Citation
1-Methylcylopropene treatment efficacy in preventing ethylene perception and ripening in tact and fresh-cut 'Galia' melon and 'Sunrise Solo' papaya fruits

Material Information

Title:
1-Methylcylopropene treatment efficacy in preventing ethylene perception and ripening in tact and fresh-cut 'Galia' melon and 'Sunrise Solo' papaya fruits
Creator:
Ergun, Muharrem
Publication Date:
Language:
English
Physical Description:
xiv, 185 leaves : ill. ; 29 cm.

Subjects

Subjects / Keywords:
Horticultural Science thesis, Ph.D ( lcsh )
Dissertations, Academic -- Horticultural Science -- UF ( lcsh )
Genre:
bibliography ( marcgt )
theses ( marcgt )
non-fiction ( marcgt )

Notes

Thesis:
Thesis (Ph.D.)--University of Florida, 2003.
Bibliography:
Includes bibliographical references.
General Note:
Printout.
General Note:
Vita.
Statement of Responsibility:
by Muharrem Ergun.

Record Information

Source Institution:
University of Florida
Holding Location:
University of Florida
Rights Management:
The University of Florida George A. Smathers Libraries respect the intellectual property rights of others and do not claim any copyright interest in this item. This item may be protected by copyright but is made available here under a claim of fair use (17 U.S.C. §107) for non-profit research and educational purposes. Users of this work have responsibility for determining copyright status prior to reusing, publishing or reproducing this item for purposes other than what is allowed by fair use or other copyright exemptions. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder. The Smathers Libraries would like to learn more about this item and invite individuals or organizations to contact the RDS coordinator (ufdissertations@uflib.ufl.edu) with any additional information they can provide.
Resource Identifier:
030441578 ( ALEPH )

Downloads

This item has the following downloads:


Full Text
1-METHYLCYCLOPROPENE TREATMENT EFFICACY IN PREVENTING ETHYLENE PERCEPTION AND RIPENING IN INTACT AND FRESH-CUT
'GALIA' MELON AND 'SUNRISE SOLO' PAPAYA FRUITS
By
MUHARREM ERGUN
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 2003




ACKNOWLEDGMENTS
This dissertation could not have been completed without the support of many people who are gratefully acknowledged here.
My greatest debt is to Dr. Donald JI Huber who has been a dedicated advisor and mentor, and kindest friend. He provided constant guidance to my academic work and research projects. This dissertation task would not be in the current form without his insightful input and constructive criticism.
I extend my appreciation to my supervisory committee, Dr. Jerry A. Bartz, Dr. Daniel J. Cantliffe, Dr. Charles L. Guy, and Dr. Steven A. Sargent, for their academic guidance. I am very thankful to Dr. Daniel J. Cantliffe and his graduate student Juan C. Rodriguez for allowing me to use their produce 'Galia' melon in my experiments. I am very grateful to Dr. Steven A. Sargent for his constant supervision in any issues. I thank Dr. Jerry A. Bartz and his lab personnel for their guidance and help. I express my gratitude to Dr. Charles L. Guy for his welcoming assistance. I also thank James Lee for the teaching, training, and friendly, never-ending assistance in the laboratory.
I would like to express my deepest appreciation to the Ministry of Education of Turkey who has supported my scholastic and living expenses during my graduate education in the U.S.
I would like to make a special acknowledgement to my friends, graduate students, and faculties and personnel of the postharvest research group for their kindly support and help.
ii




Finally, I thank my father, Ismail, mother, Unzile, brothers, Sadik, Sezai and Recai, and sister, Meryem for their emotional and financial support not only during my Ph.D education but also throughout my life.
iii




TABLE OF CONTENTS
Page
ACKNOW LEDGM ENTS ............................................................................................... ii
LIST OF TABLES ....................................................................................................... viii
LIST OF FIGURES ........................................................................................................ ix
A B S T R A C T ................................................................................................................. x iii
I IN T R O D U C T IO N ........................................................................................................ 1
2 LITERATURE REVIEW ............................................................................................. 4
M e lo n ....................................................................................................................... 4
In tro d u ctio n ....................................................................................................... 4
H arv e st M atu rity ............................................................................................... 4
R ip e n in g P ro c e ss ............................................................................................... 5
Postharvest Storage of M elon Fruit .................................................................... 7
P o sth arv e st D isease s .......................................................................................... 7
Postharvest Disease Control .............................................................................. 8
P a p a y a ...................................................................................................................... 9
In tro d u ctio n ....................................................................................................... 9
Uses of Papaya Fruit ........................................................................................ 10
Papaya Harvest M aturity ................................................................................. 10
P ap ay a R ip e n in g .............................................................................................. I I
Postharvest Handling and Storage of Papaya ................................................... 13
C h illin g Inju ry ................................................................................................. 14
Postharvest Pathology ...................................................................................... 14
I-M ethylcyclopropene ............................................................................................ 16
In tro d u ctio n ..................................................................................................... 16
Treatment Procedures for I -M CP .................................................................... 17
I-M C P an d E thy len e ....................................................................................... 17
1 -M CP and Fruit Softening .......... ................... ............................................... 19
The Influence of Suppressed Ethylene Perception on Ripening Physiology and
B io ch e m istry ................................. .............................................................. 2 0
Physiology of Fresh-cut Produce ........... .......................................................... 25
In tro d u ctio n ..................................................................................................... 2 5
Consequences of Processing ............................................................................ 25
External and Internal Factors Contributing to Quality of Fresh-cut Fruits and
V e g e ta b le s ................................................................................................... 2 8
iv




Cell W all ................................................................................. 32
introduction.......................................................................... 32
Cell Wall Structure.................................................................. 33
Cell Wall Loosening and Growth................................................... 34
Fruit Ripening and the Cell Wall................................................... 34
The Cell Wall and Pathogen Attacks............................................... 35
3 DELAYING ETHYLENE-INDUCiBLE RIPENING PROCESS 1METHYLCYCLOPROPENE IN 'GALIA' MELON FRUIT ....................... 36
Introduction .............................................................................. 36
Materials and Methods................................................................... 37
Plant Material........................................................................ 37
l-MCP Application ................................................................. 38
Respiration and Ethylene Production .............................................. 39
Firmness Assessment................................................................ 39
Electrolyte Leakage Assessment ................................................... 39
Soluble Solids Concentration, pH and Titratable Acidity Determination....... 40 Statistical and Informal Taste Analyses ........................................... 40
Results .................................................................................. 41
1 -MCP Concentration and Efficacy................................................ 41
Respiration and Ethylene Production .............................................. 41
Firmness..................................................................... 42
Electrolyte Leakage ........................................................ 43
Soluble Solids Concentration, pH and Titratable Acidity ....................... 43
Informal Quality Analysis .......................................................... 44
Discussion............................................................................... 45
4 PHYSIOLOGICAL CHANGES IN FRESH-CUT AND INTACT 'GALIA' MELON
FRUIT WITH TREATED I1-METHYLCYCLOPROPENE ...................59
Introduction .............................................................................. 59
Materials and Methods................................................................... 61
Plant Material........................................................................ 61
I -MCP Quantification and Treatment.............................................. 61
Preparation of Fresh-cut 'Galia', and Treatment Design ........................ 62
Ethylene Analysis ................................................................... 63
Firmness Assessment................................................................ 63
Electrolyte Leakage ................................................................. 63
Pectin Efflux ............................................. ........................... 64
Quality Evaluation................................................................... 65
Microbial Counts .................................................................... 65
Experimental Design and Statistics ................................................ 66
Results and Discussion................................................................... 66
Ethylene Production................................................................. 66
Firmness Assessment................................................................ 68
Electrolyte Leakage ............................. .......................... 69
V




Pectin Efflux......................................................................... 69
Quality Evaluation................................................................... 71
Microbial Counts .................................................................... 73
5 STORAGE life EXTENTION OF PRE-RIPE AND) RIPE 'SUNRISE SOLO'
PAPAYA FRUIT BY I1-METHYLCYCLOPROPENE................................ 85
Introduction .............................................................................. 85
Materials and Methods................................................................... 86
Plant Material........................................................................ 86
1-MCP Preparation and Treatment ................................................. 87
Respiration and Ethylene Production .............................................. 88
Firmness Determination............................................................. 88
Electrolyte Efflux.................................................................... 88
Soluble Solids Concentration, pH, and Titratable Acidity....................... 89
Statistical and Informal Taste Analyses ........................................... 89
Results .................................................................................. 89
Effective I -MCP Concentration .................................................... 89
Respiration and Ethylene Production .............................................. 90
Mesocarp Firmness.................................................................. 91
Electrolyte Efflux.................................................................... 91
Soluble Solids Concentrations, Titratable Acidity and pH....................... 92
Fruit E valuation ........................................ ........92
Discussion............................................................................... 93
6 QUALITY AND STORAGE LIFE OF iNTACT AND FRESH-CUT PAPAYA FRUIT
TREATED WITH I -MCP AT THE POSTCLIMACTERIC STAGE OF
DEVELO PM EN T .... ..................................... ... ........ 109
Introduction.............................................................................. 109
Materials and Methods...............................................................111.II
Plant Material ..................................................................... 111I
I1-MCP Treatment .................................................................. 112
Fruit Preparation and Treatment Design.......................................... 112
Ethylene Production ............................................................... 113
Firmness ............................................................................ 113
Electrolyte Leakage............................................................... _114
Color and Sensory Evaluation..................................................... 114
Microbial Count.................................................................... 115
Statistical Analysis................................................................. 116
Results and Discussion .......... ...................................................... 116
Ethylene Production ............................................................... 116
Firmness Assessment .............................................................. 118
Electrolyte Leakage .......................................... ..................... 119
Color and Sensory Evaluation..................................................... 121
Microbiological Counts............................................................ 124
vi




7 CELL WALL MODIFICATION IN4 POSTCLIMACTERIC FRESH-CUT AND
INTACT PAPAYA FRUIT WITH AND WITHOUT 1
METHYLCYCLOPROPENE ........................................135
Introduction.............................................................................. 135
Materials and Methods ................................................................. 138
Plant Material and I -MCP Treatment ............................................ 138
Ethanol-insoluble Solids........................................................... 139
Total Soluble Sugars and Polyuronides........................................... 140
Sequential Fractionation of Cell Wall Materials ................................. 140
Hemnicelullosic Polysaccharide Extraction ....................................... 140
Compositional Analysis of Cell Wall Polymers ................................. 141
Results ................................................................................. 142
Ethanol-insoluble Solids and Total Soluble Sugars.............................. 142
Polyuronides and their Sequential Fraction ...........................142
Hemicellulosic Polysaccharides................................................... 144
Compositional Analysis of Cell Wall Polymers ................................. 144
Discussion .............................................................................. 145
8 SUMMARY AND CONCLUSION ......................................162
Influence of Ethylene-action Inhibition on Ripening of 'Galia' Melon Fruit ...... 162
Influence of Ethylene-action Inhibition on Ripening of 'Sunrise Solo' Melon
Fruit.............................................................................. 163
Cell Wall Modification of 'Sunrise Solo' Papaya Fruit in Response to Fresh-cut
Processing and I -M CP ................................. ..........164
LIST OF REFERENCES............................ ........................................ 166
BIOGRAPHICAL SKETCH .............................................................. 185
vii




LIST OF TABLES
Table pge
2-1. 1 -M CP-induced effects on ripening fruits .............................................................. 21
4-1. Microbial counts (CFU g-1 fresh weight) for intact ripe 'Galia' fruit with and
without 1-MCP and fresh-cut fruit derived from intact ripe fruit treated with and
w ithout I-M CP during storage at 5 'C ........................................................... 84
6-1. Microbial counts (CFU g' fresh weight) for intact postclimacteric 'Sunrise Solo'
papaya fruit pre-treated with 2.5 ptL L-1 1-MCP (IM) and air (control, IC), and for
fresh-cut postclimacteric fruit derived from either the intact air-treated (FCC) or the intact 1-MCP-treated (FCM) fruit during storage at 5 'C .................................... 134
7-1. Ethanol insoluble solids (EIS) and total soluble sugars (TSS) of intact three-quarter
ripe papaya fruit treated with and without 1 -MCP and fresh-cut fruit derived from
intact three-quarter ripe fruit treated with and without I -MCP papaya during
sto ra g e ............................................................................................................. 14 9
7-2. Polyuronide composition of intact three-quarter ripe papaya fruit treated with and
without I -MCP and fresh-cut fruit derived from intact three-quarter ripe fruit
treated with and without 1-MCP papaya during storage ...................................... 150
7-3. Neutral hemicellulose and pectin residue composition of intact three-quarter ripe
papaya fruit treated with and without 1-MCP and fresh-cut fruit derived from intact three quarter ripe fruit treated with and without I -MCP papaya during storage... 152
7-4. Neutral sugar composition of intact three-quarter ripe papaya fruit treated with and
without I -MCP and fresh-cut fruit derived from intact three-quarter ripe fruit
treated with and without 1-MCP papaya during storage ...................................... 154
viii




LIST OF FIGURES
Figure page
3-1. Unpared fruit firmness of green 'Galia' fruit treated with air (control), 0.09, 0.9 jiL
L and 9 [tL 1-M CP during storage at 15 'C .................................................. 50
3-2. Respiration and ethylene production of green (A) and yellow (B) 'Galia' melon fruit
treated with 1.5 gL L- 1-MCP and air (control) during storage at 20 C .............. 51
3-3. Mesocarp firmness of green (A) and yellow (B) 'Galia' melon fruit treated with 1.5
VL L- 1-MCP and air (control) during storage at 20 'C .................................... 52
3-4. Electrolyte leakage of green (A) and yellow (B) 'Galia' melon fruit treated with 1.5
[tL L-1 1-MCP and air (control) during storage at 20 'C .................................... 53
3-5. Soluble solids concentration of green (A) and yellow (B) 'Galia' melon fruit treated
with 1.5 [L L"' I-MCP and air (control) during storage at 20 C ...................... 54
3-6. Titratable acidity of green (A) and yellow (B) 'Galia' melon fruit treated with 1.5
ljL L- 1-MCP and air (control) during storage at 20 'C .................................... 55
3-7. The pH of green (A) and yellow (B) 'Galia' melon fruit treated with 1.5 tL L- 1MCP and air (control) during storage at 20 C ................................................. 56
3-8. 'Galia' fruit harvested at the pre-ripe stage (green surface) were treated with 1.5 1tL
L- 1-MCP or air (control) and then stored for 13 days at 20 'C ........................ 57
3-9. 'Galia' fruit harvested at the ripe stage (yellow surface) were treated with 1.5 l-1L L-'
1-MCP or air (control) and then stored for 7 days at 20 'C ................................ 58
4-1. Ethylene production for intact ripe 'Galia' fruit with and without 1-MCP and freshcut fruit derived from intact ripe fruit treated with and without I -MCP during
sto rag e at 5 'C ...................................................................................................... 7 6
4-2. Mesocarp firmness for intact ripe 'Galia' fruit with and without I-MCP and freshcut fruit derived from intact ripe fruit treated with and without I -MCP during
sto ra g e at 5 'C ...................................................................................................... 7 7
4-3. Electrolyte leakage from mesocarp tissues of intact ripe 'Galia' fruit with and
without I -MCP and fresh-cut fruit derived from intact ripe fruit treated with and
w ithout 1-M CP during storage at 5 'C .............................................................. 78
ix




4-4. Pectin efflux from mesocarp tissues of intact ripe 'Galia' fruit with and without 1MCP and fresh-cut fruit derived from intact ripe fruit treated with and without 1M C P during storage at 5 'C ........................................................................... 79
4-5. Color parameters, L* lightness (A), hue angle (B) and chroma (C) for ripe 'Galia'
fruit skin treated with and without 1-MCP (1 jiL L) and the color parameters (D, E and F) for intact ripe fruit with and without I -MCP and fresh-cut fruit derived from intact ripe fruit treated with and without I -MCP during storage at 5 'C ........ 80
4-6. Mesocarp water soaking percentage (A) and sensory evaluation (B) for intact ripe
'Galia' fruit with and without 1-MCP and fresh-cut fruit derived from intact ripe
fruit treated with and without I -MCP during storage at 5 'C ............................. 81
4-7. Ripe fresh-cut 'Galia' fruit derived from intact ripe fruit treated with (FC-MCP) and
without I -MCP (FC-CNT) and then stored for 4 days at 5 'C .......................... 82
4-8. Ripe fresh-cut 'Galia' fruit derived from intact ripe fruit treated with (FC-MCP) and
without I -MCP (FC-CNT) and then stored for 10 days at 5 C ............................. 83
5-1. Mesocarp firmness for 'Sunrise Solo' fruit treated with air (control), 0.9 taL L1, and
9 jtL U' I-MCP at 20 to 30% skin yellowing ripening stage and subsequently
stored for 19 day s at 15 'C .................................................................................. 100
5-2. Respiration and ethylene production for 'Sunrise Solo' fruit treated with air
(control) and 9 ,iL L" 1-MCP at the pre-ripe (A) and ripe (B) stage and
subsequently stored at 20 'C ............................................................................... 10 1
5-3. Mesocarp firmness for 'Sunrise Solo' fruit treated with air (control) and 9 PtL L- IMCP at the pre-ripe and ripe stage and subsequently stored at 20 C .................. 102
5-4. Electrolyte leakage for 'Sunrise Solo' fruit treated with air (control) and 9 PL L IMCP at the pre-ripe and ripe stage and subsequently stored at 20 'C ................. 103
5-5. Soluble solids concentration (SSC) for 'Sunrise Solo' fruit treated with air (control)
and 9 pL L- I -MCP at the pre-ripe (A) and ripe (B) stage and subsequently stored
a t 2 0 C ............................................................................................................. 10 4
5-6. Titratable acidity for 'Sunrise Solo' fruit treated with air (control) and 9 iL L-1 IMCP at the pre-ripe and ripe stage and subsequently stored at 20 'C ................. 105
5-7. The pH for 'Sunrise Solo' fruit treated with air (control) and 9 PaL L", 1-MCP at the
pre-ripe (A) and ripe (B) stage and subsequently stored at 20 0C ........................ 106
5-8. Pre-ripe 'Sunrise Solo' fruit treated with 9 [.L L-I I-MCP or air (control) and then
sto red fo r 7 day s at 20 'C .................................................................................... 107
5-9. Ripe 'Sunrise Solo' fruit treated with 9 IAL L- I-MCP or air (control) and then
stored for 3 days at 20 OC .................................................................................... 108
x




6-1. Ethylene production for intact postclimacteric 'Sunrise Solo' papaya fruit pretreated with 2.5 IL -1 1-MCP and air (control), and for fresh-cut postclimacteric
fruit derived from the either intact air-treated or the intact 1 -MCP-treated fruit
du ring sto rag e at 5 'C ......................................................................................... 12 6
6-2. Mesocarp firmness for intact postclimacteric 'Sunrise Solo' papaya fruit pre-treated
with 2.5 lL L-' 1-MCP and air (control), and for fresh-cut postclimacteric fruit
derived from either the intact air-treated or the intact 1-MCP-treated fruit during
sto rag e at 5 C .................................................................................................... 12 7
6-3. Electrolyte leakage (% of total) for intact postclimacteric 'Sunrise Solo' papaya fruit
pre-treated with 2.5 L L1 1-MCP and air (control), and for fresh-cut
postclimacteric fruit derived from either the intact air-treated or the intact I -MCPtreated fruit during storage at 5 C ...................................................................... 128
6-4. Color parameters for intact postclimacteric 'Sunrise Solo' papaya fruit skin pretreated with 2.5 jiL L- 1-MCP and air (control), and for the intact fruit flesh and
fresh-cut fruit derived from either the intact air-treated or the intact I -MCP-treated
fruit during storage at 5 C ................................................................................. 129
6-5. Fresh-cut postclimacteric 'Sunrise Solo' papaya fruit derived from either intact
postclimacteric air-treated (FCC) or intact postclimacteric I -MCP-treated fruit
(FCM ) and then stored for 10 days at 5 C .......................................................... 130
6-6. Pitting of postclimacteric 'Sunrise Solo' papaya fruit pre-treated with 2.5 g.L L- IM CP and air (control) during storage at 5 C ...................................................... 131
6-7. Water soaking (A) and sensory evaluation (B) for intact postclimacteric 'Sunrise
Solo' papaya fruit pre-treated with 2.5 iL L-1 1-MCP and air (control), and for
fresh-cut postclimacteric fruit derived from either the intact air-treated or the intact 1-M CP-treated fruit during storage at 5 C ......................................................... 132
6-8. Fresh-cut postclimacteric 'Sunrise Solo' papaya fruit derived from either intact
postclimacteric air-treated (FCC) or intact postclimacteric I -MCP-treated fruit
(FCM ) and then stored for 6 days at 5 C ............................................................ 133
7-1. Molecular mass distribution of water-soluble polyuronides of intact three-quarter
ripe papaya fruit treated with and without I-MCP and fresh-cut fruit derived from
intact three quarter-ripe fruit treated with and without I -MCP at day 0 (o), 6 (e),
a n d 10 (V ) ......................................................................................................... 15 7
7-2. Molecular mass distribution of CDTA-soluble polyuronides of intact three-quarter
ripe papaya fruit treated with and without 1-MCP and fresh-cut fruit derived from
intact three-quarter ripe fruit treated with and without 1 -MCP at day 0 (o), 6 (e),
a n d 10 ( V ) ......................................................................................................... 1 5 9
7-3. Molecular mass distribution of Na2CO3-soluble polyuronides of intact three-quarter
ripe papaya fruit treated with and without 1-MCP and fresh-cut fruit derived from
xi




intact three quarter-ripe fruit treated with and without I -MCP at day 0 (o), 6 a n d 10 ( V ) ......................................................................................................... 1 6 1
xii




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
1-METHYLCYCLOPROPENE TREATMENT EFFICACY IN PREVENTING ETHYLENE PERCEPTION AND RIPENING IN INTACT AND FRESH-CUT
'GALIA' MELON AND 'SUNRISE SOLO' PAPAYA FRUITS By
Muharrem Ergun
August 2003
Chair: D.J. Huber
Major Department: Horticultural Sciences
The objectives of this study were to determine the physiological responses of intact and fresh-cut melon (Cucumis melo L. var. reticulatus L. Naud. cv. Galia) and papaya (Caricapapaya, L. var. Sunrise Solo) fruits in which ethylene perception was blocked through application of 1-methylcyclopropene (1-MCP). The whole fruits were treated with 1-MCP (melon, 1 or 1.5 1iL L-1; papaya, 2.5 or 9 tL L-1) for 24 h at 20 oC and stored at 20 oC or processed and stored at 5 C.
Inhibition of ethylene perception via application of 1-MCP delayed the onset of the respiratory and ethylene climacterics and reduced maximum respiration and ethylene production rates of 'Galia' fruit ripened at 20 oC. Softening of intact and fresh-cut 'Galia' melon was significantly reduced by 1-MCP, regardless of application time (ripening stage). Yellowing of the fruit surface during ripening was strongly and somewhat irreversibly delayed in 1 -MCP-treated 'Galia'.
xiii




Papaya fruit ('Sunrise Solo') treated with 1-MCP exhibited delayed initiation of the respiratory and ethylene climacterics and suppressed ethylene production during ripening. Softening was significantly delayed in fresh-cut and intact fruit. Papaya treated with IMCP retained higher levels of titratable acidity compared with non-1-MCP-treated fruit throughout ripening. The color change of the fruit surface from green to yellow was significantly but temporarily inhibited in 1-MCP-treated fruit.
Cell wall modification was studied in intact and fresh-cut ripe papaya fruit stored for 10 days at 5 'C. Water-soluble polyuronides represented the major pectic fraction followed by the CDTA (1,2 cyclohexylenedinitrilotetraacetic acid)- and Na2CO3-soluble fractions irrespective of I -MCP. Both hemicellulosic and pectic polysaccharides in intact and fresh-cut fruit showed some changes but this trend was slightly or not affected by 1MCP. Neutral sugars from pectins and hemicelluloses including galactose, glucose and xylose decreased in both intact and fresh-cut fruit regardless of I -MCP. Generally, either intact fruit or fresh-cut fruit pre-treated with I -MCP exhibited little or no significant changes compared with fruit not treated with 1 -MCP.
The studies presented herein have shown that 1-MCP has potential for extending the useful storage life of intact and fresh-cut melon and papaya fruits by delaying ethylene inducible ripening process.
xiv




CHAPTER 1
INTRODUCTION
'Galia' (Cucumis melo L. var. reticulatus L. Naud. cv. Galia) fruit has excellent
flavor and aroma characteristics; however, storage life is limited to 2-3 weeks even at low temperatures. The storage life of papaya (Carica papaya L.) fruit is also short due to its inherently high respiration rate, delicate skin, and high water content. Papaya fruit can be harvested at the mature green stage (10% to 20% yellow skin), however, and maintained at 10 to 12 C for periods of up to 2 weeks. Approaches restricting ethylene synthesis, such as controlled atmosphere, have proven that storage life of melon and papaya fruits can be extended. Another, a more facile approach to extending the storage life and quality of harvested melon and papaya has been through the application of 1methylcyclopropene (I -MCP), a potent anti-ethylene compound. I -MCP is very volatile and antagomism ethylene effectively in the range of parts per billion (ppb) to parts per million (ppm). Treated fruit do not contain residue of 1 -MCP. I -MCP has been reported to delay or reduce ethylene-inducible effects on a variety of fruits. The investigation of the efficacy of 1-MCP to maintain quality of both the melon and papaya fruits will provide information value to designing and exploring new postharvest applications that contribute to reduce postharvest losses during market preparation, storage, transport, or at the wholesale, retail, or consumer level.
Fresh-cut produce is one of the new food-processing methods that is increasing in popularity. Featuring fresh-like quality in a ready to use package, fresh-cut produce has become very popular. Fresh-cut produce, however, can easily become unacceptable in a




2
few days because its tissues no longer retain their protective epidermal and cuticular layers, and become ruptured and damaged. Wounding associated with the preparation fresh-cut produce is responsible for rapid loses in appearance, aroma, flavor, firmness/texture, and resistance to microbial degradation. The knowledge of fresh-cut processing on either a theoretical or practical basis is quite limited since many fruits and vegetables have not been explored as fresh-cut commodities. Tropical fruits are highpriority commodities to be explored as fresh-cut produce due to their perishable characteristics and sensitivity low temperatures. For example, papaya is very susceptible to mechanical damage, pest attacks and diseases, has a storage life of less than one week under ambient tropical conditions.
Melon (CIwiimis melo L. var. inodorus and reticulatus L. Naud.) is one of the most popular fresh-cut products; however, information about its behavior and characteristics after fresh-cut processing is limited to observations on discoloration, offodor development, and water soaking. The behavior of fresh-cut fruits generally parallels that of wounded tissues and is affected by type of tissue, stage of maturity, extent of wounding, temperature, oxygen, ethylene, carbon dioxide, water vapor pressure, and microorganisms. The immediate response to wounding is cell rupture and loss of tissue integrity due to water loss and mix of sequestered enzymes and substrates, followed by physiological and biochemical changes including accelerated ethylene production. Ethylene, a stress-related hormone, may influence the events leading to texture loss and deterioration in fresh-cut fruits. Therefore, inhibition of ethylene synthesis or action may enhance the storage life of fresh-cut produce and also help to understand the




3
physiological changes that distinguish the behavior of fresh-cut fruit as compared with these events as they occur in ripening, intact fruit.
The primary objectives of the work reported herein were to examine the potential use of ethylene-perception inhibition for control of ripening in pre-ripe and ripe melon and papaya fruits, and to evaluate the effects of ethylene action on the postharvest qualities of either intact or fresh-cut melon and papaya fruits by measuring several physiological and biochemical properties. Ethylene action was manipulated through use of 1-methylcyclopropene, a strong and persistent inhibitor of ethylene action.




CHAPTER 2
LITERATURE REVIEW
Melon
Introduction
The melon (Cucumis melo L. var. inodorus and reticulatus L. Naud. ) plant, a
member of the Cucurbitaceae, is thought to have originated from the tropical regions of Africa and the Middle East (Seymour and McGlasson, 1993). 'Galia' (Cucumis melo L. var. reticulatus L. Naud. cv. Galia) was bred on the basis of the green-flesh qualities of 'Ha'Ogen', introduced from Hungary to Israel, at the Newe Ya'ar research center in the mid-60's (Karchi, 200). The melon plant is generally monoecious but occasionally andromonoecious (McGlasson and Pratt, 1963). Cucumis melo specie are classified into two main groups including reticulatus, the netted or muskmelon fruit types, and inodorus, the smooth-skinned or honeydew fruit types (Seymour and McGlasson, 1993). Melon fruit is categorized as an inferior berry whose edible flesh is derived from the placentae or mesocarp (Seymour and McGlasson, 1993). Melon fruits show high variation in flesh color (from green to orange), skin color (from white or green to orange or gray), skin texture (from smooth to netted), and size (Seymour and McGlasson, 1993). Some common melon types marketed in the United States are Western U.S., Eastern U.S., Charentais, LSL, Galia, Ananas, Honeydew, and Casaba (Zheng and Wolf, 2000). Harvest Maturity
The abscission properties are the most useful criteria for estimating harvest maturity in muskmelon types whereas the abscission layer does not develop in
4




5
honeydew-type melons until they are over-ripe (Pratt et al., 1977). Therefore, external color (green to white), peel texture (hairy to smooth), aroma, fruit density (low to high) and soluble solids are also used to verify harvest maturity of melon fruits (Portela and Cantwell, 1998). Melon fruit should have a minimum of 10% soluble solids concentration before harvest (Bianco and Pratt, 1977). Skin color of 'Galia' fruit can be used as a maturation index. Fallik et al. (2001) categorized 6 maturity indices for 'Galia' based on skin color: (1) dark green, (2) green (3) light yellow with green, (4) light yellow, (5) yellow, (6) dark yellow to orange).
Ripening Process
Ethylene. Muskmelon fruit types abscise at or near the climacteric (Altman and Corey, 1987) whereas honeydew types abscise after completion of the respiratory climacteric (Pratt et al., 1977). Exogenous ethylene treatment induces ripening in netted melons depending on maturity, temperature, treatment duration, and ethylene concentration (Seymour and McGlasson, 1993). Exogenous ethylene may be applied to non-netted types to achieve uniform ripening of fruit that have sufficient soluble solids content (Seymour and McGlasson, 1993). Since melons have no polymeric carbohydrate reserves such as starch, postharvest ethylene treatments do not enhance soluble solids content in harvested fruit of any maturity class (Bianco and Pratt, 1977). Orange-fleshed melon fruit (mostly netted types) produce higher ethylene levels than green- or whiteflesh types (Zheng and Wolf, 2000). Melon fruit with a netted rind have higher ethylene production than do smooth types (Zheng and Wolf, 2000).
Carbohydrates. Both in netted and honeydew type melons, sugar accumulation reaches its maximum after full maturity (Seymour and McGlasson, 1993). Soluble solids




6
in melon fruits may accumulate to values as high as 17% (Bianco and Pratt, 1977), with sucrose and fructose comprising the most prevalent sugars (Hubbard et al., 1990).
Structural polysaccharides. An increase in soluble pectin, a decrease in pectin size, loss of galactosyl residues, and changes in size of hemicelluloses represent the most evident features of cell wall changes during ripening of melon fruits (Gross and Sams, 1984; McCollum et al., 1989). Galactosidases, pectin esterase, and cellulase are thought to be responsible for cell wall degradation (Gross and Sams, 1984; Lester and Dunlap, 1985; McCollum et al., 1989).
Organic acids. Citric and malic acids are the major organic acids in most melon fruits (Leach et al., 1989: Flores et al., 2001). Artes et al. (1999) reported that titratable acidity (% citric acid equivalents) ranged from approximately 0.50 ('Galia') to 0.14 ('Tendral') in 4 different varieties of muskmelons.
Pigments. In orange-fleshed muskmelons, the following pigments have been found: 1-carotene (84.7 %), 6-carotene (6.8 %), a-carotene (1.2 %), phytoene (1.5 %), lutein (1 %), violaxanthin (0.9 %), and traces of other carotenoids (Seymour and McGlasson, 1993). During muskmelon ripening, pigment accumulation initiates in the placentae, progressing outward through the mesocarp (Reid et al., 1970). In green-fleshed types such as 'Galia', however, the carotenoid content in exocarp and mesocarp does not change significantly during ripening (Flugel and Gross, 1982).
Volatiles. The volatile ester profiles of ripe muskmelon and honeydew type
melons are very similar except for ethyl butyrate, which is more abundant in muskmelons (Yabumoto et al., 1978). The following volatiles have been reported to be representative of muskmelons: ethyl-2-methyl butyrate, ethyl butyrate, hexanoate, hexyl acetate, 3-




7
methyl butyl acetate, benzyl acetate, (Z)-6-nonenyl acetate, (E)-6-nonenol, (Z,Z)-3, 6nonadienol and (Z)-6-nonenal (Yabumoto et al., 1978; Wyllie and Leach, 1990). Butyl acetate (5), 2-methyl-butyl acetate (6), and hexyl acetate (9) are the most abundant volatiles in 'Galia' type melons (Fallik et al., 2001). Postharvest Storage of Melon Fruit
Melon fruit are typically stored at 7 to 10 oC whereas storage below 7 C may cause chilling injury, especially for honeydew types (Lipton and Aharoni, 1979; Hardenburg et al., 1986). Controlled atmosphere conditions of 3% 02 and 10% CO2 at 7 oC, and 2% 02 and 10% CO2 at 3 oC have been shown to delay ripening and extend storage life of honeydew melon fruit (Hardenburg et al., 1986). Portela and Cantwell (1998) reported that fresh-cut honeydew and muskmelon fruit stored at 5 oC for 12 days maintained their appearance and color but exhibited a 50% decline in firmness. O'Connor-Shaw et al. (1994) reported that the storage life of fresh-cut honeydew-type melons was limited to 14 days while the storage life of fresh-cut muskmelon types was 4 days at 4 oC. Postharvest Diseases
Sour rot (Galactomyces geotrichum (Buti. & Peter) Redh. & Mall.; Geotrichum candidum Lk.), Rhizopus rot (Rhizopus stolonifer (Ehrenb.:Fr.) Vuill.), Fusarium rot (Fusarium spp.), Trichothecium rot (Tricho/hecium roeum (Pers.:Fr.) Link), Botrytis rot (Botryotiniafuckelina (de Barry) Whetzel; Botrytis cinerea Pers.:Fr.), Lasiodiplodia rot (Botryosphaeria rhodina (Cooke) Arx; Lasiodiplodia theobromae (Pat.) Griff. & Maubl.), and anthracnose (Glomerella lagenarium F. Stevens.; Colletotrichum orbiculate (Berk. & Mont.) Arx; Gloeosporium orbiculare Berk. & Mont; Colletotrichum lagenarium (Pass.) Ellis & Halst; Gloeosporium lagenarium (Pass.) Sacc.) are the most common postharvest diseases in melon fruit (Sommer et al., 1992). Netted types are very




8
susceptible to fungal diseases since organisms may locate easily in the skin net and enter the fruit through mechanical breaks or abrasions (Seymour and McGlasson, 1993).
Wounds and fresh stem-scars are likely sources of ingress for sour rot
(Galactomyces geotrichum (Butl. & Peter) Redh. & Mall.; Geotrichum candidum Lk.; Sommer et al., 1992). The fungus is easily spread by wind, rain, and vinegar flies (Drosophila spp.; Sommer et al., 1992). Rhizopus (Rhizopus stolonifer (Ehrenb.:Fr.) Vuill.) colonizes fruits via mechanical injuries and stem scars very rapidly at high temperatures (Sommer et al., 1992). Fusarium (Fusariumn spp.) frequently originates from soil and becomes active when fruit ripen. Disease progression of Fusarium is typically rather slow and fruit losses do not become excessive (Sommer et al., 1992). Spores of anthracnose (Colletotrichum orbiculate (Berk. & Mont.) Arx; Gloeosporium orbiculare Berk. & Mont; Colletotrichum lagenarium (Pass.) Ellis & Halst; Gloeosporium lagenarium (Pass.) Sacc.) are distributed by water, wind, insects, or handling. As fruit mature and ripen, latent anthracnose becomes active and causes sunken and black lesions (Sommer at al., 1992)
Postharvest Disease Control
Maintaining good physiological condition of the melon plant during growth and proper handling of fruit during harvesting, transportation and storage are essential in preventing or controlling postharvest deterioration and decay (Qi et al., 1988; Teitel et al., 1989; Aharoni et al., 1993; Seymour and McGlasson, 1993; O'Connor-Shaw et al., 1996; Fallik et al., 2000). Immersing fully ripe 'Galia' fruit in hot water (52 oC) for 2 min may provide antifungal protection for a limited period (Teitel et al., 1989). A combined treatment of a hot water rinse and brushing can improve the general appearance and maintain quality of melon fruit as well as also reduce postharvest decay. According to




9
Ayhan et al. (1998), 200 ppm free chlorine performed well in reducing microbial growth in intact and fresh-cut muskmelon fruit. Honeydew melons (intact or fresh-cut) may also be dipped in 150-ppm chlorinated water for 5 min to control decay development (Qi et al., 1988).
Papaya
Introduction
Papaya (Carica papaya L.) is an herbaceous plant and a member of the family
Caricaceae. The plant is limited to the region within a latitudinal range of 32N and 32'S (Morton, 1987). The plant may have female, male or hermaphroditic flowers (Nakasone, 1986; Morton, 1987). The flower type determines the final size and shape of the fruit. Fruit from bisexual flowers are usually pyriform in shape with a small seed cavity and thick wall of firm flesh (mesocarp). On the other hand, fruit from female plants are nearly round or oval, and of relatively thin flesh (Morton, 1987). Fruit shape is usually spherical to oblong and fruit are generally composed of five longitudinal carpels united around a large central cavity wherein seeds are attached to placental tissue by 0.5-10 cm stalks (Morton, 1987). Fruit with less than five carpels are long and cylindrical, resembling cucumber fruit in shape (Nakasone, 1986). Papaya fruit range from 15 to 50 cm in length with a diameter of 10 to 20 cm (Morton, 1987), and fruit weight ranges from 30 g to 9 kg, depending on the cultivars (Nakasone, 1986). The peel is thin, usually smooth and green when immature but fairly tough, and yellow to orange when ripe. A slight injury can induce milky latex containing the proteolytic enzyme, papain (EC 3.4.22.2), to exude (Sankat and Maharaj, 1997). Flesh (mesocarp) thickness varies from 1.5 to 5 cm (Nakasone, 1986; Sankat and Maharaj, 1997). During ripening, the flesh becomes aromatic, yellow to orange or reddish-yellow, juicy, sweetish, and melon-like in flavor




10
(Morton, 1987; Sankat and Maharaj, 1997). Seeds are generally dark gray or black, covered with a transparent gelatinous aril, and have high oil and protein contents (Sankat and Maharaj, 1997).
Some of the most cultivated varieties of papaya include 'Solo', 'Sunrise Solo', 'Maradol', 'Waimanalo', 'Higgins', 'Wilder', 'Hartus Gold', 'Bettina', 'Peterson', 'Singapore Pink', and 'Cariflora' (Morton, 1987). Uses of Papaya Fruit
Ripe papaya fruit are generally consumed fresh; however, processed papaya fruit products, such as nectar and juice, are competing against fresh papaya fruit. Fruit may also be added to ice creams, sauces, used in cooked desserts, or pickled or preserved as jam (Nakasone, 1986). Green papaya can be boiled and served as a vegetable and canned in sugar syrup (Morton, 1987). Papaya fruit is a good source of vitamins A and B and an excellent source of vitamin C (Sankat and Maharaj, 1987). Carotene, thiamine, riboflavin, niacin, tryptophan, methionine and lysine are usual constituents of papaya fruit (Kimura et al., 1991). Papaya latex contains two proteolytic enzymes, papain and chymopapain (Paull, 1993). Papain is used to tenderize meat, to clarify beer, and to treat wool and silk before dyeing. Moreover, papain is used in toothpastes, cosmetics and detergents, as well as in digestion aids (Morton, 1987), and has been used to treat ulcers and to reduce swelling and fever (Morton, 1987). In India, latex from papaya fruit or seed is applied to the uterus as an irritant to induce abortion (Morton, 1987). Young leaves are cooked and consumed like spinach in India (Nakasone, 1986). Papaya Harvest Maturity
Papaya development from pollination to full ripeness requires approximately 5.5 (Hawaii) to 10.5 (Africa) months (Nakasone, 1986). Color break, sugar composition




11
(decline in sucrose, increase in glucose and fructose), and soluble solids concentration are the most useful maturity indices (Nakasone, 1986; Paull, 1993; Sankat and Maharaj, 1997). Nondestructive methods including reflectance, delayed light emission, and body transmission spectroscopy have been used to measure papaya maturity (Sankat and Maharaj, 1997). For local markets, fruit may be harvested when the skin color reaches 80% of yellow. Otherwise, fruit destined for storage or long-distance transportation are picked at the mature-green stage. The fruit must be handled properly in order to avoid injuries causing leakage of latex, which stains the fruit and reduces consumer acceptance (Morton, 1987). The latex from the peel may irritate the skin of fruit handlers; therefore, protective measures should be taken during prolonged physical contact with papaya (Morton, 1987).
Papaya Ripening
The optimum temperature for papaya ripening is between 22.5 and 27.5C (Paull, 1993). Papaya is a climacteric fruit, and the increase in ethylene production parallels respiration rate, reaching a maximum 1-2 days after (Wills and Widjanarko, 1995) or simultaneously with (Paull, 1993) the respiratory maximum. Respiration and ethylene production of mature green papaya 'Solo' fruit are below 5 mL kg1 h-1 and I ViL kg' h1, respectively; however, respiration and ethylene production increase to approximately 45 mL kg' h-1 and 7 [tL kgl h-1, respectively, during ripening at 22 'C (Paull and Chen, 1983).
Soluble carbohydrates. The principal soluble carbohydrates in papaya fruit are sucrose, glucose and fructose, with sucrose being the predominant sugar at the full ripe stage (Chan, 1979; Selvaraj and Pal, 1982). Invertase (EC 3.2.2.26) activity increases




12
during ripening, presumably causing the conversion of sucrose to fructose and glucose (Selvaraj and Pal, 1982). Papaya fruit contains low levels of starch (Selvaraj et al., 1982).
Structural polysaccharides and textural changes. Softening of papaya fruit is associated with a dramatic increase in the solubility of cell wall pectins (Paull, 1993; Lazan et al., 1995). Pectin depolymerization is also observed, occurring first in the inner mesocarp tissues (Sankat and Maharaj, 1997). Cellulase (EC 3.2.1.5), pectin methyl esterase (EC 3.1.111), xylanase (EC 3.2.1.8), polygalacturonase (EC 3.2.1.15; PG; highest in inner mesocarp) and 8-galactosidase (EC 3.2.1.23; highest in outer mesocarp) activities have been reported to increase during papaya ripening (Paull and Chen, 1983; Ali et al., 1999).
Organic acids. Citrate and malate are the predominant organic acids in papaya, but tartaric, fumaric and succinic acids have also been noted (Selvaraj et al., 1982). The concentration of total and nonvolatile acids decreases during fruit development, reaching a minimum 1.54 mEq 100g'" (fresh weight) with a pH in the range from 5.0-5.5 at the full-ripe stage (Paull, 1993). Ascorbic acid increases nearly 4-fold, reaching levels of 5.5mg-100-1 (fresh weight) during ripening (Paull, 1993). Compared with other fruit, total titratable acidity remains low during ripening, which may contribute to the sweet taste of papaya (Selvaraj et al., 1982). Non-volatile organic acids comprise 75% to 92% of total acidity (Selvaraj et al., 1982).
Pigments. Total carotenoid content of mesocarp increases up to 14-fold during papaya ripening, with levels ranging from 0.28 mg 100-' dry pulp at the mature-green stage to nearly 4 mg 100-1 dry pulp when full ripe (Selvaraj et al., 1982). 3-carotene (62%) is the predominant carotenoid in yellow-flesh cultivars whereas lycopene is the




13
major carotenoid in red-fleshed cultivars (Selvaraj et al, 1982). Lycopene constitutes 61% of the total carotenoid content of the red-fleshed 'Solo' papaya (Kimura et al., 1991). Bcryptoxanthin, 13-zeacarotene, and cryptoflavin are found in minor quantities in papaya fruit (Kimura et al., 1991).
Proteins and amino acids. Several proteases, papain, chymopapain A and B (EC 3.4.22.6), and papaya peptidase (EC 3.4.22.30), are found in papaya latex (Paull, 1993). Total protease activities in papaya mesocarp tissue decline during ripening (Paull, 1993). At last 13 free amino acids have been identified in papaya fruit (Selvaraj et al., 1982; Morton, 1987).
Volatiles. At least 199 volatiles have been identified in papaya fruit, with linalool being the most abundant (MacLeod and Pieris, 1983; Flath et al., 1990). Volatiles in the cultivar 'Solo' are comprised of up to 94% linanool followed, in declining abundance, by benzyl isothiocyanate, methyl butanoate and methyl benzoate (Flath et al., 1990). Only one volatile, methyl benzoate, is described as having papaya qualities (Paull, 1993). Postharvest Handling and Storage of Papaya
Papaya fruit have a maximum storage life of 7 days under ambient tropical
conditions (30 oC); temperatures above 32.5 oC cause abnormal ripening (An and Paull, 1990). Storage between 12 to 16 oC appears to represent the most compatible temperature range for storage. Storage below 10 to 12 'C may cause chilling injury, depending upon the maturity stage (Chen and Paull, 1986). 'Solo' fruit stored at 25 'C and 30 oC had higher total carotene and ascorbic acid, lower benzyl isothiocyanate (bitterness compound), more intense yellow peel color, and more acceptable eating attributes compared with fruit held at 20 oC (Wills and Widjanarko, 1995). This may have been the result, however, of more advanced ripening of the fruit held at the higher temperature.




14
Maharaj and Sankat (1990) reported that the best atmospheric conditions for maintaining acceptability and market quality of papaya fruit during storage were 1.5 to 2% 02 and 5% CO2 at 26 C. Plastic film wraps are more effective than waxes and other coatings in reducing water loss (Maharaj and Sankat, 1990). Chilling Injury
Chilling injury is a major physiological disorder induced by low non-freezing
temperatures (Chen and Paull, 1986; Chan, 1988). Chen and Paull (1986) reported that mature-green 'Solo' papaya stored at 7.5 C showed chilling injury symptoms after 20 days. Chilling injury symptoms of papaya fruit include epidermal discoloration of the mesocarp, development of hard areas in the flesh and around vascular bundles, enhanced mesocarp water soaking and electrolyte leakage, increased ethylene production, and increased susceptibility to decay (Chen and Paull, 1986). Postharvest Pathology
Postharvest diseases are very important in reducing market quality of papaya fruit and they are primarily responsible for the losses that occur during shipment. In Hawaii, postharvest losses of papaya fruit due to diseases extended up to 93% before 1987 depending on postharvest handling and packing procedures (Alvarez and Nishijima, 1987). Diseases are of three general types: fruit surface rots, stem-end rots, and internal infections (Alvarez and Nishijima, 1987).
Fruit surface rots. Anthracnose (Glomereila cingulata (Stonem.) Spauld. & Schr.; Colletotrichum gloeosporioides (Penz.) Arx), chocolate spot (Colletotrichum gloeosporioides spp.), dry rot (Mycosphaerella spp.), wet rot (Phomopsis caricaepapayae Petr. & Cif), Alternaria fruit spot (Alternaria alternata (Fr.) Keissler), Fusarium rot (Fusarium solanifer Snyd. & Hans.) and Guignardia spot (Guignardia spp.) are the




15
most common fruit surface pathogens found in papaya fruit (Alvarez and Nishijima, 1987; Sommer et al., 1992). Anthracnose is the major postharvest disease of papaya, and the symptoms of anthracnose (causing initially tiny, brown, superficial, watersoaked lesions that may enlarge to 2.5 cm or more in diameter) are most prominent at the fullripe stage (Alvarez and Nishijima, 1987). Chocolate spot is a surface disease and causes reddish brown lesions on the skin (Sommer et al., 1992). As fruit ripen, lesions of chocolate spot become sunken, displaying water-soaked margins (Nakasone, 1986; Sommer et al., 1992). Wet rot (Phomopsis caricae-papayae Petr. & Cif) generates soft and translucent areas on the fruit surface (Alvarez and Nishijima, 1987). Circular or oval black lesions are the symptoms of Alternaria fruit spot (Alvarez and Nishijima, 1987). Infections by Fusariumn solanifer produce small dry lesions with water soaked areas (Alvarez and Nishijima, 1987). Guignardia spot, evident as greenish-black lesions, is often seen when papaya are pretreated in water at 42 oC for at least 40 minutes (Alvarez and Nishijima, 1987).
Stem end rots. Lasiodiplodia rot (Botryosphaeria rhodina (Cooke) Arx;
Lasiodiplodia theobromae (Pat.) Griffin & Maulb.), Phytophthora rot (Phytophthora nicotianae Breda de Haan var. parasitica (Dast.) Waterh.), and Rhizopus (Rhizopus stolonifer (Her. Ex Fr.) Lind.) are among the most widespread stem end rots reported in papaya fruit (Alvarez and Nishijima, 1987; Sommer et al., 1992). Lasiodiplodia rot usually occurs at injuries to fruit skin or near fruit peduncle (Sommer et al., 1992). Rhizopus fungus invades through wounds and colonizes the entire fruit rapidly, often spreading to other fruits (Alvarez and Nishijima, 1987).




16
Internal fruit infections. Purple-stain (Erwinia herbicola (Loehnis) dye) and
internal yellowing (Enterobacter cloacae (Jordan) Hormaeche & Edwards) are the two most reported internal diseases in papaya fruit (Alvarez and Nishijima, 1987). The tissue invaded by Erwinia herbicola becomes translucent and later rots, resulting in extensive off-odors (Alvarez and Nishijima, 1987). Fruit flesh infected by Enterobacter cloacae is translucent with a bright yellow to lime-green discoloration (Alvarez and Nishijima, 1987).
Postharvest disease control. Since many infections affecting papaya during
postharvest handling become established in the field, postharvest control measures begin with choosing resistant varieties and implementing good cultural practices during fruit growth (Nakasone, 1986). After harvest, proper temperature measurement during transportation and marketing, the use of vapor/hot water treatments, and dipping in aurefungin and carnauba waxing are some of the control measures effective in controlling disease progression (Nakasone, 1986; Alvarez and Nashijma, 1987; Sommer et al., 1992; Sankat and Maharaj, 1997). Hot water treatment at 43 C to 49 C for 20 minutes has been reported to prevent/control postharvest decays (Akamine and Arisumi, 1953).
1-Methylcyclopropene
Introduction
The promotion of plant senescence by ethylene can be inhibited by a number of cyclic olefins including cyclopropene, 1-methylcyclopropene (1-MCP), 3methylcyclopropene, 1,3-dimethylcyclopropene, 3,3-dimethylcyclopropene, 1,3,3trimethylcyclopropene, 3-methyl-3-vinylcyclopropene and 3-methyl-3ethynylcyclopropene (Sisler et al., 2001). The compounds evidently compete with ethylene at the site of ethylene receptors, blocking tissue responsiveness to the growth




17
regulator (Sisler and Serek, 1997; Sisler et al., 2001). 1-MCP has been used as a tool to investigate ethylene action and tissue responses to ethylene during fruit ripening and flower senescence since it is effective in the ppb range, odorless, stable (non-explosive), and non-toxic (Sisler and Serek, 1997; Sisler and Serek, 1999). Application of 1-MCP delays ripening of climacteric fruits and flower senescence, presumably via its blocking effect on the ethylene signal transduction pathway. 1-MCP as commercial powder (active ingredient 0.14% 1-MCP) from Agrofresh, Philadelphia, PA., has been approved for use apple fruit.
Treatment Procedures for 1-MCP
1-MCP can be applied to plant tissues as a gas. The concentration required to inhibit ethylene action decreases as the exposure period increases (Serek et al., 1995a). Actively growing vegetative tissues and abscission layers, some of which involve mitotic activity, may need higher I-MCP concentrations (Sisler and Serek, 1997). Sisler et al. (1997) reported that at higher temperatures less I-MCP is required. Very low quantities of 1-MCP (20 nL L") were effective in extending the storage life of cut flowers including Rosa hybrida, Begonia, and Kalanchoe by preventing bud and flower abscission, leaf abscission, and flower senescence (Serek et al., 1994). The duration of the prophylactic period varies from plant to plant. Some cut flowers, banana and tomato fruits remain insensitive to ethylene almost for 12 days at 24 'C (Sisler and Serek, 1997). 1-MCP and Ethylene
Sisler et al. (1997) proposed that I-MCP binds to a metal in the ethylene receptor and would thus compete with the ethylene receptor, maintaining the active form of the receptors until ethylene concentration becomes adequate, new receptors are synthesized, or released from the receptor sites (Sisler and Serek, 1997). However, reports have




18
indicated that 1-MCP may bind to other receptors showing homology to ethylene receptors and/or it may not permanently attach to the ethylene receptors (based upon the continuous presence of I -MCP that further improved storage life of pak choy and broccoli compared to daily application of 1-MCP) (Abble et al., 2002). Furthermore, Jiang et al. (1999b) reported that the Km (substrate concentration at half the maximum velocity) for 1-MCP (17 nL L") was lower than that for ethylene (96 nL U') for control of banana softening, suggesting that 1-MCP has a stronger affinity than ethylene for the ethylene-binding sites.
A consistently observed effect of I-MCP treatment with climacteric fruit is the dramatically reduced level of ethylene production. 1-MCP treatment of tomato fruit decreased transcript abundance for the enzymes 1 -aminocyclopropene- 1 -carboxylate oxidase (ACO) (EC 4.2. 1.3) and ACC synthase (ACS) (EC 4.4.14); however, ACC content did not change (Nakatsuka et al., 1997). Peach fruit exhibited suppressed ethylene production, ACO activity, and accumulation of PP-ACSI mRNA in response to 1-MCP treatment (Mathooko et al., 2001). 1-MCP inhibited the ethylene-induced triple response in arabidopsis seedlings (Hall et al., 2000). ETRI and ERSI (ethylene response sensors) showed nearly identical sensitivity to I-MCP in arabidopsis, suggesting the ethylene-binding sites of ETRI and ERSI have similar affinities for ethylene (Hall et al., 2000). Accumulation of ACO mRNA during storage of'Flavortop' nectarine was inhibited by 1-MCP, and this inhibition persisted during post-storage ripening (Dong et al., 2001).
In a few reports, fruits including grapefruit (Mullins et al., 2000) and strawberry (Tian et al., 2001) were noted to show increased ethylene production in response to 1-




19
MCP treatment. However, both grapefruit and strawberry are non-climacteric fruits in which the triggering end regulation of the ripening process as a whole does not require ethylene unlike climacteric fruits. Furthermore, pre-treatments of citrus leaves and leaf explants with I -MCP induced ethylene production upon transfer of the leaves to air (Zhong et al., 2001). The reason for higher ethylene production could be stress-related ethylene production, regulation of ethylene production, and/or excessive ethylene production due to loss of ethylene feedback control mechanisms (Mullins et al., 2000). 1-MCP and Fruit Softening
One of the most commonly reported effects of treating fruit with 1-MCP is the dramatic decline in the rate of softening, presumably a consequence of reduced accumulation of specific, ethylene-induced cell wall enzymes (Huber et al., 2003). The accumulation of polygalacturonase and cellulose (EC 3.2.1.5) was significantly delayed and suppressed in 1-MCP-treated avocado fruit (Feng et al., 2000; Jeong et al., 2002). However, eventually 1-MCP-treated avocado fruit softened eventually as high as nontreated fruit, which indicates that PG and cellulose are not essential for softening of avocado fruit (Feng et al., 2000; Jeong et al., 2002). The solubilization and degradation of polyuronides of avocado fruit was significantly reduced and delayed by 1-MCP application as well (Jeong et al., 2003). The mRNA abundance of PG and pectinesterase (EC 3. 1. 1.11) during storage of I -MCP-treated 'Flavortop' nectarine was reduced, and inhibition of PG expression persisted during post-storage ripening (Dong et al., 2001). In contrast to the general inhibition of accumulation of cell wall enzymes in response to 1MCP treatment, the accumulation of endoglucanase (EC 3.2.1.4 ) and its transcript in 'Flavortop' nectarine were enhanced by I -MCP and inhibited by ethylene at all stages ripening (Dong et al., 2001). The nectarine fruit with I-MCP showed severe flesh




20
woolliness (sot dry texture) and reddening, and lower juice compared to ethylene-treated fruit, and the authors proposed that these disorders may be enhanced by the high level of expression of endoglucanase (Dong et al., 2001). 1-MCP treatment decreased the mRNA abundance of expansin 1 in mature green or ripe tomato fruit (Hoeberichts et al., 2002). Since it is believed that expansin is stimulated by ethylene, expansin may contribute tomato fruit softening at early stage of ripening (Hoeberichts et al., 2002). The Influence of Suppressed Ethylene Perception on Ripening Physiology and Biochemistry
Table 2-1 summarizes from the literature some of the physiological and
biochemical responses (PBR) of fruits in which ethylene perception suppressed by 1MCP. PBR are divided into three groups (columns) as reduced or delayed, increased and unaltered. Some of PBR of a crop are listed in three columns because of the different sources possibly caused by cultivar and ripeness stage differences.




Table 2-1. 1-MCP-induced effects on ripening fruits Fruit Reduced or delayed PBR Increased PBR Unaltered PBR References
Apple Ethylene production, respiration, Respiration, soluble Respiration, soluble (Fan and Mattheis,
(Malus sylvestris L.) softening, color change, loss of solids, and internal solids, and loss of 1999; Fan et al., 1999; titratable acidity and water loss, injury titratable acidity Watkins et al., 2002;
decay, aroma production, Jiang and Joyce, 2002;
coreflush, and scald Saftner et al., 2003)
Apple (fresh-cut) Ethylene production, respiration, (Jiang and Joyce, 2002)
(Maus sylvestris L.) softening, and color change. Apricot Ethylene production, respiration, Days to ripen (Fan et al., 2000; Dong
(Prunus softening, color change, et al., 2002)
titratable acidity, decay, and
armeniaca L.) aroma production
aroma production




Table 2-1. Continued
Fruit Reduced or delayed PBR Increased PBR Unaltered PBR References
Banana Ethylene production, Ethylene production, (Sisler et al., 1996;
(Musa sp. AAA softening, peel color change, and uneven skin color Golding et al., 1998; Jiang
group) chlorophyll loss, and aroma development et al., 1999a; Jiang et al.,
production 1999b)
Grapefruit Degreening Ethylene production Decay (Mullins et al., 2000)
(Citrus paradisi)
Mango Softening, and color change Days to ripen Soluble solids, (Jiang and Joyce, 2000;
(Mangifera titratable acidity, Hofman et al., 2001)
indica L.) weight loss, and
rots
Nectarine Ethylene production, and Woolliness and Respiration (Dong et al., 2001)
(Prunus persica softening discoloration
Lindl.)




Table 2-1. Continued
Fruit Reduced or delayed PBR Increased PBR Unaltered PBR References
Papaya Ethylene production, respiration, Days to ripen, Soluble solids (Hofman et al., 2001;
(Carica papaya L.) softening, and color change. soluble solids, rots, Jacomino et al.,
anthracnose, and 2002)
skin blemishes.
Pear Ethylene production, softening, and (Lelievre et al., 1997;
(Pyrus communis L.) water loss Baritell et al., 2001)
Peach Ethylene production, ACS and ACO Mesocarp browning (Mathooko et al.,
(Prunuspersica L.) activities, respiration, softening, and 2001;Fan et al., 2002)
loss of titratable acidity.
Pineapple Ethylene production, color change, (Selvarajah et al.,
(Ananas comosus loss of soluble solids, and chilling 2001)
Merr.) injury.




Table 2-1. Continued
Fruit Reduced or delayed PBR Increased PBR Unaltered PBR References
Plum Ethylene production, respiration, Browning Color change (Abdi et al., 1998; Dong
(Prunums salicina softening, color change, loss of et al., 2002)
L.; Prumnis x titratable acidity, aroma
domestic L) production, browning, and decay
Strawberry Ethylene production, softening, Ethylene Respiration, and (Ku et al., 1999; Tian et
(Fragaria x color change, decay, phenolics, production, and decay al., 2000; Jiang et al.,
ananassa Duch) and phenylalanine ammonia-lyase decay 2001)
(PAL)
Tomato Ethylene production, ACO and Soluble solids (Nakatsuka et al., 1997;
(Lycopesicon ACS, color development, phytoene Wills and Ku, 2002;
esculentumrn L.) synthase, expansin, respiration, Hoeberichts et al., 2002;
loss oftitratable acidity, softening Mostofi et al., 2003)




25
Physiology of Fresh-cut Produce
Introduction
The processing or preparation of lightly processed (fresh-cut) fruits and vegetables can be defined as washing, sorting, trimming, peeling, skinning or chopping of horticultural commodities in a manner that does not reduce fresh-like quality (Rolle and Chism, 1987; O'Conner-Shaw et al., 1994). The preparation can result stress similar to that found in wounded tissues (Brecht, 1995). Excessive water loss, synthesis of secondary compounds, higher ethylene production and respiration, softening, browning and degreening are examples of such behaviors shown by fresh-cut produce (Rolle and Chism, 1987; Miller, 1992). These behaviors plus temperature, relative humidity and atmospheric composition can greatly influence quality maintenance of fresh-cut produce (King and Bolin, 1989).
Consequences of Processing
The physiology of fresh-cut produce is similar to that of wounded tissues (Brecht, 1995). The process necessary for fresh-cut produce (fresh-cut processing) requires abrasion, peeling, slicing, chopping and shredding (O'Conner-Shaw et al., 1994). Each of the previous steps can generate stress conditions in living tissues. Since fruits and vegetables remain viable after the fresh-cut processing, their behavior is generally comparable to plants exposed to stress conditions in nature such as wind damage. This behavior includes enhanced ethylene and respiration rates, wound-healing processes (synthesis of secondary compounds, suberization and lignification), biochemical changes (membrane changes, browning and degreening) and physical changes (softening and water loss; Rolle and Chism, 1987; Miller, 1992; Brecht, 1995).




26
Ethylene production and respiration. Wounding caused by fresh-cut processing may accelerate ethylene production and respiration (Rolle and Chism, 1987). In climacteric fruits, wounding causes more ethylene production in the preclimacteric and climacteric periods than in the postclimacteric period (Brecht, 1995). Respiration of fresh-cut produce generally rises with temperature depending on the severity of damage during processing. Higher ethylene production due to wounding may result in an increase in respiration rate as well (Rolle and Chism, 1987). Additionally, starch break down and oxidation of fatty acids may contribute to this respiratory inclination (Miller, 1992).
Secondary metabolism. Wounding causes tissues to synthesize secondary compounds, which are mostly related to wound-healing and defense mechanism processes. These secondary compounds may affect aroma, appearance, nutritive value, and safety of fresh-cut produce (Brecht, 1995). Some of these secondary compounds are phenolics, flavonoids, terpenoids (Sakai and Nakagawa, 1988), alkaloids, glucosinolates, and long-chain fatty acids and alcohols (Miller, 1992). Wounding increases the activities and transcripts of the following enzymes associated with the secondary compounds: PAL (EC 4.1.3.5) (Fritzemeier et al., 1987; Liang et al., 1989), 4-coumarate:CoA ligase (EC
6.2.1.13) (Fritzemeier et al., 1987), chalcone synthase, 3-deoxy-D-arabinoheptulosonate-7-phosphate synthase (EC 4.2.3.4) (Miller, 1992), peroxidases (EC
1.11.1.6) (Bostock et al., 1987; Miller and Thomas, 1989), and stilbene synthase (EC
2.3.1.95) (Vornam et al., 1988).
Structural changes of the tissue surface may occur as a consequence of the
wound-healing process. Desiccation of the wounded surface is the first observable change of fresh-cut produce (Varoquax and Wiley, 1994). Desiccation is followed by suberin and




27
lignin production and deposition in cell walls, and is possibly proceeded by periderm occurrence beneath the suberin layer in many tissues for example potato tuber, bean pod, and cucumber pericarp (Burton, 1982).
Browning and degreening. Browning results from enzymatic oxidation of phenols and polyphenols (Ahvenainen, 1996). Polyphenol oxidase (EC 1.14.18.1), PAL, tyrosine ammonia lyase (EC 4.1.99.2), cinnamic acid -4-hydroxylase (EC 1.14.13.11), lipoxygenase (EC 1.13.11.13) and catechol oxidase (EC 1.1.3.14) are the enzymes that likely cause enzymatic browning (Ahvenainen, 1996).
Increased ethylene production and loss of membrane integrity may start rapid
chlorophyll degradation due to induction of chlorophyll-degrading enzymes (Varquaux and Wiley, 1994). The enzymes responsible for the chlorophyll degradation are chlorophyll oxidase, chlorophyllase (EC 3.1.1.15), lipolytic acid hydrolase, and other peroxidases (EC 1.11.1.6) (Varquaux and Wiley, 1994).
Membrane changes. Wounding causes cellular disruption, leading to
decompartmentation of enzymes and substrates (Rolle and Chism, 1987). Wounding enhances the activities of lipid acyl hydrolyse (act like phospholipase D), polyphenol oxidase (Ikediobi et al., 1989) and lipoxygenase (Lulai, 1988; Ikediobi et al., 1989). These enhanced enzyme activities may result in increases in free fatty acids and free radicals that are toxic to many cellular processes and capable of causing organelle inactivating proteins and lysis (Brecht, 1995). Additionally, excessive ethylene production enhances permeability of membranes and reduces phospholipid biosynthesis (Watada et al., 1990). For example, in fresh-cut carrot, total phospholipids and phospahatidic acid increased but phosphatidylcholine decreased (Picchioni et al., 1994).




28
Picchioni et al. (1994) also found that rough endoplasmic reticulum numbers increase in fresh-cut carrot, which may be correlated to lipid synthesis and the enzyme induction process.
Textural and cell wall changes. Firmness loss is immediate and faster in wounded tissues due to cell rupture and loss of tissue integrity (Miller, 1992). Cell wall enzyme activity may be accelerated by wounding (Miller, 1992; Karakurt and Huber, 2002), which may contribute extensive softening in fresh-cut tissues. To illustrate, Karakurt and Huber (2002) found that PG and at- (EC 3.21.22) and P-galactosidase activity (EC 3.2.1.23) was higher in fresh-cut papaya fruit compared to intact fruit at 5 OC. The enhanced cell wall enzyme activity, thus, causes depolymerization of pectic and hemicellulosic polyuronides, which may result in further textural changes in fresh-cut tissues.
Dehydration. Fresh-cut processing causes interior tissues to be exposed to air and to increase in evaporation rate which results in water loss. Dehydration at the cut surface is sometimes obligatory to control microbial growth; however, it may provoke undesirable visual appearances such as color fading in carrot skin (Watada et al., 1996). External and Internal Factors Contributing to Quality of Fresh-cut Fruits and Vegetables
Wounded tissues rapidly deteriorate and senesce. Hence, minimizing the negative consequences of wounding is a crucial step that affects storage life and maintenance of interior and exterior qualities of fresh-cut produce. These qualities are greatly affected by morphological, physiological, environmental, pathological and practical factors.
Raw product. Since fresh-cut fruits and vegetables are already at the table-ripe
edible stage, they should have excellent interior and exterior qualities. Fresh-cut produce




29
should also have superior characteristics such as slower ripening rate, good texture and flavor qualities and less sensitivity to chilling injury and microorganisms than their counterparts because they are more perishable compare to intact produce (Watada et al., 1996).
Temperature. Fresh-cut fruits and vegetables should be held at lower temperatures to slow down metabolic activity. Lower temperatures are also necessary to control microbial growth. However, most of tropical and some subtropical commodities are chill sensitive; therefore, storage at a lower temperature may lead to chilling injuries. On the other hand, keeping fresh-cut produce at lower temperatures suppresses the development of chilling injury symptoms for a limited period (Watada and Qi, 1999).
Relative humidity. Relative humidity of the atmosphere of fresh-cut produce
should be higher to reduce extensive water loss (Schlimme, 1995). Edible or non-edible coating and proper packing may reduce water loss from fresh-cut produce (Watada et al., 1996; Schlimme, 1995). In many cases, water loss in fresh-cut produce results from epidermal membrane deterioration. Particularly, at higher temperatures where the water vapor deficit is large, the water loss hastens in fresh-cut produce (Watada et al., 1996).
Controlled atmosphere and modified-atmosphere packing. Reduced oxygen
and elevated carbon dioxide levels are basic practices of controlled atmosphere condition. The response of fresh-cut produce stored in controlled atmosphere is different from that of intact produce; therefore, fresh-cut produce should be stored differently and separately. On the other hand, because of the short handling period, controlled atmosphere may not be economically applicable for fresh-cut produce (Watada et al, 1996). Gas compositions




30
in film-packed and edible-coated fresh-cut produce can be modified and this modification may extend storage life of fresh-cut produce (Watada et al., 1996; Schlimme, 1995).
Packing and edible coating. Fresh-cut produce may be packed or coated to reduce mechanical damage and water loss, to identify produce and to carry information to consumer (Schlimme, 1995). Nevertheless, packing may cause an increase in temperature that evokes higher respiration and ethylene production rate. Therefore, ethylene must be excluded or absorbed by ethylene absorbents such as charcoal and palladium chloride (Schlimme, 1995). Edible films reduce moisture loss, limit gas exchange, retard ethylene production and keep aroma inside (Ahvenainen, 1996). Lipids, resins, polysaccharides and proteins are the basic components of edible films (Baldwin et al., 1995). Some coatings may carry some additives that can prevent discoloration and microbial growth by serving as antioxidants and/or anti-microbial agents (Baldwin et al., 1995). Some of these additives are sucrose polyesters of fatty acids, sodium salts of carboxymethylcellulose, carrageenan and chitosan (Ahvenainen, 1996).
Chemical application. Chemical applications are mostly used for reducing decay and browning, and retaining firmness in fresh-cut produce. Chlorine is the standard sanitizing agent for fresh-cut produce in proper concentrations (100-300 ppm; pH, 7). Higher chlorine concentrations (> 500 ppm) may cause fresh-cut produce to discolor, equipment to corrode and aromatic hazardous chloramines to form (Hurst, 1995). Hong and Gross (1998) reported that sodium hypochlorite caused some physiological and biochemical alterations in fresh-cut tomato fruit (higher electrolyte leakage and ethylene production). Sulphating agents are the most common chemicals for inhibiting browning reactions. In addition to sulphites, ascorbic acid, sodium dehydroacetic acid, potassium




31
sorbate, citric acid, zinc, chloride and calcium chloride, resorcinol derivatives, and carbon dioxide and carbon monoxide are used as anti-browning agents (Ahvenainen, 1996).
Microorganism. Microorganisms readily grow on and in fresh-cut produce, and some of them may be detrimental to human being such as Escherichia coli. The following microorganisms have been found in fresh-cut produce: mesophilic bacteria, lactic acid bacteria, coliforms and fecal coliforms, yeast and molds, and pectinolytic microflora such as Pseudomonasfluorescens and Xanthomonas maltophila (Nguyen-the and Carlin, 1994). Mesophilic microflora is the largest population followed by lactic acid bacteria (Watada et al., 1996). Moreover, some food-borne microorganisms have been reported in fresh-cut produce that includes Listeria monocytogenes, Yersinia enterocolitica, Aeromonas hydrophila (Nguyen-the and Carlin, 1994; Alfred, 1994), Staphylococcus aureus (Nguyen-the and Carlin, 1994), Escherichia coli (Nguyen-the and Carlin, 1994; Alfred, 1994), Salmonella spp. (Nguyen-the and Carlin, 1994), Clostridium botulinum (Alfred, 1994), Bacillus cereus, Giardia lamblia (Beuchat, 1995), Shigella ssp., and Plesiomonas shigelloides (Alfred, 1994). The hepatitis A and Norwalk agent virus also have been reported in fresh-cut produce (Beuchat, 1995).
Microbial growth, particularly of human pathogens, may be inhibited by competing bacteria such as lactic acid bacteria or naturally existing antimicrobials released during fresh-cut processing (Luna-Guzman and Barrett, 2000). Low temperature is one of the most effective methods to control and prevent microbial growth (Nguyenthe and Carlin, 1994) while some of the organisms can survive in low temperatures such as Listeria, Yersinia and Aeromonas (Alfred, 1994).Washing fresh-cut produce with chlorine solution (up to 300 ppm) is another effective way to impede development of




32
microorganisms (Nguyen-the and Carlin, 1994). This process cannot completely eliminate all microorganisms because microorganisms can survive when they are inside tissues where disinfectants cannot penetrate (Watada et al., 1996). Washing fresh-cut produce in trisodium phosphate is one of the other effective ways to control microbiological growth (Beuchat, 1995). Ozone, a strong oxidant, is also used for its lethal activity upon microorganisms at microgram per milliliter concentrations (Beuchat, 1995). In addition to chemical solutions, organic antagonism, gamma irradiation, boiling in water and edible coatings containing biochemical agents are also used for the control of microbial growth (Watada et al., 1996; Nguyen-the and Carlin, 1994). Irradiation is a safe, effective and hazard-free antimicrobial method (Farkas, 1998). Campyolcbacter, Yersinia, Vibrio and Escherichia coh have low resistance to ionizing radiation (Farkas, 1998). Slow ripening and senescence induced by restricted ethylene action or synthesis may extend the storage life of fresh-cut produce: in a less ripe condition, the growth of most opportunistic microorganisms would be expected to be retarded since they tend to grow most rapidly on senescent tissues (Zagoray, 1999).
Cell Wall
Introduction
The plant cell wall is an important structure that determines cell shape, connects cells to each other, provides essential mechanical strength and ridges and acts as vital barrier against abiotic and biotic invaders such as insects and dusts. The chemical and physiological structure of the plant cell wall varies widely from plant group to plant group and from cell type to cell type. The plant cell wall is a dynamic structure that changes during the life cycle of a cell.




33
Cell Wall Structure
The plant cell wall is constructed by a very complex but highly organized
composite of many different polysaccharides, proteins and aromatic substances. The cell wall consists of three main divisions; the primary cell wall, the middle lamella and the secondary cell wall (Carpita and McCann, 2000). The primary cell wall, born in the cell plate during cell division, is capable of growth by expansion; the middle lamella forms the interface between adjacent cells; and secondary cell wall builds up upon the primary cell wall when the cells mature and are no longer growing (Goldwin, 1983; Brett and Waldron, 1996). The model of cell wall structure is thought be by three coextensive networks: the cellulose-hemicellulose framework, the pectic matrix, and a network of structural proteins (Carpita and Gibeaut, 1993). Cellulose, the most abundant plant polysaccharide, exists in the form of microfibrils that are an unbranched p (1-4) linked polymer of D-glucose strengthened by hydrogen bonds (Goodwin, 1983; Carpita and MacCann, 2000). Pectins are a mixture of heterogeneous and highly hydrated polysaccharides, rich in D-galacturonic acid. Pectins consist of 6 different polymers rhamnogalacturonan I, rhamnogalacturonan II, homogalacturonan, arabinan, galactan, and arabinogalactan I (Carpita and MacCann, 2000). Hemicelluloses are a class of polysaccharide, hydrogen-bonded to cellulose microfibrils (Carpita and MacCann, 2000). The two major hemicellulosic polymers are xyloglucans and glucomannans in flowering plants; the other polymers include xylans, mannans, galactomannans and arabinogalactan II, callose, and 31,3 and 31,4-glucans. Proteins such as extension and expansin, and phenolics such as lignin and ferulic acid are part of the plant cell structure (Reiter, 1994).




34
Cell Wall Loosening and Growth
Cell growth is provided by expansion or elongation that creates an irreversible increase in cell volume. The cell wall must change its structure to expand or elongate: cell wall loosening is probably the primary event in this process followed by continued deposition of new materials. Cellulose microfibril orientation controls cell expansion or elongation and decides the plane of elongation (Carpita and MacCann, 2000). The multinet growth hypothesis explains displacement of the microfibrils during growth. New microfibrils deposited in strata on the inner surface of the cell wall in mostly transverse orientation replace older ones that are pushed towards the outer layers of the cell wall and reoriented in the direction of the cell elongation (Carpita and MacCann, 2000). The acidgrowth hypothesis proposes that auxin causes lower pH conditions by pumping proton, which activates apoplast-localized growth-specific hydrolyses that cleave the loadbearing bonds that join cellulose microfibrils to other polysaccharides (Cosgrove, 2000). This cleavage produces a loosening in the cell wall as well as water uptake leading an increase in cell size (Coscgrove, 2000). Two kinds of enzymes xyloglucan endotransglycosylase (EC 2.4.1.72) and expansins are thought to be involved in cell wall loosening.
Fruit Ripening and the Cell Wall
The textural changes during fruit ripening are thought to be related to alterations in cell wall structure (Huber, 1983; Tucker and Grierson, 1987). The changes are mostly correlated with structure and composition of pectic components (Seymour et al., 1987). Solubilization and depolymerization of pectins (Fischer and Bennett, 1991) and hemicelluloses (Lashbrook et al., 1997) during ripening are frequently related to cell wall loosening and disintegration. Cell wall modifications have been extensively studied in




35
tomato fruit, and early reports indicated that pectin degradation by PG represented the model of fruit softening; however, PG-antisense tomato fruit revealed that pectin degradation is not essential for fruit ripening (Smith et al., 1988; Giavannoni, 1990). The other major changes during ripening occur in hemicellulose content. Xyloglucan, the chief hemicellulose in dicotyledonous plants, undergoes depolymerization in most fruits, including tomato (Sakurai and Nevins, 1993). Besides the depolymerization of both pectic and hemicellulosic polyuronides, there is a loss of neutral sugar from neutral pectins, primarily galactose and arabinose (Tucker, 1993). The Cell Wall and Pathogen Attacks
The plant cell wall fortifies cells against attacks from microorganisms and even other plants. Callose and lignin are thought to act as a physical barrier, blocking fungal penetration into plant cells (Hammond-Kosack and Jones, 2000). Hydroxyproline-rich glycoproteins contribute to defense against fungal attacks by cross-linking to the cell wall matrix and initiating additional lignin formation. PG-inhibiting proteins restrain PG activity, originated from pathogens, which is also a part of defense mechanisms (Hammond-Kosack and Jones, 2000). Oligosaccharides derived from the cell walls of fungi and plants, including 3-glucans, chitin, chitosan, and pectin, are inducers of the synthesis of a wide spectrum of defensive chemicals in plant tissues (Ryan, 1988). The oligosaccharides are generated at infection or wound sites and may be early signals to activate genes whose products, such as antibiotic phytoalexins, extensins, proteinase inhibitors, pathogenesis-related proteins and lignin, enhance the plant's defense system against pathogens and herbivores (Ryan, 1988).




CHAPTER 3
DELAYING ETHYLENE-INDUCIBLE RIPENING PROCESS 1METHYLCYCLOPROPENE IN 'GALIA' MELON FRUIT Introduction
Ethylene has been known to regulate fruit ripening and softening in climacteric fruits (Lelievre et al., 1997). Accidental exposure to ethylene or natural ethylene production can reduce the postharvest life of climacteric fruits by accelerating ripening and senescence (Reid, 1985). The ethylene inducible effect, however, can be delayed/prevented by some ethylene antagonists or ethylene action inhibitors including silver thiosulphate (STS), 2-5 norbornadiene, diazocyclopentadiene, and 1methylcyclopropene (1-MCP) (Sisler and Serek, 1997; Sisler and Serek, 1999). Commercial use of STS in cut flowers is being considered in some countries due to Ag' heavy metal in STS complex (Sisler and Serek, 1997). 1-MCP, therefore, seems to be the most practical ethylene action inhibitor due to its stability, activity in low concentration, and non-toxic and odorless properties (Sisler and Serek, 1997; Sisler and Serek, 1999). The ability of 1-MCP to inhibit ethylene action in apple fruit resulted in promising commercial development (Saftner et al., 2003). Furthermore, studies with 1-MCP confirmed that post-storage life and quality of tomato fruit at early and advanced stage of ripening can be improved by I-MCP (5, 10, 20 and 100 ptL L-1 for 24 h at 20 oC, Wills and Ku, 2002; 50 to 150 nL L" 1-MCP for 20 h at 20 oC, Hoeberichts et al., 2002).
'Galia' fruit (Cucumis melo var. reticulatus L. Naud. cv. Galia) is a climacteric fruit in which ripening is achieved by the help of ethylene, and ethylene and respiratory
36




37
climacterics (Seymour and McGlasson, 1993). The fruit has excellent flavor and aroma characteristics; however, storage life of 'Galia' fruit, harvested an early stage of ripening (green peel color), is limited to 2-3 weeks even at low temperatures (Aharoni et al., 1993; Fallik et al., 2001). Restriction of ethylene synthesis has proved that storage life of melon fruit can be extended. For example, 'Galia' fruit at an early stage of ripening held in controlled atmosphere of 10% CO2 and 10% 02 with an ethylene absorbent, potassium permanganate, for 14 days at 6 'C plus an additional 6 days at 20 'C had higher quality (reduced fruit softening and decay) than control fruit stored in controlled atmosphere only (Aharoni et al., 1993).
The present study was performed to characterize the physiological responses of 'Galia' melon fruit to 1-MCP treatment and determine whether 1-MCP treatment could be effective as a postharvest application for the extension of the storage period or storage life of pre-ripe or ripe 'Galia' fruit.
Materials and Methods
Plant Material
'Galia' plants were grown according the growing techniques and production
practices established by Shaw et al. (2001) in Greenhouse Facilities at the University of Florida Horticultural Farm near Gainesville, FL in spring 2001. Temperatures were recorded every 15 min at various locations in the greenhouse using thermocouples and a datalogger (CR-IO Campbell Scientific, Inc., N. Logan, UT). No additional heating or cooling units installed in the greenhouse. Fruit were harvested at two stages of maturity, green (GRN, early stage of ripening) and yellow (YLW, advanced stage of ripening) according a color chart (1, dark green; 2, green; 3, light yellow with green; 4, light yellow; 5, yellow; 6, dark yellow to orange) reported by Fallik et al. (2001). The




38
harvested fruit were transferred to the Postharvest Horticulture Laboratory of Gainesville. The fruit were then selected on the basis of uniformity of size and freedom from defects; afterwards, the fruit were gently brushed, washed with tap water (23 C), dipped into 200 VLL' chlorinated water for 1 min and air-dried. 1-MCP Application
A commercial powder formulation provided by Agrofresh (active ingredients
0.14%) Philadelphia, Penn. was used to generate 1-MCP. I -MCP was released from three g of the powder to the vapor phase by adding 50 mL deionized water, generating a 7.5 mLL- concentrated stock in a 136-mL sealed vial. I-MCP concentration (1 mL) in the headspace of the vial was measured using a gas chromatograph (GC) (Hewlett Packard 5890 II GC; Avondale, PA) furnished with a 80-100 mesh Chromosorb PAW stainless steel column (1.8 m x 3.18 mm i.d.; Supelco, Bellefonte, PA). Injector, oven, and detector (FID) temperatures were set at 150, 150 and 200 0C, respectively. Isobutylene gas, which has a FID response similar to that of 1-MCP (Jiang et al., 1999) was used as a standard. Approximately a 10-mL sample of vial headspace gas was injected into a 179-L metal chamber containing a 50-L void space, yielding a final I-MCP concentration of 1.5 VL L- and held for 24 h at 20 C. The treatment containers were vented for 5 minutes, resealed, and reinjected with fresh I -MCP at 6-h intervals. Control fruit were kept under identical condition.
1-MCP concentration and efficacy was investigated in a preliminary experiment in which GRN fruit were treated with air (control), 0.09, 0.9 and 9 PL U1 1-MCP for 24 h at 20 C and stored at 15 0C.
Respiration and Ethylene Production




39
Air-(control) and 1-MCP-treated fruit were placed in airtight plastic containers (1 fruit per container) (3.6 L) and sealed for 1 h at 20 C. Respiration and ethylene production from each the treatment (5 replications) was determined by measuring the CO2 and C2H4 concentration in the headspace of the containers. For CO2, 0.5-mL headspace gas sample was injected to a GC (Gow-Mac, Bridge Water, NJ) equipped with thermal conductivity detector, and for C2H4, 1-mL headspace gas sample was infused into the Hewlett-Packard-5890 GC fitted with a flame ionization detector. Firmness Assessment
Mesocarp fruit firmness was measured on opposite sides of (two equidistant points on the equatorial region) pared fruit using an Instron Universal Testing Instrument (Model 441-C8009, Canton, MA). The probe (convex, II mm diameter) located at zero force and contacted with the pared fruit surface was driven to a depth of 10 mm with a crosshead speed of 50 mm min'. Firmness data was expressed as the maximum force Newton (N) acquired during penetration. All tests were conducted with fruit pulp temperature of 20 'C.
Electrolyte Leakage Assessment
Five mesocarp cylinders were removed the equatorial position of a fruit with an 8mm diameter cork borer. From each cylinder, I disk (8 x 8 x 8 mm3) was excised by the same cork borer, yielding to a total of 5 disks per fruit. The disks were briefly rinsed with deionized water and blotted dry on a slightly moistened Whatman filter paper. The disks (five per fruit) were then incubated in 15 mL of 500 mM mannitol for 6 h in a capped polypropylene tube. Conductivity was measured with a conductivity bridge (YSI-3 IA, Yellow Springs, OH) furnished with a conductivity cell (3403, Yellow Springs, OH) immediately after addition of the bathing solution to the disks and the end of the




40
incubation period. The aliquot removed from the bathing solution for the conductivity measurement was added back to the bathing solution. The disks and bathing solution were then stored at -20 'C for at least 24 h, thawed, boiled in water for 30 min, cooled to room temperature, and conductivity measurement was measured once more. The electrolyte leakage was expressed as percentage of the conductivity of total tissue electrolytes.
Soluble Solids Concentration, pH and Titratable Acidity Determination
Soluble solids concentration (SSC), titratable acidity (TA), and pH were quantified using a digital refractometer (Abbe Mark-10480, Buffalo, NY), a Fisher-395 dispenser (Fisher 395, Pittsburg, OH) equipped with an electrometer (Fisher 380, Pittsburgh, PA), and a digital pH meter (Corning, NJ), respectively. Mesocarp tissue (80 g) was macerated with a mortar and pestle and centrifuged at 27,200 RFC for 10 min at 21 'C. Fruit juice collected from the macerated/centrifuged tissue was used for SSC and pH measurement. For TA, 6-g fruit juice was titrated with 0.1 N NaOH to an end point of pH 8.2, and TA was expressed as percentage of malic acid using the volume of mL NaOH recorded from the dispenser.
Statistical and Informal Taste Analyses
General linear model program of SAS (SAS institute, Carey, NC) and Duncan's Multiple Range Test were performed for Completely Randomized Designs. Informal taste analyses to determine the edible stage on fruit surface and flesh appearance, odor, flavor and texture quality were performed by untrained personnel of the postharvest research group of University of Florida.




41
Results
1-MCP Concentration and Efficacy
The firmness of GRN control fruit decreased from 66.7 to 6.3 N while fruit treated with 0.09 iL L- 1-MCP only softened from 67.8 to 11.3 N, fruit treated with 0.9 piL L' 1-MCP from 67.1 to 17 N and fruit treated with 9 gL L- 1-MCP 70.3 to 18.1 N over a 21-day period at 15 'C (Figure 3-1). The decrease in firmness from day 1 to 21 was over 10-fold in the control whereas approximately 6-fold in 0.09 iL L'-1-MCP-treated fruit and 4-fold in both 0.9 tL L'-1-1-MCP- and 9 pL L- 1-MCP-treated fruit. The firmness level of fruit treated with 9 [tL L-1 1-MCP did not result in a significant difference in firmness relative to the fruit treated with 0.9 VL L-' I-MCP from on days 9 through 21, indicating that the saturation level of I-MCP is between 0.9 to 9 [L L' 1-MCP. Therefore, with the evaluation of previous 1-MCP-related publications, the present studies were performed with 1.5 ptL L- 1-MCP. Respiration and Ethylene Production
Except for the climacteric rise, respiration of both control and 1-MCP-treated fruit decreased during storage (Figure 3-2A). However, GRN control fruit reached its respiratory climacteric peak at day 6 (9.4 mL kg-1 h-') as equal with 1-MCP-treated fruit at day 15 (6 mL kg- h-'), resulting in an 11-day delay in the climacteric respiratory peak and a 36% reduction of the magnitude of respiratory climacteric peak. Ethylene production from GRN control fruit increased rapidly, reached a peak at day 3 (7.8 pIL kg-I h-'), and then decreased while ethylene climacteric of GRN 1-MCP-treated fruit started to peak at day 3 and reached its maximum at 9 days (2.7 p.L.kg' h-1), leading to a 6-day delay and 65% reduction in the magnitude of climacteric ethylene peak (Figure 3-2A). The ethylene production by GRN 1-MCP-treated fruit was statistically lower relative to




42
GRN control on days 1 through 5 when ethylene climacteric of GRN control fruit occurred.
Respiration of YLW control and 1-MCP-treated fruit gradually decreased during storage, with no statistical differences between the two treatments (Figure 3-2B). Ethylene production in both YLW control and 1-MCP-treated fruit also declined during storage. The ethylene production of YLW I -MCP-treated fruit showed a 56% decrease from the first day of storage (5.4 p-L kg-l L-1 at day 1) to the last day (2.4 gL kg-1 L-' at day 11) whereas YLW control fruit nearly a 90% decrease from day 1 (3.8 iL kg" L-1) to day 9 (0.4 1iL kg1 L), resulting a difference between treatments after day 3. YLW fruit treated with 1-MCP produced higher ethylene after day 3 while the ethylene rate was continued to decrease in YLW control fruit. Firmness
Firmness of either GRN control or 1-MCP-treated fruit declined during storage as shown in Figure 3-3A. GRN control fruit soften very quickly within first 5 days, losing 66% of their original firmness, while GRN 1-MCP-treated lost only 46%. At the last day of storage of GRN control (day 13), GRN control maintained only 6% of their initial value while GRN 1-MCP-treated fruit 20%, from then on, 1-MCP-treated fruit remained relatively firm and preserved 10% their initial firmness at the end of their storage (day 21).
Firmness of YLW control fruit and I-MCP-treated fruit was not significantly
different from each other during the first 2 days of storage (Figure 3-3B). After 2 days of storage, firmness of the YLW control fruit sharply decreased but not that of the YLW 1MCP-treated fruit. Softening of both treatments remained unchanged from day 5 to 9, from then on, that of YLW l-MCP-treated showed a sharper decline. Within 5 days,




43
YLW control fruit softened from 16.5 to 4.9 N (a 70% loss) while YLW l-MCP-treated fruit from 18 to 12.8 N (a 29% loss). At the end of the storage life (YLW control, day 9; YLW 1-MCP, day 11), YLW control maintained only 30% of their initial firmness while YLW 1-MCP-treated fruit 43%.
Electrolyte Leakage
A continuous increase in electrolytes released from mesocarp tissue of either GRN control or GRN I -MCP-treated fruit was observed until day 13. The treatments peaked their maxima of 35.8% (control) and 28.5% (1-MCP) at day 13 (Figure 3-4A). GRN control displayed statistically higher leakage rates than GRN I -MCP-treated fruit after day 3. Electrolyte efflux of GRN l-MCP-treated fruit slightly decreased from day II to 21. YLW control fruit showed an increasing electrolyte leakage through day 7; afterwards, showing a minimal decrease (Figure 3-4B). Electrolytes of YLW l-MCPtreated fruit slightly increased through day 11 as well (Figure 3-4B). The maxima of electrolyte efflux of YLW control fruit was 36.7% at day 7 whereas in YLW I-MCPtreated fruit 27.9% at day 11, resulting a significant difference between the two treatments after day 5.
Soluble Solids Concentration, pH and Titratable Acidity
Soluble solids of either GRN control or GRN l-MCP-treated fruit showed very
little change, with no differences between treatments, and averaged from 8.1% to 8.9% as shown in Figure 5A. SSC in YLW control fruit slightly decreased during storage whilst in YLW 1 -MCP-treated fruit somewhat increased but magnitude of change and differences were unremarkable, and the soluble solids ranged from 10 and 11 % (Figure 35B).




44
TA of GRN control increased a little until day 5, after that point, moderately
decreased; however, TA of GRN 1-MCP-treated fruit remained unchanged starting on days 5 through the end of storage (Figure 3-6A). Neither differences nor changes of TA of GRN control and I -MCP-treated fruit were noted during storage. TA of either YLW control or I -MCP-treated fruit slightly increased during storage though a minimal decrease was observed at the end (Figure 3-6B). YLW l-MCP-treated fruit had significantly higher TA than the control on days 7 through 9.
The pH of GRN control slightly decreased until day 7, and then, increased;
however, in 1-MCP-treated fruit did not show a unique pattern as illustrated in Figure 37A. In either YLW control fruit or I-MCP-treated fruit, pH very slightly decreased while a small peak was noted at the end (Figure 3-7A). The magnitude of changes and differences in pH of either GRN or YLW fruit treated with and without I-MCP was unremarkably low.
Informal Quality Analysis
The color change of fruit surface from green to yellow was deferred in GRN 1MCP-treated fruit (Figure 3-8). The color change of fruit skin from green to greenishyellow was also deferred in YLW 1-MCP-treated fruit (Figure 3-9). The edible stage (determined by the informal quality analysis with the help of firmness and color evaluation data) lasted on days 5 through 9 for GRN control and on days 13 through 19 for I-MCP-treated fruit, leading a 4-day delay in edibility and a 40% extension of edible stage. YLW control fruit persisted their edibility through day 5 whereas YLW I-MCPtreated fruit through day 9, representing almost a two-fold extension (80%). Fruit exhibited < 4 N firmness were not edible. Neither YLW nor GRN fruit treated with and without I -MCP did show significant external and internal decay occurrences.




45
Discussion
Treatment with 0.9 gL L- 1 -MCP significantly improved firmness retention of GRN 'Galia' fruit relative to 0 and 0.09 i.L L- 1-MCP concentration at 15 C. Increasing 1-MCP concentration from 0.9 to 9 tL U' did not confer additional benefit upon firmness of GRN 'Galia' fruit. In a previous study, charentais melon fruit exposed to I jL L' I -MCP (for 24 h at 22 C) stored at 2 C for 16 days and rewarmed for additional 5 days at 22 C became insensitive to low-temperature damage (estimated by visually rating the extend of the fruit surface pitting and browning) compared to non-I-MCPtreated fruit (Ben-Amor et al., 1999). Thereby, we propose that the commercial 1-MCP concentration for 'Galia' melon fruit would be approximately I to 1.5 1tL L' for 24 h at 20 C.
'Galia' fruit is characterized by a classic climacteric ethylene and respiration pattern (Figures 3-2A and 2B). The maximum ethylene production rate of 'Galia' fruit was below 10 gL kg' h-' during ripening, a comparable result noted by Zheng and Wolf (2002) at 24 C. Respiration of'Galia' fruit ranged from 6 to 13 mL kg' h- and declined during ripening excluding climacterics. Our results indicate 'Galia' fruit is an inferior ethylene producers; the ethylene climacteric occurs earlier than the respiratory climacteric during ripening.
1-MCP suppressed both ethylene production and ethylene climacterics in GRN 'Galia' fruit, indicating that I-MCP efficiently binds the ethylene receptors, thereby, limiting the positive feedback regulation of ethylene production in 'Galia' fruit during ripening. 1-MCP delayed both ethylene and respiratory climacteric rise of'Galia' fruit by 6 and 11 days, respectively. Similarly, preclimacteric Charentais muskmelon melon fruit exposed I VtL L- for 24 h at 14 C exhibited a delay in ethylene climacteric peak (4 days)




46
relative to non-1-MCP-treated fruit stored and measured at 14 0C (Chatenet et al., 2000). YLW 'Galia' control fruit showed a declining ethylene rate compared with 1-MCPtreated fruit, resulting a higher ethylene production in YLW 1-MCP-treated fruit. A possible explanation of this observation is: blocking ethylene binding sites of YLW 'Galia' fruit by 1-MCP may cause an interference between ethylene and the ethylene control mechanism, consequently, ethylene fails to perceive the quantity of ethylene production, and is being continued to synthesized (Mullins et al., 2000; Zhong et al., 2001).
1-MCP delayed the respiratory climacteric and suppressed its magnitude in GRN 'Galia' fruit, proving that ripening of melon fruit is strongly regulated by ethylene (Flores et al., 2001). The respiration of YLW 1-MCP-treated fruit, however, was not affected by 1-MCP, which implies respiration might not be directly related to senescence or overripening in melon fruit (Saltveit, 1993; Bower et al., 2002).
Softening of GRN 'Galia' fruit strongly deferred by I-MCP, consisting with the fact that most fruits exposed to 1-MCP at the early stage of ripening showed firmness retention relative to non-l-MCP-treated fruit (Fan et al., 1999, Jiang et al., 1999; Jeong et al., 2002; Wills and Ku, 2002). 1-MCP delayed loss of firmness in YLW 'Galia' fruit (at the advanced stage of ripening) as well. Apple (0.7 1iL L1 I-MCP for 16 h at 20 'C; Mir et al., 2001 or 10 pL L' I-MCP for 6 h at 20 'C; Jiang and Joyce, 2002) and tomato (treated with 50 150 nL L"1 1-MCP for 20 h at 20 'C; Hoeberichts et al., 2002) fruit at advanced stage of ripening treated with 1-MCP also showed delayed softening. Thus, the softening process in melon fruit even at the advanced stage of ripening is regulated by ethylene (Lelievre et al., 1997; Flores et al., 2001).




47
Electrolyte efflux, a measurable symptom of membrane damage (Marongoni et al., 1996), of both GRN and YLW 'Galia' fruit treated with and without 1-MCP increased during storage. The increase in electrolytes during ripening has been previously reported for muskmelon type melon fruit (Lester and Stein, 1996; Lacan and Baccou, 1996). The increase in leakage of both GRN and YLW 'Galia' fruit was repressed by 1-MCP, showing that membrane deterioration during melon fruit ripening is regulated by ethylene. Ethylene has been reported to stimulate the activities of free-radical-producing enzymes that contribute membrane deterioration (Paliyath and Droillard, 1992). To date only one study has been reported for the effects of I -MCP upon electrolyte efflux: petunia flower corollas treated with 150 nL L- I-MCP for 6 h at 22 'C after 12 .IL LC2H4 application displayed lower leakage rates compared to the corollas treated with ethylene; however, direct application of I -MCP (no pre-ethylene treatment) did not affected electrolyte leakage (Serek at al., 1995b).
Soluble solids concentration in either GRN or YLW 'Galia' fruit was not
significantly affected by I-MCP since muskmelon fruit types have little or no starch reserve (Seymour and McGlasson, 1993). The effects of I-MCP upon TA and pH were minimal. 1-MCP caused slightly higher TA in YLW fruit while did not have a significant effect on GRN fruit. Higher TA due to I-MCP application has been noted for tomato fruit at an advanced stage of ripening (5 to 100 kL L- 1-MCP for 2 h at 20 'C; Wills and Ku, 2002). 1-MCP had no influence upon pH of either GRN or YLW 'Galia' fruit. The color change of GRN 'Galia' fruit surface from green to yellow in YLW fruit were deferred by 1-MCP, which confirms that loss of chlorophylls and increase in carotenoids are ethylene-dependent process in melon fruit (Flores et al., 2001). The inhibitory effect of I-




48
MCP upon color change or development has been reported for most climacteric fruits at an early stage of ripening (Golding et al., 1998; Jiang and Joyce, 2000; Jeong et al., 2002). Fruits treated at the advanced stage of ripening responded to 1-MCP by deferring color change/development as well such as 'Golden Delicious' apple at 4 'C (1 10 [tL U 1-MCP for 6 h at 20 'C; Jiang and Joyce, 2002) and tomato at 20 0C (50 to 150 nL L' 1MCP for 2 h at 20 'C; Hoeberichts et al., 2002).
1-MCP significantly extended the edible stage of both GRN and YLW 'Galia' fruit by 40 and 80%, respectively. One of the affirmative effects of 1-MCP is the extended storage life for most fruits treated at the early stage of ripening (Hofman et al., 2001). Recently, apple (Mir et al. 2001; Pre-Aymard et al., 2002; Jiang and Joyce, 2002) and tomato (Wills and Ku, 2002; Hoeberichts et al., 2002) fruit at an advanced stage of ripening has been reported to respond to 1-MCP by improving their shelf life. Thus, overripening or senescence in climacteric fruits can be delayed by l-MCP. Coriander leaf senescence, as assessed by chlorophyll and protein loss, was significantly delayed by IMCP (Jiang et al., 2002). Tucker and Brady (1987) and Smith et al. (1989) earlier reported that silver thiosulphate arrested tomato ripening once initiated. Thereby, climacteric fruit ripening from the early to the advanced stage of ripening necessitates ethylene.
In summary, I-MCP extended storage and storage life of'Galia' fruit at different stages of maturity. Therefore, the use of 1-MCP seems to be a novel postharvest application that has commercial potential for melon shippers, retailers and even consumers. The affirmative effects of I -MCP upon ripe fruit would benefit for fresh-cut fruit industry as well. Our results demonstrate 'Galia' fruit was strongly benefited from 1-




49
MCP application; however, 'Galia' fruit does not represent all types of melon fruit. Thus, future studies are needed involving different melon types and cultivars.




50
80
--- Control 70 --- 0.09 tL L- 1-MCP
-0-- 0.9 itL L-1 1-MCP 60 9 pL L 1-MCP
S50 40
E
30 20 10
0
0 3 6 9 12 15 18 21 24
Days
Figure 3-1. Unpared fruit firmness of green 'Galia' fruit treated with air (control), 0.09,
0.9 IL L-1 and 9 RtL" 1-MCP during storage at 15 C. Vertical bars are
standard deviations of means.




51
20 30
--- Control CO2 AA 25
15 -o-- 1-MCP CO2
SControl C2H4 20
-a-- 1-MCP C2H4
10 15
10
5
52 111 11 pB 3
20
15
8
2 10
4
5
10
0 I I.III. 0
0 2 4 6 8 10 12 14 16 18 20 22
Days
Figure 3-2. Respiration and ethylene production of green (A) and yellow (B) 'Galia'
melon fruit treated with 1.5 pL L' 1-MCP and air (control) during storage at
20 'C. Vertical bars represent standard deviation of the means (n = 5).




52
50
A 40
- Control
30 --- 1-MCP
20
10
0
~50
40
30
20
0 2 4 6 8 10 12 14 16 18 20 22
Days Figure 3-3. Mesocarp firmness of green (A) and yellow (B) 'Galia' melon fruit treated
with 1.5 pL L- 1-MCP and air (control) during storage at 20 C. Vertical bars
represent standard deviation of the means (n = 5).




53
45
A
40 35
30 25
20 Control
--- I-MCP
15
45 '40 B
S35
30 25 20 15
10....
0 2 4 6 8 10 12 14 16 18 20 22
Days
Figure 3-4. Electrolyte leakage of green (A) and yellow (B) 'Galia' melon fruit treated
with 1.5 tL L-1 I-MCP and air (control) during storage at 20 'C. Vertical bars
represent standard deviation of the means (n = 5).




54
20
A
15
-5 Control
---- I-MCP
10
0
20
B
~15
2 10
5
0 2 4 6 8 10 12 14 16 18 20 22
Days Figure 3-5. Soluble solids concentration of green (A) and yellow (B) 'Galia' melon fruit
treated with 1. 5 [tL L-1 I-MCP and air (control) during storage at 20 'C.
Vertical bars represent standard deviation of the means (n =5).




55
0.20
A Control
0.15 I-MCP
0.10
0.05 : 0.00 0.20
o 0.15
0.10 0.05
0.00 .. .. .
0 2 4 6 8 10 12 14 16 18 20 22
Days
Figure 3-6. Titratable acidity of green (A) and yellow (B) 'Galia' melon fruit treated with
1.5 gL L- 1-MCP and air (control) during storage at 20 C. Vertical bars
represent standard deviation of the means (n = 5).




56
7.0
Control A
6.5 1-MCP
6.0
5.5
7.0
B
6.5
6.0
55 I I i i i i i
0 2 4 6 8 10 12 14 16 18 20 22
Days
Figure 3-7. The pH of green (A) and yellow (B) 'Galia' melon fruit treated with 1.5 pL U
1-MCP and air (control) during storage at 20 C. Vertical bars represent
standard deviation of the means (n = 5).




57
1-MCP
........CONTROL
Day 13
Figure 3-8. 'Galia' fruit harvested at the pre-ripe stage (green surface) were treated with
1.5 utL L-1' 1-MCP or air (control) and then stored for 13 days at 20 oC.




58
Figure 3-9. 'Galia' fruit harvested at the ripe stage (yellow surface) were treated with 1.5
u4L L"' 1-MCP or air (control) and then stored for 7 days at 20 oC.




CHAPTER 4
PHYSIOLOGICAL CHANGES IN FRESH-CUT AND INTACT 'GALIA' MELON
FRUIT WITH TREATED 1-METHYLCYCLOPROPENE Introduction
The increase in consumer demand for fresh-cut produce has prompted increased
research interest in devising and implementing methods for improving and prolonging the quality of these highly perishable products. Fresh-cut processing involves several steps including peeling, and cutting, shredding, etc. The physical injury attendant to fruit processing initiates a series of events such as increased respiration and ethylene production, stimulated phenol metabolism, and increased enzyme activities (Rolle and Chism, 1987; King and Bolin, 1989). The secondary events resulting from wounding contribute to the challenge for improving the keeping quality of fresh-cut produce. The storage life of fresh-cut commodities can also be compromised by the proliferation of microorganisms including mesophilic microflora, lactic acid bacteria, coliforms and fecal coliforms, yeasts and other fungi, and pectinolytic microflora (Nguyen-the and Carline, 1994). Low temperature has been used to preserve quality and extend storage life of fresh-cut produce. Although cold storage retards many biological processes in fresh-cut produce, events leading to tissue softening and deterioration continue at low temperature especially for fresh-cut fruits. Fresh-cut melon, one of the most popular fresh-cut fruit (International Fresh-cut Produce Association, 2003), is not an exception for these freshcut fruits displaying rapid tissue softening and deterioration (Lamikanra et al., 2000). Postharvest applications such as dipping fruit slices/cubes in dilute hypochlorite 50 1tL
59




60
L"1 total available chlorine (pH 6) (Ayhan et al., 1997), in 2.5% calcium chloride solution (Luna-Guzman and Barrett, 2000), or storing in controlled atmosphere of 2% 02 + 10% CO2 at 5 'C and 4% 02 + 10% CO2 at 10 'C (Qi et al., 1998) have been reported to improve and extend storage life of fresh-cut melons. To date, no studies have been reported upon fresh-cut 'Galia' melon fruit while other melon types especially muskmelon and honeydew fruit have been often studied as fresh-cut produce. It has been reported that storage life of fresh-cut honeydew melon were limited to 11 days at 4 'C whereas storage life of fresh-cut muskmelon 4 to 6 days at 4 or 5 'C (O'Connor-Shaw et al., 1994; Qi et al., 1998).
'Galia' melon is a climacteric fruit (Seymour and McGlasson, 1993); thus,
ripening process is ethylene-mediated. The ethylene-mediated effects on climacteric fruit can be significantly delayed by the use of ethylene binding inhibitors. One of these, Imethylcyclopropene (1-MCP; Sisler and Serek, 1997) has been shown to extend the storage life and period of optimum quality of apple (Jiang and Joyce, 2002) and tomato (Wills and Ku, 2002; Ku et al., 2002) fruits at advanced stages of ripening. Exposure of fresh-cut postclimacteric apple fruit before or after cut to 1-MCP (1 or 10 1IL L1 for 6 h at 20 'C) improved their storage life by reducing softening and color change of epidermal tissue (loss of green color) at 4 'C (Jiang and Joyce, 2002). Thus, we proposed that freshcut 'Galia' fruit should benefit from I-MCP application as well. The objectives of this study were to investigate responses of fresh-cut ripe (derived from postclimacteric intact fruit subject to treatments) versus intact ripe 'Galia' fruit to I -MCP.




61
Materials and Methods
Plant Material
'Galia' plants were grown according the growing techniques and production
practices established by Shaw et al. (2001) in Greenhouse Facilities at the University of Florida Horticultural Farm near Gainesville, FL in spring 2002. Temperatures were recorded every 15 min at various locations in the greenhouse using thermocouples and a datalogger (CR-10 Campbell Scientific, Inc., N. Logan, UT). No additional heating or cooling units installed in the greenhouse. 'Galia' fruit were harvested at three-quarter to full-slip stage and transferred to the postharvest facilities at the University of Florida in Gainesville. The fruit were selected for uniform size (approximately 1200 to 1300 g), external color (yellow) and netting development. Ethylene production and respiration rate for the fruit at the time of harvest was approximately 2 gL kg' h-1 and 10 mL kg- h- at 20 'C, respectively, and soluble solids concentrations were about II to 12%. The fruit were gently washed with tap water, immersed in 200-[tL L- chlorinated water for I min (23 'C), and air-dried before transferring to 20 'C for 1-MCP application. 1-MCP Quantification and Treatment
The source of 1-MCP was Agrofresh commercial powder (active ingredient
0.14% I-MCP) from Agrofresh, Philadelphia, PA. Three g powder were dissolved in 50mL demonized water of a 136-mL vial; afterwards, the vial was sealed with a septum and incubated on oscillating shaker for 2 h at room temperature. 1-MCP concentration in the vial headspace was measured using a gas chromatograph (Hewlett Packard-5890, Avondale, PA) equipped with a 80-100 mesh Chromosorb PAW stainless steel column (1.8 m x 3.18 i.d.; Supelco, Bellefonte, PA) at an injector, oven and detector (FID) temperature of 150, 150 and 200 'C, respectively. Isobutylene gas was used as standard to




62
calculate 1-MCP concentration (Jiang et al., 1999). Approximately 7,5 mL L-' 1-MCP stock in the headspace of the vial was generated from the 3-g powder. Vial-headspace gas sample (7.5 mL) was injected into a 174-L metal chamber having a 56.5-L void volume, yielding a final 1-MCP concentration of I jL L-, and maintained for total exposure period of 24 h at 20 C. The metal chamber was vented for 5 min at 6-h intervals and reinjected with fresh 1 -MCP avoid CO2 accumulation. Control fruit was kept under similar condition.
Preparation of Fresh-cut 'Galia', and Treatment Design
The fruit were transferred from 20 'C to a 5 0C facility that had been sanitized using 200 VL L'-chlorinated water prior to use. After a I-h period to allow temperature equilibration, the blossom and pedicle ends of the fruit were removed, and the fruit were longitudinally (from the pedicel and to the stem end) cut into 2.5 cm slices using a plastic Bread Slicer (Cuope-Pain). The slices were peeled and cut into cubes (2.5 x 2.5 x 2.5 cm3, 15 to 16 g) using a double bladed knife. The cubes were then flushed with a sterile isotonic mannitol solution (500 mM) using a squeeze bottle, and placed in non-airtight plastic containers (1.7 L, FridgeSmart) that has built-in grid on the bottom lifts (9 cubes/container). A total of 60 containers (30 each for I -MCP and control fresh-cut tissue) were used in this experiment, and 10 of these (5 each of each treatment) were removed at 2-day intervals for quality evaluation. Additionally, 80 intact fruit (40 each of control and I -MCP-treated fruit) were stored along with the fresh-cut tissue at 5 C. The
4 treatments included, fresh-cut tissue derived from intact ripe fruit pre-treated with air (FC-CNT: fresh-cut control), fresh-cut tissue derived from intact ripe fruit pre-treated with 1-MCP (FC-MCP: fresh-cut I-MCP), intact ripe fruit pre-treated with air (IF-CNT:




63
intact fruit control), and intact ripe fruit pre-treated with 1-MCP (IF-MCP: intact fruit IMCP).
Ethylene Analysis
Ethylene production was measured at room temperature every other day enclosing fresh-cut and intact fruit in plastic containers (0.9 L and 3.6 L, respectively) allowing ethylene to accumulate for 2 h at 2-day interval at 5 'C. Nine cubes per fruit for FC-CNT or FC-MCP and I fruit for IF-CNT or IF-MCP were placed in the airtight containers prior to sampling. A I-mL headspace sample was withdrawn by a hypodermic syringe through a rubber septum, ethylene production was measured using a GC (Hewlett Packard 5890 II, Avondale, PA) equipped with a flame ionization detector. The carrier gas (Nitrogen) was 30 mL min'. Oven, injector and detector temperature was 70, 200 and 250 'C, respectively.
Firmness Assessment
Mesocarp firmness of a fruit cube was measured using an Instron Universal
Testing Instrument (Model 4411, Canton, MA) equipped with a 5-kg load cell and an 8mm convex probe at 20 'C. During firmness measurement, intact fruit or fresh-cut fruit containers were kept coolers. Intact fruit prior to firmness measurements were diced into cubes using the procedures described above for fresh-cut processing. The probe was positioned at zero force contact with a fruit cube surface, and driven to a depth of 10 mm at a crosshead speed of 50 mm min-'. Firmness data are reported as the maximum force (Newton) recorded during penetration.
Electrolyte Leakage
Mesocarp disks (5 discs per cube or fruit), in 8 mm diameter and 8 mm thickness, were removed from centermost part of either fresh-cut tissue or intact fruit with an 8-mm




64
diameter cork borer, rinsed with deionized water, blotted dry, and incubated in 15 mL of 500 mM mannitol for I h in capped polypropylene tubes. Incubations were conducted at room temperature, on an oscillating shaker set at 1.4 cycle sec1. The conductivity of the bathing solution was measured at the end of the 1-hour incubation using a conductivity bridge (YSI-3 IA, Yellow Springs, OH) equipped with a conductivity cell (Model 3403, Yellow Springs, OH). The aliquot removed for the conductivity measurement was added back to the bathing solution. The disks and bathing solution were then stored -20 'C for at least 24 h, thawed, and heated in a boiling water bath for 30 min, cooled to room temperature, and conductivity again measured. Electrolyte leakage was expressed as a percent of total conductivity estimated from the frozen/heated samples. Pectin Efflux
Five mesocarp cylinders were removed from the mid section of fresh-cut tissue or an equatorial section of an intact fruit using a 15-mm diameter cork borer. The cylinders were then sliced into disks perpendicularly from the long axis of a cylinder using a double bladed razor in which a razor was mounted from the other razor by 10 mm. Each disk, 15 mm diameter and 10 mm thick, weighed approximately 5 g. The disks (5 per replication) were briefly rinsed with distilled water, blotted dry, and incubated in 10 mL of 500 mM sucrose on an oscillating shaker for 6 h at room temperature. Afterwards, the bathing solution was filtered through a Whatman GF/C filter, and 40 mL of 95% ethanol was added to the filtrate. The filtrates were centrifuged at 2,000 g for 20 min at 4 'C and the supernatant was discarded. The pellet was washed with 40 mL of 80% ethanol (2 times) and dissolved in 5 mL distilled water, and total uronic acid was determined by the m- phenylphenol method (Blumenkrantz and Asboe-Hansen, 1973).




65
Quality Evaluation
Fruit surface and flesh color were measured using a chromameter (Minolta-CR200, Japan). The results were presented as lightness (L*, representing the lightness or grey scale), hue angle (the dimension of color that specifies a position on a color wheel of 3600, with 00, 900, 1800 and 270' representing the hues red, yellow, green and blue, respectively) and chroma (distinguishing the difference from a grey shade to a pure hue) values. Water soaking on the flesh was expressed as the percentage of areas of a cube with a 5% interval (total 5 cubes for each treatment) using only the top face. Five intact fruit or five fresh-cut cubes from each treatment were evaluated for mesocarp water soaking. Informal descriptive analysis was used to profile the quality of either fresh-cut cubes and intact fruit flesh by untrained personnel, evaluating appearance, odor, texture and flavor (O'Connor-Shaw et al., 1994) according to the following hedonic chart: 1, poor; 2, poor-good; 3, fair; 4, good-excellent; and 5, excellent. The informal descriptive analyses were done immediately after removing fresh-cut or intact fruit from the 5-C cold room at days 0, 2, 4, 6, 8 and 10. The samples were tested under white light and at the room temperature, 23 C, with testing at least 3 samples for each treatment. Microbial Counts
Fruit tissue (5 g) was removed with a flame-sterilized cork borer (21.5 mm
diameter) and knife from innermost part of a fruit or with a flame-sterilized knife from a fruit cube (approximately 1/3 of a cube) on sterilized aluminum foil in an air-circulated fume. The fruit tissue then was incubated in a 45-mL sterile phosphate buffered solution (PBS), pH 7. The bathing solution and fruit tissue were vortexed at high speed using a vortex (Fisher-Genie 2, Scientific Industries Inc., Bohemia, NY) for I min. Afterward, a series of dilutions were prepared using sterile PBS as needed. Total aerobic,




66
Enterobacteriaceae, yeasts and other fungi, total coliforms, and lactic acid bacteria counts were made using 1 mL inoculum of the bathing solution. The plates and incubation conditions for each count were: total aerobic count, 3M Petrifilm aerobic count plate (3M Microbiology Products, St. Paul, MN), 3 days at 30 C; Enterobacteriaceae, 3M Petrifilm Enterobacteriaceae count plate, 1 day at 30 C; yeast and other fungi, 3M Petrifilm yeast and mold count plate, 5 days at 25 C; total coliforms, 3M Petrifilm coliform count plate,
1 day at 30 'C; and lactic acid bacteria, 3M Petrifilm aerobic count plate anaerobic incubation for 2 days at 30 C in a 1.9-L airtight plastic container with an anaerobic system envelope (Gas Pak, Becton and Dickinson Co., Cockeysville, MD). The plates were prepared in an air-circulated hood after 0 (immediately after dicing), 5 and 10 days at room temperature, and microbial counts were reported as colony forming units per gram of tissue (CFU g-).
Experimental Design and Statistics
The experiments were conducted in a randomized complete-block design, using 3 to 5 replications per treatment. Statistical procedures were performed using the PC-SAS software package. Differences between means were determined using the Duncan Mean Comparison Test.
Results and Discussion
Ethylene Production
Ethylene production of fresh-cut cubes of ripe 'Galia' fruit stored at 5 oC (FCCNT, 10.4 jtL kg' h'; FC-MCP, 8.5 1iL kg-1 h') was at least 4-fold higher than that of intact fruit (IF-CNT, 1.9 ltL kg- h-'; IF-MCP, 1.6 pL kg' h-1) at day 1 (Figure 4-1). Ethylene production of both fresh-cut fruit and intact fruit declined during storage; however, intact fruit displayed statistically lower ethylene production and declining rates




67
relative to fresh-cut fruit on days 1 through 7. Ethylene production declined approximately 5-fold in FC-CNT (from 10.3 to 2.1 VaL kg1 h-) and 4-fold in FC-MCP (from 8.5 to 1.9 VtL kg"1 h-1) over a 9-day period. Ethylene production in intact fruit declined approximately 2 to 3 folds in both IF-CNT (from 1.9 to 0.8 kgl h') and IF-MCP (from 1.6 to 0.6 kgl h). Generally, ethylene production of intact ripe 'Galia' fruit was under 2 p.L kg' h-' at 5 'C, which is similar to the case for ethylene production rate of intact ripe or postclimacteric muskmelon fruit held at 5 'C (I to 3 jiL kgl h-1; LunaGuzman et al., 1999). These authors, however, reported slightly higher ethylene production rates in intact ripe muskmelon fruit (approximately from I to 3 gtL kg" h-1) compared with fresh-cut ripe muskmelon fruit (approximately from 0.5 to 2 [tL kg1 hf) during 12 days of storage. Fresh-cut 'Galia' fruit in this study had higher ethylene production rates than intact 'Galia' fruit, likely due to both the wound response, stressrelated ethylene production in wounded cells (Rolle and Chism, 1987), and increased surface area exposed to the atmosphere after dicing, facilitating oxygen diffusion to interior cells (Zagory et al., 1995). The decrease in ethylene production in intact and fresh-cut ripe 'Galia fruit during storage is in agreement with the findings of LunaGuzman et al. (1999), who reported that intact or fresh-cut ripe muskmelon fruit stored at
5 'C exhibited declining ethylene production rates during the first 7 days of storage.
In the present study, the effect of 1-MCP on ethylene production was insignificant though some small differences were noted at day 1 between IF-CNT and IF-MCP. However, fresh-cut postclimacteric 'Golden Delicious' apple fruit treated with 1-MCP (1 or 10 pL L- for 6 h at 20 'C) before or after cutting had lower ethylene production relative to non-l-MCP-treated fruit (fruit sample sealed in glass jars at 20 'C) after 5 or




68
10 days of storage (Jiang and Joyce, 2002). The low ethylene production of fresh-cut and intact 'Galia' fruit at 5 C may be due to the low storage temperatures employed and/or the inherently low ethylene production of'Galia' fruit relative to other muskmelon types such as both preclimacteric 'Giant Perfection' and 'Iroquois' that, have ethylene rate of over 80 VL kg1 h1 (Zheng and Wolf, 2000). In contrast, the ethylene production rate of intact ripe 'Galia' fruit is under 6 p-L kg-' h-1 during ripening at 20 'C (Chapter 3). Firmness Assessment
As shown in Figure 4-2, both fresh-cut and intact ripe 'Galia' fruit regardless of 1-MCP softened moderately during storage. IF-CNT and FC-CNT lost about 31% and 22% of their original firmness, respectively, after 10 days while IF-MCP and FC-MCP softened 16% and 11%, respectively. At day 10, IF-MCP (10.6 N) had 22% higher firmness compared to IF-MCP (8.7 N), and FC-MCP (11. 6 N) had 32% higher firmness than FC-CNT (8.5 N). Firmness loss in either fresh-cut tissue derived from ripe I-MCPtreated fruit, or in intact ripe fruit treated with I-MCP was significantly lower compared with either fresh-cut ripe control or intact ripe control fruit during storage, proving that melon fruit softening is an ethylene-dependent process (Flores et al., 2001). Recent studies have shown that 1-MCP can delay softening of climacteric fruits when applied at advanced stages of ripening. The deferred softening has been shown for apple (1 or 10 [IL L 1-MCP for 6 h at 20 'C; Jiang and Joyce, 2002) and tomato (50 to 150 nL Ll, for 24 h at 20 'C; Hoeberichts et al., 2002). Additionally, fresh-cut postclimacteric 'Golden Delicious' apple fruit treated with 1-MCP (1 and 10 pL L1 for 6 h at 20 'C) before or after cutting showed significant firmness retention relative to fresh-cut control fruit after
5 or 10 days at 4 'C (Jiang and Joyce, 2002).




69
Electrolyte Leakage
Electrolyte leakage, a estimate of membrane permeability and integrity
(Marangoni et al., 1996), slightly increased in both intact ripe and fresh-cut ripe 'Galia' fruit during storage (Figure 4-3). The increase in leakage of both IF-CNT (from 13.1% to 14.3%) and IF-MCP (from 12.3% tol4.1%) was below 15% on days 0 through 10 while the increase FC-CNT (from 14.9 to 20.7) was 38% and in FC-MCP (from 14.24 to 18) was 26%. 1-MCP did not suppress the leakage increase in either intact or fresh-cut fruit. However, intact ripe 'Galia' fruit treated 1-MCP (1.5 pL L-1) displayed slightly lower leakage during a limited time (on days 7 through 9) compared with intact ripe control at 20 'C (chapter 3). Fresh-cut ripe 'Galia' fruit showed slightly higher leakage compared with intact ripe fruit during storage at 5 C, which supports the fact membrane permeability increases in response to wounding (Portela and Cantwell, 2001). Membrane permeability is a common feature of senescing organs and ripening fruit (Lester and Stein, 1993; Flores et al., 2001). Increased membrane permeability results in a loss of cellular components and accumulation of liquid in intercellular spaces (Saltveit, 1997). Similar to fresh-cut ripe 'Galia' fruit, increased leakage (measured at 22 'C) for fresh-cut climacteric/postclimacteric muskmelon fruit (approximately a 15% increase) was reported by Portela and Cantwell (2001) on days 0 through 12 stored at 5 'C. Portela and Cantwell (2001) further noted that fresh-cut muskmelon discs prepared by a blunt cork borer caused slightly higher leakage (22%) than fresh-cut fruit prepared by a sharp borer since the blunt borer resulted in more severe damage in tissues relative to the sharp borer. Pectin Efflux
Pectin efflux values from mesocarp disks of both FC-CNT (18.8 g kg-1 fresh
weight) and FC-MCP (19.1 g kg- f.w.) were 2-fold higher than those of IF-CNT (9.1 g




70
kg'1 f.w.) or IF-MCP (9.3 g kg'1 fw.), respectively, at day 2, and the efflux remained over
2 folds throughout the remainder of storage (Figure 4-4). Pectin efflux from mesocarp disks of FC-CNT increased by 25% and that of FC-MCP by 22% on days 2 thorough 4; afterwards, the efflux decreased by 15% in FC-CNT and by 11% in FC-MCP through day 10 compare to the values at day 2. However, efflux from mesocarp disks of both IF-CNT and IF-MCP did not change during storage, with having the approximate average efflux of 9.3 g kg-1 f.w. Pectin efflux from mesocarp disks was unaffected by prior 1-MCP treatment in either fresh-cut or intact ripe 'Galia' fruit; however, pectin efflux increased significantly in fresh-cut ripe fruit relative to intact ripe fruit, indicating that pectin degradation may be affected by wounding. The insignificance of I -MCP upon pectin efflux was reported for pericarp disks of ripe tomato fruit treated with 1-MCP (4.8 iL L-_' for 24 at 18 oC) and stored for 2 to 3 weeks at 5 'C (Almeida, 1999). The wounding effect upon pectin degradation was noted for fresh-cut ripe papaya fruit by Karakurt and Huber (2003), who reported that the levels of water and CDTA-soluble polyuronides increased significantly within 24 h after cutting and remained higher (over 50%) compared to intact ripe fruit during 8 days of storage at 5 'C. Higher uronic acid efflux in fresh-cut 'Galia' fruit may be the result of increased activities of cell wall enzymes including polygalacturonase and P3-galactosidase. (Miller et al., 1987; Karakurt and Huber, 2003). Recently, Moctezuma et al. (2003) reported the TBG4 (tomato D-galactosidase) gene was up-regulated by ethylene while the TBG6 gene was down-regulated by ethylene in ripe tomato fruit. Additionally, I-MCP (1 tL L-1 for 12 h at 20 'C) has been shown to decrease the activity of pectinmethylesterase, a-mannosidase and 3-glucosidase in ripe 'SanCastrese' apricot fruit at 20 'C (Botondi et al., 2003).




71
Quality Evaluation
No changes in color parameters of hue angle and L* (lightness) of the skin of both IF-CNT and IF-MCP were noted during storage (Figure 4-5A and 5B), indicating that the original skin color of intact fruit regardless of 1 -MCP remained unchanged. However, the skin color intensity became duller during storage, as evaluated by the development of chroma value (Figure 4-5C). Flesh color and intensity/purity of either FC-CNT or FCMCP remained unchanged during storage, as estimated by L*, hue angle and chroma (Figure 4-5D, 5E and 5F). Flugel and Gross (1982) observed relatively low levels of chlorophyll and carotenoids in the flesh of 'Galia' fruit with a gradual decrease in both types of pigment during ripening, which might explain the insignificant color change and insignificant variation among treatments noted here.
Water soaking in both FC-CNT and FC-MCP 'Galia' fruit increased with storage while the flesh of either IF-CNT or IF-MCP showed no sign of the disorder (Figure 46A). The extent of water soaked areas in FC-CNT was 44% after 10 days while that of FC-MCP was only 15%, leading a significant difference between these treatments from day 8 to 10. All 4 treatments had excellent quality at day 0, as assessed by sensory evaluation (Figure 4-6B).The score for sensory of IF-CNT or IF-MCP never dropped below 5 (excellent) during storage while FC-CNT scored 2.8 (poor-good to fair) at day 10 (Figure 4-8). On days 4 through 8 the sensory scores for FC-CNT fruit were below 4 (fair to good-excellent) whereas that of FC-MCP fruit were over 4 (good-excellent to excellent), which indicates the limit of acceptability, the average keeping quality, of fresh-cut ripe 'Galia' fruit was 4 days (Figure 4-7) at 5 'C while that of fresh-cut ripe fruit derived from intact ripe 1-MCP-treated fruit was 8 days at 5 'C.




72
Changes in either mesocarp or fruit skin color of'Galia' melon fruit is due a
gradual decrease in both chlorophyll and carotenoid content during ripening (Flugel and Gross, 1982). The color data 'Galia' fruit during storage showed no color changes (based on hue angle) in either fruit skin or mesocarp were recorded regardless of treatment, which may indicate ripening progress was deferred by the low temperature, 5 'C. Water soaking of fresh-cut 'Galia' fruit increased with storage time and water soaking of freshcut fruit was significantly higher than those of intact 'Galia' fruit on days 6 through 10. 1-MCP did not have a significant effect on water soaking of fresh-cut 'Galia' fruit during most of the storage period but significantly suppressed the increase in water soaking noted during the period from 8 through 10 days. In contrast, 1-MCP (1 pl L' for 24 h) did not affect water soaking in Charentais melon flesh, treated at the preclimacteric stage, after 35 days of storage at 14 'C (Chatenet et al. 2000). The authors further reported that water soaking in Charentais melon mesocarp during the late stage of ripening was not an ethylene-inducible event based on the absence of expression of genes encoding 1aminocyclopropane- 1-carboxylic acid synthase and 1 -aminocyclopropane- I -carboxylic acid oxidase. Water soaking in fresh-cut 'Galia' fruit, however, seems to be an ethylenedependent process which may be related to the stage of ripeness and/or wounding. Chatenet et al. (2000) attributed the water soaking phenomenon to a depletion of cell wall calcium and Karakurt and Huber (2002) to ethylene-inducible changes in membrane permeability. Lipolytic enzymes including lipoxygenase and phospholipase D are like involved in wound-induced degradation of membrane lipids (Karakurt and Huber, 2003). Lipoxygenase can contribute to the membrane permeability by involving production of




73
reactive oxygen species, participating in peroxidative reactions (Huber et al., 2001), and inactivating protein synthesis (Karakurt and Huber, 2003) Microbial Counts
Total aerobic load in fresh-cut ripe 'Galia' fruit increased sharply during days 5 to 10 of storage at 5 C, with FC-CNT having higher counts (1.8 x 103 CFU g-') than FCMCP (1.5 x 103 CFU g-) at day 10 (Table 4-1). Total aerobic population of intact ripe fruit were very low compared to fresh-cut fruit and showed a very slim increase through day 10 (Table 4-1). Total coliforms or other fungi in either fresh-cut or intact fruit were negligible throughout storage (Table 4-1). The Enterobacteriaceae population in FC-CNT and FC-MCP increased significantly during storage, and FC-CNT showed statistically higher counts (8.7 x 101 CFU g-1) than FC-MCP (5.2 x 10 CFU g-1) at day 10 (Table 4-1). Enterobacteriaceae were almost undetectable in intact fruit over the 10-day storage period (Table 4-1). There was also an increase in lactic acid bacteria of both fresh-cut and intact fruit; however, fruit with 1-MCP (IF-MCP) or derived l-MCP-treated fruit (FC-MCP) displayed higher counts compared to fruit without I -MCP at day 10 (IF-CNT and FCCNT; Table 4-1). Yeasts accumulated significantly in fresh-cut ripe fruit on days 5 through 10 (FC-CNT, 1.2 x 103 CFU g-1; FC-MCP, 1.1 x 103 CFU g-'), with no variations between the two treatments while yeasts in intact fruit were imperceptible (Table 4-1).
The significant increase in microbe populations of fresh-cut 'Galia' arose after day 5, which is in agreement with the finding of Luna-Guzman and Barrett (2000) who noted a significant raise of total aerobic counts in non-l-MCP-treated fresh-cut ripe muskmelon fruit (sanitized with 50 1iL L'-chlorinated water) after 4 days of storage at 5 'C. Luna-Guzman and Barrett (2000) recorded total aerobic counts ranging from approximately 2 x 102 to 9 x l09 CFU g-1, plated at days 4, 8 or 12. These values are




74
quite high compared to the values noted herein for fresh-cut ripe 'Galia' fruit. Higher total aerobic (from 1.4 x 104 to 8.8 x 104 CFU g-'), lactic acid bacterium (from 1.4 x 102 to 7.2 x 102 CFU g-1) and Enterobactericium (from 7.8 x 103 to 3.2 x 104 CFU g-) counts were also documented in muskmelon fruit (undefined ripeness stage) stored 11 days at 4 'C by O'Connor-Shaw et al. (1994). However, Ayhan et al. (1998) reported a similar or slightly lower microbial count fresh-cut muskmelon fruit (undefined ripeness stage) (10 x 101 CFU cm2 for total aerobic counts; 1 x 101 CFU cm2 for yeast; and 1 x 101 CFU cm2 for other fungi) washed with chlorinated water (50 [tL L1 total available chlorine) and sealed with modified atmosphere (95% nitrogen and 5% oxygen) stored 10 days at 2.2 'C. Low microbial population (1.5 x 103 to 3 x 103 CFU g-1 for total aerobic count and l x 102 CFU g for yeast count) in pre-ripe/ripe non-I -MCP-treated fresh-cut muskmelon fruit stored for 6 days at 5 'C (not washed previously with chlorinated water) was also noted by Portela and Cantwell (2001). Portela and Cantwell (2001) attributed the low microbial count in the fresh-cut muskmelon to strict sanitation procedures during freshcut processing. 1 -MCP slightly suppressed total aerobic and Enterobactericium count increase in fresh-cut 'Galia fruit whereas lactic acid bacterium growth was promoted. Even so, the lactic acid bacterium population was very low in all treatments and at all times of evaluation, with thinking the maximum total limit for microbial growth for fresh-cut vegetables (is this value for vegetables of relevance to fruits?) is 5 x 107 CFU g 1 (Francis et al., 1999). The 'Solo' papaya variety treated with 25 jiL U' I-MCP at 20 C for 14 h at mature green stage and stored at 20 C for approximately for 20 days showed slightly higher symptoms of stem rots, body black rots, and anthracnose compared to untreated fruit lasted approximately 5 days (Hofman et al., 2001). Hofman et al. (2001)




75
attributed the slight increase in the symptoms of the diseases in 1-MCP-treated fruit to a reduction in antifungal concentrations due to extended storage life Jiang et al. (2001) found that ripe 'Everest' strawberry fruit treated with 500 to 1000 nL L1 1-MCP (24 h at 20 C) displayed accelerated leak disease development at 20 'C compared to non-I-MCPtreated fruit; however, 1-MCP at 100 and 250 nL L-1 delayed the onset of the decay. Tomato plant lines expose to 10 nL L- 1-MCP (24h at 18 to 24 'C) resulted in a significant increase in Botrytis cinerea frequency in the cultivars 'Moneymaker' and 'Castlemart' but no increase in 'Pearson' (Diaz et al., 2002). Hence, the effects of I -MCP upon microbial growth are complex and depend on the type of microorganisms, I -MCP concentration, cultivars, and tissue type and development.
In summary, the storage life of fresh-cut melon fruit derived from intact ripe 'Galia' fruit treated with I-MCP was extended by 4 days compared to fresh-cut fruit, derived from intact ripe non-I -MCP-treated fruit that lasted 4. Thus, I -MCP can be considered a safe potential growth regulator or conditioner for fresh-cut melon fruit alternative to calcium chloride/lactate, ethylene absorbent or controlled atmosphere. Finally, the natural resistance of melon fruit to microbial growth can be supported by using sanitized equipments, containers, spaces and low temperature.




76
12
--0-- Intact control 10 Intact I-MCP
---- Fresh-cut control ,- 8 _L-- Fresh-cut I-MCP
6
." 4
2 0
1 3 5 7 9
Days
Figure 4-1. Ethylene production for intact ripe 'Galia' fruit with and without 1-MCP and
fresh-cut fruit derived from intact ripe fruit treated with and without I -MCP
during storage at 5 C. Vertical bars represent standard deviation of the means
(n = 5).




77
18 16 14
__12 10 S 8
-h
6O---- Intact control
--G- Intact I-MCP
4 -v-- Fresh-cut control
-v--- Fresh-cut I-MCP
2
0 2 4 6 8 10
Days
Figure 4-2. Mesocarp firmness for intact ripe 'Galia' fruit with and without 1-MCP and
fresh-cut fruit derived from intact ripe fruit treated with and without I -MCP
during storage at 5 C. Vertical bars represent standard deviation of the means
(n = 5).




78
30
---- Intact control Fresh-cut control
25 Intact I-MCP Fresh-cut 1-MCP
20
'
-15 S10
5 0
0 2 4 6 8 10
Days
Figure 4-3. Electrolyte leakage from mesocarp tissues of intact ripe 'Galia' fruit with and
without I -MCP and fresh-cut fruit derived from intact ripe fruit treated with and without I -MCP during storage at 5 C. Vertical bars represent standard
deviation of the means (n = 5).




79
30 25
- 20
-4-- Intact control --- Fresh-cut control 15 ---- Intact I-MCP Fresh-cut I-MCP
.E 10 __ _ _ _ __ _ _
5
01
0 2 4 6 8 10
Days
Figure 4-4. Pectin efflux from mesocarp tissues of intact ripe 'Galia' fruit with and
without I -MCP and fresh-cut fruit derived from intact ripe fruit treated with and without 1-MCP during storage at 5 C. Vertical bars represent standard
deviation of the means (n = 5).




80
75 80
70 75
49 49
65 70 ,J
6 65 "
Q 65
.= 60 .. 60 ..P
A5-o- Intact control D
50 -0-- Intact I-MCP 50
--v-- Fresh-cut control
60 --v-- Fresh-cut 1-MCP 40
35
50 3
30
40 25
20
3 0 1 5
B E
20 .. .o 1
100 1... !20
95
90
85 110E
80 105
75 C F
70 .. .oo10
0 2 4 6 8 10 0 2 4 6 8 10
Days
Figure 4-5. Color parameters, L* lightness (A), hue angle (B) and chroma (C) for ripe
'Galia' fruit skin treated with and without 1-MCP (1 VL LI) and the color
parameters (D, E and F) for intact ripe fruit with and without I -MCP and
fresh-cut fruit derived from intact ripe fruit treated with and without I -MCP
during storage at 5 C. Vertical bars represent standard deviation of the means
(n = 5).




81
60
A 7
50
-4- Inatc control 40 -0-- Intact 1-MCP
-v- Fresh-cut control 30 ---v- Fresh-cut 1-MCP
0
10
0 5
4 B
4
S3
2
1
0
0 2 4 6 8 10
Days Figure 4-6. Mesocarp water soaking percentage (A) and sensory evaluation (B) for intact
ripe 'Galia' fruit with and without 1 -MCP and fresh-cut fruit derived from
intact ripe fruit treated with and without 1 -MCP during storage at 5 C.
Vertical bars represent standard deviation of the means (n = 5). When bars
absent, the value for standard deviations was within the dimension of the
symbol.




82
i~i ...........,
FC-CNT M FC-MCP
Figure 4-7. Ripe fresh-cut 'Galia' fruit derived from intact ripe fruit treated with (FC-1MCP) and without 1-MCP (FC-CNT) and then stored for 4 days at 5 oC.




83
F.
FC-CNT FC-MCP
Figure 4-8. Ripe fresh-cut 'Galia' fruit derived from intact ripe fruit treated with (FCMCP) and without 1-MCP (FC-CNT) and then stored for 10 days at 5 oC.




84
Table 4-1. Microbial counts (CFU g- fresh weight) for intact ripe 'Galia' fruit with and
without I -MCP and fresh-cut fruit derived from intact ripe fruit treated with and without 1-MCP during storage at 5 oC. IF-CNT, intact control; IF-MCP, intact control with 1 -MCP; FC-CNT, fresh-cut control fruit without 1 -MCP;
and FC-MCP, fresh-cut fruit derived from intact I -MCP-treated fruit.
Total aerobic count Total coliforms
Day Day
Treatment 0 5 10 Treatment 0 5 10
IF-CNT 4.7 a 0.7 b 8.0 c IF-CNT 0 a 0 a 3.3 a
IF-MCP 0 a 0 b 11.3 c IF-MCP 0 a 0 a 3.3 a
FC-CNT 6.0 a 2.0 b 1.8 x 103 a FC-CNT 0 a 0 a 5.3 a
FC-MCP 6.0 a 15.3 a 1.5 x 103 b FC-MCP 0 a 0 a 4.0 a
Enterobacteriaceae Lactic acid bacteria
0 5 10 0 5 10
IF-CNT 0 a 0 b 0.7 c IF-CNT 0.7 a 0.7 a 6.7 b
IF-MCP 0 a 0 b 4.7 c IF-MCP 0.7 a 0 a 22.7 a
FC-CNT 0 a 26.0 a 86.7 a FC-CNT 0 a 0.7 a 6.7 b
FC-MCP 0 a 0 a 52.7 b FC-MCP 4.7 a 2.0 a 22.0 a
Yeasts Other fungi
0 5 10 0 5 10
IF-CNT 0 a 0 a 1.3 b IF-CNT 0 a 0 a 2.7 a
IF-MCP 0 a 0 a 2.0 b IF-MCP 0 a 0 a 1.3 a
FC-CNT 0 a 3.3 a 1.1 x 103a FC-CNT 2.0 a 0 a 0 a
FC-MCP 0 a 0 a 1.1 x 103a FC-MCP 0.7 a 0.7 a 4.0 a
Means (n = 3) followed by the same letter within a column are not significantly different, P < 0.05.




CHAPTER 5
STORAGE LIFE EXTENTION OF PRE-RIPE AND RIPE 'SUNRISE SOLO'
PAPAYA FRUIT BY 1-METHYLCYCLOPROPENE Introduction
The storage life of papaya fruit under tropical conditions (30 'C) is limited due to their high respiration rate, delicate skin, and high water content (Sankat and Maharaj, 1997). Papaya fruit harvested at the color break stage can be kept for periods of up to 16 days at 10 to 16 'C (Sankat and Maharaj, 1997). Furthermore, papaya fruit can be harvested at the mature green stage; however, according to Hawaiian grade standards, the fruit must have at least 6% surface yellow coloration to meet the minimum grade requirement of 11.5% soluble solids (Akamine and Goo, 1971). For local markets, papaya fruit are harvested at the full-ripe stage (full yellowish-orange surface coloration; Morton, 1987) and stored at temperatures above 7 'C. Storage below 7 to 10 'C may cause low-temperature injuries depending on the variety and maturity stage (Paull and Chen, 1983; Sankat and Maharaj, 1997).
Approaches to extending the postharvest quality and duration of the chill-sensitive papaya have included the use of controlled-atmosphere storage, polymeric films and wax coating, and gamma irradiation (Sankat and Maharaj, 1997). For example, Maharaj and Sankat (1988) reported that papaya fruit harvested at the color break stage and stored under controlled atmosphere conditions of 1.5 to 2% 02 and 5% CO2 at 16 'C remained acceptable for 17 to 29 days. Another, more facile approach to extending the storage life and quality of harvested papaya fruit has been through the application of 185




86
methylcyclopropene (1-MCP), a potent anti-ethylene compound (Sisler and Serek, 1997). In recent years some very effective agents for blocking ethylene action have been discovered by Sisler and coworkers, and four of them are extensively used in scientific investigation: 2,5-norbornadiene, trans cyclooctene, diazocyclopentadiene and 1-MCP (Sisler and Serek, 1999). Silver thiosulphate is used widely as commercial non-organic ethylene action inhibitor for cut flowers potted plants (Sisler and Serek, 1997). SincelMCP has no detectable odor, minimal phototoxic properties, and is stable and active at very low concentrations, it has been the favored compound among the inhibitors of ethylene responses (Sisler and Serek, 1997; Sisler and Serek, 1999). Recently, it is or has been reported that 1-MCP improved the storage life and quality of fruits treated prior to or during ripening including apple (Fan et al., 1999; Pre-Aymard et al., 2002) and tomato (Wills and Ku, 2002; Hoeberichts et al., 2002). 1-MCP (90 or 270 nL L-1) extended the storage life of 'Sunrise Solo' papaya fruit treated at an early stage of ripening from 4 to 6 days at 20 oC (Jacomino et al., 2002). Furthermore, 25 pL L1' I-MCP treatment increased the number of days to ripening from approximately 5 to 20 days for 'Solo' fruit treated at commercial harvest maturity (Hofman et al., 2001).
The objective of this study was to examine the physiological responses and quality of papaya fruit treated withl-MCP at the pre-ripe and full-ripe stages of maturation.
Materials and Methods
Plant Material
Papaya fruit (Carica papaya, L. var. 'Sunrise Solo') originating from Belize (no thermal or wax treatment) and were obtained from Brooks Tropicals Inc., Homestead, FL. 'Sunrise Solo' variety was chosen due to its year-round availability. After transfer to the postharvest facilities in Gainesville, fruit were selected on the basis of uniformity of




Full Text

PAGE 1

1-METHYLCYCLOPROPENE TREATMENT EFFICACY IN PREVENTING ETHYLENE PERCEPTION AND RIPENING TN TNT ACT AND FRESH-CUT GALIA' MELON AND SUNRISE SOLO PAPAYA FRUITS By MUHARREM ERGUN 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 2003

PAGE 2

ACKNOWLEDGMENTS This dissertation could not have been completed without the support of many people who are gratefully acknowledged here My greatest debt is to Dr Donald J. Huber who has been a dedicated advisor and mentor, and kindest friend He provided constant guidance to my academic work and research projects This dissertation task w ould not be in the current form without his insightful input and constructive criticism. I extend m y appreciation to my supervisory committee Dr Jerry A. Bartz Dr Daniel J. Cantliffe Dr. Charles L. Gu y, and Dr. Steven A. Sargent for their academic guidance I am very thankful to Dr Daniel J. Cantliffe and his graduate student Juan C. Rodriguez for allowing me to use their produce Galia melon in m y experiments I am very grateful to Dr Steven A. Sargent for his constant supervision in any issues I thank Dr Jerry A. Bartz and his lab personnel for their guidance and help. I e x press my gratitude to Dr Charles L. Guy for his welcoming assistance I also thank James Lee for the teaching training and friendl y, ne v er-ending assistance in the laboratory I would like to e x press m y deepest appreciation to the Ministr y of Education of Turkey who has supported my scholastic and living expenses durin g my graduate education in the U.S I would like to make a special acknowledgement to my friends graduate students and faculties and personnel of the postharvest research group for their kindl y support and help II

PAGE 3

Finally I thank my father Ismail mother Onzile, brothers Sadtk, Sezai and Recai, and sister, Meryem for their emotional and financial support not only during my Ph D education but also throughout my life. 111

PAGE 4

TABLE OF CONTENTS ACKNOWLEDGMENTS ...... .. ...... .. .. ....... ... .. .. .. ... .. ... ..... . .. .. ... ......... .. .. .. ... .. ii LIST OF TABLES ... ... ... .. ................................ ... .. .. .... ... ... ... .. .. ... ... .. ..... .. .. .. viii LIST OF FIGURES . .. .. .... .. ......... ... .......................... ....... .......... .... . .... .. .. ...... .... ix ABSTRACT .. ... .. ... ... ... ..... .. .. .......... ... .. ... ........... .................. . . ... .. ... ... ......... xiii 1 INTRODUCTION .. .... ............ .. .................................. ....... ... ...... . ...... .. ... .. ... . .. 1 2 LITERATURE REVIEW ....... .......... ... ... ...................... ... ...... ... .. ....... .. ...... .. .. .. 4 Melon .. ............ ........................ .. ... ... ... .. ... .. ... .. ..... .... .. ...... .. . ......... ............. 4 Introduction .......... .. ....... . .. ... ....... .. .. .... .. .... ...... .. . ..... ... ...... ...... ........ 4 Harvest Maturity .... .. .. .. ... ... ...... ... .. ... .. ... .. .. . .. .. ... .. .. .. ... .. ..... ........ 4 Ripening Process ................ ............... .. .. .. .. .. ... .. .. ..... .... .. .. ... .. .. .. .. .... 5 Postharvest Storage of Melon Fruit ... .. ... .. .. .. . .... ... .. ....... .. ... ... ... ...... .. .. 7 Postharvest Diseases ........... .. .... ..... . .... .. .... .. ... . .. ...... ............ ... ... . ...... 7 Postharvest Disease Control .. .. ......... .... ... ... .. ... . .. ... .. .. .. .... .............. .. ..... 8 Papaya ....... ... .. .. .................. .. . .. ... .. .. ..... .... ...... .. ...... .... ... .. ... .. . ... .. .. ... .. 9 Introduction ................. .. .. .... .... ... .. .... .. .. ....... ........ .. . .. .... .. .. .. ..... .. ... 9 Uses of Papaya Fruit.. ..... .. .. .. . . .. .. .. .. .... .... .. .... ... ..... .. .. ..... ... .. .... I 0 Papaya Harvest Maturity ... .. .. .. .. . . ... ... ....... .. ... .. .. .... ... ... . . .... 10 Papaya Ripening .................... ... ........................ ..... .. . ... .. ..... .. .. ... .. .... 11 Post harvest Handling and Storage of Papaya ... .... ... . .. ... ....... .. .. ... ... ... .. .... 13 Chilling Injury .. .. .... .. .. .. .. .. .. .. . ................ .. . ... .. .. .... ..... .. .. .. .. .. .. 14 Postharvest Pathology .. .......... ... ... ... ... ... .............. ... ... .. ...... ... ... ...... .... 14 1-Methylcyclopropene . .. .. .... .. .. .. .. ... ................. .. .. .... .. .. .... ... ...... .. ... .... .. .. 16 Introduction ....... .... .. ....... ......... ..... .... ... ... .......... .. . .. ...... .. .... ... .. ... .... 16 Treatment Procedures for 1-MCP .... .. ... ....... .. ... . ...... .. .... ... .. .... ... ..... 17 l-MCP and Ethylene ............. .. ....... ... .. ...... .. .. .. ... .. ... .... .. ....... .. .. .. .. .. 17 1-MCP and Fruit Softening ... ................. .................... ............................. ... 19 The Influence of Suppressed Ethylene Perception on Ripening Ph y siology and Biochemistry ................. .. .. ...... . ......... .... ........ .. .. ... .... ... ... .. ...... ... ... 20 Physiology of Fresh-cut Produce .... ... ... .... ... ... ......... ... ... ................ ... ... .. 25 Introduction ................... .... .. ... . .. ............ ....... .. ......... . ... .. . .. . .... 25 Consequences of Processing ..... ..... ............ ......... .. .. .. .............. .. ....... .... 25 External and Internal Factors Contributing to Quality of Fresh-cut Fruits and Vegetables ..... ... ...... ..... ... ..... ... ... .... .......... ..... ....... ... ..... ... ... ..... ... .... 28 IV

PAGE 5

Cell Wall ...... ........ . ....... ................... .............. .. .. .... ..... .. .. ... ............... ..... .... 32 Introduction .... .. .... .. .... .. ..... ........ ... ....... .............. ... ... .... ... .... ... .. ..... ... .... 32 Cell Wall Structure ..... ......... .. ............ .. .. .... ... .. ....... ... .. .. ................... 33 Cell Wall Loosening and Growth ............. .. .. .. ..... ...... ... .. .. .. .. .... ... ........ 34 Fruit Ripening and the Cell Wall ............... ... ..... .. ... .......... .. ..... ..... ... ........... 34 The Cell Wall and Pathogen Attacks .. .. ... ... ........... .................... ... .... .. .... 35 3 DELAYING ETHYLENE-INDUCiBLE RIPENING PROCESS 1METHYLCYCLOPROPENE IN 'GALIA' MELON FRUIT ... .. .... ... .... .. ... ..... 36 Introduction ..... ... .................. ... .... ...... . ............ ... ..... ...................................... 36 Materials and Methods ..... ... .. .. ..... .. .. ... .. .. ... ... .. . .. . .... .. .... .... ... ...... .. .. 37 Plant Material ............ ...... ... .. .. .. ... . .. .. .. . .... .... .. ... .... .. .. .. ... .. ......... 37 1-MCP Application ..................................................... .. .. .......... ... ...... ... ..... 38 Respiration and Ethylene Production ..... ...... .... .............. ...... ........... ...... .. .. 39 Firmness Assessment. ........ ....... ... ... .. .. .. .. ............ ..... ... ............... ....... .. .. ... 39 Electrolyte Leakage Assessment .. .. .. .. .. .. ... .......................................... .......... 3 9 Soluble Solids Concentration, pH and Titratable Acidity Determination . ..... 40 Statistical and Informal Taste Analyses ................................ ... .. ...................... 40 Results .............................................. .... .. .... ...... ... ... ... .... .. .. .... ............. ... ...... 41 1-MCP Concentration and Efficacy .......... .................. ... .... .............. .. ... ........ 41 Respiration and Ethylene Production ............ .. ........ .............. .. .......... .... ........ .41 Firmness .............. ................ .. .. ................. ... ...... ... .. .. .. .. .. .. .. ...... .. ........... 42 Electrolyte Leakage .......... .. ...................... ....... ... ........... .. ... ... ... ..... .. ...... ... 43 Soluble Solids Concentration, pH and Titratable Acidity .... .. ......... ...... ... ...... 43 Informal Quality Analysis ............ .. .................. ................... .. ... ... .. .. .... .. ... 44 Discussion .. ... ...... ............ ... .. .. .. ..... .. .. .... .. .. ...... .. .. .. .. .. .. . . ... .. .... .... .... 45 4 PHYSIOLOGICAL CHANGES IN FRESH-CUT AND INTACT 'GALIA' MELON FRUIT WITH TREATED 1-METHYLCYCLOPROPENE ..... .... ... ........ ... .... ...... 59 Introduction ............................ ... ..... ... ............... .......... .... ...... .... .. ... ................... 59 Materials and Methods .. .. ... .. . .. .. .. .. .. ... .. .. .. .. .. .. ... .. .... .... ... .. ......... ........... 61 Plant Material ........................ ................. ... .. .. ..... .. .. .. .... ... .... ... ... .... ... . ... 61 1-MCP Quantification and Treatment ........ ... .... ................. .. ........ .... ........ ...... 61 Preparation of Fresh-cut Galia ', and Treatment Design .... ................. ..... .. .... 62 Ethylene Analysis ......................... ................. .......... .. .. .. ................... ..... ........ 63 Firmness Assessment. ..... .. ..... ....... .... ... .. .. .. ... . ... ... .. . ... .. .... .. .. ... 63 Electrolyte Leakage .......... ................... .... .......... .............. .. ................... .... ... 63 Pectin Effiux ..................... .. ........... .. ..... .. .. .. ...... ..... ... ............... .. .................. 64 Quality Evaluation ........ .... ...... .. .. .. .. ...................... ................. ...... .. ........ 65 Microbial Counts ... .................. .. ...... ...... ............ ... ................. ... .... ... .. ..... .. 65 Experimental Design and Statistics ... .. ... .. .. ..... .. .. .. .. .. .. . ... ... .. . ... .. .. ...... 66 Results and Discussion . .............. ........... .. ........ .. .. .. .. ... ..... ... .. ..... ..... ...... ..... ... 66 Ethylene Production ... .. .. .... .......................... .. ................ .... ............ ... .... ...... 66 Firmness Assessment. .. .. .. .. .. ... .. .......... .. ...... ... ... .. .... .. ............. ... ........ ... ... 68 Electrolyte Leakage .. .. .... ... ..... ... ... ........... ... .. ... .. .. .. .. ... .. .. . ... ... ... .. ...... 69 V

PAGE 6

Pectin Effiux .. . .. ..... ................... .... .. ... .. .. .. ... .... ........................ ... ..... ... .. 69 Quality Evaluation ...... .. ... .. ... .. .... .. .. .... .. .......... ............... .... .. .... ........... ... 71 Microbial Counts ... .... .. .. ... . .. .. ...... .. . ... .. .. .. .. . .. .. .. ... ... ... ... .. .... .... 73 5 STORAGE life EXTENTION OF PRE-RIPE AND RIPE SUNRISE SOLO' PAP A YA FRUIT BY 1-METHYLCYCLOPROPENE ..................................... .... 85 Introduction .. ...... ...... .... ... .. .. .. .. .. ................ .... ... .. .... ...... ... ... .. .. .. ... ...... .. .. 85 Materials and Methods ... .. .. ... ....... .... ...... ...... ..... .. .. .. .......... .... .................. ........ 86 Plant Material .. .... ...... .. ................... ... .. ... . .. .. ... .. .. ..... .. .. ... ... .. .. .. .. .. 86 1-MCP Preparation and Treatment. ........ ... .. ... ..... .. ... . .. .. ... ... .. .. ... .. 87 Respiration and Ethylene Production ............ .... .. .. .. ... . ... .. ... .. .. . ... .... 88 Firmness Determination ........................ ...... ..... . ... ....... ......... ... ............ ... 88 Electrolyte Effiux ... ..................... ......... .. .... ................. .. .......... ... .......... .. .. 88 Soluble Solids Concentration pH and Titratable Acidity .. ...... ...... ....... .. .... ..... 89 Statistical and Informal Taste Analyses .. ...... .. ..... ...... .. .. ...... .. .. .. .. . .. ... ...... 89 Results .... .. .... ... ... .. .. ... .. .. .. ............ .. ........... .. .. .. ... ... ... .. .. ... .. .............. ...... 89 Effective 1-MCP Concentration ............ ....... ...... ......... ..... .. ..... .. ..... .. .......... 89 Respiration and Ethylene Production .......... .. ..... .. .. ... .. .. ... ... .. ..... .. ... ... 90 Mesocarp Firmness ............................... ........... .. .. .... .................. .... .... ........ 91 Electrolyte Effiux .. .. ........................................ .. .. .... .. .. ... ...... ... ... .. ............. 91 Soluble Solids Concentrations Titratable Acidity and pH ... .... ... ... ... ... .......... 92 Fruit Evaluation ......... .......... .................. .. ...... .. ................... .. ....... ....... .... .. 92 Discussion ....... ............ .. .. .. ... .. .. .. .. ... ... . .. .. .. ... .. .. .. ..... . ... .. ... ...... ......... 93 6 QUALITY AND STORAGE LIFE OF INT ACT AND FRESH-CUT PAP A YA FRUIT TREATED WITH 1-MCP AT THE POSTCLIMACTERIC ST AGE OF DEVELOPMENT ... .. .. .. ..... ..... ... .. ... ............... ... .......... ....... .. ...... .. ..... .. ... ... 109 Introduction ...... ................. .. .. .. .. ...... .. .. .. ... . .. .. ... .. ....... .... .. .. ... .. ... .... 109 Materials and Methods ....... .. ...... ... .............. .. ..... .. .. .............. .. ... .......... ..... 111 Plant Material ..... .... .... ... ... .... ... ... ................................. ... ... .... ... .. ......... 111 1-MCP Treatment .................. ... .. ........... .. .... .. .. .. ..... .. .. ...... ..... .. ............ 112 Fruit Preparation and Treatment Design .... ... ... ... .. .. ... .. .... ....... ....... .. ...... 112 Ethylene Production .................... .. .. ....... .... .......... .......... ...... ...... ...... ...... 113 Firmness ..................... ... ...... ... .......... .... . ...... ... .......... .. .... ... ...... .. .... 113 Electrolyte Leakage ... .. ...... .. .. ..... ... ... .. .. .... . .. .. ... .. .... ... .. .. .. .. ... .. .... 114 Color and Sensory Evaluation ... .. ... .. ... .. ... .... .......... .. .................. ...... .. ..... 114 Microbial Count .............. ... .... ..... ...... .. ... .. .. ..... ... .. ... ..... ..... .. .... .. ... 115 Statistical Analysis ........ ....... .. ... .. .. ... ... ... ... .... .. .. .. ..... .. .. . ... .. . ..... 116 Results and Discussion .. ... .... .... ... .. .............. .... ................. ...... ...... .................. 116 Ethylene Production ...... ... ........ ........... ... ....... ....... ................. ... ... ...... .... 116 Firmness Assessment. ... .. .. ... .. .. ... ........ .. .. ... .. .. ... .. ......... ....... .. ...... 118 Electrolyte Leakage ...... .... .. ................ ...... ... .. ... ... ... .. . ... .......... . ....... 119 Color and Sensory Evaluation ........ ....... .......... .... ... .. ... ..... ..... ...... ...... .. .... 121 Microbiological Counts .............. .. .. .. ... ....... .. .. .. ......... ... ..... .. ............ ... ... 124 VI

PAGE 7

7 CELL WALL MODIFICATION IN POSTCLIMACTERIC FRESH-C U T AND INTACT PAPAYA FRUIT WITH AND WITHOUT 1METHYLCYCLOPROPENE ............... ... ...... .. .......... ...... .... ... ...... ... ......... 13 5 Introduction ..................... ..... .................................. ... .. .. .. ........... .... ................... 13 5 Materials and Methods .. .. .. ...... ... .. ............ ...... .. ... .. ... ... .. ........ .. .. ....... .. .. 138 Plant Material and 1-MCP Treatment ........ .. ...... .......... .......... ..................... 138 Ethanol-insoluble Solids ..... .... .. .. .. ........... .. .. .. ........ ... ... ...... .... ..... .... .. ... .. 139 Total Soluble Sugars and Polyuronides ........ ...... ...... ..... .... .. .. ... .... ................. 140 Sequential Fractionation of Cell Wall Materials .... .. ...... .. .... .. .. .. ................ .. .. 140 Hemicelullosic Polysaccharide Extraction ............................. .. ........ .... .. .... .. .. 140 Compositional Anal y sis of Cell Wall Polymers .. .. ..... .... .... ..... .. .. ..... .. .. .. ....... 141 Results ................. ........ ............ .. .. ... ... ....................... . .. ... .. .. .. ... . .. 142 Ethanol-insoluble Solids and Total Soluble Sugars .......... .. ..... .... .... ...... ....... 142 Polyuronides and their Sequential Fraction .................. .. .. ........... ...... .... .. ...... 142 Hemicellulosic Polysaccharides .. .. .. .. ........... ... .... .. ........... . .. .... .. ... ... ........ 144 Compositional Analysis of Cell Wall Polymers .. ... .. .. ... .. .... .. .. .. .. ...... .. .......... 144 Discussion .............................. ........ .. ..... ... ............... ...... ... .. .... .. .. ... .. .. .... 145 8 SUMMARY AND CONCLUSION ............ .. .. .. .......... .. .... ...... ... ...... .. .... .. .. ..... .. .... 162 Influence of Ethylene-action Inhibition on Ripening of Galia Melon Fruit .. .. .... 162 Influence of Ethylene-action Inhibition on Ripening of Sunrise Solo Melon Fruit .... ... ... ........ ...... .. .. . .. ....... ....... .. ............. .... ....... ...... ..... .. ..... .... .. .. .. 163 Cell Wall Modification of Sunrise Solo Papa y a Fruit in Response to Fresh-cut Processing and 1-MCP ..................................... .... ... ....... ............... .... .. .... .. 164 LIST OF REFERENCES ...... ... ... ............... .. .. ... .. .. ....... .. .. .... ... ... . ... . .. .. 166 BIOGRAPHICAL SKETCH ..... .... ... ..... ....... ........... ...... .. .. .... .. .. .. .... .. ...... .. ...... ... 185 Vil

PAGE 8

LIST OF TABLES 2-1. 1-MCP-induced effects on ripening fruits .... .. .. ........ .. ........................ ........... ..... 21 4-1 Microbial counts (CFU g1 fresh weight) for intact ripe Galia fruit with and without 1-MCP and fresh-cut fruit derived from intact ripe fruit treated with and without 1-MCP during storage at 5 C. ..... .... .. .. .. ......... .. .. ...... .. ..... .. ..... .. 84 6-1. Microbial counts (CFU g1 fresh weight) for intact postclimacteric Sunrise Solo' papa y a fruit pre-treated with 2. 5 L L1 1-MCP (lM) and air ( control IC ), and for fresh-cut postclimacteric fruit deri v ed from either the intact air-treated (FCC) or the intact 1-MCP-treated (FCM) fruit during storage at 5 C ... ............................... 134 7-1. Ethanol insoluble solids (EIS) and total soluble sugars (TSS) of intact three-quarter ripe papaya fruit treated with and without 1 MCP and fresh-cut fruit derived from intact three-quarter ripe fruit treated with and without 1-MCP papaya during storage ................. .. .. .. .. ...................................................... ....... .................. 149 7-2 Polyuronide composition of intact three-quarter ripe papa y a fruit treated with and without 1-MCP and fresh-cut fruit deri v ed from intact three-quarter ripe fruit treated with and without 1-MCP papa y a during storage .................................... 150 7 3. Neutral hemicellulose and pectin residue composition of intact three-quarter ripe papa y a fruit treated w ith and without 1-MCP and fresh-cut fruit derived from intact three quarter ripe fruit treated with and without 1-MCP papaya during stora g e ... 152 7-4 Neutral sugar composition of intact three-quarter ripe papa y a fruit treated with and without 1-MCP and fresh-cut fruit derived from intact three-quarter ripe fruit treated with and without 1-MCP papaya during s torage ...................... ............... 154 VIII

PAGE 9

LIST OF FIGURES Figure 3-1. U~pared fruit_prmness of ween Galia fruit/reated with air (control) 0 09 0 9 L L and 9 L 1-MCP durmg storage at 15 C. ............ ....................................... 50 3-2. Respiration and ethylene production of green (A) and yellow (B) 'Galia' melon fruit treated with 1. 5 L L1 1-MCP and air ( control) during storage at 20 C. .............. 51 3-3. Mesocarp firmness of green (A) and yellow (B) Galia melon fruit treated with 1.5 L L" 1 1-MCP and air ( control) during storage at 20 C. ................................ .. .... 52 3-4 Electrolyte leakage of green (A) and yellow (B) 'Galia melon fruit treated with 1 5 L L1 1-MCP and air (control) during storage at 20 C ........... .. ........................... 53 3-5 Soluble solids concentration of green (A) and yellow (B) Galia melon fruit treated with 1 5 L L1 1-MCP and air (control) during storage at 20 C .. .. ....................... 54 3-6. Titratable acidity of green (A) and yellow (B) Galia melon fruit treated with 1 5 L L" 1 1-MCP and air (control) during storage at 20 C. ......... .. .... ......... .. ............. 55 3-7 The pH of green (A) and yellow (B) Galia melon fruit treated with 1 5 L L1 1MCP and air (control) during storage at 20 C. ......................... ....... .. .... .... ... ....... 56 3-8 Galia' fruit harvested at the pre-ripe stage (green surface) were treated with 1 5 L L1 1-MCP or air ( control) and then stored for 13 days at 20 C. .................. .. .. ..... 57 3-9. 'Galia fruit harvested at the ripe stage (yellow surface) were treated with 1 5 L L" 1 1-MCP or air (control) and then stored for 7 days at 20 C. ........... .. .......... .. .......... 58 4-1. Ethylene production for intact ripe Galia fruit with and without 1-MCP and fresh cut fruit derived from intact ripe fruit treated with and without 1-MCP during storage at 5 C ... ... ... ..................... .................. . .... ........... .. ..................... ..... 76 4-2. Mesocarp firmness for intact ripe Galia' fruit with and without 1-MCP and fresh cut fruit derived from intact ripe fruit treated with and without 1-MCP during storage at 5 C. .. .. .. .. .. .. .. .. .. .. .. .. .. .. . .. . .. .. .. .. .. .. .. .. .. .. .. .. .. ........ .. 77 4-3 Electrolyte leakage from mesocarp tissues of intact ripe Gali a fruit with and without 1-MCP and fresh-cut fruit derived from intact ripe fruit treated with and without 1-MCP during storage at 5 C. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. ..... .... .......... 78 IX

PAGE 10

4-4 Pectin effiux from mesocarp tissues of intact ripe Galia fruit with and without 1MCP and fresh-cut fruit derived from intact ripe fruit treated with and without 1MCP during storage at 5 C. ... ....... ...... .. .. ... .... ....... .. ... .... ... ...... ... ........ ..... 79 4-5 Color parameters, L lightness (A) hue angle (B) and chroma (C) for ripe Galia fruit skin treated with and without 1-MCP (1 L L1 ) and the color parameters (D, E and F) for intact ripe fruit with and without 1-MCP and fresh-cut fruit derived from intact ripe fruit treated with and without 1-MCP during storage at 5 C. .......... .... 80 4-6 Mesocarp water soaking percentage (A) and sensory evaluation (B) for intact ripe 'Galia' fruit with and without 1-MCP and fresh-cut fruit derived from intact ripe fruit treated with and without 1-MCP during storage at 5 C. ...................... ... ...... 81 4-7 Ripe fresh-cut 'Galia fruit derived from intact ripe fruit treated with (FC-MCP) and without 1-MCP (FC-CNT) and then stored for 4 days at 5 C. ... .. ..... .... ............... 82 4-8 Ripe fresh-cut Galia fruit derived from intact ripe fruit treated with (FC-MCP) and without 1-MCP (FC-CNT) and then stored for 10 days at 5 C. ............... ...... .. .. 83 5-1 Mesocarp firmness for 'Sunrise Solo fruit treated with air (control) 0 9 L L" 1 and 9 L L 1 1-MCP at 20 to 30% skin yellowing ripening stage and subsequently stored for 19 days at 15 C. .. .. ....... .. .. ............................................. ........ .. .... I 00 5-2 Respiration and ethylene production for 'Sunrise Solo fruit treated with air (control) and 9 L L1 1-MCP at the pre-ripe (A) and ripe (B) stage and subsequently stored at 20 C. .... .. .. .. ................... .......... ......... ..... .... ... ... .. .. 10 I 5-3 Mesocarp firmness for Sunrise Solo fruit treated with air (control) and 9 L L1 1MCP at the pre-ripe and ripe stage and subsequently stored at 20 C. ............. 102 5-4 Electrolyte leakage for Sunrise Solo fruit treated with air ( control) and 9 L L1 1MCP at the pre-ripe and ripe stage and subsequently stored at 20 C ..... .. .. ...... I 03 5-5 Soluble solids concentration (SSC) for Sunrise Solo' fruit treated with air (control) and 9 LL-' 1-MCP at the pre-ripe (A) and ripe (B) s tage and subsequently stored ~20~ .................................... ... .. ... .. .. .. ..... ................ .............. .. ... ...... ..... )~ 5-6 Titratable acidity for Sunrise Solo' fruit treated with air (control) and 9 LL' 1MCP at the pre-ripe and ripe stage and subsequently stored at 20 C. ........... ... 105 5-7 The pH for Sunrise Solo' fruit treated with air (control) and 9 L 1 1-MCP at the pre-ripe (A) and ripe (B) stage and subsequently stored at 20 C. .......... .. ....... I 06 5-8. Pre-ripe Sunrise Solo fruit treated with 9 LL' 1-MCP or air (control) and then stored for 7 days at 20 C. .... .. .. .... ... ............... ............ ..... ..... ...... .. .. ............. I 07 5-9 Ripe Sunrise Solo fruit treated with 9 LL' 1-MCP or air (control) and then stored for 3 days at 20 C. .............. ..... ...... .................. ... ........ .... ..... .. ......... ... 108 X

PAGE 11

6-1. Ethylene production for intact postclimacteric 'Sunrise Solo' papaya fruit pre treated with 2 5 L L1 1-MCP and air (control), and for fresh-cut postclimacteric fruit derived from the either intact air-treated or the intact 1-MCP-treated fruit during storage at 5 C. ... ...................................... .. .. .... ..... .. .... .. ..... ... . .. ... .. 126 6-2 Mesocarp firmness for intact postclimacteric 'Sunrise Solo' papaya fruit pre-treated with 2 5 L L1 1-MCP and air (control), and for fresh-cut postclimacteric fruit derived from either the intact air-treated or the intact 1-MCP-treated fruit during storage at 5 C. ....................... .......................... ....... .. ... .. ....... .. 127 6-3 Electrolyte leakage(% of total) for intact postclimacteric Sunrise Solo' papaya fruit pre-treated with 2 5 L L1 1-MCP and air (control), and for fresh-cut postclimacteric fruit derived from either the intact air-treated or the intact 1-MCPtreated fruit during storage at 5 C. .............. .. ................................................... 128 6-4. Color parameters for intact postclimacteric Sunrise Solo' papaya fruit skin pre treated with 2 5 L L 1 1-MCP and air (control) and for the intact fruit flesh and fresh-cut fruit derived from either the intact air-treated or the intact 1-MCP-treated fruit during storage at 5 C. ...................... ............ .......... ......... ..... .. .... ... .. .. .. 129 6-5 Fresh-cut postclimacteric 'Sunrise Solo' papaya fruit derived from either intact postclimacteric air-treated (FCC) or intact postclimacteric 1-MCP-treated fruit (FCM) and then stored for IO days at 5 C. ............... .. .......... ..... .. ... ...... ... ..... 130 6-6 Pitting of postclimacteric Sunrise Solo' papaya fruit pre-treated with 2.5 L L 1 1MCP and air ( control) during storage at 5 C. .... .... .... .. ......... .. .. . .. ... ..... .... ..... 131 67. Water soaking (A) and sensory evaluation (B) for intact postclimacteric Sunrise Solo' papaya fruit pre-treated with 2.5 L L1 1-MCP and air (control), and for fresh-cut postclimacteric fruit derived from either the intact air-treated or the intact 1-MCP-treated fruit during storage at 5 C ...... .. ........ ...... ... ... .... ..... ....... .... .... .... 132 6-8. Fresh-cut postclimacteric Sunrise Solo papaya fruit derived from either intact postclimacteric air-treated (FCC) or intact postclimacteric 1-MCP-treated fruit (FCM) and then stored for 6 days at 5 C. ............ . . . . ....... ............ 133 7-1. Molecular mass distribution of water-soluble polyuronides of intact three-quarter ripe papaya fruit treated with and without 1-MCP and fresh-cut fruit derived from intact three quarter-ripe fruit treated with and without 1-MCP at day 0 ( o ), 6 ( ) and 10 (T) .... .. .. ..... ..... .... .. .... ... ... . ... ... .................. ....... .. .. .... .. .. ... ..... ..... 157 7-2 Molecular mass distribution of CDT A-soluble polyuronides of intact three-quarter ripe papaya fruit treated with and without 1-MCP and fresh-cut fruit derived from intact three-quarter ripe fruit treated with and without 1-MCP at day 0 ( o ) 6 ( ), and 10 ( T) ....................................... ... .......... .. ... ...... ........... ......... 15 9 7-3. Molecular mass distribution ofNa 2 CO 3 -soluble polyuronides of intact three-quarter ripe papaya fruit treated with and without 1-MCP and fresh-cut fruit derived from XI

PAGE 12

intact three quarter-ripe fruit treated with and without 1-MCP at day O (o), 6 and 10 (T) ...................................... .. ............................................................... 161 XII

PAGE 13

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 1-METHYLCYCLOPROPENE TREATMENT EFFICACY IN PREVENTING ETHYLENE PERCEPTION AND RIPENING IN INT ACT AND FRESH-CUT 'GALIA' MELON AND 'SUNRISE SOLO' PAPAYA FRUITS By Muharrem Ergun August 2003 Chair: D J. Huber Major Department: Horticultural Sciences The objectives of this study were to determine the physiological responses of intact and fresh-cut melon (Cucumis me/a L. var. reticulatus L. Naud. cv. Galia) and papaya (Carica papaya, L. var Sunrise Solo) fruits in which ethylene perception was blocked through application of 1-methylcyclopropene (1-MCP). The whole fruits were treated with 1-MCP (melon, 1 or 1.5 L L1 ; papaya, 2 5 or 9 L L1 ) for 24 hat 20 C and stored at 20 C or processed and stored at 5 C. Inhibition of ethylene perception via application of 1-MCP delayed the onset of the respiratory and ethylene climacterics and reduced maximum respiration and ethylene production rates of 'Galia' fruit ripened at 20 C. Softening of intact and fresh-cut 'Galia' melon was significantly reduced by 1-MCP, regardless of application time (ripening stage). Yellowing of the fruit surface during ripening was strongly and somewhat irreversibly delayed in 1-MCP-treated 'Galia'. XIII

PAGE 14

Papaya fruit ( Sunrise Solo ) treated with 1-MCP exhibited delayed initiation of the respiratory and ethylene climacterics and suppressed ethylene production during ripening. Softening was significantly delayed in fresh-cut and intact fruit. Papaya treated with 1MCP retained higher levels of titratable acidity compared with non-1-MCP-treated fruit throughout ripening The color change of the fruit surface from green to yellow was significantly but temporarily inhibited in 1-MCP-treated fruit. Cell wall modification was studied in intact and fresh-cut ripe papaya fruit stored for 10 days at 5 C. Water-soluble polyuronides represented the major pectic fraction followed by the CDT A (1,2 cyclohexylenedinitrilotetraacetic acid)and Na 2 CO 3 -soluble fractions irrespective of 1-MCP Both hemicellulosic and pectic polysaccharides in intact and fresh-cut fruit showed some changes but this trend was slightly or not affected by 1MCP Neutral sugars from pectins and hemicelluloses including galactose glucose and xylose decreased in both intact and fresh-cut fruit regardless of 1-MCP Generally either intact fruit or fresh-cut fruit pre-treated with 1-MCP exhibited I ittle or no significant changes compared with fruit not treated with 1-MCP. The studies presented herein have shown that 1-MCP has potential for extending the useful storage life of intact and fresh-cut melon and papaya fruits by delaying ethylene inducible ripening process XIV

PAGE 15

CHAPTER 1 INTRODUCTION 'Galia' (Cucumis melo L. var. reticulatus L. Naud. cv. Galia) fruit has excellent flavor and aroma characteristics; however storage life is limited to 2-3 weeks even at low temperatures. The storage life of papaya (Carica papaya L.) fruit is also short due to its inherently high respiration rate delicate skin, and high water content. Papaya fruit can be harvested at the mature green stage ( 10% to 20% yellow skin) however and maintained at 10 to 12 C for periods ofup to 2 weeks Approaches restricting ethylene synthesis, such as controlled atmosphere, have proven that storage life of melon and papaya fruits can be extended Another, a more facile approach to extending the storage life and quality of harvested melon and papaya has been through the application of 1methylcyclopropene (1-MCP), a potent anti-ethylene compound 1-MCP is very volatile and antagomism ethylene effectively in the range of pa,ts per billion (ppb) to parts per million (ppm) Treated fruit do not contain residue of 1-MCP 1-MCP has been reported to delay or reduce ethylene-inducible effects on a variety of fruits. The investigation of the efficacy of 1-MCP to maintain quality of both the melon and papaya fruits will provide information value to designing and exploring new postharvest applications that contribute to reduce postharvest losses during market preparation storage, transport, or at the wholesale retail or consumer level. Fresh-cut produce is one of the new food-processing methods that is increasing in popularity Featuring fresh-like quality in a ready to use package fresh-cut produce has become very popular Fresh-cut produce, however, can easily become unacceptable in a

PAGE 16

2 few days because its tissues no longer retain their protective epidermal and cuticular layers, and become ruptured and damaged Wounding associated with the preparation fresh-cut produce is responsible for rapid loses in appearance, aroma, flavor, firmness/texture and resistance to microbial degradation The knowledge of fresh-cut processing on either a theoretical or practical basis is quite limited since many fruits and vegetables have not been explored as fresh-cut commodities. Tropical fruits are high priority commodities to be explored as fresh-cut produce due to their perishable characteristics and sensitivity low temperatures. For example, papaya is very susceptible to mechanical damage pest attacks and diseases has a storage life of less than one week under ambient tropical conditions Melon (Cucumis melo L. var inodorus and reticulatus L. Naud.) is one of the most popular fresh-cut products; however, information about its behavior and characteristics after fresh-cut processing is limited to observations on discoloration off odor development, and water soaking The behavior of fresh-cut fruits generally parallels that of wounded tissues and is affected by type of tissue stage of maturity extent of wounding, temperature, oxygen, ethylene, carbon dioxide, water vapor pressure, and microorganisms The immediate response to wounding is cell rupture and loss of tissue integrity due to water loss and mix of sequestered enzymes and substrates followed by physiological and biochemical changes including accelerated ethylene production Ethylene a stress-related hormone may influence the events leading to texture loss and deterioration in fresh-cut fruits. Therefore, inhibition of ethylene synthesis or action may enhance the storage life of fresh-cut produce and also help to understand the

PAGE 17

3 physiological changes that distinguish the behavior of fresh-cut fruit as compared with these events as they occur in ripening, intact fruit. The primary objectives of the work reported herein were to examine the potential use of ethylene-perception inhibition for control of ripening in pre-ripe and ripe melon and papaya fruits, and to evaluate the effects of ethylene action on the post harvest qualities of either intact or fresh-cut melon and papaya fruits by measuring several physiological and biochemical properties Ethylene action was manipulated through use of 1-methylcyclopropene, a strong and persistent inhibitor of ethylene action

PAGE 18

CHAPTER2 LITERATURE REVIEW Melon Introduction The melon (Cucumis melo L. var inodorus and reticulatus L. Naud ) plant, a member of the Cucurbitaceae, is thought to have originated from the tropical regions of Africa and the Middle East (Seymour and McGlasson 1993) Galia ( C ucumis melo L. var. reticulatu s L. Naud cv. Gali a) was bred on the basis of the green-flesh qualities of 'Ha Ogen ', introduced from Hungary to Israel at the Newe Ya ar research center in the mid-60 s (Karchi, 200). The melon plant is generally monoecious but occasionally andromonoecious (McGlasson and Pratt 1963) C u c umi s m e lo specie are classified into two main groups including reticulatus the netted or muskmelon fruit types and inodorus the smooth-skinned or honeydew fruit types (Seymour and McGlasson, 1993) Melon fruit is categorized as an inferior berry whose edible flesh is derived from the placentae or mesocarp (Seymour and McGlasson 1993). Melon fruits show high variation in flesh color (from green to orange) skin color (from white or green to orange or gray) skin texture (from smooth to netted) and size (Seymour and McGlasson 1993). Some common melon types marketed in the United States are Western U.S. Eastern U.S. Charentais LSL Galia, Ananas Honeydew and Casaba (Zheng and Wolf, 2000). Harvest Maturity The abscission properties are the most useful criteria for estimating harvest maturity in muskmelon types whereas the abscission layer does not develop in 4

PAGE 19

5 honeydew-type melons until they are over-ripe (Pratt et al. 1977) Therefore, external color (green to white), peel texture (hairy to smooth), aroma, fruit density (low to high) and soluble solids are also used to verify harvest maturity of melon fruits (Portela and Cantwell, 1998) Melon fruit should have a minimum of 10% soluble solids concentration before harvest (Bianco and Pratt, 1977). Skin color of 'Galia' fruit can be used as a maturation index Fallik et al. (2001) categorized 6 maturity indices for 'Galia' based on skin color : (1) dark green, (2) green (3) light yellow with green (4) light yellow (5) yellow, (6) dark yellow to orange) Ripening Process Ethylene. Muskmelon fruit types abscise at or near the climacteric (Altman and Corey 1987) whereas honeydew types abscise after completion of the respiratory climacteric (Pratt et al. 1977). Exogenous ethylene treatment induces ripening in netted melons depending on maturity, temperature treatment duration and ethylene concentration (Seymour and McGlasson 1993). Exogenous ethylene may be applied to non-netted types to achieve uniform ripening of fruit that have sufficient soluble solids content (Seymour and McGlasson 1993). Since melons have no polymeric carbohydrate reserves such as starch postharvest ethylene treatments do not enhance soluble solids content in harvested fruit of any maturity class (Bianco and Pratt, J 977). Orange-fleshed melon fruit (mostly netted types) produce higher ethylene levels than greenor white flesh types (Zheng and Wolf, 2000). Melon fruit with a netted rind have higher ethylene production than do smooth types (Zheng and Wolf, 2000) Carbohydrates. Both in netted and honeydew type melons sugar accumulation reaches its maximum after full maturity (Seymour and McGlasson 1993) Soluble solids

PAGE 20

6 in melon fruits may accumulate to values as high as 17% (Bianco and Pratt, 1977) with sucrose and fructose comprising the most prevalent sugars (Hubbard et al. 1990). Structural polysaccharides An increase in soluble pectin a decrease in pectin size, loss of galactosyl residues, and changes in size of hemicelluloses represent the most evident features of cell wall changes during ripening of melon fruits (Gross and Sams 1984 ; McCollum et al., 1989). Galactosidases pectin esterase and cellulase are thought to be responsible for cell wall degradation (Gross and Sams, 1984; Lester and Dunlap 1985 ; McCollum et al. 1989) Organic acids. Citric and malic acids are the major organic acids in most melon fruits (Leach et al. 1989 : Flores et al., 2001) Artes et al. (1999) reported that titratable acidity(% citric acid equivalents) ranged from approximately 0 50 ('Galia') to 0 14 ( Tendral ) in 4 different varieties of muskmelons Pigments 1n orange-fleshed muskmelons the following pigments have been found : ~-carotene (84 7 %) 8-carotene (6 8 %) a-carotene (1 2 %) phytoene (1.5 %) lutein (I%) violaxanthin (0 9 %), and traces of other carotenoids (Seymour and McGlasson 1993) During muskmelon ripening, pigment accumulation initiates in the placentae progressing outward through the mesocarp (Reid et al. 1970) In green-fleshed types such as Galia ', however the carotenoid content in exocarp and mesocarp does not change significantly during ripening (Flugel and Gross 1982) Volatiles. The volatile ester profiles of ripe muskmelon and honeydew type melons are very similar except for ethyl butyrate which is more abundant in muskmelons (Yabumoto et al., 1978) The following volatiles have been reported to be representative of muskmelons: ethyl-2-methyl butyrate ethyl butyrate hexanoate hexyl acetate 3

PAGE 21

7 methyl butyl acetate, benzyl acetate, (Z)-6-nonenyl acetate, (E)-6-nonenol (Z Z)-3 6nonadienol and (Z)-6-nonenal (Yabumoto et al., 1978; Wyllie and Leach, 1990) Butyl acetate (5), 2-methyl-butyl acetate (6), and hexyl acetate (9) are the most abundant volatiles in 'Galia' type melons (Fallik et al. 2001). Postharvest Storage of Melon Fruit Melon fruit are typically stored at 7 to 10 C whereas storage below 7 C may cause chilling injury, especially for honeydew types (Lipton and Aharoni, 1979; Hardenburg et al., 1986) Controlled atmosphere conditions of 3% 0 2 and 10% CO 2 at 7 C, and 2% 02 and 10% CO 2 at 3 C have been shown to delay ripening and extend storage life of honeydew melon fruit (Hardenburg et al., 1986) Portela and Cantwell (1998) reported that fresh-cut honeydew and muskmelon fruit stored at 5 C for 12 days maintained their appearance and color but exhibited a 50% decline in firmness. O Connor-Shaw et al. (1994) reported that the storage life of fresh-cut honeydew-type melons was limited to 14 days while the storage life of fresh-cut muskmelon types was 4 days at 4 C. Postharvest Diseases Sour rot (Galactomyces geotrichum (Butl. & Peter) Redh & Mall.; Geotrichum candidum Lk.), Rhizopus rot (Rhizopus stolonifer (Ehrenb.:Fr.) Yuill.) Fusarium rot (Fusarium spp ), Trichothecium rot (Trichothecium roeum (Pers :Fr.) Link) Botrytis rot (Botryotiniafuckelina (de Barry) Whetzel; Botrytis cinerea Pers.:Fr.) Lasiodiplodia rot (Botryosphaeria rhodina (Cooke) Arx; Lasiodiplodia theobromae (Pat.) Griff & Mau bl.), and anthracnose ( Glomerella lagenarium F Stevens.; Colletotrichum orbiculate (Berk & Mont.) Arx; Gloeosporium orbiculare Berk & Mont; Colletotrichum lagenarium (Pass ) Ellis & Haist; Gloeosporium lagenarium (Pass .) Sacc ) are the most common postharvest diseases in melon fruit (Sommer et al. 1992) Netted types are very

PAGE 22

8 susceptible to fungal diseases since organisms may locate easily in the skin net and enter the fruit through mechanical breaks or abrasions (Seymour and McGlasson, 1993). Wounds and fresh stem-scars are likely sources of ingress for sour rot (Galactomyces geotrichum (Butl. & Peter) Redh. & Mall.; Geotrichum candidum Lk.; Sommer et al., 1992) The fungus is easily spread by wind rain, and vinegar flies (Drosophila spp.; Sommer et al., 1992). Rhizopus (Rhizopus stolonifer (Ehrenb : Fr.) Yuill.) colonizes fruits via mechanical injuries and stem scars very rapidly at high temperatures (Sommer et al., 1992). Fusarium (Fusarium spp ) frequently originates from soil and becomes active when fruit ripen. Disease progression of Fusarium is typically rather slow and fruit losses do not become excessive (Sommer et al., 1992). Spores of anthracnose (Colletotrichum orhiculate (Berk & Mont.) Arx ; Gloeosporium orhiculare Berk. & Mont; Colletotrichum lagenarium (Pass.) Ellis & Haist; Gloeosporium lagenarium (Pass.) Sacc.) are distributed by water, wind insects, or handling As fruit mature and ripen, latent anthracnose becomes active and causes sunken and black lesions (Sommer at al., 1992) Postharvest Disease Control Maintaining good physiological condition of the melon plant during growth and proper handling of fruit during harvesting, transportation and storage are essential in preventing or controlling postharvest deterioration and decay (Qi et al., 1988; Teitel et al., 1989; Aharoni et al., 1993; Seymour and McGlasson, I 993; O'Connor-Shaw et al., 1996; Fallik et al. 2000) Immersing fully ripe 'Galia' fruit in hot water (52 C) for 2 min may provide antifungal protection for a limited period (Teitel et al. 1989) A combined treatment of a hot water rinse and brushing can improve the general appearance and maintain quality of melon fruit as well as also reduce post harvest decay. According to

PAGE 23

9 Ayhan et al. (1998), 200 ppm free chlo r ine performed well in reducing microbial growth in intact and fresh-cut muskmelon fruit. Honeydew melons (intact or fresh-cut) may also be dipped in 150-ppm chlorinated water for 5 min to control decay development (Qi et al., 1988) Papaya Introduction Papaya (Carica papaya L.) is an herbaceous plant and a member of the family Caricaceae The plant is limited to the region within a latitudinal range of 32N and 32S (Morton, 1987). The plant may have female, male or hermaphroditic flowers (Nakasone 1986; Morton, 1987) The flower type determines the final size and shape of the fruit. Fruit from bisexual flowers are usually pyriform in shape with a small seed cavit y and thick wall of firm flesh (mesocarp) On the other hand fruit from female plants are nearly round or oval and of relatively thin flesh (Morton 1987). Fruit shape is usually spherical to oblong and fruit are generally composed of fi v e longitudinal carpels united around a large central cavity wherein seeds are attached to placental tissue by 0 5-10 cm stalks (Morton 1987). Fruit with less than five carpels are long and c y lindrical resembling cucumber fruit in shape (Nakasone 1986) Papa y a fruit range from 15 to 50 cm in length with a diameter of 10 to 20 cm (Morton 1987), and fruit weight ranges from 30 g to 9 kg depending on the cultivars (Nakasone I 986) The peel is thin usually smooth and green when immature but fairl y tough and yellow to orange when r i pe A slight injury can induce milky latex containing the proteolytic enzyme papain (EC 3 .4 22 2) to exude (Sankat and Maharaj 1997). Flesh ( mesocarp) thickness varies from I 5 to 5 cm (Nakasone, 1986 ; Sankat and Maharaj 1 9 97) During ripening the flesh becomes aromatic yellow to orange or reddish-yellow juic y, sweetish and melon-like in flavor

PAGE 24

10 (Morton, 1987; Sankat and Maharaj, 1997). Seeds are generally dark gray or black, covered with a transparent gelatinous aril and have high oil and protein contents (Sankat and Maharaj 1997). Some ofthe most cultivated varieties of papaya include 'Solo', Sunrise Solo' 'Maradol', 'Waimanalo', 'Higgins', 'Wilder', 'Hartus Gold', 'Bettina', 'Peterson', 'Singapore Pink', and 'Cari flora (Morton, 1987). Uses of Papaya Fruit Ripe papaya fruit are generally consumed fresh; however, processed papaya fruit products, such as nectar and juice, are competing against fresh papaya fruit. Fruit may also be added to ice creams sauces, used in cooked desserts or pickled or preserved as jam (Nakasone 1986). Green papaya can be boiled and served as a vegetable and canned in sugar syrup (Morton, 1987) Papaya fruit is a good source of vitamins A and B and an excellent source of vitamin C (Sankat and Maharaj 1987) Carotene, thiamine riboflavin, niacin, tryptophan methionine and lysine are usual constituents of papaya fruit (Kimura et al., 1991 ) Papaya latex contains two proteolytic enzymes papain and chymopapain (Paull 1993) Papain is used to tenderize meat to clarify beer and to treat wool and silk before dyeing. Moreover, papain is used in toothpastes cosmetics and detergents as well as in digestion aids (Morton, 1987), and has been used to treat ulcers and to reduce swelling and fever (Morton, 1987) In India, latex from papaya fruit or seed is applied to the uterus as an irritant to induce abortion (Morton, 1987) Young leaves are cooked and consumed like spinach in India (Nakasone 1986). Papaya Harvest Maturity Papaya development from pollination to full ripeness requires approximately 5 5 (Hawaii) to 10.5 (Africa) months (Nakasone 1986). Color break sugar composition

PAGE 25

11 (decline in sucrose increase in glucose and fructose) and soluble solids concentration are the most useful maturity indices (Nakasone, 1986; Paull 1993 ; Sankat and Maharaj 1997) Nondestructive methods including reflectance delayed light emission and body transmission spectroscopy have been used to measure papaya maturity (Sankat and Maharaj, 1997) For local markets fruit may be harvested when the skin color reaches 80% of yellow. Otherwise, fruit destined for storage or long-distance transportation are picked at the mature-green stage The fruit must be handled properly in order to avoid injuries causing leakage of latex which stains the fruit and reduces consumer acceptance (Morton 1987) The latex from the peel may irritate the skin of fruit handlers ; therefore, protective measures should be taken during prolonged physical contact with papaya (Morton, 1987) Papaya Ripening The optimum temperature for papaya ripening is between 22.5 and 27.5C (Paull 1993) Papaya is a climacteric fruit and the increase in ethylene production parallels respiration rate, reaching a maximum 1-2 days after (Wills and Widjanarko 1995) or simultaneously with (Paull 1993) the respiratory maximum Respiration and ethylene production of mature green papaya 'Solo fruit are below 5 mL kg1 h1 and 1 L kg1 h1 respectively ; however respiration and ethylene production increase to approximately 45 mL kg 1 h. 1 and 7 L kg 1 h. 1 respectively during ripening at 22 C (Paull and Chen 1983) Soluble carbohydrates The principal soluble carbohydrates in papaya fruit are sucrose glucose and fructose with sucrose being the predominant sugar at the full ripe stage (Chan, 1979 ; Selvaraj and Pal 1982) Invertase (EC 3 2.2 26) activity increases

PAGE 26

12 during ripening, presumably causing the conversion of sucrose to fructose and glucose (Selvaraj and Pal, 1982). Papaya fruit contains low levels of starch (Selvaraj et al., 1982) Structural polysaccharides and textural changes. Softening of papaya fruit is associated with a dramatic increase in the solubility of cell wall pectins (Paull, 1993; Lazan et al., 1995) Pectin depolymerization is also observed, occurring first in the inner mesocarp tissues (Sankat and Maharaj, 1997) Cellulase (EC 3 2 1. 5), pectin methyl esterase (EC 3.1.111), xylanase (EC 3.2 1.8), polygalacturonase (EC 3.2.1.15 ; PG ; highest in inner mesocarp) and 13-galactosidase (EC 3.2 1.23; highest in outer mesocarp) activities have been reported to increase during papaya ripening (Paull and Chen, 1983; Ali et al. 1999) Organic acids. Citrate and malate are the predominant organic acids in papaya but tartaric, fumaric and succinic acids have also been noted (Selvaraj et al. 1982). The concentration of total and nonvolatile acids decreases during fruit development reaching a minimum 1.54 mEq 1 oog1 (fresh weight) with a pH in the range from 5.0-5.5 at the full-ripe stage (Paull, 1993) Ascorbic acid increases nearly 4-fold reaching levels of 5 5mg-1001 (fresh weight) during ripening (Paull 1993) Compared with other fruit total titratable acidity remains low during ripening, which may contribute to the sweet taste of papaya (Selvaraj et al. 1982). Non-volatile organic acids comprise 75% to 92% of total acidity (Selvaraj et al. 1982) Pigments Total carotenoid content of mesocarp increases up to 14-fold during papaya ripening, with levels ranging from 0.28 mg 1001 dry pulp at the mature-green stage to nearly 4 mg 1001 dry pulp when full ripe (Selvaraj et al. 1982) P-carotene (62%) is the predominant carotenoid in yellow-flesh cultivars whereas lycopene is the

PAGE 27

13 major carotenoid in red-fleshed cultivars (Selvaraj et al 1982). Lycopene constitutes 61 % of the total carotenoid content of the red-fleshed 'Solo' papaya (Kimura et al., 1991). f3cryptoxanthin, ~-zeacarotene and cryptoflavin are found in minor quantities in papaya fruit (Kimura et al., 1991). Proteins and amino acids Several proteases, papain chymopapain A and B (EC 3.4 22.6), and papaya peptidase (EC 3.4 22.30) are found in papaya latex (Paull 1993). Total protease activities in papaya mesocarp tissue decline during ripening (Paull, 1993) At last 13 free amino acids have been identified in papaya fruit (Selvaraj et al. 1982; Morton, 1987). Volatiles At least 199 volatiles have been identified in papaya fruit with linalool being the most abundant (Macleod and Pieris, 1983; Flath et al. 1990). Volatiles in the cultivar Solo' are comprised ofup to 94% linanool followed in declining abundance, by benzyl isothiocyanate, methyl butanoate and methyl benzoate (Flath et al. 1990) Only one volatile, methyl benzoate is described as having papaya qualities (Paull 1993) Postharvest Handling and Storage of Papaya Papaya fruit have a maximum storage life of 7 days under ambient tropical conditions (30 ; temperatures above 32.5 C cause abnormal ripening (An and Paull, 1990). Storage between 12 to 16 C appears to represent the most compatible temperature range for storage Storage below IO to 12 C may cause chilling injury depending upon the maturity stage (Chen and Paull, 1986). Solo fruit stored at 25 C and 30 C had higher total carotene and ascorbic acid lower benzyl isothiocyanate (bitterness compound), more intense yellow peel color and more acceptable eating attributes compared with fruit held at 20 C (Wills and Widjanarko, 1995). This may have been the result, however of more advanced ripening of the fruit held at the higher temperature.

PAGE 28

14 Maharaj and Sankat (I 990) reported that the best atmospheric conditions for maintaining acceptability and market quality of papaya fruit during storage were 1.5 to 2% 0 2 and 5% CO 2 at 26 C. Plastic film wraps are more effective than waxes and other coatings in reducing water loss (Maharaj and Sankat, 1990) Chilling Injury Chilling injury is a major physiological disorder induced by low non-freezing temperatures (Chen and Paull 1986; Chan, 1988) Chen and Paull (1986) reported that mature-green 'Solo papaya stored at 7 5 C showed chilling injury symptoms after 20 days. Chilling injury symptoms of papaya fruit include epidermal discoloration of the mesocarp, development of hard areas in the flesh and around vascular bundles enhanced mesocarp water soaking and electrolyte leakage increased ethylene production, and increased susceptibility to decay (Chen and Paull 1986) Postharvest Pathology Postharvest diseases are very important in reducing market quality of papaya fruit and they are primarily responsible for the losses that occur during shipment. In Hawaii postharvest losses of papaya fruit due to diseases extended up to 93% before 1987 depending on postharvest handling and packing procedures (Alvarez and Nishijima 1987). Diseases are of three general types : fruit surface rots stem-end rots and internal infections (Alvarez and Nishijima, 1987) Fruit surface rots Anthracnose (Glom e r e lla cingulata (Stonem.) Spauld & Sehr .; C olletotrichum gloeo s porioides (Penz ) Arx) chocolate spot ( C oll e t o tri c hum gloeosporioides spp ), dry rot (Mycosphaerella spp ) wet rot (Phomop s is carica e papayae Petr. & Cif.) Alternaria fruit spot (Alt e rnaria alternata (Fr ) Keissler), Fusarium rot (Fusarium solanif e r Snyd & Hans ) and Guignardia spot (Guignardia spp ) are the

PAGE 29

15 most common fruit surface pathogens found in papaya fruit (Alvarez and Nishijima, 1987; Sommer et al., 1992) Anthracnose is the major postharvest disease of papaya, and the symptoms of anthracnose ( causing initially tiny, brown, superficial, watersoaked lesions that may enlarge to 2 5 cm or more in diameter) are most prominent at the full ripe stage (Alvarez and Nishijima, 1987). Chocolate spot is a surface disease and causes reddish brown lesions on the skin (Sommer et al., 1992) As fruit ripen, lesions of chocolate spot become sunken, displaying water-soaked margins (Nakasone, 1986 ; Sommer et al., 1992) Wet rot (Phomopsis caricae-papayae Petr. & Cif.) generates soft and translucent areas on the fruit surface (Alvarez and Nishijima, 1987) Circular or oval black lesions are the symptoms of Alternaria fruit spot (Alvarez and Nishijima, 1987). Infections by Fusarium solanifer produce small dry lesions with water soaked areas (Alvarez and Nishijima, 1987). Guignardia spot, evident as greenish-black lesions, is often seen when papaya are pretreated in water at 42 C for at least 40 minutes (Alvarez and Nishijima, 1987). Stem end rots Lasiodiplodia rot (B01tyosphaeria rhodina (Cooke) Arx; Lasiodiplodia theobromae (Pat.) Griffin & Maulb ), Phytophthora rot (Phytophthora nicotianae Breda de Haan var. parasitica (Dast.) Waterh.), and Rhizopus (Rhizopus stolonifer (Her Ex Fr.) Lind ) are among the most widespread stem end rots reported in papaya fruit (Alvarez and Nishijima 1987 ; Sommer et al. 1992). Lasiodiplodia rot usually occurs at injuries to fruit skin or near fruit peduncle (Sommer et al. 1992). Rhizopus fungus invades through wounds and colonizes the entire fruit rapidly, often spreading to other fruits (Alvarez and Nishijima, 1987).

PAGE 30

16 Internal fruit infections Purple-stain (Erwinia herhicola (Loehnis) dye) and internal yellowing (Enterohacter cloacae (Jordan) Hormaeche & Edwards) are the two most reported internal diseases in papaya fruit (Alvarez and Nishijima 1987) The tissue invaded by Erwinia herhicola becomes translucent and later rots resulting in extensive off-odors (Alvarez and Nishijima, 1987) Fruit flesh infected by E nterohacter cloacae is translucent with a bright yellow to lime-green discoloration (Alvarez and Nishijima, 1987) Postharvest disease control. Since many infections affecting papaya during postharvest handling become established in the field postharvest control measures begin with choosing resistant varieties and implementing good cultural practices during fruit growth (Nakasone, 1986) After harvest proper temperature measurement during transportation and marketing, the use of vapor/hot water treatments and dipping in aurefungin and carnauba waxing are some of the control measures effective in controlling disease progression (Nakasone 1986 ; Alvarez and Nashijma 1987 ; Sommer et al. 1992 ; Sankat and Maharaj, 1997) Hot water treatment at 43 C to 49 C for 20 minutes has been reported to prevent / control postharvest decays (Akamine and Arisumi 1953) 1-Methylcyclopropene Introduction The promotion of plant senescence by ethylene can be inhibited by a number of cyclic olefins including cyclopropene 1-methylcyclopropene ( 1-MCP), 3methylcyclopropene 1 3-dimethylcyclopropene 3,3-dimethylcyclopropene, 1,3,3trimethylcyclopropene, 3-methyl-3-vinylcyclopropene and 3-methyl-3ethynylcyclopropene (Sisler et al. 2001 ) The compounds evidently compete with ethylene at the site of ethylene receptors, blocking tissue responsiveness to the growth

PAGE 31

17 regulator (Sisler and Serek, l 997; Sisler et al., 2001 ). 1-MCP has been used as a tool to investigate ethylene action and tissue responses to ethylene during fruit ripening and flower senescence since it is effective in the ppb range, odorless, stable (non-explosive), and non-toxic (Sisler and Serek, 1997; Sisler and Serek, 1999). Application of 1-MCP delays ripening of climacteric fruits and flower senescence, presumably via its blocking effect on the ethylene signal transduction pathway 1-MCP as commercial powder (active ingredient 0 14% 1-MCP) from Agrofresh, Philadelphia, PA has been approved for use apple fruit. Treatment Procedures for 1-MCP 1-MCP can be applied to plant tissues as a gas The concentration required to inhibit ethylene action decreases as the exposure period increases (Serek et al., I 995a). Actively growing vegetative tissues and abscission layers, some of which involve mitotic activity may need higher 1-MCP concentrations (Sisler and Serek, 1997) Sisler et al. (1997) reported that at higher temperatures less 1-MCP is required Very low quantities of 1-MCP (20 nL L1 ) were effective in extending the storage life of cut flowers including Rosa hyhrida Begonia, and Kalanchoe by preventing bud and flower abscission leaf abscission and flower senescence (Serek et al., 1994). The duration of the prophylactic period varies from plant to plant. Some cut flowers, banana and tomato fruits remain insensitive to ethylene almost for 12 days at 24 C (Sisler and Serek, 1997) 1-MCP and Ethylene Sisler et al. ( 1997) proposed that 1-MCP binds to a metal in the ethylene receptor and would thus compete with the ethylene receptor maintaining the active form of the receptors until ethylene concentration becomes adequate, new receptors are synthesized, or released from the receptor sites (Sisler and Serek, 1997). However reports have

PAGE 32

18 indicated that 1-MCP may bind to other receptors showing homology to ethylene receptors and/or it may not permanently attach to the ethylene receptors (based upon the continuous presence of 1-MCP that further improved storage life of pak choy and broccoli compared to daily application of 1-MCP) (Abbie et al., 2002). Furthermore, Jiang et al. (1999b) reported that the Km (substrate concentration at half the maximum velocity) for 1-MCP (17 nL L1 ) was lower than that for ethylene (96 nL L1 ) for control of banana softening, suggesting that 1-MCP has a stronger affinity than ethylene for the ethylene-binding sites A consistently observed effect of 1-MCP treatment with climacteric fruit is the dramatically reduced level of ethylene production 1-MCP treatment of tomato fruit decreased transcript abundance for the enzymes J-aminocyclopropene-1-carboxylate oxidase (ACO) (EC 4.2 1 3) and ACC synthase (ACS) (EC 4.4 14) ; however ACC content did not change (Nakatsuka et al., 1997). Peach fruit exhibited suppressed ethylene production, ACO activity and accumulation of PP-A CS J mRNA in response to 1-MCP treatment (Mathooko et al., 2001). 1-MCP inhibited the ethylene-induced triple response in arabidopsis seedlings (Hall et al., 2000) ETR 1 and ERS 1 ( ethylene response sensors) showed nearly identical sensitivity to 1-MCP in arabidopsis suggesting the ethylene-binding sites of ETRl and ERS I have similar affinities for ethylene (Hall et al. 2000). Accumulation of ACO mRNA during storage of Flavortop nectarine was inhibited by 1-MCP and this inhibition persisted during post-storage ripening (Dong et al., 2001). In a few reports fruits including grapefruit (Mullins et al., 2000) and strawberry (Tian et al., 200 I) were noted to show increased ethylene production in response to I

PAGE 33

19 MCP treatment. However, both grapefruit and strawberry are non-climacteric fruits in which the triggering end regulation of the ripening process as a whole does not require ethylene unlike climacteric fruits Furthermore, pre-treatments of citrus leaves and leaf explants with 1-MCP induced ethylene production upon transfer of the leaves to air (Zhong et al. 2001 ) The reason for higher ethylene production could be stress-related ethylene production, regulation of ethylene production, and/or excessive ethylene production due to loss of ethylene feedback control mechanisms (Mullins et al., 2000). 1-MCP and Fruit Softening One of the most commonly reported effects of treating fruit with 1-MCP is the dramatic decline in the rate of softening presumably a consequence of reduced accumulation of specific, ethylene-induced cell wall enzymes (Huber et al., 2003) The accumulation ofpolygalacturonase and cellulose (EC 3 2.1.5) was significantly delayed and suppressed in 1-MCP-treated avocado fruit (Feng et al. 2000 ; Jeong et al. 2002) However eventually 1-MCP-treated avocado fruit softened eventually as high as non treated fruit which indicates that PG and cellulose are not essential for softening of avocado fruit (Feng et al., 2000 ; Jeong et al. 2002). The solubilization and degradation of polyuronides of avocado fruit was significantly reduced and delayed b y 1-MCP application as well (Jeong et al. 2003) The mRNA abundance of PG and pectinesterase (EC 3 1 1 11) during storage of 1-MCP-treated Flavortop nectarine was reduced, and inhibition of PG expression persisted during post-storage ripening (Dong et al. 2001 ) In contrast to the general inhibition of accumulation of cell wall enzymes in response to 1MCP treatment the accumulation of endoglucanase (EC 3 2 1.4) and its transcript in 'Flavortop' nectarine were enhanced by 1-MCP and inhibited by ethylene at all stages ripening (Dong et al. 200 I). The nectarine fruit with 1-MCP showed severe flesh

PAGE 34

20 woolliness (sot dry texture) and reddening, and lower juice compared to ethylene treated fruit and the authors proposed that these disorders may be enhanced by the high level of expression of endoglucanase (Dong et al. 2001) 1-MCP treatment decreased the mRNA abundance of expansin 1 in mature green or ripe tomato fruit (Hoeberichts et al., 2002) Since it is believed that expansin is stimulated by ethylene expansin may contribute tomato fruit softening at early stage ofripening (Hoeberichts et al. 2002) The Influence of Suppressed Ethylene Perception on Ripening Physiology and Biochemistry Table 2-1 summarizes from the literature some of the physiological and biochemical responses (PBR) of fruits in which ethylene perception suppressed by 1MCP PBR are divided into three groups (columns) as reduced or delayed increased and unaltered Some of PBR of a crop are listed in three columns because of the different sources possibly caused by cultivar and ripeness stage differences

PAGE 35

Table 2-1. 1-MCP-induced effects on ripening fruits Fruit Reduced or delayed PBR Increased PBR U nattered PBR References Apple Ethylene production respiration Respiration, soluble Respiration, soluble (Fan and Mattheis (Ma/us sylvestris L.) softening color change loss of solids, and internal solids and loss of 1999 ; Fan et al. 1999 ; titratable acidity and water loss lnJUry titratable acidity Watkins et al., 2002; decay aroma production Jiang and Joyce, 2002 ; coreflush and scald Saftner et al., 2003) Apple (fresh-cut) Ethylene production respiration, (Jiang and Joyce, 2002) (Ma/us sylvestris L.) softening, and color change N ...... Apricot Ethylene production respiration Days to ripen (Fan et al. 2000; Dong (Prunus softening, color change et al. 2002) titratable acidity decay and armeniaca L.) aroma production

PAGE 36

Table 2-1 Continued Fruit Reduced or delayed PBR Increased PBR U nattered PBR References Banana Ethylene production Ethylene production (Sisler et al. 1996; (Musa sp AAA softening peel color change and uneven skin color Golding et al. 1998; Jiang group) chlorophyll loss and aroma development et al. 1999a ; Jiang et al. production 1999b) Grapefruit Degreening Ethylene production Decay (Mullins et al. 2000) ( C itru s paradi s i) N Mango Softening and color change Days to ripen Soluble solids (Jiang and Joyce 2000 ; N (Man g if e ra titratable acidity Hofman et al. 2001) indica L.) weight loss and rots Nectarine Eth y lene production and Woolliness and Respiration (Dong et al. 2001) (Pnmu s p e r s ica softening discoloration Lindi.)

PAGE 37

Table 2-1 Continued Fruit Papaya ( C ari c a p a pa y a L.) Reduced or delayed PBR Ethylene product i on respiration softening and color change Pear Ethylene production softening and (P y ru s co mmuni s L.) water los s Increased PBR Days to ripen soluble solids rots anthracnose and skin blemishes Peach Ethylene production ACS and ACO Mesocarp browning (Pr u nu s p ers i c a L.) activities respiration softening and loss of titratable acidity Pineapple ( A nana s co m os u s Merr. ) Eth y lene production color chan g e loss of soluble s olids and chilling mJur y. U nattered PBR References Soluble solids (Hofman et al., 2001 ; Jacomino et al. 2002) (Lelievre et al. 1997 ; Baritell et al. 2001) (Mathooko et al. 2001 ; Fan et al. 2002) (Sel v arajah et al. 2001) N w

PAGE 38

Table 2-1 Continued Fruit Reduced or delayed PBR Increased PBR U nattered PBR References Plum Ethylene production respiration Browning Color change (Abdi et al., 1998 ; Dong (Prunu s salicina softening color change loss of et al. 2002) L.; Prunu s x titratable acidity aroma dom es tica L) production browning and decay Strawberry Ethylene production softening, Ethylene Respiration and (Ku et al., 1999 ; Tian et (Fragaria x color change decay phenolics production and decay al. 2000; Jiang et al. anana ss a Duch) and phen y lalanine ammonia-lyase decay 2001) N (PAL) Tomato Ethylene production ACO and Soluble solids (Nakatsuka et al. 1997 ; ( L yc op es i c on ACS color development phytoene Wills and Ku 2002 ; e s c ul e 11t11m L.) synthase expansin respiration Hoeberichts et al. 2002 ; loss of titratable acidity softening Mostofi et al., 2003)

PAGE 39

25 Physiology of Fresh-cut Produce Introduction The processing or preparation of lightly processed (fresh-cut) fruits and vegetables can be defined as washing sorting trimming peeling skinning or chopping of horticultural commodities in a manner that does not reduce fresh-like quality (Rolle and Chism 1987 ; O Conner-Shaw et al. 1994) The preparation can result stress similar to that found in wounded tissues (Brecht 1995) Excessive water loss synthesis of secondary compounds, higher ethylene production and respiration, softening, browning and degreening are examples of such behaviors shown by fresh-cut produce (Rolle and Chism, 1987; Miller 1992) These behaviors plus temperature relative humidity and atmospheric composition can greatly influence quality maintenance of fresh-cut produce (King and Bolin 1989) Consequences of Processing The physiology of fresh-cut produce is similar to that of wounded tissues (Brecht, 1995). The process necessary for fresh-cut produce (fresh-cut processing) requires abrasion peeling, slicing chopping and shredding (O Conner-Shaw et al., 1994) Each of the previous steps can generate stress conditions in living tissues. Since fruits and vegetables remain viable after the fresh-cut processing their behavior is generally comparable to plants exposed to stress conditions in nature such as wind damage This behavior includes enhanced ethylene and respiration rates, wound-healing processes (synthesis of secondary compounds suberization and lignification) biochemical changes (membrane changes, browning and degreening) and physical changes (softening and water loss ; Rolle and Chism 1987; Miller 1992 ; Brecht, 1995)

PAGE 40

26 Ethylene production and respiration. Wounding caused by fresh-cut processing may accelerate ethylene production and respiration (Rolle and Chism I 987). In climacteric fruits, wounding causes more ethylene production in the preclimacteric and climacteric periods than in the postclimacteric period (Brecht 1995) Respiration of fresh-cut produce generally rises with temperature depending on the severity of damage during processing Higher ethylene production due to wounding may result in an increase in respiration rate as well (Rolle and Chism, 1987). Additionally, starch break down and oxidation of fatty acids may contribute to this respiratory inclination (Miller, I 992). Secondary metabolism. Wounding causes tissues to synthesize secondary compounds which are mostly related to wound-healing and defense mechanism processes. These secondary compounds may affect aroma, appearance, nutritive value, and safety of fresh-cut produce (Brecht 1995) Some of these secondary compounds are phenolics flavonoids terpenoids (Sakai and Nakagawa 1988) alkaloids glucosinolates and long-chain fatt y acids and alcohols (Miller 19 9 2) Wounding increases the activities and transcripts of the following enzymes associated with the secondary compounds : PAL (EC 4 1 3 5) (Fritzemeier et al. 1987 ; Liang et al. 1989) 4-coumarate : CoA ligase (EC 6 2 1 13) (Fritzemeier et al. 1987) chalcone synthase 3-deoxy-D-arabino heptulosonate7-pho s phate synth a se (EC 4.2 .3 .4) (Miller 19 9 2) pero x ida s es (EC 1 11 1.6) (Bostock et al. 1987 ; Miller and Thomas 198 9 ) and stilbene synthase (EC 2.3 1.95) (Vornam et al. 1988) Structural changes of the ti s sue surface may occur as a consequence of the wound-healing process Desiccation of the wounded surface is the first observable change of fresh-cut produce (Varoquax and Wiley, 19 9 4) Desiccation is followed by suberin and

PAGE 41

27 lignin production and deposition in cell walls, and is possibly proceeded by periderm occurrence beneath the suberin layer in many tissues for example potato tuber bean pod, and cucumber pericarp (Burton, I 982) Browning and degreening Browning results from enzymatic oxidation of phenols and polyphenols (Ahvenainen, 1996) Polyphenol oxidase (EC 1.14 18 1), PAL, tyrosine ammonia lyase (EC 4 I 99 2) cinnamic acid -4-hydroxylase (EC 1.14.13.11), Ii poxygenase (EC I 13 I 1. I 3) and catechol oxidase (EC 1. 1 3 14) are the enzymes that likely cause enzymatic browning (Ahvenainen 1996). Increased ethylene production and loss of membrane integrity may start rapid chlorophyll degradation due to induction of chlorophyll-degrading enzymes (Varquaux and Wiley, 1994) The enzymes responsible for the chlorophyll degradation are chlorophyll oxidase, chlorophyllase (EC 3. 1.1 15) lipolytic acid hydro lase and other peroxidases (EC 1.11 1 6) (Varquaux and Wiley 1994) Membrane changes Wounding causes cellular disruption leading to decompartmentation of enzymes and substrates (Rolle and Chism 1987). Wounding enhances the activities of lipid acyl hydrolyse (act like phospholipase D) polyphenol oxidase (Ikediobi et al., 1989) and lipoxygenase (Lulai, I 988 ; lkediobi et al., 1989) These enhanced enzyme activities may result in increases in free fatty acids and free radicals that are toxic to many cellular processes and capable of causing organelle inactivating proteins and lysis (Brecht, 1995) Additionally, excessive ethylene production enhances permeability of membranes and reduces phospholipid biosynthesis (Watada et al. 1990) For example, in fresh-cut carrot, total phospholipids and phospahatidic acid increased but phosphatidylcholine decreased (Picchioni et al., 1994).

PAGE 42

28 Picchioni et al. (1994) also found that rough endoplasmic reticulum numbers increase in fresh-cut carrot which may be correlated to lipid synthesis and the enzyme induction process. Textural and cell wall changes Firmness loss is immediate and faster in wounded tissues due to cell rupture and loss of tissue integrity (Miller 1992) Cell wall enzyme activity may be accelerated by wounding (Miller 1992 ; Karakurt and Huber 2002), which may contribute extensive softening in fresh-cut tissues To illustrate Karakurt and Huber (2002) found that PG and a(EC 3 21.22) and ~-galactosidase activity (EC 3 2.1.23) was higher in fresh-cut papaya fruit compared to intact fruit at 5 C. The erihanced cell wall enzyme activity, thus causes depol y merization of pectic and hemicellulosic polyuronides which may result in further textural changes in fresh-cut tissues Dehydration Fresh-cut processing causes interior tissues to be exposed to air and to increase in evaporation rate which results in water los s. Deh y dration at the cut surface is sometimes obligatory to control microbial growth ; however it may provoke undesirable visual appearance s such as color fading in carrot skin (Watada et al. 1996) External and Internal Factors Contributing to Quality of Fresh-cut Fruits and Vegetables Wounded tissues rapidl y deteriorate and senesce Hence minimizing the negative consequences of wounding is a crucial step that affects storage life and maintenance of interior and exterior qualities of fresh-cut produce These qualities a re greatly affected b y morphological physiological environmental pathological and practical factors Raw product. Since fresh-cut fruits and vegetables are already at the table-ripe edible stage they should have excellent interior and exterior qualities Fresh-cut produce

PAGE 43

29 should also have superior characteristics such as slower ripening rate good texture and flavor qualities and less sensitivity to chilling injury and microorganisms than their counterparts because they are more perishable compare to intact produce (Watada et al. 1996) Temperature Fresh-cut fruits and vegetables should be held at lower temperatures to slow down metabolic activity Lower temperatures are also necessary to control microbial growth. However most of tropical and some subtropical commodities are chill sensitive; therefore storage at a lower temperature may lead to chilling injuries On the other hand keeping fresh-cut produce at lower temperatures suppresses the development of chilling injury symptoms for a limited period (Watada and Qi, 1999) Relative humidity Relative humidity of the atmosphere of fresh-cut produce should be higher to reduce extensive water loss (Schlimme 1995) Edible or non-edible coating and proper packing may reduce water loss from fresh-cut produce (Watada et al. 1996 ; Schlimme 1995) In many cases water loss in fresh-cut produce re s ults from epidermal membrane deterioration. Particularly at higher temperatures where the water vapor deficit is large the water loss hastens in fresh-cut produce (Watada et al. 1996) Controlled atmosphere and modified-atmosphere packing Reduced oxygen and elevated carbon dio x ide le v els are basic practices of controlled atmosphere condition The response of fresh-cut produce stored in controlled atmosphere is different from that of intact produce ; therefore fresh-cut produce should be stored differently and separately On the other hand because of the short handling period controlled atmosphere may not be economically applicable for fresh-cut produce (Watada et al 1996). Gas compositions

PAGE 44

30 in film-packed and edible-coated fresh-cut produce can be modified and this modification may extend storage life of fresh-cut produce (Watada et al., 1996 ; Schlimme 1995) Packing and edible coating Fresh-cut produce may be packed or coated to reduce mechanical damage and water loss, to identify produce and to carry information to consumer (Schlimme, 1995) Nevertheless, packing may cause an increase in temperature that evokes higher respiration and ethylene production rate Therefore, ethylene must be excluded or absorbed by ethylene absorbents such as charcoal and palladium chloride (Schlimme 1995) Edible films reduce moisture loss, limit gas exchange retard ethylene production and keep aroma inside (Ahvenainen 1996) Lipids resins, polysaccharides and proteins are the basic components of edible films (Baldwin et al. 1995) Some coatings may carry some additives that can prevent discoloration and microbial growth by serving as antioxidants and/or anti-microbial agents (Baldwin et al. 1995) Some of these additives are sucrose polyesters of fatty acids, sodium salts of carboxymethylcellulose, carrageenan and chitosan (Ahvenainen 1996). Chemical application Chemical applications are mostly used for reducing decay and browning, and retaining firmness in fresh-cut produce. Chlorine is the standard sanitizing agent for fresh-cut produce in proper concentrations (100-300 ppm ; pH 7). Higher chlorine concentrations ( > 500 ppm) may cause fresh-cut produce to discolor equipment to corrode and aromatic hazardous chloramines to form (Hurst 1995) Hong and Gross (I 998) reported that sodium hypochlorite caused some physiological and biochemical alterations in fresh-cut tomato fruit (higher electrolyte leakage and ethylene production) Sulphating agents are the most common chemicals for inhibiting browning reactions 1n addition to sulphites, ascorbic acid sodium dehydroacetic acid potassium

PAGE 45

31 sorbate, citric acid, zinc, chloride and calcium chloride, resorcinol derivatives, and carbon dioxide and carbon monoxide are used as anti-browning agents (Ahvenainen 1996) Microorganism Microorganisms readily grow on and in fresh-cut produce, and some of them may be detrimental to human being such as Escherichia coli The following microorganisms have been found in fresh-cut produce: mesophilic bacteria, lactic acid bacteria coliforms and fecal coliforms yeast and molds, and pectinolytic microtlora such as Pseudomonas fluores c ens and Xanthomonas maltophila (Nguyen-the and Carlin, 1994) Mesophilic microflora is the largest population followed by lactic acid bacteria (Watada et al., 1996) Moreover some food-borne microorganisms have been reported in fresh cut produce that includes Li s teria monoc y togenes Y e rsinia enterocolitica A e romona s hydrophila (Nguyen-the and Carlin 1994 ; Alfred 1994), Staphylococ c us aureu s (Nguyen-the and Carlin 1994) E s c h e richia c oli (Nguyen-the and Carlin 1994 ; Alfred, 1994), Salmonella spp. (Nguyen-the and Carlin 1994), C lostridium botulinum (Alfred 1994) Bacillus cer e us Giardia /amblia (Beuchat 1995), Shigella ssp ., and Ple s iomonas shig e l/oides (Alfred 1994) The hepatitis A and Norwalk agent virus also have been reported in fresh-cut produce (Beuchat 1995). Microbial growth particularly of human pathogens, may be inhibited by competing bacteria such as lactic acid bacteria or naturally existing antimicrobials released during fresh-cut processing (Luna-Guzman and Barrett 2000) Low temperature is one of the most effective methods to control and prevent microbial growth (Nguyen the and Carlin 1994) while some of the organisms can survive in low temperatures such as Listeria, Y e r s inia and A e romona s (Alfred 1994) Washing fresh-cut produce with chlorine solution (up to 300 ppm) is another effective way to impede development of

PAGE 46

32 microorganisms (Nguyen-the and Carlin, 1994). This process cannot completely eliminate all microorganisms because microorganisms can survive when they are inside tissues where disinfectants cannot penetrate (Watada et al., 1996) Washing fresh-cut produce in trisodium phosphate is one of the other effective ways to control microbiological growth (Beuchat 1995) Ozone a strong oxidant is also used for its lethal activity upon microorganisms at microgram per milliliter concentrations (Beuchat, 1995) In addition to chemical solutions, organic antagonism, gamma irradiation boiling in water and edible coatings containing biochemical agents are also used for the control of microbial growth (Watada et al. 1996 ; Nguyen-the and Carlin, 1994) Irradiation is a safe effective and hazard-free antimicrobial method (Farkas 1998) Campyolchacter, Yersinia, Vibrio and Es cherichia coli have low resistance to ionizing radiation (Farkas 1998) Slow ripening and senescence induced by restricted ethylene action or synthesis may extend the storage life of fresh-cut produce : in a less ripe condition the growth of most opportunistic microorganisms would be expected to be retarded since they tend to grow most rapidly on senescent tissues (Zagoray, 1999) Cell Wall Introduction The plant cell wall is an important structure that determines cell shape connects cells to each other provides essential mechanical strength and ridges and acts as vital barrier against abiotic and biotic invaders such as insects and dusts. The chemical and physiological structure of the plant cell wall varies widely from plant group to plant group and from cell type to cell type The plant cell wall is a dynamic structure that changes during the life cycle of a cell.

PAGE 47

33 Cell Wall Structure The plant cell wall is constructed by a very complex but highly organized composite of many different polysaccharides, proteins and aromatic substances The cell wall consists of three main divisions ; the primary cell wall, the middle lam ell a and the secondary cell wall (Carpita and McCann, 2000) The primary cell wall, born in the cell plate during cell division is capable of growth by expansion; the middle lamella forms the interface between adjacent cells; and secondary cell wall builds up upon the primary cell wall when the cells mature and are no longer growing (Goldwin, 1983 ; Brett and Waldron, 1996). The model of cell wall structure is thought be by three coextensive networks: the cellulose-hemicellulose framework, the pectic matrix and a network of structural proteins (Carpita and Gibeaut, 1993) Cellulose the most abundant plant polysaccharide, exists in the form of microfibrils that are an unbranched B (1-4) linked polymer ofD-glucose strengthened by hydrogen bonds (Goodwin, 1983 ; Carpita and MacCann, 2000) Pectins are a mixture of heterogeneous and highly hydrated polysaccharides rich in D-galacturonic acid Pectins consist of 6 different polymers rhamnogalacturonan I rhamnogalacturonan II, homogalacturonan arabinan galactan, and arabinogalactan I (Carpita and MacCann 2000). Hemicelluloses are a class of polysaccharide, hydrogen-bonded to cellulose microfibrils (Carpita and MacCann 2000) The two major hemicellulosic polymers are xyloglucans and glucomannans in flowering plants ; the other polymers include xylans, mannans, galactomannans and arabinogalactan II, callose and p 1,3 and p 1 4-glucans Proteins such as extension and expansin and phenolics such as lignin and ferulic acid are part of the plant cell structure (Reiter, 1994).

PAGE 48

34 Cell Wall Loosening and Growth Cell growth is provided by expansion or elongation that creates an irreversible increase in cell volume The cell wall must change its structure to expand or elongate : cell wall loosening is probably the primary event in this process followed by continued deposition of new materials Cellulose microfibril orientation controls cell expansion or elongation and decides the plane of elongation (Carpita and MacCann 2000) The multinet growth hypothesis explains displacement of the microfibrils during growth New microfibrils deposited in strata on the inner surface of the cell wall in mostly transverse orientation replace older ones that are pushed towards the outer layers of the cell wall and reoriented in the direction of the cell elongation (Carpita and MacCann, 2000). The acid growth hypothesis proposes that auxin causes lower pH conditions by pumping proton, which activates apoplast-localized growth-specific h y drolyses that cleave the load bearing bonds that join cellulose microfibrils to other polysaccharides (Cosgrove 2000) This cleavage produces a loosening in the cell wall as well as water uptake leading an increase in cell size (Coscgrove 2000). Two kinds of enzymes xyloglucan endo transglycosylase (EC 2.4 1 72) and expansins are thought to be involved in cell wall loosening. Fruit Ripening and the Cell Wall The textural changes during fruit ripening are thought to be related to alterations in cell wall structure (Huber 1983 ; Tucker and Grierson 1987) The changes are mostly correlated with structure and composition of pectic components (Seymour et al. I 987) Solubilization and depolymerization of pectins (Fischer and Bennett, 1991) and hemicelluloses (Lashbrook et al. 1997) during ripening are frequently related to cell wall loosening and disintegration Cell wall modifications have been extensively studied in

PAGE 49

35 tomato fruit, and early reports indicated that pectin degradation by PG represented the model of fruit softening; however, PG-anti sense tomato fruit revealed that pectin degradation is not essential for fruit ripening (Smith et al., 1988; Giavannoni, 1990) The other major changes during ripening occur in hemicellulose content. Xyloglucan, the chief hemicellulose in dicotyledonous plants, undergoes depolymerization in most fruits, including tomato (Sakurai and Nevins, 1993). Besides the depolymerization of both pectic and hemicellulosic polyuronides, there is a loss of neutral sugar from neutral pectins, primarily galactose and arabinose (Tucker 1993). The Cell Wall and Pathogen Attacks The plant cell wall fortifies cells against attacks from microorganisms and even other plants Callose and lignin are thought to act as a physical barrier blocking fungal penetration into plant cells (Hammond-Kosack and Jones 2000) Hydroxyproline-rich glycoproteins contribute to defense against fungal attacks by cross-linking to the cell wall matrix and initiating additional lignin formation. PG-inhibiting proteins restrain PG activity originated from pathogens, which is also a part of defense mechanisms (Hammond-Kosack and Jones 2000) Oligosaccharides derived from the cell walls of fungi and plants, including ~-glucans, chitin, chitosan, and pectin are inducers of the synthesis of a wide spectrum of defensive chemicals in plant tissues (Ryan, 1988) The oligosaccharides are generated at infection or wound sites and may be early signals to activate genes whose products, such as antibiotic phytoalexins, extensins, proteinase inhibitors pathogenesis-related proteins and lignin, enhance the plant s defense system against pathogens and herbivores (Ryan 1988)

PAGE 50

CHAPTER3 DELA YING ETHYLENE-INDUCIBLE RIPENING PROCESS 1METHYLCYCLOPROPENE IN 'GALIA' MELON FRUIT Introduction Ethylene has been known to regulate fruit ripening and softening in climacteric fruits (Lelievre et al., 1997) Accidental exposure to ethylene or natural ethylene production can reduce the postharvest life of climacteric fruits by accelerating ripening and senescence (Reid, 1985). The ethylene inducible effect however, can be delayed/prevented by some ethylene antagonists or ethylene action inhibitors including silver thiosulphate (STS), 2-5 norbornadiene, diazocyclopentadiene and 1methylcyclopropene ( 1-MCP) (Sisler and Serek, 1997; Sisler and Serek, 1999) Commercial use of STS in cut flowers is being considered in some countries due to Ag + heavy metal in STS complex (Sisler and Serek, 1997) 1-MCP, therefore, seems to be the most practical ethylene action inhibitor due to its stability, activity in low concentration, and non-toxic and odorless properties (Sisler and Serek, 1997 ; Sisler and Serek 1999) The ability of 1-MCP to inhibit ethylene action in apple fruit resulted in promising commercial development (Saftner et al., 2003). Furthermore, studies with 1-MCP confirmed that post-storage life and quality of tomato fruit at early and advanced stage of ripening can be improved by 1-MCP (5, 10, 20 and 100 LL' for 24 hat 20 C, Wills and Ku, 2002; 50 to 150 nL L1 1-MCP for 20 hat 20 C, Hoeberichts et al., 2002). Galia' fruit (Cucumis me/a var. reticulatus L. Naud cv Galia) is a climacteric fruit in which ripening is achieved by the help of ethylene, and ethylene and respiratory 36

PAGE 51

37 climacterics (Seymour and McGlasson, 1993). The fruit has excellent flavor and aroma characteristics; however storage life of 'Galia' fruit harvested an early stage of ripening (green peel color), is limited to 2-3 weeks even at low temperatures (Aharoni et al., 1993; Fallik et al. 2001) Restriction of ethylene synthesis has proved that storage life of melon fruit can be extended. For example, 'Galia' fruit at an early stage of ripening held in controlled atmosphere of 10% CO 2 and 10% 0 2 with an ethylene absorbent, potassium permanganate, for 14 days at 6 C plus an additional 6 days at 20 Chad higher quality (reduced fruit softening and decay) than control fruit stored in controlled atmosphere only (Aharoni et al. 1993) The present study was performed to characterize the physiological responses of 'Galia' melon fruit to 1-MCP treatment and determine whether 1-MCP treatment could be effective as a postharvest application for the extension of the storage period or storage life of pre-ripe or ripe 'Galia' fruit. Materials and Methods Plant Material Galia' plants were grown according the growing techniques and production practices established by Shaw et al. (2001) in Greenhouse Facilities at the University of Florida Horticultural Farm near Gainesville FL in spring 200 I Temperatures were recorded every 15 min at various locations in the greenhouse using thermocouples and a datalogger (CR-10 Campbell Scientific Inc. N. Logan UT). No additional heating or cooling units installed in the greenhouse Fruit were harvested at two stages of maturity green (GRN early stage of ripening) and yellow (YLW advanced stage of ripening) according a color chart (1 dark green ; 2 green ; 3 light yellow with green ; 4 light yellow ; 5, yellow; 6 dark yellow to orange) reported by Fallik et al. (200 I) The

PAGE 52

38 harvested fruit were transferred to the Postharvest Horticulture Laboratory of Gainesville The fruit were then selected on the basis of uniformity of size and freedom from defects ; afterwards the fruit were gently brushed washed with tap water (23 dipped into 200 LL" 1 chlorinated water for 1 min and air-dried 1-MCP Application A commercial powder formulation provided by Agrofresh (active ingredients 0 14%) Philadelphia, Penn was used to generate 1-MCP 1-MCP was released from three g of the powder to the vapor phase by adding 50 mL deionized water generating a 7 5 mL L1 concentrated stock in a 136-mL sealed v ial. 1-MCP concentration (1 mL) in the head space of the vial was measured using a gas chromatograph (GC) (Hewlett Packard 5890 II GC ; Avondale PA) furnished with a 80-100 mesh Chromosorb PAW stainless steel column (I 8 m x 3 18 mm i d .; Supelco Bellefonte PA) Injector oven and detector (FID) temperatures were set at 150 150 and 200 C respectively Isobutylene gas which has a FID response similar to that of 1-MCP (Jiang et al 1999) was used as a standard Approximately a 10-mL sample of vial headspace gas was injected into a 179-L metal chamber containing a 50-L void space y ielding a final 1-MCP concentration of 1 5 L L1 and held for 24 h at 20 C. The treatment containers were vented for 5 m i nutes resealed and reinjected with fresh 1-MCP at 6-h intervals Control fruit were kept under identical condition. 1-MCP concentration and efficacy was in v estigated i n a preliminary e x periment in which GRN fruit were treated with air (control) 0 09 0 9 and 9 LL' 1-MCP for 24 hat 20 C and stored at 15 C. Respiration and Ethylene Production

PAGE 53

39 Air-( control) and 1-MCP-treated fruit were placed in airtight plastic containers ( 1 fruit per container) (3 6 L) and sealed for l hat 20 C. Respiration and ethylene production from each the treatment (5 replications) was determined by measuring the CO 2 and C 2 H 4 concentration in the headspace of the containers For CO 2 0 5-mL headspace gas sample was injected to a GC (Gow-Mac, Bridge Water NJ) equipped with thermal conductivity detector, and for C2H4, 1-mL headspace gas sample was infused into the Hewlett-Packard-5890 GC fitted with a flame ionization detector Firmness Assessment Mesocarp fruit firmness was measured on opposite sides of (two equidistant points on the equatorial region) pared fruit using an lnstron Universal Testing Instrument (Model 441-C8009 Canton, MA) The probe (convex 11 mm diameter) located at zero force and contacted with the pared fruit surface was driven to a depth of 10 mm with a crosshead speed of 50 mm min 1 Firmness data was expressed as the maximum force Newton (N) acquired during penetration All tests were conducted with fruit pulp temperature of 20 Electrolyte Leakage Assessment Five mesocarp cylinders were removed the equatorial position of a fruit with an 8mm diameter cork borer From each cylinder I disk (8 x 8 x 8 mm 3 ) was excised by the same cork borer yielding to a total of 5 disks per f ruit. The di s ks were briefly rinsed with deionized water and blotted dry on a slightly moistened Whatman filter paper The disks (five per fruit) were then incubated in 15 mL of 500 mM mannitol for 6 h in a capped polypropylene tube Conductivity was measured with a conductivity bridge (YSI-31 A Yellow Springs OH) furnished with a conductivity cell (3403 Yellow Springs OH) immediately after addition of the bathing solution to the disk s and the end of the

PAGE 54

40 incubation period. The aliquot removed from the bathing solution for the conductivity measurement was added back to the bathing solution The disks and bathing solution were then stored at -20 C for at least 24 h, thawed boiled in water for 30 min, cooled to room temperature, and conductivity measurement was measured once more. The electrolyte leakage was expressed as percentage of the conductivity of total tissue electrolytes Soluble Solids Concentration, pH and Titratable Acidity Determination Soluble solids concentration (SSC), titratable acidity (TA), and pH were quantified using a digital refractometer (Abbe MarkI 0480, Buffalo, NY) a Fisher-395 dispenser (Fisher 395, Pittsburg, OH) equipped with an electrometer (Fisher 380, Pittsburgh, PA) and a digital pH meter (Corning NJ), respectively Mesocarp tissue (80 g) was macerated with a mortar and pestle and centrifuged at 27,200 RFC for l O min at 21 C. Fruit juice collected from the macerated/centrifuged tissue was used for SSC and pH measurement. For TA 6-g fruit juice was titrated with 0 1 N NaOH to an end point of pH 8 2 and TA was expressed as percentage of malic acid using the volume of mL NaOH recorded from the dispenser. Statistical and Informal Taste Analyses General linear model program of SAS (SAS institute Carey NC) and Duncan s Multiple Range Test were performed for Completely Randomized Designs Informal taste analyses to determine the edible stage on fruit surface and flesh appearance, odor, flavor and texture quality were performed by untrained personnel of the post harvest research group of University of Florida

PAGE 55

41 Results 1-MCP Concentration and Efficacy The firmness of GRN control fruit decreased from 66 7 to 6 3 N while fruit treated with 0.09 L L1 1-MCP only softened from 67 8 to 11.3 N fruit treated with 0 9 L L1 1-MCP from 67 1 to 17 N and fruit treated with 9 L L1 1-MCP 70. 3 to 18 1 N over a 21-day period at 15 C (Figure 3-1). The decrease in firmness from day I to 21 was over 10-fold in the control whereas approximately 6-fold in 0 09 L L1 -1-MCP-treated fruit and 4-fold in both 0. 9 L L1 -1-MCPand 9 L L1 1-MCP-treated fruit. The firmness level of fruit treated with 9 L L1 1-MCP did not result in a significant difference in firmness relative to the fruit treated with 0 9 L L1 1-MCP from on days 9 through 21, indicating that the saturation level of 1-MCP is between 0 9 to 9 L L1 1-MCP Therefore with the evaluation of previous 1-MCP-related publications the present studies were performed with 1.5 L L1 1-MCP. Respiration and Ethylene Production Except for the climacteric rise respiration of both control and 1-MCP-treated fruit decreased during storage (Figure 3-2A). However GRN control fruit reached its respiratory climacteric peak at day 6 (9.4 mL ki 1 h1 ) as equal with 1-MCP-treated fruit at day 15 (6 mL kg1 h1 ), resulting in an 11-day delay in the climacteric respiratory peak and a 36% reduction of the magnitude of respiratory climacteric peak. Ethylene production from GRN control fruit increased rapidly reached a peak at day 3 (7 8 L kg 1 h1 ), and then decreased while ethylene climacteric ofGRN 1-MCP-treated fruit started to peak at day 3 and reached its maximum at 9 days (2 7 L.kg1 h1 ) leading to a 6-day delay and 65% reduction in the magnitude of climacteric ethylene peak (Figure 3-2A) The ethylene production by GRN 1-MCP-treated fruit was statistically lower relative to

PAGE 56

42 GRN control on days 1 through 5 when ethylene climacteric of GRN control fruit occurred Respiration ofYLW control and 1-MCP-treated fruit gradually decreased during storage, with no statistical differences between the two treatments (Figure 3-2B) Ethylene production in both YL W control and 1-MCP-treated fruit also declined during storage The ethylene production ofYLW 1-MCP-treated fruit showed a 56% decrease from the first day of storage (5.4 L kg1 L1 at day 1) to the last day (2.4 L kg1 L1 at day 11) whereas YLW control fruit nearly a 90% decrease from day 1 (3 8 L kg1 L1 ) to day 9 (0.4 L kg1 L1 ) resulting a difference between treatments after day 3 YL W fruit treated with 1-MCP produced higher ethylene after day 3 while the ethylene rate was continued to decrease in YL W control fruit. Firmness Firmness of either GRN control or 1-MCP-treated fruit declined during storage as shown in Figure 3-3A. GRN control fruit soften very quickly within first 5 days, losing 66% of their original firmness while GRN 1-MCP-treated lost only 46% At the last day of storage ofGRN control (day 13) GRN control maintained only 6% of their initial value while GRN 1-MCP-treated fruit 20% from then on 1-MCP-treated fruit remained relatively firm and preserved I 0% their initial firmness at the end of their storage ( day 21 ). Firmness of YL W control fruit and 1-MCP-treated fruit was not significantly different from each other during the first 2 days of storage (Figure 3-3B) After 2 days of storage, firmness of the YL W control fruit sharply decreased but not that of the YL W 1MCP-treated fruit. Softening of both treatments remained unchanged from day 5 to 9, from then on, that of YL W l-MCP-treated showed a sharper decline Within 5 days

PAGE 57

43 YLW control fruit softened from 16.5 to 4 9 N (a 70% loss) while YLW 1-MCP-treated fruit from 18 to 12 8 N (a 29% loss) At the end of the storage life (YLW control, day 9; YLW 1-MCP, day 11) YLW control maintained only 30% of their initial firmness while YLW 1-MCP-treated fruit 43% Electrolyte Leakage A continuous increase in electrolytes released from mesocarp tissue of either GRN control or GRN 1-MCP-treated fruit was observed until day 13 The treatments peaked their maxima of35.8% (control) and 28 5% (1-MCP) at day 13 (Figure 3-4A) GRN control displayed statistically higher leakage rates than GRN 1-MCP-treated fruit after day 3 Electrolyte efflux of GRN 1-MCP-treated fruit slightly decreased from day 1 I to 21. YL W control fruit showed an increasing electrolyte leakage through day 7 ; afterwards showing a minimal decrease (Figure 3-48) Electrolytes of YL W 1-MCP treated fruit slightly increased through day 11 as well (Figure 3-48) The maxima of electrolyte efflux ofYLW control fruit was 36.7% at day 7 whereas in YLW 1-MCP treated fruit 27 9% at day 11 resulting a significant difference between the two treatments after day 5 Soluble Solids Concentration, pH and Titratable Acidity Soluble solids of either GRN control or GRN 1-MCP-treated fruit showed very little change, with no differences between treatments and averaged from 8 I% to 8 9% as shown in Figure 5A. SSC in YLW control fruit slightly decreased during storage whilst in YL W l-MCP-treated fruit somewhat increased but magnitude of change and differences were unremarkable, and the soluble solids ranged from IO and 1 I% (Figure 35B)

PAGE 58

44 TA of GRN control increased a little until day 5, after that point moderately decreased; however, TA ofGRN l-MCP-treated fruit remained unchanged starting on days 5 through the end of storage (Figure 3-6A). Neither differences nor changes of TA of GRN control and 1-MCP-treated fruit were noted during storage TA of either YL W control or 1-MCP-treated fruit slightly increased during storage though a minimal decrease was observed at the end (Figure 3-6B) YL W 1-MCP-treated fruit had significantly higher TA than the control on days 7 through 9. The pH of GRN control slightly decreased until day 7 and then, increased; however, in 1-MCP-treated fruit did not show a unique pattern as illustrated in Figure 37 A. In either YLW control fruit or 1-MCP-treated fruit pH very slightly decreased while a small peak was noted at the end (Figure 37 A) The magnitude of changes and differences in pH of either GRN or YLW fruit treated with and without 1-MCP was unremarkably low. Informal Quality Analysis The color change of fruit surface from green to yellow was deferred in GRN 1MCP-treated fruit (Figure 3-8). The color change of fruit skin from green to greenish yellow was also deferred in YL W 1-MCP-treated fruit (Figure 3-9) The edible stage (determined by the informal quality analysis with the help of firmness and color evaluation data) lasted on days 5 through 9 for GRN control and on days 13 through 19 for 1-MCP-treated fruit leading a 4-day delay in edibility and a 40% extension of edible stage. YLW control fruit persisted their edibility through day 5 whereas YLW 1-MCP treated fruit through day 9, representing almost a two-fold extension (80%). Fruit exhibited < 4 N firmness were not edible Neither YL W nor GRN fruit treated with and without 1-MCP did show significant external and internal decay occurrences

PAGE 59

45 Discussion Treatment with 0 9 L L1 1-MCP significantly improved firmness retention of GRN 'Galia' fruit relative to O and 0 09 L L1 1-MCP concentration at 15 C. Increasing 1-MCP concentration from 0.9 to 9 L L1 did not confer additional benefit upon firmness of GRN 'Galia' fruit. In a previous study, charentais melon fruit exposed to 1 L L1 1-MCP (for 24 h at 22 C) stored at 2 C for 16 days and rewarmed for additional 5 days at 22 C became insensitive to low temperature damage ( estimated by visually rating the extend of the fruit surface pitting and browning) compared to non-1-MCP treated fruit (Ben-Amor et al., 1999) Thereby, we propose that the commercial 1-MCP concentration for Galia melon fruit would be approximately 1 to 1 5 L L1 for 24 hat 20 C. Galia fruit is characterized by a classic climacteric ethylene and respiration pattern (Figures 3-2A and 2B) The maximum ethylene production rate of Galia fruit was below IO L kg1 h1 during ripening a comparable result noted by Zheng and Wolf (2002) at 24 C. Respiration of Galia fruit ranged from 6 to 13 mL kg1 h1 and declined during ripening excluding climacterics. Our results indicate Galia fruit is an inferior ethylene producers; the ethylene climacteric occurs earlier than the respiratory climacteric during ripening 1-MCP suppressed both eth y lene production and ethylene climacterics in GRN Galia' fruit indicating that 1-MCP efficiently binds the ethylene receptors thereby limiting the positive feedback regulation of ethylene production in Galia fruit during ripening 1-MCP delayed both ethylene and respiratory climacteric rise of Galia fruit by 6 and 11 days, respectively Similarly, preclimacteric Charentais muskmelon melon fruit exposed 1 L L1 for 24 hat 14 C exhibited a dela y in ethylene climacteric peak (4 days)

PAGE 60

46 relative to non-1-MCP-treated fruit stored and measured at 14 C (Chatenet et al. 2000) YLW 'Galia' control fruit showed a declining ethylene rate compared with 1-MCP treated fruit, resulting a higher ethylene production in YL W 1-MCP-treated fruit. A possible explanation of this observation is: blocking ethylene binding sites of YL W 'Galia' fruit by 1-MCP may cause an interference between ethylene and the ethylene control mechanism, consequently ethylene fails to perceive the quantity of ethylene production, and is being continued to synthesized (Mullins et al., 2000 ; Zhong et al. 2001) 1-MCP delayed the respiratory climacteric and suppressed its magnitude in GRN Galia' fruit, proving that ripening of melon fruit is strongly regulated by ethylene (Flores et al., 2001 ) The respiration of YL W 1-MCP-treated fruit, however, was not affected by 1-MCP, which implies respiration might not be directly related to senescence or over ripening in melon fruit (Saltveit, 1993 ; Bower et al. 2002). Softening ofGRN Galia fruit strongly deferred by 1-MCP consisting with the fact that most fruits exposed to 1-MCP at the early stage of ripening showed firmness retention relative to non-1-MCP-treated fruit (Fan et al. 1999 Jiang et al., 1999; Jeong et al., 2002; Wills and Ku, 2002) 1-MCP delayed loss of firmness in YL W Galia fruit (at the advanced stage of ripening) as well Apple (0 7 L 1 1-MCP for 16 hat 20 C; Mir et al. 2001 or 10 L L1 1-MCP for 6 hat 20 C; Jiang and Joyce 2002) and tomato (treated with 50 150 nL L1 1-MCP for 20 hat 20 C; Hoeberichts et al. 2002) fruit at advanced stage of ripening treated with 1-MCP also showed delayed softening. Thus the softening process in melon fruit even at the advanced stage of ripening is regulated by ethylene (Lelievre et al., 1997 ; Flores et al. 2001)

PAGE 61

47 Electrolyte efilux a measurable symptom of membrane damage (Marangoni et al. 1996) of both GRN and YLW 'Galia' fruit treated with and without 1-MCP increased during storage. The increase in electrolytes during ripening has been previously reported for muskmelon type melon fruit (Lester and Stein 1996; Lacan and Baccou, 1996) The increase in leakage of both GRN and YLW Galia' fruit was repressed by 1-MCP showing that membrane deterioration during melon fruit ripening is regulated by ethylene Ethylene has been reported to stimulate the activities of free-radical-producing enzymes that contribute membrane deterioration (Paliyath and Droillard 1992). To date only one study has been reported for the effects of 1-MCP upon electrolyte efilux : petunia flower corollas treated with 150 nL L. 1 l-MCP for 6 hat 22 C after 12 L L 1 C 2 H4 application displayed lower leakage rates compared to the corollas treated with ethylene; however direct application of 1-MCP (no pre-ethylene treatment) did not affected electrolyte leakage (Serek at al. 1995b ) Soluble solids concentration in either GRN or YLW Galia fruit was not significantly affected by 1-MCP since muskmelon fruit types have little or no starch reserve (Seymour and McGlasson, 1993) The effects of 1-MCP upon TA and pH were minimal. 1-MCP caused slightly higher TA in YLW fruit while did not have a significant effect on GRN fruit. Higher TA due to 1-MCP application has been noted for tomato fruit at an advanced stage of ripening (5 to 100 L L1 1-MCP for 2 hat 20 ; Wills and Ku 2002) 1-MCP had no influence upon pH of either GRN or YLW Galia fruit. The color change of GRN Galia fruit surface from green to yellow in YLW fruit were deferred by 1-MCP, which confirms that loss of chlorophylls and increase in carotenoids are ethylene-dependent process in melon fruit (Flores et al. 2001 ). The inhibitory effect of I

PAGE 62

48 MCP upon color change or development has been reported for most climacteric fruits at an early stage ofripening (Golding et al., 1998; Jiang and Joyce 2000; Jeong et al., 2002) Fruits treated at the advanced stage of ripening responded to 1-MCP by deferring color change/development as well such as Golden Delicious' apple at 4 C (1 IO L L1 1-MCP for 6 hat 20 C; Jiang and Joyce, 2002) and tomato at 20 C (50 to 150 nL L1 1MCP for 2 hat 20 C; Hoeberichts et al., 2002). 1-MCP significantly extended the edible stage of both GRN and YLW 'Galia' fruit by 40 and 80%, respectively One of the affirmative effects of 1-MCP is the extended storage life for most fruits treated at the early stage of ripening (Hofman et al. 200 I) Recently, apple (Mir et al. 2001; Pre-Aymard et al. 2002 ; Jiang and Joyce, 2002) and tomato (Wills and Ku, 2002 ; Hoeberichts et al., 2002) fruit at an advanced stage of ripening has been reported to respond to 1-MCP by improving their shelf life Thus, over ripening or senescence in climacteric fruits can be delayed by 1-MCP. Coriander leaf senescence as assessed by chlorophyl I and protein loss was significantly delayed by 1MCP (Jiang et al., 2002). Tucker and Brady ( I 987) and Smith et al. ( I 989) earlier reported that silver thiosulphate arrested tomato ripening once initiated Thereby climacteric fruit ripening from the early to the advanced stage of ripening necessitates ethylene In summary, 1-MCP extended storage and storage life of Galia fruit at different stages of maturity. Therefore the use of 1-MCP seems to be a novel post harvest application that has commercial potential for melon shippers retailers and even consumers. The affirmative effects of 1-MCP upon ripe fruit would benefit for fresh-cut fruit industry as well. Our results demonstrate Galia' fruit was strongly benefited from I

PAGE 63

49 MCP application ; however Galia fruit does not represent all t y pes of melon fruit. Thus future studies are needed involving different melon types and cultivars

PAGE 64

80 70 60 50 z
PAGE 65

51 20 30 _.._ Control CO2 AA 25 15 -01-MCP CO2 _,._ Control C2H4 20 -61-MCP C2H4 10 15 10 5 .... .... I I 5 .c .c .... .... I I !l 0 .:ii: 0 i. .l 20 30 e "" N u3B :i= 0 N U 16 25 u 20 12 15 8 10 4 5 0 0 0 2 6 8 10 12 14 16 18 20 22 Days Figure 3-2 Respiration and ethylene production of green (A) and yellow (B) Gali a melon fruit treated with 1.5 LL' l-MCP and air (control) during storage at 20 C. Vertical bars represent standard deviation of the means (n = 5).

PAGE 66

52 50 A 40 -+Control 30 -o1-MCP 20 10 z "' 0 "' 50 = = I. B ri: 40 30 20 10 0 -,....----,----------------.--..--....--------r 0 2 4 6 8 10 12 14 16 18 20 22 Days Figure 3-3. Mesocarp firmness of green (A) and yellow (B) Galia melon fruit treated with 1.5 L L1 1-MCP and air (control) during storage at 20 C. Vertical bars represent standard deviation of the means (n = 5).

PAGE 67

45 A 40 35 30 25 20 = Cl.I 15 t).() e-= .:.= 45 e-= Cl.I B 40 0 c.l 35 30 25 20 15 10 0 53 2 4 6 8 10 12 Days 14 ____..._ Control ---0-1-MCP 16 18 20 22 Figure 3-4 Electrolyte leakage of green (A) and yellow (B) Galia melon fruit treated with 1.5 L 1 1-MCP and air (control) during storage at 20 C. Vertical bars represent standard deviation of the means (n = 5).

PAGE 68

54 20 ---------------------------A 15 -----Control -o-1-MCP 10 = 5 = -~ = Q, 0 = 0 20 B "' 0 "' Q, ::c 15 .E 0 rJ1 10 5 o.....,. ___________ ..,..._....., ___________ ......,. 0 2 4 6 8 10 12 14 16 18 20 22 Days Figure 3-5 Soluble solids concentration of green (A) and yellow (B) Galia melon fruit treated with 1 5 L 1 1-MCP and air (control) during storage at 20 C. Vertical bars represent standard deviation of the means (n = 5)

PAGE 69

55 0.20 ---------------------------A ---Control 0.15 -o1-MCP 0.10 0.05 = -;;.-. "= 0.00 "CS ;; 0.20 Q,I ::c B .. 0.15 0.10 0.05 0.00 -t-..... --------------------------11 0 2 4 6 8 10 12 14 16 18 20 22 Days Figure 3-6 Titratable acidity of green (A) and yellow (B) Galia' melon fruit treated with 1 5 LL' 1-MCP and air (control) during storage at 20 C. Vertical bars represent standard deviation of the means (n = 5).

PAGE 70

56 7.0 --------------------------eControl A 6.5 -o1-MCP 6.0 5.5 = c. 7 .0 ...,__....,.. ______ ...,. ____ ..,.... __,. __ ,.._ ______ --I B 6.5 6.0 5.5 _____ ___,; ______________ _... ___________ 0 2 4 6 8 10 Days 12 14 16 18 20 22 Figure 3-7 The pH of green (A) and y ellow (B) Galia melon fruit treated with 1.5 LL1 1-MCP and air (control) during storage at 20 C. Vertical bars represent standard deviation of the means (n = 5).

PAGE 71

57 Figure 3-8. 'Galia' fruit harvested at the pre-ripe stage (green surface) were treated with 1.5 L L1 1-MCP or air (control) and then stored for 13 days at 20 C.

PAGE 72

58 Figure 3-9. 'Galia' fruit harvested at the ripe stage (yellow surface) were treated with 1.5 L L1 1-MCP or air (control) and then stored for 7 days at 20 C.

PAGE 73

CHAPTER4 PHYSIOLOGICAL CHANGES IN FRESH-CUT AND INT ACT 'GALIA' MELON FRUIT WITH TREATED 1-METHYLCYCLOPROPENE Introduction The increase in consumer demand for fresh-cut produce has prompted increased research interest in devising and implementing methods for improving and prolonging the quality of these highly perishable products. Fresh-cut processing involves several steps including peeling and cutting shredding, etc The physical injury attendant to fruit processing initiates a series of events such as increased respiration and ethylene production, stimulated phenol metabolism, and increased enzyme activities (Rolle and Chism, 1987; King and Bolin, 1989) The secondary events resulting from wounding contribute to the challenge for improving the keeping quality of fresh-cut produce The storage life of fresh-cut commodities can also be compromised by the proliferation of microorganisms including mesophilic microflora, lactic acid bacteria coliforms and fecal coliforms, yeasts and other fungi and pectinolytic microflora (Nguyen-the and Carline, 1994). Low temperature has been used to preserve quality and extend storage life of fresh-cut produce Although cold storage retards many biological processes in fresh-cut produce, events leading to tissue softening and deterioration continue at low temperature especially for fresh-cut fruits. Fresh-cut melon one of the most popular fresh-cut fruit (International Fresh-cut Produce Association 2003), is not an exception for these fresh cut fruits displaying rapid tissue softening and deterioration (Lamikanra et al, 2000) Postharvest applications such as dipping fruit slices / cubes in dilute hypochlorite 50 L 59

PAGE 74

60 L1 total available chlorine (pH 6) (Ayhan et al. 1997), in 2 5% calcium chloride solution (Luna-Guzman and Barrett, 2000), or storing in controlled atmosphere of 2% 0 2 + 10% CO 2 at 5 C and 4% 0 2 + 10% CO 2 at 10 C (Qi et al. 1998) have been reported to improve and extend storage life of fresh-cut melons To date, no studies have been reported upon fresh-cut Galia melon fruit while other melon types especially muskmelon and honeydew fruit have been often studied as fresh-cut produce It has been reported that storage 1 i fe of fresh-cut honeydew melon were Ii m ited to 11 days at 4 C whereas storage life of fresh-cut muskmelon 4 to 6 da y s at 4 or 5 C (O Connor-Shaw et al. 1994 ; Qi et al. 1998) Galia melon is a climacteric fruit (Seymour and McGlasson 1993) ; thus ripening process is ethylene-mediated The ethylene-mediated effects on climacteric fruit can be significantly delayed by the u s e of ethylene binding inhibitor s. One of these, 1methylcyclopropene (1-MCP ; Sisler and Serek 1997) has been shown to extend the storage life and period of optimum quality of apple (Jiang and Joyce 2002) and tomato (Wills and Ku 2002 ; Ku et al. 2002) fruits at advanced stages ofripening Exposure of fresh-cut postclimacteric apple fruit before or after cut to 1-MCP ( 1 or 10 L L1 for 6 h at 20 C) improved their storage life by reducing softening and color change of epidermal tissue (loss of green color) at 4 C (Jiang and Jo y ce 2002). Thus we proposed that fresh cut Galia fruit should benefit from 1-MCP application as well. The objectives of this study were to investigate responses of fresh-cut ripe (derived from postclimacteric intact fruit subject to treatments) versus intact ripe Galia fruit to 1-MCP

PAGE 75

61 Materials and Methods Plant Material Galia' plants were grown according the growing techniques and production practices established by Shaw et al. (2001) in Greenhouse Facilities at the University of Florida Horticultural Farm near Gainesville, FL in spring 2002. Temperatures were recorded every 15 min at various locations in the greenhouse using thermocouples and a datalogger (CR-IO Campbell Scientific, Inc N Logan, UT). No additional heating or cooling units installed in the greenhouse Galia fruit were harvested at three-quarter to full-slip stage and transferred to the postharvest facilities at the University of Florida in Gainesville The fruit were selected for uniform size (approximately 1200 to 1300 g), external color (yellow) and netting development. Ethylene production and respiration rate for the fruit at the time of harvest was approximately 2 L kg1 h1 and 10 mL ki 1 h1 at 20 respectively and soluble solids concentrations were about 11 to 12%. The fruit were gently washed with tap water, immersed in 200-L L1 chlorinated water for 1 min (23 and air-dried before transferring to 20 C for 1-MCP application 1-MCP Quantification and Treatment The source of 1-MCP was Agrofresh commercial powder (active ingredient 0 14% 1-MCP) from Agrofresh Philadelphia, PA Three g powder were dissolved in 50mL deionized water of a 136-mL vial ; afterwards the vial was sealed with a septum and incubated on oscillating shaker for 2 hat room temperature 1-MCP concentration in the vial headspace was measured using a gas chromatograph (Hewlett Packard-5890 Avondale, PA) equipped with a 80-100 mesh Chromosorb PAW stainless steel column (1.8 m x 3 18 i d ; Supelco, Bellefonte, PA) at an injector oven and detector(FID) temperature of 150 150 and 200 respectively lsobutylene gas was used as standard to

PAGE 76

62 calculate 1-MCP concentration (Jiang et al. 1999). Approximately 7 5 mL L1 1-MCP stock in the head space of the vial was generated from the 3-g powder. Vial-head space gas sample (7 5 mL) was injected into a 174-L metal chamber having a 56 5 L void volume yielding a final 1-MCP concentration of 1 L L1 and maintained for total exposure period of 24 hat 20 C. The metal chamber was vented for 5 min at 6-h intervals and reinjected with fresh 1-MCP avoid CO 2 accumulation Control fruit was kept under similar condition Preparation of Fresh-cut 'Galia', and Treatment Design The fruit were transferred from 20 C to a 5 C facility that had been sanitized using 200 L L1 -chlorinated water prior to use After a 1-h period to allow temperature equilibration the blossom and pedicle ends of the fruit were removed, and the fruit were longitudinally (from the pedicel and to the stem end) cut into 2 5 cm slices using a plastic Bread Slicer (Cuope-Pain) The slices were peeled and cut into cubes (2 5 x 2 5 x 2 5 cm 3 15 to 16 g) using a double bladed knife The cubes were then flushed with a sterile isotonic mannitol solution (500 mM) using a squeeze bottle and placed in non-airtight plastic containers (I. 7 L FridgeSmart) that has bui It-in grid on the bottom lifts (9 cubes / container) A total of 60 containers (30 each for 1-MCP and control fresh-cut tissue) were used in this experiment, and l O of these (5 each of each treatment) were removed at 2-day intervals for quality evaluation Additionally 80 intact fruit (40 each of control and 1-MCP-treated fruit) were stored along with the fresh-cut tissue at 5 C. The 4 treatments included fresh-cut tissue derived from intact ripe fruit pre-treated with air (FC-CNT: fresh-cut control), fresh-cut tissue deri v ed from intact ripe fruit pre-treated with 1-MCP (FC-MCP : fresh-cut 1-MCP) intact ripe fruit pre-treated with air (IF-CNT:

PAGE 77

63 intact fruit control) and intact ripe fruit pre-treated with 1-MCP (IF-MCP: intact fruit 1MCP) Ethylene Analysis Ethylene production was measured at room temperature every other day enclosing fresh-cut and intact fruit in plastic containers (0 9 Land 3.6 L, respectively) allowing ethylene to accumulate for 2 h at 2-day interval at 5 C. Nine cubes per fruit for FC-CNT or FC-MCP and 1 fruit for IF-CNT or IF-MCP were placed in the airtight containers prior to sampling A 1-mL headspace sample was withdrawn by a hypodermic syringe through a rubber septum eth y lene production was measured using a GC (Hewlett Packard 5890 II Avondale PA) equipped with a flame ionization detector The carrier gas (Nitrogen) was 30 mL min 1 Oven injector and detector temperature was 70 200 and 250 respectively Firmness Assessment Mesocarp firmness of a fruit cube was measured using an Instron Universal Testing Instrument (Model 4411 Canton MA) equipped with a 5-kg load cell and an 8mm convex probe at 20 C. During firmness measurement intact fruit or fresh-cut fruit containers were kept coolers Intact fruit prior to firmness measurements were diced into cubes using the procedures described above for fresh-cut processing The probe was positioned at zero force contact with a fruit cube s urface and driven to a depth of IO mm at a crosshead speed of 50 mm min 1 Firmness data are reported a s the maximum force (Newton) recorded during penetration Electrolyte Leakage Mesocarp disks (5 discs per cube or fruit) in 8 mm diameter and 8 mm thickness were removed from centermost part of either fresh-cut tissue or intact fruit with an 8-mm

PAGE 78

64 diameter cork borer, rinsed with deionized water blotted dry, and incubated in 15 mL of 500 mM mannitol for 1 h in capped polypropylene tubes Incubations were conducted at room temperature, on an oscillating shaker set at 1.4 cycle sec 1 The conductivity of the bathing solution was measured at the end of the I-hour incubation using a conductivity bridge (YSl-3 lA, Yellow Springs, OH) equipped with a conductivity cell (Model 3403, Yellow Springs, OH). The aliquot removed for the conductivity measurement was added back to the bathing solution. The disks and bathing solution were then stored -20 C for at least 24 h, thawed, and heated in a boiling water bath for 30 min, cooled to room temperature and conductivity again measured Electrolyte leakage was expressed as a percent of total conductivity estimated from the frozen/heated samples Pectin Efflux Five mesocarp cylinders were removed from the mid section of fresh-cut tissue or an equatorial section of an intact fruit using a 15-mm diameter cork borer. The cylinders were then sliced into disks perpendicularly from the long axis of a cylinder using a double bladed razor in which a razor was mounted from the other razor by IO mm Each disk, 15 mm diameter and 10 mm thick, weighed approximately 5 g The disks (5 per replication) were briefly rinsed with distilled water, blotted dry, and incubated in 10 mL of 500 mM sucrose on an oscillating shaker for 6 hat room temperature Afterwards, the bathing solution was filtered through a What man GF/C filter, and 40 mL of 95% ethanol was added to the filtrate The filtrates were centrifuged at 2,000 g for 20 min at 4 C and the supernatant was discarded. The pellet was washed with 40 mL of 80% ethanol (2 times) and dissolved in 5 mL distilled water, and total uronic acid was determined by the mphenylphenol method (Blumenkrantz and Asboe-Hansen, 1973).

PAGE 79

65 Quality Evaluation Fruit surface and flesh color were measured using a chromameter (Minolta-CR200, Japan) The results were presented as lightness (L *, representing the lightness or grey scale), hue angle (the dimension of color that specifies a position on a color wheel of 360 with 0, 90 180 and 270 representing the hues red, yellow, green and blue respectively) and chroma (distinguishing the difference from a grey shade to a pure hue) values. Water soaking on the flesh was expressed as the percentage of areas of a cube with a 5% interval (total 5 cubes for each treatment) using only the top face Five intact fruit or five fresh-cut cubes from each treatment were evaluated for mesocarp water soaking Informal descriptive analysis was used to profile the quality of either fresh-cut cubes and intact fruit flesh by untrained personnel evaluating appearance odor, texture and flavor (O'Connor-Shaw et al., 1994) according to the following hedonic chart : I poor ; 2, poor-good; 3 fair ; 4, good-excellent ; and 5, excellent. The informal descriptive analyses were done immediately after removing fresh cut or intact fruit from the 5-C cold room at days 0, 2 4, 6 8 and I 0. The samples were tested under white light and at the room temperature, 23 with testing at least 3 samples for each treatment. Microbial Counts Fruit tissue (5 g) was removed with a flame-sterilized cork borer (21 5 mm diameter) and knife from innermost part of a fruit or with a flame-sterilized knife from a fruit cube (approximately 1 / 3 of a cube) on sterilized aluminum foil in an air-circulated fume. The fruit tissue then was incubated in a 45-mL sterile phosphate buffered solution (PBS), pH 7. The bathing solution and fruit tissue were vortexed at high speed using a vortex (Fisher-Genie 2 Scientific Industries Inc., Bohemia, NY) for I min. Afterward, a series of dilutions were prepared using sterile PBS as needed Total aerobic

PAGE 80

66 Enterobacteriaceae yeasts and other fungi total coliforms and lactic acid bacteria counts were made using 1 mL inoculum of the bathing solution The plates and incubation conditions for each count were: total aerobic count 3M Petrifilm aerobic count plate (3M Microbiology Products, St. Paul MN) 3 days at 30 C ; Enterobacteriaceae 3M Petrifilm Enterobacteriaceae count plate I day at 30 C; yeast and other fungi, 3M Petrifilm yeast and mold count plate, 5 days at 25 C ; total coliforms 3M Petrifilm coliform count plate 1 day at 30 C ; and lactic acid bacteria 3M Petrifilm aerobic count plate anaerobic incubation for 2 days at 30 Cina 1 9-L airtight plastic container with an anaerobic system envelope (Gas Pak, Becton and Dickinson Co Cockeysville MD) The plates were prepared in an air-circulated hood after 0 (immediately after dicing) 5 and 10 days at room temperature, and microbial counts were reported as colon y forming units per gram of tissue (CFU g). Experimental Design and Statistics The experiments were conducted in a randomized complete-block design using 3 to 5 replications per treatment Statistical procedures were performed using the PC-SAS software package. Differences between means were determined using the Duncan Mean Comparison Test. Results and Discussion Ethylene Production Ethylene production of fresh-cut cubes ofripe Galia fruit stored at 5 C (FCCNT 10.4 L kg' h1 ; FC-MCP, 8 5 L kg" 1 h 1 ) was at least 4-fold higher than that of intact fruit (IF-CNT 1.9 L kg' h1 ; IF-MCP 1 6 L kg' h" 1 ) at day 1 (Figure 4-1 ) Ethylene production of both fresh-cut fruit and intact fruit declined during storage ; however, intact fruit displayed statistically lower ethylene production and declining rates

PAGE 81

67 relative to fresh-cut fruit on days 1 through 7 Ethylene production declined approximately 5-fold in FC-CNT (from 10 3 to 2 1 L kg 1 1 ) and 4-fold in FC-MCP (from 8 5 to 1 9 L kg" 1 h 1 ) over a 9-day period Ethylene production in intact fruit declined approximately 2 to 3 folds in both IF-CNT (from 1 9 to 0 8 kg" 1 h1 ) and IF-MCP (from 1 6 to 0 6 kg" 1 h1 ) Generally, ethylene production of intact ripe Galia' fruit was under 2 L kg 1 h. 1 at 5 which is similar to the case for ethylene production rate of intact ripe or postclimacteric muskmelon fruit held at 5 C (1 to 3 L kg 1 h ; Luna Guzman et al. 1999) These authors however reported slightly higher ethylene production rates in intact ripe muskmelon fruit (approximatel y from I to 3 L kg 1 h1 ) compared with fresh-cut ripe muskmelon fruit (appro x imatel y from 0 5 to 2 L kg 1 h1 ) during 12 days of storage Fresh-cut Galia fruit in this study had higher ethylene production rates than intact Galia fruit likely due to both the wound response stress related ethylene production in wounded cells (Rolle and Chism 1987) and increased surface area exposed to the atmosphere after dicing facilitatin g o x ygen diffusion to interior cells (Zagory et al., 19 9 5) The decrease in ethylene production in intact and fresh-cut ripe Galia fruit during storage is in agreement with the findings ofLuna Guzman et al. ( 1999), who reported that intact or fresh-cut ripe muskmelon fruit stored at 5 C exhibited declining ethylene production rates during the first 7 days of storage In the present study the effect of 1-MCP on ethylene production was in s i g nificant though s ome small differences were noted at day I between IF-C N T and IF-MCP However fresh-cut postclimacteric Golden Delicious apple fruit treated with 1-MCP (1 or 10 L L 1 for 6 h at 20 C) before or after cutting had lower eth y lene production relative to non-1-MCP-treated fruit (fruit sample sealed in glass jars at 20 C) after 5 or

PAGE 82

68 IO days of storage (Jiang and Joyce 2002). The low ethylene production of fresh-cut and intact 'Galia' fruit at 5 C may be due to the low storage temperatures employed and/or the inherently low ethylene production of 'Galia' fruit relative to other muskmelon types such as both preclimacteric Giant Perfection and Iroquois that have ethylene rate of over 80 L kg" 1 1 (Zheng and Wolf, 2000) In contrast, the ethylene production rate of intact ripe 'Galia' fruit is under 6 L kg' h1 during ripening at 20 C (Chapter 3). Firmness Assessment As shown in Figure 4-2 both fresh-cut and intact ripe Galia fruit regardless of 1-MCP softened moderately during storage. IF-CNT and FC-CNT lost about 31 % and 22% of their original firmness respectively after 10 days while IF-MCP and FC-MCP softened 16% and 11%, respectively. At day 10 IF-MCP (10 6 N) had 22% higher firmness compared to IF-MCP (8 7 N), and FC-MCP (11 6 N) had 32% higher firmness than FC-CNT (8.5 N) Firmness loss in either fresh-cut tissue derived from ripe 1-MCP treated fruit or in intact ripe fruit treated with 1-MCP was significantly lower compared with either fresh-cut ripe control or intact ripe control fruit during storage proving that melon fruit softening is an ethylene-dependent process (Flores et al. 2001 ) Recent studies have shown that 1-MCP can delay softening of climacteric fruits when applied at advanced stages of ripening. The deferred softening has been shown for apple (1 or IO L L 1 1-MCP for 6 hat 20 C; Jiang and Joyce, 2002) and tomato (50 to I 50 nL L, for 24 h at 20 ; Hoeberichts et al. 2002) Additionally, fresh-cut postclimacteric Golden Delicious apple fruit treated with 1-MCP (l and IO L L1 for 6 h at 20 C) before or after cutting showed significant firmness retention relative to fresh-cut control fruit after 5 or IO days at 4 C (Jiang and Joyce 2002)

PAGE 83

69 Electrolyte Leakage Electrolyte leakage, a estimate of membrane permeability and integrity (Marangoni et al., 1996), slightly increased in both intact ripe and fresh-cut ripe 'Galia' fruit during storage (Figure 4-3) The increase in leakage of both IF-CNT (from 13.1% to 14 3%) and IF-MCP (from 12 3% to14 1%) was below 15% on days 0 through 10 while the increase FC-CNT (from 14.9 to 20.7) was 38% and in FC-MCP (from 14.24 tol8) was 26%. 1-MCP did not suppress the leakage increase in either intact or fresh-cut fruit. However, intact ripe Galia' fruit treated 1-MCP (1.5 L L1 ) displayed slightly lower leakage during a limited time (on days 7 through 9) compared with intact ripe control at 20 C (chapter 3) Fresh-cut ripe 'Galia' fruit showed slightly higher leakage compared with intact ripe fruit during storage at 5 C, which supports the fact membrane permeability increases in response to wounding (Portela and Cantwell, 2001 ) Membrane permeability is a common feature of senescing organs and ripening fruit (Lester and Stein, 1993; Flores et al. 2001). Increased membrane permeability results in a loss of cellular components and accumulation of liquid in intercellular spaces (Saltveit, 1997). Similar to fresh-cut ripe Galia fruit increased leakage (measured at 22 C) for fresh-cut climacteric / postclimacteric muskmelon fruit (approximately a 15% increase) was reported by Portela and Cantwell (2001) on days 0 through 12 stored at 5 C. Portela and Cantwell (200 I) further noted that fresh-cut muskmelon discs prepared by a blunt cork borer caused slightly higher leakage (22%) than fresh-cut fruit prepared by a sharp borer since the blunt borer resulted in more severe damage in tissues relative to the sharp borer. Pectin Effiux Pectin effiux values from mesocarp disks of both FC-CNT ( 18.8 g kg1 fresh weight) and FC-MCP (19 1 g ki 1 f.w.) were 2-fold higher than those of IF-CNT (9.1 g

PAGE 84

70 ki 1 f.w ) or IF-MCP (9 3 g ki 1 f. w.), respectively at day 2 and the eftlux remained over 2 folds throughout the remainder of storage (Figure 4-4) Pectin eftlux from mesocarp disks ofFC-CNT increased by 25% and that ofFC-MCP by 22% on days 2 thorough 4 ; afterwards, the eftlux decreased by 15% in FC-CNT and by 11 % in FC-MCP through day 10 compare to the values at day 2. However, eftlux from mesocarp disks of both IF-CNT and IF-MCP did not change during storage, with having the approximate average eftlux of9.3 g kg1 f.w Pectin eftlux from mesocarp disks was unaffected by prior 1-MCP treatment in either fresh-cut or intact ripe Galia' fruit ; however pectin eftlux increased significantly in fresh-cut ripe fruit relative to intact ripe fruit indicating that pectin degradation may be affected by wounding. The insignificance of 1-MCP upon pectin eftlux was reported for pericarp disks of ripe tomato fruit treated with 1-MCP ( 4 8 L L1 for 24 at 18 C) and stored for 2 to 3 weeks at 5 C (Almeida 1999) The wounding effect upon pectin degradation was noted for fresh-cut ripe papaya fruit by Karakurt and Huber (2003) who reported that the levels of water and CDT A-soluble polyuronides increased significantly within 24 h after cutting and remained higher (over 50%) compared to intact ripe fruit during 8 days of storage at 5 C. Higher uronic acid eftlux in fresh-cut Galia fruit may be the result of increased activities of cell wall enzymes including polygalacturonase and p-galactosidase (Miller et al., 1987 ; Karakurt and Huber 2003). Recently Moctezuma et al. (2003) reported the TBG4 (tomato P-galactosidase) gene was up-regulated by ethylene while the TBG6 gene was down-regulated by ethylene in ripe tomato fruit. Additionally 1-MCP (I L L1 for 12 hat 20 C) has been shown to decrease the activity of pectinmethylesterase, a-mannosidase and p-glucosidase in ripe SanCastrese apricot fruit at 20 C (Botondi et al. 2003)

PAGE 85

71 Quality Evaluation No changes in color parameters of hue angle and L* (lightness) of the skin of both IF-CNT and IF-MCP were noted during storage (Figure 4-SA and SB) indicating that the original skin color of intact fruit regardless of 1-MCP remained unchanged However, the skin color intensity became duller during storage, as evaluated by the development of chroma value (Figure 4-SC). Flesh color and intensity / purity of either FC-CNT or FC MCP remained unchanged during storage, as estimated by L hue angle and chroma (Figure 4-SD, SE and SF) Flugel and Gross (1982) observed relatively low levels of chlorophyll and carotenoids in the flesh of Galia' fruit with a gradual decrease in both types of pigment during ripening, which might explain the insignificant color change and insignificant variation among treatments noted here Water soaking in both FC-CNT and FC-MCP Galia fruit increased with storage while the flesh of either IF-CNT or TF-MCP showed no sign of the disorder (Figure 46A) The extent of water soaked areas in FC-CNT was 44% after 10 days while that of FC-MCP was only IS, leading a significant difference between these treatments from day 8 to 10 All 4 treatments had excellent quality at day 0 as assessed by sensory evaluation (Figure 4-6B) The score for sensory of JF-CNT or IF-MCP never dropped below S (excellent) during storage while FC-CNT scored 2 8 (poor-good to fair) at day 10 (Figure 4-8) On days 4 through 8 the sensory scores for FC-CNT fruit were below 4 (fair to good-excellent) whereas that of FC-MCP fruit were over 4 (good-excellent to excellent) which indicates the limit of acceptability the average keeping quality, of fresh-cut ripe Galia fruit was 4 days (Figure 4-7) at S C while that of fresh-cut ripe fruit derived from intact ripe 1-MCP-treated fruit was 8 days at S 0 C.

PAGE 86

72 Changes in either mesocarp or fruit skin color of Galia melon fruit is due a gradual decrease in both chlorophyll and carotenoid content during ripening (Flugel and Gross, 1982) The color data Galia fruit during storage showed no color changes (based on hue angle) in either fruit skin or mesocarp were recorded regardless of treatment which may indicate ripening progress was deferred by the low temperature, 5 C. Water soaking of fresh-cut 'Galia fruit increased with storage time and water soaking of fresh cut fruit was significantly higher than those of intact Galia fruit on days 6 through I 0 1-MCP did not ha v e a significant effect on water s oaking of fresh-cut Galia fruit during most of the storage period but significantl y suppressed the increase in water soaking noted during the period from 8 through 10 day s. In contrast 1-MCP (1 I L 1 for 24 h) did not affect water soaking in Charentais melon flesh treated at the preclimacteric stage after 3 5 days of storage at 14 C (Chatenet et al. 2000) The authors further reported that water soaking in Charentais melon mesocarp during the late stage of ripening was not an ethylene-inducible event based on the absence of expression of genes encoding aminocyclopropane-1-carboxylic acid synthase and l-aminocyclopropane-1-carboxylic acid oxidase Water soaking in fresh-cut Galia fruit however seems to be an ethylene dependent process which may be related to the s tage of ripeness and / or wounding Chatenet et al. (2000) attributed the water soaking phenomenon to a depletion of cell wall calcium and Karakurt and Huber (2002) to ethylene-inducible changes in membrane permeability. Lipolytic enzymes including lipoxygenase and phospholipase Dare like involved in wound-induced degradation of membrane lipids (Karakurt and Huber 2003) Lipoxygenase can contribute to the membrane permeabilit y b y involving production of

PAGE 87

73 reactive oxygen species, participating in peroxidative reactions (Huber et al. 200 I) and inactivating protein synthesis (Karakurt and Huber 2003) Microbial Counts Total aerobic load in fresh-cut ripe 'Galia' fruit increased sharply during days 5 to IO of storage at 5 C, with FC-CNT having higher counts (I .8 x 10 3 CFU g1 ) than FC MCP (1.5 x 10 3 CFU i 1 ) at day 10 (Table 4-1). Total aerobic population of intact ripe fruit were very low compared to fresh-cut fruit and showed a very slim increase through day 10 (Table 4-1 ). Total coli forms or other fungi in either fresh-cut or intact fruit were negligible throughout storage (Table 4I). The Enterobacteriaceae population in FC-CNT and FC-MCP increased significantly during storage, and FC-CNT showed statistically higher counts (8.7 x 10 1 CFU g1 ) than FC-MCP (5.2 x 10 CFU g1 ) at day 10 (Table 4-1) Enterobacteriaceae were almost undetectable in intact fruit over the I 0-day storage period (Table 4-1). There was also an increase in lactic acid bacteria of both fresh-cut and intact fruit; however fruit with 1-MCP (IF-MCP) or derived 1-MCP-treated fruit (FC-MCP) displayed higher counts compared to fruit without 1-MCP at day 10 (TF-CNT and FC CNT; Table 4-1 ). Yeasts accumulated significantly in fresh-cut ripe fruit on days 5 through 10 (FC-CNT, 1.2 x 10 3 CFU g1 ; FC-MCP, 1.1 x 10 3 CFU g1 ) with no variations between the two treatments while yeasts in intact fruit were imperceptible (Table 4-1 ) The significant increase in microbe populations of fresh-cut Galia arose after day 5, which is in agreement with the finding of Luna-Guzman and Barrett (2000) who noted a significant raise of total aerobic counts in non-1-MCP-treated fresh-cut ripe muskmelon fruit (sanitized with 50 L L-' -chlorinated water) after 4 days of storage at 5 0 C. Luna-Guzman and Barrett (2000) recorded total aerobic counts ranging from approximately 2 x I 0 2 to 9 x I 0 9 CFU g1 plated at days 4, 8 or 12 These values are

PAGE 88

74 quite high compared to the values noted herein for fresh-cut ripe Galia fruit. Higher total aerobic (from 1.4 x 10 4 to 8 8 x 10 4 CFU g1 ) lactic acid bacterium (from 1.4 x 10 2 to 7 2 x I 0 2 CFU g1 ) and Enterobactericium (from 7 8 x 10 3 to 3.2 x 10 4 CFU g 1 ) counts were also documented in muskmelon fruit (undefined ripeness stage) stored 11 days at 4 ~C by O'Connor-Shaw et al. (1994) However Ayhan et al. (1998) reported a similar or slightly lower microbial count fresh-cut muskmelon fruit (undefined ripeness stage) (10 x 10 1 CFU cm 2 for total aerobic counts; 1 x 10 1 CFU cm 2 for yeast ; and I x I 0 1 CFU cm 2 for other fungi) washed with chlorinated water (50 L L1 total available chlorine) and sealed with modified atmosphere (95% nitrogen and 5% oxygen) stored IO days at 2 2 0 C. Low microbial population (1 5 x 10 3 to 3 x 10 3 CFU i' for total aerobic count and I x 10 2 CFU g 1 for yeast count) in pre-ripe / ripe non-1-MCP-treated fresh-cut muskmelon fruit stored for 6 days at 5 C (not washed previously with chlorinated water) was also noted by Portela and Cantwell (200 I). Portela and Cantwell (200 I) attributed the low microbial count in the fresh-cut muskmelon to strict sanitation procedures during fresh cut processing 1-MCP slightly suppressed total aerobic and Enterobactericium count increase in fresh-cut Galia fruit whereas lactic acid bacterium growth was promoted. Even so the lactic acid bacterium population was very low in all treatments and at all times of evaluation with thinking the maximum total limit for microbial growth for fresh-cut vegetables (is this value for vegetables of relevance to fruits ? ) is 5 x I 0 7 CFU g 1 (Francis et al. 1999) The 'Solo papaya variety treated with 25 L L1 1-MCP at 20 C for 14 h at mature green stage and stored at 20 C for approximately for 20 days showed slightly higher symptoms of stem rots body black rots and anthracnose compared to untreated fruit lasted approximately 5 days (Hofman et al., 2001) Hofman et al. (2001)

PAGE 89

75 attributed the slight increase in the symptoms of the diseases in 1-MCP-treated fruit to a reduction in anti fungal concentrations due to extended storage life Jiang et al. (2001) found that ripe Everest' strawberry fruit treated with 500 to 1000 nL L1 1-MCP (24 h at 20 C) displayed accelerated leak disease development at 20 C compared to non-1-MCP treated fruit ; however, 1-MCP at 100 and 250 nL L1 delayed the onset of the decay Tomato plant lines expose to 10 nL L1 1-MCP (24h at 18 to 24 C) resulted in a significant increase in Botryti s cinerea frequency in the cultivars Moneymaker and Castlemart but no increase in Pearson' (Diaz et al. 2002) Hence, the effects of 1-MCP upon microbial growth are complex and depend on the type of microorganisms 1-MCP concentration cultivars and tissue type and de v elopment. In summary the storage life of fresh-cut melon fruit derived from intact ripe 'Galia fruit treated with 1-MCP was extended by 4 days compared to fresh-cut fruit, derived from intact ripe non-1-MCP-treated fruit that lasted 4 Thus 1-MCP can be considered a safe potential growth regulator or conditioner for fresh-cut melon fruit alternative to calcium chloride / lactate ethylene absorbent or controlled atmosphere Finally the natural resistance of melon fruit to microbial growth can be supported by using sanitized equipments containers spaces and low temperature

PAGE 90

12 10 8 ... I .c ... I 6 l)f) -;_ -4 = u 2 0 76 1 3 5 Days ____..._ -0-TIntact control Intact 1-MCP Fresh-cut control Fresh-cut 1-MCP 7 9 Figure 4-1 Ethylene production for intact ripe Galia fruit with and without 1-MCP and fresh-cut fruit derived from intact ripe fruit treated with and without 1-MCP during storage at 5 C. Vertical bars represent standard deviation of the means (n = 5)

PAGE 91

18 16 14 12 z "' 10 "' C e 8 i.. ri: 6 4 2 0 0 _._ Intact control -oIntact 1-MCP 77 -TFresh-cut control Fresh-cut 1-MCP 2 4 Days 6 8 10 Figure 4-2 Mesocarp firmness for intact ripe Galia fruit with and without 1-MCP and fresh-cut fruit derived from intact ripe fruit treated with and without 1-MCP during storage at 5 C. Vertical bars represent standard deviation of the means (n = 5).

PAGE 92

78 30 ---Intact control -TFresh-cut control 25 -oIntact 1-MCP -vFresh-cut 1-MCP ':le. e 20 t){) C: ..::.:: C: 15 .... ,>-, 0 l,,, 10 .... 5 0 0 2 4 6 8 10 Days Figure 4-3 Electrolyte leakage from mesocarp tissues of intact ripe Galia fruit with and without 1-MCP and fresh-cut fruit derived from intact ripe fruit treated with and without 1-MCP during storage at 5 C. Vertical bars represent standard deviation of the means (n = 5)

PAGE 93

79 30 25 c..: 20 "';' I:){) .::a:: I:){) -TFresh-cut control 15 i< = ---0Intact 1-MCP -?Fresh-cut 1-MCP e .5 10 f i= Q =-5 0 0 2 4 6 8 10 Days Figure 4-4 Pectin efflux from mesocarp tissues of intact ripe Galia fruit with and without 1-MCP and fresh-cut fruit derived from intact ripe fruit treated with and without 1-MCP during storage at 5 C. Vertical bars represent standard deviation of the means (n = 5)

PAGE 94

80 75 ------------------------80 "S.o C 55 --Intact control A 50 -----------oIntact 1-MCP -TFresh-cut control 60 ...... --...--------, -vFresh-cut 1-MCP 50 40 = :c 30 B D 75 ... 70 ..:i "' 65 C 60 :J 55 ________ .._ 50 ---------40 E 35 30 "S.o C 25 = 20 :C 15 20 ---------------' JOO ....... ----.--...--..---....-----. ._ _____________ 10 95 90 E e 85 .c U 80 75 C 70 ....., ___________ __, 0 2 4 6 8 10 Days 0 F 2 4 6 8 JO 120 115 110 105 100 <:IS E 0 i.. .c u Figure 4-5 Color parameters L lightness (A), hue angle (B) and chroma (C) for ripe Galia' fruit skin treated with and without 1-MCP (I L L l) and the color parameters (D E and F) for intact ripe fruit with and without 1-MCP and fresh-cut fruit derived from intact ripe fruit treated with and without 1-MCP during storage at 5 C. Vertical bars represent standard de v iation of the means (n = 5)

PAGE 95

60 50 ,-.., = 40 --ell = 30 ,:,: 0 "' i.. 20 .... ,:,: 10 0 5 4 3 0 u rJJ 2 1 81 A --------Inatc control -o--Intact 1-MCP Fresh-cut control ---'vFresh-cut 1-MCP B 0 -L--...-------------~P"----.,.--------' 0 2 4 6 8 10 Days Figure 4-6. Mesocarp water soaking percentage (A) and sensory evaluation (B) for intact ripe 'Galia' fruit with and without 1-MCP and fresh-cut fruit derived from intact ripe fruit treated with and without 1-MCP during storage at 5 C. Vertical bars represent standard deviation of the means (n = 5). When bars absent the value for standard deviations was within the dimension of the symbol.

PAGE 96

82 Figure 4-7. Ripe fresh-cut Galia' fruit derived from intact ripe fruit treated with (FC-1MCP) and without 1-MCP (FC-CNT) and then stored for 4 days at 5 C.

PAGE 97

83 Figure 4-8. Ripe fresh-cut 'Galia' fruit derived from intact ripe fruit treated with (FC MCP) and without 1-MCP (FC-CNT) and then stored for 10 days at 5 C.

PAGE 98

84 Table 4-1. Microbial counts (CFU g1 fresh weight) for intact ripe Galia' fruit with and without 1-MCP and fresh-cut fruit derived from intact ripe fruit treated with and without 1-MCP during storage at 5 C. IF-CNT, intact control ; IF-MCP, intact control with 1-MCP; FC-CNT, fresh-cut control fruit without 1-MCP ; and FC-MCP, fresh-cut fruit derived from intact 1-MCP-treated fruit. Total aerobic count Total coliforms Dai: Dai: Treatment 0 5 10 Treatment 0 5 10 IF-CNT 4.7 a 0 7 b 8 0 C IF-CNT 0 a 0 a 3 3 a IF-MCP 0 a O b 11.3 C IF-MCP 0 a 0 a 3 3 a FC-CNT 6.0 a 2 0 b J 8 X 10 3 a FC-CNT O a 0 a 5 3 a FC-MCP 6 0 a 15 3 a 1 5 X 10 3 b FC-MCP O a O a 4 0 a Enterobacteriaceae Lactic acid bacteria 0 5 10 0 5 10 IF-CNT 0 a O b 0 7 C lF-CNT 0 7 a 0 7 a 6 7 b IF-MCP 0 a O b 4 7 C IF-MCP 0 7 a 0 a 22 7 a FC-CNT 0 a 26 0 a 86 7 a FC-CNT 0 a 0.7 a 6 7 b FC-MCP 0 a O a 52.7 b FC-MCP 4 7 a 2 0 a 22 0 a Yeasts Other fungi 0 5 10 0 5 10 IF-CNT 0 a O a 1.3 b IF-CNT 0 a 0 a 2.7 a IF-MCP 0 a 0 a 2 0 b IF-MCP 0 a 0 a 1 3 a FC-CNT 0 a 3 3 a I Ix 10 3 a FC-CNT 2 0 a 0 a 0 a FC-MCP 0 a 0 a l l x l 0 3 a FC-MCP 0 7 a 0.7 a 4.0 a Means (n = 3) followed by the same letter within a column are not significantly different P :S 0.05

PAGE 99

CHAPTER 5 STORAGE LIFE EXTENTION OF PRE-RIPE AND RIPE SUNRISE SOLO' PAPAYA FRUIT BY l-METHYLCYCLOPROPENE Introduction The storage life of papaya fruit under tropical conditions (30 C) is limited due to their high respiration rate, delicate skin, and high water content (Sankat and Maharaj, 1997) Papaya fruit harvested at the color break stage can be kept for periods of up to 16 days at 10 to 16 C (Sankat and Maharaj, 1997) Furthermore papaya fruit can be harvested at the mature green stage ; however according to Hawaiian grade standards the fruit must have at least 6 % surface yellow coloration to meet the minimum grade requirement of 11.5% soluble solids (Akamine and Goo I 971 ) For local markets papaya fruit are harvested at the full-ripe stage (full yellowish-orange surface coloration ; Morton 1987) and stored at temperatures above 7 C. Storage below 7 to IO C may cause low-temperature injuries depending on the variety and maturit y stage (Paull and Chen, 1983; Sankat and Maharaj 1997). Approaches to extending the post harvest quality and duration of the chill-sensitive papaya have included the use of controlled-atmosphere storage polymeric films and wax coating and gamma irradiation (Sankat and Maharaj 1997) For example Maharaj and Sankat (1988) reported that papaya fruit harvested at the color break stage and stored under controlled atmosphere conditions of 1 5 to 2% 0 2 and 5% CO 2 at 16 C remained acceptable for 17 to 29 days Another more facile approach to extending the storage life and quality of harvested papaya fruit has been through the application of I85

PAGE 100

86 methylcyclopropene ( 1-MCP), a potent anti-ethylene compound (Sisler and Serek, 1997). In recent years some very effective agents for blocking ethylene action have been discovered by Sisler and coworkers, and four of them are extensively used in scientific investigation : 2 5-norbornadiene tran s cyclooctene diazocyclopentadiene and 1-MCP (Sisler and Serek, 1999) Silver thiosulphate is used widely as commercial non-organic ethylene action inhibitor for cut flowers potted plants (Sisler and Serek 1997). Since 1MCP has no detectable odor minimal phototoxic properties, and is stable and active at very low concentrations, it has been the favored compound among the inhibitors of ethylene responses (Sisler and Serek 1997 ; Sisler and Serek 1999). Recently it is or has been reported that 1-MCP improved the storage life and qualit y of fruits treated prior to or during ripening including apple (Fan et al. 199 9; Pre-A y mard et al. 2002) and tomato (Wills and Ku 2002 ; Hoeberichts et al. 2002) 1-MCP (90 or 270 nL L1 ) extended the storage life of Sunrise Solo papa y a fruit treated at an early stage of ripening from 4 to 6 days at 20 C (Jacomino et al. 2002). Furthermore 25 L L1 1-MCP treatment increased the number of days to ripening from approximatel y 5 to 20 days for Solo fruit treated at commercial harvest maturity (Hofman et al. 200 I) The objective of this study was to examine the ph y siolo g ical responses and qualit y of papaya fruit treated withl-MCP at the pre-ripe and full-ripe stages of maturation Materials and Methods Plant Material Papaya fruit ( C ari c a papa y a L. var. Sunrise Solo ) originating from Belize (no thermal or wax treatment) and were obtained from Brooks Tropicals Inc Homestead FL. Sunrise Solo variety was chosen due to its year-round availability After transfer to the postharvest facilities in Gainesville fruit were selected on the basis of uniformity of

PAGE 101

87 size, freedom from defects, and graded according to surface color. Fruit at the stage of pre-ripe (PRP ; 10 to 20% surface yellow coloration) and ripe (RP ; 70 to 80% surface yellow coloration) were employed in these studies The fruit were gently brushed and washed with tap water, dipped in 200 L L1 chlorinated water for 1 min, and then rinsed with tap water and dried 1-MCP Preparation and Treatment Three g EthylBloc powder (Floralife Inc Walterboro, SC) were dissolved in 50 mL deionized water in a 136-mL glass vial and sealed with a septum The vial was placed on an oscillating shaker for 2 h. 1-MCP concentration in the vial headspace was measured using a gas chromatograph (Hewlett Packard-5890, Avondale PA) equipped with an SP-1700 column (Supelco Bellefonte, PA). Injector, oven and detector (FID) were maintained at 150, 150 and 200 C respectively Isobutylene gas that has a FID detector response similar to that of 1-MCP (Jiang et al. 1999) was used as an external standard Three g EthylBloc powder in 50 mL deionized water generated 1 -MCP levels in the 137-ml vial of approximately 27 mL L1 Headspace samples (3 3 ml) ofthis gas were injected into sealed 18 9-L buckets having approximately 10-L free space yielding a final 1-MCP concentration of9 L L1 and maintained for a total exposure period of 18 hat 20 C (20 fruits per bucket) At 6-h intervals the treatment containers were vented for 5 minutes, resealed and injected with fresh 1-MCP Air-treated fruit ( 1-MCP free, controls) were maintained under identical storage conditions 1-MCP concentration and efficacy was investigated in a preliminary experiment in which fruit (20 to 30% skin yellowing) were treated with air (control) 0 9 and 9 L L1 1-MCP for 24 hat 20 C and stored at 15 C.

PAGE 102

88 Respiration and Ethylene Production Following the 1-MCP or air treatment, individual fruits (5 fruit/treatments) were sealed in 2-L airtight plastic containers for 1 h CO 2 in 0 5 ml headspace samples was measured using a Gow-Mac GC (Bridge Water, NJ) equipped with a Porapak-Q column. Ethylene in 1 ml headspace samples was measured with a Hewlett Packard-5890 GC equipped with an activated alumina column Firmness Determination Firmness was measured at two equidistant points on the equatorial region of each fruit using an Instron Universal Testing Instrument (Model 44 l 1-C8009 Canton MA) fitted with a 5 kg load cell and an 8-mm convex probe The probe was positioned at zero force contact with the pared fruit surface, and driven to a depth of IO mm at a crosshead speed of 50 mm min1 Firmness data are expressed as the maximum force (N) attained during penetration Electrolyte Efflux Five cylinders of mesocarp tissue were removed from the equatorial region of each fruit using an 8-mm diameter cork borer. From each cylinder one disk (8 mm diameter and thickness) was excised from the centermost portion. Disks (5 per fruit) were rinsed briefly with deionized water to remove loosely adhering tissue and then blotted on moistened Whatman filter paper The five disks were placed in 15 mL of 500 mM mannitol and the initial conductivity of the bathing solution was measured using an YSI3 lA conductivity bridge equipped with a conductivity cell (Model 3403 Yellow Springs OH). The disks and bathing solution were incubated on an oscillating shaker (1.4 cycles per second) at room temperature for 7 hand conductivity of the bathing solution was again measured Total electrolyte content was determined after freezing (24 h at 20

PAGE 103

89 thawing, and heating the disks and bathing solutions in a boiling water bath for 30 min Electrolyte efilux was expressed as a percentage of total tissue electrolytes Soluble Solids Concentration, pH, and Titratable Acidity Frozen fruit samples (80 g) were ground using a mortar and pestle and centrifuged at 27,200 RFC for 10 min at 21 C. Soluble solids concentration (SSC) in the supernatant was determined using a digital refractometer (Abbe Mark-I 0480, Buffalo NY), and titratable acidity (TA) using a Fisher-395 dispenser and 380 electrometer (Pittsburgh PA) Six g of juice were titrated with 0.1 N NaOH to an end point of pH 8 2. TA was calculated from the volume of mL NaOH added and expressed as% malic acid equivalents Statistical and Informal Taste Analyses General linear model program of SAS (SAS institute Carry, NC) and Duncan s multiple range tests were performed for Completely Randomized Designs. Informal taste analyses to determine the edible stage on fruit surface and flesh appearance, odor flavor and texture quality were performed by untrained personnel of the postharvest research group of University of Florida Results Effective 1-MCP Concentration Experiments employing different 1-MCP concentrations (0 9 to 9 L L1 ) were conducted with fruit at 20 to 30% skin yellowing ripening stage to determine the effectiveness of 1-MCP at affecting the ripening metabolism of papaya with emphasis on fruit firmness Firmness retention of the papaya fruit treated with 1-MCP and stored at 15 C was the highest in response to 9 L L1 (5.2 N) followed by 0.9 nL L 1 (5 1 N) and control (4.3 N) respectively at the final day (day 19) of storage (Figure 5-1) Since

PAGE 104

90 firmness retention was of particular interest the present studies were performed using 1MCP at 9 L L1 Respiration and Ethylene Production Respiration showed an increase after day 5 for both treatments, reaching a maximum of23.8 mL ki 1 h1 at day 6 for control and 25.4 mL kg1 h1 at day 8 for 1MCP-treated, representing a 2-day delay in the clim a cteric respiratory peak (Figure 52A) Ethylene production in PRP control fruit increased through day 5 and reached a peak of 1 81 L ki 1 h1 at day 5 while in 1-MCP-treated fruit remained unchanged during the first 4 days of storage thereafter exhibiting a slow, continuous increase through day IO (Figure 5-2A) Maximum ethylene p ro duction of PRP 1-MCP-treated fruit remained below 1.5 L ki 1 h1 ; however, as evident in Figure 5-2A, a clear ethylene climacteric peak was not observed. The onset of the e thylene rise and the maximum ethylene production for PRP fruit treated with 1-MCP was delayed 3 and 5 days respectively compared with the control. Both control and 1-MCP-treated RP fruit exhibited nearly linear trends for respiration during storage remaining under 25 ml kg1 h1 (Figure 5-28) The respiration of RP 1-MCP-treated fruit averaged about 20 to 25% lower than control fruit during the 6to 8-day storage period. Ethylene production of fruit treated with 1-MCP at the RP stage was significantly reduced by 1-MCP through the initial 4 d of storage thereafter attaining values comparable to the control (Figure 5-28) ln RP control fruit ethylene production remained nearly constant during storage. Consistent with the fact that RP fruit were post-climacteric at the start of the experiment, ethylene production during storage did not exceed 1 L kg1 h 1 In contrast, ethylene production in both control and fruit

PAGE 105

91 treated with 1-MCP at the PRP stage reached maximum values of between 1 5 and 1 9 L kg" 1 h1 Mesocarp Firmness Mesocarp firmness of PRP control and 1-MCP-treated fruit declined through day 5 (Figure 5-3A) Thereafter, control fruit continued to soften while the firmness of the 1MCP-treated fruit remained relatively constant through day 8 The firmness of PRP control fruit declined 52% (from 14.1 to 6.8 N) within 9 days of storage compared with 30% (from 15 1 to I 0 5) for 1-MCP-treated fruit over the same time period The firmness ofPRP 1-MCP-treated fruit (8 8 N) at day 11 was comparable in magnitude to that of control fruit (8.1 N) at day 5. After 11 days PRP 1-MCP-treated fruit retained about 58% of their original firmness The consistent but insignificant differences in firmness at the first measurement (day I) likely reflects a slight divergence in firmness values during the 24 h 1-MCP treatment during which time fruit were held at 20 C. Firmness of papaya fruit treated with and without 1-MCP at the RP stage gradually declined but the decrease was significantly attenuated in the 1-MCP-treated fruit (Figure 5-3B) Within 2 days the firmness of RP control fruit had declined about 22% (from 5. 7 to 4.4 N); thereafter, the rate of softening declined through day 5 ( 4 09 N) with the rate again increasing through day 6 (2 91 N) at which time RP control fruit had lost approximately 50% of their initial firmness values Firmness loss in 1-MCP-treated fruit was only about 15% After 8 days fruit treated with 1-MCP at the RP stage retained 84% of their initial firmness (5. 76 N at day I 4 81 N at day 8) Electrolyte Efflux Electrolyte effiux (% of total) of PRP control and 1-MCP-treated fruit gradually increased during storage (Figure 5-4A) ; however in neither treatment were trends

PAGE 106

92 indicative of significant leakage increases during ripening Between PRP control and 1MCP-treated fruit, total electrolyte leakage ranged from about 17 to 22% Even so, PRP 1MCP-treated fruit displayed statistically lower leakage values from days 5 to 9. Electrolyte leakage of RP fruit treated with or without 1-MCP showed no clear trend during storage and no differences between the treatments (Figure 5-48) Soluble Solids Concentrations, Titratable Acidity and pH Soluble solids concentration of PRP control and 1-MCP-treated fruit displayed similar trends during storage and no significant differences were noted between treatments (Figure 5-5A). The trend of SSC in RP fruit showed a similar pattern between control and 1-MCP-treated fruit (Figure 5-58) TA of both PRP control and 1-MCP treated fruit gradually increased until day 7, followed by a slight decline in control fruit and little further change in 1-MCP treated fruit (Figure 5-6A) At 5 days of storage and thereafter, the TA values were significantly higher in PRP 1-MCP treated fruit compared with control fruit. TA of RP control and 1-MCP-treated fruit also rose until day 4, and then decreased The TA of RP control was significantly higher than that of RP 1-MCP treated fruit during most of storage (Figure 5-68) PRP 1-MCP-treated fruit had significantly higher pH values relative to control fruit after day 1 of storage through day 9; however the magnitude of the differences was unremarkable with both treatments displaying pH values near 5 (Figure 57 A) In RP fruit pH gradually decreased though not significantly from values near 5 0 on day I to values from 4 77 to 4.87 on day 6 (Figure 57B) Fruit Evaluation Based on informal quality analysis assessed from peel and pulp color aroma, texture, and flavor (O Connor-Shaw et al. 1994) by untrained laboratory personnel, the

PAGE 107

93 period of table-ripe edibility (fruit suitable for consumption) persisted from days 4 through 7 for PR control fruit and on days 6 through 10 for 1-MCP-treated fruit representing an average shelf-life extension of 25 % RP control fruit were edible through 3 days of storage whereas fruit treated with 1-MCP at the RP stage were still edible on day 6, representing a doubling of useful shelf-life or table-ripe edibility 1-MCP delayed the surface color change from green to yellow in both PRP and RP fruit treated with 1MCP (Figure 5-8 and 5-9). No external decay was evident in either PRP control or 1MCP-treated fruit until day 7 ; thereafter some fruit displayed external decay. Five of 30 PRP control fruit (16 6%) and 3 of30 PRP 1-MCP-treated fruit (10%) were removed form the experiment due to decay throughout storage Decay was primarily evident as stem-end rot which becomes more prominent in papaya as the fruit ripen (Al v arez and Nishijima 1987). Decay incidence in RP fruit was negligible during storage Discussion The response of either PRP or RP Sunrise Solo fruit to 9 L L 1 1-MCP was significant for most measured parameters In a much lower concentration (90 or 270 nL L1 1-MCP 12 hat 20 Sunrise Solo fruit at the breaker stage and pre-climacteric stage responded to 1-MCP by extending their storage life from 4 to 6 and from 2 to 4 days respectively at 20 C (Jacomino et al. 2002) Hofman et al. (2001) reported that Solo fruit treated with 25 L C 1 1-MCP (14 hat 20 C) at commercial harvest maturity increased the days to reach the edible soft stage from about 5 to 20 days at 20 C. Hofman et al. (2001) also speculated that effective 1-MCP concentrations might be lower than 25 L L1 In our findings the storage life of PRP papaya treated with 9 L L1 1-MCP was 11 days while the control 9 days Thus in terms of shelf-life extension the response of papaya fruit to 1-MCP appears to be clo s ely linked to concentration and appears to reflect

PAGE 108

94 a high saturation level for maximum Wills and Ku (2002) reported that higher concentration of 1-MCP (20 or 100 L L1 ) achieved a greater increase in postharvest life ofripe tomato fruit at 20 C relative lower 1-MCP concentrations ( 1 5 or 10 L L1 ) The maximum response of 1-MCP to papaya fruit in the upper concentration range (over 1 L L1 ) might be related to features of the ethylene receptors : saturation of the ethylene receptors either requires higher concentrations of 1-MCP or exposures longer than 24 h. PRP Sunrise Solo' fruit displayed a typical climacteric pattern with a peak respiration rate of23 8 mL kg1 h1 and ethylene production rate of 1.81 L kg1 h1 at 20 0 C. These values are in general agreement with those of Paull and Chen (1997) who reported similar respiration rates (approximately 20 mL kg 1 h1 ) and similar or slightly higher ethylene production (1 to 4 L kg1 h1 ) in Sunset papaya at the color break stage at 22 C. In the papaya cultivar Solo the maximum respiration and ethylene production were documented at 71.5 mL ki 1 h1 and 5 5 L kg1 h 1 at 20 C (Wills and Widjanarko 1995) Thus, the present data for respiration and ethylene production confirm previously published results for respiration and ethylene production of papaya fruit and further report on the ethylene and respiration responses of 1-MCP-treated papaya fruit at the ripe stage The onset of climacteric respiration and ethylene production in PRP fruit were significantly delayed by 1-MCP Ethylene production of PRP papaya fruit treated with 1MCP was delayed significantly reaching production levels comparable to control fruit after about 10 days of storage The recovery in ethylene production may be due to formation of new ethylene binding sites (Sisler and Serek 1997) or release of 1-MCP from the receptors Other explanations include the non-permanence dissociation of 1

PAGE 109

95 MCP from ethylene receptors, competition with ethylene and potential binding of 1MCP to receptors showing homology with the ethylene receptors (Able et al., 2002). Able et al. (2002) reported that multiple applications of 1-MCP had no further impact on storage life of broccoli florets Delayed climacteric ethylene production and respiratory in PRP fruit in response to 1-MCP has been noted for various fruits treated before the onset of ripening including banana (Jiang et al., 1999 ; I L L1 24 hat 24 C) at 20 C and avocado (Jeong et al., 2002; 0.45 L L1 24 hat 20 C) at 20 C. 1-MCP suppressed both respiration and ethylene production in RP papaya fruit. Ethylene production of RP 1-MCP-treated papaya fruit recovered to control values after 5 days whereas respiration remained suppressed Similarly Golden Delicious apple (Jiang and Joyce, 2002) and tomato (Wills and Ku 2002) fruits treated with 1-MCP at a late stage of ripening displayed reduced ethylene production and respiration compared with non 1-MCP-treated fruit. The delayed respiratory climacteric peak in PRP and persistent respiration in RP papaya fruit treated with 1-MCP may imply that ethylene and respiration are not tightly linked during papaya fruit ripening ln detached tomato (Saltveit, 1993) and muskmelon (Bower et al., 2002) fruits, respiratory climacteric has been found be not an essential part of ripening. Treatment with 1-MCP delayed softening in both PRP and RP papaya which indicates that softening in papaya fruit is dependent upon ethylene action throughout the ripening process Lelievre et al. (1997) stated that softening of climacteric fruits is regulated by ethylene Electrolyte efllux, an indication of membrane damage in senescing tissues (Marangoni et al., 1996) increased slightly in PRP papaya fruit but in not PR fruit during storage. Similar results regarding increased electrolyte efllux during papaya fruit

PAGE 110

96 npenmg was reported by Chan et al. ( 1985) who found that papaya fruit (harvested at the color break stage) initially stored at 10 C followed by storage at 24 C showed a significant increase in electrolyte leakage. 1-MCP significantly suppressed the increase in electrolyte leakage in PRP papaya fruit, which may serve as evidence that ethylene action is involved in mechanisms contributing to membrane catabolism (Kuo and Parkin, 1989; Faragher et al., 1986) 1-MCP had no influence on soluble solids concentration levels in either PRP or RP papaya fruit indicating that ethylene responsiveness is not involved in soluble solids accumulation in ripening papaya fruit. Hofman et al. (200 I) documented a small but significant increase in soluble solids of Solo papaya treated with 1-MCP (control 10.09%; 1-MCP, 11.47) at the edible soft stage The control Solo' fruit reached the eating stage in 4 to 5 days, while 1-MCP-treated fruit required nearly in 21 days which may explain the small but significance differences noted in their studies. Treatment of Delicious and Fuji apple with 1-MCP (0.8 to I L L1 for 12 to 16 hat 20 to 24 C) caused higher SSC compared to control at 0 C after 6 to 7 months (Fan et al. 1999) treatment of Elberta peach harvested pre-climacteric period with 1-MCP (0 5 mL L1 for 4h at 20 C) resulted in higher SSC compared to control at 20 C (Fan et al. 2002) as well. Ethylene balance could determine the way tissues respond to changes in soluble solid concentration, for example partitioning carbohydrate into metabolism or into storage. In muskmelon fruit a diminution of biosynthetic enzymatic activities sucrose synthesis and an increase invertase acid activity attributed to ethylene level (Hubbard et al. 1989) which further supports the idea of ethylene involvement in soluble solid accumulation

PAGE 111

97 The results indicate that 1-MCP suppressed the increase in TA in papaya fruit regardless of ripening stage These finding are in contrast to reports where the TA of fruits including Gala' apple (Fan et al., 2001; 0 5 L L1 23 hat 20 C) at 20 C and Royal Zee plum (Dong et al., 2002 ; I L kg1 h1 20 hat 20 C) at 0 C was enhanced by 1-MCP when applied at an early stage of ripening, and where the decrease in TA of tomato (Wills and Ku, 2002) and Anna' apple (Pre-Aymard et al. 2002 ; 0.1 or I L ki 1 h 1 1-MCP, 24 h, room temperature) was either suppressed or unaffected by 1-MCP when applied at a late stage of ripening. Flores et al. (2001) reported that aminocyclopropene-1-carboxylic acid oxidase antisense muskmelon displayed higher citric acid compared to wild type. The result from PRP and RP papaya fruit and others cited above indicate that ethylene may involve directly or indirectly organic acid metabolism The surface color change from green to yellow in both PRP and RP papaya fruit was delayed by l-MCP Similar results from fruits treated with 1-MCP at an early stage of ripening have been reported for banana (Golding et al. 1998; 45 L L 1 1-MCP, for 1 hat the room temperature) and avocado fruits (Jeong et al., 2002) Pre-Aymard et al. (2002) found that 1-MCP delayed the surface color change from green to yellow in Anna apple treated with 1-MCP late in ripening The delayed color change from green to yellow in papaya fruit treated with 1-MCP supports the conclusion of Lelievre et al. (1997) who documented that color production can be either ethylene-dependent or independent according to the type pigments and the fruit species. 1-MCP significantly extended the acceptable edible period of papaya fruit irrespective of stage of maturity at the time of treatment. PRP control fruit reached the

PAGE 112

98 acceptable edible stage at day 4 that persisted through day 7 while PRP control attained an edible condition at day 6 persisting through day 10 and a 25-percent increase in response to 1-MCP. The edible stage ofRP fruit lasted 3 days while RP-1-MCP-treated fruit 6 days indicating a 3-day extension in storage life Jacornino et al. (2002) found papaya fruit treated with 1-MCP at the color break stage lasted longer compared with control fruit (control, 4 days ; 1-MCP 6 days). Additionally Hofman et al. (2001) reported that 1-MCP treatment increased the number of days to the ripe stage defined as the time at which fruit attained firmness readings of 5 to 7 N of Solo papa y a-treated at commercial maturity (mature-green stage) by 325% (from 4.8 to 20.6 days) l-MCP-treated PRP fruit showed less decay compared with non-1-MCP-treated PRP fruit. During storage 16 6% of PRP control fruit were eliminated from the experiment due to decay while onl y 10 % of PRP 1-MCP Stern-end rot which can develop rapidly in ripe fruit was the primar y indication of decay (Alvarez and Nishijima 1987). The reduced incidence of decay in fruit treated with 1-MCP at the PRP stage might be due to a consequence of the reduced rate of ripening Decay incidence was unremarkably low in either RP control or RP 1-MCP-treated fruit probably due to the fact that RP fruit had been selected based on external quality prior to the 1-MCP treatment. Hofman et al. (2002) noted that Solo fruit treated with 25 L L1 1-MCP at the commercial maturity stage showed higher disease symptoms (stem black rots, body black rots and anthracnose) after approximately 20 days of storage than non-1-MCP treated fruit after 4 to 5 days of storage at 20 C. The authors speculated that delaying ripening by 1-MCP may result in fruit close to full ripe stage having lower concentrations of endogenous anti fungal compounds and higher incidence of decay

PAGE 113

99 1-MCP has been reported to reduce the rate of over-ripening by delaying firmness loss and color change in Anna (Pre-Aymard et al. 2002) and Golden Delicious' (Jiang and Joyce 2002) apple and by extending postharvest storage life in the tomato fruit (Wills and Ku 2002) Coriander leaf senescence as assessed in terms of chlorophyll and protein loss was significantly retarded by 1-MCP (Jiang et al. 2002) Furthermore earlier work demonstrated that silver thiosulphate arrested tomato ripening (Tucker and Brady 1987; Smith et al. 1989). 2 5-norbornadiene a competitive ethylene action inhibitor has been reported to interrupt petal sen es cence of carnation flower a s well (Peiser, 1989 ; Wang and Woodson 1989) The results from RP Sunrise Solo fruit show quite conclusively that ethylene is required throughout the ripening process (Tucker and Brady 1987 ; Smith et al. 1989 ; Hoeberichts et al. 2002) In conclusion our results indicate that inhibition of ethylene action in Sunrise Solo' papaya fruit by 9 L L1 1-MCP is sufficient to extend the table-ripe edibility at either the pre-ripe or ripe stage by 25 % and I 00 % at 20 C re s pectivel y. Use of 1-MCP in combination with low temperature and controlled atmosphere storage has potential to extend storage life of papaya fruit for a longer period. In consideration of the perishable nature and inherentl y short storage life of papaya and other tropical fruits 1-MCP treatment should provide a number of alterna t ive h a ndling and shipping options These data should be of considerable interest to the papa y a and other tropical fruit industries which is a tremendous development for tropical fruit industry

PAGE 114

100 10 8 6 4 --Control 2 -00.9 L L1 1-MCP ---T9 L L 1 l-MCP 0 -..... -------------..... -------.-----..... 1 3 5 7 9 11 13 15 17 19 Days Figure 5-1. Mesocarp firmness for Sunrise Solo fruit treated with air (control) 0 9 L L1 and 9 L L1 1-MCP at 20 to 30% skin yellowing ripening stage and subsequently stored for 19 days at 15 C. Vertical bars represent standard deviations of the means (n = 5).

PAGE 115

IOI 30 5 A 25 4 20 -+-Control C 2 3 15 -o1-MCP CO2 _._ Control C 2 H 4 2 10 --t::.-1-MCP c 2 1 5 :.c: :.c: -;-;el) 0 0 el) .:i:: .:i:: .J 30 5 .J e :::1. B 's:t N o= 0 25 4 N u u 20 3 15 2 10 5 1 0 0 0 2 4 6 8 IO 12 Days Figure 5-2 Respiration and ethylene production for Sunrise Solo fruit treated with air (control) and 9 L L-1 1-MCP at the pre-ripe (A) and ripe (B) stage and subsequently stored at 20 C. Vertical bars represent standard deviation of the means (n = 5).

PAGE 116

102 21 A 18 _._ Control ---01-MCP 15 12 9 6 z 3 = e 10 .!:: B 8 6 4 2 0 _,_ ___ ....,. ____ ,.... ___ ....., ____ ..,... ___ ....., ___ ____,, 0 2 4 6 8 10 12 Days Figure 5-3. Mesocarp firmness for Sunrise Solo fruit treated with air (control) and 9 L L1 1-MCP at the pre-ripe (A) and ripe (B) stage and subsequently stored at 20 0 C. Vertical bars represent standard deviation of the means (n = 5)

PAGE 117

28 26 24 22 20 ,_ 18 = Q,I 16 .:a:: ] 14 28 0 I. 26 C.I Q,I &1 24 22 20 18 16 14 0 _._ Control -01-MCP 2 4 103 6 Days A B 8 10 12 Figure 5-4 Electrolyte leakage for Sunrise Solo fruit treated with air (control) and 9 L L1 1-MCP at the pre-ripe (A) and ripe (B) stage and subsequently stored at 20 0 C. Vertical bars represent standard deviation of the means (n = 5)

PAGE 118

C u rJ). rJ). 104 15 --------------------------A -----Control 14 -o1-MCP 13 12 11 15 B 14 13 12 11 10 -,...----,.----....------,.----.,....-----,.---___,, 0 2 4 6 Days 8 10 12 Figure 5-5. Soluble solids concentration (SSC) for 'S unrise Solo' fruit treated with air (control) and 9 L L1 1-MCP at the pre-ripe (A) and ripe (B) stage and subsequently stored at 20 C. Vertical bars represent standard de viatio n of the means (n = 5).

PAGE 119

= .... .s::: "C ;; < .... lo, .... 105 0.18 --------------------------A 0.16 _._ Control ---01-MCP 0.14 0.12 0.10 0.18 B 0.16 0.14 0.12 0.10 0.08 ...,_ ___ ...,. ____________ ....., ____ .,.... ___ ..., 0 2 4 6 Days 8 10 12 Figure 5-6. Titratable acidity for Sunrise Solo fruit treated with air (control) and 9 LL1 1-MCP at the pre-ripe (A) and ripe (B) stage and subsequently stored at 20 0 C. Vertical bars represent standard deviation of the means (n = 5)

PAGE 120

106 5.2 A 5.1 5.0 4.9 4.8 4.7 --Control -o1-MCP 4.6 = 5.2 C. 5.1 8 5.0 4.9 4.8 4.7 4.6 4.5 0 2 4 6 8 10 12 Days Figure 5-7. The pH for Sunrise Solo fruit treated with air (control) and 9 LL' 1-MCP at the pre-ripe (A) and ripe (B) stage and subsequently stored at 20 C. Vertical bars represent standard deviation of the means (n = 5). When bars absent the value for standard deviations was within the dimension of the symbol.

PAGE 121

107 Figure 5-8 Pre-ripe 'Sunrise Solo' fruit treated with 9 L L1 1-MCP or air (control) and then stored for 7 days at 20 C.

PAGE 122

108 Figure 5-9. Ripe 'Sunrise Solo fruit treated with 9 L L1 1-MCP or air (control) and then stored for 3 days at 20 C.

PAGE 123

CHAPTER6 QUALITY AND STORAGE LIFE OF INT ACT AND FRESH-CUT PAP A YA FRUIT TREATED WITH 1-MCP AT THE POSTCLIMACTERIC STAGE OF DEVELOPMENT Introduction Fresh fruit processing described as cutting, slicing dicing peeling trimming of agricultural commodities in a fresh-like stage (O'Connor-Shaw et al, 1994) has increased in popularity over the past 9 years and continues to increase in popularity along with fresh produce in general (International Fresh-cut Produce Association 2002) In the United States the sales of fresh-cut fruits and vegetables have increased from $3 3 billion in 1994 to $11 billion in 2000 with sales projected to increase to $15 billion in 2005 (International Fresh-cut Produce Association, 2002) Due to the extensive tissue damage involved in fresh-cut processing fresh-cut produce can deteriorate rapidly becoming unacceptable in a few days (Rolle and Chism, 1987; King and Bolin 1989) This represents a dramatic loss in storage life compared with the intact commodity and illustrates the need to explore alternative procedures for prolonging the storage life of fresh-cut fruits and vegetables The inherent high perishability of certain commodities including ripe tropical fruits is particularly problematic and they are high-priority commodities to be explored as fresh-cut products For example, papaya fruit are very susceptible to mechanical damage and diseases and papaya fruit harvested at the mature green stage typically exhibit a storage life of less than one week under ambient tropical conditions (Sankat and Maharaj 1987) For local markets papaya fruit are harvested at the full-ripe stage (full yellowish-orange surface coloration ; Morton 1987) and stored at 109

PAGE 124

110 temperatures above 7 C. Storage below 7 to IO C may cause low-temperature injuries depending on the variety and maturity stage (Paull and Chen, 1983 ; Sankat and Maharaj 1997) Since papaya fruit have a limited shelf life, their fresh-cut products also exhibit a short storage life depending on temperature and maturity stage ranging from 2 to 7 days at 3, 4 or 6 C (O'Connor-Shaw et al. 1994; Teixeira et al., 2001) Paull and Chen (1997) reported that Solo' papaya fruit at 55-80% skin yellowing was judged as the best stage for fresh-cut processing based on flesh firmness edible flesh and seed separation from placenta Wounding due to fresh-cut processing can induce certain physiological and biochemical changes that result in a reduction in storage life of For instance ethylene production increases significantly in many fresh-cut fruits and vegetables (Rolle and Chism 1987) Another consequence of wounding is increased susceptibility to pathogenic microorganisms (Varoquaux and Wiley 1994) most likely caused by removal of protective outer tissues. The tissue response to microorganisms may also contribute to fermentative alcohol or lactic acid production (Varoquaux and Wiley 1994) which may contribute to the reduction in storage life of fresh-cut produce The exact role or consequence of the increased ethylene production in fresh-cut commodities is not known, but in view of the requirement for continued eth y lene responsiveness throughout ripening (Watada et al. 1990) it seems logical to assume that fresh-cut fruits should also respond to the gas If ethylene does pla y a role either adverse or beneficial, in the deterioration of fresh-cut produce, effective inhibitors of ethylene action, including 1-methylcyclopropene (Sisler and Serek 1997) provide excellent tools for addressing these questions. 1-MCP is a volatile cyclic olefin, effective in the ppb to

PAGE 125

111 ppm range and leaving undetectable residual levels in fumigated tissues (Sisler and Serek, 1997) 1-MCP has been reported to reduce ethylene responses and improve the storage life of fruits at advanced stages of ripening including apple (Mir et al. 2001; Pre Aymard et al. 2002 ; Jiang and Joyce 2002) and tomato (Wills and Ku, 2002 ; Hoeberichts et al. 2002). Since fresh-cut fruit will typically not continue to ripen post processing, in large part due to the low temperatures employed for storage, optimum quality can be achieved only with fruits that are nearly ripe at the time of processing. The objectives of the current study were to determine the storage Ii fe of fresh-cut papaya tissue derived from fruit treated with 1-MCP at the full-ripe, postclimacteric stage of development. Materials and Methods Plant Material Papaya ( C ari c apapaya, L var Sunrise Solo ) fruit originating from Brazil (no thermal and wax treatment) were obtained from C-Brand Tropicals Inc ., Homestead, FL Fruit were transferred to the postharvest storage facilities in Gainesville FL on the day of receipt and stored at 20 C for 1 day The fruit were then selected on the basis of uniformity of size and freedom from defects and graded according to surface color as an estimate of ripeness Afterward the fruit were gentl y brushed washed with tap water immersed in 200 L L" 1 chlorinated water for I min dried and transferred 20 C. Fruit at the postclimacteric stage (based on data from chapter 5 ; approximately 70 to 80% surface yellow coloration) were employed in this stud y The firmness ethylene production and respiration of the fruit measured at 20 C immediately prior to 1-MCP treatment was 6 8 N 0.8 L kg 1 h 1 and 22 mL kg 1 h 1 respectively

PAGE 126

112 1-MCP Treatment 1-MCP as Agrofresh commercial powder (active ingredient 0 14% 1-MCP) was acquired from the manufacturer (Agrofresh, Philadelphia PA). Three g of Agrofresh powder were placed in a 136-mL glass vial along with 50 mL of deionized water. The vial was sealed with a septum and incubated on an oscillating shaker for 2 h The concentration of 1-MCP in the vial headspace was measured using a gas chromatograph (Hewlett-Packard; Model 5890 Avondale PA) equipped with a 1/8 80-100 mesh Chromosorb PAW stainless steel column (1.8 m x 3 18 mm i.d .; Supelco Bellefonte PA) Injector oven and detector (FID) were set to 150, 150 and 200 C respectively Isobutylene gas, which has a FID response similar to that of 1-MCP (Jiang et al. 1999) was used as a standard. 1-MCP levels in the stock preparation were approximately 7 500 L L1 in the headspace. Headspace gas sample (3 3 mL) were injected into a 18 9-L plastic airtight bucket having I 0-L void volume (20 fruit per bucket) yielding a 1-MCP concentration of2.5 L L 1 The final 1-MCP concentration of2.5 L L1 was maintained for a total exposure period of 24 h at 20 C. The bucket was vented and reinjected with fresh 1-MCP gas at 6-h intervals Control fruit were sealed in similar containers but without 1-MCP. Fruit Preparation and Treatment Design Papaya fruit following treatment with 1-MCP or air ( control) were transferred to a 5-C room that had been sanitized (200 L L1 -chlorinated water) prior to fresh-cut processing After a I h period at 5 C to allow temperature equilibration both the blossom and pedicel ends of each fruit were removed and the fruit longitudinally cut into 1 5 cm thick slices (from the pedicel end to the stem end) The two outermost slices were peeled and cut into pieces approximating slices with approximate dimensions of 1 5 x

PAGE 127

113 3.5 x 4 cm and weighing 15 to 20 g using a plastic Bread Slicer (Coupe-Pain). Afterward the slices were rinsed quickly with a sterile isotonic mannitol solution (500 mM) using a squeeze bottle, and then placed in I. 7-L vented plastic containers that had built-in grids on the bottom lifts (FridgeSmart Tupperware Co ., St. Paul MN). The treatments included fresh-cut tissue derived from intact fruit pre-treated with air ( control, FCC), fresh-cut tissue derived from intact fruit treated with 2. 5 L L1 1MCP (FCM), intact fruit pre-treated with air (IC) and intact fruit pre-treated with 1-MCP (FCM). At selected intervals during storage at 5 ethylene production, mesocarp firmness, electrolyte leakage, color sensory changes, and microbial growth were measured for both fresh-cut and intact fruit. Ethylene Production Ethylene production was measured by placing individual fruit or fruit slices ( 4 slices per fruit) in 1 9-L and 950-mL plastic containers respectively The containers were sealed for 2 hat 5 C, and C 2 H 4 in the containers was measured using a Hewlett Packard gas chromatograph (5890) equipped with an activated alumina SS column and flame ionization detector at room temperature The carrier gas (Nitrogen) was 30 mL min1 Oven injector and detector temperature was 70 200 and 250 C, respectively Firmness Fruit firmness in mesocarp tissue of intact and fresh-cut papaya, kept in a cooler, was measured with an Instron Universal Testing Instrument (Model 4411 Canton MA) fitted with an 8-mm convex probe and 5-kg load cell at 20 C. Intact fruit prior to firmness measurements were sliced using the procedures described for obtaining fresh cut slices from intact fruit. The probe was placed at zero force contact with the fruit surface, and penetrated to a depth of I 0-mm at a crosshead speed of 50 mm min 1 Data

PAGE 128

114 are reported as the maximum force (Newton) generated during penetration of the tissue slices. Electrolyte Leakage Five cylinders of mesocarp tissue were removed from the equatorial region of each fruit or from a fresh-cut slice using an 8-mm diameter cork borer From each cylinder, one disk (8 mm diameter and 8 mm thickness) was excised from the centermost portion Disks (5 slices per fruit) were rinsed with deionized water and blotted on a slightly moistened Whatman filter paper The disks were then incubated in 15 mL of 500 mM mannitol on an oscillating shaker at room temperature for I h followed by a conductivity measurement of the bathing solution The aliquot removed from the bathing solution for the conductivity measurement was returned to the bathing solution Conductivity was measured using a conductivity bridge (Y-3 1 A Yellow Springs OH) equipped with a conductivity cell (model 3403 Yellow Springs OH) Afterward the bathing solution and disks were stored at -20 C for at least 24 h thawed and placed into a boiling water bath for 30 min, cooled to room temperature and conductivity of the bathing solution was measured again. Electrolyte leakage was expressed as percentage of the total tissue electrolytes estimated from the frozen/heated samples Color and Sensory Evaluation Fruit skin (equatorial region) and flesh (centermost mesocarp) color were assessed as lightness (L ; representing the lightness or grey scale) hue angle (the dimension of color that specifies a position in a color wheel of 3 60 with 90 180 and 270 representing the hues red, yellow green and blue respectively) and chroma (distinguishing the difference from a pure hue to a grey shade) values using a chromameter (Minolta-CR-200, Japan) Informal descriptive analysis by untrained

PAGE 129

115 personnel was used to profile the quality of fresh-cut and intact fruit, evaluating appearance, odor, texture and flavor (O Connor-Shaw et al. 1994) according to the following hedonic scale: I, poor ; 2 poor-good; 3, fair ; 4, good-excellent; and 5 excellent. Intact fruit showing surface-pitting were graded from 0 to 20 by 5% increments, 0 for 0%, l for 5% 2 for I 0%, and so on Water soaking on the flesh was expressed as the percentage of areas of a slice (upright surface) with a 5% interval (total 5 slices for each treatment). Intact fruit expressing water soaking were graded from Oto 20 indicating a 5% increase, 0 for 0%, 1 for 5% and so on. Five intact fruit or five slices of a container from each treatment was evaluated for mesocarp water soaking. Informal descriptive analysis was used to profile the qualit y of either fresh-cut cubes and intact fruit flesh by untrained personnel, evaluating appearance, odor texture and flavor (O'Connor-Shaw et al. 1994) according to the following hedonic chart: I poor ; 2, poor good; 3 fair; 4 good-excellent ; and 5 excellent. The informal descriptive analyses undertaken immediately after removing fresh-cut and intact fruit from the 5-C cold room at days 0,2 ,4 6 8 and 10 at under white light and the room temperature 23 with testing at least 3 samples for each treatment. Microbial Count Fruit tissue (5 g) was removed with a flame-sterilized cork borer (21 5 mm diameter) and knife from innermost part of a fruit or with a flame-sterilized knife from a fruit slice (approximately 1 / 3 to 1 / 4 of a cube) on sterilized aluminum foil in an air circulated fume The fruit tissue then was incubated in a 45-mL sterile phosphate buffered solution (PBS), pH 7. The PBS and fruit tissue were then vortexed at high speeds for I min using a vortex (Fisher-Genie 2 Scientific Industries Inc Bohemia NY) followed by subsequent I 0-fold dilutions using sterile PBS as needed Total

PAGE 130

116 aerobic, Enterobacteriaceae, yeasts and other fungi, total coliforms and lactic acid bacteria counts were made using 1 mL inoculum of the PBS. The plates and incubation conditions for each count were: total aerobic count, 3M Petrifilm aerobic count plate (3M Microbiology Products St. Paul, MN), 3 days at 30 C ; Enterobacteriaceae, 3M Petrifilm Enterobacteriaceae count plate I day at 30 C; yeasts and other fungi 3M Petrifilm yeast and other fungi count plate, 5 days at 25 C; total coliforms 3M Petrifilm coliform count plate, 1 day at 30 C; and lactic acid bacteria, 3M Petrifilm aerobic count plate anaerobic incubation for 2 days at 30 C in a I. 9-L airtight plastic container with a Gas Pak anaerobic system envelope (Becton Dickinson and Co Cockeysville, MD) The plates were prepared in an air circulated hood after 0 (immediately after dicing) 5 and I 0 days at room temperature, and microbial counts were reported as colony forming units per gram of tissue (CFU g1 ) Statistical Analysis General linear model program of SAS (SAS institute Carey NC) and Duncan s multiple range taste were performed for randomized complete block design in which treatments (FCC FCM, IC and IM) were blocks. Results and Discussion Ethylene Production Differences in ethylene production rates (at 5 C) were insignificant among treatments at day I as shown in Figure 6-1 : IC, 38.4; IM, 24 7 ; FCC 31 .5; FCM 3 I .4 nL ki 1 h1 The ethylene production rates increased during storage and reached values of 64.4 (IC), 47.8 (IM), 41.4 (FCC) and 34 7 nL kg1 h1 (FCM) at day 9. The increases in ethylene production rates from day I to 9 were 70% for IC 94% for IM, 32% for FCC and 11% for FCM. The ethylene production of both fresh-cut postclimacteric and intact

PAGE 131

117 postclimacteric Sunrise Solo fruit at 5 C was lower than the values reported by Paull and Chen (1997) for preclimacteric and postclimacteric Sunset papaya cultivar These authors reported that ethylene production of halved, unpeeled and deseeded preclimacteric and postclimacteric papaya fruit was approximately 1 L kg 1 h1 at 4 C. Postclimacteric 'Sunrise Solo' fruit produce less than l L ki 1 h1 ethylene at 20 C (Chapter 5), which is consistent with the observation that Sunrise Solo is a low-ethylene producer. Paull and Chen ( 1997) noted that ethylene production of the halved and deseeded preclimacteric papaya fruit was approximately 5 fold higher than that of intact preclimacteric fruit after 2-day of storage at 22 C. The ethylene production of FCC and FCM did not differ significantly nor did that of IC and IM, however, FCC and FCM had slightly lower ethylene production compare to IC and IM collectively Similarly Artes et al. ( 1999) reported slightly higher ethylene production in fresh-cut preclimacteric tomato fruit relative to intact preclimacteric tomato fruit at 2 C but the authors noted a 5-fold higher ethylene production than intact fruit at 10 C (Artes et al. I 999) The ethylene production of either fresh-cut or intact postcli macteric Sunrise Solo fruit was not affected by 1-MCP at 5 C. However postclimacteric Golden Delicious apple treated with 1-MCP (IO L L1 for 6 hat 20 C) before or after fresh-cut processing showed lower ethylene production measured at 20 compared with fresh cut fruit treated with air at 4 C (Jiang and Joyce 2002). Jacomino et al. (2002) reported that intact preclimacteric Sunrise Solo treated with 1-MCP (90 or 270 nL L1 for 12 hat 20 C) also showed suppressed ethylene production compared to non-1-MCP-treated fruit at 20 C. The very low ethylene production (below 65 L kg 1 h" 1 ) of either fresh-cut or intact 'Sunrise Solo' fruit at 5 C may explain the insignificant ethylene production rates

PAGE 132

118 between Sunrise Solo fruit or slices with 1-MCP and fruit or slices without 1-MCP. Another reason for the insignificant ethylene production of' Sunrise Solo fruit with 1MCP and without 1-MCP might be release of 1-MCP from the receptor sites. Papaya fruit are chill-sensitive; and the storage temperatures used in this study are below the chill threshold for papaya (Chen and Paull, 1986) Storage of either fresh cut or intact papaya at sub-threshold temperatures might perturb the cell membrane system resulting in conformational changes in the ethylene receptors that may influence the tenacity of 1MCP-receptor interactions. Postclimacteric Sunrise Solo fruit treated with 9 LL' 1MCP for 24 h recovered their suppressed ethylene production (measured at 20 C) in 5 days at 20 C (chapter 5), which may support the idea that 1-MCP binding is not irreversible and is possibly released with time Firmness Assessment Mesocarp firmness values for all treatments (IC, IM FCC and FCM) decreased during storage (Figure 6-2) The extent of softening was consistently and significantly higher in the fresh cut compared with the intact fruit. During the 10-day storage period IC softened from 5 6 to 4.1 N (a 26% decline) and lM from 7 1 to 5 9 N (a 15% decline) Firmness of FCC decreased approximately 50%, from 4 7 to 2.4 N during the first 2 days of storage Afterward, the rate of softening of FCC declined with firmness reaching values of 1 22 N after IO days Firmness of fresh cut tissue from 1-MCP-treated fruit (FCM) was significantly retained, declining only 19% (from 5.6 to 4 6 N) during the first 2 days reaching a low of2 6 Nat day 10 FCC lost 74 % of their original firmness during the 10-day storage while FCM declined only 53% Initially FCM had 27% higher firmness than FCC and the percentage of firmness difference between IC and IM

PAGE 133

119 increased 92 % at day 2. At day 10 the firmness value of fruit slices with 1-MCP was mor e 2-fold higher than that of fresh-cut control fruit. The augmented softening of fresh-cut postclimacteric 'Sunrise Solo' papaya fruit tr e ated \ V ith or without 1-MCP might be due to over activation of cell-wall enzymes For example wounding stimulated polygalacturonase, and aand ~-galactosidase activities in fresh-cut 'Sunri s e Solo fruit (at 60 to 70% yellow surface color) compared with intact fruit (Karakurt and Huber, 2002). These enzymes in addition to xylanase (Paull and Chen 1987) might collectively contribute to softening of low-temperature stored fresh cut papaya and of intact papaya fruit during normal ripening as well. 1-MCP delayed or r e duced the rate of softening in both intact and fresh-cut postclimacteric 'Sunrise Solo papa y a which is similar to the case for fresh-cut, postclimacteric 'Golden Apple' treated with 10 L L" 1 1-MCP (6 h at 20 C) before or after slicing and stored at 4 C (Jiang and Joy c e 2002) The delayed softening in intact postclimacteric fruit treated with 1-MCP or fre s h-cut fruit derived from 1-MCP-treated intact postclimacteric fruit suppo1ts the fact that ethylene is involved in the softening of climacteric fruit (Lelievre et al., 1997) even under stress (e.g storage below the critical minimum temperature wounding caused by processing). The deferred softening as a result of 1-MCP treatment has been also reported fo r intact po s tclimacteric 'Redchief Delicious' apple fruit subjected to either one or multiple applications of 1-MCP (0.7 L L 1 for 16 hat 0, 5 10, 15 and 20 C; Mir et al., 2001) and tomato fruit treated with 1-MCP at the orange red stage (50 to 150 nL L1 for 24 hat 20 ; Hoeberichts et al. 2002) at 20 C. Electrolyte Leakage A continuous increase of electrolyte efflux in FCC and FCM tissue was observed during l 0 days of storage at 5 C (Figure 6-3). The increase in electrolyte leakage of FCC

PAGE 134

120 and F CM followed similar trends w1til day 8, after which time leakage of FCC tissue was si g nificantl y higher than that of FCM. Total electrolyte leakage of FCC increased from 15 3 % to 38.8% during the 10-day storage period while FCM increased from 15.3% to 25.0 % In sharp contrast to the fresh-cut tissues intact fruit (IC and IM), showed no changes in ion leakage during storage (Figure 6-3). Between IM and IC electrolyte leakage ranged from lows of 14.0% (IM at day 0), comparable to day 0 fresh cut tissue, to a high of 17 .8% (IM at day 10) over the 10-day period. Electrolyte leakage is considered to be a n indirect measure of cell membrane damage (Marangoni et al. 1996), which may contribute to fruit softening through a loss in cell turgor. Karakmi and Huber (2002) reported that fresh-cut papaya fruit at slightly less ripe stage (60 to 70% yellow color) cau se d incr e ased acti v ities of lipoxygenase and phospholipase D, suggesting that the s e en z yme s may assist in membrane breakdown and, consequently, the more rapid softening of fresh-cut fruit. Increased ion leakage is considered to be a common symptom of chilling injury (CI) in papaya fruit (Chen and Paull 1986). As is true for papaya fruit mo s t chill-sensitive fruits show increased ion leakage in response to prolonged low temp e rature storage (Saltveit, 2000). The effects of 1-M CP on leakage of postcli macteric 'Sunris e Solo' fruit were minimal and significant only for fresh-cut fruit derived from 1MCP treated intact fruit. The suppressed electrolyte leakage of fresh-cut tissue derived from 1-MCP-treated fruit thorough day 10 might be due to the inhibitory effects of 1MCP upon chilling injury. Preclimacteric Charentais melon fruit exposed to 1 L L" 1 1MCP (for 24 h at 22 C) stored at 2 C for 16 days followed by transfer to 22 C for 5 days were insensitive to low-temperature damage (estimated by visually rating the extent of surface pitting and browning) compared to non-1-MCP-treated fruit (Ben-Amor et al.

PAGE 135

121 1999). 1-MCP, furthermore, suppressed chilling injury symptom (internal browning) in pineapple ( I L L1 18 h at 20 C) relative to non-1-MCP-treated fruit stored at 10 C for 4 weeks (Selvarajah et al. 2001) and avocado fruit (100 L L1 for 24 or 48 hat 20 C) (mesocarp discoloration) compared with non-MCP-treated fruit at 5 C after 4 weeks (Pesis et al., 2002) Color and Sensory Evaluation L and hue angle values for IC and IM did not change significantly during 10 days at 5 indicating no visible color changes over fruit surface (Figure 6-4A and 4B). Chroma, however decreased slightly during IO days storage for both IC and TM suggesting that the epidermal color for both treatments became dull (Figure 6-4C). None of the skin color parameters (L hue angle or chroma) in IC and IM papaya were significantly affected by 1-MCP No significant changes in mesocarp color as evaluated by of hue angle, were observed for any of the 4 treatments (IC IM FCC and FCM) during storage (Figure 64E) L and chroma of FCC and FCM however slightly decreased during the 10-day storage implying that the color became duller during storage (Figure 6-4D and 4F) Through the last day of storage ( day I 0) L and chroma of FCC significantly decreased compared to FCM meaning that the loss of color intensit y or purity might have been delayed by 1-MCP (Figure 6-5) A beneficial effect of 1-MCP on the color of fresh-cut fruit was also reported for postclimacteric Golden Delicious apple (Jiang and Joyce 2002) These authors reported that the epidermal tissue of fresh-cut apple treated with 1MCP (1 or 10 L L1 for 6 hat 20 C) before or after cutting wa s greener after 10 days of storage at 4 C than fresh-cut fruit treated with air.

PAGE 136

122 Pitting of the fruit surface a chilling injury symptom in papaya (Chen and Paull, 1986), was observed in the present studies but was not influenced by 1-MCP (Figure 66) The overall average pitting percentage for IC and IM was 27% and 21. 8% respectively. Water soaked areas of mesocarp tissue ofIC was 0% at day O and 6 5% at day 10 and those ofIM 0% at day Oto 2 5% at day 10 (Figure 6-7A) FCC and FCM mesocarp tissue displayed a 96% (from 0% to 96%) and 76% (from 0% to 76%) increase in water soaked areas respectively during the 10 day of storage (Figure 67 A) The extent of water soaking of FCC mesocarp tissue was 54% at day 4 while the incidence in FCM was only 30%. FCC exhibited significantly higher water soaking incidence than FCC on da y s 8 through 10 The flesh ofIC and IM scored over 4 (good-excellent to excellent) for sensory evaluation during most of the storage period whereas FCC and FCM showed a dramatic decline to as low as 1 2 (FCC ; poor to poor-good) at day 10 (Figure 67B). At day 2 sensory evaluation ofFCM was 5 (excellent) whereas FCC was below 4 (fair to good excellent) By day 6 of storage the sensory scores for that ofFCM was 3.2 (fair to good excellent) compared with 2 2 (poor-good to fair) for FCC (Figure 6-8) Storage life FCC was limited to 2 days due to a dramatic decline in sensory evaluation while that ofFCM limited 6 days for the same reason Advanced ripening of papaya is associated with development of y e llowish-orange coloration of the surface (Akamine and Goo 1971) involving primarily a loss of chlorophyll (Sanxter et al. 1992). Sanxter et al. (1992) reported that mature green papaya skin had the highest chlorophyll and total carotenoids content and chlorophylls in skin consistently decreased through the full ripe stage while total carotenoids showed a

PAGE 137

123 minimal decrease The initial skin color (estimated by hue angle) of intact, postclimacteric Sunrise Solo' fruit regardless of 1-MCP treatment remained unchanged during storage Storage at 5 C seems to be very effective at delaying color change irrespective of 1-MCP treatment by slowing down chlorophyll degradation Pitting of the fruit surface was not extensive in either IC or IM postclimacteric Sunrise Solo fruit, which may due to immediate evaluation of fruit upon removal of the fruit from 5 C since chilling injury symptoms are accelerated upon transfer of injured fruit to a higher temperature (Chan, 1998) Fresh-cut Sunrise Solo fruit regardless of 1-MCP treatment showed an increase in water soaking during storage However fresh-cut fruit derived from 1-MCP-treated fruit exhibited less increase in water soaking relative to fresh-cut control on days 8 through 10 which may support that ethylene contributes water soaking in papaya fruit. The ethylene inducible water soaking was reported for watermelon fruit at 18 or 20 C by Elkashif and Huber (1988) and Karakurt and Huber (2000) In contrast Chatenet et al. (2000) reported water soaking in Charentais melon mesocarp during the late stage of ripening was not an ethylene-inducible event as evaluated due to no change in the expression of genes encoding ACS and ACO Chatenet et al. (2000) also documented 1-MCP (1 I L1 for 24 h) did not prevent water soaking in Charentais melon fruit treated at the preclimacteric stage after 35 days of storage at 14 C. Water s oaking in c rease in fresh cut 'Sunrise Solo' fruit during storage at 5 C may be caused by a depletion of cell wall calcium (Chatenet et al. 2000) and / or ethylene-inducible membrane permeability changes (Karakurt and Huber 2000)

PAGE 138

124 Microbiological Counts No changes were noted in intact fruit in terms of total aerobic count during storage, regardless of 1-MCP treatment (Table 6-1 ). However, population of aerobic organisms on fresh-cut fruit irrespective of 1-MCP were increased from 1 73 x 10 1 (at day 0) to 2.72 x 10 3 CFU g1 (at day 10) for FFC and from 1.47 x 10 1 (at day 0) to 3 04 x 10 3 (at day 10) for FCM (Table 6-1 ) The number of total coliforms Enterobacteriaceae and other fungi were almost undetectable in all treatments during storage (Table 6-1 ) Both IC and FCC showed a slight but significant increase in lactic acid bacteria through day 5 then the two treatments displayed a decrease trough day I 0 However lactic acid bacterium count of both IM and FCM initially decreased through 5 and then increased through day 10 (Table 6-1 ). Only FCC and FCM displayed significant yeast enumeration increase during storage and the population of yeast were 9.73 x 10 1 and 9.13 x 10 1 CFU i 1 for FCC and FCM respectively at day I 0, with no significant differences between the two treatments (Table 6-1 ). Our microbial counts were low compared to findings of O'Connor-Shaw et al. (1994), who reported total aerobic counts of climacteric / postclimacteric papaya cubes (no 1-MCP and no chlorine treatment) varied from 1.4 x 10 4 to l 7 x l 0 7 CFU i 1 after 4 days of storage at 4 C. On the other hand Teixeira et al. (200 I) reported a total aerobic count of 1 x l 0 3 in fresh cut climacteric / postclimacteric papaya fruit (no 1-MCP ; washed with 200-L L1 chlorinated water) after 7 days of storage at 9 C. The lower microbial count in our experiment may be due to very strict sanitation practices during and after fresh-cut processing Teixeira et al. (2001) also stated that hygienic care adopted during processing resulted in low microbial counts in fresh-cut papaya fruit. 1-MCP had no significant effect upon microbial growths except for lactic acid bacterium the growth of which seemed to be

PAGE 139

125 promoted by 1-MCP; however, the population of lactic acid bacteria was insignificantly low in all treatments even at day 10 The Solo variety treated with 25 L 1 1-MCP 20 ~C at mature green stage and stored at 20 C for approximately for 20 days showed slightly higher symptoms of stem rots, body black rots, and anthracnose compared to untreated fruit lasted approximately 5 days (Hofman et al. 2001 ) Hofman et al. (200 I) attributed the slight increase in the symptoms of the diseases in 1-MCP-treated fruit to a reduction in anti fungal concentrations due to extended storage life Jiang et al. (200 I) reported that ripe Everest' strawberry fruit treated with 500 to I 000 nL 1 1-MCP (24 h at 20 C) displayed accelerated leak rot disease development at 20 C compared to untreated fruit; however 1-MCP at 100 and 250 nL L. 1 delayed the onset of the decay Tomato plant lines expose to IO nL L. 1 1-MCP for 24 h at 18 to 24 C resulted in a significant increase in Bottytis cinerea frequency in the cultivars Moneymaker' and 'Castlemart' but no increase in Pearson' (Diaz et al. 2002) Hence, the effects of 1-MCP upon microbial growth are complex and depend on the type of microorganisms 1-MCP concentration cultivars, and tissue type and development. To conclude, 1-MCP delayed quality loss of fresh-cut postclimacteric papaya fruit derived from intact postclimacteric fruit treated with 1-MCP resulting in a 4-day extended shelf life Thus, 1-MCP is a safe and inexpensive postharvest application for delaying quality loss and extending storage life of fresh-cut postclimacteric Sunrise Solo fruit at 4 C. Microbial proliferation in fresh-cut postclimacteric papaya fruit would not be a problematic factor contributing either tissue softening or quality loss during the storage life if the necessary precautions are taken.

PAGE 140

1 26 120 --Intact con t rol 100 --0Intac t 1MC P -TF resh-cut control .... I Fresh-cut 1M CP .c 80 -v-.... I 60 = "" = M 40 u 20 0 1 3 5 7 9 D ays F i g u re 6-1. Ethy l ene p ro d uction for intact postclimacteric Sunrise Solo papaya fruit pre t r eated with 2 5 L 1 1-MC P and air (co nt ro l ) and for fres hc ut p os t c li macteric f ru it d er i ve d from t he eit h er intact a i r treate d o r th e intac t 1MCP-trea t ed f ru it d u rin g storage at 5 C. Vertica l bars r epresents s t a nd ar d d ev iati o ns o f th e m ea n s ( n = 5)

PAGE 141

12 10 8 z r,} r,} 6 = e i.. ri: 4 2 0 0 127 2 4 Days ____..._ Intact control --oIntact 1-MCP -----Y-Fresh-cut control Fresh-cut 1-MCP 6 8 10 Figure 6-2. Mesocarp firmness for intact postclimacteric Sunrise Solo papaya fruit pre treated with 2 5 L 1 1-MCP and air (control), and for fresh-cut postclimacteric fruit derived from either the intact air-treated or the intact 1MCP-treated fruit during storage at 5 C. Vertical bars are standard deviations of means (n = 5)

PAGE 142

60 50 = 40 1)1) .::t: 30 >-. 0 .. 20 .::t: 10 0 0 _.__ Intact control -oIntact 1-MCP 128 -YFresh-cut control -vFresh-cut 1-MCP 2 4 Days 6 8 10 Figure 6-3 Electrolyte leakage(% of total) for intact postclimacteric Sunrise Solo papaya fruit pre-treated with 2. 5 L L 1 1-MCP and air ( control) and for fresh-cut postclimacteric fruit derived from either the intact air-treated or the intact 1-MCP-treated fruit during storage at 5 C. Vertical bars are standard deviations of means (n = 5).

PAGE 143

80 70 ..;i "" "" 60 = ..., -= 1:)1) 50 40 110 100 "'SJl = c-= 90 = ::c 80 70 70 60 c-= e 0 50 .. -= u 40 30 A ..... ----...----~ 129 _._ Intact control -oIntact 1-MCP ----TFresh-cut contro l D .....,.----,,.....-...--------4 -vFresh-cut 1-MCP i-,.--....,..----...-.C F 0 2 4 6 8 10 0 2 4 6 8 10 Days Days 70 60 ..;i "" 50 "" = ..., -= 1:)1) 40 30 70 60 50 "'SJl = c-= = 40 ::c 30 60 50 40 c-= e 0 .. 30 -= u 20 10 Figure 6-4. Color parameters for intact postclimacteric Sunrise Solo papaya fruit skin pre-treated with 2.5 L L" 1 1-MCP and air (control) and for the intact fruit flesh and fresh-cut fruit derived from either the intact air-treated or the intact 1-MCP-treated fruit during storage at 5 C. Vertical bars are standard deviations of means (n = 5)

PAGE 144

130 Figure 6-5. Fresh-cut postclimacteric 'Sunrise Solo' papaya fruit derived from either intact postclimacteric air-treated (FCC) or intact postclimacteric 1-MCP treated fruit (FCM) and then stored for 10 days at 5 C.

PAGE 145

131 100------------------------80 "$, 60 -OD C .... .... ii: 40 20 ____._ Intact control --0Intact 1-MCP 0-----------------------------0 2 4 Days 6 8 10 Figure 6-6 Pitting of postclimacteric Sunrise Solo papaya fruit pre-treated with 2 5 L L" 1 1-MCP and air (control) during storage at 5 C. Vertical bars are standard deviations of means (n = 5)

PAGE 146

132 _..__ Intact control A 100 -0Intact 1-MCP -TFresh-cut control 80 Fresh-cut 1-MCP = = 60 ; .:ii= Q "' l,,, 40 QJ ,= 20 0 5 B 4 QJ 3 l,,, Q rJ':J 2 l 0 0 2 4 6 8 10 Days Figure 67. Water soaking (A) and sensory evaluation (B) for intact postclimacteric Sunrise Solo papaya fruit pre-treated with 2 5 L L1 1-MCP and air ( control), and for fresh-cut postclimacteric fruit derived from either the intact air-treated or the intact 1-MCP-treated fruit during storage at 5 C. Vertical bars are standard deviations of means (n = 5)

PAGE 147

133 Figure 6-8. Fresh-cut postclimacteric 'Sunrise Solo' papaya fruit derived from either intact postclimacteric air-treated (FCC) or intact postclimacteric 1-MCP treated fruit (FCM) and then stored for 6 days at 5 C.

PAGE 148

134 Table 6-1. Microbial counts (CFU 1 fresh weight) for intact postclimacteric Sunrise Solo' papaya fruit pre-treated with 2 5 L L" 1 1-MCP (IM) and air (control, IC) and for fresh-cut postclimacteric fruit derived from either the intact air treated (FCC) or the intact 1-MCP-treated (FCM) fruit during storage at 5 C Total aerobic counts Total coliforms Dey Dey Treatment 0 5 10 Treatment 0 5 ---------------IC 0 a 0 a 0 7 a IC 0 a 0 a IM FCC FCM IC IM FCC FCM IC IM FCC FCM 0a 0a 0 7a 17.3 b 42 0 b 2 7 x 10 3 b 14.7 b 36 0 b 3 0 x 10 3 b Enterobacteriaceae 0 0 a 0 a 1.3 a 0 7 a Yeasts 0 0a 0a 0a 0a 5 0a 0a 0a 0a 5 0a 0 a 0 7 a 0a 10 0a 0a 4 0 a 0a 10 0a 0a 97 3 b 91.3 b IM FCC FCM IC IM FCC FCM IC IM FCC FCM 0a 0a 0a 0a 0a 0a Lactic acid bacteria 0 2 0 a 1 3 a 11 3 b 9 3 b 5 5 3 a 0 7 a 26 7 b 2 7 a Other fungi 0 0a 0a 0a 0a 5 0a 0a 0a 0a 10 0a 0a 3 3 a 0 7 a 10 0 7 a 6 7 b 12 0 C 18 0 C 10 0a 0a 0 7 a 0a Means (n = 3) followed by the same letter within a column are not significantly different, P :S 0 05

PAGE 149

CHAPTER 7 CELL WALL MODIFICACTION IN POSTCLIMACTERIC FRESH-CUT AND INT ACT PAP A YA FRUIT WITH AND WITHOUT 1-METHYLCYCLOPROPENE Introduction Fresh-cut produce are extremely fragile and perishable produce relative to their intact counterpart Fresh-cut produce exhibit significant differences in terms of physiological behavior relative to their intact counterpart even though the quality and sensory of fresh-cut produce are somehow similar to intact produce The behavior includes enhanced ethylene and respiration rates wound-healing processes (synthesis of secondary compounds suberization and lignification) biochemical changes (membrane changes, browning and degreening) and physical changes (softening and water loss ; Rolle and Chism I 987; Miller, 1992 ; Brecht 1995). This behavior can greatly influence quality maintenance of fresh-cut fruits associated mostly rapid texture loss The rapid texture loss in fresh-cut produce has not been clarified yet. However, a number studies have been shown that the rapid texture loss possibly results from cell wall and / or membrane damage For example the activities of some cell wall enzymes have been reported to increase in response to wounding (Dumville and Fry 2000 ; Huber et al. 200 l ; Karakurt and Huber 2003) The senescence-delaying influence of Ca + on shredded carrots (Picchioni et al., I 996) and increased juice leakage in fresh-cut melon fruit (Cartaxo et al., 1997) supports a role of cell wall and membranes in the deterioration of fresh-cut produce 135

PAGE 150

136 Textural modifications during fruit ripening are related to changes in cell wall structure (Huber, 1983; Tucker and Grierson, 1987). The changes are mostly correlated with structure and composition of pectic components (Seymour et al., 1987). Solubilization and depolymerization of both pectins (Fischer and Bennett, I 991) and hemicelluloses (Lashbrook et al.,, 1997) during ripening are frequently associated with cell wall loosening and disintegration. Cell wall modifications have been extensively studied in tomato fruit, and early reports indicated that pectin degradation by polygalacturonase (PG) represented the model of fruit softening (Crookes and Grierson 1983); however, analysis of PG-anti sense tomato fruit (Smith et al. ,, 1988; Giavannoni, 1990) revealed that this enzyme was not a significant contributor to tomato softening Brummel and Harpester (200 I) suggested that pectin metabolism might contribute to textural changes during tomato fruit ripening whereas modifications to the cellulose/matrix glycan network mainly to softening while each obviously affects each other. Xyloglucan, the primary hemicellulose in dicotyledonous plants, also undergoes depolymerization in most fruits (Sakurai and Nevins 1993) In addition to the depolymerization of both pectin and hemicellulose, fruit softening is accompanied by a loss of neutral sugars, primarily galactose and arabinose, from pectic and hemicellulosic polysaccharides (Tucker, 1993) Papaya fruit softening is accompanied by pectin hydrolysis and modification of hemicelluloses (Zhao et al.,, 1996; Paull et al.,, 1999; Karakurt and Huber, 2002) and increases in the activities of polygalacturonase (PG EC 3.2.1.15; Paull and Chen, 1983; Karakurt and Huber, 2002) xylanase (EC 2.4.1.207; Paull and Chen, 1983) and galactosidase (EC 3 2.1 23; Ali et al., 1998) The levels of water-soluble (Zhao et al.,

PAGE 151

137 1996; Paull et al., 1999), chelator-soluble (Lazan et al., 1995; Paull et al., 1999) and alkali-soluble polyuronides have been reported to increase during papaya ripening (Lazan et al., 1995 ; Zhao et al., 1996; Ali et al., 1998 ; Paull et al. 1999) A continuous or temporary increase in the activities of PG, pectin methylesterase (PME. EC 3 .1.1.11 ), xylanase and a(EC, 3 .2 .1 22) and P-galactosidases has been cited during papaya fruit ripening as well (Paull and Chen 1983; Lazan et al., 1995; Ali et al., 1998). 1-methylcyclopropene (1-MCP), an ethylene action inhibitor (Sisler and Serek 1997) has been shown to delay ripening of postclimacteric apple (Pre-Aymard et al. 2002 ; Jiang and Joyce, 2002), tomato (Wills and Ku 2002 ; Hoeberichts et al., 2002), and apricot fruits (Botondi et al., 2003) Sunrise Solo' papaya fruit treated with 1-MCP at an early stage of ripening displayed delayed softening and color change (Jacaomino et al. 2002) Recently, 1-MCP has been shown to modify cell wall enzyme activities in a number of fruits including avocado nectarine, tomato and apricots Accumulation of both PG and cellulase (EC 3 2.1.5) activities were delayed by 1-MCP treatment in avocado fruit (Feng et al. 2000; Jeong et al. 2002) Less extensive molecular mass downshifts of polyuronides and alkali-soluble hemicelluloses including xyloglucan application were reported for 1-MCP-treated avocado (Jeong et al. 2002) In Flavortop' nectarine, 1-MCP treatment suppressed transcript abundance and activities of PG and PME during ripening whereas accumulation of endoglucanase activity and transcript abundance was enhanced by 1-MCP (I L C 1 1-MCP for 20 hat 20 C, Dong et al. 2001) 1-MCP treatment decreased mRN A abundance of exp I in mature green or ripe tomato fruit (Hoeberichts et al., 2002), and 1-MCP slightly reduced the activities of PME a-mannosidase (EC

PAGE 152

138 3 2 1 113) and ~-glucosidase (EC 3 2 1 21) in San Castrese apricot stored harvested at an advanced ripening stage at 20 C (Botondi et al., 2003) In the present study we have examined the cell wall disintegration of fresh-cut and intact postclimacteric papaya fruit in which ethylene perception was suppressed by 1MCP Materials and Methods Plant Material and 1-MCP Treatment Papaya fruit (Carica papaya L. 'Sunrise Solo ) originated from Brazil were purchased from C-Brand Tropicals Inc Homestead FL. The fruit were transferred the Postharvest Horticulture Laboratory at the University of Florida within 24 h of arrival at the packinghouse. The fruit were maintained at 20 C until the majority of the fruit reached the desired ripeness stage (postclimacteric 70% to 80% yellow surface color (Wills and Widjanarko 1995) The average firmness, ethylene production, and respiration of the fruit was 6.8 N 0.8 L kg1 h1 and 22 mL CO 2 kg1 h1 respectively at 20 C. The fruit were gently brushed dipped in 200 L L1 -chlorinated water for 1 min air-dried and placed in 174-L metal cambers for 1-MCP treatment. The fruit were treated four times at 6-h interval with 2 5 L.L1 of 1-MCP generated from Agrofresh powder (active ingredient 0 14%; Philadelphia PA) for 24 hat 20 C. 1-MCP was measured using a gas chromatograph (Hewlett Packard-5890 II ; Avondale, PA) equipped with a 80-100 mesh Chromosorb PAW stainless steel column (1.8 m x 3. 18 mm i.d. ; Supelco Bellefonte PA) with injector oven and detector (FID) set at 150 150 and 200 C, respectively Isobutylene gas which has an FID response similar to that of 1-MCP (Jiang et al. 1999), was used as a standard Control fruit were kept under identical conditions with the exception of 1-MCP gassing The fruit were then transferred to facilities at 5 C

PAGE 153

139 for fresh-cut processing. After I hat 5 C to allow temperature equilibration, the blossom and pedicel ends of each fruit were removed, and the fruit longitudinally cut using a Bread Slicer (Coupe-Pain China) into 1 5-cm thick slices The two outermost slices were peeled and cut into pieces (1.5 cm x 3.5 cm x 4 cm) weighing I 5 to 20 g. Afterward, the slices were rinsed with sterile isotonic mannitol (500 mM) using a squeeze bottle, and then the slices were placed in I. 7 L vented plastic containers (FridgeSmart Tupperware Co., St. Paul, MN) The treatments included: fresh-cut tissue derived from intact fruit pre treated with 1-MCP (FCM), fresh-cut tissue derived from intact fruit pre-treated fruit with air as fresh-cut control (FCC), intact fruit pre-treated with 1-MCP (IC) and intact fruit pre-treated with air as intact control (IM) The fresh-cut and intact fruit were stored for 10 days at 5C. At the indicated intervals, fresh-cut and intact fruit were removed from storage and stored at -30C until analyzed. Prior to freezing, intact fruit were peeled and cut into slices as described above Ethanol-insoluble Solids Approximately 80 g of pai1ially thawed mesocarp tissue derived from fresh-cut and intact papaya fruit were combined with 420 mL of 95% ethanol, macerated with a Polytron homonogizer (Kinematica Kriens-Luzen Switzerland) for 2 min refluxed in a boiling water bath for 20 min, and filtered through glass fiber filter paper (Whatman GF/C) in an aspiration flask and washed with 95% cold ethanol. The residue was transferred to 200 mL of chloroform / methanol (I : I v / v) and incubated with stirring for 30 min. The suspensions were filtered (GF / C) and washed 300 mL of acetone The ethanol insoluble solids (EIS) were oven-dried at 43 C for 5 h and stored in a desiccator at room temperature.

PAGE 154

140 Total Soluble Sugars and Polyuronides Partially thawed mesocarp tissue (2 g) derived from fresh-cut or intact fruit in 20 mL of 95% ethanol was homogenized with the Polytron homonogizer for 30 sec The homogenate was held at -20 C for a minimum of 2 hand were then centrifuged at 3430 g for 5 min Aliquots of the supernatant (0.5 mL) were used for measuring total soluble sugar (TSS) levels using the procedure the phenol-sulfuric assay, described by Dubois et al. (1956). Total polyuronide content in the EIS samples (7 g) was determined using the hydroxydiphenol assay (Blumenkrantz and Asboe-Hansen 1973) Sequential Fractionation of Cell Wall Materials Water-, CDT A-( 1 2 cyclohexylenedinitrilotetraacetic acid) and Na2CO 3 -soluble pectins were extracted by suspending 30 mg of EIS in 7 mL distilled water 50 mM CDTA plus 50 mM Na-acetate pH 6 5, and 50 mM Na 2 CO 3 sequentially at room temperature Suspensions incubated on an oscillating shaker ( 1.4 cycle min1 ) for 4 h were filtered through a Whatman GF / C filter paper in an aspiration flask Pol y uronides in aliquots were measured (Blumenkrantz and Asboe-Hansen 1973) Pectins solubilized in water COTA and Na 2 CO 3 (0.5 mg galacturonic acid equivalent) were run on a Sepharose CL-4B column ( 1 5 cm x 28 cm; Sigma Chemical Co ., St. Louis MO) equilibrated with 200 mM ammonium acetate at pH 5 0 Fractions of 2 mL were collected at a flow rate of 40 mL h1 and 0 5 mL of each fraction was used for the determination polyuronide content. Dextran 2 000 and glucose (Sigma St. Louis, MO) were used to determine the void (V 0 ) and total (Vi) volumes of the column Hemicellulosic Polysaccharide Extraction For pectin removal approximately 200 mg EIS in 500 mL Na-phosphate (40 mM ph 6 8) were heated in a boiling water bath for 20 min cooled filtered through Miracloth

PAGE 155

141 and washed with I L distilled water sequentially Excessive water was removed from the residue and the residue was transferred into 200 ml of 80% ethanol filtered through Miracloth transferred into 200 mL of 50% chloform / 50% methanol and filtered through Miracloth sequentially The residue was then transferred into 200 mL of acetone to remove chloroform/methanol and filtered through GF/C under aspiration using Buchler funnel system with additional acetone wash. The residue was oven-dried at 43 C for 5 h. For hemicellulose extraction the dried residues (50 mg) were suspended in 5 mL 4% KOH/0 02% NaBH 4 o v ernight at room temperature The suspension was centrifuged at 1 300 g at room temperature for IO min, and the supernatant was removed from the pellet and saved at 5 C. The pellet was resuspended in I mL 4% KOH/0 02% NaBH 4 centrifuged as described above, and supernatant was removed The supernatant was added into the previous supernatant. The remaining pellet was subject to the same procedure described above while using 24% KOH/0 02% NaBH 4 instead of 4 % KOH/0 02% NaBH4 The combined supernatants were neutralized over ice with concentrated acetic acid Hemicellulosic polysaccharides in the neutralized samples were determined by using the hydroxydiphenol and phenol-sulfuric assa y Compositional Analysis of Cell Wall Polymers EIS (2 mg) were used for glycosyl composition analysis using a GC (HewlettPackard 5490 II, Avondale, PA) on a 25-m cross-linked 5% phenylmethyl silicone capillary column (Hewlett Packard 0.2 mm i.d. 0 33 M film thickness). Myo-inositol was added as internal standard The EIS samples were hydrolyzed in 2 N trifluoroacetic acid for I h at 120 C. The cooled hydrolytes were then reduced and acetylated as described below (Blakane y et al 1983). The resulting monosaccharides were reduced with 0 66 M sodium borohydride in I N ammonium hydroxide overnight at 25 C. The

PAGE 156

142 samples were acidified with Dowex SOW (Sigma, St. Louis, MO) and the resin was removed by filtration through a syringe fitted with GC/C filter paper The samples were dried and subsequently washed three times with methanol and once with ethanol before derivatization. The sugars were converted into acetyl derivatives in the presence of 0 2 mL of acetic acid anhydride and 0 2 mL of pyridine for I hat 100 C. After cooling to room temperature the samples were dried under a gentle stream of air, washed with toluene three times, solubilized in methylene chloride, and injected into a gas chromatography (Hewlett Packard 5890 II, Avondale PA) The chromatograph was run at 210 C for 5 min increased to 230 C within 10 min held at 230 C for 5 min Results Ethanol-insoluble Solids and Total Soluble Sugars The EIS yield remained unchanged in all treatments, having no variations among treatments during storage (Table 7-1 ) Assay of total soluble sugars (TSS) using the phenol sulfuric acid method was used primarily to quantify glucose sucrose and xylose in the ethanol homogenate. TTS significantly decreased in all treatments over the 10-day period (Table 7-1) The decreases in TS were approximately 30 %, 33 %, 36% and 35% for IC IM FCC and FCM respectively Polyuronides and their Sequential Fraction The water-soluble fraction constituted the majorit y of pectins followed by CDT A and Na 2 CO 3 as illustrated in Table 7-2 The pectic polymers recovered in solutions of water chelator and dilute alkali compromised approximately more than 50% of total polymers recovered in EIS at day 0 2 6 or 10 Water-soluble pectins consisting of more than 30% of the total polyuronide content increased in all treatments throughout storage and by day 10 the levels of water-soluble pectins had increased 17 % for IC 14 % for IM

PAGE 157

143 13% for FCC, and 8% for FCM compared to the levels at day 0. The increase in water soluble pectins was similar for both fresh-cut and intact fruit regardless of 1-MCP. The CDT A-soluble polyuronides of intact fruit (IC and IM) did not change during storage while that of fresh-cut fruit (FCC and FCM) increased significantly (8% for FCC and 22% for FCM) from day O to I 0 On days 2 through 10, fresh-cut fruit (FCC and FCM) yielded higher chelator-soluble pectins compared to intact fruit (IC an IM) The alkali soluble pectins increased in both intact (IC, 30%; IM, 27%) and fresh-cut (FCC, 28% ; FCM, 19%) fruit over the 10-day period At day 2 IM and FCM showed higher alkali soluble pectin content relative to IC and FCC, collectively. Total polyuronide levels consistently decreased for all treatments during storage (IC 15% ; IM 15% ; FCC 19%, and FCM, 20%), with both FFC and FCM exhibiting slightly higher decline compared with both IC and IM Gel permeation chromatography of water-soluble pectins from all treatments is shown in Figure 7-1. Water-soluble polyuronides from IM and FCM showed negligible changes in mol mass during storage while IC and FCC demonstrated slightly higher changes especially in the levels of intermediate mo) mass polymers The mo) mass of chelator-soluble pectins also exhibited significant changes during storage (Figure 7-2) As was noted for the water-soluble pectins the decrease in the levels of intermediate mol mass polymers was slightly higher in FFC and FCM compared with TC and IM Alkali soluble polyuronides exhibited mol mass downshifts involving a decrease in the levels of intermediate mass pol y mers in all treatments and a more extensive decline in the recovery of higher molecular mass polymers in FCC and FCM (Figure 7-3).

PAGE 158

144 Hemicellulosic Polysaccharides The levels of neutral hemicelluloses and pectins extracted with 4% and 24% KOH from EIS are shown in Table 7-3. Weakly bound neutral hemicellulosic polysaccharides (extracted with 4% KOH) significantly declined (IC, 24%; IM 16%; FCC, 27% ; and FCM 26%) in all treatments while no differences were noted among treatments during storage Pectin content of the 4% KOH extracted polysaccharides also decreased (25% for IC 35% IM, 28% FCC and 28% FCM) during storage Strongly bound hemicelluloses (extracted with 24% KOH) showed no variations among treatments and no changes during storage Pectin levels in the 24% KOH extract however declined significantly in IC (22%) and IM (24%) during storage whereas the levels did not change in FCC and FCM The decrease in the pectin content of intact fruit (IC and IM) resulted in a significant difference at day 10 between intact (IC and IM) and fresh-cut (FCC and FCM) fruit. Compositional Analysis of Cell Wall Polymers Noncellulosic neutral sugar composition of EIS derived from the treatments is shown in Table 7-4. Analysis of the neutral sugars in EIS revealed that the predominant non-cellulosic neutral sugar in ripe papaya fruit is galactose followed by glucose xylose rhamnose mannose, and arabinose respectively Rhamnose showed no changes in any treatments during storage whereas the proportional quantity of arabinose increased (30% for IC 21% for IM 32 for FCC and 26% for FCM) Xylose levels decreased by 30% (IC) 24% (IM) 27% (FCC) and 26% (FCM). IM yielded higher xylose compared with IM at days 2 and 6, and FCM had higher xylose levels in its EIS than FCC on days 2 through 10 No significant changes in man nose levels were observed for any of the treatments All treatments showed a decline in galactose level during storage: IC 26% ;

PAGE 159

145 IM, 20%; FCC, 30%; and FCM 30% Galactose levels in FCC and FCM were lower than those measured for IC and IM. Glucose declined significantly during storage, and by day 10, the decline in glucose quantity was 13%, 10%, 17% and 17% for IC IM FCC and FCM, respectively, compared with the day 0 values. Discussion The EIS recoveries exhibited no changes during 10 days of storage and they were not affected by either fresh-cut processing or 1-MCP However it has been reported that the yield of cell wall material using ethanol decreased significantly during ripening of papaya fruit at 25 C (from color break to I 00% yellow skin color ; Paull et al 1999) The insignificant change in EIS content in the present study was possibly due to storage temperature (5 C) plus the fact that fruit were nearly ripe (70-80% yellow skin color) at the start of the experiment. Total polyuronide levels in EIS decreased significantly during IO days of storage in both intact and fresh-cut fruit and were not significantly affected in response to 1-MCP treatment. The decrease in total pectin content was more prominent in fresh-cut fruit confirming the findings of Karakurt and Huber (2003) who reported a significant reduction in total pectin content of fresh-cut papaya fruit (60 to 70% yellow surface color)during storage at 5 C. The decrease in total polyuronides suggests that at least some component of these polysaccharides are depolymerized to monomer and other small oligomers which, due to their solubility in ethanol, are not recovered in EIS preparations Water-soluble polyuronides constituted the majority of polyuronides in EIS followed by the CDT Aand Na 2 CO 3 -soluble fractions and all showed an increase during storage except for the CDT A-soluble polyuronides of intact fruit (TC and IM) The changes in all three pectic fractions of fresh-cut fruit (FCC and FCM) suggests a possible

PAGE 160

146 role of polyuronide metabolism in the rapid tissue softening reported for fresh-cut papaya fruit (Karakurt and Huber, 2003) In addition to the increase in polyuronide solubility in the intact and fresh-cut fruit, polyuronides exhibited mol mass downshifts during storage. The lower mo! mass of polyuronides from fresh-cut compared with intact fruit could arise from depolymerization or largely from increased solubility of inherently smaller polymers Most fleshy fruits show an increase in pectin solubility during ripening (Huber et al, 2001) attributed primarily to cell wall including PGs Karakurt and Huber (2003) reported greater levels of PG activity in fresh-cut compared with intact fruit supporting the involvement of PG in polyuronide solubility and depolymerization in papaya fruit. Paull et al. ( 1999) have attributed the mol mass downshifts in papaya polyuronides during ripening to increases in PG activity Pectin solubility and depolymerization in stored intact and fresh-cut papaya fruit was not significantly altered in response to 1-MCP treatment. This observation suggests that that polyuronide solubility and depolymerization in papa y a fruit is possibly ethylene independent at the final stages of ripening Nevertheless PG activity during avocado fruit ripening was lowered by 1-MCP at as low as 30 nL L1 (Feng et al. 2002 ; Jeong et al. 2002) Consisting with the decrease of PG activity in 1-MCP treated avocado fruit pectin solubility and depolymerization during ripening was greatly suppressed by 1-MCP (Jeong et al. 2002) 1-MCP also affected a-galactosidase activity by delaying decline in the activity during avocado ripening while it did not affect p-galactosidase level (Jeong et al. 2002). 1-MCP, moreover, reduced levels of a-and P-galactosidase in pre-ripe Ceccona apricot fruit whereas 1-MCP did not affect the activities of aand P-galactosidase in ripe San Castrese' apricot fruit (Botondi et al., 2003) The data for ripe San Castrese imply

PAGE 161

147 that polyuronide solubility and depolymerization during advanced ripening may be ethylene-independent or no longer respond to 1-MCP The works presented by Jeong et al. (2002) and Botondi et al. (2003) showed that pectin methyl esterase activity seemed to be not significantly affected by 1-MCP during either avocado or apricot ripening. 1-MCP effect upon expansin was also recorded by Hoeberichts et al. (2002) who found that 1MCP decreased the mRNA abundance of expansin 1 (EXP) in mature green breaker orange, and red ripe tomato fruit (Hoeberichts et al. 2002). Significant changes were evident in neutral hemicellulosic polysaccharides derived from papaya fruit during storage The strongly bound hemicelluloses extracted with 24% KOH contained high levels of pol y saccharides relative to the weakly bound hemicelluloses extracted with 4 % KOH. The change in hemicellulosic polysaccharides might be due to the increase in a and P-galactosidase activities Beta-galactaosidases / galactans have been linked with pectin and hemicellulose solubility and depolymerization in several fruit during ripening including papaya (Lazan et al. 1995 ; Rose et al. 1998 ; Karakurt and Huber 2003), avocado (De Yeau 1993) and melon fruit (Ranwala et al. 1992) Neutral hemicelluloses showed no changes in response to fresh-cut proce s sing or 1-MCP treatment. The pectin composition extracted with 4 % KOH from EIS were higher than the pectin composition extracted with 24% KOH and weakly pectin s in both fresh cut and intact fruit and strongly bound pectins in only intact fruit declined during storage. The decrease in pectin content is possibly due to PG activit y which has been reported to increase during ripening of papaya fruit (Paull and Chen I 983 ; Lazan et al. 1995 ; Karakurt and Huber, 2003)

PAGE 162

148 Consistent with the involvement of galactosidases / -galactanases in depolymerization and solubility of hemicelluloses, galactose, glucose and xylose, the predominant neutral sugars in EIS derived papaya fruit, decreased significantly during storage. Significant changes in neutral sugars in EIS composition were minimal in response to 1-MCP treatment. I -MCP treatment enhanced the loss of xylose but did not significantly affect the levels of glucose galactose man nose and arabinose Since a marked decrease in galactose content was noted for fresh-cut fruit it is possible that the enzymes contributing to degalactosidation of polyuronides and hemicelluloses may be up-regulated by wounding (Karakurt and Huber 2003) Pectin solubilization may result from the loss of galactosyl residues in the form of the galactose-rich side chains of rhamnogalacturonans (Seymour et al., 1990 Redgewell et al., 1992) Loss of galactans has been demonstrated to accompany increased solubilization of polyuronides (Gross and Wallner 1979, Kim et al. 1991) It is concluded that modifications of cell wall polyuronides and hemicelluloses in fresh-cut ripe papaya fruit showed similar patterns to those in intact ripe papaya fruit ; although, few minor differences were observed between fresh-cut and intact fruit. The effect of 1-MCP is minimal in inhibiting the solubility and depolymerization of polyuronides and hemicelluloses of both fresh-cut and intact ripe papaya fruit at 5 C.

PAGE 163

149 Table 7-1 Ethanol insoluble solids (EIS) and total soluble sugars (TSS) of intact three quarter ripe papaya fruit treated with and without 1-MCP and fresh-cut fruit derived from intact three-quarter ripe fruit treated with and without 1 MCP papaya during storage IC intact control fruit ; IM Intact l-MCP-treated fruit FCC, fresh-cut control fruit ; and FCM fresh-cut fruit with 1-MCP. Day 0 2 6 10 0 2 6 10 Ethanol insoluble solids (EIS) (mg gf.w ) IC IM FCC 28 03 a 26.43 a 27.44 a 25 94 a 25 69 a 25 54 a 25 24 a 25.43 a 25 76 a 26 34 a 25 53 a 25 .6 5 a Total soluble sugar (TSS) (mg gf.w ) 147 31 a 144.45 a 140 58 a 106 27 a 1 0 4 92 a I 05 86 a 98 81 a 96 19 a 99 86 a 102 58 a 98 03 a 93 12 ab FCM 27 12 a 26 36 a 26 18 a 25 6 5 a 142 12a 112 50 a 116 98 a 94 76 a a: means (n = 3) in the same row with s ame letters were not s ignificantly different at P '.S 0.05 TSS were measured in gluco s e equi v alent s.

PAGE 164

150 Table 7-2 Polyuronide composition of intact three-quarter ripe papaya fruit treated with and without 1-MCP and fresh-cut fruit derived from intact three-quarter ripe fruit treated with and without 1-MCP papaya during storage IC intact control fruit; IM Intact 1-MCP-treated fruit FCC fresh-cut control fruit ; and FCM fresh-cut fruit with 1-MCP Day 0 2 6 10 0 2 6 10 0 2 6 10 Water-soluble polyuronides (g mg EIS) IC 127 78a 137 16a 135 33 a 149 29 a IM 128 24 a 130 05 a 136 54 a 146 34 a FCC 132 25 a 137 21 a 138 66 a 149 10 a CDT A-soluble polyuronides (g mg EIS) 54 65 a 51.14 b 54.33 b 53.53 b 53 70 a 49 55 b 49 70 b 52 21 b 56.75 a 63.41 a 60.47 a 61 14 a Na2C0 3 -soluble polyuronides (g mg" ElS) 24 70 a 23 73 b 30 00 a 32 04 a 25 88 a 32 30 a 33 17 a 32 80 a 23 10 a 25 80 b 28 1 O ab 29 58 a FCM 131.63 a 139 84 a 137 87 a 142 76 a 53.01 a 63 57 a 61.43 a 64 65 a 26 74 a 30 29 a 30 77 a 31.72 a a : means (n = 3) in the same row with same letters were not significantly different at P :S 0 05

PAGE 165

151 Table 7-2. Continued Total polyuronides (g mi EIS) Day IC IM FCC FCM 0 432.67 a 428 65 a 425 25 a 422 23 a 2 381.35 a 387.35 a 368 15 a 362 50 ab 6 363 94 a 373 06 a 357 69 ab 356 92 ab 10 363.31 a 365 33 a 345 35 ab 339 67 b a: means (n = 3) in the same row with same letters were not significant l y different at P :S 0 05

PAGE 166

152 Table 7-3. Neutral hemicellulose and pectin residue composition of intact three-quarter ripe papaya fruit treated with and without 1-MCP and fresh-cut fruit derived from intact three quarter ripe fruit treated with and without l-MCP papaya during storage IC, intact control fruit; IM Intact 1-MCP-treated fruit, FCC, fresh-cut control fruit; and FCM, fresh-cut fruit with 1-MCP Fraction 4%KOH 24% KOH Day 0 2 6 10 0 2 6 10 0 2 6 10 Neutral hemicelluloses (g mg EIS) IC I 0.49 a 10.30 a 9 89 a 7.99 a IM 10 01 a 10 89 a 10 50 a 8.40 a Pectins (g mg EIS) 5 13 a 5 21 a 4.54 a 3 86 a 5 20 a 4 17 b 4 07 a 3 35 a FCC 11. 11 a 10 01 a 9 09 a 8 16 a 4 95 a 4 80 a 4 36 ab 3 57 a Neutral hemicelluloses (g mg EIS) 24 90 a 22 76 a 22 24 a 21.48 a 23 12 a 22 95 a 22 24 a 22 32 a 22 34 a 24 88 a 22 73 a 22 78 a FCM I 1.03 a 10 05 a 9 73 a 8 19 a 5 01 a 4 32 a 4.06 a 3.59 a 22 00 a 24 38 a 23 00 a 21.18 a a : means (n = 3) in the same row with same letters were not significantly different at P S 0 05

PAGE 167

153 Table 7-3. Continued Fraction Days Pectins (g mg EIS) 24%KOH IC IM FCC FCM 0 2 85 a 2 95 a 3 12 a 3 04 a 2 2.26 a 2.41 a 2 80 a 2 71 a 6 2 30 a 2 35 a 2 75 a 2 71 a 10 2.21 b 2 26 b 2 68 a 2.65 a a : means (n = 3) in the same row with same letters were not significantly different at P '.S 0 05

PAGE 168

154 Table 7-4. Neutral sugar composition of intact three-quarter ripe papaya fruit treated with and without 1-MCP and fresh-cut fruit derived from intact three-quarter ripe fruit treated with and without 1-MCP papaya during storage. IC, intact control fruit; IM, Intact 1-MCP-treated fruit, FCC, fresh-cut control fruit; and FCM, fresh-cut fruit with 1-MCP. Rhamnose (g mgEIS) Day IC IM FCC FCM 0 35 89 a 38.25 a 36 65 a 36.46 a 2 35.74 a 36.79 a 35.60 a 36.84 a 6 38.31 a 37 50 a 34 80 a 38.42 a 10 38 62 a 41 24 a 37 71 a 39 24 a Arabinose (g mg EIS) 0 18.65 a 21.37 a 19.19 a 17.48 a 2 17.45 a 20.34 a 20.46 a 17 66 a 6 22 60 a 21.68 a 24.47 a 18 27 a IO 24.08 a 25. 78 a 25.26 a 22.06 a Xylose (g mgEIS) 0 88.46 a 85 23 a 90.56 a 85 27 a 2 73.40 a 61.23 b 69 00 a 55 19 b 6 70.46 a 64 32 b 68 26 a 54 95 C IO 62 04 a 65 33 a 66 33 a 49 08 b a : means (n = 3) in the same row with same letters were not significantly different at P :S 0 05.

PAGE 169

155 Table 7 4 Continued. Mannose ( g mg EIS) D ay IC IM FCC FCM 0 24 52 a 25 89 a 23 97 a 24.49 a 2 22 7 1 a 23 92 a 24 7 6 a 22 81 a 6 22 06 a 24 94 a 24 63 a 25 58 a 10 25 .1 2 a 27 72 a 26 32 a 25.42 a Ga l actose (g mg EIS) 0 1 1 4 2 1 a 1 1 0.42 a 108.08 a 1 07 16 a 2 85 04 a 88 33 a 75.48 C 75 38 C 6 8 7 74 a 89 54 a 80 7 9 b 80.48 b 10 88 .7 6 a 86 96 a 83 36 b 8 1 .45 b G l ucose (g mg EIS) 0 102 27 a 104 04 a 106 18 a I 07 00 a 2 96 80 a 99 32 a 95 35 a 97 24 a 6 95 7 4 a 98 54 a 90 79 a 88.48 a 10 89 25 a 94 0 7 a 88 84 a 89 72 a a : mea n s ( n = 3) in t h e same row w i th same l etters were not significa n t l y different a t P '.S 0 05

PAGE 170

Figure 7-1. Molecular mass distribution of water-soluble polyuronides of intact three quarter ripe papaya fruit treated with and without 1-MCP and fresh-cut fruit derived from intact three quarter-ripe fruit treated with and without 1-MCP at day 0 (o) 6 and 10 Polyuronides (0 5 mg galacturonic acid equivalents) were applied to CL-4B-200 (l.5 x 28 cm) column operated with a mobile phase of 200 mM ammonia acetate pH 5 0 Individual fractions were analyzed for polyuronides Data for each fraction were e x pressed as a percentage of the total polyuronides V 0 void volume ; Vi total volume

PAGE 171

157 15 V 0 I Vt! 12 Intact contol 9 --Day 0 6 ---0-Day 6 _,,,_ Day 10 3 0 15 Intact 1-MCP 12 -9 "C C, i.. C, ;;.. 6 0 u C, i.. 3 .... 0 .... .... 0 0 0 15 '-' "' Fresh-cut control :-g 12 u (,: u 9 C 0 i.. 6 3 0 15 Fresh-cut 1-MCP 12 9 6 3 0 0 20 40 60 80 Elution volume (mL)

PAGE 172

Figure 7-2 Molecular mass distribution of CDT A-soluble polyuronides of intact three quarter ripe papaya fruit treated with and without 1-MCP and fresh-cut fruit derived from intact three-quarter ripe fruit treated with and without 1-MCP at day 0 (o) 6 and 10 (T) Polyuronides (0 5 mg galacturonic acid equivalents) were applied to CL-4B-200 (1 5 x 28 cm) column operated with a mobile phase of 200 mM ammonia acetate pH 5 0 Individual fractions were analyzed for polyuronides Data for each fraction were expressed as a percentage of the total polyuronides V 0 void volume ; Vi, total v olume

PAGE 173

159 10 Vo Vt 8 Intact control --eDay 0 6 ---o--Day 6 4 -TDaylO 2 0 10 Inatct 1-MCP 8 6 "O C) :.. 4 C) ;> 0 (.) C) 2 :.. 0 0 '0 10 e Fresh-cut control '-" 8 V, (.) 6 (.) C 4 0 :.. ;:, 2 0 10 Fresh-cut 1-MCP 8 6 4 2 0 0 20 40 60 80 Elution volume (mL)

PAGE 174

Figure 7-3 Molecular mass distribution ofNa 2 CO 3 -soluble polyuronides of intact three quarter ripe papaya fruit treated with and without 1-MCP and fresh-cut fruit derived from intact three quarter-ripe fruit treated with and without 1-MCP at day 0 ( o ) 6 ( ) and 10 ( T ).Polyuronides (0 5 mg galacturonic acid equivalents) were applied to CL-4B-200 (1 5 x 28 cm) column operated with a mobile phase of 200 mM ammonia acetate pH 5 0 Individual fractions were analyzed for polyuronides Data for each fraction were expressed as a percentage of the total polyuronides V 0 void volume ; V. total volume

PAGE 175

161 12 Vo Vt 10 Intact control 8 ---Day 0 6 ---0Day 6 ----TDay 10 4 2 0 12 Intact 1-MCP 10 8 ,..-... 6 '0 Q,; l. 4 Q,; ;;. 0 CJ 2 Q,; l. c,: 0 .... 0 .... 12 '0 Fresh-cut control e 10 '-' "' '0 8 CJ c,: CJ 6 = 0 4 l. ;::i 2 0 12 10 Fresh-cut 1-MCP 8 6 4 2 0 0 20 40 60 80 Elution volume (mL)

PAGE 176

CHAPTER 8 SUMMARY AND CONCLUSION The objectives of the research reported in this study were to characterize physiological responses of both Galia melon and Sunrise Solo' papaya fruits treated with the ethylene action antagonist 1-methylcyclopropene ( 1-MCP) The results indicated that inhibition of ethylene action in these fruits by 1-MCP delayed the rate of ripening of both pre-ripe fruit and fruit in which ripening had been initiated In all cases the use of 1-MCP significantly extended storage and shelf life The study has also shown that most many of the physiological changes associated with Galia and Sunrise Solo fruit ripening including softening pigment changes ethylene production and respiration require functional ethylene re s pon s iveness. Influence of Ethylene-action Inhibition on Ripening of Galia' Melon Fruit Galia' fruit is climacteric, with the ethylene climacteric peak occurring prior to the respiratory climacteric 1-MCP delayed th e onset and peak activity of both respiration and ethylene production and suppressed both respiration and e th y l e ne production during ripening at 20 C. Respiration and ethylene production of ripe fruit decreased during over-ripening at 20 C. The decrease in eth y lene production of ripe fruit was inhibited by 1-MCP while the decrease in respiration was not. Fresh-cut processing (wounding ; in ripe fruit) initially promoted eth y lene production at 5 C that was not significantly affected by prior treatment with 1-MCP Softening during ripening or over-ripening at 20 C was significantly delayed by 1-MCP 1-MCP also delayed softening in both fresh-cut and intact fruit stored at 5 C. Membrane deterioration measured by electrolyte efflux, was 162

PAGE 177

163 evident during ripening and over-ripening and was partially inhibited by 1-MCP irrespective of ripeness stage. Soluble solids accumulation was not affected by 1-MCP during ripening; pH and titratable acidity were slightly or not affected by 1-MCP at 20 ~C The change in skin surface color of 'Galia melon from green to yellow during ripening at 20 C was delayed by 1-MCP however, the color development at 5 C was not influenced by 1-MCP The effect of 1-MCP upon microbial growth was minimal, complex and dependent on ripening stage and type of microorganism l-MCP affected most of the physiological parameters measured in Galia' fruit indicating that 1-MCP efficiently binds ethylene receptors thereby restraining the positive feedback regulation of ethylene regardless of ripening stage Therefore 1-MCP significantly extended storage and storage life of both intact pre-ripe and ripe Galia fruit and storage life of fresh-cut ripe Galia fruit. Influence of Ethylene-action Inhibition on Ripening of 'Sunrise Solo' Papaya Fruit Sunrise Solo papaya fruit exhibited a typical climacteric pattern with peak ethylene production occurring prior to the respiratory climacteric. 1-MCP delayed the initiation of the respiratory and ethylene climacteric, and suppressed ethylene production during ripening at 20 C. 1-MCP had no influence however on ethylene production of either intact or fresh-cut fruit held at 5 C. Wounding resulting from fresh-cut processing also did not affect ethylene production of fresh-cut fruit at 5 C. 1-MCP deferred firmness loss in intact pre-ripe, and intact ripe and fresh-cut ripe fruit (at 5 1-MCP treatment delayed membrane damage during ripening but not during over-ripening 1-MCP slightly suppressed membrane deterioration of wounded tissue through the end of storage at 5 C as well Titratable acidity of 1-MCP-treated fruit was significantly lower during ripening/over-ripening compared with non-1-MCP-treated fruit whereas soluble solids

PAGE 178

164 levels were not influenced by 1-MCP 1-MCP caused a slight increase in pH during ripening at 20 C though the magnitude was minimal The color change from green to yellow of the fruit surface was delayed by 1-MCP during ripening and over-ripening at 20 C. The effects of 1-MCP upon flesh color were minimal at 5 C and only significant at day IO when 1-MCP-treated fresh-cut fruit displayed higher lightness and chroma values (more intense and brighter color) compared with control (no 1-MCP) fruit. 1-MCP did not have a significant effect upon microbial growth at 5 C with the exception of lactic acid bacteria, whose growth appeared to be promoted by 1-MCP The magnitude of this promotion, however was so small as to not be a major concern in fresh-cut fruit maintained under proper temperature management. 1-MCP arrested ripening and over ripening by restricting several physiological characteristics mentioned above proving conclusively that ethylene responsiveness is required throughout ripening Thus, inhibition of ethylene action by 1-MCP was sufficient to extend the postharvest life and windows of edibility for Sunrise Solo regardless of ripening stage Cell Wall Modification of 'Sunrise Solo' Papaya Fruit in Response to Fresh-cut Processing and 1-MCP The recoveries of ethanol insoluble solids did not change during IO days of storage at 5 C and was not influenced by either fresh-cut processing or 1-MCP treatment. On the other hand total uronic acids decreased significantly during the IO days of storage and were not affected by processing or 1-MCP treatment. Water-soluble polyuronides represented the primary pectic fraction followed by CDT A and Na 2 C0 3 respectively Water-and alkali-soluble polyuronides increased in both intact and fresh-cut fruit irrespective of 1-MCP during storage Intact fruit did not exhibit an increase in CDT soluble polyuronides while fresh-cut showed an increase Despite the increase in the

PAGE 179

165 solubility of all pectic fractions, the mol mass of polyuronides changed only s l ightly during storage indicating that inhibition of ethylene action (1-MCP) resulted i n only minor effects on the depolymerization of water chelator and alkali-soluble polyuronides Significant changes were observed in hemicellulosic polysaccharides during storage The 4% KOH fraction contained more than 30% uronic acids (UA) ; however in the 24% KOH fraction UA content was minimal. The greatest proportion of total sugars was extracted by 24% KOH. However consistent with the decrease in total polyuronides, UA content of the 24% KOH fraction decreased significantly with or without 1-MCP or processing Consistent with the involvement of galactosidases / galactans in depolymerization and solubility of hemicelluloses galactose glucose and xylose the predominant non-cellulosic neutral sugars in papaya decreased significantly during storage. Significant changes in neutral sugars were also evident in response to 1-MCP treatment. 1-MCP treatment enhanced the loss of xylose but did not significantly affect the change in glucose galactose mannose and arabinose lt is concluded that cell wall polyuronides and hemicelluloses show significant solubility and depolymerization in response to wounding and the influence of ethylene action inhibition ( 1-MCP) is minimal at affecting the solubility and depol y merization of polyuronides and hemicelluloses

PAGE 180

LIST OF REFERENCES Abdi, N W.B. McGlasson, P. Holford, M Williams, and Y. Mizrahi 1998. Responses of climacteric and suppressed-climacteric plumbs to treatment with propylene and 1-methylcyclopropene. Postharvest Biol. Technol. 14 : 29-39. Abe, K. and A.E. Watada I 991. Ethylene absorbent to maintain quality of lightly processed fruits and vegetables J. Food Sci 56:1493-1496. Adams D O and S F. Yang 1979. Ethylene biosynthesis : identification of aminocyclopropene-1-carboxylic acid as intermediate in the conversion of methionine to ethylene Proc Natl. Acad Sci USA 76 : 170-174 Aharoni Y., A. Copel, and E Fallik I 993 Storing Galia' melons in a controlled atmosphere with ethylene absorbent. Hort Science 28(7) : 725726 Ahvenainen, R I 996 New approaches in improving the storage life of minimally processed fruit and vegetables Trends Food Sci Technol. 7 : 179-187 Akamine E K. and T. Arusimi I 953. Control of postharvest storage decay of fruits of papaya (Carica papaya L.) with special reference to the effect of hot water Proc Amer. Soc Hort Sci 61: 270-274. Akamine E K. and T. Goo 1969 Effects of controlled atmosphere storage of fresh papayas ( C arica papaya L. var. Solo) with special reference to shelf-life extension of fumigated fruits Hawaii Agric Exp. Stn. Bull. I 44 Akamine, E.K. and T. Goo 1971 Relationship between surface color development and total soluble solids in papaya HortScience 14 : 138-139 Alfred R F 1994 A review of the microbiological safety of fresh salads S C.P O A technical bulletin from Silliker laboratorie s, September Ali Z M. N Shu-Yih, R. Othman, L-Y Goh and H. Lazan 1998. Isolation, characterization of papaya fi-galactanases to cell wall modification and fruit softening during ripening Physiol. Plant. I 04: I 05-115 Almeida, D.L.F. 1999 Cell wall metabolism in ripening and chilled tomato fruit as influenced by the pH and mineral composition of the apoplast. PhD Diss ., Dept of Horticultural Sciences, University of Florida, Gainesville 166

PAGE 181

167 Altman, S.A. and KA. Corey 1987 Enhanced respiration of muskmelon fruits by pure oxygen and ethylene. Scientia. Hort 91 :275-281 Alvarez, AM. and W.T. Nishijima 1987 Postharvest diseases of papaya. The Amer Phytopathol. Soc. 71 (8) : 681-686 An, J.F. and R.E Paull. 1990 Storage temperature and ethylene influence on ripening of papaya fruit. J. Amer. Soc Hort. Sci 115(6):949-953 Andre, L.B., G M. Hyde, J.K. Fellman, and J. Varith 2001. Using 1-MCP to inhibit the influence of ripening in impact properties of pear and apple tissue. Postharvest Biol. Technol. 23 : 153-160 Artes F. M.A. Conesco S Hernandez and M.I. Gil. 1999. Keeping quality of fresh-cut tomato. Postharvest Biol. Technol. 17153-162 Ayhan, Z., G W. Chism, and E.R. Richter. 1998 The storage life of minimally processed fresh cut melons J. Food Quality 21 :29-40. Baldwin, E A. M O. Nisperos-Carriedo, and R A. Baker 1995 Edible coatings for lightly processed fruits and vegetables. HortScience 30:35-38 Baritelle, AL. G M. Hyde, J.K. Fellman, and l Varith 2001 Using 1-MCP to inhibit the influence of ripening in impact properties of pear and apple tissues. Postharvest Biol. Technol. 23 : 153-163 Beuchat L.R 1995 Pathogenic microorganisms associated with fresh produce. J Food Protection 59:204-216 Bianco V. and H.K. Pratt. 1977 Compositional changes in muskmelon during development and in response to ethylene treatment. J. Amer. Soc. Hort. Sci. 102(2) : 127-133 Blakaney AB., P Harris R. Henry and B A Stone 1983 A simple and rapid preparation of alditol acetates for Monosaccharide analysis Carbohydrate Res 113 : 291-299. Blumenkrantz, N. and G Asboe-Hansen. 1973. New method for quantitative determination of uronic acids Anal. Biochem 54:484-489 Bostock, R M ., C.S Thomas J.M Ogawa R.E Rice, and J.K. Uyemoto 1987 Relationship of wound-induced peroxidase activity to epicarp lesion development in maturing pistachio fruit. Physiol. Biochem 77 : 275-281.

PAGE 182

168 Bover, J., P. Holford, A. Latche, and J-C. Pech. 2002. Culture conditions and detachment of the fruit influence the effect of ethylene on the climacteric respiration of melon PostharvestBiol. Technol. 26:135-146 Brecht, J.K. 1995 Physiology of lightly processed fruits and vegetables. HortScience 30 : 18-22. Brett, C. and K. Waltron. 1996 Physiology and biochemistry of plant cell walls. London Chapman and Hall. Brummell, D.A. M.H Harpster, P M. Civello, J.M Plays, and A. Bennett. 1999 Modification of expansin protein abundance in tomato fruit alters softening and cell wall polymer metabolism during ripening The Plant Cell 11 :2203-2216 Burns, J.K. 1995 Lightly processed fruit and vegetables : Introduction to colloquium. HortScience 30 : 14 Burton W.G 1982 The physiological implications of structure : water movements, loss and uptake, p 43-68 In: W.G. Burton (ed) Postharvest Physiol. Food Crops Longman Inc New York Cameron A.C., P C. Talasila, and D W Joles 1995 Predicting film permeability for modified atmosphere packing of lightly processed fruits and vegetables HortScience 30 : 25-34. Cameron, A.C. and M.S. Reid 200 I 1-MCP blocks ethylene-inducible petal abscission of P e largonium peltatum but the effect is transient. Postharvest Biol. Technol. 22 : 169-177 Carpita, N C. and D M Gibeaut. 1993 Structural models of primary cell walls in flowering plants: consistency of molecular structures with the physical properties of the wall during growth. Plant J. 3 : 1-30 Carpita N and M. MaCann 2000 The cell wall p 52-108. In: B Buchanan, W Gruissem, and R Jones (eds ) Biochemistry and molecular biology of plants American Society of Plant Physiologists Rockville Maryland Carrinton C.M.S., C.L. Greve, and J M Labavitch 1993 Cell wall metabolism in ripening fruit. Plant Physiol. I 03 :429-434 Chan, H. T. 1979 Sugar composition of papayas during fruit development. HortScience 14(2): 140141 Chan, H.T., S Tam, and S T. Seo. 1981. Papaya polygalacturanase and its role in thermally injured ripening fruit. J Food Sci 46: 190-197.

PAGE 183

169 Chan H.T., S San x ter, and H.M. Couey 1985 Electrolyte leakage and ethylene production induced by chilling injury of papayas HortScience 20(6) : 1070-1072 Chan HT. 1988. Alleviation of chilling injury in papayas HortScience 23(5):868-869 Chan H. T. 1991. Ripeness and tissue depth effects on heat inactivation of papaya ethylene-forming enzyme J. Food Sci. 56(4) : 996-998 Chatenet, C-Du, A. Latche E. Olmos M Charpenteau R Renjeva J.C. Peach and A. Graziana. Spatial-resolved analysis of histological and biochemical alterations induced by water-soaking in melon fruit. Physiol. Plant. 110:248-255 Chen, N M and R.E. Paull. 1986 Development and prevention of chilling injury in papaya fruit. J. Amer Soc. Hort Sci 111 : 639-643 Choi J-R. H-J Bang, S-J ., Hwang J-Y Hong Y-K. Kim, and J-M Lee 200 I. Effects of MCP treatment on postharvest skin-color change s in bl a cks pined cucumber cultivars HortSci e nce 36:4 6 7 Civello P M ., A.LT. Powell A. Sabehat and A.B Bennet. 1 99 9 An expansin gene expressed in ripening strawberr y fruit. Plant Phy s iol. 121 : 1273-1279 Cosgrove DJ 2000 Expansive growth of plant cell plants Plant Physiol. Biochem 3 8: I 09-124 Crane K.E. and C.W Ross 1986 Effects of wounding on c y tokinin activity in cucumber cotyledons Plant Physiol. 82: 1151-11 5 2 Dauny P T. and D C Joyce. 2002 1-MCP improves storability of Queen Co x' and Bramle y' apple fruit HortScience 37(7) : I 082-1085 De Veau E.J.1. 1983 Degradation and solubilization of pectin by r-galactosidases purified from avocado mesocarp Physiol. Plant. 87 : 27 9 -285 Devon Z 1989 Quality maintenance in fresh fruit s and vegetables by controlled atmosphere s, p 174-187 In : J.J Jen ( e d.) Qualit y fa c tors of fruit s and vegetable s. ACS s y mposium se rie s (Sept e mber 15-30 1 9 88) Am e rican Chemical Societ y, Washington, DC. Dong L. H-W Zhou L.S Sonego A. Lers and S Lurie 200 I Ethylene involvement in the cold storage of Flavortop nectarine. Postharvest Biol. Technol. 23: I 05-115. Dong L. S Lurie and H-W Zhou. 2002 Effect of 1-methycyclopropene on ripening of Canino apricots and Royal Zee plums Postharvest Biol. Technol. 24 135-145

PAGE 184

170 Dubois, M K.A. Gilles, J.K. Hamilton, P A. Rebers and F Smith 1956 Calorimetric method for determination of sugars and related substances Anal. Chern 28:350356. Durnville, J.C and S. Fry 2000 Uronic acid-containing oligosaccharins : Their biosynthesis, degradation and signaling roles in non-diseased plant tissues Plant Physiol. Biochem 38 : 15-140 Elkashif, M and D J. Huber 1998 Enzymic hydrolysis of placental cell wall pectins and cell separation (Citrullis lanatus) fruits exposed to ethylene. Physiol. Plant. 73:432-439. Fallik, E., Y. Aharoni, A Copel, V. Radov, S. Tuvia-Alkalai, B. Horev 0 Yekutieli, A Wiseblurn, and R Rage 2000 Reduction of postharvest losses ofGalia melon by a short hot-water rinse. Plant Pathol. 49:333-338 Fallik, E. S Alkali-Tuvia B. Horev A Copel V. Rodov Y. Aharoni, D Ulrich and H. Schulz 2001. Characterization of Galia melon aroma by GC and mass spectrometric sensor measurement after prolonged storage Postharvest Biol. Technol. 22 : 85-91 Faragher J D ., S. Mayak, and E.J. Watchel. 1986 Changes in physical properties of cell membranes and their role in senescence of rose flower petals Acta Hort 181 : 3 71375. Fan X. and J.P Mattheis 1999 Methyl jasmonate promotes apple fruit degreening independently of ethylene action HortScience 34(2) : 3 I 0-312 Fan, X., S.M Blankenship, and J.P. Mattheiss 1999. 1-Methylcyclopropene inhibits apple ripening Journal of the American society for horticultural sciences 124(6):690-695 Fan X., L. Argenta, and J.P Matthies 2000 Inhibition of ethylene action by 1rnethylcyclopropene prolongs storage life of apricots. Postharvest Biol. Technol. 20:135-145 Fan X. and J P Mattheis 200 I 1-Methylc y clopropene and storage temperature influence responses of Gala apple fruit to gamma irradiation Postharvest Biol. Technol. 23 : 143-151. Fan X., L. Argenta and J.P Mattheis 2002 Interactive effects of 1-MCP and temperature on Elberta peach quality HortScience 37(1) : 1 3 4-138 Feng, X., A Apelbaum, E.C. Sisler, and R Gore. 2000 Control of ethylene responses in avocado fruit with l-rnethylcyclopropene. Postharvest Biol. Technol. 20: 143-150

PAGE 185

171 Fischer, R L. and AB. Bennett. 1991 Role of cell wall hydrolyses in fruit ripening Annu Rev Plant Physiol. Plant. Mol. Biol. 42 : 350-356. Flath, R A D M. Light, E B. Jang T.R Moon, and J.O John 1990 Headspace examination of volatile emission from ripening papaya (Carica papaya L. Solo Variety). J. Agric Food Chem. 38: I 060-1063 Flores, F.B., M. Concepcion, M.C. Martinez-Madrid, F.J Sanchez-Hidalgo, and F. Romojaro 2001. Differential, rind and pulp ripening of transgenic antisense ACC oxidase melon Plant Physiol. Biochem 39 :3 7-43. Flugel, M and J. Gross. 1982 Pigment and plastid changes in mesocarp and exocarp of ripening muskmelon, C ucumis me/o cv Galia. Angew Bot. 56 : 393-406 Forney C. 1990 Ripening and solar exposure alter polar lipid fatty acid composition of 'Honey Dew' muskmelons Hort Science 23 : 2457-2461 Francis, G.A C. Thomas, and D. O Beirne 1999 The microbiological safety of minimally processed vegetables In J. Food Sci Tech no I. 34( I) : 1-22 Fritzemeier, K-H, C. Cretin, E. Kombrink, F. Rohwer J. Taylor, D Schell and K. Holbrook. 1987 Transient induction of phenylalanine ammonia-lyase and 4coumarate : CoA ligase mRNAs in potato leaves infected with virulent or avirulent races of Phytopthora i11.festa11s Plant Physiol. 85 : 34-41 Giovannoni, J.J ., D DellePenna AB ., Bennett, and R L. Fischer. 1989 Expression ofa chimeric polygalacturonase gene in transgenic rin (ripening inhibitor) tomato fruit results in polyuronide degradation but not fruit soften in g. Plant Cell I : 53-63 Golding, J.B ., D Shear S.G. Wyllie and W B. Mcglasson 1998 Application of 1-MCP and propylene to identify ethylene-dependent ripening process in mature banana fruit. Postharvest Biol. Technol. 14 : 87-98 Gonzales-Aguilar G.A J. Fortiz, R Cruz R Baez and C.Y Wang 2000 Methyl jasmonate reduces chilling injury and maintains post harvest quality of mango fruit. J Agr Chem. 48 : 515-519 Goodwin T.W 1983 The plant cell wall, p 55-91 In : T.W Goodwin (ed ). Introduction to plant biochemistry. Pergamon Press Ltd UK. Grierson, D. and G.A. Tucker I 983. Timing of ethylene and polygalacturanose synthesis in reaction to the control of tomato fruit ripening Plant Physiol. 157174-179. Gross K.C. and C.E Sams. 1984 Changes in cell wall neutral sugar composition during fruit ripening: A species survey. Phytochem 23 : 2457-2461

PAGE 186

172 Guerzoni M.E., A Gianotti, M R Corbo, and M. Sinigaglia 1996 storage life modeling for fresh cut vegetables Postharvest Biol. Technol. 9 : 195-207. Hammond-Kosack, K.J and D G Jones 2000. Responses to plant pathogens, p 11021139 In : B. Buchanan W. Gruissem, and R. Jones (eds ) Biochemistry and molecular biology of plants American Societ y of Plant Physiologists Rockville Maryland. Hardenburg, R.E., A.E Watada, and C.Y Wang 1986 The commercial storage of fruits vegetables mad florists and nursery stocks U S D A. Agric Handbook No 66 : 133 Harris, D R., J.A. Seberry R.B.H Wills, and L.J. Spohr 2000 Effect of fruit maturity of 1-methylcyclopropene to delay the ripening of bananas. Post harvest Biol. Technol. 20 : 303-308 Hoeberichts F A. L.H W Van Der Plas, and E.J Waitering 2002 Eth y lene reception is required for expression of tomato ripening-related genes and associated physiological changes even at advanced stag e s of ripening Post harvest Biol. Tech no I. 26 : 125-133 Hofman P J., M Jobin-Decor G F Meiburg A.J. Macnish and D C Joyce 2001. Ripening and quality responses of avocado custard apple mango and papaya fruit to 1-methylcyclopropene Austral. J. Expt. Agric 41 : 567-572 Hong J.H. and K.C. Gross 1998 Surface sterilization of whole tomato fruit with sodium hyperclorite influences subsequent postharvest behavior of fresh-cut slices PostharvestBiol. Technol. 13 : 51-58 Hong J.H and K.C. Gross 2000 Involvement of eth y lene in de v elopment of chilling injury in fresh-cut tomato slices during cold storages J Amer Soc. Hort Sci. 125 (6):736-741 Hong J.H D.J Mills, C.B Coffman J.D Anderson M J. Camp and K.C. Gross 2000 Tomato cultivation systems affect subsequent quality of fresh-cut fruit slices J. Amer Soc Hort Sci 125(6) : 729-735 Hubbard N.L ., SC. Huber, and D.M Pharr 1989 Sucro s e phosphate synthase and acid invertase as determinants of sucrose concentration in developing muskmelon ( C ucumis m e lo L.) Plant Physiol. 91 : 1527-1534 Hubbard N L D M Pharr SC. Huber 1990 Sucrose metabolism in ripening muskmelon fruit as affected by leaf area J. Amer Soc Hort Sci 115 : 798-802 Huber D.J 1983 Polyuronide degradation and hemicellulose modification in ripening tomato fruit. J Arn. Soc Hort Sci. I 08 405-40 9.

PAGE 187

173 Huber D.J 1984 Strawberry fruit softening : The potential roles of polyuronides and hemicelluloses J. Food Sci 49 : 1310-1315 Huber D.J 1991. Acidified phenol alters tomato cell wall pectin solubility and calcium content. Phytochem 30 : 2523-2527. Huber, D.J Y Karakurt, and J. Jeong, 200 I Pectin degradation in ripening and wounded fruits Brazilian J. Plant Physiol. 13 : 224-241 Huber D.J., J. Jeong, and L C. Mao. 2003. Softening ofripening fruits in response to 1methylcyclopropene applications Acta Hort (In press) Hurts W C. 1995 Sanitation of lightly processed fruits and vegetables HortScience 30 : 22-24 Ikediobi C.O R L. Chevarajan and A.I. Ukoha. 1989 Biochemical aspects of wound healing in yams (Di osc or e a spp). J. Sci. Food Agr 48: 131-139. International Fresh-cut Produce Association 2002 Fresh-cut facts < http :// www fresh cuts.org ./ fcf html. (12 February 2003) Jacomino AP ., R A. Kluge and A. Brackmann 2002 Amadur e cimento e s ene s cencia de mamso com 1-metilciclopropeno Scientia Agricola 59(2) : 303-308 Jeong J. D.J Huber and S Sargent, 2002 Influence of 1-meth y lcyclopropene (1MCP0 on ripening and cell-wall matrix polysaccharides of avocado (P e rs e a Am e rica) fruit. Postharvest Biol. Technol. 25, 241-256 Jiang Y. D C. Joyce and A. J. Macnish 1999a Extension of the storage life banana fruit by 1-methylcycloppropene In combination with pol y ethylene bags Postharvest Biol. Technol. 16:187 1 9 3 Jiang Y. D C. Joy c e and A.J. Macnish 1 99 9b Responses of banana fruit to treatment with 1-methylcycloprpen e. Plant Growth R e gul. 28:77-82 Jiang Y. D.C. Joyce and A.J. Macnish 2000 Effect of abscisic acid on banana fruit ripening in relation to the role of ethylene J. Plant Growth Re g ul. 1 9: I 06-111 Jiang Y. and J. Fu. 2000 Ethylene regulation of fruit ripening: Molecular aspects Plant Growth Regulat 30 : 193-2000 Jiang, Y and D C. Joyce 2000 Effect s of 1-meth y lc y clopropene alone and in combination with polyeth y lene bag s on the po s t harvest life of mango Annu Appl. Biol. 137 : 321-327

PAGE 188

174 Jiang Y. D C. Joyce and L.A. Terry 2001. 1-M e h y lc y clopropene treatment affects strawberry fruit decay Postharvest Biol. Technol. 23 : 227-232 Jiang, Y. and D C. Joyce 2002 1-Methylcyclopropene treatment effects on intact and fresh-cut apple J. Hort Sci Biotechnol. 77 (I) : 19-21. Jiang, W ., Q Sheng, X-J. Zhou M-J. Zhang, and X-J. Liu 2002. Regulation of detached coriander leaf senescence by 1-methylcyclopropene and ethylene Post harvest Biol. Technol. (in press) Jocelyn K.C. K.A. Hadfield J.M. Labavitch, and AB Bennett. I 998. Temporal sequence of cell wall disassembly in rapidly ripening melon fruit Plant Physiol. 117:345 361. Jones T.M and P Albersheim 1991 Acidified phenol alters tomato cell wall pectin solubility and calcium content. Photochem 30 : 2523-2527 Karakurt Y. and D.J Huber 2002 Cell wall-degrading enzymes and pectin solubility and depolymerization in immature and ripe watennelon (Citrullis lanatus) fruit in response to exogenous ethylene Phy s iol. Plant. 116:398-405 Karakurt Y. and D.J Huber 2003 Activities of several membrane and cell-wall hydrolases, ethylene bios y nthetic enz y mes and cell wall polyuronide degradation during low-temperature storage of intact and fresh-cut papaya ( C arica papa y a) fruit. Postharvest Biol. Technol. (in press) Karchi Z. 200 Development of melon culture and breeding in Israel. Acta Hort 510 : I 317 Kimura M ., D B Roriguez-Ama y a and S.M Yokoyama. 199 I Cultivar differences and geographic effects on the carotenoid composition and vitamin A value of papaya. Lebensmittel-Wissenchaft Technol. 24:415-418 King, AD. and H.R Bolin. 1989 Physiological and microbiological storage stability of minimally processed fruits and vegetables Food Tech no I. 43 : 132-136 King G A. and E M O'Donoghue 1995 Unrevealing senescence : new opportunities for delaying the inevitable in harvested fruit and vegetables Trends Food Sci. Technol .. 6 : 385-389 Ku, V.V.V and R.B.H. Wills 1999. Effect of 1-methylcyclopropene on the storage life of broccoli Postharvest Biol. Technol. 17 : 127-132 Ku, V V V ., R B.H. Wills and S Ben-Yehoshua 1999 1-methylcyclopropene can differentially affect the postharvest life of strawberries exposed to ethylene HortScience 34( I): 119-120

PAGE 189

175 Lashbrook C.C., D A. Brummell J K.C. Rose and AB ., Bennett. 1997 Non-pectolytic cell wall metabolism during fruit ripening. In : Giovannoni J J (Ed ) Fruit Ripening Molecular Biology. Harwood Academic, Reading, UK Lazan, H. M.K. Selamat, and Z M Ali 1995 Beta-galactosidase polygalacturonase and pectin esterase in differential softening and cell wall modification during papaya fruit ripening. Physiol. Plant. 95: 106-112 Leach, D.N., V Sarafis, R Spooner-Hart, and S G. Willey 1989 Chemical and biological parameters of some cultivars C ucumi s m e lo Acta Hort. 247 : 353-357 Lelievre, J-M A. Latche B. Jones M Bouzayen and J-C Pech 1997 Ethylene and fruit ripening. Physiol. Plant. 1001:727739. Leshem Y. and A. Halevy I 988 Plant senescence p 54-83 In : A. Leshem, A. Halevy and J. Frenkel (eds ) Process and control of plant senescence Elseiver New York 1986 Lester G 1988. Comparison of Honey Dew and netted muskmelon fruit tissues in relation to storage life HortScience 23( I) : I 80-182 Lester G E and J.R Dunlap 1985 Physiological changes during development and ripening of Perlita' muskmelon fruits Scientia Hort 26 323-331 Lester G. and E Stein. 1993 Plasma membrane physicochemical changes during maturation and postharvest storage of muskmelon fruit. J Amer Soc Hort Sci 118(2) : 223-227. Liang X.W ., M Dron C.L. Cramer R.A. Dixon and C.J Lamb 1989 Differential regulation of phenylalanine ammonia lyase genes during development and by environmental stress J. Biol. Biochem 264 : 14486-144 9 2 Leverentz B ., W S Conway W.J Janisiewicz R A. Saftner, and M J. Camp 2002 Effect of combining MCP treatment heat treatment, and bi control on the reduction ofpostharvest decay of Golden Deli c ious apples Po s tharve s t Biol. Technol. (in press) Lipton, W.J and Y. Aharoni 1979 Chilling injury and ripening of Honey Dew muskmelons stored at 2 5 or 5 C after ethylene treatment at20 C. J. Amer. Soc Hort Sci I 04 : 327-330 Lukasik J. M L. Bradley T.M Scott W-Y Hsu S R Farrah and M L. Tamplin 2000. elution detection and quantification of Polio I Bacteriohages Salmonal/ e Montevido, and Esch e ria coli O 157 H7 from seeded strawberries and tomatoes J. Food Protection 64(3):292-297

PAGE 190

176 Lulai, E.C. 1988 Induction of lipoxygenase activity increases a response to tuber wounding. Amer. Potato J. 65:490. Luna-Guzman, I., M. Cantwell, and D M Barrett. I 999 fresh-cut muskmelon: effects of CaCh dips and heat treatments on firmness and metabolic activity Postharvest Biol. Technol. 17 : 201-213. Luna-Guzman, I. and D M. Barrett. 2000 Comparison of calcium chloride and calcium lactate effectiveness in maintaining shelf stability and quality of fresh-cut muskmelons Postharvest Biol. Technol. 19 :6 172. Lurie, S and R. Ben-Arie. 1983 Microsomal membrane changes during the ripening of apple fruit. Plant Physiol. 73 636 638. Macleod, A.J. and N M. Pieris 1983 Volatile components of papaya (Carica papaya L.) with particular reference to glucosinolate products J Agric Food Chem. 31: I 005I 008. Macnish A.J., D C. Joyce, P.J Hofman D.H. Simons and M S Reid 2000. 1methylcyclopropene treatment efficacy in preventing ethylene perception in banana fruit and grevi lle a and waxflower flowers Austral J. Agr. Expt. Bot. 40:471-481 Maharaj R. and C.K. Sankat. 1990. Storability of papayas under refrigerated and controlled atmosphere. Acta Hort. 269 : 375-383 Marangoni, A.G ., T. Palma and D.W Stanley. 1996 Membrane effects in postharvest physiology Postharvest Biol. Technol. 7 : 193-217 Mathooko, F M Y. Tsunashima W Z.O Owino Y. Kubo and A Inaba 200 I Regulation of genes encoding ethylene enzymes in peach (Prunus persica L.) fruit by carbon dioxide and 1-methyelcyclopropene Postharvest Biol. Technol. 21 :265 281. McCarthy M A. and R H Matthews 1994. Nutritional quality of fruits and vegetables subject to minimal process p 313-326 In : R.C. Wiley (ed ). Minimally processed refrigerated fruits and vegetables. Chapman and Hall, New York. McColl um T.G ., D.J Huber and D.J Cantliffe 1989 Modification of polyuronides and hemicellulose during muskmelon fruit softening. Physiol. Plant. 76:3003-308 McGlasson W B. and H.K. Pratt. 1963 Fruit set patterns and fruit growth in muskmelon ( C ucumis melo L. var. reticulates Naud) Proc Amer. Soc Hort Sci. 83;495-505

PAGE 191

177 Miccolis, V. and M.E. Salveit. 1995. Influence of storage period and temperature on the postharvest characteristics of six melon (Cucumis melo L. inodorous group) cultivars. Postharvest Biol. Technol. 5:211-219 Miller A.R., J.P. Dalmasso, and D Kretchman 1987. Mechanical stress, storage time, and temperature influence cell wall-degrading enzymes, firmness, and ethylene production by cucumbers. J. Amer. Soc Hort. Sci. 112(4):666-671. Miller, A.R. and T.J. Kelly 1989. Mechanical stress stimulates peroxidase activity in cucumber fruit. Hort Science 24( 4):650-652. Miller, AR 1992 Physiology, biochemistry and detection of bruising (mechanical stress) in fruits and vegetables Postharvest News and Information 3 (3):53N-58N MirN.A., E. Curell, N Khan, M Whitaker and R M. Beaudry 2001 Harvest maturity, storage temperature, and 1-MCP application frequency alter firmness retention and chlorophyll fluorescence of Redchief Delicious apples. J. Amer. Soc Hort Sci 126(5) : 618-624 Mactezuma E ., L. D. L. Smith, and K. C. Gross. 2003 Effect of ethylene on mRN A abundance of three ~ galactosidase genes in wild type and mutant tomato fruit. Postharvest Biol. Technol. (in press) Morton J.F 1987. The papaya, p 336-346 In : J F Morton (ed ) Fruits of warm climates. Media, Incorporated Greensboro N .C. Mostofi, Y., P M.A. Toivonen H Lessani M Babalar and C. Lu 2003 Effects of 1methycyclopropene on ripening of greenhouse tomatoes at three storage temperatures Postharvest Biol. Technol. (in press) Muller R ., E.C. Sisler, and M Serek 2000 Stress induced ethylene production ethylene biding and the response to the ethylene action inhibitor 1-MCP in miniature roses Scientia Hort. 83 :51-59. Mullins E.D ., T.G. McCollum, and R .E. McDonald 2000 Consequences on ethylene metabolism of inactivating the ethylene receptor sites in diseased non-climacteric fruit. Postharvest Biol. Technol. 19: 155-164 Nakasone, H.Y 1986 Papaya p 227-301. In: S P. Moose (ed ). Handbook of fruit set and development CRC press Boca Raton FL. Nakatsuka, A. S Shiomi Y. Kubo and A. Inaba I 997 Expression and internal feedback regulation of ACC synthase and ACC oxidase genes in ripening tomato fruit. Plant Cell Physiol. 38(10) : 1103-J I 10.

PAGE 192

178 Nguyen-the C. and F Carlin 1994 The microbiology of minimally processed fresh fruits and vegetables. Critical Rev Food Sci Nut. 34(4) : 371-401. O Connor-Shaw R.E. R. Roberts, AL Ford, and S.M Nottingham 1994 storage life of minimally processed honeydew kiwifruit papaya pineapple and muskmelon J. Food Sci. 59 (6) : 1202-1216 O Connor-Shaw R.E. R. Roberts A.L. Ford and S M Nottingham 1996. Changes in sensory quality of sterile muskmelon dice stored in controlled atmosphere J. Food Sci 61(4) : 847-851 Paull, R.E. and N.J Chen. 1983 Postharvest variation in cell wall-degrading enzymes of papaya (Carica papaya L.) during ripening Plant Physiol. 72 : 382-385 Paull R.E 1993 Pineapples and papa y as p. 303-323 In : G Seymour and G Tucker (eds ) Biochemistry of fruit ripening Published by Chapman Hall London UK Paull RE and N.J. Chen I 997 Minimal processing of papa y a ( C ari c a papa ya L.) and the physiology of halved fruit. Postharvest Biol. and Technology 12:93-99 Paull, R.E, K. Gross and Y. Qiu 1999. Changes in papaya cell walls during fruit ripening Postharvest Biol. Technol. I 6:79-89 Peiser, P 1989 Effect 2,5-norbornadiene upon ethylene biosynthesi s in midclimacteric carnation flowers Plant Physiol. 90 : 21-24 Perez, S. K. Mazeau, C. Catherine and H-du Penhoat. 2000 The three-dimensional structure of the pectic polysaccharides Plant Physiol. Biochem 38:37-55 Picchioni G.A. A.E. Watada B.D Whitaker and A. Reyes 1996. Calcium delays senescence-related membrane lipid changes and increase net synthesis of membrane lipid components in shredded carrots. Postharvest Biol. Technol. 9 : 235-245. Picchioni G A. A.E. Watada S Ro y, B D Whitaker and W P Wergin 1994 Membrane lipid metabolism cell permeability and ultrastructural changes in lightly processed carrots J Food Sci 59(3) : 5 9 7-601 Porat, R. B Weiss L. Cohen A. Daus, R Goren and S Droby 1999 Effect s of ethylene and 1-methylcycloprpene on the postharvest qualities of Shamouti oranges Postharvest Bio I Tech no I 15 : I 5 5163 Portela S I. and M.I. Cantwell. 1998 Quality changes of minimally processed honeydew melons stored in air or controlled atmosphere Postharvest Biol. Technol. 14 : 351357.

PAGE 193

179 Portela, S l. and M l. Cantwell. 2001. Cutting blade sharpness affects appearance and other quality attributes of fresh-cut muskmelon melon J. Food Sci 66(9): 12651270 Pratt H.K. J.D. Goeschl and F W Martin 1977. Fruit growth and development ripening and the role of ethylene in Honey Dew' muskmelon J. Amer Soc Hort Sci. 102 : 203-210 Pre-Aymard, C. A. Weksler, and S. Lurie. 2002 Responses of' Anna' a rapidly ripening summer apple to 1-methylcyclopropene. Postharvest Biol. Technol. (in press) Qi, L., T. Wu and A.E. Watada, 1988 Quality changes of fresh-cut honeydew melons during controlled atmosphere storage J. Food Quality 22 : 513-521 Ranwala, AP ., C. Suematsu, and H Masuda 1992 The role of p-galactosidases in the modification of cell wall components during muskmelon fruit ripening Plant Physiol. 100: 1318-1325 Reid, M.S T.H. Lee H.K. Pratt and C.O Chichester. 1970 Chlorophyll and carotenoids changes in developing muskmelon J Amer Soc Hort Sci 95 : 946-948 Reid M S 1985 Ethylene in postharvest technology pp 68-74 In : A.A. Kader R F Kasmire, F G Mitchell and M.S. Reid (eds) Postharvest Technology of Horticultural Crops UC at Berkley Reyes, V G. 1996 Improved preservation systems for minimally processed vegetables Food Austral. 48 (2) : 87-90 Riter W D 1994 Structure synthesis, and function of the plant cell wall p. 995-988 In : E M. Meyerowitz and C.R. Somverville (eds ) Arabidopsis Cold Spring Hrabor Press Robin S G. Seymour, and G A. Tucker 1989 Inhibition of cell wall degradation by silver (I) ions during ripening of tomato fruit. J. Plant Ph y siol. 134 : 524-516 Rolle R.S. and G.W Chism 1987 Physiological consequences of minimally processed fruits and vegetables J Food Quality IO 157-177 Roming W R. 1995. Selection of cultivars for lightl y processed fruits and vegetables. HortScience 30:38-40 Rose, J.K.C., H.H. Lee, and AB Bennett. 1997 Expression of a divergent expansin is fruit-specific and ripening-regulated Proc Natl. Acad Sci USA 94:5955-5960.

PAGE 194

180 Rose, J.K.C., K.A. Hadfield J.M Labavitch, and A.B Bennett. 1998 Temporal sequence of cell wall disassembly in rapidly ripening melon fruit. Plant Physiol. 117:3455361. Ryan, C.A. 1988 Oligosaccharides as recognition signals for the expression of defensive genes in plants. Biochem 27 : 8879-8883 Saftner, R.A. J.A. Abbot, and W S. Convay 2003 Effects of 1-methylcyclopropene and heat treatments on ripening and postharvest decay in Golden Delicious apples J. Amer Soc Sci 128(1) : 120-127 Sakai T. and Y. Nakagawa 1988 Diterpenic stress metabolites from cassava roots. Photochem 27:3769-79 Sakurai N. and D.J Nevins. 1993 Changes in physiological properties and cell polysaccharides of tomato (L y persicum e s c ult e nlum) pericarp tissues Pysiol. Plant. 89 : 681-686 Saltveit M E. I 993. Internal carbon dioxide and ethylene levels in ripening tomato fruit attached to or detached from the plant. Physiol. Plant. 89 : 204-210 Salveit M E. 1999. Effect of ethylene on quality of fresh fruit and vegetables Postharvest Biol. Technol. 15 : 279-292 Salveit M E 2000. Wound induced changes in phenolic metabolism and tissue browning are altered by heat shock. Postharvest Biol. Technol. 21 :61-69 Salveit M.E. and L.L. Morrris 2000 Chilling injury of horticultural crops ln: C. Y Wang (ed) CRC Press Inc Boca Rotan FL Sankat, C.K. and R Maharaj. 1997 Papaya p 167-189. In: S Mitra (ed.). Postharvest physiology and storage of tropical and subtropical fruits CAB International, Wallinford, Oxon UK Sanxter S S ., H. Yamamoto D G Fisher and H T. Chan 1992 Development and decline of chloroplas t in exocarp of C arica papaya. Can J Bot. 70:364-373 Sasser, M.J 1990 Identification of bacteria through fatty acid analysis Pp I 99-204 In : Methods in Phytobacteriology, Z Klement and D Sands (eds ) Academia Kiado Budapest Hungary Selvarajah, Y., D K. Pal D Subranmanyam, and P.A. Iyer I 982. Changes in the chemical composition of four cultivars of papaya ( C aricapapa y a L.) during growth and development J. Amer. Hort Sci. 57: 135-143.

PAGE 195

181 Selvarajah, S., A.O. Bauchot, and P. John. 200 I. Internal browning in cold-stored pineapples is suppressed by a postharvest application of 1-methyclcyclopropene. Postharvest Biol. Technol. 23 : 167-170. Schlimme, V. D. 1995. Marketing lightly processed fruits and vegetables. HortScience 30:15-17. Serek, M E.C. Sisler, and M S Reid 1994. Novel gaseous ethylene binding inhibitor prevents ethylene effects in potted flowering plants J. Amer Soc Hort Sci. 199(6) : 1230-1233 Serek, M. and E.C Sisler I 995 Effect of 1-methylcyclopropene ( 1-MCP) on the vase life and ethylene responses of cut flowers. Plant Growth Regul. I 6 : 93-97. Serek M ., E.C. Sisler T. Tsipora and S Mayak 1995a 1-methylcyclopropene prevents bud, flower and leaf abscission ofGeralton waxflower HortScience 30(6) : 1310 Serek, M ., G. Tamari E C. Sisler and A. Borochov 1995b Inhibition of ethylene induced senescence symptoms by 1-methylcycyloprpopene a new inhibitor of ethylene action Physiol. Plant. 94 : 229-232 Seymour G ., S Harding, A. Taylor G Hobson and G Tucker 1987 Polyuronide solubilisation during ripening of normal and mutant tomato fruit Phytochem 29 : 171-1976 Seymour, G B. and W B McGlasson. I 993 Melons p 273-290 In : G Seymour, J Taylor and G Tucker (eds) Biochemistry of fruit ripening Chapman and Hall Cambridge University Press UK Shellie, K.C. 200 I. Reduced ethylene concentration and post harvest quality of transgenic netted melon (Cucumis melo L.) expressing S-adenosylmethionine hydrolase HortScience 36(3):467. Sisler, E.C. and M Serek. 1997 Inhibitors of ethylene responses in plants at the receptor level: Recent developments. Physiol. Plant. 100 : 577-582 Sisler E.C. and M Serek. 1999 Compounds controlling the eth y lene receptor Bot. Bull. Acad Sin 40 : 1-7. Sisler, E.C. M Serek K-A. Rohn, and R Goren 200 I The effect of chemical structure on the antagonism by cyclopropenes of ethylene responses in banana Plant Growth Regul. 33 : 107-110 Shaw N.L. D.J Cantliffe, and B.S. Taylor. 200 I Hydroponically produced 'Galia' muskmelon-What s the secret ? Proc. Fla. State Hort. Soc 114:288-293.

PAGE 196

182 Smith, C.J.S. C.F. Watson, J. Ray C.R Bord P C. Morris W Schuch and G. Grierson, 1988. Anti sense RNA inhibition of polygalacturonase gene expression in transgenic tomatoes. Nature 334:724-726 Smith C.S 2000. Carbohydrate chemistry, p 73-111 In : P.J Lea and RC. Leegood (eds.). Plant biochemistry and molecular biology John Wiley and Sons Ltd Baffins lane, Chichester, West Sussex PO19 IUD, England. Sommer, N.F., R.J Fortlage and D C. Edwards 1992 Postharvest diseases of selected commodities, p.117-160 In : A.A Kader (ed ) Postharvest technology of horticultural crops. Division of Agriculture and Natural Resources University of California Oakland CA. Song, J, M S Tian D R. Dilley, and R M Beaudry 1997 Effect of 1-MCP on apple fruit ripening and volatile production. HortScience 32 : 536 Teitel, D C. Y. Aharoni, and R Barkai-Golan 1989 The use of heat treatments to extent the storage life of Galia melons J Hort Sci. 64(3) : 367-372 Teixeira G H.A J.F Durigan B-H Mattiuz and O Djr Rossi 200 I. Processamento minimo de mamao 'Formaso '. Cienc Technol. Aliment Campinas. 21 ( I ):47-50 Tian M S ., S Prakash H.J. Elgar, H Young, D.B Burmeister and G S Ross 2000 Response of strawberry fruit to 1-methylcycylopropene ( 1-MCP) and ethylene Plant Growth Regul. 32 : 83-90 Trebitsh T. E.E Goldshmidt and J. Riov. 200 I Ethylene induces d e n o va synthesis of chlorophyllase a chlorophyll degrading enz y me in C itrus fruit peel. Proc Natl. Acad Sci USA 90:9441-9445 Tucker G.A. and C.J Brad 1987 Silver ions interrupt tomato fruit ripening J. Plant. Physiol. 127 : 165-169 Tucker, G A. and D Grierson 1987 Fruit ripening. In : Davis D (Ed ) Biochemistry of Plants vol. 12 Academic Press New York pp. 265-318 Tucker G.A. 1993. Texture changes p 17-24. In : G Seymour, J Taylor and G Tucker (eds.). Biochemistry of fruit ripening 1 s t edition Chapman and Hall Cambridge University press UK Varoquaux, P and R C. Wiley 1994 Biological and biochemical changes in minimally processed refrigerated fruit and vegetables p 226-268 In : RC. Wiley (ed ). Minimally processed refrigerated fruits and vegetables Chapman and Hall, N Y.

PAGE 197

183 Vorbam, B., H. Schon, and H. Kindl. 1988. Control of gene expression during induction of cultural peanut cells : mRNA levels, protein synthesis and enzyme activity of stilbene synthase. Plant Mol. Biol. 10:215-243 Wakabayashi, K. 2000. Changes in cell wall polysaccharides during fruit ripening. J. Plant Res 133:231-237 Wang, H. and W R. Woodson. 1989 reversible inhibition of ethylene action and interruption of petal senescence in carnation flower by norbornadiene Plant Physiol. 89: 434-438. Watada, E.A. K. Abe, and N. Yamauchi 1990 Physiological activities of partially processed fruits and vegetables Food Technol. XX: 116, 118, 120-122. Watada, E.A., NP Ko, and D A. Minott. 1996 Factors affecting quality of fresh-cut horticultural products. Postharvest Biol. Technol. 9: 115-125 Watada, E.A. and L. Qi I 999 Quality of fresh-cut produce Postharvest Biol. Tech no I. 15 : 210-205 Watkins C.B., J.F Nock, and B.D. Whitaker. 2000. Responses of early, mid and late season apple cultivars to postharvest application of 1-mathylcyclopropene under air and controlled atmosphere storage conditions. Postharvest Biol. Technol. 19 : 17-32 Wei C.l. L. Look and J.R Kirk 1985 Use of chlorine in the food industry Food Technol. 1 : 107-115 Whitaker, B.D 1991 Growth conditions and ripening influence plastid and microsomal membrane lipid composition in bell paper fruit. J Amer. Soc Hort Sci 116:528688 Whitaker, B.D. 1995 Lipid changes in mature-green bell pepper fruit during chilling at 2 degrees Celsius and after transfer to 20 degrees Celsius subsequent to chilling Physiol. Plant. 93 :683-688 Wills, R B.H and S B Widjanarko 1995. Changes in physiology composition and sensory characteristics of Australian papaya during ripening Austural. J Expt Agr. 35 : 1173-1176 Wills, R B.H. and V V V. Ku. 2002. Use of 1-MCP to extend the time to ripen of green tomatoes and post harvest life of ripe tomatoes Post harvest Biol. Tech no I. 26 : 8595 Wilson C. and W.J Lucas 1988 Wounding and the regulation of apoplasmic retrieval in source leaf tissue of Spinacia o/ e racea L. J. Expt. Bot. 39 : 529-542

PAGE 198

184 Wyllie S.G. and D N. Leach I 990 Aroma volatiles of C ucumis m e lo cv Golden Crispy. J. Agric Food Chem. 38:2042-2044 Yabumoto, K. M. Yamaguchi, and W G Jennings 1978 Production of volatile compounds by muskmelon, C ucumis melo. Food Chem. 3 : 7-15 Zagory D 1999. Effects of post-processing handling and packing on microbial populations Postharvest Biol. Tech no I. I 5 : 313-321. Zauberman, G ., Y. Fuchs, I. Rot and A Wexler J 988 Chilling injury peroxidase, and cellulase activities in the peel of mango fruit at low temperature HortScience 23 (4) : 732-733 Zhao M ., J. Moy, and R E Paull. 1996 Effect of gamma-irradiation on ripening papaya pectin Postharvest Biol. and Technol. 8:209-222 Zheng X .. Y and D Wolf 2000 Ethylene production shelf-life and evidence of RFLP polymorphisms linked to ethylene genes in melon ( C ucumis m e lo L.) Theor Appl. Genet IO I : 613-624 Zhong G Y. M. Huberman X.Q Feng E C. Sisler D Holland and R Goren 2001. Efect of 1-methylcyclopropene on ethylene-induced abscission in citrus Physiol. Plant. 1 I 3 : 134-141. Zink D L. I 997 The impact of consumer demands and trends on food processing Emerging Infectious Dis. 3(1): 1-3

PAGE 199

BIOGRAPHICAL SKETCH Muharrem Ergun was born on March 20 1971 in Kanya, Turkey He received his primary and secondary education in Konya, graduating from Gazi High School in 1988 He attended Ege University, Izmir Turkey, and was awarded a BS degree in horticulture in 1992 In 1997 he entered a graduate program (in the Horticultural Sciences Department) at the University of Nebraska Lincoln where he was awarded an MS degree in 1998 Upon completing his PhD degree he will be employed as an association professor at a university in Turkey. 185

PAGE 200

I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully ade uate, in scope and quality as a dissertation for the degree of Doctor of P 1 a1r Professor of Horticu : mal Science I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate in scope and quality as a dissertation for the degree of Doctor of Philosophy. I /t~ ( J Jerr f A. Bart Associate Professor of Plant Pathology I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality as a dissertation for the degree of Doctor of Philosophy. ~ Daniel J. Cantliffe I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate in scope and quality, as a dissertation for the degree of Doctor of Philosophy. Charles L. Guy Professor of Horticultural Sci I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality as a dissertation for the degree of Doctor of Philosophy. L.dk./ Steven 7 A. Sargent 7 Professor of Horticultural Science

PAGE 201

This di ssertat ion was submitted to the Gradu a te Faculty of the College of Agr i c ultu ra l and Life Sciences and to the Graduate School and was accepted as partial fu l fi llm e nt of the r e quir eme nts for the degree of Doctor of Phil osop h Aug u s t 2003 ~. ( ); Dean College of Ag ric Sciences De a n Graduate School