Classification of vegetable oils by high-resolution <Superscript>13</Superscript>C NMR spectroscopy using chromatographically obtained oil fractions

¹³C NMR spectra of oil fractions obtained chromatographically from 109 vegetable oils were obtained and analyzed to evaluate the potential use of those fractions in the classification of vegetable oils and to compare the results with the NMR analysis of complete oils. The oils included the following: virgin olive oils from different cultivars and regions of Europe and north Africa; “lampante” olive, refined olive, refined olive pomace, hazelnut, rapeseed, high-oleic sunflower, corn, grapeseed, soybean, and sunflower oils; and mixtures of virgin olive oils from different geographical origins. Oils were divided into two sets of samples. The training set (98 samples) was employed to select the variables that resulted in significant discrimination among the different oil classes. By using stepwise discriminant analysis, more than 98% of correct validated assignments were obtained; these results were confirmed when applied to the test set (11 blind samples). Results suggest that the use of oil fractions considerably increases the discriminating power of NMR in the analysis of vegetable oils.     

Stability and radical‐scavenging activity of heated olive oil and other vegetable oils

The effect of heating at 180 °C on the antioxidant activity of virgin olive oil (VOO), refined olive oil (ROO) and other vegetable oil samples (sunflower, soybean, cottonseed oils, and a commercial blend specially produced for frying) was determined by measuring the radical‐scavenging activity (RSA) toward 1,1‐diphenyl‐2‐picrylhydrazyl radical (DPPH^?). The RSA of the soluble (polar) and insoluble (non‐polar) in methanol/water fractions of olive oil samples was also measured. The stability of heated oils was assessed by determining their total polar compound (TPC) content. VOO was the most thermostable oil. Total polar phenol content and the RSA of VOO heated for 2.5 h decreased by up to 70 and 78%, respectively, of their initial values; an up to 84% reduction in RSA of VOO polar and non‐polar fractions also occurred. Similar changes were observed in the RSA of ROO and its non‐polar fraction after 2.5 h of heating. The other oils retained their RSA to a relatively high extent (up to 40%) after 10 h of heating, but in the meantime they reached the rejection point (25–27% TPC). The results demonstrate that VOO has a remarkable thermal stability, but when a healthful effect is expected from the presence of phenolic compounds, heating has to be restricted as much as possible.     

Evaluation of MALDI-TOF/MS Technology in Olive Oil Adulteration

Adulteration of extra virgin olive oil (EVOO) by addition of other vegetable oils or lower-grade olive oils is a common problem of the oil market worldwide. Therefore, we developed a fast protocol for detection of EVOO adulteration by mass spectrometry fingerprinting of triacylglycerol (TAG) profiles based on MALDI-TOF/MS. For that purpose, EVOO TAG profiles were compared with those of edible sunflower oil and olive oil composed of refined olive oil and virgin olive oils. Adulteration of EVOO was simulated by addition of sunflower and mixture of refined olive oil and virgin olive oils at 1, 10 and 20% w/w. Results of mass spectrometry TAG profiling were compared with routinely assessed K values for identification of adulteration. MALDI-TOF/MS technology coupled with statistical analysis was proven as useful for detection of adulteration in EVOO at a rate down to 1%. In contrast, standard spectrophotometric methods failed to identify minor adulterations. In addition, the ability of MALDI-TOF/MS in detection of adulteration was tested on EVOO samples from different geographical regions. Results demonstrated that MALDI-TOF/MS technology coupled with statistical analysis is able to distinguish adulterated oils from other EVOO.     

Use of high-resolution 13C nuclear magnetic resonance spectroscopy for the screening of virgin olive oils

¹³C Nuclear magnetic resonance (NMR) spectra of 104 oil samples were obtained and analyzed in order to study the use of this technique for routine screening of virgin olive oils. The oils studied included the following: virgin olive oils from different cultivars and regions of Europe and north Africa, and refined olive, “lampante” olive, refined olive pomace, high-oleic sunflower, hazelnut, sunflower, corn, soybean, rapeseed, grapeseed, and peanut oils, as well as mixtures of virgin olive oils from different geographical origins and mixtures of 5–50% hazelnut oil in virgin olive oil. The analysis of the spectra allowed us to distinguish among virgin olive oils, oils with a high content of oleic acid, and oils with a high content of linoleic acid, by using stepwise discriminant analysis. This parametric method gave 97.1% correct validated classifications for the oils. In addition, it classified correctly all the hazelnut oil samples and the mixtures of hazelnut oil in virgin olive oil assayed. All of these results suggested that ¹³C NMR may be used satisfactorily for discriminating some specific groups of oils, but to obtain 100% correct classifications for the different oils and mixtures, more information than that obtained from the direct spectra of the oils is needed.     

Assessment of olive oil adulteration by reversed-phase high-performance liquid chromatography/amperometric detection of tocopherols and tocotrienols

A method involving reversed-phase high-performance liquid chromatography with amperometric detection has been developed for the analysis of tocopherols and tocotrienols in vegetable oils. The sample preparation avoids saponification. Recoveries of α-tocotrienol and γ-tocotrienol in extra virgin olive oil were 97.0 and 102.0%, respectively. No tocotrienols were detected in olive, hazelnut, sunflower, and soybean oils, whether virgin or refined. However, relatively high levels of tocotrienols were found in palm and grapeseed oils. This method could detect small quantities (1–2%) of palm and grapeseed oils in olive oil or in any tocotrienol-free vegetable oil and might, therefore, help assess authenticity of vegetable oils.     

Low molecular weight polypeptides in virgin and refined olive oils

Twenty-eight virgin olive oils—from different regions of Spain and prepared from olive drupes of different varieties—and six refined olive oils were analyzed to determine the presence of proteins in these oils. All oils studied showed the presence of proteins in the range of 7–51 μ/100 g of oil. There were no significant differences in protein content in oils from different varieties or between virgin or refined oils. In addition, all oils exhibited analogous amino acid patterns, suggesting a similarity among protein fractions obtained from different oils. A polypeptide with an apparent M.W. of 4600 Da was common to the isolated protein fractions. These results suggest that this polypeptide is a previously unknown minor component in olive oils. No clear influence of this component on oil stability was observed when oil stabilities were estimated as a function of phenol, tocopherol, phosphorus, and protein contents of the oils.     

Frying Quality Characteristics of French Fries Prepared in Refined Olive Oil and Palm Olein

The objective of this study was to compare two oils with different polyunsaturated/saturated (P/S) fatty acid ratios, refined olive oil (P/S 0.75) and palm olein (P/S 0.25), in frying French fries. The chemical qualities of the oil residues extracted from the French fries were assayed for five consecutive batches fried at 1-h intervals. The levels of total polar compounds, free fatty acids, p-anisidine value and phytosterol oxidation products (POPs) were elevated in French fries fried in both oils. The level of total polar compounds increased from 4.6 in fresh refined olive oil to 7.3% in final batches of French fries. The corresponding figures for palm olein were 9.8–13.8%. The level of free fatty acid in fresh refined olive oil increased from 0.06 to 0.11% in final products. These figures for palm olein were 0.04–0.13%. The p-anisidine value increased from 3.7 to 32.8 and 2.5 to 53.4 in fresh oils and in final batches of French fries in refined olive oil and palm olein, respectively. The total amount of POPs in fresh refined olive oil increased from 5.1 to 9.6 μg/g oil in final products. These figures were 1.9 to 5.3 μg/g oil for palm olein.     

Thermoxidative stability of triacylglycerols from mutant sunflower seeds

Thermoxidative stability was evaluated in triaclyglycerols (TAG) from the oils of the mutant sunflower lines CAS-3, CAS-4, and CAS-8 (with a high percentage of stearic acid), CAS-5 (with a high percentage of palmitic acid), all from standard highlinoleic genetic backgrounds, and the mutant sunflower line CAS-12 (with a high percentage of palmitic acid), from a high-oleic genetic background. These oils contained unusually high contents of TAG molecular species with one or two saturated fatty acids at the sn-1,3 positions. Purified total TAG devoid of tocopherols were subjected to controlled thermoxidative treatment at 180°C. Polymerized TAG were determined at 2-h intervals for 10 h. After this time, total polar compounds, oxidized TAG monomers, TAG dimers, and TAG oligomers were determined. TAG from highly saturated sunflower oils with levels of linoleic acid similar to those found in conventional sunflower oils (40–50%) showed enhanced thermal stability. In these TAG, the amount of polar compounds formed during the thermoxidative treatment was similar to that formed in the high oleic acid line. Excellent results were obtained for the TAG of the CAS-12 oil, which had the highest thermal stability, producing half the amount of polar compounds as the conventional line and less than two-thirds that of the high-oleic line.     

Determination of sterols by capillary column gas chromatography. Differentiation among different types of olive oil: Virgin,refined, and solvent-extracted

Compositional analysis of the sterol fraction of olive oil can be used to assess the degree of purity of the oil and the absence of admixture with other plant oils. This determination also permits characterization of the type of olive oil in question: virgin, refined, or solvent-extracted. In the present work, 130 samples of olive oil were analyzed, the sterol fractions were separated from the unsaponifiable fraction by silica gel plate chromatography, and later they were analyzed as the trimethylsilyl ether derivatives by capillary column gas chromatography. From the results obtained, it was concluded that this methodology is able to differentiate among virgin, refined, and solvent-extracted olive oils. Stigmasterol, clerosterol, Δ5-avenasterol, Δ7-stigmasterol, and Δ7-avenasterol permit the differentiation of the three types of oil from one another. Campesterol, Δ5, 23-stigmastadienol, β-sitosterol, and Δ5,24-stigmastadienol permit the differentiation of only two oils from each other but confirm the conclusions obtained for other sterols. Correlations between the different sterols of virgin, refined, and solven-extracted olive oil also have been obtained.     

Stability to oxidation of virgin olive oils as related to olive conditions: Study of polar compounds by chemometric methods

Polar compounds of virgin olive oils were analyzed. They influence oil flavor and aroma and improve the shelf-life of the oil. The orthodiphenolic fraction is particularly significant for oil stability because of its antioxidative activity. A relationship between the composition of the whole fraction of polar compounds and the state of health of the olives was established. For this purpose, oil samples were obtained from olives that had reached different degrees of ripeness and that had been affected by Dacus oleae infestation differently. The polar compounds were then analyzed by high-performance liquid chromatography. The data set was studied by means of chemometric methods. Partial least squares regression was used to obtain models that show a significant correlation between composition of the oil’s polar compounds and conditions of the olives sampled. In particular, compounds with antioxidative activity were directly linked with the state of health of the olives. The models obtained allow tracing of the state of health of the olives sampled through analysis of the polar fraction of virgin olive oil with a high degree of accuracy, and thus prediction of the oil’s expected shelf life.     

Evaluation of medium polarity materials isolated from fried edible oils by RP‐HPLC

Some frying by‐products of medium polarity called medium polarity materials (MPMs) were isolated by reversed‐phase high‐performance liquid chromatography (RP‐HPLC) from three different cooking oils used for frying during the domestic successive deep‐frying of potatoes. The cooking oils investigated were virgin olive oil, sunflower oil and a vegetable shortening oil. The relative RP‐HPLC increments of the MPM fractions showed a significant correlation to the total polar material and to the polymerised triacylglycerol increment. They could be used as a new method for the assessment of fried oil deterioration. The capillary gas chromatography/mass spectrometry analysis revealed two main groups of peaks for the MPM fractions, which are almost identical in the three examined oils. This indicates that the MPM constituents rather result from the triglycerides than from minor constituents of the oils.     

A high-field1H nuclear magnetic resonance study of the minor components in virgin olive oils

High-field (600 MHz) nuclear magnetic resonance (NMR) spectroscopy was applied to the direct analysis of virgin olive oil. Minor components were studied to assess oil quality and genuineness. Unsaturated and saturated aldehyde resonances, as well as those related to other volatile compounds, were identified in the low-field region of the spectrum by two-dimensional techniques. Unsaturated aldehydes can be related to the sensory quality of oils. Other unidentified peaks are due to volatile components, because they disappear after nitrogen fluxing. The statistical analysis performed on the intensity of these peaks in several oil samples, obtained from different olive varieties, allows clustering and identification of oils arising from the same olive variety. Diacylglycerols, linolenic acid, other volatile components, water, acetic acid, phenols, and sterols can be detected simulteneously, suggesting a useful application of high-field NMR in the authentication and quality assessment of virgin olive oil.     

Analysis of the wax ester fraction of olive oil and sunflower oil by gas chromatography and gas chromatography-mass spectrometry

The wax ester fractions of solvent-extracted sunflower oil and “extra virgin” olive oil were obtained by solid-phase extraction and subsequently subjected to gas-chromatographic and gas chromatographic-mass spectrometric analysis. The comprehensive qualitative analysis of these fractions, which was carried out by the interpretation of mass spectral data, revealed several types of wax esters. In olive oil, shortchain, even-numbered wax esters, saturated and unsaturated long-chain, even-numbered wax esters, benzyl esters, and the diterpenic esters phytyl and geranylgeranyl ester (the latter as a minor component) are present. With the exception of benzyl esters, all these esters occur in sunflower oil as well, but in considerably different amounts compared to those in olive oil. Whereas unsaturated wax esters are present in a negligible amount, diterpenic esters, mainly geranylgeranyl esters, represent the major part of the wax ester fraction.     

Wax composition of sunflower seed oils

Waxes are natural components of sunflower oils, consisting mainly of esters of FA with fatty alcohols, that are partially removed in the winterization process during oil refining. The wax composition of sunflower seed as well as the influence of processing on the oil wax concentration was studied using capillary GLC. Sunflower oils obtained by solvent extraction from whole seed, dehulled seed, and seed hulls were analyzed and compared with commercial crude and refined oils. The main components of crude sunflower oil waxes were esters having carbon atom numbers between 36 and 48, with a high concentration in the C₄₀−C₄₂ fraction. Extracted oils showed higher concentrations of waxes than those obtained by pressing, especially in the higher M.W. fraction, but the wax content was not affected significantly by water degumming. The hull contribution to the sunflower oil wax content was higher than 40 wt%, resulting in 75 wt % in the crystallized fraction. The oil wax content could be reduced appreciably by hexane washing or partial dehulling of the seed. Waxes in dewaxed and refined sunflower oils were mainly constituted by esters containing fewer than 42 carbon atoms, indicating that these were mostly soluble and remained in the oil after processing.     

Improved method for the determination of wax esters in vegetable oils

In this work, a modified International Olive Council (IOC) method for wax determination involving a double‐adsorbent layer of silica gel and silver nitrate‐impregnated silica gel is presented (SN method). Column chromatography by the SN method did not show retention of wax esters standards with an even number of carbon atoms (C34–C44), observing recovery percentages higher than 90% even for unsaturated wax esters. All wax fractions were lower by the SN method than by the IOC method, resulting in a percentage decrease in the total wax content (olive oils: 20–50%, crude sunflower oil: 38%, crude soybean oil: 58% and crude grape seed oil: 13%). Olive oils analysed by the SN method showed increases of up to 27% in C40 relative percentage with respect to the IOC method. Additionally, decreases were observed by the SN method in the relative percentages for odd‐carbon atom waxes for the seed oils in comparison to the IOC method (crude sunflower oil: 27%, crude soybean oil: 28% and crude grape seed oil: 13%). The main advantages of the proposed modification consist in its easy implementation and a better determination of wax esters (C34–C60) by controlling their complete recovery and removing interfering substances. The method is suitable for quality control and for authentication of olive oil and seed oils as well as in processing monitoring. Practical applications: The proposed method is useful in the quality, authentication and processing control of fruit and seed oils. Moreover, it can be an important tool for vegetable oil industries to control the efficiency of the wax separation process to prevent turbidity in the refined oil.     

Effects of Filtration on the Quality Properties of Extra Virgin Olive Oils during Storage

In this study, the effects of filtration on quality parameters, chemical characteristics, antioxidant activity, and oxidative stability (OS) of Turkish olive oils during the storage period of 12 months were investigated. The olive oil free acidity (% oleic acid per 100 g of olive oil) (free fatty acidity, FFA), peroxide values (PV) (meq O₂ kg⁻¹ oil), and UV spectrophotometric indices (K₂₃₂ and K₂₇₀ measurements) were used for evaluating the quality parameters of olive oils. α-tocopherol analysis, total phenolic content (TPC), total chlorophyll and carotenoid analyses, and 2,2-diphenyl-1-picrylhydrazyl (DPPH•) radical-scavenging activity (RSA) assays were carried out. Chromatographic methods were applied to determine the fatty-acid and triacylglycerol (TAG) composition, the content of methyl and ethyl esters (FAEE and FAME), and the content of fatty acids of olive oils. Univariate and multivariate statistical methods were performed to evaluate results. Univariate data analysis results showed that filtration of Ayvalık, Memecik, and Domat olive oils had no considerable influence on quality parameters, antioxidant compounds, FAEE and FAME, antioxidant activity, and OS, except TPC (P < 0.05). A significant difference between the samples was determined regarding storage times of the olive oils. Principal component analysis (PCA) analysis revealed that olive oils were grouped according to storage periods of the olive oils regarding fatty-acid and triacylglycerol (TAG) composition while there was no clear separation among the samples according to the filtration process. However, qualitative and quantitative changes took place on minor and major components of olive oils during the storage period.     

Wax analysis of vegetable oils using liquid chromatography on a double-adsorbent layer of silica gel and silver nitrate-impregnated silica gel

A chromatographic method is described to measure the crystallizable wax content of crude and refined sunflower oil. It can also be applied to any other vegetable oil. The preparative liquid chromatography step on a glass column containing a silica gel adsorbent superimposed upon a silver nitrate-impregnated silica gel support is used to isolate a wax fraction which is then analyzed by gas chromatography. The recovered wax fraction contains, in addition to the crystallizable waxes, hydrocarbons and other compounds with gas chromatographic retention times corresponding to waxes with chain lengths C₃₄−C₄₂. These compounds are short-chain saturated waxes in fruit oils, such as grapeseed and pomace. In seed oils such as sunflower, soybean or peanut, the compounds initially referred to as “soluble esters” are identified as monounsaturated waxes, esters of long-chain saturated fatty acids, and a monounsaturated alcohol, mainly eicosenoic alcohol. Such waxes are absent from corn or rice bran oils.     

Detection of pressed hazelnut oil in admixtures with virgin olive oil by analysis of polar components

Analysis of the polar fraction from virgin olive oil and pressed hazelnut oil by high-performance liquid chromatography showed marked differences in the chromatograms of the polar components in the two oils. Six commercial samples of pressed hazelnut oil and 12 samples of virgin olive oil (or blended olive oil including virgin olive oil) were analyzed. The phenolic content of the pressed hazelnut oil samples was 161±6 mg·kg⁻¹. Inspection of the chromatograms showed that the pressed hazelnut oil extracts contained a component that eluted in a region of the chromatogram that was clear in the olive oil samples, and consequently this component could be used to detect adulteration of virgin olive oil by pressed hazelnut oil. The component had a relative retention time of 0.9 relative to 4-hydroxybenzoic acid added to the oil as an internal standard. The ultraviolet spectrum of the component showed a maximum at 293.8 nm, but the component could not be identified. Analysis of blends of oils showed that adulteration of virgin olive oil by commercial pressed hazelnut oil could be detected at a level of about 2.5%.     

Free and Esterified 4,4′-dimethylsterols in Hazelnut Oil and their Retention During Refining Processes

Free and esterified forms of sterols provide detailed information on the identity and the quality of vegetable oils. In this study, 4,4′-dimethylsterols in free and esterified forms were investigated in hazelnut and virgin olive oils. Moreover, a sample of solvent-extracted hazelnut oil was refined at the laboratory to monitor the effects of processing on the levels of 4,4′-dimethylsterols. Generally, the level of total 4,4′-dimethyslterols was higher in the esterified form (49–68%) compared with that in free form (32–51%) of these compounds in the hazelnut oil. In virgin olive oil samples, cycloartenol and 24-methylenecycloartanol were present in higher amounts in free forms (70–80%) than in esterified forms (20–30%). Among the refining processes, degumming, deodorization, neutralization and bleaching, only neutralization and bleaching considerably reduced 4,4′-dimethylsterols. In fully refined hazelnut oil, 18 and 37% of lupeol and an unknown compound X in the esterified form were lost, respectively. The loss of these two compounds in the free form was considerably higher, 26 and 72%, respectively. GC–MS analysis showed that adulteration of olive oil with a sample of fully refined hazelnut oil could be detected at a level as low as 2% by tracing lupeol in total or only in esterified forms of 4,4′-dimethylsterols. Further studies on the levels of free and esterified 4,4′-dimethylsterols and their retention during refining processes are anticipated in hazelnut cultivars from different origins.     

Sterol fractions in hazelnut and virgin olive oils and 4,4′-dimethylsterols as possible markers for detection of adulteration of virgin olive oil

Reports on the methylsterol fractions of hazelnut oils are scarce. The objectives of this study were to characterize methylsterols in hazelnut and virgin olive oils and to study the possibility of detection of adulteration of virgin olive oils. In hazelnut oils, 4-desmethylsterols were present in higher proportions (86 to 91%) than in virgin olive oils where this fraction was ca. 50% of the total sterol. In the 4-monomethylsterol fraction, citrostadienol was the major component in both kinds of oils followed by cycloeucalenol and obtusifoliol in virgin olive oils, and obtusifoliol in hazelnut oils. 24-Methylenecycloartanol was predominant in both kinds of oils in the 4,4′-dimethylsterols. For the first time, δ-amyrin was tentatively identified by comparing published mass spectral data in the analyzed samples of both kinds of oils. An unknown compound X (containing a lupane skeleton) and lupeol were detected only in the 4,4′-dimethylsterols fraction of hazelnut oils at a level of 2–8 and 6–10%, respectively. GC-MS analysis showed that adulteration of virgin olive oil by hazelnut oil could be detected at a level less than 4% by using these two compounds as possible potential markers.