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Postharvest Biology and Technology 94 (2014) 58–65

Contents lists available at ScienceDirect

Postharvest Biology and Technology

journa l h om epa ge : www.elsev ier .com/ locate /postharvbio

evelopment of flavor-related metabolites in cherimoya (Annonaherimola Mill.) fruit and their relationship with ripening physiology

aniel A. Manríqueza,∗, Pablo Munoz-Robredob, Orianne Gudenschwagerb,aula Robledob, Bruno G. Defilippib

AgroFresh, South Cone, Avenida Américo Vespucio 100, piso 6, Santiago, ChileUnidad de Postcosecha, Instituto de Investigaciones Agropecuarias (INIA-La Platina), Casilla 439/3, Santiago, Chile

r t i c l e i n f o

rticle history:eceived 3 December 2013ccepted 6 March 2014

eywords:AT

a b s t r a c t

Flavor is one of the most important attributes of fresh fruit for the consumer, and is affected by severalfactors, including genotype, maturity stage, and environmental conditions. Flavor-related metaboliteswere characterized in two important cherimoya varieties, cv. Concha Lisa and cv. Bronceada, during fruitripening. The most important sugars present were glucose, fructose and sucrose, and only fructose andglucose increased during ripening. The most important acids were tartaric, malic and citric acids, and all

sterslavorruit quality

increased as ripening progressed. Overall aroma profile was mainly determined by esters and terpenes inboth varieties. Ester compounds such as ethyl hexanoate, butyl butyrate and hexyl propanoate increasedduring ripening. The activity of alcohol acyl transferase also increased during fruit ripening concomitantwith ester accumulation. Terpenes, such as � and �-pinene, showed a reduction during ripening, whereasothers, such as myrcene and limonene, increased.

© 2014 Elsevier B.V. All rights reserved.

. Introduction

The cherimoya (Annona cherimola Mill.) is a subtropical fruit ofhe genus Annona. The most important cherimoya varieties grownn Chile are ‘Concha Lisa’ and ‘Bronceada’. In the cherimoya, as inther Annona species, flavor, defined primarily by aroma, sweetnessnd sourness, is the most important attribute at the consumer levelDíaz, 1991; Palma et al., 1993; Gardiazabal and Cano, 1999).

As in other climacteric fruit, many of the changes related to qual-ty occur during fruit ripening. Ethylene plays an important role inhe coordination of many of these changes. In the cherimoya, respi-ation exhibits a particular pattern during ripening, described by aouble-sigmoid curve. The first increase occurs soon after harvestnd is followed by a plateau, which in turn is followed by a secondeak. Both the respiration rate and ethylene production are veryigh during ripening, with ethylene production increasing at theeginning of the plateau observed for respiration (Paull et al., 1983;ills et al., 1984; Martínez et al., 1993; Palma et al., 1993; Alique

nd Oliveira, 1994; Gutiérrez et al., 1994). Many of the changeselated to organoleptic quality, such as softening, sugar and acidncreases and aroma biosynthesis, appear to begin after the first

∗ Corresponding author. Tel.: +56 2 4404831; fax: +56 2 4404831.E-mail address: manriquez@agrofresh.com (D.A. Manríquez).

ttp://dx.doi.org/10.1016/j.postharvbio.2014.03.004925-5214/© 2014 Elsevier B.V. All rights reserved.

respiratory peak, earlier than the increase in ethylene production.Sugars and acids influence flavor properties of cherimoya, impart-ing sweetness and sourness, respectively. Malic acid has been foundto be the predominant acid, with tartaric and citric acids present atlower concentrations. The development of sweetness is associatedwith an increase in the content of soluble solids. This increase isrelated primarily to an increase in sugars, which results from theconversion of starch into simple sugars such as fructose, glucoseand sucrose.

Aroma is one of the most important attributes at the consumerlevel in many fruit including cherimoya. Esters, alcohols and ter-penes are the most important compounds in the aroma profile forAnnona species (Idstein et al., 1984; Wyllie et al., 1987; Iwaoka andZhang, 1993; Ferreira et al., 2009; Pino et al., 2003). The role of eth-ylene in aroma development has previously been described in manyfruit (Defilippi et al., 2004; El-Sharkawy et al., 2005; Manríquezet al., 2006). Regarding the synthesis of esters, ethylene can regu-late the activity of two key enzymes involved in volatile production,alcohol dehydrogenase (ADH) and alcohol acyltransferase (AAT).AAT regulates the last step in ester biosynthesis (Defilippi et al.,2004; El-Sharkawy et al., 2005; Manríquez et al., 2006).

This work contributes to a better understanding of the relation-ships among ethylene, the respiratory pattern and the changes inflavor-related metabolites during ripening in two varieties of che-rimoya grown in Chile. Within volatile production, new insights

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bout the relationship between ester biosynthesis and AAT enzymectivity in both varieties are provided.

. Materials and methods

.1. Plant material and ripening conditions

The cherimoya (Annona cherimola Mill.) varieties ‘Concha Lisa’nd ‘Bronceada’ were harvested at maturity from a commercialrchard located in the Valparaiso region, Chile. Immediately afterarvest, the fruit were transported to the Postharvest Laboratory athe Institute for Agricultural Research (INIA-La Platina) in Santiago,hile. The fruit were placed at 20 ◦C and 50% R.H. until they reachedhe ripe (ready-to-eat) stage and underwent senescence. Each day,

sample of fruit was collected to measure the respiration rate, eth-lene production, and quality parameters, as described below. Forhe assays of volatiles, sugars, organic acids and enzyme activity,ulp tissue was frozen with liquid nitrogen and stored at −80 ◦Crior to analysis.

.2. Maturity parameters

The flesh firmness of peeled whole fruit, expressed in terms ofewtons (N), was measured on opposite sides of the fruit with

penetrometer (Effegi, Milan, Italy). At harvest, a 4 mm plungeras used, and an 8 mm probe was used when the fruit approached

he ripe stage. The total soluble solids (TSS) were measured with aanual temperature-compensated refractometer (ATC-1E, Atago,

okyo, Japan) in a sample of the juice; the results were expressed as percentage (%). The titratable acidity (TA) was obtained throughhe titration of 10 mL of juice with 0.1 N NaOH until the organiccids were neutralized to pH 8.2–8.3. In this case, the results werexpressed as a percentage of malic acid equivalents.

.3. Ethylene production and respiration rate

Ethylene production and the respiration rate were measuredor intact fruit with a static system. On each sampling date, sevenruit were weighed and placed in 2.6 L airtight jars. The jars wereealed and kept at 20 ◦C for 30–60 min prior to measurement. Theoncentrations of carbon dioxide (mg CO2 kg−1 h−1) and ethylene�L C2H4 kg−1 h−1) in the jar headspace were then determinedsing a gas analyzer (PBI-Dansensor Checkmate 9900, Ringsted,enmark) and a gas chromatograph (Shimadzu 8A, Tokyo, Japan)quipped with a flame ionization detector (FID), respectively. TheC-FID was equipped with a Supelco 80/100 Porapak costumolumn (75 cm × 5 mm × 3 mm). The injector and detector temper-tures were 150 ◦C and 40 ◦C, respectively. The oven temperatureept at 40 ◦C, and the nitrogen was used as the carrier gas at8.08 kPa.

.4. Sugar and acid extraction and measurement

The samples to be analyzed for sugars and organic acids wererepared from a homogeneous sample of 60 g of tissue per fruit,nd six replicates per variety at each sampling time were consid-red for both metabolites. Sugars and organic acids were analyzedccording to the method of Pérez et al. (1997). Briefly, 10 g of tissueas homogenized in a fruit crusher (Polytron) with 25 mL of cold

5% ethanol for 3–5 min. The sample was centrifuged at 12,000 rpmor 20 min and vacuum-filtered through two layers of Whatman N◦

paper. The solution was brought up to a volume of 50 mL with

0% ethanol. An aliquot of 10 mL was then dried under a nitro-en stream at 50 ◦C. The residue was dissolved in 2 mL of 0.2 N2SO4 with 0.05% EDTA. The sample was loaded onto an activatedep-Pak C-18 cartridge, and the eluate was collected. The sample

y and Technology 94 (2014) 58–65 59

was washed thoroughly with an additional 4 mL of the solution.The eluate was filtered through a 0.45 �m filter and analyzed byHPLC. For quantification, calibration curves were designed based onstandards for each compound. Calibration curves for d-(−)-fructose(Sigma–Aldrich, USA), d-(+)-glucose and sucrose (Supelco Analyt-ical, USA) were used to quantify the sugars. Calibration curves forcitric acid, d-malic acid (Supelco Analytical, USA) and d-(−)-tartaricacid (Fluka Analytical, USA) were used to quantify the acids.

The sugars were analyzed using a chromatography system com-posed of an ELSD (Evaporative Light Scattering Detector) detector,a Sedex 60 lt ELSD (Sedere) and an LC-NET II/ADC interface (JASCO,Japan). The separation of the sugars was performed with a Kro-masil 100 5NH2 amino column (250 mm × 4.6 mm) (AkzoNobel,Bohus, Sweden) with a mobile phase of 77% acetonitrile and 23%HPLC-grade water, which had been degassed and ultrasonicated.The conditions during the analysis were held constant, with a flowrate of 1.8 mL min−1 for 12 min at 20 ◦C under a pressure of 13.2 kPa.The injection volume was 20 �L.

Organic acids were analyzed in a chromatography systemwith an L-4250A UV-VIS ultraviolet detector (Merck-Hitachi, USA)to measure the absorbance at 195 nm with a D-6000 interface(Merck-Hitachi, Tokyo, Japan). The separation of the acids was per-formed using a Symmetry C-18 column (4.6 mm × 250 mm, 5.0 �m)(Waters, Ireland). The data were analyzed with D-7000 HSM soft-ware. The mobile phase used was 0.0085 N H2SO4 that had beendegassed and ultrasonicated. The conditions during the analysiswere held constant, with a flow rate of 0.4 mL min−1 for 24 min at20 ◦C at an average pressure of 8.4 kPa. The injection volume was20 �L.

2.5. Volatile extraction and quantification

Different volatile compounds present in the fruit pulp were ana-lyzed based on a sample of 8 g of pulp frozen in liquid nitrogen andhomogenized in 16 mL of 2 mM sodium fluoride with a homog-enizer (Ultra-Turrax, Staufen, Germany). The homogenized tissuewas filtered through four layers of cheesecloth and centrifuged at20,000 × g for 20 min at 4 ◦C. A total of 10 mL of supernatant wasfiltered (Whatman paper N◦ 2); 9.8 mL of filtrate was collected, and0.2 mL of 1-octanol was used as an internal standard (1 �L mL−1).The volatile compounds were extracted with 10 mL of pentane andvortexed for 1.5 min, and the pentanolic phase was concentrated.

The quantification of the compounds was performed with gaschromatography (GC); 1 �L of the concentrated pentanolic phasewas injected into the GC equipped with a Clarus 500 flame ion-ization detector (FID) (Perkin Elmer, Shelton, USA). The GC-FIDwas equipped with a Supelco SPB-5 cross-linked polyethylene gly-col column (30 m × 0.25 mm × 0.25 �m). The injector and detectortemperatures were 250 ◦C. The oven temperature was programmedto increase from 40 ◦C for 1 min to 60 ◦C for 1 min at a rate of2 ◦C min−1 and finally to 190 ◦C for 5 min at a rate of 10 ◦C min−1.Nitrogen was used as the carrier gas at 100 kPa.

The compounds were identified by comparing the reten-tion times with those of authentic standards. Quantification wasperformed using calibration curves that were made by adding stan-dards from each of the quantified volatiles to water, and using theratios of standard and aroma volatiles to the internal standard (1-octanol) for the calculations.

2.6. Alcohol acyltransferase (AAT) activity of cherimoya fruitcrude protein extract

The total protein was extracted from cherimoya pulp cv. Bron-ceada using the method described by El-Sharkawy et al. (2005)with modifications. Five grams of mesocarp tissue with 1 mL ofextraction buffer (250 mM Tris/HCl, pH 7.5, 1 mM DTT) was ground

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echanically in liquid nitrogen. The protein extract was thawed ince and centrifuged at 45,000 × g for 20 min at 4 ◦C. The supernatanthase was desalted with SephadexTM G-25 columns (Amershamiosciences, Sweden) and eluted with buffer (Tris–HCl 50 mM, pH.5, 10% (v/v) glycerol, 1 mM DTT). Total proteins were quanti-ed according to the method of Bradford (Bradford, 1976). AATctivity was assayed in a 500 �L total volume containing 300 �Lf the soluble fraction of the protein extract, 2 mM alcohol (ethanolr 1-butanol) and 250 �M acyl-CoA. The sample was brought upo the final volume with elution buffer (Tris–HCl 50 mM, pH 7.5,0% (v/v) glycerol, 1 mM DTT). The mixture was incubated at 30 ◦Cor 30 min. Immediately after the reaction, 20 �L of the internaltandard (1-octanol 0.5 �L L−1) was added to the mixture. Theolatile compounds produced by the reaction were extracted with00 �L of pentane and vortexed for 1.5 min. The pentanolic phaseas concentrated, and the volatile quantification was performed

s described above.

.7. Data analysis

Each fruit was considered an experimental unit. For maturity

nd physiological parameters seven fruit were analyzed per dayuring ripening at 20 ◦C. For sugars, organic acids, and enzymectivity 6 replicates were considered. Differences between ana-yzed parameters were statistically evaluated using an analysis of

ig. 1. Respiration rate, ethylene production and firmness during fruit ripening at 20 ◦C fohow standard deviations of the mean. Arrow indicates beginning of the ready to eat stag

y and Technology 94 (2014) 58–65

variance (ANOVA) and the mean comparisons between varietieswere determined by Student’s t-test at p < 0.05 using JMP 10 (SASInstitute Inc., Duxbury, USA) statistical program (Maalekuu et al.,2006).

3. Results

3.1. Ethylene production and respiration rate

The ‘Concha Lisa’ and ‘Bronceada’ fruit showed an increasein ethylene production during ripening and higher rates wereobserved for both varieties when the fruit reaching a ready-to-eatstage. The respiration rate in both varieties followed a double-sigmoid curve. The first peak occurred immediately after exposureto 20 ◦C in ‘Concha Lisa’ and after 3 days in ‘Bronceada’. A plateauin the respiration rate was observed after this first increase. Theplateau was longer in ‘Concha Lisa’ than in ‘Bronceada’. The lastphase of the curve was characterized by a second increase, whichcoincided with the maximum level of ethylene production in ‘Con-cha Lisa’ (Fig. 1A).

3.2. Ripening and quality parameters

Pulp firmness was very high at harvest, reaching values of 131 Nand 125 N in ‘Concha Lisa’ and ‘Bronceada’ fruit, respectively. Later,

r cherimoya cv. Concha Lisa (A) and cv. Bronceada (B). Bars at each sampling pointe.

D.A. Manríquez et al. / Postharvest Biology and Technology 94 (2014) 58–65 61

F cv. Brt

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ig. 2. Soluble solids and sugar changes during ripening of cv. Concha Lisa (A) andhe mean.

rapid softening was observed in both varieties after 1 day at 20 ◦C.his change coincided with an increase in ethylene production inoth varieties (Fig. 1A and B). As expected, increases in the TSS andA were observed during ripening (Figs. 2 and 3). The ready-to-at stage was mainly determined when the fruit reached a pulbrmness value below 10 N, i.e. after 3 and 4 days for ‘Concha Lisa’nd ‘Bronceada’, respectively. Similarly, the TA at the ready-to-eattage was three times greater than at harvest. These increases inSS and TA levels were observed during the entire process of fruitipening. The maximum values were reached after 5 and 4 days inoncha Lisa’ and ‘Bronceada’, respectively (Figs. 2 and 3).

Fructose and glucose were the main sugars identified (Fig. 2).hese sugars showed the highest concentrations during ripeningnd followed a pattern similar to that observed for the TSS. The con-entration of sucrose was higher during the first days of ripeningnd then decreased after 2 and 3 days at 20 ◦C for ‘Concha Lisa’ and

Bronceada’, respectively. This decrease was greater in the ‘Conchaisa’ fruit (Fig. 2). The analysis of the acids showed that malic and

itric acids were predominant, and their concentrations increaseduring ripening. In ‘Concha Lisa’, malic acid was the predominantcid during the first 3 days at 20 ◦C, whereas citric acid was pre-ominant during the final stage of ripening. In contrast, citric acid

onceada cherimoyas (B). Bars at each sampling point show standard deviations of

had the highest concentration in ‘Bronceada’ fruit throughout theripening period. Tartaric acid was present in both varieties, butits concentration was very low from harvest to the last stages ofripening (Fig. 3).

3.3. Changes in volatiles

The volatile profile in both varieties was dominated by esters,terpenes and alcohols. These groups of compounds representedbetween 78% and 83% of the volatiles occurring at the ready-to-eatstage in ‘Concha Lisa’ and ‘Bronceada’ fruit, respectively (data notshown). Based on odor activity values (OAV) reported in the liter-ature, i.e. the ratio of the concentration and odor threshold of eachindividual compound, the compounds with the highest OAV occur-ring in ripe fruit were the esters ethyl octanoate, ethyl hexanoate,ethyl butyrte, butyl butyrate, hexyl butyrate, hexyl propanoate,methyl decanoate, ethyl heptanoate and isobutyl isobutyrate, andthe terpenes �-pinene, linalool, myrcene, �-pinene, limonene and

caryophyllene. In Table 1 we provide the odor thresholds for thevolatiles considered in this study (Guth, 1997; Ferreira et al., 2000;Culleré et al., 2004; Pet‘ka et al., 2006; Gomez-Miguez et al., 2007;Loscos et al., 2007). The concentrations of these compounds and

62 D.A. Manríquez et al. / Postharvest Biology and Technology 94 (2014) 58–65

Fig. 3. Titratable acidity and acid changes during ripening in cv. Concha Lisa (A) and cv. Bronceada cherimoyas (B). Bars at each sampling point show standard deviations ofthe mean.

Table 1Changes in volatiles (�mol kg−1) during the ripening of cv. Concha Lisa cherimoyas.

Odor threshold (mg L−1) Harvest Days at 20 ◦C

1 2 3 4 5

Ethyl butyrate 20a 0.066bb 0.070ab 0.131ab 0.106ab 0.217ab 0.318aEthyl hexanoate 14 0.016a 0.045a 0.075a 0.055a 0.167a 0.396aEthyl heptanoate 2.2 0.099b 0.161b 0.094b 0.091b 0.124b 6.236aEthyl octanoate 2.0 1.819a 1.648ab 0.727c 1.884a 1.373abc 0.712bcButyl butyrate 100 0.326b 0.269b 0.249b 0.278b 0.408b 2.231aHexyl butyrate 250 0.304b 0.334b 0.394b 0.311b 0.451b 2.915aHexyl propanoate 8 0.196b 0.281b 0.425b 0.815a 0.222b 0.129bMethyl decanoate 2 3.010b 3.259b 9.493a 2.525b 2.188b 2.686bLinalool 6 9.016a 6.703a 2.424a 1.800b 2.187b 1.552b�-Pinene 6 0.236a 0.185ab 0.041c 0.072bc 0.025c N.D.�-Pinene 140 0.134a 0.040b 0.007b 0.012b 0.011b 0.014bMyrcene 14 0.074ab 0.063b 0.049b 0.084b 0.110b 0.151aLimonene 10 0.296ab 0.232ab 0.137b 0.701ab 0.471ab 2.049aCaryophyllene 64 0.367ab 0.670a 0.264b 0.385ab 0.328ab

a Odor thresholds from literature (Guth, 1997; Ferreira et al., 2000; Culleré et al., 2004; Pet‘ka et al., 2006; Gomez-Miguez et al., 2007; Loscos et al., 2007).b Values are means of six replicates. Means within the same compound followed by different letters are significantly different (ANOVA, Student’ t-test, p < 0.05).

D.A. Manríquez et al. / Postharvest Biology and Technology 94 (2014) 58–65 63

Table 2Changes in volatiles (�mol kg−1) during the ripening of cv. Bronceada cherimoyas.

Odor threshold (�g L−1) Harvest Days at 20 ◦C

1 2 3 4 5 6

Ethyl butyrate 20a N.D.b 0.055 N.D. N.D. N.D. N.D. N.D.Ethyl hexanoate 14 0.143cb 0.032b N.D. 0.050b 0.130b 0.740b 2.426aEthyl heptanoate 2.2 0.107ab 0.049b 0.026b 0.132ab 0.177ab 0.124ab 0.327aEthyl octanoate 2.0 1.377a 0.908a 1.411a 1.070a 1.035a 1.181a 2.220aButyl butyrate 100 0.297b 0.329b 0.218b 0.250b 0.246b 4.153b 15.771aHexyl butyrate 250 0.350b 0.379b 0.382b 0.383b 0.369b 0.779b 2.004aHexyl propanoate 8 0.169c 0.594bc 0.706abc 1.141ab 1.564a 1.323ab 0.852abcMethyl decanoate 2 4.232a 3.640a 4.433a 3.860a 3.402a 3.455a 5.501aLinalool 6 2.537b 4.804a 1.924bc 1.057cd 1.067cd 0.590d 0.887cd�-Pinene 6 0.091b 0.095a N.D. N.D. N.D. N.D. N.D.�-Pinene 140 0.028b 0.078a N.D. N.D. N.D. N.D. N.D.Myrcene 14 0.123a 0.054a N.D. 0.557a 0.042a 0.129a 0.074aLimonene 10 0.285b 0.742b 2.013b 0.576b 0.419b 0.448b 0.588bCaryophyllene 64 0.457a 0.386a 0.476a 0.542a 0.361a 0.408a 0.759a

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ences in ovarian development and maturity, as described for A.muricata (Biale et al., 1965; Broughton and Tan, 1979; Reginato,1980; Paull, 1982; Bruinsma and Paull, 1984; Brown et al., 1988;

a Odor thresholds from literature (Guth, 1997; Ferreira et al., 2000; Culleré et al.,b Non-detected.c Values are means of six replicates. Means within the same compound followed

heir change during ripening were different for each compoundnd variety. However, a similar pattern was observed for certainompounds in both varieties, although the actual concentrationsiffered (Tables 1 and 2). Esters such as ethyl hexanoate and ethylutyrate showed an increase in their concentrations during ripen-

ng (Tables 1 and 2). This increase coincided with the increasen ethylene production that occurred after the plateau in respira-ion in both varieties. Other compounds, such as butyl butyrate,howed an increase in concentration at the end of ripening whenhe fruit were soft and reached the ready-to-eat stage and the res-iration rates were the highest. Another pattern observed was inexyl propanoate, whose concentration increased after 3 and 4ays at 20 ◦C in ‘Concha Lisa’ and ‘Bronceada’, respectively. Somether esters, such as ethyl octanoate and methyl decanoate, wereresent during the entire ripening process. Their concentrationsid not show a clear pattern of change (Tables 1 and 2). Amongerpenes, linalool and �- and �-pinene concentrations decreaseduring ripening (Tables 1 and 2). In the ‘Concha Lisa’ fruit, theoncentrations of myrcene and limonene increased after 5 dayst 20 ◦C, when the fruit were at the ready-to-eat stage. However,hese compounds showed a peak in their concentrations after 2–3ays at 20 ◦C in the’ Bronceada’ fruit (Tables 1 and 2). The increase

n the concentration of caryophyllene during ripening also differedetween the varieties. The maximum concentration in ‘Concha Lisa’ccurred after 1 day at 20 ◦C, whereas in ‘Bronceada’ caryophyl-ene concentration reached a maximum at the ready-to-eat stageTables 1 and 2).

.4. Alcohol acyltransferase activity

To evaluate AAT activity, a combination of two pairs of sub-trates was assayed in the crude protein extract from pulp extractedrom cherimoyas cv. Bronceada in different ripening stages. In bothairs of substrates, an increase in AAT activity was observed dur-

ng ripening (Fig. 4). This activity was highest at the later stagesf ripening. The increase in AAT activity began after 2 or 3 days at0 ◦C and coincided with the increase observed in ethylene pro-uction during the plateau in respiration rate and when the rate ofoftening was higher (Figs. 1 and 4).

. Discussion

The high rate of respiration and the double-sigmoid patternbserved during ripening in both varieties has been described forther cherimoya varieties. These characteristics are also known in

Pet‘ka et al., 2006; Gomez-Miguez et al., 2007; Loscos et al., 2007).

ferent letters are significantly different (ANOVA, Student’s t-test, p < 0.05).

other species of the genus Annona, such as Annona muricata andAnnona squamosa. This particular pattern could be related to theseparation of the fruit from the plant at harvest and/or to differ-

Fig. 4. Alcohol acyl-transferase activity of crude protein extracts of cherimoyacv. Bronceada during ripening. (A) Measured acyl-transferase activity for ethanoland hexanoyl-CoA and (B) measured acyl-transferase activity for 1-butanol andbutanoyl-CoA.

6 Biolog

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gram quantities of protein using the principle of protein–dye binding. Anal.Biochem. 72, 248–254.

4 D.A. Manríquez et al. / Postharvest

ahoz et al., 1993; Martínez et al., 1993; Palma et al., 1993). The ratef ethylene production was high, and a single peak was observeduring ripening. In both varieties, an increase in ethylene produc-ion coincided with the plateau observed in respiration (Reginato,980; Paull, 1982; Bruinsma and Paull, 1984; Brown et al., 1988;ahoz et al., 1993; Martínez et al., 1993; Palma et al., 1993). Asn other climacteric fruit, such as tomato (Lycopersicon esculen-um) and melon (Cucumis melo), ethylene appears to be involvedn the coordination of particular processes during fruit ripeningGiovannoni, 2001; Alexander and Grierson, 2002; Pech et al., 2004,008). In both varieties, a high rate of softening was observed dur-

ng the initial days at 20 ◦C. This high softening rate coincidedith the increase in ethylene production. The high rate of soft-

ning could be related to the regulation by ethylene of the genesnvolved in cell wall disassembly (Alique and Oliveira, 1994; Lahozt al., 1993; Alique and Zamorano, 2000; Li et al., 2009). Espinoza2005) showed that the ethylene antagonist 1-methylcyclopropene1-MCP) reduced endogenous ethylene production and delayedoftening in ‘Concha Lisa’ cherimoyas both during ripening in coldtorage and during the shelf-life of the fruit.

The increase in TSS observed in both cultivars could be aesult of the high level of starch present in the fruit at harvest.he hydrolysis of the starch during cold storage is the principalource of sugars. This process increases the TSS concentration inhe fruit, a phenomenon also observed in other climacteric fruit,uch as apples (Malus × domestica) and kiwifruit (Actinia deliciosa)Reginato, 1980; Knee, 1993; Lahoz et al., 1993; Martínez et al.,993; Mitchell, 1994; Goni et al., 2008). In apples and kiwifruit,tarch hydrolysis has been shown to be ethylene independent. Thencrease in TSS from the beginning of the ripening process indicateshat the process of starch hydrolysis in cherimoya is also ethylenendependent (Lahoz et al., 1993; Martínez et al., 1993; Manríquezt al., 2006; Watkins, 2006). Glucose and fructose, the most impor-ant sugars in terms of concentration, showed an increase duringipening. This pattern was similar to that found for the concentra-ion of TSS. A decrease in sucrose was observed after 3 days at roomemperature in both varieties, as described for other Annona speciesAlique and Oliveira, 1994; Alique and Zamorano, 2000). In contrasto the pattern found in other climacteric fruit, an increase in TAuring ripening was observed in both cherimoya varieties duringruit ripening. This increase reached a peak at the ready-to-eat stageReginato, 1980; Wills et al., 1984; Martínez et al., 1993; Alique andliveira, 1994; Alique and Zamorano, 2000). Malic and citric acidsere the most abundant organic acids in the fruit during ripen-

ng. Citric acid was the most important in terms of concentrationn both varieties. The changes in its concentration during ripeningould be regulated by signals other than ethylene, as described forpples (Lau et al., 1986; Blankenship and Sisler, 1989; Fan et al.,999).

Esters and terpenes were two important groups that contributedo the aroma profile in both varieties. Esters are an important groupf compounds in the aroma profile of other cherimoya varietiesIdstein et al., 1984; Iwaoka and Zhang, 1993; Ferreira et al., 2009).he increase in concentration observed for many of the estersresent in both varieties began during the plateau in the respirationate, at the same time as ethylene production, and also occurredhen the fruit began to soften. The highest concentration occurred

t the ready-to-eat stage. The activity of AAT, a key enzyme in esteriosynthesis, showed an increase during fruit ripening similar tohat of many of the esters. Similar studies in other cherimoya vari-ties and in other Annona species found that the development ofroma in the fruit begins when the plateau in respiration occurs

nd when softening of the fruit is evident (Wills et al., 1984; Palmat al., 1993). Ethylene production increased at this point in the ‘Con-ha Lisa’ and ‘Bronceada’ fruit. The biosynthesis of esters and theole of ethylene as a coordinator of this process have been studied

y and Technology 94 (2014) 58–65

and described for many climacteric fruit, including apples, melonsand bananas (Musa spp.) (Golding et al., 1999; Fellman et al., 2000;Defilippi et al., 2005; El-Sharkawy et al., 2005). The biosynthesis ofesters in cherimoya is also modulated by ethylene. Ethylene worksat the molecular and enzymatic level to modulate the expressionof particular genes coding for AAT and ADH (Flores et al., 2002;Defilippi et al., 2005, 2009; El-Sharkawy et al., 2005; Manríquezet al., 2006). However, the lack of an increase associated with ethyl-ene production during ripening in certain compounds could resultfrom the coordination of their synthesis by other factors. In thestrawberry (Fragaria × ananassa), a non-climacteric fruit, ethylenedoes not play a key role in the coordination of ripening, and cer-tain AATs have been described as ethylene independent (Pérezet al., 1996; Aharoni et al., 2000). In this study, changes in ter-penes were observed to follow different patterns of increase duringfruit ripening. However, the role of ethylene in modulating differ-ent pathways of terpene synthesis is still not clear in climactericand non-climacteric fruit (Herianus et al., 2003; Ninio et al., 2003;Sharon-Asa et al., 2003; Sitrit et al., 2004; Harb et al., 2008).

5. Conclusions

In the cherimoya, certain processes appear to be regulated byethylene during the ripening of the fruit. These processes includesoftening and portions of the biosynthesis of the compoundsresponsible for the aroma of the fruit. However, other processesassociated with ripening appear to be regulated by other sig-nals. The plateau observed in the respiration rate during ripeningappears to be an important period of fruit ripening because thisplateau is associated with many of the changes associated with fla-vor, including softening, an increase in acidity, starch degradationand aroma biosynthesis. To understand the processes associatedwith flavor, it will be necessary to achieve a better understandingof the role of ethylene as a coordinator of aroma biosynthesis incherimoya fruit.

Acknowledgments

This work was funded by Fondecyt Grant N◦ 11090098. Wethank Pontificia Universidad Católica de Valparaiso, ResearchStation La Palma, for providing the fruit for the trials. We thankProf. Dr. Jean Claude Pech and Dr. Alain Latché from INP-ENSATFrance for the critical review of this paper.

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