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Innovhub – Stazioni Sperimentali per l’IndustriaAzienda Speciale della Camera di Commercio di MilanoAbout detection of animal Squalene/Squalane in vegetable pro-ducts used in the cosmetic field

Evoluzione della normativa per la classificazione merceologicadegli oli d’oliva

Physico-chemical, nutritional and sensory characterization ofVerdeña, Verdilla and Royal varieties olive oilInfluence of water quality used in irrigation on the sensoryproperties of olive oil

Analysis of some Algerian virgin olive oils by headspace solidphase micro-extraction coupled to gas chromatography/massspectrometryThe quality of polish spreadable fats especially with emphasis oftrans isomers contentCongressi

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GENNAIO/MARZO 2012 – ANNO LXXXIX

SSoommmmaarriioo

Editoriale

A. Gasparoli, C. Mariani, M.E. Gaboardi, G. Morchio, G. SantusF. Caponio, L. Conte, C. Summo,V.M. Paradiso, G. Pedone, T. GomesM. Benito, M. Abenoza, R. Oria, A.C. Sánchez-Gimeno A.K. Alsaed, Kh.M. Al-Ismail, S. Ayub, R. Ahmad, S. Abdel-QaderS. Nigri, R. Oumeddour, X. Fernandez

A. Żbikowska, M. Kowalska, J. RutkowskaNotiziario

ORGANO UFFICIALE DELLA DIVISIONE SSOG DI INNOVHUB STAZIONI SPERIMENTALI PER L’INDUSTRIA

AZIENDA SPECIALE DELLA CAMERA DI COMMERCIO DI MILANO



Organo ufficiale della Divisione SSOG di Innovhub Stazioni Sperimentali per l’IndustriaAzienda Speciale della Camera di Commercio di Milano

GENNAIO/MARZO 2012 ISSN 0035-6808 RISGARD 89 (1) 1-72 (2012) - Poste Italiane s.p.a.Spedizione in Abbonamento Postale -70% Finito di stampare nel mese di Marzo 2012

RISG

LA

RIVISTA ITALIANA

DELLE

SOSTANZE GRASSE

12012

La RIVISTA ITALIANA DELLE SOSTANZE GRASSE è l’organo ufficiale della Divisione SSOG di Innovhub

Stazioni Sperimentali per l’Industria Azienda Speciale della

Camera di Commercio di Milano. Ha periodicità trimestrale e la scientificità dei contenuti è garantita

da un Comitato Internazionale di Referee.Pubblica lavori originali e sperimentali di autori

italiani ed esteri riguardanti la chimica, la biochimica, l’analisi e la tecnologia nei settori:

sostanze grasse e loro derivati, tensioattivi, detersivi, cosmetici, oli minerali.

Include metodi di analisi in inchiesta pubblica relativi ai settori di competenza, il Notiziario coninformazioni su congressi, notizie in breve e libri.La Rivista viene consultata in Italia dalle industrieproduttrici di oli e grassi alimentari ed industriali,

dalle industrie chimiche, da laboratori di enti statali,da istituti di ricerca e facoltà universitarie. E’ inoltre

distribuita all’estero in vari Paesi come Spagna,Grecia, Francia, Germania, Tunisia, Nigeria, Congo, Polonia, Romania, Bulgaria, Russia,

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da dove provengono diversi lavori scientifici.

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L A R I V I S TA I TA L I A N A D E L L E S O S TA N Z E G R A S S E - V O L . L X X X I X - G E N N A I O / M A R Z O 2 0 1 2

Squalene and its hydrogenated product Squalane are highly sought raw materials by thecosmetic industries for their functional characteristics. Besides a general preference by the cosmetic industry for ingredients of vegetable vs. ani-mal origin, vegetable Squalene is most required since it is a co-product of vegetable oilproduction (or better, refining process), mainly olive oil (Olea Europaea), while animalSqualene is exclusively obtained by protected marine species (particularly little sizesharks that live in deep water sea), and is therefore considered unethical by many userssince sharks are slaughtered in (almost) totally illegal way (only for removing their liverwhile what remains of their body is thrown back into the sea!). Especially during these last years we observed on the market Squalene/Squalane withvery different prices: this may suggest a possible addition of animal origin product tothe vegetable one. Several years ago, analytical techniques were developed based on the determination ofsome minor compounds, such as sterols and neoformation hydrocarbons, that allowed todifferentiate products of animal and vegetable origin. However, innovative production technologies, together with continuous improvements ofclean-up techniques, including the possible addition of vegetable unsaponifiables (espe-cially obtained by deodorizer distillates from refining of corn and sunflower seed oil) madeit very difficult to differentiate mixtures of animal and vegetable origin. In this work werevisited and updated this issue by using a combination of analytical techniques employedalso in previous works by introducing preparative High Pressure Size Exclusion liquidChromatography (HPSEC). This new analytical procedure is structured as follows : 1. preventive screening by thin layer chromatography (TLC) in order to check the class

of compounds, where detectable; 2. direct analysis by gas chromatography - mass spectrometry (GC-MS) in order to

highlight the presence of neoformation hydrocarbons and/or sterols, whereas the con-centration at which these compounds were present would allow the detection;

3. separation by glass column chromatography in order to isolate and concentrate the frac-tions corresponding to neoformation hydrocarbons and/or the sterols, followed by gaschromatography - mass spectrometry (GC-MS) analysis of the separated fractions;

4. where the separation of neoformation hydrocarbons from the matrix shows particulardifficulties related to the extreme similarity of chromatographic major component (thecase of Squalanes), liquid chromatography HPSEC is used for isolating and concen-trating the fraction to be investigated;

5. separation by Silver nitrate of the fraction corresponding to the neoformation hydro-carbons, in order to eliminate possible interference of products with a number of dou-ble bonds higher than those found in the Steradienes.

The results show that the differentiation between animal and vegetable Squalene and itshydrogenated derivative Squalane can be made through the detection of markers of theanimal (Cholesterol) and vegetable (Sitosterol) origins, of their dehydroxylated derivatives(Steradienes), and of their corresponding hydrogenated products (Stanols and Steranes).

About detection of animalSqualene/Squalane in vegetableproducts used in the cosmetic field

A. Gasparoli1C. Mariani1M.E. Gaboardi1G. Morchio2

G. Santus3*

1 Divisione SSOG - Innovhub Stazioni Sperimentali perl’Industria - Azienda Specialedella Camera di Commercio diMilano2 Technoil C.S., Imperia3 Amedeo Brasca & C. Srl,Origgio (VA)

*CORRESPONDENCE TO:e-mail: [email protected]

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Sulla possibile individuazione di Squalene/Squalano animale nei corrispet-tivi prodotti vegetali utilizzati nel settore cosmeticoLo Squalene e il suo derivato idrogenato Squalano sono materie prime molto ricercate dall’in-dustria cosmetica per le loro caratteristiche funzionali.Oltre ad una generale preferenza dell’industria cosmetica per ingredienti di origine vegetaleanziché animale, lo Squalene vegetale è maggiormente richiesto in quanto è un coprodottodella lavorazione/raffinazione degli oli vegetali, prevalentemente dell’olio d’oliva (Olea Europea)mentre l’utilizzo dello Squalene animale, provenendo esclusivamente da specie ittiche protette(in particolare squali di piccole e medie dimensioni che vivono in acque profonde), è conside-rato non etico da molti utilizzatori, poiché la cattura avviene in modo totalmente illegale. La presenza sul mercato, da alcuni anni, di Squalene/Squalano con quotazioni molto diverse,ha indotto ad ipotizzare una possibile aggiunta del prodotto di origine animale a quello di ori-gine vegetale. Diversi anni orsono, sono state messe a punto tecniche analitiche basate suldosaggio di alcuni componenti minori, quali gli steroli e gli idrocarburi di neoformazione, chehanno consentito di differenziare prodotti di origine animale da quelli di origine vegetale.Tuttavia, l’impiego di innovative tecnologie di produzione, insieme a continui miglioramentidelle tecniche di purificazione e la possibile aggiunta di insaponificabili vegetali ottenuti, inparticolare, dalla lavorazione degli oli di semi di mais e girasole, hanno reso molto difficile ladifferenziazione di miscele e/o commistioni tra la provenienza animale e quella vegetale. Inquesto lavoro abbiamo riaffrontato la problematica attraverso tecniche analitiche coordinate,introducendo, accanto alle tecniche utilizzate anche nei precedenti lavori, la cromatografialiquida ad esclusione sterica preparativa.L’iter analitico seguito si è così articolato:1. screening preliminare mediante cromatografia su strato sottile, per verificare le classi di

composti presenti, laddove rilevabili;2. analisi diretta per gascromatografia-spettrometria di massa (GC-MS), allo scopo di evi-

denziare la presenza di idrocarburi di neoformazione e/o di steroli, laddove la concentra-zione alla quale questi composti erano presenti ne consentisse la rilevazione;

3. separazione mediante cromatografia su colonna, per isolare e concentrare le frazioni cor-rispondenti agli idrocarburi di neoformazione e/o agli steroli, sottoponendo successiva-mente le frazioni separate ad analisi mediante gascromatografia-spettrometria di massa;

4. laddove la separazione degli idrocarburi di neoformazione dalla matrice presentasse diffi-coltà particolari, legate all’estrema similarità cromatografica del componente principale(caso degli Squalani), utilizzo della cromatografia liquida HPSEC per isolare e concentra-re la frazione da indagare;

5. separazione mediante Argento Nitrato della frazione corrispondente agli idrocarburi di neo-formazione, allo scopo di eliminare eventuali interferenze di composti aventi numero didoppi legami superiore a quelli presenti negli steradieni.

Dai risultati ottenuti emerge che la differenziazione tra origine animale e vegetale delloSqualene e del suo derivato idrogenato Squalano, può essere effettuata attraverso la ricer-ca dei marker di origine animale (Colesterolo) e del mondo vegetale (Sitosterolo) e deiloro derivati deidrossilati, gli steradieni, nonchè dei loro corrispondenti prodotti idroge-nati, gli stanoli e gli sterani.

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INTRODUCTION

Squalene and its totally hydrogenated productSqualane are raw materials highly sought by cos-metic and dermofarmaceutical markets for theirfunctional properties.Squalene is a highly unsaturated hydrocarbonand a precursor of Sterols. It can have both veg-etable and animal origin since it is contained bothin several vegetable oils (olive, amaranth, and inlittle amount, also in sunflower and corn, etc.),and in the liver of some sharks, as, for instance,the ones that live in deep water sea.Vegetable Squalene and Squalane are moreappreciated by consumers since they comes bythe processing (or better, as co-productsobtained by the refining process) of vegetable oils(especially olive oil), while animal Squalene isexclusively obtained by protected fish speciesthat (even if subject to severe controls) are illegal-ly slaughtered, especially in Horn of Africa andIndian Ocean, with the aim to recover only theirliver (it contains around 50% fat matters, of which70-80% is Squalene).As a consequence of what above reported, andby considering the higher easiness and shortnessof its extraction/production cycle, animal Squa-lene shows a very lower cost compared to thevegetable one.The use of animal origin Squalene and Squalaneis generally considered as unethical by most ofconsumers and producers of cosmetic products.Squalene/Squalane can be often found on themarket with very different quotations, and this haslead to the hypothesis that some commercialmaterials may contain animal origin product,which is less expensive than the vegetable prod-uct.Recently two research groups [1, 2] explainedindependently the possibility of quantitative deter-mination of Squalene/Squalane origin, by employ-ing 13C/12C stable isotope ratio, reported as δ13C,evaluated by Isotope Ratio Mass Spectrometry(IRMS) related to an analyzer for high purity Squa-lene/Squalane or to gas chromatographic/burningsystem for low purity (>80 %) samples and forproducts obtained by finished cosmetic formula-tions [2].However, although being highly quantitativelyaccurate, this technology could show some prac-tical problems for employment in traditional analy-sis Laboratories, especially as a methodology forroutine Quality Control by producers and users,due to its high specialization and its poor spreadon national and international territory.A few years ago [3, 4] some analytical technolo-gies (based on dosing minor compounds assterols and neo-formation hydrocarbons) were

introduced in order to differentiate animal originproducts from vegetable ones.However, continuous improvements of productiontechnologies, together with new clean-up tech-niques, and also by considering potential additionof unsaponifiables of vegetable origin (comingfrom refining process of corn and sunflower oils),caused higher difficulty of differentiation, particu-larly for animal Squalene/Squalane mixtures invegetable ones.In this Research we dealt with this problemthrough coordinated analytical techniques, byintroducing (together with the traditional technolo-gies already used in previous works about thissubject) the preparative High Pressure SizeExclusion liquid Chromatography (HPSEC).

MATERIALS

- 5 samples by the market, declared as vegetableSqualene, with numbers from 1 to 5,

- 6 samples by the market, declared as animalSqualene, with numbers from 6 to 11,

- 5 samples by market, declared as vegetableSqualane, with numbers from 12 to 16,

- 3 samples by the market, declared as animalSqualane, with numbers from 17 to 19,

- 5 unknown samples, with numbers from 20 to 24,- 1 mixture prepared by us of vegetable Squalene,

containing 20% animal Squalene, reported as Mix,- 3 mixtures prepared by us of vegetable

Squalane, containing, respectively, 17,0-2,0-5,5% of animal Squalane, reported as 17, 12, 5,5

- silica gel plates 20x20 cm, 0,25 mm thickness,- silica gel for chromatography 7734 Merck, acti-

vated at 160°C for 4 hours, with 5% water added,- rotary evaporator- n hexane residue grade,- diethyl ether residue grade,- tetrahydrofuran seccosolv,- anhydrous pyridine,- sylon BFT,- glass columns [3],- hydrophilic cotton degreased with hexane.

METHODS

PRELIMINARY QUALITATIVE SEPARATION BY THIN LAYERCHROMATOGRAPHY (TLC)Silica gel plates 20x20 cm, 0,25 mm thickness,have been used, by employing hexane - diethylether (95:5 ratio) mixture as elution product.1st indicator: 2,7 dichlorofluorescein, 0,2% alco-holic solution,2nd indicator: diluted sulphuric acid.

GASCHROMATOGRAPHY – MASS SPECTROMETRY (GC-MS)A Fisons MD 800 mass spectrometry has been

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employed, linked with a Fisons 8000 gaschro-matographic apparatus,- fused silica column SE 52, 25 m lenght, i.d.

0,32 mm, 0,1 mm film thickness,- oven temperature: 80°C (1’) to 210°C,

with 20°C/min increase, then to 280°C, with 5°C/min increase,

- on-column mode injection,- carrier gas: He, at 50 kPa,- interface temperature: 300°C,- apparatus temperature: 200°C,- ionization for electronic impact: 70eV

COLUMN CHROMATOGRAPHYAround 1,3 g of sample are weighed inside a 25ml round-bottom flask and diluted with hexane;during the preparative phase we proceed as pre-cised in [5], while during separative phase weproceed as here below reported: 1st elution: hexane:- 20 ml of hexane containing the sample are

adsorbed and they are separately collected,- elution is continued with hexane, by collecting

a 50 ml fraction.2nd elution mixture: hexane – diethyl ether in 9:1ratio; 250 ml of eluate are collected,3rd elution product: diethyl ether; 200 ml are col-lected.The different fractions are concentrated by rotaryevaporator, they are weighed and submitted tosuccessive analysis.

CHROMATOGRAPHY ON SILVER NITRATE COLUMNProcedure according to [6] has been followed.

ANALYSIS OF STEROL FRACTIONThe fraction eluted by glass column with diethylether is fractioned by thin layer chromatography(TLC).Silica gel plates with 0,25 mm thickness havebeen used, previously treated with methanolicKOH, as reported in [7], by employing hexane –diethyl ether (50:50 ratio) mixture and, as indica-tor, 2,7 dichlorofluorescein, 0,2% alcoholic solu-tion.The band corresponding to sterols is isolated,extracted from silica, derivatized as reported in[7] and then analyzed by gaschromatography –mass spectrometry.

SEPARATION BY HPSEC (High Pressure Size Exclusionliquid Chromatography)Agilent 1100 isocratic liquid chromatographicapparatus has been used- Agilent 1047 detector with refraction index,

kept at 35°C temperature,- two columns connected in series, maintained

at 35°C:- PL gel 5µ 100 Å, 300 mm lenght, Ø 7,5 mm,- PL gel 5µ 100 Å, 300 mm lenght, Ø 7,5 mm.

- injector: 20 µl loop - elution solvent: tetrahydrofuran at 0,8 ml/min.

RESULTS AND DISCUSSION

The scheme reported in Figure 1 summarizes theanalytic path followed by the analysis of the testedsamples; for unknown samples, we proceededaccording to indications supplied by thin layerchromatography and by preliminary gas-chromato-graphic analysis. An assessment by thin layer chromatography hasbeen carried out for all samples: these results arereported by Figure 2 that shows as hydrocarbonfraction (corresponding to reference Squalene orSqualane) is the predominant component.This Figure highlights also that in the n. 6 and 10animal Squalene samples, there is a triglyceridecomponent, by supplying a significant informationfor next treatment of these samples.As this scheme reports, we proceeded - with theexception of the above mentioned two samples -with direct analysis of samples by gaschromatog-raphy – mass spectrometry (GC-MS), in order todetect specific markers (sterols and/or neo-forma-tion hydrocarbons), possibly contained at such aconcentration that may be directly detectable.

Figure 1 - Analytic path followed in samples analysis

Subsequently, for the samples that did not give a sig-nificant result with direct analysis, we employed a dif-ferent procedure, according to each sample type.Particularly, Squalene samples have been fraction-ated by glass column chromatography, as reportedin Methods part, thus obtaining a hydrocarbonfraction and a polar fraction.Hydrocarbon fraction has been analyzed by GC-MS, to search neo-formation hydrocarbons; polarfraction has been eluted by thin layer chromatogra-

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phy in order to isolate the sterol fraction, which isthen analyzed by GC-MS.For Squalane samples, instead, we employed sep-aration – by preparative HPSEC – of compoundswith lower molecular weight, that have been subse-quently analyzed by GC-MS. Separation by preparative HPSEC has been car-ried out by using a blend – made of α-Colestane, αand β-Stigmastane, α and β-Campestane – that isused both for detecting elution zone of these com-

Figure 3 - HPSEC separation of Squalane – α-Cholestane

Figure 2 - Thin layer chromatography of all samples

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pounds, and for evaluating the correspondencebetween recovered products and the compositionof the used blend. Figure 3 reports HPSEC separation and Figure 4shows the run obtained through GC-MS by theanalysis of collected fraction: in the lower part thetotal path is reported, while the upper part showsthe run obtained by setting 217 m/z ion, specific ofthese compounds.Here below we report the results we obtained by

Figure 4 - GC-MS chromatogram obtained by the analysis of α-colestane (1), α (2) and β (3)-campestane and α (4) and β (5)-stigmastanemixture, HPSEC fraction collected; down: total ion, upper:m/z 217

analyzing all the samples.

VEGETABLE SQUALENESSample n. 1: direct GC-MS analysis detects thepresence of only Stigmastadiene.This result is also confirmed by assessmentthrough GC-MS of hydrocarbon fraction that is iso-lated by glass column separation.Figure 5 reports the result obtained by GC-MSanalysis, by showing the whole run in the central

part, and in the upper part the path got by isolating368 and 396 m/z ions, related respectively toCholestadiene and Stigmastadiene: only Stigmas-tadiene is detected and its mass spectrum isreported in the lower part of this figure while thepolar fraction does not show Cholesterol.Samples n. 2, 3, 4 and 5: also for these samplesonly Stigmastadiene is confirmed.Figure 6, underlines GC-MS run related to the

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Figure 5 - Sample 1: result obtained by GC-MS of Hydrocarbon fraction

hydrocarbon fraction of Sample n. 3 (lower part ofthis figure) and the profile obtained by isolating 368and 396 ions while the upper part of this figureshows only Stigmastadiene compound.

ANIMAL SQUALENESSample n. 6: as already above indicated, it showsa triglyceridic fraction, and therefore a preliminarglass column separation of different fractions of

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Figure 6 - Sample 3: result obtained by GC-MS of Hydrocarbon fraction

this sample is required. By assessment of the hydrocarbon fraction, wecan detect Cholestadiene, whereas polar fractioncontains Cholesterol, as Figure 7 shows.Sample n. 7: Figure 8 reports GC-MS run (upperpart) and mass spectrum (lower part) of the peakwith RT 13,669, referred to Cholestadiene.Samples n. 8 and 9: also these two products con-firm only Cholestadiene content.

Sample n. 10: also here there is a triglyceridicfraction and therefore it is necessary to proceedwith glass column separation of these com-pounds.The polar fraction shows Cholesterol content, aswe can see by Figure 9 that shows the run got byGC-MS analysis of this fraction after derivatiza-tion with silyl reagent: in fact both by specific 368m/z ion and through mass spectrum assessment,

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Figure 8 - Sample 7: GC-MS result

Figura 7 - Sample 6: GC chromatogram of sterolic fraction

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Figure 9 - Sample 10: result by GC-MS of polar fraction

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Figure 10 - Sample 11: GC-MS result of hydrocarbon fraction

Cholesterol is detected.Sample n. 11: Figure 10 reports GC-MS chro-matogram of the hydrocarbon fraction (middlepart) and the run got by isolating 368 m/z ionreferred to Cholestadiene, whereas the lower partshows mass spectrum of this peak.

Figure 11 highlights the run obtained by analysis ofderivatized polar fraction; it shows Cholesterol bothby specific 368 m/z ion (up part of this figure), andalso through the peak spectrum with RT of Choles-terol (lower part). The middle part reports the fullscan path.

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Figure 11 - Sample 11: GC-MS result of polar fraction


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VEGETABLE SQUALANESSample n. 12: neo-formation hydrogenated hydro-carbons are detected, by carrying out direct GC-MS analysis of this sample.Figure 12 reports in the middle part the whole GC-MSrun, whereas in the upper part it shows the path got

Figure 13 - Sample 12: GC-MS result of polar fraction

by isolating 217 ion, characteristic of Steranes com-pounds, and in the lower part the mass spectrum ofthe peak with RT 15,268, referred to Stigmastane.The polar fraction of this sample shows also (Figure13) hydrogenated Sterols: the upper part of this fig-ure reports the GC-MS run, and the lower part

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highlights the spectra of α and β-Sitostanol.Sample n. 13: only vegetable Steranes are detect-ed in this product.Sample n. 14: it is prepared by employing HighPressure Size Exclusion liquid Chromatography(HPSEC); the lower part of Figure 14 reports thepath obtained by GC-MS analysis of the isolated

Figure 14 - Sample 14: GC-MS result of HPSEC fraction

fraction. The upper part highlights the run got byisolating 217 m/z ion, typical of Steranes. Sample n. 15: this sample was declared as fromvegetable origin, but it is possible to see also Ster-anes compounds, coming from Cholestadienehydrogenation, as Figure 15 reveals.Sample n. 16: only vegetable Steranes are noted.

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20



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ANIMAL SQUALANESSample n. 17: direct analysis of this sample, as Fig-ure 16 shows, underlines (by setting 217 m/z ion) αand β-Cholestane.Sample n. 18: the same result (as n. 17 up sample)is detected for this product.Sample n. 19: for this sample it was suitable toenrich Steranes fraction by HPSEC. The upper partof Figure 17 shows the run got by fraction collect-


ed in HPSEC, by isolating 217 m/z ion, and thelower part reports the mass spectrum ofCholestane.

UNKNOWN SAMPLESSample n. 20: this product is cleaned-up byemploying a glass column chromatography inorder to remove alkanes; it shows simultaneouslyCholestadiene and Stigmastadiene, as Figure 18

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Figure 18 - Sample 20: GC-MS result of Hydrocarbon fraction

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Figure 19 - Sample 21: GC-MS of HPSEC fraction

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Figure 20 - Sample Mix: gascromatografic analysis of fraction purified by AgNO3

Figure 21 - Sample 23: GC-MS result of hydrocarburic fraction

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reports: its middle part underlines the full scan run,and its upper part shows the path got by isolating368 m/z ion, typical of Cholestadiene and 396 m/zion, specific of Stigmastadiene. In the lower partwe can see the mass spectrum referred toCholestadiene. Sample n. 21: the hydrocarbon fraction is enriched

by HPLC and it shows – besides Stigmastane –also Cholestane, as Figure 19 reports.It is interesting to note that other peaks are detectedin this sample: their structure could be referred toshort chain triglycerides. Sample n. 22: different trials did not allow todefinetely detect animal origin products: for this


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Figure 23 - Sample 24: GC chromatogram of sterolic fraction

Figure 24 - trace of GC-MS (ion 357) of fraction obtained by HPSEC of:Squalane vegetable (upper), mixture of Squalane vegetable with Squalane animal 17% (medium) and 12% (down)

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reason we prepared a mixture (Mix) made of 20%animal Squalene (by employing sample n. 8) and80% vegetable Squalene (by using sample n. 1).Gaschromatographic analysis of hydrocarbon frac-tion – cleaned-up by Silver nitrate – gave origin tothe result showed by Figure 20, where Cholestadi-ene is detected, therefore confirming the presenceof an animal product.Sample n. 23: Figure 21 – that reports GC-MS runsof hydrocarbon fraction after glass column purifica-tion – underlines both Cholestadiene and Stigmas-tadiene contents.Sample n. 24: GC-MS analysis of the fraction sep-arated by HPSEC shows Stigmastadiene content(Fig. 22), whereas the polar fraction confirms Cho-lesterol compound (Fig. 23).In order to verify the detection limit of animalSqualane added to the vegetable one, some mix-tures were prepared (namely enriched/spiked) withdecreasing contents of animal Squalane.With this aim, we used n. 14 vegetable Squalanesample, respectively supplemented 17, 12 and 5,5%animal Squalane (sample n. 19), and proceededaccording to what experimented in this Research.The obtained results are reported in Figures 24 and25, where Cholestane content is detected in thesethree different mixtures.

CONCLUSIONS

The analytical procedure employed in this Researchis structured in this following way:1. preliminary screening, by employing thin layerchromatography (TLC), in order to verify if differentcompound fractions are detectable;2. direct analysis by gaschromatography – massspectrometry (GC-MS), in order to detect neo-forma-tion hydrocarbons and/or sterols, if their concentrationallows to detect them;3. separation by glass column chromatography inorder to isolate and concentrate the fractions referredto neo-formation hydrocarbons and/or sterols, thensubmission of these separated fractions to analysisby GC-MS; 4. if separation of neo-formation hydrocarbons fromraw product shows particular difficulties related toextreme chromatography similarity of the main com-pound (as for Squalanes), then liquid chromatogra-phy HPSEC is employed in order to isolate and con-centrate the fraction to be detected;5. separation, by using Silver nitrate, of the fractionreferred to neo-formation hydrocarbons in order toremove potential interference compounds with moredouble bonds than Steradienes.The study of these samples allowed to confirm that

Figure 25 - Trace GC-MS (ion 357) of HPSEC fraction of Squalane vegetabl (upper), and your mixture with Squalane animal 5,5% (down)

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discovered concepts in previous studies [1, 2] stillshow their validity.However, the procedure to follow in order to isolateand then concentrate these markers needs absolute-ly to employ more sophisticated methodologies.Both Silver nitrate chromatography and HPSEC werefirstly introduced for analyzing these samples andthey allowed to get positive results.In the global view of declared origin samples thisResearch highlighted that a Squalane sample (n. 15)has not a vegetable origin (as declared), since itshows Steranes compounds that come fromCholestadiene hydrogenation (Fig. 15).The results got by analyzing unknown samplesappeared particularly relevant: in fact, samples n. 20,21 and 23 are a mixture of both vegetable and animalorigin products for the simultaneous presence ofCholestadiene and Stigmastadiene in Squalene sam-ples n. 20 and 23, and of Cholestane and Stigmas-tane in Squalane sample n. 21.Sample n. 22 did not show any animal origin com-pounds.Therefore, we prepared a mixture with 20% animalSqualene; we set this limit since we estimated thatbelow such value, the addition of animal product intovegetable one may not be further profitable. Samples n. 2 and 8 have been used since they wererather difficult in their specific analysis. The presence of animal substance in this mixture isshowed through the analysis of the hydrocarbon frac-tion purified on Silver nitrate column. About the mixtures of vegetable/animal Squalane thefollowing considerations can be outlined.It is known that vegetable fatty matters contain littleamounts of Cholesterol, with a possible resultingincrease of the minimum detection level of animalproducts, in comparison to the vegetable ones. Thisis due to the fact that the origin of little amounts ofCholestadiene is likely occurring during refiningprocess: they should follow the same path of Squa-lene, by decreasing sensitivity and perhaps by givingorigin to wrong positives.In this Research (see Fig. 24, upper part and n. 25,lower part) we observed that n. 14 vegetable Squa-lene does not contain any Cholestane trace comingfrom Cholestadiene hydrogenation. For this reason this sample was used as a basis forthe mixtures, by adding to it known amounts of animalSqualane (sample n. 19): the obtained samples (mix-tures n. 17, 12 and 5,5) were analyzed according tothe methodology developed by this Research.As the referred Figures can show, animal Squalane isdetectable also at 5,5% minimum level.Obviously this limit will be linked to possible purifica-tion processes or to other specified and aimed tech-nological treatments which the product can be sub-jected to: however, in our opinion, by considering thereached sensitivity of this Method, they may not be

employed anymore, given a negative ratio costs/ben-efits.Another important aspect of this Research is that thevegetable Squalane sample, analyzed byHPSEC/GC-MS and used as a basis for the mixtures,does not show any trace of Cholestane.It is known that vegetable Squalene mainly comes bythe phase of neutralizing distillation and/or deodoriza-tion of olive oil refining process: this product shouldshow Cholestadiene since – by tables of olive oil com-position – up to 0,5% Cholesterol in sterol fraction isreported.Since we did not detect Cholestanes coming fromCholestadiene hydrogenation, this can point to thefact that Cholesterol mainly could result from contam-ination of materials used in the analysis.In overall terms, according to what above reported, itderives that the differentiation between animal andvegetable origin of Squalene and its hydrogenatedproduct Squalane can be carried out through thedetection of specific markers of the animal(Cholestanol) and vegetable realms (Sitosterol), oftheir dehydroxylate derivatives – Steradienes – and oftheir related hydrogenated products – Stanols andSteranes.Since it employs relatively more traditional and acces-sible equipment, this proposed analytical procedurecan therefore represent a reliable integrative or alter-native methodology to recently developed isotopictechniques, e.g. for use as a standard/routine QualityControl analysis by Laboratories of both producersand buyers.

BIBLIOGRAPHY

[1] P. Jame, H. Casabianca, M. Batteau, P.Goelink, V. Salomon, Differentiation of the originof Squalene and Squalane using stable iso-topes ratio analysis. SOFW-Journal 136, 1/2(2010)

[2] F. Camin, L. Bontempo, L. Ziller, C. Piangiolino,G. Morchio, Stable isotope ratios of carbonand hydrogen to distinguish olive oil from sharksqualene-squalane. Rapid Commun. MassSpectrom. 24, 1810-1816 (2010)

[3] A. Gasparoli, C. Mariani, M.G. Fedrigucci,Squalano Differenziazione tra origine vegetalee origine animale. Riv. Ital. Sostanze Grasse73, 293-302 (1996)

[4] A. Gasparoli, S. Tagliabue, C. Mariani, Fito-squalene genuinità di una materia prima utiliz-zata nel settore cosmetico. Riv. Ital. SostanzeGrasse 75, 241-245 (1998)

[5] IUPAC 2.507[6] Consiglio Oleicolo Internazionale DOC/T20

Doc16 rev.2[7] Metodo di analisi NPC Ga IV-4 (2002) (Norme

Prodotti Cosmetici)

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Questa rassegna ha l’obiettivo di offrire una trattazione organica dei diversi atti legislativiemanati nel tempo per disciplinare la classificazione merceologica degli oli d’oliva. Laprima forma di regolamentazione del settore risale, in Italia, al 1890; attualmente la clas-sificazione merceologica degli oli d’oliva è disciplinata dal Regolamento CEE 2568/91 eda molteplici regolamenti ad integrazione e/o modifica e/o abrogazione di parti di esso. Aconclusione, sono prospettate le tendenze future di regolamentazione del settore degli olid’oliva nell’Unione Europea.

Evolution of regulations regarding the commercial classes of olive oilsThe aim of this paper is to offer a comprehensive overview of the evolution of the nation-al and European legislative framework regarding the commercial classes of olive oils. Theearliest rule in Italy dates back to 1890, while the current commercial classes have beenset by the EEC Regulation 2568/91 and its further several amendments. Finally, futuretrends for European Regulations have been proposed.

Evoluzione della normativa per la classificazione merceologica

degli oli d’oliva

F. Caponio1*

L. Conte2

C. Summo1

V.M. Paradiso1

G. Pedone1

T. Gomes1

1 Dipartimento di Biologia eChimica Agro-Forestale ed

Ambientale (DIBCA), Universitàdegli Studi “Aldo Moro”

di Bari, Italy2 Dipartimento di Scienze degliAlimenti, Università degli Studi

di Udine, Italy

*CORRISPONDENZA AUTOREUniversità degli Studi

Dept. DIBCASezione di Scienze e Tecnologie AlimentariVia Amendola 165/aI-70126 Bari, Italy.

Fax: +39 080 5442235e- mail:

[email protected]

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1. INTRODUZIONE

Per la sua rilevanza socio-economica e per le suepeculiari caratteristiche che lo contraddistinguonodagli altri oli vegetali, l’olio di oliva è da lungotempo oggetto di particolare attenzione da partedel legislatore. La prima forma di regolamentazio-ne del settore risale, in Italia, al 1890.Attualmente la classificazione merceologica deglioli d’oliva è disciplinata, nel nostro Paese, comemembro della UE, dal Regolamento CEE 2568/91 eda molteplici regolamenti ad integrazione e/omodifica e/o abrogazione di parti di esso. Lo scopo di questa rassegna è quello di offrire una

trattazione organica dei diversi atti legislativi ema-nati nel tempo per disciplinare la classificazionemerceologica degli oli d’oliva.

2. EVOLUZIONE NORMATIVA IN MATERIADI CLASSIFICAZIONE DEGLI OLI DI OLIVA

Le Figure 1 e 2 mostrano rispettivamente l’evolu-zione ad oggi della normativa in materia di classifi-cazione degli oli di oliva rispettivamente a livellonazionale e comunitario.

2.1. LA “PREISTORIA”La prima forma di regolamentazione dell’olio di

Figura 1 - Evoluzione della normativa in materia di oli d’oliva a livello nazionale.

Figura 2 - Evoluzione della normativa in materia di oli d’oliva a livello comunitario.

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oliva risale al 1890 con il Regio Decreto Legge n.7045 [1]. Tale decreto, formulato ai fini della tuteladell’igiene e della sanità pubblica, all’art. 114 proi-biva “la vendita a scopo alimentare di grassi ani-mali e vegetali alterati per irrancidimento o prove-nienti, rispettivamente, da animali affetti da morbiinfettivi o da semi putrefatti” ed ancora, all’art 115,“il commercio con il nome di olio e grasso, seguitodalla designazione di derivazione o provenienza,di un prodotto diverso da quello indicato con taledenominazione, o di un prodotto guasto o sofistica-to con sostanze estranee che ne diminuiscano ilpotere alimentare o che siano nocive”. Inoltre, l’art.117 imponeva che “le mescolanze di oli di oliva,con quelli di altra derivazione” dovevano “essereposte in commercio con il nome di questi ultimi”. Èpossibile constatare che pur senza proporre unaclassificazione e senza prevedere l’impiego dialcun indice analitico, già alla fine del XIX secolo siiniziavano ad affrontare le problematiche relativealla qualità e genuinità degli oli e grassi alimentari.Una prima bozza di classificazione si ebbe agliinizi del secolo scorso con la Legge n. 136 del 5aprile 1908 [2], che distingueva gli “oli di olivagenuini” dagli “oli di oliva miscelati”. Questi ultimierano costituiti da una miscela di oli di oliva con olidi semi o altre sostanze oleose: si focalizzava,quindi, l’attenzione sulla tutela della genuinità deglioli di oliva senza, tuttavia, che per questi vi fosseuna definizione specifica. Con il Regio Decreto Legge n. 2033 del 15 ottobre1925 [3] – poi convertito in legge (Legge 18 marzo1926 n. 562) e che aveva lo scopo della repressio-ne delle frodi nella preparazione e nel commerciodi sostanze di uso agrario e di prodotti agrari – siebbe la prima definizione dell’olio di oliva intesocome “il prodotto ottenuto dalla lavorazione dell’oli-va (Olea europaea) senza aggiunta di sostanzeestranee o di oli di altra natura”. Inoltre, tale decre-to puntualizzava alcuni aspetti circa le miscele dioli di oliva ed oli di semi. In particolare, era defini-to come “olio miscelato” la miscela di olio di olivaed altri oli vegetali quando questi ultimi non supe-ravano il 50%, mentre la denominazione “olio disemi” era riservata sia agli oli diversi da quelli dioliva sia alle miscele nelle quali gli oli di oliva rap-presentavano meno del 50%. Il Regio DecretoLegge, inoltre, consentiva “la vendita e il commer-cio per uso commestibile degli oli di oliva deodora-ti, disacidificati o comunque raffinati, purché noncontengano sostanze estranee aggiunte per cor-reggerne il colore od altro carattere”, mentre vieta-va la vendita “per uso commestibile di oli di sansa,di oli rancidi e di oli sensibilmente difettosi o altera-ti”. Esso, infine, introduceva l’obbligo per chiunqueintendesse fabbricare o mettere in commercio olivegetali commestibili diversi da quelli di oliva, difarne denunzia per iscritto al Sindaco.

Successivamente con il Regio Decreto n. 1361dell’1 luglio 1926 [4], che approvava il Regolamen-to per l’esecuzione del R.D.L. n. 2033, si stabilì chegli oli commestibili non dovessero contenere piùdel 4% di acidità totale, espressa in acido oleico, eche non dovessero presentare all’esame organo-lettico “odori disgustosi, come di rancido, di putri-do, di fumo, di muffa, di verme, ecc.”. Allo scopo diaccertarne la natura, fu inoltre introdotto l’impiegodi altri saggi analitici (“indice rifrattometrico, nume-ro di iodio, indice termico1 e ricerche di oli diversida quelli di oliva”), pur non fornendo alcuna indica-zione riguardo ai limiti. Inoltre, il Regio Decretoincludeva tra gli oli raffinati destinati alla venditaper uso commestibile anche gli oli estratti dallesanse di oliva, purché privi di qualsiasi sostanzaestranea e di tracce del solvente eventualmenteadoperato. Tali oli dovevano essere commercializ-zati come oli di seconda lavorazione.Il Regio Decreto Legge n. 2316 del 30 dicembre1929 [5], che modificava parzialmente il RegioDecreto Legge n. 2033, vietava la preparazione ela vendita di miscele tra oli di oliva e altri oli vege-tali commestibili ed obbligava all’impiego delladenominazione “olio di seme” per la commercializ-zazione di tutti gli oli vegetali commestibili adesclusione di quelli di oliva. Questi, inoltre, prima diessere commercializzati dovevano essere addizio-nati con il 5% di olio di sesamo a reazione croma-tica caratteristica.La Legge n. 378 del 16 marzo 1931 [6], che sosti-tuiva l’art. 24 del Regio Decreto Legge n. 2033,consentiva la vendita e il commercio per uso com-mestibile sia degli oli di oliva deodorati, disacidifi-cati o comunque raffinati sia degli oli estratti dallesanse. Questi ultimi dovevano essere messi incommercio con la denominazione di “oli di sansacommestibili”, purché addizionati con il 5% di oliodi sesamo e privi di sostanze estranee aggiunteper correggerne il colore od altro carattere.

2.2. NASCE L’OLIO DI OLIVA VERGINEI decreti fino a quel momento emanati definivanouna prima embrionale classificazione degli olicommestibili, che in realtà si concretizzava in unadifferenziazione tra oli di oliva, oli di sansa comme-stibili ed oli di semi.Successivamente con il Regio Decreto Legge n.1986 del 27 settembre 1936 [7] fu introdotta la“Classificazione ufficiale degli oli di oliva”. Taledecreto rappresenta una pietra miliare nella classi-ficazione degli oli di oliva, determinando il passag-

1 Presumibilmente il legislatore si riferiva all’indice termosol-forico.

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gio dalla semplice distinzione tra oli di semi, oli disansa commestibili ed oli di oliva alla più comples-sa discriminazione delle varie tipologie di oli dioliva sulla base dell’acidità percentuale (Tab. I). È possibile constatare che il termine “vergine”compariva per la prima volta nella denominazionedi una classe commerciale di oli d’oliva. Inoltre, ilRegio Decreto vietava a partire dall’1 gennaio 1937la vendita, la detenzione per la vendita e la com-mercializzazione, per il consumo diretto, di oli condenominazione diverse da quelle riportate in tabel-la ed abrogava la Legge n. 378 del 1931.Con la seconda guerra mondiale e le difficoltà deldopoguerra, trascorsero ben 24 anni prima del-l’emanazione di un’altra legge in materia di classi-ficazione di oli di oliva. La Legge n. 1407 del 13novembre 1960 [8] “Norme per la classificazione ela vendita degli oli di oliva” tornò a modificare laclassificazione degli oli d’oliva (Tab. II). Per laprima volta comparve la denominazione “extra ver-gine” per identificare la categoria degli oli d’oliva dimaggiore pregio. La Legge n. 578 del 5 luglio 1961 [9] modificò ladefinizione dell’olio di oliva rettificato e dell’olio disansa di oliva rettificato, autorizzando tra i proces-si fisici di raffinazione solo quelli che non causava-no alterazioni all’olio più profonde di quelle appor-tate dal processo agli alcali. Parallelamente veni-

vano approvati i “Metodi ufficiali di analisi per gli olie i grassi” (Decreto Ministeriale 22 aprile 1959 [10],Decreto Ministeriale 26 novembre 1963 [11] – sup-plemento 1 – e Decreto Ministeriale 20 dicembre1971 [12] – supplemento 2 – con l’istituzione di unelenco ufficiale per la lotta contro le frodi, il cuisistematico aggiornamento era affidato al Ministe-ro per l’Agricoltura e le Foreste (Legge 1407/1960).

2.3. L’EUROPA SI METTE D’ACCORDOIn seguito al trattato di Bruxelles, che nel 1965 rea-lizzava una prima forma di coordinamento tra le treComunità europee (CECA, CEEA e CEE), venivaemanato il Regolamento CEE n. 136 del 22 settem-bre 1966 [13], che attuava a livello europeo l’Orga-nizzazione Comune dei Mercati (OCM) nel settoredei grassi. L’allegato di tale regolamento “Denomi-nazione e definizioni di cui all’articolo 35”, definival’olio d’oliva vergine (o puro olio d’oliva vergine)come “l’olio d’oliva naturale ottenuto soltantomediante processi meccanici, compresa la pres-sione, esclusa qualsiasi miscela con oli di altranatura o con olio d’oliva ottenuto con altro proces-so”. In Tabella III è riportata la classificazione deglioli d’oliva in base a tale Regolamento. L’aciditàpercentuale continuava ad essere l’unico parame-tro analitico impiegato per la discriminazione dellediverse tipologie di oli d’oliva vergini.

Tabella I - Classificazione degli oli di oliva in base al Regio Decreto Legge n. 1986 del 27 settembre 1936.

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Seguirono numerosi Regolamenti che introdusseroaltri parametri analitici utili a discriminare le variecategorie di oli d’oliva. In particolare, si sussegui-rono: il Regolamento CEE n. 177 del 7 novembre 1966[14] che introduceva un ulteriore saggio analitico2,per la distinzione fra i diversi oli d’oliva che hannosubito un processo di raffinazione;

il Regolamento CEE n. 618 del 29 marzo 1972 [15],che abrogava il Regolamento CEE 177/1966 edindicava i limiti per il coefficiente di estinzione spe-cifica a 270 nm ed il contenuto in β-sitosterolo, ai

Tabella II - Classificazione degli oli di oliva in base alla Legge n. 1407 del 13 novembre 1960.

Tabella III - Classificazione degli oli di oliva in base al Regolamento CEE 136/66 alla sua emanazione.

2 Il metodo descritto nell’Allegato al Regolamento corrispon-de a quello successivamente definito come saggio di Bellier.

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fini della commercializzazione degli oli d’oliva; il Regolamento CEE n. 1058 del 18 maggio 1977[16], che definiva come olio d’oliva vergine quelloche conteneva un tenore di acidi grassi liberi nonsuperiore al 3% ed un coefficiente di estinzionespecifica a 270 nm (K270) non superiore a 0,25(0,11 dopo passaggio su allumina) con una varia-zione, in prossimità di 270 nm, non superiore a0,01. Inoltre, tale Regolamento introduceva: i) ilsaggio di Vizern modificato per la rivelazione deglioli di sansa negli oli d’oliva, ii) la ricerca dei sapo-ni per evidenziare l’alcalinità degli oli dovuta a trat-tamenti di neutralizzazione, iii) la determinazionedella composizione degli acidi grassi saturi in posi-zione 2 del trigliceride per la ricerca della presen-za di olio riesterificato, iv) il metodo per la determi-nazione del tenore in olio d’oliva delle sanse;il Regolamento CEE n. 3132 del 28 dicembre 1978[17], che introduceva i limiti degli altri steroli pre-senti negli oli d’oliva;il Regolamento CEE n. 1858 del 30 giugno 1988[18], che modificava il Regolamento CEE1058/1977 ed introduceva la determinazione deltenore in tetracloroetilene per la discriminazionedegli oli d’oliva. Si tornò a modificare la denominazione degli olid’oliva con il Regolamento CEE n. 1915 del 2 luglio1987 [19] (Tabella IV). Tale regolamento definiva glioli vergini di oliva come “gli oli ottenuti dal fruttodell’olivo soltanto mediante processi meccanici oaltri processi fisici, in condizioni, segnatamente ter-miche, che non causano alterazioni all’olio, e chenon hanno subito alcun trattamento diverso dallavaggio, dalla decantazione, dalla centrifugazionee dalla filtrazione, esclusi gli oli ottenuti mediantesolvente o con processi di riesterificazione e qual-siasi miscela con oli di altra natura”. Le principalinovità contenute nel Regolamento riguardavanoessenzialmente l’innalzamento del tenore massimo

dell’acidità per la categoria commerciale dell’oliodi oliva vergine e l’introduzione dei limiti di aciditàpercentuale sia per gli oli d’oliva e di sansa d’olivaraffinati sia per le loro miscele con gli oli di olivavergini. Inoltre, compariva per la prima volta inmaniera esplicita, nella definizione delle classicommerciali, l’esclusione degli oli vergini lampantitra quelli ammessi nella costituzione delle miscelecon i raffinati.

2.4. I NOSTRI GIORNIL’emanazione del Regolamento CEE n. 2568dell’11 luglio 1991 [20] ha rappresentato una deci-sa innovazione rispetto ai Regolamenti precedenti.Tale Regolamento aveva lo scopo principale di rior-ganizzare la normativa comunitaria in materia di olid’oliva, oltre a uniformare le diverse metodicheanalitiche riportandole in un unico testo di legge. Ilregolamento introduceva l’obbligatorietà dell’anali-si sensoriale ai fini della commercializzazione deglioli d’oliva vergini (Tabella V). Negli anni successivi sono stati introdotti altri para-metri analitici per meglio differenziare le diversecategorie di oli d’oliva, quali: il contenuto in isome-ri trans degli acidi grassi insaturi (RegolamentoCEE n. 1429 del 26 maggio 1992) [21]; il contenu-to in cere in sostituzione di quello degli alcoli alifa-tici (Regolamento CEE n. 183 del 29 gennaio 1993)[22]; il contenuto in stigmastadieni (RegolamentoCE n. 656 del 28 marzo 1995) [23]; la valutazionedei triacilgliceroli con ECN 42, mediante il calcolodella differenza tra contenuto teorico e contenutosperimentale (HPLC) (Regolamento CE n. 2472dell’11 dicembre 1997, integrato dal RegolamentoCE n. 282 del 3 febbraio 1998) [24, 25]; il contenu-to in 2-gliceril monopalmitato (Regolamento CE n.702 del 21 giugno 2007) [26] e il contenuto in metiled etil esteri degli acidi grassi (Regolamento UE n.61 del 24 gennaio 2011) [27].

Tabella IV - Classificazione degli oli di oliva in base al Regolamento CEE 1915/87.

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Successivamente alla loro emanazione, sia ilRegolamento 2568/1991 sia i regolamenti cheintroducevano gli altri parametri analitici sono statirivisti e modificati dal legislatore al fine di tutelare laqualità e genuinità degli oli d’oliva. In particolare, il Regolamento CE n. 1989/2003 [28]ha nuovamente modificato la classificazione deglioli di oliva, eliminando la categoria commercialedell’olio di oliva vergine corrente. Con lo stessoRegolamento è stato introdotto lo schema di cam-pionamento per oli d’oliva confezionati e lo schemadecisionale per la verifica della conformità di uncampione di olio d’oliva alla categoria dichiarata.Inoltre, sono stati oggetto di modifiche i parametridi seguito riportati:Valutazione sensoriale: i) il Regolamento CEE n. 1683/1992 [29] ha

apportato modifiche al vocabolario specificoutilizzato per l’analisi sensoriale;

ii) il Regolamento CEE 3288/1992 [30] ha previstola costituzione, da parte degli Stati membri, dicomitati di assaggio incaricati del controllo uffi-

ciale delle caratteristiche sensoriali degli oli; iii) i Regolamenti CE n. 2632/1994 e n. 2527/1995

[31, 32] hanno modificato la modalità di espres-sione dei risultati della valutazione sensoriale;

iv) il Regolamento CE n. 796/2002 [33] ha modifi-cato ulteriormente le modalità di esecuzione edi espressione della valutazione sensorialedegli oli. Per adeguare il sistema di valutazionesensoriale COI alle norme ISO si è passatiall’adozione di una scala astrutturata lineare eall’espressione dei risultati organolettici in ter-mini di “mediana del fruttato” e “mediana deidifetti”;

v) il Regolamento CE n. 1989/2003 [28] ha modifi-cato il valore limite della mediana del difetto perl’olio di oliva lampante (Md > 2,5);

vi) il Regolamento CE n. 640/2008 [34] ha modifi-cato il valore limite della mediana dei difetti del-l’olio di oliva vergine (da < 2,5 a < 3,5) ed haaggiornato la procedura per la valutazioneorganolettica degli oli ed il vocabolario ufficiale.Inoltre, tale regolamento ha definito le condizio-

Tabella V - Caratteristiche degli oli d’oliva in base al Regolamento CEE 2568/91 alla sua emanazione

M = massimo; m = minimo. Per classificare diversamente un olio o dichiararlo non conforme per la purezza è sufficiente che uno solo dei parametri non rientri nei limiti fissati. Ai fini della consta-tazione della purezza, qualora il K270 superi il limite della categoria corrispondente, si deve procedere alla determinazione del K270 dopo passaggio su allumina. (1) Limite massimo complessivo per i composti rilevati dal rilevatore a cattura di elettroni. Per i componenti accertati singolarmente il limite massimo è 0,10 mg/kg. (2) Delta-5-23-stigmastadienolo+clerosterolo+beta-sitosterolo+sitostanolo+delta5-avenasterolo+delta-5-24-stigmastadienolo. (3) Nel caso di oli con acidità > 3,3% se dopo passaggio su allumina si ottiene K270 > 0,11 si deve effettuare la prova di raffinazione prevista dall’allegato XIII.


Tabella VI - Caratteristiche degli oli d’oliva in base al Regolamento UE 61/2011.

È sufficiente che una sola caratteristica non sia conforme ai valori indicati perché l’olio venga cambiato di categoria o dichiarato non conforme riguardo la sua purezza. (1) Somma degli isomeri che potrebbero (o meno) essere separati mediante colonna capillare.(2) O quando la mediana del difetto è inferiore o uguale a 3,5 e la mediana del fruttato è uguale a 0. (3) Gli oli con un tenore di cera compreso tra 300 mg/kg e 350 mg/kg sono considerati olio di oliva lampante se gli alcoli alifatici totali sono pari o inferiori a 350mg/kg o se la percentuale di eritrodiolo ed uvaolo è pari o inferiore a 3,5%. (4) Gli oli con un tenore di cera compreso tra 300 mg/kg e 350 mg/kg sono considerati olio di sansa di oliva greggio se gli alcoli alifatici totali sono superiori a 350mg/kg o se la percentuale di eritrodiolo ed uvaolo è superiore a 3,5%. (5) Tenore di altri acidi grassi (%): palmitico 7,5 - 20,0; palmitoleico 0,3 - 3,5; eptadecanoico ≤ 0,3; eptadecenoico ≤ 0,3; stearico 0,5 - 5,0; oleico 55,0 - 83,0; linoleico 3,5 - 21,0. (6) Somma di: delta-5-23-stigmastadienolo+clerosterolo+beta-sitosterolo+sitostanolo+delta-5-avenasterolo+delta-5-24-stigmastadienolo. (*) - per l’olio di oliva lampante, i corrispondenti valori limite possono non essere rispettati simultaneamente

- per gli oli di oliva vergini, l’inosservanza di almeno uno di questi valori limite comporta il cambiamento di categoria, pur rimanendo classificati in una delle categorie di oli di oliva vergini.

(**) per tutti gli oli di sansa di oliva i corrispondenti valori limite possono non essere rispettati simultaneamente. (#) NdA. Benché il Legislatore non abbia ritenuto opportuno specificarlo, il valore del ∆ECN42 va espresso come valore assoluto.

36

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ni per l’utilizzo facoltativo in etichetta degli attri-buti positivi (fruttato, amaro, piccante), con lapossibilità di specificare fruttato verde o fruttatomaturo e la relativa intensità: intenso, medio oleggero. Infine, ha consentito l’uso dei terminiequilibrato e dolce, in relazione al rapporto delvalore della mediana dei diversi attributi positi-vi.

Determinazione della trilinoleina:i) il Regolamento CEE n. 1996/1992 [35] ha

apportato modifiche alla determinazione dellatrilinoleina nell’olio di sansa greggio;

ii) il Regolamento CEE n. 620/1993 [36] ha aggior-nato i limiti massimi in trilinoleina dell’olio disansa di oliva greggio e raffinato e dell’olio disansa oliva, portandoli rispettivamente a 0,7%,0,6% e 0,6%;

iii) il Regolamento CE n. 2472/1997 [24] nell’alle-gato 1 non prevede più i limiti per tale determi-nazione.

Esame spettrofotometrico nell’ultravioletto:i) il Regolamento CEE n. 1429/1992 [21] ed i

Regolamenti CE n. 656/1995 e n. 1989/2003[23, 28] hanno modificato i limiti delle diversecategorie di oli d’oliva.

Determinazione degli alcoli alifatici/cere: i) il Regolamento CEE n. 183/1993 [22] ha modifi-

cato il valore limite per gli oli di oliva vergini(valore massimo 250 mg/kg) ed ha introdotto unvalore minimo per l’olio di sansa di oliva (> 350mg/kg);

ii) il Regolamento CE 796/2002 [33] ha portato illimite massimo per l’olio di oliva vergine lam-pante a 300 mg/kg ed ha introdotto il valoreminimo per l’olio di sansa di oliva greggio e raf-finato (> 350 mg/kg).

Determinazione degli steroli:i) il Regolamento CE n. 656/1995 [23] ha modifi-

cato il valore limite del brassicasterolo (da 0,2%a 0,1%) per tutte le categorie di olio;

ii) il Regolamento CE 2472/1997 [24] ne ha ripor-tato il valore massimo per gli oli di sansa a0,2%.

Determinazione della composizione acidica:i) il Regolamento CE n. 656/1995 [23] ha modifi-

cato il valore limite degli acidi: miristico (M0,05%), arachico (M 0,6%), eicoseinoico (M0,4), beenico ad eccezione degli oli di sansa (M0,2%) e lignocerico (M 0,2%);

ii) il Regolamento CE n. 1989/2003 [28] ha modifi-cato il valore limite dell’acido linolenico (M1,0%) ed ha introdotto dei range per il tenore dialtri acidi grassi.

Determinazione degli isomeri trans: i) il Regolamento CE n. 656/1995 [23] ha modifi-

cato il valore limite per le categorie di oli vergi-ni di oliva commestibili sia per gli isomeri tran-soleici che per la somma di translinoleici e tran-

slinolenici (da M 0,03% a M 0,05%).Determinazione dell’eritrodiolo ed uvaolo:i) il Regolamento CE n. 796/2002 [33] ha modifica-

to i limiti previsti per gli oli di sansa di oliva greg-gio e raffinato, portandoli da m 12 a > 4,5%.

Determinazione dell’acidità libera: i) il Regolamento CE n. 1989/2003 [28] ha modifi-

cato il valore limite di acidità dell’olio extra ver-gine di oliva (≤ 0,8%), dell’olio di oliva verginelampante (> 2,0%), degli oli di oliva raffinato edi sansa di oliva raffinato (≤ 0,3%) e degli oli dioliva e di sansa di oliva (≤ 1,0%).

Determinazione degli acidi grassi in posizione 2del trigliceride:

i) il Regolamento CE n. 1989/2003 [28] ha modifi-cato i limiti per le diverse categorie di olio d’oli-va.

Determinazione degli stigmastadieni:i) il Regolamento CE n. 702/2007 [26] ha modifi-

cato il valore limite per gli oli extra vergine e ver-gine di oliva (da 0,15 mg/kg a 0,10 mg/kg).

La Tabella VI riporta gli indici ed i relativi limiti pre-visti ad oggi per la classificazione merceologicadegli oli d’oliva.

3. TENDENZE FUTURE DI REGOLAMENTA-ZIONE DELL̓UNIONE EUROPEA NEL SET-TORE DEGLI OLI DʼOLIVA

Una grande importanza viene attribuita alla realeetà dell’olio, che può essere valutata grazie alladeterminazione del rapporto tra le forme 1,2/1,3dei diacilgliceroli, esiste da molti anni un metodoNGD e da un po’ meno tempo un metodo DGF.Questa valutazione risulta già applicata spesso alivello di capitolati e transazioni commerciali,dando per acquisita la indubbia validità della suabase scientifica, resta da armonizzare un metodo,o, per lo meno, da rendere disponibili metodi affi-dabili e con caratteristiche di confidenza parago-nabili.Un’ulteriore parametro considerato è la valutazionedella frazione dei polifenoli, per la quale il COI hastandardizzato un metodo HPLC. La situazione deipolifenoli è ambigua: non costituiscono un parame-tro di legge, tuttavia sono riportati nei disciplinaridelle DOP che sono pubblicati sulle Gazzette Uffi-ciali e recepiti da Leggi. Sembra comunqueimproponibile fissare un limite per questo parame-tro, considerando l’ampio range di variazione chesi registra anche solo all’interno del nostro Paese.Un altro aspetto riguarda la necessità di armoniz-zazione tra i metodi comunitari ed i metodi adotta-ti ed utilizzati a livello mondiale. Attualmente la ten-denza è di riportare tutto nell’ambito della ISO epertanto a livello COI si sta lavorando in collabora-zione con la ISO per una completa armonizzazionedei due metodi.

38


Essendo la norma ISO una norma tecnica, essariporta le caratteristiche di affidabilità dei metodi(ripetibilità, riproducibilità e loro deviazioni stan-dard relative) e risulta in tal modo utilissima perl’accreditamento delle prove a norma ISO 17025 esuccessive modifiche ed integrazioni, mentre iregolamenti comunitari non riportano questi dati.La sostanziale identità dei metodi ISO e di quellicomunitari renderebbe di fatto disponibili le carat-teristiche dei metodi anche per quelli CEE, confe-rendo ad essi maggiore robustezza e migliori pos-sibilità di essere sostenuti in ambito di contenziosi.Un’ulteriore richiesta dei consumatori e dei produt-tori riguarda, infine, la possibilità di certificazionedell’origine, ad oggi basata su aspetti meramentedocumentali o su valutazioni di tipo chemiometriconon validate da alcun Ente.La questione non è di poco conto e non appare disemplicissima risoluzione, in letteratura moltissimiautori hanno applicato queste tecniche a varieclassi di composti [37-42], e la Società Tedescaper lo Studio delle Sostanze Grasse (DGF) com-mercializza addirittura un software (Oil Inspektor)dedicato a questo scopo. L’estrema variabilità incomposizione degli oli di oliva e la crescente diffu-sione in nuove zone climatiche non agevola questoapproccio.Il problema è stato affrontato anche applicando tec-niche differenti, quali la risonanza magnetica nuclea-re [43-47] e lo studio degli isotopi stabili [48-50].

BIBLIOGRAFIA

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CE n. 1989 del 6 novembre 2003. [29] Gazzetta Ufficiale delle Comunità Europee n.

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[32] Gazzetta Ufficiale delle Comunità Europee n.258 del 28 ottobre 1995. Regolamento CE n.2527 del 27 ottobre 1995.

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[39] S. Vichi, L. Pizzale, L.S. Conte, S. Bux-aderas, E. Lopez-Tamames, Solid phasemicroextraction in the analysis of virgin oliveoil volatile fraction: characterisation of virginolive oils from two distinct geographicalareas of northern Italy. J. Agric. Food Chem51, 6572-6577 (2003).

[40] D. Ollivier, J. Artaud, C. Piantel, J.P. Durbec,M. Guerere, Differentiation of French virginolive RDOs by sensory characteristics, fattyacid and triacylglycerol composition andchemometrics. Food Chem. 97, 382-393(2006).

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of olive oils. Eur. J. Lipid Sci. Technol. 109,72-78 (2007).

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[43] R. Sacchi, L. Mannina, P. Fiordiponti, P.Barone, L. Paolillo, M. Patumi, A. Segre,Characterization of Italian extra virgin oliveoils using 1H-NMR spectroscopy. J. Agric.Food Chem. 46, 3947-3951 (1998).

[44] M.D. Guillen, A. Ruiz, High resolution 1Hnuclear magnetic resonance in the study ofedible oils and fats. Trends Food Sci. Tech.12, 328-338 (2001).

[45] L. Mannina, G. Dugo, F. Salvo, L. Cicero, G.Ansanelli, C. Calcagni, A. Segre, Study of thecultivar-composition relationship in Sicilianolive oils by GC, NMR, and statistical meth-ods. J. Agric. Food Chem. 51, 120-127 (2003).

[46] M. D’Imperio, L. Mannina, D. Capitani, O.Bidet, E. Rossi, F.M. Bucarelli, G.B. Quaglia,A. Segre, NMR and statistical study of oliveoils from Lazio: a geographical, ecologicaland agronomic characterization. FoodChem. 105, 1256-1267 (2007).

[47] N. Araghipour, J. Colineau, A. Koot, W.Akkermans, J.M.M. Rojas, J. Beauchamp, A.Wisthaler, T.D. Märk, G. Downey, C. Guillou,L. Mannina, S.V. Ruth, Geographical originclassification of olive oils by PTR-MS. FoodChem. 108, 374-383 (2008).

[48] F. Angerosa, L. Camera, S. Cumitini, G.Gleixner, F. Reinero, Carbon stable isotopesand olive oil adulteration with pomace oil. J.Agric. Food Chem. 46, 4179-4184 (1998).

[49] J.E. Spangenberg, S. A. Macko, J. Hunziker,Characterization of olive oil by carbon iso-tope analysis of individual fatty acids: impli-cation for authentication. J. Agric. FoodChem., 46, 4179-4184 (1998).

[50] J.E. Spangenberg, N. Ogrinc, Characteriza-tion of olive oils from Slovenia and Croatia. Ann. Series Historia Naturalis, 9, 1-4 (1999)

Ricevuto, 27 Luglio 2011Accettato, 31 Agosto 2011

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Verdeña Olive oil variety is most important cultivar grown in the Huesca Somontano areawhere it reaches more than 50% of the production of olive oil. It is used as a table oil andfor oil extraction, usually being blended with other varieties, however the properties ofmonovarietal olive oils have not been studied. In this work the physicochemical, nutrition-al and sensory properties of this cultivar plus two similar varieties, Royal and Verdilla havebeen studied. These three olive oils were all extra virgin with different fatty acid profiles,plus other nutritional compounds such as phenols, tocopherols, and colour and pigmentcontent. The olive oils all had different sensory profiles, characterized by the green attrib-ute of the Verdeña olive oil.Key words: olive oil, physico-chemical quality, sensory, Verdeña, Verdilla, Royal

Caratterizzazione chimico-fisica, nutrizionale e sensoriale dell’olio di olivadelle varietà Verdeña, Verdilla e RoyalLa varietà di olive Verdeña è la più importante della zona Somontano Huesca e conta piùdel 50% della produzione. Viene utilizzata per la stagionatura e l’estrazione di olio, di solito mescolato con altre varie-tà di olio d’oliva. Nonostante ciò le proprietà dei loro oli di oliva monovarietali non sonostate studiate. In questo lavoro abbiamo studiato le proprietà chimico-fisiche, nutrizionali e sensoriali diquesti oli di oliva e di quelli ottenuti da varietà simili come la Royal e la Verdilla.Gli oli extra vergini di oliva sono stati studiati e sono stati differenziati i profili degli acidigrassi e degli altri composti nutrizionali come fenoli, tocoferoli così come il colore e pig-menti.Gli oli di oliva hanno mostrato diversi profili sensoriali caratterizzati dall’attributo verdedell’olio d’oliva Verdeña.Parole chiave:: Olio di oliva, qualità fisico-chimiche, sensoriale, Verdeña, Verdilla,Royal

Physico-chemical, nutritional andsensory characterization ofVerdeña, Verdilla and Royalvarieties olive oil

M. BenitoM. AbenozaR. OriaA.C. Sánchez-Gimeno*

Tecnología de los Alimentos, Facultad de VeterinariaUniversidad de Zaragoza(Spain)

*CORRESPONDING AUTHORDr.ssa Ana Cristina Sánchez GimenoTecnología de los AlimentosFacultad de VeterinariaUniversidad de ZaragozaMiguel Servet, 17750013 Zaragoza (Spain)Tel: 34- 976-761000 ext 4149Fax: 34- 976-761590e-mail: anacris@unizar

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1. INTRODUCTION

Olive oil is obtained from olives through mechani-cal procedures or other physical methods, in spe-cial thermal conditions to keep the olive oil quality,using the following operations: washing, decanta-tion, centrifugation and filtration [1]. This product isstrictly olive oil and has a large nutritional, sensoryand commercial quality and is a main ingredient ofthe Mediterranean diet. Nutritionally, olive oil is veryinteresting because of its high monounsaturatedfatty acid content (oleic acid) and its antioxidants,phenols, and vitamin E. The quality of olive oil depends on the type of olive,climate, soil conditions, harvesting method, olivematurity, extraction procedure and agronomic fac-tors [2,3,4]. In Spain, the main producer of olive oil, there areseveral olive varieties with different physico-chem-ical, nutritional and sensorial properties. Fromthese olives it is possible to obtain monovarietalolive oils with special and typical characteristics.The Aragón region is sixth in olive oil productionwith 58000 hectares dedicated to olive trees pro-ducing 15000 tons per year of olive oil, reaching15% of agronomy production in some areas suchas Bajo Aragon, in the Aragon region. Here, oliveoil production is traditionally important because ofthe presence of some native olives such asEmpeltre, which has been given the protected des-ignation of origin (PD) Bajo Aragón. The olive oilproduction in Aragon is distributed in three areas:Alto Aragón and Cinca River, Iberic Somontanoand Jalón River, and Bajo Aragón and Belchite.These areas have some native varieties associated[5]. One of these varieties is Verdeña, the mainvariety (50%) in the middle area-Somontano land-of Huesca city. In this area the Verdeña variety cov-ers 2000 hectares, being used as table olives andolive oil [6], however there is no information aboutthe properties of their monovarietal olive oils. Otherminor varieties in some Aragon areas are similar toVerdeña, like Verdilla and Royal; some authorshave described these varieties as identical toVerdeña [5,6].The aim of this work is to study the physico-chem-ical, nutritional and sensory properties of theVerdeña, Verdilla and Royal olive oils.

2. MATERIALS AND METHODS

SAMPLINGVerdeña (Olea Europea Viridula) [5] variety oliveswere handpicked from olive trees from Somontanoarea of Barbastro (Huesca). Verdilla and Royal vari-eties olives were also handpicked on the samedate in Calatayud (Zaragoza). The samples ofeach variety were picked from three orchards. For

each sample, 50 olives were picked from each treein a total of 60 trees per orchard. Only healthy fruitswere handpicked and immediately transported tothe laboratory.

MATURITY INDEX AND MORPHOLOGIC PARAMETERS OFTHE OLIVE FRUITSThe olives were carefully blended and 100 olives ofeach cultivar randomly taken for determining thematurity index (MI) as a function of fruit colour ofboth skin and pulp [7]. In this system a group of100 olives was separated into different categoriesin function of the colour of the skin and pulp: 0-Deep green; 1-Yellow green; 2-Turning colour withreddish spots; 3-Turning colour with red or lightpurple colour in all the fruit; 4-Black, without colourunder the skin; 5-Black, with colour in less than thehalf of the pulp; 6-Black, with colour in more thanthe half, but not in the stone; 7-Black, with colour inall the pulp. After that the MI was calculated as fol-lows:MI = (ax0+bx1+cx2+dx3+ex4+fx5+gx6+hx7)/100a,b,c,d,e,f,g, and h are fruit number of each cate-gory. Five olives from each variety were also randomlyselected in order to calculate the average weightand diameter. Then the skin, stone and pulp of fiveolives of each variety were separated using ascalpel in order to determine the percentages rep-resented by these parts of the fruit as a whole.

OIL EXTRACTIONAn Abencor© analyzer (MC2 System, Sevilla,Spain) was used for oil extraction [8]. The unit con-sists of three essential elements: the mill, the ther-mo-beater, and the pulp centrifuge. After process-ing, the oil was decanted, filtered through a cellu-lose filter, poured into dark glass bottles, andstored in the dark.

INDUSTRIAL OIL YIELDThe industrial oil yield was expressed as a percent-age of fresh olive paste weight using the equation:Oil yield = (V x d/W) x 100; where V is the volume of olive oil obtained (ml), d isthe density of the olive oil (0.915 g/ml), W is theweight of olive paste used.

ANALYTICAL DETERMINATIONSDeterminations of the regulated physico-chemicalquality parameters (free acidity, peroxide valueand UV absorption characteristics, K270 and K232),were carried out following the analytical methodsdescribed in Regulation EEC/2568/91 of theCommission of the European Union [9]. Free acidity, given as % of oleic acid, was deter-mined by titration of a solution of oil dissolved inethanol/ether (1.1) with 0.1 M potassium hydroxide

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ethanolic solution.Peroxide value, expressed in milliequivalents ofactive oxygen per kilogramme of oil (meq/kg) wasdetermined as follows: a mixture of oil and chloro-form-acetic acid was left to react with a solution ofpotassium iodide in darkness; the free iodine wasthen titrated with a sodium thiosulfate solution. K270 and K232 extinction coefficients were calculat-ed from absorption at 270 and 232 nm respective-ly, with a UV spectrophotometer Unicam 6405using a 1% solution of oil in cyclohexane and apath length of 1 cm.

DETERMINATION OF FATTY ACIDSThe fatty acid composition of the olive oil sampleswas determined by gas chromatography using amodified fatty acid methyl ester (FAME) method asdescribed by Frega and Bocci [10]. The FAMEwas prepared by vigorous shaking of a solution ofeach olive oil sample in n-hexane and 2N methano-lic potassium hydroxide. Chromatographic analy-ses were performed using a Hewlett Packard 5890gas chromatograph equipped with a flame ioniza-tion detector and a split-splitless injector. Theexperimental conditions used were: DB-225 col-umn (30 m x 0.25 mm i.d x 0.15 µm film thickness)(J&W Scientific, Agilent). The injector and detectortemperatures were maintained at 250°C. The oventemperature was programmed from 190°C (1 min)to 210°C at 4°C/min and maintained for 5 min,then heated to 215°C at 3°C/min and finally anisotherm for 18 minutes; the carrier gas was nitro-gen. Commercial mixtures of fatty acid methylesters were used as reference data for the relativeretention times.

α-TOCOPHEROL DETERMINATIONA solution of oil in hexane was analyzed by HPLC(HP series 1100) with a Zorbax SB-C18 phasereverse column (Agilent), which was eluted withacetonitrile/water (98:2 v/v) at a flow rate of 1ml/min. A photodiode array detector (G1315B,serie 1100) was used. Chromatograms were regis-tered at 295 nm.

TOTAL PHENOLS DETERMINATIONTotal phenols were extracted by a Solid PhaseExtraction System using C18 Isolute columns. A modification of the method of Favati et al. [11]was used. Colorimetric total phenols determinationwas done using the Folin and Ciocalteau method.Gallic acid was used as standard.

SENSORY ANALYSIS The sensory analyses of the samples were carriedout by selected and trained panellists fromAragon´s accredited panel and the ZaragozaFaculty of Veterinary Science, according to the

method described in Regulation EEC/796/2002[12]. The intensities of the positive (fruity, bitter andpungent) and negative (fusty, winey, musty, muddy,rancid, metallic and other) attributes were evaluat-ed for each oil sample, on a non-structured, 10 cmscale anchored by its origin. The presence of otherbouquets (sweet, ripened fruits, green) was alsostudied to define the sensory profile of the oliveoils. A spider graph was employed to obtain theprofiles for each oil.

COLOUR AND PIGMENTS MEASUREMENTThe entire visible spectra (380-770 nm) at 2 nmintervals was measured and recorded using aspectrophotometer Avantes Ava Spec 1024 afterzeroing the apparatus with n-hexane. Chlorophyll and carotenoid compounds (mg/kg)were determined using the method described byMínguez-Mosquera et al. [13]. This method con-sists of a quantitative estimation of chlorophyll andcarotenoid content by measuring the olive oilabsorbance at 670 nm 470 nm. The CIELAB colourcoordinates [14] of the olive oils were determinedfrom the spectra. Illuminant 65 was chosen as wellas observer CIE64. The following colour coordi-nates were determined: lightness (L*), redness (a*,red-green) and yellowness (b*, yellow-blue). Inaddition, the hue angle, which describes the hue orcolour (h*) [h* = tan-1(b*/a*)] and the saturationindex or chroma (C*) [C* = (a*2 + b*2) 0.5] thatdescribes the brightness or vividness of a colourwere determined.

STATISTICAL ANALYSISStatistical analysis was performed using the GraphPad Prisma 5.0 program. Significant differencesamong the varieties were determined using theANOVA and Bonferroni test (p<0.05) to discrimi-nate among the mean values.

3. RESULTS AND DISCUSSION

The results of the maturity index, morphologicindex and industrial oil yield of the olive oil of thethree varieties are shown in Table I. Maturity indexvalues were similar for the three varieties. For mor-phologic parameters, Verdilla olives had the higherweights. The highest pulp content was for olives ofVerdeña and this would be related to a higherindustrial oil yield. The differences in morphologicparameters were not significant in some cases,thus not considered to be useful for olive varietiescharacterization. These differences depend direct-ly on the harvest, climatic conditions, and otherfactors.The physico – chemical parameters of the studiedolive oils are shown in Table II. In all cases, theolive oils were classified as extra virgin olive oil if

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we use the limits established in European regula-tions [9]. In Table III we can see the fatty acid pro-file of the different olive oils. For the three olive oils,the percentage of the different fatty acids was with-in the regulatory limits [15] for extra virgin olive oil(linolenic ≤ 1%, arachidic ≤ 0.6% and gadoleic

≤ 0.4%) Verdeña olive oil had a palmitic acid con-tent (saturated) two times higher than the linoleicacid content (polyunsaturated). This behaviourwas described for Picual olive oil [16]. This indi-cates a good relationship unsaturated/saturated(not too high) and consequently the oxidation risk

Table I - Maturity index, morphologic index and industrial oil yield of the studied olives

Table II - Physico-chemical parameters of the studied olive oils

Table III - Fatty acid profile of the studied olive oils

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would be lower. MUFAs (monounsaturated fattyacids) are of great importance because of theirnutritional properties and role on the oxidative sta-bility of the olive oils, specially oleic acid [17]. Thehighest content of oleic acid was obtained inVerdilla olive oils corresponding to a lower contentin linoleic acid. The oleic acid content for the otherolive oils was similar. In all the varieties this contentwas around 80%, but for linolenic acid it was lessthan 1%. The linoleic acid content was higher inRoyal variety and similar to that described for theFrantoio Italian variety [18]. The MUFAs (monoun-saturated fatty acid) content was lower in Verdeñavariety and had significant differences to the othertwo varieties. The higher content for these fattyacids was obtained for Verdilla variety. The PUFAs(polyunsaturated fatty acids) content was lower inVerdilla variety in comparison with that obtained inRoyal variety. In this case significant differenceswere also obtained. Finally, SFAs (saturated fattyacids) content was higher in Verdeña variety thanin the other olive oils.In contrast, the oleic/linoleic acid ratio was inter-mediate for Verdeña variety, giving the higher valuefor Verdilla and the lower for Royal variety. In all thecases this value was above the minimum value of7. This relationship is useful for olive varieties char-acterization and for stability interpretation [19, 20]. For nutritional characterization, the total phenolscontent is shown in Table IV. The stability of oliveoil oxidation is directly related to this value [21, 22].In general, phenols content depends on the extrac-tion system, irrigation, or olive variety [23]. The val-ues obtained in this study ranged from 120 to 190mg/Kg and this means that these olive oils wouldhave a low phenol content, like other varieties fromAragon (Empeltre) [24]. The lower values wereobtained for Verdilla variety olive oil. Royal varietyhad the highest content of the three varieties.

The alpha-tocopherol contents for the olive oilsstudied are shown in Table IV. The tocopherol con-tent, especially alpha tocopherol, in conjunctionwith the total phenols content contribute to theoxidative stability of the olive oil and thus to theirshelf life [25]. The values ranged from 90.97 mg/Kgto 104.7 mg/Kg. These levels are in concordancewith other values described for virgin olive oils (50-200 mg/Kg) [26]. The Royal variety olive oil had ahigher content than the Verdeña variety.The chlorophyll and carotenoid content and thecolour parameters of the olive oils studied areshown in Table IV. The pigments quantity dependson the olives’ maturity and variety. The valuesobtained for chlorophyll and carotenoids had signif-icant differences for the three varieties and are con-sistent with the values obtained for lightness. Theselast values were the higher for the Verdeña olive oils(having the lower pigment contents). The values forpigments have also a concordance with the colourintensity values (C*). This value was the lower forVerdeña variety. The other parameters had also sig-nificant differences for the olive oils of the three vari-eties. The positive values for b* parameter indicat-ed the yellow colour for these olive oils.The results of sensory analysis of the olive oils areshown in Figure I. The olive oils studied do notpresent negative attributes. This result, in additionto the ones shown in Table II will confirm their clas-sification, as extra virgin olive oil. The sensory pro-files of the three olive oils were different. TheVerdeña olive oil was fruity and had importantgreen notes. These characteristics made this oliveoil a monovarietal one (not very useful) or thatcould be used in coupages (assembly) by beingmixed with sweeter olive oils like Empeltre. TheRoyal variety olive oil had characteristic spicynotes and Verdilla variety olive oil had both sweetand spicy notes.

Table IV - Total phenol, α-tocopherol, chlorophyll and carotenoid contents and colour parameters (L*, a*, b*, h*, C*) of the studied olive oils

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4. CONCLUSIONS

The characterization of the Verdeña, Verdilla andRoyal varieties olive oils were carried out. In allcases, interesting physico-chemical, nutritionaland sensory properties have been discovered.From the different results obtained for the threevarieties of olive oil, we could conclude that theyare really different varieties and not just a denomi-nation question. Therefore more studies are need-ed to further our knowledge.

5. ACKNOWLEDGMENTS

We thank Zaragoza University and Aragon Govern-ment for helping us to buy the Abencor instrument.We also thank the Aragon Government for giving afellowship to Marta Benito.

REFERENCES

[1] R. Aparicio, J. Harwood, Manual del aceite deoliva. AMV Ediciones. Mundiprensa. (2003).

[2] M. Benito, R. Oria, A.C. Sánchez-Gimeno,Influencia del retraso en el procesado de lasaceitunas tras la recolección, en parámetrosfísico-químicos y nutricionales del aceite deoliva de la variedad Racimilla, Grasas yAceites 60, 382-387 (2009).

[3] M.D. Salvador, F. Aranda, S. Gómez-Alonso,G. Fregapane, Influence of fruit ripening onCornicabra virgin olive oil quality. A study offour crop seasons. Food Chemistry 73, 45-53 (2001).

[4] G. Beltran, M.P. Aguilera, C. Del Río, S. Sán-chez, M. Martínez, Infuence of fruit ripeningon the natural antioxidant content of Hoji-blanca virgin olive oils. Food Chemistry 89,207-215 (2005).

[5] J.M. Priego, Las variedades del olivo en Ara-gón y Rioja. Servicio de Publicaciones Agrí-colas. Ministerio de Fomento. DirecciónGeneral de Agricultura y Montes (1930).

[6] J. Viñuales Andreu, Variedades de olivo delSomontano. Instituto de Estudios Altoarago-neses (2007).

[7] M. Hermoso, M. Uceda, A. García, B.Morales, M.L. Frías, A. Fernández, Elabora-ción de aceite de oliva de calidad. Conseje-ría de Agricultura y Pesca, Serie Apuntes5/92. Sevilla. España (1991).

[8] J.M. Martínez-Suárez, E. Muñoz Aranda, J.Alba Mendoza, A. Lanzón Rey, Informesobre la utilización del Analizador de Rendi-mientos Abencor. Grasas y Aceites 26, 379-385 (1975).

[9] EEC. Reglamento (CEE) nº 2568/91 relativoa las características de los aceites de oliva yde los aceites de orujo de oliva y sobre susmétodos de análisis, J. Eur. Comun. L248,1-82 (1991).

[10] N. Frega, F. Bocci, L’analisi rápida dell’olio dioliva (3) (28). Laboratorio 2000, Italia (2001).

[11] F. Favati, G. Caporale, M. Bertuccioli, Rapiddetermination of phenol content in extra virginolive oil. Grasas y Aceites 45, 68-70 (1994).

[12] EEC. Reglamento (CE) nº 796/2002 quemodifica el Reglamento (CEE) nº 2568/91relativo a las características de los aceitesde oliva y de los aceites de orujo de oliva y

Figure 1 - Sensory profile of the studied olive oils

46


sobre sus métodos de análisis. J. Eur.Comun, L 128, 8-22 (2002).

[13] M.I. Mínguez-Mosquera, L. Rejano-Navarro,B. Gandul-Rojas, A.H. Sánchez-Gómez, J.Garrido-Fernández. Color-pigment correlationin virgin olive oil. Journal of the American OilChemists Society 68, 332-336 (1991).

[14] CIE. Colorimetry. Publication 15.2. CentralBureau, Vienna (1986).

[15] EEC. Reglamento (CE) nº 1989/2003 de laComisión, de 6 de noviembre de 2003 quemodifica el Reglamento (CEE) nº 2568/91relativo a las características de los aceitesde oliva y de los aceites de orujo de oliva ysobre sus métodos de análisis. J. Eur.Comun. L295:57 (2003).

[16] M.V. Moreno Romero, F. Espinola Lozano,Características y parámetros de calidad delaceite de oliva virgen Picual de Jaén. Alimen-tación, equipos y tecnología 4, 45- 48 (2004).

[17] M.C. Aguilera, M.C. Ramírez-Tortosa, M.D.Mesa, A. Gil. Do MUFA and PUFA have ben-efitial effects on development of cardiovas-cular disease? S.G. Pandai Ed. Recentresearch developments in lipids. Advancesin Lipid Research 369-390 (2000).

[18] M. Uceda, M. Hermoso, La calidad del acei-te de oliva en El cultivo del olivo, D. Barran-co, R. Fernández- Escobar y L Rallo (eds).MundiPrensa, Madrid, p. 589-614 (2001).

[19] A.K. Kiritsakis, G.D. Nauos, Z. Polymenou-poulos, T. Thomai, E.Y. Sfakiotakis, Effect offruit storage conditions on olive oil quality.Journal of the American Oil Chemists Socie-ty 75, 721- 724 (1998).

[20] L. Roda, Contribución de los ácidos grasosy de los compuestos antioxidantes del acei-

te de oliva virgen a su estabilidad oxidativa.Trabajo Fin de Carrera. Universidad de Sevi-lla (1997).

[21] F. Gutiérrez, B. Jiménez, A. Ruíz, A. Albi,Effect of olive ripeness on the oxidative sta-bility of virgin olive oil extracted from the vari-eties Picual and Hojiblanca. Journal of Agri-cultural and Food Chemistry 47, 121-127(1999).

[22] M.D. Salvador, F. Aranda, G. Fregapane,Contribution of chemical components ofCornicabra virgin olive oils to oxidative sta-bility. A study of three successive crop sea-sons. Journal of the American Oil Chemist´sSociety 76, 427-432 (1999).

[23] M. Solinas, Analisi HRGC delle sostanzefenoliche di oli vergini di oliva in relazione algrado di maturazione e alla varietá delleolive. Rivista Ital. Sostanze Grasse 64, 255-262 (1987).

[24] M.S. Gracia Gómez, Composición químicade distintas calidades de aceites de olivavirgen de la variedad Empeltre en el BajoAragón. Grasas y Aceites 52, 52- 58 (2001).

[25] R. Mateos, M.M. Domínguez, J.L. Espartero,A. Cert, Antioxidant effect of phenolic com-pounds, alpha-tocopherol and other minorcomponents in virgin olive oil. Journal ofAgricultural and Food Chemistry 51, 7170-7175 (2003).

[26] J. Mataix Verdú, E. Martínez de Vitoria,Bases para el futuro. El aceite de oliva. Ed.Consejería de Agricultura y Pesca. Junta deAndalucía. Sevilla (2001).

Received, January 24, 2011Accepted, February 11, 2011

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This study was conducted to evaluate the influence of the quality of water used in irrigat-ing ‘Nabali Muhasan’ olive trees (Olea europaea L.) grown in an orchard in the north-eastof Jordan on the sensory attributes of the produced olive oil. The obtained results revealedthe high salinity of the well water used in this experiment. On the other hand, the proper-ties of rain water were significantly better than the standards for ground water, whereasreclaimed wastewater achieved the Jordanian standard, except for Na+ and HCO3ˉ. Oliveoils made from trees that received well and rain water, both in 2006 or in 2007 crop sea-sons were free from any defect and were classified as extra virgin olive oil. On the otherhand, the negative attributes fusty, musty, winey and muddy were detected in olive oilsfrom trees receiving reclaimed wastewater in the 2006 crop year; whereas only two nega-tive attributes i.e fusty and musty were detected in the 2007 crop season. However, moreresearch is needed to clarify the suitability of reclaimed wastewater for irrigating olivetrees.Keywords: Olive oil, reclaimed wastewater, quality, irrigation, sensory properties,

Influenza della qualità delle acque usate per l’irrigazione sulle proprietàsensoriali dell’olio d’olivaQuesto studio è stato condotto per valutare l’influenza della qualità delle acque, utilizzatein ‘Nabali Muhasan’ per l’irrigazione degli alberi d’olivo (Olea europaea L.) coltivati in unfrutteto nel nord-est della Giordania, sulle caratteristiche sensoriali dell’olio d’oliva pro-dotto.I risultati ottenuti in questo esperimento hanno rivelato l’elevata salinità delle acque dipozzo utilizzate.D’altra parte, le proprietà delle acque piovane sono state significativamente migliori rispet-to a quelle delle acque superficiali mentre le acque reflue depurate hanno raggiunto glistandard giordani ad eccezione di Na+ e HCO3ˉ.Gli oli d’oliva ricavati dalle olive di alberi irrigati con acque di pozzo e acque piovane nellestagioni delle colture 2006 e 2007, sono esenti da difetti e sono stati classificati come olioextra vergine di oliva.Invece, gli attributi negativi riscaldo, muffa, avvinato e morchio, sono stati individuati inoli di oliva ottenuti da alberi che hanno ricevuto irrigazioni, nelle stagioni delle colture2006, con acque reflue depurate, mentre solo due attributi negativi cioè riscaldo e muffasono stati rilevati in oli d’oliva ottenuti nelle colture 2007.Tuttavia, sono necessarie ulteriori ricerche per chiarire l’idoneità delle acque reflue depu-rate per l’irrigazione delle piante di olivo.Parole chiave:: Olio di oliva, acque reflue depurate, qualità, irrigazione, proprietà senso-riali

Influence of water quality used inirrigation on the sensory

properties of olive oil

A.K. Alsaed1*Kh.M. Al-Ismail1

S. Ayub2

R. Ahmad3

S. Abdel-Qader1

1Department of Nutrition and Food Technology, Faculty of Agriculture,University of Jordan

Amman, Jordan2National Center for

Agricultural Research and Extension, Jordan

3Industrial Chemistry Center, The Royal Scientific Society,

Amman, Jordan

*CORRESPONDING AUTHOR: A.K. Alsaed

e-mail: [email protected]: 00962 6 5332536

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INTRODUCTION

Jordan is facing a drastic water shortage problemand is classified among the poorest countries withregard to water availability. Consequently, one ofthe strategies to be adopted to alleviate the watershortage problem in the country is to use treatedsewage water (reclaimed wastewater) for irrigationpurposes. As a result, some wastewater treatingplants were established to treat the wastewater andto reuse it in irrigating fruit trees, including olives, inthe last few years [1].There is a shortage of the basic knowledge regard-ing most of the scientific aspects of locally pro-duced olive oil i.e its sensory characteristics [2],factors affecting its quality and the effect of irriga-tion water on its quality. It is well established thatvarious factors contribute to olive oil quality. Thosefactors include general climate, soil conditions, fer-tilizer, fruit maturity, processing and storage condi-tions, etc.Methodology for the evaluation of the sensory qual-ity of olive oil have been described by manyauthors [3]. General basic vocabulary to be usedin the sensory analyses of olive oil was given by theInternational Olive Oil Council. The IOOC alsoissued several standards describing the sensorymethods to be used for assessing the sensoryquality of olive oil and how to analyze the resultsstatistically [2, 4, 5, 6]. According to the IOOC standards [4], sensoryproperties of olive oil are being classified to posi-tive and negative attributes. The positive attributesinclude fruity, bitter and pungent. Negative attrib-utes include fusty/muddy sediment, musty humid,winey-vinegary acid-sour, metallic, rancid andother negative attributes.It has been established that either reducing orincreasing the quantity of water used in irrigatingolive trees affects the quality of olive oil. For exam-ple, it was reported that whereas intermediate irri-gation levels of 40 and 57% evapotranspirationproduced olive oils with fairly higher fruitiness thanthe bitterness and pungency intensity levels,increasing the irrigation level to 71% produced oilsthat had very little bitterness or pungency [7]. Fur-thermore, the influence of water stress and the tim-ing of irrigation of olive trees of the cultivar “Picual”on the quality of oil produced was studied [8]. Itwas found that the different irrigation treatmentspositively affected the yield and the oils producedfrom rainfed trees contained the highest polyphe-nolics, bitterness and stability compared with theother irrigation treatments. The effect of irrigation ofolive trees with different water volumes on the char-acteristics of olive fruits and the produced olive oilswere also studied [9]; it was found that oilsobtained from olive trees that received rain water or

were irrigated at a level of 100% evapotranspira-tion. were free from any sensory defects and wereclassified as extra virgin olive oil. A recent studyregarding the effect of irrigation of olive groves withtreated municipal wastewater on the microbiologi-cal quality of soil and fruits was conducted [10]. Itwas reported that no significant contamination wasrecorded on fruits harvested by irrigated olivetrees. The effect of different irrigation strategies onthe composition and quality of “Cornicabra” virginolive oil was studied [11]. Their results showed nostatistically significant differences in the positivesensory attributes including bitterness, betweenthe olive oils obtained under rain-fed and irrigationconditions.Due to the scarcity of water resources in arid andsemi-arid areas, reclaimed wastewater reuseappears to be the most suitable solution [12].Reclaimed wastewater reuse provides additionalwater resources, ensures the balance of naturalwater cycle and protects the environment. Further-more, such reclaimed wastewater when reused inagriculture brings more benefits that resulting in anincrease in crop yield and the improvement of irri-gation efficiency. For example, irrigating olive treeswith wastewater resulted in a yield increase of 50%[13]. It was reported that the application of treatedurban wastewater enhanced olive productivity, lim-ited alternate bearing and allowed the productionof safe-high value olive yields, having excellent fruittechnological parameters and produced good oilquality [14]. In addition, municipal wastewater sub-jected to simplified treatments can be used as bothirrigation water and a source of mineral elements.As for the infection risk among fruit trees, olive iswell fit for wastewater irrigation because its harvestperiod is about one month after the last waterapplication. Finally, it was concluded that irrigationwith treated wastewater did not affect oil qualityparameters significantly and no dangerous faecalcontamination was recorded on the fruit [14]. The effect of different irrigation strategies on thecomposition and quality of Cornicabra virgin oliveoil was also studied [11]. Different irrigation treat-ments, based on regulated deficit irrigation (RDI),100% and 125% evapotranspiration, and rain-fedas control, were applied to a traditional oliveorchard (cv. Cornicabra) in a randomized com-plete-block design with four replications. The aver-age olive production of the trees grown under rain-fed conditions was much lower, about 35%, thanthat obtained by applying the different irrigationtreatments studied, between which practically nodifference were observed. The total phenol contentwhich affects the sensory bitterness in the oils,decreased significantly as the amount of suppliedwater increased. Notably, one of the RDI strategiesproduced olive oil similar in composition and qual-

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ity to that obtained by 100% evapotranspiration butwith reduced water usage.A comparative study was conducted to evaluatethe influence of seven different levels of water usedin irrigating olive trees grown in Sacramento Valleyof California on olive oil quality [15]. Water wasapplied differentially by drip irrigation at rates rang-ing from 15-107% evapotranspiration. Theobtained results showed that total polyphenol lev-els and oxidative stability decreased as the treesreceived more water. Oil sensory properties offruitiness, bitterness and pungency all declined inoils made from trees receiving more water. The low-est irrigation levels produced oils that were charac-terized by excessive bitterness, very high pun-gency, and woody, herbaceous flavor. Intermediateirrigation levels (40% to 57% evapotranspiration)produced oils with balance, complexity, and char-acteristic artichoke, grass, green apple, and someripe fruit flavors. Higher irrigation levels producedrelatively bland oils with significantly less fruitinessand almost no bitterness or pungency.The effect of irrigation with three types of water(rain water, fresh well water and reclaimed waste-water) on the olive oil chemical quality (acidity, per-oxide, total phenols, sterols and fatty acids profile)is documented in another paper [16]. The aim ofthis study was to determine the effect of waterquality used in irrigating olive trees (rain water,fresh well water and reclaimed wastewater) on thesensory properties of the olive oil. Therefore, theoverall objective of this two-component olive oilsensory/chemical quality study was to find the suit-ability of reclaimed wastewater for irrigation of olivetrees. As mentioned before, many factors affectolive oil quality; attention was paid to stabilize allthose factors except quality of water used in irrigat-ing olive trees i.e. the olive trees were in the sameorchard, having the same climate, fertilizer, fruitmaturity, storage and processing conditions etc.

MATERIALS AND METHODS

EXPERIMENTAL DESIGN, OLIVE ORCHARD AND IRRIGA-TION TREATMENTSThe experiment was carried out during 2006 and2007 growing seasons (from March to Novem-ber), using 15-year old ‘Nabali Muhasan’ olivetrees planted at a 3x6 m distance (density of 550trees/ha). The orchard is located in Alhashemiaarea in the north-east of Jordan. Soil texture issilty clay; the summer temperature in this areaoften exceeds 30°C and the average winter rain-fall is about 100-200 mm annually.The experiment was designed as a randomizedblock with three treatments. Each treatment con-sisted of 20 trees in two rows. All 20 olive trees ineach treatment used for harvest were irrigated

with the same amount of water (300 l) twicemonthly except for the 20 trees on the rain waterexperiment, which was considered as the control.Here only 75 l water was given to each olive treefor sustainability purposes, the same as it is prac-ticed by the neighboring olive farmers. Irrigationtreatments began on July 16 and continued until15 November resulting in 8 irrigations. The select-ed trees were irrigated with reclaimed wastewater(treated wastewater) which was taken from aneighboring treating plant, fresh well water andrain water. The total harvest for the 20 trees foreach treatment was taken and used for oil extrac-tion.

ANALYTICAL DETERMINATIONS IN IRRIGATION WATER Three samples of each types of water (wellwater (WW), rain water (RW) and treated waste-water (TWW)) were collected in 2006 and 2007and analyzed according to standard methods[17]. The measured parameters were pH, elec-trical conductivity (EC), total suspended solids(TSS), total dissolved solids (TDS), and Ca++,Mg++, Na+, Cl¯, HCO3¯, SO4

=, Fe, Zn, Mn, Cd, Cu,Pb, and Hg.

OLIVE FRUITS HARVESTING AND PRESSINGThe 20 olive trees for each treatment were har-vested by hand during the 1st week of Decemberof each year of the experiment period. Olive fruitswere sent to the mill in a 25 kg ventilated plasticboxes. Olives were stored at ambient temperature(15-20˚C) for a maximum duration of 48 hrs. Theolive fruits were pressed in a quality certified 3-phase centrifugal system commercial olive mill(Al-Zyoud oil mill, (Rapenelli, Foigno, Italy)) whichis only 1 km from the olive orchard. Olive leaveswere removed by a blower; the fruits were washedand then crushed to a paste. Malaxation was car-ried at 28±2˚C. Water was then added followedby decantation and finally centrifugation. Olive oilsamples were poured into 16 kg coated tin con-tainers.

SENSORY EVALUATION OF OLIVE OIL SAMPLESSensory evaluation was carried on the olive oilsamples after two months of storage at ambienttemperature by 12 trained olive oil tasters usingthe official International Olive Oil Council (IOOC)testing methodologies [2, 4, 5, 6]. The panel ofassessors from the Department of Nutrition andFood Technology at the Faculty of Agriculture ofthe University of Jordan was recognized by IOOC. Olive oil samples were tested by the panel twiceon two different days using a sensory testing lab-oratory, having booths designed according to theIOOC standard. The new version (issued in 2007)of the IOOC sensory profile sheet was used for oil

50


evaluations (Fig. 1). Each taster was placed in thebooth allotted to him and was given the profilesheet to fill in and the olive oil sample to examine,and he was asked to mark the intensity for eacholive oil attribute on a scale from 0-10, where 0.0means that the attribute such as fruity is of very lowintensity, while 10 means that it is of very highintensity. At the end of the session, the filled insheets were collected by the panel leader and theaverages of the median for each olive oil attribute,as well as the classification of the oil, were deter-mined using a special computer program providedby the IOOC. The program is based on the IOOCstandard for the organoleptic assessment of virginolive oil where olive oil is classified to several class-es according to the defect level. Olive oil shall beclassified in the extra virgin category when themedian of the defects is equal to 0 and the medianof the fruity attribute is more than 0; in the virgincategory the median of the defects is more than 0and less than or equal to 3.5 and the median of thefruity attribute is more than 0; in the ordinary virgincategory the median of the defects is more than3.5 and less than or equal to 6.0 or when the medi-an of the defects is less than or equal to 3.5 and themedian of the fruity attribute is equal to 0; in thelampante virgin category the median of the defectsis more than 6.0.In an attempt to confirm the effect of reclaimedwastewater on the sensory properties of olive oil,

additional three samples from oils obtained in the2006 harvesting season from trees receivingreclaimed wastewater were examined.

STATISTICAL ANALYSISThe mean of each tasting session for each olive oilsample for the three treatments (two samples/treat-ments) was calculated. Data were analyzed usingone-way analysis of variance (ANOVA) [18]. Meanswere compared using Duncan’s multiple rangetests. Significance was set at (p ≤ 0.0 5). Themeans for each sensory olive oil attribute was alsocalculated using the IOOC statistical computerprogram [6] and accordingly the classification ofthe oil was determined.

RESULTS AND DISCUSSION

CHARACTERISTICS OF IRRIGATION WATERChemical and physical characteristics of well water(WW), rain water (RW), and treated wastewater(TWW) are shown in Table I. It is clear from data inTable I, that the levels of many quality parameters(EC, TDS, Ca++, Mg++, Na+, Cl¯, and SO4

=) of WW didnot achieve the Jordanian Standards (JS) forground water [12]. The high levels of these param-eters indicate the high salinity of this type of water(WW). Such nonconformity may have some nega-tive effect on the sensory quality of the olive oil[19]. However, it is well established that olive treescould tolerate a relatively high concentration ofsalinity (TDS). On the other hand, and as it isexpected, the properties of RW were significantlybetter than the national standards for both groundwater and treated waste water. Regarding theTWW, it can be observed that its propertiesachieved the JS for TWW except for Na+ andHCO3¯.

EFFECT OF HARVESTING SEASON AND IRRIGATIONWATER QUALITY ON THE SENSORY PROPERTIES OF THEOBTAINED OLIVE OILTable II lists the olive oil sensory properties asaffected by the different types of irrigation waterduring the two crop seasons studied in 2006 and2007. It is obvious from data in Table II that oliveoils made from trees that received the well and rainwater both in 2006 or in 2007 crop seasons werefree from any defect (having no negative attributesi.e. fusty, musty, winey, muddy, rancid, metallic,heated) and accordingly were classified as extravirgin olive oil. On the other hand, the negativeattributes fusty, musty, winey and muddy weredetected in olive oils made from trees receivingreclaimed wastewater in the 2006 crop year; onlytwo negative attributes i.e fusty and musty weredetected in 2007 crop season and as a result, theolive oil from the reclaimed treatment was classi-

Figure 1

51


fied as ordinary virgin in 2006 crop season and vir-gin in the 2007 crop season. The appearance ofthese defects might be ascribed to the composi-tion of the reclaimed wastewater.Olive oils in Table II were obtained using a com-mercial mill. It can also be observed that in 2007crop season the reclaimed wastewater treatmentproduced oils with better positive sensory attrib-utes in terms of fruity, bitter and pungent compared

with the 2006 crop season . In 2007, the oil made from well water irrigated trees(in spite of its high salinity (EC 4.47, Table I) hadthe highest flavor intensity of all the treatments forfruitiness (3.71), bitterness (2.38), and pungency(2.63) (Table II). Such results agree with thosereported by another study [19] which revealed thatolive trees of the cv. Barnes grown in Al-Naqabdesert of Palestine and irrigated with moderately

Table I - Properties and criteria of water used in irrigation of olive trees

Table II - Effect of type of water used in irrigation and harvesting year on the sensory attributes of olive oils produced by a commercial press

*) Means of triplicate; **) The letters a, b, c and d are used to refer to the presence or absence of significant differences between means. Means ineach row followed by the same letter are not significantly different at 95% confidence.

* ) Means of triplicate; **) The letters a, b, c and d are used to refer to the presence or absence of significant differences between means. Means ineach row followed by the same letter are not significantly different at 95% confidence.

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saline water (4.2 EC) produced oils of relativelyhigh quality. Rain water treatment also produced oils character-ized with high positive sensory attributes wheretheir figures did not differ significantly from those ofwell water treatment. Sensory results for the rainwater olive oil treatment were very similar to thoseobtained by other studies [9, 11], i.e the obtainedolive oils under the conditions of those studies wereclassified as extra virgin and oils from irrigatedregime were less bitter and less pungent than thoseof the rain-fed oil. However, higher values for fruiti-ness, bitterness and pungency were obtained forthe rain water olive oil by another study [11]. Suchvariation might be attributed to differences in olivecv., sensory panel and or agro-climatic conditions.It is apparent from data in Table II that the harvest-ing year affected significantly the sensory proper-ties of the produced olive oil. The 2007 crop sea-son was characterized by higher values for thestudied positive attributes and lower values for thestudied negative attributes (fusty, musty, muddyand winey) as compared to 2006 crop season.These results agree with those reported in anotherstudy [15] where the effect of irrigation levels fortwo crop seasons 2002 and 2003 on the sensoryand chemical properties of olive oil was studied.

EFFECT OF RECLAIMED WASTEWATER ON THE SENSORYPROPERTIES OF OLIVE OILRegarding the effect of reclaimed wastewater onthe sensory properties of olive oil, additional threesamples from oils obtained in the 2006 harvestingseason from trees receiving reclaimed wastewaterwere examined and the results are presented inTable III. It is highlighted that the obtained resultsassure the negative effect of reclaimed wastewateron the sensory attributes of the produced olive oil.Furthermore, data in Table III show that the oilmade from trees receiving reclaimed wastewater

was less intense in all positive sensory attributes(fruity, bitter and pungent) compared to oils madefrom trees receiving rain-fed or well water (Table II).The decrease in the intensity of fruity properties inthe 2006 season might have been due to the rela-tively lengthy duration between pressing and timeof conducting the sensory evaluation where it wasabout 2 months compared to 2 weeks for the 2007season. There were also the drawbacks of thereclaimed wastewater composition on the sensoryproperties of olive oil. However, no publishedresults about the effect of treated wastewater onthe sensory properties of olive oil are available inthe literature for comparison purposes. According-ly, future research work should focus on the effectof irrigation with treated wastewater on the sensoryproperties of produced olive fruits as well as otherfruits or vegetables.

ACKNOWLEDGMENT

The researchers thank the Deanship of ScientificResearch of University of Jordan for their financingof this project. Due regards for Alfaqueh, A. profes-sor of human nutrition at the University of Jordan forreviewing the manuscript.

CONCLUSION

Olive oils made from trees that received the well andrain water both in 2006 or in 2007 crop seasonswere free from any defect and accordingly wereclassified as extra virgin olive oil. On the other hand,some negative attributes such as fusty, and mustywere detected in olive oils made from trees receivingreclaimed wastewater and as a result, the olive oilfrom the reclaimed treatment was classified as ordi-nary virgin in 2006 crop season and virgin in the2007 crop season. The appearance of these defectsmight be ascribed to the composition of the

Table III - Effect of irrigation with reclaimed wastewater on the sensory attributes of olive oil

*) Means of triplicate ** ) The letters a, b, c and d are used to refer to the presence or absence of significant differences between means. Means in each row followed bythe same letter are not significantly different at 95% confidence.

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reclaimed wastewater which exhibited nonconformi-ties regarding the concentration of Na+ and HCO3¯.The achieved results also showed that the oil com-ing from olive trees receiving reclaimed wastewaterwas less intense in all positive sensory attributes(fruity, bitter and pungent) compared to oils madefrom trees receiving rain-fed or well water.It is apparent from the obtained data that the har-vesting year affected significantly the sensoryproperties of the produced olive oil. The 2007 cropseason was characterized by higher values for thestudied positive attributes and lower values for thestudied negative attributes (fusty, musty, muddyand winey) as compared to 2006 crop season.However, further research work on the effect of irri-gation with treated wastewater on the sensoryproperties of olive oil as well as the sensory prop-erties of other fruits or vegetables is needed.

REFERENCES

[1] S. Al-Shdiefat, S. Ayoub, K. Jamjoum. Effectof irrigation with reclaimed wastewater onsoil properties and olive oil quality. JordanJournal of Agricultural Sciences; 5 (2) 128-141 (2009).

[2] Y. Tawalbeh, A. Alsaed, K. Al-Ismail. Influ-ence of olive harvesting dates on the senso-ry and chemical characteristics of the Jor-danian olive oil. Olivebioteq. 329-334, Nov.5-10, Mazara del Vallo, Marsala, Italy (2006).

[3] IOOC International Olive Oil Council.. Senso-ry analysis of olive oil method for theorganoleptic assessment of virgin olive oil.COI\T.20\Doc. no. 15\Rev.2 (2007).

[4] IOOC International Olive Oil Council. Senso-ry analysis of olive oil standard, sensoryanalysis: general basic vocabulary,COI/T.20\Doc No. 4. Madrid, Spain (1987a).

[5] IOOC. International Olive Oil Council. Senso-ry Analysis of Olive Oil Standard. Generalmethodology for the organoleptic assess-ment of virgin olive oil. COI\T.20\Doc. no.13\Rev.1 (1996).

[6] IOOC. International Olive Oil Council. Senso-ry analysis of olive oil standard. Glass for oiltesting, COI/T.20\Doc No.5. Madrid, Spain(1987b).

[7] P. M. Vossen, M.J. Berenguer, S. R. Grattan,V.S. Cornell. The influence of different levelsof irrigation on the chemical and sensoryproperties of olive oil. Acta Horticulture. 791,439-444 (2008).

[8] A. Castro, P. Fernandez, F. Aguilera, J.A.

Orgaz, B. Garcia. Oil quality and response toirrigation in traditional olive orchards. Olive-bioteq. November 5-10 Mazara del Vallo,Marsala, Italy (2006).

[9] M. Patumi, R.d’Andria, V. Marsilio, G. Fonta-nazza, G. Morelli, B. Lanza. Olive and oliveoil quality after intensive monocone olivegrowing (cv. Kalamata) in different irrigationregimes. Food Chemistry 77, 27-34 (2002).

[10] A.M. Palese, V. Pasquale, G. Celano, G.Figliuolo, S. Masi, C. Xiloyannis. Irrigation ofolive groves in southern Italy with treatedmunicipal wastewater: Effect on microbio-logical quality of soil and fruits. Agr. Ecosys.and Environ. 129, 43-51 (2009).

[11] A. Gomez-Rico, M.D. Salvador, A. Moriana,D. Perez, N. Olmedilla, F. Ribas, G. Fregapa-ne. Influence of different irrigation strategiesin a traditional Corincabra cv. olive orchardon virgin olive oil composition and quality.Food Chem 100, 568-578 (2007).

[12] M. Andre. Wastewater reuse in localized irri-gation. EAU & Irrigation: Water & IrrigationNews, October Issue (2003).

[13] A. Lopez, A. Police, A. Lonigro, S. Masi, A.M.Palese, G.L. Cirelli, A. Toscano, R. Passino.Agricultural wastewater reuse in southernItaly. Desalination 187, 323-334 (2006).

[14] Palese A., G. Celano, G. Figliuolo, S. Masi,C. Xiloyannis . Olivebioteq. November 5-10Mazara del Vallo, Marsala, Italy (2006).

[15] P. Berenguer, M.Vossen, S.R. Grattan,J.H.Connell, V.S. Polito. Tree irrigation levelsfor optimum chemical and sensory proper-ties of olive oil. Horticultural. Science. 41,427-432 (2006).

[16] K. M. Al-Ismail, A. K. Alsaed, R. Ahmad, S.Ayoub. Influence of irrigation water qualityon the chemical properties of olive oil. Riv.Ital. Sotanze Grasse 87, 84-89 (2010).

[17] A.D. Eaton, L.S. Clesceri, A.E. Greenberg.Standard Methods for the Examination ofWater and Wastewater. APHA, AWWA, WEF,19th edn. Washington DC. (1995).

[18] Statistical Analysis System. SAS Users Guide,SAS Institute Inc. Cary, NC, US (1994).

[19] Z. Wiesman, D. Itzhak, N. Ben Dom. Opti-mization of saline water level for sustainableBarnea olive and oil production in desertconditions. Scientia Horticulturae 100, 257-266 (2004).

Received, November 25, 2010Accepted, January 31, 2011

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The volatile profile of some Algerian virgin olive oils was established using headspacesolid phase micro-extraction (HS-SPME), coupled to GC-MS and GC-FID. Seventy-eightcompounds were identified and quantified. The results showed that the most importantcontributors to Algerian olive oil aroma are C6 aldehydes, alcohols and esters. These com-pounds are produced through lipoxygenase pathway. Significant differences in the propor-tion of volatile constituents from oils of different varieties and geographical origins wereproved. The results indicate that genetic factor and geographical origins influence thevolatile production.Key words:: virgin olive oil, solid phase micro-extraction, volatile profile, lipoxygenasepathway

Analisi di alcuni oli di oliva vergini algerini con micro-estrazione a fase soli-da con spazio di testa accoppiata alla gascromatografia/spettrometria dimassaÈ stato valutato il profilo della frazione aromatica volatile di alcuni oli di oliva vergini alge-rini utilizzando la micro estrazione in fase solida con spazio di testa (HS-SPME), accop-piato alla GC-MS e GC-FID. Settantotto composti sono stati identificati e quantificati. I risultati hanno mostrato che i composti più importanti al contributo dell’aroma dell’oliod’oliva algerino erano le C6 aldeidi, gli alcoli e gli esteri. Questi composti sono prodotti attraverso il meccanismo della lipossigenasi. Sono state dimostrate differenze significative nella percentuale delle componenti volatili inoli di diverse varietà e origini geografiche.I risultati indicano che il fattore genetico e le origini geografiche influenzano la produzio-ne dei composti volatili.Parole Chiave:: olio d’oliva vergine, micro-estrazione in fase solida, profilo volatile, per-corso della lipossigenasi

Analysis of some Algerian virginolive oils by headspace solid phasemicro-extraction coupled to gaschromatography/massspectrometry

S. Nigria

R. Oumeddoura*X. Fernandezb

a Laboratoire d’AnalysesIndustrielleset Génie des Matériaux,Université 08 mai 1945 Guelma- Algeriab Laboratoire de Chimie desMolécules Bioactives et desArômes, UMR CNRS 6001, Faculté desSciences de Nice Sophia-Antipolis, France

*CORRESPONDING AUTHORProf. Rabah OumeddourLaboratoire d’Analyses Industrielleset Génie des MatériauxUniversité 08 mai 1945 GuelmaB.P 401 Guelma 24000 AlgeriaTel.: +213 662123098Fax: +213 37207268.e-mail address: [email protected]

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1. INTRODUCTION

Olea europea L. is one of the most important cropsin Mediterranean countries especially Spain, Italy,Greece, Tunisia and Algeria. Virgin olive oil is oneof many vegetable oils, which are consumed with-out refining and it preserves its typical aroma [1, 2].A large increase in demand for virgin olive oil ofgood quality is due to its health virtues andorganoleptic properties [3]. Several works demon-strated that some non volatile compounds are relat-ed in a direct and an indirect way to the positiveeffects on the human health [4, 5]; whereas thesensory attributes are directly ascribable to thestrong stimulation of the human sensory receptorsby both volatile and some non volatile compoundspresent in virgin olive oil [6-8].The identification of the compounds contributing tothe aroma of virgin olive oil is considered to be akey for quality and authentication control. In fact,volatile compounds of olive oil are of great interestsince they are related to its quality and are used todetect adulteration and rancidity or to determinethe variety of olive used [9-12].The characteristic virgin olive oil aroma dependson many volatile compounds that derive from thedegradation of polyunsaturated fatty acids througha chain of enzymatic reactions known as the lipoxy-genase (LOX) pathway [13]. In addition to C6 com-pounds, the volatile fraction of virgin olive oil showsa reasonable amount of C5 alcohols and C5 car-bonyl compounds. The detection of these com-pounds suggested the presence of an additionalbranch of the LOX pathway leading to the produc-tion of C5 compounds. This additional branch isactive during olive oil aroma biogeneration.The volatile composition of olive oils depends onthe level and activity of the enzymes involved invarious pathways, which are genetically deter-mined [14]. Other factors that influence thevolatiles are: climate, soil type [15, 16], degree ofripeness of the fruit [17], olive storage and oilextraction process [18].Several compounds that contribute to the aroma ofolive oil have been identified; these include aseries of saturated aldehydes that range from C6 toC12. The aldehydes content in green and blackolives are generally in the range of 50 and 75%respectively of the total volatile compounds [19].Fedeli [20] and Montedero, Bertuccioli and Anichi-ni [19] classified the aroma compounds of olive oilsas aliphatic and aromatic hydrocarbons, aliphatictriterpenic alcohols, aldehydes, ketones, ethers,esters, furan and thiophene derivatives.The formation of volatile compounds in olive fruit isrelated to cell destruction. An enzymatic processoccurs that involves hydrolysis and oxidation [6].The reactions proceed at a high rate, depending

on pH and temperature. 13-Hydroperoxy-9,11-octadecadienoic acid is formed by the enzymaticoxidation of linoleate. Subsequently, Hexenal isformed by the action of the enzyme aldehyde-lyase. Finally, 1-hexanol is formed by an enzymereduction process. In a different process, (Z)-3-hexenal and (E)-2-hexenal are formed by means ofaldehyde-lyase. Further reduction leads to the for-mation of (Z)-3-hexenol, (E)-3-hexenol and 1-hexenol [19]. Some products deriving from sugarfermentation and amino acid transformation werealso found. They were ethanol, ethyl acetate, aceticacid [9] and branched aldehydes, alcohols andacids. The latter are thought to be produced bymoulds during olive oil storage.Minor volatile compounds were observed in the vir-gin olive oil, among them, the hydrocarbons:octane and octene and the aldehydes: heptanal,octanal, nonanal, (E)-2-heptenal and 2,4-heptadi-enal isomers which are due to the autoxidationreactions that inevitably start after the virgin oliveoil has been extracted [21].Several analytical procedures have been used toseparate, identify and quantify the volatile compo-nents that characterize olive oil aroma [9]. Head-space solid-phase micro-extraction (HS-SPME)has been introduced as an alternative to thedynamic headspace technique and as a samplepreconcentration method prior to chromatographicanalysis [22]. HS-SPME is a rapid, sensitive andsolvent-free sampling technique developed byArthur and Pawliszyn [23] for water analyses andthen applied to food flavor analysis. The suitabilityof HS-SPME for qualitative and quantitative analy-sis of virgin olive oil volatile fraction has recentlybeen evaluated [21] and other authors [24-26]reported qualitative data obtained by applying thistechnique to virgin olive oil.In Algeria, the olive oil sector plays an importantrole. In 2006, the area covered by olive trees was263,352 HA with a production of 196,258 tons (17.8l oil/quintal of olives). The oliviculture is concentrat-ed mainly in the middle of the country, the kabyliewith 58.4% of the total oliviculture area [27]. To ourknowledge, this is the first study of aroma chemicalcomposition of the Algerian olive oils by HS-SPME-GC-MS. We present here the characterization bymeans of analyses of the volatiles of some varietiesgrown in Algeria. These varieties come from thesouth, northeast and middle of the country.

2. MATERIALS AND METHODS

2.1 OLIVE OIL SAMPLESSix virgin olive oil samples, extracted from olives ofthe Chemlel, Azeradj and Blanquette varieties fromdifferent regions of Algeria were used for the inves-tigation of this study. Olives were picked by hand at

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a known ripening degree. The olives were washedand deleafed, crushed with hammer crusher andthe paste mixed at 25°C for 30 min, centrifugedwithout addition of warm water and then trans-ferred into glass bottles.A and B are respectively Chemlel and Azeradj ofOued Souf in the south, C and D are Blanquette ofGuelma and Heliopolis in the northeast and E andF are Chemlel of Tizi Ouzou and Bejaia in the mid-dle of Algeria (Table I).

2.2 ANALYSIS OF VOLATILE COMPOUNDSVolatile profile of virgin olive oils were establishedusing SPME and gas chromatography coupled tomass spectrometry.Solid-phase micro-extraction (SPME) was used asa technique for headspace sampling of olive oils.For SPME analyses, Supelco SPME devices coat-ed with divinylbenzene/carboxen/polydimethyl-siloxane (DVB/CAR/PDMS) 50/30 µm was used[28]. Before use, fiber was conditioned as recom-mended by the manufacturer. 5 g of olive oil was placed in a 10 ml glass vialclosed with PTFE/silicone septum. Before extrac-tion, stabilization of the headspace in the vial wasobtained by equilibration for 60 min at 25°C. Afterthe equilibration time, the fiber was exposed to thehead space for 90 min [28] at room temperature at25°C; after exposure, the fiber was thermally des-orbed into GC and left in the inject port (equippedwith a 0.75 mm i.d. inlet liner) for 4 min. The injec-tor was set at 250°C and operated in the splitlessmode for 4 min. For each sample two types of analyses were car-ried out separately. GC analyses were carried outusing an Agilent 6890 Gas Chromatographequipped with a Flame Ionization Detector FID andwith fused silica capillary columns HP-1 (polydi-methylsiloxane 50 m × 0.2 mm i.d., film thickness:0.33 µm). The carrier gas was the helium (constantflow: 1 mL/min). The column temperature programwas as follows: 60 to 250°C at 2°C/min and thenheld isothermally for 20 min. The FID temperaturewas set at 250°C.

Each oil was analyzed using an Agilent 6890 GasChromatograph coupled to an Agilent 5973 MassSelective Detector (quadrupole), with fused silicacapillary columns HP-1 (polydimethylsiloxane 50 m × 0.2 mm i.d., film thickness: 0.5 µm ). Ovenconditions were the same as above for GC. Thetemperature of ion source and the transfer linewere 170 and 280°C; energy ionization, 70 eV;electron ionization mass spectra were acquiredover the mass range 35-350 atomic mass units.Before sampling, the fiber was reconditioned for 5min in the injection port at 250°C, and blank runswere carried out periodically during the study.

2.3 IDENTIFICATIONQualitative analysis of the volatile fraction was per-formed by means of HS-SPME/GC-MS. Volatilecompounds present in Algerian virgin olive oilswere identified by computer matching their massspectra with reference mass spectra with those ofthe laboratory library (wiley6N, Nist98). Then, com-paring their GC retention indices (RI) on apolar col-umn, determined relative to the retention times of aseries of n-alkanes (C5-C28) with those reported inthe Literature. The relative proportions of the con-stituents were obtained by FID peak area normal-ization. Three replicates were performed for eachsample; the average of these values and the stan-dard deviation were determined for each compo-nent identified.

3. RESULTS AND DISCUSSION

The aroma of olive oil is attributed to aldehydes,alcohols, esters, hydrocarbons, ketones, furans,and probably, other as yet unidentified volatilecompounds.Hexanal, (E)-2-hexenal, 1-hexenol, (Z)-3-hexenoland 3-methyl butanol are found in most virgin oliveoils in Europe. A previous study of Italian, Spanishand Moroccan extra virgin olive oil confirmed therichness of C6 volatile compounds in Italian oils butshowed that they were poor in fruity esters. Thefruity esters, ethyl isobutyrate, ethyl butyrate, ethyl2-methybutyrate, ethyl 3-methybutyrate and ethylcyclohexylcarboxylate were rich in Moroccan extravirgin olive oil [29].The headspace of Tunisian virgin olive oils wereparticularly rich in C6 aldehydes (Hexanal, (E)-2-hexenal and (Z)-3-hexenal) [26, 30].A list of different compounds identified by HS-SPME-GC-MS of Algerian virgin olive oil is reportedin Table II. Relative amounts of individual com-pounds are based on peak areas obtained withoutFID response factor correction.Ninety-two compounds were isolated, and seventy-eight were identified representing about 90% of thetotal amount. The number of identified compounds

Table I - Origin and variety of the six olive oils

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Table II - Compounds identified by HS-SPME/GC-MS in the six Algerian virgin olive oils

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continuous Table II

†) Compounds are listed in order of their elution time from a HP-1 column. Compositional values less than 0.1% are denoted as traces (tr). Presen-ce of a compound is indicated by its GC/FID percentage, without FID response factor correction; with standard deviation and absence of any is indi-cated by ND. ‡) RI = retention indices are determined on HP-1 column using the homologous series of n-alkanes. RIs < 900 were determined by GC-MS. A and B are respectively Chemlel and Azeradj of Oued Souf; C and D are Blanquette of Guelma and Heliopolis; E, F are Chemlel of Tizi Ouzou and Bejaia respectively

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depends on the quality of the virgin olive oil and themethodology adopted for their determination, thetemperature and the time used for obtaining thevolatile fraction [31]. The major compounds identified were hexanal, (E)-2-hexenal, (E)-2-hexenol, 1-hexenol and (Z)-3-hexenol.In addition, the aroma of Algerian virgin olive oilscontains a reasonable amount of pentene dimmersand C5 compounds (Table II). The presence ofthese compounds indicates the existence of anadditional branch of the LOX pathway, which leadsto production of C5 compounds through the alkoxyradical already proven in soy seeds. Similar resultswere observed for Tunisian virgin olive oils [2].The SPME analysis of Chemlel oils of Oued Soufallowed the identification among the main volatiles:hexanal 21.5%, (E)-2-hexenal 12.6%, (E)-2-hexenol9.6%, 1-hexenol 4.8% and formic acid 2.8%.The major constituents of Azeradj of Oued Soufwere (E)-2-hexenal 39.7%, hexanal 9.7%, (Z)-3-hexenol 3.4% and pentan-3-one 3.2%. The majorconstituents that characterize the olive oil head-space of Blanquette of Guelma were hexanal39.3%, pentan-3-one 4.9%, octane 4.8% and 1-hexanol 3.6%. The most important constituents ofBlanquette of Heliopolis were (E)-2-hexenal 4.6%,hexanal 12.9%, (E)-2-hexenol 8.8%, and 1-hexenol8.4%.The main constituents of volatile fraction of Chem-lel of Tizi Ouzou oils were octane 17%, which isdue to the autoxidation reactions [32], hexanal14.6%, 1-hexanol 13.2%, pentane 3.6%, 6-methyl-5-heptene-one 3.3% and nonanal 2.7%. The major constituents of Chemlel of Bejaia wereesters. Ethyl butyrate 15.0%, hexanal 13.1%,octane 11.2% and ethanol 5.9%. In all oil samples analyzed, the most representativeC6 compounds were aldehydes which accountedfor 87.9% to 40.6% respectively of the whole C6

fraction of Azeradj of Oued Souf and Chemlel ofBejaia region. Furthermore, the percentage of alco-hols differed according to the cultivar. It rangedfrom 47.3 to 11.4%, respectively in Chemlel of TiziOuzou and Blanquette of Guelma oils. Esters, alsodiffered from a cultivar to another; varying from46.9% to 0%, respectively in Chemlel of Bejaia andAzeradj of Oued Souf (Figure 1).The nature of the cultivar can be evidenced by adifferent concentration of C6 compounds arisingfrom the enzymatic oxidation of linolenic acid of oils(Table III).The results of Table III prove that the genetic effectrelated to the cultivar is one of the most importantaspects of the volatile composition of olive oil. It is worth mentioning that a non-identified compo-nent represented by 1203 retention indice is pres-ent in large amounts in Oued Souf olive oil.The geographical origin of olive growing can affect

A and B are respectively Chemlel and Azeradj of Oued Souf; C and D are Blanquette of Guelma and Heliopolis ; E and F are Chemlel of Tizi Ouzouand Bejaia respectively.

Table III - Composition of C6 compounds of oils from different Algerian cultivars

Figure 1 - Distribution of C6 Aldehydes, Alcohols and Esters in sixAlgerian olive oils. A and B are respectively Chemlel and Azeradj of Oued Souf; C and D areBlanquette of Guelma and Heliopolis; E and F are Chemlel of Tizi Ouzou andBejaia

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volatile composition of olive oils obtained by thesame cultivar. For example, the Chemlel in threedifferent regions: Oued Souf in south, Tizi Ouzouand Bejaia in the middle of countryl contain differ-ent terpenes. Chemlel of Oued Souf is character-ized by the presence of α-pinene. While γ-ter-pinene and para-cymene are the compounds thatcharacterized the volatile fraction of Chemlel of TiziOuzou but Chemlel Bejaia oil is characterized bythe presence of α-copaene.

4. CONCLUSION

The application of HS-SPME to the analysis ofsome Algerian virgin olive oils allowed the detec-tion of significant differences in the proportion ofvolatile constituents from oils of different varietiesand geographical origins.Seventy-eight compounds were isolated and iden-tified by GC-MS representing about 90% of thetotal amount.The most representative volatile compounds ofAlgerian olive oils are C6 aldehydes and alcoholsproduced enzymatically through the lipoxygenasepathway. The percentages of these products areknown. In addition, concerning LOX compounds,the aroma of Algerian virgin olive oils contains rea-sonable amount of pentene dimmers and C5 com-pounds.The results indicate that the genetic factor andenvironmental conditions influence the volatile pro-duction.

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[29] J. Reiners, W. Grosch, Odorants of virginolive oils with different flavor profiles, J.Agric. Food Chem. 46, 2754-2763 (1998)

[30] W. Zarrouk, B. Baccouri, W. Taamalli, A.Trigui, D. Daoud, M. Zarrouk, Oil fatty acidcomposition of eighteen Mediterranean oliveoil varieties cultivated under the arid condi-tions of Boughrara (southern Tunisia).Grasas y Aceites 60(5), 498-506 (2009)

[31] F. Angerosa, M. Servili, R. Selvaggini, A.Taticchi, S. Esposto, G.F. Montedero, Volatilecompounds in virgin olive oil: Occurrenceand their relationship with the quality. J.Chromatogr. A 1054, 17-31 (2004)

[32] R. Przybylski, N.A.M. Eskin. In: K. Warner,N.A. Michael, I.L. Champaign, ed. Methodsto measure volatile compounds and the fla-vor significance of volatile compounds.Methods to Assess quality and solubility ofoils and fat-containing foods. AOCS Press,USA, 107-132 (1994)

Received, November 23, 2010Accepted, January 24, 2011

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Eighteen margarines (6 stick, 12 tub) marketed in Poland were analyzed for their fatty acidcomposition including trans fatty acids (TFA), peroxide value, anisidine value, and acidvalue. The content of SFA, cis PUFA, cis MUFA in investigated fats was 39.7, 20.4, 32.9for stick, and for tub 32.2, 26.1, 39.5%, respectively. The content of TFA varied from 0-7.9% for tub and 0-10.9% for stick products. 58% of tub margarines contained to 0.7%TFA. This suggested they were formulated from interesterified or blended fats and oils.Compared with the products formulated in Czech and Turkey margarines marketed inPoland contained less TFA, but more than Danish and Austria margarines.PV ranged 0.1-2.0 for stick margarines and higher content was noticed for tub margarines0.2-4.1 meqO/kg (except for one sample with 10.32). The highest amounts of secondaryoxidative products were found in stick margarines especially where the amounts of PUFAwere higher. The TFA content in Poland has been reduced during the last several years.Keywords: Quality of European food - fatty acid composition – TFA – spreadable fats -lipid peroxidation products

La qualità delle margarine spalmabili prodotte in Polonia con particolareriferimento agli acidi grassi transSono state analizzate diciotto margarine (6 confezionate in tubo, 12 in vaschetta) per laloro composizione in acidi grassi tra cui acidi grassi trans (TFA), numero di perossidi evalore di acidità e di p-anisidina.Il contenuto di SFA, cis PUFA, cis MUFA nei grassi in esame era rispettivamente 39,7 -20,4 - 32,9% per quelli confezionati in tubo e 32,2 - 26,1 - 39,5% per quelli confezionatiin vaschetta.Il contenuto di TFA variava da 0 a 7,9% per i prodotti in vaschetta, e da 0 a 10,9% per iprodotti in tubo. Il 58% delle margarine in vaschetta conteneva lo 0,7% di TFACiò ha suggerito che i prodotti sono stati formulati con grassi e oli interesterificati o mis-celati. Rispetto ai prodotti formulati nella Repubblica Ceca e in Turchia, le margarine com-mercializzate in Polonia contenevano meno TFA, ma il contenuto era maggiore di quellecommercializzate in Danimarca e Austria.Il PV variava da 0,1 a 2,0 per margarine confezionate in tubo e un contenuto più alto, da0,2 a 4,1 meqO/kg, è stato rilevato per le margarine confezionate in vaschette (adeccezione di un campione con 10.32).I valori più elevati di prodotti di ossidazione secondaria sono stati rilevati in margarineconfezionate in tubo, in particolare quando le quantità di PUFA erano più elevate.Il contenuto TFA in Polonia è stato rilevato nel corso degli ultimi anni.PPaarroollee cchhiiaavvee: Qualità dei prodotti alimentari europei, composizione in acidi grassi, TFA,grassi da spalmare, prodotti della perossidazione lipidica.

The quality of polish spreadablefats especially with emphasis oftrans isomers content

A. �bikowskaa

M. Kowalskab*J. Rutkowskac

Warsaw University of LifeSciences (SGGW) aFaculty of Food SciencesbFaculty of Material Science,Technology and Design,Technical University in RadomcFaculty of Human Nutrition andConsumer Sciences Warsaw

*CORRESPONDING AUTHORM. KowalskaFaculty of Material Science,Technology and Design, Technical University in Radom27 Chrobrego Street26-600 Radom, Poland tel/fax 48 48 3617547 e-mail address: [email protected]

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1. INTRODUCTION

Margarines and shortenings are major sources oftrans fatty acids (TFA) in the diet. TFA occurringduring the hydrogenation of cis unsaturated veg-etable oils and marine oils allows the fatty acids topack together more closely than their correspon-ding cis isomers, resulting in improved functionalproperties, including harder texture and longershelf life [29].Hydrogenation is a process that reduces the rela-tive unsaturation of the oils and promotes geomet-ric and positional isomerization. Formation of transisomers affects the physical and chemical proper-ties of the final products, as trans isomers havehigher melting points and greater stability than cisones [21]. However, several published reportshave indicated that TFA have adverse effects onserum cholesterol, triacylglycerol (TAG) levels, andcoronary heart disease [11]; [30]; [31]. Further-more, trans isomers of fatty acids can interfere withthe metabolism of fatty acids – cis isomers valuablefrom the nutritional point of view [7]. It is claimedthat trans isomers are able to build into phospho-lipid membranes. This may change the propertiesof these membranes so as to cause neoplastic dis-eases. But so far there is no evidence that wouldconfirm these suggestions.Therefore, there is concern about the consumptionof products having high TFA, and efforts are beingundertaken to produce zero-trans products byalternative techniques to partial hydrogenation, forexample chemical and enzymatic interesterifica-tion, and fractionation of fats [18]; [21]; [26]; [27]. In response to these reports, many health profes-sional organizations have recommended reducedconsumption of food containing TFA. On January 1, 2003, Canada became the firstcountry in the world to introduce labelling of thecontent of trans fatty acids (TFA) in food products[30]. In July 2003, the US Food and Drug Adminis-tration issued regulations requiring the labelling oftrans fatty acids on packaged foods on or beforeJanuary 1, 2006 [11]. On July 11, 2003, the Food and Drug Administra-tion (FDA) published its regulations on nutritionlabelling. They require that TFA content bedeclared on the nutrition label of conventionalfoods and dietary supplements on a separate lineimmediately under the line for the content of satu-rated fatty acids, to be effective by January 1,2006. The FDA has decided not to distinguishbetween industrially produced trans fatty acidsand TFA of ruminant origin. Consequently dairyproducts will be labelled with the content of TFA[35].In the EU processed and packaged food should belabelled with a list of ingredients ranked according

to the amounts in grams in the products. If theproducts contain some partially hydrogenated fat,it should appear on the label, but not how muchTFA it contains. If food is provided with a healthclaim concerning its content of industrially pro-duced TFA, e.g. “free of trans fatty acids”, a decla-ration is required of the fat composition in the prod-uct, stating the amounts of mono-, poly-, and satu-rated fatty acids [30]. In March 2003, after consultations with the memberstates of the EU, the Danish Government decidedthat oils and fats with more than 2% content ofindustrially produced TFA would not be sold inDenmark after January 1, 2004 [2]. The objective of this study was to determinate thequality of chosen Polish trade spreadable fats fromthe year 2008, with special emphasis on the con-tent of fatty acids.

2. EXPERIMENTAL PART

2.1. MATERIALSIn Europe margarines/spreadable fats are regulat-ed and harmonized by European Council Regula-tion [9] No. 2991/94 of December 1994 layingdown standards for spreadable fats. Whereas themain aspect of the products is their fat content, theproducts are basically separated into three majorgroups according to their fat composition. The sec-ond group of the aforementioned classificationcomprises products in the form of a solid, mal-leable emulsion, principally of the water-in-oil type,derived from solid and/or liquid vegetable and/oranimal fats, suitable for human consumption, with amilk-fat content of not more than 3% of the fat con-tent. This group is separated into four categoriesaccording to their fat content: I – margarine (80-90% fat content),II – three-quarter-fat margarine (60-62),III – half-fat margarine (39-41),IV – fat spreads X% (<39; 41-60; 62-80%). X – any fat content between the indicated values.The tested margarines are consumed by the major-ity of the Polish population. 18 different brands ofmargarines were purchased from several super-markets in Poland in 2008 (Table I). 6 stick mar-garines (1-6) with fat content from 69.8 to 80.2%and 12 soft margarines (7-18) with fat content from25.6 to 60.3% were investigated. All manufacturers(A-F) produce their spreadable fats in Poland(Table I).

2.2. PREPARATION OF FAT SOLUTIONAll samples were melted in an oven at 50°C toobtain the fat phase. This phase was removed bycentrifugation and dried with anhydrous sodiumsulfate. All samples were frozen at -22°C untilanalysis.

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2.3. FATTY ACIDS COMPOSITIONPreparation of fatty acid methyl esters (FAMEs).Fatty acids were converted into their methyl estersaccording to the ISO official method [14].Gas chromatography analyses (GC) accordingISO standards [13]. The FAMEs were determinedusing a Shimadzu 17A gas chromatograph (Shi-madzu Co., Kyoto, Japan) equipped with a flameionization detector (FID). Analyses were performedwith an SP2560 column (100 m x 0.25 mm). The ini-tial temperature of 140°C was maintained for 5 min,then raised to 240°C at a rate of 4°C /min, and keptat 240°C for 10 min. The split ratio was 1:100, andthe carried gas was helium. The injector and detec-tor temperatures were 230 and 240°C respectively.FAMEs were identified by comparing their relativeand absolute retention times to those of authenticstandards of FAME obtained from Sigma ChemicalCo. All of the quantification was done by a built-indata-handing program provided by the manufac-turer of the gas chromatograph. The FA composi-tion was reported as a relative percentage of thetotal peak area.

2.4. QUALITY OF EXTRACTED FATS Peroxide value. The peroxide value (PV) was calcu-lated using the formula PV=[1000x(Vs-Vb)x N]/W,where Vs and Vb are the volumes of Na2 S2O3 usedfor titration of sample and blank, respectively, N isnormality of Na2 S2O3 and W is the amount of sam-ple (g). The PV is expressed as milliequivalents(meq) of active oxygen per kg of fat sampleaccording to ISO 3960 [12].Anisidine value. The anisidine value (AnV) wasdetermined colorimetrically according to ISO 6886[16].Acid value. The acid value (AV) was determinedaccording to ISO 660 [15].

3. RESULTS WITH DISCUSSION

Fatty acid composition of the samples is given inTable II. As seen from this table, various oils wereused to formulate spreadable fats. The relativeamounts (percentage of total fatty acids) of saturat-ed (SFA), cis-monounsaturated (MUFA), cis-polyunsaturated (PUFA), trans-fatty acids (TFA),the n-6/n-3 families ratio and P/S ratio of the 18

brands are listed in Table III.Margarines were high in palmitic acids (16:0) andwere in the range 15.9-23.9% (stick margarines)and 14.3-27.1% (tub margarines), as with Turkishmargarines [17], Pakistani margarines [5] and Por-tuguese margarines [33]. On the other hand, sam-ples 3, 5 (stick margarines) and 12, 15, 16, 17 (tubmargarines) contained high amounts of lauric(12:0) and myristic acids (14:0), and they were lowin TFA, with the exception of sample 3. Theseresults suggested that margarine samples wereproduced by blending palm oil fractions or coconutoil with hydrogenated vegetable fats. The contentof palmitic, myristic, and lauric acids (average28.4%), which have atherogenic potential [28], wassmaller than in Pakistan [5], similar to Portugal [33]but slightly higher than in Denmark (24%) [19].The amount of total SFA was high, between 30.9and 60.8 (stick margarines) and from 25.4 to 39.5%(spreadable fats). The total SFA content was high-er than other values recently reported by others forsimilar samples in Canada [27], France [4], andAustria [34], but corresponded to results in Portu-gal presented by Torres et al. [33]. The main cis-MUFA, oleic acids, ranged from 28.0to 38.5 (stick) and 25.7 to 52.4 (tub). The highestlevels of oleic acids were found in soft margarines;samples 7, 11, 15, 16 (Table II) probably have ahigh oleic sunflower oil content.Ovesen et al. [24] divided the margarines into threecategories based on their linoleic acid (LA) con-tent: margarines with less than 20% LA were hardmargarines; margarines with 20-40% LA weresemisoft margarines, and those with more than40% LA were soft margarines. According to thisclassification samples 2, 3, 4, 5 (intended for bak-ing from the producer’s view), but not sample 1, arehard margarines, but although the tub margarinesare intended for spreading on bread in the produc-er’s opinion, 5 samples (7,11,15,16; 18) shouldhave been classified in the hard margarines group(Table II). A similar situation was noted in Turkey byTekin et al. [32].The soft margarines, as generally expected, had ahigher content of PUFA than the hard margarines.EFA are included in the PUFA group, which is ofmajor importance for the biological and nutritionalvalue of these products. The average PUFA con-

Table I - Margarines used in the present investigation

65


Table II -

Fat

ty ac

id co

mpo

sition

(as p

ercen

tage

of to

tal fa

tty ac

ids) o

f Poli

sh sp

reada

ble fa

ts (m

ean±

SD)

* C

22:0

for 3

-0.2

% a

nd fo

r 4 -0

.3%

, 8-0

.3, f

or 1

3 -0

.6, 1

8 –

0.2%

, nd

– no

t det

ecte

d (<

0.1

%),

66


Table IV - Comparison of the main group of FA content of current margarines with those reported for Poland for the last 10 years

tent (20.4%) for soft-type Polish margarines washigher than those of recent results reported fromPakistan (17.5%) [5], but much lower than resultsreported from Turkey (32.4%) [32]. The hard mar-garines had PUFA values between 12.5 and 47.0%(average 26.1) and were lower than those reportedfor Turkish hard margarines (17.8) [32], but higherthan hard margarines from Pakistan (6.2%) [5].Regardless of the kind of margarines the PUFAcontent in margarines sold in Poland amounted to24% (average), less than in margarines sold in Por-tugal (32.7%) [33] and in Austria (31.4%) [34]. Onthe basis of values of PUFA it can be seen that mar-garines in Poland are not produced from oils rich inPUFA. Linolenic acid (ALA) amounts ranged from0.7 to 3.5% (stick margarines) and 0.1 to 4.3% (tubmargarines). 11 brands had less than 0.5% of ALA.Higher amounts of linolenic acids are probably dueto the incorporation of added soybean oil in manu-facturing process of margarines.The TFA content is the sum of quantified transmonounsaturated fatty acids with 18 (18:1) carbonand the geometry of linoleic acids (C18:2: 9t/12t9c/12t 9t/12c where c and t are cis and trans,respectively). The total TFA content of the spreadable fats variedbetween nd (not detected) and 10.9%. The C18:1trans acid was the major trans isomer, having awide variation, from nd to 10.9% for all 18 mar-garine samples. The isomeric pattern of C18:1 TFAin margarines is formed by technical hydrogena-tion.

Investigated spreadable fats samples containedsmall amounts of C18:2 trans acids. No producersdeclared the content of TFA on the packaginglabel.TFA content of hard margarines ranged betweennd and 10.9% of the total FA content, whereas theTFA of soft margarines ranged from nd to 7.9%.TFA were not found only in one out of 6 stick mar-garines, whereas 7 out of 12 tub margarines con-tained low levels of TFA (nd - 0.7%). But it isimportant to point out that in the United States TFAcontent of soft products was higher and rangedfrom 2.6 to 14.6% of the total FA content [32]. The mean values of total TFA content of hard andsoft margarines combined in Poland (3.7%) werelower than those reported in Pakistan (6.8%) [5],in the Czech Republic (9.7%) [6] and Turkey(16.5) [32], while recent data revealed that thepresented values in this work are higher thanthose from Denmark (0.7) [19], Austria (1.6) [34],and Portugal (2.5%) [33]. But the mean value ofTFA in soft margarines was lower and averaged2.1%. It is important to point out that 5 samples ofmargarines had no TFA (they had no partiallyhydrogenated fat). However, in Poland mar-garines are prepared from partially hydrogenatedvegetable oils, so variable amounts of trans iso-mers were present.TFA content in the product depends not only onthe kind of fat but also on the fatty matter contentin the margarine. Some parts of the analyzed mar-garines were fat reduced products, below 80%

*n6/n3 ratio (C18:2 and C18:3 were used for this calculation as the main representatives)

Table III - Fatty acid profile (%), P/S ratio and n-3/n-6 of different margarines

67


fat. TFA values (g/100g) were calculated in rela-tion to the total fat content of margarines (Figure1). Published data indicated a 50% increase incardiovascular disease due to hydrogenated TFAintake [23]. A survey of TFA content of tub margarines/spreadsin Poland was reported by Daniewski et al. [8] andBalas [3]. A reduction of TFA content was notedfrom 1998 to 2008 (Table IV). It was found thatTFA content in stick margarines sold in Poland in1998 ranged from 0.0 to 35.3%. However, in thepresent work TFA content in stick margarinesranged from nd to 10.9%. A reduction of TFA wasalso found in tub margarines. As can be seenfrom Table IV, it can be concluded that the warn-ings of nutritionists and also the knowledge aboutthe content of TFA in food meant that the produc-ers had to introduce modern technologies to formmodified fats without TFA. A similar situation wasreported by List et al. [20] in the US. From 1992 to1999, the overall trans reduction ranged fromabout 25% to 82%, with an average reduction ofnearly 56%. The 1992 data indicate an average ofnearly 20% of TFA in soft tub products, whereasthe 1999 data show less than 9% of TFA. Stickproducts have shown decreased TFA over thepast decade – trans acids averaged 26.8% in1989, but by 1999 the average had dropped to16.9%, a 37% overall reduction [1]. The literature data of TFA content of margarinesover a 5-year period (1998-2002) show someinteresting trends in oil processing [1]; [11]; [19];[24]; [27]; [32]. Low-trans products are arbitrarilydefined as those having 5% or less TFA, whilezero-trans products may have small amounts (1-2%). Of all the samples reported, 38% may beconsidered zero- or low-trans. The largest num-bers of samples reported include those fromCanada and the U.S.

TFA is practically absent from the margarine mar-ket in Denmark [19]. Matsuzaki et al. [22] analyzedmargarines from 11 countries (Europe, U.S.) fortheir TFA and other compositional data. Their dataconfirmed that any decrease in TFA is achieved atthe expense of increased SFA content. Ratnayake et al. [27] reported that over the lasttwo decades margarine manufacturers havetaken measures to gradually lower the TFA con-tent in their products. This trend is most likely aresult of the public pressure on margarine pro-ducers to reduce TFA in their products.It is possible to claim that the highest changesappeared in the content of trans isomers of ana-lyzed margarines in comparison to literature data.The range in the content of SFA as for mono- andPUFA acids showed the same level in years 1998-2008.The ratio of polyunsaturated to saturated fattyacids (P/S) in two of the six stick margarines test-ed and two of twelve soft margarines was <0.5.Generally about 33% of tested margarines haveP/S <0.5, but in Austria about 55% do so [34]. In1998 only 22% of Polish margarines had P/S val-ues of <0.5 [8]. The recommended P/S ratio is 1and causes elevated plasma cholesterol andtriglyceride levels. High levels of cholesterol andtriglycerides are associated with the increasedincidence of coronary heart diseases, atheroscle-rosis and coronary artery diseases [34]. PUFA are acids which are recommended from thenutritional point of view. The ratio of these acids infood is also important. International experts rec-ommended a reduction of n-6 fatty acids and anincrease of n-3 fatty acids. In the present work themain drawback is the high n-6/n-3 ratio for someanalyzed brands. One stick margarine containeda ratio of 41:1. Other hard margarines have an n-6/n-3 ratio in the 4:1 and 11:1 range. Eight tub

Figure 1 - Content of trans fatty acids in product (margarines)

68


brands contained ratios between 40:1 and 246:1.Four soft brands more popular in Poland have ann-6/n-3 ratio between 3:1 and 15:1. In the presentstudy mean n-6/n-3 is 82 (from 3 to 246) while inPortuguese margarines the mean is 81 (from 3 to383) [33].The oxidative quality of investigated margarineswas determined from the values of primary andsecondary oxidation products and also from thevalues of free fatty acids. Analyzed margarineshad the same or comparable expiry date. The acid values (AV) were similar in all kinds ofmargarines (stick, tub) and ranged from 0.17 to0.34 mgKOH/g. Bigger differences were observed in oxidativechanges of fats. As concerning the primary per-oxide products measured by PV their contentranged from 0.1 to 2.0 for stick margarines andhigher content was noted for tub margarines: from0.2 to 4.1 meqO/kg. Generally no dependencebetween the content of fatty acids on the PV valuewas found, although in one of the tub margarinesas much as 10.32 meqO/kg was recorded. Thesedifferences may be caused by the quality of rawmaterials or the method of storage of margarines.Anisidine value (AnV) was determined to describethe secondary oxidative changes of fats.Independently of kind of margarines (stick, tub)AnV were from 2.5 to 9.2 The number of second-ary products of oxidation was below 3 in 67% ofanalyzed stick and only 17% of tub margarines,which proved the good quality of the fat [25]. Thehighest amounts of secondary oxidative productswere found in stick margarines, especially wherethe amount of PUFA was the highest (samples 1,6). On the other hand, the lowest amounts of sec-ondary oxidative products were observed forsamples (tub margarines) with low content ofPUFA (samples 15, 16). These observations confirm earlier research car-ried out on the mechanism of oxidation of fattyacids. It was claimed that hydroperoxides arisingin the oxidation of linolenic acid could decom-pose more easily than those which had arisen inthe oxidation of oleic acid [10]; [36].In general, the spectrum of TFA found in the ana-lyzed margarine samples is associated withincreased risk of cardiovascular disease wherethey are negatively correlated with plasma HDL-cholesterol concentration and positively correlat-ed with plasma LDL-cholesterol level. TFA canalso interfere with growth and development dur-ing the first 6 months of life. Also there is a lot ofconflicting evidence concerning the role of TFA insome cancers, in type 2 diabetes, strokes andfood sensitivities. So in Poland there is an urgentnecessity to make people aware of how fattyacids, especially TFA, can affect the quality of

food and in turn people’s health. Several possibilities exist to reduce TFA levels infoods and thus reduce intakes. Fats can be mod-ified in the following ways: using interestification,modifying the hydrogenation process such as byuse of electrocatalysis (lower temperature), pre-cious metal catalysts or supercritical fluid statesand also genetically modifying the fatty acid pro-file, e.g. to produce higher oleic acid level. InPoland a good solution would be to introduce theregulatory approach. Customer education cam-paigns and food labels will be important tools toprovide consumers with factual information oncurrent levels of TFA in foods, and how they canreduce their intake of TFA.

ACNOWLEDGEMENTS

This work was supported by Ministry of Scienceand Higher Education, grant No. N N 312 200035.

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[27] W.M.N. Ratnayake, G. Pelletier, R. Holly-wood, S. Bacler, D. Leyte, Trans fatty acidsin Canadian margarines: recent trends. J.Am. Oil Chem. Soc. 75, 1587-1594 (1998).

[28] S. Ray, D.K. Bhattacharyya, Comparativenutritional quality of plasmstearin-liquidoilblends and hydrogenated fat (vanaspati). J.Am. Oil Chem. Soc. 73, 617-622 (1996).

[29] M. Sommerfeld, Trans unsaturated fatty acidsin natural products and processed foods.Progress in Lipid Research 22, 221-223 (1983).

[30] S. Stender, J. Dyerberg, Influence of transfatty acids on health. An. Nutr. Metab. 48,61-66 (2004).

[31] K. Sundram, M.A. French, M.T. Clandinin,Exchanging partially hydrogenated fat forpalmitic acid in the diet increases LDL-cho-lesterol and endogenous cholesterol synthe-sis in normocholesterolemic women. Eur. J.Nutr. 42, 188-194 (2003).

[32] A. Tekin, M. Cizmeci, H. Karabacak, M.Kayahan, Trans FA and solid fat content ofmargarines marketed in Turkey. J. Am. OilChem. Soc. 79, 443-445 (2002).

[33] D. Torres, S. Casel, M. Beatriz, P.P. Oliveira,Fatty acids composition of Portuguese spread-able fats with emphasis on trans isomers. Eur.Food Res. Technol. 214, 108-111 (2002).

[34] K.H. Wagner, E. Auer, I. Elmadfa, Content oftrans fatty acids in margarines, plant oils, friedproducts and chocolate spreads in Austria,Eur. Food Res. Technol. 210, 237-241 (2000).

[35] M.P. Yurawecz, FDA requires mandatorylabelling of trans fat. Inform 15, 184 (2004).

[36] A. Zbikowska, M. Kowalska, Influence oftrans unsaturated fatty acids content onchemical changes in the shortening duringbaking and storage of cakes. Pol. J. FoodNutr. Sci. 57, 451-455 (2007).

Received, November 8, 2010Accepted, March 1, 2011

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Annual meetings

World Biofuels Markets Congress &Exhibition

Rotterdam,The Netherlands, 13 -15 marzo 2012Il World Biofuels Markets Congress & Exhibitiontorna a Rotterdam nel 2012, per riunire ancora unavolta l’intera filiera dell’industria dei biocarburanti alfine di ampliare i rapporti commerciali.La fiera-congresso prevede come parte principaledell’ordine del giorno relazioni presentate daesperti leader nel settore. Sono inoltre previste ses-sioni su: biocarburanti per aviazione, biocombusti-bili, biodiesel globale, piattaforme di bioraffineria,colture energetiche, bioetanolo globale, alghe, car-burante per il trasporto di biogas, mercati emer-genti, progetti finanziari e investimenti. World Biofuels Markets Conference si svolgerà incontemporanea (13-14 marzo) con altri due eventileader a livello mondiale del settore: • Biopower Generation - Scaling up biomassa e

produzione di calore • Bio-based Chimicals - Accelerare lo sviluppo, la

commercializzazione e lo sviluppo del mercato.Per ulteriori informazioni e aggiornamenti collegar-si al sito:

www.worldbiofuelsmarkets.com

PRINCIPLES & PRACTICE OF COSME-TIC SCIENCE - AN INTERACTIVERESIDENTIAL COURSE

Wessex Hotel, Bournemouth, BH2 5EU, UK11-16 March 2012

Il corso, di fama internazionale, è al servizio dell’in-dustria cosmetica da oltre 40 anni.Esperti provvederanno ad informare sulle novitàriguardanti i temi più rilevanti del settore.Argomenti trattati:- Project management and working in team –Susan Hurst (MiDAS Consultants)

- Product development – Ged O’Shea (The BootsCompany plc)

- Basic surface & colloid science – Kevan Hatch-man (Mclntyre (Rhodia))

- Perfumery – Jonathan Gray (Mane Ltd)- Safety – Barbara Hall (Sureconsult Ltd)- Legislation – Emma Meredith (CTPA)- Skin & Skin care products – John Woodruff (Cre-

ative Developments)- Naturals – Tony Dweck (Dweck Data)- Preservation in a manufacturing environment –Sarah Gladstone (Schülke)

- Processing - John Tainton (Unilever USSC)- Quality/GMP – Neil Jarvis (The Body Shop Inter-

national PLC)

- Rheology – Steve Goodyer (Anton Paar)- Product advertising and claim support – SteveBarton (Oriflame Research and DevelopmentLtd)

- Packaging – Anne Emblem (London College ofFashion)

- Hair & Hair products – Bob Hefford (IndependentCosmetic Advice Ltd)

- Cosmetic Colours Dental & APD Products – TonyMorton (The Body Shop International PLC)

Per informazioni dettagliate contattare:Society of Cosmetic Scientists

e-mail: [email protected] collegarsi al sito:

http://www.scs.org.uk/principles-and-practice

HPCI CONGRESSBombay Exhibition Centre, Mumbai, 15-16 March 2012La manifestazione HPCI che si terrà a Mumbai nellegiornate del 15 e 16 marzo 2012 riunisce molti deimaggiori esperti nelle tecniche di formulazione deicosmetici e nei prodotti per la cura personale e dellacasa, professionisti che si occupano di ricerca e cheoperano nel settore lavanderia e detergenti, orienta-ti a diffondere le novità sulle tecnologie e le informa-zioni all’avanguardia sul settore. Il programma formativo 2012 è stato arricchito conl’iniziativa di una nuova conferenza: la Indian deter-gents Conference studiata appositamente permigliorare le conoscenze e per stare al passo conle tendenze e le tecniche di applicazione.I seminari presentati dalle aziende espositriciriguarderanno le questioni relative alle formulazionidei prodotti. Le aziende avranno l’opportunità di promuovere leprestazioni dei loro prodotti in applicazioni specifiche.Al momento non sono disponibili ulteriori informazio-ni. Per aggiornamenti visitare regolarmente il sito:

www.hpci-congress.com

OFI–OILS & FATS INTERNATIONALTURKEY 2012Istanbul Lutfi Kirdar Convention & Exhibition Centre,

Turkey, 17-18 April 2012L’annuale fiera Ofi Medio Oriente presenta le ulti-me novità nelle attrezzature, tecnologie e servizidel settore Oli e Grassi dal punto di vista tecnico,commerciale e scientifico. L’evento interessa leseguenti aree commerciali: impianti e attrezzatu-re, prodotti chimici, detergenti e tensioattivi, gras-si, trasporti, stoccaggio, ispezione e analisi, stru-mentazione, biodiesel.Per informazioni e aggiornamenti collegarsi al sito:

www.oilsandfatsinternational.com

CONGRESSINo

tiziar

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NotiziarioFOOD SAFETY SUMMIT – EXPO ANDCONFERENCE

Washington DC, April 17-19, 2012La Conferenza tratterà temi riguardanti la sicurez-za alimentare.I partecipanti avranno l’opportunità di acquisireutili informazioni e conoscere le ultime innovazio-ni in materia di sicurezza alimentare.Per aggiornamenti collegarsi al sito:

www.foodsafetysummit.com/

IN-COSMETICS EXHIBITION ANDCONFERENCEFira Barcelona, Barcelona, Spain, 17-19 April 2012

La In-cosmetics exhibition and conference è un’im-portane grande esposizione e conferenza del set-tore cosmetico. Occasione per conoscere le ultimetendenze e incontrare esperti del settore.Annotare l’evento. Per ulteriori informazioni con-tattare:

e-mail: [email protected]

13th INTERNATIONAL EXHIBITIONON SURFACTANT AND DETERGENTShanghai Everbright Convention and Exhibition Cen-

ter, China, April 28-30, 2012Dopo aver ottenuto risultati soddisfacenti con lamanifestazione di Shanghai del 27-29 Aprile2011, gli organizzatori vogliono ripetere l’eventoanche nel 2012.Sarà occasione per gli espositori per presentarenuovi prodotti, materie prime, soluzioni, tecnolo-gie e applicazioni, dando così ai partecipanti, perlo più fornitori e industriali, una prospettiva globa-le sulle tendenze emergenti e sull’andamento delsettore.Per informazioni:

e-mail: [email protected]

103rd AOCS ANNUAL MEETING & EXPOLong Beach Convention Center - Long Beach,

California, USA, April 29-May 2, 2012Il meeting annuale AOCS & Expo è leader mondia-le nell’ambito della scienza e del forum commer-ciale sui grassi, oli, tensioattivi e materiali relativi. Il programma dell’incontro sarà caratterizzato dasessioni poster, presentazioni orali, corsi brevi,una fiera e collegamenti in rete con oltre 1.600esperti di 60 paesi.Il Comitato Organizzatore di AOCS ha individuatole aree di interesse, di seguito riportate, sulla basedei risultati dell’indagine del 2011 effettuata tra i

partecipanti agli incontri annuali:- carburanti da fonti rinnovabili, incluse le alterna-

tive e le tecnologie di produzione;- sostenibilità, compresa la tecnologia “verde”, il

ciclo di vita e la questione energetica;- lipidi Funzionali (struttura, applicazione, fonda-

menti e ricadute sulla salute; - acidi grassi omega-3 (ossidazione dei lipidi,

nutrizione, antiossidanti e shelf-life); - tensioattivi e detergenti (analisi, metodologie). Per ulteriori informazioni collegarsi alla paginaweb:

http://annualmeeting.aocs.org/index.cfm

2012 HPCI EXHIBITION AND CONFE-RENCE HOME AND PERSONAL CAREINGREDIENTS EXHIBITION AND CON-FERENCE

Istanbul, 23-24 May 2012L’evento prevede un ampio spazio per gli esposito-ri. Più di 160 aziende presenteranno i loro prodottie servizi ad un pubblico altamente qualificato.Durante la manifestazione verranno presentateconferenze scientifiche e seminari tecnici.Per informazioni e aggiornamenti collegarsi al sito:

www.hpci-congress.com

10th CONGRESS OF THE ISSFALWestin Bayshore Hotel, Vancouver, Canada, 26-30 May 2012ISSFAL International Society for the Study of FattyAcids and Lipids è una società scientifica interna-zionale fondata nel 1991, che conta 500 membriesperti nel settore, provenienti da oltre 40 paesi.Il Comitato organizzatore della Società Scientificaè lieto di ospitare il 10° Congresso, che si terrà alWestin Bayshore Hotel di Vancouver dal 26 al 30maggio 2012, per lo Studio degli effetti sulla salu-te del contenuto di acidi grassi e lipidi nella dieta. Il Comitato garantirà che le sessioni affrontino temirelativi ai progressi scientifici della ricerca, a livellointernazionale.Il Congresso fornirà l’opportunità di rinnovareconoscenze e stabilire nuovi contatti di networking.Per informazioni aggiornate collegarsi al sito:

www.issfal.org/conferences/vancouver-2012

BIO INTERNATIONAL CONVENTION Boston, Massachusetts, USA, June 18-21, 2012

Torna a Boston dal 18 al 21 giugno 2012, presso ilBoston Convention & Exhibition Center la fierainternazionale BIO International Convention. Per informazioni aggiornate:

http://convention.bio.org

72


Notiz

iario ACHEMA

Francoforte sul Meno, Germania, 18-22 giugno 2012La Achema fiera di Francoforte è la mostra inter-nazionale sulle tecnologie chimiche, biotecnolo-gie e sulla protezione ambientale. Circa 4.000espositori provenienti da numerosi paesi presen-teranno i loro prodotti e servizi nei settori dellatecnologia di laboratorio, di analisi, biotecnologie,impianti, pompe, compressori, accessori, tecni-che di sicurezza, sicurezza sul lavoro. ACHEMA èquindi il forum mondiale per l’ingegneria chimica,i processi industriali e le innovazioni; punto diincontro di esperti e dirigenti a livello internazio-nale.Segreteria Organizzativa:

Dechema e.V.Theodor-Heuss-Allee 25

60486, Francoforte sul Meno (0)Tel: +49 (0)69 75640

Fax: +49 (0)69 7564201Email: [email protected]

URL: http://www.dechema.de

IFT 12 ANNUAL MEETING AND FOODEXPO

Las Vegas, NV, 25-28 June, 2012Dal 25 al 28 Giugno 2012, a Las Vegas, NV, siterrà l’Annual Meeting & Food Expo.Evento annuale che riunisce esperti di scienze etecnologie alimentari nel settore industriale.Imperdibile opportunità di: - Aggiornarsi sugli ultimi sviluppi della scienza

degli alimenti e sulle sue applicazioni;- scoprire le ultime tendenze di consumo;- vedere, gustare e sperimentare i nuovi prodotti

e le nuove tecnologie;- stabilire nuove relazioni commerciali;- ampliare le proprie competenze e conoscenze

di base;- stabilire nuovi contatti di networking;- acquisire idee ed innovazioni che avranno un

impatto diretto sul lavoro e sullo sviluppo azien-dale.

Per ulteriori informazioni:http://www.am-fe.ift.org/cms/

COSI 20128th Coatings Science International2012Noordwijk, The Netherlands, 25 June - 29 June 2012

Benvenuti al più importante congresso europeosulla Scienza e Tecnologia dei rivestimenti.Questa conferenza è rivolta a ricercatori e mana-ger della ricerca, attivi nel campo della Scienza edella Tecnologia dei rivestimenti sia del mondoindustriale che accademico. Gli specialisti di polimeri, materiali e vernici chimi-che; così come chimici, formulatori, progettisti eresponsabili del settore troveranno in questa con-ferenza un’ampia piattaforma di scambio riguardogli ultimi sviluppi. Per informazioni:

http://www.coatings-science.com/index.html

La SSOG organizza ogni anno due

Circuiti di Correlazione Oli Vegetali tra i diversi laboratori del settore oleario

Per informazioni: INNOVHUB Azienda Speciale della Camera di Commercio di Milano Divisione SSOG - Via Giuseppe Colombo 79 Milano Tel. 02.7064971 Fax 02.2363953 e-mail: [email protected]



Organo ufficiale della Divisione SSOG di InnovhubStazioni Sperimentali per l’IndustriaAzienda Speciale della Camera di Commercio di Milano

GENNAIO/MARZO 2012ISSN 0035-6808 RISGARD 89 (1) 1-72 (2012) - Poste Italiane S.p.a.Spedizione in Abbonamento Postale -70%Finito di stampare nel mese di Marzo 2012

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