ANNOextranet.innovhub-ssi.it/allstd/risg/2015-RISG 1.pdf · aqueous samples by HPLC with UV and evaporative detectors is described. The procedure The procedure is based on a Liquid-Liquid

ANNO

Organo ufficialedella Divisione SSOG di InnovhubStazioni Sperimentali per l’industriaAzienda Specialedella Camera di Commercio di Milano

2015

RISG

92

LA

RIVISTAITALIANA

DELL

SOSTANZEGRASSE

E

GENNAIO/MARZO 2015ISSN 0035-6808 RISGARD 92 (1) 1-80 (2015)Poste Italiane S.p.a. - Spedizione in Abbonamento Postale - 70%Finito di stampare nel mese di Giugno 2015

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INNOVHUB - Stazioni Sperimentali per l’Industria Azienda Speciale della Camera di Commercio di Milano

www.innovhub-ssi.it

LLLaaabbbooorrraaatttooorrriiiooo dddiii aaannnaaallliiisssiii dddeeegggllliii ooollliii vvveeegggeeetttaaallliii eee gggrrraaassssssiii aaannniiimmmaaallliii

Il laboratorio svolge principalmente attività analitica conto terzi e sviluppa, su richiesta, nuovi metodi per eseguire analisi particolari per le quali non esistano ancora procedure validate.

L’attività di analisi e di ricerca si applica su diverse tipologie di prodotti che comprendono: - Semi, frutti oleaginosi e sostanze grasse da esse estratte per analisi di composizione e di caratterizzazione; - Oli d'oliva e oli di sansa d'oliva secondo il Regolamento CE 2568/91 e successivi aggiornamenti; - Oli di semi, grassi vegetali e animali (burro, strutto, olio di pesce), semilavorati e prodotti finiti alimentari; - Oli e grassi animali e vegetali, loro intermedi e derivati impiegati come biocombustibili liquidi secondo la norma

UNI/TS 11163:2009; - Sottoprodotti di lavorazione delle sostanze grasse e derivati (es: lecitine, oleine, paste di degommazione); - Farine animali non destinate al consumo umano (di 1a e 2a categoria) per la ricerca del tracciante GTH (trieptanoato di

glicerina) secondo il Reg CE 1774/2002; - Farine e idrolizzati proteici (determinazione di masse molecolari, di amminoacidi liberi e totali, solforati e triptofano,

proteine); - Mangimi animali per la ricerca della presenza di grassi animali aggiunti (colesterolo), per analisi di composizione e di

contaminanti metallici. - Prodotti alimentari per la ricerca degli allergeni mediante saggi immunologici (ELISA) e per la determinazione dei grassi

vegetali diversi dal burro di cacao nei prodotti di cacao e di cioccolato destinati all'alimentazione umana

Dr.ssa Liliana Folegatti Responsabile Settore Qualità/Genuinità (Componenti principali)

Tel. 02.70649772 – e-mail: [email protected]

Sito web: www.innovhub-ssi.it100,00 200,00

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ORGANO UFFICIALE DELLA DIVISIONE SSOG DI INNOVHUB

STAZIONI SPERIMENTALI PER L’INDUSTRIA

AZIENDA SPECIALE DELLA CAMERA DI COMMERCIO DI MILANO

E–mail: [email protected] – Sito web: www.ssog.it

100,00 200,00umero singolo 30,00

ORGANO UFFICIALE DELLA DIVISIONE SSOG DI INNOVHUB STAZIONI SPERIMENTALI PER Lí INDUSTRIA

AZIENDA SPECIALE DELLA CAMERA DI COMMERCIO DI MILANO

SSOG_1_ok:Layout 1 22-02-2013 9:01 Pagina 1

direttore responsabile: M. Surdiredazione: F. paparella

GraFiCa, iMpaGinazione e staMpa

Grafiche parole Nuove srlVia Garibaldi 58 - Brugherio

1duemi laquindic iGENNAIO/MARZO 2015 - ANNO XCII

[email protected]

abbonaMenti e [email protected]

Sommario

D. Mariani, E. Trimigno, G. Pallotti, S. Polesello, S. Capri

3 Nota tecnica - Determinazione di tensioattivi anionici e non ionici in matrici acquose mediante cromatografia liquida ad alta prestazione (HPLC) accoppiata a rivelatori UV ed evaporativi

P. Bondioli, L. Della Bella, G. Rivolta, S. Faragò, A. Boschi, S. Beretta

11 Study of biodiesel solid contaminants by means of Scan Electron Microscosopy (SEM)

D. Baglio, M.R. Porta, L. Folegatti

17 Comparazione dei differenti sistemi di iniezione gascromatografici per la rilevazione di grassi vegetali diversi dal burro di cacao nei cioccolati fondenti

A.A. Giuliani, A. Cichelli, L. Tonucci, N. d’Alessandro

25 Chlorophyll photosensitized oxidation of virgin olive oil: a comparison between selected unsaturated model esters and real oil samples

B. Marongui, M.M. Özcan, A. Rosa, M.A. Dessi, A. Piras

39 Short note - Monitoring of the fatty acid compositions of some olive oils

P. Catania, M. Vallone, D. Planeta, P. Febo

43 Instrumental evaluation of the texture of cv. Nocellara del Belice table olives

C. Guillaume, R. Ravetti 53 Technical note - Technological and agronomical factors affecting sterols in Australian olive oils

M.M. Özcan, L. Altinöz, D. Arslan, A. Ünver

61 Short note - Physical and chemical characteristics of oils of some olive varieties in Turkey

Notiziario 69Indice annata 2014 79

Comitato di redazione

P. BONDIOLI settore tecnologie olearie e oleochimiche

L. FOLEGATTI settore sostanze grasse e proteine vegetali

S. TAGLIABUE settore cosmetica

G. GASPERINI settore prodotti vernicianti

P. ROVELLINI settore qualità/genuinità (micronutrienti e sicurezza

alimentare)

D. MARIANI settore detersivi e tensioattivi

M. SALA settore lubrificanti

Comitato di Referee

R. APARICIO Istituto de la Grasa y sus Derivados – Siviglia (E)

G. CONTARINI Istituto Lattiero Caseario - Lodi

L. CONTE Dipartimento di Scienza degli Alimenti – Università di Udine

G. DONATI Istituto Superiore Sanità – Roma

A. FABERI Ministero delle Politiche Agricole Alimentari e Forestali – Roma

C. GIGLIOTTI Dipartimento di Scienze Biomediche e Biotecnologiche –

Università di Brescia

F. LACOSTE Institut des Corps Gras – ITERG – Pessac (F)

G. LERCKER Dipartimento di Scienze Alimentari – Università di Bologna

L. MANNINA Facoltà di Agraria – Università degli Studi di Campobasso

R. SACCHI Dipartimento Scienze Alimentari – Università Federico II – Portici

(NA)

C. SCESA Corso di Laurea in Tecniche Erboristiche – Facoltà di Farmacia –

Università di Urbino

M. SERVILI Dipartimento di Scienze Economico-Estimative e degli Alimenti –

Università di Perugia

L. SISTI Henkel – Divisione Tensioattivi – Lomazzo (CO)

Ö. TOKUŞOĞLU Celal Bayar University - Engineering Faculty – Manisa Turkey

Indexed and Abstracted in:• Thomson Scientific Service: Science Citation Index Expanded

(SciSearch), Journal Citation/Science Edition, Current Contents/Clinical Medicine

• Chemical Abstracts• Elsevier Bibliographic Databases: SCOPUS• FSTA – Food Science and Technology Abstract (IFIS Publishing – UK)

IMPACT FACTOR 2014: 0,392

La RIVISTA ITALIANA DELLE SOSTANZE GRASSEè 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. Pubblica un Notiziario con informazioni su congressi,

notizie in breve e libri.La Rivista viene distribuita e consultata in Italia dalle

industrie produttrici ed esportatrici di oli e grassi alimentari ed industriali, dalle industrie chimiche, da laboratori di enti statali, da istituti di ricerca e facoltà

universitarie, da dove provengono diversi lavori scientifici.

È inoltre distribuita all’estero in vari Paesi come Spagna, Principato di Monaco, Canada, Paesi

Bassi, Svizzera, Slovenia, Regno Unito, Turchia, Lussemburgo, Malaysia, Grecia, Francia, Germania, Tunisia, Nigeria, Congo, Polonia, Romania, Bulgaria,

Russia, Stati Uniti, Brasile, Cina, Giappone.

La rivista itaLiana deLLe sostanze grasse - voL. XCii - gennaio/Marzo 2015

3

D. Mariani1*

E. Trimigno1

G. Pallotti1

S. Polesello2

S. Capri3

1INNOVHUB-SSIAzienda Speciale della

Camera di Commercio di MilanoDivisione SSOG - Milano

2IRSA-CNR, Brugherio (MB)

3IRSA-CNR, Roma

(*) CORRESPONDING AUTHOR:D. Mariani

Divisione SSOG Via Giuseppe Colombo 79

20133 Milano, Italy.e-mail: [email protected]

nota tecnicadeterminazione di tensioattivi anionici

e non ionici in matrici acquose mediante cromatografia liquida ad alta prestazione

(HplC) accoppiata a rivelatori UV ed evaporativi

Il metodo descrive una procedura analitica per determinare tensioattivi anionici e non ionici in campioni acquosi mediante HPLC accoppiata a rivelatori UV ed evaporativi previo arricchimento degli analiti mediante estrazione liquido-liquido (LLE) o in fase solida (SPE). L’utilizzo di un sistema di rivelatori in serie consente di determinare gli analiti in miscele anche complesse.

Determination of anionic and non-ionic surfactants in aqueous matrices by high-performance liquid chromatography (HPLC) with UV and evaporative detectors An analytical procedure for the determination of anionic and non-ionic surfactants in aqueous samples by HPLC with UV and evaporative detectors is described. The procedure is based on a Liquid-Liquid (LLE) or a Solid Phase Extraction (SPE).A system of serially connected detectors allows to determine the analytes even in complex mixtures.


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INTRODUZIONE

I tensioattivi sono composti organici costituiti da un gruppo funzionale lipofilo, solitamente una catena idrocarburica (R) e da gruppi funzionali idrofili. Sono solubili in acqua e in grado di abbassare la tensione superficiale di un liquido (tensione all’interfaccia tra due liquidi o tra un solido e un liquido). In base alle caratteristiche del gruppo idrofilo i tensioattivi si sud-dividono in anionici (carica negativa), non ionici (sen-za carica), cationici (carica positiva), anfoteri (carica sia positiva che negativa), e in sottoclassi per affinità chimica nell’ambito della stessa classe (Grafico 1).L’identificazione e la quantificazione di tensioatti-vi commerciali in campioni ambientali è operazione complessa in quanto si tratta di composti con ca-ratteristiche chimiche diverse, presenti sotto forma di miscele di decine-centinaia di omologhi, oligomeri e isomeri. Ciò ha determinato, sia a livello nazionale che internazionale, la diffusione di metodi ufficiali per la determinazione routinaria di tensioattivi basati su tecniche colorimetriche o potenziometriche, prece-dute da estrazioni più o meno selettive. Detti meto-di, oltre ad essere poco affidabili, specie nel caso di matrici complesse, non forniscono alcuna informa-zione sulla presenza di più tensioattivi appartenenti

alla stessa classe, né sulla composizione di omolo-ghi od oligomeri o sulla lunghezza della catena idro-fobica.Questa limitazione dei metodi ufficiali è riconosciuta anche a livello normativo dall’Unione Europea che, nel Regolamento n. 648/2004 relativo ai detergenti (CE 648/2004), afferma che per i tensioattivi che non reagiscono ai metodi colorimetrici “o qualora sembri più opportuno per ragioni di efficienza e di esattezza si applicano adeguate analisi strumentali specifiche, quali la cromatografia in fase liquida ad alta pressio-ne (HPLC) o la cromatografia in fase gassosa (GC)”.Essendo notoriamente i metodi chimici citati (MBAS per i tensioattivi anionici e BIAS per i non ionici) po-tenzialmente interferiti da molte sostanze e non es-sendo possibile, con accettabile approssimazione, essere a conoscenza delle eventuali interferenze presenti nelle matrici da analizzare, la procedura tra-mite HPLC risulta più affidabile ed efficace in caso di determinazione di tensioattivi in matrice dalla com-posizione poco nota e più completa in quanto in grado di evidenziare la presenza contemporanea di molteplici specie di tensioattivi (non solo anionici e non ionici) qualora presenti nella matrice analizzata.Nel seguito viene riportata una proposta di metodo per la determinazione di tensioattivi anionici e non io-

Grafico 1 - Principali classi e sottoclassi di tensioattivi

Tabella I - Condizioni operative utilizzate per l’analisi in HPLC Fase stazionaria

Colonna Acclaim Surfactant Plus 3 m (4,6 150 mm)

Eluente Miscela binaria acetonitrile/ammonio acetato 100 mM, pH=5 “Loop” 5 µL Flusso eluente 0,6 mL/min Volume di iniezione 5 L Rivelatore UV (225 nm)

ELSD Temperatura di nebulizzazione: 30°C Temperatura di evaporazione: 90°C Gas di trasporto: 0,90 litro standard/min (slm) oppure CAD Controllo della temperature di nebulizzazione: ON Temperatura di nebulizzazione: 30°C Frequenza di acquisizione dei dati: 2Hz Filtro: non inserito

ANIONICI NON IONICI

Carbossilati Solfonati Solfati Etossilati eterei Etossilati esterei

Alchilarilsolfonati (LAS) Alchensolfonati (AOS) Alcansolfonati (SAS) Vari Alchilsolfati Etossisolfati Olii solfatati Vari Olii solfonati

Alcoli etossilati Alchilfenoli etossilati Acidi etossilati Esteri di polialcoli etossilati

CATIONICI

Etossilati amminici Etossilati Ammidici Alchilammine ammonio quaternario

Altri derivati dell’azoto

Etanolammidi Ammidi etossilate Amminoossidi Ammine etossilate


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nici in campioni acquosi mediante HPLC accoppiata a rivelatori UV ed evaporativi, previo arricchimento degli analiti mediante estrazione liquido-liquido (LLE) o in fase solida (SPE).L’utilizzo di un sistema di ri-velatori in serie consente di determinare gli analiti in miscele anche complesse.

1. PRINCIPIO DEL METODO

Il metodo prevede la determinazione di tensioattivi anionici e non ionici nelle acque mediante cromato-grafia liquida ad alta prestazione (HPLC) accoppiata ad opportuni rivelatori (UV, fluorescenza, “Evaporative Light Scattering Detector” ELSD, o “Charged Aerosol Detector” CAD), anche disposti in serie. La colonna cromatografica utilizzata è composta da catene alchi-liche, gruppi amminici terziari e gruppi ammidici polari che consentono differenti meccanismi di separazione degli analiti: fase inversa, scambio anionico e intera-zioni dipolo-dipolo. I tensioattivi anionici vengono ri-tenuti principalmente grazie ad interazioni di scambio ionico e ripartizioni idrofobiche, mentre i tensioattivi non-ionici vengono separati grazie ad interazioni idro-fobiche e dipolo-dipolo.I campioni acquosi vengono preventivamente sotto-posti ad estrazione liquido-liquido (LLE) o liquido-soli-do su cartucce per estrazione a fase solida (SPE) e gli estratti ottenuti, concentrati di volume, sono analizzati mediante HPLC. Il riconoscimento dei picchi nei cro-matogrammi degli estratti si effettua confrontando il loro tempo di ritenzione con quello dei picchi di una soluzione di riferimento multicomponente.

2. CAMPO DI APPLICAZIONE

Il metodo è in grado di determinare ciascuna singo-la specie di tensioattivo eventualmente presente in campioni di acque di scarico urbane ed industriali, in acque superficiali e sotterranee e, in generale, in qual-siasi soluzione acquosa di interesse.Il campione, ove non già disponibile in adeguata con-centrazione di tensioattivi, deve essere avviato alla fase di concentrazione prima della determinazione mediante HPLC. Tale fase mira all’ottenimento di una soluzione contenente almeno 100 mg/kg di ciascu-na specie tensioattiva che si desidera determinare. Pertanto il limite inferiore di determinazione (LOD) è dipendente dal fattore di concentrazione che viene applicato in fase di preparazione del campione.

3. INTERFERENZE E CAUSE DI ERRORE

Normali interferenti possono essere quei composti organici che danno luogo, durante l’analisi cromato-grafica, a picchi con tempi di ritenzione coincidenti a quelli dei composti in esame. Solventi, reattivi, vetreria, contaminazione dell’am-biente di lavoro ed ogni trattamento del campione

possono causare la presenza di picchi interferenti e/o alterazioni della corrente di fondo del rivelatore con conseguenti difficoltà di interpretazione del tracciato cromatografico. Pertanto, al fine di essere sicuri che tutti i materiali utilizzati siano esenti da interferenze nelle condizioni operative adottate, è buona norma, sia all’inizio dell’indagine che periodicamente, sotto-porre all’intera procedura uno o più “bianchi” sosti-tuendo al campione acquoso acqua ultrapura.Nel caso di evidenza di interferenze, individuarne la provenienza analizzando ogni singolo passaggio della procedura e procedere alla loro eliminazione. Data la diffusione quasi ubiquitaria dei tensioattivi si richiede una particolare cautela nella scelta dei reattivi e dei solventi, con continue verifiche di bianchi.Pulire tutta la vetreria utilizzata tenendola a bagno in HCl al 10% per almeno un’ora e quindi sciacquarla con acqua ultrapura. Evitare l’uso di detergenti per il lavaggio della vetreria. Prima dell’utilizzo effettuare una pulizia finale con acetone.

4. CAMPIONAMENTO E CONSERVAZIONE DEL CAMPIONE

I campioni, prelevati in bottiglie di vetro, possibilmen-te scuro, sono conservati al buio ad una temperatura di 4°C dopo essere stati addizionati con l’1% (v/v) di formaldeide al 37% per prevenire la degradazione batterica. La preconcentrazione deve essere effettua-ta entro 24 ore dal momento del prelievo affinché non si alteri la composizione chimica.

5. APPARECCHIATURE

5.1 Normale vetreria da laboratorio5.2 Apparecchiature per estrazione liquido-liquido

(LLE)5.2.1 Cilindri graduati (250-1000mL)5.2.2 Imbuti separatori (500-1000 mL)5.2.3 Cilindro da 25 mL5.2.4 Micropipetta a stantuffo (da 100 a 1000 µL)5.2.5 Omogeneizzatore meccanico per provette5.3 Apparecchiature per estrazione (SPE)5.3.1 Cartucce per estrazione in fase solida SPE (es.

“C18 endcapped”) Per l’estrazione liquido-solido sono utilizzate

colonnine monouso costituite da un “housing” esterno, generalmente in propilene, contenen-te il materiale adsorbente e da setti per il con-tenimento del materiale adsorbente in vetro sinterizzato.

La C18 è una fase adsorbente apolare, in grado di trattenere soprattutto composti apolari o de-bolmente polari, costituita da catene idrocar-buriche con il gruppo ottadecilico legato al gel di silice, e disattivazione per “endcapping” dei silanoli residui. Scegliere una quantità di fase sufficiente per trattenere gli analiti.


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5.3.2 Sistema di filtrazione per cartucce SPE (SPE work station) 1

(1) È possibile effettuare l’estrazione mediante SPE an-che senza l’utilizzo di un sistema automatico usando lo stesso protocollo indicato di seguito. La strumentazione automatica, tuttavia, garantisce un risparmio di tempo e assicura prestazioni elevate per quanto riguarda il recu-pero.

5.4 Membrane filtranti in fibra di vetro, 0,7 µm di porosità nominale.

5.5 Bilancia tecnica, risoluzione 0,1 g.5.6 Bilancia analitica, risoluzione 0,1 mg.5.7 Dispositivo per l’erogazione di azoto gassoso,

purezza 5.05.8 HPLC HPLC con pompa a gradiente (0,2-10 mL/min)

dotato di autocampionatore o valvola di inie-zione manuale e di forno per il riscaldamento della colonna analitica. L’HPLC è equipaggia-to con un rivelatore UV ed un rivelatore ELSD (“Evaporative Ligth Scattering Detector”) o un CAD (“ Charged Aerosol Detector”).

5.9 Colonna a fase stazionaria inversa con grup-po polare incorporato.

Si consiglia l’uso di una colonna cromatogra-fica, la cui superficie di scambio sia composta da catene alchiliche, gruppi amminici terziari e gruppi ammidici polari (tipo “Acclaim Surfac-tant Plus” 3 µm, 4,6 x 150 mm). Se disponibi-le, si consiglia l’uso della rispettiva precolon-na.

Cambiando natura o dimensioni della colonna analitica utilizzata dovranno essere modificati i parametri analitici adottati secondo specifi-che indicazioni fornite dalla ditta produttrice.

6. REATTIVI

6.1 Acqua ultrapura.6.2 Acetone per residui di pesticidi.6.3 Cloroformio (CHCl3).6.4 Metanolo (MeOH) per HPLC grado gradien-

te. 6.5 Acetonitrile per HPLC grado gradiente.6.6 Formaldeide al 37%.6.7 Acido cloridrico concentrato (d = 1,2 g/mL)6.8 Ammonio acetato (CH3CO2NH4) di grado

standard per cromatografia.6.9 Soluzione concentrata di ammonio acetato

(1 M). Pesare 77,08 g di ammonio acetato, essic-

cato per 2 ore a 100°C; sciogliere in acqua e diluire a 1 litro con acqua in matraccio tarato. La soluzione è stabile per sei mesi in bottiglia di polietilene, polipropilene o vetro.

6.10 Soluzione di ammonio acetato 100 mM, pH 5

Diluire 10 volte la soluzione concentrata (100 mL in 1 L) in un matraccio tarato. Portare il pH di questa soluzione a 5 con acido acetico concentrato.

6.11 Soluzioni di riferimento Preparare in acqua cromatografica soluzioni

di riferimento concentrate (0,5-1 g/L) di ten-sioattivo anionico e non ionico. Le soluzioni multicomponente aventi concentrazioni com-prese nell’intervallo 0-500 mg/L sono otte-nute per diluizioni successive in acqua cro-matografica delle soluzioni concentrate. Le soluzioni vanno conservate a 4°C.

Tabella II - Caratteristiche del gradiente utilizzato per l’analisi in HPLC

Tempo (minuti)

Acetonitrile (%)

Ammonio acetato (%)

0 35 65 1.0 35 65 8.0 85 15

15.0 85 15 15.1 35 65 23.0 35 65

Figura 1 - Cromatogramma di una miscela di 4 LAS di sodio (tensioattivo anionico), ottenuto nelle condizioni di Tabella I con rivelatore CAD


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7. PROCEDIMENTO

7.1 Trattamento preliminare La filtrazione dei campioni d’acqua è condot-

ta nel caso si ricorra all’estrazione in fase so-lida (SPE) per rimuovere i solidi sospesi che andrebbero a otturare la fase della cartuccia SPE. Questa operazione viene condotta solo quando la concentrazione di solidi sospesi ri-sulta maggiore di 50 mg/L. Impiegare mem-brane filtranti in fibra di vetro (5.4) e verificare con l’uso di bianchi l’assenza di contaminanti.

7.2 Estrazione liquido-liquido (LLE) Trasferire almeno 200 mL di campione tal qua-

le nell’imbuto separatore e procedere all’estra-zione liquido-liquido in almeno due fasi suc-cessive, utilizzando aliquote minime da 25 mL ciascuna di CHCl3 ed unendo le fasi clorofor-miche in un beacker.

Concentrare l’estratto cloroformico con debole riscaldamento in corrente d’azoto fino raggiun-gere un quantità di pochi mL; trasferire quanti-tativamente in una provetta e procedere con la concentrazione dell’estratto.

Lavare il beacker con due aliquote da 5 mL di cloroformio e trasferire in provetta.

Portare a secco, riprendere il residuo con 1 mL di acqua ultrapura e agitare vigorosamente uti-lizzando un agitatore per provette se disponi-bile. Iniettare quindi il campione in HPLC.

7.3 Estrazione SPE Qualora si utilizzi un sistema automatico di

estrazione SPE, seguendo le istruzioni del ma-nuale fornito a corredo dell’apparecchiatura,

posizionare la cartuccia per SPE, condizionare la stessa con MeOH ed in seguito con acqua ultrapura (25 mL di ciascun solvente, velocità di flusso 5 mL/min).

Caricare sulla cartuccia utilizzata una quantità di campione tale da consentire l’ottenimento di una soluzione finale di concentrazione adegua-ta. Impostare i parametri dell’apparecchiatura affinché la fase di caricamento sia eseguita ad una velocità compresa fra i 5 ed i 10 mL/min, di seguito far eseguire una fase di essicazione della cartuccia in corrente di azoto per almeno 5 minuti.

Eluire con 8 mL di MeOH alla velocità di 1 mL/min raccogliendo l’estratto in una provetta a fondo conico.

Evaporare il solvente, riprendere il residuo con 1 mL di acqua ultrapura ed iniettare il campio-ne in HPLC.

7.4 Analisi A titolo esemplificativo si riportano di seguito le

condizioni cromatografiche adottate nell’analisi di estratti di campioni reali (Tab. I, Tab. II). I rela-tivi cromatogrammi sono riportati nella Figura 1 e Figura 2.

Sia il pH della soluzione di ammonio acetato (Tab. I) sia il gradiente (Tab. II) sono puramente indicativi e possono essere modificati in fun-zione delle diverse caratteristiche del sistema cromatografico utilizzato e della eventuale pre-senza di picchi interferenti. Si deve comunque mantenere una sufficiente risoluzione tra i pic-chi (si può tollerare una sovrapposizione tra i picchi che non superi il 10% dell’altezza misu-

Figura 2 - Cromatogramma di una miscela di alcool etossilato C13-C15 10 EO (tensioattivo non ionico) ottenuto nelle condizioni di Tabella I con rivelatore CAD


8

rata dalla base del picco)*. Il campione di acqua opportunamente con-

centrato, come descritto ai paragrafi 7.2 o 7.3, viene iniettato nel sistema cromatografico.

(*) Ciascun picco è costituito da un insieme di isomeri più o meno risolti. Per questo motivo non si può ottenere un picco cromatografico di forma gaussiana.

8. CALCOLI

Qualora i tensioattivi rilevati siano identificabili con uno degli standard di riferimento, la loro quantificazione è da esprimere utilizzando tale riferimento.Se invece non fosse possibile eseguire questo pas-saggio, perché i picchi rilevati hanno distribuzione di-versa dal riferimento, il risultato andrà espresso utiliz-zando un riferimento appartenente alla stessa classe di tensioattivi che appaia più confacente alla risposta analitica qualitativa individuata.Per le sostanze che rispondono all’UV l’analisi quan-titativa viene effettuata sulla base di opportune curve di taratura, area-concentrazione, ottenute riportando in ascissa la concentrazione della soluzione di riferi-mento e in ordinata la media delle aree dei picchi delle soluzioni di riferimento. Per quanto riguarda l’utilizzo del rivelatore “Evapora-tive Light Scattering Detector” (ELSD), a causa della risposta non lineare di molte delle molecole tensioat-tive, potenziali analiti, si consiglia di effettuare la de-

terminazione quantitativa iniettando, in coda all’ana-lisi del campione, uno o più soluzioni di riferimento in grado di dare un segnale il più possibile simile a quello dell’analita di interesse.L’uso, in alternativa, di un rivelatore CAD (Charged Aerosol Detector) permette di ovviare alla limitazione nell’intervallo dinamico, intrinseca alla tecnica ELSD.

9. QUALITÀ DEL DATO

9.1 Accuratezza Soluzioni sintetiche preparate miscelando un

tensioattivo non ionico (alcol etossilato C11-7EO) ed uno anionico (Sodio alchilbenzensolfonato) alla concentrazione di circa 1 mg/kg ciascuno, in acqua di rete, sottoposte ad estrazione in fase solida, hanno fornito dati di recupero me-dio del 94,6% per l’anionico e del 100,1% per il non ionico.

Campioni reali di acque di scarico o di proces-so, esenti da tensioattivi, dopo aggiunta di ten-sioattivi anionici e/o non ionici nella misura di 1-2 mg/kg hanno fornito dati di recupero non superiori all’85% applicando entrambe le pro-cedure di preconcentrazione (LLE ed SPE).

9.2 Precisione La precisione associata alla sola determinazio-

ne cromatografica, senza la variabilità dovuta all’omogeneità del campione o alla tecnica di estrazione, è stata valutata dal coefficien-te di variazione, CV = (scarto tipo/valore me-dio) × 100, di una serie di ripetizioni di soluzioni di riferimento effettuate in giorni diversi. Per diversi tensioattivi e con i due metodi di rivela-zione il CV non è mai superiore al 3%.

Nota - Si consiglia ai laboratori di attivare, in accordo con le norme internazionali più recenti, dei programmi di controllo formale sulla qualità dei dati prodotti. Ciò si può realizzare verificando le proprie prestazioni attraverso ana-lisi effettuate, ad intervalli regolari di tempo, su campioni certificati prodotti da organismi internazionali e su materiali

Grafico 1 - Principali classi e sottoclassi di tensioattivi

Tabella I - Condizioni operative utilizzate per l’analisi in HPLC Fase stazionaria

Colonna Acclaim Surfactant Plus 3 m (4,6 150 mm)

Eluente Miscela binaria acetonitrile/ammonio acetato 100 mM, pH=5 “Loop” 5 µL Flusso eluente 0,6 mL/min Volume di iniezione 5 L Rivelatore UV (225 nm)

ELSD Temperatura di nebulizzazione: 30°C Temperatura di evaporazione: 90°C Gas di trasporto: 0,90 litro standard/min (slm) oppure CAD Controllo della temperature di nebulizzazione: ON Temperatura di nebulizzazione: 30°C Frequenza di acquisizione dei dati: 2Hz Filtro: non inserito

ANIONICI NON IONICI

Carbossilati Solfonati Solfati Etossilati eterei Etossilati esterei

Alchilarilsolfonati (LAS) Alchensolfonati (AOS) Alcansolfonati (SAS) Vari Alchilsolfati Etossisolfati Olii solfatati Vari Olii solfonati

Alcoli etossilati Alchilfenoli etossilati Acidi etossilati Esteri di polialcoli etossilati

CATIONICI

Etossilati amminici Etossilati Ammidici Alchilammine ammonio quaternario

Altri derivati dell’azoto

Etanolammidi Ammidi etossilate Amminoossidi Ammine etossilate

Tabella II - Caratteristiche del gradiente utilizzato per l’analisi in HPLC

Tempo (minuti)

Acetonitrile (%)

Ammonio acetato (%)

0 35 65 1.0 35 65 8.0 85 15

15.0 85 15 15.1 35 65 23.0 35 65

Figura 1 - Cromatogramma di una miscela di 4 LAS di sodio (tensioattivo anionico), ottenuto nelle condizioni di Tabella I con rivelatore CAD


9

di riferimento non certificati (carte di controllo). Caratteriz-zato il materiale di riferimento non certificato in termini di valore medio ed incertezza ad esso associata, è possibile verificare gli scostamenti di misure giornaliere condotte in parallelo con l’insieme dei campioni incogniti da determi-nare.

BIBLIOGRAFIA

S. Capri, S. De Angelis, L. Patrolecco, S. Pole-[1] sello, S. Valsecchi. Determinazione del nonilfe-nolo e nonilfenoli mono- e di-etossilati in acque superficiali, Notiziario IRSA dei Metodi Analitici, 2-8 aprile (2004).IRSA. Criteri e limiti per il controllo dell’inquina-[2] mento delle acque. Quad. Ist. Ric. Acque 75, 385-40, (1986).T. Knepper, D. Barcelo, P. Voogt. Analysis and [3] fate of surfactants in the aquatic environment, Comprehensive Analytical Chemistry, XL, Else-

vier, Amsterdam (2003).X. Liu, C.A. Pohl, J. Weiss. New polar-embed-[4] ded stationary phase for surfactant analysis. J. Chromatogr. A 1118, 29-34, (2006).S. Polesello, A. Polesello, S. Guenzi, C. Roscio-[5] li. Strumenti per il laboratorio chimico-biologico - Vol. II Le tecniche separative, Editore: Tecni-che Nuove, Milano, pp 280, ISBN 978-88-481-1943-6, (2007).Regolamento (CE) N. 648/2004 del Parlamento [6] Europeo e del Consiglio del 31 marzo 2004 re-lativo ai detergenti, Gazzetta ufficiale dell’Unione europea, 8.4.2004 L 104/1.S. Valsecchi, E. Lietti, S. Polesello. Determina-[7] zione di nonilfenolo (4-NP) e di alchilbenzene solfonati lineari (LAS) in acque e matrici solide ambientali. Notiziario IRSA dei Metodi Analitici, 2-9 agosto (2008).

Ricevuto e Accettato, 9 dicembre 2013


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La Rivista Italiana delle Sostanze Grasse La Rivista Italiana delle Sostanze Grasse (RISG) welcomes research, experimental or technological papers, short communications, review articles, on edible and industrial oils and fats of vegetable and animal origin, soaps, detergents, surfactants, cosmetics and toiletries, mineral oils, lubricants. The manuscript will be assessed by a team of referee whose opinion is essential for the article for to be accepted for publication. We ask you to indicate three names of qualified experts as a referee. The Authors publishing their manuscripts on our Journal are authorized to upload the abstract as well as the full text version in .pdf format, on their page of Research Gate, increasing the diffusion and visibility of the results.

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The editorial staff reserves the right to edit the manuscript. Major changes, however, will be made only with authors permission. Papers publication is free of charge, except: a) if the figures have to be redrawn because of poor reproduction b) if considerable changes have to be made of the text c) if the numbers and/or size of tables exceed the standard agreed on by the Editorial Committee. Before publication the corresponding author will receive offprint in .pdf file for correction. Corrections must be returned within 48 hours. After publication of the paper the corresponding author will receive one complimentary copy of the Journal and the reprint as a PDF file.

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aut

hors

inst

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ions


11

P. Bondioli1*

L. Della Bella1

G. Rivolta1

S. Faragò2

A. Boschi2

S. Beretta2

INNOVHUB-SSIAzienda Speciale della

Camera di Commercio di Milano1Divisione SSOG - Milano

2 Divisione SSS - Milano

(*) CORRESPONDING AUTHOR:Paolo Bondioli

Divisione SSOG Via Giuseppe Colombo 79

20133 Milano, Italy.e-mail: [email protected]

study of biodiesel solid contaminants by means of scan electron Microscosopy

(seM)

The presence of solid particles in biodiesel renewable fuel represents one of the main problems for the in field use either as a neat product or in blends with mineral diesel fuel. The presence of solid particles is regulated in EN 14214 specifications as “Total Contamination” parameter, set at the value of 24 mg/kg max, as determined according to the EN 12662:2014 Standard. Several studies are available about the possible correlation between total contamination and filter plugging behavior in terms of FBT (filter blocking test) or CSFBT (cold soak filter blocking test) also in view of the presence of the typical organic contaminants such as Steryl Glucosides and Saturated Monoglycerides. In this paper we wish to discuss the results obtained by evaluating the solid material retained on the EN 12662:2014 glass fiber filter by means of Scan Electron Microscopy (SEM) instrument. The solid particles on the filter were evaluated in terms on morphology, particle size and possible chemical identification, by coupling the SEM results with other diagnostic techniques such as X Rays for inorganic constituents and GC-FID.Some SEM pictures of different contamination situations are shown and discussed also in comparison with the classical physical-chemical characterization data of the same biodiesel samples.

Studio dei contaminanti solidi del biodiesel mediante Microscopia Elettronica a ScansioneLa presenza di particelle solide nel combustibile rinnovabile biodiesel rappresenta uno dei principali problemi per l’utilizzo in campo sia come prodotto puro che in miscela con gasolio tradizionale. La presenza di particelle solide è regolata nella specifica Europea del biodiesel EN 14214 mediante il parametro “Contaminazione Totale”, per il quale è previsto un valore di 24 mg/kg max, determinato secondo EN 12662:2014. Numerosi studi sono disponibili sulla correlazione tra il valore di contaminazione totale ed altri parametri, quali ad esempio il valore FBT (Filter Blocking Test) o CSFBT (Cold Soak FBT), anche in considerazione della presenza di contaminanti organici tipici del biodiesel, quali come sterilglicosidi e monogliceridi degli acidi grassi saturi. In questo articolo vengono discussi i risultati ottenuti valutando per mezzo del microscopio elettronico a scansione (SEM) il materiale solido trattenuto sui filtri in fibra di vetro utilizzati per la prova EN 12663:2014. Le particelle solide sul filtro sono state valutate in termini di morfologia, dimensioni delle particelle e identificazione chimica, accoppiando i risultati SEM con quelli ottenuti con altre tecniche quali Raggi X per i componenti inorganici e GC-FID. Alcune immagini ottenute al SEM sono discusse in comparazione con i classici dati di caratterizzazione chimico-fisica ottenuti sui medesimi campioni di biodiesel.


12

INTRODUCTION

Biodiesel is a renewable, biodegradable, alternative fuel prepared by means of a transesterification reac-tion of natural oils and fats. In practice, biodiesel is a mixture of fatty acids methyl esters (FAME) containing traces of starting material (triglycerides), reaction in-termediates (mono- and diglycerides of fatty acids), reaction by-products (glycerol) and a number of diffe-rent substances generally known as “minor compoun-ds”. In this last family, free and esterified sterols, free and acylated steryl glucosides (SG and ASG respec-tively), fatty alcohols, waxes, tochopherols, terpenic hydrocarbons, etc. are but some constituents of such a complicate pattern of minor compounds as can be found in a natural oil-derived biodiesel. According to the different feedstocks, quality and concentration of different minor constituents may vary dramatically. Not all listed molecules have an impact on biodiesel performance, but some of them, if not carefully moni-tored, can cause some problems in operation and in analysis. Some examples of these facts are: i) Steryl Glucosides (SG), having a very low solubility in biodiesel, while they are soluble in the original oil along with the related molecule ASG. Furthermore, during transesterification reaction, ASGs are dea-cylated and the final steryl glucoside concentration increases, leading to the formation of a solid material that is insoluble in biodiesel. We can estimate that the SG concentration in biodiesel does not pose evident precipitation problems up to around 20 mg/kg at am-bient temperature. Several problems related to the presence of SG were reported some years ago, contemporarily with the in-creased use of palm and soy oils as a feedstock for biodiesel production [1 - 3];ii) Waxes, in a past when sunflower oil was used in Southern Europe for biodiesel production some pro-blems were recorded in terms of filter plugging due to waxes;iii) Squalene, an ubiquitous terpenic hydrocarbon, does not give raise to problems in biodiesel use, but causes problems during the analytical evaluation of monoglyceride content according to EN 14105:2011 standard. In some situations, Squalene can be con-fused with glycerol monostearate, causing an out-of-spec situation. Bondioli et al. [4] described this fact for biodiesel samples prepared from Palmoil Distilled Fatty Acids (PFAD) and suggested a possible solution for doubtful cases.Within the group of reaction intermediates the most problematic class is represented by Saturated Mono-glycerides (SM), mainly glycerol monoesters of palmi-tic and stearic acid. These substances have high mel-ting points and high polarity: for this reason SM have the tendency to become insoluble during biodiesel storage in particular if temperature is low. The final result in this case too is filter clogging. The problem of the presence of SM in biodiesel became evident when

oils and fats having high concentrations of Palmitic and/or Stearic acid, such as palm oil and animal fats in general, came in use as a feedstock in high con-centrations because of their more competitive price. For the correct monitoring of these products, some interesting analytical procedures recently appeared in international literature [5, 6] based on SPE-GC-FID and GC-MS techniques, but a standardized procedu-re for SM evaluation is still under development.The co-relation existing between Total Contamination, Filter Blocking Test (FBT) and Cold Soak Filter Blocking Test (CSFBT) after several years of strong activity car-ried out by a number of important laboratories and researchers is still to be demonstrated. In other words, for some samples a correlation between laboratory re-sults and in-field performance is clear, but for others it is not. One possible explanation of this effect can be found if we consider that solid particles in biodiesel may have a different nature and origin. We must also remember that the original meaning of Total Contami-nation determination was to evaluate the cleanliness of the fuel in terms of the amount of dirt contaminants such as the so called “rust and dust”. In fact, this me-thod comes from long-term experience on diesel fuel but, when transferred on biodiesel, some problems might arise. The presence of already listed SM and SG can have an impact on the final result, even though they cannot be regarded as solid contaminants ac-cording to the classic definition. Moreover, as these molecules have a very high polarity degree, they have a different solubility behavior in neat biodiesel, when biodiesel is in mixture with diesel fuel and when bio-diesel is diluted in a solvent, such as in the case of the new EN 12662:2014 standard for total contamination. The entry into force of this new standard introducing a new procedure for biodiesel represented for us the occasion to carry out a study about the nature of the solid contaminants that can be retained on the filter used in this procedure. Only few examples of studies carried out on biodiesel solid contaminants are cur-rently available [7]. In this paper we are presenting our results obtained by analyzing the material retained on EN 12262:2014 filters at the end of analytical test. The results obtained by means of Scan Electron Microsco-pe (SEM) were also coupled with results obtained by more classical analytical data.

MATERIALS AND METHODS

Biodiesel samples were obtained from an Italian Company. The properties of the samples were wi-thin the limits set by the European specification for biodiesel EN 14214:2014. Looking at the fatty acids composition we can say that the samples represent a mixture of different feedstocks such as palm, soy and rapeseed oil. The main compositional parameters are listed in Table I. Before delivery, the supplier analyzed the samples for total contamination using the old pro-cedure set forth by EN 12662:2008. After the arrival


13

in our laboratory the samples were analyzed again using the new standard. The results obtained are a little higher than the ones obtained using the previous procedure. Ester content (EN 14103:2011), Free and Total Gly-cerol, mono-, di- and triglycerides (EN 14105:2011) were measured according to the existing standards, while fatty acids composition was determined accor-ding to ISO 5508:1990, steryl glucosides were eva-luated by means of SPE-GC-FID procedure using Cholesteryl glucoside as an internal standard. The new EN 12662:2014 procedure contains some new unit operations with respect to the old one. In short, the sample preparation starts with the storage of the sample for 2 hours at 60°C in a thermostatic oven to remove the previous “thermal history” of the material before sampling. The sample is then proces-sed by means of weighing in a suitable container. A dilution with solvent (Heptane 75-Toluene 25) follo-wed by 2 hours in quiet at ambient temperature is preliminary to the quantitative filtration. After proper washing, the weighing of the filter allows to calculate the final result.The filters as obtained from EN 12662:2014 test were sent to the SEM evaluation. The samples were pre-pared for Electronic Microscopy, according to the fol-lowing procedure: For SEM analysis a Tescan (Brno, Czech Republic) instrument mod. MIRA 3 FEG-SEM, field emission scanning electron microscope, was used. The elec-tronic microscope can operate in High Vacuum (HV) mode, with residual pressure of 0,005 Pa, for the eva-luation of conductive samples. Non conductive sam-

ples can be processed after a preliminary metalliza-tion procedure. Non conductive samples can also be processed in Low Vacuum mode (residual pressure between 5 and 150 Pa) avoiding metallization. The used detectors were Secondary Electron detector (SE) and Back Scattered Electron detector (BES). The electron microscope is also equipped with a microa-nalysis detector EDS XFlash 6/10 (Quantum version, Bruker, Milano, Italy), which allows to get information about the metal composition of the surface. Repre-sentative aliquots were taken, installed on an alumi-num stub, metalized in Gold Palladium and analyzed in SEM-FEG, HV mode.The short path distillation test was carried out by me-ans of UIC (Alzenau-Hoerstein, Germany) mod. KDL 5 unit. Evaporator Temp. 140°C, Feed temperature 60°C, Residue discharge temperature 80°C, Con-denser temperature 30°C, residual pressure 1 mbar, feed rate 800 g/h. The yield in distilled product was higher than 95% in mass.

Table I – Main composition parameters of samples used for this research

Sample 19 Sample 40 Free Glycerol, % m/m N. D. 0.002 Monoglycerides, % m/m 0.27 0.29 Diglycerides, % m/m 0.10 0.09 Triglycerides, % m/m < 0.01 < 0.01 Fatty acids composition % Lauric Myristic Palmitic Palmitoleic Eptadecanoic Eptadecenoic Stearic Oleic Linoleic Linolenic Eicosanoic Eicosenoic Behenic Erucic

0.05 0.17 8.94 0.32 0.06 0.13 2.40

56.08 22.59 7.07 0.49 1.06 0.29 0.33

0.11 0.24 9.79 0.32 0.07 0.17 2.23

57.14 20.14 7.74 0.48 1.03 0.27 0.27

Saturated Monoglyceride (calculated), % m/m

0.03 0.04

Steryl Glucosides, mg/kg 28 41 Total contamination, mg/kg 19 40

Figure 1 –

Figure 2 -

– Desert rose lik

- Multilayer solid

ke solids on the

d (x 7250)

e filter (x 6120)

Figure 1 –

Figure 2 -

– Desert rose lik

- Multilayer solid

ke solids on the

d (x 7250)

e filter (x 6120)


14

RESULTS AND DISCUSSION

The evaluation of the solids retained on the test filter by means of SEM allowed to draw some interesting remarks about the nature and the morphology of said solids.First of all, two different solids showing a different sha-pe were detected on filters coming from both sam-ples. The two main forms identified correspond at Desert Rose-like solids (Fig. 1) and at Multilayer solids (Fig. 2). These crystals were present in both evaluated samples. Other minor, simpler solids were also pre-sent on the filters. Actually the question was about the nature of these precipitates: Are they of inorganic or organic products? In order to try and find an answer for this question, filters were carefully washed with se-veral milliliters of a mixture chloroform/methanol in a 2:1 volume ratio. In our intention, this solvent could dissolve all organic substances leaving practically un-changed the inorganic fraction of solid material. The chloroform/methanol solution obtained from filters cle-aning was collected in a test tube. The solvent was

finally removed under inert gas and the solid analyzed by means of GC-FID under the experimental condition of EN 14105:2011. The sample was TMS derivatized before injection. Looking at the path reported in Figure 3 we can observe that for the two samples (A and B), the main constituents of the solid contaminants are represented by a mixture Steryl glucosides, and also glycerol (main peak on the left side of the path) is present in detectable amounts. Also interesting is the observation that in both cases monoglycerides were not detected (retention time between 10 and 15 mi-nutes). With this simple experience it was possible to demonstrate that the main solid contaminant in this case was represented by a mixture of SG, surely co-ming from the original oil. Also very interesting was the remark that no total and/or saturated monoglycerides could be detected. At this point of the research we could evaluate two different possibilities: i) an estima-ted concentration of 300-400 mg/kg of saturated mo-noglycerides does not contribute to the total conta-mination or ii) the standard test as implemented does not allow for this detection. We shall come back on

Figure 3 derivatizat

– GC-FID pathtion)

hs of material rrecovered from

m filters after w

ashing with chhloroform/metha

anol 2:1 mixture (after TMS


15

this problem later in the paper. The solid fraction retai-ned on the filter after solvent washing represents the inorganic fraction of total contaminants. As an estima-tion, we can say that this fraction represented approx. the 10% of total solid contaminants. Using the SEM instrument coupled with an Energy Dispersive X-ray (EDX) spectrometer it is also possible to evaluate the real composition of these products. Some interesting color pictures can be taken where different salts have colors, according to their different metal content. In every case the most abundant elements detected were Sodium, Calcium, Magnesium, Aluminum and Iron. We can suppose that the first three metals are coming from chemicals (namely Na methoxyde) and starting material, while Al and Fe belong to plant le-akage. One interesting issue still remained unanswered, con-cerning the impact of saturated monoglycerides on total contamination test.For this reason a sample of distilled biodiesel was prepared by means of a short path distillation test. In practice, short path distillation allows to isolate nearly pure FAME, leaving in the undistilled residue mono-, di-, triglycerides as well as the different unsaponifia-ble constituents. This was the right base stock for saturated monoglyceride additivation. Starting from

a pure glycerol alpha monononadecanoate reference material a blend containing 2000 mg/kg of saturated monoglyceride in distilled biodiesel was prepared. The dissolution of monoglyceride and the homogenization of the sample was carried out at 70°C under stirring. The mixture was subdivided into two fractions: the first quantity was treated in the oven for 2 hours t 60°C, according to the EN 12662:2014 standard, while the second quantity was left in the laboratory at ambient temperature for the same period. The Figure 5 clearly shows that the sample maintained in oven is perfectly limpid, while the ambient temperature sample shows a high turbidity. The same situation remained after di-lution with the heptane/toluene solvent. The final re-sult was that the sample prepared according to the proposed method did not leave any residue on the filter surface (Fig. 6A), while for the unheated sample a very high contamination level was detected. The cor-responding filter was highly contaminated (Fig. 6B).

CONCLUSIONS

The results obtained with this research activity allo-wed to increase the knowledge about solid contami-nants in biodiesel.

Figure 4 – Inorganic contaminants left on the filter after washing with chloroform/methanol mixture 2:1

Figure 5 – C19 monoglyceride solution in distilled biodiesel. Upper picture: sample still hot (60 °C), lower picture: sample at ambient temperature


16

In fact it was demonstrated that filters prepared accor-ding to the standard method for total contamination (EN 12662:2014) contain both organic and inorganic contaminants. In our samples organic contaminants represented approx. 90% of total. Using complemen-tary techniques it was possible to demonstrate that the organic fraction is represented by Steryl Gluco-sides. No saturated monoglycerides were detected. On the other side an additional experiment carried out on distilled biodiesel spiked with saturated mono-glycerides, and demonstrated that under the required experimental conditions saturated monoglycerides remain soluble in the solvent/sample mixture and are not retained by the filter. Now two big questions emerge that need to be an-swered: 1. Are saturated monoglycerides a real problem for the in field use of biodiesel either as a neat fuel or in

blend with diesel fuel to meet the EN 590 diesel fuel specification?2. The actually available standard for total contami-nation measurement is a suitable standard to pro-perly check the fuel quality or is it influenced by the changes in solubility of minor constituents as a con-sequence of the dilution with the heptane/toluene sol-vent mixture?About the contamination by inorganic products, we demonstrated that, in the evaluated samples, it has a minor impact, in terms of quantity. Two different cate-gories of inorganic contaminants were detected:1. Na, Mg and Ca salts, coming from the feedstock and from the alkaline catalyst used for transesterifi-cation;2. Fe, Zn and Al salts and particles deriving from the plant used for biodiesel processing.

REFERENCES

[1] P. Bondioli, N. Cortesi, C. Mariani, Identification and quantification of Steryl Glucosides in bio-diesel. Eur. J. Lipid Sci. Technol. 110, 120-126 (2008)

[2] V. van Hoed, N. Zyaykna, W. de Greyt, J. Maes, R. Verhè, K. Deemeestere, Identification and oc-currence of steryl glucosides in palm and soy biodiesel. J. Am. Oil Chem. Soc. 85, 701-709 (2008)

[3] R. A. Moreau, K.M. Scott, M.J. Haas, The iden-tification and quantification of steryl glucosides in precipitates from commercial biodiesel, J. Am. Oil Chem. Soc. 85, 761-770 (2008)

[4] P. Bondioli, L. Della Bella, G. Rivolta, Evaluation of monoglyceride content in biodiesel samples prepared from PFAD (Palmoil Fatty Acids Di-stilled). Riv. Ital. Sostanze Grasse 89, 240-246 (2012)

[5] P. Bondioli, L. Della Bella, G. Rivolta, Evaluation of total and saturated monoglyceride content in biodiesel at low concentration. Eur. J. Lipid Sci. Technol. 115, 576-582 (2013)

[6] B. Pieber, S. Schober, C. Goebl, M. Mittelbach, Novel sensitive method for the determination of steryl glucosides in biodiesel by gas-chromato-graphy/mass spectroscopy. Journal of Chroma-tography A 1217 (42), 6555-6561 (2010)

[7] L. Hirschegger, S. Schober, M. Mittelbach, Effi-cient and sensitive method for the quantification of saturated monoacylglycerols in biodiesel by gas chromatography-mass spectrometry. Eur. J. Lipid Sci. Technol. 116, 89-96 (2014)

[8] S. Camerlynck, J. Chandler, B. Hornby, I. Van Zuylen, FAME filterability: understanding and so-lutions. SAE Int. J. Fuels Lubr. 5 (3) (2012)

Received and Accepted, November 26, 2014

Figure 6A12662:201

Figure 6B12662:201sample

A - Filter after f14 standard

B - Filter after f14 standard.

filtration of sam

filtration of samMixture prepa

mple according

mple according ared with unh

to EN

to EN

heated


17

D. BaglioM.R. Porta

L. Folegatti*

INNOVHUB-SSI Azienda Speciale della

Camera di Commercio di MilanoDivisione SSOG – Milano

(*)CORRISPONDENZA AUTORE:Dr.ssa Liliana Folegatti

Divisione SSOGVia G. Colombo, 79

20133 MilanoE-mail: [email protected]

Tel. +39 02 70649772

Comparazione dei differenti sistemi di iniezione gascromatografici per la

rilevazione di grassi vegetali diversi dal burro di cacao nei cioccolati fondenti

Il presente studio ha lo scopo di valutare l’impiego di diverse tecniche di iniezione in gascromatografia (iniezione on-column, split e PTV) per l’analisi della componente trigliceridica del burro di cacao.La comparazione è stata effettuata iniettando un materiale di riferimento certificato di burro di cacao e valutando i fattori di risposta strumentali dei principali trigliceridi. Inoltre è stata valutata anche la risoluzione cromatografica tra alcune coppie critiche di trigliceridi.I risultati ottenuti hanno dimostrato che la tecnica di iniezione on-column è quella maggiormente consigliata ma non fornisce risultati statisticamente differenti rispetto alle altre due tecniche split e PTV. Ciò solo dopo aver effettuato un’attenta ottimizzazione delle condizioni gascromatografiche al fine di ridurre al minimo le discriminazioni a carico della componente trigliceridica del burro di cacao.Successivamente, sono stati analizzati diversi campioni di cioccolato fondente allo scopo di identificare la presenza di grassi estranei al burro di cacao. Il metodo di analisi utilizzato è stato quello proposto dai ricercatori del IRMM. Quasi tutti i campioni analizzati contengono solo burro di cacao ad eccezione di un unico campione che è risultato essere addizionato con grassi vegetali. Ulteriori verifiche sono comunque necessarie allo scopo di confermare tale risultato e l’utilizzo di metodi aggiuntivi all’analisi trigliceridica può fornire un valido supporto.

Comparison of different injection techniques in gas chromatography to detect cocoa butter equivalents in plain chocolatesThe present study is aimed evaluating the use of different injection techniques in gas chromatography (on-column, split and PTV injection) for the analysis of the triglyceride fraction of cocoa butter. The comparison was performed by injecting a certified reference material of cocoa butter and evaluating the instrumental response factors of the main triglycerides. In addition, the chromatographic resolution between some critical pairs of triglycerides was evaluated. The results obtained showed that the on-column injection was the most recommended technique but it did not provide statistically different results compared to the other two techniques, split and PTV. This only after a careful optimization of GC conditions in order to minimize the discrimination against the triglyceride components of the cocoa butter. Several samples of plain chocolates were analyzed in order to identify the presence of cocoa butter equivalents. The method of analysis used was the one proposed by the researchers of IRMM. Almost all the samples analyzed contained only cocoa butter with the exception of a single sample which was found to be added with vegetable fats. Further investigations were still needed in order to confirm this result and the use of additional analytical methods could provide valuable support.


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1. INTRODUZIONE

Il cioccolato è uno degli alimenti più antichi consuma-ti dall’uomo e più diffusi al mondo. Il burro di cacao (CB), il grasso estratto dai semi di cacao della specie Theobroma cacao, è l’ingrediente più costoso nella formulazione del cioccolato [1]. I semi di cacao sono coltivati principalmente in Africa orientale, in Ameri-ca centrale e meridionale e in Asia sud/occidentale e note sono le differenze chimico-fisiche del burro di cacao delle differenti origini geografiche. Tali diffe-renze sono imputabili alla composizione trigliceridica e il burro di cacao di provenienza asiatica contiene una percentuale maggiore di trigliceridi monoinsaturi rispetto al burro di provenienza americana ed africa-na e mostra quindi una durezza maggiore. Il burro di cacao americano contiene la percentuale maggiore di trigliceridi di- e polinsaturi, mentre il burro di cacao africano si posiziona nel mezzo. Le proprietà uniche del CB sono dovute alla composizione della sua fra-zione trigliceridica, ed in particolare i 2-oleilglicero-li dell’acido palmitico e stearico (POP, POS e SOS) che formano una struttura cristallina beta-polimorfa durante il processo di produzione del cioccolato, fa-vorendo la dispersione degli altri ingredienti.Considerando il fatto che il CB è l’ingrediente più co-stoso del cioccolato e la sua disponibilità è variabile nel tempo sono stati ricercati dei sostituti, completa-mente o in parte, impiegando altri grassi vegetali [2]. Dal punto di vista legislativo, la Direttiva CE 2000/36 [3] disciplina l’impiego e l’etichettatura dei grassi ve-getali aggiunti al cioccolato e dei prodotti ad esso correlati. Per prodotti commerciali con la denomina-zione di cioccolato è consentito esclusivamente utiliz-zare grassi ricavati da un determinato tipo di materie prime. Tali grassi devono peraltro essere compatibili con il burro di cacao e non possono essere sottopo-sti a modifica per via enzimatica. La massima quan-tità consentita di questi grassi è pari al 5% (calcolata sull’intera ricetta) e deve essere dichiarata in etichet-ta con la dicitura “grassi vegetali”.I grassi vegetali sono, singolarmente o miscelati, equivalenti al burro di cacao (CBE) e devono rispon-dere ai seguenti criteri: sono grassi vegetali non con-tenenti acido laurico, ricchi di trigliceridi monoinsaturi simmetrici di tipo POP, POS, SOS; sono mescola-bili in qualunque proporzione con il burro di cacao e compatibili con le sue proprietà fisiche (punto di fusione e temperatura di cristallizzazione, velocità di fusione, necessità di trattamento di tempra); inoltre sono ottenuti esclusivamente mediante procedimen-to di raffinazione e/o frazionamento ed è esclusa la modificazione enzimatica della struttura del triglice-ride.A norma di tali criteri possono essere utilizzati i se-guenti grassi vegetali, ricavati dalle piante sotto elen-cate:1. Burro d’illipé, sego del Borneo o Tengkawang

(Shorea spp);

2. Olio di palma (Elaeis guineensis, Elaeis olifera);3. Grasso e stearina di Shorea robusta, sal (Shorea

robusta);4. Burro di karité (Butyrospermum parkii);5. Burro di cocum (Garcinia indica);6. Nocciolo di mango (Mangifera indica).In Italia questa direttiva è stata recepita dal Decreto Legislativo n. 178 del 12 giugno 2003 pubblicato sul-la Gazzetta Ufficiale n. 165 del 18 luglio 2003 [4].Il problema di rilevare e quantificare i grassi vegetali estranei al CB è stato investigato per anni [5]. Esi-stono diversi metodi di analisi basati principalmente sull’impiego della gascromatografia e della cromato-grafia liquida e che analizzano gli acidi grassi, i tri-gliceridi e alcuni componenti minori (steroli, prodotti di degradazioni degli steroli e terpeni [6, 7]). I metodi più utilizzati analizzano la frazione trigliceridica del CB mediante GC o HPLC [8-10]. Nel 1980 due gruppi di ricercatori capitanati rispetti-vamente da Fincke e da Padley e Timms hanno pro-posto due metodi di calcolo basati sulla composizione trigliceridica per rilevare la presenza di CBE in prodotti a base di cioccolato [11, 12]. L’ipotesi, supportata dai dati sperimentali, era che il contenuto di C50 e C54 in campioni genuini di CB aveva una relazione lineare, quando la frazione trigliceridica era normalizzata, cioè C50+C52+C54=100%. Successivamente fu dimo-strato che i CBE avevano un contenuto di C52 infe-riore a quello del CB e che quindi le miscele CBE/CB deviavano dal comportamento lineare sopra esposto. Quindi l’aggiunta di CBE al cioccolato portava ad avere un contenuto di C50 nel campione superiore a quello calcolato applicando l’equazione matematica. L’unica eccezione a questa regola era l’aggiunta di burro di illipè a causa della sua composizione triglice-ridica molto simile a quella del CB.Altri ricercatori raggiunsero risultati simili riguardo la relazione lineare tra i trigliceridi C50 e C54 nel CB (Po-dlaha, 1984; Simoneau, 2000; Young, 1984; Barca-rolo e Anklam, 2001, 2002) [13].Metodi di analisi successivi si basarono sull’analisi della frazione trigliceridica ottenuta mediante analisi gascromatografica ad elevata temperatura ma con l’utilizzo di colonne mediamente polari, portando quindi alla separazione dei trigliceridi sulla base del peso molecolare e del grado di insaturazione [8, 9, 14, 15]. In questo caso riportando in grafico %POP vs. %SOS la differenza tra CB e CBE era maggior-mente evidenziata.La gascromatografia ad elevata temperatura per l’analisi dei trigliceridi è una tecnica critica in quanto può causare delle discriminazioni a carico dei trigli-ceridi, specialmente nel processo di introduzione del campione [14-19]. Il presente studio ha lo scopo di investigare l’effetto delle diverse tecniche di iniezione in gascromatogra-fia (iniezione split, on-column e PTV) sul recupero dei diversi componenti la frazione trigliceridica del burro di cacao. Inoltre applicando il metodo di analisi pro-


19

posto dai ricercatori dell’istituto IRMM della Comunità Europea [20, 21] per il cioccolato fondente e divenu-to metodo ISO [22, 23] e AOCS [24] si sono voluti analizzare diversi campioni di cioccolato acquistati sul mercato italiano.

2. PARTE SPERIMENTALE

2.1 MATERIALI E REAGENTITutti i reagenti impiegati sono di qualità analitica rico-nosciuta e l’acqua è distillata o demineralizzata o di equivalente purezza.L’isottano (grado GC), l’etere di petrolio (grado p.a., intervallo di punto di ebollizione tra 40-60°C), l’acido cloridrico 37% (grado p.a.) sono forniti dalla Sigma-Aldrich (Milano, IT). Lo standard di burro di cacao certificato (IRMM-801) è distribuito dal IRMM, Belgio [25].I campioni di cioccolato fondente sono stati acquistati nei supermercati e comprendono sia prodotti di mar-che note che primi prezzi.

2.2 STRUMENTAZIONELa frazione trigliceridica è stata separata impiegan-do una colonna capillare RTX-65TG (30 m, 0.25 mm i.d., 0.10 µm spessore del film) della Restek montata su un gascromatografo Agilent 7890 A con iniettore on-column e split/splitless e rilevatore FID. Il software di gestione dello strumento era la Chemstation della Agilent.Per le iniezioni split: la temperatura dell’iniettore era di 370°C, il gas di trasporto era elio al flusso costante di 1.8 ml/min, e il rapporto di splittaggio era di 1:10. Il volume iniettato era di 1 µl. La programmata di tem-peratura del forno era di 340°C per 1 min, poi 1°C/min fino a 360°C mantenuta per 3 minuti. La durata complessiva dell’analisi era di 24 minuti. Il rilevatore FID era mantenuto alla temperatura di 370°C.Per le iniezioni on-column: il gas di trasporto era elio alla pressione di 270 kPa. Il volume iniettato era di 0.5 µl. La programmata di temperatura del forno era di 80°C per 1 min, poi 30°C/min fino a 340°C mante-nuta per 30 minuti. La durata complessiva dell’analisi era di 40 minuti. Il rilevatore FID era mantenuto alla temperatura di 360°C.Per le analisi in PTV, la colonna è stata montata su un gascromatografo DANI GC Master con iniettore PTV e rilevatore FID. Il software di gestione dello strumen-to era il Clarity della Dani Instruments.La temperatura dell’iniettore PTV era di 100°C per 0.5 min, poi 500°C/min fino a 360°C per 5 minuti, il gas di trasporto era elio al flusso costante di 1.6 ml/min, e il rapporto di splittaggio era di 1:10. Il volume iniettato era di 1 µl. La programmata di temperatura del forno era di 100°C per 1 min, poi 35°C/min fino a 340°C, poi 5°C/min fino a 360°C mantenuta per 15 minuti. La durata complessiva dell’analisi era di 27 minuti. Il rilevatore FID era mantenuto alla temperatu-ra di 370°C.

2.3 PREPARAZIONE DEL CAMPIONELa preparazione dei campioni di cioccolato fondente è stata eseguita in accordo al metodo AOAC 970.20 [26].L’estrazione del grasso dal cioccolato è stata esegui-ta in accordo al metodo AOAC 963.15 [27] nel caso in cui era richiesta l’analisi quantitativa dei CBE pre-senti.Per le analisi qualitative sulla presenza di CBE in ag-giunta al burro di cacao è stato impiegato un metodo rapido di estrazione della parte lipidica. In particolare, circa 4 g di cioccolato fuso a 55°C sono stati estratti con 10 ml di isottano. Il campione è stato sciolto, agi-tato e centrifugato. Dopo la separazione delle fasi, il cioccolato è stato estratto per altre due volte con 10 ml di isottano. Le fasi organiche sono state riunite ed evaporate in corrente di azoto.Il grasso ottenuto, sia esso estratto dal cioccolato o il materiale di riferimento IRMM-801, è stato scaldato a circa 55°C in stufa fino a completo scioglimento. Successivamente si sono pesati circa 0.2 g di grasso in un matraccio tarato da 20 ml e si è portato a volu-me con isottano. I campioni sono stati ulteriormente diluiti prelevando 1 ml di soluzione, ponendola in un matraccio tarato da 10 ml e portando a volume con isottano. Tale soluzione aveva una concentrazione di circa 1 mg/ml ed era pronta per l’analisi gascroma-tografica.

2.4 ESPRESSIONE DEI RISULTATIIl metodo di analisi proposto dai ricercatori del IRMM e divenuto poi metodo ISO e AOAC si basa sulla iden-tificazione e quantificazione dei 5 principali trigliceridi del burro di cacao. L’applicazione di formule mate-matiche permette poi di rilevare la presenza di CBE in aggiunta al CB nei cioccolati fondenti [20].L’identificazione dei principali trigliceridi è stata ese-guita iniettando il materiale di riferimento certificato di burro di cacao (IRMM-801) ed i principali cinque trigli-ceridi presenti sono: 1,3-dipalmitoil-2-oleoil-glicerolo (POP), 1-palmitoil-2-oleil-3-stearoil-glicerolo (POS), 1-palmitoil-2,3-dioleil-glicerolo (POO), 1,3-distearoil-2-oleoil-glicerolo (SOS) e 1-stearil-2,3-dioleil-glicerolo (SOO). Un esempio di cromatogramma ottenuto iniettando la miscela standard IRMM-801 è riportato in Figura 1.Mediante l’iniezione del materiale di riferimento IRMM-801, eseguita nelle medesime condizioni sperimentali dei campioni, sono stati calcolati i fattori di risposta per i cinque trigliceridi POP, POS, POO, SOS e SOO. Il calcolo ha previsto la determinazione dell’area % relativa dei 5 composti e mediante la massa % ripor-tata nel certificato di analisi del materiale di riferimento [25], la determinazione dei fattori di risposta.Il metodo di riferimento sviluppato dai ricercatori del IRMM prevede che per i campioni di burro di cacao genuini vale l’equazione 1:

POP = 43.734 – 0.733 × SOS


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con una deviazione standard residua di 0.125 e dopo aver normalizzato i risultati, cioè POP+POS+SOS=100%. Per il 99% dei casi, un burro di cacao genuino soddi-sfa l’equazione 2:

POP < 43.734 – 0.733 × SOS + 2.326 × 0.125

Un valore di POP superiore al teorico indica che il burro di cacao in esame non è genuino. Questa pro-cedura permette di rilevare la presenza minima del 2% di CBE in burro di cacao, che equivale ad una percentuale del 0.4% nel cioccolato (assumendo una quantità di grasso del 20%).Se il campione analizzato risultasse non genuino è possibile calcolare la percentuale di CBE presente in miscela con il burro di cacao e la percentuale nel cioccolato utilizzando le formule matematiche svilup-pate dai ricercatori del IRMM [21].Tali formule permettono il calcolo della percentuale di CBE nel burro di cacao e nel cioccolato applicando il metodo della regressione lineare PLS (Partial Least Squares) mediante l’equazione 3:

% CBE nel burro di cacao = = 37.439 + 1.175 × POP – 1.939 × POS – 0.121 ×

POO + 0.982 × SOS – 0.097 × SOO

La percentuale di CBE nel cioccolato è calcolata me-diante l’equazione 4:

% sostanza grassa × % CBE nel burro di cacao% CBE nel cioccolato = _____________________________________

100

dove:% sostanza grassa = % grassi estratti dal cioccolato applicando il metodo AOAC 963.15% CBE nel burro di cacao = valore trovato mediante l’equazione 3.

3. RISULTATI E DISCUSSIONE

3.1 COMPARAZIONE DELLE DIVERSE TECNICHE DI INIEZIONE DEL CAMPIONE IN GC

I metodi gascromatografici pongono il problema della degradazione dei componenti insaturi dei trigliceridi alle alte temperature impiegate (> 300°C), special-mente in fase di introduzione del campione. Molti autori hanno pubblicato degli studi sull’effetto della tecnica di iniezione in gascromatografia sul recupero dei diversi componenti la frazione trigliceridica di un grasso. Grob [19] ha pubblicato uno studio dettagliato su questo aspetto evidenziando come la tecnica on-column sia la migliore per questa tipologia di analisi, mentre l’iniezione per vaporizzazione (split e splitless) provocava una scarsa precisione e causava la degra-dazione del campione stesso. Questo effetto era più evidente per i componenti poli-insaturi dei trigliceridi rispetto agli analoghi mono-insaturi e saturi. Geeraert e Sandra [14, 18] hanno ottenuto risultati molto riproducibili per l’analisi dei trigliceridi del bur-ro di cacao impiegando un iniettore “movable on-column injector”. Questa modifica ha permesso di isolare l’iniettore dal forno, potendo quindi effettuare le analisi a temperature più elevate mantenendo l’inie-zione del campione a freddo. Il risultato era stato la

Figura 1 - Profilo trigliceridico della miscela standard IRMM-801 ottenuto mediante iniezione split nelle condizioni specificate nel testo. Identificazione dei principali trigliceridi: 1-PPP, 2-MOP, 3-PPS, 4-POP, 5-PLP, 6-PSS, 7-POS, 8-POO, 9-PLS, 10-PLO, 11-SSS, 12-SOS, 13-SOO, 14-SLS+OOO, 15-SOA. Abbreviazioni: P = acido palmitico, M = acido miristico, O = acido oleico, S = acido stearico, L = acido linoleico, A = acido arachico, nella struttura del trigliceride.

min16 18 20 22 24 26 28

pA

30

40

50

60

70

80

90

1 2 3

4

5 6

7

8 9

10 11

12

13 14

15


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riduzione dei tempi di analisi e la possibilità di risolvere miscele complesse di trigliceridi come nel caso del burro e degli oli vegetali (oli di palma, olio di caffè, olio di lino, etc.).I ricercatori del IRMM [15] hanno pubblicato i risultati di due circuiti interlaboratorio per la determinazione della composizione trigliceridica del burro di cacao mediante analisi gascromatografica e impiegando tre diverse tecniche di iniezione: on-column, split e PTV. Le conclusioni a cui sono giunti sono state che la tec-nica di iniezione on-column era quella maggiormente consigliata, ma non forniva risultati oggettivamente migliori rispetto agli altri due sistemi split e PTV per questa specifica matrice. Questi risultati non devono essere interpretati come universalmente validi e solo un’attenta ottimizzazione delle condizioni strumenta-li può ridurre la perdita dei singoli trigliceridi ma non eliminarla del tutto. Nel caso del burro di cacao, esso è costituito principalmente da trigliceridi aventi un nu-mero di atomi di carbonio e grado di insaturazione simile; quindi le discriminazioni in fase di iniezione possono essere ridotte al minimo. Per confermare questi risultati nel nostro laboratorio abbiamo impiegato tre diversi sistemi di iniezione del campione montati su due gascromatografi differenti: uno era dotato di iniettore split e on-column, mentre il secondo di iniettore PTV. La colonna cromatografi-ca utilizzata per la separazione era sempre la stessa (RTX-65TG). Nel metodo di analisi proposto dai ricercatori del IRMM non erano riportate le condizioni strumentali standard da utilizzare, ma esse dipendono da diver-si fattori quali il tipo di gascromatografo, la colonna analitica, il tipo di iniettore etc. I parametri strumentali devono essere ottimizzati al fine di soddisfare deter-minate condizioni di risoluzione dei picchi e di fattori di risposta del rivelatore FID.La comparazione tra le varie tecniche di iniezione è stata effettuata analizzando il materiale di riferimento IRMM-801 e confrontando i fattori di risposta speri-mentali del rivelatore FID per i trigliceridi POP, POS, POO, SOS e SOO. I risultati sono riportati in Tabella I dove per ogni tipo di iniettore si riporta il valore medio di 6 determinazioni e la deviazione standard relativa (in parentesi).I fattori di risposta strumentale per i cinque principali trigliceridi devono essere compresi nell’intervallo tra 0.8 e 1.2. Come si può notare dai dati della Tabella I per tutti i sistemi di iniezione questa condizione è verificata e il valore della deviazione standard relativa è inferiore al 3%.Inoltre si può notare come i fattori di risposta aumen-tino con l’aumentare del tempo di ritenzione croma-tografico del trigliceride, passando da POP a SOO. Questo aumento lineare è stato studiato e verificato da vari ricercatori ed è attribuito a dei fattori discrimi-natori della colonna cromatografica e in parte del si-stema di iniezione a carico dei trigliceridi a più elevato grado di insaturazione [14, 15].

Un secondo requisito del metodo di analisi era la separazione di alcune coppie critiche di trigliceridi e in particolare le coppie POS/POO e SOS/SOO. Per queste coppie era richiesta una separazione croma-tografica di almeno 1.0. I risultati ottenuti per i tre di-versi sistemi di iniezione sono riportati in Tabella II, dove si può notare che anche questo requisito è am-piamente soddisfatto.I risultati ottenuti in questa fase di comparazione delle diverse tecniche di iniezione hanno evidenziato come un’attenta ottimizzazione dei parametri strumentali ha portato ad avere delle prestazioni equivalenti per l’analisi del burro di cacao, indipendentemente dalla tecnica di iniezione impiegata.

3.2 ANALISI DEI CAMPIONI DI CIOCCOLATO FONDENTE DEL MERCATOAllo scopo di valutare il metodo di analisi per la ricer-ca dei grassi diversi dal burro di cacao nei cioccolati fondenti sono stati acquistati una serie di campioni appartenenti a note marche e a primi prezzi.Per ogni campione è stata eseguita l’estrazione della parte grassa applicando il metodo rapido di analisi e si è poi analizzata la frazione trigliceridica mediante gascromatografia con iniezione on-column e split.La lista dei campioni di cioccolato analizzati è ripor-tata in Tabella III, insieme al valore di sostanza grassa estratta con il metodo veloce in comparazione al va-lore riportato in etichetta.La sostanza grassa estratta con il metodo veloce dif-ferisce rispetto a quella dichiarata in etichetta di una quantità variabile dal 0.4% al 10%, sempre in positi-vo. La sostanza grassa ottenuta applicando il metodo di riferimento AOAC 963.15 fornisce dei risultati mi-gliori ed in particolare per il campione A 45.8% e per il campione B 39.0% in perfetto accordo con il valore dichiarato in etichetta. La sostanza grassa estratta dai vari campioni di cioc-colato è stata poi analizzata nelle condizioni riportate nella parte sperimentale del presente lavoro allo sco-po di identificare la presenza di grassi estranei al bur-

Tabella I - Fattori di risposta FID sperimentali dei trigliceridi per le varie tecniche di iniezione: valore medio di 6 determinazioni e deviazione standard relativa (in parentesi)

Tabella II - Risoluzione cromatografica per le coppie di trigliceridi per le varie tecniche di iniezione

Risoluzione cromatografica Coppia di trigliceridi On-column Split PTV

POS/POO 1.14 1.31 1.49 SOS/SOO 1.32 1.53 1.60

Tabella III - Lista dei campioni di cioccolato sottoposti ad analisi

Campioni % cacao minimo

dichiarato in etichetta Grassi dichiarato in etichetta (g/100 g)

Grassi determinati sperimentalmente (g/100 g) Altri ingredienti dichiarati

A 72 45.4 46.2 Aromi B 70 39.4 43.5 Lecitina di soia, aromi C 50 30.0 31.7 Lecitina, vaniglia, vanillina D 50 30 31.4 Lecitina di soia, aromi E 52 -- 33.1 Lecitina di soia F 50 31.8 32.5 Lecitine di semi di girasole, vaniglia G 60 36.6 39.0 Lecitina di soia, estratto di vaniglia H 74 41.0 43.0 Lecitine di semi di girasole, vaniglia I 71 42 43.1 Baccelli di vaniglia L 72 44.0 44.2 Lecitina di soia, estratto di vaniglia

M 53 32 34.5

Edulcorante (lattitolo), inulina, lecitina di girasole, estratto di

vaniglia, edulcoranti (aspartame, acesulfame K)

Tecnica di iniezione Trigliceride On-column Split PTV

POP 0,88 (0.5%) 0,89 (0.4%) 0,87 (0.4%) POS 0,98 (0.6%) 0,97 (0.1%) 0,96 (0.2%) POO 0,81 (1.5%) 1,04 (1.7%) 0,98 (2.8%) SOS 1,13 (1.0%) 1,10 (0.4%) 1,13 (0.4%) SOO 1,16 (2.7%) 1,17 (0.7%) 1,20 (2.9%)




POS/POO 1.14 1.31 1.49 SOS/SOO 1.32 1.53 1.60






M 53 32 34.5






22

ro di cacao. Tutti i campioni acquistati dichiaravano in etichetta l’uso esclusivo di burro di cacao come ingrediente.I campioni sono stati analizzati in duplicato e per ognuno si è verificato che la differenza tra le due re-pliche rientrasse nel limite di ripetibilità riportato nel metodo del IRMM, e più precisamente:% POP nell’intervallo tra 18.99 – 25.37, r = 0.514% POS nell’intervallo tra 43.76 – 47.73, r = 0.293% SOS nell’intervallo tra 30.87 – 33.80, r = 0.621dopo aver normalizzato i dati %POP+%POS+%SOS = 100%.

In Tabella IV sono riportati i dati delle analisi on-co-lumn dei campioni di cioccolato. Per ogni campione si riportano i valori di %POP, %POS, %SOS misurati e di %POP calcolata utilizzando le formula matema-tiche proposte dal metodo IRMM e la decisione se è burro di cacao genuino.In Tabella V sono riportati gli stessi parametri per le iniezioni split.Come si può notare dal confronto tra le due Tabelle, i due sistemi di iniezione forniscono risultati paragona-bili per tutti i campioni analizzati. Inoltre essi risultano essere quasi tutti composti da burro di cacao genu-




POS/POO 1.14 1.31 1.49 SOS/SOO 1.32 1.53 1.60






M 53 32 34.5





Tabella IV - Risultati dei campioni di cioccolato per le iniezioni on-column

Tabella V – Risultati dei campioni di cioccolato per le iniezioni split

Campioni %POP misurato %POS misurato %SOS misurato %POP calcolato Genuino

Prova A Prova B Prova A Prova B Prova A Prova B Prova A Prova B Prova A Prova B A 19,14 19,20 47,59 47,44 33,28 33,37 19,63 19,57 Si Si B 19,88 20,10 48,21 47,99 31,91 31,91 20,63 20,64 Si Si C 19,99 19,98 47,75 47,89 32,25 32,13 20,39 20,47 Si Si D 19,81 19,48 47,40 47,24 32,79 33,29 19,99 19,62 Si Si E 19,37 19,36 47,28 47,13 33,34 33,51 19,59 19,46 Si Si F 19,32 19,46 47,32 47,43 33,35 33,10 19,58 19,76 Si Si G 21,88 21,61 47,52 47,51 30,60 30,88 21,59 21,39 No No H 19,82 19,94 47,45 47,43 32,73 32,63 20,03 20,11 Si Si I 21,11 20,90 47,81 47,63 31,08 31,47 21,24 20,96 Si Si L 20,57 20,67 47,82 48,05 31,61 31,27 20,86 21,10 Si Si M 19,96 19,82 47,48 47,30 32,56 32,88 20,16 19,92 Si Si



Tabella IV - Risultati dei campioni di cioccolato per le iniezioni on-column

Tabella V – Risultati dei campioni di cioccolato per le iniezioni split






23

ino ad eccezione del campione G che è risultato es-sere addizionato. L’analisi di questo campione è stata ripetuta fornendo sempre lo stesso risultato.A questo punto abbiamo proceduto al calcolo della percentuale di CBE nel cioccolato G determinando la sostanza grassa secondo il metodo di riferimento AOAC 963.15 e applicando le formule matematiche sviluppate dai ricercatori del IRMM.Nel caso del cioccolato G si ha che la sostanza gras-sa estratta è stata del 34.0% e quindi la %CBE nel burro di cacao corrisponde al 1.78% che equivale al 0.61% nel cioccolato. Tale valore è inferiore al limite del 5% riportato dalla Direttiva 2000/36, ma non è dichiarato in etichetta come richiede la legislazione.Questo metodo permette di rilevare e quantificare la presenza di grassi estranei al burro di cacao, ma non fornisce indicazioni sulla tipologia di tali grassi. Altri metodi di analisi, basati sulla rilevazione dei prodotti di degradazione degli steroli forniscono risultati quali-tativi sulla presenza di CBE nel burro di cacao senza fornire una quantificazione e identificazione dei grassi vegetali eventualmente aggiunti.

4. CONCLUSIONI

La tecnica di iniezione on-column per l’analisi gascro-matografica dei trigliceridi del burro di cacao è quella maggiormente consigliata, ma non fornisce risultati statisticamente migliori rispetto agli altri due sistemi split e PTV per questa specifica matrice. Questi risul-tati sono validi a condizione di effettuare un’attenta ottimizzazione dei parametri gascromatografici per tutte e tre le tecniche di iniezione impiegate.Le analisi dei campioni di cioccolato fondente effet-tuate determinando la frazione trigliceridica allo sco-po di rilevare la presenza di grassi diversi dal burro di cacao hanno permesso di applicare il metodo di analisi proposto dai ricercatori di IRMM. Tutti i cioc-colati sono risultati contenere solo burro di cacao ad eccezione di un campione. Ulteriori approfondimenti sono però necessari al fine di evitare dei falsi positivi e l’analisi dei composti di degradazione degli steroli e dei terpeni potrebbe essere una valida aggiunta per confermare il risultato ottenuto.

Ringraziamenti

Si vuole ringraziare la Dani Instruments per aver mes-so a disposizione le loro conoscenze tecniche e aver fornito il materiale cromatografico, in particolare la colonna analitica, necessari allo svolgimento del pre-sente studio.

BIBLIOGRAFIA

[1] M. Lipp, C. Simoneau, F. Ulberth, E. Anklam, C. Crews, P. Brereton, W. De Greyt, W. Schwack, C. Wiedmaier, Composition of genuine cocoa butter and cocoa butter equivalents, J. Food

Composition and Analysis 14, 399-408 (2001).[2] M. Lipp, E. Anklam, Review of cocoa butter and

alternative fats for use in chocolate – Part A. Compositional data, Food Chemistry 62, 73-97 (1998).

[3] Direttiva 2000/36/CE del Parlamento Europeo e del Consiglio del 23 giugno 2000 relativa ai prodotti di cacao e di cioccolato destinati all’ali-mentazione umana, GU L 197 del 3.8.2000, pag. 19-25.

[4] Decreto Legislativo n. 178 del 12 giugno 2003, Attuazione della direttiva 2000/36/CE relativa ai prodotti di cacao e di cioccolato destinati all’ali-mentazione umana, GU n.165 del 18-7-2003.

[5] M. Lipp, E. Anklam, Review of cocoa butter and alternative fats for use in chocolate – Part B. Analytical approaches for identification and determination, Food Chemistry 62, 99-108 (1998).

[6] M. Jee, Oils and fats authentication, Blackwell publishing, CRC press (2002).

[7] C. Crews, R. Calvet-Sarrett, P. Brereton, The analysis of sterol degradation products to de-tect vegetable fats in chocolate, JAOCS 74, 1273-1280 (1997).

[8] C. Simoneau, P. Hannaert, E. Anklam, Detection and quantification of cocoa butter equivalents in chocolate model systems: analysis of trigliceride profiles by high resolution GC, Food Chemistry 65, 111-116 (1999).

[9] C. Simoneau, M. Lipps, F. Ulberth, E. Anklam, Quantification of cocoa butter equivalents in mixture with cocoa butter by chromatographic methods and multivariate data evaluation, Eur. Food Res. Technol. 211, 147-152 (2000).

[10] M. Buchgraber, F. Ulberth, E. Anklam, Cluster analysis for the systematic grouping of genu-ine cocoa butter and cocoa butter equivalent samples based on trigliceride patterns, J. Agric. Food Chem. 52, 3855-3860 (2004).

[11] F.B. Padley, R. E. Timms, the determination of cocoa butter equivalents in chocolate, J. Am. Oil Chem. Soc. 57, 286-293 (1980).

[12] C.C. Young, The interpretation of GLC triglic-eride data for the determination of cocoa but-ter equivalents in chocolate: a new approach, J. Am. Oil Chem. Soc. 61, 576-581 (1984).

[13] I. Bohacenko, Z. Kopicova, J. Pinkrova, Cho-colate authenticity control concerning complian-ce with the conditions for adding cocoa butter equivalents as laid down by Directive 2000/36/EC, Czech. J. Food Sci. 23, 27-35 (2005).

[14] E. Geeraert, P. Sandra, Capillary GC of triglyce-rides in fats and oils using a high temperature phenylmethylsilicone stationary phase. Part II. The analysis of chocolate fats, J. Am. Oil Chem. Soc. 64, 100-105 (1987).

[15] M. Buchgraber, F. Ulberth, E. Anklam, Interla-boratory evaluation of injection techniques for


24

trigliceride analysis of cocoa butter by capilla-ry gas chromatography, J. Chromatogr. 1036, 197-203 (2004).

[16] A.A. Carelli, A. Cert, Comparative study of the determination of triacylglycerol in vegetable oils using chromatographic techniques, J. Chroma-togr. 630, 213-222 (1993).

[17] T. Řezanka, P. Mareš, Determination of plant triaylglycerols using capillary gas chromatogra-phy, high-performance liquid chromatography and mass spectrometry, J. Chromatogr. A 542, 145-159 (1991).

[18] E. Geeraert, P. Sandra, D. De Schepper, On-column injection in the capillary gas chromato-graphic analysis of fats and oils, J. Chromatogr. 279, 287-295 (1983).

[19] K. Grob, Evaluation of injection techniques for triglycerides in capillary gas chromatography, J. Chromatogr. 178, 387-392 (1979).

[20] M. Buchgraber, E. Anklam, Validation of a me-thod for the detection of cocoa butter equiva-lents in cocoa butter and plain chocolate. Re-port on the validation study, Report EUR 20685 EN (2003).

[21] M. Buchgraber, E. Anklam, Validation of a me-

thod for the quantification of cocoa butter equi-valents in cocoa butter and plain chocolate, Re-port EUR 20777 EN (2003).

[22] ISO 23275-1:2006 - Animal and vegetable fats and oils. Cocoa butter equivalents in cocoa but-ter and plain chocolate - Part 1: Determination of the presence of cocoa butter equivalents

[23] ISO 23275-2:2006 - Animal and vegetable fats and oils. Cocoa butter equivalents in cocoa but-ter and plain chocolate - Part 2: Quantification of cocoa butter equivalents.

[24] AOCS Official Method Ce 11-05 - Determination of Cocoa Butter Equivalents in Cocoa Butter and Plain Chocolate.

[25] R. Koeber, M. Buchgraber, F. Ulberth, R. Barca-rolo, A. Bernreuther, H. Schimmel, E. Anklam, The certification of the content of five triglyce-rides in cocoa butter, Report EUR 20781 EN (2003).

[26] AOAC Official method 970.20 – Cacao products – Preparation of laboratori sample.

[27] AOAC Official method 963.15 – Fat in cacao products – Soxhlet extraction method.

Ricevuto e Accettato, 3 dicembre 2014


25

A.A. Giuliani A. Cichellia

L. Tonuccib

N. d’Alessandroc*

a Dipartimento DEC Università “G. d’Annunzio” di Chieti-Pescara, Pescara

b Dipartimento di Scienze Filosofiche, Pedagogiche ed

Economico-QuantitativeUniversità “G. d’Annunzio”

di Chieti-Pescara, Chieti

c Dipartimento di Ingegneria e Geologia Università “G. d’Annunzio” di Chieti-Pescara, Pescara

(*) CORRESPONDING AUTHORtelephone: 0039 0871 3555365

E-mail: [email protected]

Chlorophyll photosensitized oxidation of virgin olive oil: a comparison between

selected unsaturated model esters and real oil samples

The effects of chlorophyll on photooxidation of virgin olive oil was evaluated. A purified chlorophyll fraction from spinach leaves was used as a photosensitiser to oxidise tiglic acid, as a model unsaturated compound that is useful to tune reaction conditions. Oleic and linoleic acid methyl esters were used to test photosensitised oxygenation. Virgin olive oil samples with the added spinach chlorophyll were photooxidised and the formation of hydroperoxides was monitored by 13C NMR. The virgin olive oil showed a behaviour similar to oleic acid methyl esters, as the olive oil contains more than 75% oleic acid. Headspace gas chromatography-mass spectrometry analysis of the irradiated reaction mixtures showed several short-chain products for the linoleic acid methyl esters, while for the oleic acid methyl esters, octane was the only product that was detected in any quantity. The analysis of photooxidised virgin olive oil confirmed the presence of octane, which can therefore be proposed as a marker for an irradiation step of the oil.Keywords: olive oil, chlorophyll, photosensitised oxidation, singlet oxygen, hydroperoxide

Ossidazione di olio vergine d’oliva foto sensibilizzata da clorofilla: un confronto tra esteri insaturi modello e campioni reali di olioNel presente studio è stato valutato l’effetto della clorofilla sulla foto ossidazione dell’olio vergine d’oliva. Una frazione purificata di clorofilla, ottenuta da foglie di spinaci, è stata utilizzata come foto sensibilizzatore per ossidare l’acido tiglico, un composto insaturo modello, utile per mettere a punto le condizioni di reazione. Gli esteri metilici dell’acido oleico e dell’acido linoleico sono stati usati per testare l’ossigenazione foto sensibilizzata. Sono stati foto ossidati anche campioni di olio vergine d’oliva con aggiunta di clorofilla, ottenuta dagli spinaci, e la formazione degli idroperossidi è stata monitorata registrando gli spettri 13C NMR. L’olio vergine d’oliva, che contiene più del 75% di acido oleico, come aspettato, ha mostrato un comportamento simile a quello dell’estere metilico dell’acido oleico. Dall’analisi con spazio di testa gascromatografia-massa delle miscele di reazione irradiate si osservano diversi prodotti a corta catena derivanti dall’estere metilico dell’acido linoleico, mentre per l’estere metilico dell’acido oleico l’ottano è stato l’unico prodotto osservato. L’analisi dell’olio vergine d’oliva foto ossidato ha confermato la presenza di ottano che, dunque, può essere proposto come marker per uno step di irradiazione dell’olio.Parole chiave: olio d’oliva, clorofilla, ossidazione foto sensibilizzata, ossigeno singoletto, idroperossido


26

INTRODUCTION

Fresh, genuine and good quality virgin olive oil (VOO) contains a large number of minor components, like phenols, tocopherols, saturated and unsaturated long-chain hydrocarbons, short-chain compoun-ds (mainly alcohols, esters, hydrocarbons, carbonyl derivatives), sterols, carotenoids, chlorophylls, and many other compounds at very low levels. However, among these classes of compounds, only chlorophyl-ls and carotenoids interact with visible light, hence in-fluencing both the oil colour and its stability during light exposure [1, 2, 3]. Upon exposure of VOO to light, the relative stability of the chlorophylls and ca-rotenoids are extremely different, as the chlorophylls rapidly degrade into colourless products [4, 5], whi-le the carotenoids often remain the sole pigments in olive oils, therefore changing the colour from green to yellow [6]. The dominant mechanism of action of the carotenoids against photooxidation appears to be a quenching of excited sensitiser molecules, as well as of singlet oxygen (1O2) [7]. On the other hand, the chlorophylls are the sole photosensitisers in VOOs [8, 9, 10] and they can produce 1O2 via a type II process (Scheme 1) [11, 12, 13].The chlorophylls are low-polarity pigments that be-long to the chlorin family, and they have a complexed magnesium ion and a phytol chain (diterpene) linked to the periphery of a tetrapyrrolic macrocycle [14]. In olive oil the chlorophyll profile includes chlorophylls a and b, along with other demetallated and dephyty-lated derivatives, such as pheophytins and pheo-phorbides, respectively [15, 16, 17]. The content of pheophytin a and b is reported to increase during the extraction and storage of VOOs [18], such that pheo-phytin a is the main pigment, with levels ranging from 2.06 mg kg-1 to 37.06 mg kg-1, followed by pheo-phytin b (0.05-9.72 mg kg-1) and chlorophyll b (0.00-5.19 mg kg-1) [19]. The mechanism of 1O2 photogeneration can be sum-

marised as follows: the absorption of a quantum of light converts the chromophore to its excited singlet state (1Chl*) which can undergo intersystem crossing to produce the triplet state (3Chl*). The interaction between 3Chl* and molecular oxygen can occur via a type II process, to generate 1O2.1O2 is an electrophylic species that can readily react with unsaturated derivatives via cycloadditions (4+2 or 2+2) or ene reactions. Although 1O2 has often been evoked as responsible for fatty-acid deterioration [20], only recently its presence has been observed in actual samples of VOO and measured directly, using luminescence emission at 1268 nm [21].This photochemical reaction between unsaturated fatty acids and 1O2 leads to the formation of hydrope-roxides, which can easily decompose to give short-chain oxygenated and hydrocarbon species. There-fore chlorophyll-sensitised photooxidation of lipids results in rapid deterioration of olive oil; i.e., a change in colour and the development of undesirable odour and flavour constituents, thus decreasing its nutritio-nal and economic value [22].Olive oil triglycerides are formed by saturated (main-ly palmitic and stearic) and mono-unsaturated acids (mainly oleic acid; from 65% to 85%), while linoleic acid is the only polyunsaturated fatty acid present in reasonable amounts (around 10%). Despite the large number of studies showing olive oil thermo- oxidation [23] and the formation of the short-chain molecules that are responsible for the typical unpleasant aroma (of rancid oil) [24], there are only a few studies on the photo-oxidation of olive oil in the literature [25, 26].In the present study, we investigated chlorophyll as a 1O2 photosensitiser in ene reactions under irradiation by visible light. We first tested a model reaction that used chlorophyll-photogenerated 1O2 and tiglic acid methyl ester, a substrate that is often used as a probe for ene reactions [27]. After this preliminary investi-gation, we monitored the fate of the most important unsaturated fatty acids in VOO, namely oleic (55% to

Scheme 1 - Photosensitized generation of singlet oxygen (*: excited state)


27

85%) and linoleic acids (8% to 13%), as their methyl ester derivatives, in the presence of oxygen and chlo-rophyll under induced light and thermal stress. Finally, we carried out the same using VOO as the double-bond source. Our aim was to try to understand the effects of light and chlorophyll photosensitisation on the oxidative deterioration of VOO through the direct spectroscopic detection of hydroperoxide compoun-ds (using nuclear magnetic resonance [NMR]) and through the monitoring of volatiles.

EXPERIMENTAL

MATERIALSFreshly made VOO samples (oil-1, oil-2, oil-3 and oil-4) were obtained as a generous gift from a local oil mill (L’Olivicola Casolana, Casoli, Italy). Spinach (Spinacia oleracea, variety Inermis) was purchased from a local market. Tiglic acid, triolein and oleic and linoleic acid methyl esters were purchased from Sigma-Aldrich. Ti-glic acid methyl ester was prepared by treating a known aliquot of the free acid with etherate diazomethane. All other reagents were purchased from Aldrich.

INSTRUMENTSNMR spectra were recorded using a Bruker Avance 300 spectrometer (7.05 Tesla) equipped with a high-resolution multinuclear probe that operated in the range of 30 MHz to 300 MHz. The 1H and 13C spectra (1H NMR,13C NMR) were run without any treatment, in an NMR tube (5 mm); tetramethylsilane was used as a reference. Free induction decays were acquired at 22°C using the standard zg (zgdc for 13C NMR) pulse sequence (Bruker-made) with a spectral width of -0.5 ppm to 12.5 ppm (0-240 ppm for 13C NMR). A 90°C excitation pulse (7.6 µs for 1H, 5 µs for 13C NMR) and 1 s and 5 s relaxation delays (1H NMR,13C NMR, re-spectively) were used to collect 64 scans (12000 for 13C NMR).UV visible spectra were recorded using a Jenway 6505 UV/Vis system. Before the acquisition, the re-action mixtures were diluted 200-fold with 2,2,4-tri-methylpentane, directly into the 3 ml high-precision quartz cell (made of Suprasil® quartz; Hellman), and the spectra were acquired over the wavelength range of 400 nm to 750 nm.The gas chromatography (GC) apparatus (model 6890; Hewlett Packard) was equipped with a split-splitless injector, a free induction decay detector, and a poly(alkylene glycol) capillary column (SPB® PUFA, 30 m, i.d. 0.32 mm, f.t. 0.20 µm). The acquisition pa-rameters for fatty-acid methyl ester detection were: constant injector pressure for all analyses, 35 kPa; injector temperature, 250°C; detector temperature, 280°C; initial temperature, 120°C (1 min), then 8°C/min up to 160°C (kept for 15 min), and 20°C min-1 up to 250°C (kept for 4.5 min), for a total acquisition time of 30 min. In split mode (split ratio, 1:10), 1 µl of the organic ether solution was injected.

For the GC-mass spectroscopy (GC-MS) a gas-chromatograph (Thermo Scientific Focus series) was coupled to an ISQ mass-selective detector operating in electron-impact mode at 70 eV. The GC-MS was equipped with a split-splitless injection system and a TR-5 MS (cross-linked, 5% phenyl methyl siloxa-ne) capillary column (30 m length, 0.25 mm diameter, 0.25 µm film thickness) (Thermo Scientific Inc.; Walt-ham, USA), with helium as carrier gas. The acquisition parameters for the detection of the volatile derivati-ves were: injections made in split mode (ratio, 1:10); constant injector pressure for all analyses, 20 kPa; injector temperature, 250°C; source temperature, 250°C; transfer line temperature, 250°C; mass ran-ge, 40-300 u; initial temperature, 60°C (1.5 min), then 15°C min-1 up to 80°C (kept for 0 min), and 30°C min-1 up to 250°C (kept for 2.5 min), for a total acquisition time of 11 min. The headspace fraction (300 µl) was thermostated at 70°C for 5 min and injected.A photochemical multirays apparatus (Helios Ital-quartz) was used for the photochemical reactions, which contained ten UV lamps of 15 W power each that emitted light at around 450 nm to 500 nm. Tran-sparent vials (10 mL) were used for the irradiation of the solutions. To better preserve the neo-formed hydroperoxides, the experiments were carried out at temperatures <20°C, using a thermostated water bath inside the photochemical multirays apparatus.

CHLOROPHYLL ISOLATIONFresh, washed and drained spinach leaves (fresh weight, 250 g) were pounded in a mortar with 1 l cold acetone (-20°C) and 150 mg Na2HPO4 H2O, and then filtered through two sheets of filter paper (Whatman 41) into a Buchner funnel under reduced pressure. The filtrate was treated with 175 ml dioxane and 150 ml water, which was added drop-wise under magnetic stirring, until a green precipitate was for-med. This mixture was placed in a freezer (-20°C) for 12 h, to allow sedimentation of the adduct containing chlorophyll. The top clear yellow solution was care-fully decanted, and the lower, dark-green, suspen-sion containing the solid chlorophyll-dioxane-hydrate complex was collected by filtration through filter paper (Whatman 42). This was then dissolved with acetone until the filter was colourless. The crude chlorophyll extract was completely dried by evaporation under reduced pressure at <30°C. The resulting pigments were dissolved in a small amount of hexane/diethyl ether (70:30, v/v) and loaded onto a silica gel column (silica gel 60; 230-400 mesh). The carotenoids were eluted first with hexane/diethyl ether (70:30, v/v), and then the polarity of the eluent was increased to elute pheophytin a, chlorophylls a and b, and xanthophylls. The purity of each fraction was analysed using thin layer chromatography, 1H NMR and 13C NMR. The chromatographic fraction that contained mainly ch-lorophyll a was dissolved in CH2Cl2 to obtain a 3000 mg l-1 solution ready for use in the experiments.


28

Sample preparation for photo and thermal oxidationSamples of tiglic acid methyl ester (0.50 g), oleic acid methyl ester (OAME) (0.50 g), linoleic acid methyl ester (LAME) and VOO (0.50 g) were put into 10-ml glass vials, to which chlorophyll a (3000 mg l-1) was ad-ded. The solvent was removed under a nitrogen gas flow, and the sample vials were sealed (air-tight) with rubber caps. Photosensitised oxidation experiments were carried out at 35°C using the above-mentioned photochemical multi-ray apparatus. A VOO sample (0.50 g) without added chlorophyll was used as the control and was irradiated under the same condi-tions. For thermal oxidation experiments, VOO sam-ples with and without added chlorophyll were placed in an oven at 100°C.

Hydroperoxide analysisThe tiglic acid, OAME, LAME and VOO hydroperoxi-des were directly detected by 1H NMR and 13C NMR spectroscopy after addition of 600 µl CDCl3 to the reaction mixture.

Volatile compounds detectionPhoto-induced and thermo-induced volatile com-pounds were identified by static headspace sampling as the extraction technique, coupled to GC-MS. For the photooxidation, static headspace sampling was carried out after heating the closed vial of the pho-tooxidised mixture in a water bath at 70°C for 5 min. Volatiles from photosensitised oxidation of VOO wi-thout or with added chlorophyll a were analysed at 1, 2, 4, 6, 12 and 22 h, with the thermo-induced volatiles analysed at 30, 60, 90, 120,150, 180, 240, 270, 360 and 390 min. The data are expressed as relative signal intensities obtained by the total ion chromatogram.

Spectrophotometric quantitation of total chlorophyll contentThe chlorophyll content was performed by vis spec-trophotometry at 630, 670 and 710 nm following the methodology reported in literature [28].

Statistical analysisAll experiments were performed in triplicate using fre-shly prepared samples and were reported as calcu-lated means (chromatographic areas) and standard deviations.


The chlorophyll-photosensitised ene reaction of tiglic acid methyl ester was carried out in a homogeneous phase upon irradiation with visible light of a CH2Cl2 solution of the α, β-unsaturated ester (10 mM) in the presence of spinach chlorophyll (3000 mg l-1); the hy-droperoxide and the corresponding alcohol derivative were monitored by 1H NMR. The mass balance (con-version of reactants vs. yield of products) was strictly respected, and of the two potential isomers, only one was detected (3-hydroperoxide), in good agreement with Stensaas and coworkers [29], for the analogous reaction performed in deuterated methanol or ben-zene solutions, who reported a maximum yield of only 3% for the 2-hydroperoxide derivative (Scheme 2). The reaction was conducted until the green colour of the chlorophyll solutions became light brown (20 h; Fig. 1), with maximum conversion of the starting unsaturated substrate of 8%. To better understand the nature of the green pigments contained in our spinach extracts, we recorded the 1H NMR spec-tra: by comparing the relative areas of the aldehydic (around 10.8-10.9 ppm) and the methinic (α, β and δ, between 8.5 and 9.8 ppm) signals of the raw chlo-rophyll extract it was possible to confirm that chloro-phyll a and pheophytin a were the dominant species, in agreement with data published previously [30].The photooxidation of OAME and LAME was car-ried out under analogous experimental conditions as above (visible light and chlorophyll extract). The course of the reaction was again monitored by 1H NMR, even if in this case 13C NMR was more conclu-sive. 1O2 oxidation of oleic acid is known to produce only two hydroperoxide isomers, while the same reaction with linoleic acid leads to the formation of four isomers [31]. Here, for OAME and LAME, the 13C NMR of the reaction mixtures showed signals of 9-[OOH]-OAME and 10-[OOH]-OAME (Fig. 2) and 9-, 10-, 12- and 13-[OOH]-LAME (Fig. 3), respectively. The hydroperoxide isomer distribution of OAME was 47% (9-[OOH]-OAME) vs 53% (10-[OOH]-OAME), while for LAME, the non-conjugated 10-[OOH]-LAME and 12-[OOH]-LAME appeared to be slightly less intense that the two conjugated ones, namely 9-[OOH]-LAME and 13-[OOH]-LAME (for both, the data was obtained as means of signal intensities

3Chl

1Chl

0Chl Ground state

1O2

3O2

Inter System Crossing

Fluorescence

Phosphorescence

Photosensitizer Oxygen

Energy

Scheme 1 - Photosensitized generation of singlet oxygen.

CH3

O

H3C

H

H3C-O

CDCl3

CH2

O

H3C-O

H3CHOO

H

CH3

OOH

O

H3C-O

H

H2C

major minor

vis lightChl

1O2

+

Scheme 2 - Photosensitised oxidation of tiglic acid methyl ester by use of chlorophyll.


29

from five different 13C NMR analyses). This finding is in very good agreement with data reported previously [32] for OAME and for LAME [33] who used HPLC to define ratios of 48.9:51.1 for 9-[OOH]-OAME and 10-[OOH]-OAME, and of 32:17:17:34 for 9-,10-,12- and13-[OOH]-LAME, respectively.Finally, we irradiated a genuine sample of VOO (oil-1) in the absence and presence of the chlorophyll extract, and followed this again by 13C NMR for the

formation of hydroperoxides. Considering the fatty-acid composition of the oil-1 sample, which showed a content of 75% oleic acid (Tab. I), we expected a hydroperoxide distribution comparable to that seen for the OAME samples. Indeed, the signals of the vinyl carbon atom of the 9-oleil hydroperoxides and 10-oleil hydroperoxides appeared as the dominant signals after 22 h irradiation in both supplemented and genuine (Fig. 4) oil-1 samples. Although the chemical

Figure 1 - UV Vis spectra of spinach chlorophyll extract solutions (3000 mg kg-1 in CH2Cl2) after 0,1, 2, 3, 6 and 22 hours irradiation.

0

0,1

0,2

0,3

0,4

0,5

0,6

0,7

0,8

400 450 500 550 600 650 700 750

Abso

rban

ce

Wavelength (nm)

0 to 22 hours of irradiation time

Figure 2 - 13C NMR spectrum of spinach chlorophyll-supplemented OAME sample after 22 hours of irradiation time.


30

shifts of the hydroperoxide unsaturated carbons of OAME and VOO were not perfectly superimposable, we clearly noted that on irradiating a chlorophyll-sup-plemented pure triolein sample, the triglycerides were slightly shifted compared to the analogous signals of OAME (Fig. 5). To confirm the results obtained upon hard supplementation with chlorophyll extract we carried out further photooxidation experiments using a set of three real samples of VOOs (oil-2, oil-3 and oil-4) which were selected according to the chloro-phyll amounts ranging from 14.3 (oil-4) to 38.5 (oil-2) mg kg-1 of pheophytin a /oil (Tab. II). As expected, the 13C NMR analysis showed that the relative intensity of

the signals of the vinyl carbon atom of the 9-oleil hy-droperoxides and 10-oleil hydroperoxides depended on the chlorophyll amounts (Fig. 4).We also investigated the generation of volatile com-pounds upon irradiation of solutions containing the two concentrated unsaturated methyl esters and oil-1, each one respectively in the absence and pres-ence of the chlorophyll extract. These analyses were performed by GC-MS, by sampling the headspace of the reaction mixture at the end of the photoreac-tions; i.e., after the disappearance of the green colour from the solutions (which, as noted above, became light brown), and after leaving the reaction mixture at 70°C for 5 min, to both facilitate the degradation of the hydroperoxides and to avoid the formation of other oxidation products that were not derived from the photooxidation pathway.OAME irradiated without chlorophyll did not show any volatiles, while the chlorophyll-supplemented sample showed a very simple GC profile (only octane was detected in any appreciable amounts; Fig. 6). Other aldehydes were detected, like heptanal, octanal, non-anal and decanal, but only in very tiny amounts (Tab. III). Octane was undoubtedly the most abundant de-rivative, which continuously accumulated during the photosensitised oxidation (at 20°C, for up to 22 h ir-radiation), and it was derived from the breakdown of 10-[OOH]-OAME; it was also reported to be the domi-nant product when oleic acid was irradiated in the presence of the methylene blue photosensitiser [34].To better understand the trend of the photooxidation,

Figure 3 - 13C NMR spectrum of spinach chlorophyll-supplemented LAME sample after 22 hours of irradiation time.

Table I - Fatty acid composition of oil-1 sample (from Leccino cultivar)

Fatty acid % Tetradecanoic C14:0 0.15 Hexadecanoic C16:0 9.36 9-Hexadecenoic C16:1 0.64 Eptadecanoic C17:0 0.14 10-Eptadecenoic C17:1 0.2 Octadecanoic C18:0 1.82 9-Octadecenoic C18:1 75.08 11-Octadecenoic C18:1 1.95 9,12-Octadecadienoic C18:2 9.13 Eicosanoic C20:0 0.42 6,9,12-Octadecenoic C18:3 0.67 9-Eicosenoic C20:0 0.26 Docosanoic C22:0 0.11 Tetracosanoic C24:0 0.07


31

we compared the photochemical experiments with the thermal experiments conducted at high tempera-ture (100°C) in the dark and in the presence of chlo-rophyll (Tab. IV). A completely different distribution of volatiles was observed. Here, there were consider-able amounts of saturated and unsaturated carbonyl derivatives; namely, heptanal, octanal, nonanal, de-canal and trans-2-undecenal. These results obtained with chlorophyll-supplemented OAME samples are not completely unexpected, as its photooxidation leads to only two hydroperoxides (9-[OOH] and 10-[OOH]), thus strongly reduce the number of potential volatile derivatives. On the other hand, the thermal oxidation led to four hydroperoxides, thus increasing the number of volatile derivatives from their degra-dation. As only octane accumulates in the reaction mixture, while the expected degradation products of 9-[OOH]-OAME (mainly, e.g., for 1-nonene and t-2-decenal) were not detected at all, this cannot be explained only on the basis that 10-[OOH]-OAME is present in slightly larger amounts (53%; see previous data by NMR). Probably the 10-isomer gives rise to octane via a kinetically favoured pathway, while the

9-isomer degrades indistinctly to many compounds (i.e., through polymerisation pathways), without the formation of any dominant volatile reaction product. A similar finding was also noted in a previous study [35], which indicated octane as the dominant marker of an olive oil sample stored for a long time under exposure to visible light.When the LAME samples were irradiated in the pres-ence of chlorophyll, almost all of the expected satu-rated and unsaturated alcohols and aldehydes were found, from C-5 to C-10 (Fig. 7). However, together with a large pool of short-chain products, all of these were in comparable amounts, and hexanal was by far

Figure 4 - 13C NMR spectra of VOOs and spinach chlorophyll-supplemented oil-1 after 22 hours of irradiation time.

Chlorophyll-supplemented oil-1

oil-2

oil-1

oil-3

oil-4

Table II - Total chlorophyll content of the set of genuine VOO samples used for the photooxidation experiments

VOO samples Total chlorophyll content (mg/kg pheophytin a)

oil-1 18.8 oil-2 38.5 oil-3 15.5 oil-4 14.3


32

Figure 5 - 13C NMR spectrum of a triolein sample with 3000 mg kg-1 of chlorophyll from spinach, after 22 hours irradiation.

140 120

Octa

ne

D

ioxan

e (fro

m Ch

l spin

ach e

xtrac

t)

Figure 6 - Headspace GC-MS of an OAME sample with 3000 mg kg-1 of chlorophyll from spinach, after 22 hours irradiation.


33

the most abundant. If we consider the scheme of the hydroperoxide formation (see Scheme 3), it can be seen that four hydroperoxides can be formed by the photochemical pathway (9- 10-, 12-, 13-[OOH]), and this explains the large number of degradation prod-ucts found. For comparison purposes, we also used LAME to perform an experiment in the dark at 100°C

and in the presence of chlorophyll. Unfortunately, in this case, there were no meaningful differences, as we also observed a large number of volatile compounds in the thermal experiment, in spite of only two poten-tial hydroperoxides available for the oxidation path-way in the dark, as reported recently in another study [36]. We tried to record the 13C NMR of the thermal

Table III - Irradiated oil-1 (supplemented and not supplemented), OAME and LAME samples; data are expressed as relative total ion chromatogram signal intensities

tr: detected only by the extraction technique.

oil-1 oil-1 OAME LAME Chl Chl Chl Pentanal 4.38±0.12 18.82±0.14 -- 9.68±0.31 1-Pentanol -- -- -- 3.63±0.16 Octane 2.63±0.13 7.32±0.10 7.57±0.09 -- Hexanal 10.95±0.21 13.24±0.08 -- 96.20±0.20 t-2-Hexenal 4.38±0.18 4.53±0.16 -- 2.78±0.09 2-Heptanone -- -- -- 2.78±0.21 Heptanal 3.28±0.17 4.25±0.19 0.35±0.11 3.63±0.22 1-Heptanol 3.28±0.14 4.18±0.17 tr 2.66±26 2-Heptenal 5.47±0.24 10.80±0.20 -- 21.78±0.26 1-Octen-3-ol 4.38±0.28 6.62±0.10 -- 8.47±0.10 2-Pentylfurane 2.63±0.20 4.18±0.09 -- 6.29±0.15 Octanal 5.47±0.21 8.36±0.27 tr 3.63±0.13 t-2-Octenal 3.94±0.16 4.88±0.09 -- 15.73±0.12 Nonanal 5.48±0.14 12.20±0.10 0.22±0.10 -- Methyl octanoate -- -- -- 10.90±0.10 Decanal 5.26±0.20 6.27±0.27 tr 3.63±0.19 2,4-Decadienal -- -- -- tr 8-Oxo-methyl-octanoate -- -- -- 10.09±0.15 t-2-Undecenal 5.91±0.15 10.80±0.27 -- 24.20±0.07 8-Hydroxy-methyl-octanoate -- -- -- 6.05±0.20 9-Oxo-methyl-nonanoate -- -- -- 53.84±0.16

Table IV - Thermo experiments on oil-1 (supplemented and not supplemented), OAME and LAME samples; data are expressed as relative total ion chromatogram signal intensities

oil-1 oil-1 OAME LAME Chl Chl Chl Pentanal 5.32±0.25 66.10±0.12 n.d.§ 32.72±0.09 1-Pentanol -- 7.27±0.13 -- 16.36±0.05 Octane -- -- 3.75±0.13 -- Hexanal 11.86±0.19 11.90±0.35 4.63±0.09 163.60±0.20 t-2-Hexenal 10.03±0.15 5.29±0.16 -- 4.09±0.11 2-Heptanone 2.74±0.07 15.20±0.15 -- 8.18±0.17 Heptanal 3.04±0.09 2.31±0.13 3.62±0.32 4.09±0.26 1-Heptanol 2.43±0.15 1.98±0.12 7.12±0.12 4.09±0.13 2-Heptenal 2.74±0.29 1.65±0.19 -- 20.45±0.08 1-Octen-3-ol 2.13±0.21 -- -- 8.18±0.09 2-Pentylfurane 2.74±0.15 2.51±0.18 -- 16.36±0.24 Octanal 2.58±0.04 1.98±0.18 4.07±0.13 -- t-2-Octenal 1.82±0.26 1.98±0.10 2.71±0.19 8.18±0.15 Nonanal 4.26±0.09 2.64±0.12 5.42±0.17 -- Methyl octanoate -- -- 3.62±0.10 12.27±0.07 Decanal 3.95±0.21 1.98±0.10 4.52±0.17 -- 2,4-Decadienal -- -- -- -- 8-Oxo-methyl-octanoate -- -- 4.07±0.24 8.18±0.13 t-2-Undecenal 6.69±0.11 3.31±0.18 4.07±0.31 4.09±0.20 8-Hydroxy-methyl-octanoate -- -- -- 8.18±0.33 9-Oxo-methyl-nonanoate -- -- 4.52±0.17 20.45±0.12

§ it appears partially superimposed to the final part of the solvent peak; this makes extremely difficult an accurate detection, at least for lower concentrations.


34

oxidised LAME samples, but the data were not easy to explain as at 100°C the hydroperoxides rapidly re-arrange and decompose. Indeed, it is important to recall that the bis-allyl radical, which is formed in the dark with LAME, is definitely lower in energy than the allyl radical with OAME [37]. Our opinion is that in the case of LAME, the radical thermal reaction cannot be completely avoided, even if the irradiation is carried out at low temperatures.The photosensitised oxidation of oil-1 led to a series of volatile derivatives, as reported in Table III. It is not easy to interpret the two different irradiation experi-ments, as qualitatively the product distributions are not particularly different. However, following careful analysis of the values obtained from the two photo-oxidation experiments using VOO without and with spinach chlorophyll extract at several irradiation times, we noted that octane appears more abundant in the chlorophyll-supplemented oil-1. This finding is worth noting, as with VOO thermal oxidation, octane has never been detected. In addition to octane, with the chlorophyll-supplemented photooxidation we observed increased 2-heptenal, nonanal and trans-2-undecenal, with almost the same increased per-centages. These results are in agreement with the concept that the formation of the OAME and LAME hydroperoxides occurs at comparable rates during the photo-oxidation, with the course of the reaction

little was influenced by the number of double bonds or the type of allylic sites. In fact the decomposi-tion of hydroperoxides of LAME is easier than that of hydroperoxides of OAME, but the photosensitized oxidation of both has a comparable rate, because of the very high reactivity of the singlet oxygen. As con-firmation, we observed that 2-heptenal from the 12-OOH of linoleic acid had noticeably increased, while hexanal increased by only 20%, compared with the non-supplemented irradiated oil-1 sample.Comparing the thermo-oxidised oil-1 experiments in the case of the chlorophyll-supplemented samples, there were decreasing amounts of all of the other vol-atiles, apart from pentanal and 2-heptanone. These data indicate that chlorophyll limited the formation of hydroperoxides, thus indirectly contributing to the re-duction in the total amounts of volatile compounds. On the other hand, the increases of pentanal and 2-heptanone might be attributed to a thermo-deg-radation step on the impurities present in the whole chlorophyll spinach extract.The GC-MS analyses of the set of four genuine sam-ples of irradiated VOOs, strongly evidenced the pres-ence of octane which is always found in appreciable amounts. Although this qualitative response is un-doubtable, we could not observe a linearity between the increase of chlorophyll content with the octane amount. However, plotting the 13C signal intensities

Figure 7 - Headspace GC-MS of an LAME sample with 3000 mg kg-1 of chlorophyll from spinach, after 22 hours irradiation.


35

vs the total chlorophyll amounts, it appears clearly the existing correlation between the photogenerated hy-droperoxides and the chlorophyll content (Fig. 8).

CONCLUSION

In the present study, we investigated the effects of visible light and chlorophyll photosensitisation on the oxidation of VOO, not only by monitoring the volati-les distribution, but also through direct spectroscopic detection (NMR) of hydroperoxide compounds.Based on the 13C NMR data, we can state that, du-ring irradiation at low temperature, the presence of chlorophyll in VOO samples enhances the formation of hydroperoxides, which accumulate with time. The 13C NMR analyses of irradiated samples allowed the identification of the specific hydroperoxide isomers from the 1O2 oxidation of the most abundant fatty acid present, namely oleic acid. The same analyses on thermo-oxidised VOO samples conducted in the presence of chlorophyll did not show the expected hydroperoxide isomer distribution due essentially to the occurrence of a radical pathway, which confirms the high decomposition rate of hydroperoxides.By comparing the volatiles detected in the photooxi-dised and thermo-oxidised VOO samples, we noted

Figure 8 - Correlation of 13C signal intensities of photogenerated hydroperoxides (9- and 10-) vs the total chlorophyll amounts of analysed VOOs.

0,000

0,005

0,010

0,015

0,020

0,025

0,030

0,035

0,040

0,045

0 10 20 30 40 50

relat

ive in

tensit

y of 13

C sig

nal

Total chlorophyll content (mg/kg)

CO

O CH3

CO

O CH3

OOHC

O

O CH3

OOH

CO

O CH3

CO

O CH3

OOH

CO

O CH3

OOHC

O

O CH3

OOH

CO

O CH3

OOH

CO

O CH3

OOH

CO

O CH3

OOH

3 3

3 3 3 3

3

3

3 3

3

3 3 3 3

OAME

10-HP-OAME

11-HP-OAME

9-HP-OAME

8-HP-OAME

13-HP-LAME

12-HP-LAME

10-HP-LAME

9-HP-LAME

LAME

Scheme 3 - Hypothesized hydroperoxides coming from the oxidation (thermo- and photo-) of OAME and LAME.


36

similar product distributions, apart from octane, which was detected only with the photooxidation. Thus, the presence of octane strongly suggests a participation of 1O2 in the oxidative pathway of the VOO sample.Another useful marker for the involvement of 1O2 is trans-2-heptenal, which comes from the 12-OOH of linoleic acid. Indeed, there was a noticeably increa-se in trans-2-heptenal in the VOO sample irradiated in the presence of chlorophyll, and these data were confirmed by the significant values observed with the irradiation of LAME in the presence of chlorophyll.For the thermo-oxidation of VOO, there were much lower amounts of total volatiles in the chlorophyll-supplemented samples, thus suggesting that large amounts of chlorophyll can scavenge 3O2, thus decre-asing the hydroperoxide formation rate, and hence the total volatile evolution at high temperature in the dark.

Acknowledgments

The authors are grateful to the “Consorzio di Ricerca per l’Innovazione Tecnologica, la Qualità e la Sicu-rezza degli Alimenti (ITQSA) S.C.R.L.” (CIPE funding 20.12.04; DM 28497) for financial support.

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Received November 15, 2013Accepted February 4, 2014

Biblioteca


39

B. Maronguia

M.M. Özcanb*

A. Rosac

M. Assunta Dessic

A. Pirasa

F. Al Juhaimid

aDipertimento di Scienze ChimicheUniversita degli Studi di Cagliari

Italy

bDepartment of Food EngineeringFaculty of Agriculture

Konya, Turkey

cDipartimento di Biologia SperimentaleSezione di Patologia Sperimentale

Universita delgi Studi di CagliariCagliari, Italy

dDepartment of Food Science and Nutrition,

College of Food and Agricultural Sciences,

King Saud University,Riyadh-Saudi, Arabia

(*) CORRESPONDING ADDRESS:Dr. M.M. Özcan

Department of Food EngineeringFaculty of Agriculture

Selcuk University42031 Konya, TurkeyTel:+90.332.2232933

Fax:+90.332.2410108Email: [email protected]

short noteMonitoring of the fatty acid

compositions of some olive oils

The major fatty acids of olive oils were oleic, palmitic and linoleic acids. Oleic acid is the main monounsaturated fatty acid (from 60.81%; Ayvalık cv to 68.98%; Sarıulak cv). Linolenic acid (from 0.70%; Gemlik cv to 0.90%; Uslu cv) was found to be the lowest in all the variety of oils; while-palmitoleic acid and oleic acid contents in the oil samples changed between 0.93% to 1.05% and between 60.81% to 68.98% respectively. Omega-6 contents of olive oils were found ranging 94.42 µg/mg to 161.63 µg/mg. Omega-3 contents were found at low levels (8.05 µg/mg to 9.60 µg/mg). According to statistical analysis, fatty acid compositional differences among the oils studied were significant, indicating a varietal effect on olive oil quality.Key words: olive oil content, GC, fatty acid composition

Monitoraggio delle composizioni di acidi grassi di alcuni oli d’olivaI principali acidi grassi degli oli di oliva sono gli acidi oleico, palmitico e linoleico. L’acido oleico è il principale acido grasso monoinsaturo (da 60.81% Ayvalık cv a 68.98% Sarıulak cv). L’acido linolenico è stato trovato basso in tutte le varietà di oli (da 0.70% Gemlik cv a 0.90% Uslu cv); mentre i contenuti di acido palmitoleico e di acido oleico nei campioni di olio variavano rispettivamente tra lo 0.93% e l’1.05% e tra il 60.81% e il 68.98%. I contenuti di omega-6 degli oli di oliva sono stati rilevati tra 94.42 µg/mg e 161.63 µg/mg.I contenuti di omega-3 sono stati trovati a livelli bassi (8.05 µg/mg - 9.60 µg/mg). Secondo l’analisi statistica le differenze di composizione degli acidi grassi tra gli oli studiati sono risultate significative, indicando un effetto varietale sulla qualità dell’olio di oliva.Parole chiave: contenuto di olio d’oliva, GC, composizione in acidi grassi


40

1. INTRODUCTION

Oils are extracted either by crushing or by solvent ex-traction using a suitable solvent from seed or fruits. The olive tree (Olea europaea L.) is one of the most important crops in the Mediterranean countries, espe-cially Spain, Italy and Greece. Virgin olive oil, due to its use without refining, shows very interesting nutritional and sensorial properties, being one of the pillars of the so called Mediterranean diet. Its fatty acid com-position, monounsaturated fatty acids, and its natu-ral antioxidants provide advantages for health [1-4]. Olive trees have been grown along the Aegean and Mediterranean coasts of Turkey for over 8.000 years. This has had an important effect on a wide range of olive growing regions and olive fruit cultivars in Turkey today [5, 6]. The nutrient characteristics of olive oil are well known due to the high energetic value and high content of monounsaturated fatty acids. A nutritionally favourable saturated/unsaturated fatty acids ratio and the presence of minor constituents with antioxidant properties are very important for he-alth nutrition [7]. The aim of this study was to monitor the amount of the fatty acid composition of Turkish olive oils obtained from several provinces.

2. MATERIAL AND METHOD

2.1. MATERIALTurkish olive varieties (Domat, Gemlik, Uslu, Ayvalık and Sarıulak) used in this research were collected manually from the olive trees growing in Mersin (Sil-ifke), Balıkesir (Edremit) and Manisa provinces. The Herbarium number of specimens were OO4, OO2, OO7, OO5 and OO9 for Domat, Gemlik, Uslu, Ayvalık and Sarıulak, respectively. Olive fruits were transfer-red by using cool bags, and kept frozen (-18°C) until the analyses. Triolein, trilinolein, fatty acids, fatty acid methyl esters, and desferal (deferoxamine mesylate salt) were purchased from Sigma-Aldrich (Milan, Italy). All solvents used were also purchased from Sigma-Aldrich. Methanolic HCl (3N) was purchased from Su-pelco (Bellefonte, PA).

2.2 METHOD

2.2.1 Oil ExtractionThe laboratory mill was used to prepare the olive oil samples in the Department of Food Engineering La-boratory, Faculty of Agriculture. About 1-2 kg of olives were crushed with a hammer mill and slowly mixed for 35 min. The paste was centrifuged in thin layers for oil extraction. This oil was filtered, and transferred into dark bottles, and added into nitrogen. Oil sam-ples were kept at -18°C during experiment period.

2.2.2 Determination of fatty acid Separation of methyl esters was obtained by mild sa-ponification [8] as follows: 3 mg of oils were dissolved

in 5 ml of EtOH and 100 µL of desferal solution (25 mg/ml of H2O), 1 ml of a water solution of ascorbic acid (25% w/v), and 0.5 ml of 10 N KOH were added. The mixtures were left in the dark at room tempera-ture for 14 h. After an addition of 10 ml of n-hexane and 7 ml of H2O, samples were centrifuged for 1 h at 900 rpm. The hexane phase was collected and, after a further addition of n-hexane to the mixtures, sam-ples were acidified with 37% HCl to pH 3-4 and then centrifuged for 1 h at 900 rpm. The hexane phase (saponifiable fraction) with free fatty acids was collec-ted and the solvent was evaporated. A portion of the dried residue was dissolved in CH3CN with 0.14% CH3COOH (v/v) and aliquots of the samples were in-jected into the HPLC system. An aliquot of dried fatty acids was methylated with 1 mL of methanolic HCl (3 N) [9] for 30 min at room temperature. After an ad-dition of 4 ml of n-hexane and 2 ml of H2O, samples were centrifuged for 20 min at 900 rpm. The hexane phase with fatty acid methyl esters was collected, the solvent was evaporated, the residue was dissolved in n-hexane and aliquots of the samples were injected into the GC system. The recovery of fatty acids during saponification was calculated by using an external standard mixture prepared dissolving 1 mg of triolein and trilinolein in EtOH and processed as samples. All solvents evaporation was performed under vacuum.

2.2.3. GC AnalysesFatty acid methyl esters were measured on a gas chromatograph Hewlett-Packard HP-6890 (Hew-lett-Packard, Palo Alto, USA) with a flame ionisation detector and equipped with a cyanopropyl methyl-polysiloxane HP-23 FAME column (30 m × 0.32 mm × 0.25 µm) (Hewlett-Packard). Nitrogen was used as the carrier gas at a flow rate of 2 ml/min. The oven temperature was set at 175°C; the injector tempera-ture was set at 250°C; and the detector temperature was set at 300°C. The fatty acid methyl esters were identified by comparing the retention times to those of standard compounds. The composition of individual fatty acid was calculated as a percentage of the total amount of fatty acids (%), using the Hewlett-Packard A.05.02 software.

2.2.4 Statistical analyses Results of the research were analysed for statistical significance by analysis of variance [10].

3. RESULTS AND DISCUSSION

Fatty acid compositions of several olive oils are pre-sented in Table I. It has been seen that the differen-ce of olive cultivars are highly effective on the oleic, linoleic and palmitic acids. The results show that all studied olive oils are rich with unsaturated fatty acids (more than 80%). The major fatty acid of oils was oleic acid with more than 60.0% followed by linoleic acid (9.87% to 18.21%) and palmitic acid (13.09%


41

to 13.31%). Moreover, palmitoleic and linolenic acids are between 0.76% to 1.05% and 0.7% to 0.90%, respectively. These fatty acids give a nutritional and medicinal importance for these olive oils. Indeed, li-noleic and linolenic acids have a beneficial role for the human body [11]. These results show that some fatty acids of olive oils showed similarities with soy-bean, corn, coconut, peanut, sunflower and rapese-ed oils [12].Unsaturated fatty acids composition (µg/mg oil) by GC of olive oils are given in Table II. While palmito-leic acid contents of olive samples changed between 5.09 µg/mg to 7.88 µg/mg oil, oleic acid contents ranged between 533.58 µg/mg to 626.63 µg/mg oil. Omega-6 contents of olive oils were found between 94.42 µg/mg to 161.63 µg/mg oil. Omega-3 contents were found at low levels (8.05 µg/m to 9.60 µg/mg).In a study about the olive cultivars in Turkey, the value of palmitic acid was found to be much lower in Gemlik cultivar and the value of stearic acid was found to be much higher in Kilis cultivar [13]. Ollivier et al. [14] reported that the palmitic, stearic, oleic, linoleic and linolenic acid contents values are in an

order of 8.49% to 13.72%, 2.11% to 2.6%, 66.36% to 79.39%, 5.82% to 11.85% and 0.61% to 0.65%. Aparicio and Luna [15] determined oils which are ob-tained from Coratina, Koroneiki and Picual cultivars and established 9.7% to 11.6% palmitic, 2.2% to 2.4% stearic, 78.1% to 80.3% oleic, 4.8% to 5.7% linoleic and 0.4% to 0.8% linolenic acids. Olive com-position is influenced by environmental and cultivar differences. These differences might be due to the different materials analyzed, varietal differences and maturation [16].

4. CONCLUSION

According to statistical analysis, fatty acid composi-tional differences among the oils studied were signi-ficant, indicating a varietal effect on olive oil quality. These results are in agreement with the findings of other authors [13, 17-21]. Fatty acid compositions of olive oils have shown differences among varieties [22]. Olive composition is influenced by environmental and cultivar differences. These differences might be due to the different materials analyzed, varietal diffe-

1

Table I - Fatty acid compositions by GC of olive oils (%).

*Mean and Standard deviation of 2 samples (n:2) SFA,saturated fatty acids; MUFA, monounsaturated fatty acids; PUFA, polyunsaturated fatty acids

Table II - Unsaturated fatty acid compositions by GC of olive oils (µg/mg oil).

Fatty acids Domat Gemlik Uslu Ayvalık Sarıulak 16:1 n-7 7.67±0.17* 6.79±0.46 7.88±0.64 5.09±0.57 5.29±0.11 18:1n-9 613.18±8.80 614.17±0.99 605.13±11.75 533.58±10.53 626.63±1.18 Omega-6 96.03±0.87 117.16±1.56 113.56±3.57 161.63±2.42 94.42±1.60 Omega-3 8.05±0.16 8.43±0.73 9.60±0.22 8.88±0.31 8.35±0.59

*Mean and Standard deviation of 2 samples (n:2)

Fatty acids Domat Gemlik Uslu Ayvalık Sarıulak 12:0 0.02±0.00* 0.01±0.00 0.01±0.00 0.01±0.00 0.02±0.00 14:0 0.07±0.05 0.07±0.01 0.05±0.02 0.05±0.01 0.08±0.05 16:0 13.31±0.11 13.09±0.03 13.15±0.41 13.29±0.10 13.11±0.01 16:1 n-7 1.05±0.01 0.93±0.00 1.03±0.01 0.78±0.04 0.76±0.00 18:0 2.28±0.10 2.22±0.00 2.12±0.06 2.67±0.04 2.77±0.00 18:1 n-7 1.41±0.04 1.34±0.09 1.38±0.15 1.40±0.12 1.30±0.02 18:1n-9 68.16±0.56 65.68±0.37 66.49±0.81 60.81±0.03 68.98±0.08 18:2 n-6 10.32±±0.05 12.05±0.05 12.09±0.17 18.21±0.00 9.87±0.07 18:3n-3 0.77±±0.00 0.70±0.02 0.90±0.01 0.83±0.03 0.75±0.00 20:0 0.39±0.00 0.45±0.07 0.45±0.02 0.47±0.05 0.54±0.00 20:1n-9 0.29±0.03 0.29±0.00 0.29±0.00 0.30±0.01 0.33±0.00 SFA 16.07±±0.17 15.85±0.12 15.79±0.47 16.49±0.00 16.52±0.02 MUFA 70.91±0.54 68.23±0.28 69.20±0.98 63.29±0.18 71.36±0.07 PUFA 11.09±0.04 12.75±0.07 12.99±0.19 19.05±0.03 10.62±0.07

1

Table I - Fatty acid compositions by GC of olive oils (%).

*Mean and Standard deviation of 2 samples (n:2) SFA,saturated fatty acids; MUFA, monounsaturated fatty acids; PUFA, polyunsaturated fatty acids

Table II - Unsaturated fatty acid compositions by GC of olive oils (µg/mg oil).

Fatty acids Domat Gemlik Uslu Ayvalık Sarıulak 16:1 n-7 7.67±0.17* 6.79±0.46 7.88±0.64 5.09±0.57 5.29±0.11 18:1n-9 613.18±8.80 614.17±0.99 605.13±11.75 533.58±10.53 626.63±1.18 Omega-6 96.03±0.87 117.16±1.56 113.56±3.57 161.63±2.42 94.42±1.60 Omega-3 8.05±0.16 8.43±0.73 9.60±0.22 8.88±0.31 8.35±0.59

*Mean and Standard deviation of 2 samples (n:2)

Fatty acids Domat Gemlik Uslu Ayvalık Sarıulak 12:0 0.02±0.00* 0.01±0.00 0.01±0.00 0.01±0.00 0.02±0.00 14:0 0.07±0.05 0.07±0.01 0.05±0.02 0.05±0.01 0.08±0.05 16:0 13.31±0.11 13.09±0.03 13.15±0.41 13.29±0.10 13.11±0.01 16:1 n-7 1.05±0.01 0.93±0.00 1.03±0.01 0.78±0.04 0.76±0.00 18:0 2.28±0.10 2.22±0.00 2.12±0.06 2.67±0.04 2.77±0.00 18:1 n-7 1.41±0.04 1.34±0.09 1.38±0.15 1.40±0.12 1.30±0.02 18:1n-9 68.16±0.56 65.68±0.37 66.49±0.81 60.81±0.03 68.98±0.08 18:2 n-6 10.32±±0.05 12.05±0.05 12.09±0.17 18.21±0.00 9.87±0.07 18:3n-3 0.77±±0.00 0.70±0.02 0.90±0.01 0.83±0.03 0.75±0.00 20:0 0.39±0.00 0.45±0.07 0.45±0.02 0.47±0.05 0.54±0.00 20:1n-9 0.29±0.03 0.29±0.00 0.29±0.00 0.30±0.01 0.33±0.00 SFA 16.07±±0.17 15.85±0.12 15.79±0.47 16.49±0.00 16.52±0.02 MUFA 70.91±0.54 68.23±0.28 69.20±0.98 63.29±0.18 71.36±0.07 PUFA 11.09±0.04 12.75±0.07 12.99±0.19 19.05±0.03 10.62±0.07


42

rences and different maturation indices. As a result, the present study should be considered an important step in providing information for researchers for fur-ther studies on olive oils.

Acknowledgements

This study was supported by Selçuk University Scien-tific Research Project (S.Ü.-BAP, Konya-Turkey).

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[2] F. Visioli, C. Gali, F. Barnet, A. Mattei, R. Petelli, G. Galli, D. Caruso, Olive oil phe-nolics are dose-dependently absorbed in humans. Federation of Eur. Biochem. Soc. 468, 159-160 (2000).

[3] E. Martinez de Victoria, M. Manas, El aceite de oliva en la dieta y salud humanas. In: D. Barran-co, R. Fernandez-Escobar, & L. Rallo (eds.), El cultivo del olivo (pp. 663-684). Madrid: Mundi-Prensa, (2001).

[4] M.P. Aguilera, G. Beltran, D. Ortega, A. Fernan-dez, A. Jimenez, M. Uceda, Characterisation of virgin olive of Italian olive cultivars: Frantoio and Leccino, grown in Andalusia. Food Chem. 89, 387-391(2005).

[5] S. Gürbüz, N. Kiran-Ciliz, O. Yenigun, Cleaner production implementation through process modifications for selected SMEs in Turkish oli-ve oil production. J. Cleaner Prod. 12, 613-621 (2004).

[6] M. Andjelkovic, S. Acun, V. Van Hoed, R. Verhe, J. Van Camp, Chemical composition of Turkish olive oil-Ayvalık. J.Am. Oil Chem. Soc. 86, 135-140 (2009).

[7] E. Finotti, C. Beye, N. Nardo, G.B. Quaglia, C. Milin, J. Giacometti, Physico-chemical characte-ristics of olives and olive oil from two mono-cul-tivars during various ripening phases. Nahrung/Food 45, 350-352(2001).

[8] A. Rosa, A. Atzeri, M. Deiana, M.P. Melis, D. Loru, A. Incani, B. Cabboi, M.A. Dessì, Effect of aqueous and lipophilic mullet (Mugil cephalus) bottarga extracts on the growth and lipid profile of intestinal Caco-2 cells. J. Agric. Food Chem. 59, 1658-1666 (2011).

[9] W.W. Christie, Preparation of ester derivatives of fatty acids for chromatographic analysis. In Ad-vantage in Lipid Methodology – Two; Christie; W.W., Ed.; The Oily Press: Dundee, Scotland; pp. 69-111(1993).

[10] H. Püskülcü, F. Ikiz, Introduction to Statistic Bil-gehan Press, p. 333, Bornova, Izmir,Turkey. (in Turkish) (1989).

[11] C. Letawe, M. Bone, G.E. Pierard, Digital image analysis of the effect of topically applied linoleic acid on acne microcomodones. Clin. Experim. Derm. 23, 56-58 (1998).

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[13] K. Tanılgan, M.M. Özcan, A. Ünver, Physical and chemical characteristics of five Turkish olive (Olea europea L.) varieties and their oils. Grasas y Aceites, 58 (2), 142 (2007).

[14] D. Ollivier, J. Artaud, C. Pinatel, J.P. Durbec, M. Guerere, Differentiation of French virgin olive oil RDOs by sensory characteristics, fatty acid and triacylglycerol compositions and chemometrics. Food Chem. 97 (3), 382-393 (2006).

[15] R. Aparicio, G. Luna, Characterisation of mono-varietal virgin olive oils. Eur. J. Lipid Sci. Technol. 104, 614-627 (2002).

[16] G. Beltran, C. Del Rio, S. Sanchez, L. Martinez, Influence of harvest date and crop yield on the fatty acid composition of virgin olive oils from cv. Picual. J. Sci. Food Agric. 52, 3434-3440 (2004).

[17] G. Schiratti, Presentacifin del estudio sobre la influencia de las variedades ambientales, agron-fimicas y tecnolfigicas en las caracteristicas y niveles de los componentes menores del aceite de oliva virgen extra. Olivae 79, 38-40 (1999).

[18] L. Cerretani, A. Bendini, A. Del Caro, A. Piga, V. Vacca, M.F. Caboni, T.G. Toschi, Preliminary characterisation of virgin olive oils obtained from different cultivars in Sardinia. Eur. Food Res. Technol. 222, 354-361 (2006).

[19] B. Baccouri, S. Ben Temime, W. Taamalli, D. Da-oud, M. M’sallem, M. Zarrouk, Analytical charac-teristics of virgin olive oils from two new varieties obtained by controlled crossings on Meski va-riety J. Food Lipids 14, 19-34, (2007a).

[20] B. Baccouri, S. Ben Temime, E. Campeol, P. Cioni, D. Daoud, M. Zarrouk, Application of so-lid-phase microextraction to the analysis of vo-latile compounds in virgin olive oils from five new cultivars. Food Chem. 102, 850-856, (2007b).

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Received March 27, 2013Accepted June 6, 2013


43

P. CataniaM. Vallone *

D. PlanetaP. Febo

Dipartimento di Scienze Agrarie e Forestali - Università degli Studi di

Palermo, Italy

(*) CORRESPONDING AUTHOR: Dipartimento di Scienze Agrarie e

Forestali, Università degli Studi, Viale delle Scienze Ed. 4,

90128 Palermo, Italye-mail: [email protected]

tel. +39 91 23865609

instrumental evaluation of the texture of cv. nocellara del belice table olives

The Nocellara del Belice (Olea europaea L.) is the most important double-aptitude olive variety in western Sicily (Italy), it is one of the most important domestic varieties both for the production and the marketed quantities. Nowadays there is a lack of studies on its mechanical properties through instrumental analysis able to provide quantitative measurements of its flesh texture which is a very important quality attribute for the consumer.The aim of this study is to evaluate the texture of Nocellara del Belice table olives, packed in glass jars, through a mechanical system allowing us to continuously measure and record the compression force along the profile of the flesh of the fruit. For this purpose, we evaluated the influence of the retention period on the texture of olive fruits in brine produced in three different years, supporting the results with the kinesthetic properties evaluated by sensory analysis. Our results show that in the three years of production we studied, olives packed immediately after processing (with “Castelvetrano” system) preserve their texture better than olives packed six months later. Our study found that the packaging of table olives in glass jars containing covering liquid reduces the tissues of the drupe from softening than long-term storage in drums in contact with the brine.Keywords: mechanical properties, table olives, texture.

Valutazione strumentale della consistenza di olive da tavola della cv. Nocellara del BeliceLe olive della varietà Nocellara del Belice (Olea europaea L.), rappresentano, dal punto di vista produttivo e dei volumi commercializzati, una delle realtà più significative in Italia. Ad oggi non sono stati condotti studi sulle loro proprietà meccaniche tramite analisi di tipo strumentale in grado di fornire misure quantitative della consistenza della polpa. Quest’ultimo rappresenta un attributo qualitativo molto importante ai fini del gradimento del prodotto da parte del consumatore. Obiettivo di questo studio è stato quello di valutare la consistenza delle olive da tavola della varietà Nocellara del Belice, confezionate in barattoli di vetro, mediante l’applicazione di un sistema meccanico che ha consentito di misurare e registrare in continuo i valori di forza di compressione lungo tutto il profilo della polpa. É stata valutata l’influenza del periodo di conservazione sulla consistenza di drupe in salamoia prodotte in tre annate diverse supportando i risultati ottenuti con la valutazione delle proprietà cinestetiche, tramite analisi sensoriale.I risultati hanno mostrato che, nelle tre campagne di produzione prese in esame, le olive confezionate subito dopo il processo di deamarizzazione (eseguito con il metodo “Castelvetrano”) conservano meglio la loro consistenza rispetto a quelle confezionate sei mesi dopo. Dallo studio è emerso che il confezionamento delle olive da tavola in barattoli di vetro contenenti il liquido di governo consente di ridurre il rammollimento dei tessuti della drupa rispetto alla lunga conservazione delle stesse olive nei fusti a contatto con la salamoia. Parole chiave: olive da tavola, consistenza, proprietà meccaniche.


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1. INTRODUCTION

The Nocellara del Belice (Olea europaea L.) is the most important double-aptitude olive variety in we-stern Sicily (Italy) and obtained the recognition of tra-demarks PDO (Protected Denomination of Origin). It is widely used as a table olive and, with an average annual production of 25,000 t, it is one of the most important domestic varieties both for the production and the marketed quantities.The texture of table olives is a very important quality attribute deriving from the size of the cell wall, mid-dle lamella and fibrous tissues, which determine the mechanical properties of the drupe together with the cell turgor. Mechanical tests for determining the consistency of the pulp of olives have been performed by several au-thors, while still making a point or discrete measure-ments and obtaining average values of some textural properties of the fruit. The maximum shear force of the pulp showed decreasing values with an increasing de-gree of maturity on the Manzanilla variety in [1]. In [2] it was observed a reduction of the maximum puncture force of the pulp during ripening. The authors were among the first to carry out a study aimed at making an objective assessment of the olive fruit texture using a penetrometer. Similar tests were carried out by Mafra et al. [3] by measuring, in particular, the force required to insert a 2 mm diameter probe 5 mm into the flesh of olives having different degrees of maturation; in this case the fruit mesocarp was cut around the equator and positioned vertically. Puncture and compression tests showed tissue softening induced by ripening.Kiliçkan and Güner [4] studied the physical proper-ties and mechanical behavior of olive fruits under compression loading using a device having a fixed base plate and a moving parallel plate, a driving unit, and the data acquisition system composed of a dy-namometer, amplifier and XY plotter. In this study the mechanical behavior of olive fruits and pits was expressed in terms of rupture force, rupture energy, and specific deformation required for initial rupture of olive fruit and pit along different compression axes. The results allowed the authors to identify the X-axis (the longitudinal axis through the hilum containing the major dimension) as the direction where the highest rupture force occurs. The authors also found that the rupture force, rupture energy, and specific deforma-tion increases as deformation rate and fruit size and pit size increase for all compression axes along where the tests were performed.As for table olives, Cardoso et al. [5] studied the ef-fects of dry-salt processing on the textural properties and cell wall polysaccharides of cv. Thasos black oli-ves. Mechanical properties of olive fruits were studied for the skin and flesh, cutting a strip 5 mm wide and approximately 20 mm long, whose thickness varied from 0.05 to 0.3 mm that were arranged between the two metal plates of a texture analyzer with a 5 kg load

cell and a test speed of 0.05 mm s-1. The research demonstrated that in processed olives, the tissue was approximately 4.5 times stronger and also more deformable up to failure and stiffer than that from the raw olives.Despite the cv. Nocellara del Belice table olives repre-sent one of the most important varieties in Italy from the production point of view, there is a lack of studies on their mechanical properties through instrumental analysis able to provide quantitative measurements of certain properties that can be also evaluated by sensory analysis.The standard for sensory analysis of table olives is-sued by IOOC (International Olive Oil Council) in 2008, recently revised in November 2011, establishes the criteria necessary for sensory analysis and the proce-dure for the qualitative classification of the product [6]. Lanza et al. [7] evaluated chemical, nutritional, sen-sory and textural characteristics of table olives cul-tivar Intosso d’Abruzzo before and after processing for consumption in order to enhance the value of this fruit from a nutritional point of view. Texture analysis was carried out through a compression test pressing each olive with a flat cylindrical plunger of 1 cm Ø, using a weight-force of 500 g and a test time of 30 s. Fruits of this variety are particularly solid, so the de-terioration suffered by tissues after processing does not compromise the edibility of the finished product. This was related to the perception of kinesthetic sen-sations thought sensory analysis that gave medium-high hardness and crunchiness values. However, in this study the texture of the pulp is evaluated in a sin-gle point, without investigating the possible variation throughout the thickness of the pulp.The relationship between the kinesthetic and mecha-nical characteristics of a given product was highlighted in [8]; the authors pointed out that a texture profile analysis of the product is correlated with its crunchi-ness. Therefore, the instrumental and sensory analy-sis seem to be two different approaches to the same problem: the study of the crunchiness of products that have to meet with the consumers’ appreciation [9].The aim of this study is to evaluate the texture of No-cellara del Belice table olives, packed in glass jars, through a mechanical system allowing us to conti-nuously measure and record the compression force along the profile of the flesh fruit. For this purpose, we evaluated the influence of the retention period on the texture of olive fruits in brine produced in three diffe-rent years, supporting the results with the kinesthetic properties evaluated by sensory analysis.

2. MATERIALS AND METHODS

2.1 PLANT MATERIALThe experimental tests were performed in April 2013 in the Agricultural Mechanics laboratory at the De-partment of Agricultural and Forest Sciences of the University of Palermo, on Nocellara del Belice table


45

olives which were representative samples taken from batches of the years 2010, 2011 and 2012. Olives were supplied by Oleificio Asaro Company (Partanna, Trapani, Italy). The olives we used in the experimental tests were manually harvested each year in October and stored in 30.00 l drums at a salt concentration of 6.5%. Olive debittering was obtained with a typical west Sicilian method called the “Castelvetrano” sy-stem, also called “dolcificata” in the local language. According to this method, olive fruits, graded by size, are usually placed in 200-220 l plastic drums, treated with lye overnight and after 12 hours 5-8 kg of salt are added. After 15 days of storage the product is ready [10]. The fruits were packaged in glass jars having 488 g gross weight, 300 g net weight and 160 g drained weight. The package contains the following ingre-dients: water, salt, citric acid, lactic acid and ascorbic acid, with a salt concentration of 4%. The stabilization was performed with pasteurization at a temperature of 75°C for 8 minutes by using a tunnel pasteurizer.

Two different treatments were carried out for each year, differing for the time between olive processing and packaging (Fig. 1); in treatment named A, olives were packaged immediately after processing, while letter B indicates olives packaged six months later (Tab. I). Therefore, the tests we carried out were: tests 1A and 1B for year 2010, tests 2A and 2B for year 2011, and tests 3A and 3B for year 2012.

2.2 MECHANICAL TESTS FOR TEXTURE EVALUATION The fruit and pit mass were measured by an electronic balance to an accuracy of 0.01 g (ORMA, BC4000S - Italy). The fruit and pit diameter and the flesh thick-ness were measured by a digital micrometer caliper to an accuracy of 0.01 mm.The mechanical test to evaluate olive fruit texture was performed compressing the olives by using a mecha-nical dynamometer (Imada DPS 5R - USA) connected to an electronic stand (IMADA MX2-500N - L) and a PC for data download (Fig. 2).The fruit compression was obtained by means of a cylindrical steel plate of 16 mm in diameter, whose surface was disposed orthogonally with respect to the minor axis of the olive (Fig. 3). The test speed was set at 0.125 mm s-1 and was kept constant during the tests. The use of an electronic stand connected to the dynamometer, allowed us to measure the com-pression force every 0.25 s. Each trial was stopped when the compression force reached the value of 200 N to avoid pit crushing, that was not relevant in our research. At the end of the mechanical test, the

Table I - Experimental tests

Table II - Olive fruit dimensions and mass

Quantity Value a Fruit diameter [mm] 20.99 ± 0.91 Pit diameter [mm] 9.55 ± 0.49 Mesocarp thickness [mm] 11.44 ± 0.94 Fruit mass [g] 6.78 ± 0.86 Pit mass [g] 0.96 ± 0.12 Mesocarp mass [g] 5.82 ± 0.85

a Numeric values are means ± standard error of thirty replicates.

Test Year Date of packaging 1A 2010 October 2010 1B 2010 April 2011 2A 2011 October 2011 2B 2011 April 2012 3A 2012 October 2012 3B 2012 April 2013

Figure 1 – Olives processing steps for the experimental tests

Figure 2 - Electronic balance (ORMA, BC4000S - Italy), mechanical dynamometer (Imada DPS 5R - USA), electronic stand (IMADA MX2-500N - L) and PC for data download

Manual harvest

Storage at ambient conditions

Pasteurization at industrial scale

Packaging in glass jars

Processing with “Castelvetrano“ system


Test A - Packaging in glass jars immediately

after processing

Test B – Storage in brine for six months


46

pit was removed and cleaned from the mesocarp tis-sues, and its mass and diameter were determined.The mesocarp was divided into three layers, named I, II and III from the outside towards the inside of the flesh, in which we studied the values of compression force (Fig. 4). The force value obtained in correspon-dence of the lower limit of each layer has been identi-fied. The division of the flesh into three layers allowed us to check the flesh consistency from the skin to the pit. The tissues of the fruit, in fact, suffer a softening from the outer to the inner cells both during the sto-rage period, when they are in contact with the brine, and within the jar in contact with the covering liquid.

The application of a compression force on the fruit allowed us to evaluate the flesh consistency in terms of hardness of the tissues. The a priori division of the mesocarp into three layers was carried out in order to study the effects of the conservation process near to the fruit skin (layer I), in the center of the pulp (layer II) and in the vicinity of the pit (layer III). This kind of subdivision was applicable to all the olives we analy-zed because the fruits were preliminarily calibrated. Mechanical tests were repeated three times for treat-ment using thirty olives for each of them

2.3 SENSORY EVALUATION OF TABLE OLIVES Sensory analysis was carried out according to the Method of Sensory Analysis of Table Olives [6]; this standard applies to the fruit of the cultivated olive tree (Olea europaea L.) which has been suitably treated or processed and which is offered for trade and for final consumption as table olives.The olives were evaluated by a panel of ten selected and trained tasters. The tests were performed in a test room using a glass for olive oil tasting, a watch-glass to cover the tasting glasses, while metal tongs were used to get olive samples. This sensory work has fo-cused its attention mainly on kinesthetic sensations rather than gustatory sensations in order to associa-te them to the mechanical strength of the olive fruit flesh. Gustatory attributes are salty, bitter and acid. Salty is the basic taste produced by aqueous solu-tions of substances such as sodium chloride, that are essential in olive processing; bitter is the basic taste produced by dilute aqueous solutions of substances such as quinine or caffeine; acid is the basic taste produced by dilute aqueous solutions of most acid substances, such as tartaric acid, citric acid.Also kinesthetic sensations are divided into three groups: hardness, fibrousness and crunchiness. Hardness indicates the mechanical textural attribute relating to the force required to attain the deformation of a product or for an object to penetrate it. It is eva-luated by compressing the product between the te-eth (solids) or between the tongue and palate (semi-solids). Fibrousness is the geometric textural attribute relating to the perception of the shape and the orien-tation of particles in a product. Fibrousness refers to the elongated conformation of the particles, oriented in the same direction. It is evaluated by perceiving the fibers between the tongue and palate when chewing the olive. Crunchiness is the attribute relating to the noise produced by friction or fracture between two surfaces. It is related to the force required to fracture a product with the teeth and is determined by com-pressing the fruit between the molars. The sample of table olives intended for sensory analysis, were repre-sentative of homogenous batches in accordance with the sampling rules.

2.4 STATISTICAL ANALYSISData were analyzed using analysis of variance and

Figure 1 – Olives processing steps for the experimental tests

Figure 2 - Electronic balance (ORMA, BC4000S - Italy), mechanical dynamometer (Imada DPS 5R - USA), electronic stand (IMADA MX2-500N - L) and PC for data download

Manual harvest

Storage at ambient conditions


Packaging in glass jars

Processing with “Castelvetrano“ system


Test A - Packaging in glass jars immediately

after processing

Test B – Storage in brine for six months

Figure 3 - Olive fruit during the compression test

Figure 4 - Olive section with the identification of the three layers of the mesocarp

Figure 3 - Olive fruit during the compression test

Figure 4 - Olive section with the identification of the three layers of the mesocarp


47

t-test at the 95.0% confidence level to compare the two different treatments of each year. The analysis in-volved the force values obtained in correspondence of the lower limit of each layer of the mesocarp. Also with reference to the sensory assessment of the olives, analysis of variance and t-test at the 95.0% confiden-ce level were applied. Statistical analyses were carried out by means of Statgraphics centurion XV version (Statpoint inc., USA, 2005) software package.


3.1 COMPRESSION TESTSThe characteristics of the olive fruits are summarized in Table II. In particular, the mesocarp thickness and mass respectively represent 55% and 86% of the drupe. The consumer highly appreciates this kind of

fruit flesh whose consistency in terms of hardness re-presents a qualitative aspect of fundamental impor-tance; flesh firmness, in fact, enhances the fragrance of the edible part.Figures 5, 6 and 7 show the average values of com-

Table I - Experimental tests

Table II - Olive fruit dimensions and mass

Quantity Value a Fruit diameter [mm] 20.99 ± 0.91 Pit diameter [mm] 9.55 ± 0.49 Mesocarp thickness [mm] 11.44 ± 0.94 Fruit mass [g] 6.78 ± 0.86 Pit mass [g] 0.96 ± 0.12 Mesocarp mass [g] 5.82 ± 0.85

a Numeric values are means ± standard error of thirty replicates.

Test Year Date of packaging 1A 2010 October 2010 1B 2010 April 2011 2A 2011 October 2011 2B 2011 April 2012 3A 2012 October 2012 3B 2012 April 2013

Figure 5 - Compression force curves for tests 1A and 1B, 2010 harvest. Letter A indicates olives packaged immediately after processing, B indicates olives packaged six months later. Data are reported as means of thirty replicates


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pression force through the mesocarp for all the tests. The compression force increases throughout the thickness of the mesocarp reaching the maximum value, in the vicinity of the pit, to about 5.66 mm from the skin. The maximum values of the compression force agree with those obtained in [4], where the au-thors had a mean value of 72.00 N olives with physi-cal characteristics similar to our variety. It is plain that the compression force is greater in test 1A, 2A and 3A whose olives were packaged immediately after processing, than tests 1B, 2B and 3B whose olives were packaged 6 months later. It follows that the date of packaging affects the consistency of the flesh of olives.The compression force values obtained in correspon-dence with the lower limit of each layer of the me-

socarp are shown in Figures 8, 9 and 10 for all the treatments.The compression force values for the olives harvested in 2010 always show statistically significant differen-ces between the different layers of the mesocarp both in test 1A and in 1B (Fig. 8). In fact, the compression force values increase about 35% from layer I to layer II and about 41% from layer II to layer III in test 1A. In test 1B, where olives were packaged six months after processing, the compression force increased about 66% from layer I to layer II and about 39% from layer II to layer III. No statistically significant differences were found between tests 1A and 1B in layer I and layer II, on the contrary layer III where test 1A was about 26% higher than test 1B.The compression force values for the olives harve-


Figure 8 - Compression force in the three layers of the mesocarp for tests 1A and 1B, 2010 harvest. Letter A indicates olives packaged immediately after processing, B indicates olives packaged six months later. Data are reported as means of thirty replicates

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sted in 2011 always show statistically significant dif-ferences between the different layers of the mesocarp both in test 2A and 2B (Fig. 9). The compression for-ce values show a 71% increase going from layer I to layer II and a 37% increase going from layer II to layer III in test 2A. In test 2B the compression force values increase about 78% from layer I to layer II and 47% from layer II to layer III. Comparing the compression force values in tests 2A and 2B, it comes out that the-re are no statistically significant differences between the mean values of layer I. On the contrary, we found statistically significant differences between tests 2A and 2B both in layer II and III, with test 2A values al-ways higher than test 2B values (about 26% in layer II

and 12% in layer III).Also for the olives harvested in 2012, the compres-sion force values always show statistically significant differences between the different layers of the meso-carp both in test 3A and 3B (Fig. 10). In test 3A, the-se values show a 78% increase going from layer I to layer II and a 38% increase from layer II to layer III; in test 3B the compression force increases about 26% from layer I to layer II and 49% from layer II to layer III. Comparing tests 3A and 3B, we always found stati-stically significant differences in the three layers of the mesocarp; test 3A values were always higher than test 3B values (of about 36% in layer I, 26% in layer II and 11% in layer III).



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We can affirm that olives packed immediately after processing suffer from the softening of the tissues less than olives packed six months later, leaving out of consideration the year of production. The lower compression resistance of the fruits packed six mon-ths after processing is caused by the prolonged con-tact of the drupe with the brine during the storage period.Our study also showed that, among the olive fruits packaged immediately after processing, those of the most recent production, year 2013, retained their hardness in all the three mesocarp layers than those produced in 2012, in which the flesh hardness was guaranteed in layers II and III of the mesocarp and those produced in 2011, where the flesh hardness was guaranteed only in the third layer of the meso-carp.

3.2 SENSORY ANALYSIS Sensory analysis results are useful to highlight the dif-ferent characteristics of the examined olives both from gustative and kinesthetic point of view, this aspect is the most relevant to the present study (Tab. III).The olive sample of test 1A was evaluated mildly salty while it appeared to be slightly acid, the kinesthetic sensations concerning hardness was held at a me-dium-low level as crunchiness and fibrousness. Sam-ple 1B is slightly bitter, acid and salty sensations ap-peared to be medium while the kinesthetic sensations regarding hardness, crunchiness and fibrousness were low. Sample 2A appeared to be average salty and slightly bitter and acid, the kinesthetic sensations of hardness, crunchiness and fibrousness were eva-luated insufficient. Sample 2B was slightly bitter while the sensations of acid and salty appeared to be me-dium as the kinesthetic sensations. Sample 3A was evaluated middle bitter, the acid sensation was light, the kinesthetic sensations were relevant. Sample 3B appears to be an average of salty, slightly bitter and acid, the kinesthetic sensations were evaluated more than satisfactory.The results of the sensory analysis, with particular reference to the kinesthetic sensations of hardness, crunchiness and fibrousness therefore agree with the results of the instrumental analysis. In particular, note

that hardness and crunchiness values are always hi-gher in tests A than tests B in the three years we con-sidered them. Comparing compression test data and sensory data we found that the compression force values are confirmed by the kinesthetic sensations.

4. CONCLUSION

This study focused on the texture evaluation of No-cellara del Belice table olives, through a mechanical system allowing us to continuously measure and re-cord the compression force along the profile of the flesh fruit. These data were supported by the kines-thetic sensations evaluated by sensory analysis. In the three years of production we studied, olives packed immediately after processing preserve their texture better than olives packed six months later. Our study found that the packaging of table olives in glass jars containing covering liquid reduces the tis-sues softening of the drupe than long-term storage in drums in contact with the brine. Packaging the entire production of the year immediately after processing, on the one hand would reduce the time in which the product would have to be consumed, on the other hand it would maintain the layers of the drupe with a much more consistent improvement of the kinesthe-tic sensations.The results obtained from this study go in the same direction as those obtained by other authors [11, 12] affirming that the mechanical characteristics of the drupe are closely related to its processing and sto-rage.In conclusion we can say that the first results on tex-ture evaluation of Nocellara del Belice table olives, create new scenarios on their storage methodology for prolonging the shelf life of the product.

Acknowledgments

This study was supported by Fondi di Ateneo per la Ricerca by the University of Palermo.The authors are grateful to Oleificio Asaro Company (Partanna, Trapani, Italy) for providing the table olives used to perform the experimental tests.

Table III - Sensory evaluation results (ANOVA and t-test for p=0.05) a

Sensation Test 1A 1B 2A 2B 3A 3B

Salty 3.3±1.6 a 4.1±1.5 a 4.7±1.8 a 4.6±1.6 a 4.6±1.5 a 4.7±1.6 a Bitter 3.0±1.3 a 3.3±1.8 a 3.4±1.3 a 3.6±2.0 a 3.9±1.8 a 3.7±1.6 a Acid 4.0±1.0 a 4.8±1.8 a 3.3±1.2 a 4.4±2.1 a 3.4±1.7 a 4.0±1.9 a Hardness 3.7±0.2 a 2.9±0.4 b 5.6±1.1 a 4.1±0.5 b 6.9±1.1 a 6.1±0.8 b Fibrousness 4.2±1.7 a 3.9±0.9 a 4.9±1.4 a 4.3±1.9 a 6.3±1.7 a 6.2±1.8 a Crunchiness 3.9±0.5 a 3.1±0.3 b 5.7±1.1 a 4.2±0.8 b 7.6±1.1 a 6.6±1.0 b

a Data are means + st.dev.; n = 10. Different letters in the row indicate statistically significant differences between the two treatments of the same year.


51

REFERENCES

M.J. Fernàndez Díez, Texture of table olives and [1] pimentos, J. Texture Stud. 10, 103-116 (1979).A. Brighigna, V. Marsilio, M. De Angelis, Realiz-[2] zazione di un penetrometro per la determinazio-ne della consistenza della polpa delle olive, Ann. Ist. Sper. Elaiotec. 8, 3-15 (1978-80).I. Mafra, B. Lanza, A. Reis, V. Marsilio, C. Camp-[3] estre, M. De Angelis, M.A. Coimbra, Effect of ripening on texture, microstructure and cell wall polysaccharide composition of olive fruit (Olea europaea), Physiol. Plant. 111, 439–447 (2001).A. Kiliçkan, M. Güner, Physical properties and [4] mechanical behavior of olive fruits (Olea euro-paea L.) under compression loading, J. Food. Eng. 87, 222-228 (2008).S.M. Cardoso, I. Mafra, A. Reis, D.M. R. Geor-[5] get, A.C. Smith, K.W. Waldron, M.A. Coimbra, Effect of dry-salt processing on the textural properties and cell wall polysaccharides of cv. Thasos black olives, J. Sci. Food. Agric. 88, 2079-2086 (2008).IOOC (International Olive Oil Council). Sensory [6] Analysis of Table Olives. COI/OT/MO N°1/Rev.2 November 2011. Madrid: IOOC (2011).B. Lanza, M.G. Di Serio, E. Iannucci, F. Russi, P. [7] Marfisi, Nutritional, textural and sensorial char-

acterisation of Italian table olives (Olea europaea L. cv. ‘Intosso d’Abruzzo’), Int. J. Food Sci. Technol. 45, 67-74 (2010).M. Saeleaw, G. Schleining, A review: Crispness [8] in dry foods and quality measurements based on acoustic–mechanical destructive techniques, J. Food Eng. 105, 387-399. (2011).G. Roudaut, C. Dacremont, B. Valles Pamies, B. [9] Colas, M. Le Meste, Crispness: a critical review on sensory and material science approaches, Trends Food. Sci. Tech. 13, 217-227 (2002).F.V. Romeo, A. Piscopo, A. Mincione, M. Poi-[10] ana, Quality evaluation of different typical table olive preparations (cv Nocellara del Belice), Gra-sas Aceites 63, 19-25 (2012).D.M.R. Georget, A.C. Smith, K.W. Waldron, Ef-[11] fect of ripening on the mechanical properties of Portuguese and Spanish varieties of olive (Olea europaea L.), J. Sci. Food Agric. 81, 448-454 (2001).D.M.R. Georget, A.C. Smith, K.W. Waldron, L. [12] Rejano, Effect of ‘Californian’ process on the texture of Hojiblanca olive (Olea europaea L.) harvested at different ripening stages, J. Sci. Food Agric. 83, 574-579 (2003).

Received October 1, 2013Accepted November 29, 2013

Laboratorio

di analisi sensoriale

dell’Olio di

Oliva vergine

INNOVHUB - Stazioni Sperimentali per l’Industria Azienda Speciale della Camera di Commercio – Milano

Divisione SSOG – Dr.ssa Silvia Tagliabue Responsabile Laboratorio Analisi Sensoriale

Tel.: 02.706497.78 - Fax: 02.2363953 - E-mail: [email protected]

Il Reg. UE 1348 /2013 (modifica del Reg. CEE 2568/1991) stabilisce i parametri chimico-fisici e i metodi per il controllo di qualità dell’olio di oliva. La valutazione organolettica (Panel test), introdotta nel regolamento comunitario, concorre alla definizione della qualità dell’olio e alla classificazione merceologica di appartenenza. Il Regolamento classifica l’olio di oliva vergine nelle categorie:

OLIO EXTRA VERGINE DI OLIVA OLIO DI OLIVA VERGINE OLIO DI OLIVA LAMPANTE

in funzione dell’intensità del fruttato, della presenza e dell’intensità di eventuali difetti. Fornisce inoltre indicazioni sulle caratteristiche organolettiche per l’etichettatura facoltativa. La valutazione organolettica è qualificata da un livello di affidabilità paragonabile a quello delle prove analitiche e viene eseguita da un panel di assaggiatori selezionati e addestrati, avvalendosi di tecniche statistiche per il trattamento dei dati. Il nostro Panel è riconosciuto dal MiPAF (Ministero delle Politiche Agricole Alimentari e Forestali) come comitato di assaggio incaricato del controllo ufficiale delle caratteristiche degli oli di oliva vergini e degli oli DOP e IGP e dal COI (Consiglio Oleicolo Internazionale). La valutazione organolettica è accreditata da ACCREDIA. Il Panel è al servizio dell’industria, di consorzi di produzione, di enti certificatori e della grande distribuzione.


53

C. Guillaume*L. Ravetti

Modern Olives Laboratory Services Victoria, Australia

(*) CORRESPONDING AUTHOR:Claudia Guillaume P. O. Box 92-Lara

3212 Victoria, Australia+61 3 5272 9500 +61 3 5272 9599

[email protected]

technical notetechnological and agronomical factors affecting sterols in australian olive oils

Sterols are important lipids related to the purity of the oil and broadly used for checking its genuineness. Recent analyses have identified that some Australian olive oils would not meet international standards for sterols individual components. Several research works would indicate that there are some significant correlations between cultural and processing practices and sterols content and composition. In this work the horticultural and processing practices that may have an impact on the sterol content and profile of the most important Australian varieties were analysed. The information generated with this study aims to solve a legislation problem as well as maximising the nutritional and health benefits of the Australian olive oils. The evaluation was undertaken using three different varieties and the processing practices evaluated were: Irrigation, fruit size, maturity, malaxing time, malaxing temperature and delays between harvest and process. The total content of sterols and their composition in olive oil is strongly influenced by genetic factors and year. Processing practices particularly affect triterpene dialcohols and stigmasterol while horticultural practices and fruit characteristics tend to affect more significantly other sterols such as β-sitosterol, sitostanol, Δ5-avenasterol and Δ7-avenasterol.

Fattori tecnologici ed agronomici che influenzano gli steroli negli oli di oliva australianiGli steroli sono lipidi importanti relativi alla purezza dell’olio e ampiamente utilizzati per verificarne la genuinità. Recenti analisi hanno individuato che alcuni oli di oliva australiani non avrebbero rispettato gli standard internazionali di alcuni singoli steroli. Diversi lavori di ricerca avrebbero indicato che ci sono alcune correlazioni significative tra pratiche culturali e di trasformazione e contenuto e composizione di steroli. In questo lavoro sono state analizzate le pratiche orticole e di trasformazione, che possono avere un impatto sul contenuto di steroli e il profilo delle più importanti cultivar australiane. Le informazioni generate con questo studio mirano a risolvere un problema di legislazione, così come massimizzare i benefici nutrizionali e salutistici degli oli di oliva australiani. La valutazione è stata effettuata utilizzando tre diverse cultivar e le pratiche di trasformazione valutate sono state: l’irrigazione, pezzatura dei frutti, maturazione, tempo e temperatura di gramolatura e ritardi tra la raccolta e il processo.Il contenuto totale di steroli e la loro composizione nell’olio di oliva sono fortemente influenzati da fattori genetici e dall’annata. Le pratiche di lavorazione incidono particolarmente sui dialcoli triterpenici e sullo stigmasterolo mentre le pratiche orticole e le caratteristiche del frutto tendono ad incidere più significativamente su altri steroli come il β-sitosterolo, sitostanolo, Δ5-avenasterolo e Δ7-avenasterolo.


54

INTRODUCTION

THE SITUATION IN AUSTRALIA It has been previously reported that sterol compo-sition and total sterol content would be affected by cultivar, crop year, degree of fruit ripeness, storage time of fruits prior to oil extraction, processing and by geographical factors [1-5]. It has been found that a significant amount of samples of largely cultivated varieties in Australia do not meet international stan-dards as regards sterols. The Australian olive industry is currently targeting this problem. Research projects [6] comprehensively cover the area of cultivar and en-vironment characterisation. This study complements this research by analysing the horticultural and pro-cessing practices that may have an impact on the sterol content and profile of the most important Au-stralian cultivars as well as generating biochemical and genetic information for a better understanding of the dynamics of sterols in olive oil. Recent analyses have identified that Australian olive oils have signifi-cant amount of sterols. Some Australian olive oils do not meet international standards for total content of sterols or for certain minor components [6]. The cultivar Barnea oils, in particular, contain up to 5.8% Campesterol [6]. Some Frantoio oil samples have shown extremely low total sterol levels, barely above or even under the minimum 1,000 ppm established as international limit. It is extremely important to point out that Barnea oil represented 41% and Frantoio oil 26% of the olive oils produced in Australia in 2006 [7]. Also is important to highlight the importance of phytosterols for human nutrition [12-15].

OBJECTIVES

This work complements previous research by analy-sing the technological and agronomical practices that may have an impact on the sterol content and profile of the most important Australian cultivars. The infor-mation generated aims to solve a legislation issue, and also to maximise the nutritional value of the Au-stralian olive oils.By determining the influence of major horticultural and olive oil processing practices on total sterols and their composition in different olive cultivars, growers and processors will be better prepared to plan the ma-nagement and process of their fruit, minimising the amount of oil that does not meet international criteria, and maximising the nutritional value of their product.

METHODOLOGY

HORTICULTURAL AND PROCESSING TRIALSThe evaluation of horticultural and olive oil processing practices on total sterols and their composition was undertaken in commercial groves in Victoria. The se-lected groves are: Boort Estate (Boort, Victoria) and Boundary Bend Estate (Boundary Bend, Victoria).

The management and climatic conditions of each grove during the trial period was recorded. All groves had automatic weather stations, which enabled tem-perature, humidity, wind speed, radiation and rainfall to be recorded. Considering that most physiological aspects related to sterol formation and ripening pro-cesses in the fruit are related to one or more of those parameters, it is considered that the available infor-mation was appropriate for an adequate evaluation of the final results.The trials were conducted following a proper statisti-cal design. Fruit from three different varieties (Fran-toio, Barnea and Picual) with clearly different sterol profiles were crushed. The significant differences between those varieties that justified their selection are well documented [7], [6]. Frantoio: average total sterol levels of 1,490 ppm and average campesterol of 3.05%.Barnea: average total sterol levels of 1,700 ppm and average campesterol of 4.50%.Picual: average total sterol levels of 1,500 ppm and average campesterol of 3.40%. The fruit was processed in an experimental olive oil mill (Abencor®). The Abencor® bench top extraction system imitates the process used by the industry to extract olive oil. It consists of a hammer mill, a thermo-mixer and a centrifuge. The Abencor® system provi-des a fast and inexpensive means to obtain a sample of oil, operating in accordance with a well stablished method. The oil extraction efficiency index attained is close to the industrial efficiency to be achieved in an industrial plant for most varieties. The quantity of oli-ves used ensures that the sample is fully representati-ve. Oil obtained is usually enough to perform organo-leptic and chemical tests. The processing conditions were the standards for this extraction method apart from the variations applied while evaluating malaxing temperatures and malaxing times [8]The agronomical and processing practices evalua-ted were: Irrigation, fruit size, maturity, malaxing time, malaxing temperature, delays between harvest and process and storage time. Irrigation: Kc (Crop Factor) of 0.74 during the oil ac-cumulation period (January-April) (Normal treatment); Kc of 0.32 during the oil accumulation period (1/2 X) and Kc of 1.48 during the oil accumulation period (2 X).Fruit size: fruit of the different varieties was classified with a table olive fruit grader into three standard si-zes. For Barnea: Small (<2.00 grams), medium (2.00-3.00 grams), large (>3.00 grams). For Frantoio: Small (<1.40 grams), medium (1.40-2.00 grams), large (>2.00 grams). For Picual: Small (<2.20 grams), me-dium (2.20-3.20 grams), large (>3.20 grams).Maturity: fruit from the three varieties was harvested at three different times, two to three weeks between harvests. Maturity was measured using the maturity index developed by the CIFA Alameda del Obispo, Spain [5]. The fruit from the early harvest typically


55

showed a MI between 1.00 and 2.00, from the mid-dle harvest between 2.50 and 3.50 and from the late harvest between 4.00 and 5.00.Malaxing time: three malaxing times were utilised: 30 minutes (Standard), 15 minutes (1/2 X) and 60 minu-tes (2 X).Malaxing Temperature: three malaxing temperatures were utilised: 25°C (Standard), 15°C (Cold) and 35°C (Hot).Delays between harvest and process: three times between harvest and processing were applied: Im-

mediate processing (<12 hours), medium processing (36-48 hours) and delayed processing (72-84 hours).Storage time: all samples processed were analysed immediately after processing, 6 months later and 12 months later.Three replicates of each treatment were processed. Each replicate typically consisted of two mixing units of 700 grams of olive paste each. Replicates of each treatment were processed during the 2007 and the 2008 seasons. All samples were evaluated by duplicate.

Table I - Sterol and Triterpene Dialcohol Concentrations1 (values as % total sterols) of oils processed from fruit with maturity index of <2.00, 2.00-4.00 and >4.00

< 2 2 - 4 > 4 Std. Err. F 2 Significance Cholesterol 0.18 a 0.13 b 0.12 b 0.014 2.055 0.130 24-Methilene cholesterol 0.17 b 0.22 a 0.24 a 0.012 3.375 0.038 Campesterol 3.91 b 3.92 b 4.03 a 0.071 0.300 0.740 Campestanol 0.17 b 0.17 b 0.20 a 0.007 1.930 0.150 Stigmasterol 0.75 b 0.77 b 0.83 a 0.020 1.478 0.230 Δ7-Campesterol 0.22 a 0.08 b 0.11 b 0.016 7.445 0.001 Δ7-Stigmastenol 0.37 a 0.31 c 0.34 b 0.017 1.116 0.330 Apparent β-Sitosterol3 93.81 a 93.89 a 93.61 b 0.067 1.613 0.200 Δ5.23-Stigmastadienol 0.10 a 0.05 b 0.11 a 0.021 0.796 0.450 Clerosterol 0.91 b 0.97 a 1.00 a 0.019 2.146 0.120 β-sitosterol 87.00 a 84.99 b 84.58 b 0.217 14.880 0.000 Sitostanol 0.95 a 0.58 b 0.56 b 0.023 65.190 0.000 Δ5-Avenasterol 4.43 b 6.83 a 6.78 a 0.221 16.560 0.000 Δ5.24-Stigmastadienol 0.42 c 0.47 b 0.57 a 0.024 3.701 0.028 Δ7-Avenasterol 0.40 b 0.55 a 0.53 a 0.016 10.700 0.000 Erythrodiol + Uvaol 1.16 a 1.02 b 0.92 c 0.028 6.424 0.002 Total Sterols (in ppm) 1728.99 c 1915.09 a 1853.32 b 27.783 4.105 0.019

(1) Mean sample size = 36. Means followed by the same roman letter within each row do not present significant differences (Duncan's mutiple range test = 0.05). (2) F tests the effect of the maturity index. (3) Apparent β-Sitosterol = Δ5.23-stigmastadienol + clerosterol + β-sitosterol + sitostanol + Δ5-avenasterol + Δ5.24-stigmastadienol.

Table II - Sterol and Triterpene Dialcohol Concentrations1 (values as % total sterols) of oils processed from fruit of small, medium and large size within each variety

Small Medium Large Std. Err. F 2 Significance Cholesterol 0.12 a 0.13 a 0.14 a 0.012 0.130 0.880 24-Methilene cholesterol 0.17 c 0.22 b 0.28 a 0.013 6.519 0.002 Campesterol 4.12 a 3.85 b 3.85 b 0.081 1.228 0.300 Campestanol 0.25 a 0.14 b 0.18 b 0.013 7.878 0.001 Stigmasterol 0.62 b 0.86 a 0.78 a 0.026 7.764 0.001 Δ7-Campesterol 0.23 a 0.26 a 0.19 a 0.031 0.343 0.710 Δ7-Stigmastenol 0.34 b 0.36 b 0.39 a 0.011 2.147 0.120 Apparent β-Sitosterol3 93.66 a 93.83 a 93.48 a 0.087 1.326 0.270 Δ5.23-Stigmastadienol 0.03 a 0.02 a 0.03 a 0.004 1.454 0.240 Clerosterol 1.14 b 1.25 a 1.22 a 0.033 0.935 0.400 β-sitosterol 86.88 a 85.66 b 83.52 c 0.226 28.680 0.000 Sitostanol 0.86 a 0.65 b 0.51 c 0.018 68.650 0.000 Δ5-Avenasterol 4.25 c 5.83 b 7.74 a 0.205 43.360 0.000 Δ5.24-Stigmastadienol 0.60 a 0.57 a 0.55 a 0.031 0.241 0.790 Δ7-Avenasterol 0.51 b 0.47 b 0.73 a 0.023 14.480 0.000 Erythrodiol + Uvaol 1.19 a 1.12 b 0.89 c 0.031 10.330 0.000 Total Sterols (in ppm) 1998.16 a 2002.29 a 1947.13 b 23.947 0.544 0.580

(1) Mean sample size = 36. Means followed by the same roman letter within each row do not present significant differences (Duncan's mutiple range test = 0.05). (2) F tests the effect of the fruit size. (3) Apparent β-Sitosterol = Δ5.23-stigmastadienol + clerosterol + β-sitosterol + sitostanol + Δ5-avenasterol + Δ5.24-stigmastadienol.


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ANALYTICAL METHODOLOGY

The sterols analyses were conducted according to the official method IOC/T.20/N°10/Rev. 1 [11]. The sterol fraction was analysed by an Agilent Technology 6890N GC system, Agilent Technology 7683B series injector with a split inlet and flame ionisation detector managed by Agilent ChemStation. The analytical column was a DB-5 5% phenyl-methyl-siloxane stationary phase (30m × 0.25mm × 0.25 µm). The gas chromatographic conditions were as follows: Inlet temperature: 280°C; oven temperature 267°C; detector temperature: 290°C; split ratio: 30:1; amount injected 1: l. Hydrogen was used as gas carrier at a flow rate of 1.2 ml/min. Sterols were quantified using 5α-cholestan-3β-ol as internal standard.The data subjected to a statistical analysis was assessed through an analysis of variance using the SAS version 8.02 (SAS Institute Inc., Cary, NC, USA). Separation of the means was obtained using the least square means test and significant differences will be defined at P≤0.05. Every aspect was analysed separately. No interactions were evaluated in this project.

ADDITIONAL TRIALSIn addition to the previously detailed and initially planned processing and growing parameters, Modern Olives Laboratory Services conducted further studies associated with the sterol composition of the oil extracted from different tissues of the fruit (Exocarp or skin; mesocarp or flesh and endocarp or pit/seed) and the sterol characteristics of oils produced from pitted olives in comparison with normal whole olives.In the first case, fruit from the Barnea variety was

carefully peeled with a sharp scalpel removing the skin and external (<1mm) flesh layer and pitted. The skin, the crushed pit and the rest of the flesh components were weighed separately, dried at 100°C until constant weight and then treated with solvent utilising the Soxhlet method in order to obtain the oil present.In the case of the pitted olives, several large samples of Barnea fruit were pitted and processed through the Abencor® system in comparison with batches of entire fruit from the same variety. This trial was also conducted over two years.


EFFECT OF MATURATION INDEX ON STEROL COMPOSITIONThe evolution of sterols and triterpene dialcohols during maturation is presented in Table I. β-sitosterol, sitostanol, Δ7-campesterol, Δ5-avenasterol and Δ7-avenasterol are significantly (P<0.001) affected by maturity index. Among them sitostanol is the one most affected (F value of 65.2). β-sitosterol, sitostanol and Δ7-campesterol decrease during ripening, while Δ5-avenasterol and Δ7-avenasterol significantly increase. This result agrees with other research [2].

EFFECT OF FRUIT SIZE ON STEROL COMPOSITIONCampestanol, stigmasterol, β-sitosterol, sitostanol, Δ5-avenasterol, Δ7-avenasterol and erythrodiol and uvaol are significantly affected by fruit size. While campestanol, β-sitosterol, sitostanol and erythro-diol + uvaol significantly decrease with fruit size, Δ5-avenasterol, sitosterol and Δ7-avenasterol increase (Tab. II). Erythrodiol and Uvaol are components mainly

Table III - Sterol and Triterpene Dialcohol Concentrations1 (values as % total sterols) of oils processed from fruit of receiving three different irrigation regimes: ½ X, X and 2 X ½ X X 2 X Std. Err. F 2 Significance Cholesterol 0.22 a 0.12 c 0.17 b 0.018 2.636 0.076 24-Methilene cholesterol 0.40 a 0.27 b 0.26 b 0.015 9.700 0.000 Campesterol 4.03 a 3.95 a 3.83 b 0.066 0.844 0.430 Campestanol 0.31 a 0.25 b 0.23 b 0.012 4.364 0.019 Stigmasterol 0.92 a 0.80 b 0.75 b 0.020 7.102 0.001 Δ7-Campesterol 0.15 b 0.24 a 0.16 b 0.018 2.810 0.065 Δ7-Stigmastenol 0. 54 a 0.51 a 0.41 b 0.009 24.350 0.000 Apparent β-Sitosterol3 92.85 c 93.42 b 93.73 a 0.079 13.200 0.000 Δ5.23-Stigmastadienol 0.16 a 0.08 b 0.09 b 0.018 2.084 0.130 Clerosterol 1.58 a 1.12 b 1.10 b 0.062 7.361 0.001 β-sitosterol 83.96 c 85.29 a 84.69 b 0.203 3.737 0.027 Sitostanol 0.73 a 0.66 b 0.72 a 0.018 1.543 0.220 Δ5-Avenasterol 5.81 b 5.79 b 6. 59 a 0.177 2.284 0.110 Δ5.24-Stigmastadienol 0.59 a 0.49 b 0.55 a 0.023 1.927 0.150 Δ7-Avenasterol 0.60 a 0. 48 b 0.49 b 0.013 10.530 0.000 Erythrodiol + Uvaol 0.93 a 0.99 a 0.93 a 0.033 0.358 0.700 Total Sterols (in ppm) 1933.71 a 1851.10 b 1992.07 a 29.511 1.954 0.150

(1) Mean sample size = 36. Means followed by the same roman letter within each row do not present significant differences (Duncan's mutiple range test = 0.05). (2) F tests the effect of the irrigation regime during oil accumulation. (3) Apparent β-Sitosterol = Δ5.23-stigmastadienol + clerosterol + β-sitosterol + sitostanol + Δ5-avenasterol + Δ5.24-stigmastadienol. Table IV - Sterol and Triterpene Dialcohol Concentrations1 (values as % total sterols) of oils processed at malaxing times of 15, 30 and 60 minutes 15 min 30 min 60 min Std. Err. F 2 Significance Cholesterol 0.15 a 0.14 a 0.13 a 0.015 0.121 0.890 24-Methilene cholesterol 0.26 a 0.25 a 0.22 b 0.007 2.471 0.089 Campesterol 4.00 a 3.99 a 3.85 b 0.062 0.583 0.560 Campestanol 0.23 a 0.20 b 0.19 b 0.011 1.658 0.200 Stigmasterol 0.89 c 0.97 b 1.07 a 0.028 3.490 0.034 Δ7-Campesterol 0.13 a 0.13 a 0.09 b 0.012 1.082 0.340 Δ7-Stigmastenol 0.30 b 0.32 b 0.41 a 0.017 4.198 0.018 Apparent β-Sitosterol3 93.58 a 93.55 a 93.60 a 0.057 0.008 0.920 Δ5.23-Stigmastadienol 0.17 a 0.14 b 0.13 b 0.023 0.239 0.820 Clerosterol 0.82 b 0.85 a 0.86 a 0.020 0.317 0.730 β-sitosterol 85.23 a 84.84 b 84.67 b 0.206 0.633 0.530 Sitostanol 0.62 a 0. 63 a 0.63 a 0.011 0.004 0.970 Δ5-Avenasterol 6.29 b 6.62 a 6.79 a 0.203 0.522 0.590 Δ5.24-Stigmastadienol 0.45 b 0.47 b 0.52 a 0.029 0.406 0.670 Δ7-Avenasterol 0.49 a 0.46 b 0.48 a 0.014 0.506 0.600 Erythrodiol + Uvaol 0.81 c 1.05 b 1.17 a 0.031 14.810 0.000 Total Sterols (in ppm) 1710.33 b 1813.10 a 1794.17 a 24.160 1.732 0.180

(1) Mean sample size = 36. Means followed by the same roman letter within each row do not present significant differences (Duncan's mutiple range test = 0.05). (2) F tests the effect of the malaxing time. (3) Apparent β-Sitosterol = Δ5.23-stigmastadienol + clerosterol + β-sitosterol + sitostanol + Δ5-avenasterol + Δ5.24-stigmastadienol.


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located in the skin of the olives, which explains the smaller amount of them in large size fruits.

EFFECT OF IRRIGATION ON STEROL COMPOSITIONThe analysis of the effect of irrigation on sterol and triterpene dialcohols concentrations is presented in Table III. 24-methilene cholesterol, stigmasterol, Δ7-stigmastenol, apparent β-sitosterol, clerosterol and Δ7-avenasterol are amongst the significantly affected compounds. It is noteworthy that while stigmasterol and Δ7-stigmastenol decrease with higher levels of ir-rigation, apparent β-sitosterol significantly increases.

EFFECT OF MALAXING TIME ON STEROL COMPOSITION

The malaxing time at the paste preparation stage is a very important parameter of good manufacturing practice. As indicated in Table IV, erythrodiol + uvaol were the only components to be significantly affected (P<0.001) by malaxing time. Stigmasterol and Δ7-stigmastenol also show to be affected but to lesser extent (P<0.04). It was expected that these compo-nents increase with more malaxing time, considering that they are skin components.

Table III - Sterol and Triterpene Dialcohol Concentrations1 (values as % total sterols) of oils processed from fruit of receiving three different irrigation regimes: ½ X, X and 2 X ½ X X 2 X Std. Err. F 2 Significance Cholesterol 0.22 a 0.12 c 0.17 b 0.018 2.636 0.076 24-Methilene cholesterol 0.40 a 0.27 b 0.26 b 0.015 9.700 0.000 Campesterol 4.03 a 3.95 a 3.83 b 0.066 0.844 0.430 Campestanol 0.31 a 0.25 b 0.23 b 0.012 4.364 0.019 Stigmasterol 0.92 a 0.80 b 0.75 b 0.020 7.102 0.001 Δ7-Campesterol 0.15 b 0.24 a 0.16 b 0.018 2.810 0.065 Δ7-Stigmastenol 0. 54 a 0.51 a 0.41 b 0.009 24.350 0.000 Apparent β-Sitosterol3 92.85 c 93.42 b 93.73 a 0.079 13.200 0.000 Δ5.23-Stigmastadienol 0.16 a 0.08 b 0.09 b 0.018 2.084 0.130 Clerosterol 1.58 a 1.12 b 1.10 b 0.062 7.361 0.001 β-sitosterol 83.96 c 85.29 a 84.69 b 0.203 3.737 0.027 Sitostanol 0.73 a 0.66 b 0.72 a 0.018 1.543 0.220 Δ5-Avenasterol 5.81 b 5.79 b 6. 59 a 0.177 2.284 0.110 Δ5.24-Stigmastadienol 0.59 a 0.49 b 0.55 a 0.023 1.927 0.150 Δ7-Avenasterol 0.60 a 0. 48 b 0.49 b 0.013 10.530 0.000 Erythrodiol + Uvaol 0.93 a 0.99 a 0.93 a 0.033 0.358 0.700 Total Sterols (in ppm) 1933.71 a 1851.10 b 1992.07 a 29.511 1.954 0.150

(1) Mean sample size = 36. Means followed by the same roman letter within each row do not present significant differences (Duncan's mutiple range test = 0.05). (2) F tests the effect of the irrigation regime during oil accumulation. (3) Apparent β-Sitosterol = Δ5.23-stigmastadienol + clerosterol + β-sitosterol + sitostanol + Δ5-avenasterol + Δ5.24-stigmastadienol. Table IV - Sterol and Triterpene Dialcohol Concentrations1 (values as % total sterols) of oils processed at malaxing times of 15, 30 and 60 minutes 15 min 30 min 60 min Std. Err. F 2 Significance Cholesterol 0.15 a 0.14 a 0.13 a 0.015 0.121 0.890 24-Methilene cholesterol 0.26 a 0.25 a 0.22 b 0.007 2.471 0.089 Campesterol 4.00 a 3.99 a 3.85 b 0.062 0.583 0.560 Campestanol 0.23 a 0.20 b 0.19 b 0.011 1.658 0.200 Stigmasterol 0.89 c 0.97 b 1.07 a 0.028 3.490 0.034 Δ7-Campesterol 0.13 a 0.13 a 0.09 b 0.012 1.082 0.340 Δ7-Stigmastenol 0.30 b 0.32 b 0.41 a 0.017 4.198 0.018 Apparent β-Sitosterol3 93.58 a 93.55 a 93.60 a 0.057 0.008 0.920 Δ5.23-Stigmastadienol 0.17 a 0.14 b 0.13 b 0.023 0.239 0.820 Clerosterol 0.82 b 0.85 a 0.86 a 0.020 0.317 0.730 β-sitosterol 85.23 a 84.84 b 84.67 b 0.206 0.633 0.530 Sitostanol 0.62 a 0. 63 a 0.63 a 0.011 0.004 0.970 Δ5-Avenasterol 6.29 b 6.62 a 6.79 a 0.203 0.522 0.590 Δ5.24-Stigmastadienol 0.45 b 0.47 b 0.52 a 0.029 0.406 0.670 Δ7-Avenasterol 0.49 a 0.46 b 0.48 a 0.014 0.506 0.600 Erythrodiol + Uvaol 0.81 c 1.05 b 1.17 a 0.031 14.810 0.000 Total Sterols (in ppm) 1710.33 b 1813.10 a 1794.17 a 24.160 1.732 0.180

(1) Mean sample size = 36. Means followed by the same roman letter within each row do not present significant differences (Duncan's mutiple range test = 0.05). (2) F tests the effect of the malaxing time. (3) Apparent β-Sitosterol = Δ5.23-stigmastadienol + clerosterol + β-sitosterol + sitostanol + Δ5-avenasterol + Δ5.24-stigmastadienol.

Table V - Sterol and Triterpene Dialcohol Concentrations1 (values as % total sterols) of oils processed at temperatures of 18°C, 28°C and 38°C 18ºC 28ºC 38ºC Std. Err. F 2 Significance Cholesterol 0.17 a 0.21 a 0.21 a 0.015 0.764 0.470 24-Methilene cholesterol 0.26 a 0.26 a 0.25 a 0.008 0.208 0.810 Campesterol 3.95 a 3.98 a 3.93 a 0.064 0.004 0.960 Campestanol 0.21 a 0.22 b 0.21 a 0.008 0.213 0.810 Stigmasterol 0.89 b 0.95 b 1.12 a 0.035 4.439 0.014 Δ7-Campesterol 0.13 b 0.09 b 0.18 a 0.013 3.798 0.026 Δ7-Stigmastenol 0.34 b 0.39 b 0.38 b 0.012 1.526 0.220 Apparent β-Sitosterol3 93.59 a 93.35 a 93.22 a 0.066 2.706 0.071 Δ5.23-Stigmastadienol 0.23 a 0.24 a 0.28 a 0.034 0.174 0.840 Clerosterol 0.98 a 0.99 a 0.99 a 0.019 0.003 0.970 β-sitosterol 84.21 a 84.05 a 84.33 a 0.191 0.179 0.840 Sitostanol 0.66 a 0.69 a 0.68 a 0.012 0.658 0.520 Δ5-Avenasterol 6.96 a 6.85 a 6.45 a 0.190 0.657 0.520 Δ5.24-Stigmastadienol 0.57 a 0.53 a 0.49 a 0.023 1.047 0.350 Δ7-Avenasterol 0.53 b 0.62 a 0.55 b 0.019 2.044 0.130 Erythrodiol + Uvaol 0.86 c 1.01 b 1.25 a 0.034 13.980 0.000 Total Sterols (in ppm) 1669.97 c 1806.86 b 1924.26 a 25.487 9.656 0.000

(1) Mean sample size = 36. Means followed by the same roman letter within each row do not present significant differences (Duncan's mutiple range test α = 0.05). (2) F tests the effect of the malaxing temperature. (3) Apparent β-Sitosterol = Δ5.23-stigmastadienol + clerosterol + β-sitosterol + sitostanol + Δ5-avenasterol + Δ5.24-stigmastadienol. Table VI - Sterol and Triterpene Dialcohol Concentrations1 (values as % total sterols) of oils extracted from fruit within 12 hours of harvesting, 48 hours from harvesting and 120 hours from harvesting

<12hs 48hs 120hs Std. Err. F 2 Significance

Cholesterol 0.08 a 0.08 a 0.06 b 0.009 0.815 0.450 24-Methilene cholesterol 0.25 a 0.25 a 0.23 a 0.007 0.435 0.650 Campesterol 3.90 a 3.88 a 3.90 a 0.063 0.001 0.990 Campestanol 0.21 a 0.17 b 0.15 b 0.006 9.638 0.000 Stigmasterol 0.98 b 1.07 b 1.31 a 0.037 8.241 0.001 Δ7-Campesterol 0.07 b 0.07 b 0.14 a 0.011 5.816 0.004 Δ7-Stigmastenol 0.37 a 0.33 b 0.31 b 0.011 2.568 0.082 Apparent β-Sitosterol3 93.56 a 93.63 a 93.42 b 0.045 1.934 0.150 Δ5.23-Stigmastadienol 0.21 a 0.25 a 0.12 b 0.025 2.517 0.086 Clerosterol 0.98 a 0.95 b 0.94 b 0.011 1.084 0.340 β-sitosterol 84.25 a 84.66 a 84.80 a 0.220 0.552 0.580 Sitostanol 0.65 a 0.62 b 0. 61 b 0.011 1.018 0.360 Δ5-Avenasterol 6.90 a 6.62 b 6.50 b 0.215 0.303 0.740 Δ5.24-Stigmastadienol 0.58 a 0.52 b 0.47 b 0.026 1.218 0.300 Δ7-Avenasterol 0.58 a 0.54 a 0.48 b 0.017 2.630 0.077 Erythrodiol + Uvaol 1.00 a 1.11 b 1.39 c 0.032 15.670 0.000 Total Sterols (in ppm) 1817.20 a 1802.15 a 1776.41 a 18.0228 0.432 0.650

(1) Mean sample size = 36. Means followed by the same roman letter within each row do not present significant differences (Duncan's mutiple range test = 0.05). (2) F tests the effect of the delay between harvesting and processing. (3) Apparent β-Sitosterol = Δ5.23-stigmastadienol + clerosterol + β-sitosterol + sitostanol + Δ5-avenasterol + Δ5.24-stigmastadienol.


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EFFECT OF MALAXING TEMPERATURE ON STEROL COMPOSITIONSimilarly to malaxing time, processing temperature is another important parameter during the olive oil manufacturing process. Correspondingly, erythrodiol + uvaol were significantly affected (P<0.001) by ma-laxing temperature and stigmasterol was one of the few sterols being affected (P:0.014) (Tab. V). Similar to malaxing time, it was expected that the higher the malaxing temperature, the higher would be the ex-traction of components from the skin/outer layer of flesh. This is in agreement with other research work

[2]. Additionally, the total level of sterols was signi-ficantly affected (P<0.001) by this processing para-meter showing increasing values at higher malaxing temperatures.

EFFECT OF DELAY BETWEEN HARVEST AND PROCESS ON STEROL COMPOSITIONSimilar to the other processing parameters evaluated, the delay between harvest and processing significan-tly affected the percentage of erythrodiol + uvaol and campestanol (P<0.001) (Tab. VI). Erythrodiol + uvaol level increased with longer days between harvesting

Table V - Sterol and Triterpene Dialcohol Concentrations1 (values as % total sterols) of oils processed at temperatures of 18°C, 28°C and 38°C 18ºC 28ºC 38ºC Std. Err. F 2 Significance Cholesterol 0.17 a 0.21 a 0.21 a 0.015 0.764 0.470 24-Methilene cholesterol 0.26 a 0.26 a 0.25 a 0.008 0.208 0.810 Campesterol 3.95 a 3.98 a 3.93 a 0.064 0.004 0.960 Campestanol 0.21 a 0.22 b 0.21 a 0.008 0.213 0.810 Stigmasterol 0.89 b 0.95 b 1.12 a 0.035 4.439 0.014 Δ7-Campesterol 0.13 b 0.09 b 0.18 a 0.013 3.798 0.026 Δ7-Stigmastenol 0.34 b 0.39 b 0.38 b 0.012 1.526 0.220 Apparent β-Sitosterol3 93.59 a 93.35 a 93.22 a 0.066 2.706 0.071 Δ5.23-Stigmastadienol 0.23 a 0.24 a 0.28 a 0.034 0.174 0.840 Clerosterol 0.98 a 0.99 a 0.99 a 0.019 0.003 0.970 β-sitosterol 84.21 a 84.05 a 84.33 a 0.191 0.179 0.840 Sitostanol 0.66 a 0.69 a 0.68 a 0.012 0.658 0.520 Δ5-Avenasterol 6.96 a 6.85 a 6.45 a 0.190 0.657 0.520 Δ5.24-Stigmastadienol 0.57 a 0.53 a 0.49 a 0.023 1.047 0.350 Δ7-Avenasterol 0.53 b 0.62 a 0.55 b 0.019 2.044 0.130 Erythrodiol + Uvaol 0.86 c 1.01 b 1.25 a 0.034 13.980 0.000 Total Sterols (in ppm) 1669.97 c 1806.86 b 1924.26 a 25.487 9.656 0.000

(1) Mean sample size = 36. Means followed by the same roman letter within each row do not present significant differences (Duncan's mutiple range test α = 0.05). (2) F tests the effect of the malaxing temperature. (3) Apparent β-Sitosterol = Δ5.23-stigmastadienol + clerosterol + β-sitosterol + sitostanol + Δ5-avenasterol + Δ5.24-stigmastadienol. Table VI - Sterol and Triterpene Dialcohol Concentrations1 (values as % total sterols) of oils extracted from fruit within 12 hours of harvesting, 48 hours from harvesting and 120 hours from harvesting

<12hs 48hs 120hs Std. Err. F 2 Significance

Cholesterol 0.08 a 0.08 a 0.06 b 0.009 0.815 0.450 24-Methilene cholesterol 0.25 a 0.25 a 0.23 a 0.007 0.435 0.650 Campesterol 3.90 a 3.88 a 3.90 a 0.063 0.001 0.990 Campestanol 0.21 a 0.17 b 0.15 b 0.006 9.638 0.000 Stigmasterol 0.98 b 1.07 b 1.31 a 0.037 8.241 0.001 Δ7-Campesterol 0.07 b 0.07 b 0.14 a 0.011 5.816 0.004 Δ7-Stigmastenol 0.37 a 0.33 b 0.31 b 0.011 2.568 0.082 Apparent β-Sitosterol3 93.56 a 93.63 a 93.42 b 0.045 1.934 0.150 Δ5.23-Stigmastadienol 0.21 a 0.25 a 0.12 b 0.025 2.517 0.086 Clerosterol 0.98 a 0.95 b 0.94 b 0.011 1.084 0.340 β-sitosterol 84.25 a 84.66 a 84.80 a 0.220 0.552 0.580 Sitostanol 0.65 a 0.62 b 0. 61 b 0.011 1.018 0.360 Δ5-Avenasterol 6.90 a 6.62 b 6.50 b 0.215 0.303 0.740 Δ5.24-Stigmastadienol 0.58 a 0.52 b 0.47 b 0.026 1.218 0.300 Δ7-Avenasterol 0.58 a 0.54 a 0.48 b 0.017 2.630 0.077 Erythrodiol + Uvaol 1.00 a 1.11 b 1.39 c 0.032 15.670 0.000 Total Sterols (in ppm) 1817.20 a 1802.15 a 1776.41 a 18.0228 0.432 0.650

(1) Mean sample size = 36. Means followed by the same roman letter within each row do not present significant differences (Duncan's mutiple range test = 0.05). (2) F tests the effect of the delay between harvesting and processing. (3) Apparent β-Sitosterol = Δ5.23-stigmastadienol + clerosterol + β-sitosterol + sitostanol + Δ5-avenasterol + Δ5.24-stigmastadienol.

Table VII - Sterol and Triterpene Dialcohol Concentrations1 (values as % total sterols) of oils processed from fruit in two different years

2007 2008 Std. Err. F 2 Significance

Cholesterol 0.21 a 0.07 b 0.006 126.500 0.000 24-Methilene cholesterol 0.23 b 0.25 a 0.004 2.974 0.085 Campesterol 3.98 a 3.90 a 0.027 1.617 0.200 Campestanol 0.22 a 0.16 b 0.003 57.680 0.000 Stigmasterol 1.09 a 0.84 b 0.013 72.520 0.000 Δ7-Campesterol 0.16 a 0.08 b 0.005 43.330 0.000 Δ7-Stigmastenol 0.29 b 0.41 a 0.006 91.050 0.000 Apparent β-Sitosterol3 93.37 a 93.76 b 0.025 45.300 0.000 Δ5.23-Stigmastadienol 0.33 a 0.01 b 0.011 223.800 0.000 Clerosterol 0.93 b 0.95 a 0.007 0.961 0.330 β-sitosterol 85.16 a 84.44 a 0.087 11.460 0.001 Sitostanol 0.64 b 0.67 a 0.006 2.838 0.093 Δ5-Avenasterol 5.94 b 7.06 a 0.085 30.970 0.000 Δ5.24-Stigmastadienol 0.38 b 0.63 a 0.010 120.100 0.000 Δ7-Avenasterol 0.49 b 0.54 a 0.007 8.120 0.005 Erythrodiol + Uvaol 0.95 a 1.18 a 0.013 62.640 0.000 Total Sterols (in ppm) 1767.92 b 1834.06 a 9.854 7.625 0.006

(1) Mean sample size = 216. Means followed by the same roman letter within each row do not present significant differences (Duncan's mutiple range test = 0.05). (2) F tests the effect of the year. (3) Apparent β-Sitosterol = Δ5.23-stigmastadienol + clerosterol + β-sitosterol + sitostanol + Δ5-avenasterol + Δ5.24-stigmastadienol. Table VIII - Sterol and Triterpene Dialcohol Concentrations1 (values as % total sterols) of oils processed from fruit of three different varieties: Frantoio, Picual and Barnea

Frantoio Picual Barnea Std. Err. F 2 Significance

Cholesterol 0.16 a 0.14 b 0.13 b 0.006 1.980 0.140 24-Methilene cholesterol 0.27 b 0.29 a 0.18 c 0.005 67.540 0.000 Campesterol 3.39 c 3.53 b 4.88 a 0.028 3125.000 0.000 Campestanol 0.23 a 0.21 b 0.17 c 0.004 16.760 0.000 Stigmasterol 0.92 b 1.07 a 0.73 c 0.013 76.050 0.000 Δ7-Campesterol 0.12 b 0.16 a 0.16 a 0.008 2.978 0.052 Δ7-Stigmastenol 0.40 a 0.39 a 0.34 b 0.006 9.235 0.000 Apparent β-Sitosterol3 93.87 a 93.80 a 92.96 b 0.028 157.700 0.000 Δ5.23-Stigmastadienol 0.21 a 0.15 b 0.05 c 0.010 26.870 0.000 Clerosterol 1.08 a 1.11 a 0.92 b 0.014 18.050 0.000 β-sitosterol 82.57 b 86.09 a 85.94 a 0.088 368.000 0.000 Sitostanol 0.69 a 0.62 b 0.70 a 0.007 16.120 0.000 Δ5-Avenasterol 8.61 a 5.47 b 4.93 c 0.083 451.000 0.000 Δ5.24-Stigmastadienol 0.75 a 0.39 b 0.42 b 0.011 174.600 0.000 Δ7-Avenasterol 0.66 a 0.44 c 0.48 b 0.007 127.100 0.000 Erythrodiol + Uvaol 1.08 a 1.01 b 1.05 a 0.013 2.037 0.130 Total Sterols (in ppm) 1855.44 b 1731.69 c 1968.92 a 10.629 47.510 0.000

(1) Mean sample size = 216. Means followed by the same roman letter within each row do not present significant differences (Duncan's mutiple range test = 0.05). (2) F tests the effect of the variety. (3) Apparent β-Sitosterol = Δ5.23-stigmastadienol + clerosterol + β-sitosterol + sitostanol + Δ5-avenasterol + Δ5.24-stigmastadienol.


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and processing. Stigmasterol was also affected by the delay between harvest and processing (P=0.001) increasing accordingly.

EFFECT OF THE YEAR ON STEROL COMPOSITIONThe variations of sterols and triterpene dialcohols between the two years are presented in Table VII. Most of these compounds are significantly affected by the season.

EFFECT OF THE CULTIVAR ON STEROL COMPOSITIONThe effect of the cultivar on sterol and triterpene dial-cohols composition is presented in Table VIII. It is im-portant to point out that the cultivar has shown the most significant level of effect on the different sterols. This is in line with other authors [9, 10, 17]. Only cho-lesterol and Δ7-campesterol had levels of significan-ce higher than 0.001. Campesterol, β-sitosterol and

Δ7-avenasterol were the most affected with F values of 3125, 368 and 451 respectively. Erythrodiol + uva-ol were not significantly affected by cultivar. This also is in agreement with the statements above where we relate the skin components with processing practices instead of genetics factors. This is not in agreement with other research work [3].

STEROL COMPOSITION IN THE DIFFERENT FRUIT TISSUESAs it is indicated in Table IX, the vast majority of the oil (>75%) comes from the flesh with similar proportions of the remaining oil being contributed by the pit/seed and by the skin/outer layer of flesh. Significant diffe-rences were observed regarding certain sterols and associated substances. Stigmasterol and total sterols were significantly higher in the oil produced from the pit/seed fraction, while Δ7-Stigmastenol and Erythro-

Table VII - Sterol and Triterpene Dialcohol Concentrations1 (values as % total sterols) of oils processed from fruit in two different years

2007 2008 Std. Err. F 2 Significance

Cholesterol 0.21 a 0.07 b 0.006 126.500 0.000 24-Methilene cholesterol 0.23 b 0.25 a 0.004 2.974 0.085 Campesterol 3.98 a 3.90 a 0.027 1.617 0.200 Campestanol 0.22 a 0.16 b 0.003 57.680 0.000 Stigmasterol 1.09 a 0.84 b 0.013 72.520 0.000 Δ7-Campesterol 0.16 a 0.08 b 0.005 43.330 0.000 Δ7-Stigmastenol 0.29 b 0.41 a 0.006 91.050 0.000 Apparent β-Sitosterol3 93.37 a 93.76 b 0.025 45.300 0.000 Δ5.23-Stigmastadienol 0.33 a 0.01 b 0.011 223.800 0.000 Clerosterol 0.93 b 0.95 a 0.007 0.961 0.330 β-sitosterol 85.16 a 84.44 a 0.087 11.460 0.001 Sitostanol 0.64 b 0.67 a 0.006 2.838 0.093 Δ5-Avenasterol 5.94 b 7.06 a 0.085 30.970 0.000 Δ5.24-Stigmastadienol 0.38 b 0.63 a 0.010 120.100 0.000 Δ7-Avenasterol 0.49 b 0.54 a 0.007 8.120 0.005 Erythrodiol + Uvaol 0.95 a 1.18 a 0.013 62.640 0.000 Total Sterols (in ppm) 1767.92 b 1834.06 a 9.854 7.625 0.006

(1) Mean sample size = 216. Means followed by the same roman letter within each row do not present significant differences (Duncan's mutiple range test = 0.05). (2) F tests the effect of the year. (3) Apparent β-Sitosterol = Δ5.23-stigmastadienol + clerosterol + β-sitosterol + sitostanol + Δ5-avenasterol + Δ5.24-stigmastadienol. Table VIII - Sterol and Triterpene Dialcohol Concentrations1 (values as % total sterols) of oils processed from fruit of three different varieties: Frantoio, Picual and Barnea

Frantoio Picual Barnea Std. Err. F 2 Significance

Cholesterol 0.16 a 0.14 b 0.13 b 0.006 1.980 0.140 24-Methilene cholesterol 0.27 b 0.29 a 0.18 c 0.005 67.540 0.000 Campesterol 3.39 c 3.53 b 4.88 a 0.028 3125.000 0.000 Campestanol 0.23 a 0.21 b 0.17 c 0.004 16.760 0.000 Stigmasterol 0.92 b 1.07 a 0.73 c 0.013 76.050 0.000 Δ7-Campesterol 0.12 b 0.16 a 0.16 a 0.008 2.978 0.052 Δ7-Stigmastenol 0.40 a 0.39 a 0.34 b 0.006 9.235 0.000 Apparent β-Sitosterol3 93.87 a 93.80 a 92.96 b 0.028 157.700 0.000 Δ5.23-Stigmastadienol 0.21 a 0.15 b 0.05 c 0.010 26.870 0.000 Clerosterol 1.08 a 1.11 a 0.92 b 0.014 18.050 0.000 β-sitosterol 82.57 b 86.09 a 85.94 a 0.088 368.000 0.000 Sitostanol 0.69 a 0.62 b 0.70 a 0.007 16.120 0.000 Δ5-Avenasterol 8.61 a 5.47 b 4.93 c 0.083 451.000 0.000 Δ5.24-Stigmastadienol 0.75 a 0.39 b 0.42 b 0.011 174.600 0.000 Δ7-Avenasterol 0.66 a 0.44 c 0.48 b 0.007 127.100 0.000 Erythrodiol + Uvaol 1.08 a 1.01 b 1.05 a 0.013 2.037 0.130 Total Sterols (in ppm) 1855.44 b 1731.69 c 1968.92 a 10.629 47.510 0.000

(1) Mean sample size = 216. Means followed by the same roman letter within each row do not present significant differences (Duncan's mutiple range test = 0.05). (2) F tests the effect of the variety. (3) Apparent β-Sitosterol = Δ5.23-stigmastadienol + clerosterol + β-sitosterol + sitostanol + Δ5-avenasterol + Δ5.24-stigmastadienol. Table IX - Fruit Composition (oil distribution)

Table X - Sterols profile of the oil obtained from different part of the fruit

Cholesterol

(%) Campesterol

(%) Stigmasterol

(%)

Δ 7 Stigmastenol

(%)

β Sitosterol (%)

Δ 5 Avenasterol

(%)

E+U (%)

Total Sterols (ppm)

Skin 0.000 4.820 0.990 0.900 89.440 1.580 9.180 1.842.4 Flesh 0.000 5.140 0.740 0.160 89.770 1.750 0.630 2.596.5 Pit/Seed 0.000 4.680 1.210 0.360 88.290 1.530 0.440 4.991.0 Total 0.000 5.020 0.856 0.261 89.441 1.691 1.286 3024.450

Weight (% of total)

Oil (% fresh content)

Oil (% of total)

Oil (% of origin)

Skin 7.7% 33.5% 2.6% 12.3% Flesh 71.0% 22.6% 16.1% 76.4% Pit/Seed 21.3% 11.2% 2.4% 11.4% Total 100.0% 21.0% 100.0%

Table IX - Fruit Composition (oil distribution)

Table X - Sterols profile of the oil obtained from different part of the fruit

Cholesterol

(%) Campesterol

(%) Stigmasterol

(%)

Δ 7 Stigmastenol

(%)

β Sitosterol (%)

Δ 5 Avenasterol

(%)

E+U (%)

Total Sterols (ppm)

Skin 0.000 4.820 0.990 0.900 89.440 1.580 9.180 1.842.4 Flesh 0.000 5.140 0.740 0.160 89.770 1.750 0.630 2.596.5 Pit/Seed 0.000 4.680 1.210 0.360 88.290 1.530 0.440 4.991.0 Total 0.000 5.020 0.856 0.261 89.441 1.691 1.286 3024.450

Weight (% of total)

Oil (% fresh content)

Oil (% of total)

Oil (% of origin)

Skin 7.7% 33.5% 2.6% 12.3% Flesh 71.0% 22.6% 16.1% 76.4% Pit/Seed 21.3% 11.2% 2.4% 11.4% Total 100.0% 21.0% 100.0%


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diol + Uvaol were particularly higher in the skin/outer flesh fraction, as shown in (Tab. X). This difference can explain why the levels of those sterols tend to incre-ase in the final oil produced when processing con-ditions deteriorate, particularly associated to higher malaxing times, temperatures or time delays betwe-en harvesting and crushing. The relatively constant proportion of Campesterol and β-Sitosterol would confirm that those two sterols could not be used as quality indicators and there is relatively little influenced by processing conditions.

STEROL COMPOSITION FROM PITTED VERSUS ENTIRE FRUITNo statistically significant differences were observed in any of the sterols between the oils produced by crushing the entire fruit (conventional method) versus crushing the pitted olives. While this commercial pro-duction technique may have an impact on other oil chemical parameters, it failed to produce significant changes in the sterol composition or in the total sterol levels of the final oils as shown in Table X

CONCLUSION

According to the variables studied in this research, it has been observed that (i) the main components of the sterol fraction, β-sitosterol, Δ5-avenasterol and campesterol [16], are not affected by irrigation, they are only affected by fruit size and maturity index, (ii) total sterols are not affected by agronomical factors, only are affected by malaxing temperature; which was expected due the higher extraction of oil; (iii) Eri-throdiol + uvaol are affected by fruit size, malaxing time, malaxing temperature and delay in time from harvest to processing, those results were expected, due that the triterpene dialchohols are located in the skin of the olives, as shown also in this research when analysing the sterols composition in the different fruit tissue. Also was found that erythrodiol and uvaol are not affected by the cultivar.

Acknowledgments

Special thanks to Rural Industries Research and De-velopment Corporation for their financial support of this project as well as to the Australian Olive Associa-tion and Boundary Bend Limited.

REFERENCES

A. Ranalli, F. Angerosa, Integral Centrifuges [1] for Olive Oil Extraction. The Qualitative Characteristics of Products. JAOCS 73, N° 4, 417-422, (1996).A. Koutsaftakis, et al. Effect of Extraction System, [2] Stage of Ripeness, and Kneading Temperature on the Sterol Composition of Virgin Olive Oils. JAOCS 76 (12), 1477-1481, (1999).

R. Aparicio, G. Luna, Characterisation of [3] monovarietal virgin olive oils. Eur. J. Lipid Sci. Technol. 104, 614-627, (2002). A. Ranalli, et al., Sterol and alcohol components [4] of seed, pulp and whole olive fruit oils. Their use to characterise olive fruit variety by multivariates. J. Sci. Food Agric. 82, 854-859, (2002).D. Boskou, et al., Olive Oil: Chemistry and [5] Technology. AOCS Monograph Series on Oilseeds. Illinois, USA (2006). R. Mailer, The Natural Chemistry of Australian [6] Extra Virgin Olive Oil. Rural Industries Research and Development Corporation. Canberra, Australia (2007) L. Ravetti, Horticultural Review of Australian [7] Olive Industry. Australian Olive Association Field Days. Canberra, Australia (2006).Abencor Olive Analysing System: Hammer Mill, [8] Themo-Mixer and Centrifuge MC2 Ingenieria y Sistemas, S.L.V. Paganuzzi, Utilidad de la determinación [9] de esteroles y eritrodioles para individualizar el origen de los aceites de oliva vírgenes por métodos quimiométricos. Olivae 16, 19-22, (1987). R. Aparicio, M. Morales, V. Alonso, Authentication [10] of European virgin olive oils by their chemical compounds, sensory attributes and consumers’ attitudes. J. Agric. Food Chem. 45, 1076-1083, (1997).International Olive Council. Trade standard [11] applying to olive oils and olive pomace oils. Madrid, Spain. Available online at: http://www.internationaloliveoil.org/. (2006).G. Assmann, U. Wahrburg, Health effects of the [12] minor components of olive oil, http://www.food-info.net/uk/products/olive/olive06.htm (2006)D. Boskou, et.al., Olive Oil: Minor constituents [13] and health. CRC Press Taylor & Francis Group. Chapter 4, 51-52, (2009).R. Rodriguez-Rodriguez, M.D. Herrera, J.S. Pe-[14] rona, V. Ruiz-Gutierrez, Potential vasorelaxant effects of oleanolic and erythrodiol, two triterpenoids contained in orujo olive oil, on rat aorta, Br. J. Nutr. 92, 635-642, (2004).J. Quilez, P. Garcia-Lorda, J. Salas-Salvado, [15] Potential uses and benefits of phytosterols in diet: present situation and future directions, Clin. Nutr. 22, 343-351, (2003). A. Kornfeld, 4-demethyl, 4-monomethyl and [16] 4,4-dimethylsteorls in some vegetable oils. Lipids 16, 306-314, (1981).R. Rivera del Alamo, G. Fregapane, F. Aranda, et [17] al., Sterol composition of cornicabra virgin olive oil: the campesterol content exceeds the upper limit of 4% established by EU Regulations. Food Chem. 84, 533-537, (2004).

Received September 13, 2012Accepted January 31, 2014


61

M.M. Özcana*

L. Altınözb

D. Arslana

A. Ünvera

aSelçuk UniversityFaculty of Agriculture

Department of Food EngineeringKonya, Turkey

bFood Engineering Şifa Olive oils and Cosmetic

Industry Commercial Ltd, Şti, Altınözü/Hatay, Turkey

(*) CORRESPONDING AUTHOR Dr. M. M. Özcan

E-mail: [email protected] Tel.: +90 332 2232933Fax: +90 332 2410108

short notephysical and chemical characteristics

of oils of some olive varieties in turkey

This study was carried out on the monovarietal virgin olive oils from three olive varieties (Saurani, Halhalı and Karamani) growing in the Mediterranean region of Turkey. Saurani oil showed high oleic acid levels rising to 78% especially in both the harvest dates. At the same time, saturated fatty acids were found at lower concentrations in Saurani oils. The total phenolic contents of the oils can be considered medium-high (within 125.9-404.8 mg gallic acid /Kg). Karamani oil had a lower total phenol value (125.9 mg GA/Kg) than those of the other variety of oils. Viscosity values changed between 55.775 and 62.175 m.Pa and the brightness values of Karamani oils were lower than the other variety of oils. Key words: olive oil, fatty acid composition, phenol, color indices

Caratteristiche fisiche e chimiche degli oli di alcune varietà di olive TurcheQuesto studio è stato condotto sugli oli di oliva vergini monovarietali provenienti da tre varietà di olive (Saurani, Halhali e Karamani) coltivate nella regione mediterranea della Turchia. L’olio Saurani ha mostrato elevati livelli di acido oleico aumentando fino al 78% soprattutto nelle due epoche di raccolta. Allo stesso tempo, sono stati trovati in concentrazioni più basse gli acidi grassi saturi.I contenuti totali di fenoli in questi oli possono essere considerati medio-alti (da 125,9 a 404,8 mg acido gallico/kg). L’olio Karamani aveva un contenuto totale di fenoli più basso (125.9 mg GA/Kg) rispetto alle altre varietà di oli. I valori di viscosità variavano tra 55,775 e 62,175 m.Pa ed i valori di trasparenza erano più bassi rispetto alle altre varietà di oli.Parole chiave: olio di oliva, composizione acidi grassi, fenoli, indici di colore.


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1. INTRODUCTION

Olive trees grow in the Aegean, Mediterranean, Mar-mara and Southeast Anatolia Regions in Turkey. In the Southeast Anatolia Region, Hatay is the first place which produces the olive trees. The chemical charac-terization of olive oils from both Mediterranean and non-Mediterranean regions is essential for conduct-ing a geographical characterization of the oil [1].It is estimated that there are 130 million olive trees in Turkey. Of these, 71% are in the Aegean and Mar-mara Regions, and 26% are in the Mediterranean and the Southeast Anatolia Regions. The remaining 3% of Turkish olive cultivars are distributed throughout dif-ferent areas of the country including the Black Sea Region, with variations in climate and soil giving the oils their specific characteristics [2]. The quality of olive oil depends on the different olive cultivars, growing applications, harvest period, geo-graphical and ecological conditions of the growing place and the procedure of the oil extraction [3]. In the harvest period; sensorial properties have impor-tant effects on the quality of oil, oil yield and oil stabil-ity [4]. Late harvest causes too much damage to the potential fruit and/or oil quality during the harvest and the processing. Early harvested fruit produces a high phenol concentration of oil to provide the highest bit-terness and toughness [5]. Fatty acid composition of the oil is affected from the factors known as cultivar, climate, latitude, location and the maturation phase as in the oil accumulation period of the olive [4]. The increasing popularity of olive oil has been main-ly attributed to its high content of oleic acid, which may affect the plasma lipid/lipoprotein profiles, and its richness in phenolic compounds acting as natural antioxidants, which may contribute to the prevention of human diseases [6, 7]. Maturity is one of the most important factors associated with the quality evalua-tion of fruits and vegetables [8-10]. The variation of the color of olives is marked by a series of transformations that occur during the ripening process [10, 11]. As ri-pening progresses, photosynthetic activity decreases [11], while the amount of oil increases [12]. In addition, harvest timing can have a significant effect on the oil quality. This illustrates the need to determine the qua-lity of olive oil from a range of harvest times and culti-vars to establish an optimum harvest time [13].The aim of this work is to determine some physico-chemical properties of Halhalı, Karamani and Saurani oil-bearing olive cultivars which are in different loca-tions from Hatay.

2. MATERIAL AND METHOD

2.1. MATERIALOlive varieties (Halhalı, Saurani and Karamani) used in this research were collected from the Hatay prov-ince. Olives were collected in two different harvest periods; first period was between 15 September to

1 October, second period was between 20 October to 1 November. The laboratory mill was used to pre-pare the olive oil samples in the Department of Food Engineering Laboratory, Faculty of Agriculture. About 1-2 kg of olives were crushed with a hammer mill and slowly mixed for 35 min. The paste was centrifuged in thin layers for oil extraction. This oil was filtered, and transferred into dark bottles, and added into nitrogen. Oil samples were kept at –80°C. Halhali and Saurani varieties are used for both oil extraction and table ol-ive production, while Karamani is more often utilized in table olive production

2.2. METHOD 2.2.1. Dimension of fruit20 fruit’s width, height and thickness were evaluated with a digital compass (Mitutoyo Corp. Model no SC-6, Japan); and the averages were given in millime-ters.

2.2.2.Oil extractionThe olives were washed, and crushed with a hammer crusher. The paste was pressed with a manuel press (stainless steel) and the oil was extracted by means of a laboratory basket centrifuge (6,000Xg for over 5 min) without the addition of warm water and then trans-ferred into dark coloured glass bottles. All samples were stored at 4°C in darkness using amber glass bottles without headspace until the analysis [14].

2.2.3. Assessment of instrumental colorA colorimeter (Minolta Chroma meter CR 400 (Minolta Co., Osaka, Japan)) was used to assess the oil color and the CIELAB colorimetric system was applied. Each time 20 ml of samples were put into a glass Petri dish, and the liquid probe of the instrument was immersed into the dish sitting on the white tile, and readings of the CIE lab coordinates were recorded. The L*, a*, b* values are the average of the ten read-ings. The colour brightness coordinate L* measures the whiteness value of a colour and ranges from black at 0 to white at 100. The chromaticity coordinate a* measures red when positive and green when nega-tive, and chromaticity coordinate b* measures yellow when positive and blue when negative [15, 16].

2.2.4. Oil sample analysisFree fatty acids and peroxide values of olive oils were determined following the analytical methods de-scribed in the Regulations of the European Econo-mic Commission [17]. Free fatty acids (oleic %) were determined by titration of a solution of oil dissolved in ethanol/ether (1:1, v/v) with 0.1 mol L−1 potassium hydroxide ethanolic solution. For the peroxide value (meq kg−1), a known weight of olive oil was dissolved in a mixture of acetic acid/chloroform (3:2 v/v), and a saturated solution of KI (1 ml) was then added. The liberated iodine was titra-ted with sodium thiosulphate solution in the presence


63

of starch as an indicator.

2.2.5. Extraction of phenolic components from olive oil2 g of oil was weighed into the centrifuge tubes, add-ing 1.0 ml n-hexane and 2.0 ml CH3OH – water to it (95:5; v/v). Mixture was blended with vortex in 2 min and centrifuge at 3000 dev/min 5 min. Phase of methanol was separated and the extraction was re-peated at two periods. Extracts were combined and filtrated with 0.45 µm filter (AIM Syringe Filter PTFE) [18, 19]. Total phenolic content was determined by the Folin-Ciocalteu method [20]. Quantification was performed with the hydrolysed samples. Results were expressed as mg gallic acid /kg samples.

2.2.6. Fatty acid analysisFor the determination of fatty acid composition of the oils, fatty acid methyl esters were prepared from olive oil, using a cold saponification [21]. The fatty acids were converted to fatty acid methyl esters before the analysis by shaking a solution of 0.2 g oil and 3 mL of hexane with 0.4 mL of 2 N methanolic potassium hydroxide. A Shimadzu (Kyoto, Japan) gas chromato-graph, equipped with a flame ionization detector and a split/splitless injector, was employed. Separations were made on a Teknokroma TR-CN100 (Barcelona, Spain) fused-silica capillary column (60 m × 0.25 mm i.d. × 0.20 µm film thickness). The carrier gas was nitrogen, with a flow rate of 1 mL/min. The tempera-tures of the injector, the detector and the oven were held at 220°C, 250°C and 210°C, respectively. The injection volume was 1 µL. Peaks were identified by comparing their retention times with those of authen-tic reference compounds (Sigma–Aldrich, St. Louis, MO, USA).

2.2.7. Statistical AnalysisThis study was planned with 3 varieties, 2 different treatments, 3 replications. The results are given as the mean values (n:3). In addition, Duncan’s multiple range tests were used to determine significant diffe-rences among the data. Statistical analysis was carri-ed out using the SPSS 10.0 statistic program [22].


3.1. THE WIDTH AND HEIGHT OF THE PROPERTIES OF OLIVE FRUITSThe variance analysis results regarding the width and height of the properties of the olive fruit are given in

Table I. The Duncan multiple comparison test results, which show the effect of the cultivar difference and harvest period on the width and height properties are given in sequence in Table II and Table III Sequen-tially in Table IV and Table V, the effect of the inter-action of the cultivar and different harvest period on the width and height was presented. According to the variance analysis results, it has been observed that the differences of the olive cultivars and harvest pe-riod effect the width and height at p<0.01 level and that the interaction of the cultivar difference × harvest period was in affective on the width but effective on height at p<0.01 level (Tab. I). According to the Dun-can multiple comparison test results, the effect of the of difference of olive cultivars on width and height was given in Table II. Width values, depending on culti-var difference, varied from 14.545 to 15.717 mm. It’s been detected that the cultivar having the maximum width value is the Saurani. Height values are between 18.740 and 22.272 mm and this makes the Saurani cultivar the one with the maximum height value. Sau-rani was been evaluated as the biggest fruit among the cultivars. According to the Duncan multiple com-parison test results the effect of the harvest period on width and height is given in Table III. It’s been shown that width and height values had a little amount of de-crease in the second harvest period. Related interac-tion data were given in Table IV and Table V. As seen from the interactions, Saurani has the biggest fruit in

1

Table I - The variance analyze results belong to the width and height of olive fruit

VK S.D Width (mm) Height (mm)

KO F KO F Difference of Cultivar 2 14.729 22.323** 142.228 89.868** Harvest Period 1 25.854 39.186** 26.602 16.809** CD × HT int 2 1.632 2.473ns 18.797 11.877** Mistake 114 0.660 1.583

**p<0.01, * p<0.05, KO: correlation; F: Factor

Table II - The Duncan multiple comparison test results which shows the effect of difference olive cultivars on the width and height

*Mark with the same letters is not different from each other as in statically.

Table III - Duncan multiple comparison test results which is effect of harvest period on the width and height properties

Difference of Harvest Period

Width (mm) Length (mm)

1. Harvest period 15.505a* 21.358a 2. Harvest period 14.577b 20.417a


Cultivars Width (mm) Height (mm) Halhalı 14.545b* 18.740b Saurani 15.717a 22.272a Karamani 14.860b 21.650a

1













1














64

terms of width, Karamani has the second biggest fruit and Halhalı has the smallest size of fruit samples. If we consider height in the first harvest period, cultivars are listed from the biggest to the smallest, Karamani - Saurani - Halhalı, in the second harvest period we can see that Saurani surpasses Karamani.

3.2. SOME PHYSICAL AND CHEMICAL PROPERTIES OF OLIVE OILVariance analysis results that belong to the physical and chemical properties of olive oil are given in Table VI. Duncan multiple comparison test results, which show the effect of the cultivar difference and harvest period on some physical and chemical properties of oil is given in sequence in Table VII and Table VIII. In Table IX, Table X, Table XI and Table XII, the effect of interaction of the cultivar difference × harvest period on viscosity, free fatty acid, peroxide values and phe-nolic contents of oils has been presented. According to the variance analysis results, differences of olive cultivars had an effect on the viscosity, free fatty acid, peroxide value and phenolic content at p < 0.01 level and the harvest period is effective on the viscosity, free fatty acid , peroxide value and phenolic content at p < 0.01 level. It has also been observed that the interaction of the cultivar difference × harvest period is effective on the viscosity at p < 0.05 level and is ef-fective on free fatty acid, peroxide and phenolic con-tent at p < 0.01 level (Tab. VI)Table VII presents the Duncan multiple comparison test results which show the effect of the cultivar dif-ference on some physical and chemical properties of olive oils. Viscosity values change between 55.775 and 62.175 mPa and the Halhalı cultivar has the high-est value of viscosity. Free fatty acids values changed between 0.905% and 2.648%. In addition, Karamani 2

Table IV - Interaction of cultivar difference harvest period effect on the width (mm)

Cultivars 1. Harvest period 2. Harvest period Halhalı 15.075b 14.015b Saurani 15.955a 15.450a Karamani 15.485ab 14.235b


Table V - Interaction of cultivar difference harvest period effect on the height (mm)

Cultivars 1. Harvest period 2. Harvest period Halhalı 18.650b* 18.830b Saurani 22.540a 22.005a Karamani 22.885a 20.415a


2

Table IV - Interaction of cultivar difference harvest period effect on the width (mm)

Cultivars 1. Harvest period 2. Harvest period Halhalı 15.075b 14.015b Saurani 15.955a 15.450a Karamani 15.485ab 14.235b


Table V - Interaction of cultivar difference harvest period effect on the height (mm)

Cultivars 1. Harvest period 2. Harvest period Halhalı 18.650b* 18.830b Saurani 22.540a 22.005a Karamani 22.885a 20.415a


3

Tab

le VI

- Th

e res

ults o

f var

ience

analy

sis of

some

phys

ical a

nd ch

emica

l cha

racte

ristic

s of o

live o

ils

*p<0

,01, *

*p<0

,05; K

O:co

rrelat

ion; F

:Fac

tor

Sour

ces o

f Va

riatio

n SD

Vi

scos

ity (m

Pa)

Fatty

acid

(%)

L A

B Pe

roxid

e valu

e (m

eq/K

g)

Phen

olics

(mg

GA/K

g)

KO

F KO

F

KO

F KO

F

KO

F KO

F

KO

F Di

feren

ce of

Cu

ltivar

2 43

.301

13.69

6**

3.420

31

3.497

** 2.7

19

0.62n

s 0.1

43

1.824

ns

0.924

3.5

46ns

55

1.226

48

2.706

** 93

733.7

14

6614

.112*

*

Harve

st pe

riod

1 10

.830

3.425

ns

5.320

48

7.701

** 0.2

58

0.059

ns

0.012

0.1

54ns

3.8

87

14.91

5**

344.1

12

301.3

77**

2512

4.986

17

72.88

9**

VS×H

T int

2

18.00

7 5.6

96*

3.321

30

4.424

** 1.1

69

0.266

ns

0.168

2.1

47ns

0.7

78

2.984

ns

324.6

18

284.2

67**

1113

6.334

78

5.811

** Mi

stake

6

3.162

0.0

11

4.386

0.0

78

0.261

1.1

42

14.17

2


65

oil has been detected as the cultivar with the highest free fatty acids value. In color values, there isn’t much difference among cultivars. If we look at the peroxide values of the cultivars, we can see that the Saurani is the cultivar with the lowest value. The Karamani culti-var has a noticable value of peroxide and has a great amount that can be seen from the table. If we look at

the total phenol contents of all the cultivars, the Sau-rani cultivar has the highest value of phenolic material when compared to the other two cultivars and this value is 404.82 mg gallic acid/Kg. Karamani oil had a lower total phenol value (125.9 GA/Kg) than those of the other varieties of oils.Table VIII presents the multiple comparison test re-sults which show the effect of the harvest period on some physical and chemical properties of olive oil. In the first harvest period, viscosity was found to be 60.367 mPa, in the second harvest period it was esta-blished at 58.467 mPa. Free fatty acid was found to be 0.932% in the first harvest period. This increases to 2.263% in the second harvest period. There is not a significant change in L, a and b values. In the first harvest period, the peroxide value was found to be 15.925 meq/Kg and this value increases to 26.635 meq/Kg in the second harvest period. Besides this, the total phenol content was found to be 274.667 mg GA/Kg in the first harvest period and 183.152 mg GA/Kg in the second harvest period.Table IX presents the effect of interaction of the cul-tivar difference × harvest period on the viscosity contents of the oils. In the first harvest period, the viscosity values of the Halhalı, Saurani and Karamani cultivars were found to be between 54.950 mPa and 63.600 mPa and the Karamani had the highest vis-cosity value, as for second period the viscosity values were found to be between 56.600 mPa and 61.800 mPa and the Halhalı cultivar had the highest viscosity value. Table X presents the effect of interaction of the cultivar difference × harvest period on the free fatty acid contents of the oils. In the first harvest period, free fatty acid values of the Saurani and Karamani cul-tivars were very close to each other and these values were found to be 0.855% and 0.935% in sequence, whereas the free fatty acid value of the Halhalı cultivar

4

Table VII - Duncan multiple comparison test result that shows the cultivar differences effect on the physical and chemical properties of oil

Cultivars Viscosity (mPa) FFA (%) L a b

Peroxide value

(meq/Kg)

Total Phenol (mg gallic acid/Kg)

Halhalı 62.175a* 1.240b 31.470a 0.550a 3.633a 16.958b 155.953b Saurani 55.775b 0.905c 30.453a 0.778a 4.460a 12.315c 404.820a Karamani 60.300a 2.648a 29.838a 0.403a 4.470a 34.567a 125.955c

* Mark with the same letters is not different from each other as in statically

Table VIII - Duncan multiple comparison test result that shows harvest period effect on to the physical and chemical properties of oil

Difference of Harvest

period Viscosity (mPa) FFA (%) L a b

Peroxide value

(meq/Kg)

Total Phenol (mg

gallic acid/Kg)

1.Harvest period 60.367a* 0.932b 30.440a 0.608a 4.757a 15.925b 274.667a 2.Harvest period 58.467a 2.263a 30.733a 0.545a 3.618a 26.635a 183.152b

* Mark with the same letters is not different from each other as in statically.

Table IX - Interaction of cultivar difference harvest period effect on the viscosity contents of oils (mPa)

Cultivars 1. Harvest period 2. Harvest period Halhalı 62.550a* 61.800a Saurani 54.950b 56.600a Karamani 63.600a 57.000a

* Mark with the same letters is not different from each other as in statiscally.

4



Peroxide value

(meq/Kg)







Peroxide value

(meq/Kg)

Total Phenol (mg

gallic acid/Kg)






4



Peroxide value

(meq/Kg)







Peroxide value

(meq/Kg)

Total Phenol (mg

gallic acid/Kg)






5

Table X - The interaction of cultivar difference harvest period effects on the free fatty acid contents of oils (oleic acid %)

Cultivars 1. Harvest period 2. Harvest period Halhalı 1.005a* 1.475b Saurani 0.855a 0.955c Karamani 0.935a 4.360a


Table XI - The interaction of cultivar difference harvest period effect on the peroxide value contents of oils (meq/Kg oil)

Cultivars 1. Harvest period 2. Harvest period Halhalı 16.530a* 17.385b Saurani 12.430b 12.200c Karamani 18.815a 50.320a


Table XII - The interaction of cultivar difference harvest period effects on the phenolic material contents of oils (mg GA/Kg)

Cultivars 1. Harvest period 2. Harvest period Halhalı 197.090c* 114.815b Saurani 400.275a 409.365a Karamani 226.635b 252.75c


5











66

was 1.005%. In the second harvest period the free fatty acid values were found to be between 0.955% and 4.360%. There was a significant increase in the rate in the Karamani cultivar. Table XI presents the ef-fect of interaction of the cultivar difference × harvest period on the peroxide value contents of the oils. The peroxide values of the oils were found to be between 12.430 and 18.815 meq/Kg in the first harvest period, and the Karamani cultivar had the highest peroxide value. During the second harvest period the peroxide values were found to be between 12.200 and 50.320 meq/Kg and the Karamani again had the highest per-oxide value. Table XII presents the effect of interaction of the cultivar difference × harvest period on the pheno-lic contents of the oils. The total phenol contents of the Halhalı, Saurani and Karamani were found to be 197.090, 400.275 and 226.635 mg gallic acid/Kg, respectively, in the first harvest period, and were at 114.815, 409.365 and 252.75 mg gallic acid/Kg in the second periods. As can be seen in the table, Sau-rani has the highest values of phenolic material con-tents among the cultivars. Moussa et al. [23] found the free fatty acids to be over 0.55% - 0.62% in the olive oil. The peroxide value was found to be between 7.3 - 18.1 meq/Kg [24]. By the way of combine the different methods in Greece, where olive oils are tak-en from the olive fruits, the peroxide values are found to be between 6.0 - 47.7 meq/Kg [25]. With respect to luteolin and apigenin, the flavone compounds iden-tified but not quantified in Cornicabra virgin olive oil were described by Vazquez-Roncero et al. [26]. They are formed by hydrolysis of the glucosides present in the olive pulp [27].

3.3. FATTY ACID COMPOSITION OF OLIVE OILSComposition of fatty acid determined with the gas chromatography of olive oils was presented in Table XIII.All cultivars contained high levels of oleic acid in the early and late harvest period. In the late harvest pe-riod, the palmitic acid content decreases in the Halhalı cultivar and increases in the other two cultivars. In the late harvest period, the palmitoleic acid content de-creases in the Halhalı and Saurani cultivars, and incre-ases in the Karamani. As for the stearic acid contents; from the highest to the lowest, Karamani, Saurani and Halhalı are in sequence. In the late harvest period, we can see that stearic acid contents of oils extrac-ted from the first two cultivars decrease and stearic acid contents of the oil extracted from the other one increases. In the late harvest period we can see that linoleic acid contents of oils extracted from the first two cultivars increase and linoleic acid contents of oils extracted from the other one decreases. Finally, if we look at the contents of the arachidic acid; from the highest to the lowest the order is Saurani, Karamani and Halhalı. In the late harvest period we can see that arachidic acid content of oils extracted from the first two cultivars decreases and arachidic acid content of oil extracted from the other increases. Palmitic acid is the highest acid value after the oleic acid content in the cultivars of Halhalı and Saurani, and linoleic acid is of the Karamani cultivar. The lowest fatty acid con-tent of the Halhalı cultivar is in the early harvest period and is found to be arachidic acid, in the late harvest period it is established to be palmitoleic acid.In a study about with the olive cultivars in Turkey, the value of palmitic acid was found to be lower in the Gemlik cultivar and the value of stearic acid was found to be higher in the Kilis cultivar [28]. In a study that was done by Ollivier et al. [29], it was found that the palmitic, stearic, oleic, linoleic and linolenic ac-ids contents values are in an order of 8.49-13.72%, 2.11-2.6%, 66.36-79.39%, 5.82-11.85% and 0.61-0.65% . Aparicio and Luna [30] search oils taken from Coratina, Koroneiki and Picual cultivars are found to be the proportional contents of essential fatty acids of oils i.e. 9.7-11.6% palmitic, 2.2-2.4% stearic, 78.1-80.3% oleic, 4.8-5.7% linoleic and 0.4-0.8% linolenic. Yazıcıoğlu and Karaali [31] report that the contents of essential fatty acids in the oils are 14.3% palmitic, 4-12% stearic, 64.05% oleic and 15-53% linoleic.

5










6

Table XIII - Fatty acid compositions of olive oil which olive cultivars from Halhalı, Saurani and Karamani (%) Cultivars Harvest periods Palmitic Oleic Palmitoleic Stearic Linoleic Arachidic

Halhalı 1.Harvest 10.6 74.4 - 4.5 9.0 0.4 2. Harvest 10.3 74.5 - 4.1 9.2 0.7

Saurani 1. Harvest 6.5 78.5 0.6 4.7 7.9 0.7 2. Harvest 7.7 78.0 0.5 5.2 7.0 0.6

Karamani 1. Harvest 9.2 74.0 0.5 5.0 9.9 0.5 2. Harvest 9.8 73.0 0.6 5.6 10.0 0.4



67

According to statistical analysis, fatty acid composi-tional differences among the oils studied were signifi-cant, indicating a varietal effect on the olive oil quality. These results are in agreement with the findings of other authors [32-35]. Fatty acid compositions of olive oils show differences among the varieties [36]. Olive composition is influenced by environmental and culti-var differences. As a result, the present study should be considered an important step in providing infor-mation to researchers for further studies on olive oils. These results are valuable in determining the accep-table times of the harvest to ensure that phenols are within the limits for the virgin olive oil. The appropriate harvest timing based on the scientific criterios is a key factor in the determination of the balance between oil quality and quantity.

Acknowledgement

This work was supported by Selçuk University Scien-tific Research Project (S.Ü.-BAP, Konya-TURKEY).

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Received June 10, 2013Accepted September 13, 2013


69

Notiziario• • • • • • • • • • • • • • CONGreSSi

EUBCE 201523rd European Biomass Conference and ExhibitionJune 1-4, 2015 - Vienna, AustriaThe European Biomass Conference and Exhibition (EUBCE) is a world class annual event which, since 1980, is held at different venues throughout Europe.It is Europe’s largest international conference focused on biomass combining a highly-respected internatio-nal scientific conference with an industrial exhibition and gathers participants from research, industry, po-licy and business of biomass.

It highlights progress in research, technological •development and production processes.It brings together all key specialists to make it the •most important international platform for dialogue between research, industry, research and industry, and policy in the biomass sector.The EUBCE is the event in which the members of •the bioenergy community can get a broad pictu-re of the situation and trends emerging in today’s market.The Conference provides a high-level scientific •programme and parallel events which attract par-ticipants from a wide-ranging background: resear-chers, engineers, technologists, standards orga-nisations, financial institutions, policy makers and decision makers.This event is supported by European and interna-•tional organizations. The Technical Programme is coordinated by DG Joint Research Centre of the European Commission.

Bioenergy in Integrated Energy SystemsDeployment of renewables at a very large scale re-quires impact-advanced strategies and technologies for energy system integration. Development and de-monstration of such solutions should be done in the broad context of the transition to a sustainable ener-gy system, where Bioenergy is accompanied by other renewables and a changing range of conventional energy technologies.This Focus Session aims to bring together energy sy-stem specialists, experts from the renewable energy sectors and other stakeholders to present and di-scuss experiences, plans and options for advanced system integration.

Present at the EUBCE Conference. An opportunity for the bioenergy industryBased on the success and positive feedback regar-ding the initiative in the past edition of the conference, the EUBCE Executive Committee has decided to set-up a specific Industry dedicated section in the Pro-gramme in order to promote the interest and needs

of the bioenergy sector and establish a platform to influence the market deployment of new innovative bioenergy technologies while at the same time ad-dressing key policy initiatives.The aim is to create a balance in the event between the scientific content and the industry contributions to ensure the coverage of the entire bioenergy value chain.All industry experts and players are invited to con-tribute and to submit abstracts, propose workshops or events, suggest industry speakers and make con-structive recommendations to serve the needs of the bioenergy industry.This is an opportunity for the bioenergy industry to reinforce the 23rd EUBCE Conference programme!For updates:ht tp: / /www.eubce.com/Home.404.0.html#.VFd0OTSG_Co

28th Nordic Lipidforum SymposiumJune 3-6, 2015 - Reykjavik IcelandThe Nordic Lipidforum is a professional arena for pe-ople interested in lipids in the five Nordic countries, Norway, Sweden, Finland, Denmark and Iceland. The forum should benefit both scientists and compa-nies involved by having a common meeting place and a system for exchange of knowledge.The Nordic Lipidforum was formally founded in 1969.Key points in the Nordic Lipidforum activities are:

Organize a contact network for a Nordic collabo-•ration in the lipid areaPromote applied research and technology for in-•dustrial application of lipids, fats and oils with a special focus on the Nordic raw materials such as fish and other marine oils, rapeseed, camelina and flaxseed oil.Provide information network playground for Nor-•dic and international meetings, job opportunities in academia, research institutes and/or industry, etc.Provide a forum for exchanging of ideas and in-•formationIncrease international visibility of Nordic research •and industry in the lipid field.To inspire talented employees to increase their •competence in lipid science and development.

For information and updates:http://www.lipidforum.info/

1st Sustainable Oils & Fats International Congress June 16 - 17, 2015 – Paris, FranceYou have certainly noted that the sustainability con-cept has ramped up over the past years in the oils & fats industry. A strong social and environmental awa-reness is rising from producers (palm, soy, rapeseed,


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tional congresses are granting at best one session on sustainability. Others are focusing on a specific source, such as sustainable palm or responsible soy. We, at FAT & Associés, have decided to organize an international event to gather all industrial actors and decision makers around a 360° view of the sustaina-ble oils & fats market. The 1st Sustainable Oils & Fats International Congress (SOFIC) will take place on 16 & 17 June 2015 in Paris, France. This global event aims at assembling diverse views and answering a rather provocative question “The Sustainable Oils in Question: Miracle or Mirage?”. You are part of the sustainable value chain, or would like to understand how far the phenomenon would expand: join us and share your experience with the community!

Tuesday, June 16SESSION 1 - SOURCING AND PRODUCING SU-STAINABLE OILS & FATS

11.00 - 11.30 Transforming the Market to Make •Sustainable Palm Oil the Norm - Inke Van Der SLUIJS (RSPO, the Netherlands)11.30 - 12.00 Responsible Soy Market Overview - •Lieven CALLEWAERT (RTRS, Belgium)12.00 - 12.30 Oil Crops: Opportunities for Impro-•vements in Productivity, Environmental Quality and Human Wellbeing - Romano DE VIVO (SYNGEN-TA, Switzerland)

SESSION 2 - CREATING, MAINTAINING AND CON-TROLLING SUSTAINABLE SUPPLY CHAIN

14.00 - 14.30 Strengths and Weaknesses of Vo-•luntary International Standards for Sustainability: The RSPO Case - Alain RIVAL (CIRAD, Indonesia)14.30 - 15.00 Including smallholders in palm oil •sustainability efforts - Eric SERVAT (RAINFOREST ALLIANCE, UK) 15.00 - 15.30 Sustainability Challenges in Produc-•tion of Soy Oil & Opportunities for Supply Chain Actors - Gert Van Der BIJL (SOLIDARIDAD, the Netherlands)

SESSION 3 - ECONOMIC ANALYSIS AND PROFITA-BILITY

16.00 - 16.30 Demonstrating Sustainability - Andy •GREEN (BM TRADA Certification, UK)16.30 - 17.00 Does Sustainability make a differen-•ce in Consumer Price? - Olivier DELANNOY (FAT & Associés, France)17.00 - 17.30 Valuing the Economic and Natural •Capital Benefits of Sustainable Palm Oil Practices - Richard MATTISON (TRUCOST PLC, UK)

Wednesday, June 17SESSION 4 - DEMAND AND USAGE IN FOOD AND NON-FOOD APPLICATIONS

09.00 - 09.30 Sustainable Palm Oil, Challenges & •Solutions from a Major Brand and Retailer - Fiona

WHEATLEY (MARKS and SPENCER, UK) and Jo-nathan HORRELL (MONDELEZ, UK)09.30 - 10.00 The German Market on the Move •towards a 100% Certified Palm Oil: Progress and Constraints - Daniel MAY (GERMAN FORUM FOR SUSTAINABLE PALM OIL, Germany)10.00 - 10.30 Sharing Sustainability Palm Oil De-•rivates Sourcing: L’Oréal Purchasing Strategy - Frédéric NIOLA (L’Oréal, France)

SESSION 5 - CONSUMER PERCEPTION AND RAI-SING AWARENESS AMONG CONSMERS

14.00 - 14.30 Loci of sustainable palm oil con-•sumption: consumers, brands, values or markets? - Simona D’ANTONE (KEDGE Business School, France)14.30 - 15.00 Are Consumers Concerned about •Palm Oil? Evidence from a Lab Experiment - Ste-phan MARETTE (INRA, France)15.30 - 16.00 ROUNDTABLE DISCUSSION: The •Sustainable Oils in Question: Miracle or Mirage? 16.00 Closing Remarks & Conclusions

For information: http://www.fat-associes.com/wp-content/uploads/2014/12/SOFIC_Brochure.pdfE-mail: [email protected]

Personal & Home Care, Cosmetics & Foods Industry Focus at the Tech-Connect World Innovation ConferenceJune 15-18, 2015 – Washington DC USAThe consumer products and food industries are dri-ven by innovations in materials, and in natural and synthetic materials characterization and processing: from texture, rheology, and product appearance to active delivery and shelf-life.Join industry partners and applied research leader-ship accelerating the development and deployment of new material solutions into products and society.http://www.techconnectworld.com/World2015/in-dustry/PersonalHomeCareCosmeticsFoods_Indus-try.html

Industrial Biotechnology CongressMilestones of Innovative Scientific Research in Biotechnology & its Industrial ApplicationsAugust 10-12, 2015 - Birmingham, United KingdomHilton Birmingham Metropole HotelIndustrial Biotechnology Congress is a remarkable event which brings together a unique and internatio-nal mix of Biotechnologists, Industrialists, Scientists, Clinicians, Leading universities and Research Insti-tutions making the congress a perfect platform to share experience, foster collaboration across industry and academia, and evaluate emerging technologies across the globe. The theme of congress “Milestones of Innovative Scientific Research in Biotechnology &


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Notiziarioits Industrial Applications” reflects the emerging pro-gress being made in biotechnology industry. Industrial biotechnology also called as white biotechnology is a key enabling technology to realize a bio economy that uses biological resources as an input to industrial processes.Industrial Bio-2015 officially welcomes all the par-ticipants across the globe to attend the Industrial Biotechnology Congressduring August 10-12, 2015 at Birmingham, United Kingdom.Conference Highlights

Biotechnology and its Applications•Industrial Microbiology and Enzyme Technology•Microbial and Biochemical Technology•Metabolomics and Genomics Research•Food Processing, Functional Foods and Health•Biotechnology and Pharmaceutical Industry•Tissue Engineering and Biomaterials•Plant Extracts and Commercial Uses•Environmental Biotechnology•Biotechnology in Healthcare Industry•Stem Cell Therapeutics - Commercialization•Nanobiotechnology•White Biotechnology and Economy•

Congress Secretariat:5716 Corsa Ave., Suite 110, West LakeLos Angeles, CA 91362-7354, USA Ph: +1-650-268-9744, Fax: +1-650-618-1414E-mail [email protected]

4th International Conference and Exhibition on Food Processing & TechnologyFood Technology: Trends and Strategies for In-novation of Sustainable FoodsAugust 10-12, 2015 - London, UKOMICS Group feel blissful to invite you all to attend “4th International Conference and Exhibition on Food Processing & Technology (Food Technology-2015)” which is going to be held during August 10-12th, 2015 on a theme “Food Technology: Trends and Strategies for Innovation of Sustainable Foods”.This meet enables a common platform for the partici-pants to discuss their research in order to establish a scientific network between the academia and indust-ry leading to foster collaboration and to evaluate the emerging issues, technologies and innovations leads to explore new possibilities and improving the existed opportunities.The prevention of diet-related diseases is one of the new societal challenges of the 21st century. In Oc-tober 2011, the world population passed the 7 bil-lion mark. Such growth will put a massive strain on the global food supply. These factors alone make the production and distribution of food a critical issue for the 21st century. London’s food sector is worth a massive £17bn with small and medium food busines-ses providing the majority of the industry’s 300,000 jobs. In 2011, 25 countries together accounted for

90% of UK food supply. Just over half of this (51.8%) was supplied domestically from within the UK.Food Technology-2015 is designed to offer compre-hensive range of sessions that includes breaking inno-vations in Food Science, Preservation, Quality Stan-dard and Systems Management, Food Processing and Packaging Technology, Nutrition and Nutritional Management, Food and Health, Application of Food Technology, Nutritional Deficiencies and Nutraceuti-cals, Sustainable Food Security, Food Nanotechnolo-gy and Food Biotechnology.Conference Highlights:

Breaking Innovations in Food Science•Functional & baby foods•Non-thermal Food Preservation Technologies•Packaging processes, materials & components•Food regulatory affairs & sensory analysis•Nutritional Deficiencies and Nutraceuticals•Sustainable Food Security•Food fortification•

For information and updates:http://foodtechnology.conferenceseries.com/

2nd High Oleic Oil Congress - HOC 2015September 2-4, 2015 – Paris, France The High Oleic Oils Congress (HOC) is the only event dedicated to the fast growing high oleic oil market. High oleic (HO) oil is a niche market with its own co-des and unclear rules for new entrants: premium pri-ces for grain and oil, closed contracts between pro-ducers and traders, and regular unbalances between supply and demand.The objective of the congress is to explain the HO oil market from demand to supply. We will describe the current state of the HO oil market and analyze key market topics of direct commercial relevance to industry participants, while giving an overview of the entire value chain.The congress also provides participants with an ex-cellent opportunity to network with other key players in the HO oils industry.For information and updates:http://higholeicmarket.com/

Oils+Fats September 16-18, 2015 – Messe München´s MOCGermanyOils+fats: the trade fair for your success!Oils+fats is the world’s only Trade Fair for Business, Technology and Innovations in the field of vegeta-ble as well as animal oils and fats.2015 will be the sixth time it takes place in Mu-nich. This is where the industry meets, which makes it an absolute must!Vegetable and animal oils and fats comprise a mar-ket whose significance continues to increase. As the world’s only Trade Fair for Business and Technolo-


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the latest Innovations and showcases current tren-ds, products and services. As in the past, the three-day exhibition at the MOC Veranstaltungscenter Mün-chen will show that it is worthwhile! In 2013, oils+fats demonstrated yet again that exhibitors and visitors share the same opinion. A total of 2,221 visitors from 110 countries attended the fair, and the share of inter-national visitors at oils+fats was 58 percent. That is why exhibitors also appreciate oils+fats as a unique opportunity to meet with customers from throughout Europe. Companies were particular prai-seworthy of the visitors’ extraordinary technical ex-pertise. Visitors consider oils+fats a success: According to a representative survey conducted by GMM Gelszus Messe-Marktforschung, 84% of visitors are planning to attend oils+fats again. For information and updates:http://www.oils-and-fats.com/en/Home

7th International Conference on Informa-tion and Communication Technologies in Agriculture, Food and Environment (HAI-CTA 2015)September 17-20, 2015 - Kavala, GreeceHAICTA 2015 aims to bring together professional, experts and researchers working on Information and Communication Technologies in Agriculture, Food and Environment. Furthermore, another emphasis is put on the applicability of ICT solutions to real indus-try cases and the respective challenges.TopicsWe are accepting submissions of original research papers and posters for the main program and the doctoral consortium in all areas of ICT in Agriculture, Food and Environment including but not limited to:

Decision Support Systems•Information Systems•Database Systems•Environmental Modelling and Simulation•Environmental Control Systems•Environmental Impact Assessment•e-Waste Management & Clean Technologies•Environmental Design & Policy•Issues in ICT Adoption•New technologies in Ecosystems Management, •Forestry and Natural ResourcesInformation Systems and Wild Life Management •& ProtectionWood Technology & Wood Products•Flora and Fauna Management using ICTs•ICT in Climate Change and Global Warming•ICT in Cultivation and Pastoral Areas•ICT in Rural Development•Innovative Technologies in Agrotourism & Ecot-•ourisme-Business, e-Commerce, e-Government, e-•

Learning and e-ServicesGIS and Applications•Spatial Analysis & Landscape Planning•Internet Marketing, Internet of Things and Web •2.0Mobile/wireless Systems & Networks•Ontology and Web Semantic•Sensor and Wireless Network Applications•Mobile/Wireless Applications in Agriculture•Novel RFID applications•Precision Farming Systems, VR Technologies•Green IC Technologies•Traceability•Supply Chain Management & Logistics•Environmental Education•

23rd IFSCC Conference 2015September 21-23, 2015 - ZürichThe Swiss organization of cosmetic chemists SWISS SCC is organizing the conference in 2015 in Zurich. In 2015 it is exactly 20 years ago that the last IFSCC conference was held in Switzerland. It took place in Montreux at the Lake Léman with a beautiful view to the Alps. We looked back to that conference. Its title Facts and illusions in cosmetics gave us the inspiration for the new scheme in 2015. Time has changed and facts have gained importance with changing legislation for cosmetic products and their ingredients (REACH) and many new developments in the area of analytics and product testing whether in vitro, ex vivo or in vivo.The new conference title became “More Facts, Less Illusions”.We are convinced that some more illusions will be transferred into facts and that there is much more to come in the future. The conference venue will be the Conference Center ZurichWe are looking forward to welcoming you in Switzer-land soon.for information on the scientific program: www.ifscc2015.com

13th Euro Fed Lipid Congress “Fats, Oils and Lipids: New Challenges in Technology, “Quality Control and Health”September 27-30, 2015 - Florence, Italyhosted by SISSGThe Italian Society for Fats and Oils Research-es (Società Italiana per lo Studio delle Sostanze Grasse - SISSG) is proud to host the Euro Fed Lipid congress 27-30 September 2015 in Florence. The symposium will be hosted in Firenze Fiera Congress and Exhibition Center located inside the 18th century Villa Vittoria, at walking distance from the historical centre of one of the most beau-tiful towns of Italy, rich of artistic masterpieces,


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Notiziariowith an ancient history rich of arts and science. Palazzo Vecchio, Piazza della Signoria, Ponte Vec-chio, the Dome, Galleria degli Uffizi are world wide well known symbols of this ancient town, where Leonardo da’ Vinci began his work, be-fore moving to Milan and other Europeans towns. Tradition in hosting science in Florence dates back to 1753, when the “Accademia dei Georgofili” was established, the headquarter is in the Uffizi Gallery building. With such a background, the choice for the 2015 Euro Fed Lipid congress in Italy was really easy! Science, however is still alive in Florence and its re-gion, Tuscany, with different University (Florence itself, Pisa, Siena) and a number of research centre (Nation-al Research Council of Italy - CNR) where research is actively carried out on several topics, enclosing Agri-cultural and Food Science which are the basis of the topics to which Euro Fed Lipid looks to. Tuscany agricultural landscape is well known for food production and some of these like extra virgin olive oil will be one of the topics of Euro Fed Lipid congress, however, within the frame of a developed agricultural and food industry production, other topics like Plant lipids and oilseeds as well as Animal Science will surely meet interests outside of the “traditional” Euro Fed Lipid attendants.Social program could surely ensure a number of in-teresting possibilities while the traditional gala dinner will be hosted at Palazzo Borghese, a wonderful an-cient residence. Symposium topics will be Analytics, Authenticity & Lipidomics, Bioscience, Biocatalysi & Biochemistry, Lipid oxidation & Antioxidants, Lipids in animal Science, Health and Nutrition, Microbial & Algae lipids, Oliseeds, Plant breeding & Plant lipids, Oleochemistry & Biodiesel, Olive oil, Physical chemis-try, Processing, sustainability & Industrial innovation.The Scientifc Committee, established by involving scientists from Europe and outside and SISSG will be delighted to welcome an huge number of Lipid Sci-entist in Florence! Plenary Lectures:

European Lipid Science Award Lecture•European Lipid Technology Award Lecture•Chevreul Medal Lecture•Wilhelm Normann Medal Lecture•

Main Topics/Keynote Lectures:Analytics, Authenticity, Lipidomics•Bioscience, Biocatalysis, Biochemistry: Romas •Kazlauskas, University of Minnesota, St. Paul/MN, USA “How the Ser-His-Asp Catalytic Triad Cata-lyzes Different Reactions”Lipid Oxidation and Antioxidants•Lipids in Animal Science•Health and Nutrition: Andrea Poli, Nutrition Foun-•dation of Italy, Milano, Italy, “Omega-6 Fatty Acids and Health: an Unbiased Critique of the Available Evidence”Microbial and Algae Lipids: Antonio Molinaro, •

University of Napoli, Italy “Microbial Cell Wall Li-poglycans as Keywords in the Dialogue with the Eukaryotic HostOil Seeds, Plant Breeding and Plant Lipids•Oleochemistry, Biodiesel•Olive Oil•Palm Oil•Physical Chemistry•Processing and Sustainability•

http://www.eurofedlipid.org/meetings/florence2015/index.php

Practical Short Course on Vegetable Oil Processing and Products of Vegetable Oil/BiodieselOctober 4-8, 2015 - College Station, TexasOrganized by theFood Protein Research & Development CenterFats and Oil ProgramTexas A&M Engineering Experiment StationThe Texas A&M University SystemCollege Station, TX 77843-2476 U.S.A. Objectives of Short CourseTrain production personnel in principles and practices of:

New methods in vegetable oil refining and pro-•cessingLatest methods in bleaching, hydrogenation, in-•teresterification, and deodorization of major veg-etable oilsProduction of biofuels•Production of non-trans fats•Filtration Systems•

Who Should AttendThis short course is a must attend for anyone involved in the field of Vegetable Oil Processing and interested in the latest developments in bleaching, hydrogena-tion, interesterification, deodorization, production of biodiesel and non-trans fats.- Plant Managers and Engineers- R &D Personnel- Sales and Marketing Personnel- Quality Control and Quality Assurance Personnel- Application ScientistsFor additional technical information, write, call, fax or e-mail to:Dr. M.S. AlamHead, Fats and Oils Program Food Protein R&D Center 2476 TAMU, Texas A&M University System College Station, Texas 77843-2476 U.S.ATel: 979-845-2740, Fax: 979-845-2744E-mail: [email protected] registration inquiries contact:Marcy Bundick Short Course CoordinatorFood Protein R&D CenterPhone: (979) 845-2741, Fax: (979) 845-2744E-mail: [email protected]


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The Premier Oil Palm event is back!October 6-8, 2015 - Kuala Lumpur Convention Centre, Ma-laysia Organized by: Malaysian Palm Oil Board Ministry of Plantation Industries & CommoditiesThe grand MPOB International Palm Oil Congress and Exhibition (PIPOC) with five concurrent Confe-rences will examine and discuss the many facets of the oil palm industry.PIPOC 2013 was attended by more than 2200 parti-cipants from 48 countriesPIPOC 2015 features 5 concurrent Conferences, na-mely:•Agriculture,Biotechnology&Sustainability•Chemistry,ProcessingTechnology&Bio-Energy•Food,Lifestyle&Health•Oleo&SpecialtyChemicals•GlobalEconomics&MarketingAnother attraction of the Congress is an Evening Fo-rum on Current Issues.You may opt to be a:speaker, poster presenter or participant or your orga-nisation may:•EXHIBITyourproductsand/orservices•ADVERTISEintheSouvenirProgrammeoftheCon-gress 6 - 8 October 2015After two years, it is timely to update your knowledge and information on the developments in the R&D of oil palm. It will be a platform for participants to interact and share information in all areas pertaining to the oil palm/palm oil industry.This bi-annual Congress provides a platform that showcases the latest advances in the industry.A grand exhibition with a total floor space of more than 2000 m2 and 300 booths will showcase many new technologies and information to increase the productivity of your business.Technical tours to an oil palm plantation, palm oil mill, refinery and R&D facilities will also be arranged. A golf tournament is also in store for participants and golf enthusiasts.So, be part of the event and don’t miss this opportu-nity to update and get yourself networked.For information and updates:[email protected]

Shaping the Future of Food Safety,TogetherMilan - Italy, 14-16 October 2015EFSA has announced the details for the Scientific Conference being organised on the occasion of the World EXPO 2015, which has food as its central the-me. Representatives from the scientific and risk as-sessment community as well as risk managers and risk communicators from in and outside Europe are

invited to attend.The conference will focus on two major themes – Assessment Science; and Science, Innovation and Society – and will be organised in plenary and bre-akout sessions, with the latter covering the following topics:

Open risk assessment•Data: co-creating added value•Key challenges in scientific advice - Weighing evi-•dence and assessing uncertaintiesNutrition challenges ahead•Novel chemical hazard characterisation approa-•chesMicrobiological risk assessment - Challenges and •opportunitiesDrivers for emerging issues in animal and plant he-•alth - The global nutrition at riskAdvancing environmental risk assessment•Expertise for the future•

Conference website: http://www.efsaexpo2015.eu/

SODEOPEC2015Soaps, Detergents, Oleochemicals, and Per-sonal Care October 27-30, 2015 - Miami, Florida, USAyou are invited to attend to SODEOPEC2015.On behalf of the organizing committee, AOCS, invite you to attend SODEOPEC2015, the eighth in a seri-es of successful meetings. Continuing the tradition of covering the four interrelated areas of soaps, deter-gents, oleochemicals, and personal care, the meeting will deliver “practical solutions for tomorrow’s challen-ges”.Presentations:• The State of the IndustriesChair: Tom Branna, Happi, USANews you can use to improve the product line and bottom line!

Keeping Healthy. - Look at the critical role that proper hygiene plays in maintaining human health around the world. Room to Grow. - Past results are no guarantee of future performance, but analysts paint a bright fu-ture for the industries.Now Smell This! - Learn how fragrance trends are shaking up the business.Let’s Get Small. - Start-ups play a crucial role in bringing innovation to market.Big and Powerful. - See how a multinational corpo-ration views the landscape.

• Sustainability in ActionChair: Brian Sansoni, American Cleaning Institute, USACompanies throughout the cleaning product sup-ply chain are challenged with greater demands for transparency on how they operate sustainably and responsibly. This session will explore the drive for su-


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Notiziariostainability improvements at the company and indust-ry levels, plus how it affects society at-large.• The Analytics of SODEOPECChair: George A. Smith, Huntsman Performance Products, USAThe session will cover the manufacturing and testing of detergent products for home and personal care applications. Different analytical methods for deter-mining product specifications will be surveyed, along with statistical quality control (SQC) techniques for optimizing the manufacturing process and product quality. The session will also discuss preparation of soil swatches for assessing the performance of laun-dry detergent formulations and different test methods for laundry, liquid dish, and hard surface cleaning applications. Use of image analysis and colorimetric determinations for optimizing the performance pro-perties of finished products will also be discussed.• Contract ManufacturingChair: David P. Hempson, Marietta Corporation, USA- Evolution of Contract Manufacturing in the Personal Care

Industry-A 30-year PerspectiveDavid P. Hempson, Senior Vice President Business Development, Marietta Corporation, USA.An in-depth review of the contract manufacturing in-dustry from an insider’s view – how the industry has changed in the areas of selling strategies, customer expectations, and contract manufacturer capability. An overview of the direction of the industry and how market forces will shape the landscape as the con-tract manufacturing industry continues to evolve.- Industry Association and Their Impact on the Contract

Manufacturing IndustryLisa Shambro, Executive Director, Foundation for Strategic Sourcing, USA.An overview of the history of the Foundation for Stra-tegic Sourcing –what compelled the formation of the F4SS; what is the role of the association in bridging the gap between customers and suppliers (contract manufacturers) in the areas of networking, establi-shment of industry standards, thought leadership, and continuous improvement.- Regulatory Controls and the Advent of Good

Documentation Practices, Analytical Testing, Microbiological Testing in the Contract Manufacturing Environment

Chris Calhoun, Senior Vice President Quality and Regulatory Affairs, Marietta Corporation, USA.How have expectations changed in the area of regu-latory compliance for contract manufacturers – what drove the changes and how has the industry respon-ded? Where is the industry headed with regard to re-gulatory compliance expectations?- The Future of Contract Manufacturing-GloballyPanel discussion: D. Hempson, L. Shambro, and C. CalhounA round table discussion leveraging the presented topics as to where we see the contract manufactu-

ring industry headed – as large food and consumer products companies look for agility, speed to market, and divest manufacturing operations, how will the contract manufacturing industry adapt?• From Solids to LiquidsChair: Jose Manuel Tamayo, Complexityless Solu-tions, LLC, USA- Flexible Formulation for Soaps to Optimize Cost Jose Manuel Tamayo, Complexityless Solutions, LLC, USA.This interactive session will use a proven model to op-timize the cost of bar soap formulation, based on the fats and oils market price and alternative raw material availability. This lecture also provides an overview of the key process fundamentals required to optimize and improve the manufacturing plant’s flexibility and output.- Computer Monitoring System for Soap Dryers and

Finishing LinesPablo Felipe Quintero, Hada S.A., Colombia.- Above and Beyond Bars - Welcome to Liquids

TechnologyJose Manuel Tamayo, Complexityless Solutions, LLC, USAWhen moving from solids - bar soap to liquids - there are a lot of challenges to overcome to obtain a clear picture of what is key to make this move more effec-tive and productive. Such as reducing the cost im-plications in formula and manufacturing processing. This presentation will analyze a basic formula to make body wash, highlighting the key cost drivers as well as the equipment required, then overview the market trends for liquid hand soap and body wash products in the USA.- Liquid Detergents Technology - What You Need to KnowJames Cush, Independent Home Care Supply Chain Consultant, USA.Consumer product giants, or young, growing contrac-tor manufacturers, in the household liquid detergent arena are subject to many factors which make serv-ing the marketplace complex. This, in turn, requires an in–depth analysis of configuring the manufacturing operation to be flexible and efficient all while address-ing the demands of your customers. This presenta-tion will discuss many factors which need to be con-sidered for liquid detergent production both now and in the future.- Innovative New Ingredients and Technologies for

Personal CareShyam Gupta, Bioderm Research, USAReview the exciting new and unusual ingredients and technologies for personal care. Today’s topics of current high consumer interest are skin rejuvenation, acne, skin clarification, and skin brightening. The pre-sentation will also include a discussion on the poten-tial, albeit futuristic, application of blue-sky pathways such as topical growth factors, stem cell therapies, senescense, autophagy, apoptosis, and mitochon-dria in personal care research. The emerging tech-


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iario nologies in this presentation have the potential for the

development of innovative, on-the-horizon skin care formulations.For information: Meeting Manager: Connie Hilson [email protected]

World Congress on Oils & Fats and 31st ISF Lectureship Series“Evoluzione, innovazione e sfide per un Futuro Sostenibile” October 31 - November 4, 2015 - Rosario ArgentinaIl Congresso è l’nvito ad uno scambio e ad un aggior-namento delle conoscenze nel campo di applicazione di oli e grassi rivolto a specialisti, ricercatori, profes-sionisti, studenti e aziende.Sta emergendo una nuova coscienza: lavorare per ri-durre al minimo l’impatto ambientale attuando un pia-no che si rivolgerà all’uso efficiente delle risorse; as-sumere un ruolo forte nella sensibilizzazione di questo problema attraverso i canali di comunicazione.L’obiettivo è quella di organizzare un evento per la-sciare il segno.Per informazioni sul programma scientifico:http://www.asagaworldcongress.org.ar/index.php/es/

AOCS Oils and Fats World Market Update 201512-13 November 2015 - Dublin, IrelandThe Convention Centre Dublin “The AOCS Oils and Fats World Market Update 2015 is designed to help you stay ahead of the compe-tition. Network and learn about the latest industry trends, unique opportunities, and practical tools and techniques designed to strengthen your business.”Mark your calendar for this premier leadership event created for oils and fats senior executives, traders, suppliers, producers, and processors from food and non-food companies around the world.

SCS FORMULATE 201517-18 November 2015SCS Formulate is the UK’s largest event of its kind, focusing on raw materials, ingredients and formula-tion services for personal care and cosmetic prod-ucts. It’s a unique opportunity to see the new, the in-novative, the proven, the everyday and the obscure – everything you need to create, make and market cosmetics for today and tomorrow.www.ifscc2015.comSave the date

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2015

RISG LA RIVISTA ITALIANA

DELLE SOSTANZE GRASSE

DIVISIONE

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La Rivista Italiana delle Sostanze Grasse La collaborazione a La Rivista Italiana delle Sostanze Grasse è aperta a tutti gli studiosi, ricercatori e tecnici, italiani ed esteri, che riferiscano su studi originali a carattere sperimentale, tecnologico o divulgativo, su oli e grassi alimentari ed industriali di origine vegetale o animale, detersivi, tensioattivi, prodotti cosmetici, oli minerali, prodotti vernicianti e sui loro impieghi nel settore alimentare, mangimistico e dell’industria in genere. Nella rubrica «Comunicazioni brevi» possono essere accolte brevi comunicazioni sui primi risultati di ricerche in corso. Tutti i lavori ricevuti vengono esaminati da un Comitato di referee al cui parere é subordinata l’accettazione per la pubblicazione. Nella lettera di presentazione del lavoro chiediamo di indicare tre nominativi di esperti qualificati come referee. Gli autori che presentano i lavori sulla nostra rivista sono autorizzati a pubblicare l’estratto del lavoro, in formato PDF e in versione full text , sulla propria pagina di Research Gate, aumentando così la diffusione e la visibilità dei risultati delle proprie ricerche scientifiche.

Compilazione dei lavori Gli Autori devono inviare i lavori con 2 file distinti (testo e tabelle/figure) via e-mail, in Word per Windows. L'articolo deve contenere: 1) Introduzione (i motivi della ricerca e i riferimenti alla letteratura) 2) Parte sperimentale (con una descrizione dettagliata della metodologia applicata) 3) Risultati e discussione.

Gli Autori sono invitati ad attenersi strettamente alle disposizioni indicate. Testo (font: arial, font size: 10). Gli autori devono inviare i lavori via e-mail, in Word per Windows. Si chiede di inviare il testo, le eventuali tabelle e le figure con files separati. I lavori devono essere corredati dei rispettivi brevi riassunti in italiano ed inglese e da un ampio sommario in inglese quando il testo del lavoro è in italiano, in italiano se il lavoro è in lingua inglese. I lavori devono essere completi di riferimenti a tabelle e figure. La prima pagina deve contenere: - il titolo del lavoro, - i nomi e cognomi degli autori, - il riferimento all’Ente di appartenenza e città; per l’autore corrispondente anche indirizzo,

e-mail e numero di telefono. Se presentati in lingua inglese i lavori devono essere scritti in linguaggio corretto, chiaro e conciso. Bibliografia: La bibliografia, posta sempre al termine dell’articolo, deve essere numerata progressivamente tra parentesi quadre; i numeri di riferimento devono essere inseriti nel testo tra parentesi quadre. Per ogni riferimento bibliografico vanno indicati nell’ordine: - nomi degli autori (iniziale del nome, cognome per intero); - titolo della pubblicazione; - nome della rivista (per esteso od opportunamente abbreviato); - numero del volume in corsivo; - numero della prima ed ultima pagina dell’articolo; - anno solare (tra parentesi) Ad esempio: [1] O. Rossi, A. Bianchi. Titolo della pubblicazione. Riv. ItaI. Sostanze Grasse 70, 52-56 (1993).

Figure, illustrazioni e tabelle (font: arial narrow, font size: 9): Le tabelle vanno numerate progressivamente con numeri romani. Le figure e illustrazioni vanno numerate progressivamente con numeri arabi. le figure ed illustrazioni devono essere in bianco e nero; devono presentare linee nitide e marcate. Le diciture e le didascalie devono essere nella stessa lingua dell’articolo. Le figure e le illustrazioni verranno ridotte in misura 8 cm (verificare pertanto che all’interno le scritte, così ridotte, risultino leggibili); se estremamente dettagliate verranno pubblicate in misura di 16,5 cm (controllare comunque che risultino leggibili). Di regola le bozze di stampa sono inviate agli Autori una sola volta. Gli Autori non hanno spese di pubblicazione. Sono tuttavia a loro carico: - le spese per il rifacimento delle figure, qualora gli originali dessero riproduzioni scadenti - le spese per sensibili modifiche apportate all’atto della revisione delle bozze.

Dopo la pubblicazione verrà inviata agli Autori una copia della rivista e il file in formato PDF dei lavori come estratto.

LA RIVISTA ITALIANA DELLE SOSTANZE GRASSE

Redazione: Via Giuseppe Colombo, 79 - 20133 Milano - Italy e-mail: [email protected] - www.innovhub-ssi.it

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ADEMILUYI A.O. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59AKDOWA E.P. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 187ALFEI B. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35AL-OKBI S.Y. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47AMARAL J.S. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 261AMMAR N.M. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47BAGLIO D. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3BALZANO M. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 167BARBIERI S. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103BENDINI A. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103BENDINI A. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 147BERARDINELLI A. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 147BOSELLI E. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 167BOUBA A.A. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 187BOUCHEFFA S. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 177BROGI P. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31CANE A. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31CAVALIERI A. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3CEVOLI C. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 147CONTE L. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21DE CESAREI S. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3DE MAURI A. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31DESANTIS D. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31DESOKY A.H. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47DI BLASI A. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31DI GIACINTO L. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35DI GIACINTO L. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117DI LORETO G. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35DI LORETO G. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117DI SERIO M.G. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117EL BAKRY H.F. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47FALADE A.O. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59FALCONE P.M. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 167FIORI F. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 167FOLEGATTI L. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3FRANCESCHI C. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31FREGA N.G. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75FREGA N.G. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 153FREGA N.G. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 167FUSARI P. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3FUSARI P. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15GALLINA TOSCHI T. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21GALLINA TOSCHI T. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103GALLINA TOSCHI T. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 147GERTZ C. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 241GIANSANTE L. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35GIANSANTE L. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117GIUFFRÉ A.M. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 221

GUILLAUME C. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 241HAMED I.M. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47HELAL A.M. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47IANNUCCI E. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117JUHAIMIC F.AL. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 255LACCHERI E. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 147LANZA B. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117LEONARDI M. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31LERCKER G. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77LINDER M. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129LUNETTI L. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31MAFRA I. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 261MAINA R. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 211MARIANI C. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21MATTHÄUS B. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 255MEAHNNI A.E. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 187MOHAMED D.A. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47MORADI L. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103MOZZON M. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 153NAVOLIO M. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31OBOH G. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59ÖZCAN M.M. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 255PACETTI D. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 153PACI V. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31PALAGANO R. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103RAGNI L. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 147RAVETTI L. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 241RENNA M. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31ROVELLINI P. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3ROVELLINI P. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15ROVELLINI P. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 177RUGGERI P. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 211RUSSI F. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117SALA M. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 211SERANI A. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31SOARES S. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 261TAGLIABUE S. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21TAMENDJARI A. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 177 TAORMINA F. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 211TCHOUANGUEP MBIAPO F. . . . . . . . . . . . . . . . . . . . . . . 129TENYANG N. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129TIENCHEU B. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129VALLI E. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103VALLI E. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 147VENTURINI S. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3VENTURINI S. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15VENTURINI S. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 177VILLENEUVE P. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129W.H. EL-REFFAEI . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 187WOMENI H.M. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129


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Pag.

Caratterizzazione chimica della farina ottenuta dopo la spremitura a freddo dei semi di Cannabis sativa L. 3

Nota breve. Oli di semi: profilo trigliceridico e qualità nutrizionale 15

Alchil esteri e composti correlate in oli d’oliva vergini: loro evoluzione nel tempo 21

Nota tecnica. Considerazioni tecniche in merito all’abbassamento del limite degli stigmastadieni negli oli di olive extra vergini e vergini.Evidenze analitiche emerse dal test inter-laboratorio 31

Alchil esteri ed altri indicatori per la tutela della qualità e della genuinità degli oli extra vergini italiani 35

Egyptian rice bran oil: chemical analysis of the main phytochemicals 47

Effect of thermal oxidation on the physico-chemical properties, malondialdehyde and carotenoid contents of palm oil 59

La scuola dell’Università di Bologna sulla rete scientifica nel settore dei lipidi 77

Quality evaluation of sunflower and hazelnut cold-pressed oils by sensory approach 103

Evaluation of the nutritional value of oven-dried table olivers (cv. Majatica) processed by the Ferradina style 117

The chemical composition, fatty acid, amino acid profiles and minerals content of six fish species commercialized on the Wouri river coast in Cameroon 129

The study of measurement systems for the assessment of basic qualitative and compositional parameters, based on the interaction of electromagnetic fields with oil products 147

Approccio tecnologico innovative nella produzione degli oli di semi 153

Effects of malaxation time on the quality of extra virgin olive oil from the Ascolana tenera olive variety 167

Composition and antioxidant activity of some Algerian wild extra virgin olive oils 177

Enzyme aqueous extraction of Moringa oleifera and Canola seed oils and their effect on physiochemical characteristics and stability 187

Nota Tecnica. Lubrificanti. Corrispondenza tra metodi analitici (gennaio-dicembre 2014) 211

Variation in triacylglycerols of olive oils produced in Calabria (Southern Italy) during olive ripening 221

Pyropheophytin a and 1,2 di-acyl-glycerols in natural olive oils under different storage conditions over time 241

Some physico-chemical properties and composition in wild olive (Olea europaea L. subsp. oleaster) fruit and oil 255

Assessing the variability of the fatty acid profile and cholesterol content of meat sausages 261

• • • • • • • • • • • • • NOTiZiariOEditoriale

Dal 1963 al 2013, da Umberto Pallotta, Pompeo - Capella ed Edoardo Turchetto in poi: i lipidi in 50 anni di ricerca 75

Indice annata 2013 71

In Biblioteca - Libri

E-Book – Sustainable Development in chimical - engineering: Innovative technologies.V. Piemonte, M. De Falco, A. Basile 199E-Book – Biosurfactants. From genes to - applications. G. Soberón-Chavéz 200

- Notizie in breveSicurezza alimentare: i metalli pesanti negli alimenti - e nei mangimi animali 200Mangimi : approfondimenti e novità normative.- Assalzoo, Bologna, 8 maggio 2013 202Convegno Nazionale “1963-2013 i lipidi in 50 anni di - ricerca”. Ancona, 10-11 ottobre 2013 202Federazione Europea per lo Studio dei Lipidi di - (Eurofedlipid). Antalya, Turchia 27-30 ottobre 2013 202I grassi in cucina e nei trasformati alimentari. Torino, - 26 Marzo 2014, Lab. Chimico CCIAA 2022- ed. Convegno Food Pack Today. Milano 27 febbraio 2014 276Riunione Codex Alimentarius – Comitato Contaminanti - negli alimenti. Roma 25 marzo 2014 277Accademia dei Georgofili - Inaugurazione 261° anno - accademico. Firenze 25 marzo 2014 277Riunione CEN TC 19/JWG1 – Biodiesel analytical - methods. Amsterdam 22 settembre 2014 27767- th plenary meeting of the Scientific Panel on Contaminants in the food chain (CONTAM-EFSA). Parma 30 settembre - 2 ottobre 2014 277Workshop on Biolubricants Marketing, Uses and - Chemistry. Milano 9-10 ottobre 2014 277Riunione degli esperti chimici – Consiglio Oleicolo - Internazionale COI. Madrid 2-3 ottobre 2014 278IV Workshop – Laboratori Nazionali di Riferimento - (LNR) per metalli pesanti negli alimenti e mangimi e additivi nei mangimi. Torino, 6-7 novembre 2014 278

Il Settore tecnologie olearie e oleochimiche di INNOVHUB–SSI – Divisione SSOG è attivo da circa trenta anni ed è stato creato con lo scopo di fornire assistenza e servizi alle industrie che producono, trasformano o utilizzano sostanze grasse a scopo alimentare o industriale. In particolare: Collaborazione con le industrie interessate allo

sviluppo di nuovi prodotti e processi o per l’ottimizzazione di quelli attualmente esistenti;

Consulenza per quesiti di tipo tecnologico che si possono presentare nella gestione delle attività produttive dell’industria di estrazione, raffinazione, trasformazione, impiego di sostanze grasse e derivati.

Partecipazione a Progetti di Ricerca Nazionali ed Internazionali

Formazione di personale tecnico mediante corsi specifici anche in loco

Messa a punto di metodiche analitiche ad hoc

Prodotti sottoposti ad analisi Biodiesel (FAME – Fatty Acids Methyl Esters) Miscele gasolio/biodiesel Biolubrificanti a base lipidica Glicerolo a diverso stadio di raffinazione e di

diversa origine Poligliceroli Sostanze grasse non convenzionali (lipidi

rigenerati, lipidi da alghe, microrganismi e insetti)

DISTILLATORE MOLECOLARE UIC mod. KDL 5

Contatto Paolo Bondioli

Settore tecnologie olearie e oleochimiche Tel. +39 02 70649765

E-mail: [email protected]

INNOVHUB - Stazioni Sperimentali per l’IndustriaAzienda Speciale della Camera di Commercio di Milano

Divisione SSOGVia Giuseppe Colombo 79 - 20133 MILANOTel. +39 02 7064971 - Fax +39 02 2363953

e-mail: [email protected] - sito web: www.innovhub-ssi.it

Documents

ANNOextranet.innovhub-ssi.it/allstd/risg/2015-RISG 1.pdf · aqueous samples by HPLC with UV and evaporative detectors is described. The procedure The procedure is based on a Liquid-Liquid