Thermally induced isomerization of linoleic acid and α‐linolenic acid in Rosa roxburghii Tratt seed oil

Abstract Rosa roxburghii seed oil is obtained from the seeds left following pressing of the juice from R. roxburghii fruit. The total oil content of R. roxburghii seed was around 9.30%. The fatty acid profile of the oil was determined by gas chromatography (GC). Among the 11 fatty acids identified in the oil, seven were unsaturated fatty acids (UFAs) (92.6%); four were saturated fatty acids (SFAs) (7.17%). Then, the kinetics of formation of trans‐fatty acids was studied by GC. Heat treatment of R. roxburghii seed oil showed an increase in the relative percentage of linoleic acid and α‐linolenic acid isomers with increasing temperature and time. The formation of linoleic acid and α‐linolenic acid isomers followed a zero‐order reaction. The presence of O2 enhanced the isomerization of these UFAs. In addition, the rate constants and activation energies for the geometrical isomerization of UFAs in R. roxburghii seed oil were presented. Overall, R. roxburghii seed oil contains high UFA contents. Heating temperature and duration and the presence of O2 should be considered to reduce the formation of trans‐fatty acids during thermal treatment of R. roxburghii seed oil.

Thus, R. roxburghii seed oil was of high quality in terms of its high UFAs contents.
It is well known that heat treatment such as frying can cause profound changes in the chemical composition of oils (Cui et al., 2017;Li et al., 2012). UFAs can be isomerize into more stable trans-fatty acids (TFAs) by thermal treatment (Christy & Arachchi, 2016;Christy et al., 2009), which is shown to accumulate in liver, heart, and many other organs (Ganguly et al., 2016), causing increased risks of coronary heart disease and type-2 diabetes (Ascherio et al., 1999;Papantoniou et al., 2010). Kinetics of geometrical isomerization of UFAs has been reported by several research groups. The geometrical isomerization of linoleic and α-linolenic acid in heated soybean oil followed a first-order reaction (Gerčar & Šmidovnik, 2002). Heat treatment of sunflower oil showed an increase in the relative percentage of trans-linoleic acid with increasing temperature and time and the formation of trans-linoleic acid isomers followed zero-order kinetics (Mateos et al., 2010). Studies on the thermally induced isomerization kinetics of 9c,12c linoleic acid in triacylglycerol showed that the consumption of trilinolein followed a second-order reaction and the formation reactions of cis,trans, trans,cis and trans,trans isomers followed zero-order kinetics and dependent on both heating temperature and total heating time (Guo et al., 2016).
Rosa roxburghii seed oil contains high amount of unsaturated fatty acids; however, till now, no scientific study on the kinetics of isomerization of linoleic and α-linolenic acids during heat treatment has been published. The objective of the present study is to (a) determine the composition of fatty acids in R. roxburghii seed oil and (b) investigate the kinetics of geometrical isomerization of unsaturated fatty acids in R. roxburghii seed oil in the presence of air (O 2 ) or in nitrogen (N 2 ) atmosphere during heat treatment using GC. The kinetic parameters determined for the thermally induced cis-trans isomerization may provide insight into methods for controlling isomerization reactions and manipulating isomeric yield ratios.

| Extraction of R. roxburghii seed oil
Rosa roxburghii seeds harvested in autumn 2019 were provided by Guizhou Lvyuan Food Co. Ltd. One hundred gram of pulverized R. roxburghii seeds was ultrasonic extracted in hexane with a solidliquid ratio of 1:3. The extraction temperature was 45°C, ultrasonic power was 200 W, and extraction time was 30 min. The extraction procedure was repeated for three times. After that, the extraction solutions were combined and evaporated to a constant weight using a rotary evaporator. The organic solvent residue was further removed via nitrogen stream. The extracted oil was then decolorized with kaolin prior to GC analysis and heat treatment.

| Thermal processing
Each 4 ml microglass ampoule bottle was filled with 100 mg of R. roxburghii seed oil. The ampoules with air or nitrogen (N 2 , 5 ml/min, 5 min) in the head space were sealed with a propane-oxygen flame.
The sealed oil samples were then heated in a silicone oil bath at 180, 200, 220, 230, or 240°C (±2°C) for regular time intervals. After heat treatment, the samples were cooled to room temperature before further analysis.

| Preparation of fatty acid methyl esters
Fatty acid methyl esters were prepared according to a previous study (Guo et al., 2015). Briefly, 100 mg of the R. roxburghii seed oil sample and 0.05 ml of 2 mol/L methanolic KOH were mixed thoroughly using a vortex. Then, the vials were centrifuged at 4,000 × g for 10 min. The supernatants were dried with anhydrous magnesium sulfate. After that, 20 μl of the dried supernatant was diluted to 1 ml with isooctane. One microliter of the supernatant was injected into a gas chromatograph (GC) for fatty acid analysis.

| GC-FID analysis
GC-FID was applied to quantify fatty acid in soybean oil during heat treatment (Guo et al., 2015;Liu et al., 2021). GC (TRACE 3000, Thermo Fisher Inc.) was equipped with an ionic liquid SLB-IL111 column (100 m × 0.25 mm × 0.2 mm), and a flame ionization detector (FID, Thermo Fisher Inc.). Helium (99.999%) was used as the carrier gas with a flow rate of 1.0 ml/min. Column temperature program was as follows: 60°C (5 min), 60-160°C at 25°C/min, 160°C (5 min), 160-225°C at 1.5°C/min, and 225°C (15 min). The temperature for the injector was 230°C. The injection volume was 1 μl with a split ratio of 1:10. Fatty acids were identified by comparison of retention time with those of commercial standards. The quantitation of the fatty acids was performed using external standard method. The content of fatty acid was expressed as g/100 g of R. roxburghii seed oil.

| Statistical analysis
Results were expressed as mean ± standard deviation of at least three independent experiments. Statistical analysis was carried out using ANOVA by Prism 5.0, GraphPad Software. The significance of differences was calculated using the Student's paired t test. pvalue < .05 was considered statistically significant.

| Fatty acid composition
The oil in R. roxburghii seed was ultrasonic extracted with hexane.
The total oil content of R. roxburghii seed was around 9.30%, which is lower compared with seeds of fruit such as chakeberry (19.3%) and blackcurrant (22.0%), and similar to those of grape seed (around 10%) and rosehip seed ( The fatty acid profile of R. roxburghii seed oil was determined by GC ( Figure 1). Fatty acid compositions of the R. roxburghii seed were illustrated in Table 1. Among the 11 fatty acids identified in R. roxburghii seed oil, 7 were unsaturated fatty acids (92.6%); four were saturated fatty acids (7.17%). The most abundant fatty acids were three unsaturated fatty acids including linoleic acid (52.0%), αlinolenic acid (20.1%), and oleic acid (20.1%). These values were close to those obtained by previous study (linoleic acid: 41.68%-74.34%; α-linolenic acid: 25.44%-41.11%; oleic acid: 12.74%-17.68%) (Shi et al., 2013;Wang & Chen, 1994;Zhang et al., 2007). Moreover, the unsaturated fatty acid content of R. roxburghii seed oil was more than rapeseed oil, peanut oil, soybean oil, and especially pig oil and safflower seed oil (Ma et al., 2011). In addition, the unsaturated fatty acid content of R. roxburghii seed oil was similar with olive oil which was characterized by high unsaturated fatty acid content (around 90%). Rosa roxburghii seed oil contains higher PUFA (72.12% vs. 6.0%-15.9%) but lower MUFA (20.47% vs. 64.4%-81.0%) compared with olive oil (Maggio et al., 2009). It is worth mentioning that the high amount of unsaturated fatty acids especially linoleic and αlinolenic acid makes R. roxburghii seed oil susceptible to oxidation.
Yet, these unsaturated fatty acids were of high nutritional values and F I G U R E 1 Gas chromatography (GC) spectrum of FAME composition of R. roxburghii seed oil TA B L E 1 Fatty acid composition of R. roxburghii seed oil  (Oomah et al., 2000).

| Analysis of trans-fatty acids in heated R. roxburghii seed oil by GC
The gas chromatography spectrum of linoleic acid isomers or αlinolenic acid isomers was presented in Figure 2a,b, respectively.
The isomerization of double bonds requires rotational energy (Tsuzuki, 2010). A range of trans-linoleic and trans-linolenic acids were observed in thermal-treated R. roxburghii seed oil in the presence of air. The amount of each trans-fatty acid (TFA) increased with increasing temperature and time ( Figure 2). The cis-trans isomerization of C18:2-9c,12c induced the formation of C18:2-9c,12t, whereas the trans isomers of C18:3-9c,12c,15c was C18:3-9c,12c,15t in R. roxburghii seed oil heated at a relative low temperature (180°C) for 4 hr (Figure 2c). The content of C18:3-9c,12c,15t was higher than C18:2-9c,12t. These results suggested that unsaturated fatty acids with more double bond are more likely undergo thermal isomerization and double bond closest to the methyl end is more likely to be thermally isomerized. Moreover, isomers with two trans double bonds such as C18:2-9t,12t, C18:3-9t,12c,15t, C18:3-9t,12t,15c, and C18:3-9t,12c,15t were formed in R. roxburghii seed oil heated at 220 or 240°C for 48 hr but not in R. roxburghii seed oil heated at 180°C for 4 or 48 hr, suggesting that the formation of isomers with two trans double bonds requires more energy than the formation of isomers with one trans double bond (Li et al., 2012).

| Kinetics for the formation of C18:2-9c,12t, C18:2-9t,12c, and C18:2-9t,12t isomers
The concentration evolution plots for C18:2-9c,12t, C18:2-9t,12c, and C18:2-9t,12t during heating of R. roxburghii seed oil were shown in Figure 3a-c (oil heated in the presence of air) and Figure 4a-c (oil heated in N 2 atmosphere). The relationships show that the formation of C18:2-9c,12t, C18:2-9t,12c, and C18:2-9t,12t followed zero-order kinetics in all tested heating temperature in the presence of air or in the presence of N 2 , which is in agreement with the reports of Guo (Guo et al., 2016). The amount of isomer formation can be calculated assisted the isomerization of linoleic acid. The E a for the formation of the 9c,12t isomer was much lower than that of the 9t,12c isomer.
9t,12t isomer has higher E a than 9t,12c isomer in the same conditions, further confirming that double bond closest to the methyl end is much easier to be thermally isomerized and the formation of isomers with two trans double bonds requires more energy than the formation of isomers with one trans double bond (Li et al., 2012). In agreement with our findings, C18:2-9t,12t isomer was reported to be formed from C18:2-9c,12t and C18:2-9t,12c in a previous study (Jiang et al., 2017). In addition, the E a values for the formation of the isomers in presence of N 2 atmosphere were much higher than that in the presence of air in the same heating temperature, further confirming that O 2 enhanced the isomerization of linoleic acid. C18:3-9c,12c,15t,  C18:3-9t,12c,15c, C18:3-9c,12t,15c, and C18:3-9c,12t,15t isomers
The E a values for the formation of the isomers in presence of N 2 atmosphere were much higher than that in the presence of air in the same heating temperature, suggesting that O 2 promoted the isomerization of linoleic acid. The E a value for the formation of C18:3-9c,12t,15c was much higher than the E a value for the formation of C18:3-9c,12c,15t and 9t,12c,15c in samples in the presence of air or in the presence of N 2 , indicating that double bound at C12 position was the most difficult one to be thermally isomerized forming single trans-fatty acids. In addition, compared Tables 2 and 3 we can find that geometrical isomerization of α-linolenic acid requires less energy than linoleic acid, which could be one explanation for the formation of trans-linolenic acid at a relative lower temperature than trans-linoleic acid as mentioned in Section 3.2.

| CON CLUS ION
Rosa roxburghii seed, which is generally a waste material, is a good natural resource of high-quality edible oil due to the fact that R. roxburghii seed oil contains high amount of UFAs (92.83%). Heat treat- Overall, R. roxburghii seed oil could be used as high-quality edible oil because of its high UFAs contents. Heating temperature and duration and the presence of O 2 should be considered to reduce the formation of trans-fatty acids during thermal treatment R. roxburghii seed oil.

CO N FLI C T O F I NTE R E S T
The authors confirm that they have no conflicts of interest with respect to the work described in this manuscript.