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.     

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.     

Pigment metabolism of 'Sikitita' olive ( Olea europaea L.): a new cultivar obtained by cross-breeding

The new olive cultivar 'Sikitita' was obtained from a cross between the 'Picual' and 'Arbequina' varieties. 'Sikitita' was selected for its features, making it particularly suited to high-density olive hedgerow orchards. From the standpoint of chloroplast pigment metabolism, the fruits of the 'Picual' and 'Arbequina' varieties have significant differences. It is therefore extremely interesting to analyze the descendants of both cultivars. With regard to chlorophyll catabolism, 'Sikitita' has proven to be a cultivar with low pigmentation and low levels of chlorophyllase activity. This is contrary to the findings obtained to date, where varieties with low pigmentation are a consequence of high chlorophyllase activity ('Arbequina') and highly pigmented fruits are due to low chlorophyllase activity ('Picual'). 'Arbequina' was, until recently, the only cultivar described that had developed a carotenogenic process, despite its anthocyanic ripening. However, from its father ('Arbequina'), the 'Sikitita' cultivar has inherited the pool of enzymes necessary to esterify xanthophylls at the chromoplast level. This makes 'Sikitita' a very interesting cultivar, with potential chemotaxonomic differences (such as esterified xanthophylls in the olive oils), and demonstrates the interest in genetic improvement programs for olive cultivars with different organoleptic characteristics.     

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.     

Parent and harvest year effects on near-infrared reflectance spectroscopic analysis of olive (Olea europaea L.) fruit traits

The influence of parent and harvest year on the determination of oil, moisture, oleic acid, and linoleic acid contents in intact olive fruit was studied by near-infrared spectroscopy (NIRS). Spectral data from 400 to 1700 nm were recorded on 437 fruit samples collected in 1996 and 1997 from seedling plants derived from three different female parents. Partial least squares models were developed using samples for each year and for each female parent separately and were validated against the other groups. Calibration models were accurate enough to predict all constituents in new samples from a different female parent but were not transferable across years. However, a calibration equation of sufficient accuracy was obtained from the combined data set (r values of 0.94, 0.93, 0.84, and 0.88 and RMSECV values of 1.33, 1.88, 4.73, and 2.91 for oil, moisture, oleic acid, and linoleic acid contents, respectively). These results demonstrate the utility of NIRS as a selection tool in olive breeding programs.     

Characterization of polyphenol oxidase from the Manzanilla cultivar (Olea europaea pomiformis) and prevention of browning reactions in bruised olive fruits

The crude extract of the polyphenol oxidase (PPO) enzyme from the Manzanilla cultivar (Olea europaea pomiformis) was obtained, and its properties were characterized. The browning reaction followed a zero-order kinetic model. Its maximum activity was at pH 6.0. This activity was completely inhibited at a pH below 3.0 regardless of temperature; however, in alkaline conditions, pH inhibition depended on temperature and was observed at values above 9.0 and 11.0 at 8 and 25 degrees C, respectively. The thermodynamic parameters of substrate oxidation depended on pH within the range in which activity was observed. The reaction occurred according to an isokinetic system because pH affected the enzymatic reaction rate but not the energy required to carry out the reaction. In the alkaline pH region, browning was due to a combination of enzymatic and nonenzymatic reactions that occurred in parallel. These results correlated well with the browning behavior observed in intentionally bruised fruits at different temperatures and in different storage solutions. The use of a low temperature ( approximately 8 degrees C) was very effective for preventing browning regardless of the cover solution used.     

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.     

Comparative study on phenolic content and antioxidant activity during maturation of the olive cultivar Chemlali from Tunisia

For the first time the identification and quantification of phenolic compounds of the Olea europaea L. cv. Chemlali olive were carried out to examine their profile during maturation. The phenolic composition was studied by using reverse-phase high-performance liquid chromatography during all steps of fruit development. Oleuropein is the abundant phenolic compound in Chemlali olive, and its concentration increases during maturation. An indirect relationship between oleuropein content in olive fruit and hydroxytyrosol was observed. Weak changes in the amounts of the other phenolic monomers and flavonoids were also observed. The total phenolic content varied from 6 to 16 g/kg expressed as pyrogallol equivalents. Its highest level was found at the last maturation period. The antioxidant capacity of olive extracts was evaluated by measuring the radical scavenging effect on 1,1-diphenyl-2-picrylhydrazyl. The IC(50) values of the olive extract ranged from 3.2 to 1.5 microg/mL. There was a correlation between antioxidant activity and total phenolic content of samples. The antioxidant activity increased with maturation. This could be attributed to the increase of the tolal phenol level with fruit development.     

Adsorption of olive leaf (Olea europaea L.) antioxidants on silk fibroin

The adsorption isotherms of oleuropein and rutin were evaluated at different temperatures, pH values, and solid/liquid ratios. The experimental data of adsorption isotherms were well fitted to a Langmuir model. The maximum adsorption capacities were determined as 108 mg of oleuropein/g of silk fibroin and 21 mg of rutin/g of silk fibroin. After adsorption of oleuropein and rutin, the antioxidant capacity of silk fibroin increased from 1.93 to 3.61 mmol of TEAC/g. Silk fibroin also gained antimicrobial activity against Staphylococcus aureus and Klebsiella pneumoniae after adsorption of olive leaf antioxidants. In a desorption process, 81% of rutin and 85% of oleuropein were removed from the adsorbent surface in 70% aqueous ethanol solution. Consequently, silk fibroin was found to be a promising biomaterial for the production of functional food or dietary supplements and for the purification of oleuropein and rutin from olive leaf extracts.     

Isolation and characterization of the antioxidant component 3,4-dihydroxyphenylethyl 4-formyl-3-formylmethyl-4-hexenoate from olive (Olea europaea) leaves

Storage of olive (Olea europaea) leaves for 22 h at 37 degrees C in closed plastic bags caused the content of a nonglycosidic secoiridoid, 3,4-dihydroxyphenylethyl 4-formyl-3-formylmethyl-4-hexenoate (3,4-DHPEA-EDA) to rise from 15% to 50% of the phenolic extract with corresponding falls in the content of oleuropein and two oleuropeindials, which were identified as precursors of 3,4-DHPEA-EDA. Pure product was isolated from one set of stored olive leaves in a 0.16% yield. Storage of olive leaves under various conditions showed that the moisture present in closed plastic bags was important for the formation of 3,4-DHPEA-EDA. The time taken to reach the maximum concentration of the product varied widely for different samples of olive leaves, with a shorter time for the sample with lower initial oleuropein content. The oleuropeindial precursors of the product were readily hydrolyzed to carboxylic acid derivatives, which have been identified by NMR. The antiradical activity of 3,4-DHPEA-EDA, evaluated by scavenging of 2,2-diphenyl-1-picrylhydrazyl radicals, was comparable to that of alpha-tocopherol.     

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.     

Chlorophyll and carotenoid patterns in olive fruits, Olea europaea Cv. arbequina

In olive fruits of the cultivar Arbequina, the chlorophyll pigments decrease significantly throughout ripening, while the carotenoids decrease more gradually and discontinuously. There is no degradation of the carotenoid fraction in stages before complete ripeness. The presence of esterified xanthophylls exclusively in this variety suggests that the chloroplast pigment metabolism is different from that in other olive varieties studied previously. There are increases of specific carotenoids, violaxanthin, neoxanthin, antheraxanthin, lutein epoxide, and esterified xanthophylls between the light green and yellowish green ripening stages. Such increases are related to the detection of precursor carotenoids (phytofluene and xi-carotene) in the yellowish green stage. Chlorophyllides (a and b) and alpha-carotene have also been detected exclusively in this variety. Quantitatively, the drastic change in color between light green and yellowish green ripening stages characteristic of this variety can be explained by the considerable reduction found in the chlorophylls/carotenoids ratio. The study of the pigments present in skin and pulp has shown that the pattern of carotenoid distribution differs depending on the fruit part concerned.     

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.     

Nondestructive evaluation of anthocyanins in olive (Olea europaea) fruits by in situ chlorophyll fluorescence spectroscopy

Anthocyanins (Anths) in olive (Olea europaea L.) fruits at different degrees of pigmentation were assessed nondestructively by measuring chlorophyll fluorescence (ChlF). The method is based on the comparison of the ChlF excitation spectra from olives with different pigmentation from green to green-red, reddish-purple, and purple. The logarithm of the ratio between the fluorescence excitation spectra (logFER) from two different colored zones gave the difference in the absorption spectrum between them. The absorbance spectrum derived from the logFER between a red olive and the same olive devoid of the skin showed the typical Anth green band (at 550 nm). It matched that recorded by microspectrophotometry on a single pulp cell and the in vitro absorbance spectrum of the olive skin extract. As expected, the in vivo Anths absorption maximum increased in intensity going from less to more mature olives and was higher in the sun-exposed olive side with respect to the sun-shaded side. Absolute quantitative nondestructive determination of Anths for each olive sample was obtained by the logFER calculated for two excitation wavelengths, 550 and 625 nm, of ChlF at 740 nm. Going from green to purple skin colors, the Log[ChlF(625)/ChlF(550)] was fairly well-correlated to the extract Anths concentration. Finally, the relationship between the Anths and the other main phenolics present in the olives analyzed by high-performance liquid chromatography was evaluated. The main result was a net increase of verbascoside with increasing Anths content. On the basis of our results, the development of a new rapid and noninvasive method for the monitoring of olive development and ripening can be envisaged.     

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.     

Genetic differentiation in the olive complex (Olea europaea) revealed by RAPDs and RFLPs in the rRNA genes

We assessed the genetic differentiation of the Mediterranean olive from its wild relatives found in different geographic areas (Mediterranean, Asia, Africa) using eighty RAPDs revealed with eight primers. Variance analysis (AMOVA) enabled us to estimate the overall genetic differentiation parameters between wild populations. Oleasters from the Near East and Turkey were discriminated from the other Mediterranean populations. Olea laperrinei, O. maroccana and O. cerasiformis were the taxa the most related to the Mediterranean olive. In contrast, O. africana was shown to be the most genetically distant taxa from the Mediterranean olive. However, we characterised hybrid trees between these two taxa. Significant trends between genetic and geographic distances were met within the subspecies cuspidata and within the Mediterranean olive. A genetic diversity gradient was observed in both subspecies europaea and cuspidata. These results are in agreement with a mechanism of differentiation by distance in the O. europaea complex, but another non-exclusive mechanism could also be gene flow between differentiated taxa. Furthermore, we characterised the discriminating power of each RAPD to recognise the different taxa using intraclass correlation coefficients. Lastly, IGS-RFLPs enabled us to assess rDNA polymorphisms on a sub-sample of individuals. On the basis of these data, a low interspecifc differentiation was found. This suggests a recent genetic divergence between the different taxa of the O. europaea complex or the occurrence of gene flow during favourable periods or because human displacements. All the olive cultivars were genetically related to the oleaster populations supporting that Mediterranean is the olive domestication area.     

Relationships between oxidative stability, triacylglycerol composition, and antioxidant content in olive oil matrices

In olive oils, relationships between oxidative stability, glyceridic composition, and antioxidant content were investigated. Lipid matrices, obtained by purification of olive and high-oleic sunflower oils, were spiked with hydroxytyrosol, alpha-tocopherol, and mixtures of them and then subjected to oxidation in a Rancimat apparatus at 100 degrees C. At the same concentration of antioxidants, induction time (IT) decreased as the unsaturation rate of the matrix increased, but only fair correlations were found with fatty acid composition. Oxidative susceptibility (OS(TAG)) was calculated as a function of the relative oxidation rate of the triacylglycerols, and a linear relationship-IT (h) = (a + b)OS(TAG)-between induction time and this parameter showed a good correlation coefficient (r > 0.990, p < 0.001). In the case of matrices with a single antioxidant, origin ordinate (a) and slope (b) can be calculated as a function of the antioxidant concentration. In matrices spiked with mixtures of hydroxytyrosol and alpha-tocopherol, a simple relationship between the coefficients a and b and the concentration of antioxidants cannot be established because additive and subtractive effects occur depending on the relative concentrations of both antioxidants. However, approximate values for these coefficients can be obtained, allowing the estimation of the oil stability. In various olive oils, an acceptable agreement was found between the IT experimentally determined and that calculated from the oil composition. These results confirmed that the Rancimat stability of olive oils mainly depends on triacylglycerol composition and concentrations of o-diphenols and alpha-tocopherol.     

Proteomic analysis of up-accumulated proteins associated with fruit quality during autumn olive (Elaeagnus umbellata) fruit ripening

Fruit ripening is a complex phenomenon that makes berries attractive and also determines their nutritional value. Autumn olive ( Elaeagnus umbellata Thunb.) fruit is a rich source of many human health-related nutrients. The changes in pericarp color are initiated at early developmental stages, coinciding with the fast increase in fruit size. Fruit quality traits with special emphasis on soluble sugars, organic acids, lycopene, and total protein contents were assayed during the fruit ripening. In the fully ripe fruit, glucose and fructose were the principal sugars, malic acid was the most abundant organic acid, and lycopene concentration was extremely high. A proteomic analysis was used to identify up-accumulated proteins induced by the ripening. Among 63 up-accumulated protein spots, 43 were successfully identified by MALDI-TOF/TOF-MS. All 43 proteins were novel for autumn olive, and 8 were first reported in the fruit. Twenty-one proteins of known function were involved in sugar metabolism, citric acid cycle, isoprenoid metabolism, fatty acid synthesis, and protein hydrolysis. The possible roles of these 21 accumulated proteins in autumn olive fruit quality are discussed.     

Relationship between apple peel and the whole fruit antioxidant content: year and cultivar variation

Antioxidants are usually considered with regard to plant defense mechanisms due to the oxidative stress or their importance for human health or both. In the present research, a comprehensive study was made to test the relationship between the antioxidant content in apple peel and the whole fruit in two growing seasons. Antioxidants were mainly localized in the apple peel, but cultivars exhibited very high biodiversity in the distribution pattern. High or very high correlation coefficients between apple peel glutathione reductase and catalase activity as well as ascorbate, glutathione, and anthocyanins concentrations and the whole fruit were discovered on the basis of both fresh and dry weight basis in two growing seasons: 2004 and 2005. In the case of ascorbate peroxidase activity and phenolics or flavonol contents, it was proven only in 2005. It seems that apple peel could be a good marker of health values as well as antioxidant potential of apple fruit. Additional arguments, as compared to the previous study, were incorporated to support the suggestion of glutathione reductase as worth considering as an environmental stress marker and/or signalling molecule.     

Changes in the phenolic composition of virgin olive oil from young trees (Olea europaea L. cv. Arbequina) grown under linear irrigation strategies.

This study reports the HPLC profiles of phenolic compounds of virgin olive oils obtained from young olive trees (Olea europaea L. cv. Arbequina) and how the application of a linear irrigation strategy affected these. Hydroxytyrosol, tyrosol, vanillic acid, vanillin, 4-(acetoxyethyl)-1,2-dihydroxybenzene, p-coumaric acid, the dialdehydic form of elenolic acid linked to hydroxytyrosol and to tyrosol, lignans, and the oleuropein aglycon were found in all the oils. Hydroxytyrosol, tyrosol, vanillic acid, and p-coumaric acid contents in the oils were unaffected by linear irrigation. The concentration of lignans was lower in the oils from the least irrigated treatment and the concentration of vanillin increased as the amount of irrigation water applied to olive trees increased. However, 4-(acetoxyethyl)-1,2-dihydroxybenzene, the dialdehydic form of elenolic acid linked to hydroxytyrosol and to tyrosol, and the oleuropein aglycon, all of them hydroxyphenyl derivatives, decreased as the level of irrigation water increased. The latter three compounds represented the most considerable part of the phenolic fraction of the oils and they were shown to be correlated to the oxidative stability, the bitter index (K(225)), and the bitter, pungent, and sweet sensory attributes. Linear irrigation strategy changed the profile of the oil phenolic compounds and, therefore, changed both the organoleptic properties and the antioxidant capacity of the product.