Study of the Chemical Composition of Olive Oil According to Its Mode of Extraction and Its Age from the Olive Tree

Journal of Environmental Treatment Techniques

2020, Volume 8, Issue 3, Pages: 1075-1080

J. Environ. Treat. Tech.

ISSN: 2309-1185

Journal web link: http://www.jett.dormaj.com

Study of the Chemical Composition of Olive Oil

According to Its Mode of Extraction and Its Age

from the Olive Tree

Miloudi Hilali *, Nadia Maata , Hanae El Monfalouti and Badr Eddine Kartah

Laboratory of Plant Chemistry and Organic and Bioorganic Synthesis, Faculty of Science, University Mohamed-V, Av. Ibn Battouta, BP 1014 Agdal-

Rabat, Morocco

Official Laboratory for Chemical Analysis and Research 25, Rue Nichakra Rahal, Casablanca, Morocco

Received: 12/04/2020

Accepted: 29/06/2020

Published: 20/09/2020

Abstract

A comparative study of the chemical composition of olive oil was carried out in the Massmouda district of Ouezzane, a city in

northern Morocco. It was made in order to study the influence of the extraction temperature and the age of the olive tree on the

chemical composition of olive oil. To carry out this work, we selected four samples of olive oil of the same kind (Moroccan

Picholine), from the same place and extracted at different temperatures, but these samples were collected from olive trees of different

ages. For this we performed physico-chemical analyzes such as acidity, peroxide index, absorbance in ultraviolet, fatty acids, sterols

and triglycerides. The result of this work shows that the temperature of olive oil extraction can increase the peroxide index, acidity, the

percentage of oleic fatty acid (C18: 1), the percentage of stigmasterol, and the percentages of triglycerides OOO, POO and can

decrease the percentage of fatty acid such as: C18: 0, C18: 2 (vitamin F) and the percentages of triglycerides LLL, LOL, OLO, PLO.

The results of the chemical composition according to the age of the olive tree reveal that the percentages of oleic acid (C18: 1), the

percentages of the triglycerides LLL, OOO are decreased with the increase in the age of the olive tree. On the other hand, our study

demonstrates that the percentage of linoleic acid C18: 2 (vitamin F) is increased with the increase of the age of the tree. In the end our

study proved the high quality of olive oil extracted by cold mechanical pressing.

Keywords: Olive, Fatty acids, Sterols, Triglycerides, Extraction temperature, Olive age

Olive oil is practically the only vegetable oil that can be

Introduction¹

consumed in its raw form without prior treatment.

Appreciated for its flavor and nutritional characteristics, it is

known for its multiple virtues in the prevention of diabetes,

high blood pressure [4], certain cancers and aging [5]. It is

also used in the pharmaceutical and cosmetic fields [6].

Making the pillar of the Mediterranean diet, several studies

have been carried out, on the fruit and leaves, to confirm the

ancestral virtues attributed to it [7-9]. This study is part of the

continuity of the research series performed by The Laboratory

of Plant Chemistry, Organic and Bio-organic Synthesis on

vegetable oils. To enhance olive oil and improve its nutritional

effect we followed some steps: (a) the study of the chemical

composition of olive oil according to the age of the olive tree;

The olive tree is a Mediterranean fruit tree that belongs to

the Oleaceae family. This tree produces olives; a fruit

consumed in various forms and from which olive oil is

extracted. In Morocco, the main cultivated fruit species is the

olive of the popular variety "Moroccan Picholine" [1] which

occupies an area of 560,000 ha. Olive groves contribute to

employment in rural areas with 11 million working days

annually. Olive production reaches the approximate figure of

60,000 T, generates 50,000 T of olive oils and 90,000 T of

industrial table olives [2]. The olive tree develops in four

periods [3].

•

and flowering. The olive tree settles, expands but produces

nothing.

Period of youth (1-7 years): it is the period of growth, size

(

b) the study of the chemical composition of olive oil

according to its mode of extraction and (c) to accomplish this

work, 4 olive samples belonging to the same region of

Morocco (the town of Ouezzane Douar Ghnioua) and of the

same variety "Moroccan Picholine" were selected.

•

Period of entry into production (7-35 years): it is in a way

the adolescence period of the tree which prepares for the

establishment of regular and significant productions.

•

Adult period (35-100 years): full production period (yield of

5 to 25 kg of olives per tree). The olive tree is in the prime of

life.

2 Materials and methods

2.1 Presentation of the study area

•

Period of senescence (beyond 150 years): end of the tree's

productive life, little by little it produces less. Carpenter

branches die, trunk bursts.

The study region is the city of Ouezzane which belongs to

the southern margins of the Jebala area whose major tribes

bordering the city are: Masmouda, Rhouna, Ghzaoua and Beni

Mesara. The Ouezzane region, in the North of Morocco,

extends over an area of 1861.2 km², and has an altitude of 614

meters [10] (figure 1).

Corresponding author: Miloudi Hilali, Laboratory of Plant

Chemistry and Organic and Bioorganic Synthesis, Faculty of

Science, University Mohamed-V, Av. Ibn Battouta, BP 1014

Agdal-Rabat, Morocco. Email address: hilali400@yahoo.com.

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Journal of Environmental Treatment Techniques

2020, Volume 8, Issue 3, Pages: 1075-1080

Figure 1: Presentation of the study area

Table 1: Origin and method of extraction of the 4 samples

N° Sample The age of the tree

The region

Ghanioua

Ghniuoua

Extraction mode

10 years

20 years

40 years

Extracted by mechanical cold pressing 25 °C

Same sample 3 but extracted by mechanical hot press 50°C

.2 Biological material

In the present work, 4 different samples were selected and

characteristics and the chemical composition of all the

samples are determined (acidity index, peroxide index, cis and

trans fatty acids, triglycerides and sterols). The oils are

analyzed according to the methods of analysis already

described in the international olive oil advisory literature [12].

collected through the mode of extraction from olive trees

having different ages or coming from the same region of

Ouezzane commune of Massmouda Douar Ghanioua (the city

of Ouezzane north of Morocco) during the month of

December 2019. Table 1 provides information on the origin,

method of extraction and age of the olive tree from each

sample of olive oil.

2.4 Physico-chemical analyzes of oils

The acidity expressed as a percentage of oleic acid and the

peroxide index were measured according to standardized

methods, respectively the French standard [13], and the

French standard [14]. The specific extinction coefficients in

the ultraviolet at 232 nm and 270 nm (K232) and (K270).

They are calculated respectively from the absorption at 232

and 270 nm according to the French method [15], using a

VARIAN type spectrophotometer.

.3 Preparation of different samples of olive oil

Only the mechanical pressing extraction method was used

to extract olive oil. It was done in the Amal cooperative (olive

oil extraction cooperative province of Ouezzane, Morocco)

according to extraction methods already described [11],

avoiding chemical and enzymatic reactions which could

change its natural composition. The extraction method

includes four main operations: cleaning the fruit (defoliation,

washing the olives), preparing the dough (grinding, kneading),

separation of the solid phase (pomace) and liquid phase (oil

and water vegetation), separation of the liquid phase (oil and

water from vegetation). Olive oil is prepared in two different

ways: (a) samples 1,2,3: the olive oil is extracted by

mechanical cold pressing at 25 °C (the exit temperature of the

olive oil after extraction) and (b) sample 4: same olive fruit as

sample 3 but the olive oil is extracted by mechanical hot

pressing at 50 °C (the outlet temperature of the olive oil after

extraction). These oils are then analyzed directly after

extraction at the Official Laboratory of Analysis and Chemical

Research of Casablanca (LOARC). The physico-chemical

2.5 Analysis of cis fatty acids

The fatty acid composition was determined after

transformation into methyl esters obtained by trans-

esterification of the triglycerides with methanolic potassium

hydroxide. The methyl esters of fatty acids in the samples of

olive oils are obtained according to the French international

standard method [16]. Then, these esters were analyzed by gas

chromatography according to the conditions described in ISO

5508: 1990, using a VARIAN chromatograph with flame

ionization detector (FID), equipped with a capillary column

(CPWAX) 30 m long and 0.25 mm inside diameter. The oven

temperature is set at 200 °C, and that of the injector at 220 °C.

The carrier gas used is helium at 1.2 ml / min and the volume

of the injection is 1 µl, leakage (split on) at ratio: 15%.

076

Journal of Environmental Treatment Techniques

2020, Volume 8, Issue 3, Pages: 1075-1080

.6 Triglyceride analysis

3.2 Analysis of the physico-chemical characteristics

To 0.15 g of olive oil is added 0.5 ml of hexane and 15 ml

of a mixture of hexane / diethyl ether (87/13). This solution is

poured into a cartridge of supelco brand containing 0.5 g of

silica gel previously activated with hexane. The fraction of

triglycerides is thus separated from diglycerides and

monoglycerides. It is collected in a 100 ml flask. It is

subjected to analysis after evaporation of the solvent and

dilution with 1.5 ml of acetone. The triglycerides are analyzed

by HPLC on a column in the reverse phase of C18 (250 mm ×

All the acidity values observed are less than 1%. This

result shows that virgin olive oil is characterized by low

acidity (acidity of olive oil must be less than or equal to 2%)

[19]. The acidity results show that the acidity of four samples

is higher (0.93%) than the acidity of sample 3 (0.7%) (3 and 4

same sample but extracted at two different temperatures).

These results suggest that the extraction temperature may

influence the acidity values of olive oil. The temperature

therefore appears as a parameter influencing the acidity value

of olive oil. Indeed, the acidity value of the oil samples

prepared from mechanical cold pressing is uniformly lower

than those of oils prepared from mechanical hot pressing.

The results of the peroxide index of the four samples of

olive oil show that the samples have a peroxide index of less

than 10 meq O2 / kg (peroxide index of olive oil ≤ 20 meq O2

/ kg) (COI / T.20 / DOC. No 42-2 / Rev.2 - 2017). It is quite

likely that the value of the peroxide index observed in our four

samples is lower. This is related to the freshness of the oil. In

fact, it is extracted directly after the recovery of the olive fruit

and analyzed after its extraction (the analyzes were made after

15 days). The result of the peroxide index shows that the

peroxide index of sample 4 is higher. Indeed, this sample is

extracted by mechanical pressing at a higher temperature (50

°C). This result clearly indicates that some components of

olive oil are extremely sensitive to oxidation [20]. The high

peroxide content is observed for sample 4. This is linked to

the extraction temperature. Our result is consistent with the

literature which indicates that extraction methods, geographic

origin and climatic factors influence the chemical

characteristics of oils. In a study carried out in Italy and taking

into account two different methods of storing olive oil, it was

established that the peroxide index which represents one of

the quality parameters of olive oil increased rapidly and above

tolerated threshold according to Torres and Maestri [21]. On

the other hand, Kiritsakis [22], considers that the place of

culture has no significant influence on these analytical

parameters (acidity, UV absorbances and peroxide index).

This author points out that the deterioration in the quality of

olive oil is rather fundamentally affected by factors damaging

the fruit such as attack by parasites (flies) or the use of

improper systems of harvesting as well as transport and

storage of olives. Table 3 shows the results of the acidity

value and the peroxide index of 4 samples.

.6 mm, Φ silica 5 μm), according to the IUPAC method N°

.0 324. The HPLC device is equipped with an HP

refractometric detector 10 47A. The elution is carried out with

a mixture (acetonitrile / acetone) (v / v) with a flow rate of 0.5

ml / min during the analysis time (90 min).

.7 Determination of the composition and nature of total

sterols [18]

Weigh 2.5g of olive oil in a 20ml flask. 25 ml of a

potassium hydroxide solution (1N ethanol) is added to it. The

flask is heated to boiling under reflux for 30 min until the

solution becomes clear. Then add 25 ml of distilled water to

stop the reaction. The extraction of the unsaponifiable is

carried out using 75 ml of hexane or petroleum ether. The

organic phase undergoes a series of washing with 15 ml of

mixture (water / ethanol 95°) (90/10) in a separatory funnel.

The hexane phase is transferred from the top of the vial to a

00ml flask. After evaporation of the solvent using a rotary

evaporator, the unsaponifiable material is recovered. The

unsaponifiable matter, diluted with 300 μl of hexane or

petroleum ether, is filtered. The unsaponifiable is obtained

according to standard NFT 50-205. It is fractionated by high

performance liquid chromatography (HPLC) on a silica

column (25cm × 4 mm). The HP device is equipped with a

05 nm-254 nm UV detector. The eluent is an isooctane /

isopropanol mixture (99/1), the flow rate of which is 1.2 ml /

min. The duration of the analysis is 15 min, and the sterol

fraction recovered according to standard NF 12228 May 1999,

is evaporated to dryness. Sterols are transformed into silylated

derivatives (TMS) using

hexamethyldisilazane (HMDS) and trimethylchlorosilane

TMCS), (9/1/1), (v / v / v). The pyridine evaporates to

mixture of pyridine,

(

dryness and the silylated derivative is diluted with 60 μl of

heptane or hexane. TMS sterols are analyzed by gas

chromatography (CPG) on an apolar column (Chroma pack)

(30m × 0.32mm, ID: 0.25 µm, phase: CPSIL8CB). The HP

Table 3: Results of acidity and peroxide index

Hewlett packard 6890 series GC system chromatograph is

equipped with a FID detector (T °: 300 °C). The carrier gas is

nitrogen and its flow rate is 1ml / min (P.E: 8.6 bar). The

analysis is carried out in temperature programming (200 °C up

to 270 °C with a speed of 10 °C / min and an isotherm at 270

N° sample

Acidity

0.84%

0.90%

0.7%

Peroxide index meq O / kg

0.93%

°C for 35 min).

.3 Determination of absorbance in ultraviolet

The specific extinction of olive oil was determined at 247

Results and Discussion

.1 The yield of the extraction

nm, 270 nm and 266 nm. In general, the values found vary

between 0.1410 to and 0.1650 to 270 nm. The specific

extinction of all the samples is lower (lower than 0.1757). It is

a virgin olive oil prepared cleanly from olive fruit. We found

that the specific extinction values of sample 4 prepared from

olive fruit by mechanical hot pressing have values higher than

sample 3 (same batch). From this result, it was concluded that

extraction by mechanical hot pressing can increase the

specific extinction values. This result clearly shows that there

is a formation of carbon-carbon bonds or carbon-oxygen

bonds in the form of secondary auto-oxidation products

during hot extraction. All of these compounds cause an

increase in absorption in the region between 225 nm and 325

From these results, since the temperature of the extraction

(50 °C) increases it is quite possible that the yield of the

extraction of the olive oil is higher. Consequently we found a

difference from 5 to 6 liters of olive oil in each 100 kg of olive

fruit if the extraction has been hot.

Table 2: The yield of olive oil as a function of the temperature

of the extraction

N° Sample

Oil yield in 100kg / liter

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Journal of Environmental Treatment Techniques

2020, Volume 8, Issue 3, Pages: 1075-1080

nm. The result of the absorbance in the ultraviolet is collated

in table 4.

groups together the results obtained for the four samples. It

appears from this result that the percentage of trans oleic,

linoleic and linoleinic acid (C18: 1, C18: 2 and C18: 3),

(elaidic acid) in virgin olive oil is very low and does not give

any information on extraction methods or age. The presence

of trans fatty acids in "virgin" olive oils, suitable for

consumption, is an indication of the fraudulent presence of

refined oil. For this reason, the content of trans fatty acids has

been limited by the standard to 0.05% [27] both for elaidic

acid and for the sum of the trans isomers of linoleic acids and

linolenic.

.4 Analysis of cis fatty acids

The fatty acid composition of the different oils was

determined after methylation of the oil and analysis of the

methyl esters by gas chromatography on a capillary column

[23, 24]. Table 4 groups together the results obtained for the

four samples. he fatty acid composition of olive oil

corroborates with data from the standards (COI / T.20 / DOC.

No 42-2 / Rev.2 - 2017). Olive oil contains 86% unsaturated

fatty acids. It is of the oleic – linoleic type and contains 13%

of essential fatty acids: linoleic acid (11 to 13%) (Vitamin F).

This acid is said to be essential because it cannot be

synthesized by the body and must be provided by food.

Unsaturated fatty acids play an essential role in the prevention

of cardiovascular diseases, and the omega 6 family (such as

linoleic acid) is vital for the growth of children [25]. Olive oil

is rich in oleic acid C18:1 Its oleic acid content makes olive

oil particularly interesting in regulating cholesterol. Studies in

progress seem to show that ingesting 2 tablespoons a day of

olive oil for a month could significantly lower blood

cholesterol levels. The other fatty acids present are: palmitic

C16: 0 (9 to 10%) and stearic C 18: 0 (2.5 to 3%). The

percentage of linolenic acid (C18: 3) in olive oil does not

exceed 1%. The presence in olive oil of long chain fatty acids

such as C20: 0 (0.3%), C20: 1 (0.3%) is noted. Comparing

samples 3 and 4 (same sample but the extraction temperature

is different) we found that the percentage of stearic acid (C18:

3.6 Triglyceride analysis

The triglycerides of the different olive oil samples

analyzed by high performance liquid chromatography are

grouped in Table 7

Analysis of the triglyceride fraction of olive oil by HPLC

allowed the separation of the individual triglycerides. The

predominance of OOO triglycerides is noted (41.2% to

45.7%). POO (19.3 to 20.6%), OLO (18.9 to 20.3%), LOL

(4%), PLO (5.5 to 7.1%). It should also be noted that the

majority of the oleic and linoleic acids occupy the Sn-2

position. Our results are in agreement with data from the

literature [26, 28] which indicate that the triglycerides OOO,

POO, OLO, LOL, PLO are predominant in olive oil.

Triglyceride results show that extraction temperature can

influence the compositions of the triglycerides. We have also

found that the extraction by mechanical hot pressing decreases

the percentages of triglycerides such as LLL, LOL, OLO and

PLO. Analysis of the triglyceride fraction shows that the age

parameter of the olive tree decreases the percentage of

triglycerides LLL and OOO. Our results are in agreement with

data from the literature [29].

) and linoleic acid (C18: 2) is reduced because of hot

extraction. This is probably due to a deterioration of the fatty

acid during the hot extraction. These variations can be

considered as useful markers to ensure the extraction method.

From this study we noticed that the percentage of linoleic acid

(C18: 2) is increased with the increase in the age of the olive

3.7 Analysis of sterols

tree (samples 1, 2 and 3). The variation in fatty acid resulting

from our samples is consistent with studies which indicate that

the percentage of oleic acid may influence the climate [26].

The sterol composition of the different olive oil samples

was determined by gas chromatography after silylation of the

sterol fraction. The latter is obtained by fractionating the

unsaponifiable of olive oil by HP on a normal phase. The

various sterols encountered were identified by gas

chromatography. Table 8 summarizes the results obtained for

the 4 selected samples.

.5 Analysis of trans fatty acids

The trans fatty acid composition of the different oil

samples was determined after methylation of the oil and

analysis of the methyl esters by gas chromatography. Table 6

Table 4: Determination of absorbance in ultraviolet

N° sample

absorbance in 247 nm

absorbance in 270 nm

absorbance in 266 nm

Extinction

0.1529

0.1392

0.1290

0.1435

0.1650

0.1513

0.1410

0.1554

0.1757

0.1619

0.1544

0.1669

0.0007

0.0008

0.0007

0.0002

Table 5: The results of cis guys-acids depending on the temperature of the extraction and the age of the tree

N° sample/ fatty acid

C16:0

C16:1

C17:0

C17:1

C18:0

C18:1

C18:2

C18:3

Retention time

11.284

11.932

12.709

13.332

14.192

14.886

15.797

17.116

8.92

0.58

0.04

2.65

73.94

12.23

0.84

0.32

0.39

10.08

0.80

0.04

0.05

2.78

71.40

13.23

0.88

0.33

0.36

9.32

0 .60

0.05

0.04

3.02

72.17

13.04

0.97

0.33

0.36

9.16

0.58

0.03

0.05

2.56

74.83

11.20

0.86

0.32

0.37

C20:0

C21:1

17.324

18.092

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Journal of Environmental Treatment Techniques

2020, Volume 8, Issue 3, Pages: 1075-1080

Table 6: the result of the trans fatty acid composition according to the temperature of the extraction and the age of the tree

N° sample

C18:1trans (TR13.03min)

0.03%

0.02%

0.00%

0.03%

0.02%

0.03%

0.02%

%C 1%C18:2trans+%C18:3trans (TR13.96min)

Table 7: The triglyceride composition according to the temperature of the extraction and the age of the tree

Sample/TG

LLL

OLL

LOL

OLO

PLO

OLO

PLO

PLP

OOO

POO

POP

TR min

32.57

36.34

44.71

47.55

49.59

59.41

66.44

74.22

81.67

92.60

106.49

Sample 1

0.71

0.11

4.27

0.45

19.83

6.13

0.56

44.26

19.33

1.63

Sample 2

0.52

4.54

1.38

20.34

7.13

0.82

41.18

20.57

1.67

Sample 3

0.53

0.57

4.41

1.62

19.64

6.61

1.08

42.10

19.88

0.94

Sample 4

0.27

0.1

3.53

1.69

0.60

18.89

5.53

0.37

45.69

20.39

2.22

(P: palmitic acid, L:linoleic acid, O: oleic acid)

Table 8: Composition in sterols of the 4 samples.

N° Samples/ Sterols

Cholesterol

Campesterol

Stigmasterol

Delta-7-stigmasterol

β-sitosterol

TR min

30.23

33.32

34.33

35.79

36.57

37.04

38.64

Sample 1

0.12

2.89

2.34

1.01

81.80

10.92

0.64

Sample 2

0.12

2.73

2.45

1.05

83.10

9.73

0.61

Sample 3

Sample 4

0.18

2.89

2.22

1.03

82.75

10.10

0.60

0.15

2.85

1.64

1.11

82.56

10.70

0.62

Delta-5-avenasterol

Delta-5-stigmasterol-stadineol- Tosterol-

The sterolic composition is in accordance with the data in

OOO, POO, LLL, LOL, OLO, PLO. These results agree with

those reported in the literature. This shows that the extraction

method influences the dietary qualities of olive oil. On the

other hand, the results of the chemical composition according

to Olivier's age do not give any significant variation between

the results obtained.

the literature [30]. These are essentially β-sitosterol. The main

products are β-sitosterol and Delta-5-avenasterol. Their

proportion varies respectively between 81.80% and 83.10%,

and from 9.73 and 10.92%. The content of campesterol found

in olive oil varies between 2.7% and 2.9%. The result shows

that the percentage of cholesterol varies from 0.12% to 0.18%.

This value is consistent with the standards which indicates

that the percentage of cholesterol in virgin olive oil must be

less than 0.5%. The result of the sterols shows that the

percentage of stigmasterol is higher in sample 4 which is

extracted by mechanical hot pressing. In spite of these results,

we have not found a significant variation on the sterol

composition of the different samples according to the

temperature of the extraction and age of olive trees.

Acknowledgements

The authors thank the director of the official Casablanca

chemical analysis and research laboratory. Special thanks

must be addressed to the head of the fat section in the official

chemical analysis and research laboratory in Casablanca.

Ethical issue

Authors are aware of, and comply with, best practice in

publication ethics specifically with regard to authorship

Conclusion

(avoidance of guest authorship), dual submission,

As part of the promotion of olive oil, we conducted a

manipulation of figures, competing interests and compliance

with policies on research ethics. Authors adhere to publication

requirements that submitted work is original and has not been

published elsewhere in any language.

comparative study of the different physico-chemical

parameters of olive oil as a function of the extraction

temperature and its olive age. Our study shows that the

extraction method appears to be a parameter influencing the

acidity value and the index of peroxide in olive oil. Indeed,

the acidity values and the peroxide index are higher in sample

Competing interests

The authors declare that there is no conflict of interest that

would prejudice the impartiality of this scientific work.

which was prepared from mechanical hot pressing. This

result suggests that the temperature of the extraction may

influence the acidity values and the peroxide index. The

analysis of fatty acids and sterol composition are in

accordance with data from the literature. The main products

are β-sitosterol (83%) and Δ-5-avenasterol (10%). The results

related to the chemical compositions show that the

temperature of the extraction by mechanical hot pressing can

influence the percentage of sterol (cholesterol), the percentage

of fatty acid and the percentages of triglycerides such as:

Authors’ contribution

All authors of this study have a complete contribution for

data collection, data analyses and manuscript writing

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