Effect of Tocopherols and Phytosterol on Color Reversion of MCT

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1 Food Sci. Technol. Res., 19 (6), , 213 Note Effect of Tocopherols and Phytosterol on Color Reversion of MCT Yuquan Zhang 1,2, Xuebing Xu 1, Yuanrong Jiang 1 and Xingguo Wang 2* 1 Wilmar (Shanghai) Biotechnology Research & Development Center Co., Ltd, NO. 118 Gaodong Road, Pudong New District, Shanghai, China 2 State Key Laboratory of Food Science and Technology, Synergetic Innovation Center of Food Safety and Nutrition, School of Food Science and Technology, Jiangnan University, China Received June 5, 213; Accepted July 9, 213 Tocopherols and phytosterols were added to medium chain triacylglycerol (MCT) to study their effect on color reversion of vegetable oils. Four samples with, 6,, 1, and 14, mg/kg phytosterols were heated at 15 for 12 h. No significant reduction of phytosterols and obvious color change were found in the four samples. Five samples with, 1,, 2,, 5,, 1, mg/kg tocopherols were also heated at 15 for 12 h. About 2 5% tocopherols were absent after heating. Reduction of total tocopherol was found linearly related to color darkening. The most significant reduction is α-tocopherol. Reduction of α-tocopherol correlated more with color darkening than that of γ-tocopherol. The conclusion is phytosterol has no effect on color reversion of oils and reduction of α-tocopherol was attributed to be the most important cause of color reversion. Keywords: vegetable oil, MCT, color reversion, phytosterols, tocopherols Abbreviations: MCT: Medium chain triacylglycerol, ΔToc: Reduction of total content of tocopherols, ΔC: Increasing of Platinum-Cobalt scale Introduction The color of most refined oils is pale yellow or amber. Some vegetable oils are getting darker during storage. This phenomenon is called the color reversion of oil. Color reversion happens often in some refined vegetable oils, like soybean oil, rapeseed oil and so on. In China, liquid vegetable oils are largely consumed as cooking oil. They are normally packed in transparent bottles and the color change of oil is obvious and highly concerned by consumers. Unlike that in western countries, solid fats or emulsions, which color is lighter and stabler, are consumed more than vegetable oils. The change of color of solid fats and emulsions is almost imperceptible. In this way, color stability of refined oils is more important in Asia and color reversion has always been avoided. Since western countries have no such issue, not much research has been done and most of them have been conducted by Asian researchers in earlier years. Almost no new reports appeared in recent three decades in this field. The research of *To whom correspondence should be addressed. wxg12@hotmail.com color reversion is mostly found on cotton seed oil (Swift et al., 1944), soybean oil (Harada and Komoda, 197) and so on. The minor components in corn oil are abundant, including pigments, high content of tocopherol and phytosterol (Moreau et al., 27). The pigment degradation for color reversion was found to relate to the lightening of corn oil in the process of corn oil recently (Knockaert et al., 212). Oxidation and polymerizing of tocopherol was investigated by several scientists in cotton seeds oil (Swift et al., 1944) and soybean oil (Chu and Lin, 199). Raw material quality, moisture, tocopherol were found to be associated with color reversion. The decrease in γ-tocopherol was accompanied by an increase in dimeric oxidation products. Formation of dimeric oxidation products of γ-tocopherol was proposed to be the reason of color reversion (Komoda et al., 1966). In China, color reversion of corn oil has brought most quality problems in cooking oil industry. The color of freshly refined corn oil is very pale and may fluctuate afterward (Sun et al., 21). However the mechanism of color reversion is far from thorough understanding, especially corn oil. In corn oil, tocopherols and phytosterols comprise two of the highest

2 1128 amount of minor components. This research is aimed to study the effect of tocopherols and phytosterols on color reversion of corn oil during heating. To eliminate the effect of fatty acids and other minor components in the oil, medium chain triacylglycerol (MCT) was used because it contains almost no unsaturated fatty acids and minor components like tocopherols, pigments and phytosterols. Platinum-Cobalt Scale was used to monitor color change because it is sensitive to light color. Materials and Methods Materials All materials used were obtained from the manufacturers. MCT was obtained from BASF (China) Company Ltd.. Tocopherols, phytosterols and fatty acid profiles were measured (Table 1). COVI-OX T-9 (T-9), mixed natural tocopherols, was supplied by Cognis Corporation (Illinois, USA). The ratio of typical composition of α-, β-, γ-, and δ-tocopherol is 16.7%, 1.4%, 64.1% and 17.8%, respectively; Phytosterol was obtained from BASF (China) Company Ltd., mixed with natural phytosterol, with the typical phytosterol composition of 25.9%, 25.1%, 43.5% of campesterol, stigmasterol and β-sitosterol. Tocopherol analysis Oil samples added with T-9 were measured by HPLC according to AOCS Ce 8-89 using an Agilent 12 instrument fitted with the column ZORBAX SIL (Agilent, mm, 5 μm). Phytosterol analysis Oil samples added with Phytosterol (BASF (China) Company Ltd.) were measured by GC according to the method of ISO 12228:1999 using an Agilent 789A instrument fitted with the column HP-5 (Agilent, 3 m.32 mm.25 μm). Oil samples preparation T g was added into g MCT to prepare 1, mg/kg tocopherol MCT sample and ultrasounded for 2 min using Ultrasonic Cleaner SK72HP (Shanghai Kudos Ultrasonic Instrument Co., Ltd.) to dissolve tocopherols sufficiently. 1, mg/kg tocopherol MCT sample was diluted with MCT into 1, mg/kg, 2, mg/kg, 5, mg/kg tocopherol MCT samples. Four samples were named as MCT-1,Toc, MCT- Table 1. Fatty acid profiles and the other minor components. Item Content Total tocopherols (mg/kg) ND Total phytosterols (mg/kg) ND Fatty acid profiles (%) C8: 52.6 C1: 46.7 C12:.5 Others.2 ND - Non-detectable. Y. Zhang et al. 2,Toc, MCT-5,Toc and MCT-1,Toc. Phytosterols g was added into g MCT to prepare 14, mg/kg phytosterol MCT, followed by heating to 4 and ultrasounded for 5 min for dissolving phytosterols sufficiently. Then, 14, mg/kg phytosterol MCT sample was diluted with MCT into 6, mg/kg, 1, mg/kg MCT samples. Four samples were named as MCT- 6,PS, MCT-1,PS and MCT-14,PS. Oven Test Schaal oven test was conducted in 15 referring to AOCS Cg Oil samples (15 g) was placed into amber bottles and capped. Seven bottles of each oil sample were putted in Schaal oven (15 ). One bottle of each kind of the oil sample was taken out at each point of time (1, 2, 4, 6, 8, 1, 12 h) for measurement. Color determination Color determination was according to method Td 1b-64 of the American Oil Chemists Society (29). Briefly, each oil samples, placed into 1 mm optical path spectrophotometer cell, was tested Platinum- Cobalt Scale (Hazen) in triplicate used Lovibond PFX 995 Tintometer (Tintometer Group, UK). Statistical analysis Each reported value is the mean of triplicate determinations. The statistical analysis of the data, Student s t-test, correlation and regression were carried out with SPSS 17. software. Pearson correlation was used to compare the significant difference between the samples. Results and Discussion Effect of phytosterols on color of MCT at 15 Generally, the content of phytosterols in refined corn oil ranged from 6, 13, mg/kg (Shahidi, 25). In this study, 6,, 1, and 14, mg/kg phytosterols were added into MCT and heated at 15 for 12 h. During heating, no significant reduction of phytosterols was observed in all three samples (Fig. 1). Content of phytosterol ( 1 3 mg/kg) MCT - 6, PS MCT - 1, PS MCT - 14, PS Fig. 1. Content of phytosterols in MCT during heating.

3 Effect of Tocopherols and Phytosterol on Color Reversion of MCT Color of MCT with phytosterols was monitored. The color of MCT remained very stable and no darkening or lightening happened (Fig. 2). This result was consistent with Harada s result published in 1967 (Komoda et al., 1967). More and more reports on degradation and oxidation of phytosterols in oils have come out in recent years. Whether the phytosterols oxides could result in darkening of oil was not found in the literatures. The oxidation of phytosterol depend ed on the saturation of oils. At 15, the more saturated of the oil, the more the absolute oxidation of phytosterols (Oehrl et al., 21). Although MCT in this study is totally saturated, no obvious oxidation of phytosterol was observed. This indicates that color reversion of vegetable oils may not relate to phytosterols in them. Effect of overall tocopherols reduction on color of MCT at 15 The tocopherols used in this study contain α-, MCT - Toc MCT - 1,Toc MCT - 2, Toc MCT - 5, Toc MCT - 1, Toc Pt-Co color scale (Hazen) MCT - PS MCT - 6, PS MCT - 1, PS MCT - 14, PS Fig. 2. Pt-Co color scale of MCT sample with different content of phytosterol during heating. Pt-Co color scale (Hazen) Fig. 3. Pt-Co color scale of MCT sample with different content of tocopherol during heating R 2 = MCT - 1, Toc R 2 = MCT - 5, Toc R 2 = MCT - 2, Toc R 2 = MCT - 1, Toc Fig. 4. Correlation between reduction of tocopherols and color darkening of MCT during heating.

4 113 contributed to color darkening. This mechanism was then developed by Lai in 1989 (Lai et al., 1989). Tocored and other two isomers were characterized by HPLC and proved to convert to color-reverted substances after bleaching and converted back to the precursors during deodorization. In all these publications, γ-tocopherol was attributed to be the only cause of color reversion. However, in this study, an interested observation was that among all four isomers, reduction of α-tocopherol was the most significant. Although the reduction of α- and γ-tocopherol was both linearly related to the color enhancing significantly (P <.1), their correlation significance was different. By comparing the pearson correlation (Table 2), it was concluded that reduction of α-tocopherol correlated more with color darkening than that of γ-tocopherol. This discovery is different from those in the literatures before. Alternation of α-tocopherol contributing was neglected before. According to the research of Yamauchi (1997), oxidation of α-tocopherol created quinones, with similar structure of γ-tocored. γ-tocored is intermediβ-, γ- and δ-isomers. Four MCT samples with 1,, 2,, 5,, 1, mg/kg tocopherols were heated for 12 h. After heating, total tocopherols content decreased in all samples. The color of the four oil samples increased significantly (P <.5) after heating (Fig. 3). The more tocopherols added, the more the color darkened. To figure out the effect of tocopherols on color darkening of MCT, correlation between reduction of total content of tocopherols (ΔToc) and increasing of Platinum-Cobalt Scale (ΔC) was studied. It was found in all four samples, ΔC was linearly related to ΔToc highly (Fig. 4). The R 2 is.93,.95,.85 and.95, respectively. This means that the presence of tocopherols is correlated to color change in vegetable oils and may be one of the causes of color reversion. This conclusion is consistent with the literatures before (Harada et al., 196; Chu and Lin, 199). Effect of tocopherol isomers on color of MCT at 15 Content of each isomer of tocopherol was monitored during heating. After heating, the content of the four isomers was all lowered. Among them, the absence of α-tocopherol is the highest. In MCT-1,Toc, MCT-2,Toc MCT-5,Toc and MCT-1,Toc, the reduction is 7.3%, 8.3%, 11.3% and 1.4%, respectively. In the tocopherols used, more than half is γ-tocopherol. Its reduction in all the samples during heating is obvious. Reduction of δ-tocopherol could be negligible (Fig. 5). The content of β-tocopherol is considerably low. Therefore, the contribution of reduction α- and γ-tocopherol will be explained in details. β- and δ-tocopherol were not included in this discussion. In some previous literatures, color reversion of vegetable oils was attributed to the degradation product of oxidized dimeric of γ-tocopherol. Komoda proposed that γ-tocopherol was first oxidized to dimer and then tocored. Tocored was regarded as the precursor of oxidized compounds which Y. Zhang et al. Fig. 5. Reduction of tocopherol isomers after heating for 12 h. Table 2. Pearson correlation of reduction of α- and γ-tocopherol with color darkening of MCT. MCT-1Toc MCT-2Toc MCT-5Toc MCT-1Toc Δα-Toc Δγ-Toc ΔC Pearson Correlation.981**.936** Sig. (2-tailed)..2 ΔC Pearson Correlation.99**.963** Sig. (2-tailed)..1 ΔC Pearson Correlation.979**.879** Sig. (2-tailed)..9 ΔC Pearson Correlation.996**.959** Sig. (2-tailed)..1 ** Correlation is significant at the.1 level (2-tailed).

5 Effect of Tocopherols and Phytosterol on Color Reversion of MCT ate product of oxidation and the precursor of compounds with intense color. Also, Kulas and Ackman (21) found that α-tocopherol is more susceptible to be oxidized than γ-tocopherol. These might be the reasons that α-tocopherol contributed more to color reversion. Further research will be conducted to monitor the precursor or the reversion mechanism. Conclusion Although oxidation of phytosterols has been stressed recently, its effect on the color change of MCT when heated to 15 was not observed in this study. It might not be the cause of the color reversion of vegetable oils. Tocopherols were reduced during heating and the reduction of them was linearly related to color darkening. Among four tocopherol isomers, the content of β-tocopherol was very low and reduction of δ-tocopherol could be ignored. The reduction of α- and γ-tocopherol was linearly related to color darkening during heating significantly (P <.1). The significance of α-tocopherol was higher than γ-tocopherol. This strongly indicated that α-tocopherol may contribute more to the color reversion of vegetable oils. This is different from the literatures before in which the effect of γ-tocopherol was stressed. Mechanism of effect of α-tocopherol on color reversion of vegetable oils is in progress. Acknowledgement This study was financially supported by Wilmar (Shanghai) biotechnology R&D center Co., LTD. References American Oil Chemists Society, A.O.C.S. (29). Official Methods and Recommended Practices of the American Oil chemists Society,Official methods and Recommended Practices.AOCS Press.Champaign, Illinois, U.S.A. Chu, Y. and Lin, W. (199). Effect of soybean pretreatment on the color quality of soybean oil. J. Am. Oil Chem. Soc., 67, Harada, I. and Komoda, M. (197). Color Reversion of Edible Oils. J. Jpn. Oil Chem. Soc., 19, Harada, I., Saratani, Y. and Ishikawa, M. (196). Studies on the color reversion of refined and deodorized soybean oil. Part VI : 1131 The influence of tocopherol, carotenoid and sterol isolated from soybean oil on the color reversion of refined soybean oil. J. Agric. Chem. Soc. Jpn., 34, Knockaert, G., Pulissery, S.K., Lemmens, L., Van Buggenhout, S., Hendrickx, M. and Van Loey, A. (212). Carrot β-carotene degradation and isomerization kinetics during thermal processing in the presence of oil. J. Agric. Food Chem., 6, Komoda, M., Onuki, N. and Harada, I. (1966). Studies on cause of color reversion of edible soybean oil and its prevention; Part I. Relation between the moisture of soybeans and the quantity of tocopherol in them. Agric. Biol. Chem., 3, Komoda, M., Onuki, N. and Harada, I. (1967). Studies on cause of color reversion of edible soybean oil and its prevention; Part II. Tocored as a precursor of color reversion of soybean oil. Agric. Biol. Chem., 31, Kulas, E. and Ackman, R.G. (21). Different tocopherols and the relationship between two methods for determination of primary oxidation products in fish oil. J. Agric. Food Chem., 49, Lai, M., Lin, W., Chu, Y., Chen, S.Y., Kong, K. and Chen, C. (1989). The mechanism of color reversion in soybean salad oil. J. Am. Oil Chem. Soc., 66, Moreau, R.A., Johnston, D.B. and Hicks, K.B. (27). A Comparison of the levels of lutein and zeaxanthin in corn germ oil, corn fiber oil and corn kernel oil. J. Am. Oil Chem. Soc., 84, Oehrl, L.L., Hansen, A.P., Rohrer, C.A., Fenner, G.P. and Boyd, L.C. (21). Oxidation of phytosterols in a test food system. J. Am. Oil Chem. Soc., 78, Shahidi, F. (25). Edible Oil and Fat Products:Chemistry, Properties, and Health Effects. In Bailey s Industrial Oil and Fat Products, 6 th ed. by John Wildy & Sons, Chichester. pp Sun, X., Gao, Y., Chen, F., Hu, M. and Yang, B. (21). Study on color reversion of corn oil. Cereals & Oils, Swift, C.E., Mann, G.E. and Fisher, G.S. (1944). Gamma-tocopherol as a precursor of a red quinoid substance developed in cottonseed oil during oxidation. Oil & Soap, 21, Yamauchi, R. (1997). Vitamin E: Mechanism of its antioxidant activity. Food Sci. Technol. Int., 3,