Variation Quality and Kinetic Parameter of Commercial n-3 PUFA-Rich Oil during Oxidation via Rancimat

Different biological sources of n-3 polyunsaturated fatty acids (n-3 PUFA) in mainstream commercial products include algae and fish. Lipid oxidation in n-3 PUFA-rich oil is the most important cause of its deterioration. We investigated the kinetic parameters of n-3 PUFA-rich oil during oxidation via Rancimat (at a temperature range of 70~100 °C). This was done on the basis of the Arrhenius equation, which indicates that the activation energies (Ea) for oxidative stability are 82.84–96.98 KJ/mol. The chemical substrates of different oxidative levels resulting from oxidation via Rancimat at 80 °C were evaluated. At the initiation of oxidation, the tocopherols in the oil degraded very quickly, resulting in diminished protection against further oxidation. Then, the degradation of the fatty acids with n-3 PUFA-rich oil was evident because of decreased levels of PUFA along with increased levels of saturated fatty acids (SFA). The quality deterioration from n-3 PUFA-rich oil at the various oxidative levels was analyzed chemometrically. The anisidine value (p-AV, r: 0.92) and total oxidation value (TOTOX, r: 0.91) exhibited a good linear relationship in a principal component analysis (PCA), while oxidative change and a significant quality change to the induction period (IP) were detected through an agglomerative hierarchical cluster (AHC) analysis.


Introduction
Fish oil accounts for less than 1% of all the global edible oil produced, but it is the main source of n-3 PUFA, specifically, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) [1]. These oils are becoming increasingly popular with consumers in view of the clear evidence of the health benefits of n-3 PUFA for individuals with cardiovascular conditions, including their beneficial role as antithrombotic, anti-inflammatory, and hypolipidemic fatty acids [2,3]. The n-6/n-3 ratio in a person's dietary intake is also an important consideration due to its influences on cardiovascular health and inflammation, with high intake of n-6 PUFA potentially attenuating the known profitable effects of n-3 PUFA. Due to the nutritional changes described above in the Western diet, the n-6/n-3 ratio has now increased to falls between 10 and 20. The dietary recommendations regarding n-3 PUFA intake are, thus, of increasing importance [4].
In Europe, the recommended daily intake of EPA and DHA is 450 mg per day, that is, around 3 g per week, while the WHO/FAO recommends daily consumption of 250 mg (for primary prevention) to 2 g (for secondary prevention) of EPA and DHA to prevent cardiovascular conditions [5]. In contrast, the FDA and American Dietetic Association suggest a minimum intake of close to 500 mg/day to prevent coronary health diseases [6]. In recent years, consumers have identified n-3 PUFA supplements as options for reducing the probability of illness and avoiding expensive medical bills, so that the

Kinetic Analysis
Given the uncertainty regarding the best temperature conditions for accelerated methods, the oxidative stability of n-3 PUFA-rich oils was studied using the Rancimat test with temperatures ranging from 70 • C to 100 • C. The IP for the lipid oxidation of n-3 PUFA-rich oils at different temperatures are presented in Figure 1. For use of the Rancimat test at temperatures of 70 • C, 80 • C, 90 • C, and 100 • C, the induction times were 15.4, 6.8, 3.4, and 0.92 h, respectively, for the VA; 15.9, 7.6, 3.6, and 0.97 h, respectively, for the SuF; and 4.8, 2.3, 1.01, and 0.47 h, respectively, for the SiF. The temperature used affects the degree of oxygen solubility in a given oil sample, with the oxygen solubility decreasing by almost 25% for each 10 • C rise in temperature [19]. Generally, the induction time was halved with each 10 • C increase in temperature. Previous research has shown that the PIs (at 50~80 • C) of fish oils without antioxidants ranged from 24.3~0.6 h, and that the IP of the same oils ranged from 52.3~2.4 h when 400 ppm of α-tocopherol were added [20].
Many studies have shown that the Rancimat test can be used to determine and evaluate the kinetic parameters of oils. The determination of such kinetic parameters is valuable for the purpose of distinguishing the origins of various oils, for characterizing the differences or similarities in the oils, and for predicting the oxidative stability of oils under various storage conditions [17,19]. In this study, there was semi-logarithmic relationship with Equation (1) for all the oil samples, including a linear dependency with good correlation of determination, with R 2 being 0.979 for the VA, 0.977 for the SuF, and 0.998 for the SiF (Figure 1).

Kinetic Analysis
Given the uncertainty regarding the best temperature conditions for accelerated methods, the oxidative stability of n-3 PUFA-rich oils was studied using the Rancimat test with temperatures ranging from 70 °C to 100 °C. The IP for the lipid oxidation of n-3 PUFA-rich oils at different temperatures are presented in Figure 1. For use of the Rancimat test at temperatures of 70 °C, 80 °C, 90 °C, and 100 °C, the induction times were 15.4, 6.8, 3.4, and 0.92 h, respectively, for the VA; 15.9, 7.6, 3.6, and 0.97 h, respectively, for the SuF; and 4.8, 2.3, 1.01, and 0.47 h, respectively, for the SiF. The temperature used affects the degree of oxygen solubility in a given oil sample, with the oxygen solubility decreasing by almost 25% for each 10 °C rise in temperature [19]. Generally, the induction time was halved with each 10 °C increase in temperature. Previous research has shown that the PIs (at 50~80 °C) of fish oils without antioxidants ranged from 24.3~0.6 h, and that the IP of the same oils ranged from 52.3~2.4 h when 400 ppm of α-tocopherol were added [20].
Many studies have shown that the Rancimat test can be used to determine and evaluate the kinetic parameters of oils. The determination of such kinetic parameters is valuable for the purpose of distinguishing the origins of various oils, for characterizing the differences or similarities in the oils, and for predicting the oxidative stability of oils under various storage conditions [17,19]. In this study, there was semi-logarithmic relationship with Equation (1) for all the oil samples, including a linear dependency with good correlation of determination, with R 2 being 0.979 for the VA, 0.977 for the SuF, and 0.998 for the SiF (Figure 1). The Ea value is of interest for the properties of oils, which demonstrates the delay of the initial oxidation reaction due to the bond scission that takes place to form primary oxidation products [21]. Table 1 shows Ea values of the assayed oils were 96.98 kJ/mol for the VA, 96.97 kJ/mol for the SuF, and 82.84 kJ/mol for the SiF ( Table 1). The Ea is influenced by unsaturated number of oil samples, as the Ea seems to be lower for oils with higher PUFA levels. According to the reference, the Ea values of DHA and EPA ethyl esters (of 95-97% purity), which were in the range of 52.1~62.4 kJ/mol [22]. However, this is contradicted by the example of the VA assayed in this study. Specifically, while the PUFA content of the VA was higher than those of the other tested oils, because the process of molecular distillation can increase the IP of VA, it had a higher Ea value than the other oils [23]. The Ea value is of interest for the properties of oils, which demonstrates the delay of the initial oxidation reaction due to the bond scission that takes place to form primary oxidation products [21]. Table 1 shows Ea values of the assayed oils were 96.98 kJ/mol for the VA, 96.97 kJ/mol for the SuF, and 82.84 kJ/mol for the SiF ( Table 1). The Ea is influenced by unsaturated number of oil samples, as the Ea seems to be lower for oils with higher PUFA levels. According to the reference, the Ea values of DHA and EPA ethyl esters (of 95-97% purity), which were in the range of 52.1~62.4 kJ/mol [22]. However, this is contradicted by the example of the VA assayed in this study. Specifically, while the PUFA content of the VA was higher than those of the other tested oils, because the process of molecular distillation can increase the IP of VA, it had a higher Ea value than the other oils [23].

Monitoring Substrate Variants
As shown in Table 2 The literature on this topic indicated that DHA could be efficiently synthesized in microalgae via an anaerobic pathway involving polyketide synthases [11]. The combination of fatty acids in edible oil is the most important factor in determining the oil's oxidation stability. Processing sophistication and antioxidants can improve the oxidation stability of commercial products. The levels of fatty acids change at 80 • C under the Rancimat test ( Figure 2), with the difference in the PUFA amounts of the VA, SuF, and SiF blends being −16.60%, −13.11%, and −2.76% at the 100% oxidation level, and −29.43%, −24.08%, and −16.61% at the 125% oxidation level, mainly as a result of DHA degradation. The difference in the SFA amounts of the VA, SuF, and SiF blends were 1.01%, 4.11%, and 2.29% at the 100% oxidation level, and −0.67%, 4.34%, and 12.36% at the 125% oxidation level. It is known that the thermal treatment of oils and fats generates hydroperoxide breakdown of any fatty acids with a chain shorter than ten carbon atoms, such that such breakdown can be considered a chemical indicator of the fat degradation grade [24]. This study was similar to such results, as SFA formation appeared to be correlated with PUFA loss, and with monounsaturated fatty acid (MUFA) increases and decreases. level, mainly as a result of DHA degradation. The difference in the SFA amounts of the VA, SuF, and SiF blends were 1.01%, 4.11%, and 2.29% at the 100% oxidation level, and −0.67%, 4.34%, and 12.36% at the 125% oxidation level. It is known that the thermal treatment of oils and fats generates hydroperoxide breakdown of any fatty acids with a chain shorter than ten carbon atoms, such that such breakdown can be considered a chemical indicator of the fat degradation grade [24]. This study was similar to such results, as SFA formation appeared to be correlated with PUFA loss, and with monounsaturated fatty acid (MUFA) increases and decreases. Tocopherols are the most abundant antioxidants in n-3 PUFA concentrates, because of their capacity to inhibit hydroperoxides and C-3 aldehydes [25]. In commercial products, tocopherol can be used independently or in combination with other compounds, such as ascorbic acid palmitate, lecithin, and catechin, and has shown significantly in fish oil stabilization. On the other hand, the structure conformation also affects the physical property of tocopherol, as γ-and δ-tocopherol have thermal resistance. Hydroperoxyl radical-scavenging activity occurs in the order of α-> β-> γ-> δ-tocopherol [26]. As shown in Figure 2, half of the total tocopherols of the tested n-3 PUFA-rich oils was lost at the 50% oxidation level under the Rancimat test, while 90% of the total was lost at the 100% oxidation level under the Rancimat test. The fact that the tocopherol content dropped very quickly under the Rancimat test conditions could be due mainly to the very high susceptibility of this molecule to oxidation to tocopherol quinones at high temperatures, which diminishes the protection of unsaturated fatty acids against oxidation [27].

Monitoring Oxidation Products
Lipid oxidation products have negative impacts on the flavor and odor of sensory parameters, Tocopherols are the most abundant antioxidants in n-3 PUFA concentrates, because of their capacity to inhibit hydroperoxides and C-3 aldehydes [25]. In commercial products, tocopherol can be used independently or in combination with other compounds, such as ascorbic acid palmitate, lecithin, and catechin, and has shown significantly in fish oil stabilization. On the other hand, the structure conformation also affects the physical property of tocopherol, as γand δ-tocopherol have thermal resistance. Hydroperoxyl radical-scavenging activity occurs in the order of α-> β-> γ-> δ-tocopherol [26]. As shown in Figure 2, half of the total tocopherols of the tested n-3 PUFA-rich oils was lost at the 50% oxidation level under the Rancimat test, while 90% of the total was lost at the 100% oxidation level under the Rancimat test. The fact that the tocopherol content dropped very quickly under the Rancimat test conditions could be due mainly to the very high susceptibility of this molecule to oxidation to tocopherol quinones at high temperatures, which diminishes the protection of unsaturated fatty acids against oxidation [27].

Monitoring Oxidation Products
Lipid oxidation products have negative impacts on the flavor and odor of sensory parameters, which can, in turn, have harmful effects on human health. The progress of lipid oxidation can be evaluated by the monitoring of a diverse series of primary, secondary, and tertiary oxidation products over time. The quality standards of GOED require specific levels of acid value (AV, ≤3 mg KOH/g), POV (≤5 meq/kg), p-AV (≤20), and TOTOX (≤26) throughout the stated lifetime of a product [15]. We found that the VA and SuF tested in this study met their stated label claims for GOED (Table 3). POV and AV are used in the industry's on-line quality control index. POV has a significant correlation to the off-flavour compounds created during the initial oxidation. AV represents the content of free fatty acids which are easily oxidized to hydro-peroxides [28].
The EC regulations suggest that absorbances at 234 nm (K234), 270 nm (K270), and 280 nm (K280) be used to measure oils according to their oxidated products such as ethylenic diketones, conjugated ketodienes, and the dienal formation of conjugated dienes and trienes. K234 is a primary oxidation index that has been found to be closely related to hydroperoxide content [29]. In addition, the measurement of UV absorbance at 270 and 280 nm has been used previously in analyzing edible oils. Absorption at these wavelengths is mainly due to secondary oxidation. In a spectral analysis conducted for this study (Table 3), we found that the absorbances of 10 mg/mL of VA, SuF, and SiF were 3.33, 14.79, and 7.58, respectively, at K234; 1.12, 0.66, and 1.78, respectively, at K270; and 1.08, 0.51 and 1.48, respectively, at K280. Most of them increased as the oxidation levels increased. The K234 of SuF had high values at the beginning, a finding which can be attributed to the residual oxidated products or fat-soluble compounds that were produced from the simplified refining process. In the literatures, hierarchical cluster was showed that could apply to grouping basis on quality, sensory attributes and refined level [30,31]. The AHC analysis was applied to identify clusters of samples with similar oxidation properties. Three main clusters were extracted (Figure 3). Cluster 1 contained the 0-100% oxidation levels of the VA, the 0-75% oxidation levels of the SuF, and the 0-75% oxidation levels of the SiF. Cluster 2 contained the 100% oxidation levels of the SuF and SiF. Cluster 3 contained the 125% oxidation levels of the VA, SuF, and SiF. These results showed that there are significant changes of quality in the IP of a given oil when it is subjected to the Rancimat test. These results were similar to those of a previous study reported by our team, which oxidized volatiles of n-3 PUFA [32].
With regard to the quality standard change at 80 • C under the Rancimat test, according to the PCA analysis, two dimensions were extracted and together account for approximately 91.51% of the variability from the original data ( Figure 3). In our results, we observed p-AV (r: 0.92), TOTOX (r: 0.91), K270 (r: 0.88), and POV (r: 0.87) with PAC1; and K234 (r: 0.83) and conjugated dienes (CVD, r: 0.81) with PCA2. The POV and p-AV are measures of primary oxidation and secondary oxidation, respectively. The TOTOX value gives an overall indication of the complete oxidation status of oil, and consists of the combination of the POV and the p-AV values to determine the oxidation level of the oil. Correlations between the quality parameters and spectral analysis is that K234, with AV, POV, p-AV, and TOTOX is low (R < 0.5), while K270 and K280 with acid value (AV), POV, p-AV, and TOTOX is high (R > 0.7). These results were based on measuring the formation of secondary oxidation products; for example, p-AV and K270 are available to support the AV evaluation of frying oil quality, and aldehyde molecules were a common marker for K270, K280, and p-AV [28,33].

Rancimat Test
The oxidative stability index of the n-3 PUFA-rich oil was previously determined at four different temperatures (70 °C, 80 °C, 90 °C, and 100 °C) as the induction period (hours) that was recorded using a Rancimat 743 apparatus and a 5 ± 0.05 g sample of oil with an air flow of 10 L/h. Then, oil samples were oxidized at 80 °C for periods of time that corresponded to 25%, 50%, 75%, 100%, and 125% of their respective induction periods. The oil samples that were used to determine the oxidative stability were also analyzed for their volatile oxidation compounds. The IP of the n-3 PUFA-rich oils were automatically recorded and taken as the break point of the plotted curves (the intersection point of the two extrapolated parts of the curve).

Kinetic Data Analysis
The kinetic parameters were determined according to the method previously utilized as reported in [17]. The IP of the oil samples were automatically recorded and taken as the break point of the plotted curves (the intersection point of the two extrapolated parts of the curve). A kinetic rate constant was taken as the inverse of the IP (k, h −1 ).
Temperature coefficients (T Coeff, K −1 ) were determined from the slopes of the lines generated

Rancimat Test
The oxidative stability index of the n-3 PUFA-rich oil was previously determined at four different temperatures (70 • C, 80 • C, 90 • C, and 100 • C) as the induction period (hours) that was recorded using a Rancimat 743 apparatus and a 5 ± 0.05 g sample of oil with an air flow of 10 L/h. Then, oil samples were oxidized at 80 • C for periods of time that corresponded to 25%, 50%, 75%, 100%, and 125% of their respective induction periods. The oil samples that were used to determine the oxidative stability were also analyzed for their volatile oxidation compounds. The IP of the n-3 PUFA-rich oils were automatically recorded and taken as the break point of the plotted curves (the intersection point of the two extrapolated parts of the curve).

Kinetic Data Analysis
The kinetic parameters were determined according to the method previously utilized as reported in [17]. The IP of the oil samples were automatically recorded and taken as the break point of the plotted curves (the intersection point of the two extrapolated parts of the curve). A kinetic rate constant was taken as the inverse of the IP (k, h −1 ).
Temperature coefficients (T Coeff, K −1 ) were determined from the slopes of the lines generated by regressing ln(k) vs. the absolute temperature (T, K): where a and b are the equation parameters. Activation energies (Ea, kJ/mol) and pre-exponential or frequency factors (A, h −1 ) were determined from the slopes and intercepts, respectively, of the lines generated by regressing ln(k) vs. 1/T using the Arrhenius equation: where k is the reaction rate constant or reciprocal IP (h −1 ), and R is the molar gas constant (8.3143 J/mol K).

Analysis of Tocopherol
Each oil sample (0.1 g) was diluted with 2-propanol to a volume of 10 mL and filtered through an MS nylon syringe filter with a 0.45 µm pore size directly to vials and then immediately analyzed using an HPLC system. Aliquots of 10 µL of the filtrate were injected into the injection port and analyzed with HPLC (Hitachi L-2130 pump, Hitachi, Tokyo, Japan). The remaining procedures were carried out as previously reported [34] using an HPLC attached to a detector L-2400 UV and a Hitachi L-2130 pump. An RP-18GP250 Mightysil column (l = 250 mm; i.d. = 4.6 mm; thickness = 0.32 µm; Kanto Chemical Co., Inc., Tokyo, Japan) was used for separation. The same mobile phase and elution conditions were adopted. The calibration curves were, respectively, established for tocopherol by plotting the peak area vs. each corresponding concentration, from which quantitations of the standards were achieved.

Fatty Acid Analysis
The n-3 PUFA-rich oil was analyzed for its fatty acid composition via GC/FID. The triacylglycerols were converted to methyl esters using the AOCS Official Method Ce 2-66 [35]. The methyl esters were separated using a column that was coated with DB-23 (30 m × 0.25 mm × 0.25 µm, Agilent, Palo Alto, CA, USA), and helium was used as the carrier gas at a flow rate of 1.0 mL/min. The oven temperature was initially held for 8 min at 200 • C, and then increased at 10 • C/min to 220 • C and then held there for 40 min. The FID was maintained at 270 • C, and the injector (split mode 1:40, 4 mm liner) was maintained at 250 • C. The contents of the fatty acids were determined using the normalization method, with heneicosanoic methyl ester used as an internal standard to quantitation.

Quality Analytical Determination
The POV and UV spectrophotometric were measured using the analytical methods described in European Regulation EEC 2568/91 [36]. The CDA measures the formed from PUFA during lipid oxidation according to the AOCS method Ti la-64 [35]. The p-AV measures secondary oxidation products, such as 2-alkenal and 2,4-alkadienal. The p-AV of each sample was determined according to the AOCS method Cd 18-90 [35]. The AV was determined using a titration with 0.1 N potassium hydroxide alcoholic solution.

Statistical Analysis
The data reported were obtained from triplicate measurements of each sample and were expressed as means. The data were subjected to an AHC analysis with squared Euclidean distances. Subsequently, the data were analyzed using PCA combined with VARIMAX rotation. For the AHC and PCA analysis, XLSTAT software (version 2010.2.01, Addinsoft Deutschland, Andernach, Germany) was used.

Conclusions
In this work, the Rancimat test was applied to the analysis of the oxidation properties of n-3 PUFA-rich oil. The results of the present study indicated: (1) the degree of unsaturation in fatty acids is not the only parameter for assessing oil quality, which is mainly influenced by commercialization. The oxidation stability of n-3 PUFA-rich oils is improved by commercialization, especially with respect to antioxidant protection; and (2) chemometric applications have also clarified the nature of the differences in correlation among oxidative and chemical parameters. The IP of primary and secondary oxidation product formations represented the quality changes as detected via AHC analysis. Not all quality parameters increased linearly through PCA analysis, which can allow for the selection of a proper and adequate method for a particular application. It is recommended for p-AV, TOTOX, K270, and POV to be used when carrying out quality control for n-3 PUFA-rich oils.