Oxidative stability of olive oil during the thermal process: Effect of Pistacia khinjuk fruit oil

ABSTRACT

The oxidative stability of refined olive oil with incorporated Pistacia khinjuk fruit oil (PKFO; 0.5%, 1%, 2%, 5%, and 10%) during thermal processing at 170ºC for 8 h was evaluated. The conjugated diene values, carbonyl values, acid values, oil/oxidative stability indices, and total tocopherol content were measured during thermal processing. Olive oil containing 0.5% PKFO was identified as the most oxidative stable oil followed by oils containing 100 ppm TBHQ and 1, 5, 10, and 2% PKFO. No significant difference between samples of olive oil containing 100 ppm TBHQ and 1% PKFO was observed. Thus, it was concluded that PKFO at levels lower than 1% could provide stronger antioxidation activity in comparison with TBHQ (the strongest syntactic antioxidant used in the food industry). Moreover, reduction in tocopherol compounds during thermal processing was higher in olive oil containing TBHQ as compared to those in pure olive oil.

KEYWORDS:

• Antioxidant compounds
• Antioxidant activity
• Oxidative stability
• Olive oil
• Pistacia khinjuk fruit oil

Introduction

Lipid oxidation involves unfavorable reactions in edible oils often regarded as one of the leading causes of shelf life reduction.^[1] Thus, the primary function of antioxidants is prevention of lipid oxidation in products containing edible oils and fats, Recently, a large number of investigations have been conducted to identify and determine application of natural antioxidants which could provide antitumor, antimutagenic, and anti-atherosclerotic properties^[2–4]. Moreover, some harmful side effects of synthetic antioxidants on human health such as the formation of the harmful toxic and cancer-related compounds have been documented.^[5–6]. The wild Pistacia species are commonly known as Pistacia khinjuk and Pistacia atlantica in Iran.^[2,4] The high oxidative stability of P. khinjuk fruit oil has been reported previously.^[4] Likewise, the extracted oils from P. khinjuk kernel and hull oils have exceptional oxidative stability.^[7,8] However, as far as we know no research has been conducted to evaluate the incorporation of P. khinjuk fruit oil in edible oils as a natural antioxidative agent. Thus, the current study was designed to investigate the influence of P. khinjuk fruit oil (PKFO) incorporation on the oxidative stability of olive oil during thermal processing.

Materials and methods

Materials

P. khinjuk fruit samples (three replications) were collected from Meimand forest, Fars province, Iran (summer, 2015). Refined olive oil samples were purchased from local market in Shiraz province, Fars, Iran. The samples were stored at 4ºC until the day of the experiment. All standards, chemicals, and solvents were purchased from the Sigma-Aldrich (St. Louis, MO) and Merck Companies (Darmstadt, Germany).

Oil extraction

P. khinjuk fruits were shade-dried (at ambient temperature) then powdered using a mill. The prepared powder was mixed with normal hexane (1:4) and was shaken for 48 h in the dark. Solvent residue was then removed using a vacuum pump at 40ºC for 6–12 h.^[8,9]

Purification and preparation of pure olive oil

In order to evaluate the antioxidative effect of PKFO, the majority of normally occurring antioxidant compounds found in refined olive oil were eliminated using column chromatography. For this purpose, 250 g of refined olive oil was passed through an aluminum oxide 60 column (100 g, active, neutral; previously activated at 200ºC for 3 h). The alumina column and collection vessels were wrapped with aluminum foil, and the oil was drawn through the column by suction without solvent.^[10]

Preparation of various samples of olive oil

To provide the simulated conditions of thermal processing, various olive oil samples containing 0.5%, 1%, 2%, 5%, and 10% of PKFO and 100 ppm of the TBHQ as a synthetic antioxidant were prepared to be compared with pure olive as a control.

Oil oxidation

Samples of olive oil (250 g) were poured into Erlenmeyer flasks and placed into a hot paraffin bath in an oven (170ºC) for 8 h. At 60 min intervals, samples (20 g) were taken and stored at –18ºC for further analysis.^[2,4,7,8]

Fatty acid composition

Fatty acid composition of the oil samples was determined by gas–liquid chromatography and is reported in relative area percentages. Fatty acids were transesterified into their corresponding Fatty acid methyl esters (FAMEs) by vigorous shaking of a solution of the oil in hexane (0.3 g in 7 mL) with 2 mL of 7 M methanolic potassium hydroxide at 50^oC for 10 min. The FAMEs were identified using an HP-5890 gas chromatograph (Agilent, Palo Alto, CA) equipped with a CP-FIL 88 (Supelco Inc., Bellefonte, PA) capillary fused silica column (60 m × 0.22 mm I.D., 0.2 mm film thickness) and the flame ionization detector. Nitrogen was used as a carrier gas at flow rate of 0.75 mL/min. The oven temperature was maintained at 198°C and that of the injector and the detector at 250°C.^[4]

Unsaponifiable matter (USM) content

Five grams of oil was mixed with 50 mL 1 N ethanolic KOH and subsequently heated at 95̊ºC for 1 h. The mixture was cooled then 100 mL distilled water was added and mixed. The obtained solution was extracted twice using 100 mL diethyl ether using a decanter funnel. During each extraction, the upper organic layer was collected and washed twice with 75 mL distilled water and once with 100 mL 0.5 N ethanolic KOH and finally neutralized with 100 mL distilled water. Organic layers were then removed and dried using NA₂SO₄, afterward was filtered and evaporated to dryness in a vacuum oven at 45ºC. For further purification of USM, compounds were dissolved in chloroform, filtered and finally evaporated at 45ºC under vacuum.^[11]

Total sterols (TS) content

Compounds were separated using gas chromatography (GC) equipped with an SE 54 CB column (Macherey–Nagel, Duren, Germany; 50 m long, 0.25 mm ID, 0.25 µm film thickness) also Betulin was applied as a standard. Operating parameters were as follows: hydrogen as a carrier gas, split ratio 1:20, injection and detection temperature adjusted to 320°C, temperature program, 240–255°C at 4°C/min.^[12]

Total tocopherol (TT) content

TT compounds in the oils were determined using a high-performance liquid chromatograph (HPLC) (WATERS, Alliance system, USA) with a Spherisorb column (25 cm×4 mm i.d., WATERS, USA) packed with silica (5 µm particle size) and a fluorescence detector operating at an excitation wavelength of 290 nm and an emission wavelength of 330 nm. The mobile phase was hexane/isopropanol (98.5:0.5 v/v) at a flow rate of 1 mL/min. Tocopherols in test samples were verified by comparison of retention times with those of reference standards.^[13]

Total phenolics (TP) content

TP content was determined as described by Capannesi et al. TP content was determined spectrophotometrically using the Folin–Ciocalteau reagent.^[14]

Oxidation parameters

The Conjugated Diene Value (CDV) was measured as described by Saguy et al. In this method, each oil sample was mixed with HPLC grade hexane (1:600), and the absorption was measured spectroscopically at 234 nm.^[15] Carbonyl Value (CV) of the oils was measured using the method developed by Endo et al. using 2-propanol and 2,4-decadienal as solvent and standard, respectively.^[16] The Oil/Oxidative Stability Index (OSI) was measured using a Rancimat device (Metrohem, model 743). For each test, 3g of oil was used, and temperature and airflow velocity of the device were set at 120ºC and 15 L/h, respectively.^[17] The Acid Value (AV) was determined according to the AOCS Official Method Cd 3d-63.^[18] The following equation was used to estimate resistance of various olive oil samples to increase in CDV, CV, and AV or reduction of OSI and TT during thermal processing:

X= Percentage of increase of CDV, CV, and AV or percentage of reduction of OSI of various olive oil samples during thermal processing compared to t0

Statistical analysis

Data from the experiments of the chemical properties of studied oils in the present investigation were analyzed in triplicate as a complete randomized design (RCD). In the subsequent experiments, the effects of heat processing on these oils were based on a factorial randomized complete design (RCD) with three replications (the variable in these experiments included oil samples and processing time). All data were subjected to analysis of variance (ANOVA). ANOVA and regression analysis were evaluated using the MStatC and SlideWrite software, respectively. The mean comparison was performed by Duncan multi-step test to determine the significance of the differences (p < 0.05).

Result and discussion

Chemical properties

Chemical properties of PKFO and pure olive oil are shown in Table 1. Regarding fatty acid composition, both oils contain common fatty acids in vegetable oils such as palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, and linolenic acid whose content in PKFO and olive oil were 19.8, 4.8, 2.1, 59.2, 13.4, 0.7; and 13.1, 0.7, 3.3, 71.5, 9.4, and 0.6%; respectively (arachidic acid and gadoleic acid were seen in olive oil at 0.6 and 0.4% concentration).

Table 1. Chemical composition of Pistacia khinjuk fruit and olive oils.*

Parameter	Pistacia khinjuk fruit oil	Refined olive oil
Fatty acid (%)
C16:0	19.8 ± 0.7^a	13.1 ± 0.5^b
C16:1	4.8 ± 0.3^a	0.7 ± 0.2^b
C18:0	2.1 ± 0.2^b	3.3 ± 0.3^a
C18:1	59.2 ± 1.6^b	71.5 ± 1.5^a
C18:2	13.4 ± 0.5^a	9.4 ± 0.2^b
C18:3	0.7 ± 0.1^a	0.6 ± 0.2^a
C20:0	—–	0.6 ± 0.15
C20:1	—–	0.4 ± 0.1
SFA	21.9 ± 0.6^a	17 ± 0.7^b
MUFA	64 ± 1.3^b	72.6 ± 1.8^a
PUFA	14.1 ± 0.8^a	10 ± 0.5^b
USM content (% of oil)	1.5 ± 0.2^a	1.2 ± 0.3^a
TT content (mg /kg oil)	639.83 ± 14.3^a	350 ± 1.2^b
TP content (mg gallic acid /kg oil)	85.23 ± 2.4^a	26.5 ± 4.5^b
TS content (mg /kg oil)	2167.5 ± 23.2^a	1076.3 ± 17.4^b

*Mean ± SD (standard deviation) within a row with the same lowercase letters are not significantly different at p < 0.05. SFA, saturated fatty acids; MUFA, monounsaturated fatty acids; PUFA, polyunsaturated fatty acids; USM, unsaponifiable matter; TT, total tocopherols; TP, total phenolics; TS, Total sterols.

The USM as active antioxidant agents includes tocopherol, polyphenol, sterol, and hydrocarbons and can be regarded as one of the quality indices of edible oils. USM content in vegetable oils is usually in the range of 0.5%–2.5% and occasionally up to 5–6%.^[19] USM content in PKFO and olive oil were 1.5% and 1.2%; respectively (Table 1). Tocopherols such as vitamin E can be attributed to the major proportion of USM in edible oils demonstrating antioxidative properties as well as beneficial health effects.^[19] TT contents of PKFO and olive oil were 639.83 and 350 mg/kg, respectively (Table 1). TT content in walnut, cottonseed, canola, olive, palm, peanut, soybean and sunflower oils have been reported to be 1500, 830–900, 690–695, 30–300, 360–560, 330–450, 900–1400, and 630–700 mg/kg.^[20] TT content of PKFO was close to those of canola and sunflower oils. Higher portions of Δ-tocopherol and γ-tocopherol endow stronger antioxidant activity compared to the higher portion of α-tocopherol and β-tocopherol.^[21] TP content in PKFO and olive oil were 85.23 and 26.5 mg/kg, respectively (Table 1). The polyphenols could provide desirable antioxidative properties, in addition to biological activities in organisms and critical roles in preventing diseases.^[22,23]

Oxidative stability test

To evaluate the effect of PKFO on oxidative stability of olive oil, pure olive oil as a control, olive oils containing 0.5, 1, 2, 5, and 10% of PKFO and 100 ppm of TBHQ were subjected to thermal processing at 170°C for 8 h. Then, the variations in CDV, CV, AV, OSI, and TT were evaluated.

The changes of CDV during 8 h of thermal processing at 170°C are presented in Table 2. At t₀, no significant difference in CDV among the samples was recorded. An increasing trend in CDVs during thermal processing was observed. The amount of increase in CDVs for pure olive oil and olive oil samples containing 0.5, 1, 2, 5, and 10% of PKFO compared with unheated oils were 158.6, 55.3, 78.6, 125.8, 51.5, 53.7, and 47.5%, respectively. The addition of various concentrations of PKFO to olive oil could prevent formation of CDV. No significant difference was observed between the increase in the amount of CDV in olive oils containing 0.5% and 10% PKFO. In other words, the efficiency of addition of a slight amount of PKFO (0.5%) against production of primary oxidation was equal to olive oil containing 10% PKFO and similar to oil samples containing 5% PKFO and 100ppm TBHQ. In comparison with TBHQ in pure form, PKFO is not a pure antioxidant; thus olive oil containing 0.5% PKFO was selected as the best sample based on the CDV evaluation.

Table 2. Changes in conjugated diene value (CDV, mmol L^–1) of olive oil as affected by PKFO and TBHQ during heat processing at 170°C.*

Time (hour)	Pure olive oil	Olive oil samples containing
		PKFO					TBHQ
		0.5%	1%	2%	5%	10%	100 ppm
0	11.5 ± 0.04^a	11.5 ± 0.04^a	11.51 ± 0.04^a	11.52 ± 0.04^a	11.55 ± 0.04^a	11.54 ± 0.04^a	11.5 ± 0.03^a
2	19.23 ± 0.06^a	12.84 ± 0.04^f	14.23 ± 0.02^e	16.17 ± 0.02^c	10.47 ± 0.03^g	16.72 ± 0.03^b	14.51 ± 0.02^d
4	20.34 ± 0.08^a	15.32 ± 0.03^b	14.64 ± 0.04^c	20.41 ± 0.04^a	12.39 ± 0.03^f	14.27 ± 0.03^e	14.54 ± 0.04^d
6	31.34 ± 0.07^a	18.00 ± 0.05^c	16.01 ± 0.03^e	27.26 ± 0.03^b	16.01 ± 0.02^e	12.03 ± 0.04^f	16.5 ± 0.04^d
8	29.74 ± 0.05^a	17.86 ± 0.05^d	20.55 ± 0.06^c	26.01 ± 0.03^b	17.49 ± 0.02^e	17.83 ± 0.04^d	16.96 ± 0.02^f

*Mean ± SD (standard deviation) within a row with the same lowercase letters are not significantly different at p < 0.05. PKFO, P. khinjuk fruit oil; TBHQ, tert-butylhydroquinone.

The variations in CV during 8 h of thermal processing at 170 ̊ºC are shown in Fig. 1. The same behavior as CDV regarding t₀ was observed. During thermal processing, CVs exhibited an increasing trend. The increase in CV for pure olive oil and olive oil samples containing 0.5, 1, 2, 5, and 10% of PKFO compared with unheated oils were 136.4, 41.6, 46.8, 138, 133.7, 187.4, and 77.6%, respectively. It was clearly observed that addition of various concentrations of PKFO resulted in different effects on the increase in CV. More notable is that the highest increment in carbonyl compounds was observed in olive oil containing 10% PKFO. Moreover, the significant difference between the increment in CV level in olive oil samples with 2% and 5% PKFO and pure oil was not observed. The olive oil containing 0.5% PKFO, was chosen as the most stable sample in term of CV, followed by olive oil containing 1% PKFO and 100 ppm TBHQ, respectively; suggesting a high antioxidative activity of PKFO against secondary oxidation.

Figure 1. Changes in carbonyl value (CV, µmol/g) of olive oil as affected by PKFO and TBHQ during heat processing at 170°C. A: pure olive oil; B: olive oil containing 0.5% PKFO; C: olive oil containing 1% PKFO; D: olive oil containing 2% PKFO; E: olive oil containing 5% PKFO; F: olive oil containing 10% PKFO; G: olive oil containing 100 ppm TBHQ. PKFO, Pistacia Khinjuk fruit oil; TBHQ, tert-butylhydroquinone.

The changes in AV are presented in Table 3. AVs of some samples were significantly different at t₀. During thermal processing, AV showed an increasing trend. Increasing of AV during thermal processing or frying can be correlated with hydrolysis of triacylglycerols or presence of carboxylic groups in oxidative or polymeric products.^[1] As can be seen, the incorporation of various concentrations of PKFO appeared to effectively inhibit the production of AV compared to pure olive oil. The lowest increase in amount of AV was observed in olive oil containing 5% PKFO (33.9%), followed by olive oil with 100 ppm TBHQ (35.7%), olive oil with 1% PKFO (36.9%), olive oil with 0.5% (41.4%), olive oil with 10% PKFO (42%; respectively), and olive oil containing 2% PKFO and pure olive oil (70.5 and 70.8%, respectively). Among the measured amounts, only the difference between pure olive oil and oil containing 2% PKFO was not significant.

Table 3. Changes in acid value (AV, mg/ml) of olive oil as affected by PKFO and TBHQ during heat processing at 170°C.*

Time (hour)	Pure olive oil	Olive oil samples containing
		PKFO					TBHQ
		0.5%	1%	2%	5%	10%	100 ppm
0	0.24 ± 0.02^c	0.25 ± 0.02^c	0.26 ± 0.03^bc	0.27 ± 0.03^bc	0.33 ± 0.04^b	0.42 ± 0.03^a	0.24 ± 0.03^c
2	0.26 ± 0.01^d	0.28 ± 0.03^d	0.28 ± 0.01^d	0.35 ± 0.02^c	0.38 ± 0.01^b	0.53 ± 0.02^a	0.27 ± 0.02^d
4	0.31 ± 0.03^c	0.31 ± 0.02^c	0.28 ± 0.02^c	0.38 ± 0.03^b	0.39 ± 0.03^b	0.54 ± 0.01^a	0.31 ± 0.03^c
6	0.37 ± 0.02^d	0.32 ± 0.02^e	0.36 ± 0.02^c	0.41 ± 0.02^b	0.41 ± 0.02^b	0.57 ± 0.04^a	0.31 ± 0.02^e
8	0.41 ± 0.02^c	0.35 ± 0.02^d	0.34 ± 0.03^d	0.46 ± 0.03^b	0.44 ± 0.03^bc	0.6 ± 0.05^a	0.33 ± 0.02^d

*Mean ± SD (standard deviation) within a row with the same lowercase letters are not significantly different at p < 0.05. PKFO, P. khinjuk fruit oil; TBHQ, tert-butylhydroquinone.

The Rancimat test is the most popular accelerated method to evaluate oxidative stability of edible oils and lipids.^[1,24] The results obtained for OSI changes are presented in Fig. 2. The addition of various concentrations of PKFO into unheated pure olive oil enhanced OSI of oil samples compared to pure olive oil. The highest OSI at t₀ was noted for the olive oil containing 100ppm of TBHQ (9.51 h), followed by olive oils containing 10%, 5%, 2%, 1%, 0.5% PKFO, and pure olive oil (8.23, 6.88, 5.87, 5.52, 5.28, and 3.35 h, respectively). A descending trend was observed for OSI variation in various oil samples during thermal processing with the greatest decrease observed for TBHQ-containing samples. Reduction of the amount of OSI for pure olive oil and olive oil samples containing 0.5, 1, 2, 5, and 10% of PKFO after the thermal process compared to unheated oils were 79.1, 75.6, 73, 87.6, 63.8, 59.9, and 79.2%, respectively. It should be noted that there was no significant difference between reduction in the amount of OSI of olive oil containing 100 ppm of TBHQ and that of pure olive oil which was lower than that of olive oil samples containing PKFO (except for samples containing 2% of PKFO). In accordance with other tests, the results obtained in the Rancimat test showed that the antioxidant activity of PKFO was much higher than that of TBHQ, the synthetic antioxidant.

Figure 2. Changes in the oil/oxidative stability index (OSI, h) of olive oil as affected by PKFO and TBHQ during heat processing at 170°C. A: pure olive oil; B: olive oil containing 0.5% PKFO; C: olive oil containing 1% PKFO; D: olive oil containing 2% PKFO; E: olive oil containing 5% PKFO; F: olive oil containing 10% PKFO; G: olive oil containing 100 ppm TBHQ.PKFO, Pistacia khinjuk fruit oil; TBHQ, tert-butylhydroquinone.

Antioxidant compound (TT and TS) changes

The results obtained for TT are shown in Table 4. The highest concentration of primary TT was observed in samples containing 10% and 5% of PKFO, followed by the remaining samples. TT changes showed a descending trend during thermal processing. Reduction in the amount of TT for pure olive oil and olive oil samples containing 0.5, 1, 2, 5, and 10% of PKFO after the thermal process compared to unheated oils were 91.4, 81.2, 86, 85.3, 83.5, 79.9, and 95.2%, respectively. As can be seen, the addition of various concentrations of PKFO into olive oil had a positive effect on prevention of TT decrease. The decline in TT values in olive oil samples containing TBHQ was higher than that of pure olive oil. This finding confirms the harmful effect of synthetic antioxidants because, apart from their adverse effects, these antioxidants degrade their natural counterparts such as tocopherols. Regarding the critical roles of tocopherols in retention of oxidative stability in edible oils as well as nutritional value, lower reduction of such compounds can always be considered as an important goal of the oil industry experts.

Table 4. Changes in total tocopherol (TT, mg/kg oil) content of olive oil as affected by PKFO and TBHQ during heat processing at 170°C.*

Time (hour)	Pure olive oil	Olive oil samples containing
		PKFO					TBHQ
		0.5%	1%	2%	5%	10%	100 ppm
0	350 ± 12.1^b	351.5 ± 10.5^b	352.9 ± 9.8^b	355.8 ± 10.8^b	364.5 ± 12.5^ab	379.0 ±11.8^a	350 ± 8.9^b
2	301.2 ± 7.9^a	280.2 ± 9.7^b	270.2 ± 6.3^bc	260.2 ± 9.9^c	300.2 ± 14.3^a	300.9 ± 10.2^a	250.1 ± 7.6^d
4	225.6 ± 8.9^ab	204.5 ± 8.7^c	221.2 ± 5.4^b	180.2 ± 8.7^d	240.1 ± 10.5^a	235.5 ± 8.7^a	124.5 ± 8.8^e
6	97.2 ± 7.6^d	132.5 ± 7.7^ab	104.5 ± 2.8^c	110.4 ± 2.9^c	130.4 ± 4.9^b	142.2 ± 6.7^a	62.5 ± 7.7^e
8	30 ± 3.03^d	66.2 ± 6.1^b	49.5 ± 3.4^dc	52.3 ± 3.6^c	60.1 ± 1.5^b	76.2 ± 3.2^a	16.9 ± 2.3^e

*Mean ± SD (standard deviation) within a row with the same lowercase letters are not significantly different at p < 0.05. PKFO, P. khinjuk fruit oil; TBHQ, tert-butylhydroquinone.

The variations of TS are presented in Table 5. TS changes showed a decreasing trend during thermal processing. The lowest reduction rate of TS content can be correlated to samples containing 0.5 and 1% of PKFO and 100ppm of TBHQ (45.4–47.4%), followed by olive oil samples containing 5, 10 and 2% of PKFO and pure olive oil, respectively (57.3, 64.8, 65.4, and 76.4%). Moreover, the addition of PKFO to olive oil had positive effects on prevention of TS decrease.

Table 5. Changes in total sterol (TS, mg/kg oil) content of olive oil as affected by PKFO and TBHQ during heat processing at 170°C.*

Time (hour)	Pure olive oil	Olive oil samples containing
		PKFO					TBHQ
		0.5%	1%	2%	5%	10%	100ppm
0	1076.3 ± 15.5^d	1081.7 ± 25.6^d	1097.6 ± 18.7^cd	1117.1 ± 8.6^c	1177.7 ± 10.9^b	1260.8 ± 25.5^a	1070.8 ± 15.3^d
2	841 ± 18.3^e	1010 ± 12.2^b	952.4 ± 13.3^d	800.5 ± 16.5^f	1008.4 ± 10.2^b	1050.7 ± 14.9^a	980.6 ± 12.2^c
4	550.4 ± 12.2^f	850.1 ± 9.8^b	721.8 ± 14.5^d	609.1 ± 14.2^e	900 ± 13.6^a	920.2 ± 7.5^a	825.3 ± 13.8^c
6	520.5 ± 11.6^d	705.9 ± 14.8^a	640.4 ± 12.6^b	500 ± 17.1^d	604 ± 17.5^c	712.3 ± 16.5^a	620.5 ± 18.2^bc
8	254.1 ± 13.3^e	585.2 ± 11.1^a	574.3 ± 16.7^a	385.9 ± 10.7^d	498.2 ± 11.4^b	446.3 ± 14.2^c	584.5 ± 14.7^a

*Mean ± SD (standard deviation) within a row with the same lowercase letters are not significantly different at p < 0.05. PKFO, P. khinjuk fruit oil; TBHQ, tert-butylhydroquinone.

Evaluation the relation between antioxidant compounds and oxidative stability

In order to the interpret the oxidative stability tests results, the resistance of various oil samples to an increase of CDV, CV, AV, and a decrease of OSI were calculated and the mean resistance to oxidation was estimated (Table 6). Olive oil containing 0.5% PKFO (1.99) was determined as to be the most resistant sample to oxidation, followed by samples containing 1% PKFO and 100 ppm of TBHQ (1.87 and 1.87, respectively), samples containing 5% of PKFO (1.8), samples with 10% PKFO (1.61), and oil samples containing 2% PKFO and pure olive oil (1.02 and 1.01, respectively). PKFO in amounts higher than 1% had lower antioxidant activity compared to those containing 0.5% and 1% PKFO which is due to their peroxidative properties. In some cases, by increasing the antioxidant concentration, instead of increasing the antioxidative activity, the peroxidative effect occurs which enhances the oxidation process.^[1,25]

Table 6. Resistance of different olive oil samples against changes in oxidation factors during heat processing at 170°C.*

Parameter	Pure olive oil	Olive oil samples containing
		PKFO
		0.5%	1%	2%
Resistance against CDV increase	0.63 ± 0.03^f	1.81 ± 0.04^c	1.27 ± 0.02^d	0.79 ± 0.03^e
Resistance against CV increase	0.73 ± 0.02^d	2.41 ± 0.04^a	2.14 ± 0.03^b	0.72 ± 0.03^d
Resistance against AV increase	1.41 ± 0.03^f	2.42 ± 0.04^d	2.71 ± 0.03^c	1.42 ± 0.05^e
Resistance against OSI reduction	1.26 ± 0.03^d	1.32 ± 0.02^c	1.37 ± 0.04^c	1.14 ± 0.04^e
Mean resistance against oxidation	1.01 ± 0.02^e	1.99 ± 0.04^a	1.87 ± 0.03^b	1.02 ± 0.03^e
	Olive oil samples containing
	PKFO		TBHQ
	5%	10%	100 ppm
Resistance against CDV increase	1.94 ± 0.04^b	1.86 ± 0.03^c	2.11 ± 0.05^a
Resistance against CV increase	0.75 ± 0.04^d	0.53 ± 0.02^e	1.29 ± 0.04^c
Resistance against AV increase	2.95 ± 0.04^a	2.38 ± 0.05^d	2.80 ± 0.04^b
Resistance against OSI reduction	1.57 ± 0.04^b	1.67 ± 0.03^a	1.26 ± 0.02^d
Mean resistance against oxidation	1.80 ± 0.02^c	1.61 ±.03^d	1.87 ± 0.04^b

*Means ± SD (standard deviation) within a row with the same lowercase letters are not significantly different at p < 0.05. PKFO, P. khinjuk fruit oil; CDV, conjugated diene value, CV, carbonyl value, AV, acid value, OSI, oil/oxidative stability Index, TBHQ, tert-butylhydroquinone.

To investigate the relation between antioxidant compounds of the examined oils and their oxidative stability, the chart of remaining TT and TS (TT₈ + TS₈) content at the end of the 8 h heating process at 170ºC against the final CDV, CV and AV (CDV₈ + CV₈ + AV₈) was drawn (Fig. 3). As can be inferred from Fig. 3, a good relation between TT₈ + TS₈ content and CDV₈ + CV₈ + AV₈ was observed, and they were well correlated (R² = 0.9715). By increasing the TT₈ + TS₈ content, CDV₈ + CV₈ + AV₈ was decreased. The highest oxidative stability was observed in olive oil containing 0.5% PKFO which had the highest remaining antioxidant compounds at the end of the heating process, followed by oil samples containing 1% PKFO and 100 ppm of TBHQ, samples containing 5% of PKFO, oil samples with 10% PKFO, oil samples containing 2% PKFO and pure olive oil, respectively.

Figure 3. Correlation between remaining TT and TS (TT₈+TS₈) content of olive oil samples after the 8 h thermal process at 170ºC and their final CDV, CV and AV(CDV₈+CV₈+AV₈).AV, acid value; CDV, conjugated diene value; CV, carbonyl value; TS, total sterols; TT, total tocopherols.

Conclusion

The results of these oxidative stability evaluations indicated that during thermal processing, PKFO in amounts lower than 1%, resulted in higher oxidative stability in olive oil compared to TBHQ. Since PKFO, compared to TBHQ, is not a pure antioxidant and had stronger antioxidant activity at 0.5% and 1% concentrations than TBHQ (the strongest widely used antioxidant in the food industry), this oil can be proposed as a natural antioxidant with high antioxidant activity and thermal stability which could make it an effective alternative for synthetic antioxidants such as TBHQ whose harmful effects on health is well documented.