1068 Eur. J. Lipid Sci. Technol. 2008, 110, 1068–1077 Research Paper Effect of phytosterol structure on thermal polymerization of heated soybean oil Jill Kristine Winkler and Kathleen Warner U.S. Department of Agriculture, Agricultural Research Service, National Center for Agricultural Utilization Research, Peoria, USA* This study determined the effect of phytosterol structure, including the degree of unsaturation and the presence of an ethylidene group in the side chain, on the thermal polymerization of heated soybean oil. Indigenous tocopherols and phytosterols were removed from soybean oil by molecular distillation. Pure phytosterols were added back to the stripped soybean oil at concentrations of 0.5, 1.0, and 5 mg/g oil (0.05, 0.1, and 0.5 wt-%). These oils were heated at 180 7C over a period of 8 h, and triacylglycerol dimers and polymers, fatty acid composition, and residual phytosterol content were determined. None of the phytosterols prevented triacylglycerol dimer and polymer formation when used at 0.5 mg/g; however, phytosterols with two or more double bonds, regardless of the presence of an ethylidene group in the side chain, provided slight protection when added at 1 mg/g. Ergosterol addition at 5 mg/g reduced polymer formation by 16–20% compared to the control oil, but at this level none of the other phytosterols provided protection of any practical significance. Thus, under the conditions used for this heating study, the degree of phytosterol unsaturation was more important for its anti-polymerization activity than the presence of an ethylidene group. Keywords: Ethylidene side chain / Fucosterol / Phytosterols / Thermal polymerization / Vegetable oils Received: April 7, 2008; accepted: June 9, 2008 DOI 10.1002/ejlt.200800089 1 Introduction monly used to reduce the PUFA in these oils, thereby increasing the oil stability for frying. However, hydrogenating Deep-fat frying is one of the most common uses for edible oils can cause the formation of fatty acids with trans double vegetable oils in the food industry. In 2006, about 7.8 billion bonds [3], and consumption of trans fatty acids is believed to lbs of fats and oils were used for baking and frying in the USA increase the risk of coronary heart disease [4]. Thus, with alone [1], which accounts for around 30% of total U.S. fat and increasing knowledge of the effects of trans fatty acids on oil consumption. Oil used for deep-fat frying is subjected to health, recent legislation requiring labeling of trans fatty acids high temperatures, moisture from foods, and migration of in foods, as well as bans in certain U.S. cities on artificially other compounds from foods that could act as pro-oxidants. produced trans fatty acids in restaurant foods, hydrogenation Vegetable oils high in polyunsaturated fatty acids (PUFA), is no longer the most desirable solution. Antioxidants such as such as regular soybean and sunflower oil, are considered tert-butyl hydroquinone (TBHQ) and the anti-foaming agent unsuitable for commercial deep-fat frying. During frying, the dimethylsilicone are often added to oils to increase their fry- PUFA are highly susceptible to oxidative damage, resulting in life; however, many antioxidants are less effective at frying off-flavors and -odors, smoking and foaming of the frying oil, temperatures because they quickly degrade or may volatilize and production of oxidized and polymerized lipids that may [2]. Several varieties of vegetable oils with modified fatty acid be detrimental to human health [2]. Hydrogenation is com- composition, such as low-linolenic soybean oil or mid- and high-oleic sunflower oil and high-oleic or low-linolenic canola oil, have been introduced as more stable alternatives without Correspondence: Jill K. Winkler, U.S. Department of Agriculture, Agricul- tural Research Service, National Center for Agricultural Utilization Re- * Mention of trade names or commercial products in this publication search, 1815 N. University St. Peoria, IL 61604, USA. is solely for the purpose of providing specific information and does E-mail: [email protected] not imply recommendation or endorsement by the U.S. Department Fax: 11-309-6816340 of Agriculture. © 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.ejlst.com Eur. J. Lipid Sci. Technol. 2008, 110, 1068–1077 Effect of sterol structure on thermal polymerization 1069 the need for hydrogenation. Vegetable oils with modified fatty acid composition are produced through genetic modification, traditional plant breeding, blending of polyunsaturated oils with more saturated oils, or interesterification [5]. While fatty acid composition is probably the most important factor in determining oil stability to frying, the composition of minor components such as tocopherols is also an important deter- minant [6]. Thus, there is increasing interest in the role that natural minor oil constituents such as tocopherols and phy- tosterols have on stability to frying. Phytosterols are triterpene compounds, similar in struc- ture to cholesterol, that are found ubiquitously in plants as well as yeast, mold, and other fungi [1]. Phytosterols are the most dominant class of compounds in the unsaponifiable material of vegetable oils [7]. The three most abundant phytosterols found in commodity vegetable oils include b- sitosterol, campesterol, and stigmasterol. Several other ster- ols, such as D5- and D7-avenasterol as well as brassicasterol, are also common in vegetable oils, but are usually found in lower concentrations. Phytosterols, such as D5-avenasterol, its isomer fucosterol, as well as vernosterol and citrostadie- nol, have been found in several studies to protect oils sub- jected to frying temperatures from oxidative degradation, while other, more abundant phytosterols have been shown to have no activity [8–11]. For example, Sims et al. [8] reported slight protective activity of fucosterol, moderate activity of D7-avenasterol, and high activity of vernosterol in Figure 1. Structures and common names for the phytosterols protecting heated safflower oil from decreasing in iodine used in this study as well as several other phytosterols (D5- and value (IV). They also tested ergosterol, lanosterol, b-sitos- D7-avenasterol) with ethylidene structures in the side chains. terol, stigmasterol, and cholesterol and found them all to be International Union of Pure and Applied Chemistry-International ineffective. Gordon and Magos [9] also reported protection Union of Biochemistry (IUPAC-IUB, 1989) numbering is shown for b-sitosterol. from reduction in IV by fucosterol and D5-avenasterol added to technical triolein heated to 180 7C. The protective phytosterols have in common an ethylidene group in their side chain (Fig. 1). Gordon and Magos [9] hypothesized We recently reported that a commercial mixture of pure that lipid free radicals react quickly with the unhindered phytosterols reduced thermal polymerization of soybean oil allylic methyl hydrogens in the ethylidene side chain to form triacylglycerols, but not high-oleic sunflower oil triacylglycer- stable tertiary free radicals that are slow to react further, ols, when added at levels ranging from 1 to 2.5% [13]. Since thus interrupting the chain of autooxidation like a classic the mixture was mainly composed of b-sitosterol, stigmas- chain-breaking antioxidant. There is scarce data, such as terol, and campesterol, with only small amounts of D5-ave- isolation of phytosterol side chain oxidation products, to nasterol, it did not appear that the anti-polymerization activity support this hypothesized mechanism. Some non-steroidal was conferred by the D5-avenasterol. Rather, it seemed to be compounds with an ethylidene group, such as undecylenic an effect of the higher total phytosterol concentration, and we acid, linalyl acetate, and linalyl oleate, were shown to also hypothesized that at the higher concentrations some of the have protective activity in oils heated to high temperatures phytosterols may have reacted with radicals and peroxides of [10, 12]. However, Onal-Ulusoy [12] also found that terpe- linoleic and linolenic acids, and thus terminated their further nyl oleates, both with and without an ethylidene structure, reaction. In order to further determine the anti-polymeriza- had antioxidant activity; so the activity of linalyl oleate could tion activity of phytosterols as related to their structure and not be solely attributed to the ethylidene group. In the liter- number of double bonds, the goal of the present study was to ature, phytosterols with an ethylidene group have been compare the effect of phytosterols with zero to three double referred to as “antioxidants” and “polymerization inhibi- bonds, or an ethylidene group in the side chain, on thermal tors”, despite the fact that in these studies oxygen con- polymerization of soybean oil triacylglycerols. Pure sitostanol, sumption was not followed, the mechanism(s) for phytos- sitosterol, stigmasterol, fucosterol, brassicasterol, and ergos- terol activity were not determined, and in most cases, the terol (Fig. 1) were added to soybean oil that had its native degree of triacylglycerol polymerization was not determined. tocopherols and phytosterols removed [stripped soybean oil © 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.ejlst.com 1070 J. K. Winkler and K. Warner Eur. J. Lipid Sci. Technol. 2008, 110, 1068–1077 (SSBO)]. The treated oils were heated for 8 h at 180 7C. The 2.4 Heating study effect of these phytosterols on polymerized triacylglycerol (PTAG) formation and loss of PUFA was investigated. The oils were thawed at room temperature and aliquots (1 g) from each treatment were weighed into four, 136100 mm open-mouth borosilicate tubes and placed in a test tube rack 2 Materials and methods that was immersed in a circulating hot oil bath maintained at 180 6 1 7C. The remaining oil was reserved frozen (–20 7C) 2.1 Materials for analysis. Two tubes per treatment were removed after 4 and 8 h. After cooling for 5 min, the oil was transferred to Hexane-extracted, alkali-refined, bleached and deodorized vials, covered with argon in the headspace and frozen until soybean oil (SBO) was obtained from a commercial pro- analyzed.