High-performance liquid chromatography evaluation of phenols in olive fruit, virgin olive oil, vegetation waters, and pomace and 1D- and 2D-nuclear magnetic resonance characterization

Phenolic compounds are the most important antioxidants of virgin olive oil. This paper reports on the application of solid phase extraction (SPE) in the separation of phenolic compounds from olive fruit, olive oil, and by-products of the mechanical extraction of the oil and the complete spectroscopic characterization by nuclear magnetic resonance of demethyloleuropein and verbascoside extracted from olive fruit. SPE led to a higher recovery of phenolic compounds from olives than did liquid/liquid extraction. SPE also was used to separate phenolic compounds from pomaces and vegetation waters. Phenylacid and phenyl-alcohol concentrations in extracts obtained from SPE and liquid/liquid extraction were not significantly different (P<0.05). The recovery of the dialdehydic form of elenolic acid linked to 3,4-(dihydroxyphenyl)ethanol and an isomer of oleuropein aglycon, however, was low.     

Evolution of phenols and pigments in extra virgin olive oil from irrigated super‐intensive orchard

Phenolic compounds have a high importance in olive oil because of their effect on shelf life and sensory properties. This study reports on the HPLC profiles of the phenolic compounds of virgin olive oils obtained from Arbequina olives from the harvesting in a super‐intensive orchard under a linear irrigation system. In addition, phenolic content, carotenoid and chlorophyllic pigments, and oxidative stability were analyzed. Total phenol content and 3,4‐DHPEA‐EDA increased up to a maximum throughout the ripening process. The simple phenols tyrosol and hydroxytyrosol acetate increased throughout the ripening process, however, there was not found a clear trend in hydroxytyrosol content. Minor constituents such as vanillic acid and p‐coumaric acid increased up to a maximum and then decreased, since vanillin decreased progressively throughout the time of harvest. 3,4‐DHPEA‐EDA and lignans were present in considerable amounts in the studied samples, while oleuropein aglycone was present in a low amount. Total phenol content and oil stability followed the same trend throughout the study, so a very good correlation was established between them. Total secoiridoids and, specifically, 3,4‐DHPEA‐EDA seemed to be responsible for oil stability. The pigment content decreased during ripening, and not a positive correlation was found between pigments and oil stability. Practical applications : The results can be used to determine the best time for harvesting in order to obtain olive oils with different phenols and pigment contents. This is important for sensory characteristics of the olive oils and also for olive oil stability.     

Bioactive Compounds of Portuguese Virgin Olive Oils Discriminate Cultivar and Ripening Stage

The presence of different bioactive compounds in virgin olive oil affects its nutritional, oxidative and sensorial properties. Phenolic compounds are olive endogenous bioactive compounds highly susceptible to degradation. Olive endogenous oxidoreductases, mainly polyphenol oxidases (PPO) and peroxidases (POD), may play an important role on the profile of bioactive compounds in olive oil by promoting oxidation of phenolic compounds. The aim of this study was to evaluate if changes on PPO and POD activities in olive fruits from two Portuguese cultivars (Olea europaea, cv ‘Cobrançosa’ and cv ‘Galega Vulgar’) are related with the composition of their olive oils, especially phenolic compounds. Pattern recognition techniques [principal component analysis (PCA), cluster analysis (CA), and discriminant analysis (DA)] were used for multivariate data analysis. Olive oils characterized by their FA composition were grouped by cultivar. When olive oils were characterized by their phenolic composition, green pigments, and enzymatic activities in fruits, they could be discriminated by olive ripening stage. Along ripening, PPO activity was only detected in the fruit mesocarp of both cultivars and POD activity was mainly detected in the seeds. The POD activity, as well as vanillin and gamma‐tocopherol contents in olive oil increased with the ripening index. Conversely, higher PPO activity in fruits at early ripening stages together with higher levels of total phenols, green pigments, beta‐tocopherol, hydroxytyrosol and p‐coumaric acid in olive oils were observed. The ripening stage of fruits showed to be a key factor on the amount and profile of bioactive compounds of olive oil.     

Evolution of phenolic compounds in virgin olive oil during storage

Phenolic compounds are of fundamental importance to the shelf life of virgin olive oils because of their antioxidative properties. In this paper, the evolution of simple and complex olive oil phenols during 18 mon of storage is studied by high-performance liquid chromatography (HPLC) analysis. The olive oils under examination were from various olive cultivars, harvested in two sectors in the same region at different stages of ripeness. The findings indicate that it is not the variety but rather the ripeness of the olives and the soil and climate that influence the phenol composition of virgin olive oil. In addition, a positive correlation was found between the age of the oils and the tyrosol to total phenols ratio. Lastly, gas chromatography-mass spectrometry analysis confirmed that the unidentified peaks detected by HPLC were of a phenolic nature.     

Air exposure time of olive pastes during the extraction process and phenolic and volatile composition of virgin olive oil

The time of exposure of olive pastes to air contact (TEOPAC) during malaxation was studied as a processing parameter that could be used to control endogenous oxidoreductases, such as polyphenoloxidase, peroxidase, and lipoxygenase, which affect virgin olive oil quality. Phenolic and volatile compounds were analyzed in the oils obtained using progressive TEOPAC at three ripening stages of olives. Multivariate statistical analysis was applied to the raw data. The phenolic concentration of virgin olive oil progressively decreased with increasing IEOPAC. On the contrary, a positive relationship was found with the concentration of several volatile compounds responsible for virgin olive oil aroma. The effect of TEOPAC, however, was strictly related to fruit ripening.     

The olive oil oxygen radical absorbance capacity (DPPH assay) as a quality indicator

The antioxidant properties of some single components and the total antioxidant activity of extra‐virgin olive oil have been evaluated by the oxygen radical absorbance capacity (ORAC) method. The total ORAC of the extra‐virgin olive oil was found to be positively correlated with the concentration of total polyphenols, which are important to the shelf life of the product. Among the single phenolic compounds studied, gallic acid showed a higher ORAC than caffeic acid and oleuropein, while among the derivates of oleuropein, hydroxytyrosol was found to be the most active compound among all the phenols studied. The total ORAC of commercial olive oils differed according to the concentration of total polyphenols. The total ORAC of extra‐virgin olive oil was constant during 1 year of storage in rational conditions, whereas it worsened dramatically in olive oil damaged by the lipase‐producing yeast Williopsis californica or by lipase from Pseudomonas spp. The study accomplished on the oily fraction of the fruits before harvesting demonstrated that the total ORAC of the oil of under‐ripe green olives is higher compared to that shown by mature fruits; therefore, through the choice of the harvesting time, it is possible to define also the future content of polyphenols of the oil. The total ORAC test, together with other analyses, can be considered as a qualitative parameter that can contribute to the expression of technological and health virtues of extra‐virgin olive oil.     

Flavor components of olive oil—A review

The unique and delicate flavor of olive oil is attributed to a number of volatile components. Aldehydes, alcohols, esters, hydrocarbons, ketones, furans, and other compounds have been quantitated and identified by gas chromatography-mass spectrometry in good-quality olive oil. The presence of flavor compounds in olive oil is closely related to its sensory quality. Hexanal, trans-2-hexenal, 1-hexanol, and 3-methylbutan-1-ol are the major volatile compounds of olive oil. Volatile flavor compounds are formed in the olive fruit through an enzymatic process. Olive cultivar, origin, maturity stage of fruit, storage conditions of fruit, and olive fruit processing influence the flavor components of olive oil and therefore its taste and aroma. The components octanal, nonala, and 2-hexenal, as well as the volatile alcohols propanol, amyl alcohols, 2-hexenol, 2-hexanol, and heptanol, characterize the olive cultivar. There are some slight changes in the flavor components in olive oil obtained from the same oil cultivar grown in different areas. The highest concentration of volatile components appears at the optimal maturity stage of fruit. During storage of olive fruit, volatile flavor components, such as aldehydes and esters, decrease. Phenolic compounds also have a significant effect on olive oil flavor. There is a good correlation between aroma and flavor of olive oil and its polyphenol content. Hydroxytyrosol, tyrosol, caffeic acid, coumaric acid, and p-hydroxybenzoic acid influence mostly the sensory characteristics of olive oil. Hydroxytyrosol is present in good-quality olive oil, while tyrosol and some phenolic acids are found in olive oil of poor quality. Various off-flavor compounds are formed by oxidation, which may be initiated in the olive fruit. Pentanal, hexanal, octanal, and nonanal are the major compounds formed in oxidized olive oil, but 2-pentenal and 2-heptenal are mainly responsible for the off-flavor.     

Effect of natural antioxidants in virgin olive oil on oxidative stability of refined,bleached, and deodorized olive oil

The factors influencing the oxidative stability of different commercial olive oils were evaluated. Comparisons were made of (i) the oxidative stability of commercial olive oils with that of a refined, bleached, and deodorized (RBD) olive oil, and (ii) the antioxidant activity of a mixture of phenolic compounds extracted from virgin olive oil with that of pure compounds andα-tocopherol added to RBD olive oil. The progress of oxidation at 60°C was followed by measuring both the formation (peroxide value, PV) and the decomposition (hexanal and volatiles) of hydroperoxides. The trends in antioxidant activity were different according to whether PV or hexanal were measured. Although the virgin olive oils contained higher levels of phenolic compounds than did the refined and RBD oils, their oxidative stability was significantly decreased by their high initial PV. Phenolic compounds extracted from virgin olive oils increased the oxidative stability of RBD olive oil. On the basis of PV, the phenol extract had the best antioxidant activity at 50 ppm, as gallic acid equivalents, but on the basis of hexanal formation, better antioxidant activity was observed at 100 and 200 ppm.α-Tocopherol behaved as a prooxidant at high concentrations (>250 ppm) on the basis of PV, but was more effective than the other antioxidants in inhibiting hexanal formation in RBD olive oil.o-Diphenols (caffeic acid) and, to a lesser extent, substitutedo-diphenols (ferulic and vanillic acids), showed better antioxidant activity than monophenols (p- ando-coumaric), based on both PV and hexanal formation. This study emphasizes the need to measure at least two oxidation parameters to better evaluate antioxidants and the oxidative stability of olive oils. The antioxidant effectiveness of phenolic compounds in virgin olive oils can be significantly diminished in oils if their initial PV are too high.     

Polyphenol oxidase from eggplant reduces the content of phenols and oxidative stability of olive oil

Activity of the polyphenol oxidase (PPO) from eggplant fruit (Solanum melongena L.) on phenolic compounds of an extra virgin olive oil (EVOO) was studied. In standardized reaction solutions, the eggplant PPO, isolated in the laboratory, depleted completely chlorogenic and caffeic acids, oleuropein, and verbascoside, while the levels of hydroxytyrosol reduced by half. Conversely, no activity of the PPO was observed on the gallic and protocatechuic acids nor on mono‐phenols, such as tyrosol and the p‐coumaric, o‐coumaric, and ferulic acids. PPO activity on phenols extracted from eggplant fruit and EVOO confirmed the enzyme substrate specificity and caused a significant decrease in the measure of total phenols and o‐diphenols. Similarly, PPO crude extract caused a significant decrease of polyphenols directly in the EVOO. Moreover, maximum degradation of EVOO polyphenols was observed when olive oil was homogenized with eggplant fruit pulp to form a cream‐like purée. In fact, immediately after the preparation, total phenols and o‐diphenols of the olive oil recovered from the eggplant‐oil purée were decreased by ～80% and 100% compared to those of the initial EVOO. As a consequence, the oxidative stability of the recovered oil was ～60% lower than that of the initial EVOO. In conclusion, in the preparation of vegetable preserves, a residual activity of phenol oxidase may adversely affect the quality and shelf life of the extra virgin olive oil used as covering.     

The effect of malaxation temperature on the virgin olive oil phenolic profile under laboratory‐scale conditions

The relationship between olive paste malaxation temperature and the concentration of olive oil hydrophilic phenols (HP), i.e. simple phenols, secoiridoids and lignans, was investigated. Malaxation experiments were performed at laboratory scale for 45 min at 21, 24, 27, 30, 33 and 36 °C. A significant (p <0.05) increment of total phenols concentration was found with a maximum at 27 °C, whereas for higher temperatures (30–36 °C) a progressive decrement was observed. A similar pattern was recorded approximately for all the secoiridoid compounds, i.e. a quasi‐linear increment of concentrations with increasing temperature until 30 °C, followed by a marked decrease in correspondence with the higher malaxation temperature (33 and 36 °C). The amount of simple phenols increased linearly with increasing temperature and no decrements were observed up to the maximal temperature investigated (36 °C), while no significant differences were found for lignans. A small increment of peroxide values and total chlorophyll was recorded as a function of the increasing malaxation temperature, whereas no differences were observed in the free acidity. The results highlight that there is not a univocal relationship between HP concentration and malaxation temperature. An equilibrium between degradation (chemical and biochemical oxidation and hydrolysis) and transfer (partitioning) phenomena was hypothesized.     

Characterization of the Volatile, Phenolic and Antioxidant Properties of Monovarietal Olive Oil Obtained from cv. Halhali

Volatile and phenolic compositions of olive oil obtained from the cv. Halhali were investigated in the present study. Fruits were harvested at the optimum maturity stage of ripeness and immediately processed with cold press. Simultaneous distillation/extraction (SDE) with dichloromethane was applied to the analysis of volatile compounds of olive oil. Sensory analysis showed that the aromatic extract obtained by SDE was representative of olive oil odour. In the olive oil, 40 and 44 volatile components were identified and quantified in 2010 and 2012 year, respectively. The total amount of volatile compounds was 18,007 and 19,178 μg kg^?1 for 2010 and 2012, respectively. Of these, 11 compounds in the 2010 and 12 in the 2012 harvest presented odour activity values (OAVs) greater than 1, with 1‐octen‐3‐ol, ethyl‐3‐methyl butanoate, (E)‐2‐heptenal and (E,Z)‐2,4‐decadienal being those with the highest OAVs in olive oil. The high‐performance liquid chromatographic method coupled with diode‐array detection was used to identify and quantify phenolic compounds of the olive oil. A total of 14 phenolic compounds in both years were identified and quantified in olive oil. The major phenolic compounds that were identified in both years were hydroxytyrosol, tyrosol, elenolic acid, luteolin, and apigenin. Antioxidant activity of olive oil was measured using the DPPH and ABTS methods.     

Natural antioxidants and volatile compounds of virgin olive oils obtained by two or three‐phases centrifugal decanters

Research has been carried out to ascertain the influence of different centrifugal decanters employed in olive process on oil yields and qualitative characteristics and composition of volatile compounds of virgin olive oil. Tests were performed in an olive oil mill equipped with centrifugal decanters at two or three‐phases. Results show that oil yields were similar and oils extracted from good‐quality olives do not differ in free fatty acids, peroxide value, UV absorptions and organoleptic assessment. Total phenols and o‐diphenols content as well as induction time values are higher in oils obtained by the centrifugal decanter at two‐phases, because it requires less quantity of water added to olive paste in comparison to the three‐phases centrifugal decanter. The amount of water added determines the dilution of the aqueous phase and lowers the concentration of the phenolic substances more soluble in vegetable waste water. Due to the partition equilibrium law the concentration of the same substances consequently diminishes in the oil. In this research, the coefficient of the partition equilibrium of total phenols between oil and vegetable water has been calculated and discussed. No significant difference occurred, due to the different decanters employed, in the average values of the volatile components of the head‐space of oils.     

Investigation of the Effects of Virgin Olive Oil Cleaning Systems on the Secoiridoid Aglycone Content Using High Performance Liquid Chromatography–Mass Spectrometry

Phenolic compounds are useful markers to control olive oil technological processes, including the virgin olive oil (VOO)/water separation after olive oil extraction. In this investigation, VOO extracted from olives of cv. Coratina using a mild oil/water separator called the hydrocyclone sedimentation system (Hydroil) was compared with VOO obtained using a conventional vertical centrifuge separator (Cenoil), which is mostly used in the modern olive oil industry. Secoiridoid aglycones were selected, among phenolic compounds, as markers and analyzed using reversed‐phase liquid chromatography coupled to linear quadrupole ion‐trap mass spectrometry with electrospray ionization in the negative mode. VOO samples obtained using the Hydroil system were found to contain significantly higher levels of secoiridoid aglycones, compared to the Cenoyl‐type samples. In particular, the total content of the aglycones of decarboxymethyl oleuropein, decarboxymethyl ligstroside, ligstroside, and oleuropein, expressed in terms of oleuropein, was estimated as 35.40 ± 0.80 mg kg^?1, compared to 8.06 ± 0.41 mg kg^?1 in the Cenoil samples (n = 3). Since no significant difference in residual water (P < 0.05) was found between the two types of VOO samples, the higher amount of secoiridoids obtained for Hydroil‐type ones was explained by the lower extent of oxidation occurring during the mild oil/water separation achieved using the Hydroil system.     

Antioxidant activity of tocopherols and phenolic compounds of virgin olive oil

The antioxidant effects of hydrophilic phenols and tocopherols on the oxidative stability in virgin olive oils and in purified olive oil have been evaluated. Total hydrophilic phenols and the oleosidic forms of 3,4-dihydroxyphenolethanol (3,4-DHPEA) were correlated (r=0.97) with the oxidative stability of virgin olive oil. On the contrary, tocopherols showed low correlation (r=0.05). Purified olive oil with the dialdehydic form of elenolic acid linked to 3,4-DHPEA, an isomer of oleuropeine aglycon, and 3,4-DHPEA had good oxidative stability. A synergistic effect was observed in the mixture of 3,4-DHPEA and its oleosidic forms with α-tocopherol in purified olive oil by the Rancimat method at 120°C.     

Effect of irrigation applied to olive trees (Olea europaea L.) on phenolic compound transfer during olive oil extraction

The main objective of this research was to determine the extent to which irrigation practices affect the partitioning of phenolic compounds between olive paste, pomace, olive oil and wastewater. The current paper also aimed to study the effect of technological natural micro‐talc (NMT) addition during the oil extraction process on the partitioning of the phenolic compounds between solid and liquid phases. The results obtained in this study showed that irrigation applied to olive trees let to a considerable decrease in the phenol content of the olive paste. The water status of the trees affected the phenol synthesis in the olive fruit, and consequently the phenol content of the olive paste, more than the partitioning of the phenolic compounds during the olive oil extraction process. The most remarkable point of the phenol partitioning was related to the simple phenols. While in the samples from non‐irrigated trees the greater proportion of these phenols partitioned into the pomace, in samples from irrigated trees most of them were lost in the wastewater. After comparison of the results obtained from the experiments with and without NMT addition, it was concluded that the use of that co‐adjuvant did not significantly alter either the phenolic profile of the oil phase obtained or the content of the individual phenolic compounds.     

Comparative study of olive oil quality from Chemlali Sfax versus Arbequina cultivated in Tunisia

The effects of the geographical region on the behavior of the Arbequina olive cultivar (cv) cultivated in the south of Tunisia (in the arid zone of Sfax) was compared to an autochthonous cultivar (Chemlali Sfax). Various olive parameters were analyzed, such as ripening index, pulp/stone ratio, oil contents, and sensory profiles. Most of the quality indices and fatty acid composition showed significant variations among olive cultivars. Arbequina cv is characterized by high oil yield with a less total phenols and pigments content than Chemlali Sfax cv. Cielab spectrophotometer coordinate L*, b*, and a* values show a great difference in olive oil colors. In spite of their low oleic acid contents, autochthonous cultivar presented a higher induction time (6.82 and 2.68 h for Chemlali and Arbequina, respectively) and high contents of phenolic compounds (158.28 and 110.27 mg/kg for Chemlali Sfax and Arbequina, respectively). The most important compounds identified were oleuropein aglycon (45.50 mg/kg), hydroxytyrosol (3.68 mg/kg), 1‐acetoxypinoresinol (6.23 mg/kg) in Chemlali Sfax oil and hydroxytyrosol glucoside (25.15 mg/kg), tyrosol (12.51 mg/kg), and oleuropein aglycon (30.60 mg/kg) in Arbequina oil. Chemlali Sfax also possessed a very bitter taste, whereas the Arbequina had a sweet taste amongst its attributes. The principal component analysis of the results indicated that the geographical region has significantly affected the olive oil quality.     

Vertical centrifugation of virgin olive oil under inert gas

Virgin olive oil vertical centrifugation under inert gas (IVC) was compared with conventional vertical centrifugation in contact with air (CVC). IVC gives a strong reduction of the oil oxygenation in terms of reduced dissolved oxygen concentration and oxidative indexes (peroxide values and K232). Minor compounds (chlorophyll and total phenols) and volatile compounds were not affected by the treatment. Practical applications: A technical implementation of inertized vertical separators appears a practicable solution to preserve virgin olive oil quality traits. This can be easily implemented both by the mill owner with homemade adjustment of the existing machines and by plant industrial manufacturers.     

Phenolic composition of virgin olive oils from cross breeding segregating populations

The evaluation and characterization of segregating populations is a critical step in olive breeding programs. In this work, phenolic profiles of virgin olive oils (VOOs) from segregating populations obtained by cross breeding in Cordoba (Spain) have been evaluated. Genotypes obtained from open pollination of the cultivar Manzanilla de Sevilla, and from crosses between the cultivars Arbequina × Arbosana, Picual × Koroneiki and Sikitita × Arbosana were tested. The phenolic composition was determined after liquid–liquid extraction with 60:40 v/v methanol–water and subsequent chromatographic analysis with ultraviolet (UV) detection of both absorption and fluorescence in a sequential configuration. Results for all studied compounds showed high degree of variability between genotypes, with a higher range of variation than the observed for the genitors. Most of the observed variability was attributable to differences in genotypes within crosses rather than among crosses. Some issues related to breeding strategies are discussed. Practical applications: Phenolic compounds are considered to be of paramount importance for the assessment of virgin olive oil quality due to their contribution to the nutraceutical and sensory profile of this natural food. This study focuses on the evaluation of the content of phenolic compounds in olive oils originated from cross breeding in an olive breeding program (Cordoba, Spain). This step is crucial to determine the range of variation of phenolic compounds and the selection of interesting genotypes with higher composition in total phenols or in an individual phenol targeted at a breeding program.     

A high-field1H nuclear magnetic resonance study of the minor components in virgin olive oils

High-field (600 MHz) nuclear magnetic resonance (NMR) spectroscopy was applied to the direct analysis of virgin olive oil. Minor components were studied to assess oil quality and genuineness. Unsaturated and saturated aldehyde resonances, as well as those related to other volatile compounds, were identified in the low-field region of the spectrum by two-dimensional techniques. Unsaturated aldehydes can be related to the sensory quality of oils. Other unidentified peaks are due to volatile components, because they disappear after nitrogen fluxing. The statistical analysis performed on the intensity of these peaks in several oil samples, obtained from different olive varieties, allows clustering and identification of oils arising from the same olive variety. Diacylglycerols, linolenic acid, other volatile components, water, acetic acid, phenols, and sterols can be detected simulteneously, suggesting a useful application of high-field NMR in the authentication and quality assessment of virgin olive oil.     

Hydrophilic antioxidants of virgin olive oil. Part 1: Hydrophilic phenols: A key factor for virgin olive oil quality

Virgin olive oil (VOO) consumption is increasing all over the world due to its excellent organoleptic and nutraceutical properties. These beneficial traits stand from a prominent and well‐balanced chemical composition, which is a blend of major (98% of total oil weight) and minor compounds including antioxidants. The main antioxidants are phenolic compounds, which can be divided into lipophilic and hydrophilic phenols. While lipophilic phenols such as tocopherols can be found in other vegetable oils, most hydrophilic phenols in olive oil are exclusive of the Olea europaea species endowing it with a chemotaxonomic interest. This review is focused on VOO antioxidant profile and, particularly, on hydrophilic phenols that are divided into different sub‐families such as phenolic acids and alcohols, hydroxy‐isochromans, flavonoids, secoiridoids, lignans and pigments. Analytical methods for qualitative and/or quantitative determination of these compounds are assessed. The implementation of efficient sample preparation protocols, separation techniques such as liquid chromatography, GC and capillary electrophoresis, as well as detection techniques such as ultraviolet absorption, fluorescence or MS are critical to succeed in the quality of the results. The effects of hydrophilic phenols on increasing VOO stability, its nutraceutical interest and organoleptic properties are also considered.