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Ultrasonics Sonochemistry 15 (2008) 590–597

Influence of bleaching by ultrasound on fatty acids andminor compounds of olive oil. Qualitative and quantitative analysis

of volatile compounds (by SPME coupled to GC/MS)

W. Jahouach-Rabai a, M. Trabelsi a,*, V. Van Hoed b, A. Adams a, R. Verhe b,N. De Kimpe b, M.H. Frikha a

a Laboratoire de chimie appliquee: Heterocycles, Corps gras et Polymeres, Faculte des Sciences de Sfax, Route Soukra Km 3.5, 3018 Sfax, Tunisiab Department of Organic Chemistry, Faculty of Bioscience Engineering, Ghent University, Coupure Links 653, B-9000 Ghent, Belgium

Received 16 December 2006; received in revised form 14 April 2007; accepted 16 June 2007Available online 22 June 2007

Abstract

The effects of bleaching using high power ultrasound (20 kHz) on the quality of olive oil were considered in this study, in order toverify the modifications that can occur in fatty acid composition and minor compounds. During the treatment of olive oil under ultra-sonic waves, a rancid odour has been detected. Treated olive oils show no significant changes in their chemical composition but thepresence of some volatile compounds, due to ultrasonic treatment. Some off-flavour compounds (hexanal, hept-2-enal and 2(E),4(E)-decadienal) resulting from the sonodegradation of olive oil have been identified. A wide variety of analytical techniques (GLC, HPLCand GC/MS) were used to follow the quality of bleached olive oils with ultrasonic waves by the determination of the amounts of certainminor compounds such as sterols and tocopherols. Steradienes, resulting from the dehydration of sterols, were detected with small quan-tities especially in severe conditions of sonication. Solid phase micro-extraction (SPME) coupled to gas chromatography was known tobe a sensitive technique to follow changes in the oxidative state of vegetable oils by measuring the amount of volatile materials producedduring the refining process.� 2007 Elsevier B.V. All rights reserved.

Keywords: Bleached olive oil; Ultrasound; Solid phase micro-extraction; Flavours; Off-flavours

1. Introduction

Ultrasound technology can be very useful for minimalprocessing, because of the instantaneous transfer of acous-tic energy to the food product. This is resulting in a reduc-tion of the total processing time, higher throughput andlower energy to the food product consumption [1,2]. Thecombination of the classic technique of bleaching edibleoils with ultrasound gave excellent results. In fact, theaction of ultrawaves is mainly mechanical towards solidbodies, leading to fragmentation or surface activation bymicrojets of liquid, during the implosion or the fragmenta-

1350-4177/$ - see front matter � 2007 Elsevier B.V. All rights reserved.

doi:10.1016/j.ultsonch.2007.06.007

* Corresponding author. Tel.: +216 74274923; fax: +216 74676606.E-mail address: Mah.Trabelsi@fss.rnu.tn (M. Trabelsi).

tion of bubbles. These actions favour also the impregnationof oil (reagent) on a solid catalytic support by action of amicromixture.

The process of bleaching with acid activated clays in theedible oil industry is generally thought to be a combinationof catalytic action and equilibrium adsorption. Theremoval of pigments and various trace constituents fromtriglyceride oils by adsorption is one of the most importantsteps in the processing of edible oils. Adsorbent perfor-mance is limited thermodynamically by competition forthe adsorbent surface. An ideal adsorbent should thereforehave high affinity for components to be removed and essen-tially no affinity for valuable components [3].

As well known, virgin olive oil has a delicate and uniqueflavour. Many volatile compounds responsible for this

Table 1Variation of bleaching parameters

Olive oil samples T (�C) Bleaching time (min)

A0a 30 13

AI 40 20AII 70 20AIII 40 30AIV 40 45

a Ai (0, I, II, III, IV) bleached olive oils with ultrasound.

W. Jahouach-Rabai et al. / Ultrasonics Sonochemistry 15 (2008) 590–597 591

flavour originate from the degradation of triacylglycerols,polyunsaturated fatty acids (18:2, 18:3) or phospholipids[4]. Other compounds are derived from autoxidation offatty acids. In fact, a large number of volatiles can beformed during autoxidation of unsaturated acyl lipidsand many of them are causing off-flavours. This phenome-non, which concerns oils rich with unsaturated bonds, haslimited the use of ultrasound as a commercial technique forthe production of edible fats [5].

Volatile and semi-volatile materials are present in thecrude oil as natural fragrances and oxidation by-products.Deterioration products such as alcohols, aldehydes,ketones, alkanes, esters, and short chain acids are formedas secondary metabolites from the hydroperoxides ofunsaturated fatty acids, under the influence of the hightemperature, oxygen, metals or other pro-oxidants [6].According to previous studies [5,7,8], the sonochemistryis directly related to cavitation. The collapse of the ultra-sound-induced cavities can produce very high local temper-atures and pressures, which have been suggested toproduce oxidation of fats and oils resulting in the break-up of pre-existing lipo(hydro)peroxides. Especially, oilshaving more unsaturated bonds C–C are more susceptibleto off-flavours when sonicated.

The aim of this work was to determine the effect of thebleaching process with high power ultrasonic waves onolive oil. To put in evidence the efficiency of the bleachingprocess using ultrasonic waves, the effect of two parame-ters: bleaching time and temperature, has been studied. Avery important bleaching index (about 89%) was released,which was reached during only 13 min and at a tempera-ture of 30 �C (optimal conditions). The effect of ultrasoundon oil quality (sensory and physico-chemical properties ofolive oil) has been investigated. A wide variety of analyticaltechniques were applied (GC determination of fatty acidsand minor components such as sterol composition andHPLC determination of steradienes and tocopherols). Inaddition, the other purpose of this work was to evaluatethe sensory changes in the volatile fraction of virgin oliveoil that occur as a consequence of the ultrasound treatment(bleaching). Solid phase micro-extraction followed by GC/MS analysis was used to monitor the quality and the oxida-tive stability of insonated edible oils.

2. Materials and methods

2.1. Materials

The olive oil used for experiments was obtained fromSios-Zitex refinery in Sfax (Tunisia). All chemicals usedwere of analytical grade.

2.1.1. Bleaching process by ultrasound

Sonication was achieved at low frequencies of 20 kHz.An ultrasonic generator type Bioblock Scientific 750 W isfitted with an immersible probe that is dipped in the liquidat the top of the tank and emits the sound vibration into

the solution via a titanium alloy rod 13 mm diameter.The reactor is a cylindrical jacket glass vessel (volume200 ml). In all experiments the volume of the used oil(100 ml of olive oil) was bleached with 1 g of Tunisianbleaching earths (acid activated clay prepared in optimalconditions with classic process published in our previouswork [9]).

To put in evidence the efficiency of the bleaching processby ultrasonic waves, two parameters were considered: tem-perature and bleaching time. The reaction was performedin the following conditions: temperature: 30–70 �C; bleach-ing time: 13–45 min; bleaching earths: 1% (g/g); amplitude:28% (Table 1).

2.2. Analytical procedure

Determination of the physico-chemical parameters ofthe oil (free fatty acids and peroxide value) were carriedout according to the analytical methods described, respec-tively, by AOCS official method Ca 5a-40 [10] and by COIofficial method [11].

The determination of fatty acid composition was carriedout following the analytical method described by AOCS(AOCS Ce 1e-91 and Ce 1c-89 [12]. A gas chromatographHP (5890 series II) equipped with flame ionisation detectorhas been used. Methyl esters of fatty acids were analysedon a CPSIL capillary column (25 m, 0.25 mm internaldiameter, 0.2 lm film thickness). The oven temperaturewas programmed from 100 to 150 �C at 4.8 �C/min, thenfrom 150 to 170 �C at 1 �C/min and then held at 170 �Cfor 10 min.

Total sterol analysis was carried out according to amodified official method [13]. Gas chromatographic sepa-rations were performed on a HP 6890 series gas chromato-graph, equipped with capillary colomn EC5 30M, 0.25 mm,0.25 lm using helium as a carrier gas with a velocity of20 cm/s and a split ratio of 1:10. A temperature programwith injection at 285 �C, with hold for 30 min and risingat 10 �C/min–300 �C with 10 min hold, to separate the dif-ferent components. Detection was done by flame ionisationwith the detector temperature set at 360 �C. Identificationof sterols was performed according to the relative retentiontime reported in the COI analysis method [14]. Steradieneanalysis was carried out according to the AOCS Officialmethod Cd 26–96 [15]. Liquid chromatographicseparations were performed on a HPLC chromatograph

592 W. Jahouach-Rabai et al. / Ultrasonics Sonochemistry 15 (2008) 590–597

(KONTRON 422), on a colomn Lichrospher RP18 (parti-cle size 5 lm, 250 mm · 4.6 mm id), flow rate:1 ml/min.Detector: UV at 235 nm.

The composition of tocopherols was determined byHPLC on a normal phase [16]. Phosphorous, iron, calciumand magnesium content was determined by inductive cou-pled plasma (ICP), according to the analytical methodAOCS ca. 20–99 [17].

Volatile compounds were analysed with GC/MS after asolid phase micro-extraction procedure (SPME). Two ml ofbleached olive oil was placed in a 10 ml headspace vial fit-ted with a silicone septum which was placed in a water bathat 40 �C under magnetic stirring for 30 min. Volatiles aresimultaneously trapped on a SPME fibre (50/30 lm divinylbenzene/carboxen/polydimethylsiloxane coating, Supelco,Bellefonte, PA). After trapping, desorption of volatilestakes place in a cis-4 PTV (Gerstel) injector at 250 �C. Sep-aration of compounds began immediately. A HewlettPackard 6890 GC/MS system is used for a chromato-graphic separation on a HP5-MS capillary column(30 m · 0.25 mm · 0.25 lm film thickness). GC analyseswere performed using the following conditions: carriergas He; flow rate 1.2 ml/min; injection temperature250 �C; oven temperature programmed from 40 to 140 �Cat 3 �C/min; then from 140 to 220 �C at 10 �C/min andholding at 220 �C; the ionisation mode used was electronicimpact at 70 eV.

Identification of the components was achieved fromtheir linear retention indices, determined with reference toa homologous series of alkanes, and by a comparison oftheir mass spectral fragmentation patterns with thosestored in the data bank (Wiley 6th and the NIST MassSpectral Library-1998).

All analyses were performed in triplicates and the meanvalues were reported.

3. Results and discussion

When olive oil is treated by ultrasound, under 20 kHz,in different conditions of bleaching, we observed a modifi-cation of the colour and sensory qualities of oil after a per-iod of time longer than 20 min (development of manyodours such as rancid, metallic and fishy. . .). The influenceof ultrasonic treatment on fatty acid composition and onminor compounds has been detailed.

3.1. Ultrasound effects on fatty acid composition

Table 2 shows a comparison between the fatty acid com-position of untreated and treated olive oils with ultrasoundat 20 kHz in different conditions. The comparison betweenthe acid composition of crude oil (A) and bleached oils,showed no significant modification after sonication. Asmall decrease was noted in C 18:2 for the oil AIV becauseof the temperature and the increase of the reaction time.This decrease in the amount of linoleic acid (case ofbleached oil AIV), confirms that oxidation has occurred.

Increasing the temperature and the bleaching time inducedhigher ultrasonic degradation of olive oil.

Generally, lipid degradation could occur by hydrolysisor oxidation. Triacylglycerols may be hydrolyzed into freefatty acids; this phenomenon is commonly known ashydrolytic rancidity. Triacylglycerols and free fatty acidscould be oxidized into volatile compounds also influencingtaste [2]. According to previous results, it was shown thatlinoleic acid is more susceptible to oxidation than oleic acidand would thus be oxidized preferentially before oleic acid[18].

The ultrasound oxidation of edible oils was attributed tocavitation, i.e. micro-mechanical shocks, that affect struc-tural and functional components up to the point of lipidoxidation and deterioration. Cavitation is the formation,growth and, sometimes, the implosion of micro-bubblescreated in a liquid when ultrasound waves propagatethrough it. The collapse of the bubbles leads to energyaccumulation in hot spots where high temperature andpressures have been measured [19]. This phenomenon cancause lipid oxidation through two mechanisms, which canact alone or combined. The first one is purely thermal,due to high temperatures achieved during cavitation, andthe second one is due to free radicals generated bysonolysis.

The cavitation only occurs when the bleaching time ishigher than 20 min. This explains why the effect is only seenin the most severe conditions.

The two parameters SFA (saturated fatty acids) andUFA (unsaturated fatty acids) confirm that there are noconsiderable modifications in the fatty acid compositionaccording to the international standards for refined oilsfor human consumption.

3.2. Ultrasound effects on quality parameters: FFA and

peroxide value

To evaluate the consequences of ultrasound on the qual-ity of bleached oils, two parameters have been determined(free fatty acids and peroxide value). The results of the clas-sical analyses are summarised in Table 3, where a compar-ison between untreated olive oil and oils treated withultrasound in different conditions is presented. Analyseshave been conducted on the typical characteristics of theoil.

Concerning the FFA, there was no major differencebetween the untreated and ultrasound-treated oils. Adecrease in peroxide value was observed after bleachingwith ultrawaves in the conditions of oil AI from5.80 meq. O2/kg oil to reach 4.86 meq. O2/kg (which wereoptimised before). Afterwards, the peroxide valueincreased with the increase of bleaching time (AIII andAIV) and of the temperature (AII). Those increases in PVindicated the rapid rate of primary oxidation in the caseof oils treated in the most severe conditions. Both durationof bleaching time and heating are responsible for the PVincreases in olive oil treated with ultrasound. In fact, PV

Table 2Ultrasound effects on fatty acid compositiona of bleached olive oils

Olive oil 16:0 16:1 18:0 18:1n9 18:1n7 18:2 18:3 20:0 20:1 SFAc UFA c

A (untreated olive oil) 18.70 2.24 2.58 52.14 5.76 17.55 0.54 0.32 0.13 21.60 78.36(0.92) (0.98) (0.40) (0.48) (0.38) (1.20) (0.19) (0.04) (0.20) (0.9) (0.9)

A0b 18.73 2.24 2.58 52.12 5.72 17.54 0.54 0.32 0.13 21.63 78.29

(0.85) (1.02) (0.35) (0.43) (0.37) (1.12) (0.18) (0.05) (0.22) (0.9) (0.9)AI 18.73 2.26 2.60 52.38 5.40 17.55 0.55 0.34 0.14 21.67 78.28

(0.86) (0.95) (0.38) (0.45) (0.35) (1.20) (0.20) (0.06) (0.25) (0.9) (0.9)AII 18.73 2.27 2.58 52.37 5.42 17.58 0.54 0.32 0.14 21.63 78.32

(0.89) (0.98) (0.31) (0.40) (0.37) (1.22) (0.20) (0.06) (0.20) (0.9) (0.9)AIII 18.68 2.24 2.58 52.56 5.43 17.50 0.56 0.34 0.15 21.60 78.44

(0.79) (0.99) (0.34) (0.41) (0.35) (1.23) (0.22) (0.09) (0.19) (0.9) (0.9)AIV 18.75 2.24 2.61 52.48 5.58 17.40 0.54 0.34 0.14 21.70 78.34

(0.93) (1.00) (0.38) (0.50) (0.37) (1.20) (0.19) (0.07) (0.26) (0.9) (0.9)

a Data are means of triplicates, and SD (standard deviation) is given in parentheses.b Ai (0, I, II, III, IV) bleached olive oils with ultrasound.c SFA: saturated fatty acids (16:0, 18:0 and 20.0); UFA: unsaturated fatty acids (16:1, 18:1, 18:2, 18:3 and 20:1).

Table 3Ultrasound effects on olive oil parameters

FFAa,b (% oleic acid) PVb (meq. O2/Kg oil)

A (untreated) 3.80 5.80AI

c 3.83 4.86AII 3.85 6.11AIII 3.84 6.20AIV 3.85 6.75

a Free fatty acid.b PV: Peroxide value; Data are means of triplicates.c Ai (0, I, II, III, IV) bleached olive oils with ultrasound.

W. Jahouach-Rabai et al. / Ultrasonics Sonochemistry 15 (2008) 590–597 593

increased from 5.80 meq. O2/kg for untreated oil (A) to6.11 meq. O2/kg measured after the increase of temperature(AII), and then increased more with the bleaching time untila value of 6.75 meq. O2/kg oil was reached.

Simultaneous formation and decomposition of hydro-peroxides could be responsible for the rancid and metallictaste detected immediately after bleaching by ultrasonicwaves in the most severe conditions (AIII and AIV).

The activated bleaching earth decomposes the hydroper-oxides and thus numerous volatile and semi-volatile sub-stances are found in the bleached oil (by applying highlyactivated earth). Applying SPME, the lipid degradationcan be characterized both by the number and by theamount of the formed compounds.

According to Patterson [20], the increase in the earthdosage reduces the peroxide value. The advantage in thebleaching process using ultrasound, is to minimize thequantity of bleaching earths with the obtention of animportant bleaching index during a very short time know-ing that the optimal bleaching index (about 89%) wasobtained after 13 min, with a quantity of 1% (g/g) and ata temperature of 30 �C.

The fatty acid composition (GC), FFA and the peroxidevalue, allowed to conclude that oils bleached with ultra-sonic waves do not undergo considerable physico-chemicalmodifications and remain corresponding to the interna-tional standards for refined oils for human consumption.

3.3. Solid phase micro-extraction (SPME): an analytical

method to monitor lipid per-oxidation during the bleaching

process

Virgin olive oil has a delicate and unique flavour. Manyvolatile compounds responsible for this flavour derive fromthe autoxidation of fatty acids. But they are not the onlycompounds that contribute to the delicate flavour of thevirgin olive oils. A large number of volatiles can be formedduring autoxidation of unsaturated acyl lipids and many ofthem can cause off-flavours. Pentane, pentanal, hexanaland 2,4-decadienal have been proposed as indicators sincetheir formation runs parallel to the formation of the com-pounds which cause the off-flavours [21].

The initial volatiles, many of them responsible for thepleasant sensory characteristics of the oil (and producedmainly through biochemical pathways), disappeared inthe first 20 min, and the formation of off-flavours, pro-duced through oxidative pathways, gradually increased.The main volatile compounds possibly responsible foroff-flavours were identified, and their evolution during theoxidative process was studied.

The oxidative state of the bleached oils was determinedby collecting and quantifying the volatile compounds fromthe headspace of the oil sample by applying SPME, cou-pled to GC/MS. This technique allows the control of oxi-dation stage of vegetable oils by the determination(qualitative and quantitative) of compounds responsiblefor the rancidity.

During the experimental procedures, a rancid odour wasdetected only for oils bleached by ultrasonic waves during along period and after increasing the temperature. A metal-lic and rancid odour was detected after sonication. It is wellknown that the major cause of the degradation of lipidspresent in food products (vegetable oils) is oxidation. Forthis reason, the deterioration of bleached oils by ultrasonicwaves has been studied by determination of physico-chem-ical values and olfactory properties. These off-flavoursappear only after 20 min of bleaching.

594 W. Jahouach-Rabai et al. / Ultrasonics Sonochemistry 15 (2008) 590–597

Table 4 shows the composition of the identified com-pounds extracted from the headspaces of the samples ofbleached oils with ultrawaves. The compounds hexanal(3), heptanal (7), 2(Z)-heptenal (9), octanal (12), D-limo-nene (13), nonanal (19), 4-ethylphenol (23) were detectedin the olive oil before and after bleaching. Furthermore,2(E),4(E)-decadienal (28) was only formed when the oilwas bleached in the conditions of oils AI, AII and AIII,where relative severe conditions were applied. Only inthe bleached oil AII (high temperature 70 �C), all the vol-atile compounds were detected. In the case of oils A0, AII,AIII and AIV, the most characteristic compounds werehexanal, nonanal and 4-ethylphenol, whereas in the caseof the oil AI, hexanal, nonanal and octanal were the mostimportant.

The metallic and rancid odour detected after bleachingby ultrasonic waves was due to the formation of 2(Z)-hept-

Table 4Peak area of identified volatile compoundsa in treated and untreated olive oil

Numbers Volatile compounds Odour description

1 Butanal2 Toluene3 Hexanal Fatty, pungent, grassy4 1,3-Octadiene5 Styrene6 1-Nonene7 Heptanal8 1,3-Nonadiene9 2(Z)-Heptenal Fishy, sweet10 6-Methyl-5-hepten-2-one11 1-Decene12 Octanal Citruslike13 D-Limonene Fresh, sweet14 1,3-Hexadiene15 1,3,7-Octatriene16 2(E)-Octenal Fatty-nutty17 1-Undecene18 Undecane19 Nonanal Fatty, waxy, citrus20 5-Undecene21 Ethyl cyclohexane carboxylate22 2(E)-Nonenal Cucumber-tallowy23 4-Ethylphenol24 1-Dodecene25 Decanal26 4-Ethyl,2-methoxyphenol27 1-Tridecene28 2(E),4(E)-Decadienal Fatty, deep, fried, citrus29 n-Pentadecane30 n-Hexadecane31 n-Heptadecane

Total peak area by SPME (E + 9)

a Data are means of triplicates.b Ai (0, I, II, III, IV) bleached olive oils with ultrasound.c Percentage of total peak area (%).

enal which is more intense in the case of treated oils A0, AI

and AIV, although it is in relatively small quantities, and itgives a fishy-sweet odour. In addition, there is formation ofsmall quantities of 2(E),4(E)-decadienal only in oils AI, AII

and AIII, which give a deep-fried odour. 2-Heptenal isobtained by the decomposition of linoleate 12-OOH. Hep-tanal originated from the decomposition of linoleate 13/1-OOH, and octanal from oleate 1-OOH [22], which werewith a high concentrations in the case of the highest tem-perature (70 �C). It can be concluded from the results thatboth parameters temperature and bleaching time stronglyinfluence the composition and the amount of volatile com-pounds formed during the process.

The compounds hexanal, nonanal, 2(E)-heptenal, oct-anal and 2(E),4(E)-decadienal have been identified as off-flavours and it was noted that 2,4-decadienal appearedonly after 20 min of treatment with ultrawaves. These

s

A (crude) A0 (%)b,c AIc AII

c AIIIc AIV

c

Bleaching time (min)

– 13 20 20 30 45

Temperature (�C)

– 30 40 70 40 40

4.27 – – – 3.2 4.227.16 4.42 1.75 2.62 0.89 0.80

54.89 29.19 34.87 34.94 23.57 29.16– 1.36 – 2.20 – 1.891.17 1.57 – 0.93 0.73 0.41

– 2.86 3.99 3.85 2.59 2.651.14 3.89 6.04 5.44 4.27 3.2

– 0.79 1.83 1.40 – 0.645.45 5.89 5.43 4.49 8.65 5.501.57 2.41 – 2.81 1.93 2.52

– 3.48 5.99 3.77 4.16 4.182.56 4.98 9.67 5.80 6.12 4.731.97 1.75 2.05 1.74 1.24 1.420.50 – – 0.59 1.23 0.79

– 0.39 – 0.32 – –1.55 3.48 2.74 1.44 2.51 2.04

– 1.64 2.98 1.61 2.59 1.950.33 0.41 – 0.66 0.43 0.363.99 7.85 9.58 9.94 15.67 13.67

– – – 0.15 0.18 0.140.64 0.75 – 0.36 0.33 0.360.47 1.30 2.70 3.09 4.57 4.41

10.63 17.42 6.58 8.09 9.06 9.51– 1.12 – 0.87 1.09 1.19– – – – 2.32 1.861.65 2.81 2.26 1.59 1.63 1.69

– 0.17 – 0.42 0.75 0.58– – 0.54 0.25 0.28 –– – – 0.17 – –– – 0.61 0.21 – –– – 0.30 0.11 – –

1.76 1.31 3.00 2.60 1.95 2.05

0

200

400

600

800

1000

1200

1400

1600

0 10 20 30 40 50

Bleaching time (min)

Hex

anal

(pea

k ar

ea)/n

onan

al (p

eak

area

)

Fig. 2. Hexanal (peak area)/nonanal (peak area) vs. bleaching time.

W. Jahouach-Rabai et al. / Ultrasonics Sonochemistry 15 (2008) 590–597 595

volatile compounds contribute, respectively, to grassy-pun-gent, fatty, fishy-sweet, citruslike, tallow-fruity, oxidizednotes. They result from the degradation of linoleic acidand sterols. Aliphatic ketone formed by autoxidation ofunsaturated fatty acids, was contributed to the undesirableflavours of treated olive oils [21,23]. It was noted that therancidity is discovered for the most severe conditions (tem-perature 70 �C and bleaching time 45 min). Increasing thetemperature (AI) and bleaching time (AII, AIII and AIV)induced higher ultrasonic degradation of olive oil. Theresults confirm that solid phase micro-extraction combinedwith gas chromatography is sensitive enough to followchanges in the oxidative state of vegetable oils by measur-ing the amount of volatile compounds produced during therefining process.

The activated bleaching earth decomposes the hydroper-oxides and this can explain the presence of numerous vol-atile and semi-volatile substances (off-flavours) in thebleached oils (by applying severe ultrasonic conditions).Applying SPME, the lipid degradation can be character-ized both by the number and by the amount of the formedcompounds. The amount of volatile compounds calculatedrelatively to the total peak area are given in Table 4.

An increase of the total amount of volatile compoundswas noted after a 20 min of bleaching with the increaseof temperature, which disappeared in severe conditions ofbleaching process (Fig. 1). The increase of the temperaturemore than 40 �C was at the origin of the decomposition offlavour compounds (Table 4).

The ratio hexanal/nonanal was used to differentiatebetween oxidized and good-quality bleached olive oil sam-ples. It was calculated for different conditions and plottedversus bleaching time. Initially, hexanal was very high,while nonanal was detected at trace level, but in thecourse of bleaching those compounds had differentamounts (Fig. 2, Table 4). In addition, the resultsreported previously show that the effect of the tempera-ture is more intense and rapid on the appearance andthe transformation of flavour compounds than the effectof ultrasound.

As it was proved, hexanal and D-limonene are typicalindicators for lipid oxidation [21] and they were found in

0.00E+00

5.00E+08

1.00E+09

1.50E+09

2.00E+09

2.50E+09

3.00E+09

3.50E+09

0 10 20 30 40 50

Bleaching time (min)

Tota

l pea

k ar

ea b

y SP

ME

Fig. 1. Influence of bleaching time on volatile compounds (at T = 40 �C).

all considered samples with different contents. The amountof hexanal decreased before 20 min of treatment and thenincreased with the increase of bleaching time (Fig. 3). Incontrast, the amount of hexanal decreased at high temper-ature (70 �C) which can be contributed to its decomposi-tion. Concerning D-limonene, there is no significantmodifications (Table 4).

As the main biochemical pathway, the lipoxygenase cas-cade [24], promotes the information of C6 volatile com-pounds from linoleic and linolenic hydroperoxides, ratherthan the C9 compounds, nonanal was formed at trace levelsin crude olive oil. As it was mentioned above, Fig. 3showed different behaviours followed by nonanal and hex-anal during bleaching process in different conditions. Infact, the initial amount of hexanal decreased enormouslybefore a treatment of 20 min, then the concentrationincreased, produced by the breakdown of linoleate 13-OOH and the nonanal arise from the oleate 9-OOH. Inaddition, the appearance of 2(E),4(E)-decadienal after20 min of treatment was noted, which increase with theincrease of a bleaching time less than 30 min. Accordingto Kochhar, the formation of 2(E),4(E)-decadienal is dueto the decomposition of 9-OOH hydroperoxide [18,25].Secondary decomposition of 2(E),4(E)-decadienal leads tohexanal formation which explains the increase of hexanalafter a bleaching time of 45 min and the total disparition

0

10

20

30

40

50

60

0 10 20 30 40 50Bleaching time (min)

Hex

anal

and

non

anal

pea

k ar

ea

Hexanal peak area

Nonanal peak area

Fig. 3. Hexanal and nonanal variation vs. bleaching time (at T = 40 �C).

Table 5Effect of bleaching by ultrasound in the formation of Stigmasta-3,5-diene

Total sterolsa (mg/100 g) Stigmasta-3,5-dienea (mg/kg)

A (crude oil) 514.09 –A0

b 504.27 0.08AI 425.45 0.20AII 451.00 0.28AIII 492.05 0.33AIV 439.85 0.50

a Data are means of triplicates.b Ai (0, I, II, III, IV) bleached olive oils with ultrasound.

Table 6An overview of a-tocopherol, phosphorous (P), Iron (Fe), Calcium (Ca)and Magnesium (Mg) contentsa of bleached olive oils

a-Tocopherols(ppm)

P(ppm)

Fe(ppm)

Ca(ppm)

Mg(ppm)

A (untreatedoil)

140.00 0.05 0.55 2.24 0.18

Aob 132.24 0.03 0.50 0.81 0.03

AI 66.50 0.20 0.55 0.81 0.25AII 62.30 0.19 0.68 0.80 0.27AIII 48.35 0.06 0.44 0.89 0.19AIV 56.40 0.18 0.85 0.96 0.45

a Data are means of triplicates.b Ai (0, I, II, III, IV) bleached olive oils with ultrasound.

596 W. Jahouach-Rabai et al. / Ultrasonics Sonochemistry 15 (2008) 590–597

of 2(E),4(E)-decadienal. Table 4 shows that nonanal andhexanal followed a similar behaviour during bleaching ata temperature less than 40 �C, then the concentration ofnonanal increased with the increase of temperature.

The modifications in flavour compounds and peroxidevalue after treatment in different conditions agreed withthe evolution of the oxidation in the case of bleached oliveoil in the most severe conditions (maxima of bleaching timeand temperature). It was concluded that the effect ofbleaching by ultrasound during short time (less than13 min) permitted to exploit the efficiency of sonochemistryand to avoid the phenomenon of degradation of flavourcompounds.

As it was considered in previous studies [5,26], the effi-ciency of ultrasonic technique in food processing was lim-ited by lipid oxidation, especially in the case of using oilshaving high amounts of unsaturated acids, they are moresusceptible to produce off-flavours when sonicated. In thiswork, we have proved that ultrasound at low frequency(20 kHz) and a power of 28%, can be useful for bleachingduring short time to avoid oxidation of fats.

3.4. Influence of ultrasound treatment on sterols and

steradiene formation

Stigmastadienes (the most important is stigmasta-3,5-diene) are dehydration compounds of b-sitosterol andbelong to the sterenes that are the wider family of dehydra-tion compounds from sterols. They are produced duringthe refining process, mainly during bleaching and deodoris-ing steps [15]. The influence of the bleaching processparameters (bleaching time, temperature and ultrasonicwaves) on the steradienes was studied by HPLCdetermination.

Generally, the steradiene level significantly increasesafter bleaching vegetable oil with an acid-activated clays.The steradiene content is dependent on the bleaching con-ditions applied together with ultrasound. The use of ultra-sonic waves has allowed to avoid the phenomenon ofdehydration of the b-sitosterol, which is the most abundantcompound in the sterolic fraction.

The bleaching earth that was most active for pigmentremoval was also causing the most severe b-sitosterol dehy-dration. Both effects can be partly due to adsorption andpartly to a stigmastadiene formation [27]. The bleachingearths had a major catalytic effect on stigmastadiene for-mation. However, in the case of the use of ultrasonicbleaching process, the dehydration phenomenon of b-sitos-terol is very low and increases with the bleaching time.

A very small decrease of the total sterols (Table 5)resulting from the bleaching operation especially in the caseof oil A0 (bleached during only 13 min): it results from thedehydration of sterols to steradienes or their degradation incompounds of rancidity. A very small quantity of 3,5-stig-mastadienes was noted and the highest amount is detectedin the oil AIV bleached relatively in the most severeconditions.

3.5. Influence of ultrasonic treatment on tocopherols and

element composition (phosphorous, iron, magnesium and

calcium)

Tocopherols present in vegetable oils, play a very impor-tant role as an antioxidant [16]. The decrease in thea-tocopherol content (Table 6) noted after bleaching, isprobably due to their decomposition or their oxidationunder the effect of ultrasound or bleaching earths. In con-trast, a very small decrease of the content was noted in thecase of bleached oil A0 which was bleached during a veryshort time. The considerable decrease of the content of toc-opherols can be contributed to their decomposition or theiroxidation under the effect of ultrasound, in combinationwith the effect of temperature (bleached oil AIII). In fact,owing to their antioxidant activity, tocopherols are alteredto yield a number of products such as quinones, dimers, tri-mers and epoxides [25]. This explains the loss of tocophe-rols in samples bleached in the severe conditions.

The presence of small quantities of metals, after bleach-ing operation handicaps the classic process. Therefore, inthis work the presence of these contaminants was moni-tored. These elements play a role in the process of oxida-tion of vegetable oils and especially iron which should bekept as low as possible. The decrease of calcium contentafter the bleaching operation is due to its adsorption onbleaching earths.

Generally, bleaching reduce significantly the phospho-rous and iron content [15], in contrast we noted an increaseof the content of iron in oils AII and AIV (Table 6). In fact,

W. Jahouach-Rabai et al. / Ultrasonics Sonochemistry 15 (2008) 590–597 597

the small increases in the quantities of Fe and Mg detectedafter bleaching can result from the fragmentation ofbleaching clays or the conversion of the molecules of phos-pholipids under the effect of ultrawaves. The contents ofdetected metals remain corresponding to the internationalstandards.

4. Conclusion

Ultrasound assisted food processing is a potential alter-native to conventional processes: it is rapid, clean, andresults in less product loss, improvements in food qualityand relatively the conservation of vitamins.

The main advantage of this technique is the decrease ofthe bleaching time, because when it is given sufficient time,any fat or oil will deteriorate even if handled under idealconditions. As it is well known, bleaching is the most crit-ical step of the refining process, resulting from the use ofacid bleaching earths, mainly the effects of bleaching timeand temperature (which are relative to the power of ultra-waves). In consequence, the bleaching process usingultrasound allows to avoid the harmful effects of theacid-activated earths by minimizing the bleaching timeand the temperature.

However, this study has revealed that the flavour ofolive oils is partly deteriorated by ultrasound after longtreatment. Also, thermooxidation can be the origin of theevolution of the volatile compounds responsible forbleached olive oil flavour during oxidative deterioration.As indicated previously, the ultrasound oxidation of edibleoils is probably attributed to cavitation, i.e. micro-mechan-ical shocks, that influence structural and functional compo-nents up to the point of lipid oxidation and deterioration.

SPME was successfully applied for monitoring thedecomposition of hydroperoxides during bleaching. Fur-thermore, SPME combined with GC/MS gives access todetailed information about the volatile compounds formedin the bleaching operation. It was concluded that the effectof bleaching by ultrasonic waves during a short time (lessthan 13 min) permitted to decrease the phenomenon ofdegradation of flavour compounds. In addition, theincrease of peroxide value and the amount of hexanal afterbleaching during 45 min can be explained by oxidation ofoleic acid and further degradation to hexanal via its oxida-tion products.

The bleaching process using ultrasound leads to weaklosses in a-tocopherols and to the formation of small quan-tities of stigmastadienes, which result from the dehydrationof sterols mainly b-sitosterol. In consequence, for the stud-ied quality parameters, there is no considerable chemicalalteration of olive oil was detected after ultrasonic treat-

ment. In fact, olive oil is not highly susceptible to oxidationbecause the linolenic acid and polyunsaturated fatty acidcontent is not high, so low linolenic acid in olive oil devel-ops less reversion flavour.

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