High-performance size-exclusion chromatography studies on the formation and distribution of polar compounds in camellia seed oil during heating

High-performance size-exclusion chromatography studies on the formation and distribution of polar compounds in camellia seed oil during heating 采用高效体积排阻色谱技术研究油茶籽油在加热过程中极性化合物的形成及其组分分布 1 first-author 1 2 1 corresp ‡Corresponding Authors ‡Corresponding Authors †E-mail:jianglianzhousci@163.com18245153561@163.com †E-mail:jianglianzhousci@163.com18245153561@163.com jianglianzhousci@163.com 1 corresp ‡Corresponding Authors ‡Corresponding Authors †E-mail:jianglianzhousci@163.com18245153561@163.com †E-mail:jianglianzhousci@163.com18245153561@163.com jianglianzhousci@163.com 3 College of Food Science, Northeast Agricultural University, Harbin 150030, China College of Food Science, Northeast Agricultural University, Harbin 150030, China Department of Nutrition and Food Science, Taif University, Taif 888, Kingdom of Saudi Arabia Department of Nutrition and Food Science, Taif University, Taif 888, Kingdom of Saudi Arabia Shanghai Grain Science Research Institute, Shanghai 200333, China Shanghai Grain Science Research Institute, Shanghai 200333, China Camellia seed oil (CSO) is rich in oleic acid and has a high number of active components, which give the oil high nutritional value and a variety of biological activity. The aim of the present study was to determine the changes in the content and distribution of total polar compounds (TPC) in CSO during heating. TPC were isolated by means of preparative flash chromatography and further analyzed by high-performance size-exclusion chromatography (HPSEC). The TPC content of CSO increased from 4.74% to 25.29%, showing a significantly lower formation rate as compared to that of extra virgin olive oil (EVOO) and soybean oil (SBO) during heating. Furthermore, heating also resulted in significant differences ( P < 0.05) in the distribution of TPC among these oils. Though the content of oxidized triacylglycerol dimers, oxidized triacylglycerol oligomers, and oxidized triacylglycerol monomers significantly increased in all these oils, their increased percentages were much less in CSO than those in EVOO, indicating that CSO has a greater ability to resist oxidation. This work may be useful for the food oil industry and consumers in helping to choose the correct oil and to decide on the useful lifetime of the oil. 目的 2 测定加热过程中油茶籽油中极性化合物及其组分的变化，同时与其它常用油脂进行对比，评估油茶籽油作为煎炸用油的优势。 创新点 2 首次在油茶籽油中采用制备型硅胶柱层析-高效 体积排阻色谱技术，监测了极性化合物总量及其组分分布在加热过程中的变化规律，预估油茶籽油的使用寿命，为煎炸用油的选择提供科学理论依据。 方法 2 取等量市售油茶籽油、橄榄油、大豆油和棕榈油，在180 °C分别加热2、4、6、8和10小时并收集样品。实验结束后，首先利用制备型硅胶柱层析分离测定总极性化合物含量的变化，并采用高效体积排阻色谱技术对分离得到的极性化合物组分进行定量分析。 结论 2 油茶籽油在加热过程中所形成的极性化合物总量（TPC）呈线性增加趋势，其形成速率显著低于橄榄油和大豆油（图1）。随着加热时间的延长，油茶籽油中的氧化甘油三酯（ox-TGM）、氧化甘油三酯二聚物（TGD）和氧化甘油三酯多聚物（TGO）的含量显著增加（ P < 0.05）。此外，ox-TGM和TGD在油茶籽油中的形成速率明显低于其在橄榄油和大豆油中的形成速率，然而这三种油脂之间的TGO含量的变化不存在显著差异（图4）。综上所述，油茶籽油可作为一种具有开发前景和健康食用价值的煎炸用油。 Camellia seed oil Polar compounds High-performance size-exclusion chromatography Oxidation 油茶籽油 极性化合物 高效体积排阻色谱技术 氧化 Introduction 1 Camellia oleifera Abel, an ornamental plant widely distributed in China and Western countries (Zeb, 2012 2012 ), has recently received great attention as a source of edible oil. Camellia seed oil (CSO), obtained from its seeds, is rich in oleic acid and contains many active compounds (Wang X.Q. et al., 2014 2014 ). It is found to have suitable nutritional characteristics and medical values (Salinero et al., 2012 2012 ; Wang Y.F. et al., 2014 2014 ), and is sometimes termed as “Oriental olive oil”. So far, most studies on CSO have been focused on chemical characterization (Su et al., 2014 2014 ; Wang X.Q. et al., 2014 2014 ), especially some active components and antioxidant capacity, the extraction (Li et al., 2013 2013 ; Lim et al., 2013 2013 ), refining process (Na, 2008 2008 ), and adulteration (Hai and Wang, 2006 2006 ). Few studies have been reported on its formation of degradation compounds during heating. However, heating treatments are an essential process for edible oil. During heat treatment, vegetable oils undergo hydrolysis, oxidation, and thermal decomposition, resulting in the formation of total polar compounds (TPC), which are of larger molecular weight and higher polarity than general triacylglycerols (Guillén and Uriarte, 2013 2013 ). These compounds can be separated from the oil and the levels of their contents are considered to be the most reliable index of the quality of the used oils (Farhoosh and Tavassoli-Kafrani, 2010 2010 ), and is the basis of the international regulations limiting the use of degraded oils for human consumption (Correia et al., 2015 2015 ). Further analysis via high-performance size-exclusion chromatography (HPSEC) can divide TPC into various classes of substances, namely, oxidized triacylglycerol dimers (TGD), oxidized triacylglycerol oligomers (TGO), oxidized triacylglycerol monomers (ox-TGM), diacylglycerols (DAG), and free fatty acids (FFA) to assess the level of oxidative and hydrolytic degradation of the oils. Even when of similar overall content level, TPC will vary significantly in the distribution of type (Farhoosh and Tavassoli-Kafrani, 2010 2010 ). Moreover, these polar components differed not only in molecular weight or pro-oxidant property (Paradiso et al., 2010 2010 ; Gomes et al., 2011 2011 ) but also in toxicological characteristics (Wenci, 2014 2014 ), and thus it is important to know the contribution of each of them to the total alteration. It is noteworthy that the hydrolysis involves breakage of the ester bond with the formation of FFA, DAG, glycerols, and monoglycerides, while the oxidation and thermal decomposition take place in the unsaturated acyl groups of the triacylglycerols (Dobarganes et al., 1988 1988 ). It is difficult to avoid the formation of TPC in oils during heating. However, their formation rates are significantly influenced by the fatty acid composition of the oil (Martín-Polvillo et al., 2004 2004 ) and the amount and type of natural antioxidants in the oil (Farhoosh and Tavassoli-Kafrani, 2010 2010 ). Therefore, it is possible to select the oil with strong antioxidant properties to reduce the formation rate of TPC. As an edible oil, CSO has a high content of total phenolics (65.9 mg gallic acid equivalents per kilogram of oil (mg GAE/kg)), which give it good performance during heating. The aim of this paper is to study the formation of TPC in CSO during heating. Changes in the distribution of TPC as well as comparison with common edible oils, such as extra virgin olive oil (EVOO), soybean oil (SBO), and palm oil (PO) are also reported. Materials and methods 1 Materials 2 The oils for this study, CSO, EVOO, SBO, and PO, were acquired from a local supermarket. The tetrahydrofuran (THF, Merk Corporation, USA) used for the HPSEC analysis was of chromatography grade, and was purchased from Tedia Company (Fairfield, USA). Glass plates (20 cm×20 cm) for thin-layer chromatography were coated with silica gel (without fluorescence indicator) at 0.25 mm layer thickness. The developing solvent was a mixture of petroleum ether (boiling range from 30 to 60 °C), diethyl ether, and acetic acid (70/30/2, v/v/v). Phosphomolybdic acid solution (100 g/L) in ethanol was applied as spray. All other chemical solvents used were of analytical grade, and were purchased from Shengda Chemical Reagent Co., Lid. (Heilongjiang, China). Thermal oxidation experiment 2 Thermal oxidation was carried out under strictly controlled conditions using an electric heater, equipped with an intelligent digital display and a magnetic stirrer (ZNCL-T/100 ml/150, Zhengzhou, China). The samples were treated under identical conditions. Equivalent samples of oils ((100±0.01) g) were weighed accurately into round-bottomed flasks of the same volume (100 ml), and inserted in the electric heater previously heated to (180±1) °C. Throughout the heating process no oil was replenished. Oil samples were periodically (every 2 h) taken in duplicate, and refrigerated at −40 °C until studied in order to avoid the continuation of the degradation process. Fatty acid composition 2 The fatty acid composition was carried out by an Agilent 6890-5973 gas chromatography-mass spectrometer (GC-MS; Agilent Technologies, CA, USA) analysis according to Li et al. ( 2013 2013 ). Separation of TPC 2 The separation of TPC from oil samples was performed using preparative flash chromatography (CHEETAH MP200, Bonna Agela Technology Company, China) according to the method of Cao et al. ( 2013 2013 ) with some modifications. In order to effectively separate polar compounds, two preparation flash silica columns (silica gel: 20 g; grain diameter: 40–60 μm) were used in series. The connected flash columns were flushed with the eluent A (petroleum ether:ethyl ether=87:13, v/v) for 10 min. Then the oil sample (1 g), dissolved in petroleum ether (5 ml), was injected into the column by a 10-ml syringe. After elution of the non-polar components with 250 ml of eluent A, the polar compounds were recovered with 500 ml of eluent B (ethyl ether). The eluent velocity was set at 25 ml/min. Then, the polar compounds dissolved in eluents B were subsequently submitted to rotary evaporation (45 °C) and vacuum drying (40 °C, 0.1 MPa). The obtained polar compounds were weighed to calculate their content based on the weight of each oil sample and were further analyzed. The elution of non-polar compounds and polar compounds was completed within one hour, greatly shortening the time needed (approximately 7 h) for silica gel column chromatography. The coefficient of variation of this separation method was less than 4% (Cao et al., 2013 2013 ). The efficacy of separation was checked by thin layer chromatography according to the International Union of Pure and Applied Chemistry (IUPAC) Standard Method 2.507 (Paquot and Hautfenne, 1987 1987 ). Distribution of TPC by the HPSEC 2 The determination of distribution of TPC in oil samples involves the use of HPSEC for their separation, identification, and quantification. The chromatographic system consisted of Waters 2695 high-performance liquid chromatographic system, 2414 differential refractive index detector (RI), a 10-μl injector loop, a Styragel gel chromatography guard column, and a series of two Styragel HR 0.5 volume exclusion gel columns (7.8 mm×300 mm, grain diameter 5 µm, aperture 10 nm, Waters company). Before injection, the obtained TPC from the previous procedure were dissolved in THF at the concentration of 10 mg/ml, and filtered through a membrane (0.22 μm). In line with the method of the previous report (Cao et al., 2013 2013 ), the filtered solution was introduced through the injector loop (10 μl). The temperature of both the columns and testing pool was set at 35 °C. The THF was used as the mobile phase for HPSEC at a flow rate of 0.7 ml/min. Peaks on the chromatograms were identified by SL-105 polystyrene standard relative molecular weight. TGO, TGD, ox-TGM, DAG, and FFA of the oils were quantified with the peak area normalization method. All the determinations were carried out in triplicate. Statistical analysis 2 Linear regression analysis of the data obtained from the separation of TPC of the oils was performed in Origin 8.0. Comparison between the means was made by applying one-way analysis of variance (ANOVA) using the statistical program SPSS Version 16.0 for Windows. A value of P < 0.05 indicated statistical significance, using Duncan’s multiple range tests. Results 1 Fatty acid composition 2 The major fatty acids and their contents in CSO as well as EVOO, SBO, and PO are listed in Table 1 1 . CSO mainly contained palmitic, stearic, oleic, and linoleic acids. The total content of unsaturated fatty acids (UFA) was 85.97%, which was a little higher than that in EVOO and SBO. In particular, the content of oleic acid in CSO was up to 76.26%, more than that in EVOO. However, palmitoleic and linolenic acids were not detected in CSO but found in EVOO. The highest linoleic acid was observed in SBO, whereas the highest content of palmitic acid and the highest saturated fatty acids (SFA) were observed in PO. a Sum of major saturated fatty acids; b Sum of major monounsaturated fatty acids; c Sum of major polyunsaturated fatty acids; d Sum of major unsaturated fatty acids; Total unsaturated fatty acids to total saturated fatty acids ratio Oil Palmitic acid (%) Palmitoleic acid (%) Stearic acid (%) Oleic acid (%) Linoleic acid (%) Linolenic acid (%) CSO 10.52±0.03 – 2.89±0.04 76.26±0.05 9.73±0.06 – EVOO 14.77±0.05 1.39±0.04 5.32±0.06 66.78±0.05 8.67±0.08 1.08±0.03 SBO 11.30±0.04 – 4.66±0.07 19.76±0.06 53.68±0.04 9.94±0.05 PO 34.83±0.05 0.23±0.02 5.13±0.06 46.23±0.03 11.67±0.07 0.47±0.01 7 Oil ∑SFA a (%) ∑MUFA b (%) ∑PUFA c (%) ∑UFA d (%) ∑UFA/∑SFA e e 7 CSO 13.41±0.07 76.26±0.05 9.73±0.06 85.99±0.03 6.41±0.03 EVOO 20.09±0.02 68.16±0.08 9.75±0.10 77.91±0.04 3.88±0.01 SBO 15.96±0.06 19.76±0.06 63.63±0.03 83.39±0.04 5.22±0.02 PO 39.96±0.01 46.46±0.04 12.13±0.08 58.59±0.04 1.47±0.01 Effect of heating time on the content of TPC 2 A heating of 10 h caused a considerable augmentation in the weight percentages of TPC in CSO, as well as in the three other edible oils (Fig. 1 1 ). It was observed that the weight percentages of TPC from all the selected oils and the heating time fit linear equations. As predicted, the formation rate of the oils showed diversity due to their differences in fatty acid composition. However, despite the highest ratio (6.41) of UFA/SFA among these oils and higher TPC content before heating, CSO presented a much lower rate of increase of TPC content compared to that in EVOO and SBO. In addition, the content of TPC in all the oils was up to 24% or more after heating for 10 h. Error bars indicate standard deviations for three replicates Distribution of TPC 2 The representative chromatogram of TPC obtained from CSO before and after heating is presented in Fig. 2 2 . Five main peaks, eluting in inverse order of molecular weight, were resolved: TGO, TGD, ox-TGM, DAG, and FFA. Peaks: 1, oxidized triacylglycerol oligomers (TGO); 2, oxidized triacylglycerol dimers (TGD); 3, oxidized triacylglycerol monomers (ox-TGM); 4, diacylglycerols (DAG); 5, free fatty acids (FFA). MW: molecular weight As is clearly seen from Fig. 2 2 , heating caused a significant shift of the HPSEC of the polar fractions isolated from CSO after heating for 10 h. Quantitative results of the relative (% of polar content) and absolute (% (w/w) on oil) contents for different groups of alteration products throughout the heating process are given in Tables 2 2 and 3 3 , respectively. Table 2 2 shows that the relative contents of TGO, TGD, ox-TGM, DAG, and FFA in CSO before heating were 5.22%, 17.62%, 20.32%, 46.58%, and 3.47%, respectively. Heating for 10 h resulted in a significant ( P < 0.05) increase of the relative contents of TGO, TGD, and ox-TGM in CSO, respectively up to 20.93%, 34.11%, and 33.89%, indicating the extent of oxidative degradation in CSO was aggravated by heating. This result also suggested that the formation rate of TGO was the fastest, followed by TGD then ox-TGM during the heating of CSO. Moreover, their absolute contents in CSO were also increased in this order (Table 3 3 ). It is noteworthy that the relative content of ox-TGM in CSO reached a steady state after heating for 6 h. In addition, heating also resulted in a significant ( P < 0.05) decrease of the relative contents of DAG and FFA in CSO (Table 2 2 ). TPC: total polar compounds; TGO: oxidized triacylglycerol oligomers; TGD: oxidized triacylglycerol dimmers; ox-TGM: oxidized triacylglycerol monomers; DAG: diacylglycerols; FFA: free fatty acids. All data are relative contents in TPC (w/w), and are expressed as mean±standard deviation of triplicate determinations; values in the same column bearing different letters are significantly different ( P < 0.05) 2 Heating time (h) 5 Relative content (%) * * TGO TGD ox-TGM DAG FFA 0 5.22±0.24 f 17.62±0.51 f 20.35±0.33 d 46.58±1.04 a 3.47±0.35 a 2 8.64±0.33 e 22.03±0.59 e 24.45±0.36 c 36.42±0.91 b 2.83±0.26 b 4 11.81±0.35 d 25.63±0.69 d 27.98±0.49 b 28.61±0.81 c 2.26±0.25 c 6 15.77±0.26 c 31.21±0.58 c 33.41±0.51 a 15.58±0.77 d 1.68±0.22 d 8 18.22±0.29 b 32.71±0.64 b 33.65±0.48 a 12.46±0.67 e 1.13±0.19 e 10 20.93±0.27 a 34.11±0.57 a 33.89±0.44 a 9.07±0.52 f 0.62±0.12 f All data are absolute contents in CSO (w/w), and are expressed as mean±standard deviation of triplicate determinations; values in the same column bearing different letters are significantly different ( P < 0.05). For abbreviation see Table 2 2 2 Heating time (h) 5 Absolute content (%) * * TGO TGD ox-TGM DAG FFA 0 0.25±0.03 f 0.83±0.06 f 0.96±0.08 f 2.20±0.16 c 0.17±0.03 b 2 0.72±0.10 e 1.83±0.14 e 2.03±0.18 e 3.02±0.24 ab 0.24±0.05 ab 4 1.42±0.13 d 3.07±0.29 d 3.36±0.35 d 3.43±0.32 a 0.27±0.06 a 6 2.32±0.19 c 4.60±0.36 c 4.93±0.40 c 2.31±0.34 c 0.25±0.06 ab 8 3.72±0.21 b 6.67±0.35 b 6.86±0.40 b 2.55±0.32 bc 0.23±0.06 ab 10 5.33±0.22 a 8.69±0.33 a 8.63±0.36 a 2.32±0.26 c 0.16±0.04 b In terms of the absolute contents of these polar components, TGO, TGD, and ox-TGM increased significantly ( P < 0.05) with prolonged heating, while small changes were observed in DAG and FFA (Table 3 3 ), suggesting that a high extent of oxidative degradation and a low level of hydrolytic degradation of CSO occurred during heating. Fig. 3 3 describes the change in the relative contents of oxidative products and hydrolytic products in EVOO, SBO, and PO, as well as comparison with that in CSO. Interestingly, for the relative contents of ox-TGM, a decreasing trend was observed only in SBO, whereas a continuously increasing throughout approximately 6 h, followed by a tendency to reach a near-steady state in later successive heating, was observed both in CSO and EVOO. Moreover, it was also observed that ox-TGM and TGD in CSO presented a slower formation rate than that in EVOO. However, the formation rate of TGO was a little higher in CSO than in EVOO. Error bars indicate standard deviations for three replicates The evolution of the absolute contents of these polar components in the oils was also illustrated and compared, as shown in Fig. 4 4 . It appears that the rates of increase of ox-TGM content and TGD content were much slower in CSO than in EVOO during heating, although higher values of ox-TGM content and TGD content were found in the former before heating. Only small differences were found in TGO content when considering the wide variation in the ratio of UFA/SFA of the oils assayed, as shown in Table 1 1 . Error bars indicate standard deviations for three replicates Discussion 1 As a new food resource, CSO is economically important for human consumption and has been widely used for making cooking oil and medicines (Su et al., 2014 2014 ). In application, heating is inevitable. During heating, the oil is prone to undergo lipid oxidation, resulting in the degradation and alteration of triacylglycerol and the formation of new compounds with polarity (Ng et al., 2014 2014 ). Under the same conditions, the formation rate of TPC in the oil is mainly dependent on its fatty acids composition and the content of antioxidant. However, the natural antioxidant presented in the oil is lost rapidly during heating and its loss was independent of the degree of oil unsaturation (Barrera-Arellano et al., 2002 2002 ). In our study, the content of TPC in oils was intensified progressively with processing time (Fig. 1 1 ). Despite this, CSO exhibited a higher stability due to the presence of high amounts of oleic acid. The distinct formation rate of TPC was found in the selected oils, demonstrating that the fatty acid composition has an apparent impact on the thermal oxidation, hydrolysis, and polymerization of oils. This result is inconsistent with the previous report, in which Marmesat et al. ( 2012 2012 ) stated that the levels of all the new compounds formed during thermoxidation, i.e., polymers, polar compounds, and polar fatty acids, strongly depended on the degree of oil unsaturation. Research has shown that TPC isolated from thermally oxidized oils are toxic to laboratory animals (Pantzaris, 1998 1998 ). Therefore, it has been recommended that the thermally oxidized oils containing more than 24%–27% of TPC content should be discarded (Caldwell et al., 2011 2011 ; Correia et al., 2015 2015 ). Assuming that the limit of acceptance for the TPC content is 25%, the time required to reach this limit was considered as a measure of oil’s useful lifetime (Marmesat et al., 2012 2012 ). From the calculated measures and the linear equations displayed in Fig. 1 1 , CSO showed a higher thermal stability (10.25 h) than that of EVOO (9.82 h) or SBO (8.61 h). From these results, it can be seen that CSO was more resistant to oxidation degradation than EVOO and SBO. In the case of PO, due to its high content of SFA, it was considered to be the most stabile (11.90 h) cooking oil. Although the suitability of PO for heating and frying has been widely demonstrated in many studies, the PO was unfavorable in view of health aspects (Matthäus, 2007 2007 ). Considering the requirement for dietary fatty acid and the formation rate of TPC, CSO exhibited a better lifecycle and is more suitable than EVOO and SBO for use as cooking oil. Analysis of the TPC enables an overall evaluation of both hydrolytic and oxidative degradation of edible oils and fats (Caponio et al., 2007 2007 ). Further application of HPSEC to the isolated polar compounds allowed differentiation of the main groups of compounds present and establishment of the relative influence of three degradation pathways predominant in heating: hydrolysis, polymerization, and oxidation (Romero et al., 1995 1995 ). Significant peak separation enabled determination of the amounts of TGD, TGO, ox-TGM, DAG, and FFA (Fig. 2 2 ). Oxidized triglyceride polymers (TGPs), including TGD and TGO, are a reliable index of secondary oxidative degradation of the oil, because they are stable and not influenced by processing conditions. On the contrary, ox-TGM are triglycerides having higher polarity than unaltered triglycerides, and mainly consist of triglyceride hydroperoxides as well as triglycerides in which the hydroperoxydic functional group has been transformed in the alcoholic or carbonyl group (Gomes et al., 2012 2012 ). The ox-TGM can give information about the primary oxidation level of oil. Finally, DAG and FFA constitute reliable parameters for a proper evaluation of the real level of hydrolytic degradation of refined oils (Caponio et al., 2011 2011 ). Note that differences in distribution of each polar component can be related to differences in the nutritional value of oils. DAG and FFA are the common compounds originating during lipid metabolism but TGP and ox-TGM contain altered acyl groups which would weaken the nutritional properties (Romero et al., 1995 1995 ). Therefore, it is necessary to determine the contribution of each of them to the total alteration. In this study comparing the distribution of each polar component before and after heating, a significant change was found, indicating that heating induced the breakage and reform of triglycerides. As shown in Tables 2 2 and 3 3 , the increased percentage of TGO content based on the weight of TPC from CSO was considerably higher than that of TGD. This is in accordance with a previous report, suggesting a higher tendency for formation of TGO than of TGD, although TGD was the principal component of TPC throughout the heating process (Farhoosh and Tavassoli-Kafrani, 2010 2010 ). Moreover, in contrast to other oils, the increased percentage of TGO based on the weight of TPC in CSO was the lowest after heating for 10 h (Fig. 3 3 ). This might be attributable to the low content of linoleic acid, which can cause polymerization (Su et al., 2014 2014 ). These results demonstrate that CSO has a higher ability to resist polymer formation than the other oils. In terms of thermally oxidized products, i.e., ox-TGM, its amount based on the weight of TPC in CSO and EVOO also increased continuously throughout approximately 6 h, followed by a tendency to reach a near-steady state in later successive heating (Fig. 3 3 ). This might be due to ox-TGM not only being present in primary oxidation compounds but also participating in the formation of polymerization compounds after prolonged heating (Martín-Polvillo et al., 2004 2004 ). Because ox-TGM were not final products of oxidation (Gomes et al., 2012 2012 ), further degradation can be induced by processing. However, in SBO, it was consistently decreasing, and in PO, it was significantly ( P < 0.05) increasing throughout the successive heating. This difference could be associated with the oxidation stage, which the oil undergoes. Given its pro-oxidant activity (Gomes et al., 2011 2011 ) and cytotoxicity (Wenci, 2014 2014 ), the higher contribution of ox-TGM with regard to the other polar components altered by heating also should be noted. As reported by Arroyo et al. ( 1992 1992 ), a decreasing tendency and a fluctuant tendency for the relative and absolute contents of DAG and FFA, respectively, were observed. This is in agreement with the present study. Moreover, low amounts of FFA added to purified olive oil caused a further increase in the content of ox-TGM and TGP, suggesting a pro-oxidant activity of FFA (Paradiso et al., 2010 2010 ). Likewise, DAG was also proved to have a pro-oxidant effect on SBO. Thus, there should be strict control of their content in the oil (Mistry and Min, 1988 1988 ). With regard to the safe limits of polar compounds, it was observed that the content of polar compounds of all oils was higher than 24% after heating for 10 h. Due to the toxicological characteristics of TGP (including TGD and TGO), some researchers reported that oils containing 10% TGP should be discarded (Farhoosh and Tavassoli-Kafrani, 2010 2010 ). Other researchers reported that the recommended and widely accepted limits are 12% for TGP (Weisshaar, 2014 2014 ). Further harmonization of regulations on the quality control of oxidized oil is desirable. In this study, it was observed that the content of TGP was 10.39% for CSO after heating for 8 h, whereas that of TPC was 20.41%, i.e., less than 24%. This fact further confirmed the more severe or restrictive requirement of oil quality for TGP content. Thus, it is essential to take account of both the contents of TPC and TGP as the standard when judging the useful lifetime of the oil. Conclusions 1 In summary, this study provides useful information on the formation and distribution of TPC in CSO. CSO had a much lower formation rate of TPC compared to EVOO and SBO. 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This article does not contain any studies with human or animal subjects performed by any of the authors. 10.1631/jzus.B1600173 Project supported by the National Natural Science Foundation of China (No. 31430067), the Northeast Agricultural University Innovation Foundation for Postgraduate of Heilongjiang Province (No. yjscx14058), and the National High-Tech R & D Program (863) of China (No. 2013AA102104) 2016-04-17 2016-10-18 2016-09-16 2022-01-05 Copyright © Zhejiang University and Springer-Verlag Berlin Heidelberg 2016 2016 Journal of Zhejiang University-SCIENCE B (Biomedicine and Biotechnology) 11 17