Storage Stability of Arauco Virgin Olive Oil: Evolution of Its Quality Parameters and Phenolic and Triterpenic Compounds under Different Conservation Conditions.

The storage conditions are very critical to minimize hydrolytic and oxidative reactions of virgin olive oils (VOOs). These reactions are logically influenced by the composition of the VOO, so that each variety may have a specific behavior. The aim of this study was to evaluate changes in quality parameters and in the phenolic and triterpenic profile of Arauco VOOs, a unique local variety from Argentina, after storage under different conditions. The effects of exposure to light (darkness and light), temperature (24 and 40 °C), packaging material (polyethylene (PET) and dark glass), and headspace (air and N2 atmosphere) were investigated for 76 days. A reduction in total phenolic compounds was observed after storage treatments, but all samples still complied with the EFSA health claim after the different handlings. Overall, the results revealed that the preservation of the oils in PET appears adequate, with improved stability when N2 was used in the headspace, along with darkness and low temperature. The study of phenolic profiles showed that substances previously reported as possible markers of olive oil aging, such as hydroxytyrosol and an isomer of decarboxymethyl oleuropein aglycone, also have a similar behavior during the aging of Arauco variety oil. Interestingly, some evidence was found that another oleuropein-derived compound (oleuropein aglycone isomer 3) could also be used as an aging marker.

Evolution of the metabolic profile of virgin olive oil during deep-frying: Assessing the transfer of bioactive compounds to the fried food.

Deep-frying in virgin olive oil (VOO) is favorable due to its desirable composition and high content of bioactive compounds that can be transferred to fried food. The main goal of this work was to investigate the evolution of VOO metabolic profile during consecutive deep-frying cycles and assess the transfer of metabolites to French fries. The evolution of 56 compounds was monitored by two complementary liquid chromatography methods, using mass spectrometry, diode array, and fluorescence detectors. Sterols and lignans were remarkably stable (greater than 70 % retention in frying oil). Seven out of the ten compounds' classes identified in the oil were transferred to the fried food. Potatoes fried in Arbequina oil from Brazil incorporated the highest amounts of VOO minor components, among the analyzed samples, and sterols presented the highest transfer rate. French fries were enriched by VOO bioactive compounds during deep-frying, especially on the first two days, improving their nutritional value.

Polycyclic aromatic hydrocarbons in edible oils: An overview on sample preparation, determination strategies, and relative abundance of prevalent compounds.

Polycyclic aromatic hydrocarbons (PAHs) are food contaminants whose presence in foodstuffs is especially alarming due to their carcinogenic character. These substances are highly lipophilic and thus, unsafe levels of these compounds have been found in edible fats and oils. Efficient methodologies to determine such molecules in lipidic matrixes are therefore essential. In this review, a detailed description of the analytical methods for the determination of PAHs in vegetable oils from the last 15 years has been provided. Particular emphasis has been placed on innovative sample treatments, which facilitate and shorten the pretreatment of the oils. Finally, results from recent investigations have been reviewed and studied in depth, in order to elucidate which PAHs are most commonly found in vegetable oils.

Study of the minor fraction of virgin olive oil by a multi-class GC-MS approach: Comprehensive quantitative characterization and varietal discrimination potential.

For the first time, a multi-class GC-MS method was applied to perform the quantitative-profiling of the minor fraction of VOOs (considering >40 compounds) in a single run. This comprehensive methodology has demonstrated a remarkable profiling ability on five groups of compounds (phenolic and triterpenic compounds, tocopherols, sterols and free fatty acids) with wide range of polarities/volatilities and chemical entities. After the complete analytical validation of the method, 32 VOO samples from eight different cultivars (some of them very scarcely studied before) were analyzed and the quantitative results were subjected to both non-supervised and supervised multivariate statistics for testing the capability of the determined VOO minor compounds to discriminate the varietal origin of the samples. Typical compositional profiles were defined for each cultivar and promising potential varietal markers were pointed out. The models built to discriminate Cayon and Maurino samples from the rest exhibited the best quality parameters. The relative levels of tocopherols together with characteristic concentration of luteolin, ß-sitosterol and tyrosol were, for instance, the most specific features of Cayon VOOs.

Production of Amphidinols and Other Bioproducts of Interest by the Marine Microalga Amphidinium carterae Unraveled by Nuclear Magnetic Resonance Metabolomics Approach Coupled to Multivariate Data Analysis.

This study assessed the feasibility of an NMR metabolomics approach coupled to multivariate data analysis to monitor the naturally present or stresses-elicited metabolites from a long-term (>170 days) culture of the dinoflagellate marine microalgae Amphidinium carterae grown in a fiberglass paddlewheel-driven raceway photobioreactor. Metabolic contents, in particular, in two members of the amphidinol family, amphidinol A and its 7-sulfate derivative amphidinol B (referred as APDs), and other compounds of interest (fatty acids, carotenoids, oxylipins, etc.) were evaluated by altering concentration levels of the f/2 medium nutrients and daily mean irradiance. Operating with a 24 h sinusoidal light cycle allowed a 3-fold increase in APD production, which was also detected by an increase in hemolytic activity of the methanolic extract of A. carterae biomass. The presence of APDs was consistent with the antitumoral activity measured in the methanolic extracts of the biomass. Increased daily irradiance was accompanied by a general decrease in pigments and an increase in SFAs (saturated fatty acids), MUFAs (monounsaturated fatty acids), and DHA (docosahexaenoic acid), while increased nutrient availability lead to an increase in sugar, amino acid, and PUFA ω-3 contents and pigments and a decrease in SFAs and MUFAs. NMR-based metabolomics is shown to be a fast and suitable method to accompany the production of APD and bioactive compounds without the need of tedious isolation methods and bioassays. The two APD compounds were chemically identified by spectroscopic NMR and spectrometric ESI-IT MS (electrospray ionization ion trap mass spectrometry) and ESI-TOF MS (ESI time-of-flight mass spectrometry) methods.

Cardioprotective Effect of a Virgin Olive Oil Enriched with Bioactive Compounds in Spontaneously Hypertensive Rats.

Olive oil and its derivatives have been described to exert beneficial effects on hypertensive states and cardiovascular disease prevention. We studied the effects of chronic consumption of extra virgin olive oil (EVOO), enriched in bioactive compounds from olive fruit and leaves, on blood pressure, endothelial function, oxidative and inflammatory status, and circulating cholesterol levels, in spontaneously hypertensive rats (SHR). Thirty SHR were randomly assigned to three groups: a control untreated SHR group, an SHR group (1 mL/rat/day) of a control olive oil (17.6 mg/kg of phenolic compounds), and an SHR group (1 mL/rat/day) of the enriched EVOO (750 mg/kg of phenolic compounds) for eight weeks. Ten Wistar Kyoto rats (WKY) were included as healthy controls. Long-term administration of the enriched EVOO decreased systolic blood pressure and cardiac hypertrophy, and improved the ex vivo aortic endothelial dysfunction measured in SHR. Moreover, enriched oil supplementation reduced the plasma levels of Angiotensin II and total cholesterol, and the urinary levels of endothelin-1 and oxidative stress biomarkers, while pro-inflammatory cytokines were unaffected. In conclusion, sustained treatment with EVOO, enriched in bioactive compounds from the olive fruit and leaves, may be an effective tool for reducing blood pressure and cholesterol levels alone or in combination with pharmacological anti-hypertensive treatment.

Evaluating the reliability of specific and global methods to assess the phenolic content of virgin olive oil: Do they drive to equivalent results?

Despite the huge number of different published methodologies, there is an open debate regarding which one is the most convenient analytical strategy for the determination of phenolic compounds from virgin olive oils. Diverse technical issues together with the disparity of criteria regarding results expression cause a lot of confusion. Herewith, a systematic comparison between specific (a powerful and LC-MS method) and global methodologies (the Folin-Ciocalteau (FC) colorimetric assay, the International Olive Council (IOC) method and hydrolysis plus HPLC-DAD) has been carried out. Thus, these strategies have been applied to the analysis of 50 extra virgin olive oils (covering all the possible quantitative ranges of these substances). This is the first time in which the individual LC-MS quantification of so many phenolic substances is included in this kind of comprehensive comparison. The outcomes of all the strategies have been thoroughly confronted and their equivalence (or divergence) has been carefully evaluated, establishing possible correspondence factors. The LC-MS individual determination with the pure standard of every analyte represented the ideal situation; when only the commercially available standards were used, a drastic change was observed in the absolute concentrations of oleuropein derivatives (in terms of hydroxytyrosol). Total phenolic content (summing individual levels) proved to be higher (1.9-3.0 times when data was expressed in mg/kg) than the values given by the three non-specific methods (with R2 from 0.84 to 0.90). In any case, the IOC method, the FC assay and the hydrolysis approach could be considered as feasible strategies when a global value is pursued. Good correlations between their results were found (R2 > 0.89), with the following equivalence factors: FC(mg caffeic acid/kg) ≈ 0.60 IOC(mg TY/kg); IOC(mg TY/kg) ≈ 1.27 Sum acid hydrolysis(mg TY+HTY/kg); FC(mg HTY/kg) ≈ 1.04 Sum acid hydrolysis(mg TY+HTY/kg).

Deep insight into the minor fraction of virgin olive oil by using LC-MS and GC-MS multi-class methodologies.

Several analytical methods are available to evaluate virgin olive oil (VOO) minor compounds; however, multi-class methodologies are yet rarely studied. Herewith, LC-MS and GC-MS platforms were used to develop two methods capable of simultaneously determine more than 40 compounds belonging to different VOO minor chemical classes within a single run. A non-selective and highly efficient liquid-liquid extraction protocol was optimized for VOO minor components isolation. The separation and detection conditions were adjusted for determining phenolic and triterpenic compounds, free fatty acids and tocopherols by LC-MS, plus sterols and hydrocarbons by GC-MS. Chromatographic analysis times were 31 and 50 min, respectively. A comparative assessment of both methods in terms of analytical performance, easiness, cost and adequacy to the analysis of each class was carried out. The emergence of this kind of multi-class analytical methodologies greatly increases throughput and reduces cost, while avoiding the complexity and redundancy of single-chemical class determinations.

Characterization of phenolic extracts from Brava extra virgin olive oils and their cytotoxic effects on MCF-7 breast cancer cells.

The aim of the present work was to evaluate the phenolic profile of the 'Brava' extra virgin olive oil and assess its potential as a "natural adjuvant" in combination with chemotherapy treatment. The total phenol content of the phenolic extracts was 764 mg gallic acid equivalents/kg and the total antioxidant capacity was 2309, 1881 and 2088 µM trolox equivalents/kg determined by Diphenyl-1-picrylhydrazyl free radical method, Ferric Reducing Antioxidant Power and Trolox Equivalent Antioxidant Capacity assay, respectively. Secoiridoids comprised 83% of the total phenolic compounds. The main secoiridoid from oleuropein was the main isomer of oleuropein aglycone (74 mg/kg). The main secoiridoid from ligstroside was the main isomer of ligstroside aglycone (214 mg/kg). These phenolic extracts showed a significant decrease in cell viability on MCF-7 breast cancer cells in a dose and time dependent manner. 48 h-treatments with different concentrations of the extracts induced intracellular ROS generation and cell death.

Nutraceutical Potential of Phenolics from 'Brava' and 'Mansa' Extra-Virgin Olive Oils on the Inhibition of Enzymes Associated to Neurodegenerative Disorders in Comparison with Those of 'Picual' and 'Cornicabra'.

The increasing interest in the Mediterranean diet is based on the protective effects against several diseases, including neurodegenerative disorders. Polyphenol-rich functional foods have been proposed to be unique supplementary and nutraceutical treatments for these disorders. Extra-virgin olive oils (EVOOs) obtained from 'Brava' and 'Mansa', varieties recently identified from Galicia (northwestern Spain), were selected for in vitro screening to evaluate their capacity to inhibit key enzymes involved in Alzheimer's disease (AD) (acetylcholinesterase (AChE), butyrylcholinesterase (BuChE) and 5-lipoxygenase (5-LOX)), major depressive disorder (MDD) and Parkinson's disease (PD) (monoamine oxidases: hMAO-A and hMAO-B respectively). 'Brava' oil exhibited the best inhibitory activity against all enzymes, when they are compared to 'Mansa' oil: BuChE (IC50 = 245 ± 5 and 591 ± 23 mg·mL-1), 5-LOX (IC50 = 45 ± 7 and 106 ± 14 mg·mL-1), hMAO-A (IC50 = 30 ± 1 and 72 ± 10 mg·mL-1) and hMAO-B (IC50 = 191 ± 8 and 208 ± 14 mg·mL-1), respectively. The inhibitory capacity of the phenolic extracts could be associated with the content of secoiridoids, lignans and phenolic acids.

Development and validation of LC-MS-based alternative methodologies to GC-MS for the simultaneous determination of triterpenic acids and dialcohols in virgin olive oil.

Pentacyclic triterpenes are minor, but very relevant compounds found in virgin olive oil (VOO). A rapid and reliable LC-MS method for determining the triterpenic acids and dialcohols (after ultrasound assisted extraction) from VOO has been developed, giving an alternative to the widely used GC (FID/MS) methodologies. The analytical parameters of the proposed method were exhaustively checked, establishing limits of detection (from 1 to 95µg/l) and quantification, precision (RSD values for inter-day repeatability were found between 4.2 and 7.3% considering area values), trueness (within the range 92.7 and 100.5%) and evaluating possible matrix effect (which was no significant). The method was applied to the analysis of six triterpenic compounds in 11 monovarietal VOOs and the results compared with the quantitative GC-MS data. Moreover, the direct injection (after a simple dilution) of the samples into the LC-MS system was also tested, in an attempt to proffer an even simpler sample treatment.

Impact of industrial hammer mill rotor speed on extraction efficiency and quality of extra virgin olive oil.

Crushing is a key step during olive oil extraction. Among commercial crushers, the hammer mill is the most widely used due to its robustness and high throughput. In the present work, the impact of hammer mill rotor speed on extraction yield and overall quality of super-high-density Arbosana olive oils were assessed in an industrial facility. Our results show that increasing the rotor speed from 2400rpm to 3600rpm led to a rise in oil yield of 1.2%, while conserving quality parameters. Sensory analysis showed more pungency with increased rotation speed, while others attributes were unaffected. Volatile compounds showed little variation with the differences in crusher speed; however, total phenols content, two relevant secoiridoids, and triterpenoids levels increased with rotor speed. Hammer mill rotor speed is a processing variable that can be tuned to increase the extraction efficiency and modulate the chemical composition of extra virgin olive oil.

Olive oil authentication: A comparative analysis of regulatory frameworks with especial emphasis on quality and authenticity indices, and recent analytical techniques developed for their assessment. A review.

Over the last decades, olive oil quality and authenticity control has become an issue of great importance to consumers, suppliers, retailers, and regulators in both traditional and emerging olive oil producing countries, mainly due to the increasing worldwide popularity and the trade globalization of this product. Thus, in order to ensure olive oil authentication, various national and international laws and regulations have been adopted, although some of them are actually causing an enormous debate about the risk that they can represent for the harmonization of international olive oil trade standards. Within this context, this review was designed to provide a critical overview and comparative analysis of selected regulatory frameworks for olive oil authentication, with special emphasis on the quality and purity criteria considered by these regulation systems, their thresholds and the analytical methods employed for monitoring them. To complete the general overview, recent analytical advances to overcome drawbacks and limitations of the official methods to evaluate olive oil quality and to determine possible adulterations were reviewed. Furthermore, the latest trends on analytical approaches to assess the olive oil geographical and varietal origin traceability were also examined.

Metabolic profiling approach to determine phenolic compounds of virgin olive oil by direct injection and liquid chromatography coupled to mass spectrometry.

A LC-MS method involving direct injection of extra-virgin olive oil (EVOO) - after a simple dilution - for determining its phenolic compounds has been developed. Optimization of the most appropriate solvent for sample dilution, selection of the optimum oil/solvent ratio, and establishment of column cleaning strategy and maximum number of injections were some of the most relevant steps. Then, the analytical parameters of the method were evaluated, establishing LOD (from 3.3 to 31.6µg/L) and LOQ, precision (RSD values for inter-day repeatability were found between 3.49 and 6.12%), and trueness (within the range 89.9-102.3% for 1.0mg/L) and checking possible matrix effect (which was no significant). Three kinds of calibration were used: external standard, standard addition and calibration in a phenols-free matrix, which was subsequently applied to quantify the phenolic compounds in 16 EVOOs (from 6 cultivars). A total of 21 compounds were determined without the need of using any extraction protocol.

Assessing the varietal origin of extra-virgin olive oil using liquid chromatography fingerprints of phenolic compound, data fusion and chemometrics.

High Performance Liquid Chromatography (HPLC) with diode array (DAD) and fluorescence (FLD) detection was used to acquire the fingerprints of the phenolic fraction of monovarietal extra-virgin olive oils (extra-VOOs) collected over three consecutive crop seasons (2011/2012-2013/2014). The chromatographic fingerprints of 140 extra-VOO samples processed from olive fruits of seven olive varieties, were recorded and statistically treated for varietal authentication purposes. First, DAD and FLD chromatographic-fingerprint datasets were separately processed and, subsequently, were joined using "Low-level" and "Mid-Level" data fusion methods. After the preliminary examination by principal component analysis (PCA), three supervised pattern recognition techniques, Partial Least Squares Discriminant Analysis (PLS-DA), Soft Independent Modeling of Class Analogies (SIMCA) and K-Nearest Neighbors (k-NN) were applied to the four chromatographic-fingerprinting matrices. The classification models built were very sensitive and selective, showing considerably good recognition and prediction abilities. The combination "chromatographic dataset+chemometric technique" allowing the most accurate classification for each monovarietal extra-VOO was highlighted.

Potential of LC Coupled to Fluorescence Detection in Food Metabolomics: Determination of Phenolic Compounds in Virgin Olive Oil.

A powerful chromatographic method coupled to a fluorescence detector was developed to determine the phenolic compounds present in virgin olive oil (VOO), with the aim to propose an appropriate alternative to liquid chromatography-mass spectrometry. An excitation wavelength of 285 nm was selected and four different emission wavelengths (316, 328, 350 and 450 nm) were simultaneously recorded, working therefore on "multi-emission" detection mode. With the use of commercially available standards and other standards obtained by semipreparative high performance liquid chromatography, it was possible to identify simple phenols, lignans, several complex phenols, and other phenolic compounds present in the matrix under study. A total of 26 phenolic compounds belonging to different chemical families were identified (23 of them were susceptible of being quantified). The proposed methodology provided detection and quantification limits within the ranges of 0.004-7.143 µg·mL(-1) and 0.013-23.810 µg·mL(-1), respectively. As far as the repeatability is concerned, the relative standard deviation values were below 0.43% for retention time, and 9.05% for peak area. The developed methodology was applied for the determination of phenolic compounds in ten VOOs, both monovarietals and blends. Secoiridoids were the most abundant fraction in all the samples, followed by simple phenolic alcohols, lignans, flavonoids, and phenolic acids (being the abundance order of the latter chemical classes logically depending on the variety and origin of the VOOs).

Comparing two metabolic profiling approaches (liquid chromatography and gas chromatography coupled to mass spectrometry) for extra-virgin olive oil phenolic compounds analysis: A botanical classification perspective.

Over the last decades, the phenolic compounds from virgin olive oil (VOO) have become the subject of intensive research because of their biological activities and their influence on some of the most relevant attributes of this interesting matrix. Developing metabolic profiling approaches to determine them in monovarietal virgin olive oils could help to gain a deeper insight into olive oil phenolic compounds composition as well as to promote their use for botanical origin tracing purposes. To this end, two approaches were comparatively investigated (LC-ESI-TOF MS and GC-APCI-TOF MS) to evaluate their capacity to properly classify 25 olive oil samples belonging to five different varieties (Arbequina, Cornicabra, Hojiblanca, Frantoio and Picual), using the entire chromatographic phenolic profiles combined to chemometrics (principal component analysis (PCA) and partial least square-discriminant analysis (PLS-DA)). The application of PCA to LC-MS and GC-MS data showed the natural clustering of the samples, seeing that 2 varieties were dominating the models (Arbequina and Frantoio), suppressing any possible discrimination among the other cultivars. Afterwards, PLS-DA was used to build four different efficient predictive models for varietal classification of the samples under study. The varietal markers pointed out by each platform were compared. In general, with the exception of one GC-MS model, all exhibited proper quality parameters. The models constructed by using the LC-MS data demonstrated superior classification ability.

Evaluation of gas chromatography-atmospheric pressure chemical ionization-mass spectrometry as an alternative to gas chromatography-electron ionization-mass spectrometry: avocado fruit as example.

Although GC-APCI-MS was developed more than 40 years ago this coupling is still far from being a routine technique. One of the reasons explaining the limited use of GC-APCI so far is the lack of spectral database which facilitates the identification of the compounds under study. The first application of a very recently developed GC-APCI database to identify as many compounds as possible in a complex matrix such as avocado fruit is presented here. The results achieved by using this database has been checked against those obtained using traditional GC-EI-MS and a comparison of the MS signals observed in both ionization sources has been carried out. 100 compounds belonging to different chemical families were identified in the matrix under study. Considering the results of this study, the wide range of application (in terms of polarity and size of analytes) and the robustness of APCI as interface, the high quality of TOF spectra, and our library as a publicly available resource, GC-APCI-TOF MS is definitively a valuable addition to the "metabolomics toolbox".

Uptake and metabolism of olive oil polyphenols in human breast cancer cells using nano-liquid chromatography coupled to electrospray ionization-time of flight-mass spectrometry.

Polyphenols from extra virgin olive oil (EVOO), a main component of the Mediterranean diet, have demonstrated repeatedly anti-tumor activity in several in vitro and in vivo studies. However, little is known about the efficiency of the absorption process and metabolic conversion of these compounds at cellular level. In this study, a nano liquid chromatography-electrospray ionization-time of flight mass spectrometry (nanoLC-ESI-TOF MS) method was developed to study the cellular uptake and metabolism of olive oil phenols in JIMT-1 human breast cancer cells. After incubation for different time periods with EVOO-derived phenolic extracts, culture media, cytosolic fraction and solid particles fraction were separated and analyzed. Most of the free phenols, mainly hydroxytyrosol, its secoiridoid derivatives, and the flavonoid luteolin, disappeared in the culture media in different ways and at different times. Besides, several metabolites were detected in the culture media, fact that may indicate absorption and intracellular metabolism followed by rapid cellular export. Low intracellular accumulation was observed with only traces of some compounds detected in the cytosolic and solid particles fractions. Methylated conjugates were the major metabolites detected, suggesting a catalytic action of catechol-O-methyl transferase (COMT) in cancer cells.

Gas chromatography-atmospheric pressure chemical ionization-time of flight mass spectrometry for profiling of phenolic compounds in extra virgin olive oil.

A new analytical approach based on gas chromatography coupled to atmospheric pressure chemical ionization-time of flight mass spectrometry was evaluated for its applicability for the analysis of phenolic compounds from extra-virgin olive oil. Both chromatographic and MS parameters were optimized in order to improve the sensitivity and to maximize the number of phenolic compounds detected. We performed a complete analytical validation of the method with respect to its linearity, sensitivity, precision, accuracy and possible matrix effects. The LODs ranged from 0.13 to 1.05ppm for the different tested compounds depending on their properties. The RSDs for repeatability test did not exceed 6.07% and the accuracy ranged from 95.4% to 101.5%. To demonstrate the feasibility of our method for analysis of real samples, we analyzed the extracts of three different commercial extra-virgin olive oils. We have identified unequivocally a number of phenolic compounds and obtained quantitative information for 21 of them. In general, our results show that GC-APCI-TOF MS is a flexible platform which can be considered as an interesting tool for screening, structural assignment and quantitative determination of phenolic compounds from virgin olive oil.