Utilization of Jujube Fruit (Ziziphus mauritiana Lam.) Extracts as Natural Antioxidants in Stability of Frying Oil

Abstract

The effects of ultrasound-assisted, supercritical CO₂ and solvent extraction methods on antioxidant activity of Ziziphus mauritiana (Lam.) extracts were investigated using 2,2-diphenyl-1-picrylhydrazyl and β-carotene assays. Ethanol-water extract of the jujube that was obtained with ultrasound-assisted extraction had the highest antioxidant activity. Ultrasound-assisted extraction was the most effective method on extraction of phenolic compounds. The effect of ultrasound extract in stability of soybean oil was compared to synthetic antioxidants by measuring total polar compounds, carbonyl value, peroxide value, free fatty acids, oxidative stability index, and conjugated dienes and trienes values. Results showed that ultrasound ethanol-water extract at 600 ppm had a higher stabilization efficiency than commercial antioxidants butylated hydroxyanisole, butylated hydroxytoluene, and tertiary butylhydroquinone.

Keywords:

• Antioxidant activity
• Frying
• Ultrasound-assisted
• Supercritical CO₂
• Solvent extraction
• Ziziphus mauritiana

INTRODUCTION

Fried foods are major part of home foods and in fast-food restaurants. So, deep frying is one of the most important unit operations in the food processing industry.^[1] Deep frying causes changes in the flavor, color, texture, and nutritional quality of fried foods. The type of oil, frying time, and temperature have significant effects on the quality of fried foods.^[2] Deep frying at high temperatures (160–200°C) and in the presence of oxygen and moisture causes important chemical reactions, such as hydrolysis, oxidation, cyclization, and polymerization.^[3] Volatile and non-volatile compounds are formed in vegetable oils during deep frying. Volatile compounds are removed from oil and non-volatile compounds accumulate in the oil. Non-volatile compounds are produced primarily by thermal oxidation and polymerization of unsaturated fatty acids.^[4] Non-volatile compounds are often polar and of high molecular weight which have toxic effects on humans and animals.^[5]

The synthetic antioxidants such as butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), and tertiary butylhydroquinone (TBHQ) are often used to increase the oxidation stability and shelf life of oils.^[6] BHT and BHA have been reported to have toxic and carcinogenic effects.^[7] However, despite the high antioxidant activity of TBHQ, its use has been prohibited in some countries, such as Japan and Canada. Studies have shown that plants are rich sources of antioxidant compounds, such as flavonoids, phenolic compounds, tocopherols, carotenoids, and tannins.^[8] Therefore, research on natural antioxidants as alternatives to the synthetics has been increasing in recent years.

Jujube (Ziziphus mauritiana Lam.) is a member of the Rhamnaceae family. The tree (lotus) grows in arid and semi-arid regions.^[9] The lotus originated from Central Asia and is widely cultivated in Zimbabwe, Thailand, India, and Malaysia.^[10] Lotus height is about 10 meters and its leaves are small and heart-shaped, and are also painted with three prominent veins. Its fruit is round with dark green skin color in young stage which turns to light green or yellow–green in the mature stage. The flesh is white to yellow–white and turns red with skin shrinkage in the ripe stage.^[11] This fruit has pharmaceutical properties and is a traditional medicine to help improve blood circulation, the nervous system, as well as a relief to insomnia. Ziziphus mauritiana fruit has good antioxidant property due to the presence of phenolic, flavonoid, and tocopherol compounds.^[9]

Therefore, the aim of this work was to compare the effects of solvent extraction, ultrasound-assisted, and supercritical CO₂ extraction techniques on antioxidant activity of jujube (Ziziphus mauritiana) fruit extracts. The study also attempted to evaluate the antioxidant potential of jujube fruit extracts during oxidation of soybean oil by measuring both the primary and secondary oxidation products and to compare its antioxidant activity with commonly employed synthetic antioxidants.

MATERIALS AND METHODS

Materials

Jujube (Ziziphus mauritiana) fruits were collected from fields in Bandar Abbas in the Hormozgan province, Iran. Refined, bleached, and deodorized soybean oil with no added antioxidant was supplied by Rana (Gorgan, Iran) and was stored at –20°C until analysis. All reagents used in the experiments were of analytical grade and obtained mostly from Sigma Chemical Co. (St. Louis, MO, USA). Also, solvents were purchased from Merck (Darmstadt, Germany).

Natural Antioxidant Preparations

The jujube fruits (10 kg) were sun-dried and powdered in a grinder to reach 40-mesh and then were packed and stored at –20°C until the extraction of antioxidant. In solvent extraction, dried powders of sample (20 g) were mixed with 100 mL of solvents (ethanol-water [1:1], water, and ethanol). The mixtures were stirred in a shaker at 160 rpm away from light at room temperature for 48 h. After extraction, the extracts were filtered and solvents evaporated using a rotary evaporator (Heidolph, Schwabach, Germany) at 50°C. The concentrated extracts were stored until testing at –20°C.^[12]

A Suprex MPS/225 Multipurpose system (Roth Scientic, Basingstoke, Hampshire, UK) in the supercritical CO₂ extraction mode was used for the extraction of phenolic compounds. In this method, extractions of 20 g of dried powders of jujube were accomplished with 100 mL of solvent at 35°C, 100 bar, for 30 min. The extract was filtered and solvent evaporated. The concentrated extract was stored at –20°C until testing.^[13]

The extraction of phenolic compounds from mixture of solvents (100 mL) and powdered sample (20 g) by ultrasonic treatment was performed with a power of 100 W ultrasonic bath (KQ2200E, Kunshan Ultrasonic Instrument Co., Jiangsu, China). The mixtures were sonicated for 30 min at 35°C. The extracts were filtered and solvents evaporated. The concentrated extracts were stored in a freezer.^[14]

Total Phenolic Content

The content of phenolic compounds in the extracts was determined with Folin-Ciocalteu reagent following the colorimetric method adapted by Taghvaei et al.^[15] Measurements were carried out in triplicate and calculations were based on a calibration curve obtained with gallic acid. The contents of total phenols were expressed as mg of gallic acid equivalents per g of dry weight ([DW] (mg GAE.g^–1 DW).

2,2-diphenyl-1-picrylhydrazyl (DPPH)^• Radical Scavenging Activity

DPPH radical scavenging activity of extracts was measured according to the method of Xu and Chang^[16] with little modifications. The DPPH^• solution was prepared by dissolving 5.9 mg of DPPH^• in ethanol (100 mL). Accurately, 3.8 mL of DPPH^• ethanolic solution was added to 0.2 mL of fruit extracts. The mixture was shaken vigorously for 1 min and left to stand at room temperature in the dark for 30 min. Absorbance was measured against the blank reagent at 517 nm. All determinations were carried out in triplicate. The radical scavenging activity was calculated according to Eq. (1) below:

(1)

A0 and A1 are the absorbance of blank and sample, respectively.

Inhibition of β-Carotene Bleaching

Lipid peroxidation inhibition activity was determined using the β-carotene bleaching assay as described by Lagha-Benamrouche and Madani.^[17] The extract (0.2 mL) was added to 5 mL of β-carotene/linoleic acid solution. The absorbance of the samples was measured at 470 nm at initial time (T = 0) against a blank, consisting of an emulsion without β-carotene. The remaining samples were placed in dark for 24 h. Then, the absorbance of each sample was measured at 470 nm. The radical inhibition activity was calculated according to Eq. (2) below:

(2)

where the Abss²⁴ is the absorbance of the antioxidant after 24 h, Absc²⁴ is the absorbance of control after 24 h, Abss⁰ is the absorbance of the antioxidant at t = 0 and Absc⁰ is the absorbance of the control at t = 0.

Frying Process

The jujube fruit extract in levels of 600 and 800 ppm, BHA, BHT, and TBHQ in level of 100 ppm were added to soybean oil. Soybean oil without antioxidant addition was used as a negative control. Oil sample (2.5 L) was placed in a fryer oven of 2.5 L capacity (Tefal model 1250, France) and heated at 185 ± 5°C for 24 h. A batch of 20 g of potato pieces (7.0 cm × 0.5 cm × 0.3 cm) was fried for 7 min. After time of heating (0, 4, 8, 12, 16, 20, and 24) 20 g of frying oil samples were stored until testing at –20°C.^[18]

Total Polar Compounds (TPC), Carbonyl Value (CV), and Peroxide Value (PV)

The TPC were determined according to the method described by Schulte.^[19]

The CV of the oil samples was measured according to the method developed by Endo et al.^[20] using 2-propanol and 2,4-decadienal as solvent and standard, respectively. The results were expressed in micromols of 2, 4-decadienal per gram of oil.

PV expressed in milliequivalents of active oxygen per kilogram (meq O₂/kg of oil) was determined according to Shantha and Decker.^[21]

Conjugated Dienes (CD), and Trienes (CT), and Free Fatty Acids (FFAs) Content

The contents of CD and CT were calculated according to the method described by Urbančič, et al.,^[22] which is based on the measurement of absorbance solution at 234 and 270 nm for CD and CT, respectively.

The FFA expressed as free oleic acid percentage, was determined by titration of an accurate sample solution, dissolved in ethanol/ether (1:1, v/v), with 0.1 M sodium hydroxide solution, using phenolphthalein as indicator.^[23]

Oxidative Stability (Rancimat)

A Metrohm 743 Rancimat instrument (Herisan, Switzerland) was used in the experiment. Air supply was maintained at 15 L/h and the temperature was kept at 110°C.^[24]

Statistical Analysis

Each analysis was carried out in triplicate. The data were subjected to analysis of variance (ANOVA) and the significant differences between means were determined by Duncan’s multiple range test (p < 0.05) using SPSS statistical software (version 19; SPSS Inc., Chicago, IL, USA).

RESULTS AND DISCUSSION

Extraction Yield

Figure 1a shows the extraction yield of jujube fruit (Ziziphus mauritiana) which range from 17.56 to 45.71%. Results showed that the ethanol-water (1:1) was the most effective solvent for extraction of bioactive compounds compared to water and ethanol. This is in accordance with the findings of Hemwimol et al.^[25] The extraction yields of different techniques in descending order were: ultrasound-assisted > solvent extraction > supercritical CO₂. This shows that ultrasound-assisted extraction was the best technique for the extraction of bioactive compounds from the Ziziphus mauritiana. Our results concurred with previously published results such as the works by Hemwimol et al.^[25] and Rodríguez-Rojo et al.^[26] that reported the ultrasound-assisted extraction method was more effective in extraction of plant materials compared to other techniques.

FIGURE 1 (a) Extraction yield (%) of jujube extracts; (b) Total phenolic contents of jujube extracts. Solvent extracts of jujube by ethanol (SJE), water (SJW) and ethanol-water (SJEW). Ultrasound-assisted extracts of jujube by ethanol (UJE), water (UJW), and ethanol-water (UJEW). Supercritical CO₂ extracts of jujube by ethanol (CJE), water (CJW), and ethanol-water (CJEW).

Total Phenolic Content

Polyphenolic compounds are widely distributed in different parts of plants^[27] and reports showed that there is a positive relation between total phenolic content and antioxidant activity in many plant species.^[28] There were significant differences (p < 0.05) between phenolic compounds of extraction techniques with different solvents (Fig. 1b). There were various phenolic contents in the jujube fruit extracts ranging from 285.74 to 664.72 mg GAE/g. Jujube extract was previously reported to have total phenolic compounds in the range of 1321.98 mg^[29] to 2352.5 mg GAE/100 g.^[30] The composition of fruits and phenolic content depend on the genetic and environmental factors as well as post-harvest processing conditions.^[31] Our results indicated that the ethanol-water (1:1) had better effects on extraction of phenolic compounds compared to other solvents in the same extraction conditions. Also, we found that UJEW had a greater effect on the extraction of phenolic compounds. Goli et al.^[32] and Yasoubi et al.^[33] reported similar results indicating that the ultrasound-assisted extraction was the most effective method in extraction of phenolic compounds in comparison with supercritical CO₂ and solvent extraction techniques.

DPPH^• Radical Scavenging Activity

In the reaction of free radical DPPH^• with antioxidant species, antioxidant inhibits and stabilizes the free radical which causes color to change from purple to yellow, and decreases the absorbance which was monitored using a spectrophotometer at 517 nm.^[34] Figure 2a shows the ability of jujube fruit extracts to scavenge DPPH^• radical as inhibition percentage at concentrations of 200 to 800 ppm. In this assay similar to previously published results, such as the works by Chirinos et al.^[35] and Akkol et al.,^[36] the results indicated that scavenging free radicals increased as concentrations of the extracts increased, which was due to the increasing amount of phenolic compounds at higher concentrations of the extracts. With increasing concentrations of phenolic compounds the number of hydroxyl groups available in the reaction medium increased. Therefore, the possibility of hydrogen donation to free radicals increased.^[37] The results showed UJEW at concentrations of 200 to 800 ppm had the highest DPPH^• radical-scavenging capacity between other extracts. This was in agreement with González-Montelongo et al.^[38] who mentioned that ethanol-water (1:1) extract was the best to scavenge DPPH^• free radical. Also, our results indicated that the ultrasound had better performance on antioxidant activity of jujube in scavenging of free radicals. These results concurred with previously published results, such as the work by Rodríguez-Rojo et al.,^[26] in which they evaluated effects of different extraction methods on antioxidant activity of rosemary extract.

FIGURE 2 (a) Scavenging activities of jujube extracts against DPPH^• radicals; (b) Antioxidant activities of jujube extracts in β-carotene/linoleic acid system. Solvent extracts of jujube by ethanol (SJE), water (SJW) and ethanol-water (SJEW). Ultrasound-assisted extracts of jujube by ethanol (UJE), water (UJW), and ethanol-water (UJEW). Supercritical CO₂ extracts of jujube by ethanol (CJE), water (CJW), and ethanol-water (CJEW).

β-Carotene Bleaching Assay

In this assay, β-carotene is oxidized by free radicals derived from the oxidation of linoleic acid, which can better simulate the food system compared to DPPH assay.^[39] The antioxidant activities of the jujube fruit extracts were measured by bleaching of β-carotene which is illustrated in Fig. 2b. The extracts obtained from different methods and solvents, showed different degrees of antioxidant activity. Ethanol-water extract of jujube fruit that extracted by ultrasound showed maximum antioxidant activities with significant differences than other solvents. This result was in agreement with González-Montelongo et al.^[38] who reported that banana peel extract obtained by ethanol-water (1:1) showed higher antioxidative activity than water and ethanolic extracts in the β-carotene/linoleic acid system. In general, supercritical CO₂ extraction method had comparatively poor on the antioxidant activity of jujube, whereas samples extracted by UJEW technique at different concentrations had the highest inhibitory effects in prevention of β-carotene oxidation. Our results were in agreement with previously published results of Esmaeilzadeh Kenari et al.^[40]

From the evaluation of the antioxidant properties of extracts in vivo (DPPH and β-carotene assay) it is realized that ultrasound-assisted extraction of jujube by ethanol-water at 600 and 800 ppm had the highest antioxidant activity. Therefore, UJEW was used to assess its effect on stability of soybean oil during frying process.

Changes in TPC

Determination of TPC is one of the most important tests and a valid criterion for assessing the thermal oxidative degradation of the oils during deep frying (Stier 2013). Table 1 shows changes in TPC content of the oil samples during deep frying process.^[41] In many European countries the TPC value is considered as a major oil degradation indicator and it is acceptable at a maximum of 25–27% for used frying oil.^[5] Fresh soybean oil had a TPC content of 5.62%, reflecting the good quality of the oil used, as TPC content of unused oils normally ranges between 0.4 and 6.4%.^[42] TPC content increased during frying and had high correlation coefficient with frying time. This result concurred with the results of Matthäus,^[43] Bansal et al.,^[44] and Osawa et al.^[45] The correlation equation and correlation coefficient in soybean oils containing 600 and 800 ppm of extract, BHT, BHA, TBHQ, and SBO (soybean oil as a control) were y = 5.209x – 0.594; R² = 0.978, y = 6.195x – 2.841; R² = 0.972, y = 6.726x – 2.32; R² = 0.981, y = 6.414x – 2.18; R² = 0.981, y = 6.254x + 2.958; R² = 0.973, y = 8.116x – 1.698; R² = 0.995, respectively.

TABLE 1 Changes in total polar compounds (TPC) content, carbonyl value (CV), and peroxide value (PV) of soybean oil with and without jujube fruit antioxidant during deep frying at 180°C

	TPC (w/w %)
	UJEW	UJEW	BHT	BHA	TBHQ
Time (h)	600 ppm	800 ppm	100 ppm	100 ppm	100 ppm	SBO
0	5.62 ± 0.87a	5.62 ± 0.87a	5.62 ± 0.87a	5.62 ± 0.87a	5.62 ± 0.87a	5.62 ± 0.87a
4	11.62 ± 0.71b	9.53 ± 0.64a	12.87 ± 0.58c	11.65 ± 0.55b	10.34 ± 0.70a	16.24 ± 0.27d
8	12.71 ± 1.01a	14.18 ± 0.37bc	15.22 ± 0.33c	15.08 ± 0.48bc	14.11 ± 0.41b	22.47 ± 0.55d
12	19.15 ± 0.12a	21.45 ± 0.64b	24.46 ± 0.76c	23.62 ± 0.56c	18.25 ± 0.65a	30.67 ± 0.65d
16	24.55 ± 0.68a	26.33 ± 0.86b	29.17 ± 0.40c	27.43 ± 0.93b	27.51 ± 0.89b	38.25 ± 0.58d
20	30.03 ± 0.35a	32.15 ± 0.47b	37.29 ± 0.83d	35.41 ± 0.57c	36.48 ± 0.80d	45.52 ± 0.54e
24	42.02 ± 0.38a	44.31 ± 0.22b	47.47 ± 0.53d	45.53 ± 0.51c	42.10 ± 0.62a	56.59 ± 0.50e
			CV (µmol/g)
0	13.75 ± 0.49a	13.75 ± 0.49a	13.75 ± 0.49a	13.75 ± 0.49a	13.75 ± 0.49a	13.75 ± 0.49a
4	19.23 ± 0.25b	17.68 ± 0.68a	22.56 ± 0.69c	21.45 ± 0.97c	19.06 ± 0.33b	25.68 ± 0.88d
8	25.14 ± 0.26a	25.73 ± 0.60a	29.36 ± 0.30c	27.48 ± 0.59b	27.55 ± 0.49b	38.66 ± 0.63d
12	30.44 ± 0.29a	33.61 ± 0.46c	35.58 ± 0.44d	37.14 ± 0.44e	32.25 ± 0.61b	49.54 ± 0.54f
16	34.95 ± 0.30c	36.73 ± 0.57d	42.34 ± 0.42e	33.27 ± 0.21b	31.48 ± 0.45a	47.29 ± 0.36f
20	38.14 ± 0.52a	46.50 ± 0.36c	49.68 ± 0.43e	44.72 ± 0.49b	48.19 ± 0.54d	55.33 ± 0.41f
24	42.73 ± 0.14a	44.41 ± 0.70b	55.21 ± 0.14d	52.08 ± 0.47c	51.70 ± 0.26c	64.72 ± 0.30e
			PV(meq/kg)
0	0.55 ± 0.03a	0.55 ± 0.03a	0.55 ± 0.03a	0.55 ± 0.03a	0.55 ± 0.03a	0.55 ± 0.03a
4	2.15 ± 0.12b	1.87 ± 0.06a	2.21 ± 0.06d	2.27 ± 0.11b	1.73 ± 0.06a	3.54 ± 0.05c
8	2.68 ± 0.06ab	2.75 ± 0.04b	3.75 ± 0.06d	3.24 ± 0.05c	2.62 ± 0.06a	4.26 ± 0.07e
12	3.46 ± 0.06a	3.98 ± 0.06b	4.32 ± 0.06c	4.47 ± 0.10d	4.01 ± 0.07b	5.73 ± 0.49e
16	4.22 ± 0.06a	4.70 ± 0.06b	5.01 ± 0.09d	4.21 ± 0.10c	4.72 ± 0.07b	5.54 ± 0.09e
20	5.02 ± 0.08b	5.38 ± 0.07c	4.42 ± 0.04a	5.65 ± 0.04d	5.62 ± 0.05d	7.44 ± 0.03e
24	5.01 ± 0.10c	5.12 ± 0.10d	4.02 ± 0.08a	5.16 ± 0.05d	4.70 ± 0.06b	6.25 ± 0.04e

Means ± SD; Significant differences in a same row are shown by different letters (p < 0.05); UJEW: ultrasound-assisted extract of jujube by ethanol-water (1:1), SBO: soybean oil with no added antioxidant.

The TPC of the soybean oils containing UJEW of 600 and 800 ppm, BHT, BHA, TBHQ, and SBO after 24 h of frying were 42.02, 44.31, 47.47, 45.53, 42.10, and 56.59%, respectively. These results showed that SBO reached 25% TPC (degradation limit for regulation purposes) after 10.2 h of deep-frying cycles, while the oil with the addition of UJEW 600 and 800 ppm, BHT, BHA, TBHQ reached 25% TPC after 16.01, 15.1, 12.06, 13.8, and 15.12 h deep frying cycles. Therefore, the thermo-oxivative stability of soybean oil increased significantly in the presence of extracts compared to control oil. During frying time observed the rate increase in the amount of TPC in 600 ppm of UJEW was relatively slower than that of other oil samples and later reached the critical limit (25%). Therefore, the rate of discard of oil samples were as follows:

These results were in agreement with previous studies results such as the works by Casarotti and Jorge^[46] and Urbančič et al.^[22] which indicated that rosemary extract was more effective in reducing polar compounds production in frying oil compared to synthetics antioxidants.

Changes in CV

Changes in CV of the oil samples during frying process are shown in Table 1. The CV is an appropriate method for evaluation of frying oil quality because carbonyl compounds are the cause for undesirable flavors and reducing nutritional value of fried foods. CV measures secondary products of oxidation such as aldehydes and ketones.^[20] On the basis of the National Standards of Japan frying oils containing more than 50 µmol/g of CV should be discarded, because undesirable changes have been occurred in their flavor.^[47] The result showed the CV of a set of oil samples increased and reached a maximum value during frying and then decreased. The result concurred with previously published results of Farhoosh and Kenari^[48] and Farhoosh and Tavassoli-Kafrani.^[24] According to Farhoosh and Kenari^[48] during the prolonged frying period carbonyl compounds may convert to new compounds that are not measurable with CV test. There were no significant differences between the initial CV of the oil samples (13.75 µ mol/g). The CV for soybean oil containing 600 and 800 ppm of UJEW was lower than critical limit (51 µ mol/g) during frying process. Whereas, the oil containing BHT, BHA, and TBHQ after 24 h and SBO after 20 h reached the maximum CV (55.21, 52.08, 51.70, and 64.72 µmol/g, respectively). Therefore, UJEW at 600 ppm showed higher antioxidant activity compared to other oil samples in prevention of carbonyl compounds increase during frying process.

Changes in PV

Table 1 shows changes in PV of the oil samples during deep frying process at 180°C. PV is used to assess primary products of oxidation (hydroperoxide) in oils and fats.^[3] Initial PV for all samples was lower than 2 meq O₂/kg. The PV of fresh oil should be less than 2 meq O₂/kg.^[49] It was generally observed that the extract significantly reduced PV of oils compared to control oil (p < 0.05). There was an initial increase in PV of SBO and BHA from 0 to 12 h, for BHT and TBHQ from 0 to 16 h, for UJEW at 600 and 800 ppm from 0 to 20 h and after that the rate slowed down. Peak values for PV were obtained and are as follows: BHT (5.01 meq O₂/kg after 16 h); BHA (5.65 meq O₂/kg after 20 h); UJEW at 600 ppm (5.02 meq O₂/kg after 20 h); UJEW at 800 ppm (5.38 meq O₂/kg after 20 h); TBHQ (5.62 meq O₂/kg after 20 h); and SBO (7.44 meq O₂/kg after 20 h).

The PV decreased in oil samples after the peak was reached. The PV results were in agreement with previously published results of Casal et al.^[1] and Farhoosh et al.^[23] A rapid rise in PV of soybean oils containing BHT, BHA, TBHQ, and SBO showed that they were more sensitive to oxidation degradation. UJEW at 600 ppm indicated greater ability to prevent an increase in PV as compare to the other oil samples. The PV test is not a valid parameter to assess oils’ oxidative changes during deep frying process, because peroxidase under frying conditions are unstable and are converted into other compounds, such as carbonyl and aldehyde, that cause PV abatement.^[50]

Changes in CD and CT

Changes in CD which represent the degree of production of primary oxidation products are shown in Table 2. The CD values of soybean oil treated with UJEW at 600 and 800 ppm were significantly different from the control oil, and also from the oils with added BHA, BHT, and TBHQ. The CD values in oils containing 800 ppm of UJEW and TBHQ with no significant difference (p > 0.05) at 4 h and in oil with 600 ppm of UJEW at other frying times (8, 12, 16, 20, and 24 h) were lower than other oil samples. At the end of 24 h frying process the CD values for the soybean oil containing 600 and 800 ppm of UJEW, BHT, BHA, and TBHQ were 15.65, 17.08, 17.77, 17.46, and 16.53 (mmol/L), respectively.

TABLE 2 Changes in conjugated diene (CD) and triene (CT) values of soybean oil with and without jujube fruit antioxidant during deep frying at 180°C

	CD (mmol/L)
	UJEW	UJEW	BHT	BHA	TBHQ
Time (h)	600 ppm	800 ppm	100 ppm	100 ppm	100 ppm	SBO
0	3.20 ± 0.02a	3.21 ± 0.02a	3.21 ± 0.02a	3.22 ± 0.02a	3.22 ± 0.02a	3.20 ± 0.02a
4	7.51 ± 0.06c	6.44 ± 0.12a	7.35 ± 0.03b	7.48 ± 0.04c	6.35 ± 0.05a	10.55 ± 0.05d
8	9.42 ± 0.07a	10.07 ± 0.05c	10.57 ± 0.05e	10.33 ± 0.03d	9.84 ± 0.06b	12.36 ± 0.05f
12	10.43 ± 0.05a	11.7 ± 0.04b	12.02 ± 0.07d	11.84 ± 0.06c	11.66 ± 0.12b	15.87 ± 0.08e
16	12.71 ± 0.06a	13.84 ± 0.02c	14.35 ± 0.07e	14.18 ± 0.03d	13.45 ± 0.04b	17.44 ± 0.08f
20	14.32 ± 0.04a	15.75 ± 0.05c	16.83 ± 0.07e	16.25 ± 0.07d	15.26 ± 0.04b	20.81 ± 0.06f
24	15.67 ± 0.05a	17.08 ± 0.04c	17.77 ± 0.05e	17.46 ± 0.04d	16.53 ± 0.02b	23.47 ± 0.07f
			CT (mmol/L)
0	0.74 ± 0.03a	0.75 ± 0.03a	0.74 ± 0.03a	0.75 ± 0.03a	0.76 ± 0.03a	0.75 ± 0.03a
4	2.75 ± 0.03c	2.43 ± 0.03a	2.83 ± 0.04d	2.90 ± 0.04d	2.53 ± 0.06b	4.28 ± 0.04e
8	3.17 ± 0.05a	3.45 ± 0.05b	4.67 ± 0.05d	4.58 ± 0.06c	3.46 ± 0.05b	5.60 ± 0.04e
12	4.84 ± 0.06a	5.25 ± 0.03b	6.73 ± 0.03e	6.42 ± 0.04d	5.57 ± 0.05c	8.40 ± 0.06f
16	5.44 ± 0.03a	6.83 ± 0.08c	7.31 ± 0.03e	7.02 ± 0.13d	6.22 ± 0.04b	9.15 ± 0.08f
20	6.77 ± 0.12a	8.14 ± 0.08c	8.65 ± 0.04e	8.39 ± 0.08d	7.83 ± 0.05b	11.47 ± 0.05f
24	8.54 ± 0.06b	9.36 ± 0.05c	9.76 ± 0.04e	9.53 ± 0.05d	8.40 ± 0.04a	13.23 ± 0.02f

Means ± SD; Significant differences in a same row are shown by different letters (p < 0.05); UJEW: ultrasound-assisted extract of jujube by ethanol-water (1:1), SBO: soybean oil with no added antioxidant.

The CT values of soybean oils treated with antioxidants were significantly different from the control oil (Table 2). The CT value of control oil at the end of 24 h of frying was greater than that of oils treated with jujube fruit extracts and synthetic antioxidants. Generally, CT values for soybean oil containing UJEW at 600 and 800 ppm BHT, BHA, and TBHQ were 5.54, 9.36, 9.76, 9.53, and 8.4 (mmol/L), respectively. Therefore, the UJEW at 600 ppm indicated a greater ability to reduce the production of conjugated compounds compared to other oils treated with antioxidants. These results were approved by previous studies of Casarotti and Jorge^[46] and Urbančič et al.^[22] In the present study, similar to those reported by Chirinos et al.^[35] and Bou et al.,^[51] the CD and CT values increased with increasing frying time. Abdulkarim et al.^[5] showed that due to the formation of CD and CT absorption increase is proportional to the uptake of oxygen and formation of peroxides during early stages of oxidation. However, the increase of CD was considerably higher compared to the CT, which will be specifically due to the high content of linoleic acid in soybean oil.

Changes in FFA Content

Changes in the FFA content of the oil samples are shown in Table 3. The FFA is used as an indicator for assessment of oil deterioration during frying.^[5] There was no significant difference between initial FFA (0 h) of oil samples. However, in all samples FFA showed a trend to increase from the beginning of the frying period to the end of experiment, similar to results reported by Kim and Choe^[52] and Wang et al.^[53] The FFA content at the end of 24 h of frying for soybean oil containing UJEW at 600 and 800 ppm BHT, BHA, TBHQ, and SBO reached to 3.52, 4.22, 4.02, 3.66, 3.73, and 6.41%, respectively. However, the values of oil samples treated with natural and synthetic antioxidants were found to be significantly (p < 0.05) lower than the control oil. These results indicated that natural antioxidants in UJEW of 600 ppm protected soybean oil from hydrolysis better than synthetic antioxidants. Our results concurred with the results of Casarotti and Jorge^[46] and Urbančič et al.^[22] which examined the antioxidant effect of rosemary extract in soybean and sunflower oil.

TABLE 3 Changes in free fatty acids (FFA) content and oxidative stability index (OSI) of soybean oil with and without jujube fruit antioxidant during deep frying at 180°C

	FFA (%)
	UJEW	UJEW	BHT	BHA	TBHQ
Time (h)	600 ppm	800 ppm	100 ppm	100 ppm	100 ppm	SBO
0	0.05 ± 0.00a	0.05 ± 0.00a	0.05 ± 0.00a	0.05 ± 0.00a	0.05 ± 0.00a	0.05 ± 0.00a
4	0.85 ± 0.01a	0.85 ± 0.01a	1.18 ± 0.02d	1.05 ± 0.04c	0.94 ± 0.01b	1.22 ± 0.01e
8	1.52 ± 0.03b	1.22 ± 0.03a	1.98 ± 0.03d	1.74 ± 0.04c	1.22 ± 0.03a	2.04 ± 0.04e
12	1.95 ± 0.05a	2.20 ± 0.05c	2.84 ± 0.03e	2.46 ± 0.04d	2.11 ± 0.04b	3.51 ± 0.06f
16	2.55 ± 0.05a	2.73 ± 0.02b	3.25 ± 0.06e	2.94 ± 0.03d	2.84 ± 0.06c	4.26 ± 0.04f
20	3.17 ± 0.03a	3.44 ± 0.06b	4.32 ± 0.08c	3.28 ± 0.04a	3.21 ± 0.08a	5.35 ± 0.03d
24	3.52 ± 0.02a	4.22 ± 0.04d	4.02 ± 0.04c	3.66 ± 0.06b	3.73 ± 0.03b	6.41 ± 0.09e
			OSI (h)
0	4.92 ± 0.04a	4.83 ± 0.07ab	4.65 ± 0.11b	4.65 ± 0.13bc	4.59 ± 0.13bc	4.44 ± 0.18c
4	4.72 ± 0.07a	4.68 ± 0.12a	4.42 ± 0.80b	4.37 ± 0.05b	4.36 ± 0.04b	2.25 ± 0.03c
8	4.47 ± 0.05a	4.45 ± 0.06a	4.28 ± 0.07b	4.22 ± 0.06b	4.18 ± 0.06b	1.52 ± 0.04c
12	4.26 ± 0.06a	4.23 ± 0.05a	4.01 ± 0.06c	4.01 ± 0.05b	3.85 ± 0.06b	1.20 ± 0.06d
16	3.95 ± 0.08a	3.87 ± 0.05a	3.36 ± 0.06d	3.48 ± 0.04c	3.24 ± 0.06b	0.98 ± 0.07e
20	3.68 ± 0.09a	3.55 ± 0.07b	2.05 ± 0.06c	2.87 ± 0.10d	2.92 ± 0.05c	0.72 ± 0.05e
24	3.50 ± 0.09a	3.22 ± 0.04b	2.11 ± 0.07c	2.24 ± 0.05e	2.35 ± 0.06d	0.48 ± 0.03f

Means ± SD; Significant differences in a same row are shown by different letters (p < 0.05); UJEW: ultrasound-assisted extract of jujube by ethanol-water (1:1), SBO: soybean oil with no added antioxidant.

Changes in Oxidative Stability Index (OSI)

Changes in the OSI of oils during deep-frying process are shown in Table 3. The Rancimat method is often used for predicting oxidative stabilities of oils under deep frying conditions. The oxidation stability of soybean oil was greatly improved in the presence of extracts. Similar to results reported by Nor et al.^[3] and Casal et al.^[1] it was observed that stability of oil samples decreased gradually during frying process. Change percentage of oils containing BHA from 4 to 8 h (3.55%), 16 to 20 (6.4%), and UJEW at 600 ppm from 0 to 4 (2.07%), 8 to 12 (4.92%), 12 to 16 (7.84%), and 20 to 24 (5.14%) was lower than that of other oil samples, respectively. The rate of oxidation of oil samples was as follows:

Therefore, results indicated that the maximum stability in soybean oil was obtained by addition of 600 ppm of UJEW. OSI results were in agreement with previously published results such as the work of Taghvaei et al.^[15] and Aladedunye and Matthäus.^[28]

CONCLUSION

In this study, natural antioxidants extracted from jujube (Ziziphus mauritiana) using ultrasound-assisted extraction method with ethanol-water were found to be the most active in DPPH^• radical scavenging capacity and β-carotene-linoleic acid assays. In terms of TPC, CV, PV, OSI, FFAs, CD, and CT our results also confirmed that the protection offered by ultrasound-assisted extraction is better than that of widely used synthetic antioxidants such as BHA, BHT, and TBHQ. Therefore, it is suggested that the best method for the extraction of phenolic compounds from jujube is ultrasound-assisted extraction method. Jujube extract showed acceptable oxidation prevention activity in soybean oil and could be used as a natural antioxidant in oil and oil-based products.