Accepted Manuscript

Postharvest vapour heat treatment as a phytosanitary measure influences thearoma volatiles profile of mango fruit

Sukhvinder Pal Singh, Manpreet Kaur Saini

PII: S0308-8146(14)00701-8DOI: http://dx.doi.org/10.1016/j.foodchem.2014.05.009Reference: FOCH 15790

To appear in: Food Chemistry

Received Date: 19 January 2014Revised Date: 3 May 2014Accepted Date: 5 May 2014

Please cite this article as: Singh, S.P., Saini, M.K., Postharvest vapour heat treatment as a phytosanitary measureinfluences the aroma volatiles profile of mango fruit, Food Chemistry (2014), doi: http://dx.doi.org/10.1016/j.foodchem.2014.05.009

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Postharvest vapour heat treatment as a phytosanitary measure influences the aroma 1

volatiles profile of mango fruit 2

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Sukhvinder Pal Singh*and Manpreet Kaur Saini

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National Agri–Food Biotechnology Institute (NABI), Mohali 160 071, Punjab, India 5

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Running title: Aroma volatiles in mango fruit treated with vapour heat 8

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* Corresponding author

Tel.: +91 172 2290123; fax: +91 172 4604888

E–mail address: spsingh@nabi.res.in; sukhvinder.pal.singh@gmail.com (S.P. Singh)

ABSTRACT 22

Our objective was to determine the influence of postharvest vapour heat treatment (VHT) on 23

qualitative and quantitative measurement of aroma volatiles during fruit ripening in mango 24

(cv. Chausa) using gas chromatography-mass spectrometry (GC-MS). VHT (48 °C for 20 25

min) accelerated the process of fruit ripening leading to edible-soft stage within 4 days after 26

heat treatment against 8 days in control. Reversible inhibition of aroma volatiles emission 27

was observed in heat-treated fruit, with a significant alteration in aroma volatiles profiles at 28

different stages of fruit ripening. The heat-induced increase in the rate of fruit ripening 29

proceeded with a significant lag in the emission of aroma volatiles. The suppression of aroma 30

volatiles at ripe stage in heat-treated fruit might adversely impact the consumer acceptance of 31

fruit. 32

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Keywords: Flavour; GC-MS, Heat treatment; PCA; Quarantine; Ripening; Volatile 34

compounds 35

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1. Introduction 47

Aroma is an important attribute determining fruit quality and consumer acceptance of mango 48

fruit (Singh & Singh, 2012). A diverse range of chemical compounds, including 49

monoterpenes, sesquiterpenes, esters, lactones, alcohols, aldehydes and ketones contributes to 50

the aroma volatiles profile in mango fruit (Lalel, Singh & Tan, 2003a; MacLeod & Snyder, 51

1985; Pandit, Chidley, Kulkarni, Pujari, Giri, & Gupta, 2009; Pino, Mesa, Muñoz, Martí, & 52

Marbot, 2005). Monoterpenes and sesquiterpenes are the major volatile compounds, 53

representing 70–90% of the total volatiles depending upon the cultivar. The composition and 54

concentrations of aroma volatiles in mango fruit are influenced by various factors, such as 55

cultivar (Pandit et al., 2009; Pino et al., 2005), rootstock (Vazquez-Luna, Rivera-Cabrera, 56

Perez-Flores, & Diaz-Sobac, 2011), growing location (Kulkarni, Chidley, Pujari, Giri, & 57

Gupta, 2012), fruit ripening (Lalel et al., 2003a), harvest maturity (Lalel, Singh, & Tan, 58

2003b), storage atmosphere composition (Lalel & Singh, 2006) and postharvest treatments 59

(Chidley, Kulkarni, Pujari, Giri, & Gupta, 2013; Dang, Singh, & Swinny, 2008). 60

Quarantine regulations in several mango importing countries such as Australia, Japan, 61

and New Zealand require the fresh fruit to undergo a postharvest vapour heat treatment 62

(VHT) in order to be accepted for import. The objective of VHT is to eliminate the risk of 63

entry of insect pests associated with the fruit into importing countries’ territories. VHT 64

involves the use of hot air saturated with water vapour to heat the fruit core to a specified 65

temperature and hold that temperature for a defined period to ensure that all target insect 66

pests are destroyed (Jacobi, MacRae, & Hetherington, 2001). The temperature–time 67

combinations for disinfestation protocols are determined on the basis of lethality to all stages 68

of the pest life cycle. For instance, Indian mangoes are required to undergo pre-export VHT 69

with fruit core temperature ≥47.5 °C for ≥20 min for Japan (APEDA, 2007), 46.5 °C for 30 70

min or 47.5 °C for 20 min for Australia (Biosecurity Australia, 2011), and ≥48.0 °C for ≥20 71

min for New Zealand (Biosecurity New Zealand, 2012). 72

Severity and type of heat treatment (vapour heat, forced-air heat and hot water dip) 73

depend upon the objective of treatment and heat tolerance of a commodity (Lurie, 1998). 74

Generally, the heat treatments aimed to achieve insect pest disinfestations are more severe 75

than those applied to control disease and to improve chilling tolerance. Postharvest heat 76

treatments including VHT are known to influence mango fruit quality, depending on several 77

factors such as cultivar, harvest maturity, fruit size, preharvest growing conditions, 78

temperature–time regime, pre-treatment conditioning and postharvest environmental factors 79

(Jacobi et al., 2001). Most studies related to the effects of heat-based quarantine treatments 80

on mango have focused on physiological responses and physical injuries, which eventually 81

determine the appearance and eating quality (Heather, Corcoran, & Kopittke, 1997; Jacobi & 82

Giles, 1997; Jacobi et al., 2001; Mitcham & McDonald, 1993). Currently, no information is 83

available on the effect of heat-based insect pest control treatments on aroma volatiles 84

emission in mango fruit. 85

Postharvest heat treatments are known to affect the aroma volatiles production in 86

fruits such as apple (Escalada & Archbold, 2009; Fallik, Archbold, Hamilton-Kemp, 87

Loughrin, & Collins 1997) and orange (Obenland, Arpaia, Austin, & MacKey, 1999; 88

Obenland, Collin, Sievert, & Arpaia, 2012). Dang et al. (2008) reported that hot water dip 89

treatment (52 °C for 10 min) conducted for postharvest disease control in ‘Kensington Pride’ 90

mango did not impact the aroma volatiles production in ripe fruit. The treatment in this report 91

was not as heat intensive as required for VHT (≥47.5 °C fruit core temperature for ≥15–20 92

min) and hot water dip treatment (≥48.0 °C for 60–90 min depending upon the fruit weight) 93

protocols accepted for phytosanitation purposes. Also, the volatiles emissions were measured 94

only in ripe mangoes (Dang et al., 2008), while reversible inhibitory effects of heat treatment 95

on volatile emission have been reported in apple fruit (Fallik et al., 1997). It was 96

hypothesised that the measurement of aroma volatiles production in a time-course spanning 97

different stages of fruit ripening would provide better understanding of the heat treatment 98

effects on flavour quality of fruit. Furthermore, the consumer-driven high-value export 99

markets demand superior quality fruit with special attention to aroma. The objective of this 100

study was to determine if VHT might affect qualitative and quantitative profiles of aroma 101

volatiles during fruit ripening in mango. 102

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2. Material and methods 104

2.1. Fruit material 105

The experiment was conducted on a commercial Indian mango cultivar, ‘Chausa’, a late-106

maturing cultivar. Mature unripe fruit at hard–green stage were harvested in morning hours 107

from a commercial orchard located at Saharanpur (latitude 30.61 ° N, longitude 77.91 ° E), 108

Uttar Pradesh, India. The orchard was about 20 years old maintained under integrated 109

management practices. The fruit were harvested with 3–4 cm long stems, placed in plastic 110

crates (~15 kg) and were transported to the packing house within 1 h. Fruit were sorted for 111

uniformity of shape, colour and size, and blemished or diseased fruit were discarded. The 112

fruit were desapped by cutting the stems to < 0.5 cm and keeping the fruit upside down for 113

sap drainage for about 4 h. The desapped fruit were washed with sodium hypochlorite 114

(0.01%), rinsed with clean water and then allowed to dry in air. The fruit were randomised 115

and segregated into two lots for VHT and untreated control. 116

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2.2. Vapour heat treatment (VHT) 118

The VHT was conducted following the standard operating procedures developed by the 119

Agricultural and Processed Products Export Development Authority (APEDA) of India and 120

approved by the importing countries. The VHT facility was accredited by the quarantine 121

authorities of India, Australia and Japan. To conduct VHT, fruit were held in single layers in 122

perforated plastic crates, which were palletised and loaded into the VHT chamber (Techno 123

Reinetsu Co., Ltd, Kagoshima, Japan). To monitor temperature rise during VHT, a thermal 124

probe was inserted in the innermost part of the fruit pulp (n = 6). The positioning of probes 125

was at the top, middle and bottom crates of the pallet inside the VHT chamber. The 126

temperature of the treatment chamber was increased up to 50 °C to raise the temperature of 127

the innermost fruit pulp to 48.0 °C and thereafter maintained at 48.0 °C for 20 minutes and 128

then cooled in the chamber by tap water from a built-in shower. It took 180 minutes to 129

increase the innermost pulp temperature from 30 °C to 48 °C and the total treatment duration 130

was 205 min Temperature–time profile depicting core temperature increase in mango fruit 131

pulp during VHT is shown in Supplementary Figure 1. The vapour heat temperatures and 132

durations achieved during this study (46.5 °C for 45 min or 47.5 °C for 30 min or 48 °C for 133

20 min) could meet the regulatory requirements of importing countries such as Australia, 134

Japan and New Zealand. Following VHT, fruit were transferred to a room maintained at 18–135

20 °C for ~4 h. The fruit were then single-layer packed in the fibre board boxes having 136

ventilation holes protected with nylon mesh. Finally, the fruit were transported to the 137

laboratory in an air-conditioned vehicle within 4 h. After receiving in laboratory, fruit were 138

held under ambient conditions (26.1 ± 1.8 °C; RH 49.3 ± 4.6 %) for 8 days. The control and 139

heat-treated fruit were sampled after 4 and 8 days at ambient conditions. The fruit were 140

peeled and pulp was cut into small cubes. The pulp cubes representing all parts of the fruit (5 141

fruit per replicate) were pooled to make a representative sample and there were three 142

replicates in both heat–treated and control fruit. A 2.5-g mango pulp homogenate prepared 143

using Ultraturrax™ homogeniser (T25, IKA India Pvt Ltd., Bangalore, India) was combined 144

with 10 ml saturated NaCl in a 15-ml glass vial (Supelco Co., Bellefonte, PA), screw capped 145

with PTFE/silicone septa and were stored at −80 °C before analysis. 146

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2.3. Fruit ripening assessments 148

The changes in fruit skin colour and firmness as indicators of fruit ripening were subjectively 149

and objectively measured during ripening at ambient conditions for 8 days. The subjective 150

assessments were made daily for 8 days (10 fruit/replicate; three replications). For skin 151

colour, visual assessment was performed using an arbitrary 1 to 5 scale (1 = 100% green, 2 = 152

75% green, 3 = 50 green/yellow, 4 = 75% yellow, and 5 = 100% yellow), as described by 153

Shorter and Joyce (1998). Subjective firmness of individual fruits was non-destructively 154

evaluated daily by hand during the ripening period, using the rating scale from 1 to 5 (hard to 155

oversoft) described by Shorter and Joyce (1998): 1 = hard, 2 = sprung, 3 = slightly soft, 4 = 156

eating soft, and 5 = oversoft. 157

The instrumental measurements of skin colour and firmness were conducted at 0, 4 and 8 158

days during ripening under ambient conditions (10 fruit/replicate; three replications). Skin 159

colour was measured at four different points around the equatorial region of each fruit using a 160

colour meter (ColourFlex EZ, Hunter Associates laboratory, Inc., USA) which provided CIE 161

L*, a* and b* values. The b* value represents bluish to yellowish, as the value increases from 162

negative to positive, indicating skin yellowing associated with fruit ripening. Fruit firmness 163

was measured using a texture analyser (TA–HD plus; Stable Microsystems Ltd, Surrey, UK) 164

interfaced to a computer with Exponent® software. An 8-mm thick probe, with a 100-kg load 165

cell on, punctured the peeled fruit at a crosshead speed of 2 mm sec−1 to 5 mm depth. Each 166

fruit was punctured on both sides at the equatorial region. The firmness was expressed in 167

newton (N). 168

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2.4. Sample preparation and extraction of aroma volatile compounds 170

Headspace solid-phase microextraction (HS-SPME) technique was used for extraction of 171

aroma volatiles from the fruit pulp tissue. A preliminary experiment was conducted to 172

optimise the minimum time and temperature for equilibration of the compounds between pulp 173

tissue and headspace. The sample vials were removed from the deep freeze (−80 °C), allowed 174

to thaw at ~22 °C and then incubated in a hot water bath for a period of 60 min at 65 °C. A 2-175

cm fused-silica SPME fibre coated with 100 µm polydimethylsiloxane was inserted into the 176

headspace of the vial and exposed for 30 min using a manual SPME device (Supelco). The 177

fibre was thermally desorbed in the injection port (splitless mode) for 10 min at 240 °C. 178

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2.5. GC-MS analysis of aroma volatiles 180

The separation of volatile compounds was achieved using an Agilent 7890A gas 181

chromatograph (GC, Agilent Technologies Pvt. Ltd., Chandigarh, India) instrument equipped 182

with a SPB–5 MS capillary column (60 m length, 250 µm i.d., 1.0 µm film thickness; 183

Supelco), coupled with a triple quadrupole mass spectrometer (7000B QQQ, Agilent 184

Technologies). The column oven was programmed to increase at 5 °C min–1

from the initial 185

40 to 100 °C, followed by 4 °C min‒1

from 100 to 230 °C and then finally ramped at 5 °C to 186

250 °C, held for 15 min, with a total run time of 65 min. Helium was used as carrier gas at 187

flow rate of 1.5 ml min‒1. Transfer line, ion source, and quadrupole temperatures were set at 188

280, 230 and 150 °C, respectively. The mass spectrometer was used in scan mode between 189

m/z 50 and 500 using electronic impact ionization at 70 eV. 190

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2.6. Identification and quantification of aroma volatiles 192

Volatile compounds were identified by comparison of their mass spectra with library entries 193

(NIST Mass Spectral Library, version 2.0d; National Institute of Standards and Technology, 194

Gaithersburg, MA) and with those of authentic standards. The comparison of linear retention 195

indices (Kovats indices, calculated in relation to a homologous series of straight-chain 196

alkanes) of sample compounds with those of authentic standards was an additional approach 197

to identification. 198

The quantification of volatile compounds was conducted using linear regression models 199

based on the mass spectrometric responses of the external standards. The diluted solutions of 200

authentic external standards belonging to the same chemical class (e.g., monoterpenes) were 201

mixed together with internal standard (1-pentanol) and subjected to HS-SPME-GC-MS 202

analysis under similar conditions as described for fruit samples. The data were normalised 203

against the response of internal standard for both samples and external standards. The peak 204

areas obtained from 5–6 concentrations of standards were used to construct the calibration 205

curves and to quantitate each compound using MassHunter Quantitative Analysis Software 206

(version B.05.01). The concentrations of volatile compounds were expressed as µg/100 g 207

fresh weight of fruit pulp. 208

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2.7. Statistical analysis 210

Data were subjected to a two-way analysis of variance (ANOVA) using GenStat Release 12.1 211

(VSN International Ltd., Hemel Hempstead, UK). The effects of treatment and ripening 212

period and their interactions on aroma volatiles and fruit ripening parameters were assessed 213

within ANOVA and the least significant differences (LSD) were calculated at the 5 % level 214

of significance after a significant F–test. The validity of statistical analysis was ensured by 215

checking all the assumptions of ANOVA. Prior to multivariate statistical analysis, data were 216

centred to adjust for differences in the offset between high and low abundant volatiles. The 217

centred data were loge transformed to reduce heteroscedasticity and then subjected to 218

principal component analysis (PCA) and hierarchical cluster analysis (HCA), using 219

MultiExperiment Viewer (MeV) software (version 4.8.1; Dana-Farber Cancer Institute, 220

Boston, MA). A heat map based on the quantitative changes in volatiles was also generated 221

using the MeV software. 222

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3. Results 224

3.1. Fruit ripening 225

Postharvest VHT accelerated the process of fruit ripening in mango fruit, as evident from the 226

changes in skin colour and flesh firmness (Fig. 1). The increase in skin colour rating and b* 227

value during fruit ripening indicated skin yellowing of mango fruit (Fig. 1A & B). The 228

magnitude of increase in skin colour rating scale and b* value reflecting skin yellowing was 229

higher in VHT fruit compared to that in the control. Similarly, VHT fruit exhibited 7-fold 230

decrease in flesh firmness compared to 2.6-fold decrease in control fruit during the first 4 231

days at ambient conditions (Fig. 1D). The VHT fruit reached eating soft stage (>75% yellow 232

skin) in 4 days at ambient conditions, as compared to control fruit which turned semi-ripe 233

during the same period. The control fruit took about 7–8 days for ripening, while the heat-234

treated fruit became over-ripe by then. The objective measurements of skin colour and flesh 235

firmness were consistent with the subjective assessments (Fig. 1). 236

No adverse effect of VHT on external and internal fruit quality was observed in terms 237

of common heat-induced symptoms, such as lenticel browning, internal breakdown and 238

cavity formation in pulp. The number of days to reach edible ripe stage differed in VHT and 239

control fruit, but the flesh colour of both was similar at the ripe stage (Supplementary Fig. 2). 240

The sensory evaluation by an untrained panel of eight scientists offered with both VHT and 241

control fruit at ripe stage revealed that the VHT fruit lacked in characteristic aroma of 242

‘Chausa’ mango. As a result, the overall acceptability of VHT fruit at the ripe stage, despite a 243

good score in appearance quality, was comparatively less than that of the control (data not 244

shown). 245

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3.2. Total aroma volatiles 247

The VHT suppressed total aroma volatiles production in mango fruit on the day of treatment, 248

but it resumed with the progression of fruit ripening (Fig. 2A). During the first 4 days, the 249

total volatiles emission associated with faster fruit ripening increased in heat-treated fruit, 250

whereas the increase in volatiles production in control fruit was statistically non-significant. 251

The total volatiles production in heat-treated fruit at ripe stage on the 4th day was lower than 252

that in the control fruit which were ripe on the 8th day. However, the emission of total 253

volatiles surged in heat-treated fruit at over-ripe stage on the 8th day. 254

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3.3. Sesquiterpenes 256

Sesquiterpenes were observed to be the major aroma volatiles contributing about 80–86% of 257

the total volatiles production during different stages of fruit ripening in ‘Chausa’ mango 258

(Supplementary Fig. 3). The effects of VHT on the levels of total sesquiterpenes showed a 259

similar trend to those of the total aroma volatiles, except the control fruit did not show any 260

significant change during fruit ripening for 8 days (Fig. 3B). The concentration of total 261

sesquiterpenes in heat-treated fruit was significantly lower than that in the untreated fruit on 262

the day of treatment. Regarding individual sesquiterpenes, the concentrations of trans-263

caryophyllene, α-humulene, aromadendrene, ledene, alloaromadendrene, α–gurjunene, and 264

isoledene were determined. The trans–caryophyllene was found to be the major sesquiterpene 265

and its emission was reversibly inhibited by VHT (Table 1). The concentration of trans-266

caryophyllene increased 1.7- and 1.2-fold during ripening in the untreated and treated fruit, 267

respectively. α–Humulene, the second major sesquiterpene was suppressed greatly by VHT 268

on the day of treatment. The control fruit did not show any significant increase in the 269

concentration of α–humulene during fruit ripening. In comparison with Day 0, the heat-270

treated fruit showed 3- and 10-fold increase in its concentration of α-humulene on the 4th

and 271

8th

day of fruit ripening, respectively. The concentration of aromadendrene did not differ 272

significantly between treated and untreated fruit on day of heat treatment, but it increased 273

several folds during fruit ripening, leading to 1.8-fold lower concentration in VHT fruit than 274

in the control at the ripe stage (Table 1). A significant decrease in ledene concentration was 275

observed immediately after heat treatment. However, its concentration increased significantly 276

during fruit ripening in heat–treated fruit, which is contrary to its decrease in control fruit. 277

Fruit ripening caused a significant decrease in concentration of alloaromadendrene in control 278

fruit, but heat–treated fruit sustained higher levels of its emission during ripening. The heat–279

induced acceleration of fruit ripening leading to over-ripe fruit by 8th day showed several fold 280

increase in the concentrations of major sesquiterpenes, such as trans-caryophyllene, α-281

humulene, and aromadendrene (Table 1). 282

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3.4. Monoterpenes 284

The contribution of monoterpenes to the total aroma volatiles production ranged between 4–285

9%, depending upon the treatment and stage of fruit ripening (Supplementary Fig. 3). No 286

significant differences in the concentrations of total monoterpenes in heat-treated and control 287

fruit were observed on Day 0 (Fig. 2C). The VHT fruit showed significantly lower 288

concentration of total monoterpenes at the ripe stage compared to control fruit. The principal 289

monoterpene compound in ‘Chausa’ mango, α–terpinolene, was significantly influenced by 290

heat treatment (Table 1). The VHT inhibited the production of α-terpinolene to a greater 291

extent leading to no significant increase during fruit ripening for the first 4 days, while its 292

concentration was very high at over-ripe stage on the 8th

day. However, control fruit did not 293

show significant difference in the concentration of α-terpinolene during unripe (0 day) and 294

semi-ripe (4 day) stages, but exhibited 3-fold increase at the ripe stage (8th day). The effect of 295

VHT on the concentration of δ-3-carene showed a trend similar to that of α-terpinolene. The 296

concentrations of other monoterpenes, such as limonene, camphene, 2-carene, α-297

phellandrene, and α-pinene were not significantly influenced by heat treatment and fruit 298

ripening in mango fruit. VHT promoted the evolution of 4-terpineol and carvone immediately 299

after treatment. However, the concentration of 4-terpineol at the ripe stage was lower in 300

VHT fruit than in the control, while no significant differences in concentrations of carvone 301

were observed. Geranyl acetate concentration decreased in response to heat treatment and 302

fruit ripening without any significant differences in heat-treated and control fruit at the ripe 303

stage. In comparison with ripe stage, the heat-treated fruit at over-ripe stage showed several-304

fold increase in evolution of various monoterpenes, such as α-terpinolene, δ-3-carene, 305

limonene, γ-terpinene, α-phellandrene, myrcene, and ocimene (Table 1). 306

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3.5. Esters 308

Esters constituted about 2–6% of the total aroma volatiles production in ‘Chausa’ mango 309

pulp, depending upon the treatment and fruit ripening (Supplementary Fig. 3). VHT 310

promoted the evolution of total esters on Day 0, followed by a significant decrease during the 311

first 4 days of ripening and then resumed to initial level on Day 8 (Fig. 2D). No significant 312

difference in the total esters production was observed at ripe stage of heat-treated and control 313

fruit. Amongst esters identified and quantified, ethyl hexanoate, ethyl octanoate, ethyl 314

dodecanoate and neryl acetate were present in higher concentrations compared to other esters 315

contributing to fruit aroma (Table 2). The concentrations of ethyl dodecanoate, hexyl acetate 316

and neryl acetate were significantly higher in VHT fruit compared to control on Day 0, while 317

concentrations of other esters, such as ethyl acetate, ethyl butyrate, ethyl hexanoate, ethyl 318

heptanoate, ethyl octanoate, methyl butyrate, methyl hexanoate, ethyl benzoate, ethyl 319

caprylate, methyl nonanoate, were unaffected by VHT. In comparison with the control, neryl 320

acetate and nopyl acetate were present in higher concentration in heat-treated fruit at the ripe 321

stage. The over-ripe VHT fruit also showed higher concentration of different esters on Day 8 322

(Table 2). 323

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3.6. Aldehydes, alkanes, ketones and others 325

The concentrations of benzaldehyde and hexanal were significantly enhanced by VHT on 326

Day 0 and sustained at higher level at ripe stage in heat-treated fruit on the 4th day compared 327

to control on the 8th day (Table 2). Contrarily, octanal concentration was suppressed by VHT 328

and remained significantly lower at ripe stage in VHT fruit than the control. The alkanes 329

identified and quantified in the pulp tissue included cycloheptane, tridecane, pentadecane, 330

and hexadecane. The concentrations of cycloheptane, tridecane and hexadecane were not 331

influenced by heat treatment at unripe and ripe stages in both heat-treated and control fruit. 332

The levels of pentadecane were significantly lower in heat-treated fruit on the day of 333

treatment and also on Day 4 compared to control fruit (8th

day). The concentrations of 2,3-334

pentanedione and 6-methyl-5-hepten-2-one were not influenced by heat treatment and fruit 335

ripening in ‘Chausa’ mango. Benzothiazole and linalool oxide levels increased during fruit 336

ripening, irrespective of treatment, but their concentrations were lower at ripe stage on Day 4 337

in heat-treated fruit than in the control on Day 8 (Table 2). 338

339

3.7. Multivariate analysis and data visualisation 340

The PCA score plot based on aroma volatiles data showed segregation of different groups of 341

VHT and control fruit, depending upon the fruit ripening stage (Fig. 3A). Principal 342

component 1 (PC1), 2 (PC2) and 3 (PC3) accounted for 43.73%, 23.69%, and 14.59% of the 343

data variance, respectively. The first two PCs collectively could explain 67.42% of the total 344

variance (Fig. 3A) .The control and VHT fruit groups at unripe stage, though segregated to 345

some extent, were present in the same quadrant on the day of treatment. The VHT and 346

control fruit at 4 days showed segregation to greater extent revealing differences in their 347

ripening stages. The aroma volatiles profile of over-ripe VHT fruit led to its segregation from 348

the ripe control fruit after 8 days at ambient conditions. The HCA showed clustering of 349

control and VHT fruit at different stages of ripening into two principal clusters, one 350

representing over-ripe VHT fruit at 8 days and another showing all remaining groups (Fig. 351

3B). The ripe VHT fruit at 4 days and control at 8 days clustered together in the HCA which 352

supports the observations from PCA score plot (Fig. 3A) and fruit ripening data on firmness 353

and skin colour (Fig. 1). The comparative quantitative changes in the aroma volatiles profiles 354

of VHT and control fruit during different stages of fruit ripening are shown in the heat map 355

(Fig. 4). 356

357

4. Discussion 358

To achieve phytosanitary security against the regulated insect pests in Australia, Japan and 359

New Zealand, Indian mangoes to be exported to these countries must be heated to 48 °C fruit 360

core temperature and held for more than 20 min. Results indicated that VHT accelerated skin 361

yellowing and fruit softening in ‘Chausa’ mangoes (Fig. 1). The rate of fruit ripening in heat-362

treated fruit was almost double to that in untreated fruit. Various types of heat treatments 363

including VHT have been reported to promote fruit softening and skin yellowing in mango 364

cultivars, such as ‘Kensington Pride’ (Jacobi & Giles, 1997), ‘Tommy Atkins’, and ‘Keitt’ 365

(McGuire, 1991; Pesis et al., 1997). Fruit physiological processes such as respiration rate 366

remained elevated for about 4–6 days in heat-treated ‘Keitt’ mangoes (Mitcham & 367

McDonald, 1993). Similar to ‘Keitt’, heat stress might have caused a significant increase in 368

respiration rate of ‘Chausa’ mangoes resulting in faster fruit ripening. The increased rates of 369

degradation of chlorophyll pigments in the skin and synthesis of carotenoids and up–370

regulation of the activities of cell wall hydrolytic enzymes, such as pectin methyl esterase and 371

polygalacturonase, may be responsible for faster fruit ripening in response to heat treatment 372

(Jacobi et al., 2001). Contrarily, VHT (50 °C for 240 min) has also been reported to 373

reversibly inhibit the rate of fruit ripening for about 3 days in ‘Tommy Atkins’ mangoes, due 374

to suppression of activity of ethylene biosynthesis enzyme, ACC oxidase, colour 375

development and softening in the inner mesocarp (Mitcham & McDonald, 1997). The 376

variable effects of heat treatments on fruit physiology and ripening behaviour may arise due 377

to multiple factors, such as temperature–time regime, cultivar, fruit size, maturity, preharvest 378

and postharvest conditions (Jacobi et al., 2001; Lurie, 1998). 379

More than 300 aroma volatiles have been identified in various mango cultivars (Lalel 380

et al., 2003a; MacLeod & Snyder, 1985; Pandit et al., 2009; Pino et al., 2005) and the 381

variability in these compounds is reported to be dependent on several preharvest and 382

postharvest factors (Chidley et al., 2013, Dang et al., 2008; Kulkarni et al., 2012; Lalel et al., 383

2003a, 2003b; Lalel and Singh, 2006; Vazquez–Luna et al., 2011). The diversity of aroma 384

volatiles identified and quantified in various mango cultivars offers a challenge for 385

developing and adopting a comprehensive procedure for maximum coverage of these 386

compounds. The extraction of aroma volatiles in mango has been accomplished by 387

employing organic solvents (Chidley et al., 2013; Pandit et al., 2009; Pino et al., 2005) and 388

solvent–free techniques (Dang et al., 2008; Lalel et al., 2003a; Lalel & Singh, 2006). 389

However, the solvent–free method using HS-SPME has been extensively used for extraction 390

of aroma volatiles, due to its ease, sensitivity, and superiority in extraction efficiencies. The 391

extraction of aroma volatiles by HS-SPME from ‘Chausa’ mango resulted in qualitative and 392

quantitative determination of 50 aroma volatiles, compared with the solvent extraction 393

method (Pandit et al., 2009), which yielded 28 compounds in the same cultivar. Our data 394

show that the most abundant group of volatile compounds in ‘Chausa’ cultivar belonged to 395

sesquiterpene class, which constituted about 80–86 % of the total volatiles, followed by 396

monoterpenes (4–9%), esters (2–6%) and aldehydes, alkanes, ketones, and others (5–11%) 397

which is partially in agreement with a previous study in which sesquiterpenes (55%) were the 398

dominant class of volatiles, followed by monoterpenes (33%), lactones (6.4%) and aldehydes 399

and ketones (6%) (Pandit et al., 2009). The higher number and concentrations of 400

sesquiterpenes in our data may be attributed to the solvent-free HS-SPME technique, which is 401

more effective in extraction of sesquiterpenes than the solvent extraction method followed by 402

Pandit et al. (2009). All these aroma volatiles have been previously reported in various 403

cultivars of mango fruit (Lalel et al., 2003; Pandit et al., 2009; Pino et al., 2005), but several 404

compounds like aromadendrene, ledene, alloaromadendrene, α–gurjunene, and isoledene 405

have been identified and quantified for the first time in ‘Chausa’ mango. A few 406

sesquiterpenes, such as longifolene, longicyclene, longipinene, longidione, and germacrene D 407

identified in ‘Chausa’ mango by Pandit et al. (2009) were not detected in our study. 408

Monoterpenes profile of ‘Chausa’ mango in our data agrees well with the previous study on 409

aroma volatiles profiling in Indian mango cultivars. 410

The total aroma volatiles production was reversibly inhibited immediately after heat 411

treatment in mangoes (Fig. 2A) which is in agreement with the findings of Fallik et al. 412

(1997) in apple fruit subjected to heat treatment (38 °C for 4 days). In comparison with 413

control fruit on the 8th day, the VHT fruit at ripe stage produced lower amount of aroma 414

volatiles on the 4th day (Fig. 2A). However, the emission increased significantly as the heat-415

treated fruit became over-ripe on the 8th

day. The data clearly indicated that the recovery of 416

aroma volatiles synthesis in heated fruit was not parallel to the ripening-related changes in 417

skin colour and flesh softening. Physiological and biochemical alterations in response to heat 418

could be responsible for the delayed recovery of volatile synthesis mechanism. The increase 419

in aroma volatiles production during fruit ripening in mango has been reported to be 420

synchronous with skin yellowing and flesh softening (Lalel et al., 2003a). No significant 421

impact of hot water dip treatment (52 °C for 10 min) on aroma volatiles production in 422

‘Kensington’ mangoes has been reported (Dang et al., 2008), which is contrary to our results 423

because the hot water dip treatment conducted for postharvest disease control was very mild 424

compared to our study that was focused on application of VHT for insect disinfestation (fruit 425

core temperature 48 °C for 20 min). The temporary inhibition of volatiles in ‘Golden 426

Delicious’ apples was observed by heat treatment (38 °C for 4 days), but eventually 427

recovered to even higher levels than non-heated fruit after 6 weeks of storage at 1 °C (Fallik 428

et al., 1997). Contrastingly, heat treatment for the same duration in ‘Redchief Delicious’ 429

apples stored for 4 weeks at 4 °C resulted in a drastic reduction in the total esters in heat-430

treated fruit (Escalada & Archbold, 2009). The discrepancies in the reports might have arisen 431

due to the time of measurement of aroma volatiles. Our data show that volatile emissions in 432

heat-treated mangoes at over-ripe stage (8th

day) were much higher than at the ripe stage (4th

433

day). The comparison of volatiles profiles at different fruit ripening stages appears to be more 434

appropriate rather than the number of days after treatment and transfer from cold storage as 435

mentioned in previous studies (Fallik et al., 1997; Escalada & Archbold, 2009). 436

In addition to the total volatiles, VHT altered the concentrations and composition of 437

aroma volatiles profiles both at unripe and ripe stages. The emission of total sesquiterpenes, 438

the major aroma volatiles contributors, was significantly inhibited by VHT on the day of 439

treatment, while the effect on total monoterpenes was non-significant (Fig. 2B & C). On the 440

day of heat treatment, the suppression of emission of sesquiterpenes such as trans–441

caryophyllene (sweet, floral, dry wood and clove leaf oil-like odour) and α-humulene (fresh, 442

green and floral odour; MacLeod & Snyder, 1985) was observed. The negative effects of heat 443

on principal aroma volatiles such as esters in apple (Escalada & Archbold, 2009; Fallik et al., 444

1997) and monoterpenes in oranges (Obenland et al., 1999) have been previously reported. 445

The heat–induced alteration in the enzyme systems that catalyse the synthesis of volatile 446

compounds has been speculated by Fallik et al. (1997). Interestingly, the levels of esters such 447

as ethyl dodecanoate and neryl acetate increased immediately after heat treatment (Table 2). 448

The heat treatment might have stimulated β-oxidation of fatty acids as the biosynthesis of 449

fatty acids, the precursors of esters, has been found to be high during ripening of mango 450

(Lalel et al., 2003a). In another study, forced-air heating of navel orange to core temperatures 451

of 44 °C and then holding at that temperature for 100 min has also been reported to enhance 452

the concentrations of esters like ethyl propaonate, methyl butanoate, ethyl hexanoate and 453

ethyl butanoate, concomitant with the loss of freshness in heat-treated oranges (Obenland et 454

al., 2012). 455

From a consumer perception perspective, the aroma volatiles profile is important at 456

the ripe stage. The heat-treated fruit at the ripe stage showed lower concentration of total 457

monoterpenes (Fig. 2C), especially α-terpinolene, and total esters (Fig. 2D), which could 458

have potentially a negative impact on the fruit flavour. α–Terpinolene has floral, sweet, and 459

pine–like aroma notes (Engel & Tressl, 1983), whereas the esters are known for their 460

characteristic fruity odour, coinciding with eating-ripe stage in mango (Lalel et al., 2003a). 461

Over-ripeness in heat-treated fruit showed several-fold increase in the concentrations of 462

sesquiterpenes (trans-caryophyllene, α-humulene and aromadendrene), monoterpenes (α-463

terpinolene, δ-3-carene, limonene, γ-terpinene, α-phellandrene, myrcene, and ocimene), 464

benzothaizole and linalool oxide (Tables 1 & 2). The upsurge in emission of volatiles at over-465

ripe stage in heated fruit may be attributed to the delay in recovery of aroma volatiles 466

synthesis as a detrimental effect of heat. The multivariate statistical analysis of the 467

quantitative data on aroma volatiles also showed that the aroma quality of VHT fruit at ripe 468

stage was closer to that of the semi-ripe control fruit after 4 days at ambient conditions (Fig. 469

3). The segregation of VHT and control fruit could be achieved through PCA and HCA, 470

facilitating data visualisation in terms of aroma-volatiles contribution to different stages of 471

fruit ripening (Fig. 3). 472

In conclusion, VHT accelerated the process of fruit ripening and resulted in fruit 473

reaching edible-soft stage within 4 days after heat treatment against 8 days in control. The 474

physiological effects of quarantine treatment in terms of faster rate of fruit ripening have 475

implications on limited time period availability for transport and distribution of this cultivar 476

to the export markets. The heat-induced logarithmic increase in the rate of fruit ripening 477

proceeded with a significant lag in the emission of aroma volatiles. Postharvest VHT as a 478

phytosanitary measure caused reversible inhibition of aroma volatiles emission in ‘Chausa’ 479

mango, with a significant alteration in aroma volatiles composition at different stages of fruit 480

ripening. The suppression of aroma volatiles at eating-soft stage coincident with skin 481

yellowing may have a deterrent effect on the consumer acceptance of the fruit. The 482

phytosanitary protocols for mango fruit have evolved over several years of research and 483

policy interventions and thus have limited scope for amendment. However, future research is 484

required to minimise the adverse effect of heat-based phytosanitary treatment on flavour 485

quality of fresh mango fruit, through manipulation of other factors such as harvest maturity, 486

storage and ripening conditions, in order to consistently deliver a high quality mango fruit to 487

the consumer. 488

489

Acknowledgements 490

We are thankful to Mr. Narendra Malik for facilitating VHT at mango packing house, 491

Saharanpur, Uttar Pradesh. We acknowledge technical support from Mr. Jagdeep Singh and 492

financial support from NABI, Department of Biotechnology, Government of India. 493

494

References 495

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temporal changes in the volatile profile of Alphonso mango upon exogenous ethylene 497

treatment. Food Chemistry, 136, 585–594. 498

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methods and cold storage on volatiles, color development and fruit quality in ripe 500

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HortScience, 44(6):1637–1640 507

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Heat treatment temporarily inhibits aroma volatile compound emission from Golden 509

Delicious apples. Journal of Agricultural and Food Chemistry, 45, 4038–4041. 510

Heather, N. W., Corcoran, R. J., & Kopittke, R. A. (1997). Hot air disinfestation of 511

Australian ‘Kensington’ mangoes against two fruit flies (Diptera: Tephritidae). 512

Postharvest Biology and Technology, 10, 99–105 513

Jacobi, K., Giles, J. E., MacRae, E. A., & Wegrzyn, T. (1995). Conditioning ‘Kensington’ 514

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Jacobi, K. K., MacRae, E. A., & Hetherington, S. E. (2001). Postharvest heat disinfestation 520

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ripening, quality and biosynthesis of aroma volatile compounds in ‘Kensington Pride’ 528

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5184–5188. 547

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APEDA (2007). Guidelines for export of Indian mangoes to Japan. Agricultural & processed food 570

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Government of India. 572

http://www.apeda.gov.in/apedawebsite/announcements/guidelines_exportmangoestoja573

pancdversion.doc Accessed on 11 Jan 2014. 574

Biosecurity Australia (2011). Revised conditions for importing fresh mango fruit from India, final 575

report. Biosecurity Australia, Canberra, Australia. 576

http://www.daff.gov.au/__data/assets/pdf_file/0011/1901567/Mangoes_from_India-577

Final_revised_conditions.pdf Accessed on 11 Jan 2014. 578

Biosecurity New Zealand (2012). Import health standard: fresh fruit/vegetables. Mangoes, 579

(Mangifera indica) from India. http://www.biosecurity.govt.nz/files/ihs/mango-in.pdf 580

Accessed on 11 Jan 2014. 581

582

583

584

585

586

587

588

589

590

591

592

593

594

595

596

597

Figure captions 598

Fig. 1. Effects of VHT on (A) subjective skin colour rating (1, 100% green; 5 = 100% 599

yellow), (B) skin colour b* value, (C) subjective fruit firmness (1, hard; 2, sprung; 3, slightly 600

soft; 4, eating soft; 5, overly soft), and (D) flesh firmness during ripening at ambient 601

conditions for 8 days. n = 30 (10 fruit × 3 replications). The bars in the right panel, showing 602

mean values, bearing the same letter(s) are not significantly different (p ≤ 0.05). Fruit 603

ripening stages: 0 day, unripe in both control and VHT; 4 day, semi–ripe in control and ripe 604

in VHT; 8 day, ripe in control and over–ripe in VHT. 605

606

Fig. 2. Effects of VHT on the concentrations of (A) total aroma volatiles, (B) sesquiterpenes, 607

(C) monoterpenes and (D) esters in the pulp of mango fruit during ripening at ambient 608

conditions. The bars, showing mean values, bearing the same letter(s) are not significantly 609

different (p ≤ 0.05). Fruit ripening stages: 0 day, unripe in both control and VHT; 4 day, 610

semi–ripe in control and ripe in VHT; 8 day, ripe in control and over-ripe in VHT. 611

612

Fig. 3. Principal component analysis (PCA) and hierarchical cluster analysis (HCA) of the 613

aroma volatiles of VHT and control fruit during different stages of fruit ripening. (A) Score 614

plot for the first two principal components of PCA and (B) clustering pattern based on HCA. 615

616

Fig. 4. Heat map showing quantitative changes in aroma volatiles data for VHT and control 617

fruit during different stages of fruit ripening. 618

619

Figure 1.

0

1

2

3

4

5

Skin

co

lou

r(1

-5 s

cale

)

Control VHT

LSD (P≤0.05)

A

0

1

2

3

4

5

0 1 2 3 4 5 6 7 8

Fir

mn

ess

(1-5

scale

)

Ripening period (days)

LSD (P≤0.05)

C

0

30

60

90 Firm

ness (N

)

Ripening period (days)

b

aa

c c

d

D

0 4 8

0

20

40

60b

* valu

eVHT Control

b

aa

cc

cB

Figure 2

0

300

600

900

1200

Ses

qu

iterp

en

es

µg

.100 g

-1F

W

b

a

b bb

c

B

0

30

60

90

120

Mo

no

terp

en

es

µg

.100 g

-1F

W

aa

a a

b

cC

0

5

10

15

20

0 4 8

Este

rsµ

g.1

00 g

-1F

W

Ripening period (days)

aba

b

cc

a

D

0

300

600

900

1200

To

tal

aro

ma v

ola

tile

sµ

g.1

00 g

-1F

W

Control VHT

b

a

bb

c

dA

Figure 3

VH

T 0

day

Co

ntr

ol

0 d

ay

Co

ntr

ol

4 d

ays

VH

T 4

days

VH

T 8

da

ys

Co

ntr

ol

8 d

ays

0.85

0.92

1.0

A

B

Figure 4

Minimum Maximum

Table 1. Effects of VHT on the concentrations of different sesquiterpenes and monoterpenes (µg 100g-1

FW) in the pulp of mango fruit during ripening

at ambient conditions for 8 days

* Identification by mass spectra and Kovats index with authentic standards. Kovats index on SPB–5 MS

™ column.

† Fruit ripening stage was determined by changes in skin colour and flesh firmness (refer to Fig. 1).

Day 0 Day 4 Day 8

Compound* Kovats

RI

Control

(unripe)†

VHT

(unripe)

Control

(semi–ripe)

VHT

(ripe)

Control

(ripe)

VHT

(over–ripe)

Sesquiterpenes

trans–Caryophyllene 1465 243.1b 186.7a 243.2b 234.9b 287.3c 406.8d

α–Humulene 1467 100.7b 31.18a 118.8b 92.61b 110.5b 318.1c

Aromadendrene 1475 8.09a 8.25a 40.27b 48.21b 86.88c 181.1d

Ledene 1508 46.27d 21.79b 26.47b 37.13c 19.72a 45.17d

Alloaromadendrene 1496 3.87d 1.96c 0.86a 1.57bc 0.71a 1.29b

α–Gurjunene 1412 0.07a 0.06a 0.11a 0.12a 0.43b 0.50c

Isoledene 1408 0.28a 0.23a 0.24a 0.25a 0.25a 0.24a

Table(s)

Monoterpenes

α–Terpinolene 1102 12.69a 6.47a 11.33a 4.34a 35.59b 92.46c

δ–Car–3–ene 1024 1.09a 0.37a 0.75a 1.62a 3.95b 16.22c

Limonene 1033 0.44a 0.45a 0.64a 0.65a 2.17a 2.33a

γ–Terpinene 1070 0.19a 0.18a 0.19a 0.18a 0.23a 0.37b

Camphene 967 0.09a 0.07a 0.07a 0.07a 0.08a 0.08a

Car–2–ene 1014 0.20a 0.19a 0.20a 0.20a 0.27a 0.22a

α–Phellandrene 1017 0.15a 0.15a 0.15a 0.17a 0.18a 3.32a

α–Pinene 948 0.22a 0.22a 0.24a 0.22a 0.28a 0.28a

β–Pinene 995 0.19a 0.16a 0.16a 0.16a 0.17a 0.61b

Myrcene 992 0.45a 0.42a 0.45a 0.43a 0.45a 2.94b

Ocimene 1049 0.16a 0.16a 0.19a 0.19a 0.16a 0.58b

p–Cymene 1036 0.19a 0.19a 0.27b 0.20a 0.24a 0.63c

4–Terpineol 1179 1.75a 3.54ab 5.17b 10.80c 12.96d 3.17ab

Carvone 1265 0.93a 1.49b 2.75c 1.46b 1.49b 4.10d

Geranyl acetate 1364 8.95c 5.51b 8.46c 1.03a 0.74a 0.61a

Table 2. Effects of VHT on the concentrations (µg 100g-1

FW) of esters, aldehydes, alkanes, ketones, and others in the pulp of mango fruit during

ripening at ambient conditions for 8 days

Day 0 Day 4 Day 8

Compound* Kovats

RI

Control

(unripe)†

VHT

(unripe)

Control

(semi–ripe)

VHT

(ripe)

Control

(ripe)

VHT

(over–ripe)

Esters

Ethyl acetate 628 0.08a 0.08a 0.08a 0.07a 0.08a 0.08a

Ethyl butyrate 796 0.22a 0.23a 0.23a 0.23a 0.24a 0.23a

Ethyl hexanoate 993 6.50c 7.33c 4.80b 1.47a 1.44a 6.75c

Ethyl heptanoate 1092 0.11a 0.13a 0.16b 0.17b 0.27d 0.24c

Ethyl octanoate 1200 2.36ab 1.58a 4.49c 2.53ab 1.89a 3.20b

Ethyl dodecanoate 1589 1.10b 3.13d 0.22a 2.55cd 7.02e 2.59cd

Hexyl acetate 1006 0.16a 0.22bc 0.16a 0.24c 0.18ab 0.17a

Methyl butyrate 720 0.16a 0.13a 0.12a 0.14a 0.13a 0.14a

* Identification by mass spectra and Kovats index with authentic standards. Kovats index on SPB–5 MS

™ column.

† Fruit ripening stage was determined by changes in skin colour and flesh firmness (refer to Fig. 1).

Methyl hexanoate 1000 0.47c 0.48c 0.20a 0.23ab 0.26b 0.23ab

Octyl acetate 1149 0.22ab 0.30c 0.19a 0.24b 0.22ab 0.21ab

Ethyl benzoate 1183 0.23a 0.23a 0.24ab 0.27bc 0.29c 0.24ab

Ethyl caprylate 1191 0.24a 0.21a 0.23a 0.23a 0.21a 0.20a

Methyl benzoate 1104 0.47c 0.21a 0.23ab 0.23ab 0.30b 0.24ab

Methyl nonanoate 1221 0.19a 0.14a 0.70c 0.52b 0.12a 0.16a

Neryl acetate 1363 1.54b 3.61c 1.25ab 3.19c 0.82a 1.63ab

Nopyl acetate 1445 0.20b 0.12a 0.28c 0.30c 0.21b 0.31c

Aldehydes

Benzaldehyde 973 2.25a 8.35c 19.55e 29.12f 10.73d 4.30b

Octanal 1002 2.55c 1.54b 1.50b 0.64a 1.29b 0.82a

Hexanal 800 0.03a 0.09b 0.17c 0.18c 0.10b 0.11b

Alkanes

Cycloheptane 808 0.08a 0.08a 0.32a 0.08a 0.08a 0.33a

Tridecane 1297 0.14a 0.11a 0.83b 1.38c 2.28c 0.14a

Pentadecane 1497 0.25b 0.07a 1.26d 0.28b 1.55e 0.66c

Hexadecane 1590 0.14a 0.11a 0.83b 1.38c 2.28d 1.14bc

Ketones

2,3–Pentendione 701 1.58a 0.96a 0.32a 1.22a 1.04a 0.15a

Cyclohexanone 901 1.35a 1.17a 5.22c 1.54a 3.94b 1.70a

6–Methyl–5–

heptenone

985 3.41a 3.23a 2.61a 1.62a 1.31a 1.40a

Others

Benzothiazole 1259 0.98a 1.20a 5.64b 10.51c 12.26d 13.67d

Linalool oxide 1099 0.81a 1.40ab 2.18b 4.36c 5.62d 4.76cd

33

Highlights 28

• Vapour heat treatment (VHT) accelerated the process of fruit ripening in ‘Chausa’ 29

mango fruit. 30

• Volatiles emission was not synchronous with ripening in heat-treated fruit. 31

• Reversible inhibition of aroma volatiles was caused by VHT. 32

• Heat-treated fruit at ripe stage were deficient in terpenes and esters. 33

• Volatiles emission in heat-treated fruit upsurged at over-ripe stage. 34

35

36

37