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Rapid extraction and determination of phenols in extra virgin olive oil
G. Charalambous (Ed.), Food Flavors: Generation, Analysis and Process Influence © 1995 Elsevier Science B.V. All rights reserved
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Rapid extraction and determination of phenols in extra virgin olive oil. F. Favati, G. Caporale, E. Monteleone and M. Bertuccioli Dipartimento di Biologia, Difesa e Biotecnologie Agro-Forestali, Sez. Scienze e Tecnologie Alimentari, Universita degli Studi della Basilicata, Via N. Sauro 85, 85100 Potenza, Italy Abstract Solid Phase Extraction (SPE) was utihsed as an alternative technique for the rapid recovery of the phenolic fraction from virgin olive oil. Different kind of sorbent materials (Cig, CN and Florisil) were tested and the validity of the SPE methodology was tested against two commonly utilised phenol extraction techniques. Statistical analysis of the analytical data showed that SPE can represent a reliable alternative to the traditional procedures. SPE with CN columns was also utihsed as an enrichment technique for the HPLC analysis of the phenolic fraction in 12 virgin olive oils coming from Greece, Italy and Spain. Eleven unidentified peaks were found to be always present in the chromatograms of the 12 oils. The relationships between these peaks and the total phenol content, the oil oxidative stability and some of the perceived properties of the virgin olive oilflavour(bitter and astringent) were statistically evaluated.
1. INTRODUCTION The overall quality of olive oil is influenced by several factors, and among these autoxidation represents a process that can deeply affect the flavour and stability of the oil. However, autoxidation can be partially inhibited by the presence of natural antioxidant as tocopherols and polyphenols. Compared to other vegetable oil, the tocopherol content in olive oil is relatively low, 100-200 ppm (1,2); conversely, polyphenol content can usually range from 50 to 500 ppm expressed as cafifeic acid (3). The olive fiuit contains 2-5% of polyphenols, essentially as glucosides, and the main phenolic compound is oleuropein, an heterosidic ester of elenohc acid and
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3,4-dihydroxyphenylethanol. Oleuropein content increases during the growth stage of the finit and then decreases in the ripening stage (4). During the oUve oil extraction process, mainly in the pressing and malaxing stage, oxidation and hydrolysis reactions cause the appearance of simpler phenolic compound. Among the identified phenolic compound are: tyrosol, hydroxytyrosol, homo vanillic acid, vanillic acid, /7-hydroxyphenilacetic acid, protochatechuic acid, p-hydroxybenzoic acid, caffeic acid, siringic acid, ferulic acid, /7-coumaric acid, and cimiamic acid (5,6). Tyrosol and hydroxytyrosol are the most abundant, however the latter seems to have a much higher antioxidant activity (7-10). Besides its role in the oil stability, the importance of the polyphenol fraction arises also from its contribution to the flavour and nutritional value of the extra virgin olive oil (3,11,12). The study of the phenolfractionin the olive oil is therefore of great importance and much research has been and is carried out on this group of compounds. One of the critical point in the assessment of the total phenol content in the virgin olive oil, or in the identification of the single components, is the extraction procedure of the polyphenols from the oil. Nowadays the recovery of the polyphenols is mainly based on liquid-liquid partitioning techniques that are long and tedious. Solid phase extraction (SPE) methodology is currently intensively developed for the isolation or concentration of analytes from a wide variety of substrates. SPE has already been utilised in olive oil analysis for the extraction of pigments (13) and for the extraction of nonvolatile bitter constituents (14,15). The objective of this work was to evaluate the vahdity of a rapid phenol extraction method based on SPE in comparison with two other currently utilised methods. Three popular types of sohd support, Cjg (octadecyl-bonded sihca), Florisil (Mg2Si03) and Cyano (CN) were tested for their capacity of retaining the analytes of interest, therefore allowing their extraction from the virgin olive oil. The method was then utilised in a case study where the total phenol content was correlated to the sensory evaluation of virgin olive oils coming from different countries. Furthermore, the oxidative stability of these oils was correlated to the HPLC analysis data of the phenolic extracts obtained by SPE. 2. MATERIALS and METHODS 2.1 Reagents and standards All reagents were analytical or HPLC grade and were obtained from Carlo Erba Reagenti (Milano, Italy). Phenol standards 2-(p-Hydroxyphenil)-3 ethanol and cinnamic acid were purchasedfromAldrich Chem. Co. (Milano, Italy), while protochatechic acid, p-hydroxybenzoic acid, vanillic acid, caffeic acid, siringic
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acid, /?-coumaric acid and ferulic acid were obtained from Sigma Co. (Milano, Italy); gallic acid, utilised as internal standard in the HPLC analyses was purchased from Fluka Chimica (Milano, Italy). The peaks were identified on the basis of the retention time and taking their spectra over the 190-367 nm range. 2.2 Phenol extraction procedures 2.2.1 Method 1 The oil (50 g) was diluted in 50 mL of hexane and the solution was washed three times with 30 mL of a methanol/water solution (60:40). Each time the mixture was shaken for 2 min before allowing the two phases to separate. The methanolic extracts were then washed with 50 mL of hexane before being combined and brought up to 100 mL in a volumetric flask (Figure 1). 2.2.2 Method 2 Ten mL of a methanol/water solution (80:20) plus 0.2 mL of Tween 20 (Sigma Co., Milano, Italy) were added to 10 g of oil and mixed with an Ultra-Turrax T25 at 15,000 rpm for 1 min. The methanolic extract was then separated by centrifiigation at 5,000 rpm for 10 min and recovered with a pasteur pipette. This procedure was repeated two more times and the 3 extracts combined. In order to separate any remaining oil, the solution was stored at -20°C for 24 h and then the methanolic extract was transferred to a 25 mL volumetric flask and brought up to volume (Figure 2). 2.2.3 Methods Phenols were extracted from the oil utilising a sohd phase extraction cartridge (6 mL volume) packed with Ig of Cig material (J.T. Baker, Milano, Italy). The oil (1 g) was diluted in 10 mL of hexane and loaded onto the column previously conditioned with 2x5 mL methanol and 2x5 mL of hexane. In order to remove the non polar fraction, the column was washed with 3x5 mL of hexane and the recovery of the analytes of interest was obtained eluting with 2x5 mL of methanol. The extract was collected in a 10 mL volumetric flask and brought up to volume (Figure 3). 2.2.4 Method 4 Phenols were extracted from the oil utilising a sohd phase extraction cartridge (6 mL volume) packed with Ig of CN or Florisil material (Varian, Segrate-Mi, Italy). The column was conditioned with 4x5 mL methanol and 2x5 mL of hexane. Afterwards, 5 g of virgin olive oil, diluted in 50 mL of hexane, were loaded onto the column. Because of the large sample volume, a 25 mL empty
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50 g of Oil + 50 mL of Hexane
^
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Phenol extraction with 30 mL of MeOH/H20 (60:40)
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^
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^
Extracted phenols - Total required time: 2 hours
Figure 1. Scheme of the main steps of the polyphenol extraction method 1.
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10 g of Oil + 10 mL of MeOH/H20 (80:20) + 0.2 mL of Tween 20 ^
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Extracted phenols - Total required time: 25 hours
Figure 2. Scheme of the main steps of the polyphenol extraction method 2.
434
Conditioning of a SPE Cjg column (1 g of sorbent material) with 2 x 5 mL of MeOH and 2 x 5 mL ofHexane ^
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Extracted phenols - Total required time: 15 minutes
Figure 3. Scheme of the main steps of the polyphenol extraction method 3.
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Conditioning of a SPE CN or FL column (1 g of sorbent material) with 4 x 5 mL of MeOH and 2 x 5 mL of Hexane
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Extracted phenols - Total required time: 30 minutes
Figure 4. Scheme of the main steps of the polyphenol extraction method 4.
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reservoir (Varian, Segrate-Mi, Italy) was attached to the cartridge. In order to remove the non polar fraction, the column was washed with 25 mL of hexane and the recovery of the analytes of interest was obtained eluting with 4x5 mL of methanol. The extract was collected in a 20 mL volumetric flask and brought up to volume (Figure 4). 2.3 Phenol content determination The phenol content of the extracts obtained with the different procedures was determined colorimetrically as follows: 2 mL of the phenol containing solution were transferred into a 20 mL volumetric flask and 5 mL of distilled water were added, followed by 0.5 mL of Folin-Ciocalteau reagent (Carlo Erba Reagents, Milano, Italy). After 3 min, 4 mL of a 10% solution of sodium carbonate were added, the volume was brought up to 20 mL with distilled water and the flask was stored in the dark. After 90 min the solution was filtered through a 0.2 |Lun filter (Alltech, Milano, Italy) and the absorbance read at 765 nm. The total phenol content of the samples was expressed as |ig tyrosol/g oil. 2.4 Oil samples The proposed SPE extraction methodology was initially tested utiUsing a commercial mixture of refined seed oils (Friol-OIO, Unil. It., Milano, Italy) not containing any detectable amount of phenols. For the tests the oil was spiked with 3 known amount of tyrosol, namely 110, 330 and 550 jug tyrosol/g oil. Table 1 Main characteristics of the olives utihsed to produce some of the tested oils. Country of origin
Greece Italy Spain Spain
Variety
Coroneiki Coratina Picual Arbequina
Ripening stage Unripe
Ripe
Overripe
+ + +
+ + + +
+ + + +
4-
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Further tests were conducted on olive oil samples coming from Greece, Spain and Italy. The oils were obtained from olives of selected varieties and at 3 different ripening stages (Table 1). The olives were delivered to local extraction plants and processed within 24 hours. Another set of olive oil samples was produced in our pilot plant. Olives of the Maiatica variety at a ripe stage were processed in six different ways according to the scheme reported in Table 2. A disk mill was utihsed for the milling process, pressing was performed at 250 atm and a disk centrifiige was utihsed for the separation and recovery of the oil. Table 2 Scheme oiMaiatica olives processing procedures. Procedure Leaf n. removal
1 2 3 4 5 6
+ + + + + +
Milling
+ + + + +
Malaxing at 30°C (min)
10 20 35 50
Centrifiigation Pressing 250 atm for 45 (4,,000 rpm) min
+ + + 4-
+ +
+ 4-
+ + + +
2.5 Stability test A Rancimat apparatus (Methrohm Co. Basel, Switzerland) was utilised to conduct the accelerated automatic Swift test. The air flow was set at 20 L/h and the temperature at 120°C. The results were expressed as induction time (hours). 2.6 HPLC analysis The phenolic extracts were analysed utilising a Varian HPLC system comprising a 9010 pump and a 9065 Polychrom UV diode array detector (Varian, Segrate-Mi, Italy). The methanolic extracts were injected onto a Supelcosil LC18 5|Lim column (150 mm x 4.6 mm) (Supelchem, Milano, Italy) equipped with a guard column holding a 10 mm x 4.6 mm Cjg guard cartridge (Alltech, Milano,
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Italy). The elution of the analytes from the column was obtained with a slight modification of the method of Montedoro et al. (5). The mobile phase consisted of acetic acid 0.2% in water (pH 3.1) and methanol; the two eluents were mixed according to the gradient reported in Table 3. The flow rate was set at 1 mL/min and the detection of the phenols was achieved at 278 nm. Standard curves were obtained injecting 20 |LIL of a solution containing the following compounds: tyrosol, cinnamic acid, protochatechic acid, phydroxybenzoic acid, vanillic acid, cafifeic acid, siringic acid, /7-coumaric acid, ferulic acid and gallic acid; the latter was utilised as internal standard. Calibration curves were constructed for concentrations of each of the above compounds rangingfrom0.5 to 10 ng/|LiL. Table 3. Mobile phase gradient conditions utilised in the HPLC analysis of the phenolic extracts. Solvent A = acetic acid 0.2% in water (pH 3.1); solvent B = methanol. Step
Time
1 2 3 4 5 6 7
0 2 10 20 30 60 65
Flow (mL/min) Solvent A (%) Solvent B(%)
][ ][ ]I ][ ]I ]I ]I
95
95 75 60 50 0 0
5 5 25 40 50 100 100
2.7 Sensory analysis The intensity of the attributes bitter and astringent was determined on the Italian, Spanish and Greek oils. After an adequate training period, the panel (12 judges) evaluated the 12 oils in triphcate according to an incomplete randomised block design (16). The samples were randomly presented, utihsing 20 mL of oil in a 50 mL glass, and 4 oils were evaluated in each session with the intensity of each attribute being measured on a scale of 1 to 9.
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2.8 Statistical analysis ANOVA analysis was performed on the data utilising the STATGRAPHICS software program. The multivariate analysis of the relationship among the data was carried out according to the Latent Variable analysis (PLS) utihsing the statistical package UNSCRAMBLER EX Ver. 4.0 (CAMO Co, Norway). 3. RESULTS and DISCUSSION The extraction procedures actually utilised for the recovery of the phenol fractionfromvirgin olive oil are long and tedious. A literature survey showed that they are mainly based on the use of methanol as the extracting solvent, eventually added with water in different percentages. However, there is not an agreement on the optimal ratio methanol/water and in some cases also ethanol was utilised instead of methanol (5-7, 9-11, 17-21). Among the procedures found in the literature, two extraction methods were chosen in order to test the validity of SPE for polyphenols recovery. These methods are reported in Figures 1 and 2. Nowadays, SPE methodology is more aad more applied for the isolation or concentration of analytes from a wide variety of substrates. However, there are no reports in the literature evaluating the vahdity of this technique for the polyphenols extractionfromvirgin olive oil. Octadecyl-bonded silica has been utilised as the sorbent material in the recovery of non-volatile bitter constituents from virgin olive oil (14,15); fiirthermore, Cjg cartridges have been employed for the isolation of the polyphenol fraction in wine (22). On the basis of these data, in a previous work (23) we chose to test this kind of sorbent for the extraction of polyphenols from virgin olive oil. SPE was initially performed utihsing columns containing Ig of Ci8 sorbent material and the first part of the study was devoted to the optimisation of the SPE parameters, like solvents to be utilised, maximum amount of oil that could be loaded, maximum amounts of analytes that could be retained, etc. The optimised extraction methodology, named Method 3, is reported in Figure 3. Further studies were then conducted, utilising tyrosol as model compound, to assess the vahdity of the proposed method. A commercial mixture of refined seed oil (Friol-OIO), not containing any detectable amount of phenols, was utilised to test this SPE procedure vs. Methods 1 and 2. After spiking with known amount of tyrosol (110, 330 and 550 ^ig tyrosoL/g of oil), the Friol was extracted according to the three methodologies. The extraction efficiencies were assessed and compared applying the following procedure: for each spiking level five analyses were performed independently by two operators, for a total of ten rephcates for
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each tyrosol concentration and for each extraction methodology. The acquired data were then statistically analysed by ANOVA as reported in Table 4. The results showed that for a tyrosol content ranging from 110 to 550 fig /g oil, the three methods gave comparable results. Therefore the SPE method was tested also on commercial samples of extra virgin olive oil coming from different countries. Also in this case the SPE method was evaluated in comparison with Methods 1 and 2 (Table 4). The analytical results indicated that the total phenol content in olive oil could be reliably assessed utilising the proposed SPE methodology;fiirthermore,the time required for the extraction of phenols with Method 3 is significantly shorter and in comparison with the two other methods is also possible to handle several samples at the same time, especially utilising the commercially available multi-extraction units for SPE (23). To understand the influence phenols on the characteristics of the virgin olive oil, it is of major important to be able to discriminate and identify the various compounds that constitute the phenohcfraction.In order to achieve this goal the phenols are generally extracted according to Method 1 or 2 and then analysed by HPLC technique. However, prior to the analysis the extract must be concentrated and this is usually accomplished working at reduced pressures and at mild temperatures. Perrin reports that this is a very critical step, during which from 35 to 75% of the phenols, and specially the hydroxytyrosol, can be degraded (8). Because of these problems, it was tried to directly utilise the extracts obtained by Ci8 SPE for the assessment of the phenohc composition by HPLC analysis, but the amoimt of phenols in the extract was not high enough to allow the analytical determination without concentrating the sample. In order to have an higher phenol concentration in the extract it would have been necessary to load onto the Cig cartridge much more oil, but this was not possible because the retention capability of the column would have been exceeded. Other type of sorbent materials have found utilisation in the extraction of polar analytes from lipid matrices and among these we tested Florisil and CN type of cartridges. In order to obtain an effective concentration procedure, maximisation of the amount of the oil sample and minimisation of the eluting solvent are required and working with commercial cartridges holding 1 g of sorbent material the best result were obtained when the CN phase was used. The extraction method was then optimised for a cartridge loading of 5 g of virgin olive oil (Figure 4). In order to test the procedure, the polyphenol content of virgin olive oils coming from 3 different countries was assessed. For each oil the analysis was performed on samples produced from olives at different ripening stage, for a total of 12 samples (Table 1). Further analysis were also conducted on oil samples
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produced from olives of the Maiatica cultivar processed in 6 different ways (Table 2). The analysis results are reported in Table 5 were the data obtained with the Method 4 are tabulated with those obtained determining the total phenol content on the same samples but according to the procedure of Method 2. The results show that the two methods are comparable and only for the oils having an higher phenol content there is a slight difference in the results. However, Method 2 gave lower recoveries also in the study conducted utilising tyrosol as model compound, as reported in Table 4. Table 5 Total polyphenol content as determined by Method 2 and Method 4 in virgin olive oils. Numbers nQ?^ Maiatica cultivar refer to the olive processing procedure as reported in Table 2. Total polyphenols (|ig tyrosol/g oil) Cultivar
Coratina Coratina Coratina Coroneiki Coroneiki Coroneiki Picual Picual Picual Arbequina Arbequina Arbequina Maiatica (\) Maiatica (2) Maiatica (3) Maiatica (4) Maiatica (5) Maiatica (6)
Ripening stage
unripe ripe overripe unripe ripe overripe unripe ripe overripe unripe ripe overripe npe ripe ripe npe ripe ripe
Method 2
Method 4
302 272 251 112 150 147 172 174 153 60 50 39 108 240 252 223 234 212
447 390 328 117 144 144 183 190 142 47 35 16 90 234 286 236 230 173
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Because of the amount of oil extracted, the phenol concentration in the extracts obtained utilising the CN cartridge was much higher compared to that of the extracts recovered utilising the Cjg cartridge. Therefore the CN extracts could analysed without any concentration. All the extracts obtained from the above oils were analysed by HPLC and in Figure 5 is shown the chromatogram of one of the Greek oils (A). As a comparison, a chromatogram of the extract obtained with Method 2 is shown in the same Figure (B). The chromatographic profiles are very similar, with only slight differences. In the chromatograms of the tested oil it was not possible to identify any peak corresponding to the available standards; however, it was possible to point out the constant presence of certain peaks in all the chromatograms. These peaks are identified in Figure 5 (A) with numbers from 1 to 11. As already mentioned, the polyphenols can have a positive influence on the resistance of the oil to oxidative processes. Therefore the relationship between the assessed polyphenol content values and the oxidative oil stability determined with the Rancimat was studied. In Table 6 the total phenol content of the 12 oils is reported with the determined Rancimat values. In Figure 6 the Rancimat value of the 12 oils is plotted as a fimction of the total polyphenol content; linear regression was performed on these values and the low correlation coefficient shows that the oxidative stability is not correlated to the total phenol content, at least in the tested oils. This is because not all the compounds of the olive oil that react with the Folin-Ciocalteau reagent can be directly related to the oil stability. The relationship between the 11 detected peaks and oil stability, as well as total polyphenol content, was analysed using the Latent Variable analysis (PLSl). The values relative to the area counts of the 11 detected peaks were set as "x matrix," while total polyphenol content and Rancimat values were the "y vector". Figure 7 shows that the stability of the tested oils, represented by the Rancimat values, is related only to the peak identified by the number 1, while the total phenol content is strictly related to the peaks defined with number 3,2, 5 and 4 (Figure 8). The total variation explained by the two models with two factors was 50% and 95% respectively. The relationship between peak number 1 and the oxidative stability of the oils is represented in Figure 9; the correlation between the data is quite high with a correlation coefficient of 0.8. Polyphenols are important also for their contribution to some of the perceived properties of the virgin olive oil flavour, like bitter and astringent. The relationship between the 11 chosen peaks ("x matrix") and the intensity of the attributes bitter and astringent ("y matrix") was studied by PLS2 metiiod. As shown in Figure 10, the bitter intensity is related to the peak number 6, while the astringent intensity is related to peaks 2,3,4 and 5.
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Figure 10. PLS relationship: selected chromatographic peaks and selected perceived properties {bitter and astringent) of the virgin olive oil flavour.
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4. CONCLUSIONS The use of SPE for the recovery of the polyphenolfractionfromvirgin olive oil can represent a valuable alternative to the traditional extraction procedures. The results have shown that Cig cartridges can be utilised if the final goal is a rapid determination of the total polyphenol content in the oil. SPE can also be applied for the concentration of the analytes of interest, and in this case CN cartridges should be utihsed. This extraction methodology, besides being rapid, minimises possible degradation process that could affect the polyphenols; this is important because this study has pointed out that a relationship exists between certain components that are found in the phenohc extract and some important characteristics of the virgin olive oil (oxidative stability and perceived properties). Further studies will be conducted with the aim to chemically identify these components.
5. REFERENCES 1 E. Tiscomia, M. Forina and F. Evangehsti, Riv. Ital. Sost. Grasse, 59 (1982) 519 2 F.D. Gunstone, J.L. Harwood and F.B Padley (eds). The Lipid Handbook, Chapman and Hall, New York, 1986. 3 A. Vazquez Roncero, Rev. Fran?. Corps Gras, 25 (1978) 21. 4 J. Amiot, A. Fleuriet and J. J. Macheix, Phytochemistry, 28 (1989) 67. 5 G. Montedoro, M. Servih, M. Baldioh and E. Miniati, J. Agric. Food Chem., 40(1992)1571. 6 M. Akasbi, D. W. Shoeman and A. Saari Csallany, JAOCS, 70 (1993) 367. 7 T. Gutfinger, JAOCS, 58 (1981) 966. 8 J.L. Perrin, Rev. Fran9. Corps Gras, 39 (1992) 26. 9 M. Tsimidou, G. Papadopoulos and D. Boskou, Food Chem., 45 (1992) 141. 10 M.LI Forcadell, M. Comas, X. Miquel and M.C. De La Torre, Rev. Fran?. Corps Gras, 34 (1987) 547. 11 N. Cortesi and Fedeh E., Riv. Ital. Sost. Grasse, 60 (1983) 341. 12 G. Charalambous and G.E. Inglett (eds), Flavor of Foods and Beverages Chemistry and Technology, Ac. Press, New York, 1978, 247-281. 13 M.I. Minguez-Mosquera, B. Gandul-Rojas and M.L. Gallardo-Guerrero, J. Agric. Food Chem., 40 (1992) 60. 14 F. Gutierrez, M.A. Albi, R. Palma, J.J. Rios and J. M. Olias, J. Food Sc, 54 (1989) 68.
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15 F. Gutierrez Resales, S. Perdiguero, Gutierrez R. and J.M. Olias, JAOCS, 69 (1992)394. 16 M.A. Amerine and E.B. Roessler, Wines: their sensory evaluation, W. H. Frem Company, New York, 1983. 17 E. Ragazzi and G. Veronese, Riv. Ital. Sost. Grasse, 50 (1973) 443. 18 V. Vacca, P. Fenu, M. A. Franco and G. Sferlazzo, Riv. Ital. Sost. Grasse, 70 (1993) 595. 19 G. Papadopoulos and D. Boskou, JAOCS, 68 (1991) 669. 20 M. Brenes-Balbuena, P. Garcia-Garcia and A.Garrido-Femandez, J.Agric. Food Chem., 40 (1992) 1192. 21 P. Goupy, A. Fleuriet, M.J. Amiot and J.J. Macheix, J.Agric. Food Chem., 39(1991)92. 22 R. Di Stefano and S. Guidoni, Vignevini, 16 (1989) 47. 23 F. Favati, G. Caporale and M. Bertuccioh, Grasas y Aceites, in press.
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Rapid extraction and determination of phenols in extra virgin olive oil. F. Favati, G. Caporale, E. Monteleone and M. Bertuccioli Dipartimento di Biologia, Difesa e Biotecnologie Agro-Forestali, Sez. Scienze e Tecnologie Alimentari, Universita degli Studi della Basilicata, Via N. Sauro 85, 85100 Potenza, Italy Abstract Solid Phase Extraction (SPE) was utihsed as an alternative technique for the rapid recovery of the phenolic fraction from virgin olive oil. Different kind of sorbent materials (Cig, CN and Florisil) were tested and the validity of the SPE methodology was tested against two commonly utilised phenol extraction techniques. Statistical analysis of the analytical data showed that SPE can represent a reliable alternative to the traditional procedures. SPE with CN columns was also utihsed as an enrichment technique for the HPLC analysis of the phenolic fraction in 12 virgin olive oils coming from Greece, Italy and Spain. Eleven unidentified peaks were found to be always present in the chromatograms of the 12 oils. The relationships between these peaks and the total phenol content, the oil oxidative stability and some of the perceived properties of the virgin olive oilflavour(bitter and astringent) were statistically evaluated.
1. INTRODUCTION The overall quality of olive oil is influenced by several factors, and among these autoxidation represents a process that can deeply affect the flavour and stability of the oil. However, autoxidation can be partially inhibited by the presence of natural antioxidant as tocopherols and polyphenols. Compared to other vegetable oil, the tocopherol content in olive oil is relatively low, 100-200 ppm (1,2); conversely, polyphenol content can usually range from 50 to 500 ppm expressed as cafifeic acid (3). The olive fiuit contains 2-5% of polyphenols, essentially as glucosides, and the main phenolic compound is oleuropein, an heterosidic ester of elenohc acid and
430
3,4-dihydroxyphenylethanol. Oleuropein content increases during the growth stage of the finit and then decreases in the ripening stage (4). During the oUve oil extraction process, mainly in the pressing and malaxing stage, oxidation and hydrolysis reactions cause the appearance of simpler phenolic compound. Among the identified phenolic compound are: tyrosol, hydroxytyrosol, homo vanillic acid, vanillic acid, /7-hydroxyphenilacetic acid, protochatechuic acid, p-hydroxybenzoic acid, caffeic acid, siringic acid, ferulic acid, /7-coumaric acid, and cimiamic acid (5,6). Tyrosol and hydroxytyrosol are the most abundant, however the latter seems to have a much higher antioxidant activity (7-10). Besides its role in the oil stability, the importance of the polyphenol fraction arises also from its contribution to the flavour and nutritional value of the extra virgin olive oil (3,11,12). The study of the phenolfractionin the olive oil is therefore of great importance and much research has been and is carried out on this group of compounds. One of the critical point in the assessment of the total phenol content in the virgin olive oil, or in the identification of the single components, is the extraction procedure of the polyphenols from the oil. Nowadays the recovery of the polyphenols is mainly based on liquid-liquid partitioning techniques that are long and tedious. Solid phase extraction (SPE) methodology is currently intensively developed for the isolation or concentration of analytes from a wide variety of substrates. SPE has already been utilised in olive oil analysis for the extraction of pigments (13) and for the extraction of nonvolatile bitter constituents (14,15). The objective of this work was to evaluate the vahdity of a rapid phenol extraction method based on SPE in comparison with two other currently utilised methods. Three popular types of sohd support, Cjg (octadecyl-bonded sihca), Florisil (Mg2Si03) and Cyano (CN) were tested for their capacity of retaining the analytes of interest, therefore allowing their extraction from the virgin olive oil. The method was then utilised in a case study where the total phenol content was correlated to the sensory evaluation of virgin olive oils coming from different countries. Furthermore, the oxidative stability of these oils was correlated to the HPLC analysis data of the phenolic extracts obtained by SPE. 2. MATERIALS and METHODS 2.1 Reagents and standards All reagents were analytical or HPLC grade and were obtained from Carlo Erba Reagenti (Milano, Italy). Phenol standards 2-(p-Hydroxyphenil)-3 ethanol and cinnamic acid were purchasedfromAldrich Chem. Co. (Milano, Italy), while protochatechic acid, p-hydroxybenzoic acid, vanillic acid, caffeic acid, siringic
431
acid, /?-coumaric acid and ferulic acid were obtained from Sigma Co. (Milano, Italy); gallic acid, utilised as internal standard in the HPLC analyses was purchased from Fluka Chimica (Milano, Italy). The peaks were identified on the basis of the retention time and taking their spectra over the 190-367 nm range. 2.2 Phenol extraction procedures 2.2.1 Method 1 The oil (50 g) was diluted in 50 mL of hexane and the solution was washed three times with 30 mL of a methanol/water solution (60:40). Each time the mixture was shaken for 2 min before allowing the two phases to separate. The methanolic extracts were then washed with 50 mL of hexane before being combined and brought up to 100 mL in a volumetric flask (Figure 1). 2.2.2 Method 2 Ten mL of a methanol/water solution (80:20) plus 0.2 mL of Tween 20 (Sigma Co., Milano, Italy) were added to 10 g of oil and mixed with an Ultra-Turrax T25 at 15,000 rpm for 1 min. The methanolic extract was then separated by centrifiigation at 5,000 rpm for 10 min and recovered with a pasteur pipette. This procedure was repeated two more times and the 3 extracts combined. In order to separate any remaining oil, the solution was stored at -20°C for 24 h and then the methanolic extract was transferred to a 25 mL volumetric flask and brought up to volume (Figure 2). 2.2.3 Methods Phenols were extracted from the oil utilising a sohd phase extraction cartridge (6 mL volume) packed with Ig of Cig material (J.T. Baker, Milano, Italy). The oil (1 g) was diluted in 10 mL of hexane and loaded onto the column previously conditioned with 2x5 mL methanol and 2x5 mL of hexane. In order to remove the non polar fraction, the column was washed with 3x5 mL of hexane and the recovery of the analytes of interest was obtained eluting with 2x5 mL of methanol. The extract was collected in a 10 mL volumetric flask and brought up to volume (Figure 3). 2.2.4 Method 4 Phenols were extracted from the oil utilising a sohd phase extraction cartridge (6 mL volume) packed with Ig of CN or Florisil material (Varian, Segrate-Mi, Italy). The column was conditioned with 4x5 mL methanol and 2x5 mL of hexane. Afterwards, 5 g of virgin olive oil, diluted in 50 mL of hexane, were loaded onto the column. Because of the large sample volume, a 25 mL empty
432
50 g of Oil + 50 mL of Hexane
^
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<=i
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^
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^
Extracted phenols - Total required time: 2 hours
Figure 1. Scheme of the main steps of the polyphenol extraction method 1.
433
10 g of Oil + 10 mL of MeOH/H20 (80:20) + 0.2 mL of Tween 20 ^
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Figure 2. Scheme of the main steps of the polyphenol extraction method 2.
434
Conditioning of a SPE Cjg column (1 g of sorbent material) with 2 x 5 mL of MeOH and 2 x 5 mL ofHexane ^
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^
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^
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Figure 3. Scheme of the main steps of the polyphenol extraction method 3.
435
Conditioning of a SPE CN or FL column (1 g of sorbent material) with 4 x 5 mL of MeOH and 2 x 5 mL of Hexane
^
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^
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Figure 4. Scheme of the main steps of the polyphenol extraction method 4.
436
reservoir (Varian, Segrate-Mi, Italy) was attached to the cartridge. In order to remove the non polar fraction, the column was washed with 25 mL of hexane and the recovery of the analytes of interest was obtained eluting with 4x5 mL of methanol. The extract was collected in a 20 mL volumetric flask and brought up to volume (Figure 4). 2.3 Phenol content determination The phenol content of the extracts obtained with the different procedures was determined colorimetrically as follows: 2 mL of the phenol containing solution were transferred into a 20 mL volumetric flask and 5 mL of distilled water were added, followed by 0.5 mL of Folin-Ciocalteau reagent (Carlo Erba Reagents, Milano, Italy). After 3 min, 4 mL of a 10% solution of sodium carbonate were added, the volume was brought up to 20 mL with distilled water and the flask was stored in the dark. After 90 min the solution was filtered through a 0.2 |Lun filter (Alltech, Milano, Italy) and the absorbance read at 765 nm. The total phenol content of the samples was expressed as |ig tyrosol/g oil. 2.4 Oil samples The proposed SPE extraction methodology was initially tested utiUsing a commercial mixture of refined seed oils (Friol-OIO, Unil. It., Milano, Italy) not containing any detectable amount of phenols. For the tests the oil was spiked with 3 known amount of tyrosol, namely 110, 330 and 550 jug tyrosol/g oil. Table 1 Main characteristics of the olives utihsed to produce some of the tested oils. Country of origin
Greece Italy Spain Spain
Variety
Coroneiki Coratina Picual Arbequina
Ripening stage Unripe
Ripe
Overripe
+ + +
+ + + +
+ + + +
4-
437
Further tests were conducted on olive oil samples coming from Greece, Spain and Italy. The oils were obtained from olives of selected varieties and at 3 different ripening stages (Table 1). The olives were delivered to local extraction plants and processed within 24 hours. Another set of olive oil samples was produced in our pilot plant. Olives of the Maiatica variety at a ripe stage were processed in six different ways according to the scheme reported in Table 2. A disk mill was utihsed for the milling process, pressing was performed at 250 atm and a disk centrifiige was utihsed for the separation and recovery of the oil. Table 2 Scheme oiMaiatica olives processing procedures. Procedure Leaf n. removal
1 2 3 4 5 6
+ + + + + +
Milling
+ + + + +
Malaxing at 30°C (min)
10 20 35 50
Centrifiigation Pressing 250 atm for 45 (4,,000 rpm) min
+ + + 4-
+ +
+ 4-
+ + + +
2.5 Stability test A Rancimat apparatus (Methrohm Co. Basel, Switzerland) was utilised to conduct the accelerated automatic Swift test. The air flow was set at 20 L/h and the temperature at 120°C. The results were expressed as induction time (hours). 2.6 HPLC analysis The phenolic extracts were analysed utilising a Varian HPLC system comprising a 9010 pump and a 9065 Polychrom UV diode array detector (Varian, Segrate-Mi, Italy). The methanolic extracts were injected onto a Supelcosil LC18 5|Lim column (150 mm x 4.6 mm) (Supelchem, Milano, Italy) equipped with a guard column holding a 10 mm x 4.6 mm Cjg guard cartridge (Alltech, Milano,
438
Italy). The elution of the analytes from the column was obtained with a slight modification of the method of Montedoro et al. (5). The mobile phase consisted of acetic acid 0.2% in water (pH 3.1) and methanol; the two eluents were mixed according to the gradient reported in Table 3. The flow rate was set at 1 mL/min and the detection of the phenols was achieved at 278 nm. Standard curves were obtained injecting 20 |LIL of a solution containing the following compounds: tyrosol, cinnamic acid, protochatechic acid, phydroxybenzoic acid, vanillic acid, cafifeic acid, siringic acid, /7-coumaric acid, ferulic acid and gallic acid; the latter was utilised as internal standard. Calibration curves were constructed for concentrations of each of the above compounds rangingfrom0.5 to 10 ng/|LiL. Table 3. Mobile phase gradient conditions utilised in the HPLC analysis of the phenolic extracts. Solvent A = acetic acid 0.2% in water (pH 3.1); solvent B = methanol. Step
Time
1 2 3 4 5 6 7
0 2 10 20 30 60 65
Flow (mL/min) Solvent A (%) Solvent B(%)
][ ][ ]I ][ ]I ]I ]I
95
95 75 60 50 0 0
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2.7 Sensory analysis The intensity of the attributes bitter and astringent was determined on the Italian, Spanish and Greek oils. After an adequate training period, the panel (12 judges) evaluated the 12 oils in triphcate according to an incomplete randomised block design (16). The samples were randomly presented, utihsing 20 mL of oil in a 50 mL glass, and 4 oils were evaluated in each session with the intensity of each attribute being measured on a scale of 1 to 9.
439
2.8 Statistical analysis ANOVA analysis was performed on the data utilising the STATGRAPHICS software program. The multivariate analysis of the relationship among the data was carried out according to the Latent Variable analysis (PLS) utihsing the statistical package UNSCRAMBLER EX Ver. 4.0 (CAMO Co, Norway). 3. RESULTS and DISCUSSION The extraction procedures actually utilised for the recovery of the phenol fractionfromvirgin olive oil are long and tedious. A literature survey showed that they are mainly based on the use of methanol as the extracting solvent, eventually added with water in different percentages. However, there is not an agreement on the optimal ratio methanol/water and in some cases also ethanol was utilised instead of methanol (5-7, 9-11, 17-21). Among the procedures found in the literature, two extraction methods were chosen in order to test the validity of SPE for polyphenols recovery. These methods are reported in Figures 1 and 2. Nowadays, SPE methodology is more aad more applied for the isolation or concentration of analytes from a wide variety of substrates. However, there are no reports in the literature evaluating the vahdity of this technique for the polyphenols extractionfromvirgin olive oil. Octadecyl-bonded silica has been utilised as the sorbent material in the recovery of non-volatile bitter constituents from virgin olive oil (14,15); fiirthermore, Cjg cartridges have been employed for the isolation of the polyphenol fraction in wine (22). On the basis of these data, in a previous work (23) we chose to test this kind of sorbent for the extraction of polyphenols from virgin olive oil. SPE was initially performed utihsing columns containing Ig of Ci8 sorbent material and the first part of the study was devoted to the optimisation of the SPE parameters, like solvents to be utilised, maximum amount of oil that could be loaded, maximum amounts of analytes that could be retained, etc. The optimised extraction methodology, named Method 3, is reported in Figure 3. Further studies were then conducted, utilising tyrosol as model compound, to assess the vahdity of the proposed method. A commercial mixture of refined seed oil (Friol-OIO), not containing any detectable amount of phenols, was utilised to test this SPE procedure vs. Methods 1 and 2. After spiking with known amount of tyrosol (110, 330 and 550 ^ig tyrosoL/g of oil), the Friol was extracted according to the three methodologies. The extraction efficiencies were assessed and compared applying the following procedure: for each spiking level five analyses were performed independently by two operators, for a total of ten rephcates for
440
each tyrosol concentration and for each extraction methodology. The acquired data were then statistically analysed by ANOVA as reported in Table 4. The results showed that for a tyrosol content ranging from 110 to 550 fig /g oil, the three methods gave comparable results. Therefore the SPE method was tested also on commercial samples of extra virgin olive oil coming from different countries. Also in this case the SPE method was evaluated in comparison with Methods 1 and 2 (Table 4). The analytical results indicated that the total phenol content in olive oil could be reliably assessed utilising the proposed SPE methodology;fiirthermore,the time required for the extraction of phenols with Method 3 is significantly shorter and in comparison with the two other methods is also possible to handle several samples at the same time, especially utilising the commercially available multi-extraction units for SPE (23). To understand the influence phenols on the characteristics of the virgin olive oil, it is of major important to be able to discriminate and identify the various compounds that constitute the phenohcfraction.In order to achieve this goal the phenols are generally extracted according to Method 1 or 2 and then analysed by HPLC technique. However, prior to the analysis the extract must be concentrated and this is usually accomplished working at reduced pressures and at mild temperatures. Perrin reports that this is a very critical step, during which from 35 to 75% of the phenols, and specially the hydroxytyrosol, can be degraded (8). Because of these problems, it was tried to directly utilise the extracts obtained by Ci8 SPE for the assessment of the phenohc composition by HPLC analysis, but the amoimt of phenols in the extract was not high enough to allow the analytical determination without concentrating the sample. In order to have an higher phenol concentration in the extract it would have been necessary to load onto the Cig cartridge much more oil, but this was not possible because the retention capability of the column would have been exceeded. Other type of sorbent materials have found utilisation in the extraction of polar analytes from lipid matrices and among these we tested Florisil and CN type of cartridges. In order to obtain an effective concentration procedure, maximisation of the amount of the oil sample and minimisation of the eluting solvent are required and working with commercial cartridges holding 1 g of sorbent material the best result were obtained when the CN phase was used. The extraction method was then optimised for a cartridge loading of 5 g of virgin olive oil (Figure 4). In order to test the procedure, the polyphenol content of virgin olive oils coming from 3 different countries was assessed. For each oil the analysis was performed on samples produced from olives at different ripening stage, for a total of 12 samples (Table 1). Further analysis were also conducted on oil samples
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Coratina Coratina Coratina Coroneiki Coroneiki Coroneiki Picual Picual Picual Arbequina Arbequina Arbequina Maiatica (\) Maiatica (2) Maiatica (3) Maiatica (4) Maiatica (5) Maiatica (6)
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Method 2
Method 4
302 272 251 112 150 147 172 174 153 60 50 39 108 240 252 223 234 212
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Because of the amount of oil extracted, the phenol concentration in the extracts obtained utilising the CN cartridge was much higher compared to that of the extracts recovered utilising the Cjg cartridge. Therefore the CN extracts could analysed without any concentration. All the extracts obtained from the above oils were analysed by HPLC and in Figure 5 is shown the chromatogram of one of the Greek oils (A). As a comparison, a chromatogram of the extract obtained with Method 2 is shown in the same Figure (B). The chromatographic profiles are very similar, with only slight differences. In the chromatograms of the tested oil it was not possible to identify any peak corresponding to the available standards; however, it was possible to point out the constant presence of certain peaks in all the chromatograms. These peaks are identified in Figure 5 (A) with numbers from 1 to 11. As already mentioned, the polyphenols can have a positive influence on the resistance of the oil to oxidative processes. Therefore the relationship between the assessed polyphenol content values and the oxidative oil stability determined with the Rancimat was studied. In Table 6 the total phenol content of the 12 oils is reported with the determined Rancimat values. In Figure 6 the Rancimat value of the 12 oils is plotted as a fimction of the total polyphenol content; linear regression was performed on these values and the low correlation coefficient shows that the oxidative stability is not correlated to the total phenol content, at least in the tested oils. This is because not all the compounds of the olive oil that react with the Folin-Ciocalteau reagent can be directly related to the oil stability. The relationship between the 11 detected peaks and oil stability, as well as total polyphenol content, was analysed using the Latent Variable analysis (PLSl). The values relative to the area counts of the 11 detected peaks were set as "x matrix," while total polyphenol content and Rancimat values were the "y vector". Figure 7 shows that the stability of the tested oils, represented by the Rancimat values, is related only to the peak identified by the number 1, while the total phenol content is strictly related to the peaks defined with number 3,2, 5 and 4 (Figure 8). The total variation explained by the two models with two factors was 50% and 95% respectively. The relationship between peak number 1 and the oxidative stability of the oils is represented in Figure 9; the correlation between the data is quite high with a correlation coefficient of 0.8. Polyphenols are important also for their contribution to some of the perceived properties of the virgin olive oil flavour, like bitter and astringent. The relationship between the 11 chosen peaks ("x matrix") and the intensity of the attributes bitter and astringent ("y matrix") was studied by PLS2 metiiod. As shown in Figure 10, the bitter intensity is related to the peak number 6, while the astringent intensity is related to peaks 2,3,4 and 5.
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Loadings
0.6-
BITTER
0.4-
peak10 peak6
0.2ASTRING 0peakl peakl: peak8
Pe^i^
-0.2peaks -0.4] M
-0.2
I 1 1 1 1 1 1 1 1 1 1 1 I 1 1 M
-0.1
0
1 1 1 1 1 1 1 1 1 1 1 r 1 I 1 1 1 1 1 1 1 1 1 M
0.1
0.2
0.3
1 1 1 1 1 1 1 1 1 1 1 M
0.4
0.5
1 1 1 I I 1 1 [ T 1 1 1 I I 1 1 I I
0.6
sta2 a, factor: <1,2> <1,2>
Figure 10. PLS relationship: selected chromatographic peaks and selected perceived properties {bitter and astringent) of the virgin olive oil flavour.
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4. CONCLUSIONS The use of SPE for the recovery of the polyphenolfractionfromvirgin olive oil can represent a valuable alternative to the traditional extraction procedures. The results have shown that Cig cartridges can be utilised if the final goal is a rapid determination of the total polyphenol content in the oil. SPE can also be applied for the concentration of the analytes of interest, and in this case CN cartridges should be utihsed. This extraction methodology, besides being rapid, minimises possible degradation process that could affect the polyphenols; this is important because this study has pointed out that a relationship exists between certain components that are found in the phenohc extract and some important characteristics of the virgin olive oil (oxidative stability and perceived properties). Further studies will be conducted with the aim to chemically identify these components.
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