Comparative study of microsatellite profiles of DNA from oil and leaves of two Tunisian olive cultivars

The potential of microsatellites in traceability of Tunisian oils was assessed by comparison of the genetic profile for six SSR markers of DNA extracted from oil and leaves of the two Tunisian major olive cultivars: Chemlali and Chetoui. Different protocols of DNA extraction from oil were tested and compared and we found that the most reproducible protocol in terms of successful microsatellites amplification was the Qiagen QIAamp DNA Stool extraction Kit, which we slightly modified. After amplification and automatic genotyping of six SSRs, it has been shown that the pattern of the DNA purified from a monovarietal oil did not correspond exactly to the profile of the same cultivar leaves’ DNA. In fact, we found evidence for the presence in the oil of alleles originating from the pollinators, which are present in the genome of the seed embryo. These alleles appear as minor peaks and could be easily distinguished from the mother tree’s alleles, so they do not interfere with oil traceability.     

Characterization and authenticity of virgin olive oil (<Emphasis Type="Italic">Olea europaea</Emphasis> L.) cultivars by microsatellite markers

Traceability and authenticity of olive oils have become a major instrument to guarantee the consumer’s protection. Recently, determination of adulteration of olive oils is based on the analysis of simple sequence repeats. In this study, we evaluate the feasibility of identifying above cultivars and the derived oils by the analysis of SSR markers. To this purpose, 22 virgin olive oils were prepared in the laboratory and DNA was extracted using a commercial kit, and 8 SSR markers were analyzed. The obtained results indicated that the comparison of the genetic profile of DNA extracted from oil and leaves of the cultivars studied concluded different genetic profiles in some cultivars when studied for their genome as well as for their oils. These additional alleles in oil’s profile appear as minor peaks can be originating from the pollinators and which are present in the genome of the seed embryo. Hierarchical classification of varieties based on similarity measures and clustering was globally inconsistent with the grouping of varieties by end use and the geographical origin. Finally, we set up a categorization means to discriminate all the types of oil among them with the minimum of markers (three markers).     

Load of polycyclic aromatic hydrocarbons in edible vegetable oils: importance of alkylated derivatives

The presence of polycyclic aromatic hydrocarbons (PAHs) has been studied in different samples of olive oil, extra virgin olive oil, and refined seed oils. A high number of PAHs have been found, with a wide range of molecular weights and in concentrations that are high or even very high compared with the data obtained by other authors, especially in the seed oils. Among the PAHs identified, more than half are alkylated compounds, which account for the major part of the total PAH concentration in some of the samples. The total PAH concentrations in olive oils and extra virgin olive oils are similar, but the former present a higher proportion of heavy PAHs than the latter. The seed oils, in general, have much higher concentrations than the different types of olive oil and their PAH profiles are different. One of the olive oil samples exhibited a PAH distribution similar to that observed in olive pomace oil, suggesting possible adulteration. These data reveal that, in some cases, PAH profile provides useful information in relation to the possible origin of the contamination. We also observed large differences in PAH distribution between oils with the same label but from different batches. PAHs with varying degrees of carcinogenicity have been identified in all the samples, including benzo[a]pyrene, although this PAH was identified neither in the extra virgin olive oils nor in two of the seed oil samples.     

Applicability of SSR markers to the traceability of monovarietal olive oils

BACKGROUND: To protect the features and authenticity of food products, the European Commission enforces two certification labels: Protected Designation of Origin (PDO) and Protected Geographical Indication (PGI). EEC Regulation No. 510/2006 imposes criteria for labelling, production and commercialisation of olive oil. Since plant genotype is a major determinant in establishing the PDO and PGI labels, methods to ascertain the varieties present in a batch of olive oil are essential in validating product conformity. The traceability of olive oil can be assessed through simple sequence repeat (SSR) co‐dominant markers targeted to specific regions of DNA from olive cultivars. RESULTS: Twenty‐one monovarietal olive oils were analysed with nine nuclear and two shortened SSRs. For each marker the correspondence of allelic profile with the reference cultivar, the reproducibility of profiles in different DNA extractions and the polymorphism information content were determined. CONCLUSION: The results showed that using a panel of SSR markers such as those described in this paper allows one to make a reliable attribution of an olive oil to a specific cultivar. Copyright © 2011 Society of Chemical Industry     

A Cost-Efficient and Simple Plant Oil DNA Extraction Protocol Optimized for DNA-Based Assessment of Product Authenticity

DNA-based assays offer precision in ascertaining the species/cultivar origin of agro-food products. Yet, obtaining DNA of sufficient quality and quantity is the main challenge while performing DNA-based food authentication analyses. The aim of the present work was to standardize a cost-efficient, easy-to-apply, yet effective plant oil DNA isolation protocol that allows reliable downstream PCR-based analyses. Because capillary electrophoresis (CE) separation of species/cultivar discriminating genomic fragments is a widely adopted approach in food genomics, a CE system was utilized in order to assess the performance of the proposed cetyl trimethyl ammonium bromide (CTAB)-based protocol. A plastid intergenic spacer and a nuclear olive gene were used as targets in order to evaluate the amplificability of DNA extracted with the CTAB-based protocol. The plastid barcode not only allowed assessing the reproducibility of PCR amplifications from the extracted oil DNA samples (olive, hazelnut, corn, rapeseed, cottonseed, and soybean oils) but also proved successful in discriminating all tested oil crop species based on amplified fragment length polymorphisms. Moreover, the barcode assay proved successful in correctly identifying the tested olive oil: cottonseed oil blends as admixtures of the two oil species. Thus, it was also feasible to demonstrate the potential of the barcode sequence as a discriminatory analyte to detect adulteration in plant oils. In addition, application of a CAPS (cleaved amplified polymorphic sequence) assay designed to genotype a nuclear SNP (single nucleotide polymorphism) marker resulted in the successful identification of the two single-cultivar olive oils included in the study. As a result of the present work, it was feasible to standardize a reliable and cost-efficient DNA extraction protocol that works well with both unrefined (olive and hazelnut) and refined (corn, rapeseed, cottonseed, and soybean) oils.     

Contribution of olive seed to the phenolic profile and related quality parameters of virgin olive oil

BACKGROUND: Conflicting results have been reported about the effect of fruit de‐stoning on the virgin olive oil (VOO) phenolic profile. The aim of the present study was to determine whether olive seed plays any role in the synthesis of this oil phenolic fraction. RESULTS: Increases of around 25% of total phenolic compounds were observed in oils obtained from de‐stoned olive fruits in three main Spanish cultivars. To investigate the involvement of olive seed in determining the phenolic profile of VOO, whole intact olive fruits were added with up to 400% olive stones. Excellent regression coefficients were found in general for the decrease of total phenolic compounds and, particularly, of o‐diphenolics in the resulting oils. On the other hand, it was found that olive seed contains a high level of peroxidase (POX) activity (72.4 U g^?1 FW), accounting for more than 98% of total POX activity in the whole fruit. This activity is able to modify VOO phenolics in vitro, similar to the effect of adding stones during VOO extraction. CONCLUSION: Olive seed plays an important role in determining VOO phenolic profile during the process to obtain an oil that seems to be associated with a high level of POX activity. Copyright © 2007 Society of Chemical Industry     

Influence of genetic matrix and crop year on chemical and sensory profiles of Italian monovarietal extra‐virgin olive oils

BACKGROUND: Commercial virgin olive oils belonging to the cultivars (Bosana, Carolea, Coratina, Frantoio, Itrana, Leccino, Moraiolo, Peranzana, Piantone di Mogliano and Ravece) most represented at the Italian National Review of Monovarietal olive oils (Rassegna Nazionale Italiana degli oli Monovarietali) were considered. The evaluation of the influence of the cultivar and of the crop year as well as their interaction on oil composition were statistically analysed by a complete factorial design by principal components analysis and by linear discriminant analysis. RESULTS: In fatty acids composition, the effect of the cultivar and crop year and their interaction were highly significant. The statistical analysis showed that the sensory attributes (olive fruity, grassy, fresh almond, artichoke, tomato, aromatic herbs, bitter and pungent) were strongly influenced by the cultivar. The prevalent effect of the cultivar on the sensory profile was also demonstrated by the low or absent level of significance observed in the crop year. CONCLUSION: The construction of a databank based on a large number of samples, which is available at URL http://www.olimonovarietali.it , has contributed to the reduction of the variable effects involved in the oil production process. Knowledge of the chemical and sensory profiles of the Italian monovarietal olive oils could start a certification process of these oils, thus giving greater guarantees about their origin. Copyright © 2010 Society of Chemical Industry     

Pigment profile and chromatic parameters of monovarietal virgin olive oils from different Italian cultivars

The aim of this study was to characterise the pigment profile (chlorophylls and carotenoids) of monovarietal virgin olive oils from six Italian olive cultivars (Ghiacciolo, Giarraffa, Sant’Agatese, Biancolilla, Nocellara del Belice and Gentile di Larino), from three Italian olive-growing regions (Emilia-Romagna, Sicily and Molise). The results show that the qualitative study of the pigment chlorophyll and carotenoid fractions of virgin olive oils demonstrated a common pattern that did not depend on the olive cultivar or growing region. Besides these, mutatoxanthin was only detected in oils from the Nocellara del Belice and Gentile di Larino cultivars, and pheophorbide a was only detected in oils from the Ghiacciolo and Sant’Agatese cultivars. However, significant quantitative differences were observed in the composition of the pigment fractions. The principal component discriminate analysis showed that the first two discriminate functions of the statistical analysis explained 78.9% of the total variance. The plot of discriminate functions showed an effect of the olive cultivar and growing area on the chlorophyll and carotenoid contents of the virgin olive oils. The results of this study are a contribution to the pigment characterization of the Italian monovarietal olive oils, which should be completed with more samples obtained from different crop seasons and different growing areas.     

Sterolic composition of Chétoui virgin olive oil: Influence of geographical origin

The sterol profile of Tunisian virgin olive oils produced from Chétoui cultivar, the second main variety cultivated in the north of the country, grown under different environmental conditions, was established by gas chromatography using a flame ionisation detector. More than ten compounds were identified and characterised. As expected for virgin olive oil, the main sterols found in all Chétoui olive oils were β-sitosterol, Δ5-avenasterol, campesterol and stigmasterol. Cholesterol, 24-methylenecholesterol, clerosterol, campestanol, sitostanol, Δ7-stigmastenol, Δ5,24-stigmastadienol, and Δ7-avenasterol were also found in all samples, but in lower amounts. Most of these compounds are significantly affected by the geographical origin. The majority of the Chétoui virgin olive oils analysed respected EC Regulation No. 2568, and in all cases total sterol amounts were higher than the minimum limit set by legislation, ranging from 1017 to 1522 mg/kg.     

Evolution and perspectives of cultivar identification and traceability from tree to oil and table olives by means of DNA markers

In recent years, an increasing number of typicality marks has been awarded to high‐quality olive oils produced from local cultivars. In this case, quality control requires effective varietal checks of the starting materials. Moreover, accurate cultivar identification is essential in vegetative‐propagated plants distributed by nurseries and is a pre‐requisite to register new cultivars. Food genomics provides many tools for cultivar identification and traceability from tree to oil and table olives. The results of the application of different classes of DNA markers to olive with the purpose of checking cultivar identity and variability of plant material are extensively discussed in this review, with special regard to repeatability issues and polymorphism degree. The characterization of olive germplasm from all countries of the Mediterranean basin and from less studied geographical areas is described and innovative high‐throughput molecular tools to manage reference collections are reviewed. Then the transferability of DNA markers to processed products – virgin olive oils and table olives – is overviewed to point out strengths and weaknesses, with special regard to (i) the influence of processing steps and storage time on the quantity and quality of residual DNA, (ii) recent advances to overcome the bottleneck of DNA extraction from processed products, (iii) factors affecting whole comparability of DNA profiles between fresh plant materials and end‐products, (iv) drawbacks in the analysis of multi‐cultivar versus single‐cultivar end‐products and (v) the potential of quantitative polymerase chain reaction (PCR)‐based techniques. © 2016 Society of Chemical Industry     

Sterol and alcohol components of seed,pulp and whole olive fruit oils. Their use to characterise olive fruit variety by multivariates

Sterols, triterpene dialcohols, long‐chain aliphatic alcohols and higher triterpene alcohols were determined in three fruit oil kinds (seed, pulp and whole fruit oils). Seven major Italian olive fruit varieties from the same environment were considered. Results of this research suggested that the compositional data concerning the above analytical fractions were effective in discriminating between seed and pulp oils. The seed oil fraction did not substantially modify the sterol and alcohol composition of the whole fruit oil (mixture of seed and pulp oils), the percentage weight of the seed (～2%) being far lower than that of the pulp (～85%) (whole fruit weight basis). Based on the concentrations of the above components and using appropriate statistical parametric or non‐parametric multivariate techniques, the genetic origin (olive variety) of the three fruit oil kinds was characterised. © 2002 Society of Chemical Industry     

A rapid screening for adulterants in olive oil using DNA barcodes

A distinctive methodology is developed to trace out the mixing into olive oil, which is marketed every year with 20% or more fraudulent oils. Such adulteration has been difficult to differentiate using fatty acid analysis and other available current techniques, as chemically fatty acids are same regardless of their source. The total genomic DNA isolated from olive oil, contaminated with canola and sunflower was analysed for single nucleotide polymorphism (SNP) variation in noncoding spacer region between psbA-trnH and partial coding region of matK of plastid genome. These DNA regions were amplified by PCR using specific primers and resulting DNA sequences were matched to the predetermined consensus DNA barcode sequences of canola and sunflower for discerning the contaminations in olive oil samples. The matching of an adulterant DNA sequence with their respective DNA barcode revealed the mixing of canola and sunflower oil into olive is simpler way and the combined approach of molecular biology and bioinformatics technology can be used as an inexpensive method for ensuring the purity of olive. This plastid based molecular DNA technology can be used for rapid detection of adulteration easily up to 5% in olive oil.     

A procedure for olive oil traceability and authenticity: DNA extraction,multiplex PCR and LDR–universal array analysis

Traceability of olive oils is relevant not only in assessing their origin, but also in protecting against frauds. Here, we present an improvement of the assay previously developed for the genotyping of forty-nine frequently grown Mediterranean olive cultivars by ligation detection reaction (LDR)/universal array (UA), refining the entire procedure in order to address DNA extracted from monovarietal olive oils. Firstly, a simple and robust protocol to extract amplifiable DNA from olive oil was developed. Then, the SNP-containing DNA sequences were simultaneous amplified by multiplex PCR and used on a LDR-UA platform, which gave precise and accurate genotype results. Thirteen out of the seventeen investigated SNPs were amplifiable in multiplex PCR, and were sufficient to unequivocally discriminate the forty-nine cultivars. The availability of this semi-automated SNP genotyping assay should help food testing laboratories to verify the origin and authenticity of monovarietal extra-virgin olive oils. C. Consolandi and L. Palmieri equally contributed to this research paper.     

Oxidative stability of olive oil after food processing and comparison with other vegetable oils

The use of olive oil showed an important protection of meat and potatoes when compared with other vegetable oils, with sunflower oil samples being oxidised after 60 min of processing at 180 °C. Olive oil samples were not oxidised, independently of the olive oil quality used. Shelf life was longer for extra-virgin olive oil containing samples and this fact was positively correlated with their higher phenolic content. The radical-scavenging activity of extra-virgin olive oil was higher than for other olive oil samples and was also positively correlated with the phenolic content of the oil. Seed oil antioxidants showed little capacity in delaying the oxidative degradation of seed oils and meat processed with them. However, tocopherol content and the identity of tocopherols present in the oil were shown to have a more important role in the oxidative stability of seed oils than the fatty acid composition.     

Pigment profile and colour of monovarietal virgin olive oils from Arbequina cultivar obtained during two consecutive crop seasons

Monovarietal virgin olive oils are labelled with the olive varieties giving them their distinctive character. There are numerous studies focussed on the characterisation and quantification of the minor fractions of virgin olive oils that have generated databases on varietal olive oils. However, few studies have focussed on the components of the pigment fraction of virgin olive oils. The aim of this work was to quantify the components of the chlorophyll and carotenoid fractions of the monovarietal virgin olive oils from the Arbequina cultivar, growing in the Spanish area of Catalonia, during two consecutive crop seasons. Additionally the pigment changes occurring during 24 months of oil storage were evaluated. The results of this study showed minor qualitative differences between monovarietal virgin olive oils from two consecutive seasons. The quantitative differences could be attributed to the harvest period in each season rather than to the year’s weather conditions. Storage of the monovarietal virgin olive oils probably caused an important loss of the chlorophyll fraction, mainly chlorophyll a, during the first 6 months of storage. On the other hand, the carotenoid fraction was very stable and the retention of provitamin A was close to 80%, even after 24 months of storage.     

Storage-time effects on olive oil DNA assessed by Amplified Fragments Length Polymorphisms

In this study Amplified Fragments Length Polymorphisms (AFLPs) analysis was applied on DNA extracted from different monovarietal olive oils. The aim was to study how the length of storage after milling of the oil can affect the use of DNA as an analyte for molecular traceability. Results, all assessed by statistical analyses, showed that the authentication of olive oil with molecular methods should be performed within a month from olive oil production. After this period, a significant decrease of quality of DNA extracted from olive oil was observed, with a consequent loss of information, that can affect the reliability of the results.     

Quality assessment of edible vegetable oils used as liquid medium in canned tuna

An experimental investigation was conducted to assess the quality of vegetable oils used as liquid medium in preserved fish. Sixteen tuna preserves were tested, including two in extra virgin olive oil, nine in olive oil (refined olive oil plus virgin olive oil) and five in refined seed oil. Next to the traditional routine analyses prescribed by the EEC Regulation N° 2568/91 (useful for a preliminary characterisation of oils used as liquid medium), the content of trans isomers of unsaturated fatty acids was determined and the analysis of polar compounds was performed by HPSEC (high performance size-exclusion chromatography). The acid composition showed the presence of highly unsaturated fatty acids, typical of fish lipids. Trans isomers were always absent in extra virgin olive oils, whereas they were always present in olive oils and in refined seed oils. Triglyceride oligopolymers, oxidised triglycerides and diglycerides, measured by means of the HPSEC analysis of polar compounds, ranged within 0.13-1.07%, 0.51-2.36% and 0.96-4.02%, respectively. The HPSEC analysis of polar compounds and the determination of trans isomers enabled better assessment of the quality of different types of oils used as liquid medium for canned tuna.     

Saturated hydrocarbon content in olive fruits and crude olive pomace oils

Olive fruits contain an n-alkane series of saturated hydrocarbons mainly in the pulp. Lower amounts of a complex mixture of paraffins, unresolved by gas chromatography (UCM – unresolved complex mixture), have been found in cuticle, stone (woody shell and seed), olive leaves, and talc used as an aid to olive oil extraction. The amounts of both kinds of hydrocarbons are related to the olive cultivar and are transferred to oils in a proportion depending on the oil-obtaining process (centrifugation or solvent extraction). In olive oil obtained by centrifugation, only n-alkanes were detected. However, in olive oil extracted by second centrifugation, small amounts of UCM paraffins were detected together with the n-alkanes. Olive pomace oils showed a very variable content of both types of hydrocarbons according to the different obtaining process, such as double centrifugation, solvent extraction or centrifugation followed by solvent extraction. ‘White mineral oil’ used in oil extraction machinery is the source of the high concentrations of UCM paraffins found in some olive and olive pomace oils. In the case of second centrifugation olive oil, a maximum limit of 50 mg kg^?1 of UCM is suggested, whereas in the case of crude olive pomace oil, it amounts to 250 mg kg^?1 plus an additional minimum of 1.0 for the n-alkanes/UCM ratio.     

Genetic and chemical assessment of Arbequina olive cultivar grown in Córdoba province,Argentina

BACKGROUND: Thirty‐eight accessions of olive (Olea europaea L.) originating from Córdoba province (Argentina) and preliminarily identified as belonging to the Arbequina variety were genotyped using AFLP (amplified fragment length polymorphism) DNA markers. Also, the oil chemical composition was studied during three consecutive crop years. The objectives of the work were (a) to investigate genetic intra‐cultivar diversity and (b) to evaluate the oil chemical composition and compare it with that of Arbequina oil produced in Spain. RESULTS: The 19 primer combinations employed to perform the AFLP analysis produced 98 polymorphic bands. A reduced genetic heterogeneity was obtained, confirming that (a) the selected accessions belong to the Arbequina variety and (b) the traditional vegetative propagation practice has caused low genetic erosion in this variety cultivated in Córdoba. The main features that characterise the Argentinian Arbequina oils studied are the lower content of oleic acid and higher levels of phenolics and high‐molecular‐weight volatile compounds compared with those found in Spanish Arbequina oils. CONCLUSION: In spite of the small proportion of intra‐cultivar variability, the Arbequina variety grown in Argentina produces oils with different chemical traits from those obtained in the original Spanish growing region. These differences can be attributed mainly to the particular environmental conditions of the olive‐growing areas in these countries. Copyright © 2008 Society of Chemical Industry     

The potential of plastid trnL (UAA) intron polymorphisms for the identification of the botanical origin of plant oils

This work describes a methodology for the identification of the botanical origin of plant oils emphasising on the detection of adulteration of olive oil with sesame oil. This methodology comprises a PCR-based assay that exploits the polymorphisms found in the plastid genome combined with a capillary electrophoresis system to discriminate a range of 11 plant species commonly used for oil extraction on the basis of differential length of their corresponding PCR amplicons. The assay takes advantage of universal primers that amplify a region from the trnL (UAA) intron of every plant species. The results showed that amplicons from all species can be accurately discriminated apart from olive and avocado. Single-base primer extension was then proposed as an additional methodology to discriminate the two species and to confirm the results of the former approach. These assays were successfully applied on olive and sesame oils, thus confirming the validity of this approach.