The effect of malaxation temperature on the virgin olive oil phenolic profile under laboratory-scale conditions

Research Paper

The effect of malaxation temperature on the virgin olive oilphenolic profile under laboratory-scale conditions

Alessandro Parenti1, Paolo Spugnoli1, Piernicola Masella1 and Luca Calamai2

1 Dipartimento di Ingegneria Agraria e Forestale, Facoltà di Agraria, Università degli Studi di Firenze,Florence, Italy

2 Dipartimento di Scienza del Suolo e Nutrizione della Pianta, Facoltà di Agraria,Università degli Studi di Firenze, Florence, Italy

The relationship between olive paste malaxation temperature and the concentration of olive oil hydrophilicphenols (HP), i.e. simple phenols, secoiridoids and lignans, was investigated. Malaxation experimentswere performed at laboratory scale for 45 min at 21, 24, 27, 30, 33 and 36 7C. A significant (p ,0.05)increment of total phenols concentration was found with a maximum at 27 7C, whereas for higher tem-peratures (30–36 7C) a progressive decrement was observed. A similar pattern was recorded approxi-mately for all the secoiridoid compounds, i.e. a quasi-linear increment of concentrations with increasingtemperature until 30 7C, followed by a marked decrease in correspondence with the higher malaxationtemperature (33 and 36 7C). The amount of simple phenols increased linearly with increasing temperatureand no decrements were observed up to the maximal temperature investigated (36 7C), while no signifi-cant differences were found for lignans. A small increment of peroxide values and total chlorophyll wasrecorded as a function of the increasing malaxation temperature, whereas no differences were observed inthe free acidity. The results highlight that there is not a univocal relationship between HP concentrationand malaxation temperature. An equilibrium between degradation (chemical and biochemical oxidationand hydrolysis) and transfer (partitioning) phenomena was hypothesized.

Keywords: Hydrophilic phenols profile / Malaxation temperature / Olive oil / Partition coefficient

Received: December 13, 2007; accepted: March 26, 2008

DOI 10.1002/ejlt.200700307

Eur. J. Lipid Sci. Technol. 2008, 110, 735–741 735

1 Introduction

The quality of extra-virgin olive oil (VOO) is strictly related tothe presence of hydrophilic phenols (HP), a group of sec-ondary plant metabolites with peculiar sensory and healthproperties [1–3]. The major HP compounds identified andquantified in VOO belong to three main classes: simple phe-nols (hydroxytyrosol and tyrosol), secoiridoids (ligstrosideand oleuropein aglycons, and their respective decarboxylateddialdehyde derivatives), and lignans [(1)-pinoresinol and(1)-1-acetoxy-pinoresinol] [4]. Several studies have proventhe relationship between the concentration of these anti-

oxidant compounds in VOO and the technological extractionconditions [5]. The extraction process of VOO, consistingonly of physical methods, includes olive crushing, malaxation(mixing) of the pastes and separation of the oil phase.Malaxation is a crucial step of the process as it is necessary toobtain satisfactory extraction yields [6] and because it largelyaffects the qualitative and quantitative composition of HP [5].The influence of malaxation time [7] and temperature [8–14]on VOO overall quality was widely investigated, but contrast-ing results have been reported on the effects of temperature onthe HP concentration. Several authors found a negative rela-tionship between malaxation temperature and HP concentra-tion [8–10] whereas others found more HP in VOO when thetemperature was increased [11–14]. The aim of this work wasto ascertain, under controlled laboratory-scale conditions, therelationship between the malaxation temperature and theconcentration of the three main HP classes: simple phenols,secoiridoids and lignans. Some authors [15, 16] underline the

Correspondence: Alessandro Parenti, Dipartimento di IngegneriaAgraria e Forestale, Facoltà di Agraria, Università degli Studi di Firenze,Piazzale Cascine 15, 50144 Firenze, Italy.E-mail: [email protected]: 139 055 3288316

© 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.ejlst.com

736 A. Parenti et al. Eur. J. Lipid Sci. Technol. 2008, 110, 735–741

differences that usually occur between laboratory-scale andindustrial-scale experiments with specific regard to the quan-tification of the phenolic fraction, thus the difficulty to extra-polate from laboratory outcomes to predict the practicalresults that could be obtained at industrial scale. On the otherhand, the laboratory approach allows a better understandingof the guiding principle of a specific process or phenomenon,independently of the practical applications, and could giveuseful basic information especially when conflicting results arepresent in the literature.

2 Materials and methods

2.1 Olives

The trials were performed using a batch of 3.6 kg of drupes(cv Frantoio), harvested in early November 2006 near Flor-ence, Italy. The fruits were in good sanitary conditions and inan advanced ripeness state (i.e. over 90% of the drupes werefully black). The initial olive batch was homogenized and thendivided into equal aliquots of 0.2 kg (a total of 18 malaxationtrials were carried out) with the aim of reducing the differ-ences in the starting material among different trials. The olivedrupes were stored in aerated fruit baskets and refrigerated at5 6 1 7C until use, to minimize any possible alteration phe-nomena. The malaxation experiments were run as soon aspossible, i.e. within 3 days after the harvest (six trials per day).On each day, six subsamples of 0.2 kg were treated as de-scribed hereafter.

2.2 Malaxation apparatus

The olive drupes were processed into paste with a laboratoryextrusion mill. As this step generates a local temperatureincrement (about 4 7C) of the olive paste that would affect theenzyme activity, before each test the olives were heated from5 7C (storage refrigeration temperature) to a level near themalaxation temperature, i.e. from 5 to 17 7C for malaxation at21 7C, from 5 to 20 7C for malaxation at 24 7C, and so on.This procedure also allowed performing the malaxation at theselected temperature from the beginning of each trial. A rota-tional torque rheometer (Rheolab MC1, Paar-PhysicaGmbH, Germany) was used to perform the malaxation trials.For this purpose, the instrument was configured for use as aconcentric cylinder rheometer but replacing the inner cylinderwith a steel rotating shaft equipped with three series of bentpalettes (purposely designed for this experiment) for obtain-ing an effective malaxation of the olive paste. The outercylinder, which corresponded to the malaxation chamber, hadan internal total volume of 0.23 L. The instrument wasequipped with a liquid temperature control system (TEZ180/MC1) enabling a precise regulation of the sample tempera-ture. A Pt 100 resistance thermometer was used to measurethe temperature either in the heat exchange fluid (water), in

the measuring cup wall (malaxation chamber) or directly inthe sample.

2.3 Experimental procedure

The malaxation experiments were performed at laboratoryscale for 45 min at temperatures of 21, 24, 27, 30, 33 and36 7C. Each malaxation trial was repeated in triplicate. Aftermalaxation, the pastes were centrifuged (Beckman JA21 lab-oratory centrifuge at 100006g) and the recovered oils wereseparated and stored at –20 7C in 50-mL plastic Falcon tubesuntil analyzed. The experiment was completed in 3 days:During each day the entire range of temperature was investi-gated (i.e. one replicate consisting of six trials). The effects oftreatments were evaluated through one-way ANOVA andLSD post-hoc tests.

2.4 Chemical analyses

Free acidity and peroxide value (PV) measurements werecarried out according to the European Official Method ofAnalysis [17].

The total chlorophyll concentration in oil was obtainedfrom the following formula [18]:

Total chlorophyll = 345.36[A670 – (A630 1 A710)/2]/Lwhere total chlorophyll is expressed in mg/kg as pheophytin a,L is the optical path in mm, and A670, A630 and A710 are ab-sorbance units at 630, 670 and 710 nm, respectively.

Total hydrophilic phenols were extracted by liquid-liquidpartition with an 80 : 20 methanol/water solution. The totalphenol content of the extract was determined by the Folin-Ciocalteau spectrophotometric method at 765 nm, using gal-lic acid as the calibration standard [19].

Liquid-liquid extraction of phenolic compounds forHPLC analyses was performed following the procedure ofCortesi et al. [20] with some small changes as previouslydescribed by Parenti et al. [21].

The method is based on direct extraction of the phenolicminor polar compounds from olive oil by means of a methanolsolution and subsequent quantification by HPLC with UVdetection at 280 nm. Syringic acid was used as the internalstandard. Briefly, the extraction was performed with an80 : 20 methanol/water solution on a 200-mg aliquot of VOO,after the addition of 100 mL of an 80 : 20 methanol/water so-lution of syringic acid at 20 mg/L as internal standard. Thefinal extractant volume was 1 mL. The samples were agitatedon a vortex mixer for 3 min, let sit for 15 min and again agi-tated on a vortex mixer for 3 min. Then the oil fraction (theheaviest) was separated by centrifugation (10 min at10,0006g) and the clear supernatant containing the hydro-philic phenols was injected into the HPLC system.

The HPLC system consisted of a Perkin-Elmer 410 quat-ernary pump, a Series 200 autosampler and a 235C UV-DADdetector. Analytical conditions were: HPLC column: Phe-nomenex Synergy C18 4.66615 cm; injection volume:


Eur. J. Lipid Sci. Technol. 2008, 110, 735–741 Malaxation temperature and hydrophilic phenols 737

20 mL; solvent: pH 2.5 H2O/acetonitrile gradient as describedby Cortesi et al. [20]; wavelength: 280 nm.

Individual phenolic component identification wasachieved by their retention time. Quantification and content,expressed in mg/kg, were calculated by measuring the sum ofthe areas of the related chromatographic peaks according tothe retention time and calculated as tyrosol equivalents. Acalibration curve of tyrosol was constructed using syringicacid as the internal standard. Figure 1 shows a typical chro-matogram of the phenols in an olive oil sample characterizedby individual components. The identified phenolic com-pounds were: (i) simple phenols: tyrosol, (p-hydroxy-phenyl)ethanol (p-HPEA), hydroxytyrosol, (3,4-dihydroxy-phenyl)ethanol (3,4-DHPEA); (ii) secoiridoids: dialdehydicform of decarboxymethyl elenolic acid linked to 3,4-DHPEA(3,4-DHPEA-EDA), dialdehydic form of decarboxymethylelenolic acid linked to p-HPEA (p-HPEA-EDA), oleuropeinaglycon (3,4-DHPEA-EA), ligstroside aglycon (L-agl);(iii) lignans: (1)-1-acetoxypinoresinol, (1)-pinoresinol.

3 Results and discussion

Free acidity, PV and total chlorophyll concentrations weremeasured during the experiment for a better monitoring of theoil quality as affected by increasing malaxation temperature.As showed in Table 1, no significant differences were recorded

in the free acidity, whereas a small but in some cases signifi-cant increment of PV was recorded as a function of theincreasing malaxation temperature, probably as a result of anintensification of the primary oxidation processes. As forchlorophylls, the concentration increased with the olive pastemalaxation temperature (Table 1). This factor probably pro-moted the release of pigments from the vegetable tissues, sothat greater amounts of them dissolved into the oil phase.These results are in accordance with what has already beenreported by Ranalli et al. [13].

Since the experiments were planned with a small and ho-mogeneous batch of fruits (harvested from the same tree) andthe same laboratory-scale apparatus in really controlled con-ditions, any difference was exclusively attributed to the dif-ferent malaxation temperatures. The trend of total HP (col-orimetric method) with respect to malaxation temperature isreported in Fig. 2. A bell-shaped curve with a significant (p at0.05, as stated by ANOVA and LSD test) increment of phenolconcentration was found, with a maximum at 27 7C. Forhigher temperatures, a progressive decrement was observed,so that the total amount of HP seems to be negatively affectedby the malaxation temperature in the range of 30–36 7C.These results partially confirm what has been reported byRanalli et al. [13] who found a significant increment of totalHP in the range of 20–35 7C. However, in that case the max-imal concentration was reached at 30 instead of 27 7C. Asimilar trend was observed for the secoiridoid compounds

Figure 1. HPLC chromatograms (at 280 nm) of phenolic extracts from virgin olive oil: (1) 3,4-dihydroxyphenyl)ethanol (3,4-DHPEA); (2) (p-hydroxyphenyl)ethanol (p-HPEA); (3) syringic acid (internal standard); (4) dialdehydic form of decarboxymethylelenolic acid linked to 3,4-DHPEA (3,4-DHPEA-EDA); (5) dialdehydic form of decarboxymethyl elenolic acid linked to p-HPEA(p-HPEA-EDA); (6) lignans, (1)-1-acetoxypinoresinol, (1)-pinoresinol); (7) 3,4-DHPEA-EA, oleuropein aglycon; (8) ligstrosideaglycon.



Table 1. Free acidity, PV and total chlorophyll concentrations as afunction of malaxation temperature.

Tempera-ture [7C]

Free acidity [%] PV [meqO2/kg] Chlorophyll [mg/kg]

mean SD mean SD mean SD

21.00 0.35a 0.02 6.20b,c 0.36 9.51c 1.0424.00 0.36a 0.01 5.80c 0.30 8.24c 0.9227.00 0.32b 0.01 5.83c 0.78 12.62b 1.0730.00 0.31b,c 0.02 6.83a,b 0.31 14.53a 0.6933.00 0.36a 0.02 6.87a,b 0.25 14.86a 1.4336.00 0.34a,b 0.01 7.17a 0.32 16.06a 0.77

# Mean values within columns with different letters indicate signifi-cant differences at p ,0.05 (LSD test).

(Fig. 3), i.e. the prevalent class of HP in VOO [5] whose mo-lecular structure is characterized by the presence of eitherelenolic acid or elenolic acid moieties. Particularly, the mostabundant compounds of this group, i.e. the dialdehydic formof elenolic acid linked to 3,4-DHPEA (3,4-DHPEA-EDA) orto p-HPEA (p-HPEA-EDA), and an isomer of the oleuropeinaglycon (3,4-DHPEA-EA), originate from the breakdown ofthe two major phenolic constituents of the olive fruit, oleur-opein and ligstroside [5]. Also, ligstroside aglycon was gen-erally detected in VOO [5]. As reported in Fig. 3, approxi-mately the same trend was recorded for all the consideredcompounds, i.e. a quasi-linear increment of concentrationswith increasing temperature until 30 7C, followed by a markeddecrease in correspondence with the highest malaxation tem-peratures (33 and 36 7C). 3,4-DHPEA-EDA was the mostabundant compound and accounted for over 60% of the totalsecoiridoids. Further, a temperature increase between 21 and30 7C afforded a fourfold concentration of this compound inthe VOO. No significant differences were found between 33and 36 7C for all the compounds.

Olive paste can be considered as a heterogeneous systemin a dynamic state, i.e. composed of three different phases(one solid and two liquid phases) which are subject to chemi-cal-physical changes during malaxation deriving from boththe mechanical mixing action [22] and a combination ofchemical and biochemical reactions (both enzymatic and non-enzymatic hydrolysis and oxidation) [5, 10]. With reference tothe HP, the activation of endogenous b-glucosidases duringcrushing catalyzes the hydrolysis of oleuropein, demethylo-leuropein and ligstroside to their respective aglyconic forms[23, 24]. During mixing, these last compounds, i.e. 3,4-DHPEA-EDA, p-HPEA-EDA and 3,4-DHPEA-EA, arereleased from the solid phase and partitioning between thewater and oil phases occurs according to their relative affinitiestoward these phases [25, 26]. The proportions of antioxidantsresiding in the three different phases (oil, water, and solids)depend, in turn, on the relative polarities of the antioxidants,the presence of surface-active natural compounds (e.g. phos-

Figure 2. Total phenols concentration (colorimetric method) as afunction of malaxation temperature. Data are means 6 standarddeviations of three independent replicates. Error bars with differentletters indicate significant differences among mean values atp ,0.05 (LSD test).

pholipids), the composition and relative amounts of thephases, and the temperature. At the same time, the oxidativereactions catalyzed by endogenous oxidoreductases (such aspolyphenoloxidases and peroxidases) can promote the oxi-dation of HP [23, 24]. Therefore, the final concentration ofHP in VOO probably depends on the equilibrium amongtransfer and oxidation phenomena, i.e. the release-degrada-tion ratio. In our study, both total HP and secoiridoid con-centrations increased with the temperature until 27 and30 7C, respectively, and then sharply decreased. It is likelythat at the higher temperature the degradation rate of thesecompounds increased and therefore their concentration in thefinal oil was lower. By contrast, Parenti et al. [14] in labora-tory-scale extraction experiments under sealed conditionsfound a linear increase of all the considered phenolic fractionswithout decline at temperatures up to 55 7C. This apparentcontradiction can be explained in terms of different oxidationof the phenolic fractions, as it has been recently demonstratedthat olive pastes produce large amounts of CO2 and a rapidO2 depletion occurs during malaxation [27, 28], which mayhave inhibited oxidation in those experiments conducted withno gas exchange. The same rationale may also explain thecontinuous increase in total phenolic content observed byParenti et al. [29] in plant-scale experiments, due to the largeamounts of olive pastes subjected to the malaxation treat-ment, which may have yielded huge volumes of CO2 whichprotect, at least partially, the phenolic components from oxi-dation. A different pattern was recorded for p-HPEA and 3,4-DHPEA, i.e. the two main compounds of the simple phenolsgroup. As reported in the literature [5], the concentration ofthese compounds is generally very low in comparison to the



Figure 3. Secoiridoid concentration as a function of malaxation temperature. Data are means 6 standarddeviations of three independent replicates. Error bars with different letters indicate significant differencesamong mean values at p ,0.05 (LSD test).

other HP classes. In our experiment, the occurrence of thesecompounds varied in the range 1–7 and 4–15 mg/kg for 3,4-DHPEA and p-HPEA, respectively. Despite these low con-centrations, a significant influence of the malaxation temper-ature could be highlighted, as shown in Fig. 4. The amount ofboth simple phenolic alcohols increased linearly with increas-ing temperature and no decrements were observed up to themaximal temperature investigated (36 7C). These findings arein agreement with the results achieved by Ranalli et al. [13]and according to what was hypothesized before, i.e. thehydrolytic processes may be accelerated at elevated tempera-tures, and the formation of these compounds, which involveglycosidic bond cleavage, could explain their trend. This is inline with earlier observations on the formation of phenoliccompounds, such as p-HPEA, from higher-molecular-weightphenolic compounds [13, 30, 31]. The trend of lignans as afunction of malaxation temperature is reported in Fig. 5. Thepresence of this HP fraction, with (1)-pinoresinol and (1)-1-acetoxypinoresinol as major components, has recently beendescribed in olive oil [32, 33]. Under our HPLC conditions,the two lignans co-elute and, consequently, the results report-ed in Fig. 5 correspond to the sum of the two compounds. Nosignificant differences were found between the different levelsof malaxation temperature. According to Artajo et al. [26], thisresult could be explained considering the lipidic character ofthese compounds and their lower antioxidant activity as com-pared to other HP [5]. On the basis of these considerations, wecan suppose that the lignan concentrations are only slightlyrelated to the equilibrium among transfer and oxidation phe-nomena (release-degradation ratio), but rather their con-centrations are relatively independent of the malaxation tem-perature.

The amounts of total phenols determined according to theFolin-Ciocalteau method were highest at 27 7C whereas themaximal concentration of secoiridoids was observed at 30 7C

Figure 4. Simple phenols concentration as a function of malaxa-tion temperature. Data are means 6 standard deviations of threeindependent replicates. Error bars with different letters indicatesignificant differences among mean values at p ,0.05 (LSD test).

and a constant concentration (lignans) or continuous increasein concentration was observed for the other phenol classes.This apparent discrepancy may be explained by a poor speci-ficity of the Folin-Ciocalteau reaction. In fact, the methodrelies on the reduction of the phosphomolybdic/phospho-tungstic acid complex (Mo61/W6–). Many reducing agentsother than poliphenolic compounds may achieve this reduc-tion, including sugars, aromatic amines, sulfur dioxide,ascorbic acid, organic acids, and Fe(II), as well as non-phe-nolic organic substances [34, 35]. It is therefore possible that adifferent extraction/solubility of some interfering compoundsmay have occurred at different temperatures.



Figure 5. Lignans concentration as a function of malaxation tem-perature. Data are means 6 standard deviations of three inde-pendent replicates. Error bars with different letters indicate signifi-cant differences among mean values at p ,0.05 (LSD test).

4 Conclusion

These results indicate that the malaxation temperaturestrongly affects the HP concentration in VOO. At the sametime, it is also clear that there is not a univocal relationshipbetween the two parameters. The final HP concentrationseems to be the result of the equilibrium between degrada-tion and transfer phenomena. Hence, the temperatureprobably exerts two contrasting effects during pastemalaxation. On the one side, it accelerates the degradationphenomenon, and on the other side, it improves the solu-bility of HP compounds in the oil phase. Servili et al. [5]discussed the conflicting results reported in the literature interms of reduced oxygen concentration in the pastes duringprocessing, i.e. as the result of inhibited activity of PPO andPOD in the pastes and, consequently, of reduced oxidationreactions. However, it is probable that oxidation is not theonly factor involved in the change of the above-mentionedequilibrium. Further, the release and distribution of HP be-tween water and oil phases, i.e. the transfer phenomenon, isprobably related to the intensity of the mechanical mixingaction and the extent of the contact between the solid andthe oil phase. In this context, future investigations shouldconsider the possible effect of parameters such as mixingspeed during malaxation or mixing shaft shape on the HPtransfer.

Conflict of interest statement

The authors have declared no conflict of interest.

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The effect of malaxation temperature on the virgin olive oil phenolic profile under laboratory-scale conditions