Lipoxygenase activity in olive (Olea europaea) fruit

The present work was designed to characterize lipoxygenase activity in olive fruit pulp, in order to determine its significance in the biosynthesis of virgin olive oil aroma. Lipoxygenase activity has been detected in particulate fractions of enzyme extracts from olive pulp subjected to differential centrifugation. The activity in different membrane fractions showed similar properties, with optimal pH in the range of 5.0–5.5 and a clear specificity for linolenic acid, which was oxidized at a rate double that of linoleic acid under the same reaction conditions. The enzyme preparations displayed very low activity with dilinoleoyl phosphatidylcholine, suggesting that olive lipoxygenase acts on nonesterified fatty acids. The enzyme showed regiospecificity for the Δ-13-position of both linoleic and linolenic acid, yielding 75–90% of Δ-13-fatty acid hydroperoxides. Olives showed the highest lipoxygenase activity about 15 wk after anthesis, with a steady decrease during the developmental and ripening periods. Olive lipoxygenase displayed properties that support its involvement in the biogenesis of six-carbon volatile aldehydes, which are major constituents of the aroma of virgin olive oil, during the process of oil extraction.     

Oxidative responses in soybean foliage to herbivory by bean leaf beetle and three-cornered alfalfa hopper

Variation in induced responses in soybean is shown to be dependent, in part, upon herbivore species. Herbivory by the phloem-feeding three-cornered alfalfa hopper caused increases in the activities of several oxidative enzymes including lipoxygenases, peroxidases, ascorbate oxidase, and polyphenol oxidase. Bean leaf beetle defoliation caused increased lipoxygenase activity, but had little effect upon peroxidase, polyphenol oxidase, ascorbate oxidase, or trypsin inhibitor levels in either field or greenhouse studies. In one field experiment, prior herbivory by the bean leaf beetle subsequently reduced the suitability of foliage to the corn earwormHelicoverpa zea. The contribution of these findings to emerging theories of insect-plant interactions is discussed.     

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.     

Differential induction of tomato foliar proteins by arthropod herbivores

The effects of mechanical and chemical damage and three types of biotic damage on the activities of four foliar proteins of the tomato plant (Lycopersicon esculentum Mill var. Castlemart) were assayed. Proteinase inhibitor, polyphenol oxidase, peroxidase, and lipoxygenase activities were assayed in damaged leaflets and compared with activities in undamaged leaflets. These proteins are putative plant defenses in tomato. Differential induction of these proteins by the various damage-treatments was demonstrated, such that different subsets of the four proteins were induced by different types of damage. This study clearly demonstrates the ability of plants to respond differentially to different types of damage. Possible mechanisms for this differential induction and the implications of differential induction for plant defense are discussed.     

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.     

Endogenous microflora in turbid virgin olive oils and the physicochemical characteristics of these oils

Cloudy olive oil, the fresh olive juice, is an intermediate form before full precipitation of freshly produced olive oil. Some consumers prefer it because they consider it as more natural and less processed. The cloudy form can persist for several months. The oil is a sort of dispersion/suspension system which can be also described as a micro‐emulsion/suspension. Water micro‐droplets were found to have a size ranging from 1 to 5 µm. Cloudiness is due to the low water content and the presence of natural emulsifiers in the oil. The suspension is formed by solid particles (5–60 µm) deriving from the olive fruit. They are present in small amounts (12–460 mg/kg oil). In the newly produced olive oil, containing 0.17–0.49% water, a number of microorganisms of different types (bacteria, yeasts, moulds) were found to survive, but at very low concentrations (<3 log cfu/mL oil). They originate from the exterior of the fruit (epiphytic microflora) and their presence is considered natural. Their enzyme activities do not seem to affect the quality of the final product.     

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.     

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.     

Rapid evaluation of olive oil quality by NIR reflectance spectroscopy

NIR spectroscopy calibrations have been developed for a range of quality parameters in olive oil, including FFA, PV, polyphenol content, induction time, chlorophyll, and the major FA. A set of 216 olive oil samples from throughout the Australian olive-growing areas were used to provide a representative range of quality. The variation in the oils tested virtually covered the range of the chemical standard limits described by the International Olive Oil Council. A FOSS NIRSystems^® 6500 spectrophotometer with a liquid cell holder was used. Multiple correlation coefficients squared (R ²) for minor components stearic acid (0.86), and linolenic acid (0.85) were relatively low because the concentration range is very narrow compared with the reproducibility of the reference method. However, the major FA, oleic (0.99) and linoleic (1.00), FFA (0.97), and chlorophyll (0.98) provided high levels of accuracy. All of the parameters measured were sufficiently accurate for routine screening of olive oil.     

Chemical Composition of Turkish Olive Oil––Ayvalik

Although large amounts of olive oil are produced in Turkey, not much information on its chemical composition is available in the literature to date. The aim of this study was to evaluate the chemical composition of commercial olive oils produced from the Ayvalik olive cultivar in Canakkale, Turkey. Five different samples corresponding to the olive oil categories of extra virgin (conventional, extra virgin olive oil (EVOO), and organic extra virgin olive oil (OGOO) production), virgin olive oil (OO-1), ordinary virgin olive oil (OO-2) and refined olive oil (RFOO) were evaluated. Olive oils were collected from two consecutive production years. According to the free fatty acids, the absorbance values (K₂₃₂ and K₂₇₀), and peroxide values of all the samples conformed to the European standards for olive oil. The level of oleic acid was in the range of 68–73%; while the linoleic acid content was significantly lower in the refined olive oils. The tocopherol and polyphenol content was in the lower range of some European olive oils. However, pinoresinol was a major phenolic compound (5–77 mg/kg depending on the oil category). Its content was markedly higher than in many other oils, which would be a useful finding for olive oil authentication purposes.     

Chemical composition of commercial cornicabra virgin olive oil from 1995/96 and 1996/97 crops

The chemical composition of commercial Cornicabra virgin olive oils (n=65) was studied, as was its relationship with oil quality and the influence of the extraction method and production year. The main characteristics of these olive oils were: oxidative stability 53 ± 24 h, mean polyphenol content 162 ± 57 mg/kg (as gallic acid), oleic acid 80.8 ± 0.9%, linoleic acid 4.6 ± 0.6%, and campesterol 4.3 ± 0.1%, which is peculiar to this variety. No clear differences in composition were observed with respect to the different extraction systems (dual-phase/triple-phase decanters and pressure), although oils produced by the dual-phase decanter showed higher oxidative stability and polyphenol content. There were significant differences in major fatty acids and sterols according to the production year.     

Malaxing temperature affects volatile and phenol composition as well as other analytical features of virgin olive oil

Three Italian olive varieties (Caroleo, Leccino and Dritta) were processed by centrifugation in the oil mill. The olive paste was kneaded at 20, 25, 30 and 35 °C. The results achieved revealed that the oil content in green volatiles from lipoxygenase pathway (including C₅ and C₆ compounds and especially unsaturated C₆ aldehydes) decreased progressively as the kneading temperature increased, dropping markedly at 35 °C. The content of phenols, o‐diphenols and secoiridoids showed an opposite trend, but the temperature of 35 °C was critical also for them, as it was for the majority of the other components, analytical parameters and indices related to quality, typicality and genuineness. In general, an increasing kneading temperatures increased the release of oil constituents from the vegetable tissue. This factor also affected the oil extraction yields. The best overall results were achieved by malaxing the olive paste at 30 °C. In fact, this temperature level led to achieving both pleasant green virgin olive oils and satisfactory oil extraction outputs.     

Effect of Nitrogen Availability on Expression of Constitutive and Inducible Chemical Defenses in Tomato, Lycopersicon esculentum

Young tomato plants (Lycopersicon esculentum) grown in sand in a greenhouse and subjected to different fertilization regimes were used to test the effects of nitrogen availability on constitutive levels of phenolics and on constitutive and inducible activities of polyphenol oxidase and proteinase inhibitors. Theories that emphasize physiological constraints on the expression of phytochemicals predict an increase in levels of carbon-based allelochemicals under moderate nitrogen stress but predict, under the same conditions, an attenuation of chemical responses involving nitrogen-containing compounds such as proteinase inhibitors and polyphenol oxidase. We found that nitrogen availability had a strong effect on constitutive levels of phenolics; weaker effects on constitutive polyphenol oxidase activity, constitutive proteinase inhibitor activity, and inducible polyphenol oxidase activity; and no effect on inducible proteinase inhibitor activity. These results point to a need for the integration of theories that emphasize physiological influences on secondary metabolism with those that emphasize ecological influences on secondary metabolism and suggest that current theories of plant defense do not adequately account for enzymatic and proteinaceous defenses against arthropods.     

Stability of refined olive oil and olive‐pomace oil added by phenolic compounds from olive leaves

Refined olive oil and olive‐pomace oil were enriched with olive leaf phenolic compounds in order to enhance its quality and bring it closer to virgin olive oil. The changes that occurred in the concentrations of pure oleuropein, oleuropein aglycone, hydroxytyrosol acetyl and α‐tocopherol at 400 µg/kg of oil during the storage of refined olive oil and olive‐pomace oil under accelerated conditions (50 °C) were investigated. In a period of 4 months, α‐tocopherol decomposed by 75% whereas less than 40% of the phenols were lost. During storage, enzymatic olive leaf extract hydrolysate that contains two major compounds, hydroxytyrosol and oleuropein aglycone showed the highest antioxidant activity and the lowest detected stability, followed by oleuropein. The oleuropein in olive leaf extracts exhibited similar degradation profiles, reducing by 60–50% and 80% for the olive oil and olive‐pomace oil in 6 months, respectively. The acetylated extract, however, displayed a loss of 10 and 5% in olive oil and olive‐pomace oil, respectively. In the fatty acid composition, an increase in oleic acid and a decrease in linoleic acid were observed. The antiradical activities of the olive oil and olive‐pomace oil enriched with olive leaf phenolic compounds at 400 ppm showed that enzymatic hydrolysate extract had the highest protective effect against oil oxidation. Based on the Rancimat method, the oils with added leaf enzymatic hydrolysate extract had the lowest peroxide value and the highest stability. After 6 months of storage and at 120 °C, the oxidative resistance of refined olive oil and olive‐pomace oil reached 0.71 and 0.89 h, respectively, whereas that of the non‐enriched samples fell to zero.     

The role of endogenous amphiphiles on the stability of virgin olive oil-in-water emulsions

Stable virgin olive oil-in-water emulsions were prepared using total endogenous surface-active components derived from oil as emulsifying agents, and the interfacial properties of the emulsion droplets were examined. The amount of oil extracted into the aqueous buffer increased with buffer pH, with the most stable emulsions being formed at pH 7.5. Light microscopy of the emulsions revealed the presence of spherical droplets with diameters ranging from 1.5 to 3 μm. Their surface was negatively charged at pH 7.5, as confirmed by the effect of ions and polycations. Potassium chloride, Ca²⁺, and spermine induced rapid aggregation (as monitored by the turbidity change and by light microscopy), showing their maximal effect at 1 M, 4 mM, and 60 μM, respectively. Papain treatment of the emulsion particles rapidly induced particle aggregation, suggesting the destruction of stabilizing structural olive oil proteins. Unlike papain, treatment with phospholipase C did not result in an appreciable turbidity change. Treatment with soybean lipoxygenase slightly increased the turbidity of the emulsion. The interaction of linoleate-Tween 20 mixed micelles with emulsion droplets produced turbidity, which was maximal at a neutral pH, whereas interaction with proteolyzed and lipoxygenase-treated droplets induced both a significant increase in turbidity and a red shift to a different absorption maximum of the system as compared with those of the untreated emulsion.     

Low molecular weight polypeptides in virgin and refined olive oils

Twenty-eight virgin olive oils—from different regions of Spain and prepared from olive drupes of different varieties—and six refined olive oils were analyzed to determine the presence of proteins in these oils. All oils studied showed the presence of proteins in the range of 7–51 μ/100 g of oil. There were no significant differences in protein content in oils from different varieties or between virgin or refined oils. In addition, all oils exhibited analogous amino acid patterns, suggesting a similarity among protein fractions obtained from different oils. A polypeptide with an apparent M.W. of 4600 Da was common to the isolated protein fractions. These results suggest that this polypeptide is a previously unknown minor component in olive oils. No clear influence of this component on oil stability was observed when oil stabilities were estimated as a function of phenol, tocopherol, phosphorus, and protein contents of the oils.     

Exogenous jasmonates simulate insect wounding in tomato plants (Lycopersicon esculentum) in the laboratory and field

Wounding increases the levels and activities of several defense-related proteins in the foliage of the tomato plant,Lycopersicon esculentum Mill. Evidence indicates that two of these responses, the systemic increases in polyphenol oxidase and proteinase inhibitors, are regulated by an octadecanoid-based signalling pathway which includes the wound hormone, jasmonic acid. It is not known whether other responses to wounding are also regulated by this same signalling pathway. In this paper, we show that application of jasmonates (jasmonic acid or its volatile derivative, methyl jasmonate) in low concentrations to foliage of young tomato plants induced, in a dose-dependent manner, the same protein responses-polyphenol oxidase, proteinase inhibitors, lipoxygenase, and peroxidase-as doesHelicoverpa zea Boddie feeding. Application of jasmonic acid to a single leaflet of four-leaf tomato plants induced these four proteins in a spatial pattern nearly identical to that produced by localized feeding ofH. zea. Exogenous jasmonic acid also decreased suitability of foliage for the beet armyworm,Spodoptera exigua Hubner in the laboratory. Based on these results, we conducted an experiment to measure the effects of jasmonic acid spray under field conditions. We provide the first evidence that jasmonic acid spray on field plants induces production of chemical defenses above the levels found in unsprayed controls. Exogenous jasmonic acid sprayed on plants in agricultural plots increased levels of polyphenol oxidase and proteinase inhibitors. Because application of jasmonic acid induces these defensive compounds at low concentrations in a manner similar to natural wounding, it may prove to be a useful tool for stimulating plant resistance to insects in the field.     

Impact of Olive Harvesting Date on Virgin Olive Oil Volatile Composition in Four Spanish Varieties

The unique sensory characteristics of extra virgin olive oil (EVOO) depend upon its volatile composition. This work investigates the impact of olive fruit harvesting time and growing location on the volatile composition of the obtained EVOO, on four typical Spanish olive varieties (Cornicabra, Picual, Castellana, Manzanilla Cacereña). Several growing locations within the Madrid region (Spain) are studied to assess the natural variability attributed to the environmental factors. Aroma compounds are analyzed by solid-phase microextraction coupled with gas-chromatography and mass spectrometry, and sensory analysis. A considerable different behavior is observed depending on the olive variety and ripening stage. Statistically significant differences are obtained for volatile compounds biosynthesized from the lipoxygenase pathway and other fatty acid metabolism routes, which results in significant differences in their aroma profiles. Practical applications: These results have practical applicability for the olive oil industry and regulatory bodies. For example, for protected designation of origin EVOOs the aroma profile needs to be consistent over different production lots. The outcome of this research is of interest to the olive oil industry to get a better insight into the expected variability and interactions among cultivars, small pedoclimatic differences within the same broader area, and the harvesting date on the sensory and volatile profile of the resulting EVOO.     

A laboratory study of the bleaching process in stigmasta-3,5-diene concentration in olive oils

The bleaching effect was simulated in pilot plant by measuring the influence of temperature (40, 50, 60, 70, 80, and 90°C), time (5, 10, 15, 20, 25, and 30 min), and concentration of solid adsorbent [1.5 and 8% (w/w) of Tonsil supreme NFF] on stigmasta-3,5-diene (STIG) obtained by dehydration of steroidal compounds. Conditions were chosen to simulate those used in industrial operations. The presence of refined oils in extra virgin olive oil can be detected by these newly formed steroid hydrocarbons. Experimental results indicated that STIG did not exceed an imposed limit of 0.15 mg/kg in extra virgin olive oil, when oils were bleached with 1.5% earth at temperatures ≤80°C for 30 min in admixed to oils sold as virgin. A large proportion of the adulterations were not detectable by the official methods. Color determinations (CIE-1931) chromatic coordinates) were replicated on a refined oil and in admixed extra virgin olive oil. Color of olive oil was not significantly affected by mixing with refined oil (≤20%).     

Intensification of Mass Transfer in the Extraction Process with a Nanofluid Prepared in a Natural Deep Eutectic Solvent

The phytotoxic residues after olive oil production and residue leaves after pruning contain valuable bioactive compounds. A natural deep eutectic solvent (NADES) and an ethanol-water mixture were used for polyphenol extraction from olive pomace and leaves. Type of solvent, extraction temperature, and particle size of the leaves were optimized. NADES demonstrated better efficiency in the extraction process than conventional solvents; with higher temperature and smaller fraction of olive leaves, a higher yield of polyphenols was obtained from leaves. Extraction with a nanofluid as solvent was carried out. A stable nanofluid was prepared from NADES by adding Al₂O₃ nanoparticles. A higher yield was obtained with nanofluid from leaves, while an improvement of polyphenol extraction was achieved after the removal of oil from olive pomace.