Functional Characterization of Four Olive Squalene Synthases with Respect to the Squalene Content of the Virgin Olive Oil.

The release of new olive cultivars with an increased squalene content in their virgin olive oil is considered an important target in olive breeding programs. In this work, the variability of the squalene content in a core collection of 36 olive cultivars was first studied, revealing two olive cultivars, 'Dokkar' and 'Klon-14', with extremely low and high squalene contents in their oils, respectively. Next, four cDNA sequences encoding squalene synthases (SQS) were cloned from olive. Sequence analysis and functional expression in bacteria confirmed that they encode squalene synthases. Transcriptional analysis in distinct olive tissues and cultivars indicated that expression levels of these four SQS genes are spatially and temporally regulated in a cultivar-dependent manner and pointed to OeSQS2 as the gene mainly involved in squalene biosynthesis in olive mesocarp and, therefore, in the olive oil. In addition, the biosynthesis of squalene appears to be transcriptionally regulated in water-stressed olive mesocarp.

Stepwise strategy based on 1H-NMR fingerprinting in combination with chemometrics to determine the content of vegetable oils in olive oil mixtures.

1H NMR fingerprinting of edible oils and a set of multivariate classification and regression models organised in a decision tree is proposed as a stepwise strategy to assure the authenticity and traceability of olive oils and their declared blends with other vegetable oils (VOs). Oils of the 'virgin olive oil' and 'olive oil' categories and their mixtures with the most common VOs, i.e. sunflower, high oleic sunflower, hazelnut, avocado, soybean, corn, refined palm olein and desterolized high oleic sunflower oils, were studied. Partial least squares (PLS) discriminant analysis provided stable and robust binary classification models to identify the olive oil type and the VO in the blend. PLS regression afforded models with excellent precisions and acceptable accuracies to determine the percentage of VO in the mixture. The satisfactory performance of this approach, tested with blind samples, confirm its potential to support regulations and control bodies.

Olive oil mixtures. Part one: Decisional trees or how to verify the olive oil percentage in declared blends.

The commercialization of declared blends of olive oil and seed oil is something long approved by the European Union. There, the olive oil percentage must be at least 50% if the producer aims to advertise its presence on the front label, i.e., somewhere other than in the ingredients list. However, the Regulation did not propose any method to verify such proportion. For this purpose, we recommend the use of decisional trees, being the parameters under study those in which the greatest differences between olive and seed oils are shown: triacylglycerols, acyclic saturated hydrocarbons, free sterols, and tocopherols. In this way, to guarantee the presence of olive oil at 50%: i) palmitodiolein must be above 11-15%; ii) the ß/γ-tocopherol ratio must be below 2.4; iii) the alkane sum C21-C25 should be higher than 3.5-6%; and iv) the total sterol content cannot surpass 2400 mg/kg.

Characterization of glyceridic and unsaponifiable compounds of Sacha inchi (Plukenetia huayllabambana L.) oils.

This work deals with the characterization of the main glyceridic and unsaponifiable components of oils obtained from Sacha inchi (Plukenetia huayllabambana L.) seed ecotypes collected during two harvests in the Department of Amazonas in Peru. The seed-oil yield was 30.3-41.2%; standing out are the high percentages of the ω3- and ω6-fatty acids series whose ranges lie within those of the present Regulation for Sacha inchi oils. Triacylglycerols with even equivalent carbon number (ECN; 36-42) were the main components. Minor glyceridic polar compounds such as oxidized triglycerides, diglycerides, monoglycerides, and free fatty acids were determined by high-performance size exclusion chromatography. The low campesterol/stigmasterol ratio (1:6), unusual in the majority of vegetable oils, stands out. Regarding aliphatic hydrocarbons, these oils showed a particular profile for the saturated series of odd and even carbon atom numbers. According to our results Sacha inchi P. huayllabambana oils can be offered as a good alternative to P. volubilis, the species mainly commercialized for this vegetable oil.

Comparative study of phytosterol derivatives in monovarietal olive oils.

Plant sterols and their derivatives are minor compounds that have been extensively studied in vegetable oils, mainly in olive oil, where they are closely related with its identity. The objective of this work is to determine the content of free and esterified steryl glucosides and their profiles in olive oil in relation to different geographical situation of olive orchards, cultivar, farming modality, and sampling time. The orchards under study were located in the outer ring of the submetropolitan area of Madrid (Spain), where olives from Cornicabra, Manzanilla Cacereña, Manzanilla Castellana, and Picual varieties were grown under traditional and organic modes, and harvested in four different samplings. Conclusions state that cultivar, farming mode, and light exposure do not have outstanding effects, whereas pedoclimate might affect the steryl glucoside presence in a substantial way. Further studies are being carried out presently in order to confirm such statement. Also glucoside derivative profiles are discussed, and reasons for differences with results in previous studies pointed out.

Rapid determination of olive oil oxidative stability and its major quality parameters using vis/NIR transmittance spectroscopy.

This paper reports the determination of the olive oil stability index (OSI) by multivariate models from the visible and near-infrared spectrum. The technique proposed is rapid and nondestructive and can be used as a multiparametric method. Moreover, it does not require specific instrumentation, and it is environmentally friendly. The determination of the OSI using the Rancimat instrument was used as a reference method. Predictive visible and near-infrared (vis/NIRS) models were obtained from partial least squares (PLS) for the OSI, showing satisfactory performance in independent tests as proven by the R(2) values of 0.93 and 0.94 from the calibration and the residual predictive deviation (RPD) of the external validations of 3.30 and 3.00, respectively. Predictive models for the determination of free fatty acids, peroxide value, and conjugated dienes were also developed, and their satisfactory performances were demonstrated by RPDs of 3.14, 2.84, and 2.56; hence, its multiparametric determination together with OSI would be possible.

On the glucoside analysis: simultaneous determination of free and esterified steryl glucosides in olive oil. Detailed analysis of standards as compulsory first step.

This work covers two important gaps in the field of micronutrient databases: herein we describe a short and easy protocol that allows the analysis of both free and esterified steryl gulcosides in olive oil. By utilising accurate quantitative methods we achieve a better understanding of olive oil composition and health promoting properties. The procedure consists of isolating the fraction of interest through solid phase extraction, and using gas chromatography-flame ionisation detection for both identification and quantification of the derivatised species. Additionally, mass-spectrometry detection has been utilised for confirming the identity of the individual esterified steryl glucosides in some cases. The method's limit of detection has been set at 0.37mg/kg for each free steryl glucoside and 0.20mg/kg for each esterified steryl glucoside, whereas the recoveries are around 96% and 77%, respectively. Finally, we provide a complete analysis of the commercial standard for esterified steryl glucosides, since such information was not yet available.

Polycyclic aromatic hydrocarbons in spanish olive oils: relationship between benzo(a)pyrene and total polycyclic aromatic hydrocarbon content.

Samples of Spanish virgin olive oils (VOOs) from different categories, origins, varieties, and commercial brands were analyzed by HPLC with a programmable fluorescence detector to determine the content of nine heavy polycyclic aromatic hydrocarbons (PAHs): benzo(a)anthracene, chrysene, benzo(e)pyrene, benzo(b)fluoranthene, benzo(k)fluoranthene, benzo(a)pyrene, dibenzo(a,h)anthracene, benzo(g,h,i)perilene, and indeno(1,2,3-c,d)pyrene. Samples of olive pomace and crude olive pomace oils were also investigated. Benzo(a)pyrene concentrations were below the allowed limit in the European Union (2 microg/kg) in 97% of the VOO samples. Only those samples coming from contaminated olive fruits or obtained in oil mills with highly polluted environments exceeded this value. High correlation coefficients (<0.99) were obtained between the contents of benzo(a)pyrene and the sum of the nine PAHs for all of the analyzed categories, suggesting that benzo(a)pyrene could be used as a marker of the content of these nine PAHs in olive oils.

Alkyl esters of fatty acids a useful tool to detect soft deodorized olive oils.

Fatty acid alkyl esters (FAAEs) are a family of natural neutral lipids present in olive oils and formed by esterification of free fatty acids (FFAs) with low molecular alcohols. Inappropriate practices during the olive oil extraction process and bad quality of the olive fruits promote their formation. Quantification can be done by isolation with a silica gel solid phase extraction cartridge followed by analysis on a gas chromatograph equipped with a programmed temperature vaporizer injector using a polar capillary column. The application of the method to more than 100 Spanish olive oils from different categories, varieties, and geographical origin allowed for establishing the average content of FAAEs and distinguishing the Spanish protected denomination of origin (PDO) and extra virgin olive oils from other categories of olive oils. Those other categories of oils can be subjected to a mild refining process, which leads to blending with extra virgin olive oils. Studies on low quality oils subjected to mild refining showed that FAAEs remain after that process. Thereby, blends of extra virgin olive and mildly refined low quality olive oils can be detected by their alkyl ester concentrations.

Sources of contamination by polycyclic aromatic hydrocarbons in Spanish virgin olive oils.

The presence of polycyclic aromatic hydrocarbons (PAHs) in virgin olive oils results from contamination on olive skins and the oil itself during processing. Determination of nine PAHs was carried out by isolation of the hydrocarbon fraction and subsequent clean-up by solid phase extraction, followed by RP-HPLC analysis using a programmable fluorescence detector. Contamination of olive skins depends directly on environmental pollution levels and inversely on fruit size. In the oil mill, PAHs levels were increased by contamination from combustion fumes during the extraction process. Other procedures, such as washing or talc addition during extraction, did not affect PAHs levels. High concentrations of PAHs were only found as a consequence of accidental exposure to contamination, such as direct contact of olives with diesel exhaust and oil extraction in a polluted environment.

Relationships between oxidative stability, triacylglycerol composition, and antioxidant content in olive oil matrices.

In olive oils, relationships between oxidative stability, glyceridic composition, and antioxidant content were investigated. Lipid matrices, obtained by purification of olive and high-oleic sunflower oils, were spiked with hydroxytyrosol, alpha-tocopherol, and mixtures of them and then subjected to oxidation in a Rancimat apparatus at 100 degrees C. At the same concentration of antioxidants, induction time (IT) decreased as the unsaturation rate of the matrix increased, but only fair correlations were found with fatty acid composition. Oxidative susceptibility (OS(TAG)) was calculated as a function of the relative oxidation rate of the triacylglycerols, and a linear relationship-IT (h) = (a + b)OS(TAG)-between induction time and this parameter showed a good correlation coefficient (r > 0.990, p < 0.001). In the case of matrices with a single antioxidant, origin ordinate (a) and slope (b) can be calculated as a function of the antioxidant concentration. In matrices spiked with mixtures of hydroxytyrosol and alpha-tocopherol, a simple relationship between the coefficients a and b and the concentration of antioxidants cannot be established because additive and subtractive effects occur depending on the relative concentrations of both antioxidants. However, approximate values for these coefficients can be obtained, allowing the estimation of the oil stability. In various olive oils, an acceptable agreement was found between the IT experimentally determined and that calculated from the oil composition. These results confirmed that the Rancimat stability of olive oils mainly depends on triacylglycerol composition and concentrations of o-diphenols and alpha-tocopherol.

Determination of esters of fatty acids with low molecular weight alcohols in olive oils.

A simple and precise analytical method was developed for the simultaneous determination of squalene and methyl, ethyl, propyl, and butyl esters of fatty acids present in olive and olive pomace oils. A fraction containing squalene and fatty acid alkyl esters was isolated from the oil by solid phase extraction on silica gel cartridges and quantitatively analyzed by gas chromatography. A modification of the procedure allowed the isolation of squalene and esters separately. Repeatability and recovery of the method were good. The method was applied to extra and lampant virgin olive oil categories and also to oils obtained from olive pomace by second centrifugation and solvent extraction. Extra virgin olive oils contained low amounts of fatty acid methyl and ethyl esters, while oils obtained from altered olive or olive pomace showed high concentrations of fatty acid alkyl esters, mainly ethyl esters. Correlation between oil acidity and ethyl esters concentration was poor.