7 Olive Oil David Firestone
United States Food and Drug Administration Washington, DC
1. INTRODUCTI INTRODUCTION ON AND HISTORY HISTORY Olive oil, an important component in the diet of Mediterranean people, is obtained by mechanical extraction from the fruit of the Olea europaea L. tree, which belongs to the Olive family, comprises some 400 species, and thrives in temperate and tropical climates (1). Of the 35 species in the genus Olea, mainly of African, Indian, and Australian origin, O. europaea is the only Mediterranean species. Although its origin is not known, one theory is that it originated in ancient Iran and Turkestan, spreading westward to Anatolia, Syria, and Israel along commercial and migratory routes (2). Olives appeared in Israel about 45,000 years ago (1). Charred pieces of olive wood have been found in excavations at Lower Boker-Har Hanegev in layers dating to 42,980 B.C. Both charred wood and carbonized stones have been found in many archeological sites in Israel dating from 8000 B.C. onward, and indirect evidence suggests the use of wild olives (O. oleaster ) by humans as early as the seventh millennium B.C. (3). It is not known whether the carbonized stones and charred wood obtained from Chalcolithic (fourth millennium B.C.) and Early Bronze Age (2900–2700 B.C.) sites represented cultivated or wild olives. Olive farming and an olive oil industry appear to have been well established through throughout out the region region border bordering ing the Medit Mediterra erranea nean n from from Palest Palestine ine and Syria Syria to Greece in the middle and late Bronze Age (4). Olive farming in Palestine and Syria Bailey’s Bailey’s Industrial Oil and Fat Products, Sixth Edition, Six Volume Set. Edited Edited by Fereidoon Fereidoon Shahidi. Shahidi. Copyright Copyright # 2005 John Wiley & Sons, Inc.
303
304 30 4
OLIVE OIL
increased dramatically at the turn of the first millennium B.C. (2). An olive oil industry became well established in Palestine, and the export of olive oil from Palestine to Egypt is documented in Old Kingdom Egypt. Olive cultivation provided materials useful as a lamp fuel, lubricants, and body ointments; the fruit was easily cured by salting, and the wood was used for carpentry and fuel. Later, the olive fruit became a source of edible oil. The manufacturer of olive oil became a mass production industry during the Israelite period when processing methods improved (3). In Judea, oil presses generally consisted of large stone beds with a collecting vat in the center of the pressing surface. A beam, which acted as a lever and was weighted down by several stones, was used for pressing. The end of the beam was anchored to a wall behind the press (niche wall) with a special niche stone. Olives were crushed in a rectangular basin by a roller, which an operator set into forward and backward motion by means of an attached shaft. A typical Iron Age industrial site is that of the seventh century B.C. biblical town of Timnah (Tel Batach), which was a center of olive oil production along with other towns in the Tel Aviv area (5). The oil presses of the town were constructed similarly to those of other Iron Age sites in the area. Each press consisted of a crushing basin with two pressing vats on either side. Olives were crushed in the basin with stone rollers, each of which had wooden handles fitted into sunken depressions at the sides. The crushing basin was a shallow trough made of one large chalk stone. Each pressing vat contained a large stone with a flat top and an inner hollowed space for collecting the oil. Because there was no means of draining the oil from the vet, pottery jugs were used to withdraw the oil. Baskets with crushed olives were pressed by wooden beams anchored at one end to niches in the wall; the other end of each beam was pressed down with three heavy stone weights (Figure 1). Olive growing reached Cyprus and the Aegean area around the sixteenth century B.C. As Renfrew (6) pointed out, the olive was one of three important constituents, along with the vine and domesticated wheat, that contributed to the emergence of civilization in the Aegean region. Oil production and trade played important roles in the Minoan–Mycenaen economy of Crete and main-land Greece in the second millennium B.C. (7). Olive oil was used in the manufacture of scented perfumes and unguents in the palace industries of Crete and Mycenae. Wild rather than cultivated olives were apparently preferred for Aegean perfume and unguents because of the low fat content of the wild olive. Initially, olives were harvested by beating the trees with flails (6). After harvesting, the olives were drenched in hot water and pressed to extract the oil. The oil was separated from the water in a vat from which the water was drawn off, and then stored in jars similar to those used to hold wine. Oil was used locally for lighting, hygi hygien enic ic purp purpos oses es (to (to clea clean n the the body body), ), and and as food food,, espe especi cial ally ly for for cook cookin ing. g. Mycenaen documents suggest that scented olive oil was used for religious purposes and as a body ointment for the rich (8). The earliest evidence of olive oil extraction in Cyprus dates to about 1300 B.C. (9). (Wild olives grew on the island at least as early as 4000 B.C.) An olive press (probably a lever and weight press) found at a Maroni excavation site consisted
INTRODUCTION AND HISTORY
305
Figure 1. Oil press at Tel Batach (biblical Timnah), 7 km west of Beth-Shemesh, Israel. Isometric plan and sections (5). [Reprinted with kind permission of the editors of Olive oil in Antiquity (5).]
306
OLIVE OIL
of a large rectangular trough (pressing bed) set on a mudbrick platform and sloping downward. The trough is a flat stone with channels cut to meet at a small projection, permitting the liquid to pour off into a jar standing on the floor below the press. Other presses of the late Roman period found on the island were of the lever and screw type in which the horizontal beam is immobile while the screw presses down on the pressing bed. The screw was already used for pressing in Italy in the first century B.C., initially as a lever and screw press and then in a direct frame press (10). Olive orchards continued to be extensively cultivated in Palestine throughout the Byzantine and Arab periods (11). The chain of mountains from the upper Galilee to Hebron were covered with olive trees. Olive oil was used regularly for food and cooking as well as for lighting and manufacture of soap by boiling the oil with ashes. During the early period under the Umayyides (661–750 A.D.) and Abbasides (750–1258 A.D.), oil surpluses were exported from Palestine by land to neighboring countries. With revival of maritime commerce under the Fatimids (909–1171 A.D.), oil was transported to Egypt and other countries by boat. Phoenician settlements in the Mediterranean basin introduced olive farming into Sicily, Sardinia, southern France, and Spain (2). The Greeks later spread farming independently of the Phoenicians, reintroducing the olive into Sicily. The Romans spread olive farming throughout their territories and used the olive tree in their land reclamation projects, particularly in North Africa where they instituted olive farming and other projects to reclaim desert areas. Although of variable quality, olive oil was a staple food and an important industrial product in Roman times. Olive growing continued to prosper in the Mediterranean region until the fifth century A.D., when the Roman Empire was invaded from the north and maritime routes were closed (2). Olive farming was later revived with commercial development of Venice and other maritime republics during the Renaissance. In 1709, olive growing entered a new modern age when all of Europe was hit with a deep cold spell and new orchards were planted to replace those destroyed by the cold weather. As modern farming techniques evolved, large-scale state enterprises were begun and olive farming reached a peak in the first half of the nineteenth century.
2. STATISTICS AND DEFINITIONS Currently, more than 95% of the world’s olive trees grow in the Mediterranean Basin. About 81% of total olive production comes from the European Community (EC) (Spain, Italy, Greece, Portugal, and France), with the Near East contributing, ca 7% and North Africa supplying about 11%. The remaining 1% is of American origin, chiefly from Argentina, Mexico, Peru, and the United States (Table 1). Olive oil consumption is growing in the developed countries that produce little or no olive oil (Table 2). The fruit of the olive tree is an egg-shaped drupe, consisting of a pericarp and an endocarp. The pericarp includes an epicarp (skin) of variable thickness according to the variety, and a mesocarp (pulp) surrounding the endocarp (woody pit) in which
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307
a
TABLE 1. World Production of Olive Oil (Thousand Metric Tons).
Country Algeria Argentina Cyprus EC Croatia Israel Jordan Lebanon Morocco Palestine Syria Tunisia Turkey Australia Egypt USA Iran Libya Mexico Yugoslavia Serbia and Montenegro World Total a
1997/98 15.0 8.0 1.5 2,116.5
1998/99
1999/00
2000/01
2002/03 2003/04 2001/02 (prov.) (est.)
1.0 1.0 3.0 6.0 2.0 0.5
54.5 6.5 2.5 1,707.0 5.0 4.5 21.5 7.0 65.0 5.5 115.0 215.0 170.0 0.5 0.5 1.0 2.5 8.0 2.5 1.0
33.5 11.0 3.5 1,878.5 9.0 2.5 6.5 5.0 40.0 2.0 81.0 210.0 70.0 0.5 2.5 1.0 2.5 7.0 1.0 1.0
26.5 4.0 5.5 1,940.5 5.5 7.0 27.0 6.0 35.0 20.0 165.0 130.0 175.0 1.0 0.5 0.5 3.0 4.0 1.5 0.5
25.5 10.0 6.5 2,463.5 5.0 3.5 14.0 5.0 60.0 18.0 92.0 35.0 65.0 1.0 1.5 0.5 2.5 7.0 2.0 0.5
16.5 11.0 7.0 1,942.5 7.0 9.0 28.0 6.0 45.0 21.5 165.0 70.0 160.0 2.0 5.0 1.0 1.5 6.5 2.5 0.5
40.0 22.0 7.0 2,307.0 3.0 2.5 11.5 4.0 80.0 5.0 110.0 180.0 60.0 3.0 2.0 1.0 4.0 6.5 2.5 0.5
8.5 2,465.5
7.5 2,402.5
6.5 2,374.5
7.5 2,565.5
7.5 2,825.5
7.5 2,515.0
7.5 2,859.0
3.0 14.0 3.5 70.0 9.0 70.0 93.0 40.0
Source: International Olive Oil Council (IOOC).
the seed is enclosed. The yield per hectare is about 2.45 tons. Oil yield per 100 kg of fruit is 19.6 kg (based on yields in Italy during the past 10 years). In addition to oil, the pulp and epicarp contain a variety of natural components soluble in the oil. As will be seen later, the oil is obtained from the olive by a variety of techniques, always physical, leaving a residue (pomace) that contains up to 8% oil, which is then extracted by solvent (usually hexane) and named pomace oil. Because of the behavior of the solvent, solvent-extracted oil contains more minor components at higher levels than those found in physically extracted oil. This provides the basis for designating pomace oil as a commercial product distinct from virgin oil (obtained only by mechanical means) or refined (lower grade) virgin oil mixed with virgin oil (olive oil, Riviera type). The following internationally recognized definitions of oils derived from olives and available on the market were promulgated by the International Olive Oil Council (IOOC) (12): 1. Olive oil is that oil produced by extraction of the fruit of the olive tree (Olea Europaea Sativa Hoffman et Link) to the exclusion of oils obtained using solvents or reesterification processes and of any mixture with oils of other
308
OLIVE OIL a
TABLE 2. Olive Oil Consumption (Thousand Metric Tons).
Country
1997/98
Algeria Argentina Cyprus EC Croatia Israel Jordan Lebanon Morocco Palestine Syria Tunisia Turkey
31.5 8.0 2.0 1,705.5
Australia Brazil Chile Egypt USA Iran Libya Mexico Yugoslavia/Serbia and Montenegro Other producing countries Saudi Arabia Canada Japan USSR/Russia Switzerland Taiwan Other nonproducing countries Total World a
1998/99
1999/00
2000/01
2002/03 2003/04 2001/02 (prov.) (est.)
6.5 19.0 8.0 55.0 5.5 95.0 52.0 85.5 2,073.5 17.5 29.0
44.0 8.0 2.5 1,709.0 4.0 9.5 19.0 9.0 55.0 4.0 88.0 49.0 85.0 2,086.0 24.0 23.5
42.0 7.0 4.0 1,728.0 8.5 12.5 9.0 8.0 55.0 4.0 90.0 60.0 60.0 2,088.0 25.5 25.0
26.0 6.0 5.0 1,835.0 6.5 13.5 17.0 8.0 45.0 8.0 110.0 58.0 72.5 2,210.5 31.0 25.0
25.0 5.5 5.5 1,894.0 5.0 14.5 20.0 7.0 60.0 10.0 86.0 28.0 55.0 2,215.5 27.5 22.5
16.0 5.5 6.0 1,904.5 6.0 14.5 25.0 7.0 55.0 12.0 100.5 30.0 55.0 2,237.0 31.0 20.0
39.0 6.0 6.0 1,932.0 3.0 13.5 15.5 7.0 70.0 12.0 115.0 60.0 40.0 2,319.0 31.0 21.0
1.0 142.5 4.0 7.0 4.5
1.0 151.0 2.5 16.0 5.0
1.5 169.5 2.5 11.0 5.0
1.0 194.5 3.0 7.0 6.5
1.5 188.5 2.0 8.0 8.0
3.5 190.0 2.0 8.5 10.0
2.5 195.0 3.5 8.5 10.0
0.5
1.0
1.0
0.5
0.5
0.5
0.5
13.5 219.5 5.0 17.5 34.0 1.5 5.5 4.5
12.5 236.5 5.5 18.5 28.5 2.0 6.0 7.0
13.0 254.0 4.5 23.0 27.0 3.0 8.0 6.0
13.0 281.5 4.0 24.5 30.0 4.0 8.0 8.0
14.0 272.5 5.0 24.0 31.5 4.0 9.0 6.5
14.5 280.0 7.0 24.0 32.5 6.0 10.0 5.5
14.5 286.5 7.5 24.5 33.0 7.0 10.0 6.0
20.5 88.5 2,381.5
23.0 90.5 2,413.0
29.0 100.5 2,442.5
20.0 98.5 2,590.5
38.0 118.0 2,606.0
38.5 123.5 2,640.5
38.5 126.5 2,732.0
Source International Olive Oil Council (IOOC).
kinds. In no case shall the designation ‘‘olive oil’’ be used to refer to olive– pomace oils. A. Virgin olive oil is the oil obtained from the fruit of the olive tree solely 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.
STATISTICS AND DEFINITIONS
309
Virgin olive oil fit for consumption as is (and can be designated as ‘‘natural’’) includes: a. Extra virgin olive oil: virgin olive oil that has an organoleptic rating of 6.5 or more as determined by the IOOC method (13) and a free acidity, expressed as oleic acid, of not more than 1 g per 100 g. b. Fine virgin olive oil: virgin olive oil that has an organoleptic rating of 5.5 or more and a free acidity, expressed as oleic acid, of not more than 1.5 g per 100 g. c. Semifine virgin olive oil (or ordinary virgin olive oil): virgin olive oil that has an organoleptic rating of 3.5 or more and a free acidity, expressed as oleic acid, of not more than 3.3 g per 100 g. (This class of olive oil is normally traded in bulk for blending purposes.) B. Virgin olive oil not fit for human consumption, also designated as lampante virgin olive oil: virgin olive oil that has an organoleptic rating of less than 3.5 and/or a free acidity, expressed as oleic acid, of more than 3.3 g per 100 g. This class of olive oil is used to produce refined olive oil or is intended for technical (nonfood purposes). C. Refined olive oil: olive oil obtained from virgin olive oils by refining methods that do not lead to alterations in the original triglyceride structure. D. Olive oil: the oil consisting of a blend of refined olive oil and virgin olive oil in various proportions. 2. Olive–pomace oil: the oil obtained by solvent extraction of olive–pomace and not including any oil obtained by a reesterification procedure or any mixture with other kinds of oils. (The various categories of olive–pomace oil are described below.) A. Crude olive–pomace oil: olive–pomace oil intended for refining to produce a product (as B, below) suitable for human consumption, or intended for technical purposes. B. Refined olive–pomace oil: the oil obtained from crude olive–pomace oil by refining methods that do not lead to alterations in the original triglyceride structure. C. Olive–pomace oil: a blend of refined olive–pomace oil and virgin olive oil (any A, B, or C). In no case may this be called ‘‘olive oil.’’ Because the yearly production of olive oil is variable, low-production years can follow years of high production. Therefore, it is customary to record average values (Table 1).
310
OLIVE OIL
3. EXTRACTION TECHNOLOGY Ripe olives contain a variety of components, including water, oil, sugars, proteins, organic acids, and cellulose. Olive cultivars with medium-size fruits generally provide the best oil yields. The pulp-to-kernel ratio of olives for oil production ranges from 4:1 to 8:1. The epicarp contains a number of components of relatively high polarity that are not removed by mechanical extraction and remain in the pomace. Removal of these components along with the oil by solvent extraction of the pomace accounts for the higher unsaponifiable content of olive–pomace oil. Most of the oil (96–98%) is in the pulp along with most of the water ‘‘vegetation water’’ (VW), which accounts for 40–60% of the weight of the fruit. The woody pit inside the mesocarp holds a seed whose oil is more unsaturted than the mesocarp (pulp) oil because of a higher content of linoleic acid. The ratio of fruit oil to seed oil is 50:1. The approximate chemical composition of olive fruit is as follows: water 52.4%; oil 19.6%; proteins 1.6%; sugars 19.1%; cellulose 6.8%; and ash 1.5%. Oil yield and quality depend on the cultivar of olive tree, ratio of the various anatomical parts, and levels of minor components as well as growing conditions and health of the trees. Soil moisture is very important during fruit development. Harvesting of fruit for oil production begins in the middle of autumn and lasts until the end of February. In some regions, it begins earlier, and in other locales, it lasts until March. Accordingly, differences in oil quality and composition can be expected along with variations caused by climatic and soil conditions. Variations in quality are chiefly related to the levels of minor components and flavor compounds, acidity, and the presence of mono- and diglycerides (14–16). Analytical and organoleptic data show that oil content is lower at the beginning than at the end of the harvesting period, but it is of higher quality (15). Harvesting technology is very important for production of high-quality oil. Olives should be collected as soon as they reach optimal maturity; however, it is dif ficult to have mechanical collection devices available where and when needed. In addition, because of the conformation of the tree branches, strong adherence of the fruit to the tree, and limited accessibility, most olives are picked by hand. Another harvesting procedure is to wait until the olives drop naturally and then collect the fruit with a system of nets. When the ripening period is delayed, both this procedure and handpicking are used. Although attempts have been made in the past to use chemicals to influence dropping time, chemicals are seldom used. Mechanical devices must be used with caution so that neither the tree nor the branches are damaged. When mechanical devices are used, the olives are caught in nets to avoid contact with the ground and damage to the fruit. Under optimum conditions, the olives are transferred from the nets to cages (usually plastic), forming layers not higher than 30 cm each, and the olives are sent promptly to the extraction plant. In most regions of Italy and Greece, cages are stored no more than 3 to 5 days before extraction. This procedure ensures
EXTRACTION TECHNOLOGY
311
high-quality oil if climatic conditions were good, the trees received proper care, and the fruit was not damaged by pests. If proper precautions are not taken and the olives are collected in large batches and held in piles several meters high, the fruit may be damaged. The enzymes released will cause hydrolytic and oxidative transformations resulting in off-flavors that affect the quality of the oil. Even with low acidity, such oils will have an unpleasant taste not acceptable for virgin oil and will have to be refined. Because of the difference in price between virgin and refined oils, economic losses to the farmer can be high. Three systems are used for mechanical extraction of oil from the olive fruit: pressure processing (Figure 2); centrifugation (Figure 3); and adhesion filtering (Figure 4) (17). Pressing is the oldest and most often used method for olive oil extraction. High-speed rotating machines are used for centrifugation extraction. With adhesion filtration, a series of steel plates or blades are dipped into olive paste; when withdrawn, the oil drips off the blades. Several processing steps are required before extraction. The fruit must first be cleaned to eliminate branches and leaves and any extraneous materials that might damage plant equipment. The fruit is then washed to remove dirt and agricultural contaminants, and finally crushed and milled to a coarse paste (Figure 5). During the last step, enzymatic action breaks up the bitter components and reduces the level of peppery constituents while increasing the amount of minor polar components
Figure 2. Pressure extraction of oil. 1, movement of the rack; 2, movement of the oil; A, mobile head; B, fixed head.
312
OLIVE OIL
Figure 3. Centrifuge for oil extraction from olive – pomace.
and tocopherols in the oil. If enzymatic action is prolonged, the minor polar components break down into water-soluble compounds that are removed from the oil, causing the loss of much of the antioxidant strength of the oil. Milling releases the oil from the oil-bearing cells and helps smaller droplets of oil to merge into larger drops, thus preparing the fruit for the following extraction step. A solid residue and vegetation water are produced during extraction in addition to oil (Figure 6). The vegetation water must be purified before discharge into a municipal sewer. Waste water has been used to grow yeast, to produce butanol using microorganisms, to isolate anthocyanin compounds for use in the food industry, and to produce steam.
Figure 4. Diagram of adhesion extraction of oil.
EXTRACTION TECHNOLOGY
313
Figure 5. Flow diagram of steps to prepare olives for extraction of oil.
Figure 6. Flow diagram of olive oil extraction and processing to yield olive oil products and byproducts.
314
OLIVE OIL
Figure 7. Flow diagram of solvent extraction of pomace.
Efforts are being made to reduce waste water by recycling in the milling process and to decrease its environmental pollution by treatment with biological or physical processes prior to its discharge (22). A number of alternative technologies are available for waste water purification (18–21); however, they are costly and dif ficult to apply. If suitable for consumption, the oil is centrifuged after extraction to eliminate solid impurities and residual water. If the free fatty acid content is too high or organoleptic properties are unsatisfactory, the oil is refined. At the solvent extraction plant, the cake (pomace) containing up to 8% residual oil is dried in a rotary kiln before proceeding to the solvent extraction unit, usually a semicontinuous system (Figure 7). The extracted pomace oil is always refined. Spent cake is used as fuel or is separated into two fractions, the pulp (including skin) and the pit. In addition to use as fuel, the pit is occasionally used to produce fiberboard (23).
4. REFINING OF OLIVE OILS Olive oil refining is carried out in either of two ways: by alkali refining, generally used for animal and vegetable oils and fats; or by physical refining, a technology not usually used for seed oils. Flow diagrams of the two procedures are shown in Figures 8 and 9.
REFINING OF OLIVE OILS
Figure 8. Flow diagram of alkali re fi ning.
Figure 9. Flow diagram of physical re fin ing.
315
316
OLIVE OIL
In the first procedure, the oil is treated with dilute acid to precipitate the gums (phosphatides and proteinaceous material), which are separated by settling or centrifugation. Phosphoric acid and citric acid are the two most common degumming agents. After degumming, the oil is neutralized (alkali refined) either in a batched or continuous system. Batch neutralization is currently preferred because centrifuging of only the settled soap fraction lowers the neutralization coef ficient values, thereby shortening the washing time of the oil. The separated soap solution is acidified with sulfuric acid to recover the free fatty acids (containing 30–40% triglycerides) for industrial applications. The alkali-refined oil is then bleached under vacuum with mixtures of various adsorbents (bleaching earth or clay and sometimes small amounts of activated carbon) and filtered by any of a number of available filter presses occasionally equipped with a solvent system for recovering oil entrained in the bleaching earth. The bleached oil is deodorized in a semicontinuous or continuous deodorizer operating at a vacuum of less than 2 mm Hg. The final step involves mixing refined oil with virgin oil to improve the organoleptic and keeping properties of the oil. A good olive oil will contain at least 20% virgin oil, but the product must, of course, meet consumer preference, which sometimes requires a very light flavor and taste. With physical refining, the oil is first degummed and bleached and then fed to a continuous distillation (deodorization) unit, which removes the free fatty acids (92– 95%) and volatiles. The refined oil is blended as above. Frequently, distillation is stopped before removal of all of the free fatty acids, and the oil is alkali refined to remove the remainder of the free fatty acids. This procedure has the advantage of eliminating oxidation byproducts and pro-oxidant metals, thus improving product stability.
5. REFINING OF POMACE OIL The technologies adopted for refining pomace oil are based primarily on physical refining because the acidity of pomace oils is about 10% (expressed as oleic acid). Because degumming of pomace oil requires more drastic conditions than those for pulp oil, larger amounts of acidulant are used (phosphoric acid is preferred), and occasionally, the precipitate (gum) that entrains a high proportion of oil is centrifuged to recover the oil. Larger amounts of bleaching earths are required to remove the intense green color of the oil. Additional processing of the bleached oil usually follows the same procedures described for physical refining of olive oil, including incomplete distillation (deodorization) followed by alkali refining of the partially deodorized oil. Dewaxing (winterization) of pomace oil is mandatory because of its high content of waxes (olive oil may also be winterized, especially if it is used to produce margarine or mayonnaise). Higher melting point triglycerides are also removed. Winterization can be carried out after bleaching or following partial deodorization
OLIVE OIL COMPONENTS
317
and alkali neutralization (alkali refining). If alkali neutralization is performed at a low temperature, winterization can be carried out simultaneously. A continuous apparatus is generally used for winterization (Figure 9) coupled with continuous filtering units. The winterization oil is then blended with virgin oil to restore the oil’s antioxidant properties.
6. OLIVE OIL COMPONENTS Glycerides account for at least 97% of a virgin oil if the acidity is disregarded. The free fatty acid content is used to distinguish the various classes of virgin oil, from extra virgin to lampant. It must be emphasized that virgin olive oil is a natural product and therefore subject to variations in composition, both qualitative and quantitative. The origin, cultivar, extraction technology, state of ripening of the fruit, climatic conditions, and rainfall all influence biosynthesis within the fruit and, therefore, the composition and quality of the oil. The fatty acid composition of olive oil is shown in Table 3, which lists typical compositions of European, Turkish, and African (Tunisian) oil as well as IOOC limits (12). Differences in composition are due chiefly to linoleic, linolenic, and palmitic acid content. Olive oils from Argentina resemble those from Tunisia. The triglyceride composition of European, Turkish, and Tunisian olive oils is shown in Table 4 (main glycerides are shown). Fatty acid distribution in the triglycerides follows the 1,3-random, 2-random rule (24–26). Several classes of minor components are present in virgin olive oil. The structure, concentration, and number of these substances are characteristic of virgin oils. Some are minor glyceridic components (MGCs); others fall into other categories as listed below.
TABLE 3. Fatty Acid Composition of Olive Oil (%).
Acid
CAN
European
Turkish
Tunisian b
Limits (12)
Palmitic Palmitoleic Heptadecanoic Heptadecenoic Stearic Oleic Linoleic Linolenic Arachidic Eicosenoic Behenic Lignoceric
16 : 0 16 : 1 17 : 0 17 : 1 18 : 0 18 : 1 18 : 2 18 : 3 20 : 0 20 : 1 22 : 0 24 : 0
8.4 0.7 0.1 0.1 2.5 78.0 8.3 0.8 0.5 0.3 0.1 0.2
12.1 0.7 0.2 0.2 3.1 71.3 10.6 0.7 0.4 0.3 0.2 0.2
15.3 1.6 0.1 0.1 2.1 62.5 16.5 0.8 0.5 0.3 0.1 0.1
7.5–20.0 0.3–3.5 0.0–0.3 0.0–0.3 0.5–5.0 55.0–83.0 3.5–21.0 0.3–0.9 0.2–0.6 0.1–0.4 0.0–0.2 0.0–0.2
a b
a
CAN ¼ Carbon atom number. Typical values for Tunisian olive oil analyzed during 1994.
318
OLIVE OIL
TABLE 4. Main Triglycerides of Olive Oil (%).
ECNa
Triglyceride b
42 44
LLL, TLOd, TLPd LLOd TOOd , LLP LOOd LOPd, PLP OOO POO POP SOO SOP OSS, PSS
46 48
50 52
European
Turkish
Tunisian c
0.5 2.4 2.6 13.3 8.0 39.9 26.0 — 5.1 1.0 0.8
0.8 3.2 2.9 13.8 9.7 34.0 24.4 — 5.1 1.4 —
1.6 10.6 1.7 16.0 16.2 23.2 22.0 5.1 4.3 1.2 0.5
a
ECN ¼ equivalent carbon number. L ¼ C18 : 2; T ¼ 18 : 3; O ¼ C18 : 1; P ¼ C16 : 0; S ¼ C18 : 0. c Typical values for Tunisian olive oil analyzed in 1994. d Mixture of isomers. b
Hydrocarbons Tocopherols Linear short chain alcohols and their esters Linear long chain alcohols and their esters Sterols and their esters a-Methyl sterols Monohydroxytriterpenes Dihydroxytriterpenes Triterpenic acids Phytol Geranylgeraniol Phenols and related compounds Flavor components Methyl and ethyl esters Other components
6.1. Minor Glyceridic Components Monoglycerides (MGs) and diglycerides (DGs) in the olive fruit are caused by enzymatic hydrolysis of the triglycerides and incomplete triglyceride biosynthesis (16). In general, DGs are more abundant than MGs. Determination of DG concentration is useful for evaluating oil freshness and time of fruit harvesting because the DG level is strongly related to climatic influences. DG concentration can even be used to determine the source of an oil, even a refined oil, because the DG content of edible virgin olive oils differs from that of high acidity oils or solvent-extracted oils. Phospholipids are essentially absent from olive oil.
OLIVE OIL COMPONENTS
319
6.2. Nonglyceridic Minor Components Hydrocarbons. Both even- and odd-chain n-paraf fins, including branched-chain (iso and anteiso) compounds, which are minor components of the hydrocarbon fraction, are present in virgin olive oil. The polyunsaturated triterpenic hydrocarbon squalene, and biochemical precursor of sterols, is the main component of the hydro carbon fraction. The squalene content of olive oil ranges from 150 to 700 mg per 100 g (27–30). b-Carotene is also present in olive oil as are aromatic hydrocarbons, including benzenoid, napthalenic, and more complex aromatic hydrocarbons (30–37). Linear Short Chain Alcohols and Their Esters. Methanol and ethanol esters of the fatty acids present in olive and in the same proportions as in the olive are present among the volatile compounds in virgin olive oil (31–37). Straight Long Chain Alcohols. Linear long-chain alcohols with carbon numbers between C22 and C32 are present in olive oil both free and esterified (waxes). The components are abundant in the epicarp of the fruit and concentrate in solvent extracted oil. Phytol, probably derived from biodegradation of chlorophyll, is also present along with geranyl (38). Cyclic Monohydroxy Compounds. Triterpenic tetra- and pentacyclic monohydroxy compounds are characteristic of olive oils (34–49). The following compounds have been shown to be present, accompanied by small amounts of lanosterol and obtusifoliol:
Tetracyclic: Pentacyclic:
cycloartenol 24-methylene cycloartanol a-amyrin b-amyrin
Methylsterols (4-desmethyl triterpenes) and sterols (4,4-di-desmethyl triterpenes) present in olive oils are derived from the tetracyclic alcohols. The following methyl sterols (4a-methyl-7-cholesten-3b-ol compounds) are present: 24-methylene, 24-methyl-, 24-ethylidene, and 24-ethyl. The main sterols of olive oil are (40, 43, 45–66) campesterol, stigmasterol, clerosterol, b-sitosterol, sitostanol, and d-5-avenasterol. These are accompanied by small amounts of cholesterol (max. 0.5%), brassicasterol (max. 0.1%), 24-methylenecholesterol (max. 0.5%), campestanol (max. 0.5%), d-5,24,-stigmastadienol (max. 1%), d-7-stigmastenol (max. 0.5%), and d-7-avenasterol (max. 1.1%). Analysis of the sterol fraction isolated from the unsaponifiable fraction is very important, as will be seen later, for determining the authenticity of the oil. The triterpenes and sterols are present both as free alcohols and as fatty acid esters (46, 47). Cyclic Dihydroxy Compounds. Pentacyclic triterpenes in olive oil include 3b,17b-dihydroxy-12-oleanene (erythrodiol) and its parent compound uvaol, obtained largely from the epicarp and therefore characteristic of solvent extracted oils (42, 65).
320
OLIVE OIL
Triterpenic Acids. The following pentacyclic mono- and dihydroxy triterpenic acids are present in virgin olive oil (35, 43, 44): 3b-hydroxy-17-carboxy-d-12-oleanene (oleanolic acid); 3b,2a-dihydroxy-17-carboxy-d-12-oleanene (maslinic acid); 3b-hydroxy-17-carboxy-d-12-ursene (ursolic acid); 2a,3b-dihydroxy-17-carboxyd-12-ursene (2a-hydroxyursolic acid); and deoxyursolic acid (structure not fully elucidated). Chlorophylls. Both chlorophyll a and chlorophyll b are present in olives and are partially extracted into the oils. Flavor Components. Olive oil volatiles contain at least 100 compounds (33–37) in several categories: hydrocarbons (5 compounds), aliphatic alcohols (13 compounds), terpenic alcohols (4 compounds), aldehydes (27 compounds), ketones (8 compounds), ethers (2 compounds), furans (3 compounds), thiophenes (6 compounds), and esters (29 compounds).
6.3. Minor Polar Components The olive mesocarp contains a number of phenolic and polyphenolic compounds and their esters, small amounts of which are present in olive oil (35, 43, 44). These include monohydroxy- and dihydroxy-phenylethanol, including tyrosol and other phenols and a series of carboxy–phenols, including caffeic, o-coumaric, p-coumaric, cinnamic, ferulic, gallic, p-hydroxybenzoic, protocatechuic, sinapic, syringic, and vanillic acids. Benzoic and cinnamic acids are produced by hydrolysis of flavonoids. The hydroxyphenyl–ethanols arise from hydrolysis of oleoeuropein. Their esters are responsible for the bitterness and pepperlike sensation occasionally dominant in the taste of olive oils. Olive oil contains a-tocopherol in the range of 12–190 mg/kg. According to one report (43), olive oil tocopherols were found to consist of 88.5% a-tocopherol, 9.9% b- þ g-tocopherol, and 1.6% d-tocopherol. Tocopherol content can be used to detect adulteration of olive oil with seed oils.
7. ANALYSIS OF OLIVE OILS Olive oil is initially examined to determine purity, then to place it in the proper category, and finally to establish its quality.
7.1. Determination of Purity Sterol Composition. Sterol analysis involves preparation of the unsaponifiable fraction, fractionation by thin-layer chromatography (TLC), and gas chromatographic analysis of the TMS derivatives (66). The following limits apply to all types of olive oil (12):
ANALYSIS OF OLIVE OILS
Sterol
321
Sterol Fraction (%)
Cholesterol Max. 0.5 Brassicasterol Max. 0.1 Campesterol Max. 4.0 Stigmasterol Less than 4.0 Max. 0.5 d-7-Stigmastenol The sum of the following sterols must be more than 93.0% of the sterol function: b-Sitosterol d-5-Avenasterol d-5,23-Stigmastadienol Clerosterol Sitostanol d-5,24-Stigmastadienol
Total Sterol Content. The gas liquid chromatographic method for sterol determination using an internal standard (cholestanol) is used to calculate the absolute (total) sterol content of an oil (68, 69). Gravimetric, enzymatic, colorimetric, and liquid chromatographic methods have also been reported (69). Limits (mg/100 g) are as follows (12): virgin olive oil, refined olive oil, and olive oil (mixture of refined and virgin) >100; crude olive–pomace oil >250; and refined olive–pomace oil, olive oil and olive–pomace oil (mixture) >180. Fatty Acid Composition. Olive oil triglycerides are converted into methyl esters, and the methyl esters are analyzed by gas–liquid chromatography (GLC) (70, 71). The limits of genuine olive oil are as follows (% m/m) (12):
Acid
CANa
Minimum
Maximum
Myristic Palmitic Palmitoleic Heptadecanoic Heptadecenoic Stearic Oleic Linoleic Linolenic Arachidic Eicosenoic Behenic Lignoceric
14:0 16:0 16:1 17:0 17:1 18:0 18:1 18:2 18:3 20:0 20:1 22:0 24:0
— 7.50 0.30 — — 0.50 55.00 3.50 — — — — —
0.05 20.00 3.50 0.30 0.30 5.00 83.00 21.00 0.90 0.60 0.40 0.20 0.20
a
CAN ¼ carbon atom number.
Saturated Fatty Acids in Position 2 of the Triglycerides. Hydrolysis with pancreatic lipase is followed by thin-layer chromatographic isolation of the monoglyceride fraction, which is converted to methyl esters. The methyl esters are analyzed
322
OLIVE OIL
by GLC (72, 73). Maximum acceptable level is the sum of palmitic and stearic acid (% m/m) (12): Virgin olive oil Refined olive oil Olive oil (mixture of refined and virgin) Crude olive–pomace oil Refined olive–pomace oil
1.5 1.8 1.8 2.2 2.2
Absolute Difference Between Found and Theoretical Equivalent Carbon Number (ECN) 42 (Trilinolein) Values. The triglyceride composition of the oil is determined by high-performance liquid chromatography (HPLC) (74). (A chromatogram of an olive oil sample (ECN 42, 0.8%) is shown in Figure 10.) The theoretical triglyceride composition is calculated with a Lotus 123 program provided by the IOOC. The maximum difference of theoretical ECN 42 vs. ECN 42 found is calculated. (ECN ¼ CN-2n, where CN is the carbon number and n is the number of double bonds.) The maximum difference between the real and theoretical ECN content
Figure 10. HPLC chromatogram of olive oil triglycerides. Column: LC-18, 200 Â 4 :6 mm i.d.; mobile phase : acetone : acetonitrile (60 : 40, v/v); fl o w rate : 0.75 mL/min; refractive index detector; oven and detector temperature : 40 C. IUPAC Method 2.324 (72) with injection of 10- mL test sample diluted 1 : 20 with acetone. ECN 42, 0.8% of total glycerides.
ANALYSIS OF OLIVE OILS
323
of olive oils and olive–pomace oils should be 0.3 and 0.5, respectively. This procedure avoids errors because of miscalculation of trilinolein alone (75). Trans-Fatty Acid Content. Trans-fatty acids arise during refining of vegetable oils as well as during hydrogenation, or from attempts to eliminate the sterol fraction of seed oils with a fatty acid composition similar to that of olive oil. Methyl esters are analyzed by capillary column GLC (76, 77). The following limits (% m/m) are mandatory (12):
Oil Virgin olive oil Lampant olive oil Refined olive oil Olive oil (mixture of refined and virgin) Crude olive–pomace oil Refined olive–pomace oil Olive–pomace oil and olive oil mixture
18:1 trans
18:2 trans þ 18:3 trans
<0.03 0.10 0.20 0.20 0.20 0.40 0.40
<0.03 0.10 0.30 0.30 0.10 0.35 0.35
7.2. Differentiation Between Olive Oil and Olive –Pomace Oil Wax Content. Olive oil fatty acid esters of straight chain alcohols (wax esters present in solvent extracted olive–pomace oil are isolated by column chromatography on silica gel (LC) and quantitated by GLC to determine if olive–pomace oil is present in olive oil (78). LC separation of the wax esters can be replaced with HPLC to automate the separation step and improve reliability and repeatability (79). Limits for content of C40 þ C42 þ C44 þ C46 wax esters (mg/kg) are as follows (12):
Virgin olive oil Lampant olive oil Refined olive oil Olive oil (mixture of refined and virgin)
250 350 350 350
Dihydroxyterpene Alcohol Content. Olive–pomace oil contains relatively high levels of erythrodiol, uvaol, and wax esters. Erythrodiol and uvaol (total diol) content is determined by the same procedure as that used for sterol analysis (80, 81). Limits for total diol content (as % of total sterols) are as follows:
Virgin olive oil Lampant olive oil Refined olive oil Olive oil (mixture of refined and virgin)
4.5 4.5 4.5 4.5
324
OLIVE OIL
7.3. Differentiation Between Virgin and Refined Olive Oil and Detection of Refined Olive Oil and Seed Oils in Virgin Olive Oil Concentration of Stigmasta-3,5-Diene. When olive oil and seed oils are refined, stigmasta-3,5-diene is produced by dehydration of b-sitosterol, the parent sterol (82). Refined olive oils contain significant amounts of stigmasta-3,5-diene (3– 100 mg/kg) not present in any significant amount in virgin olive oils. Refined seed oils also contain significant amounts of steroidal hydrocarbons, including campesta-3,5-diene and stigmasta-3,5,22-triene in addition to stigmasta-3, 5-diene. The relative amounts of these steroidal hydrocarbons can be used to detect refined seed oils or seed oils desterolized for the purpose of adulterating olive oil. Isolation of the hydrocarbon fraction from the unsaponifiables by column chromatography on silica gel followed by GLC is used to determine the concentration of stigmasta-3,5-diene and accompanying hydrocarbons (83, 84). A chromatogram of the hydrocarbon fraction from an olive oil is shown in Figure 11.
Figure 11. Capillary GLC of the hydrocarbon fraction of olive oil (blend of re fi ned and virgin olive oil). Column: DB-5, 25 m  0 :25 mm i.d., 0.2- mm fi lm thickness; split ratio; 1 : 15; temperature program: 235 C, 6 min; 20 C/min; 285 C fi nal temperature; injector: 300 C; detector; 320 C, 1, cholesta-3,5-diene (internal standard); 2, stigmasta-3,5-diene.
ANALYSIS OF OLIVE OILS
325
A chromatogram of the hydrocarbon fraction from an olive oil admixed with desterolized, refined seed oil is shown in Figure 12. Ratios of stigmasta-3,5-diene to campesta-3,5-diene (R1) and stigmasta-3,5-diene to stigmasta-3,5,22-triene (R2) are determined when the level of stigmasta-3,5-diene exceeds 4 ppm (12).
Oil
Stigmasta-3,5-diene (ppm)
Virgin olive oil Lampant olive oil Refined olive oil Olive oil Crude olive–pomace oil Refined olive–pomace oil Pomace and olive oil mixture
0.1 0.5 50.0 50.0 0.5 120.0 120.0
R1 a
R2b;c
— — !15 !15 !15 !15 !15
— — !15 !15 !15 !15 !15
a
R1 ¼ ratio of stigmasta-3,5-diene to campesta-3,5-diene. R2 ¼ ratio of stigmasta-3,5-diene to campesta-3,5,22-triene. c Provisional limits. b
However, a July 1994 IOOC report (84) noted that the R1 and R2 values of many Italian and Greek olive oils were considerably lower than those proposed by the IOOC (12) and that the composition of steroidal hydrocarbons should be identical to that of the sterols from which they are derived when the R1 and R2 ratios are used to identify extraneous oils in refined olive oil. UV Absorption at 268 nm. K (1%, 1 cm) and related value, d-K , are useful for readily classifying olive oil quality according to the following values (12, 85): Oil
K 270 nm
Extra virgin olive oil Virgin olive oil (fine) Virgin olive oil (semifine) Lampant olive oil Refined olive oil Olive oil Crude olive–pomace oil Refined olive–pomace oil Pomace and olive oil mixture
0.25 0.25 0.30 No limits 1.10 0.90 No limits 2.00 1.70
a
d-K a
0.01 0.01 0.01 No limits 0.16 0.15 No limits 0.20 0.18
d-K ¼ K 268 À ð½K 262 þ K 274=2Þ.
Both K and d-K are altered when oxidation products are present. In this case, the oil is dissolved in hexane and passed through an alumina column before measurement of K and d-K .
326
OLIVE OIL
Figure 12. Capillary GLC of the hydrocarbon fraction of olive oil admixed with a desterolized seed oil (GLC column and operating conditions as described for Figure 11). 1, Cholesta-3, 5-diene (internal standard); 2, campesta-3,5-diene; 3, stigmasta-3,5,22-triene; 4, stigmasta-3, 5-diene.
7.4. Quality Parameters Organoleptic Characteristics. Organoleptic properties of virgin oil can be determined by a ‘‘panel test’’ (13, 86), which gives results that are often controversial. Organoleptic testing is currently undergoing revision. Currently the IOOC is preparing a draft method for the organoleptic assessment of virgin olive oil using a designation of origin (DO) code. It is intended for use by DO authorities to ensure that the oil meets requirements (87). The panel test method is based on examination of virgin oil by a panel of 8 to 12 trained personnel who grade various characteristics and defects that are then converted into a number score. The following scores apply to various grades of virgin olive oil:
Extra virgin olive oil Fine virgin olive oil Semifine virgin olive oil Lampant virgin olive oil
>6.5 >5.5 >3.5 <3.5
ANALYSIS OF OLIVE OILS
327
Free Fatty Acid Content. Free fatty acid content (expressed as % oleic acid) (88) is used to define the various grades of virgin olive oil (12):
Extra virgin olive oil Fine virgin olive oil Semifine virgin olive oil Lampant virgin olive oil
<1.0 <1.5 <3.3 >3.3
Refined olive oil and mixtures have the following limits (12): Refined olive oil Olive oil Refined olive–pomace oil Olive–pomace and olive oil
0.3 1.5 0.3 1.5
Olive oil and mixtures of olive–pomace and olive oil have higher free fatty acid contents because they are generally mixed with virgin olive oils of high acidity. Peroxide Value (PV). PV (expressed in meq per kg oil) (89) allowed for various grades of olive oil is as follows (12): Extra virgin, fine, and semifine virgin olive oil Refined olive oil Olive oil Refined olive–pomace oil Pomace oil and olive oil mixture
20 10 15 10 15
Virgin olive oil contains components that interfere with conventional PV determination. Even freshly expressed olive oil has PV values of about 10, and under certain climatic conditions (dry weather), the PV value can be higher than 10. Tocopherol Content. Tocopherols can be determined by colorimetry or GLC (90), or by HPLC (91, 92). Added tocopherols are not permitted in virgin olive oils and crude olive–pomace oils (12). Added a-tocopherol is allowed in refined olive oil, olive oil, refined olive–pomace oil, and olive–pomace oil to restore natural tocopherol lost during refining with a maximum level of 200 mg/kg of total a-tocopherol in the final product (12). Impurities. Water content (93) of virgin olive oil should not exceed 0.2% (m/m); for refined oil and mixtures (olive oil, olive–pomace and olive oil), the maximum value is 0.1%; for lampant olive oil, 0.3%; for crude olive–pomace oil, 1.5% (12). Allowable hydrocarbon (hexane, petroleum ether) residues (94) are as follows (% m/m): Extra virgin, fine, and semifine virgin olive oil Refined olive oil, olive oil Refined olive–pomace oil, olive–pomace and olive oil
0.10 0.05 0.05
328
OLIVE OIL
The occurrence of mg/kg to mg/kg amounts of tetrachloroethylene in some olive oils (95) led to an EEC regulation limiting the tetrachloroethylene content of olive oil and products containing olive oil to not more than 0.1 mg/kg, as determined by a head space/electron capture GLC method (96). Maximum allowable contents of iron and copper (97) are 3 ppm and 0.1 ppm, respectively. Smoke point (98) is a function of acidity level in the oil. The smoke point for olive oil generally ranges from 150 C to 163 C.
7.5. Combined Gas Chromatography–Mass Spectrometry (GC/MS) GC/MS is a powerful tool for identification and confirmation of the various components of olive oil. With GC/MS in the selective ion mode, unresolved GC peaks can be identified and accurately quantitated. For example, an apparent b-sitosterol peak in the sterol fraction was resolved into clerosterol (m/z 218) and Á-5-avenasterol (m/z 314), and both sterols measured regardless of inadequate GC resolution. Italian and Spanish olive oil from the 1991–1992 crop year contained a very high level of 9,19-cyclolanosterol (>400 mg/kg), which was not found with the standard method for sterol analysis. Two isomers of this sterol were identified by GC/MS of the unsaponifiable fraction, and their levels were found to be inversely proportional to the levels of b-sitosterol in the oils. GC/MS of the unsaponifiable fraction with high-resolution GC capillary columns provides a relatively rapid means of checking product purity and the identity of individual components. Thus, triterpene diols were identifiable at m/z 203, a-tocopherol at m/z 165, squalene at m/z 69, cholesterol at m/z 386, and brassicasterol, characteristic of canola oil and other Brassica oils, at m/z 398.
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330
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