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Effects of α-tocopherol, β-carotene and ascorbyl palmitate on oxidative stability of butter oil triacylglycerols
Food Chemistry 123 (2010) 622–627
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Food Chemistry journal homepage: www.elsevier.com/locate/foodchem
Effects of a-tocopherol, b-carotene and ascorbyl palmitate on oxidative stability of butter oil triacylglycerols Ihsan Karabulut * Department of Food Engineering, Inonu University, 44280 Malatya, Turkey
a r t i c l e
i n f o
Article history: Received 24 August 2009 Received in revised form 22 March 2010 Accepted 29 April 2010
Keywords: Butter Triacylglycerol a-Tocopherol b-Carotene Ascorbyl palmitate Oxidation Peroxide value p-Anisidine value
a b s t r a c t Butter oil triacylglycerols (BO-TAGs), free of antioxidants, including b-carotene, were obtained via sequential treatments with activated carbon (AC) adsorption and alumina column chromatography. a-Tocopherol, b-carotene and ascorbyl palmitate (AP) were added to BO-TAGs, individually, or in different combinations. An accelerated oven-oxidation test was carried out at 60 °C to determine the most effective dosages of the antioxidants. Among the antioxidants evaluated, a-tocopherol was found to be the most effective, at the concentration of 50 lg/g. To determine the possible synergism between the antioxidants, binary or ternary combinations of a-tocopherol, b-carotene and AP were added to BO-TAGs at concentrations of 50, 5, and 50 lg/g, respectively. Ternary combinations of these antioxidants were significantly better in retarding oxidation than were binary blends of a-tocopherol with b-carotene or AP. However, a prooxidant effect was observed, especially when b-carotene and AP were used individually or in binary combination. Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction Fats and oils are important functional components of foods. They contribute to flavour, odour, colour and texture. However, lipid oxidation occurs during storage or processing and leads to depletion in the quality of food products, resulting in shortening of shelf life, reducing their nutritional quality (Frankel, 1991; St. Angelo, 1996). When lipids are exposed to environmental factors, such as air, light and temperature, autoxidation reactions start to produce undesirable flavours, rancid odours, discoloration and other forms of deterioration. The primary autoxidation products are hydroperoxides, which have no taste or flavour, while their degradation products, called secondary oxidation products, do have detectable tastes and flavours (Choe & Min, 2006). a-Tocopherol is a primary antioxidant, functioning by terminating free-radical chain reactions, by donating hydrogen or electrons to free radicals and converting them to more stable products (Frankel, 1998). b-Carotene is an oil-soluble and natural pigment of many oils, such as palm, olive and butter oils. In addition to its use as a food colouring agent, b-carotene also has a strong antioxidant activity. It has been shown to protect lipids from autoxidation by reacting with peroxyl radicals, thereby inhibiting propagation and promoting termination of the oxidation chain * Tel.: +90 4223410010; fax: +90 4223410046. E-mail address: [email protected] 0308-8146/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2010.04.080
reaction (Britton, 1995). b-Carotene retards potentially harmful oxidative reactions by trapping free radicals and quenching singlet oxygen (Yanishlieva, Aitzemüller, & Raneva, 1998). Ascorbic acid is a secondary antioxidant that can be broadly classified as an oxygen scavenger/singlet oxygen quencher; it reacts with free oxygen and removes it in a closed system (Perricone et al., 1999). Ascorbyl palmitate (AP) is a synthetically-derived fat/oil-soluble ester of ascorbic acid. It was reported that AP is not only a synergist, with a-tocopherol, but also a radical-type inhibitor of lipid autoxidation (Beddows, Jagait, & Kelly, 2001; Marinova & Yanishlieva, 1992). Triacylglycerols (TAGs) of edible oils are usually used to evaluate the anti- and/or prooxidant activities of synthetic and natural antioxidants. In order to determine the effects of antioxidants in oils, TAGs free of antioxidants have been produced and used in some model oxidation studies (Fuster, Lampi, Hopia, & Kamal-Eldin, 1998; Haila, Lievonen, & Heinonen, 1996; Isnardy, Wagner, & Elmafda, 2003; Lampi & Piironen, 1998; Lampi, Piironen, Hopia, & Koivistoinen, 1997). Components of mixed antioxidant systems can contribute to the inhibition of oxidation, with the resulting antioxidant activity reflecting either additive or synergistic effects of the components. Due to uncompleted application of stripping, cooperative actions of b-carotene, naturally present in the oil with added antioxidants, have been neglected in reported studies (Lampi & Piironen, 1998; Lampi et al., 1997). Therefore, the aim of this study was to compare the effectiveness of a-tocopherol, b-carotene and AP, individually,
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or in combination, on oxidative state of butter oil triacylglycerols (BO-TAGs) under accelerated oxidation conditions. Primary oxidation products were measured by peroxide value (PV) and secondary oxidation products were measured by p-anisidine value (AV). 2. Materials and methods 2.1. Reagents Butter was purchased from local markets. Organic solvents and neutral aluminium oxide were purchased from Merck (Darmstadt, Germany). b-Carotene (Type I, 95%), p-anisidine and AP were purchased from Sigma–Aldrich (Steinheim, Germany). Activated carbon (AC) was prepared from waste apricots in our laboratory, as described in a previous report (Basar, 2006). 2.2. Methods 2.2.1. Preparation of BO-TAGs BO was produced by melting of butter at 55 °C, and filtering through a plain filter paper coated with anhydrous sodium sulphate at the same temperature. BO was kept at 18 °C, until used in subsequent procedures. Fatty acid composition (mol%) of BO was determined by a GC technique as follows: C4: 1.8, C6: 2.2, C8: 1.2, C10: 2.9, C12: 3.6, C13: 0.1, C14: 12.1, C14:1: 1.4, C15: 1.4, C15:1: 0.5, C16: 35.4, C16:1: 2.2, C17: 0.6, C17:1: 0.4, C18: 9.0, C18:1: 24.9, C18:2: 1.9, C18:3: 0.3, and C20:1: 0.2. Tocopherol and b-carotene contents of BO were determined by normal phase HPLC with a Phenomenex Luna Silica column (4.6 mm i.d. 250 mm, 5 lm). Separation was based on isocratic elution with n-hexane (99%) and iso-propanol (1%) at 292 nm (Karabulut, Topcu, Yorulmaz, Tekin, & Ozay, 2005). Tocopherol and b-carotene contents were determined as follows: a-tocopherol, 22.1 lg/g; b-tocopherol, 0.5 lg/g; d-tocopherol, 2.6 lg/g; c-tocopherol, 1.4 lg/g; b-carotene, 3.7 lg/g. To obtain pure BO-TAGs, b-carotene and tocopherols were removed from BO by the same procedure as described in a previous report (Karabulut, Topcu, Akmil-Basar, Onal, & Lampi, 2008). To remove b-carotene, BO (60 g) was mixed with AC (6 g) in a flask (100 ml) connected to a rotary evaporator at 60 °C for 300 min. Thereafter, BO was stripped to remove pro- and antioxidants to obtain purified BO-TAGs, applying the method of Lampi, Dimberg, and Kamal-Eldin (1999) with the following modifications. A double-jacketed glass chromatography column (300 9 28 mm i.d.) was packaged with a slurry of 150 g neutral aluminium oxide (activated at 100 °C for 16 h and 220 °C for 8 h) and conditioned with 250 ml of n-hexane. The mixture, containing AC and BO, obtained from an adsorption experiment, was suspended in an equal volume of n-hexane and loaded onto the column. The column, which was maintained at 37 °C by circulating water from a water bath, was eluted with 150 ml of n-hexane. In order to prevent light-induced oxidation of the purified BO-TAGs, the column and the collecting flask were wrapped with aluminium foil. The solvent was removed with a rotary evaporator at 37 °C under vacuum. The BO-TAGs were then dried under nitrogen and stored at 18 °C for further experiments. It was determined that the BO-TAGs contained no tocopherols or b-carotene. 2.2.2. Oxidation At the first stage of the oxidation experiments, 5.0 g aliquots of liquid BO, BO-TAGs (control), and BO-TAGs containing antioxidants alone (different concentrations of a-tocopherol, b-carotene and AP) were placed in 2.4 cm (i.d.) vials (15 ml) and oxidised in an oven set at 60 °C ± 1. To prepare BO-TAGs spiked with antioxidants, aliquots of solutions of a-tocopherol in acetone, b-carotene in chloro-
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form and AP in acetone, at known concentrations, were added to the BO-TAGs. The solvents were evaporated under a stream of nitrogen. Concentrations of a-tocopherol (10, 25 and 50 lg/g), bcarotene (5, 10, 25 and 50 lg/g) and AP (5, 50, 100 and 200 lg/g) were selected, based on the results of numerous preliminary studies in which concentrations of the antioxidants were evaluated. Two vials, one for each sample, were withdrawn at definite time intervals (2, 4, 6, 8, 16 and 32 days for BO and BO-TAG + a-tocopherol mixtures; 2, 4, 6 and 8 days for BO-TAG, BO-TAG + b-carotene and BO-TAG + AP mixtures) to assess the oxidation status. Flow charts, showing the experimental design, including oxidation times and antioxidant dosages, are summarised in Fig. 1. After determination of effective dosages that provide the lowest PV and AV, binary or ternary mixtures of antioxidants, at effective dosages with BO-TAGs, were subjected to oxidation under the same conditions. 2.2.3. Measurements of oxidation status Primary oxidation products (hydroperoxides) were determined by PV measurements. Approximately 1 ± 0.1 g of oil was weighed and subjected to iodometric determination of PV according to Official Method Cd 8-53 (AOCS, 1997). Formation of secondary oxidation products was measured by AV according to Official Method Cd 18-90 (AOCS, 1997). Duplicate measurements were conducted for each vial. 2.2.4. Statistics SPSS version 9.0 was used to perform statistical calculations. Significant differences in the means of PVs and AVs between BO and BO-TAG, with and without antioxidants, were determined by using a least significant difference test and analysis of variance procedure (p < 0.05). 3. Results and discussion BO-TAGs, free of antioxidants, including tocopherols and b-carotene, partial glycerides and free fatty acids, were obtained via sequential treatments, using AC adsorption and alumina column chromatography. After this treatment, no detectable levels of PV and AV were found in BO-TAGs. When the fatty acid composition of the BO-TAG is taken into account, unsaturated fatty acids, including oleic, linoleic and linolenic acids, were destroyable molecules in the BO-TAGs. Thus, BO-TAGs began to oxidise at the beginning of oxidation. Compared to the PV of the BO, significantly higher values were observed (p < 0.05) for BO-TAGs throughout the oxidation experiment, as shown in Fig. 2. Similarly, Lampi and Piironen (1998) have reported that PV of the BO-TAGs reached 32 meq/kg at the 16th day of oxidation, monitored at 40 °C. As shown in Fig. 2, the increasing trend observed for AV results is very similar to that obtained in PV experiments. Fast oxidation of BOTAGs has to be a consequence of the instability of the hydroperoxides. As hydroperoxides decomposed, new reactive species were formed which catalysed oxidation (Lampi & Piironen, 1999). Both PV and AV indicated that the BO had a resistance to oxidation due to its own (naturally occurring) antioxidants. While the induction period of BO was between 16 and 32 days, there was no apparent induction period for BO-TAGs, as shown in Fig. 2. Fig. 3 shows the effects of a-tocopherol, b-carotene and AP, individually, on oxidation, monitored by PV and AV under the same conditions as with BO and BO-TAGs, with the exception of oxidation times. There was a significant difference (p < 0.05) between PV and AV of three antioxidants when they were added to BO-TAGs at the same levels. Compared to b-carotene and AP, a higher oxidation time is needed to reach an induction period for a-tocopherol. As shown in Fig. 3A, among the antioxidants tested, a-tocopherol
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Fig. 1. Schematic presentation of experimental design, including purification of BO-TAG, oxidation times and antioxidant dosages.
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was found to be the effective antioxidant in BO-TAGs at levels of, 25 and 50 and 50 lg/g for 8, 16, and 32 days, respectively. Due to the higher antioxidative potential of a-tocopherol, oxidation time was lengthened to 32 days (same as that of BO). Oxidation of the 10 lg/g a-tocopherol-containing sample was drastically accelerated compared with the 25 and 50 lg/g samples after the 4th day. There were no significant differences (p > 0.05) in oxidation level between the 25 and 50 lg/g a-tocopherol-containing samples up to the 16th day. However, at the end of the oxidation experiment (32nd day), the 50 lg/g concentration gave the best scores for both of PV and AV measurements. These findings are in accordance with previous reports (Heinonen, Haila, Lampi, & Piironen, 1997; Lampi & Piironen, 1998; Lampi et al., 1997). Conflicting results have been reported for the effect of a-tocopherol on lipid oxidation. The relative antioxidant activity of tocopherols depends on temperature, lipid composition, physical state (bulk phase or emulsion), and concentration (Huang, Frankel, & German, 1994). It has
been reported that increased levels of a-tocopherol can result in increased levels of a-tocopheroxyl radicals, which can initiate processes of lipid peroxidation by themselves (Rietjens et al., 2002). We did not find any prooxidant effect of a-tocopherol in the range of evaluated concentrations. However, b-carotene did not inhibit oxidation of BO-TAGs and promoted formation of lipid hydroperoxides (Fig. 3B) and secondary oxidation products (Fig. 3E) during oxidation. PV and AV increased with the increasing oxidation period, at all of concentrations tested. A strong relationship has been reported between the antioxidative effect of b-carotene as a chain-breaking agent and oxygen tensions in the lipid environment. At higher oxygen tensions, b-carotene loses its activity and plays a role as prooxidant (Burton & Ingold, 1984). This conclusion was repeated in our study for the b-carotene concentrations tested. Compared to the higher concentrations, 5 and 10 lg/g of b-carotene addition caused lower PV and AVs. It was reported that a higher concentration of b-carotene promotes formation of epoxy
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Fig. 3. Changes in the PV and AV of BO-TAGs during oxidation at 60 °C with the addition of a-tocopherol, b-carotene and AP at different concentrations.
derivatives, which is accompanied by release of an alkoxyl radical that may carry on the chain oxidation (Kennedy & Liebler, 1992). In addition, more b-carotene peroxyl radicals are formed at higher concentrations of b-carotene. Both of these compounds trigger the oxidation chain reactions, which may explain the increase of PV and AV at higher b-carotene concentrations. AP was added to BO-TAGs at concentrations of 5, 50, 200 and 200 lg/g. There were no significant differences (p > 0.05) between PV and AV with these concentrations at the end of the 2nd and 4th days of oxidation, while the values for the 6th and 8th days were significantly different (p < 0.05). Similar to the findings obtained with b-carotene addition, a dose dependent prooxidant effect was observed for AP additions. Primary (Fig. 3C) and secondary (Fig. 3F) oxidation products formed just after the beginning of the oxidation experiments. By contrast, it has been reported that the addition of AP to oils containing arachidonic, docosapentaenoic and docosahexaenoic acids, at higher concentrations (1200 lg/g), lengthened the induction period, as also did a-tocopherol at 800 lg/g concentration (Bartee, Kim, & Min, 2007). In the second stage of the study, synergism between the antioxidants was assessed by using binary or ternary combinations of antioxidants at the most effective dosages The most effective dosages were determined to be 50, 5 and 50 lg/g for a-tocopherol, b-
carotene and AP, respectively, when they were used individually in oxidation experiments. To provide a comparable assessment for the PVs and AVs of BO and BO-TAG + antioxidants, the same oxidation time was chosen (32 days). The effects of antioxidant combinations on the PV and AV of BOTAGs are presented in Figs. 4 and 5, respectively. PV and AV of the binary mixtures of antioxidants were found to be significantly different (p < 0.05). A strong synergistic effect was detected for the binary mixtures of a-tocopherol and AP, while the b-carotene and AP mixture had no effect on the oxidation of the BO-TAGs. It is well established that tocopherol and ascorbic acid/ascorbyl palmitate exert a synergistic antioxidant effect in a number of systems and this interaction is based on radical exchange reactions between the two antioxidants (Beddows et al., 2001; Meyer, 1996). Several antioxidant mechanisms have been proposed for the action of ascorbic acid/ascorbyl palmitate in oils. These include singlet oxygen quenching, metal chelating, and free-radical-scavenging potential resulting from regeneration of oxidised tocopherols (Beddows et al., 2001; Let, Jacobsen, & Meyer, 2007). During the synergistic interaction of a-tocopherol-AP, tocopherols are spared at the expense of AP during oxidation. In other words, AP is used to regenerate tocopherols. AP donates hydrogen to the tocopheroxyl radical formed by a-tocopherol, donating hydrogen to the lipid
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Peroxide value (meq/kg)
626
Peroxide value (meq/kg)
230 180
for BO-TAGs with the ternary blends of antioxidants. Consequently, it appears that, to improve the oxidative state of BO, this combination may be used in butter manufacture. In conclusion, this study will contribute to better understanding of the antioxidant mechanisms of a-tocopherol, b-carotene and AP during oxidation of BO and BO-TAGs. Observations of the study can be summarised as follows:
8 6 4 2 0
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Days Fig. 4. Changes in the PV of BO-TAGs with binary and ternary mixtures of atocopherol (50 lg/g), b-carotene (5 lg/g) and AP (50 lg/g) during oxidation at 60 °C. (}) [a-Tocopherol + b-carotene]; (h) [a-tocopherol + AP]; (D) [b-carotene + AP]; () [a-tocopherol + b-carotene + AP]; (s) BO.
1. a-Tocopherol, individually, effectively protects BO-TAGs better than do AP and b-carotene. 2. A prooxidant effect is observed when the AP and/or b-carotene are added to BO-TAGs, individually at certain concentrations. 3. b-Carotene behaves as an antioxidant, for inhibiting lipid peroxidation, when it is used along with a-tocopherol. 4. The highest antioxidant protection is provided by ternary blends of a-tocopherol (50 lg/g), b-carotene (5 lg/g) and AP (50 lg/g) during oxidation of BO-TAGs. Acknowledgement
70 4 4 p-Anisidine value
p-Anisidine value
60 50 40 30
This study was supported by Inonu University, Directorate for Scientific Research (Project No. 2007/26).
3 3 2 2 1 1 0
References 0
8
16 Days
24
32
20 10 0 -10 0
8
16
24
32
Days Fig. 5. Changes in the AV of BO-TAG with binary and ternary mixtures of atocopherol (50 lg/g), b-carotene (5 lg/g) and AP (50 lg/g) during oxidation at 60 °C. (}) [a-Tocopherol + b-carotene]; (h) [a-tocopherol + AP]; (D) [b-carotene + AP]; () [a-tocopherol + b-carotene + AP]; (s) BO.
radical (Beddows et al., 2001). So, among the binary mixtures tested, a-tocopherol and AP cooperatively provided the best protection, as shown in Figs. 4 and 5. Compared to the additions of a-tocopherol and b-carotene, individually, to BO-TAGs, their combination synergistically decreased the level of oxidation products better. This result is in accordance with reports of the antioxidative behaviour of b-carotene. In the presence of sufficient concentrations of other antioxidants, carotenoids may behave as antioxidants, whereas they present a prooxidant character in the absence of other antioxidants (Palozza, 1998). Carotenoids may exert a radical-trapping function by acting cooperatively with tocopherols. A synergism between bcarotene and a-tocopherol has been reported for inhibiting lipid peroxidation in biological membranes. This effect has been attributed to the rapid consumption of a-tocopherol compared to b-carotene. Via that mechanism, a-tocopherol is supposed to protect bcarotene from oxidation (Palozza & Krinsky, 1992). Compared to the effects of a-tocopherol, b-carotene and AP addition, individually, or in combination, on the oxidative state of BO-TAGs, higher scores for antioxidant protection were obtained with ternary mixtures of them. Meanwhile, the PV and AV of BOTAGs + ternary blends of antioxidants were considerably lower than that of BO (p < 0.05). PV and AV of BO reached to 136 meq/ kg and 32.0, at the 32nd day oxidation, respectively. Whereas, these values were measured as 7.38 meq/kg and 3.33, respectively,
AOCS (1997). Official and Recommended Methods of the American Oil Chemists’ Society. Champaign, IL: American Oil Chemists’ Society. Bartee, S. D., Kim, H. J., & Min, D. B. (2007). Effects of antioxidants on the oxidative stability of oils containing arachidonic, docosapentaenoic and docosahexaenoic acids. Journal of the American Oil Chemists’ Society, 84, 363–368. Basar, C. A. (2006). Applicability of the various adsorption models of three dyes adsorption onto activated carbon prepared waste apricot. Journal of Hazardous Materials, 135, 232–241. Beddows, C. G., Jagait, C., & Kelly, M. J. (2001). Effect of ascorbyl palmitate on the preservation of a-tocopherol in sunflower oil, alone and with herbs and spices. Food Chemistry, 73, 255–261. Britton, G. (1995). Structure and properties of carotenoids in relation to function. FASEB Journal, 9, 1551–1558. Burton, G. W., & Ingold, K. U. (1984). b-Carotene: An unusual type of lipid antioxidant. Science, 244, 569–573. Choe, E., & Min, D. B. (2006). Mechanisms and factors for edible oil oxidation. Comprehensive Reviews in Food Science and Food Safety, 5, 169–186. Frankel, E. N. (1991). Recent advances in lipid oxidation. Journal of the Science of Food and Agriculture, 54, 495–511. Frankel, E. N. (1998). Antioxidants. In E. N. Frankel (Ed.), Lipid oxidation (2nd ed., pp. 129–166). Dundee, Scotland: The Oily Press Ltd. Fuster, M. D., Lampi, A.-M., Hopia, A., & Kamal-Eldin, A. (1998). Effects of a- and ctocopherols on the autooxidation of purified sunflower triacylglycerols. Lipids, 33, 715–722. Haila, K. M., Lievonen, S. M., & Heinonen, M. (1996). Effects of lutein, lycopene, annatto, and c-tocopherol on autoxidation of triglycerides. Journal of Agricultural and Food Chemistry, 44, 2096–2100. Heinonen, M., Haila, K., Lampi, A.-M., & Piironen, V. (1997). Inhibition of oxidation in 10% oil-in-water emulsions by b-carotene with a- and c-tocopherols. Journal of the American Oil Chemists’ Society, 74, 1047–1052. Huang, S. W., Frankel, E. N., & German, J. B. (1994). Antioxidant activity of a- and ctocopherols in bulk oils and in oil-in-water emulsions. Journal of Agricultural and Food Chemistry, 42, 2108–2114. Isnardy, B., Wagner, K.-H., & Elmafda, I. (2003). Effects of a-, c-, and d-tocopherols on the autoxidation of purified rapeseed oil triacylglycerols in a system containing low oxygen. Journal of Agricultural and Food Chemistry, 51, 7775–7780. Karabulut, I., Topcu, A., Akmil-Basar, C., Onal, Y., & Lampi, A.-M. (2008). Obtaining butter oil triacylglycerols free from b-carotene and a-tocopherol via activated carbon adsorption and alumina-column chromatography treatments. Journal of the American Oil Chemists’ Society, 85, 213–219. Karabulut, I., Topcu, A., Yorulmaz, A., Tekin, A., & Ozay, D. S. (2005). Effects of the industrial refining process on some properties of hazelnut oil. European Journal of Lipid Science and Technology, 107, 476–480. Kennedy, T. A., & Liebler, D. (1992). Peroxyl radical scavenging by b-carotene in lipid bilayers. Journal of Biological Chemistry, 267, 4658–4663. Lampi, A.-M., Dimberg, L. H., & Kamal-Eldin, A. (1999). A study on the influence of fucosterol on thermal polymerization of purified high oleic sunflower triacylglycerols. Journal of the Science of Food and Agriculture, 79, 573–579. Lampi, A.-M., & Piironen, V. (1998). a- and c-Tocopherols as efficient antioxidants in butter oil triacylglycerols. Fett/Lipid, 100, 292–295.
I. Karabulut / Food Chemistry 123 (2010) 622–627 Lampi, A.-M., & Piironen, V. (1999). Dissimilarity of the oxidations of rapeseed and butter oil triacylglycerols and their mixtures in the absence of tocopherols. Journal of Agricultural and Food Chemistry, 79, 300–306. Lampi, A.-M., Piironen, V., Hopia, A., & Koivistoinen, P. (1997). Characterization of the oxidation of rapeseed and butter oil triacylglycerols by four analytical methods. LWT – Food Science and Technology, 30, 807–813. Let, M. B., Jacobsen, C., & Meyer, A. S. (2007). Ascorbyl palmitate, c-tocopherol, and EDTA affect lipid oxidation in fish oil enriched salad dressing differently. Journal of Agricultural and Food Chemistry, 55, 2369–2375. Marinova, E. M., & Yanishlieva, N. V. (1992). Inhibited oxidation of lipids III: On the activity of ascorbyl palmitate during the autoxidation of two types of lipid systems in the presence of a-tocopherol. Fett Wissenschaft Technologie – Fat Science Technology, 12, 448–452. Meyer, A. S. (1996). Fate of the synergistic antioxidant system ascorbic acid, lecithin, and tocopherol in mayonnaise: Partition of ascorbic acid. Journal of Food Lipids, 3, 139–147.
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Palozza, P. (1998). Pro-oxidant actions of carotenoids in biological systems. Nutrition Reviews, 56, 257–265. Palozza, P., & Krinsky, N. I. (1992). b-Carotene and a-tocopherol are synergistic antioxidants. Archives of Biochemistry and Biophysics, 297, 184–187. Perricone, N., Nagy, K., Horvath, F., Dajko, G., Uray, I., & Zs-Nagy, I. (1999). The hydroxyl free radical reactions of ascorbyl palmitate as measured in various in vitro models. Biochemical and Biophysical Research Communications, 262, 661–665. Rietjens, I. M. C. M., Boersma, M. G., de Haan, L., Spenkelink, B., Awad, H. M., Cnubben, N. H. P., et al. (2002). The pro-oxidant chemistry of the natural antioxidants vitamin C, vitamin E, carotenoids and flavonoids. Environmental Toxicology Pharmacology, 11, 321–333. St. Angelo, A. J. (1996). Lipid oxidation in foods. Critical Reviews in Food Science and Nutrition, 36, 175–224. Yanishlieva, N. V., Aitzemüller, K., & Raneva, V. G. (1998). b-Carotene and lipid oxidation. Fett/Lipid, 100, 444–462.
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Effects of a-tocopherol, b-carotene and ascorbyl palmitate on oxidative stability of butter oil triacylglycerols Ihsan Karabulut * Department of Food Engineering, Inonu University, 44280 Malatya, Turkey
a r t i c l e
i n f o
Article history: Received 24 August 2009 Received in revised form 22 March 2010 Accepted 29 April 2010
Keywords: Butter Triacylglycerol a-Tocopherol b-Carotene Ascorbyl palmitate Oxidation Peroxide value p-Anisidine value
a b s t r a c t Butter oil triacylglycerols (BO-TAGs), free of antioxidants, including b-carotene, were obtained via sequential treatments with activated carbon (AC) adsorption and alumina column chromatography. a-Tocopherol, b-carotene and ascorbyl palmitate (AP) were added to BO-TAGs, individually, or in different combinations. An accelerated oven-oxidation test was carried out at 60 °C to determine the most effective dosages of the antioxidants. Among the antioxidants evaluated, a-tocopherol was found to be the most effective, at the concentration of 50 lg/g. To determine the possible synergism between the antioxidants, binary or ternary combinations of a-tocopherol, b-carotene and AP were added to BO-TAGs at concentrations of 50, 5, and 50 lg/g, respectively. Ternary combinations of these antioxidants were significantly better in retarding oxidation than were binary blends of a-tocopherol with b-carotene or AP. However, a prooxidant effect was observed, especially when b-carotene and AP were used individually or in binary combination. Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction Fats and oils are important functional components of foods. They contribute to flavour, odour, colour and texture. However, lipid oxidation occurs during storage or processing and leads to depletion in the quality of food products, resulting in shortening of shelf life, reducing their nutritional quality (Frankel, 1991; St. Angelo, 1996). When lipids are exposed to environmental factors, such as air, light and temperature, autoxidation reactions start to produce undesirable flavours, rancid odours, discoloration and other forms of deterioration. The primary autoxidation products are hydroperoxides, which have no taste or flavour, while their degradation products, called secondary oxidation products, do have detectable tastes and flavours (Choe & Min, 2006). a-Tocopherol is a primary antioxidant, functioning by terminating free-radical chain reactions, by donating hydrogen or electrons to free radicals and converting them to more stable products (Frankel, 1998). b-Carotene is an oil-soluble and natural pigment of many oils, such as palm, olive and butter oils. In addition to its use as a food colouring agent, b-carotene also has a strong antioxidant activity. It has been shown to protect lipids from autoxidation by reacting with peroxyl radicals, thereby inhibiting propagation and promoting termination of the oxidation chain * Tel.: +90 4223410010; fax: +90 4223410046. E-mail address: [email protected] 0308-8146/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2010.04.080
reaction (Britton, 1995). b-Carotene retards potentially harmful oxidative reactions by trapping free radicals and quenching singlet oxygen (Yanishlieva, Aitzemüller, & Raneva, 1998). Ascorbic acid is a secondary antioxidant that can be broadly classified as an oxygen scavenger/singlet oxygen quencher; it reacts with free oxygen and removes it in a closed system (Perricone et al., 1999). Ascorbyl palmitate (AP) is a synthetically-derived fat/oil-soluble ester of ascorbic acid. It was reported that AP is not only a synergist, with a-tocopherol, but also a radical-type inhibitor of lipid autoxidation (Beddows, Jagait, & Kelly, 2001; Marinova & Yanishlieva, 1992). Triacylglycerols (TAGs) of edible oils are usually used to evaluate the anti- and/or prooxidant activities of synthetic and natural antioxidants. In order to determine the effects of antioxidants in oils, TAGs free of antioxidants have been produced and used in some model oxidation studies (Fuster, Lampi, Hopia, & Kamal-Eldin, 1998; Haila, Lievonen, & Heinonen, 1996; Isnardy, Wagner, & Elmafda, 2003; Lampi & Piironen, 1998; Lampi, Piironen, Hopia, & Koivistoinen, 1997). Components of mixed antioxidant systems can contribute to the inhibition of oxidation, with the resulting antioxidant activity reflecting either additive or synergistic effects of the components. Due to uncompleted application of stripping, cooperative actions of b-carotene, naturally present in the oil with added antioxidants, have been neglected in reported studies (Lampi & Piironen, 1998; Lampi et al., 1997). Therefore, the aim of this study was to compare the effectiveness of a-tocopherol, b-carotene and AP, individually,
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or in combination, on oxidative state of butter oil triacylglycerols (BO-TAGs) under accelerated oxidation conditions. Primary oxidation products were measured by peroxide value (PV) and secondary oxidation products were measured by p-anisidine value (AV). 2. Materials and methods 2.1. Reagents Butter was purchased from local markets. Organic solvents and neutral aluminium oxide were purchased from Merck (Darmstadt, Germany). b-Carotene (Type I, 95%), p-anisidine and AP were purchased from Sigma–Aldrich (Steinheim, Germany). Activated carbon (AC) was prepared from waste apricots in our laboratory, as described in a previous report (Basar, 2006). 2.2. Methods 2.2.1. Preparation of BO-TAGs BO was produced by melting of butter at 55 °C, and filtering through a plain filter paper coated with anhydrous sodium sulphate at the same temperature. BO was kept at 18 °C, until used in subsequent procedures. Fatty acid composition (mol%) of BO was determined by a GC technique as follows: C4: 1.8, C6: 2.2, C8: 1.2, C10: 2.9, C12: 3.6, C13: 0.1, C14: 12.1, C14:1: 1.4, C15: 1.4, C15:1: 0.5, C16: 35.4, C16:1: 2.2, C17: 0.6, C17:1: 0.4, C18: 9.0, C18:1: 24.9, C18:2: 1.9, C18:3: 0.3, and C20:1: 0.2. Tocopherol and b-carotene contents of BO were determined by normal phase HPLC with a Phenomenex Luna Silica column (4.6 mm i.d. 250 mm, 5 lm). Separation was based on isocratic elution with n-hexane (99%) and iso-propanol (1%) at 292 nm (Karabulut, Topcu, Yorulmaz, Tekin, & Ozay, 2005). Tocopherol and b-carotene contents were determined as follows: a-tocopherol, 22.1 lg/g; b-tocopherol, 0.5 lg/g; d-tocopherol, 2.6 lg/g; c-tocopherol, 1.4 lg/g; b-carotene, 3.7 lg/g. To obtain pure BO-TAGs, b-carotene and tocopherols were removed from BO by the same procedure as described in a previous report (Karabulut, Topcu, Akmil-Basar, Onal, & Lampi, 2008). To remove b-carotene, BO (60 g) was mixed with AC (6 g) in a flask (100 ml) connected to a rotary evaporator at 60 °C for 300 min. Thereafter, BO was stripped to remove pro- and antioxidants to obtain purified BO-TAGs, applying the method of Lampi, Dimberg, and Kamal-Eldin (1999) with the following modifications. A double-jacketed glass chromatography column (300 9 28 mm i.d.) was packaged with a slurry of 150 g neutral aluminium oxide (activated at 100 °C for 16 h and 220 °C for 8 h) and conditioned with 250 ml of n-hexane. The mixture, containing AC and BO, obtained from an adsorption experiment, was suspended in an equal volume of n-hexane and loaded onto the column. The column, which was maintained at 37 °C by circulating water from a water bath, was eluted with 150 ml of n-hexane. In order to prevent light-induced oxidation of the purified BO-TAGs, the column and the collecting flask were wrapped with aluminium foil. The solvent was removed with a rotary evaporator at 37 °C under vacuum. The BO-TAGs were then dried under nitrogen and stored at 18 °C for further experiments. It was determined that the BO-TAGs contained no tocopherols or b-carotene. 2.2.2. Oxidation At the first stage of the oxidation experiments, 5.0 g aliquots of liquid BO, BO-TAGs (control), and BO-TAGs containing antioxidants alone (different concentrations of a-tocopherol, b-carotene and AP) were placed in 2.4 cm (i.d.) vials (15 ml) and oxidised in an oven set at 60 °C ± 1. To prepare BO-TAGs spiked with antioxidants, aliquots of solutions of a-tocopherol in acetone, b-carotene in chloro-
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form and AP in acetone, at known concentrations, were added to the BO-TAGs. The solvents were evaporated under a stream of nitrogen. Concentrations of a-tocopherol (10, 25 and 50 lg/g), bcarotene (5, 10, 25 and 50 lg/g) and AP (5, 50, 100 and 200 lg/g) were selected, based on the results of numerous preliminary studies in which concentrations of the antioxidants were evaluated. Two vials, one for each sample, were withdrawn at definite time intervals (2, 4, 6, 8, 16 and 32 days for BO and BO-TAG + a-tocopherol mixtures; 2, 4, 6 and 8 days for BO-TAG, BO-TAG + b-carotene and BO-TAG + AP mixtures) to assess the oxidation status. Flow charts, showing the experimental design, including oxidation times and antioxidant dosages, are summarised in Fig. 1. After determination of effective dosages that provide the lowest PV and AV, binary or ternary mixtures of antioxidants, at effective dosages with BO-TAGs, were subjected to oxidation under the same conditions. 2.2.3. Measurements of oxidation status Primary oxidation products (hydroperoxides) were determined by PV measurements. Approximately 1 ± 0.1 g of oil was weighed and subjected to iodometric determination of PV according to Official Method Cd 8-53 (AOCS, 1997). Formation of secondary oxidation products was measured by AV according to Official Method Cd 18-90 (AOCS, 1997). Duplicate measurements were conducted for each vial. 2.2.4. Statistics SPSS version 9.0 was used to perform statistical calculations. Significant differences in the means of PVs and AVs between BO and BO-TAG, with and without antioxidants, were determined by using a least significant difference test and analysis of variance procedure (p < 0.05). 3. Results and discussion BO-TAGs, free of antioxidants, including tocopherols and b-carotene, partial glycerides and free fatty acids, were obtained via sequential treatments, using AC adsorption and alumina column chromatography. After this treatment, no detectable levels of PV and AV were found in BO-TAGs. When the fatty acid composition of the BO-TAG is taken into account, unsaturated fatty acids, including oleic, linoleic and linolenic acids, were destroyable molecules in the BO-TAGs. Thus, BO-TAGs began to oxidise at the beginning of oxidation. Compared to the PV of the BO, significantly higher values were observed (p < 0.05) for BO-TAGs throughout the oxidation experiment, as shown in Fig. 2. Similarly, Lampi and Piironen (1998) have reported that PV of the BO-TAGs reached 32 meq/kg at the 16th day of oxidation, monitored at 40 °C. As shown in Fig. 2, the increasing trend observed for AV results is very similar to that obtained in PV experiments. Fast oxidation of BOTAGs has to be a consequence of the instability of the hydroperoxides. As hydroperoxides decomposed, new reactive species were formed which catalysed oxidation (Lampi & Piironen, 1999). Both PV and AV indicated that the BO had a resistance to oxidation due to its own (naturally occurring) antioxidants. While the induction period of BO was between 16 and 32 days, there was no apparent induction period for BO-TAGs, as shown in Fig. 2. Fig. 3 shows the effects of a-tocopherol, b-carotene and AP, individually, on oxidation, monitored by PV and AV under the same conditions as with BO and BO-TAGs, with the exception of oxidation times. There was a significant difference (p < 0.05) between PV and AV of three antioxidants when they were added to BO-TAGs at the same levels. Compared to b-carotene and AP, a higher oxidation time is needed to reach an induction period for a-tocopherol. As shown in Fig. 3A, among the antioxidants tested, a-tocopherol
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was found to be the effective antioxidant in BO-TAGs at levels of, 25 and 50 and 50 lg/g for 8, 16, and 32 days, respectively. Due to the higher antioxidative potential of a-tocopherol, oxidation time was lengthened to 32 days (same as that of BO). Oxidation of the 10 lg/g a-tocopherol-containing sample was drastically accelerated compared with the 25 and 50 lg/g samples after the 4th day. There were no significant differences (p > 0.05) in oxidation level between the 25 and 50 lg/g a-tocopherol-containing samples up to the 16th day. However, at the end of the oxidation experiment (32nd day), the 50 lg/g concentration gave the best scores for both of PV and AV measurements. These findings are in accordance with previous reports (Heinonen, Haila, Lampi, & Piironen, 1997; Lampi & Piironen, 1998; Lampi et al., 1997). Conflicting results have been reported for the effect of a-tocopherol on lipid oxidation. The relative antioxidant activity of tocopherols depends on temperature, lipid composition, physical state (bulk phase or emulsion), and concentration (Huang, Frankel, & German, 1994). It has
been reported that increased levels of a-tocopherol can result in increased levels of a-tocopheroxyl radicals, which can initiate processes of lipid peroxidation by themselves (Rietjens et al., 2002). We did not find any prooxidant effect of a-tocopherol in the range of evaluated concentrations. However, b-carotene did not inhibit oxidation of BO-TAGs and promoted formation of lipid hydroperoxides (Fig. 3B) and secondary oxidation products (Fig. 3E) during oxidation. PV and AV increased with the increasing oxidation period, at all of concentrations tested. A strong relationship has been reported between the antioxidative effect of b-carotene as a chain-breaking agent and oxygen tensions in the lipid environment. At higher oxygen tensions, b-carotene loses its activity and plays a role as prooxidant (Burton & Ingold, 1984). This conclusion was repeated in our study for the b-carotene concentrations tested. Compared to the higher concentrations, 5 and 10 lg/g of b-carotene addition caused lower PV and AVs. It was reported that a higher concentration of b-carotene promotes formation of epoxy
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Fig. 3. Changes in the PV and AV of BO-TAGs during oxidation at 60 °C with the addition of a-tocopherol, b-carotene and AP at different concentrations.
derivatives, which is accompanied by release of an alkoxyl radical that may carry on the chain oxidation (Kennedy & Liebler, 1992). In addition, more b-carotene peroxyl radicals are formed at higher concentrations of b-carotene. Both of these compounds trigger the oxidation chain reactions, which may explain the increase of PV and AV at higher b-carotene concentrations. AP was added to BO-TAGs at concentrations of 5, 50, 200 and 200 lg/g. There were no significant differences (p > 0.05) between PV and AV with these concentrations at the end of the 2nd and 4th days of oxidation, while the values for the 6th and 8th days were significantly different (p < 0.05). Similar to the findings obtained with b-carotene addition, a dose dependent prooxidant effect was observed for AP additions. Primary (Fig. 3C) and secondary (Fig. 3F) oxidation products formed just after the beginning of the oxidation experiments. By contrast, it has been reported that the addition of AP to oils containing arachidonic, docosapentaenoic and docosahexaenoic acids, at higher concentrations (1200 lg/g), lengthened the induction period, as also did a-tocopherol at 800 lg/g concentration (Bartee, Kim, & Min, 2007). In the second stage of the study, synergism between the antioxidants was assessed by using binary or ternary combinations of antioxidants at the most effective dosages The most effective dosages were determined to be 50, 5 and 50 lg/g for a-tocopherol, b-
carotene and AP, respectively, when they were used individually in oxidation experiments. To provide a comparable assessment for the PVs and AVs of BO and BO-TAG + antioxidants, the same oxidation time was chosen (32 days). The effects of antioxidant combinations on the PV and AV of BOTAGs are presented in Figs. 4 and 5, respectively. PV and AV of the binary mixtures of antioxidants were found to be significantly different (p < 0.05). A strong synergistic effect was detected for the binary mixtures of a-tocopherol and AP, while the b-carotene and AP mixture had no effect on the oxidation of the BO-TAGs. It is well established that tocopherol and ascorbic acid/ascorbyl palmitate exert a synergistic antioxidant effect in a number of systems and this interaction is based on radical exchange reactions between the two antioxidants (Beddows et al., 2001; Meyer, 1996). Several antioxidant mechanisms have been proposed for the action of ascorbic acid/ascorbyl palmitate in oils. These include singlet oxygen quenching, metal chelating, and free-radical-scavenging potential resulting from regeneration of oxidised tocopherols (Beddows et al., 2001; Let, Jacobsen, & Meyer, 2007). During the synergistic interaction of a-tocopherol-AP, tocopherols are spared at the expense of AP during oxidation. In other words, AP is used to regenerate tocopherols. AP donates hydrogen to the tocopheroxyl radical formed by a-tocopherol, donating hydrogen to the lipid
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Peroxide value (meq/kg)
626
Peroxide value (meq/kg)
230 180
for BO-TAGs with the ternary blends of antioxidants. Consequently, it appears that, to improve the oxidative state of BO, this combination may be used in butter manufacture. In conclusion, this study will contribute to better understanding of the antioxidant mechanisms of a-tocopherol, b-carotene and AP during oxidation of BO and BO-TAGs. Observations of the study can be summarised as follows:
8 6 4 2 0
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80 30 -20 0
8
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Days Fig. 4. Changes in the PV of BO-TAGs with binary and ternary mixtures of atocopherol (50 lg/g), b-carotene (5 lg/g) and AP (50 lg/g) during oxidation at 60 °C. (}) [a-Tocopherol + b-carotene]; (h) [a-tocopherol + AP]; (D) [b-carotene + AP]; () [a-tocopherol + b-carotene + AP]; (s) BO.
1. a-Tocopherol, individually, effectively protects BO-TAGs better than do AP and b-carotene. 2. A prooxidant effect is observed when the AP and/or b-carotene are added to BO-TAGs, individually at certain concentrations. 3. b-Carotene behaves as an antioxidant, for inhibiting lipid peroxidation, when it is used along with a-tocopherol. 4. The highest antioxidant protection is provided by ternary blends of a-tocopherol (50 lg/g), b-carotene (5 lg/g) and AP (50 lg/g) during oxidation of BO-TAGs. Acknowledgement
70 4 4 p-Anisidine value
p-Anisidine value
60 50 40 30
This study was supported by Inonu University, Directorate for Scientific Research (Project No. 2007/26).
3 3 2 2 1 1 0
References 0
8
16 Days
24
32
20 10 0 -10 0
8
16
24
32
Days Fig. 5. Changes in the AV of BO-TAG with binary and ternary mixtures of atocopherol (50 lg/g), b-carotene (5 lg/g) and AP (50 lg/g) during oxidation at 60 °C. (}) [a-Tocopherol + b-carotene]; (h) [a-tocopherol + AP]; (D) [b-carotene + AP]; () [a-tocopherol + b-carotene + AP]; (s) BO.
radical (Beddows et al., 2001). So, among the binary mixtures tested, a-tocopherol and AP cooperatively provided the best protection, as shown in Figs. 4 and 5. Compared to the additions of a-tocopherol and b-carotene, individually, to BO-TAGs, their combination synergistically decreased the level of oxidation products better. This result is in accordance with reports of the antioxidative behaviour of b-carotene. In the presence of sufficient concentrations of other antioxidants, carotenoids may behave as antioxidants, whereas they present a prooxidant character in the absence of other antioxidants (Palozza, 1998). Carotenoids may exert a radical-trapping function by acting cooperatively with tocopherols. A synergism between bcarotene and a-tocopherol has been reported for inhibiting lipid peroxidation in biological membranes. This effect has been attributed to the rapid consumption of a-tocopherol compared to b-carotene. Via that mechanism, a-tocopherol is supposed to protect bcarotene from oxidation (Palozza & Krinsky, 1992). Compared to the effects of a-tocopherol, b-carotene and AP addition, individually, or in combination, on the oxidative state of BO-TAGs, higher scores for antioxidant protection were obtained with ternary mixtures of them. Meanwhile, the PV and AV of BOTAGs + ternary blends of antioxidants were considerably lower than that of BO (p < 0.05). PV and AV of BO reached to 136 meq/ kg and 32.0, at the 32nd day oxidation, respectively. Whereas, these values were measured as 7.38 meq/kg and 3.33, respectively,
AOCS (1997). Official and Recommended Methods of the American Oil Chemists’ Society. Champaign, IL: American Oil Chemists’ Society. Bartee, S. D., Kim, H. J., & Min, D. B. (2007). Effects of antioxidants on the oxidative stability of oils containing arachidonic, docosapentaenoic and docosahexaenoic acids. Journal of the American Oil Chemists’ Society, 84, 363–368. Basar, C. A. (2006). Applicability of the various adsorption models of three dyes adsorption onto activated carbon prepared waste apricot. Journal of Hazardous Materials, 135, 232–241. Beddows, C. G., Jagait, C., & Kelly, M. J. (2001). Effect of ascorbyl palmitate on the preservation of a-tocopherol in sunflower oil, alone and with herbs and spices. Food Chemistry, 73, 255–261. Britton, G. (1995). Structure and properties of carotenoids in relation to function. FASEB Journal, 9, 1551–1558. Burton, G. W., & Ingold, K. U. (1984). b-Carotene: An unusual type of lipid antioxidant. Science, 244, 569–573. Choe, E., & Min, D. B. (2006). Mechanisms and factors for edible oil oxidation. Comprehensive Reviews in Food Science and Food Safety, 5, 169–186. Frankel, E. N. (1991). Recent advances in lipid oxidation. Journal of the Science of Food and Agriculture, 54, 495–511. Frankel, E. N. (1998). Antioxidants. In E. N. Frankel (Ed.), Lipid oxidation (2nd ed., pp. 129–166). Dundee, Scotland: The Oily Press Ltd. Fuster, M. D., Lampi, A.-M., Hopia, A., & Kamal-Eldin, A. (1998). Effects of a- and ctocopherols on the autooxidation of purified sunflower triacylglycerols. Lipids, 33, 715–722. Haila, K. M., Lievonen, S. M., & Heinonen, M. (1996). Effects of lutein, lycopene, annatto, and c-tocopherol on autoxidation of triglycerides. Journal of Agricultural and Food Chemistry, 44, 2096–2100. Heinonen, M., Haila, K., Lampi, A.-M., & Piironen, V. (1997). Inhibition of oxidation in 10% oil-in-water emulsions by b-carotene with a- and c-tocopherols. Journal of the American Oil Chemists’ Society, 74, 1047–1052. Huang, S. W., Frankel, E. N., & German, J. B. (1994). Antioxidant activity of a- and ctocopherols in bulk oils and in oil-in-water emulsions. Journal of Agricultural and Food Chemistry, 42, 2108–2114. Isnardy, B., Wagner, K.-H., & Elmafda, I. (2003). Effects of a-, c-, and d-tocopherols on the autoxidation of purified rapeseed oil triacylglycerols in a system containing low oxygen. Journal of Agricultural and Food Chemistry, 51, 7775–7780. Karabulut, I., Topcu, A., Akmil-Basar, C., Onal, Y., & Lampi, A.-M. (2008). Obtaining butter oil triacylglycerols free from b-carotene and a-tocopherol via activated carbon adsorption and alumina-column chromatography treatments. Journal of the American Oil Chemists’ Society, 85, 213–219. Karabulut, I., Topcu, A., Yorulmaz, A., Tekin, A., & Ozay, D. S. (2005). Effects of the industrial refining process on some properties of hazelnut oil. European Journal of Lipid Science and Technology, 107, 476–480. Kennedy, T. A., & Liebler, D. (1992). Peroxyl radical scavenging by b-carotene in lipid bilayers. Journal of Biological Chemistry, 267, 4658–4663. Lampi, A.-M., Dimberg, L. H., & Kamal-Eldin, A. (1999). A study on the influence of fucosterol on thermal polymerization of purified high oleic sunflower triacylglycerols. Journal of the Science of Food and Agriculture, 79, 573–579. Lampi, A.-M., & Piironen, V. (1998). a- and c-Tocopherols as efficient antioxidants in butter oil triacylglycerols. Fett/Lipid, 100, 292–295.
I. Karabulut / Food Chemistry 123 (2010) 622–627 Lampi, A.-M., & Piironen, V. (1999). Dissimilarity of the oxidations of rapeseed and butter oil triacylglycerols and their mixtures in the absence of tocopherols. Journal of Agricultural and Food Chemistry, 79, 300–306. Lampi, A.-M., Piironen, V., Hopia, A., & Koivistoinen, P. (1997). Characterization of the oxidation of rapeseed and butter oil triacylglycerols by four analytical methods. LWT – Food Science and Technology, 30, 807–813. Let, M. B., Jacobsen, C., & Meyer, A. S. (2007). Ascorbyl palmitate, c-tocopherol, and EDTA affect lipid oxidation in fish oil enriched salad dressing differently. Journal of Agricultural and Food Chemistry, 55, 2369–2375. Marinova, E. M., & Yanishlieva, N. V. (1992). Inhibited oxidation of lipids III: On the activity of ascorbyl palmitate during the autoxidation of two types of lipid systems in the presence of a-tocopherol. Fett Wissenschaft Technologie – Fat Science Technology, 12, 448–452. Meyer, A. S. (1996). Fate of the synergistic antioxidant system ascorbic acid, lecithin, and tocopherol in mayonnaise: Partition of ascorbic acid. Journal of Food Lipids, 3, 139–147.
627
Palozza, P. (1998). Pro-oxidant actions of carotenoids in biological systems. Nutrition Reviews, 56, 257–265. Palozza, P., & Krinsky, N. I. (1992). b-Carotene and a-tocopherol are synergistic antioxidants. Archives of Biochemistry and Biophysics, 297, 184–187. Perricone, N., Nagy, K., Horvath, F., Dajko, G., Uray, I., & Zs-Nagy, I. (1999). The hydroxyl free radical reactions of ascorbyl palmitate as measured in various in vitro models. Biochemical and Biophysical Research Communications, 262, 661–665. Rietjens, I. M. C. M., Boersma, M. G., de Haan, L., Spenkelink, B., Awad, H. M., Cnubben, N. H. P., et al. (2002). The pro-oxidant chemistry of the natural antioxidants vitamin C, vitamin E, carotenoids and flavonoids. Environmental Toxicology Pharmacology, 11, 321–333. St. Angelo, A. J. (1996). Lipid oxidation in foods. Critical Reviews in Food Science and Nutrition, 36, 175–224. Yanishlieva, N. V., Aitzemüller, K., & Raneva, V. G. (1998). b-Carotene and lipid oxidation. Fett/Lipid, 100, 444–462.