Chemical Composition and Lipid Fraction Characteristics ofAleppo Pine (Pin us halepensis Mill.) Seeds Cultivated in

Tunisia

S. Cheikh-Rouhou,' B. Hentati,' S. Besbes,':" C. Blecker,3 C. Deroanne" and H. Attia'

1 Unité Analyses Alimentaires, Ecole Nationale d'Ingénieurs de Sfax, Route de Soukra 3038 Sfax, Tunisia2 Unité de Biotechnologie et Pathologie, Institut Supérieur de Biotechnologie de Sfax, Route de Soukra 3038 Sfax,

Tunisia"Unitë de Technologie des Industries Agro-alimentaires, Faculté Universitaire des Sciences Agronomiques de

Gembloux, passage des Déportés 2, 5030 Gembloux, Belgium

The proximale composition of Aleppo pine (Pinus halepensis Mill.) seeds cultivated in Bizerta (Tunisia)were investigated for their fatty acid composition, and their thermal and physical properties of lipid frac-tion. The proximate analysis of pine seeds showed the following composition (on a dry-weight basis):protein 22.7%, oil 43.3%. ash 8.3% and total carbohydrate 25.7%. Potassium. magnesium and calciumwere the predominant mineraI elements present in the seeds and reached, together, about 1%. Oleic andlinoleic acids were the major unsaturated fatty acids (27.3 and 48.8%, respectively), while the main satu-rated one was palmitic acid (8.75%). Myristic. rnyristoleic. palmitoleic, margaric, margaroleic, stearic,linolenic, arachidic, eicosenoic, eicosadienoic eicosatrienoic behenic and lignoceric acids were alsodetected. Thermal profile of Pinus halepensis Mill. seed oil, determined by its DSC melting curve,showed that the en tire liquefaction of the Aleppo pine seed oil occurs at -8°C. CieLab colour para-meters (L", a*. b*). oxidative stability by Rancimat test and viscosity were also determined. Physico-chernical properties of the oil include: saponification nurnber J 90, peroxide value 3.18. iodine index 1J 7,and a low acidity of 0.61 %. Results suggested that the production of oil from Pinus halepensis seedscould provide a potential use in food, pharmaceutical, cosmetics and other non-food industries.

Key Words: Pinus halepensis Mill. seeds, oil, fatty acids. thermal profile

INTRODUCTION

Aleppo pine (Pinus halepensis), described by Millerin 1768. is a circummediterranean species (Nahal,1986). This woody and perennial pine tree, cultivatedin the Mediterranean area is useful for reforestation(Le Thiec and Manninen, 2003). Pinus halepensisspecies belonging to the Pinaceae family and is themost widely distributed pine in this region. where itcovers more than 25,000 km2 (Maestre et al., 2003). Themain Aleppo pine forests are found in Spain, France

*To whom correspondence should be sem(e-mail: besbes.stêvoila.jrï.Received 9 November 2005; revised 23 January 2006.

Food Sei Tech Int 2006: 15(5):407-416© 2006 SAGE PublicationsISSN:1082-0132DO 1: 10.117711082013206069910

and North Africa (Raeda and Arianoutsou, 2000)especially in Algeria and Tunisia where they constitutea clump (Nahal, 1986). ln Tunisia, Aleppo pine coversnear 300,000ha from the mountains to the south ofMejerda and to the west of Zaghouan, to the centralregion of Tunisia (Bizerta, Ben Arous. Nabeul.Zaghouan, Beja, Jendouba, El Kef, Kairouan andKasserine). Pinus species are essentially grouped assoft pines (haploxylon) and hard pines (diploxylon).Pinus halepensis Mill. belongs to diploxylon pinegroups, which produce the most cornmercialised pineseeds in the world. ln tact, these seeds, lodged in largecones, are highly nutritious and very appreciated byconsumers.

Aleppo pine seeds are traditionally used throughoutTunisia, and other lslamic countries, for preparing asweet pudding of ground pine seeds. called 'Assida-Zgougou'. Recently. it has also been employed as aningredient in ice-creams and candies. A few workshave been published on the Tunisian Aleppo pine

408 S. CHEIKH-Rou HOU ET AL.

seeds (Nasri and Triki, 2004) which focus on the fattyacid composition, the unsaponifiable fraction and thephenolic compounds of the corresponding oil, howevernot on their detailed composition and physicochemicalproperties.

The aim of this work was to study the chemical com-position of Pinus halepensis seeds cultivated in Tunisiaand to determine the fatty acid composition andphysico-chemical properties of their lipid fraction.

MATERIALS AND METHODS

Materials

Seeds

Aleppo pine seeds iPinus halepensis Mill.) werepurchased (20 kg) from a herbal market in Bizerta(Tunisia), where they were harvested. Seeds weredirectly stored at 15°C for a maximum of 3 days andthen c1eaned manually to remove foreign matter. Seedsamples were separately milled in a heavy-duty grinderfor 4min to pass 1-2mm screens and then fractionedinto 40 powdered samples (500g) which are preservedin hermetic bags at -20°C until analysis.

Methods

Oil extraction and preservation

Aleppo pine seeds (50 g) were placed in a dark flask(capacity = 1L) and homogenised with 250mL ofhexane. The mixture was moved for 4 h in a shaker(Selecta, Spain) at 180 U/min, and then centrifuged for15min at 1,000 X g. The extraction was conducted at24 ± 1"C. The supernatent was filtered through aWhatman No. 2 filtering paper. The extraction pro-cedure was repeated twice and the collected solventwas removed using a rotary evaporator at 40°C. Theseed oil obtained was finally drained under a stream ofnitrogen and then stored in a freezer ( - 20°C) for sub-sequent physico-chemical analyses.

Analytical Methods

Ail analytical determinations were performed atleast in triplicate. Values were expressed as themean ± standard deviation.

Chemical Analysis of Powdered SeedsThe dry matter was determined according to the

Association of Officiai Analytical Chemists (AOAC,1990).

Oil was extracted from 15g of Aleppo pine seedpowder in a Soxhlet extractor for 8 h using hexane as a

solvent at 45°C. The result was expressed as the per-centage of lipids in the dry matter of seed powder.

Total protein was determined by the Kjeldahlmethod. Protein was ca1culated using a nitrogen con-version factor of 5.30 for pine nut seeds (Greenfieldand Southgate, 1992; Nergiz and Dënmez, 2004).

To remove organic matter, about 0.5 g of powderedseed samples were incinerated in the muffle furnace at550°C for about 12h. The ashes were dissolved inHNO, (Larrauri et al., 1996) and the minerai con-stituents (Ca, Na, K, Mg, Fe, Zn, Cu and Mn) weredetermined using an atomic absorption spectropho-tometer (Hitachi Z 6100, Japan). The phosphoruscontent was analysed by the phosphomolybdovanadatemethod (AOAC, 1990). The total ash was expressed asa percentage of dry matter.

The carbohydrate content was estimated by dif-ference of me an values and dry matter content.

Analysis of Oil ExtractAOCS officiai methods (American Oil Chemists'

Society, AOCS, 1997) were used to evaluate the perox-ide, iodine, saponification and acidity values (methodsnumber Cd 8-53, Cd 3d-63, Cd 3-25 and Cd 3d-63, respec-tively). Determination of refractive index (at 20°C) wasdetermined with a refractometer (Abbe-Zeiss).

Km and Km extinction coefficients were calculatedfrom absorption at 232 and 270nm, respectively, with aUV spectrophotometer (Secoman, Anthelie 70 MI0291, No. 344, France) using a 1% solution of oil incyclohexane and a path length of 1cm.

Chlorophyll content was determined by spectropho-tometry following the methodology described byMinguez et al. (1991).

Total phenols, expressed as gallic acid (mg/kg of oil)were determined colorimetrically at 725 nm usingFolin-Ciocalteau reagent as previously done by Gutfin-ger (1981) on virgin olive oil.

Viscosity was measured with a Stress Tech Rheolog-ica Rheometer (Rheologica Instruments AB, Lund,Sweden). Measurements were performed at 25°C witha steel cene-plate (C40/4) under a constant shear rateof lOO/s.

Absorbance of oil solutions in hexane was measuredwith a UV-240 spectrophotometer (Shimadzu-Corpo-ration, Kyoto, Japan).

The CieLab coordinates (L *, a*, b*) were measuredwith a spectrophotometer MS/Y-2500 (Hunterlab, Inc.,Reston, VA, USA), calibrated with a white tile (Besbeset al., 2004a). Absorbance of oil solutions in hexane wasmeasured with a spectrophotometer UV -240 (ShimadzuCorporation, Kyoto, Japan) with light of wavelengthsbetween 205 and 800nm (Besbes et al., 2004a).

Oxidation stability was evaluated by measuring theoxidation induction time, with the use of the Rancimat679 apparatus (Metrohm AG, Herison, Switzerland).A flow of air (15 Llh) was bubbled through the oil

Composition of Aleppo Pine (Pinus halepensis Mill.) Seeds

Table 1. Chemical composition of Pinus halepensis and Pinus pinea seeds.

409

Composition Pin us pinea seeds'Pinus ha/epensis seeds (mean :!: SO, n = 3)

Ory matter (%)Oil (g/100g)

Crude protein (g/loog)Ash (g/100g)

Total carbohydrate (g/100g)

94.8:!:0.143.3:!: 0.522.7:!: 0.58.3:!:0.1

25.7:!:0.2

94.944.931.64.5

13.9

'Nergiz and Dônmez (2004).

Table 2. Minerais of Pin us halepensis seeds compared to other seeds.

Mineral content of seeds (mg/kg)

Minerais Pinus halepensis(mean :!: SO, n =3)

Pinus otnee'Xylopia aethiopica' Oeglet Nour'

PotassiumMagnesium

CalciumPhosphorus

SodiumIron

CopperZinc

Manganese

6171 :!:12.03303:!:9.81167:!:4.9568:!:0.8

69.6:!:0.1271 :!: 1.8

22.5:!: 0.1134.9:!: 0.451.3:!:0.1

8676.31266.5

13020.91840.0

68.5203.5

43.538.5

Nd

229051738868310423

NdNdNd

71303250

1385120

117102

156469

'Barminas et al. (1999); 'Nergiz and Dônmez (2004); 'Besbes et al. (2004a). Nd: not determined.

(2.5 g) heated at lOO°C and the volatile degradationproducts were trapped in distilled water, increasing thewater conductivity. The induction time was defined asthe time necessary to reach the inflection point of theconductivity curve (Halbault et al., 1997).

Fatty acid composition was analysed by gas-liquidchromatography after derivatisation to fatty acidmethyl esters (FAMEs) with 2M KOH in methanol atroom temperature according to the IUPAC standardmethod (IUPAC, 1992). Analyses of FAMEs werecarried out with a Hewlett-Packard 5890 Series Il gaschromatograph (H. P. Co., Amsterdam, The Nether-lands) equipped with a hydrogen flame ionisationdetector and a capillary column: HP Inovax cross-linked PEG (30 m X 0.32 mm X 0.25 J-Lm film). Thecolumn temperature was programmed from 180 to240°C at 5°C/min and the injector and detector tem-peratures were set at 250°C. Identification and quan-tification of FAMEs was accomplished by comparingthe retenti on times of peaks with those of pure stand-ards purchased from Sigma and analysed under thesame conditions. The results were expressed as a per-centage of individu al fatty acids in the lipid fraction.

Thermal characteristics of Pinus halepensis seed oilwas performed using a modulated differential scanningcalorimeter (OSC 2920 Modulated OSC-TA Instru-ments, Newcastle. OE. USA). Oil samples (2±0.lOmg)were weighed directly in a OSC-pan (SFl-Aluminium,TA Instrument Tll024). The samples were quicklycooled to - 50°C with a speed of 15°C/min, maintainedal this temperature for 15min and heated to 90°C with

a heating speed of 15°C/min. The same operation(cooling and heating) was repeated and the OSC ther-mographs were recorded during the second melting.An empty OSC-pan was used as reference. The instru-ment was calibrated for temperature and heat flowusing eicosane (Tp = 36.80°C, H = 247.70J/g) anddodecane (Tp = -9.65 "C, H = 216.73J/g). Solid fatcontent was determined from the OSC melting curveaccording to Oeroanne (1977).

RESULTS AND DISCUSSION

Chemical Composition of Aleppo Pi ne Seeds

The average composition of Pinus halepensis seeds isshown in Table 1. The moisture content and the amountof oil were 5.15% and 3.34%, respectively. Quite similarresults were reported by Nergiz and Dënmez (2004) forPinus pinea seed oil. Wolff and Bayard (1995) reportedthat the oil content of sorne varieties of pine seedsvaried from 31 to 68%, however Nasri and Triki (2004)found that Tunisian Pinus halepensis Mill. contained34.63%, an amount lower than our results for the samespecies. ln general, pine seeds are rich in oils, theircontent varies due to differences in species and environ-mental factors (Nergiz and Dënmez, 2004). The averageprotein content of the seeds (22.66%) was lower thanthat reported for Pinus pinea by those authors.

Aleppo pine seeds minerai profile was compared tothose of other seeds such as Pinus pinea, Xylopia

410 S. CHEIKH-RoUHOU ET AL.

aethiopica and Phoenix dactylifera L. (Table 2). Pinushalepensis seeds contained significant amounts ofimportant minerai elements (Table 2, macro and microconstituents). Potassium was the most abundantelement in the Pinus halepensis seeds, followed bymagnesium and calcium. The other elements, indescending order by quantity, were p, Fe, Zn, Na, Mnand Cu. Pinus halepensis seeds seemed to bave loweramounts of phosphorus and sodium tban thosereported for Deglet Nour and Pinus pinea seeds, alsocalcium and copper were lower th an those for Xylopiaaethiopica, while magnesium, iron and zinc weredetected to be the highest, compared with thosereported for ail the oil seeds studied. Pinus halepensisseeds provide appreciable amounts of microelements(Mn, Zn, Cu and Fe) from a nutritional point of view.Tbey could significantly con tribu te to the daily require-ments of ail the elements studied.

Profile of Aleppo Pine Seed Oil

As previously studied, the lipid fraction was foundto be the most abundant in Pinus halepensis seeds.Consequently, we were interested in determining fattyacid composition, and thermal and physical profiles ofAleppo pine seed oil.

Fatty Acid Composition

Essential fatty acids and their long-chain derivativesare important structural elements of cel! membranesand are essential for the formation of new tissues(McKevith,2005).

Fatty acid composition of Pinus halepensis seed oil(Table 3) showed that linoleic and oleic acids accountfor more than 76% of the total fatty acids. Pinushalepensis seed ail is rich in unsaturated fatty acids.This result was in accordance with those reported forPinus pinea seed ail (Nergiz and Dônmez, 2004; Nasriet al., 2005). Previous studies showed that the oils ofPin us varieties contained oleic and linoleic acids at rel-atively high levels (Sagrero-Nieves, 1992; Wolff andMarpeau, 1997) in agreement with our results. ln thisstudy, saturated fatty acids (SAFA) accounted for16.8% of total fatty acids. Among them, the main satu-rated acids were palmitic, stearic and behenic, withminute amounts of arachidic, lignoceric, myristic andmargaric. The high polyunsaturated fatty acids(PUFA) ta SAFA ratio of 3: 1 is attributed ta a highlevel of PUF A. This is a health benefit, as consumptionof diets high in PUF A have been shawn ta have aninverse correlation to coronary heart disease (Thomp-son et al., 1993). Therefore, the consumption of theseseeds in sufficient amounts could con tribu te positivelyta human health.

The w6/w3 ratio is very important in terms of humannutrition. An estimate of the ratio in our diet 100 yearsaga was between 3:1 and 5:1 (Guilliams, 2000) and forthe ideal daily consumption varies from 2:1 ta 4:1(Eramus, 1993). Pinus halepensis seed oil showed alower w6/w3 ratio compared to Pinus pinea seed ail(-10:1 as against -70:1). This ratio is very variable inusual vegetable oils used in human food, such as oliveail, sunflower ail, soybean oil, palm oil and corn ail(13:1; 199:1; 7:1; 45:1 and 82:1, respectively; Guilliams,2000).

Fatty Acid (FA)

Table 3. Fatty acid composition of Pin us halepensis seed oil.*

Pinus pinea seed oil'Pinus halepensis seed oil(g/100g of total FA, mean ± SO, n = 3)

Myristic C,,,oMyristoleic C14,

Palmitic C'6'0

Palmitoleic C'6"

Margaric C17•0

Margaroleic C17"

Stearic C, s.oOleic C, e.,

Linoleic C,.,Linolenic C, ••3Arachidic C200

Eicosenoic C,o,Eicosadienoic C20.,

Eicosatrienoic C203

8ehenic C'2'O

Lignoceric C24•0

SAFAMUFAPUFA

0.09±0.020.08±0.018.75±0.510.22±0.110.08±0.01

Tr4.32±0.1827.3 ± 1.748.8± 2.44.86±0.360.62±0.010.83±0.060.42 ± 0.050.49 ±0.032.89 ± 0.230.18±0.1116.8± 1.328.4 ± 2.354.2 ± 3.4

0.05 ± 0.004Nd

6.49±0.0780.224 ± 0.03

TrNd

3.47 ±0.138.6±0.647.6±0.30.68±0.0010.54 ± 0.030.79±0.06

NdNd

0.13±0.013.02 ± 0.0213.7±0.239.6±0.748.3±0.3

'Nergiz and D6nmez (2004). SAFA: saturated fatty acids; MUFA: monounsaturated fatty acids; PUFA: polyunsaturated fatty acids; Tr: trace (,;;0.05%); Nd: Notdetected.

Composition ofAleppo Pine (Pinus halepensis Mill.) Seeds 411

melting points. Pinus halepensis seed oil exhibited amelting peak (-29.37 0C), a melting enthalpy (52.5J/g)and an onset temperature (-42.3 °C, Table 4), valueslower than those reported by Besbes et al. (2004a) fordate seed oils. This was due to the higher PUFA inAleppo pine seed oil (54.2 as against 14.0 for Aleppopine and Deglet Nour seed oils, respectively).

Solid fat content decreased when temperatureincreased (Figure 2) and remained constant for tem-peratures above -8°C, that corresponded to the entireliquefaction of Aleppo pine seed oil. Liquefation ofDeglet Nour seed oil (Besbes et al., 2004a) began at15°C. This can be explained by the fact that DegletNour seed oil had a higher SAFA content than theAleppo pine seed oil (44.3 as against 16.8, respec-tively).

Thermal Properties

DifferentiaI scanning calorimetry (DSC) is a fastand direct way to assess the quality of oil (Gloria andAguilera, 1998). Any endothermic or exothermic eventis registered as a peak in the chart, and its area is pro-portional to the enthalpy gained or lost, respectively.Pinus halepensis seed oil exhibited a thermogram withthree thermal structural transitions between - 39.83and -12.05°C, indicative of crystalline melting (Figure1). The obtained peaks were asymmetric and may indi-cate the presence of three components, having differ-ent weights. The first small endothermic peak at- 39.83 °C may represent the melting of the unstable (ï

crystal form, followed by the crystallisation of theunstable W form and the last one may correspond tothe more stable ~ form. This hypothesis should be sup-ported later by the X-ray diffraction metbod to identifythe crystalline forms. The fact that thermogramsseemed to correspond to a number of compoundshigher than those clearly shown, suggests the presenceof mixed glyceride groups with different melting pointsin the used conditions (Herrera and Anon, 1991).After total solidification, mixed glyceride crystalsshould be formed by intersolubility. They are associ-ated in different crystalline groups with different

0.0

-0.5

-2.0 +---~~-~~~-~~-~~~-~è-60 -40 -20 0 20 40 60

Temperature (OC)

Figure 1. Melting thermogram of Aleppo pine seedoil.

Table 4. Thermal parameters, from ose meltingcurves, of Pin us ha/epensis seed oil.

Parameter Pinus halepensis(mean ± 3D, n =3)

Peak transition temperatures (0G)T1T2T3

-39.83 ± 0.18-29.37±0.18-23.36 ± 0.31

Onset temperature (OC)Peak temperature (0G)

Total melting enthalpy (J!g)

-42.3± 1.3-29.37±0.18

52.5±3.3

Colour

Aleppo pine seed oil showed lower L *, a* and b*values than those reported for Deglet Nour seed oil(57.31, -1.82 and 30 as against 66, -0.4 and 55, forAleppo pine and Deglet Nour seed oils, respectively;Besbes et al., 2004a). This means that Pinus halepensisseed oil was slightly darker th an Deglet Nour seed oil.Such a colour seems to attract consurners (Hsu andChung, 1998). Tbe CieLab (L*, a*, b*) values of othervegetable oils, such as palm, soybean, sunfiower, oliveand corn ranged from, 63.4 to 69.5, 3.8 to 4.4 and 9.2 to10.4, respectively (Hsu and Yu, 2002). Pinus halepensisseed oil b* value was higher than those of other veg-etable ails. Pin us halepensis seed oil was more yellow-coloured than vegetable oils studied by Hsu and Yu(2002). This may suggest the presence of more yellowpigments (carotenoids) in Pinus halepensis seed oil.Aleppo pine seed oil showed another colour particular-ity: Hunter a* negative value (-1.82) was markedlylower than the Hunter a* value of common vegetableoils.

Pinus halepensis seed oil showed some absorbancein the UV-C (100-290nm) with a maximum of 2.39units at 210nm for oil diluted to 1:800, in the UV-B

100

80

~ 60<n:2 400CI)

20

0-40 -20

Temperature ("C)

-20 o-30

Figure 2. Solid-temperature curve for Aleppo pineseed oil determined by ose.

412 S. CHEIKH-RoUHOU ET AL.

(290-320nm) ranging from 0.3 to 0.1 for oil diluted to1:100. and UV-A (320-400nm) ranging from 0.1 to 0.02for oil diluted to 1:100. Therefore, Aleppo pine seed oilmay provide protection against both UV-A (an originof oxidative stress to skin) and UV-B. The opticaltransmission of Pinus halepensis seed oil, especially inthe UV range (290-400nm) was comparable to that ofdate seed and raspberry seed oils. This is comparableto titanium dioxide preparations as a sun protectionfactor for UV -B (SPF) and a protection factor for UV-A (PFA) (Besbes et al., 2004a; Oomah et al., 2000).Pinus halepensis seed oil also contained more yellowcolouring than raspberry seed oil studied by Oomah etal. (2000). The latter is characterised by a lowerabsorbance than Aleppo pine seed oil (absorbanceranging from 0.08 to 0.11 against 0.17 to 0.19 at440-460 nrn, respectively, for oils diluted to 1:10). Thisconfirmed the results of colour measurements. Yellowcolours, due parti y to carotenoids, are beneficial, sincethey simulate the appearance of butter without the useof primary colorants, such as carotenes and annatos,commonly used in the oil and fat industry (Oomah etal., 2000).

Oxidative Stability

Oxidation is the main problem affecting oils, leadingto aldehyde production which imparts strong disagree-able flavours and odours, referred to as rancidity.Generally, the rate of oxidation will de pend on thedegree of unsaturation of the oil and its temperature asweil as the presence of antioxidants (McKevith. 2005).Oils deteriorate after an induction period in which rel-atively small changes occur (Gordon, 1991). Stability,although is not a standard parameter of qua lity, isuseful to provide information about the hypotheticalshelf-life of an oil (Aparicio et al., 1999).

Stability, expressed as the oxidation induction time,was about 24.5 h for Pinus halepensis seed oil (Table 5).This oxidation time is lower than that reported byBesbes et al. (2004a) for date seed oil (Deglet Nourcultivar). Such difference may be explained by the factthat Pinus halepensis seed oil contained more PUFAthan date seed oil (Deglet Nour cultivar) (54.2 asagainst 14, respectively). The latter also has a higherantioxidant content (phenolic compounds) (526 asagainst 186 f.lg/g for date seed and Pinus halepensisseed oils, respectively, Table 5) which mainly de ter-mines a greater resistance to auto-oxidation (Perrin,1992: Tsirnidou et al., 1992; Baldioli et al., 1996). Phe-nolics in vegetable oils may generally exert a beneficialeffect on their oxidative stability (Farag et al., 2003).Aparicio et al. (1999) mentioned a significant correla-tion of phenols, oleicllinoleic ratio and tocopherolswith oil stability, and it was also observed betweentotal phenol content and oxidative stability by Ranci-mat (Gurfinger, 1981; Salvador et al., 2001). Phenolic

Table 5. Oxidation induction time and viscosity ofseed oil from Pin us ha/epensis and Deglet Nour seed

oils.

Pinus halepensis(mean ± SD, n =3)

Deglet Nour'

Induction time (h)Viscosity (mPa.s)

44.818.3

24.4± 2.57.17±O.05

'Besbes et al. (2004a).

compounds would contribute around 50% to thestability of olive oil, while the oleic/linoleic ratio wouldreach only 27%. The contribution of total tocopherolsand colouring pigments (chlorophylls and carotenoids)were around 9 and 13%, respectively (Aparicio et al.,1999). The oxidative stability of other vegetable oils,such as canola, coconut, corn, grapeseed, olive, pean ut,safflower, soybean and sunflower ranged from 6.1.11.25,4.75,2.07,4.85,5.07,2.27.2.22 and 3.25h, respec-tively at 110°C (Tan et al., 2002). This shows that Pinushalepensis seed oil oxidative stability was higher thanthat of most oleic-linoleic vegetable oils.

Viscosity

Viscosity of Pinus halepensis seed oil (Table 5) waslower than that reported by Besbes et al. (2004a) fordate seed oil (Deglet Nour variety) (7.17mPa.s asagainst 18.3mPa.s), which may be explained by the factthat the latter contained less UFA (65.45 as against82.6, for Deglet Nour and Pinus halepensis seed oils,respectively). It is worth noting that the viscosity ofPinus halepensis seed oil is lower than that of most veg-etable oils (26mPa.s, 47.3mPa.s and 49.4mPa.s, forraspberry, safflower and grape seed oils, respectively(Oomah et al., 2000». The viscosity of olive oilreported by Lalas and Tsakins (2002) varies from 45 to80mPa.s.

Sorne Quality Pararneters

Pinus halepensis seed oil has a relatively higherphenol content (Table 6) compared to most edible oilsexcept for olive oil, which is considered as a rich sourceof phenolic compounds in the Mediterranean diet. Sal-vador et al. (2001) reported that total phenolic contentmeasured by the Folin-Ciocalteau method rangedfrom 19 to 380mg/kg, and from 124 to 516mg/kgaccording to Nissiotis and Tasioula-Margari (2002).The amount of phenols in crude seed oils is an import-ant factor when evaluating the quality of the oitbecause these compounds have been correlated withcolour, the shelf-life of oit and, in particular, its resis-tance to oxidation (Cinquanta et al., 1997). Pinushalepensis seed oil could be considered as a potentialsource of natural phenolic compounds. Despite their

Composition of Aleppo Pine (Pinus halepensis Mill.) Seeds

Table 6. Physicochemical characterisation of Pinushalepensis seed oil.

Parameter Pinus haJepensis(mean ± SD, n = 3)

Polyphenols (as mg gallic acid/kg of oil)Chlorophyll (mg/kg)

Refractive index (at 20°C)Acidity (% oleic acid)

Saponification index (mg of KOH/g of oil)lodine index (g of IJ100g of oil)

Peroxide index (meq 02/kg of oil)E232

E270

186± 190.07 ±0.06

1.4750±0.01000.61 ±0.08190±13117± 10

3.18±0.050.51 ±0.01

1.5±0.31

participation in conferring specifie ftavour to oil(Caponio et al., 1999), dietary phenolic compoundsmight have a positive effect in the prevention of coro-nary heart disease and cancer (Owen et al., 2000; Tuckand Hayball, 2002) a protective and a therapeuticpotential in oxidative damage-related pathologies(Auger et al., 2004).

The chlorophyll pigments (Table 6) are importantquality parameters because they correlate with colour,which is a basic attribute for evaluating oil quality (Sal-vador et al., 2001). These pigments are involved inauto-oxidation and photo-oxidation mechanisms(Gutierrez et al., 1992; Mfnguez-Mosquera et al., 1990).Interesse et al. (1971) reported that the oxidativestability of olive oil is greatly affected by the presenceof chlorophylls and their derivatives, especially in thepresence of light as they have the ability to transferenergy from light into chemical molecules. The chloro-phyl! content of Pinus halepensis seed oil is lower thanthat reported by Aparicio et al. (1999) for virgin oliveoil (0.07 as against 9.32-12.67I-lg/g, respectively).

Refractive index of Aleppo pine seed oil (1.4750)was similar to that of olive oils studied by Lalas andTsakins (2002) and date seed oils studied by Besbes etal. (2004b). The saponification value of 190 is compara-ble to Xylopia aethiopica oil (Barminas et al., 1999).The lower acidity of Pinus halepensis seed oil showedthat it might be edible and could have a long shelf-life.It is known that the increase of free acidity is mainlydue to enzyme activity (Boskow, 1996). The iodineindex of 117 indicates that Pinus halepensis seed oil is ahighly unsaturated oil and suggests that it con tains highlevels of oleic and linoleic acids, as previously shown inTable 3. The iodine value of Pinus halepensis seed oil issituated inside the interval range of the values men-tioned by Tan et al. (2002) in sorne edible oils (9.37 tolA5 g of 1/100 g oil), It provides a measure of degree ofoil unsaturation and a means of predicting shelf-life.Lower iodine value me ans a more stable oil with alonger shelf-life. Peroxide value (3.18meq Oikg of oil)of Pin us halepensis seed oil is lower than that of canola(6.68meq O/kg of oil), grapeseed (10.6 meq/kg), olive

413

(8.5meq/kg), peanut (6.82meqlkg), safflower(5.07meq/kg) and sunftower (9.1meq/kg) oils (Tan et al.,2002). Salvador et al. (2001) reported that less than 5%of the oils had a peroxide value higher than the upperlimit of 20 established for the 'extra-virgin' olive oil

Aleppo pine seed oil showed low absorptivity valuesat 232 and 270nm, thus containing little amounts ofprimary (hydroperoxides) and secondary oxidationproducts. Compared to Nigella seed oil studied byRamadan and Môrsel (2004), absorptivities at 232 and270nm of Pinus halepensis seed oil were relativelylower (0.51 and 1.5 as against 3.6 and 1.2, respectively).

The results revealed that Aleppo pine (Pinushalepensisï seeds are a rich source of many importantnutrients that appear to have a very positive effect onhuman health. The seed lipids of the investigatedsamples were rich in linoleic acid, which has a benefi-cial effect on blood lipids, lowering blood pressure andserum cholesterol. They constitute a good alternativesource of essential fatty acids compared with comrnonvegetable oils and could contribute to the overalldietary intake of the minerai elements studied. Thisunique fatty acid composition, relatively high polyphe-nol content and quality, and hence high protectionagainst oxidative stress, relatively good shelf-life andother desirable physicochemical characteristics lead tomore diverse and novel applications of Aleppo pineseed oil in the food, pharmaceutical, cosmetic andother non-food industries.

ACKNOWlEDGEMENT

The authors thank ML Mohamed Hammami for hisvaluable help in spectrometrie and chromatographieanalysis.

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