Healthy blends of high linoleic sunflower oil with selected cold pressed oils: Functionality, stability and antioxidative characteristics

Industrial Crops and Products 43 (2013) 65–72

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Healthy blends of high linoleic sunflower oil with selected cold pressed oils: Functionality, stability and antioxidative characteristics Mohamed Fawzy Ramadan ∗ Agricultural Biochemistry Department, Faculty of Agriculture, Zagazig University, Zagazig 44519, Egypt

a r t i c l e

i n f o

Article history: Received 13 March 2012 Received in revised form 7 July 2012 Accepted 9 July 2012 Keywords: Healthy oils Nigella sativa Cuminum cyminum Coriandrum sativum Syzygium aromaticum

a b s t r a c t The consumption of health-promoting products such as cold pressed oils may improve human health and prevent certain diseases. Blends (10% and 20%, w/w) of cold pressed oils including black cumin oil (BC), cumin oil (Cum), coriander oil (Cor) and clove oil (Clo) with high linoleic sunflower oil (SF) were formulated. Oxidative stability (OS) and radical scavenging activity (RSA) of SF and blends stored under oxidative conditions (60 ◦ C) for 8 days were studied. By increasing the proportion of cold pressed oils in SF, linoleic acid level decreased, while tocols level increased. Progression of oxidation was followed by measuring peroxide value (PV), p-anisidine value (Av), conjugated dienes (CD) and conjugated trienes (CT). Inverse relationships were noted between PV as well as Av and OS at termination of storage. Levels of CD and CT in SF and blends increased with increase in time. Cold pressed oil blends gave about 70% inhibition of DPPH• radicals. Oxidative stabilities of oil blends were better than SF, most likely as a consequence of changes in fatty acids and tocols’ profile, and minor bioactive lipids found in cold pressed oils. © 2012 Elsevier B.V. All rights reserved.

1. Introduction Lipid oxidation is an important problem for food and cosmetic industry. This is relevant when the lipidic substrates are composed of unsaturated or polyunsaturated fatty acids (PUFA) that are sensitive to oxidation. Antioxidants’ effectiveness depends on their chemical reactivity (as radical scavengers or metal chelators), interaction with food components, environmental conditions (e.g., pH and concentration) and physical location of the antioxidant in food systems (Lucas et al., 2010). Oxidation imparts undesirable flavors and aromas, compromises the nutritional quality of oils, and leads to the induction of toxic compounds. Lipids involved in the oxidation process contain unsaturated fatty acids and long chain PUFA; however, other unsaturated lipids such as sterols do become oxidized. The oxidation of edible fats and oils can be controlled by application of antioxidants and using processing techniques that minimize loss of tocopherols and other antioxidants (Miraliakbaru and Shahidi, 2008). Sunflower oil (SF) with high levels of PUFA is one of the main oils used for cooking and frying. These oils, however, is not quite suitable for frying due to oxidation at elevated temperatures (Anwar et al., 2007). Therefore, the use of more stable frying oils of comparatively low price would be desirable. To overcome the problem of poor oxidative stability (OS) of traditional oils, ways of

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reducing the unstable PUFA content and increasing natural antioxidants were sought. One way to improve the OS of these oils is by blending with oils of high-oleic acid contents and/or high antioxidants’ levels (Mariod et al., 2005; Anwar et al., 2007; Ramadan et al., 2008). The need for widely usable and easily available bioactive lipids and natural antioxidants continues to grow. Over the last few years, increased interest in cold pressed oils has been observed as these oils have high nutritive properties. The cold pressing procedure is becoming an interesting substitute for conventional practices because of consumers’ desire for natural and safe food products (Parry et al., 2006; Lutterodt et al., 2010). Cold pressing is a technology for seed oil production, which involves no heat treatment or chemical treatments. Cold pressing also involves no refining process and may contain a higher level of lipophilic phytochemicals including natural antioxidants. Black cumin (BC), cumin (Cum), coriander (Cor) and clove (Clo) are traditional food spices and are commonly used in the food industry because of their special aroma as well as their health-promoting properties. Bioactivities of these spices are most attributed to a number of phenolic compounds and fat-soluble bioactives (tocols, sterols and polar lipids). Few reports have been published on cold pressed BC oil, but it is hard to find any data on cold pressed Cum, Cor and Clo oils. Nigella sativa (BC) seed components have also been used to prepare functional cosmetic and dietary supplemental products. Studies were conducted on pharmacological properties of BC essential oil (Ramadan, 2007; Lutterodt et al., 2010). BC seed oil is rich in essential fatty acids as well as bioactive phytosterols and

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tocopherols (Ramadan and Moersel, 2002; Ramadan, 2007). Cumin (Cuminum cyminum L.) is the second most popular spice in the world after black pepper. The proximate composition of the seeds indicates that they contain fixed oil (ca. 10%), protein, cellulose, sugar, mineral elements and volatile oil (Li and Jiang, 2004). Cumin seeds contain volatile oil (1–5%), which has been the subject of previous studies (Oroojalian et al., 2010). However, data on C. cyminum lipids are limited (Shahnaz et al., 2004; Rebey et al., 2012). Coriander (Coriandrum sativum L.) fruit-seeds contain high levels of petroselinic acid (6 -cis-octadecenoic acid, 18:1n − 12) as part of triacylglycerols. Coriander seed oil had recently been investigated and the results indicated that crude seed oil is highly promising edible oil with high levels of bioactive lipids (Ramadan et al., 2008). Clove (Syzygium aromaticum, Myrtaceae) is cultivated as a spice in many tropical countries. For oil production, clove buds are brought to European and American distilleries. Clove oil is frequently used in perfumery and medicine, but the largest part by far is used in flavorings (Zheng et al., 1992). The clove species have been demonstrated to produce a wide variety of potentially useful compounds that include sesquiterpenes, tannins, and triterpenoids. It is well-known that clove possesses a phenolic compound, 4-allyl2-methoxyphenol, commonly called eugenol. Eugenol acts as an antioxidant on oleogenous foods, as an anticarminative, antispasmodic, antiseptic in pharmacy, and also as an antimicrobial agent (Farag et al., 1989; Miyazawa and Hisama, 2003). Blending vegetable oils can increase the levels of bioactive lipids and natural antioxidants in the blends and improve nutritional value at affordable prices. Oil blends has been a common permitted practice in the many countries. Lately, it has permitted to manufacture and market blended oils containing commonly edible oil mixed with unconventional oil (Ramadan et al., 2008). Cold pressed oils are a good source of beneficial components, such as antioxidative phenolic compounds and other health-beneficial phytochemicals. As a continuation of efforts in developing healthy oils rich in health beneficial components, the present study was designed to investigate the effects of blending cold pressed BC, Cum, Cor and Clo with SF on the OS, functionality and radical scavenging activity of high linoleic SF. There is no such previous studies yet been conducted on the blending of cold pressed oils with SF. This report might serve as a milestone toward development of healthy blended oils with improved OS and nutritional value.

2. Materials and methods 2.1. Materials Cold pressed black cumin (N. sativa), cumin (C. cyminum), coriander (C. sativum) and clove (S. aromaticum) oils were obtained from a local market (Zagazig, Egypt). Refined, bleached and deodorized SF was purchased from a local market (Zagazig, Egypt). Eight oil blends were formulated by blending SF with cold pressed BC, Cum, Cor and Clo oils in proportions of 9:1 and 8:2 (w/w). The oils were thoroughly mixed to form uniform blends at room temperature. Standards used for tocols (␣-, ␤-, ␥- and ␦-tocopherol and tocotrienols) were purchased from Merck (Darmstadt, Germany). 1,1-Diphenyl-2-picrylhydrazyl (DPPH, approximately 90%) was purchased from Sigma (St. Louis, MO, USA). Galvinoxyl was from Aldrich (Milw., WI, USA). Toluene of HPLC grade was used throughout the antiradical test. All solvents and reagents from various suppliers were of the highest purity needed for each application and used without further purification.

2.2. Methods 2.2.1. Fatty acids composition of SF, cold pressed oils and oil blends Fatty acids and tocopherols of SF and oil blends were analyzed using GLC and HPLC according to Ramadan et al. (2006b, 2010). Fatty acids were transesterified into FAME using N-trimethylsulfoniumhydroxide (Macherey–Nagel, Düren, Germany). In brief, 10 mg of oil sample was dissolved in 500 ␮L of tert-butyl methyl ether then 250 ␮L of TMSH was added and the mixture was vortexed for 30 s before injection. FAME was identified on a Shimadzu GC-14A equipped with flame ionization detector (FID) and C-R4AX chromatopac integrator (Kyoto, Japan). The flow rate of the carrier gas helium was 0.6 mL/min and the split value with a ratio of 1:40. A sample of 1 ␮L was injected in a Supelco SPTM -2380 (Bellefonte, PA, USA) capillary column (30 m × 0.25 mm × 0.2 ␮m film thickness). The injector and FID temperature was set at 250 ◦ C. The initial column temperature was 100 ◦ C programmed by 5 ◦ C/min until 175 ◦ C and kept for 10 min at 175 ◦ C, then 8 ◦ C/min until 220 ◦ C and kept for 10 min at 220 ◦ C. A comparison between the retention times of the samples with those of reference compounds mixture, run on the same column under the same conditions, was made to facilitate identification. 2.2.2. Tocols profile of SF and oil blends For tocols analysis, a solution of 250 mg of oil in 25 mL n-heptane was directly used for the HPLC. The HPLC analysis was conducted using a Merck Hitachi low-pressure gradient system, fitted with an L-6000 pump, a Merck-Hitachi F-1000 Fluorescence Spectrophotometer (the detector wavelength was set at 295 nm for excitation, and at 330 nm for emission) and a D-2500 integration system. Twenty microliters of the samples were injected by a Merck 655A40 Autosampler onto a Diol phase HPLC column 25 cm, 94.6 mm ID (Merck, Darmstadt, Germany) using a flow rate of 1.3 mL/min. The mobile phase used was n-heptane/tert-butyl methyl ether (99:1, v/v). 2.2.3. Extraction and purification of the phenolic compounds from cold pressed oils Aliquots of oil (2 g) were dissolved in n-hexane (5 mL) and mixed with 10 mL methanol–water (80:20, v/v) in a glass tube for 2 min in a vortex. After centrifugation at 3000 rpm for 10 min, the hydroalcoholic extracts were separated from the lipid phase by using Pasteur pipette then combined and concentrated in vacuo at 30 ◦ C until a syrup consistency was reached. The lipidic residue was redissolved in 10 mL methanol–water (80:20, v/v) and the extraction was repeated twice. Hydroalcoholic extracts were redissolved in acetonitrile (15 mL) and the mixture was washed three times with n-hexane (15 mL each). Purified phenols in acetonitrile were concentrated in vacuo at 30 ◦ C then dissolved in methanol for further analysis. 2.2.4. Characterization of phenolic compounds in cold pressed oils Aliquots of phenolic extracts were evaporated to dryness under nitrogen. According to Ramadan et al. (2003) the residue was redissolved in 0.2 mL water and diluted (1:30) Folin–Ciocalteu’s phenol reagent (1 mL) was added. After 3 min, 7.5% sodium carbonate (0.8 mL) was added. After a further 30 min, the absorbance was measured at 765 nm using UV-260 visible recording spectrophotometer (Shimadzu, Kyoto, Japan). Gallic acid was used for the calibration and the results of triplicate analyses are expressed as parts per million of gallic acid. UV spectrum (200–400 nm) of 1% oil in 2,2,4-trimethylpentane was recorded using a Shimadzu UV-260 spectrophotometer (Kyoto, Japan).

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Table 1 Fatty acid composition (relative content, %) of SF and cold pressed oils.

C 10:0 C 12:0 C 14:0 C 16:0 C 16:1 C 18:0 C 18:1n − 12 C 18:1n − 9 C 18:2n − 6 C 18:3 C 20:2 C 22:0 C 22:1 C24:0

SF

Black cumin oil

Cumin oil

Coriander oil

Clove oil

N.D. N.D. N.D. 7.00 ± 0.22 0.40 ± 0.09 3.80 ± 0.15 N.D. 26.5 ± 0.99 61.1 ± 1.25 0.20 ± 0.02 N.D. 0.80 ± 0.09 0.10 ± 0.06 0.30 ± 0.02

N.D. 0.06 ± 0.01 0.19 ± 0.05 11.7 ± 0.25 0.20 ± 0.05 3.70 ± 0.14 N.D. 24.7 ± 1.66 55.8 ± 3.12 0.94 ± 0.05 2.66 ± 0.02 N.D. N.D. N.D.

0.09 ± 0.01 0.03 ± 0.01 0.06 ± 0.01 8.06 ± 0.30 0.13 ± 0.03 5.16 ± 0.19 41.2 ± 2.14 4.97 ± 1.09 39.5 ± 2.19 0.80 ± 0.06 N.D. N.D. N.D. N.D.

N.D. 0.33 ± 0.05 0.09 ± 0.02 7.82 ± 0.19 0.26 ± 0.09 4.97 ± 0.12 47.9 ± 2.22 N.D. 36.6 ± 2.30 1.66 ± 0.08 N.D. N.D. 0.27 ± 0.03 N.D.

2.76 ± 0.09 0.07 ± 0.01 0.06 ± 0.01 8.58 ± 0.24 0.36 ± 0.05 6.59 ± 0.19 N.D. 39.4 ± 1.53 40.2 ± 2.10 1.99 ± 0.05 N.D. N.D. N.D. N.D.

Results are given as the average of triplicate determinations ± standard deviation. N.D., not detected.

2.2.5. Accelerated oxidation experiment of SF and oil blends (Schaal oven test) SF and oil blends were placed in a series of transparent glass bottles having a volume 20 mL each. The bottles were filled with 10 g of SF and/or oil blends and left open. The oxidation reaction was accelerated in a forced-draft air oven T6 (Heraeus Instruments GmbH; Hanau, Germany) set at 60 ± 2 ◦ C for up to 2, 4 and 8 days. Immediately after storage period, oil samples were withdrawn for triplicate analyses.

2.2.6. Analytical procedures for monitoring oxidative stability The progress of the oxidative deterioration of the oil blends during storage was followed by measuring at regular intervals changes in peroxide value (PV) and p-anisidine value (Av) according to the official methods of the American Oil Chemists’ Society (1995). The absorptivity values at 232 and 270 nm were recorded in spectrophotometry (Shimadzu UV-260 visible recording spectrophotometer, Kyoto, Japan) following the analytical methods described by IUPAC (1979), method II.D.23. The contents of conjugated diene (CD) and trienes (CT) were expressed as absorptivities of the 1% oil in 2,2,4-trimethylpentane. 2.2.7. Radical scavenging activity (RSA) toward DPPH• radical The RSA of SF and oil blends during oven test was assayed with DPPH• radicals dissolved in toluene according to Ramadan et al. (2003). RSA and presence of hydrogen donors in SF and oil blends during accelerated oxidation test were examined by reduction of DPPH• in toluene. Toluene solution of DPPH• radicals was freshly prepared at a concentration of 10−4 M. The radical, in the absence of antioxidant compounds, was stable for more than 2 h of normal kinetic assay. For evaluation, 10 mg of SF or oil blends sampled during Schaal oven test (in 100 ␮L toluene at room temperature) were mixed with 390 ␮L toluene solution of DPPH• radicals and the mixture was vortexed for 20 s at ambient temperature. Against a blank of toluene without DPPH• , the decrease in absorption at 515 nm was measured in 1 cm quartz cells after 60 min of mixing using UV-260 visible recording spectrophotometer (Shimadzu, Kyoto, Japan). RSA toward DPPH• was estimated from the differences in the absorbance of toluenic DPPH• solution with or without sample (control) and the inhibition percent was calculated from the following equation: % inhibition =

 Ac − As  Ac

× 100

where Ac is absorbance of control and As is absorbance of test sample

2.2.8. Radical scavenging activity toward galvinoxyl radicals Miniscope MS 100 ESR spectrometer (Magnettech GmbH, Berlin, Germany) was used throughout the analysis (Ramadan et al., 2010). Experimental conditions were as follows: measurement at room temperature; microwave power, 6 db; centerfield, 3397 G, sweep width 83 G, receiver gain 10 and modulation amplitude 2000 mG. Ten milligram of oil (in 100 ␮L toluene) was allowed to react with 100 ␮L of toluene solution of galvinoxyl (0.125 mM). The mixture was stirred on a vortex stirrer for 20 s then transferred into 50 ␮L micro pipette (Hirschmann Laborgeräte GmbH, Ederstadt, Germany) and the amount of galvinoxyl radical inhibited was measured exactly 60 s after the addition of the galvinoxyl radical solution. The galvinoxyl signal intensities were evaluated by the peak height of signals against a control. A quantitative estimation of the radical concentration was obtained by evaluating the decrease of the ESR signals in arbitrary units after 1 min incubation using KinetikShow 1.06 Software program (Magnettech GmbH, Berlin, Germany). The reproducibility of the measurements was ±5 as usual for kinetic parameters. All work was carried out under subdued light conditions. All the experiments were repeated at least thrice when the variation on any one was routinely less than 5%. All experimental procedures were performed in triplicate and the mean values (±standard deviation) were given. 3. Results 3.1. Fatty acids composition of SF, cold pressed oils and oil blends Fatty acid composition of SF and cold pressed oils is presented in Table 1. The major fatty acids in SF and cold pressed oils were linoleic, oleic, palmitic and stearic as well as petroselinic acid in Cor and Cum oils. The fatty acid profile of BC and Cor is in agreement with Ramadan et al. (2003). Cold pressed oils under study contained much lower amounts of saturated fatty acids (SFA) than that of cold-pressed cardamom seed oil (30.8 g/100 g total fatty acids) and comparable to that of 13.8 and 15.9 g/100 g total fatty acids found in the cold pressed milk thistle and roasted pumpkin seed oils, respectively (Parry et al., 2006). While, saturated fat level was higher than that of 7.4–9.7 g/100 g total fatty acids in the cold-pressed parsley, onion, hemp, mullein, and cranberry seed oils (Lutterodt et al., 2010), cold pressed oils contained significant level of monounsaturated fatty acids (MUFA) with a range of 24.7–47.9 g/100 g total fatty acids, which is comparable to the cold pressed hemp, cranberry, blueberry, onion, and milk thistle seed oils but was much lower than that of 81% and 82% in the cold-pressed carrot and parsley seed oils. Cold pressed oils had high levels of PUFA content (Table 1). This PUFA content was comparable to that in the

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Table 2 Fatty acid composition (relative content, %) of oil blends.

C 10:0 C 12:0 C 14:0 C 16:0 C 16:1 C 18:0 C 18:1n − 12 C 18:1n − 9 C 18:2n − 6 C 18:3 C 20:2 C 22:0 C 22:1 C24:0

SF:BC (9:1, w/w)

SF:BC (8:2, w/w)

SF:Cum (9:1, w/w)

SF:Cum (8:2, w/w)

SF:Cor (9:1, w/w)

SF:Cor (8:2, w/w)

SF:Clo (9:1, w/w)

SF:Clo (8:2, w/w)

N.D. 0.01 ± 0.001 0.02 ± 0.003 7.46 ± 0.66 0.34 ± 0.09 3.76 ± 0.22 N.D. 26.3 ± 0.99 60.57 ± 2.91 0.26 ± 0.13 0.27 ± 0.12 0.68 ± 0.22 0.07 ± 0.01 0.25 ± 0.03

N.D. 0.01 ± 0.001 0.04 ± 0.001 7.93 ± 0.62 0.33 ± 0.08 3.76 ± 0.26 N.D. 26.1 ± 0.79 60.05 ± 2.54 0.34 ± 0.12 0.53 ± 0.11 0.61 ± 0.19 0.06 ± 0.01 0.22 ± 0.09

0.01 ± 0.001 N.D. 0.01 ± 0.001 7.10 ± 0.55 0.34 ± 0.09 3.91 ± 0.22 4.12 ± 0.24 24.3 ± 0.77 58.94 ± 2.16 0.25 ± 0.11 N.D. 0.68 ± 0.15 0.07 ± 0.01 0.25 ± 0.07

0.02 ± 0.001 0.01 ± 0.001 0.01 ± 0.001 7.20 ± 0.24 0.31 ± 0.04 4.05 ± 0.31 8.24 ± 0.32 22.2 ± 0.85 56.78 ± 2.55 0.31 ± 0.10 N.D. 0.61 ± 0.17 0.06 ± 0.01 0.22 ± 0.08

N.D. 0.03 ± 0.001 0.01 ± 0.001 7.07 ± 0.71 0.35 ± 0.07 3.89 ± 0.21 4.80 ± 0.12 23.8 ± 0.99 58.65 ± 2.10 0.33 ± 0.11 N.D. 0.68 ± 0.13 0.10 ± 0.02 0.25 ± 0.07

N.D. 0.07 ± 0.009 0.02 ± 0.001 7.16 ± 0.59 0.34 ± 0.09 4.01 ± 0.19 9.59 ± 0.16 21.2 ± 0.86 56.21 ± 2.28 0.48 ± 0.19 N.D. 0.61 ± 0.11 0.12 ± 0.02 0.22 ± 0.06

0.28 ± 0.03 0.01 ± 0.001 0.01 ± 0.001 7.15 ± 0.62 0.36 ± 0.06 4.05 ± 0.12 N.D. 27.7 ± 0.55 59.01 ± 2.71 0.37 ± 0.18 N.D. 0.68 ± 0.10 0.07 ± 0.01 0.25 ± 0.05

0.55 ± 0.07 0.01 ± 0.001 0.01 ± 0.001 7.31 ± 0.36 0.36 ± 0.04 4.33 ± 0.16 N.D. 29.0 ± 0.49 56.92 ± 2.26 0.55 ± 0.16 N.D. 0.61 ± 0.17 0.06 ± 0.01 0.22 ± 0.07

Results are given as the average of triplicate determinations ± standard deviation. N.D., not detected.

cold-pressed cranberry (67.6 g/100 g), onion (64–65 g/100 g), milk thistle (61 g/100 g), and blueberry (69 g/100 g) seed oils, but lower than that in the cold pressed red raspberry, marionberry, hemp, boysenberry, and mullein seed oils with a PUFA content of 73–86 g/100 g total fatty acids (Parry et al., 2006). The main fatty acids in the oil blends were also linoleic, oleic, palmitic and stearic acids as presented in Table 2. Blending of cold pressed oils with SF non-significantly modified the levels of main fatty acids in the blends. Due to blending of Cor, Cum and Clo oils, changes were noted in the contents of C18:1 and C18:2 of blended oils (Table 2). For example, blending of Cor resulted in increases from 26.9% to 29.1% and 31.2% in the MUFA contents of SF:Cor blends 9:1 and 8:2, respectively (Fig. 1). On the other side, the contents of PUFA were decreased from 61.3% to 59.0% and 56.7%, respectively. The changes in SFA as a result of blending SF with cold pressed oils were very small (Fig. 1). In general, it could be noted that the impact of blending SF with BC on fatty acid profile was not as marked as that of the blending with Cor, Cum and Clo oils.

3.2. Tocols composition of SF, cold pressed oils and oil blends Tocols in vegetable oils are believed to protect PUFA from peroxidation and improve OS of the oil (Ramadan et al., 2007). Results of qualitative and quantitative composition of tocols are summarized in Table 3. SF contained high amount of total tocopherols (3852 mg/kg), wherein ␣-, ␤- and ␥-tocopherols were measured in levels of 3604, 162 and 59.9 mg/kg, respectively. Cor, Cum and Clo oils were characterized by extremely high levels of total tocols especially tocotrienols. BC oil contained high level of ␤-tocotrienol, while ␥-tocopherol, ␣-tocotrienol and ␦tocotrienol were measured in high levels of Cor, Cum and Clo oils. Tocols profile, especially ␣- and ␥-tocopherols and tocotrienols, of SF was totally enriched upon blending with cold pressed oils (Table 3). ␣- and ␥-tocopherols proved to be the major tocopherols in vegetable oils and fats. ␥-Tocopherol occurred in highest concentrations in camelina, linseed, cold-pressed rapeseed and corn oil (Schwartz et al., 2008). Kallio et al. (2002) mentioned that ␣tocopherol is the most efficient antioxidant of tocopherol isomers, while ␤-tocopherol has 25–50% of the antioxidative activity of ␣-tocopherol, and ␥-isomer 10–35%. Other study stated that ␥tocopherol serves as a better in vitro antioxidant than ␣-tocopherol (Miraliakbaru and Shahidi, 2008). 3.3. Phenolics profile of cold pressed oils

Fig. 1. Levels of SFA, MUFA and PUFA in percentages in SF and oil blends.

Total phenolic contents (TPC) of cold pressed oils were evaluated since phenolics may act as antioxidants and protect lipids from peroxidation. Cold pressed oils differed in their TPC values, which accounted for 3.5, 3.9, 4.3 and 5.9 mg gallic acid equivalents (GAE) per gram of BC, Cum, Cor and Clo oil, respectively. The TPC value was higher than that of 1.73–2.0 mg GAE/g oil for the cold pressed red raspberry, blueberry, and boysenberry seed oils and that of 1.8–3.4 mg GAE/g oil for the cold-pressed parsley, onion, cardamom, mullein, and milk thistle seed oils (Parry et al., 2006). These data suggest that cold pressed oils contain significant level of phenolic components, which may partially contribute to the oil stability under the accelerated oxidative conditions. The oil stability is correlated not only with the total amount of phenolics, but also with the presence of selected phenols. To assist in characterizing phenolic compounds in cold pressed oils, absorption ranges were scanned between 200 and 400 nm. The UV spectra of all cold pressed oils (Fig. 2) exhibited absorption maxima at 280 nm. Clo oil had the highest absorption maxima followed by Cor, Cum and BC oils, respectively. The absorption maximum at 280 nm may be due to the presence of p-hydroxybenzoic acid and flavone/flavonol derivatives (Ramadan et al., 2003).

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Table 3 Levels of tocols (mg/kg) in SF, cold pressed oils and oil blends.

Sunflower (SF) Black cumin Cumin Coriander Clove SF:BC (9:1) SF:BC (8:2) SF:Cum (9:1) SF:Cum (8:2) SF:Cor (9:1) SF:Cor (8:2) SF:Clo (9:1) SF:Clo (8:2)

␣-T

␣-T3

␤-T

␥-T

␤-T3

␥-T3

␦-T

␦-T3

3604.5 64.225 831.29 45.951 14890 3191.0 2696.9 2767.6 2670.6 3154.0 2605.1 5718.3 7564.9

8.2830 47.602 1147.7 1327.1 1110.9 5.9510 17.108 112.35 142.56 133.45 238.32 14.533 25.637

162.61 53.889 38.682 38.766 55.960 149.56 139.60 130.88 129.61 147.57 123.04 143.56 114.04

54.980 208.94 4012.2 3944.1 4184.3 60.271 76.899 703.33 860.08 399.62 728.57 362.20 1302.3

N.D. 1195.084 42.715 40.101 55.456 162.767 338.592 3.2 5.6 3.1 5.5 4.7 7.6

13.857 N.D. 62.094 507.29 84.960 12.056 11.599 17.668 23.857 48.093 85.606 14.204 29.193

6.947 14.480 82.191 110.22 186.41 12.447 8.591 23.234 23.801 18.680 47.199 47.180 184.53

1.353 14.309 9205.0 10056 9498.7 0.8990 1.1140 1.0150 2.7930 500.09 900.42 464.00 1500.5

Results are given as the average of triplicate determinations. N.D., not detected.

3.4. Oxidative stability of SF and oil blends 3.4.1. Changes in tocols profile during Schaal oven test The main function of tocols is as a radical-chain breaking antioxidant in membranes and lipoproteins as well as in foods. Fig. 3A

Fig. 2. Ultraviolet scans (200–400 nm) of cold pressed BC (1), Cum (2), Cor (3) and Clo (4) oils.

presents the changes in the levels of ␣-tocopehrol in SF and oil blends after 8 days of storage under oxidation conditions. It can be seen that SF:Clo contained the highest level ␣-tocopehrol followed by SF:Cor, SF:Cum, SF and SF:BC, respectively. The changes in total tocols contents of oil blends during Schaal oven experiment are shown in Fig. 3B. SF:Clo still had the highest levels of total tocols during the heating experiments followed by SF:Cor, SF:Cum, SF and SF:BC, respectively. It was clear that heating during Schaal oven test did not affect greatly the levels of tocols in SF blends and even after 8 days of storage the levels of total tocols were still extremely high especially in SF:Clo and SF:Cor blends. It could be stated that levels

Fig. 3. (A) HPLC chromatogram of tocols profile in SF and oil blends after 8 days of Schaal oven test and (B) changes in total tocols of SF and oil blends during oven test. Values given are the mean of three replicates and error bars show the variations of three determinations in terms of standard deviation.

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proportion of 9:1 and 8:2 (w/w), respectively. At the end of storage period, SF control had the highest PV (17.6 mequiv./kg) and was oxidized rapidly. p-Anisidine value (Av) plays an important role in the oxidation process of edible oil and fats. It is based on the reactivates of the aldehyde carbonyl bond on the p-anisidine amine group, leading to the formation of a Schiff base (Mohdaly et al., 2010): R CH O + H2 N  OMe → R CH N  OMe (Schiff base) + H2 O The Av, which measures the unsaturated aldehydes (principally 2-alkenals and 2,4-dienals) in oils, was determined by reacting p-anisidine with the oil in isooctane and the resultant color was measured at 350 nm. During autoxidation at 60 ◦ C in the dark (Fig. 4B), the SF oil blends were more stable. Control SF had the highest Av and was oxidized rapidly. Av increased significantly throughout the storage time, wherein the Av of SF reached a maximum of 30 from an initial value of 1.2 after 8 days of storage. Different patterns of oxidation were observed in SF oil blends. After 8 days of oven test, values for SF:Clo, SF:Cor and SF:Cum were significantly lower than those for SF:BC and SF. The results demonstrated that Clo, Cor and Cum had a potent OS. The difference in antioxidant activity may be accounted for on the basis of chemical structures. The results demonstrated that SF oil blends had higher antioxidant activity than SF.

Fig. 4. Changes in PV (A) and Av (B) of SF and oil blends during oven test. Values given are the mean of three replicates and error bars show the variations of three determinations in terms of standard deviation.

of tocols measured in oil blends may contribute to the stability of the oils toward oxidation. 3.4.2. Peroxide value (PV) and p-anisidine value (Av) Peroxide value (PV) is widely used assay for the measurement of oxidative rancidity in oils and fats. Hydroperoxide is the primary product of lipid oxidation; therefore, determination of PV can be used as oxidative index during the early stage of lipid oxidation (Ramadan and Moersel, 2004). The PV calculated for SF and oil blends are shown in Fig. 4A. The initial PV of SF was 1.1 mequiv. O2 /kg oil. Addition of cold pressed oils to the SF resulted in a marked decline in their PV during storage, thus showing enhancement of the OS of SF. On the basis of PV, the OS of SF and oil blends varied significantly, with the blends enriched with Clo (9:1 and 8:2, w/w) are being most stable. The PV clearly showed that as the storage time increased the OS of the SF and oil blends decreased (Fig. 4A). Results show that PV of oil samples increased with increase in storage period and followed the order: SF:Clo (8:2, w/w) < SF:Clo (9:1, w/w) < SF:Cor (8:2, w/w) < SF:Cor (9:1, w/w) < SF:Cum (8:2, w/w) < SF:Cum (9:1, w/w) < SF:BC (8:2, w/w) < SF:BC (9:1, w/w) < SF. Both Clo enriched blends had a much lower PV than that of blends enriched with BC over the entire storage period. PV in Clo and Cor enriched blends increased at lower level over 8 days, whereas the peroxides accumulated at relatively higher amounts in Cum and BC enriched blends in comparison with SF. The PV after 8 days were 8.10 and 7.55 mequiv. O2 /kg for SF blends enriched with Clo in proportion of 9:1 and 8:2 (w/w), respectively. At the end of storage experiment, PV recorded 13.6 and 13.2 mequiv. O2 /kg for SF blends enriched with BC in

3.4.3. Ultraviolet absorptivity Measurement of CD and CT is a good parameter for the determination of OS of oils. Formation of hydroperoxides is coincidental with conjugation of double bonds in PUFA, measured by absorptivity at the UV spectrum (Ramadan and Moersel, 2004). Lipids containing methylene-interrupted dienes or polyenes show a shift in their double bond position during oxidation. The resulting CD exhibit intense absorption at 232 nm, similarly CT absorb at 270 nm. The increase in CD and CT contents is proportional to the uptake of oxygen. The greater the levels of CD and CT in oil the lower will be the OS (Mohdaly et al., 2010). Absorptivity at 232 nm and 270 nm in SF and oil blends, due to the formation of primary and secondary products of oxidation, showed a pattern similar to that of the PV and Av (Fig. 5A and B). Absorptivity at 232 nm increased gradually with the increase in time, due to the formation of CD (Fig. 5A). Formation of aldehydes, ketones (rancid off-flavor compounds) and other oxidation products followed by an increase in absorptivity at 270 nm (Fig. 5B). The variation of absorptivity at 270 nm, due to the formation of CT as well as unsaturated ketones and aldehydes, presented a pattern similar to that of absorptivity at 232 nm. Again, the OS of SF:Clo, SF:Cor, SF:Cum and SF:BC blends were better during the oven test and UV absorptivity showed this phenomenon. The high content of conjugated oxidative products in SF can be attributed to its high linoleic acid content (61.1% of total fatty acids), which is readily decomposed to form conjugated hydroperoxides. SF:Clo blends contained the lowest level of conjugated oxidative products and these results are in agreement with the PV and Av. 3.4.4. Radical scavenging activity toward DPPH• and galvinoxyl radicals Previous research on the application of free radicals to the study of the autoxidation processes included crude vegetable oils and deep-fried oils (Ramadan et al., 2006a). The method is also valid for oil fractions with different polarity such as neutral lipids, glycolipids and phospholipids (Ramadan et al., 2003). In this study, the ability of SF and oil blends to prevent lipid peroxidation was screened using DPPH• and galvinoxyl radicals. From the data presented in Fig. 6 it can be seen that blending cold pressed oils with

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SF increased antiradical action of SF. After 8 days of storage under oxidative conditions, the impact of cold pressed oils as additives on SF oxidation was strong and gave 10–70% inhibition of DPPH• radicals, respectively (Fig. 6A). ESR measurements showed also the same pattern and similar profile of deactivating galvinoxyl radicals was obtained during oven test (Fig. 6B). After 8 days, 72% of galvinoxyl radicals were quenched in SF:Clo (8:2, w/w), while the SF:BC (8:2, w/w) was able to quench 12%. The strong RSA of SF blends might be due to the high levels of bioactive tocols and phenolics found in cold pressed oils.

4. Discussion

Fig. 5. Absorptivity at 232 nm (A) and 270 nm (B) of SF and oil blends during oven test. Values given are the mean of three replicates and error bars show the variations of three determinations in terms of standard deviation.

Fig. 6. Scavenging effect at 60 min incubation time of SF and oil blends on DPPH• (A) and galvinoxyl (B) radical as measured by changes in absorbance values at 515 nm and ESR. Values given are the mean of three replicates and error bars show the variations of three determinations in terms of standard deviation.

Oxidative reactions limit the shelf life of fresh and processed foodstuff and are a serious concern in food industry. Synthetic antioxidants such as butylated hydroxyanisole, butylated hydroxytoluene, propyl gallate and tert-butylhydroquinone are very effective in the protection of unsaturated oils and are therefore, used as potential inhibitors of oxidative reaction. However, growing concern over the safety of synthetic antioxidants has led to an increased interest in exploration of effective natural antioxidants. Moreover, foods rich in natural antioxidants play an essential role in the prevention of cardiovascular diseases and cancer. Antioxidants are often added to food to prevent the radical chain reactions of oxidation and they act by inhibiting the initiation and propagation steps, consequently delaying the oxidation process. The effectiveness of an antioxidant mainly relies on its chemical reactivity but it is also important how it interacts with other food components, their concentration and especially their physical location in different homogeneous or heterogeneous food systems. Cold pressing is a technology for seed oil production, which involves no heat treatment or solvent. Cold pressed oil involves no refining process and may contain a higher level of lipophilic compounds including natural antioxidants. Thus, crude vegetable oils are usually oxidatively more stable than the corresponding refined and processed oils. Apart from their OS depends on the fatty acid composition and the presence of minor components such as tocols, carotenoids, metal ions, polar lipids and the initial amount of hydroperoxides. Statistics regarding the composition of SF, cold pressed oils and oil blends are given in Tables 1–3. A striking feature of cold-pressed oils was the relative high level of PUFA and MUFA. From the health point of view, MUFA have been shown to lower “bad” LDL cholesterol (low density lipoproteins) and retain “good” HDL cholesterol (high density lipoproteins). This is in fact the major benefit of olive oil over the highly polyunsaturated seed oils, wherein PUFA reduce both the “bad” as well as the “good” serum cholesterol levels in our blood (Ramadan et al., 2010). The fatty acid composition of oil can be indicator of its OS and nutritional quality. The SF and oil blends have different fatty acid profile. Petrocelinic acid (C18:1n − 12) was the fatty acid marker of Cor and Cum oils, while linoleic (C18:2n − 6) and oleic (C18:1n − 9) were the major fatty acids in BC and Clo oils. A slower rate of increment in PV and Av of SF blends might be attributed to the higher amounts of oleic acid (less susceptible to oxidation) and linoleic acids (more prone to oxidation). The reported rates of oxidation of C18:1 and C18:2 are of the order of 1:12 (Miraliakbaru and Shahidi, 2008). Tocochromanols (vitamin E) are the most important lipid soluble antioxidants found in mammalian cells, and they represent an essential nutrient for human health. These molecules are important for scavenging free radicals and inhibiting lipid peroxidation in biological membranes. The tocochromanols comprise eight chemically distinct compounds that are separated into tocopherols and tocotrienols, according to saturation of the hydrophobic tails. Tocopherols contain a fully saturated tail, whereas tocotrienols have three unsaturated double bonds. The different tocopherol and

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tocotrienol isomers, that is, alpha (␣), beta (␤), gamma (␥), and delta (␦), are distinguished by the locations of methyl groups on the chromanol ring. Tocopherols are abundant in common vegetable oils; whereas tocotrienols are present only in cereal grains such as barley, rice bran, oat, wheat germ, rye, and palm oil (Sen et al., 2006). In recent years, tocotrienols have received much more attention than tocopherols, because they exhibit a different suite of biological effects and because tocotrienols show greater antioxidant activity. For example, ␣-tocotrienol is more effective at reducing lipid peroxidation than ␣-tocopherol. This elevated antioxidant activity is due to higher efficiency recycling from chromanoxyl radicals, more uniform distribution in cell membranes, and better interactions with lipid radicals. Tocotrienols have shown promise in a number of treatment areas, including lowering blood cholesterol and preventing stroke induced brain damage, as well as exhibiting some anti-inflammatory and antiangiogenesis properties (Schaffer et al., 2005). They show potent anticancer activity in various human cancer cells including prostate, breast, and colon, and tocotrienol isomers will suppress up to 50% of the proliferation of breast tumor cells both in vitro and in vivo (Miyazawa et al., 2009). These biological properties have led to the inclusion of tocotrienols in a broad spectrum of dietary supplements, functional foods, and nutraceuticals, as well as their use for cosmeceutical applications. However, tocopherols are the only tocochromanols found in popular oil crops such as soybean, sunflower, corn, and cotton. Thus, it is important to develop healthy oils that contain tocotrienols to meet the surging demand for products containing this nutrient. The results demonstrated that SF blends enriched with selected cold pressed oils had a potent OS and blending Clo, Cor, Cum and BC caused a decrease in peroxide levels during incubation for 8 days. Aside from fatty acid and tocols profile, factors, such as oxygen concentration, metal contaminants, lipid hydroxy compounds, enzymes and light may also influence the OS of oil. 5. Conclusions As food habits worldwide are based on deep fried and baked foods, oxidative-resistant oils are needed. Conventionally available cooking oils cannot fulfill this requirement; rather, they may cause serious health disorders due to the generation of hazardous oxidation products. This requirement can be conveniently met through the blending process. Cold pressed oils have been part of a supplemental diet in many parts of the world and their consumption is also becoming increasingly popular in the non-producer countries. Yet these oils rich in bioactive lipids (phenolics and tocols) may bring nutraceutical and functional benefits to food systems. At different concentrations of cold pressed oils, OS of high-linoleic SF was enhanced. Furthermore, blends enriched with cold pressed oils had strong RSA. The optimal levels of cold pressed oils enrichment will depend on the actual application. It is anticipated that commercial exploitation of cold pressed vegetable oil blends will soon be realized. References Anwar, F., Hussain, A.I., Iqbal, S., Bhanger, M.I., 2007. Enhancement of the oxidative stability of some vegetable oils by blending with Moringa oleifera oil. Food Chem. 103, 1181–1191. AOCS, 1995. Official Methods and Recommended Practices of the American Oil Chemists’ Society, 4th ed. AOCS, Champaign, IL, USA. Farag, R.S., Badei, A.Z.M.A., Hewedi, F.M., El-Baroty, G.S.A., 1989. Influence of thyme and clove essential oils on cottonseed oil oxidation. J. Am. Oil Chem. Soc. 66, 800–804.

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