Thermal stability of lipoxygenase and hydroperoxide lyase from olive fruit and repercussion on olive oil aroma biosynthesis

Thermal stabilities of main enzymes involved in the biosynthesis of virgin olive oil (VOO) aroma through the lipoxygenase (LOX) pathway were studied in crude enzymatic preparations. Kinetic parameters of thermal inactivation for LOX were determined graphically and were shown to be compatible with the presence of two LOX isoenzymes (LOXlab and LOXres) having different thermal stabilities and displaying relative activities of 88 and 12% each. Data on hydroperoxide lyase (HPL) suggest the existence of just one HPL isoform. Thermal stabilities of LOX and HPL enzymatic activities in crude preparations seem to explain the observed decrease of volatile contents in VOO aroma as a consequence of heat treatments of olive fruit. Moreover, differences in thermal stability of LOXlab and LOXres would justify the distinct pattern of reduction of C6 and C5 compound contents observed in the aroma of these oils.     

Role of olive seed in the biogenesis of virgin olive oil aroma

Results obtained in a set of experiments point to an effective participation of olive seeds in the biosynthesis of olive oil aroma through the lipoxygenase pathway during the extraction process to produce virgin olive oil. Data showed that olive seeds should contain enzymatic activities metabolizing 13-hydroperoxides other than hydroperoxide lyase, giving rise to a net decrease in the content of C6 unsaturated aldehydes during the olive oil extraction process. Olive seeds seem also to supply this process with alcohol dehydrogenase activity, being more specific for saturated C6 aldehydes and not acting on C5 alcohols. Moreover, olive seeds would be responsible for the biosynthesis of 30-50% esters during the olive oil extraction process of intact fruits. Thus, olive seeds would afford a load of alcohol acyltransferase activity that might be quite unspecific in terms of substrate, producing any kind of esters.     

Characterization of the lipoxygenases in some olive cultivars and determination of their role in volatile compounds formation

Enzymatic extracts from olive pulp (Olea europea L.) were used to characterize lipoxygenase (LOX) activity in order to determine its role in the biogenesis of the volatile compounds that influence the aroma of extra virgin olive oil. The LOX activity was tested spectrophotometrically at an optimal pH of 6.0 in three olive cultivars, Ascolana Tenera, Kalamata, and FS17. The trend of the LOX activity was determined as a function of pH and temperature; the kinetic constants of the enzyme were also determined. The highest LOX activity was observed in the FS17 fruit, which had the highest concentrations of C(5) and C(6) compounds (aldehydes, alcohols, and ketones), followed by Kalamata and Ascolana T., respectively. Given the direct relationship between enzymatic activity and the quantity of aromas measured in the fruit, it is hypothesized that olive LOX is involved in the formation of C(5) and C(6) volatile compounds. To study the mechanism of the movement of the aromas from the fruit to the oil, which was obtained by simple mechanical extraction, the headspace of the oil for each cultivar was analyzed as well as the aromatic composition in order to compare it with the aromas of the fruit.     

Effect of olive stoning on the volatile and phenolic composition of virgin olive oil

Olive stoning during the virgin olive oil (VOO) mechanical extraction process was studied to show the effect on the phenolic and volatile composition of the oil. To study the impact of the constitutive parts of the fruit in the composition of olive pastes during processing, the phenolic compounds and several enzymatic activities such as polyphenoloxidase (PPO), peroxidase (POD), and lipoxygenase (LPO) of the olive pulp, stone, and seed were also studied. The olive pulp showed large amounts of oleuropein, demethyloleuropein, and lignans, while the contribution of the stone and the seed in the overall phenolic composition of the fruit was very low. The occurrence of crushed stone in the pastes, during malaxation, increased the peroxidase activity in the pastes, reducing the phenolic concentration in VOO and, at the same time, modifying the composition of volatile compounds produced by the lipoxygenase pathway. The oil obtained from stoned olive pastes contained higher amounts of secoiridoid derivatives such as the dialdehydic forms of elenolic acid linked to (3,4-dihydroxyphenyl)ethanol and (p-hydroxyphenyl)ethanol (3,4-DHPEA-EDA and p-HPEA-EDA, respectively) and the isomer of the oleuropein aglycon (3,4-DHPEA-EA) and, at the same time, did not show significant variations of lignans. The stoning process modified the volatile profile of VOO by increasing the C6 unsaturated aldehydes that are strictly related to the cut-grass sensory notes of the oil.     

Influence of the decrease in oxygen during malaxation of olive paste on the composition of volatiles and phenolic compounds in virgin olive oil

The sensory and health properties of virgin olive oil (VOO) are highly related to its volatile and phenolic composition. Oxygen control in the pastes during malaxation may be a new technological parameter to regulate enzymatic activities, such as polyphenoloxidase, peroxidase, and lipoxygenase, which affect the phenolic and volatile composition of VOO. In this work, we monitored CO2 and O2 concentrations during industrial-scale olive paste malaxation with various initial O2 concentrations within the malaxer headspace. Results show that the O2 concentration in the malaxer headspace did not affect CO2 production during processing, whereas a strong influence was observed on the changes of the phenolic composition of olive pastes and VOOs, with high correlation coefficient for the total phenols (R = 0.94), especially for oleuropein and demethyloleuropein derivatives (R = 0.81). In contrast, aroma production during malaxation was minimally affected by the O2 concentration in the malaxer headspace.     

Changes in virgin olive oil quality during low-temperature fruit storage

'Frantoio' olive fruits were stored at low temperature (4 +/- 2 degrees C) for 3 weeks to investigate the effect of postharvest fruit storage on virgin olive oil quality. Volatile compounds and phenolic compounds explained the changes in sensory quality that could not be explained with quality indices (FFA, PV, K232, and K270). Increases in concentrations of ( E)-2-hexenal and hexanal corresponded to positive sensory quality, whereas increases in ( E)-2-hexenol and (+)-acetoxypinoresinol were associated with negative sensory quality. Volatile and phenolic compounds were also indicative of the period of low-temperature fruit storage. Oleuropein and ligstroside derivatives in olive oil decreased with respect to storage time, and their significant ( p < 0.05) change corresponded to changes in bitterness and pungency. ( Z)-2-Penten-1-ol increased during low-temperature fruit storage, whereas 2-pentylfuran decreased. Changes in volatile compounds, phenolic compounds, quality indices, and sensory notes indicated that virgin olive oil quality was lost within the first week of low-temperature fruit storage and regained at 2 weeks. This research suggests that low-temperature olive fruit storage may be beneficial, with a possibility of increasing oil yield and moderating the sensory quality of virgin olive oils. This study demonstrates that deeper insights into virgin olive oil quality changes during low-temperature fruit storage may be gained by studying volatile and phenolic compounds in addition to quality indices and physical appearance of the fruit.     

Factors limiting the synthesis of virgin olive oil volatile esters

The aim of the present work was to establish the limiting factors affecting the biosynthesis of volatile esters present in virgin olive oil (VOO). Oil volatile fractions of the main Spanish olive cultivars, Arbequina and Picual, were analyzed. It was observed that acetate esters were the most abundant class of volatile esters in the oils, in concordance with the high content of acetyl-CoA found in olive fruit, and that the content of C6 alcohols is limited for the synthesis of volatile esters during the production of VOO. Thus, the increase of C6 alcohol availability during VOO production produced a significant increase of the corresponding ester in the oils in both cultivars at two different maturity stages. However, the increase of acetyl-CoA availability had no effect on the VOO volatile fraction. The low synthesis of these C6 alcohols seems not to be due to a shortage of precursors or cofactors for alcohol dehydrogenase (ADH) activity because their increase during VOO production had no effect on the C6 alcohol levels. The experimental findings are compatible with a deactivation of ADH activity during olive oil production in the cultivars under study. In this sense, a strong inhibition of olive ADH activity by compounds present in the different tissues of olive fruit has been observed.     

Cultivar differences on nonesterified polyunsaturated fatty acid as a limiting factor for the biogenesis of virgin olive oil aroma

The relationship between the content of nonesterified polyunsaturated fatty acids and the contents of oil aroma compounds that arise during the process to obtain virgin olive oil (VOO) was studied in two olive cultivars, Picual and Arbequina, producing oils with distinct aroma profiles and fatty acid compositions. Results suggest that the biosynthesis of VOO aroma compounds depends mainly on the availability of nonesterified polyunsaturated fatty acids, especially linolenic acid, during the process and then on the enzymatic activity load of the lipoxygenase/hydroperoxide lyase system. Both availability of substrates and enzymatic activity load seem to be cultivar-dependent.     

The activity of healthy olive microbiota during virgin olive oil extraction influences oil chemical composition

The activity of olive microbiota during the oil extraction process could be a critical point for virgin olive oil quality. With the aim to evaluate the role of microbiological activity during the virgin olive oil extraction process, just before oil extraction freshly collected healthy olive fruits were immersed in contaminated water from an olive mill washing tank. The oils extracted were then compared with control samples from the same batch of hand-picked olives. The presence of lactic and enteric bacteria, fungi and Pseudomonas on the surface of olives was proved to be much higher in washed than in control olives, with increments in cfu/g between 2 and 3 orders of magnitude. The biogenesis of volatile compounds and the extraction of olive polyphenols and pigments were significantly influenced by the microbiological profile of olives even without any previous storage. In most cases the effect of olive microbiota on oil characteristics was greater than the effect exerted by malaxation time and temperature. Oils from microbiologically contaminated olives showed lower amounts of C5 volatiles and higher levels of C6 volatiles from the lipoxygenase pathway and some fermentation products. On the other hand, a decrease of chlorophylls, pheophytins, xanthophylls and the ratio chlorophyll/pheophytin was observed in these oils. Likewise, the microbiological activity during oil extraction led to significantly lower amounts of polyphenols, in particular of oleuropein derivatives. These differences in olive oil chemical composition were reflected in oil sensory characteristics by the decrease of the green and bitter attributes and by the modification of the oil color chromatic ordinates.     

Synthesis of volatile compounds of virgin olive oil is limited by the lipoxygenase activity load during the oil extraction process

The aim of this work was to determine whether the lipoxygenase (LOX) activity is a limiting factor for the biosynthesis of virgin olive oil (VOO) volatile compounds during the oil extraction process. For this purpose, LOX activity load was modified during this process using exogenous LOX activity and specific LOX inhibitors on olive cultivars producing oils with different volatile profiles (Arbequina and Picual). Experimental data suggest that LOX activity is a limiting factor for the synthesis of the oil volatile fraction, this limitation being significantly higher in Picual cultivar than in Arbequina, in line with the lowest content of volatile compounds in the oils obtained from the former. Moreover, there is evidence that this limitation of LOX activity takes place mostly during the milling step in the process of olive oil extraction.     

Volatile compounds characterizing Tunisian Chemlali and Chétoui virgin olive oils

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

Characterization of volatiles in virgin olive oil produced in the Tunisian area of Tataouine

The Tataouine province in southern Tunisia is well known for its severe pedoclimatic conditions. Using solid phase microextraction (SPME) and gas chromatography (GC), coupled to flame ionization and mass spectrometer detectors, we characterized virgin olive oils from Chemlali Tataouine, Fakhari Douirat, Zarrazi Douirat, and Dhokar Douirat varieties, which grow in the harsh arid region of Tataouine. Significant differences in the proportions of volatile constituents from oils of different varieties were detected. The results showed that lipoxygenase products were generally the major metabolites of the volatile fraction, and (E)-Hex-2-enal was the principal compound characterizing the olive oil headspace for most samples, though the absolute levels varied greatly, never exceeding 76.45 and 32.16%, respectively. The C5 compounds were unusually abundant, comprising 42.97% of the total lipoxygenase products and a remarkably high level of penten dimers. Each autochthonous variety could thus be differentiated according to the percentage of each metabolite.     

Characterization of alcohol acyltransferase from olive fruit

Alcohol acyltransferase catalyzes the esterification of volatile alcohols with acyl-CoA derivatives to produce volatile esters typically present in the aroma of some fruits. This enzyme was detected in extracts from the pericarp tissues of ripe olive fruits using hexanol and acetyl-CoA as the substrates. Alcohol acyltransferase showed a very low activity level in these fruits, with an optimum pH value at 7.5 and high K(m) values for hexanol and acetyl-CoA. The substrate specificity of this enzyme for various alcohols was also studied. The involvement of the studied enzyme in the biogenesis of the volatile esters present in the aroma of virgin olive oil was discussed.     

Comparison of static headspace, headspace solid phase microextraction, headspace sorptive extraction, and direct thermal desorption techniques on chemical composition of French olive oils

Static headspace (SHS), headspace solid phase microextraction (HS-SPME), headspace sorptive extraction (HSSE), and direct thermal desorption (DTD) were applied to the analysis of four French virgin olive oils from Corsica. More than 60 compounds were isolated and characterized by GC-RI and GC-MS. SHS was not suited to the characterization of olive oil volatile compounds because of low sensitivity. The SPME and HSSE techniques were successfully applied to olive oil headspace analysis. Both methods allow the characterization of volatile compounds (mainly C(6) aldehydes and alcohols), which contribute significantly to the "green" flavor note of virgin olive oils. The PDMS stir bar showed a higher concentration capacity than a DVB/CAR/PDMS SPME fiber due to the higher volume of polymeric coating. DTD was a very good tool for extracting volatile and especially semivolatile compounds, such as sesquiterpenes, but requires a significant investment like that for HSSE. Finally, SPME may be a more appropriate technique for routine quality control due to its operational simplicity, repeatability, and low cost.     

Detection of rancid defect in virgin olive oil by the electronic nose

A sensor array of 32 conducting polymer sensors has been used to detect the rancid defect in virgin olive oils. A training set, composed of admixtures of a Portuguese virgin olive oil with different percentages (0-100%) of a rancid standard oil, was used for the selection of the best sensors classifying correctly the samples. Information on volatile compounds responsible for rancidity and the sensory evaluation of samples by assessors were used for explaining the mathematical selection of sensors. A tentative calibration, using unsupervised procedures (PCA and MDS) and a nonlinear regression, was carried out, with the training set, and later confirmed with a test set with which rancid commercial samples of different varieties were used to spike a Greek extra virgin olive oil at low levels of rancidity (0.5-6%).     

Thermal inactivation kinetics of recombinant proteins of the lipoxygenase pathway related to the synthesis of virgin olive oil volatile compounds

The aim of this work was to characterize the thermal inactivation parameters of recombinant proteins related to the biosynthesis of virgin olive oil (VOO) volatile compounds through the lipoxygenase (LOX) pathway. Three purified LOX isoforms (Oep2LOX1, Oep1LOX2, and Oep2LOX2) and a hydroperoxide lyase (HPL) protein (OepHPL) were studied. According to their thermal inactivation parameters, recombinant Oep1LOX2 and Oep2LOX2 could be identified as the two LOX isoforms active in olive fruit crude preparations responsible for the synthesis of 13-hydroperoxides, the main substrates for the synthesis of VOO volatile compounds. Recombinant Oep2LOX1 displayed a low thermal stability, which suggests a weak actuation during the oil extraction process considering the current thermal conditions of this industrial process. In addition, recombinant OepHPL could be identified as the HPL activity in crude preparations. The thermal stability was the highest among the recombinant proteins studied, which suggests that HPL activity is not a limiting factor for the synthesis of VOO volatile compounds.     

Oxygen concentration affects volatile compound biosynthesis during virgin olive oil production

The effect of O 2 concentration on oil volatile compounds synthesized during the process to obtain virgin olive oil (VOO) was established. The study was carried out either on the whole process or within the main steps (milling and malaxation) of this process with two olive cultivars, Picual and Arbequina, at two ripening stages. Data show that O 2 control during milling has a negative impact on VOO volatile synthesis. This effect seems to depend on cultivar and on the ripening stage in cultivar Picual. Because most VOO volatiles are synthesized during olive fruit crushing at the milling step, O 2 control during malaxation seems to affect just slightly the volatile synthesis. The highest effect was observed when control of O 2 concentration was performed over the whole process. In this case, the content of volatile compounds of oils obtained from both cultivars and ripening stages showed quite similar trends.     

Bovine serum albumin produces a synergistic increase in the antioxidant activity of virgin olive oil phenolic compounds in oil-in-water emulsions

Virgin olive oil is valued for its flavor, but unacceptable off-flavors may develop on storage in food products containing this oil due to oxidation. The oxidative stability of oil-in-water emulsions containing bovine serum albumin (BSA) and virgin olive oil phenolic compounds was studied. Four oil-in-water emulsions with and without BSA and phenols isolated from virgin olive oil were prepared. These model systems were stored at 60 degrees C to speed up lipid oxidation. Primary and secondary oxidation products were monitored every three days. Peroxide values and conjugated diene contents were determined as measures of the primary oxidation products. p-Anisidine values and volatile compounds were determined as measures of the secondary oxidation products. This latter determination was carried out by headspace solid-phase microextraction coupled with gas chromatography. Although olive oil phenolic compounds and BSA contributed some antioxidant activity when present as individual additives, the combination of BSA with phenols in an emulsion showed 58-127% synergy, depending on which analytical method was used in the calculation. The emulsion containing phenolic compounds and BSA showed a low level of deterioration after 45 days of storage at 60 degrees C.     

Solid-phase microextraction in the analysis of virgin olive oil volatile fraction: characterization of virgin olive oils from two distinct geographical areas of northern Italy

SPME was employed to characterize the volatile profile of virgin olive oils produced in two geographical areas of northern Italy: the region of the Gulf of Trieste and the area near Lake Garda. There are as yet no data on the headspace composition of virgin olive oils from these regions, characterized by particular conditions of growth for Olea europaea. Using the SPME technique coupled to GC-MS and GC-FID, the volatile components of 42 industrially produced virgin olive oil samples were identified and the principal compounds quantitatively analyzed. Significant differences in the proportion of volatile constituents from oils of different varieties and geographical origins were detected. The results suggest that besides the genetic factor, environmental conditions influence the volatile formation.