Eur. J. Lipid Sci. Technol.

2010, 112, 13891392

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Short Communication Nitrogen stripping to remove dissolved oxygen from extra virgin olive oil
Piernicola Masella1, Alessandro Parenti1, Paolo Spugnoli1 and Luca Calamai2
1

` Dipartimento di Economia, Ingegneria, Scienze e Tecnologie Agrarie e Forestali, Facolta di Agraria, ` degli Studi di Firenze, Firenze, Italia Universita 2 ` Centro Interdipartimentale di Spettrometria di Massa, Universita degli Studi di Firenze, Polo Scientico, Firenze, Italia

The stripping treatment of extra virgin olive oil (EVOO) by nitrogen gas to remove dissolved oxygen (DO) was tested immediately after the oil production. Dissolved oxygen was measured before and after stripping, as well as one week later along with chemical analyses with the aim to assess the effects of the stripping treatment on EVOO quality. Stripping gave a great reduction of DO, of ca 50%. Both stripped (SO) and non-stripped (non-SO) oil samples showed a fast DO consumption up to zero in seven days. At this time, the non-SO samples showed signicant higher peroxide value probably as a consequence of the initial higher DO concentration that gives a greater formation of free radicals. A slightly lower concentration of total phenols was recorded for SO samples. A slight but signicant decrease was recorded for only (E)-hex-2-enal concentration within the volatile compounds.
Keywords: Dissolved oxygen / Nitrogen stripping / Olive oil quality / oxidation

Received: December 11, 2009 / Revised: May 20, 2010 / Accepted: June 25, 2010 DOI: 10.1002/ejlt.200900277

1 Introduction
Extra virgin olive oil (EVOO) plays a crucial role in the Mediterranean diet as it is the primary source of fat intake [1]. The characteristic aroma, taste, color, and nutritive properties of this product distinguish it from other edible vegetable oils [2]. Several studies were performed in the last years to establish the relationships between EVOO quality and the processing technology [3, 4]. Recently, it was shown that the main processing steps contribute differently to the amount of dissolved oxygen (DO) in EVOO and that the nal vertical centrifugation, aimed to clean the oil from small amounts of residual vegetative water and impurities, afforded the highest oxygenation effect [5]. Moreover, the DO that was shortly consumed by the oil in a few days seems to act as a starter for the subsequent autoxidation reactions. This was conrmed by the faster quality decay kinetics during shelf-life

of the oils with higher DO concentration [5]. Ottaviani et al. [6] showed that the occurrence of radicals in the oils was strictly related to the amount of DO and the possible use of bubbling nitrogen in to the oil was tested at laboratory scale to remove oxygen, nevertheless the effects of this treatment on EVOO quality were not evaluated. A similar attempt [7] was performed in a later study where the effect of nitrogen stripping on the oil quality was tested only with respect to peroxide value at lab-scale experiment. The aim of this work was to evaluate the effectiveness of nitrogen stripping to remove DO oxygen from EVOO at pilot scale immediately after production. The effects of the treatment on the EVOO overall quality were assessed.

2 Materials and methods

2.1 Experimental procedure
The quality effects of stripping nitrogen to remove DO from EVOO were tested at pilot scale. Nine trials were performed at a continuous 2-phase extraction system model Jumbo2 (Pieralisi, Italy) by processing a previously homogenized olives batch cv. Moraiolo of about 4500 kg, i.e., 500 kg in each trial. Drupes were manually harvested near Florence (Italy) in early December 2009. They were in good sanitary conditions and in advanced state of ripeness. EVOO was
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Correspondence: Dr. Piernicola Masella, Dipartimento di Economia, ` Ingegneria, Scienze e Tecnologie Agrarie e Forestali, Facolta di Agraria, ` Universita degli Studi di Firenze, Piazzale Cascine 15, 50144 Firenze, Italy E-mail: piernicola.masella@unifi.it Fax: 39-055-3288316 Abbreviations: DO, dissolved oxygen; EVOO, extra virgin olive oil; nonSO, non-stripped oil; SO, stripped oil

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produced within 24 h after harvesting. The oil (20 L) was sampled directly after the vertical centrifuge, i.e., immediately after separation, in a 25 L cylindrical (diameter 300 mm, height 600 mm) stainless steel container. Furthermore, two samples were withdrawn for the measurement of DO concentration and for the successive quality analyses, respectively. For this purpose, the latter sample was stored under dark in a hermetically sealed green screw cap glass bottle (250 mL) with a negligible headspace. Stripping was performed on the oil in the stainless steel container by bubbling dry nitrogen gas at ow rate of 1 L/min for 20 min. These treatment conditions, i.e., nitrogen ow rate and stripping time, were adopted as in previous lab scale trials [7] a ratio of 1:1 between treated oil volume and the total gas stripping volume allowed a strong DO concentration reduction (about 74% of the initial value). A nitrogen gas cylinder (200 atm) equipped with a pressure regulator and a stainless steel porous sparger placed at the bottom of the container was used for stripping. After stripping the DO concentration was immediately measured, then the oil was placed in a green screw cap glass bottle (250 mL) hermetically sealed with a negligible headspace and stored under dark until chemicals analyses. The same procedure was adopted for all the trials so to obtain eighteen samples to compare, i.e., 9 samples of non-stripped oil (non-SO) versus 9 samples of stripped oil (SO). As previous laboratory experience showed the DO was quickly consumed by the oil in 7 days [5], one week after the stripping treatment the measurement of DO concentration was repeated on the collected oil samples. At this time all the samples showed a DO concentration close to zero, thus the qualitative chemical analyses were performed. Thus, the effects of DO consumption on the oil quality as well as of the stripping treatment were assessed.

GC-MS analysis of volatile compounds was performed using an Agilent 7890 GC equipped with an Agilent 5975C inert XL MSD quadrupole mass spectrometer, after automated SPME sampling with PDMS/DVB/Carboxen 30 mm ber [11].

2.3 Sensory evaluation

Sensory evaluation of EVOO samples was performed by means of a panel consisting of by 10 panelists, trained to distinguish between similar samples. At this purpose ve preliminary sessions were performed with oil samples presenting low and high intensity of the main sensory descriptors of olive oil, both positive (fruity, bitter, pungency) or negative attributes (rancid, fusty, vinegary). A triangular sensory test (ISO 4120:2004, 2004) was applied to identify sensory differences among non-SO and SO samples.

2.4 Statistical analysis

Statistical signicance of the investigated treatment was evaluated through paired t-test with eight degrees of freedom, i.e., nine pairs to compare between non-SO versus SO samples.

3 Results and discussion

The DO into a lipid matrix is co-substrate of the autoxidation reactions and the amount of free radicals formed is strictly related to the amount of DO available [6]. Studies concerning a direct measurement of DO concentration in EVOO are rather limited. Ottaviani et al. [6] reported values ranging between 2.6 and 4.6 mg/L, measured on fresh and old oil samples of different origin. Sacchi et al. [12] recorded values between 1 and 2.5 mg/L and discuss these differences in terms of more or less intense stirring undergone by oils on different bottling lines. Parenti et al. [5] measured considerably higher concentrations, i.e., about 9 mg/L, on EVOO samples collected immediately after extraction. As reported in Table 1 the DO concentrations measured during the present experiment are slightly lower, i.e., a mean value of about 5.4 mg/L. Probably, this variability in the DO contents was dependent on different conditions such as the types of systems used for the oil extraction along with the related operational conditions as well as on the practices, e.g., ltration and bottling, that are performed before the DO measurements and which should imply a different degree of oxygenation. As reported in Table 1 the stripping treatment allowed a marked signicant removal of DO, i.e., a percentage reduction of 50%. One week after production, at the time of chemical analyses, both the non-SO and SO samples showed the same DO concentration that was close to zero, i.e., an average concentration of about 0.008 mg/L. This indicates that in both the cases fast oxygen consumption occurs. This result conrms what was already showed by
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2.2 Chemical analyses

The DO concentration in EVOO was measured by a portable oxygen analyzer model InPro 9500-M701 (Mettler-Toledo S.p.A, Italy). Each determination was carried out in duplicate at the same temperature, either heating at 288C or refrigerating the sample by means of a water bath. The instrument was calibrated with reference to the atmospheric pressure before each new measurement. The concentrations were expressed as milligram per liter. Free acidity (FA), peroxide value (PV), and UV specic extinction coefcients were determined according to the European ofcial method of analysis (European Regulation EEC 2568/91, 1991) [8]. Total chlorophylls and carotenes concentration (mg/kg) were determined spectrophotometrically according to MinguezMosquera et al. [9]. Total hydrophilic phenols were extracted by liquidliquid partition with an 80:20 methanol/water solution. The total phenols content of the extract was determined by the FolinCiocalteau spectrophotometric method at 765 nm, using gallic acid as the calibration standard [10].
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Table 1. Effects of stripping treatment on EVOO standard quality indices, DO, minor components concentration and volatiles compounds concentration
Parameter DO (mg/L) FA (%) PV (meqO2/kg) K232 K270 DK Carotenes (mg/kg) Chlorophylls (mg/kg) Total phenols (mg/kg) Volatiles compounds (mg/kg) Ethyl acetate 3-Hexen-1-ol acetate Methyl salicylate Pentanal Pent-1-en-3-one Hexanal (E)-Hex-2-enal Pent-1-en-3-ol Hexyl alcohol (Z)-Hex-3-enol Total
a)

Non-SOa) 5.42 (0.75) 0.3 (0.02) 7.32 (0.91) 1.17 (0.04) 0.09 (0.00) 0.01 (0.00) 6.66 (0.61) 6.64 (1.9) 336.37 (63.94) 320 (143.7) 2705.56 (1529.58) 1053.33 (433.47) 137.78 (50.94) 727.78 (161.85) 936.67 (203.9) 24903.33 (3439.75) 1062.22 (165.74) 1964.44 (720) 1698.89 (331.57) 35510 (2111.66)

SOa) 2.48 (0.88) 0.3 (0.04) 6.23 (0.99) 1.16 (0.02) 0.09 (0.00) 0.01 (0.00) 6.77 (0.69) 7.56 (2.41) 310.26 (67.25) 338.89 (124.14) 2977.78 (1770.97) 1032.22 (430.14) 138.89 (48.59) 692.22 (202.59) 865.56 (203.54) 21044.44 (5250.75) 1130 (130.77) 2211.11 (715.53) 1850 (391.22) 32281.11 (4650.08)

Mean differencea) 2.94 (0.93) 0.00 (0.04)ns 1.59 (0.54) 0.01 (0.06)ns 0.00 (0.00)ns 0.00 (0.00)ns 0.11 (0.49)ns 0.92 (2.66)ns 26.11 (8.45) 18.89 (110.05)ns 272.22 (576.97)ns 21.11 (74.41)ns 1.11 (34.8)ns 35.56 (293.9)ns 71.11 (127.22)ns 3858.89 (3480.78) 67.78 (144.46)ns 246.67 (676.76)ns 151.11 (383.09)ns 3228.89 (3180.47)

Data are mean of 9 independent replicated experiments; SDs are showed in brackets; mean differences are signicantly different from 0 according to paired t-test ( p 0.05, p 0.01, ns, not signicant).

Parenti et al. [5] but it is in disagreement with Sacchi et al. [12], which reported much slower DO consumption kinetics, i.e., the DO concentration reduction in the oil from an average value of about 1.8 mg/L to a steady value of about 0.5 mg/L in 90 days. This discrepancy could be related both to the differences of the oils used for the tests and to the higher initial DO concentration of the oils used in the present experiment that could imply faster oxygen consumption. Surely, further studies are necessary to better understand the DO consumption kinetics as related to the EVOO physical-chemical characteristics and the storage conditions. One week after production, i.e., at a zero DO concentration, the SO showed a signicantly lower PV (about 22%) probably because of lesser formation of free radicals [6]. On the contrary, no signicant differences were recorded for both K232 and K270 specic extinction coefcients. In agreement with Kanner and Rosenthal [13] these last indexes were affected in a marked way only in advanced phases of oxidation, so that an immediate effect of the tested treatment was not detectable. Pigment concentrations, of both chlorophylls and carotenes, were not signicantly affected by stripping (Table 1), whereas total hydrophilic phenols compounds showed slightly lower concentration in the SO. According to Ottaviani et al. [6] we can suppose that the molecular motion created by the bubbling nitrogen favors collisions between radicals and antioxidants, so to determine the radicals scavenge and the consequent decrement of
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phenols. Therefore, the nal effect was a slight reduction in phenols in the SO. As we could expect the stripping treatment results to a loss of aromatic compounds (Table 1). Signicant differences were detected both for total volatiles concentration and for (E)-hex-2-enal. However, the recorded decrease was rather limited as it accounted for about 1015% of the control value and according to the results of the triangular sensory test (Table 2) it was not detectable by a sensory point of view. In fact, the panelists were able to
Table 2. Triangle test on non-SO and SO paired samples
Pairs of samples 1 2 3 4 5 6 7 8 9 Mean Proof assayed 10 10 10 10 10 10 10 10 10 10

Successesa) 7 5 2 4 3 3 5 2 4 4

Significance 0.05 ns ns ns ns ns ns ns ns ns

ns, Not signicant. a) Minimum number of success at p 0.05 7.

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signicantly distinguish between non-SO and SO only for one out of the nine pairs of samples, while on the average only four out of the ten panelists were able to identify a difference between the assayed samples.

4 Conclusions
The stripping treatment results to two divergent effects on EVOO quality. On one side, it determines a negligible loss of phenolic and aromatic compounds. On the other side, it allows a substantial reduction in the DO concentration and peroxides formation. The effectiveness of stripping the oil by nitrogen to expel DO was proven and suggests the possible implementation of this treatment at the end of EVOO extraction chain. The authors have declared no conict of interest.

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[4] Angerosa, F., Servili, M., Selvaggini, R., Taticchi, A. et al. Volatile compounds in virgin olive oil: occurrence and their relationship with the quality. J. Chromatogr. A 2004, 1054, 1731. [5] Parenti, A., Spugnoli, P., Masella, P., Calamai, L., Inuence of the extraction process on dissolved oxygen in olive oil. Eur. J. Lipid Sci. Technol. 2007, 109, 11801185. [6] Ottaviani, M. F., Spallaci, M., Cangiotti, M., Bacchiocca, M., Ninfali, P., Electron paramagnetic resonance investigations of free radicals in extra virgin olive oils. J. Agric. Food Chem. 2001, 49, 36913696. [7] Parenti, A., Spugnoli, P., Recchia, L., Masella, P., Stripping technique to improve olive-oil conservability. in: Proceedings of the XXX CIOSTA-CIGR V Congress, D.E.I.A.F.A. ` Universita degli studi di Torino (Italy), 2003, pp. 14551458. [8] European Commission Regulation. EEC/1989/2003 of 6 November 2003 amending Regulation (EEC) No. 2568/91 on the Characteristics of olive oil and olive-pomace oil and on the relevant methods of analysis. Off. J. Eur. Communities 2003, L295, 5777. [9] Mnguez-Mosquera, M. I., Gandul-Rojas, B., Garrido Fernandez, J., Gallardo-Guerrero, M. L., Pigments present in virgin olive oil. J. Am. Oil Chem. Soc. 1990, 67, 192196. [10] Capannesi, I., Palchetti, Mascini, M., Parenti, A., Electrochemical sensor and biosensor for polyphenols detection in olive oils. Food Chem. 2000, 71, 553562. [11] Masella, P., Parenti, A., Spugnoli, P., Calamai, L., Inuence of vertical centrifugation on extra virgin olive oil quality. J. Am. Oil Chem. Soc. 2009, 86, 11371140. [12] Sacchi, R., Savarese, M., Del Regno, A., Paduano, A. et al. Shelf life of vegetable oils bottled in different scavenging polyethyleneterephthalate (pet) containers. Package Technol. Sci. 2008, 21, 269277. [13] Kanner, J., Rosenthal, I., An assessment of lipid oxidation in foods. Pure Appl. Chem. 1992, 64, 19591964.

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