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Ethanol in olive fruit. Changes during ripeness Gabriel Beltran, Mohamed Aymen Bejaoui, Antonio Jimenez, and Araceli Sánchez-Ortiz J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b01453 • Publication Date (Web): 22 May 2015 Downloaded from http://pubs.acs.org on May 26, 2015

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Journal of Agricultural and Food Chemistry

‘Ethanol in olive fruit. Changes during ripeness’ Gabriel Beltrán*, Mohamed A. Bejaoui, Antonio Jimenez, Araceli Sanchez-Ortiz

IFAPA Center Venta del Llano, Junta de Andalucia. P.O. Box 50, 23620 Mengibar, Jaén. Spain

* Corresponding author: Dr. Gabriel Beltrán Tel: +34 671532213; Fax: +34 953 366 380; Email: [email protected]

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ABSTRACT

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Ethanol is one of the precursors of ethyl esters, the virgin olive oil quality parameter

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for extra category recently adopted by the European Union and International Olive Oil

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Council. Although ethyl esters content has great importance for virgin olive oil

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classification, the origin of ethanol is not clear. A possible source of ethanol may be

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the own olive fruit while remains on the tree. Variation of fruit ethanol content during

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ripening was studied for three different olive cultivars: ‘Picual’, ‘Hojiblanca’ and

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‘Arbequina’. Ethanol was measured in fruit homogenates by HS-SPME-GC-FID. The

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ethanol content varied between 0.56 and 58 mg/kg. ‘Hojiblanca’ fruits showed the

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highest ethanol concentration. For all the cultivars, ethanol content of fruit increased

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during ripening process although a clear cultivar dependent effect was observed since

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‘Hojiblanca’ fruits showed the most important raise. Therefore, results indicated that

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ethanol can be accumulated during fruit maturation on the olive tree.

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Keywords: Olea europaea L .; olive fruit; ethanol; ripening; cultivar

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INTRODUCTION

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Olive (Olea europaea L.) growing has great importance in the Mediterranean basin

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since is the base of two important food industries: Table olives and virgin olive oil

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production. Even although it has great importance information about the raw material

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characteristics is scarce, mainly in olives for oil mill use.

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In this way, ethanol in olive fruit is a metabolite that has achieved great importance for

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the last two years since is one of the precursors of ethyl esters. Ethyl esters is a virgin

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olive oil quality parameter adopted recently by both European Commission and

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International Olive Oil Council1, 2 to discriminate ‘extra virgin olive oil ‘ from healthy

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and high quality olive fruits. In fact, for oil classification into ‘extra virgin’ category

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ethyl esters content must be lower than 40 mg/kg. This limit will be reduced in

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sequence for the next crop years: 35 mg/kg in 2014/15 and finally, 30 mg/kg for the

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crop year 2015/16.

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Although a close relationship between virgin olive oil quality and ethyl esters was

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described,3-5 their biosynthesis and the source of ethanol as ethyl ester precursor are

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not clear and have to be analysed.

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Conte et al.6 proposed that ethanol can be solely produced by fermentation during

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virgin olive oil extraction and storage process. However a possible source of ethanol

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may be the synthesis in the own olive fruit. The production of ethanol in olives may be

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originated on the tree, during fruit collection, transport and/or in the postharvest

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treatments until processing. However to our best of knowledge there are no data for

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ethanol content in olive fruit and its possible biosynthesis from the olive tree to the oil

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mill.

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Ethanol in fruits is formed by the enzyme alcohol deshydrogenase (ADH) (EC 1.1.1.1),

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being described for oranges and pears an increase of ethanol production during

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maturation.7,8 Considering the fruits as source of ethanol, has been described as during

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ripening on the tree are activated reactions to produce aroma and other components

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requiring the synthesis of anaerobic metabolites, among them ethanol.9

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Furthermore, ethanol is accumulated in different fruits that remain for long periods on

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the tree as oranges and grapefruits. In these cases the concentration of ethanol

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showed a fast increase during their permanence on the tree.10 Litchi fruits increased

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the ethanol production during maturation on tree.11 Peaches and nectarines showed

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increases in aroma volatiles and ethanol during maturation.12

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Ethanol can be accumulated in over-ripe and senescent fruits than remain on the tree

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for a long period. In apple cultivars although small amount of ethanol can be detected,

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it can be synthetized at higher concentrations in over-ripe and senescent fruits.13

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Olive is a non-climateric fruit that usually remains for long periods on the tree until

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harvesting. This period can oscillate between 5 and 9 months, depending of olive

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cultivar, including fruit development, ripening, and overmaturation. The mean ripening

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period described for World Olive Germoplasm Bank cultivars varied between 53 and

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69 days for the earliest and latest olive cultivars, respectively.14 However, a

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considerable part of the olive production can remain on the tree until overmaturation

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(2-3 months) because of adverse climatic conditions during harvesting period or high

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crops. Thus is of great importance determining how ethanol content varies in fruit

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during ripening process in order to evaluate if ethanol is synthetized on the tree and

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then, harvesting date may have effect in the levels of ethanol in fruit, olive paste and

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finally in the virgin olive oil ethanol and ethyl esters content.

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The objective of this work was to study the ethanol content in olive fruit and its

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variation during ripeness. To achieve this aim three olive cultivars were selected by

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their importance in the world olive oil production: ‘Picual’, ‘Hojiblanca’ and

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‘Arbequina’ and acetaldehyde content, as ethanol precursor, was analysed too.

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MATERIALS AND METHODS

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Plant Material. The study was carried out for the crop year 2012/13. Olive trees of

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three olive cultivars: ‘Picual’, ‘Hojiblanca’ and ‘Arbequina’ were selected by their crop

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load homogeneity (3-4). Two trees were selected for each cultivar. The twenty-five-

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year-old trees were grown in the Wold Olive Germplasm Collection of IFAPA Center

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Venta del Llano in Mengibar, Jaen. The trees were spaced 7*7 m and grown under

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irrigation and traditional techniques.

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Olive samples (2 kg) were collected from each tree and cultivar at three harvesting

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dates for ‘Picual’, ‘Hojiblanca’ and ‘Arbequina’. In addition for ‘Arbequina’ more

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harvesting dates were included because of the stepped maturation of its fruits. Fruit

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ripening index was measured according to the method the method proposed by the

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Estacion de Olivicultura y Elaiotecnia.15 The fruit sampling dates and ripening index are

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shown in Table 1. After harvesting, the fruits were washed using milliQ water, dried

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with filter paper and processed immediately (15 min) in order to avoid any alteration .

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Fruit homogenates. To measure the ethanol content a fruit homogenate approach was

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followed.16 For this purpose, 4 g of olive fruit mesocarp, from at least 20 olive fruits,

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were homogenized with 8 mL of distilled water by means of a homogenizer Ultra-

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Turrax T-25 at the highest speed (24 000 rpm) for 2 min. After an equilibrium period

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of 5 min at 25 °C, homogenate aliquots of 2 mL were taken into 10 mL vials containing

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2 mL of a saturated CaCl2 solution to deactivate the enzyme systems, which were

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sealed and stored at −18 °C unKl analysis. Two homogenates were prepared in

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duplicate for each olive sample.

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Analysis of ethanol and acetaldehyde. Solid-phase microextraction (SPME) followed by

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GC-FID were used to analyze the ethanol and acetaldehyde in the samples studied

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according to the method described by Sanchez–Ortiz et al.16 Briefly, homogenate

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samples were conditioned to room temperature and then placed in a 10 mL vial fitted

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with a silicone septum heater at 40 °C. After 10 min of equilibrium time, ethanol from

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headspace was adsorbed by exposing the solid-phase microextraction (SPME) fiber

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DVB/Carboxen/PDMS 50/30 μm 1 cm (Supelco Co., Bellefonte, PA) for 50 min at 40 °C

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in the headspace of the sample, and then retracted into the needle and immediately

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transferred and desorbed for 5 min into the injection port of a gas chromatograph

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equipped with an FID.

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Ethanol and acetaldehyde were analysed using a Varian CP 3800 GC equipped with a

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Supelcowax 10 capillary column (30 m x 0.25 mm, 0,25 μm, Sigma-Aldrich Co. LLC).

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Operating conditions were as follows: He was the carrier gas; injector and detector at

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250 °C; and column held for 5 min at 40 °C and then programmed at 4 °C min−1 to 200

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°C. Compound identification was carried out on an ISQ single quadrupole MS, Thermo

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Fisher Scientific, Austin, Texas, USA) operating in EI mode (70 eV) under identical

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conditions for GC-FID, matching against the Wiley/NBS Library, and by GC retention

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time against standard. Quantification was performed using individual calibration

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curves in the matrix (olive mesocarp homogenate). Results were expressed as mg per

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kilogram of fruit.

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Data analysis. Data for harvesting dates were shown as mean value and standard

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deviation whereas for cultivar content was expressed as mean value and standard

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error. ANOVA analysis was performed to establish the effect of cultivar and ripening

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stage considering the three common harvesting dates for the three olive cultivars.

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Tukey’s test was applied to establish differences between means p:0.05. Statistical

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analyses were performed with Statistix 8.0 software.

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RESULTS AND DISCUSSION

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ADH activity catalyses the reversible reduction of aldehydes to alcohols using reduced

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pyridine nucleotides as cofactors. In previous works ADH activity was measured only in

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crude extracts prepared from acetone powders of olive mesocarp and seed tissues

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whereas it was not detected in crude extracts from fresh tissues.16 In order to

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determine the effect of homogenisation process in ethanol synthesis, a previous

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experiment was carried out. Fruit frozen tissue was ground in a cold blender

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containing CaCl2 at 0.33 g.g–1 tissue to inhibit enzyme activities during homogenization

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comparing with the method used in this work. Both methods showed similar content

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of ethanol (data not shown).

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Ethanol content in fruit varied depending on olive cultivar. For ‘Hojiblanca’ fruits

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ethanol concentration varied between 6 and 58 mg/kg whereas ‘Picual’ cultivar

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oscillated from 0.56 to 2.90 mg/kg. ‘Arbequina’ olives showed an ethanol content that

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ranged between 1.5 and 11.5 mg/kg.

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When ANOVA analysis was performed could be observed as the olive cultivar was the

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main responsible of the variability for ethanol concentration although the harvesting

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date showed similar values (Table 2). Among the olive cultivars analysed, ‘Hojiblanca’

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showed the highest ethanol content in fruit and ‘Picual’ achieved the lowest

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concentration (Table 3). Significant differences between olive cultivars were obtained

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(P. 0.05). These differences in ethanol content between cultivars were described for

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apples and nectarines previously.16 There are not previous works describing

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differences in olive ethanol content between cultivars. The differences observed in this

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work may be explained by the ADH content/activity in different olive cultivars as

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described for ‘Coratina’ and ‘Carolea’ olive cultivars for C6 volatile alcohols.18 In our

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case, the levels of ethanol in fruits of different cultivars corresponded to similar

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differences for its precursor the acetaldehyde (data not shown).

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Harvesting date showed a highly significant effect on ethanol content in olive fruits

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(Table 2). During olive fruit ripening, ethanol concentration increased slightly at the

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beginning of maturation and then, a fast increase was detected (Figure 1). Although

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’Arbequina’ has a fruit stepped maturation and was studied for more harvesting dates

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the behaviour was similar to the other cultivars. In the first raise, ethanol showed an

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increase comprised between 23% for ‘Picual’ cultivar and 62% for ‘Hojiblanca’. For the

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last harvesting date the fastest increases were detected; the increases obtained varied

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around 70% for the three olive cultivars. Between the first sample collection and the

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final one, ethanol content rose around 80% for ‘Picual’ and ‘Arbequina’ whereas

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‘Hojiblanca’ fruits achieved an increase of 89%. Therefore, ‘Hojiblanca’ fruits achieved

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the highest ethanol content during fruit maturation. These differences between

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cultivars in the biosynthesis of ethanol may be explained by the high significance

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obtained for the interaction between olive cultivar and harvesting date in the ANOVA

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(Table 2). Ethanol is formed from acetaldehyde by the enzyme alcohol dehydrogenase

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(ADH). Salas et al.17 described an increase of ADH activity from 13 weeks after

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flowering up to 25 weeks after flowering when fruits are still green. After this point

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ADH activity decreased during the ripening process. Therefore the increase of fruit

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ethanol content observed for the three cultivars may be not explained by a higher ADH

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activity since the period analysed corresponded to a reduction of ADH activity.

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Both acetaldehyde and ethanol have been shown to accumulate in various fruit (both

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climacteric and non-climacteric) that remain on the tree for long periods.9 The increase

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in anaerobic respiration in over-mature fruit may be explained by a reduction of

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mithocondrial activity in its tissues. In our case both of them are accumulated in the

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fruit during maturation although ethanol showed a faster increase giving a reduction

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of the ratio acetaldehyde/ethanol (Figure 2). This ratio can be used as indicator of fruit

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anaerobic respiration.

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The increase of ethanol content observed for the three cultivars was similar to those

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described for both climacteric and non-climateric fruits during the ripening and their

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permanence on the tree for long periods

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was not achieved.

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although in our case over-maturation

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The results confirm that ethanol is produced naturally in the olive fruits whereas

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remains on the tree since for this work their processing was immediate. Ethanol

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content in olives had a very important genetic component since significant differences

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were observed between cultivars. ‘Hojiblanca’ fruits showed the highest ethanol

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concentration. During fruit ripening ethanol concentration rose, showing a slight

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increase at the beginning and a fast raise for the last harvesting date. Ethanol

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production during fruit ripening was affected by the cultivar too. Therefore ethanol

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was accumulated in the olive fruit during its permanence on tree as a result of

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anaerobic respiration. Further works should be focused on the formation of ethanol

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from olive tree to virgin olive oil and how ethyl esters are synthetized.

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REFERENCES

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(1) European Commission. EU 1348/2013 of 16 December 2013 amending Regulation

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No 2568/91/EEC on the characteristics of olive oil and olive-residue oil and on the

227

relevant methods of analysis. Off. J. Europ. Comm. 2013, L338, 31-67.

228 229

(2) International Olive Oil Council. Trade standard applying to olive oils and olivepomace oils. COI 2013/T15/NC No 3/Rev. 17.

230

(3) Pérez-Camino, M. C.; Cert, A.; Romero-Segura, A.; Cert-Trujillo, R.; Moreda, W. Alkyl

231

esters of fatty acids a useful tool to detect soft deodorized olive oils. J. Agric. Food

232

Chem. 2008, 56, 6740–6744.

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(4) Biedermann, M.; Bongartz, A.; Mariani, C.; Grob, K. Fatty acid methyl and ethyl

234

esters as well as wax esters for evaluating the quality of olive oils. Eur. Food Res.

235

Technol. 2008, 228, 65-74.

236 237

(5) Mariani, C.; Bellan, G. On the possible increase of the alkyl esters in extra virgin olive oil. Riv. Ital. Sost. Grasse 2011, 88, 3-10.

238

(6) Conte, L.; Mariani, C.; Gallina Toschi, T.; Tagliabue, S. Alkyl esters and related

239

compounds in virgin olive oils: Their evolution over time. Riv. Ital. Sost. Grasse 2014,

240

91, 21-29

241 242 243 244

(7) Bruemmer, J.H.; Roe, B. Pyruvate dehydrogenase activity during ripening of ‘Hamlin’ oranges. Phytochemistry 1985, 24, 2105-2106. (8) Chervin, C.; Truett, J.K. Alcohol deshydrogenase expression and alcohol production during pear ripening. J. Am. Soc. Hortic. Sci. 1999, 124, 71-75.

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(9) Pesis, E. The role of the anaerobic metabolites, acetaldehyde and ethanol, in fruit

246

ripening, enhancement of fruit quality and fruit deterioration. Postharvest Biol.

247

Tech. 2005, 37, 1-19.

248 249

(10) Davis, P.I. Relation of ethanol content of citrus fruits to maturity and storage conditions. Proc. Fla. State Hort. Soc. 1970, 84, 217-222.

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(11) Pesis, E.; Dvir, O.; Feygenberg, O.; Ben-Arie, R.; Ackerman, M.; Lichter, A.

251

Production of acetaldehyde and ethanol during maturation and modified

252

atmosphere storage of Litchi fruit. Postharvest Biol. Tech. 2002, 14, 99-106.

253

(12) Lavilla, T., Recasens, I.; Lopez, M.L. Production of volatile aromatic compounds in

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Big Top nectarines and Royal Glory peaches during maturity. Acta Hortic. 2001, 553,

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233-234.

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(13) Nichols, W. C.; Patterson, M.E. Ethanol accumulation and poststorage quality of

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‘Delicious’ apples during short term, low O2, Ca storage. Hortscience 1987,33, 992-

258

994.

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(14) Barranco, D.; Rallo, L. Epocas de floracion y maduracion. In Variedades de Olivo en

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España; Rallo, L., Barranco, D., Caballero, J.M., del Rio, C., Martin, A., Trujillo, I.,

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Eds.; Junta de Andalucia-MAPA-Ediciones MundiPrensa: Madrid, Spain, 2005; pp.

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281-292.

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(15) Beltran, G.; Uceda, M.; Hermoso, M.; Frias, L. Maduracion. In El cultivo del olivo;

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Barranco, D., Fernandez-Escobar, R., Rallo, L., Eds.; Junta de Andalucia- Ediciones

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MundiPrensa, Madrid, Spain, 2004; pp. 158–183.

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(16) Sanchez-Ortiz, A.; Romero-Segura, C.; Gazda, V.E.; Graham, I.A.; Sanz, C.; Perez,

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A.G. Factors limiting the synthesis of virgin olive oil volatile esters. J. Agric. Food

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Chem. 2012, 60, 1300-1307.

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(17) Salas, J.J.; Sanchez, J. Alcohol dehydrogenases from olive (Olea europaea) fruit. Phytochem. 1998, 48, 35-40.

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(18) Iara, D.L.; Bruno, L.; Macchione, B.; Tagarelli, A.; Sindona, G.; Giannino, D.; Bitonti,

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M.B.; Chiapetta, A. The aroma biogenesis-related Olea europaea Alcohol

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Dehydrogenase gene is developmentally regulated in the fruits of two O. europaea

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L. cultivars. Food Res. Int. 2012, 49, 720-727.

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(19) Zerbini, P.E.; Giudetti, G.; Rizzolo, A.; Grassi, M. Harvest and quality indexes of peach. Inf. Agrar. 2001, 57, 57-60.

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Note

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This work was supported by the projects FEDER-INIA RTA 2010-00013-C02-01 from

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Spanish government and FEDER P10-AGR6099 from Junta de Andalucia.

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FIGURE CAPTIONS

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Figure 1. Changes during the ripening process in ethanol concentration of fruits from

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the olive cultivars: ‘Picual’, Hojiblanca’ and ‘Arbequina’.

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Figure 2. Changes during the ripening process in the ratio Acetaldehyde/Ethanol of

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fruits from the olive cultivars: ‘Picual’, Hojiblanca’ and ‘Arbequina’.

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Table 1. Sampling Dates of Olive Fruits from ‘Picual’, ‘Hojiblanca’ and ‘Arbequina’ Olive Cultivars.

Olive cultivar ‘Picual’ ‘Hojiblanca’ ‘Arbequina’

11 Sept (14 WAF)

0

a

16 Oct (21 WAF) 0 0 0.3

24 Oct (22 WAF)

1.0

a

13 Nov (26 WAF) 1.8 1.5 2.0

a

20 Dec b (30 WAF ) 2.4 2.2 2.4

a

Harvesting dates common for the three olive cultivars b WAF: Weeks after flowering

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Table 2. Partial Mean Squares from Analysis of Variance for the Effect of Cultivar and Harvesting Date on Fruit Ethanol Content from the Olive Cultivars: ‘Picual’, ‘Hojiblanca’ and ‘Arbequina’.

Source Cultivar Harvesting date(HD) Cultivar*HD Error Total

DFa 2 2 4 9 17

MSb 942.828 938.539 353.892 0.805

SSToc 36.36 36.20 27.30 0.14

P 0.0000 0.0000 0.0000

a

Degrees of freedom Mean squares c Partial mean squares for the effect, expressed as percentage, of the total corrected sum of the squares. b

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Table 3. Mean Ethanol Content of Olive Fruits from ‘Picual’, ‘Hojiblanca’ and ‘Arbequina’ Olive Cultivars.

Olive cultivar ‘Picual’ ‘Hojiblanca’ ‘Arbequina’ a

Ethanol (mg/kg) 3.4 ± 1.7 ac 26.1 ± 10.2 a 5.7 ± 1.9 b

Different letters indicate significant differences between means for p value of 0.05

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Figure 1.

60

55

50

Ethanol (mg/kg)

20

15 11-Sept 16-Oct

10

24-Oct 13-Nov 5

20-Dec

0 'Picual'

'Arbequina'

'Hojiblanca'

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Figure 2.

Acetaldehyde/Ethanol

1.0 0.8 11-Sept 0.6

16-Oct 24-Oct

0.4

13-Nov 20-Dec

0.2 0.0 'Picual'

'Arbequina'

'Hojiblanca'

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