Changes in phenolic content of olive during maturation

*Correspondent: Fax: 1612 6933 2737; email: [email protected]

Changes in phenolic content of olive during maturation

Danielle Ryan1, Kevin Robards1* & Shimon Lavee2

Correspondent: K. Robards, School Of Science And Technology, Charles Sturt University, PO Box 588, Wagga Wagga2678, Australia

Summary Qualitative and quantitative data are presented for the phenolic content of cvs Manzanilloand Cucco based on separation by high performance liquid chromatography, HPLC, withultraviolet, fluorescence and mass spectrometric detection. Oleuropein is the principalphenolic compound in olive and its concentration changed significantly during fruit devel-opment. Changes in the content of tyrosol, ligstroside and verbascoside were alsoobserved but these were relatively smaller.

Keywords Chromatography, mass spectrometry.

Introduction

The phenolic content of olive fruits is importantfor a variety of reasons (Esti et al., 1998; Ryanet al., 1998). The phenolics constitute a complexmixture in both fruit and oil although there aresome notable differences in composition betweenthe two that are attributed to a series of chemicaland enzymatic alterations of some phenols duringoil extraction. These modifications include hydrol-ysis of glycosides by glucosidases, oxidation ofphenolic compounds by phenoloxidases and poly-merisation of free phenols (Duran, 1990).

A considerable amount of data has beenamassed on the phenolic content of olive oil usinghigh resolution techniques (Cimato et al., 1990;Alessandri et al., 1994; Deidda et al., 1994; Garciaet al., 1996). Significantly less work has been con-ducted on the phenolic composition of the fruit(Ryan & Robards, 1998) and yet it is the fruitwhich is fundamentally the more important.Changes in phenol content during fruit develop-ment are important and it is desirable to have anindex of maturation which can be related to fruitcomposition. In the 6–8 months following flower-ing the olive attains its maximum fruit weight.This is followed by change in fruit colour andassociated physiological modifications, with the

appearance of the purplish-black olive fruit indi-cating the end of olive morphology.

Two degrees of maturation are recognized(Shulman & Lavee, 1976) in olive fruits, namelygreen maturation and black maturation. Forexample, green maturation is characterized by areduction in chlorophyll content in conjunctionwith fruit softening and an increase in oil content.A further reduction in chlorophyll content isapparent in the black maturation phase, alongwith a significant increase in CO2 accumulation,ethylene secretion and anthocyanin content, thelatter of which are responsible for black fruitcolouring, and are classified as phenolics. Amiotet al. (1989) have included a third phase in olivedevelopment, aptly named the growth phase,which occurs prior to that of green maturation,during which the accumulation of oleuropein(Fig. 1) occurs. In contrast, four stages of matu-ration have been identified by Garcia et al. (1996),which quite simply correspond to the apparentchanges in fruit colour and anthocyanin content atthe green, spotted, purple and black stage of olivematuration.

Cimato et al. (1990) showed that with fruitripening, hydrolysis of components with ‘highermolecular weight’ occurred, with the formation oftyrosol and hydroxytyrosol. Thus, the concentra-tion of tyrosol and hydroxytyrosol was also shownto increase with the harvesting period, which hasbeen correlated with an evident reduction in four

International Journal of Food Science and Technology 1999, 34, 265–274

© 1999 Blackwell Science Ltd

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unidentified, but presumably phenolic compo-nents. The majority of research on the relationshipbetween phenolics and olive development concernsoleuropein, which is known to be the most promi-nent and significant individual phenolic compo-nent of olive pulp, reaching concentrations of upto 14% on a dry weight basis in young Picholineolives (Amiot et al., 1986). The concentration ofoleuropein declines with fruit maturity (Amiotet al., 1989) and is accompanied by the accumula-tion of two compounds, namely demethyloleu-ropein and elenolic acid glycoside, of which, onlythe former is phenolic. The work of Amiot et al.(1986, 1989) who examined the phenolic profile ofolive fruit as a function of physiological develop-ment is notable. Esti et al. (1998) have also report-ed data for the phenolic content of olives duringmaturation but their results were restricted to anexamination of changes during a relatively shortperiod of harvest-time.

The present paper presents an investigation ofthe phenolic content of two olive cultivars as

determined by liquid chromatography usingphotodiode array detection and electrospray ion-ization mass spectrometry. Changes in the pheno-lic content of the drupes were examined fromabout 1 week prior to green maturation untilblack maturation had been reached. The cultivarswere chosen because they represented extremes ofbehaviour under the prevailing agronomic condi-tions. The ultimate aim of this work is to provideinformation that will assist in the design of agro-nomic, harvesting and processing practices thatwill ‘optimize’ (not necessarily maximize) the phe-nolic content of olive products. There has been nocomparable study of olives grown in the SouthernHemisphere and yet agricultural conditions inAustralia are ideally suited to olive production.

Experimental

Reagents

Reagents from the following sources were usedwithout further purification: methanol (EMScience, New Jersey, USA), acetic acid (AjaxChemicals, Sydney), Folin and Ciocalteu’s PhenolReagent 2.0 N (Merck, Germany), hexane (BiolabScientific, Mallinckrodt, Paris, Kentucky) andhydrochloric acid (32%) (Ajax Chemicals,Sydney). Phenolic standards were obtained as fol-lows: oleuropein from Extrasynthese (Genay,France); caffeic, chlorogenic, p-coumaric andvanillic acids from Sigma Chemical Co.; tyrosolfrom Aldrich Chemical Co. and phenol(Analytical Reagent, Mallinckrodt, Paris,Kentucky) from Mallinckrodt. All water used forHPLC analyses, sample and standard preparationwas purified using reverse osmosis. Acetone sup-plied by Ajax Chemicals was used for cleaningpurposes.

Standards

A stock standard was prepared in methanol water(50 1 50 v/v) containing caffeic acid, 25 m5 g ml21;p-coumaric acid, 25 mg ml21; oleuropein, 350 mgml21; tyrosol, 100 mg ml21 and vanillic acid, 25 mgml21. The stock standard was diluted appropriate-ly and filtered (0.45 mm) prior to HPLC or LC-MS injection. The specified concentration ratiosensured approximately equal UV detectorresponse (peak height) for each compound.

Phenolic contents of olives D. Ryan


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Figure 1 Chemical structure of representative phenoliccompounds found in olive fruit.

Sample preparation and storage

Olive samples were picked randomly from trees ofdifferent cultivars (Manzanillo and Cucco) froman approximately 60–year–old olive grove inWagga Wagga at various stages of physiologicaldevelopment as reflected by skin colouration.Olive samples of at least 50 grams were selectedfor each fruit colour. The olives were refrigeratedat 4 ºC prior to processing. The fruit was hand-pitted and freeze dried over a 2–3 day intervalaccording to the moisture content of the fruitsamples. The freeze dried olives were blended intoa fine powder using a general purpose electricblender. The powdered samples were stored inscrew top plastic jars that were kept in desiccatorsprior to analysis which was performed as soon aspracticable, although there were no notablechanges observed in freeze-dried samples storedfor up to four months.

Details of the analytical procedure have beenreported previously (Ryan et al., 1999) and onlythe salient features are presented. For RPLCanalysis, powdered olive sample (1 g) was recon-stituted with sodium carbonate solution (5 ml; 1M) which was swirled and left to sit for 15 min-utes at ambient temperature (optimized in theranges 10–60 min and ambient to 60 ºC). Thismixture was then filtered using a buchner funnelapparatus fitted with Advantec 55 mm filter paperand a hand pump. The olive mass was recoveredand transferred to the same conical flask to whicha further 5 ml portion of carbonate solution wasadded. The flask was again swirled, left to standfor 15 minutes and filtered using fresh filterpaper. Both filtrates were combined and trans-ferred to a 25 ml separating funnel. The com-bined filtrate was washed once with hexane (5 ml)to remove lipoidal material and the aqueousphase recovered and adjusted to a pH of approx-imately 4 with hydrochloric acid (4–5 ml; 3 M).The acidified extract was subjected to solid-phaseextraction (SPE) using a Waters reversed phase(C18) Sep-Pak cartridge that had been condi-tioned with methanol (6 ml) followed by nano-pure water (6 ml). Phenolic compounds wereeluted using methanol + water (5 ml; 50 1 50v/v). Sample extracts were collected in screw topscintillation vials and stored at 4 ºC where neces-sary prior to RPLC analysis. The eluate was

diluted (1:10) with the same solvent and filteredusing 0.45 mm Cameo 25AS acetate filters andTerumo plastic syringes before being injected intothe RPLC system. Peak identification was basedon retention time and spiking of sample extractswith authentic materials in conjunction with spec-tral data.

Total phenol measurement

Total phenols were determined following reactionwith Folin-Ciocalteu reagent as follows (Shahidi& Naczk, 1995). Eluent from SPE (0.5 ml, afterappropriate dilution but usually 1:10) or a phe-nolic standard was mixed with Folin-Ciocalteureagent (5 ml, 1:10 diluted with nanopure water)and sodium carbonate solution (4 ml, 1 M). Themixture was heated for 15 minutes at 45 °C in awater bath. Absorbance of the solutions was mea-sured at 765 nm against a reagent blank using aPye Unicam PU 8800 UV/VIS spectrophotometer(Cambridge, UK) and quartz cuvettes (1 cm).Total phenol values are expressed as mg equiva-lents of phenol per gram dry mass of olivesample.

High performance liquid chromatography

HPLC analyses were performed using a PerkinElmer binary LC pump 250 equipped with a20 ml loop injector. A Perkin Elmer LC-235Diode Array detector and a Perkin Elmer LC-240Fluorescence detector connected in series servedto monitor the column eluent. The HPLC systemwas interfaced to a model number DCM-1488ELasernet Computer Systems computer with 20MByte hard disk. Chromatograms were generat-ed using an LX-300 printer.

Gradient elution (Gradient I) was used with aVarian C18 column (4.0 mm 3 15 cm; 4.5 mm)for routine analyses where Solvent A is methanol1 acetic acid (100 1 1 v/v) and Solvent B is water1 acetic acid (100 1 1 v/v) at a constant flow rateof 1.0 ml min21. The gradient comprized an initialoptimization at 20%A followed by a linearincrease to 80%A over 30 min, 5 min isocraticoperation, increasing to 100%A over 15 min, holdfor 5 min and return to initial conditions. Theeluent was monitored by UV at 280 nm (sensitiv-ity 0.1) whilst an excitation wavelength of 280 nm



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and an emission wavelength of 320 nm were usedfor fluorescence detection. The mobile phaseswere degassed under vacuum using Alltech Nylon66 membranes, 47 mm 3 0.45 µm and continu-ously sparged with high purity helium (60 kPa)during analysis to prevent resaturation by air.

Liquid chromatography-mass spectrometry

Sample extracts were analyzed using a HewlettPackard model 1090 liquid chromatograph and aQuattro II quadrupole mass spectrometer (MS)(Micromass, Altrincham, Cheshire, UK) by elec-trospray ionization (ESI). A Waters C18 column(2 mm 3 15 cm) thermostatted at an oven tem-perature of 35 ºC was used. HPLC grade methanol(Mallinckrodt) with 0.1% formic acid and Milli Qwater with 0.1% formic acid served as Solvents Aand B, respectively for the gradient elution pro-gram (Gradient II). This involved an initial iso-cratic development for 5 min at 20%A followed bya linear increase to 80%A at 35 min, 100%A at 37min, isocratic operation for 3 min and return toinitial conditions over 2 min. An injection volumeof 10 ml and a constant flow rate of 0.200 ml min21

was used for each analysis with a split ratio ofapproximately 10:1 (UV detector:MS). The UVwas monitored at 280 nm and the UV trace wasacquired by the Masslynx Data System(Micromass) along with the mass spectral data.

Results and discussion

Ripening patterns of cvs Cucco andManzanillo olives

Fruit from the two cultivars matured at signifi-cantly different rates with that from the Cuccotree reaching black maturation prior to that ofthe Manzanillo fruit (Table 1). The proportion of

purple and black fruit contained in the Cuccosamples beyond the third harvesting date alwaysexceeded the corresponding proportions in theManzanillo samples. Collection of Cucco sampleswas suspended after the fifth harvesting datewhen black maturation had been attained. Of thetwo varieties, Cucco fruit (average mass at greenmaturation 5 g) was consistently larger than thatof Manzanillo (3 g).

Chromatographic analysis

The diversity of phenolic compounds in olivesand the utility of both ultraviolet and fluores-cence detection is illustrated by the chro-matograms of Fig. 2. This also demonstrates theimportance of correct choice of detection wave-length although photodiode array detection elim-inated this problem (in UV work) and alsofacilitated the identification of eluted peaks (Ryanet al., 1999). However, the direct coupling of LCand electrospray ionization MS offered a distinctadvantage in this regard. The data generated bythe combined system provided unparalleledopportunities for compound identification usingboth retention times and the mass spectra thatgave structural information without the necessityof isolating individual compounds.

Extracts from several olive samples were exam-ined by LC-MS (Gradient II conditions) usingESI in the positive and negative ion modes to gen-erate Total Ion Current (TIC) chromatograms(Fig. 3). The latter were generally not as welldefined as those obtained with UV or fluorescencedetection. However, the presence or otherwise ofparticular phenols was readily determined byextraction of mass spectra and use of extractedion mass chromatograms (computer-generatedplots of the abundance of a specific ion extracted



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Harvest number Date* Days from first harvesting Cucco Manzanillo

1 March 12 0 G G2 April 2 21 G G3 April 9 28 G G4 April 16 35 P G5 April 24 43 B M6 May 5 54 — P7 May 13 62 — B*

*Year 1997.

Table 1 Dates on which fruit wereharvested showing the dominantfruit colour at the respectiveharvests. Collection of fruit on aparticular date was restricted tothe dominant colour. Fruit colouridentified as G, green; M, greenwith purple spots; P, purple; B, black

from the TIC chromatogram) (Fig. 4). Thus, theelution of oleuropein at 16.5 minutes usingGradient II was confirmed by the presence of veryclean and distinct peaks in both the positive andnegative ion mass chromatograms at m/z 541 andm/z 539, respectively with a sodium adduct at m/z563 in the positive ion mass chromatogram. Moreimportantly, the related compounds, elenolic acidand elenolic acid glucoside which did not absorbin the 280–340 nm range were identified from themass spectral traces. Other species identified inthis fashion are listed in Table 2. Nevertheless, theidentification of some phenolic compounds insample extracts was inconclusive (see Table 2).This is largely a factor of the low levels of manyof the title compounds in olive fruit. Selective con-centration of sample extracts using SPE would beexpected to facilitate the identification process aswould Selected Ion Monitoring (SIM) LC-MS inplace of TIC. Such processes are being investigat-ed for future applications.

From a consideration of chromatogramsobtained under various detection conditions, UV

at 280 nm and fluorescence detection at 320 nm(excitation 280 nm) were adopted for routine pro-filing. Chromatographic profiles using these con-ditions were qualitatively similar for the two olivevarieties at all stages of maturation. This is illus-trated in Fig. 5 which shows the phenolic profileof Manzanillo and Cucco olives harvested at greenmaturation. Distinction between UV chro-matograms at 280 nm of the two varieties howev-er can be made on quantitative differences in thedistribution of phenolics observed between 12 and18 minutes. Thus, chromatograms of extracts ofManzanillo were characterized by a collection ofthree major peaks at the approximate retentiontimes of 12 (verbascoside), 14 (oleuropein) and 18(ligstroside) minutes, whilst Cucco chromatogramsonly exhibited two major peaks in this region cor-responding to oleuropein and ligstroside.Verbascoside was rarely detected in Cucco samplesand then only at certain stages of development. Incontrast, verbascoside was present in allManzanillo samples at all stages of maturity, butits concentration varied greatly between fruits har-



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UV detection at 340 nm Fluorescence detection at 380 nm (excitation 340 nm)

Fluorescence detection at 320 nm (excitation 280 nm)UV detection at 280 nm

Figure 2 Chromatograms obtained with gradient I comparing various methods of detection for the elution of an extractof green Manzanillo fruit. Detection was achieved by UV absorption at 280 or 340 nm (a and c, respectively) or byfluorescence at 320 nm (excitation 280 nm) or 380 nm (excitation 340 nm) (b and d, respectively). Chromatograms areshown in the region from 0 to

vested from different regions (see Fig. 5). Theoleuropein content of Manzanillo fruit was higherthan that of Cucco fruit at all stages of maturationin accordance with the observations of Amiotet al. (1986) who noted an inverse relationshipbetween fruit size and oleuropein content. In con-trast, the inverse relationship between oleuropeinand verbascoside (Amiot et al., 1986) was not sub-stantiated in the present work. Tyrosol was pre-sent in Cucco fruit at a considerably reduced levelto that in Manzanillo, unlike ligstroside which wasrelatively more abundant in the Cucco fruit.

Maturation Results

This represents a preliminary report of the effectsof maturation on the phenolic content of olivefruit of the two cvs Manzanillo and Cucco. It isrestricted to the four compounds, oleuropein,tyrosol, verbascoside and ligstroside. Changes in

content of other phenolic compounds includingthe flavonoids during maturation were insignifi-cant by comparison. Data were collected as chro-matographic peak areas (from UV at 280 nm withthe exception of tyrosol) per gram dry mass ofolive versus degree of maturation. Results areexpressed as peak area units rather than concen-tration because two of the compounds were notavailable as standards. In such circumstances,peak area represents a preferable method (Cimatoet al., 1990) of expressing results. Similarly, theuse of dry fruit mass values avoids problems asso-ciated with analyte dilution as a result of fruitgrowth. In the case of tyrosol, some UV datawere unreliable due to interference by an uniden-tified co-eluting species which did not fluoresceand hence, fluorescence peak areas are reportedfor tyrosol.

One of the difficulties associated with olivematuration studies is the precise identification of



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Figure 3 Chromatograms of oliveextracts of cv Manzanillocomparing (Top to Bottom) UVat 280 nm and total ion current(negative and positive ion mode)for detection. Elution conditions:

the various physiological stages. The use of har-vest date versus change in phenolic content makesno allowance for the vastly different rates of mat-uration of the olive fruit on the same tree. In the

present work, fruit colour rather than harvestingdate was used to indicate the extent of matura-tion, and green, spotted, purple and black olivesamples were analyzed in isolation. This method



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Figure 4 Representative extractedmass chromatograms for Fig. 2 at(top to bottom) m/z 539(oleuropein; negative ion); 523(ligstroside; negative ion); 541(oleuropein; positive ion); 525(ligstroside; positive ion).Chromatograms are shown in theregion from 5 to 45 min.

is believed to be more suitable for such investiga-tions since different coloured fruits are known tobe chemically distinct, particularly with respect tophenolic compounds (Amiot et al., 1986; Garcia

et al., 1996). Amiot et al. (1986) used harvestingdate as a measure of fruit maturation, but indi-cated the timing of black maturation. However,no details were provided regarding the proportion



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Table 2 Phenolic compounds identified in olives using RPLC and LC-MS

Compound Molecular Retention Retention Major ESI2 Major ESI1 Sodium

mass time (minutes) time (minutes) peaks peaks adduct

Gradient I Gradient II

Tyrosol 138 3.6 Ip-Coumaric acid 164 9.9 IHydroxytyrosol 168 IVanillic acid 168 6.1 ICaffeic acid 180 6.8 IHomovanillic acid 182 1.3 181 183 205Ferulic acid 194 1.1 193 n.d. 217Syringic acid 198 1.1 197Chlorogenic acid 354 7.0 n.d.Quercetin-3-rhamnoside 448 14.2 447 449Ligstroside 524 18.0 18.9 523 525, 363, 345 547Oleuropein 540 14.5 16.5 539 541 n.d.Rutin 610 IVerbascoside 624 12.0 13.7 623, 241 625 647Verbascoside – isomer 624 13.4 15.3 623, 241 625 647Elenolic acid 242 10.3 241 243 265Elenolic acid glucoside 6.4 241 265

I 5 mass spectral data were inconclusive; n.d. not detected.

Fluorescence chromatograms

UV chromatograms

Manzanillo I

Manzanillo I

Manzanillo II

Manzanillo II

Cucco

Cucco

Figure 5 Chromatographic profiles of Manzanillo and Cucco fruit at green maturation. Fruit from a second Manzanillotree grown in a different location are shown for comparison. Chromatograms were obtained with gradient I and eitherUV detection at 280 nm (attenuation 800) or fluorescence detection at 320 nm (attenuation 600). Chromatograms are

of olives which were black and the use of wholefruit during the first four weeks of sampling anddivision of pulp and seed in later samplings is apotential problem as the phenolic content of pulpand seeds can vary substantially. Cimato et al.(1990) also failed to provide any details concern-ing fruit colours and have simply based their con-clusions on three samples collected at differentharvesting dates. In the present paper, data arediscussed at a given harvest date for the majorityfruit colour. For example, as shown in Table 1,Cucco data for harvest 3 were collected for greenfruit although the tree also contained some spot-ted, purple and black fruit.

The oleuropein content of Cucco fruit at initialharvest was 22 6 4 mg g21 (all data are reportedon a dry mass basis) and then showed a progres-sive decrease with maturation from green,through purple and black fruit. Indeed, oleu-ropein was not detected in Cucco fruit at blackmaturation on day 43. In contrast, the oleuropeincontent of Manzanillo showed an initial slightdecrease from 60 mg g21 but then increased bysome 50% from 50 6 5 mg g21 2 100 6 5 mg g21

between harvests 2 and 3 (covering a period ofseven days) before decreasing again consistentwith the findings of Amiot et al. (1986, 1989) whoobserved a decline in oleuropein content withfruit maturity. The change in oleuropein contentwas reflected by a corresponding change in totalphenols which increased from 20 6 2 mg g21

(expressed as mg equivalents phenol per gram drymass) to 150 mg g21 between harvests 2 and 3 fol-lowed by a rapid decline to 20 mg g21 at harvest4 with a slight monotonous decrease thereafter.The apparent discrepancy between oleuropeincontent and total phenols is attributed to the non-specificity of the latter measurement and also tothe method of expressing results as mg equiva-lents of phenol. The increase in oleuropein con-tent during the early stages of development hasbeen attributed to a growth phase (Amiot et al.,1989) that occurs prior to that of green matura-tion and is characterized by an accumulation ofoleuropein. A closer examination of the changesoccurring during this short period is warrantedand should include an examination of elenolicacid glucoside which is not present prior to greenmaturation (Amiot et al., 1989). In the case of theCucco samples, the results suggest that harvesting

commenced too late to observe the growth phasewith its rapid accumulation of oleuropein. Thetotal phenol content of the Cucco fruit was con-stant throughout maturation at 10 6 2 mg g21.

Manzanillo fruits exhibited a gradual decreasein verbascoside with maturation (peak area countper gram dry mass was 1000 at commercial greenmaturation decreasing to 300 at black matura-tion) while in Cucco fruits verbascoside was notinitially detected. However, in this variety, ver-bascoside started to accumulate after day 21,peaked (200 area counts) and then decreased onceagain to an undetectable level at black matura-tion. No significant change in tyrosol concentra-tion was observed in Manzanillo fruits withmaturation whereas the tyrosol concentration inCucco fruits decreased by approximately 50% byharvest at day 28 before increasing again at blackmaturation to be approximately equivalent to theinitial concentration found in green fruit sampledon the first harvesting date. In olive oil, however,the content of tyrosol and hydroxytyrosol hasbeen reported (Cimato et al., 1990) to increasewhen the oil was extracted from more maturefruit. This difference might be due to the differ-ent material analyzed, varietal differences and theuse of mixed samples by Cimato et al. (1990) con-taining fruits of different maturation indices.

The ligstroside content of Manzanillo olivesremained relatively constant although there was aslight but significant peak at approximately 35days. In contrast, the ligstroside content of Cuccofruits increased by approximately 40% betweenharvests on days 21 and 28. This increase wasaccompanied by a corresponding increase in ver-bascoside and a decrease in tyrosol content.

Acknowledgements

The assistance of Dr Daniel Jardine, MacquarieUniversity, Sydney and the financial support ofRural Industries and Horticultural Research andDevelopment Corporations, Australia is grate-fully acknowledged.

References

Alessandri, S., Cimato, A., Mattei, A. & Modi, G.(1994). Harvesting period and variations in Tuscanolive oil composition: a multivariate approach. ActaHorticulturae, 356, 233–238.



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Amiot, M. J., Fleuriet, A. & Macheix, J.-J. (1986).Importance and evolution of phenolic compounds inolive during growth and maturation. Journal ofAgricultural and Food Chemistry, 34, 823–826.

Amiot, M.-J., Fleuriet, A. & Macheix, J.-J. (1989).Accumulation of oleuropein derivatives during olivematuration. Phytochemistry, 28, 67–69.

Cimato, A., Mattei, A. & Osti, M. (1990). Variation ofpolyphenol composition with harvesting period. ActaHorticulturae, 286, 453–456.

Deidda, P., Nieddu, G., Spano, D., Bandino, G., Orru,V., Solinas, M. & Serraiocco, A. (1994). Olive oilquality in relation to environmental conditions. ActaHorticulturae, 356, 354–357.

Duran, R. M. (1990). Relationship between thecomposition and ripening of the olive and quality ofthe oil. Acta Horticulturae, 286, 441–451.

Esti, M., Cinquanta, L. & La Notte, E. (1998). Phenoliccompounds in different olive varieties. Journal of

Agricultural and Food Chemistry, 46, 32–35.Garcia, J. M., Seller, S. & Perez-Camino, M. C. (1996).

Influence of fruit ripening on olive oil quality. Journalof Agricultural and Food Chemistry, 44, 3516–3520.

Ryan, D., Robards, K. & Lavee, S. (1998). Assessment ofquality in olive oil. Olivae, 72, 23–41.

Ryan, D. & Robards, K. (1998). Phenolic compounds inolives. Analyst, 123, 31R–44R.

Ryan, D., Robards, K. & Lavee, S. (1999).Determinatioin of phenolic compounds in olives byreversed phase chromatography and mass spectrometry,Journal of Chromatography, 832, 87–96.

Shahidi, F. & Naczk, M. (1995). Methods of analysis andquantification of phenolic compounds. In: FoodPhenolics: sources, chemistry, effects and applications.Lancaster, PA: Technomic.

Shulman, Y. & Lavee, S. (1976). Endogenous cytokininsin maturing Manzanillo olive fruits. Plant Physiology,57, 490–492.



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