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This study was planned to evaluate the stability of four extracted edible oils namely extra virgin olive oil (EVOO), moringa oil (MO), apricot kernel oil (AKO) and sunflower oil (SO). The oils were analyzed in a new manner by applying modern non-traditional instrumental methods of analysis, along with traditional ones as a reference to test the potency of the new analytical methods, in order to identify the relation between their composition and stability, with great stress to investigate what components are responsible to stability. The oils were exposed to some familiar physical and chemical determinations such as color and refractive indices; acidity; saponification value; unsaponifiable matter %; iodine and peroxide values; ultraviolet absorbencies at 232, 270, 266, 274 nm; chlorophyll and carotenoids; as well as, the oxidative stability by Rancimat method and the antioxidant activity of phenolic extracts of oils by DPPH method. The analytical methods extended to more sophisticated instrumental methods of analysis such as chromatographic separation and determination techniques involved gas chromatography (GC) with flam ionization detector in order to investigate the composition of fatty acids and sterols content of the oils. High performance liquid chromatography (HPLC) was also used to investigate the composition of polyphenols, flavonoids and tocopherols which proved their main causes of oils stability. Fourier transform infra red (FTIR) and nuclear magnetic resonance (NMR) spectroscopic determinations technique were employed and applied as a potent, nondestructive and effective analytical tools to investigate the oxidative stability of the oils. FTIR spectroscopy determined the functional groups with their relative transmittances according to their concentrations in samples and their characteristic fingerprints. It also, determined the attributes of some functional groups to stability and monitoring the oxidation processes by following the changes in the spectra upon oxidation.
NMR investigate the differences between the tested oils to evaluate their oxidative stability 1H-NMR helped in the identification of the protons of the main components of oils to show the difference in oils structures along with 13C-NMR that helped in the identification of carbon skeleton of oils and their main components of carbonyl, olefinic, glyceryl and aliphatic carbons. The obtained results are summarized as follows:
1- Physical and chemical characteristics of oils:
- The refractive index (RI) of oils increased in auto-oxidation and can be used to evaluate oils rancidity. The order of the obtained increasing RI values was in relation to linoleic acid (LO) content in tested oils except of EVOO due to the differences in structure between oils. MO had the least RI (1.4621) with the highest stability, followed by AKO (1.4647), then EVOO (1.4696), and at least SO having the highest RI (1.4739). Corresponding to 0.75, 23.13, 11.04 and 52.20% of LO, respectively.
- MO showed the highest red color value (3.6) followed by EVOO (2.2) then AKO (0.6) and SO (0.2) which correlated with the carotenoids content, where MO had the highest value followed by EVOO then AKO and SO, being 4.81, 3.21, 0.16 and 0.05 mg/kg, respectively. The blue color appeared only for EVOO and MO, with the highest value (3.2) for EVOO due to its highest chlorophyll content (2.78 mg/kg) comparable to MO (0.54 mg/kg) which had a low blue color index (0.5).
- The acidity of the oils formed by hydrolysis and oxidation, as well as, the peroxide value was too small to affect the quality of the tested oils.
- MO showed slightly higher saponification value (194.70) which reflect the short acyl chain of fatty acids, followed by AKO (193.50) then EVOO (186.10) and at least SO (183.00).
- EVOO characterized by the highest percentage of unsaponifiable matter (2.19%), followed by MO (1.90%), AKO (1.80%) then SO (1.36%), indicating the higher stability of EVOO and MO than others.
- The highest IV (116.96) was recorded for SO that was high unsaturated followed by AKO (105.37), then EVOO (81.46), and at least MO (73.65). The lower IV, the higher the resistance of oil oxidation, thus this value ordered the oils according to their stability as follows: MO ˃ EVOO ˃ AKO ˃ SO.
- MO had the lowest absorption band at 232 nm followed by that of EVOO, then AKO, indicating the highest antioxidant effectiveness and the lower concentration of linoleate. However, SO gave rise to this intensity having relatively the highest value due to conjugated dienoic acid in this oil and its higher polyunsaturation. Thus, this UV absorption band arranged the oils according to their stability as: MO ˃ EVOO ˃ AKO ˃ SO. The UV-absorption at 266 nm of EVOO was very small value, while for other oils were relatively higher. Nevertheless, all the measured wavelengths for all the tested oils were considered as small values.
- It was noticed that the relative ratios of PUFA/MUFA, C16:0/C18:2, C18:1/C18:2, C18:2/C18:3 and [C18:2 + C18:3 / C18:1] were correlated with the stability and ordered the tested oils as MO ˃ EVOO ˃ AKO ˃ SO according to Rancimat method.
- EVOO had the highest content of chlorophyll followed by MO then SO and AKO. While, MO had the highest concentration of carotenoids followed by EVOO then AKO and SO, which reflect their stability and correlated positively with the oils oxidative stability measured by Rancimat, where, MO ˃ EVOO ˃ AKO ˃ SO.
- EVOO had the highest total tocopherols and polyphenols followed by MO then AKO and SO, which arranged the oils descendingly according to their stability according to the order obtained by chlorophyll compounds.
- MO had the highest induction period (IP) being 190.00 hr indicating superior resistance to oxidation, followed by EVOO (59.11 hr) then AKO (26.60 hr). However, SO recorded only 7.45 hr.
- The highest resistance of MO to oxidation followed by EVOO was due to their less linoleate, higher percentage of oleate and unsaponifiable matter and the antioxidant content of tocopherols, flavonoids, sterols, phenolics, chlorophyll and carotenoids.
- The high stability of EVOO is mainly due to its relatively low degree of PUFA and its antioxidant activity of the unsaponifiable components, tocopherols, flavonoids and phenolic compounds which also showed synergistic effect with each other.
- AKO had a middle IP having relatively high stability due to its higher percentage of OL, but had higher LO and lower antioxidant content compared to MO and EVOO.
- The lowest IP of SO was due to its higher PUFA and lower antioxidant content and the higher degree of unsaturation.
- It was observed a decrease in the IP of the oils after removing their polyphenols that reflect the antioxidant activity of these compounds in the oils. It could be also observed that polyphenols were a main antioxidant in EVOO which content the highest value among other oils, and approached half of its IP. However, MO which had the highest stability included other factors other than polyphenols.
- The phenolic extract from EVOO had the highest value of radical scavenging capacity (92.91%) followed by MO (48.82%), AKO (45.18%) then SO (25.18).
- The highest percentage of β-sitosterol was found in EVOO followed by AKO, SO then MO. β-sitosterol besides Δ5-avenasterol may be mainly responsible for olive oil stability. It seems to be also a main factor in AKO and SO oxidative stability. MO contained the highest percentages of stigmasterol, campesterol and Δ5-avenasterol among other oils which may be in totally responsible for its oxidative stability.
- The most abundant tocopherol in both EVOO and SO was α-tocopherol; and in AKO was γ-tocopherol, whereas, in MO was δ-tocopherol which its antioxidant activity exceeds that of α- and γ-tocopherol, and was contribution to the highest resistance to oxidation, and making clear the long IP of this oil.
2- Evaluation of oils stability using FTIR spectroscopy
- All the fresh studied oils showed many similarities between their FTIR spectral data. However, some variations were found, which successfully led to the differentiation between oils.
- The observed shifts of absorption peaks around 3005 cm-1 ordered the oils where the lowest shift of the peak was related to the highest stability as follows: MO (3003.59 cm-1) ˃ EVOO (3004.55 cm-1) ˃ AKO (3005.52 cm-1) ˃ SO (3006.48 cm-1). This arrangement reflect the same order of oils stability evaluated by Rancimat method, and also correlated to the PUFA content of corresponding oils.
- The oil with lowest value of PUFA will have the lowest shift of absorption peak around 3005 cm-1 and will show the highest stability. In the same way, the PUFA arranged these oils as: MO (0.96%) ˃ EVOO (11.84%) ˃ AKO (23.56%) ˃ SO (52.55%).
- Also, the higher in intensity of this peak around 3005 cm-1, the highest in unsaturated fatty acids. from this relation, it was easy to predict the order and the appropriate values of the unsaturated fatty acids of edible oils.
- When the oil shows a peak around 3470 cm-1, it is an indication that the oil is non-oxidized.
- MO which was evaluated as an oil of the highest stability by the Rancimat method, showed the lowest shifts of absorption peaks around 1236, 1162 and 1118 cm-1 followed by EVOO, then AKO and SO.
- The oxidized olive oil (Ox-OO) showed the similar peak at 1236 cm-1 with much higher intensity than the double of fresh EVOO.
3- Evaluation of oils stability using NMR spectroscopy:
3.1- 1H-NMR spectral data analysis of fresh oils:
- In addition to main components, the studied oils contained minor components which are important because they show antioxidant stability. These components were present in high enough concentration to be detected by 1H-NMR, and the signals of their protons did not overlap with those of the main lipid components.
- The chemical shifts of solvent peaks around 2.5 and 3.3 ppm ordered the oils stability, where the lesser shift was corresponded to the oil of higher stability. Accordingly, the order of oils stability obtained as follows: MO ˃ EVOO ˃ AKO ˃ SO, agreed with the order of obtained by Rancimat method and with the order of tocopherols concentration as powerful natural antioxidants present in the oils.
- The higher oxidative stability of MO and EVOO than other oils is mainly due to their relatively low degree of fatty acid unsaturation and the antioxidant activity of some of the unsaponifiable components such as sterols. MO showed the presence of both stigmasterol and β-sitosterol from the signals at 0.662 and 0.784 ppm, respectively. However, EVOO showed a peak at 0.780 ppm assigned to β-sitosterol which is an important marker in olive oil analysis.
- The higher oxidative stability of MO over EVOO could be attributed to their fatty acid composition, both had higher OL content, EVOO contained LO and LN which were more readily undergoes oxidation and degradation than OL. These acids were in much lower quantities in MO. The higher stability was also due to other constituents of non-glyceride fraction that possess antioxidant properties. In addition, MO was the most rich in aliphatic chains.
3.2- 1H-NMR spectral data analysis of stored oils after 6 months:
- The same peaks in fresh EVOO were still present in the stored oil. The slow and slight changes in oil stability caused some shifts in chemical assignments of 1H-NMR signals.
- Additional peaks were appeared in 1H-NMR spectrum of EVOO after 6 months. The most important peak was at 2.255 ppm attributed to free fatty acids due the hydrolytic degradation of triglycerides, thereby increasing the acidity of the oil.
- It was observed some differences between 1H-NMR spectra of fresh MO, AKO and SO and their oils after storage for 6 months including the disappearance of some peaks and appearance of new peaks; also, some shifts were denoted in the spectra of the stored oils, attributed to free fatty acids and indicating the effect of hydrolytic degradation of acyl groups and the formation of new compounds, as well as conjugated systems.
3.3- 1H-NMR spectral data analysis of EVOO after 60 months:
- Some chemical shifts of the 1H-NMR signals of some compounds appeared after 6 months were still unmoved after 60 months, such as intensity of β-sitosterol signal.
- The 1H-NMR spectrum of stored oil showed slight changes in intensity of triglycerides and of other components to lower intensities compared to fresh oil.
- The appearance of new signals in oxidized oil stored for 60 months were aroused due to the different kinds of alcohols, the free fatty acids and diglycerides resulted from the oxidation and degradation of the primary oxidative products. These compounds having protons whose signals in the 1H-NMR spectrum did not overlap with any other, making it possible to identify.
- Combining the results of FA analysis and the oxidative stability of oils by Rancimat method, in general, the lower the content in
polyunsaturated groups in the oil, the higher its resistance to degradation, however, other factors, such as the presence of minor antioxidant components, play an important role in degradation role of the oil.
- The 1H-NMR spectroscopy is a very useful technique in the study of several aspects of edible oils and fats. It has the advantages that it is not destructive, requires a very small sample whose preparation is very simple and it takes little time.
3.4- 13C-NMR spectral data analysis of fresh oils:
- No data were obtained from the 13C-NMR spectroscopy of EVOO and SO dissolved in DMSO-d6. However, the results obtained from 13C-NMR spectra of MO and AKO were very poor in data; the DMSO-d6 solvent occupied already all the spectra except a very few peaks appeared in aliphatic and glycerol regions.
- Thus, the use of DMSd6 was not suited as a solvent for the 13C-NMR analysis of the studied oils. Another trial was carried out. When using the deuterated chloroform as a solvent, a large number of signals were spread over a wide range of chemical shifts.
- The sole CDCl3 as a solvent showed a triplet chemical shifts around 77 ppm.
- Four spectral regions were observed in 13C-NMR spectra that belong to four different groups of carbon resonances. The first one contained the carbonyl resonances of fatty acids; the second region involved resonances of the olefinic carbons of the unsaturated fatty acids; in the third region, the signals of glycerol backbone carbons appeared; and the fourth one comprised signals of aliphatic carbons.
- MO had the lowest chemical shifts of solvent peaks which reflect the highest stability of this oil among all other studied oils.
- In the first region, ranging from 172-174 ppm, it was observed the absence of peak resonating at 173 ppm characteristic of saturates (S) in AKO and SO, where it appeared in both EVOO and MO at 173.303 and 173.277 ppm, respectively. These finding agreed with the GC analysis of fatty acids.
- In the second region, ranging from 124-134 ppm, two peaks at 127.858 and 128.018 ppm assigned to LO were shown only in SO 13C-NMr spectrum, that agreed with the GC analysis of FA, where, linoleic acid was the major fatty acid in SO (52.20%).
- Also, the absence of two peaks at 129.650 and 129.790 ppm in SO, while they present in other oils indicating that they had higher content of OL and lower content of LO than SO. EVOO showed also two peaks at 130.177 and 130.280 ppm indicating its highest content in LN and considerable LO among other oils. However, MO had no peak at 128.173 ppm as three other oils indicating the lowest content of LO.
- The signals of glycerol backbone carbons appeared in the third region between 60 and 72 ppm showed that all the oils had similar peaks with differences in values corresponding to their shifts. MO had the least chemical shifts followed by EVOO, then AKO and SO, this reflects the order of oils stability that it was the same obtained by Rancimat method.
- In the fourth region, from 10 to 35 ppm that comprises signals of aliphatic carbons, SO showed LO chains more than EVOO, MO and AKO that agreed with GC analysis of fatty acids of oils.
- 13C-NMR technique is particularly useful in distinguishing between mono-, di- and tri-glycerides, as a strong discriminating power to detect the specific components and free fatty acids.
- When combining the results from 1H-NMR it was observed the absence of diacylglycerols in EVOO which mainly arise either from incomplete triacylglycerol biosynthesis or from limited lipase action on triacylglycerols