Determination of squalene in olive oil using fractional crystallization for sample preparation

A simple, rapid method for the determination of squalene in virgin olive oil was developed using RP-HPLC with detection at 208 nm. Fractional crystallization from methanol/acetone (7∶3, vol/vol) was applied to obtain squalene in the liquid fraction of the oil prior to HPLC. Elution of squalene was then carried out isocratically with acetone/acetonitrile (40∶60 vol/vol) within 11 min. The detection limit was 23 mg/kg, and the limit of quantification 79 mg/kg. The precision of the crystallization procedure (CV%=3.76, n=7) and the mean recovery (92.5 and 81.5% for the 7,000 and 700 mg/kg levels of addition, respectively) were satisfactory. The method is easily applicable to fulfill future needs for nutrition labeling.     

The contents of proteins and phospholipids in cloudy (veiled) virgin olive oils

Extra virgin olive oil is produced in the form of a “suspension–dispersion” which can persist for several months before full deposition of a residue. Many consumers and chefs prefer unfiltered raw olive oil because it looks thicker and richer in flavors. The nature of the material in the suspension–dispersion is poorly described. The presence of proteins has been connected with the appearance of the “veiled” oil and also with its oxidative stability, although there are discrepancies in the literature with regard to their levels. The level of phosphorus, a measure of phospholipids, is also poorly studied. This work aims at quantifying proteins and phospholipids in cloudy olive oil. For the analysis of proteins, a practical method is used that can be applied for routine analysis. The proteins are precipitated with acetone and determined colorimetrically using the Bradford method suitably modified to measure protein dye‐binding at low concentrations. Twenty three virgin and one refined olive oil samples from different places in Greece were all found to have protein levels below 2.5 mg/kg. In most of the samples, values were lower after filtration of the cloudy oils. In the refined oil samples, protein was hardly determined (value ≤0.1 mg/kg). Phosphorus levels ranged from 0.8 to 4.8 mg/kg. These correspond to approximately 21–124 mg/kg of phospholipids. The results are discussed in relation to the oxidative and physicochemical stability of the veiled oils.     

Rapid FTIR determination of water,phenolics and antioxidant activity of olive oil

A rapid Fourier transformed infrared (FTIR) attenuated total reflectance (ATR) spectroscopic method was applied to the determination of water content (WC), total phenol amount (TP) and antioxidant activity (ABTS ^{. +}) of virgin olive oils (VOO) and olive oils. Calibration models were constructed using partial least squares regression. Oil samples with WC ranging from 289 to 1402 mg water/kg oil, with TP from 46 to 877 mg gallic acid/kg oil and with ABTS ^{. +} from 0 to 5.7 mmol Trolox/kg oil were considered for chemometric analysis. Better results were obtained when selecting suitable spectral ranges; in particular, from 2260 to 1008 cm^?1 for WC, from 3610 to 816 cm^?1 for TP and from 3707 to 1105 cm^?1 for ABTS ^{. +}. Satisfactory LOD values by the FTIR‐chemometric methods were achieved: 9.4 (mg/kg oil) for WC; 12.5 (mg gallic acid/kg oil) for TP, and 0.76 (mmol Trolox/kg oil) for ABTS ^{. +}. The evaluation of the applicability of these analytical approaches was tested by use of validation sample sets (n = 16 for WC, n = 11 for TP and n = 14 for ABTS) with nearly quantitative recovery rates (99–114%). The FTIR–ATR method provided results that were comparable to conventional procedures. Practical applications : The presented method is based on ATR–FTIR in combination with multivariate calibration methodologies and permits a simultaneous evaluation of important quality parameters of VOO (WC, TP and ABTS ^{. +}). This approach represents an easy and convenient means for monitoring olive oil quality with the advantage of ease of operation, speed, no sample pretreatment and no consumption of solvents. The data obtained with this method are comparable to those obtained using the official reference method. Therefore, the technique is highly plausible as an alternative to the standard procedure for routine analysis or control at‐line of production processes.     

Determination of Mineral Oil-Saturated Hydrocarbons (MOSH) in Vegetable Oils by Large Scale Off-Line SPE Combined with GC-FID

A method based on an off-line large-scale solid phase extraction (SPE) approach combined with conventional gas chromatographic-flame ionization detection (GC-FID) was developed to determine the mineral oil-saturated hydrocarbons (MOSH) in vegetable oils. A large-scale SPE column loaded with 10 g of activated silica gel impregnated with 1% silver nitrate which was used to retain lipids and olefins in vegetable oils and the MOSH in the oil samples was eluted with hexane. Then 2 μL concentrated solution was splitlessly injected into a common GC-FID instrument. The quantification limit reached 2.5 mg/kg when the MOSH fraction was concentrated to 0.1 mL. The accuracy of this procedure, as assessed by measuring the recoveries from spiked oil samples, was higher than 80%. This procedure was applied to analyze the MOSH in 38 commercial vegetable oils from Chinese market, which was the first survey of mineral oil contaminant in Chinese edible oils. The oil samples contaminated with different levels of MOSH, among which, 15 samples contained no mineral oils and 3 samples were contaminated with more than 50 mg/kg of MOSH. The highest contamination level was found in one of rice oils, in which the concentration of MOSH was up to 713.36 mg/kg. Of the 9 types of oils analyzed, camellia oil contained MOSH ranging between 6.76 and 78.49 mg/kg, averaging 46.72 mg/kg, indicating a higher contamination level than other types of oils. The results suggested that it is necessary to routinely detect mineral oil contamination in vegetable oils for food safety.     

On the quality control of ‘olive paste’, a specialty based on olives and olive oil

‘Olive paste’ is a preserved food gaining popularity as a gourmet product. Its quality depends on that of the major ingredients, table olives (green or black) and virgin olive oil, as well as on the changes occurring to the constituents of the latter during preparation and storage. In this view, our attention was focused on the characteristics of the lipid fraction (l.f.) of a great number of commercial products after a careful search in retail markets and the web. Ultraviolet absorbance values (K₂₃₂, K₂₇₀) of the l.f., a criterion set for edible and non‐edible olive oil oxidative status due to paste heat treatment during pasteurization, could not support the label information regarding the quality of the oil used. On the contrary, the content of α‐tocopherol (?250 mg/kg l.f. or ?50 mg/kg paste) was a strong indication of good‐quality major ingredients. Within each brand, consistency with labeling was checked through squalene (higher content in products containing higher amounts of olive oil) or β‐carotene determination (higher levels in preparations containing red pepper). For green olive paste samples, the values of the ratio pheophytin a/pyropheophytin a may be used to monitor the shelf life of the product. The findings support routine quality control of the new product.     

Development of steryl ester analysis for the detection of admixtures of vegetable oils

The steryl ester content and composition of 28 samples from 10 vegetable oil types have been determined by isolation of the steryl esters by high-performance liquid chromatography and analysis by gas chromatography. The oils can be classified into oils with a high content (>4000 mg/kg) of steryl esters (corn and rapeseed); oils with a medium content (1400–2400 mg/kg) of steryl esters (sunflower oil and high-oleic sunflower oil); and oils with a low content (<1200 mg/kg) of steryl esters (safflower, soybean, cottonseed, groundnut, olive, and palm oils). The composition of the steryl ester fraction varies to a greater extent for different oil types than for different varieties of the same oilseed. The developed method is promising for authentication of some oils, and is particularly suitable for detecting admixtures of low levels of corn or rapeseed oils.     

Characterization of a 13-lipoxygenase from virgin olive oil and oil bodies of olive endosperms

Olive oil is one of the oldest known vegetable oils, and it is almost unique in that it can be consumed without any refining treatment. One of its most important quality problems is oxidative rancidity due to the oxygenation of polyenoic fatty acids and formation of compounds that derive from these fatty acid hydroperoxides. Beside autoxidation, lipoxygenases (LOXs) were suggested to be involved in this process. Here we show, that approximately 1.6% of all linoleic acid (LA) molecules within olive oil samples had been converted into LOX-derived (13S,9Z,11E)-13-hydroperoxy-9,11-octadecadienoic acid (13-HPODE) as determined by ¹H NMR- and HPLC analysis. LOX activity tests indicated the occurrence of an active 13-LOX exhibiting a pH optimum between pH 5.5 and 6.0. Furthermore, this enzyme preferentially metabolized free fatty acids. In order to elucidate the origin of this LOX, we analyzed olive endosperms for LOX forms. Chromatography of total protein extracts of the tissue showed LOX activity almost exclusively associated with a high molecular mass fraction. Light microscopic inspection, as well as the calculated phosphate, neutral lipid, and protein content of this fraction, suggested that this fraction may contain oil bodies and that LOX activity was associated with their membrane. This LOX activity had a pH optimum of 6.0. Activity assays at various temperatures indicated a significant catalytic efficiency of the enzyme up to 55°C. HPLC analysis of LA oxygenation products within the lipid fraction and of activity tests of isolated oil bodies showed that the LOX present in mature olive endosperm oil bodies was, as the enzyme from olive oil, a linoleate 13-LOX preferentially active on free LA. We suggest, that this oil body LOX from olive endosperm, is the one detected originally in olive oil and may survive at least in part olive oil production.     

Detection of adulteration of olive oil by argentation thin layer chromatography

Minute amounts of adulterant seed oils in olive oil can be detected by GLC analysis of fatty acids of the polyunsaturated triglyceride fraction, obtained by TLC on silver nitrate impregnated silica gel. Every possible effort was made to avoid any critical or time consuming manipulation in this method in order to develop it as a routine testing procedure. A complete analysis is possible in less than 2 hr and the detection of as low as 2% adulteration by other seed oils is accurate and reliable.     

Influence of operation variables on quality parameters of olive husk oil extracted with CO<Subscript>2</Subscript>: Three-step sequential extraction

Supercritical CO₂ extraction is a viable alternative process for the extraction of high-quality oil from olive husk (also known as olive pomace), a residue obtained in the production of olive oil. We analyzed the effect of pressure (100–300 bar), temperature (40–60°C), solvent flow (1–1.5 L/min), and particle size (0.30–0.55 mm) on four important quality parameters of the oil extracted with CO₂: tocopherol concentration, extinction coefficients at 232 and 270 nm, and saponification value. Response surface methodology was used to obtain mathematical expressions related to the operating variables and parameters studied. Results from these experiments were also used to design a three-step sequential CO₂ extraction procedure to obtain a higher-quality extract. The optimal operational sequence consisted of a first extraction step at 75 bar for 1 h using 1% (vol/vol) ethanol modifier, followed by a second extraction stage at 350 bar for 2.5 h without ethanol and a third step, also at 350 bar, for 2.5 h but using ethanol. These extraction conditions obtained an intermediate fraction of oil with 64% yield and all normal parameters according to European Commission food legislation. This fraction is suitable without any further refining. On the contrary, the oils obtained by hexane extraction and by conventional supercritical CO₂ extraction at optimal conditions are suitable for human consumption after further refining. This last finding may result in improved economics of the sequential CO₂ extraction process compared to the conventional extraction method with hexane.     

Quantification of free and esterified steryl glucosides in vegetable oils and biodiesel

Steryl glucosides (SG) are minor components that dramatically modify the low temperature performance of fatty acid methyl esters (FAME) used as biodiesel. SG are naturally present in vegetable oils but they may also be the result of the transesterification of esterified steryl glucosides (ESG). These are present in vegetable oils at a level of a few hundred milligrams per kilogram, depending on the nature of the feedstock. We developed an analytical method to quantify SG and ESG in vegetable oils and in FAME. The purification of SG and ESG was performed by liquid chromatography on silica gel, and the analysis of the trimethylsilyl derivatives was achieved by gas chromatography and flame ionization detection. The filterability of biodiesel is affected when the SG content is higher than 20 mg/kg. Therefore, the sensitivity of this new method is adapted for this purpose since the quantification limit is 10 mg/kg of SG and ESG. The recoveries are acceptable, between 75% and 90% depending on the species and content, and the reproducibility relative standard deviation, evaluated at 10%, is comparable to other studies.     

Potential of selected Portuguese cultivars for the production of high quality monovarietal virgin olive oil

The effect of cultivar and ripeness stage on the potential nutritional value of monovarietal extra virgin olive oils (MEVOOs) obtained from Cordovil, Carrasquinha, Verdeal, and Negrinha do Freixo cultivars was investigated. MEVOOs produced were characterized by high oleic acid (72–83%), tocopherol (182–530 mg/kg), and phenolic compounds (326–1110 mg/kg) content and by a similar polyphenolic profile. 1‐Penten‐3‐one was found to be the compound with the highest contribution for the aroma of the four MEVOO, related to bitter, pungent, and leaf attributes. MEVOO from Verdeal cultivar showed the best performance in terms of the composition: the highest yield of oil, the highest content of oleic acid, high tocopherol, polyphenol and sterol content, and the lowest content of linoleic acid. These characteristics give to these MEVOO not only a great oxidative stability but also interesting properties from the health point of view. MEVOO obtained with fruits at the maturity index of around 4 were in general richer in beneficial minor compounds. MEVOO produced were discriminated by variety and ripeness stage, using a stepwise linear discriminant analysis. This discrimination will in the future enable the prevention of adulteration of these monovarietal olive oils with specific nutritional composition with other olive oils. Practical implications: High‐quality MEVOOs have recently been introduced in the market, which for growers is a practical way to differentiate and increase the commercial value of extra virgin olive oil. The quantification of major and minor olive oil compounds in monovarietal olive oils represents an objective way of predicting the sensory characteristics, stability, and potential health benefits of the oils, as well as preventing their adulteration with other olive oils. This study will help in the selection of olive varieties during the maintenance or development of new olive orchards and also to select optimum harvest period for these varieties, in order to obtain MEVOOs with the maximum quality and health benefits for consumers.     

Comparative study of olive oil quality from Chemlali Sfax versus Arbequina cultivated in Tunisia

The effects of the geographical region on the behavior of the Arbequina olive cultivar (cv) cultivated in the south of Tunisia (in the arid zone of Sfax) was compared to an autochthonous cultivar (Chemlali Sfax). Various olive parameters were analyzed, such as ripening index, pulp/stone ratio, oil contents, and sensory profiles. Most of the quality indices and fatty acid composition showed significant variations among olive cultivars. Arbequina cv is characterized by high oil yield with a less total phenols and pigments content than Chemlali Sfax cv. Cielab spectrophotometer coordinate L*, b*, and a* values show a great difference in olive oil colors. In spite of their low oleic acid contents, autochthonous cultivar presented a higher induction time (6.82 and 2.68 h for Chemlali and Arbequina, respectively) and high contents of phenolic compounds (158.28 and 110.27 mg/kg for Chemlali Sfax and Arbequina, respectively). The most important compounds identified were oleuropein aglycon (45.50 mg/kg), hydroxytyrosol (3.68 mg/kg), 1‐acetoxypinoresinol (6.23 mg/kg) in Chemlali Sfax oil and hydroxytyrosol glucoside (25.15 mg/kg), tyrosol (12.51 mg/kg), and oleuropein aglycon (30.60 mg/kg) in Arbequina oil. Chemlali Sfax also possessed a very bitter taste, whereas the Arbequina had a sweet taste amongst its attributes. The principal component analysis of the results indicated that the geographical region has significantly affected the olive oil quality.     

Identification and classification of olive oils by high-resolution13C nuclear magnetic resonance

Unsaponifiable matter from 19 olive and olive pomace oils were studied by high-resolution¹³C nuclear magnetic resonance spectroscopy. Their spectra showed characteristic peaks that corresponded to molecular substructures rather than the individual constituents present in the unsaponifiable matter. The presence of squalene and other hydrocarbons, sterols and triterpenic alcohols, in addition to other groups of minor compounds, were observed. Based on the analysis of these spectra, it was possible to distinguish among different grades of olive oils by using stepwise discriminant analysis. This direct method of analysis is suggested to be used in artificial neural networks to define oil identity and quality.     

Detection of adulteration of olive oil with seed oils by a combination of column and gas liquid chromatography

Samples of virgin olive oil and refined seed oils, as well as mixtures of olive oil with 10 and 5% seed oils were fractionated by column chromatography on silicic acid impregnated with ammoniacal silver nitrate. It was possible to isolate a characteristic fraction enriched in polyunsaturated triglycerides. Its linoleic acid content in pure olive oil never exceeds 9.3%, whereas in pure seed oils, it varies between 38.1 and 70.1%; in mixtures of olive oil with 10 and 5% of seed oils, the respective values are 22.3–38.2% and 15.6–32.1%. The oleic-to-linoleic acid ratios of the same fraction are more than 7.6 (olive oil), 0.2–0.8 (seed oils), 1.1–2.0 (olive oil with 10% seed oils) and 1.4–3.6 (olive oil with 5% seed oils). These analytical values may be used as a safe criterion for the eventual adulteration of olive oil with seed oils. This work was taken in part from the doctoral dissertation of S. Passaloglou-Emmanouilidou.     

An efficient extraction and clean‐up procedure for pesticide determination in olive oil

This work reports a simple, rapid, and effective extraction method based on liquid–liquid extraction (LLE) followed by matrix solid‐phase dispersion‐sonication for detection, identification and quantification of multiclass pesticides in virgin olive oil using liquid chromatography mass spectrometry (LC‐QTOF‐MS). LLE to extract pesticide residues in virgin olive oil was performed in order to study the centrifugation efficiency to obtain high recovery yield and low co‐extract fat residue in the final extract. Different suitable parameters of MSPD procedure were evaluated, such as nature of dispersing phase, clean‐up adsorbent, and volume of eluting solvent (acetonitrile) in different extraction conditions, with or without sonication. The best results were obtained using 5 g of virgin olive oil, 2 g of PSA as dispersant sorbent, 2 g of Florisil/GCB (70:30 w/w) as clean‐up sorbent, and 15 mL of acetonitrile as eluting solvent under conditions of 15 min ultrasonic bath at RT. Method validation was performed in order to study sensitivity, linearity, precision, and accuracy. Average recoveries ranged between 73.7 and 104.2% with relative SDs 5.3–13.4% at three concentration levels (25, 50, and 100 µg/kg). Detection and quantification limits ranged from 1.5 to 5 µg/kg and 3 to 9 µg/kg, respectively.     

Characterization and chemometric study of crude and refined oils from table olive by‐products

Table olive processing produces defective fruits and the conditioning operations give rise to solid by‐products which are processed to obtain oil. In this study, the most relevant characteristics of crude oils extracted from table olive by‐products were high average acidity values (4.5%, green olives; 8.1%, ripe olives), ECN42 values of 0.34 (green olives) and 0.10 (ripe olives), while 2‐mono‐palmitin averaged 0.92%. The overall content of sterols was 2257 mg/kg (green olives) and 1746 mg/kg (ripe olives), while the concentration of cholesterol was 36 mg/kg (green olives) and 19 mg/kg (ripe olives). The effect of refining was mainly reflected by a decrease in acidity and sterols. Although most characteristics were in agreement with the established regulation for olive oil, the overall trans fatty acid content, the low apparent β‐sitosterol content, and the relatively high cholesterol content prevented their inclusion into classes of crude or refined lampante or pomace olive oils, not even into the vegetable oil category. Therefore, the oils analyzed should be considered for non‐edible purposes. The physicochemical characteristics used for chemometric discrimination permitted discrimination among types of oils (crude, 100%; physically refined, 90%; chemically refined, 100%), elaboration styles (green and ripe olives, 100%) and cultivars (Gordal, Manzanilla, Hojiblanca and Cacereña, 100%), with the sterol composition being the most useful parameter for discrimination.     

GxE Effects on Tocopherol Composition of Oils from Very Old and Genetically Diverse Olive Trees

Tocopherols are compounds with high biological activity, beneficial for human health that can be found in vegetable oils like olive oil, contributing for its resistance to oxidation. In this work, the tocopherol contents of olive oils extracted from centenarian olive trees of six cultivars (cvs. Lentisca, Madural, Rebolã, Redondal, Verdeal, and Verdeal Transmontana) were evaluated during five consecutive crop seasons (2013–2017). Three tocopherol isoforms (α-, β- and γ-tocopherols) were detected in all analyzed olive oils, and their content varied significantly with the cultivar and year of production. The highest amounts were found in cv. Lentisca (456 ± 122 mg/kg olive oil), while the lowest were observed in cv. Verdeal (179 ± 45 mg/kg olive oil). Crop year was the most influential factor, with the highest contents observed in 2013 and lowest in 2014. Principal component analysis and hierarchical clustering analysis helped differentiate olive oils according to cultivar or production year. These data suggest that tocopherol composition may serve as a chemical marker to distinguish the subject cultivar olive oils from centenarian trees either by olive cultivar or by crop year, being some cultivars identified as potential candidates for guaranteeing the production of olive oils rich in these compounds.     

Effect of location on virgin olive oils of the two main Tunisian olive cultivars

The olive oil content in phenolic compounds depends on the variety of the fruit used for its extraction as well as on the predominant climate conditions in the tree cultivation area. Here, we report on the characterization of virgin olive oil samples obtained from fruits of the main Tunisian olive cultivars Chemlali and Chétoui, grown in three different Tunisian locations, Zaghouan (North), Sousse (Center) and Sfax (South). Chétoui olive oil samples obtained from fruits of olive trees cultivated in Zaghouan and Chemlali olive oil samples obtained from fruits of olive trees cultivated in Sousse were found to have a higher mean total phenol content (1004 and 330 mg/kg, respectively). Olive oil samples obtained from fruits of both cultivars had different phenolic profiles and a higher content in 3,4‐DHPEA‐EDA when the olive trees were cultivated in Zaghouan. Both olive cultivars were found to have different responses to environmental conditions. Chétoui olive oil showed decreased oxidative stability when the fruits were obtained from olive trees cultivated in the center of Tunisia (34.8 h) and in Sfax (16.17 h). Furthermore, statistical data showed that the phenolic composition and oxidative stability of Chétoui olive oil varied more by location than those of Chemlali olive oils.     

Quality characteristics of olive leaf‐olive oil preparations

Olive leaf‐olive oil preparations were obtained by vigorous mixing at various levels of addition (5, 10, 15%w/w) of new or mature leaves. After removal of the plant material via centrifugation, quality and sensory characteristics of the preparations were determined. Oxidative stability (120°C, 20 L/h) and DPPH radical scavenging were increased ～2–7 fold depending on the level of leaves used due to enrichment with polar phenols, mainly oleuropein, and a‐tocopherol. The extraction process affected the chlorophyll content and organoleptic traits as indicated by acceptability and preference tests (n = 50). Forty‐four % of the panelists identified a strong pungency in preparations with 15% w/w new leaves. Fifty‐four % of them identified a bitter taste in those with 15% w/w mature leaves, which was attributed to high levels of oleuropein (～200 mg/kg oil). Olive leaf‐olive oil preparations had interesting properties regarding antioxidants present that can attract the interest of a functional product market. Practical applications: The wider use of olive oil and derived products is highly desirable. In this sense, the current study presents data that support introduction to the market of a new specialty olive oil based solely on olive tree products (olive oil and leaves). Thus, in addition to olive oil and olive paste, a new product, that is an olive oil enriched with olive leaf antioxidants, especially oleuropein produced via a “green” technique (mechanical means instead of extraction with organic solvent) can be made available for consumers.     

Stability of refined olive oil and olive‐pomace oil added by phenolic compounds from olive leaves

Refined olive oil and olive‐pomace oil were enriched with olive leaf phenolic compounds in order to enhance its quality and bring it closer to virgin olive oil. The changes that occurred in the concentrations of pure oleuropein, oleuropein aglycone, hydroxytyrosol acetyl and α‐tocopherol at 400 µg/kg of oil during the storage of refined olive oil and olive‐pomace oil under accelerated conditions (50 °C) were investigated. In a period of 4 months, α‐tocopherol decomposed by 75% whereas less than 40% of the phenols were lost. During storage, enzymatic olive leaf extract hydrolysate that contains two major compounds, hydroxytyrosol and oleuropein aglycone showed the highest antioxidant activity and the lowest detected stability, followed by oleuropein. The oleuropein in olive leaf extracts exhibited similar degradation profiles, reducing by 60–50% and 80% for the olive oil and olive‐pomace oil in 6 months, respectively. The acetylated extract, however, displayed a loss of 10 and 5% in olive oil and olive‐pomace oil, respectively. In the fatty acid composition, an increase in oleic acid and a decrease in linoleic acid were observed. The antiradical activities of the olive oil and olive‐pomace oil enriched with olive leaf phenolic compounds at 400 ppm showed that enzymatic hydrolysate extract had the highest protective effect against oil oxidation. Based on the Rancimat method, the oils with added leaf enzymatic hydrolysate extract had the lowest peroxide value and the highest stability. After 6 months of storage and at 120 °C, the oxidative resistance of refined olive oil and olive‐pomace oil reached 0.71 and 0.89 h, respectively, whereas that of the non‐enriched samples fell to zero.