Comparative Evaluation of Different Targeted and Untargeted Analytical Approaches to Assess Greek Extra Virgin Olive Oil Quality and Authentication.

Extra virgin olive oil (EVOO) is a key component of the Mediterranean diet, with several health benefits derived from its consumption. Moreover, due to its eminent market position, EVOO has been thoroughly studied over the last several years, aiming at its authentication, but also to reveal the chemical profile inherent to its beneficial properties. In the present work, a comparative study was conducted to assess Greek EVOOs' quality and authentication utilizing different analytical approaches, both targeted and untargeted. 173 monovarietal EVOOs from three emblematic Greek cultivars (Koroneiki, Kolovi and Adramytiani), obtained during the harvesting years of 2018-2020, were analyzed and quantified as per their fatty acids methyl esters (FAMEs) composition via the official method (EEC) No 2568/91, as well as their bioactive content through liquid chromatography coupled to high resolution mass spectrometry (LC-HRMS) methodology. In addition to FAMEs analysis, EVOO samples were also analyzed via HRMS-untargeted metabolomics and optical spectroscopy techniques (visible absorption, fluorescence and Raman). The data retrieved from all applied techniques were analyzed with Machine Learning methods for the authentication of the EVOOs' variety. The models' predictive performance was calculated through test samples, while for further evaluation 30 commercially available EVOO samples were also examined in terms of variety. To the best of our knowledge, this is the first study where different techniques from the fields of standard analysis, spectrometry and optical spectroscopy are applied to the same EVOO samples, providing strong insight into EVOOs chemical profile and a comparative evaluation through the different platforms.

Evaluation of Olive Oil Quality with Electrochemical Sensors and Biosensors: A Review.

Electrochemical sensors, sensor arrays and biosensors, alongside chemometric instruments, have progressed remarkably of late, being used on a wide scale in the qualitative and quantitative evaluation of olive oil. Olive oil is a natural product of significant importance, since it is a rich source of bioactive compounds with nutritional and therapeutic properties, and its quality is important both for consumers and for distributors. This review aims at analysing the progress reported in the literature regarding the use of devices based on electrochemical (bio)sensors to evaluate the bioactive compounds in olive oil. The main advantages and limitations of these approaches on construction technique, analysed compounds, calculus models, as well as results obtained, are discussed in view of estimation of future progress related to achieving a portable, practical and rapid miniature device for analysing the quality of virgin olive oil (VOO) at different stages in the manufacturing process.

Isotopic Traceability (13C and 18O) of Greek Olive Oil.

In recent years, isotopic analysis has been proven a valuable tool for the determination of the origin of various materials. In this article, we studied the 18O and 13C isotopic values of 210 olive oil samples that were originated from different regions in Greece in order to verify how these values are affected by the climate regime. We observed that the δ18O isotopic values range from 19.2 ‱ to 25.2 ‱ and the δ13C values range from -32.7 ‱ to -28.3 ‱. These differences between the olive oils' isotopic values depended on the regional temperature, the meteoric water, and the distance from the sea. Furthermore, we studied the 13C isotopic values of biophenolic extracts, and we observed that they have same capability to differentiate the geographic origin. Finally, we compared the isotopic values of Greek olive oils with samples from Italy, and we concluded that there is a great dependence of oxygen isotopes on the climatic characteristics of the different geographical areas.

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.

Multivariate approaches for stability control of the olive oil reference materials for sensory analysis - part I: framework and fundamentals.

BACKGROUND: Virgin olive oil is the only food product for which sensory analysis is regulated to classify it in different quality categories. To harmonize the results of the sensorial method, the use of standards or reference materials is crucial. The stability of sensory reference materials is required to enable their suitable control, aiming to confirm that their specific target values are maintained on an ongoing basis. Currently, such stability is monitored by means of sensory analysis and the sensory panels are in the paradoxical situation of controlling the standards that are devoted to controlling the panels. RESULTS: In the present study, several approaches based on similarity analysis are exploited. For each approach, the specific methodology to build a proper multivariate control chart to monitor the stability of the sensory properties is explained and discussed. CONCLUSION: The normalized Euclidean and Mahalanobis distances, the so-called nearness and hardiness indices respectively, have been defined as new similarity indices to range the values from 0 to 1. Also, the squared mean from Hotelling's T2 -statistic and Q2 -statistic has been proposed as another similarity index. © 2018 Society of Chemical Industry.

The anticancer and antiobesity effects of Mediterranean diet.

Cancers have been the leading cause of death worldwide and the prevalence of obesity is also increasing in these few decades. Interestingly, there is a direct association between cancer and obesity. Each year, more than 90,000 cancer deaths are caused by obesity or overweight. The dietary pattern in Crete, referred as the traditional Mediterranean diet, is believed to confer Crete people the low mortality rates from cancers. Nevertheless, the antiobesity effect of the Mediterranean diet is less studied. Given the causal relationship between obesity and cancer, the antiobesity effect of traditional Mediterranean diet might contribute to its anticancer effects. In this regard, we will critically review the anticancer and antiobesity effects of this diet and its dietary factors. The possible mechanisms underlying these effects will also be discussed.

Virgin olive oil quality of hedgerow 'Arbequina' olive trees under deficit irrigation.

BACKGROUND: Regulated deficit irrigation (RDI) is used in hedgerow olive orchards to achieve a sustainable balance between water savings, tree vigor and oil production. Its effects on the presence of compounds responsible for the taste of the oil and its nutritional value are controversial. The present 3-year study was conducted in an 'Arbequina' orchard (1667 trees ha-1 ) under a full irrigation (FI) treatment (470.1 mm year-1 of water) and two RDI treatments scaled to replace 60% and 30%, respectively, of FI. The quality parameters, antioxidant contents and volatiles of the extracted virgin olive oil (VOO) were analyzed. RESULTS: In general, oils from the 30% RDI treatment had higher contents of pigments and phenolic compounds, a higher oleic/linoleic ratio and the highest oxidative stability, despite their lower tocopherol content. FI oils showed higher (E)-2-hexenal, 1-penten-3-one, ocimene, E-2-pentenal and pentene dimer contents than 30RDI oils, but lower contents of (E)-2-pentenol and volatile esters. CONCLUSION: The results of the present study suggest that a RDI strategy supplying 30% of the total irrigation needs induces an increase in natural antioxidants in VOO. Neither yield, nor the rest of the quality parameters were affected by the reduced irrigation. However, abundant autumn precipitation can over-ride these effects of 30% RDI treatment on oil quality. © 2016 Society of Chemical Industry.

Olive oil authenticity studies by target and nontarget LC-QTOF-MS combined with advanced chemometric techniques.

Food analysis is continuously requiring the development of more robust, efficient, and cost-effective food authentication analytical methods to guarantee the safety, quality, and traceability of food commodities with respect to legislation and consumer demands. Hence, a novel reversed-phase ultra high performance liquid chromatography-electrospray ionization quadrupole time of flight tandem mass spectrometry analytical method was developed that uses target, suspect, and nontarget screening strategies coupled with advanced chemometric tools for the investigation of the authenticity of extra virgin olive oil. The proposed method was successfully applied in real olive oil samples for the identification of markers responsible for the sensory profile. The proposed target analytical method includes the determination of 14 phenolic compounds and demonstrated low limits of detection ranging from 0.015 µg mL-1 (apigenin) to 0.039 µg mL-1 (vanillin) and adequate recoveries (96-107 %). A suspect list of 60 relevant compounds was compiled, and suspect screening was then applied to all the samples. Semiquantitation of the suspect compounds was performed with the calibration curves of target compounds having similar structures. Then, a nontarget screening workflow was applied with the aim to identify additional compounds so as to differentiate extra virgin olive oils from defective olive oils. Robust classification-based models were built with the use of supervised discrimination techniques, partial least squares-discriminant analysis and counterpropagation artificial neural networks, for the classification of olive oils into extra virgin olive oils or defective olive oils. Variable importance in projection scores were calculated to select the most significant features that affect the discrimination. Overall, 51 compounds were identified and suggested as markers, among which 14, 26, and 11 compounds were identified by target, suspect, and nontarget screening respectively. Retrospective analysis was also performed and identified 19 free fatty acids. Graphical Abstract Development of a novel RP-LC-ESI-QTOFMS analytical method employing target, suspect and non-target screening strategies coupled to advanced chemometric tools for the investigation of markers responsible for the sensory profile of extra virgin olive oil and guarantee authenticity.

How 'ground-picked' olive fruits affect virgin olive oil ethanol content, ethyl esters and quality.

BACKGROUND: Olives dropped on the ground naturally sometimes are not separated from those fresh and healthy collected from the tree for harvest and processing. In this work we compared the quality, ethanol content and bioactive components of virgin olive oils from ground-picked olives, tree-picked fruits and their mixture. RESULTS: Ground-picked olives produced 'Lampante' virgin olive oils; these are of a lower quality category, because of important alterations in chemical and sensory characteristics. Ethyl esters showed the highest values, although under the regulated limit. The mixture of ground and tree-picked olives gave oils classified as 'virgin' because of sensory defects, although the quality parameters did not exceed the limits for the 'extra' category. Ethanol content showed a significant increase in the oils from ground- picked olives and their mixture with respect to those from tree-picked fruits. Furthermore, bioactive compounds showed a significant decrease as fruit quality was poorer. CONCLUSION: Ground-picked olives must be harvested and processed separately since they produce low-quality virgin olive oils with sensory defects and lower concentrations of bioactive compounds. The higher acidity and ethanol concentration observed in oils from ground-picked fruits or their mixture may help ethyl ester synthesis during storage. © 2015 Society of Chemical Industry.

Comparison Between Different Flavored Olive Oil Production Techniques: Healthy Value and Process Efficiency.

Three different flavoring methods of olive oil were tested employing two different herbs, thyme and oregano. The traditional method consist in the infusion of herbs into the oil. A second scarcely diffused method is based on the addition of herbs to the crushed olives before the malaxation step during the extraction process. The third innovative method is the implementation of the ultrasound before the olive paste malaxation. The objective of the study is to verify the effect of the treatments on the quality of the product, assessed by means of the chemical characteristics, the phenol composition and the radical scavenging activity of the resulting oils. The less favorable method was the addition of herbs directly to the oil. A positive effect was achieved by the addition of herbs to the olive paste and other advantages were attained by the employment of ultrasound. These last two methods allow to produce oils "ready to sell", instead the infused oils need to be filtered. Moreover, the flavoring methods applied during the extraction process determine a significant increment of phenolic content and radical scavenging activity of olive oils. The increments were higher when oregano is used instead of thyme. Ultrasound inhibited the olive polyphenoloxidase, the endogenous enzyme responsible for olive oil phenol oxidation. This treatment of olive paste mixed with herbs before malaxation was revealed as the most favorable method due to the best efficiency, reduced time consumption and minor labor, enhancing the product quality of flavored olive oil.

The effect of water stress on super-high- density 'Koroneiki' olive oil quality.

BACKGROUND: Over the last two decades, the area of cultivated super-high-density olive orchards has increased rapidly. Water stress is an important tool in super-high-density orchards to reduce tree growth and promote suitability for overhead mechanical harvesters. Little is known regarding the effect of water stress in super-high-density orchards on oil quality parameters. In this study the effect of irrigation rate on oil quality parameters was evaluated in a six-year-old super-high-density 'Koreneiki' olive orchard for five consecutive seasons. Five water status levels, determined by irrigating in order to maintain various midday stem water potential threshold values (-1.5, -2, -2.5, -3 and -4 MPa), were applied during the oil accumulation stage. RESULTS: The MUFA/PUFA ratio and free fatty acid content generally decreased as a function of increasing tree water stress. In most seasons a reduction in polyphenols was found with decreasing irrigation level. Peroxide value was not affected by the water stress level. CONCLUSION: The present study demonstrates that limiting irrigation and exposure of olive trees to water stress in a super-high-density orchard lowers free fatty acid content and therefore benefits oil quality. However, the decreased MUFA/PUFA ratio and the reduction in polyphenol content that were also found under increased water stress negatively influence oil quality.

Attitudes towards Olive Oil Usage, Domestic Storage, and Knowledge of Quality: A Consumers' Survey in Greece.

Consumption of unbranded olive oil obtained in bulk has previously been reported to be very high in Greece, underlining the need to investigate knowledge regarding its health attributes and storage practices, two areas that can affect oil quality. This study aimed to investigate Greek consumers' use and choice of olive oil, their knowledge about its quality, as well as domestic storage practices of olive oil. A cross-sectional survey was conducted in a representative sample of 857 Greek households that consume olive oil, using a previously validated questionnaire. Most participating households use olive oil produced by themselves or by their extended family or friends (60.3%), and only 27.4% purchase branded olive oil, while 57% reported using extra virgin olive oil (EVOO). Only 38.4% of the respondents reported optimal domestic storage practices to maintain olive oil quality, with a significant greater percentage of non-producers group compared to olive oil producers. In all areas of Greece, the higher the knowledge of olive oil quality, the higher the probability of consumers selecting EVOO and perceiving olive oil price as low. The present survey highlights the need to heighten consumers' knowledge of olive oil attributes and correct storage practices and awareness about branded EVOO and its superior quality.

Evaluating the Variability in the Phenolic Concentration of Extra Virgin Olive Oil According to the Commission Regulation (EU) 432/2012 Health Claim.

The health benefits of extra virgin olive oil (EVOO) are associated to its fatty acids profile (with predominance of oleic acid) and to the minor components that include phenols, among others. Phenols are responsible for the only health claim of olive oil reported in the Commission Regulation (EU) 432/2012. Here, we have applied a liquid chromatography-tandem mass spectrometry method to determine the most abundant phenols included in the health claim (with special emphasis on secoiridoids) in 1239 EVOO samples produced in two consecutive agronomical seasons. The predominant cultivars in Spain ("Picual", "Arbequina", "Hojiblanca", and "Cornicabra") were evaluated. We also studied the influence of harvesting date and orchard location on the EVOO phenolic concentration. A great variability in phenolic content, from 1 to 2850 mg/kg, was found in these EVOOs, and not all of them (4.6 and 23.1% in the two seasons) reported a concentration above 250 mg/kg to certify the health claim.

Homogeneity assessment of reference materials for sensory analysis of liquid foodstuffs. The virgin olive oil as case study.

Sensory properties are critical characteristics that determine quality and can be evaluated by trained tasting panels. The panels function as multi-sensor measuring instrument and need the use of reference materials (RMs) for training. The homogeneity between units packaged from a batch of RM can be evaluated by gas chromatography coupled to flame ionization detection (GC-FID), using this instrumental technique as an alternative to sensory analysis. For this purpose, the fingerprint methodology is applied, taking into account that the homogeneity assessment will be based on evaluating the similarity between the fingerprints of the fraction of volatile organic compounds acquired from samples representative of the batch. The proposed methodology is applied with good results to evaluate the homogeneity of several RMs for sensory analysis of virgin olive oil samples, using similarity indices, control charts and exploratory analysis of multivariate data to observe the grouping RM and fingerprint regions representative of each defect.

Chemical Profiles of Twenty-three Monovarietal Olive Oils Produced in Liangshan Region of China.

The main objective of this study was to differentiate twenty-three monovarietal olive oils produced in China (Azappa, Thiaki, Leucocarpa ovoid, manzanillo de labata, etc.) by studying their physicochemical properties, fatty acid profiles and oxidative stability. The majority of analytical indicators had statistically significant differences (p < 0.05). The tested cultivars produced excellent olive oils with an appreciable amount of natural antioxidants and chemical composition within the legal range. Our results demonstrate that Leucocarpa ovoid oil was characterized by high mean levels of total phenols (401.8 mg/kg), oleic acid (79.62%) and oxidative stability (18.64 h). Additionally, five types of olive oil were selected due to their high overall quality. Investigation of virgin olive oils indicates that the differences in the fatty acid profiles, phenols, sterols and tocopherols may be caused by genetic factors. The principal components analysis (PCA) results showed a strong differentiation between various cultivars in regard to their fatty acid composition and phenol, sterol and tocopherol levels. These components can be effective tools for efficient comparison and differentiation of these cultivars. To the best of our knowledge, this is the first work on the differentiation of the chemical constituents of virgin olive oils.

Effect of storage on quality parameters and phenolic content of Italian extra-virgin olive oils.

The quality of extra virgin olive oils is affected mainly by hydrolytic and oxidative reactions. The present paper investigated the changes of major and minor components and oxidation indices of three monovarietal extra virgin olive oils after 18 months of storage at room temperature and in dark glass bottles conditions. After storage, the basic quality parameters such as free acidity, peroxide values, extinction coefficients, fatty acids composition, chlorophyll and carotenoid content, did not exceed the upper limits set by European Community Regulations for extra-virgin olive oils. Given the importance of the phenolic fraction, UHPLC-HESI-MS metodology was used. A decrease in 3,4-DHPEA-EDA (oleacin) and p-HPEA-EDA (oleochantal) was detected whereas, an increase of tyrosol and hydroxytyrosol was measured as a consequence of degradation of ligstroside and oleuropein derivatives. Based on the results it is possible to observe the high nutritional value of the studied oils even after 18 months of conservation.

Stimulated Brillouin scattering in combination with visible absorption spectroscopy for authentication of vegetable oils and detection of olive oil adulteration.

Vegetable oils provide high nutritional value in the human diet. Specifically, extra virgin olive oil (EVOO) possesses a higher price than that of other vegetable oils. Adulteration of pure EVOO with other types of vegetable oils has attracted increasing attentions. In this work, a stimulated Brillouin scattering (SBS) combined with visible absorption spectroscopy method is proposed for authentication of vegetable oils and detection of olive oil adulteration. The results provided here have demonstrated that the different vegetable oils and adulteration oils exhibit significant differences in normalized absorbance values of two relevant wavelengths (455 and 670 nm) and frequency shifts of SBS. The normalized absorbance values of all spectra at the two relevant wavelengths of 670 nm and 455 nm linearly decrease with the increase of the adulteration concentration. The Brillouin frequency shifts exponentially increase with the increase of the adulteration concentration. Due to non-destructive and requiring no sample pretreatment procedure, this method can be effectively employed for authentication and detection of oils adulteration.

A robustness study of calibration models for olive oil classification: Targeted and non-targeted fingerprint approaches based on GC-IMS.

The dual separation in gas chromatography-ion mobility spectrometry generates complex multi-dimensional data, whose interpretation is a challenge. In this work, two chemometric approaches for olive oil classification are compared to get the most robust model over time: i) an non-targeted fingerprinting analysis, in which the overall GC-IMS data was processed and ii) a targeted approach based on peak-region features (markers). A total of 701 olive samples from two harvests (2014-2015 and 2015-2016) were analysed and processed by both approaches. The models built with data samples of 2014-2015 showed that both approaches were suitable for samples classification (success >74%). However, when these models were applied for classifying samples from 2015-2016, better values were obtained using markers. The combination of data from the two harvests to build the chemometric models improved the percentages of success (>90%). These results confirm the potential of GC-IMS based approaches for olive oil classification.

Evaluation of changes in the quality of extracted oil from olive fruits stored under different temperatures and time intervals.

Chilling and freezing injuries of olives harvested in geographically high elevated locations may affect the quality of olive, and subsequently lead to oil with a poor quality. This study was aiming to investigate the influence of whether changes and freezing condition on the quality of extracted olive oil. The olive Koroneiki cultivar obtained from two origins was stored at three different temperatures (20, 5 and -18 °C) before oil extraction and common analyses (oil yield, acidity, peroxide value, thiobarbitorik acid value, total phenolics level, and color) were carried out in different time intervals (0, 20, 40, and 60 days) in order to assess the olive oil quality. Our data revealed that longer storage times significantly (P < 0.05) decreased the quality of olive oil. The fruits remained at 20 °C provided the lowest oil quality in all parameters. For example, the acidity of olive oil at 20 °C was 177% higher than samples kept at 5 °C after 20 days of olive storage. The general trend for comparing the quality parameters of extracted oils from olives kept at different temperatures was -18 > 5 > 20 °C. No significant negative effect (P < 0.05) was found for the samples kept at -18 °C, compared to 5 °C. Also, the geographical source of olive had a statistically significant influence (P < 0.05) on the quality of olive oil.

Validation of Fluorescence Spectroscopy to Detect Adulteration of Edible Oil in Extra Virgin Olive Oil (EVOO) by Applying Chemometrics.

Due to high price and nutritional values of extra virgin olive oil (EVOO), it is vulnerable to adulteration internationally. Refined oil or other vegetable oils are commonly blended with EVOO and to unmask such fraud, quick, and reliable technique needs to be standardized and developed. Therefore, in this study, adulteration of edible oil (sunflower oil) is made with pure EVOO and analyzed using fluorescence spectroscopy (excitation wavelength at 350 nm) in conjunction with principal component analysis (PCA) and partial least squares (PLS) regression. Fluorescent spectra contain fingerprints of chlorophyll and carotenoids that are characteristics of EVOO and differentiated it from sunflower oil. A broad intense hump corresponding to conjugated hydroperoxides is seen in sunflower oil in the range of 441-489 nm with the maximum at 469 nm whereas pure EVOO has low intensity doublet peaks in this region at 441 nm and 469 nm. Visible changes in spectra are observed in adulterated EVOO by increasing the concentration of sunflower oil, with an increase in doublet peak and correspondingly decrease in chlorophyll peak intensity. Principal component analysis showed a distinct clustering of adulterated samples of different concentrations. Subsequently, the PLS regression model was best fitted over the complete data set on the basis of coefficient of determination (R2), standard error of calibration (SEC), and standard error of prediction (SEP) of values 0.99, 0.617, and 0.623 respectively. In addition to adulterant, test samples and imported commercial brands of EVOO were also used for prediction and validation of the models. Fluorescence spectroscopy combined with chemometrics showed its robustness to identify and quantify the specified adulterant in pure EVOO.