Effect of Thymus vulgaris and Bunium persicum essential oils on the oxidative stability of virgin olive oil

Natural antioxidants are becoming a major focus because natural food ingredients are safer than synthetic types. The aim of this study was to investigate the protective effects of Thymus vulgaris and Bunium persicum essential oils (EO) on the oxidation of virgin olive oil (VOO) during accelerated storage. The antioxidant activities of EOs were compared with those of α-tocopherol and BHT. GC/MS analyses revealed that thymol (28.50%), p-cymene (27.14%), carvacrol (18.36%), and γ-terpinene (4.97%) are the main components of T. vulgaris EO, while cuminaldehyde (32.81%), γ-terpinene (16.02%) and p-cymene (14.07%) are the main components of B. persicum EO. Both EOs provided protection for the VOO, inhibiting the formation of primary and secondary oxidation products although T. vulgaris EO showed greater protection against the oxidation process than B. persicum EO. The effect of T. vulgaris essential oil on the oxidation inhibition of VOO was similar to that of BHT. α-Tocopherol showed no measurable effect on improving the oxidative stability of VOO. This study suggests that T. vulgaris and B. persicum EOs can be used to improve the oxidative stability of VOO.


INTRODUCTION
Virgin olive oil (VOO) is highly appreciated for its delicious taste and aroma, as well as for its nutritional properties.Its nutritional benefits are primarily related to its fatty acid composition, mainly due to the high content of oleic acid and also to the balanced ratio of saturated and polyunsaturated fatty acids (PUFAs).Furthermore, olive oil presents considerable amounts of natural antioxidants (Moldão-Martins et al., 2004).During storage, oxidation reactions reduce the high nutritional value of VOO and modify its characteristic flavor through the development of offflavors derived from hydro peroxide decomposition products (Morales et al., 1997).
Among the most usual strategies to inhibit lipid oxidation in vegetable oils, the addition of antioxidants has been practiced for decades.However, recent studies claim that synthetic antioxidant compounds could pose possible hazards and carcinogenic effects (Sasaki et al., 2002).Moreover, according to the Codex Alimentarius Commission, synthetic antioxidants are not permitted for use in VOO (Codex, 2011).Furthermore, natural antioxidants such as tocopherols and their derivatives, which can be used as alternatives to BHA and BHT, exhibit little effectiveness in some systems and also increase manufacturing costs.Consequently, there is the need to identify alternative natural and safe sources of antioxidants to be incorporated into food products.These safer sources of antioxidants can be especially of plant origin, where relevant research has notably increased in recent years.
Buniumpersicum is an important aromatic plant that belongs to the Apiaceae family.It originates from central Asia to North India.The γ-Terpinene, cuminaldehyde, p-cymene and limonene are major components of B. persicum EO (Mazidi et al., 2012).Thymus vulgaris (also known as common thyme) is a member of the Labiatae family.Dried plant materials of T. vulgaris contain 1 -2.5% EO.Meanwhile, Thymol, carvacrol, p-cymene, and γ-terpinene are the main components of T. vulgaris EO (Golmakani and Rezaei, 2008).
Essential oils (EOs) obtained from aromatic plants have received considerable attention in the current era of concerns for food safety.For instance, a report claims that Carumcopticum EO (0.075%) is more effective than BHA and BHT (0.02%) in retarding the oxidation of sunflower oil (Hashemi et al., 2014).Furthermore, Inanc and Maskan (2014) reported that carvacrol can significantly improve the oxidative stability of palm oil in comparison with the control sample.Also, the antioxidant activity of carvacrol is known to be similar to that of BHT.Ruberto and Baratta, (2000) reported that γ-terpinene (which is the major component of B. persicum EO) shows a very high antioxidant activity.More specifically, γ-terpinene has a comparable activity to that of α-tocopherol.
The objective of this study is to compare the effects of T. vulgaris and B. persicum EOs on the oxidation of VOO during accelerated storage.The antioxidant activities of EOs are compared with those of α-tocopherol and BHT.

Materials
Dried seeds of B. persicum and the dried aerial parts of T. vulgaris were purchased from a local market in Shiraz, Iran.The genus and species of both plants were confirmed by experts from the Herbarium of Biology Department at Shiraz University, Shiraz, Iran.VOO was supplied from the Etka Oil Company (Rudbar, Iran).All chemicals used in this research were of analytical grade and were purchased from Merck (Darmstad, Germany) and Sigma-Aldrich (St. Louis, MO, USA).

Extraction of EO
Fifty grams of each plant sample were mixed with 500 mL of distilled water.They were hydrodistillated for 3 h using a Clevenger-type apparatus (Golmakani and Rezaei, 2008).The final yields of T. vulgaris and B. persicum EOs were reported here to be 2.13±0.04%and 1.92±0.32,respectively.EO samples were dried over anhydrous sodium sulphate and stored in sealed vials at -18 °C until further use.

GC Analysis of EO
The identification of EO constituents, known as a qualitative analysis, was made using a GC (7890A, Agilent Technologies, Santa Clara, CA) which was coupled with a mass spectrometer (5975C, Agilent Technologies, Santa Clara, CA) operating at 70 eV ionization energy, 0.5 s/scan, and a mass range of 35-400 atomic mass units (amu), equipped with a HP-5MS capillary column (5% Phenyl Polysilphenylene-siloxane; 30 m length; and 0.25 mm internal diameter; 0.25 μm film thickness, Agilent Technologies, Santa Clara, CA).One μL of the EO sample was injected into the GC/MS in split mode (split ratio: 1/100).Helium was used as the carrier gas with a flow rate of 0.9 mL/min.The injector and detector temperatures were at 280 °C.The oven temperature was programmed to start at 60 °C and gradually heated up to a temperature of 210 °C at a rate of 3 °C/min.Thereafter, the rate of temperature elevation was such that the temperature increased by 20 °C/min until the point of 240 °C was reached, whereupon the temperature was held constant for 8.5 min.The MSD ChemStation Software (G1701EA, E.02.01.1177,Agilent Technologies, Santa Clara, CA) was employed to analyze the mass spectra and chromatograms.The compounds were identified by comparing their mass spectral fragmentation patterns with those stored in the data bank (Wiley/NBS) and with mass spectral data derived from the relevant literature (Golmakani and Rezaei, 2008;Hashemi et al., 2014;Mazidi et al., 2012;Moldão-Martins et al., 2004;Shahsavari et al., 2008;Zeng et al., 2011).In addition, a quantitative analysis of EO constituents was made under the same chromatographic conditions using a GC, coupled with a flame ionization detector (FID).The relative data for percentages were obtained from the electronic integration of chromatogram peak areas.

Determination of EO antioxidant activity
The antioxidant activities of the EOs were evaluated based on the free radical scavenging capacity and their reducing power.

Free radical scavenging capacity
The free radical scavenging capacity of the EO samples were measured using DPPH o (2,2-diphenyl-1-picrylhydrazyl radical) as described by Mazidi et al. (2012).The IC 50 value is defined as the concentration of the antioxidant which is required to inhibit 50% of the DPPH° activity.Here, the IC 50 value was determined through graph plotting, by considering the percentage of the remaining DPPH° against the EO concentrations.

Ferric reducing assay
The ferric reducing power of EOs and that of the positive control (L-ascorbic acid) were determined here according to the method of Ardestani and Yazdanparast (2007).The reducing power was measured by reducing the Fe (III) to Fe (II).One mL of each EO solution (100-10000 mg•L −1 ) was mixed with 2.5 mL of sodium phosphate buffer (0.2 M, pH 6.6) and 2.5 mL of 10 g•L −1 potassium ferricyanide (K 3 Fe(CN) 6 ).The mixture was incubated at 50 °C for 20 min, whereupon 2.5 mL of 100 g•L −1 trichloro acetic acid was added to the mixture and centrifuged for 10 min at 3000 g.The upper layer of solution (2.5 mL) was mixed with 2.5 mL of distilled water and 0.5 mL of FeCl 3 (1 g•L −1 ).The absorbance was measured by the spectrophotometer at 700 nm.Generally, a higher absorbance value indicates a higher reducing power.Results were expressed as mg ascorbic acid equivalents per gram of sample.

Cupric ion reducing assay
The cupric ion reducing power of the EOs and the positive control (L-ascorbic acid) were determined here in a test tube by mixing together 1 mL of CuCl 2 solution (10 mM), 1 mL neocuproinemethanolic solution (7.5 mM) and 1 mL ammonium acetate aqueous buffer solution (1 M).EO sample solutions (0.50 mL, 100-10000 mg•L −1 ) and H 2 O (0.60 mL) were added to the initial mixture so that the final volume reaches 4.10 mL.The tubes were stoppered and, after 30 min, the absorbance was recorded against the blank at 450 nm (Apak et al., 2008).Results are expressed as mg of ascorbic acid equivalents per gram of sample.

Determination of initial characteristics of VOO
The initial characteristics of VOO were determined by measuring its chemical and physical properties as follows.

Determination of free fatty acid content
Free fatty acid content was determined according to the AOCS official method (Cd 3d-63) and was reported as a percentage of oleic acid (AOCS, 2000).

Determination of fatty acid composition of VOO
Fatty acid methyl esters were prepared here according to the method described by Golmakani et al. (2012).The composition and types of fatty acids in the VOO sample was analyzed using a GC system (SP-3420A, Beijing Beifen-Ruili Analytical Instrument, Beijing, China) which is a device equipped with a split/splitless injector, a flame ionization detector (FID) and a BPX70 capillary column (Bis-cyanopropylsiloxane-silphenylene, 120 m × 0.25 mm internal diameter; 0.25 μm film thickness, SGE Analytical Science, Melbourne, Australia).The temperatures of the column, injector and detector were set at 198 (isothermal), 250 and 300 °C, respectively.Nitrogen was used as the carrier gas.One μL of fatty acid methyl esters was then injected into the column with a split ratio of 1:10 accordingly.The fatty acids in the VOO were identified according to the retention times for standard fatty acids injected under the same operating conditions.The quantities of fatty acids were measured by calculating their relative peak areas.

Determination of total phenolic content
Here, total phenols were isolated from a solution of oil in hexane by triple-extraction with watermethanol (60:40 v/v).The amounts of phenols were estimated using the Folin-Ciocalteu reagent at 725 nm.Results were expressed as mg of gallic acid per grams of VOO (Casal et al., 2010).

Determination of chlorophyll and carotenoid contents
Chlorophyll and carotenoid contents were determined at 470 and 670 nm, respectively, according to the method described by Minguez-Mosquera et al. (1990).

Determination of oxidation indices of VOO
The peroxide value (PV) was determined according to the AOCS official method (Cd 8-53) and was expressed as meq O 2 •kg −1 VOO.The p-Anisidine value (AV) was determined using the AOCS official method (Cd 8-53) and was expressed as mg•kg −1 VOO (AOCS, 2000).The TOTOX value (total oxidation value; AV + 2 PV) is used as an empirical measure of the relevant precursors, the nonvolatile carbonyls, present in the processed oils.The TOTOX value can also be used as a measure of any further oxidation products developed after storage (Frankel, 2012).The K 232 and K 268 extinction coefficients were determined according to the AOCS official method (ch 5-91) by measuring the absorbance of a pertinent solution (1% concentration) in isooctane at 232 and 268 nm, with 1 cm of pass length (AOCS, 2000).

Accelerated storage of VOO
Here, the EOs of T. vulgaris and B. persicum were added to the VOO at a concentration of 1000 mg•L −1 .The BHT and α-tocopherol at 100 mg•L −1 concentration are added to the VOO.For the control group, a sample with no added antioxidants was used.VOO samples (70 mL) were then kept in open amber bottles in an incubator at 70±1 °C for 42 days.The PV, AV, K 232 and K 268 were measured weekly.Also, the chlorophyll and carotenoids were measured every two weeks, as they have been in this research.
The induction period of PV (IP pv ) is commonly considered as the number of days required for a sample to reach a PV of 20 meq O 2 •kg −1 , which is beyond the maximum permitted limit, when a samlpe consequently loses the classification of VOO category (Hashemi et al., 2014;IOC, 2015).
Generally, the IP K232 and IP K268 are considered as the number of days required to reach the upper legal limits of K 232 and K 268 (with a K 232 value of 2.6 and K 268 value of 0.25).These are established by the International Olive Council (IOC) for VOO (IOC, 2015).
The Average percentage of difference was calculated according to eq. ( 1) The effectiveness of the antioxidant, also known as the stabilizing effect, is defined as the induction period extension (IPE) according to eq. ( 2) (Abramovic and Abram, 2006).

Statistical analysis
All experiments were performed in triplicate and the data were reported as mean values of the measurements while presenting the standard deviation values in tables and the standard deviation bars in figures.A general linear model (GLM) procedure from SAS (Statistical Analysis Software, version 9.1; SAS Institute Inc. Cary, NC) was used for the comparison of mean values.The simple regression equations for the chemical variables that were obtained from the storage study of VOO (PV, K 232 , and K 268 ) were calculated by Microsoft Office Excel 2010.

GC analysis of EO
The chemical compositions of T. vulgaris and B. persicum EOs are presented in Table 1.The total numbers of chemical constituents identified in the EOs were measured to be 24 for B. persicum and 29 for T. vulgaris EO.The main components of B. persicum EO were cuminaldehyde (32.81%) and monoterpene hydrocarbons (γ-terpinene (16.02%) and p-cymene (14.07%)).Previous reports suggest that the antioxidant activity of γ-terpinene is significantly higher than that of p-cymene (Ruberto and Baratta, 2000).The T. vulgaris EO was characterized mainly by monoterpene phenols (thymol (28.50%) and carvacrol (18.36%)) and also by their corresponding monoterpene hydrocarbon precursors (p-cymene (27.14%) and γ-terpinene (4.97%)).Similarly, Golmakani and Rezaei (2008) reported that thymol, carvacrol, p-cymene, and γ-terpinene were the major compounds of T. vulgaris EO.On the contrary, camphor was not detected in our samples, but had been found as the main component of T. vulgaris EO from the plant source in Eastern Morocco (Imelouane et al., 2009).According to Ruberto and Baratta (2000) thymol and carvacrol possessed stronger antioxidant activity than camphor.

Determination of antioxidant activity of EOs
Antioxidants can scavenge radical species by hydrogen donation, which causes a decrease in DPPH° absorbance at 517 nm (Zeng et al., 2011).The radical scavenging capacity of T. vulgaris and B. persicum EOs are presented in Table 2.Both EOs managed to reduce the stable, purple-colored radical DPPH° into yellow-colored DPPH-H, reaching IC 50 values of 4.15 mg•mL −1 for B. persicum EO and 0.50 mg•mL −1 for T. vulgaris EO.There was no significant difference between the IC 50 value of T. vulgaris EO with that of BHT, whereas the IC 50 value of B. persicum EO was significantly higher than BHT.However, in previous studies, the radical scavenging capacity of B. persicum EO (IC 50 value of 0.88 mg mL −1 ) was significantly higher than that of T. vulgaris EO (IC 50 value of 8.9 mg•mL −1 ) and lower than that of BHT and α-tocopherol (IC 50 values of 0.58 and 0.2 mg•mL −1 , respectively) (Fazel et al., 2007;Shahsavari et al., 2008;Zeng et al., 2011).
Ferric ion (Fe 3+ ) and cupric reduction is often used as an indicator of electron-donating activity, which is an important mechanism of phenolic antioxidant action, and can be strongly correlated with other antioxidant properties (Apak et al., 2008;Zhang et al., 2010).The reducing powers of T. vulgaris and B. persicum EOs are presented in Table 2.Both results obtained from ferric and cupric reduction assays showed nearly the same outcome.T. vulgaris and B. persicum EOs showed some degree of hydrogen-donating capacity, but the capacities were, as expected, inferior to ascorbic acid.This is in agreement with the radical scavenging capacity results that T. vulgaris EO showed stronger reducing power than B. persicum EO.These results indicate that EOs rich in phenolic monoterpenes are more potent reductants and radical scavengers than those rich in monoterpene hydrocarbons.

Initial characteristics of VOO
The initial characteristics of VOO are presented in Table 3.The oxidative and hydrolytic integrity of the oil was confirmed by the low PV, K 232 , and K 268 values and by the low contents of free fatty acids which were below the upper legal limit established by IOC for VOO.The VOO contained high amounts of oleic acid (68.21%) and an appropriate ratio of monounsaturated fatty acid (MUFA)-to-PUFA.It also contained considerable amounts of phenolic compounds (286.30±1.16µg gallic acid equivalents g VOO −1 ) in the beginning of the assay.

Measurement of PV, AV, and TOTOX values
Primary oxidation products, namely hydroperoxides, were determined by PV measurement.Changes in the PVs of VOO samples during storage at 70 °C are illustrated in Figure 1.The PV of the control increased gradually until the 35 th day, indicating the high resistance of VOO to oxidation due to its own (naturally occurring) antioxidants and low unsaturation level.However, the rate of hydroperoxide formation of the control increased sharply after reaching a PV of 38.44 meq O 2 •kg −1 .This phase (after reaching a PV value of 38.44 meq O 2 •kg −1 ) indicated an accelerated degradation process.VOO supplemented with BHT, T. vulgaris EO, and B. persicum EO showed lower PVs in comparison with that of the control throughout the storage period.In the initial stages of oxidation, BHT and T. vulgaris EOs appear to be slightly more effective than B. persicum EO.However, the PVs of the samples containing BHT and T. vulgaris EO were significantly lower than that of B. persicum EO at the end of storage.This phenomenon may be due to the fact that natural antioxidants in the VOO are consumed at the initial stage of oxidation.Also, this result may be related to the fact that the different magnitudes of antioxidant activities observed among the various antioxidants are more evident at later stages of oxidation.The PV of the sample containing α-tocopherol was slightly lower than that of the control during the entire period of the experiment.The IP PV of the control, BHT, α-tocopherol, T. vulgaris EO,and B. persicum EO samples were 16.86,27.08,18.05,25.13,and 22.49 days,respectively.The anisidine test is designed to measure high molecular weights of saturated and unsaturated carbonyl compounds in triacylglycerols (Frankel, 2012).Changes in AVs of VOO samples during storage at 70 °C are illustrated in Figure 1.During the storage, the increasing trend observed for AV was very similar to that obtained for PV.
The control sample exhibited the highest AVs during the entire period of the experiment.Adding T. vulgaris and B. persicum EOs offered protection to the VOO, inhibiting the formation of undesirable flavors emanating from secondary lipid oxidation processes.Similar to the PV results, the effects of natural and synthetic antioxidants on delaying the formation of secondary oxidation products was clearly determined at the end of storage, and the order of inhibitory effects of natural and synthetic antioxidants was BHT > T. vulgaris, EO > B. persicum, EO > α-tocopherol.
The TOTOX value is an indicator of primary and secondary oxidation products.The results of the TOTOX values of the VOO samples during storage at 70 °C are presented in Figure 1.BHT, T. vulgaris EO, and B. persicum EO all reduced the formation of primary and secondary oxidation products in VOO by 78.51, 69.01, and 53.99%, respectively, when considered at the end of storage.This indicates good capacity of both EOs to inhibit the oxidative process.Thymol and carvacrol are the major components of T. vulgaris EO, while γ-terpinene is one of the major components of B. persicum EO.These components have been reported to exhibit antioxidant properties (Ruberto and Baratta, 2000).Thymol and carvacrol are primary antioxidants which either delay or prevent the initiation step by reacting with a lipid-free radical or prevent the propagation step by reacting with the peroxy or alkoxy radicals (Yanishlieva et al., 1999), thereby retarding VOO oxidation.It has been previously reported that thymol is a better antioxidant in lipids than carvacrol, due to the greater steric hindrance capability of its phenolic group (Yanishlieva et al., 1999).The antioxidant activity of γ-terpinene is attributed to the presence of methylene groups in monoterpene hydrocarbons which is strongly active and may compete with the activated methylene in C-11 of linoleic acid (Ruberto and Baratta, 2000).
α-Tocopherol showed no measurable effect in reducing the TOTOX value of VOO.α-tocopherol is reported to possess slight degrees of antioxidant activity and can even be pro-oxidative at times.These results depend on the chemical concentrations and the temperatures applied (Marinova and Yanishlieva, 1992;Schuler, 1990).The threshold value for α-tocopherol as a pro-oxidant in extra VOO oxidation was 60 to 70 ppm during storage at 37 and 75 °C (Deiana et al., 2002).Also, it has been reported that the pro-oxidant activity of α-tocopherol tends to decrease as the temperature increases, even at its high levels of concentration (Marinova and Yanishlieva, 1992).

Measurement of K 232 and K 268
The formation of conjugated dienes in fats or oils gives rise to an absorption peak at 232 nm in the ultraviolet region (Frankel, 2012).Changes in K 232 of the VOO samples during storage at 70 °C are presented in Figure 2. A significant difference (p<0.05) in K 232 was observed between the control and the samples containing T. vulgaris EO and B. persicum EO.This indicates the significant antioxidant effect of both EOs (p<0.05).The K 232 value of BHT, T. vulgaris EO, and B. persicum EO exhibited identical increasing trends in the first 28 days of storage.After that, the K 232 value of the sample which contained B. persicum EO increased faster and reached 6.21±0.09at the end of storage, whereas K 232 of BHT and T. vulgaris EO reached 4.2±0.98 and 5.3±0.07,respectively.The α-tocopherol had no measurable effect on causing a decrease in the formation of the conjugated dienes of VOO during storage at 70 °C.
Changes in K 268 of VOO samples are due to the formation of conjugated trienes (Figure 2).In line with the K 232 results, the levels of conjugated trienes at the end of storage were of lowest value in samples containing BHT followed by T. vulgaris and B. persicum EO, while the highest levels were found in the control and the sample containing α-tocopherol.
The durations of time that were required to reach the upper legal limits of K 232 (IP K232 ) and K 268 (IP K268 ) during storage at 70 °C are presented in Table 4.As expected, there was a strong correlation between IP PV and IP K232 (R 2 =0.985; y=0.432x+3.232).It is also understood that a strong correlation exists between IP PV and IP K268 (R 2 =0.967; y=0.444x+4.277).Moreover, IP K232 and IP K268 correlated directly with each other (R 2 =0.987; y=1.028x-0.589).However, in all samples, IP K232 and IP K268 values were lower than that of IP PV (with the average percentage of difference being 41.51 and 48.19%, respectively).Also, it was separately observed that K 232 and K 268 reached the upper legal limits of 2.6 and 0.25, respectively, almost concurrently.This indicates that the monitoring of VOO oxidation in terms of K 232 or K 268 at 70 °C will lead to the maintenance of stability in the VOO with regard to relatively similar quantities.
IP PV , IP K232 , and IP K268 were increased by the four antioxidants used in this study by approximately the same order as described before (BHT > T. vulgaris, EO > B. persicum EO > α-tocopherol).However, in all of the VOO samples under study, the IPE PV value was higher than the values for the IPE K232 and IPE K268 measurements.This indicates that both natural and synthetic antioxidants are more effective in protecting MUFA than PUFA.

Determination of chlorophyll and carotenoid contents
Chlorophyll compounds play an important role in the oxidative stability of VOO due to their antioxidant nature in the dark and their pro-oxidant activity in the presence of light (Criado et al., 2008).Carotenoids can act as primary antioxidants by trapping free radicals.They may also act as secondary antioxidants by quenching the singlet oxygen (Liebler, 1993).The chlorophyll contents of VOO samples during accelerated storage are presented in Figure 3.At the end of the storage period, all samples showed a substantial loss in chlorophyll content.
At the end of storage, the chlorophyll contents of VOO samples containing BHT, T. vulgaris EO and B. persicum EO ultimately decreased by 70.53%, 78.94% and 83.58%, respectively, whereas the chlorophyll content of the sample containing α-tocopherol and the control decreased by 89.68 and 90.91%, respectively.Carotenoid fractions decreased faster than the chlorophyll fraction during oxidation.It is commonly documented that the presence of oxygen and free radicals might accelerate the degradation rate of carotenoids.It is believed that the oxidation of carotenoids depends on the simultaneous oxidation of unsaturated fats (Criado et al., 2008).Thus, both oxygen and the presence of free radicals could explain the drastic decrease in carotenoid contents after a short period of storage.BHT, T. vulgaris EO, and B. persicum EO significantly retarded carotenoid degradation in the treated samples, compared with the control samples and also compared with those containing α-tocopherol.Nonetheless, at the end of storage, there were 28.37, 20.47%, and 13.49% carotenoids remaining in the samples containing BHT, T. vulgaris EO, and B. persicum EO, respectively, whereas the content of carotenoids was almost completely degraded in the control group and in the sample which contained αtocopherol.

CONCLUSION
According to the results observed in the present research, the inclusion of T. vulgaris and B. persicum EOs in VOO can retard the lipid oxidation process, thereby delaying the increase in adverse chemical quality parameters (PV, AV, K 232 , and K 268 ) and protecting the chlorophyll and carotenoid contents of VOO.Also, those EOs rich in phenolic monoterpenes  Grasas Aceites 67 (4), October-December 2016, e162.ISSN-L: 0017-3495 doi: http://dx.doi.org/10.3989/gya.0337161(T.vulgaris EO) were found here to be more effective than those rich in monoterpene hydrocarbons (B.persicum EO).The effect of T. vulgaris EO on retarding the oxidation of VOO in this study was found to be similar to that of BHT.However, BHT is not permitted to be incorporated into VOO.The α-tocopherol had only a small effect on improving the oxidative stability of VOO.Generally, T. vulgaris and B. persicum EOs can be used as potential natural antioxidants for extending the shelf life of VOO.Further studies at ambient temperatures would be required to determine the actual shelf life of VOOs containing plant EOs.

Figure 1 .
Figure 1.Changes in (a) peroxide values, (b) p-anisidine values, and (c) TOTOX values of virgin olive oil samples during accelerated storage at 70 °C.

Figure 2 .
Figure 2. Changes in (a) K 232 and (b) K 268 of virgin olive oil samples during accelerated storage at 70 °C.

Figure 3 .
Figure 3. Changes in (a) chlorophyll content and (b) carotenoid content of virgin olive oil samples during accelerated storage at 70 °C.

Table 1 (
Continued) a Not detected.

Table 2 .
Radical scavenging capacity and reducing power of Thymus vulgaris and Bunium persicum essential oils (EOs) Values given are the means of three replicates ± standard deviation.In each row, means with different letters are significantly different (p < 0.05).

Table 3 .
Initial characteristics of virgin olive oil (VOO)

Table 4 .
Duration required to reach the upper legal limits of K 232 (IP K232 ) and K 268 (IP K268 ) for virgin olive oil samples during storage at 70 °C