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.     

Study of the cultivar-composition relationship in Sicilian olive oils by GC,NMR, and statistical methods

The aim of this research is to find if there is direct evidence relating the fatty acid composition of olive oils to specific cultivars grown within a well-limited geographical region. To group olive oils according to their own cultivars,(13)C high-field nuclear magnetic resonance (NMR) and gas chromatography (GC) were used to analyze 60 extra virgin olive oils from the same Italian region (southwestern Sicily) obtained from four monovarietal cultivars. The (13)C NMR spectrum provides information about glycerol triesters of olive oils, i.e., about the acyl composition of major components and about the fatty acids' positional distribution on the glycerol moiety. GC gives the complete fatty acid profile of olive oil samples. Selection of NMR and GC peaks on the basis of their sensitivity to the different cultivars was performed by using multivariate analysis of variance (MANOVA). Principal component analysis, tree clustering analysis, multidimensional scaling (MDS), and linear discriminant analysis (LDA) were then performed on the MANOVA-selected peaks. Results obtained from (13)C NMR and GC techniques combined with the multivariate statistical procedure are in good agreement and prove the usefulness of fatty acids analysis to group the monovarietal olive oils belonging to the same cultivars. Grouping of olive oils according to their cultivars occurs for particular (13)C resonances all belonging to fatty chains in the sn 1,3 position of the glycerol moiety.     

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

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

Determination of phospholipids in olive oil by 31P NMR spectroscopy

A nondestructive analytical method based on NMR spectroscopy was developed for the determination of phospholipids in olive oil. The phospholipids extracted from virgin olive oil with a mixture of ethanol/water (2:1 v/v) were identified and quantified by high resolution (31)P NMR spectroscopy. The main phospholipids found in olive oil were phosphatidic acid, lyso-phosphatidic acid, and phosphatidylinositol. Validation of the (31)P NMR methodology for quantitative analysis of phospholipids in olive oil was performed. Sensitivity was satisfactory with detection limits of 0.25-1.24 mumol /mL. In addition, the composition of fatty acids in phospholipids model compounds and those in olive oil samples was estimated by employing one- and two-dimensional (1)H NMR. The results indicated that the fatty acid composition in phospholipids and triacylglycerols of olive oil was similar.     

Triacylglycerol and fatty acid compositions of French virgin olive oils. Characterization by chemometrics

There is no data concerning the fatty acid and triacylglycerol composition of French virgin olive oil. Thus, these compositions were determined using 564 samples coming from four olive harvests (1998-1999 to 2000-2001). Among these 564 samples, 372 came from the four main French cultivars (Aglandau, Cailletier, Picholine, and Salonenque) and from both of the oldest French protected designations of origin: "Nyons" (cv. Tanche) and "Vallée des Baux". The fatty acid compositions took the different isomeric monounsaturated fatty acids (C16:1 and C18:1) into account. The eicosenoic acid is gondoic acid (20:1n-9) and was determined by dimethyl disulfide adduct using GC/MS. The use of propionitrile as a mobile phase for the HPLC analysis of the triacylglycerols led to better resolutions between triacylglycerols than those resolutions obtained with the mix of solvents recommended by the normalized method (acetone/acetonitrile). Of the samples, 88 had a 9-heptadecenoic acid level (17:1n-8) higher than 0.3% and 33 had a linolenic acid level higher than 0.9%, which are maximal values accepted by the International Olive Oil Council and the European Union. A linear discriminant analysis was carried out on 372 samples with the SAS system and particularly with STEPISC and CANDISC procedures. Variables (n = 37) representing the different fatty acids, the sum of saturated, monounsaturated, and polyunsaturated fatty acids, squalene, and triacylglycerols were used, thus allowing us to classify samples into six groups defined with 100% of well classified samples. These results constitute an original data bank that can be used to identify the origin of virgin olive oils.     

分提橄榄油中主要脂肪酸成分傅里叶变换红外光谱法测量（英）

为了测量从橄榄油中分提的高、低熔点油脂的脂肪酸成分,在4 000～600 cm-1 的范围测量了31个含有不同脂肪酸成分植物油的傅里叶变换红外光谱,用于建立偏最小二乘（PLS）回归分析校正模型。在油脂的傅里叶变换红外光谱变量和脂肪酸组成变量之间建立了交叉验证的PLS校正模型。为了校正油酸和亚油酸含量,在4?000～600 cm-1 的频率范围,经平滑,二阶导数,规范化处理的红外光谱获得了最好的交叉验证校正模型和最佳的预测结果。PLS校正模型预测结果表明,与高熔点橄榄油（油酸,72.29%,亚油酸,9.98%）相比,低熔点橄榄油含有较高的油酸含量和亚油酸含量（油酸,77.46%,亚油酸,12.51%）,预测的结果与气相色谱测量的结果有很好的一致性。建立的PLS校正模型预测橄榄油的不饱和脂肪酸含量具有较好的相关性。该研究为分提油脂质量的判别评价提供了便捷的方法。     

Fatty acid, triacylglycerol, and phytosterol composition in six Tunisian olive varieties

The physicochemical and stability properties as well as the fatty acid, triacylglycerol, sterol, and triterpenic dialcohol compositions of Tunisian olive oil varieties were analyzed. On the basis of our results, we classified all of the monovarietal oils into the extra virgin category. Oleic and linoleic acids were the most useful fatty acids to discriminate three cultivars, Neb Jmel, Chétoui, and Ain Jarboua, from the others. Of the six monovarietal virgin olive oils analyzed, the main triacylglycerols were OOO, POO, PLO plus SLL, and OLO, which was expected given the high oleic acid and low linoleic and linolenic acids content observed in total fatty acids. In total, these accounted for more than 80% of the total HPLC chromatogram peak area. The main sterols found were beta-sitosterol, Delta5-avenasterol, and campesterol. The statistical analysis showed significant differences between oil samples, and the obtained results showed a great variability in the oil composition between cultivars, which is influenced exclusively by genetic factors.     

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

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

Geographical characterization of italian extra virgin olive oils using high-field (1)H NMR spectroscopy.

1H high-field nuclear magnetic resonance (NMR) was used to analyze 216 extra virgin olive oils collected in three years (1996, 1997, and 1998) in different Italian areas in order to evaluate the potential contribution of this technique to the geographical characterization of olive oils. A statistical procedure performed on the intensity of selected NMR peaks has been proposed. Tree clustering analysis of NMR data performed without any a priori hypothesis showed the existence of reliable parameters able to group the olive oils according to the location of olive oil production. Linear discriminant analysis applied to selected NMR parameters of olive oils of the same year of production allowed the grouping of samples according to their geographical origin with only very few errors. Moreover, a satisfactory grouping is reached by combining the NMR data of olive oils from two different years (1996 and 1997). Operating on appropriate sampling, a careful analysis of data yielded the conclusion that the place of olive production could be singled out as a discriminating factor regardless of the cultivars from which the olive oils are derived.     

Geographical characterization of greek virgin olive oils (cv. Koroneiki) using 1H and 31P NMR fingerprinting with canonical discriminant analysis and classification binary trees

This work deals with the prediction of the geographical origin of monovarietal virgin olive oil (cv. Koroneiki) samples from three regions of southern Greece, namely, Peloponnesus, Crete, and Zakynthos, and collected in five harvesting years (2001-2006). All samples were chemically analyzed by means of 1H and 31P NMR spectroscopy and characterized according to their content in fatty acids, phenolics, diacylglycerols, total free sterols, free acidity, and iodine number. Biostatistical analysis showed that the fruiting pattern of the olive tree complicates the geographical separation of oil samples and the selection of significant chemical compounds. In this way the inclusion of the harvesting year improved the classification of samples, but increased the dimensionality of the data. Discriminant analysis showed that the geographical prediction at the level of three regions is very high (87%) and becomes (74%) when we pass to the thinner level of six sites (Chania, Sitia, and Heraklion in Crete; Lakonia and Messinia in Peloponnesus; Zakynthos). The use of classification and binary trees made possible the construction of a geographical prediction algorithm for unknown samples in a self-improvement fashion, which can be readily extended to other varieties and areas.     

Detection of the presence of hazelnut oil in olive oil by FT-raman and FT-MIR spectroscopy

The detection of the presence of refined hazelnut oil in refined olive oil at low percentages is still a challenge with the current official standards. FT-Raman and FT-MIR spectroscopies have been used to determine the level of detection of the presence of hazelnut oil in olive oil. Spectroscopic analysis has been made not only with the entire oil but also with its unsaponifiable matter. Univariate and multivariate statistical models have been designed with this objective. This study shows that a complete discrimination between olive and hazelnut oils is possible and that adulteration can be detected if the presence of hazelnut oil in olive oil is >8% and if the blends are of Turkish olive and hazelnut oils. The limit of detection is higher when the blends are of edible oils from diverse geographical origins.     

Effect of olive ripeness on the oxidative stability of virgin olive oil extracted from the varieties picual and hojiblanca and on the different components involved

The initial stability of virgin olive oil depends on various factors, among which are the variety and the degree of fruit ripeness. The former, which genetically determines the composition of the olive and its oil, also marks, to some extent, its stability. However, oil stability changes as the olive ripens, so it is obvious that the degree of ripeness is an important factor. The oils were obtained by the Abencor system. Acidity, peroxide index, UV absorption at 232 and 270 nm, sensory analysis, fatty acid composition, tocopherols, phenolic compounds, orthodiphenolic compounds, sterols, pigments, and oxidative stability were determined, and the results were analyzed statistically. During ripening there was a decrease in all of the parameters studied except linoleic acid, Delta-5-avenasterol, and oil content, which increased. Virgin oils showed very good correlation between stability and the concentrations of total phenols, o-diphenols, tocopherols, chlorophyll pigments and carotenoids, linoleic and linolenic acids, total sterols, beta-sitosterol, and Delta-5-avenasterol.     

Chemical authentication of extra virgin olive oil varieties by supervised chemometric procedures.

This work has focused on discriminating extra virgin olive oils from Sabina (Lazio, Italy) by olive fruit variety (cultivar). A set of oils from five of the most widespread cultivars (Carboncella, Frantoio, Leccino, Moraiolo, and Pendolino) in this geographical area was analyzed for chemical composition using only the Official Analytical Methods, recognized for the quality control and commercial classification of this product. The obtained data set was converted into a computer-compatible format, and principal component analysis (PCA) and a method based on the Fisher F ratio were used to reduce the number of variables without a significant loss of chemical information. Then, to differentiate these samples, two supervised chemometric procedures were applied to process the experimental data: linear discriminant analysis (LDA) and artificial neural network (ANN) using the back-propagation algorithm. It was found that both of these techniques were able to generalize and correctly predict all of the samples in the test set. However, these results were obtained using 10 variables for LDA and 6 (the major fatty acid percentages, determined by a single gas chromatogram) for ANN, which, in this case, appears to provide a better prediction ability and a simpler chemical analysis. Finally, it is pointed out that, to achieve the correct authentication of all samples, the selected training set must be representative of the whole data set.     

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).     

Influence of harvest date and crop yield on the fatty acid composition of virgin olive oils from cv. Picual

In this study was analyzed the effect of crop year and harvesting time on the fatty acid composition of cv. Picual virgin olive oil. The study was carried out during the fruit ripening period for three crop seasons. The mean fatty acid composition of Picual oils was determined. The oils contained palmitic acid (11.9%), oleic acid (79.3%), and linoleic acid (2.95%). The content of palmitic acid and saturated fatty acids decreased during fruit ripening while oleic and linoleic acids increased. The amount of stearic and linolenic acids decreased. The amount of saturated acids, palmitic and stearic, and the polyunsaturated acids linoleic and linolenic was dependent on the time of harvest, whereas the amount of oleic acid varied with the crop year. The differences observed between crop years for both palmitic and linoleic acid may be explained by the differences in the temperature during oil biosynthesis and by the amount of summer rainfall for oleic acid content. A significant relationship was observed between the MUFA/PUFA ratio and the oxidative stability measured by the Rancimat method.     

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.     

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.     

Effects of fly attack (Bactrocera oleae) on the phenolic profile and selected chemical parameters of olive oil

The phenolic fraction of virgin olive oil influences both its quality and oxidative stability. One of the principal threats of the quality of olive fruit is the olive fly ( Bactrocera oleae) as it alters the chemical composition. The attack of this olive pest has been studied in order to evaluate its influence on the quality of virgin olive oil (free acidity, peroxide value, fatty acid composition, water content, oxidative stability, phenols, and antioxidant power of phenolic fraction). The study was performed using several virgin olive oils obtained from olives with different degrees of fly infestation. They were acquired in different Italian industrial mills from the Abruzzo region. Qualitative and quantitative analyses of phenolic profiles were performed by capillary electrophoresis-diode array detection, and electrochemical evaluation of the antioxidant power of the phenolic fraction was also carried out. These analyses demonstrated that the degree of fly attack was positively correlated with free acidity ( r = 0.77, p < 0.05) and oxidized products ( r = 0.58, p < 0.05), and negatively related to the oxidative stability index ( r = -0.54, p < 0.05) and phenolic content ( r = -0.50, p < 0.05), mainly with secoiridoid compounds. However, it has been confirmed that the phenolic fraction of olive oil depends on several parameters and that a clear correlation does not exist between the percentages of fly attack and phenolic content.     

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.     

Volatile compounds characterizing Tunisian Chemlali and Chétoui virgin olive oils

A total of 33 virgin olive oil samples of the two main Tunisian cultivars, Chemlali and Chétoui, were characterized by their volatile compounds. The olive oil samples were obtained from olives harvested at four stages of ripeness in costal and inland farms of different geographical places. Major volatiles, mostly C6 and C5 compounds produced from linolenic and linoleic acids through the lipoxygenase cascade, were quantified by solid-phase microextraction-gas chromatography. Mathematical procedures allowed for the determination of the volatiles that not only are able to discriminate the olive oils by their olive cultivar (hexanal, E-2-hexenal, and total ketones) and ripeness (pentanal and 1-penten-3-one) but also contribute to their distinctive aroma. Finally, an electronic nose based on metal oxide sensors was checked for a rapid and at-line implementation of Tunisian olive oil varietal traceability. The classification of the samples by the sensors was explained by their sensitivity to volatiles E-2-hexanal, hexanal, 1-penten-3-one, ethanol, and Z-3-hexenol. Multivariate procedures of discriminant analysis and principal component analysis were used in the study.