Oxidative stability of olive oil after food processing and comparison with other vegetable oils

Food Chemistry 121 (2010) 1177–1187

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Oxidative stability of olive oil after food processing and comparison with other vegetable oils Lisete Silva a,b, Joana Pinto a,b, Joana Carrola b, Fátima Paiva-Martins a,b,* a b

Centro de Investigação em Química, Faculdade de Ciências, Universidade do Porto, Rua do Campo Alegre, No. 687, 4169-007 Porto, Portugal Departamento de Química, Faculdade de Ciências, Universidade do Porto, Rua do Campo Alegre, No. 687, 4169-007 Porto, Portugal

a r t i c l e

i n f o

Article history: Received 3 September 2009 Received in revised form 2 December 2009 Accepted 1 February 2010

Keywords: Olive oil Vegetable oil Roast processing Polyphenols Tocopherol Antioxidant activity

a b s t r a c t The use of olive oil showed an important protection of meat and potatoes when compared with other vegetable oils, with sunflower oil samples being oxidised after 60 min of processing at 180 °C. Olive oil samples were not oxidised, independently of the olive oil quality used. Shelf life was longer for extra-virgin olive oil containing samples and this fact was positively correlated with their higher phenolic content. The radical-scavenging activity of extra-virgin olive oil was higher than for other olive oil samples and was also positively correlated with the phenolic content of the oil. Seed oil antioxidants showed little capacity in delaying the oxidative degradation of seed oils and meat processed with them. However, tocopherol content and the identity of tocopherols present in the oil were shown to have a more important role in the oxidative stability of seed oils than the fatty acid composition. The presence of food showed a protective effect on the oils, with oil samples processed without food showing a higher level of oxidation than the oil samples processed in the presence of food. All polyphenolic components of olive oils decreased in concentration with the thermal treatment and this decrease was dramatic in the presence of food. During processing, two new compounds were found in olive oil samples and their concentration was higher for samples containing a higher initial polyphenolic content. The content in tocopherols was not so dramatically affected by the thermal treatment as was the polyphenolic content. Moreover, a sparing effect of food was, however, observed with the tocopherol content of samples which probably contributes to the better oxidative stability of these samples. Ó 2010 Elsevier Ltd. All rights reserved.

1. Introduction Free radicals are thought to be responsible for several pathological processes, such as cancer (Hertog et al., 1995), atherosclerosis (Steinberg, 1995), and cellular damage associated with ageing (Ashok & Ali, 1999). Therefore, the consumption of non-oxidised foods and dietary antioxidants seems to play an important role in protecting against these degenerative events. Extra-virgin olive oil (EVOO), produced by applying mechanical pressure on the fruits of Olea europea L., in addition to its high proportion of monounsaturated fatty acids, i.e. oleic acid, and the modest presence of polyunsaturated fatty acids, contains natural antioxidants such as tocopherols, carotenoids, sterols, and phenolic compounds (Boskou, 1996). Olive oil hydrophilic extracts contain a large number of phenolic compounds including phenyl-alcohols, such as 3,4dihydroxyphenylethanol (3,4-DHPEA or hydroxytyrosol) and phydroxyphenylethanol (p-HPEA or tyrosol) as well as phenyl-acids.

* Corresponding author. Address: Departamento de Química, Faculdade de Ciências, Universidade do Porto, Rua do Campo Alegre, No. 687, 4169-007 Porto, Portugal. Tel.: +351 220402556; fax: +351 220402559. E-mail address: [email protected] (F. Paiva-Martins). 0308-8146/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2010.02.001

Oleosidic forms of 3,4-DHPEA, in particular the dialdehydic form of elenolic acid linked to 3,4-DHPEA (3,4-DHPEA-EDA), an isomer of oleuropein aglycone (3,4-DHPEA-EA) and the dialdehydic form of elenolic acid linked to p-HPEA (p-HPEA-EDA) have been identified as the major secoiridoid compounds of virgin olive oil (Montedoro et al., 1993). Nowadays, different kinds of vegetable oils can be obtained in the market. Consumers, much more conscientious of the influence of food on their health, want to understand, and above all, to know the advantages of using olive oil instead of other less expensive vegetable oils and if there are advantages in using extra-virgin olive oil instead of olive oil in their cooking. However, these questions remain unanswered for most cooking conditions or, at least, are not supported by experimental data. The effects of phenols and tocopherols on the oxidative stability in EVOO during storage and heating processes have been extensively evaluated but these studies do not make any comparison with other vegetable oils and/or are performed in the absence of food (Aparicio, Roda, Albi, & Gutiérrez, 1999; Baldioli, Servili, Perreti, & Montedoro, 1996; Beltrán-Maza, Jiménez-Márquez, García-Mesa, & Frías-Ruiz, 1998; Brenes, García, Dobarganes, Velasco, & Romero, 2002; CarrascoPancorbo et al., 2007; Gutierrez-Rosales, Rios, & Gomez-Rey,

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Table 1 Analytical parameters of tested oils.

b

Acidity (%) Peroxide value (meq/kg)b 12:0 (%)a,c 14:0 (%)a,c 16:0 (%)c 16:1 (%)c 18:0 (%)c 18:1 (%)c 18:2 (%)c 18:3 (%)c SFAc MUFAc PUFAc a-Tocopherol (mg/kg)c c-Tocopherol (mg/kg)a,c d-Tocopherol (mg/kg)a,c Polyphenols (mg/kg)a,c

EVOO

VOO

OO

SFO

CO

SO

PNO

Meat fat

0.3 2.1 nd nd 11.2 0.46 3.8 77.4 6.8 0.4 15.0a 77.8a 7.2a 112a nd nd 315a

0.7 3.6 nd nd 11.9 0.75 3.6 76.1 7.1 0.6 15.5a 76.8a 7.6b 113.9a nd nd 198b

0.2 3.4 nd nd 12.8 0.82 3.6 75.5 6.7 0.6 16.3b 76.3a 7.3a 23.7b nd nd 130c

0.2 3.8 nd nd 7.4 0.00 3.4 35.0 54.0 0.0 10.8c 35.0b 54.0c 265.2c nd nd nd

0.2 3.2 nd nd 14.1 0.13 2.3 33.5 49.4 0.6 16.4b 33.6c 50.0d 60.6d 393.9a 33.0a nd

0.2 2.9 nd nd 12.7 0.00 3.7 24.8 54.0 4.8 16.4b 24.8d 58.8e 33.8e 348.8b 166.3b nd

0.2 3.1 nd nd 13.0 0.04 3.0 50.4 33.4 0.1 16.1b 50.5e 33.5f 46.1f 66.7c 22.8c nd

– 2.5 0.6 4.5 28.7 4.9 21.1 35.4 4.1 0.7 55.9d 39.3f 4.8g 0.81g nd nd nd

Different superscripts within a line indicate samples that were significantly different (p < 0.05). a nd, not detected. b SD < 5%. c SD < 3%.

2003; Kalantzakis, Blekas, Pegklidou, & Boskou, 2006; Pellegrini, Visioli, Buratti, & Brighenti, 2001; Tsimidou, Papadopoulos, & Boskou, 1992). In fact, there is not much data that reports the contents of these compounds after domestic heating of the oil in the presence of foods or if this presence may influence the stability of oils and its components in a negative or positive way, depending on the food used. Furthermore, most studies concerning the influence of food in olive oil stability and olive oil polyphenols only concern deep frying or pan frying conditions (Andrikopoulos, Kalogeropoulos, Falirea, & Barbagianni, 2002; Chiou et al., 2007, 2009; GómezAlonso, Fregapane, Salvador, & Gordon, 2003; Kalogeropoulos, Chiou, Mylona, Ioannou, & Andrikopoulos, 2007; Ramírez, Morcuende, Estévez, & Cava, 2004; Salta, Kalogeropoulos, Karavanou, & Andrikopoulos, 2008). The behaviour of olive oil phenolic compounds during food roast processing has, to our knowledge, never been reported. In Portugal, food is roasted mainly in olive oil and sunflower oil but other oils such soy oil, are now in use. The aim of this study was to study the changes in the phenolic content of extra-virgin olive oil following roasting operation in the presence of food and compare the benefits of its use with the use of other vegetable oils and other olive oil grades. Roasting meat and potatoes together with virgin olive oil is among the most important techniques employed in domestic and industrial food preparation in Mediterranean countries. This processing technique is characterised by a big surface area exposed to air, therefore being very prone to oxidation. 2. Materials and methods 2.1. Materials Samples of extra-virgin olive oil (EVOO), virgin olive oil (VOO), olive oil (OO), sunflower oil (SFO), soy oil (SO), corn oil (CO) and peanut oil (PNO) were supplied by a local company. In the case of olive oils, a mixture of two EVOO, a mixture of two VOO and mixture of two OO were used in order to obtain an EVOO sample, a VOO sample and an OO sample with a similar fatty acid profile. Analytical characteristics of these oils are shown in Table 1. 2.2. Fatty acid analysis Fatty acid methyl esters were prepared from the oils catalysed by boron trifluoride in methanol and analysed by gas chromatogra-

phy. A DBWAX capillary column (30 m  0.32 mm i.d., film thickness 0.25 lm) was used for separation. The oven temperature was held for 2 min at 180 °C and subsequently increased to 240 °C at 5 °C/min. The injector and detector temperatures were 220 and 240 °C, respectively. Peaks were identified by comparing retention time of authentic standards. All determinations were performed in duplicate. 2.3. Processing conditions Beef (150 g, cube shape) or 150 g of potatoes (six quarters of potatoes) were processed in 60 g of each oil, poured into 15 cm diameter glass beakers, in an oven at 180 °C until the inner temperature of meat reached 180 °C (60 min). Control samples without food were also processed in the same conditions. Each sample was performed in triplicate. After processing at 180 °C, the oil and foods were separated, weighed and frozen at 20 °C under vacuum. Oil uptake by food was estimated by weighing the separated oil in the glass beakers before and after processing. Five grams of each oil sample were kept for 1 month at 4 °C. 2.4. Isolation of lipids from meat and potatoes samples Lipids from 10 g of fresh and cooked meat (at 30 days of 20 °C vacuum storage in a plastic bag) were extracted by 150 ml hexane with a Soxhlet apparatus for an hour (hot extracted lipids) or by two portions of 150 ml of hexane by homogenisation (cold extracted lipids). Anhydrous sodium sulphate was added to the separated lipid layer to remove the residual water. The lipid extract was evaporated, and concentrated using a rotary evaporator (Rotavapor, R110, Buchi, Flawil, Switzerland). Lipids from potatoes were also extracted by homogenisation with two portions of 150 ml of hexane following the same procedure used for meat. 2.5. Level of oxidation The level of oxidation of oil samples and meat lipids was determined by the conjugated dienes content (CD) (AOCS Official Method Ti 1a-64), p-anisidine value (AV) (AOCS Official Method Cd 1890) and by the oil total polar compounds (TPC) content determined by silica column chromatography, following IUPAC method 2.507. The level of oxidation of oil samples was also assessed after the storage at 4 °C for 30 days.

L. Silva et al. / Food Chemistry 121 (2010) 1177–1187

2.6. Isolation and determination of tocopherols from oils Tocopherols were extracted by a methanol/isopropanol mixture and quantified by reverse phase HPLC with a diode-array detector connected in series with a fluorescence detector programmed at the excitation and emission wavelengths of 290 and 330 nm, respectively. The flow rate was 1 ml/min; the mobile phase used was 2% acetic acid (pH 3.1) in water (A), methanol (B) and isopropanol (C) for a total running time of 70 min, and the gradient changed as follows: 95% A/5% B for 2 min, 40% A/60% B for 8 min, 100% B for 20 min, 100% B for 10 min, 40% B/60% C for 10 min, 40% B/60% C for 15 min, 100% C for 2 min and this was kept constant until the end of the experiment. To carry out the quantification, three standard calibration curves were made using reference tocopherols (a, d and c). All calibration curves showed good linearity in the studied range of concentrations. a-, d- and c-Tocopherols were purchased from Sigma–Aldrich (Lisbon). 2.7. Isolation and determination of phenolic compounds from oils Mixtures of phenolic compounds were obtained from oils by SPE extraction following the procedure described by Mateos et al. (2001). The composition of these extracts was determined by HPLC. 2.8. Isolation and determination of phenolic compounds from aqueous phases Aqueous phases from meat samples were separated from the oil by centrifugation after processing and the volume measured. An aliquot was then extracted by dichloromethane and, after solvent evaporation, the residue was dissolved in 500 ll of methanol. The composition of these extracts was also determined by HPLC.

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analysis time of 70 min, according to the method previously described (Paiva-Martins & Pinto, 2008). Samples were analysed using 20 ll of each solution dissolved in methanol. Solvents were HPLC grade. To carry out the quantification, seven standard calibration curves were made using reference compounds: chlorogenic acid, caffeic acid, tyrosol, hydroxytyrosol, hydroxytyrosol acetate, oleuropein, 3,4-DHPEA-EA and 3,4-DHPEA-EDA. All calibration curves showed good linearity in the studied range of concentrations. These reference compounds were quantified using the calibration curves of their corresponding standards at 280 nm; other simple phenol compounds were quantified using the calibration curve of hydroxytyrosol at 280 nm; (+)-pinoresinol, (+)-1-acetoxypinoresinol and other secoiridoid derivatives (ligstroside aglycon) were quantified with the calibration curve of oleuropein obtained at 280 nm. Total polyphenolic content was quantified using the calibration curve of caffeic acid. For the LC–MS analysis, a Finnigan Surveyor with both a PDA detector and a MS detector was used. The flow rate used was 0.5 ml min 1; the mobile phase used was also a mixture of 0.1% formic acid (pH 3.1) in water (A) and methanol (B) with a total analysis time of 100 min, according to the method previously described (Paiva-Martins & Pinto, 2008). Peak identification and quantification were performed by comparison of retention times using a standard solution containing reference polyphenols. Mass spectrometry analysis was performed using a Finnigan LCQ DECA XP MAX detector, equipped with an API source, using an electrospray ionisation (ESI) probe. The capillary temperature and voltage used were 180 °C and 3 V, respectively, and spectra were obtained in negative ion mode. When the molecular ion was detected, the MS2 spectrum was obtained using a relative energy collision of 27. 2.11. Determination of the antioxidant activity of extracts. Shaal oven test

2.9. Isolation and determination of phenolic compounds from potatoes Polyphenols were extracted from potatoes with methanol as previously described by Chiou et al. (2007). The composition of these extracts was also determined by HPLC with detection at 280 nm. All experiments were performed in duplicate.

Oil samples were oxidised in an oven at 60 °C. Progress of oxidation was monitored by determination of the conjugated dienes content (CD) (AOCS Official Method Ti 1a-64) and p-anisidine value (AV) (AOCS Official Method Cd 18-90). 2.12. DPPH test

2.10. Calibration curves and quantification of phenols by HPLC and HPLC–MS. Reference compounds Hydroxytyrosol was synthesised from 3,4-dihydroxyphenylacetic acid (Sigma–Aldrich, Quimica-S.A., Madrid, Spain) according to the procedure of Baraldi, Simoni, Manfredini, and Menziani (1983). Hydroxytyrosol acetate was synthesised from hydroxytyrosol according to the procedure of Gordon, Paiva-Martins, and Almeida (2001). Oleuropein was purchased from Extrasynthese (Genay, France). The oleuropein aglycon 3,4-DHPEA-EA was obtained from oleuropein by enzymatic reaction using b-glycosidase (Fluka, Buchs, Switzerland) according to the procedure of Limiroli et al. (1995). The dialdehydic form of elenolic acid linked to hydroxytyrosol (3,4-DHPEA-EDA) was obtained from olive leaves according to the procedure of Paiva-Martins and Gordon (2001). Chlorogenic acid and caffeic acid were purchased from Sigma–Aldrich (Lisbon). The HPLC system comprised of a Merck Hitachi chromatograph with a Merck Hitachi L-6200 Intelligent Pump and a 250 mm  4.6 mm Waters Spherisorb ODS2 5 lm column (Supelco Inc.), coupled to a Merck Hitachi L-4200 UV–vis detector and components were detected at 280 nm with elution at room temperature. The composition of samples was determined by HPLC using a flow rate of 1 ml min 1 and, as a mobile phase, a mixture of 0.1% formic acid (pH 3.1) in water (A) and methanol (B) with a total

1,1-Diphenyl-2-picrylhydrazyl (DPPH) radical was used as a stable radical (Brand-Williams, Cuvelier & Berset, 1995). Each extract solution (20 ll) was added to 0.28 ml of a 0.08 mM methanolic 1,1-diphenyl-2-picrylhydrazyl radical solution. The decrease in absorbance was determined at 515 nm for 15 min. The exact initial 1,1-diphenyl-2-picrylhydrazyl radical concentration in the reaction was calculated from a calibration curve. The percentage of remaining DPPH radical was calculated for each extract tested. In the case of standards, a-, b-, c- and d-tocopherols (Sigma–Aldrich, Lisbon), several concentrations were tested and the change in absorbance with time was plotted. From this graph, the percentage of 1,1-diphenyl-2-picrylhydrazyl radical remaining was determined a number of times. The values were transferred onto another graph showing the percentage of residual DPPH radical as a function of the molar ratio of phenolic compound to DPPH radical. Antiradical activity was defined as the relative concentration of standard required to lower the initial DPPH concentration by 50% [EC50 (mol/l)]. 2.13. Statistical analysis SPSS 16.0 software was used for statistical analysis by one-way analysis of variance (ANOVA, with Tukey’s HSD multiple comparison) with the level of significance set at p < 0.05.

800

U P Pt M

700 600 500

Tocopherols/ mg kg -1

L. Silva et al. / Food Chemistry 121 (2010) 1177–1187

Total Tocopherols/ mg kg -1

1180

α γ δ

500 400 300 200 100 0

400 300 200 100 0

EVOO

VOO

OO

SFO

CO

SO

PNO

3. Results and discussion 3.1. Tocopherol content in processed oil samples Tocopherols act as antioxidants by trapping the hydroperoxide intermediates and stopping the autoxidation chain reaction. Differences in relative amounts of tocopherols are important: a-tocopherol affects human nutrition and health aspects, while ctocopherol shows a strong activity in seed protecting compounds, like fatty acids. On the other hand, the antioxidant activity of tocopherols in bulk oils is greatly dependent on its concentration. Huang, Frankel, and German (1994) observed that at 100 ppm, atocopherol exerted the best antioxidant activity in corn oil when compared with c-tocopherol. However, at concentrations higher than 250 ppm, a-tocopherol began to show prooxidant effects and the tocopherol with the best activity at concentrations up to 1000 ppm was the c-tocopherol (Huang et al., 1994). d-Tocopherol, besides being less active than other tocopherols at lower concentrations (100–1000 ppm), did not show any prooxidant activity in corn oil, even at 5000 ppm (Huang, Frankel, & German, 1995). The amount of tocopherols was very high in SFO, CO and SO (Table 1). All seed oils show a preponderance of c-tocopherol except sunflower oil where a-tocopherol is the most important. Extra-virgin olive oils always show a relatively high level of a-tocopherol and in these particular olive oils samples other tocopherols were not detected. Usually high amounts of tocopherols are associated with a high PUFA content and in fact soybean oil with the higher content in PUFAs was also the oil with the highest content in tocopherols, especially of c-tocopherol. For CO and SO samples, the decrease of c-tocopherol was faster than the decrease of a-tocopherol (Fig. 1, insert). Some studies (Huang et al., 1994, 1995) have shown that at this level of concentration found in seed oils, the order of activity is c > a > d, which is in accordance with these results. In the presence of c-tocopherol, a sparing effect can be observed on a-tocopherol. Food showed a sparing effect on the tocopherol content (Fig. 1) in EVOO, VOO, SFO and PNO samples, which probably contributes to the better oxidative stability of these samples. It was also interesting to note that the content in tocopherols was not as dramatically affected by the thermal treatment as was the polyphenolic content.

Total Phenols/ mg kg -1

Fig. 1. Tocopherol content of samples. Mean (error bars represent standard deviation) of triplicates. U, unprocessed oils; P, oils processed at 180 °C; Pt, oils processed at 180 °C with potatoes; M, oils processed at 180 °C with meat. The insert in this figure shows the contribution of the different tocopherols (a, c and d) to the total tocopherol content of each seed oil sample in the bottom.

350 a 300 250 200 150 100 50 0

U P Pt M

b b c

c

c

c cd

EVOO

cd

VOO

d d d OO

Fig. 2. Total phenolic content of samples. Mean (error bars represent standard deviation) of triplicates. U, unprocessed oils; P, oils processed at 180 °C; Pt, oils processed at 180 °C with potatoes; M, oils processed at 180 °C with meat. Different superscripts indicate samples that were significantly different (p < 0.05).

3.2. Phenolic content in processed oil samples All polyphenolic components decreased in concentration with the thermal treatment (Fig. 2). In the absence of food, phenolic alcohols were the phenols more affected and lignans the ones less affected (Fig. 3). An increase of hydroxytyrosol and tyrosol concentration with the thermal treatment, which is reported by some authors (Brenes et al., 2002), was not observed. Heating EVOO in the absence of food, resulted in a relatively minor loss of the secoiridoids 3,4-DHPEA-EDA (25%) and 3,4-DHPEA-EA (40%), compared to that in VOO (80% and 70%, respectively) and OO (90% and 98%, respectively). This result may point out the importance of the initial concentration of phenolic compounds in the oil. Moreover, the presence of different quantities of the several polyphenolic compounds in the oil will interfere with the relative stability of each other. In the absence of food, 3,4-DHPEA-EA showed to be less stable than 3,4-DHPEA-EDA, which is in accordance with previous results obtained by other authors (Allouche, Jiménez, Gaforio, Uceda, & Beltran, 2007). However, in the presence of food, a dramatic loss of 3,4-DHPEA-EDA (98%) and 3,4-DHPEA-EA (70%) was observed (Fig. 3) in EVOO samples. Apparently, the decrease in the concentration of phenolic compounds in the presence of food was more dramatic in the oil richer in these compounds. Chiou et al. (2007)

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Concentration mg / kg

140

140

Hy-EDA

120 100

a Hy-EA

120

a

100

b

80 60

60

40

40

d

20

b

80

c c

d

e

c

c c

e 20 c c

c

c

b

d

cd de e e de

0

EVOO

Concentration mg / kg

100

VOO

OO

a b

80

EVOO

100

VOO

80

Py+Ty-EDA

60

OO

PyAc

60

d

40

40

d d c c

20

c c c

a c c 20

b b b

ab b bc c

b

c c c

0

Concentration mg / kg

EVOO 14

VOO

a a

12

VOO

b

d

b b

12

Ty

b

4

b

2

8

a

a b cc

c2

c

HyAc

10

8 6

OO

14

10

8

4

EVOO

12

Hy

10

6

OO 14

a

6

b

b

4 a a

cc

cc

a

2

b ab b b

a

0

EVOO

VOO U

OO

EVOO P

VOO

OO Pt

EVOO

VOO

OO

M

Fig. 3. Changes in the phenolic composition of the samples. Mean (error bars represent range of two determination for each duplicate). Hy, hydroxytyrosol; Ty, tyrosol; EDA, 3,4-DHPEA-EDA; EA, 3,4-DHPEA-EA; Py, pinoresinol; AcPy, acetoxypinoresinol. Different superscripts indicate samples that were significantly different (p < 0.05).

reported the retention of polyphenolic compounds by French fries processed in oils enriched with olive leaf extract. Therefore, since the decrease in phenol concentration in the oils could be due to the diffusion of these compounds into foods, we investigated the content of potatoes in olive polyphenolic compounds by HPLC. In the methanolic extracts obtained from potatoes processed in EVOO (Fig. 4) secoiridoids were not found and only unidentified compounds with low retention time could be observed. However, a small amount of chlorogenic acid could be determined (Table 2). Only a small increase in the total phenolic content of potatoes processed with EVOO could be observed but this increase, mainly due to olive oil absorbed on the surface of potatoes, was not enough to explain the loss of phenolic compounds in these samples. In fact, the oil uptake by potatoes (150 g) was estimated to be 8.4 ± 0.9 g. According to the phenolic concentration found in the olive oil processed in the presence of potatoes, this amount of oil should contain around 0.7 mg of total phenolic compounds and this value is in accordance with the one found in potatoes. Since phenolic compounds did not diffuse into food samples, the elemental content of potatoes and meat must have made a huge contribution to the loss of phenolic compounds. In fact, both potatoes and meat are very rich in important elements, such as iron. Depending on the cultivar, iron can be found in potatoes in a concentration higher than 25 ppm (dry matter) and copper can also be present in a concentration of up to 10 ppm (dry matter) (Bethke & Jansky, 2008). Small concentrations, such as 0.4 ppm, of these ions

Table 2 Polyphenolic content of raw and cooked potatoes. Potato sample

Raw Processed in EVOO Processed in VOO Processed in OO

Polyphenolic content of raw and cooked potatoes TPP (mg CAE/100 g potato)

Chlorogenic acid (mg/100 g potato)

0.23 (0.13)a 0.43 (0.17)b

0.17 (0.06)a 0.04 (0.02)b

0.21 (0.08)a

0.05 (0.02)b

0.18 (0.07)a

0.03 (0.02)b

Mean (standard deviation in parentheses) of two determinations in three independent experiments. Superscripts within a column indicate samples that were significantly different (p < 0.05). TPP, total polyphenols determined by HPLC with detection at 280 nm; CAE, caffeic acid equivalents.

have been shown to have dramatic effects on the antioxidant activity of these polyphenolic compounds in emulsions and bulk oils because they increase the rate of phenolic compound destruction by oxidation and this rate varies depending on the compound (PaivaMartins & Gordon, 2002, 2005; Paiva-Martins, Mangiricão, & Gordon, 2007). During the polyphenolic determination by HPLC of the olive oils, two new peaks arose in the chromatogram, unknown 1 and unknown 2 (Fig. 4). Peaks from unknown 1 were more intense

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L. Silva et al. / Food Chemistry 121 (2010) 1177–1187

HO

[A]

O

COOCH3 O

[B]

[M-A]- (m/z 195) [M-A-B]- (m/z 139)

O Elenolic acid derivative PM: 242 m/z-: 241

1

120

mv

7

4

100

U1

80

U2 5

60

2

9

40

8

6

3

Unknown 1/ Relative area

20 0 0

10

20

30 30

40

50

(Fig. 4) for 4-formyl-3-[methyloxycarbonyl-2-oxo)ethyl]-hex-4enoic acid. In the presence of food, an increase in the hydrolytic degradation of secoiridoids (probably 3,4-DHPEA-EA and 4-HPEAEA) was observed, probably enhanced by small amounts of water leaked from foods or occurring at the surface of food. This result was in accordance with the lower concentration of these secoiridoids found in the oil from samples containing foods. Nevertheless, as already stated, the increase in the concentration of hydroxytyrosol and tyrosol was not observed, suggesting that these compounds are rapidly consumed by the oil as a response to the high temperature. Compound 2 was only detected in samples containing food and its peaks were more intense for olive oil samples with higher polyphenolic content. Compound 2 could not be identified because other compounds, such as the methanolic 4-DHPEA-EDA derivative (m/z = 335; other fragments: m/z 199, 155, 111), co-eluted. However it showed an usual basal fragment of m/z = 221 in the negative ion mode spectra. Chlorogenic acid (3-O-caffeoylquinic acid) constitutes about 90% of the total phenolic compounds of potato tubers (Dao & Friedman, 1992). This compound was not, however, identified in the phenolic extract of oils processed with potatoes. Nevertheless, since this compound also has a high antioxidant activity (Kayano, Kikuzaki, Fukutsuka, Takahiko, & Nakatani, 2002), it could have been consumed in the protection of oils. In fact a spare effect on tocopherol in most of the samples containing potatoes was observed (Fig. 1). During the extraction of tocopherols from oils by a mixture of methanol/isopropanol, other compounds are also extracted such as polyphenolic compounds. However, no phenolic compounds were revealed by the HPLC chromatogram of these extracts except, as expected, for olive oil samples. 3.3. Level of oxidation and stability of processed oil samples

Time / min 3000

U P Pt M

2500 2000 1500 1000 500

0

EVOO

VOO

OO

Fig. 4. Concentration of unknown 1 in samples (relative area of the chromatogram picks), HPLC chromatogram of EVOO phenolic extract before (grey line) and after processing (black line) in the presence of potatoes, HPLC chromatogram of polyphenolic extract obtained from potatoes processed in EVOO (- - -), mass spectra in full-scan mode and mass spectra in product ion scan mode of m/z 241 (MS2) and proposed negative electrospray ionisation fragmentation scheme for the unknown secoiridoid derivative 1. 1, hydroxytyrosol; 2, tyrosol; 3, hydroxytyrosol acetate; U1, unknown 1; 4, 3,4-DHPEA-EDA; U2, unknown 2; 5, 4-DHPEA-EDA; 6, acetoxypinoresinol; 7, 3,4-DHPEA-EA; 8, 4-DHPEA-EA; 9, chlorogenic acid.

for olive oil samples with higher polyphenolic content and, within each olive oil grade, in samples containing food. The deprotonated molecule [M H] (m/z = 241) was identified in full-scan mode as the only peak for unknown 1. The UV spectra of the unknown 1 revelled a peak at 232 nm, corresponding to p ? p* transitions of an a,b-unsaturated aldehydic group and another peak at 280 nm corresponding to n ? p* transitions of carbonyl groups. Since the strong peak at 240 nm, characteristic of a a,b-unsaturated ester group substituted at the b position with a O-alkyl group was not present, unknown 1 could not be elenolic acid (Mr = 242) but one of its isomers. Mass spectra in production scan mode of m/z 241 proved useful in confirming the proposed fragmentation scheme

It has been reported that the consumption of products from oxidised fats seems to be involved in several pathological conditions (Esterbauer, 1993). Thus, there is considerable interest in the evaluation of the oxidation state of oils after processing. The level of oxidation of processed oils was assessed by the conjugated diene content (%CD) to determine primary oxidation products, by the p-anisidine value determination (AV) to determine secondary oxidation products and by the oil TPC content to determine the oxidative polymerisation degree. Levels of TPC in unheated and heated oil samples for short periods of time are usually very low. Usually oils reach the rejection limit (set at 25–27% TPC) after more than 10 h of deep fry heating (180 °C). Since the conjugate diene and carbonyl compound content and the total antioxidant content of oil samples change drastically long before the oils became thermally abused in terms of TPC (TPC = 25–27%), %CD and AV determinations were found to be more relevant to assess differences between oil sample level of oxidation. Furthermore, polymeric polar compounds are more easily formed in deep frying operations where the surface of contact with air oxygen is very low. Since higher absorbance values at 233 nm are expected, depending on their characteristic chemical composition, for some of the oils, the difference between the absorbance obtained for each sample minus the absorbance obtained for the unprocessed sample of each oil was used to evaluate the increase in conjugated diene content. In fact, some of the samples may decrease their absorbance at this wavelength due to the loss of volatile unsaturated compounds during the thermal processing but an increase in these values can only be due to an increase in the conjugated diene content. Therefore, on the basis of the increase in the conjugated diene content, the use of olive oil showed an important pro-

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0.60

Δ %CD

0.50

c c

P Pt M

0.40

d

d

0.30

d

0.20 0.10

a

b b

a

a b b

ababab

b

0.00 -0.10

ROO

OO

EVOO

a

SFO

CO

a

ab

ba SO

PNO

70 60

P Pt M

Δ AV

50 40 30 20 10 0 -10-

ROO

OO

EVOO

SFO

CO

SO

PNO

b

14

ΔTPC / %

12

P Pt M

10 8

c

c d

6 4

a a a

a

a a

a a a

a

a f f

f e

e e

2 0

ROO

OO

EVOO

SFO

CO

SO

PNO

Fig. 5. Conjugated diene content, AV and TPC content of samples after processing. Values are related to the initial CD content, AV or TPC of oils. Mean (error bars represent standard error) of triplicate processed samples. P, oil processed at 180 °C; Pt, oil processed at 180 °C with potatoes; M, oil processed at 180 °C with meat. Different superscripts indicate samples that were significantly different (p < 0.05).

Table 3 1,1-Diphenyl-2-picrylhydrazyl radical-scavenging effects of tocopherols after various reaction times (15, 60 and 250 min). Compound

a-Tocopherol d-Tocopherol c-Tocopherol

Time 15 minB

Time 60 minB

EC50A

EC50A

a

0.25 (±0.01) 0.31b (±0.01) 0.42c (±0.01)

Number of reduced radicals 2.0 1.6 1.2

a

0.25 (±0.01) 0.30b (±0.01) 0.41c (±0.01)

Time 250 minB Number of reduced radicals

EC50A

Number of reduced radicals

2.0 1.6 1.2

0.24a (±0.01) 0.30b (±0.01) 0.40c (±0.01)

2.1 1.6 1.2

Superscripts within a column indicate samples that were significantly different (p < 0.05). A EC50 expressed as mol of antioxidant/mol of 1,1-diphenyl-2-picrylhydrazyl radical. B Mean (standard deviation in parentheses) of four determinations.

tection of food when compared with other oilseeds (Fig. 5). Furthermore, the p-AV and levels of TPC in unheated and heated olive oil samples (Table 3) also show a remarkable stability of these samples against hydroperoxide degradation and oxidative polymerisation. Most of the other oilseed samples had a higher level of oxidation, with all sunflower oil samples being oxidised after processing at 180 °C (Fig. 5) and already showing an important percentage of TPC (Fig. 5). The high a-tocopherol content in sunflower oil sam-

ples (Table 1) did not prevent the oxidation of those samples much richer in linoleic acid probably because, at this concentration, atocopherol exhibits some prooxidant activity (Huang et al., 1995). However, PNO samples, with a low content in tocopherols, showed a lower level of oxidation probably because of their lower content of PUFAs (Table 1) and a a-tocopherol content in the range in which this tocopherol is able to exert its maximum antioxidant activity (Huang et al., 1994, 1995). SO samples, very rich in PUFAs, were the ones with the highest content in tocopherols, in particular

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1,4 U P Pt M

1,2

Δ%CD

1,0 0,8

c c c c

c b

b

c b

b

b

b

b

0,6 0,4 0,2

a a a a

a a a a

a

a a

0,0 EVOO

VOO

OO

CO

SO

PNO

U P Pt M

U P Pt M

U P Pt M

CO

SO

PNO

100

U P Pt M

80

Δ AV

60 40 20 0

-20

U P Pt M

U P Pt M

EVOO

VOO

U P Pt M OO

Fig. 6. Conjugated diene content and AV of unprocessed and processed samples after 15 days of storage at 60 °C. Values are related to the initial CD content or AV of oils. Mean (error bars represent standard error) of triplicate stored samples. U, unprocessed oils; P, oils processed at 180 °C; Pt, oils processed at 180 °C with potatoes; M, oils processed at 180 °C with meat. Different superscripts indicate samples that were significantly different (p < 0.05).

in the most active tocopherols in the range of concentrations present in the oils, c and d, which probably contributes to the low level of oxidation of these samples after processing. The presence of food showed a protective effect on most of the oils, with oil samples processed without food showing a higher level of oxidation than the oil samples processed in the presence of food. Actually, for CO and PNO, only the oil samples processed without food were oxidised after processing. In the case of the PNO sample, its high level of oxidation was probably related to the total destruction of tocopherols with the thermal treatment but no explanation could be found in the case of the CO sample, which still had high tocopherol content. The AV and TPC determinations, however, confirmed these results (Fig. 5). At the end of meat roast processing there is always a significant amount of Maillard reaction products (MPRs) at the surface and therefore they may have contributed to the observed results. It is well known that the Maillard reaction influences the oxidative stability of food products. However, the exact nature of the antioxidants formed is not yet well known. The most commonly recognised MRPs with antioxidative activity are the high molecular weight melanoidins. Furthermore, melanoidin has also exhibited a synergistic effect with tocopherol, BHA, and BHT, in inhibiting the autoxidation of linoleic acid. Chlorogenic acid of potato tubers, due to its high antioxidant activity (Kayano et al., 2002), must have contributed to the protection of oils. It was odd to observe that olive oil samples processed with food, besides containing lower polyphenolic concentration, showed a better stability than the olive oil samples processed without food. In these samples, however, a higher concentration of tocopherol was observed. Olive oil (EVOO, VOO and OO) samples and the oilseed samples that were not oxidised after processing were stable after a month of storage at 4 °C (data not shown). To determine the oxidative sta-

bility of oils after processing and therefore, the antioxidant capacity of the remaining antioxidants in the oils, samples were oxidised in the dark at 60 °C. After 15 days at 60 °C (Fig. 6), all oilseed samples and only OO samples processed without food were oxidised, anticipating that the antioxidative system is being depleted earlier in these samples than in VOO and EVOO samples. The low phenolic content in the OO sample and the absence of tocopherol in the OO and PNO samples may have contributed to this result (Figs. 1 and 2). The level of oxidation of OO, SO and PNO samples processed in the presence of potatoes and meat were lower (Fig. 6), as already observed for these samples after processing. Once more, the AV (Fig. 6) determinations confirmed these results. After 25 days, EVOO samples were still with a low level of oxidative degradation but VOO samples processed in the presence of foods, in contrast with seed oils, showed more oxidative degradation than the samples processed without food (Fig. 7). In the case of olive oil samples the remaining phenolic content seems to play an important role in their stability. According to the literature, lipid oxidation should be much faster in cooked meat than in fresh meat, due to the fact that cooking induces an acceleration of oxidative processes as a result of the high temperatures reached, destruction of cellular structures and interactions of lipids and prooxidants and the release of non-haem iron. The mean oil absorption by meat ranged from 7% to 11% depending on the oil type. In general, meat samples processed in olive oil were richer in oil but differences between means did not show statistical significance (data not shown). However, all types of olive oil, but not all seed oils, were shown to protect processed meat and potatoes after storage at 20 °C (Fig. 8). In general, the level of oxidation of lipids extracted from meat and potatoes were similar to the level of oxidation of the correspondent oils (Fig. 8). However, lipids extracted from meat processed in SO were not stable during the lipid extraction by hot hexane. This meat, besides

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c 1,8 1,6 1,4 1,2

Δ % CD

d

U P Pt M

1,0 0,8

b b a a a a

a

b b

a

0,2 0,0

EVOO

VOO

OO

d

U P Pt M A

50 40 30 20

d e c

c

c b

b b

10 a a a

b

ab a

0

EVOO

0,6 0,4

60

VOO

OO

Fig. 9. Changes in the antioxidant activity of olive oil phenolic extracts assessed by the DPPH assay after 15 min of reaction. Mean (error bars represent standard deviation) of quadruplicates. U, unprocessed olive oils; P, processed olive oils at 180 °C; Pt, processed olive oils at 180 °C with potatoes; M, processed olive oils at 180 °C with meat; A, aqueous phase from meat samples. Different superscripts indicate samples that were significantly different (p < 0.05).

100

U P Pt M

Δ AV

80 60 40 20 0 -20

EVOO

VOO

OO

Fig. 7. Conjugated diene content and AV of unprocessed and processed olive oil samples (EVOO, VOO and OO) after 25 days of storage at 60 °C. Values are related to the initial CD content or AV of oils. Mean (error bars represent standard error) of triplicate stored samples. U, unprocessed oils; P, oils processed at 180 °C; Pt, oils processed at 180 °C with potatoes; M, oils processed at 180 °C with meat. Different superscripts indicate samples that were significantly different (p < 0.05).

Δ% CD

1.2

M2 Pt

0.6 0.4 0.2 0

c

M1

1.0 0.8

a a ab

a a ab

d

a a b

-0.2 -0.4 EVOO VOO OO

e de abab b

SFO CO

b ab ab ab b

SO

PNO

Fig. 8. Conjugated diene content of oil extracted from meat samples after processing and storage for 30 days at 20 °C and of oil extracted from potatoes samples after processing and storage for 4 months at 20 °C. M1 – oil extracted from meat by hot hexane (Soxhlet apparatus). M2 – oil extracted from meat by cold hexane (by homogenisation). Pt – oil extracted from potatoes by cold hexane (by homogenisation). Values are related to the initial CD content of meat lipids or related to the initial CD content of used oils in the case of potatoes samples. Mean (error bars represent standard error) of triplicates. Different superscripts indicate samples that were significantly different (p < 0.05).

the low level of oxidation after processing, would probably not be stable after a further heating process. 3.4. Determination of the radical-scavenging activity of extracts Several authors have determined the radical-scavenging activity of oils spectrophotometrically by measuring the disappearance

of the 1,1-diphenyl-2-picrylhydrazyl radical (DPPH) at 515 nm (Espin, Soler-Rivas, & Wichers, 2000; Gómez-Alonso et al., 2003; Kalantzakis et al., 2006; Pellegrini et al., 2001). It has been reported that the radical-scavenging activity, measured by this method, decreased rapidly during heating or frying potatoes (Gómez-Alonso et al., 2003; Pellegrini et al., 2001). However, besides its lower concentration after processing, polyphenolic extracts from olive oil still kept their high radical-scavenging activity (Fig. 9) and this activity positively correlates with sample phenolic content. Polyphenolic extracts from aqueous phases also showed an important radical-scavenging activity, which may have contributed to the stability of meat samples. When the more lipophilic extracts, containing tocopherols, were tested, peculiar results were observed. Some extracts showed higher radical-scavenging activity after processing than before. Moreover, some flotation during the time of reaction could be observed (data not shown). On the other hand, instead of a decrease, an increase of absorbance intensity of the DPPH radical solution after addition of extracts from EVOO and SFO samples was observed. Since the EVOO extract also contains polyphenols besides tocopherol, and therefore a decrease in the absorbance should be expected, this increase could only be obtained if coloured compounds absorbing at 515 nm were formed during the reaction with the DPPH radical. In order to understand if tocopherols could produce such coloured compounds, a DPPH test was performed with pure standards of a-, d- and c-tocopherols. As expected for atocopherol, none of the other tocopherols showed such behaviour with the DPPH radical, with the intensity of colour decreasing during the reaction with the DPPH radical solution (Table 3). However, other compounds such as chlorophylls, squalene, and carotenoids, which were not quantified in this study, may interfere in this reaction. In fact, the polyene structure of carotenoids provides a chromophoric system which leads to interference in the DPPH method currently using the 515 nm wavelength. Therefore, the value of the absorbance at this wavelength at the reaction plateau step is due to the carotenoid, to the DPPH and possibly also to the reaction products. Since the higher the percentage of DPPH remaining, the lower the radical-scavenging activity of the carotenoid, all the measures of antioxidant activity using this wavelength are therefore underestimated (Goupy, Hugues, Boivin, & Amiot, 1999). However, in most of the studies the comparison is done between the radicalscavenging capacities of different oils and therefore this interference does not create peculiar results. Also the use of this method to compare fresh oils and highly oxidised oils may also not show this interference. In order to avoid this interference, some authors (Nomura, Kikuchi, Kubodera, & Kawakami, 1997) have used a blank of solo carotenoid at the beginning of the reaction, but in our case this correction was not useful because of the bleaching of the

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carotenoids during the course of the reaction. Moreover, Nomura et al. (1997) reported that in contrast to the anoxic reactions, under aerobic conditions, only a part of the carotenoid fucoxanthin consumed the DPPH radical and the degree of reaction fluctuated with repeated trials and this partial and unstable reactivity of fucoxanthin with DPPH suggested that fucoxanthin was partially oxidised by molecular oxygen with the aid of DPPH. Kalantzakis et al. (2006) reported only a reduction of 11–30% of the initial radical-scavenging activity of several oils (some of them were SO, SFO) after 2.5 h of thermal treatment at 180 °C. Even after 10 h of treatment, oils still kept a high radical-scavenging activity (16–30% of the initial radical-scavenging activity) and this was correlated by the authors with the initial tocopherol content. However, these authors also comment that it was odd to observe such a high radical-scavenging activity in oils so heavily oxidised. In the present study, a high radical-scavenging activity for processed seed oil containing samples was also observed. However, and since these samples were only processed for 1 h, this activity was even higher than for unprocessed samples. Polyphenolic compounds were not detected in these more lipophilic seed oil extracts, and therefore, the radical-scavenging activity observed with them should correlate with their tocopherol content. However, during processing, compounds with radical-scavenging activity must have been formed and this activity did not correlate, either with the tocopherol content or with sample oxidative stability. 4. Conclusion After roast processing, all sunflower samples were oxidised. In contrast, all olive oil samples were not oxidised, independently of the olive oil quality used. However, the shelf life was longer for samples containing extra-virgin olive oil and this fact apparently correlated positively (R = 0.64) with the higher phenolic content of these samples. The radical-scavenging activity of EVOO was higher than for other olive oil samples and also positively correlated (R = 0.87) with the phenolic content of the oil. Besides all olive oil samples not exhibiting deleterious properties to human consumption, samples containing higher amounts of radical-scavenging compounds are probably capable of exerting an extra beneficial biological activity. Seed oil antioxidants showed little capacity in delaying the oxidative degradation of oils and meat processed with them. However, tocopherol content and the identity of tocopherols present in the oil were shown to have a more important role in the oxidative stability of seed oils than the fatty acid composition. In fact, the excellent behaviour of SO samples, the oil with the higher degree of unsaturation, is a proof of the complexity of oxidation and how important the antioxidant/prooxidant species balance present in the oils is. This study also brings to attention the differences observed between ideal conditions and more realistic processing conditions, since food brings dramatic changes in the stability of oils and some of their components, such as polyphenols and tocopherols. References Allouche, Y., Jiménez, A. M., Gaforio, J., Uceda, M., & Beltran, G. (2007). How heating affects extra virgin olive oil quality indexes and chemical composition. Journal of Agricultural and Food Chemistry, 55, 9646–9654. Andrikopoulos, N. K., Kalogeropoulos, N., Falirea, A., & Barbagianni, M. N. (2002). Performance of virgin olive oil and vegetable shortening during domestic deepfrying and pan-frying of potatoes. International Journal of Food Science and Technology, 37, 177–190. Aparicio, R., Roda, L., Albi, M. A., & Gutiérrez, F. (1999). Effect of various compounds on virgin olive oil stability measured by rancimat. Journal of Agricultural and Food Chemistry, 47, 4150–4155. Ashok, B. T., & Ali, R. (1999). The aging paradox: Free radical theory of aging. Experimental Gerontology, 34, 293–303.

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