Application of real-time PCR on the development of molecular markers and to evaluate critical aspects for olive oil authentication

Olive oil authentication using DNA-based markers is becoming very important. qRT-PCR was an efficient tool in investigating the utility of PCR primers for olive oil authentication allowing to discard primers with low PCR efficiency. It also allows investigating of the relative effectiveness among four DNA isolation methods and therefore qRT-PCR would be useful for further optimisation of the DNA extraction protocols. The number of target molecules for the amplification of 80 bp amplicons was higher than that of 200 bp. Therefore the amplicon size should be optimised for olive oil authentication since the higher the number of templates the greater the probability of successful amplification. On conclusion, qRT-PCR is a useful tool in the development of molecular markers for olive oil authentication and it should be used for the optimisation of critical parameters such as the amplicon size and the DNA extraction method.     

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.     

Sensory and physicochemical changes occurring during storage of Galician chorizo in oil

 Changes in the physicochemical and sensory properties of Galician chorizos stored in olive oil, sunflower oil or pork fat were monitored over a 19-week period. The results of analysis of variance showed that the storage medium had a significant effect on some variables. Specifically, moisture content, water activity, acid value and cohesiveness were lowest in chorizos stored in sunflower oil; acid value and pH were highest in chorizos stored in olive oil; external smoky and paprika odours were lowest, and after-taste intensity highest, in chorizos stored in pork fat. Acid value, peroxide index, hardness, colour, external odour intensity, perceived hardness and acid flavour all increased significantly during storage, while pH and water activity declined. In all three storage media, external rancid odour and rancid after-taste were first detected at week 17, when the peroxide index peaked. The overall rating was best for chorizos stored in sunflower oil. Received: 9 September 1997 / Revised version: 24 November 1997     

Sensory and physicochemical changes occurring during storage of Galician chorizo in oil

 Changes in the physicochemical and sensory properties of Galician chorizos stored in olive oil, sunflower oil or pork fat were monitored over a 19-week period. The results of analysis of variance showed that the storage medium had a significant effect on some variables. Specifically, moisture content, water activity, acid value and cohesiveness were lowest in chorizos stored in sunflower oil; acid value and pH were highest in chorizos stored in olive oil; external smoky and paprika odours were lowest, and after-taste intensity highest, in chorizos stored in pork fat. Acid value, peroxide index, hardness, colour, external odour intensity, perceived hardness and acid flavour all increased significantly during storage, while pH and water activity declined. In all three storage media, external rancid odour and rancid after-taste were first detected at week 17, when the peroxide index peaked. The overall rating was best for chorizos stored in sunflower oil. Received: 9 September 1997 / Revised version: 24 November 1997     

Detection of olive oil using the Evagreen real-time PCR method

A sensitive real-time PCR method using the novel fluorescence stain Evagreen was established for detection of olive oil. First, a comparative study was made of two different methods for the recovery of high quality DNA from oil samples. Thereby, the Promega wizard DPSF kit proved to be the most suitable for recovery of DNA from oil samples. Second, an olive-specific pair of primers was designed based on the sequence of an olive plasma intrinsic protein cDNA sequence. Experiments with 13 samples including olive, soybean, sesame, sunflower seed, pumpkinseed, walnut, rapeseed, rice, peanut, maize, pig, chicken and fish confirmed that this pair of primers is highly specific for olive. Additional experiments indicated that the absolute detection limit of our test is 0.07 ng olive DNA and that 0.5% olive DNA can be detected against background of 2 ng/μl sesame DNA.     

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.     

Detection of DNA in virgin olive oils extracted from destoned fruits

Characterization of genetic identity using DNA extracted from olive oil has the potential to facilitate assessment of origin and varietal conformity. Such a prospect is particularly interesting in light of the increased regional spread of olive cultivars and their various contributions to olive oil mixtures for certification of denomination of origin. Towards this goal, we have devised a reliable method for extracting DNA from virgin olive oil that was utilized on monovariety oils from the single, self-sterile cultivar ‘Ogliarola salentina’. We show that DNA purified from oil can be used for microsatellite analysis and that the profile of DNA purified from a monovariety oil corresponds to the profile of DNA purified from the leaves of the same cultivar. While DNA from the pollinators present in the genome of the seed embryo, could potentially contain alleles not present in the genome fruit pulp, invalidating the molecular traceability of olive oil, we show for the first time that there is no contamination of seed embryo DNA in a monovariety oil. Thus, this molecular assay is applicable for monovariety olive oils.     

Shelf life predictions for packaged olive oil using flavor compounds as markers

The experimentally-derived amounts of five selected flavor compounds, namely hexenal, 2-pentyl furan, (E)-2-heptenal, nonanal, and (E)-2-decenal, all produced during the oxidation of extra virgin olive oil packaged in various storage conditions (glass/PET/PVC bottles; 15/30/40 °C temperature; light or dark conditions) for one year, were used in a mathematical model for calculating the probability that the olive oil would not have reached the end of its shelf life (P_safe) after a certain storage period time. The storage times corresponding to probabilities of 70%, 50% and 30% were also calculated. On the basis of these results, an optimal group of flavor compounds were selected that were highly correlated to the degradation factors (storage conditions), and therefore the P_safe, of the oil. These flavor compounds could then be used as markers to identify the cause of the oxidative degradation (the storage history) of the olive oil.     

The detection of the presence of hazelnut oil in olive oil by free and esterified sterols

Genuine olive and hazelnut oils from diverse geographical origins, as single varieties and blends, were mixed at different percentages and analysed by the method based on the quantification of free and esterified sterols. Two formulas based on three sterols (Campesterol, Δ⁷-stigmastenol and Δ⁷-avenasterol) together with empirical decision rules were able to detect the presence of hazelnut oil in olive oil when the percentage of the former was more than 6–8%, although this figure was much lower in the most of the adulterations. Results of univariate and multivariate statistical procedures based on the analysis of 116 samples are presented in support of the method efficiency.     

Physico-Chemical Properties,Composition, and Oxidative Stability of Olive and Soybean Oils Under Different Conditions

Commercial olive and soybean oils have been analyzed periodically in order to evaluate the influence of storage conditions on quality. The following parameters were determined every 10 days of storage: peroxide, saponification and acid values, absorption coefficients K₂₇₀ and K₂₃₂, phenols, chlorophylls and carotenoids contents, DPPH scavenging ability, and total antioxidant activity. Edible oils were exposed to daylight and stored at room temperature in different containers with the same surface area of exposure to air either in clear glass or opaque glass bottles. Changes in oils properties were also examined after the addition of Vit-E as an antioxidant in oil samples. The results showed a gradual loss of quality during storage, especially in clear glass bottles. The addition of the antioxidant, however, significantly reduced the deterioration and oxidation rate in the oil. The best containers for commercial packing of oil were opaque glass bottles containing antioxidant products.     

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.     

Relationship between flavour descriptors and overall grading of analytical panels for virgin olive oil

An analytical panel is used to evaluate the quality of virgin olive oil. Two regression procedures—step-wise multiple regression and regression on principal components—have been used to construct equations that relate profile to overall grading of 57 samples of virgin olive oil collected from all over Spain. Every equation has an adjusted R² > 0.94. Forty new samples evaluated by another panel were used to verify the models. Regression on the principal components correctly classified 87.5% of samples, against 95% classified by step-wise multiple regression. Kurtosis, outliers, multicollinearity and singularity were also studied before building the regression models.     

Stability of encapsulated olive oil in the presence of caffeic acid

The aim of this study was to investigate the influence of microencapsulation and addition of the phenolic antioxidant caffeic acid (CA) on the storage stability of olive oil. Olive oil in the absence or presence of 300 ppm CA was encapsulated in 1.5% w/w sodium alginate shells. Encapsulated oil (with/without added CA) and unencapsulated oil were stored at 20 or 37 °C for 30 days and then subjected to stability and quality evaluation based on peroxide value (PV), p-anisidine value (p-AV), Totox value, free fatty acid (FFA), total extractable phenolic content (TEPC), and fatty acid composition. The CA addition increased the stability and TPC of the final oil product. Oxidation changes were generally slower in the encapsulated oil samples. Both encapsulation and addition of CA preserved unsaturated fatty acids (UFAs) including C18:1 (omega-9 FA), C18:2 (omega-6 FA) and C18:3 (omega-3 FA). We conclude that the current oil encapsulation method using alginate microspheres could be a feasible approach to increasing olive oil stability. The addition of CA to olive oil not only provides additional protection to the oil, but also improves the nutritional values of the final oil product in terms of elevated TEPC and desired UFAs.     

Gas chromatography screening of bioactive phytosterols from mono-cultivar olive oils

Phytosterols (PS) from nine samples of olive oil from Olea europaea L., the Carolea, Cassanese and Coratina mono-cultivars, have been analyzed by gas chromatography. Coratina virgin olive oil (VOO) from the month of November showed highest contents of β-sitosterol (5491 mg kg⁻¹) and Δ⁵ avenasterol (1767 mg kg⁻¹). Olive pomace oil had the lowest total content of all PS, when compared to VOO. These results suggests that, PS can be an important regulatory factor for the functional quality of olive oil along the agro-industry chain from the orchard to nutraceutical.     

Determination of copper with 5,5-dimethylcyclohexane-1,2,3-trione 1,2-dioxime 3-thiosemicarbazone in olive oils by adsorptive stripping square wave voltammetry

In the present paper a method for the determination of Cu in olive oil samples by adsorptive stripping square wave voltammetry (Ad-SSWV) is presented. It has been proven that Cu reacts with 5,5-dimethylcyclohexane-1,2,3-trione 1,2-dioxime 3-thiosemicarbazone, DCDT, in strongly acid media giving rise to a complex. In Ad-SSWV the complex Cu–DCDT experiments an adsorptive reductive process which promotes the appearance of a peak at −0.570 V. The extraction process of Cu from olive oil is carried out with hot concentrated HCl. Calibration graph has been constructed from 0 to 35 ng mL⁻¹ and the detection limit was 0.49 ng mL⁻¹. The method has been applied to commercial olive oils samples and the amounts of Cu found are very similar to those obtained when samples are analysed by AAS.     

Phenolic compounds in virgin olive oils: influence of the degree of olive ripeness on organoleptic characteristics and shelf-life

 An experimental investigation was performed on virgin olive oils from Coratina and Ogliarola salentina cultivars extracted from green, partially blackened and totally blackened olives in order to evaluate changes in the organoleptic properties and in the shelf-life of the oil. The aim of the investigation was to identify the optimal stage of olive maturation for each cultivar to produce higher quality oils. Routine analyses in accordance with EC regulation no. 2568/91, the determination of phenols by HPLC and induction times were carried out. On the whole, the simple phenolic compounds increased as darker olives were used whereas the hydrolysable phenolic compounds decreased. In both the cultivars, total phenols and induction times were significantly higher in the oils obtained from green olives than in oils from totally blackened olives. Hence, it seems advisable to delay olive harvesting for cultivars usually yielding bitter to pungent oils (e.g. Coratina), but to anticipate harvesting for olives producing sweet tasting oils (e.g. O. Salentina). This would produce less bitter Coratina oils, with organoleptic features which may better fulfil consumers' expectations, and O. Salentina oils more resistant to oxidation. Received: 5 June 2000     

Low-fat frankfurters enriched with n−3 PUFA and edible seaweed: Effects of olive oil and chilled storage on physicochemical,sensory and microbial characteristics

This article reports a study of the physicochemical, sensory and microbiological characteristics of low-fat (10%) and n−3 PUFA-enriched frankfurters as affected by addition of seaweed (5% Himanthalia elongata), partial substitution (50%) of animal fat by olive oil and chilled storage (41 days at 2 °C). The presence of seaweed improved water and fat binding properties, reduced (P < 0.05) lightness and redness and increased (P < 0.05) the hardness and chewiness of low-fat frankfurters enriched with n−3 PUFA. The effect of olive oil on those characteristics was less pronounced than that of seaweed. Replacing pork backfat with olive oil in frankfurters produced acceptable sensory characteristics, similar to control, while addition of seaweed resulted in less acceptable products, due mainly to the special flavour of the seaweed. Formulation and storage time affected the total viable count and lactic acid bacteria count. Frankfurters containing olive oil and seaweed had the highest total viable count from day 14 of storage, with lactic acid bacteria becoming the predominant microflora.     

Partial purification and characterization of peroxidase from olives (<Emphasis Type="Italic">Olea europaea</Emphasis> cv. Koroneiki)

The enzyme peroxidase (POD) activity was extracted from olives (Olea europaea cv. Koroneiki) and was partially purified by ammonium sulfate fractionation and gel permeation chromatography (Sephacryl S 300). Further characterization of the POD was performed using the ammonium sulfate purified fraction. POD showed a molecular mass of 44 ± 2 kDa and it expressed catalytic activity with 2,2′-azino-bis(3-ethylbenz-thiazoline-6-sulfonic acid) (ABTS), N,N-dimethyl-p-phenylenediamine (DMPD) and some olive fruit phenols. However, the enzyme was found ineffective as regards the oxidation of oleuropein, the major polyphenol of olives, as well as with coumaric, ferulic, ascorbic and p-hydroxy benzoic acids. pH optimum of the peroxidase-catalyzed oxidation depended on the substrate used and it varied from 4.0 to 6.0. Olive peroxidase shows high thermal stability. Oleuropein, the major polyphenol of olives, drastically inhibited ABTS peroxidation by the POD preparation with an IC₅₀ value of 47 μM. The presence of POD enzyme activity in virgin olive oil samples was also confirmed.     

Microsatellite markers to identify specific alleles in DNA extracted from monovarietal virgin olive oils

The suitability of DNA present in olive oil for PCR analysis has been reported by several authors. However, low concentration, degradation, and the possible presence of additional alleles due to paternal contribution in oils extracted from entire drupes, should be taken into consideration when comparing the amplification profiles of leaves with the corresponding oils for varietal traceability purposes. The aim of this work was to assess, by capillary electrophoresis of microsatellite markers, the phenetic relationships among seven certified Olea europaea L. cultivars, and to verify the genomic equality between leaf DNA and the corresponding monovarietal oil DNA. Moreover, the aim was to establish an identification key to distinguish all the types of oil among them with the minimum number of markers. The results referred to oil DNA, obtained in 70 PCR experiments, confirmed the possibility, in 85.7% of cases, of extracting DNA suitable for the analysis of microsatellites. Ninety percent of the successful amplifications led to identical patterns for leaves and oil DNA. Cima di Melfi shared only 20% of the alleles with the other cultivars. Toscanina and Leccino, showed the highest similarity (about 60%). A single microsatellite was able to distinguish the seven types of oil.