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A. Cecchetti, S. De Cesarei, 143 Determinazione delle masse molecolari di piccoli peptidi edM. Cardillo idrolizzati proteici mediante elettroforesi capillare con la

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Comitato scientifico di refereeR. APARICIO: Istituto de la Grasa y sus Derivados – Siviglia (E)B. BERRA: Istituto di Fisiologia – Facoltà di Farmacia – Università di MilanoF. CAMURATI: MonzaA. CERT: Instituto de la Grasa y sus Derivados – Siviglia (E)E. CHRISTOPOULOU: Hellenic Republic Ministry of Finance – G.S. of Consumer –Directorate Technical Control – Atene (Gr)G. CONTARINI: Istituto Lattiero Caseario – LodiL. CONTE: Dipartimento di Scienza degli Alimenti – Università di UdineN. CORTESI: MilanoG. DONATI: Istituto Superiore Sanità – RomaH.J. FIEBIG: Federal Research Centre for Nutrition and Food – Institute for Lipid Research – Münster (D)C. GIGLIOTTI: Dipartimento di Scienze Biomediche e Biotecnologiche – Università di BresciaK. GROB: Kantonales Laboratorium – Zurigo (CH)F. LACOSTE: Institut des Corps Gras – ITERG – Pessac (F)G. LERCKER: Dipartimento di Scienze Alimentari – Università di BolognaL. MANNINA: Facoltà di Agraria – Università degli Studi di CampobassoR. SACCHI: Dipartimento Scienze Alimenti - Università Federico II - Portici (NA)C. SCESA: Corso di Laurea in Tecniche Erboristiche – Facoltà di Farmacia – Università di UrbinoM.SERVILI: Dipartimento di Scienze Economico-Estimative e degli Alimenti - Università di PerugiaL. SISTI: Henkel – Divisione Tensioattivi – Lomazzo (CO)E. TISCORNIA: GenovaT. ZELINOTTI: Roma

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IM PA C T FA C T O R 2007: 0,244

Determinazione delle massemolecolari di piccoli peptidi ed

idrolizzati proteici medianteelettroforesi capillare con la tecnica

Dynamic Sieving of SDS - ProteinComplexes

A. CECCHETTI, S. DE CESAREI,M. CARDILLO

STA Z I O N E SP E R I M E N TA L E P E R L E

IN D U S T R I E D E G L I OL I E D E I GR A S S I ,MILANO

PER LA DETERMINAZIONE DELLE MASSE MOLECOLARI DI PEPTIDI ED IDROLIZZATI PROTEICI ABASSA MASSA MOLECOLARE (Mr 2.500-17.000 DALTON), È STATA IMPIEGATA LA TECNICADSCE MEDIANTE ELETTROFORESI CAPILLARE. I TAMPONI PER LA SEPARAZIONE SONO STATIPREPARATI IN LABORATORIO: UN POLIMERO È STATO INTRODOTTO NEL TAMPONE DI SEPARA-ZIONE IN MODO DA CREARE, ALL’INTERNO DEL CAPILLARE STESSO, UN SISTEMA DINAMICO DISETACCI SPECIFICI PER LA SEPARAZIONE DI PROTEINE A BASSA Mr. SONO STATE STUDIATE LEPRESTAZIONI DELLE DIFFERENTI PREPARAZIONI POLIMERICHE IN RELAZIONE AL TIPO DI POLI-M E RO U T I L I Z Z ATO, A L LA S UA CO NC E N T R A Z I O N E, A L LA CO NC E N T R A Z I O N E D E L TA M P O N E D ICORSA ED ALLA CONCENTRAZIONE DEL MODIFICATORE ORGANICO AGGIUNTO. I TAMPONI PREPARATI NON SONO STATI IDONEI PER LA DETERMINAZIONE DI Mr AL DI SOTTODI 2500 DALTON.

DETERMINATION OF THE MOLECULAR WEIGHT OF PEPTIDES AND HYDROLYZEDPROTEINS BY CAPILLARY SIEVING ELECTROPHORESISDSCE TECHNIQUE BY CAPILLARY SIEVING ELECTROPHORESIS WAS USED TO DETERMINE LOWM O L E C U LA R W E I G H T ( Mr 2 . 5 0 0 - 17.000 DA LTO N) O F P E P T I D E S A N D H Y D RO LY Z E D P RO-TEINS, USING DIFFERENT POLYMERS AS A SIEVING MEDIUM IN THE BUFFER UNDER OPTIMI-ZED CONDITIONS. SIEVING BUFFERS PREPARED WERE UNSUITABLE FOR THE SEPARATION OFPEPTIDES AND HYDROLYZED PROTEIN AT Mr UNDER 2.500 DALTON.

CORRISPONDENZA: DR.SSA MARINA CARDILLO,E-MAIL: [email protected]

L A R I V I S T A I T A L I A N A D E L L E S O S T A N Z E G R A S S E - V O L . L X X X V I - L U G L I O / S E T T E M B R E 2 0 0 9

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INTRODUZIONE

P roteine a bassa massa molecolare (Mr), piccolipeptidi ed idrolizzati proteici di origine vegetale, tro-vano un largo impiego nella preparazione di pro-dotti cosmetici [1-3], mentre quelli di origine anima-le vengono per lo più impiegati in agricoltura comefertilizzanti [4]. La conoscenza delle masse moleco-lari relative è importante ai fini di un loro adeguatoimpiego. Per la determinazione delle Mr di pro t e i n e ,in alternativa alla tecnica tradizionale di elettro f o re s isu gel di acrilammide con sodio dodecilsolfato( S D S - PAGE), lunga e macchinosa, [5,6] viene utiliz-zata la tecnica “Dinamic Sieving of SDS-Pro t e i nComplexes” (DSCE) mediante Elettro f o re s iC a p i l l a re. Tale tecnica utilizza tamponi di separazio-ne contenenti polimeri, tali da cre a re un sistemadinamico di setacci all’interno del capillare. Aseconda del tipo di polimero impiegato, della sualunghezza e della sua concentrazione, si ottengonod i ff e renti tipi di setacci, specifici per la separazionedi peptidi nel range relativo alle rispettive Mr.

In un precedente lavoro [7], per la separazione dicomposti ad alta Mr (14.000-200.000 Dalton) erastata messa a punto una metodica perE l e t t ro f o resi Capillare mediante la tecnica DSCE,impiegando un tampone commerciale contenenteun sistema polimerico specifico per la separazionedi composti in tale range di Mr. In questo lavoro lostudio è indirizzato alla preparazione in laboratoriodi tamponi contenenti diff e renti sistemi polimerici,quali destrano e polietilenglicole, adatti a separarep roteine, peptidi ed idrolizzati a bassa Mr, nelrange 2.512 -16.949 Dalton, poiché in commerc i o ,attualmente, non sono reperibili tamponi che off ro-no tali prestazioni.

Le preparazioni polimeriche sono state studiatein relazione alla diff e rente concentrazione dei poli-meri, alla diff e rente forza ionica dei tamponi utilizza-ti ed alla diff e rente concentrazione del glicero l oaggiunto come modificatore organico.

PARTE SPERIMENTALEStrumentazione

La determinazione delle masse molecolari (Mr)dei campioni in esame è stata eseguita con la tec-nica SDS Protein Complexes by Dynamic Sieving(DSCE) mediante Elettro f o resi Capillare Biofocus®

3000 System (Bio-Rad) dotata di rivelatore UV- v i s i-bile, collegata ad un computer.

Materiali• acido benzoico, 2-mercaptoetanolo e sodio

dodecil solfato (SDS) forniti da Merck;• acido borico, tris idro s s i m e t i l - a m m i n o m e t a n o

( Tris), Tris-HCl, destrano (Mr 70.000, 500.000,2.000.00), polietilenglicole (PEG) a Mr 35.000, gli-cerolo forniti da Fluka;

• miscela di peptidi standard ottenuta da mioglobi-na di cavallo, fornita da SIGMA, costituita da unaserie di peptidi aventi le seguenti Mr: 2.512 -6.214 - 8.159 -10.701- 14.404 -16.949;

• serina con Mr 105, arginina con Mr 174, glutatio-ne con Mr 307 forniti da Merck;

• campioni analizzati: idrolizzati proteici di farine diorigine vegetale ed animale.

MetodiCondizioni operative dello strumento

Per le sperimentazioni condotte con la tecnicaDSCE in presenza di SDS, sono state utilizzate leseguenti condizioni operative:• c a p i l l a re in silice fusa non rivestito, della lunghezza

di 25 cm (distanza dal detector 20 cm) avente und i a m e t ro interno (ID) di 50 µm BioCap T M (Bio Rad);

• tempo di iniezione di 100 psi/sec;• voltaggio di 15 KV; • polarità da negativa a positiva; • lunghezza d’onda di 220 nm; • tempo di corsa 20 min; • la temperatura del capillare è mantenuta tra 22-

24 °C per minimizzare la diffusione delle bande edassicurare una separazioni di Mr effettiva.

Prima di ogni corsa sono stati effettuati 5 cicli dipre-iniezione per lavare e riequilibrare il capillare:- 600 sec H2O - 600 sec NaOH 0,1 N- 300 sec H2O- 300 sec tampone di lavaggio senza polimero - 600 sec tampone di corsa con polimero.

Preparazione della miscela standard e del campione

La miscela di peptidi standard ed i campioni inesame, prima della determinazione elettro f o re t i c a ,sono stati sottoposti ad una procedura che conver-te le proteine in complessi SDS-proteine.

PREPARAZIONE DELLA MISCELA STANDARD

10 ml della soluzione standard (concentrazione di20 mg/ml) sono stati posti in una microprovetta da


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0,5 ml alla quale sono stati aggiunti 10 ml di acidobenzoico (1mg/ml), 5 ml di 2-mercaptoetanolo, 100ml di tampone di solubilizzazione del campione e15 ml di acqua bidistillata. La microprovetta è stataimmersa in un bagnomaria ad una temperatura di100°C per 10 minuti. Dopo raffreddamento, la solu-zione è stata centrifugata per 2 minuti ed analizzata.

PREPARAZIONE DEI CAMPIONI

I campioni sono stati preparati in maniera analogaalla soluzione standard ad una concentrazione fina-le di proteine di 1 mg/ml.

Le soluzioni preparate possono essere conserva-te a -20°C e riutilizzate, dopo scongelamento, almomento dell’analisi.

Preparazione dei tamponi di solubilizzazione e

di separazione

• Il tampone di solubilizzazione del campione ècostituito da tris-HCl 100 mM a pH 9 con aggiun-ta di 1% SDS.

• I tamponi di separazione sono costituiti da tam-pone tris-borato a pH 8,4 con concentrazionevariabile da 0,3 – 0,5 M, SDS 0,1 % e glicero l oaggiunto in percentuale dal 10% al 12 %. Come polimeri sono stati utilizzati destrano a dif-

ferenti Mr e polietilenglicole a Mr di 35.000, discioltinel tampone nelle percentuali del 10% e del 12%.

RISULTATI E DISCUSSIONE

I polimeri ed i tamponi impiegati nel presente studiosono stati scelti sulla base di dati pubblicati [8-10].

Le prove preliminari di messa a punto delle condi-zioni ottimali di separazione, in funzione delle diffe-renti preparazioni, sono state condotte sulla misceladi peptidi standard con Mr 2.512- 16.949 Dalton.

Dopo le prime sperimentazioni in cui sono statiutilizzati entrambi i polimeri, destrano e PEG, si èdeciso di utilizzare come polimero di separazionesolo il destrano, poiché il PEG ad alte concentrazio-ni è risultato poco solubile nei tamponi.

Relativamente alle preparazioni polimeriche intro-dotte nei tamponi, tra i parametri che influenzano laseparazione dei peptidi sono stati studiati: la diffe-rente Mr del destrano, la concentazione del destra-no, la percentuale di glicerolo aggiunta e la forzaionica dei tamponi impiegati.

Le prove condotte con destrano a basse Mr

hanno evidenziato un basso potere di risoluzione

dei picchi con una sovrapposizione degli stessi. Nelpresente lavoro vengono riportati solamente i risul-tati ottenuti dalle diff e renti prove che utilizzanodestrano con Mr di 2.000.000.

La composizione dei sistemi polimerici pre p a r a t ied utilizzati per le prove è riportata nella Tabella I.

Ef f e t to della co n ce nt razione del destrano con Mr 2 . 0 0 0 . 0 0 0sulla separazione di SDS-peptidi

Gli elettro f e rogrammi riportati nella Figura 1 (A, B)mostrano la relazione tra i tempi di migrazione dei pep-tidi (TM) e la concentrazione del destrano nei tamponidi separazione. Nella soluzione senza destrano non siottiene nessuna eluizione dei picchi entro i primi 20minuti di eluizione. Quando si incrementa la concentra-zione del destrano nel tampone, migliora la risoluzionedei picchi, indicando che la concentrazione del polime-ro di separazione influenza significativamente il poteredi separazione: più è alta la concentrazione del destra-no, più è alto il potere di separazione per i peptidi; siosserva inoltre un aumento del tempo di migrazione.

Effetto della concentrazione del glicerolo sulla separazione diSDS-peptidi

È stato studiato l’effetto del glicerolo sulla separa-zione di SDS-peptidi, comparando i risultati dellaseparazione ottenuta quando si utilizzano le solu-zioni polimeriche senza e con aggiunta di gliceroloin quantità crescenti: se non viene usato il glicerolo iTM diminuiscono, si osserva una sovrapposizionedei picchi con una separazione di 4 peptidi invece

Tabella I - Composizione dei sistemi polimerici preparati in labora-torio che utilizzano destrano con Mr di 2.000.000Tampone tris-borato a pH 8.4 con glicerolo al 10%, senza aggiunta

di destrano

Tampone tris-borato a pH 8.4 con destrano al 12%, senza aggiunta

di glicerolo

Tampone tris-borato a pH 8.4 0.3 M Destrano 10%-glicerolo 10%

Destrano 10%-glicerolo 12%












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di sei, mentre con l’aumentare delle percentuali diglicerolo si ottiene la separazione completa dei seipicchi. L’aggiunta di glicerolo minimizza la diffusionedei picchi, ritarda il TM migliorando la risoluzione deipicchi (Fig. 2 A,B,C).

Ef f e t to della co n ce nt razione della forza ionica del tamponesulla separazione di SDS- peptidi

L’aumento della forza ionica del tampone, da 0,3M a 0,4 M, porta ad una migliore risoluzione deipicchi, mentre si ottiene una separazione meno effi-ciente con tampone 0,5 M. I risultati ottenuti dalle

Figura 1 - Ef f e t to delle differe nti co n ce nt razioni di destrano sullaseparazione dei SDS-peptidi. Tampone tris- borato 0,3 M, pH 8, 4, gli-cerolo 10% . A) destrano 10%, B) destano 12%

Figura 2 - Ef f e t to delle differe nti co n ce nt razioni del glice rolo sullas e p a razione di SDS-peptidi. Tampone tris-borato 0,3 M, pH 8,4,destrano 10%. A) glicerolo 0, B) glicerolo 10%, C) glicerolo 12%

A A

B B

C


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Figura 3 - Ef f e t to delle differe nti co n ce nt razioni del tampone tris-b o rato sulla separazione di SDS. Tampone tris-borato a pH 8,4,d e s t rano 12%, glice rolo 10%. A) tampone 0,3 M, B) tampone 0,4 M,C) tampone 0,5 M

Figura 4 – Ef f e t to delle alte co n ce nt razioni di destrano e glice ro l osulla separazione di SDS-peptidi in relazione all’a u m e nto della forz aionica del tampone. Tampone tris-borato pH 8,4. A) tampone 0,3 M,d e s t rano e glice rolo 12%, B) tampone 0,4 M, destrano e glice ro l o12%, C) tampone 0,5 M, destrano e glicerolo 12%

A

B

C

A

B

C


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I valori dei parametri utilizzati per la costruzionedella retta di calibrazione sono riportati nella TabellaII; la Figura 7 riporta la retta di calibrazione costruitacon tali valori.

Le Mr dei peptidi relativi ai campione di idrolizzatip roteici estrapolate dalla retta di calibrazione sonoriportate nelle Tabelle III e IV.

Tabella II - Valori dei parametri utilizzati per la costruzione dellaretta di calibrazione

TM acido benzoico TM TMn Log Mr Mr

5,2 6,92 1,34 3,40 2,512

5,2 7,10 1,37 3,79 6,214

5,2 7,69 1,48 3,91 8,159

5,2 7,85 1,51 4,03 10,710

5,2 8,32 1,60 4,16 14,404

prove condotte sono riportati nella Figura 3 (A,B,C).Quando si utilizzano contemporaneamente alte

p e rcentuali di destrano e di glicerolo (12%), e siaumenta la forza ionica del tampone, non si ottieneun miglioramento nella risoluzione dei picchi (Fig.4A,B,C). In tali condizioni operative, spesso si verifical’ostruzione del capillare a causa dell’aumento dellaviscosità del tampone ed occorre aumentare iltempo di lavaggio del capillare con il tamponesenza polimero.

Il tempo di lavaggio del capillare è un parametromolto importante da considerare, per questo tipo diseparazioni.

Determinazione delle Mr nel range 2.512- 16.949 Dalton deicampioni di farine vegetali ed animali

Sulla base dei risultati ottenuti utilizzando la miscelas t a n d a rd, per determinare le Mr di peptidi ed idro l i z z a t ip roteici di campioni di origine vegetale ed animale,sono state scelte le condizioni operative che impieganola preparazione polimerica costituita da destrano al12%, glicerolo al 10% e tampone tris-borato con forzaionica 0,4 M a pH 8,4.

Esempi di elettro f e rogrammi relativi a due campionitra quelli esaminati sono riportati nelle Figure 5 e 6.

Le Mr incognite dei campioni sono state calcolate,sulla base dei relativi tempi di migrazione (TM), dall’e-quazione della retta di calibrazione, costruita con i valoridei log Mr dei peptidi dello standard, in funzione dei lororispettivi tempi di migrazione normalizzati (TMn) rispettoall’acido benzoico, utilizzato come riferimento intern o .

Figura 5 – El e t t ro f e ro g ramma re l at i vo ad un idro l i z z ato pro te i co diorigine vegetale. TM acido benzoico=5,4

Figura 6 - El e t t ro f e ro g ramma re l at i vo ad un idro l i z z ato pro te i co diorigine animale. TM acido benzoico=5,4

Figure 7 – Retta di calibrazione relativa alla miscela standard di pep-tidi 2512-16949 Dalton


149

non sulla base delle relative Mr, ma solamente sullabase del loro rapporto carica/ massa.

CONCLUSIONI

I tamponi contenenti destrano come sistema poli-merico, con effetto setacciante all’interno del capil-lare, preparati in laboratorio, sono stati utilizzati perla separazione di piccoli peptidi ed idrolizzati protei-ci sulla base delle rispettive Mr, nel range 2.512-16.949 Dalton, impiegando la tecnica DSCE. Ilsistema polimerico costituito da tampone a concen-trazione 0,4 M, 12% destrano e 10 % glicerolo èquello che ha prodotto la migliore separazione, intale range di Mr, dei peptidi in campioni di idrolizzatiproteici di origine vegetale e animale.

La stessa preparazione polimerica, alle stesseconcentrazioni, non è risultata adatta per la deter-minazione di Mr < a 2500 Dalton. E’ importante sot-tolineare che la separazione di proteine in base alleMr con la tecnica elettroforetica DSCE dipende daltipo di setaccio creato all’interno del tampone, chesepara le proteine solo in un definito range di Mr.

BIBLIOGRAFIA

[1]G. Secchi, Clinics in Dermatology, 26, pag. 321-325, Elsevier (2008)

[2]G. Zocchi, Handbook of Cosmetic Science andTechnology. Informa Healthcare, 3a edition, 2009

[3]J.A. Swift, S.P. Chahal, N.I. Challoner, J.E.Parfrey, Journal of Cosmetic Science, 51 (3) 193-203 (2000)

[4]J.M. Choi, P. V.Nelson, American Society forHorticultural Science, 121, 634-638 (1996)

[5]A.L. Shapiro, E. Vinuela, J.B. Maizel, Biochem.Biophys. Res. Commun., 28, 815 -820 (1967)

[6 M. Osborn, K. Weber, J. Biol. Chem., 244, 4406-4412 (1969)

[7]M. Cardillo., A. Cecchetti, S. De Cesarei, Riv. Ital.Sostanze Grasse 8 2 (maggio/giugno) 123-128(2005)

[8]Y. Zhang, H.K. Lee, Sam F. Y. Li, Journal ofChromatography A., 744, 249-257 (1999)

[9]M.D. Zhu, D. L. Hansen, S. Bund, F. Gannon,Journal Chromatography, 480, 311 (1989)

[10] K. Ganzler, K.S. Greve, A. S. Greve, A. S.Cohen, B.L. Karger, A. Guttman, N. C. Cook,Anal. Chem., 64, 2665 (1992)

La risoluzione del minimo picco, nelle condizionioperative descritte, è risultato essere circa 2.500Dalton. Sono state eseguite successivamente alcu-ne prove per cercare di determinare Mr inferiori, uti-lizzando il sistema polimerico di separazione nelrange 2.512-16.949 Dalton, facendo però riferimen-to ad una retta di calibrazione costruita con stan-dard a Mr nel range 105-1.759 Dalton. Ogni stan-dard è stato iniettato nel capillare separatamente.

Nella tabella V sono riportati, per ogni componen-te dello standard, la Mr, il relativo log, il TM ed ilTMn rispetto al TM dell’acido benzoico.

Dai dati ottenuti si osserva che, per la maggiorparte dei piccoli peptidi, i tempi di migrazione nor-malizzati si sovrappongono con quelli dei peptididella miscela standard con Mr più alte.

Tali risultati indicano che, nelle condizioni operati-ve descritte, i peptidi con Mr < a 2.500 Dalton nonsubiscono l’effetto setacciante del sistema polimeri-co creato all’interno del capillare; essi vengono eluiti

Tabella V - Valori dei parametri utilizzati per la costruzione dellaretta di calibrazione nel range 105-1.759 Dalton

TM acido TM TMn log Mr Mr

benzoicoSerina 9,29 10,16 1,09 2,02 105Arginina 9,91 13,78 1,39 2,24 174Glutatione 9,44 15,63 1,66 2,49 307Peptide 1 9,45 14,25 1,51 2,78 604Peptide 2 10,52 18,23 1,73 2,99 970,9Peptide 3 10,49 26,70 2,55 3,24 1759,6

Tabella IV - Mr dei peptidi re l ativi al campione idro l i z z atoproteico animaleTM acido TM TMn m c log M r Mr

benzoico

5,4 6,09 1,13 1,6474 1,5239 3,38 2409

5,4 6,05 1,29 1,6474 1,5239 3,64 4407

5,4 7,48 1,36 1,6474 1,5239 3,81 6395

5,4 7,73 1,43 1,6474 1,5239 3,88 7623

Tabella III - Mr dei peptidi relativi al campione idrolizzato proteicovegetaleTM acido TM TMn m c log M r Mr

benzoico5,4 5,56 1,03 1,6474 1,5239 3,22 1660 *5,4 6,11 1,13 1,6474 1,5239 3,39 24435,4 0,93 1,28 1,6474 1,5239 3,64 43465,4 7,61 1,41 1,6474 1,5239 3,85 70075,4 7,84 1,45 1,6474 1,5239 3,92 8235

* Valore fuori della retta di calibrazione

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THE PRESENT STUDY WAS CARRIED OUT TO ESTIMATE THE EVOLUTION OF OLIVE OIL BIOPHE-NOLS DURING THE RIPENING PROCESS. THE WORK WAS FOCUSED ON FOUR ALGERIAN OLIVEVA R I E T I E S (CH E M L A L, BO U G H E N F O U S, BL A N Q U E T T E and TA K E S R I T). FO R T H E A NA LYS I S,COLORIMETRIC AND HPLC METHODS WERE USED. THE RESULTS SHOWED THAT BOTH MATU-R AT I O N A N D C U LT I VA R S I N F LU E NC E D S I G N I F I CA N T LY T H E Q UA N T I TY A N D T H E Q UA L I TY O FOLIVE OIL PHENOLIC FRACTION. DURING OLIVE MATURATION, ALL VARIETIES REGISTERED AD E C R E A S E O F TOTA L B I O P H E NO LS, H OW E V E R I T I S V E RY I M P O R TA N T TO U N D E R L I N E T H ATTHIS DECREASE DIFFERED IN THE AMOUNT FROM ONE CULTIVAR TO ANOTHER. CV. CHEMLALHAD THE LEAST IMPORTANT DIMINUTION.KEYWORDS: OLIVE MATURATION, BIOPHENOLS, ALGERIAN OLIVE OIL

EVOLUZIONE DEI COMPOSTI BIOFENOLICI IN OLI VERGINI DI OLIVA DURANTE LAMATURAZIONE DELLE OLIVE DI CULTIVARS ALGERINEIL PRESENTE LAVORO È STATO CONDOTTO CON LO SCOPO DI VALUTARE L’EVOLUZIONE DEI BIO-F E NO L I N E G L I O L I D I O L I VA D U R A N T E I L P RO C E S S O D I M AT U R A Z I O N E. IL LAVO RO È STATOFO CA L I Z Z ATO S U Q UAT T RO VA R I E TÀ A LG E R I N E (CH E M L A L, BO U G H E N F O U S, BL A N Q U E T T E eTAKESRIT). PER LE ANALISI SONO STATI UTILIZZATI SIA I METODI COLORIMETRICI CHE HPLC.I RISULTATI HANNO MOSTRATO CHE SIA LA MATURAZIONE, SIA LA CULTIVAR INFLUENZAVANOS I G N I F I CAT I VA M E N T E LA Q UA N T I TÀ E LA Q UA L I TÀ D E L LA F R A Z I O N E F E NO L I CA D E G L I O L I D IO L I VA. DU R A N T E LA M AT U R A Z I O N E D E L L E O L I V E, T U T T E L E VA R I E TÀ S U B I VA NO U N D E C R E-M E N TO D E I B I O F E NO L I TOTA L I, Q UA N T I TAT I VA M E N T E D I F F E R E N T E DA U NA C U LT I VA R A L L’ALTRA. LA CV. CHEMLAL MOSTRAVA LA DIMINUZIONE MENO SIGNIFICATIVA.PAROLE CHIAVE: MATURAZIONE DELLE OLIVE, BIOFENOLI, OLI DI OLIVA ALGERINI

Evolution of biophenolic compoundsin virgin olive oil during olive ripening of Algerian cultivars

R. LARIBI1*, P. ROVELLINI2,L. DEFLAOUI1, A. AIDLI1, S. METTOUCHI1, L. ARRAR3,A. TAMENDJARI1

1FACULTY OF NATURE AND LIFE SCIENCES, LA BO R ATO RY O F A P P L I E D B I O CH E M I ST RY,A/MIRA UNIVERSITY, BEJAIA, ALGERIA2STA Z I O N E SP E R I M E N TA L E P E R L E

IN D U S T R I E D E G L I OL I E D E I GR A S S I ,MILAN, ITALY3LABORATORY OF APPLIED BIOCHEMISTRY,DEPARTMENT OF BIOLOGY, FERHAT ABBAS

UNIVERSITY, SETIF, ALGERIA

*) CORRESPONDING AUTHOR:

LARIBI RAHIMA, FACULTY OF NATURE AND LIFE SCIENCES,DEPARTMENT OF FOOD SCIENCES, A/MIRA UNIVERSITY,

BEJAIA 06000, ALGERIA.

TEL. +213 34 21 43 33 À 35,FAX +213 34 21 60 98

E-MAIL: [email protected]


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1. INTRODUCTION

Biophenols, present in virgin olive oil, constitute awide class of secondary metabolites, derived fromoleuropein and ligstroside, which have an importantrole in human health. In recent years the interest inphenolic compounds has increased, consideringtheir potent biological activities [1, 2].

Olive oil is one of the food-stuffs which has attrac-ted considerable attention as a source of biophe-nols [3-6]. They contribute to the flavor, stability andnutritional value of oil [7, 8].

The olive oil content of phenolic compoundsdepends on the cultivar, climatic conditions, degre eof maturation and the technology used for oil extrac-tion [9 - 12]. Maturation degree is one of the factorswhich are studied by several works, especially theinfluence on evolution of phenolic compounds eitherin olives [13 - 15], or in olive oil [15-19].

A c c o rding to Uceda and Frias [20], there are fourolive ripening stages which are, green, spotted, pur-ple and black. During ripening, the concentration ofbiophenolic compounds pro g ressively increases untilit reaches a maximum at the ‘spotted’ and ‘purple’pigmentation stage, after which it decreases [21-23].

D i ff e rent studies focused on biophenolic com-pounds of virgin olive oil, were conducted in particu-lar on the oxidized biophenols [24], secoiridoidsacids [25], lignanes and flavonoids [26]. However,t h e re has been only a little re s e a rch on phenoliccompounds of Algerian olive oil varieties. The aim ofthis work was to characterize the biophenol profile ofAlgerian olive oil varieties and to determine how theolive maturation influenced them.

2. MATERIAL AND METHODS

2.1 Fruit harvest and sorting out of olivesThe study was carried out during the 2006/2007

olive collection season. Olive fruit samples from fourvarieties (Chemlal, Boughenfous, Blanquette de

Guelma and Ta k e s r i t) were manually collected fro molive trees in two diff e rent locations. Olive fruits fro mC h e m l a l v a r i e t y, the most widespread in Algeria( m o re than 50% of the olive groves), were harvestedin the region of Tazmalt (Bejaia) in east-centralAlgeria. Boughenfous, Blanquette and Ta k e s r i t v a r i e-ties were collected from an olive orc h a rd maintainedby ITAFV of Ta k a r i e t z, located in Sidi Aich, Bejaia.

After harvest, olive fruits were immediately tran-

sported to the laboratory and then hand-picked atspotted, purple and black stages during ripening.For Chemlal and B o u g h e n f o u s varieties, a supple-mentary green stage was designed.

2.2 Olive oil extraction Virgin olive oil samples were obtained using a

laboratory oil mill (Levi-Deleo-Lerogsame), consi-sting of three basic elements: a hammer crusher,thermobeater (mixer) and a pulp centrifuge. Olivefruits were milled in the hammer crusher, and thenthe olive paste was kneaded for 30 mn with warmwater addition (50 ml was added to 920 g of paste).After the vertical centrifugation, the oil was collectedand left to decant. The oil samples were stored inamber glass bottles at 4°C in darkness withoutheadspace until analysis.

2.3 Solid phase ext ra ction (SPE) of phenolic compounds forspectrophotometric determination

Extraction of phenols was performed by a solid-phase extraction (SPE) according to Favati et al.[27], using an octadecyl C18 cartridge (J.T. Backer,Milan, Italy). 1 g of olive oil was dissolved in n-hexa-ne (10 ml) and deposited on cartridge pre v i o u s l ywashed with 2*5 ml of MeOH and 2*5 ml of n-hexa-ne. The elution steps were: n-hexane (3*5 ml) toremove lipophilic compounds, methanol (2*4 ml) toremove the polar fraction. The methanolic solutionwas used to measure total phenolic compoundsand o-diphenols.

2.4 Spectrophotometric determination of total phenolsThe total phenol content of extract was determi-

ned by the Folin-Ciocalteu spectro p h o t o m e t r i cmethod at 765 nm, using gallic acid for extern a lstandard calibration curve. The analysis was repea-ted three times for each extract (n=3).

2.5 Spectrophotometric determination of o-diphenolsAccording to Mateos et al. [28], 0.5 ml of phenolic

extract, obtained from olive oil by solid-l iquidextraction, was dissolved in 5 ml of methanol( C H3OH)-water (1:1, v/v); a mixture of 4 ml of thesolution and 1ml of a 5 % solution of sodiummolybdate dihydrate in EtOH-water (1:1, v/v) wasshaken vigorously. After 15 mn, the absorbance at370 nm was measured using caffeic acid for thecalibration curve with a glass cuvette. The analysiswas repeated three times for each extract (n=3).


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analyzed by HPLC with UV detector at 280 nm.The HPLC system used consisted of C18

Spherisorb ODS-2 reversed column (5 µm, 250 mm4.6 mm). Elution was performed at a flow rate of 1ml/mn following a gradient, composed of a mixtureof water and orthophosphoric acid (99,8 : 0,2 v/v)(solvent A), MeOH (solvent B) and acetonitril (sol-vent C): from 96 % (A) – 2 % (B) - 2% (C) to 0% (A)- 50% (B) - 50% (C) in 60 mn. The last gradient waskept for 10 mn. The successively gradient was:from 0 % (A) – 50 % (B) – 50 % (C) to 96 % (A) – 2% (B) - 2% (C) in 2 mn and then kept for 10 mn.

2.6 Chromatographic determination of phenolic compoundsA solution of internal standard (1 ml of 0.015

mg/ml of syringic acid in water/ MeOH (20:80 v/v)was added to the sample of anhydride virgin oliveoil (2 g). The mixture was shaken by vortex (30 s).5ml of extraction solution of water and MeOH(20:80 v/v) were added. The obtained mixture wasagitated for 1mn, extracted for 15mn in an ultraso-nic bath and then centrifuged at 5000 rpm for 25mn [29].

The upper phase was filtered with a 0.45 µmPVDF syringe filter. 20 µl of the filtered solution were

Table I – Total phenols and o-diphenols changes during maturation in different Algerian varieties of virgin olive oil

Determinations StagesVarieties

Chemlal Boughenfous Blanquette Takesrit

Total phenols (mg/kg) Green 628.72 ± 2.91 514.39 ± 4.77 ND ND

Spotted 553.41 ± 4.32 293.36 ± 1.68 444.17 ± 14.90 268.33 ± 0.97

Purple 447.14 ± 4.60 130.23 ± 1.60 381.17 ± 2.20 193.23 ± 0.36

Black 410.87 ± 4.23 77.63 ± 2.29 121.54 ± 8.27 82.08 ± 4.80

o-diphenols (mg/kg) Green 56.36 ± 1.82 16.45 ± 0.63 ND ND

Spotted 61.71 ± 0.46 48.17 ± 0.30 60.19 ± 0.62 32.62 ± 0.17

Purple 9.18 ± 2.14 4.94 ± 0.46 57.36 ± 0.63 16.25 ± 0.69

Black 13.93 ± 0.79 11.91 ± 0.46 14.94 ± 0.46 4.44 ± 0.34

ND: not determined

Figure 1 - Evolution of total phenol content of Algerian virgin olive oil during ripening stages


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the results showed a decreasing trend of o -d i p h e n o l scontent, during the ripening pro g ress for B l a n q u e t t e

and Ta k e s r i t varieties. Yousfi et al. [30] had reported ad e c reasing of o -diphenols content of oil during matu-ration of A r b e q u i n a and Picual olive varieties.

The decrease of total phenols and o -d i p h e n o l scontents of virgin olive oil with olive maturation pro-g ress has been already mentioned by Cerretani e tal. [31] for cv. Nostrana di Brisighella. In the samestudy these authors had reported an increase of o-

diphenol in the last but one stage, with a ripenessindex value of 4.03, for cv. Ghiacciolo.

3.2 HPLC profiles of biophenol The results obtained in this study, concerning the

HPLC profiles of biophenols [24] of Algerian olive oilvarieties, evidenced that this fraction was largelyinfluenced by the variety and the ripening degree.

For the total phenolic compounds content (TableII, Figure 3: a and b), we observed that C h e m l a l

variety contained the greatest quantity for all matu-ration stages. Concerning the spotted stage,Takesrit variety registered the smallest quantity. Forpurple and black stages, the lowest values werenoticed for Boughenfous variety.

3. RESULTS AND DISCUSSION

3.1 Total phenols and ortho-diphenols contentsAs shown in Table I and Figure 1, the total phenols

content in virgin olive oil decreased from the green toblack stage for C h e m l a l a n d Boughenfous and fro mspotted to black stages for Ta k e s r i t and B l a n q u e t t e

varieties. This demonstrated that the maturation pro-cess generated a decreasing of phenol content in vir-gin olive oil. The C h e m l a l olive oil had the highestvalues for all the considered stages. On the otherhand and with exception of the black stage, the oliveoils obtained from Ta k e s r i t p resented the lowest phe-nol content. Only B o u g h e n f o u s and Ta k e s r i t, at theblack stage, re g i s t e red a content value which wasless than 100 mg/kg. Examining the decreasing per-centage of total phenols, we found that B o u g h e n f o u s

had the highest one (84.90 %), followed byB l a n q u e t t e (72.63 %), then Takesrit (69.41 %) andC h e m l a l came in the last position (34.64 %).

The oils from the spotted stage re g i s t e red the maxi-mum score of o -diphenols for all varieties (Table I andF i g u re 2). For C h e m l a l and B o u g h e n f o u s v a r i e t i e s ,after an increase, we observed a decrease of o -d i p h e-nols content from spotted to black stages. In contrast,

Figure 2 - Evolution of o-diphenols content of Algerian virgin olive oil during olive ripening stages


155

those indicated by Oliveras-Lopez et al. [36].As shown in Table II and Figure 4, hydro x y t y ro s o l

was found at low concentration, it constitutedbetween 0.34 % and 2.80 % of total biophenols.C o n c e rning its evolution during maturation, hydro x y t y-rosol contents of C h e m l a l variety at initial harvest was5 mg/kg, then showed a slight increase from green tospotted stages. This increase was followed by ad e c rease through purple and black stages.

However, in Blanquette and Takesrit varieties, nochanges in hydro x y t y rosol concentration wereobserved from spotted to purple stages. After that,there was a decrease from purple to black stagesestimated at 57.14 % and 40 % respectively forBlanquette and Takesrit. Boughenfous oils exhibiteda decrease from green to spotted stages, estimatedat 50 %, it maintained the same concentration inpurple stage and then demonstrated a decrease.

On the other hand, the data also indicated a lowconcentration of tyrosol (Table II and Figure 4) butgreater than hydroxytyrosol. It ranged between 0.64and 19.56 % with regard to total biophenols.

C o n c e rning the evolution during the maturation pro-cess, there were two diff e rent types of behavior. ForC h e m l a l, B o u g h e n f o u s and B l a n q u e t t e varieties, tyro-sol content increased from the first stage to purplestage and then showed a decrease, whereas, Ta k e s r i t

variety exhibited a gradual decrease in tyrosol contentwith maturation pro g ress. So, for C h e m l a l,B o u g h e n f o u s and B l a n q u e t t e, the purple stage coin-cided with the greatest quantity of tyrosol but forTa k e s r i t, it coincided with the spotted stage. Theresults obtained did not agree with Cimato et al. [37],who had reported that the content of tyrosol andh y d ro x y t y rosol increased when the oil was extractedf rom more mature fruits. As reported by Ryan et al.[38], this diff e rence might be due to the varietal factor.

The data shown in Table III illustrate the re s u l t sobtained for biophenol profiles in Algerian virgin oliveoil at diff e rent maturation stages. As can be seen,t h e re exist highlighted diff e rences between diff e re n tvirgin olive oil samples. Oleuropein derivatives werethe main compounds found in C h e m l a l a n dB l a n q u e t t e virgin olive oil varieties. The oil extractedf rom Ta k e s r i t variety presented the highest contentof secoiridoid acids, consisting of elenolic anddecarboxymethyl-elenolic acids [25]. For these thre evarieties, ligstroside derivatives contents came insecond posit ion. For the oi l extracted fro mB o u g h e n f o u s v a r i e t y, there was a predominance of

However, there was a decrease of the amount ofphenols during maturation for all the varieties. Themost important point was the decreasing rate.Passing from the first stage to black stage the mostimportant decrease was for B o u g h e n f o u s ( 8 4 . 0 8%), followed by Blanquette (77.37 %), then Takesrit

(62.99 %) and lastly, we found Chemlal (41.99 %).The results obtained show that the speed of varia-tion of olive oil phenols through the olive maturationcould also be linked to the variety.

The decreasing of total phenols of oil during theolive maturat ion can be explained by:- decreasing of enzymatic activity of L-phenylalani-

ne ammonia-lyase, which is responsible of pheno-lic compounds synthesis [32];

- increasing of hydrolytic activity of enzymes (este-rase and glucosidase) [33];

- oxidation of phenolic compounds by pero x i d a s e[34] and polyphenoloxidase (PPO) [35].H y d ro x y t y rosol and tyrosol are the main simple

phenols found in all the virgin olive oils [36]. Differentauthors have already studied a lot of olive varietiesindicating that the content of these two compoundsranged between 5 % and 23 %, with regard to totalbiophenols.

In our study, we observed the presence ofhydroxytyrosol and tyrosol in all virgin olive oil varie-ties, but their quantities were less important than

Table II - Evolution of total biophenols, hydroxytyrosol and tyrosolconcentrations (mg/kg) with HPLC analysis, in different Algerian cul-tivar extra virgin olive oils

Varieties Stages Total Hydroxytyrosol Tyrosol

biophenols

(mg/kg) (mg/kg) (mg/kg)

Chemlal Green 631 5 4

Spotted 598 6 7

Purple 491 5 9

Black 366 3 8

Blanquette Spotted 473 7 9

Purple 372 7 10

Black 107 3 6

Takesrit Spotted 254 1 14

Purple 155 1 10

Black 94 0.6 8

Boughenfous Green 400 2 7

Spotted 298 1 12

Purple 114 1 21

Black 46 0.3 9


156

Figure 3 (a and b) - Comparison between the total biophenols content, analysed by HPLC, of four Algerian virgin olive oil varieties during diffe-rent maturation stages

l i g s t roside derivatives, followed by secoiridoid acids.Several authors had reported that the secoiridoidicderivatives of oleuropein and ligstroside are often themain phenolic compounds in fresh olive oil [39 - 42].

In Figure 5, it can be seen that the maturationprogress spanned a decrease of oleuropein deriva-tives quantity in all studied varieties. We noted only

one increase of these compounds with 16.66 %,which was between the purple and black stages ofBoughenfous variety. Chemlal variety contained themost important concentration of oleuropein derivati-ves for all stages and this class of biophenols weremore present in this variety than the other analyzedbiophenols. Concerning the level of decrease of


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health significance as reported by several works[25, 36, 41, 42, 44]

Luteolin and apigenin are usually present in extravirgin olive oil in a very low amount (less than 2-3 %of the total polyphenols [42]. In general, the flavo-noids were the least present compounds amongthe analyzed biophenol. The highest value was 31mg/kg in Chemlal and Boughenfous varieties. Thisbiophenol class re g i s t e red diff e rent speed duringmaturation progress: Boughenfous recorded a dimi-nution whereas Chemlal registered an increase. Onthe other hand, we found an increase followed by ad e c rease of these compounds for Ta k e s r i t a n dBlanquette varieties. This latter had the low level (4mg/kg) at black stage but Ta k e s r i t totalized themost important percentage of flavonoids withregard to total biophenols (15.95 % at black stage).

The lignans, a class of phenolic compounds,were found in notable concentration with regard tothe total polyphenol and C h e m l a l had raised themost important contents at all stages, about 13 %of the total biophenols.

For this class, in Chemlal, Takesrit and Blanquette

t h e re was a pro g ressive decrease during matura-t ion, the rate of diminut ion was re s p e c t i v e l y41.17%, 76.47% and 90%. B o u g h e n f o u s, which

o l e u ropein derivatives between green and blackstages, we noted the most important diminution inB o u g h e n f o u s variety (91.35 %) and the leastdecrease was reading in Chemlal variety (44.97 %).

For ligstroside derivatives, (Figure 5) there was ad e c rease during maturation pro g ress except forBoughenfous variety where a slight increase of 1.42% was found between oils extracted from gre e nand spotted stages. Once again, C h e m l a l v a r i e t yhad the most important contents of these com-pounds, except at the spotted stage, the thre eothers stages re g i s t e red a high level of ligstro s i d ederivatives. By passing from the first stage to theblack stage, results showed that the maturationdecreased the ligstroside derivatives levels by 81.51%, 68.06 %, 63.73 % and 53.95 % respectively inB o u g h e n f o u s, B l a n q u e t t e, Ta k e s r i t and C h e m l a l

olive oil varieties.These results agree with those obtained by

Baccouri et al. [43] concerning the decrease ofsecoiridoids (aglycon of oleuropein and ligstro s i d e )contents during maturation process.

Our results indicated a small quantity of flavo-noids between 2 % to 16 % with regard to the totalbiophenols, confirming this class as a minor consti-tuent of olive oil phenolic fraction, but with the same

Figure 4 - Comparison between the hydroxytyrosol and tyrosol contents, analysed by HPLC, of four Algerian virgin olive oil varieties during diffe-rent ripening stages


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Table III - Contents of major biophenol compounds of Algerian virgin olive oil (mg/kg), by HPLC analysis, with regard to cultivar and fruit ripeningstages

Varieties Stages Oleuropein derivatives Ligstroside derivatives Lignans Flavonoids Secoiridoid acids Biophenols oxidized(mg/kg) (mg/kg) (mg/kg) (mg/kg) (mg/kg) (mg/kg)

Chemlal Green 249 215 85 20 80 56Spotted 240 191 74 29 112 56Purple 203 147 60 29 77 45Black 137 99 50 31 117 43

Boughenfous Green 81 211 44 31 76 25Spotted 25 214 17 6.4 57 12Purple 6 87 3 3.6 22 8Black 7 39 4 5.2 6 7

Takesrit Spotted 72 91 34 13 95 35Purple 40 63 15 16 83 16Black 16 33 8 15 59 16

Blanquette Spotted 262 119 30 14 47 35Purple 204 74 27 18 30 33Black 31 38 3 4 21 8

Figure 5 - Comparison of olive oil biophenols with regard to cultivar and fruit ripening stages of Algerian varieties

had the most important decrease of lignans (90.90%), presented a decrease from green to purple sta-ges followed by an increase evaluated to 33.33 %between purple and black stages.

For secoiridoid acids, with the exception ofChemlal variety, the three others varieties had a pro-g ressive decrease during maturation. The smallest

diminution (37.89 %) was provided by Takesrit, butthe highest decrease (92.10 %) was in theBoughenfous variety.

C o n c e rning the class of oxidized biophenol, theresults showed a decrease of these compoundswith the maturation pro g ress for all varieties. Themost important content was highlighted in Chemlal


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variety for all stages. With regard to the total biophenols, Takesrit had

totalized the most important percentages for allmaturation stages. For the decreasing levelsbetween the first and the last stages, we noted23.21 %, 54.28 %, 72.00 % and 77.14 % respecti-vely for C h e m l a l, Ta k e s r i t, B o u g h e n f o u s a n dBlanquette varieties.

CONCLUSION

This study, undertaken on Algerian virgin olive oilsvarieties has provided a new proof that both matu-ration and cultivars influence significantly the quan-tity and the quality of the olive oil phenolic fraction.

The results obtained show that cv. Chemlal con-tains the most important contents of total biophe-nols at all maturation stages. C v. B l a n q u e t t e p re-sents the highest content of o-diphenols especiallyh y d ro x y t y rosol, which may signify that this varietyhas a good stability to oxidation.

During olive maturation, there was a decrease oftotal biophenols in all varieties. Yet it is very importantto underline that the levels of decrease are diff e re n tf rom one cultivar to another. C v. C h e m l a l had theleast important diminution (41.68 %) and cv.B o u g h e n f o u s had the most important one (84.08 %).

The maturation also influenced the biophenol pro-files of oil. Generally, by passing from the first to thelast stage, there is a decrease of the most impor-tant class of biophenols (oleuropein and ligstrosidederivatives) for all studied varieties. Lignans and oxi-dized biophenols had also re g i s t e red a decre a s eduring maturation. For C h e m l a l and Ta k e s r i t, itshould be underlined that there was an increase offlavonoids content during maturation.

There is a predominance of oleuropein derivativesfor cvs. Chemlal and Blanquette, but we observed ap redominance of ligstroside derivatives for cv.B o u g h e n f o u s. The four studied varieties have anappreciable content of lignans. This latter class andflavonoids are well presented in cv. Takesrit.

These results suggest that the biophenol profilescould be used in varietal characterization.

On the whole, the green and spotted stages coin-cided with the most important content of biophe-nols, but for organoleptic reasons (bitterness attri-bute), the olive harvesting should be made at thepurple stage, which is characterized by an appre-ciable content of biophenol.

AcknowledgmentThe authors would l ike to thank the ITA F V

Institute of Takarietz (Bejaia, Algeria).

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Received March 22nd, 2009, accepted June 22nd 2009


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TH E T R A D I T I O NA L D I E TS O F ME D I T E R R A N E A N P E O P L E R E F L E C T D I ST I NC T C U I S I N E S A N D C U L I-NA RY P R AC T I C E S B U T NAT U R A L LY H AV E A G R E AT D E A L I N CO M M O N, A PA R T I C U LA R CA S E I S T H ET R A D I T I O NA L SI C I L I A N D I E T D O M I NAT E D BY T H E H I G H CO N S U M P T I O N O F V I RG I N O L I V E O I L,C I T RU S F RU I T A N D A M O D E R AT E CO N S U M P T I O N O F W I N E. AN T I OX I DA N TS R E P R E S E N T A CO M-M O N E L E M E N T I N T H E S E FO O D S A N D M AY B E I M P O R TA N T M E D I ATO R S O F T H E B E N E F I C I A LE F F E C T O F T H I S D I E T. TH E P R E S E N T PA P E R R E V I E WS T H E R E S U LTS O B TA I N E D O N T H E CO N T E N TI N SI C I L I A N V I RG I N O L I V E O I LS A N D W I N E S O F A N T I OX I DA N T CO M P O U N D S I NC LU D I NG S E L E-N I U M, F LAVO NO I D S, R E SV E R AT RO L A N D OT H E R P H E NO L I C CO M P O U N D S. RE D W I N E SA M P L E SF RO M T H E M O ST I M P O R TA N T SI C I L I A N AU T H O C TO NO U S A N D A L LO CH TO NO U S VA R I E T I E S A N DV I RG I N O L I V E O I LS R E P R E S E N TAT I V E O F A L L T H E PDO Z O N E S O F T H E I S LA N D W E R E I NC LU D E DI N T H I S ST U DY. PO LY P H E NO LS A NA LYS I S I N W I N E A N D V I RG I N O L I V E O I L SA M P L E S W E R EP E R FO R M E D BY HPLC-MS A N D GC-MS/MS, R E S P E C T I V E LY, W H E R E A S T R AC E S E L E N I U ML E V E LS W E R E M E A S U R E D I N O L I V E O I LS BY CAT H O D I C ST R I P P I NG CH RO NO P OT E N T I O M E T RY. TH EO B TA I N E D R E S U LTS GAV E E V I D E NC E T H AT SI C I L I A N R E D W I N E A N D V I RG I N O L I V E O I L A R E A NI M P O R TA N T S O U RC E O F A N T I OX I DA N T CO M P O U N D S, T H U S CO N T R I B U T I NG TO T H E H E A LT H B E N E-F I TS O F T H E SI C I L I A N D I E T.KE Y W O R D S: V I RG I N O L I V E O I LS, P H E NO L I C CO M P O U N D S, S E L E N I U M, SI C I L I A N D I E T, R E D W I N E

OLI DI OLIVA VERGINI E VINI ROSSI SICILIANI: UNA RICCA FONTE DI COMPOSTIANTIOSSIDANTI NELLA DIETA MEDITERRANEALA DIETA MEDITERRANEA, PRINCIPALMENTE BASATA SUL CONSUMO DI OLIO DI OLIVA, PASTA,LEGUMI, FRUTTA, VERDURA, PESCE E VINO, È ASSOCIATA SPESSO AD UNA BASSA INCIDENZADI MALATTIE CARDIOVASCOLARI. LA TRADIZIONALE DIETA SICILIANA È CARATTERIZZATA DA UNR E G O LA R E CO N S U M O D I O L I O E X T R A V E RG I N E D I O L I VA, AG RU M I E V I NO RO S S O, A L I M E N T ICHE CONTENGONO COMPOSTI CON PROPRIETÀ ANTIOSSIDANTI CHE GIOCANO UN IMPORTANTERUOLO COME AGENTI BENEFICI PER LA SALUTE.IL LAVO RO P R E S E N TA DAT I R E LAT I V I A L CO N T E N U TO I N CO M P O ST I A N T I O S S I DA N T I Q UA L IP O L I F E NO L I, F LAVO NO I D I, R E SV E R AT RO LO E S E L E N I O I N CA M P I O N I D I V I N I E O L I D I O L I VAPRODOTTI IN SICILIA. IN PARTICOLARE I CAMPIONI DI VINO APPARTENGONO A DIFFERENTIVA R I E TÀ AU TO C TO N E E A L LO C TO N E CA R AT T E R I ST I CH E D E L LA SI C I L I A, I N V E C E I CA M P I O N I D IOLIO D’OLIVA SONO RAPPRESENTATIVI DI TUTTE LE DOP SICILIANE. LE ANALISI DEI POLIFE-NOLI NEI CAMPIONI DI VINO E DI OLIO SONO STATE ESEGUITE RISPETTIVAMENTE MEDIANTEHPLC-MS E GC MS/MS; LE ANALISI DEL SELENIO NELL’OLIO D’OLIVA MEDIANTE CRONO-POTENZIOMETRIA IN STRIPPING CATODICO. I RISULTATI DI QUESTO STUDIO, CHE PUÒ ESSERECO N S I D E R ATO U N P R I M O A P P RO C C I O A L L A C A R AT T E R I Z Z A Z I O N E D E L L E P RO D U Z I O N ISICILIANE, EVIDENZIANO CHE I VINI ROSSI E GLI OLI D’OLIVA SICILIANI SONO UN’IMPORTANTEFO N T E D I CO M P O ST I A N T I O S S I DA N T I, CO N T R I B U E N D O CO S Ì A L L E P RO P R I E TÀ SA LU T I ST I CH EDELLA DIETA SICILIANA.PAROLE CHIAVE: OLI DI OLIVA VERGINI, COMPOSTI FENOLICI, SELENIO, DIETA SICILIANA, VINOROSSO

Sicilian virgin olive oils and red wines:a potentially rich source of

antioxidant compounds in theMediterranean diet

G. MO DUGO1, L. LA PERA1*,G. DI BELLA1, V. LO1, D. POLLICINO1,G.L. LA TORRE1, T.M. PELLICANÒ2

1 DEPT. OF FOOD AND ENVIRONMENTAL SCIEN-

CE, UNIVERSITY OF MESSINA, ITALY

2 DEPT. OF CH E M I ST RY, UNIVERSITY OF CA LA-

BRIA (UNICAL), ARCAVACATA DI RENDE - COSEN-

ZA, ITALY

*CORRESPONDING AUTHOR:

tel/fax: +39 (0) 90/ 6765436

e-mail: [email protected]


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1. INTRODUCTION

In the past two decades, numerous biochemicaland clinical studies have provided consistent evi-dence of the healthy propert ies of theMediterranean diet [1, 2, 3]. This diet is based on alow to moderate consumption of dairy pro d u c t sand meat while is rich in vegetables, fruit, legumes,grain, fish and vegetable lipids, particularly virginolive oil, which does not have the same cholesterolraising effect as saturated lipids.

Thus the traditional Mediterranean diet is rich inoleic acid, ω-3 fatty acids, B-group vitamins, fibres,and various antioxidants, but low in cholestero l ,saturated and polyunsaturated lipids.

Several epidemiological studies and clinical trialshave provided evidence that the Mediterranean dietis associated with a lower incidence of certain chro-nic diseases including coronary heart disease(CHD), neurodegenerative diseases, and cancers(colon and prostate), for which the involvement ofan uncontrolled free radical generation has beenhypothesized [4-7].

T h e re is growing scientific evidence that dietaryantioxidants, which are able to neutralise oxygenfree radicals, may be a critical mediator of the bene-ficia l effects of the Medi terranean diet [8] .Confirmatory evidence for this hypothesis comesfrom the inverse associations between great adhe-rence to the Mediterranean diet [9-11] and deathdue to cancer of the colon, breast, prostate andovary [3, 12], as well as atherosclerosis and coro-nary heart disease [7, 13, 14].

Virgin olive oil (VOO) and red wine have an impor-tant role in the Mediterranean diet and re p re s e n trich sources of antioxidant compounds. The antioxi-dants identified in red wine include phenolic acids,flavonols, catechins and anthocyanidins. [15-17].The prevalent classes of hydrophilic phenols foundin VOO are phenolic alcohols, phenolic acids, flavo-noids, lignans and secoiridoids. Secoiridoids inclu-ding aglycon derivatives of oleuropein, demethylo-leuropein and ligustroside, that are present in olivefruit, are the most abundant phenolic antioxidantsof VOO [18-20]. Several authors found a stro n grelationship between some sensory attributes asthe “bitterness” of olive oil and its secoiridoids con-tent [21-23]. Among VOO lignans, the most impor-tant compounds are ( + ) - 1 - a c e t o x y p i n o resinol and( + ) - p i n o resinol. These lignans, which are potent

antioxidants, are absent in seed oils and virtuallyabsent in refined virgin oils [24, 25]. More o v e rrecent studies have evidenced the presence ofselenium, an antioxidant microelement, in virginolive oils [26].

The purpose of the present work is to provide anoverview of the findings related to the presence ofantioxidant compounds in VOO and red wine pro-duced in Sicily, one of the biggest VOO and wineproducing region in Italy.

1.1 Sicilian wine and virgin olive oilSicily is the largest island in the Mediterranean

Sea, the mild climate and fertile soil of the seasideplains create conditions for one of the most succes-sful agriculture economies in Italy. Virgin olive oil andwines are of the utmost importance in Sicily, botheconomically and nutritionally. Over the last fewyears the vast universe that is Italian wine has reva-lued the products of Sicily. Sicily accounts for atleast 17 % (8.7 million of hl) of all wine making inItaly, and the most of these wines (about 90%) arenow protected by the CDO (Contro l l e dDenomination of Origin) regulation. Four per cent ofSicilian wine and must production is exportedabroad, particularly to France (75%), Spain (12 %),the UK (5 %), but there are also overseas consu-mers such as USA, Canada, Japan, though withonly a small percentage. In recent years, wine hasbeen one of the few Sicilian agricultural products tosee a rise in export levels, both in quantity and ave-rage unit value compared to previous years [27].The production and consumption of virgin olive oilmostly concerns the Mediterranean countries.Spain is the major producer (620000 T per year),followed by Italy (500000 T per year), Gre e c e ,Portugal, Tunisia and Turkey. The amount of olive oilconsumed in Greece is 18 kg per year per capita, inItaly 13 kg per year per capita and in Spain 11 kgper year per capita [28]; due to the incre a s i n gpopularity of the Mediterranean diet, in which virginolive oil is the major lipid component, its consump-tion is expanding to non-producing countries suchas the United States, Canada, and Japan. In Italytwenty-seven PDO virgin olive oils from diff e re n tregions were produced.

Sicily is the third Italian region in olive oil pro d u c t i o n .Almost all the Sicilian provinces are interested in oliveg rowing and each zone produces particular olive culti-vars. At present Sicily produces four PDO virgin olive


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H y d ro x y t y rosol is an ortho-diphenolic compoundand seems to be among the most important phe-nols; it is present in free form and also as a consti-tuent of complex molecules (i.e. oleuropein). It hasbeen observed that virgin olive oil phenolic com-pounds inhibit the in vitro oxidation of LDL, a pro-cess which in vivo is thought to enhance the athe-rogenicity of these lipoproteins [34, 35].

The French have low coronary diseases mortalitywith high saturated lipid consumption [34]; this epi-demiological observation is known as the “Fre n c hparadox” and is normally attributed to the con-sumption of red wine. Red wine is a rich and con-centrated source of polyphenolic substances; themajor polyphenols in wine include phenolic acids,anthocyanins, tannins, and other flavonoids.

Epidemiological studies have shown that themoderate consumption of wine prevents card i o v a-scular pathologies thus reducing the risk of mortality[35]. The phenolic compounds are contained in skin,seed and flesh of grapes and are extracted fro mwine (especially red) during vinification process. Theconcentrations of these compounds may depend ona number of factors: variety of the grapes, climaticconditions, ripening stage of the grapes, agro n o m i cpractices and wine-making technology, the year andthe period of the wine conservation [36].

The 3,5,4'- trihydroxystilbene (re s v e r a t rol) is oneof the most studied polyphenolic compounds foundin red wine; it is a phytoalexin synthesized by plantsin response to injury or fungal attack. Resveratro lexist in cis and trans isomeric forms, and their gly-cosides derivatives, known as piceid [37, 38].

Many biological effects have been attributed toresveratrol such as activation of estrogen receptors,inhibition of platelet aggregation, modification ofhepatic apolipoprotein, and inhibition of ciclooxige-nase 2 enzyme activity; all these effects pointtoward the possibility that resveratrol may contribu-te to the cardio-protective effect offered by red wineconsumption [39, 40].

1.3. Selenium Selenium is present in food matrices in many

biological forms as selenite, selenate, elementalselenium, selenocysteine, selenoproteins; it actsas an antioxidant. Selenoenzymes, particularly glu-tathione peroxidase, play an important role in pro-tecting cells from the oxidative damage, thus pre-venting cardiovascular diseases and other patho-

oils: Monti Iblei, Valli Trapanesi, Val di Mazara,Valdemone, whereas Monte Etna and Valle del Belicea re in the process of obtaining the mark [29].

1.2 Phenolic compounds in virgin olive oil and wineThe particular climate of the Mediterranean area,

characterized by hot summers and mild winters,has allowed the growth of plants such as olive treesand grape vines whose fruits require a high quantityof antioxidant molecules, to protect themselvesf rom the noxious effect of prolonged sunlight irra-diation. In fact the synthesis of pigments such aspolyphenols is activated by white light irradiationand result in dark-coloured fruits [30].

A diet rich in fresh fruits, virgin olive oils and cha-racterized by a moderate consumption of wine,grants an elevated intake of those antioxidant com-pounds which may transpose their biological activi-ties from the fruit to the human body [31, 32].

Phenolic compounds in foods originate from oneof the main classes of secondary metabolites inplants. This class of substances can be classifiedinto two groups: a) flavonoids that includes the fla-vonols (myricetin, quercetin, kaempferol, rhamnetin,isorhamnetin); flavones, flavanones, flavan-3-ols ((-)-epicatechins, (+)-catechins and the procyanidins B1and B2) and anthocyanins as malvidin etc. b) other

phenolic compounds that include gallic acid, proto-catechuic acid, vanillic acid, syringic acid, thehydroxycinnamic acids as caffeic acid, ferulic acid,p-coumaric acid and the stilbenes trans-resveratrol,c i s- re s v e r a t rol, t r a n s- re s v e r a t rol-O-glucoside, c i s-re s v e r a t rol-O-glucoside and oligomers. Althoughphenolic compounds are present in virtually all plantfoods, their levels vary enormously among dietsdepending on the type and quantity of plant foods.

The phenolics, like other compounds such ash y d rocarbons, chlorophylls, carotenoids, tocophe-rols, re p resent the “minor components” of virgin oliveoil and their concentration depends on several fac-tors including the zone of provenience, the soil, thec u l t i v a r, the degree of ripeness, production and sto-rage pro c e d u re of the oil; furthermore the polypheno-lic fraction provides the typical pungent taste anda roma of extra-virgin olive oil [33]. Virgin olive oil phe-nols are a complex mixture of compounds that inclu-de 3,4-dihydroxyphenylethanol (hydro x y t y rosol), 4-h y d roxyphenylethanol (tyrosol), 4-hydro x y p h e n y l a c e-tic acid, protocatechuic acid, syringic acid, vanillicacid, caffeic acid, and p-coumaric acid.


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logies such as cancer brought on by oxidatives t ress. Selenium is also involved in the pro d u c t i o nof the active thyroid hormone, in the muscle func-tion, in the re p roduction process and in the immu-ne response to some infections. It enters the foodchain through plants that take it up from the soil,so the availability of selenium strictly depends onlocation and season.

For an adult the RDA is 50-70 µg / d a y, howeverconcentrations higher than 400 µg/day may beco-me toxic [41-43]. Some authors studied the corre-lation between the presence of selenium in diff e re n tfood matrices like garlic, nuts and cereals and theirregion of provenience [44-46], whereas very fewdata are found in literature about the presence oftrace selenium concentration in virgin olive oils [47,48]. As reported above, Sicily accounts for fivePDO olive growing zones and Sicilian olive tre egermplasm is very rich and diff e rent, it comprisesseveral olive varieties characterized by very intere-sting physiological and nutritional pro p e r t i e s .T h e re f o re it is of great concern to study the possi-ble statistical correlation among the presence ofselenium in Sicilian olive oils, the olives variety andthe zone of pro v e n i e n c e .

2. MATERIALS AND METHODS

2.1 Samples - Virgin olive oils. Samples re p resentative of the

most important olive growing zones of Sicily wereincluded in this study in order to have furtherinformation about their micro-constituent compo-sition. Selenium analysis were performed on fiftyvirgin olive oil samples from the crop year 2001p roduced in d iff e rent PDO (Pro t e c t e dDenomination of Origin) zones of Sicily from diffe-rent varieties. The studied zones were: “Va l l iTrapanesi” (Cerasuola and Crastu varieties); “Va ldi Mazara”(Biancolil la cv); “Valle del Belice”(Nocellara del Belice cv); “Va l d e m o n e ”(Santagatese and Minuta cultivars); “Monti Iblei”( Tonda Iblea and Moresca varieties) and MonteEtna (Nocellara Etnea cv). Phenolic compoundsw e re determined in 16 samples of Sicilian virginolive oils from the varieties Nocellara del Belice,Santagatese and Cerasuola produced in the cropyear 2001 in three diff e rent PDO zones of theregion: Valle del Belice, Valdemone and Va l l iTrapanesi. All the olive oil samples were stored in

corked dark glass bottles at 4°C until the analysis.The analyses were performed within two months.The analysis of selenium was also performed onc o m m e rcial seed oils (peanut, soybean, sun-f l o w e r, corn, grapestone, mixed seeds) purc h a-sed in a supermarket on April 2002.

- Red wines. Phenolic compounds were also deter-mined in 22 red wine samples from allocthonousand autochthonous variet ies (Nero d’Av o l a ,Merlot, Petit-Ve rdot, Cabernet, Syrah), pro d u c e din different crop seasons (1999- 2002). The sam-ples were stored in corked dark glass bottles at4°C until the analysis.

2.2 Reagent and instrumentation All the reagents and the instrumentation used in

this study were described in previous papers [26,29, 49-52] .

The analytical methods performed were the fol-lowing: - phenolics compounds determination in red wines

and virgin olive oils

The qualitative and quantitative composition ofphenolic compounds in Sicilian red wine sampleswere investigated by a new and recent HPLC-MSmethod, using APCI and ESI probe. This methodallowed the identification of 24 phenolic com-pounds in wine samples with detection limits ran-ging from 0.002 mg/L and 0.16 mg/L and preci-sion within 5% (expressed as RSD% of nine mea-s u rements) [29, 49, 50]. Phenolic compounds inSicilian olive oils were analyzed by GC- MS andGC-MS/MS [51] after the extraction with metha-nol: water 80:20 and derivatization with bis (tri-methylsilyl)trifluoracetamide and trimethylchloro s i-lane (BSTFA:TMCS 99:1). This method allowedthe detection of numerous compounds and 23w e re identified; in particular 4-(acetoxyethyl)-1-h y d roxybenzene, 3,4-dihydro x y p h e n y l a c e t a l d e h y-de, syringaldehyde and the cis form of ferulic acidwere identified in olive oils for the first time.

- Selenium determination in oils

Cathodic stripping potentiometric analysis wasused to determinate selenium levels in Sicilian vir-gin olive oils and seed oils [53]. Selenium wasextracted from samples by concentrated hydro-gen peroxide and hydrochloric acid tre a t m e n t ,then the cathodic stripping potentiometric deter-mination was carried out. Cathodic stripping


167

c h ronopotentiometry for trace selenium analysisin vegetable oils ensured high precision (2.8 %,expressed as RSD% of nine measurements) andaccuracy (95.5-97.7%, expressed as re c o v e r yfactors), furthermore detection limit of 0.6 µg / k gwas achieved [26].

2.3 Statistical analysisThe statistical elaboration of the data was perfor-

med by STATISTICA package for Windows (version5.1, 1997). The analysis of variance (ANOVA) wasperformed both grouping VOO according to theirzone of provenience (Valli Trapanesi, Val di Mazara,Valle del Belice, Valdemone, Monti Iblei and MonteEtna) - and according to their ol ive var iety(Cerasuola, Crastu, Biancoli lla, Nocellara delBelice, Santagatese, Minuta, Tonda Iblea Morescaand Nocellara Etnea) to value if geographical andgenetic factors significantly influenced the contentsof selenium in VOO.

3. RESULTS AND DISCUSSIONS

3.1 Polyphenols T h e re was a wide range of phenol concentrations in

selected wines. The concentrations of all the studiedpolyphenolic compounds in Sicilian red wines strictlydepends on the grape variety; the obtained re s u l t sp rovide evidence that gallic acid, tyrosol, pro c y a n i d i nB1, (+)-catechin and (-) epicatechin were the mostabundant polyphenolic compounds in the analyzedsamples. In particular, samples from Merlot variety pre-sented the highest mean values: 86.26±19.56 mg/Lgallic acid, 33.19±8.94 mg/L tyrosol, 76.87±33.18mg/L procyanidin B1, 52.44 ±24.81 mg/L (+)-catechinand 71.99±31.35 mg/L (-) epicatechin. The averagevalue of t r a n s -re s v e r a t rol in Sicilian red wines(0.75±0.42 mg/L) appeared to be lower than thosereported for wines from France and Greece [54-56].

Among Sicilian red wines, those produced fro mMerlot variety presented the highest amount of

Table I - Polyphenols concentration range found in Sicilian virgin olive oils and red wine

Virgin olive oil mg/kg Red wine mg/L

Salicylic acid 0.02-0.09

Vanillin 0.09-0.38

Tyrosol 12.40-33.20 Tyrosol 4.50-156.4

4-(Acetoxyethyl)-1-hydroxybenzene 2.57-5.29 Caffeic acid ethyl ester 0.20-4.20

4-Hydroxybenzoic acid 0.12-0.64 Protocatechuic acid ethyl ester nd-1.70

Vanillic alcohol 0.02-0.24 Ethyl gallate 0.30-56.30

3,4-Diidroxyphenylacetaldehyde 0.38-0.94 Gallic acid 3.90-103.90

Omovanillic alcohol 0.56-1.86

Siringic aldehyde 0.11-0.78

Idroxytyrosol 16.70-30.40 Idroxytyrosol 0.20-15.50

Vanillic acid 1.23-5.11 Vanillic acid 0.70-16.50

2-Cumaric Acid 0.06-0.35 cis-Resveratrol nd-1.60

4-(Acetoxyethil)-1,2-hydroxybenzene 4.12-9.54 trans-Resveratrol 0.10-6.50

Protocatechuic acid 0.03-0.41 Protocatechuic acid 1.10-18.90

Syringic acid 0.05-0.36 Syringic acid 0.90-28.30

cis-Ferulic acid 0.02-0.12

4-Cumaric Acid 0.71-1.67 4-Cumaric Acid 0.50-29.70

Ferulic acid 0.44-1.49 Ferulic acid 0.10-2.70

Caffeic acid 0.32-1.21 Caffeic acid 1.20-38.10

Ligstroside aglycon –dialdehydic form 12.70-192.0

Ligstroside aglycon (elenolic acid linked to tyrosol) 2.85-12.70

Dialdehydic form of elenolic acid linked to 3-methoxy-4-hydroxyphenilethanol 0.22-2.08

Oleuropein aglycon - dialdehydic form 13.20-158.0

Oleuropein aglycon (elenolic acid linked to hydroxytyrosol) 3.04-10.40


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t r a n s re s v e r a t rol (1.37±0.80 mg/L) and those pro-duced by mixing Cabernet, Syrah and Petit-Verdotvarieties the lowest (0.11±0.80 mg/L). Cis-resvera-t rol was found in the studied wines at lower con-centration than trans-resveratrol, the mean amountsdetected in Merlot, Nero d’Avola and in the blendedsamples were 0.75±0.82 mg/L, 0.11±0.06 mg/Land 0.13±0.10 mg/L, re s p e c t i v e l y. C i s -piceid wasusually found at a lower concentration than t r a n s -piceid, and their levels exceeded those of the freeisomers and reached maximal concentrations of3.74 and 1.97 mg/L, respectively.

Table I shows that tyrosol and hydro x y t y rosol arethe principal free phenols found in Sicilian virgin oliveoils; their concentration ranges were 12.40-33.20mg/kg and 16.70-30.40 mg/ kg, re s p e c t i v e l y. Amonglinked phenols, the dialdehydic forms of ligstro s i d eaglycon and oleuropein aglycon showed the highestconcentration ranges, 12.70-192.0 mg/kg and13.20-158.0 mg/kg re s p e c t i v e l y. Furthermore Table Ip rovides evidence that some phenolic compoundsa re present both in VOO and in red wines. In particu-

lar hydro x y t y rosol in virgin olive oils presented higherconcentration ranges with respect to red wines; whe-reas protocatechuic acid, syringic acid and caff e i cacid levels are significantly higher in red wines.

3.2 Selenium Selenium levels in all the studied red wine sam-

ples were lower than the instrumental detectionlimits (<1µg/L). The content of selenium found in thestudied Sicilian virgin olive oils, ranged from 3.0 to122.9 µg/kg. The obtained results evidenced thatolive oil samples from the same cultivar had a simi-lar content of selenium.

The maximum mean value of selenium was foundfor samples of virgin olive oil from Santagatese culti-var; while samples from Biancolilla, Moresca andNocellara Etnea varieties showed the lowest meanselenium amounts (Fig. 1). The analysis of variance( A N O VA) performed on data expressing seleniumconcentrations, gave evidence that both the diff e-rences between the cultivars (p=0.00004, F=7.58)and between the zone of provenience (p=0.01,

Figure 1 - Mean concentration of selenium in Sicilian virgin olive oils from different zones and different varieties


169

F=3.4) were statistically important. The analysis ofvariance confirmed that both genetic factors (culti-var) and geographic factors (olive-growing zone),may influence the content of selenium in the studiedSicilian virgin olive oils.

M o reover the average selenium concentrationfound in this work is in the range of selenium levelsfound in Greek (2.0±1.0 µg/kg) and Fre n c h(220.0±52.0 µg/kg) virgin olive oils [46,47] .

Due to the diffusion of seed oils worldwide, it wasi n t e resting to compare selenium concentration ofSicilian olive oils with those found in commercial seedoils (peanut, soybean, sunflower, corn, grapestone,mixed seeds). The obtained results gave evidencethat seed oils had higher selenium levels than Sicilianolive oils; in particular concentrations ranging fro m99.5 to 460 µg/kg were detected, with a mean level of267.4 µg/kg against 35 µg/kg of Sicilian virgin olive oil.

3.3 Nutritional consideration T h e re are problems in assessing human intake of

polyphenols, and relating this to effects on cancer orc a rdiovascular pathologies risks. Wine and virgin oliveoils are essential components of the Mediterraneandiet, and their moderate consumption may be one ofthe factors responsible for the lower incidence ofdiseases characterized by oxidative stress amongMediterranean population. The total phenol content ofvirgin olive oil has been reported numerous times inl i t e r a t u re, it varies between 88 mg/kg and 206 mg/kgin Spanish VOO[57], from 55 to 125 mg/kg in Gre e kVOO [58], from 81 to 394 mg/kg and from 228 to 490mg/kg in Italian VOO produced in Tuscany [59] andApulia regions [60], re s p e c t i v e l y.

A c c o rding to the results of our study the totalphenol content in Sicilian VOO ranged from 117 to390 mg/kg with samples from Nocellara del Belicevariety showing the highest amount and samplesf rom Cerasuola variety the lowest. Considering allthese values reported in literature, an estimate ofthe mean amount of olive oil phenolics consumedper person per year in Greece is 1.6 kg, in Spain1.7 kg, and among Italian regions is in Tuscany 3.1kg, in Apulia 4.7 kg and 3.3 kg in Sicily. As reportedin Table II, of all the studied phenolics tyro s o l ,i d ro x y - t y rosol, vanillic acid, protocatechuic acid,syringic acid, p-coumaric acid, ferulic acid, and caf-feic acid are present both in red wines and olive oilsp roduced in Sicily. Considering a moderate dailyconsumption of olive oils and wine, 30 g and 0.2 L

respectively, red wine provides a significantly higheramounts of phenols compared to olive oil. The con-sumption of 0.2 L of red wine and 30 g of olive oilresults in the ingestion of 0.4-1 mg/L and 0.9-31.2mg/kg of tyrosol, 0.5-0.9 mg/L and 0.04-3.1 mg/kgof hydroxy-tyrosol, <0.01 mg/L and 0.2-3.8 mg/kgof protocatechuic acid, <0.04 mg/L and 0.24-7.6mg/kg of caffeic acid, respectively.

On the basis of the obtained results, a daily con-sumption of 0.20 L of Sicilian wine supply about 0.02-1.3 mg/L of t r a n s -re s v e r a t rol. A daily consumption of30 g of olive oil for a 70 kg adult supply a maximum of1.6 µg of Se that re p resents the 2.3 RDA%.

4. CONCLUSION

Part of the beneficial effect of the Mediterraneandiet may be due to the presence of antioxidant“minor components” in typical foods of theMediterranean diet including olive oils, fruit, vegeta-bles and a moderate consumption of wine. Theobtained data provides evidence that Sicilian re dwine and virgin olive oil are a rich source of antioxi-dant compounds as polyphenols, flavonoids, resve-r a t rol and selenium; there f o re a correct consump-tion of these typical foods may contribute to thehealth benefit of the Sicilian diet.

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Table II - Dietary daily intake of polyphenols from a 30 g consump-tion of olive oil and 0.2 L of red wines

Olive oil Dietary intake Wine Dietary intake

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[56]. P. Jeandet, R. Bessis, B.F. Maume, P. Meunier,D. Peyron, P. Trollat, Journal of Agriculturaland Food Chemistry, 43, 316-319 (1995)

[57]. F. Saura-Calixto, I. Goni, Food Chemistry, 94,

442-447 (2006). [58]. K. Papadopoulos, T. Triantis, E. Yannakopou-

lou, A. Nikokavoura, D. Dimotikali, AnalyticaChimica Acta, 494, 41-47 (2003)

[59]. M. Bonoli, M. Montanucci, T. Toschi Gallina, G.Lercker, Journal of Chromatography A, 1011,163-172 (2003).

[60]. F. Caponio, V. Alloggio, T. Gomes, FoodChemistry 64, 203-209 (1999).

OLI DI OLIVA V E RGINI E VINI ROSSI SICI-LIANI: UNA RICCA FONTE DI CO M P O S T IA N T I O S S I DANTI NELLA DIETA MEDITER-RANEA

La dieta Mediterranea, principalmente basata sul

consumo di olio di oliva, pasta, legumi, frutta, ver-

dura, pesce e vino, è associata spesso ad una

bassa incidenza di malattie cardiovascolari. La tra-

dizionale dieta siciliana è caratterizzata da un rego-

lare consumo di olio extra vergine di oliva, agrumi e

vino rosso, alimenti che contengono composti con

proprietà antiossidanti che giocano un importante

ruolo come agenti benefici per la salute.

Il lavoro presenta dati relativi al contenuto in com-

posti antiossidanti quali polifenoli, flavonoidi, resve-

ratrolo e selenio in campioni di vini e oli di oliva pro-

dotti in Sicilia. In particolare i campioni di vino

appartengono a differenti varietà autoctone e alloc-

tone (Nero d’Avola, Merlot, Petit-Verdot, Cabernet,

Syrah) caratteristiche della Sicilia, invece i campioni

di olio d’oliva sono rappresentativi di tutte le DOP

siciliane.

Le analisi dei polifenoli nei campioni di vino e di olio

sono state eseguite rispettivamente mediante

HPLC-MS e GC MS/MS; le analisi del selenio nel-

l’olio d’oliva mediante cronopotenziometria in strip-

ping catodico. Questo studio, che può essere con-

siderato un primo approccio alla caratterizzazione

delle produzioni Siciliane, evidenzia che i vini rossi e

gli oli d’oliva siciliani sono un’importante fonte di

composti antiossidanti, contribuendo così alle pro-

prietà salutistiche della dieta siciliana.

In particolare, la concentrazione dei composti poli-

fenolici studiati nei vini siciliani dipende dalla varietà

del vitigno; i risultati ottenuti dimostrano che l’acido

gallico, il tirosolo, la procianidina B1, la (+)-catechi-

na e la (-) epicatechina sono i fenoli più abbondanti.

Inoltre tra i vini siciliani analizzati, quelli della varietà


172

Merlot presentano il più alto contenuto di trans-

resveratrolo.

Negli oli d’oliva siciliani, i polifenoli liberi più rappre-

sentativi sono tirosolo e idrossitirosolo mentre le

forme dialdeidiche del ligustroside aglicone e dell’o-

leuropeina aglicone, sono i polifenoli legati più

abbondanti. La concentrazione di selenio è inferiore

ai limiti di rivelabilità strumentale in tutti i vini studia-

ti, è stata invece osservata una correlazione inte-

ressante tra il contenuto di selenio negli oli d’oliva

siciliani, la zona di provenienza e la cultivar.

Ricevuto 29/1/2009,

accettato 12/3/2009


173

TH I S WO R K R E F E R S TO T H E Q UA L I TAT I V E A N D S E N S O RY CH A R AC T E R I ST I C S O F A TU N I S I A NM I NO R O L I V E VA R I E TY (C V. MA R SA L I N E) W I T H R E GA R D S TO T H E I R R I GAT I O N R E G I M E A N D T H EC U LT I VAT I O N S I T E. MA R SA L I N E F RU I TS W E R E CO L L E C T E D F RO M TWO D I F F E R E N T R E G I O N S I NT H E NO R T H WE ST O F TU N I S I A. SA M P L E S W E R E A LS O T E ST E D I N BOT H A R A I N-F E D CO N T RO LA N D A N I R R I GAT I O N R E G I M E. TH E M O R P H O LO G I CA L CH A R AC T E R I ST I C S W E R E A NA LY Z E D A N D O NT H E O B TA I N E D O I LS Q UA L I TY I N D I C E S, M A J O R A N D M I NO R CO M P O U N D S, OX I DAT I V E STA B I L I TYA N D O RGA NO L E P T I C AT T R I B U T E S W E R E A L L CA R R I E D O U T. PR E L I M I NA RY R E S U LTS CO N F I R M E DT H AT MA R SA L I N E I S A G O O D P I CK L I NG O L I V E VA R I E TY, A N D T H E O I LS O F T H I S M I NO R C U LT I VA RH A D A N I N T E R E ST I NG O L I V E O I L CO M P O S I T I O N. TH E I R R I GAT I O N R E G I M E H A D A P O S I T I V EI N F LU E NC E O N A N T I OX I DA N T CO M P O U N D S A N D CO N S E Q U E N T LY O N T H E OX I DAT I V E STA B I L I TY,W H I L E T H E G E O G R A P H I C A R E A M O D I F I E D T H E MA R SA L I N E FAT TY AC I D CO M P O S I T I O N.KE Y W O R D S: MI NO R VA R I E TY; OL I V E O I L CO M P O S I T I O N; S E N S O RY CH A R AC T E R I ST I C S;IRRIGATION; CULTIVATION SITE

INFLUENZA DEL REGIME IDRICO E DELL’AREALE DI COLTIVAZIONE SULLECARATTERISTICHE QUALITATIVE E SENSORIALI DI UNA VARIETÀ DI OLIVA TUNISINAMINORE (CV. MARSALINE)QUESTO LAVORO HA COME OBIETTIVO LO STUDIO DEGLI ASPETTI QUALITATIVI E DELLE CARAT-T E R I ST I CH E S E N S O R I A L I D I O L I P ROV E N I E N T I DA O L I V E A P PA R T E N E N T I A D U NA VA R I E TÀTUNISINA MINORE (CV. MARSALINE) IN RELAZIONE ALL’AREA DI COLTIVAZIONE E AL REGIMEI D R I CO A D OT TATO. I F RU T T I S O NO STAT I CO LT I VAT I I N D U E D I V E R S E R E G I O N I D E L NO R D-OVEST DELLA TUNISIA E SOTTOPOSTI SIA AD UN REGIME DI IRRIGAZIONE NATURALE CHE ADU NO CO N T RO L LATO. SO NO STAT E Q U I N D I E F F E T T UAT E A NA L I S I S U L L E CA R AT T E R I ST I CH EM O R FO LO G I CH E D E L L E O L I V E, M E N T R E G L I O L I OT T E N U T I S O NO STAT I S OT TO P O ST I A L L ED E T E R M I NA Z I O N I D E I P R I NC I PA L I PA R A M E T R I D I Q UA L I TÀ P R E V I ST I P E R G L I O L I V E RG I N I.SO NO STAT I VA LU TAT I, I NO LT R E, I CO M P O N E N T I M I NO R I, LA STA B I L I TÀ O S S I DAT I VA E G L IATTRIBUTI SENSORIALI. I R I S U LTAT I P R E L I M I NA R I H A N NO CO N F E R M ATO CH E LA C U LT I VA R MA R SA L I N E S I P R E STA B E N EA L LA R AC CO LTA E D I NO LT R E G L I O L I OT T E N U T I H A N NO P R E S E N TATO U N’I N T E R E S SA N T E CO M P O-S I Z I O N E CH I M I CA NO NCH É P RO F I LO S E N S O R I A L E. IL R E G I M E D I I R R I GA Z I O N E CO N T RO L LATO H ACO N D I Z I O NATO P O S I T I VA M E N T E I L Q UA N T I TAT I VO D I CO M P O ST I A N T I O S S I DA N T I N E L L’O L I O E, D ICO N S E G U E N Z A, LA S UA STA B I L I TÀ O S S I DAT I VA, M E N T R E LA D I V E R SA A R E A G E O G R A F I CA D IP RO D U Z I O N E H A M O D I F I CATO S I G N I F I CAT I VA M E N T E LA CO M P O S I Z I O N E I N AC I D I G R A S S I.PAROLE CHIAVE: VARIETÀ MINORE, COMPOSIZIONE OLIO DI OLIVA, CARATTERISTICHE SENSO-RIALI, IRRIGAZIONE, AREALE DI COLTIVAZIONE

Influence of irrigation and site ofcultivation on qualitative and sensory

characteristics of a Tunisian minorolive variety (cv. Marsaline)

O. BACCOURI1, M. GUERFEL1,M. BONOLI-CARBOGNIN2, L. CERRETANI2*, A.BENDINI2,M. ZARROUK1 AND D. DAOUD1*

1 )L A BO R ATO I R E C A R A C T É R I SAT I O N E T

QUALITÉ DE L’HUILE D’OLIVE, CE N T R E D E BI OT E CH NO LO G I E D E BO R J-CÉDRIA, HAMMAM-LIF, TUNISIA2 )DI PA R T I M E N TO D I SC I E N Z E D E G L I

AL I M E N T I, UN I V E R S I TÀ D I BO LO G NA,CESENA (FC), ITALY

*CORRESPONDING AUTHORS:


and [email protected]

Tel. +390547338121, Fax: +390547382348


174

INTRODUCTION

The Tunisian genetic resources of olives are sub-stantial. Indeed, more than fifty varieties and ecoty-pes are scattered through diff e rent regions of thecountry [1]. Among them two main varieties are pre-dominant, Chétoui in the northern regions andChemlali in the central and southern areas [2]. Thislatter contributes up to 80 % of national olive oilproduction. Its oil is characterized by relatively lowlevels of oleic acid and high levels of palmitic andlinoleic acids [2]. In addition to the principal varie-ties, several secondary or minor cultivars have only“farm” diffusion (neglected varieties). A good exam-ple is the case of Marsaline cultivar, a minorTunisian variety growing in the region of Siliana(North West of the country) (Figure 1).

The biodiversity is a re s o u rce of scientific value thatmust be protected by increasing the national collec-tions. It also has a big economic interest because itcan contribute to the success of the Tunisian olivep roducts (table olives and oils) in the world and the-re f o re it must be valorized. Accurate chemical andsensorial information concerning these olive cultivarswould greatly enhance the Tunisian table and olive oilp roduction. Marsaline is well-known as a pickling olivev a r i e t y, also its oil is highly appreciated by local con-sumers due to its unique aroma and delicate flavour.

On the other hand, the Tunis ian cl imate isMediterranean, characterized by little or no rainfallduring the most critical phenological phases foryield formation. Therefore, for table olives, irrigationis particularly important since it assures better vege-tative and re p roductive growth and guarantees acontinuous, homogeneous increase of fruit size,together with a positive effect on fruit uniformity [3].H o w e v e r, contradictory data are available on theeffect of the water supply on the chemical composi-tion and the sensory characteristics of virgin olive oil(VOO) [2-4]. It is widely known, that the olive oilcomposition determines its intrinsic quality that islinked to several factors. Cultivar, environment andagricultural techniques can affect the fruit physio-l o g y, whereas processing and storage conditionscan influence the oil composition [2-7].

Concerning the first points, this paper intends togive firstly a contribution to the characterization ofMarsaline olive fruit and oil, and secondly, to deter-mine the effects of the water supply and thegrowing area on the characteristics of this variety.

MATERIAL AND METHODS

Plant materialMarsaline olive samples were collected, at the

m a t u re stage, from three diff e rent farms in theNorth West of Tunisia. Samples obtained from BouArada farm were cultivated in rainfall regime ( E 1 ).Olive samples (E2 and E3) coming from the sameregion of El Aroussa, were also tested in a rain-fedc o n t rol ( E 2 ) and under irrigation regime ( E 3 ) ( F i g u re1). Under the latter condition, the water re q u i re-ments were calculated using a methodology basedon the crop evapotranspiration (ETc) proposed bythe United Nations Food and Agr icul tureOrganization [8]. After harvesting, the olives werewashed, deleafed, and then transported to thelaboratory where they were immediately transfor-med (Abencor system, MC” Ingenierias y sistemas,Sevilla, Spain). This extraction technology wasre p roduced at laboratory scale following the samephases of industrial processes: milling, malaxation,centrifugation, and decanting. Obtained oil samplesw e re stored without filtration at 9 °C in darknessusing amber glass bottles without headspace priorto analysis.The assays were carried out at least int r i p l i c a t e .

Morphological aspects of olive oilAt the mature stage, 200 olives (3 replicates for

each orc h a rd) were picked randomly and then splitinto two samples, one put in an oven to dry, the otherused for fresh fruit and pit analysis. Average fruit wei-ght was determined, after removing and cleaning thestones. Pulp, stone weights and the ratio betweenpulp and stone were also re c o rded. The water con-tent was obtained after drying the fruits.

Quality indicesFree acidity, peroxide value and UV spectrophoto-

metric indices (at 232 and 270 nm) were determi-ned according to the European Communities officialmethods [9]. All parameters were determined in tri-plicate for each sample.

Oil ContentFor oil content determination, 40 g of olive fruits

were dried in an oven at 80 °C to constant weight.The dry olives were extracted with n-hexane using aSoxhlet apparatus. The results were expressed aspercentage of dry matter (DM).


175

Fatty acid compositionFAMEs (fatty acid methyl esters) analysis was

performed using a GC (HP Company, Wi l m i n g t o n ,DE, USA) equipped with a flame ionization detector(FID). The analyses were carried out after alkalinetreatment; this was obtained by mixing 0.05 g of oildissolved in 1 mL of n-hexane with 1 mL of 2 Npotassium hydroxide in methanol [10]. FAMEs wereseparated and quantified with a Hewlett-Packardmodel 4890D gas chromatograph equipped with a30 m x 0.25 mm x 0.25 µm film thickness fusedSil ica capil lary column (HP-Innowax; AgilentTechnologies, Germantown, MD, USA) coupled to aflame ionization detector (column temperature210°C). The FID detector was at 240 °C. The initialoven temperature was kept at 120 °C for 1 min andraised to 240 °C at a rate of 4.0 °C/min and main-tained for 4 min. Nitrogen was used as carrier gasat 1 mL/min with split injector system (split ratio1:100).

Extraction of the phenolic fractionThe phenolic fraction was extracted from the oil

samples by a liquid/liquid extraction method, accor-ding to Pirisi et al. [11].

Spectrophotometric determination of total phenolsThe total phenol contents of the extracts were

determined according to the Folin-Ciocalteu spec-trophotometric method at 750 nm [12], using a gal-lic acid calibration curve (r2 = 0.998) (Lambda 25s p e c t ro p h o t o m e t e r, PerkinElmer, Waltham, MA).The results were expressed as milligrams of gallicacid per kilogram of oil. The spectro p h o t o m e t r i canalysis was repeated at least three times for eachtype of extract.

Determination of o-diphenolsA 0.5 mL sample of each phenolic extract was

dissolved in 5 mL of methanol/water (1:1, v/v), and4 mL of the resulting solution was added to 1 mL ofa 5 % solution of sodium molybdate dihydrate inethanol/water (1:1, v/v) and shaken vigorously. After10 min it was centrifuged for 3 min at 1490 g, theabsorbance at 370 nm of upper layer was measu-red using the calibration curve of gallic acid (r2 =0.997). The results were expressed in milligrams ofgallic acid per kilogram of oil. The spectrophotome-tric analyses were repeated at least three times foreach type of extract.

Fi g u re 1 - Regions of Bou Arada (E1) and El Aroussa (E2– E3) inTunisia


176

Chromatographic analysis of tocopherols1 g of oil sample was dissolved in 10 mL n- h e x a n e

and extracts were filtered through a 0.45 µm nylon fil-t e r. α-, β- and γ- t o c o p h e rols (α-toc, β-toc, and γ- t o c ,respectively) were determined by HPLC HP 1100Series (Agilent Technologies, Palo Alto, CA, USA)instrument equipped with a photodiode array detec-tor set at 295 nm. The separation of tocopherols wasobtained using a Phenomenex column, Luna, CN100A (CA, USA) (150 mm, 4.6 mm ID) under isocraticconditions with n- h e x a n e / d i c h l o romethane (95: 5, v/v)as the mobile phase at a flow rate of 1 mL/min. Theinjection volume was 20 µL. Analyses were carriedout at room temperature, and the total run time was10 min. Three calibration curves were constructedwith standard solutions of each compound (α-, β- t o c ,and γ-toc, r2 = 0.999, 0.986 and 0.999, re s p e c t i v e l y )and used for quantification. The results are expre s s e din mg α, β, γ-toc per kilogram of oil.

Rancimat assayOxidative stability was evaluated by the Rancimat

method [13]. Stability was expressed as the oxida-tion induction time (h), measured with the Rancimat743 apparatus (Metrohm, Switzerland), using an oilsample of 3.5 g warmed to 101.6 °C and an air flowof 10 L h- 1. Assays were carried out in triplicate.

Sensory analysis Sensory analysis was performed by a fully trained

analytical taste panel for v irgin ol ive oi l ofDipartimento di Scienze degli Alimenti of Universitàdi Bologna (recognized by the Italian Ministry forA g r i c u l t u re, Food and Forestry Policy-Mipaaf).Quantitative descriptive analysis (QDA) was appliedin order to identify diff e rent sensory profiles betweentested VOO [14]. Each taster was asked to identifyolfactive and gustatory characteristics in VOO sam-ples (specifying the diff e rence in terms of major orminor presence of bitter and/or pungent attributes).Each oil sample was analyzed by 10 tasters duringt h ree diff e rent sessions using the sensory ballotreported in Cerretani et al. [15]. The sample setsw e re randomly distributed among the assessors.

Statistical analysisAll data were analyzed using SPSS r. 11.0.0 statisti-

cal software (SPSS Inc., Chicago, IL, USA). The signifi-cance diff e rences at 5 % level among means wasdetermined by one-way ANOVA, using Tu k e y ’s test.

RESULTS AND DISCUSSION

Morphologic olive aspects Both the average fresh fruit weight and the pulp

proportion are important characteristics of the pick-ling olive varieties. Marsaline samples cultivated inrain-fed regime, as well as in Bou Arada (E1) and ElAroussa farms (E2), showed high fruit fresh weightreaching 5 g. Under the irrigation regime in ElA roussa farm (E3), average olive weight incre a s e dremarkably to 9 g (Table I).

The pulp/stone ratio was about 7.3 in rain-fed;under the irrigation regime this parameter incre a-sed considerably and reached 9.1 (Table I). Patumiet al. [16] have already reported a positive effect ofwater supply on fruit volume for some Italian varie-ties, highlighting the importance of the irrigationpractices for pickling cultivars.

The fruits obtained from irrigated trees (E3) hadhigher water content (69 %) than olives subjectedto rain-fed conditions (57 %) (E1 and E2). Alves etal. [17] reported that fruit moisture depends on thee n v i ronmental conditions and cultural practicessuch as irrigation.

The Marsaline pomologic aspects showed thatthe fruit is big and fleshy, so this cultivar could bec o n s i d e red as a good table olive variety.

Table I - Morphologic olive aspects and oil co nte nts of Marsalinesamples

Samples Samples Samples

collected collected collected

from E1 from E2 from E3

Average fruit

fresh weight (g) 5±0.5b 5±0.3b 9±0.2a

Pulp/stone 7.3±0.4b 7.3±0.6b 9.1±0.3a

Water

content (%) 57±2.0b 57±1.7b 69±1.9a

Oil content

(% DM) 47±1.8b 47±1.6b 51±2.0a

E1: Bou Arada farm where olive trees were cultivated in rain-fed regime.

E2: orchard in the region of El Aroussa, where olive trees were cultivated in

rain-fed control.

E3: irrigated orchard in the same region of El Aroussa.

Mean ± SD (n = 3), a-c Different letters in the same row indicate significantly

different values (Tukey’s test, p<0.05).


177

Oil ContentThe olive oil contents were expressed as percent

of DM (Table I). The Marsaline oils were characteri-zed by high oil yield (>46 % DM) according to theclassification of Tous and Romero [18] and evenexceed Chemlali oil content (40 %) [19]. Moreover,Marsaline oil contents were fairly enhanced by theirrigation regime but were not influenced by thegeographical area. In fact, oil yield of the irrigatedfarm (E3) was higher (51 %) than that obtained inrain fed conditions (E1 and E2) (47 %). Other stu-dies showed that olive oil yield is positively affectedby the fruit flesh humidity during harvesting time[20]. It has also been reported that the stage ofmaturity of fruits has significant effects on oil yield[19]. Ben Temime et al. [21] showed that the geo-graphic area affected the oil content of the secondmain Tunisian ‘‘Chétoui” variety.

Quality Indices Taking into account that these olive oils were pro-

duced by a laboratory scale mill that slightly differswith respect to industrial mills [22], all the obtainedand analyzed oil samples (Table II) showed lowvalues for the physicochemical parameters evalua-ted (acidity ≤ 0.8 %; peroxide index ≤ 20 meqO2/kg; K270 ≤ 0.22; K232 ≤ 2.5; ΔK ≤ 0.01), with allof them falling within the ‘‘extra virgin’’ category(EVOO), as stated by Regulation EEC Reg. 2568/91[9] (Table II). This finding could be attributed to thefact that tested olives were collected at an adequa-te maturity stage, then immediately processed andconsequently were not exposed to serious hydroly-

tic and oxidative damage. Nevertheless, a slight risein free acidity values was observed in samplesobtained from irrigated orc h a rd (E3). This behaviorcan be explained by the increase in enzymatic acti-v i t y, especially lipolytic enzymes in olives obtainedf rom irrigated trees. In fact, fruits having importantmoisture seem to be more sensitive to pathogenicinfections and mechanical damage [23].

Moreover, Table II shows that there were no stati-stically significant differences in peroxide values andextinction coefficients between all the studied oils.These results are in accordance with other workscarried out by our re s e a rch group [2]. In fact,p e roxide value and UV characteristics were notinfluenced by the irrigation of Chétoui trees. Otherauthors [24] reported that cultivar or origin area hadno significant influence on the quality indices, whichare basically affected by factors causing damage tothe fruits (e.g., olive fly attacks or improper systemsof harvesting, transport and storage of olives).

Fatty acid compositionThe identified and quantified fatty acids were: pal-

mitic (C1 6 : 0), palmitoleic (C1 6 : 1), stearic (C1 8 : 0), oleic( C1 8 : 1), linoleic (C1 8 : 2), linolenic (C1 8 : 3) and arachidicacid (C2 0 : 0) (Table III). The fatty acid composition ofthe Marsaline oils covered the range expected forolive oil by the International Olive Oil Council and theEEC Regulation [9, 25-26]. The monounsaturatedfatty acids have great importance because of theirnutritional implication and effect on oxidative stability.The oleic acid percentage, related to the total fattyacid fraction (% TFA), ranged from 71.5 to 76.9 % intested oils. Marsaline cultivar has a higher oleic acidcontent compared to Chemlali [2].

Palmitic and linoleic acid levels varied between9.4-12 % and 6.9-11.1 %, re s p e c t i v e l y. The con-tents of these two fatty acids were lower than thoseof Chemlali (Table III). The stearic acid is an impor-tant saturated fatty acid. Its content ranged from 2.9and 4.9 %, and thus was always within the rangere q u i red for this fatty acid in olive oil (0.5-5) [9, 25-26]. Marsaline oil samples have a good fatty acidcomposition characterized by higher percentage ofoleic acid and lower levels of palmitic and linoleicacids than Chemlali and some other secondaryTunisian VOOs [19]. On the other hand, the fattyacid composition of Marsaline oils showed variationsdepending on the zones of plantation. For instance,the oleic/linoleic acid ratio of Marsaline oils obtained

Table II - Quality parameters of Marsaline virgin olive oils

Oils from E1 Oils from E2 Oils from E3 Norm*[9]

Free acidity 1 0.3±0.04b 0.4±0.03b 0.6±0.02a ≤ 0.8

Peroxide value 2 16±2a 15±2a 13.5±3ab ≤ 20

K 2323 1.5±0.3a 1.1±0.7a 1.2±0.6a ≤2.5

K 2703 0.18±0.1a 0.09±0.2a 0.12±0.1a ≤0.22

ΔK 3 0.002±0.003a 0.001±0.002a 0.005±0.003a ≤ 0.01

E1: Bou Arada farm where olive trees were cultivated in rain-fed regime

E2: orchard in the region of El Aroussa, where olive trees were cultivated in rain-

fed control


Mean (n = 3), a-c Different letters in the same row indicate significantly diffe-

rent values (Tukey’s test, p<0.05)

* For extra virgin olive oil; 1 acidity as g oleic acid/100g of oil; 2 peroxide index as

meq O2/kg; 3 K232, K270 and ΔK as nm.


178

f rom Bou Arada farm was higher than those pro d u-ced in El Aroussa region (E2, E3). This result wassimilar to that found by Ben Temime et al. [21].These authors reported that the geographic are acan modify the fatty acid composition of Chétouiolive oil. On the other hand, the irrigation tre a t m e n tdid not affect Marsaline fatty acid composition. Incontrast, previous works carried out by our re s e a rc hg roup reported that Chétoui VOO obtained in rain-fed conditions had a statistically significant highercontent of oleic acid, while EVOO from irrigatedt rees showed a higher content of linoleic acid [2].

Total phenols and o-diphenols The amount of phenolic compounds are an impor-

tant factor able to indicate the quality of VOO becauseof their involvement in its resistance to oxidation andits sensory taste characteristics such as bitter andpungent [27]. Amounts of total phenols and o- d i p h e-nols of Marsaline EVOO ranged from 48.5 to 101.7and from 20.3 to 73.8 mg kg- 1, respectively (Table III).

The content of these compounds in Marsaline oilswas significantly affected by the irrigation regime. Infact, samples obtained from irrigated trees had lowerlevels of total phenols and o-diphenols than non irri-gated ones (Table III). This behavior had already beenobserved for Chétoui cultivar produced by the same

laboratory scale mill and under the same technologi-cal conditions [2]. With re g a rds to water availability, itis generally agreed that the level of phenolic com-pounds is higher in oils obtained from dro u g h t - s t re s-sed crops than in those from irrigated crops [3-4]. Theobserved diff e rences in phenol concentration in theoils could be a consequence of the diff e rent waters t ress level of olives from rain-fed to irrigation condi-tions that involve changes in the activity of enzymesresponsible for phenolic compound synthesis, suchas L-phenylalanine ammonia-lyase whose activity isg reater under higher water stress conditions [28].

On the other hand, results showed that the pro d u c-tion site did not cause any significant variation in thetotal contents of phenols and o-diphenols in Marsalineolive oil. On the contrary, Ben Temime et al. [24] foundthat phenols were influenced by the site of pro d u c t i o nhaving diff e rent climate and soil parameters.

Tocopherol compoundsTogether with polar phenolic compounds,

tocopherols are responsible for the oxidative stabi-lity of olive oil and, there f o re, for its shelf life withspecial emphasis on α- t o c o p h e rol [27]. Table IIIreported that in Marsaline samples, and as expec-ted for extra virgin olive oil [29], α-tocopherol is byfar the most abundant isoform of vitamin E. The

Table III - Chemical composition of Marsaline virgin olive oils

Oils from E1 Oils from E2 Oils from E3 Chemlali [2] Norm [9, 25-26]

C16 :01 9.35±0.5c 11.99±1.2b 11.99±1b 19.33±0.6a 7.5 - 20

C16 :11 0.87±0.3c 0.79±0.5c 1.35±0.6b 2.64±0.3a 0.3 - 3.5

C18 :01 4.91±0.3a 3.32±0.5b 2.88±0.5b 2.22±0.3c 0.5 - 5

C18 :11 76.87±0.9a 71.53±1.2b 71.80±1.9b 60.32±0.7c 55 - 83

C18 :21 6.90±0.4c 11.09±0.8b 10.90±0.7b 14.05±0.5a 3.5 - 21

C18 :31 0.64±0.2ab 0.69±0.2ab 0.70±0.1ab 0.71±0.1ab < 0.9

C20 :01 0.45±0.1ab 0.59±0.1ab 0.38±0.2ab 0.51±0.1ab ≤ 0.6

C18 :1/C18 : 2 11.14±1a 6.45±0.9b 6.59±1.3b 4.29 ±0.8c

Total phenols2 101.7±5.3b 99.7±8.1b 48.5±6.2c 190.1±4.3a -

o-diphenols2 73.8±8.9a 65.3±6.1a 20.9±3.2b 59.6±6.7a -

α-tocopherols3 161.8±3.2c 271.3±5.1a 223.7±4.3b 259.8±4.6ab -

Total tocopherols3 180.3±7.6c 298.9±6.1a 250.1±8.5b 300.5±5.3a -

Oxidative stability4 32.1±1.2ab 28.5±2.9ab 17.3±1.1c 34±1.3a -

E1: Bou Arada farm where olive trees were cultivated in rain fed regime.

E2: orchard in the region of El Aroussa, where olive trees were cultivated in rain-fed control.


Mean ± SD (n = 3), a-c Different letters in the same row indicate significantly different values (Tukey’s test, p<0.05)1 as percentage of total fatty acids; 2 as mg of gallic acid kg-1 of oil; 3 as mg of tocopherols kg-1 of oil; 4 as hours.


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total tocopherol contents of Marsaline VOO (180.3-298.9 mg kg-1), covered the range expected for anolive oil of good quality (100-300 mg kg-1) [30-31].

As reported in Table III, the origin of the oil affectsthe tocopherol content significantly. Indeed, the oilsfrom El Aroussa had higher α- and total tocopherolcontent than samples from Bou Arada re g i o n .These results are in agreement with Ben Temime etal. [24] findings. More o v e r, the water supply toMarsaline trees affected tocopherol contents in thetested oils. In fact, similar to the polar phenolst rend, samples subjected to the irrigation re g i m ehad lower levels of this natural antioxidant than non-irrigated one. A previous paper suggested thattocopherol content is highly variety-dependent [2].

Oxidative stabilityOxidation stability is an important property of olive

oil quality. Oxidative stability of Marsaline VOO rangedf rom 17.3 to 32.1 h (Table III). Compared to otherTunisian cultivars, this local variety seems to havemedium stability values. For instance, Abaza et al. [6]reported the mean stability of the seven principalTunisian olive oils ranged between 11.5 (Gerboui) and45.6 h (Sayali). The highest stability value was obser-ved for samples obtained in rain-fed conditions. Thiscan be explained by the richness in phenol com-pounds, more exactly in o-diphenols and tocophero l s( Table III). In accordance with literature [2], strong cor-relations were found between the oxidative stabilityand the content in polar phenols (data not shown).

Sensory analysisSensory appraisal of the Marsaline samples

according to Cerretani et al. [15] failed to show anydefects in the tested oils that are classified as extravirgin (EVOO) independently of irrigation tre a t m e n tand plantation area. The aroma of tested EVOOw e re characterized by a high-medium fruitiness( t h rough both orthonasal and re t ronasal analysis).M o re o v e r, samples had typical medium-low valuesof bitterness and pungency, with no diff e re n c e samong the irrigation treatments in the pungencycharacteristic. The bitter sensation had slightlylower values in the irrigated farm according to thephenolic contents. Furthermore, pleasant secon-dary flavours of fresh grass and ripe fruits were per-ceived in Marsaline oils which are particularly prizedby local people for their fruity aroma and lack of bit-terness and sharpness (sweet oils).

CONCLUSION

This work is the first evaluation of a neglected andlocal olive variety generally used as a table olive (cv.Marsaline) that has the potential to produce goodquality EVOO. Indeed, the Marsaline variety hadexcellent nutritional characteristics in terms of fattyacid composition, evidenced by a high percentageof oleic acid in addition to good levels of antioxi-dants, particularly tocopherols. Moreover, the repor-ted results suggested that the oleic/linoleic ratio ofMarsaline olive oils was affected by the geographi-cal area of cultivation, while the irrigation treatmentaffected widely their antioxidant contents and con-sequently their oxidative stability.

AbbreviationsDM: Dry Matter; EVOO: Extra Virgin Olive Oil; FA M E s :

Fatty Acid Methyl Esters; VOO: Virgin Olive Oil.

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Received 28/1/2009, accepted 6/3/2009


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TH E TOTA L M E T H A NO L, P E T RO L E U M E T H E R, CH LO RO FO R M, E T H Y L AC E TAT E E X T R AC TS A N D T H EAQ U E O U S R E S I D U E F RO M AS P H O D E L I N E L U T E A O F BU LGA R I A N O R I G I N W E R E E VA LUAT E D FO RT H E I R A N T I OX I DA N T E F F E C T I V E N E S S I N CO NC E N T R AT I O N R A NG E 0 .1-0.5% D U R I NG OX I DAT I O NO F T R I AC Y LG LYC E RO LS O F LA R D ( TGL), T R I AC Y LG LYC E RO LS O F S U N F LOW E R O I L ( TGSO) A N DNAT I V E S U N F LOW E R O I L AT 100°C. TH E CH LO RO FO R M A N D E T H Y L AC E TAT E E X T R AC TS M A N I F E ST E D A N E XC E L L E N T A N T I OX I DA N TAC T I V I TY D U R I NG OX I DAT I O N O F TGL A N D TGSO. TH E A N T I OX I DAT I V E E F F E C T I NC R E A S E DW I T H R I S I NG CO NC E N T R AT I O N O F T H E E X T R AC TS. TH E CH LO RO FO R M A N D E T H Y L AC E TAT EE X T R AC TS AT 0.5% CO NC E N T R AT I O N I M P ROV E D T H E OX I DAT I O N STA B I L I TY O F NAT I V E S U N-F LOW E R O I L I N A M U CH LOW E R D E G R E E. FO R FA ST S C R E E N I NG O F A N T I OX I DAT I V E CO M P O U N D S,A T H I N LAY E R CH RO M ATO G R A P H I C M E T H O D WA S U S E D. IT WA S E STA B L I S H E D T H AT T H E 1, 8 –D I H Y D ROX YA N T R AQ U I NO N E S D I D NOT E X H I B I T A N T I OX I DAT I V E P RO P E R T I E S. TH E 2 -AC E TY L- 1 -H Y D ROX Y- 8 -M E T H OX Y- 3 -M E T H Y L NA P H T H A L E N E WA S FO U N D TO B E AC T I V E A S A N T I OX I DA N T.KE Y W O R D S:, A N T I OX I DA N T E F F E C T I V E N E S S, AS P H O D E L I N E L U T E A, LA R D, S U N F LOW E R O I L,1, 8 -D I H Y D ROX YA N T H R AQ U I NO N E S, H Y D ROX Y NA P H T H A L E N E D E R I VAT I V E S

PRORPIETÀ ANTIOSSIDANTI DI ASPHODELINE LUTEA DI ORIGINE BULGARAL’E F F E T TO A N T I O S S I DA N T E D E G L I E ST R AT T I A L M E TA NO LO, E T E R E D I P E T RO L I O, C LO RO FO R-MIO, ACETATO DI ETILE ED I RESIDUI ACQUOSI DI ASPHODELINE LUTEA DI ORIGINE BULGARA ÈSTATO VALUTATO A CONCENTRAZIONI 0,1-0,5% DURANTE L’OSSIDAZIONE DI TRIGLICERIDI DILARDO (TGL), TRIGLICERIDI DI OLIO DI GIRASOLE (TGSO) E OLIO DI GIRASOLE TAL QUALE A100°C. GLI ESTRATTI AL CLOROFORMIO ED ACETATO DI ETILE HANNO MANIFESTATO ECCEL-L E N T E AT T I V I TÀ A N T I O S S I DA N T E D U R A N T E L’O S S I DA Z I O N E D I TGL E TGSO. L’E F F E T TOA N T I O S S I DA N T E AU M E N TAVA AU M E N TA N D O LA CO NC E N T R A Z I O N E D E G L I E ST R AT T I. GL IESTRATTI AL CLOROFORMIO ED ACETATO DI ETILE ALLA CONCENTRAZIONE DI 0,5% MIGLIO-RAVANO LA STABILITÀ ALL’OSSIDAZIONE DELL’OLIO DI GIRASOLE IN GRADO MOLTO MINORE.PE R U NO S C R E E N I NG V E LO C E D E I CO M P O ST I A N T I O S S I DA N T I È STATO U SATO U N M E TO D OTLC. SI È STA B I L I TO CH E L’ 1, 8 -D I D RO S S I A N T R ACH I NO N E NO N P R E S E N TA P RO P R I E TÀANTIOSSIDANTI. SI È OSSERVATO CHE IL 2-ACETIL-1-IDROSSI-8-METOSSI-3-METILNAFTALENEPRESENTA ATTIVITÀ ANTIOSSIDANTE.PAROLE CHIAVE: PROPRIETÀ ANTIOSSIDANTI, ASPHODELINE LUTEA, LARDO, OLIO DI GIRASO-LE, 1,8-DIIDROSSIANTRACHINONE, DERIVATI DI IDROSSINAFTALENE

Antioxidant properties of Asphodeline lutea of Bulgarian origin

I. LAZAROVA, E. MARINOVA, G.TODOROVA-NIKOLOVA, I. KOSTOVA

IN ST I T U T E O F ORGA N I C CH E M I ST RY W I T H

CE N T R E O F PH YTO CH E M I ST RY - BU LGA R I A N

ACA D E M Y O F SC I E NC E S SO F I A - BU LGA R I A

CORRESPONDENCE: EMMA M. MARINOVA, INSTITUTE OF

ORGANIC CHEMISTRY WITH CENTRE OF PHYTOCHEMISTRY,BULGARIAN ACADEMY OF SCIENCES, 1113 SOFIA,

BULGARIA;

PHONE: +359 2 9606 178; FAX: +359 2 8700 225;E-MAIL: [email protected]

OR [email protected]


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INTRODUCTION

Many plants are reported to contain a wide varietyof antioxidant compounds such as phenolic com-ponents. They are considered beneficial for humanhealth, decreasing the risk of degenerative diseasesby the reduction of the oxidative stress and the inhi-bition of the macromolecular oxidation [1 – 3].

The genus Asphodeline belongs to the familyAsphodelaceae, subfamily A s p h o d e l o i d e a e. Thepresence of 1,8-dihydroxyanthraquinones based ona chrysophanol unit is a characteristic feature of thissubfamily [4].

Asphodeline lutea is a perennial plant growing in theMediterranian region. The only report found on thisplant species reveals the presence of 1,8-dihy-d roxyanthraquinones, flavonoids and chlorogenic acid[5, 6]. Data on the medicinal use of A. lutea a re notavailable. However, the edible use of its roots, shootsand flowers has been reported [7]. The ancientG reeks roasted the roots like potatoes and ate themwith salt and oil or mashed them with figs. The rawf resh flowers are very decorative and tasty addition tothe salad, while the young shoots are eaten cooked.The existing literature data on the antioxidative capa-bility of A. lutea a re very limited [6] (Fig. 1).

Figure 1 – Plants of Asphodeline lutea

The objectives of the present study were: (I) toevaluate the antioxidant activity of extracts from A.

lutea in two pure lipid systems with different degreeof unsaturation, i.e. triacylglycerols of lard (TGL) andtriacylglycerols of sunflower oil (TGSO), and in nati-ve sunflower oil; (II) to test the antioxidative proper-ties of some anthraquinones isolated from the chlo-roform extract and to determine some other com-ponents that act as antioxidants.

The work is connected with our interest in findingnew plant resources of health improving products.

MATERIALS AND METHODS

Plant materialThe roots of A. lutea (Asphodelaceae) were col-

lected in June 2005 near the town of Pern i k ,Bulgaria. A voucher specimen is deposited at theherbarium of the Institute of Botany, BulgarianAcademy of Sciences, Sofia.

MaterialsC o m m e rcially available samples of lard and sunflower

oil were used. TGL and TGSO were obtained by clea-ning commercially available lard and sunflower oil sam-ples from pro- and antioxidants and trace metals byadsorption chromatography [8]. Briefly, the lipid sub-strates (100 g in 1000 ml distilled hexane) were passedt h rough a (2 cm i.d.) column filled with 70 g alumina(type 507C, neutral, activity stage II, Fluka, Buchs,Switzerland) activated at 180°C for 4 h. The obtainedt r i a c y l g l y c e rols were collected in nitrogen in the darkand were stored under nitrogen at -20°C for no morethan 10 days. The TGL and TGSO were found to con-tain undetectable amounts of tocopherols (HPLC, <0.5ppm) and iron and copper (atom absorption spectro-s c o p y, <0.01 and 0.001 ppm, respectively). Contro loxidation experiments at 80°C in the presence of0.01% and 0.02% citric acid demonstrated that thechelating agent had no effect on the oxidation kinetics.The initial peroxide value of the sunflower oil was 4.2meq/kg, and of the TGL and TGSO were zero .

Methods

ExtractionThe dried roots (0.70 kg) of A. lutea were extrac-

ted with methanol (3 times x 24 h x 2.7 l) at roomt e m p e r a t u re, than concentrated in vacuum to givethe crude methanol extract (TE, 22 g). Solvent-sol-vent partition of this extract using petroleum ether,c h l o roform and ethylacetate aff o rded the corre-sponding petroleum ether (A, 4.3 g), chloroform (B,5.2 g) and ethylacetate (C, 0.5 g) extracts andaqueous residue (D, 15.2 g).

The chloroform extract was subjected to liquidvacuum chromatography (LVC) on silica gel usingPE: CHCl3 (20:1 → 1:1) to give fractions F1-F4.

Lipid sample preparationLipid samples containing different concentrations

of the investigated samples (0.1, 0.2 and 0.5%)


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High performance liquid chromatographyThe tocopherol content of sunflower oil was

determined by normal - phase HPLC [10], using aM e rck - Hitachi (Darmstadt, Germany) apparatusequipped with a L - 6000 pump and a Merc kHitachi F - 1050 fluorescence detector (λe x = 295nm, λe m = 330 nm). A column with Nucleosil SI -50, 250 x 4 mm (Macherey - Nagel, Düre n ,Germany), and hexane:dioxane (96:4) elutionsystem with a rate of 1 ml/min was used.

Oxidation proceduresOxidation was carried out at 100°C (0.2°C) in the

dark by blowing air through the samples (2 g) at a rateof 50 ml/min. Under these conditions the pro c e s stook place in a kinetic regime, i.e. at a sufficiently highoxygen concentration at which the diffusion rate didnot influence the oxidation rate. The process was fol-lowed by withdrawing samples (ca 0.1 g) at varioustime intervals and subjecting them to iodometricdetermination of the primary oxidation pro d u c t s( p e roxides) concentration, i . e . the peroxide value (PV)[11]. The coefficient of variation for PV determinationwas 7-8% irrespective of the measured value. Kineticcurves of peroxide accumulation were plotted.

Kinetic evaluation of the antioxidant actionThe effectiveness of the additives in the lipid

systems was estimated on the basis of the induc-tion period (IP) which was determined by themethod of the tangents. That means the tangentsa re applied to the two parts of the kinetic curves[12]. The coefficient of variation ranged from 6 -13% and was inversely related to the inductionperiod. The reported IP values result from thre eindependent experiments. The effectiveness wasexpressed as stabilization factor F:

F = IPadd/IP0

where IPadd is the induction period in the presenceof the tested additive, and IP0 is the inductionperiod of the control sample.

Thin layer chro m ato g raphic method for dete r m i n ation ofantioxidants

The sample (for preparative chromatography 20mg or spots) was applied on the TLC plate (Silicagel G, layer thickness 0.25 mm). Chloroform wasused as a solvent system for chromatography. Afterdrying the chromatograms they were sprayed withlard solution (3% in hexane) and allowed to stand at

St a n d a rd compounds of chr ysophanol (1), asphodeline (2),1 , 1 ’, 8 , 8 ’, 1 0 - p e nt a hyd roxy- 3 , 3 ’ -d i m e t hy l - 1 0 , 7 ’ - b i a nt ra ce n e- 9 , 9 ’, 1 0 ’ -trione (3), 2-ace ty l - 1 , 8 -d i m e t h oxy- 3 - m e t hy l n a p hthalene (4), 2-acetyl-1-hydroxyl-8-methoxy-3-methylnaphthalene (5) and 2-acetyl-8-methoxy-3-methylnaphthaquinone (6), available in our laboratorywere used in this study. These compounds were isolated from A. luteaand identified by detailed examination of their UV, IR, 1D and 2D NMRspectra. A publication on the isolation and structure elucidation of 1 -6 and other components of A. lutea is already in press [9].

were prepared by adding aliquots of their solutionsin acetone, or ethanol to a weighed amount of thelipid substrates, followed by removal of the solventwith nitrogen. For control, samples without addedantioxidant were used.

Gas chromatographyThe fatty acid compositions of the starting oils

( l a rd and sunflower oil) were determined by gaschromatography of their methyl esters using a PyeUnicam instrument, model 304, equipped with adual flame-ionization detector and a glass capillarycolumn (30 m x 0.2 mm id) coated with SILAR 10C(Supelco Inc., Bellefonte, PA). The carrier gas wasnitrogen at a flow rate of 14 ml/min. The temperatu-re was maintained at 165°C for 5 min, and thenincreased to 200°C by 2°C/min.


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room temperature for 24 h. After that, the platesw e re sprayed first with 1% aqueous solution ofpotassium iodide, and, after being allowed to standfor 5 minutes in the dark, with 1% solution of starchin 1% acetic acid. The zones with antioxidativeeffect remained white on a violet background [13].

Thin layer chro m ato g raphic ex a m i n ation of ext ra cts andcompounds for detection of antioxidants

The total methanol extract (TE), the chloro f o r mextract (B) and fractions F1-F4 were tested for theirantioxidant activity by applying the above describedTLC method [13]. Only TE, B and F3 showed activezones. A very large zone with antioxidative eff e c twas observed in F3. This fraction was subjected tocomparative TLC examination using standard com-pounds 1 - 6 and the compounds with Rf valuescorresponding to the zone with antioxidative effectdetermined as 4 and 5.

F u r t h e r, the individual compounds 4, 5 and 6were also tested using the same TLC method [13]and only 5 was found to be active.

GC-MS analysis of the chloroform extract of the active zoneThe silica gel of the active zone was scraped off,

eluted with chloroform and subjected to GC-MSanalysis. Three main components C 1 - C 3 w e reobserved in the chloroform extract: component C1

– Rt 25.317 min, MS - m/z 230 (M+ .), 215 (100),200 (60), 187 (8), 171(4), 159 (8), 43 (6); compo-nent C2 – Rt 27.041 min, M+. at m/z 244 was iden-tical with 2-acetyl-1,8-dimethoxy–3-methylnaphtha-lene (4); component C 3 – Rt 30.451 min, M+ . a tm/z 230 was identical with 2-acetyl-1-hydro x y - 8 -methoxy-3-methylnaphthalene (5).


Since the type and unsaturation degree of thelipid medium may strongly influence the inhibitinge ffect of the antioxidants [14] we investigated thestabilizing effects of the substrates studied in puri-fied triacylglycerols of lard (TGL) and of sunflower oil(TGSO). As established [15], during the initial stageof autoxidation of TGSO the linoleate moieties aloneare attacked by oxygen and peroxide radicals, whe-reas both the linoleate and oleate in TGL participatein chain generation and chain propagation.

The fatty acid compositions of TGL and TGSOwere as follows:

• TGL - myristate 2%, palmitate 23%, palmitoleate2%, stearate 16%, oleate 50%, linoleate 7%

• TGSO - palmitate 5%, stearate 5%, oleate 23%,linoleate 67%.

• The sunflower oil contained 655 ppm α- t o c o p h e-rol, 26 ppm β- t o c o p h e rol, and 5 ppm γ- t o c o p h e rol.

Antioxidant activity of extracts from A. luteaThe total methanol (TE), petroleum ether (A), chlo-

roform (B), ethyl acetate (C) extracts and theaqueous residue (D) from A. lutea w e re evaluatedfor their antioxidant effectiveness in concentrationrange 0.1-0.5% during oxidation of TGL, TGSO andnative sunflower oil at 100°C.

Antioxidant activity in TGLBy way of example, Figure 2 illustrates the kinetic

curves of peroxide accumulation during oxidation ofTGL in the presence of 0.5% of the extracts. Afterp rocessing all the kinetic curves obtained, the valuesfor the stabilization factor (F) were determined. Theresults are presented in Figure 3. The stabilization fac-tor determines how many times the oxidation stabilityof the lipid system increases in the presence of anantioxidant. A value of F = 1 corresponds to noantioxidant activity. It was assumed that a value of Fover 10 corresponds to excellent antioxidant activityand value under 2 – to weak one. Figure 3 shows thatthe chloroform (B) and ethyl acetate (C) extracts mani-fested an excellent antioxidant activity, the total

Figure 2 - Kinetic curves of peroxide accumulation during the oxida-tion of TGL at 100°C in the presence of 0.5% extracts from A. lutea: 0 –control sample; 1 –total methanol extract (TE); 2 – petroleum etherextract (A); 3 - chloroform extract (B); 4 - ethyl acetate extract (C); 5 -aqueous residue (D)


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Figure 5. It is to be seen that stabilization factors ofthe extracts in TGSO are close to these in TGL. Acomparison of the inhibiting action of the most acti-ve extracts (TE, B and C) in both lipid systems arep resented in the Figure 6. A comparison with theeffectiveness of BHT, α-tocopherol and ferulic acidat a concentration of 0.02% is given. The re s u l t sshow that chloroform and ethyl acetate extractsare especially suitable for the inhibition of the oxida-tion process of the lipid systems.

Figure 3 - Dependence of the stabilization factor (F) during the oxi-dation of TGL on the concentration of the extracts from A. lutea. Thenumbers stand for the corresponding extracts in Figure 2.

Figure 4 - Kinetic curves of peroxide accumulation during the oxida-tion of TGSO at 100°C in the presence of 0.5% extracts from A. lutea.The numbers stand for the corresponding extracts in Figure 2.

Figure 6 - Stabilization factor (F) of total methanol (TE) (1 – 0.2%; 1’– 0.5%); chloroform (B) (3 – 0.2%; 3’– 0.5%) and ethyl acetate (C) (4– 0.2%; 4’ – 0.5%) extracts from A. lutea during oxidation of TGL andTG S O. For comparison F for BHT, α- to co p h e rol (Toc) and ferulic acid(FA) (0.02%) are given.

Figure 5 - Dependence of the stabilization factor (F) during the oxi-dation of TGSO on the concentration of the extracts from A. lutea. Thenumbers stand for the corresponding extracts in Figure 2.

methanol (TE), and petroleum ether (A) extracts - amoderate one, while the aqueous residue (D) had noe ffect. The antioxidative effect increased with risingconcentration of the extracts.

Antioxidant activity in TGSOF i g u re 4 shows the kinetic curves of pero x i d e

accumulation during oxidation of TGSO in the pre-sence of 0.5% of the extracts. The results for thestabilization factors obtained are presented in


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Antioxidant activity in native sunflower oil (SO)The results obtained have demonstrated that only

0.5 % of the chloroform and ethyl acetate extractsimproved the oxidation stability of native sunfloweroil. Figure 7 shows the kinetic curves of pero x i d eaccumulation during oxidation of SO in the presen-ce of these extracts. After processing the kineticcurves the values for stabilization factors weredetermined. They are 1.4 for chloroform extract and1.2 for ethyl acetate extract. Previous investigations[16-18] have shown that it is much difficult toimprove the oxidative stability of native sunflower oilby adding antioxidants because of the optimumconcentration of natural ant ioxidants (mainlytocopherols).

Antioxidant activity of some individual componentsIn order to determine some of the compounds

with antioxidative activity, a TLC investigation [13] ofthe chloroform extract of A. lutea was undertaken.A good correlation between the Rf values of theactive zone with these of 2-acetyl-1,8-dimethoxy-3-methylnaphthalene (4) and 2-acetyl-1-hydro x y - 8 -methoxy-3-methylnaphthalene (5) was observed.

In another experiment the individual compounds 4and 5 w e re subjected to the same TLC experiment[13] and only the 2-acetyl-1-hydro x y - 8 - m e t h o x y - 3 -methylnaphthalene (5) was found to be active. Thisresult suggests that the presence of a hydro x y lg roup is a structural re q u i rement for the antioxidativeactivity of the naphthalene derivatives. Other authors

have also reported antioxidative properties for someh y d roxynaphthalene compounds [19, 20].

It has been also found that under the same expe-rimental conditions [13] the naphthaquinone deriva-tive (6) did not demonstrate antioxidative properties.

GC-MS analysis of the chloroform extract of theactive zone revealed 3 main components. Two ofthem were identified as 2-acetyl-1,8-dimethoxy-3-methylnaphthalene (4) and 2-acetyl-1-hydro x y - 8 -methoxy-3-methylnaphthalene (5). The mass spec-trum of the third component C 1 (Rt 30.451 min) wasvery similar to that of 5 and suggested it to be ah y d roxyl naphthalene derivative isomeric to 5. Thepeaks at m/z 43 and m/z 187 (M+. – 43) indicate ap resence of COMe unit, while that at m/z 171 (m/z200 – CHO) - a phenolic OH group. The peak at m/z159 arises from a loss of CO from the ion at m/z 187.In the absence of 1D and 2D NMR data the structureof C1 is difficult to be unambiguously determined.

Our earlier studies showed that chrysophanol (1),asphodeline (2) and 1,1’,8,8’,10-pentahydroxy-3,3’-dimethyl-10,7’-biantracene-9,9’,10’-trione (3) arethe main anthraquinone components of A. lutea [9].It was of interest to study their antioxidative activityas to the best of our knowledge compounds 2 and3 were not tested in this connection.

The data in the literature concerning antioxidativep roperties of anthraquinones are contradictory.Initiated peroxidation of linoleic acid was inhibitede ffectively by anthraquinone, 1,2-dihydro x y a n t h r a q u i-none and 1,8-dihydroxyanthraquinone [21]. Yen et al.[22] found that alizarin, aloe-emodin, rhein and emo-din inhibited the peroxidation of linoleic acid in theo rder alizarin>aloe-emodin>rhein>emodin>anthra-quinone, while chrysophanol accelerated it .A c c o rding to Vargas et al. emodin, aloe-emodin, andrhein scavenge reactive oxygen and free-radical spe-cies (emodin>rhein>aloe-emodin) [23]. Emodin wasfound similar to quercetin and kaempferol in inhibitings u p e roxide radical generation and second to them ininhibiting lipid peroxidation [24]. However, the re s u l t sof Cai et al. [25] showed that anthraquinone, emodinand aloe-emodin do not have a radical scavenginga c t i v i t y. These authors reported potent activity forpurpurin, pseudopurpurin and alizarin, and pointedout the importance and the influence of ortho diphe-nolic structure in anthraquinones on their radical sca-venging activity [25]. This idea is supported by thevery good antioxidant activity exhibited by morindoneand alizarin [26], and alaternin [27]. Huang et al. [28]

Figure 7 - Kinetic curves of peroxide accumulation during the oxida-tion of native sunflower oil at 100°C in the presence of 0.5% extracts.The numbers stand for the corresponding extracts in Figure 2.


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p roposed that two hydroxyl groups arranged in eitherthe ortho- or meta-position in the C ring of anthraqui-none nucleus are re q u i red for such derivatives to inhi-bit lipid peroxidation.

In the present investigation anthraquinones 1 – 3

were evaluated during TGL and TGSO oxidation at100°C and found to be inactive. Our results are inline with the findings of Cai et al. [25] as the studiedcompounds 1 – 3 lack ortho-dihydroxy structure.

CONCLUSIONS

1.The chloroform and ethyl acetate extracts fro mAsphodeline lutea manifested an excellent antioxi-dant activity during autoxidation of TGL andTGSO. The antioxidative effect increased withrising concentration of the extracts. The chlo-roform and ethyl acetate extracts at 0.5% con-centration improved the oxidation stability of nati-ve sunflower oil in a much lower degree (stabiliza-tion factors 1.4 and 1.2 respectively).

2.The 1,8 – dihydroxyantraquinones (1 – 3) did notexhibit antioxidative properties. This result sup-ports the observation of Cai et al. [25] that onlyantraquinones having ortho-dihydroxy unit in theirstructure possess potent antioxidative activity.

3 .The 2-acetyl-1-hydro x y - 8 - m e t h o x y - 3 - m e t h y l-naphthalene (5 ) was found to be active asa n t i o x i d a n t .

AcknowledgmentsThe authors are grateful to the National Council

for Scientific Research in Bulgaria for the partialfinancial support under contract TK-X-1601.

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dants on the stability of triacylglycerols andmethyl esters of fatty acids of sunflower oil.Food Chemistry, 54, 377-382 (1995).

[9] G. To d o rova-Nikolova, I . Lazarova, B.Mikhova, I . Kostova, Naphtha lene andnaphthaquinone components of A s p h o d e l i n e

lutea. Chem. Nat. Comp., 2009 in press.[ 1 0 ] S. Ivanov, K. Aitzetmüller, Untersuchungen über

die To c o p h e rol- und To c o t r i e n o l z u s a m-mensetzung der Samenöle einiger Ve r t reter derFamilie Apiaceae. Fat Sci. Technol., 9 7, 24-29( 1 9 9 5 ) .

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[13] E. Marinova, N. Yanishlieva, A thin layer chro-matographic method for the rapid determina-tion of antioxidants in mixtures and estimationof their activity towards lipids. Comm. Dept.Chem. Bulg. Acad. Sci. 19, 524-527 (1986).

[14] N. Yanishlieva, E. Marinova, Natural antioxi-dants in lipid oxidation. Bulg. Chem. Commun.35, 79-91 (2003).

[15] N. Yanishlieva, A. Popov, La spectrophotomé-trie ultraviolette en tant que méthode d’esti-mation de l’êtat d’oxydation des lipides unsa-turés. Rev. Franç. Corps Gras 2 0, 11-26(1973).

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[18] N. V. Yanishlieva, E. M. Marinova, Antioxidativee ffectiveness of some natural antioxidants insunflower oil. Z. Lebensm. Unters. Forsch.203, 220-223 (1996).

[19] L . T. Ng, C.-C. Lin, C.M. Lu, antioxidativeeffects of 6-methoxysorigenin and its derivati-ves from Rhamnus nakaharai. Chem. Pharm.Bull., 55, 382-484 (2007).

[20] I.D. Yoo, B.S. Yun, I.K. Lee, I.J. Ryoo, D.-GChoung, K.H. Han, Three naphthalenes fro mroot bark of Hibiscus syriacus. Phytoche-mistry, 47, 799-802 (1998).

[21] J. Cui, Z.L. Li, X.Y. Hong, Studies on theantioxidant of anthraquinone derivatives anda-carotene. Acta Chim. Sinica, 62, 1095-1100(2004).

[22] G.C. Yen, P.D. Duh, D.Y. Chuang, Antioxidantactivity of anthraquinones and anthrone. FoodChem., 70, 437-441 (2000).

[23] F. R. Vargas, Y. H. Diaz, K. M. Carbonell,Antioxidant and scavenging activity of emodin,aloe-emodin, and rhein on free-radical and

reactive oxygen species. Pharmac. Biology,42, 342-348 (2004).

[24] T. B. Ng, Y. Lu, C. H. K. Cheng, Z. Wa n g ,Antioxidant activity of compounds from themedicinal herb Aster tatar icus . Comp.Biochem. Physiol. – C Toxic. Pharmac., 1 3 6,109-115 (2003).

[25] Cai, M. Sun, J. Xing, H. Corke, AntioxidantPhenolic constituents in roots of Rheum offici-

nale a n d Rubia corditofolia: Structure - r a d i c a lscavenging relationships. J. Agric. FoodChem., 52, 7884-7890 (2004).

[26] N. H. Ismail, H. Mohamad, A. Mohidin, N.H.Lajis, Antioxidant activity of anthraquinonesfrom Morinda elliptica. Nat. Prod. Sci., 8, 48-51 (2002).

[27] J. S. Choi, H. Y. Chung, H. A. Jung, H. J.Park, T. Yokozava, Comparative evaluation ofantioxidant potential of alaternin (2-hydro x y e-modin) and emodin. J. Agric. Food Chem., 48,6347-6351 (2000).

[28] S.S. Huang, S.F. Yeh, C.Y. Hong, Effect ofanthraquinone derivatives on lipid peroxidationin rat heart mitochondria: Structure - a c t i v i t yrelationship, J. Nat. Prod., 5 8, 1365-1371(1995).

Received February 11th, 2009,

accepted February 24th, 2009


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FIVE BURGER FORMULATIONS (BEEF, CHICKEN, MIXED BEEF AND CHICKEN (50:50), BEEFW I T H O L I V E O I L A N D CH I CK E N W I T H O L I V E O I L) W E R E P R E PA R E D. TH E O B J E C T WA S TOSTUDY THE EFFECTS ON TBARS (THIOBARBITURIC ACID REACTIVE SUBSTANCES VALUES),FAT TY AC I D S, CH O L E ST E RO L, A N D 7 -K E TO CH O L E ST E RO L O F R E P LAC I NG B E E F O R CH I CK E NFAT W I T H O L I V E O I L, O R M I X I NG CH I CK E N M E AT W I T H B E E F. TBARS O F R AW SA M P L E SINCREASED AFTER ONE MONTH OF STORAGE AND THEN DECLINED. GR I L L I NG T H E B U RG E R S H A D D I F F E R E N T A S P E C TS O N T BARS VA LU E S, T H E R E WA S A NI NC R E A S E I N BOT H CH I CK E N FO R M U LAT I O N S, A N D A D E C R E A S E I N T H E B E E F SA M P L E S.HOW E V E R T H E R E WA S NO C L E A R E F F E C T O F G R I L L I NG O N M I X E D SA M P L E S. MI X I NGCH I CK E N W I T H B E E F O R R E P L A C I NG FAT W I T H O L I V E O I L I NC R E A S E D T H E I RU N SAT U R AT E D/SAT U R AT E D R AT I O. MUFA A N D P U FA D E C R E A S E D G R A D UA L LY D U R I NGSTORAGE, BUT THEY INCREASED AFTER GRILLING. BY ADDING OLIVE OIL TO THE BEEF ANDCHICKEN BURGERS, THE CHOLESTEROL CONTENT DECREASED, FURTHERMORE CHOLESTEROLOXIDATION WAS NOT INFLUENCED BY STORAGE OR GRILLING.KEYWORDS: CHOLESTEROL OXIDATION, LIPID OXIDATION, MEAT BURGER, FATTY ACIDS.

VALUTAZIONE DI ALCUNE CARATTERISTICHE CHIMICHE DI DIFFERENTIFORMULAZIONI DI HAMBURGERSO NO STAT I P R E PA R AT I 5 T I P I D I H A M B U RG E R (D I M A N Z O, D I P O L LO, M I S C E LA M A N Z O-POLLO 50:50, MANZO CON OLIO DI OLIVA E POLLO CON OLIO DI OLIVA) PER STUDIARE L’EF-FETTO DELLA SOSTITUZIONE DEL GRASSO DI MANZO E DI POLLO CON OLIO DI OLIVA O L’EF-F E T TO D E L LA M I S C E LA Z I O N E D I CA R N E D I M A N Z O CO N CA R N E D I P O L LO, S U I VA LO R I D ISOSTANZE REATTIVE ALL’ACIDO TIOBARBITURICO (TBARS), SUGLI ACIDI GRASSI, SUL COLE-STEROLO E SUL 7-CHETOCOLESTEROLO. I VALORI DI TBARS NEI CAMPIONI SONO AUMEN-TATI DOPO UN MESE DI STOCCAGGIO E QUINDI SONO DIMINUITI.LA COTTURA AL GRILL HA AVUTO L’EFFETTO DI VARIARE I VALORI DI TBARS; QUESTI SONOAUMENTATI NEI CAMPIONI DI POLLO E SONO DIMINUITI NEI CAMPIONI DI MANZO. NON SI ÈR I L E VATO U N CH I A RO E F F E T TO G R I G L I A N D O I CA M P I O N I M I ST I. MI S C E LA N D O CA R N E D IP O L LO CO N CA R N E D I M A N Z O O S O ST I T U E N D O LA PA R T E G R A S SA CO N O L I O D’O L I VA S I ÈOTTENUTO L’AUMENTO DEL RAPPORTO INSATURI/SATURI. MUFA E PUFA SONO DIMINUI-TI GRADUALMENTE DURANTE LO STOCCAGGIO MA SONO AUMENTATI DOPO LA COTTURA ALG R I L L. L’AG G I U N TA D I O L I O D I O L I VA H A D I M I N U I TO I L CO N T E N U TO D I CO L E ST E RO LO N E ICA M P I O N I D I P O L LO E M A N Z O. IL P E R I O D O D I STO C CAG G I O E LA COT T U R A A L G R I L L NO NHANNO INFLUENZATO L’OSSIDAZIONE DEL COLESTEROLO.PAROLE CHIAVE: OSSIDAZIONE DEL COLESTEROLO, OSSIDAZIONE LIPIDICA, HAMBURGER DICARNE, ACIDI GRASSI.

Evaluation of some chemicalproperties of different

burger formulations

KHALLED M. AL-MRAZEEQ, KHALID M. AL-ISMAIL, BASEM M. AL-ABDULLAH

DE PA R T M E N T O F NU T R I T I O N A N D FO O D

TE CH NO LO GY, FAC U LTY O F AG R I C U LT U R E,TH E UN I V E R S I T Y O F JO R DA N, AM M A N,JORDAN.

CORRESPONDENCE: [email protected]


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INTRODUCTION

More and more convenience foods, such as beefor chicken burgers, are being consumed in restau-rants nowadays. These products should contain20-30% of fat to give the desirable succulence andtexture [1].

The fat of beef burger is characterized by its highcontent of saturated fatty acids, which improves itsoxidative stability during storage and cooking. Onthe other hand, excess triglycerides in plasma cal-led hypertriglyceridemia are linked to the occurren-ce of coronary artery disease in humans. Chickenfat contains appreciable levels of di- and polyunsa-turated fatty acids, and it is known that the con-sumption of food rich in di- and polyunsaturatedfatty acids may depress the level of saturated trigly-ceride and cholesterol and provide the body withthe needed essential fatty acids [2].

However, the high content of these fatty acids infood, such as burgers, lowers the oxidative stabilityduring storage and cooking. Reducing the fat levelin burgers is difficult and results in the reduction ofsome of its sensorial properties such as tendernessand flavor intensity [3].

Complete or partial replacement of burger fat withoil rich in monounsaturated fatty acids, such asolive oil, may improve the oxidative stability ofchicken burger and the nutritional value of beef bur-gers. Another approach that could improve thequality of beef and chicken burgers is the partialreplacement of beef meat with chicken meat.

Little information has been published in literature

on the effect of partial replacement of beef meatwith chicken meat and partial replacement of beeftallow or chicken fat with olive oil on the chemicaland sensory properties of burgers. There f o re, thisstudy aims at studying the effect of partial replace-ment of beef tallow and chicken fat with olive oil,the effect of partial replacement of beef fat andmeat with chicken meat and fat (50:50) and thee ffect of grilling of the above mentioned diff e re n tformulations on some chemical properties of a fre-shly prepared and stored burger.


ChemicalsSqualane, 7-ketocholesterol, cholesterol and the

s t a n d a rd of fatty acid methyl esters were fro mSigma (St Louis, MO, USA), analytical grade diethylethyl ether, chloroform and n-hexane were fro mTEDIA Company, Inc ( Fairfield, USA), KOH andTBA were from Merck (Germany), HPLC grademethanol was from Schar lau Chemie S.A(Barcelona, Spain).

Burgers preparationThe raw materials (meat and olive oil) used in the

p reparation of burgers were purchased from thelocal market.

The combinations of beef and chicken burgerswith beef fat, chicken fat and olive oil shown in Ta b l eI were pre p a red as follows: samples were pre p a re din the Meat Technology Laboratory at the Universityof Jordan. The fresh meat was coarsely ground in a

Table I - Cholesterol content (mg/100g fat) for the raw and grilled burger samples during storage

Characteristic Time of storage (month) *Treatment

Beef Chicken Mixed Beef with olive oil Chicken with olive oil

0 a333.87 a462.10 a391.67 a157.70 a193.43

Raw 1 a331.27 a461.67 a390.66 a156.61 a193.03

3 a331.30 a460.27 a390.47 a155.73 a192.00

**Means c332.15 a461.35 b390.9 e156.68 d192.82

0 a331.73 a460.13 a390.27 a156.47 a191.13

Grilled 1 a330.23 a459.37 a389.30 a154.92 a191.07

3 a330.93 a459.11 a389.13 a154.67 a190.28

**Means c330.96 a459.54 b389.5 e155.35 d190.83

Each value is the mean of three replicates.

* Values within the same column with same subscripts are not significantly (p> 0.05) different according to LSD.

** Values within the same row with different superscripts denote significant differences (p< 0.05) between treatments according to LSD.


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Hobart mincer (Hobart, London, UK) through an 8mm plate and then water was added and thoro u g h l ymixed. The level of fat was adjusted to about 15%by the addition of beef or chicken fat or olive oil (thislevel of fat is usually used in the production of bur-gers in Jordan). Fat adjustments were carried outafter determining the actual level of the fat in them i x t u re using Soxhlet method (AOAC, 1995) [4]. Thep re p a red mixtures were then shaped into (100 g)patties using a household burger mould press (MeatTechnology Laboratory, University of Jordan).

Some of the freshly pre p a red burger samplesw e re taken and analyzed for some chemical pro-perties before and after grilling. The remaining sam-ples were kept in a freezer (at -18°C), and after oneand three months of storage (this period is theexpected shelf life of this products), samples wereanalyzed for some chemical properties before andafter grilling.

Chemical and instrumental analysisThiobarbituric acid reactive substances values

(TBARS) - The extent of lipid oxidation was determi-ned by the TBARS method of Faustman, et al. [5].One burger from each treatment before and aftergrilling were analyzed. Ten gram portions of burgerw e re combined with 25 ml of 20% trichloro a c e t i cacid (TCA) and 20 ml warmed distilled water, andhomogenized using a stomacher (Model AES,France, Laboratory) for 30 sec. The homogenatewas filtered through Whatman No.1 filter paper and2 ml of the filtrate were combined with 2 ml of 0.02M aqueous 2-thiobarbituric acid (TBA) in a testtube. The tubes were incubated at 22°C in the darkfor 20 h. At the end, the absorbance of the resultingsolution was measured at 532 nm using Spectrophotometer (Biotech Engineering Management CO.Ltd, UK) . The TBARS number was expressed asmg of malondialdehyde/kg of sample using a con-version factor of 7.8 [6].

Lipid extractionTotal lipid extraction was carried out according to

the modified Folch method [7]. A 30-g of raw orcooked sample was added to 200 ml of a chlo-roform/methanol (CHCl3/CH3OH) solution (1:1, v/v),homogenized for 30 sec and placed in an oven for20 min at 60°C, and then 100 ml of CHCl3 w e readded to the mixture. This gave a final ratio of 2:1(v/v) of CHCl3/ C H3OH. The mixture was again

homogenized for 1 min and filtered to eliminate thesolid residue, which consisted mostly of pro t e i n s .The filtered fraction was added to 100 ml of 1MKCl, and was left overnight at 4°C in a refrigerator.After phase separation, the chloroform phase wasevaporated using rotary evaporator and the lipidfraction was stored at - 20°C.

De te r m i n ation of sat u rate d, monounsat u rated and polyun-saturated fatty acids

Fatty acid methyl esters (FAMEs) of the burgersamples were prepared according to Chritophersonand Glass method [8]. Briefly: 50 mg of lipid extractwere weighed, dissolved in 1 ml hexane (GC grade)and mixed by vortex for 1 min. A 200 µl of 2 M-potassium hydroxide prepared in anhydrous metha-nol were added and mixed for 30 sec until the solu-tion became clear, and then 200 µl of acetic acidwere added and mixed for 30 sec.

The prepared methyl esters were analyzed usingcapillary GLC column (Restek, Rtx-225, USA, cros-sbond 50%-cyanopropylmethyl 50%-phenylmethylpolysiloxane, 60 m, 0.25 mm/D, 0.25 µm df) imme-diately after esterification by injection 1 µl of thehexane layer through the injection port of the GLC(model GC-2010, Shimadzu Inc., Kyoto, Japan).The FAMEs were injected after adjusting the GLCconditions; column oven temperature was 180°Cfor 10 min, increased to 200°C 5°C/min and kept at200°C for 5 min, then increased to 210°C 3°C/minand kept at 210°C for 20 min. Injector temperaturewas 250°C, flame ionization detector temperaturewas 260°C, flow rate 1.2 ml/min N2, and split ratioused was 70. The fatty acids methyl esters (FAMEs)w e re identified using chromatogram of fatty acidsstandard.

Cholesterol and cholesterol oxidation products (COPs) deter-mination

Cold saponification and extraction were carriedout according to the method used by Sander, et al.[9]. 150 mg of the lipid extract were weighed accu-rately into a 25 ml screw capped test tube, 200 µlof squalane solution as internal standard and 15 mlof 1 M KOH in methanol were added to the sample,the mixture was shaken until it became free ofdispersed fat particles.

Saponification was conducted at room tempera-t u re for 18 to 20 h. 10 mls of distilled water wereadded to the saponified mixture, which was tran-


192

sferred to 100 ml separatory funnel fitted with Tefloncap. Non saponifiables were extracted three timeswith 10 ml of diethyl, and the pooled diethyl etherextracts were washed once with 5 ml of 0.5 M KOHin water, and then with 5 ml of distilled water untilthe washing solution became colorless with phe-nolphthalein.

After drying by shaking with anhydrous sodiumsulfate (Na2S O4), the extracts were filtered usingWhatman No. 1 filter paper. The Na2SO4 and the fil-ter paper were washed twice with 5 ml diethyl etherto minimize the losses due to the transfer steps.The combined filtrates were concentrated to about1 ml in a vacuum evaporator and freed of solventusing nitrogen (ultra pure) after being transferre dinto a 5 ml vial, then trimethylsilylation solution wasadded to the dried nonsaponifiables extracts.

The trimethylsilyl derivatives (TMS) of cholestero land cholesterol oxides were carried out according tothe method used by Pie, et al. [10]. TMS solutionwas pre p a red by mixing five volume of pyridine, twovolume of hexamethyldisilazane and one volume oft r i m e t h y l c h l o rosilane. The dried nonsaponifiablesextracts were dissolved in 0.5 ml TMS solution andmixed for 1 min by vortex mixer. The vial was placedin a water bath at 40°C for 20 min, and then TMSsolution was evaporated using extra pure nitro g e ngas. The derivatized TMS cholesterol and cholestero loxides were redissolved in 1 ml hexane (GC grade)and mixed for 30 sec in vortex mixer then centrifugedfor 5 min. The derivatized sterols were analyzedusing capillary GLC column (Restek, USA, cro s s b o n d5%-diphenyl 95%-dimethyl polysiloxane, 30 m, 0.25mm/D, 0.1 µm df) immediately after trimethylsilylationby injection 1 µl of the hexane layer through the injec-tion port of the GLC. The GLC conditions used were :column oven temperature was 200°C for 2 min,i n c reased to 260°C 2°C/min and kept at 260°C for12 min. Injector temperature was 270°C, detectort e m p e r a t u re was 280°C, flow rate 1.2 ml/min N2,and split ratio used was 40. The cholesterol andCOPs peaks were identified compared with theretention time of the re f e rence standard s .

GrillingBurgers were cooked directly from the fro z e n

state by contact grilling on a preheated electric grill(LT15, Ankara, Turkey) for 20 mins until the browncolor of cooked appearance was reached (grillingt e m p e r a t u re 115°C, internal temperature 75°C for

15 sec). The upper part of the grill was closed andd i rectly came into contact with the burgers. Theinternal temperature of the burgers was monitoredby a thermocouple (Digitec, Modena, Italy) placedat the geometric center of the sample.

Statistical analysisStatistical analysis of data was carried out using stati-

stical analysis system package (SAS Inc., 2000) [11].The data obtained were analyzed using Split PlotDesign to study the effect of treatments on the TBARSvalues, cholesterol, and fatty acids profile results.


Oxidative rancidity measured by TBARS testThe results of the effect of formulation, storage

and grilling on TBARS of burger treatments expres-sed as mg malondialdehyde/kg meat are shown inF i g u res 1 and 2. The figures show that the initialTBARS values of the beef sample expressed as mgmalondialdehyde/kg meat (2.26 mg/kg) were abouttwo times greater than those of the chicken sample(1.21 mg /kg). These results reflect the quality of theraw materials, which in the case of beef already hada high initial degree of peroxidation. Inappro p r i a t estorage conditions of meat, together with the actionof light, oxygen and the presence of myoglobineprobably accelerated the oxidation [12].

The TBARS values for all treatments incre a s e dsignificantly (P≤ 0.05) during the first month of sto-rage then declined during the next two months ofstorage except for the beef treatment. The decrea-se in TBARS values was notable in chicken withand without the addition of olive oil, followed bybeef with olive oil. For example, the decrease inTBARS values of chicken and beef with olive oil atthe end of the experiment was about 60% and49%, re s p e c t i v e l y, of their TBARS values at onemonth of storage. These results might be explainedby the fact that the fatty acids of these sampleshave a higher degree of unsaturation when compa-red with those of beef.

Kayaard and Gök [13] reported that “soudjouks”prepared with the addition of olive oil in place of ani-mal fat were more susceptible to lipid oxidation, asthe TBARS values increased with respect to theamount of olive oil incorporated into the formulation.On the other hand, the decrease in TBARS valuesnoticed at the end of the storage period was 85,


193

60, 47, 18 and 3% for chicken with ol ive oil,chicken, beef with olive oil, mixed and beef tre a t-ments, respectively. These results agree with thosereported by Severini, et al. [14] who found thatTBARS values of pork salami with olive oil werehigher than the basic formulation with pork backfatduring drying. After 15 days of storage, TBARS ofall samples increased then decreased at the end ofstorage. The Authors ascribed this behavior to thecombination of aldehydes with other compoundsand to the loss of volatile aldehydes.

Nassu et al. [15] evaluated the effect of naturalantioxidant on fermented goat sausages, finding

that TBARS values of goat sausages increased atthe beginning of storage and then began to decrea-se. They concluded that malonaldehyde did notaccumulate as a stable end product. Grose et al.[16] attributed the decline of TBARS value in beefpatties during storage to the slower rate of auto-oxidation of the unsaturated fatty acids after thre emonths of frozen storage and to the instability ofmalonaldehyde. Additionally, further oxidation ofmalonaldehyde and other short chains by-productsmay have yielded organic alcohol and acids, whichwere not measurable by the TBARS method [17].

Ansorena and Astiasarán [18] studied the effect of

Figure 1 - Effect of storage period on TBARS values of the raw burger samples.

Figure 2 - Effect of grilling on TBARS values of the burger samples.


194

storage and packaging on fatty acid compositionand oxidation in dry fermented sausages made withadded olive oil and antioxidants. They observed thehighest TBARS value after two months of storagefor control sausages (75% pork meat and 25% porkbackfat) aerobically packed, then after five monthsof storage TBARS values showed a re m a r k a b l edecrease. They attributed this decrease to the reac-tion of malonaldehyde with proteins and sugars.

The effect of grilling on TBARS values was diffe-rent, since TBARS values of both beef tre a t m e n t ssignificantly (P≤0.05) decreased, while those of bothchicken treatments significantly (P≤0.05) increased,w h e reas those of the mixed treatment were notclear. For example, the TBARS of cooked beef andchicken samples that were stored for one monthre p resent about 40% and 234%, re s p e c t i v e l y, oftheir TBARS value before cooking. These re s u l t sagreed with Shin, et al. [19] who found that TBARSvalues of the cooked prerigor and postrigor beefw e re lower than the raw one, while the TBARSvalues of the cooked pork were relatively higherthan the raw one. They explained that by the factthat pork contains more unsaturated fatty acidsthan beef. Al-Ismail [20] reported that TBARSvalues of chicken shawerma were higher than thoseof beef samples. He attributed this finding to thefact that chicken fat contains higher levels of PUFA,which are prone to a higher level of oxidation.

Cholesterol and cholesterol oxidesCholesterol contents of all treatments are shown

in Table I. Mean values of the raw treatments indica-ted that there were significant (P≤0.05) diff e re n c e samong all treatments. Cholesterol content of theraw chicken sample (461.35 mg/100g fat) wassignificantly (P≤0.05) higher than that of the beefsample (332.15 mg/100g fat). The mixed samplehad a cholesterol content between those of chickenand beef samples (390.93 mg/100g fat). The repla-

cement of the added beef and chicken fat with oliveoil significantly (P≤0.05) reduced the level of chole-s t e rol. The cholesterol level of beef and chickensamples with olive oil was reduced by about 53%and 58% re s p e c t i v e l y. This result was confirmedwith Kayaard and Gök [13] who found that the cho-l e s t e rol content decreased in”soudjouk” pre p a re dwith 40% and 60% olive oil, depending on theamount of the incorporated olive oil in the formula-tion. Marquez, et al. [21] found that using peanut oilto replace 60% of the beef fat in frankfurters contai-ning 29% fat, reduced the cholesterol content bymore than 35%. By using olive, cottonseed and soyoils, Paneras et al. [22] obtained frankfurters (10%fat) with up to 59% less cholesterol compared tonormal frankfurters containing 30% animal fat.Muguerza et al. [23] replaced pork fat in sausagewith pre-emulsified olive oil, and found that pro-ducts with 20, 25 and 30% olive oil re p l a c e m e n thad significantly lower cholesterol levels.

Storage time and grilling did not affect the chole-sterol contents of all the treatments, when calcula-ted on the fat basis (mg cholesterol/100g fat). Therew e re no significant diff e rences in cholesterol con-tents between the raw and the grilled samples, orbetween the fresh and stored samples thro u g h o u tthe entire storage period. These results were confir-med by the level of 7-ketocholesterol, which is usedin this study as an indicator to determine the degreeof cholesterol oxidation, since no detectableamount of 7-ketocholesterol in all raw and grilledsamples was observed. This indicates that storageand grilling did not affect the stability of cholesterolagainst oxidation, which could be explained by thefact that grilling conditions were not severe, as themaximum temperature of grilling was about 75°Cand the time of grilling did not exceed 20 min.F u r t h e r m o re, there was a low oxygen level as theupper part of the grill was closed and directly cameinto contact with the burgers, moreover, it has been

Table II - Cholesterol content (mg/100g burger) for the raw and grilled burger samples during storage

Characteristic *Treatment


Raw c50.12aa70.82a

b59ae23.78a

d29a

Grilled c38.76ba56b

b45.42be17.77b

d23b

• Each value is the mean of three replicates.

* Means in the same row with the different subscripts denote significant differences among treatments of burger (p< 0.05) according to LSD.

** Means in the same column with different superscripts denote significant differences among raw and grilled burger samples (p< 0.05) according to Duncan test.


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reported that cholesterol showed high oxidationstability at temperatures below 100°C [24].

These results are consistent with the observationsof other authors, who did not find any increase inthe 7-ketocholesterol content of meat, raw orcooked. Baggio and Bragagnolo [25] found that noc h o l e s t e rol oxides were formed in any of the fourprocessed meat products analyzed during storage,either at room temperature or under re f r i g e r a t i o n .Beef purchased from a local market and cookedhamburger from a fast food restaurant did not con-tained 7-ketocholesterol [26]. However, cholestero lcontent calculated on the burgers basis (mg chole-s t e rol/100g burger) showed a lower cholestero lcontent in the grilled samples compared to the rawones (Table II). The reduction was about 23, 21, 23,25 and 21% for beef, chicken, mixed, beef witholive oil and chicken with olive oil samples, respecti-vely. This reduction might be due to the loss of fatduring cooking. These results agree withRodriguez-Estrada, et al. [7] who found that theamount of cholesterol in the raw hamburger sam-ples calculated on the hamburger basis, was signifi-cantly higher than that found in the cooked ones.

Saturated, mono and poly unsaturated fatty acids contents The effect of formulation, grilling and storage

period on SFA, MUFA and PUFA contents of the

burgers is illustrated in Table III. The data indicatedthat different patterns were observed depending onthe type of burger formulation, grilling and storageperiod. As expected, fatty acid composition of bur-gers reflected the fatty acid composition of the tis-sues and the fat used for their manufacturing. Asshown in our results and due to the fact thatchicken contains less saturated and more unsatura-ted fatty acids than beef, the manufacture of burgerformula by mixing of chicken with beef was adop-ted in this study as a strategy for changing the fattyacid profile of beef and chicken burgers. The addi-tion of chicken meat and fat to beef produced bur-gers with a higher nutritional value than beef bur-gers, due to the increase in its MUFA and PUFAwhich include essential fatty acids, and the decrea-se of SFA contents. The increase of MUFA andP U FA in mixed treatments compared to the beeftreatment was about 11% and 473%, respectively,and the decrease in SFA was about 24%. On theother hand, the addition of beef meat and fat tochicken burger enhanced its oxidative stability byincreasing SFA by 32% and decreasing PUFA con-tent by 34%, approximately.

P U FA are easily prone to oxidation generatingshort chain compounds that deteriorate the sensoryp roperties of the meat products [27].These re s u l t sconfirmed those obtained by Estévez et al. [28]

Table III - Effect of formulation, storage and grilling on the saturated (SFA), monounsaturated MUFA) and di- and polyunsaturated fatty acids(PUFA) (g/100g Fat) of the burger samples

Characteristic Time

of storage *Treatment**

(month)


Raw Grilled Raw Grilled Raw Grilled Raw Grilled Raw Grilled

SFA 0 a58.54aa53.70b

a33.42aa33.11b

a44.20aa40.11b

a27.20aa24.61b

a23.40aa19.48b

1 a58.28aa53.75b

a33.50aa33.25b

a44.57aa40.24b

a26.93aa24.90b

a23.61aa19.50b

3 a58.89aa53.63b

a33.76aa33.57b

a44.61aa40.49b

a27.13aa24.92b

a23.72aa19.73b

MUFA 0 a38.73ba41.13a

a47.56aa47.50a

a42.95ba46.69a

a59.65ba64.43a

a62.60ba68.71a

1 b38.11bb39.99a

b45.87ab45.76a

b41.70bb46.02a

b56.90bb63.46a

b60.19bb67.72a

3 c35.35bc36.50a

c35.17bc40.60a

c36.58bc38.72a

c47.22bc54.50a

c50.59bc57.52a

PUFA 0 a2.16ba4.06a

a18.92aa18.69a

a12.39ba12.86a

a9.4ba9.82a

a13.26aa12.87a

1 b1.53bb2.47a

b17.82ba18.55a

b11.93ab11.42b

b7.29ab7.93a

b12.59ba12.77a

3 c1.15bb 2.69a

b17.64ba18.42a

c11.29ac10.79b

c5.94bb7.77a c10.34b

b12.02a

• Each value is the mean of three replicates.

* Values within the same column with different subscripts are significantly (p< 0.05) different according to LSD.

** Values within the same row with different superscripts denote significance differences (p< 0.05) among raw and grilled sample according to Duncan test.


196

who manufactured frankfurters by replacing 10% fatfrom white pigs with fat from Iberian pigs which hadsignificantly higher amounts of MUFA, and loweramounts of SFA and PUFA compared to those ofwhite pigs. The Authors found that this meat mixingsignificantly reduced SFA and PUFA and increasedthe oleic acid and MUFA compared to those ofwhite pigs. St. John et al. [29] increased theM U FA / S FA ratio in low-fat frankfurters using thelean meat and fat from pigs fed elevated levels ofcanola oil that contain 64% oleic acid.

Another strategy for changing the fatty acid profileof meat products rather than meat mixing is thereplacement of animal fats by vegetable oils. Oliveoil is a vegetable oil whose MUFA content is veryhigh, since the MUFA, PUFA and SFA are about72%, 10% and 13%, respectively. In our study theaddition of olive oil in place of beef and chicken fatchanged the fatty acids composition of the beefand chicken burgers.

The decrease in SFA of beef sample was about54%, whereas the increase in MUFA and PUFA con-tents was about 54% and 339%, re s p e c t i v e l y, oftheir original contents in beef fat. On the other hand,the increase in MUFA was about 32%, whereas thed e c rease in SFA and PUFA contents was about30% of their original contents in chicken fat. Thed e c rease in SFA contents in these burger sampleswas due to the decrease in myristic, palmitic andstearic acid contents, while the increase in MUFAwas due mainly to oleic acid, since the addition ofolive oil decreased the palmitoleic acid contents.

The increase in PUFA content of beef sample wasmainly due to the increase in linoleic and to a lesserextent to the increase in linolenic content. On theother hand, the decrease in PUFA content inchicken sample resulted from the decrease in thelinoleic content. Similar results were reported whenolive oil was added to the Turkish “soudjouk” [13],dry fermented sausage [18] and for the fermentedsausage [23]; myristic, palmitic, palmitoleic andstearic acid contents decreased, oleic and linoleicacid contents increased compared to the contro ltreatments.

The storage period had no effect on SFA, sincethe level remained constant during the entire periodof storage for all treatments, while there was a gra-dual and significant decrease of MUFA and PUFA inall treatments during and specially at the end of sto-rage. This might be due to the oxidation eff e c t ,

which results in decomposition and degradation ofunsaturated fatty acids. The data in Table III indica-ted that the decline in MUFA and PUFA contents atthe end of storage of chicken with olive oil (≈ 1 9and 22%, respectively) was less than in beef witholive oil (≈ 21 and 37%, respectively). The decline inMUFA of the chicken with olive oil sample was lessthan in the chicken sample without olive oil (≈ 26%),however, beef with olive oil showed a greater decli-ne in MUFA when compared to beef with tallow (≈9%). In the case of PUFA, the decrease in their con-tents in beef with olive oil was less than in beef withtallow (≈ 47%), while chicken samples showed areverse trend, since the decline in PUFA contents ofchicken was about 8% compared to 22% inchicken with olive oil.

As for the mixed treatment, the MUFA and PUFAcontents were preserved to a higher extent than insamples with olive oil. For MUFA, the effect of sto-rage was greater on oleic acid (C18:1) than on pal-mitoleic acid (C16:1) this could be due to the abun-dant amount of oleic acid compared to palmitoleic,whereas in PUFA the effect of storage was greateron l inolenic acid (C18:3) than on linoleic acid(C18:2), most likely due to the fact that linolenicacid is more unsaturated than linolenic, so it will bem o re susceptible to oxidation. This decrease inM U FA and PUFA contents could be attributed totheir oxidation during storage, since no antioxidantsw e re added. However, Ansorena and Astiasarán[18] reported a decrease in oleic acid and PUFAcontents of pork sausage (25% pork backfat)during 5 months of storage period due to oxidation,while no significant effect of storage was observedon these acids in sausages with olive oil and sausa-ges with olive oil treated with antioxidants.

Conchillo et al. [30] studied the effect of storageon the fatty acid profile of chicken breast under diff e-rent conditions (refrigeration at 4°C for 6 days, orf reezing at -18°C for 3 months, in aerobic or vacuumconditions); they found that MUFA decreased duringrefrigeration storage and were not affected duringf rozen storage. Baggio and Bragagnolo [25] re p o r-ted that the fatty acid composition of diff e rent meatp roducts (jerked beef, Italian-type salami, chickenmortadella and Chester mortadella) did not changeunder diff e rent conditions (refrigeration at 4°C forchicken and Chester, and room temperature at 25°Cfor jerked beef and salami). This could be due to theadded antioxidant (sodium erythorbate, spices and


197

natural condiments) in the formulations. N u e rnberg et al. [31] found that storage of pork

chops for 144 hours under refrigeration at 4°C cau-sed a significant decrease of the PUFA proportion,and an increase in SFA and MUFA. Grilling signifi-cantly decreased SFA, and increased MUFA con-tents of all treatments, except for MUFA contents ofchicken sample where it remained constant. PUFAcontents, in general, increased in most samples,but in some cases there was no clear trend. Thedecline in SFA was mainly due to a decrease in pal-mitic and stearic acid contents, while the incrementin MUFA was mainly due to oleic acid. Ono et al.[32] in ground beef, and Scheeder et al. [33] inbeef patties found a significant decrease of totalS FA and an increase of total PUFA with cooking,while MUFA were not affected. These authors sta-ted that unsaturated fatty acids were less affectedby cooking because they belonged to the membra-ne structure to a higher extent than saturated fattyacids, leading to a lower decrease of PUFA by diffu-sion during cooking [32,33]. Rodriguez-Estrada etal. [7] detected an increase of PUFA with cookingthe hamburgers, while they did not observe diff e-rences among the percentage of SFA and MUFA ofraw and cooked hamburgers.

Conchillo et al. [30] studied the effect of cookingon the fatty acid profile of chicken breast; theyfound that frying in sunflower oil increased signifi-cantly the MUFA and PUFA/SFA ratios in compari-son with the raw samples as a consequence ofabsorption of sunflower oil. However, roasting didnot significantly affect these ratios. They re p o r t e dthat modif icat ions of fatty acids during meatcooking could be related to three mechanisms: oxi-dation, loss of fatty acids by diffusion (in roasting) orfatty acid exchange between the meat and oil (infrying) [30]. Nuernberg et al. [31] found that grillingof pork chops slightly affected the fatty acid com-position, but there was only a significant increase inPUFA proportion.

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Received October 27th, 2008,accepted December 5th 2008


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THE EFFECT OF TEMPERATURE AND TIME ON THE BLEACHING OF THE OIL FROM BLIGHIAUNIJUGATA BAK WAS STUDIED USING SURFACE ACTIVE CLAY. THE STUDY WAS CARRIED OUTAT A TEMPERATURE RANGE OF 60-180°C OVER A PERIOD OF 0-90 MIN. THE ADSORPTIONOF PEROXIDES WAS ADEQUATELY MODELED BY ARRHENIUS TYPE EQUATION AND DESCRI-B E D BY T H E F I R ST-O R D E R K I N E T I C. TH E AC T I VAT I O N E N E RGY FO R B L E ACH I NG AT 12 0 ° CAND 45 MIN WAS 136.604 CAL/MOLE FOR THE ARIL (AL) AND 47.245 CAL/MOLE FORTHE KERNEL (KL). THE BLEACHING RESULTED IN AN IMPROVEMENT IN THE PHYSICO-CHE-MICAL PROPERTIES OF THE OIL AS WELL AS REMOVAL OF SOME TRACES OF TOXIC METALS.KEY WORDS: ADSORPTION; CHARACTERIZATION; KINETICS; PEROXIDATION; PHYSICO-CHEMI-CAL PROPERTIES, BLIGHIA UNIJUGATA

EFFETTO DELLA DECOLORAZIONE SULLA CARATTERIZZAZIONE, MINERALI E VITAMINELIPOSOLUBILI DELL’OLIO DEI SEMI DI BLIGHIA UNIJUGATAL’E F F E T TO D E L LA T E M P E R AT U R A E D E L LA D U R ATA S U L LA D E CO LO R A Z I O N E D E L L’O L I O D E ISEMI DI BLIGHIA UNIJUGATA È STATO STUDIATO USANDO TERRE ATTIVE. LO STUDIO È STATOEFFETTUATO AD UN RANGE DI TEMPERATURA DA 60 A 180°C E PER UNA DURATA DI 0-90M I N U T I. L’A D S O R B I M E N TO D E I P E RO S S I D I H A S E G U I TO I L M O D E L LO D E L L’E Q UA Z I O N E D IARRHENIUS ED È STATO DESCRITTO CON LA CINETICA DI PRIMO ORDINE. L’ENERGIA DI ATTI-VA Z I O N E P E R LA S B I A NCA A 120°C E 45 M I N U T I E R A 13 6 , 6 04 CA L/M O L P E R AL E47,245 CAL/MOL PER KL. IL PROCESSO DI DECOLORAZIONE HA DATO LUOGO AL MIGLIO-RAMENTO DELLE CARATTERISTICHE CHIMICO-FISICHE DELL’OLIO E ALLA RIMOZIONE DI TRAC-CE DI METALLI TOSSICI DALL’OLIO STESSO.PA R O L E C H I A V E: A D S O R B I M E N TO, CA R AT T E R I Z Z A Z I O N E, C I N E T I CH E, P E RO S S I DA Z I O N E,CARATTERISTICHE CHIMICO-FISICHE, BLIGHIA UNIJUGATA

Kinetics of the effect of bleaching onthe characterization, mineral

nutrients and fat soluble vitamins ofBlighia unijugata bak seed oil

A. ADEWUYI*, R.A. ODERINDE, I. A. AJAYI

DE PA R T M E N T O F CH E M I ST RY, IN D U ST R I A L

CH E M I ST RY U N I T, UN I V E R S I TY O F IBA DA N,IBADAN, OYO STATE, NIGERIA

*CORRESPONDING AUTHOR: A. ADEWUYI

E-MAIL: [email protected]


200

INTRODUCTION

Seed oils are important food products and a lot ofattention has been focused on the analysis of theirphysico-chemical composition and application in thefood industry. At present there is a great interest ini m p roving the quality of the underutilized seed oils.

Crude oils contain various substances that maygive an undesirable flavour, colour or influence qua-lity. The nonglyceride components contribute practi-cally all the colour and flavour, other substancessuch as free fatty acids, waxes, colour particles,mucilaginous materials, phospholipids, caro t e n o i d sand gossypol (a yellow pigment found only in cot-tonseed oil) contribute to the undesirable propertiesin oils used for edible and to some extent for indu-strial purposes. These extraneous substances mayimpart objectionable properties to the oil [1] whichhowever could be improved refining, resulting in amore nutrious product with a better shelf life [2].

Blighia unijugata bak seed oil is one of theseunderutilized seed oils whose quality needs impro-ving. Blighia unijugata is a small tree though someti-mes reaching 35 m height, it has attractive red orpinkish yellow fruit and it is planted so as to giveuseful shade [3].

In a previous paper the proximate composition ofthe seed and physico-chemical characterization ofthe bak was reported [4]. In this ongoing study in tonew oils, we have considered the kinetic study ofbleaching to improve the quality of this seed oil.


Sample preparationSeeds of Blighia unijugata were collected from the

University of Ibadan, Oyo State, Nigeria. They wereidentified at the Herbarium Unit, Botany Departmentof the University. The seeds were manually separa-ted from the aril (AL) and cracked in order to remo-ve the kernel (KL). AL and KL were ground separa-tely in a laboratory mill and stored in a cellophanebag at 4°C prior to analysis.

Physico-chemical analysisOil was extracted from KL and AL using a Soxhlet

extractor with petroleum ether (40-60°C) for 10 h[5]. The extracted oils were immediately analyzedfor iodine value, peroxide value, saponificationvalue, acid value and unsaponifiable matter by

methods described by the Association of Off i c i a lAnalytical Chemists [6]. Estimation of the percenta-ge free fatty acids as oleic acid followed the methoddescribed by Cock and Rede [7].

The refractive indices of the oils (35°C) weredetermined with an Abbe refractometer [8] and thespecific gravity measurements were also carried outat room temperature (35°C) using specific gravitybottles. Visual inspection was used to note thestate and colour of the oils at room temperature [9].

Lipid classesLipid classes were separated on 0.75 mm plates

(20 x 20 cm) coated on a silica gel (Merck). Platesw e re developed vertically in an 80: 20: 1 volume mix-t u re of petroleum ether (95%): diethylether (92%): ace-tic acid (99%) obtained from Sigma-Aldrich ChemicalCo., Steinheim, Germany. They were developeda c c o rding to the method of Oboh and Oderinde [10].

Isolation of unsaponifiables10 g of the oil were dissolved in 200 ml of 2M-

ethanol potassium hydroxide and refluxed for 1 h.The reaction mixture was later diluted to 400 mlwith distilled water and transferred into a 1L separa-ting funnel. The unsaponifiables were then extrac-ted three times with 100 ml diethylether. The etherextract was first washed with 100 ml aqueous solu-tion of 0.5 M potassium hydroxide in order to remo-ve any residual fatty acids. This was further washedand cleaned 5 times with 100 ml distilled water anddried over anhydrous sodium sulphate. The solutionwas filtered and dried [11].

Separation of unsaponifiablesC h l o roform solution (50%) of the unsaponifiable

material (30 mg/plate) was then applied uniformlyalong the line from the edge of the 20 x 20 cm platecoated with a 0.55 mm layer of silica gel and deve-loped three times with hexane/ethylacetate (6:1,v/v) as mobile phase. The developed plates weredried and visualized at 254 nm with ultraviolet radia-tion. Three zones corresponding to n-alkanes, triter-pene alcohols and sterols were marked care f u l l yscrapped and extracted with petroleum ether [12].

Determination of vitaminsThe procedures of Bogumila and Marek [13] were

adapted for separation and quantification of the fatsoluble vitamins (A, D and E) in the oil using HPLC


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(1100 series, Agilent) equipped with a thermostatedcolumn compartment ( G1316A Germany), variablewavelength detector (G1314A),quaternary pump(G1311A,Germany) and a degasser (G1379A).Theautomated system (HPLC) is dr iven by aChemstation software. The mobile phase was waterand acetonitrile (5:95 v/v). The flow rate was 1.5ml/min. The vitamins were monitored using a UVdetector at 210 nm and 35°C.

Mineral determinationMetals - lead, cadmium, copper, zinc, iro n ,

magnesium, calcium, sodium, potassium and man-ganese were determined. This was achieved bydigesting the samples using 5 ml (2:1) of 69.40%(w/w) nitric acid and 90.00% (w/w) perchloric acid[14]. These metals were analyzed by atomicabsorption spectrophotometry (Perkin-Elmer,GMBH, Ueberlingen, Germany).

Bleaching1:1 mixture of activated carbon and surface acti-

ve clay was used for the bleaching. 35g of the oilwas warmed up and the bleaching mixture wasgradually added. The flask was corked and thet e m p e r a t u re maintained while varying time. The oilwas filtered hot through a sintered glass funnelusing a suction pump.

Heat treatmentHeat treatment of Blighia unijugata bak oil was

carried out by heating the oil up to 180°C for aperiod of 0-90 min. The peroxide value, re f r a c t i v eindex, acid value and the iodine value were determi-ned respectively at 60°C, 120°C and 180°C usingthe Association of Official Analytical Chemistsmethod. The oil was recharacterized after 120°Cand 45 min of treatment.

Kinetic calculationsA general reaction rate expression for the blea-

ching kinetic can be written as follows [15, 16]: -d[C]\dt = K[C]m

C is the quantitative value of the concentration ofthe molecule under consideration,

K is the reaction rate constant and ‘m’ is theorder of the reaction.

For first order reaction where m = 1 the equationcan be written as: In ([Ct] \ [Co]) = -Kt

[ Co] is the concentration of the reactants underconsideration at time zero and

[Ct] is the concentration of the reactants at time‘t’. Arrhenius relationship of the reaction rate to tem-

perature is generally given as:K =Ao exp (-Ea\ RT)where Ea is the activation energy of the reaction, R is the gas constant,

Table I - Effect of bleaching on PV, IV, RI and AV of AL

Temperature (°C) Time (min) PV (mg active O2/kg) IV (mg iodine/g) RI (25°C) AV (mg KOH/g)

60 30 8.2 ± 0.4a 8.6 ± 1.6a 1.5590a 14.2 ± 0.1a

45 8.0 ± 1.2a 8.2 ± 1.0b 1.5600a 14.0 ± 0.1b

60 7.8 ± 0.8b 8.8 ± 0.4c 1.5650b 13.5 ± 0.5c

75 7.6 ± 0.3c 9.2 ± 0.5d 1.5680c 12.8 ± 0.1d

90 7.4 ± 1.0d 9.3 ± 1.2d 1.5680c 11.2 ± 0.3e

120 30 4.4 ± 0.5a 9.1± 1.2a 1.5670a 9.8 ± 0.3a

45 4.2 ± 0.4b 10.0 ± 5.4b 1.5840b 6.7 ± 0.2b

60 4.0 ± 0.1b 10.2 ± 3.0c 1.5890c 6.8 ± 0.5b

75 3.8 ± 1.2c 10.7 ± 2.1d 1.5900c 7.0 ± 0.1c

90 3.6 ± 0.8c 9.9 ± 4.4e 1.5800d 7.2 ± 0.1d

180 30 4.0± 0.2a 10.1 ± 1.4a 1.5800a 7.6 ± 0.2a

45 3.8 ± 0.5b 10.0 ± 2.0a 1.5890b 8.0 ± 0.1b

60 3.9 ± 0.1c 9.8 ± 1.0b 1.5720c 8.2 ± 0.2c

75 3.6 ± 1.0d 10.5 ± 0.8c 1.5970d 8.5 ± 0.1d

90 3.4 ± 0.5e 10.5 ± 1.1c 1.5980d 8.5 ± 0.1d

Values are mean ± standard deviation of triplicate determinations. Data in a column with different superscript letters are statistically different (P≤ 0.05).

PV: Peroxide Value, IV: Iodine Value, RI: Refractive Index, AV: Acid value


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T is absolute temperature and Ao is a pre-exponential constant.Each experiment was performed in triplicates and

the average values were taken for the parametersdetermined. Kinetic data were analysed by regres-sion analysis using MS Excel.

Statistical analysisAll data were analyzed by one-way analysis of

variance (ANOVA) and least significant diff e re n c e sbetween treatment means were determined byDuncan’s multiple range test [17] with significant dif-ferences measured at P < 0.05.


Ef f e ct of bleaching on AL and KL after te m p e rat u re- t i m etreatment

The rate of adsorption of peroxides in the oils ofAL and KL increases as temperature and timeincrease as shown in Table I and Table II respecti-vely. At 180°C, the value of peroxide initially decrea-sed at 30 min and 45 min but later increased at 60min. This may be explained by the result of the sti-mulation of peroxidation at a high temperature. Thisshows that the prolonged heating of these oils atthis high temperature (180°C) makes it undergothermal degradation resulting in oxidative rancidity,

formation of hydroperoxides and other products ofdegradation that can liberate volatile compounds.These latter compounds reduced as time increasedwhich may be due to the ability of the clay and acti-vated carbon to adsorb the peroxides as they wereformed at this temperature and time.

The formation of this peroxide was also re f l e c t e din the acid value obtained as this also increased atthis temperature and time. This increase may be dueto the hydrolysis of the glycerides present in the oils.For the other conditions of temperature and timeadopted, the acid value reduced over time exceptfor the treatment at 180°C which shows an incre a s eat 60, 75 and 90 min which may be due to theh y d rolysis of the glyceride bonds at this temperatureresulting in the liberation of the fatty acids. The iodi-ne values obtained after bleaching also incre a s e dwith time. This suggests the gain of unsaturation inthe fatty acids of the triacylglycerols, or removal ofextraneous materials which would have shielded theunsaturation of these oils. This gain of unsaturationwas also reflected by the increase in the value of therefractive index obtained after bleaching.

Ef f e ct of bleaching on phy s i co-chemical pro p e rty of AL and KLThe result of the effect of bleaching on the physi-

co-chemical properties of AL is presented in TableIII while that of KL is presented in Table IV.

Table II - Effect of bleaching on PV, IV, RI and AV of KL

Temperature (°C) Time (min) PV (mg active O2/kg) IV (mg iodine/g) RI (25°C) AV (mg KOH/g)

60 30 4.6 ± 0.5a 7.1 ± 1.3a 1.5520a 16.1 ± 0.1a

45 4.2 ± 0.2b 7.2 ± 1.5b 1.5550b 14.2 ± 0.5b

60 4.0 ± 0.6c 7.2 ± 0.6b 1.5800c 12.0 ± 0.5c

75 3.8 ± 0.5d 7.6 ± 1.5c 1.5850d 11.6 ± 0.2d

90 3.6 ± 0.2d 8.0 ± 2.2d 1.5990e 10.2 ± 0.5e

120 30 3.1 ± 0.6a 8.0 ± 2.0a 1.6000a 9.8 ± 0.1a

45 2.9 ± 0.2b 8.5 ± 4.2b 1.6510b 3.1 ± 0.4b

60 2.7 ± 0.1c 8.5 ± 3.5b 1.6520c 3.2 ± 0.2b

75 2.5 ± 0.2c 8.7 ± 2.8c 1.6540d 3.3 ± 0.5c

90 2.3 ± 0.1d 8.7 ± 3.2c 1.6540d 3.2 ± 0.3c

180 30 2.8 ± 0.2a 7.8 ± 1.7a 1.6000a 9.6 ± 0.5a

45 2.6 ± 0.5b 8.5 ± 3.7b 1.6500b 8.0 ± 0.2b

60 2.7 ± 0.3c 8.8 ± 2.1c 1.6550c 8.2 ± 0.2c

75 2.4 ± 0.1d 8.5 ± 2.8d 1.6500b 8.5 ± 0.1d

90 2.2 ± 0.2e 8.8 ± 1.5c 1.6550c 8.5 ± 0.3d

Values are mean ± standard deviation of triplicate determinations. Data in a column with different superscript letters are statistically different (P≤ 0.05).

PV- Peroxide Value, IV- Iodine Value, RI- Refractive Index, AV – Acid value


203T h e re is a significant diff e rence between the crude andthe bleached oils of AL and KL. All the parametersdetermined decreased after bleaching except for theiodine value and the refractive index. There is a majord e c rease in the free fatty acid value of the oils. Itd e c reased from 9.10 ± 0.30% to 2.30 ± 0.10% in KLwhile in AL it decreased from 7.00 ± 0.10% to 3.30 ±0.10%. This reduction is an indication of a lesser stimu-lation of oxidative deterioration of these oils by enzyma-tic and chemical oxidation to form off-flavour compo-nents. This also makes it fall within the re q u i red limit forcooking oils [18], giving it a useful potential in the foodi n d u s t r y. The peroxide value of AL reduced from 10.50± 0.61 Mg O2/g oil to 4.22 ± 0.40 Mg O2/g oil. Thet reatment reduced this value by adsorbing some of thep e roxides making them lower than values stipulated forrancid oils [8]. These extraneous compounds negati-vely influence the oils by reducing the value. Theadsorption was also noticed in the case of the unsapo-nifiable matters which are mainly sterols, the value of

Table IV - Effect of bleaching on the physico-chemical properties ofKL at 120°C and 45 min

Parameter Crude Bleached

Free fatty acids (%) 9.1 ± 0.3a 2.3 ± 0.1b

Acid value (mg KOH/g) 18.0 ± 0.1a 3.1 ± 0.4b

Saponification value (mg KOH/g) 217.0 ± 0.1a 212.0 ± 1.1b

Iodine value (mg iodine/g) 7.0 ± 1.0a 8.5 ± 4.2b

Peroxide value (mg active O2/kg) 6.8 ± 0.2a 2.9 ± 0.2b

Specific gravity 0.8480 ± 0.1a 0.7983 ± 0.1b

Refractive index 1.5530a 1.6510b

Unsaponifiable matter (%) 0.7 ± 0.0a 1.0 ± 0.6b

Values are mean ± standard deviation of triplicate dete r m i n at i o n s. Data in a row

with differe nt letters are statistically differe nt acco rding to DMRT (P ≤ 0.05).

Table III - Effect of bleaching on the physico-chemical properties ofAL at 120°C and 45 min

Parameter Crude BleachedFree fatty acids (%) 7.0 ± 0.1a 3.3 ± 0.1b

Acid value (mg KOH/g) 15.0 ± 0.3a 6.7 ± 0.2b

Saponification value (mg KOH/g) 300.0 ± 0.4a 291.0 ± 5.1b

Iodine value (mg iodine/g) 8.7 ± 1.5a 10.0 ± 5.4b

Peroxide value (mg active O2/kg) 10.5 ± 0.6a 4.2 ± 0.4b

Specific gravity 0.8500 ± 0.0a 0.8016 ± 0.0b

Refractive index 1.5580a 1.5890b

Unsaponifiable matter (%) 3.0 ± 0.0a 2.8 ± 0.1b

Values are mean ± standard deviation of triplicate dete r m i n at i o n s. Data in a row

with differe nt letters are statistically differe nt acco rding to DMRT (P ≤ 0.05).

the unsaponifiable being reduced in both oils. On the other hand there was an increase in the

values of the iodine value obtained for KL and AL.This increase might be due to the removal of satu-rated substances which may have masked theunsaturated ones. This increase is also reflected inthe refractive index value which also incre a s e dsince double bonds (unsaturation) increase therefractive index of organic compounds by reducingthe angle of refraction in relation to the angle of inci-dence. The higher the unsaturation the greater theeffect of reducing the refraction angle.

Ef f e ct of bleaching on unsaponifiable mat te r, lipid classesand fat soluble vitamins of AL and KL

Table V and Table VI show the effect of bleachingon the unsaponifiable composition and lipid classesof AL and KL respectively.

The removal of impurities and other substances fro mthe oils might account for the increase noticed in thet r i a c y l g l y c e rol content of the oils [2]. Bleaching selecti-

Table VI - Effect of bleaching on the lipid classes (%) of AL and KL at120°C and 45 min

Parameter AL AL KL KL

(Crude) (Bleached) (Crude) (Bleached)Polar lipids 1.2 ± 0.9a 0.5 ± 1.2b 0.6 ± 0.7c 0.2 ± 0.5dSterols 3.6 ± 0.5a 1.2 ± 1.0b 2.0 ± 0.3c 0.8 ± 0.1dDiacylglycerols 0.9 ± 1.2a 1.8 ± 0.1b 1.8 ± 0.9c 2.1 ± 0.5b

M o n o a cy l g l yce ro l s 2.0 ± 0.4a 3.1 ± 1.0b 0.8 ± 0.5c 1.2 ± 0.7d

Triacylglycerols 88.3 ± 0.8a 91.7 ± 0.8b 90.8 ± 0.2c 94.4 ± 0.5d

Hydrocarbons 0.8 ± 0.6a 0.3 ± 0.5b 1.9 ± 0.4c 0.8 ± 0.1d

Free fatty acids 3.2 ± 0.0a 1.4 ± 0.2b 2.1 ± 0.7c 0.5 ± 0.2d

Values are mean ± standard deviation of triplicate dete r m i n at i o n s. Data in a rowwith differe nt letters are statistically differe nt acco rding to DMRT (P ≤ 0.05).

Table V - Effect of bleaching on the unsaponifiable composition (%)of AL and KL at 120°C and 45 min

ALMethod Triterpene Sterols n-Alkanes Unidentified

alcohols componentsCrude 20.8 ± 0.6a 40.1 ± 1.5a 13.4 ± 0.2a 25.7 ± 0.4a

Bleached 22.0 ± 0.1b 34.0 ± 0.7b 11.2 ± 0.5b 32.8 ± 0.4b

KLMethod Triterpene Sterols n-Alkanes Unidentified

alcohols componentsCrude 18.1 ± 1.1a 38.5 ± 0.8a 12.6 ± 0.1a 30.8 ± 1.0a

Bleached 21.6 ± 0.5b 28.5 ± 0.1b 10.0 ± 0.7b 39.9 ± 1.0b

Values are mean ± standard deviation of triplicate dete r m i n at i o n s. Data in acolumn with differe nt letters are statistically differe nt acco rding to DMRT (P ≤ 0.05).


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vely removes pigments, oxidative products, tracemetals and sulphur from the oil [19]. The monoacylgly-c e rol and the diacylglycerol content of these oils alsoi n c reased. The polar lipid content of AL decreased fro m1.20 ± 0.90% to 0.50 ± 1.20 while that of KL decre a-sed from 0.60 ± 0.70% to 0.20 ± 0.50%.

This reduction in the polar lipids and increase int r i a c y l g l y c e rol content of these oils is of great impor-tance, indicating the possibility of their application inthe food industry. There is reduction in the hydro c a r-bon content, triterpene alcohol and sterol in the stu-died oils, this might be as a result of the surface

Figure 1 - HPLC of the fat soluble vitamins of AL (Crude)Vitamin A (Retention time) = 3.543 min, Vitamin D (Retention time) = 7.468 min, Vitamin E (Retention time) = 8.315 min

Figure 2 - HPLC of the fat soluble vitamins of AL (Bleached)Vitamin A (Retention time) = 3.586 min, Vitamin D (Retention time) = 7.702 min, Vitamin E (Retention time) = 8.578 min


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adsorption of the clay. A reduction in the polar lipid,s t e rol and increase in glycerol (tri, di and mono) ofthese oils after treatment shows their possible role inp reventing athero s c l e rosis which is the underlyingcause of heart attack, stroke and peripheral vasculardiseases [20]. Work is under way on the identificationof the fractions that were not identified. The effect ofbleaching on the fat soluble vitamins of AL and KL isshown in Figures 1-4. The summary of the change in

the concentrations of these vitamins is also pre s e n t e din Table VII. The concentration of these fat soluble vita-mins is low in the oils. Vitamin D which promotes pha-gocytosis, anti-tumor activity, and immunomodulatoryfunctions was the least in AL while vitamin E was theleast in concentration in KL. The concentration ofthese vitamins increased after treatment in AL but onlyvitamin A was found to increase in KL after bleaching.These oils are not good sources of vitamin A, D and E.

Figure 3 - HPLC of the fat soluble vitamins of KL (Crude)Vitamin A (Retention time) = 3.567 min, Vitamin D (Retention time) = 7.538 min, Vitamin E (Retention time) = 8.378 min

Figure 4 - HPLC of the fat soluble vitamins of KL (Bleached)Vitamin A (Retention time) = 3.574 min, Vitamin D (Retention time) = 7.452 min, Vitamin E (Retention time) = 8.260 min


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Ef f e ct of bleaching on the nutritional and tra ce metal co m-position of AL and KL

Sodium was the most abundant metal in AL whilehad the most potassium in KL as shown in Table VIII.

The concentration of the macro metals decre a s e dsligthly in the two oils while the micro metals decre a-sed in a higher amount. There is significant diff e re n-ce in the concentration of the trace metals betweenthe crude and the bleached oils. The reduction inthe concentration of the trace metals was highshowing the affinity and adsorption capacity of thisclay for the metals. Cu was reduced from 0.70 ±0.01 ppm to 0.01 ± 0.30 ppm in AL and in KL it wasreduced from 1.00 ± 0.01 ppm to 0.20 ± 0.01. Pbwas found to be 1.57 ± 0.04 ppm in AL which wasnot detected after treatment where as in KL it wasreduced from 2.90 ± 0.05 ppm to 0.95 ± 0.50 ppm.

This reduction makes these oils safer because of thelow concentration of this toxic trace metal. Fe plays animportant role in blood cell chemistry since it is animportant part of the hemoglobin. The level of Fe in AL

showed that it can serve as good source of Fe espe-cially in anemic patients. This concentration alsod e c reased slightly after treatment from 99.07 ± 0.09ppm to 95.20 ± 0.10 ppm in AL and in KL it was re d u-ced from 24.17 ± 0.01 ppm to 20.00 ± 0.06 ppm.Mn, which is an essential component of co-enzymesimportant in growth and photosynthesis, was alsofound in appreciable concentration and within the tole-rable limit [21]. Cd was found in a small amount inboth AL and KL. The bleaching process removed thismetal totally from the oils signifying that it can be usedto make the oils free of toxic trace metals.

Kinetic data for the bleaching of AL and KLA first order bleaching was followed in order to

obtain the kinetic data. ‘In ([C]t/ [ C ]o)’ was plottedversus‘t’, from which rate constant, ‘K’ was calcula-ted as the slop. The results of these plots are pre-sented in Figure 5 (for KL) and Figure 6 (for AL).

Figure 5 - First order plot of the adsorption of peroxide in KL

Figure 6 - First order plot of the adsorption of peroxide in AL

T1/2, which is the time taken for half peroxide tobe removed from the oil by 50% of its original valuewas calculated from the rate constant as ‘0.693/k’.The rate constant and T1/2 is shown in Table IX for

Table V I I - Ef f e ct of bleaching on fat soluble vitamins (ppm) of ALand KL at 120°C and 45 min

Vitamins AL (Crude) AL (Bleached) KL (Crude) KL (Bleached)

A 1.1703 1.2500 0.2485 0.2490

D 0.0176 0.2115 0.2251 0.2225

E 0.0275 0.0334 0.1570 0.1548

Table VIII - Nutritionally valuable and trace metals (ppm) of AL andKL at 120°C and 45 min

A L K L

M e t a l Cr u d e B l e a c h e d Cr u d e B l e a c h e d

N a 396.72 ± 0.02a 404.50 ± 0.07a 348.40 ± 0.04b 355.30 ± 0.80b

K 354.00 ± 0.21a 340.80 ± 1.00a 866.66 ± 0.58b 855.10 ± 1.20b

Ca 240.50 ± 0.05a 210.10 ± 0.10a 858.73 ± 0.03b 801.40 ± 0.50b

Mg 192.57 ± 0.11a 140.50 ± 0.50b 228.50 ± 0.08c 212.60 ± 0.50c

Fe 99.07 ± 0.09a 95.20 ± 0.10a 24.17 ± 0.01b 20.00 ± 0.06b

Cu 0.70 ± 0.01a 0.01 ± 0.30b 1.00 ± 0.01a 0.20 ± 0.01c

Z n 31.17 ± 0.01a 28.00 ± 0.50a 33.40 ± 0.01a 28.50 ± 0.60a

M n 103.33 ± 0.52a 92.30 ± 0.10b 55.00 ± 0.03c 44.10 ± 0.20d

P b 1.57 ± 0.04a N D 2.90 ± 0.05b 0.95 ± 0.50c

Cd 1.00 ± 0.01a N D 0.73 ± 0.01b N D

Average concentration + standard deviation of triplicate determinations (ppm)

(mg/kg). Data in a row with different letters are statistically different according

to DMRT (P ≤ 0.05). ND; Not detected


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AL and Table X for KL. The activation energy, Ea(cal M- 1) was calculated as a product of gas con-stant, R (1.987 cal M-1K-1) and the slop of the graphobtained by plotting ‘InK’ versus ‘1/T’. The linearnature of the plot obtained in Figure 7 gave the acti-vation energy of the adsorption of peroxide in AL as136.604 cal/mole while the linear nature of Figure 8gave the activation energy of the adsorption ofperoxide in KL to be 47.245 cal/mole.

In conclusion, the effect of temperature and time onthe bleaching of the oil from Blighia unijugata bak wasstudied using surface active clay. This bleaching pro-cess showed a first-order kinectic with the Arrheniusplot yielding a straight line with a slop equivalent toactivation energy of 136.604 cal/mole for AL and47.245 cal/mole for KL at 120°C and 45min. Thebleaching method applied led to an improvement inthe physico-chemical properties of the treated oil aswell as removal of toxic trace metals.

AcknowledgementThe authors would like to thank the Department

of Chemistry, University of Ibadan for allowing theuse of materials and equipments.

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Table X - Rate constant and half–life of adsorption of peroxide in KL

Temperature (°C) Regression equation T1/2(min)

for adsorption (R2)

60 Y = -0.0039x – 0.2900 (0.98) 177.70

120 Y =-0.00500x – 0.6254 (0.99) 138.60

180 Y = -0.0038x – 0.7571 (0.90) 182.37

Table IX - Rate constant and half –life of adsorption of peroxide in AL

Temperature (°C) Regression equation T1/2(min)

for adsorption (R2)

60 Y = -0.0017x – 0.1955 (0.99) 407.65

120 Y =-0.0034x – 0.7619 (0.99) 203.82

180 Y = -0.0025x – 0.8867 (0.90) 277.20

Figure 7 - Arrhenius plot for the adsorption of peroxide in AL

Figure 8 - Arrhenius plot for the adsorption of peroxide in KL


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[10] F. J Oboh, R. A. Oderinde, Analysis of the pulpand pulp oil of Tucum, A s t r o c a ryum vulgare.Food Chemistry, 30, 277 – 287 (1988).

[11] K. O. Adebowale, C. O. Adedire, Chemicalcomposition and insecticidal properties ofunderutilized Jatropha curcas seed oil. AfricanJournal of Biotechnology, 5 (10) pp. 901–906(2006).

[12] O. A. Kayode, O. O. Mike, A. A. Yemisi, F. A.Dawodu, Evaluation of the nutritive and func-tional properties of AKEE APPLE (Blighia sapi-

d a). Journal of Tropical Forestry Research 1 7(1) 160–169 (2001).

[13] K. Bugumila, G. Marek, Effect of menadione(vitamin K3) addition on lipid oxidation andt o c o p h e rols content in plant oils. Nahrung/Food 47 (1) 11-16 (2003).

[14] O. A. Almoaruf, O. B. Muibat, A. O. Isiaka, O.A. Nureni, Heavy trace metals and macro n u-trients status in herbal plants of Nigeria. FoodChemistry 85, 67-71 (2003).

[15] H. S. Ramaswami, F. R. Van De Voort, S.Ghazada, Analysis of TDT and Arrhenius

method for handling process and kinetic data.J o u rnal of Food Science, 5 4, 1322-1326(1989).

[16] M. A. Van Boekel, Statistical aspect of kineticmodeling for food science problems. Journalof Food Science, 477-485 (1996).

[17] D. B. Duncan, Multiple range and multiple Ttests. Biometrics II, pp. 1- 42 (1955).

[18] E. N. Onyeika, G. N. Acheru, Chemical com-position of selected Nigerian oil seeds andphysico-chemical properties of the oil extracts.Food Chemistry, 77, 431 – 437 (2002).

[19] Y. Ruili, L. Guowei, L. Anlin, Z. Jianliang, S.Yonghui, Effect of antioxidant capacity onblood lipid metabolism and lipoprotein lipaseactivity of rats fed with a high fat diet. Nutrition22, 1185 – 1191 (2006).

[20] M. E. Badeae, M. S, Kawther, A. A. K. Abou-Arab, Quality estimation of some contaminantsin commonly used medicinal plants in theEgyptian market. Food Chemistry, 6 7, 357-363 (1999).

[21] M.Y. Jung, S. I. T Yoon, D. B Min, Effects ofp rocessing steps on the content of minorcompounds and oxidation of soybean. Journalof the American Oil Chemists Society 66, 118– 120 (1989).

Received on December 2nd, 2008,accepted on February 5th, 2009

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XIII Giornate del Comitato Italiano

Derivati Tensioattivi

Bologna 11-12 giugno 2009

La 13° edizione delle Giornate di studio del ComitatoItaliano Derivati Tensioattivi si è tenuta a Bologna neig i o rni 11 e 12 giugno, presso Palazzo Gnudi, anticoedificio del 1600 situato nel cuore della città, re s t a u-rato e dotato di tutte le attre z z a t u re idonee all’orga-nizzazione di congressi e manifestazioni.B reve il convegno, 1 giorno e mezzo, ma denso dii n t e ressanti lavori sui problemi più attuali e che hannocatturato la costante attenzione dei partecipanti.Dopo l’apertura della manifestazione tenuta dal pre-sidente CID Ing. Agostino Zatta, la Dr.ssa MilenaNaldi, esperta di arte, ha tenuto un’intere s s a n t econferenza su Bologna, la sua storia, i suoi palazzi,il suo sviluppo nei secoli.La prima delle due key lectures in programma èstata tenuta dal Dr. Vittorio Maglia, Dire t t o re delC e n t ro Analisi Economiche ed intern a z i o n a l i z z a z i o-ne di Federchimica, su un argomento dei più attualiper il mondo intero: la situazione economica gene-rale con particolare focus sul comparto industriale edei consumiCon dati statistici il Dr. Maglia ha esaminato inmodo obiettivo i vari punti, il calo di produzione, lacontrazione dei consumi, chiedendosi: quandousciremo dalla crisi e come ne usciremo? E’ comin-ciata la ripresa? Per l’industria il peggio sembra allespalle. La caduta si è arrestata e si inizia a risalire. In Italia non si è avuta un’enorme crisi dei consumi.L’inflazione non è più un problema ed il calo dell’in-flazione alimenta la fiducia delle famiglie. Da unpunto di vista pessimistico si può comunque ipotiz-zare che molte conseguenze di questa situazione sip e rcepiranno in un prossimo futuro portandoci adoverne sopportare le conseguenze per anni.La seconda key lecture è stata tenuta dalla Dr.ssaMilena Presutto, del Dipartimento Tecnologie perl’Energia, Fonti Rinnovabili e Risparmio Energeticodell’Enea ed ha riguardato l’efficienza energetica neip rocessi di lavaggio con macchine ecologiche, abasso consumo di acqua ed energia e alta resa.Le varie relazioni succedutesi ed i poster espostihanno incanalato il dibattito tecnico sull’avvenutar i c e rca di nuove materie prime e nuove tecnologieper ottenere prodotti che stiano al passo con lenecessità di mercato, che soddisfino le aspettatives e m p re più pressanti dei consumatori con unosguardo comunque attento alle problematiche lega-

te all’impatto ambientale. Numerosi gli espositori italiani ed internazionali pre-senti che, in ambienti raccolti, limitrofi alla sala con-f e renze, hanno potuto seguire le varie relazioni edi n t r a t t e n e re gli ospiti interessati alle loro pro p o s t eanche durante le pause dei lavori. I partecipanti hanno avuto infine modo di apprezza-re anche la cordialità e la buona cucina tipiche emi-liane durante i lunch organizzati nel chiostro delPalazzo e durante la cena sociale tenutasi pre s s ouna tenuta agricola sulle colline bolognesi.I lavori del congresso sono raccolti in un CD ROMdistribuito a tutti i partecipanti. Chi fosse interessatopuò richiederlo a

Segreteria CID – Via Giuseppe Colombo, 79 – 20133 Milano

Tel. 02.2367216 – e-mail: [email protected]

www.ciditalia.it

1st MS Food Day

Parma, 2-3 dicembre 2009

Il primo convegno sulla spettrometria di massaapplicata al settore degli alimenti, organizzato dallaSocietà Barilla in collaborazione con la Divisione diS p e t t rometria di Massa e con i l GruppoInterdivisionale Chimica degli Alimenti della SocietàChimica Italiana, si terrà presso la Società Barilla,Via Mantova 166, Parma.Nell’industria alimentare ed agraria la ricerca è sem-pre più indirizzata alla sviluppo di metodi rapidi perl’analisi di materie prime e prodotti finiti. In questocontesto si deve constatare come i progressi dellemetodiche in spettrometria di massa, combinateanche con tecniche di separazione, abbiano contri-buito ad un notevole salto di qualità. Infatti, grazieanche a significativi investimenti in fatto di risorseumane e strumentazione avanzata, la spettrometriadi massa porta un contributo fondamentale allavalutazione dei prodotti alimentari da diversi punti divista che vanno dalla sicurezza, alla caratterizzazio-ne, alla tracciabilità ed agli aspetti nutrizionali comepure alla validazione di metodi di analisi alternativi.Scopo del convegno è di mettere in evidenza lepotenzialità applicative della spettrometria di massaper le grandi e medie industrie.Il programma, articolato in comunicazioni orali,poster e conferenze divulgative, sarà occasione diapprendimento e di aggiornamento sulle applicazio-ni di questa tecnica nel settore alimentare e perscopi innovativi.Il programma finale e l’elenco dei relatori saranno

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comunicati con una circolare nel mese di settembre2009 e inseriti nel sito web della conferenza.Gli abstract delle comunicazioni e dei poster dovran-no pervenire entro il 10 ottobre 2009; potrannocomunque essere accettati poster last minute pre v i aa p p rovazione da parte del Comitato scientifico. Nella sessione di chiusura della conferenza saràassegnato il 1st MS Food Day Best Poster Award allavoro più innovativo.Lingua ufficiale della manifestazione sarà l’italiano,saranno comunque accettati lavori in inglese essen-do previsto un servizio di traduzione simultanea.Il modulo per la presentazione di abstract è reperibi-le nel sito:h t t p : / / w w w. s o c . c h i m . i t / d i v i s i o n i / S d M / 1 M S - F o o d D a y.

Per ogni altra informazioni: 1st MS-Food Day Organising

Secretariat

Elena Bergamini, Dante Catellani, Barilla G.R. F.lli Spa –

Research Labs, Via Mantova 166, 43100 Parma

Tel. +39.0521.263231 oppure +39.0521.262946

Fax +39.0521.263452 – e-mail: e.bergamini@barilla it

oppure [email protected]

Attività Società Bulgara dei Chimici

Cosmetologi

Nei giorni 4 e 5 giugno 2009 la Società Bulgaradei Chimici Cosmetologi ha ricevuto la visita delp rof. Johann Wieckers e del dr. Luigi Rigano chehanno tenuto conferenze nei rispettivi campi diesperienza. Questa visita è stata resa possibile dalp rogramma che International Federation Societiesof Cosmetic Chemists ha istituito nel 2007 adA m s t e rdam: Ecaterina Merica Cosmetic EducationP ro g r a m m e .Questo programma consente a paesi i cui studiosinormalmente non hanno la possibilità di partecipa-re ai congressi IFSCC, di ricevere dei re l a t o r iesperti che tengano corsi di istruzione.Per quest’anno sono state scelte la Romania e laB u l g a r i a .L’evento si è tenuto a Plovdiv dove ha sede lamaggior parte delle industrie cosmetiche bulgaretra le quali la più antica la Alen Mak AG, le maggio-ri, Solvex CP Ltd e Rosa Impex Ltd ed altre menoimportanti ma che stanno sviluppandosi veloce-mente come BodiD Ltd e Biofresh Ltd.Le date del 4 e 5 giugno sono state scelte perc h écoincidevano con la conferenza Iasi in Romania,organizzata dalla Società Rumena dei ChimiciCosmetologi. Inoltre nello stesso periodo si teneva

il Festival bulgaro delle rose. Durante la prima giornata il prof. Wieckers hatenuto una serie di lezioni sulla pelle, sulle pro-prietà di penetrazione dei prodotti cosmetici, sulleformulazioni, micro-emulsioni, gels.Il secondo giorno il dr. Rigano ha trattato delle for-mulazioni di emulsioni cosmetiche, tenendo pre-senti la sicurezza dei prodotti, le valutazioni senso-riali e tutti i fattori per una equilibrata formulazionedei pro d o t t i .L’esperienza è stata giudicata molto positiva daparte dei tecnici bulgari che hanno potuto appre n-d e re da esperti del settore nozioni che sarannomolto utili per il loro lavoro futuro .

Per maggiori informazioni: General Secretary IFSCC,

G.T. House, 24/26 Rothesay Road, Luton, Beds. LU1 1QX,

England, er-mail: [email protected]

2009 Annual general meeting of

Society of Cosmetic Scientists

Il 61° meeting annuale della Società britannica deiChimici Cosmetologi si é tenuta a Londra il 28 mag-gio 2009. Nell’occasione è stato nominato il consi-glio direttivo della Società per il periodo 2009/2010,così composto:Presidente: Dene Godfrey (S. Black) Vice presidente Chris Flower (CTPA Ltd) Segretario onorario Clare Henderson (SPC) Tesoriere onorario Tony Gough (ISP Europe)Membri del consiglio: Barbara Brockway (IMCD UKLtd), Richard Crombie (Cognis Care Chemicals),Barry Winslett (IMCD UK Ltd)Continueranno la col laborazione il pre s i d e n t euscente Judi Beerling, i membri del consiglio:A n d rei Goodwin (Rahn UK), Chris Gummer (CidesSolutions), Emma Meredith (CTPA), Joyce Ry a n ,Richard Summers (S Black), John WoodruffS e g retaria generale Lorna Weston, assistente allasegreteria Gem Bektas

Per informazioni sull’attività di Society of Cosmetic Scientists:

e-mail [email protected] - www.scs.org.uk

World Congress on oils and fats

and 28th ISF Congress

Nell’ambito della manifestazione di cui al titolo chesi terrà a Sydney dal 27 al 30 settembre 2009,American Oil Chemists Society organizza brevi corsisu argomenti specifici nei giorni che precedono eseguono il congresso.

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Practical edible oil refining short course:

process optimisation, equipment and tech-

nology selection, and specialty oils

26-27 settembre 2009

• S. Sefa Koseoglu (Filtration and MembraneWorld LLC, USA), Opening re m a r k s

• A. Richards (Food Science Australia, Australia)The chemistry of fats and oils

• K . P. Eickhoff (GEA Westfalia, D)Review of current degumming technologies

• K . - P. Eickhoff (GEA Westfalia, D)P rocess automation and remote condit ionmonitoring for centrifuges

• G. Boemer (ÖHMI Engineering, D)Bleach ing techno log ies and potent ia ls insavings of bleaching earth

• N. Andria (Süd Chemie, D)Bealching cost calculation

• F. Veldkamp (Amafilter-LFC, NL)How to deal with filtration pro b l e m s

• H.Ch. Holm (Novozymes, DK)Enzymatic degumming and interesterification ofoi ls and fats: fundamentals, enzymes, andindustrial applications

• F. Galhardo (Bunge, USA)P rocess optimization studies on enzymaticdegumming using PLA and PLC enzymes

• M. Kellens (DeSmet-Ballestra, B)Considerations on winterization and fractiona-t i o n

• K. Carlson (Crown Iron Works Inc., USA)Deodorizer design and optimization

• I. Debruyne (ID&A, B)Mechanism of oxidation and quality criteria infrying and cooking oils

• G. Hatfield (Bunge North America, Canada)Critical considerations in processing cruderapeseed and canola oils

• A. Marangoni (Universiity of Guelph, Canada)Review of fat crystallization and structure

• C. Wijesundera (Food Science Australia)New healthy vegetable oil pro d u c t s

• D. Bell (Connell Wagner Ltd, New Zealand)Fish oil (EPA and DHA) and its refining to achie-ve products used by the food and naturalhealth products industries

• R. Verhe (University of Gent, B)Separation of minor components of oils andf a t s

• L. Eyres (Eyres Commerc ia l Group, New

Z e a l a n d )Boutique oils in New Zealand: avocado, olive,flax and boutique oils

• R. Daniell (Nutrizeal, New Zealand)Global business and opportunities for supercriticalcarbon dioxide processing of lipid materials

• P. Gerstenberg (Gerstenberg Schröder A/S, DK)Critical parameters and points in margarinep roduction: how to identify and solve pro b l e m s

Lipid oxidation and antioxidant short course

26-27 settembre 2009

• E. Frankel (Univ. of California, Davis, USA)F ree radical oxidation and methods to determi-ne oxidative stability

• E. FrankelC o n t rol of oxidation and antioxidants

• E. FrankelBiological lipid oxidation

• F. Dionisi (Nestlé Research Center, Losanna,C H )Quality and safety implications of lipid oxidation

• E. FrankelMechanisms of lipid oxidation and antioxidantsin multi-phase emulsion systems

• K. Wa rner (USDA, ARS, NCAUR, Peoria, USA)I m p roving the quality and functionality of vege-table oils

• L. Inturrisi (Cargil Australia, Melbourn e )C o n t rolling oxidation during vegetable oil pro-c e s s i n g

• A, Sut ton (Kemin Food Ingred ients , DesMoines, USA)The multifunctional role of antioxidants: mat-ching consumer desires with practical solutions

• B. Forrest (Danisco Australia Ltd, Botany)Natural extracts as alternatives to syntheticsa n t i o x i d a n t s

Olive oil short course

30 settembre

• P. MillerWelcome and intro d u c t i o n

• Ch. Gertz Methods of determining quality, orig in andauthenticity of olive oil

• Speaker TBCThe nutritional diff e rence between extra virginand refined olive oils

• R. MailerOlive oil quality – natural chemistry of Australianoils and survei llance results from Australian

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s u p e r m a r k e t s• Speaker TBC

Trade issues, quality, substitution and globalmarket dynamics for olive oil

• C. GuillaumeThe Australian olive oil market – quality andauthenticity of olive oils labelled as extra virgin

• M. TsimidouNon-regulatory quality considerations for extra vir-gin olive oils

Il giorno 26 settembre sarà tenuto inoltre il sim-posio dal titolo

Deep frying Symposium

Il programma sarà il seguente• R. Mai ler ( ISF2009 Organis ing Committee

C h a i r m a n )O p e n i n g / w e l c o m e

• Overwiew of deep-fraying• R. Smith (CSIRO Food Science, Melbourn e ,

A u s t r a l i a )P roduction of frying fats

• Ch. Gertz (Chemisches Untersuchungsamt,Hagen, D)Fundamentals of deep frying

• Ch. Rose (Melbourne, Australia)Factors affecting oil fry life/ degradation mecha-n i s m s

• P. Bordi (Center for Food Innovat ion, PennState Univ., USA)Chemistry of deep frying

• C. W ijesundera (CSIRA Food, Melbourn e ,A u s t r a l i a )Specialty oils for frying

• K. Sundram (Malaysian Pa lm Oi l Counci l ,S e l a n g o r, Malaysia)Frying with palm – Opportunities and issues

• P. Henness (Brisbane, Australia)Industrial fryers – Design and operation

• J. Richard (Henny Penny, Paris, F)What food service operators want: existing andnew technologies

• L. Inturissi (Cargill, Melbourne, Australia)Frying oils: maintenance of quality

• C. Rose (Melbourne, Australia)Monitoring/evaluation of used frying oil

• Ch. Gertz (Chemisches Unters. Hagen, D)Regulatory and HACCP re q u i rements in thefrying industries

Per la registrazione on line consultare il sito del congresso

www.isfsydney2009.com

CAOCS – 23rd Meeting of Canadian

Section of the AOCS

Toronto, Canada 4-6 ottobre 2009

Il 23° meeting della Sezione canadese di AOCS siterrà presso i l Méridien King Edward Hotel diToronto.Tema generale: Fats and oils functionality in proces-sed foods: from the fundamental to the applied.Sono previste le seguenti sessioni di lavoro- Healthy developments in fats and oils- Innovations in oilseed crops- P rocessed foods applications: bakery, dairy and

snack foods- Industrial and non-food applications

Per informazioni dettagliate consultare il sito

www.aocs.org/meetings/CAOCS09

Oppure contattare Dérick Rousseau, Ryerson University,

Toronto, ON, Canada


Pharmac India 2009 – Pharma and

healthcare exhibition

Ahmedabad, 7-9 novembre 2009

L’industria farmaceutica in India è in grande espan-sione con un aumento previsto per il 2012-2013 del21% annuo.L’esposizione verterà sui seguenti settori:Formulazioni farmaceutiche; Prodotti ayurvedici ederivati dalle erbe; Prodotti nutraceutici e dietetici;P rodotti cosmetici; Medicinali veterinari; Macchinariper l’industria farmaceutica; Aromi e fragranze;Reagenti diagnostici; Eccipienti, additivi alimentari,estratti naturali; Ingredienti farmaco attivi; Materiali emacchinari di imballaggio; Ricerca e sviluppo, meto-di di analisi; Attre z z a t u re e prodotti chimici da labora-torio; Associazioni, stampa; Prodotti biofarmaceutici.

Per informazioni dettagliate sull’esposizione:

Orbitz Exhibitions Pvt Ltd, Ms Cogita Panchal, e-mail:

[email protected] - www.pharmacindia.com

Sommario - INNOVHUB SSI

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