LIPIDS

OF OLIVES

ENZO FEDELI Departnwrit qf’ Food Science,

Lliiiuersity of Milan,

Italy

CONTENTS 57 . 58 58 58 59 59 60 60 60 60 62 62 63 63 64 64 64 66 67 68 68 69 71 72 72

1. INTRODUCTION Il.

LIPID

CONTENT

OF OLMS

A. Fatty acid composition of olive oil B. Glyceride composition of olive oil C. Nonglyceridic components of olive oil I. Hydrocarbons 2. Fatty acid esters 3. Tocopherols 4. Aliphatic alcohols 5. Monohydroxy triterpenes 6. 4a-Methylsterols 7. Sterols 8. Dihydroxy triterpenes 9. Hydroxy triterpenic acids IO. Chlorophylls 1 I. Phospholipids D. Flavoring components of olive oil 111.WATER-SOLUBLE OLIVE COMPONENTS A. Phenols and carboxy-phenols B. Polyphenols C. Sugars. polyhydroxy compounds and hydroxy acids IV. EXTKA~TION AND PUKIFICATION OF OLIVE OIL V. OLIVE

OIL

VI. NUTRITIONAL

ANALYSIS

PKOPEKTIES OF OLIVE

OIL

REFERENCES

1. INTRODUCTION

The olive is the fruit produced by several sub-varieties, differing from country to country, of the genus Olea ruropaea L., of the Oleaceae family. Olive tree cultivation, a traditional farming activity, is still very important in the Mediterranean basin, which accounts for 95% of the total cultivated area. The geographical distribution of 0. europaea production is shown in Table 1. The social and economical importance of Olea ruropaea in ancient times pervaded the classical literature born around the primary civilization which blossomed in that region, but olive oil is still one of the most important fats in the diet of a large amount of Mediterranean populations. Currently (1974) olive growing extends over a total area of approximately 13,300 acres (5382 ha), with about 500 million trees. Olive production ranges from 260 to 90 pounds per acre (291-101 kg/ha). The production of olives and olive oil has nearly doubled in the course of the last TABLE

1. Geographic Distribution

of Olive Production

Europe 84”;

Asia 5.50/,

Africa 8.5%

Spain 42r0 Italy 249, Greece 12” Portugal 6%; Cyprus France USSR Yugoslavia

Turkey 3”,; Tunisia SS(, Syria Algeria Lebanon Morocco Israel Libya Jordan Egypt Iraq South Africa Iran Japan 57

America 27; Argentina 1.9% USA Mexico Uruguay Chile Peru

58

Enzo

Fedeli

50 years. and average yearly production has soared from 700,000 tons (711.000 t) of oil in 1918-1922 to 1,350,000 tons (1,372,OOOt) in 1970-1974, the increase being mostly due to the expansion of the olive-growing area. Most of the oil produced by pressing the olives is consumed without any refining whatsoever as long as the acidity and organoleptic characters are satisfactory-otherwise the oil is refined and blended with virgin (unrefined) oil. The oil extracted with solvent is also refined and blended with virgin oil before consumption. A large amount of olives is preserved and consumed as such (table olives).

II.

LIPID

CONTENT

OF

OLIVES

The fruit of the Olea etrropaea tree is an ovoidal drupe in which it is possible to distinguish the epicarp (1.5-3.5x of the fruit weight), mesocarp (or pulp, 65-839/,) and kernel (or pit, 13-30x of fruit weight). From 96 to 984,0 of the oil is located in the pericarp (mesocarp + epicarp), and 2-4x is contained in the kernel or pit. The total amount of oil obtained (by solvent extraction) varies from 15 to 35x, depending on varieties. The fruit contains anywhere between 25 and 607: water, known as “vegetation water”. The average chemical composition of the olive is as follows: water SO%, oil 22x, proteins 1.6x, sugar 19.1%, cellulose 5.8% and ash 1.5%. The average yield from 10 t of fruit is 1.99 t of pericarp oil and 0.04 t of kernel oil. Unless otherwise stated, only pericarp oil will be considered in this paper. A. Fatty Acid Compositiort of Olive Oil The fatty acids normally present in olive oil are indicated in Table 2 (gas-liquid chromatography determinations), with the minimum and maximum amounts of each. As for other vegetable oils, percentages vary depending on sub-varieties and climate conditions

1~33~79~90~104~125~130

Besides the acids shown in Table 2, according to Amelotti,’ elaidic acid (t 9-18: 1) would also be present in trace amounts, but Tiscornia and Bertini,123 after examining sixty samples of different origins, found no trace of this substance in the virgin oil. B. Glyceride Conzpositioil of Olive Oil The fatty acids are distributed in olive oil triglycerides according to the 1,3 random, 2 random rule observed for the majority of vegetable oils. The following glycerides predominate over the others (13): PO0 (18.4%) SO0 (5.1%), POL; (5.9%) 000 (43.5X), OOL (6.8%). A great deal of work has been done to determine the amount of palmitic acid in the 2-position of the glycerides, as the basis for a method used to determine authenticity of both virgin and refined oil. As they examined virgin oils of various origins, Bertini TABLE

Italy: Acid 14:o l6:O l6:l l7:O 17:1 l8:O l8:l l8:2 l8:3 20:o 2O:l 22:o

Liguria %

traces 8.7-l I.1 0.7-0.4 traces traces I .8-3.4 79-82.2 6.6-4.2 0.3-0.8 0.2-0.5 0. I-O.2 traces

2. Fatty

Italy:

Sicily %

traces 10.4-17.5 0.24.4 0.1-1.2 traces 2.34.4 66-76.7 5.0-12.6 0.3-1.1 0.2-1.3 -

Acid

Composition

Tunisia %

of Olive

Oils

Variety (Italy.

Coratina South) 0,,D

Variety Oliarola S. (Italy, South) :,,

traces 18.3 2.4

traces 10.3 0.7

traces 16.3 2.4

2.5 56.4 18.9 0.8 0.5

2.3 78.1 7.2 0.6 0.4 0.3

1.9 66.5 II.5 0.7 0.4 0.3

Lipids of olives

59

and Tiscornia came to the conclusion that the 2-monoglycerides prepared by pancreatic lipase hydrolysis never contain more than 1.6% palmitic acid.76s86*118*122,123 C. Nonglyceridic

Components of Olive Oil

Like other vegetable oils, olive oil contains, together with glycerides, several chemical compounds, which are present in small amounts and are referred to as “minor components”. Because of their low concentration in the oil, minor components were originally investigated in the unsaponifiable fraction of the oil, obtained .by eliminating the fatty acids through saponification. Such a destructive method, however, does not allow study of minor components which are destroyed by saponification. In an attempt to recover intact compounds, a crystallization-procedure has been successfully used.36*68 By fractional crystallization in a polar solvent (acetone or ethyl acetate) at low temperatures ( - 15 to - 6O”C), the glycerides are eliminated, leaving a concentrate of the minor components in the mother liquor, from which distinct classes of compounds are separated by thin-layer chromatography (TLC) and analyzed by the classical methods of organic chemistry. Quantitative analysis of certain classes of minor components (sterols, triterpenic alcohols) is amongst the chief methods for determining an oil’s authenticity, or for distinguishing between expressed and solvent-extracted oils. The latter subject will be further dealt with later. Minor oil components are described here in order of increasing polarities. 1. Hydrocarbons Using silicic-acid column chromatography, Capella and FedeliL9 were able to isolate the hydrocarbon fraction from the unsaponifiable portion of the oil and the fraction was analyzed by GLC using standard compounds for comparison. Both even and odd n-paraffins ranging from Cl 1 to C3e, were thus found in virgin olive oil. Branched hydrocarbons, probably with iso- and ante-iso-structures, are also present, though in a very low concentration. While investigating the volatile constituents of olive oil (see Flavor section), Fedeli identified by GLC mass spectrometry (MS) several benzenoid hydrocarbons whose structures were determined by synthesis and by comparison of spectroscopic properties. In Chart 1 are listed the components so identified. Naphthalene and naphthalene derivatives are also present. Other polycyclic aromatic hydrocarbons were identified by Ciusa and Nebbia using spectrofluorimetry24 in the hydrocarbon fraction after separation on an alumina column; phenanthrene, pyrene, fluoranthrene,

LH I

n:4,11

II

n:&

CH,-CH-LCHJmCH,

+

n:6,10

+CHs

CH3

dH, P

n:3,12

CH$CHJ,CH,

CHJCH,)“CH,

+CHs

E

EI

n:4,11

3 PI

n:5,lO

CHART 1. Aromatic hydrocarbons found in olive oil.

Enzo Fedeli

60

l,Zbenzanthracene, crisene and perilene were also identified in virgin oil, their pg/l. concentrations being 0.20, 0.027, 0.14, 0.07, 0.05 and 0.12, respectively. The polyunsaturated iriterpenic hydrocarbon squalene, precursor of the sterol biosynthesis, is the main component of the hydrocarbon fraction (1.5 mg/kg).35 b-carotene is also present in an indefinite amount (33&3690 y/kg) in the oi1.57*58*131 2. Fatty Acid Esters

Aliphatic alcohols as well as sterols and triterpenic alcohols are found in virgin olive oil in both free and esterified forms. Esters of n-aliphatic alcohols (CZ7-C3& of sterols (p-sitosterol, campesterol, stigmasterol) and of triterpenic alcohols (cycloartenol, 24 methylene-cycloartanol) with the acids shown in Table 3 were detected by Fedeli and Jacini6* when analyzing the nonglyceride fraction. Methanol and ethanol esters have also been found in the volatile fraction, and will be discussed later. Mono- and diglycerides in small amounts were also demonstrated.68 TABLE 3. Fatty Acid Compositions of Non-Glyceridic

14:o 16:O 16:l 18:0 18:l IS:2 IS:3 20:o 2O:l

Esters

A

Sample B

C

10.6 3.0 0.5 49.5 22.9 0.7 1.4 8.5

2.2 5.9 1.8 0.7 41.4 26.8 0.5 9.0 9.5

0.2 5.5 0.6 0.4 89.4 2.5 0.3 0.5 0.2

A: Esters of long chain fatty alcohols, sterols and triterpenes. B: Mostly esters of triterpenic alcohols. C: Ethyl and methyl esters.

3. Tocopherols

The composition of the tocopherol mixture present in olive oil was determined by Losi and Piretti” using GLC methods. It was found to consist of 88.5% a-tocopherol, 9.9% j?- + y-tocopherol and 1.6% &tocopherol. The total amount of tocopherols in olive oil is controversial. Kofler’ *,I3 reported it as 300 mg/kg, whereas, in fifty-two samples of various origins (from Italy and Tunisia) Vitagliano and Turri131 found a maximum of only 187 mg/kg, and even lower values were reported by other workers.5*9s62*’ l9 As suggested by Gracian and Arevalo,61 such discrepancies may be explained by the progressive destruction tocopherols undergo. Values as low as 5-30 mg/kg were determined by Fedeli in high-acidity commercial oils. 4. Aliphatic Alcohols

As demonstrated by Fedeli, virgin oil contains no appreciable amount of n-aliphatic saturated alcohols, although it contains their esters (see above). Actually, the free alcohols previously referred to can be detected by GLC in the unsaponifiable fractions. Phytol was identified, too,68 probably originating from the decomposition of chlorophyll contained in the oil. 5. Morwhydroxy

Triterpenes

Several triterpenic alcohols are present in olive oil, some of them as esterified compounds, some of them free.(j* By isolating the triterpenic alcohol’s fraction from the unsaponifiable matter by GLC and comparing the retention times of the trimethysylyl

Lipids

FIG.

1. GLC

analysis

of olives

of the triterpenic

61

fraction

of olive

oil.

derivatives (TMS), cycloartenol (CA), a- and fi-amyrin5’ (Chart 2, I, III, IV) could be identified. The structure of 24-methylene-cycloartenol (24 MCA) (Chart 2, II) was evidenced by isolation and chemical degradation. 36s3 The structures of CA and MCA were demonstrated by MS;37 the GLC camp osition of the triterpenic fraction of olive oil is described in Fig. 1. In addition to the components listed above, Fedeli46,52 was able to show the presence, in the unsaponifiable fraction, of trace amounts of two alcohols-lanosterol (Chart 2, V) and obtusifoliol (4cr, lQ-dimethyl, 24methylene-A8-cholesten-3/I-01) (Chart 2, VI) after they were isolated by TLC and identified by GLC-MS. Obtusifoliol has likewise been found by Itoh in olive oil.

CHART

2. Monohydroxy

triterpenes

of olive

oil.

62

Enzo Fedeli

HoJq5e?.. CHART 3. 4u-Metilsterols of olive oil.

6. 4a-Methylsterols

As in other vegetable oils, a small fraction whose polarity in TLC is very similar to that of sterols could be isolated in olive oil as well, its composition being identified by GLC-MS.46*52 The following are normal constituents of this fraction: 4a-methyl, 24-methylene-A’-cholestene-3/I-01, 4a-methyl, 24-methyl-A’-cholestene-3P-01, 4a-methyl, 24-ethylidene-A’-cholestene-3/I-ol, and 4a-methyl, 24-ethyl-A’-cholestene-3/I-01, (Chart 3, I. II. III, IV). Using GLC-MS, Fedeli and Cortesi 45 have demonstrated in the 4a-methylsterol fraction an unknown component having two double bonds, one in the side chain and one in the ring D. They were unable, however, to isolate and fully characterize it. 7. Sterols The adoption of GLC for sterol analysis gives excellent results insofar as identification and quantitative determination of the components of the sterol mixture present in olive oil are concerned. Using chemical methods, in 1961 Capella and de Zotti’* indicated /I-sitosterol (24-ethyl-A’-cholestene-3fi-01) (Chart 4, I) as the main component of the sterol fraction (0.1-0.2’? of the oil) obtained by column chromatography of the unsaponifiable matter. The identification of the other sterols, stigmasterol (24-ethyl-A5bz2-cholestene-3/I-01, Chart 4, II) and campesterol (24-methyl-AS-cholestene-3/I-01, Chart 4, III),

HO CtimT 4. Sterols of olive oil.

Lipids

63

01‘ olives

was made possible only by GLC of the TMS derivatives after separation by column chromatography or TLC2’*” With reference to the unsaponifiable fraction and to GLC analysis on low polarity stationary phases (SE-30 or JXR), the values for the three sterols found in olive oil are shown in Table 4, along with regional and seasonal mean variations. The sterol fraction, however, is far more complex than low polarity phase GLC analysis indicates. Working on OV-17 columns and on refined oil, Fedeli and Mariani52 found two other components, besides the sterols already mentioned, which eluted after p-sitosterol. The second of these components was found to have the same retention time as A’-stigmasterol, with which, though, it should not be identified, as Itoh did.65 In point of fact, this particular sterol is probably an artifact produced during the refining process. The other sterol is also present in virgin oil. The occurrence of many compounds, from squalene to the tetracyclic terpenic hydroxy compounds, to 4a-methylsterols and to sterols bound by structural relationships might indeed shed some light on the biosynthetic sequences occurring within the vegetable cell. TABLE

Origin

4. Composition

of Olive

/W.itosterol, %

Stigmasterol, %

Virgin oil* Italy Spain Tunisia Morocco Husk oilt Italy Spain Tunisia * kO.3 t +0.6

seasonal seasonal

variation variation

Oil

Sterols Campesterol, %

95.6 96.1 95.2 95.8

1.3 1.1 I .4 1.6

3.1 2.8 3.4 2.6

94.0 94.8 94.1

2.4 1.9 1.7

3.6 3.3 3.6

and and

kO.5 regional + I.2 regional

variation. variation.

8. Dihydros~~ Triterpenes

The presence of a dihydroxy pentacyclic triterpenic compound, erythrodiol (38, 17fl dihydroxy A’ 2-oleanene, Chart 5, I), was reported in husk oil by Kotakis,74 and was subsequently characterized by mass spectrometry of its TMS derivative.89 As noted by Fedeli and Mariani, 51 the GLC peak of erythrodiol is invariably accompanied by a smaller one, denoting the presence of a component whose properties are very similar to those of the major constituent. GLC and GLC-MS comparison with the alcohol from LiALH, reduction of ursolic acid evidenced the two components to be one and the same thing. and the unknown peak was consequently identified as uvaol (3fl. 17/3 dihydroxy-A12-ursene, Chart 5, II). Quantitation of erythrodiol and uvaol by GLC provides the basis for differentiation between expressed and solvent-extracted oi1.89 9. Hydroxy

Triterpenic Acids

The presence of the pentacyclic hydroxy triterpenic acid, oleanolic acid (3b-hydroxy, Chart 5, III), in olive oil has long been 17-carboxy, A’ 2-oleanene known. 1oo.102*1l’s’ 13.’ 28 A bihydroxy acid (3fl-2a-dihydroxy, 17-carboxy, A’ 2-oleanene, Chart 5, IV) called maslinic acid was recently extracted by Caglioti from olive husk.1o-13 The same substance had been independently discovered by Vioque and Morris.12* The two acids are present in olive oil. Ursolic (3/3-hydroxy, 17-carboxy, A12-ursene, Chart 5, V) and betulinic (3/%hydroxy, 17-carboxy, A12-lupene, Chart 5, VI) were supposed to be present in virgin olive oil according to Thiers,“! who has not, however, been able to this moment to supply any evidence to substantiate his

Enzo

64

Fedeli

Ho&~&ooH CHART

5. Dihydroxy

triterpenes

and

triterpenic

pentacyclic

acids

of olive

oil

contention. By isolating the acidic fraction of virgin oil and analyzing the mixture after TLC separation by GLC and GLC-MS, the following acids could be identified, besides maslinic and oleanolic acids: ursolic, a deoxyursolic and 2cr-hydroxy ursolic (2cr, 3fi-hydroxy, 17-carboxy, A12-ursene) acids.67 10. Chloroplzylls

Chlorophylls a and h, both of them easily degraded to pheophytins. are also contained in olive oil, their presence being due to both biological and technological factors.129+131 In freshly-produced virgin oils, the sum total of chlorophylls a and h ranges from 1 to 10 ppm (pheophytins from 0.2 to 24 ppm), and tends to decrease with time. The presence of chlorophylls in virgin oil, unless the latter is protected from the action of light, considerably affects its storage 1ife.40s41 11. Phosplzolipids

Like many other fruit fats, olive oil contains a small amount of phospholipids, the proportion ranging from 40 to 135 ppm.‘29a’31 As demonstrated by Vioque and Maza,12’ a larger phosphatide amount is contained in the kernel, its constituents being phosphatidylcholine and phosphatidylethanolamine. In the phospholipids mentioned, oleic acid is the most important fatty acid. The olive pulp also contains sterolglucosides, mono- and digalactosyldiglycerides, cerebrosides and su1pho1ipides.‘26 none of which is extracted when the oil is expressed. D. Flavoring Comporwts

of Olive Oil

Most olive oil is consumed as such, whether in the form of virgin oil or of a blend of refined and virgin oils, because the organoleptic properties which are imparted to it by a large number of pleasant flavoring substances make it particularly palatable.

Lipids TABLE Hydrocarbons Naphtalene [I] Ethylnapthtalene [I] Dimethynaphtalene [I] Acenaphtenr [I] n-Octane [I] [2] Aromatic hydrocarbons (see Chart I)

[I]

Aliphatic hydroxy compounds Methanol [3] Ethanol [2] [3] Methylpropane-l-01 [7] [3] I-Pentenc-01 [2] 3-Methylbutane-I-ol 121 2-Methylbutanc-l-01 [Z] [3] cis-3-Hexene-l-01 [2] Hcxane-l-01 [2] [3] rrurrs-2-Hexenel-01 [2] [3] Heptanel-01 [2] Octane-l-01 [I] [2] Nonane-l-01 [I] [Z] 2-Phenylethane-l-01 [I] [2] Terpenic hydroxy I .X-Cineole [2] Linalol [Z] I-Terpineol [I] Lavandulol [I]

compounds

[Z]

Aldehydes Ethane-l-al [Z] [3] h-Propane-al [?I 3-Methylbutane-l-al [2] 2-Methylbutane-l-al [2] n-Butane-1 -al [I] n-PentaneI -al [I] [2] nuns-3-Pentene-I-al [2] Pentene- I -al (prob. cis-2) [?I rt-Hexane-I -al [2] [I] cis-l-HexenI -al [2] n-uns-2-Hesen-l -al [2] n-HeptaneI -al [I] [2] 2.CHexadiene-l-al [2] HepteneI-al(prob. cis-2) [2] rrcrrts-l-Heptene-l-al [2] Benzaldehyde [I] [Z] h-Octane-l-al [I] [Z] 2.4Heptadiene-l-al [2] (two isomers) Iruns-2-Octene-l-al [I] [Z] rr-Nonane-l-al [I] [2] trutls-2-Nonene-l-al [I] [2] Z.CnonadieneI -al [Z] truns-2-Decene-I-al [I] [2] 2.4-Decadien-l-al [Z] (two isomers) rrurrs-2-UndecenI -al [I] [Z] [I] [2] [3] C41

5. Flavor

65

of olivcs

Components

of Olive

Oil

Ketones Acetone [3] 3-Metbylbutanc-2-one Pcntan-3-one [2] [3] Hexan-2-one [2] 2-Methyl-2-hepten-6-one Octane-2-one [7] Nonane-‘-one [2] Acetophenone [2] Ethers Methoxybenzene I .2-Dimethoxybcnzene

[I]

[Z]

[2]

[2] [2]

Furane derivatives 2-Propylfurane (2 isomers) 2-jr-Pcntyl-Smethyl-furane 2-n-Propyl-dihydrofurane

[I] [I] [I]

Thiophene derivatives 2-lsopropenylthiophenc [I] 2-Ethyl-5-hexylthiophenc [I] 2,SDiethylthiophcne [I] 2-Ethyl-Shexyldihydrothiophene 2-Ethyl-S-methyldihydrothiophene 2-Octyl-5-methyl-thiophene [I] Esters Ethylacetate [2] [3] Ethylpropionate [Z] Methylbutirdte [2] Ethyl 2-Mcthylpropionate [2] 2-Methyl-I-propylacetate [2] Methyl 3-Methylbutyrate [2] Ethyl hutyrate [2] Propyl propionatc [2] Methyl pentanoate [2] Ethyl 2-methylbutyrate [Z] Ethyl 3-methylbutyrate [2] I -Propyl-2-methylpropionate [2] 3-Methyl I-butylacetate [2] 2-Methyl-I-propyl l-methylpropionate Methyl hexanoate [I] [I!] cis-3-Hesenyl acetate [2] Methyl heptanoate [I] [2] Methyl octanoate [I] [3] Ethyl benzoate [Z] Ethyl octanoate [I] [2] Methyl salycilate [I] I-Octyl acetate [2] Ethyl phenylacetate [2] Ethyl nonanoate [I] Ethyl decanoate [I] Ethyl heptanoate [I] Ethyl palmitate [I] [4] Methyl oleate [I] [4] Methyl linoleate [I] [3]

[I] [I]

[2]

Fedeli er ~I/.“‘~~-‘~~‘~“’ Flath er (11.‘~ Lerkrr er (I/.” awl,r,w.q4 N

Until recently, relatively few works had been devoted to investigating the chemical composition of the volatile components of olive oil. Fedeli,39~43~47~49Flath,56 Nawar93994 and Lercher,” after isolating these flavoring components and analyzing them by GLC-MS, demonstrated the presence of the following classes of constituents, all of them individually listed in Table 5: aliphatic and aromatic hydrocarbons, aliphatic and

66

Enzo Fedeli

terpenic alcohols, aldehydes, ketones, ethers, esters, furan and thiopene derivatives. Particularly complex are Jhe aromatic hydrocarbons (see Chart 1) and the aldehyde fractions, most of which probably originate from oxidative degradation of the oil. From the papers mentioned, it may be noted that a unique experimental technique was used by each worker to recover the volatile products from the oil. Fedeli,43*49 for example, employed two different methods, one of which was based on the co-distillation of the flavoring substances in ethyleneglycol vapor under reduced pressure, the volatile components then being extracted in small amounts of pentane, after which GLC-MS analyses were performed directly on the concentrated pentane extract and on fractions obtained by preparative GLC or TLC. The second method employed continuous falling film distillation under high vacuum, the volatile material being recovered in traps cooled by a mixture of acetone and dry ice, after which the flavor substances were analyzed both with and without TLC or GLC fractioning. Steam was used by Flathj6 to strip the aromatic constituents of a California virgln oil in a stream of nitrogen, after which the substances were extracted with hexane. The Loev and Goodman” dry column method was then employed to fractionate the volatile constituents before analyzing them by GLC-MS. Lerker7’ and Nawar93a94 submitted the oil to high vacuum bulk distillation and used traps cooled by liquid nitrogen to recover the flavoring components. Each of the methods in question had its own advantages and shortcomings, and the results were probably also influenced by the origin of the oil each worker used. Investigating such relations as exist between an oil’s aroma composition and its organoleptic properties is one of the most interesting aspects of the research on olive oil volatiles. An approach in this direction was made by Gutierrez96*97 who, however, confined himself to a GLC analysis of the head space above the oil and used autoxidized samples (a method that may also be used for quality determination), with no attempt to identify any of the volatile components.

III. WATER-SOLUBLE

OLIVE

COMPONENTS

Olives contain a large amount of water, known as “vegetation water”, which is squeezed out together with the oil and is usually removed by centrifugal separation. The average composition of vegetation water is shown in Table 6, but phenols, polyphenols and bitter substances are also normally found as constituents of vegetation water. Simple, as well as complex, phenol structures are found in olives. As the oil is processed, they are distributed between the product’s organic and aqueous phases. A great deal of work is still being done in this fascinating field, most of which being devoted to investigating the phenolic fraction that is retained in the oil and, as demonstrated by Cantarelli and Montedoro’ 4*15 may favorably affect the stability of virgin oil to oxidation. The totality of complex phenols with different structures and the majority of simple ones present in olives, however, go to enrich the aqueous fraction (vegetation water). TABLE

6. Mean Composition tation Waters4

Component Water Sugars Nitrogen derivatives Organic acids Polyhydroxy compounds Pectin Oil Salts

of Vege0, 10 83.2 2.8 I s2.4 o.I-1.5 1.0-1.5 1.0-1.5 0.03-1.0 1.8

Lipids of olives

67

CH,CH,OH

cH30@oH

P

HocH&;;$ccH3)3 6H

m

91 CHART

6H

6. Phenolic components of vegetation water.

A. Phenols and Cat-boxy-phenols The presence of phenolic components in olives was postulated by Cruess,32 as well as by Joslyn and Smith, 6g but no experimental proof was produced to substantiate such a contention; P(4-hydroxyphenyl)ethanol (Chart 6, I), /l(3,4-dihydroxy-phenyl)ethanol (Chart 6, II) and o-hydroxyphenol (pyrocathekin) were subsequently isolated by Ragazzi and Veronese lo6 from the vegetation water of ripe olives. The presence of the first two constituents was confirmed by Fedeli,3**42 who isolated the phenol fraction by chemical and TLC methods, then used GLC and GLC-MS to analyze the mixtures obtained by resorting to the formation of their trimethysylil ethers and ethyl ethers. In addition to the two constituents above, P(3-hydroxy phenyl) ethanol, P(3-hydroxy, 4-methoxy phenyl) ethanol; /?(3,4-dihydroxy. 5-methoxy phenyl) ethanol; 3,5-dihydroxymethylen-l-phenol and 2,6-di-tertbutyl-4-hydroxyphenol (Chart 6, III, IV, V, VI, VII, resp.) were also identified by Fedeli in the mixture. Phenols having a carboxylic function are present in olives and were found in their vegetation water. Caffeic (3,4-dihydroxycynnamic) and protocatechic (3,4-dihydroxybenzoic) acids (Chart 7, I and II, resp.) were identified by Ragazzi,lo6 gallic (3,4,5trihydroxybenzoic) CH=CH

-COOH

HO&H

COOH

HOA:H

CH*CH-COOH

COOH

HO&H

COOH

COOH

OH

OH

OCH,

CH,-CH,-COOH

CH,-COOH

OH

OH

CHART 7. Carboxylic phenols of vegetation water.

Enzo

68

Fedeli

acid by Canzonieri,’ 6;1’ and ferulic (3-methoxy, 4-hydroxy cynnamic) acid by Cantarelli (Chart 7, III and IV, resp.).14 The presence of these acids was confirmed by Fedeli4’ who isolated from vegetation water a mixture which was subsequently converted into the methyl esters and then into the ethoxy derivatives at the hydroxylic functions and analyzed using GLC-MS by comparison with synthetically-prepared reference compounds. Other hydroxy acids were identified by the same worker. i.e. 3-methoxybenzoic; 4-hydroxybenzoic; 4-hydroxy cynnamic; 4-hydroxy-3-methoxybenzoic (vanillic); 3.4.5-trimethoxy benzoic; 3,4-dihydroxydihydrocynnamic (dihydrocaffeic) and 3,4,5-trihydroxy phenylacetic acids (Chart 7, V, VI, VII, VIII, IX, X, XI, resp.).

As they ripen, the fruits of Olea europcrea develop a number of pigments, which isolated a red-brown pigment, account for their increasingly dark color. Musajo”v9’ an anthocyanin which he called “oleocyanin”, probably a ramnoglucoside, having the following structure: 0-

6H

6H

Two brown pigments, cyanidin-3-monoglucoside and cyanidin-3-diglucoside, were recently extracted by Cantarelli,‘4 who also postulated the presence of leucoanthocyanidins in the unripe fruits. In addition to this, certain flavonoids were tentatively identified by the same worker14 in the ripe fruits. Immature olives contain a bitter substance, called oleoeuropein (approximately 2”/6 of the fruit weight), isolated by Borquelot and Vintilesco’ in 1908. A structure was attributed to oleoeuropein by Panizzi,98*99 who described it as follows: ?COCH, -

CH,-CH,-COO-CH,-C,H

F

?Glucose CH 6

C- -CH dCOCH,

0 OH + OH

whereas Shasha and Lybowitz

proposed : l ‘&I ’ 6

but there is no proof of these two constituents being one and the same substances. Pharmacological properties (as a hypotensive agent) have been attributed”**’ to oleoeuropein. Several phenols are also present in the leaves and exudates of the Olea europaea tree. 16.17.71.105.124 C. Sugars, Polyhydrosy

Corupour~ds and Hydrosy

Acids

Several sugars are present in the vegetation water of olives, their amounts varying from 2 to 4% of the pulp weight (8% of the vegetation water). Glucose, fructose, mannose and galactose were identified by Marcelet,8’ whereas xylose and arabinose were evi-

Lipids

of olives

69

dented by Nunez and Spiteri”’ using paper chromatography. GLC of the TMS derivatives of the sugars present in the mother liquor remaining after isolation of phenols and phenolic acids led Fedeli38*42 to identify the following constituents: arabinose, CXand /&glucose, fructose, ramnose, xylose and galactose. Other sugars, too, are present, though only as minor components. A relatively high proportion (733%) of mannitol is present in the unripe fruit, and decreases as the fruit ripens.3’*“0*’ ” Acetic, oxalic, malonic, fumaric, glyceric, lactic, tartaric, malic, tricarballilic and citric acids were found in vegetation waters.j9*“’ Olives washing 1 Washedolives grinding I Olive paste extraction 1 Virginoil -due + Vegetotion

grinding water

1 drying Dry residue Solvent 1extraction

Centrifuging 1 VegDgin water to waste

1

Quality evaluation

-

Low grodeoil refining bleach. deodor. I Refined oil

Refined

husk oil

Blend withvirginoil

oil

FIG. 2. Olive

EXTRACTION

Husk oil refining bleach. I deod.

Htghgradeoil

Blend wdh virgin

IV.

Spe7 (to waste)

oil

AND

oil extraction

PURIFICATION

OF

OLIVE

OIL

As long as its organoleptic properties are satisfactory and its acidity is low, olive oil is consumed unrefined. An oil’s economical value is therefore closely related to its quality, and technological problems involved in olive-oil processing are so typical they are associated with no other oil-yielding produce. The method traditionally used for olive oil extraction is illustrated in Fig. 2. Three types of products are obtained-virgin, refined and husk oils-each being distinct in both quality and economical value. Yield rates of both virgin and refined oils are largely determined by a number of factors-tree age. seasonal conditions, pest infestation. ripeness, harvesting practice (i.e. if the olives are knocked from the tree by beating, or hand-picked), etc. For information on these factors closely related to farming reference should be made to specialized publications. s3*‘O’ Because the ratio of economical values of virgin to refined oil is 1.5 to 1, olive-growing profitability is determined to a considerable extent by how much of either product can be obtained by pressing. The fact should not be overlooked that refining increases cost, lowers quality by raising acidity from 4 to IS’%, and causes losses from 1.1 to 2.5x, depending on oil quality and tree variety. Referring now to Fig. 2. the first step in oil extraction is that of washing the olives to remove dirt, stones and other impurities that may adhere to the fruits. In modern washing machines, a fast-moving stream of water is used, the impurity-laden water being subsequently separated, filtered and recycled.” The olives are then crushed, usually in a two-step process. They are first coarsely ground, and the pits crushed in a hammer mill, after which the pomace is finely homogenized preparatory to pressing. Both operations can be performed by a single machine. but the two steps are still separated.” Crushing and grinding are meant to break down the pits, pulp fibers and

Enzo Fedeli

70

seeds, while leaving the individual cells intact. The traditional batch method, employing hydraulic plate presses, is still largely in use21*22 because the yields are higher than those obtained with the newer extraction methods, also due to the introduction of highly sophisticated equipment. The pomace is normally fed directly from the crusher to the hydraulic press plates, each of which is covered with a filtering diaphragm, then the plate pile is inserted into the press for extraction. Pressures ranging from 3500 to 5700 psi (24-39 MPa) are attainable with modern hydraulic oil presses, and pressure is applied gradually because acidity tends to rise as higher pressures are applied. Continuous pressing systems are also utilized, though normally for second pressing only. Vegetation water and mucillaginous matters are removed from the oil-water mixture coming from the press using a centrifugal clarifier, the product then being forced through a filter press, brilliantly clear oil thus being obtained. Other oil-extracting methods have been considered, some of them having already been adopted in commercial practice. One such method is based upon the different surface strength of oil and water as they come in contact with a stainless steel blade, causing the oil to stick to the blade. The equipment uses 6000 such blades, which are dipped into the pomace to a depth of * in. (9.525 mm), the oil being then gently removed from the blades.2’*22 Good quality oils can be obtained by this procedure. A continuous horizontal centrifugal separator rotating at 1200-1500 rev/min is also used to separate the oil from the pomace in the presence of water. The new methods do offer certain advantages in terms of higher oil yields and lower manpower requirements, but they are not likely to completely replace the traditional press method.2’*22 After pressing, residual solids (husk) still contain anywhere from 5 to 9% oil and 15 to 30% water. Before being submitted to solvent extraction, they are ground in a hammer mill or disc mill to break down the cake coming from the press and make the material uniformly textured. After the residual humidity is reduced in a rotary kiln, the oil is extracted using hexane in a continuous or batch extractor.22 As husks are stored, however, the action of pulp enzymes causes oil acidity to rise (up to 20 or NoOH

Crude oil

Neutral oil P M D T

FIG.

= = = =

Flowmeter Mixing Settling Distilling

Fatty acids FT = Froctioning ET = Hexane tank PT = Polar solvent

3. Flowsheet of a neutralization

tank

plant, carried out in solvents.

Lipids

of olives

71

even 25”,,). and some oxidation also occurs, so that the oil must be refined. After this last extraction. the residue has no commercial value, not even as animal food.22 Good quality oil, i.e. one with good organoleptic characters and less than 4% acidity, is bottled or canned for direct human consumption. The remainder is thoroughly refined and then blended with variable amounts of virgin oil. Neutralization of low-acidity oil involves no particular problem, and is normally performed by continuous plants where soaps are separated and the oil itself is centrifugally clarified. High-acidity oils (mostly husk oils) require appropriate neutralization employing either chemical or physical methods. Chemical processing is still based on the use of sodium hydroxide as the neutralizing agent, dissolved, however, in a polar solvent (acetone or isopropylic alcohol), the oil being diluted in a nonpolar solvent (hexane).4 The process is described in Fig. 3. In physical refining,3 the difference in volatility between oil and free fatty acids is used to neutralize the oil. The oil is generally degummed and bleached before it is deacidified, a process which also has deodorizing effects (see Fig. 4). A convenient method for recovering husk oil might be esterification with glycerine, but such a process is outlawed in most countries. Oil Degumming (H,PO, or H,SO,

1

I Degummed oil Bleaching earth I Bleached oil

w

High vacuum distillation FIG. 4. Scheme of oil refining

Finished oil by distillation.

Oils neutralized by chemical method are subsequently bleached in the presence of active bleaching earth. Because of the high value of oil which is retained by the earth, filtration plants of this kind are usually equipped with solvent-extraction lines. Deodorization is the last refining step, requiring no particular comment. Refined olive oils are usually dewaxed before bottling by means of crystallization tanks at a temperature of 7-8°C. Crystallization occurs in 24-36 hr, after which the oil is filtered, and the residual solid glycerides used for industrial purposes. Solvent winterization methods are also employed, resulting in higher yields and lower manpower costs. Virgin olive oil possesses good stability to autoxidation because of its iodine value and because of the presence of the several phenolic antioxidant compounds that have been previously described. Its chlorophyll contents, however, make it quite sensitive to photochemical autoxidation, as demonstrated by Fedeli,40*41 who determined autoxidation rates in the presence of light and in the dark. This is the reason why canned oil has a longer storage life than bottled oil. V. OLIVE

OIL

ANALYSIS

The same methods are normally used for olive oil analysis as for other commercial oils.25 Specific methods, however, have been developed, to determine an oil’s authenticity and/or its commercial value, and they are extensively used. Tests to identify genuine olive oil, in addition to conventional methods and fatty acid assessment by GLC, may consist of determining the sterol composition2’ or palmitic acid content in the 2-position of the glycerides.‘* Quantitative analysis of sterol composition is done on the oil’s unsaponifiable matter first, the latter being prepared by conventional methods and deacidified by passing on an alumina column. The sterol fraction is then isolated by fractioning on a Silica Gel G plate (using heptane-acetone as the eluent), whereupon the

Enzo

72

Fedeli

sterols are converted into their TMS derivatives and analyzed by GLC on SE-30 or JXR (3%). Under standard conditions, a virgin olive oil has a /3-sitosterol content of 94% or higher, this substance being associated with stigmasterol and campesterol; whereas the minimum p-sitosterol contents of refined oils are 93%. To determine the palmitic acid content in position 2 of the glycerides, the oil is deacidified if necessary and it is then submitted to pancreatic lipase hydrolysis under standard conditions.28 The monoglyceride fraction is isolated by TLC and the fatty acid composition determined by GLC of the methyl esters obtained by transesterification. Genuine olive oil never contains more than 2% palmitic acid in position 2. , Oils obtained by solvent extraction may be distinguished from expressed oils by such simple procedures as the Bellier-Carocci-Buzi reaction26730 or the Vizern Test or, better still, by GLC determination of their erythrodiol contents.64 The procedure is similar to the one used for sterol analysis. U.V. analysis may also be used to classify commerical oils with respect to autoxidation, by determining E:‘& at 232 and 268 pm or by employing the relationship* AK = K268 -

K2c32

+ 2

Km.

Other tests are also employed for detecting foreign oils in olive oil. VI.

NUTRITIONAL

PROPERTIES

OF

OLIVE

OIL

From the standpoint of its acidic and glyceride composition, olive oil appears to possess excellent nutritional properties associated with a high degree of oxidative stability because of its low iodine value and the presence of several antioxidants. Olive oil is well absorbed by both animals and humans; absorption values of 98% were measured in rats by Thomasson’ and in infants by Giovannini and Cevini.59 Such findings having been confirmed by other workers as well. Such a good absorption factor is associated with a high degree of digestibility. Taking olive oil digestibility, as 100 Perettilo3 found only coconut oil to be just as digestible, whereas sunflower and corn oils rated only 83 and 48, respectively. Olive oil exhibits a more effective cholagogic action upon the liver than most vegetable oils, as demonstrated by Mastrilli and Stocchi,‘3*60~84~88~108~109 and confirmed by several other workers. In human nutrition, Bronte-Stewart’ was the first to attribute to olive oil a possible hypocholesterinemic activity, which was confirmed by Demarne,34 Gounelle,60 and others7,34*63*8’ whereas a low atherogenicity index was reported by Leonardi. (Received June 30, 1975) REFERENCES I. 2. 3. 4. 5. 6. I. 8. 9. 10. Il. 12. 13. 14. 15. 16. 17.

AMELOTTI, G., DAGHRTA, A.. GRIECO, D. and MARTIN, K. Riu. ital. Sosran;e Grasse 50. 30 (1973). ANONYMOUS Riu. ital. Sostanze Grasse 40. 108 (1963). BERNARDMI, E. Oil and Fat Technology, Teenologie, Rome, pp. 544546, 1973. BERNARDMI, E. Oil and Fat Technology, Tecnologie, Rome, pp. 630641, 1973. BERTONI, M. M. and CATTANEO. P. An. Assoc. quim. argent. 47, 52 (1959). BORQUELOT, E. and VITILESCO, J. C.r. hebd. Sianc. Acad. Sci. Paris 147, 533 (1908). BOZEK, J., BUZINA, R. and MIKIC, F. Am. J. c/in. Nutr. 5. 285 (1957). BRINTE-STEWART, B., ANTONIS, A., EALIS, L. and BROCK, J. F. Lancer i. 521 (1956). BUNYAN, J., GREM, J., MAMALIS, P. and MARKINKIEWICZ, R. Nature 179. 418 (1957). CAGLIOTI, L. and CAINELLI, G. Tetrahedron 18, 1061 (1962). CAGLum. L.. CAINELLI, G. and MINLJTILLI, F. Atri Accad. naz. Lincei Rc. 29. 544 (1960). CAGLIOTI, L., CAMELLI, G. and MINLITILLI, F. Gazz. chim. ifal. 71, 1387 (1961). CAGLIOTI, L., CAINELLI, G. and MINIJTILLI, F. Chimica lad. Milan0 43, 278 (1961). CANTARELLI, C. Riu. ital. Sostanze Grasse 38, 69 (1961). CANTARELLI, C. and MONTEWRO, G. Riv. ital. Sosranze Grasse 46, I15 (1969). CANZONERI, F. Gaze. cbim. ital. 27. 1 (1897). CANZONERI, F. Gazz. chim. ital. 36, 372 (1906).

Lipids

of olives

73

18. CAPELLA, P., DE Z~TTI, G., RICCA, CR. S., VALENTINI, A. F. and JACINI, G. J. Am. Oil Chem. Sot. 37. 564 (1960). 19. CAPELLA, P., FEDELI, E., CIRIMELE, M. and JACINI, G. Riu. ital. Sostanze Grasse 40, 603 (1963). 20. CAPELLA, P., FEDELI. E., CIRIMELE, M., LANZANI, A. and JACINI, G. Riu. ital. Sostanze Grasse 40. 660 (1963). 21. CAROLA, C., FEDELI, E. and JACINI, G. Riu. ital. Sostanze Grasse 41, 343 (1964). 22. CAROLA, C.. FEDELI, E. and JACINI, G. Riu. ital. Sostanze Grasse 41, 463 (1964). 23. CHARBONNIER, A. Epatologia 6, 391 (1961). 24. CIUSA, W.. NEBBV\, G., BUCCELLI, A. and VOLWNES, E. Riu. ital. Sostanze Grasse 42, 175 (1965). 25. COMM. TECNICA GOVERN. Norme Italiane per il controllo dei grassi e dei deriuati (NGD), Staz. Sper., ed. Oli e Grassi, Milano. 26. COMM. TECNICA GOVERN. Ibid. Method. Ba-V-4-1957. 27. COMM. TECNICA GOVERN. Ibid. Method. Ba-111-13-1971. 28. COMM. TECNICA GOVERN. Ibid. Method. Ba-IV-40-1971. 29. COMM. TECNICA GOVERN. Ibid. Method. Ba-V-11-1971. 30. COMM. TECNICA GOVERN. Ibid. Method. Ba-V-3-1971. 31. ()REUSS, W. V. Ind. Engng Chem. 33, 300 (1941). 32. CREUSS, W. V. and SIJGIHARA, J. Archs Biochem. 16, 39 (1948). 33. CUCURACHI, A. Riu. ital. Sostanze Grasse 41. 18 (1965). 34. DEMARNE. M. Dactylo-Sorbonne (1960). 35. DRUMMOND, J. and THORBJANARSON, T. Analyst 60. 23 (1935). 36. FEDELI, E. Reuuefr. Cps gras 15, 281 (1968). 37. FEDELI, E. Atti 2” Conv. Spettrometria di Mass, Ispra, Italy, 1971. 38. FEDELI, E. Rendic Simpos. Fondaz. C. (CISALPINO GOGLIARDICA, ed.) Erba, Milan, p. 155 (1972). 39. FEDELI, E., BARONI, D. and JACINI, G. Riu. ital. Soslanze Grasse 50. 38 (1973). 40. FEDELI, E. and BRILLO, A. Riu. ital. Sostanze Grasse 52. 88 (1975). 41. FEDELI, E. and BRILLO, A. Riu. ital. Sostanze Grasse 52. 109 (1975). 42. FEDELI, E. and Cwuwn, F. 12th World Congress, ISF, Paper 75, Milan, Sept. 2-7, 1974. 43. FEDELI, E., CAMUKATI, F., CORTESI, N., FAVINI, G., CIRIO, V. and VITA, G. 12th World Congress, ISF, Paper 117, Milan, Sept. 2-7, 1974. 44. FEDELI, E.. CAMIJRA~, F. and JACINI. G. Rio. ital. Sostanze Grasse 48. 565 (1971). 45. FEDELI, E. and CORTESI, N. 12th World Congress, ISF, Abstract 122. Milan. Sept. 2-7. 1974. 46. FEDELI, E., CORTESI, N., MARIANI, C., BARONI, D. and JACINI, G. Scienza Tee. Alimenti 3, 143 (1974). 47. FEDELI, E., FAVINI, G., BARONI, D. and JACINI, G. Chimica Ind. Milan0 55, 681 (1973). 48. FEDELI. E. and JACINI. G. Riu. ital. Sostanze Grasse 44. 393 (1967). 49. FEDELI, E. and JACINI, G. Chimica Ind. Milan0 52. 16i (1970). 50. FEDELI, E., LANZANI, A., CAPELLA, P. and JACINI, G. J. Am. Oil Chem. Sot. 43. 254 (1966). 51. FEDELI, E. and MARUNI, C. Riu. ital. Sostanze Grasse 50. 164 (1973). 52. FEDELI, E. and MARIANI, C. Riu. ital. Sostanze Grasse 51, 129 (1974). 53. FIECCHI, A., CAPELLA, P., FEDELI, E., LANZANI, A. and JACINI, G. Ricerca Scient. 36, 1316 (1966). 54. FIESTAS, J. Grasas Aceit. 9, 249 (1958). 55. FIESTAS. J. Grasas Aceit. 10. 30 (1959). 56. FLATH, R. A., FORREY, R. R. and GUADAGNI, D. G. J. Agric. Fd Chem. 21, 948 (1973). 57. FUHRUANN. R. J. Rech. Cent. natn Rech. scient. 5. 325 (1954). 58. FUHRUANN. R. Revue jir. Cps gras 2, 237 (1955). 59. GIOVANNINI, M. and CEVINI, G. Dietol. Dietoter. 7. 13 (1963). 60. GOUNELLE, H. Atti II Conv. Int. Lipidi Alim., Rimini, 1961. 61. GRACIAN, J. and AREVALO, G. Grasas Aceit. 16, 278 (1965). 62. HERTING, 0. C. and DRURY, E. I. E. J. Nutr. 81, 335 (1963). 63. HOLNER, M., KRIEUBER, C. and WENGER, R. Virchows Arch. path. Anat. Physiol. 333. 210 (1960). 64. IST. POLIGR. DELLO STA~O. Gazz. Ufl Repub. ital., 134, 3635 (1973). 65. ITOH, I., TAMURA, T. and MATSUMOTO, T. J. Am. Oil Chem. Sot. SO, 300 (1963). 66. ITOH, T., TAMURA, T. and MATSUMOTO, T. J. Am. Oil Chem. Sot. SO1 122 (1973). 67. JACINI, G. and FEDELI, E. Rc. 1st. lamb. Sci. Lett. 106, 446 (1972). 68. JACINI, G., FEDELI, E. and LANZANI, A. J. Ass. 08~. Anal. Chem. 50, 84 (1967). 69. JOSLIN, M. A. and SMITH, C. J. B. Mitt. Kloster, Obstbau Cl. Garten, Sep. B. 4, 141 (1954). 70. JULIUS BIJLSMA, J. A. Pharm. Weekbl. Ned 96, 417 (1961). 71. KOERNER, G. and CANELU-ITI, N. Rc. 1st. lamb. Sci. Len. 15, II, 654 (1882). 72. KOFLER, M. He/u. chim. Acra 28, 26 (1945). 73. KOFLER, M. He/u. chim. Acta 30, 1053 (1947). 74. KOTAKIS, Gr. Reuuefr. Cps gras. 14, 143 (1967). 75. LEONARDI, G., MORO, V., RODRIGUEZ, R. and MARTELLI, C. Atti III Conv. Int. Lipidi Alim., Rimini (1962). 76. LEONE, A. M. and LAMPARELLI, F. Ind. Agric 6, 477 (1968). 77. LERKER. G.. CAPELLA. P., and DESERTI, P. L. Scienza Tee. Alimenti 3. 299 (1973). 78. LOEV. i. and GOODMAN, M. M. Chem. Ind. Lond. 2026 (1967). 79. Lost. G. and PALLOTTA. U. Riu. ital. Sostanze Grasse 38. 425 (1966). 80. Lo& G. and PIRETTI, A. Riu. ital. Sostanze Grasse 47, 493 (19?0). 81. MALMROS, H. and WIEGAND, G. Lancet i, 1 (1957). 82. MARCELET, M. C.r. hebd. SPanc. Acad. Sci. 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Lipids of olives.

LIPIDS OF OLIVES ENZO FEDELI Departnwrit qf’ Food Science, Lliiiuersity of Milan, Italy CONTENTS 57 . 58 58 58 59 59 60 60 60 60 62 62 63 63 64 6...
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