Original article Fatty acid composition and biological activities of volatiles from fruits of two Tunisian olive cultivars Faten Brahmi, 1 Samia Dabbou, 1 Guido Flamini, 2 Hayet Edziri, 3 Maha Mastouri 3 & Mohamed Hammami 1 * 1 Laboratoire de Biochimie UR ‘Nutrition Humaine et De´sordres Me´taboliques’ Faculte´ de Me´decine, Rue Avicenne, 5019 Monastir, Tunisie 2 Dipartimento di Chimica Bioorganica e Biofarmacia via Bonanno 33 56126 Pisa, Italy 3 Laboratory of Microbiology, CHU, F. Bourguiba, Monastir, Tunisia 5000, Tunisia (Received 22 September 2010; Accepted in revised form 17 February 2011) Summary This study investigates volatile compounds, fatty acid composition and antioxidant, antibacterial and antifungal activities of fruits from two olive cultivars (Chemlali and Neb Jmel). Fatty acid profiles varied significantly between cultivars (cvs) where Neb Jmel seem to have higher contents of palmitic and oleic acids (16.4% and 66.4%, respectively) and lower of linoleic acid (9.4%). The volatile profile indicated the apparent difference between cvs. In fact, the main components detected in Chemlali cv. were (E,E)-2,4-decadienal (23.0%), (E,Z)-2,4-decadienal (14.9%) and nonanal (6.7%), while 3-ethenylpyridine (15.5%), (E)-2-decenal (14.4%) and (E)-2-undecenal (7.0%) were the major components in Neb Jmel cv. Furthermore, these volatiles were subjected to screening for their possible antioxidant activities where volatiles from Neb Jmel fruits were found to be better. Results presented here may suggest that the volatiles fraction of two cultivars possess antimicrobial and antifungal activities. Keywords Antibacterial, antifungal, antioxidant, fruits, fatty acids, Olea europaea L., volatile compounds. Introduction Aromatic plants are frequently used in traditional med- icine as possessing biological activities. Chief amongst these are their antimicrobial, antifungal and antioxidant properties (Burits et al., 2001; Lee et al., 2003). With a growing interest in the use of mixtures of natural volatile compounds, isolated by steam distillation, in both the food and the pharmaceutical industries, a systematic examination of plant extracts for these properties has become increasingly important (El-Ghorabl et al., 1999). Olea europaea L. (Oleaceae) has been widely used since antiquity in folk medicine in European Mediterranean islands and countries such as Spain, Italy, France, Greece, Morocco, Turkey and Tunisia. In fact, many references have cited the medicinal use of this plant since ancient times (Hansen et al., 1996). Olive tree, belonging to the family of Oleaceae, is one of the most important fruit trees in Mediterranean countries, where it cover about eight million ha, accounting for almost 98% of the world crop. This demonstrates the great economic and social impor- tance of this crop and the possible benefits derived from the utilisation of any of it’s by products (Tabera et al., 2004). Its fruit is a drupe similar to that of a cherry, peach, or apricot. The stone fruit is the raw material for the production of olive oil and table olives. Volatile compounds are low molecular weight com- pounds (< 300 Da) which vapourise readily at room temperature. Some volatile compounds reach the olfac- tory epithelium, dissolve into the mucus and may bond with olfactory receptors to give an odour sensation (Angerosa, 2002). Volatile compounds are not produced in significant amounts during fruit growth but arise during the climacteric stage of ripening. During the climacteric period fruits produce ethylene, inducing biochemical, physical and chemical changes and an increase in some protein and enzyme activities (Kalua et al., 2007). In olives, the climacteric phase corresponds to a period when oil extracted from drupes gives an elevated oil quality that is rich in aromatic volatile compounds (Ranalli et al., 1998). Although, in the literature, a lot is known about aroma compounds in olive oil, very little is known about the quali-quantitative composition of volatile compounds in olives, whose matrix is pro- foundly different from that of olive oil. The aroma of virgin olive oil results from a complex mixture of volatile compounds that can be analysed and quantified by gas chromatography-mass spectrometry (Morales et al., 1995). Among such compounds, six carbon aldehydes (hexanal, 3(Z)-hexenal and 2(E) hexe- nal), alcohols (hexanol, 3(Z)-hexenol and 2(E)-hexenol), and their acetyl esters (hexyl acetate and 3(Z)-hexenyl acetate), make up to 80% of total volatile compounds in all the different oils (Morales et al., 1995; Dabbou et al., *Correspondent: Fax: +216 73 460 737; e-mail: [email protected]International Journal of Food Science and Technology 2011, 46, 1316–1322 1316 doi:10.1111/j.1365-2621.2011.02616.x Ó 2011 The Authors. International Journal of Food Science and Technology Ó 2011 Institute of Food Science and Technology
Transcript
Original article
Fatty acid composition and biological activities of volatiles from
Aromatic plants are frequently used in traditional med-icine as possessing biological activities. Chief amongstthese are their antimicrobial, antifungal and antioxidantproperties (Burits et al., 2001; Lee et al., 2003). With agrowing interest in the use of mixtures of natural volatilecompounds, isolated by steam distillation, in both thefood and the pharmaceutical industries, a systematicexamination of plant extracts for these properties hasbecome increasingly important (El-Ghorabl et al., 1999).Olea europaea L. (Oleaceae) has been widely used since
antiquity in folk medicine in European Mediterraneanislands and countries such as Spain, Italy, France,Greece,Morocco, Turkey and Tunisia. In fact, many referenceshave cited the medicinal use of this plant since ancienttimes (Hansen et al., 1996). Olive tree, belonging to thefamily ofOleaceae, is one of themost important fruit treesin Mediterranean countries, where it cover about eightmillion ha, accounting for almost 98% of the world crop.This demonstrates the great economic and social impor-tance of this crop and the possible benefits derived fromthe utilisation of any of it’s by products (Tabera et al.,2004). Its fruit is a drupe similar to that of a cherry, peach,or apricot. The stone fruit is the raw material for theproduction of olive oil and table olives.
Volatile compounds are low molecular weight com-pounds (< 300 Da) which vapourise readily at roomtemperature. Some volatile compounds reach the olfac-tory epithelium, dissolve into the mucus and may bondwith olfactory receptors to give an odour sensation(Angerosa, 2002).Volatile compounds are not produced in significant
amounts during fruit growth but arise during theclimacteric stage of ripening. During the climactericperiod fruits produce ethylene, inducing biochemical,physical and chemical changes and an increase in someprotein and enzyme activities (Kalua et al., 2007). Inolives, the climacteric phase corresponds to a periodwhen oil extracted from drupes gives an elevated oilquality that is rich in aromatic volatile compounds(Ranalli et al., 1998). Although, in the literature, a lot isknown about aroma compounds in olive oil, very little isknown about the quali-quantitative composition ofvolatile compounds in olives, whose matrix is pro-foundly different from that of olive oil.The aroma of virgin olive oil results from a complex
mixture of volatile compounds that can be analysed andquantified by gas chromatography-mass spectrometry(Morales et al., 1995). Among such compounds, sixcarbon aldehydes (hexanal, 3(Z)-hexenal and 2(E) hexe-nal), alcohols (hexanol, 3(Z)-hexenol and 2(E)-hexenol),and their acetyl esters (hexyl acetate and 3(Z)-hexenylacetate), make up to 80% of total volatile compounds inall the different oils (Morales et al., 1995; Dabbou et al.,
International Journal of Food Science and Technology 2011, 46, 1316–13221316
doi:10.1111/j.1365-2621.2011.02616.x
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2009; Issaoui et al., 2010). Evidence indicates that, inolive fruits, the C5–C6 compounds, which are alsoconstituents of the aroma of many fruits, vegetables andtheir products, are produced mainly from polyunsatu-rated fatty acids through a cascade of biochemicalreactions commonly known as the lipoxygenase (LOX)pathway (Angerosa et al., 1999; Sanchez & Salas, 2000),whereas the formation of higher molecular weight com-ponents occurs through chemical pathways (Frankel,1985). LOX activity is high at early stages of olive fruitdevelopment. This phenomenon suggests that in olivetrees, as in other plants, the LOX enzyme is important inthe physiological response to stress. LOX activitydecreases during fruit ripening and this phenomenoncould explain, at least in part, the lower amounts of C6volatiles present in oils prepared from olives harvested atadvanced stages of maturation (Salas et al., 1999).In Tunisia, olive culture is one of the most important
agricultural activities. Olive plantations count about 57million trees, where the Chemlali olive is the mostwidespread and dominant olive cultivar (Issaoui et al.,2007; Dabbou et al., 2009).This paper intends to first characterise the volatile
profiles of two Tunisian varieties of Olea europaea(Chemlali and Neb Jmel cultivars); secondly, to deter-mine the fatty acid composition of the studied freshfruits; finally, to evidence evaluate their antioxidant,antibacterial and antifungal activities.
Materials and methods
Plant material
Fresh fruits of two Tunisian cultivars (Chemlali and NebJmel) ofOlea europaeaL.were collected at fullmaturity ofthe season 2008–2009 from the coastal region ofMahdia –the centre of Tunisia. Voucher specimens have beendeposited in the Herbarium of the Laboratory ofBiochemistry, Faculty of Medicine of Monastir, Tunisia.
Analysis of fatty acids
Triplicate sub-samples of 0.5 g were extracted using themodified method of Bligh & Dyer (1959). Thus, fruitsamples were kept in boiling water for 10 min toinactivate lipase (Douce, 1964) and then ground man-ually using a mortar and pestle. A chloroform ⁄methanol(Analytical Reagent, LabScan, Ltd, Dublin, Ireland)mixture (1:1, v ⁄v) was used for total lipid extraction.After washing with water and centrifugation at 3000 gfor 10 min, the organic layer containing total lipids wasrecovered and dried under a nitrogen stream. Total fattyacids (TFA) were methylated by using sodium methox-ide solution (Sigma, Aldrich, St. Louis, MO, USA)according to the method of Cecchi et al. (1985). Methylheptadecanoate (C17:0) was used as an internal stan-
dard. Those fatty acids methyl esters (FAMEs) obtainedwere subsequently analysed.
Extraction of volatiles and GC–MS analysis
Each sample (500 g) of the fresh fruits was subjected tohydrodistillation for 3 h using a Clevenger-type appa-ratus (Clevenger, 1928). The volatiles obtained aftertrapping in diethyl ether were dried over anhydroussodium sulphate, evaporated and concentrated under agentle stream of N2 and stored at 4 �C until analysis.GC analyses were accomplished with an HP-5890
series II instrument equipped with HP-Wax and HP-5capillary columns (30 m · 0.25 mm, 0.25 lm film thick-ness), working with the following temperature program:60 �C for 10 min, ramp of 5 �C min)1 to 220 �C;injector and detector temperatures, 250 �C; carrier gas,nitrogen (2 mL min)1); detector, dual FID; split ratio,1:30; injection, 0.5 lL. The identification of the compo-nents was performed, for both columns, by comparisonof their retention times with those of pure authenticsamples and by means of their linear retention indices(LRI) relative to the series of n-hydrocarbons.Gas chromatography-electron impact mass spectrom-
etry analyses were performed with a Varian CP 3800 gaschromatograph (Varain, Inc., Palo Alto, CA, USA)equipped with a DB-5 capillary column (Agilent Tech-nologies Hewlett-Packard, Waldbronn, Germany;30 m · 0.25 mm; coating thickness · 0.25 lm) and aVarian Saturn 2000 ion trap mass detector.
Antioxidant activity
DPPH radical scavenging assay
Radical scavenging activity was determined by a spec-trophotometric method based on the reduction of amethanol solution of DPPH as reported by Blois (1958).One millilitre of various concentrations of the volatilesin methanol was added to 1 mL of a 0.004% methanolsolution of DPPH. The mixture was shaken vigorouslyand left to stand at room temperature for 30 min in thedark. Then, the absorbance was measured with a UV–vis spectrophotometer (Secommam, U-1789, France), at517 nm against a blank.Concentration providing 50% inhibition (IC50) was
calculated from the graph plotting inhibition percentageagainst sample concentration. Butylated hydroxytoluene(BHT) was used as positive control.
ABTS+ radical cation scavenging
The ABTS+ radical cation scavenging activity of eachvolatile fraction and ascorbic acid (control) was deter-mined according to the literature (Yvonne et al., 2005).In brief, 5.0 mL of a 7.0 mm ABTS solution was
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overnight in the dark treated with 88.0 lL of a 140 mm
potassium persulfate solution to yield the ABTS+ radicalcation. Prior to use in the assay, the ABTS+ radicalcation was diluted with ethanol for an initial absorbanceof about 0.700 (ratio of 1:88) at 734 nm, with 30 �C. Theantioxidant capacity of volatile fraction was expressed asIC50, the concentration necessary for 50% reduction ofABTS+.
Antibacterial and antifungal activity
Microbial strainsThe employed bacterial strains were Enterococcus fae-calis ATCC 29212, Staphylococcus aureus ATCC 27950,Escherichia coli ATCC 25922 and Pseudomonas aeru-ginosa ATCC 27950. In the case of yeasts, Candidaglabrata ATCC 90030, C. kreusei ATCC 6258, C.parapsilosis ATCC 22019 and C. albicans ATCC 90028strains were used.
Micro-well dilution assayMinimum inhibitory concentration (MIC) values weredetermined by micro-titre plate dilution method (Sahinet al., 2004). The inocula of the bacteria and yeasts wereprepared from 12 h broth cultures and suspensions wereadjusted to 0.5 McFarland standard turbidity. Thevolatile fractions were first dissolved in 10% DMSO andthen diluted to the highest concentration (10 mg mL)1)to be tested, and then serial twofold dilutions were madein 10 mL sterile test tubes containing nutrient broth.Results were expressed in microgram per millilitre
(Zacchino, 2001; Smania et al., 2006).
Statistical analysis
All parameters analysed were carried out in triplicate.The results are reported as mean values of threerepetitions and standard deviation. Significant differ-ences among varieties studied were determined byTukey’s test b (P < 0.001), using the SPSS program,release 11.0 for Windows (SPSS, Chicago, IL, USA).
Results and discussion
Fatty acid composition
The distribution of fatty acids, from the two olivecultivars studied, is presented in Table 1. Resultsshowed significant statistical variations (P = 0.01) inthe saturated and polyunsaturated fatty acids levels butthese variations were variety dependant since cultivarsare planted in the same pedoclimatic conditions andharvested at the same date. These findings are inaccordance with previous works showing that the fattyacid composition is one of the characteristics predom-inantly genetically determined, with a lesser impact of
the environmental factors (Hrncirik & Fritsche, 2005).For the two studied cultivars, palmitic acid content washigh (> 15%). Oleic acid, the main monounsaturatedfatty acid in olives showed higher content in Neb Jmelolives (66.4%) than Chemlali (61.5%). Monounsatu-rated fatty acids are very important for the final productstability and for their nutritional properties where NebJmel olives were more abundant (70%). On the con-trary, the higher content in polyunsarurated fatty acidswas found in Chemlali olives (16.45%) which proved thehigher O ⁄L ratio in Neb Jmel olives (8.22%).
Volatile compounds
The volatile composition ofO. europaea L. Chemlali andNeb Jmel fruits are reported in Table 2 and Fig. 1. Infact, fifty-six compounds were identified by GC ⁄MSanalysis, representing approximately the same totalvolatile compounds content (92% of the total peakarea). Both volatile fractions were characterised by a veryhigh content of oxygenated non terpene derivatives with75.3% to 59.8% of the total peak areas for Chemlali andNeb Jmel fruits, respectively (Fig. 1). Furthermore, thepercentage of total Monoterpene hydrocarbons, Oxy-genated monoterpenes, Sesquiterpene hydrocarbons,Oxygenated monoterpenes, Phenylpropanoids, Non-ter-pene hydrocarbons, Oxygenated non terpene derivatives,
Table 1 Fatty acid composition (%) evaluated in the fruits samples
from the two olive cultivars grown in the area studied
Chemlali Neb Jmel
Palmitic acid 15.26 ± 0.40 16.39 ± 0.16*
Margaric acid 0.16 ± 0.02 0.07 ± 0.00*
Stearic acid 2.27 ± 0.02 1.80 ± 0.01**
Arachidic acid 0.86 ± 0.47 0.88 ± 0.69
Behenic acid 0.58 ± 0.55 0.14 ± 0.03
Lignoceric acid 0.08 ± 0.01 0.28 ± 0.01**
SFA 19.20 ± 0.56 19.55 ± 0.51
Palmitoleic acid 2.15 ± 0.20 3.03 ± 0.05**
Margaroleic acid 0.23 ± 0.08 0.15 ± 0.07
Oleic acid 61.44 ± 0.36 66.37 ± 0.59**
Gadoleic acid 0.46 ± 0.18 0.50 ± 0.30
Erucic acid 0.03 ± 0.00 0.19 ± 0.07*
MUFA 64.30 ± 0.45 70.09 ± 0.49**
Linoleic acid 15.61 ± 0.14 9.39 ± 0.08**
Linolenic acid 0.84 ± 0.03 0.80 ± 0.01
PUFA 16.45 ± 0.11 10.19 ± 0.09**
O ⁄ L ratio 3.94 ± 0.01 8.22 ± 2.01*
Values are the means of the three different VOO samples
polyunsaturated fatty acids; O ⁄ L, Oleic ⁄ Linoleic ratio.
*P < 0.05; **P < 0.01.
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Nitrogen derivatives and Sulphur derivatives differedaccording to the cultivar (Fig. 1). The major compoundfamilies found in the volatile matrix were the same for thetwo studied cultivars (Oxygenated non terpene deriva-tives, Nitrogen derivatives and Non-terpene hydrocar-bons). In fact, the cultivar Chemlali was morerepresentative by (E,E)-2,4-decadienal (23%), (E,Z)-2,4-decadienal (14.9%), Nonanal (6.7%), (E)-2-decenal(4.7%) and Benzaldehyde (4%) whereas the most abun-dant components of Neb Jmel olives were 3-ethenylpyri-dine (15.5%), (E)-2-decenal (14.4%), (E)-2-undecenal(7%), (E,Z)-2,4-hexadienal (5.6%), (Z)-2-heptenal (5%),Benzaldehyde (4.5%) and (E)-2-octenal (4%).Comparing the two olive cultivars collected at the same
date and grown under the same pedoclimatic conditionsshowed a great qualitative and quantitative difference inthe volatile fruit contents. These results confirmed thateach cultivar can be distinguishable from the other asdemonstrated by aroma of many kinds of citrus fruits(Shaw, 1979) and jack (Artocarpus heterophyllus) fruits(Maia et al., 2004). Only in Chemlali fruits were 3-methylbutanol acetate, diethyl disulfide, benzyl alcohol,4-methylbenzaldehyde, camphor, 1-nonanol, methylchavicol, Dodecane, 2-ethylbenzaldehyde, cis-p-mentha-1(7), 8-dien-2-ol, nonanoic acid, (E)-anethole, 1-tride-cene, Carvacrol, Globulol and 6-methyl-5-hepten-2-oneto be found. In fact, the presence of 6-methyl-5-hepten-2-one is attributed to the activity of the microorganisms(Pseudomonas sp.) present in olives (Issaoui et al., 2009).Furfural, a-pinene, Phenol, (E)-2-undecenal and pen-
tadecane were present only in Neb Jmel olive (Table 2)which indicated that aroma compounds accumulatedifferently depending on the cultivar and that metabo-lites accumulation depends from the genetically deter-
Table 2 Volatile compoundsa (%) evaluated by GC-MS in the fruits
samples from the two olive cultivars grown in the area studied
Volatile compounds LRIb Chemlali cv. Neb Jmel cv.
Furfural 800 – 2.2
(E,Z)-2,4-hexadienal 801 2.1 5.6
(E)-3-hexenol 855 0.8 3.9
1-hexanol 868 1.6 1.5
3-methylbutanol acetate 870 0.8 –
Heptanal 904 Trc 1.1
diethyl disulfide 906 0.8 –
a–pinene 936 – 1.1
(Z)-2-heptenal 938 0.7 5.0
Benzaldehyde 957 4.0 4.5
3-ethenylpyridine 970 3.1 15.5
Phenol 977 – 2.3
3-octanone 985 – Tr
6-methyl-5-hepten-2-one 987 0.7 –
(E,Z)-2,4-heptadienal 1016 3.6 1.1
Octanal 1002 Tr Tr
(Z)-3-hexenyl acetate 1005 Tr 2.4
(E,E)-2,4-heptadienal 1012 1.4 1.2
1-hexyl acetate 1009 Tr Tr
1,8-cineole 1034 Tr –
benzyl alcohol 1036 1.7 –
3-octen-2-one 1042 – Tr
Phenylacetaldehyde 1045 1.4 2.3
(E)-2-octenal 1064 0.8 4.0
1-octanol 1069 1.1 1.8
cis-linalool oxide (furanoid) 1074 – Tr
trans-linalool oxide (furanoid) 1087 – Tr
4-methylbenzaldehyde 1093 2.0 –
Linalool 1098 0.7 1.2
Nonanal 1103 6.7 3.3
phenylethyl alcool 1111 2.5 Tr
Camphor 1147 0.7 –
(E)-2-nonenal 1165 0.8 1.0
1-nonanol 1170 0.9 –
4-terpineol 1180 Tr –
a–terpineol 1188 Tr –
methyl chavicol 1196 0.6 –
Dodecane 1200 0.6 –
Decanal 1206 Tr –
(E,E)-2,4-nonadienal 1218 Tr Tr
2-ethylbenzaldehyde 1221 2.2 –
cis-p-mentha-1(7),8-dien-2-ol 1230 0.6 –
(E)-2-decenal 1266 4.7 14.4
nonanoic acid 1278 0.8 –
(E)-anethole 1283 1.3 –
1-tridecene 1291 0.9 –
(E,Z)-2,4-decadienal 1295 14.9 1.2
Carvacrol 1298 0.8 –
4-vinylguaiacol 1316 0.8 Tr
(E,E)-2,4-decadienal 1318 23.0 3.5
(E)-2-undecenal 1349 – 7.0
(E)-b-damascenone 1384 0.7 1.8
Tetradecane 1400 0.8 1.3
Pentadecane 1500 – 2.5
Globulol 1585 0.6 –
Hexadecane 1600 – Tr
Total identified volatile compounds 92.2 92.7
aPercentages obtained by FID peak area normalisation (HP-5 column).bLRI (DB-5 column).cTr < 0.1%.
0
10
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40
50
60
70
80
Vola
tile
com
poun
ds (%
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M HO M S H O M PH
N-T H
O N-T
D N D S D
Chemlali fruitsNeb Jmel fruits
Figure 1 Composition according to compound families of the volatiles
obtained from fruits of O. europaea L. Chemlali and Neb Jmel. MH,
oonoterpene hydrocarbons; OM, oxygenated monoterpenes; SH,
sesquiterpene hydrocarbons; OM, oxygenated monoterpenes; PH,
genated non terpene derivatives; ND, nitrogen derivatives; SD, sulphur
derivatives.
Composition and biological activities of olive F. Brahmi et al. 1319
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mined fruit enzyme composition. These results provedthe findings of Angerosa et al. (1999). The olive volatilefraction of Neb Jmel cultivar contained about twice asmuch Oxygenated non terpene derivatives as Nitrogenderivatives (59.8% vs. 25%) whereas they prevailed onNitrogen derivatives in Chemlali olive, which wereproduced in very small amounts (75.3% vs. 7.8%,respectively).Although there are surely different biogenesis path-
ways of volatile compounds between olive fruits andolive oil, it is indeed important to consider someenzymatic pathways which occurs in olive oil flavourcompounds biosynthesis. Furthermore, main volatilecompounds are produced through biogenic pathways ofthe olive fruit, such as the LOX cascade, and fatty acid oramino acid metabolism (Morales & Tsimidou, 2000). Infact, C5 and C6 aldehydes and alcohols and theircorresponding esters are the most volatile compoundspresent in olive oils and are formed by polyunsaturatedfatty acids through a sequence of enzymatic reactionscollectively known as the LOX pathway (Feussner &Wasternack, 2002). LOX enzymes are widespread in theplant and animal kingdoms. They catalyse the oxygen-ation of polyunsaturated fatty acids containing a 1,4-Z,Zpentadiene moiety using molecular oxygen. The LOXpathway is critical in olive fruit for the formation ofvarious flavours or scent components of virgin olive oil(Williams & Harwood, 2000). It is well-known thatlipoxigenases, after their release owing to the disruptionof fruit cells during the milling in olive oil produce 9- and13-hydroperoxides of linolenic and linoleic acids (Ange-rosa, 2002; Ridolfi et al., 2002). These processes give riseto a wide variety of volatile compounds that constitutethe profile of the two cultivars as we found out somecompounds which occur in it, as showed by the presenceof 1-hexanol, and cis-3-hexen-1-ol (Table 2).The amounts of C6 aldeydes, alcohols and their
acetate (LOX products) are mainly related to thedifferent olive cultivar (Angerosa et al., 1999, 2000;Aparicio & Luna, 2002) while other aldeydes such astrans-2-heptenal and n-nonanal are known to be pro-duced during oxidation (Morales et al., 1997).In addition, data showed that (Z)-3-hexenyl acetate
was present in volatile fraction of Neb Jmel cultivar butdetected in trace levels in Chemlali cultivar. This ester issynthesised by alcohol acyltransferase within the LOXpathway. These elaborated results indicated a strictdependence of olive volatile fraction on the enzymaticstore, which is genetically determined (Table 2).
Antioxidant activity
Antioxidant activity of the fresh fruits volatiles of the twoOlea europaea L. cultivars has been determined by twodifferent tests namely DPPH and ABTS+ radical-scav-enging assays. All of the data is presented in Table 3.
Free-radical- scavenging activityIn essence, the antioxidants react with the stable freeradical i.e. 1,1-diphenyl-2-picrylhydrazyl (deep violetcolour) and convert it to 1,1-diphenyl-2-picrylhydrazinewith discoloration. The degree of discoloration indicatesthe free radical scavenging potentials of the sample ⁄antioxidant and it has been found that known antiox-idant such as cysteine, glutathione, ascorbic acid,tocopherol, polyhydroxy aromatic compounds (hydro-quinone, pyrogallol, etc.) reduce and decolorise 1,1-diphenyl-2-picrylhydrazyl by their hydrogen donatingability (Blois, 1958). Free radical scavenging propertiesof the fresh fruits volatiles of Chemlali and Neb Jmelcultivars are presented in Table 3. The volatile fractionof Neb Jmel cultivar (IC50 = 3353.5 lg mL)1) showedhigher scavenging ability on DPPH radicals thanChemlali (IC50 = 5649 lg mL)1), and were lower thanthat of synthetic antioxidant BHT (IC50 = 181.2lg mL)1).
ABTS+ radical-scavengingThe antioxidant capacity of volatiles was expressed asIC50 (concentration of antioxidant required to quench50% of the stable free radical), which was used toacquire the optimised extraction condition. In thepresent study, as showed in Table 3, the weakest radicalscavenging activity was exhibited by the volatile fractionof Chemlali cv. (13 585.62 lg mL)1). Antioxidant activ-ity of the fresh fruits volatile of cv. Neb Jmel wassuperior to the Chemlali cv. with an IC50 value of5396.34 lg mL)1. On the other hand, none of thesamples showed activity as strong as the positive controlascorbic acid (7.45 lg mL)1).The volatile fraction of the cultivars contained
antioxidative compounds, namely linalool and a–pinenewhich were reported to possess a strong antioxidantactivity (Ruberto & Baratta, 2000). The quantitativedifferences in the amounts of these components mightalso explain the differences between the activities of thetwo volatile fractions. Summarising, volatiles from thefresh fruits of Neb Jmel cv. exhibited better antioxidantactivity than those of Chemlali cv. (Table 3). Conse-quently, both ABTS+ and DPPH radicals, cultivarinfluenced the antioxidant activities.
Table 3 Antioxidant activity of the volatile fractions from fresh fruits
Composition and biological activities of olive F. Brahmi et al.1320
International Journal of Food Science and Technology 2011 � 2011 The Authors
International Journal of Food Science and Technology � 2011 Institute of Food Science and Technology
Antibacterial activity
Results of antibacterial activity assays showed that thevolatile fractions from two cultivars of Olea europaea L.had inhibitory effects on the growth of 50% of studiedbacteria (Table 4). In fact, the volatile of the fresh fruitsof Chemlali cv. endowed with a stronger activity againstS. aureus (MIC78 lg mL)1). While, the volatiles comingfrom fresh fruits of Chemlali and Neb Jmel cvs. showedmoderate antimicrobial activity against P. aeruginosawith an MIC of 312 and 625 lg mL)1, respectively. Onthe one hand, the fruit volatile of Neb Jmel cv. demon-strated no significant antimicrobial activity against S.aureus (MIC 1250 lg mL)1). On the other hand, thevolatiles of the twoOlea europaea L. cultivars showed noantimicrobial activity against E. faecalis and E. coli.The variation of the antimicrobial activity could be
correlated to chemical composition variability (Burt,2004). The existence of some antimicrobial constituentssuch as linalool (Bassole et al., 2003), carvacrol (Rotaet al., 2008) and a-pinene (Dorman & Deans, 2000),combined with other minor constitutes might beinvolved in improving overall antimicrobial activity ofvolatile fractions.
Antifungal activity
As shown in Table 4, C. glabrata showed best suscep-tibility towards the volatile fraction of Chemlali cv. witha MIC value of 156 lg mL)1 (Table 4) followed byC. albicans and C. kreusei MIC 312 lg mL)1. On theother hand, the fruits volatiles of Neb Jmel cv. presentedmoderate activities against C. kreusei. No antifungalactivity was detected against C. albicans and C. glabratafor the volatile of the fresh fruits of Neb Jmel cultivar.All in all, the antimicrobial and antifungal efficacy of
volatile fractions is the consequence of interactionbetween minor and major components and conditioned
by the activity of their components (Kalemba &Kunicka, 2003; Yu et al., 2004).
Conclusion
In conclusion, our results confirm that volatile com-pounds and fatty acid composition accumulated differ-ently according to the cultivar. Regarding the studiedvarieties, the cultivar is the important factor influencingthe antioxidant activity of the olive. As far as it concernsthe antimicrobial and antifungal activities, it should benoted that the studied samples showed minor differencesin their activities dependently from their genetic originand chemical consistency.
Acknowledgments
This research was supported by a grant from the‘‘Ministere de l’Enseignement Superieur et de la Recher-che Scientifique’’ UR03ES08 ‘‘Nutrition Humaine etDesordres Metaboliques’’ and ‘DGRST-USCR-Spec-trometrie de masse. Part of this work was carried out atthe Dipartimento di Chimica Bioorganica e Biofarmacia,Universita’di Pisa, via Bonanno 33, 56126 Pisa, Italy.
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