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.     

Detection of the presence of refined hazelnut oil in refined olive oil by fluorescence spectroscopy

The fluorescence spectroscopy technique has been tested as regards its ability to differentiate between refined hazelnut and olive oils. Classification of these oils based on their excitation-emission fluorescence spectra data (spectral range 300-500 nm of the excitation spectra at lambdaem=655 and spectral range 650-900 of the emission spectra at lambdaex=50 nm) was performed using principal component analysis and artificial neural networks. Both methods provided good discrimination between the refined hazelnut and olive oils. The results have also pointed out the possibilities of a spectrofluorimetric method joined to multivariate analysis, to differentiate refined oils, and even to detect the presence of refined hazelnut oils in refined olive oils at percentages higher than 9%.     

Comparison of analytical methodologies based on 1h and 31p NMR spectroscopy with conventional methods of analysis for the determination of some olive oil constituents

The present study was designed to assess the agreement between analytical methodologies based on 1H and 31P NMR spectroscopy and conventional analytical methods (titration, gas chromatography, and high performance liquid chromatography) for measuring certain minor and major constituents (free acidity, fatty acids, iodine value, and phenolic compounds) of olive oil. The standard deviations of the NMR method were comparable to those of the conventional methods, except perhaps those of the total hydroxytyrosol and total tyrosol. Linear regression analyses showed strong correlations between NMR and conventional methods for free acidity, total hydroxytyrosol, total tyrosol, total diacylglycerols, (+)-pinoresinol, (+)-1-acetoxypinoresinol, and apigenin; good correlations for linoleic acid, free hydroxytyrosol, and free tyrosol; and weak correlations for oleic acid, linolenic acid, saturated fatty acids, and luteolin. Furthermore, a method comparison study was conducted and the agreement between NMR and conventional methods was evaluated by using the Bland and Altman statistical analysis. The distribution of the data points in the bias plot showed that 96.4% and 100% of the measurements of free acidity and iodine value, respectively, were within the limits of agreement of the two methods. For the remaining constituents of olive oil, the percentage of measurements, located within the limits of agreement, ranged from 94% to 98.5%.     

Development of a phenol-enriched olive oil with both its own phenolic compounds and complementary phenols from thyme

Besides affecting the oil's sensorial characteristics, the presence of herbs and spices has an impact on the nutritional value of the flavored oils. The aim of the study was to develop a new product based on the phenol-enrichment of a virgin olive oil with both its own phenolic compounds (secoiridoid derivatives) plus additional complementary phenols from thyme (flavonoids). We studied the effect of the addition of phenolic extracts (olive cake and thyme) on phenolic composition, oxidative stability, antioxidant activity, and bitter sensory attribute of olive oils. Results showed that flavonoids from thyme appeared to have higher transference ratios (average 89.7%) from the phenolic extract to oil, whereas secoiridoids from olive presented lower transference ratios (average 35.3%). The bitter sensory attribute of the phenol-enriched oils diminished with an increase of the concentration of phenols from thyme, which might denote an improvement in the consumer acceptance.     

Cluster analysis applied to the exploratory analysis of commercial spanish olive oils by means of excitation-emission fluorescence spectroscopy

Olive oil fluorescence is related to oil composition. Here it is shown that the natural clustering of different types of commercial Spanish olive oils depends on their fluorescence excitation-emission matrices (EEMs). Fifty-six commercial samples of olive oil (29 virgin olive oils, 20 pure olive oils, and 7 olive-pomace oils) were used. The clustering method was hierarchical agglomerative clustering using the Euclidean distance as a similarity measure and the average linkage. Two spectral ranges were considered (which either contained the fluorescence peak of the chlorophylls or did not), and various methods for preprocessing the fluorescence spectra were compared. The oils were clearly distinguished using the unfolded EEMs measured between lambda(ex) = 300-400 nm and lambda(em) = 400-600 nm. The optimal preprocessing was normalization of the unfolded spectra followed by column autoscaling. Also shown are the advantages of using second-order data (EEMs) instead of first-order data (a single fluorescence spectrum) for each sample.     

Origin of French virgin olive oil registered designation of origins predicted by chemometric analysis of synchronous excitation-emission fluorescence spectra

The authentication of virgin olive oil samples requires usually the use of sophisticated and very expensive analytical techniques, so there is a need for fast and inexpensive analytical techniques for use in a quality control methodology. Virgin olive oils present an intense fluorescence spectra. Synchronous excitation-emission fluorescence spectroscopy (SEEFS) was assessed for origin determination of virgin olive oil samples from five French registered designation of origins (RDOs) (Nyons, Vallée des Baux, Aix-en-Provence, Haute-Provence, and Nice). The spectra present bands between 600 and 700 nm in emission due to chlorophylls a and b and pheophytins a and b. The bands between 275 and 400 nm in emission were attributed to alpha-, beta-, and gamma-tocopherols and to phenolic compounds, which characterize the virgin olive oils compared to other edible oils. The chemometric treatment (PLS1) of synchronous excitation-emission fluorescence spectra allows one to determine the origin of the oils from five French RDOs (Baux, Aix, Haute-Provence, Nice, and Nyons). Results were quite satisfactory, despite the similarity between two denominations of origin (Baux and Aix) that are composed by some common cultivars (Aglandau and Salonenque). The interpretation of the regression coefficients shows that RDOs are correlated to chlorophylls, pheophytins, tocopherols, and phenols compounds, which are different for each origin. SEEFS is part of a global analytic methodology that associates spectroscopic and chromatographic techniques. This approach can be used for traceability and vindicates the RDOs.     

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.     

Cultivar differences on nonesterified polyunsaturated fatty acid as a limiting factor for the biogenesis of virgin olive oil aroma

The relationship between the content of nonesterified polyunsaturated fatty acids and the contents of oil aroma compounds that arise during the process to obtain virgin olive oil (VOO) was studied in two olive cultivars, Picual and Arbequina, producing oils with distinct aroma profiles and fatty acid compositions. Results suggest that the biosynthesis of VOO aroma compounds depends mainly on the availability of nonesterified polyunsaturated fatty acids, especially linolenic acid, during the process and then on the enzymatic activity load of the lipoxygenase/hydroperoxide lyase system. Both availability of substrates and enzymatic activity load seem to be cultivar-dependent.     

Verification of silage type using near-infrared spectroscopy combined with multivariate analysis

The ability to authenticate the feed given to animals has become a major challenge in animal production, where the diet fed to the animal is one of the most important production factors affecting the composition of milk and meat from cattle, sheep, and goats. Hence, there is currently an increased consumer demand for information on herbivore production factors and particularly the animal diet. The aim of this study was to evaluate the reliability and accuracy of near-infrared (NIR) reflectance spectroscopy as a tool to verify and authenticate the type of silage used as fed for ruminants. Grain silage (GrS, n = 94), grass and legume silage (GLegS, n = 121), and sunflower silage (SunS, n = 50) samples were collected from commercial farms and analyzed in the visible and NIR regions (400-2500 nm) in a monochromator instrument in reflectance. Principal component analysis (PCA), partial least-squares discriminant analysis (PLS1-DA), and linear discriminant analysis (LDA) models were used as methods to verify the different silage types. The classification models based on the NIR data correctly classified more than 90% of the silage samples according to their type. The results from this study showed that NIR spectra combined with multivariate analysis could be used as a tool to objectively authenticate silage samples used as a feed for ruminants.     

Front-face fluorescence spectroscopy as a rapid and nondestructive tool for differentiating various cereal products: a preliminary investigation

The potential of intrinsic fluorescence spectroscopy was investigated for differentiating between processed grains (flours, pasta, and semolinas) of different wheat cereal products. A total of 59 samples (e.g., 20 complete Kamut, semicomplete Kamut, and soft wheat flours, 28 pasta, and 11 semolinas manufactured from complete Kamut, semicomplete Kamut, and hard wheat flours) were analyzed by front-face fluorescence spectroscopy. Tryptophan fluorescence spectra were scanned between 305 and 400 nm on samples following excitation at 290 nm. The principal component analysis (PCA) performed on flour spectra clearly differentiated complete Kamut and semicomplete Kamut samples from those produced from complete and semicomplete soft wheat flours. The PCA performed on pasta spectra discriminated samples manufactured from complete Kamut and complete hard wheat flours from those made with semicomplete Kamut and semicomplete hard wheat flours. The best discrimination was obtained from tryptophan spectra recorded on semolinas since the four groups were well discriminated. Correct classification amounting to 61.9% was obtained for pasta spectra. A better classification was obtained for flour and semolina spectra since correct classification amounted to 86.7% and 87.9%, respectively. Front-face fluorescence spectroscopy has the potential to be a rapid, low-cost, and efficient method for the authentication of cereal products.     

Multielement X-Ray fluorescence analysis of natural waters by using a preconcentration technique with ion exchange resins

The analysis of trace element in different kinds of waters is described, using a preconcentration technique on ion exchange loaded filter papers. The filters were analyzed employing an energy dispersive X-ray fluorescence system characterized by radioisotopic excitation sources and gas proportional or semiconductor detectors. The detection limits of the method were experimentally determined and the elemental levels in typical waters were measured.     

Kinetics of diglyceride formation and isomerization in virgin olive oils by employing 31P NMR spectroscopy. Formulation of a quantitative measure to assess olive oil storage history

Diacylglycerol isomers and free acidity were determined for five extra virgin olive oils of different initial acidities by employing a facile (31)P NMR methodology as a function of storage time and storage conditions. The kinetic treatment of the hydrolysis of triacylglycerols (TGs) and the isomerization of 1,2-diacylglycerols (1,2-DGs) to 1,3-diacylglycerols (1,3-DGs) during storage of 18 months at ambient temperature in the dark and light and at 5 degrees C in the dark showed that the isomerization is strongly dependent on the rate of the TGs hydrolysis, the initial free acidity (H(0)) of the virgin olive oil samples, and storage conditions. Although the time-evolution of the diacylglycerols (DGs) depends on the TGs hydrolysis, the ratio D of the concentration of 1,2-DGs to the total amount of DGs was found to be independent of this factor. From the kinetic expression of the ratio D, a quantitative measure was formulated that allows the estimation of the storage time or age of virgin olive oils. Application of this quantitative measure to several olive oil samples of known and unknown storage history resulted in a very good agreement with respect to the actual storage time for up to 10-12 months of storage. For a longer storage period, where the isomerization of DGs is close to its equilibrium state, the calculated age index is only indicative.     

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.     

Comparative study of methods for DNA preparation from olive oil samples to identify cultivar SSR alleles in commercial oil samples: possible forensic applications

Virgin olive oil is made from diverse cultivars either mixed or single. Those ensure different tastes and typicity, and these may be also enhanced by the region of production of cultivars. The different olive oil labels correspond to their chemical composition and acidity. Labels also may correspond to a protected origin indication, and thus, such oils contain a given composition in cultivars. To verify the main cultivars used at the source of an olive oil sample, our method is based on DNA technology. DNA is present in all olive oil samples and even in refined oil, but the quantity may depend on the oil processing technology and oil conservation conditions. Thus, several supports were used to retain DNA checking different techniques (silica extraction, hydroxyapatite, magnetic beads, and spun column) to prepare DNA from variable amounts of oil. At this stage, it was usable for amplification through PCR technology and especially with the magnetic beads, and further purification processes were checked. Finally, the final method used magnetic beads. DNA is released from beads in a buffer. Once purified, we showed that it did not contain compounds inhibiting PCR amplification using SSR primers. Aliquot dilution fractions of this solution were successfully routinely used through PCR with different SSR primer sets. This enables confident detection of eventual alien alleles in oil samples. First applied to virgin oil samples of known composition, either single cultivars or mixtures of them, the method was verified working on commercial virgin oil samples using bottles bought in supermarkets. Last, we defined a protocol starting from 2 x 40 mL virgin olive oil, and DNA was prepared routinely in about 5 h. It was convenient to genotype together several loci per sample to check whether alleles were in accordance with those of expected cultivars. Thus, forensic applications of our method are expected. However, the method needs further improvement to work on all oil samples.     

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.     

Classification of edible oils by employing 31P and 1H NMR spectroscopy in combination with multivariate statistical analysis. A proposal for the detection of seed oil adulteration in virgin olive oils

A combination of (1)H NMR and (31)P NMR spectroscopy and multivariate statistical analysis was used to classify 192 samples from 13 types of vegetable oils, namely, hazelnut, sunflower, corn, soybean, sesame, walnut, rapeseed, almond, palm, groundnut, safflower, coconut, and virgin olive oils from various regions of Greece. 1,2-Diglycerides, 1,3-diglycerides, the ratio of 1,2-diglycerides to total diglycerides, acidity, iodine value, and fatty acid composition determined upon analysis of the respective (1)H NMR and (31)P NMR spectra were selected as variables to establish a classification/prediction model by employing discriminant analysis. This model, obtained from the training set of 128 samples, resulted in a significant discrimination among the different classes of oils, whereas 100% of correct validated assignments for 64 samples were obtained. Different artificial mixtures of olive-hazelnut, olive-corn, olive-sunflower, and olive-soybean oils were prepared and analyzed by (1)H NMR and (31)P NMR spectroscopy. Subsequent discriminant analysis of the data allowed detection of adulteration as low as 5% w/w, provided that fresh virgin olive oil samples were used, as reflected by their high 1,2-diglycerides to total diglycerides ratio (D > or = 0.90).     

Proton transfer reaction-mass spectrometry (PTR-MS) headspace analysis for rapid detection of oxidative alteration of olive oil

Olive oil has been characterized by rapid proton transfer reaction-mass spectrometry (PTR-MS) headspace analysis without any concentration of the volatiles or pretreatment of the samples. Comparison of extra virgin and defective (rancid) samples, as described by a panel of sensory judges, and the monitoring of thermo-oxidation processes are discussed. Multivariate analysis of PTR-MS data has been carried out and cross-validated, providing (i) reliable classification models for extra virgin oil as opposed to defective oil and (ii) calibration models able to predict independently thermo-oxidative degradation and the corresponding peroxide value. PTR-MS fragmentation patterns of volatiles considered in this study are also reported.     

The potential of glauconitic sandstone as a potassium fertilizer for olive plants

This study evaluated the potential of glauconitic sandstone as a fertilizer for supplying potassium to plants. The glauconite sandstone (Maraveh, Iran), as analyzed by X-ray fluorescence, was composed of 2.24% potassium oxide plus high contents of silicon, aluminum and ferric oxide. One-year old olive trees, Olea europaea L., were grown in sand or hydroponic culture in a greenhouse under three potassium treatments. Modified Hoagland nutrient solutions based on potassium treatments including 0.5 mM K⁺, 5 mM K⁺ and 400 g glauconitic sandstone powders (per 10 L in hydroponics and per 2.5 L in sand instead of K⁺ supply) were used in both cultures. Plants grown under the three different potassium treatments did not show any potassium deficiency symptoms. In the sand culture, growth and potassium content were higher in plants fed with 5 mM potassium than with the other two potassium treatments. Growth retardation and decreased potassium content in plants fed with 0.5 mM potassium were more severe in the hydroponic culture than in the sand culture. However, plants fed with 400 g glauconitic sandstone showed higher growth in the hydroponic culture than the sand culture. Thus, glauconitic sandstone has the ability to release potassium and can be utilized in combination with other potassium fertilizers.