Ethanol in Olive Fruit. Changes during Ripening - Journal of

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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]

ACS Paragon Plus Environment

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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

6

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

7

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

8

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

13

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

18 19 20 21 22 23 24 25 26 27 28 29 30 31 32


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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

38

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

138

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

139

time against standard. Quantification was performed using individual calibration

140

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

141

kilogram of fruit.

142 143

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

148

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

152

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

153

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

157

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

159

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).

178 179

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.

206 207

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

210

was not achieved.

10, 19

although in our case over-maturation

211 212

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

226

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.

233

(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.

245

(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.

250

(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

254

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

255

233-234.


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

257

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

258

994.

259

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

260

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

261

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

262

281-292.

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

264

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,

267

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

268

Chem. 2012, 60, 1300-1307.

269 270

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

271

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

272

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

273

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

274

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

275 276

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

277 278

Note

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

280

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

281 282 283 284 285 286 287


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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’.

292 293 294

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

295

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

296 297 298 299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331


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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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For Table of Contents Only


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Ethanol in Olive Fruit. Changes during Ripening - Journal of

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