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POSTHARVEST HEAT STRESS AND SEMI-PERMEABLE FRUIT COATING TO IMPROVE QUALITY AND EXTEND SHELF LIFE OF CITRUS FRUIT DURING AMBIENT TEMPERATURE STORAGE

MD. GOLAM FERDOUS CHOWDHURY

A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA

2018

Dedicated to my beloved parents, and my family

ACKNOWLEDGMENTS

First and foremost, I would like to thank and express my gratitude to Almighty

Allah who gave me patience, and kept my good health towards this achievement. I would like to thank my advisor, Professor Dr. Jeffrey K. Brecht for his encouraging role as mentor and for his invaluable guidance, support and all kinds of assistance through all those years.

I would like to thank my co-chair and member of my dissertation supervisory committee, Dr. Mark A. Ritenour, for his valuable assistance and support during my study and dissertation research. I would like to thank the other members of my dissertation supervisory committee, Dr. Steven A. Sargent, Dr. Charles A. Sims, and Dr.

M. Miaruddin for their help and support through this work.

I would like to thank the postharvest family, Senior Biological Scientists Kim

Cordasco and Adrian Berry, Dr. Ramadhani, Dr. Mildred, and Dr. Angelos Deltsidis, Dr.

Alvin Cheng, JBT Company for providing coatings, Dr. Jinhi Bai, also Sara Marshall for their help and support. I would like to thank and express my gratitude to the BARI PHT

Divisional scientists and staff for their cordial assistance. I would like to thank the BARI authority and MoA, Govt. of Bangladesh, who approved my deputation leave.

Last, but not least, I would like to thank my Sponsor, as this work was made possible by the generous Support of the American people through the United States

Agency for International Development (USAID)-as part of the Feed the Future Initiative, under CGIAR fund (Award No. BFS-G-11-00002), and the predecessor fund the Food

Security and Crisis Mitigation II grant (Award number EEM-G-00-04-00013). The opinions expressed herein are those of the author(s) and do not necessarily reflect the views of the USAID or the United States Government.

TABLE OF CONTENTS

Page

ACKNOWLEDGMENTS...... 4

LIST OF TABLES ...... 9

LIST OF FIGURES...... 16

LIST OF ABBREVIATIONS ...... 21

ABSTRACT ...... 24

CHAPTER

1 INTRODUCTION...... 26

2 LITERATURE REVIEW...... 29

Origin and Cultivars of Citrus Fruits ...... 29 Citrus Fruits Production Trends in the World and in Bangladesh ...... 31 Citrus Fruits Production Trends in the World ...... 31 Citrus Production Trends in Bangladesh ...... 31 Nutritional Importance of Citrus Fruits ...... 35 Nutritional Composition in Citrus Fruits ...... 35 Volatile Compounds in Citrus Fruits ...... 35 Postharvest Physiology of Citrus Fruits...... 41 Postharvest Treatment to Citrus Fruits ...... 43 Postharvest Fungicide Treatment ...... 43 Postharvest De-greening Treatment...... 45 Physiological Indicators of Tolerance versus Stress from Heat Treatment ...... 46 Tolerance of Fruits to Heat Stress ...... 46 Benefits of Hot Water Stress Treatment to Fruits...... 47 Effect of Heat Stress Treatment on Chilling Sensitive Fruits ...... 49 Effect of Heat Stress Treatment on Decay Control ...... 50 Effects of Heat Stress Temperature and Atmosphere Modification on Fruits .... 51 Modifying the Atmosphere Around and Within Produce by Restricting Gas Diffusion: Modified Atmosphere Packaging (MAP) and Coatings ...... 53 The Role of Anaerobic Metabolites of Produce in Each Variety/Condition ...... 55 Effect of Anaerobic Stress on Ethylene Production and Fruit Softening ...... 57 Production of Off-flavor in Response to Anaerobic Stress...... 59 Fermentative Metabolism and Fruit Disorders ...... 60 Effect of Heat Stress Treatment on Sensory Quality ...... 65 Semi-permeable Coatings for Produce ...... 67 Fruit Coatings to Create Internal Modified Atmospheres ...... 68

Benefits of Wax Coatings to Maintain Quality and Reduce Physiological Disorders of Fruits ...... 70 Main Matrix Constituents for Edible Wax Coating Formulation ...... 72

3 USING HOT WATER IMMERSION AND EDIBLE COATINGS TO MAINTAIN PHYSICOCHEMICAL ATTRIBUTES AND SHELF LIFE OF NAVEL AND VALENCIA ORANGES ...... 75

Materials and Methods ...... 78 Experiment 1 -- Temperatures...... 78 Experiment 2 – Internal Atmospheres ...... 80 Experiment 3 – Optimal Imersion ...... 82 Experiment 4 – Optimal Combination of Temperature and Immersion...... 84 Experiment 5 – Respiration Rate and Ethylene Production...... 85 Results ...... 87 Determination of the Temperature at Which Maximal Respiration Rate and Ethylene Production Occurs in Fresh Navel Orange Fruit...... 87 Internal Atmospheres of Valencia Oranges During Storage in Controlled Atmosphere (CA) Condition...... 87 Determination of the Most Suitable Temperature(S) and Time(S) for Hot Water Immersion to Create a Potentially Beneficial Internal Modified Atmosphere (MA) in Orange Fruit ...... 88 Effect of Combined Harvest on O2, CO2, and C2H4 for Lower Water Immersion Temperature with Higher Duration...... 89 Effect of Combined Harvest on Internal O2, CO2, and C2H4 for Higher Water Immersion Temperatures with Shorter Durations ...... 91 Comparison of Fruit Internal Atmosphere between Navel and Valencia Orange ...... 93 Peel Injury Incidence and SeverityfFor Valencia Oranges During 3 Weeks Storage...... 94 Internal Temperature Profiles of Navel Oranges Using the Best Combination of Hot Water Treatment Temperature and Immersion Duration ...... 94 Respiration Rate and Ethylene Production of Navel Oranges During Storage at Different Temperatures after Immersion in 45oc Water with or without Application of Edible Wax Coating...... 95 Discussion ...... 97 Chapter Summary ...... 102

4 USE OF HOT WATER IMMERSION AND EDIBLE COATINGS TO MAINTAIN QUALITY AND SHELF LIFE OF NAVEL AND VALENCIA ORANGES DURING SIMULATED AMBIENT TEMPERATURE STORAGE...... 127

Materials and Methods ...... 129 Experiment 1 – Effect of Immersion...... 129 Experiment 2 – Effect of Hot Water Treatment ...... 129 Experimental Procedure ...... 130

Chemical Analyses ...... 134 Statistical Analysis ...... 139 Results ...... 141 Ambient Water Immersion of Un-Coated Navel Orange Fruit Quality and Shelf Life During Simulated Ambient Temperature Storage...... 141 Hot Water Treatment and Wax Coating on Quality and Shelf Life of Navel and Valencia Oranges During Simulated Ambient Temperature Storage. ... 141 Comparison of Two Cultivars with 4-Combination (Navel Vs Valencia) ...... 148 Comparison of Two Cultivars with 5-Combination (Navel Vs Valencia) ...... 150 Gas Exchange into Orange Fruit...... 151 Discussion ...... 153 Chapter Summary ...... 155

5 SENSORY ACCEPTABILITY OF NAVEL AND VALENCIA ORANGE FRUIT TREATED WITH HOT WATER IMMERSION AND EDIBLE COATINGS DURING STORAGE ...... 205

Materials and Methods ...... 206 Hedonic Scale ...... 207 Just About Right Scale...... 207 Statistical Analysis ...... 208 Results ...... 208 Navel Oranges ...... 208 Valencia Oranges ...... 210 Discussion ...... 214

6 USE OF HOT WATER IMMERSION AND EDIBLE COATING TO MAINTAIN QUALITY AND SHELF LIFE OF SWEET ORANGE AND POMELO FRUIT DURING SIMULATED AMBIENT TEMPERATURE STORAGE IN BANGLADESH ...... 224

Materials and Methods ...... 226 Sample Collection and Experimentation ...... 226 Fruit Peel Appearance and Fruit Firmness Resistance ...... 227 Nutritional Quality Study ...... 228 Sensory Quality Evaluation ...... 229 Results ...... 230 Effect of Hot Water Treatment and Carnauba Wax Coating on Improving Quality Retention of Sweet Orange (BARI Malta 1) During Ambient Storage...... 230 Effect of Hot Water Treatment and Carnauba Wax Coating on Improving Quality Retention of Pomelo Fruit (Var. Commercial) During Ambient Storage...... 233 Pomelo Fruit ...... 238 Sensory Quality Evaluation ...... 239 Discussion ...... 239

Sweet Orange...... 239 Pomelo Fruit ...... 242 Sensory Quality ...... 243 Chapter Summary ...... 244

7 OVERALL CONCLUSIONS AND RECOMMENDATIONS ...... 269

APPENDIX

A SENSORY EVALUATION BALLOT OF ORANGE FRUITS ...... 273

B SENSORY EVALUATION BALLOT OF SWEET ORANGE (BARI MALTA 1) AND POMELO FRUIT (UNKNOWN COMMERCIAL)...... 276

LIST OF REFERENCES ...... 279

BIOGRAPHICAL SKETCH ...... 308

LIST OF TABLES

Table page

3-1 Determining the most suitable hot water immersion temperature(s) and time(s) to create an internal modified atmosphere (MA) inside Navel orange fruit...... 104

3-2 Mean values ± SE for internal atmospheres of Navel oranges treated with hot water at 40oC for different durations...... 104

3-3 Analysis of variance table for combined harvest effect of Valencia orange on O2, CO2, and C2H4 for lower water immersion temperature with longer duration...... 105

3-4 Effect of harvest x hot water temperature on CO2 for Valencia orange...... 105

3-5 Effect of harvest x duration of hot water treatment on O2 for Valencia orange. . 105

3-6 Effect of harvest x duration of hot water treatment on C2H4 for Valencia orange...... 106

3-7 Effect of hot water temperature x duration on O2 for Valencia orange...... 106

3-8 Effect of hot water temperature x duration on CO2 for Valencia orange...... 106

3-9 Effect of hot water temperature x duration on C2H4 for Valencia orange...... 106

3-10 Effect of hot water temperature x treatment duration x harvest on O2 for Valencia orange...... 107

3-11 Effect of hot water temperature x treatment duration x harvest on C2H4 for Valencia orange...... 107

3-12 Analysis of variance table for combined harvest effect of Valencia orange on O2, CO2, and C2H4 for higher water immersion temperature with shorter duration...... 108

3-13 Effect of harvest x hot water immersion temperature on O2 for Valencia orange...... 108

3-14 Effect of harvest x hot water immersion temperature on CO2 for Valencia orange...... 108

3-15 Effect of harvest x hot water immersion temperature on C2H4 for Valencia ...... 109

3-16 Effect of harvest x treatment duration of hot water immersion on O2 for Valencia orange...... 109

3-17 Effect of harvest x treatment duration of hot water immersion on CO2 for Valencia orange...... 109

3-18 Effect of harvest x treatment duration of hot water immersion on C 2H4 for Valencia orange...... 109

3-19 Effect of hot water temperature x duration of hot water immersion on O2 for Valencia orange...... 110

3-20 Effect of hot water temperature x duration of hot water immersion on C2H4 for Valencia orange...... 110

3-21 Effect of hot water temperature x duration of hot water immersion x harvest on O2 for Valencia orange...... 110

3-22 Effect of hot water temperature x duration of hot water immersion x harvest on C2H4 for Valencia orange...... 111

3-23 Percentage peel injury and peel injury severity of hot water treated Valencia orange during 3 weeks of storage at 250C with 85±5%RH...... 112

4-1 Fruit and treatment conditions used for six studies evaluating the effects of a 30 minutes hot water dip and different edible wax coatings on quality and shelf life of Navel and Valencia orange during simulated ambient temperature storage...... 157

4-2 Analysis of variance table for combined effect of cultivar, storage period, water temperature, and wax coatingz on soluble solids content (SSC), total titratable acidity (TTA), soluble solids content (SSC)/total titratable acidity (TTA), ascorbic acid content (ASC), and total carotenoids content (TCC)z...... 158

4-3 Analysis of variance table for combined effect of cultivar, storage period, water temperature, and wax coating on weight loss (WL), total phenolic compounds (TP), antioxidant capacity (ATC), lightness value (L*), chroma value (C), and hue angle (H) z...... 159

4-4 Analysis of variance table for combined effect of cultivar, storage period, water temperature, and wax coating on compression (COM), puncture (PUN), and oleocellosis (OLC) z...... 160

4-5 Combined effect of cultivar-storage interaction for 3rd harvest of Navel and Valencia orange on SSC, TTA, SSC/TTA, and ASC...... 161

4-6 Combined effect of cultivar-storage interaction for 3rd harvest of Navel and Valencia orange on TCC, WL, TP, and ATC...... 161

4-7 Combined effect of cultivar-storage interaction for 3rd harvest of Navel and Valencia orange on L*, COM, and OLC...... 162

4-8 Combined effect of cultivar-treatment temperature interaction for 3rd harvest of Navel and Valencia orange on TTA, SSC/TTA ratio, ASC, and ATC...... 162

4-9 Combined effect of cultivar-treatment temperature interaction for 3rd harvest of Navel and Valencia orange on L*, C, H, and PUN...... 162

4-10 Combined effect of cultivar-surface coating interaction for 3rd harvest of Navel and Valencia orange on SSC, TTA, SSC/TTA, TCC, and WL...... 163

4-11 Combined effect of cultivar-surface coating interaction for 3rd harvest of Navel and Valencia orange on L*, C, H, PUN, and OLC...... 163

4-12 Combined effect of storage-water temperature interaction for 3rd harvest of Navel and Valencia orange on ASC, ATC, WL, COM, and OLC...... 164

4-13 Combined effect of storage-surface coating interaction for 3rd harvest of Navel and Valencia orange on SSC, SSC/TTA, ATC, and WL...... 164

4-14 Combined effect of storage-surface coating interaction for 3rd harvest of Navel and Valencia orange on C, COM, PUN, and OLC...... 165

4-15 Combined effect of water temperature- surface coating interaction for 3rd harvest of Navel and Valencia orange on ASC, TCC, and WL...... 165

4-16 Combined effect of water temperature-surface coating interaction for 3rd harvest of Navel and Valencia orange on ATC, LG, HU, and COM...... 166

4-17 Combined effect of cultivar-storage-water temperature interaction for 3rd harvest of Navel and Valencia orange on SSC, ATC, COM, and OLC...... 167

4-18 Combined effect of cultivar-storage-surface coating interaction for 3rd harvest of Navel and Valencia orange on TCC, ATC, and WL...... 168

4-19 Combined effect of cultivar-storage-surface coating interaction for 3rd harvest of Navel and Valencia orange on COM, PUN, and OLC...... 169

4-20 Combined effect of water temperature-storage-surface coating interaction for 3rd harvest of Navel and Valencia orange on SSC/TTA, ATC, and COM...... 170

4-21 Combined effect of cultivar-water temperature-surface coating interaction for 3rd harvest of Navel and Valencia orange on SSC, TCC, ATC, and WL...... 171

4-22 Combined effect of cultivar-water temperature-surface coating interaction for 3rd harvest of Navel and Valencia orange on L*, C, H, PUN, and OLC...... 172

4-23 Effect of cultivar – water temperature – wax coating – storage time interaction for 3rd harvest of Navel and Valencia orange on weight loss (WL). . 173

4-24 Effect of cultivar– water temperature – wax coating – storage time interaction for 3rd harvest of Navel and Valencia orange on antioxidant capacity (ATC)..... 174

4-25 Analysis of variance table for combined effect of cultivar, harvest, storage period, and wax coating on SSC, TTA, SSC/TTA, ASC, and TCC during 3 weeks of storage at 25oC with 85%RH z...... 175

4-26 Analysis of variance table for combined effect of cultivar, harvest, storage period, and wax coating on WL, TP, ATC, L*, C, and H angle value during 3 weeks of storage at 25oC with 85%RHz...... 176

4-27 Analysis of variance table for combined effect of cultivar, harvest, storage period, and wax coating on COM force, PUN force, and OLC force during 3- week of storage at 25oC with 85%RHz...... 177

4-28 Effect of cultivar x Harvest x Treatment x Storage time interaction for early, mid & late harvest of Navel and Valencia orange on total soluble solids content (SSC) z...... 178

4-29 Effect of cultivar x Harvest x Treatment x Storage time interaction for early, mid & late harvest of Navel and Valencia orange on total titratable acid content (TTA)z...... 179

4-30 Effect of cultivar x Harvest x Treatment x Storage time interaction for early, mid & late harvest of Navel and Valencia orange on ascorbic acid content (ASC)z...... 180

4-31 Effect of cultivar x Harvest x Treatment x Storage time interaction for early, mid & late harvest of Navel and Valencia orange on weight loss (WL)z...... 181

4-32 Effect of cultivar x Harvest x Treatment x Storage time interaction for early, mid & late harvest of Navel and Valencia orange on compression (COM)z...... 182

4-33 Effect of cultivar x Harvest x Treatment x Storage time interaction for early, mid & late harvest of Navel and Valencia orange on puncture force (PUN)z. .... 184

4-34 Effect of cultivar x Harvest x Treatment x Storage time interaction for early, mid & late harvest of Navel and Valencia orange on oleocellosis (OLC)z...... 185

4-35 Analysis of variance table for combined effect of cultivar, harvest, storage period, and treatment on SSC, TTA, SSC/TTA ratio, ASC, and TCC during 3- week of storage at 25oC with 85%RHz...... 186

4-36 Analysis of variance table for combined effect of cultivar, harvest, storage period, and treatment on WL, TP, ATC, L*, C, and H angle value during 3- week of storage at 25oC with 85%RHz...... 187

4-37 Analysis of variance table for combined effect of cultivar, harvest, storage period, and treatment on COM, PUN, and OLC during 3-week of storage at 25oC with 85%RHz...... 188

4-38 Effect of cultivar x Harvest x Treatment x Storage time interaction for mid & late harvest of Navel and Valencia orange on total titratable acid (TTA)Z...... 189

4-39 Effect of cultivar x Harvest x Treatment x Storage time interaction for mid & late harvest of Navel and Valencia orange on vitamin C (ASC)z...... 190

4-40 Effect of cultivar x Harvest x Treatment x Storage time interaction for mid & late harvest of Navel and Valencia orange on weight loss (WL)z...... 191

4-41 Effect of cultivar x Harvest x Treatment x Storage time interaction for mid & late harvest of Navel and Valencia orange on antioxidant capacity (ATC)z...... 192

4-42 Effect of cultivar x Harvest x Treatment x Storage time interaction for mid & late harvest of Navel and Valencia orange on oleocellosis (OLC) forcez...... 193

4-43 Major volatile compounds detected in sweet orange fruit...... 194

5-1 Sensory ratings of Navel oranges from Harvest 1 that received ambient or hot water immersion treatments followed by application or not of different fruit coatings with weekly evaluations over 3 weeks of ambient storage...... 218

5-2 Sensory ratings of Navel oranges from Harvest 2 that received ambient or hot water immersion treatments followed by application or not of different fruit coatings with weekly evaluations over 3 weeks of ambient storage...... 219

5-3 Sensory ratings of Navel oranges from Harvest 3 that received ambient or hot water immersion treatments followed by application or not of different fruit coatings with weekly evaluations over 3 weeks of ambient storage ...... 220

5-4 Sensory ratings of Valencia oranges from Harvest 1 that received ambient or hot water immersion treatments followed by application or not of different fruit coatings with weekly evaluations over 3 weeks of ambient storage...... 221

5-5 Sensory ratings of Valencia oranges from Harvest 2 that received ambient or hot water immersion treatments followed by application or not of different fruit coatings with weekly evaluations over 3 weeks of ambient storage...... 222

5-6 Sensory ratings of Valencia oranges from Harvest 3 that received ambient or hot water immersion treatments followed by application or not of different fruit coatings with weekly evaluations over 3 weeks of ambient storage...... 223

6-1 Analysis of variance table for sweet orange fruit (var. BARI Malta 1) for fruit weight loss (WL), fruit firmness (FF), soluble solids content (SSC), and total titratable acidity (TTA) during 3 weeks storage at 25oC...... 246

6-2 Analysis of variance table for sweet orange fruit (var. BARI Malta 1) for soluble solids content (SSC)/total titratable acidity (TTA), ascorbic acid content ASC) and total carotenoid content (TCC) during 3 weeks storage at 25oC...... 246

6-3 Combined effect of storage x harvest location interactions on weight loss (WL), fruit firmness (FF), soluble solids content (SSC), SSC/total titratable acidity (TTA) ratio, ascorbic acid content (ASC), and total carotenoid content (TCC) of sweet orange (var. BARI Malta 1)...... 247

6-4 Combined effect of storage x treatment interactions on weight loss (WL), and fruit firmness (FF) of sweet orange (var. BARI Malta 1)...... 248

6-5 Combined effect of treatment-harvest location interactions on weight loss (WL), whole fruit firmness (FF), and SSC/TTA ratio of sweet orange (var. BARI Malta 1) during 3 weeks storage...... 249

6-6 Combined effect of storage-treatment x fruit harvest location interaction on weight loss (WL), whole fruit firmness (FF), SSC/TTA ratio of sweet orange (var. BARI Malta 1) during 3 weeks storage...... 250

6-7 Sensory ratings for the treatments for 2 harvest locations (HL) during 3 weeks storage of sweet orange (BARI Malta 1)...... 252

6-8 Analysis of variance table of pomelo fruit for total soluble solids content (SSC), total titratable acidity (TTA), for soluble solids content (SSC), SSC/TTA ratio, and total carotenoid content (TCC) during 3 weeks storage..... 254

6-9 Analysis of variance table of pomelo fruit (unnamed commercial var.) for fruit weight loss (WL), whole fruit firmness (FF), and ascorbic acid content (ASC) during 3 weeks storage...... 254

6-10 Combined effect of storage x harvest location interactions of pomelo fruit on weight loss (WL), whole fruit firmness (FF), soluble solids content (SSC), ascorbic acid content (ASC), and total carotenoids content (TCC) during 3 weeks storage...... 255

6-11 Combined effect of storage x treatment interactions of pomelo fruit on SSC, TTA, SSC/TTA ratio, TCC, fruit WL, ASC, and FF during 3 weeks storage...... 256

6-12 Effect of treatment-harvest location interactions of pomelo fruit on total carotenoids content (TCC) during 3 weeks storage...... 257

6-13 Combined effect of treatment- fruit harvest location-storage interaction on SSC, TTA, TCC, and ASC during 3 weeks storage...... 257

6-14 Sensory evaluation by overall likability score and JAR scale among the treatments for 2 harvest locations (HL) during 3 weeks storage of pomelo fruit...... 259

LIST OF FIGURES

Figure page

2-1 Map showing the major citrus growing regions in Bangladesh. Reprinted with permission from, http://www.maps-of-the-world.net/maps/maps-of-asia/maps- of-bangladesh/small-administrative-map-of-bangladesh.jpg...... 33

2-2 Area and production of oranges from 2010-2011 to 2014-15 (BBS, 2015) ...... 34

2-3 Production of different Citrus fruit types in Bangladesh, 2014-15 (BBS, 2015) ... 34

3-1 Sample preparation for respiration and ethylene measurement of Navel oranges at room air temperatures ranging from 25oC to 60oC, with 5oC increments. Photo courtesy of author...... 115

3-2 Fruit in containers are connected in series for CA storage of Valencia orange during 3 weeks storage at 5oC or 25oC. Photo courtesy of author...... 115

3-3 Fruit in plastic container showing insertion of the collection tube into fruit to collect internal gas samples. Photo courtesy of author...... 115

3-4 Apparatus for immersing fruit into hot water at different temperatures and for different durations and collecting internal gas samples. Photo courtesy of author...... 116

3-5 Squirrel data logger set up for measurement of temperature & exposure time at different tissue layer of orange fruit immersed in hot water at 45oC for 1 hour. Photo courtesy of author...... 116

3-6 Measurement of respiration rate presented by (Mean+SE) for a) fruit not changed with storage temperature, and b) fruit changed with storage temperature increased from 25oC to 60oC with 5oC increment of fresh Navel orange fruit when each set temperature in storage chamber and fruit inside tissue temperature were similar...... 117

3-7 Internal atmosphere of Valencia orange stored in CA of 5% O2 plus 5% CO2 at 5oC during 3 weeks storage...... 117

3-8 Internal atmosphere of Valencia orange stored in CA of 5% O2 plus 10% CO2 at 5oC during 3 weeks storage...... 118

3-9 Internal atmosphere of Valencia orange stored in CA of 5% O2 plus 5% CO2 at 25oC during 3 weeks storage...... 118

3-10 Internal atmosphere of Valencia orange stored in CA of 5% O2 plus 10% CO2 at 25oC during 3 weeks storage...... 119

3-11 Internal atmosphere in Valencia oranges stored in ambient air (25oC) during 3 weeks storage ...... 119

3-12 Internal gas concentrations of Navel oranges immersed into 40oC water for different durations...... 120

3-13 Internal gas concentrations of Valencia oranges immersed into 40oC water for different durations (Harvest 1)...... 120

3-14 Internal gas concentrations of Valencia oranges immersed into 40oC water for different durations (Harvest 2)...... 121

3-15 Valencia oranges dipping into different water temperature with different duration (Harvest 1)...... 121

3-16 Valencia oranges dipping into different water temperature with different duration (Harvest 2)...... 122

3-17 Mean value with SE of internal O2 of Navel (NAV) and Valencia (VAL) oranges treated with hot water at 40oC or 45oC for 30 min or 20 min...... 122

3-18 Mean value with SE of internal CO2 of Navel (NAV) and Valencia (VAL) oranges treated with hot water at 40oC or 45oC for 30 min or 20 min...... 123

3-19 Mean value with SE of internal C2H4 of Navel (NAV) and Valencia (VAL) oranges treated with hot water at 40oC or 45oC for 30 min or 20 min...... 123

3-20 Temperature profile at different tissue layer of orange fruit submerged in hot water at 45oC for 1 hr...... 124

3-21 Temperature reached in different tissue layers during hot water treatment of 45 oC for 30 or 60 min...... 124

3-22 Effect of storage temperature on respiration rate of Navel oranges after treated into hot water +no coating vs 45oC water immersion + no coating...... 125

3-23 Effect of temperature on respiration rate of Navel orange after treated into ambient water + polyethylene vs hot water + polyethylene...... 125

3-24 Effect of temperature on respiration rate of Navel orange after treated into ambient water + carnauba vs hot water + carnauba...... 126

3-25 Effect of temperature on respiration rate of Navel orange after treated into ambient water + shellac vs hot water + shellac...... 126

4-1 Postharvest treatment of Navel oranges from harvest to storage. Photo courtesy of author...... 194

4-2 Navel orange storage after 3 weeks at 25oC with 85%RH. Photo courtesy of author...... 195

4-3 Valencia orange storage after 3 weeks at 25oC with 85%RH. Photo courtesy of author...... 195

4-4 Internal O2 in four different treatments (AMW+NC, HW+PL, HW+CR, HW+SH) of Navel orange fruit during 3-week of storage at 25oC with 85%RH. 196

4-5 Internal CO2 in four different treatments (AMW+NC, HW+PL, HW+CR, HW+SH) of Navel orange fruit during 3-week of storage at 25oC with 85%RH. 196

4-6 Internal C2H4 in four different treatments (AMW+NC, HW+PL, HW+CR, HW+SH) of Navel orange fruit during 3-week of storage at 25oC with 85%RH . 197

4-7 Internal O2 in four different treatments (AMW+NC, HW+PL, HW+CR, HW+SH) of Valencia orange fruit during 3-week of storage at 25oC with 85%RH ...... 197

4-8 Internal CO2 in four different treatments (AMW+NC, HW+PL, HW+CR, HW+SH) of Valencia orange fruit during 3-week of storage at 25oC with 85%RH ...... 198

4-9 Internal C2H4 in four different treatments (AMW+NC, HW+PL, HW+CR, HW+SH) of Valencia orange fruit during 3-week of storage at 25oC with 85%RH ...... 198

4-10 Internal O2 in five different treatments (AMW+NC, HW+NC, HW+PL, HW+CR, HW+SH) of Navel orange fruit during 3-week of storage at 25oC with 85%RH...... 199

4-11 Internal CO2 in five different treatments (AMW+NC, HW+NC, HW+PL, HW+CR, HW+SH) of Navel orange fruit during 3-week of storage at 25oC with 85%RH...... 199

4-12 Internal C2H4 in five different treatments (AMW+NC, HW+NC, HW+PL, HW+CR, HW+SH) of Navel orange fruit during 3-week of storage at 25oC with 85%RH...... 200

4-13 Internal O2 in five different treatments (AMW+NC, HW+NC, HW+PL, HW+CR, HW+SH) of Valencia orange fruit during 3-week of storage at 25oC with 85%RH...... 200

4-14 Internal CO2 in five different treatments (AMW+NC, HW+NC, HW+PL, HW+CR, HW+SH) of Valencia orange fruit during 3-week of storage at 25oC with 85%RH...... 201

4-15 Internal C2H4 in five different treatments (AMW+NC, HW+NC, HW+PL, HW+CR, HW+SH) of Valencia orange fruit during 3-week of storage at 25oC with 85%RH...... 201

4-16 Internal O2 in with/without HW and with/without coating (AMW=25oC, HW=45oC, NC=No coating, PL=Polyethylene, CR=Carnauba, SH=Shellac) in Navel orange fruit during 3-week of storage at 25oC with 85%RH ...... 202

4-17 Internal CO2 in with/without HW and with/without coating (AMW=25oC, HW=45oC, NC=No coating, PL=Polyethylene, CR=Carnauba, SH=Shellac) in Navel orange fruit during 3-week of storage at 25oC with 85%RH ...... 202

4-18 Internal C2H4 in with/without HW and with/without coating in (AMW=25oC, HW=45oC, NC=No coating, PL=Polyethylene, CR=Carnauba, SH=Shellac) in Navel orange fruit during 3-week of storage at 25oC with 85%RH ...... 203

4-19 Internal O2 in with/without HW and with/without coating in (AMW=25oC, HW=45oC, NC=No coating, PL=Polyethylene, CR=Carnauba, SH=Shellac) Valencia orange fruit during 3-week of storage at 25oC with 85%RH ...... 203

4-20 Internal CO2 in with/without HW and with/without coating (AMW=25oC, HW=45oC, NC=No coating, PL=Polyethylene, CR=Carnauba, SH=Shellac) in Valencia orange fruit during 3-week of storage at 25oC with 85%RH ...... 204

4-21 Internal C2H4 in with/without HW and with/without coating (AMW=25oC, HW=45oC, NC=No coating, PL=Polyethylene, CR=Carnauba, SH=Shellac) in Valencia orange fruit during 3-week of storage at 25oC with 85%RH ...... 204

6-1 Postharvest treatment of sweet orange (BARI Malta 1) from harvest to storage. Photo courtesy of author...... 261

6-2 Sweet orange (BARI Malta 1) storage after 3 weeks at ambient temperature. Photo courtesy of author...... 261

6-3 Pomelo fruit (var.unknown commercial) storage after 3 weeks at ambient temperature. Photo courtesy of author...... 262

6-4 Effect of storage duration at ambient conditions on lightness (L*) of peel for BARI Malta-1 sweet orange fruit harvest from location 1. AMW = ambient water; HW = hot water; CR = Carnauba; NC = no coating...... 262

6-5 Effect of storage duration at ambient conditions on lightness (L*) of peel for BARI Malta-1 sweet orange fruit harvest from location 2. AMW = ambient water; HW = hot water; CR = Carnauba; NC = no coating...... 263

6-6 Effect of storage duration at ambient conditions on a* (-greenness to + redness) value of peel for BARI Malta-1 sweet orange fruit harvest from

location 1. AMW = ambient water; HW = hot water; CR = Carnauba; NC = no coating...... 263

6-7 Effect of storage duration at ambient conditions on a* (-greenness to + redness) value of peel for BARI Malta-1 sweet orange fruit harvest from location 2. AMW = ambient water; HW = hot water; CR = Carnauba; NC = no coating...... 264

6-8 Effect of storage duration at ambient conditions on b* (-blueness to +yellowness) value of peel for BARI Malta-1 sweet orange fruit harvest from location 1. AMW = ambient water; HW = hot water; CR = Carnauba; NC = no coating...... 264

6-9 Effect of storage duration at ambient conditions on b* (-blueness to +yellowness) value of peel for BARI Malta-1 sweet orange fruit harvest from location 2. AMW = ambient water; HW = hot water; CR = Carnauba; NC = no coating...... 265

6-10 Effect of storage duration at ambient conditions on lightness (L*) of peel for pomelo fruit harvest fruit from location 1. AMW = ambient water; HW = hot water; CR = Carnauba; NC = no coating...... 265

6-11 Effect of storage duration at ambient conditions on lightness (L*) of peel for pomelo fruit harvest from location 2. AMW = ambient water; HW = hot water; CR = Carnauba; NC = no coating...... 266

6-12 Effect of storage duration at ambient conditions on a* (-greenness to + redness) value of peel for pomelo fruit harvest from location 1. AMW = ambient water; HW = hot water; CR = Carnauba; NC = no coating...... 266

6-13 Effect of storage duration at ambient conditions on a* (-greenness to + redness) value of peel for pomelo fruit harvest from location 2. AMW = ambient water; HW = hot water; CR = Carnauba; NC = no coating...... 267

6-14 Effect of storage duration at ambient conditions on b* (-blueness to +yellowness) value of peel for pomelo fruit harvest from location 1. AMW = ambient water; HW = hot water; CR = Carnauba; NC = no coating...... 267

6-15 Effect of storage duration at ambient conditions on b* (-blueness to +yellowness) value of peel for pomelo fruit harvest from location 2. AMW = ambient water; HW = hot water; CR = Carnauba; NC = no coating...... 268

LIST OF ABBREVIATIONS oC degree Celsius

4MP 4-methylpyrazole

AA acetaldehyde

ACC aminocyclopropane-1-carboxylic acid

ACP anaerobic compensation point

ADH alcohol dehydrogenase

AMW ambient water

ASC ascorbic acid content

ATC total antioxidant capacity

C chroma

C2H4 ethylene

CA controlled atmosphere cm centimeter

COM compression

CR carnauba d day

EP extinction point

FAO Food and Agricultural Organization

FF fruit firmness

FIP fermentation induction point

FL Florida hr hour

H hue angle

HL harvest location

HPMC hydroxypropylmethylcellulose hr

HSP heat shock protein

HTFA high temperature forced air

HW hot water

HWT hot water treatment

IMZ imazalil

IRREC Indian River Research & Education Center

JAR just about right kg kilogram kPa kilopascals

L liter

L* lightness

LDH lactate dehydrogenase

LOL lower oxygen limit

MA modified atmosphere

MAP modified atmosphere packaging mg milligram mL milliliter

MT metric tons

NAV Navel

NC no coating

O2 oxygen

OLC oleocellosis

ORAC oxygen radical absorbance capacity

PG polygalacturonase

PL polyethylene ppm parts per million

PUN puncture

RH relative humidity

ROS reactive oxygen species

RQ respiratory quotient

RQB respiration quotient breakpoint

S storage s seconds

SH shellac

SSC soluble solids content

TCC total carotenoid content

TBZ thiabendazole

TP total phenolics

TTA total titratable acidity

USAID United States Agency for International Development

VAL Valencia

WL weight loss

Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy

POSTHARVEST HEAT STRESS AND SEMI-PERMEABLE FRUIT COATING TO IMPROVE QUALITY AND EXTEND SHELF LIFE OF CITRUS FRUIT DURING AMBIENT TEMPERATURE STORAGE

Md. Golam Ferdous Chowdhury

May 2018

Chair: Jeffrey K. Brecht Cochair: Mark A. Ritenour Major: Horticultural Sciences

Plants respond to sublethal stresses such as heat treatments by upregulating their antioxidant system, increasing resistance to subsequent stress. Submersion in hot water (HW) and fruit coatings result in development of internal modified atmospheres

(MA) that help maintain quality. The aim of this research was to utilize HW treatments plus fruit coatings for citrus fruits to stimulate the antioxidant system and create beneficial internal MA to better maintain postharvest quality when refrigerated storage is not available.

Based on preliminary research with Florida ‘Washington Navel’ oranges, immersion in 45ºC water for 30 min resulted in the most extreme internal atmosphere without causing external peel injury. In subsequent research, ‘Washington Navel’ and

‘Valencia’ oranges (Citrus sinensis) from commercial groves near Fort Pierce, FL, were harvested at commercial maturity and treated with HW, coatings [polyethylene (PL)-, carnauba (CR)-, or shellac (SH)-based] applied or not (NC), and stored for 3 weeks at

25ºC and 85% RH. The HW+CR best maintained near-optimum internal MA (5-10% O2 and CO2) and significantly reduced weight loss (WL) compared to other treatments.

Coatings also improved peel appearance (lightness, chroma and hue), fruit firmness, and peel puncture resistance during storage. The HW+CR significantly increased antioxidant capacity during storage compared to other treatments. Sensory panelists preferred the overall quality of HW+CR and HW+NC fruit among the treatments. They also rated the flavor and overall quality of CR fruit higher than NC fruit, or fruit with PL or

SH coatings. Shellac coating resulted in the lowest sensory rating, corresponding to elevated internal ethylene (0.2 to 0.4 versus 2 ppm) after 3 weeks, suggesting a stress response (<3% O2 and >15% CO2).

In a companion study in Bangladesh using ‘Bari’ sweet orange and pomelo

(Citrus grandis) fruit, HW+CR again significantly reduced fruit WL and decay during storage, and CR also better maintained fruit peel appearance, firmness, and sensory quality.

The positive results seen for orange and pomelo in both Florida and Bangladesh suggests that this approach to maintaining postharvest citrus quality is widely applicable to diverse citrus varieties and growing conditions, particularly areas where refrigerated storage is unavailable.

CHAPTER 1 INTRODUCTION

Citrus fruits rank first among fruits with respect to commercial importance and are grown in more than 80 countries in the world (Ladaniya, 2008). The average world production of citrus fruits from 2002 - 2011 was 1.17 x 108 MT, with an average area under production of 8,375,842 hectares (FAOSTAT, 2013). The major world producers are Brazil, United States, China, Mexico, Spain, India, Italy, Egypt, Nigeria, Japan and

Morocco (Wardowski et al., 2006; Ladaniya, 2008; FAOSTAT, 2013). The average production in the United States from 2002 to 2011 was 11,666,970 MT, with an average production area of 361,670 hectares (FAOSTAT, 2013). Citrus is the main tree fruit crop in the world with a tremendous economic, social and cultural impact in our society

(Iglesias et al. 2007). With increases in production, the increase in availability of the fruit and its supply requires preservation in terms of quality and quantity.

Citrus fruits are nonclimacteric, with persistently low respiration and ethylene production rates during ripening, and do not undergo any major softening or compositional changes after harvest. The fruit can therefore normally be stored for relatively long periods of 6–8 weeks at their optimum storage temperatures (Kader,

2002). However, two major problems limit the long-term storage capability of citrus fruits: the first is pathological breakdown, leading to decay; the second is physiological breakdown, resulting in the appearance of various physiological disorders such as creasing, splitting, puffing, and peel pitting as commonly occurring phenomena (Agusti et al., 2002). Most physiological disorders of citrus fruits are manifested as physical defects appearing on the surface (i.e., peel) and typically are related to the rupture of oil glands, phytotoxic injury to tissues, and subsequent water loss. Fruit with even very

slight splits or cracks will rot eventually. Moreover, respiration rate is increased in fresh produce after harvesting when the product is subject to physical stress, which can result in compositional changes and reduced postharvest life (Sandhya, 2010).

Postharvest losses of fruits and vegetables are more serious in developing countries than in developed countries. In the case of citrus fruits, postharvest losses in developing countries were estimated to be 23-33% (Coursey, 1983). Physiological deterioration of fruits and vegetables during storage depends largely on temperature control and humidity control, with the former primarily acting by reducing the rate of fruit and vegetable metabolism. Due to insufficient electrical power produced in developing countries (including Bangladesh) for refrigeration to be widely available, alternative technologies are necessary for extending storage life of agricultural produce. The metabolic rate of fresh fruits and vegetables can be reduced by many postharvest techniques including low temperature, controlled atmosphere (CA) storage, hypobaric storage, and modified atmosphere (MA). Heat treatments using hot water have also shown promise for improving sensory attributes and extending shelf life. One way other than temperature control that may be used to slow down the respiration rate and fruit metabolism is to modify the fruit internal atmosphere.

Modified atmosphere within the fruit can be created by either modification of the atmosphere surrounding the fruit, as in modified atmosphere packaging (MAP), or by applying a semipermeable coating on the fruit surface. The MA is usually chosen to create the most extreme modification of oxygen (O2) and carbon dioxide (CO2) possible within the fruit without inducing fermentative metabolism. However, sometimes anaerobic metabolites produced in fruit, such as acetaldehyde (AA) and ethanol, can

help to improve postharvest fruit quality (Pesis, 2005). On the other hand, several authors have established a relationship between the heat-shock response, anaerobic metabolism, and protection provided against different types of stress. According to

Schoffl et al. (1998) heat stress induces a cellular response that is able to protect both the cell itself and the whole organism from severe damage. Many researchers have reported the use of high temperatures (thermal shock) to maintain quality and extend fruit shelf life (Lurie, 1998; Murray, 1992; Sabehat et al., 1996; cited in Polenta et. al.,

2006). So, both thermal shock and MA can be used for producing desired anaerobic metabolites that can improve fruit postharvest quality including sensory attributes, nutritional value and shelf life of citrus fruits.

Therefore, considering the above situation, the following objectives were proposed for dissertation research.

1. to identify critical and optimum heat stress treatment level for quality citrus,

2. to evaluate the physiological changes during thermal treatment prior to

development of visible injury symptoms of citrus fruits,

3. to study the interactions of physiological changes regarding anaerobic

metabolites for maintaining fruit quality of citrus;

4. to identify the optimum condition for producing and maintaining desired levels of

anaerobic metabolites in terms of heat stress treatment, storage temperature and

MA;

5. commercial application of effective heat stress treatment and MA conditions for

quality citrus fruits.

CHAPTER 2 LITERATURE REVIEW

Origin and Cultivars of Citrus Fruits

Citrus fruits are native to the tropical and subtropical areas of Asia and originated in what are now the South Asian countries of China, India, and Malaysia (Bartholomew and Sinclair, 1952; Gmitter and Hu, 1990; Ramana et al., 1981; Scora, 1975; Sinclair,

1961). Sweet oranges and mandarins are indigenous to China and lemon was originally grown in India. Citrus grows mostly on either side of a belt around the equator surrounding tropical and subtropical areas of the world from 35°N and 35°S latitudes

(Ramana et al., 1981; UNCTAD, 2004). Citrus fruits are grown all over the world in more than 140 countries and were first introduced to America by Spanish and Portuguese, with the first citrus groves in Florida appearing about 1655 and in California about 1769

(Liu et al., 2012).

Citrus is the main tree fruit crop in the world with a tremendous economic, social and cultural impact in our society (Iglesias et al., 2007). With increases in production, the increase in availability of the fruit and its supply requires preservation in terms of quality and quantity.

Citrus fruit are botanically considered hesperidia, a particular kind of berry having a leathery rind and divisions internally called segments. It belongs to six genera - Citrus,

Fortunella, Poncirus, Microcitrus, Eremocitrus, and Clymenia. The structure of a hesperidium is very complex compared with the typical berry fruit such as grape (Vitis spp.), drupe [peach (Prunus), mango (Mangifera), coconut (Cocos)], blueberry

(Vaccinium), all of which are derived from the ovary proper, as the citrus fruit develops from a superior ovary (one lacking non-ovarian tissues) with axile placentation. Flavedo,

albedo, segments, seeds, central axis, and vascular bundles are several distinct tissues apparent when citrus fruit are cut longitudinally or transversely. Citrus fruits are classified as nonclimacteric due to their low ethylene production and declining respiration pattern. But citrus fruits respond to exogenous ethylene (Eaks, 1970) or stress such as rough handling (Grienson et al., 1971), increasing temperature, longer storage period, fungal invasion, chilling temperature, and senescence, which cause the respiration rate to increase. However, application of wax coating on the peel surface of citrus fruits plays a significant role in keeping the produce stable by reducing respiration rate and maintaining a beneficial internal modified atmosphere (MA) in the fruit. Chilling injury of orange fruit can be prevented by waxing, due to increased CO2 level into fruit

(Palou et al., 2015). However, peel pitting disorder is associated with low O2 level in the fruit, which is related to the use of coatings with too much resistance to O2 diffusion

(Petracek et al, 1998). In this case, optimum amount of plasticizer used in coatings increases O2 permeability into fruit. Stem end rind breakdown is related to drying conditions and water loss, so after harvest, rapid application of coatings is used to reduce or minimize water loss (Ritenour, 2016).

Citrus fruits are generally comprised of an outer peel or rind made up of the epidermis, the flavedo, the albedo, and vascular bundles. The inner flesh has segments, usually aligned and situated around the soft central core of the fruit and wrapped by a thin segment membrane called the septum. Small and densely packed sacs contain juice and seeds fill the segments, and the citric together with a complex mix of other acids, oils, and sugars contained in the juice provide the flavor profile (Albrigo and

Carter, 1977; Ranganna et al., 1983).

Citrus Fruits Production Trends in the World and in Bangladesh

Citrus Fruits Production Trends in the World

Among fruit crops, citrus ranks number one in worldwide commercial production, with sweet orange comprising about 60% of this (Ladaniya, 2008). In 2015-2016, total fresh orange production was 47.08 million MT and the major producing countries were

Brazil (14.32 million MT), China (6.90 million MT), European Union (6.24 million MT),

United States (5.36 million MT), Mexico (4.40 million MT), Egypt (2.93 million MT),

Turkey (1.80 million MT), and South Africa (1.56 million MT) (USDA, January 2017).

In the United States, the major citrus production areas are Florida, California,

Arizona, and Texas (Jackson, 1991; Ladaniya, 2008; Saunt, 1990; Wardowski et al.,

2006). In the United States, 2016-2017 orange production is predicted to decline by

470,000 tons to 4.9 million as citrus greening disease (Candidatus liberibacter spp.) continues to reduce Florida production (USDA, January 2017). Overall, Florida accounts for nearly 60% of U.S. production and California for about 40%.

Florida citrus production is dominated by sweet orange (83.6%) followed by grapefruit (12.6%) and specialty fruit such as tangerines and tangelos and a small amount of lemons. Most Florida oranges (95%) are used for processing, whereas

California oranges are mainly used for fresh consumption. Exports are downward slightly while consumption is forecast 8% higher in 2015-2016 (USDA, January 2017).

So, fresh processing is decreasing due to insufficient production of citrus fruits in

Florida.

Citrus Production Trends in Bangladesh

In Bangladesh, citrus is grown on a small scale on commercial plantations and also in backyard orchards and small holdings in different regions of the country (Figure 2-1).

The major citrus fruits are sweet orange (Malta, mandarin), lime, lemon, pomelo, and other exotic citrus fruits (Figure 2-2). In the sub-continent, juicy sweet oranges were locally introduced with the name of Malta fruit. This fruit is very popular in Asian countries including Bangladesh. Malta fruit have a thin peel with juicy segments.

Consumers like the fruit for its aroma, flavor, and sweet-sour taste. Sweet orange consumption is increasing as fresh consumption and processed juice because of consumers demand though production is very limited. Substantial amounts of sweet oranges are imported from foreign countries such as China, Pakistan, Bhutan, India,

Australia, and some African nations. Home production increased in the last 10 years and growers are interested in producing more quality sweet oranges because of consumer preference and the market price. In 2014-2015, the total production of citrus fruits in Bangladesh was 157.24 thousand metric tons (MT) from 78.83 thousand acres

(Figure 2-2) (BBS, 2015). According to BBS (2015), the major citrus production in 2014-

2015 was in lime and lemon (68.72 thousand MT) followed by pomelo fruit (63.22 thousand MT) (Figure 2-3). Those fruits are generally used in the processing industry to produce different fruit drinks, juices, and nectars. In addition, fresh processed juice such as fresh squeezed and pasteurized juices in different concentrations made from citrus fruits have a significant demand by the consumers around the country for their nutritional value.

Figure 2-1. Map showing the major citrus growing regions in Bangladesh. Reprinted with permission from, http://www.maps-of-the-world.net/maps/maps-of- asia/maps-of-bangladesh/small-administrative-map-of-bangladesh.jpg.

Area ('00' acres) Production ('000' MT)

157.35 157.24 160 148.09 150.21 136.76 140 120 100 78.83 80 65.60 60.00 59.69 62.37 60 40 20 0 2010-2011 2011-2012 2012-2013 2013-2014 2014-2015

Figure 2-2. Area and production of oranges from 2010-2011 to 2014-15 (BBS, 2015)

80 68.72 63.22 60

40 21.34 20 3.95 Production ('000' MT) ('000' Production 0 Sweet Lime and Pomelo Other citrus orange lemon fruit Citrus fruit

Figure 2-3. Production of different Citrus fruit types in Bangladesh, 2014-15 (BBS, 2015)

However, within the last few years, sweet orange (i.e., Malta) and pomelo are now the fruit most in demand for fresh consumption in the country, served as fresh cut, fruit salad, and fruit cocktail. But lack of adequate postharvest technology and handling practices to maintain the quality and shelf life of these fruit are now the main concern for

growers, shippers, handlers and processors. After harvest, growers are forced to sell their fruit at undesirable prices due to insufficient storage technology and fruit decay.

Farmers are not able to cool the fruit or maintain low temperatures during storage and marketing or utilize controlled atmosphere storage facility because of insufficient and expensive electrical equipment. Therefore, affordable technologies are needed to support the fresh citrus industry to ensure delivery of quality fruit.

Nutritional Importance of Citrus Fruits

Nutritional Composition in Citrus Fruits

The major nutritive value of citrus fruits comes from vitamin C (Nagi, 1980).

Citrus fruits also contain significant amounts of macronutrients such as sugars and dietary fiber, and micronutrients such as folate, thiamin, niacin, vitamin B6, riboflavin, pantothenic acid, potassium, calcium, phosphorus, magnesium, and copper, which are essential for a healthy human diet (Economos and Clay, 1999; Rouseff and Nagy,

1994). It is stated from epidemiological studies and other research that citrus fruits have bioactive compounds such as phytochemicals, limonoids, flavonoids, and carotenoids, which are beneficial for human health. These compounds existing in citrus fruits prevent chronic diseases (Liu, 2003; Silalahi, 2002; Steinmetz and Potter, 1991; Yao et al.,

2004) such as cardiovascular disease (Clinton, 1998; Ford and Giles, 2000), cancer

(Nishino, 1997; Steinmetz and Potter, 1996), neurological deficits (Youdim et al., 2002), and osteoporosis (Yang et al., 2008).

Volatile Compounds in Citrus Fruits

Many research reports indicate that many flavor volatile compounds exist in citrus, both in the edible tissue and the peel components. Volatile compounds are released or changed in different ways such as by increasing temperature (Nisperos-

Carriedo and Shaw, 1990), fruit maturity, ruptured peel, and changing tissue components (Ladaniya, 2008). Citrus fruits have more than 300 volatiles and oils such as terpene hydrocarbons, esters, aldehydes, ketones, alcohols, and organic acids

(Parez-Cacho and Rouseff, 2008).

The flavor of fresh oranges is comprised of sugars, acids, and volatile compounds (Obenland et al., 1999). Aroma flavor compounds are considered to be essential constituents such as alcohols, aldehydes, esters, and hydrocarbons

(Nisperos-Carriedo and Shaw, 1990). Citrus fruits have a complex mixture of volatile components which together identify their flavor (Hinterholzer and Schieberle, 1998). The volatile composition of orange fruit depend on maturity and postharvest conditions, and their perception is influenced by the soluble solids content/titratable acid (SSC/TA) ratio and peel color, resulting in characteristic fruit flavor (Obenland et al., 2009).

According to Shaw et al. (1992), in citrus fruit low O2 ramps up production of a large number of flavor volatiles such as ethanol, ethyl acetate, ethyl butyrate, AA, 1- butanol, acetone, 1,1-diethoxyethane, octanol, and ethyl 3-hydroxyhex-anoate. If O2 and

CO2 exchange is hampered between the fruit and its environment, then low O2 inside the fruit can result in a trend of increased anaerobic respiration, which results in development of off-flavor caused by ethanol and AA accumulation (Hagenmaier and

Shaw, 2002; Shaw et al., 1990).

Different citrus fruits including Navel and Valencia sweet oranges naturally accumulate flavor volatiles such as AA and ethanol (Ahmad and Khan, 1987; Cohen et al., 1990; Hagenmaier, 2002) when they are exposed to longer storage (David et al.,

2008). Moshonas and Shaw (1989) stated that flavor decreases during storage, but

undesirable compounds increase. Moshonas and Shaw (1994) observed 46 flavor volatiles in fresh orange juices extracted from different varieties of Florida and California oranges. Barbieri et al. (1996) noticed higher flavor volatile components in terms of the ratio between terpenes (except limonene) and sesquiterpenes in Valencia orange compared to blood orange juices.

Shellac coatings are less permeable to O2 and CO2 than other common coatings and therefore, flavor volatile compounds increase in the fruit during the storage period

(Baldwin et al., 1995). In shellac-coated Valencia oranges, the ethyl acetate content increased about 200% during 7 days of storage at 21°C, and by another 100% during the next 14 days, whereas ethyl butyrate, ethanol, and methanol increased in lesser amounts in the fruit (Baldwin et al., 1995).

Nisperos-Carriedo et al. (1990) used five different coatings in the storage of pineapple oranges at 21°C for 12 days and found five potential beneficial volatile components increased to influence the flavor of the fruit. Baldwin et al. (1995) showed that some flavor volatiles positively influence and others are negatively influence orange flavor.

Bitterness is an important flavor component that influences consumers’ overall acceptance of any fresh fruit. The quantity of bitterness differs in orange fruit according to variety/cultivar, maturity, and flavor. Limonin and a triterpenoid, dilactone, are crucial to bitter flavor perception and they are detected generally in all citrus fruits (Higby,

1938), although their concentration levels are below the taste thresholds in most mature citrus cultivars (Rouseff et al., 2009). Concentrations of the important bitterness component, limonin, generally trends downward if the fruit become more mature,

because of the conversion of the A-ring lactone to the tasteless limonin glucoside (Fong et al., 1989). Limonene has a fresh, minty, citrusy aroma that mostly influences orange flavor (Moshonas and Shaw, 1994; Tonder et al., 1998). In fresh market fruit, freshness and likeability decreased as a result of storage due to presence of limonene, while ethyl butanoate and ethyl hexanoate flavor compounds strongly increased (Obenland et al,

2008). The aromas of both the latter compounds are sweet with sweet, fruity essence, which affects orange flavor (Buettner and Schieberle, 2001). If those compounds increase, even though both are considered positive flavors, it could lead to changes in the balanced fruit aroma. Sensory threshold is vital to know the contribution of a volatile aroma compound to the overall flavor. The number of volatiles in the fruit tissue or juice that exist below the sensory threshold may still have additive or synergistic effects which complement the overall flavor compounds.

Heat treatment was reported to positively modify the flavor volatiles in oranges

(Obenland et al., 2009). Application of heat treatment delayed ripening and reduced volatiles during cold storage of climacteric tomato fruit (McDonald et al., 1996). This was possibly due to heat-induced alteration in the enzyme systems that catalyze synthesis of the volatile compounds in the fruit.

Heat treatment sometimes creates an adverse effect on flavor volatiles in fruit

(Schirra and D’hallewin, 1997). According to Obenland et al. (1999) navel oranges treated with heat stress by high temperature forced air (HTFA) showed a significant changed of key volatiles that resulted in a decline in orange flavor. Similar results were found in other research conducted by Obenland et al. (2012).

Heat stress can also increase flavor volatiles in fruit. Heat is a very effective medium to kill insects and pathogens, but excess heat can injure fruit tissue. Some research has shown that heat stress applied for insect control (fruit fly) negatively affected flavor volatiles in (Texas ‘N33’ Navel orange) fruit (Shellie and Mangan, 1998) but no significant difference were apparent in SSC or TA (Shellie and Mangan, 1994).

However, no particular explanation or concrete results are available to explain the possible effect of heat treatments on specific flavor compounds (Obenland et al., 1999).

HTFA treatment applied to fresh oranges to reduce Mexican fruit fly infestation resulted in a 49% loss in limonene as compared to untreated fruit (Obenland et al.,

1999). Similar results were obtained for Navel orange. High amounts of limonene present in orange juice indicated a negative effect on fruit flavor, but very much less or no effect on SSC or TA was found in fruit tissue or juice (Shellie and Mangan, 1994).

Consumer Preference of Citrus Fruits

Sensory evaluation is conducted to evaluate human perception such as appearance, flavor, and texture attributes of any food product. This evaluation is used in consumer test panels to determine preference or liking and determine overall consumer acceptability, and in trained panels to describe or characterize a food product in order to develop a sensory profile.

Consumers prefer citrus fruit such as Navel and Valencia oranges for their flavor, taste, nutritional attributes, and sensory quality. Good quality orange fruit will be mature and fully ripe that are firm, have good size and shape, and have uniform color distributed around the surface. Peel appearance first attracts the customers to purchase the fruit, so fruit must be decay free and without physiological disorders, exhibit no

defects or injuries, and have no blemishes. Consumers also select attractive packaging such as different sizes of well-vented polyethylene, net bags, plastic mesh bags, corrugated fiber board cartons, and plastic film sealed packets to carry orange fruit.

Fruit maturity indices are important for quality citrus fruit intended for fresh consumption as well as for processing, and it indicates acceptable color, soluble solids content (SSC), titratable acidity (TTA), sugar and acid (SSC/TTA) ratio, and juice content. Fresh, mature fruit are full of natural aroma and taste that is pleasing and attractive to the consumers.

Taste is also considered to be the most important internal quality attribute for fresh consumption of any fruits. Taste is comprised of sour and sweet that is related to titratable acidity (TTA) and soluble solids content (SSC). Fruit taste is defined by

SSC/TTA ratio (Blanke, 1996) and a specific variety of fruit taste originates from their unique content of other chemical compounds (Abu-Khalaf and Bennedsen, 2004).

According to Auerswald et al. (1999), sugars, acids, aroma volatiles, amino acids, and amines all are combined to produce the overall flavor of fruit. Orange juice flavor is varied due to the combined effects of fruit cultivar, maturity, and temperature conditions

(Rouseff et al., 2009).

Valencia orange is the single most widely grown sweet orange cultivar worldwide

(Saunt, 1990) and consumers prefer it for its attractive color, high levels of total soluble solids content, and juice content. Therefore, it is in demand by processors and is often blended with lower quality juices to increase palatability (Davies and Albrigo, 1994).

Navel oranges are consumed mostly as fresh fruit and is considered a dessert orange

(Saunt, 1990). It particularly contains a high level of limonin, which results in bitterness

development in juices, therefore, Navels are not always suitable for juice processing.

Navel oranges are characterized by small size, and by secondary fruit embedded in the stylar end of the primary fruit (Stover et al., 2005).

Postharvest Physiology of Citrus Fruits

Most physiological disorders of citrus fruits are manifested as physical defects appearing on the surface (i.e., peel) and typically are related to the rupture of oil glands, phytotoxic injury to tissues and subsequent water loss. Fruit with even very slight splits or cracks will rot eventually. Moreover, respiration rate is increased in fresh produce after harvesting when the product is subject to physical stress, which can result in compositional changes and reduced postharvest life (Sandhya, 2010).

Citrus Fruits Maturation and Maturity Indices. Fruit maturity determines the harvesting time and different fruits have different maturity standards. Fruit maturity is determined by evaluating physicochemical parameters and sensory quality attributes of fruit. During maturation and fruit ripening, significant physicochemical changes are observed in citrus fruits such as increases in total sugar, decreases in citric acid and

ascorbic acid content, change of fruit peel color, and increase in fruit size. Acid and sugar levels present in orange fruit are important taste attributes to evaluate the flavor balance between sweet and sour that are commonly part of maturity standards.

Citrus fruits are consumed as fresh or processed upon harvesting without any significant change in composition (Bain, 1958; Harding, 1947; Ramana et al., 1981;

Sinha et al., 1962). During maturation and fruit ripening, significant physicochemical changes are observed in citrus fruits such as increases in total sugar, decreases in citric acid and ascorbic acid content, change of fruit peel color, and increase in fruit size.

The United States Department of Agriculture was authorized by the California

Citrus Industry in 1915 to standardize maturity for citrus fruit by using the ratio of

SSC/TTA (Chace, 1917). The minimum maturity standard of California Navel orange is determined by a ratio of SSC to TTA of 8:1 (°Brix: %), which has been shown to provide good eating quality for consumers (Pehrson and Ivans, 1988). It is also required that the fruit have at least 25% yellow-orange color on the peel surface and at least 90% of the whole lot (California Department of Food and Agriculture, 2003). A similar protocol is now used in Australia (Boyd, 2014) by following the California index and in New

Zealand following Laurenson (2015) for harvest maturity.

Pehrson and Ivans (1988) determined that for early season Navel oranges, the sugar: acid ratio of 8:1 is too low for acceptable flavor. It seems the flavor is not fully described by SSC and TTA alone, and aroma volatiles are changing during Navel orange maturation, which also affects the orange flavor. Regarding maturity determination, a few sensory panel tests have been conducted in California, Texas,

Nevada, and New York to determine consumer acceptability and it was noticed that

consumers preferred the SSC/TTA ratio of above 8:1 for their purchasing of orange fruit

(Ivans and Feree, 1987; Pehrson and Ivans, 1988) although SSC: TTA ratio does not always correlate fruit sweetness or tartness (Jordan et al., 2001). Obenland et al. (2009) have suggested that the minimum SSC/TTA ratio probably needs to be increased to a level that meets with consumer acceptance.

Fruit maturity may also be determined by identifying flavor volatiles that act as quality markers in Navel orange (Obenland et al., 2009). There are other, simple, nondestructive, methods that are also useful for determining fruit maturity by the measurement of rind color and fruit firmness (Olmo et al., 2000), as well as more sophisticated instruments such as near infrared spectroscopy that are more difficult to use successfully because of citrus’ thick-skinned peel (Nicolai et al., 2007).

Postharvest Treatment to Citrus Fruits

Postharvest Fungicide Treatment

Globally postharvest disease is a major concern in the citrus industry (Eckert and

Eaks, 1989). Postharvest fungicides are used to reduce decay of citrus fruit. Application of postharvest fungicides must provide effective response to residue loading, protection against pathogens such as green mold and stem end rot, and control of infections after treatment (Brown, 1984). Different types of fungicide are applied in aqueous solutions or suspensions.

Imazalil (IMZ) and thiabendazole (TBZ) are two systemic fungicides applied to control a vast range of fungal diseases of citrus fruits (Tomlin, 1994). They reduce decay by inhibiting the development of latent infections of the stem-end rot fungi, by inactivating spores in fresh wounds, by protecting the peel from infection at injury sites, and by inhibition of sporulation of Penicillium on the surface of decaying fruit and the

contact spread of several diseases (Eckert, 1977). The applied fungicides do not penetrate beyond the peel surface of the fruit to a significant extent, so the pathogen must be completely inactivated before its hyphae grow beyond the wounded site

(Eckert, 1977). The residue load of IMZ or TBZ on citrus fruits depends on temperature, concentration, and exposure time (Smilanick et al., 1997). Fungicide residue is increased when temperature is warmer and concentration is higher, with longer exposure of treated fruit (Erasmus et al., 2011).

TBZ is stable under normal conditions of formulation and in postharvest use. It is applied to harvested Navel or Valencia oranges in bins before the degreening treatment. After harvest, fruit in bins are drenched with a suspension of around 1000 ppm TBZ mixed in water for 30 seconds and maintained at pH 8 before being placed in the degreening room (Eckert et al., 1969).

Imazalil is applied to citrus fruits immediately after harvest for controlling green mold (Penicillium digitatum) (Eckert, 1995; Smilanick et al., 2005). Imazalil is used in different ways on oranges. One method is, after harvest, fruit bins are drenched with

IMZ mixed with water either prior to or on the packingline. Fruit may be dipped into a fungicide dip tank, or the IMZ solution may be sprayed on the fruit surface, or used as a drench, or mixed with wax coatings (Kaplan and Dave, 1979; McCornack et al., 1977).

Both TBZ and IMZ fungicides are also widely used in packinghouse treatments in citrus fruit (Eckert and Eaks, 1988). Thiabendazole is more effective than IMZ fungicide to control decay in Navel and Valencia oranges. Imazalil works effectively as a control against green mold (Penicillium digitatum), including benzimidazole-resistant strains, and against sporulation, but it is less effective in controlling stem-end rot and ineffective

against sour rot and brown rot, whereas TBZ works effectively against them (Wardowski and Brown, 1991). In the industry, both of these fungicides are used as a combined treatment for decay control. But, they may not perform appropriately, because IMZ– resistant isolates of Penicillium digitatum have been noticed after their combined application (Bus et al., 1991; Holmes and Eckert, 1992) and they may lead to double resistance (Eckert et al., 1994).

Postharvest De-greening Treatment

Citrus fruits are non-climacteric as per their respiration patterns, and produce low amounts of ethylene during ripening (Kader, 2002). However, they respond to exogenous ethylene exposure, resulting in hastening of the advancement in peel appearance known as degreening through both chlorophyll degradation and carotenoid biosynthesis (Iglesias et al., 2007; Matsumoto et al., 2009; Sdiria et al., 2012; Shemer et al., 2008). In the citrus industry, exogenous ethylene exposure is used to induce degreening of fruit that are at an early maturity stage (Cajuste and Lafuente, 2007).

Degreening is a common and commercial practice in the world that is used to change citrus fruit peel color by exposure to ethylene gas immediately after harvest. It is important for citrus fruits to have desirable peel color during the early season when nighttime temperature is not sufficiently low (7-130C) to stimulate chlorophyll degradation. During the early harvest season, citrus fruits such as Navel oranges are often of acceptable internal maturity, but possess very poor color, which does not appeal to consumers for taste or purchasing fruit (Smilanick et al., 2006).

The effectiveness of the degreening treatment depends on ethylene concentration, temperature, relative Humidity, weather condition, fruit cultivar, peel permeability and external color of the fruit. In case of weather condition, California has

cool arid climate where low temperature exists at night that is favorable for peel chlorophyll breakdown for citrus fruits, whereas Florida’s climate is warm and humid and this is not favorable for early harvesting of citrus fruits, resulting in more need for degreening treatment. The difference in optimal degreening between the two regions are mainly based on the climatic conditions under which the fruits develop in the field as well as incidence of diseases such as green mold caused by Penicillium digitatum. The environment is optimal in California for the development of green mold during degreening, whereas in Florida, it is more likely that an increase the Diplodia stem end rot may occur during the early season due to warm and Humid conditions (McCornack,

1972; Smilanick et al., 2006). In addition, research has shown that degreening, if not managed properly, can enhance senescence-related physiological disorders, including rind breakdown in Clementine mandarin (Cronjie et al., 2011).

The recommended Florida degreening conditions are: exposure to ethylene at 1 to 5 ppm at 90-95% relative Humidity and 27°C to 29°C. Ventilation of one air change per hour is needed to keep CO2 conc. below 0.1% and an air circulation rate of 100 ft3/min per 900-lb bin are also recommended. Whereas, in California, recommended degreening conditions are: ethylene at 5 to 10 ppm at 90% relative Humidity and 20°C to 25°C. Ventilation of 1 to 2 air changes per hour or sufficient to keep CO2 below 0.1% and an air circulation rate of one room volume pre minute are also recommended

(Ritenour et al., 2003).

Physiological Indicators of Tolerance versus Stress from Heat Treatment

Tolerance of Fruits to Heat Stress

Several authors have established a relationship between the heat-shock response and the protection provided against different types of stress. According to

Schoffl et al., (1998) the heat stress induces a cellular response that is able to protect both the cell itself and the whole organism from severe damage. Bierkens (2000) and

Neumann et al. (1994) described that a single stress (dehydration, heavy metals, oxidative stress) induced a response able to exert a cross protection against any other stress.

Benefits of Hot Water Stress Treatment to Fruits

Postharvest heat treatment is one option that can be used commercially in fresh commodities for improving sensory attributes and shelf life. Many researchers have reported the use of high temperatures (thermal shock) to maintain quality and extend fruit shelf life (Lurie, 1998; Murray, 1992; Sabehat et al., 1996). Heat treatments have been successfully used for commodities like mango (Mangiferea indica; Heather et al.,

1997; Paull and Armstrong, 1994; USDA, Animal and Plant Health Inspection Service,

2012), strawberry (Fragaria ananassa; Civello et. al., 1997), avocado (Persea americana; Florissen et al., 1996), and apple (Malus domestica; Lurie and Klein, 1990).

The medium used to heat a commodity may influence its tolerance to heat. Hot water is preferred for most applications since water is a more efficient heat transfer medium than air.

Postharvest heat treatment is being used for disinfestation and disinfection of an increasing variety of crops, including fresh flowers, fruits, and vegetables (Lurie, 1998;

Soto-Zamora et al., 2005). During the past few years, there has been growing interest in the use of heat treatment to control insect pests, prevent fungal rots, or retard or minimize commodity response to temperature extremes (Lu et al., 2007). Part of this interest relates to the growing pressure from consumers to reduce the quantities of postharvest chemicals used against pathogens and insects.

Heat treatment is considered a relatively safe physical treatment that can be used as an alternative to chemical control (Lu et al., 2008). The susceptibility of freshly harvested produce to postharvest diseases increases during prolonged storage as a result of physiological changes that enable pathogens to develop in the fruits (Lu et al.,

2010). Treatments in the form of hot water, hot air, or vapor heat have been developed to control postharvest decay and insect infestations in a wide range of fresh produce

(Barkai-Golan and Phillips, 1991; Coates and Johnson, 1993; Porat et al., 2000).

The success of a heat treatment depends on the existence of a sufficient difference between the heat tolerance of the host and the pathogen. If temperatures are too high or the treatment period too long, a range of negative effects on quality can occur on the host, of which the most common are higher levels of water loss, skin discoloration, increased susceptibility to contaminating microorganisms, and a decrease in shelf life (Barkai-Golan and Phillips, 1991).

Controlled heat stress can be delivered to postharvest commodities by the application of hot water, hot air, and vapor heat or microwave energy. Hot water treatment is widely applied as a postharvest treatment to extend fresh produce quality, to control diseases, as a quarantine requirement (Ferguson et al., 2000) to enhance resistance to other stresses (Paull and Chen, 2000), and to avoid the use of chemicals in postharvest applications; the mechanisms involved may be related to generation of reactive oxygen species (ROS), activation or inhibition of functional proteins, and the production of phytohormones in response to heat stress (Lurie, 1998).

Hot-water treatments are characterized by exposure to high temperatures of from about 40°C up to 60°C for short times: from 120 minutes at lower temperatures within

the effective range to as short as 20 seconds at the highest temperatures (Lurie, 1998;

Porat et al., 2000). Furthermore, the hot water can contain chemicals such as thiabendazole, imazalil, sulfur dioxide (Lurie, 1998), and GRAS compounds such as ethanol (Lurie, 1998), sodium carbonate (Palou et al., 2002) or acetic acid (Radi et al.,

2010) to enhance the fungicidal potential.

It has been reported that the uniformity in the response of horticultural commodities to a heat treatment depends on the plant organ size, shape, and morphology, since heat transfer depends on the contact surface (Paull and Chen,

2000). Furthermore, many commodities have shown signs of heat injury when heat treatments are not optimized. This has been attributed to tissue damage and micro- wounds (Ferguson et al., 2000); such heat injury has been observed in mangoes (Joyce et al., 1993) and in tomatoes (Iwahashi et al., 1999).

Effect of Heat Stress Treatment on Chilling Sensitive Fruits

Low-temperature storage is used widely to extend the postharvest life of horticultural products. Most tropical and subtropical fruits and vegetables are subject to chilling injury if stored at low temperatures. Low temperature discontinues the metabolic process in the fruit, which induces impaired ripening, pitting, skin discoloration, tissue decomposition, internal or surface browning, membrane leakage, dry texture, lack of flavor and aroma, lower resistance to mechanical injury, and increased susceptibility to microbial infections.

Optimal exposure to heat stress induces protection of the commodity from chilling temperature. Some types of citrus fruit can be very susceptible to chilling injury, so much research has been done to understand the physiological mechanisms that are involved in heat-induced cold tolerance. According to Bassal and El-Hamahmy (2011),

chilling injury of ‘Navel’ and ‘Valencia’ oranges treated with hot water immersion at 41°C for 20 min declined by up to 16%, weight loss was reduced, and the juice percentage,

SSC/TA ratio and ascorbic acid content were retained. ‘Fortune’ mandarin was treated with hot air at 37°C for 1-2 days prior to a single (16 days) or double (32 days) quarantine treatment at 1.5°C followed by 4 days at 20°C. The results showed that there were no changes in the surface peel color, firmness, weight loss, vitamin C, acidity, flavonoids, and antioxidant capacity (Lafuente et al., 2011).

Effect of Heat Stress Treatment on Decay Control

Heat stress can be a very effective method for decay control. Karabulut et al.

(2010) found that control of brown rot (Monilinia fructicola) was achieved for stone fruit such as plums (Prunus domestica) and nectarines (Prunus persica var. nectarina) treated with hot water immersion at 60°C for 60 sec. The disease incidence was reduced from 80% to less than 2% in plums, and from 100% to less than 5% in nectarine fruit. In the case of peaches (Prunus persica), application of hot water at 40°C for 10 min, reduced decay from Monilinia fructicola, which was directly associated with an increase in intracellular ROS, mitochondrial dysfunction, and a decrease in ATP (Liu et al., 2012).

Hot water treatment induced disease resistance of papaya fruit. Fruit dipped into hot water at 54°C for 4 min controlled the fungal pathogen Colletotrichlm gloeosporioides in the fruit peel of papaya by inducing defense-related proteins (Li et al., 2013). It also significantly inhibited anthracnose and stem-end rot, effectively delayed fruit softening, but slightly increased peel fruit color. Moreover, it was suggested that HW caused the fruit wax to melt so that the melted wax created a mechanical barrier against pathogen penetration into fruit.

Yuan et al. (2013) observed similar results for muskmelon. Those findings were for hot water immersion at 53°C for 3 min. Fruit treated with 53°C water for 3 min had reduced decay incidence of Trichothecium roseum, Alternaria alternata, Fusarium spp., and Rhizopus stolonifer. The HW cleaned the surface of the fruit, melted the epicuticular waxes, and covered and sealed the stomata. It also promoted the defense-related enzymes phenylalanine ammonia lyase, cinnamate-4-hydroxylase, 4-coumarate: CoA ligase, polyphenoloxidase, and peroxidase. The HW increased the antifungal compounds, cinnamic, coumaric, caffeic and ferulic acids. The treatment also stepped up the phenolic compounds, flavonoids, lignin, and hydroxyproline-rich glycoproteins, and it maintained fruit firmness by suppressing the activities of cell-wall degrading enzymes.

Effects of Heat Stress Temperature and Atmosphere Modification on Fruits

Temperature is a vital factor regulating plant growth and development. Plants counter the effects of higher temperature and reduce heat stress related damage using signaling pathways that change in response to changes in the ambient temperature and then adjust metabolism and cell function by acclimation, or by thermotolerance (Kotak et al., 2007; Mittler et al., 2012). One way in which plants respond to heat stress is by the production of heat shock proteins (HSPs). Heat stress affects the stability of proteins, membranes, RNA species, and cytoskeletal structures, and alters the efficiency of enzymatic reactions in the cell, causing a state of metabolic imbalance (McClung and

Davis, 2010; Ruelland and Zachowski, 2010; Suzuki et al., 2011), which causes the accumulation of toxic compounds, such as ROS. The increases of ROS trigger antioxidant processes that are part of the heat stress response (Konigshofer et al.,

2008; Mittler et al., 2012).

Heat treatments can delay or prevent the development of chilling injury (Lurie,

1998) in fruit because of the presence of HSPs in the tissue (He et al., 2012; Sevillano et al., 2010; Yi et al., 2006). HSPs increase during heat stress but disappear rapidly with the return to ambient temperature, which slows down metabolic process (Sabehat et al.,

1996) and plants are protected from cold injury.

Heat stress also alters the normal program of protein synthesis and cellular metabolism during the treatment, which can help prevent disorders or slow fruit ripening. Application of heat stress quickly slow down the disassociation of polyribosomes and protein synthesis, that latter which re-initiates with a new set of proteins and HSPs (Ferguson et al., 1994) that inhibit the ripening process of the fruit stored at low temperature, but ripening processes will resume when the fruit are rewarmed.

Atmosphere modification (reduced O2 and elevated CO2) is usually applied to fruits during storage or transport in order to further reduce the rate of tissue metabolism beyond what can be achieved by reduced temperature and thus further slow the rate of various degradative quality changes postharvest (Kader, 1986). Atmosphere modification may also be perceived by the plant tissue as a mild stress, with effects on fruit metabolism that are similar to those detailed above for heat stress (Eason et al.,

2007) in addition to directly limiting the supply of oxygen for generation of reactive oxygen species (Toivonen, 2003). Controlled and modified atmospheres (CA and MA) also inhibit ethylene biosynthesis and action (Kanellis et al., 2009) and slow the growth of decay pathogens (El-Goorani and Sommer, 1981).

Modifying the Atmosphere Around and Within Produce by Restricting Gas Diffusion: Modified Atmosphere Packaging (MAP) and Coatings

Packaging technology can contribute to maintenance of appropriate postharvest quality of fresh produce. MAP technology has been successfully used to maintain postharvest quality and to prolong the storage period of many fruits and vegetables. By creating higher CO2 and lower O2 concentrations in the surrounding atmosphere of the commodities, decay, respiration rate, ethylene production, and enzymatic activity can be controlled, resulting in improved maintenance of postharvest quality (Caleb et al., 2012;

Kader and Watkins, 2000). MAP may also prevent weight loss and fruit shriveling by creating a higher relative humidity in the surrounding environment of the products

(Zagory et al., 1989). The efficacy of MAP is reliant on not only the product, but also importantly on the gas permeability and thickness of the polymeric film used, since this plays a vital role in establishing an appropriate atmosphere within the package.

According to Somboonkaew and Leon (2010), MAP successfully prolonged shelf- life of assorted harvested litchi (Litchi chinensis) fruit. Use of MAP for sweet cherry

(Prunus avium) showed that while fruit stored in polyethylene film retained firmness, off- odors and off-flavors associated with anaerobic respiration increased (Gerhardt and

Wright, 1948; Gerhardt et al., 1957). Meheriuk et al. (1995) suggested that sweet cherry shelf life may benefit from MAP, but success in practice is dependent on achieving low ppkgO2 and/or high ppkgCO2 that suppress mold growth, respiration, and other metabolic processes without stimulating anaerobic respiration.

Sevillano et al. (2009) reported that MA has been shown to reduce chilling injury

(CI) symptoms in cantaloupe (Cucumis melo), tomato (Solanum lycopersicum), pepper

(Capsicum annuum), banana (Musa × paradisiaca), avocado and mango. However,

packaging technology (like perforated films) that doesn’t generate MA also reduces CI symptoms but, in this case, the higher RH inside the package has a beneficial effect in reducing symptoms related to water loss like skin pitting (Meir et al., 1995).

In MAP, increases in temperature lead to further reduced O2 and increased CO2 levels and consequently increases in the levels of AA and ethanol. The higher the storage temperature of sweet cherry (over a range from 0 to 25°C) in polyethylene packaging, the higher the respiratory quotient (RQ), leading to more AA and ethanol being produced, and shorter shelf life (Petracek et al., 2002). In litchi fruit stored in

MAP, transfer to 20°C for 3 days shelf life following 1 month at 2°C, caused an extreme atmosphere to develop and a dramatic increase in the production of AA and ethanol

(Pesis et al., 2002).

Coating white grapefruit (Citrus × paradisi) with wax containing shellac increased pitting on the peel, the severity of which increased with decreasing internal O2 levels

(below 4%), and was also associated with high AA and ethanol production (Petracek et al., 1998). In mango cv. Tommy Atkins, the levels of AA and ethanol were much higher in fruit coated with NutraSeal than in those coated with carnauba wax, a difference that affected the fruit taste (Baldwin et al., 1999). A similar effect on taste was found in mandarin (Citrus reticulata) for which the highest ethanol levels tended to coincide with the highest internal CO2 and low flavor scores (Hagenmaier, 2002). Ethanol content of stored, coated mandarins was independent of the initial ethanol content of the uncoated fruit, which suggests that it is impossible to predict the build-up of ethanol after coating on the basis of the initial ethanol content. However, it is possible to predict how much

ethanol will accumulate during storage in the internal atmosphere of uncoated fruit

(Hagenmaier, 2002).

The Role of Anaerobic Metabolites of Produce in Each Variety/Condition

Both climacteric and non-climacteric fruits produce the fermentative metabolites

AA and ethanol, even while aerobic respiration is active, with the amounts depending mainly on their genetic characteristics and on the storage conditions. The O2 level at which fermentation starts and ethanol tends to accumulate was named the Pasteur point (Fidler and North, 1971), but more recently has been referred to as the lower oxygen limit (LOL) (Beaudry, 1993) or fermentation induction point (FIP) (Petracek et al., 2002). Other designations include the extinction point (EP; Blackman, 1928, cited in

Peppelenbos and Oosterhaven, 1998), the anaerobic compensation point (ACP;

Boersig et al., 1988), and the respiration quotient breakpoint (RQB: Gran and Beaudry,

1993). The ACP and RQB are not directly related to fermentation rates but are derived from gas exchange rates (Peppelenbos and Oosterhaven, 1998). Whatever its designation, this parameter differs among different commodities.

Optimal compositions of controlled atmosphere (CA) and modified atmosphere

(MA) for fresh produce vary according to the species, its maturity or ripeness stage, the temperature and the duration of exposure. The O2 for CA or MA is often considered to be optimal when respiration rates are minimized without development of fermentation

(Banks et al., 1993). It is not the purpose of this review to cover all the work on AA and ethanol accumulation in relation to MA and CA. Kader (1997) in his review summarized the CA requirements and recommendations for fruit other than apples and pears in order to avoid fermentation damages, and two more recent reviews cover the question

of maintaining optimal atmospheric conditions for fruits and vegetables without causing

adverse effects because of fermentation processes (Brecht et al., 2003; Saltveit, 2003).

In every fruit or vegetable subjected to low O2 there is a threshold below which fermentation damage is liable to occur, and the tolerance of commodities to low O2 and high CO2 levels is influenced by combinations of cultivar, temperature, and duration of exposure. It is well documented that deciduous fruits can withstand low O2 storage much better than other fruit, such as those from sub-tropical or tropical origins, but there also may be a lot of variability within a species. For example, in apples, differences between ‘Delicious’ and ‘Granny Smith’ in their peel anatomy can cause differences in gas exchange and the accumulation of anaerobic metabolites under various coatings

(Bai et al., 2003). Among various apple cultivars, tolerance to low O2 ranges from an

LOL of 0.7% for ‘Delicious’ and 0.9% for ‘Law Rome’ to approximately 1.9% for

‘Mclntosh’ fruit (Gran and Beaudry, 1993).

It has been shown that, in apples, the intercellular air space volume, peel porosity, and resistance to gas diffusion are factors that determine the occurrence of

CO2 injury during storage (Watkins et al., 1997). Argenta et al. (2002) showed that ‘Fuji’ apples that had smaller intracellular air spaces suffered from water-core in the tissue, and the more severe the water-core, the higher the levels of internal AA and ethanol. In citrus fruits, which are sensitive to low O2 during storage, ‘Valencia’ oranges produced much more ethanol than ‘Marsh Seedless’ grapefruit at 20°C (Norman, 1977).

Some fruit that are very aromatic, such as banana (Musa × paradisiaca; Hyodo et al., 1983), strawberry (Fragaria x ananassa; Ke et al., 1991; Pesis and Avissar, 1990), feijoa (Acca sellowiana; Pesis et al., 1991), guava (Psidium guajava; McGuire and

Hallman, 1995), melon (Cucumis melo; Choi et al., 2001) and mango (Baldwin et al.,

1999), can produce a lot of volatiles, including AA and ethanol, and, under certain storage conditions, a lot of off flavors. On the other hand, other fruit can withstand exposure to much higher levels of AA and ethanol without generating any off-flavors

(Pesis, 2005).

The optimal atmospheric conditions for CA, MA and MAP that have been

recommended for oranges are approximately 5% O2 plus 5-10% CO2 at 5 to 10 ºC

(Kader, 1997), resulting in delayed senescence, better firmness retention, better flavor,

reduced water loss, inhibition of chilling injury, and reduced peel breakdown (Chase,

1969; Davis et al., 1973; Kader, 1997; Shaw et al., 1990).

Effect of Anaerobic Stress on Ethylene Production and Fruit Softening

Acetaldehyde and ethanol have been shown to be capable of retarding senescence and inhibiting ethylene production in plants, as demonstrated for ethanol applied to cut carnation flowers (Dianthls caryophyllus; Heins, 1980, cited in Pesis, 2005

; Wu et al., 1992). Ethanol has been reported to inhibit color development and lycopene production in whole tomato fruit (Kelly and Saltveit, 1988) and to inhibit ethylene production in tomato pericarp disks (Saltveit and Mencarelli, 1988).

Acetaldehyde, and not ethanol, appears to be responsible for inhibition of ethylene production under anaerobic conditions. Incubation of mango discs with 1- aminocyclopropane-1-carboxylic acid (ACC), the precursor of ethylene, caused a dramatic increase in ethylene production, which was completely inhibited by 1.0% AA but not by 1.0% ethanol (Burdon et al., 1996). The inhibition of ethylene in ACC-treated halved grapes (Vitis vinifera) was found only with AA application, whereas ethanol at the same concentrations did not produce that effect (Pesis and Marinansky, 1992). Banana

ripening was delayed by AA vapor, but not by ethanol vapor (Hewage et al., 1995). In mango, ethylene production was negligible in AA-treated fruit even on day 10 following harvest (Prasad et al., 1999).

Acetaldehyde has been shown to reduce activity of cell wall degrading enzymes such as polygalacturonase (PG) in peach and nectarine (Lurie and Pesis, 1992), PG in tomato (Pesis and Marinansky, 1993) and PG, cellulase, and β-galactosidase in avocado (Dori et al., 1995; Pesis et al., 1998; Ritenour et al., 1997).

In tomato disks treated with 4-methylpyrazole (4MP), an inhibitor of alcohol dehydrogenase (ADH), the enzyme catalyzing the interconversion of ethanol and AA,

Beaulieu et al. (1997) showed that ripening was inhibited only when AA was formed from ethanol, and that when ethanol conversion to AA was inhibited by 4MP, the ripening process was not affected. Acetaldehyde has been shown to be the active agent inducing the effects of ethanol in carrot (Daucus carota) cells (Perata and Alpi, 1991) and in cut flowers (Podd and van Staden, 1999). These latter authors suggested that in cut flowers the toxic effect of AA on ethylene synthesis occurs at the key step of ACC synthase activity. Addition of ACC to avocado disks (in situ) showed that AA reduced ethylene production by inhibiting ACC oxidase activity (Pesis et al., 1998).

However, small amounts of AA (0.04%) can enhance ripening and ethylene production in kiwifruit (Actinidia chinensis; Mencarelli et al., 1991). Also in mango disks, low concentrations of AA (0.1 and 0.5%) or ethanol (0.5–1.0%) vapor stimulated the production of ethylene (Burdon et al., 1996). Beaulieu and Saltveit (1997) concluded that inhibition or promotion of tomato ripening by AA or ethanol vapor was concentration dependent. These results confirm that AA is a very reactive molecule, and this has been

further demonstrated in vitro where it is able to elicit non-enzymatic ethylene production from ACC (Beaulieu et al., 1998).

Production of Off-flavor in Response to Anaerobic Stress

For many fruits, reduction of O2 inside the fruit is accompanied by fermentation

and accumulation of anaerobic off-flavors related to the production of ethanol and AA.

When the O2 concentration is lowered below the Pasteur point, anaerobic metabolism

induces accumulation of AA and ethanol, which can lead to the development of off-

flavors (Fidler and North, 1971). This has been demonstrated in fruit such as strawberry

(Ke et al., 1991; Pesis and Avissar, 1990), cherry (Prunus avium; Meheriuk et al., 1997;

Petracek et al., 2002), muskmelon (Cucumis melo; Choi et al., 2001), litchi (Pesis et al.,

2002) and ‘Oroblanco’ (Citrus paradisi × C. maxima; Porat et al., 2003). Thus, the

anaerobic conditions should be applied carefully, and with attention to the species and

variety of the fruit. Subtropical fruit are among the most sensitive to anaerobiosis

damage, and are less tolerant to low O2 atmospheres, as has been shown in oranges

(Norman, 1977), papaya (Carica papaya; Yahia et al., 1992) and mango fruit (Lizada,

1992).

Off-flavors were also found in strawberries kept in low O2 or treated with AA, and

were correlated with the concentrations of ethanol, ethyl acetate, and AA in the juice

(Ke et al., 1991; Pesis and Avissar, 1990). Severe off-odors, mainly from compounds

that contain sulfur, such as methanethiol, are produced by broccoli when it is held under

anaerobic conditions, and can develop after 2–3 days in MAP (low O2 atmosphere in

polyethylene) (Forney et al., 1991).

Fermentative Metabolism and Fruit Disorders

A physiological disorder is an interruption of the normal plant development

(Morrow and Wheeler, 1997). This definition can also cover harvested plant organs; physiological disorders can originate from preharvest conditions like nutritional imbalances, environmental conditions, and agricultural practices. However, other physiological disorders can be caused by storage conditions such as atmosphere composition, temperature and relative Humidity (Kader, 2002).

A major question that was asked more than three decades ago by Smagula and

Bramlage (1977) - “Acetaldehyde accumulation: Is it a cause of physiological deterioration of fruits?” - is still an open question. Up to now there is no clear answer as to whether AA or ethanol is responsible for water cored ‘Fuji’ apples or for water soaked mesocarp in netted melon (Argenta et al., 2002; Nishizawa et al., 2002). Moreover, there are indications that AA and ethanol are produced in apples because of CO2 injury

(Fernandez-Trujillo et al., 2001). The incidence and severity of brown core in pear

(Pyrus calleryana) increased with increasing CO2, and it was suggested that brown core in pear is not directly correlated with fermentation, but directly with oxidative damage

(Pinto et al., 2001). No significant browning disorder was observed in ‘Braeburn’ apples exposed to ethanol vapor at low temperature, although, the amounts of ethanol, AA, and ethyl acetate in the apple tissue did increase with increasing ethanol vapor concentration and with longer treatment duration (Jamieson et al., 2003).

On the other hand, in other fruit, such as citrus and other tropical and subtropical fruits, accumulation of AA and ethanol causes severe damage. For example, peel discoloration in banana was associated with AA treatment (Hewage et al., 1995). In citrus, it is well documented that accumulation of AA and ethanol in the fruit can cause

severe injury (Cohen et al., 1983, 1990; Norman, 1977). Pitting in white grapefruit increased with decreasing internal O2 levels (below 4%), which were correlated with high AA and ethanol production (Petracek et al., 1998). In avocado cv. Ettinger, higher levels of anaerobic volatiles were correlated with higher mesocarp discoloration, but it is not clear if AA and ethanol were the cause or the result of this damage or coincident with it (Feygenberg et al., 2005).

Product tolerance to low O2 (1-4%) concentration depends on both morphological and metabolic adaptations that are both species and tissue specific (Ratcliffe, 1995).

During O2 limitation, energy metabolism switches from respiration to fermentation, leading to disorders like necrotic and discolored tissues, off odors and off tastes (Kader et al., 1989), suggesting that they have a direct relationship. Kader (1986) stated that decarboxylation of pyruvate to AA through to ethanol results in the development of off- flavors and tissue breakdown. However, under aerobic conditions, ethanol is detected as a normal constituent of apples and many other fruits (Bender et al., 2000; Ke et al.,

1993; Wilkinson and Fidler, 1973).

Many metabolic studies of the survival of plant tissues in the absence of oxygen are focused on the possible toxicity of the main fermentation end-products, lactic acid,

AA, and ethanol (Perata and Alpi, 1993; Ricard et al., 1994). Davies et al. (1974) proposed a regulatory role for lactate during the shift from oxidative to fermentative pathways, through decreasing cell pH and thereby increasing ADH activity. Different researchers, however, have found that the production of lactate is not well matched with the initial fall in the cytoplasmic pH (Ratcliffe, 1995). Also, the occurrence of lactic acid

fermentation prior to the induction of alcoholic fermentation is not universally present in plants (Perata and Alpi, 1993).

Regulation of cytosolic pH is considered to be the major determinant of plant tissue survival in anoxia (Ricard et al., 1994), and attempts to understand the time dependence of cytoplasmic pH in an anoxic tissue need to take into account the full range of events that potentially could influence pH (Ratcliffe, 1995). Lactic acid is sometimes found in harvested fruits and vegetables (Andreev and Vartapetian, 1992;

Ke et al., 1993), but the concentrations found are always lower than ethanol concentrations. Lactate is thought to be toxic at lower concentrations than ethanol

(Perata and Alpi, 1993), so that lactate accumulation may still be responsible for the damage occurring under prolonged anoxia (Richard et al., 1994). Low lactate dehydrogenase (LDH) activity may contribute to the survival under anoxia if lactate production acts to cause acidosis (Perata and Alpi, 1993).

Alcoholic fermentation is always found in plant tissues exposed to anoxia (Perata and Alpi, 1993) and ethanol is often its most abundant product (Pfister-Sieber and

Brandle, 1994; Ricard et al., 1994). Kader (1986) suggested there is also a direct relation between ethanol and tissue disorders. Crawford (1967) proposed that evolution of flood tolerance in plants depended on decreased ethanol production, reducing its presumed toxic effects. Later ethanol was found to be toxic indeed, but only at very high concentrations that are never found in plant tissues (Perata and Alpi, 1993). Many plant tissues can accommodate ethanol concentrations much higher than those found in nature (Kennedy et al., 1992). In contrast, research on ADH null mutants demonstrated that ADH activity is essential for extended survival of maize during anoxia (Kennedy et

al., 1992). Therefore, the assumption that ethanol is toxic under certain conditions is not generally accepted (Pfister-Sieber and Brandle, 1994), making unlikely a direct relationship between concentrations of ethanol and storage disorders of fruits and vegetables.

Acetaldehyde accumulation has emerged as a central mechanism for anaerobic disorders in fruit (Jones, 1989). Already in 1925, AA was shown to be more toxic to plants than ethanol (Thomas, 1925). Perata and Alpi (1991) did not observe toxic effects of exogenously added ethanol in carrot cells when ethanol oxidation to AA was prevented by also adding an ADH inhibitor.

Acetaldehyde appears to affect the secondary metabolism and developmental processes of plant tissues in anaerobic conditions (Ricard et al., 1994). Aldehydes in general are known to be reactive towards proteins with uncharged amino acids (Chervin et al., 1996), thereby interfering with the functioning of essential enzymes. Unclear is whether AA can normally accumulate in high enough quantities to cause damage. It is interesting to note that, for yeasts and bacteria, the strains most tolerant to ethanol have an excess of ADH, such that AA is not allowed to accumulate (Jones, 1989).

Effect of Heat Stress Treatment on Physicochemical and Nutritional Quality

Heat stress treatment can play a significant role in quality control of the produce.

Besides the direct effects of heat exposure on the antioxidant system and other metabolic processes, heat treatments can modify quality changes of produce through the effect of the time and temperature of treatment on the internal product atmosphere in ways that can benefit ripening and postharvest quality. Plants are living organisms that are involved in biological activities in their life cycle even after harvest. Some

activities are desirable, and result in adequate firmness, attractive color, proper fruit size, weight, and biochemical changes such as sugar/acid ratio, vitamin C, phenolic compounds, antioxidant capacity, and volatile compounds. Slowing down respiration and delaying senescence are indispensable to maintenance of quality and shelf life

(Escrinbano and Mitcham, 2014), either for short time storage as a link between harvest and marketing or longer storage to extend postharvest life for distant marketing.

Mandarin fruit treated with hot air at 40°C for 2 days showed a significant increase in the amount of fructose and glucose and decrease in citric acid (Chen et al.

2012), which improved flavor quality. Heat stress may be applied to citrus fruits for controlling disease and insects (Wang et al., 2010), and to improve resistance to chilling injury (Erkan et al., 2005; Schirra et al., 2004). Navel oranges treated with hot air at

46°C for 4.5 h exhibited significantly decreased TA and increased SSC: TTA ratio

(Shellie and Mangan, 1998). Similarly, hot water immersion at 41°C for 20 min or 50°C for 5 min reduced chilling injury in Washington Navel and Valencia Late oranges, but there were no effects on SSC, TTA, ascorbic acid or sugars (Bassal and El-Hamahmy,

2011). Exposure of peaches to 39°C for 3 days resulted in citric and malic acid being reduced by 20% and 50%, respectively, and larger amounts of fructose and glucose were noticed in treated fruit, but there was no effect on sucrose content (Lara et al.,

2009). The higher temperature produced about a 40% increase in respiration rate and organic acids are preferred as a respiratory substrate, which explains the loss of citrate and malate.

Heat treatment affects physicochemical quality of banana fruit. When two different varieties of banana, ‘Bari Kola’ and ‘Sabri Cola’, were exposed to six different

combinations of hot water temperatures and immersion times, it was found that the bananas treated with combinations of 53°C for 9 min or 55°C for 7 min had higher lightness (L*), SSC, acidity, and β-carotene than untreated fruit control fruit (Amin and

Hossain, 2013), whereas ascorbic acid degraded during storage. ‘Gros Michel’ fruit immersed in hot water at 50°C for 10 min showed delayed degreening, and maintained higher pulp firmness, and increased phenolics and flavonoids compared to control fruit

(Ummarat et al, 2011). However, the fruit peel was injured at temperatures higher than

57°C for 7 to 9 mins. Similar trends were observed for ‘Red Fuji’ apples subjected to hot air at 45°C for 3 hr, which developed the highest total phenolics content and antioxidant capacity, while ‘Golden Delicious’ apples lost TTA because of apparent higher sensitivity to heat treatment (Li et al., 2013).

Effect of Heat Stress Treatment on Sensory Quality

Consumer decisions for produce purchasing or consumption around the world depend on excellent postharvest quality, especially the first appearance, which encompasses sensory quality. Heat treatment can influence sensory quality of a commodity, an effect which can be determined by sensory test panelists.

Heat stress applied as hot air at 42°C for 18 hr inhibited peel spotting on the surface of ‘Sucrier’ bananas while maintaining acceptable fruit flavor, which was evaluated by a sensory panel (Kamdee et al., 2009).

Effective heat stress treatment demonstrated better sensory quality for ‘Venus’ nectarines. Jemric and Fruk (2013) treated ‘Venus’ nectarines with hot water immersion at 48°C for 6 to12 min. Nectarines treated for 12 min performed better in terms of sensory quality for texture, aroma, taste, SSC/TTA ratio, firmness, and general appearance after 2 weeks of storage.

‘Gala’ apples are among the most popular in the world due to good sensory qualities and heat stress improved Gala fruit sensory profiles compared with untreated control. Hot air treatment was applied to delay softening of ‘Gala’ apple by Shao et al.

(2012) who treated the fruit with a combination of hot air (38°C for 4 days) and 1% chitosan coating, which suppressed ethylene production and respiration rate, and inhibited fungal development. The overall sensory score showed the highest consumer acceptance after storage for fruit treated with heat stress before coating. But, severe heat stress damage was found on the fruit treated with heat stress after coating formation.

Waxing application before heat treatments can have negative effects on sensory quality of orange fruit. Wax coating was applied to Navel oranges either before or after the APHIS T103-b-1 high temperature forced-air treatment protocol (44°C for 100 min) for disinfestation of Anastrepha spp. fruit flies (Obenland et al., 2012). Application of hot air resulted in changes in flavor and volatile compounds. A significant loss in flavor quality occurred after the treatment though the SSC/TTA ratio increased. Therefore, off- flavor developed in the fruit because of reduction in orange sweetness. Waxing restricted the exit of volatile compounds from the fruit, which resulted in formation of off- flavors in the fruit; four esters were enhanced by the heat treatment and two of them, ethyl hexanoate and ethyl butanohate, induced loss in flavor quality.

Heat stress accelerated production of aroma volatiles in mango fruits. Singh and

Saini (2014) treated ‘Chausa’ mangoes with several vapor heat time-temperature combinations including 46.5 to 48.0°C for 45 to 20 min, after 8 days of storage at 26°C.

The treatments enhanced fruit ripening, as evidenced by skin color and flesh firmness.

However, the treated fruit had less characteristic mango aroma as judged by a sensory

panel. The heat stress treatment altered the volatile composition and the ripe fruit flavor.

At the ripe stage, lower concentrations of total monoterpenes, especially α- terpinolene,

and total esters were presented in the heat-treated mango fruit.

Semi-permeable Coatings for Produce

Edible coatings for fresh produce consist of a thin layer of protective material that

is added by dipping or spraying on the surface of the product (Gennadios and Weller,

1994; Ukai et al., 1976). Coating applications have been used in fresh commodities for

a long time to prevent quality loss and extend the shelf life of the commodities by

modifying their surface properties. The first fruit coating formulations were suspensions

of oils or waxes in water that were used to reduce dehydration. Research indicated that

China introduced wax coatings in the 12th and 13th centuries to citrus fruits (Hardenburg,

1967).

Fresh fruit are coated to improve surface gloss, reduce abrasion, minimize solute leakage, establish a barrier to moisture loss, change O2 and CO2 levels inside the commodity, and modulate ripening and senescence. Edible coating formulations are prepared from a variety of materials such as lipids, polysaccharides, and proteins, alone or in combination (Kester and Fennema, 1986; Ukai et al., 1976). The other components are added with the coating formulation such as plasticizers, emulsifiers, reinforcements, additives and solvents (water, alcohols) to complete the coating matrix.

Edible coatings may control the internal gas atmosphere of citrus fruits, thus minimizing fruit respiration rate (Park, 1999) and may serve as a barrier to water vapor, reducing moisture loss and delaying fruit dehydration (Baldwin et al., 1995). Coatings

may also improve textural quality and help retain volatile flavor compounds as well as reducing microbial growth (Debeaufort et. al., 1998).

Fruit Coatings to Create Internal Modified Atmospheres

Edible coating is a promising technology to keep orange fruit firm and fresh while

preserving the internal quality by creating a beneficial internal atmosphere. Wax

coatings have the potential to achieve benefits for orange fruits by maintaining an

internal atmosphere in the fruit that stabilizes the produce and thereby promotes

extended shelf life (Baldwin et al., 1995). Edible coatings may also decrease

physiological disorders via the modification of internal atmosphere (BenYehoshla,

1969).

Gas exchange rate in fruit depends on external factors such as O2, CO2, C2H4,

and temperature as well as internal factors such as species, cultivars, and growth state

(Kluge et al., 2002) and physical factors, primarily barriers to gas diffusion in the form of

cuticle, peel and applied edible coatings. Proper gas exchange into produce extends

shelf life. Commodities can eventually transfer to partial anaerobic respiration at around

1-3% O2 (Guilbert et al., 1996; McHugh and Senesi, 2000; Park et al., 1994) by

modification of internal gases, so the gas exchange must be properly managed to avoid

that.

Surface coatings have been used extensively on many fruits and vegetables to improve cosmetic features, to reduce water loss, and to achieve internal MA benefits to delay ripening (Amarante and Banks, 2000; Banks et al., 1993, 1997; Hagenmaier and

Baker, 1993; Smith et al., 1987). The optimization of surface coatings involves retention of postharvest quality with acceptable levels of risk and to avoid fermentation and physiological disorders (Amarante and Banks, 2000; Banks et al., 1993, 1997).

Generally, the effect of reduced O2 and/or elevated CO2 on reducing respiration rate and processes linked to respiration, as well as ethylene synthesis and action, have been assumed to be the primary reasons for the beneficial effects of CA/MA on fruits and vegetables (Kader et al., 1989).

In the citrus industry, wax coatings are applied primarily to reduce weight loss

from transpiration (Kaplan, 1986). Coatings can also form a barrier against O2 and CO2

diffusion, resulting in a modified internal atmosphere that can help to reduce respiration

rate by the produce (Saftner, 1999). When a fruit is harvested, there is typically a

temporaty upsurge in the consumption of O2 and the production of CO2, resulting in

metabolic loss and accelerating the fruit’s progression to eventual senescence (Dhall,

2013). Edible coatings that create internal MA are important for fresh produce because,

while a commodity respires continuously after harvest, resulting in significant

postharvest qualitative and quantitative loss, this process can be inhibited by the MA.

Citrus fruits are commonly coated with fruit waxes that reduce the gas exchange between fruit and atmosphere, resulting in elevated internal CO2 concentration and reduced weight loss (Durand et al., 1984; Farooqi et al., 1988; Hasegawa and Iba,

1980; Meheriuk and Porritt, 1972;). According to Park (1999) formulated edible coatings may provide additional protection against contamination by microorganisms while serving a similar role as MA storage in modifying internal gas composition.

Coatings decrease firmness loss and moisture loss (Avena-Bustillos et al., 1997;

Li and Barth, 1998) from the produce and control respiration rate (Banks, 1984),

ethylene production (Baldwin et al., 1995; Banks, 1984), and reduce metabolism and

oxidation rates (Li and Barth, 1998). Citrus fruits are commonly coated with fruit waxes

that reduce the gas exchange between fruit and atmosphere, resulting in elevated internal CO2 concentration and reduced weight loss (Durand et al., 1984; Farooqi et al.,

1988; Hasegawa and Iba, 1980; Meheriuk and Porritt, 1972;). According to Park (1999) formulated edible coatings may provide additional protection against contamination by microorganisms while serving a similar role as MA storage in modifying internal gas composition.

Benefits of Wax Coatings to Maintain Quality and Reduce Physiological Disorders of Fruits

Edible coatings can suppress physiological disorders by reducing moisture and solute migration, gas exchange, respiration, and oxidative reaction rates (Baldwin et al.,

1996; Park, 1999). Coating also may be designed to carry active substances such as anti-browning agents, flavors, nutrients, and antimicrobial compounds that work against pathogen growth (Pranoto et al., 2005).

Wax coating can reduce chlorophyll loss (Banks, 1984), and control microbial growth (Baldwin et al., 1995), as well as improve the fruit peel appearance (Davis and

Hofmann, 1973) such as increased shininess on the surface of Navel and Valencia oranges. Proteins, lipids and polysaccharides are the main constituents of coatings and act as a barrier with regard to water vapor, O2, CO2 and lipid transfer in food systems

(Guilber et., 1996; Morris, 1982). Coating may also provide additional nutrients and enhance sensory attributes, and include quality-enhancing antimicrobials to the fresh produce (Guilber et al., 1996).

Flavor sensitivity is associated with high-gloss coatings. For commercial purpose, packinghouse is needed for uniform coating application with good gloss on the surface of citrus fruit (Hagenmaier and Shaw, 2002). A commercial packing line is associated

with washing, rinsing, waxing, drying, sorting, grading, and then packing into boxes.

High gloss coating comprise of shellac with wood rosin exhibit much lower internal O2

and higher CO2 compared to polyethylene or carnauba wax (Hagenmaier and Baker,

1994).

Edible coatings have been studied for extending shelf life of some citrus fruits

(Han et al., 2004; Park, 1999; Ribeiro et al., 2007; Vargas et al., 2006). The extent to which coatings restrict the air exchange of fruits, depends not only on the surface properties of the fruits, but also how a coating is distributed over the surface of the fruit, especially whether it forms a continuous layer or penetrates into pores (Hagenmaier and

Baker, 1993). For uncoated fruit, the holes associated with lenticels, stomata, stem scars, and injuries are probably the main pathway for gas exchange, but for coated fruit, on the other hand, it is possible that these holes are filled or bridged over by the coating

(Burg, 1990).

Research findings showed that polysaccharides coating from hydroxypropylmethylcellulose (HPMC) extended shelf life with sensory quality of oranges up to 7 weeks at ambient temperature of 27±3°C and RH of 50-65% (Adetunji et al., 2012). Polysaccharides, proteins, lipids and their combinations may be used as coating materials for fresh produce (Baldwin et al., 1995). Chitosan (1,4-linked2-amino-

2-deoxy--d-glucan), a derivative of chitin, has excellent film-forming and antimicrobial functions and has been successfully used to control quality loss of some fruits such as fresh strawberries, raspberries (Rubus idaeus; Han et al., 2004; Park et al., 2005;

Ribeiro et al., 2007; Vargas et al., 2006), fresh-cut water chestnut (Trapa natans; Pen and Jiang, 2003) and sliced mango fruits (Chien et al., 2007), and many other fruits and

vegetables (Lin and Zhao, 2007). SemperfreshTM, a commercial coating product of sucrose-fatty acid ester, was reported to effectively decrease weight loss of hardy kiwifruit (Actinidia arguta; Fisk et al., 2008), cherry (Yaman and Bayoindirli, 2002), summer squash (Cucurbita pepo; Kaynas and Ozelkok, 1999), and extend shelf life of pineapple (Ananas comosus) for up to 5 weeks by preventing moisture loss

(Nimitkeatkai et al., 2006). Sodium alginate is a natural linear polysaccharide and has many attractive physical and biological properties, such as moisture retention, gel- forming capability, and good biocompatibility (Pei et al., 2008).

Coating fruit with various waxes that influence their internal O2 atmosphere may lead to AA and ethanol accumulation and production of off-flavors, especially in subtropical fruits such as, guava, mango and citrus (Baldwin et al., 1999; Hagenmaier,

2002; McGuire and Hallman, 1995). In mango cv. Tommy Atkins coated with NutraSeal, the levels of AA and ethanol were much higher than in fruit coated with carnauba wax, and these higher levels affected the fruit taste (Baldwin et al., 1999).

Main Matrix Constituents for Edible Wax Coating Formulation

The main matrix components used in coating applications are hydrocolloids,

lipids, and their composite mixtures (Poverenov et al., 2014). Protein and

polysaccharide films and coatings generally exhibit excellent barrier properties against

O2 and aroma movement in or out of the fruit, but have high water vapor permeability

(Baldwin et al., 2012). The composite coatings are produced by combining two or more

constituents. Different types of lipids, waxes (beeswax, candelilla, carnauba), free fatty

acids, fatty alcohols, fatty acids and sucrose esters, edible terpene resins such as

shellac, and paraffins have been used to increase surface gloss and limit water loss for

citrus fruits (Lawrence and Iyengar, 1983; Paredes-López et. al., 1974; Park et al.,

2014; Warth, 1986).

Different ingredients are incorporated into edible coating matrices to improve or modify their internal functionality. Plasticizers are commonly used in polysaccharide and protein-based coatings to reduce brittleness (Han and Gennadios, 2005; Oz and

Ujukanli, 2012; Sothornvit and Krochta, 2005). Water, glycerol, propylene glycol, sorbitol, sucrose, polyethylene glycol, fatty acids, and monoglycerides are added with plasticizers in different proportions (15-40% to the main matrix) to make edible coatings

(Sothornvit and Krochta, 2005).

Plasticizers enhance film flexibility and susceptibility to humidity and decrease strength and barrier properties against moisture and O2 (Vanin et al., 2005). Good gas exchange or permeability depends on the amount of added plasticizer. Higher amount of plasticizer provide greater O2 permeability (Park and Chinnan, 1990), which results in elevated internal O2 and decreased internal CO2 in the fruit. However, the minimal amounts of plasticizer used in wax coating formulations provide optimum peel appearance and optimum flavor (Hagenmaier, 2004).

Wax coatings, including carnauba wax, shellac wax, polyethylene wax, candelilla, and beeswax, are mostly used for fruits and vegetables to improve the quality and enhance the shelf life. Carnauba wax is prepared from palm leaves (Copoernica cerifera), and has a very high melting point. It is used to increase toughness and luster.

Good quality of fatty acid is important for ammonia-based coatings to get low turbidity, which determines the quality of the coating.

Wax coatings for citrus fruits may be made from various waxes, oleic acid or fatty acids, plasticizer (aqueous ammonia as morpholine), and water (Hagenmaier and

Baker, 1994), with a micro-emulsion being prepared by one of three different methods:

1) wax to water, 2) water to wax, and 3) direct pressure.

Lipid coatings (paraffin, beeswax) are used as fruit coatings to protect against moisture loss and control respiration, and they improve peel appearance by generating a shine on the surface of fruits such as Navel and Valencia oranges (Kester and

Fennema, 1986).

Polyethylene wax is produced by the oxidation of polyethylene and is used primarily to make emulsion coatings. Edible waxes are applied as barrier films to gas and moisture. Applicable of thin layer wax coating provide a good humidity barrier for the citrus fruit such as carnauba, candelilla, and bee wax.

Shellac is not a “generally regarded as safe” (GRAS) substance and as such is only permitted as an indirect food additive in coatings (Shit and Shah, 2014). It is mostly used in coatings for the pharmaceutical industry and also some indirect food additive uses (Hernandez, 1994). Its use on fruit like citrus is primarily because shellac contributes excellent gloss properties to coatings. However, use of shellac coatings on fruit is problematic because it can create dangerously low internal O2 and high internal

CO2 and C2H4 (Hagenmaier and Baker, 1993). Shellac coating increases prevalence of postharvest pitting during storage of citrus fruits, including Valencia and Navel oranges

(Petracek et al., 1998).

CHAPTER 3 USING HOT WATER IMMERSION AND EDIBLE COATINGS TO MAINTAIN PHYSICOCHEMICAL ATTRIBUTES AND SHELF LIFE OF NAVEL AND VALENCIA ORANGES

Hot water treatment is a type of heat stress treatment that has been used and evaluated for many years to reduce decay and to control insect infestation, mainly in fruit (Kader and Arapia, 1992; Erkan et al, 2005). It has been successfully applied for many fruits and is favored for its low impact on the environment. Application of hot water treatment has been used in many countries successfully to control potential decay of citrus fruits. In Florida, the most prevalent decay organism of citrus fruits is stem-end- rots (Lasiodiplodia theobromae and Phomopsis citri). Penicillium species invade citrus tissue through wounds, while the stem-end-rot organisms develop latent infections within the button tissue that are more protected from physical and chemical treatments

(Ritenour et al., 2013). Florida grapefruit (Citrus paradisi Macf.) exposed to hot water from 56 to 62oC for 20 s exhibited subsequent reduction of mold (Penicillium) and CI

(Ritenour et al., 2003).

Heat treatments have been primarily and widely applied to fruits and flowers in order to control pathogens and insect pests, to increase resistance to chilling injury (CI;

Paull and Jung Chen, 2000), as an insect quarantine treatment (Ferguson et al., 2000), to avoid the use of chemicals (Lurie, 1998), and to preserve product quality. It has been proposed that sublethal, reversible heat stress stimulates the fruit antioxidant system and heat shock proteins, which improves the fruit’s capacity to tolerate subsequent stresses (Schoffl et al., 1998). For example, heat stress treatment may induce fruit tolerance to cold temperatures and thus reduce the development of CI symptoms during

cold storage. Oranges are subtropical fruits that are known to be susceptible to CI development during cold storage.

The uniformity in the response of horticultural commodities to a heat treatment depends on the plant organ, size, shape, and morphology, since heat transfer depends on the contact surface (Paull and Chen, 2000). Moreover, many commodities show signs of heat injury when heat treatments are not optimized. This has been attributed to and micro wounds (necrotic tissue), which caused decay development, and also resulted in internal cavity development (Ferguson et al., 2000; Jacobi and Wong, 1992;

Lurie, 1998); such heat injury has been observed in mangoes (Joyce et al., 1993) and in tomatoes (Iwahashi et al., 1999). Depending on the commodity, different temperature and time combinations are effective to prolong shelf life and quality. But, extreme heat stress produces structural damage (Paull and Chen, 2000) in many produce items such as tomato fruit. Brecht et al. (1999) observed visible surface damage (“scald”) in tomatoes treated with hot water over 50oC for more than 10 minutes, injury that was also detected as electrolyte leakage.

Temperature is the most important environmental factor in the postharvest life of horticultural commodities because of its effect on biological activity such as respiration.

Every 10oC temperature increase causes a 2 to 3-fold increase in respiration rate and reduces shelf life of the produce by 1/2 to 2/3. But extreme temperature such as >40oC can result in drastically reduced respiration, eventually resulting in tissue reaching a temperature referred to as the “thermal death point.” Generally, in immature plant parts, respiration is very high during the early stages of development and it decreases with maturation and steadily after harvest with slower decline in mature fruit crops and faster

in vegetative tissues and immature plant organs. Faster reduction of respiration is associated with the depletion of respirable substrates (Kader and Salveit, 2003).

Initially, respiration rate typically accelerates up to 35 - 40oC, and it then decreases with further temperature increase and longer exposure time (Lurie and Klein, 1991).

Respiration involves enzymatic oxidation of organic substrates coupled with energy production. Respiratory rate is inversely proportional to produce storage life: the higher respiration of produce, the shorter is the storability. Respiration is ramped up in fresh produce after harvesting if the produce is subject to physical stress, resulting in compositional changes and reduced shelf life (Sandhya, 2010). Modified atmosphere established quickly after harvest inhibits respiration and thus helps extend storage life.

A prolonged heat treatment can increase respiration of produce, although respiration usually returns to normal levels after a non-injurious heat treatment (Paull and Chen, 2000). According to Mitcham and McDonald (1993), mango fruit respiration increased up to 5-fold during treatment when fruit were treated with forced-air at 48oC for 5 hours, but the fruit recovered their normal respiration shortly after being transferred to 20oC. Similarly, an effective heat stress treatment can modify the fruit internal atmosphere by increasing respiration. Increased temperature during hot water treatment raises the fruit respiration rate without occurrence of gas exchange, resulting in the internal CO2 increasing whereas internal O2 decreases. It was found that grapefruit treated with hot water at 48oC for 3 hours had increased internal concentration of CO2

(to 21 kPa) and O2 decreased to as low as 3 kPa (Shellie and Mangan, 2000).

The beneficial effects of MA are basically related to reduce metabolism of the stored produce under altered atmospheric storage conditions (Kader, 1986). Generally,

O2 levels are reduced and CO2 levels are elevated, which results in a proportional reduction in respiratory activity (Kader, 1986). Under optimum low O2 atmospheres, both the changes in O2 consumption and CO2 release remain proportional with no alteration of the respiratory quotient (RQ). In contrast, under increased CO2 atmospheres, the CO2 production is affected more than O2 consumption, thus leading to differences in RQ under different CO2 levels (Knee, 1973). Elevated CO2 may also have a beneficial effect for the commodity in reducing or minimizing physiological disorders or slowing down loss of fruit firmness. The optimal atmospheric conditions that have been recommended for oranges are approximately 5% O2 plus 5-10% CO2 (Kader, 1997).

Considering the above, a series of preliminary experiments were conducted to test the hypothesis that a hot water treatment and subsequent wax coating will create and maintain a modification of the internal atmosphere that will allow successful storage of orange fruit at ambient temperature for sufficient duration to allow marketing.

Therefore, the objectives of this part of my dissertation were to determine a non- injurious combination of hot water immersion temperature and duration of immersion to stimulate respiration without causing peel injury of citrus fruits, when coupled with proper fruit coating, to create and maintain a beneficial internal modified atmosphere that will better maintain physicochemical attributes and shelf life during ambient temperature storage.

Materials and Methods

Experiment 1 -- Temperatures

Determination of the temperature at which maximal respiration rate and ethylene production occurs in fresh Navel orange fruit. Washington Navel oranges

(Citrus sinensis, L.) were harvested and collected in August 2014 from a commercial

grower in the Fort Pierce area and taken to the UF/IFAS Indian River Research and

Education Center (IRREC). After that, fruit were immersed in a solution of 1,000 ppm thiabendazole (TBZ) fungicide for 30 s. Then the fruit were placed in a degreening room at 85oF for ethylene treatment at 3 ppm with 85-90% relative humidity (RH) for 24 hrs.

After degreening, the fruit were stored at 5oC for 3 days before being transported to the

UF/IFAS Horticultural Sciences Departmental in Gainesville. There, fruit of uniform size

(approximately 230 g), without blemishes, defects or injuries were used for the experiment. Fruit were weighed and held in 3.2-liter glass jars, each containing 3 fruit

(one replicate; Figure 3-1), and then CO2 and ethylene production measured at different temperatures. This experiment was conducted to help direct future planned hot water immersion experiments by determining the range of water temperatures that could be used to induce elevated respiration rates during immersion that would significantly change the fruit internal atmospheres.

Prior to respiration and ethylene measurements, jar lids were open and the fruit allowed to equilibrate for 6 hours with the treatment air temperature (until treatment air temperature equilibrated with the fruit pulp, which was determined earlier by measurements made with thermistor probes and recorded by a data logger); the temperatures ranged from 20 to 60oC with 5oC increments. Two procedures were used in consecutive experiments for measurement of respiration and ethylene. The first time the experiment was conducted, one batch of fruit was used throughout the temperature increases and, for the second the experiment, a different batch of fruit was used for each temperature.

For measurement of respiration and ethylene, the glass jars with temperature- equilibrated fruit were sealed and the void volume gas was sampled after 30, 45, and 60 min from each glass jar with a 3-ml syringe. A 2.5 ml-gas sample was injected into a

Varian CP-3800 GC (Varian Inc., Palo Alto, CA) equipped with a thermal conductivity detector (TCD) and a pulse discharge helium ionization detector (PDHID) to measure the concentrations of CO2 and C2H4, respectively. Via an automated sample-loop and valve system, a 1-ml portion of the injected sample passed through Hayesep Q

Ultimetal (1 m × 3.18 mm) [particle size 149–177 μm (80/100 mesh)] and Molsieve 13

(1.5 m × 3.18 mm) [particle size 149–177 μm (80/100 mesh)] columns (Varian) coupled in series to the TCD for O2 and CO2 determination. A 1-ml portion of the gas passed through two Hayesep Q Ultimetal (1 m × 3.18 mm) [particle size 149–177 μm (80/100 mesh)] columns (Varian) coupled in series to the PDHID for ethylene determination. The carrier gas (helium) flow rate was 0.35 ml s−1. The injector was set to 220°C, the oven to

50°C, the TCD to 175°C, and the PDHID to 120°C. The respiration and ethylene production rates were calculated as the respective amounts of CO2 and C2H4 evolved by the fruit based on unit fresh weight using the following equations:

Respiration rate calculation: Ethylene production rate calculation: ml CO2/kg-hr = % CO2 x K µlC2H4/kg-hr = ppm C2H4 x K where, K = ml (void)/(kg x hr x100) where, K = ml (void)/(kg x hr x100)

Experiment 2 – Internal Atmospheres

Internal atmospheres of un-waxed and waxed Valencia oranges during storage in controlled atmosphere (CA) conditions. This experiment was conducted to determine the internal fruit atmosphere when orange fruit are stored in recommended

CA conditions. The experiment included a typical refrigerated storage temperature (5oC)

and a higher temperature (25oC) to simulate ambient temperature storage. Valencia oranges were harvested from a commercial grove in Fort Pierce, FL in March 2015 and were transported to Gainesville and then the fruit stored at 5oC in a Horticultural

Sciences Department storage room until used. Uniform size fruit were randomly selected for each treatment and exposed to 5oC or 25oC for an hour prior to application of the atmosphere treatments. Two different CA treatments (5%O2+5%CO2 and

5%O2+10%CO2) were selected for the study and the control treatment was air. Three,

1-L plastic containers holding one fruit (replicate) each were connected to the gas flow in series (Figure 3-2).

Holes (15 mm diameter) were drilled into each container in the middle of the bottom, front, and back sides to allow insertion of a rubber tube with gasket to keep the container air tight. The top of the container was closed with a snap-tight lid and was positioned lid side down on the rack of the CA chamber. Approximately 10-cm lengths of glass tubing were cut using an electric cutting machine and a tube inserted into the opposite sides of each plastic container top and into the blossom end of the fruit to collect the internal atmosphere of the fruit (Figure 3-3). Before being placed into the containers, the fruit peel at the blossom end was carefully removed so that the underlying fruit tissue (segments) was not disrupted. The required flows of gases to create the CA treatments were continuously supplied from pressurized tanks to all containers for 3 weeks storage using a gas mixing board with manifolds and needle valves. The gas mixtures were humidified by bubbling the gas flow through water.

Fruit internal gas measurement. Internal CO2 and O2 values were determined during 3 weeks of storage at 5oC or 25oC and 80±5%RH. A syringe needle was inserted

into the glass tube through a rubber septa from each of the fruit, and 1 ml of internal gas was collected. To increase the sample size for injection onto the GC, which required a

2.5-ml sample size, first the 1-ml internal fruit sample was injected into a 5.5-ml glass container sealed with a rubber serum stopper that had been previously flushed with N2 gas (60ml/min). Next, 1.5 ml N2 gas was added to the glass container and then a 2.5-ml sample was collected from the container by another syringe to inject onto the GC columns (Varian) and O2, CO2, and C2H4 levels were analyzed. This procedure allowed a 2.5-ml gas sample to be collected without creating a negative pressure in the syringe that would cause dilution of the unknown sample by air in an uncontrolled manner.

Finally, internal atmospheres (O2, CO2, and C2H4) were calculated from calibration curves at each sampling interval, and expressed as percentages (vol/vol).

Experiment 3 – Optimal Imersion

Determination of the most suitable temperature(s) and duration(s) for hot water immersion to create a potentially beneficial internal modified atmosphere

(MA) in orange fruit. Five replicated experiments were conducted in 2014-2015 (Table

3-1) to determine the optimum time-temperature combination of orange fruit to create an extreme internal MA (similar to under CA conditions) with/without causing peel injury by measuring the internal atmosphere of hot-water-treated fruit. 2-3 fruit were placed in a clamshell and used a rubber band so that it would n’t be lossen. At the top of the chamshell 2 to 3 hole were made to be inserted a 1-ml syringe needle into the blossom end for each fruit. When set temperature reached into water then fruit dipped into water hot water and submersion time counted for each batch of fruit. Atfter completing dipping time, 1-ml internal gas was collected by the attached syringe needle for each fruit still

fruit submerged into hot water. Collected gas sample was kept into a 5.5 ml glass container for further gas analysis by GC (Figure 3-4).

For all five repeats of the experiment, orange fruits (Navel and Valencia) were collected from commercial groves near Fort Pierce and transported to Gainesville for conducting the experiments in the Horticultural Sciences Department of the University of

Florida. Transported fruit were stored at 5oC until used for the study and fruit were exposed to ambient laboratory temperature (approximately 24oC) for 2-3 hours prior to initiating the experiment. The first study was conducted in August 2014 to select appropriate hot water immersion durations for a 40oC hot water treatment temperature.

There were seven different durations: 30, 40, 50, 60, 70, 80, and 90 min (Table 3-1).

The hot water temperature was selected based on Experiment 1, which indicated that

40oC provided the desired elevated respiration rate of Navel orange fruit without any peel injury. The control treatment was immersion in 25oC water. Five to eight fruit were randomly selected for each treatment to measure fruit internal atmospheres, and another 24 fruit were used to evaluate peel injury or percentage decay during 3 weeks storage at 25oC with 85±5%RH following the hot water treatments.

The second and fourth studies were conducted with two different harvests of

Valencia oranges in March 2015. Lower hot water treatment temperatures (30o, 35o,

40oC) with longer hot water immersion times (60, 70, 80 min), plus 45oC for 20, 30 and

40 min (fourth Study), were used. Those experiments utilized a completely randomized design (CRD) with 2 factors (treatment temperature and water immersion time). Fruit internal atmospheres were measured during hot water immersion and fruit were evaluated for peel injury during subsequent storage as previously described.

Similarly, the third and fifth studies were conducted with two different harvests of

Valencia oranges in April 2015 to evaluate higher temperatures with shorter durations of water immersion. For those experiments, three different temperatures (45o, 50o, 55oC) with three different durations (10, 15, 20 min) were used. Three replications with 5 to 8 fruit each were used to measure the internal fruit atmospheres, and 18 oranges were considered for peel injury/decay evaluation during the 3-week storage period (25oC with

85±5%RH).

Fruit internal atmospheres were measured during hot water immersion and fruit peel injury was evaluated during subsequent storage with assistance from a plant pathologist in the Plant Pathology Department of the University of Florida. Incidence of injured fruit was expressed as the percentage of the fruit that were affected, and peel injury severity was determined using a 5-point rating scale (1 = No injury, 2 = slight injury, up to 15% brown skin area; 3 = moderate injury, 16% to 50% brown skin area; 4

= severe injury, 51% to 85% brown skin area; 5 = extreme injury, 86% to 100% brown skin area) according to Ke and Kader (1990). The above scale was used for all studies except the first study. Subjective evaluation of peel injury and the measurement of fruit internal atmospheres were also done immediately after hot water immersion of orange fruit.

Experiment 4 – Optimal Combination of Temperature and Immersion

Internal temperature profiles of Navel oranges using the best combination of hot water temperature with immersion time. This experiment was conducted in

December 2015 for tracking the temperature profile of Navel oranges inside the fruit tissue at different layer depths during hot water immersion treatment. A Squirrel data logger with “SQ2020” thermocouples (Grant Instruments, Cambridge LTD, Shepreth,

Cambridgeshire, UK) was used for the experiment (Figure 3-5). Before setting up the equipment, Navel oranges that had been held at 5oC were exposed to room temperature (25oC) for adequate time to equilibrate. Then, uniform size (approximately

240 g) orange fruit were selected and seven thermocouples were used for two sets of fruit (6 thermocouples to measure fruit temperatures plus one thermocouple to measure water temperature). First, the fruit diameter was measured using a slide caliper and the location of the fruit center and the midpoint (half way between the fruit center and surface) were estimated to insert thermocouples. For measurement of the “surface” temperature of the fruit, the peel thickness was also estimated so as to add a thermocouple at the midpoint of the fruit peel. After estimating the center, midpoint, and surface of fruit, one thermocouple was inserted into the fruit at each of those points and the entry holes sealed with silicon glue to fix the thermocouples in place and so that water would not enter. Two rubber bands per fruit were used to immobilize the fruit and thermocouples in a plastic commercial clamshell container that had sufficient opening on the surface to allow water entry inside the container and to maintain uniform water temperature. The water bath was filled with water and the temperature set at 45oC as well as being monitored by a portable thermometer. When the temperature reached the desired temperature, two sets of individual fruit were immersed for 30 min and 60 min.

Experiment 5 – Respiration Rate and Ethylene Production

Determination of respiration rate and ethylene production of Navel oranges at different storage temperatures after immersion in 45oC water with or without application of edible coatings. This experiment was conducted in November 2015 for measurement of respiration rate and ethylene production of Navel orange fruit treated with/without hot water and with/without coating application to determine the effect of hot

water treatment with or without coating on fruit respiration during storage. Hot water and coating treatments were conducted at IRREC, Fort Pierce by using the Postharvest

Packingline facility and then the fruit were transported to the Horticultural Sciences

Department, University of Florida, Gainesville for the measurement of respiration.

For this study, uniform size fruit with no blemishes, defects or injuries were sorted from stored Navel orange fruit and the fruit were randomized. The water immersion treatments were applied in a stainless steel tank (31 x 18 x 19 inch), which was used to submerge the fruit for 30 min in water maintained at 25 °C or 45 °C with rapid stirring. The fruit were then brush-washed with detergent and either coated with polyethylene-, carnauba-, or shellac-based coatings (JBT Corp., Lakeland, FL, USA) using a hand spray applicator on a semi-commercial citrus packingline at the UF/IFAS

IRREC in Ft. Pierce. Treated fruit were then exposed to hot air for surface drying on the packingline.

The fruit were collected in plastic crates and carried to the Postharvest Biology and Technology lab in the Horticultural Sciences Department, University of Florida,

Gainesville and placed into storage overnight at 5°C. The following day, the fruit were allowed to equilibrate to 25°C and uniform size orange fruit were selected and randomly assigned to each treatment. Three replications/treatment with each replication having 3 fruit were weighed and held in 3.2-liter glass jars for the measurement of respiration and ethylene production, which was done following the procedure that was described in the materials and methods of Experiment 1.

Results

Determination of the Temperature at Which Maximal Respiration Rate and Ethylene Production Occurs in Fresh Navel Orange Fruit

As shown in Figure 3-6, fruit respiration rate significantly increased up to 50oC and then respiration declined at 55 and 60oC, at which point most of the fruit were peel injured because of extreme fruit temperature. The initial respiration rate of fresh orange at 25oC was 19.57 mlCO2/kg-hr and the maximum respiration measured at 50oC for the same fruit transferred to progressively higher temperatures was 98.02 mlCO2/kg-hr. It was also noticed that there was no significant fruit peel injury found from 40oC to 45oC room air temperature, which had maximal respiration rate of 75.34 mlCO2/kg-hr. Similar trends were observed when the fruit samples were changed for respiration measurements at each air temperature. In this case, respiration increased almost 6-fold from 25oC to 50oC, which was a 2- to 3-fold greater increase at each temperature that observed for fruit that were repeatedly measured. The maximal respiration rate was observed at 45oC (295.91 mlCO2/kg-hr) and 50oC (305.48 mlCO2/kg-hr) and then respiration rate declined drastically up to 60oC (119.22 mlCO2/kg-hr) and a significant amount of fruit peel injury was noted.

Internal Atmospheres of Valencia Oranges During Storage in Controlled Atmosphere (CA) Condition

As shown in Figure 3-7, when Valencia oranges were stored in a CA of 5% O2 +

5% CO2 at 5oC, the fruit internal atmosphere did not change for up to 3 weeks. Initially the internal O2 levels of fruit were similar to those of the external CA, increasing to

6.32% O2 after 1 week and then declining to 5.08% O2 after 3 weeks of storage at 5oC.

Similarly, the internal CO2 of the orange fruit was maintained slightly higher than the amount applied in the CA (5% CO2) during 3 weeks at 5oC storage temperature. It was

noted in Figure 3-8, that when Valencia oranges were exposed to 5% O2 + 10% CO2 CA at 5oC, the internal O2 was about the same (~5% O2) during 3 weeks of storage and there was only a small increase of CO2 above the amount provided by the CA (10%

CO2) during 3 weeks at 5oC storage temperature.

As shown in Figure 3-9, when Valencia oranges were exposed to a CA of 5% O2

+ 5% CO2 at 25oC, the internal O2 of orange fruit was drastically reduced to about 3% in comparison with the level of O2 in the CA (5%), but the opposite trend was observed for the fruit internal CO2, after 3 weeks of storage at 25oC. The internal CO2 of orange fruit rapidly increased and was about 3 times the CO2 concentration supplied by the CA

(5%) during 3 weeks at 25oC. In the case of orange fruit exposed to CA of 5% O2 + 10%

CO2 at 25oC, similar trends were observed in the fruit internal atmosphere during the 3 weeks storage period as noted in Figure 3-10.

As observed in Figure 3-11 for control fruit exposed to ambient air, the internal fruit atmospheres were above 20% O2 and below 2% CO2 and those levels were maintained during 3 weeks of storage. Blue mold grew on the stem end of the air control orange fruit at 25oC, presumably contributing somewhat to the atmosphere that developed in the fruit.

Determination of the Most Suitable Temperature(S) and Time(S) for Hot Water Immersion to Create a Potentially Beneficial Internal Modified Atmosphere (MA) in Orange Fruit

As shown in Table 3-2, Navel orange fruit treated with hot water at 40oC and different water immersion times of 30, 40, 50, 60, 70, 80, or 90 min performed significantly different in terms of internal atmosphere of % O2 and % CO2, but there were insignificant amounts of ethylene measured in the fruit after HW. The maximum amount of %O2 was found in control fruit (20±09.88%) that were immersed in ambient

water followed by fruit treated with hot water at 40oC for 50 min (9.29±0.64%), whereas the lowest concentration of O2 was observed in treated fruit immersed in 40oC water for

90 min (3.57±1.77%). The highest %CO2 was exhibited in the treatments of 40oC for 60 min (28.25±0.64b), 40oC for 70 min (29.67±0.50b), and 40oC for 80 min (26.86±0.51b) and 40oC for 90 min (35.43±0.27a). However, internal %CO2 levels closer to what would be found in recommended CA conditions were present in fruit treated instantly with hot water at 40oC for 30min (18.51±1.62c) without any peel injury followed by 40oC for

50min (6.07±1.65d), and 40oC for 40min (4.68±0.04d) (Table 3-2). In the case of internal C2H4, there were no significant differences between treatments although concentrations varied from 1.46±0.09 ppm at 40oC +90 min to 0.63±0.10 ppm in non- treated control fruit.

Effect of Combined Harvest on O2, CO2, and C2H4 for Lower Water Immersion Temperature with Higher Duration

For the combined analysis of Valencia Harvest 1 and Harvest 2 (Table 3-3), there was a significant harvest effect on internal CO2 and C2H4 concentrations. There was a significant harvest X temperature interaction for CO2, but not for O2 and C2H4 (P<0.05).

The only difference in amount of CO2 observed between harvests was at 40oC, which was 19.14% in Harvest 1 and 28.66% in Harvest 2 (Table 3-4). In the case of harvest X treatment duration, there were highly significant interactions (P<0.001) for O2 and C2H4, but not for CO2. For O2, the only difference between harvests was seen in the 60-min treatment time, for which the O2 was 4.52% in Harvest 1 and 7.40% in Harvest 2 (Table

3-5). In contrast, C2H4 was lower in Harvest 1 than in Harvest 2 for all treatment durations (Table 3-6). It might be happened due to two different harvests of Valencia orange collected from two different locations.There were significant interactions of

treatment temperature X duration for all three of the internal gases (O2, CO2, and C2H4).

The highest concentrations of O2, CO2, and C2H4 were observed in 30oC + 60 min

(7.93%) (Table 3-7), in 40oC + 70 min (25.23%) (Table 3-8), and 35oC + 60 min (0.76 ppm) (Table 3-9), respectively. In the case of the harvest X water immersion temperature X duration interaction (Table 3-3), O2 (P<0.05) and C2H4 (P<0.0001) exhibited significant differences. The highest amount of O2 (10.37%) was noticed in fruit from Harvest 2 treated at 30oC for 60 min and the lowest amount (3.41%) was observed in the same Harvest 2, but for fruit treated at 40oC for 60 min (Table 3-10), and for the

C2H4 concentration, Harvest 2 fruit treated at 35oC for 60 min (1.31 ppm; Table 3-11).

As noticed in Figure 3-12 for Navel orange treated into hot water at 40oC for 30,

40, 50, 60, 70, 80, or 90 min, %CO2 elevated and %O2 declined with hot water immersion time increased. The maximum %CO2 increased with the range of 27 to 35% during fruit treatment with hot water at 40oC for 60, 70, 80, and 90 min where extreme peel injury were observed. However, fruit treated at 40oC+30 min exhibited desired or expected internal atmposphere (~5% O2, ~17% CO2) into fruit without any peel injury.

As shown in Figure 3-13 for Valencia Harvest 1, increasing hot water temperature and treatment duration resulted in increased % CO2 and decreased %O2, but a negligible amount of C2H4 was observed. However, when comparing hot water treatment temperatures (30oC, 35oC, and 40oC) there were significant differences for

%O2, %CO2, and ppm C2H4 (P<0.05) and also similar trends were noticed for hot water immersion duration (60 min, 70 min, and 80 min), except for % CO2. On the other hand, there were no significant interactions of temperature and treatment duration for % O2,

and % CO2, but C2H4 concentration in fruit exhibited significant differences between treatments (P<0.05).

For Valencia Harvest 2 (Figure 3-14), the effect of hot water immersion on internal CO2 and O2 concentrations was more extreme than in Harvest 1 (Figure 3-13).

However, there were similar amounts of C2H4 found in all the treated fruit (Figure 3-13).

Effect of Combined Harvest on Internal O2, CO2, and C2H4 for Higher Water Immersion Temperatures with Shorter Durations

As shown in Table 3-12, the harvest X hot water immersion temperature interaction was highly significant for the internal gases (O2, CO2, and C2H4) (P<0.001).

The maximum amount of O2 (10.80%) was noticed in Harvest 1 fruit treated at 55oC

(Table 3-13). Similarly, the highest amount of CO2 (18.85%) was observed at 55oC for

Harvest 2 fruit, which was followed by Harvest 1 fruit at 50oC (14.23%CO2) (Table 3-14).

In Table 3-15, the most extreme amount of C2H4 observed (1.70 ppm) was in Harvest 2 fruit treated at 55oC, which was followed by Harvest 2 fruit at 50oC (0.34 ppm). In the case of the harvest X treatment duration interaction (Table 3-12) there were highly significant differences (P<0.001) in the amount of O2 and significance difference for CO2 and C2H4 observed among the treatments (P<0.05). The highest amount of O2 (9.60%) was present in Harvest 1 fruit from the 20-min treatment duration, which was followed by Harvest 2 fruit from the 20-min treatment duration (8.51%) (Table 3-16). Similarly, the maximum amount of CO2 (16.44%) was present in Harvest 2 fruit from the 20-min duration treatment and the lowest amount (10.40%) was observed in Harvest 2 fruit from the 10-min treatment duration (Table 3-17). In the case of C2H4, the highest amount (0.99 ppm) was found in Harvest 2 fruit from the 20-min duration of hot water treatment (Table 3-18).

As observed in Table 3-12, the interaction of temperature X hot water immersion duration, the amounts of O2 (P<0.0001) and C2H4 (P<0.05) were significantly affected for Valencia orange. The highest amount of O2 (12.29%) was observed in the 55oC for

20 min treatment (Table 3-19), which was followed by 55oC for 10 min (6.27%), and the lowest amount of O2 (4.18%) was at 50oC for 15 min. Similarly, the highest amount of

C2H4 (1.19 ppm) was noticed in fruit treated at 55oC for 20 min (Table 3-20) followed by

55oC for 10 min (1.07 ppm).

For the interaction of harvest X hot water treatment temperature X immersion duration for Valencia oranges, there were significant differences in terms of the amounts of O2 and C2H4 among treatments (Table 3-12). In Table 3-21, the maximum amount O2

(18.23%) was observed in Harvest 1 for the 55oC for 20 min treatment and the lowest amount (3.74%) was in Harvest 1 for 50oC for 15 min. In the case of C2H4, the most extreme amount (2.15 ppm) was observed in fruit from Harvest 2, treatment 55oC for 20 min and Harvest 2, 55oC for 10 min (1.85 ppm), which was followed by Harvest 2 for

55oC for 15 min (1.10 ppm; Table 3-22) resulted peel injury or surface scalding, and peel color degradation.

As shown in Figure 3-15 for Harvest 1 Valencia oranges, increasing the water temperature from 45 to 55 oC and the treatment duration from 10 to 20 min increased %

CO2 and decreased % O2, but had a negligible effect on C2H4 concentration compared to control fruit. In the case of hot water treatment (45oC, 50oC, and 55oC), it was found that internal O2 and CO2 were significantly affected (P<0.001), but C2H4 was not. Also, considering the treatment duration (10 min, 15 min, 20 min), only O2 concentrations were significantly different among the treatments. The interaction of hot water treatment

X treatment duration was highly significant (P<0.001) for % O2 (Table 3-12). The highest amount of CO2 (18.29%) was observed in the treatment 55oC for 20 min for Harvest 1, but the O2 in that treatment was over 10% (Figure 3-15). However, several other treatments (55oC for 10 or 15 min; 50oC for 10, 15 or 20 min) developed at or near 15%

CO2 plus O2 around 5 to 7%.

Similar trends were observed for Valencia Harvest 2 (Figure 3-16). It was observed that there was significant CO2 increase (17-22 %) in the treatments 50oC for

10 min, and 55oC for 10, 15, 20 min. However (~10%) O2 present in 45oC for 10 min, and in 50oC for 20 min which was higher compared to 5%O2. O2 below 5% present in

50oC for 10 min, and in 50oC for 15 min whereas control treatment exhibited maximum

(~20%) O2 (Table 3-15). It was found that both temperature and treatment duration exhibited highly significant effects on internal gases in the fruit and their interaction was also significant except for the amount CO2. The highest amount of C2H4 (~2.2 ppm) were observed in 55oC for 20 min and in 55oC for 10 min (Figure 3-16) for harvest 2 of

Valencia orange.

Comparison of Fruit Internal Atmosphere between Navel and Valencia Orange

The desired internal O2 of <5% was found in Valencia orange fruit treated at 45oC for 30 min (Figure 3-17); those fruit were also without any peel injury. In the case of internal

CO2, orange fruit from the same treatment (45oC for 30 min) were observed to develop just under 20% internal CO2 (Figure 3-18). The highest amount of C2H4 (~1 ppm) was noted in Navel oranges treated at 40oC for 30 min, about 3.5-fold more than in the other treatments with Valencia oranges (Figure 3-19).

Peel Injury Incidence and SeverityfFor Valencia Oranges During 3 Weeks Storage

As shown in Table 3-23, no peel injury of Valencia oranges was observed immediately after hot water treatment or after 1 week storage at 25oC with 85±5%RH.

After 2 weeks, more than 50% fruit injured for 35oC+70 min, 55oC+15 min, 55oC+20 min duration due to extreme water temperature with duration. However, after 3-weeks of storage100% oranges were noted peel injury for longer water dipping (30oC+80 min),

35oC+60 min, 35oC+70 min). Fruit submerged into hot water with longer duration injured

86 to 100% fruit surface area affected by diplodia stem end rot, blue mold, phomopsis etc. No severe peel injury was noticed for 45oC+30 min but control fruit had 50% moderately severe and and 83.33% severe peel injury after 2 and 3 weeks, respectively. Control fruit exhibited some blue molds with blemishes and developed pale or darker color on the surface at the end of 3 weeks because of water loss.

Internal Temperature Profiles of Navel Oranges Using the Best Combination of Hot Water Treatment Temperature and Immersion Duration

Fresh Navel oranges were treated with hot water at 45oC and the temperature profile at the surface, mid, and center parts were recorded for 1 hr (Figure 3-20). The

“surface” of the fruit (peel midpoint) reached 45oC at between 20 and 30 min without any peel injury and noted shiny surface color. In the case of the midpoint tissue temperature, it had reached 39oC after 30 min and was just below 45oC after 60 min of hot water treatment (Figure 3-20). There was a 6-7-min lag before the center temperature began to increase and the temperature reached about 36oC after 30 min and 44oC after 60 min (Figure 3-21).

Respiration Rate and Ethylene Production of Navel Oranges During Storage at Different Temperatures after Immersion in 45oc Water with or without Application of Edible Wax Coating

As shown in Figure 3-22, respiration rate rose slowly in fruit as the storage temperatures were increased from 25oC to 40oC following either ambient water or hot water treatment with no coating, but respiration began to significantly increase at higher temperatures. For storage temperatures of 45oC and higher, respiration of ambient water-treated fruit with no coating was higher than that of hot water-treated fruit with no coating. The maximum respiration was found at 50oC (198.04 ml CO2/kg-hr,113.55 ml

CO2/kg-hr) and 55oC (220.72 ml CO2/kg-hr,130.20 ml CO2/kg-hr) in both ambient water- and hot water-treated fruit without coating, but a significant amount of fruit peel injury occurred at those air temperatures, which damaged the fruit tissue. Treatment with hot water reduced respiration rate in storage as compared to ambient water treated fruit.

Therefore, for both ambient water and hot water immersion, the most effective and optimum respiration rate for developing extreme internal modified atmosphere could be claimed for fruit without coating that were stored at 45oC (85.95 ml CO2/kg-hr, 65.60 ml

CO2/kg-hr) due to lack of peel injury.

In Figure 3-23 for ambient water + polyethylene versus hot water + polyethylene, there were similar respiration rates for fruit stored at from 25oC to 35oC, but an increased respiration trend was found for storage at from 40oC to 55oC in both treatments. It was also shown that 28 to 38% higher respiration occurred in AMW+PL coated fruit compared to HW+ PL coated fruit stored at from 45oC to 55oC. The most extreme respiration rates were noticed at 50oC (216.90 mlCO2/kg-hr, 140.84 ml CO2/kg- hr) and 55oC (278.03 ml CO2/kg-hr, 173.68 ml CO2/kg-hr) in both AMW + PL and HW+

PL treated fruit because of increase water temperature exposure. However, it was

observed that a significant number of fruit at the higher storage temperatures developed peel injury. Fruit stored at 40oC (56.28 ml CO2/kg-hr, 52.55 mlCO2/kg-hr) and 45oC

(92.56 ml CO2/kg-hr, 66.61 ml CO2/kg-hr) exhibited elevated respiration in both AMW +

PL, and HW+PL without any significant amount of fruit peel injury.

As shown in Figure 3-24, it was observed that for temperatures from 25oC to

45oC, a similar pattern of increasing respiration happened both in AMW+CR and

HW+CR coated fruit. After that, the respiration increased drastically at 50oC and 55oC and the effect of storage temperature on respiration rate deviated greatly for AMW+CR coated fruit (279.70 ml CO2/kg-hr, 268.78 ml CO2/kg-hr) versus HW+CR coated fruit

(160.10 ml CO2/kg-hr, 35.94 ml CO2/kg-hr). It was also noticed that respiration declined significantly from 50 and 55oC to 60oC for AMW+CR, and between 50 and 55oC for

HW+CR; a significant amount of fruit peel injury also occurred in fruit stored at 55 or

60oC. Considering absence of peel injury, the optimum performance in terms of stimulating respiration by HW treatment was for HW+CR coated fruit stored at 45oC.

As shown in Figure 3-25, respiration rate increased steadily and similarly from

25oC to 45oC for both AMW+SH and HW+SH coated fruit. It was also found that, unlike the other treatments, respiration rate increased more in AMW+SH coated fruit compared to HW+SH coated fruit stored at 50oC. Respiration rate declined for fruit stored at 55 or 60oC compared to 50oC because of higher temperature exposure. Fruit treated with HW at 45oC and coated with shellac coating initiated fermentative metabolism based on the alcohol odor that was noticed, but without any peel injury. The maximum respiration was observed at 50oC in both AMW+SH (174.02 ml CO2/kg-hr) and HW+SH (247.49 ml CO2/kg-hr) coated fruit and it was also noticed that significant

fruit peel injury occurred due to extreme heat stress resulted higher amount %CO2. Fruit stored at 40oC (83.86 mlC O2/kg-hr) or 45oC (156.14 ml CO2/kg-hr) exhibited elevated respiration in HW+SH coated fruit without any peel injury.

Discussion

Fruit respiration pattern varies depending on the type of fruit, and knowledge of the maximum respiration rate of produce can assist in determining the optimum postharvest temperature for that particular commodity. Respiration rate is an indication of metabolic activity, which also indicates the shelf life of the produce. In this study, respiration rate of fresh Navel oranges increased with increasing temperature to a peak before declining with and without changing the fruit used for measurement of respiration at different temperatures.

The initial respiration rate of fresh Navel oranges at 25oC was 19.57 ml CO2/kg- hr, which was similar to the rate for Valencia orange respiration (19.2 ml CO2/kg-hr) measured by Eaks (1970). The peak respiration measured when fruit were not changed between measurements at different temperatures was 98.02 ml CO2/kg-hr at 50oC

(Figure 3-6) whereas when different fruit were measured at each temperature, the peak respiration rate was 3-fold higher at 50oC (305.48 ml CO2/kg-hr). The respiration rates at 50oC were 5- and 15-fold higher than at 25oC for repeatedly measured and single measurement fruit, respectively. This difference between respiration rates measured using the different procedures may have been due to more stress being experienced by fruit that were suddenly exposed to elevated temperatures compared to those fruit that were gradually exposed to higher temperatures in 5-degree increments over the course of a day. Similar results were found for mango fruit respiration, which increased 5-fold during a forced hot-air treatment at 48oC (Mitcham and McDonald, 1993). Alternatively,

it is possible that internal O2 and CO2 concentrations within the fruit became modified during the repeated high temperature exposures, inhibiting the fruit respiration (Banks et al., 1993).

Fruit respiration declined rapidly above 50oC and severe injury was observed on the peel surface because of the extreme heat stress, as has been reported for numerous fruit types exposed to high temperatures (Sharp, 1994). Inhibition of respiration rate at extreme high temperature probably reflects heat-related inhibition of enzymes controlling the rates of key metabolic pathways. Similar trends were observed in other fruit in research conducted by Djioua et al. (2009). Respiration reduction with extended exposure to extreme high temperature can also be related to the depletion of respirable substrate (Kader and Salveit, 2003). Fruit peel discolored or became pale with a moist surface in heat-injured orange fruit, reflecting injury and death of the outermost peel cells.

Valencia oranges stored at 5oC in CA of 5% O2 + 5% CO2 or 5% O2 + 10% CO2, which was near the recommended CA for citrus fruit storage (Kader et al., 1989), had internal atmospheres of about 5% O2 + 6% CO2 (Figure 3-7) and 5%O2 + 11%CO2, respectively, without any apparent injury (Figure 3-8) after 3 weeks of storage. The small differences between external and internal gas concentrations shows that the orange peel is not a major barrier to gas exchange. However, Valencia oranges stored at 25oC in the same CA conditions exhibited lower O2 (<3%) and higher CO2 (~13%)

(Figure 3-9 & Figure 3-10) after 3 weeks of storage. Those fruit also developed peel injury and blue mold and Diplodia stem end rot. The appearance of peel injury and decay with the moderately more extreme internal atmosphere suggests the limit of

atmosphere tolerance had been reached at the higher temperature. It has been shown that CA tolerance is less at higher compared with lower temperatures due to initiation of anaerobic respiration and associated accumulation of AA and ethanol for oranges (Ke and Kader, 1990) and other fruits (Ke and Kader, 1992).

Heat stress treatment can create beneficial modified internal atmospheres in fruit that extend their shelf life. Five different studies were conducted to determine the most suitable treatment temperature and water immersion time combination that would be potentially beneficial in extending storage by creating an MA in the orange fruit. In the first study, it was noticed that Navel oranges treated with 40oC water for different immersion times (30, 40, 50, 60, 70, 80, or 90 min) were significantly different in terms of the internal gas concentration. Navel oranges submerged for 30 min at 40oC developed about 6% O2 and about 18% CO2 internally (Figure 3-11) without any peel injury. According to Chang et al. (1983), high CO2 can enhance anaerobic metabolism, and at high temperature and with high respiration rate, there may be a greater demand for O2, resulting in anaerobic respiration at relatively high O2 concentrations. Increasing water immersion time (from 40 to 90 min) decreased the O2 and elevated the CO2 rapidly. There were also higher amounts of C2H4 produced (about 1.5 ppm) in those fruit, suggesting a stress response. Longer exposure times also resulted in peel injury and accumulation of AA and ethanol during the storage period.

From another two studies of Valencia orange using 30, 35 or 40oC water for 60,

70, or 80 minutes (Figures 3-12 and 3-13) ), there was less increase in internal CO2 and decrease in O2 compared to the previous experiment for these similar durations of immersion in lower water temperatures. Another two studies using higher water

temperatures of 45, 50 and 55oC and shorter durations of 10, 15, and 20 minutes

(Figures 3-14 and 3-15) showed similar elevated CO2 and decreased O2 along with increased C2H4 (1.19 ppm), and the fruit peel became injured because of the heat stress. These results illustrate that different combinations of hot water treatment temperatures and times may result in development of similar internal atmospheres in orange fruit, but eventually high temperatures become physically injurious to outer fruit tissues even with short exposure times. Similar results have been found during development of hot water treatments for many different fruit types (Lurie, 1998). In this work with Navel and Valencia oranges, the combination of 45oC water immersion for 30 minutes provided the greatest internal modification of O2 and CO2 without either peel injury or indication of stress by elevated internal C2H2 (Figures 3-16, 3-17, 3-18).

With HW immersion, heat is transferred from the water to the fruit surface and then is conducted through the fruit flesh to the center of the fruit (Jordan, 1993). The water-to-fruit-surface heat transfer is faster than from the fruit surface to the center of the fruit. The temperature profile in the different tissue layers of Navel oranges immersed in 45oC water for 30 min and 60 min showed that there was a 6-7-min lag before the fruit center temperature began to increase and the center temperature reached about 36oC after 30 min and 44oC after 60 min (Figure 3-21). Thus, at the end of the 45oC for 30 min HWT used throughout the rest of this study, the peel temperature was 45oC for essentially the entire treatment time, while the fruit center temperature rose from about 25oC to 36oC over the last 23-24 min of the HWT. Similar trends were observed in the temperature profile of mango fruit treated with different HWT temperatures (Couey, 1989; Jordan, 1993; Stewart et al., 1990).

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It was further shown that for storage temperatures of 45oC and higher, respiration of AMW-treated fruit was higher than that of HW-treated fruit, with or without coatings

(Figures 3-22 through 3-25). Thus, the AMW-treated fruit must have had higher metabolic rate than the HW-treated fruit. The slower metabolic rate of fruit following

HWT may have been due to a direct effect of the heat on fruit metabolism (Lurie, 1998) or due to the more extreme internal MA that developed in the HW-treated fruit (Figures

3-11 through 3-15), since MA and CA have been consistently shown to reduce fruit metabolic rates as indicated by reduced respiration (Banks et al., 1993). The only exception to this observation was for HW+SH coated fruit at 50oC, for which the respiration rate was higher for the HW-treated fruit than for the AMW-treated fruit

(Figure 3-25). It is likely that the HW+SH fruit initiated fermentative metabolism during the HWT, which was maintained during 50oC storage based on the alcohol odor that was noted. Reduction of O2 inside the fruit can be accompanied by fermentation and accumulation of off-flavors associated with ethanol and AA production (Fidler and North,

1971). Shellac-coated orange fruit were observed by Hagenmaier and Baker (1994) to have higher gloss than with PL or CR coating, but they also developed lower internal O2 and higher CO2 concentrations, which resulted in fermentative off-odors. Similar results have also been shown for a few other fruits and vegetables such as strawberry (Ke et al., 1991; Pesis and Avissar, 1990). In contrast to SH-coated oranges, the fruit treated with HW at 45oC for 30 min and either left uncoated or coated with CR or PL developed apparently beneficial internal MA without any evidence of fermentative metabolism or peel injury.

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Chapter Summary

In this study, it was shown that the maximal respiration rate without any peel injury of Navel orange fruit occurred with a HWT of 45ºC for 30 min. The respiration rate declined when oranges were stored at air temperatures above 50oC, and peel injury developed, which was useful in designing the hot water immersion treatments because it suggests that HWT at those higher temperatures would also likely be injurious.

Maximum accumulation of internal CO2 without any injury during HW immersion occurred at 45oC for 30 min duration. That treatment also reduced O2 without significant induction of anaerobic respiration, which produces very high CO2 along with ethanol and AA.

Valencia oranges stored at 5oC in a CA of 5% O2 + 5% CO2 or 5% O2 + 10% CO2 developed internal MA that was similar to the external O2 and CO2 levels; however, while internal O2 was lower and CO2 higher in the same CA combinations at 25oC, peel injury was observed, suggesting that the oranges did not tolerate the more extreme internal O2 (~3%) and CO2 (~13%) during 3 weeks storage at 25oC. Those results helped in selection of potential hot water immersion time-temperature combinations by including measurements of internal O2 and CO2 levels to identify those HWTs that resulted in potentially beneficial internal MA.

Navel oranges receiving a 45oC for 30 min HWT exhibited the desired internal

MA (about 5% O2 + 17% CO2) in the fruit at the end of the treatment without developing any peel injury. Valencia oranges treated with lower water temperatures for longer exposure times showed that internal CO2 was elevated and O2 was reduced with increasing treatment temperature and water immersion time, reaching levels above the tolerance limits recommended for CA storage of citrus fruit. Similar trends were shown

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in another study with Valencia oranges immersed in higher water temperatures with shorter durations. Higher water temperatures accelerated the transition from aerobic to anaerobic respiration, and peel appearance deteriorated due to the heat stress, resulting in injury to 86 to 100% of the fruit surface area, as well as the appearance of

Diplodia stem end rot and blue mold rot during storage. In contrast, no severe peel injury was noticed for the 45oC for 30 min treatment. The peels of control fruit immersed in AMW turned a pale or darker color due to water loss after 3 weeks storage while

HWT maintained peel appearance.

The temperature profile in the different tissue layers of Navel oranges immersed in 45oC water for 30 min and 60 min showed that there was a 6-7-min lag before the fruit center temperature began to increase and the center temperature reached about

36oC after 30 min and 44oC after 60 min.

For storage temperatures of 45oC and higher, the respiration of AMW-treated fruit was higher than that of HW-treated fruit, with the exception of SH-coated fruit, which initiated fermentative metabolism due to extreme internal MA.

In conclusion, immersion in HW at 45oC for 30 min created a beneficial internal

MA in orange fruit without any peel injury, and this temperature-time combination exhibited maximal reduction of respiration rate, greater decay control, and enhanced fruit peel appearance after HWT and during ambient or higher temperature storage.

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Table 3-1. Determining the most suitable hot water immersion temperature(s) and time(s) to create an internal modified atmosphere (MA) inside Navel orange fruit. Water Temperature Emersion Durations Tested (min) (oC) Study 1 Study 2 Study 3 Study 4 Study 5 Ambient (Control) 30 60, 70, 80 60, 70, 80 35 60, 70, 80 60, 70, 80 30, 40, 50, 40 60, 70, 80, 60, 70, 80 60, 70, 80 90 45 10, 15, 20 20, 30, 40 10, 15, 20 50 10, 15, 20 10, 15, 20 55 10, 15, 20 10, 15, 20

Table 3-2. Mean values ± SE for internal atmospheres of Navel oranges treated with hot water at 40oC for different durations. Treatment (oC) Duration (min) O2 (%) CO2 (%) C2H4 (ppm) 40 30 6.01±0.25bcz 18.51±1.62c 0.92±0.08 40 7.57±0.35bc 4.68±0.04d 0.88±0.05 50 9.29±0.64b 6.07±1.65d 1.15±0.41 60 6.18±0.70bc 28.25±0.64b 0.70±0.15 70 5.34±0.99bc 29.67±0.50b 1.36±0.02 80 8.38±1.49bc 26.86±0.51b 1.36±0.53 90 3.57±1.77c 35.43±0.27a 1.46±0.09 Control - 20.09±0.88a 5.10±0.94d 0.63±0.10 ***y *** ns Z Means within a column denoted by the same letter of each column or each row for specific parameters combined analysis do not differ significantly according to Tukey’s Test (P<0.05). yns, *, **, ***, ****, not significant, significant at P<0.05, P<0.01, P<0.001, or highly significant at P<0.0001 respectively according to Tukey test.

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Table 3-3. Analysis of variance table for combined harvest effect of Valencia orange on O2, CO2, and C2H4 for lower water immersion temperature with longer duration. Main effect O2 (%) CO2 (%) C2H4 (ppm) Harvest (H)y nsz **** **** Water temperature (T)x **** **** ** Duration (D)w * ** **** Interaction: H x T ns ** ns H x D *** ns **** T x D * ** ** H x T x D * ns **** zns, *, **, ***, ****, not significant, significant at P<0.05, P<0.01, P<0.001, or highly significant at P<0.0001 respectively according to Tukey test. yH, two harvests of Valencia orange in 2015. xT, water temperature of hot water treated oranges at three different temperatures (30, 35, 40oC). w D, three durations (60, 70, 80 mins) of hot water treatment.

Table 3-4. Effect of harvest x hot water temperature on CO2 for Valencia orange. CO2 (%) Harvest 30oC 35oC 40oC Harvest 1 9.08 dz 12.47 cd 19.14 b Harvest 2 13.49 cd 15.00 bc 28.66 a Z Means within a column denoted by the same letter are not significant difference. Statistical analysis performed by SAS software and, Tukey’s HSD at 5% level of significance used for multiple comparison test.

Table 3-5. Effect of harvest x duration of hot water treatment on O2 for Valencia orange. O2 (%) Harvest 60 min 70 min 80 min Harvest 1 4.52 cz 5.39 abc 6.74 ab Harvest 2 7.40 a 4.73 bc 5.87 abc Z Means within a column denoted by the same letter are not significant difference. Statistical analysis performed by SAS software and, Tukey’s HSD at 5% level of significance used for multiple comparison test.

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Table 3-6. Effect of harvest x duration of hot water treatment on C2H4 for Valencia orange. C2H4 (ppm) Harvest 60 min 70 min 80 min Harvest 1 0.20 cz 0.24 c 0.14 c Harvest 2 0.98 a 0.60 b 0.46 b Z Means within a column denoted by the same letter are not significant difference. Statistical analysis performed by SAS software and, Tukey’s HSD at 5% level of significance used for multiple comparison test.

Table 3-7. Effect of hot water temperature x duration on O2 for Valencia orange. Water temperature O2 (%) (oC) 60 min 70 min 80 min 30 7.93 az 6.65 abc 6.11 abcd 35 6.30 abcd 3.88 cd 6.94 ab 40 3.66 d 4.65 bcd 5.67 abcd Z Means within a column denoted by the same letter are not significant difference. Statistical analysis performed by SAS software and, Tukey’s HSD at 5% level of significance used for multiple comparison test.

Table 3-8. Effect of hot water temperature x duration on CO2 for Valencia orange. Water temperature CO2 (%) (oC) 60 min 70 min 80 min 30 10.37 dz 10.51 d 12.98 cd 35 9.03 d 13.50 cd 18.68 bc 40 23.04 ab 25.23 a 23.44 ab Z Means within a column denoted by the same letter are not significant difference. Statistical analysis performed by SAS software and, Tukey’s HSD at 5% level of significance used for multiple comparison test.

Table 3-9. Effect of hot water temperature x duration on C2H4 for Valencia orange. Water temperature C2H4 (ppm) (oC) 60 min 70 min 80 min 30 0.57 abz 0.38 bcd 0.21 d 35 0.76 a 0.38 bcd 0.43 bcd 40 0.46 bc 0.51 bc 0.27 cd Z Means within a column denoted by the same letter are not significant difference. Statistical analysis performed by SAS software and, Tukey’s HSD at 5% level of significance used for multiple comparison test.

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Table 3-10. Effect of hot water temperature x treatment duration x harvest on O2 for Valencia orange. Water temperature O2 (%) (oC) Harvest 60 min 70 min 80 min 30 Harvest 1 5.48 bcz 7.77 abc 7.52 abc Harvest 2 10.37 a 5.53 bc 5.70 abc

35 Harvest 1 4.17 bc 3.88 bc 7.22 abc Harvest 2 8.43 ab 3.88 bc 6.65 abc

40 Harvest 1 3.91 bc 4.52 bc 5.48 bc Harvest 2 3.41 c 4.78 bc 5.25 bc ZStatistical analysis performed by SAS software and, Tukey’s HSD at 5% level of significance used for multiple comparison test.

Table 3-11. Effect of hot water temperature x treatment duration x harvest on C 2H4 for Valencia orange. Water temperature C2H4 (ppm) (oC) Harvest 60 min 70 min 80 min 30 Harvest 1 0.17 fz 0.09 f 0.13 f Harvest 2 0.96 ab 0.66 bcde 0.28 ef

35 Harvest 1 0.20 f 0.38 cdef 0.13 f Harvest 2 1.31 a 0.38 cdef 0.72 bcd

40 Harvest 1 0.24 f 0.24 f 0.17 f Harvest 2 0.67 bcde 0.77 bc 0.37 def ZStatistical analysis performed by SAS software and, Tukey’s HSD at 5% level of significance used for multiple comparison test.

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Table 3-12. Analysis of variance table for combined harvest effect of Valencia orange on O2, CO2, and C2H4 for higher water immersion temperature with shorter duration. Main effect O2 (%) CO2 (%) C2H4 (ppm) Harvest (H)y nsz ns **** Water temperature (T)x *** **** **** Duration (D)w **** ** ** Interaction : H x T **** *** **** H x D **** * ** T x D **** ns ** H x T x D ** ns * zns, *, **, ***, ****, not significant, significant at P<0.05, P<0.01, P<0.001, or highly significant at P<0.0001 respectively according to Tukey test yH, two harvests of Valencia orange in 2015 xT, water temperature of hot water treated orange at three different temperatures (55, 50, 45oC). w D, three durations (10, 15, 20 mins) of hot water treatment

Table 3-13. Effect of harvest x hot water immersion temperature on O2 for Valencia orange. O2 (%) Harvest 45oC 50oC 55oC Harvest 1 5.74 bz 5.23 b 10.80 a Harvest 2 7.44 b 6.28 b 5.66 b Z Means within a column denoted by the same letter are not significant difference. Statistical analysis performed by SAS software and, Tukey’s HSD at 5% level of significance used for multiple comparison test.

Table 3-14. Effect of harvest x hot water immersion temperature on CO2 for Valencia orange. CO2 (%) Harvest 45oC 50oC 55oC Harvest 1 7.93 cz 14.73 b 13.81 b Harvest 2 8.11 c 12.53 b 18.85 a Z Means within a column denoted by the same letter are not significant difference. Statistical analysis performed by SAS software and, Tukey’s HSD at 5% level of significance used for multiple comparison test.

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Table 3-15. Effect of harvest x hot water immersion temperature on C2H4 for Valencia orange. C2H4 (ppm) Harvest 45oC 50oC 55oC Harvest 1 0.14 bz 0.26 b 0.23 b Harvest 2 0.11 b 0.34 b 1.70 a Z Means within a column denoted by the same letter are not significant difference. Statistical analysis performed by SAS software and, Tukey’s HSD at 5% level of significance used for multiple comparison test.

Table 3-16. Effect of harvest x treatment duration of hot water immersion on O2 for Valencia orange. O2 (%) Harvest 10 min 15 min 20 min Harvest 1 6.68 bcz 5.48 c 9.60 a Harvest 2 8.51 ab 4.98 c 5.88 c Z Means within a column denoted by the same letter are not significant difference. Statistical analysis performed by SAS software and, Tukey’s HSD at 5% level of significance used for multiple comparison test.

Table 3-17. Effect of harvest x treatment duration of hot water immersion on CO2 for Valencia orange. CO2 (%) Harvest 10 min 15 min 20 min Harvest 1 11.55 bz 12.62 ab 12.30 b Harvest 2 10.40 b 12.64 ab 16.44 a Z Means within a column denoted by the same letter are not significant difference. Statistical analysis performed by SAS software and, Tukey’s HSD at 5% level of significance used for multiple comparison test.

Table 3-18. Effect of harvest x treatment duration of hot water immersion on C 2H4 for Valencia orange. C2H4 (ppm) Harvest 10 min 15 min 20 min Harvest 1 0.24 cz 0.18 c 0.20 c Harvest 2 0.68 b 0.48 bc 0.99 a Z Means within a column denoted by the same letter are not significant difference. Statistical analysis performed by SAS software and, Tukey’s HSD at 5% level of significance used for multiple comparison test.

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Table 3-19. Effect of hot water temperature x duration of hot water immersion on O2 for Valencia orange. Water temperature O2 (%) (oC) 10 min 15 min 20 min 45 8.33 bz 5.41 bc 6.03 bc 50 8.20 b 4.18 c 4.90 c 55 6.27 bc 6.12 bc 12.29 a Z Means within a column denoted by the same letter are not significant difference. Statistical analysis performed by SAS software and, Tukey’s HSD at 5% level of significance used for multiple comparison test.

Table 3-20. Effect of hot water temperature x duration of hot water immersion on C 2H4 for Valencia orange. Water temperature C2H4 (ppm) (oC) 10 min 15 min 20 min 45 0.14 cdz 0.15 cd 0.10 d 50 0.19 cd 0.21 cd 0.51 bc 55 1.07 a 0.64 b 1.19 a Z Means within a column denoted by the same letter are not significant difference. Statistical analysis performed by SAS software and, Tukey’s HSD at 5% level of significance used for multiple comparison test.

Table 3-21. Effect of hot water temperature x duration of hot water immersion x harvest on O2 for Valencia orange. Water temperature O2 (%) (oC) Harvest 10 min 15 min 20 min 45 Harvest 1 6.35 bcdz 5.41 bcd 5.45 bcd Harvest 2 10.30 b 5.41 bcd 6.61 bcd

50 Harvest 1 6.85 bcd 3.74 d 5.11 cd Harvest 2 9.55 bc 4.61 cd 4.69 cd

55 Harvest 1 6.85 bcd 7.30 bcd 18.23 a Harvest 2 5.69 bcd 4.93 cd 6.35 bcd Z Means within a column denoted by the same letter are not significant difference. Statistical analysis performed by SAS software and, Tukey’s HSD at 5% level of significance used for multiple comparison test.

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Table 3-22. Effect of hot water temperature x duration of hot water immersion x harvest on C2H4 for Valencia orange. Water temperature C2H4 (ppm) (oC) Harvest 10 min 15 min 20 min 45 Harvest 1 0.17 cz 0.15 c 0.10 c Harvest 2 0.10 c 0.15 c 0.09 c

50 Harvest 1 0.28 c 0.22 c 0.28 c Harvest 2 0.10 c 0.19 c 0.73 bc

55 Harvest 1 0.28 c 0.17 c 0.23 c Harvest 2 1.85 a 1.10 b 2.15 a Z Means within a column denoted by the same letter are not significant difference. Statistical analysis performed by SAS software and, Tukey’s HSD at 5% level of significance used for multiple comparison test.

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Table 3-23. Percentage peel injury and peel injury severity of hot water treated Valencia orange during 3 weeks of storage at 250C with 85±5%RH. Water temperature Dipping time Storage Injured fruit Peel injury (oC) (min) (week) (%) severityz 30 60 0 - 1 1 - 1 2 33.33 1.17 3 50.00 2.67

70 0 - 1 1 - 1 2 50.00 3 3 66.67 4.67

80 0 - 1 1 - 1 2 50.00 2.83 3 100 4.83

35 60 0 - 1 1 - 1 2 33.33 2.33 3 100 4.83

70 0 - 1 1 - 1 2 66.67 2.67 3 100 5.00

80 0 - 1 1 - 1 2 50.00 1.33 3 83.33 4.50

40 60 0 - 1 1 - 1 2 50.00 1.67 3 83.33 4.50

70 0 - 1 1 - 1 2 33.33 1.33 3 83.33 4.50

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Table 3-23. Continued. Water temperature Dipping time Storage Injured fruit Peel injury (oC) (min) (week) (%) severityz 80 0 - 1 1 - 1 2 33.33 1.33 3 83.33 3.33

45 10 0 - 1 1 - 1 2 16.67 1.17 3 33.33 2.33

15 0 - 1 1 - 1 2 16.67 1.17 3 33.33 1.67

20 0 - 1 1 - 1 2 16.67 1 3 33.33 2.83

50 10 0 - 1 1 - 1 2 16.67 1.17 3 50.00 2.83

15 0 - 1 1 - 1 2 16.67 1.17 3 66.67 3

20 0 - 1 1 - 1 2 16.67 1.17 3 66.67 3

55 10 0 - 1 1 - 1 2 50.00 1.67 3 83.33 4

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Table 3-23. Continued. Water temperature Dipping time Storage Injured fruit Peel injury (oC) (min) (week) (%) severityz 15 0 - 1 1 16.67 1.17 2 66.67 2.17 3 83.33 2.83

20 0 - 1 1 16.67 1.67 2 66.67 2.67 3 83.33 3

40 30 0 - 1 1 - 1 2 33.33 1 3 33.33 2.5

40 0 - 1 1 - 1 2 50.00 3 3 50.00 3

50 0 - 1 1 - 1 2 33.33 1 3 50.00 2.5 45 30 0 - 1 1 - 1 2 16.67 1 3 16.67 1

40 0 - 1 1 - 1 2 16.67 1.17 3 33.33 1.17

Control - 0 - 1 1 - 1 2 50.00 2.5 3 83.33 3 zPeel injury severity evaluated by stem end injury rating scale: 1 = No injury; 2 = slight injury up to 15% brown skin area; 3 = moderate injury, 16% to 50% brown skin area; 4 = severe injury, 51% to 85% brown skin area; 5 = extreme injury, 86% to 100% brown skin area.

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Figure 3-1. Sample preparation for respiration and ethylene measurement of Navel oranges at room air temperatures ranging from 25oC to 60oC, with 5oC increments. Photo courtesy of author.

Figure 3-2. Fruit in containers are connected in series for CA storage of Valencia orange during 3 weeks storage at 5oC or 25oC. Photo courtesy of author.

Figure 3-3. Fruit in plastic container showing insertion of the collection tube into fruit to collect internal gas samples. Photo courtesy of author.

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Figure 3-4. Apparatus for immersing fruit into hot water at different temperatures and for different durations and collecting internal gas samples. Photo courtesy of author.

Figure 3-5. Squirrel data logger set up for measurement of temperature & exposure time at different tissue layer of orange fruit immersed in hot water at 45oC for 1 hour. Photo courtesy of author.

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Fruit not changed with temperature Fruit changed with temperature

350

hr) - 300 250 200 150 100 50

Respiration (mlCO2/kg Respiration 0 25 30 35 40 45 50 55 60 Temperature (oC)

Figure 3-6. Measurement of respiration rate presented by (Mean+SE) for a) fruit not changed with storage temperature, and b) fruit changed with storage temperature increased from 25oC to 60oC with 5oC increment of fresh Navel orange fruit when each set temperature in storage chamber and fruit inside tissue temperature were similar.

5%O2 5%CO2

5 Internal atmospher (%O2, %CO2) Internal (%O2, atmospher 0 1 2 3 Storage period (week)

Figure 3-7. Internal atmosphere of Valencia orange stored in CA of 5% O2 plus 5% CO2 at 5oC during 3 weeks storage.

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15 5%O2 10%CO2

Internal atmospher (%O2, %CO2) Internal(%O2, atmospher 0 1 2 3 Storage period (week)

Figure 3-8. Internal atmosphere of Valencia orange stored in CA of 5% O2 plus 10% CO2 at 5oC during 3 weeks storage.

20 5%O2 5%CO2

Internal Internal atmosphere (%O2, %CO2) 1 2 3 Storage period (week)

Figure 3-9. Internal atmosphere of Valencia orange stored in CA of 5% O2 plus 5% CO2 at 25oC during 3 weeks storage.

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20 5%O2 10%CO2

5 Internal Internal atmosphere (%O2, %CO2)

0 1 2 3 Storage period (week)

Figure 3-10. Internal atmosphere of Valencia orange stored in CA of 5% O2 plus 10% CO2 at 25oC during 3 weeks storage.

25 %O2 %CO2

10 Internal Internal atmosphere (%O2, %CO2) 5

0 1 2 3 Storage period (week)

Figure 3-11. Internal atmosphere in Valencia oranges stored in ambient air (25oC) during 3 weeks storage

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40 O2 (%) CO2 (%) C2H4 (ppm) 35 30 25 20 15 10 5

0 Inter. Inter. atm.(%O2, %CO2, ppmC2H4)

Water immersion treatment

Figure 3-12. Internal gas concentrations of Navel oranges immersed into 40oC water for different durations.

40 O2 (%) CO2 (%) C2H4 (ppm) 35 30 25 20 15 10 5

0 Inter. atm.(%O2, %CO2,ppmC2H4)

Water immersion tempereature and time combination

Figure 3-13. Internal gas concentrations of Valencia oranges immersed into 40oC water for different durations (Harvest 1).

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40 O2 (%) CO2 (%) C2H4 (ppm) 35 30 25 20 15 10 5

0 Inter. atm.(%O2, %CO2, ppmC2H4)

Water immersion temperature and time combination

Figure 3-14. Internal gas concentrations of Valencia oranges immersed into 40oC water for different durations (Harvest 2).

40 O2 (%) CO2 (%) C2H4 (ppm) 35 30 25 20 15 10 5

Inter. atmos. (%O2, %CO2, ppmC2H4) 0

Water immersion tempereature and time combination

Figure 3-15. Valencia oranges dipping into different water temperature with different duration (Harvest 1).

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40 O2 (%) CO2 (%) C2H4 (ppm) 35 30 25 20 15 10 5

0 Inter. atmos.(%O2, %CO2, ppmC2H4)

Water immersion temperature and time combination

Figure 3-16. Valencia oranges dipping into different water temperature with different duration (Harvest 2).

25 O2 (%) 20

%O2 in NAV and VAL orange 0 NAV-40C+30min VAL-40C+30min VAL-45C+30min VAL-45C+20min Treatment

Figure 3-17. Mean value with SE of internal O2 of Navel (NAV) and Valencia (VAL) oranges treated with hot water at 40oC or 45oC for 30 min or 20 min.

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25 CO2 (%) 20

%CO2 in NAV %CO2 inNAV and VAL orange 0 NAV-40C+30min VAL-40C+30min VAL-45C+30min VAL-45C+20min Treatment

Figure 3-18. Mean value with SE of internal CO2 of Navel (NAV) and Valencia (VAL) oranges treated with hot water at 40oC or 45oC for 30 min or 20 min.

1.20 C2H4 (ppm) 1.00

0.80

0.60

0.40

0.20

C2H4 in NAV C2H4 NAV in and VAL orange 0.00 NAV-40C+30min VAL-40C+30min VAL-45C+30min VAL-45C+20min Treatment

Figure 3-19. Mean value with SE of internal C2H4 of Navel (NAV) and Valencia (VAL) oranges treated with hot water at 40oC or 45oC for 30 min or 20 min.

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35 C) ° 30 Center 25 Midpoint 20

Temperature ( Temperature 15 Surface 10

0 0 10 20 30 40 50 60 Time (min)

Figure 3-20. Temperature profile at different tissue layer of orange fruit submerged in hot water at 45oC for 1 hr.

45 degC+30 min 45 degC+60 min

50 C) o 40

Fruit temperature ( temperature Fruit 0 Surface Mid Center Fruit tissue layer

Figure 3-21. Temperature reached in different tissue layers during hot water treatment of 45 oC for 30 or 60 min.

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350

hr) Ambient water + no coating - 300 Hot water + no coating 250 200 150 100

50 Respiration (mlCO2/kg Respiration 0 25 30 35 40 45 50 55 60 Temperature (oC)

Figure 3-22. Effect of storage temperature on respiration rate of Navel oranges after treated into hot water +no coating vs 45oC water immersion + no coating.

- 350 Ambient water + polyethylene 300 Hot water + polyethylene 250 200 150hr) 100

50 Respiration (mlCO2/kg Respiration 0 25 30 35 40 45 50 55 60 Temperature (oC)

Figure 3-23. Effect of temperature on respiration rate of Navel orange after treated into ambient water + polyethylene vs hot water + polyethylene.

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- 350 Ambient water +carnauba 300 Hot water + carnauba 250

200 hr) 150 100

50 Respiration (mlCO2/kg Respiration 0 25 30 35 40 45 50 55 60 Temperature (oC)

Figure 3-24. Effect of temperature on respiration rate of Navel orange after treated into ambient water + carnauba vs hot water + carnauba.

350 Ambient water +shellac hr) - 300 Hot water + shellac 250 200 150 100

50 Respiration (mlCO2/kg Respiration 0 25 30 35 40 45 50 55 60 Temperature (oC)

Figure 3-25. Effect of temperature on respiration rate of Navel orange after treated into ambient water + shellac vs hot water + shellac.

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CHAPTER 4 USE OF HOT WATER IMMERSION AND EDIBLE COATINGS TO MAINTAIN QUALITY AND SHELF LIFE OF NAVEL AND VALENCIA ORANGES DURING SIMULATED AMBIENT TEMPERATURE STORAGE

Proper temperature management is important for horticultural commodities to extend shelf life and maintain postharvest quality. Optimum temperature assists in reducing decay and chilling injury (CI) of many fruits and vegetables (Fallik, 2004; Porat et al,

2000). Higher than optimum temperatures reduce shelf life and may have an adverse effect on flavor quality. In horticultural crops, the most prevalent method for maintaining postharvest quality and shelf life is rapid cooling and maintaining the lowest optimal temperature along with high relative humidity (Ghaouth et al., 1991). Cold storage is one of the most widely used technologies to slow respiration and other metabolic processes to preserve postharvest life of horticultural products (Wang, 1994). However, orange fruit are susceptible to CI when exposed to temperatures of less than 2–5oC. In most developing countries, temperature control is difficult because of insufficient power or interrupted electricity supply. Also, low temperature storage is often not economically feasible due to high equipment, infrastructure, and per capita electricity cost (Li and Yu,

2000). Creation of a modified atmosphere (MA) within produce, involving reduced O2 and elevated CO2, may be an alternative way to prolong produce shelf life under ambient conditions by retarding respiration and delaying senescence (Drake et al.,

1987).

Postharvest heat treatments are physical treatments that induce respiration, ethylene production, reduces CI, and can control decay. The associated biochemical and physiological changes result in the development of a MA within the products. In many cases, treating fruit with hot water (Fallik, 2004; Schirra and D’hallewin, 1997)

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also stimulates antioxidant systems (Martinez-Tellez and Lafuente, 1997; Sala and

Lafuente, 2000). Ivory mango treated with hot water (50oC for 10 min, 60oC for 1 min, or

70oC for 5 sec) experienced improved quality compared to the control, with the 60oC for

1-min treatment being optimal and associated with inhibited respiration, electric conductivity, malondialdehyde content, and increased total phenols and flavonoids

(Wang et al., 2017). Heat treatments inhibited softening (Paull and Chen, 2000), enhanced shelf life and improved postharvest quality of a number of fruits such as apple, peach, strawberry, citrus fruit, sweet pepper and mango (Bustamante et al. 2012;

Dotto et al. 2011; Jacobi et al. 2001; Porat et al. 2003). Because of these beneficial effects, heat treatments are currently used commercially to prolong quality and shelf life of fresh produce (Ferguson et al., 2000).

Surface coatings containing plasticizer are used as protective materials on fruit and can be beneficial for maintaining the quality of fresh commodities. Edible coatings extend shelf life by reducing water loss, maintaining appearance, and by modifying produce internal atmospheres. Coatings also can increase resistance to gas diffusion

(Pinheiro et al., 2012), but with potentially adverse effects on flavor if gas exchange is too severely inhibited (Mannheim and Soffer, 1996; Petracek et al., 1995). Fruit peel appearance is important to consumers, and coatings can help preserve produce color and texture, retain volatile flavor compounds, and control microbial growth on the commodity, prolonging shelf life. Coatings are used as supplements to refrigeration in many produce items to modulate the effects of non-optimal temperature exposure and reduce produce deterioration (Petracek et al., 1998). Oranges and mangoes coated with

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commercial waxes exhibited less weight loss and extended shelf life compared with control samples (Mathur and Shrivastava. 1955; Davis and Hofmann, 1973).

The objectives of this part of my dissertation were to determine the effects of hot- water immersion (45oC for 30 min) and three different edible wax coatings on the internal atmosphere, quality, and shelf life of Navel and Valencia oranges during simulated ambient temperature storage.

Materials and Methods

Experiment 1 – Effect of Immersion

Effect of ambient water immersion of un-coated Navel orange fruit quality and shelf life during simulated ambient temperature storage. The experiment was conducted on October 19, 2015 at the Horticultural Sciences Department, University of

Florida, Gainesville. Navel oranges were collected from a commercial grove in Fort

Pierce and were randomly distributed among treatments for the study. Then oranges were immersed in ambient (~25oC) water for 30 min in a mini stainless-steel water tank.

Control fruit were not immersed. Fruit were transported to Gainesville on the same day and stored at 25oC with 80±5% RH for 3 weeks. The purpose of the study was to determine if there is any effect of ambient water immersion on oranges, in terms of shelf life and storage quality.

Experiment 2 – Effect of Hot Water Treatment

Effect of hot water treatment and wax coating on quality and shelf life of

Navel and Valencia oranges during simulated ambient temperature storage.

Washington Navel and Valencia oranges were harvested from commercial groves in the

Fort Pierce area and transported to the Indian River Research and Education Center

(IRREC) where they were immersed for 30 sec in a solution of 1,000 ppm thiabendazole

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(TBZ) fungicide the same day. Navel oranges were harvested on October, 2015,

November, 2015, and January, 2016, representing early-, mid-, and late-season fruit, respectively (Table 4-1). For degreening (first Navel harvest only), fruit were exposed to

3 ppm C2H4 in a degreening room at 29oC and 90-95% relative humidity (RH) for 3 days. Valencia oranges were harvested in March 2016, April 2016, and May 2016, also representing early-, mid-, and late-season fruit, respectively. The purpose of the experiment was to use hot water immersion followed by coating application to first, increase the respiration rate and stimulate the antioxidant system in fruit during immersion and, second, create and maintain a beneficial internal MA within Navel and

Valencia oranges for improving quality and shelf life during simulated ambient temperature storage.

Experimental Procedure

Application of hot water immersion and waxing on a semi-commercial packing line. Uniform-size Navel and Valencia orange fruit were selected for each experiment, then the fruit were grouped into three batches as per the experimental design (Table 4-1). For Navel oranges, the first harvest (N=960) utilized four treatments, the second harvest (N=1200) utilized five treatments, and the third harvest utilized eight treatments (N=1500) in a factorial arrangement where with/without hot water and with/without coating were considered as factors. For all three batches (N=1500) of

Valencia oranges there were eight treatments in the same factorial arrangement as the third harvest of Navel oranges. Each treatment was comprised of 30 fruit where 10 fruit were randomly considered as a replicate. Fruit were completely submerged for 30 min in rapidly stirred water to stabilize the water temperature at 45oC; control fruit were submerged in ambient water (~25oC) for 30 min.

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Following application of the water immersion treatments, the fruit were run over a rotating brush bed for 1 min adding water with detergent soap (Fruit Cleaner 395, JBT

Food Tech, California) to remove dirt and microorganisms from the fruit surface (Figure

4-1). The fruit were then rinsed with potable water for 1 min before 1 min of forced-air drying on a brush bed. Another set of brush rollers were wetted using a hand sprayer with commercial carnauba (CR; EF9000), shellac (SH; SF 590HS), or polyethylene (PL;

EF 4000) fruit coatings (JBT Corporation, California) separately for a 1 min application on the fruit surface. Spray was also applied to the surface of rotating fruit as they passed over the waxing bed to achieve uniform coverage. The fruit were then dried for

2-3 min using fans blowing air at 52±2oC continuously around the fruit. After drying, fruit were weighed, sorted, graded and then kept in labeled plastic crates to designate treatments. Fruit were transported using a minivan and maintained at room temperature

(15±2oC) during transportation (~5 hours) to Gainesville. On the same day, fruit were sorted and randomly distributed into each batch (study) as per treatment and kept at the

Horticultural Sciences Departmental storage room at simulated temperature (25oC with

80±5%RH) for 3 weeks. The physio-chemical and sensory quality were studied initially and at weekly intervals for up to 3 weeks of storage.

Fruit weight loss. Weight loss was determined on each of 10 fruit per replicate and expressed as a percentage of weight change compared to the initial weight just after treatment. The fruit were stored at 25oC and 80±5%RH and were weighed at weekly intervals for up to 3 weeks of storage.

Fruit peel color. Orange peel color was measured using a hand held Minolta

Chroma meter (Chroma meter CR-400, Konica Minolta Sensing Inc., Japan) using the

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CIE color system on the L*, h*, C* color space. Fruit were marked with circles at two equidistant points on the equator of each fruit. Two measurements were then taken per fruit, one on each marked side of the fruit at weekly intervals for up to 3 weeks storage.

Tristimulus values (X, y, z) were converted to hue angle (h*) and chroma color value

(C*). The actual perceived color (h*) was defined by tan-1(b*/a*) and expressed in degree, where 0o, 90o, 180o, 270o represent red, green, yellow, and blue, respectively.

Chroma color value (C*) where, C*=ѵ(a*)2+(b*)2, is the vividness (intensity) of the color represented by numbers ranging from 0 (center=gray) to 60 of the color solid radius for any given color hue angle (Francis, 1995; Konica Minolta, 2007; Machado et al., 2015;

McGuire, 1992).

Fruit compression (COM) and peel puncture (PUN). Fruit firmness was measured according to Plaza et al. (2004) with some modifications, using a computer controlled texture analyzer machine (TA.HD.plus Texture Analyzer, Stable Micro

Systems Ltd., New York, U.S.A.). The machine was equipped with a 500-kg load cell with a 3-mm flat tipped-cylinder probe attached for COM or PUN and was calibrated before estimating the fruit firmness. For both tests, the texture analyzer was set at probe pre-test, test, and post-test speeds of 20, 5, and 20 mm s-1, respectively. Force deformation curves were made for compressing fruit to 8 mm and for puncturing the fruit peel to 10 mm and estimated in Newtons. Thirty fruit per treatment were measured comprising three replicates of 10 fruit each. Each fruit was placed on its equator on a sample platform of the machine and the firmness data was recorded.

Fruit peel oleocellosis (OLC). Similar sized fruit (n=24), without any defects, were randomly selected from each treatment and evaluated for susceptibility to OLC.

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The amount of pressure to release the oil from the oil glands of orange was determined by the rind oil release pressure (RORP) method according to Wardowski et al., (1997) using a tissue paper and pressure tester (Force Dial FDK 20, Wagner instruments, Italy) fitted with a 3/8 inch cylindrical tip. The average force (in Newtons) per treatment required to release oil was calculated.

Fruit internal gas measurement. Concentrations of CO2 and O2 within individual fruit were determined weekly during 3 weeks of storage at 25oC with 80±5%

RH. Internal gas samples were extracted by inserting a syringe needle into the blossom end of a fruit submerged in water and withdrawing the gas sample using a gas-tight syringe (MR-GT, SGE Co., Australia). The gas samples were analyzed by gas chromatography (GC) according to Zhang et al. (2011). Initial analysis of internal gases in orange fruit was conducted just after hot water immersion and application of coating.

The analysis was conducted at the USDA-ARS lab in Fort Pierce, FL. Oxygen, carbon dioxide, and ethylene levels were analyzed using GC (Hawlet Packard 5890 series II) equipped with a thermal conductivity detector (TCCD, O2, CO2) and a pulse discharge helium ionization detector (PDHID, ethylene). The injection port temperature was

198oC, the oven temperature was 50oC, and the detector temperature 200oC. Five to eight fruit were used per treatment to measure internal atmospheres. Fruit were immersed in water and 5-ml glass syringes with 38-mm needles were used to collect internal gases from orange fruit, which were immediately injected onto the GC. The internal gases were analyzed using a calibration curve at each sampling interval and were expressed as percentage of O2 and CO2, and ppm for C2H2. Similarly, weekly measurements of internal atmospheres in orange fruit were conducted at the

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Postharvest Biology and Technology GC lab. Five to eight fruit were used per treatment to collect internal gas. Gas samples of 1 ml were collected from each fruit and injected into a 5.5-ml glass container that had been previously flushed with N2 gas (60ml/min).

Next, 2.5 ml of N2 gas was added to the glass container to increase the gas volume and then a 2.5-ml sample was collected using another syringe for injection onto a GC (CP-

3800, Varian Inc., Palo Alto, CA) equipped with a TCCD and a PDHID. The injection port temperature was 220oC, the oven temperature was 50oC, and the detector temperature 175oC for the TCCD and 120 oC for the PDHID. The sample for

CO2 determination passed through Hayesep Q Ultimetal (1 m × 3.18 mm) [particle size

149–177 μm (80/100 mesh)] and Molsieve 13 (1.5 m × 3.18 mm) [particle size 149–

177 μm (80/100 mesh)] columns (Varian) coupled in series to the TCCD. The gas for ethylene determination passed through two Hayesep Q Ultimetal (1 m × 3.18 mm)

[particle size 149–177 μm (80/100 mesh)] columns (Varian) coupled in series to the

PDHID. The carrier gas (helium) flow rate was about 0.35 mL s−1. Internal atmospheres

(O2, CO2, and C2H2) were calculated from calibration curves as before.

Chemical Analyses

Soluble solids content (SSC). A composite fruit tissue sample of 10 orange fruit was taken per replicate and blended in an electric blender (Model HBB908, Hamilton

Beach Commercial Inc. China); the blended samples were then homogenized (OMNI

GLH, the Homogenizer Company Inc., USA) for 30 s. After homogenizing, the samples were centrifuged (Thermos Scientific, Model Sorvall LYNX 400 label 2, Osterode am

Harz, Germany) at 13,200 x gn for 20 min in a JA-20 rotor (4oC) to precipitate tissue solids, and the clear supernatant was filtered through cheesecloth. A few drops of the resulting clear juice were taken (three different times) to measure SSC using an automatic,

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temperature-compensated refractometer (AMetek, Ultra Precision Technologies,

Reichart Inc., Model r2 i300, New York, USA) and expressed as degrees Brix.

Total titratable acidity (TTA). Titratable acidity of clarified juice was measured using an automated titrator (719S Titrino, Metrohm, Riverview, FL, USA) with 6.0 ml of extracted juice mixed with 50 ml of deionized (DI) water. The samples were then titrated with 0.1 N sodium hydroxide (Fisher) to an end point of pH 8.2 and the acidity expressed as percentage of citric acid. The sample pH was measured at the beginning of the titration and the end point of the titration was pH 8.2 to reproduce historical results performed with the AOAC method 22.058 (AOAC International, 1995).

Fruit ascorbic acid (ASC) content. Ascorbic acid content was determined according to AOAC method 967.21 (AOAC International, 1995). Tissue samples (2.0 g) were blended and homogenized as for SSC and TA measurements, but with 20 mL of

6% metaphosphoric acid (MPA) certified ACS in 2N acetic acid certified ACS plus

(Fisher). The homogenized samples were frozen at -20oC until analysis. During analysis, the samples were thawed at room temperature and then centrifuged for 20 min at 13,200 x gn in a JA-20 rotor (4oC) to precipitate tissue solids, and the clear supernatant was filtered through cheesecloth. Each sample from 10-fruit composite sample was analyzed in triplicate for each replicate sample. Aliquots of 1 ml clear supernatant were transferred to test tubes for analysis. Then, one drop of 0.2% 2,6- dichlorophenol indophenol (Sigma) was added to each test tube, which was incubated at room temperature for 1 h.

After incubation at room temperature, 1.0 ml of 2% thiourea reagent grade and

500 µL of 2% 2,4-dinitrophenyl hydrazine (Sigma) in 9N H2SO4 (Fisher) solution was

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added to each sample and the samples vortexed for a few seconds. The tubes were covered with marbles and, except for the blank, incubated again in a 60oC water bath for 3 h. Blank samples were kept at room temperature without addition of 2% dinitrophenyl hydrazine in 9N H2SO4.

After the second incubation, the test tubes were immediately chilled in ice water and 500 µL of 2% dinitrophenyl hydrazine in 9N H2SO4 was added to the blank. Then,

2.5 ml of 95% H2SO4 certified ACS plus was added to all of the test tubes, which were immediately vortexed for 10-15 seconds and placed back into the ice water.

Sample aliquots of 250 µl were multichannel pipetted into a 96-well plate and the absorbance was read at 540 ɳm in a microplate reader (PowerWave XS2, Biotek). A calibration curve was prepared using L-ascorbic acid certified ACS (Fisher) as the standard and then the ascorbic acid concentration for each of the sample was calculated. The results were expressed as mg/100 g of fruit tissue dry weight.

Total carotenoid (TCC) content. For TCC content, fruit flavedo was separated carefully from the albedo of the orange fruit rind using a sharp knife. The flavedo part of the rind and fruit carpel tissue was blended and homogenized separately. Blended and homogenized samples (2.0 g) of each replicate were weighed for carotenoid extraction

(Tee, 1991). Total carotenoid extraction was done according Lee and Castle (2001) with some modifications. The samples were placed in 50-ml graduated orange cap centrifuge tubes and 20 ml of extracting chemicals (Hexane: Ethanol, 50:50 v/v) were added before storing the sample in a freezer at -30oC for at least 1 hour prior to analysis. During analysis, the mixture was thawed, agitated, and then centrifuged at

29,000 x gn for 20 min (F14-14 X 50cy rotor, Thermo Scientific, USA) at 2oC. The tubes

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were shaken to extract all the carotenoids in the tissue then left to stand for 5 min to get clear separation. The top layer of hexane containing the carotenoid fraction was removed and transferred into 50-ml centrifuge tubes using a 3-ml transfer pipette and the tubes covered. The sample residues were rinsed with ethanol-hexane mixture (1:9) and deionized water was used to remove any remaining carotenoids. The sample were stored in the -30oC freezer for at least 1 hour, and then placed in crushed ice. The volume of recovered hexane was adjusted to 15 to 25 ml using hexane. For the blank, only hexane was used in the microplate. TCC content of each fruit tissue sample was calculated by the following equation: (AVx106)/ (A1%x100G), where A = absorbance,

V = total volume used (e.g., 25 ml), A1% = 2500, and G = sample in grams.

Total phenolic (TP) content. The TP content was analyzed with the Folin-

Ciocaltew reaction reported by Singleton and Rossi (1965) using gallic acid anhydrous certified power (Fisher) as the standard in a calibration curve between 0.1 to 1 mg/ml.

For this procedure, 1.0 ml of hydrophilic fraction was diluted in 2.0 ml of DI water. Then a duplicate 0.4 ml of sample (diluted hydrophilic fraction) was pipetted into test tubes.

Now 2.5 ml of 0.2 N Folin-Ciocalteu reagent (Sigma) was added and subsequently 2.0 ml of 7.5% sodium carbonate certified ACS (Fisher). The tubes were then capped and mixed with a vortex. Finally, the samples were incubated in a water bath at 45oC for 15 min. For the absorbance reading, sample aliquots of 250 µl were pipetted into a 96-well plate and absorbance read at 765 ɳm in a microplate reader (PowerWave XS2, Biotek).

The results were expressed as mg gallic acid equivalents (GAE)/100 g of fruit tissue dry weight.

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Antioxidant capacity (ATC) analysis. The ATC of orange fruit tissue was determined by the oxygen radical absorbance capacity (ORAC) assay according to the method described by Cao et al. (1993) and modified by Ou et al. (2001) and Huang et al. (2002), and Prior et al. (2003). The ORAC values for hydrophilic fractions were only measured, since the lipophilic fraction was reported to be negligible in most citrus species (Huang et al., 2002; Legua et al., 2014). Fruit tissue of ten whole fresh fruit were blended together for each replicate and 45 g of composite tissue sample weighed and then frozen and stored at -30oC. For sample preparation, the thawed samples were homogenized and then centrifuged at 13,200 x gn, 4oC for 20 min and the supernatant filtered through four layers of cheese cloth into a 20-ml vial. A 20-µl aliquot of the tissue sample was diluted with 10 ml of 75 mM of potassium phosphate buffer. The 75 mM potassium phosphate buffer was prepared using monosodium phosphate monohydrate certified ACS (Fisher Scientific Inc., MA, USA) and dipotassium phosphate anhydrous certified ACS (Fisher Scientific Inc., MA, USA) and adjusted to pH 7.4. Using the 75 mM potassium phosphate buffer as a solvent, 0.4 µM, 153 mM, and 1 mM of sodium fluorescein, 2,2´-azobis (2-amidinopropane) dihydrochloride (AAPH), and Trolox®, 6- hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (Acros Organics, Geel,

Belgium) were prepared. Trolox was used as a standard to generate a calibration curve of 0-100 µM. Aliquots of 150 µl of 0.4 µM sodium fluorescein laboratory grade (Fisher

Scientific Inc., MA, USA) was pipetted into 60 inner wells of the 96-well black plate and the outer 36 wells of the plate were filled with 200 µl distilled water. In duplicate, 25 µl of

1 mM Trolox standard or 25 µl of diluted sample was added to each well with sodium fluorescein solution. The plate was then incubated at 37oC for 15 min in a

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spectrofluorometer microplate reader with an automated injector (FLx800, Biotek). By the end of the incubation, the reaction was initiated following an automatic injection of

25 µl of AAPH into each well. The machine was set at 485 ɳm and 528 ɳm excitation and emission, respectively, with kinetic readings generated on the computer and ORAC values calculated based on the below equation. The results were expressed as µmol

Trolox equivalents (TE) per 100 g of fresh tissue weight.

AUC = 0.5+f5/f4+f6/f4+f7/f4+……….+f59/f4+f60/f4

Net ORAC = AUCsample - AUCblank.

Where, AUC = Area under the curve

Statistical Analysis

The physiochemical attributes were measured initially and also at weekly intervals during 3 weeks of simulated ambient storage. The quality and physicochemical data were analyzed using analysis of variance based on proc GLM (SAS Institute Inc.

Version 9.3, Cary, NC, USA) using a CRD design, CRD with factorial with different cultivars (C), harvest (H), different treatment temperature (T), and storage period (S) as the main effects. The sensory data were analyzed using SAS software and mean separation of each parameter was based on Tukey’s test at P<0.05.

Fruit volatile compounds. Analysis of volatile compounds was done according to Obenland et al. (2011). A frozen tissue sample of 2.5 ml was thawed and 1-pentanol added as an internal standard to a final concentration of 490 µg L-1. A total of three samples per treatment were measured with each sample being a pooled tissue sample from 10 fruit as a composite sample. Volatile analysis was conducted by solid phase micro extraction (SPME) with a 75 µm carboxen/polydimethylsiloxane fiber (Supelco, St.

Louis, MO, USA) using a Gerstel MPS-2 robotic system (Linthicum, MD, USA). The

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Gerstel system initiated a sample’s analysis by transferring the sample from the cooled

(4oC) holding tray into the heated (40oC) agitator where the sample was allowed to warm for 15 min. The SPME fiber was inserted into the head space of the vial to trap volatiles for 30 min at 40oC with an agitator speed of 4.2 s-1. After volatile trapping, the

SPME fiber was removed from the vial and desorbed for 2 min at 280oC in the splitless inlet of an Agilent 7890 GC coupled with a 5975 mass selective detector (Agilent, Palo

Alto, CA). The column was an Agilent HP-5ms ultra-inert (30 m x 0.25 µm film thickness). Identification of the volatiles was made by comparison to Wiley/NBS library spectra and retention times of standards. Comparison of retention indices to published values was used to obtain additional confirmation of identities. Quantification was done by calibration curves generated from standards added to deodorized orange fruit juice and adjusted, with the resulting values based upon the amount of the internal standard

(1-pentanol) present (Obenland et al., 2011).

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Results

Ambient Water Immersion of Un-Coated Navel Orange Fruit Quality and Shelf Life During Simulated Ambient Temperature Storage.

There were no significant effects observed from immersing orange fruit in ambient water in terms of shelf life and storage quality, except fruit immersed fruit being slightly cleaner.

Hot Water Treatment and Wax Coating on Quality and Shelf Life of Navel and Valencia Oranges During Simulated Ambient Temperature Storage.

Comparison of two cultivars with 8-combination (Navel vs Valencia) -

Cultivar-storage interaction. Using the mid- and late-harvests of both cultivars, Tables

4-2, 4-3, and 4-4 shows the ANOVA main factor effects and interactions. The cultivar- storage interaction were significant for SSC, TTA, SSC/TTA, ASC, and TCC (Table 4-

2), for WL, T&P, ATC, and L* (Table 4-3), and for COM and OLC (Table 4-4). The maximum SSC was observed in Navel oranges (13.60 a) (Table 4-5) whereas for TTA content, the highest levels were noted in Valencia orange (0.59 a) after 1 week storage and then a slight decrease thereafter due to water loss or acid converted to sugar.

Similar trends were found for SSC/TTA ratio during the 3-week storage duration (Figure

4-2). In Table 4-5 from combined analysis of ASC, the highest amount was observed in

Navel oranges (63.27 a) after 2 weeks storage and the lowest amount was observed in

Valencia orange (44.62 c) after 3 weeks storage (Figure 4-3). After 3 weeks storage, the highest TCC was observed in Valencia oranges (4.99 a), followed by Navel oranges

(4.37 b).The lowest TCC was found in Navel oranges (2.13 d) after 2 weeks storage

(Table 4-6).

The lowest WL was observed in Valencia oranges after 1 week storage and maximum (6.25 a) was noticed after 3 weeks (Table 4-6). T&P increased during

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storage, with the highest amounts observed in Valencia oranges after 3 weeks of storage (228.90 a) and the lowest in Navel oranges before storage (37.94 c) (Table 4-

6). The maximum ATC was observed in Navel oranges after 1 week storage (1649.30 a), followed by Navel oranges after 3 weeks storage (1504.50 ab) (Table 4-6). The lowest ATC was noted in Valencia oranges after 2 weeks storage (1137.70 e). The L* of

Navel oranges were higher than Valencia oranges (Table 4-7).

COM force declined slightly during storage with the lowest value noted in both cultivars after 3 weeks of storage (Table 4-7).

Cultivar-treatment temperature interaction. There were significant cultivar by treatment temperature interactions for TTA, SSC/TTA, ASC, and TTC (Table 4-2), ATC,

L*, C, and H (Table 4-3), and for PUN (Table 4-4). The maximum TTA was noted in

Valencia oranges immersed in water at 45oC for 30 mins (0.52 a), and lowest in Navel oranges (0.45 c) immersed in ambient water (25oC) for 30 min (Table 4-8The highest

SSC/TTA was observed in Navel oranges immersed in ambient (30.29 a) or 45oC water

(29.17 a) compared to Valencia oranges. Similar trends were noted for ASC (Table 4-8).

The highest ATC was observed in Navel oranges immersed in 45oC water for 30 min, and lowest in Valencia orange immersed in ambient water for 30 min (Table 4-8).

Navel oranges immersed in water at 45oC for 30 min exhibited the highest L* (68.04 a) and C (71.17 a), and Valencia oranges exhibited the highest H (81.71 a) (Table 4-9).

PUN values were significantly higher in Valencia oranges compared to Navel oranges, regardless of immersion treatment (Table 4-4).

Cultivar-Surface coating interaction. There were significant cultivar by surface coating interactions for SSC, TTA, SSC/TTA, and TCC (Table 4-2), WL, L*, C, H, PUN,

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and OLC (Table 4-3), and for PUN and OLC (Table 4-4). The highest SSC was observed in NC Navel orange fruit (13.92 a) and PL-coated fruit (13.85 a), followed by

CR- and SH-coated fruit (Table 4-10). The highest TTA was observed in NC (0.52 a) and SH-coated (0.52 a) Valencia oranges (Table 4-10). All coated Navel oranges contained the highest SSC/TTA (Table 4-10). , Valencia oranges had greater TCC than

Navel oranges (Table 4-10).

The highest WL was observed in NC Valencia oranges (Table 4-10), while both

Navel (1.66 f) and Valencia (1.62 f) oranges coated with CR exhibited the least WL. The highest L* was observed in Navel oranges with PL- (69.74 a) and SH- (69.94 a) coated fruit (Table 4-11). Similar trends were noted for C. The greatest H was observed in CR- coated Valencia oranges (83.42 a), followed by SH-coated fruit (80.94 b) of the same cultivar (Table 4-11). The greatest PUN was observed with SH-coated Valencia oranges

(33.62 a), followed by NC Valencia oranges (31.37 b). The highest OLC was observed in CR-coated Valencia fruit (55.16 a) (Table 4-11).

Storage-water temperature interaction. There were significant storage by water temperature interactions for ASC (Table 4-2), WL, and ATC (Table 4-3), and for

COM and PUN (Table 4-4). The highest ASC was observed in fruit immersed in water at

45oC (59.53 a) (Table 4-12). The greatest WL was observed in fruit immersed in ambient water (25oC) after 3 weeks of storage, and the greatest ATC (1693.40 a) was observed in fruit immersed in 45oC water after 1 week of storage (Tables 4-3 and 4-12).

Fruit immersed in 25oC water had the highest COM before storage (127.54 a), followed by the same fruit after 1 week of storage (107.92 b) and fruit immersed in 45oC

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water after 1 week of storage (106.65 b) (Table 4-12). The greatest PUN force was observed on fruit immersed in 25oC water after 3 weeks of storage (29.98 a).

Storage-surface coating interaction. , There were significant storage by surface coating interactions for SSC and SSC/TTA (Table 4-2), for WL, ATC, and C

(Table 4-3), and for COM, PUN, and OLC force (Table 4-4). The maximum SSC was observed in PL-coated fruit (13.21 a) after 3 weeks of storage (Table 4-13), followed by

NC fruit (12.97 ab) after 1 week of storage. The greatest SSC/TTA was observed in PL-

(30.38 a) and CR-coated (30.27 a) fruit after 3 weeks of storage, followed by CR-coated fruit (29.52 ab) after 2 weeks of storage (Table 4-13).

The highest WL was observed in NC fruit after 3 weeks of storage (7.36 a) and

SH-coated fruit (7.40 a) (Table 4-13). The greatest ATC was found in NC fruit after 1 week of storage (1675.20 a), followed by PL-coated fruit (1523.50 ab) after 1 week of storage (Table 4-13).

The highest C was observed in PL-coated fruit (69.21 a), followed by SH-coated fruit (68.82 ab) after 1 week of storage (Table 4-14).

The greatest COM values were observed in CR-coated fruit before storage

(131.76 a) (Table 4-4). The greatest PUN force was observed on SH-coated fruit (31.04 a) after 1 week of storage, followed by SH-coated fruit after 3 weeks of storage (30.72 b) (Table 4-14). The greatest OLC was observed in SH-coated fruit before storage

(53.60 a) (Table 4-14).

Water temperature-surface coating interaction. There was significant temperature by coating interactions for ASC and TCC (Table 4-2). The highest ASC was observed NC fruit immersed in 25oC water for 30 min (54.38 a), followed by CR-

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coated fruit immersed in 45oC water for 30 min (54.06 ab) (Table 4-15). As shown in

Table 4-15, the highest amount of TCC was observed in PL-coated fruit immersed in water at 25oC for 30 min (3.87 a), followed by NC fruit immersed in water at 45oC for 30 min (3.76 ab).

There were also significant interactions for in WL, ATC, L*, and H (Table 4-3), and for COM (Table 4-4). The highest WL was observed in SH-coated fruit immersed in water at 25oC for 30 min (4.14 a) and in NC fruit (4.11 a) (Table 4-15). The greatest

ATC was found in NC fruit immersed in water at 45oC for 30 min (1605.80 a), followed by PL-coated fruit immersed in water at 45oC for 30 min (1453.80 ab). Similarly, the highest L* was observed in PL- (67.56 a) and SH- (67.51 a) coated fruit immersed in water at 45oC for 30 min(Table 4-16). The highest H angle was observed in CR-coated fruit immersed in water at 45oC for 30 min (80.37 a), followed by SH-coated fruit (79.05 ab) (Table 4-16). The maximum COM was observed in PL- (113.48 a) and CR- (119.83 a) coated fruit immersed in 25oC water for 30 min, and CR-coated fruit immersed in hot water at 45oC for 30 min (112.59 a) fruit (Table 4-16).

Cultivar-storage-water temperature interaction. Only SSC (Table 4-2), ATC

(Table 4-3), COM (Table 4-4), and OLC experienced significant cultivar by storage by water temperature interactions. Navel orange immersed in water at 25oC or 45oC for 30 min exhibited higher SSC after 3 weeks of storage compared to Valencia orange (Table

4-17). Almost all fruit from both cultivars immersed in hot water experienced increased

ATC during 3-week storage compared to fruit immersed in ambient water (Table 4-17).

The greatest COM force (137.13 a) was observed in Navel oranges before storage that had been immersed in 25oC water for 30 min. The highest OLC force (57.57 a) was

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observed in Valencia orange immersed in 25oC water for 30 min and stored for 1 week

(Table 4-17).

Cultivar-storage-surface coating interaction. TCC (Table 4-2), WL (Table 4-

3), ATC, COM (Table 4-4), PUN, and OLC experienced significant cultivar by storage by fruit coating interactions. TCC increased during storage, with the greatest TCC (5.66 a) observed in NC Valencia orange after 3 weeks of storage. The greatest WL was observed in NC Valencia orange after 3 weeks of storage (8.76 a). The highest ATC was observed in NC Navel oranges after 1 week of storage (1811.90 a), followed by PL- coated Navel oranges after 1 week of storage (1771.07 ab) (Table 4-18).

In Table 4-19, the highest COM force was observed in CR-coated Navel oranges before storage (147.82 a), followed by CR-coated Valencia oranges after 1 week of storage (130.95 ab). The greatest PUN force was observed in SH-coated Valencia oranges after 1 (30.45 a) and 2 (35.19 a) weeks of storage (Table 4-19). Similarly, the highest OLC force was observed in CR-coated Valencia oranges after 1 week of storage (58.15 a). followed by SH-coated (56.94 ab) and NC (56.77 ab) Valencia oranges after 1 week of storage, and PL-coated Valencia oranges before storage (56.10 ab) (Table 4-19).

Storage-water temperature-surface coating interaction. Only SSC/TTA

(Table 4-2), ATC (Table 4-3), and COM (Table 4-4) experienced significant storage by water temperature by fruit coating interactions. The greatest SSC/TTA (31.80 a) was observed in PL-coated fruit immersed in 25oC water (31.80 a), followed by CR-coated fruit (31.56 ab) after 3 weeks of storage (Table 4-20). The greatest ATC was observed in NC fruit immersed in 45oC water (2129.05 a) after 1 week of storage (Table 4-20).

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The greatest COM force was observed in PL-coated fruit immersed in 25oC water

(143.96 a), followed by CR-coated fruit also immersed in 25oC water (138.61 ab) (Table

4-20).

Cultivar-water temperature-surface coating interaction. SSC and TCC (Table

4-2), WL, ATC, L*, C, and H (Table 4-3), and PUN and OLC (Table 4-4) all experienced significant cultivar by water temperature by fruit coating interactions The highest SSC was observed in NC Navel oranges immersed in 25oC water (14.10 a) (Table 4-21). The highest TCC was observed in PL-coated Valencia oranges immersed in 25oC water

(5.30 a) (Table 4-21)

The highest ATC was exhibited in NC Navel oranges immersed in 45oC water

(1832.50 a), followed by PL-coated Navel oranges immersed in 45oC water (1584.90 ab) (Table 4-21). The greatest WL was observed noted from NC Valencia orange immersed in 25oC water (4.90 a) and lowest from CR-coated fruit of both cultivars

(Table 4-21). SH-coated Navel oranges immersed in 45oC water developed the highest

L* (72.35 a). Similar trends were observed for CR-coated fruit (Table 4-22). CR-coated

Valencia oranges immersed in 45oC water exhibited the greatest H values among the treatments (85.11 a) (Table 4-22).

SH-coated Valencia oranges immersed in 45oC water (33.40 a) or 25oC water

(33.83 a) had the highest PUN force among the treatments (Table 4-22). The highest C value was observed on coated Navel oranges immersed in 45oC water (73.93 – 75.08).

Cultivar-storage-water temperature-surface coating interaction. Only WL and ATC showed four-way interactions (Tables 4-2; 4-3; 4-4).

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The lowest WL in Navel oranges was observed from CR-coated fruit submerged in 25oC for 30 min (1.37 i) after 1 week of storage, and the greatest WL was observed in

SH-(6.57 - 7.84 a) coated fruit immersed in either temperature, and NC-coated fruit

(5.89 a) immersed in 45oC water, all after 3 weeks of storage (Table 4-23) In case of

Valencia oranges immersed in 45oC water for 30 min, the greatest WL was observed in

NC (8.15 a) and SH (7.65 a) fruit after 3 weeks of storage, and the lowest WL was exhibited by CR-coated fruit (0.80 hi). immersed in 25oC water for 30 min. The greatest

WL was observed in NC fruit (9.38 a), followed by SH-coated fruit (7.54 b) after 3 weeks of storage, with the least WL coming from CR-coated fruit (0.92 i) (Table 4-23).

As shown in Table 4-24, the greatest ATC was found in CR-coated Navel oranges immersed in 45oC water for 30 min (2289.11 a) after 1 week of storage. The lowest ATC was observed in NC Navel oranges (1219.39 e) after 2 weeks of storage.

Similarly, PL-coated Navel orange immersed in 25oC water for 30 min had the highest

ATC (1767.92 a) after 2 weeks of storage, and the lowest ATC (972.93 e) was found in

SH-coated fruit before storage (Table 4-24). In Valencia oranges treated at 45oC for 30 min, NC fruit had the highest ATC (1968.99 a) after 1 week of storage, and SH-coated fruit exhibited the lowest ATC (969.23 c) before storage. SH-coated Valencia oranges immersed in 25oC water for 30 min had the highest ATC (1589.75 a) after 3 weeks storage, and SH-coated fruit had the lowest (931.69 b) after 2 weeks of storage (Tables

4-23 and 4-24).

Comparison of Two Cultivars with 4-Combination (Navel Vs Valencia)

Cultivar-harvest-storage-surface coating interaction. As shown in Table 4-25,

Table 4-26, and Table 4-27, SSC, TTA, ASC, WL, COM, PUN, and OLC all showed significant four-way interactions.

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The greatest SSC of Navel oranges for early-H1, mid-H2, and late-H3 were

AMW+NC (10.80 a) after 3 weeks, AMW+NC (11.20 a) after 3 weeks, and AMW+NC

(14.70 a) after 2 weeks among the treatments, respectively (Table 4-28). Similar trends were observed for Valencia oranges except for H2 fruit. Initially, SSC was higher in

AMW+NC fruit (13.90 a) and remained similar after 2 weeks storage.

The maximum ASC of Navel orange for early- H1, mid-H2, and late-H3 were

AMW+NC (53.30 a) after 3 weeks, HW+PL (66.87 a) initially storage, AMW+NC (72.27 a) after 2 weeks among the treatments, respectively (Table 4-30). But for Valencia orange, the highest SSC was found in HW+SH (129.16 a), HW+SH (91.08 a), and

HW+CR (57.70 a) fruit after 2 weeks at H1, H2, and H3 respectively.

The least WL from Navel oranges was observed in AMW+NC (6.34 a) and

HW+SH (6.10 a), followed by HW+PL (4.77 b) and HW+CR (2.69 e) after 3 weeks of storage at H 1 (Table 4-31). Similar results were noted for other harvests of Navel orange as well as Valencia orange after 3 weeks of storage.

The largest COM was observed in H1, H2, and H3 fruit that received treatments of HW+CR (119.71 a) after 3 weeks, HW+CR (174.94 a) initially harvest, and HW+CR

(140.00 a) fruit before storage (Table 4-32). For Valencia orange, the greatest COM force was observed from HW+PL (128.27 a) fruit before storage, AMW+NC (104.79 a) fruit before storage, and HW+CR (131.70 a) fruit after 1 week of storage.

For PUN force of Navel orange at early, mid and late harvests, the greatest PUN force were from HW+SH (35.17 a) fruit after 2 weeks storage of H 1, AMW+NC (28.41 a) fruit after 2 weeks of H2, and AMW+NC (26.68 a) & AMW+NC (26.81 a) fruit for H3 fruit. The greatest PUN force for Valencia oranges were from HW+CR (39.95 a) fruit of

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H1 before storage, HW+SH (33.46 a) fruit before storage, and HW+SH (32.71 a) fruit after 2 weeks of storage of H2, and AMW+NC (38.33 a) fruit after 3 weeks of storage of

H3 fruit.

HW+SH coated Navel oranges had the highest OLC force after 1 week of storage in H1 (70.96 a) and H2 (54.05 a) and H3 (54.12 a) before storage (Table 4-34).

The OLC was highest in Valencia orange stored for 3 weeks in HW+CR coated fruit

H1(51.67 N), H2(47.82 N), and H3(53.41 N), followed by HW+PL fruit for all 3 harvests

H1(48.00 N), H2(43.41 N), and H3(51.09 N).

Comparison of Two Cultivars with 5-Combination (Navel Vs Valencia)

Cultivar-harvest-storage-temperature surface coating interaction. As shown in Table 4-35, Table 4-36, and Table 4-37 there were no significant four-way interactions for most of the quality attributes except TTA, ASC, WL, ATC, and OLC.

The highest TTA of Navel orange mid-H2, and late-H3 were from HW+NC fruit

(0.75 a) after 1 week of storage, and from AMW+NC fruit (0.50 a) before storage (Table

4-38). In Valencia oranges, the highest TTA were observed in AMW+NC (0.82 a) fruit after 3 weeks storage of H2 and AMW+NC (0.64 a) fruit before storage.

The highest ASC for Navel oranges for mid-H2, were from HW+NC (66.87 a) fruit and HW+CR (59.78 a) fruit before storage, and from HW+CR (56.68 a) fruit after 3 weeks storage (Table 4-39). The highest ASC for H3 of Navel orange was from

AMW+NC (72.27 a) fruit after 2 weeks storage. For Valencia oranges, the highest ASC for H2 was from HW+SH (98.10 a) fruit after 2 weeks storage and AMW+NC (53.24 a) fruit before storage of H3 fruit.

As stated in Table 4-40, the minimum WL of Navel orange was observed in

HW+CR coated fruit for H2 (2.68%) and H3 (2.33%) after 3 weeks storage. Similar

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results were noted for Valencia orange fruit after 3 weeks storage at ambient temperature.

The highest ATC in Navel oranges were noticed in HW+CR (1370.71 a) for H2 fruit, and HW+NC (2289.11 a) for H3 after 1 week storage (Table 4-41). For Valencia orange, the maximum ATC was in HW+SH (2349.76 a) after 2 weeks for H2 harvest, and HW+NC (1968.99 a) after 1 week for H3 harvest fruit.

As observed in Table 4-42 for OLC force of Navel oranges, the maximum OLC force were HW+NC (54.77 a) for H2 and HW+SH (54.12 a) for H3 harvest fruit before storage. Similarly, for Valencia orange OLC force was highest before storage, and then declined during storage.

Gas Exchange into Orange Fruit

Internal atmosphere (%O2) in Navel vs Valencia. As shown in Figure 4-4, initially %O2 was higher in AMW+NC (~21%) coated Navel oranges and after 3 weeks, while it was at about 16% immediately after HWT and coating, the percentage O2 dropped to below 8%, with a bit of an increase after 3 weeks of storage. After 3 weeks storage, HW+NC, HW+PL, and HW+CR fruit all exhibited below 15% O2, whereas

HW+SH exhibited extremely low O2 (<5%) (Figure 4-10). Similar trends were observed with the Valencia oranges for AMW+NC fruit, but HWT and coated fruit experienced lower internal O2 levels below 5% initially, increasing during storage to 13-17% in HWT fruit, with SH-coated remaining low (<5%) (Figure 4-7, 4-13 & 4-19).

Internal atmosphere (%CO2) into Navel vs Valencia. As shown in Figure 4-5 for Navel orange, initially %CO2 was higher in HWT with coated fruit (8-13% CO2) than in AMW+NC (~2% CO2) fruit after 3 weeks (<5% CO2). Similar trends were noted in

Figure 4-11 for the 5-combination treatments. In Figure 4-11, the highest CO2

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concentrations were observed in HW+CR coated fruit (~ 7% CO2) after 3 weeks storage. Initially after HWT and coating, fruit increased internal CO2 (~15%), but decreased during storage, except in HW/without HW and with SH/without SH coated fruit (Figure 4-17). Among the treatments, CR coated fruit maintained desired internal

CO2 (5-8%) during storage.

Initially, HW treated Valencia fruit increased internal CO2 (13-17%) and during storage those levels tended to decrease (Figure 4-8). After 3 weeks storage, HW+CR coated fruit exhibited what is considered a desirable internal CO2 level (~6%). As shown in Figure 4-14, initially storage CO2 level was below 3% and then increased significantly among the treatments. Among the treatments, HW+CR coated fruit increased CO2

(~16%) after 3 weeks storage. Similar trends were observed in Figure 4-20 for 8- combination treatments.

Internal atmosphere (ppm C2H4) into Navel vs Valencia. Initially, C2H4 levels within Navel orange fruit were below 0.5 ppm (Figure 4-6). After 3 weeks storage, the maximum C2H4 concentration was found in fruit of HW+SH (~1.30 ppm). Similar trends were observed in Figure 4-12 & 4-18.

AMW+NC Valencia oranges initially produced little C2H4 (<1.00 ppm) and during storage for 3 weeks, relatively little C2H4 was produced, with a maximum produced after

3 weeks from in the HW+SH treatment (~0.80 ppm). Similar trends were observed in

Figure 4-15 & 4-21 for C2H4 in Valencia orange during storage.

Volatile compounds. As shown in Table 4-43, twenty two major volatile were detected during storage. The major volatile compounds were observed were identified as limonene, naphthalene, butanoic acid, hexanoic acid, ethyl ester, octanoic acid,

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acetamide, etc. Data is not presented for total volatile compounds or the amount of each flavor volatile compounds.

Discussion

Hot water immersion treatments significantly reduced O2 concentrations within fruit, which subsequently increased again between week 0 and week 1 for Valencia, and were maintained in both varieties during the 3 weeks of storage. Fruit immersed in

AMW retained high O2 at week 0, but by week 1 had declined 6-8% in Navel oranges, and 3-4% in Valencia oranges. After the first week in storage, O2 level did not change.

Immersion of Navel and Valencia oranges in 45°C water for 30 mins, resulted in significantly reduced internal O2 and elevated CO2 during storage compared to fruit immersed in AMW, perhaps because the heat treatment melted and redistributed the natural cuticular waxes, restricting gas diffusion (Porat et al., 2003). Gas exchange into fruit depends on the rate of diffusion through fruit cuticle, pores, and applied coatings

(Banks et al., 1993) with coatings representing an additional barrier that can result in modification of the internal atmosphere in the fruit (Hagenmaier and Baker, 1994).

According to Ben-Yehoshla (1969), edible coatings may decrease physiological disorders such as CI via modification of the internal atmospheres. Petracek et al. (1998) found that CR- and PL-based coatings were more effective than SH-based coatings for control of citrus pitting, which is a physiological disorder caused by extreme internal MA.

They found that PL-coated grapefruit had variable internal gas concentrations that usually fell somewhere between CR-coated and NC fruit. Shellac-coated fruit experienced the most extreme internal Mas and pitting. Application of a SH-based coating to white grapefruit reduced the internal O2 to <4% and increased the CO2 to

>5% (Petracek et al., 1995).

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In the present study, after HWT (45oC for 30 min), the internal atmosphere of

HW+CR fruit during storage at ambient conditions was between that of the HW+PL and

HW+SH fruit. After HWT, coated oranges developed internal MAs associated with reduced fruit decay and increased postharvest quality during 3 weeks storage at ambient conditions. Carnauba-coated fruit performed better in terms of fruit peel appearance and decay control than the other coating treatments and the NC control, probably because the internal MA in the CR-coated fruit was most beneficial.

The internal MA in PL-coated fruit was less extreme than in CR-coated fruit likely resulting in PL being less effective than CR in maintaining fruit quality during ambient storage. In contrast, the internal MA in SH-coated fruit was more extreme than in CR- coated fruit, resulting in fruit injury. As shown in later sensory testing (see Chapter 5), most consumers preferred HW+CR fruit and disliked the HW+SH fruit due to off-flavors, reinforcing the conclusion that SH coating resulted in synthesis of fermentative metabolites in the fruit. These results are in agreement with Saftner (1999), who demonstrated that choice of the correct coating determines whether the resulting semipermeable barrier against O2 and CO2 diffusion results in formation of a beneficial internal MA, which can reduce fruit respiration, maintain postharvest quality, and extend storage life.

Valencia oranges contained 2-fold higher TCC and higher firmness value (COM,

PUN, OLC) compared to Navel oranges, but Navel oranges had higher sugar:acid ratio than Valencia oranges. It might be that there was more acid consumption in Navel oranges during storage due to higher respiration. The AMW+NC and HW+SH coated fruit had higher WL loss for both Navel and Valencia orange. The HW+SH coated fruit

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exhibited extreme internal MA and significant decay was noted during storage, suggesting injury. Thus, higher WL for NC fruit was probably due to insufficiency of the natural coating to restrict WL, while SH-coated fruit probably lost water due to peel injury and has been shown to inherently allow more water loss than carnauba-based coatings (Bai et al., 2002).

Late harvested Navel oranges had higher SSC compared to fruit harvested at early or mid harvest, and mid harvested Valencia oranges exhibited higher SSC compared to early and late harvested Valencia oranges. Variations in SSC during the harvest season are probably due to slightly different environmental conditions (i.e., climate, soil, cultural practices, etc.) of the different source grove locations and changing climatic conditions that affect the balance between photosynthesis and translocation of photosynthates to the fruit. Otherwise, there was little effect of variety or harvest found in this study with regard to the response of oranges to HWT and coatings.

This could be an important result because it suggests that the HWT of 45oC for 30 min coupled with CR coating that was shown in this study to improve the postharvest performance of oranges stored under ambient conditions may be widely applicable to other citrus varieties and other growing conditions.

Chapter Summary

The HW+CR treatment created and maintained a near-optimum internal atmosphere of 5-10% O2 plus 5-12% CO2; it also reduced WL and increased ATC during storage compared with other treatments. After 3 weeks of ambient storage, CR- coated fruit had 3-fold less WL compared to NC fruit. The HW+CR treatment also significantly improved fruit peel appearance (lightness, chroma and hue), and FF during

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storage. There were few significant temperature, coating, or storage effects on SSC,

ASC, and TP, but ATC was increased by HWT.

HW+SH resulted in more extreme internal atmosphere (<3% O2 and >15% CO2) along with elevated internal ethylene (2 ppm), which is indicative of stress, after 3 weeks of ambient storage.

Besides developing an intermediate internal MA compared to the other coatings,

CR-coated fruit also developed good external color, maintained good internal quality

(Brix, acid, and ratio), maintained the greatest firmness, and accumulated among the highest ATC after 3 weeks. Between the two cultivars, Valencia had higher TCC, were firmer, and had lower hue angle value.

Finally, this study demonstrated a potential commercial benefit of this HWT and

CR coating for maintaining citrus fruit quality in developing countries where refrigerated storage is unavailable.

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Table 4-1. Fruit and treatment conditions used for six studies evaluating the effects of a 30 minutes hot water dip and different edible wax coatings on quality and shelf life of Navel and Valencia orange during simulated ambient temperature storage. Parameter Study 1 Study 2 Study 3 Study 4 Study 5 Study 6 Fruit variety Washingt Washingt Washingt Valencia Valencia Valencia on Navel on Navel on Navel Harvesting October November January March 14, April 10, May 08, date 19, 2015 10, 2015 06, 2016 2016 2016 2016 Average 294 g 326 g 262 g 196 g 181 g 193 g fruit size Postharvest Yes No No No No No degreening Treatment 4 5 8 8 8 8 AMWz+N AMW+NC Factorial Factorial Factorial Factorial C, , HW+NC, AMW/HW AMW/HW AMW/HW AMW/HW HW+PL, HW+PL, , , , , HW+CR, HW+CR, NC/PL/C, NC/PL/C, NC/PL/C, NC/PL/C, HW+SH HW+SH R/SH R/SH R/SH R/SH zAMW = Ambient water (~25oC, 30 min); HW=Hot water (45oC, 30 min); NC=No coating; PL=Polyethylene coating; CR=Carnauba coating; SH=Shellac coating

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Table 4-2. Analysis of variance table for combined effect of cultivar, storage period, water temperature, and wax coatingz on soluble solids content (SSC), total titratable acidity (TTA), soluble solids content (SSC)/total titratable acidity (TTA), ascorbic acid content (ASC), and total carotenoids content (TCC)z. Main effect SSC TTA (% SSC/TTA ASC TCC (%)y citric acid) ratio (mg/100g) (mg/100g) Cultivar (C) ****x **** **** **** **** Storage (S) **** **** **** **** ** Temperature (T) * * ns ns ns Wax coating (W) **** *** *** ns *** Interaction: C x S * **** **** ** **** C x T ns *** ** ** ns C x W *** * ** ns ** S x T ns ns ns *** ns S x W ** ns ** ns ns T x W ns ns ns * * C x S x T * ns ns ns ns C x S x W ns ns ns ns * S x T x W ns ns * ns ns C x T x W ** ns ns ns ** C x S x T x W ns ns ns ns ns z Combined effect for 3rd harvest of both Navel and Valencia oranges; with/without HW at 45oC/25oC for 30 min immersion; with/without coating (NC=No coating, PL=Polyethylene, CR=Carnauba, SH=Shellac) y Fruit composition: Measured initially after treatment and at weekly intervals for 3 weeks storage. x ns, *, **, ***, ****, not significant, significant at P<0.05, P<0.01, P<0.001, or highly significant at P<0.0001 respectively according to Tukey test.

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Table 4-3. Analysis of variance table for combined effect of cultivar, storage period, water temperature, and wax coating on weight loss (WL), total phenolic compounds (TP), antioxidant capacity (ATC), lightness value (L*), chroma value (C), and hue angle (H) z. Main effect WL (% TP ATC (Trolox L* C H FW) y (mg/100g) µmol/100g) Cultivar (C) **** x **** **** **** **** **** Storage (S) **** ** **** ns * **** Temperature (T) **** ns **** **** **** **** Wax coating (W) **** ns * **** **** **** Interaction: C x S **** * ** ** ns ns C x T ns ns * **** **** * C x W **** ns ns **** **** **** S x T ** ns **** ns ns ns S x W **** ns * ns ** ns T x W **** ns * *** ns ** C x S x T ns ns **** ns ns ns C x S x W **** ns * ns ns ns S x T x W ns ns **** ns ns ns C x T x W **** ns * *** * ** C x S x T x W ** ns **** ns ns ns z Combined effect for 3rd harvest of both Navel and Valencia orange treated with/without HW at 45oC/25oC for 30 min immersion, and with/without coating (NC=No coating, PL=Polyethylene, CR=Carnauba, SH=Shellac) followed by 3 weeks storage at 25oC with 85%RH. y Physicochemical fruit quality: Measured initially after treatment and at weekly intervals for 3 weeks storage. x ns, *, **, ***, ****, not significant, significant at P<0.05, P<0.01, P<0.001, or highly significant at P<0.0001 respectively according to Tukey test.

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Table 4-4. Analysis of variance table for combined effect of cultivar, storage period, water temperature, and wax coating on compression (COM), puncture (PUN), and oleocellosis (OLC) z. Main effect COM (N) y PUN (N) OLC (N) Cultivar (C) **** x **** **** Storage (S) **** ** **** Temperature (T) **** **** **** Wax coating (W) **** **** **** Interaction: C x S **** ns **** C x T ns **** ns C x W ns **** **** S x T ** ** ns S x W * * **** T x W * ns ns C x S x T ** ns *** C x S x W **** *** ** S x T x W ** ns ns C x T x W ns ** ** C x S x T x W ns ns ns z Combined effect for 3rd harvest of both Navel and Valencia orange treated with/without HW at 45oC/25oC for 30 min immersion, and with/without coating (NC=No coating, PL=Polyethylene, CR=Carnauba, SH=Shellac) followed by 3 weeks storage at 25oC with 85%RH. y Chemical composition: Measured initially after treatment and at weekly intervals for 3 weeks storage. x ns, *, **, ***, ****, not significant, significant at P<0.05, P<0.01, P<0.001, or highly significant at P<0.0001 respectively according to Tukey test.

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Table 4-5. Combined effect of cultivar-storage interaction for 3rd harvest of Navel and Valencia orange on SSC, TTA, SSC/TTA, and ASC. Cultivar Storage SSC (%) TTA (% SSC/TTA ASC (week) citric acid) ratio (mg/100g) Navel 0 13.22 az 0.47 c 28.11 bc 53.20 b 1 13.60 a 0.45 c 30.02 ab 51.77 b 2 13.49 a 0.45 c 30.31 a 63.27 a 3 13.60 a 0.45 c 30.49 a 58.57 a

Valencia 0 10.79 c 0.53 b 20.89 e 45.40 c 1 11.10 c 0.59 a 19.06 e 45.77 c 2 11.60 b 0.46 c 25.82 d 53.03 b 3 11.61 b 0.44 c 26.90 cd 44.62 c zMeans within a column denoted by the same letter for SSC, TTA, SSC/TTA ratio, and ASC of each column for combined analysis of both cultivars for 3rd harvest and all treatment combinations do not differ significantly according to Tukey’s Test (P<0.05).

Table 4-6. Combined effect of cultivar-storage interaction for 3rd harvest of Navel and Valencia orange on TCC, WL, TP, and ATC. Cultivar Storage TCC WL (% TP (mg/100g) ATC (Trolox (week) (mg/100g) FW) µmol/100g) Navel 0 2.59 cz - 37.94 c 1210.00 de 1 2.54 c 2.27 d 40.31 c 1649.30 a 2 2.13 d 3.98 c 47.88 c 1396.70 bc 3 2.52 cd 5.12 b 47.13 c 1504.50 ab

Valencia 0 4.37 b - 176.77 b 1216.70 de 1 4.63 ab 1.71 e 199.94 ab 1384.10 bc 2 4.77 ab 5.12 b 196.55 ab 1137.70 e 3 4.99 a 6.25 a 228.90 a 1319.70 cd zMeans within a column denoted by the same letter for TCC, WT, TP, and ATC of each column for combined analysis of both cultivars for 3rd harvest and all treatment combinations do not differ significantly according to Tukey’s Test (P<0.05).

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Table 4-7. Combined effect of cultivar-storage interaction for 3rd harvest of Navel and Valencia orange on L*, COM, and OLC. Cultivar Storage (week) L* COM (N) OLC (N) Navel 0 66.98 az 121.29 a 47.17 d 1 66.80 a 95.66 cd 43.89 e 2 66.63 a 80.51 e 38.98 f 3 66.16 a 91.88 d 34.23 g

Valencia 0 62.20 b 112.90 ab 54.22 b 1 62.57 b 114.97 a 56.98 a 2 62.72 b 103.96 bc 50.82 c 3 62.95 b 91.03 d 50.43 c zMeans within a column denoted by the same letter for L*, COM, and OLC of each column for combined analysis of both cultivars for 3rd harvest and all treatment combinations do not differ significantly according to Tukey’s Test (P<0.05).

Table 4-8. Combined effect of cultivar-treatment temperature interaction for 3rd harvest of Navel and Valencia orange on TTA, SSC/TTA ratio, ASC, and ATC. Cultivar Water TTA (% SSC/TTA ASC ATC (Trolox temperature (oC) citric acid) ratio (mg/100g) µmol/100g) Navel 25 0.45 c z 30.29 a 55.11 b 1287.00 b 45 0.46 bc 29.17 a 58.30 a 1593.20 a

Valencia 25 0.49 b 22.65 b 48.28 c 1173.30 c 45 0.52 a 22.69 b 46.13 c 1355.80 b zMeans within a column denoted by the same letter for TTA, SSC/TTA, ASC, and ATC of each column for combined analysis of both cultivars for 3rd harvest and all treatment combinations dipped into 45oC or 25oC water for 30 min do not differ significantly according to Tukey’s Test (P<0.05).

Table 4-9. Combined effect of cultivar-treatment temperature interaction for 3rd harvest of Navel and Valencia orange on L*, C, H, and PUN. Cultivar Water temperature (oC) L* C H PUN (N) Navel 25 65.24 bz 66.94 b 74.06 c 26.05 b 45 68.04 a 71.17 a 74.56 c 23.11 c

Valencia 25 63.02 c 60.29 c 79.83 b 31.12 a 45 62.20 d 58.22 d 81.71 a 30.58 a zMeans within a column denoted by the same letter for L*, C, H, and PUN of each column for combined analysis of both cultivars for 3rd harvest and all treatment combinations dipped into 45oC or 25oC water for 30 min do not differ significantly according to Tukey’s Test (P<0.05).

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Table 4-10. Combined effect of cultivar-surface coating interaction for 3rd harvest of Navel and Valencia orange on SSC, TTA, SSC/TTA, TCC, and WL. Cultivar Wax SSC (%) TTA (% SSC/TTA TCC WL (% FW) coating citric acid) ratio (mg/100g) Navel NC 13.92 az 0.47 bc 29.73 a 2.47 d 3.27 c PL 13.85 a 0.45 c 30.81 a 2.46 d 2.51 e CR 13.21 b 0.45 c 29.63 a 2.41 d 1.66 f SH 12.93 b 0.45 c 28.76 a 2.44 d 3.93 b

Valencia NC 11.16 c 0.52 a 21.88 c 4.90 ab 4.53 a PL 11.49 c 0.51 ab 23.42 bc 5.00 a 2.94 d CR 11.12 c 0.46 c 24.99 b 4.33 c 1.62 f SH 11.33 c 0.52 a 22.38 c 4.53 bc 4.08 b zMeans within a column denoted by the same letter for SSC, TTA, SSC/TTA, TCC, and WL of each column for combined analysis of both cultivars for 3rd harvest and all treatment combinations and wax coatings do not differ significantly according to Tukey’s Test (P<0.05).

Table 4-11. Combined effect of cultivar-surface coating interaction for 3rd harvest of Navel and Valencia orange on L*, C, H, PUN, and OLC. Cultivar Wax coating L* C H PUN (N) OLC (N) Navel NC 58.37 ez 58.45 d 72.58 e 24.61 e 43.57 de PL 69.74 a 73.85 a 74.39 d 25.42 de 41.64 e CR 68.51 b 71.66 b 75.11 d 21.49 f 34.20 f SH 69.94 a 72.27 ab 75.15 d 26.80 d 44.86 d

Valencia NC 59.18 e 54.73 e 80.45 b 31.37 b 51.09 c PL 64.96 c 63.90 c 78.26 c 28.78 c 54.05 ab CR 63.29 d 59.19 d 83.42 a 29.63 bc 55.16 a SH 63.02 d 59.20 d 80.94 b 33.62 a 52.15 bc zMeans within a column denoted by the same letter for L, C, H, PUN, and OLC of each column for combined analysis of both cultivars for 3rd harvest and all treatments combination and wax coatings after water treatment do not differ significantly according to Tukey’s Test (P<0.05).

163

Table 4-12. Combined effect of storage-water temperature interaction for 3rd harvest of Navel and Valencia orange on ASC, ATC, WL, COM, and OLC. Storage Water temp. ASC ATC (Trolox WL (% COM (N) PUN (N) (wk) (oC) (mg/100g) µmol/100g) FW) 0 25 48.40 cdz 1146.50 de - 127.54 a 27.28 bcd 45 50.20 cd 1280.20 cd - 106.65 b 26.98 bcd

1 25 51.70 bc 1340.00 bc 2.06 e 107.92 b 28.78 ab 45 45.84 d 1693.40 a 1.94 e 102.71 bc 27.81 bcd

2 25 56.77 ab 1072.90 e 4.74 c 96.75 cd 28.28 abc 45 59.53 a 1461.50 b 4.45 d 87.72 d 26.17 d

3 25 49.90 cd 1361.30 bc 5.84 a 94.24 cd 29.98 a 45 53.29 bc 1463.00 b 5.53 b 88.67 d 26.43 cd zMeans within a column denoted by the same letter for ASC, ATC, WT, COM, and PUN of each column for combined analysis of both cultivars for 3rd harvest and all treatment combinations do not differ significantly according to Tukey’s Test (P<0.05).

Table 4-13. Combined effect of storage-surface coating interaction for 3rd harvest of Navel and Valencia orange on SSC, SSC/TTA, ATC, and WL. Storage Wax SSC (%) SSC/TTA ATC (Trolox WL (% (wk) coating ratio µmol/100g) FW) 0 NC 11.84 ez 23.37 d 1316.30 bcd - PL 12.34 bcde 25.15 cd 1204.40 de - CR 11.85 e 25.48 cd 1283.90 bcde - SH 11.98 de 24.01 d 1048.90 e -

1 NC 12.97 ab 25.82 cd 1675.20 a 2.34 f PL 12.48 abcde 24.85 cd 1523.50 ab 1.81 g CR 11.98 de 23.98 d 1396.70 bcd 1.13 h SH 11.97 de 23.53 d 1471.40 abc 2.68 ef

2 NC 12.53 abcde 26.32 bcd 1242.90 cde 5.91 b PL 12.66 abcd 28.08 abc 1249.50 cde 4.08 d CR 12.53 abcde 29.52 ab 1267.10 bcde 2.45 f SH 12.43 abcde 28.33 abc 1309.90 bcde 5.94 b

3 NC 12.83 abc 27.71 abc 1434.60 abcd 7.36 a PL 13.21 a 30.38 a 1394.90 bcd 5.01 c CR 12.28 bcde 30.27 a 1449.00 abcd 2.98 e SH 12.12 cde 26.41 bcd 1370.00 bcd 7.40 a zMeans within a column denoted by the same letter for SSC, SSC/TTA, ATC, and WL of each column for combined analysis of both cultivars for 3rd harvest and all treatment combinations do not differ significantly according to Tukey’s Test (P<0.05).

164

Table 4-14. Combined effect of storage-surface coating interaction for 3rd harvest of Navel and Valencia orange on C, COM, PUN, and OLC. Storage (wk) Wax coating C COM (N) PUN (N) OLC (N) 0 NC 55.29 fz 109.82 bcd 26.51 efg 51.47 a PL 68.25 abc 123.11 ab 26.75 efg 51.35 a CR 65.25 d 131.76 a 26.08 fg 46.34 b SH 65.92 cd 103.69 de 29.18 abcde 53.60 a

1 NC 55.89 ef 96.71 def 27.56 cdefg 51.12 a PL 68.82 ab 105.91 cde 28.27 abcdef 51.19 a CR 65.62 cd 120.02 abc 26.31 efg 46.77 b SH 66.30 bcd 98.61 def 31.04 a 52.65 a

2 NC 57.20 ef 77.59 g 27.75 bcdefg 44.58 bcd PL 69.21 a 97.61 def 26.47 efg 45.25 bc CR 66.06 cd 102.10 de 24.78 g 43.49 bcd SH 66.67 abcd 91.64 efg 29.90 abcd 46.29 b

3 NC 57.98 e 78.74 g 30.13 abc 42.13 cd PL 69.22 a 91.88 efg 26.91 defg 43.58 bcd CR 64.77 d 110.95 bcd 25.08 g 42.11 cd SH 64.06 d 84.24 fg 30.72 ab 41.50 d zMeans within a column denoted by the same letter for C, COM, PUN, and OLC of each column for combined analysis of both cultivars for 3rd harvest and all treatment combinations do not differ significantly according to Tukey’s Test (P<0.05).

Table 4-15. Combined effect of water temperature- surface coating interaction for 3rd harvest of Navel and Valencia orange on ASC, TCC, and WL. Water temperature Wax ASC TCC WL (% FW) (oC) coating (mg/100g) (mg/100g) 25 NC 54.38 a z 3.61 abc 4.11 a PL 50.08 c 3.87 a 2.74 c CR 50.69 bc 3.41 bc 1.65 d SH 51.62 abc 3.35 c 4.14 a

45 NC 50.84 bc 3.76 ab 3.69 b PL 53.09 abc 3.58 abc 2.71 c CR 54.06 ab 3.33 c 1.63 d SH 50.86 bc 3.63 abc 3.86 b zMeans within a column denoted by the same letter for ASC, TCC, and WL of each column for combined analysis of both cultivars for 3rd harvest and all treatment combinations do not differ significantly according to Tukey’s Test (P<0.05).

165

Table 4-16. Combined effect of water temperature-surface coating interaction for 3rd harvest of Navel and Valencia orange on ATC, LG, HU, and COM. Water Wax ATC (Trolox L* H COM (N) temperature (oC) coating µmol/100g) 25 NC 1228.60 d z 58.53 d 76.09 d 92.79 bc PL 1232.30 d 67.14 ab 76.48 cd 113.48 a CR 1268.40 cd 65.40 c 78.16 bc 119.83 a SH 1191.30 d 65.45 c 77.04 cd 100.35 b

45 NC 1605.80 a 59.02 d 76.94 cd 88.64 c PL 1453.80 ab 67.56 a 76.17 d 95.77 bc CR 1430.00 bc 66.40 b 80.37 a 112.59 a SH 1408.50 bc 67.51 a 79.05 ab 88.74 c zMeans within a column denoted by the same letter for ATC, L*, H, and COM of each column for combined analysis of both cultivars for 3rd harvest and all treatment combinations do not differ significantly according to Tukey’s Test (P<0.05).

166

Table 4-17. Combined effect of cultivar-storage-water temperature interaction for 3rd harvest of Navel and Valencia orange on SSC, ATC, COM, and OLC. Cultivar Storage Water SSC (%) ATC (Trolox COM (N) OLC (N) (week) temp. (oC) µmol/100g) Navel 0 25 13.40 az 1064.9 ef 137.13 a 49.32 d 45 13.04 a 1355.1 cd 105.46 bcdef 45.02 ef

1 25 13.66 a 1556.1 abc 97.16 cdefg 45.71 e 45 13.53 a 1742.4 a 94.15 efg 42.06 fg

2 25 13.43 a 1145.8 def 82.96 gh 38.83 g 45 13.55 a 1647.7 a 78.06 h 39.14 g

3 25 13.72 a 1381.5 bcd 92.85 efgh 34.37 h 45 13.49 a 1627.6 ab 90.91 fgh 34.09 h

Valencia 0 25 10.63 d 1228.2 def 117.96 b 55.17 ab 45 10.94 d 1205.3 def 107.83 bcde 53.26 bc

1 25 11.08 cd 1123.8 def 118.67 b 57.57 a 45 11.13 cd 1644.4 a 111.26 bc 56.39 ab

2 25 11.83 bc 1000.0 f 110.55 bcd 53.41 bc 45 11.38 bcd 1275.3 de 97.38 cdefg 48.24 de

3 25 11.93 b 1341.1 cd 95.62 defg 50.90 cd 45 11.28 bcd 1298.2 def 86.43 gh 49.96 cd zMeans within a column denoted by the same letter for SSC, ATC, COM, and OLC of each column for combined analysis of both cultivars for 3rd harvest and all treatment combinations do not differ significantly according to Tukey’s Test (P<0.05).

167

Table 4-18. Combined effect of cultivar-storage-surface coating interaction for 3rd harvest of Navel and Valencia orange on TCC, ATC, and WL. Cultivar Storage Wax TCC ATC (Trolox WL (% FW) (week) coating (mg/100g) µmol/100g) Navel 0 NC 2.64 dz 1377.43 bcdef - PL 2.68 d 1112.64 ef - CR 2.61 d 1234.38 def - SH 2.45 d 1115.54 ef -

1 NC 2.45 d 1811.90 a 2.56 gh PL 2.42 d 1771.07 ab 2.08 h CR 2.60 d 1478.47 abcde 1.40 i SH 2.68 d 1535.57 abcd 3.03 fg

2 NC 2.34 d 1352.03 cdef 4.56 e PL 2.13 d 1378.57 bcdef 3.53 f CR 2.03 d 1445.27 abcde 2.33 h SH 2.04 d 1410.93 abcde 5.48 d

3 NC 2.45 d 1688.09 abc 5.95 cd PL 2.62 d 1448.27 abcde 4.43 e CR 2.42 d 1458.26 abcde 2.91 g SH 2.58 d 1423.47abcde 7.20 b

Valencia 0 NC 4.49 bc 1255.26 def - PE 4.36 bc 1296.15 cdef - CR 4.16 c 1333.39 cdef - SH 4.47 bc 982.17 f -

1 NC 4.69 abc 1538.46 abcd 2.13 h PL 5.22 ab 1275.85 def 1.54 i CR 4.31 bc 1314.91 cdef 0.86 j SH 4.31 bc 1407.23 abcde 2.33 h

2 NC 4.77 abc 1133.67 def 7.25 b PL 5.22 ab 1120.41 ef 4.64 e CR 4.55 bc 1088.92 ef 2.57 gh SH 4.53 bc 1207.62 def 6.40 c

3 NC 5.66 a 1181.02 def 8.76 a PL 5.18 ab 1341.49 cdef 5.59 d CR 4.29 bc 1439.80 abcde 3.06 fg SH 4.82 abc 1316.55 cdef 7.59 b zMeans within a column denoted by the same letter for TCC, ATC, and WL of each column for combined analysis of both cultivars for 3rd harvest and all treatment combinations do not differ significantly according to Tukey’s Test (P<0.05).

168

Table 4-19. Combined effect of cultivar-storage-surface coating interaction for 3rd harvest of Navel and Valencia orange on COM, PUN, and OLC. Cultivar Storage Wax COM (N) PUN (N) OLC (N) (week) coating Navel 0 NC 109.80 bcdef z 22.99 jklmn 50.03 defghi PL 127.32 abc 25.58 fghijklm 46.60 hijk CR 147.82 a 20.67 n 37.69 lmn SH 100.25 defghi 27.29 cdefghij 54.35 abcd

1 NC 83.87 ghij 25.62 fghijkl 45.47 ijk PL 97.89 defghi 27.71 cdefghij 46.32 hijk CR 109.10 bcdef 22.50 klmn 35.40 no SH 91.77 efghi 26.63 48.36 efghi deafghijkl 2 NC 63.67 j 24.58 hijklmn 42.22 jkl PL 89.63 fghi 24.10 ijklmn 38.14 lmn CR 76.30 ij 20.86 mn 33.50 no SH 92.45 efghi 27.06 42.08 klm cdefghijk 3 NC 76.26 ij 25.25 ghijklmn 36.54 mn PL 93.24 efghi 24.32 hijklmn 35.50 no CR 113.82 bcdef 21.93 lmn 30.22 o SH 84.19 ghij 26.25 efghijkl 34.67 no

Valencia 0 NC 109.84 bcdef 30.04 bcdef 52.91 abcdef PL 118.90 bcd 27.93 cdefghi 56.10 ab CR 115.71 bcde 31.50 abc 55.00 abcd SH 107.14 bcdefg 31.08 abcd 52.85 abcdef

1 NC 109.54 bcdef 29.51 bcdefg 56.77 ab PL 113.92 bcdef 28.85 bcdefgh 56.06 abc CR 130.95 ab 30.13 bcdef 58.15 a SH 105.46 cdefgh 35.45 a 56.94 ab

2 NC 91.52 efghi 30.92 abcde 46.95 ghijk PL 105.59 cdefgh 28.85 bcdefgh 52.36 bcdefg CR 127.91 abc 28.69 bcdefghi 53.49 abcde SH 90.84 fghi 32.75 ab 50.50 cdefghi

3 NC 81.23 hij 35.00 a 47.72 fghij PL 90.51fghi 29.50 bcdefg 51.67 bcdefg CR 108.08 bcdefg 28.22 bcdefghi 54.01 abcd SH 84.29 ghij 35.19 a 48.33 efghi z Means within a column denoted by the same letter for COM, PUN, OLC of each column for combined analysis of both cultivars for 3rd harvest and all treatment combinations do not differ significantly according to Tukey’s Test (P<0.05).

169

Table 4-20. Combined effect of water temperature-storage-surface coating interaction for 3rd harvest of Navel and Valencia orange on SSC/TTA, ATC, and COM. Water Storage Wax SSC/TTA ratio ATC (Trolox COM (N) temperature (week) coating µmol/100g) (oC) 25 0 NC 22.00 g z 1189.53 cdefg 116.77 bcd PL 25.88 cdefg 1187.85 cdefg 143.96 a CR 26.46 abcdefg 1224.70 bcdefg 138.61 ab SH 23.90 defg 984.03 g 110.84 cdefg

1 NC 26.26 bcdefg 1221.31 bcdefg 95.58 defghijk PL 25.02 defg 1426.83 bcde 114.44 bcde CR 22.31 fg 1318.96 bcdefg 114.97 bcde SH 23.27 fg 1392.71 bcdefg 106.68 cdefgh

2 NC 26.46 abcdefg 1237.90 bcdefg 76.86 k PL 27.19 abcdefg 1026.68 efg 101.42 cdefghij CR 28.24 abcde 1033.17 efg 111.22 cdef SH 29.00 abcd 993.82 fg 97.51 defghijk

3 NC 28.29 abcde 1265.81 bcdefg 81.94 ijk PL 31.80 a 1287.98 bcdefg 94.09 defghijk CR 31.55 ab 1496.75 bcd 114.52 bcde SH 26.25 bcdefg 1394.51 bcdef 86.40 ghijk

45 0 NC 24.75 defg 1443.17 bcd 102.87 cdefghi PL 24.41 defg 1220.94 bcdefg 102.26 cdefghi CR 24.50 defg 1343.07 bcdefg 124.92 abc SH 24.12 defg 1113.69 defg 96.55 defghijk

1 NC 25.38 cdefg 2129.05 a 97.83 defghijk PL 24.68 defg 1620.09 b 97.37 defghijk CR 25.64 cdefg 1474.41 bcd 125.07 abc SH 23.79 defg 1550.09bc 90.55 efghijk

2 NC 26.18 bcdefg 1247.80 bcdefg 78.32 jk PL 28.97 abcd 1472.30 bcd 93.79 efghijk CR 30.80 abc 1501.01 bcd 92.99 efghijk SH 27.67 abcdef 1624.78 b 85.78 hijk

3 NC 27.13 abcdefg 1603.30 b 75.55 k PE 28.97 abcd 1501.78 bcd 89.66 fghijk CR 28.99 abcd 1401.31bcdef 107.38 cdefgh SH 26.58 abcdefg 1345.50 bcdefg 82.08 ijk zMeans within a column denoted by the same letter for SSC/TTA, ATC, and COM of each column for combined analysis of both cultivars for 3rd harvest and all treatment combinations do not differ significantly according to Tukey’s Test (P<0.05).

170

Table 4-21. Combined effect of cultivar-water temperature-surface coating interaction for 3rd harvest of Navel and Valencia orange on SSC, TCC, ATC, and WL. Cultivar Water temp. (oC) Wax Coating SSC (%) TCC (mg/100g) ATC (Trolox µmol/100g) WL (% FW) Navel 25 NC 14.10 a z 2.36 d 1282.2 cdef 3.32 cd PL 13.75 ab 2.45 d 1270.4 cdef 2.46 g CR 13.18 bc 2.46 d 1302.9 cdef 1.66 h SH 13.18 bc 2.50 d 1292.7 cdef 4.20 b

45 NC 13.74 ab 2.58 d 1832.5 a 3.22 d PL 13.96 ab 2.48 d 1584.9 ab 2.46 fg CR 13.25 bc 2.36 d 1505.3 bc 1.65 h SH 12.67 c 2.38 d 1450.1 bcd 3.65 c

Valencia 25 NC 11.24 d 4.87 ab 1175.0 ef 4.90 a PL 11.80 d 5.30 a 1194.3 def 3.03 de CR 11.19 d 4.36 bc 1233.9 def 1.64 h SH 11.23 d 4.20 c 1089.8 f 4.08 b

45 NC 11.08 d 4.94 ab 1379.2 bcde 4.17 b PL 11.18 d 4.69 abc 1322.6 cdef 2.86 ef CR 11.04 d 4.30 bc 1354.6 bcde 1.61 h SH 11.42 d 4.87 ab 1367.0 bcde 4.08 b zMeans within a column denoted by the same letter for SSC, TCC, ATC, and WL of each column for combined analysis of both cultivars for 3rd harvest and all treatment combinations do not differ significantly according to Tukey’s Test (P<0.05).

171

Table 4-22. Combined effect of cultivar-water temperature-surface coating interaction for 3rd harvest of Navel and Valencia orange on L*, C, H, PUN, and OLC. Cultivar Water temp. (oC) Wax Coating L* C H PUN (N) OLC (N) Navel 25 NC 57.18 gz 56.16 g 72.85 hi 25.31 efg 45.33 e PL 69.31 b 72.62 a 75.19 fghi 27.27 def 44.15 e CR 66.96 c 69.38 b 74.59 ghi 22.31 gh 33.98 g SH 67.53 c 69.61 b 73.61 hi 29.31 cd 44.78 e

45 NC 58.48 fg 60.73 de 72.31 i 23.90 g 41.81 ef PL 70.18 b 75.08 a 73.59 hi 23.58 gh 39.13 f CR 70.06 b 73.93 a 75.64 fgh 20.67 h 34.42 g SH 72.35 a 74.93 a 76.69 efg 24.30 fg 44.94 e

Valencia 45 NC 58.48 fg 53.19 h 81.57 bc 30.37 bc 49.87 d PL 64.94 d 63.31 cd 78.74 cde 29.49 bcd 53.36 abcd CR 62.74 e 57.83 fg 85.11 a 29.08 cd 54.06 abc SH 62.66 e 58.54 efg 81.40 bc 33.40 a 50.56 cd

25 NC 59.87 f 56.27 g 79.33 bcde 32.37 ab 52.31 bcd PL 64.98 d 64.49 c 77.77 def 28.08 cde 54.73 ab CR 63.84 de 60.55 e 81.74 b 30.19 bcd 56.27 a SH 63.38 e 59.86 ef 80.47 bcd 33.83 a 53.75 abc zMeans within a column denoted by the same letter for L*, C, H, PUN, and OLC of each column for combined analysis of both cultivars for 3rd harvest and all treatment combinations do not differ significantly according to Tukey’s Test (P<0.05).

172

Table 4-23. Effect of cultivar – water temperature – wax coating – storage time interaction for 3rd harvest of Navel and Valencia orange on weight loss (WL). Cultivar Water temp.(oC) WL (% FW) Wax coating Week 0 Week 1 Week 2 Week 3 Navel 25 NC - 2.61 gf z 4.65 c 6.01 b PL - 2.03 h 3.47 d 4.33 c CR - 1.37 i 2.33 gh 2.93 ef SH - 3.11 de 5.87 b 7.84 a

45 NC - 2.51 de 4.48 b 5.89 a PL - 2.14 e 3.59 c 4.52 b CR - 1.42 f 2.32 de 2.88 d SH - 2.94 cd 5.09 b 6.57 a

Valencia 25 NC - 2.39 g 7.82 b 9.38 a PL - 1.63 h 4.76 e 5.71 d CR - 0.92 i 2.61 fg 3.02 f SH - 2.39 g 6.41 c 7.54 b

45 NC - 1.86 fg 6.67 b 8.15 a PL - 1.44 gh 4.51 d 5.47 c CR - 0.80 hi 2.53 ef 3.10 e SH - 2.28 efg 6.38 bc 7.65 a zMeans within a column denoted by the same letter for WL of each column or each row for specific temperature for combined analysis of both cultivars for 3rd harvest and all treatment combinations do not differ significantly according to Tukey’s Test (P<0.05).

173

Table 4-24. Effect of cultivar– water temperature – wax coating – storage time interaction for 3rd harvest of Navel and Valencia orange on antioxidant capacity (ATC). Cultivar Water Wax ATC (Trolox µmol/100g) temp. coating Week 0 Week 1 Week 2 Week 3 (oC) Navel 25 NC 1004.11 dez 1334.69 bcd 1484.68 abc 1305.49 bcde PL 1025.29 de 1767.92 a 976.49 e 1311.73 bcd CR 1257.16 cde 1484.69 abc 1065.90 de 1403.76 bc SH 972.93 e 1637.01 ab 1055.95 de 1504.88 abc

45 NC 1750.76 bcd 2289.11 a 1219.39 e 2070.69 ab PL 1199.99 e 1774.21 abcd 1780.65 abcd 1584.81 bcde CR 1211.60 e 1472.24 cde 1824.64 abc 1512.76 cde SH 1258.15 de 1434.12 cde 1765.92 abcd 1342.05 cde

Valencia 25 NC 1374.95 ab 1107.93 ab 991.12 ab 1226.13 ab PL 1350.40 ab 1085.73 ab 1076.87 ab 1264.23 ab CR 1192.24 ab 1153.23 ab 1000.45 ab 1589.75 a SH 995.12 ab 1148.40 ab 931.69 b 1284.15 ab

45 NC 1135.57 bc 1968.99 a 1276.21 bc 1135.91 bc PL 1241.89 bc 1465.96 abc 1163.95 bc 1418.75 abc CR 1474.53 abc 1476.59 abc 1177.39 bc 1289.85 bc SH 969.23 c 1666.07 ab 1483.64 abc 1348.95 abc zMeans within a column denoted by the same letter for WL of each column or each row for specific temperature for combined analysis of both cultivars for 3rd harvest and all treatment combinations do not differ significantly according to Tukey’s Test (P<0.05).

174

Table 4-25. Analysis of variance table for combined effect of cultivar, harvest, storage period, and wax coating on SSC, TTA, SSC/TTA, ASC, and TCC during 3 weeks of storage at 25oC with 85%RH z. Main effect SSC (%) y TTA (% citric SSC/TTA ratio ASC (mg/100g) TCC (mg/100g) acid) Cultivar (C) **** x **** **** **** **** Harvest (H) **** **** **** **** ** Storage (S) **** **** **** **** ns Temperature-coating (T) ** **** **** **** ns Interaction: C x H *** **** **** **** **** C x S *** **** ** * *** C x T ** ns ns ns ns H x S *** **** **** ** *** H x T ** * ns *** * S x T ns ns ns ns ns C x H x S *** **** **** * *** C x H x T ** * * **** * H x S x T ns **** ** ns ns C x S x T ** * * ** ns C x H x S x T ** * ns ** ns z All 3 harvests of both cultivars for four different treatments AMW+NC, HW+PL, HW+CR, HW+SH); AMW=25oC for 30 min, HW = 45oC for 30 min; NC=No coating, PL=Polyethylene, CR=Carnauba, SH=Shellac. y Fruit composition: Initially after treatment and at weekly intervals for 3 weeks storage. x ns, *, **, ***, ****, not significant, significant at P<0.05, P<0.01, P<0.001, or highly significant at P<0.0001 respectively according to Tukey test.

175

Table 4-26. Analysis of variance table for combined effect of cultivar, harvest, storage period, and wax coating on WL, TP, ATC, L*, C, and H angle value during 3 weeks of storage at 25oC with 85%RHz. Main effect WL (% FW)y TP (mg/100g) ATC (Trolox µmol/100g) L* C H Cultivar (C) **** x **** **** **** **** **** Harvest (H) **** *** **** **** **** **** Storage (S) **** **** ** *** **** **** Temperature-coating (T) **** ns ** **** **** **** Interaction: C x H **** ns **** **** **** **** C x S **** ** ns **** **** ns C x T **** ns * * *** **** H x S **** * **** **** *** **** H x T **** ns * **** **** **** S x T **** ns ns ns **** ns C x H x S **** ns **** **** **** **** C x H x T **** ns ns **** **** **** H x S x T **** ns ns ns ns ns C x S x T **** ns ns ns ns ns C x H x S x T ** ns ns ns ns ns z All 3 harvests (Early, mid, late) of both cultivars for four different treatments (AMW+NC, HW+PL, HW+CR, HW+SH); AMW=25oC for 30 min, HW = 45oC for 30 min; NC=No coating, PL=Polyethylene, CR=Carnauba, SH=Shellac. y Fruit physicochemical quality: Initially after treatment and at weekly interval for 3 weeks storage. x ns, *, **, ***, ****, not significant, significant at P<0.05, P<0.01, P<0.001, or highly significant at P<0.0001 respectively according to Tukey test.

176

Table 4-27. Analysis of variance table for combined effect of cultivar, harvest, storage period, and wax coating on COM force, PUN force, and OLC force during 3- week of storage at 25oC with 85%RHz. Main effect COM (N)y PUN (N) OLC (N) Cultivar (C) ****x **** **** Harvest (H) **** **** **** Storage (S) **** **** **** Temperature-coating (T) **** **** * Interaction: C x H **** **** **** C x S ns ns **** C x T **** ns **** H x S * *** **** H x T * ** **** S x T *** ** **** C x H x S **** ns **** C x H x T ns * **** H x S x T * * * C x S x T ** ns ** C x H x S x T *** * **** z All 3 harvests of both cultivars for four different treatments (AMW+NC, HW+PL, HW+CR, HW+SH); AMW=25oC for 30 min, HW = 45oC for 30 min; NC=No coating, PL=Polyethylene, CR=Carnauba, SH=Shellac. y Fruit composition: Initially after treatment and at weekly intervals for 3 weeks storage. x ns, *, **, ***, ****, not significant, significant at P<0.05, P<0.01, P<0.001, or highly significant at P<0.0001 respectively according to Tukey test.

177

Table 4-28. Effect of cultivar x Harvest x Treatment x Storage time interaction for early, mid & late harvest of Navel and Valencia orange on total soluble solids content (SSC) z. Cultivar Harvest Treatment SSC (%) Week 0y Week 1 Week 2 Week 3 Navel H1 AMW+NC 9.40 bx 10.20 ab 10.47 ab 10.80 a HW+PL 9.70 b 10.27 ab 10.20 ab 10.23 ab HW+CR 9.73 ab 9.90 ab 10.17 ab 10.30 ab HW+SH 9.60 b 10.13 ab 10.03 ab 10.10 ab

H2 AMW+NC 10.77 abc 10.50 bc 10.97 ab 11.20 a HW+PL 10.67 abc 11.03 ab 11.00 ab 10.73 abc HW+CR 10.47 bc 10.40 bc 11.03 ab 10.67 abc HW+SH 10.17 c 10.63 abc 10.47 bc 10.50 bc

H3 AMW+NC 13.57 abcde 14.70 a 13.77abcde 14.37 ab HW+PL 13.37 abcde 14.13 abcd 14.17 abc 14.17 abc HW+CR 13.40 13.53 13.20 12.87 cde abcde abcde bcde HW+SH 12.77 cde 12.70 de 12.60 e 12.60 e

Valencia H1 AMW+NC 12.27 abc 11.10 cd 13.73 a 13.83 a HW+PL 9.30 d 11.70 bc 13.57 ab 13.63 ab HW+CR 11.03 cd 11.10 cd 13.33 ab 13.43 ab HW+SH 11.70 bc 11.90 abc 13.23 ab 13.30 ab

H2 AMW+NC 13.90 a 11.00 c 13.27 abc 11.40 bc HW+PL 13.43 ab 12.07 abc 12.93 abc 12.57 abc HW+CR 13.30 ab 12.47 abc 13.13 abc 12.80 abc HW+SH 13.30 ab 11.40 bc 13.10 abc 11.80 abc

H3 AMW+NC 9.80 a 11.80 ab 11.40 ab 11.97 a HW+PL 11.13 ab 10.57 ab 11.07 ab 11.97 a HW+CR 10.60 ab 11.20 ab 11.57 ab 10.80 ab HW+SH 11.20 ab 11.27 ab 11.80 ab 11.40 ab z All three harvests of both cultivars for four different treatments (AMW+NC, HW+PL, HW+CR, HW+SH); AMW=25oC for 30 min, HW = 45oC for 30 min; NC=No coating, PL=Polyethylene, CR=Carnauba, SH=Shellac. y Weight loss measured initially after treatment and at weekly intervals for 3 weeks storage. x Means within a column denoted by the same letter of each column or each row for specific temperature combined analysis do not differ significantly according to Tukey’s Test (P<0.05).

178

Table 4-29. Effect of cultivar x Harvest x Treatment x Storage time interaction for early, mid & late harvest of Navel and Valencia orange on total titratable acid content (TTA)z. Cultivar Harvest Treatment TTA (% citric acid) Week 0y Week 1 Week 2 Week 3 Navel H1 AMW+NC 0.53 abx 0.56 a 0.51 abc 0.43 cd HW+PL 0.48 abcd 0.46 bcd 0.47 abcd 0.46 bcd HW+CR 0.49 abcd 0.47 abcd 0.49 abcd 0.43 bcd HW+SH 0.57 a 0.47 abcd 0.47 abcd 0.41 abcd

H2 AMW+NC 0.40 ab 0.42 a 0.37 ab 0.36 ab HW+PL 0.35 ab 0.34 ab 0.33 ab 0.34 ab HW+CR 0.35 ab 0.35 ab 0.32 b 0.33 ab HW+SH 0.38 ab 0.36 ab 0.35 ab 0.33 ab

H3 AMW+NC 0.48 ab 0.44 ab 0.50 a 0.45 ab HW+PL 0.47 ab 0.47 ab 0.44 ab 0.48 ab HW+CR 0.44 ab 0.43 ab 0.43 b 0.42 b HW+SH 0.47 ab 0.46 ab 0.43 b 0.44 ab

Valencia H1 AMW+NC 1.14 abc 1.10 abcd 0.89 cdef 0.87 def HW+PL 1.14 ab 0.85 def 0.92 bcdef 0.91 bcdef HW+CR 1.18 a 0.76 f 0.88 def 0.73 f HW+SH 1.15 ab 1.01 abcde 1.02 abcde 0.82 ef

H2 AMW+NC 0.65 bc 0.72 abc 0.68 bc 0.82 a HW+PL 0.60 c 0.70 abc 0.72 abc 0.66 bc HW+CR 0.64 bc 0.75 ab 0.67 bc 0.76 ab HW+SH 0.65 bc 0.71 abc 0.73 ab 0.76 ab

H3 AMW+NC 0.64 a 0.60 ab 0.48 bcdef 0.48 bcdef HW+PL 0.53 abcd 0.58 abc 0.43 def 0.43 def HW+CR 0.51 abcdef 0.55 abcd 0.38 ef 0.38 f HW+SH 0.53 abcde 0.55 abcd 0.45 cdef 0.46 cdef z All 3 harvests of both cultivars for four different treatments (AMW+NC, HW+PL, HW+CR, HW+SH); AMW=25oC for 30 min, HW = 45oC for 30 min; NC=No coating, PL=Polyethylene, CR=Carnauba, SH=Shellac. y TTA determined initially after treatment and at weekly intervals for 3 weeks storage. x Mean within a column denoted by the same letter of each column or each row for specific temperature combined analysis do not differ significantly according to Tukey’s Test (P<0.05).

179

Table 4-30. Effect of cultivar x Harvest x Treatment x Storage time interaction for early, mid & late harvest of Navel and Valencia orange on ascorbic acid content (ASC)z. Cultivar Harvest Treatment ASC (mg/100g) Week 0y Week 1 Week 2 Week 3 Navel H1 AMW+NC 43.50 bcx 43.19 c 47.42 bc 53.30 a HW+PL 49.32 ab 43.83 bc 49.17 ab 48.01 abc HW+CR 46.37 bc 44.92 bc 47.65 abc 46.50 bc HW+SH 46.94 bc 44.88 bc 44.72 bc 49.11 ab

H2 AMW+NC 54.37 abc 50.84 bcd 41.09 bcd 49.54 bcd HW+PL 66.87 a 45.49 bcd 43.92 bcd 47.27 bcd HW+CR 39.78 cd 38.27 d 44.64 bcd 56.68 ab HW+SH 50.87 bcd 47.66 bcd 44.44 bcd 54.73 abc

H3 AMW+NC 49.57 bc 64.25 ab 72.27 a 62.35 ab HW+PL 59.41 abc 45.19 c 65.45 ab 58.59 abc HW+CR 51.54 bc 51.32 bc 61.04 abc 58.54 abc HW+SH 56.99 abc 48.96 bc 61.84 abc 58.59 abc

Valencia H1 AMW+NC 110.21 abc 101.69 abcd 115.20 ab 105.10 abcd HW+PL 98.55 abcd 102.17 abcd 97.62 abcd 111.17 abc HW+CR 78.82 cd 74.53 d 82.12 cd 97.92 abcd HW+SH 94.85 bcd 100.98 abcd 129.16 a 103.55 abcd

H2 AMW+NC 84.89 ab 66.09 d 77.76 abcd 69.28 cd HW+PL 78.61 abcd 75.67 bcd 85.27 ab 79.43 abcd HW+CR 76.54 bcd 80.48 abc 73.72 bcd 66.28 cd HW+SH 78.06 abcd 70.96 bcd 91.08 a 69.63 cd

H3 AMW+NC 53.24 ab 52.97 abc 48.90 abcd 42.11 bcd HW+PL 47.14 abcd 41.39 cd 51.81 abcd 46.04 bcd HW+CR 42.96 bcd 48.59 abcd 57.70 a 46.05 bcd HW+SH 43.44 bcd 40.36 d 46.65 abcd 44.73 bcd z All 3 harvests of both cultivars for four different treatments (AMW+NC, HW+PL, HW+CR, HW+SH); AMW=25oC for 30 min, HW = 45oC for 30 min; NC=No coating, PL=Polyethylene, CR=Carnauba, SH=Shellac. y ASC determined initially after treatment and at weekly intervals for 3 weeks storage. x Means within a column denoted by the same letter of each column or each row for specific temperature combined analysis do not differ significantly according to Tukey’s Test (P<0.05).

180

Table 4-31. Effect of cultivar x Harvest x Treatment x Storage time interaction for early, mid & late harvest of Navel and Valencia orange on weight loss (WL)z. Cultivar Harvest Treatment WL (% FW) Week 0y Week 1 Week 2 Week 3 Navel H1 AMW+NC - 2.08 fx 4.27 bc 6.34 a HW+PL - 1.72 f 3.37 d 4.77 b HW+CR - 0.98 g 1.76 f 2.69 e HW+SH - 2.27 ef 4.09 c 6.10 a

H2 AMW+NC - 2.74 de 4.88 bc 7.68 a HW+PL - 1.72 g 2.92 de 4.51 c HW+CR - 0.97 h 1.73 g 2.68 ef HW+SH - 2.01 fg 3.46 d 5.42 b

H3 AMW+NC - 5.89 a 2.61 d 6.01 a HW+PL - 3.59 c - 3.47 c HW+CR - 2.32 d - 2.33 d HW+SH - 5.09 b - 5.87 a

Valencia H1 AMW+NC - 2.68 d 5.04 b 6.56 a HW+PL - 1.91 e 3.58 c 4.66 b HW+CR - 1.50 e 2.80 d 3.58 c HW+SH - 2.60 d 5.05 b 6.68 a

H2 AMW+NC - 2.41 cde 4.63 b 6.97 a HW+PL - 1.61 ef 3.38 c 5.06 b HW+CR - 0.95 fg 2.09 de 2.96 cd HW+SH - 2.31 de 5.11 b 7.62 a

H3 AMW+NC - 2.39 f 7.82 b 9.38 a HW+PL - 1.44 gh 4.51 e 5.47 d HW+CR - 0.80 hi 2.53 f 3.10 f HW+SH - 2.28 fg 6.38 c 7.65 b z All 3 harvests of both cultivars for four different treatments (AMW+NC, HW+PL, HW+CR, HW+SH); AMW=25oC for 30 min, HW = 45oC for 30 min; NC=No coating, PL=Polyethylene, CR=Carnauba, SH=Shellac. y WL calculated at initially after treatment and at weekly intervals for 3 weeks storage. x Means within a column denoted by the same letter of each column or each row for specific temperature combined analysis do not differ significantly according to Tukey’s Test (P<0.05).

181

Table 4-32. Effect of cultivar x Harvest x Treatment x Storage time interaction for early, mid & late harvest of Navel and Valencia orange on compression (COM)z. Cultivar Harvest COM (N) Treatment Week 0y Week 1 Week 2 Week 3 Navel H1 AMW+NC 84.81 cdx 89.44 bcd 83.74 cd 71.11 d HW+PL 105.91 abc 106.72 abc 93.80 abcd 87.87 cd HW+CR 113.18 abc 117.66 ab 110.60 abc 119.71 a HW+SH 107.61 abc 96.42 abcd 92.01 abcd 84.34 cd

H2 AMW+NC 155.02 ab 116.72 bcd 107.10 bcd 75.23 d HW+PL 139.90 abc 133.70 abc 116.11 bcd 109.80 bcd HW+CR 174.94 a 156.66 ab 146.98 abc 134.15 abc HW+SH 129.70 abc 139.41 abc 114.27 bcd 103.16 cd

H3 AMW+NC 118.48 ab 84.64 cde 58.92 e 77.08 de HW+PL 92.17 bcd 91.32 bcd 89.98 bcd 90.13 bcd HW+CR 140.00 a 118.44 ab 66.73 de 111.26 abc HW+SH 88.58 bcde 83.74 cde 87.12 cde 86.79 cde

Valencia H1 AMW+NC 124.77 ab 104.50 abcde 95.29 bcde 88.81 cde HW+PL 128.27 a 112.80 abcd 108.15 abcde 90.73 cde HW+CR 113.72 abc 107.07 abcde 94.07 bcde 97.91 abcde HW+SH 98.51 abcde 122.52 ab 80.62 e 82.63 de

H2 AMW+NC 104.79 a 94.58 abcd 83.36 bcde 75.23 de HW+PL 92.79 abcde 90.44 abcde 84.55 bcde 79.65 cde HW+CR 102.13 ab 98.29 abc 101.39 ab 92.16 abcde HW+SH 93.84 abcde 84.51 bcde 73.86 e 78.00 de

H3 AMW+NC 115.06 abc 106.52 abcde 94.81 bcde 86.81 cde HW+PL 112.36 abcd 103.42 abcde 97.60 bcde 89.19 cde HW+CR 109.80 abcd 131.70 a 119.24 ab 103.50 abcde HW+SH 104.52 abcde 97.36 bcde 84.44 de 77.38 e

182

z All 3 harvests of both cultivars for four different treatments (AMW+NC, HW+PL, HW+CR, HW+SH); AMW=25 oC for 30 min, HW = 45oC for 30 min; NC=No coating, PL=Polyethylene, CR=Carnauba, SH=Shellac. y Compression (COM) force determined at initially after treatment and at weekly intervals for 3 weeks. x Means within a column denoted by the same letter of each column or each row for specific temperature combined analysis do not differ significantly according to Tukey’s Test (P<0.05).

183

Table 4-33. Effect of cultivar x Harvest x Treatment x Storage time interaction for early, mid & late harvest of Navel and Valencia orange on puncture force (PUN)z. Cultivar Harvest Treatment PUN (N) Week 0y Week 1 Week 2 Week 3 Navel H1 AMW+NC 33.76 abx 29.01 bc 30.70 abc 30.21 abc HW+PL 33.53 ab 28.99 bc 29.49 bc 29.25 bc HW+CR 30.95 abc 30.23 abc 28.68 bc 28.22 c HW+SH 31.29 abc 35.17 a 31.79 abc 31.70 abc

H2 AMW+NC 26.61 abc 28.41 a 24.93 abc 25.45 abc HW+PL 23.88 bc 25.02 abc 24.99 abc 24.73 abc HW+CR 25.69 abc 26.95 abc 24.92 abc 22.87 c HW+SH 27.64 ab 27.32 ab 25.06 abc 26.52 abc

H3 AMW+NC 22.72 ab 26.81 a 25.03 ab 26.68 a HW+PL 25.23 ab 25.03 ab 21.71 ab 22.36 ab HW+CR 20.79 ab 21.64 ab 19.66 b 20.58 ab HW+SH 23.18 ab 24.99 ab 25.35 ab 23.66 ab

Valencia H1 AMW+NC 36.36 ab 35.57 ab 32.32 ab 35.09 ab HW+PL 35.73 ab 34.95 ab 30.68 b 31.11 ab HW+CR 39.95 a 34.97 ab 37.09 ab 31.18 ab HW+SH 36.99 ab 34.56 ab 31.70 ab 34.37 ab

H2 AMW+NC 27.95 ab 29.27 ab 29.80 ab 29.12 ab HW+PL 30.20 ab 28.91 ab 29.20 ab 27.80 ab HW+CR 29.94 ab 30.18 ab 27.95 ab 26.41 b HW+SH 33.46 a 28.48 ab 32.71 a 29.88 ab

H3 AMW+NC 30.13 bc 28.79 c 32.21 abc 38.33 a HW+PL 29.25 c 29.99 c 29.38 c 29.33 c HW+CR 32.36 abc 30.03 bc 26.82 c 27.08 c HW+SH 31.84 abc 36.17 ab 32.66 abc 32.95 abc z All three harvests of both cultivars for four different treatments (AMW+NC, HW+PL, HW+CR, HW+SH); AMW=25oC for 30 min, HW = 45oC for 30 min; NC=No coating, PL=Polyethylene, CR=Carnauba, SH=Shellac. y Puncture (PUN) force determined at initially after treatment and at weekly intervals for 3 weeks. x Means within a column denoted by the same letter of each column or each row for specific temperature combined analysis do not differ significantly according to Tukey’s Test (P<0.05).

184

Table 4-34. Effect of cultivar x Harvest x Treatment x Storage time interaction for early, mid & late harvest of Navel and Valencia orange on oleocellosis (OLC)z. Cultivar Harvest Treatment OLC (N) Week 0y Week 1 Week 2 Week 3 Navel H1 AMW+NC 64.22 abx 59.54 bc 58.96 abc 61.09 abc HW+PL 68.51 ab 62.98 bc 64.12 bc 58.57 bc HW+CR 62.78 abc 59.72 abc 61.74 bc 57.42 c HW+SH 63.80 abc 70.96 a 65.11 abc 58.53 abc

H2 AMW+NC 49.08 abc 51.50 abc 50.62 abc 47.74 c HW+PL 52.09 abc 53.50 ab 49.67 abc 48.27 bc HW+CR 51.50 abc 49.51 abc 51.78 abc 48.66 abc HW+SH 53.53 ab 54.05 a 51.45 abc 47.21 c

H3 AMW+NC 53.18 ab 46.47 abc 43.04 cdef 38.62 cdefgh HW+PL 42.18 cdefg 43.52 cde 36.05 defgh 34.76 gh HW+CR 36.87 defgh 34.33 gh 35.15 fgh 31.33 h HW+SH 54.12 a 45.90 bc 43.94 cd 35.81 efgh

Valencia H1 AMW+NC 51.08 a 48.85 ab 47.27 ab 47.84 ab HW+PL 49.74 a 50.85 a 41.42 b 48.00 ab HW+CR 50.98 a 49.87 a 44.37 ab 51.67 a HW+SH 51.86 a 47.58 ab 45.45 ab 45.72 ab

H2 AMW+NC 52.71 ab 45.74 cd 39.57 efg 38.58 fg HW+PL 55.59 a 44.31 cde 41.82 defg 43.41 cdef HW+CR 54.71 a 44.39 cde 40.71 defg 47.82 bc HW+SH 55.00 a 40.47 efg 41.86 defg 37.01 fg

H3 AMW+NC 55.10 ab 58.33 a 48.07 de 47.74 de HW+PL 56.90 a 55.59 ab 49.87 cde 51.09 bcde HW+CR 53.75 abc 57.97 a 51.11 bcde 53.41 abc HW+SH 51.66 bcd 56.78 a 46.15 e 47.66 de z All 3 harvests of both cultivars for four different treatments (AMW+NC, HW+PL, HW+CR, HW+SH); AMW=25oC for 30 min, HW = 45oC for 30 min; NC=No coating, PL=Polyethylene, CR=Carnauba, SH=Shellac. y Oleocellosis (OLC) force determined at initially after treatment and at weekly intervals for 3 weeks. x Means within a column denoted by the same letter of each column or each row for specific temperature combined analysis do not differ significantly according to Tukey’s Test (P<0.05).

185

Table 4-35. Analysis of variance table for combined effect of cultivar, harvest, storage period, and treatment on SSC, TTA, SSC/TTA ratio, ASC, and TCC during 3- week of storage at 25oC with 85%RHz. Main effect SSC (%)y TTA (% SSC/TTA ASC TCC citric acid) ratio (mg/100g) (mg/100g) Cultivar (C) **x **** **** **** **** Harvest (H) **** **** **** **** **** Storage (S) * **** **** **** ns Treatment (T) *** **** **** ns ** Interaction: C x H **** **** **** **** **** C x S ** ns ns ** * C x T *** *** * ns ns H x S **** **** **** *** *** H x T * * ns * ns S x T ns *** * ns ns C x H x S *** **** **** ** **** C x H x T * ** ** ns ns H x S x T ** **** *** ns ns C x S x T ns **** **** ** ns C x H x S x T ns ** ns * ns z Two harvests (2nd and 3rd harvest) of both cultivars (Navel and Valencia) for five different treatments (AMW+NC, HW+NC, HW+PL, HW+CR, HW+SH); AMW=25oC for 30 min, HW = 45oC for 30 min; NC=No coating, PL=Polyethylene, CR=Carnauba, SH=Shellac. y Fruit composition: Initially after treatment and at weekly intervals for 3 weeks storage. x ns, *, **, ***, ****, not significant, significant at P<0.05, P<0.01, P<0.001, or highly significant at P<0.0001 respectively according to Tukey test.

186

Table 4-36. Analysis of variance table for combined effect of cultivar, harvest, storage period, and treatment on WL, TP, ATC, L*, C, and H angle value during 3- week of storage at 25oC with 85%RHz. Main effect WL (% FW)y TP ATC (Trolox L* C H (mg/100g) µmol/100g) Cultivar (C) ****x **** **** **** ns **** Harvest (H) ns ** **** ns **** **** Storage (S) **** *** **** **** **** **** Treatment (T) **** ns **** **** **** **** Interaction: C x H **** ns **** **** **** **** C x S **** ns ** **** **** **** C x T **** ns *** **** **** **** H x S **** ns **** **** **** **** H x T **** ns **** **** **** **** S x T **** ns *** ns ** ns C x H x S **** ns **** **** **** **** C x H x T **** ns ns **** **** **** H x S x T **** ns ** ns ns ns C x S x T **** ns **** ns ns ns C x H x S x T **** ns ** ns ns ns z Two harvests (2nd and 3rd harvest) of both cultivars (Navel and Valencia) for five different treatments (AMW+NC, HW+NC, HW+PL, HW+CR, HW+SH); AMW=25oC for 30 min, HW = 45oC for 30 min; NC=No coating, PL=Polyethylene, CR=Carnauba, SH=Shellac. y Fruit physicochemical quality: Initially after treatment and at weekly intervals for 3 weeks storage. x ns, *, **, ***, ****, not significant, significant at P<0.05, P<0.01, P<0.001, or highly significant at P<0.0001 respectively according to Tukey test.

187

Table 4-37. Analysis of variance table for combined effect of cultivar, harvest, storage period, and treatment on COM, PUN, and OLC during 3-week of storage at 25oC with 85%RHz. Main effect COM (N)y PUN (N) OLC (N) Cultivar (C) ****x **** **** Harvest (H) **** ns **** Storage (S) **** ns **** Treatment (T) **** **** * Interaction: C x H **** **** **** C x S *** * **** C x T **** ns **** H x S * * **** H x T ns ** **** S x T ** *** **** C x H x S **** ns **** C x H x T ns ns **** H x S x T ns * ns C x S x T *** ns ** C x H x S x T ns ns ** z Two harvests (2nd and 3rd harvest) of both cultivars (Navel and Valencia) for five different treatments (AMW+NC, HW+NC, HW+PL, HW+CR, HW+SH); AMW=25oC for 30 min, HW = 45oC for 30 min; NC=No coating, PL=Polyethylene, CR=Carnauba, SH=Shellac. y Fruit composition: Initially after treatment and at weekly intervals for 3 weeks storage. x ns, *, **, ***, ****, not significant, significant at P<0.05, P<0.01, P<0.001, or highly significant at P<0.0001 respectively according to Tukey test.

188

Table 4-38. Effect of cultivar x Harvest x Treatment x Storage time interaction for mid & late harvest of Navel and Valencia orange on total titratable acid (TTA)Z. Cultivar Harvest Storage TTA (% Citric acid) (week)y AMW+NC HW+NC HW+PL HW+CR HW+SH Navel H2 0 0.40 bx 0.35 b 0.35 b 0.35 b 0.38 b 1 0.42 b 0.75 a 0.34 b 0.35 b 0.36 b 2 0.37 b 0.38 b 0.33 b 0.32 b 0.35 b 3 0.36 b 0.34 b 0.34 b 0.33 b 0.33 b

H3 0 0.48 abc 0.46 abcd 0.47 abcd 0.44 abcd 0.47 abcd 1 0.44 abcd 0.48 abcd 0.47 abcd 0.43 bcd 0.46 abcd 2 0.50 a 0.48 abcd 0.44 bcd 0.43 cd 0.43 bcd 3 0.45 abcd 0.49 ab 0.48 abcd 0.42 d 0.44 abcd

Valencia H2 0 0.65 bcd 0.59 d 0.60 cd 0.64 bcd 0.65 bcd 1 0.72 abcd 0.70 abcd 0.70 abcd 0.75 ab 0.71 abcd 2 0.68 bcd 0.68 bcd 0.72 abc 0.67 bcd 0.73 ab 3 0.82 a 0.74 ab 0.66 bcd 0.76 ab 0.76 ab

H3 0 0.64 a 0.54 abcd 0.53 abcd 0.51 abcd 0.53 abcd 1 0.60 ab 0.53 abcd 0.58 abc 0.55 abc 0.55 abc 2 0.48 abcd 0.48 abcd 0.43 cd 0.38 d 0.45 bcd 3 0.48 abcd 0.44 cd 0.43 cd 0.38 d 0.46 bcd z Combined effects of two harvests (2nd and 3rd harvest) of both cultivars for five different treatments (AMW+NC, HW+NC, HW+PL, HW+CR, HW+SH); AMW=25oC for 30 min, HW = 45oC for 30 min; NC=No coating, PL=Polyethylene, CR=Carnauba, SH=Shellac. y Total titratable acid (TTA) determine at initially after treatment and at weekly intervals for 3 weeks storage. x Means within a column denoted by the same letter of each column or each row for specific temperature combined analysis do not differ significantly according to Tukey’s Test (P<0.05).

189

Table 4-39. Effect of cultivar x Harvest x Treatment x Storage time interaction for mid & late harvest of Navel and Valencia orange on vitamin C (ASC)z. Cultivar Harvest Storage ASC (mg/100g) (week)y AMW+NC HW+NC HW+PL HW+CR HW+SH Navel H2 0 54.37 abcx 51.59 abcd 66.87 a 59.78 a 50.87 abcde 1 45.49 50.84 abcdef 40.96 ef bcdef 38.27 f 47.66 abcdef 2 47.88 41.09 def abcdef 43.92 cdef 44.64 bcdef 44.44 bcdef 3 49.81 47.27 49.54 abcde abcde abcdef 56.68 a 54.73 ab

H3 0 49.57 bc 55.66 abc 59.41 abc 51.54 bc 56.99 abc 1 64.25 ab 49.57 bc 45.19 c 51.32 bc 48.96 bc 2 72.27 a 64.75 ab 65.45 ab 61.04 abc 61.84 abc 3 62.35 abc 55.67 abc 58.59 abc 58.54 abc 58.59 abc Valencia H2 0 84.89 ab 79.53 ab 78.61 ab 76.54 b 78.06 ab 1 66.09 b 76.31 b 75.67 b 80.48 ab 70.96 b 2 77.76 ab 75.94 b 85.27 ab 73.72 b 98.10 a 3 69.28 b 72.75 b 79.43 ab 66.28 b 69.63 b

H3 0 53.24 a 42.08 abcd 47.14 abcd 42.96 abcd 43.44 abcd 1 52.97 ab 39.32 abcd 41.39 abc 48.59 abc 40.36 abc 2 48.90 abcd 52.62 abcd 51.81 cd 57.79 d 46.65 bcd 3 42.11 abcd 47.08 cd 46.04 cd 46.05 d 44.73 bcd z Combined effects of two harvests (2nd and 3rd harvest) of both cultivars for five different treatments (AMW+NC, HW+NC, HW+PL, HW+CR, HW+SH); AMW=25oC for 30 min, HW = 45oC for 30 min; NC=No coating, PL=Polyethylene, CR=Carnauba, SH=Shellac. y Ascorbic acid content (ASC) determine initially after treatment and at weekly intervals for 3 weeks storage. x Means within a column denoted by the same letter of each column or each row for specific temperature combined analysis do not differ significantly according to Tukey’s Test (P<0.05).

190

Table 4-40. Effect of cultivar x Harvest x Treatment x Storage time interaction for mid & late harvest of Navel and Valencia orange on weight loss (WL)z. Cultivar Harvest Storage WL (% FW) (week)y AMW+NC HW+NC HW+PL HW+CR HW+SH Navel H2 0 - - - - - 1 2.74 dex 3.28 d 1.72 fg 0.97 g 2.01 ef 2 4.88 bc 5.06 bc 2.92 d 1.73 fg 3.46 d 3 7.68 a 7.56 a 4.51 c 2.68 de 5.42 b

H3 0 - - - - - 1 5.89 a 4.48 c 3.59 d 2.32e 5.09b 2 2.61 e - - - - 3 6.01 a 4.65 bc 3.47 d 2.33 e 5.87 a

Valencia H2 0 - - - - - 1 2.41 cde 1.87 ef 1.61 ef 0.95 fg 2.31 de 2 4.63 b 4.70 b 3.38 c 2.09 de 5.11 b 3 6.97 b 7.25 a 5.06 b 2.96 cd 7.62 a

H3 0 - - - - - 1 2.3 fg 1.86 gh 1.44 hi 0.80 ij 2.28 fgh 2 7.82 b 6.67 c 4.51 e 2.53 fg 6.38 c 3 9.38 a 8.15 b 5.47 d 3.10 f 7.65 b z Combined effects of two harvests (2nd and 3rd harvest) of both cultivars for five different treatments (AMW+NC, HW+NC, HW+PL, HW+CR, HW+SH); AMW=25oC for 30 min, HW = 45oC for 30 min; NC=No coating, PL=Polyethylene, CR=Carnauba, SH=Shellac. y Weight loss (WL) determined initially after treatment and at weekly intervals for 3 weeks storage. x Means within a column denoted by the same letter of each column or each row for specific temperature combined analysis do not differ significantly according to Tukey’s Test (P<0.05).

191

Table 4-41. Effect of cultivar x Harvest x Treatment x Storage time interaction for mid & late harvest of Navel and Valencia orange on antioxidant capacity (ATC)z. Cultivar Harvest Storage ATC (Trolox µmol/100g) (week)y AMW+NC HW+NC HW+PL HW+CR HW+SH Navel H2 0 1066.41 abx 1049.39 ab 1105.91 ab 1212.88 ab 1103.15 ab 1 923.71 b 1117.96 ab 1256.29 ab 1370.71 a 979.93 ab 2 968.25 ab 956.36 b 996.68 ab 1139.40 ab 1019.33 ab 3 944.49 b 1042.27 ab 975.44 ab 1076.81 ab 927.83 b

H3 0 1004.11 g 1750.76 1199.99 fg 1211.60 fg 1258.15 efg bcde 1 1334.69 cdefg 2289.11 a 1774.21 bcd 1472.24 1434.12 cdefg cdefg 2 1484.68 cdefg 1219.39 fg 1780.65 bcd 1824.64 abc 1765.92 bcd 3 1305.49 defg 2070.69 ab 1584.81 1512.76 1342.05 cdefg bcdef cdef Valencia H2 0 1170.99 b 1053.83 b 1628.87 ab 1130.69 b 1282.91 b 1 1433.51 b 1185.25 b 1175.28 b 1286.17 b 1370.60 b 2 1880.61 ab 1832.77 ab 1858.87 ab 1941.39 ab 2349.76 a 3 1255.63 b 1141.45 b 1565.00 ab 1826.31 ab 1638.61 ab

H3 0 1374.95 abc 1135.57 bc 1241.89 bc 1474.53 abc 969.23 c 1 1107.93 bc 1968.99 a 1465.96 abc 1476.59 abc 1666.07 ab 2 991.12 c 1276.21 bc 1163.95 bc 1177.39 bc 1483.64 abc 3 1226.13 bc 1135.91 bc 1418.75 abc 1289.85 bc 1348.95 abc z Combined effects of two harvests (2nd and 3rd harvest) of both cultivars for five different treatments (AMW+NC, HW+NC, HW+PL, HW+CR, HW+SH); AMW=25oC for 30 min, HW = 45oC for 30 min; NC=No coating, PL=Polyethylene, CR=Carnauba, SH=Shellac. y Antioxidant capacity (ATC) determined initially after treatment and at weekly intervals for 3 weeks storage. x Means within a column denoted by the same letter of each column or each row for specific temperature combined analysis do not differ significantly according to Tukey’s Test (P<0.05).

192

Table 4-42. Effect of cultivar x Harvest x Treatment x Storage time interaction for mid & late harvest of Navel and Valencia orange on oleocellosis (OLC) forcez. Cultivar Harvest Storage OLC (N) (week)y AMW+NC HW+NC HW+PL HW+CR HW+SH Navel H2 0 49.08 abcdx 54.77 a 52.09 abcd 51.50 abcd 53.53 abc 1 51.50 abcd 51.24 abcd 53.50 abc 49.51 abcd 54.05 ab 2 50.62 abcd 50.75 abcd 49.67 abcd 51.78 abcd 51.45 abcd 3 47.74 cd 48.00 cd 48.27 bcd 48.66 bcd 47.21 d

H3 0 53.18 ab 46.89 abc 42.18 cdefg 36.87 defgh 54.12 a 1 46.47 abc 44.47 bcde 43.52 cdefg 34.33 gh 45.90 abcd 2 43.04 cdefg 41.40 cdefg 36.05 efgh 35.15 efgh 43.94 bcdef 3 38.62 cdefgh 34.46 fgh 34.76 fgh 31.33 h 35.81 efgh

Valencia H2 0 52.71 ab 52.52 ab 55.59 a 54.71 a 55.00 a 1 45.74 cd 41.53 defg 44.31 cde 44.39 cde 40.47 defg 2 39.57efg 45.94 cd 41.82 defg 40.71 defg 41.86 defg 3 38.58 fg 44.84 cde 43.41 cdef 47.82 bc 37.01 g

H3 0 55.10 abcd 50.73 cdef 56.90 abc 53.75 abcde 51.66 bcdef 1 58.33 a 55.22 abcd 55.59 abcd 57.97 ab 56.78 abc 2 48.07 ef 45.82 f 49.87 def 51.11 cdef 46.15 f 3 47.74 ef 47.70 ef 51.09 cdef 53.41 abcde 47.66 ef z Combined effects of two harvests (2nd and 3rd harvest) of both cultivars for five different treatments (AMW+NC, HW+NC, HW+PL, HW+CR, HW+SH); AMW=25oC for 30 min, HW = 45oC for 30 min; NC=No coating, PL=Polyethylene, CR=Carnauba, SH=Shellac. y Oleocellosis force (OLC) determined initially after treatment and at weekly intervals for 3 weeks storage. x Means within a column denoted by the same letter of each column or each row for specific temperature combined analysis do not differ significantly according to Tukey’s Test (P<0.05).

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Table 4-43. Major volatile compounds detected in sweet orange fruit. 1. Limonene 12. 2-Hexenal 2. Naphthalene 13. 1-Hexanol 3. Butanoic acid, ethyl ester 14. Azulene 4. ß-Pinene 15. 1H-Cyclopropa[a]naphthalene 5. Hexanoic acid, ethyl ester 16. Cyclohexane 6. 1,6-Octadien-3-ol, 3,7-dimethyl- 17. 6-Octen-1-ol, 3,7-dimethyl- 7. Hexanoic acid 18. Cyclopentasiloxane 8. Octanoic acid, ethyl ester 19. 1-Octanol 9. ß-Panasinsen 20. 1,4-Cyclohexadiene 10. Acetamide 21. Oxime 11. Cyclotrisiloxane, hexamethyl- 22. Heptanal

Fungicide Fruit treated treated fruit into HW

Washing on Waxing on packaingline brush pad

Fruit storage Hot air drying (25oC with 85% RH)

Figure 4-1. Postharvest treatment of Navel oranges from harvest to storage. Photo courtesy of author.

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HW+NC AMW+NC

HW+PL AMW+PL

HW+CR AMW+CR

HW+SH AMW+SH

Figure 4-2. Navel orange storage after 3 weeks at 25oC with 85%RH. Photo courtesy of author.

Figure 4-3. Valencia orange storage after 3 weeks at 25oC with 85%RH. Photo courtesy of author.

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O2 in Navel orange during storage 25 AMW+NC HW+PL HW+CR HW+SH 20

10 in Navel in Navel orange 2 5

% % O 0 wk 0 wk 1 wk 2 wk 3 Storage (week)

Figure 4-4. Internal O2 in four different treatments (AMW+NC, HW+PL, HW+CR, HW+SH) of Navel orange fruit during 3-week of storage at 25oC with 85%RH.

CO2 in Navel orange during storage 25 AMW+NC HW+PL HW+CR HW+SH 20

in Navel orange in Navel 2 5

%CO 0 wk 0 wk 1 wk 2 wk 3

Storage (week)

Figure 4-5. Internal CO2 in four different treatments (AMW+NC, HW+PL, HW+CR, HW+SH) of Navel orange fruit during 3-week of storage at 25oC with 85%RH.

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C2H4 in Navel orange during storage 5 AMW+NC HW+PL HW+CR HW+SH 4

3 in Navel in Navel orange

4 2 H 2 1

ppm ppm C 0 wk 0 wk 1 wk 2 wk 3 Storage (week)

Figure 4-6. Internal C2H4 in four different treatments (AMW+NC, HW+PL, HW+CR, HW+SH) of Navel orange fruit during 3-week of storage at 25oC with 85%RH

O2 in Valencia orange during storage AMW+NC HW+PL HW+CR HW+SH 25

in Valencia orange fruit orange in Valencia 2 0

% % O wk 0 wk 1 wk 2 wk 3

Storage (week)

Figure 4-7. Internal O2 in four different treatments (AMW+NC, HW+PL, HW+CR, HW+SH) of Valencia orange fruit during 3-week of storage at 25oC with 85%RH

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CO2 in Valencia orange during storage 25 AMW+NC HW+PL HW+CR HW+SH 20

in Valencia orange in Valencia 5 2 0 %CO wk 0 wk 1 wk 2 wk 3

Storage (week)

Figure 4-8. Internal CO2 in four different treatments (AMW+NC, HW+PL, HW+CR, HW+SH) of Valencia orange fruit during 3-week of storage at 25oC with 85%RH

C2H4 in Valencia orange during storage 5 AMW+NC HW+PL HW+CR HW+SH 4

in Valencia orange in Valencia 4

H 1 2

ppm ppm C wk 0 wk 1 wk 2 wk 3 Storage (week)

Figure 4-9. Internal C2H4 in four different treatments (AMW+NC, HW+PL, HW+CR, HW+SH) of Valencia orange fruit during 3-week of storage at 25oC with 85%RH

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O2 in Navel orange during storage AMW+NC HW+NC HW+PL HW+CR HW+SH 25 20 15

in Navel in Navel orange 10 2

5 % % O 0 wk 0 wk 1 wk 2 wk 3 Storage (week)

Figure 4-10. Internal O2 in five different treatments (AMW+NC, HW+NC, HW+PL, HW+CR, HW+SH) of Navel orange fruit during 3-week of storage at 25oC with 85%RH

CO2 in Navel orange during storage 25 AMW+NC HW+NC HW+PL HW+CR HW+SH 20

in Navel orange in Navel 2 2 5

% % CO 0 wk 0 wk 1 wk 2 wk 3 Storage (week)

Figure 4-11. Internal CO2 in five different treatments (AMW+NC, HW+NC, HW+PL, HW+CR, HW+SH) of Navel orange fruit during 3-week of storage at 25oC with 85%RH

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C2H4 in Navel orange during storage 5 AMW+NC HW+NC HW+PL HW+CR HW+SH 4

in Navel in Navel orange 2

4 H 2 1

ppm ppm C 0 wk 0 wk 1 wk 2 wk 3

Storage (week)

Figure 4-12. Internal C2H4 in five different treatments (AMW+NC, HW+NC, HW+PL, HW+CR, HW+SH) of Navel orange fruit during 3-week of storage at 25oC with 85%RH

O2 in Valencia orange during storage AMW+NC HW+NC HW+PL HW+CR HW+SH 30 25 20 15

in Valencia orange in Valencia 10 2 5 % % O 0 wk 0 wk 1 wk 2 wk 3 Storage (week)

Figure 4-13. Internal O2 in five different treatments (AMW+NC, HW+NC, HW+PL, HW+CR, HW+SH) of Valencia orange fruit during 3-week of storage at 25oC with 85%RH

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CO2 (%) in Valencia orange during storage 25 AMW+NC HW+NC HW+PL HW+CR HW+SH 20

10 in Valencia orange in Valencia 2 5

% % CO 0 wk 0 wk 1 wk 2 wk 3 Storage (week)

Figure 4-14. Internal CO2 in five different treatments (AMW+NC, HW+NC, HW+PL, HW+CR, HW+SH) of Valencia orange fruit during 3-week of storage at 25oC with 85%RH

C2H4 (ppm) in Valencia orange during storage 5 AMW+NC HW+NC HW+PL HW+CR HW+SH 4

in Valencia orange in Valencia

4 H 2 1

0 ppm ppm C wk 0 wk 1 wk 2 wk 3

Storage (week)

Figure 4-15. Internal C2H4 in five different treatments (AMW+NC, HW+NC, HW+PL, HW+CR, HW+SH) of Valencia orange fruit during 3-week of storage at 25oC with 85%RH

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O2 (%) in Naval orange during storage 25 HW+NC HW+PL HW+CR HW+SH AMW+NC AMW+PL AMW+CR AMW+SH 20

(%) (%) fruit in orange 5

2 2 O 0 wk 0 wk 1 wk 2 wk 3 Storage (week)

Figure 4-16. Internal O2 in with/without HW and with/without coating (AMW=25oC, HW=45oC, NC=No coating, PL=Polyethylene, CR=Carnauba, SH=Shellac) in Navel orange fruit during 3-week of storage at 25oC with 85%RH

CO2 (%) in Naval orange during storage 25 HW+NC HW+PL HW+CR HW+SH AMW+NC AMW+PL AMW+CR AMW+SH 20

(%) in fruit orange (%) 2 2

5 CO

0 wk 0 wk 1 wk 2 wk 3 Storage (week)

Figure 4-17. Internal CO2 in with/without HW and with/without coating (AMW=25oC, HW=45oC, NC=No coating, PL=Polyethylene, CR=Carnauba, SH=Shellac) in Navel orange fruit during 3-week of storage at 25oC with 85%RH

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C2H4 (ppm) in Naval orange during storage 5 HW+NC HW+PL HW+CR HW+SH AMW+NC AMW+PL AMW+CR AMW+SH 4

1 C2H4 (ppm) in orange orange (ppm) C2H4 fruit in 0 wk 0 wk 1 wk 2 wk 3

Storage (week)

Figure 4-18. Internal C2H4 in with/without HW and with/without coating in (AMW=25oC, HW=45oC, NC=No coating, PL=Polyethylene, CR=Carnauba, SH=Shellac) in Navel orange fruit during 3-week of storage at 25oC with 85%RH

O2 (%) in Valencia orange during storage HW+NC HW+PL HW+CR HW+SH AMW+NC AMW+PL AMW+CR AMW+SH 25

(%) (%) fruit in orange 2 2

O 5

0 wk 0 wk 1 wk 2 wk 3 Storage (week)

Figure 4-19. Internal O2 in with/without HW and with/without coating in (AMW=25oC, HW=45oC, NC=No coating, PL=Polyethylene, CR=Carnauba, SH=Shellac) Valencia orange fruit during 3-week of storage at 25oC with 85%RH

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CO2 (%) in Valencia orange during storage HWT+NCT HWT+PL HWT+CRB HWT+SHL AMB+NCT AMB+PL 25

(%) in orange fruit orange in (%) 2 2

5 CO

0 wk 0 wk 1 wk 2 wk 3 Storage (week)

Figure 4-20. Internal CO2 in with/without HW and with/without coating (AMW=25oC, HW=45oC, NC=No coating, PL=Polyethylene, CR=Carnauba, SH=Shellac) in Valencia orange fruit during 3-week of storage at 25oC with 85%RH

C2H4 (ppm) in Valencia orange during storage HW+NC HW+PL HW+CR HW+SH AMW+NC AMW+PL AMW+CR AMW+SH 5

1 C2H4 (ppm) in orange orange (ppm) fruit C2H4 in

0 wk 0 wk 1 wk 2 wk 3 Storage (week)

Figure 4-21. Internal C2H4 in with/without HW and with/without coating (AMW=25oC, HW=45oC, NC=No coating, PL=Polyethylene, CR=Carnauba, SH=Shellac) in Valencia orange fruit during 3-week of storage at 25oC with 85%RH

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CHAPTER 5 SENSORY ACCEPTABILITY OF NAVEL AND VALENCIA ORANGE FRUIT TREATED WITH HOT WATER IMMERSION AND EDIBLE COATINGS DURING STORAGE

Sensory evaluation of food is conducted by the panelists who use their senses of sight, smell, taste, touch, and hearing for the purpose of evaluating the quality or acceptability of food products. Consumers like orange fruit for its nutritional value as well as sensory quality. Sensory qualities of orange fruit vary significantly between orange varieties, cultivars, and subgroups (Goldenberg et al., 2016). Differences in fruit color, carotenoid pigments, bioactive compounds, aromatic compounds, flavor and taste, and harvest time exist among orange subgroups, varieties, and cultivars

(Goldenberg et al., 2016).

Consumers’ decision to purchase orange fruit is highly influenced by attractive external fruit appearance, high nutritional content, juice content, flavor, and taste

(Campbell et al., 2006). In addition, consumer acceptance of orange fruit is positively correlated with high fruit sweetness and flavor, whereas acceptance negatively correlated to fruit acidity, sourness, and bitterness (Goldenberg et al., 2015). Orange fruits with high sweetness and moderate to low acidity and low bitterness are highly preferred to fruit with high sourness, bitterness, high acidity, relatively less sweetness and flavor (Raithore et al., 2015; Goldenberg et al., 2016).

Important citrus fruit quality parameters that affect sensory quality include fruit size, shape, peel color, ease of peeling, juice content, SSC, TTA, and flavor. On the other hand, juice content, SSC, and TTA are also important quality attributes for citrus for processing (Castle, 1995).

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The objective of this study was to evaluate the sensory acceptability of Navel and

Valencia orange fruit treated with hot water and edible wax coatings during simulated ambient storage.

Materials and Methods

The fruit used for sensory evaluation were Navel and Valencia oranges from the experiments described in Chapter 4. The treatments included were ambient water

(AMW)-treated fruit without coating (AMW+NC), hot water (HW)-treated fruit with no coating (HW+NC), and HW-treated fruit that had been coated with carnauba, polyethylene or shellac coating (HW+CR, HW+PL, HW+SH, respectively). There were three harvests conducted for each variety representing early, midlle and late seasons.

The fruit were obtained from farms in the Fort Pierce area and the treatments were applied at the IRREC. After treatments had been applied, the fruit were transported to

Gainesville and stored at at 25oC and 85% RH for 3 weeks, with initial plus weekly sampling during storage for conducting sensory tests.

Consumer sensory evaluation was conducted at the sensory laboratory in the

Food Science and Human Nutrition Department of the University of Florida according to

Lawless and Heyman (1988) with some modifications. A questionnaire was prepared to rate the quality attributes as well as the magnitude of the difference. Navel and Valencia oranges were treated with HW and several edible fruit coatings the day before the sensory test and taken to the laboratory for overnight storage at room temperature

(25oC) before evaluation the next morning. Twenty fruit were picked at random from the five treatments, hand peeled, segments separated and placed in one large container for mixing. Three segments were presented to panelists in small plastic cups labeled with

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3-digit random numbes. This procedure was followed for each variety and multiple harvests, weekly duing up to 3 weeks of storage at 25oC and 85% RH.

The order of presentation of the fruit segment samples was according to the

Williams and Arnold design (1984). In this case sample names, sample letter code, sample three digit blinding number, and order of presentation prepared earlier and posted to the sample preparation room. If the number of sample is 3, and the sample codes are A, B, and C then the order of sample presentation will be ABC, BAC, BCA,

ACB, CBA, and CAB. This procedure has been followed during sample evaluation by panelists. Panelists were also presented with four intact fruit for each treatment in plastic trays labeled with different 3-digit number codes and asked to rate the external fruit appearance of each treatment.

Hedonic Scale

Panelists rated the acceptability of samples in terms of overall liking, external appearance, flavor, and texture using a 9-point hedonic scale in which 1 = dislike extremely and 9 = like extremely (Appendix A). Crackers and water were provided during the test for palate cleansing between samples. A total of 75 to 90 panelists’ evaluated fruit from each harvest and storage duration for Navel oranges, and a total of

75 to 85 panelists evaluated each set of harvest and storage duration sample of

Valencia oranges.

Just About Right Scale

Following the acceptability questions, panelists were asked to quantify the fruit textural qualities, firmness, juiciness, orange flavor, and sweetness-sourness balance using the Just About Right scale from 1 to 5 (Appendix A).

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Statistical Analysis

Data analysis for sensory data was conducted independently for each of sensory attribute using analysis of variance (SAS Institute Inc., Version 9.3, Cary, NC, USA).

The experimental design was a Randomized Complete Block Design (RCBD) with replicates as blocks. Mean separation was based on Tukey’s test at P<0.05.

Results

Navel Oranges

For the first harvest of Navel oranges, there were significant differences (P<0.05) among the treatments for all quality attributes except fruit texture and sweet-sour balance at the initial storage (Table 5-1). The highest overall liking rating was for the

HW+PL fruit (6.2 a), which was followed by HW+CR fruit (5.9 ab). In terms of overall external appearance of whole fruit, most of the panelists preferred HW+CR (6.2 a) and the lowest rating was for AMW+NC fruit (5.0 c) because of darker color. Similar trends among the treatments were found for 2 weeks and 3 weeks of storage.

For Harvest 1 of Navel oranges stored for 2 weeks, there were significant differences (P<0.05) among the treatments for overall acceptability, overall appearance, overall flavor, and sweetness-sourness balance. The highest rating for overall acceptability was noted in HW+CR fruit (6.5 a), which was followed by HW+PL fruit (6.2 ab). There were no differences observed for overall texture, firmness, juiciness, or orange flavor.

Similarly, after 3 weeks of storage of Harvest 1 Navel oranges, there were significant differences (P<0.05) among the treatments except for sweet-sour balance and orange flavor. The highest ratings for overall acceptability were for AMW+NC (6.3 a), HW+PL (6.1 a), and HW+CR (6.1 a) fruit. The maximum overall appearance was

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exhibited by HW+CR (6.3 a), and the minimum was in HW-treated fruit with shellac coating (HW+SH; 5.1 c).

For Harvest 2 of Navel oranges, initially, there were no significant differences

(P<0.05) among the treatments except for overall appearance and overall texture (Table

5-2). The highest ratings for overall appearance were noted in HW+SH (5.5 a) and

HW+CR (5.3 a), and the minimum rating was again found in AMW+NC (4.2 c).

After 1 week of storage of harvest 2 Navel oranges, there were significant differences (P<0.05) among the treatments except for overall texture, juiciness, and orange flavor (Table 5-2). The highest rating for overall acceptability was noted in

HW+PL (6.2 a), and the lowest rating was in AMW+NC (5.7 b).

For Harvest 2 of Navel oranges, after 2 weeks storage, all sensory parameters were significantly different (P<0.05) among the treatments (Table 5-2). The highest ratings for overall acceptability were observed in HW+PL (6.3 a) and AMW+NC (6.1 a), and the lowest rating was in HW+SH (5.6 b). Also, coated fruit were rated higher than uncoated fruit for overall external appearance after 2 weeks of storage.

For Harvest 2 of Navel oranges stored for 3 weeks, there were significant differences (P<0.05) among the treatments for all sensory attributes (Table 5-2). The highest ratings for overall acceptability were observed in HW+CR (6.5 a) and HW+NC

(6.4 a) after 3 weeks of storage. Similar ratings were found for all treatments for overall appearance of Navel orange fruit after 3 weeks.

For Harvest 3 of Navel oranges, initially there were no significant differences among the treatments for any of the sensory attributes except overall appearance and

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overall texture (Table 5-3). The highest ratings for overall appearance were observed in

HW+SH (5.5 a) and HW+CR (5.3 a), and the lowest rating was in AMW+NC (4.2 c).

After 1 week of storage for Navel oranges from Harvest 3, there were significant differences (P<0.05) among the treatments for overall appearance, fruit firmness, and sweet-sour balance (Table 5-3). The highest rating for overall appearance was found in

HW+SH (5.5 a), and the lowest rating was in AMW+NC (4.0 c) after 1 week of storage.

For Harvest 3 of Navel oranges after 2 weeks of storage, there were significant differences (P<0.05) among the treatments for all sensory attributes (Table 5-3). The highest ratings for overall acceptability were noted in HW+PL (6.3 a) and AMW+NC (6.1 a) after 2 weeks, and all coated fruit were rated higher than uncoated fruit for overall peel appearance after 2 weeks.

For Harvest 3 of Navel oranges stored for 3 weeks, there were significant differences (P<0.05) among the treatments for all sensory attributes except overall peel appearance (Table 5-3). The highest ratings for overall acceptability were in HW+CR

(6.5 a) and HW+NC (6.4 a), and the lowest ratings were in HW+PL (5.5 b) and HW+SH

(5.5 b) after 3 weeks.

Valencia Oranges

For Valencia oranges, there were significant differences (P<0.05) among the treatments initially for overall acceptability, external appearance, flavor, texture, and firmness (Table 5-4). The highest overall acceptability was noted in HW+NC (6.5 a) and the lowest was in HW+PL (5.9 b).

For Harvest 1 of Valencia fruit evaluated after 1 week of storage, there were significant differences (P<0.05) among the treatments for all sensory attributes except juiciness and orange flavor (Table 5-4). The highest ratings for overall likability were for

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HW+CR (6.5 a), HW+PL (6.4 a), HW+NC (6.2 a), and AMW+NC (6.2 a). Similar trends were noticed for overall acceptability of Valencia oranges after 2 weeks of storage.

For Harvest 1 of Valencia oranges stored for 3 weeks, there were significant differences (P<0.05) among the treatments for overall acceptability, overall appearance, and overall flavor (Table 5-4). The highest rating for overall acceptability was for

HW+NC (6.1 a), which was followed by HW+PL (6.1 ab) and HW+CR (6.0 ab). In the case of overall appearance, HW+CR (6.4 a) and HW+PL (6.3 a) exhibited the highest ratings among the treatments. For overall flavor, the highest ratings were noted in

HW+CR (6.4 a) and HW+NC (6.4 a) after 3 weeks of storage (Table 5-4).

There were significant differences (P<0.05) among the treatments for Valencia

Harvest 2 for overall appearance, overall texture, firmness, and orange flavor (Table 5-

5). The highest ratings for overall appearance were for HW+SH (5.3 a), HW+PL (5.2 a),

HW+CR (5.1 a), and AMW+NC (6.2 a) at 0 week of storage (Table 5-5). In the case of overall texture, the highest ratings were for HW+CR (5.9 a) and AMW+NC (5.9 a). In the case of firmness, HW+PL (3.1 a) exhibited the highest ratings among the treatments.

The highest orange flavor rating was observed for HW+NC (2.7 a), and the lowest ratinge was for HW+SH (2.4 b) (Table 5-5) at 0 week of storage.

For Harvest 2 Valencia fruit evaluated after 1 week, there were significant differences (P<0.05) among the treatments for all sensory attributes except overall texture (Table 5-5). The highest overall likeability rating was observed for AMW+NC (6.3 a) and the lowest rating was for HW+PL (5.8 b) after 1 week of storage. For overall appearance, the highest ratings were for HW+SH (5.8 a) and HW+CR (5.6 a). In the case of overall flavor, HW+NC had highest rating (6.3 a) among the treatments and the

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highest firmness was for HW+PL (3.3 a) after 1 week of storage. The highest juiciness ratings were noted in HW+NC (3.0 a) and AMW+NC (3.0 a), whereas AMW+NC (2.9 a) exhibited the hightest rating for sweet-sour balance among the treatments after 1 week.

Similarly, the highest orange flavor presented for HW+NC (2.7 a) and the lowest rating was for HW+PL (2.4 b) after 1 week of storage (Table 5-5).

For Harvest 2 Valencia fruit evaluated after 2 weeks, there were significant differences (P<0.05) among the samples for all sensory attributes except for overall texture, firmness, and juiciness after 2 weeks of storage. After 3 weeks of storage, all sensory attributes for Harvest 2 Valencia oranges were significantly different among the treatments except firmness, juiciness, and orange flavor (Table 5-5).

For Harvest 3 of Valencia oranges, all sensory parameters were significantly different (P<0.05) among the treatments at time 0. The HW+NC fruit had the highest overall acceptability (6.5 a) among the treatments (Table 5-6).

For Harvest 3 Valencia fruit evaluated after 1 week, all sensory attributes were significantly different (P<0.05) among the treatments except for overall texture, juiciness, and sweet-sour balance (Table 5-6). The highest overall acceptability (6.2), and overall flavor (6.1 a) ratings were noted in AMW+NC after 1 week of storage. But the overall appearance ratings were highest for HW+CR (5.8 a), and HW+PL (5.5 a) after 1 week of storage. A similar trend was noted for Harvest 3 Valencia fruit firmness after 1 week of storage. In the case of orange flavor, the highest rating was observed for

AMW+NC (2.5 a) and the lowest rating was for HW+SH (2.2 b) after 1 week of storage

(Table 5-6).

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After 2 weeks of storage for Valencia oranges from Harvest 3, overall acceptability, overall appearance, overall flavor, and orange flavor were significantly different among the treatments (Table 5-6). The highest ratings among all the treatments for overall acceptability (6.1 a), overall appearance (6.1 a), overall flavor

(6.1a), and orange flavor (2.7 a) were observed for HW+CR (Table 5-6).

After 3 weeks of storage, all sensory attributes of Harvest 3 fruit were significantly different among the treatments except for overall texture, sweet-sour balance, and orange flavor (Table 5-6). The highest overall acceptability ratings were for

HW+CR (6.2 a), HW+NC (6.1 a), and HW+PL (6.0 a), while the lowest rating was noted in HW+SH (5.4 b). The highest overall appearance ratings were for HW+CR (6.5 a), followed by HW+PL (5.5 b) and HW+SH (5.0 b) after 3 weeks of storage (Table 5-6), while the lowest ratings were for HW+NC (3.7c) and AMW+NC (3.4c).

As shown in Chapter 4, the orange fruit that had been treated with HW and then coated with either polyethylene or carnauba created and maintained internal atmospheres during 3 weeks ambient storage that had oxygen and carbon dioxide levels that were nearest to the levels desired for quality maintenance. On the other hand, fruit that had been treated with HW and then coated with SH created and maintained internal atmospheres during 3 weeks ambient storage that had extreme, injurious levels of oxygen and carbon dioxide (<5% O2 + >10% CO2). The extreme atmosphere caused initiation of fermentative metabolism in the SH-coated oranges, resulting in ethanolic off-odors and off-flavors that were perceived by the sensory panelists according to the comments they made during the testing.

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Discussion

In this study, panelists generally preferred coated fruit over uncoated fruit and that was because WL in the uncoated fruit led to loss of gloss followed by peel shriveling. External fruit appearance is one of the most important attributes that appeals to consumers and influences their decision to purchase citrus fruit (Poole and Baron,

1996).

In the case of the first harvest of Navel oranges, panelists overall liked HW+PL fruit the most and then HW+CR fruit. But, for overall external appearance of whole fruit, panelists preferred HW+CR among the treatments. Carnauba wax has a very high melting point and has a positive effect on toughness and luster of fruit coatings.

According to Davis and Hofmann (1973) wax coating improves the fruit peel appearance including increasing the shininess of the surface of citrus fruit. Initially the

AMW+NC fruit were the least preferred by the panelists because of either the pale or darker color of the fruit peel that developed during storage.

Most of the panelists preferred HW+CR fruit because of fruit taste, flavor, and overall fruit color after 3 weeks storage. The panelists’ lowest preference was HW+SH fruit because the HW+SH fruit developed off-odor and off-flavor that was probably related to the extreme internal atmospheres that developed in the fruit (see Chapter 4).

Fruit treated with coatings that restrict gas diffusion so as to lead to severely reduced internal O2 atmosphere have been found to lead to AA and ethanol accumulation and production of off-flavors, especially in subtropical fruits such as, guava, mango and citrus (Baldwin et al., 1999; Hagenmaier, 2002; Ke and Kader, 1992; McGuire and

Hallman, 1995).

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As a whole, fruit panelists preferred the external appearance of HW+SH and

HW+CR fruit because of the shiny color and smooth surface, whereas the highest ratings for overall acceptability were observed after 3 weeks in HW+CR and HW+NC

(Table 5-2). The CR was most preferred overall because it resulted in the best combination of good appearance and good taste. As per the research results of Jemric and Fruk (2013), nectarines treated with HW immersion at 48°C for 6 to12 min performed better in terms of sensory quality for texture, aroma, taste, SSC/TA ratio, firmness, and general appearance after 2 weeks of storage. No previous research has included sensory evaluation of HW-treated Navel orange fruit.

For Valencia oranges, the highest overall likeability rating was observed for

AMW+NC and the lowest rating was for HW+PL. Water loss in AMW+NC resulted in increased SSC, which the panelists preferred, but was also accompanied by dehydration. For overall appearance, the highest ratings were for HW+SH and HW+CR, but for overall flavor, HW+NC had the highest rating among the treatments; the highest firmness rating was for HW+PL. After 1 week, the highest overall acceptability, and overall flavor ratings were noted in AMW+NC because of how the sweetness increased during storage, again, probably due to WL from the fruit. It is likely that pore spaces or holes associated with lenticels, stomata, stem scars, and injuries, which are probably the main pathway for gas exchange (Burg, 1990), are blocked when coatings are applied (Baldwin et al., 1999; Hagenmaier, 2002; Ke and Kader, 1992; McGuire and

Hallman, 1995), resulting in significant differences in WL between coated and uncoated oranges.

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Later, after 3 weeks of storage, however, the highest overall acceptability ratings were for HW+CR, HW+NC, and HW+PL, while the lowest rating was noted in HW+SH because of fruit decay due to extreme internal atmosphere modification, especially the low O2 level in the fruit. According to Petracek et al. (1998), shellac coating used on the surface of white grapefruit (Citrus × paradisi) increased pitting on the peel because of decreased internal O2 levels (<4%), which was associated with high AA and ethanol production. Low of O2 inside the fruit induces fermentation and accumulation of anaerobic off-flavors that are related to the production of ethanol and AA (Fidler and

North, 1971). The AMW+NC and HW+NC treatments were no longer preferred after 3 weeks storage because WL became excessive, resulting in peel injury and loss of juiciness into fruit. No previous research has included sensory evaluation of HW-treated

Valencia orange fruit.

It is likely that the orange fruit that received HWT and were then coated with either PL or CR had internal atmospheres during 3 weeks ambient storage with O2 and

CO2 levels that were nearest to the levels shown in previous research to be desired for quality maintenance (about 5% O2 + 10% CO2 – see Chapter 4) . On the other hand, fruit that had been treated with HW and then coated with SH had internal atmospheres during 3 weeks ambient storage that had extreme, injurious levels of O2 and CO2 (<5%

O2 + >15% CO2). The extreme atmosphere in SH-coated oranges apparently caused initiation of fermentative metabolism, resulting in ethanolic off-odors and off-flavors that the sensory panelists were able to perceive according to the comments they made during the testing. Previous work with citrus fruits has shown that CA and MA storage can help maintain better peel appearance and reduce negative changes in composition

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(Chase, 1969; Davis et al., 1973), but there have been no previous reports of sensory evaluation of MA- or CA-stored oranges.

Chapter Summary

The orange fruit that received HWT and were then coated with either PL or CR had internal atmospheres during 3 weeks ambient storage with O2 and CO2 levels that were nearest to the levels desired for quality maintenance (about 5% O2 + 10% CO2 – see Chapter 4). On the other hand, fruit that had been treated with HW and then coated with SH had internal atmospheres during 3 weeks ambient storage that had extreme, injurious levels of O2 and CO2 (<5% O2 + >15% CO2). The extreme atmosphere in SH- coated oranges caused initiation of fermentative metabolism, resulting in ethanolic off- odors and off-flavors that were perceived by the sensory panelists according to the comments they made during the testing.

In the above tests, it was shown that HW-treated orange fruit performed better than AMW-treated oranges. This was probably due to the reduction of O2 and increase of CO2 within the fruit during the HWT, which created a significant modification of the internal atmosphere environment as shown previously. Subsequent coating application, especially CR, produced an attractive peel appearance and maintained a beneficial MA within the oranges, which extended shelf life and retained quality during simulated ambient storage for 3 weeks. The taste panelists recognized this effect as improved retention of overall acceptability and overall appearance.

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Table 5-1. Sensory ratings of Navel oranges from Harvest 1 that received ambient or hot water immersion treatments followed by application or not of different fruit coatings with weekly evaluations over 3 weeks of ambient storage. Harv.z Storage duration (wk) Treatmenty OVACx OVAPx OVFLx OVTXx FRx JUCx SWSUx (1-5)v ORFx (1-5) v (1-9)w (1-9)w (1-9)w (1-9)w (1-5)v (1-5)v 1st 0 AMWz+NC 5.7 b 5.0 c 5.8 ab 6.1 3.1 ab 2.8 a 2.6 2.4 a HW+NC 6.2 a 5.6 b 6.2 a 6.1 3.2 ab 2.8 a 2.7 2.2 ab HW+PL 5.9 ab 6.2 a 5.7 ab 6.0 3.3 a 2.6 ab 2.6 2.2 ab HW+CR 5.7 b 5.5 bc 5.2 b 5.7 3.3 a 2.6 ab 2.4 2.0 b HW+SH 5.8 b 5.5 b 5.6 b 6.2 3.0 b 2.5 b 2.3 2.0 b

1 AMWz+NC 6.2 ab 6.0 a 6.1 ab 5.9 b 3.3 2.7 2.5 ab 2.2 HW+NC 6.5 a 6.5 a 6.4 a 6.3 a 3.4 2.8 2.8 a 2.3 HW+PL 5.8 b 5.5 b 5.6 b 6.1 ab 3.3 2.8 2.5 ab 2.3 HW+CR 6.3 a 5.5 bc 6.0 a 6.3 a 3.1 2.8 2.7 a 2.3 HW+SH 6.1 a 5.8ab 5.9 a 5.9 b 3.3 2.6 2.4 b 2.1

2 AMWz+NC 6.1 a 6.3 a 5.9 a 5.9 3.5 a 2.7 a 2.6 2.3 HW+NC 5.4 b 5.1 c 5.1 b 5.5 3.4 a 2.6 ab 2.5 2.2 HW+PL 5.7 b 5.0 c 5.8 ab 6.1 3.1 ab 2.6 ab 2.6 2.4 HW+CR 6.2 a 5.6 b 6.2 a 6.1 3.2 ab 2.8 a 2.7 2.3 HW+SH 5.9 ab 6.2 a 5.7 ab 6.0 3.3 a 2.5 b 2.6 2.2

3 AMWz+NC 5.7 b 5.5 bc 5.2 b 5.7 3.0 b 2.8 a 2.4 2.0 HW+NC 5.8 b 5.5 b 5.6 b 6.2 3.4 a 2.7 ab 2.3 2.3 HW+PL 6.2 ab 6.0 a 6.1 ab 6.2 3.3 ab 2.5 b 2.5 2.2 HW+CR 6.5 a 6.5 a 6.4 a 6.3 3.4 a 2.8 a 2.6 2.3 HW+SH 5.8 b 5.5 b 5.6 b 5.9 3.3 ab 2.7 ab 2.5 2.3 zHarv. = First harvest on 19 October 2015. y AMW = Ambient water (~25oC, 30 min); HW=Hot water (45oC, 30 min); NC=No coating; PL=Polyethylene coating; CR=Carnauba coating; SH=Shellac coating. xSensory attributes: OVAC = Overall acceptability, OVAP = Overall peel appearance, OVFL = Overall flavor, OVTX = Overall texture, FRM = Fruit firmness/softness, JUC = Juiciness, SWSU = Sweet sour balance, ORFL = Orange flavor. wHedonic scale: 1 = Dislike extremely, 2 = Dislike very much, 3 = Dislike moderately, 4 = Dislike slightly, 5 = Neither like nor dislike, 6 = Like slightly, 7 = Like moderately, 8 = Like very much, 9 = Like extremely. v Just About Right scale: 1 = too extreme (negative), 2 = slightly too much/little, 3 = just about right, 4 = slightly too much/little, 5 = too extreme (positive). uMeans within a column for each storage period followed by same small letter do not differ significantly according to Tukey’s test.

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Table 5-2. Sensory ratings of Navel oranges from Harvest 2 that received ambient or hot water immersion treatments followed by application or not of different fruit coatings with weekly evaluations over 3 weeks of ambient storage. Harv.z Storage duration (wk) Treatmenty OVACx OVAPx OVFLx OVTXx FRx JUCx SWSUx (1-5)v ORFx (1-5) v (1-9)w (1-9)w (1-9)w (1-9)w (1-5)v (1-5)v 2nd 0 AMWz+NC 6.0 4.2 c 5.8 6.3 ab 3.0 2.8 2.6 2.2 HW+NC 6.2 5.1 ab 6.0 6.4 a 3.2 2.9 2.6 2.4 HW+PL 5.9 4.7 b 6.0 6.1 ab 3.0 2.8 2.7 2.2 HW+CR 5.8 5.3 a 5.8 6.0 ab 3.2 2.9 2.6 2.3 HW+SH 5.9 5.5 a 5.6 5.9 b 3.2 2.8 2.5 2.3

1 AMWz+NC 5.7 b 4.0 c 5.6 b 5.9 3.2 a 2.8 2.5 b 2.2 HW+NC 6.1 ab 5.1 ab 6.0 ab 6.1 2.9 b 2.9 2.7 ab 2.4 HW+PL 6.2 a 5.0 b 6.0 ab 6.3 3.2 ab 2.7 2.9 a 2.4 HW+CR 6.1 ab 4.9 b 6.1 a 6.2 3.2 ab 2.7 2.7 ab 2.3 HW+SH 6.1 ab 5.5 a 6.0 ab 6.3 3.2 a 2.7 2.8 a 2.3

2 AMWz+NC 6.1 a 3.7 b 6.3 a 6.2 ab 2.9 b 3.0 a 2.8 ab 2.6 a HW+NC 5.9 ab 5.4 a 5.9 ab 6.1 ab 3.0 b 2.9 ab 2.7 ab 2.2 b HW+PL 6.3 a 4.9 a 6.2 a 6.3 a 2.9 b 3.0 a 2.9 a 2.4 ab HW+CR 6.1 ab 5.1 a 5.9 ab 6.1 ab 3.0 b 2.8 ab 2.8 ab 2.3 ab HW+SH 5.6 b 4.9 a 5.5 b 5.7 b 3.5 a 2.7 b 2.6 b 2.4 ab

3 AMWz+NC 6.1 ab 4.4 ab 6.1 ab 6.3 ab 2.6 c 3.0 a 2.6 b 2.5 a HW+NC 6.4 a 4.9 a 6.3 a 6.2 ab 2.8 bc 3.0 a 3.1 a 2.5 a HW+PL 5.5 b 4.3 b 5.5 b 6.1 ab 3.1 ab 2.7 b 2.7 b 2.2 b HW+CR 6.5 a 4.9 a 6.4 a 6.4 a 3.1 a 2.8 ab 2.8 b 2.3 ab HW+SH 5.5 b 4.5 ab 5.5 b 5.6 b 3.2 a 2.7 ab 2.7 b 2.2 b zHarv. = Second harvest on 10 November 2015. y AMW = Ambient water (~25oC, 30 min); HW=Hot water (45oC, 30 min); NC=No coating; PL=Polyethylene coating; CR=Carnauba coating; SH=Shellac coating. xSensory attributes: OVAC = Overall acceptability, OVAP = Overall peel appearance, OVFL = Overall flavor, OVTX = Overall texture, FRM = Fruit firmness/softness, JUC = Juiciness, SWSU = Sweet sour balance, ORFL = Orange flavor. wHedonic scale: 1 = Dislike extremely, 2 = Dislike very much, 3 = Dislike moderately, 4 = Dislike slightly, 5 = Neither like nor dislike, 6 = Like slightly, 7 = Like moderately, 8 = Like very much, 9 = Like extremely. v Just About Right scale: 1 = too extreme (negative), 2 = slightly too much/little, 3 = just about right, 4 = slightly too much/little, 5 = too extreme (positive). uMeans within a column for each storage period followed by same small letter do not differ significantly according to Tukey’s test.

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Table 5-3. Sensory ratings of Navel oranges from Harvest 3 that received ambient or hot water immersion treatments followed by application or not of different fruit coatings with weekly evaluations over 3 weeks of ambient storage Harv.z Storage Treatmenty OVACx OVAPx OVFLx OVTXx FRx JUCx SWSUx ORFx (1- duration (1-9)w (1-9)w (1-9)w (1-9)w (1-5)v (1-5)v (1-5)v 5) v (wk) 3rd 0 AMWz+NC 6.0 4.2 c 5.8 6.3 ab 3.0 2.8 2.6 2.2 HW+NC 6.2 5.1 ab 6.0 6.4 a 3.2 2.9 2.6 2.4 HW+PL 5.9 4.7 b 6.0 6.1 ab 3.0 2.8 2.7 2.2 HW+CR 5.8 5.3 a 5.8 6.0 ab 3.3 2.9 2.6 2.3 HW+SH 5.9 5.5 a 5.6 5.9 b 3.2 2.8 2.5 2.3

1 AMWz+NC 5.7 4.0 c 5.6 5.9 3.2 a 2.8 2.5 b 2.2 HW+NC 6.1 5.1 ab 6.0 6.1 2.9 b 2.9 2.7 ab 2.4 HW+PL 6.2 5.0 b 6.0 6.3 3.1 ab 2.7 2.7 ab 2.4 HW+CR 6.1 4.9 b 6.0 6.2 3.1 ab 2.7 2.7 ab 2.3 HW+SH 6.1 5.5 a 6.0 6.3 3.1 ab 2.7 2.8 a 2.3

2 AMWz+NC 6.1 ab 3.7 b 6.3 a 6.2 ab 2.9 b 3.0 a 2.8 ab 2.6 a HW+NC 5.9 ab 5.4 a 5.9 ab 6.1 ab 3.0 b 2.9 ab 2.7 ab 2.2 b HW+PL 6.3 a 4.9 a 6.2 a 6.3 a 2.9 b 3.0 a 2.9 a 2.4 ab HW+CR 6.1 ab 5.1 a 5.9 ab 6.1 ab 3.0 b 2.8 ab 2.8 ab 2.3 ab HW+SH 5.6 b 5.4 a 5.5 b 5.7 b 3.5 a 2.7 b 2.6 b 2.4 ab

3 AMWz+NC 6.1 ab 4.4 6.1 ab 6.3 ab 2.6 c 3.0 a 2.6 b 2.5 a HW+NC 6.4 a 4.9 6.3 a 6.2 ab 2.8 bc 3.0 a 3.1 a 2.5 a HW+PL 5.5 b 4.3 5.5 b 6.1 ab 3.1 ab 2.7 b 2.7 b 2.2 b HW+CR 6.5 a 4.9 6.4 a 6.4 a 3.1 a 2.8 ab 2.8 b 2.3 ab HW+SH 5.5 b 4.5 5.5 b 5.6 b 3.2 a 2.7 ab 2.7 b 2.2 b zHarv. = Third harvest on 06 January 2016. y AMW = Ambient water (~25oC, 30 min); HW=Hot water (45oC, 30 min); NC=No coating; PL=Polyethylene coating; CR=Carnauba coating; SH=Shellac coating. xSensory attributes: OVAC = Overall acceptability, OVAP = Overall peel appearance, OVFL = Overall flavor, OVTX = Overall texture, FRM = Fruit firmness/softness, JUC = Juiciness, SWSU = Sweet sour balance, ORFL = Orange flavor. wHedonic scale: 1 = Dislike extremely, 2 = Dislike very much, 3 = Dislike moderately, 4 = Dislike slightly, 5 = Neither like nor dislike, 6 = Like slightly, 7 = Like moderately, 8 = Like very much, 9 = Like extremely. v Just About Right scale: 1 = too extreme (negative), 2 = slightly too much/little, 3 = just about right, 4 = slightly too much/little, 5 = too extreme (positive). uMeans within a column for each storage period followed by same small letter do not differ significantly according to Tukey’s test.

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Table 5-4. Sensory ratings of Valencia oranges from Harvest 1 that received ambient or hot water immersion treatments followed by application or not of different fruit coatings with weekly evaluations over 3 weeks of ambient storage. Harv.z Storage Treatmenty OVACx OVAPx OVFLx OVTXx FRx JUCx SWSUx ORFx (1- duration (1-9)w (1-9)w (1-9)w (1-9)w (1-5)v (1-5)v (1-5)v 5) v (wk) 1st 0 AMWz+NC 6.3 ab 4.1 b 6.3 ab 5.9 a 3.2 abc 2.8 2.6 2.6 HW+NC 6.5 a 3.9 b 6.6 a 5.8 ab 3.1 bc 3.0 2.7 2.7 HW+PL 5.9 b 6.2 a 6.1 ab 5.4 b 3.4 a 2.8 2.6 2.6 HW+CR 6.2 ab 6.4 a 6.3 ab 5.8 ab 3.3 ab 2.9 2.5 2.7 HW+SH 6.1 ab 6.5 a 6.1 b 5.7 ab 3.0 c 2.8 2.5 2.6

1 AMWz+NC 6.2 a 4.3 b 6.2 a 5.8 ab 2.9 b 3.0 2.6 b 2.8 HW+NC 6.2 a 4.2 b 6.3 a 5.7 ab 3.0 b 2.9 2.5 b 2.9 HW+PL 6.4 a 6.5 a 6.5 a 5.8 ab 3.3 a 2.9 2.9 a 2.7 HW+CR 6.3 a 6.4 a 6.4 a 6.0 a 3.1 b 3.0 2.7 ab 2.8 HW+SH 5.1 b 6.4 a 5.1 b 5.3 b 3.1 ab 2.8 2.6 b 2.6

2 AMWz+NC 6.3 a 3.6 c 6.2 a 6.1 a 3.1 ab 2.8 2.7 a 2.8 a HW+NC 6.3 a 4.0 c 6.5 a 5.9 a 3.0 b 2.8 2.7 a 2.8 a HW+PL 6.3 a 6.5 a 6.3 a 5.9 a 3.3 a 2.8 2.8 a 2.7 a HW+CR 6.4 a 6.3 a 6.4 a 6.0 a 3.3 a 2.8 2.3 b 2.7 a HW+SH 4.2 b 5.6 b 3.9 b 4.9 b 3.0 b 2.7 2.3 b 2.3 b

3 AMWz+NC 5.9 ab 3.6 c 6.0 ab 5.6 3.2 2.8 2.6 2.5 HW+NC 6.1 a 3.5 c 6.4 a 5.6 3.1 2.9 2.8 2.6 HW+PL 6.1 ab 6.3 a 5.9 ab 5.6 3.1 2.8 2.7 2.6 HW+CR 6.0 ab 6.4 a 6.4 a 5.8 3.1 2.9 2.8 2.7 HW+SH 5.5 b 5.6 b 5.4 b 5.7 3.1 2.9 2.6 2.5 zHarv. = First harvest on 14 March 2016. y AMW = Ambient water (~25oC, 30 min); HW=Hot water (45oC, 30 min); NC=No coating; PL=Polyethylene coating; CR=Carnauba coating; SH=Shellac coating. xSensory attributes: OVAC = Overall acceptability, OVAP = Overall peel appearance, OVFL = Overall flavor, OVTX = Overall texture, FRM = Fruit firmness/softness, JUC = Juiciness, SWSU = Sweet sour balance, ORFL = Orange flavor. wHedonic scale: 1 = Dislike extremely, 2 = Dislike very much, 3 = Dislike moderately, 4 = Dislike slightly, 5 = Neither like nor dislike, 6 = Like slightly, 7 = Like moderately, 8 = Like very much, 9 = Like extremely. v Just About Right scale: 1 = too extreme (negative), 2 = slightly too much/little, 3 = just about right, 4 = slightly too much/little, 5 = too extreme (positive). uMeans within a column for each storage period followed by same small letter do not differ significantly according to Tukey’s test.

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Table 5-5. Sensory ratings of Valencia oranges from Harvest 2 that received ambient or hot water immersion treatments followed by application or not of different fruit coatings with weekly evaluations over 3 weeks of ambient storage. Harv.z Storage Treatmenty OVACx OVAPx OVFLx OVTXx FRx JUCx SWSUx (1- ORFx (1- duration (1-9)w (1-9)w (1-9)w (1-9)w (1-5)v (1-5)v 5)v 5) v (wk) 2nd 0 AMWz+NC 6.3 3.9 b 6.4 5.9 a 3.0 ab 3.0 2.7 2.6 ab HW+NC 6.2 4.0 b 6.3 5.7 ab 3.0 ab 3.0 2.9 2.7 a HW+PL 6.1 5.2 a 6.1 5.7 ab 3.1 a 2.9 2.9 2.5 ab HW+CR 6.1 5.1 a 6.0 5.9 a 3.0 ab 3.0 2.8 2.6 ab HW+SH 5.9 5.3 a 5.9 5.4 b 2.8 b 3.0 2.8 2.4 b

1 AMWz+NC 6.3 a 4.1 c 6.3 ab 5.6 2.9 b 3.0 a 2.9 a 2.6 ab HW+NC 6.2 ab 4.5 c 6.3 a 5.7 3.0 b 3.0 a 2.8 ab 2.7 a HW+PL 5.8 b 5.0 b 5.8 ab 5.5 3.3 a 2.7 b 2.8 ab 2.4 b HW+CR 6.2 ab 5.6 a 6.3 ab 5.7 3.1 ab 2.9 ab 2.8 ab 2.6 ab HW+SH 6.0 ab 5.8 a 5.8 b 5.7 2.8 b 2.9 ab 2.6 b 2.6 ab

2 AMWz+NC 6.2 a 3.7 b 6.3 a 5.6 3.1 3.0 2.9 a 2.6 a HW+NC 6.0 a 3.9 b 6.2 a 5.7 3.0 2.9 2.8 a 2.6 a HW+PL 5.7 a 5.3 a 5.8 a 5.4 3.3 2.8 2.8 a 2.5 ab HW+CR 5.8 a 5.3 a 5.9 a 5.4 3.1 2.9 2.8 a 2.5 ab HW+SH 4.8 b 5.4 a 4.8 b 5.2 3.1 2.9 2.5 b 2.2 b

3 AMWz+NC 5.9 a 4.2 c 6.0 a 5.6 ab 3.3 2.8 2.7 ab 2.3 HW+NC 6.0 a 4.3 c 6.0 a 5.7 a 3.2 2.9 2.9 a 2.6 HW+PL 5.9 a 5.2 b 6.0 a 5.7 a 3.2 2.7 2.8 ab 2.6 HW+CR 5.9 a 6.0 a 6.0 a 5.4 ab 3.3 2.7 2.8 ab 2.6 HW+SH 4.6 b 5.5 ab 4.5 b 5.0 b 3.3 2.8 2.5 b 2.4 zHarv. = Second harvest on 10 April 2016. y AMW = Ambient water (~25oC, 30 min); HW=Hot water (45oC, 30 min); NC=No coating; PL=Polyethylene coating; CR=Carnauba coating; SH=Shellac coating. xSensory attributes: OVAC = Overall acceptability, OVAP = Overall peel appearance, OVFL = Overall flavor, OVTX = Overall texture, FRM = Fruit firmness/softness, JUC = Juiciness, SWSU = Sweet sour balance, ORFL = Orange flavor. wHedonic scale: 1 = Dislike extremely, 2 = Dislike very much, 3 = Dislike moderately, 4 = Dislike slightly, 5 = Neither like nor dislike, 6 = Like slightly, 7 = Like moderately, 8 = Like very much, 9 = Like extremely. v Just About Right scale: 1 = too extreme (negative), 2 = slightly too much/little, 3 = just about right, 4 = slightly too much/little, 5 = too extreme (positive). uMeans within a column for each storage period followed by same small letter do not differ significantly according to Tukey’s test .

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Table 5-6. Sensory ratings of Valencia oranges from Harvest 3 that received ambient or hot water immersion treatments followed by application or not of different fruit coatings with weekly evaluations over 3 weeks of ambient storage. Harv.z Storage Treatmenty OVACx OVAPx OVFLx OVTXx FRx JUCx SWSUx (1- ORFx (1- duration (1-9)w (1-9)w (1-9)w (1-9)w (1-5)v (1-5)v 5)v 5) v (wk) 3rd 0 AMWz+NC 5.9 bv 3.6 b 5.9 b 5.6 b 2.9 b 2.8 ab 2.9 a 2.3 ab HW+NC 6.5 a 3.9 b 6.5 a 6.3 a 2.9 b 2.9 a 2.9 a 2.5 a HW+PL 5.9 b 5.2 a 5.7 b 5.7 b 3.2 a 2.7 b 2.7 b 2.3 ab HW+CR 6.0 ab 5.1 a 6.1 ab 5.9 ab 3.1 ab 2.9 a 2.8 ab 2.4 ab HW+SH 5.7 b 5.3 a 5.6 b 5.7 b 3.1 ab 2.8 ab 2.7 b 2.2 b

1 AMWz+NC 6.2 a 3.6 c 6.1 a 6.0 3.0 b 2.9 2.8 2.5 a HW+NC 6.0 ab 3.7 c 5.8 ab 5.6 3.0 b 2.8 2.8 2.3 ab HW+PL 5.6 b 5.5 a 5.6 b 5.5 3.3 a 2.8 2.7 2.3 ab HW+CR 5.6 b 5.8 a 5.5 b 5.7 3.3 a 2.7 2.7 2.3 ab HW+SH 5.9 ab 5.0 b 5.8 ab 5.8 3.0 b 2.9 2.8 2.2 b

2 AMWz+NC 6.1 a 3.8 c 6.0 a 5.7 3.3 2.7 2.7 2.5 ab HW+NC 6.1 a 3.9 c 6.1 a 5.9 3.0 2.8 2.9 2.4 ab HW+PL 5.6 ab 5.2 b 5.6 ab 5.6 3.2 2.7 2.7 2.3 b HW+CR 6.1 a 6.1 a 6.1 a 5.9 3.1 2.8 2.7 2.7 a HW+SH 5.3 b 4.9 b 5.2 b 5.4 3.1 2.7 2.8 2.3 b

3 AMWz+NC 5.8 ab 3.4 c 5.8 ab 5.7 3.1 b 2.7 ab 2.6 2.4 HW+NC 6.1 a 3.7 c 5.9 ab 5.7 3.2 ab 2.8 a 2.8 2.4 HW+PL 6.0 a 5.5 b 6.0 a 5.6 3.4 a 2.6 ab 2.8 2.5 HW+CR 6.2 a 6.5 a 5.4 b 5.3 3.4 a 2.5 b 2.6 2.3 HW+SH 5.4 b 5.0 b 5.5 ab 5.7 3.2 ab 2.8 a 2.7 2.3 zHarv. = Third harvest on 08 May 2016. y AMW = Ambient water (~25oC, 30 min); HW=Hot water (45oC, 30 min); NC=No coating; PL=Polyethylene coating; CR=Carnauba coating; SH=Shellac coating. xSensory attributes: OVAC = Overall acceptability, OVAP = Overall peel appearance, OVFL = Overall flavor, OVTX = Overall texture, FRM = Fruit firmness/softness, JUC = Juiciness, SWSU = Sweet sour balance, ORFL = Orange flavor. wHedonic scale: 1 = Dislike extremely, 2 = Dislike very much, 3 = Dislike moderately, 4 = Dislike slightly, 5 = Neither like nor dislike, 6 = Like slightly, 7 = Like moderately, 8 = Like very much, 9 = Like extremely. v Just About Right scale: 1 = too extreme (negative), 2 = slightly too much/little, 3 = just about right, 4 = slightly too much/little, 5 = too extreme (positive). uMeans within a column for each storage period followed by same small letter do not differ significantly according to Tukey’s test

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CHAPTER 6 USE OF HOT WATER IMMERSION AND EDIBLE COATING TO MAINTAIN QUALITY AND SHELF LIFE OF SWEET ORANGE AND POMELO FRUIT DURING SIMULATED AMBIENT TEMPERATURE STORAGE IN BANGLADESH

Introduction

Citrus (Citrus sp.) fruits are nonclimacteric, with persistently low respiration and ethylene production rates during ripening, which occurs only on the tree. Citrus fruits do not undergo any major softening or compositional changes after harvest. In

Bangladesh, citrus is grown on a small scale in commercial plantations and also in backyard orchards and small holdings in different regions of the country. Sweet orange

(Citrus sinensis; Malta) and pomelo (Citrus grandis) fruits are the most popular citrus types in the country due to their desirable good taste and flavor. In the sub-continent, juicy sweet mandarin oranges are locally known by the name of Malta fruit. Malta fruit have thin peel with juicy segments. Both Malta and pomelo are very much favored in

Asian countries.

Hot water treatment (HWT) potentially has a significant role to play in controlling the quality of produce. Heat treatments can modify quality changes by affecting the plant antioxidant system (Schoffl et al., 1998). Whe applied as hot water (HW) immersion, favorable internal atmospheres that benefit ripening and postharvest quality can be created (Ummarat et al., 2011). Reduction of respiration and delay of senescence are also potential results of heat treatment that are indispensable to maintenance of quality and shelf life (Escrinbano and Mitcham, 2014) during either short time storage, as a link between harvest and marketing, or for longer storage to extend the postharvest life of the product for distant marketing.

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Similarly, application of fruit coatings is a promising technology to keep citrus fruits firm and with shiny appearance, and to preserve their quality by modifying the internal atmosphere. Coatings have potential benefits for citrus fruits related to proper regulation of gas exchange in the fruit; an internal modified atmosphere (MA) created and maintained by the interaction of fruit respiration and a semipermeable coating can potentially stabilize the produce and thereby promote longer shelf life (Baldwin et al.,

1995). Coatings form barriers against H2O, O2 and CO2 diffusion that helps to reduce the respiration rate of the produce (Saftner, 1999). Coatings for fresh produce can reduce commodity respiration after harvest by maintaining reduced O2 and elevated

CO2 internal concentrations. The resulting reduced respiration rate slows metabolic loss and promotes a more gradual progress of senescence (Dhall, 2013).

On the other hand, continuous electricity supply and modern storage facilities are big challenges in developing countries such as Bangladesh for growers, handlers, and traders to retain fruit quality and extend storage. Moreover, the Bangladesh government has emphasized adoption of power saving technology for agricultural commodities where production of electricity is insufficient and per unit power cost is too expensive to be affordable for our producers. With a view to reduce physiological injury, enhance citrus fruit quality, and overall to generate power saving technology for agricultural producers, especially citrus growers, two research studies were conducted to determine the effect of a previously selected HWT and a surface coating of carnauba wax (CR) to retain quality and extend shelf life of sweet orange (BARI Malta 1) and pomelo fruit

(unnamed commercial variety) during storage at ambient temperature.

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Materials and Methods

Sample Collection and Experimentation

These research experiments were conducted in 2016 at the Postharvest

Technology Divisional (PHTD) Lab facility of BARI. Mature sweet oranges (BARI Malta

1) of uniform size (90-110 g, 130-150 g) were collected from two different locations with each serving as a separate experiment. Fruit from the first location were harvested on

October 24, 2016 from the Horticultural Research Center (HRC) orchard, Joydebpur,

BARI, Bangladesh, and the second harvest was on November 05, 2016 from a commercial field in Meherpur Sadar. Two additional experiments utilizing mature, uniform size (850-950 g, 950-1050 g) pomelo fruit (unnamed commercial variety) were conducted using fruit from two commercial groves in the Moulovibazar district. Fruit from the first location were harvested on October 30, 2016 and the second harvest was on

November 20, 2016. All fruit were transported by motor vehicle to the BARI PHTD lab facility, washed with ambient water (AMW) to remove dirt as well as to reduce field heat, and kept overnight in a storage chamber that was maintained at 25oC until application of treatments.

The experiments were designed as a completely randomized design with a complete factorial arrangement of treatments: with/without HW and with/without CR.

The best combination of HW temperature (45oC) and water immersion duration (30 min) and food grade CR had been determined in previous research in Florida with Valencia and Navel oranges through evaluation of physicochemical and sensory parameters.

Prior to initiating the experiments, the fruit were exposed to ambient air for 1-2 hours to equilibrate the fruit temperature with the environment. Then, the fruit were sorted and randomly distributed among the treatments and submerged into HW at 45oC or ambient

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water (AMW) for 30 min (oranges) or 45 min (pomelos) at the HW plant using the BARI

HW instrument (locally made), which uses an adjustable speed, electronically powered roller conveyor to move submerged fruit in crates along the length of a 3.11 m x 1.17 m x 1.53 m (length x width x height) trough, with the water heated using adjustable gas burners and the temperature manually monitored and controlled. Three replications (10 fruit/replicate for oranges; 8-10 fruit for pomelos) were considered for each treatment to analyze physicochemical parameters, and 15-20 orange fruit or 10-12 pomelo fruit were used for weekly sensory evaluations during 3 weeks of storage at 25oC in the aforementioned storage chamber. The humidity during storage was observed to be

75±5%RH.

Fruit washing, waxing and hot air drying. Treated fruit were washed with water containing 5% detergent (Wheel powder, BD Ltd.) on the PHTD BARI designed and fabricated brush bed @8-10 rpm for a few seconds and then rinsed thoroughly with ambient water. For uniform surface coating, a hand sprayer was used to apply CR at a rate of about 1 liter per 500 fruit for oranges and per 250 fruit for pomelos on the brush bed. Then, the fruit surfaces were dried with a hot air blower (45oC) for 2-3 minutes on a stationary table. After application of all treatments, the oranges and the pomelos were stored in the PHTD packing house facility (Figure 6-1).

Fruit Peel Appearance and Fruit Firmness Resistance

Fruit weight loss. Weight loss (WL) was determined for 30 fruit per treatment

(10 fruit/replicate) compared to the initial weight just after the HW and coating application. The fruit were weighed every week for 3 weeks and WL reported as a percentage of the initial fruit weight.

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Fruit peel color. Fruit peel color was determined using a tristimulus colorimeter

(CR-400, Minolta Corp., Japan) with 8-mm aperature and C light source at two equidistant points on the equator of each fruit using the CIE color system on the L*, a*, b* color space where L*a*b* coordinates were recorded using the D65 illuminant and a

10° standard observer as a reference system. L* is lightness; a* (-greenness to

+redness) and b* (-blueness to +yellowness) are the chromaticity coordinates. The a* and b* values were converted to hue angle and chroma.

Fruit firmness (peel puncture resistance). Fruit firmness (FF) was determined by measuring fruit peel puncture resistance using a Digital Firmness Tester (DFT 14,

Agro Technologies, France) whereby each of 8-10 fruit in a replicate was placed on a sample platform with a constant probe speed of 2 mm s-1. Resistance force was measured on opposite sides of each fruit. Fruit firmness was measured based on resistance to penetration using an 8-mm diameter flathead probe, which penetrated the fruit peel by 5 mm. The mean value of each fruit puncture resistance was calculated and expressed in N.

Nutritional Quality Study

Soluble solids content and titratable acidity. Soluble Solids Content (SSC) content was measured from a 10.0-g juice composite sample of each replicate by using a temperature-compensated automatic refractometer (Model NR151, ICT, SL) and expressed as percent. Total titratable acidity (TTA) was determined by titration with 0.1

N NaOH according to the method used by Ranganna (1986). The TTA of the fruit juice was expressed as percent citric acid equivalents.

Ascorbic acid content. Ascorbic acid content (ASC) was determined according to Ranganna (1986) using 10.0-g samples of fruit pulp blended for 2 mins in a kitchen

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food blender and homogenized with 50 mL of cold 3% metaphosphoric plus 8% acetic acid mixture. Next, the samples were filtered through Whatman No. 2 filter paper. The clear supernatant samples were collected for assaying ASC and 10 ml aliquots were titrated with 0.1% 2,6-dichlorophenolindophenol solution until the filtrate changed to a pink color that persisted for at least 15 s. The titer value was recorded for each sample.

Prior to titration, 2,6-dichlorophenolindophenol solution was calibrated using an ascorbic acid standard solution. The results were expressed as mg/100 g of fruit fresh weight

(FW).

Total carotenoid content. The estimated total carotenoid content (TCC) was determined by extraction of a 3.0-g composite fruit pulp tissue sample with acetone

(Fisher Scientific Ltd., UK) and petroleum ether. It was further purified with acetone, methanolic KOH, and distilled water. The resulting solutions were filtered with anhydrous sodium sulphate and absorbance read on a spectrophotometer (T-80, PG

Instrument Ltd., UK) at 451 ɳm against petroleum ether as a blank. A standard curve was prepared using synthetic crystalline total ß-carotene (Fluka, Germany) dissolved in petroleum ether and its optical density was measured at 451 ɳm (Alasalvar et al., 2005) and TCC was expressed as ug/100 g FW.

Sensory Quality Evaluation

The sensory acceptability of the oranges was determined by panelists after 0, 2

& 3 weeks of storage. Each treatment was assigned a three-digit random number to avoid bias among panelists. The samples were presented to panelists in different orders as followed similar method in Chapter 5 to avoid order preference among panelists. The fruit were rated by 25-40 non-experienced panelists who were asked to rate samples using 9-point hedonic scales for overall acceptability, overall peel appearance, overall

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flavor, overall texture, and fruit firmness/softness; 5-point just about right (JAR) scales were used for juiciness, sweet-sour balance, and orange flavor (Appendix B).

Statistical analysis

The nutritional quality and physical attributes were measured, and the sensory studies conducted initially and also at weekly intervals during 3 weeks of ambient storage. The quality and physicochemical data were analyzed using analysis of variance based on proc GLM (SAS Institute Inc. Version 9.3, Cary, NC, USA) using a

CRD design with harvest location (HL), HW, coating treatment (T) which were with/without HW, with/without CR, and storage period (S) as the main effects. The sensory data were analyzed using SAS software and mean separation of each parameter was based on Tukey’s test at P<0.05.

Results

Effect of Hot Water Treatment and Carnauba Wax Coating on Improving Quality Retention of Sweet Orange (BARI Malta 1) During Ambient Storage

Weight loss. There was a significant S x HL interaction for WL (Table 6-1), such that the maximum WL was consistently higher during storage for orange fruit from HL 1 than for HL 2 (Table 6-2). There was also a significant interaction of S x T (Table 6-1), such that fruit WL significantly increased during storage with higher WL exhibited in non-CR versus CR fruit. For non-CR fruit, HW also increased WL (Table 6-3). Similar results for fruit WL were noticed for the T x HL interaction during 3 weeks storage

(Table 6-1).

Hot water + CR fruit had significantly reduced WL after 3 weeks of storage

(8.17ijk) for fruit from HL 2 (P<0.05), which was followed by AMW+CR fruit from HL 1

(8.53 ij), but the greatest WL after 3 weeks storage (Figure 6-2) occurred in HW without

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CR fruit (22.99a) (Table 6-6) from HL 1, which was followed by AMW without CR fruit

(17.34b) from HL 1.

Peel appearance. The highest peel lightness value was in HW+CR fruit from HL

1 (48.52) and HL 2 (68.12) compared to other treatment combinations (Figure 6-4 &

Figure 6-5). Similar trends were observed for a* (Figure 6-6 & Figure 6-7) and b* (Figure

6-8 & Figure 6-9) during 3 weeks storage. For both HL, the orange fruit turned from greenish to yellowish and the lightness of the fruit peel increased during 3 weeks storage (Figure 6-2).

Fruit firmness (peel puncture resistance). The interactions of HW, S, and HL had significant effects on FF during storage among the treatments (Table 6-1). Initially

HW fruit were found to have higher FF and then it declined with increasing storage duration. Firmness of CR fruit decreased more slowly than non-CR fruit (Table 6-3).

After 3 weeks of storage, non-CR fruit had higher FF (2.50 bc & 2.78 ab) than CR fruit

(2.33 cd & 2.20 d) (Table 6-4), which was similar to the initial FF, suggesting that WL occurred, resulting in toughening that accompanied the fruit peel shrinkage that was noted on the surface of non-CR fruit during the storage period (Table 6-4). Similar results were exhibited in Table 6-5 & Table 6-6 in that CR assisted in reducing the decline in WL during fruit during storage and the peels of non-CR fruit eventually toughened, which was measured as greater FF by the peel puncture test used in this study.

Soluble solids content (SSC). The S x HL interaction had a significant effect on

SSC among the treatments during storage (Table 6-1). The SSC was lowest in fruit from

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HL 2 (7.18 d) after 3 weeks storage (Table 6-3) whereas the maximum SSC was in fruit from HL 1 (11.99 a). Fruit from HL 1 had higher SSC than HL 2 fruit.

Soluble solids content (SSC)/Total titratable acidity (TTA) ratio. The

SSC/TTA ratios were significantly different for S x HL, T x HL, and S x T x HL interactions of sweet oranges (Table 6-2). In terms of the S x HL interaction, the maximum SSC/TTA ratio (89.07a) (Table 6-3) was observed in fruit from HL 1 compared to HL 2. The presence of CR and application of HW generally had no effect on SSC/TTA ratio during storage for sweet orange fruit from either HL (Table 6-5 and

Table 6-6).

Ascorbic acid content (ASC). There were no significant differences for the CR effect related to S or HL. But the S x HL interaction significantly influenced ASC (Table

6-2). The highest ASC (62.10 a) was observed in fruit from HL 1 after 1 week of storage and the lowest ASC (14.44 c) was found initially in fruit from HL 2 and this trend was continued throughout the storage period (Table 6-3).

Total carotenoids content (TCC). The TCC in sweet oranges was significantly affected by S, HL, and T, but only the S x HL interaction had a significant effect on TCC

(Table 6-2). The highest amount of TCC was observed in fruit from HL 1 after 3 weeks

(19.77 a) and the lowest amount (4.53 c) was noted initially in fruit from HL 2 (Table 6-

3). The TCC significantly increased by 2-fold during storage in fruit from HL 1, but did not change in fruit from HL 2 (Table 6-3).

Sensory quality evaluation

As shown in Table 6-7, among the treatments for HL 1, most of the panelists rated CR fruit highly (7.61 a & 7.65 a) by considering the overall likability scale (0-9

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scale) after 3 weeks storage (Figure 6-2). In the case of overall fruit peel appearance, panelists extremely preferred HW+ CR fruit (7.65 a), which was followed by AMW+CR fruit. Coated fruit were shiny, smooth and had an attractive surface as per sensory taster remarks in the recorded data sheets. In the case of overall flavor, the highest scores was also observed for HW+CR (7.13 a) and AMW+CR (7.22 a) treatments after

3 weeks storage. Other attributes had similar ranks among the treatments.

In the case of HL 2, panelists ranked both CR and non-CR fruit highly considering overall acceptability, but for overall peel appearance judgment, CR fruit had higher scores (7.50 a, 7.33 ab) than non-CR fruit after 3 weeks storage (Figure 6-2). No significant differences were found in overall flavor among the treatments during storage.

In terms of FF, higher values were found in AMW+CR fruit (3.50 a) and lower FF values in HW without CR fruit (2.88 c) after 3 weeks storage. All other attributes were found to have similar scores, as evaluated by the JAR scale.

Effect of Hot Water Treatment and Carnauba Wax Coating on Improving Quality Retention of Pomelo Fruit (Var. Commercial) During Ambient Storage

Whole fruit weight loss. In terms of the S x HL interaction, the maximum WL was found in fruit from HL 2 (16.69 a), which was followed by HL 1 (13.20 b) (Table 6-

10) after 3 weeks storage. Fruit WL significantly increased during storage and the highest amount of WL was exhibited by HW without CR (17.01a) and AMW without CR

(18.18 a) fruit after 3 weeks storage (Table 6-11). Insignificant differences were noted for the HW x HL interaction and the S x HW x T interaction during 3 weeks storage at ambient conditions (Figure 6-3).

Pomelo peel appearance. The highest peel lightness was observed in HW+CR fruit from HL 1 (39.52*) (Figure 6-10 & Figure 6-11). Similar trends were observed for a*

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(Figure 6-9 & Figure 6-10) and b* (Figure 6-14 & Figure 6-15) during the storage. For both HL 1 and HL 2, the fruit turned from greenish to yellowish, but the lightness of the fruit peel changed little during storage in HW+CR fruit.

Fruit firmness (peel puncture resistance). As shown in Table 6-9, T, S, and

HL, as well as their interactions had a significant effect on FF during storage. Initially,

HW fruit were found to have higher FF, which was then reduced during storage.

Firmness of CR fruit decreased slower than non-CR fruit (Table 6-11) up to 2 weeks.

Similar results were observed in Table 6-12 & Table 6-13 for FF. Treating fruit with

HW+CR assisted in decreasing WL from the fruit during storage.

Soluble solids content (SSC). As shown in Table 6-8, the S x HL, S x T, and S x HL x T interactions had significant effects on SSC during storage among the treatments. A higher amount of SSC was found in fruit from HL 2 compared to HL 1

(Table 6-10), but there was no consistent effect of CR or HW on SSC (Table 6-11).

Total titratable acidity (TTA). Total titratable acidity was significantly different in terms of the S x HW and S x HL x HW interactions among the treatments (Table 6-8).

Hot water plus CR fruit had lower TTA during storage (Table 6-11). Lower TTA was noticed in HW+CR fruit (8.90 bcde) and the maximum was observed in HW without CR fruit (10.02 a) after 3 weeks storage (Table 6-11).

Soluble solids content (SSC)/Total titratable acidity (TTA) ratio. The S x HW interaction had a significant effect on SSC/TTA ratio during storage (Table 6-8). After 3 weeks of storage, the maximum SSC/TTA ratio was observed in HW+CR fruit (12.38 abc), which indicated TTA decreased with storage, suggesting a bit of sourness reduction would be noticed in HW+CR fruit (Table 6-11).

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Ascorbic acid content (ASC). There were significant differences due to the S x

HL, S x T, and S x HL x T interactions on ASC during storage (Table 6-9). Ascorbic acid content decreased during storage for all treatments (Table 6-11). After 3 weeks storage, the highest ASC was observed in AMW+ CR fruit (51.03 defghi) from HL 2 and the lowest was in HW+CR fruit (35.11 i) from HL 1 (Table 6-13).

Total carotenoids content (TCC). Total carotenoids were significantly affected by the S x HL, S x T, T x HL, and S x HL x T interactions (Table 6-8). Hot water treatment increased TCC early on during storage, but the effect was lost later (Table 6-

11). Total carotenoids content was higher in fruit from HL 1 (Table 6-12) and increased during storage for all treatments (Table 6-13), except TCC increased up to 1 week and after that it was slightly reduced for HL 2.

Sensory quality evaluation

As shown in Table 6-14, among the treatments for HL 1, most of the panelists rated HW+CR fruit higher than other treatments (7.3 a) by considering overall likability scale (0-9 scale) after 2 weeks storage. In the case of overall fruit peel appearance, the panelists extremely preferred HW+CR fruit (7.6 a) and HW without CR fruit (7.1 a).

Coated fruit had shiny, smooth and attractive surfaces as per sensory taster remarks in the recorded data sheets. In the case of overall flavor, higher scores were also observed for HW+CR fruit (6.9 a) and AMW+CR fruits (7.0 a) after 3 weeks (Figure 6-3).

In the case of HL 2, the panelists ranked both CR and non-CR fruit higher considering overall acceptability, but for overall peel appearance judgment, fruit treated with HW+CR had the highest score (7.5 a) among the treatments after 3 weeks storage

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(Figure 6-3). No significant differences were found in all other attributes after 3 weeks storage. Other attributes were evaluated by the JAR scale.