Changes in the HPLC phenolic profile of virgin olive oil from young trees (Olea europaea L. Cv. Arbequina) grown under different deficit irrigation strategies

The HPLC phenolic profile of virgin olive oils obtained from young olive trees (Arbequina cv.) grown under different deficit irrigation strategies was studied. Deficit irrigation (RDI) did not affect all the phenolic compounds in the same way. Lignans, vanillic acid, vanillin, and the unknown phenolic compound named P24 increased in the oils from the most irrigated treatments. The secoiridoid derivatives and the unknown phenolic compound named P19 increased in the oils from the most stressed irrigation treatments. The period of growth where a water stress significantly affects the phenolic profile of oils was between pit hardening and the first stages of fruit growth and oil accumulation, independently of the water applied during the previous period to harvest. The phenolic profile and those parameters related to phenol content, oxidative stability, and the bitter index were significantly affected only in the most severe RDI strategies. Other strategies produced important savings in irrigation requirements and an increase in the water use efficiency without noticeably affecting the phenolic profile.     

Phenolic and volatile compounds of extra virgin olive oil (Olea europaea L. Cv. Cornicabra) with regard to fruit ripening and irrigation management

This study investigated the effect of both the degree of ripening of the olive fruit and irrigation management-rain-fed, two different regulated deficit irrigations (RDI), the method proposed by the Food and Agriculture Organization of the United Nations (known as FAO), and 125 FAO (125% FAO)-on the phenolic and volatile composition of Cornicabra virgin olive oils obtained during two crop seasons. Secoiridoid phenolic derivatives greatly decreased upon increase of both irrigation and ripening, for example, the 3,4-DHPEA-EDA content decreased from 770 to 450 mg/kg through fruit ripening under rain-fed conditions and from 676 to 388 mg/kg from rain-fed conditions to FAO irrigation treatment (at a ripeness index of approximately 4). Moreover, secoiridoid derivatives of hydroxytyrosol decreased more than those of tyrosol. The levels of major volatile components decreased in the course of ripening but were higher in irrigated olive oils: for example, the E-2-hexenal content ranged between 4.2 and 2.6 mg/kg (expressed as 4-methyl-2-pentanol) over fruit maturation under rain-fed conditions and between 8.0 and 3.5 mg/kg under FAO scheduling. It is important to note that where water was applied only from the beginning of August (RDI-2), when oil begins to accumulate in the fruit, the resulting virgin olive oil presented a phenol and volatile profile similar to those of the FAO and 125 FAO methods, but with a considerable reduction in the amount of water supplied to the olive orchard.     

Changes in phenolic composition and antioxidant activity of virgin olive oil during frying

The concentration of hydroxytyrosol (3,4-DHPEA) and its secoiridoid derivatives (3,4-DHPEA-EDA and 3,4-DHPEA-EA) in virgin olive oil decreased rapidly when the oil was repeatedly used for preparing french fries in deep-fat frying operations. At the end of the first frying process (10 min at 180 degrees C), the concentration of the dihydroxyphenol components was reduced to 50-60% of the original value, and after six frying operations only about 10% of the initial components remained. However, tyrosol (p-HPEA) and its derivatives (p-HPEA-EDA and p-HPEA-EA) in the oil were much more stable during 12 frying operations. The reduction in their original concentration was much smaller than that for hydroxytyrosol and its derivatives and showed a roughly linear relationship with the number of frying operations. The antioxidant activity of the phenolic extract measured using the DPPH test rapidly diminished during the first six frying processes, from a total antioxidant activity higher than 740 micromol of Trolox/kg down to less than 250 micromol/kg. On the other hand, the concentration of polar compounds, oxidized triacylglycerol monomers (oxTGs), dimeric TGs, and polymerized TGs rapidly increased from the sixth frying operation onward, when the antioxidant activity of the phenolic extract was very low, and as a consequence the oil was much more susceptible to oxidation. The loss of antioxidant activity in the phenolic fraction due to deep-fat frying was confirmed by the storage oil and oil-in-water emulsions containing added extracts from olive oil used for 12 frying operations.     

Modulation of the biosynthesis of some phenolic compounds in Olea europaea L. fruits: their influence on olive oil quality

The phenolic composition of olive fruits (Olea europaea L.) (cv. Picual, Villalonga, Alfafarenca, and Cornicabra) grown in different areas of Spain was studied by high performance liquid chromatography-mass spectrometry. Different levels of tyrosol, catechin, p-coumaric acid, rutin, luteolin, and oleuropein were observed in the different varieties analyzed. Treating the fruit with 0.3% Brotomax 50 days after anthesis had a beneficial effect on fruit size, oil content, levels of polyphenolic compounds, and Trolox-equivalent antioxidant activity (TEAC) in all the varieties analyzed.     

Hot water dipping of olives (Olea europaea) for virgin oil debittering

Olives (Olea europaea L.) of the Manzanilla, Picual, and Verdial varieties harvested at the green mature stage of ripening were dipped in hot water at a range of temperatures between 60 and 72 degrees C for 3 min. Immediately after treatment, oils were physically extracted from the olives. Olive heating promotes a reduction of oil bitterness in direct relationship to the temperature used. Fruit heating at > or =60 degrees C for 3 min did not cause significant changes in acidity, UV absorption, peroxide index, and panel test score of the oils obtained but decreased its oxidative stability. Oils extracted from heated fruit showed higher concentrations of chlorophylls and carotenes and lower total phenol content.     

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.     

Reduction of oil bitterness by heating of olive (Olea europaea) fruits

Olives (Olea europaea) of the Manzanilla and Verdial varieties, harvested at the green mature stage of ripening, were heated at 30, 40, 45, and 50 degrees C during 24 h and at 40 degrees C during 24, 48, and 72 h, respectively. Just after treatments, oils were physically extracted from the olives. Olive heating promotes a reduction of oil bitterness in direct relationship to the time and temperature used. Fruit heating at < or =40 degrees C during 24 h did not produce significant changes of acidity, UV absorption, peroxide index, panel test score, or oxidative stability of the obtained oils. Both longer treatments at 40 degrees C and heating at >40 degrees C yielded oils with less oxidative stability. Oils obtained from olives heated at > or =40 degrees C showed higher concentrations of chlorophylls and carotenes. For each olive variety, a good correlation between oil bitterness and content of hydroxytyrosol secoiridoid derivatives was found.     

Unusual carotenogenesis in fruits with pronounced anthocyanic ripening (Olea europaea Var. Arbequina)

Fruits of the Olea europaea Arbequina variety showed a carotenogenic stage overlapping the biosynthesis of anthocyanins. At the start of ripening, the carotenoid concentration increases (20%) or is maintained (besides the esterification of xanthophylls found in this variety), compared with a decrease (30%) found in the same period in the Picual variety (used as a control that was representative of the rest of the varieties). Both the beta,beta and beta,epsilon carotenoid series were found to be implicated in this carotenogenic process. Differences in both varieties for the chlorophyll a/b ratio, chlorophylls/carotenoids ratio, and relative carotenoid composition may indicate the existence of a different metabolism of chloroplast pigments, but also indicates a different structure and function of the photosynthetic apparatus. This fact shows that the photosynthetic behavior in the Picual variety is similar to that of shaded leaves, and in the Arbequina variety is similar to that of sun leaves.     

Changes in microbial communities and carbohydrate profiles induced by the mycorrhizal fungus (Glomus intraradices) in rhizosphere of olive trees (Olea europaea L.)

The influence of inoculation of olive trees with arbuscular mycorrhizal (AM) fungi, Glomus (G) intraradices, on microbial communities and sugar concentrations, were examined in rhizosphere of olive trees (Olea europaea L.). Analyses of phospholipid and neutral lipid fatty acids (PLFA and NLFA, respectively) were then used to detect changes in microbial community structure in response to inoculation of plantlets with G. intraradices.Microscopic observations studies revealed that the extraradical mycelium of the fungus showed formation of branched absorbing structures (BAS) in rhizosphere of olive tree. Root colonization with the AM fungi G. intraradices induced significant changes in the bacterial community structure of olive tree rhizosphere compared to non-mycorrhizal plants. The largest proportional increase was found for the fatty acid 10Me18:0, which indicated an increase in the number of actinomycetes in mycorrhizal rhizosphere soil, whereas the PLFAs i15:0, a15:0, i16:0, 16:1ω7 and cy17:0 which were used as indicators of bacteria decreased in mycorrhizal treatment compared to non-mycorrhizal control treatment. A highest concentration of glucose and trehalose and a lowest concentration of fructose, galactose, sucrose, raffinose and mannitol were detected in mycorrhizal rhizosphere soil. This mycorrhizal effect on rhizosphere communities may be a consequence of changes in characteristics in the environment close to mycorrhizal roots.     

Quantitative changes in phenolic content during physiological development of the olive (Olea europaea) cultivar Hardy's Mammoth

This investigation was designed to characterize phenolic metabolism of the olive cultivar, Hardy's Mammoth, by examining its constitutive tissues. The phenolic profiles of pulp, seed, stone, and new and old season leaves were monitored over two fruiting seasons, to investigate possible relationships between tissues and phenol content and to determine the impact of alternate fruit bearing. No major qualitative differences in phenolic composition were found between the various tissues; however, distinct differences between the tissues with respect to quantifiable phenols were established. Relationships between 2-(3,4-dihydroxyphenyl)ethyl (3E,4E)-4-formyl-3-(2-oxoethyl)hex-4-enoate ester, oleuropein, and hydroxytyrosol in pulp and leaf were identified and found to be related to alternate bearing. Concentrations of 5-caffeoylquinic acid in old season leaves differed dramatically between seasons, confirming earlier studies.     

Changes occurring in phenolic compounds and alpha-tocopherol of virgin olive oil during storage

Changes occurring in the concentrations of alpha-tocopherol, total phenols, and complex phenols linked to 3,4-dihydroxyphenylethanol (fractions FII and FIV) and p-hydroxyphenylethanol (FIII) during storage of virgin olive oil under environmental conditions were studied. Under diffused light, alpha-tocopherol was decomposed by 79% in 4 months, whereas <45% of the phenols were lost during the same period. Among the phenols, FII showed the least stability, and decreased by 72% in 6 months. Total phenols, FIII, and FIV recorded reductions in the range of 57-63% in 6 months. When the oil was stored in the dark, alpha-tocopherol, total phenols, FIII, and FIV exhibited similar profiles of degradation, reducing by 39-45% in the first 6 months and 50-62% in 12 months. FII was the least stable compound in the dark and recorded a loss of 64% in 6 months and 79% in 12 months. The levels of the above antioxidants were further related to peroxide formation. Remaining levels of these compounds at PV = 20 meq/kg ranged between 50 and 73% under diffused light and between 40 and 62% in the dark.     

Irrigation effects on quality, phenolic composition, and selected volatiles of virgin olive oils cv. Leccino

Field-grown olive trees (Olea europaea L. cv. Leccino) were used over two growing seasons to determine the effect of deficit irrigation regimes on virgin olive oil (VOO) quality. Drip irrigation was managed to maintain a predawn leaf water potential (PLWP): (a) higher than -1.1 MPa (full irrigation: FI); (b) between -1.0 and -3.3 MPa (deficit irrigation: DI); (c) higher than -4.2 MPa (severe deficit irrigation: SI). The fruit yield and oil yield of DI trees were over 90% of those of FI treatments in both years, respectively, whereas yields of SI trees ranged from 61 to 76%. The irrigation regime had minor effects on the free acidity, peroxide value, and fatty acid composition of VOO. The concentrations of phenols and o-diphenols in VOO were negatively correlated with PLWP. The concentrations of the dialdehydic form of decarboxymethyl elenolic acid linked to (3,4-dihydroxyphenyl)ethanol (3,4-DHPEA-EDA), the isomer of the oleuropein aglycon (3,4-DHPEA-EA), and the dialdehydic form of decarboxymethyl elenolic acid linked to (p-hydroxyphenyl)ethanol (p-HPEA-EDA) were lower in FI than in SI treatments. The concentrations of lignans (+)-1-acetoxipinoresinol and (+)-1-pinoresinol were unaffected by the irrigation regime. The tree water status had a marked effect on the concentration of volatile compounds, such as the C(6)-saturated and unsaturated aldehydes, alcohols, and esters.     

Factors affecting the contents of iridoid oleuropein in olive leaves (Olea europaea L.)

In this study, for the first time, the impact of the genetic factor on the contents of oleuropein in olive leaves was not only evaluated but the influence exerted by the color/age of leaves (green, green-yellowish, and yellow) and the collecting period (spring or autumn) was also evaluated. A repetitive high-resolution gas chromatographic quantitation method and an accurate high-performance liquid chromatographic method were developed. These analytical methods gave results showing a highly linear relationship. Samples of olive leaves were taken from seven major Italian olive cultivars, such as Dritta, Leccino, Caroleo, Coratina, Castiglionese, Nebbio, and Grossa di Cassano. Such a vegetal raw material could actually be exploited for recovering oleuropein, considered to be a high-added value molecule. This could be converted into hydrxytyrosol, a compound known to possess strong bioactive properties. Olive leaves showed considerable contents of oleuropein, which with some cultivars were even higher with respect to those present in the corresponding olive fruits (reported in the literature). The amounts of oleuropein in the collected leaves were markedly modified by the color/age and genetic factors, whereas meaningless variations were ascribable to the quantitation method and the collecting period factors. Various chemometrics, applied to the obtained analytical data, appeared to be effective in discriminating the samples on the basis of the above-examined experimental factors, thus confirming how these should be taken into account in future industrial recovery of oleuropein from olive leaves.     

Metabolites involved in oleuropein accumulation and degradation in fruits of Olea europaea L.: Hojiblanca and Arbequina varieties

The biosynthetic pathway of oleuropein (from 7-ketologanin, oleoside-11-methyl ester, 7-β-1-d-glucopyranosyl-11-methyl oleoside, and ligstroside to oleuropein) was investigated in two fruit species of Oleaceae, namely, Arbequina and Hojiblanca. Main oleuropein precursors and their metabolites, produced by the enzymatic hydrolysis mediated by β-glucosidase, were identified and quantified to establish the oleuropein transformation pathway. Changes in the concentration of these compounds were measured by direct control of in vivo fruit tissue during their ripening. High contents of aglycones at the initial stage of the process were caused by the high activity of β-glucosidase, which supports that oleuropein biosynthesis is coupled with enzymatic hydrolysis, producing its aglycone form. The low oleuropein content at this initial stage was caused by the imbalance between catabolic and anabolic pathways, favoring the former ones. Once the main polyphenol synthesis phase was completed, the biosynthetic capacity diminished and the content of all compounds decreased. Mass balance revealed that precursors of oleuropein, which are rapidly transformed by β-glucosidase and esterases, scarcely contributed to the accumulation of oleuropein. The biosynthetic pathway proposed by Damtoft applies for both varieties, but our study reveals that the β-glucosidase enzyme is involved in oleuropein synthesis. This enzyme shows high substrate specificity to oleuropein, which consequently is degraded to its aglycone form, with diminished efficacy of oleuropein biosynthesis. Different enzymatic activities of varieties will result in oleuropein accumulation and metabolic transformation of phenols.     

Isolation and characterization of a new hydroxytyrosol derivative from olive (Olea europaea) leaves

A new secoiridoid compound was isolated from the leaves of Olea europaea. This compound, not previously identified, is the bis methylacetal of oleuropein aglycone, the 3,4-dihydroxyphenylethyl [(2,6-dimethoxy-3-ethylidene)-tetrahydropyran-4-yl]acetate (3,4-DHPEA-DETA), and was found in different olive cultivar phenolic extracts as one of the major secoiridoid components. This compound was shown to be easily transformed in acidic aqueous media into 3,4-DHPEA-EDA, the major polyphenolic compound found in olive oil, and permitted us to increase the yield of 3,4-DHPEA-EDA isolation from the olive leaf extract. The antiradical activity of this new compound, evaluated by scavenging of 2,2-diphenyl-1-picrylhydrazyl radicals, was much higher than the one found for 3,4-DHPEA-EDA or alpha-tocopherol. Results also call to attention the need for a careful identification of compounds by HPLC-MS, usually performed in acidic conditions.     

Effect of the herbicides terbuthylazine and glyphosate on photosystem II photochemistry of young olive (Olea europaea) plants

The purpose of this study was to understand the effect produced by the addition of the herbicides terbuthylazine (N(2)-tert-butyl-6-chloro-N(4)-ethyl-1,3,5-triazine-2,4-diamine) and glyphosate (N-(phosphonomethyl)glycine) on photosystem II photochemistry of young plants of Olea europaea L. under greenhouse conditions. The effect of soil amendment with an organic residue from olive oil production was also assessed. Terbuthylazine reduced the efficiency of photosystem II photochemistry of plants due to chronic photoinhibition, and this effect was counterbalanced by soil amendment with the organic waste, whereas the photosystem II photochemistry of olive plants was not affected by glyphosate or by glyphosate and organic waste addition. In this study, we have shown that the soil application of terbuthylazine is a source of indirect phytotoxicity for olive plants. We have also observed that the olive plants were not affected by higher amounts of glyphosate in the soil.     

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.     

Evaluation of the antioxidant capacity of individual phenolic compounds in virgin olive oil

Virgin olive oil has a high resistance to oxidative deterioration due to its tryacylglycerol composition low in polyunsaturated fatty acids and due to the presence of a group of phenolic antioxidants composed mainly of polyphenols and tocopherols. We isolated several phenolic compounds of extra virgin olive oil (phenyl-ethyl alcohols, lignans, and secoiridoids) by semipreparative high-performance liquid chromatography (HPLC) and identified them using ultraviolet, atmospheric pressure chemical ionization, and electrospray ionization MS detection. The purity of these extracts was confirmed by analytical HPLC using two different gradients. Finally, the antioxidant capacity of the isolated compounds was evaluated by measuring the radical scavenging effect on 1,1-diphenyl-2-picrylhydrazyl radical, by accelerated oxidation in a lipid model system (OSI, oxidative stability instrument), and by an electrochemical method.     

Influence of harvest date and crop yield on the fatty acid composition of virgin olive oils from cv. Picual

In this study was analyzed the effect of crop year and harvesting time on the fatty acid composition of cv. Picual virgin olive oil. The study was carried out during the fruit ripening period for three crop seasons. The mean fatty acid composition of Picual oils was determined. The oils contained palmitic acid (11.9%), oleic acid (79.3%), and linoleic acid (2.95%). The content of palmitic acid and saturated fatty acids decreased during fruit ripening while oleic and linoleic acids increased. The amount of stearic and linolenic acids decreased. The amount of saturated acids, palmitic and stearic, and the polyunsaturated acids linoleic and linolenic was dependent on the time of harvest, whereas the amount of oleic acid varied with the crop year. The differences observed between crop years for both palmitic and linoleic acid may be explained by the differences in the temperature during oil biosynthesis and by the amount of summer rainfall for oleic acid content. A significant relationship was observed between the MUFA/PUFA ratio and the oxidative stability measured by the Rancimat method.