Influence of cultivar and fruit ripening on olive (Olea europaea) fruit protein content, composition, and antioxidant activity

Proteins of olive fruit mesocarp are not very well-known at present. However, they have been shown to pass, at least partially, to the olive oil during its elaboration and therefore might be contributing to some of the special characteristics of this vegetable oil. In this study, protein content and composition were determined in olive fruits, cv. Arbequina and Picual, at three stages of ripening: green, spotted, and purple. Mesocarp proteins constituted 1.3-1.8% of the dry weight of the olive fruit, and cultivar and fruit ripening did not produce important changes in mesocarp protein content or composition. In addition, this composition was also similar to the amino acid composition of a 4.6-kDa polypeptide, which is the major protein component of olive oils and of oil bodies of olive fruit mesocarp, suggesting that this polypeptide is likely to be a major component of mesocarp proteins. There was, also, a relationship between the oil content of the olive fruit and the protein content determined, suggesting a stabilizing function of these proteins in the oil bodies of the olive fruit, analogously to the role suggested for oleosins. This stabilizing function does not seem to be extended to olive oils because when the polypeptides isolated were added at 20 ppm to soybean oil, the stability of the oil increased only slightly, suggesting that if these compounds play some role in the stability of the oils, this should be mostly a consequence of the possible interactions among these protein components and other olive oil antioxidant constituents.     

Factors influencing phenolic compounds in table olives (Olea europaea)

The Mediterranean diet appears to be associated with a reduced risk of several chronic diseases including cancer and cardiovascular and Alzheimer's diseases. Olive products (mainly olive oil and table olives) are important components of the Mediterranean diet. Olives contain a range of phenolic compounds; these natural antioxidants may contribute to the prevention of these chronic conditions. Consequently, the consumption of table olives and olive oil continues to increase worldwide by health-conscious consumers. There are numerous factors that can affect the phenolics in table olives including the cultivar, degree of ripening, and, importantly, the methods used for curing and processing table olives. The predominant phenolic compound found in fresh olive is the bitter secoiridoid oleuropein. Table olive processing decreases levels of oleuropein with concomitant increases in the hydrolysis products hydroxytyrosol and tyrosol. Many of the health benefits reported for olives are thought to be associated with the levels of hydroxytyrosol. Herein the pre- and post-harvest factors influencing the phenolics in olives, debittering methods, and health benefits of phenolics in table olives are reviewed.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Effect of bruising on respiration, superficial color, and phenolic changes in fresh Manzanilla olives (Olea europaea pomiformis): development of treatments to mitigate browning

The aim of the work was to study the postharvest changes in Manzanilla olives and to find treatments to mitigate damages because of bruises. The phenolic content in bruised and unbruised fruits exposed to air always decreased, but the loss in phenols and the respiratory activity were greater in bruised olives; these changes were related to the appearance of brown spots. Immersion of the picked fruits in a cold (8 °C) acidic solution (pH 3), ascorbic acid solution (100 mM), or sodium metabisulfite solution (100 mM) significantly reduced the loss in phenols in olives and led to lighter brown bruised areas. This immersion did not affect the behavior of the fruits during the lye treatment and the subsequent fermentation. In the final product, no influence on the surface color of unbruised olives was observed and there was a significant color improvement in the bruised areas of damaged olives.     

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.     

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.     

Antioxidant activity of olive pulp and olive oil phenolic compounds of the arbequina cultivar

The aim of this study was to characterize antioxidant activities of phenolic compounds that appear in olive pulp and olive oils using both radical scavenging and antioxidant activity tests. Antiradical and antioxidant activities of olive pulp and olive oil phenolic compounds were due mainly to the presence of a 3,4-dihydroxy moiety linked to an aromatic ring, and the effect depended on the polarity of the phenolic compound. Glucosides and more complex phenolics exhibited higher antioxidant activities toward oxidation of liposomes, whereas in bulk lipids aglycons were more potent antioxidants with the exception of oleuropein. Lignans acted as antioxidants only in liposomes, which could partly be due to their chelating activity, because liposome oxidation was initiated by cupric acetate. The antioxidant activity of virgin olive oil is principally due to the dialdehydic form of elenolic acid linked to hydroxytyrosol (3,4-DHPEA-EDA), a secoiridoid derivative (peak RT 36, structure unidentified), and luteolin.     

Polyphenolic content in five tuscany cultivars of Olea europaea L.

In this study polyphenolic compounds extracted from olive fruits of five registered cultivars were analyzed. A solid-liquid extraction (LSE) procedure with Extrelut cartridge (diatomaceous earth) using different eluents was developed to obtain polyphenolic compounds. HPLC-DAD and HPLC-MS methods were applied for the quali-quantitative analysis of each fraction obtained from LSE. The results of this work show that the LSE procedure with diatomaceous earth cartridge supplies a rapid and reproducible fractioning method able to obtain a quantitative recovery of all compounds and to collect fractions directly analyzed by HPLC. A comparison among different cultivars shows significant quantitative differences in some polyphenols, such as verbascoside, anthocyanic compounds, and oleuropein derivatives.     

Identification of phenolic compounds in tissues of the novel olive cultivar hardy's mammoth

A methodological approach to phenolic profiling making extensive use of LC-MS with extracted ion chromatograms was applied to extracts of five different olive tissues: pulp, seed, stone, new-season leaves, and old-season leaves. Tissue extracts of the cultivars Hardy's Mammoth, Corregiola, Verdale, and Manzanillo were analyzed by HPLC with UV and ESI MS detection. Chromatograms of samples of green Hardy's Mammoth drupes, a uniquely Australian olive cultivar, were dominated by a large, broad peak. This peak was not attributable to oleuropein, which is usually the dominant phenolic compound in green olive fruit, but the phenolic compound I. This compound was isolated by semipreparative HPLC and characterized by 1D- and 2D-NMR. Extraction studies showed that the compound was not likely to be an artifact of an enzymatic degradation process. Tritium labeling studies were used to establish a possible relationship between the biosynthesis of I and oleuropein.     

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.