Detection of extra virgin olive oil adulteration with lampante olive oil and refined olive oil using nuclear magnetic resonance spectroscopy and multivariate statistical analysis

High-field 31P NMR (202.2 MHz) spectroscopy was applied to the analysis of 59 samples from three grades of olive oils, 34 extra virgin olive oils from various regions of Greece, and from different olive varieties, namely, 13 samples of refined olive oils and 12 samples of lampante olive oils. Classification of the three grades of olive oils was achieved by two multivariate statistical methods applied to five variables, the latter being determined upon analysis of the respective 31P NMR spectra and selected on the basis of one-way ANOVA. The hierarchical clustering statistical procedure was able to classify in a satisfactory manner the three olive oil groups. Subsequent application of discriminant analysis to the five selected variables of oils allowed the grouping of 59 samples according to their quality with no error. Different artificial mixtures of extra virgin olive oil-refined olive oil and extra virgin olive oil-lampante olive oil were prepared and analyzed by 31P NMR spectroscopy. Subsequent discriminant analysis of the data allowed detection of extra virgin olive oil adulteration as low as 5% w/w for refined and lampante olive oils. Further application of the classification/prediction model allowed the estimation of the percent concentration of refined olive oil in six commercial blended olive oils composed of refined and virgin olive oils purchased from supermarkets.     

Influence of crystallization on the oxidative stability of extra virgin olive oil

The aim of this study was to evaluate the influence of the extra virgin olive oil (EVOO) physical state on the kinetics of oxidative reactions. To this purpose, EVOO was stored at increasing temperatures from 3 to 60 degrees C and the oxidation was followed by measuring both primary and secondary oxidation products. Results highlighted that crystallization plays an important role in determining EVOO stability. Below the melting point, the oxidation rate was found to be higher than that expected on the basis of the Arrhenius equation. The observed deviation from the Arrhenius equation was attributed to the physicochemical changes occurring as a consequence of phase transitions. In particular, the increase in unsaturated triacylglycerol concentration and the decrease of polyphenol content in the liquid phase surrounding fat crystals were indicated as the main factors causing the deviation. By taking into account these changes it was possible to describe the temperature dependence of the oxidation rate in the entire range of temperatures considered. This model appears to be promising in the challenge to find mathematical models able to predict the stability and, hence, the shelf life of lipid-containing foods.     

Effectiveness of microsatellite DNA markers in checking the identity of protected designation of origin extra virgin olive oil

Collina di Brindisi is an Italian extra virgin olive oil that obtained the mark of protected designation of origin (PDO) according to EC Regulation 2081/92. The varietal requirements of the official production protocol of this oil foresee that this oil is prepared from cultivar Ogliarola (minimum 70%) and other Olea europaea L. cultivars that are diffused in the production area, accounting for a maximum of 30%. The aim of this work was to verify the effectiveness of microsatellite analysis in verifying the identity of Collina di Brindisi PDO olive oil. A preliminary assessment of product's quality by means of chemical analyses was also carried out. Microsatellite analysis clarified that the generic name Ogliarola, indicated in the technical sheet of this PDO oil, actually corresponded to the Ogliarola salentina cultivar. Furthermore, the obtained results showed that the examination of a limited number of DNA microsatellites enables the identification of the Ogliarola salentina cultivar in this PDO oil.     

Novel approach to study oxidative stability of extra virgin olive oils: importance of alpha-tocopherol concentration

The stability of extra virgin olive oils is mainly due to their relatively low fatty acids unsaturation and to the antioxidant activity of some of the unsaponifiable components. We studied the activity of alpha-tocopherol in extra virgin olive oil in its natural state, using new and simple oxidizing conditions in "thin layer" and "bulk phase". Oxidation time course was monitored at 37 degrees C or 75 degrees C. A storage test was also performed. Two parameters were evaluated: depletion of alpha-tocopherol and formation of PUFA hydroperoxides, measured as conjugated dienes (CD) and peroxide value. The value of complex polyphenols was also measured. alpha-Tocopherol concentration decreased in function of time and temperature and showed a strong inverse correlation with the increase of CD. When alpha-tocopherol reached a "threshold value" of 60-70 mg/kg, a significant increase of CD formation was observed, together with a good correlation between CD and peroxide value. The initial amount of alpha-tocopherol is one of the components that influences oil oxidative susceptibility.     

FTIR spectroscopy and multivariate analysis can distinguish the geographic origin of extra virgin olive oils

This work investigates whether Fourier transform infrared spectroscopy (FTIR), in combination with multivariate analysis, can distinguish extra virgin olive oils from different producing countries. Duplicate spectra were collected from 60 oils from four European countries. Two approaches to data analysis were used as follows: first, the "whole spectrum" method of partial least squares (PLS) followed by distance-based linear discriminant analysis (LDA) applied to the PLS scores, and second, a genetic algorithm (GA) for variate selection from the raw data, followed by LDA applied to the selected subset. The PLS-LDA approach produced a cross-validation success rate of 96%, whereas the GA-LDA approach achieved a 100% cross-validation success rate, from subsets comprising only eight variates. Neither the selected variate nor the whole spectrum approach was able to offer insight into the origin of the discrimination in biochemical terms. However, FTIR analysis is rapid, and this work shows that it has the required discriminatory power to potentially offer a "black box" method of screening oils to verify their country of origin.     

Wastes generated during the storage of extra virgin olive oil as a natural source of phenolic compounds

Phenolic compounds in extra virgin olive oil (EVOO) have been associated with beneficial effects for health. Indeed, these compounds exert strong antiproliferative effects on many pathological processes, which has stimulated chemical characterization of the large quantities of wastes generated during olive oil production. In this investigation, the potential of byproducts generated during storage of EVOO as a natural source of antioxidant compounds has been evaluated using solid-liquid and liquid-liquid extraction processes followed by rapid resolution liquid chromatography (RRLC) coupled to electrospray time-of-flight and ion trap mass spectrometry (TOF/IT-MS). These wastes contain polyphenols belonging to different classes such as phenolic acids and alcohols, secoiridoids, lignans, and flavones. The relationship between phenolic and derived compounds has been tentatively established on the basis of proposed degradation pathways. Finally, qualitative and quantitative characterizations of solid and aqueous wastes suggest that these byproducts can be considered an important natural source of phenolic compounds, mainly hydroxytyrosol, tyrosol, decarboxymethyl oleuropein aglycone, and luteolin, which, after suitable purification, could be used as food antioxidants or as ingredients in nutraceutical products due to their interesting technological and pharmaceutical properties.     

Modification of volatile compound profile of virgin olive oil due to hot-water treatment of olive fruit

The effect of hot-water treatments of olive fruits before processing on the biosynthesis of virgin olive oil aroma was investigated by quantifying the variation within the major classes of volatile compounds. Data showed that hot-water treatments gave rise to changes in the volatile aroma profile of virgin olive oil from the three olive cultivars under study, Manzanilla, Picual, and Verdial. Different effects by thermal treatments were observed according to cultivar. In general, these changes are mainly due to a decrease in the contents of C(6) aldehydes and C(5) compounds. Contents of C(6) alcohols and esters remained constant or decreased slightly when the temperature of the treatment was increased. Thus, heat treatments seemed to promote a partial deactivation of the lipoxygenase/hydroperoxide lyase enzyme system, whereas other enzymatic activities, within the lipoxygenase pathway, such as alcohol dehydrogenase and alcohol acyltransferase, remained apparently unaffected as a consequence of heat treatments.     

Qualitative and semiquantitative analysis of phenolic compounds in extra virgin olive oils as a function of the ripening degree of olive fruits by different analytical techniques

Capillary electrophoresis (CE) can be effectively used as a fast screening tool to obtain qualitative and semiquantitative information about simple and complex phenolic compounds of extra virgin olive oil. Three simple phenols (tyrosol, hydroxytyrosol, and vanillic acid), a secoiridoid derivative (deacetoxy oleuropein aglycon), and two lignans (pinoresinol and acetoxypinoresinol) were detected as the main compounds in extra virgin olive oils by high-performance liquid chromatography (HPLC) and capillary zone electrophoresis (CZE). Spectrophotometric indices, radical scavenging activity, and oxidative stability of extra virgin olive oil samples obtained from olives hand-picked at different ripening degrees were statistically correlated with the CZE and HPLC quantification. The concentration of phenols in extra virgin olive oil decreased with ripeness of olive fruits. The high correlations found between CZE and the other analytical results indicate that CE can be applied as a rapid and reliable tool to routinely determine phenolic compounds in extra virgin olive oils.     

Relation between the endogenous antioxidant system and the quality of extra virgin olive oil under accelerated storage conditions

Three monovarietal extra virgin olive oils (EVOOs) were subjected to accelerated storage conditions (60 degrees C, dark) representative of the autoxidation process during shelf life. Oxidation markers, i.e., the peroxide value, conjugated dienes, the oil stability index, and minor components, were monitored. The changes in minor components, related to the stage of ongoing oxidation and expressed as a percentage of the induction period (IP), followed a similar pattern in all oils: o-diphenols diminished by the highest rate (halved within 15% of the IP), followed by alpha-tocopherol (halved within 35% of the IP). Carotenoids and chlorophylls were also affected by autoxidation, whereas squalene showed high stability (<20% loss within 100% of the IP). Polar phenols (especially o-diphenols) and alpha-tocopherol were deduced to be the most potent antioxidants of EVOO. They efficiently inhibited oxidative lipid deterioration and subsequent development of sensory defects (rancidity, discoloration), which occurred only after substantial depletion of these antioxidants. Therefore, they could also be used as markers for the oxidative status of EVOO particularly in the early stage of oxidation.     

Influence of olive maturity stage and geographical origin on some minor components in virgin olive oil of the chemlali variety

Minor compounds such as sterols, aliphatic alcohols, tocopherols, and chlorophylls in virgin olive oil from Chemlali cultivar were analyzed during the maturity process. The study concerns oils from the following three different olive growing areas of Tunisia: Sfax, Sidi Bouzid, and Enfidha. Analytical results showed that both the maturity process and the olive provenances influence the evolution of the content of these compounds.     

Protective effects of extra virgin olive oil phenolics on oxidative stability in the presence or absence of copper ions

The antioxidant activity of the phenolic fraction of extra virgin olive oil was assessed in samples that had a decreasing content of antioxidants in the presence and absence of copper ions as a catalyst of autoxidation. The oxidation process was evaluated by measuring primary and secondary oxidation products. Changes in phenols and tocopherols were investigated by high-performance liquid chromatography. Both the total phenol content and their antioxidant activity were monitored by spectrophotometric assays (with Folin-Ciocalteu and ABTS*+ reagents). The important role of phenolic compounds (particularly the o-diphenols) in protection from autoxidation was confirmed. However, the tocopherols were more quickly consumed in oils that had the lowest content of o-diphenols, which also showed evidence of an ability to chelate copper. In particular, a dramatic decrease was observed in the isomeric form of decarboxymethyl-oleuropein aglycone after addition of the metal, despite its significant increase in samples stored in the absence of copper.     

New investigation of the isothermal oxidation of extra virgin olive oil: determination of free radicals, total polyphenols, total antioxidant capacity, and kinetic data

As a follow-up of the research programs carried out by our group concerning the artificial isothermal rancidification process in extra virgin olive oil (EVOO), in the present work the trends of both the total antioxidant capacity and the total polyphenols concentration as well as the main kinetic parameters of the process during the thermal oxidation of EVOO were studied and compared. In addition, the possibility of evaluating the increase in radicals concentration during the thermal oxidation process using a superoxide dismutase biosensor was also studied. The present investigation concerning this important food product is highly topical as it refers to the state of alteration of the EVOO used for cooking or frying, as a function of the temperature reached.     

Mathematical model to predict the formation of pyropheophytin a in virgin olive oil during storage

A mathematical model has been developed that describes the changes of pyropheophytin a (pyphya) in virgin olive oil (VOO). The model has been created using multivariate statistical procedures and is used in the prediction of the stability and loss of freshness of VOO. An earlier thermokinetic study (Aparicio-Ruiz, R.; M??nguez-Mosquera, M. I.; Gandul-Rojas, B. Thermal degradation kinetics of chlorophyll pigments in virgin olive oils. 1. Compounds of series a. J. Agric. Food Chem.2010, 58, 6200-6208) that looked at the characterization of the degradation of pheophytin a (phya), the main chlorophyll compound in VOO and a precursor of pyphya, allowed the authors to obtain the kinetic parameters necessary for mathematically expressing the percentage of pyphya, according to the time and temperature of storage using the Arrhenius model. Data regarding the percentage of pyphya obtained during the actual degradation of VOO in darkness, at room temperature and with a limited supply of oxygen, has allowed the mathematical prediction model to be validated. Using average monthly temperatures in the calculation of kinetic constants, theoretical data are obtained that are generally found to be within 95% confidence levels of experimental data.     

Quality evaluation of olive oil by statistical analysis of multicomponent stable isotope dilution assay data of aroma active compounds

An instrumental method for the evaluation of olive oil quality was developed. Twenty-one relevant aroma active compounds were quantified in 95 olive oil samples of different quality by headspace solid phase microextraction (HS-SPME) and dynamic headspace coupled to GC-MS. On the basis of these stable isotope dilution assay results, statistical evaluation by partial least-squares discriminant analysis (PLS-DA) was performed. Important variables were the odor activity values of ethyl isobutanoate, ethyl 2-methylbutanoate, 3-methylbutanol, butyric acid, E,E-2,4-decadienal, hexanoic acid, guaiacol, 2-phenylethanol, and the sum of the odor activity values of Z-3-hexenal, E-2-hexenal, Z-3-hexenyl acetate, and Z-3-hexenol. Classification performed with these variables predicted 88% of the olive oils' quality correctly. Additionally, the aroma compounds, which are characteristic for some off-flavors, were dissolved in refined plant oil. Sensory evaluation of these models demonstrated that the off-flavors rancid, fusty, and vinegary could be successfully simulated by a limited number of odorants.     

Sensory properties of virgin olive oil polyphenols: identification of deacetoxy-ligstroside aglycon as a key contributor to pungency

Polyphenols are an important functional minor component of virgin olive oils that are responsible for the key sensory characteristics of bitterness, pungency, and astringency. Polyphenols were isolated from virgin olive oils by using liquid/liquid extraction and then separated by using reverse phase HPLC followed by fraction collection. The sensory qualities of the isolated polyphenols were evaluated, and almost all fractions containing polyphenols were described as bitter and astringent. However, the fraction containing deacetoxy-ligstroside aglycon produced a strong burning pungent sensation at the back of the throat. In contrast, the fraction containing the analogous deacetoxy-oleuropein aglycon, at an equivalent concentration, produced only a slight burning/numbing sensation, which was perceived more on the tongue. No other polyphenol fractions from the analyzed oils produced the intense burning sensation; thus, deacetoxy-ligstroside aglycon is the polyphenol responsible for the majority of the burning pungent sensation found in pungent extra virgin olive oils.     

Solid-phase microextraction in the analysis of virgin olive oil volatile fraction: characterization of virgin olive oils from two distinct geographical areas of northern Italy

SPME was employed to characterize the volatile profile of virgin olive oils produced in two geographical areas of northern Italy: the region of the Gulf of Trieste and the area near Lake Garda. There are as yet no data on the headspace composition of virgin olive oils from these regions, characterized by particular conditions of growth for Olea europaea. Using the SPME technique coupled to GC-MS and GC-FID, the volatile components of 42 industrially produced virgin olive oil samples were identified and the principal compounds quantitatively analyzed. Significant differences in the proportion of volatile constituents from oils of different varieties and geographical origins were detected. The results suggest that besides the genetic factor, environmental conditions influence the volatile formation.     

Determinant parameters and components in the storage of virgin olive oil. Prediction of storage time beyond which the oil is no longer of "extra" quality.

This work studies the changes in the quality indices standardized by the European Union, together with the evolution of the oxidative stability and sterols, polyphenols, alpha-tocopherol, pigments, and fatty acids contents throughout the storage of Picual and Hojiblanca olive cultivar "extra" virgin olive oils at 2 degrees C + darkness and 30 degrees C + illumination. Only two quality indices (K(270) and sensory evaluation) indicate the loss of the extra quality of the oil during storage, and there is an excellent correlation between initial stability and the time to reach the limit of K(270) > 0.25 (after which the oil quality is no longer of "extra" quality). This time can be predicted with an error of <10%, which is of great commercial interest and previously unknown. Also unknown until now is that the changes in polyphenols, pigments, and alpha-tocopherol with storage time follow first-order kinetics.     

Impact of extra virgin olive oil and ethylenediaminetetraacetic acid (EDTA) on the oxidative stability of fish oil emulsions and spray-dried microcapsules stabilized by sugar beet pectin

The influence of EDTA on lipid oxidation in sugar beet pectin-stabilized oil-in-water emulsions (pH 6, 15% oil, wet basis), prepared from fish oil (FO) and fish oil-extra virgin olive oil (FO-EVOO) (1:1 w/w), as well as the spray-dried microcapsules (50% oil, dry basis) prepared from these emulsions, was investigated. Under accelerated conditions (80 °C, 5 bar oxygen pressure) the oxidative stability was significantly (P < 0.05) higher for FO and FO-EVOO formulated with EDTA, in comparison to corresponding emulsions and spray-dried microcapsules formulated without EDTA. The EDTA effect was greater in emulsions than in spray-dried microcapsules, with the greatest protective effect obtained in FO-EVOO emulsions. EDTA enhanced the oxidative stability of the spray-dried microcapsules during ambient storage (~25 °C, a(w) = 0.5), as demonstrated by their lower concentration of headspace volatile oxidation products, propanal and hexanal. These results show that the addition of EDTA is an effective strategy to maximize the oxidative stability of both FO emulsions and spray-dried microcapsules in which sugar beet pectin is used as the encapsulant material.