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Научни трудове на Съюза на учените в България-Пловдив. Серия В. Техника и технологии, естествен ии хуманитарни науки, том XVI., Съюз на учените сесия "Международна конференция на младите учени" 13-15 юни 2013. Scientific research of the Union of Scientists in Bulgaria-Plovdiv, series C. Natural Sciences and Humanities, Vol. XVI, ISSN 1311-9192, Union of Scientists, International Conference of Young Scientists, 13 - 15 June 2013, Plovdiv.
STABILIZATION OF FLAXSEED OIL WITH DIFFERENT
ANTIOXIDANTS
Olga Teneva1* and Magdalen Zlatanov1
Department of Chemical Technology, University of Plovdiv "Paisii
Hilendarski", 24"Tsar Assen"str., Bulgaria, Plovdiv 4000 ^Corresponding author: Olga Teneva, e-mail: olga.teneva@abv.bg
ABSTRACT
Flaxseed oil is highly rich in polyunsaturated fatty acids and it is easily susceptible to auto-oxidation process. Antioxidant effects of ascorbyl palmitate, extract of rosemary and mixture of ascorbyl palmitate and extract of rosemary were examined. The oxidative stability of flaxseed oil was studied using Rancimat method, based on conductometric measurements. It was investigated as function of time at temperature 1000 C using different antioxidants. Antioxidants at the level of 0.01%-0.05% were added into the flaxseed oil. The level of 0.05% ascorbyl palmitate and 0.05% extract of rosemary was found to have higher efficiency (22.8 h and 22.3 h respectively). Combination of these antioxidants in concentration 0.05% shows similar result (20.2h). The same antioxidants used for stabilization of cosmetics increase stability more five times - Induction period is over 48h.
Key words: flaxseed oil, antioxidants, oxidative stability, ascorbyl palmitate, rosemary extract, stabilization
INTRODUCTION
Flax (Linum usitatissimum L.) is one of the oldest and multi-purpose oilseed crops cultivated in a Europe and Asia (Beltagi, 2007; Berglund, 2002). Flaxseed oil contains more than 50.0% a-linolenic acid (omega-3, essential fatty acid) (Michotte, 2011). It was found that a high content of unsaturated fatty acids leads to reduced oxidation stability of oils (Rudnik, 2001). Lipid oxidation is major factor of changes in chemical and nutritional properties of fats and oils (Yanishlieva and Popov, 1976). The addition of antioxidants improves oxidative stability of flaxseed oil. Synthetic and natural antioxidants are used in food and cosmetic industries (Bera, 2006). Ascorbyl palmitate (corresponding synthetic counterparts of ascorbic acid) was used in different concentration (Tae Kim, 2012) for stabilization of vegetable oils. There is a need to use new natural antioxidants for prevention of lipid peroxidation in lipids. Natural antioxidants have these advantages (Pokorny, 1991): acceptance by the consumers with nutraceutical value of the oil; identity with food which people have taken over hundred years.
The aim of this study is comparison efficiency of synthetic antioxidant ascorbyl palmitate, natural antioxidant extract of rosemary and mixture of both using different concentration on the increment oxidative stability of flaxseed oil.
MATERIALS AND METHODS
All solvents and reagents were of analytical grade and were used without additional purification.
Samples. The flax oil is obtained from flax seeds (Linum usitatissimum L), A900012 variety, crop 2011.
Antioxidants were provided by "Ikarov" Ltd.
Isolation of glyceride oil and determination of oil content. The oil was extracted with n-hexane (ISO 659 2009) in Soxhlet for 8 h.The solvent was partly removed in a rotary vacuum evaporator, the residue was transferred in pre-weight glass vessels and the rest of the solvent was removed under a stream of nitrogen to a constant weight to determine the oil content.
Fatty acids. The fatty acid composition of triacylglycerols was determined by gas chromatography (CG) of fatty acid methyl esters (FAME) (ISO 5508 1990). FAME were prepared by pre-esterification with sulfuric acid in methanol as catalyst (Christie, 2003) and were purified by TLC on silica gel 60 G with mobile phase hexane:acetone = 100:8 (by volume). Determination was performed on a gas chromatograph equipped with a 60 m x 0.25 mm x 25 ^m (I.D.) capillary DB-23 column (Hewlett Packard GmbH, Vienna, Austria) and a flame ionization detector. The column temperature was programmed from 130oC (hold 1 min), at 6.5oC/min to 170oC, at 3oC/ min to 215oC (hold 9 min), at 40oC/min to 230oC (hold 1 min); the injector temperature was 270oC and detector temperature was 280oC. Hydrogen was the carrier gas at a flow rate 0.8 ml/min; split was 50:1. Identification was performed by comparison of retention times with those of a standard mixture of fatty acid methyl esters subjected to GC under identical experimental conditions.
phospholipids. The quantification of phospholipids was carried out spectrophotometrically against a standard curve by measuring the phosphorous content at 700 nm after mineralization of the substance with a mixture of perchloric acid and sulphuric acid, 1:1 (by volume). The calibration curve was constructed by using a standard solution of KH2PO4. It was linear in the concentration range 1 - 130 ^g/ml (as phosphorus) (ISO 10540-1, 2003).
Sterols. Glyceride oil was hydrolized with ethanolic KOH, sterols were extracted with n-hexane and purified by thin layer chromatography with mobile phase n - hexane : diethyl ether, 1 : 1 (by volume). The quantification was carried out spectrophotometry at 597 nm against standard curve of pure P-sitosterol (Ivanov et al. (1972).
Tocopherols. High performance liquid chromatography (HPLC) (ISO 9936 2006) on a Merck-Hitachi (Merck, Darmstadt, Germany) instrument equipped with 250 mm x 4 mm Nucleosil Si 50-5 column (Merck, Darmstadt, Germany) and fluorescent detector Merck-Hitachi F 1000 was used for determination of total content. The operating conditions were as follows: mobile phase of n-hexane: dioxan, 96 : 4 (by volume), flow rate 1 ml/min, excitation 295 nm, emission 330 nm. A 20 ^l solution of crude oil (1.0%) was injected. Tocopherols were identified by comparing the retention times with those of authentic individual pure tocopherols. The tocopherol content was calculated based on the tocopherol peak areas in the sample vs. tocopherol peak area of standard tocopherol solution.
oxidative stability. The oxidative stability of oils was determined by measuring the Induction period using conductometric detection of volatile compounds (ISO 6886, 2006). A Rancimat apparatus Methrom 679 (Methrom, Herisau, Switzerland) was used at 100oC and an air flow rate
217
20 l/h.
RESULTS AND DISCUSSION General characteristics of the oil
The content of oil and the main lipid components are shown in Table 1. Table 1. Main lipid components and oil content of flaxseed oil
Flaxseed oil
Compounds Content
Oil, % 40.0
Sterols, % 0.3
Phospholipids, % 1.2
Tocopherols, mg/kg 720
Oxidative stability, h 9.6
The content of glyceride oil in the analyzed flaxseeds is in accordance to several reports (Choo et al. 2007; El-Beltagi et al. 2011; Kasote et al. 2013; Coskuner And Karababa (2007). The quantity of sterols, phospholipids and tocopherols in the oil is similar to data reported earlier (Pilat and Zadernowski 2010; Gunstone 2002; Przybylski (2005). Oxidative stability of flaxseed oil is higher than values reported earlier - 6.4 h (Rudnik et al., 2001) and 5.8 h (Szterk et al., 2010).
Fatty acid composition of triacylglycerols
Fatty acid composition of investigated triacylglycerols is presented in Table 2.
Table 2. Fatty acid composition of triacylglycerols
Fatty acid composition, %
Lauric acid (C„.n) 0,1
Myristic acid (C14.0) 2,5
Palmitic acid (C160) 9,8
Palmitoleic acid (C161) 0,1
Margaric acid (C170) 0,1
Stearic acid (C180) 4,1
Oleic acid (C181) 32,2
Linoleic acid (C1S7) 16,5
Linolenic acid (C18.3) 34,2
Arachidonic acid (C20.0) 0,4
Saturated fatty acids (SFA) 17,0
Monounsaturated fatty acids (MUFA) 32,3
Polyunsaturated fatty acids (PUFA) 50,7
The main component is linolenic acid, followed by oleic and linoleic acids. The content of linolenic acid is very close to the data announced earlier by El-Beltagi et al. (2007), Herchi et al.
(2011)-30.0-50.0 %.
The efficiency of synthetic antioxidant ascorbyl palmitate, natural antioxidant extract of rosemary and mixture of both using different concentration was examined in different concentrations (fig.1).
□ Ascorbyl palmitate
□ RLKvcmary LTtlnift
u Anlio\idaiil mixture oi" Ascorbyl Pulmihuc and Roscuiaiy extract
control 0,01 a 02 a o,(m
^P1« CouccDlratiou, %
Fig.1. Tracing oxidative stability of flaxseed oil in variation concentration of different antioxidants
The obtained results show that antioxidant effect is in correlation with concentration of antioxidants and increased gradually. The Induction period was found to be closed for both antioxidants (from 9.6 h to 22.8 h for ascorbyl palmitate and 22.3 h for rosemary extract). In practice there is increasing of oxidative stability of the oil more than two times in comparison with control sample.
Combination of two or more antioxidants works synergistically in increasing the oxidative stability of vegetable oils compared to single antioxidants (Hras et al., 2000; Azeredo et al., 2004; Omar et al., 2010). However the results show that the mixture of ascorbyl palmitate and rosemary extract at the highest concentration (0.05%) gives Induction period (20.2h) close to this when they work as single antioxidants at the same concentration (0.05% ascorbyl palmitate-22.8h and 0.05% rosemary extract-22.3h).
Since the cosmetics are to be stored for a long time (1-2 years) use of higher concentrations of antioxidants is allowed. Because of it antioxidant effect of ascorbyl palmitate and rosemay extract is investigated at concentration 0.1% and 0.2%. The results prove correlation between concentration of antioxidant and Induction period. At concentration of ascorbyl palmitate (0.1%) the Induction period is 33.1 h. At the same concentration rosemary extract gives 21.7 h. When use the same antioxidants at twice as high concentration (0.2%), they have Induction period over 48h.
CONCLUSION
The antioxidant effect is in correlation with concentration of antioxidants. Used independently in different concentration extract of rosemary has similar to ascorbyl palmitate antioxidant effect. At highest used concentrations the Induction period increases five times. Mixture of ascorbyl palmitate and rosemary extract has not synergistic antioxidant effect for protection of flaxseed oil against oxidation. When concentration of antioxidants is in borders 0.01-0.04% the highest results were observed by using of rosemary extract. For longer lipid protection ascorbyl palmitate showed significantly higher effectiveness.
ACKNOWLEDGMENTS
The investigations were carried out with the partial financial support of the Science Research Department to Plovdiv University "Paisii Hilendarski", contract SI 13FC006/2013.
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