Food and Chemical Toxicology 48 (2010) 29722979

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Food and Chemical Toxicology

journal homepage: www.elsevier.com/locate/foodchemtox

Olive oil stability under deep-frying conditions

Susana Casal a, Ricardo Malheiro b, Artur Sendas a,b, Beatriz P.P. Oliveira a, Jos Alberto Pereira b,*
a b

REQUIMTE/Servio de Bromatologia, Faculdade de Farmcia da Universidade do Porto, Rua Anbal Cunha 164, 4050-047 Porto, Portugal CIMO/School of Agriculture, Polytechnic Institute of Bragana Apartado 1172, 5301-854 Bragana, Portugal

a r t i c l e

i n f o

a b s t r a c t
The suitability of different commercial olive oil categories for domestic frying was investigated. Oil samples were taken every 3 h of frying and evaluated for free acidity, peroxide and p-anisidine values, specic extinction coefcients, oxidative stability, fatty acids, vitamin E, b-carotene and total phenols, until the total polar compounds achieved the maximum legal value (25%). All olive oils were fried during more time than the commercial vegetable oil blend taken for comparison (from 24 to 27 h, against 15 h). The extra-virgin Protected Designation of Origin (PDO) olive oil was characterized by reduced levels of oxidation and hydrolysis, and superior amounts of minor antioxidant compounds. The olive oil commercial category behaves similarly, but Cobranosa olive oils performance was slightly worse, and clearly different between years, highlighting the importance of blending different cultivars. The vegetable oil, despite containing signicantly higher amounts of vitamin E, was highly susceptible to oxidation under frying conditions when compared to all olive oils. The results also show that the chemical composition of olive oils, particularly the amount of natural antioxidants, are important parameters in their predictive behavior along the frying process, but mostly that olive oil is clearly resistant to frying conditions, independently to the commercial category chosen. 2010 Elsevier Ltd. All rights reserved.

Article history: Received 4 May 2010 Accepted 26 July 2010

Keywords: Olive oil Oxidative stability Frying Vitamin E Fatty acids Total phenols

1. Introduction Frying is one of the most popular culinary processes worldwide, both for industrial and domestic food preparation procedures. Fried products have unique organoleptic and sensorial properties, including avor, texture, and appearance, which turn them largely enjoyed by consumers. Also, this procedure considerably reduces cooking time (Snchez-Muniz and Bastida, 2006) and is regarded as inducing equal or even smaller nutrient losses when compared with other common culinary processes (Fillion and Henry, 1998). Frying can enhance the food nutritive value due to the simultaneous incorporation of important lipid components, namely vitamin E and essential fatty acids, providing that it is consumed under a balanced diet, because the lipid uptake might increase signicantly the total daily energy intake. The high temperatures used during frying, in the presence of oxygen and water, induce important chemical changes of the oils, namely by oxidation, polymerization, cyclization, and hydrolysis (Paul and Mittal, 1997; Saguy and Dana, 2003), inevitably reducing their shelf life and affecting directly the quality of the nal fried food (Kochhar, 2001). These chemical reactions are inuenced by the type and quality of the oil, the food properties, and the food/oil ratio, among other parameters (Saguy and Dana,
* Corresponding author. Tel.: +351 273303277; fax: +351 273325405. E-mail address: jpereira@ipb.pt (J.A. Pereira). 0278-6915/$ - see front matter 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.fct.2010.07.036

2003), altogether determining the frying oil performance (Andrikopoulos et al., 2002). Each vegetable oil is characterized by typical stabilities against oxidation, dependent on the fatty acids composition, particularly the insaturation degree, and the content and composition of minor compounds such as tocopherols (particularly c-tocopherol), certain sterols, hydrocarbons (squalene), carotenoids, polyphenols, and trace metals (Hoffman, 1989; Kochhar, 2001). Several studies support a relationship between the Mediterranean diet and a lower incidence of some important diseases of our century, including cancer (Assmann et al., 1997) and cardiovascular diseases (Covas, 2007). Olive oil is typically the main lipid source in the Mediterranean diet, being used as salad dressing and for frying purposes. Its benecial properties are associated with the richness in monounsaturated fatty acids, but other minor compounds also take an important part (Varela and Ruiz-Roso, 2000). The differences to other common vegetable oils are enhanced by the fact that olive oil is mostly obtained by cold pressure procedures, without being subjected to rening virgin olive oil retaining therefore higher amounts of important bioactive components of the olive fruit. More recently, however, mostly supported by economical reasons and reduced consumer information, the consumption of olive oil, a blend of rened with virgin olive oils in undeclared proportions, is increasing, together with other rened vegetable oils blends, including sunower, soybean, and high oleic acid seed oils, particularly for frying purposes.

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The objective of the present work is to compare the stability under real domestic frying conditions of several olive oils categories. The vegetable oil blend most frequently consumed in Portugal, sunower based, was also included in the study. Aware that predictions based on the oxidative stability alone generally do not correlate with the real oxidation kinetics under frying conditions, and in the presence of food (Velasco and Dobarganes, 2002), this study was performed under real domestic frying conditions. The frying media degradation was evaluated taking the actual law as rejection point, particularly the total polar compounds, while complemented by several chemical evaluations in order to support and understand the degradation patterns and potential nutritional losses.

2.3.4. Anisidine value Unsaturated aldehydes, secondary oxidation products, were estimated by the anisidine value (AV), as detailed in ISO 6885:2006. This measurement is based on the absorbance increase per g of oil, measured at 350 nm (Shimadzu UV 160A spectrophotometer, Japan), of an olive oil solution in iso-octane, before and after reaction with p-anisidine reagent in the dark. 2.3.5. K232 and K270 extinction coefcients The extinction coefcients K232 and K270 (absorption of 1% solution (m/v) in isooctane at 232 and 270 nm, respectively, with 1 cm of path length) were measured using a UV spectrophotometer (Shimadzu UV 160A spectrophotometer, Japan). 2.3.6. Oxidative stability (Rancimat) The oxidative stability was estimated by measuring the oxidation induction time, on a Rancimat apparatus (Metrohm CH series 679). Air (20 L/h) was bubbled through the oil (3.0 g) heated at 110 0.2 C, with the volatile compounds being collected in water, and the increasing water conductivity continually measured. The time taken to reach the conductivity inection was recorded. 2.3.7. Fatty acids composition Fatty acids were evaluated as their methyl esters after alkaline trans-esterication with cold methanolic solution of potassium hydroxide, according with Regulation EEC 2568/91. The methyl esters were analyzed on a Chrompack CP 9001 gas chromatograph equipped with a split-splitless injector, a FID detector, an autosampler Chompack CP-9050 and a 50 m 0.25 lm i.d. fused silica capillary column coated with a 0.19 l lm of CP-Sil 88 (Chrompack). Helium was used as carrier gas at an internal pressure of 120 kPa. The temperatures of the detector and injector were 250 C and 230 C, respectively. The split ratio was 1:50 and the injected volume 1 lL. The results are expressed in relative percentage of each fatty acid methyl ester, calculated by internal normalization of the chromatographic peak areas. A mixture of fatty acid methyl esters (Supelco 37 FAME Mix) was used for identication purposes (Sigma, Spain). 2.3.8. Tocopherol and tocotrienol compositions Tocopherols and tocotrienol measurements were based on the ISO 9936:2006 standard with the addition of tocol as internal standard. Briey, an accurate solution of oil in hexane, with tocol, was analyzed by HPLC on a silica gel column (Varian, Inertsil 5 Si, 250 mm, 3 mm i.d.; Middelburg, The Netherlands), eluting with hexane/dioxane (97:3) (v/v) at a ow rate of 0.7 mL/min. A uorescence detector (Jasco 821-FP) was used, with excitation wavelength at 290 nm and emission wavelength at 330 nm, gain 10. The concentrations were expressed as mg/kg of oil. 2.3.9. b-Carotene Carotene concentration was calculated from the UV measurements of the hexane extracts obtained in accordance with Maia et al. (2008). For the purpose, oil samples were dissolved in dimethylformamide and extracted with several portions of n-hexane. The combined solutions were concentrated under a gentle nitrogen ow, and their absorbance evaluated at 454 nm. Quantication was achieved by external standards, based on calibration curves obtained with standard solutions extracted in accordance with the samples protocol. 2.3.10. Total phenols content Total phenols were isolated from a solution of oil in hexane by triple-extraction with watermethanol (60:40 v/v), and estimated with FolinCiocalteu reagent at 725 nm. Results were expressed as mg of caffeic acid per kg of oil.

2. Materials and methods All reagents were of analytical, chromatographic or spectroscopic grade and were supplied from Merck (Darmstadt, Germany) or SigmaAldrich (St. Louis, USA). All experiments and analytical determinations were performed at least in duplicate.

2.1. Samples Five oil samples were used throughout the frying studies. One commercial extra-virgin PDO olive oil Azeite de Trs-os-Montes from the northeast of Portugal (EVOO), two monovarietal virgin olive oils from the same region (Cv. Cobranosa from two consecutive years 2004 and 2005) (COB2004 and COB2005), a commercial blend of rened olive oil and virgin olive oil labelled as olive oil (ROO), and a rened vegetable oil blend with large commercial signicance importance in Portugal, based mostly on sunower oil (SO). Pure rened olive oil, despite being mentioned as a commercial category in the EEC Regulation (2568/91), was not included in the present study because its commercialisation is not legally recognized in Portugal (Dec-Lei 16/2004). Oil samples were taken every 3 h of frying from each fryer (about 30 mL), with immediate reposition with equal amounts of fresh oil, in order to avoid an early end of the study due to low oil level. Each sample was kept under nitrogen atmosphere until analysis.

2.2. Frying conditions Domestic deep-fat electric fryers (Kenwood, DF 150) with 1.5 L capacity, designed for a maximum load of 400 g, were used for frying. Fresh potato slices (300 g), shelled shortly before frying and cut into toothpicks, were used throughout all frying sections. The fryers were regulated for 170 C, and a batch of potato slices was fried every hour, during 9 h/day, until oil discard. The end of frying assays was determined by the value of total polar compounds (max. 25%), in accordance to the Portuguese law (Portaria No. 1135/95).

2.3. Physical and chemical parameters evaluated The free acidity (FA), peroxide value (PV), coefcients of specic extinction at 232 nm and 270 nm (K232 and K270), and fatty acid composition were determined according to ofcial methods describe in EEC Reg. No. 2568/91. The other parameters were either based on international standards or literature reports as briey described below.

3. Results and discussion 3.1. Changes in the total polar compounds (TPC) In accordance with the Portuguese law and as mandatory in several countries, the legal rejection point for frying oils is 25% of TPC, including under this label the hydrolysis products (diglycerides, monoglycerides and free fatty acids), the oxidation and polymeric derivatives, all formed at temperatures below 180 C (Portaria No. 1135/95). Under real frying facilities, mostly restaurants and industries, this parameter is usually evaluated by commercial rapid tests, mostly based on colorimetric readings, which have proven to correlate well with the values obtained by ofcial standards, and allow rapid on-line measurements. All samples were evaluated before the rst frying session and at every 3 h of frying, until the 25% were achieved. All olive oils presented similar TPC amounts before frying, with 5.5% (EVOO), 7.0% (COB2004), 6.5% (COB2005), and 6.0% (ROO) (Table 1). The TPC of

2.3.1. Total polar compounds Total polar compounds estimation was based on the dielectric constant changes, measured directly on the hot oil, with a Test 265 cooking oil tester (Testo, Germany), according to the manufacturer operation guide.

2.3.2. Free fatty acids The free fatty acids (FFA), expressed as free oleic acid percentage, was determined by titration of an accurate sample solution, dissolved in ethanol/ether (1:1, v/v), with 0.1 M sodium hydroxide solution, using phenolphthalein as indicator.

2.3.3. Peroxide value The PV, expressed in milliequivalents of active oxygen per kilogram (mEq O2/ kg), was determined as follows: a mixture of oil and chloroform/acetic acid 2:3 (v/v) was left to react in the dark with saturated potassium iodine solution, and the free iodine titrated with a sodium thiosulfate standard solution.

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S. Casal et al. / Food and Chemical Toxicology 48 (2010) 29722979 Table 1 Physical and chemical characteristics of the oils during frying. Frying time (h) EVOO 0 3 6 9 12 15 18 21 24 27 COB2004 0 3 6 9 12 15 18 21 24 COB2005 0 3 6 9 12 15 18 21 24 ROO 0 3 6 9 12 15 18 21 24 27 SO 0 3 6 9 12 15 TPC (%) 5.5 7.5 8.5 11.5 13.5 15.5 18.0 20.5 22.5 25.0 7.0 8.5 11.0 14.5 16.0 19.0 21.5 23.0 25.5 6.5 8.5 11.0 14.5 16.0 19.0 21.5 22.0 25.5 6.0 8.0 9.5 12.5 14.5 16.5 19.0 21.5 23.0 26.0 13.5 17.5 18.0 20.5 23.5 27.0 FFA 0.2 0.3 0.3 0.3 0.3 0.4 0.4 0.5 0.5 0.6 0.2 0.3 0.3 0.3 0.4 0.4 0.4 0.5 0.5 0.2 0.3 0.3 0.4 0.4 0.4 0.5 0.5 0.6 0.3 0.3 0.3 0.5 0.5 0.5 0.5 0.6 0.7 0.7 0.1 0.1 0.1 0.1 0.1 0.2 PV 11 14 15 15 15 12 11 11 10 11 11 11 9 10 10 11 10 11 10 11 9 12 10 11 13 14 14 14 12 11 10 10 11 10 11 10 9 9 7 28 23 21 21 15 p-AV 6 32 41 50 52 66 58 59 59 60 3 32 42 49 55 58 62 58 64 4 32 43 50 56 61 61 62 65 6 31 44 49 50 55 56 55 61 58 10 58 92 116 144 167 K232 1.66 1.80 1.92 2.16 2.36 2.08 2.14 2.13 2.24 2.05 1.40 1.98 2.15 1.92 2.06 2.02 1.93 2.24 2.27 1.72 2.16 2.18 2.15 2.13 2.39 2.39 1.97 2.24 1.84 2.24 2.11 2.10 1.94 1.88 1.98 2.18 2.36 2.09 2.11 2.13 2.02 2.07 2.43 2.28 K270 0.20 0.90 1.01 1.03 0.99 0.97 0.95 1.02 1.01 1.02 0.15 0.64 0.88 0.95 0.89 0.89 0.97 0.91 1.04 0.14 0.82 0.90 0.96 1.00 1.05 1.05 1.08 1.13 0.33 1.06 1.10 1.10 1.05 1.02 1.04 1.06 1.10 1.06 1.10 1.99 2.11 2.24 2.63 2.51

DK
0.00 0.07 0.07 0.06 0.05 0.04 0.02 0.01 0.02 0.01 0.01 0.05 0.04 0.03 0.02 0.01 0.01 0.02 0.02 0.00 0.08 0.03 0.01 0.04 0.02 0.02 0.01 0.01 0.02 0.08 0.08 0.04 0.03 0.02 0.02 0.01 0.01 0.01 0.14 0.08 0.04 0.03 0.02 0.01

ROS (h:min) 16:24 16:00 4:44 2:56 2:27 2:31 1:59 1:54 1:59 1:59 8:51 7:06 3:23 1:48 1:45 1:42 1:42 1:16 1:14 6:01 1:49 1:47 1:09 1:11 0:54 0:51 0:45 0:42 15:18 14:30 3:19 2:22 2:08 2:06 1:38 1:53 1:56 1:51 3:55 2:57 2:39 2:42 2:20 2:19

the vegetable oil (SO) was signicantly higher (13.5%), but in accordance with similar vegetable oils commonly evaluated in the laboratory where this study was developed. The total frying time was similar for all the olive oils, ranging from 24 to 27 h (3 days), with steady increases up to the mandatory rejection point (25%) (Fig. 1). Both monovarietal extra-virgin olive oils achieved the rejection point after 24 h, followed by the ROO mixture with around 26 h (26% at the 27th h), and nally the EVOO with 27 h of frying. The vegetable oil (SO) reached the TPC limit sooner, with 27% already after the 15th frying hour. If we are detained on the formation of TPC during the consecutive frying sessions, the degradation rates are similar between all olive samples, with a 0.7% increase per hour in the EVOO, and 0.8% in all the other olive samples, without clear differences. This degradation rate was, however, slightly higher in the vegetable oil used for comparison, with 0.9% per hour. The reduced differences between all ve samples, independently of their identity, highlight that the formation of TPC must be highly dependent upon the frying conditions of the study,

namely frying temperature, type and amount of food, etc., situations that were kept controlled during this study. In order to understand the chemical factors supporting the differences ob-

Fig. 1. Changes occurred in the total polar compounds (% TPC) during the frying sessions.

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served, several parameters were evaluated and are discussed below. 3.2. Changes in free fatty acids The FFA, resulting mainly from hydrolysis of triglycerides, was evaluated by conventional acidity measurement. All the virgin olive oils presented acidity levels below 0.3% before heating, in accordance with the labelling category extra-virgin olive oils (60.8%). The vegetable oil (SO) acidity was comparatively smaller (0.1%), due to the neutralization step during rening, and entirely within the legal limits for vegetable oils, as dened in Codex Standard 210 (2009). A steady increase in the acidity was observed for all olive oils (Fig. 2), with nal values between 0.5% and 0.7%. For the vegetable oil the hydrolysis was reduced until rejection, with only 0.2% after 15 h of frying. The commercial olive oil (ROO) presented a slightly higher acidity than all the other virgin olive oils. Knowing that rening removes the free fatty acids by saponication, smaller amounts are expected for rened olive oil. Therefore, one can suppose that the virgin oil used in the blend presented a superior acidity. Despite the inexistence of mandatory rejection points for used oil regarding hydrolysis (Paul and Mittal, 1997), this parameter is some timed used in rapid commercial tests as an estimation of the rejection point. Based on the information obtained from these frying experiments, the hydrolysis was reduced at the rejection point, being a parameter with minor capacity to evaluate the true thermal degradation of frying oils. 3.3. Changes in peroxide and p-anisidine values The hydroperoxides formation is correlated with the fatty acids oxidation susceptibility and the antioxidant levels. However, hydroperoxides are transient chemical compounds and, despite being a mandatory parameter for both bottled olive and vegetable oils oxidative status, they are not always directly correlated with sample oxidation, mostly on heated samples. The p-anisidine value, despite being a more empiric determination, is well correlated with the level of secondary oxidation products, namely aldehydes, far more stable than hydroperoxides. Therefore, for an accurate estimation of the oxidation status, both parameters should be interpreted simultaneously. The results for peroxide (PV) and p-anisidine (p-AV) levels are detailed in Table 1. All unheated samples presented peroxide values within the legal limits: below 20 mEq O2/kg for virgin oils, 10 mEq O2/kg for the vegetable oil, and 15 mEq O2/kg for the blend of rened with virgin olive oil. The p-anisidine values before frying

were below 10, indicating the almost absence of secondary oxidation products in all samples under study. All olive oils presented similar peroxide levels during frying, with slight variations around the initial amounts (1011 mEq O2/ kg) due to the volatile nature of peroxides. The EVOO sample was characterized by a peroxide increase during the rst 12 h (15 mEq O2/kg), followed by a return to the initial levels. Both monovarietal olive oils extracted from Cv. Cobranosa, for 2004 and 2005, had similar behavior during the rst 12 h, but the COB2005 PV increased afterward up to the rejection point, with a nal value of 14 mEq O2/kg, in opposition to only 10 mEq O2/kg in the COB2004 sample. This observation is interesting from the chemical point of view because both samples are similar concerning their origin, but the older one (COB2004) appears to be more resistant to oxidation. The olive oil blend (ROO) presented comparatively smaller peroxide amounts, as expected by the presence of rened oil where the peroxides are eliminated, and the behavior during frying was also quite constant, being the sample with less peroxide amounts, in opposition to the hydrolysis results. The vegetable oil assumed a clearly different behavior, starting with only 7 mEq O2/kg and achieving, after 3 h, a peroxide value of 28 mEq O2/kg. From this moment, this parameter decreased until the rejection, reporting a nal peroxide value of 15 mEq O2/kg. The increased levels might be explained by the high polyunsaturated fatty acids level characteristic of sunower based formulas, mostly linoleic acid, highly susceptible to oxidation when compared to monounsaturated oils, as olive oil. As regards to p-anisidine values, all olive oil samples presented an identical evolution during the entire study, independently of the initial peroxide values and variations, with nal values from 58 to 65. COB2005 sample in particular, although presenting a higher PV, was characterized by similar p-anisidine values, indicating that the formation of secondary stable oxidation products occurs at similar rates in all olive oil samples (Fig. 3). The vegetable oil blend (SO) presented a steady increase in the p-anisidine value, from 10 to 167, representing a clear formation of secondary oxidation products when compared with the olive oil samples. It is noticed that after 3 h of frying the value of p-anisidine is identical to the values obtained by the olive oils after 15 18 h. This result attests that this vegetable oil presents a minor resistance to oxidation under frying conditions when compared to the olive oils. The higher p-anisidine and peroxide levels in the vegetable oil (SO), being related with the formation of oxidation products, can also give some insight into the higher TPC observed in this sample. The similarity in the olive samples behaviors and its opposition to the SO blend indicates that the fatty acid composition might be the main responsible.

Fig. 2. Changes occurred in free acidity values (% of free fatty acids) during frying sessions.

Fig. 3. Changes in p-anisidine value during the frying sessions.

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3.4. Changes in the specic extinction coefcient at 232 and 270 nm The ultraviolet spectrophotometric analysis, expressed as specic extinction coefcients, indicates the olive oil oxidation status by providing information on the quality, preservation state and/or changes brought about by technological processes, being mandatory for olive oils categories (Annexes II and IX in European Community Regulation EEC/2568/91). The K232 is correlated with the formation of conjugated dienes of polyunsaturated fatty acids, while the K270 values are indicative of the presence of primary and secondary oxidation products, including conjugated trienes and carbonyl compounds. The maximum values allowed for K232 and K270 are, respectively, 2.50 and 0.20 for extra-virgin olive oils, and 2.60 and 0.25 for virgin olive oil. For oils partially (olive oil) or totally rened (common vegetable oils), the K270 limit is comparatively higher (0.90) and the K232 is not evaluated. The DK value, corresponding to the variation within the 270 nm region, is also regulated for olive oils, with maximum amounts of 0.01 for virgin olive oils and 0.15 for blends. All unheated olive oil samples were within the legislation limits (Table 1). Despite the inexistence of limits regarding the rened vegetable oil (SO), the values were within those for regulated for 100% rened olive oil (K270 6 1.10 and DK 6 0.16). The specic coefcient K232 increased in the analyzed samples, experiencing slight variations around a plateau level with none exceeding the maximum legal value for this parameter, despite being regulated only for unheated samples. The K270 measurements increased above the legislation limit soon after the rst frying hours and kept constant thereafter until the end of the study, with no clear differences between all the olive oils under study. The SO also increased proportionally, but with a high reading already before frying, due to the severe conditions of the rening process. The DK values presented also similar behaviors in all oils, with initial increases followed by decreases until the end of the frying process, but surpassing the limits at the rst heating sessions. Only with the vegetable oil (SO) a continuous decline along the 15 h of frying was observed. For these spectrophotometric parameters, no relevant correlations were observed with the frying time (Table 4), except in the DK value of the vegetable oil (SO) where a very signicant negative correlation was established. 3.5. Changes in the oxidative stability The Rancimat method is frequently used for evaluating or predicting oxidative stabilities under heating conditions, known as induction time. Despite being an important tool for comparison of different vegetable oils, the results are not directly transposable to real frying conditions. Clearly distinct results were observed for all samples (Table 1). The highest oxidative stability was depicted by the EVOO, with 16 h, followed by the ROO with 15 h. Indeed, during the frying studies these samples presented similar results, with 27 h and 26 h, respectively, as previously discussed. The monovarietal samples presented a comparatively reduced stability, with only near 9 h for the COB2004 and 6 h for COB2005, but in line with the previous discussed results, with a worse performance for the fresher oil. Nevertheless, these values are smaller than expected because under real frying conditions only a slight reduction was observed, with 24 h for both samples. The vegetable oil (SO) presented a clearly reduced stability, with only 4 h, in line with the frying performance. In order to understand the changes in the oxidative stability during the frying sessions, samples were taken from each frying session and also evaluated with the Rancimat test. The oxidative stabilities of both EVOO and ROO kept almost constant after the

rst 3 h of frying, but a clear reduction was observed at the 6th h (4:44 h and 3:19 h, respectively), and after 18 h of frying their values remained constant until the end of the process (2 h). The monovarietal COB2004 presented a similar reduction trend but the COB2005 stability was reduced already in the rst sampling, after 3 h of heating, supporting its inferior quality, and in line with the previous observations. The SO, despite presenting reduced initial levels, was characterized by a smother reduction, varying between 3.6 and 2.2 h. 3.6. Changes in the fatty acids composition In order to understand the chemical issues behind the frying susceptibility observed for the samples under analysis, several parameters were evaluated. Being the fatty acids the main oils constituent and their nature, particularly the unsaturation degree, among the most important factors determining the oxidative stability, the fatty acid composition was evaluated in detail. Olive oil is, as expected, was characterized by a high degree of monounsaturated fatty acids (MUFA), particularly oleic acid, an important issue contributing to the cardiovascular health effects (Covas, 2007). All olive oil samples presented oleic acid amounts within those expected (55.083.0%, Reg. EEC 2568/91), varying from 73.4% in the COB2005 sample to 77.0% in the ROO (Table 2). The sunower based vegetable oil (SO) was characterized by a dominant polyunsaturated fraction, particularly linoleic acid (62.8%), and a comparatively low amount of oleic acid (24.8%) and linolenic acid (0.1%). All olive oil samples were also within the regulated levels regarding trans fatty acids, with reduced amounts in comparison with the olive oil mixture ROO and the SO sample. When comparing the relative percentage of fatty acids during the frying sessions, a clear reduction of unsaturated fatty acids was observed, with the consequent increase in the saturated fatty acids amount (Table 2). The decrease was particularly noticeable in the polyunsaturated fatty acids, presumably by oxidation, with the linolenic acid reduced to about half the initial amounts, despite being a minor fatty acid. The SO alterations followed the same trend, with equivalent losses for the same sampling times, despite the increased polyunsaturated amounts. A linear trans fatty acids increase with frying time was observed for all samples under study, and despite the initial higher amounts for the rened (SO) or partially rened (RO) samples, the nal values were equivalent in all samples, but still clearly within the requirement for a zero trans content per serving. Based on these observations, and despite the clear differences on the fatty acid composition of both matrices, the degradation rated appears to be similar. 3.7. Changes in the tocopherol and tocotrienol content Vitamin E, especially a-tocopherol, is an important vegetable oil component contributing to the nutritional value, both by its vitaminic action and antioxidant properties (Kiritsakis, 1998). Their presence in the oils is also important from the technological point of view due to its antioxidant action, being generally oxidized to quinidine and dimmers while protecting the fat (Hoffman, 1989). Table 3 details the changes occurred in the tocopherol and tocotrienol contents, with an increased degradation as function of the frying time. Prior frying all olive oils presented variable amounts of total vitamin E, from 139 mg/kg to 189 mg/kg, mainly a-tocopherol. These values are within the expected for olive oils. In line with the previous observations, the monovarietal oils presented lower vitamin E amounts, particularly the COB2005 sample, an important parameter for the antioxidant capability and thus the oil performance. The vegetable oil (SO) presented signicantly higher

S. Casal et al. / Food and Chemical Toxicology 48 (2010) 29722979 Table 2 Changes in the fatty acid composition during frying at 170 C (g/100 g fatty acids). Frying time (h) EVOO 0 3 6 9 12 15 18 21 24 27 COB2004 0 3 6 9 12 15 18 21 24 COB2005 0 3 6 9 12 15 18 21 24 ROO 0 3 6 9 12 15 18 21 24 27 SO 0 3 6 9 12 15 SFA 14.50 0.27 14.39 0.52 14.95 0.01 14.80 0.02 14.60 0.45 14.43 0.02 14.91 0.46 14.85 0.15 15.38 0.09 15.38 0.09 14.69 0.11 14.97 0.22 15.05 0.12 15.48 0.05 15.45 0.14 15.50 0.05 15.71 0.00 16.47 0.21 16.09 0.04 15.27 0.09 15.59 0.20 15.75 0.07 15.86 0.13 16.01 0.20 16.18 0.08 16.33 0.02 16.51 0.11 16.72 0.02 14.43 0.06 15.11 0.09 15.41 0.62 15.03 0.04 15.27 0.31 15.58 0.03 15.80 0.02 15.87 0.10 15.72 0.00 16.07 0.14 11.28 0.08 11.38 0.01 11.35 0.00 11.43 0.03 11.66 0.01 11.77 0.02 18:1 76.80 0.25 76.83 0.52 76.85 0.05 76.85 0.23 77.40 0.37 77.16 0.11 77.96 0.50 77.29 0.10 76.75 0.10 75.94 0.59 75.41 0.00 75.33 0.22 74.95 0.22 75.05 0.07 75.36 0.33 75.40 0.11 75.17 0.08 74.51 0.24 74.99 0.02 73.38 0.18 74.04 0.10 74.13 0.12 73.60 0.05 74.03 0.21 74.06 0.09 73.64 0.41 74.17 0.39 73.76 0.04 77.05 0.08 76.09 0.17 75.35 0.17 76.34 0.11 76.04 0.07 75.93 0.12 76.11 0.03 75.94 0.29 75.92 0.22 75.66 0.04 24.82 0.90 24.63 0.06 24.49 0.06 24.72 0.01 25.35 0.20 25.21 0.00 18:2 5.64 0.04 5.60 0.12 5.13 0.01 5.04 0.00 4.86 0.00 4.68 0.03 4.46 0.04 4.32 0.02 4.18 0.02 4.31 0.05 7.00 0.06 6.73 0.06 6.47 0.02 6.30 0.01 6.06 0.04 5.88 0.01 5.67 0.01 5.40 0.10 5.34 0.03 7.87 0.06 7.55 0.04 7.26 0.05 7.18 0.03 6.77 0.02 6.50 0.01 6.29 0.01 6.18 0.04 5.91 0.05 5.59 0.00 5.71 0.01 5.42 0.16 5.24 0.04 5.10 0.04 4.86 0.00 4.66 0.00 4.49 0.05 4.32 0.03 4.21 0.01 62.78 0.93 62.73 0.03 62.84 0.04 62.45 0.01 61.57 0.11 61.31 0.07 18:3 0.76 0.0 0.71 0.0 0.62 0.0 0.60 0.0 0.53 0.0 0.49 0.0 0.45 0.0 0.41 0.0 0.40 0.0 0.41 0.0 0.74 0.00 0.67 0.00 0.62 0.01 0.57 0.00 0.52 0.00 0.47 0.00 0.45 0.00 0.44 0.01 0.39 0.00 0.64 0.00 0.60 0.00 0.55 0.00 0.52 0.02 0.47 0.00 0.42 0.00 0.41 0.00 0.39 0.00 0.36 0.01 0.74 0.00 0.57 0.00 0.54 0.02 0.45 0.01 0.43 0.01 0.40 0.00 0.37 0.00 0.34 0.01 0.32 0.00 0.31 0.01 0.11 0.00 0.10 0.00 0.10 0.00 0.10 0.02 0.09 0.00 0.09 0.00 Trans 0.03 0.00 0.06 0.01 0.17 0.02 0.15 0.01 0.19 0.01 0.25 0.03 0.28 0.01 0.34 0.02 0.37 0.01 0.41 0.02 0.03 0.00 0.09 0.00 0.14 0.03 0.21 0.01 0.25 0.02 0.29 0.01 0.35 0.02 0.45 0.05 0.45 0.00 0.03 0.01 0.09 0.01 0.14 0.01 0.18 0.02 0.24 0.00 0.29 0.01 0.35 0.01 0.42 0.00 0.44 0.01 0.10 0.00 0.22 0.00 0.38 0.03 0.33 0.02 0.39 0.00 0.41 0.00 0.46 0.00 0.52 0.01 0.54 0.01 0.58 0.02 0.27 0.01 0.29 0.00 0.31 0.00 0.34 0.01 0.38 0.05 0.39 0.02

2977

amounts of total vitamin E (474 mg/kg), again with a-tocopherol as the main vitamer, as expected from a sunower based formula. Vitamin E degraded sharply in all olive oils under study (Table 3), being practically inexistent after 36 h of frying. In opposition, the vegetable oil levels were only slightly reduced, with still about 65% of the initial amounts at the rejection point. The atocopherol degraded at a faster rate in all olive oils (1548 mg/ kg/h) when compared to the vegetable oil (9 mg/kg/h). Therefore, and despite being the most important lipid antioxidant component of the oils, vitamin E seems to play a reduced part in the oil frying resistance. 3.8. Changes in the b-carotene Carotenoids are also naturally present in virgin olive oils, because only physical processes are used during extraction. Table 3 reports the changes occurred in the b-carotene contents of the oils subjected to the frying process. All samples presented distinct bcarotene amounts, within the expected for olive oil (Velasco and

Dobarganes, 2002), clearly higher in the EVOO. In accordance with the previous observations, the COB2005 sample presented lower amounts than the COB2004 sample, and even lower that the ROO sample. During frying a similar pattern was observed, with a reduction up to the 912th h, followed by a slight increase in all samples, probably from the potatoes. Only diminutive amounts of b-carotene were determined in the sunower based sample. 3.9. Total phenols content In comparison with other vegetable oils, olives and olive oils are receiving particular attention for their phenolic content given that these compounds have antioxidant and antimicrobial effects (Ocakoglu et al., 2009). Indeed, phenols are frequently described as key factors in the olive oils oxidative resistance (Gmez-Alonso et al., 2003). The amount of total phenols, expressed as mg of caffeic acid equivalents, was higher in the EVOO sample, with 204 mg/kg, followed by the ROO (154 mg/kg) and by the COB2004 and COB2005,

2978

S. Casal et al. / Food and Chemical Toxicology 48 (2010) 29722979

Table 3 Vitamin E, b-carotene and phenols oil contents during frying (mg/kg). Frying time (h) EVOO 0 3 6 9 12 15 18 21 24 27 COB2004 0 3 6 9 12 15 18 21 24 COB2005 0 3 6 9 12 15 18 21 24 ROO 0 3 6 9 12 15 18 21 24 27 SO 0 3 6 9 12 15

a-T
(mg/kg) 179.9 2.3 101.4 1.1 4.7 0.2 n.d. n.d. n.d. 151.9 3.2 8.4 0.0. 5.6 5.1 1.7 0.1 n.d. n.d. 136.8 2.5 2.1 0.0 0.1 0.0 n.d. n.d. n.d. 139.1 0.4 94.1 0.2 2.6 0.2 n.d. n.d. n.d. 432.2 9.0 413.1 0.0 372.1 0.7 350.4 0.7 322.3 2.5 291.3 0.7

a-Ttr
(mg/kg) 1.1 0.2 0.6 0.1 n.d. n.d. n.d. n.d. 1.6 1.0 n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. 1.1 0.2 n.d. n.d. n.d. n.d. n.d. 2.9 0.4 2.3 0.2 1.7 0.1 1.7 0.0 1.4 0.0 1.1 0.0

b-T (mg/kg) 1.9 0.1 0.9 0.1 n.d. n.d. n.d. n.d. 1.7 0.0 n.d. n.d. n.d. n.d. n.d. 1.2 0.2 n.d. n.d. n.d. n.d. n.d. 3.2 0.3 1.3 0.0 0.1 0.0 n.d. n.d. n.d. 21.1 0.2 19.7 0.2 18.2 0.2 17.5 0.0 16.1 0.1 15.1 0.2

b-Ttr (mg/kg) 5.7 0.2 2.0 0.1 n.d. n.d. n.d. n.d. 1.6 0.0 n.d. n.d. n.d. n.d. n.d. 1.1 0.2 n.d. n.d. n.d. n.d. n.d. 21.2 1.6 4.7 0.0 0.2 0.0 n.d. n.d. n.d. 5.6 0.1 4.5 0.0 4.3 0.0 3.5 0.0 2.3 0.4 1.6 0.2

c-T (mg/kg)
n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. 8.0 0.2 7.3 0.1 6.4 0.0 5.6 0.0 4.5 0.1 3.5 0.0

c-Ttr (mg/kg)
n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. 3.2 0.1 2.6 0.3 3.9 0.0 3.5 0.1 2.3 0.6 1.8 0.6

d-T (mg/kg) n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. 0.5 0.1 0.1 0.1 n.d. n.d. n.d. n.d. 1.2 0.1 1.0 0.1 1.0 0.0 1.0 0.1 0.8 0.1 0.7 0.1

Total vitamin E (mg/kg) 189 105 5 n.d. n.d. n.d. 157 8 6 2 n.d. n.d. 139 2 n.d. n.d. n.d. n.d. 165 100 3 n.d. n.d. n.d. 474 451 408 383 350 315

b-Carotene (mg/kg) 5.2 0.1 3.7 0.1 3.0 0.1 2.0 0.0 1.4 0.0 1.7 0.1 2.5 0.2 1.7 0.1 2.2 0.2 3.0 0.1 2.04 0.06 0.65 0.08 0.57 0.02 0.74 0.07 0.88 0.07 0.99 0.02 1.04 0.08 1.23 0.03 1.07 0.03 0.97 0.11 0.49 0.05 0.49 0.03 0.63 0.08 0.91 0.06 1.08 0.11 1.11 0.04 1.20 0.03 1.38 0.04 1.6 0.0 1.3 0.0 1.6 0.2 1.5 0.1 2.0 0.0 2.0 0.0 2.1 0.1 2.1 0.2 2.6 0.1 3.7 0.1 0.23 0.05 n.d. n.d. n.d. n.d. n.d.

Phenolsa (mg/kg) 204 17 119 29 53 3 n.d. 98 15 59 1 n.d. 63 2 50 11 n.d. 154 5 72 6 n.d. 31 1 n.d.

n.d. not detectable. Not determined. a Phenols: mg caffeic acid equiv./kg.

with only 98 and 63 mg/kg, respectively. These amounts are in accordance with the frying performances. Nevertheless, after 6 h of frying the total phenols were only quantied in the EVOO sample, being absent in all samples at the 9th h, in accordance with the data published by Gmez-Alonso et al. (2003). These results are totally comparable with the vitamin E performance, indicating that both compounds are fast degraded during the rst frying hours. Therefore, if these compounds take a part in the frying resistance towards oxidation, it must be restricted to the rst heating hours. If the Rancimat results are also compared, one can observe that while both vitamin E and phenols are present the oxidative resistance is shortly reduced, but once these compounds are degraded the oxidative resistance is signicantly reduced. The olive oil fatty acid composition, highly monounsaturated, should be the rate limiting factor determining its oxidation. The almost absence of pro-

tective phenols and the polyunsaturated nature are certainly important factors contributing to the precocious oxidation of the vegetable oil blend (SO). 3.10. Correlations The correlation between the parameters evaluated and the frying time are depicted in Table 4. Signicant correlations were observed for the TPC, as expected, for all the samples. The acidity, in opposition, was only signicant in the olive oil samples but not in the sunower blend (SO) with a reduced hydrolysis rate up to the last frying session. Despite being formed in reduced amounts, the trans fatty acids correlation with the frying time was highly signicant, independently of the samples identity. The PUFA reduction was also statis-

S. Casal et al. / Food and Chemical Toxicology 48 (2010) 29722979 Table 4 Correlation between the evaluated parameters and frying time. EVOO r TPC FA PV p-AV K232 K270 DK ROS SFA MUFA PUFA Trans FA Vitamin E b-Carotene n.s. not signicant. p 6 0.05. ** p 6 0.01. *** p 6 0.001.
* 2

2979

COB2004 p
*** ***

COB2005 p
*** ***

ROO p
*** ***

SO p
*** *** * **

r2 0.970 0.428 0.013 0.981 0.393 0.801 0.878 0.685 0.873 0.556 0.802 0.981 0.996

p
***

0.996 0.913 0.318 0.673 0.412 0.328 0.272 0.601 0.559 0.087 0.967 0.977 0.997 0.297

n.s.
** *

n.s. n.s.
** *

n.s.
*** *** **

n.s.

0.995 0.919 0.283 0.766 0.483 0.568 0.198 0.680 0.881 0.309 0.993 0.988 0.635 0.010

n.s.
** * *

n.s.
** ***

n.s.
*** ***

n.s. n.s.

0.991 0.939 0.061 0.782 0.228 0.601 0.115 0.525 0.990 0.052 0.988 0.996 0.761 0.615

n.s.
**

n.s.
*

n.s.
* ***

n.s.
*** ***

n.s.
*

0.998 0.905 0.411 0.639 0.119 0.256 0.412 0.569 0.814 0.030 0.992 0.860 0.987 0.717

n.s. n.s.
***

n.s. n.s.
* * ***

n.s.
* ** * **

n.s.
*** ***

n.s.
* *** ***

n.s.
**

tically noticeable, but with higher signicance in the olive oils when compared with the vegetable oil. Among the oxidative parameters evaluated, only the p-anisidine was well correlated with the frying hours, but with higher signicance in the vegetable oil. The vitamin E reduction was also correlated with the frying hours but only with statistically signicance in the EVOO and SO samples, mostly because of their early loss. 4. Conclusions In the present work is proved that olive oil, independently of its category label, is clearly resistant to degradation under domestic frying conditions (170 C). Among the olive oil categories, the extra-virgin PDO olive oil was more resistant under the conditions of this study. Being from the Trs-os-Montes region, this olive oil is particularly rich in Cobranosa cultivar. Nevertheless, when this cultivar was evaluated individually, the monovarietal olive oils were clearly less resistant, probably due to the lower oleic acid, vitamin E and phenol amounts. Also, the monovarietal olive oils from consecutive years presented different oxidative susceptibilities, strengthen the importance of avoiding single cultivar olive oils, a practice that has increased in the last years because of their natural chemical and sensory characteristics. The olive oil, a commercial blend of rened with virgin olive oil, traditionally cheaper, behaved similarly to the EVOO sample. This highlights that, despite the presence of undeclared portions of rened olive oil, the most important compounds dening its oxidative resistance are still present, namely vitamin E, phenols and mostly the monounsaturated triglycerides. The vegetable oil taking as comparison, despite being less expensive and enclose higher amounts of vitamin E, is decient in important bioactive compounds, like phenols, and highly susceptible to early oxidation, contributing to the ingestion of higher amounts of oxidized fatty acids. Vitamin E, phenols, and b-carotene seem to be key compounds determining the reduced loss of oxidative stability during the rst frying hours. The highly monounsaturated nature of the olive oils is a second decisive parameter for the reduced thermal degradation under real domestic frying conditions, as observed by comparison with the commercial vegetable oil blend. Conict of Interest The authors declare that there are no conicts of interest.

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