at finding correlations that can explain the presence of positive or negative sensory notes that are perceived by tasters during the virgin olive oil tasting. According to Angerosa (2002) and Angerosa et al., (2004), cultivar, geographic region, fruit maturity, processing methods and parameters influence the volatile composition and therefore the sensory characteristics of the resulting olive oils. Studies conducted by Aparicio and Luna (2002), have shown how pedoclimatic aspects together with olive ripeness, harvest of olives, and the olive extraction system determine the chemical composition and sensory descriptors which assess the quality of virgin olive oils. Agronomic and climatic aspects Aparicio et al., (1997) have studied the sensory authentication of the most marketed virgin olive oil varieties: Arbequina, Picual, Coratina, and Koroneiki. The sensory authentication has been carried out by six panels of assessors, both potential and habitual, of different nationalities (Spanish (A), Italian (B, D), Greek (C), British (E), and Dutch (F)). Panels A-C strictly followed the EC regulation (EC, 1991) and the score for each attribute was the result of the overall gustatory-olfactory-tactile perception. Panels D-F did not follow the EC regulation (EC, 1991) but the International Standards Organization (ISO) document “General Guidance for Establishing, a Sensory Profile” (Lyon and Watson, 1994). The former varieties are picked when they are completely black while the latter varieties are still green when harvested. These sensory characterizations from the volatile compounds agree with the sensory evaluation carried out by the assessors. The Arbequina variety is characterized by high values of attribute fruity (tomato and apple sensory attributes) and low values of bitter, pungent and astringent; Koroneiki, green and slightly astringent; Picual, high values of attributes fruity (tomato and artichoke) and pungent and slightly undesirable; Coratina, high values of all sensory attributes clustered inside the attribute undesirable and those inside the attributes bitter and pungent. Other sensory attributes are sweet (odour) and green olives. Ranalli et al., (2000), examining the compositional quality and sensory properties of virgin olive oil from a new olive cultivar I-77 grown in three different geographical areas (Perugia, Campobasso and Lecce), found that the pedoclimatic aspects determine the chemical composition and sensory descriptors which assess the quality of virgin olive oils. Aguilera et al., (2005) observed that the sensorial characteristics of virgin olive oils from the main Italian cultivars, “Frantoio” and “Leccino” grown in two different locations in

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Andalusia: Mengibar (Jaén) and Cabra (Córdoba), with important differences in altitude, were influenced by the environmental conditions and showed significant differences between the oils from each cultivar and location. “Leccino” oils from Cabra and Mengibar are similar: fruity, with a soft touch of apple, without other ripening fruits, with attributes such as green, bitterness, pungency, astringency and almond and slightly sweet. This profile is common for both locations although the Mengibar oils show higher intensities of bitterness, pungency and astringency. However, in Cabra, ‘Frantoio’ oils showed two different attributes: ‘wood’ and ‘fig tree’ typical of oils with high phenolic content. The description may be considered similar for the remaining sensorial attributes of ‘Frantoio’ oils. The ‘triangle test’ was applied to evaluate differences between oils from different locations. For the second crop year, there were significant differences between the oils for both cultivars from Mengibar and Cabra (p < 0.05). Ollivier et al., (2006), examining the sensory characteristics of the five registered designations of origin (RDOS) of French virgin olive oils (‘Aix-en-Provence’, ‘Haute-Provence’, ‘Nyons’, ‘Nice’, and ‘Vallée des Baux de Provence’) over a six year harvest period, found that the evaluation of fruity, bitter and pungent oils was insufficient to describe the RDOs, so it was necessary to complete the olive description with descriptive attributes proposed by the tasters. Ripeness Sensory quantitative descriptive analysis (QDA) and triangular tests were performed to establish the influence of olive ripening degree on the resulting oil organoleptic properties of Cv. Nostrana di Brisighella. The evolution of the analytical parameters studied shows that the ripeness stage of Nostrana di Brisighella olives that yields the best oil corresponds to a Jaén index value between 2.5 and 3.5. Oils produced from olives harvested within this time frame present a superior sensory profile (Rotondi et al., 2004). Quantitative descriptive sensory analysis highlighted a clear decreasing trend of the positive olive oil descriptors as the olives ripened. None of the samples of Nostrana di Brisighella oils included in this study presented any sensorial defects. Olive fruity expressed as olfactory intensity value, bitter, pungent, green-leaf, and pleasant gustative attributes showed a statistically significant higher intensity at the first stage of olive ripening (RII) when compared to the other oils’ sensory profiles. Also, pleasant flavours, mainly ascribable to grassy, artichoke, and green tomato attributes, were significantly higher in oil obtained from olives harvested at RII. In the case of the olive fruity attribute, we observed a significant decrease in oils produced from olives harvested at RIII and RIIII, whereas at the last stage of ripeness (RIIV) the intensity of the olive fruity attribute increased again, allowing the oil to reach a panel score similar to the one recorded for the oils obtained from olives at the first stage of pigmentation. Nevertheless, it is important to emphasize that the results of the triangular test confirmed the statistical results of the sensory quantitative descriptive analysis (QDA). In fact, comparison with triangular tests oils obtained from the first and fourth olive ripening stages, 100% of the assessors correctly identified all of the samples provided during the panel tests (Rotondi et al., 2004). The effect of fruit ripeness on the sensorial characteristics of the oil has been extensively investigated for several cultivars such as Cornicabra (Salvador et al., 2001). The results indicated that the total scores generally diminished at higher ripeness index (RI), although they were always higher than 6.5, the threshold value for ‘extra virgin classification. This was probably due to the observed loss in some positive attributes, especially fruitiness (from about 3.0 at RI close to 2, to 1.8 at RI of about 5). In particular, bitterness scores decreased with RI, with a correlation coefficient higher than -0.9. The oils obtained from green olives were excessively bitter according to the panelist comments. This does not imply rejection of the oil, but if the level of bitterness is too high it could cause problems for

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consumer acceptance. A high level of bitterness is a peculiar characteristic of Conicabra virgin olive oil, but according to this study, for an optimum sensory quality of this olive oil it is recommended that the bitterness score should be lower than 4.0 – 3.5. In general, the sensory characteristics of the oil indicates that the optimal ripeness corresponds to incomplete pigmentation of the fruit surface, even if it strongly depended on the individual olive cultivar. A previous study on cv. Nostrana di Brisighella showed that in order to obtain the best sensorial oil quality, the olive RI cannot exceed a value of 3.5 (Cerretani et al., 2004). Climatic conditions Climatic and agronomic conditions of olive growing can affect the sensory properties of the olive oil obtained from the same cultivar. In this context, the relationships between the water availability during fruit ripening and the sensory properties has been studied. In the study by Gucci (Gucci et al., 2004), fruits were harvested at the stage of full pigmentation of the epicarp only (black skin) or at green-black skin colour. Irrigation decreased pungency and bitterness and increased flavours, such as fruity-apple or hay-like, only of oils from black fruits. These results were confirmed in other studies (Salas et al., 1997; Tovar et al., 2001; Tovar et al., 2002; Gomez-Rico et al., 2007). As it is well-known, the intensity of sensory pungency, and especially bitterness, are related to the phenol content in the olive oil, which, as expected, was higher in oils obtained under rain-fed conditions. In all cases, a slight decrease in the intensity of these positive attributes was observed, more marked in the case of bitterness, by increasing the amount of water delivered through irrigation. This observation is very relevant from the olive quality and marketing point of view since, although bitterness is a positive sensory attribute in virgin olive oil, a high level of bitterness could cause consumers to reject the oil. Studies conducted by Berenguer et al., (2006), to evaluate the influence of seven different levels of irrigation applied to Arbequina I-18 olive (Olea europea L.) trees grown in a super-high-density orchard in the Sacramento Valley of California on oil sensory characteristics, have shown that oil sensory properties of fruitiness, bitterness and pungency all declined in oils made from trees receiving more water. The lowest irrigation levels produced oils that were characterized by excessive bitterness, very high pungency, woody and herbaceous flavors. Intermediate irrigation levels (33% to 40% ETC) produced oils with balance, complexity and characteristic artichoke, grass, green apple and some ripe fruit flavors. Higher irrigation levels lowered oil extractability and produced relatively bland oils with significantly less fruitiness and almost no pungency. Morelló et al., (2003) have studied the effect of freeze damage during the harvest period on the quality indices of olive oil from the Arbequina cultivar, as well as the influence of freeze conditions on the virgin olive oil sensory attributes. No differences were found in the quality indices of oils extracted from olives affected by freeze injuries. On the other hand, differences are detected between oils in aroma, and mouthfeel perceptual differences are detected between oils. The sensory notes of artichoke, tomato and almond were perceived in before-frost oils, and no unpleasant aroma or flavour was detected. After-frost olive oils were qualified as non-extra virgin olive oil. Defects were defined as frozen olives by some panellists, while others defined these oils as thicker, softer and with the term rancid tallow.

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4.2.2. Technological aspects Harvesting methods Nowadays olive harvesting is mechanically performed and always less frequently olives are picked by hand from the tree. However, in some areas of olive production olives are gathered from the ground by using brushes and aspirators at regular intervals of time until the end of the spring (Di Giovacchino, 2000). The appearance of a typical defect, reminiscent of “mouldy” and “earthy” tastes at the same time (Angerosa et al., 1995), can be considered as a consequence of the prolonging of contact time of fruits with the ground. They characterize oils extracted from olives fallen from trees of Dritta and Nebbio varieties. The results showed that the unpleasant flavour considerably increases with the increase of contact time between olives and ground. (Angerosa et al., 1995). Olive fruit storage Fruit storage before oil processing is not encouraged in olive oil production. Good practice in fruit handling recommends that the fruit should be processed as soon as possible after harvest, without storage (Di Giovacchino, 2000). One of the main causes of sensory defects in virgin olive oil is the storage of olive fruits into sacks or in piles before oil extraction (Morales et al., 2005). The production of different metabolites, according to the type of microorganisms from the environment (Angerosa et al., 1996b) whose development is promoted by the temperature reached in the pile and the humidity degree, gives rise to different sensory defects. First Clostridia and Pseudomonas genera develop producing branched aldehydes, branched alcohols and their corresponding acids (Angerosa et al., 1996b; Angerosa et al., 1990) of which concentrations in a few days their overstep the threshold levels for the perception of “fusty” defect (Morales et al., 2005) However sometimes, especially if the temperature is relatively high, an important growth of yeasts can occur with the production of considerable amounts of ethanol and ethyl acetate. As a consequence, the ‘winey’ defect appears. The possible presence of Acetobacter is responsible for the ‘vinegary’ defect because it promotes the production of acetic acid (Angerosa et al., 1996b; Morales et al., 2005). Processing conditions Under optimal extraction conditions, using healthy and mature olive fruits, extra virgin olive oil is always produced whatever the olive variety processed. Malaxation temperature and time are the two main parameters that can be controlled during processing to potentially change the sensory properties of the oil. Raising the temperature of the olive paste reduces viscosity, making it easier to separate and obtain high yields. However, raising the processing temperature reduces the quality of the oil (Amirante et al., 2002). A general weakening of the oil flavour is recorded by tasters, especially for walnut husk and tomato, when the malaxation temperature rises, whereas prolonged periods of malaxation cause the weakening of leaf, freshly cut grass, typical green attributes, walnut husk, bitter and pungent sensory notes (Servili et al., 1996; Angerosa et al., 2001). Research has shown that malaxing the olive paste at 30°C achieves both pleasant “green” virgin olive oil and satisfactory oil extraction outputs, but that 35°C introduces numerous defects into the oil without substantially increasing the oil yield (Morales and Aparicio, 1999; Ranalli et al., 2001). According to Angerosa et al., (2001), malaxation time, and especially temperature, negatively affected the intensity of the sensory attributes. Low temperatures and times,

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ranging between 30 and 45 mins, according to the rheology of the olive pastes, were the optimal operative conditions for the malaxation. Olive oil extracted from Corregiola grown in Australia showed very little difference between the amount of volatile compounds produced with malaxation temperatures of 25 °C or 35°C, or between malaxation times of 15 and 60 min (Tura et al., 2004). This is inconsistent with reports from Europe where temperature has been shown to affect the sensory characteristics of the resulting oils (Morales and Aparicio, 1999). Geographic and cultivar influences might explain the high processing temperature tolerance observed (Tura et al., 2004), and different processing parameters may be required in differing geographic growth regions to produce high quality virgin olive oil. It has previously been observed that the optimum processing parameters also vary with cultivar (Servili et al., 2003). Oil storage The olive oil profile changes during its storage because of the simultaneous drastic reduction of compounds and the neo-formation of some volatile compounds (Solinas et al., 1987; Frankel, 1985) responsible for some common defects known as “rancid”, “cucumber” and “muddy sediment” attributes. The most advanced oxidation stages are characterised by very high concentrations of aldehydes, contributing mainly to undesirable aromas. Other contributors are represented by unsaturated hydrocarbons, furans and ketones. Unsaturated aldehydes and ketones can be further oxidised producing new off-flavour compounds, whose presence accounts for the different nuances of the unpleasant aromas described by tasters as rancid, painty, fishy, etc. (Morales and Przybylski, 2000). The presence of the sediment consequent to unfiltered olive oil decantation during its storage can determine, under suitable conditions of temperature, the production of unpleasant compounds responsible for the typical “muddy sediment” defect due to the fermentation that produce compounds, possibly of the butyric kind (Angerosa et al., 2004). 4.2.3. Sensory properties of seed oils Most oils are obtained from oilseeds using a two-step process (extraction and refining). Oil can be extracted from seeds either by pressing (using a screw press) or by the use of solvents (mainly by percolating the solvent through the prepared seeds). The oil produced then goes through a series of refining processes to remove unwanted components, which may affect taste, smell, appearance or storage stability. One of these processes is the deodorisation, which reduces the level of FFA and removes odours, off-flavours and other volatile components from the oil to ensure that the oil has an acceptable taste and shelf-life (McKevith, 2005). After processing, oxidation is the main problem affecting oils, leading to aldehyde production, which imparts strong disagreeable flavours and odours, referred to as rancidity. Generally, the rate of oxidation will depend on the degree of instauration of the oil and its temperature as well as the presence of antioxidants. Oxidation will occur at varying rates throughout the life of the oil: during storage and distribution of the oil, during food preparation and storage of the final food product. Sensory profile of peanut, soybean, rapeseed, sunflower and corn oils

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There are very few specific studies on the sensory analysis of seed oils. The objectives of the investigation by Leveaux and Resurreccion (1996) were to determine the sensory profile of different freshly processed vegetable oils (peanut, soybean, canola) using quantitative descriptive analysis (QDA) tests. The samples evaluated in the QDA tests were prepared for sensory evaluation following the procedures described in ASTM Standard Practice for Sensory Evaluation of Edible Vegetable Oils. This standard practice details the methodology for profiling of vegetable oils. Panelists were instructed to evaluate samples for one appearance, thirteen odors, fourteen flavors and two texture attributes. The appearance attribute evaluated was color. The odor attributes evaluated were beany, buttery, cardboard, corny, fishy, fruity, hully, hydrogenated, musty, nutty, painty, waxy and weedy. The flavor attributes were the same thirteen attributes used for odor plus bitter. The texture attributes were viscosity and mouth coat. The sensory profile of these oils was the following: a) the appearance (color) of the oils was yellow with different intensities; b) the oils had very low odor intensity scores with the exception of the hydrogenated attribute, indicating a bland odor for each of the oils. Mean odor attribute scores with lower intensities, included corny, weedy, painty, waxy, fruity, cardboard, nutty, buttery, fishy; c) the oils had a very low flavour intensity except for the hydrogenated and nutty attributes. Amongst the mean of flavour attribute scores with lower intensities were the flavours corny, weedy, cardboardy, fruity, hully, musty, painty, beany, and bitter. Amongst the mean flavour attributes score with higher intensities were the flavour nutty, buttery and fishy. The fishy flavour of oils may be caused by linolenic acid, which represents 9.1 % of the fatty acids found in canola oil. This fishy note should decrease in commercial canola oil in the future as research is being done to lower the level of linolenic acid in canola oil; d) the texture attribute “viscosities” was the same in all oils. Fig. 4.4 shows a study conducted on sunflower oil. Two oils at different levels of oxidation are compared through the Student's t test. t value for the panel is higher than the theoretical t value for 3 descriptors: fruity, rancid and linseed intensities are significantly different between the 2 oils.

0

2,0

4,0buttery

fishy

linseed

rancid

fruity

grassy

Product AProduct B

significantsignificant

significant

0

2,0

4,0buttery

fishy

linseed

rancid

fruity

grassy

Product AProduct B

significantsignificant

significant

Descriptors Product A Product B t calculated Significant at 1 %

butter 0,35 0,73 -1,97 nofish 0,55 0,13 1,25 nolinseed 1,13 0,16 3,78 yesrancid 2,58 1,03 4,13 yesfruity 2,05 3,16 -3,91 yesbeany 0,09 0,22 -1,16 no

n = 13 t 1 % = 3,055ddl = 12

Descriptors Product A Product B t calculated Significant at 1 %butter 0,35 0,73 -1,97 nofish 0,55 0,13 1,25 nolinseed 1,13 0,16 3,78 yesrancid 2,58 1,03 4,13 yesfruity 2,05 3,16 -3,91 yesbeany 0,09 0,22 -1,16 no

n = 13 t 1 % = 3,055ddl = 12

Figure 4.4. Example of sensory profiles for two refined oil There are no specific papers about the sensory profile of corn oil. One study is an AOCS collaborative study by Warner and Nelsen (1996), conducted to determine the

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effectiveness of sensory analyses of volatile compound in measuring vegetable oils for level of oxidation that ranged from none to high. The descriptors evaluated were: nutty, buttery, corn, beany, hydrogenated, burnt, weedy, grassy, rubbery, melon, rancid, painty, fishy. The oxidation level of corn oils, determined by overall quality score were correct 50-60% of the times for the none-, low- and moderate-oxidation levels and 86% of the times for the high oxidation level. Most of the flavour in corn oil did not change with increasing oxidation with the exception of small increases in rancid and painty flavours as well as a high level of off-flavours (musty, bacon, lard) detected in the highly oxidized samples. Sensory profile of argan oil There are no scientific papers in literature reporting data on the sensory analysis of argan oil to date. It is possible to find a few pieces of information on the organoleptic characteristic of the oil on web sites, but they are very generic, they lack any methodology as well as scientific validation. However, a tasting of different argan oil samples has been performed in the context of the activity of “Slow Food” for the promotion of biodiversity and the culinary traditions from around the world, in collaboration with the “Moroccan Association Ibn Al Baytar” (Soracco and Boeri, 2008). Thirteen expert tasters judged nine different argan oils produced by six different cooperatives, the oils belonged to 4 groups: Group a - edible argan oils mechanical extracted Group b - edible argan oils manually extracted Group c - cosmetic argan oils mechanical extracted Group d - edible argan oils produced with berries regurgitated from Different to the edible oil, cosmetic oil is produced from untoasted kernels, but the other aspects of the production procedure are the same (Charrouf and Guillame, 2008). The characteristics of the single tested oils belonging to the same group taken into consideration were: Group a (three samples): To the eye, they are clear and have a light gilding, the colour of the oil could be more amber probably depending by a longer roasting of the kernel. The scents are quite intense, with notes of hazelnut, roasted almonds, peanuts, and/or spicy, and/or coffee; sometimes burnt probably depending by the roasting. The taste is a little bit sweet and bitter and the oil is very fluid. The retro-nasal smell has more or less the same characteristics of the smell. Group b (four samples): To the eye, they are clear or a little bit muddy, the colours are light gilding or straw. The scents are: roasted almond and hazelnut, a little bit of coffee. The taste is sweet and bitter. The oils are very fluid. Some samples have defects: A little bit fusty, fermented or wine-vinegar perceived by both nasal and retro-nasal smell. Group c (one sample): To the eye, it is clear almost brilliant. The smell is not very strong and a little bit wine-vinegar. For the retro-nasal smell prevail the rancid. Group d (one sample): The flavour is wild and unpleasant, it shows all the defects perceived tasting the oils of the other groups. For a final determination of the sensory profile of the different argan oils, more work is needed following scientific procedures. Fig. 4.4 shows a form for the sensory evaluation of argan oil (Matthaus, personal communication), with it being of utmost importance that the trained tasters use the same vocabulary to describe the product.

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This method for the sensory assessment of argan oil uses an evaluation which is both descriptive and quantitative. For good argan oils from sound seeds, attributes such as nutty and roasty have been found useful and typical. Since for the production of argan oil for pharmaceutical uses no roasting process is carried out this type of oil should have only a slight nutty taste and smell without the appearance of the attribute roasty. This attribute is typical for argan oil used for edible oils, because this type of oil is roasted either directly or indirectly. The attribute roquefort cheese often appears in argan oils especially when nuts from the digestion of goats are used for the production of oil, but up to now it is not clear whether the appearance of this attribute is a failure or typical for some kind of production. Additionally to the typical attributes several atypical off-flavours are described as rancid, wood-like, fusty, musty, yeast, burnt and bitter. For the quantitative assessment, a scale is used from 0 for not perceivable to 5 for very strongly perceivable at the level of saturation. The sensory evaluation of argan oil is carried out by at least four trained tasters and from the individual results of the tasters the median value is calculated as a result for the sample. Sensory profile of rice bran oil The literature regarding the sensory study of rice bran oil, deals with blends. Oils are blended to increase the thermal stability, to cater for consumer preference, to desired flavours in foodstuffs and nutritionally balanced fatty acid composition. In their study, Raj and collaborators (Raj et al., 2006) have investigated PO and rice bran oil (RBO) blend, the oils were procured from the market, blends were prepared by mixing 70 parts of RBO and 30 parts of PO on weight basis. A group of 10 panelists were trained over three sessions for descriptive sensory analysis. The members of the panel were drawn from the scientific staff familiar with sensory analysis techniques. The training included the development of a common lexicon of the sensory attributes in evaluation. The panelists evaluated the oils in a group but recorded the perceived attributes individually. Following this, an open discussion was held in order to reach an agreement on the appropriate descriptors, to enable the panelists to characterize the odours of base oils and oil blend.

(roasty)

othersfustyyeast-likemustyburntbitterwood-likerancidatypical attributes(Roquefort cheese)

nutty543210typical attributes

Figure 4.5. Sensory evaluation form for argan oil

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The scorecards developed for the quantitative descriptive analysis had a 15-cm scale with selected descriptors anchored at either end at 1.25 cm (detection threshold) for low intensity and 13.75 cm (saturation threshold) for high intensity. The characteristic odour notes of RBO are beany, branny and earthy. When it was blended with palm oil, the intensity of these attributes was not pronounced, while on frying these attributes became more prominent. The odour profile of the blend of RBO + PO showed that the typical fresh oil note decreased, while husk-like, earthy, branny and beany notes increased by the end of the 10th frying. In another study (Ravi et al., 2004), mustard oil (MO) or Groundnut oil (GNO) or sunflower oil (SNO) were mixed with RBO 80:20 on weight basis. The methods for evaluation were more or less the same, for blend of GNO and RBO the intensity of branny and beany notes was perceived to lessen during frying. The blends containing RBO had a distinct yellow colour, increased heated note and higher apparent viscosity. 4.2.4. Comparison of sensory properties: soybean, peanut, rapeseed/canola, sunflower and extra virgin olive oils The study by Leveaux and Resurreccion (1996) compared four oils (soybean, peanut, cottonseed and canola oils). It showed that the appearance (color) of the oils was significantly different from each other. Soybean oil was the least yellow in color, followed by peanut oil and canola oil. Cottonseed oil was the most yellowish, but still less than half the yellow of extra-virgin olive oil reference standard. Odour attributes scores, corny, weedy, painty, and waxy were not significantly different among the samples. Soybean oil was less fruity than peanut oil. Cottonseed had a hully odour, which was significantly higher than peanut and soybean oil. Nutty odour was not significantly different among the oils. The same behaviour has been observed for flavour attribute scores, corny, weedy, cardboardy, fruity, hully, musty and bitter, which were not significantly different among the samples. Cottonseed oil was more waxy than the other oils. The flavour nutty was not significantly different between the oils. Canola oil was more fishy, but the standard deviation was high as is the case with the fishy odour. The high standard deviation may be attributed to panelists working very close to their thresholds. Probably either the sensory methodology or scale needs to be re-evaluated. The fishy flavour of oils may be caused by linolenic acid. Cottonseed oil had a more hydrogenated flavour than the other three oils. The viscosities of these four oils were not significantly different. In the study by Mildner-Szkudlar et al., (2003), rapeseed, soybean, peanut, sunflower, and extra virgin olive oil were used. Sensory evaluation of the odour profiling of samples was carried out by a 10-member panel, experienced in descriptive analysis. Six odour attributes, acidic, sweet, green, floral, oxidised and hay were scaled on linear 10-cm scales anchored on both sides for the intensity as ‘‘not desired’’ and ‘‘very desired’’, respectively. All the samples investigated were subjected to PCA based on odour profiling analysis to find discrimination according to their sensory quality. All the oils were compared with each other in relation to all the used odour attributes. Oxidised, hay, acidic and sweet were the major odour attributes responsible for the differentiation. Three oils, rapeseed, peanut and sunflower were similar and almost odourless, characterised only by a weak sweet odour. Soybean oil was characterised by sweet and hay odours.

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Stored oils, rapeseed, olive, soybean and sunflower were characterised by an undesired sensory quality with oxidised and acidic attributes being predominant. Stored peanut oil was characterised by hay and flowery notes in addition to oxidised one. Fresh olive oil was perceived as the most aromatic of investigated fresh oils and described with the dominant acidic, green notes. Stored rapeseed and peanut oils oxidised more rapidly than the others. Due to the presence of natural antioxidants, the olive oil profile did not change to the extent observed in other oils. This was reflected in the close distance between stored and fresh olive oils on the PCA plot. 4.3. Relationship between Virgin Olive Oil Compounds and

Sensory Attributes The acceptability of foodstuffs by consumers is mainly explained by their sensory quality; colour and flavour being the main perceptions. While colour can be measured with instruments, flavour is a complex sensation comprising primarily smell and taste, that it is evoked by stimulation of all the oral and nasal chemosensory systems. Flavour plays an important role in nutrition as it is partly responsible for aiding in the digestion of food in humans and, furthermore, it is vital in the control of food supply, recognition, selection and acceptance. The demand for high quality crude oils over the past few years can be attributed not only to the habits of their consumers, who have spread all over the world publicizing their advantages, but also their fragrant flavour or their health benefits which paradigm is virgin olive oil. Volatile compounds are mainly responsible for aroma while phenolic compounds are considered to be related to taste sensations. The presence of these compounds gives rise to particular flavours. Many analytical procedures have been developed during recent years to carry out the analysis of volatiles (Chapter 3 in this Handbook). The huge amount of information from instruments has allowed establishing relations between chemical compounds and sensory descriptors. Thus, the profile of some chemical compounds varies in accordance with the edible oil quality. Thus, high quality oils are mainly due to volatiles produced by biogenic pathways (i.e. virgin olive oil) or controlled oxidation processes (i.e. roasted hazelnut oils) while low quality oils are qualified with off-flavour perceptions resulting of unhealthy fruits/seeds, inadequate extraction processes or, almost always, from oils that have exceeded the shelf-life. Studies devoted to explain sensory perceptions from the profile of volatiles have not been carried out in all edible oils but in a few, mostly those oils extracted under conditions that allow appreciating desirable or cherished sensory attributes. Otherwise, the studies are focused on refined edible oils and the rancid perception. 4.3.1 Contribution of different compounds to the oil sensory quality The establishment of relationships between chemical compounds and sensory attributes is the most complex aspect of the global flavour study. The great advances in instrumentation in the past decades have allowed the identification of a great number of

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chemical compounds, mostly in virgin olive oil, that have been used to explain sensory attributes by means of several multidisciplinary approaches. The first attempts were focused on finding correlations between single compounds and sensory attributes. Their findings, however, lack the global information and, hence, did not allow the explanation of sensory attributes, but only preliminary conclusions, since synergic and antagonist processes among compounds, due the lipid matrix complexity, occur. The best approaches, however, have been achieved by applying multivariate procedures to all the available information from the sensory assessment and the chromatographic analysis; investigation that has been commanded by the researchers on olive oil. Relationship between volatile and phenolic compounds and sensory attributes It is obvious that volatile compounds responsible for flavour come from the seed or the fruit, and they are direct metabolites produced in plant organs by intracellular biogenic pathways (Salas et al., 2005, 2007). Thus, the kind of volatiles and their concentration depend on the genetic factors of the species and their cultivars that are modulated during the ripening of the seeds or the fruits, and later altered in the oil extraction process and its storage. Thus, ripeness increases the activity of enzymes that lead to the accumulation of metabolites and substrates for production of volatiles, e.g. fatty acids and amino acids. Some of the volatiles characterising virgin olive oil, for example, are already present in the intact fruits while other volatiles are "secondary products" not occurring (or only in traces) in intact cells. The main precursors of volatiles are fatty acids (linoleic and α-linolenic in particular) and amino acids (leucine, isoleucine and valine). Three are the main biochemical pathways involved in the biogenesis of volatiles present in crude edible oils though there are oxidation processes (Table 4.2) from diverse fatty acids as well. There are, however, much more compounds (Table 4.3) whose formation process is still unknown and their contribution to aroma depends on their concentration. Biophenols encompass a major group of secondary plant metabolites that display a wealth of structural variety and a large diversity of significant biological activities. Despite this importance, few studies have focused on the origin and fate of phenols within the plants The vast majority of phenolic molecules owe their origin to one or more of the following blocks: erythrose-4-phosphate, phosphoenol pyruvate and/or acetyl co-enzyme A. The first two are both responsible for known shikimic acid pathway, whereas the third and its activated form (malonyl co-enzyme A) are the central point of the polyketide or acetate pathway. Acetyl co-enzyme A may also be converted into mevalonic acid which is the beginning for a wide range of terpene compounds. Shikimic acid pathway is more complex than the acetate pathway. From the formation of the typical shimikate C6C3 skeleton, three amino acids (tryptophan, phenylalanine, and tyrosine) are the most important precursors of the alkaloid synthesis. In some plants and grasses tyrosine is converted into-4-hydroxy-cinnamic acid via the action of tyrosine ammonia lyase. The final result of the entire process is the synthesis of caffeic, ferulic and sinapic acids that are precursors of lignins and many other compounds. Combination of shikimate and acetate pathways, in the form C6C3 + (C2)n – where n is usually three (C6C3C6) – generates several groups of phenolics which flavonoids are by the far the most important. Flavonoids are formed via condensation of a phenylpropane compounds with the help of malonyl co-enzyme A to the formation of chalcones. Once formed, the flavanone nuclei can undergo modifications involving pyran-4-one ring. Oxidation or reduction gives a wide variety of products like flavones, flavonols, flavonones, flavononols, flavans, and tannins. Finally, mevalonate and terpene pathways are formerly regarded as the universal route to terpenoid and steroid biosynthesis.

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Explanation of flavour perception from the volatile and phenolic profiles The explanation of aroma sensory attributes by volatile compounds has always been a temptation for researchers who have tried to give plausible explanations by applying several kinds of mathematical algorithms. Thus, correlation studies among physical, chemical, and sensory characteristics in foods have the utopian objective of replacing sensory evaluations, which are time-consuming, by physical and chemical analyses. Additionally, correlation evaluation among these characteristics assists the understanding of factors that cause food to modify its sensory profile, optimizing the production process (O'Mahoni, 1986). Because of the difficulties in sensory assessments, it is always tempting to substitute a sensory panel with instrumental measures. All the volatiles, however, do not have the same influence on the sensory assessment. Their influence depends not only on the volatile concentration but also on its odour threshold. Two procedures are used to elucidate the role of volatiles from a sensory viewpoint. GC-sniffing techniques have been widely applied to study the independent sensory impact of the different volatile compounds on sensory assessment, mostly in virgin olive oil (Morales et al., 1994). Calculation of odour activity values (OAV, ratio of concentration of volatile compound to odour threshold) is the other procedure that has been used to determine the volatiles contributing most to aroma (Guth and Grosh, 1993; Morales et al., 1996; Aparicio and Morales, 1998). This procedure is effective for the determination of the most important contributors to aroma but the presence of other volatile compounds in the matrix of the oil (see Table 4.3, for example) should not be forgotten as they can contribute to the global flavour by synergism phenomena. Multivariate statistical methods have proved to be the most powerful tool to point out these relationships and take into consideration the whole set of chemical compounds and the standard sensory attributes. Most of the studies, however, has been focused on oxidation process (§ 3.1.3) as almost all the edible oils are consumed after refining with the exception of virgin olive oil and other crude oils sold in delicatessen (e.g., hazelnut and avocado oils). Concerning crude or virgin oils, artificial neural networks (ANN), partial least squares (PLS), principal components analysis (PCA) and multidimensional scaling (MDS) have been applied to bring out inter/intra dissimilarities from datasets of volatile compounds and sensory attributes with diverse success (Aparicio et al . ,1994; Morales et al . ,1995; Servili et al . ,1995; Angerosa et al.,1996a; Angerosa, 2002). An interesting approach to the knowledge of these relationships was based on the building of a statistical sensory wheel (SSW) representing the global flavour matrix of an extra-virgin olive oils (Aparicio et al . , 1994; Aparicio and Morales, 1995).

Table 4.2. Basic information of oxidative pathways involved in the production of volatiles from fatty acids

Oleic acid (ω-9) Linoleic acid (ω-6) Linolenic acid (ω-3) Arachidonic acid (ω-6) Nonanal Octanal E-2-Undecenal Decanal E-2-Decenal Heptanal

Hexanal 2-Heptenal 2-Nonenal Propanal 2-Octenal 2,4-Nonadienal 2,4-Decadienal

2,4-Heptadienal Propanal 2-Hexanal 2-Heptenal 2-Pentenal 2-Butenal

Hexanal 2,4-Decadienal 2,4,7-Tridecatrienal 2-Heptenal 2-Octenal Pentanal 4-Decenal 2,6-Dodecadienal 3-Nonenal

The idea was not new but it was beyond the subjective procedure, in which each

flavourist placed the sensory descriptors at his convenience (Noble et al., 1987). Here,

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statistical algorithms play in important role relating chemical and sensory information. SSW used the information of 103 sensory attributes evaluated by six different European panels; all panels used quantitative descriptive analysis (QDA) in the sensory assessment. A simple but particular mathematical algorithm was used to project 85 volatile compounds, quantified in the same samples evaluated by the sensory assessors, on the plot built with the sensory attributes (Aparicio and Morales, 1995). The global sensory matrix of VOO dissected by SSW was divided into sectors representing the main perceptions produced by virgin olive oil (Aparicio and Morales, 1995): fruity, green fruity, bitter-pungent, undesirable (off-flavours), ripe-olives, ripe fruit and sweet. In between sectors some miscellanies appeared due to the change of the perception is not sudden but progressive and some zones may be the result of this progressive change. Table 4.3. Some of the volatile compounds identified in different kinds of virgin olive oils. (Morales and Tsimidou, 2000)

Form the results of SSW, it can conclude that Z-3-hexen-1-ol, Z-3-hexenal, hexyl acetate and 3-hexenyl acetate were placed in the green sector close to green sensory attributes thus emphasising their contribution to the green perception (Figure 4.4). Hexanal and pent-1-

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en-3-one were placed in the sweet sector. Various volatiles (E-2-hexen-1-ol, 1-penten-3-ol, hexan-1-ol, etc.) were placed in the undesirable sector but most of them are not usually present in high concentration in virgin olive oils. It is concerning undesirable attributes where the research has progressed much more. Today, it is known the volatiles responsible for the most remarkable defective virgin olive oils (Morales et al., 2005). Concerning individual sensory perceptions, some sensory attributes can be partially explained with some volatiles. Thus, 2-penten-l-ol can partially explain green banana attribute; E-2-hexenal contributes to almond and twig attributes; 3-methyl-2-butanal, 2-methyl-butanal seem to contribute to butter attribute; propionic acid contributes to fried oil sensory perception; artichoke might be partially explained E-3-hexenal; E-2-hexenal and E-2-penetnal contribute to bitter and astringent perceptions via retro nasal; etc. Other sensory perceptions are more complex such as tomato. Two perceptions have been identified for this sensory attribute. Sweet tomato can be explained by the combined action of 3-pentanone, 1-penten-3-one and ethyl propanoate among others. These compounds would explain the tomato sensory perception by British consumers. The other tomato perception, green-tomato, might be explained mixing 2-heptanone, 2-nonanone, 1-hexanol, and E-2-pentenal among other volatiles (Aparicio et al., 1994; Morales and Tsimidou, 2000). The relationship between sensory perceptions and volatile compounds has also been used to characterize olive oils from diverse cultivars as shown in Table 4.4.

Figure 4.4. Sensory attributes related to “green” perception (from fruity to bitter) and volatiles compounds explaining this perception [Reprinted with permission from Garcia-Gonzalez DL et al. Olive Oil. In Moreau RA, Kamal-Eldin A (eds.). Gourmet and Health-Promoting Specialty Oils. Urbana, IL: AOCS Press, 2009]

The relationship between phenolic compounds and sensory attributes is almost exclusively focused on olive (Morales and Tsimidou, 2000) and argan (Charrouf and Guillaume, 2007) oils, and several manuscripts have correlated the total concentration of

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phenols with bitterness while others have related the presence of aglycones with pungency (Tsimidou 1998; Gutiérrez et al., 2000; Servilli and Montedoro 2002), and even in the olive oil shelf-life (Aparicio et al., 1999). Explanations of tasting perceptions with individual phenols have however failed with the exception of the projection of phenols on SSW. Thus, when quantitative information of phenols, quantified in the same VOO samples, was used for SSW, the compounds were automatically projected inside bitter-pungent sector by the mathematical algorithm (Figure 4.5). Furthermore, the bitter-pungent-astringent notes though being taste and tactile perceptions are also explained by the contribution of volatiles via retro nasal perception; E-2-hexenal and E-2-pentenal for example.

Table 4.4. Concentration of volatiles characterizing varietal virgin olive oils (Luna et al., 2006a) [copyright granted]

Variety Volatile (max. in μg/kg) Variety Volatile (max. in μg/kg)

Tsounati 3-Methyl-butanal (223) Coratina E-2-Hexenal (27 435) Cornicabra Ethyl propanoate (249) Cañivano 3-Methyl butanol (5 357) Picholine 2-Methyl butanal (510) Manzanilla Z-3-Hexenyl acetate (457) Picudo 4-Methyl-pentan-2-one (899) Koroneiki Z-2-Penten-1-ol (2 052)

Picudo 1-Penten-3-one (1 689) Picual 6-Methyl-5-hepten-2-one (2 337)

Picudo Butyl acetate (455) Empeltre Hexan-1-ol (3 297) Cañivano Hexanal (5 353) Sourani Z-3-Hexen-1-ol (2 764) Zaity E-2-Methyl-2-butenal (404) Cañivano Nonan-2-one (728) Moraiolo E-2-Pentenal (3 822) Empeltre E-2-Hexen-1-ol (4 795) Imperial 1-Penten-3-ol (342)

Figure 4.5. Relationship between phenolic compounds and sensory attributes. Zoom of the statistical sensory wheel (SSW) bitter-pungent sector [Reprinted with permission from Garcia-Gonzalez DL et al. Olive Oil. In Moreau RA, Kamal-Eldin A (eds.). Gourmet and Health-Promoting Specialty Oils. Urbana, IL: AOCS Press, 2009]

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Relationship between off-flavour and sensory attributes The consumption of edible oils, crude or refining, determines the kind of off-flavours to be analysed. Most of the studies related to crude edible oils have been focused on olive oil and the defective sensory attributes defined by the International olive Council (IOC) while the studies of refined oils are almost exclusively centred on the rancid perception concerning the frying process and oil shelf-life that are aspects of interest of crude oils as well. Under optimal extraction conditions, using healthy and mature olive fruits, the production of extra-virgin olive oil is always possible whichever the olive variety processed. Only the olives attacked by pests or fell down to the ground before harvesting produce off-flavours (Angerosa et al . , 1999; Morales and Przybylski, 2000; Morales et al., 2000; Morales et al . , 2005), the rest of the defective sensory notes in the olive oils are due to an inadequate harvesting or processing or olive oil preservation (Angerosa et al . , 1996b; Morales et al . , 1997; Luna et al . , 2006b).

Lipolysis and oxidation are the processes leading to the most serious deterioration of olive oil. Lipolysis usually starts while the oil is still in the fruit, while oxidation begins after the oil is obtained from the fruit and proceeds mainly during storage (Morales et al . , 1997), both processes affect the composition and the sensory characteristics of the oil. One of the main reasons of sensory defects in virgin olive oil is the storage of olive fruits in piles before oil extraction, olives transpire during the storage so that the temperature of the pile increases. In this way, when olives are stored under inadequate conditions suffer biodegradation or bio-spoilage by micro-organisms that inevitable give rise to less quality VOO, ultimately rendering the product inedible. When oils reach high intensities of sensory defects they are classified as lampante olive oils and must undergo refining before being consumed. The decision of classifying an olive oil as lampante is based on sensory assessments. However, the sensory attributes which allow determining if an olive oil is lampante are due to the concentration of some volatiles (Table 4.5) as the profiles of an extra-virgin olive oil and a lampante virgin olive oil show (Figure 4.6). But the designation of lampante virgin olive oil is so large that there is a particular profile of volatiles for each one of the most habitual off-flavours (Morales et al., 2000; 2005). Concerning argan oils, they are qualified with slight nutty taste and smell roasted because it is consumed roasted. The attribute Roquefort cheese often appears when nuts from the digestion of goats are used for the production of oil. Additionally to the desirable attributes several off-flavours like rancid, wood-like, fusty, musty, yeast, burnt and bitter have been described. Unfortunately, there are not studies that relate the concentration of volatile compounds with the sensory perceptions. The rancid perception has traditionally been the most investigated from the volatile viewpoint in refined oils since they are almost odourless - characterised perhaps by a weak sweet and hay odours when they are fresh – but the increase of the concentration of aldehydes (E-2-pentenal, hexanal, E-2-hexenal, heptanal, E-2-heptenal, E-2-octenal, nonanal, etc) and alcohols (1-penten-3-ol, 1-pentanol, 1-hexanol, 1-octen-3-ol, 1-octanol, etc) during the shelf-life causes the rancidity perception. Thus, stored rapeseed, soybean and sunflower oils have been characterised with oxidised and acidic attributes while stored peanut oil has been characterised by hay and flowery notes in addition to the rancid sensory attribute.

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Figure 4.6. Chromatograms of volatile compounds quantified in a lampante virgin olive oil (A) and an extra-virgin olive oil (B). Note: numbers correspond to the names of the volatiles described in Table 4.5 (Morales et al., 2005) [copyright granted]

The compounds hexanal, hexenal, E-2-heptenal, E,E-2,4-heptadienal, nonanal, E,E,2,4-nonadienal, E-2-decenal, E,E-2,4-decadienal may play significant roles in the flavour characteristics of foods because of their low thresholds and specific flavour characteristics. Previous studies estimated the significance of some volatile compounds from the oxidation of soybean oil on food flavour, based on their concentrations and threshold values. For example, t,c-2,4-decadienal was the most flavourful, followed by t,t-2,4-decadienal, t,c-2,4- heptadienal, 1-octen-3-ol, n-butanal, n-hexanal, t,t-2,4- heptadienal, 2-heptenal, n-heptanal, n-nonanal, and 2-hexenal. In the study of Su and White (2004), the greater amount of hexanal, E,E-2,4-heptadienal (fresh), and hexenal may have contributed to its generally stronger grassy and fishy off-flavours. Conversely, the generally low amounts of these compounds may have resulted in generally weaker grassy and fishy off-flavours in 79%oleic and low linolenic soybean oils. The tendency for more E-2- heptenal and E,E-2,4-decadienal to be present in control and low linolenic soybean oil treatments may have caused slightly stronger rancid and fried food flavour in fresh fried cubes. Ten trained panellists identified for deodorized soybean oil many components previously separated and identified by GCMS (Kao et al., 1998). The substances and their odours were hexanal (green), cis-4-heptenal (fish oil),

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heptanal (heptanal), benzaldehyde (cherry), 2,4-heptadyenal (fruity), nonanal (slight fruity), heptanoic acid (sweaty), phenylpropanone (fruity-rose), 2-nonenal (aldehyde), 3-Methyl-2,4-nonadione (liquorice), 2,4-Decadienal (bean), Decano-γ-lactone (buttery-lactone), Menadione (spicy).

Table 4.5. Main volatile compounds identified as partially responsible for VOO off-flavours according to their odour activity value. Note: A, concentration, mg/kg, in the standard defective virgin olive oils mustiness-humidity, fusty, winey-vinegary, rancid, supplied by the International Olive Oil Council; B, odour threshold, mg/kg, determined by olfactometry with experienced assessors (Morales et al., 2005) [copyright granted]

Soybean oil has been frequently associated with a fishy flavour due to the presence of oxidation breakdown products from linolenic acid (López-Aguilar et al., 2006). Evans et al., (1965) found that the linolenic acid content needed to be decreased to less than 5% to improve the flavour quality and oxidative stability of soybean oil when they were evaluated as salad oils after storage. The target for linolenic acid needs to be even lower than 5% for frying oils because linolenic acid–containing vegetable oils such as canola and soybean are well known to produce off-flavours and odours such as fishy when they were exposed to high-temperature heating (Evans et al., 1965; Eskin et al., 1989). Deodorization of soybean oils may also result in a nutty flavour that is considered to be acceptable by consumers (Kao et al., 1998). Warner et al., (2000) examined the compositions of soybean oils that were chemically hydrogenated or electrochemically hydrogenated. The odour characteristics of these hydrogenated oils were determined after the oils were heated to frying temperatures. It was verified that hydrogenation of soybean oil introduces a characteristic aroma and flavour that can be removed by deodorization; however, the aroma can be evident when oil is heated at frying temperatures and in fried food especially in more highly hydrogenated oils. This complex aroma/flavour characteristic of hydrogenation, which has been described as waxy, fruity, flowery, and crayon-like, can be easily detected in hydrogenation plant. Hastert (1996) stated that although no particular compounds have been identified as definitely responsible for this odour/flavour, isomeric unsaturated acids probably produce the sensory response. The

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intensities of the hydrogenation odour in the electrochemical samples were at weak levels, whereas for the commercial oils the same odour had intensity levels in the moderate range. 4.3.2. Innovative instrumental characterisation of the flavours of oils: oral processing

effects Volatile flavour Flavour encompasses volatile components that are sensed in the nose (aroma), non-volatile components that are sensed on the tongue (taste) along with compounds and structures that are perceived in the mouth as mouthfeel and/or texture. The aroma stimulus depends upon the concentrations of volatile flavour compounds in the region of the olfactory epithelium. Their concentrations are affected by the release rates of the compounds from the food in the mouth (van Ruth et al., 2008). Food composition affects volatile flavour release as volatile compounds may be dissolved, adsorbed, bound, entrapped, encapsulated or diffusion-limited by other food components. The relative importance of each of these mechanisms with respect to volatile flavour release varies with the properties of the volatile compounds and the physical and chemical properties of the components in the food (Kinsella, 1988). During eating, most foods undergo considerably physicochemical changes in the mouth. Mouth movements increase the surface area exposed to the air in the mouth, which in turn enhances the release of volatiles (van Ruth et al., 2003). Saliva has a diluting effect on the flavour compounds, whereas proteins in saliva have the potential of binding flavour compounds (De Wijk and Prinz, 2005). Saliva and chewing rate/force also interact. When volatile flavour compounds have been released in the mouth their transfer to the olfactory epithelium site depends on their travel through the oral and nasal cavity. The rate of opening and closure of the velum, the valve in the back of the throat preventing fluids to enter the nasal cavity, as well as retention by mucous in the respiratory tract affect the concentration of volatiles eventually being available for perception. The perception of flavour is not a single event but a dynamic process, involving a series of events. Usually after food is taken in the mouth, there will be a typical delay before anything happens, then a sharp rise in the concentrations of the stimulating molecules at the receptors, followed by a slower decline in concentration. After swallowing, the decline will continue, possibly very slowly, allowing for a long aftertaste, until the stimulating molecules have all diffused away from the receptors (Piggott, 2000). Simultaneously, there are short-term fluctuations in the concentrations carried to the olfactory receptors, caused by breathing (Baek et al., 1999). Volatile flavour analysis For the analysis of volatile flavour, one has to consider that no study on flavour is complete unless the consumer is considered as well as the chemistry and physics of the food. The techniques available to characterize the volatile flavours of oils and fats are designed to measure two types of volatile profiles: (a) the total volatile content of a food and (b) the profile of volatiles responsible for sensory perception. The (b) group which aims at mimicking the profile of volatiles in the area of the olfactory epithelium can be divided in three subgroups: (i) headspace analysis, (ii) mouth analogues, and (iii) in vivo measurements. The first group incorporates several extraction methods, and the approach is important for determining the formation/production of volatiles flavours or sometimes to get sufficient concentrations of volatiles for identification purposes. Most of the isolation technique used for determining the total volatile content of a food use some form of extraction or distillation, or

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combinations of both. These methods utilise differences in solubility in different solvents (extraction and chromatography) and differences in vapour pressures (distillation). Headspace analysis If oil is placed into a sealed vial and allowed to stand, the volatile compounds in the sample will leave the sample and distribute over the headspace around it. The concentration of the compounds in the headspace depends on factors such as the concentration in the original sample, the volatility of the compound, the solubility of that compound in the oil, the temperature of the sample, and combination of the size of the vial and the time the sample has been in the vial. At equilibrium, the relationship between the concentration of the volatile compound in the product phase and in the vapour phase can be expressed by Henry’s law. This law states that the mass of vapour dissolved in a certain volume of solvent is directly proportional to the partial pressure of the vapour that is in equilibrium with the solution. In static headspace analysis a part of the equilibrium headspace is injected into a gas chromatograph, either by syringe or by transferring a known volume of vapour from a sample loop attached to a valve. More recently, solid-phase micro-extraction (SPME) has become popular for headspace sampling. It involves extraction of volatile compounds out of the headspace onto a fused-silica fibre coated with a polymeric phase. After equilibration, the fibre containing the adsorbed or absorbed volatiles are thermally desorbed and subjected to gas chromatography (GC) analysis (Machiels and Istasse, 2003). It should be kept in mind that a second equilibrium between headspace and fibre is involved that may affect the volatile profile. Table 4.6 shows pre-concentration and chromatographic parameters used to quantify volatiles in virgin olive oil (Garcia-Gonzalez et al., 2007) In dynamic headspace analysis, a flow of gas is passed over the food to strip off volatiles (the gas phase is continually renewed). In a separate part of the apparatus, the volatiles are trapped from the gas stream in a solid adsorbent such as charcoal, Porapak Q, Chromosorb 101-1-5, or Tenax TA. These trapped volatiles are mostly analysed by GC combined with a variety of detectors.

Table 4.6. Pre-concentration and chromatographic parameters for the quantification of virgin olive oil volatiles (García-González et al., 2007)

Pre-concentration Chromatographic conditions

Sample amount 1g Tª injector 260 ºC

Temperature 40 ºC Tª detector 280 ºC

Time equilibrium 10 min Carrier-gas H2 1 ml/min

Time adsorption 40 min Column DB-Wax (60m × 0.25mm × 0.25μm)

Stirring Yes Injector FID

Stirring speed Oven temperature programme

Tª desorption 260 ºC Temperature Ratio t isotherm t total

Time desorption 5 min 40 ºC 4 min 4 min

Standard 4-metil-2-pentanol 200 ºC 3

ºC/min 10 min 67 min

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The alternative to chromatographic techniques is based on aroma sensors (García-González and Aparicio, 2002) that are resulted competitive detecting the presence of defective sensory attributes (García-González and Aparicio, 2003, 2008; García-González et al., 2004) or by using sensors that emulate tongues to detect the presence of taste perceptions in foodstuffs (Vlasov et al, 2002) like bitterness and pungency in virgin olive oil (Busch et al., 2006). Mouth analogues Mouth analogues have been developed to mimic volatile flavour release in the mouth more precisely and to consider changes in volatile release during eating. Only a few instrumental methods have incorporated the crushing, mixing, dilution, and temperature conditions required to simulate aroma release in the mouth. An example is the apparatus reported by Lee (1986) that involved a mass spectrometer (MS) coupled with a dynamic headspace system with several small metal balls which were shaken to simulate chewing. Roberts and Acree (1995) published their ‘retronasal aroma simulator’, a purge-and-trap device with a blender as base. Naßl et al., (1995) described a ‘mouth imitation chamber’, which consisted of a thermostated 800 ml vessel with a stirrer. The authors presented their ‘model mouth system’ for the first time in 1994 (van Ruth et al., 1994). The model mouth system had a volume of 70 ml, which is similar to the volume of the mouth, was thermostated (37ºC), artificial saliva was added, and a plunger with adjustable up-and-down and circular movement rates simulated chewing movements. The analysis of the volatiles isolated in mouth analogues is carried out by GC. In vivo analysis Initially in vivo analysis volatiles originating from the expired breath of people were concentrated, e.g., by cryo-trapping or on solid carbon dioxide or Tenax (Linforth and Taylor, 1993) with subsequent GC analysis. Real-time in vivo analysis requires detection with a high temporal resolution. Taylor and co-workers have been quite successful by modifying an atmospheric pressure chemical ionisation (APCI) source of a MS, which allowed high sensitivity and rapid response times for a few ions (e.g. Linforth et al., 1999). APCI-MS is a soft ionisation technique, which adds a proton to the compound of interest and does not normally induce fragmentation. Lindinger et al., (1998) reported an online gas monitoring technique for measurement of volatile organic compounds based on proton transfer reaction mass spectrometry (PTR-MS). PTR- reactions are used to induce chemical ionization of the vapours to be analysed. The sample gas is continuously introduced into a drift tube, where it is mixed with H3O+ ions formed in a hollow cathode ion source. Volatile compounds that have proton affinities higher than water (>166.5 kcal/mol) are ionised by proton transfer from H3O+, mass analysed in a quadrupole or time-of-flight MS, and eventually detected by a second electron multiplier (Aprea et al., 2006). PTR-MS is interesting for time-resolved analysis because it (a) requires no pre-treatment of the sample, (b) allows rapid measurements (typically 0.2s/mass), (c) allows direct quantification without additional standards, and (c) is extremely sensitive. The technique has been applied for in-nose analysis, for comparison of oral processing (Aprea et al., 2006), and for spatial intranasal analysis (Frasnelli et al., 2005).

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4.4. Consumers Acceptability of Edible Oils The most important factors in motivating consumer choice are probably the preference and satiety for specific sensory features, as well as the sensory perception of organoleptic quality (Rozin et al 2004, Booth and Shepherd 1988). These aspects can be studied through the intrinsic and extrinsic factors that influence food consumption behaviour (Fandos C., 2006). Their assessment is complicated because they deal with multi-faceted factors that influence the market including attitudes, lifestyle, social status and gender. Consumer studies on mass-consumed products highlight that preference and acceptability are not only based on biological factors (age, sex, taste, etc.) (Drewnowski, 1997), but also on “immaterial” and symbolic psycho-social factors, which include personality, mood, habits, desire of belongingness and safeguarding of tradition, territory, culture, health benefits, regardless of consumer attitudes, personal competence and product expectations, information source, etc. (Bone B., 1987; Saba and Di Natale, 1998; Siri G., 2004;). Nevertheless, it has often been observed that the importance of a certain attribute of a food product differs between cultures and nationalities due to the cultural differences in the perception of food products as well as the personal value given to them. In different European countries (U.K, France, Denmarck), cross-cultural differences have shown that English consumers have a more pragmatic orientation to food, Danish consumers place a strong emphasis on an enjoyable setting for all family members, whereas taste, odour, tradition and cooking results are more important in France (Nielsen et al., 1998). 4.4.1. Factors that affect the acceptability of virgin olive oil In relation to products considered to be “natural” by the consumer such as oil, strongly associated to the territory, there is a lot of “commercial literature” that, in order to widen the market, uses industrial studies to understand consumption (Istituto per la Valorizzazione dell'Olio di Oliva: Ivoli - 2005). Regarding the definition of “natural”, the most common association is a nostalgic memory from the past and, in the case of olive oil, this factor is present due to being a determining factor in the symbolic imagination of the consumer such as factors evoking the origin of food products, thus creating a favourable hedonic expectation in familiar consumers (Rozin et al., 2004). In relation to products such as olive oil, this factor is very relevant due to extra-virgin olive oil having an important role as a typical example of a Mediterranean production whose typicality of its raw material and the manufacturing technology is strongly affected by its origin. In recent years, the European Union has recognised many protected extra virgin olive oils (protected designation of origin or protected geographical indication). According to this view, the region of origin has an indirect impact on consumer preference as a quality cue. This is a product characteristic that consumers can identify prior to consumption and is perceived as related to others attributes of the product. Several works tend to highlight that the information about the country of origin has a “tremendous influence on the acceptance and success of products” as well as geographical origin having further direct roles in determining consumer behaviour due to its symbolic and affective roles. Others studies have claimed that health benefits have had a significant influence on the collective imagination (Frost Larsen et al., 1999; Psaltopoulou et al., 2004). The multiplicity of hedonic and sensorial expectation factors (colour, taste, etc.) influence

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product acceptability (Del Giudice and D’Elia, 2001) and can be identified as intrinsic and extrinsic cues. The first cues refer to characteristics such as the physical features of the product, for instance its colour, shape, bitter, hot, sweet, viscosity, nutritional components, effects on the product due to technological processes, etc. While, the second include price, brand name, packaging, selling place, origins, typicality, etc. The “expected” and “experienced” or “perceived” quality can only be assessed through actual consumption that influences the consumer’s sensorial response (Caporale et al., 2006). With regard to the hedonic factor, this is generally “weakly” expressed through general phrases such as “it’s good” and “I like it” and can therefore, create discrepancy between expectations, perception and acceptability of the product (Drewnowski, 1997; Caporale et al., 2006). In a new consumer culture of the olive oil market, where people make many choices based on the intangible, “immaterial” and symbolic characteristics of the product, choices based on the positives health benefits, often at the centre of the collective imagination, with many studies on the effects of olive oil on the heart as well on cancer (Frost Larsen et al., 1999; Psaltopoulou et al., 2004), are relevant too. Many other studies show consumer attitudes, product expectations (Saba and Di Natale. 1998), as well as interest for brand name, price, typicality and affective factors, such as the origin of food products (with strong symbolic value role) that can directly affect the intent to purchase. “Familiar” organoleptic characteristics are also imporatnt (Del Giudice and D’Elia, 2001; Ivoli, 2005; Caporale et al., 2006; Stefani et al., 2006). If the product fulfils the “familiar” expectations of the individual, the consumption is then facilitated in a positive sense (Caporale et al., 2006). Finally, in the olive oil market, many claims to health benefits or quality components are associated to persuasive commercial factors such as packaging and selling strategies (environment, atmosphere, colours, odours, etc.). These factors may influence social consumer behaviour but they are often associated to ambiguity and inconsistent performance between personal knowledge and scientific information, making choice complex showing that factors such as discounts, promotions, exposure, distributor price, etc., are not a sign of neither health benefit nor quality factors. (Del Giudice and D’Elia, 2001; Ivoli, 2005). 4.4.2 Factors that affect the acceptability of seed oils Peanut, soybean and rapeseed/canola oils Scientific evaluations of the acceptability of seed oils have been applied to products, based on various factors (type of cooking use, fried, conservation, habits, territory, price, etc). This therefore creates many difficulties in consumer studies. It is worth taking into consideration the studies which compare products made from various types of vegetable oils (Hekmat and Haines, 2003; Stephens et al., 1997). An example is the study that compares rapeseed oil with carotino oil in fried products (Hekmat and Haines, 2003). There were no significant differences in appearance, flavour, texture, and overall quality between zucchini, shrimp, and scallop samples fried in Carotino oil versus canola oil. However, compared to carotino oil, rapeseed oil is preferred by consumers, especially when cooking, not only due to its “mild taste” but also its neutral colour, in contrast to that of carotene, which has a bright red colour due to its high level of carotenoids, with 90% in the form of alfa-beta carotene that is a precursor of vitamin A. It is also worth noting that once consumers recognize the nutritional and “health benefits” of carotene oil, they might be willing to incorporate it into their diet due to these “health” factors (Hekmat and Haines, 2003).

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The aim of the study by Stephens (Stephens et al., 1997) was to evaluate the acceptability and preference of crude oils to their dietary uses. Therefore, crude oils made from peanut, rapeseed and two soybean cultivars were stored over time at different temperatures and used in bread and salad dressings and subjected to sensory evaluation. The sensory panel (about 25 individuals) evaluated samples of white bread as well as lettuce dressed with a vinegar/oil salad dressing using a 9-point hedonic scale. This study showed that, in general, the odour of bread made with canola oil was less acceptable than that of bread with peanut or soybean oils. Soybean oil-bread was similar to that of bread with peanut oil. There were minimal differences in odour acceptability of the breads with the two soybean oils compared to peanut oil. For flavour acceptability, there was a strong disliking for bread with canola oil compared to the other oils. Studies on the effect of oilseed type on the sensory attributes of salad dressings made with crude oils, have given the following: 1) overall odour intensity of salad dressing made with either canola or peanut oil decreased with oil storage time and 2) overall odour acceptability and flavour acceptability of salad dressing made with peanut oil decreased over oil storage time and crude soybean oil, stored at 23 °C, was more acceptable in sensory evaluation than were crude peanut and canola oils. Preference ranking for breads made with the crude oils indicated that soybean 'Century 84' was the most preferred oil for use in a bread type product. Overall preference rankings for salad dressings made with crude oils were similar to the rankings of breads. No differences were noted with the breads when using preference ranking to determine if any effect occurred within an oilseed type over time. The preference rankings were significantly different over storage time for the salad dressings, only for the 'Century 84' soybean oils. In conclusion, the crude soybean oil, stored at 23°C was more acceptable in sensory evaluation than crude peanut and canola oils. The study carried out by Nielsen et al on “consumer perceptions” was an online, self-administered investigation for the U.S. Canola Association and Northern Canola Growers Association on the consumer knowledge of canola versus other oils. It highlighted the need for “further consumer education about the oil”. Several German studies have highlighted that virgin cold rapeseed oils are becoming more popular in Germany. The “Germany Society for Fat Science” (DGF) therefore created, in 2006, the DGF Rapeseed Oil Award for virgin rapeseed oil in order to bring about a better performance of this product (Matthaus et al., 2008), with it becoming more interesting for human consumption due to the added value of this product. The price of one litre of virgin edible oil ranges between 4 and 16 Euros, in comparison to 0.75 Euros for one litre of virgin rapeseed oil for fuel. Thus, the value of virgin edible rapeseed oil is very attractive for the producer and an important feature for consumers. The sensorial quality of virgin rapeseed oil is directly correlated to the value of the oil for the consumer and determines the success or failure of the product in the market due to the typical characteristic taste and smell which resembles that of asparagus, cabbage or fresh, green vegetables as well as the intensive colour of such virgin oils. Sunflower oil There are only two papers in literature that deal with the consumer study of sunflower oil. They were developed in Argentina and Brazil, two countries that have important roles in the production and exportation of sunflower oil. The first scientific study was carried out in Argentina (Ramirez et al., 2000). The aims of the study were: 1) to correlate consumer acceptability with a trained sensory panel to define the sensory failure of commercial sunflower oil; 2) to study the effect of storage temperature and light exposure on the sensory shelf life of a commercial sunflower oil showing the “shelf life” for the product stored at 45°C

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and exposed to light was 60 days and approximately the double of the days if the oil was stored in the dark (Ramirez et al.2000; Costa et al., 2000); 3) to study the acceptance of genetic modification to the plants from which the oil is derived and new technologies by European consumers (Hascim et al., 1996). On the acceptance of emerging food technologies, it has been demonstrated that packaging plays an important role in consumer food choice. It was assessed to be a “purchase intention” with sunflower oil, with the following results: 11% included respondents who made purchase decisions based on labelled illustration. These respondents expressed a greater intention to purchase products with the sunflower image on the label. 25% of the respondents based their purchase intention on information, price, and brand name. These respondents were more likely to purchase sunflower oil associated with “environmental friendly processes” and were also prepared to pay a higher price for the product. These respondents also preferred familiar brands. 19% of the respondents reacted more strongly to information as the most important contributor to purchase decision (Green and Srinivasan, 1978; Punj and Stewart, 1983). Corn oil New food habits due to new lifestyles where people eat out often lead to new alimentary trends characterised by street food, snacks, sauces, catering, happy hours, high use of industrial food, take away food, precooked food, which are all generally cooked with corn oil. The “new food behaviour” promotes globalization, also bringing obesity and food disorders (compulsions, altered feeling of hunger-satiety) and damaging traditional behaviours, with negative influence on sensory sensation especially for Mediterranean cooking which implies the use of extra virgin olive oil and the use of genetic engineering in food production. For those reasons, European consumer perceptions, associated to the risk concept and the consumption of corn oil, particularly in Europe, due to the GMO factor, are in decline (Schenkelaars, 2001; Sinemus and Egelhofer, 2007; Hoban, 2004). New biotechnologies need to be more widely accepted by the general public and EU organisations are devising new strategies to communicate the benefits of biotechnology in agriculture to a broader public (Schenkelaars, 2001; Kynda et al., 2004). The 2000 Eurobarometer (Hogan, 2000) shows the reasons for consumer concerns on GM food as "GM food is simply not necessary". Respondents thinking that food production is a useful application of biotechnology decreased from 54% (1997) to 43% (Hogan, 2000) and "the level of knowledge and familiarity with biotechnology” are not so decisive in shaping general attitudes. However, only 11% of the respondents feel adequately informed on biotechnology. The “cultural factors” and the attachment of consumers to their national food is an important factor in the process of acceptance of modern food technology (Menrad, 1999) and only 18% of the respondents judge GM labelling useless. 8% do not have an opinion and 74% favour a clear labelling of GM food (Karlheinz, 1997; Hogan, 2000). 53% of the respondents say that they would pay more for non-GM food, 36% would not or say “yes” when offered the opportunity with a price differential in favour of GM (EC Project, 2008). 4.4.3 Acceptability of argan oil Argan oil is used as a seasoning, being added to already cooked or uncooked food and should be considered an "exotic product". Between January and July 2005, on the basis of Article 5 of the Novel Food Regulation, the European Commission required new food manufacturers to submit to the Committee the ingredients in order to ensure that they are

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similar to products already on the market for the "supply chain" for the characteristics of their "processes". Among these new foods, argan oil must be "tagged" in the EU and the Committee has requested the opinion of various committees on a product derived from Argania spinosa. One of the committees consulted, the French Action Committee, has given a favourable opinion on the marketing of argan oil in Europe, while it considers the current studies insufficient to support the insertion of health claims on the label (Committee paper, 2006; Hermann, 2004). Traditional non-EU foods well known in their lands of origin but unknown to European consumers require studies on the sensory acceptability for the European food habits. Previous reports have also identified some concerns about this category of product (EU Novel Food Regulation, 2005; Hermann, 2004). Specifically with regard to argan oil, in the paper on evaluation of the product for deep-fat frying, it is stated that the oil has a high dietetic and culinary value because it contains a high percentage of unsaturated fatty acids and is rich in flavour and aroma (intrinsic factors). In addition, argan oil can substitute olive or cottonseed oils in deep-fat frying and the aroma, flavour, oxidative stability and the health benefits might ‘compensate’ for the high cost of the oil (extrinsic factors), but sensory evaluation of fried French potatoes were not studied, and additional work is needed (Yaghmur et al., 2001). The “virgin” oil is characterized by a golden colour and a hazelnut taste. Besides frying, it could also be used uncooked and eaten with bread, couscous, meat tejine, fish tapine and salads. When mixed with almonds and honey, as is the local tradition, it produces a cream called “amlou”, a high energetic value food which is generally spread on bread. (Charrouf and Guillaume, 2007; Rea, 2007). Field research has pointed out some benefits on human health, for both nutritional and food attributes as well as aesthetic ones in the cosmetics sector (Charrouf and Guillame, 2007). Argan oil may be obtained either by traditional processing or more recent mechanical procedures, which use any volatile lipophilic solvent. After evaporation of the solvent, the oil has unsatisfactory organoleptic properties compared to the traditional or press extraction. The product obtained from this process is used for cosmetic purposes. The “virgin” oil, used for the alimentary market, has purity and quality standards defined by the Moroccan Normative. At present, Tunisia is the only African country with its own organic (EU compatible) standards, certification and inspection system. While, Morocco has made some progress in developing its own standards. 4.4.4 Acceptability of rice bran oil Rice bran oil is extracted from the germ and inner husk of rice. It is popular as a cooking oil in several Asian countries. Rice bran oils are obtained by solvent extraction of commercial rice brans. There is unfortunately only one study regarding consumer studies of rice bran oil. This oil is blended to improve its thermal stability and consumer preference. Repeated frying triggers the formation of volatile and non-volatile products that have an effect on the texture of fried foods. Raj and collaborators (Raj et al., 2006) have investigated a blend constituted by 70 parts of rice bran oil and 30 parts of palm oil, evaluating fried poories obtained after 1st - 4th - 7th and 10th frying for consumer acceptance. The study was carried out on 50 trained and non-trained panellists who were familiar with the product “poori”. A 7 point hedonic scale was used. A score of 7 was given for “like very much” and a score of 1 for “dislike very much”. The cumulative frequency was calculated and an Ogive curve was drawn plotting cumulative frequency (%) versus hedonic ratings.

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The study showed that the oil blend formulation was acceptable to be used as frying media. Around 24% of the population rated it “like very much,” 38% “like moderately” and 20% “like slightly.” Because more than 80% fell into the “like” category, the product is acceptable as a frying oil.

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4.5. Concluding Remarks • It is important to distinguish between extra-virgin, virgin and blended olive oils (the last, in Italy is noted as “olio di oliva”). The first two are completely different to the blended olive oil, due to the 0.5 to 2% unsaponifiable compounds, 98 to 99.5% are fatty acids, which are esentially the same in the three oils. The refined oil, from lampante oil, during the refining process, lose all the unsaponifabile compounds, it became blended olive oil only after adding a little amount of virgin or extravirgin olive oil. The lack of unsaponifible compounds in the blended olive oils, determine the loss of organoleptic and some of the healthy properties. • 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, while there seems to be very little regarding that of seed oils. • There are a significant number of studies carried out on the sensory characterization of olive oil. In the same time, there are few published studies dealing with seed oils. • 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 there 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. • Comparison studies between virgin olive oil and seed oils, have shown that the seed refined oils generally have a bland odour and flavour, in contrast to olive oils, which are rich in both. • There are no scientific published studies on the sensory properties of argan oil to date. While, in relation to rice bran oil there are very few, which tend to study blends rather than single oil, thus making it difficult to establish the real sensory characteristics. • There are only a few studies on consumer evaluation of the many factors involved in consumer acceptance. It is important to have the appropriate strategies and actions in order to manage consumer expectations, as well as understand the determinants of the choice of oils.

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CHAPTER 5

Risk and benefits of edible oil consumption on human

health

Authors Contributors GIACCO, R.1 ABAZA, L.2

RUSSO, G.L.1 BALLUT, M.3

CHARROUF, Z.4

COMBE, N.3

DEBRUYNE, I.5

DE GIULIO, B.1

MATTHÄUS, B.6

PERRI, E.7

ROSSIGNOL-CASTERA, A.3

TEDESCO, I.1

1Institute of Food Science (ISA-CNR), Avellino (Italy) 2Centre de Biotechnologie de Borj-Cedria (CBBC), Hammam-Lif (Tunisia) 3French Institute for Fats and Oils (ITERG), Pessac (France) 4Faculté des Sciences Université Mohammed V - Agdal (FS-UMV-Agdal), Rabat (Morocco) 5ID&A, Izegem (Belgium) 6Max Rubner-Institute, Federal Research Centre for Nutrition and Food (BFEL), Münster (Germany) 7Centro di Ricerca per l’Olivicoltura e l’Industria Olearia (CRA-OLI), Rende ( (Italy)

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Abstract

One of the aims of the MAC-Oils project was to compare the risks and benefits of eight selected oil consumption on human health. The biological effects of these oils depend on their fatty acid, phytosterols and antioxidants content. Evidence from epidemiological studies suggests that replacing long-chain saturated and trans fatty acids with MUFA and PUFA in the diet is able to substantially reduce the risk of CHD. Intervention studies report that this modification decreased by 24% cardiovascular combined events in trials of at least two years’ duration. In fact vegetable oil consumption improve many cardiovascular risk factors such as lipid metabolism, insulin sensitivity, blood pressure, oxidative stress and inflammation. Both MUFA and PUFA rich oils enhance insulin sensitivity and have a comparable cholesterol lowering effect; however, olive and canola/rapeseed oils do not reduce HDL cholesterol, due to high oleic acid content, whereas sunflower, soybean and all oils rich in linoleic acid have a slightly more triglyceride lowering effect. Also phytosterols contained in high amounts in corn and rice oils contribute to reduce plasma cholesterol levels. Compared to saturated fat, consumption of the all selected vegetable oils reduce blood pressure even if the extra virgin olive oil, due to its high content in polyphenols, more clearly exhibits this beneficial property. Olive and Canola oils also enhance the resistance of LDL to oxidative modifications, however this effect is more evident for extra virgin olive oil rather than for other traditional or novel MUFA rich oils. Compared to other oils, consumption of Canola/rapeseed oil decreases plasma concentrations of inflammatory markers; in fact it contains α-linolenic acid which is a precursor of anti-inflammatory and anti-thrombotic molecules. Less evidence is available on the effect of vegetable oil consumption on cancer development. However, it has been reported that a high dietary fat intake based on a high saturated and n-6 PUFA consumption is associated with a high risk of cancer; contrarily oleic and n-3 PUFA consumption in most studies is associated with a low risk of some types of cancer. In conclusion, unsaturated fatty acids, essentially those monounsaturated, seem to play a key role not only in the prevention of cardiovascular diseases but also of certain types of cancer. However the complexity of genetic and environmental factors involved in the development of cancer prevents us from drawing a conclusion on this issue, although experimental studies in animals support a potentially favourable effect of MUFA and olive oil. The families of molecules which are generally classified as “minor” or “unsaponifiable” compounds have been described in details in Chapter 3. From a biological point of view, the most significant “minor” compounds in edible oils belong to the classes of tocopherols, sterols and phenols. The amount of tocopherols ingested, independently by the source of vegetable oils used in the diet, should be enough to sustain the daily allowance of vitamin E. Respect to the content of phenols in edible oils, an important aspect of tocopherols activity regards the potentiation of their antioxidant activity by a synergistic interaction with phenols. The eight edible oils considered in MAC-Oils study contain an average of total sterols ranging between 738 (olive oil) to 18000 mg/Kg, with rice bran and corn oils presenting the highest concentrations. The positive trend on the use of plant sterols for human health started with the demonstration that this class of compounds was found to prevent adsorption of dietary cholesterol. The actually minimum daily intake in U.S.A. has been fixed in about 1.6 g/die. The cardioprotective activity of phytosterols is due to their ability to compete with cholesterol

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for space within bile salt micelles in the intestinal lumen thereby reducing cholesterol absorption. The cholesterol-lowering activity of phytosterols does not strictly depend on the specific chemical structure of single molecules, but, more convincible, by their total uptake (quantity) and bioavailability. Scientific evidence indicates that a real benefit in lowering cholesterol may derive from a regular consumption of phytosterol-rich oils at the recommended daily intake. One of the most abundant sterol in many edible oils, β-sitosterol, possesses antiproliferative activity being able to inhibit the growth of several specific types of tumor cells in vitro and decreases the size and the extent of tumor metastases in vivo. The phenolic compounds of olive oil in the Mediterranean diet have been associated with a reduced incidence of heart disease. Hydroxytyrosol has antithrombotic activities and is a potent scavengers of superoxide anions, protecting cells lines against oxidative insults. Together with its cardioprotective activity, hydroxytyrosol is reported to exert chemopreventive effects. These results support the hypothesis that olive oil phenolic compounds may possess both cardioprotective and chemopreventive activities. However, the fundamental importance of their bioavailability and metabolism has been sometime neglected. In fact, less than 10% of phenols, or their metabolites, ingested are found in plasma, where concentrations barely reach 1 μM. The general low bioavailability of single phenolic compounds, together with the complex transformation reactions they undergo, makes difficult a cause-effect analysis. On the contrary, when ingested in the whole food, their “combinatory” effect might suggest a rationale for dietary prevention. A correct diet, as a whole, can be “preventive” without necessary focusing the interest on a single molecule. This view generated the development of “combination prevention”, intending that low doses of biologically active (cardioprotective and/or chemopreventive) agents differing in the mode of action may synergize increasing efficacy and minimize toxicity.

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5.1 Introduction Oil has been an important source of fat in human nutrition since ancient times. In Roman times only olive oil was used as cooking and dressing oil, as reported by Marrone, Pliny, Horace and Stradone. Linguistic, literary and archeological evidence indicate that the olive plant was already cultivated and oil extracted from its fruit back in the VIII and VII centuries before Christ. More recent is the use in human nutrition of vegetable oils extracted from seeds, such as sunflower, soybean, corn, peanut, canola/rapeseed, rice and argan. While the effects of olive oil on human health have been widely studied, those of other oils are less known and may therefore their use may conditioned by prejudiced opinions. Therefore, one of the aims of the MAC-oils project was to compare the risks and benefits of eight selected oil consumption on health of consumers. As reported in Chapter 3 ninety-nine percent of oils are made up of a mixture of fats (saturated, mono and polyunsaturated fatty acids, the latter classified in PUFA n-6 and PUFA n-3) and the remaining 0.5%-1% is made up of minor components such as molecules with antioxidant activity and phytosterols. Since most of the biological effects of oils on health depend on their chemical composition, the first section of this report will examine the effects of oil fatty acid component on human health while the second will examine the effects of minor component. 5.2. The Effects of the Fatty Acids in Edible Oils on Human Health All the eight vegetable oils evaluated in the MAC-oils project have a relatively low content of saturated fatty acids (SAFA) and a moderately high content of mono (MUFA) and polyunsaturated (PUFA); therefore their unsaturated/saturated fatty acid ratio (U/S) ranges an average from a minimum of 4.0 for peanut oil to a maximum of about 15.7 for canola/rapeseed oil. However, these oils are different for the proportion of MUFA (oleic acid) and PUFA n-6 (linoleic acid, an essential fatty acid). Olive and canola/rapeseed oils are the richest in MUFA (an average about 70%), sunflower, soybean and corn oils the richest in PUFA n-6 (an average 60%) and peanut, argan and rice oils are those with an intermediate MUFA and PUFA n-6 content. Canola/rapeseed and soybean oils are those with the highest content in α-linolenic acid (ALA) − an essential fatty acid of the n-3 PUFA family, however the n-6/n-3 PUFA ratio of canola is lower than that of soybean oil (on average 2.2 vs 7.7). However, it is interesting to underline that over the last decade, several soybean varieties have been developed, with a different fatty acid profile. The first of its kind is the high oleic soybean oil. These transgenic soybean varieties produce oils having from 80% to over 90% oleic acid. In addition to high oleic soybean oils, nowadays, high oleic sunflower oil varieties are available in the market. Recently, some food industries in the US have begun to produce novel varieties of soybean oils with very low linolenic acid content, even in combination with reduced or enhanced saturated fatty acid composition. Since all traditional edible oils investigated in the MAC-oil project are low in saturated fatty acids, their biological effects essentially depend on the difference in their proportion of oleic, linoleic (n-6 PUFA), α-linolenic (n-3 PUFA) acids, trans fatty acids and on the phytosterols content. According to consensus criteria to support claims for healthy foods, a critical review of scientific literature regarding each selected oil was performed and the effects of either

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whole oil or its fatty acid component on biological parameters in human, animal and in vitro studies were analysed. In particular, we considered the effects of dietary fat or oil consumption on hard endpoints like cardiovascular disease (CVD) morbidity and mortality, total mortality for any cause and cancer mortality, where applicable, or surrogate endpoints such as blood lipids, insulin sensitivity and blood glucose, blood pressure, oxidative stress and systemic inflammation. 5.2.1 Cardiovascular diseases The impact of the quality of dietary fatty acid on the risk factors for atherosclerosis in humans is supported by wealth of data. There is no direct evidence from observational studies and/or clinical trials that a specific vegetable oil consumption is related to lower risk of death for any cause. Only for olive oil are there studies that have investigated the direct effects of its consumption on CVD in humans. Epidemiological evidence Few data are available on the direct relationship between vegetable oil consumption and risk of cardiovascular events. In fact only two hospital based case-control studies have evaluated the association between olive oil consumption and risk of first non fatal myocardial infarction (MI). They provided discordant results: Fernandez-Jarne et al., (2002) reported 82% reduction of MI in the highest quintile of olive oil consumption whereas Bertuzzi et al., (2002) observed no effect. Conversely, a lot of data are available on the association of dietary fat quality and quantity and CHD prevalence and mortality. Most observational studies, particularly prospective cohort studies, show that a diet low in SAFA and trans fatty acids and rich in MUFA or PUFA is associated with lower risk of mortality for coronary heart disease (CHD) and total cardiovascular (CV) diseases (Pietinen et al., 1997; Oh et al., 2005; Kris-Ethereton et al., 1999; Kris-Ethereton et al., 2004). The Seven Countries Study was the first observational study to suggest that a MUFA rich diet, low in SAFA, protects against CHD (Keys, 1980). In fact, in this study the populations living in Mediterranean countries, where the main source of dietary fat is olive oil and consumption of SAFA is low, had a low prevalence and mortality for CHD. In contrast, some studies did not confirm the association between MUFA intake and low CHD risk ( Kromhout et al., 1995; Garcia-Palmieri et al., 1980), but their validity is limited by the failure to control for confounding variables. For what concerns the association between PUFA intake, specifically linoleic acid, and cardiovascular disease morbidity and mortality, two cross-sectional studies found that PUFA intake, adjusted for dietary SAFA, was inversely associated with cardiovascular disease mortality (Hegsted, 1998 and Artaud-Wild et al., 1993) whereas a third one found no significant association (Kromhout et al., 1995). Data from prospective studies are not consistent; in fact, some studies performed in men and women during a follow-up that ranged from six to twenty years (Garcia-Palmieri et al., 1980; Kromhout and de Lezenne Coulander 1984; Posner et al., 1991) failed to find any association between dietary PUFA intake and coronary heart disease whereas others observed a strong (Shekelle et al., 1981) or borderline (Kushi et al., 1985) significant inverse association. Also in this case the inconsistency of the finding from prospective epidemiological studies may be partially due to small sample size, use of different diet assessment methods and to the absence of adjustments for confounding

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variables. These limitations are particularly significant in the studies which did not confirm the association between MUFA intake and low CHD risk. The Nurses’ Health Study (Hu FB et. al., 1997), a more recent study performed in a large sample of women living in the USA and followed up for 14 years, reported that both MUFA and PUFA were associated with low CHD risk. Compared with equivalent energy from dietary carbohydrates, a 5% increment in energy from MUFA or PUFA reduced the risk of coronary disease by 19% and 38 %, respectively. The same population was also examined to seek a possible association between P/S ratio of a diet and risk of CHD. The multivariate analyses showed that for each 0.2-unit increment in the ratio, there was a decrease in CHD risk; therefore at the highest P/S ratio (0.72) compared to the lowest (0.23), CHD risk was 42% lower (Hu et al., 1999). These results suggest that replacing saturated fat with PUFA substantially reduces the risk of CHD. It is underlined that oils rich in PUFA are highly sensitive to oxidation and undergo a mild hydrogenation process which increases the melting point, shelf life and helps to reduce production costs. However, as drawback, this process induce a transformation of part of unsaturated fats into trans fatty acids which have detrimental effects on CHD risk factors. In fact, the results of some prospective and retrospective case-control studies which evaluated the relationship between trans fatty acid intake and CHD risk showed that the high trans fatty acid intake increase CHD risk and the pooled prospective and retrospective studies showed an increase of the relative risk of CHD by 29% (Mozaffarian et al., 2006). The negative cardiac effects induced from trans fat is confirmed by a population-based case control study of 179 cardiac arrest patients and 285 community controls. In this study higher red-cell membrane levels of trans-fatty acids, especially trans isomers from partially hydrogenated vegetable oils, were associated with a significantly increased risk of primary cardiac arrest (Leimatre et al., 2002). The association of α-linolenic acid (ALA) intake and cardiovascular endpoints has been investigated in several epidemiological studies. Two prospective studies have reported that ALA intake, adjusted for total fat and other confounding variables, significantly reduced acute myocardical infarction (AMI) by 59% (Ascherio et al., 1996) and total ischemic heart disease (CHD) by 45% (Hu et al., 1999). It is interesting that in the same cohort of the Health Professional study (Ascherio et al., 1995) the intake of fish oil was not associated with CHD risk, suggesting that the cardioprotective effects of ALA itself may be different from those of fish oil. In three cross-sectional studies, higher ALA intake (measured in the diet, plasma or adipose tissue) was inversely related to thickness of the internal and bifurcation segments of the carotid arteries (Dejousse et al., 2001), non fatal AMI (Baylin et al., 2003) and a tendency to lower risk of IHD (Lemaitre et al., 2003). Therefore, there is consistent evidence that ALA is beneficially associated with CHD morbidity and mortality. Experimental evidence suggests that the potential cardiovascular benefits of ALA can be offset by dietary linoleic acid (LA) and long chain n-3 PUFA (EPA and DHA) concentrations. However, the interactions of different types of PUFA in relation to CHD risk are not well established. These relations are of considerable public health importance, given the ubiquity of n-6 PUFA in the diet and the strong evidence for beneficial effects of ALA and long chain n-3 PUFA on CHD risk (Russo, 2009). LA and ALA are metabolized to their respective essential metabolites by alternate desaturation-elongation reactions by the same set of Δ5 and Δ6 desaturases and elongases, which are therefore in competition. Normally, Δ5 and Δ6 desaturases and elongases exhibit affinity to metabolize n-3 more than n-6 PUFA provided that both are present in optimal ratio. Depending on this ratio, different classes of eicosanoids are generated. Eicosanoids derived from n-6 PUFA have opposite metabolic properties to those derived from n-3 fatty acids. Consequently, a balanced intake of both n-6 and n-3 PUFA is essential for health. The optimal levels of these fatty acids, both absolute and relative to each other, remain uncertain.

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Estimates of minimum requirements for n-6 and n-3 PUFA are in the range of 1% to 2% energy and 0.2% to 0.5% energy, respectively, and it has been proposed that the ratio between the two series should be between 5:1 and 10:1. In an early study, a ratio of LA to ALA of 4:1 or less was shown to be optimal for the elongation of 11 g of ALA to 1 g EPA (Indu & al., 1992). In Western diets, the mean ratio of LA to ALA is about 14–16:1 instead of the presumably healthy range of 1–5:1. Mozaffarian and coworkers (2005) in their study reported that ALA intake was associated with a reduced total CHD risk irrespective of n-6 PUFA intake. Conversely, this association was influenced by the dietary intake of long chain n-3 PUFA. In fact, in the cohort of men tested, each 1g/day of ALA intake was associated with about 50% lower risk of non fatal MI and total CHD when long chain n-3 PUFA intake was very low (<100 m/day). These results suggest that ALA may reduce CHD risk and that the relative intake of n-6/n-3 fatty acids may be less important than the absolute intake of long chain n-3 PUFA. In conclusions, evidence from epidemiological studies suggests that replacing long-chain saturated fat with MUFA and PUFA is able to substantially reduce the risk of CHD without adverse effects, at least when the dietary PUFA content varies from 2% to 10% of daily energy and provided that the n-6/n-3 PUFA ratio is not too high. Intervention Studies Several studies have been conducted to evaluate whether the occurrence of CVD can be influenced by changes in the fatty acid composition of the diet, in particular, by replacing SAFA with MUFA or PUFA. However, until now, no trial has compared the effects of a MUFA with n-6 PUFA- rich diet on CVD. Among the studies evaluating the effect of replacing dietary SAFA with PUFA, on CHD events in primary prevention, without changes in total fat intake, both the Los Angeles Veteran Hospital Study (Dayton et al., 1969) and the Finnish Mental Hospital study (Turpeinen et al., 1979), showed a reduction in CHD rate in the intervention group respectively by 31% and 43% during eight and six years of follow-up; indeed, this decrease was associated with reduced plasma cholesterol levels. In both studies, the linoleic acid content in adipose tissue increased substantially, indicating compliance to diet. On the contrary, data from the Minnesota study (Frantz et al., 1989) reported no reduction in CHD although in the intervention group serum cholesterol decreased by 14%. However, the duration of this study was shorter (only 4.5 years) and the P/S ratio of the intervention diet (P/S=1.6) was below the specified goal. Two secondary prevention trials in patients with MI on a high PUFA rich diet produced controversial results. In the Oslo Diet-Heart study (Leren 1970) the PUFA rich diet decreased significantly fatal MI by 44% after eleven years. On the contrary, Morris et al., (1968), in a study using soybean oil observed a 16% reduction in serum cholesterol after six months and 12% less (non significant) coronary events after four years, but no effect on mortality for CVD. However, two angiographic studies in patients with coronary disease−the Leiden Intervention Trial (an uncontrolled study) (Arntzenius et al., 1985), which compared the effect of a diet rich in PUFA and at high P/S ratio with a vegetarian diet, and the St. Thomas Atherosclerosis Study (STARS) (Watts et al., 1992), which evaluated the effects of a diet with a moderate amount of total fat (27%) alone or associated to cholestyramine (an cholesterol-lowering medication)–reported a significantly slower progression, or in some cases an increased regression of coronary artery lesions. A quite important finding with regard to the effects of MUFA and PUFA on CHD stems from the Lyon Diet Heart Study (de Lorgeril et al., 1994). In this study, a usually recommended diet to prevent CHD was compared to a Mediterranean diet rich in MUFA and ALA (from olive oil and canola oil) and low in n-6/n-3 PUFA ratio (4:1) in relation to coronary events and death in myocardial infarction survivors. This type of diet was able to

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reduce the incidence of a second myocardial infarction as well as overall mortality by 70%. Since the Mediterranean diet not only differed in the fatty acid composition, but also contained more bread, vegetables an fruits and less SAFA and meat compared to the control diet, it is hard discriminate which of these factors contributed most to the reduction in CHD events. It is likely, however, that it was a combination of all the factors (thus also the fatty acid composition characterized by high MUFA and ALA) that contributed to the effect. There is evidence that at least ALA played a role in the reduction of risk; in fact after 4 years the plasma ALA content was significantly associated with an improved prognosis (de Lorgeril et al., 1999). A systematic review of 27 randomized controlled trials that evaluated the effects of fat reduction or substitution of SAFA with MUFA and PUFA intake on CVD risk, excluding multiple risk intervention trials, has shown that a reduction or modification of dietary fat intake decreases cardiovascular combined events by 24% but only in trials of at least two years’ duration, whereas the effect on total mortality is little (Hooper et al., 2001) (Table 5.1). The reduction of events was observed in population both with high and low cardiovascular risk. Therefore, results of overall studies seem to suggest that a partial substitution of SAFA with MUFA and PUFA and the addition of ALA in the diet reduces CHD morbidity and mortality in both primary and secondary prevention; however to better clarify the role of fat quality on cardiovascular morbidity and mortality there is need for studies comparing the effects of a MUFA with an n-6 PUFA- rich diet.

Table 5.1. Effect of reduction or modification of dietary fat intake on total and cardiovascular mortality (Meta-analysis of intervention trials) Combined CV events

(RR) Total

Mortality (RR)

All trials 0.84*

(0.72 – 0.99) 0.98

(0.86 – 1.12)

Trials with mean follow-up >2 years

0.76* (0.65 – 0.90)

0.93 (0.75 – 1.15)

*significant; ( ) 95% Confidence Interval; RR= Relative Risk. Modified from Hooper, BMJ 2001

Risk factors The mechanisms whereby vegetable oils may prevent cardiovascular disease have been investigated in a large number of intervention studies. The results strongly suggest that dietary oils are able to influence plasma lipid profile, blood glucose and insulin sensitivity, blood pressure and endothelial function and finally oxidative stress and inflammation − two new risk factors for atherosclerosis and cancer. Plasma Lipids. High blood lipid levels play an important role in the development of atherosclerosis. It has been reported that the consumption of a MUFA-rich diet did not affect total cholesterol levels whereas PUFA decreased and SAFA raised them. However, the results of trials on the effect of monounsaturated and/or polyunsaturated-rich oils on plasma cholesterol and lipoprotein levels are controversial and have been analyzed in various meta-analyses (Truswell and Choudhury, 1998). Mensik and Katan (1992) performed a meta-analysis of 27 trials showing that the consumption of MUFA compared to PUFA had a lesser LDL cholesterol lowering effect but increased HDL cholesterol to a greater extent. Clarke et

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al., (1997) in a meta-analysis of 63 trials reported that a MUFA diet produced no significant effect on total and LDL plasma cholesterol levels while these parameters were increased by SAFA and decreased by PUFA. However, results of another meta-analysis of only 14 trials provided different conclusions, as it showed that the replacement of SAFA by oils enriched in MUFA versus PUFA had similar effects on total, LDL and HDL cholesterol, whereas the PUFA-enriched oil had a slightly more triglyceride-lowering effect (Gardner and Kraemer, 1995). Therefore, the authors concluded that the hypocholesterolemic effects of replacing SAFA by either MUFA or PUFA were comparable. After the publication of these meta-analyses, other trials have been performed to compare the effects of various edible oil consumption on lipid metabolism and understand the reasons for the variability in the degree of the effects observed. In a double-blinded randomized cross-over study (3 weeks intervention period) Pedersen et al., (2000) compared the effects of olive oil with rapeseed and sunflower oil (with a higher PUFA content) and reported that the olive oil-rich diet induced a higher concentration of LDL cholesterol and higher number of LDL subfraction particles than rapeseed and sunflower oil-rich diets but had a more favourable effect on HDL-cholesterol plasma levels compared to sunflower oil. Later Perona et al., (2003) compared the effect on plasma lipid levels of the administration of two virgin olive oils of the same variety (Olea europaea var. hojiblanca) with a different triglyceride composition, showing a different effect on total and LDL cholesterol levels in a healthy elderly population. Olive oil with higher oleic acid content significantly reduced total and LDL cholesterol and this reduction was related to higher incorporation of oleic acid into plasma cholesteryl esters and phospholipids. These data suggest that the composition of bioactive components and triglyceride molecular species in different olive oil varieties may have different effects on health. As a matter of fact, the discrepancy of results on plasma cholesterol levels in human studies may depend on the different amount of dietary fat utilised in each trial, on different fatty acid patterns of oils and on interactions between those factors. Differences in non-saponifiable lipids between oils could also work in the same direction. Olive oil contains more squalene (involved in the pathway of cholesterol biosynthesis) and less phytosterols (molecules that interfere with cholesterol absorption) than other vegetable oils. The greater effectiveness in reducing total and LDL plasma cholesterol concentrations of corn oil compared to a mixture of olive/sunflower oil has been related to its high amount of unsaponifiable substances, mainly phytosterols (see also paragraph “Phytosterols” below). Few data are available on the effects on the lipid metabolism of peanut, argan and rice bran oil consumption, three oils with intermediate content of MUFA and PUFA. Kris-Etherton and al. (1999) in a cross-over study that compared the effect of a diet containing peanut oil with one containing olive oil in healthy subjects reported that both oils decreased total and LDL plasma cholesterol levels and triglyceride concentrations to the same extent without any reduction in plasma HDL cholesterol levels. The comparison of virgin argan oil with virgin olive oil in Derouiche’s study (2005) showed that only the extra virgin olive oil rich diet significantly decreased low density lipoprotein (LDL-chol) and Apo B plasma levels while only the virgin argan oil rich diet significantly decreased plasma triglyceride levels. In the Lichtenstein study (1994) the consumption of rice bran, canola, and corn oil-enriched diets induced a similar and statistically indistinguishable reduction in plasma cholesterol and LDL-cholesterol concentrations and this decrease was higher than that induced by an olive oil-enriched diet. For rice bran and corn oil the cholesterol lowering effect was also attributed to their unsaponifiable compounds rather than to differences in the fatty acid composition (Most et al., 2005). Based on this evidence which oil among those analysed in MAC-Oils projects has healthier effects on lipid metabolism? Which oil should the consumer prefer to improve lipid profile? The overall results of trials performed in humans clearly demonstrate that all

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vegetables oils, due to their fatty acid composition and amount and quality of unsaponifiable compounds are able to improve cholesterol metabolism more or less to same extent. The choice depends on metabolic characteristics of the consumer. As a matter of fact subjects with high plasma cholesterol levels and low HDL-chol should prefer oils richer in oleic acid whereas individuals with higher plasma triglyceride levels should prefer those richer in linoleic acid. Another important question concerns the effects on lipid metabolism of vegetable oils that undergo hydrogenation. In fact, the hydrogenation process produces changes in the fat composition, leading to an increase in trans fatty acids, which are associated with a higher CHD risk (Mozaffarian et al., 2006). This transformation involved both MUFA and PUFA. Replacing oils with oil margarine in stick form (a fat obtained from hydrogenated oils) in the diet significantly increases total and LDL plasma cholesterol levels and decreases HDL-cholesterol concentration; for this reason the American Heart Association (AHA) and other Health Organizations recommend to limit the intake of trans fatty acids in the diet to not more than 1% of total fat intake. Dietary fat quality also influences postprandial lipemia. Postprandial hypertriglyceridemia is an independent risk factor for CVD, and may be a better predictor of risk than fasting triglyceride levels alone, given the variations in postprandial triglyceride response, even in individuals with normal fasting triglyceride concentrations. The presence of higher levels of triglyceride-rich lipoproteins, which includes triglycerides, very-low-density-lipoproteins (VLDL), chylomicrons and their smaller, more dense remnants, and small, dense LDL-cholesterol, is associated with the process of atherogenesis (Blackburn et al., 2003). In fact, both triglyceride-rich lipoproteins and small LDL-cholesterol are slowly removed from the blood circulation, are more susceptible to oxidative modification and more apt to penetrate the arterial wall. Few are the studies in humans that have compared the effects of various types of edible oil consumption on postprandial lipemia. An oral olive oil load had a smaller effect on the magnitude of postprandial lipemia and remnant lipoproteins than the ingestion of butter (Thomsen et al, 1999) and higher than that of safflower oil (another oil rich in PUFA) (Higashi et al, 1997). Other trials that have compared the effects of n-6 PUFA with olive oil or a MUFA -rich meal showed a lower or a comparable postprandial lipemia (Tholstrup. et al, 2001). Abia (2001) reported that virgin olive oil intake also lowered postprandial plasma triglyceride-rich lipoprotein levels and accelerated their removal from blood compared to high oleic acid sunflower oil intake. Up to now there are no consistent data on the effects of various types of vegetable oil consumption on postprandial plasma triglyceride levels although the data in favour of PUFA are more than those in favour of MUFA rich oils. Blood glucose and insulin sensitivity. Insulin sensitivity has a key actor in the human body since it plays a crucial role in a number of functions and regulatory mechanisms involving intermediary metabolism, body fat deposition, blood flow in the cardiovascular system and many other functions. Reduced insulin sensitivity, insulin resistance, have been recognized as the cause of glucose metabolism abnormalities and other cardiovascular risk factors. Insulin sensitivity can be modulated by different types of dietary fatty acids. In fact, insulin resistance is associated with a specific serum lipid fatty acid pattern, characterized by increased proportions of saturated fatty acids [palmitic (16:0) and palmitoleic acids (16:1 n-7)] and reduced levels of linoleic acid (18:2 n-6). Several cross-sectional studies in humans have examined the relationship between markers of insulin resistance (plasma insulin values or serum/muscle lipid fatty acid profile) and dietary fat showing a positive association with saturated fat and a negative one with

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monounsaturated and polyunsaturated fat intake. Conversely to observational studies, the results of most intervention trials comparing the effects of a MUFA and PUFA vs. a SAFA-enriched diet did not confirm this finding (Riccardi et al., 2004). However, it is important to underline that these intervention studies were performed in very small groups of subjects and, generally for too short a period to show any effect. Instead, the KANWU Study, a trial performed on a large number of healthy subjects (162) on a dietary treatment for 3 months, is the first intervention study to show that a MUFA-enriched diet did not change insulin sensitivity whereas a saturated enriched one significantly impaired it, with a 23% difference on insulin sensitivity between the two diets in individuals consuming less than 37% energy as total fat (Vessby et al., 2001). As to the ability of PUFA to influence insulin resistance, only Summers’ study (Summers et al., 2002) reported a significant improvement in insulin sensitivity after the PUFA diet compared to the SAFA one. There are no controlled studies with adequate sample size and duration in humans that compared the effects of MUFA vs. PUFA intake on insulin sensitivity. The mechanisms linking dietary fat quality to insulin sensitivity are not completely understood; however the effects of dietary fatty acids on this biological function are believed to be mediated, at least partially, through the fatty acid composition of cell membranes. A specific fatty acids profile in cell membranes could influence insulin action through several potential mechanisms, including altered insulin receptor binding or affinity, and by influencing ion permeability and cell signalling. Therefore, the results deriving from intervention studies, suggest that the consumption of oils with high unsaturated to saturated fat ratio themselves improves or at least does not change insulin sensitivity and glycemic control in both healthy individuals and diabetic patients, provided that the energy intake is controlled. Blood pressure and endothelial functions. Several epidemiological studies have assessed the relationship between saturated, monounsaturated, polyunsaturated dietary fatty acids intake and incidence of hypertension or changes in blood pressure (BP), providing controversial results. As a matter of fact the results of studies performed in the USA show either no effect or a negative association between MUFA intake and BP, whereas those conducted in Mediterranean countries provide a positive association. In particular, the Nurses’ Health Study, a prospective study performed in the USA on middle aged women, followed for 4 years, showed no relation between total fat, saturated or unsaturated fat intake, and the risk of hypertension (Ascherio et al., 1996). Similarly, the Health Professionals’ Follow-up Study, a cohort study performed on US men aged 40-75 years followed for 4 years, did not find any association between BP and saturated, unsaturated or trans fatty acids intake (Ascherio et al., 1992). Also the Multiple Risk Factor Intervention Trial, performed on US men, showed that BP was inversely related with PUFA but not with MUFA intake (Stamler et al., 2002). Contrarily, the results of studies performed in Mediterranean populations support a protective role of MUFA on blood pressure. The Italian Nine Communities Study showed a significant inverse association between olive oil consumption and BP for both systolic and diastolic BP when normotensive men and women were analyzed separately (Trevisan et al., 1990). In the same study, the PUFA intake was associated with lower systolic BP but had no effect on diastolic BP. Another cross-sectional study, the EPIC Study (European Prospective Investigation into Cancer), performed on Greek individuals of both genders, confirmed that both the MUFA/SAFA ratio and olive oil intake were inversely associated with BP, also after adjustments for vegetable consumption and other confounding factors (Psaltopoulou et al., 2004). For each 22 g daily increase in olive oil consumption, systolic and diastolic BP decreased by an average of 0.8 and 0.3 mmHg respectively. In the SUN Study (Seguimiento

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Universidad de Navarra) at baseline, MUFA intake was associated with a reduced risk of hypertension among individuals with low consumption fruit and vegetable but not among those with higher consumption of these foods (Martinez-Gonzales et al., 2002; Alonso et a.l, 2004). However, due to their cross-sectional design, these studies do not provide information on the causal relation. More recently a prospective analysis of the SUN Study showed that olive oil consumption after 28.5 months of follow-up was inversely associated with risk of hypertension development among normotensive men but not women (Alonso et al., 2004). The controversial results provided from studies performed in the USA and those conducted in Mediterranean countries could be explained by a strong differences between Western and Mediterranean diets. Western populations derive most of their MUFA from meat, the consumption of which correlates with their saturated fat intake, whereas in the Mediterranean diet MUFA are derived essentially from olive oil and nuts, foods rich in unsaturated fats. However, results of randomized controlled trials investigating the effects of different dietary fat or oil consumption on blood pressure in humans have contributed to clarify the inconsistencies observed in the observational studies. Table 5.2 reports results of a large number of controlled intervention trials comparing the effects of different dietary fats and oils on blood pressure. The studies were performed in both normotensive and hypertensive healthy or type 2 diabetic individuals, the intervention duration ranges from 3 weeks to six months. As expected, MUFA and PUFA rich oil intake in comparison with SAFA intake decreased BP levels (Lahoz et al., 1997; Rasmussen et al., 2006). Interestingly, in the Rasmussen study (2006) the favourable effects of MUFA on diastolic BP disappeared at a total fat intake above the median (>37% of energy). These data could explain the absence of any effect of MUFA on BP in epidemiological studies performed in an American population who consumed a greater total amount of dietary fat than a Mediterranean population. No differences on BP levels were observed in the studies that compared the MUFA (from rapeseed oil) with PUFA (from sunflower oil) rich diet (Mutanen et al., 1992; Lahoz et al., 1997). On the contrary, results comparing the effects of MUFA from olive oil or virgin olive oil with PUFA from sunflower oil showed that olive oil was more effective to reduce blood pressure. As a matter of fact Thomsen et al., (1995) observed that an olive oil rich diet (30% of energy from MUFA) after 3 weeks significantly reduced systolic and diastolic BP compared to a PUFA rich diet (27% of energy from PUFA) in normotensive type 2 diabetic patients. Ferrara et al., (2000) in a double blind cross-over trial in hypertensive patients, reported that 6 months on a diet rich in extra virgin olive oil reduced the need for antihypertensive medication compared to a diet rich in sunflower oil ( 50% vs 4% reduction, respectively) and decreased systolic and diastolic blood pressure by 8 and 6 mmHg respectively. Perona et al., (2004) in a cross-over trial on hypertensive and normotensive individuals, showed that a virgin olive oil- rich diet compared to one rich in sunflower oil reduced systolic BP but not diastolic BP in hypertensive patients but had no effect in normotensive individuals. It is interesting to underline that in the Ruiz- Gutiérrez study (1996), which compared the effects on BP of two oils rich in MUFA, VOO vs high oleic sunflower oil, a more evident effect on BP reduction was observed after Virgin Olive Oil rich diet. This suggests that the effect of olive oil on BP could be mediated by compounds other than MUFA, such as the polyphenols present in virgin olive oil (see also paragraph “Phenols” below). The mechanism through which MUFA and olive oil could modify BP are not completely clear. A high MUFA intake modifies membrane phospholipids and reduces BP; as a matter of fact, olive oil consumption is associated with a higher MUFA content in plasma phospholipids. In addition, polyphenols present in olive oil, such as oleuropein, hydroxytyrosol, tyrosol and others etc., reduce the generation of reactive oxygen species; these molecules cause endothelial dysfunction, a process implicated in the pathophysiology of hypertension (Cai and Harrison,

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2000). These mechanistic findings of a beneficial effect due to the non lipidic fraction of olive oil fit well with the epidemiological evidence supporting a major benefit associated not with all MUFA, but specifically those derived from olive oil (studies conducted in Mediterranean countries), and even more from virgin olive oil, which is rich in polyphenols. Table 5.2 Intervention Trials evaluating the effect of SAFA, MUFA, PUFA dietary intake on blood pressure

Study Participants Design Intervention (duration)

Blood Pressure

Mutanen et al., 1992 29 men and 30 normotensive women (range 18-65 yrs)

Randomised cross-over

MUFA (RO) vs PUFA (SO) (3.5 weeks)

No differences

Thomsen et al., 1995 16 type 2 diabetic (mean age 59 yrs)

Randomised cross-over

MUFA (OO) vs PUFA diet (3 weeks)

↓ MUFA

Ruiz-Gutiérrez et al., 1996

16 hypertensive women (mean age 56 yrs)

Randomised parallel groups

MUFA (VOO) vs MUFA (HOSO)

(4 weeks) ↓ VOO

Lahoz et al., 1997 42 healthy subjects (age range 17-71 yrs) Cross-over

SAFA vs MUFA (OO) vs PUFA n-3

vs PUFA n-6 (5 weeks)

↑ SAFA, no differences

between MUFA and PUFA

Ferrara et al., 2000 23 hypertensive subjects (age range 25-70 yrs)

Double-blind randomised cross-over

VOO vs SO diet (6 months)

↓ VOO ↓

antihypertensive drugs

Perona et al., 2004 31 hypertensive and 31 normotensive subjects (mean age 84 yrs)

Randomised cross-over

VOO vs SO diet (4 weeks)

↓ VOO in hypertensive

subjects

Rasmussen et al., 2006

162 healthy subjects (range age 30-65 yrs)

Randomized parallel groups

SAFA vs MUFA (3 months) ↓ MUFA

SBP = Systolic Blood Pressure; DBP = Diastolic Blood Pressure; OO = Olive Oil; VOO = Virgin Olive Oil; HOSO = High Oleic Sunflower Oil; RO =Rapeseed Oil; SO = Sunflower Oil In conclusion, the results of most intervention studies support that olive oil, particularly virgin olive oil, compared to PUFA rich-oils or other MUFA rich-oil is more effective to reduce blood pressure in both normotensive and hypertensive individuals. In fact this effect is due to action of both MUFA and polyphenol compounds. Oxidative stress. The oxidative modification of LDL plays a key role in the pathogenesis of atherosclerosis, since oxidized LDL damage the arterial wall more than native LDL. Elevated concentrations of oxidized plasma LDL are positively associated with severity of acute coronary events (Weinbrenner et al., 2003), and are predictors of coronary heart diseases (CHD) in patients with CHD and in the general population (Meisinger et al., 2005). Fat quality is one of the most important factors modulating the susceptibility of LDL to oxidative modification. This effect depends on diet-induced changes in the concentration of PUFA and antioxidants in LDL. The particles rich in MUFA are less susceptible to oxidative modifications compared to those rich in n-6 PUFA (Reaven et al., 1991). Reaven (1996) compared the effects of n-6 PUFA with those of MUFA on LDL oxidation susceptibility in healthy individuals who consumed either linoleic or oleic-enriched diets or who continued their habitual American diet for 6 weeks. All participant were given α-tocopherol supplementation for at least 3 months prior to randomization into 3 dietary

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groups. The oleic-rich diet decreased the susceptibility of LDL to lipid oxidation; in addition, a high dosage of α-tocopherol did not prevent the enhanced oxidation of n-6 enriched LDL particles. This finding was confirmed also in hypercholesterolemic patients (Baroni et al., 1999) in whom a diet rich in MUFA (olive oil) compared to one rich in PUFA increased the concentration of MUFA in LDL by 11%, decreased that of PUFA by 10% , while antioxidant values remained unchanged during the study. In this study, the oleic-enriched LDL was more resistant to oxidative modification, as measured by different peroxidation parameters. The LDL oxidazibility in vitro was reported to be lower in refined olive oil consumers, intermediate in rapeseed and higher in sunflower oil consumers (Kratz et al., 2002). Therefore, the higher LDL susceptibility to oxidation of rapeseed oil respect also to refined olive oil is due to slightly higher linoleic and α-linolenic fatty acid content. In addition, linoleic acid significantly increased urinary excretion levels of 8-iso-prostaglandin 2α and decreased those of nitric oxide metabolites, without, however, inducing significant changes following the ingestion of oleic acid. In one study, an olive oil diet compared to a peanut oil rich diet showed a slower LDL oxidation rate and was associated with a lower LDL linoleic acid content (Kris-Etherton et al., 1999). The authors underlined that despite some varieties of peanut, sunflower or soybean oil have an oleic/linoleic fatty acid ratio similar to that of olive oil − compared to the latter they have a lower content of molecules (vitamins and phenolic compounds) with antioxidant properties able to contrast oxidative damages; this is especially true if the comparison is made with extravirgin olive oil. In fact the antioxidants contained in oils can influence LDL susceptibility to oxidation but this will be extensively debated in the section on polyphenols (see below). In summary, among all edible oils only olive oil combines the advantages of both lowering cholesterol level and decreasing LDL susceptibility to oxidation. Inflammation and thrombosis. Atherosclerosis is considered an inflammatory disease. Fatty acids composition and minor components of oils may modulate inflammation. Mediators of inflammation include prostaglandins, leukotrienes, thromboxanes, which are produced from the n-6 and n-3 PUFA metabolism. Under normal physiological conditions, a balance is maintained between pro- and anti-inflammatory molecules. Thus, a high intake of linoleic acid shifts the physiological state to a pro-inflammatory and pro-thrombotic state, increasing blood viscosity, vasospasm, and vasoconstriction (Bogatcheva & al., 2005). In contrast, α-linolenic acid (n-3 PUFA) derived products have an anti-inflammatory, antithrombotic, vasodilatory and hypolipidemic properties. Therefore, a high intake of sunflower, soybean and corn oil, oils particularly rich in linoleic acid, increase inflammation markers and thrombotic risk whereas oils rich in α-linolenic acid should reduce them. Table 5.3 shows some of intervention studies that evaluated the effect of a diet enriched in α-linolenic acid (from oils) versus a diet enriched in linoleic acid or in SAFA in hyperlipidemic patients or in healthy obese subjects. Compared to linoleic acid (from safflower oil) the supplementation of α-linolenic acid (from linseed oil) in hyperlipidemic patients following a typical Greek diet after 3 months significantly decreased c-reactive protein (CRP), serum amyloid A (SAA) and interleukin 6 (IL6) serum levels (Rallidis et al., 2003). The ability of ALA supplementation to decrease serum inflammatory markers was more evident when the background diet was rich in saturated fatty acids and poor in MUFA (Paschos et al., 2004). In addition, when ALA and linoleic rich diets were compared to Average American Diet (rich in SAFA and cholesterol), both were able to decrease plasma intercellular cell adhesion molecule-1 (ICAM-1) in hyperlipidemic patients but ALA diet decreased CRP, vascular cell adhesion molecule-1 (VCAM-1) and E-selectin more than the linoleic diet (Zhao et al., 2004). On the contrary, the Nelson study (2007) did not confirm that ALA enriched diet was able to decrease inflammatory markers in healthy overweight/obese

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subjects. The absence of effect could depend on young age of participants whose inflammatory factors were not elevated. Therefore, results of overall intervention trials seem to suggest that ALA intake is more effective to reduce inflammation in subjects with metabolic abnormalities and when it is consumed in the context of western diet. T here is no direct evidence of the effects of MUFA on inflammation. A protective role of oleic acid against inflammation is suggested by interventions studies where the consumption of a Mediterranean diet, rich in olive oil, in individuals at high CHD risk was able to reduce vascular and inflammation markers (Esposito et al., 2004; Estruch et al., 2006). However, these trials do not help us discriminate whether the benefits depends on olive oil or on other component of diet. Trans fatty acids consumption can have detrimental effects on inflammation. A recent observational study evaluating the relationship between the consumption of partially hydrogenated (PHVOs) and non hydrogenated vegetable oils (non HVOs) (Sunflower, Corn, Canola, Soybean and Olive oil) and markers of systemic inflammation and endothelial dysfunction, showed that the women in the highest quintile of consumption of partially hydrogenated oils intake had higher CRP, TNF-alpha, interleukin-6, and soluble intercellular adhesion molecule-1 plasma concentrations (Esmaillzadeh et al., 2008). On the contrary women who consume non hydrogenated oils showed lower plasma levels of these molecules. Therefore, consumers must account of negative effects on inflammation of partially hydrogenated oil consumption even if, nowadays, the availability on the market of sunflower and soybean oils varieties rich in oleic acid and new mild hydrogenation technologies able to reduce the trans fatty acid formation tend to overcome this risk.

Table 5.3 Effects of α-linoleic acid (ALA) intake on serum Inflammatory markers (evidence from intervention trials)

Study Participants

(age) Study Design Intervention (duration) Inflammatory

markers

Rallidis et al., 2003

76 male hyperlipidemic subjects

(mean age 51±8 yrs)

parallel groups

ALA (linseed oil) vs LA (safflower oil)

(12 weeks) ↓ CRP, SAA, IL6

Paschos et al., 2004

19 dyslipidemic (WGD) and 21 dyslipidemic subjects

(MCD) (mean age 49.4±7.3 yrs)

parallel groups

ALA in WGD vs ALA in McD (12 weeks)

↓ CRP, SAA, IL6, VCAM-1

Zhao et al., 2004 23 hypercholesterolemic

subjects (mean age 49.8 ±1.6 yrs)

cross-over AAD vs LA diet vs ALA diet (6 weeks)

↓ VCAM-1, PCR, E-selectin

Nelson et al., 2007

51 healthy obese subjects (mean age 38.5±11.1 yrs)

parallel groups

ALA vs Control diet (8 weeks) no effect

LA = Linoleic acid; AAD = Average American Diet; WGD = Western Greek Diet; MGD = Mediterranean Cretan Diet; CRP = c-reactive protein; SAA= serum amyloid A; IL6= Interleukin 6; VCAM-1=vascular cell adhesion molecule-1 For what concerns the effects of edible oil consumption on thrombosis, there is evidence that canola/rapeseed oil, due to its relatively high MUFA and ALA content, has a anti-thrombotic effects in humans. As a matter of fact the consumption of this oil has been shown to alter the fatty acid composition of plasma and platelet phospholipids. In general, rapeseed oil results in higher levels of EPA and lower concentrations of arachidonic acid in platelet phospholipids compared to sunflower, soybean or safflower oil. However, not all

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studies report higher levels of EPA (Kwon et al., 1991; Mutanen et al., 1992) or lower levels of arachidonic acid (Chan et al., 1993). Similarly, controversial data on the effect of rapeseed oil on platelet aggregation have been reported. In fact, Renaud et al., (1986) and Kwon et al., (1991) reported that rapeseed oil reduced platelet aggregation in vitro while Mutanen (1992) found that it stimulated platelet aggregation. In addition, sunflower oil was found to produce effects similar to those of canola oil in terms of platelet function (Renaud et al., 1986; Mutanen et al., 1992), clotting time and eicosanoid production (McDonald et al., 1989). The marked differences among studies may relate to the balance between n-6/n-3 fatty acid ratio in the diet. In fact, Chan (1993) reported that changes in plasma and platelet fatty acid composition varied with both the level of ALA in the diet and its ratio to linoleic acid. Also the proportions of saturated, monounsaturated and polyunsaturated fatty acids in the experimental diets were not standardized. However, there is sufficient evidence that canola/rapeseed oil or other oils rich in ALA, counter the inflammatory processes in humans. On the contrary, its protective effect against thrombotic risk has not been established and further studies are needed to clarify whether ALA intake plays a role on thrombosis in humans. To summarize, the overall reported data suggest that MUFA and PUFA rich oils have beneficial effects on lipid profile, blood pressure, endothelial function, insulin sensitivity and blood glucose control in comparison to dietary SAFA. However, compared to PUFA rich oils, MUFA are able to not reduce HDL cholesterol, to enhance the resistance of LDL to oxidative modifications, to prevent endothelial dysfunction and inflammation. Nevertheless, virgin olive oil rather than other traditional or novel MUFA oils, more evidently exhibits these beneficial properties. Trans fatty acids contained in partially hydrogenated oils have detrimental effects on lipid profile and inflammation (Table 5.4).

Table 5.4. Effects of different fatty acids on cardiovascular disease risk factors

LDL-cholesterol

HDL-cholesterol Triglycerides Blood

Pressure Insulin

sensitivity Oxidative

stress Inflammation

SAFA ↑ = =↑ ↑ ↓ ? ?

Trans-fatty acids

↑↑ ↓↓ = n.e. n.e. n.e. ↑

MUFA ↓ =↑ = ↓ ↑ ↓ ?

Linoleic acid (n-6)

↓ =↓ ↓ =↓ ↑ ↑ =↑

α-linolenic acid (n-3)

↑ =↑ =↓ =↓ = ↑ ↓

n.e not evaluated; = no effect; ↑ increase; ↓ decrease; ? uncertain 5.2.2 Cancer

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Human Studies A wealth of epidemiological evidence in humans has shown that the amount and quality of dietary fat influences the development of breast, colorectal and prostate cancer. It has been reported that a high dietary fat intake based on a high saturated and n-6 PUFA consumption is associated with a high risk of cancer; contrarily oleic and n-3 PUFA consumption in most studies is associated with a low risk of cancer. Breast cancer. Epidemiological studies performed in different countries report a positive relationship between the per capita consumption of animal fat and the incidence of breast cancer, essentially in post-menopausal women (Prentice and Sheppard, 1990), whereas this relationship is not found with olive oil. This observation is in line with a lower incidence of this type of cancer in Mediterranean countries compared to Western countries (Trichopoulou et al., 1995). However, a review of epidemiological studies indicates some evidence of a positive relationship between total fat consumption and breast cancer risk (Bingham et al., 2003) whereas others show a positive albeit non significant relationship (Cho et al., 2003) and others yet find no relation at all (Byrne et al., 2002). Contradictory results have been attributed to the different quality of dietary fat, with a high consumption of SAFA being associated with increased risk of breast cancer in several studies (Do et al., 2003) and oleic/MUFA consumption with a lower risk (Binukumar and Mathew, 2005). Case-control studies in Spain (Martin-Moreno et al., 1994), in Italy (La Vecchia et al., 1995) and Greece (Trichopoulou et al, 1995), provide evidence that olive oil consumption is related to a significant 14% to 34% reduction in breast cancer risk. Nevertheless, Saadatian-Elahi et al., (2004) observed a significant positive association between total MUFA and oleic acid consumption and breast cancer risk. The mechanisms through which oleic fatty acid or the polyphenol content of virgin olive oil may influence breast cancer risk have been investigated in experimental and in vitro studies, providing often contradictory results. Colorectal cancer. The incidence of colorectal cancer has increased in Western countries in the last three decades, with more than a third of cases being associated with diet (Roynette et al., 2004). Several epidemiological studies support a key role of high amount of dietary fat and SAFA in the development of this type of cancer while epidemiological case-control studies have demonstrated a protective effect of olive oil and n-3 PUFA consumption (Llor et al., 2003; Roynette et al., 2004; Reddy, 2004). Studies on cohorts and control cases do not report a consistent association between dietary n-6 PUFA and colorectal cancer, as opposed to experimental studies showing an increased risk of this type of cancer with high consumption of n-6 PUFA. Prostate cancer. As with breast and colon cancer, there is a positive correlation between the mortalities indices for prostate cancer and the estimated per capita intake of fat (Terry et al., 2004). Ecological analyses report a positive association between this cancer and saturated fat intake, which becomes weak when compared to MUFA and PUFA. Currently, no direct evidence relates edible oil consumption to a specific incidence in this type of cancer. In conclusion, unsaturated fatty acids, essentially those monounsaturated, seem to play a key role also in the prevention and progression of some types of cancer. However the complexity of genetic and environmental factors involved in the development of cancer prevents us from drawing a conclusion on this issue, although experimental studies in animals support a potentially favourable effect of MUFA and olive oil.

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5.2.3 Nutritional recommendations and new prospective Nutritional guidelines of the American Heart Association and other Health Organizations recommend to limit daily SAFA intake to less than 10% and keep trans-fatty acid intake as low as possible in order to reduce cardiovascular diseases and type 2 diabetes. The 2006 guidelines from the American Heart Association (AHA) (Lichtstein et al., 2006) recommend to further limit dietary SAFA intake to no more than 7% of total daily energy, trans-fatty acid intake to no more than 1% of energy and cholesterol to less than 300 mg/day. In addition the Food and Nutrition Board of the Institute of Medicine (IOM) (2002) established that an adequate intake of n-6 PUFA should be in the range of 5% to 10% of total daily energy and 0.6% to 1.2% energy as ALA. In term of grams per day, daily intake of ALA should be 1.1 and 1.6 g/day in women and men respectively and that of linoleic acid of 11 to 12 g/d for women and 14 to 16 g/d for men. Therefore, the dietary consumption of olive and canola oil – both rich in MUFA- should be encouraged as a replacement of butter (rich in SAFA), margarine (rich in trans fatty acids) and other oil rich in PUFA (soybean, peanut, sunflower oil etc) in order to reduce CVD mortality. On November 2004 and October 2006, the U.S. Federal Drug Administration (FDA) permitted a claim on olive and canola oil labels, respectively, concerning “the benefits on the risk of coronary heart disease of eating about two tablespoons (23g) of olive oil daily or one and half tablespoons (19g) of canola oil daily, due to their composition in MUFA and provided that these oils replace a similar amount of saturated fat and not increase daily energy intake. Although European Mediterranean Countries are major producers and consumers of olive oil, and several scientific reports have shown the benefits of olive oil for human health, up to now no claim for olive oil has so far been recognized by the European Community. Johnson et al., (2007) have examined the effect of replacing the oils commonly used by American adults (soybean, corn, cottonseed, palm, olive, peanut, and sunflower oil), butter and margarine with 25%, 50% and 100% levels of canola oil and canola oil-based margarine on fatty acid and cholesterol intake in a large population of 8983 subjects aged ≥20 years. Data were derived from the 1999-2002 National Health and Nutrition Examination Survey. The results indicate that replacing other vegetable oils and spreads with 25%, 50% and 100% of canola oil and canola oil-based margarine increases compliance with dietary recommendation for SAFA, MUFA, α-linolenic acid and linoleic acid, without affecting cholesterol intake in US adults. In particular, complete substitution decreased SAFA intake by 9.4%, increased MUFA and ALA by 27.6% and 73.0%, respectively and decreased n-6 PUFA and linoleic acid intakes by 32.4 and 44.9%, respectively with an n-6/n-3 PUFA ratio that passed from 9.8 to 3.1. The results of this study are clinically relevant because they suggest that fatty acid intake can be influenced through a simple recommendation to change the type of vegetable oil used for dressing and cooking. The need to limit dietary consumption of soybean, sunflower oil and other oils with high PUFA content, depends on their negative effects on human health such as the increase of LDL susceptibility to oxidation, DNA damage and endothelial dysfunction. An answer to this question is provided by introduction on the market of novel soybean and sunflower oils rich in oleic acid and low in linoleic acid and a variety of soybean oil with a lower α-linolenic acid content. Another question related to oils rich in PUFA is that these oils, being highly sensitive to oxidation, undergo a mild hydrogenation process which increases the melting point, shelf life and helps reduce production costs. However, this process has the drawback of inducing a transformation of part of PUFA into trans fatty acids which have detrimental effects on risk factors for CHD (Mozaffarian et al., 2006) and have recently been the cause of some

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controversy since some ‘fast foods’ contain high levels of these fatty acids. As reported above, trans-fatty acids increase LDL-and triglyceride levels, and lower HDL levels. The main source for trans-fatty acids in nutrition is hydrogenated fat. Therefore, the challenge to industry has been to decrease trans fatty acid levels without increasing levels of saturated fatty acids. This has posed a number of practical difficulties as the solid nature of trans fatty acids and saturated fatty acids cannot simply be replaced by monounsaturated fatty acids or polyunsaturated fatty acids as they are liquid at room temperature. In the last decades the improvement of technologies utilised for refining, bleaching and especially deodorizing has permitted to achieve a reduction of the trans fatty acid formation during processing below acceptable levels. New products with more healthy composition start to replace existing products, switching from trans fatty acids to saturated fatty acids in baked goods, but also by replacing trans and saturated fatty acids with unsaturated fats where it is possible. Health issues related to trans fatty acids, and growing consumer awareness about health, nutrition and diet are the primary drivers for more balanced fat compositions, low in trans fats and with reduced saturated fats. This was further enhanced by the mandatory Trans fat labelling as of 2006. The technical demand for heat- and oxidation-stable oils is the first question to be answered. At the same time, consumers ask for good tasting healthier foods. High oleic soybean and sunflower oils will be the alternative for partially hydrogenated frying oils with too high trans fatty acid content. Equal or better oxidation stability will come together with an equal or reduced formation of polar compounds, polymers, secondary oxidation products, FFA and POV, as well as less negative odour development than regular soybean and sunflower oil based frying oils. Finally, a question common to consumption of all edible oils, irrespective of their fatty acid composition, is that they are a source of calories. Oil provides about 890 kcal (3700 kJ) per 100g, one tablespoon of oil contains about 100-120 calories, therefore if consumed in large amount they increases the risk to develop overweight and/or obesity. Therefore, their consumption must be particularly limited (two-three tablespoons per day) in obese or overweight individuals and in those prone to developing obesity. 5.3. Effects of Minor Components in Edible Oils on Human Health 5.3.1 Tocopherols The presence of minor components in edible oils analyzed under MAC-Oils project has been largely reported and discussed during the thematic ateliers. The families of molecules which are generally classified as “minor” or “unsaponifiable” compounds have been described in details in Chapter 3 of the present Handbook. They include: tocopherols, sterols, phenols, methylsterols and triterpene alcohols, pigments (carotenoids, chlorophylls and derivates), waxes, fatty alcohols, hydrocarbons, squalene, stigmastadiene, volatiles and sulphur compounds, metals, glucosinolates. Tables 3.6-3.10 in Chapter 3 report the relative concentrations of the most abundant and well-characterized molecules in the eight oils studied during the course of the present project. From a biological point of view, the most significant “minor” compounds in edible oils belong to the classes of tocopherols, sterols and phenols.

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Looking at Table 3.6 (Chapter 3), total tocopherols in studied oils range between 200 and 2000 mg/Kg with corn and soybean with the highest concentration mostly represented by γ-tocopherols, while α-tocopherol is abundant in olive, peanut, rice and sunflower oils. The ingestion of 25-50 ml daily of edible oils corresponds to about 10-100 mg of tocopherols. These values are in the range of the Recommended Daily Allowance (RDA) of vitamin E which, for an average size adult, is 10 mg of α-tocopherol in Europe and 15 mg of α-tocopherol in US (Dutta and Dutta, 2003). We think that, independently by the source of vegetable oils used in the diet, the amount of tocopherols ingested should be enough to sustain the daily allowance of vitamin E, without considering other sources of supply. Accordingly, bioavailability studies performed in humans clearly established that plasma concentration of α-tocopherols doubles after a daily administration of 25 ml of olive oils respect to the baseline level (from about 4.1-5 to 8.4-8.6 μM). This work was performed comparing olive oils with different amount of phenols (from 0 to 800 μmol/Kg caffeic acid equivalent) (Gimeno et al., 2007). An extensive review on absorption, transport, metabolism and biological activities of vitamin E in degenerative pathologies has been prepared at the end of corn oil atelier in Paris (www.mac-oils.eu) by M. Ballut, N. Combe and A. Rossignol-Castera (ITERG, Pessac, France). Here, we want make the point that, in terms of human nutrition, the abundance and bioavailability of different tocopherols does not make a significant difference among the different oils analysed. More important is the content of vitamin E in terms of chemical stability of the product, an issue largely discussed in Chapter 2. Respect to the content of phenols in edible oils, an important aspect of tocopherols activity regards the potentiation of their antioxidant activity by a synergistic interaction with phenols. As an example, catechins and flavonols (from almond skin) and α-tocopherol synergistically increase the resistance of human LDL to oxidation as well as their bioavailability, pharmacokinetics, and in vivo antioxidant actions in hamsters (Chen et al., 2005). This effect was observed with 0.36 μM of flavonoids in combination with 5.5 μM α-tocopherol. The lag time was 289% longer than the expected additive value (Chen et al., 2005). Similarly, the synergy between polyphenols and tocopherol for the prevention of oxidation is confirmed by the positive interaction between α-tocopherol and the major components of green tea polyphenols, i.e. (–)-epicatechin (EC), (–)-epigallocatechin (EGC), (–)-epicatechin gallate (ECG), (–)-epigallocatechin gallate (EGCG) and gallic acid (GA). Addition of tea polyphenols together with α-tocopherol significantly increases the inhibition period of α-tocopherol, decreases the kinetic chain length both in the inhibition period and after the inhibition period, and decreases the oxidizability of linoleic acid. The synergistic effectiveness of the tea polyphenols can be arranged in the order EGCG >> ECG ~ EGC > GA >EC (Jia et al., 1998). With the limits imposed by this type of in vitro studies, we may suggest that the synergistic effect of vitamin E and phenols could be reproduced in those oils, such as olive and argan (Table 5.5), where a significant concentration of polyphenols is present, respect to refined, seed oils. On the opposite, all edible oils reported in Table 3.7 possessing detectable concentration of sterols and sterol esters, may benefit of the potential reduction of cholesterol (see paragraphs below), but suffer for the reduced bioavailability of β-carotene and α-tocopherol. In fact, in normocholesterolemic subject supplemented with 2.2 g plant sterol equivalents provided as either free sterols or sterol esters, the treatment reduced the bioavailability of β-carotene by 50% and α-tocopherol by 20%. The reduction in β-carotene and α-tocopherol bioavailability was significantly less with plant free sterols than with plant sterol esters (Richelle et al., 2004). To comment this study, we noted that the concentration of sterols employed corresponds to an intake of 0.5-2 liters of edible oils (see paragraphs below).

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5.3.2 Phytosterols As extensively described in Chapter 3 and summarized in Table 3.7, the eight edible oils considered in this study contains an average of total sterols ranging between 738 (olive oil) to 18000 (rice bran oil) mg/Kg, with rice bran and corn oils presenting the highest concentrations, from 5- to 7-fold higher compared to the other oils (Table 3.7). It has been also well described that phytosterol composition varies among crops, as expected, as well as within crops, depending on several genetic and environmental factors (Chapter 1). In considering the potential beneficial effects of sterols in edible oils for human health, two different issues should be addressed: 1. quantity (total amount in the diet); 2. quality (specific mechanism of action of single phytosterols). The sterol structure derives from cyclopentanoperhydrophenanthrene. They are essential constituent of cell membranes in animals and plants. While cholesterol is the sterol of mammalian cells, phytosterols are produced by plants. Plant sterols differ structurally from cholesterol by a methyl or ethyl group in their side chains and are not synthesized in the human body. The most representative members of this class of compounds are: β-sitosterol, campesterol, and stigmasterol (www.lipidlibrary.co.uk), whose concentrations, in edible oils, represent 50-80% of the total sterols (Table 3.7). Plant sterols also include phytosterol esters (the previous phytosterol mixture esterified with fatty acids). Plant sterols, although structurally similar to cholesterol, are not synthesized by the human body, and they are very poorly absorbed by the human intestine (Lichtenstein and Deckelbaum, 2001). Previous studies (Miettinen et al., 1990; Normen et al., 2000; Tilvis and Miettinen, 1986) performed with stable isotope–labelled cholesterol confirmed plant derived sterols decrease the absorption of both dietary and endogenously derived cholesterol in the human intestine. Usually, half of the cholesterol in our diet is absorbed. In contrast, the adsorption of sterols ranges from about 5-16% (campesterol about 16%, while sitosterol and β-sigmasterol about 5%). As the intake of sterols is increased, the percent absorbed actually decreases, leading to a reduced efficiency of absorption. In addition, sterols are eliminated faster than cholesterol through the bile, and combined with a lower absorption, contribute to blood levels of sterols that are about 100-fold less than cholesterol blood levels. In animal models, the rate of phytosterol adsorption is super imposable to human subjects (Sanders et al., 2000).The positive trend on plant sterols in terms of human health started in the early 1950’s, when this class of compounds were found to prevent adsorption of dietary cholesterol (Pollak, 1953a; Pollak, 1953b). Since than, a growing number of evidence supported the efficacy of sterols in lowering cholesterol (Acuff et al., 2007; Amundsen et al., 2002; Gylling et al., 2006; Hallikainen et al., 2000; Lichtenstein and Deckelbaum, 2001; Normen et al., 2000; O'Neill et al., 2005). The effective dose in humans ranges between 5 and 10 g/d, although the capacity to lowering blood cholesterol levels was observed also at lower doses (Lees et al., 1977). In 2000, the U.S. Food & Drug Administration (FDA) agency approved a health claim in which announced that “foods containing at least 0.65 g per serving of vegetable oil sterol esters, eaten twice a day with meals for a daily total intake of at least 1.3 g, as part of a diet low in saturated fat and cholesterol, may reduce the risk of heart disease” (Food and Drug Administration, 2000). Based on this claim, the actually minimum daily intake in U.S.A. has been fixed in about 800 mg of sterols (corresponding to 1300 mg/d as sterol esters) and 800 mg of stanols (corresponding to 1430 mg/d of stanol esters) (Gylling et al., 1999; Heinemann et al., 1993; Lutjohann et al., 1995).

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The cardioprotective activity of phytosterols is due to their ability to compete with cholesterol for space within bile salt micelles in the intestinal lumen thereby reducing cholesterol absorption. The understanding of the function of plant sterols in impeding cholesterol absorption has been clarified with the discovery of the adenosine binding cassette transporters, ABCG5/8, involved in the regulation of sterol absorption and secretion into the enterocyte and hepatocyte. Compared to cholesterol and other sterols, sitosterol is preferentially pumped out to the intestinal lumen by the ABCG5/8 transporters. In addition, liver can increase endogenous cholesterol production if it senses that less cholesterol is being absorbed. However, the endogenous cholesterol production does not compensate for the cholesterol-lowering effect of sterols, resulting in a net cholesterol reduction, with the result of a significant decrease of plasma cholesterol (Fernandez and Vega-Lopez, 2005). In addition, the observed inverse relation between hepatic clearance and known intestinal absorption of cholesterol, campesterol, and β−sitosterol (Sudhop et al., 2002) supports the hypothesis that the ABCG5/8 transporters regulating intestinal sterol absorption might also be involved in biliary sterol excretion. On the opposite side of the coin, some findings support the hypothesis that plant sterols might be an additional risk factor for coronary heart disease. In fact, some individuals in the population who have abnormally high absorption of plant sterols. For example, individuals homozygous for sitosterolemia absorb substantial amounts of sitosterol, with resultant hypercholesterolemia and development of xanthomas (Belamarich et al., 1990). It is not known what percentage of individuals in a given population would have this condition. Still, in the absence of more data on genetic mutations involved in sitosterolemia, this plant sterol should be used with caution in certain individuals who have a higher absorption rate of sitosterol at the present time (Fernandez and Vega-Lopez, 2005; Lichtenstein and Deckelbaum, 2001). Finally, a recent study that requires further evaluations, indicated that elevations in sitosterol concentrations and the sitosterol/cholesterol ratio appear to be associated with an increased occurrence of major coronary events in men at high global risk of coronary heart disease (Assmann et al., 2006). From the data discussed above, it appears clear that the cholesterol-lowering activity of plant sterols does not strictly depend on the specific chemical structure of single molecules, but, more convincible, by their total uptake (quantity) and bioavailability. The ability of phytosterols in preventing cardiovascular diseases, at the recommended doses, is well consolidated and supported by a clear mechanism of action (lower cholesterol concentration). However, supplementation with phytosterols and “disease preventive” diets are two distinct issues which may generate a great confusion in consumers if not clearly explained. In fact, terms as “cardioprotective” or “chemopreventive” (see below), referred to one or more compounds present in the normal diet, are commonly interpreted by healthy consumers as diets able to prevent cardiovascular diseases or cancer, respectively. Of course it is not so simple! As an example, the total amount of phytosterols present in 1 litre of sunflower oil may reach the value of about 5 g. In the optimistic case that 25-30% of them will be adsorbed, and in order to get a recommended daily dose of 1.6 g/die (stanols plus sterols, see above), each consumer should eat 1 litre of sunflower oil per day to lower cholesterol! Usually, this paradox is not clearly explained by firms selling and/or advertising on the use of healthy products or functional foods including phytosterol fortified edible oils. Similarly, the scientific community, in many cases, fails to make a fine distinction between the effects of “healthy molecules”, such as phytosterols, defined and characterized in animal and cellular models at “pharmacological” concentration and in pathological situations (affected subjects or disease-prone animals) respect to “real” concentrations of the same compounds in the diet. In the case of edible oils, erroneous or incomplete scientific and media messages may encourage the consumers to increase the intake of phytosterol-rich oils in order to obtain a

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cardioprotective benefit. This tendency may produce two negative results: 1. they do not reach the recommended dose and, consequently, the auspicated effect; 2. they do increase the intake of total fat which represents the main risk factor associated to cardiovascular diseases. Despite this criticism, scientific evidence indicates that a real benefit in lowering cholesterol may derive from a regular consumption of phytosterol-rich oils at the recommended daily intake. In fact, as mentioned above (paragraph Plasma Lipids), it has been reported that consumption of 30-35 gr of sterol-free corn oil increased cholesterol absorption by 38%. When corn oil phytosterols were added back to sterol-free corn oil at a concentration of 300 mg/test meal (a value compatible with the average amount of sterols present in commercial corn oil), cholesterol absorption was reduced by 27.9 % (Ostlund et al., 2002). This work raises further questions: 1. only phytosterol-rich oils may produce these healthy benefits; 2. in this contest, olive oil seems less effective since it limited concentration in total sterols compared to seed oils (Table 3.7). Overall, it is our opinion that more studies are necessary to define the correct balance between phytosterols concentration/bioavailability and their healthy properties following dietary consumption of edible oils. In addition to the cardioprotective effects, more recently, an increasing number of studies suggest a chemopreventive activity of phytosterols, especially β-sitosterol. Accordingly to a more modern and complete definition, chemoprevention includes the use of natural or pharmacological agents to suppress, arrest or reverse carcinogenesis, at its early stages (Russo, 2007; Sporn and Suh, 2002). The chemopreventive mechanism(s) of action of phytosterols, from a molecular point of view, is still target of intense studies and it is strictly dependent on the specific chemical structure of the molecule investigated. Recent works indicate that β-sitosterol, is the most abundant phytosterol in many edible oils, inhibits the growth of several specific types of tumor cells in vitro and decreases the size and the extent of tumor metastases in vivo (Ovesna et al., 2004). β-Sitosterol exposure promotes its enrichment in transformed cell membranes and activate apoptosis by increasing CD95/Fas levels and caspase-8 activity (Awad et al., 2007), by activating caspase-3 and induction of Bax/Bcl-2 ratio (Bu et al., 2007; Park et al., 2007), and down-regulating Akt kinase (Moon et al., 2007). These findings support the hypothesis that β-sitosterol is an effective apoptosis-promoting agent and that incorporation of more phytosterols in the diet may serve a preventive measure against cancer, at least in in vitro cellular models. In animal models, β-sitosterol, its glucoside and a mixture of both modulate the growth of estrogen-responsive breast cancer cells in ovariectomized athymic mice (Ju et al., 2004). As already discussed in previous MAC-Oils reports and reviews (Russo, 2007), here, we are dealing with a class of compounds possessing multiple biological activities. Phytosterols, in facts, seem to be both cardioprotective and chemopreventive. The chemopreventive potential of phytosterols requires further studies. Data on the anticancer properties of sterols and stanols (and their metabolites) are still limited and they mainly refer to in vitro studies on cell lines, where, usually, the effects of high concentrations of these compounds are tested. If we focus our attention on the structure-function relationship, argan oil contains compounds, such as schottenol and spinasterol totally absent in other oils and specific of this product. Unfortunately, we must register the lack of studies on the potential biological properties of those sterols specifically present in argan oil.

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5.3.3 Phenols The analysis of data reported in Table 5.5 evidences the most significant difference in terms of “minor components” in studied oils. In fact, only argan and olive oils present a detectable amount of phenolic compounds, with a concentration in olive oil about 60-fold higher than argan oil. From a qualitative point of view, syringic acid is exclusively present in argan oil, while more than 95% of phenols in argan oil are represented by ferulic acid, almost absence in olive oil where hydroxytyrosol and tyrosol constitute about 20% of the total. As stated above for phytosterols, phenols in argan oil suffers for the limited number of studies performed and for their scientific quality. As an example, a paper from Owen’s group (Khallouki et al., 2003) concludes that the high content of γ-tocopherol, squalene, oleic acid and phenols is likely to enhance the cancer prevention effects of the Moroccan diet. This work seems to be mostly based on the presence of potentially bioactive compounds, more than on real experimental data. In other words, the observation that phenols are present in argan oil and these molecules have been associated to anticancer properties, it is not sufficient, in our view, to extrapolate that argan oil possess chemopreventive properties. Key issues, such as bioavailability and doses, must be addresses before elaborating final claims (see comments below). Similarly, studies performed on cell lines suggests that the products of Argania spinosa, not only oil extracts, but also keel and cake may provide a new therapeutic avenue against proliferative diseases (Samane et al., 2006) (Bennani et al., 2007; Drissi et al., 2006). One important bias common to these works regards the pharmacological doses applied which ranged in the order of hundreds micrograms/ml, i.d., in the micromolar range with an GI-50 (defined as the concentration inhibiting growth by 50% compared to the control) for argan phenols around 70 microg/ml. We disagree with Authors conclusion based only on these data that argan oil may be interesting in the development of new strategies for prostate cancer prevention. Although controversial data are present in the Literature, the total phenolic content of olive oils ranges between 500 mg/kg (Montedoro et al., 1992; Owen et al., 2000) and 200 mg/kg (Owen et al., 2000), with values for extra virgin olive oil (VOQ) significantly higher than that for refined virgin oil (RVO). The difference in total phenolics between VOQ and RVO reflects the concentration of the major individual components. Appreciable quantities of hydroxytyrosol, tyrosol and secoirodoids have been detected in olive oils by HPLC, with differences between VOQ and RVO (Owen et al., 2000). Differently than olive oil, in seed oils, the refining procedure strongly reduces the presence of natural antioxidants. The attention of many scientists focused on the biological effects of oleuropein, hydroxytyrosol and tyrosol, although the large part of these studies are referred to in vitro and ex vivo models, which generate controversial topics concerning the bioactivity of these molecules, such as bioavailability, metabolism and doses applied (physiological versus pharmacological). The phenolic compounds of olive oil in the Mediterranean diet have been associated with a reduced incidence of heart disease. Hydroxytyrosol has antithrombotic activities since inhibits LDL oxidation (Visioli et al., 1995), platelet aggregation (Petroni et al., 1995), and endothelial cell activation (Carluccio et al., 2003) with unknown mechanism(s) involving phospholipase C activation, arachidonic acid metabolism, and reduction of hydrogen peroxide (Singh et al., 2007). Hydroxytyrosol is a potent scavengers of superoxide anions (O'Dowd et al., 2004), protecting cells lines against oxidative insults (Goya et al., 2007). It appears very interesting the comparison between the antioxidant properties of tyrosol and hydroxytyrosol. Both biophenols inhibited cell-mediated oxidation of LDL, but to a different extent (100% hydroxytyrosol vs 40% tyrosol). In addition, tyrosol was effective in inhibiting about 30% of

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ROS production only at later time-points compared to hydroxytyrosol. This behaviour was probably related to cell-biophenol interactions: hydroxytyrosol was rapidly found inside the cells (1.12+0.05ng/mg cell protein) and disappeared within 18h, while tyrosol accumulated intracellularly with time (0.68+0.09 vs 1.72+0.13 ng/mg cell protein at minute 5 and hour 18, respectively). Therefore, in spite of its weak antioxidant activity, tyrosol was effective in preserving cellular antioxidant defences, probably by intracellular accumulation. These findings give further evidence in favour of olive oil consumption to counteract cardiovascular diseases (Di Benedetto et al., 2006). Cell culture experiments suggested that the olive oil phenolics induce a significant reduction in the secretion of intercellular adhesion molecule-1 and vascular cell adhesion molecule-1, and protect against cytotoxic effects of hydrogen peroxide and oxidized LDL (Turner et al., 2005). Similarly, hydroxytyrosol, oleuropein aglycone and homovanillyl alcohol strongly reduced cell surface expression of ICAM-1 and VCAM-1 at concentrations physiologically relevant (IC50 < 1 μM) in human umbilical vascular endothelial cells (HUVEC), linked to a reduction in mRNA levels, supplying new insights on the protective role of olive oil against vascular risk through the down-regulation of adhesion molecules involved in early atherogenesis (Dell'Agli et al., 2006). The protective effects of hydroxytyrosol and tyrosol were compared also in other studies. Only hydroxytyrosol was effective at low concentrations (10 μΜ) in lowering hydroperoxides, DNA damage, and mRNA levels. Tyrosol reduced DNA oxidation only at high (>50 μΜ) concentrations and increased hydroperoxides. Therefore, hydroxytyrosol appeared the only significant antioxidant phenolic in olive oil and may be the major component accounting for its beneficial properties, while tyrosol seemed to exhibit pro-oxidant effects (only at high concentrations) (Quiles et al., 2002). Together with its cardioprotective activity, hydroxytyrosol is reported to exert chemopreventive effects. The molecule induces growth arrest and apoptosis in cell lines by inhibiting pro-survival factors (Akt/PKB pathway) and TNF-α (tumor necrosis factor)-induced NF-κB (nuclear factor-kappaB) activation (Guichard et al., 2006). This result represents only one recent example of several works reporting the chemopreventive activity of olive oil phenolic compound on malignant cell lines. The concentrations of hydroxytyrosol, which inhibited 50% of cell proliferation were approximately 50 and approximately 750 μM for HL60 and HT29, respectively. At concentrations ranging from 50 to 100 μΜ, hydroxytyrosol induced an appreciable apoptosis in HL60 cells after 24 h of incubation as evidenced by flow cytometry, fluorescence microscopy and internucleosomal DNA fragmentation. These results support the hypothesis that hydroxytyrosol may exert a protective activity against cancer by arresting the cell cycle and inducing apoptosis in tumour cells, and support its potential anticancer activity (Fabiani et al., 2002) (Della Ragione et al., 2000). Taken together, all these data on the biological effects of olive oil phenols go in same direction. Cardioprotective and chemopreventive activities are observed in the following situation: 1. at pharmacological and concentrated doses of phenols, significantly far from the dietary intake; 2. in experimental models (cell lines) who do not take in account the metabolism and bioavailability of these molecule; 3. in animal models where the metabolism of these compounds is different from a biochemical and microbiological point of view compared to humans; 4. in “not physiological” experimental models (cell lines and animals) who do not mimic a state of “prevention” since the alteration is already present (cell lines) or experimentally induced (e.g., carcinogenesis in rat or transgenic mice). In the voluminous Literature on the anticancer activity of dietary phenolic compounds, the fundamental importance of their bioavailability and metabolism has been sometime neglected. As clearly stated by the Chemoprevention Working Group (1999; Russo, 2007), one of the main features of a potential chemopreventive agent is “safety”: it must be administered at low doses.

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However, from a Literature screening, appears clear that the concentrations generally used in the scientific papers are in the range of pharmacological doses or higher, including works on olive oil phenolic compounds (see above). Bioavailability means the rate and extent to which the active ingredient or active moiety is absorbed from a drug product and becomes available at the site of action. The extent of absorption and the bioavailability of a specific molecule must be understood in order to determine what dose will induce the desired therapeutic effect. It will also explain why the same dose may cause a therapeutic effect by one route, but a toxic or no effect by another. Generally, less than 10% of phenols, or their metabolites, ingested are found in plasma, where concentrations barely reach 1 μM (Russo, 2007; Scalbert and Williamson, 2000). Intake of “biologically active” phenols from olive oil is very difficult to determine. In a recent commentary (Vissers et al., 2004), the Authors concluded that 50 g of olive oil per day provides about 2 mg or approximately 13 μomol of hydroxytyrosol-equivalents per day, corresponding to a plasma concentration of 0.06 μM, a value significantly lower from those employed in in vitro studies (hundreds of micrograms). However, it should be reported that in a different study, the ingestion of 25 ml of extra virgin olive oil resulted in a plasma concentration of 0.16 μM, higher compared to the previous study, but still from the concentration applied in vitro (Miro-Casas et al., 2003). Based on these data, it appears clear the fundamental request for human studies. An example is represented by a double-blind, randomized, crossover interventional study in which 3 olive oils with low, moderate, and high phenolic content were given as raw doses (25 ml/die) for 4 consecutive days. The conclusion is in agreement with: 1. decreased plasma oxidized LDL; 2. decreased 8-oxo-dG in mitochondrial DNA and urine; 3. decreased malondialdehyde in urine; 4. increased HDL cholesterol and glutathione peroxidase activity, in a dose-dependent manner with the phenolic content of the olive oil administered (Weinbrenner et al., 2004). However, the most significant effects were measured only in the high phenolic treatment group [Table 2 in (Weinbrenner et al., 2004)], suggesting that oil phenols are well absorbed when administered in a phenol-rich diet, but their amount in the regular diet is probably too low to produce a quantifiable and biologically significant effect on LDL oxidisability (Vissers et al., 2004). Circumstantial evidence in humans also support the protective role of olive oil intake on BP. As mentioned above (paragraph “Blood Pressure and Endothelial Functions”) the effect of olive oil on BP could be mediated by compounds other than MUFA, such as the polyphenols present in virgin olive oil. This conclusion was raised after a study in hypertensive women demonstrating a reduction of both systolic and diastolic BP after an olive oil-rich diet, but not after a high-oleic acid sunflower oil diet (Ruiz-Gutierrez et al., 1996). Although of interest, this work may generate some criticism: 1. the effect of olive oil phenols on BP is “supposed”, not “demonstrated”; 2. the subjects in the study present already a pathological condition (hypertension). In fact, a different and more recent report on hypertensive and normotensive individuals, showed that a virgin olive oil-rich diet compared to one rich in sunflower oil reduced systolic BP in hypertensive patients but had no effect in normotensive individuals (Perona et al., 2004). Oryzanol A special mention is devoted to γ-oryzanol. This term is referred to a mixture of esters of ferulic acid with triterpenealcohols and sterols. It was originally identified as a single component, but it was later determined to be a fraction containing ferulic acid esterified by triterpene alcohols or plant sterols, such as campesteryl and β-sitosterol (Kumar et al., 2009). For these reasons and for the purpose of this review, γ-oryzanol has been included in Table 5.5. Among the studied oils in MAC-Oils project, γ-oryzanol is abundantly present in corn and rice oils. Since it potent antioxidant activity and cholesterol-lowering capacity, γ-oryzanol has generated global interest in terms of human health. In fact, the molecule may act as a

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double sword compound since the presence of the two moieties: fetulic acid and sterols. Taking a part the numerous studies present in the Literature on in vitro and animal models where γ-oryzanol is administrated at pharmacological doses, falling into the problems described above, we will report two studies suggesting the potential cardioprotective and anti-inflammatory use of the molecule. Supplementation of hypercholesterolemic men with 50 g/d of rice bran oil containing 0.05-0.8 g/d of γ-oryzanol for 4 weeks was able to lower plasma cholesterol and ameliorate lipoprotein patter. This effect was attributed to the “sterol” moiety of γ-oryzanol, assuming that all ferulated sterols become de-ferulated in the gut (Berger et al., 2005). In parallel, in a model of experimentally induce colitis in mice, γ-oryzanol, at a concentration of 50 mg/kg, was able to inhibit markedly inhibited inflammatory reactions (Islam et al., 2008). The Authors concluded that the anti-inflammatory effect could be mediated by inhibition of NF-kappaB activity, which was at least partly due to the antioxidant effect of the FA moiety in the structure of γ-oryzanol. Although the large part of γ-oryzanol (> 90%) is lost during conventional refining process of crude rice bran oils, the data reported above suggest that the remaining amount of ferulate esters might be sufficient to justify their biological activity in humans. 5.3.4 Combinatory prevention - synergistic effect The general low bioavailability of single phenolic compounds, together with the complex transformation reactions they undergo, makes difficult a cause-effect analysis. On the contrary, when ingested in the whole food, their “combinatory” effect might suggest a rationale for dietary prevention. In other words, a correct diet, as a whole, can be “preventive” without necessary focusing the interest on a single molecule (Russo, 2007). As demonstrated by the examples cited above, the cardioprotective effects of olive oil phenolic compounds may be explained recurring to the “antioxidant hypothesis” in disease prevention, which suggests that a regular antioxidant diet may balance a small disequilibrium in ROS (reactive oxygen species) homeostasis occurring in “normal” cells, as a consequence of lifestyle (diet, environment, etc.). The limited availability of dietary phenols, discussed above, that significantly reduces their presence in the target cells, might not represent a limitation. In fact, low concentration of antioxidants are, probably, sufficient to “correct” low increases in intracellular ROS, delaying, in long term, the development of atherosclerotic plaques. Perhaps, this speculative explanation has the merit to reconcile low bioavailability of dietary polyphenols with disease prevention. Paraphrasing Paracelsus: “all substances are poisons … the right dose differentiates a poison from a remedy” explain some contradictory results obtained for naturally occurring chemopreventers (Russo, 2007), including biophenols from olive oils. As an example, different types of normal and malignant cells (e.g., normal gingival fibroblasts, immortalized, non-tumorigenic gingival epithelial cells, and carcinoma cells from the salivary gland), were all sensitive to phenolic compounds. The cytotoxicity increased in the following order: oleuropein aglycone>oleuropein glycoside>>tyrosol. Cytotoxicity was noted only at phenolic concentrations far exceeding those attainable after habitual consumption (Babich and Visioli, 2003). Combination treatments might represent a new strategy that can play a major role in the future of disease prevention. However, only when the clinical efficacy of a compound, or class of compounds, has been scientifically proven (e.g., stanols and sterols), it can be prescribed as supplements or drugs on a large scale population, starting from subjects at a medium-high risk for a specific disease. The association of several molecules (as naturally

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happens in some foods) might be more effective prevention, than single compounds. This idea can be circumstantially proved by the following example. A virgin olive oil phenol extract inhibited HL60 cell proliferation in a time- and concentration-dependent manner, blocked cell growth and induced apoptosis. Determination of the cell cycle distribution by flow cytometry revealed an accumulation of cells in the G(0)/G(1) phase (Fabiani et al., 2006). It is easy to verify from independent data reported in the Literature that bioactive compounds present in the same extract, such as hydroxytyrosol and to tyrosol, taken singularly and at the exact concentration present in the crude extract, do not possess the same antiproliferative activities. However, mixing them in different combinations, keeping constant the original concentrations measured in the extract, it is possible to reconstitute the anti-proliferative and apoptogenic effects of the whole extract. This view generated the development of “combination prevention”, intending that low doses of chemopreventive agents differing in the mode of action may synergize increasing efficacy and minimize toxicity (Reddy, 2000; Sporn and Suh, 2002). Paradoxically, the most effective combination chemopreventive preparation is diet. The same rationale can be applied to single bioactive phytosterol present in edible oils at relatively low concentrations and no well characterized yet (e.g., in argan oil). The numerous phenolic compounds present in olive oil make unique this type of edible oil compared to others (Perez-Jimenez et al., 2007). In fact, in terms of healthy properties, olive oil may associate the high antiatherogenic capacity of MUFA with the presence of bioactive phenols with different molecular structure, stability, solubility, metabolism and cellular uptake. These compounds may undertake competitive and/or synergistic activity that cannot be simply reproduced in an in vitro model. In view of these effects, it would appear that olive oil represents the “gold standard”, as source of dietary fats for its composition which is not shared to the same extent by other oils that are rich in oleic acid but lack its characteristic micronutrients. An exception might be represented by those edible oils enriched in MUFA and containing high concentration of sterols and or oryzanol. Rice and corn oils represents good examples. Future studies will clarify which component, between fatty acids and “minor components” in edible oils will take the lead in the controversy field of functional food. We cannot evade from the recommendation to consider edible oils primarily as “fat source”, before to consider them as source of “healthy” compounds. Keeping total fat uptake below 35-40% of the energy intake represents actually one of the main determinant for an healthy life.

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5.4. Concluding remarks • On November 2004 and October 2006, the U.S. Federal Drug Administration (FDA)

permitted a claim on olive and canola oil labels, respectively, concerning “the benefits on the risk of coronary heart disease of eating about two tablespoons (23g) of olive oil daily or one and half tablespoons (19g) of canola oil daily, due to their composition in fatty acid and provided that these oils replace a similar amount of saturated fat and not increase daily energy intake.

• The lowering cholesterol effect of various oils is comparable. • The lowering triglyceride effect is more evident with high PUFA-oils than with high

MUFA-oils. • MUFA high-oils, essentially extra virgin olive oil, improve blood pressure. • High MUFA-oils oxidize LDL-cholesterol less than high PUFA-oils. • Extra virgin olive oil induces the lowest oxidative stress in the body. • High n-3 PUFA-oils decrease markers of inflammation • Soybean and canola/rapeseed oils are the two edible oils richer in ALA, however the n-

6/n-3 PUFA ratio of latter is healthier than that of soybean oil (on average 2.2 vs 7.7). • Hydrogenation and high cooking temperature increase the amount of trans fatty acids in

all oils, however this effect is more evident for high-PUFA oils. • All oils, irrespective of their fatty acid composition, have the same caloric power. • The amount of tocopherols ingested, independently by the source of vegetable oils used in

the diet, should be enough to sustain the daily allowance of vitamin E. • The cardioprotective activity of phytosterols is due to their ability to compete with

cholesterol in the intestinal lumen thereby reducing cholesterol absorption. • The cholesterol-lowering activity of phytosterols does not strictly depend on the specific

chemical structure of single molecules, but, more convincible, by their total uptake and bioavailability.

• Scientific evidence indicates that a real benefit in lowering cholesterol may derive from a regular consumption of phytosterol-rich oils at the recommended daily intake.

• The phenolic compounds of olive oil in the Mediterranean diet have been associated with a reduced incidence of heart disease.

• Hydroxytyrosol has antithrombotic activities and antiproliferative effects, supporting the hypothesis that olive oil phenolic compounds may possess both cardioprotective and chemopreventive activities.

• In assessing the healthy properties of phenols in olive oil, the bioavailability and metabolism of this molecules have been sometime neglected. The general low bioavailability of single phenolic compounds, together with the complex transformation reactions they undergo, makes difficult a cause-effect analysis.

• The association of several molecules, as naturally happens in foods included edible oils, might be more effective, than single compounds, intending that low doses of preventive agents differing in the mode of action may synergize increasing efficacy and minimize toxicity.

• A correct diet (e.g., Mediterranean diet) as a whole, can be “preventive” since the “combinatory” effects of the bioactive compounds present in the different food.

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