Effect of a- and d-tocopherol on the oxidative stability of a mixed hydrogenated fat under frying conditions

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1 Eur Food Res Technol (2005) 221: DOI /s ORIGINAL PAPER Malgorzata Nogala-Kalucka Jozef Korczak Ibrahim Elmadfa Karl-Heinz Wagner Effect of a- and d-tocopherol on the oxidative stability of a mixed hydrogenated fat under frying conditions Received: 18 October 2004 / Revised: 14 January 2005 / Published online: 11 March 2005 Springer-Verlag 2005 Abstract Information on the antioxidative potential of d- tocopherol is scanty, in particular for frying conditions. This study was aimed at assessing the antioxidative effects and degradation of a- and d-tocopherol between 0.01 and 0.1% on the oxidation of a commercial frying fat at 160 C. Oxidation experiments were performed by assessing every 15 or 30 min the peroxide value and conjugated dienes as primary oxidation products, p-anisidine reactive products and hexanal as secondary oxidation products, as well as the stability of tocopherols. The fatty acid composition was determined after 6 h. The fat samples enriched with a-tocopherol were considerably less resistant to oxidation compared to those with d-tocopherol. Both primary and secondary oxidation parameters increased their speed of formation with a-tocopherol but not with d-tocopherol. The latter observation is confirmed for the total amount of oxidation and also for the first stage, showing a lag phase only for d-tocopherol. These antioxidative effects can be due to the higher stability of d-tocopherol compared to a-tocopherol, which is oxidized faster into tocopheryl radicals, which can participate in side reactions that result in an acceleration of the oxidation speed. Neither a-tocopherol nor d-tocopherol influenced the fatty acid pattern. The investigation showed that during mild frying conditions the tocopherol homologues displayed various antioxidant activities. The M. Nogala-Kalucka Department of Biochemistry and Food Analysis, August Cieszkowski University of Agriculture, Poznan, Poland karl-heinz.wagner@univie.ac.at Tel.: Fax: J. Korczak Department of Human Nutrition and Food Technology, August Cieszkowski University of Agriculture, Poznan, Poland I. Elmadfa K.-H. Wagner Institute of Nutritional Sciences, University of Vienna, Althanstrasse 14, 1090 Vienna, Austria less effective homologue, a-tocopherol, underwent disintegration more quickly than d-tocopherol. Key words a-tocopherol d-tocopherol Stability Lipid oxidation Primary and secondary oxidation products Hydrogenated fat Introduction The most important characteristics for a good frying shortening are flavor stability, frying stability, and oxidative stability [1]. The resistance of frying shortenings against oxidation mainly depends on their fatty acid compositions but to a certain extent on the availability and, in particular, the concentrations of substances with antioxidant or pro-oxidant properties. Generally, fats used for deep frying are usually fats of plant origin obtained by selective partial hydrogenation with further modeling of their properties by mixing the hardened oils with those possessing high and medium contents of unsaturated fatty acids [2]. One of the widely applied frying fat components is red palm oil, which is less susceptible to lipid oxidation and its composition is characterized by an equal content of saturated fatty acids (SFAs) and unsaturated fatty acids and the presence of native tocochromanols [3]. When using mild frying conditions, at the first stage of oxidation during which hydroxyperoxides are intensively generated, the biologically active compounds, such as tocopherols or tocotrienols, undergo rapid oxidation [4, 5]. Although tocopherols in general are accepted to be very important food antioxidants, their effects depend on the form, their concentrations, substrates and the test conditions [6]. In the majority of the fats used for frying, such as high-oleic sunflower, or palm oils, a-tocopherol is the main native tocopherol form [7]. Although several publications reported investigations concerning tocopherols at a low oxidation temperature, thereby considering the different tocopherol forms too [8 10], reports concerning rapid degradation of tocochromanols under accelerated oxidation conditions, e.g., frying at a high

2 292 temperature, are scarce [11 13]. Long-lasting heating, as it is usual in restaurants and sometimes in households, leads to an almost complete degradation of these substances [5, 14]. At high frying temperatures tocopherols and tocotrienols present in fats undergo degradation, but the speed depends on the composition of triacylglycerols in the frying fat. Oxidative transformations taking place during frying adversely affect the nutritional as well as the sensorial and salubrious values of the fried product. Long-term consequences of interactions between functional food components and functions in a body and interactions between components must be carefully monitored [15, 16]. In this respect data on d-tocopherols are missing, in particular for high-temperature conditions. Therefore, in the present study the activity of d-tocopherol in comparison with the mainly studied a-tocopherol under frying conditions at 160 C was assessed. Our aim was to consider the relationship between the stability of tocopherols and the formation of primary and secondary fat oxidation products. Materials and methods Materials and chemicals The substrate of the frying fat was Planta, mainly composed of partially hydrogenated rapeseed oil with some palm oil added. The main characteristics of the fat used are as follows: saturated fatty acids (SFAs), 48.2%; monounsaturated fatty acids (MUFAs), 39.1%; polyunsaturated fatty acids (PUFAs), 12.7%; peroxide value (PV), Planta was purchased in 220-g bars at a local market. Tocopherol standards were obtained commercially (Merck). All other chemicals were ACS grade or higher. Methods Fat enrichment and preparation a-tocopherol and d-tocopherol dissolved in hexane were added to a round-bottomed flask and the solvent was evaporated at 35 C in the dark. Simultaneously, the fat was melted in a water bath at 40 C and an appropriate amount was added to the flasks with the evaporated tocopherols to obtain final concentrations of 0.01, 0.05 and 0.1%. To ensure a homogeneous dissemination of the tocopherols within the sample, the flask was rotated in the water bath for 3 min. The final concentrations were monitored by high-performance liquid chromatography (HPLC) [10]. Oxidation experiments The experiment was carried out using a Rancimat 679 Metrohm (Herisau, Switzerland) at C as the heating unit under the simulated frying conditions. During heating the samples (9-g starting sample size) for analysis were drawn successively (every 15 or 30 min) and frozen at Ÿ80 C until analysis. The procedure was optimized in pre-experiments in order to be uniform and reproducible. Determination of tocopherols Tocopherol content was analyzed in duplicate with reversed-phase HPLC. The system consisted of an L-7100 pump, an L-7400 UV detector, a D-7000 interface module and a mm LiChrospher RP-18 column, all from Merck. Methanol/dichloromethane (85:15, v/v) was used as the mobile phase; the flow rate was 0.8 ml/ min. The detection wavelength was set to 295 nm. Fat samples were briefly heated at 40 C, an appropriate amount was dissolved in hexane, diluted and evaporated until dryness under vacuum, and dissolved again in the mobile phase and injected [10]. Determination of primary and secondary oxidation products In order to determine oxidative changes the PV was determined by the AOCS Cd 8-53 acetic acid chloroform procedure [17]. Conjugated dienes (CD) were observed following the AOCS official method Ti 1a-64 [18], the p-anisidine value (AV) and hexanal, as the main volatile oxidation product, were determined following the methods described in AOCS Cd [19] and by Isnardy et al. [9], respectively. Fatty acid composition The fatty acid composition of the substrate was converted into the corresponding fatty acid methyl esters (FAMEs) and analyzed by gas chromatography using an Auto-System gas chromatograph, (PerkinElmer, Vienna, Austria) equipped with a split/splitless capillary injector. FAMEs were separated by a 30 m 0.25-mm inner diameter fused silica column (RT 2330) and detected with a flame ionization detector (FID); the FID temperature was set to 250 C. The fatty acid pattern was analyzed in duplicate [20]. Statistical analysis Oxidation experiments were performed twice; the samples were assessed in duplicate. Different oxidative responses observed for different concentrations were analyzed by one-way analysis of variance using SPSS 11.0 for Windows. Differences were considered significant at p>0.05. The quality criterion of the analytical methods was the coefficient of variation (CV): CV% a-tocopherol, 3.5; d-tocopherol, 3.9; PV, 2.1; CD, 2.5; AV, 3.1; hexanal, 5.2; fatty acid pattern, 6.4. Results and discussion The stability of fats, independent of whether they are enriched with antioxidants or not, can be examined with various instrumental tests [21 24]. The selection of the most appropriate methods to evaluate the oxidative stability of a fat is conditioned by its later consumption or application in the preparation of dishes (e.g., frying, microwaving). Oxidation stability is a very important discriminant of fat quality, and is particularly significant for fats used for deep-frying because of the temperature and duration of the process. Oxidation changes in fats constitute a serious health problem as their products are involved in the process of ageing and also in the etiopathology of many diseases [25, 26]. One effective method of delaying the processes of lipid oxidation is the addition of compounds with antioxidant properties. However, the application of synthetic antioxidants is currently being strongly discussed; therefore, native antioxidants including tocochromanols are of particular interest owing to the possibility of their extensive utilization.

3 293 Fig. 1 Residual a- and d-tocopherol contents (mg/100 g fat) of added % tocopherol levels during oxidation of Planta at 160 C The present study is a follow-up of the research activities conducted a few years ago which considered the effects of tocopherols added to coconut fat, olive and linseed oils using heating and frying conditions [4, 27] followed by testing tocopherol forms in rapeseed oil triacylglycerols [10] and its emulsion [9]. The fat used in the present investigation is frequently used in households for frying or pan-frying; it is a mixture of partially hydrogenated plant oils (rapeseed oil and palm oil) characterized by a very low initial level of antioxidants. Tocopherol stability The initial concentration of tocopherols and tocotrienols, expressed as the total vitamin E activity, was 3.22 mg/ 100 g fat, which was marginal. This kind of fat is particularly interesting in respect to its fatty acid pattern the p/s ratio is In our experiments, for the sake of comparison, the two homologues a- and d-tocopherol were tested. a-tocopherol was chosen because it is the most bioactive and mainly studied form and d-tocopherol because it has been sporadically studied, although very recent results indicate that d-tocopherol is a very efficient antioxidant in a bulk oil triglyceride and its emulsion when oxidized at low temperature [9, 10]. The stability of the tocopherols during oxidation is shown in Fig. 1. After 2 h of oxidation the residual a- tocopherol was approximately 9, 19 and 21% for the 0.01, 0.05 and 0.1% additions, respectively. In contrast, between 56 and 77% of d-tocopherol was available after 2 h. After 4 h, between 30 and 50% of the initial d-tocopherol concentration remained. The absolute amount used up during oxidation was considerably different. At the additional levels of 10, 50 and 100 mg/100 g the respective amounts of a-tocopherol consumption after 2 h were 9, 40 and 79 mg/100 g, while those of d-tocopherol were 2, 16 and 44 mg/100 g, respectively. The strongest degradation for a-tocopherol was found within the first 45 min (Fig. 1). These results are in accordance with those of previous studies [9, 10] at lower oxidation temperatures, which also showed a-tocopherol to be less stable.

4 294 Fig. 2 Effects of added a- and d-tocopherols at % on the hydroperoxide formation (peroxide value, PV) in Planta oxidized at 160 C Studies by Gordon and Kourimska [5] also revealed that during frying of potato chips in rapeseed oil at 162 C a-tocopherol decomposed first. Effect of tocopherols on primary oxidation products Similar to previous observations [4, 9, 27] at lower temperatures a-tocopherol was not able to extend shelf life. Formation of peroxides was induced when using higher concentrations (Fig. 2). The same tendency was found for CD formation, which sped up with increasing a-tocopherol levels, too. However, the absolute CD levels were not high, owing to the low amount of PUFA in the frying fat. In contrast, d-tocopherol was able to decrease the speed of lipid oxidation. Between the different concentrations no increasing effect was observed; all concentrations used ( %) prolonged the time until the threshold level of PV=10 was exceeded by 100%. Particularly during the first hour of the test, in comparison with the control sample, for which the PV amounted to 9, the addition of d-tocopherol resulted in values that were half as much as that of the control for all concentrations. In the second stage of oxidation the PV in the d-tocopherol enriched sample of fat increased, but almost till the end it was lower than in the sample without addition. The effects on CD were not as powerful, owing to the remarkable amount of saturated acids in the fat being tested. The results are in accordance with recent results which were achieved at room temperature. It is well known that in particular during deep-fat frying, a complex series of reactions takes place resulting in auto-oxidation, thermal oxidation, polymerization, hydrolysis, oxidation and cyclization of the oil [28, 29]. Lipid hydroperoxides, the primary products, quickly degrade to several secondary oxidation products; however, the temperature was set to 160 C, which is a moderate frying temperature and the degradation speed was low. Furthermore, the measurement intervals of 15 min for a- tocopherol and 30 min for d- tocopherol were frequent in order to consider the first

5 295 Fig. 3 Effects of added a- and d-tocopherols at % on the p-anisidin value (p-av) of Planta oxidized at 160 C stage of lipid oxidation. The fast degradation of a-tocopherol results in side reactions of the tocopheroxyl radical formed with hydroperoxides or unoxidized fatty acids to generate more radicals. But the initial tocopherol molecule can also react with hydroperoxides formed to form peroxyl radicals [30]. Both reactions increase the speed of lipid oxidation. The free radicals formed in these side reactions may act as catalysts to reinitiate new oxidation reduction sequences. Since d-tocopherol is stabler it does not participate in these side reactions as early as a-tocopherol. Effects of tocopherols on secondary oxidation products In order to follow the generation of secondary oxidation products, hexanal, as one major aldehyde, and the AV were monitored. The secondary oxidation products are of importance when determining dynamics of the oxidation. The level of hexanal of the control fat without tocopherol additions reached 17.2, 39.1 and 55.1 mg/100 g after 2, 4 and 6 h of oxidation, respectively. Similar to the results of the primary oxidation products, a-tocopherol affected the oxidation and sped it up with increasing concentrations when considering hexanal and the AV, too (Figs. 3, 4). In particular, the highest concentration increased the formation of both parameters from the onset of oxidation. d- tocopherol only showed marginal effects; no concentration-dependent changes were determined. However, no acceleration of the speed of lipid oxidation was observed. The results of the secondary oxidation products support the results of the primary parameter: a-tocopherol accelerated the speed of lipid oxidation, whereas d-toco-

6 296 Fig. 4 Effects of added a- and d-tocopherols at % on the formation of hexanal of Planta oxidized at 160 C Table 1 The fatty acid pattern of Planta after addition of a-tocopherol (a-t) or d-tocopherol (d-t) after 6 h of oxidation at 160 C Fatty acid Initial Control 0.05% d-t 0.1% d-t 0.2% d-t 0.05% a-t 0.1% a-t 0.2% a-t Sum of SFA Sum of MUFA Sum of PUFA n-3/n p/s ratio All fatty acids contents are expressed as a percentage of the content of total fatty acids; there is no statistical difference between the enriched samples and the control. SFA saturated fatty acids, MUFA monounsaturated fatty acids, PUFA polyunsaturated fatty acids pherol remained ineffective or showed marginal antioxidative effects. Only scattered information is available on d-tocopherol; however, what is currently known is affirmed by the data presented, although some results have been obtained at lower oxidation temperatures [9, 10]. Effect of tocopherols on the fatty acid pattern The fatty acids are stable and are oxidized only in an advanced stage of lipid oxidation. In order to see possible impacts of tocopherols we compared the fatty acid pattern of the initial fat with that of the fats obtained after 6 h of oxidation (Table 1). Our aim was to remain in the oxi-

7 dation time where lipid oxidation takes place and primary and secondary oxidation parameters exceed the threshold levels. However, this time was too short to influence the fatty acid pattern; no sustained change in the fatty acids was detected, although the a-tocopherol enriched samples showed a tendency to have reduced PUFA concentrations compared with the other samples. This can be explained by the stability of the fatty acids and cannot be related to the effects of tocopherols. This result underlines the priority of changes within the lipid oxidation pathway. A change in the fatty acid pattern is a signal of an advanced stage of oxidation. The results presented here are confirmed by numerous studies proving the antioxidant properties of a-tocopherol are weaker in foods [4, 9, 10, 14, 31]. Concluding remarks Numerous determinations of the tocopherol decomposition and also the generation of primary and secondary oxidation products lead to the conclusion that soon after starting the process of frying oxidation changes take place in the fat being used in the experiment. The stability of samples with a-tocopherol added was much less in comparison to that of the fat enriched with d-tocopherol. The oxidation parameters tested confirm the slower oxidation of samples with d-tocopherol added. Only this process did not affect the fatty acid composition in individual samples. The investigation proved that the more effective inhibitor of the oxidation process was d-tocopherol; however, no concentration-dependent effects were observed. References 1. Covington RM Jr, Unger EH (1999) Canola shortenings for food application US Patent document: 5,912,041, Cargill Incorporated, Wayzata, MN, USA 2. Elmadfa I, Wagner K-H (1999) In: Boskou D, Elmadfa I (eds) Frying of food chemistry and nutrition. Technomics Publishing Company Inc., Lancaster (USA), pp Pantzaris TP (1999) In: Boskou D, Elmadfa I (eds) Frying of food chemistry and nutrition. Technomics Publishing Company Inc., Lancaster, USA, pp Wagner K-H, Elmadfa I (2000) Eur J Lipid Sci Technol 102: Gordon MH, Kourimska L (1995) Food Chem 52: Kamal-Eldin A, Appelquist L-A. (1996) Lipids 31: Bramely PM, Elmadfa I, Kafatos A, Kelly FJ, Manios Y, Roxborough HE, Schuch W, Sheehy PJA, Wagner K-H (2000) J Sci Food Agric 80: Lampi A-M, Kataja L, Kamal-Eldin A, Piironen V (1999) JAOCS 76: Isnardy B, Wagner KH, Elmadfa I (2003) J Agric Food Chem 51: Wagner K-H, Isnardy B, Elmadfa I (2004) Eur J Lipid Sci Technol 106: Barrera-Arellano D, Ruiz-Mendez V, Marquez-Ruiz G, Dobarganes C (1999) J Sci Food Agric 79: Verleyen T, Kamal-Eldin A, Dobarganes C, Verhe R, Dewettinck K, Huyghebaert A (2001) Lipids 36: Lampi A-M, Kamal-Eldin A (1998) JAOCS 75: Barrera-Arellano D, Ruiz-Mendez V, Velasco J, Marquez-Ruiz G, Dobarganes C (2002) J Sci Food Agric 82: Stuchlik M, Zak S (2002) Biomed Pap Med Fac Univ Palacky Olomouc Czech Rep 146: Frankel EN (1996) Food Chem 57: AOCS Official Methods (1998) In: Official Methods and Recommended Practices of the American Oil Chemists Society, 5edn. AOCS Press, IL, Official Method Cd, pp AOCS Official Methods (1998) In: Official Methods and Recommended Practices of the American Oil Chemists Society, 5edn. AOCS Press, IL, Method Ti, pp 1a AOCS Official Methods (1998) p-anisidin value. In: Official methods and recommended practices of the american oil chemists society, 5edn. AOCS Press, IL, USA, Official Method Cd, pp Wagner K-H, Auer E, Elmadfa I (2000) Eur Food Res Technol 210: Lingnert H, Vallentin K, Eriksson CE (1979) J Food Process Preserv 3: Nagao A, Yamazaki M (1981) Yukagaku 30: Wan JP (1995) In: Warner K, Eskin NAM (eds) Methods to assess quality and stability of oils and fat containing foods, AOCS Press, IL, USA, pp Frankel EN, Meyer AS (2000) J Sci Food Agric 80: Frankel EN (1998) In: Frankel EN (ed) Lipid oxidation, The Oily Press Ltd, Dundee, Scotland, pp Crozier G, Turini M (1996) Proc. of the 21st ISF World Congress, Prague 1995, J.P. Barnes& Associates, pp Wagner K-H, Wotruba F, Elmadfa I (2000) Eur J Lipid Sci Technol 103: Dobarganes MC, Mµrquez Ruiz G (1998) Grasas y Aceites 49: Kamal-Eldin A, Marquez-Ruiz G, Dobarganes C, Appelqvist LA (1997) J Crom A 776: Mäkinen M, Kamal-Eldin A, Lampi AM, Hopia A (2001) Europ J Lipid Sci Technol 103: Huang SW, Frankel EN, German JB (1995) J Agric Food Chem 43:

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