Comparison of fatty acid profile changes between unroasted and roasted brown sesame (Sesamum indicum L.) seeds oil

ABSTRACT

The conditions of roasting are the key step in manufacturing Sesame (Sesamum indicum L.) oil. Roasting sesame seeds changed its oil, moisture, and ash content. In oils from unroasted and roasted seeds, nine fatty acids analyzed by gas chromatography-mass spectrometry. The fatty acids composition were changed in both oils. Oleic and linoleic changed in large amount, but changes for other fatty acids were small. ¹H and ¹³C NMR analysis showed the frequency changes for both oils, the roasting condition caused to shift the frequency to lower degree.

KEYWORDS:

• Sesame oil
• roasting
• Comparison
• Fatty acids
• GC-MS, NMR

Introduction

Sesame (Sesamum indicum L.) is a member of the Pedaliaceae family.^[1] It is an economically important oil seed crop which is widely cultivated in many parts of the world, primarily in tropical and subtropical areas.^[2] It has been used as a food source extensively for thousands of years, then later as significance for edible oil, paste, cake, confectionary purposes, and flour.^[2–6] The extensive usage of sesame may be due to its stable oil content, nutritious protein, and savory nutty roasted flavor.^[7,^8] The chemical composition of sesame shows that the seed is an important source of oils (44–58%), proteins (18–25%), carbohydrates (11–13.5%), and ash (±5%).^[1,⁵,^9] Its oil has a mild taste and high unsaturated fatty acids, about 85%.^[10,^11] It is known that sesame oil is resisted to oxidative deterioration compared to other edible oils,^[10] because it is naturally contains antioxidants such as tocopherols and lignans.^[2,³,^12–14] High contents of essential linoleic and linolenic acids are another merit of sesame oil as a food source.^[15]

Roasting is a significant step in the processing of some types of foods like coffee, peanuts, and beans, which results in important physical, chemical, structural, and organoleptic changes.^[16–19] It is also the basic operation in the processing of sesame products.^[7,^10] Roasting of the sesame enhances its flavor, color, and texture and this ultimately increases the overall palatability.^[7,^20] The conventional production of sesame oil involves cleaning, roasting, grinding, cooking, and pressing, but not refining.^[21]

The roasting process introduces chemical composition changes in the sesame seeds and oil. It is reported that the oxidative stability of sesame oil depends on the roasting temperature.^[21–23] At higher roasting temperatures a strong favor is obtained, but the quality of the oil may be reduced.^[21,^24] Also the concentration of hydrocarbons varies between roasted and unroasted seed; the lower molecular weight hydrocarbons (C12–C19) are decreased, whereas the higher molecular weight hydrocarbons (C20–C32) and squalene are increased as a percent of the hydrocarbon fraction.^[20] The percentage of unsaponifiable matter is also slightly increased after the roasting process.^[13] The object of this study was therefore to compare the fatty acid profile between oils from unroasted and roasted brown sesame seeds from Kurdistan Region, Iraq using gas chromatography-mass spectrometry (GC-MS) and nuclear magnetic resonance (NMR).

Materials and method

Materials

Brown sesame seeds were purchased from a local market in Rania City, Kurdistan Region, Iraq. All chemicals and solvents used were either analytical or high-performance liquid chromatography (HPLC) grade. The chemicals n-butanol, chloroform, n-hexane, and methanol were obtained from Fisher (UK). Free fatty acid standards (~99% purity) of palmitic, palmitoleic, stearic, oleic, vaccenic, linoleic, α-linolenic, and eicosenoic acid were purchased form Sigma-Aldrich (UK).

Roasting and extraction oil

The seeds were cleaned manually to remove dust, dirt, stones, chaff, immature and broken seeds; and stored in sealed plastic bags at room temperature (25°C) to preserve the quality. Sesame seeds (500 g) were roasted (30 min) at 200°C on a hot plate (Fig. 1) then milled to 1–3 mm.

Figure 1. Sesame seeds from Kurdistan Region–Iraq; A: unroasted brown sesame seeds, B: roasted brown sesame seeds at 200°C for 30 min on a hot plate.

Ground seeds (100 g) were extracted in a conical flask (250 mL) with n-hexane (250 mL) and stirring at room temperature for 3 h. The solution was filtered, reserving the filtrate and further extracted successively in n-hexane (3 × 200 mL). All n-hexane filtrates were combined and then filtered under gravity (Whatman 150) to remove any solid particles. The n-hexane was dried over magnesium sulphate (MgSO₄) and filtered. Finally, the n-hexane was removed by rotary evaporator (Buchi R-114 Rotavapor, Switzerland) and water bath (Buchi B-481 water bath, Switzerland), at 40ºC. Residual solvent was removed under vacuum until a constant weight was obtained. The final product was golden yellow oil.

Transesterification and analysis of sesame oils

All standards and sesame oils were trans-esterified to produce fatty acid methyl esters (FAMEs). Aliquots of oil (2.0 g) were dissolved in sodium methoxide (8 mmol, 20 mL) and heated under reflux for 20 min. Deionized H₂O (50 mL) was added, and the solution extracted successively with n-hexane (3 × 100 mL). The organic extracts were dried with MgSO₄, then filtered and evaporated at 40°C. The trans-esterified samples and standards were diluted 1:1000 with n-hexane, then injected in the GC-MS device.

FAMEs of both unroasted and roasted sesame oil and standards were analyzed using a GC-MS Thermofisher Trace 1300 GC fitted with a DB-5 column (L 30 m, ID 0.25 mm, DF 0.25 mm). The injector type was a programmed temperature vaporizer, operating in split/splitless mode, with an injection volume of 1 μL at 250ºC, with a split flow of 20 mL min ⁻¹. The initial temperature was set at 150ºC, which was held for 2 min. The temperature of oven was increased at a rate of 5ºC/min until final temperature, 260ºC and then held for 5 min. The detector was a Thermofisher ITQ900. Electron impact (70 eV) ionization was used for fragmentation at a rate of two scans per second. All processing and analysis was carried out on Thermo Xcalibur, v 2.2.

NMR analysis

Sesame oil (100.0 mg) was dissolved in 0.6 mL deuterated chloroform (CDCl₃) and ¹H and ¹³C NMR spectra were obtained on the Ultra Shield TM Plus 400—Bruker 400 MHz spectrometer at 25°C. The ¹H NMR was performed at an operating frequency of 400 MHz and ¹³C NMR was at 100 MHz in NMR tube (5 mL). There was a relaxation time of 2 s for ¹H NMR and 4 s for ¹³C NMR operations. All processing and data analysis was performed on MestReNova LITE v 5.2.5.

Moisture content and ash

The moisture contents of unroasted and roasted sesame seeds were determined according to Kahyaoglu and Kaya.^[7] Five grams of seeds were heated to constant weigh at 105°C. Ash contents of unroasted and roasted sesame seeds were determined.^[7] The sesame seeds were dried, and 5.0 g placed in a porcelain crucible and heated under normal atmospheric conditions to 500ºC for 4 h in a muffle furnace (Carbolite–ELF11/148, England). The samples were cooled in a desiccator for 1 h, then the ash left in the crucible was weighed to determine percentage ash content.

Statistical analysis

There were three independent measurements for all observations with mean values ± standard deviations reported for each case. Analysis of variance (ANOVA) was performed on the data at the level of p < 0.05 to evaluate the significance of differences between treatment means.

Result and discussion

While sesame oil was extracted from 100 g of both unroasted and roasted brown sesame seeds, different oil contents were derived from the two treatments. The mean oil yields for unroasted and roasted seeds were 45.7 and 51.1 g, respectively (Table 1). This was consistent with other studies that roasting resulted in an increase in oil yield because it enhanced protein denaturation which in turn improved lipid extractability.^[13] However moisture content and ash content were negatively affected by roasting. Moisture content decreased from 4.9 g in unroasted seeds to 3.8 g in roasted seeds and ash content decreased from 5.9 g in unroasted seeds to 5.8 g in roasted seeds, in 100 g of raw seeds. Other studies showed that, as the roasting temperature and time were increased, the moisture content of sesame seeds decreased for all roasting methods. The seeds become more fractured with roasting and take on more crispy texture.^[7] Meanwhile roasting caused an increased in the percentage of unsaponifiable matter from 1.2 to 1.3 g, in 100 g of raw seeds.

Table 1. Chemical composition of unroasted and roasted brown sesame seeds, in 100 g raw seeds matter.

Component	Unroasted	Roasted
Oil content	49.1^a ± 0.8	51.1^b ± 0.7
Moisture content	4.9^a ± 0.4	3.8^b ± 0.2
Ash content	5.9^a ± 0.1	5.8^a ± 0.1
Unsaponifiables matter	1.2^a ± 0.1	1.3^a ± 0.1

All the given values are means of tree determinations ± standard deviation.

Means in a row followed by the same letters are not significantly different (p < 0.05).

The analysis of sesame oils by GC-MS needed derivatization to improve their volatility and the frequency of resolution. It could be obtained by transesterification using an alkyl ester. In the GC chromatogram (Fig. 2), nine fatty acids were determined in both sesame oils. The relative fatty acid content for both oils was listed in (Table 2). The chain lengths of fatty acids included C14, C16, C18, C20, and C22. The saturated fatty acids (SFA) present were myristic acid (C14:0), palmitic acid (C16:0), stearic acid (C18:0), arachidic acid (C20:0), and eicosadienoic acid (C22: 0). Unroasted sesame oil was comprised of 12.8% SFAs, while roasted sesame oil contained 15.9% SFAs. Roasting increased the oil content of SFAs. In both oils palmitic acid was the common fatty acid, followed by stearic acid, while the other SFAs were present in trace amounts. The monounsaturated fatty acids (MUFAs) palmitoleic acid (C16:1n-9) and oleic acid (C18:1n-9) were detected in both sesame oils. Linoleic acid (C18:2n-9, 12) and α-linolenic acid (C18:3n-9, 12, 15) were present as polyunsaturated fatty acids (PUFAs). The PUFA content was negatively affected by roasting; linoleic acid decreased from 46.1% in unroasted oil to 24.8% in roasted oil, and α-linolenic acid decreased from 0.4 to 0.2%.

Table 2. Fatty acid composition (%) of unroasted and roasted brown sesame seeds oil.

Fatty acids	(%) of fatty acids in unroasted sesame oil	(%) of fatty acids in roasted sesame oil
Myristic acid (C14:0)	0.9^a ± 0.01	0.9^a ± 0.01
Palmitic acid (C16:0)	5.6^a ± 0.9	7.1^b ± 0.9
Palmitoleic acid (C16:1n-9)	0.4^a ± 0.1	0.4^a ± 0.01
Stearic acid (C18:0)	5.3^a ± 0.8	6.8^a ± 0.6
Oleic acid (C18:1n-9)	40.3^a ± 5.3	58.7^b ± 4.7
Linoleic acid (C18:2n-9, 12)	46.1^a ± 1.1	24.8^b ± 0.9
α-Linolenic acid (C18:3n-9, 12, 15)	0.4^a ± 0.2	0.2^a ± 0.1
Arachidic acid (C20:0)	0.5^a ± 0.07	0.5^a ± 0.1
Eicosadienoic acid (C22: 0)	0.5^a ± 0.2	0.6^a ± 0.2
SFA	12.8 ± 1.98	15.9 ± 1.81
MUFA	40.7 ± 6.2	59.1 ± 4.71
PUFA	46.5 ± 1.3	25 ± 1

All the given values are means of tree determinations ± standard deviation.

Means in a row followed by the same letters are not significantly different (p < 0.05).

Figure 2. Chromatograms of GC-MS (Thermofisher) of FAMEs sesame oil; A: chromatogram of GC-MS of unroasted sesame seeds oil, B: chromatogram of GC-MS of roasted sesame seeds oil.

There have been reports on the analysis of oils by ¹H NMR. To determine oil composition olefinic carbons, ω-3 methylene carbons, allylic carbons, and β-methylene carbons were used. The ¹³C NMR was based on determination of the resonance intensities of carbon-13 nuclei of triglyceride fatty acids.^{[25] 1}H NMR indicates the approximate relative composition of FAs in the oils.^[26] To determine any changes in the oil composition between unroasted and roasted sesame we may evaluate the correlation in their olefinic region (Table 3). In the ¹H NMR spectrum of unroasted sesame oil (Fig. 3a), the integration of the olefinic region showed a multiplet at

Table 3. Chemical shifts and assignments of the characteristic resonances in the ¹H NMR spectra of unroasted and roasted crude sesame seed oil, (400 MHz in CDCl₃).

Chemical shifts for unroasted seeds (ppm)	Chemical shifts for roasted seeds (ppm)	Proton	Assignment
0.84 (m)	0.85 (m)	-CH3	Terminal methyl
0.95 (t)	0.95 (t)	-CH3 (ω3)	Terminal methyl of polyunsaturated (ω3)
1.23 (bp)	1.23 (bp)	CH2n	Methylene
1.57 (bp)	1.58 (bp)	CH2- CH2–COO	All acyl chains
2.01 (m)	2.01 (m)	CH2-CH=CH	Allylic-all unsaturated fatty acids
2.28 (t)	2.28 (t)	CH2–COO	All acyl chains
2.74 (t)	2.74 (t)	C=C-CH2C=C	Bis-allylic methylene (di-unsaturated fatty acid)
4.14–4.25 (dd)	4.13 – 4.25 (dd)	CH2O(ω1,3)	Glycerol (triacylglycerols)
5.24 (m)	5.23 (m)	CHO (ω2)	Glycerol (triacylglycerols)
5.31(m)	5.31(m)	CH=CH	Olefinic (all unsaturated fatty acid)

bp: broad peak; dd: double doublet; m: multiplet; t: triplet.

Figure 3. The ¹H NMR spectra of unroasted and roasted crude sesame seed oil at 400 MHz in CDCl₃; A: ¹H NMR spectrum of crude unroasted sesame oil, B: ¹H NMR spectrum of crude roasted sesame oil.

The ¹³C NMR resonances of the oils could be grouped into four well-defined spectral regions: carbonyl carbons ranging from 173.3 to 172.8 ppm; unsaturated carbons ranging from 132.1 to 126.8 ppm; glycerol carbons ranging from 69.1 to 61.6 ppm; and aliphatic carbons ranging from 34.5 to 13.9 ppm. All assayed oils differed in quantitative chain composition rather than in qualitative acid profile; therefore, exhibiting analogous signals, although with varying intensities which were characteristic for each oil.^[28,29] All chemical shifts of ¹³C NMR of sesame oil were listed (Table 4).

Table 4. ¹³C NMR assignments of unroasted and roasted crude Sesame oil at 100 MHz in CDCl₃. Two signals for the same olefinic environment indicate an equivalent resonance between two different olefinic carbons. Two signals for the same environment are splits in the triacylglycerol signal between the sn-1,3 or sn-2 fatty acid chains.

Chemical shifts (ppm) of unroasted Sesame seeds oil	Chemical shifts (ppm) of roasted Sesame seeds oil	Carbon	Assignment
14.27	14.28	C18 (CH3)	All acyl chains
22.75	22.75	C17 (CH2)	All acyl chains
25.01	25.01	C3	All acyl chains
25.85	25.79	C11	Diallylic
27.37	27.37	C8–1(oleyl) C8–14 (linoleyl)	Allylic
29.50–29.88	29.29–29.89	CH2n	All acyl chains
31.70	31.70	ω6	n-9, n-6
32.09	32.09	α-C2	All acyl chains
34.19	34.18	β-C2	All acyl chains
62.26	62.25	α-CH2O	Glycerol (triacylglycerols)
69.07	69.05	β–CH2O	Glycerol (triacylglycerols)
127.79	127.83	C15	Linolenyl
127.92	127.90	C10	Linolenyl
128.07	128.05	C12	Linoleyl
128.24	128.22	C10	Linoleyl
128.79	128.43	C12	Linolenyl
128.84	128.49	C13	Linolenyl
129.83	129.85	C9	Olelyl
129.98	130.10	C9	Linoleyl
130.16	130.15	C10	Olelyl
130.55	130.34	C13	Linoleyl
130.27	130.28	C9	Linolenyl
131.82	131.82	C16	Linolenyl
174.34	172.96	sn 1,3 C1	Carbonyl (triacylglycerols)
174.38	173.37	sn 2 C1	Carbonyl (triacylglycerols)

In ¹³C NMR, the carbonyl carbons of oil triglycerides appeared as two sets of resonances, the high frequency set at 1(3)-glycerol positions, whereas the low frequency was at the 2-glycerol position chains, as shown in Fig. 4.^[29] In particular, the signals from carbonyl carbons of 1(3)-chains were shifted by about 0.42 ppm for unroasted and 0.41 ppm for roasted seeds, at higher frequency from those of carbonyls of 2-chains.^[28,^30]

Figure 4. ¹³C NMR spectra of unroasted and roasted crude sesame oil at 100 MHz in CDCl₃; A: ¹³C NMR spectrum of unroasted crude sesame oil, B: ¹³C NMR spectrum of roasted crude sesame oil, showing the ¹³C assignment in Table 4.

¹³C NMR also showed the unsaturated region of fatty acid carbons between 127.79–131.81 ppm (Fig. 4). For each double bond of UFAs, symmetrical pairs of signals can be seen for each side. In both oils the chemical shifts were changed but the difference between the peaks had not changed that much, therefore the roasting condition caused to shift the frequency to lower degree. For instance the differences between carbon shifts of linoleyl chains in unroasted seeds were 0.17 ppm for C-10 to C-12 and for C-9 and C-13 was 0.45 ppm, also for oleyl chains were changed by 0.33 ppm for C-9 to C-10. However in roasted seeds the chemical shifts for linoleyl chains were 0.17 ppm for C-10 to C-12 and for C-9 and C-13 was 0.45 ppm, and for oleyl chains were changed by 0.31 ppm for C-9 to C-10. Also in ¹³C NMR the chemical shifts showing the presence of α-linolenic acid (C18:3n-9, 12, 15), as ¹H NMR shifting of the terminal methyl at (δ 0.95 ppm, t) confirmed that there is ω3-fatty acid, but it is in trace amount.^[26] The presence of the glycerol backbone of oil resulted in 1- and 2-carbon split signal for carbons in the olefinic (alkene) region (Fig. 5); therefore, each unsaturated carbon is split according to glycerol position. The distance between the pair of signals depends on its distance from the C1 environment. The non-equivalence of chemical shifts of olefinic carbons has been studied in a series of MUFAs with different number of C–C bonds separating the ester group and the double-bond. The relevant feature of these chemical shifts is that the carbons of a double-bond pair are shifted in opposite directions, it means the unsaturated carbon nearest to the chain ester group is shifted to lower frequency from the unsaturated carbon closest to the chain methyl end. The chemical shift differences which increase as the double bond moves toward the carboxyl group. The chemical shift differences which increase as the double-bond moves toward the carboxyl group, were proved to arise from a linear electric field effect. Also, it was possible to determine the proportion of UFAs at one or two positions.^[28]

Figure 5. The olefinic region 127–132 ppm of 100 MHz ¹³C NMR spectra of crude sesame oil; A: ¹³C NMR spectrum of olefinic region of crude unroasted sesame seeds oil, B: ¹³C NMR spectrum the olefinic region of crude roasted sesame seeds oil.

The glycerol carbons resonate in the spectral region from 62.26 to 69.07 ppm for unroasted seeds and for roasted seeds was 62.25 to 69.05 ppm. The chemical shift assignments were based on the assumption that acylglycerol symmetry or asymmetry determines the number of resonances and their relative intensities. The resonances of methylene and methyl carbons of saturated and unsaturated acyl chains are reported. The C-16–C-18 carbons, which were assigned using additively relationship which predicts the ¹³C chemical shifts of n-alkanes, appear at chemical shifts centered at 31.70, 22.86, and 14.28 ppm for roasted seeds also 31.70, 22.75, and 14.27 ppm for unroasted seeds. Within each set of signals, saturated, oleyl, and linoleyl chains resonate from higher to lower frequency in this order. However, an influence of the chain position on glycerol backbone was not detected.

Conclusion

Sesame (Sesamum indicum L) is an oil seed herbaceous crop that is of great economic importance due to its highly stable oil and nutritious content. The manufacture of high quality sesame oil requires that the maintenance of the optimum roasting conditions. The roasting process affected the physiochemical properties of sesame seeds and oil, measurably in oil content, moisture, ash, and unsaponifiable matter in 100 g of seeds. The oil content increased by roasting from 49.1 and 51.1 g for unroasted and roasted seeds, respectively. The moisture and ash content were decreased, the moisture content in unroasted seeds was 4.9 g decreased to 3.8 g also ash content decreased from 5.9 to 5.8 g. Unsaponifiables matters had increased slightly. The oils from unroasted and roasted seeds were analysed by GC-MS and NMR. By GC-MS analysis in both oils those fatty acids were analyzed; myristic, palmitic, palmitoleic, stearic, oleic, linoleic, α-linolenic, arachidic, and eicosadienoic acid. The fatty acid compositions of oils were changed by roasting. Oleic acid increased in high amount, it was 40.3% in unroasted seeds increased 58.7% in roasted seeds. Also, palmitic acid increased from 5.6 to 7.1% and stearic acid increased from 5.3 to 6.8%. But, linoleic acids decreased significantly, from 46.1 to 24.8% in unroasted and roasted seeds, respectively. The other fatty acids were changed in low amount. Both ¹H and ¹³C NMR analysis showed frequency changes during determination of oils. Overall, the roasting process caused to shift the frequency to lower degree. For all individual fatty acids the frequencies were changed during measurements. Finally, the procedures and laboratory analysis fall within the general patterns for other contents and general organic compounds of sesame seeds. It is apparent that roasting of sesame seeds had potential changes, further studies are needed to explore such combination effects and further investigations should focus on the changes of biological active content of sesame seeds.

Acknowledgments

The authors would like to thank technical staff of Chemistry Department at University of Raparin.

Funding

This work was financed with grants from the HCDP program in the Ministry of Higher Education and Scientific Research of Kurdistan – Iraq, No.: JUOR15.

Additional information

Funding

This work was financed with grants from the HCDP program in the Ministry of Higher Education and Scientific Research of Kurdistan – Iraq, No.: JUOR15.