Dynamic controlled atmosphere storage suppresses metabolism and enhances volatile concentrations of ‘Galaxy’ apple harvested at three maturity stages

Postharvest Biology and Technology 127 (2017) 1–13

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Dynamic controlled atmosphere storage suppresses metabolism and enhances volatile concentrations of ‘Galaxy’ apple harvested at three maturity stages Fabio Rodrigo Thewesa,* , Auri Brackmanna , Rogerio de Oliveira Aneseb , Eduarda Spagnol Bronzattoa , Erani Eliseu Schultza , Roger Wagnerc a Department of Plant Science, Postharvest Research Center, Federal University of Santa Maria, Roraima Avenue 1000, Camobi, Santa Maria 97105-900, RS, Brazil b Federal Institute of Santa Catarina (IFSC), Campus Urupema, Senadinho Road, Center, Urupema, Santa Catarina, 88625-000, SC, Brazil c Department of Food Science and Technology, Federal University of Santa Maria, Roraima Avenue 1000, Camobi, Santa Maria 97105-900, RS, Brazil

A R T I C L E I N F O

Article history: Received 2 August 2016 Received in revised form 3 January 2017 Accepted 4 January 2017 Available online xxx Keywords: Malus domestica Respiratory quotient Aroma Anaerobic metabolism Ethylene Respiration rate

A B S T R A C T

The objective of this study was to assess the interaction between four storage conditions and three maturity stages of ‘Galaxy’ apple at harvest (unripe, ripe and overripe, based on starch pattern index) on its metabolism and volatile concentrations after harvest and 9 months of storage plus 7 days of shelf life at 20  C. Storage conditions were: [1] Controlled atmosphere (CA) (1.2 kPa O2 + 2.0 kPa CO2); [2] CA + 1methylcyclopropene (1-MCP) (0.625 mL L 1); [3] Dynamic controlled atmosphere based on respiratory quotient 1.3 (DCA-RQ 1.3) + 1.2 kPa CO2 and [4] DCA-RQ 1.5 + 1.2 kPa CO2. Fruit stored under DCA-RQ 1.5 had higher concentrations of total esters and characteristic aroma volatile compounds in extracted juice. Highest ester concentrations occurred in overripe fruit stored at DCA-RQ 1.3 and DCA-RQ 1.5. 1-MCP application suppressed volatile compound production, not allowing its increment with advanced of the maturity stage and reduced the main esters after storage. DCA-RQ suppressed internal ethylene concentration, ethylene production, and respiration rate, but the low metabolism in fruit stored under DCA-RQ 1.5 did not result in lower volatile compound production. © 2017 Elsevier B.V. All rights reserved.

1. Introduction Volatile compounds are critically important for apple acceptance by consumers (López et al., 2007). These compounds change in response to fruit maturity and ripening and to storage conditions (Bangerth et al., 2012). Among the volatile compounds produced by apples, esters have a major impact on aroma. Apples of the ‘Gala’ group, such as ‘Galaxy’, have high butyl acetate, 2-methylbutyl acetate and hexyl acetate concentrations, which are the most important esters of this cultivar group (Salazar and Orozco, 2011; Both et al., 2014). These compounds increase with advancing maturity, but are suppressed by storage, showing the importance to develop a technology that allows to harvest the fruit in different maturity stages without volatile compound reduction.

* Corresponding author. E-mail address: [email protected] (F.R. Thewes). http://dx.doi.org/10.1016/j.postharvbio.2017.01.002 0925-5214/© 2017 Elsevier B.V. All rights reserved.

Controlled atmosphere (CA), is a worldwide used storage technique for apple fruit. Nevertheless, CA storage, strongly reduces ester formation (Brackmann et al., 1993; Fellman et al., 2003; López et al., 2007; Raffo et al., 2009; Lumpkin et al., 2014, 2015). Low O2 partial pressure (pO2) employed during CA storage suppresses ethylene biosynthesis and action, which are important for activity of ester forming enzymes (Burg and Burg, 1965; Yang et al., 2016). Low pO2 may also suppress volatile compound precursor production via b-oxidation and lipoxygenase (LOX) pathway (Brackmann et al., 1993; Song and Bangerth, 2003), because both pathways require O2 (Echeverría et al., 2004). 1-MCP application is often used in several countries for extending the storage period of apples, due to its ethylene action blocking (Watkins, 2006; Lee et al., 2012) and delaying fruit ripening. 1-MCP treated apples maintain higher flesh firmness (Fawbush et al., 2009; Moggia et al., 2010; Brackmann et al., 2013), titratable acidity, soluble solids (Watkins, 2006) and have lower

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incidences of superficial scald (Lurie and Watkins, 2012) and mealiness (Brackmann et al., 2014), among the benefits on the fruit quality. However, 1-MCP can negatively impacts volatile biosynthesis, especially alcohols and esters, that contributes to the characteristic apple aroma (Lurie et al., 2002; Ferenczi et al., 2006; Lee et al., 2012; Thewes et al., 2015; Yang et al., 2016). Additionally, the 1-MCP application presents additional costs for the storage operators and is not allowed for the storage of organic apples. Therefore, there is a need to develop a storage technology that can reduce the fruit metabolism similarly to the 1-MCP application, without significant volatile compounds loss maintaining the apple characteristic aroma. The apple storage based on the lowest oxygen limit (LOL) tolerated by the fruit has become available for commercial use. Three methodologies to detect the LOL during storage in dynamic controlled atmosphere (DCA) are available: ethanol production by fruit (Storex1, Swinglos1, ILOS-Plus1 and Fruit control1) (Veltman et al., 2003), fruit chlorophyll fluorescence emission (HarvestWatchTM and Fruit Observer1)(Prange et al., 2007; Wright et al., 2010, 2012) and respiratory quotient (Advanced control respiration1, Storage Control Systems Safe Pods1 and RQ store fresh1) (Gasser et al., 2008; Wright et al., 2012; Brackmann, 2015; Weber et al., 2015). According to Raffo et al. (2009), the storage of ‘Pinova’ apple under DCA based on chlorophyll fluorescence (DCA-CF), resulted in reduced main esters in comparison with CA storage (1.5 kPa O2 + 1.3 kPa CO2), but higher ester concentrations when compared with CA + 1-MCP. Nevertheless, there is no literature evaluating the effect of DCA based on respiratory quotient (DCARQ) on the volatile profile of apples. This technology induces a small level of fermentation by the fruit (Brackmann et al., 2015; Weber et al., 2015), which supply ethanol to the volatile compounds biosynthesis, resulting in increased volatile compound biosynthesis in comparison to CA and CA + 1-MCP. In oriental sweet melons, the ethanol application increased the volatile compound production, like ethyl esters, butyl acetate and hexyl acetate (Liu et al., 2012). The objective of this study was to assess the interaction between four storage conditions and three maturity stages of the fruit (unripe, ripe and overripe) on the metabolism and volatile compound concentration of ‘Galaxy’ apples after harvest and 9 months of storage plus 7 days of shelf life at 20  C.

2. Material and methods 2.1. Plant material, orchard location, harvest maturity and sample preparation ‘Galaxy’ apples (Malus domestica), a ‘Royal Gala’ sport (Okie, 1999), were harvested in a commercial orchard located in the town of Vacaria, RS, Brazil. The ‘Galaxy’ apples were grafted on M9 rootstocks at a density of 3,575 plants ha 1. The following fertilization was carried out during the growing season: 80 kg ha 1 of nitrogen and 120 kg ha 1 of potassium. Fruit were harvested at three maturity stage. The starch pattern index (SPI) (Streif, 1984) was used to differentiate maturity stages in the laboratory: Unripe apples <3.5, ripe fruit index between 3.5– 7.0, and overripe >7.0. In each harvest, SPI were determined using 3 replicates of 20 fruit, resulting in averages of 3.4, 6.5 and 9.7 units, for unripe, ripe and overripe, respectively. Fruit were transported to the Postharvest Research Center of the Federal University of Santa Maria, RS, Brazil, on the day of harvest, where fruit with any damage were eliminated and fruit randomized. Three replication of 25 fruit were used for each treatment.

2.2. Storage conditions Fruit replications were placed into 233-L galvanized iron CA chambers in which the following treatments were applied: [1] controlled atmosphere – CA (1.2 kPa O2 + 2.0 kPa CO2); [2] CA + 1MCP (0.625 mL L 1); [3] DCA-RQ 1.3 + 1.2 kPa CO2 and [4] DCA-RQ 1.5 + 1.2 kPa CO2. The storage temperature was 1.5  0.1  C and it was monitored daily during the 9 months of storage with the aid of mercury thermometers inserted inside the fruit flesh to determine the pulp temperature. Inside the storage chamber, the relative humidity (RH), was monitored manually with psycrometers and controlled by the addition of calcium chloride, which absorbed the excess of humidity inside the chamber to maintain an average relative humidity at 94  2%. 2.3. 1-MCP treatment 1-MCP treatment of fruit was carried out in the chamber with 0.625 mL L 1 1-MCP (EthylBloc1, 0.14% of active ingredient). The fruit were treated during 24 h. The air inside the chamber was circulated with a fan. This process was carried out at the storage temperature of 1.5  0.1  C. 2.4. CA and DCA

RQ setup and maintenance

During the first day of storage, the temperature was reduced down to 5.0  C and thereafter gradually down to 1.5  C, a process that took 5 days. CA and DCA-RQ conditions were applied on day 5, when fruit temperatures had reached 1.5  C. The experimental chambers were hermetically closed and the CA and DCA-RQ conditions were applied. The chambers were flushed with nitrogen until the pO2 reached 1.2 kPa for CA, and to achieve the DCA-RQ conditions, O2 was reduced to 0.5 kPa. This process was carried out over 5 days. The pCO2 was obtained by its accumulation in the storage chamber due to fruit respiration. Therefore, the CA and DCA-RQ conditions were installed from days 5 to 10, to simulate the commercial CA and DCA storage establishment. The pO2 was changed according to the fruit metabolism throughout the DCA storage period, while it was maintained constant during CA storage. The RQ was measured two times a week, to measure the LOL during the storage period, according to the method proposed by Brackmann (2015). The RQ was set at 1.3 and 1.5, and pO2 changed accordingly to maintain this RQ level (Supplementary Fig. S1). A standard deviation of 0.09 was obtained for DCA – RQ 1.3 and of 0.07 for DCA – RQ 1.5. The RQ was calculated once the chamber had been closed for 13 h between the first and second reading. The RQ was calculated by the ratio between CO2 production and O2 uptake. CA conditions were maintained according to the method of Brackmann et al. (2014). 2.5. Internal ethylene concentration (IEC) The IEC was determined according to Mannapperuma et al. (1991) on 10 fruit per replicate. The internal gas of fruit was withdrawn, and two samples (1 mL) were injected into a gas chromatograph (Varian1, model Star 3400CX) equipped with a flame ionization detector (FID) and a Porapak N80/100 column. The temperatures of the column, the injector and the detector were 90, 140 and 200  C, respectively. Results were expressed in ug L 1. 2.6. Ethylene production and respiration rate To determine the ethylene and respiration rate, 10 to 12 fruit were stowed inside a 5-L flask and hermetically closed for about

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Table 1 Internal ethylene concentration (IEC; mg L 1), ethylene production (ng kg ‘Galaxy’ apple harvested at three maturity stages (before storage). Compounds

LRI

OT (mg kg

1

1

)

s

1

), respiration rate (mg kg

1

s

1

) and volatile concentration (mg L

1

) in juice obtained from

Maturity stages (Mean  Standard deviation) Unripe

Ripe

Overripe

Matabolism* IEC Ethylene production Respiration rate

– – –

– – –

0.57  0.19 b 0.34  0.12 b 5.39  1.02 c

2.26  0.61 a 0.60  0.15 a 7.86  0.15 a

2.27  0.15 a 0.59  0.18 a 6.72  1.03 b

Esters** Methyl acetate Ethyl acetateg Ethyl propanoate Ethyl isobutanoate Propyl acetate Methyl 2-methyl butanoate 2-Methylpropyl acetate Ethyl butanoate Ethyl 2-methyl butanoate Butyl acetate 2-Methyl butyl acetate Butyl Propanoate 3-Methyl butyl acetate 4-Pentenyl acetate Butyl butanoate Z-2-Pentenyl-acetate Hexyl acetate Z-2-Hexenyl acetate Z-3-Hexenyl acetate E-3-Hexenyl acetate 5-Hexenyl-acetate E-2-Hexenyl acetate Heptyl acetate Butyl hexanoate Benzyl acetate

839 897 962 968 983 1015 1018 1042 1057 1083 1128 1137 1168 1192 1202 1241 1262 1286 1290 1294 1316 1321 1364 1394 1726

8,300a 13,500a 40f NF 2,000a 8f 66c 1a 0.06a 66a 11a 25a 2d NF 100a NF 2a NF 8c NF NF 7c NF 700e 364c

0.05  0.01 b 0.51 0.08 b 0.23  0.24 a ND 0.95  0.53 b ND 2.34  0.45 b ND 43.62  22.14 b 91.12  21.01 c 20.06  8.89 c 0.14  0.10 b 35.05  15.60 b 0.25  0.28 a 1.86  1.41 a 0.35  0.19 b 40.75  7.33 c 3.39  3.43 ab 1.35  0.80 b 5.41  4.38 b 1.49  0.98 b 0.21  0.11 b 0.36  0.40 a 0.59  0.37 b 0.42  0.24 b

0.09  0.02 b 1.01 0.14 b 0.01  0.01 a 0.02  0.01 a 1.73  0.26 b 0.36  0.34 b 3.08  0.22 b 0.02  0.01 a 19.71  0.29 b 388.19  9.76 b 159.37  12.53 b 0.08  0.04 b 39.22  4.10 b 0.06  0.02 a 2.40  0.18 a 1.16  0.13 b 420.93  19.29 b 0.44  0.05 b 3.42  0.31 b 0.17  0.06 b 5.23  0.37 b 9.43  0.72 a 0.09  0.02 a 0.07  0.01 b 1.66  0.11 b

1.39  0.11 a 5.70  1.88 a ND ND 55.98  16.80 a 2.50  0.13 a 35.95  10.33 a ND 112.28  30.69 a 4,374.08  848.58 a 1,757.62  292.90 a 1.95  0.83 a 530.47  216.97 a ND 7.43  12.87 a 14.78  2.82 a 4,331.64  1422.8 a 6.27  0.18 a 13.12  2.37 a 53.98  12.34 a 105.05  24.37 a 11.78  3.78 a 1.84  3.18 a 5.95  4.46 a 9.16  4.13 a

Alcohols 2-Propanol Ethanolg 1-Butanolg 4-Methyl-2-pentanol 3-Hexanol 2-Methyl-1-butanol 1-Pentanol 2-Methyl-2-buten-1-ol 1-Hexanol E-3-Hexen-1-ol Z-3-Hexen-1-ol E-2-Hexen-1-ol E-5-Hexen-1-ol E-1-Octen-3-ol 1-Heptanol 6-Methyl-5-hepten-2-ol 2-Ethyl 1-hexanol 1-Octanol

936 945 1162 1176 1201 1211 1249 1297 1352 1361 1380 1399 1407 1445 1454 1465 1485 1554

NF 100,000d 500a NF NF 250a 4,000a 3c 500a NF 70b 400d NF NF 3d 2,000d 270,000d 130d

0.03  0.03 a 1.14  0.74 b 7.85  2.91 b 0.67  0.62 b 0.06  0.11 a ND ND 2.17  1.67 a 94.80  19.11 b 1.46  0.70 a ND 22.24  5.77 a 0.78  0.54 a 0.27  0.19 b 0.85  0.29 b 0.34  0.30 b 0.85  0.36 b 0.25  0.10 b

0.03  0.01 a 0.32  0.05 b 33.75  8.64 b 0.31  0.10 b 0.06  0.02 a 7.17  2.34 b 2.03  0.35 a ND 93.09  17.60 b 0.46  0.06 a 0.17  0.05 b 9.26  2.24 a 1.57  0.32 a 0.21  0.01 b 1.42  0.12 b 0.21  0.04 b 0.47  0.14 b 0.65  0.06 b

ND 9.81  1.35 a 311.42  52.25 a 12.72  4.53 a 3.80  3.28 a 211.14  73.32 a 0.39  0.67 b ND 1,112.85  531.68 a 15.49  15.92 a 7.10  2.44 a 28.79  18.80 a 10.74  9.39 a 3.19  0.51 a 23.57  7.92 a 5.68  1.68 a 6.06  1.11 a 2.44  0.85 a

Aldehydes Acetaldehydeg Butanal Hexanal Z-3-Hexenal Z-2-Hexenal E-2-Hexenal

644 890 1099 1148 1205 1222

120d 37d 5a NF NF 17b

0.44  0.07 b 0.38  0.05 b 1.25  0.27 b 0.97  0.23 b 0.14  0.12 b 163.73  8.28 a

0.08  0.01 b 0.22  0.05 b 0.08  0.02 b 3.08  0.40 b ND 75.08  8.52 a

5.42  1.59 a 5.01  1.67 a 3.58  1.53 a 51.63  8.50 a 28.66  8.91 a 146.59  82.03 a

Ketones 2-Propanone 6-Methyl-5-heptene-2-one

831 1334

500,000d 50b

0.74  0.29 b 0.50  0.27 b

0.20  0.05 b 0.02  0.001 b

6.10 2.75 a 5.46  0.85 a

*Means followed by equal letters in the line do not differ by Tukey’s test at 5% of error probability. Odor threshold. References: a López et al. (2007); b Mehinagic et al. (2006); c Pino and Quijano (2012); d Leffingwell and Leffingwell, (1991); Komthong et al. (2006); g Mass spectrum and retention time comparable to standard (Positively identified). *Concentrations were calculated relative to an internal standard (3-octanol). LRI: Linear retention index; ND: not detected. NF = not found. 1

3

e

Takeoka et al. (1990);

f

4

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2 h. After that, two samples of 1 mL headspace gas were taken from the flask and injected in the gas chromatograph. The ethylene production rate was calculated taking into account the flask volume, the fruit weight, the ethylene concentration inside flask and the time of closure, results were expressed in ng kg 1 s 1. The internal gas of the same flask was circulated through an electronic gas analyzer (Schele1, model KB7), to determine the CO2 concentration inside the flask. The respiration rate was expressed as mg kg 1 s 1. 2.7. Flesh firmness Flesh firmness was evaluated by the insertion of an 11 mm penetrometer inside flesh in two opposite sides of 25 fruit, where previously the skin was removed. Results expressed in Newton (N). 2.8. Volatile compounds analysis The volatile compounds concentration were analyzed in the juice obtained from apples in the process of ripening at harvest and after nine months of storage plus 7 days of shelf life at 20  C. 2.8.1. Sample preparation 10–15 fruit were cooled down to 0  C, to reduce it metabolism. Immediately after the pulp cooling, horizontal slices of the equatorial region of fruit were taken, discharged the seeds, and centrifuged with a Juicer (Philips Walita1) under low temperature, to avoid the chemical and enzymatic oxidation of samples (the maximum juice temperature during sample preparation was 5  C) (Both et al., 2014). The time to undertake the juice of each sample was about 2 min. The juice was placed inside 100 mL amber flash and immediately frozen down to 30  C for subsequent analysis. 2.8.2. Volatile compounds quantification Samples were thawed in ambient temperature to 2  C at the day of analysis. An aliquot of 10 mL of this juice was taken, mixed with 3 g NaCl and 10 mL of 3-octanol standard solution (10 mg) inside a 20 mL vial that allowed hermetically sealing with a PTFE-coated silicone lid. From this solution, the volatile compounds were extracted via solid phase microextraction (HS-SPME) of the vial headspace. A Divinylbenzene/carboxen/polydimethylsiloxane (DVB/Car/PDMS) fibre (Supelco, 50/30 mm  20 mm) was preconditioned following the manufacturer protocol. Before the fiber exposing, the vial was submerged in a water bath at 35  C during 5 min. After 5 min, the fiber was exposed to the headspace of the sample during 60 min under constant stirring at the same temperature. Volatile compounds were quantified with a DANI1 (Dani Intruments Spa., Viale Brianza, Cologno Monzese, Italygas) gas chromatograph equipped with a flame ionization detector (FID). The fiber was thermally desorbed into the injection port for 10 min at a temperature of 250  C in a split less mode. A capillary column DN-WAX (60 m  0.25 mm  0.25 mm) allowed separating the volatile compounds. The flow rate of the carrier gas (nitrogen) was 1.0 mL min 1. The temperature ramp used during the analysis was: initial temperature 35  C held for 3 min, then a ramp of temperature of 2  C min 1 up to 80  C, thereafter, another ramp of 5  C min 1 up to 230  C and held at this temperature for 5 min. The temperature of FID detector was 230  C. To calculate the linear retention index was analyzed a series of n-alkanes in the same chromatographic conditions used to analyze the volatile compounds. This analysis was carried out according to Both et al. (2014).

2.8.3. Volatile compounds identification The volatile compounds were identified using a Shimadzu QP2010 Plus gas chromatography coupled to mass spectrometry (GC/MS; Shimadzu Corporation, Kyoto, Japan) or by standard solutions. The same chromatographic conditions described to quantify the volatile compounds were used, with helium as the carrier gas. The detector was operated in the electron impact ionization, ionization energy of +70 eV and a scan mass from 35 up to 350 m/z. The mass spectra of each compound was compared with mass spectra available in the National Institute of Standards and Technology (NIST) library and by comparing the linear retention index (LRI) with those available in the scientific literature. 2.9. Statistical analysis All data were submitted to a principal component analysis (PCA) using The Unscrambler1 X software (version 9.7, CAMO A/S, Trondheim, Norway). Before the PCA the data matrix was auto scaled for each variable in order to obtain the same weight for all variables (mean = 0 and variance = 1). A Pearson correlation among IEC, ethylene production, respiration rate and volatile compounds was carried out to show the linear relationship among the volatile compounds, ethylene and respiration. Additionally, a variance analysis (ANOVA) at 5% of error probability was carried out. Data that showed significant difference by ANOVA were subjected to the Tukey’s test at 5% error probability. 3. Results and discussion 3.1. Metabolism and volatile profile at harvest Fruit harvested at ripe and overripe maturity stages had high IEC and ethylene production, but the highest respiration rate was observed only in ripe harvested fruit (Table 1). High ethylene levels in ripe and overripe harvested fruit resulted in higher ester concentration, if the ethylene perception is not the limiting factor, especially butyl acetate, 2-methylbutyl acetate and hexyl acetate (Table 1). Additionally, overripe apples also showed a high ester precursor concentration, i.e., 1-butanol, 2methyl-1-butanol and 1-hexanol, which are precursors of the esters. These precursors originate from fatty acids via b-oxidation and LOX, with the exception 2-methyl-1-butanol. 3.2. Multivariate analysis

PCA

The multivariate analysis was carried out due to the large amount of compounds quantified and identified, to show an overview of the effect of the maturity stages and storage conditions on the volatile profile (Fig. 3 ). The PC I was important to separate the fruit stored under CA + 1-MCP, regardless of the maturity, unripe and ripe DCA-RQ 1.3 from overripe apples stored under DCA-RQ 1.3 and DCA-RQ 1.5 (Fig. 3a). The majority of the volatile compounds are in overripe apples stored under DCA-RQ 1.3 and DCA-RQ 1.5, and lower number of loadings to the storage under CA + 1-MCP (fruit of the three maturity stages) and DCA-RQ 1.3 (unripe and ripe fruit) (Fig. 3b). The PC II was important to separate the fruit stored under CA, regardless of the maturity stage, and unripe DCA-RQ 1.5 stored fruit from those stored in the other conditions (Fig. 3a). The IEC, ethylene production rate, respiration rate, 3-methylbutyl acetate, E-2-hexen-1-ol, 2-methyl-1-butanol and methyl 2-methyl butanoate are related to the fruit stored in CA (Fig. 3b). A noteworthy fact is that the largest amount of volatile compounds are not close to ethylene and the respiration rate (Fig. 3b), with the exception of the fruit that were stored under CA, as indicated by the Pearson

IEC At Harvest Ethylene Respiration IEC Ethylene CA Respiration IEC Ethylene CA + 1-MCP Respiration IEC Ethylene DCA-RQ 1.3 Respiration IEC Ethylene DCA-RQ 1.5 Respiration

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Acetaldehyde P 2-propanone Methyl acetate Butanal Ethyl acetate P 2-propano l Ethanol Ethyl propanoate Ethyl isobutanoate Propyl acetate Methyl 2-methyl butanoate 2-Methylpropyl Isobutyl acetate Ethyl butanoate Ethyl 2-methyl butanoate Butyl acetate Hexanal M 2-methylbutyl acetate Butyl Propanoate H Z-3-hexenal 1-Butanol 3-methylbu M tyl acetate 4-Methyl-2-pentanol P 4-pentenyl acetate 3-Hexanol Butyl butanoate Z-2-Hexenal M 2-methyl-1-bu tanol E-2-Hexenal Z-2-Pentenyl acetate P Z-2-penten-1-ol-ace tate O 3-octanon e P 1-pentanol Hexyl acetate Z-2-Hexenyl acetate Z-3-Hexenyl acetate E-3-Hexenyl acetate E-3-Hexen-1-ol-ac 5-Hexenyl aceetate tate 5-Hexen-1-ol-ac M 2-methyl-2-bu ten-1-ol H E-2-hexenyl acetate M 6-methyl-5-heptene-2-one 1-Hexanol E-3-hexen-1-ol H Heptyl acetate H Z-3-hexen-1-ol Butyl hexanoate H E-2-hexen-1-ol H E-5-hexen-1-ol E-1-octen-3-ol O 1-heptanol H M 6-methyl-5-hepten-2-ol 2-Ethyl 1-hexanol 1-Octanol Benzyl acetate

5

et al., 2003; Bangerth et al., 2012; Both et al., 2014; Thewes et al., 2015; Yang et al., 2016). However, the results reported in the literature were all conducted in aerobic conditions of air, CA and ULO, where the anaerobic metabolism of fruit is not significant. The extremely low pO2 employed during DCA-RQ 1.3 (0.23 kPa O2 in average) and DCA-RQ 1.5 (0.21 kPa O2 in average) resulted in a significant increment in anaerobic metabolism, especially in DCARQ 1.5. 3.3. IEC, ethylene production, respiration rate Fruit stored in CA showed higher IEC and ethylene production in comparison to the other storage conditions, regardless of the maturity stage, which resulted in lower flesh firmness (Supplementary Fig. 2). Additionally, in unripe apples, the storage under DCA-RQ 1.5 resulted in a higher IEC and ethylene production in comparison with CA + 1-MCP and DCA-RQ 1.3, but in ripe and overripe apples, no difference between the storage under CA + 1MCP, DCA-RQ 1.3 and DCA-RQ 1.5 was found for these two variables (Fig. 2a and b). The high IEC and ethylene production by fruit stored under CA may be a result of the high pO2 employed during storage (1.2 kPa). Previous studies have also found higher ethylene in CA stored fruit as compared with CA + 1-MCP (Lee et al., 2012; Bekele et al., 2015; Thewes et al., 2015) and DCA-RQ (Brackmann et al., 2015; Weber et al., 2015). Higher IEC were found in overripe apples when the fruit were stored under CA, but no difference between the maturity stages was identified when the fruit were stored under CA + 1-MCP, DCARQ 1.3 and DCA-RQ 1.5 (Fig. 2a). For ethylene production, no difference was found between the maturity stages when the fruit were stored under CA, CA + 1-MCP and DCA-RQ 1.3 conditions, however unripe fruit stored under DCA-RQ 1.5 showed higher ethylene production rate in comparison to ripe and overripe fruit (Fig. 2b). The high levels of IEC and ethylene production by CA stored fruit likely induced a high respiration rate, regardless of the maturity stage (Fig. 2c). The fruit stored under CA + 1-MCP, DCA-RQ 1.3 and DCA-RQ 1.5 did not differ in their respiration rate, regardless the maturity stage. Comparing the maturity stages in each storage condition, no difference was observed between the CA and DCA-RQ 1.5. In contrast, overripe CA + 1-MCP and DCA-RQ 1.3 stored fruit had higher respiration rate in comparison with unripe fruit, with no difference from those harvested at ripe maturity stage. 3.4. Volatile compounds analysis The GC-FID detected more than 100 volatile compounds, from which 50 were identified by the GC/MS, and some by standard solutions. Identified compounds comprised, 25 esters, 17 alcohols, 6 aldehydes and 2 ketones. These compounds were identified in all samples before (with some exceptions) and after storage, regardless of the maturity stage and storage condition. A significant interaction between maturity stage and storage conditions was identified for all these compounds.

-1

0

1

Fig. 1. Heat map showing the Pearson correlation, at harvest and after nine months of storage under CA, CA + 1-MCP, DCA-RQ 1.3 and DCA-RQ 1.5 plus 7 days of shelf life, among internal ethylene concentration (IEC), ethylene production (Ethylene), respiration rate (Respiration) and the volatile compounds concentration in juice obtained from ‘Galaxy’ apple harvested at three maturity stages.

correlation (Fig. 1). These results contract to other reports in the literature, which informed that the volatile compounds biosynthesis is closely related to the ethylene presence and respiratory activity (Brackmann et al., 1993; Song and Bangerth, 1996; Fellman

3.4.1. Total, most important and abundant, esters The esters show a key importance in apple characteristic fruity and floral flavor (Plotto et al., 1999; Plotto and McDaniel, 2001; Komthong et al., 2006; Mehinagic et al., 2006). Fruit stored under DCA-RQ 1.5 had higher total ester amount when compared to the other storage conditions, regardless of the maturity stage (Fig. 4a). On the other hand, fruit stored under CA + 1-MCP application had the lowest ester concentration, regardless of maturity stage. Fruit stored under CA and DCA-RQ 1.3 had an intermediate ester concentration between, that in CA + 1-MCP and DCA-RQ 1.5, but no difference was observed between CA and DCA-RQ 1.3, with the

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Fig. 2. Internal ethylene concentration (IEC; mg L 1) (a), ethylene production (ng kg 1 s 1) (b) and respiration rate (mg kg 1 s 1) (c) of ‘Galaxy’ apple harvested at three maturity stages and stored under CA, CA + 1-MCP, DCA-RQ 1.3, DCA-RQ 1.5 during nine months plus 7 days of shelf life at 20  C. Bars with the same lower case letter in the same maturity stage, and each bar with the same upper case letter in different maturity stages are not significantly different by Tukey’s test, at 5% probability. Error bars mean standard deviation.

exception of overripe apples, where CA stored fruit had higher total ester amount. The negative effect of 1-MCP in volatile compounds biosynthesis is widely reported in literature (Raffo et al., 2009; Thewes et al., 2015; Yang et al., 2016). Nevertheless, the present results show for the first time, that the pO2 lowering, at a partial pressure that allows a little fermentation, improved the total ester biosynthesis. A study carried out with anaerobic metabolism products application also resulted in a significant increment in total ester amount in melons (Liu et al., 2012; Jin et al., 2013). The most important esters of ‘Gala’ apples are butyl acetate, 2methylbutyl acetate and hexyl acetate (Young et al., 1996; Plotto et al., 2000; Salazar and Orozco, 2011). The pattern for the most abundant ester of ‘Galaxy’ was similar to the total esters (Fig. 5b and c). Again, fruit stored under DCA-RQ 1.5 had higher characteristic apple esters concentration, regardless of the maturity stage, indicating that fruit stored under extremely low pO2 (average of 0.21 kPa O2) maintained higher main ester concentration, in comparison to the fruit stored under CA (average of 1.2 kPa O2). This is an important practical result because extremely low pO2 maintained physical qualities, such as flesh firmness, acidity and soluble solids (Brackmann et al., 2015; Weber et al., 2015), and higher characteristic esters, as reported in the present study. Concerning the maturity stages, an increment of the esters was observed with maturity advance when the fruit were stored under CA, DCA-RQ 1.3 and DCA-RQ 1.5, but no increment was observed when the fruit were treated with 1-MCP (Fig. 4b and c). 3.4.2. Individual esters Differences of esters, between maturity stages and storage conditions were observed (Fig. 5a–z). Butyl acetate, one of the main straight-chain esters, showed a higher concentration in DCA-RQ 1.3 than in CA and CA + 1-MCP, when the fruit were harvested unripe ( Fig. 5j). However, in ripe and overripe apples, the storage under DCARQ 1.5 resulted in higher butyl acetate concentration in comparison to CA and CA + 1-MCP, with no difference from those stored under DCA-RQ 1.3, and its concentration was far above the odor threshold (Table 1). In contrast, Raffo et al. (2009), Both et al. (2014) and Lumpkin et al. (2014), reported a reduction in the butyl acetate concentration in ‘Pinova’, ‘Royal Gala’ and ‘Scartlett Spur Red Delicious’ apples, respectively, stored under extremely low pO2 in comparison to CA stored fruit. An interesting fact is that the 1-MCP application, in CA stored fruit, did not suppress the butyl acetate production, regardless of the maturity stage of the fruit (Fig. 5j). Higher butyl acetate was observed in overripe apples, regardless the storage condition, but ripe DCA-RQ 1.5 stored fruit had higher butyl acetate in comparison to unripe apples and no

difference was found between unripe and ripe harvested apples under the other storage conditions (Fig. 5j). These results agree with the ones found in the literature, which affirm that the butyl acetate emission by apples increases with advanced maturity (Brackmann et al., 1993; Song and Bangerth, 1996, 2003; Bangerth et al., 2012). Additionally, a synergism appears to occur between storage under DCA-RQ, regardless of the RQ level, and maturity advance on butyl acetate concentration in juice of ‘Galaxy’ apple ( Fig. 5j). The straight-chain esters, such as butyl acetate, need constant ethylene supply to allow its biosynthesis (Brackmann et al., 1993; Song and Bangerth, 1996; Fellman et al., 2003; Bangerth et al., 2012; Both et al., 2014; Thewes et al., 2015; Yang et al., 2016), but in the present study the correlation is negative (Fig. 1) under extremely low pO2, especially under the highest RQ level (DCA-RQ 1.5). Fruit stored under CA and DCA-RQ 1.5 had higher hexyl acetate concentration in comparison to the ones stored under CA + 1-MCP and DCA-RQ 1.3, regardless of the maturity stage (Fig. 5q). Additionally, overripe fruit stored under DCA-RQ 1.3 resulted in higher hexyl acetate production in comparison to CA + 1-MCP. The higher hexyl acetate production in CA and DCA-RQ 1.5 stored fruit is a result of higher 1-hexanol concentration (Fig. 6h), which is a hexyl ester precursor (Holland et al., 2005; Souleyre et al., 2005). An interesting fact is that the extremely low pO2 (0.21 kPa in average for DCA-RQ 1.5) resulted again in an increment in the hexyl acetate and its precursor (1-hexanol). Hexyl acetate production is closely related to ethylene production/action, b-oxidation and LOX (Brackmann et al., 1993; Song and Bangerth, 1996; Bangerth et al., 2012; Both et al., 2014; Yang et al., 2016). The hexyl acetate concentration was higher when the fruit were harvested in overripe maturity stage and stored under CA, DCA-RQ 1.3 and DCA-RQ 1.5 in comparison to unripe apples (Fig. 5q). Nevertheless, when the fruit were treated with 1-MCP no increment in hexyl acetate was observed with greater maturity. This result may be related to the lower precursor concentration (1hexanol) (Fig. 6h), as shown by Lara et al. (2006), or/and because the 1-MCP application reduced the alcohol acyltransferase (AAT) enzyme expression and activity, reducing the hexyl acetate production with the maturity advance (Defilippi et al., 2005; Yang et al., 2016). Branched-chain esters are fundamentally important for apple flavor, especially the 2-methylbutyl acetate (Brackmann et al., 1993; Both et al., 2014). Juice obtained from fruit stored under DCA-RQ 1.5 showed the highest 2-methylbutyl acetate in comparison to the other storage conditions, with the exception of overripe harvested fruit, where DCA-RQ 1.5 did not differ from

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7

7

(a) CA + Overripe

5 CA + Ripe

PC II (13.36%)

3

CA - Unripe

DCA-RQ 1.5 - Unripe

1 DCA-RQ 1.5 + Ripe CA + 1-MCP + Ripe

-1

DCA-RQ 1.3 + Ripe CA + 1-MCP + Overripe

CA + 1-MCP - Unripe

DCA-RQ 1.5 + Overripe

DCA-RQ 1.3 - Unripe

-3

DCA-RQ 1.3 + Overripe

-5 -7 -20

-15

-10

-5

0 PC I (60.28%)

1.0

5

10

15

20

(b)

IEC Ethylene

•

0.8 0.6

2-methyl-1-butanol

Hexyl acetate

3-methyl butyl acetate

Methyl 2-methyl butanoate

0.2 0.0 -0.2 -0.4

Z-2-pentenyl acetate 2-Ethyl 1-hexanol 1-heptanol 2-methyl butyl acetate 1-Hexanol E-5-hexen-1-ol Ethyl 2-methyl butanoate 1-Butanol Z-2-Hexenyl acetate Z-3-hexenal 3-Hexanol 5-Hexen-1-ol-acetate E-3-hexen-1-ol 2-methyl-2-buten-1-ol E-2-hexenyl acetate Isobutyl acetate Heptyl acetate Methyl acetate E-1-octen-3-ol Ethyl isobutanoate 4-Methyl-2-pentanol Butanal Ethyl propanoate Z-3-hexen-1-ol Acetaldehyde propyl acetate Ethanol Benzyl acetate Ethyl acetate Hexanal E-2-Hexenal Ethyl butanoate Butyl butanoate 1-Octanol Butyl Propanoate Z-2-Hexenal Z-3-Hexenyl acetate Butyl hexanoate E-3-Hexenyl acetate 2-propanone Butyl acetate 2-propanol 6-methyl-5-heptene-2-one 6-methyl-5-hepten-2-ol

E-2-hexen-1-ol

0.4

PC II (13.36%)

Respiration

4-pentenyl acetate

-0.6 -0.8 -1.0 -1 -0.9 -0.8 -0.7 -0.6 -0.5 -0.4 -0.3 -0.2 -0.1 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 PC I (60.28%)

1

1.1

Fig. 3. Scores (a) (treatments) and correlation loadings (b) (variables) plots showing the two major principal components of the metabolism and volatile profile of ‘Galaxy’ apple harvested at three maturity stages and stored under CA, CA + 1-MCP, DCA-RQ 1.3, DCA-RQ 1.5 during nine months plus 7 days of shelf life at 20  C.

CA stored fruit (Fig. 5k). According to Brackmann et al. (1993) and Echeverría et al. (2008) the branched-chain esters are lesser affected by low pO2 in comparison to the straight-chain ester. Storage under DCA-RQ 1.3 resulted in little difference from those fruit stored under CA, with the exception of overripe apples, where, fruit in DCA-RQ 1.3 had lower 2-methylbutyl acetate in relation to CA, but with higher concentration in comparison to CA + 1-MCP. All fruit stored under DCA-RQ 1.3, DCA-RQ 1.5 and CA + 1-MCP had low ethylene concentration, but the fruit stored under DCA had higher 2-methylbutyl acetate concentration in comparison to CA + 1-MCP. This result shows that under extremely low pO2 the 2methylbutyl acetate production may not stay under ethylene regulation (Fig. 1). Overripe apples had higher 2-methylbutyl

acetate concentration when the fruit were stored under CA and DCA-RQ 1.5, but when the fruit were stored under CA + 1-MCP and DCA-RQ 1.3, no difference between the maturity stages was observed (Fig. 5k). Pyruvate accumulation occurs in fruit flesh with low pO2, which can be linked to the amino acid production (Bekele et al., 2015), and can increase the a-ketovalerate, a 2-methylpropyl acetate precursor (Kochevenko et al., 2012; Sugimoto et al., 2015). Juice obtained from fruit stored in DCA-RQ 1.5 had higher 2-methylpropyl acetate concentration in comparison to the other storage conditions, regardless of the maturity stage (Fig. 5g). Additionally, the 1-MCP application resulted in lower 2-methylpropyl acetate in overripe apples in comparison to the other storage conditions. The 1-MCP

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Fig. 4. Total esters (a), most important ester according to the literature (butyl acetate, 2-methylbutyl acetate and hexyl acetate) (b) and most abundant esters in ‘Galaxy’ apple (butyl acetate, 2-methylbutyl acetate, hexyl acetate, isobutyl acetate and ethyl 2- methyl butanoate) (c) concentration (mg L 1) in juice obtained from ‘Galaxy’ apple harvested at three maturity stages and stored under CA, CA + 1-MCP, DCA-RQ 1.3, DCA-RQ 1.5 during nine months plus 7 days of shelf life at 20  C. Bars with the same lower case letter in the same maturity stage, and each bar with the same upper case letter in different maturity stages are not significantly different by Tukey’s test, at 5% probability. Error bars mean standard deviation.

application increased the valine concentration in apples (Lee et al., 2012; Bekele et al., 2015) and has little effect on the branchedchain amino acid aminotransferase (BCAT) (Yang et al., 2016), indicating that lower 2-methylpropyl acetate concentrations can be related to the lower alcohols precursor (Lee et al., 2012) and/or due to the lower AAT enzyme activity (Defilippi et al., 2005; Yang et al., 2016). Regarding the maturity stages, higher levels of 2methylpropyl acetate were observed in overripe CA and DCA-RQ 1.5 stored apples, but no difference was found between maturity stages when the fruit were stored in CA + 1-MCP and DCA-RQ 1.3. The ethyl 2-methyl butanoate was found to be the most abundant ester in juice of ‘Galaxy’ apple after storage plus 7 days of shelf life (Fig. 5i). Regardless of the maturity stage, fruit stored under CA and DCA-RQ 1.5 showed higher ethyl 2-methyl butanoate concentration in comparison to fruit stored under CA + 1-MCP and DCA-RQ 1.3. This ester attributes a sweet apple-like flavor to the fruit and is characteristic to ‘Gala’ and ‘Fuji’ apples (Echeverría et al., 2004; Mehinagic et al., 2006; Komthong et al., 2006; Echeverría et al., 2008). No differences, between the storage conditions, for methyl 2-methyl butanoate were observed in unripe harvested fruit, but in ripe and overripe apples, CA and DCA-RQ 1.3 resulted in higher concentrations in comparison to CA + 1-MCP and DCA-RQ 1.5 (Fig. 5f). This compound was higher in overripe apples when the fruit were stored under CA and DCA-RQ 1.3, but no difference between the maturity stages was observed in CA + 1-MCP and DCA-RQ 1.5 stored apples. The low pO2 reported in the present study (0.23 kPa for DCA-RQ 1.3 and 0.21 kPa for DCA-RQ 1.5) is expected to cause an increment in the volatile compounds related to the anaerobic metabolism, such as ethyl acetate (Wright et al., 2015), which are related to offflavors formation if present in large amounts. Nevertheless, this compound is also a fundamental contributor to apple flavor, if in low concentration (Wright et al., 2015). No difference was observed between the storage conditions in unripe harvested fruit for ethyl acetate, but in ripe and overripe apples, the storage under DCA-RQ 1.5 resulted in higher ethyl acetate concentration in comparison to the other storage conditions (Fig. 5b). Nevertheless, the ethyl acetate concentration, regardless storage condition and maturity stage, remained far below to its odor threshold (Table 1). This may be a result of a high ethanol concentration in DCA-RQ 1.5 stored apples (Fig. 6a). The high ethanol concentration was likely a substrate to the AAT enzyme for ethyl acetate production (Defilippi et al., 2005; Holland et al., 2005; Souleyre et al., 2005; Yang et al., 2016). Comparing the three maturity stages in each storage condition, no increment in ethyl acetate was observed in fruit that were stored under CA, CA + 1-MCP and DCA-RQ 1.3, but in DCA-RQ

1.5, the ethyl acetate concentration increased with greater maturity (Fig. 5b). The remaining esters, showed a similar pattern to those above, but their concentrations are lower. Juice from fruit treated with 1MCP generally had the lowest ester concentration and no increment occurred with advancing maturity in fruit treated with 1-MCP. Additionally, the storage under CA and DCA-RQ 1.3 and DCA-RQ 1.5 resulted in the highest esters concentration and an increment occurred with advancing maturity (Fig. 5). 3.4.3. Individual alcohols The most abundant alcohol detected in the juice of ‘Galaxy’ apples after storage was 1-hexanol (Fig. 6h). Fruit stored under CA and DCA-RQ 1.5 had the highest 1-hexanol concentration in comparison to the ones stored under CA + 1-MCP and DCA-RQ 1.3, regardless of the maturity stage. This low 1-hexanol concentration in 1-MCP treated fruit is likely a result of the low hexanal concentration in these apples (Fig. 7c). The higher 1-hexanol, in CA and DCA-RQ 1.5 can be also a result of higher LOX activity (Contreras et al., 2016; Schiller et al., 2015). Also, the high 1hexanol concentration in DCA-RQ 1.5 stored fruit go against to the results found in the current literature, which reports that its concentration is reduced as the pO2 are lowered down to 1.0 kPa (Echeverría et al., 2008), 0.4–0.6 kPa (Raffo et al., 2009) and 0.5 kPa (Both et al., 2014). High concentrations of 1-hexanol were observed in ripe and overripe apples, when the fruit were stored under CA, but when the fruit were stored under DCA-RQ 1.3 and DCA-RQ 1.5 only overripe apples had a higher 1-hexanol in comparison to unripe apples, and no difference between maturity stages was observed when the fruit were treated with 1-MCP (Fig. 6h). This result explains, in part, the absence of any increment in hexyl acetate by 1-MCP treated fruit and the highest hexyl acetate production by overripe apples stored under CA, DCA-RQ 1.3 and DCA-RQ 1.5 (Fig. 5q). The high level of 1-hexanol in overripe apples is probably a result of high free fatty acids content, especially linoleic acid (18:2), which had a high Pearson correlation with the hexanal and 1-hexanol production by apples (Contreras et al., 2016). These authors also reported that this free fatty acid could be provided by the de novo fatty acid biosynthesis, fact that may be induced by the extremely low oxygen in DCA-RQ 1.5 stored fruit in the present study. The storage treatments did not cause a difference in 1-butanol in unripe harvested apples, but in fruit that were harvested at ripe and overripe maturity stage, a high level of 1-butanol was observed in CA and DCA-RQ 1.5 stored fruit, respectively (Fig. 6b). This result

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9

Fig. 5. Esters concentration in juice from ‘Galaxy’ apple harvested at three maturity stages and stored under CA, CA + 1-MCP, DCA-RQ 1.3, DCA-RQ 1.5 during nine months plus 7 days of shelf life at 20  C. Bars with the same lower case letter in the same maturity stage, and each bar with the same upper case letter in different maturity stages are not significantly different by Tukey’s test, at 5% probability. Error bars mean standard deviation.

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Fig. 6. Alcohols concentration in juice from ‘Galaxy’ apple harvested at three maturity stages and stored under CA, CA + 1-MCP, DCA-RQ 1.3, DCA-RQ 1.5 during nine months plus 7 days of shelf life at 20  C. Bars with the same lower case letter in the same maturity stage, and each bar with the same upper case letter in different maturity stages are not significantly different by Tukey’s test, at 5% probability. Error bars mean standard deviation.

may be related to the 1-butanal concentration in DCA-RQ 1.5, especially in the overripe maturity stage (Fig. 7b). This is in contradiction with the current literature, which reports that the low pO2 suppresses 1-butanol formation in ‘Pinova’ (Raffo et al., 2009) and ‘Scarlett Spur Red Delicious’ apples (Lumpkin et al., 2014), and in juice obtained from ‘Royal Gala’ apples (Both et al., 2014). Similarly to 1-hexanol, high 1-butanol was observed in ripe and overripe fruit, when stored under CA, and in overripe fruit, when stored under DCA-RQ 1.5. However, no increment in 1butanol concentration with advancing maturity was found in fruit that were stored under CA + 1-MCP and DCA-RQ 1.3. When

analyzing the 1-butanol and butyl acetate concentrations together is noteworthy that the AAT activity shows a distinct activity between storage conditions, resulting in distinct butyl acetate production among the storage conditions. A similar response was observed for 2-methyl-1-butanol and 2methylbutyl acetate concentration, where the 2-methylbutyl acetate concentration also changed with the storage conditions more than its precursor (2-methyl-1-butanol) (Figs. 5 k and 6 e). Again, this result can be related to the AAT enzyme activity, which changes its affinity to the precursor concentration (Souleyre et al., 2005). According to these authors, the MpAAT1 enzyme affinity to

F.R. Thewes et al. / Postharvest Biology and Technology 127 (2017) 1–13

11

Fig. 7. Aldehydes (a–f) and ketones (g–h) concentration in juice from ‘Galaxy’ apple harvested at three maturity stages and stored under CA, CA + 1-MCP, DCA-RQ 1.3, DCA-RQ 1.5 during nine months plus 7 days of shelf life at 20  C. Bars with the same lower case letter in the same maturity stage, and each bar with the same upper case letter in different maturity stages are not significantly different by Tukey’s test, at 5% probability. Error bars mean standard deviation.

the precursor has distinct response in low alcohol concentration (2-methyl-1-butanol, 1-hexanol and 1-butanol, respectively) and in high alcohol concentration (1-hexanol, 1-butanol and 2-methyl1-butanol, respectively). Thereby, the MpAAT1 affinity may be changed according to the treatment and the maturity stage, resulting in a dissimilar 2-methylbutyl acetate production in relation to the precursor concentration. A high 2-methyl-1-butanol concentration was observed in overripe apples that were stored under CA, but in CA + 1-MCP, ripe and overripe fruit had higher 2methyl-1-butanol than unripe apples. Nevertheless, there was no increment in 2-methyl-1-butanol concentration with advancing maturity, in DCA-RQ 1.3 and DCA-RQ 1.5. E-2-hexen-1-ol was not affected by the storage conditions in unripe apples, but in ripe and overripe apples, the storage under CA resulted in the highest E-2-hexen-1-ol concentration, with no difference from fruit stored under DCA-RQ 1.5 in overripe apples (Fig. 7k). Fruit treated with 1-MCP again showed low E-2-hexen-1ol in ripe and overripe apples, which is a result of lower E-2hexanal concentration in these fruit (Fig. 7f). Ripe fruit stored under CA had the highest E-2-hexen-1-ol concentration, but when the fruit were stored under DCA-RQ 1.5, ripe and overripe apples had high E-2-hexen-1-ol concentration, in comparison to unripe apples. Additionally, no changes were observed with maturity when the fruit were stored under CA + 1-MCP and DCA-RQ 1.3. In the present study, the ethanol concentration was only increased when the fruit were stored under DCA-RQ 1.5, regardless of the maturity stage (Fig. 6a). The high ethanol production may be a result of higher acetaldehyde concentration, especially in ripe and overripe apples (Fig. 7a). In overripe apples, 1-MCP application suppressed the acetaldehyde (Fig. 7a) and ethanol (Fig. 6a) production by the fruit. Similar results were observed in some other apple cultivars, where the 1-MCP application also reduced the formation of acetaldehyde and ethanol (Lee et al., 2012; Thewes et al., 2015). A higher concentration of aldehydes and ketones was observed in ripe and overripe apples without 1-MCP in all storage conditions (Fig. 7). However, there was no increase with advanced maturity when the fruit were stored under CA + 1-MCP. These results show that the 1-MCP application may have suppressed the LOX and b-oxidation pathways, as suggested by Yang et al. (2016).

3.5. Changes in volatile compounds profile after storage in relation to harvest An important challenge faced by researchers and storage operators is to maintain the volatile compounds of fruit after longterm storage under CA and DCA similar to that found at harvest. A calculation to verify if the volatile compounds production increased or decreased during storage in each storage condition, to show the storage condition that maintain the volatile compounds similar or higher to the one obtained at harvest was performed. The main esters of ‘Galaxy’, ethyl 2-methyl butanoate, 2methylproppyl acetate, butyl actetate, 2-methylbutyl acetate and hexyl acetate, had distinct responses between harvest and after storage (Fig. 8). 2-methylproppyl acetate showed an increment in its concentration in unripe and ripe CA, CA + 1-MCP and DCA-RQ 1.3 stored fruit and a reduction in overripe apples stored in the same conditions. However, in DCA-RQ 1.5 storage, there was an increment in the three maturity stages and this increment was higher in comparison to the other storage conditions (Fig. 8). Another important result was obtained for ethyl 2-methyl butanoate, which increased after storage in comparison to harvest, when the fruit were stored under CA and DCA-RQ 1.5, however, in fruit that were treated with 1-MCP or stored under DCA-RQ 1.3, there was a decrease of this compound after storage, regardless maturity stage (Fig. 8). Regarding the butyl acetate, all overripe apples lost this ester during storage, regardless of the storage condition. Nevertheless, unripe and ripe apples stored under DCARQ 1.3 and DCA-RQ 1.5 had an increment in butyl acetate during storage. On the other hand, when the fruit were stored under CA and CA + 1-MCP, an increment of this compound was only verified in unripe apples (Fig. 8). Perhaps, the extremely low oxygen used during DCA-RQ 1.3 (0.23 kPa in average) and DCA-RQ 1.5 (0.21 kPa in average) allowed the biosynthesis of butyl acetate precursors, like 1-butanol, and consequently improving its production by apples stored in these conditions. 2-Methylbutyl acetate also increased in all storage conditions throughout the storage when fruit were harvested unripe and a reduction was observed when fruit were harvested at overripe maturity (Fig. 8). Nevertheless, in ripe harvested fruit, only the

12

Minimum µg L-1 Maximum

DCA-RQ 1.5

DCA-RQ 1.3

CA + 1-MCP

Legend

Unripe Ripe Overripe Unripe Ripe Overripe Unripe Ripe Overripe Unripe Ripe Overripe

CA

F.R. Thewes et al. / Postharvest Biology and Technology 127 (2017) 1–13

Acetaldehyde 2-propanone Methyl acetate Butanal Ethyl acetate 2-propanol Ethanol Ethyl propanoate Ethyl isobutanoate Propyl acetate Methyl 2-methyl butanoate 2-Methylpropyl acetate tate Isobutyl ace Ethyl butanoate Ethyl 2-methyl butanoate Butyl acetate Hexanal 2-Methylbutyl acetate Butyl propanoate Z-3-hexenal 1-Butanol 3-Methylbutyl acetate 4-Methyl-2-pentanol 4-pentenyl acetate 3-Hexanol Butyl butanoate Z-2-Hexenal 2-Methyl-1-butanol E-2-Hexenal Z-2-Pentenyl acetate 1-Pentanol Hexyl acetate Z-2-Hexenyl acetate Z-3-Hexenyl acetate E-3-Hexenyl acetate 5-Hexenyl acetate 2-methyl-2-buten-1-ol E-2-Hexenyl acetate 6-Methyl-5-heptene-2-one 1-Hexanol E-3-Hexen-1-ol Heptyl_acetate Z-3-Hexen-1-ol Butyl hexanoate E-2-Hexen-1-ol E-5-Hexen-1-ol E-1-Octen-3-ol 1-Heptanol 6-Methyl-5-hepten-2-ol 2-Ethyl-1-hexanol 1-Octanol Benzyl acetate

-4.9

7.6

-3.7

15 .5

-0.5

23 .0

-4.3

5.5

4.9

42 2.2

5.5

85 .9

-4.1

13 3.2

-0.1

5.8

0.0

0.8

-55 .2

6.7

-1.7

6.7

-28 .4 0.0

11 0.7 17 .7

-107.3

35 2.5

-3850 .8

32 8.6

2.0 -1697 .0

96 .6 42 8.8

-1.5

2.0

-50 .3

1.6

-309.9

62 .7

-528.1

-8.0

-10 .7

3.4

0.4

7.2

-3.6

2.5

2.3

48 .5

-28 .2 -211.1

1.8 45 .4

220.6

21 42.5

-13 .7

15 .9

-2.0 -4314 .2

23 .4 16 0.8

-6.0

1.1

-12 .1

4.2

-53 .1

1.7

-103.4

12 .8

-0.9

7.3

-10 .5

1.5

-3.6

3.6

-1071 .4

26 3.2

-15 .0

3.4

-1.5

4.6

-6.1

1.8

-5.4

Products of anaerobic metabolism, acetaldehyde, ethanol and ethyl acetate, had the highest increase during storage in DCA-RQ 1.5, regardless of the fruit maturity stage at harvest, with the exception of acetaldehyde in unripe harvested apples (Fig. 8). Moreover, the ethanol concentration was only reduced in overripe CA + 1-MCP stored fruit, but the acetaldehyde concentration was reduced in overripe apples stored under CA, CA + 1-MCP and DCARQ 1.3. It is noteworthy that overripe apples showed increased fermentation at harvest (Table 1), which was reduced during storage (Fig. 8). Therefore, storage under DCA-RQ 1.3 did not result in increased accumulation of anaerobic products. 4. Conclusions In summary, the storage of ‘Galaxy’ apple under DCA-RQ 1.5 resulted in a higher total ester amount and characteristic esters in juice, such as butyl acetate, ethyl 2-methyl butanoate, 2methylpropyl acetate, 2-methylbutyl acetate and hexyl acetate, in relation to conventional CA storage after 9 months of storage plus 7 days of shelf life. DCA-RQ 1.3 showed comparable characteristic ‘Galaxy’ esters to fruit stored under CA. The highest amount of esters was found in overripe apples stored under DCARQ 1.3 and DCA-RQ 1.5. Moreover, the volatile compounds biosynthesis were not correlated with ethylene production when the fruit were stored under DCA-RQ 1.5, differing from CA storage in which ethylene and volatile concentration were positively correlated. DCA-RQ 1.5 increased ethanolic fermentation, resulting in higher acetaldehyde, ethanol and ethyl acetate, but concentrations were below the odor threshold of these three compounds. 1-MCP application suppressed production of the main volatile compounds, even at an advanced maturity stage and reduced production of the major esters after storage. Both 1-MCP treatment and DCA suppressed the IEC, ethylene production and respiration rate, but the metabolism reduction in DCA-RQ 1.5 stored fruit did not result in a volatile compound suppression. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j. postharvbio.2017.01.002.

0.5

-19 .2

11 6.5

-10 .2

3.2

-2.8

0.4

-23 .3

0.7

-3.3

1.6

-5.2

2.7

-2.0

0.5

-6.9

2.5

Fig. 8. Heat map showing the difference between harvest and after storage of the volatile compounds concentration (mg L 1) in juice from ‘Galaxy’ apple harvested at three maturity stages and stored under CA, CA + 1-MCP, DCA-RQ 1.3, DCA-RQ 1.5 during nine months plus 7 days of shelf life at 20  C. Positive values (in red) show increment in the concentration after storage in relation to harvest and negative values (in blue) show reduction in the concentration after storage in relation to harvest, according to the legend. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

CA + 1-MCP stored fruit showed a reduction after storage in relation to harvest. Hexyl acetate is another ester that was low in unripe CA + 1-MCP stored fruit. Also, the lowest reduction after storage in comparison to harvest was observed in unripe and ripe CA and DCA-RQ 1.5 stored fruit. It is important to highlight that fruit treated with 1-MCP, independently of the volatile compound, showed the lowest increase, and generally a reduction in volatile compounds, compared with that at harvest (Fig. 8).

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