Oxidative stability and compositional characteristics of oil from microwave irradiated black cumin seed under accelerated oxidation condition

2.1. Materials

Black cumin seed (2.5 kg) was bought from Rajshahi, Bangladesh. The seed was cleaned, dried in the shade at ambient temperature, and preserved at 4 °C in a refrigerator. The chemicals and solvents used were of analytical grade. Thioburbituric acid was product of HiMedia Laboratories (Mumbai, India). Acetic acid and standards were bought from Sigma-Aldrich Co. (St. Louis, MO, USA). All other chemicals or reagents were purchased from Merck (Darmstadt, Germany or Mumbai, India) unless otherwise stated.

2.2. Pre-treatment and oil extraction

The cumin seed samples were spread on the Pyrex petri dishes (12 cm diameter) set on a turntable plate of the microwave oven (MS3042G, LG, China). After covering the dishes, the samples were then microwaved at a frequency 2450 MHz (capable of producing 580 W power) for 1, 2, and 3 min depending on trial results. After pre-treatment, the samples were allowed to cool to 25 °C and thoroughly mixed. Oils from untreated and treated whole black cumin seeds were obtained by pressing using a locally-made mechanical pressing machine at room temperature (27 °C). The temperature of the outflowing oil was around 40 °C. After filtering to remove particles, the oils were weighed and stored in capped glass bottles at a temperature below ̶ 15 °C for analysis.

2.3. Accelerated oxidation of oil samples

The untreated or treated BCOs (75 g) were placed in 100-mL glass beakers, and beakers were put in an incubator at 62 °C for accelerating oil oxidation. The oils were withdrawn at regular intervals of 0, 7, 14, and 21 days.

2.4. Oxidative indices

The American Oil Chemists’ Society (AOCS, 1987AOCS. 1987. Official methods and recommended practices of the American Oil Chemists’ Society. 4th edn. AOCS press, Champaign.) methods were applied to estimate free fatty acids (FFA) (method Ca 5a-40), peroxide value (PV) (method Cd 8-53), and thiobarbituric acid value (TBA) (method Cd 19-90). Following the PORIM (PORIM, 1995) test methods, specific extinctions (method p2.15) at 233 and 269 nm (E^1% ₂₃₃ and E^1% ₂₆₉) and p-anisidine value (p-AV) (method p2.4) of the oils were estimated using a spectrophotometer (T 60, PG Instruments, Leicestershire, UK). The oxidative value was determined by Holm’s equation: TOTOX = 2PV + p-AV (Wai et al., 2009Wai WT, Saad B, Lim BP. 2009. Determination of TOTOX value in palm olein using a FI- potentiometric analyzer. Food Chem. 113, 285-290. https://doi.org/10.1016/j.foodchem.2008.06.082 ).

2.5. Color development

The absorbance of a 5.0% (w/v) oil solution in chloroform was computed at 420 nm with a spectrophotometer (T 60U, PG Instruments, Leicestershire, UK) indicating an index of color formation, (Yoshida et al., 1999Yoshida H, Takagi S, Mitsuhashi S. 1999. Tocopherol distribution and oxidative stability of oils prepared from the hypocotyl of soybeans roasted in a microwave oven. J. Am. Oil Chem. Soc. 76, 915-920. https://doi.org/10.1007/s11746-999-0106-3 ).

2.6. Fatty acid composition (FAC)

The FAC was estimated after the preparation of methyl esters using the PORIM (PORIM, 1995PORIM. 1995. PORIM test methods. Palm Oil Research Institute of Malaysia, Malaysia.) test method p3.4. A gas chromatography (Clarus 590 GC PerkinElmer, USA) equipped with a flame ionization detector was used to determine the FAC oil samples. Helium gas was passed (2 mL/min) as carrier gas. Fatty acids were separated on a 0.25 mm i.d. × 30 m × 0.25 μm capillary column (Elite-FFAP). Analysis was carried out at an initial oven temperature 120 °C which was raised to 240 °C at 4 °C/min. The injector and detector temperatures were controlled at 120 °C and 250 °C, respectively. The peaks were identified by comparison with the standards (methyl arachidate, methyl behenate, methyl decanoate, methyl cis-13-docosenoate, methyl dodecanoate, methyl linoleate, methyl linolenate, methyl myristate, methyl octanoate, methyl oleate, methyl palmitate, methyl palmitoleate, methyl stearate, methyl tetracosanoate) (Sigma-Aldrich Co., St. Louis, MO, USA).

2.7. Triacylglycerol (TAG) molecular compounds

The concentrations in molecular TAG compounds present in seed oils were determined by a HPLC system (Agilent 1260 Infinity, USA) equipped with a column (50 mm x 4.6 mm i.d x 2.7 μm) packed with Poroshell 120 EC-C18 (Agilent, USA) and evaporative Light Scattering Detector (ELSD). The solvent system, acetone/acetonitrile (65:35, v/v) was used as a mobile phase at a flow rate of 1 mL/min. The concentrations in TAG species were determined by using standards (POL, OOL, POO, OLL, PLL, MOL, OOO, PLP, POP, SOO and POS, where P- palmitic, M- myristic, O- oleic, L- linoleic) (Sigma-Aldrich Co., St. Louis, MO, USA).

2.8. FT-IR spectroscopy

The FTIR spectra of oils were measured by a Fourier Transform Spectroscopy (IRAffinity- 1S, Shimadzu Corporation, Kyoto, Japan) furnished with a high-sensitivity pyroelectric detector (deuterated L-alanine doped triglycine sulphate). Samples were applied to a sodium chloride cell and periodic scans (15 scans and 4 cm⁻¹ resolution) were performed in the spectral range of 850-4000 cm^-1. The spectra were computed as absorbance values at each data point.

2.9. Statistical analysis

The data were declared as the mean and standard deviation (SD) of triplet determinations. One way analysis of variance (ANOVA) was performed, and mean values were compared at p < 0.05 significance level by Duncan’s multiple range test using IBM SPSS 22 statistics.

3.1. Oxidative indices

The concentrations of FFA in the oil samples increased significantly (p < 0.05) as a result of increasing accelerated storage time (Figure 1a). This hydrolytic degradation was noted to be biggest in the untreated sample (6.62%) with the lowest in the 3 min microwaved sample (4.12%) at the end of 21 days of storage. It also indicated that the higher initial FFA content in the sample microwaved for 3 min, did not affect the hydrolytic degradation of BCO during storage at 62 °C. Microwave pre-treatment had a significant impact on ultraviolet absorptions at 233 (E^1% ₂₃₃) and 269 nm (E^1% ₂₆₉) in the oils (Figures 1b and 1c). The absorptions were significantly (p < 0.05) enhanced for all the oils throughout the storage treatments. At the end of 21 days of storage, the concentrations in conjugated dienes and trienes were the greatest in the untreated samples, with the lowest found for the 3-min microwaved samples. The lower values for absorptions indicate better storage stability of treated samples compared to untreated samples. In addition, the concentration of conjugated diene was higher than triene in all oils expressed by the biggest value for E^1% ₂₃₃ at 233 nm. Ali et al. (2017a)Ali MA, Nargis A, Othman NH, Noor AF, Sadik G, Hossen J. 2017a. Oxidation stability and compositional characteristics of oils from microwave roasted pumpkin seeds during thermal oxidation. Int. J. Food Prop. 20, 2569-2580. https://doi.org/10.1080/10942912.2016.1244544 also followed a similar trend for pumpkin seed oil. Pre-treatment and storage conditions employed in this research affected the formation of color in the oils (Figure 1d). Browning substances were generated during microwave irradiation and storage treatment which resulted in a significant (p < 0.05) increment in absorbance at 420 nm. The absorbance values limited from 0.41 to 0.50 at 420 nm, were enhanced markedly (p < 0.05) during incubation and these increments were detected to be higher in the untreated oils. The formation of Maillard reaction products at the storage temperature might be responsible for the color increments in the oils during storage. However, longer seed pre-treatment time had a bigger impact on the further reduction in oxidative stability. Thus, the present findings support the earlier work done by Ali et al. (2017b)Ali MA, Islam MA, Othman NH, Noor AM. 2017b. Effect of heating on oxidation stability and fatty acid composition of microwave roasted groundnut seed oil. J. Food Sci. Technol. 54, 4335-4343. https://doi.org/10.1007/s13197-017-2904-1 , where they indicated that oil color increased with increasing seed pre-treatment time or heating time of a groundnut oil sample.

Figure 2a indicates the formation of unstable oxidative substances determined by peroxide value (PV) which was found to be faster in the raw sample than the treated samples during the storage oxidation. Oils from pre-treated samples (6.88 meq O₂/kg) had the lowest concentrationa in hydroperoxide than that of the raw one (11.20 meq O₂/kg) at the end of 21 days incubation. Ali et al. (2017b)Ali MA, Islam MA, Othman NH, Noor AM. 2017b. Effect of heating on oxidation stability and fatty acid composition of microwave roasted groundnut seed oil. J. Food Sci. Technol. 54, 4335-4343. https://doi.org/10.1007/s13197-017-2904-1 also found a slower increment in PV for treated groundnut seed oil compared to the unroasted sample during thermal oxidation. In addition, at the initial phase of the storage period, samples from the microwave pre-treated oils had slightly higher PV (3.40-3.90 meq O₂/kg); while in the untreated oils it was relatively low (3.17 meq O₂/kg). The higher value could be the result of the exposure time at an elevated temperature during the microwave pre-treatment of the seed samples. Microwave pre-treatment itself was reported to cause slight oil oxidation during prolonged heating (Anjum et al., 2006Anjum F, Anwar F, Jamil A, Iqbal M. 2006. Microwave roasting effects on the physico-chemical composition and oxidative stability of sunflower seed oil. J. Am. Oil Chem. Soc. 83, 777-784. https://doi.org/10.1007/s11746-006-5014-1 ). The PV and p-AV are normally used to determine the degree of lipid oxidation. The pre-treatment of seeds decreased the p-AV significantly (p < 0.05) in BCO compared to the raw samples under accelerated oxidation conditions (Figure 2b). Ali et al. (2017b)Ali MA, Islam MA, Othman NH, Noor AM. 2017b. Effect of heating on oxidation stability and fatty acid composition of microwave roasted groundnut seed oil. J. Food Sci. Technol. 54, 4335-4343. https://doi.org/10.1007/s13197-017-2904-1 reported similar results after 9 h heating of oil extracted from microwaved groundnut seed. In the present work, the p-anisidine values (p-AVs) from the lowest to the highest (10.77 to 15.36), were followed in oils pre-treated at 3, 2, 1 and 0 min after 21 days of accelerated storage. This indicates an extended shelf-life of oils produced from microwaved seeds, probably through the formation of Maillard reaction products (MRPs). Figure 2c shows the marked differences in total oxidation values (TOTOX) in the BCOs under storage conditions. The oil samples of untreated seeds displayed the highest TOTOX values, which revealed that the microwave irradiation reduced the formation of oxidative products during storage at 62 ^oC. The changes in the thiobarbituric acid (TBA) level of treated BCOs were significantly lower (p < 0.05) than that of the untreated seed oil (Figure 2d). Before the storage of oils under accelerated conditions, the TBA values at 0, 1, 2 and 3 min for pre-treated samples were 2.56, 2.87, 3.21 and 3.52, respectively, and after 21 days, the TBA values were increased to 8.64, 7.88 6.89 and 6.47, respectively. This reveals that the oil samples from untreated seeds were more susceptible to oxidation at storage temperature than the oil samples from treated seeds. In addition, a sharp increase in TBA values was detected at an earlier stage of storage followed by a decrease. This can be due to the volatilization of secondary oxidation products or their breakdown.

3.2. Fatty acid composition

The changes in fatty acid composition (FAC) in the oil may indicate its stability, physical properties and nutritional attributes. The dominant fatty acids in BCO were mainly oleic, linoleic and palmitic acids with percentages of 23.25, 57.94 and 13.43, respectively, and myristic, palmitoleic, stearic, linolenic, behenic and lignoceric present in concentrations of less than 1% (Table 1). Saturated (SFA), monounsaturated (MUFA) and polyunsaturated (PUFA) fatty acids in fresh BCO amounted to 15.01, 23.46 and 58.51% of the total fatty acids, respectively. The FAC suffered small changes upon microwave pre-treatment. During pre-treatment, the concentration in C18:2 decreased slightly; whereas the concentrations of C16:0 and C18:0 increased slightly. The PUFA degradation may cause this trend, and Ali et al. (2017a)Ali MA, Nargis A, Othman NH, Noor AF, Sadik G, Hossen J. 2017a. Oxidation stability and compositional characteristics of oils from microwave roasted pumpkin seeds during thermal oxidation. Int. J. Food Prop. 20, 2569-2580. https://doi.org/10.1080/10942912.2016.1244544 saw a similar trend in the case of pumpkin seed oil during thermal oxidation. As can be seen in Table 2, the relative content in PUFA was reduced; while that of SFA or MUFA increased in BCOs during storage treatment. Ali et al. (2017b)Ali MA, Islam MA, Othman NH, Noor AM. 2017b. Effect of heating on oxidation stability and fatty acid composition of microwave roasted groundnut seed oil. J. Food Sci. Technol. 54, 4335-4343. https://doi.org/10.1007/s13197-017-2904-1 followed a similar trend in groundnut oil during heating at 170 ^oC. However, the changes in fatty acid concentrations were lower in microwaved samples than in untreated ones during storage at 62 ^oC. Suri et al. (2019)Suri K, Singhb B, Kaur A, Madhav P, Yadav, Singh N. 2019. Impact of infrared and dry air roasting on the oxidative stability, fatty acid composition, Maillard reaction products and other chemical properties of black cumin (Nigella sativa L.) seed oil. Food Chem. 295, 537-547. https://doi.org/10.1016/j.foodchem.2019.05.140 also reported that dry air roasting slightly influenced the FAC of BCO. The greatest reduction in PUFA was recorded for fresh BCO (7.90%) and the lowest for 3-min microwaved sample (3.20%) after 21 days of storage. The present data indicate that the change in FAC in the pre-treated samples was smaller compared to the raw sample; this indicates the higher tendency of fresh sample towards the generation of oxidation products and polymerized compounds by oxidation degradation of PUFAs. In addition, the ratio of PUFA to SFA (P/S) of all samples decreases with increasing storage time, which serves to realize the status of oxidative tendency of lipids (Lee et al., 2007Lee J, Kim M, Choe E. 2007. Antioxidant activity of lignan compounds extracted from roasted sesame oil on the oxidation of sunflower oil. Food Sci. Biotechnol. 16, 981-987.). In this regard, the pre-treated samples showed the lowest P/S ratio change (decrease) which indicates that oxidative reactions progressed more rapidly in raw samples than in microwaved samples during storage.

3.3. Triacylglycerol (TAG) composition

During the accelerated oxidation test, the changes in concentration in TAG species (P, palmitic; M, myristic; O, oleic; L, linoleic) from BCO determined by HPLC are given in Figures 3 and 4. The major TAG species were LLL (21.89%), PLL (18.75%), OLL (17.22%), POL (13.22%), OOL (9.21 %) and PLP (5.06%). The species OOO, POO, and LOS were present as minor components (< 4%). However, microwave pre-treatment did not inflict changes (with few exceptions) in the TAG species of BCOs because only a few species possessing more than four double bonds were present in the TAGs. The percentages of LLL, OLL, PLL, OOL and POL in BCO decreased whilst, in most case, the percentages of PLP, OOO, and POO remained unaltered or slightly increased with increasing storage time. At the end of 21 days of storage, the changes in concentration in those TAG species were significantly lower (p < 0.05) in 3-min pre-treated samples than untreated ones. The changes in POL were not significant during storage. The most significant reduction was found in LLL (Figure 3a) among all the species. The percentage of this TAG in untreated and 3-min treated samples reduced from 21.89 to 15.57% and from 20.38 to 19.41%, respectively, after 21 days of storage at 62 ^oC. This reduction might be due to a decrease in the concentration of C18:2 by the oxidative process. In this work, good agreement between the fatty acid and TAG compositions was also noted.

3.4. Evaluation by FT-IR

The most significant spectral changes occurring in BCOs during accelerated storage conditions are shown in Figure 5. The detected functional groups responsible for the IR absorption peak (Ali et al., 2017bAli MA, Islam MA, Othman NH, Noor AM. 2017b. Effect of heating on oxidation stability and fatty acid composition of microwave roasted groundnut seed oil. J. Food Sci. Technol. 54, 4335-4343. https://doi.org/10.1007/s13197-017-2904-1 ; Lerma-Garcia et al., 2010Lerma-García MJ, Ramis-Ramos G, Herrero-Martínez JM, Simó-Alfonso EF. 2010. Authentication of extra virgin olive oils by Fourier-transform infrared spectroscopy. Food Chem. 118, 78-83. https://doi.org/10.1016/j.foodchem.2009.04.092 ; Guillen et al., 1997Guillen MD, Cabo N. 1997. Infrared spectroscopy in the study of edible oils and fats. J. Sci. Food Agric. 75, 1-11. https://doi.org/10.1002/(SICI)1097-0010(199709)75:1 ): 3008 (C-H stretching vibration of cis-double bond); 2928 and 2854 (Asymmetric and symmetric stretching vibration of CH₂, resp.); 1745 (C=O stretching vibration); 1465 (bending vibrations of the CH₂ and CH₃); and 1163 (C-O stretching vibration). The absorbance of cis-double bond at 3008 cm⁻¹ (shoulder) suffers a slow shifting toward higher values during storage. This increment can be attributed to the formation of free radicals under accelerated oxidation conditions which initiate a primary oxidation reaction in unsaturated fatty acids (Moharam et al., 2010Moharam MA, Abbas LM. 2010. A study on the effect of microwave heating on the properties of edible oils using FTIR spectroscopy. Afr. J. Microbiol. Res. 4, 1921-1927.;Belitz and Grosch, 1999Belitz H, Grosch W. 1999. Food Chemistry (2nd edn.). Heidelberg: Springer-Verlag Berlin.). This interpretation agrees with that reported in the literature (Farag et al., 1992Farag RS, Hewedp FM, Abu-Raiia SH, El-Baroty GS. 1992. Comparative study on the deterioration of oils by microwave and conventional heating. J. Food Prot. 55, 722-727. https://doi.org/10.4315/0362028X-55.9.722 ). The bands at 2928 and 2854 cm⁻¹ enhanced their intensity (absorbance) because of the surrounding chemical changes taking place due to the oxidation process. The vital peak at 1745 cm⁻¹ corresponds to the carbonyl substances generated from the decomposition of hydroperoxide during accelerated oxidation (Smith et al., 2007Smith SA, King RE, Min DB. 2007. Oxidative and thermal stabilities of genetically modified high oleic sunflower oil. Food Chem. 102, 1208-1213. https://doi.org/10.1016/j.foodchem.2006.06.058 ); the absorbance of it increased with oxidation time. The intensity of a weak peak near 1465 cm⁻¹ increased with the oxidation treatment. The intensity of the peak at 1163 cm⁻¹ related to the proportion in the sample of saturated acyl groups (Guillen et al., 1997Guillen MD, Cabo N. 1997. Infrared spectroscopy in the study of edible oils and fats. J. Sci. Food Agric. 75, 1-11. https://doi.org/10.1002/(SICI)1097-0010(199709)75:1 ), showed similar alterations under storage conditions, and increased its intensity. A similar trend was followed for the peaks at 2927, 2854, 1745, 1465 and 1161 cm⁻¹ by Valdés et al. (2015)Valdés A, Beltrán A, Karabagias I, Badeka A, Kontominas MG, Garrigós MC. 2015. Monitoring the oxidative stability and volatiles in blanched, roasted and fried almonds under normal and accelerated storage conditions by DSC, thermogravimetric analysis and ATR-FTIR. Eur. J. Lipid Sci. Technol. 117, 1199-1213. https://doi.org/10.1002/ejlt.201400384 for almonds during storage at 62 ^oC. In this research, the peak intensities of raw samples were greatly shifted compared to microwaved seed oils during storage, which indicates a clear impact of pre-treatment of BCOs. During storage, the intensities of absorbance of almost all peaks increased and these increments were bigger in fresh oils, which is attributed to the oxidative reactions proceeding more rapidly in the fresh oils than in the microwaved ones. Similar results from IR data were reported by Jan et al. (2019)Jan K, Ahmad M, Rehman S, Gani A, Khaqan K. 2019. Effect of roasting on physicochemical and antioxidant properties of kalonji (Nigella sativa) seed flour. J. Food Meas. Charact. 13, 1364-1372. https://doi.org/10.1007/s11694-019-00052-4 upon pan and microwave roasting of black cumin seeds. The results from the change in FTIR spectra are also in accordance with those shown in the changes in oxidative indices.