Cold-pressed linseed as well as refined and crude palm oils without any added antioxidants were supplied by Nourhan-Moohaya Company (Shiraz, Fars province, Iran). Acetic acid, chloroform, potassium iodide, sodium thiosulfate (Na₂S₂O₃, CAS No. 7772-98-7), iso-octane (2,2,4-trimethylpentane) (CAS No. 51685-57-5), and p-anisidine reagent (99%, CAS No. 104-94-9) were purchased from Sigma-Aldrich (St. Louis, MO). In all experiments, double-distilled water was used to avoid any unwanted reaction of impurities with oil samples. All solvents used in Gas chromatography (GC) were GC-grade solvents.

The fatty acid profile of linseed oil as well as refined and crude palm oil was accurately determined using gas chromatography (SP-3420A, Beijing Beifen-Ruili Analytical Instrument, Beijing, China) as previously described (Golmakani et al., 2020a). The PV of linseed oil as well as refined and crude palm oil was determined according to Keramat et al. (2023b) with some modifications. The oil sample was accurately weighed (5 g) and added to acetic acid and chloroform (9.8 mL, 3:2 v/v) and mixed well to prepare a uniform solution. Saturated potassium iodide (0.5 mL) was then added to the mixture dropwise for 10 min followed by the addition of double-distilled water (30 mL). The resulting solution was titrated using sodium thiosulfate (Na₂S₂O₃) (0.1 N) until the disappearance of the yellow color. A starch solution (0.5 mL, 1 %) was added to the solution as an indicator. Again, the solution was titrated while stirring using a thiosulfate solution (0.1 N) until the disappearance of the blue color. The blank solution contained all the reagents except oil. PV was calculated using Equation 1 and the results were reported as milliequivalent oxygen per Kg of oil (meq O₂/Kg oil):

where N is the normality of the thiosulfate solution (0.1 N) and W is the weight of the sample.

The AV of the oil samples was determined using the procedure described by Golmakani et al. (2018) with some modifications. The oil sample (4 g) was accurately weighed and diluted to a volume of 25 mL using iso-octane. The absorbance was then recorded at 350 nm for the solution against iso-octane as the blank. After that, 5 mL of the solution were transferred to a glass tube, and 1 mL of anisidine reagent was added to the tube. The second tube containing 10 mL of iso-octane and 1 mL of anisidine reagent was immediately prepared. After 10 min, the absorbance was recorded at 350 nm against the second tube as the blank. The AV of the oil samples was calculated according to Equation 2 and the results were reported as milligrams of p-anisidine per kilogram of oil (mg/kg).

where A1 and A2 are the absorbance of solutions before and after the reaction with anisidine reagent, respectively, and m is the mass of the sample in grams (4 g).

The total oxidation value (TOTOX) was also evaluated using Equation (3) (Amini et al., 2023).

In this study, refined and crude palm oils were added to the linseed oil to evaluate the effects of additional oil on the oxidation and quality of linseed oil. The chosen proportions of oils were 33 and 66% for both crude and refined palm oils. Each oil was added to the linseed oil according to the predetermined ratios followed by thermally stirring (40 °C, 30 min, 150 rpm) to achieve a uniform mixture of oils. Finally, the samples containing 33 % and 66 % crude palm oil were named 33C and 66C blends, respectively, and the samples containing 33 and 66% refined palm oils were named 33R and 33R, respectively. The oxidation stability of the oil mixtures was then determined using Rancimat.

Using a pre-calibrated Rancimat (Metrohm, model 743, Switzerland), oxidative stability was determined according to Jokar et al. (2022). Briefly, the oil blend was accurately weighed (3 g) and transferred to the reaction vessel. The water bath of the Rancimat apparatus was filled with distilled water and the temperature set to 80, 100, and 120 °C, while the airflow rate was set to 20 L/h. The rancimat will heat the sample to the desired temperature and pass a stream of air through it. After reaching the desired temperature, samples were placed in the heater channel. As the oil blend oxidizes, it releases volatile organic acids which are carried by the air stream into the reaction vessel, where they interact with the deionized water to form conductivity changes. Finally, the induction period (IP) was recorded after the predetermined time.

The data required for each parameter were obtained using a pre-calibrated Rancimat. These parameters include the temperature coefficient (Tc), acceleration factor (Q10), Arrhenius equation parameters (frequency factor “A” and activation energy Ea), and activated complex theory parameters (entropy ΔS and enthalpy ΔH). To determine the temperature coefficient (Tc, K^-1), acceleration factor (Q10), and shelf-life, the natural logarithm of IP (Ln (IP)) was plotted against the temperature (K). To find the shelf-life of oil samples at 25 °C. Corresponding plots were extrapolated according to Equation 4 (Golmakani et al., 2020b).

where a is the slope of the plot indicating Tc.

The Q10 factor can also be determined as Equation 5 (Golmakani et al., 2020b).

The rate constant of the oxidation reaction (k, h^-1) was then calculated according to Equation 6 (Arrhenius equation). The intercept of the equation refers to the activation energy (Ea, Kj·mol^-1), and the slope indicates the frequency factor (A, h^-1) (Golmakani et al., 2020b).

where R is the molar gas constant (8.314 j·mol^-1·K^-1).

According to the activated complex theory, the enthalpy (ΔH) and entropy (ΔS) of the oxidation reaction, which refer to slope and intercept, respectively, were calculated (Equation 7) (Toorani et al., 2019).

where R is the molar gas constant (8.314 j·mol^-1·K^-1), Kb is Boltzmann constant (1.38 × 10-23 j·K^-1), and Planck’s constant (6.63 × 10-94 js).

Finally, Gibbs free energy (ΔG) was then determined according to Equation 8 (Toorani et al., 2019).

It should be noted that T is temperature in Kelvin.

The experiments were conducted in triplicate and the results were reported as mean values. The difference between means was evaluated using one-way ANOVA and Duncan’s multiple-range test using SAS statistical software (SAS 9.4, SAS Institute, Cary, NC) with a significance level of 0.05. Plots were generated using Microsoft Excel Spreadsheet Software (Microsoft Office, 2016, Microsoft Corporation, Redmond, Washington).

The fatty acid profiles of linseed oil, crude palm oil, and refined palm oil are summarized in Table 1. Linseed oil is characterized by a high content of polyunsaturated fatty acids (PUFAs), with α-linolenic acid (an ω3 fatty acid) being the predominant fatty acid at 55.25 %. Oleic acid (21.08 %) and linoleic acid (15.15 %) were the second- and third-most abundant fatty acids, respectively. Overall, PUFAs accounted for approximately 91.5 % of the total fatty acids in linseed oil, making it highly susceptible to oxidation. Saturated fatty acids, such as palmitic acid and stearic acid, constituted only about 12 % of the total fatty acids in linseed oil. In contrast, palm oil (both crude and refined) exhibited a significantly different fatty acid profile. Palmitic acid, a saturated fatty acid, was the dominant component, accounting for 44 and 46.5 % of the total fatty acids in refined and crude palm oil, respectively. Oleic acid, a monounsaturated fatty acid, was the second-most abundant fatty acid in palm oil, representing 37 % and 38.88 % in refined and crude palm oil, respectively. Notably, palm oil contained no stearic acid or α-linolenic acid, which are present in linseed oil. Similarly, Edo et al. (2022) found that palmitic acid and oleic acid are the major fatty acids in palm oil while linolenic acid is a micro-constituent.

The blending of linseed oil with palm oil resulted in intermediate fatty acid profiles. For instance, the 33 % palm oil blends (33R and 33C) contained 36.83 and 36.8 % α-linolenic acid, respectively, which is significantly lower than the 55.25 % found in pure linseed oil. These blends also showed higher levels of palmitic acid (19.48 % and 20.31 %) and oleic acid (26.39 % and 25.83 %) compared to pure linseed oil. On the other hand, the 66 % palm oil blends (66R and 66C) exhibited even lower levels of α-linolenic acid (18.42 % and 17.89 %) and higher levels of palmitic acid (31.74 % and 33.41%) and oleic acid (31.69 % and 33.19%).

The comparison of these results highlights the significant impact of blending on the fatty acid composition of the oil mixtures. By blending linseed oil with palm oil, the proportion of PUFAs (particularly α-linolenic acid) reduced, while the proportion of saturated and monounsaturated fatty acids (palmitic acid and oleic acid) increased. This shift in fatty acid composition is expected to enhance the oxidative stability of the blends, as saturated and monounsaturated fatty acids are less prone to oxidation than PUFAs. These findings align with the observed improvements in oxidative stability, as discussed in subsequent sections, and underscore the potential of palm oil blends to extend the shelf-life of linseed oil.

PV is a measure of oil freshness and quality, which indicates the hydroperoxide content in oil, and reveals the primary products of oxidation at the early stages of the reaction. Oxidized oils can also have reduced nutritional value and may have negative health effects. It is noteworthy that PV is not the only parameter used to assess the quality of oils. Other parameters such as fatty acid profile can also provide valuable information about the quality and composition of oils (Chen and Liu, 2020). The maximum standard level of PV is reported to be 15 milliequivalent of active oxygen/kg oil for virgin and cold-pressed oils and 10 milliequivalent of active oxygen/kg oil for other types of oils and fats (Codex Alimentarius Commission 1999). As shown in Table 2, all samples exhibited PVs lower than the critical level. However, refined palm oil exhibited the lowest PV, followed by crude palm oil, and linseed (p < 0.05). The AV is also a measure of the oxidative stability of edible oils and determines the presence and extent of oxidative deterioration in oils and fats. The AV is based on the reaction of p-anisidine with the secondary oxidation products of oils and fats which are formed during the oxidation process (Golmakani et al., 2018). According to the literature, there is no specific maximum allowed AV for edible oils. Khakbaz Heshmati et al. (2022) reported that p-anisidine values below 10 (mg.kg^-1) are regarded as good-quality grades. The maximum AV was recorded for linseed oil and the minimum level was detected for refined palm oil. However, it is crucial to consider TOTOX as a determining factor for the quality of edible oils due to the fact that the TOTOX value is based on both PV and literature (Özdemir et al., 2021). It is reported that edible oils with TOTOX values higher than 10 are not good-quality grade oils (Özdemir et al., 2021). According to Table 2, the only unacceptable sample was linseed oil with a TOTOX value of 18.68. The reason why linseed oil is so susceptible to oxidation is its high content of PUFAs, specifically α-linolenic acid, as the gas chromatography results revealed. PUFAs contain multiple double bonds in their chemical structure, which makes them more vulnerable to oxidation than saturated or monounsaturated fatty acids.

In summary, the choice of PV, AV, and TOTOX as study parameters is justified by their ability to provide a comprehensive evaluation of oil quality and oxidative stability. The results of this study are consistent with previous findings, highlighting the role of fatty acid composition and refining processes in determining oxidative stability. These findings underscore the potential of palm oil blends, particularly those containing refined palm oil, to improve the shelf-life and quality of linseed oil-based products.

The induction period (IP), or oxidative stability index (OSI), is a term used in chemistry to describe the initial stage of a chemical reaction, during which the reactants are being transformed into the products. This period is characterized by a slow rate of reaction, which gradually increases until it reaches the maximum rate with a steady state. The induction period is a crucial stage of a chemical reaction, as it determines the rate and extent of the reaction. Therefore, the higher IP of a specific oil indicates the higher stability of the oil (Souza et al., 2021).

As shown in Table 3, at 80 °C, refined palm oil with an IP of 54.17 showed the highest oxidative stability among other oil samples. In contrast, linseed oil with an IP of 17.34 showed the lowest oxidative stability. Comparatively, the oxidative stability of oil samples at 80 °C was as follows: Refined palm oil > 66R blend > crude palm oil > 33R blend > 66C blend > linseed oil > 33C blend. Since the highest stability was reported for refined palm oil, the blends containing a higher content of refined palm oil, which were the 66R and the 33R blends, showed higher stability among the oil blends. The higher stability of the 33R blend compared to the 33C blend, and the 66C blend compared to the 33C blend, can be attributed to the differences in their fatty acid profiles. All blends containing 66% palm oil had substantially lower α-linolenic acid and higher palmitic acid in comparison with their 33% counterparts. At 100 ºC and 120 °C, the order of oxidative stability of oil samples was similar to that of 80 °C, except for the case of the 33C blend. The oxidative stability of the 33C blend was not significantly different from that of the linseed oil. This implies that at higher temperatures (above 100 °C), there is no difference between the oxidative stability of linseed oil and the 33C blend, and the blending approach with this proportion cannot be beneficial in terms of oxidative stability.

It is acknowledged that the presence of PUFAs in linseed oil is the main reason for its high susceptibility to oxidation. The addition of other resistant oils and making oil blends is a viable approach to exploit such beneficial oil in the human diet with acceptable stability against oxidation. Golmakani et al. (2020b) reported that blending linseed oils with corn, canola, sesame, and bitter almond oils positively improves the oxidative stability of linseed oil. The addition of palm oil to the linseed oil endows higher saturated and monounsaturated fatty acids as well as higher antioxidant compounds including β-carotene and vitamin E, which make the blend resistant to oxidation (Damanik and Murkovic, 2018). However, the results revealed that there was a significant difference between the oxidative stability of refined palm oil and crude palm oil as well as their blends. Palm oil is processed in a refinery where it undergoes various treatments to improve its quality (de Almeida et al., 2019). Degumming, alkali refining, bleaching, and deodorization (removing free fatty acids and impurities) are included in the process of refining, even though palm oil requires a lower extent of refining in comparison with other vegetable oils. In addition, palm oil is known as the richest vegetable oil in tocotrienols. On the other hand, it has been reported that the initial PV of crude palm oil is considerably removed and the contents of tocopherol and tocotrienol are not significantly decreased after the refining process (Damanik and Murkovic, 2018). Therefore, it is not unexpected that the absence of impurities and free fatty acids can be substantially effective on the oxidative stability of refined palm oil and the resultant linseed oil blends.

A key novelty of this study is the comparison between crude and refined palm oil in blends with linseed oil, which has not been extensively explored in previous research. The refining process, which includes degumming, alkali refining, bleaching, and deodorization, removes impurities and free fatty acids, while preserving beneficial compounds like tocopherols and tocotrienols. This study demonstrates that refined palm oil significantly outperforms crude palm oil in enhancing the oxidative stability of linseed oil blends, underscoring the critical role of refining in achieving superior stability.

The practical significance of this research lies in its potential applications in the food industry, where oxidative stability is crucial for extending the shelf-life of edible oils and their derivatives, such as margarines, spreads, and salad dressings. The findings provide a cost-effective and sustainable solution for utilizing linseed oil in food products without compromising stability or nutritional quality. By optimizing blending ratios and emphasizing the benefits of refined palm oil, this study offers a novel approach to improving the oxidative stability of PUFA-rich oils, addressing a key challenge in the food industry.