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,. -; LIBRARY
540194 30 Michigan State
University

 

 

 

This is to certify that the
dissertation entitled
DEVELOPMENT OF A l-METHYLCYCLOPROPENE

PACKAGE DELIVERY SYSTEM
TO CONTROL TOMATO RIPENING

presented by

Youn Suk Lee

has been accepted towards fulfillment
of the requirements for

Ph-D- degreein Eackagjng

 

Major professor

Dme March 26, 2003

 

MSU i: an Affirmative Action/Equal Opportunity Institution 0-12771

PLACE IN RETURN Box to remove this checkout from your record.
To AVOID FINES return on or before date due.
MAY BE RECALLED with earlier due date if requested.

 

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6/01 cJCIRC/Datoouopes-pJS

DEVELOPMENT OF A l-METHYLCYCLOPROPENE PACKAGE DELIVERY
SYSTEM TO CONTROL TOMATO RIPEN IN G

By

Youn Suk Lee

A DISSERTATION

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY
School of Packaging

2003

ABSTRACT

DEVELOPMENT OF A l-METHYLCYCLOPROPENE PACKAGE DELIVERY
SYSTEM TO CONTROL TOMATO RIPENING

By

Youn Suk Lee

l-methylcyclodextrin (1 -MCP) was used to prolong the freshness of tomatoes
during postharvest storage. The effect of storage temperature and l-MCP treatment
conditions on the ripening process of tomatoes was evaluated. Change in two major
tomato pigment (chlorophyll and lycopene) contents in tomatoes was also investigated.
Sorption behavior for the l-MCP/adsorbing agents was studied using inverse gas
chromatography. The specific retention volume and various thermodynamic parameters
relating to adsorption of l-MCP on the adsorbing agents were calculated. Adsorption
and l-MCP release studies from the adsorbing agents in the sachet system were
conducted to evaluate delivery of the l-MCP gas to the tomatoes under the environmental
conditions within the package.

Skin color, firmness. fruit weight, and ethylene production were used as quality
indicators for the stored tomatoes. Total chlorophyll and lyc0pene contents in tomatoes
were quantified using a specific extinction coefficient method. Sorption isotherms of
l-methylcyclopropene (l-MCP) on silica gel. Tenax-TA, and activated clay were
determined at low sorbate concentration. Sachets made from Tyvek®. paper, LDPE, and
PVA materials were fabricated to contain silica gel and activated carbon. The l-MCP

release study was performed using a closed system under two different environmental

release study was performed using a closed system under two different environmental
conditions, dry air (0%RH) and 90%RH. The partitioning of l-MCP between the
gas/polymer matrix was determined for several adsorbing agents, and in sachet materials
to estimate the adsorb ability of l-MCP in dry air at 23°C. The water and 1-MCP
permeability of the sachet pouch film were measured.

Once-a-day l-MCP treatment at 10°C was very effective in retarding changes in
the skin color of the tomatoes. Exposure of tomatoes to l-MCP gas at 10°C, using a
once-a-day method was the most effective in delaying chlorophyll degradation and
lyc0pene synthesis. The sorption isotherms followed Henry’s law, and behaved
according to the binding site theory. Silica gel had a much higher number of binding
sites for l-MCP, compared to Tenax-TA and activated clay agents. PVA sachets
containing silica gel indicated slow release of l-MCP. The amount of l-MCP released
from the PVA sachet containing silica gel at 90%RH was larger than the amount of 1-
MCP released at dry air condition.

The results showed that combination of l-MCP treatment with low storage
temperature was very effective in delaying color change in tomatoes. PVA sachets
containing silica gel have potential use for slow release of l-MCP from the experimental
results in a closed system. Delivering the l—MCP gas to the tomatoes, from the sachet
containing an adsorbing agent may help maintain the freshness of tomatoes during

postharvest storage.

ACKNOWLEDGMENTS

First of all, I would deeply like to acknowledge Dr. Bruce Harte, my adviser for
his help and patience. His encouragement and endless support enabled me to complete
my doctoral program. Without his assistance the completion of this dissertation could not
be possible.

Especially, I would like to thank Dr. Randolph Beaudry, Department of
Horticulture, as a leader of this research project for his guidance and valuable suggestions.
I would also like to thank Dr. Carl Lira, Department of Chemical Engineering, of his
valuable advice for the interaction study of l-MCP on the adsorbing agent. Special
thanks also go to my committee members, Dr. Theron Downes and Dr. Paul Singh for
their comments.

I would like to express my heartfelt thanks (sincere gratitude) to Dr. Jack Giacin
and Dr. Ruben Hernandez. Especially, I am indebted to Dr. Jack Giacin for his endless
care and encouragement.

My appreciation is extended to the financial support, the green project and the
fellowship from school of packaging for conducting this research.

I also want to thank my friends and fellow graduate students in the school of
packaging for their help. I would like to thank Dr. long-Koo Han for his encouragement.
I also thank Dr. Sastry and Mauricio Canoles in postharvest laboratory for helping me to
perform the tomato experiments.

I wish to express my gratitude to my family, especially my father, Hi Rak Lee. my
mother, Gyu Young Cho, and my parents-in-laws for their love and support. Also, I am
forever indebted to my brothers who supported me in spirit.

Finally, I would like to thank my wife, AnSook Kim, for her love, understanding
and encouragement. What’s more, my lovely daughter, J ayoung Lee, in her own way,

continuously provides love, joy, and inspiration.

TABLE OF CONTENTS

LIST OF TABLES VIII

LIST OF FIGURES ...................................................................................................... ix
SECTION I: INTRODUCTION .................................................................................... 1
SECTION 11: REVIEW OF LITERATURE .................................................................. 8
Tomato fruit ........................................................................................................ 8

Fruit ripening ...................................................................................................... 9
Pigments for color development in tomato fruits ............................................. 13
Chlorophylls .......................................................................................... 13

Lycopene ............................................................................................... 15

Ethylene ............................................................................................................ 1 7
l-methylcyclopropene (l-MCP) ....................................................................... 19

Other ethylene inhibitors ................................................................................... 22
Active packaging applications for fresh produce .............................................. 23
Inverse gas chromatography (IGC) ................................................................... 25

IGC analysis techniques ........................................................................ 26

Sorption isotherms (elution chromatography technique) .................................. 28
Specific retention volume (V°g) values and thermodynamic parameters ......... 30
Specific retention volume (V°g) ............................................................ 32
Thermodynamic parameters .................................................................. 33

Free energy of sorption (AG5) .................................................... 33

Enthalpy of sorption (AHS) ......................................................... 34

Entropy of sorption (ASS) ........................................................... 34

Adsorbing agents .............................................................................................. 35
Partitioning in air and adsorbing agent matrix .................................................. 36

Gas permeation ................................................................................................. 37
Permeability measurement methods ................................................................. 41
Gravimetric method ............................................................................... 41

Isostatic method ..................................................................................... 42
Quasi-isostatic method ........................................................................... 43

Active compound release system ...................................................................... 44

SECTION III: EFFECT OF SINGLE AND REPEATED
l-METHYLCYCLOPROPENE (l-MCP) EXPOSURES ON THE RIPENING OF
TOMATOES (LYCOPERSICON ESCULENTUM MILL.) DURING STORAGE AT

10, 15, AND 20°C ....................................................................................................... 46
Abstract ........................................................................................................................ 46
Introduction .................................................................................................................. 47
Materials and methods ................................................................................................. 48
Materials ........................................................................................................... 48
Arrangement of tomato fruit for storage ........................................................... 49
Preparation of l-MCP gas ................................................................................. 50
V

 

—

l-MCP treatments of tomato fruit ............................................................................... 51
Skin color, firmness, total weight loss, and ethylene production of tomato fruit

........................................................................................................................... 51
Statistical analysis ............................................................................................. 53
Results and Discussion ................................................................................................ 54
Skin color .......................................................................................................... 54
Firmness ............................................................................................................ 60
Total weight loss ............................................................................................... 65
Ethylene production .......................................................................................... 69
Conclusion ................................................................................................................... 74
Appendix A .................................................................................................................. 75
SECTION IV: EFFECT OF l-METHYLCYCLOPROPENE (l-MCP) ON
CHLOROPHYLL AND LYCOPENE CONTENT IN STORED TOMATOES ........ 77
Abstract ........................................................................................................................ 77
Introduction .................................................................................................................. 78
Materials and methods ................................................................................................. 80
Materials ........................................................................................................... 80
Storage of tomato fruit ...................................................................................... 80
Preparation of l-MCP and its treatment ........................................................... 81
Chlorophyll ....................................................................................................... 82
Extraction ............................................................................................. 82
Identification ........................................................................................ 82
Lycopene ........................................................................................................... 85
Extraction ............................................................................................. 85
Saponification ...................................................................................... 87
Separation ............................................................................................ 89
Identification ........................................................................................ 91
Results and Discussion ................................................................................................ 93
Chlorophyll content .......................................................................................... 92
Lycopene content .............................................................................................. 99
Conclusion ................................................................................................................. 106
Appendix B ................................................................................................................ 107

SECTION V: SORPTION STUDY OF l-METHYLCYCLOPROPENE (l-MCP) BY
SELECTED ADSORBING AGENTS USING INVERSE GAS

CHROMATOGRAPHY ............................................................................................ 108
Abstract ...................................................................................................................... 108
Introduction ................................................................................................................ 109
Materials and methods ............................................................................................... 111
Materials ......................................................................................................... 1 1 1
Preparation of chromatographic column ......................................................... 111
Preparation of l-MCP gas and standard calibration ....................................... 113
Inverse gas chromatography system ............................................................... 113
Adsorption isotherms ...................................................................................... 1 14
Other parameter .............................................................................................. 114

vi

 

Results and discussion ............................................................................................... 115

Sorption isotherms .......................................................................................... 1 16
Specific retention volume (V°g) values ........................................................... 124
Thermodynamic parameters ............................................................................ 130
Conclusion ................................................................................................................. 141
Appendix C ................................................................................................................ 142
SECTION VI: ADSORPTION AND RELEASE OF l-METHYLCYCLOPROPENE
(l-MCP) FROM SELECTED ADSORBING AGENTS .......................................... 146
Abstract ...................................................................................................................... 146
Introduction ................................................................................................................ 147
Materials and methods ............................................................................................... 149
Materials ......................................................................................................... 149
Sachet materials for l-MCP controlling release system ................................. 149
Water vapor transmission rate of the paper and Tyvek® sachet materials ...... 150
Water vapor transmission rate of the LDPE and PVA sachet materials ......... 151
Permeation measurements of l-MCP gas for sachet materials ..................... 152
GC instrumentation for l-MCP gas release analysis ...................................... 153
Partition coefficient ......................................................................................... 154
Adsorption efficiency ...................................................................................... 156
Release of l-MCP gas through the sachets with adsorbing agents ................. 157
Dynamic l-MCP gas trapping system for Tyvek® and filter paper sachet 157
l-MCP generator on dynamic trapping system .................................. 159
Direct injection of l-MCP gas for LDPE and PVA materials ........................ 160
Controlled release of 1-MCP gas from the adsorbing agents ......................... 160
Results and discussion ............................................................................................... 163
Partition coefficient ........................................................................................ 163
Permeability of l-MCP gas and water vapor through the sachet materials... 167
l-MCP adsorbing efficiency ........................................................................... 170
l-MCP release ................................................................................................. 171
Conclusion ................................................................................................................. 178
Appendix D ................................................................................................................ 180
NOMENCLATURE .................................................................................................. 184
BIBLIOGRAPHY ...................................................................................................... 186

vii

LIST OF TABLES

Table 1. Some published active packaging applications ............................................. 24

Table 2. Commercial applications for adsorbing agents .............................................. 36

Table 3. Days of storage at which the hue angle began to decrease (the breaker stage)
for single and once—a-day treated green fruit. For comparison, values of the

control tomatoes are included. ....................................................................... 56
Table 4. Weight loss of each maturity tomato at 10°C ................................................ 66
Table 5. Weight loss of each maturity tomato at 15°C ................................................ 67
Table 6. Weight loss of each maturity tomato at 20°C ................................................ 68
Table 7. Parameters of the selected adsorbing agents used as the stationary phase of
the IGC system ............................................................................................. 112
Table 8. Solubility coefficients (K) of l-MCP as a function of temperature for each
adsorbing agent ............................................................................................ 122
Table 9. Specific retention volumes (V°g) of l-MCP with each adsorbing agent as a
function of temperature ................................................................................ 124
Table 10. Gibbs’s free energy values of l-MCP at several temperatures .................. 130
Table 11. Entropy (A8,) and enthalpy (AHS) values of l-MCP at selected temperatures
..................................................................................................................... 135

Table 12. The average thickness of the test materials used to fabricate the sachet 150
Table 13. Partition coefficients of l-MCP gas between each solid matrix and air in a

closed system at 23°C ................................................................................ 163
Table 14. Partition coefficient of l-MCP gas between each sachet material and air in a
closed system at 23°C ................. < ............................................................... 166
Table 15. Water permeability of sachet materials used on the l-MCP release system
.................................................................................................................... 167
Table 16. 1—MCP gas permeability of selected sachet materials ............................... 168
Table 17. 1-MCP adsorbing efficiency of selected adsorbing agents in a closed system
.................................................................................................................... 170
Table 18. Salt solution and its corresponding relative humidity at 23°C ................... 180

viii

LIST OF FIGURES

Figure 1. The proposed l-MCP delivery mechanism in the packaging system for fresh

tomatoes .......................................................................................................... 7
Figure 2. Longitudinal structure of a tomato fruit ......................................................... 8
Figure 3. Patterns of some physiological changes during ripening of tomatoes ......... 11
Figure 4. The chemical structures of chlorophyll a and chlorophyll b ........................ 14
Figure 5. The chemical structure of lycopene .............................................................. 15
Figure 6. Ethylene biosynthesis pathway ..................................................................... 18
Figure 7. Chemical structures of ethylene and l-methylcyclopropene ........................ 20
Figure 8. Typical gas elution chromatogram showing the areas used in calculating
sorption isotherm data .................................................................................. 29
Figure 9. The storage arrangement of tomato fruits .................................................... 49
Figure 10. A schematic diagram of the apparatus for arrangement of tomato fruits in
the glass vessel ............................................................................................ 50
Figure 11. Measurements of the skin color, firmness, and ethylene production for
stored tomato fruits ..................................................................................... 53
Figure 12. Change in skin color of tomatoes at 10°C .................................................. 57
Figure 13. Change in skin color of tomatoes at 15°C .................................................. 58
Figure 14. Change in skin color of tomatoes at 20°C .................................................. 59
Figure 15. Change in tomato firmness at 10°C. ........................................................... 62
Figure 16. Change in tomato firmness at 15°C. ........................................................... 63
Figure 17. Change in tomato firmness at 20°C. ........................................................... 64
Figure 18. Ethylene production of tomatoes at 10°C ................................................... 71
Figure 19. Ethylene production of tomatoes at 15°C. .................................................. 72
Figure 20. Ethylene production of tomatoes at 20°C. .................................................. 73
Figure 21. Conversion of the durometer duro unit to the force unit ............................ 75
Figure 22. Chromatographic peak shapes of (a) l-MCP and l-butene standard, (b)
ethylene produced from the tomato and ethylene standard ........................ 76
Figure 23. Flow diagram of total chlorophyll extraction in tomatoes ......................... 84
Figure 24. Flow diagram of lycopene extraction in tomatoes ..................................... 86
Figure 25. Flow diagram of saponification of the extract samples .............................. 88
Figure 26. A schematic diagram of the lyc0pene separation system ........................... 90
Figure 27. Flow diagram of the method used to determine the lyc0pene content in
tomatoes ...................................................................................................... 92
Figure 28. Changes in total chlorophyll content of green tomatoes in storage at 10°C
..................................................................................................................... 96
Figure 29. Changes in total chlorophyll content of green tomatoes in storage at 15°C
..................................................................................................................... 97
Figure 30. Changes in total chlorophyll content of green tomatoes in storage at 20°C
..................................................................................................................... 98
Figure 31. A spectrophotometeric profile of the purified lyc0pene extracts ............... 99
Figure 32. Formation of lycopene in pink tomatoes in storage at 10°C .................... 102
Figure 33. Formation of lycopene in pink tomatoes in storage at 15°C .................... 103
Figure 34. Formation of lycopene in pink tomatoes in storage at 20°C .................... 104
Figure 35. Relation between lyc0pene and chlorophyll in the pericarp tissue for the
various treatments ...................................................................................................... 105
Figure 36. A spectrophotometeric profile of the standard chlorophyll a and b from a

spinach source ............................................................................................ 107
Figure 37. The packing materials and the stainless steel columns for the stationary

phase ......................................................................................................... 1 12
Figure 38. Adsorption of l-MCP on (a) Silica gel, (b) Tenax-TA, and (c) Activated

clay by inverse gas chromatography ......................................................... 115
Figure 39. Sorption isotherms of l-MCP on silica gel at 50, 60, 70, and 80°C ......... 118
Figure 40. Sorption isotherms of l-MCP on Tenax-TA at 50, 60, 70, and 80°C ...... 119

Figure 41. Sorption isotherms of l-MCP on activated clay at 50, 60, 70, and 80°C. 120
Figure 42. Linear plot of the log constant value (LnKs) versus the reciprocal of the

absolute temperature (1000/T, K) ............................................................. 121
Figure 43. Calculated sorption isotherms of l-MCP on each adsorbing agent at 23°C
................................................................................................................... 123
Figure 44. Specific retention volume (V°g) for silica gel as a function of l-MCP
concentration at 50, 60, 70, and 80°C ....................................................... 126
Figure 45. Specific retention volume (V°g) for Tenax-TA as a function of l-MCP
concentration at 50, 60, 70, and 80°C ....................................................... 127
Figure 46. Specific retention volume (V°g) for activated clay as a function of l-MCP
concentration at 50, 60, 70, and 80°C ....................................................... 128
Figure 47. Log specific retention volume (V°g) for l-MCP as a function of
temperature ............................................................................................... 129
Figure 48. Gibbs free energy of sorption (AG5) for silica gel as a function of l-MCP
concentration and temperature .................................................................. 132
Figure 49. Gibbs free energy of sorption (AG5) for Tenax-TA as a function of l-MCP
concentration and temperature .................................................................. 133
Figure 50. Gibbs free energy of sorption (AG5) for activated clay as a function of
l-MCP concentration and temperature ..................................................... 134
Figure 51. Entropy of sorption (A8,) for silica gel as a function of l-MCP
concentration and temperature .................................................................. 136
Figure 52. Entropy of sorption (ASS) for Tenax-TA as a function of l-MCP
concentration and temperature .................................................................. 137
Figure 53. Entropy of sorption (A8,) for activated clay as a function of l-MCP
concentration and temperature .................................................................. 138
Figure 54. Enthalpy of sorption (AHS) for each adsorbent as a function of l-MCP
concentration ............................................................................................. 140
Figure 55. Standard calibration curve of l-butene on silica gel packing column ...... 142

Figure 56. Standard calibration curve of l-butene on Tenax-TA packing column 143
Figure 57. Standard calibration curve of l-butene on activated clay packing column

................................................................................................................... 144
Figure 58. Chromatographic peaks l-butene on silica gel (3), Tenax-TA (b), and
activated clay (c) stationary phase column ............................................... 145
Figure 59. A schematic diagram of the l-MCP permeation test system ................... 153
Figure 60. A schematic diagram of the l-MCP gas trapping system used for sachets
containing adsorbing agents ...................................................................... 158
Figure 61. l-MCP gas trapping system for the sachets containing adsorbing agents
................................................................................................................... 159
Figure 62. The sachets containing adsorbing agents ready for direct injection of
l-MCP ....................................................................................................... 160
Figure 63. A schematic diagram of the closed system for 1-MCP release study at
controlled humidity conditions ................................................................. 161

Figure 64. Partition coefficient of l-MCP gas between solid matrix and air in a

closed system as a function of the amount of l-MCP injected at 23°C....165
Figure 65. l-MCP gas release from the Tyvek® sachets in a closed system at 23°C.174
Figure 66. l-MCP gas release from the paper sachets in a closed system at 23°C....175
Figure 67. 1-MCP gas release from the LDPE sachets in a closed system at 23°C...176
Figure 68. l-MCP gas release from the PVA sachets in a closed system at 23°C ..... 177

Figure 69. l-MCP gas transmission of LDPE film structure at 23°C ........................ 181

Figure 70. l-MCP gas transmission of PVA film structure at 23°C .......................... 182

Figure 71. Chromatographic peak shapes of l-MCP and l-butene on SPB-S nonpolar
capillary column ........................................................................................ 183

xi

SECTION I: INTRODUCTION

Ethylene is a major factor regulating plant development and can cause a
significant increase in the respiration rate. Ethylene is a ripening hormone for climacteric
fruits because the gaseous compound stimulates other ripening processes in the
postharvest environment. Ethylene responses include desirable reactions such as
softening, and color change in ripening. On the other hand, ethylene promotes
undesirable reactions inducing abscission and physiological damage, and tends to shorten
storage life (Lelievre, 1997a).

Tomatoes are representative of climatic fruits and can be affected by
environmental conditions such as temperature, light, relative humidity, ethylene, and C02
concentration (Ryall, 1979). Tomatoes have a moisture content over 90% and are a
nutritionally valuable source of pro-vitamin A (B—carotene) and vitamin C (ascorbic acid).
The color of the tomato is used to predict the degree of its maturity in terms of fruit
quality. Change in color is caused mostly by the loss of chlorophyll and synthesis of
lycopene throughout ripening (Watada, 1976a). Most tomato fruit is picked either at the
green mature stage for long-distance transport or the ripened stage for the fresh market.
For long-term storage or transport of the tomato fruit, 3 temperature of 13°C at 80%RH is
recommended to delay the ripening process (Haard, 1975).

In the fresh produce industry. postharvest treatments are the most important

processes used to extend freshness during storage, transport, and handling. Biochemical
and physiological changes in fresh produce depend mostly on environmental conditions.

Postharvest environmental treatments include low temperature storage under air and

controlled atmosphere (CA) and/or by treating them with chemicals which affect plant
hormone action (F loros, 1993).

Many fruits are subjected to a wide range of temperatures during storage and
transportation. Optimal storage temperatures of fruits differ because their responses to
lower temperatures vary widely dependent upon fruit variety and the required length of
storage. Lower storage temperature reduces respiration and fungal growth in fruits.
However, very low temperature treatment causes chilling injury (Efiuvwevwere, 1988),
or can stimulate autocatalytic ethylene synthesis (Lelievre, 1997b).

CA storage refers to storage under modified from the 02 and/or C02
concentrations in the atmosphere surrounding the fresh produce. CA storage is used
commonly to delay the ripening process of fruits, and can prevent certain diseases and
some disorder symptoms. Tomato ripening can be prevented by extremely low 0; or
high CO; concentrations in storage (Ratanachinakom, 1997). Temperature control is
also important because low 02 or high C02 levels do not control all diseases or disorders
effecting fruits. Thus, CA storage needs to be used with selected temperatures for certain
fruits in order to achieve ideal storage conditions.

The chemical treatments silver thiosulphate (STS), diazocyclopentadiene
(DACP), and 2,5 norbomadine (N BD), inhibit ethylene action in fresh produce. These
chemicals reduce the effects of ethylene, by blocking the receptor site that signals
ethylene action. STS, DACP, and NBD compete with ethylene for the binding receptor
and are very effective inhibitors of ethylene response. However, these inhibitors are
limited in their commercial application in the horticultural industry because of their

UUStable and explosive potential (DACP), toxicity and odor (NBD), and environmental

contamination (STS) (Serek, 1994). T hus, there has been a need to find alternatives to
these compounds.

One such alternative is 1- methylcyclopropene (l-MCP). Recently, 1-MCP has
begun to be commercially used as an agent to extend storage life of fruits and vegetables,
and flowers. This compound is a nontoxic gas, and been shown to prevent some aspects
of ripening in climacteric fruits (Fan, 1999). 1-MCP acts by inhibiting binding of the
ethylene to its receptor sites and is effective at low concentrations (parts per billion
levels) (Serek, 1994). Several studies have been conducted on the efficacy of l-MCP as
an inhibitor of ethylene action in ethylene-sensitive cut flowers, vegetables, and fruits
(Serek, 19953 and 1995b; Wills, 2002).

Pure l-MCP has the problems of its stability, and the potential explosive hazard
associated with a compressed gas (Daly, 2000). A convenient and safe means of
delivering l-MCP to fresh produce is to trap it using alpha-cyclodextrin as the molecular
encapsulation agent. This has been developed as a commercial product “Smart Fresh”.
This product can also be delivered by dissolving the complex in a suitable solvent in
order to release the l-MCP.

Another technique which can be used to deliver l-MCP gas is the addition of an
inert carrier system such as silica, talc. and dust. This produces a product that has greater
stability (Daly, 2000). In addition, use of a packaging system with appropriate
permeation properties can provide for slow release of l-MCP into the headspace of the
fresh produce to obtain the desired shelf life.

In packaging applications with an inert carrier system, a sachet may be used to

deliver thel-MCP gas to the fresh produce. The sachet technique has been developed to

extend shelf-life of food (Smith, 1995). This system includes an ethanol vapor generator
as an antimicrobial agent, and an oxygen absorbing agent to prevent oxidation and mold
growth.

The sachet technique can be used with postharvest produce containing high
moisture contents under the environmental conditions within the package. Figure 1
shows the proposed l-MCP package delivery system. The proposed delivering
mechanism utilizes a three-step process: (1) release of active molecules from the
absorbing surface layer within an inert material; (2) permeation of active molecules
through the sachet material into the free volume of the pouch; and (3) sorption of active
molecules onto the surface of the packaged produce. In this proposed mechanism, an
adsorbing agent, can absorb and/or desorb active molecules. Investigation into the
sorption behavior, and thermodynamic parameters of this interaction can provide
valuable information. Inverse gas chromatography is a very powerful technique which
can be used to characterize interactions between two-phase materials (Lloyd, 1989).
Material porosity in conjunction with material barrier properties can control the release of
active molecules from the sachet pouch matrix to the produce.

The hypotheses of the current research are the following: (1) The shelf life of a
packaged fresh tomato was extended by maintaining an established level of active
compounds such as l-methylcyclopropene. (2) The potential proposed delivery system
allowed release of l-MCP gas at an acceptable rate into the headspace of packages
containing fresh tomatoes.

The present study focuses on the efficient treatment of the 1—MCP gas for

ripening on tomatoes, and the development of a l-MCP delivery system for packaged

tomatoes. This information may be helpful in maintaining the quality of packaged

tomatoes.

Research Objectives

Objective 1: Application of l-methylcyclopropene (1-MCP), with temperature control, to

the tomato fruit during the ripening process to extend storage life.

The effects of l-MCP treatment and temperature on the ripening process of the
tomato fruit during postharvest storage were investigated. In this study, l-MCP treated
fruits using single and once-a-day applications compared to control fruits without l-MCP
treatment. The tomato fruit storage study was performed at three maturity stages, mature

green, pink, and red, and at three storage temperatures, 10, 15, and 20°C.

Objective 2: To observe change in pigment concentration in the tomato fruit treated with

l-MCP and held at various temperatures.

Changes in chlorophyll and lycopene content in tomatoes treated with l-MCP gas
during the ripening process were investigated. Total chlorophyll and lycopene contents
were quantified using a specific extinction coefficient absorbance technique. Three parts
of the tomato fruit were used to study pigment changes, the pericarp, the locular gel, and

the placenta.

Objective 3: To observe interactions between l-MCP and selected adsorbing agents using

inverse gas chromatography.

Sorption behavior and thermodynamic parameters related to l-MCP interaction
with selected adsorbing agents were studied using inverse gas chromatography.
Determination of thermodynamic parameters provided for a better interpretation of the 1-
MCP/adsorbing agent interactions. Calculation information of the specific retention
volume (V°g), free energy (AG5), isosteric enthalpy (AH,), and entropy (A5.) of sorption

were used to characterize l—MCP/adsorbing agent interactions.

Objective 4: To observe l-MCP release from the adsorbing agents and through the

packaging system.

This objective focused on the release of l-MCP from the adsorbing agents in the
sachet pouch system. The release study was performed under two conditions, dry
(0%RH) and high relative humidity (90%). The water and l-MCP permeability values of
the sachet pouch films were measured. Partition coefficients of l-MCP (Ks/3) with the
adsorbing agent, or the film phase in air were obtained as the ratio of the equilibrium

levels of l—MCP in these different media.

 

Adsorbing
agent with 1-
MCP -

l. Desorption

 

   
  

Paper or PE
’2 Permeation &

Release of l-MCP

3. Uptake

91

ii

Polyethylene bag

Uptake

Perm¥tion
l

 

 

 

 

Figure 1. The proposed l-MCP delivery mechanism in the packaging system for fresh

tomatoes.

1}

Environment

Air

SECTION II: REVIEW OF LITERATURE

1. Tomato fruit

Tomato (Lycopersicon esculentum) is an annual plant cultivated in temperate
regions or short-lived perennials in the tropics. Most tomato species are native to the
coastal areas of Ecuador, Peru, and Northern Chile (Male, 1999). The tomato fruit is a
fleshy berry having two or more cavities that contain seeds imbedded in a gelatinous
tissue that becomes a smooth and soft surface when it is fully developed to mature
conditions. The shape of the tomato fruit is dependent on cultivar; there is cherry-, pear-,
and egg-shaped fruit, but most cultivars have the globose shape. The tomato fruit

consists of pericarp, placenta tissue, and seeds (Figure 2).

locular cavity with
gelatinous tissue

      
  
 
  

pericarp

placenta

seeds

{—— exocarp

endocarp

Figure 2. Longitudinal structure of a tomato fruit

The skin of the pericarp consists of an epidermal layer which contains all three cell layers
of collenchymous tissue, with a relatively thick cuticle. The wall of the pericarp is
separated into the exocarp and endocarp. The gel is a gelatinous tissue containing the
seeds. It is a light cream to brown color and fills the locules. The placenta is composed
of parenchyma cells that surround the ovules with the seeds embedded in the gel.

Tomato fruits are climacteric, a class that also includes apples and bananas.
Climacteric products undergo a significant increase in ethylene production at the onset of
ripening. Exogenous ethylene causes the rate of ethylene production to increase and
induces a respiratory rise. The ethylene stimulates other ripening processes such as
softening, color changes, compositional changes, and aroma production. Non-climacteric
crops include fruits like citrus and grape which have relatively low respiration rates and
levels of ethylene production. During ripening, non-climacteric fruits are considered to

have an ethylene-independent metabolism in which ethylene does not promote fruit

ripening.

2. Fruit ripening

Ripening represents physiological changes in a number of biochemical pathways
including an increase in respiration, ethylene output, carotenoid synthesis, chlorophyll
degradation, production of cell wall hydrolases, and texture softening (Hobson, 1972).
The result of each physiological behavior in ripening occurs independently and is
normally an irreversible process. Figure 3 shows the general pattern of physiological

changes that occur during ripening in tomatoes. Among several physiological changes,

 

color is the most important visual standard of quality in tomatoes. The maturation
process in tomatoes develops from the mature green stage to the breaker stage, then the
full red stage with good texture, and then arriving at the final stage, in which most
tomatoes have a dark red color and the tissue is sofiened. The main factor in the color
change of fruits occurs in the transition of chloroplasts into chromoplasts which are rich
in yellow or red carotenoid pigments, and an increase in the water-soluble pigments such
as anthocyanins. Maturity of tomatoes is classified into six levels using a subjective
assessment of the external fruit color (Ryall, 1979; Edwards, 1967). The maturity stages
are: Stage 1-‘Green’: totally green color in internal and external tissues, showing no
evidence of red or yellow coloration; Stage 2-‘Breaker’, most green color, not more than
10% of the surface is yellow; Stage 3-‘Turning’, over 10 to 30% of the surface area is
yellow or pink color, but showing some green areas; Stage 4-‘Pink’, between 30 and 60%
of the surface is pink, showing no traces of green coloration; Stage 5-‘Light red’, more
than 60% of the surface is pinkish and red color; Stage 6-‘Red’, more than 90% of the

surface is red color.

 

 

 

 

 

 

 
   
 

 

 

aes-
g3

—UI—
380
809
atm—
9.3

‘+—

Egr— CO? —°°§EE
0?? Ethylene 3%
09; _i .3‘0
~30,- his

0— _o
I I l l l |
.5
’38
1: °\_‘
I 3— “‘3 9
e Starch
‘o— __o
l l J l l j
.Am-O
_. O
“‘ 8&3
I! 5%
33 a; _. Polygalacturonase — g 33%
3" s
m m
l I I I l l

 

Mature Breaker Turning Pink Lightred Red
green

Figure 3. Patterns of some physiological changes during ripening of tomatoes. (Wills,
1989)

Softening of fruit texture can be caused by the partial degradation of cells by

POlygalacturonase (PG) which is present in the cell walls, and through hydrolytic changes

in the cellular constituents. The ripening process increases the activity of
polygalacturonase (PG), causing pectin degradation in the cell walls. It also causes a
large increase in ethylene production in tomato fruits at the onset of ripening (pink color
stage). After the ripening process is under way, ethylene production reaches a peak, and
continues at a relatively high level throughout the ripening period. Nakatsuka (1997) and
Della Penna (1986) characterized the genes involved in ethylene synthesis as 1-
aminocyclopropane-l-carboxylate (ACC) synthase, and ACC oxidase. Control of fruit
ripening is important for the successful transport and marketing of fresh produce. Fruit
that ripens too early is easily damaged during transport, and produces ethylene, which
can adversely affect other commodities. Various techniques have been used to delay or
manage ripening of fresh produce, including cold storage, controlled atmosphere storage,
ethylene addition and removal, and chemical inhibitors of ethylene action. The optimum
storage temperature for ripening of the mature green tomato is approximately 15°C to
20°C. Throne (1982) reported that the firmness and surface color of stored tomatoes are
adequate quality indicators for temperatures above 12°C and below 27°C. Fruit firmness
of various tomato cultivars was significantly different at time of harvest and after storage
for 1 and 2 weeks at 19-23°C (Gormley, 1978). Temperatures less than 10°C will cause
chilling damage, and the fruit softens, color development slows, and flavor is reduced.
Mature-green tomato fruit stored for 9 days at 7°C, subsequently ripened to an acceptable
color (Efiuvwevwere, 1988). At 12 and 19°C, color development was more even than at

lower temperatures.

3. Pigments for color development in tomato fruits

Chlorophylls

Chlorophylls are the green colored pigments in photosynthetic tissues that convert
light energy into chemical energy. Chlorophylls produce carbohydrates and other
organic compounds from carbon dioxide and water in the presence of light, and release
molecular oxygen. All green plants contain the blue-green pigment chlorophyll a, and
yellow-green chlorophyll b. They occur concurrently within the same plant. The ratio
of Chlorophylls varies with growth conditions and environmental factors. Chlorophyll a
differs from chlorophyll b due to in the presence of a single methyl group instead of an
aldehyde group (Figure 4). Chlorophylls are porphyrins which contain four pyrrole rings
surrounding a central Mg2+ ion. Chlorophyll a and b contain a phytol chain with 20

carbons (C20H390H) attached to the propionyl group of a pyrrole ring.

13

 

Chlorophyll A Chlorophyll B

Figure 4. The chemical structures of chlorophyll a and chlorophyll b

The degradation of chlorophyll can occur through mechanisms such as loss of
magnesium, and loss of the phytol group, through the action of the enzyme
chlorophyllase. Loss of the phytol group results in the formation of chlorophyllide,
abstraction of both magnesium and phytol produces pheophorbide, and finally cleavage
of the porphyrin ring system by a dioxygenase results in the formation of
pyropheophorbide (Van Boekel, 2000). In many fruit tissues the loss of chlorophyll
leads to the transformation of chromoplasts from chloroplast, which has capacity to form
carotenoids. The end products of chlorophyll degradation are colorless, low-molecular-
weight compounds such as C02, NH3, and H20. Watada (1976a) estimated the total
chlorophyll content of green ‘Walter’ tomatoes by light absorbance. Chlorophyll content
changed from 13.4mg/kg in immature-green fruit to 0.3mg/kg in partially ripe fruit.

Chlorophyll is sensitive to light, heat, humidity and enzymes which can cause

degradation. Cold storage of cabbage resulted in a color change from green to white due
to chlorophyll degradation (Heaton, 1996a and b). Ethylene also increases chlorophyll
degradation and transforms the green chloroplasts to colored chromoplasts. Liu (1989)
found that 2,5-norbomadiene inhibited the loss of chlorophyll in the pericarp tissue of
‘Dahong’ tomato fruit. Controlled-atmosphere storage of tomatoes with low 02 and high

C02 slows down the degradation of chlorophyll (Sozzi, 1999).

Lycopene

Carotenoids are important natural pigments synthesized by higher plants and
microorganisms. Lycopene is the major carotene (red color) in tomatoes and may
comprise as much as 90 percent of the total carotenoids. Lycopene is an acyclic
carotenoid with 11 linear conjugated carbon-carbon double bonds, making it soluble in

lipids. Figure 5 illustrates the structural form of lycopene typically found in tomatoes.

 

All-trans Lycopene

Figure 5. The chemical structure of lycopene

l5

 

Lycopene is susceptible to oxidation by physical and chemical factors such as elevated
temperature, exposure to light, and oxygen (Nguyen, 1999). In common tomato varieties
lycopene exists predominantly in the all-trans configuration which is the most thermally
stable form. Its concentration varies significantly with ripening and in the different
varieties of tomatoes (Edwards, 1967). Matinez-Valverde (2002) reported that lycopene
content ranged from 18.60 to 64.98mg/kg in ‘Liso’ and ‘Durina’ tomato varieties,
respectively. Lycopene content of ripe tomato fruit varied between 12.1mg/kg in
‘Urbana’ and 198.6mg/kg in ‘San Marzano’ tomatoes (Edward, 1967). Lycopene is used
to predict the degree of maturity since it influences the color of tomatoes, and is found in
greater amounts in the chromoplast of plant tissues. Lycopene biosynthesis increases
dramatically during ripening at which time the color of the tomato turns from green to
white to yellow to pink to red. As chloroplasts are transformed into chromoplasts, the
Chlorophylls are degraded. Carotenoids are not uniformly distributed within fruits. The
chromoplast in the outer pericarp contains predominantly lycopene, while the gelatinous
part of the pericarp has a substantial amount of the major B-carotene (Nguyen, 1999).
The majority of carotenoids, including lycopene are very efficient singlet oxygen
quenchers (Di Mascio, 1989). Viljanen (2002) demonstrated that lycopene, B-carotene,
and lutein acted as antioxidants in photooxidation by quenching singlet oxygen which
prevented the formation of methyl linoleate hydroperoxides. Lycopene showed the
greatest quenching ability at a concentration higher than 40ppm and was more stable than
the other carotenoids tested. Lycopene is an efficient scavenger of oxygen radicals and
acts as an antioxidant against oxidative damage associated with carcinogenesis (Khachik,

1995). Dietary lycopene acts to prevent cancers of the prostate and gastrointestinal tract

16

(Clinton, 1998).

4. Ethylene

Ethylene is a plant hormone that plays an important role in many aspects of plant
growth, development, and senescence. It also stimulates ripening processes resulting in
changes in color and softening, and effects respiration of fruits. Ethylene can be used to
change the uniform color in the ripening of tomatoes, apples, and bananas. On the other
hand, this gas may shorten the storage life of fruits by accelerating the ripening process.
Ethylene is formed according to the following biosynthetic pathway in plant tissue: L-
methionine —> S-adenosyl-L-methionine (AdoMet) —+ l-aminocyclopropane-l-carboxylic
acid (ACC) —» ethylene (Adams, 1979). In this pathway, two enzymes react to regulate
ethylene production. First step is the conversion of AdoMet to ACC which is catalyzed
by ACC synthase. Second step is the oxidation of ACC to ethylene which is catalyzed
by ACC oxidase (EF E). These biosynthetic processes are designed to promote the rate
of ethylene production even when the amount of the amino acid methionine is small.
Besides converting ACC to ethylene, ACC synthase produces 5‘-methylthioadenosine
(MTA). This can then be used for the synthesis of new methionine via the Yang cycle
(Figure 6). ACC synthase plays a key role in regulating ethylene production and can
respond to many inducers such as ethylene, wounding of the fruit, temperature and
presence of metal ions such as Cd2+ and Li+. ACC oxidase activity also increases in
some plant tissues in response to internal or external factors that induce ethylene

production, thus its activity may be limiting. ACC can also be metabolized to l-

(malonylamino)cyclopropane-l-carboxylic acid (MACC), a biologically inactive end-
product catalyzed by ACC malonyltransferase. The rate of formation of MACC affects

the endogenous levels of ACC which have low ACC synthase activity.

We
R-CH-C 0. R-CH-COO- ATP
Met *PPfiP, MACC

Hco _ K B Malonyl-

AdoMet transferaseL Malonyl CoA
02 C02+HCN
.9 C_L "=°=°"=
Cyce ACC synthase +H3N COO- ACC Oxidase

02 ACC

MTA Fruit ripening Higher temp.-

more than 35°C

MTR-t -P
Cd”. Li’ . .
_ Anaeroblosrs
MTR Woundlng Free redial-
ADP Stress (heat. scavengers

Ade cold. light)

ATP Ethylene

Figure 6. Ethylene biosynthesis pathway (Yang, 1987)
s-adenosyl-L—methionine (AdoMet), 5’-methylthioadenosine (MTA), 5’-methylthioribose (MTR),
MTR-l-phosphate (MTR—l-P), 2-keto—4-methylthiobutyrate (KMB), methionine (Met), 1-

(malonylamino)cyclopropane-l-carboxylic acid (MACC), l-aminocyclopropene—l-carboxylic
acid (ACC)

It is well-known that ethylene biosynthesis is subject to positive and negative
feedback regulation in plants (Kende, 1993). Positive feedback regulation of ethylene
biosynthesis is a characteristic feature of ripening fruits. A large increase in ethylene
production is triggered by exposure to exogenous ethylene with activation of ACC
synthase and ACC oxidase. Negative feedback has been recognized in immature

climacteric fruit. Exogenous ethylene significantly inhibits endogenous ethylene

production induced by ripening (N akatsuka, 1997). The Yang cycle is responsible for

ethylene synthesis during ripening. Ethylene is the key regulatory molecule effecting

fruit ripening.

5. l-methylcyclopropene (l-MCP)

l-MCP (Figure 7) was discovered by Drs. Sylvia Blankenship and Ed Sisler of
North Carolina State University (Blankenship, 1996). It has been found to be an effective
blocking agent, and thus, inhibiting the ethylene response (Sisler, 1996). However, 1-
MCP gas is quite unstable and will react with oxygen and other organic compounds. It is
also explosive at high concentrations. Recently, l-MCP, encapsulated with alpha-
cyclodextrin, has been marketed under the trade names Ethleloc® and SmartfreshTM as a
commercial product. It acts by occupying the ethylene-binding site which prevents
ethylene from binding to the active site. l-MCP is an irreversible, nontoxic, and
odorless inhibitor of the ethylene binding and action. Treatment with l-MCP at
concentrations of less than 1 ppm eliminates the effect of ethylene on most ethylene-

sensitive plants.

19

H2

H2C=CH2 C/=\CH
H3C/

Ethylene 1-methylcyclopropene
Figure 7. Chemical structures of ethylene and l-methylcyclopropene

Ethylene antagonists, such as l-MCP can be a useful in investigating the role of
ethylene in the ripening of climacteric fruit. Although many studies have evaluated the
influence of l-MCP and its effect on ripening, its mode of inhibition relative to ethylene
is still not verified. Sisler (1997) proposed a model to describe the effect of l-MCP on
the ethylene receptor. The biologically active form of the ethylene receptor led to the
theory that binding takes place at an active site containing a metal, and that l-MCP binds
to a metal ion in the ethylene receptor. It was proposed that the competitive interaction
of ethylene with its inhibitor prevented the ethylene from binding to the site. Ethylene
binds a metal in the ethylene receptor, and electrons are withdrawn. A ligand substitu—
tion process with ethylene then induces an active response, and the ethylene is expelled
from a metal on the receptor. An active receptor complex is then formed. l-MCP
withdraws electrons in a way similar to ethylene, but since the binding action of l-MCP
is stronger than that of ethylene, it remains bound to a metal on the ethylene receptor
surrounded by ligands of unknown structure. l-MCP may effectively block the active

receptor, thus preventing the formation of an active receptor complex.

20

l-MCP has been found to reduce ethylene-related response in many fruits such as
apples, bananas, plums, kiwifruits, and strawberries. Golding (1998) reported that
treating mature green bananas with l-MCP significantly delayed the onset of ethylene
production, respiration, and aroma volatile production. Watkins (2000) showed that the
efficacy of l-MCP in apples was affected by storage conditions, and concentration level.
Kim (2001) reported that kiwifruit responded to l-MCP levels of 1, 10, and 100 ppm
(v/v) by reducing ethylene production, and fruit softening at 20°C. Higher concentrations
of l-MCP were needed to affect an ethylene inhibitor response. Mir (2001) showed that
l-MCP effectively reduced the rate of apple fruit softening with a treatment of 0.7 ppm 1-
MCP compared with the control at 0, 5, 10, 15, and 20°C storage temperature. Skog
(2001) demonstrated that l-MCP treatment decreased the firmness of plums significantly
compared to untreated fruit during postharvest storage and ripening. Porat (1999)
evaluated the effect of ethylene and l-MCP on oranges, a non climacteric fruit, during
postharvest storage. l-MCP concentrations of 50 and 100 ppb (v/v) were very effective
and markedly inhibited the de-greening process, but were ineffective in preventing
chilling injury and decay development. Jiang (1999) investigated the effect of l-MCP
on bananas packed in polyethylene bags. l-MCP with modified atmosphere (MA)

packaging markedly delayed peel color change and fruit softening during ripening.

21

6. Other ethylene inhibitors

Aminoethoxyvinylglycine (AVG) is an inhibitor of ethylene biosynthesis in plant
tissues that prevents the conversion of S—adenosylmethionine to 1-amino cyclopropane-l -
carboxylic acid (Yang, 1984). Fan (1998) reported that AVG inhibited production of
some volatile esters in climacteric apple fruit.

Diazocyclopentadiene (DACP), a light-activated compound, has been shown to
irreversibly inhibit ethylene action. DACP can inhibit softening, starch degradation,
ethylene production, and color change during fruit ripening. A single exposure of DACP
to mature green tomatoes prevented the ripening process for about 10 days at 25°C and
for 20 days at 145°C (Sisler, 1993). Exposure at five-day intervals resulted in longer
ethylene inhibition. DACP works at very low concentrations, and since a single
exposure protects the fruit for several days, it is easier to study the effects of blocking the
receptor with a minimum of side effects. However, DACP has not been selected for
commercial application because of its explosive potential (Serek, 1994).

2,5-norbomadiene (N BD) is a cyclic olefin compound, which blocks ethylene
action. It has been used to study ethylene action in stimulation of respiration, fruit
softening, and flower senescence. Stimulation of endogenous ethylene by NBD also
tends to interfere with ethylene by competing for the binding site. However, the effect of
its inhibition is not permanent because the binding is reversible. This compound also has
bad odor and toxicity concerns. Sisler (1984) demonstrated that NBD is a competitive
inhibitor of ethylene by inhibiting ethylene’s binding to its receptor. NBD blocks the

receptor, but requires continuous exposure at higher concentrations. Liu (1989) reported

22

that NBD reduced loss of chlorophyll, lycopene synthesis, and increased PG activity in
tomato fruits. NBD also inhibited ethylene production, which increased the ACC
content, and decreased the MACC content in ripening stages from green to pink colored
fruits.

Silver thiosulphate (STS) is one of the best-known ethylene inhibiting
compounds. Silver ions prevent exogenous ethylene from competing with ethylene for
the binding receptor. STS very effectively inhibits the ethylene response, and its
application prevents the deteriorative effects of ethylene on flower plants. Newman
(1998) showed that STS was effective in extending the life of ‘gypsophila’ flowers.
While STS is highly effective, it has been prohibited for commercial use in many

countries, because the silver component may cause environmental contamination.

7. Active packaging applications for fresh produce

Fresh produce consists of living tissue that respires after harvest for climacteric
tissues. High concentrations of CO2 with relatively low 02 concentrations can be
beneficial at extending freshness during storage, transportation, and distribution (Sozzi,
1999). Controlled atmosphere (CA) storage creates an environment in which the desired
atmosphere is maintained throughout the storage period. Modified atmosphere
packaging (MAP) is packaging which can result in a desirable headspace mixture of
gases (Zagory, 198 8). Maximum storage quality can be maintained by creating the
optimum concentration of gas or gas mixtures in the headspace of the food product or

produce. Active package systems include oxygen, carbon dioxide, and ethylene

23

absorbers, and materials which release antimicrobial and antioxidant compounds. Active

packaging applications are summarized in Table 1.

Table 1. Some published active packaging applications

 

 

 

 

 

 

 

 

 

 

Type Function Form Active Reference
components
Ethanol Prevent microbial Microencapsul- Silicon dioxide Ooraikul, (1990)
release growth ation, Sachets powder
Ethylene Control Sachets and Potassium Jayaraman, (1992);
absorbers horticultural Films permanganate Shorter, (1992);
produce ripening and Clay powder Hobson, (1981);
Liu, (1970)
Oxygen Prevent lipid Sachets, Films, Ascorbic acid, Day, (2002);
absorbers oxidation and and Bottle Ca(0H)2, and Brody, (2002, 2001);
mold growth closures Iron oxide Rooney, (1981)
Antioxidant Prevent lipid Films BHT, BHA, and Laermer, (1994);
release oxidation or off Tocopherol Ho, (1994)
flavor
CO2 and water Control Sachets Ca(OH)2 and Jenkins, (1991);
vapor horticultural CaCl2 Vogt, (1985)
absorbers produce freshness

 

 

Applications of modified atmosphere packaging to fresh produce have been

studied by many researchers. Examples include the gas exchange system (Zagory,

1988), the use of CaCO3 and Si02 filled microporous films (Mizutani, 1993), and

application of ceramic filled polymer films (Lee, 1992). Generally, most applications

use absorbers in sachets or films to remove or replace the gas in the atmosphere

surrounding the fresh produce in the package (Jaeger, 1999).

A gas releasing system,

which uses an ethylene inhibitor in conjunction with modified atmosphere packaging, has

not been reported. A releasing system which uses an ethylene inhibitor such as l-MCP

gas can be used as an alternative to ethylene absorbers to delay the fruit ripening process.

Absorbers can also be incorporated directly into plastic structures, such as bottle closure

24

 

liners and as films to form pouches (Teurnac, 1995). Multiple layers can help absorb or
release gas compounds on the inside of the package (Zeoli, 1983). Permeable systems
which include either sachets or a multi-layer film allows for controlled release of small

concentrations of active compounds into the target zone.

8. Inverse gas chromatography (IGC)

To develop active packaging, a permeable sachet and appropriate absorbing agent
are required to deliver an active gas compound to the surface of the postharvest fruit.
These interactions include the sorption/desoprtion of l-MCP gas on the adsorbing agents.
The physiochemical interactions of l-MCP and the adsorbing agent can be described
using a powerful tool, inverse gas chromatography.

Inverse gas chromatography (IGC) can be used to study the characteristics of the
stationary phase of specific solid materials and their interaction with sorbate molecules.
This is in contrast to the analytical gas chromatography system, where the stationary
phase is only used to separate and quantify the injected compound. A typical IGC
system is similar to a conventional gas chromatography because it separates the injected
compound. However, in IGC a specific material is mixed in the column or coated on the
inner wall with an inert supporting material. The sorbate is then injected at the inlet of
either a packed column or a capillary column. The carrier gas carries the injected
compound through the column, and its motion is delayed by interaction with the sorbant.
The peak profile of the interaction between the two phases is recorded in a

chromatogram. The information can then be used to deduce the physicochemical and

25

surface properties of a specified solid material by plotting sorption isotherms and
calculating thermodynamic parameters. IGC is a rapid and accurate way to measure
sorption properties and dynamic interaction parameters of the vapor/solid system. Many
researchers have used this technique to measure polymer surface adsorption
(Kontominas, 1994), interaction of polymer with its monomer (Demertzis, 1987;
Kontominas, 1993), transition temperature and crystallinity in a polymer structure
(Guillet, 1989), interaction of volatile flavor compounds in the food matrix (Delarue,
2000), and water sorption in plastic packaging materials or food products (Kalaouzis,
1993; Demertzis, 1989). IGC has been used to evaluate solid materials for measurement
at infinite dilution (zero surface coverage), where the adsorption isotherm is essentially
linear, and at finite concentration, where the shape of the adsorption isotherm reflects the
build-up of multi-layers on the surface (Vukov, 1989). In addition to the sorbate-
stationary phase interactions, the amount of an injected sorbate can become a significant
factor affecting the shape of the chromatogram. At high concentrations, most isotherms
become nonlinear. The elution profiles observed under high concentrations of sorbate
are often skewed and have a sharp front and diffuse tail or vice versa. At low sorbate
concentrations, these isotherms are linear and thus the elution profiles are symmetric and

easily interpreted.

IGC analysis techniques

The elution technique is the most common method and is referred to as the pulse

technique, the other is a frontal analysis method referred to as the step technique. These

26

IGC techniques are described briefly as follows:

Elution analysis involves the injection of a known amount of the sorbate
molecules into a column packed with the adsorbent materials. The injected sorbate
moves in the direction of the carrier gas with an initial velocity. The molecules of the
injected sorbate collide with the surface of the adsorbent. If there is no interaction, the
velocity remains the same throughout the column. If interaction takes place, the velocity
will be retarded and the area response and shape of the pulse will be affected depending
on the strength of the interaction. The IGC elution technique was used to determine the
sorption isotherms for a series of n-alkanes with polystyrene at 30°C (Kontominas, 1994).
Demertzis (1987) investigated the interaction of vinylidene chloride (V dC) with
vinylidene chloride (VdC-VC and VdC—AcN) copolymers using the elution technique.

Frontal analysis uses a continuous flow of carrier gas and sorbate vapor at a
constant concentration through a column packed with an adsorbing agent. After the
sorbate breaks through the stationary phase a frontal chromatogram is developed. When
the adsorbing agent in the column is saturated, the detector response reaches a maximum
step-height establishing a plateau. This frontal analysis doesn’t depend on the shape of
the response, but on the area bounded by the chromatogram not affected by the kinetic
band broadening process. The isotherm profile can be derived from frontal analysis
using the mixture stream as a function of time. Apostolopoulos (1988b) used frontal
IGC to determine the water sorption isotherms of commercial freeze-dried coffee at given
temperatures. A modified frontal IGC method was used to obtain water sorption
isotherms of sucrose and starch at 25°C (Paik, 1986). The results showed that water

sorption isotherms (for sucrose and starch) obtained by modified frontal IGC were similar

27

with those of the static equilibrium relative humidity method using saturated salt

solutions.
Sorption isotherms (elution chromatography technique)
The procedure is carried out by injecting a quantity of sorbate at the inlet of a

column containing the stationary phase. By analyzing the corresponding elution peak

(Kiselev, 1969), sorption isotherms can be calculated as follows.

16'
=—V.d 1
“Wi‘c ()

where a is the amount of the adsorbed sorbate per unit mass of adsorbing agent, W is the
amount of adsorbing agent in the column, and Vc is the retained volume (V, m3) of
sorbate at the gas phase concentration (c)

28

 

      

Recorder Signal
3’

 

h W‘aha‘fimee» -'-

 

L
2,\

° m Time '
Figure 8. Typical gas elution chromatogram showing the areas used in calculating
sorption isotherm data. An is the area limited by the non-interacting time (tm), height (h),

time axis, and the diffuse side of the sorbate peak. As is the peak area of sorbate. t0 is the
time of sorbate injection. tr is the retention time of a sorbate peak.

Figure 8 illustrates the typical plot of a gas elution chromatogram from which a
data point in the isotherm (concentration of l-MCP in the column and partial pressure)
can be calculated from symmetrical peaks. The concentration of l-MCP adsorbed in the

column, M3, is given (Kiselev, 1969) by

 

M = " (2)

where Mn is expressed in moles of sorbate gas per weight unit of adsorbing agent
(mole/g), m is the injected amount of l-MCP in moles, Aa and A5 are the areas indicated
in Figure 8, and w is the weight in grams of adsorbing agent in the column.

29

The partial pressure of the l-MCP vapor entering the gas chromatographic

detector, p, is calculated by,

m h T R
p = q c (3)
vA

 

S

where p is expressed in Pascal (Pa), q is the integrator chart speed (cm/min), h is the
recorder peak height as in Figure 8 in cm, Tc is the column temperature (K), v is the
corrected carrier gas flow rate (mL/min), and R is the universal gas constant.

Specific retention volume (V °g) values and thermodynamic parameters

A typical chromatogram for a single sorbate (As) contains a retention volume (VR)

at the sorbate retention time (t,) and an additional small peak (tm) early in the

chromatogram. The retention volume is shown in Figure 8 as the distance from the point

of injection to the peak maximum. At constant flow rate (F c), VR is given by,

VR = t, x Fe (4)

Equation 5 (McNair, 1998), relates the retention volume to the theoretical distribution

constant as follows.

30

VIR is the adjusted retention volume excluding the void volume corresponding to
the adjusted retention time (t’,). t’ r is the portion time (t,-tm) resulting from interactions
of the sorbate with the stationary phase. The small early peak (VM) represents inert air

that does not sorb in the stationary phase.

VR—VM=V,,=K,-V (5)

where the volume of the air peak (VM) is called the retention volume of an unretained
compound. VS is the volume of the stationary phase. Kd is the distribution constant.

The net retention volume (VN) represents the value obtained by subtracting the
non-retained air peak (V°M) from the portion of the sorbate (V°R) defined in equation 6

(Lundqvist, 1997).

VN=J-(VR—VM)=J-Fc-(t,—tm)=J-V,:=V,;’—V,‘; (6)

where tr is the retention time of sorbate, tm is the retention time of an unsorbed species
(air), and J is the James and Martin compressibility factor.

The corrected retention volumes (V°R and V°M) are defined as the retention
volume of sorbate and the air peak correcting for the compressibility of the carrier gas,

based on the correct flow rate.

31

V: =J-t. ~F. <7)

Vfisz-F (&

"I C

ngrnfm)-q Q)

2(mme-1

where pi is the column inlet pressure, pO is the column outlet pressure, and Fc is the
correct gas flow rate (ml/sec) according to the following,

T

r

Fc = Fr .(IL).{£_Q_—_EL] (10)

0

where Tc is the column temperature (K), Tr is the room temperature (296 K), and FIr is the
flow rate (ml/sec) measured using a soap-bubble flow meter and pw is the partial pressure
of water at room temperature present in the soap-bubble flow meter.

Specific retention volume (V 0g)

Specific retention volume (V°g) is the amount of carrier gas required to elute a
given amount of sorbate from a column containing a given weight of interacting

stationary phase at 273 K per gram of adsorbing agent (Delarue, 2000).

32

 

 

Vg,=VR—VMx273zJ-F,.(t,-r,,)x273 (11)
W T, W T,
V 273
V" =—LV—x-— 12
K W T ( )

C

where VN is the net retention volume, W is the weight of adsorbing agent in the column,
and Tc is the column temperature.

Thermodynamic parameters

Determination of the thermodynamic parameters provides a better insight into the
sorption mechanism. Thermodynamic parameters were calculated by defining specific
retention volume (V°g) at an infinite dilution.
Free energy of sorption (A0,)

The free energy indicates the adsorbing agent’s affinity for l-MCP gas in the
sorption process. It provides a means to tell whether the l-MCP sorption is stronger

(negative AG, value), or weaker (positive AG, value) (Apostolopoulos, 1983). The more

negative the values of AG,, the stronger the sorption is, AG, can be calculated as,

AG, = -—R . T, -ln K, (13)

33

Ent

Er.

hrs
S)‘

Enthalpy of sorption (AH,)

Enthalpy of sorption, called isosteric heat, represents the quantity of heat released
during sorption (Riganakos, 1989). Negative values of AH, are associated with strong

binding forces in the sorption mechanism. Positive values are an indication of stronger

self attraction than adsorption.

 

aan"
-311: 18 (14)
R a—
T

Entropy of sorption (AS,)

Entropy is related to the number of different equilibrium energy states occurring
at the l-MCP and adsorbing agent interface at a defined temperature and pressure. The
entropy (A8,) characterizes the structural properties that exist when l-MCP is absorbed.
Entropy values are useful in the interpretation of processes such as dissolution,
crystallization, and swelling which occur during l-MCP sorption (Apostolopoulos,
1988a). Ordered systems have negative AS, values, whereas more random systems have

positive values, AS, is calculated as,

AS = [M] (15)

C

where AG, is the free energy of sorption (kcal/mol), AH, is the enthalpy changes
(kcal/mole), and T, is the column temperature (K).

9. Adsorbing agents

The sorption of a gas on a solid surface may take place between the sorbate and
adsorbing agent. The sorption occurs in the gas/solid system provided that the
temperature and pressure are suitable, and the gas is capable of forming a chemical bond
with the solid (Engewald, 1991). Commercial adsorbing agents are listed in Table 2.
Silica gel is a unifomly porous adsorbent, it is an amorphous condensation of polysilicic
acid with a high specific surface area. The surface of silica gel is covered by hydroxyl
groups to various degrees (Kiselev, 1969). Silica gel is a common adsorbent and is
chemically inert, non-corrosive, and non-toxic. Activated carbon is a good adsorbent
that has a large surface area. The most widely used activated carbon has a surface area
of 800-1500m2/g. This surface area is predominantly within the micropores (Smisek,
1970). Tenax-TA is a porous polymer that is based on 2,6-diphenyl-p-phenylene oxide.

Tenax-TA has a low affinity for polar compounds such as water and high affinity for

35

non-polar compounds. The average pore size is 200nm. The density of Tenax-TA is
0.25g/cc. The specific surface area of Tenax-TA is 35m2/g (Loffe, 1984). Activated
clay has been used as an adsorbent for oil pigments (Boki, 1992; Chapman, 1992).

Pinnavaia (1983) and Lan (1994) showed the structure, properties, and applications of

various clay minerals.

Table 2. Commercial applications for adsorbing agents

 

 

 

 

 

 

Adsorbing agent Main function Application Reference
Silica gel Desiccant Food and packaged Rix, (1994); Satomi,
goods (1988)
Activated carbon Filtration of odor, Food and consumer Leggott, (2001);
contaminated air, water product Pendyal, (1999); and
Roy, (1995)
Activated clay Desiccant, filtration, Healthcare, food, Boki, (1992); Habile,
pollutant adsorbent, and packaging (1992)
additives in polymer
Tenax-TA Trapping of volatiles Water, air, and Piringer, (1993)
and semi-volatiles consumer product

 

 

 

 

11. Partitioning in air and adsorbing agent matrix

The change in sorbate concentration in a food packaging system can be described
by partition coefficients between a food, its packaging and the environment (Giacin,
1995). The interaction phenomena are particularly relevant for product development
where sorbate compounds are important. Headspace sampling methods can be used to
measure the partition coefficients between air and the matrix for aroma components in

the gas phase. Headspace sampling is generally classified into two types (Chaintreau,

36

 

1995): (1) static headspace method where the vapor phase is directly injected into a gas
chromatograph and (2) dynamic headspace method, which requires trapping of the
volatiles onto an adsorbent prior to GC injection. For headspace sampling, the
experiments should satisfy the following rules: sampling of the vapor phase at
equilibrium; no pressure change during sampling; and no dilution while sampling
(Chaintreau, 1995). Partition coefficients studied between a two-phase of food
packaging system are as follows: various volatile organic compounds between air and
water (Kolb, 1992; Amoore, 1978; Buttery, 1965); gas/solution (Nahon, 2000); between
food (solid) and air or film and air producing off-flavor from plastic packaging film
(Halek, 1988a and b; Biran, 1979a and b); water and polystylene in a polymer packaging
system (Gavara, 1996). Liang (1996) examined the characteristics of gas/particle (G/P)
partitioning of organic compounds in environmental tobacco smoke. Partition
coefficients were used to estimate diffusion. Lee (1996) investigated the sorption of
cadminum on 15 types of soil using a partition coefficient model which related the
adsorption of the Cd(II) to the soil components: iron oxides, manganese oxides,

aluminum oxides, and organic matter.

12. Gas permeation

Permeability is the rate at which a quantity of gas or vapor passes through a

surface of a film structure. The amount of permeant transported via the permeation

process, is expressed by the transmission rate or flux (F).

37

F=-% (16)

where Q is the amount permeated through area (A) during time (t).

The permeability model can be expressed by the three processes involved in permeation
such as absorption, diffusion, and desorption according to the movement of permeating
molecules through a film structure (Rogers, 1985). Permeation processes are affected by
several factors, including the nature of the polymer, including its chemical composition
and molecular structure, the nature of the permeants such as size and polarity, and the
environmental conditions, such as temperature and relative humidity. Other factors
affecting permeation include permeant concentration, polymer fillers, and plasticizers.
The linear relationship between the concentration of a permeant in the polymer
and the concentration or partial vapor pressure of permeant in the gas phase is expressed

by Henry’s law.

C=S-p (17)

where C is the concentration of gas in the polymer, p is the partial pressure of permeant,
and S is Henry’s law constant, which is the solubility coefficient.

The diffusion process of a permeant is described by Fick’s first and second laws of
diffusion (Crank, 1975). Fick’s first law describes the process in which a gas or vapor

diffuses through a polymer structure under steady state conditions.

38

F = —D(°9) (18)

where F is the flux or the rate of transfer of permeant per unit area, c is the permeant
concentration in the polymer, x is the direction of the diffusion process, t is time, and D is
the molecular diffusion coefficient.

6_c:D62c

at 55 0%
Fick’s second law shows the non-steady diffusion condition, in which the concentration
of the diffusing permeant across the polymer structure is changed over time.

The permeation of a gas through a polymer structure is generally described by the
permeability coefficient (P). The permeability coefficient is a measurement of the
permeation rate of a gas or vapor through a polymer structure at steady state which is

related to the diffiision coefficient (D) and solubility coefficient (S).

P=st am

where D is the diffusion coefficient that describes how fast the permeant molecules move
in a polymer structure in terms of a kinetic parameter. S is the solubility coefficient that
measures the concentration of permeant molecules sorbed in the polymer matrix as a
thermodynamic parameter.

39

By integrating equation 18 where D is independent by concentration of the permeant,

F=D.En_:§_2_)_ (21)

where C1 and C2 are the steady state concentrations of the permeant at the two surfaces of
the polymer film with thickness (1).

Eqn. 21 can be rewritten to Eqn. 22 by substituting Eqn. 21 for Eqn. 16.

 

 

Q=D.(Cl —32).A.t (22)
By combining Eqn. 22 and Eqn. 17, it gives,
QzD'S.(pl-p2).A.t (23)

f

Eqn. 20 and Eqn. 23 can be rewritten as Eqn. 24 to determine the permeability

 

coefficient.
P = Q ' i (24)
A - t - Ap

Ap = p2-p1, pressure difference across the film

40

13. Permeability measurement methods

Permeability, diffusion and solubility coefficient values can be determined using

the following procedures.

Gravimetric method

In the gravimetric procedure, the permeation value of a permeant through a
packaging material is calculated as the weight change of a total test cell. ASTM E—96 is
described as an example of a gravimetric procedure to determine the water vapor
transmission rate. An aluminum dish containing dried desiccant is covered with a
packaging material, weighed and placed in a constant temperature and humidity chamber.
The samples are weighed periodically, and the water vapor transmission rate is
determined by the weight gain over time. The gravimetric method in general (ASTM E-
96 procedure) has low sensitivity, especially for high barrier polymeric materials and is
applicable over a limited vapor pressure range. Among the gravimetric methods, an
electrobalance system can continually monitor over time the weight change of a test
specimen at equilibrium vapor pressure (Barr, 2000; Hernandez, 1994). An
electrobalance system among the gravimetric method has been used to measure the
sorption and diffusion of organic vapors by polymer films (Nielsen, 1994; Hemandez-
Munoz, 1998). The electrobalance method has excellent sensitivity and application over

a wide range of temperature and sorbate vapor pressures.

4l

Isostatic method

In the isostatic method, a permeation cell is used, which consists of two chambers,
divided by a test film. A permeant flows continuously through the high concentration
chamber, and an inert carrier gas flows through the low concentration chamber. The
partial pressure gradient of the permeant provides the driving force causing penetration
from the high concentration chamber to the low concentration chamber. The transmission
rate of the permeant through the test film is monitored until steady state is reached under
constant conditions of temperature and permeant vapor pressure (Hernandez, 1986). The
permeability coefficient (P) is determined from the transmission rate at steady state by

the expression:

P = —-——— (25)

where C,, is steady state concentration of permeant, in mass per unit volume; f is the rate
of carrier gas flow in the low concentration cell chamber, in volume per unit of time; I is
the film thickness; and A is the surface area.

The diffusion coefficient D is calculated using the following expression (Ziegel, 1969):

[2

D=———— 26
7.1994,, ( )

where 10.5 is the time required to reach a transmission rate value that is equal to half of

42

that at steady state.

Quasi-isostatic method

In this procedure, the permeability coefficient is obtained by quantifying the
amount of permeant accumulated in the low concentration chamber after passing through
the film (Liu, 1991; Sajiki, 1993). This method is also called the accumulation method.
The gas or vapor being tested is allowed to flow from the high concentration to the low
concentration chamber. The partial pressure gradient provides the driving force for the
permeant to pass into the low concentration chamber. The permeant in the low
concentration chamber is quantified periodically using a gas chromatograph. As the
quantity of permeant accumulates in the low concentration chamber, the rate of transfer
of permeant becomes a constant.

The permeability coefficient can be determined using the flux value at steady state

and is given by,

 

= (27)

where Q is the total quantity of permeant that has penetrated during a unit time (t). A is

the film area exposed to the permeant, l is the film thickness and Ap is the driving force
(concentration or partial pressure gradient).

The quasi-isostatic method can be used to measure the permeation rate of organic vapors

at any desired low concentration level.

43

Diffusion coefficient (D) can also be determined from the lag time. It is obtained at the
intersection between the steady state projection of the transmission curve and the time

axis (Hernandez, 1986), and expressed as:

D =_ (28)

where Bis the lag time and l is the film thickness.

14. Active compound release systems

The active compound delivery system either physically traps or chemically binds
a suitable material to release a constant quantity of the active compound. Applications
of a controlled release system are found in the drug release mechanism of many of
pharmaceutical products (Shao, 2001), herbicide release of agricultural materials (Zhu,
2001), flavor/fragrance release of consumer products (Greenblatt, 1993), and the food
ingredient release of food products (Meyers, 1998). The delivery technologies of
controlled release system include (Zeoli, 1983): encapsulation, hollow fiber, monolithic
system, and laminated structure. Encapsulation is a process that coats a polymeric
material with the active agent (liquids, solids, or gases), thus providing a barrier to
undesirable environmental or chemical interactions until the release action is desirable
(Greenblatt, 1993; Risch, 1995). The hollow fiber active agent system holds the active

agent in tiny open tubes where the agent releases by diffusion through the air layer above

44

the liquid in the solid interface (Sirkar, 1999; Zeoli, 1983). In the monolithic system, the
active agent is physically blended with polymeric or elastomeric materials and remains
dispersed within the polymer matrix after it is molded or extruded. The active agent
diffuses from the inner region to the surface of the monolithic materials as the active
agent evaporates (Opperrnan, 1999). In a laminated structure, the active agent is
impregnated in a layer between two outer plastic layers. Controlled amounts of the
active agents move from the inner layer to the surface layer and subsequently diffuse
through the plastic layer (Zeoli, 1983).

In conventional control release systems, the active agent is absorbed on materials
such as silica gel, activated carbon, or activated clay. The active agent in such systems is
released by desorption, so it cannot control the release rates of the compounds
(Patwardhan, 1983). Use of polymer barriers combined with these adsorbing materials
gives an alternative controlled release application for the active agent (Yoshida, 1979).
The release mechanism for the active agent includes two processes: a desorption process

from an adsorbing material and diffusion through a controlling polymer structure.

45

SECTION III: EFFECT OF SINGLE AND REPEATED
l-METHYLCYCLOPROPENE (1-MCP)EXPOSURES ON THE RIPENING 0F
TOMATOES (L YCOPERSICON ESCULENTUM MILL.) DURING STORAGE AT
10, 15, AND 20°C

ABSTRACT

The effects of storage temperature and l-MCP treatment on the ripening process
of tomatoes were evaluated using the following parameters: skin color, firmness, total
weight, and ethylene production. Skin color and firmness changes were measured
manually using a chromameter and a durometer, respectively. The total weight of a
tomato was measured using a digital balance. The tomato ethylene production was
measured on a single fruit using a static gas collection system. Tomatoes stored at 10°C
had longer ripening periods than tomatoes stored at 15°C and 20°C. l-MCP treatment
was very effective in retarding changes in the skin color of tomatoes at the respective
temperatures. In addition, once-a-day l-MCP treatment was more effective than an
initial single l-MCP treatment. The weight loss rate of fruit showed no significant
difference between l-MCP treatment and the untreated control. The total weight loss
rate for whole fruit at 20°C was greater than that at 10°C and 15°C. After 24 days of
treatment, the external ethylene production of fully ripened tomatoes was higher at 20°C

than at 10°C and 15°C.

46

INTRODUCTION

An important effect of ethylene on climacteric fruits is to induce the ripening
process by coordinating the expression of genes, responsible for increasing ethylene
production, respiration, color change, conversion of starch to sugars, tissue softening, and
other related metabolisms (Lelievre, 1997a). Tomato (Lycopersicon esculentum Mill.) is
a climacteric fruit that has a significant response to ethylene. The rapid ripening of fruit
after harvest limits storability and is a concern during transportation and marketing. To
counter balance this process several procedures have been used to extend postharvest life
of fruits: low temperature storage (Hall, 1961); controlled atmosphere (Sozzi, 1999); and
treatment with chemical agents such as diazocyclopentadiene (Sisler, 1994),

2, S-norbornadiene (Liu, 1989), and silver thiosulfate (Newman, 1998). However, in
fruits, such as pears, very low temperature storage can still induce ripening and produces
chilling injury. The fruit loses firmness and develops off colors (Efiuvwevwere, 1988),
or stimulates autocatalytic ethylene synthesis (Lelievre, 1997b). On the other hand,
chemical agents like 2, 5-norbomadiene and silver thiosulfate are unsafe and can cause
environmental contamination (Serek, 1993).

Recently, I- methylcyclopropene (1 —MCP) was evaluated as an agent to extend
the freshness Of horticultural produce by inhibiting ethylene production (Serek, 1994).
l-MCP is a non toxic gaseous compound that, at a very low concentration, retards the
ripening of fresh produce. Studies on l-MCP have been carried out with strawberries
(Ku, 1999), apples (Watkins, 2000), bananas (Jiang, 1999), kiwifruits (Kim, 2001), and

plums (Skog, 2001).

47

The objective of this study was to determine the effect of l-MCP on the ripening
of tomatoes during postharvest storage. The tomato fruits were treated with 1 ppm(v/v)
l-MCP for once-a-day, and applied a single time at the same l-MCP concentration
during storage. l-MCP treated fruits using single and once-a-day applications compared
to control fruits without l-MCP treatment. Quality changes were evaluated as a function
of storage time. The following quality factors were considered: fruit weight, skin color,

firmness, and ethylene production.

MATERIALS AND METHODS

Materials

‘Plum dandy’ tomatoes grown during the summer of 2000 at the MSU
Horticulture Research Center in Holt, M1 were picked by hand to observe quality changes
of the fruits in storage. The tomato fruits were classified into three development stages
of maturity by skin color based on standard maturity grading (Ryall, 1979): mature green
stage (green color tomatoes); pink stage (early ripened tomatoes); and red stage (rapidly
ripened tomatoes).

1-MCP was obtained from Floralife Inc., (Walterboro, SC) as Ethleloc® powder
with 0.14% active ingredient. l-Butene (99.9% pure), was purchased from AGA
Specialty Gas Inc., (Maumee, OH) to use in quantitative analysis of l-MCP. A Carle gas
chromatograph Series 100 AGC (Loveland, CO) equipped with a flame ionization
detector and a 30cm x 2.2mm i.d. Porapak—N stainless steel packed column (Aldeich Co.,

Milwaukee, WI) was used for the analysis. Latex-free plastic syringes, lmL, (B-D. Co.,

48

Franklin Lakes, NJ) were used to inject samples into GC, and a 20 mL polypropylene

syringe (B-D. Co., Franklin Lakes, NJ) was used for exposing tomatoes to l-MCP.

Arrangement of tomato fruit for storage

Defect-free fruits with uniform appearance in color, shape, and size were sorted,
rinsed with distilled water to remove any dirt, and dried in air. Between eight to ten
tomatoes were separated by color and placed in a 10 L glass desiccator (Figure 10). A
rubber port placed on the lid connected the inlet and outlet ventilating lines with constant
air flow of 12 mL/min which was bubbled through water as indicated in Figure 9. The
glass desiccators with tomato samples were then stored in a temperature-controlled
chamber in order to perform the storage study. The temperatures in the three chambers

were set at 10, 15, and 20:l:l°C respectively.

 

Figure 9. The storage arrangement of tomato fruits

49

{\ir gas l-MCP
Corrugated Inlet line injection port

board Red stage l

tomatoes

  
 
 

Pink stage
tomatoes

‘ —-> Gas exit

 

 

 

— -_‘——--

 

 

 

A glass vessel

 

 

 

 

 

Mature green stage
tomatoes

Figure 10. A schematic diagram of the apparatus for arrangement of tomato fruits in the
glass vessel

Preparation of 1-MCP gas

Aliquots of l-MCP as Ethleloc® powder were weighed and placed in a 500 mL
Erlenmeyer flask and closed tightly with a rubber stopper. I-MCP was completely
released from the Ethleloc® as a gas after two hours following the addition of 40 mL of
distilled water. At these conditions, the headspace volume had a concentration of 480uL
l-MCP per L. A 10 mL aliquot of the headspace volume was injected through a rubber
port on the top cover in the glass desiccator to expose the tomatoes to l pL l-MCP per L
of air. The concentration of l-MCP in the sealed desiccator was measured by GC and

quantified using a calibration curve constructed with certified pure l-butene gas. For all

measurements the gas chromatographic oven was set at 85°C.

50

l-MCP treatments of tomato fruit

Tomatoes were treated with l-MCP gas using either a single or once-a-day
application. l-MCP treatments were prepared as follows. (1) For the single application,
10 mL of headspace gas stock, containing 480 uL of l-MCP per L, was injected
manually into the glass desiccator containing the tomatoes to provide a concentration of
about l-MCP 1 uL-L". After exposing the tomatoes to l-MCP for 10 hours, humidified
air was supplied through the desiccator to allow the tomatoes to respire. (2) For the once-
a—day application, 10 mL of the same gas stock was manually injected daily with
humidified air flowing through the desiccators. The experiment was carried out, for up
to nine weeks depending on the temperature. Control fruit, not exposed to l-MCP, was

stored under a continuous stream of humidified air.

Skin color, firmness, total weight loss, and ethylene production of tomato fruit

Changes in the skin color, firmness, and total weight loss of tomatoes were
monitored periodically. Measurements were performed at marked locations on the
fruit’s surface. Skin color was measured at the one-third position from top stem on a
defect-free surface area and recorded with a chromameter Model CR-300 (Minolta,
Japan). Hue angle was used to describe the skin color change. A decrease in the value
of the hue angle represented a change in skin color from green to pink and to red.
Firmness was measured manually using a Shore® hand-held durometer (Shore Co., NY)
at the marked positions. A plunger-tip probe fitted in the durometer was pressed against

the surface tissue of the fruit and the value after 3 second was measured. The durometer

51

values were recorded between 0 and 100, a decrease in value represented a decline in
firmness, converted to Newton-mm"l unit. All experiments were performed on three
tomatoes, respectively. The total weight of a tomato was measured using a digital
balance with 10 mg sensitivity. The tomato ethylene production was measured on a
single fruit using a static gas collection system at room temperature. A tomato was
placed in a 475 mL wide mouth glass jar, purged using ethylene-free air, and sealed with
a closure containing a rubber septum at the top. After one hour, 1 mL of gas from the
headspace was extracted from the sealed glass jar using a 5 mL plastic syringe. The gas
sample was injected into the gas chromatograph for quantification. The concentration of
ethylene production was determined in triplicate for each time interval. Skin color,

firmness, and ethylene production of tomatoes were measured as shown in Figure 11.

 

(a) Skin color test (b) Firmness test (c) Ethylene production test

Figure 11. Measurements of the skin color, firmness, and ethylene production for stored
tomato fruits

52

The level of the external ethylene in a tomato was calculated by substitution into the

following equation:

C.xV,

Concentration of ethylene (uL/kghr") =
W I x T

(29)

where, T is the collecting time (hrs) of external ethylene produced by the tomato; V, is
the total headspace volume (475mL); W, is the tomato weight (g); C, is the amount of

injected ethylene (ppm, v/v), corresponding to the known level of standard ethylene using
GC

Statistical analysis

Statistical analyses were performed using SAS for Windows, version 6.08 (SAS
Institute, Cary, NC). Appropriate comparisons of l-MCP treatments and storage time on
the quality of tomatoes were made using a Tukey test for multiple comparisons by a one-

way analysis of variance (p<0.05).

53

RESULTS AND DISCUSSION

l-MCP has recently been shown to be an effective ethylene inhibitor, binding at
the ethylene receptor in climacteric fruits. In this experiment, treatment with l-MCP
greatly delayed the ripening process during storage from 10°C to 20°C. In addition, low
storage temperature at 10°C also greatly delays the biochemical ripening of tomato fruit
with l-MCP application. Color development of tomato fruit at 12°C and 19°C was much
more regular than at lower temperature where chilly conditions induced poor
development (Efiuvwevwere, 1988). Atta-Aly (1992) found that pink tomatoes showed
maximum ethylene production at 20°C.

The combination of l-MCP treatment and optimum temperature storage may
result in consistent control of color development and firmness of postharvest tomato
fruits. l ppm(v/v) of l-MCP could be sufficient to inhibit ethylene action based on the
work on tomato (Nakatsuka, 1997), strawberry (Ku, 1999), apple (Mir, 2001), and banana

(Jiang, 1999).

Skin color

Mature green tomatoes at the harvest stage showed the greatest difference in color
development with l-MCP treatment compared to control fruits (Figures 12, 13, and 14).
Thus, l-MCP treatment of green tomatoes at harvest was more effective in delaying skin
color development than at the pink and red stages.

The hue angle values of the control mature green tomatoes began to decrease

rapidly at day 10, 7, and 5 for 10°C, 15°C, and 20°C respectively. For mature green

54

tomatoes, the highest rate of color change occurred at day 18 at 10°C, day 15 at 15°C, and
day 10 at 20°C as shown in Table 3.

The results of skin color change indicate that the single l-MCP treatment greatly
retarded the ripening process. For example, at 10°C a single treatment delayed the onset
of color development for mature green stage fruit from 7 to 28 days. The rate of color
change was slower in mature green stage tomatoes treated with 1 pL per L l-MCP at one
day intervals for the once-a—day l-MCP treatment than in single l-MCP treated fruits.
The once-a-day l-MCP treatment was even more effective in delaying skin color change
up to 45 days at 10°C. l-MCP action was limited by fiingal growth and mottled
appearance on the skin of the tomatoes after 50 days.

Green tomato fruits treated with l-MCP at 10°C were most effective in delaying
color development. A single treatment with l-MCP on fruits at 20°C delayed the color
change for 15 days (when a hue angle value began to decrease to 110), compared to the
controls at 7 days.

Skin color change involves loss of chlorophyll, and synthesis of other pigments,
such as carotenoids and lycopene, during the ripening period. Thus, color change is
often used as an index of the degree of ripeness, and provides primary information about
the physiological condition of the fruit. Thorne (1982) used equations to predict the rate
of fruit surface color change at any temperature. Color and firmness changes were
adequate quality indicators and could be used to predict the storage life of ‘Nemato’
tomato fruits at temperature conditions between 12 and 27°C . A very small amount of
ethylene induced color development. The rate of color development varied depending

on the particular cultivar of the same fruit or of different species. Porat (1999) reported

55

that treatment with l-MCP effectively inhibited the effect of ethylene on orange fruit
degreening. J iang (1999) showed that treatment with l-MCP delayed the skin color
change of bananas when exposed to 0.01 -1 ppm (v/v) for 1 day. Increasing

concentrations of l-MCP were generally more effective for longer periods of time.

Table 3. Days of storage until hue angle began to decrease (the breaker stage) for single
and once-a-day treated mature green fruit. For comparison, values of the control
tomatoes are included.

 

 

 

Control Single l-MCP Once-a-day
Temperature No l—MCP Treatment Treatment l-MCP Treatment
°C Time (in days) at which color started developing (hue angle=110)
10 11 25 53
15 8 17 38
20 7 15 25

 

56

 

 

 

 

    

 

 

 

 

 

    

 

 

 

 

 

 

 

I
A 100.0 1 I\§\I
.92
8’
m 80 o
g .
13.
9 60.0
E + Control green
'02) 40 0 + Single green
' —o— Once-a-day green
20.0 _
0 1 2 4 5 7
120.0
A 100.0
9
g ..
g 80.0 ‘
1:, I I
9 60.0 I I
8
'5 + C ntrol ink
('7‘) 40.0 .0 P
—I— Single pink
_._ Once-a-day pink
20.0 .
0 1 2 4 5 7
120.0
A 100.0 + Control red
-§, + Single red
to _._ Once—a-day red
3 80.0
E
g
I I
.5 I I
x
m
20.0 , ,
0 1 2 4 5 7

Figure 12. Change in skin color of tomatoes at 10°C. The error bars represent the

standard errors of the means (n=3).

Time (weeks)

57

 

 

 

120.0

 

    

 

 

 

 

 

    

 

 

 

 

 

 

 

 

3 100.0 _
8’
as
8 80.0
E.
5 60.0
8
,5 + Control green
if) 40.0 . + Single green
+ Once—a-day green
20.0 .
0 1 2 3 4 5 6 7
120.0
A 100.0 a.
£2
2’
m
g 80.0
25
.3 60 0 + g g
.c
% 40,0 + Control pink
+ Single pink
_._ Once-a-day pink
20.0 . . . .
0 1 2 3 4 5 6 7
120.0
+ Control red
3 100° " + Single red
g, + Once-a-day red
8 80.0
.1,
.§ 60.0 J I x M
I 3'.
.5
33 40.0
20.0 . . . .
0 1 2 3 4 5 6 7

Time (weeks)

Figure 13. Change in skin color of tomatoes at 15°C. The error bars represent the
standard errors of the means (n=3).

58

120.0

 

 

 

 

 

 

 

 

 

 

 

 

 

 

\
3 100.0 .
8’
as
g 80.0 ,,
.1,
.§ 60.0 - +Control green
c + Single green
5 40.0 . +Once—a-day green
20.0
0 1 2 3 4 5
120.0
+ Control pink
3 100.0 . +Sing|e pink
g + Once-a-day pink
,3 .-
g 80'0 $t‘\
a . ... 4
.9 60.0 I
8
.E
3‘, 40.0 -
20.0
o 1 2 3 4 5
120.0
3 100.0 . +Control red
g + Single red
8 80.0 ‘ +0nce-a-dayred
3;
g 60.0
. I I
§§i¢ :i_ :i I j
:5 40.0
a)
20.0

 

 

 

Time (weeks)

Figure 14. Change in skin color of tomatoes at 20°C. The error bars represent the
standard errors of the means (n=3).

59

Firmness

Firmness in most fruits varies according to the type of fruit, and its maturity, and
represents an irreversible process once initiated. The texture of fruits is affected by the
composition of their cell walls, cellular constituents, and degree of hydration. Firmness
is affected by storage temperature and the time associated with ripening. As shown in
Figures 15, 16, and 17, both the l-MCP treated, and the control fruits showed a
constantly decreasing firmness at all temperatures.

On the initial day, firmness values were approximately 7.3 N-mm'l in the mature
green stage tomatoes, 4.2 N-mm'I in the pink stage fruits, and 3.2 N-mm'l in the red stage
fruits, all with an error of i 0.5. At 20°C the firmness of the control green stage fruit
rapidly decreased to 4.1 N-mm'l on day 10, as compared to 5.7N-mm.l in the single 1-
MCP treated, and 6.9 N-mm'l in the once-a-day l-MCP treated fi'uit. At 6.5 weeks

(10°C) once-a-day l-MCP treated stage pink fruit had a value of 3.06 N-mm", while the

control fruit was softer at 1.87 N-mm'l

. Once-a—day 1-MCP treated red stage fruit at 15
and 20°C was more firm than the controls, but had similar results to the controls at 10°C.
Both the single and the once-a-day l-MCP treatments significantly delayed the
loss of firmness in green skin tomatoes as compared to the control fruit at 20°C.
Moreover, the once-a-day l-MCP treatment was more effective in maintaining firmness
than the single treatment. Green fruits stored at 10°C were also significantly firmer than

those at 20°C for the storage period. This can be attributed to the lower storage

temperature and treatment with l-MCP.

60

At 20°C, the control pink fruit firmness began to decrease rapidly and had a value
of 1.59 N-mm'l on day 10. The firmness of the controls, at relatively low storage
temperatures of 10°C and 15°C, began to slowly decrease over the storage period
compared to storage at 20°C. Loss of firmness in the pink tomatoes was greatest at 20°C
and least at 10°C for 10 days of storage.

l-MCP treatments significantly extended the storage time. Once-a-day l-MCP
treatment was more effective than the single treatment. In addition, green fruit stored at
10°C were significantly firmer over a longer period.

During ripening of most fruits, enzymatic degradation of the cell walls results in a
significant decrease in firmness. A significant decrease in firmness is mostly caused by
loss of tissue integrity in the cell wall polysaccharides pectin and hemicellulose due to the
softening related tomato B-galactosidase (Smith, 1998).

Mir (2001) reported that l-MCP reduced the softening rate of apple fruit. The
frequency of l-MCP application affected the rate of fruit softening, with more frequent
applications resulting in a lower rate of firmness loss. Kim (2001) also reported that 1-
MCP affected the firmness of kiwifruit at 20°C. Similarly l-MCP treated tomato fruit
for both single and once-a-day l—MCP treatments significantly delayed firmness loss of

the fruit skin compared with the controls at 10, 15, and 20°C.

61

 

  
 
 

+ Control green
+ Single green

  

 

 

 

 

 

 

 

 

 

 

 

 

 

 

8
.7“ .0. Once-a-day green
E
E. 6 .
.2,
w
m
a 4 4
E
LL.
2
0 a
0 1 2 3 4 5 6 7 8
1 0
8 + Control pink
'5. + Single pink
'5 + Once-a-day pink
E
z;
2
8
.E .
u.
0
0 1 2 3 4 5 6 7 8
1 0
+ Control [Gd
8 —I— Single red
.7" + Once-a-day red
E
E. 6
24.
0)
m
2
E
u.
0 .
O 1 2 3 4 5 6 7 8

Time (weeks)

Figure 15. Change in tomato firmness at 10°C. The error bars represent the standard
errors of the means (n=3).

62

 

.. + Control green
8 . + Single green
-- —o-— Once-a-day green

Firmness (N mm 1)
&

 

0 f

 

 

o
A)
N
w
A"
or
c»
N

 

 

 

 

 

 

 

 

1 0
+ Control pink
8 _ —-I— Single pink
p + Once-a-day pink
E 1
g .
m
In
8
.E.
u.
0 .
0 1 2 3 4 5 6 7
10
+ Control red
3 + Single red
.7; + Once-a-day red
5. 6 -
.2,
83
8
E
u.

 

 

 

 

Time (weeks)

Figure 16. Change in tomato firmness at 15°C. The error bars represent the standard
errors of the means (n=3).

63

 

   
 

+ Control green
+ Single green
—0— Once-a-day green

  

 

 

 

 

 

 

 

 

 

'E
E. 6 -
E.
m
m
a 4 -‘
E
u.
2
0 . 4
0 1 2 3 4 5
10
+ Control pink
8 + Single pink
7" + Once-a-day pink
E
E. 6 ,
E .
m
U)
8
E
LL
0 .
0 1 2 3 4 5
10

 

+ Control red
8 + Single red
_._ Once-a-day red

 

F irrnness (N.mm")

0

 

.1».

 

 

 

0 1 2 3 4 5
Time (weeks)

Figure 17. Change in tomato firmness at 20°C. The error bars represent the standard
errors of the means (n=3).

64

Total weight loss

Weight loss by dehydration is an important quality factor. Moisture loss in
postharvest produce causes changes in the structure of the cell tissue (Woods, 1990).
The total weight of green tomatoes decreased slowly over the storage period at 10, 15,
and 20°C as shown in Tables 4, 5, and 6. On day 10, the average weight loss of green
fruits was about 0.64% at 10°C, 0.88% at 15°C, and 1.54% at 20°C. There were no clear
differences between the once-a—day 1-MCP treated and the control fruit. Porat (1999)
reported a difference in weight loss of the orange fruit with l-MCP treatment, compared
to the controls. At the end of 20°C storage for totally ripened fruit, total weight loss was
about 2.71 percent. At 20°C the control red fruit had a short storage period because
fungal growth and a mottled appearance on the skin caused leakage at surface cracks after

2.5 weeks.

65

Table 4. Weight loss of each maturity tomato at 10°C.

 

 

 

 

(grams)
Maturity Storage time Control Single Once-a-day
(weeks) l-MCP treatment l-MCP treatment
0 0.00:1:0.00 0.00i0.00 0.00:1:0.00
g 0.5 0.37i0.15 0.27zt0.12 02310.06
£30 0 1.5 0.90:1:0.26 0.50:1:0.10 067220.21
o 3’ 2.5 1.17i0.29 0.77i0.15 0.93:1:031
.2 m 3.5 1.533.032 0.973012 1.133031
2 4.5 1.73zt0.32 1.27i0.15 1.602t0.56
5.5 2.17i0.47 1.57:1:0.15 2.00:1:0.66
0 0.00i0.00 0.00i0.00 0.00d:0.00
o 0.5 0.40:t0.10 0.23i0.06 0.27d:0.12
g) 1.5 0.77:l:0.15 0.53i0.15 0.60i0. l 7
m 2.5 1.10i0.20 0.77:t0.21 0.87:1:0.12
7?. 3.5 1.50i0.26 1.03:0.29 0.97.2012
‘3‘ 4.5 1.731031 l.47:t0.51 1.27:0.06
5.5 26010.89 1.97i0.76 1.50:1:0.01
0 0.00i0.00 0.00i0.00 0.00i0.00
o 0.5 0.23i0.06 0.17zt0.06 03010.10
g0 1.5 0.53i0.06 04010.10 0.60i0.10
g 2.5 0.73:1:007 0.60i0.10 0.87:1:021
g 3 .5 0.97i0.05 0.83i0.06 1.13:1:023
4.5 1.03:t0.06 1.13i0.15 1.50i0.36
5.5 1.53zt0.23 1.50:1:001 2.10i0.44

 

Average :t standard deviation of each tomato (n=3) as a function of time.
All values in same row are not significantly different (p>0.05).

66

Table 5. Weight loss of each maturity tomato at 15°C.

 

 

 

 

(grams)

Maturity Storage time Control Single Once-a-day
(weeks) l-MCP treatment l-MCP treatment

0 0.001000 00010.00 00010.00
5 0.5 03010.10 02710.12 04310.12
{50 o 1.5 07710.23 07710.15 1.071040
6 g) 2.5 1.231038 1.131012 1.571049
.3 m 3.5 1.471040 1.401010 1.801062
2 4.5 1.731038 1.831012 2.271085
5.5 2.171049 N/A 2.531100
0 00010.00 00010.00 0.001000
0 0.5 0.371012 02710.06 0.271006
go 1.5 0.801020 0.771023 07310.12
w 2.5 1.331023 1.131021 1.101000
TE 3.5 1.701035 1.401026 1.301010
9‘ 4.5 2.201036 1.701026 1.731006
5.5 2.731055 2.331025 2.101010
0 00010.00 0.001000 00010.00
0 0.5 0.331015 0.271012 0.271006
g9 1.5 0.701010 06710.21 0.501010
17» 2.5 0.971015 09010.26 07710.15
E 3.5 1.301026 1.031025 0.871023
4.5 1.801030 1.271031 1.171032
5.5 N/A 2.431093 1.531040

 

Average :1: standard deviation of each tomato (n=3) as a function of time.
All values in same row are not significantly different (p>0.05).
N/A means it is not available due to a crack of tomato surface

67

Table 6. Weight loss of each maturity tomato at 20°C.

 

 

 

 

(grams)
Maturity Storage time Control Single Once-a-day
(weeks) l-MCP treatment l-MCP treatment

G 0 0.001000 0001000 0001000
8 0.5 0.431012 0.431012 06310.06
‘60 g, 1.5 1.701017 1.401017 1.571040
5 g 2.5 2.171015 1.701026 1.871040
:3 3.5 2.701020 2.101036 2.371047
4.5 N/A 2.371038 2.631042

0 00010.00 00010.00 00010.00

30 0.5 04710.12 0.571021 0.531012
3 1.5 1.401020 1.431023 1.431032
i 2.5 1.671031 1.901044 1.831042
'15: 3.5 2.131049 2.531051 2.231051
4.5 N/A N/A 2.431061

0 00010.00 00010.00 00010.00

go 0.5 0.671006 0.531012 0.471015
.3 1.5 1.331015 1.171029 1.071023
13 2.5 1.631015 1.501035 1.301026
1:: 3.5 N/A 1.931042 1.571032
4.5 N/A 2.331055 1.831038

 

Average 1 standard deviation of each tomato (n=3) as a function of time.
All values in same row are not significantly different (p>0.05).
N/A means it is not available due to a crack of tomato surface

68

Ethylene production

An increase of ethylene production in the control and l-MCP treated mature green
stage fruit was detected after 4, 12, and 27 days for 20, 15, and 10°C, respectively
(Figures 18, 19, and 20).

Ethylene production of l-MCP treated pink stage fruit was similar to the controls
at 10°C. Ethylene production of the control and l-MCP treated pink stage fruit began to
increase rapidly after 2.5 weeks at 20°C. Once-a-day l-MCP treated pink stage fruit also
showed increasing ethylene production over the length of the storage period, but had a
relatively lower production than the control and single l-MCP treated fruit at 20°C.
However, ethylene production in control pink stage fruit showed no significant difference
in comparison with the l-MCP treated fruit.

Ethylene production of the control red fruit began to slowly increase after 2.5
weeks at 10°C. Ethylene production of the control and l-MCP treated red fruit increased
rapidly over 2.5 weeks at 20°C and rose on week 4.5 at 15°C. However, ethylene
production in red fruit treated with l-MCP showed no significant difference in
comparison with the control fruit. Thus, the ethylene production of the whole fruit was
not significantly affected by l-MCP treatment. The rate of ethylene production of pink
tomato fruit increased at 20°C. This is in agreement with Atta-Aly (1992) who observed
that the highest level of ethylene production was at the pink stage of ripening in the intact
tomato fruit at 20°C. Mathooko (1995) reported that carbon dioxide and
diazocyclopentadiene treated tomato fruit reduced ethylene production rates compared to

the control fruit at 25°C. Maturity stage and storage temperature had only slight effect

69

on the ethylene production, compared to the controls throughout the experiment.
Nakatsuka (1997) examined the regulation of gene expression of the ACC synthase and
ACC oxidase in ripening tomato fruit. The activities of ACC synthase and ACC
oxidase, ACC content, ethylene production, and red color development increased as
maturity was reached in the fruit ripened from the turning stage. Nakatsuka (1997)
showed that ethylene production from tomato fruit was greatly reduced by l-MCP
treatment at the turning and pink stages. Kim (1991) reported that internal ethylene

concentration increased as tomato fruit ripened.

7O

N
UI

 

+ Control green
+ Single green
..._ Once—a-day green

'0
O

.5
U"

Ethylene production (“l/kghr")
a

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

5 .
0
O 8
25
'1; + Control pink
6: 2° + Single pink
E, —o— Once-a-day pink
g 15 ..
'3 10
a.
8
if 5 *
“J A
0 -
0 7 8
25
T: + Control red
g 2° + Single red
3 —o—— Once-a-day ned
g 15
g 10
a
g 5
i
m 0 . 5 I . 1
0 1 2 3 4 5 6 7 8

Time (weeks)

Figure 18. Ethylene production of tomatoes at 10°C. The error bars represent the standard
errors of the means (n=3).

71

N
0|

 

 

 

 

 

 

    

 

 

 

 

 

 

 

 

.7" + Control green
'5, 20 - + Single green
3 + Once-a-day green
g 15 .
3 1o .
a
2
g 5
LU
O
0 7
25
'7: + Control pink
0': 20 i + Single pink
i + Once-a-day pink
3 15 .
3 10 .
Q
8
E 5 ‘
Lu
0
0 1 2 3 4 5 6 7
25
it + Control red
0', 2O 1 .
g + Single red
6} + Once-a-day red
8 15
'8
'8 10
a
g 5 A
m
0
0 1 2 3 4 5 6 7

Time (weeks)

Figure 19. Ethylene production of tomatoes at 15°C. The error bars represent the standard
errors of the means (n=3).

72

25

 

   
  

 

 

 

 
  

 

 

 

 

 

 

 

 

 

 

 

 

 

'7’" + Control green
5. 20 . -
c» + Single green
g: + Once-a-day green
g 15
E 10 .
O.
2 .
E 5 '
Lu -
0 .
O 1 2 3 4
25 .-
2 20 -
a, - + Control plnk
é, + Single pink
c 15 + Once-a-day pink
.5;
8
3 10 ..
O.
8
E 5 *
Lu
0 1
0 1 2 3 4
25
'1:
g 20 + Control red
3 + Single red
5 15 —o— Once-a-day red
5 10 1
Q
2 ti
Lu
0 . 1
O 1 2 3 4

Time (weeks)

Figure 20. Ethylene production of tomatoes at 20°C. The error bars represent the standard
errors of the means (n=3).

73

CONCLUSION

Treatment with l-MCP, in addition to low storage temperature, showed good
potential for extending the storage and distribution life of postharvest tomatoes.
Both single and once-a day l-MCP treatments proved to be very effective in delaying
color change. However, the once-a-day l-MCP treatment resulted in longer storage
times than the single treatment. Long-term exposure provided benefits regarding to
slowing color development.

This inhibitor role requires that a continuous l-MCP treatment be applied over the
storage period, which also means that the effects will stop when l-MCP treatment is
discontinued. The information obtained in this work maybe useful in developing a

controlled-release packaging system for l-MCP treatment of tomatoes.

74

Fi

Alli)‘

N/n1rr‘l

APPENDIX A

35

 

30

25.

20

Wm

15

10

 

 

 

50 60 7o 80 90 100
Durometer (duro)

Figure 21. Conversion of the durometer duro unit to the force unit

75

Ethylene production

 

with tomatoes after 1-MCP Injecting)
Standard ethylene peak

 

‘_ 1-MCP peak (from headspace of a glass vessel

 

 

 

 

 

E4~ 1-Butene gas (1 ppm (Viv), 99.9% Agro Co.)

6

(a) (b)

Figure 22. Chromatographic peak shapes of (a) l-MCP and l-butene standard, (b)
ethylene produced from the tomato and ethylene standard using a Carle gas
chromatograph series 100 AGC equipped with a 30 cm x 2.2 mm id Porapak-N stainless
steel packed column and a flame ionization detector

76

SECTION IV: EFFECT OF l-METHYLCYCLOPROPENE (l-MCP) ON
CHLOROPHYLL AND LYCOPENE CONTENTS IN STORED TOMATOES

ABSTRACT

To study the influence of storage temperature and l-MCP treatment, changes in
two major tomato pigments were observed in the pericarp, the locular gel, and the
placenta tomato tissues during storage. Storage studies were performed at 10, 15, and
20°C. Harvested tomatoes were treated with l-MCP once at the beginning of storage or
daily during storage to observe inhibitory effects on color development. Total
chlorophyll and lycopene contents were obtained by a solvent extraction method using
the partial specific extinction coefficient. l-MCP treated tomatoes had relatively low
rates of chlorophyll degradation and lycopene synthesis during storage. Total chlorophyll
and lycopene contents in the pericarp tissue of tomatoes were affected more by the 1-
MCP treatment than those in the other tomato tissues. Exposure of tomatoes to l-MCP
gas at 10°C, using the once-a-day method was the most effective in delaying pigment

changes.

77

 

111110

10111111
1111111;

‘P

11160

11110
\181
1011

20 |

INTRODUCTION

Fruit color is one of the most important factors in determining market quality of
tomato. Change in tomato color is used as an index to represent the ripening process
during storage (Ryall, 1979). Chlorophyll degradation and carotenoid synthesis are
affected by ethylene (Lelievre, 1997a).

The most significant change in tomato pigments can be represented by the
chlorophylls and lycopene, which are the primary pigments responsible for the changes in
visual color of the intact fruit (Edwards, 1967). Levels of chlorophylls for green colored
tomatoes decreased while the ratio changes of each pigment’s level of carotenoids such
as lycopene, lutein, B-carotene, C-carotene, neurosporene, phytoene, and phytofluene
increased during the ripening process. Total chlorophyll content during ripening is in the
range of 13.4tlg/g tissue in immature green to 0.3ug/g in ripened fruit (Watada, 1976a).
Two main carotenoid pigments, B-carotene and lycopene, influence the color changes in
tomatoes. Total lycopene content of tomatoes can vary significantly with variety and

developmental stage. Edwards (1967) measured 198.6ug/g in San Marzano and

12.1pg/g in Urbana at the fully ripe stage.

Delay of color change during ripening has been achieved with short term
controlled atmospheres with high CO; and low 0; at 22°C (Ratanachinakom, 1997),
treatment with fluorescent-light exposure to diszocyclopentadiene (DACP) at 22°C
(Sisler, 1994), treatment with temperatures above 30°C (Mitcham, 1992), and ethanol
exposure of the whole tomato fruit at various maturity stages (Saltveit, 1992). Storing

the mature green tomatoes at low oxygen atmosphere (3.05kPa. 3% 02). also delayed

78

color development when compared to control fruit under atmosphere conditions at 20°C
(Kim, 1999).

l-methylcyclopropene (l-MCP) is an inhibitor of the ethylene action and can
delay color development in ripening fruits. Porat, (1999) found that “Shamouti” oranges
treated with 100 ppb(v/v) of l-MCP was very effective in inhibiting the ethylene-induced
fruit degreening process. J iang (1999) observed that treatment of bananas with 1-MCP
at the levels of 0.01-1.0ppm (v/v) delayed ripening markedly in a polyethylene bags held
at 20°C. Golding (1998) found that the application of l-MCP to mature green bananas
significantly delayed onset of peel degreening, which is associated with the ripening
process at 20°C.

Therefore, the purpose of this study was to focus on changes in the major tomato

pigments during storage after l—MCP application.

79

MATERIALS AND METHODS

Materials

‘Plum dandy’ tomatoes grown during the summer of 2001 at the MSU Southwest
Michigan Research and Extension Center in Benton Harbor, M1 were picked by hand and
sorted into two major maturity stages. The stages were defined based on surface color as
follows: (1) the mature green stage (green color tomatoes), loss of chlorophyll measured;
(2) the pink stage (early ripened tomatoes), formation of lycopene determined.

Total pigment content was based on the levels in the tomato pericarp, gel, and
placenta (Figure 2). The pericarp tissue consists of an epidermal layer with a relatively
thick cuticle. The gel is a gelatinous tissue containing the seeds. The placenta is

composed of parenchyma cell that surround the ovules with the seeds embedded in the

gel.

Storage of tomato fruit

Tomato fruits with defect-free appearance were sorted, rinsed with distilled water
to remove any dirt, and dried in air. About eighteen tomatoes were separated by color
and placed in a closed glass desiccator (10L).

Storage of tomato fruits was carried out using a ventilation system with a constant
humidified air flow of 18 ml/min through the inlet and outlet ventilating lines connected a

rubber port on the lid. Storage studies were conducted at 10, 15, and 20110C.

80

Preparation of l-MCP and its treatment

Aliquots of l-MCP as Ethleloc® powder were weighed and placed in a 500mL
Erlenmeyer flask and closed tightly with a rubber stopper. 1-MCP was completely
released from the Ethleloc® as a gas after two hours following the addition of 40mL of
distilled water. At these conditions, the headspace volume had a concentration of 480uL
l-MCP per L. A 10mL aliquot of the headspace volume was injected through a rubber
port on the top cover in the glass desiccator to expose the tomatoes to luL l—MCP per L
of air. The concentration of l-MCP in the sealed desiccator was measured by Gas

chromatography.

Tomatoes were treated with l-MCP gas using either a single or once-a-day
application. l-MCP treatments were prepared as follows. (1) For the single application,
10mL of headspace gas stock, containing 480pL of l-MCP per L, was injected manually
into the glass desiccator containing the tomatoes to provide a concentration of about 1-
MCP 1pL°L'1. After exposing the tomatoes to l-MCP for 10 hours, humidified air was
supplied through the desiccator to allow the tomatoes to respire. (2) For the once-a-day
application, 10mL of the same gas stock was manually injected daily with humidified air
flowing through the desiccators. Control fruit, not exposed to l-MCP, was stored under

a continuous stream of humidified air.

81

Chlorophyll

(1) Extraction

A harvested green tomato (pre-ripening) was prepared by extracting the pericarp,
placenta, and gel tissues. Each part was cut into small pieces. About 10 grams of the
sample were transferred to a Coors Mortar (90mm diameter) and ground using a hand
pestle for 10 minutes. After grinding, three—gram portions were transferred to a Kimax
glass tube (25mm OD x 150mm L). N -N-dimethylformamide (DMF) (5 mL) was added
to each sample. The mixture was stirred using a Vortex (Model K5506, Scientific
Industries Inc. Springfield, MA) mixer and allowed to stand for approximately 10
minutes. The extraction mixture were filtered through filter paper (Whatman 1, 110 mm
Diameter) using a Buchner Funnel. The pulp remaining in the filter paper was discarded
after squeezing to remove the solution in the tissues. An extraction solution of 1 mL was
placed into a glass cuvette for spectrophotometry analysis. The total chlorophyll content
of the extracted sample was measured using an UV-Visible Spectrophotometer (Model
U-300, Hitachi Ltd.). A flow diagram to determine the total chlorophyll in the tomato

fruits is shown in Figure 23.

(2) Identification

The chlorophyll content in N -N-dimethylformamide (DMF) solvent was analyzed
and calculated as described by Moran (1982). Its specific extinction coefficient (SEC)

was based according to the following equation:

82

A). = 81.01 (30)

where, A; is the absorbance (OD units) at a given wavelength, 8}, is the SEC of the
solution at wavelength A, c is the concentration (g'f 1) and I is the beam-path (1cm) in the
measuring cuvette.

From the absorbance, the total chlorophyll content in DMF was calculated by substitution

into the following equation:

Chlorophyll total (pg/ml) = 7.04A664 + 20.27A647 (31)

where total chlorophyll concentration is expressed in ug/mL.

83

 

Green tomatoes

,1 r— . v .
‘- row i- ‘4‘ “v *7... .32”

Pericarp. placenta. and gel tissues
Cut into small pieces
(About 10 gram)

 

 

 

 

 

 

 

 

Ground in Coors
Mortar e——~
for 10 min.

 

 

 

 

3g portions into a glass tube

 

 

 

 

5mL, N-N-
dimethylformamide r—~————e
(DMF) solvent Stirred using a
Vortex for 10 min.

 

 

 

 

 

 

 

 

 

 

The mixture

 

 

 

 

 

Filtered through
filter paper The pulp is

discarded

 

 

 

 

 

 

 

 

 

1mL. the extraction solution

1

SpectrOphotometry analysis

 

 

 

 

 

 

 

Figure 23. Flow diagram of total chlorophyll extraction in tomatoes

84

Lycopene

(1) Extraction

Tomatoes were separated into pericarp, and combined placenta and core tissues.
Each tissue sample was cut into small pieces. About 10 grams of sample were
transferred to a Coors Mortar (90mm diameter) and then ground using a hand Pestle for
10 minutes. After grinding, six gram portions were transferred to a Kimax glass tube
(25mm OD X 150mm L). 10 mL of methyl alcohol was added to each sample. The
mixture was stirred using a Vortex (Model K5506, Scientific Industries Inc. Springfield,
Mass) mixer and allowed to stand for approximately 10 minutes. An acetone-hexane
solution (1:1), 10 mL, was added to the mixture. The mixture was stirred again using the
Vortex for 1 minute and then filtered through filter paper (Whatman 1, 110mm Diameter)
using a Buchner Funnel into a 100 mL volumetric flask. The pulp remaining in the filter
paper was washed with 5 mL of hexane. The washed pulp was discarded after
squeezing. The extracts were combined and placed in a 125 mL separatory funnel.
IOOmL of distilled water was added and the mixture shaken gently for 1 minute to
remove the acetone and the methanol-water soluble substances. The hexane extract (top

phase in a separatory funnel) was collected in a glass tube (Figure 24).

85

 

An early ripening ‘
harvest tomato 1' ‘

 

 

 

9 ‘ ' .l g
1.. ~. . . .. n —.--- ,L...~..- ,.—.,-..

 

 

Pericarp skin tissue and both placenta and gel
Cut into small pieces (About 10 grams)

 

 

 

 

Ground in Coors Mortar
for 10 min.

 

 

 

 

6 gram portions
into a glass tube

 

 

 

 

10mL methyl
alcohol solvent

 

 

 

 

 

 

The mixture (1)

 

 

 

Stirred using a Vortex
for 10 min.

 

 

 

 

 

The mixture (2)

 

 

 

 

 

10mL Acetone-
hexane solution

(1:1)

 

\
The mixture (3)

 

 

 

 

 

 

 

 

 

Stirred using a Vortex
Filtered through for 10 min.
filter paper

 

 

 

 

 

 

 

 

 

5mL hexane solvent for
washing the pulp

 

+— 1—1———1 Discarded the pulp

 

 

 

 

 

 

 

\

 

The combined extracts in a 125ml separatory funnel

 

 

 

 

 

Shaken gently for 1 min.

100mL distilled water to remove the acetone

and the methanol-water
soluble substances

 

 

 

 

 

 

 

 

 

The hexane extract (top phase in a separatory funnel)

 

 

 

Figure 24. Flow diagram of lycopene extraction in tomatoes

86

(2) Saponification

The hexane extract was subjected to a saponification procedure to remove
unwanted lipids and chlorophylls (Figure 25). The hexane extract was stirred using a
Vortex mixer for 1 minute with one-eighth volume methyl alcohol saturated with
potassium hydroxide. After 30 minutes, the hypophase was removed using a 5 mL glass
pipette. 10 mL of methyl alcohol was added to wash the hexane epiphase. The extract
was then washed with 50 mL of distilled water to remove the methyl alcohol using a
separatory funnel. The hexane extract (top phase in a separatory funnel) was filtered

through anhydrous sodium sulfate using filter paper and Buchner Funnels. The hexane

extract was stored at -20°C, prior to separation.

87

 

Hexane extract

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

1mL methyl alcohol . .
saturated with Stirred usmg Vortex
potassium hydroxide mixer for 1 mlnute
Removed the
hypophase
The hexane
epiphase
10mL methyl
alcohol
The mixture
50mL distilled
water Top phase in a separatory
\ funnel
Saponied hexane
extract

 

 

 

Filtered through
anhydrous sodium
sulfate

 

 

 

 

 

‘

 

The hexane extract was stored at -20°C

 

 

 

Figure 25. Flow diagram of saponification of the extract samples

88

(3) Separation

The lycopene hexane extract was chromatographed on glass columns (2cm OD x
13cm L) packed with an adsorbent combination of Hyflo Super Cel and MgO magnesia
(1:1 by weight) mixture. Figure 27 shows a flow diagram of the lycopene separation
procedure. A 0.5 cm thick layer of anhydrous sodium sulfate was placed above the
absorbent. A 1 cm thick layer of glass fiber was placed on the bottom of the absorbent.
The inlet port of the packed column was connected to a 125 mL pyrex separatory funnel.
The outlet port of the packed column was equipped with a valve to control the flow of the
solution. It was also connected to a 125 mL pyrex filter flask to collect the solution
through the column. A continuous vacuum was generated using a tap water pump
aspirator to filter the extract. A schematic diagram of the lycopene separation system is
shown in Figure 26. The absorbent in the glass column was conditioned with 50 mL of
hexane under vacuum. After conditioning, the hexane extract was introduced into a 125
mL separatory funnel connected to the inlet port of the column.

50 mL of hexane was then added to the same separatory funnel. The hexane extract was

eluted using 50 mL of hexane.

89

(B)

(C)

. - -. AI— — __. _
--."_. n-—-‘-~~'. - .-—. _
‘1 m~.‘-n—--g" --

 

Figure 26. A schematic diagram of the lycopene separation system. (A) sample and
solvent supply system (125mL) (B) glass columns (2cm OD x 13cm L) packed with
adsorbent mixture of Hyflo Super Cel and MgO magnesia (1:1 by weight) (C) collection
flasks (125mL) (D) vacuum source

The small band of yellow color was slowly separated from the main pink band at
the top of the column. The column was then washed using 40 mL of a 3% acetone
solution in hexane, causing the small yellow band (carotenes) to separate and move down
the column ahead of the pink lycopene band that was slowly diffusing downward. The
eluted material containing the yellow band was discarded. The lycopene was washed
from the column with a solution of 50 mL of 10% acetone and 5% methanol in hexane.

The eluted lycopene solution was transferred to a separatory funnel, and washed with 50

9O

mL of distilled water to remove the acetone and methyl alcohol. The hexane extract was
then filtered through anhydrous sodium sulfate on a fritted funnel to remove water, and
made up to a volume of 50 mL with hexane. The concentration of lycopene in the

hexane solution was determined using a UV-Visible Spectrophotometer (Model U-300,

Hitachi Ltd.).

(4) Identification

The total lycopene content in the hexane solution was quantified using a Hitachi
Model U-300 spectrOphotometer. The absorbance of the lycopene was measured at a
wavelength of 472.5nm. The purified lycopene was identified by comparison to the

standard spectral lycopene (Britton, 1983).

The total lycopene content of the tomato fruit was calculated by substitution into the

following equation:

6
Ajax Vm, x10 (32)
E16: x100 xW,

 

11g lycopene/gram of tomato 1

where, A; is the measured absorbance, Vm. is the total sample volume (mL), Wt is the
tomato sample weight (g), E19010“, specific extinction coefficient (SEC) of a lycopene is
the extinction at a given wavelength and in a stated solvent of a 1% solution (i.e. 1 g
lycopene/100mL solution) in a 1cm light-path sepectrophotometer cuvette. E10810", of
lycopene is 3450 at 2:4725 nm at hexane solvent (Davies, 1965)

91

 

Glass columns packed with the adsorbent mixture of
Hyflo Super Cel and MgO magnesia (1 :1 by weight)

 

 

 

Conditioned with
Hexane _ _ 50mL hexane

-933“-

 

 

 

 

 

 

 

 

 

 

50mL of the hexane
elution

 

 

 

 

 

The small band of yellow color

was separated slowly from the

main pink band which stayed at
the top of the column.

 

 

40mL of 3% acetone in

hexane 7
r .___ -4

A small yellow band is completely
separated

 

 

 

 

 

 

 

50mL of 10% acetone

and 5% methanol in
hexane. The eluted solution

containing the
yellow band is
discarded

 

l
l

 

 

 

 

 

 

 

 

 

The hexane extract solution

 

 

 

 

50mL of distilled water
is added to remove the
hypophase

 

 

 

 

 

 

Filtered through anhydrous
sodium sulfate

 

 

 

 

Added 100% hexane FT

 

 

 

 

 

50mL lycopene in hexane solution

 

 

 

 

 

Spectrophotometer analysis

 

 

 

Figure 27. Flow diagram of the method used to determine the lycopene content in
tomatoes

92

RESULTS AND DISCUSSION

Chlorophyll content

Change in the total chlorophyll content in the tomatoes at the respective
temperatures was monitored over a period of 25 days (Figures 28, 29, and 30). The
initial chlorophyll content in mature green tomatoes was 4.63, 3.81, and 4.18 pg/g in the
pericarp, the gel, and the placenta tissue. The average chlorophyll content was 13.4pg/g
in ‘Walter’ tomatoes (Watada, 1976a) and 13.8ug/g in ’San Marzona’ tomatoes
(Edwards, 1967) at the first green fruit maturity level. This would result in the level of
total chlorophyll observed at the initial stage of mature green tomatoes being lower than
the level of total chlorophyll based on the previous studies. Possible explanations for the
lower level of observed total chlorophyll include the different tomato varieties and
harvested maturity conditions, and the application of the several chlorophyll extraction

analysis techniques.

Most of the chlorophyll was lost after the breaker stage. Sink (1974) determined
the chlorophyll content of normal (a fourth generation of Fireball x Cornell) tomato
fruits and rin fruits 30 days after pollination. Total chlorophyll content of the normal
and rin fruits were 9.43ug/g and 7.38pg/g, respectively. The fin mutant fruits contained
the lower amount of chlorophyll a and the highest amount of chlorophyll b, causing the
lower a:b ratio (1.65) when compared to normal fruits.

Storage at 10°C delayed pigment development, when compared to the higher
temperatures. Chlorophyll content in the green tomatoes at 20 °C decreased rapidly for 6

days and was not detected at 14 days. The tomatoes stored at 10 0C showed that the total

93

chlorophyll content decreased for 21 days, which means that the ripening process was
delayed by the low temperature.

Sozzi (1999) observed the chlorophyll content of the controls decreased from
20ug/g at 0 day to Zug/g at 6 day at 20°C. Tomatoes stored in air showed an immediate
decline in chlorophyll and a steady increase in total carotenoids and lycopene contents.
Wu (1972) also reported the chlorophyll content in control tomatoes under normal
atmospheric conditions at 13°C decreased from 50ug/g to less than Sug/g after 15 days.

Total chlorophyll content in the pericarp tissue of the green tomatoes with once-a-
day l-MCP treatment was shown to decrease slowly and remained similar to that at the
‘green’ color stage for 25 days, as compared to the controls without 1-MCP treatment,
and the green tomatoes with a single l-MCP treatment (Figure 28). The total
chlorophyll content in the pericarp tissue of the green tomatoes at 20 0C was 2.71ug/g at
14 days using a once-a-day l-MCP treatment, compared to the controls where
chlorophyll was not detected at 14 days at 20°C.

The total chlorophyll content in the pericarp tissue of the green tomatoes was
0.81ug/g at 14 days at 10°C with no 1-MCP treatment. Total chlorophyll content in the
pericarp tissue at 10°C using a once-a-day l-MCP treatment was 2.32ug/g at 25 days.
Thus, chlorophyll loss from tomatoes treated with once-a-day l-MCP, and stored at 10°C,
was slower than single 1-MCP treated application or storage temperature, or combination
of both. Total chlorophyll content of the green tomatoes was 4.63, 3.81, and 4.18ug/g in
the pericarp, gel, and placenta tissues at day 0. The total chlorophyll content in the
pericarp, gel, and placenta tissues of green tomatoes with no l-MCP treatment at 20°C

rapidly decreased to 1.64, 0.72, and 1.01 ug/g at 3 days (Figure 30).

94

Total chlorophyll content in the pericarp tissues was relatively higher than those
in the other tomato tissues during the storage period. The internal gel and placenta
tissues of green tomatoes have higher chlorophyll degradation rates than the pericarp
tissue. l—MCP treatment had a greater influence on color development of the pericarp
tissue. Internal gel tissue had slightly lower chlorophyll content than the pericarp and
the placenta.

Combination of l-MCP treatment with low storage temperature showed very
effective in delaying chlorophyll loss as well as the treatment with controlled atmosphere
storage. In controlled-atmosphere storage with low oxygen or elevated carbon dioxide
levels (Sozzi, 1999), tomatoes stored in 20% CO; was more effective in delaying
chlorophyll degradation (lzug/g at 6 day) than the fruit continuously stored in air. Wu
( 1972) observed the total chlorophyll content of ‘green wrap’ tomato fruits as a function
of storage at 13°C, after three sub-atmosphere pressure treatments. The loss of
chlorophyll in the tomato fruits was delayed by storage at sub-atmospheric pressure.

Tomato fruit treated at a sub-atmosphere pressure of 102mmHg did not ripen, and had a

chlorophyll content of 40ug/g at 100 days storage.

95

Total chlorophyll (“g/g) Total chlorophyll (pg/g)

Total chlorophyll (pg/g)

6.00- ._ _ _ g

l —e— Once-a-day pericarp + Single pericarp + Control pericarp
5.00

4.00 , \k

l
3.00 l
2.00 9

1.00-

 

0_00.__ . . ..__. __ ._ ._ _ _..-_ __ _ .._._1

6.00-- ,, - . 1.--,
+ Once-a-day placenta +Single placenta + Control placenta

5.00 «

4.00

3.00 4

2.00 -

 

1 .00 -4

0.00- , . - , 1 1 .. _ - .1- 1,

6.00 -

5 00 _, —e— Once-a-day gel —-I— Single gel + Control gel
4.00

3.00 . \

2.00

 

 

0.00 -— — .. _. .. 11 __ _ _ .
o 5 10 15 20 25 30

Time (days)

Figure 28. Changes in total chlorophyll content of green tomatoes in storage at 10°C

96

Total chlorophyll (pg/g)

Total chlorophyll (pg/g)

Total chlorophyll (“919)

6.00 2

5.00 + Once-a-day pericarp —-I-—~ Single pericarp + Control pericarp

     

4.00 -
3.00 j
2.00 -

1.00

 

5.00

+ Once-a-day placenta + Single placenta + Control placenta

:5
o
o

3.00 »

2.00

1 .00

 

0.00

 

30
5.00 . 1 ,

4-00 + Once-a-day gel —I-—- Single gel + Control gel

3.00

 

0.00 . . r _
0 5 10 15 20 25 30

Time (days)

 

Figure 29. Changes in total chlorophyll content of green tomatoes in storage at 15°C

97

6.0017 - 7” ' _, ' 5' ~
—e— Once-a-day pericarp —I— Single pericarp + Control pericarp

 

Total chlorophyll ( 119/9)
1" co
8 8

8

0.00 -

 

25

   

4.00 —e-— Once-a-day placenta + Single placenta + Control placenta

3.00 -

2.00 '

Total chlorophyll ( pg/g)

1.00 -

 

0.00 ~

5.00 ~ ,

—o— Once-a-day gel —I-— Singel gel + Control gel

4.00 .
3.00
2.00

1.00

 

Total chlorophyll ( uglg)

0.00 ~

 

25
Time (days)

Figure 30. Changes in total chlorophyll content of green tomatoes in storage at 20°C

98

Lycopene content

Two main carotenoids were separated from the hexane extract and identified by
spectrophotometric analysis (Figure 31). The spectral curve of the purified tomato
lycopene showed maximum absorption at 470 nm, which is a major peak strongly
indicating predominance of the lycopene (red color), with two minor peaks at 440 and

500 nm. The spectral for lycopene is similar to that published by Britton (1983).

— - - -Carotene
Lycopene

 

0.5000

0.4000 <

0.3000

0.2000 ‘

The measured absorbance(ABS)

0.1000

 

.—
- I— - -— - -‘ . I I— I -_ C C“
— - Id .

0.0000 ~ ~ -
400 430 460 490 520 550

Wavelength (nm)

Figure 31. A spectrophotometeric profile of the purified lycopene extracts

99

Changes in lycopene content in l-MCP treated and non-treated tomatoes was
observed during storage at 10, 15, and 20°C (Figures 32, 33, and 34). The initial
lycopene content in ripening tomatoes was 4.25 and 4.30ptg/g in the pericarp and the

internal tissues.

Observed lycopene level in tomatoes was within the range reported by the
lycopene pigment content of the full ripened tomato varied between 12.1ug/g in ‘Urbara’
tomatoes and 198.6ug/g in ‘San Marzano’ tomatoes (Edwards, 1967). The lycopene in
the ‘San Marzano’ tomatoes changed from Oug/g in the fully green tomatoes to
180.6ug/g in the fully ripened tomatoes (Edwards, 1967).

The lycopene content in the pericarp tissue rapidly increased during 22 days of
ripening, and reached 23.2811g/g in the fully ripened fruit at 20°C, with no l-MCP
treatment. The lycopene content at 22 days in the pericarp and internal tissues at 20°C,
with 1-MCP treatment once a day, was 5.93pg/g and 12.39ug/g, compared to the controls
with values of 23.28ug/g and 22.30ug/g. This difference is attributed to the 1—MCP
treatment, which inhibited lycopene synthesis. In addition, the lycopene concentration in
the pericarp tissue of l-MCP treated tomatoes was lower than that in the internal tissue.

It can be assumed that the pericarp tissue was affected more by the 1-MCP treatment due
to direct exposure of 1-MCP gas at the fruit surface.

The pericarp and internal tissues also showed a slow increase in the lycopene
content at 10°C, and had values of 9.21plg/g and 11.57ug/g at 29 days with no l-MCP
treatment. The lycopene content of pericarp and internal tissues on pink stage tomatoes
at 10°C, under once-a-day 1-MCP treatment, was 5.74ug/g and 7.04pg/g at 29 days,

respectively.

100

The relation between lycopene content (%) and chlorophyll content (%) was also
determined for a single and once-a-day treatment of 1-MCP (Figure 35). Once-a-day 1-
MCP treatment at 20°C showed the loss of chlorophyll much faster than the formation of
lycopene when compared to the control fruit. In contrast, a single l-MCP treatment did
show the same pattern as the results of the control fruit. The relation between lycopene
and chlorophyll at 10°C represented no significant difference for l-MCP treatments.

l-MCP applied as a single exposure or multiple tissues plays an important role as
an ethylene inhibitor, which delays tomato color change in storage. Lycopene synthesis
in the tomatoes at 10°C was slower than at 20°C. Based on the results, low temperature
storage may significantly affect the formation of lycopene and the loss of chlorophyll. It
is clear that the lower the storage temperature, the longer it takes for lycopene synthesis,
with the exception being storage at chill injury conditions. These results were similar to
that of studies by Mencarelli (1988) using tomato fruit slices under storage at low
temperature and under controlled atmospheres (CA) to slow the rate of lycopene

synthesis.

101

Lycopene (pg/g)

Lycopene (pg/g)

30.00

 

 

+ Control pericarp

 

 

 

 

 

 

 

 

 

 

 

25.00 . + Single pericarp E
-o— Oncea-day pericarp E
20.00
E
15.00 - E
10.00 ’
I1..——————!
5'00 *‘g—éf:
0.00
Time (days)
30.00 m.---..-...--,----___ , , .1 _ _
2500 + Control internal
+ Single internal
—e— Once-a-day internal
20.00
15.00
10.00 ,
5.00 ~
0.00 —
0 5 10 15 20 25 30 35 40
Time (days)

Figure 32. Formation of lycopene in pink tomatoes in storage at 10°C

102

30.00 .

Lycopene (pg/g)
E5
8

30.00

25.00

Lycopene (pg/g)
-* N
01 .0
O O
O O

—A
.0
o
o

5.00

0.00

Figure 33. Formation of lycopene in pink tomatoes in storage at 15°C

 

 

+ Control pericarp
—I— Single pericarp

 

 

 

 

 

Time (days)

103

 

+ Once-a-day pericarp /
0 5 10 15 20 25 30 35 40
r-,_-_-*.____._ -__-

+ Control internal

+ Single internal ./

—o— Once-a-day internal

—/

0 5 1 0 1 5 20 25 30 35 40

Lycopene (pg/g)

Lycopene (pg/g)

30.00 T“~"‘"'"” m.-.~ _.._-_- ...-.----._-_..~.--.. - --. -- -._.. .-a .... _

 

+ Control pericarp
25.00 —-I— Single pericarp

+ Once-a-day pericarp

20.00

 

15.00

10.00

 

 

 

 

 

 

 
 
   

 

 

 

5.00 ,
0.00
0 5 10 15 20 25 30 35 40
+ Control internal
25 00 + Singel internal
——o— Once-a-day internal
20.00 ,
1 5.00
1 0.00
5.00 .
0.00 -
0 5 10 15 20 25 30 35 40
Time (days)

Figure 34. Formation of lycopene in pink tomatoes in storage at 20°C

104

120

100 *

80

Chlorophyll (%)

20

17

120

100 4

80
60
40
20

Chlorophyll (%)

120
100
80
60
40
20

ChlorOphyll (%)

60-
40‘

 

 

10°C \

19 21 23 25

 

 

 

 

 

 

 

 

 

 

 

24 32 40 48
-------- Control
----- Single
Once-a-day
26 36 46 56

Lycopene (%)

Figure 35. Relation between lycopene and chlorophyll in the pericarp tissue for the
various treatments.

105

CONCLUSION

l-MCP delayed degradation of chlorophyll and production of lycopene.
Combination of l-MCP treatment with low storage temperature was very effective in
delaying color changes in tomatoes compared to l-MCP treatments or a low storage
temperature. Low storage temperature slows ripening of tomatoes. l-MCP also directly

affects the ethylene inhibitor sites related to pigment changes in tomatoes.

106

APPENDIX B

1.9

 

 

 

 

 

 

 

 

 

E
'E
:3
D
Q
0
o
5
D 1.1
5
§
0.9 . , , , , ,
Chlorophylla
- - . . Chlorophyll b
0.5
400 450 500 550 600 650 750

Wavelength (nm)

Figure 36. A spectrophotometeric profile of the standard chlorophylls a and b from a
spinach source (Sigma Chemical Co., St. Louis, MO).

107

SECTION V: SORPTION STUDY OF l-METHYLCYCLOPROPENE (l-MCP)
BY SELECTED ADSORBING AGENTS USING INVERSE GAS
CHROMATOGRAPHY

ABSTRACT

Sorption isotherms of l-MCP in silica gel, Tenax-TA, and activated clay were
determined using inverse gas chromatography at 50, 60, 70, and 80°C. The sorption
isotherms, which were developed at very low sorbate concentration, followed Henry’s
law, and behaved according to binding site theory. Specific retention volumes for 1-
MCP on the adsorbing agents were calculated. Silica gel had a much higher number of
binding sites, compared to Tenax-TA and activated clay agents. Thermodynamic
parameters, namely free energy, entropy, and enthalpy were also obtained as a function of
temperature for all adsorbing agents. Silica gel and l-MCP had the lowest Gibbs free
energy, whereas the enthalpy value of sorption for Tenax-TA had a greater binding
energy with a smaller number of binding sites. The entropy values for sorption for the 1-

MCP/adsorbing agents were not affected by the l-MCP concentrations and temperature.

108

INTRODUCTION

During postharvest storage, ethylene induces physiological changes in plants
including, accelerating abscission, ripening of fruits, leaf abscission, and fading of
flowers (Serek, 1995b). Managing the ethylene concentration is important to control the
quality of produce during postharvest storage. To limit or remove ethylene, absorbing
agents have been suggested during postharvest storage (Shorter, 1992). Most commercial
applications that remove ethylene from the package headspace require adsorbing agents
either in sachets or integrated into the packaging materials (Zagory, 1995). Instead of
removing ethylene by absorption, l-MCP offers a novel alternative, since it can inhibit
the ethylene action. Therefore, by properly delivering l-MCP into the package, it is
possible to prevent or retard undesirable postharvest ethylene effects. A system that can
deliver small quantities of l-MCP vapor into the headspace of a package containing fresh
produce is desirable. The aim of this work was to study the sorption behavior of l-MCP
by adsorbing agents. The sorption properties of l-MCP adsorbing agents can be
determined using inverse gas chromatography (IGC). Inverse gas chromatography is a
powerful tool that can rapidly and efficiently describe sorption behaviors (Kontominas,
1994). Determination of the thermodynamic parameters by IGC results in a complete
description of the sorption/desorption process, which can be useful in a l-MCP delivery
system.

The objectives of this study were: (a) to determine sorption isotherms for l-MCP

in silica gel, Tenax-TA, and an activated clay at several temperatures; (b) to evaluate the

109

specific retention volume of 1-MCP for each absorbing agent; and (c) to obtain the

thermodynamic parameters associated with the sorption processes.

”0

MATERIALS AND METHODS

Materials

l-methylcyclopropene (1 -MCP) was obtained from Floralife, Inc., (Walterboro,
SC) as Ethleloc® powder containing 0.14% active ingredient. l-Butene (99.9% pure)
was purchased from AGA Specialty Gas Inc., (Maumee, OH) for quantitative analysis of
l-MCP. Silica gel, grade 634-60A, 100-200 mesh was obtained from Aldrich Chemical
Co. (Milwaukee, WI), while Tenax-TA, 60-80 mesh was purchased from Alltech
Associates, Inc., (Deerfield, IL). Activated clay, a modified montrnorillonite mineral (I-
28E) was obtained from Nanocor, Inc., (Arlington Heights, IL). Activated carbon, 20-40
granular mesh was purchased from Aldrich Chemical Co. (Milwaukee, WI). Activated
carbon, 80-100 granular mesh was purchased from Alltech Associates, Inc., (Deerfield,
IL). a-cyclodextrin as a molecular encapsulation agent was purchased from Cerestar

Co., (Hammond, IN).

Preparation of chromatographic column

In the IGC technique, the solid phase is packed inside the chromatographic
column. Thus, the adsorbing agents used were packed in 300mm x 6.4mm O.D.
stainless steel chromatographic columns and secured with glass fiber stoppers placed at
both ends of the column. The amount of adsorbing agent used was determined by
weighing the difference in the column before and after packing. Characteristics of the

adsorbing agents and weights used as molecular encapsulation agents are shown in Table

III

7 The packed columns were pre-conditioned at 150°C under a stream of Helium gas for
24 hours before the chromatographic studies were conducted. Figure 37 shows the

packing materials, and the stainless steel columns used for the stationary IGC phase.

Table 7. Parameters of the selected adsorbing agents used as the stationary phase of the
IGC system

 

Weight of adsorbing 1-MCP retention time

 

Agents Characteristic agent in the column at 80°C
Grams min
Silica gel 100-200 mesh, 60A, 3.60 13.28
white color, granular
Tenax-T A 60-80 mesh, grayish 1.93 3.71
white color, granular
Activated clay I-28E, Powder, 1.68 1.33
brownish-white color
Activated 20-40mesh, black 1.01, 0.74, 0.57, 0.21 N/A
carbon“ color, granular
a-cyclodextrin“ white color, Powder 1.74, 0.45 N/A

 

*N/A = no peak was observed from the GC integrator after 4 hrs.

d l Ill .
,1 /
.-/ r//
w. IZLAUZ‘J ’ on silica gel
Ll-4I) me: Jhl‘r 7w; JAE]; 1U ILllr
-i‘.u "I
Tenax TA A: tivate eI‘d arbon Activated clay ‘

(80 100 mesh;

 

 

Figure 37. The packing materials and the stainless steel columns used for the stationary

phase

112

Preparation of l-MCP gas and standard calibration

Aliquots of l-MCP as Ethleloc® powder were weighed and placed in a 250 mL
glass serum vial sealed with aluminum top-hole caps and PTFE/Silicone 20 mm septa
(Supelco Inc, Bellefonte, PA). l-MCP was released from the Ethleloc® as gas by
addition of 60 mL of distilled water. Concentration of l-MCP was measured by
sampling 200 pL of the headspace volume using a gas tight microsyringe (Hamilton Co.,
Reno, NV) relative to a certified pure l-butene gas. The standard calibration solutions
were prepared using a serial dilution procedure using 0.05, 0.10, 0.15, and 0.20 pg

concentrations of a certified pure l-butene gas.

Inverse gas chromatography system

A Hewlett-Parkard gas chromatograph Model 5830A (Avondale, PA) equipped
with a flame ionization detector, was used for IGC measurements. Once the column was
packed with a particular adsorbing agent, it was installed in the gas chromatograph (GC).
Helium was used as the carrier gas, at a flow rate of 27.27 mL/min at 24°C. The inlet
pressure was controlled using a precision regulator and measured using a pressure gauge,
model SGVGSGC (AGA Specialty Gas Inc., Maumee, OH). The void volume of the
column was determined using non-interactive air. 100 uL of air was injected into the
column using a gas-tight syringe. This low volume of gas approached infinite dilution
concentration inside the column. The operation conditions of the gas chromatograph
were set as follows: oven temperatures, 50, 60, 70, and 80°C; inlet port: 150°C; and flame

ionization detector: 250°C.

113

Adsorption isotherms

The adsorption measurements were made using an elution chromatography
procedure. In this method, small quantities of sorbate of approximately 3x10'7 g in
0.5mL were injected at the column inlet port. By analyzing the corresponding elution

peak, sorption parameters could be calculated (section 11, page 29).

Other parameters

Specific retention volume and selected thermodynamic parameters were
calculated for each adsorbent (Kalaouzis, 1992). The specific retention volume (V °g)
defined as the net retention volume (VN) at 273 K per unit weight of adsorbing agent
(section 11, page 32). The specific retention volume (V°g) was calculated using Equation
11. The larger the V°g, the stronger the attraction between the sorbate and the stationary
phase. V°g is expressed in mL (STP) per gram, of stationary phase.

The following thermodynamic parameters were obtained using the equations in
Section 11, free energy of sorption, AGS, which measures the adsorbing agent’s affinity for
l-MCP; enthalpy of the sorption process, AHS; and entropy, ASS, which characterizes the

degree of disorder of l-MCP associated with the stationary phase as compared to l-MCP

in the gas phase.

114

RESULTS AND DISCUSSION

The adsorption of 1-MCP by silica gel, Tenax-TA, and activated clay at 80°C is
shown in Figure 38. The amount injected for silica gel, Tenax-TA, and activated clay

was 9, 6, and 4 nmole, respectively.

3.71

1.33

 

 

 

 

 

CW
3 START

(a) Silica gel (b) Tenax - TA (c) Activated Clay

Figure 38. Adsorption of 1-MCP on (a) Silica gel, (b) Tenax-TA, and (c) Activated clay
by inverse gas chromatography

The peak profiles are represented as typical chromatograms using the inverse gas
chromatography elution technique. Smaller amounts of l-MCP resulted in longer

retention times than large amounts. Peak shapes and retention times were dependent on

the amount of l-MCP injected.

115

Chromatogram peaks of l-MCP adsorption were not observed for the adsorbing
agents of activated carbon and a-cyclodextrin. It is probably because l-MCP has too
strong an affinity for these adsorbing agents. Activated carbon has a high affinity to
adsorb organic molecules of liquids or vapors because of strong interactions on the
carbon surface (Bansal, 1988). Meanwhile, Bagreev (2000) studied the adsorption of
hydrogen sulfide on activated carbons (unmodified and catalytic with introduced basic
nitrogen group) using inverse gas chromatography at infinite dilution. The adsorption of
hydrogen sulfide on noncatalytic and catalytic carbons was governed by Henry’s law.
Lopez-Garzén (1993) used inverse gas chromatography at infinite dilution for studying
the surface characteristics of several porous active carbons. The thermodynamic

parameters of adsorption of n-alkanes were determined from the net retention volume.

Sorption isotherms

Sorption isotherms of l-MCP adsorption versus l-MCP partial pressure were
determined at 50, 60, 70, and 80°C for the three adsorbing agents. The results for silica
gel, Tenax-TA, and activated clay are presented in Figures 39, 40, and 41.

These studies show that inverse gas chromatography is a useful method for

determining the sorption behavior of l-MCP gas at an infinite dilution concentration.

The results were obtained from the profile peaks of l-MCP interaction with the adsorbing
agents, silica gel, Tenax-TA, and activated clay.
Sorption isotherms of l-MCP and their respective adsorbing agents were linear,

that is, ruled by Henry’s law. They were used to predict the amount of l-MCP gas

116

adsorbed by the adsorbing agents (mole/g) at 23°C. Kontominas (1994) presented
nonlinear isotherms for a series of n-alkanes on polystyrene in the finite concentration
region. Demertzis (1987) obtained sorption isotherms of vinylidine chloride on
vinylidene chloride copolmers (V dc-VC and Vdc-AcN). Gavara (1997) determined the
sorption isotherms for four food aroma components on three polymeric materials. The
isotherms for both copolymers and three polymeric materials were linear in the low and

high vapour pressure range.

117

45

4o

35

30

20

Ma (n mole/g)

25‘

15‘

10

0.0

0.1

0.2

0.3

50 °C

0.4 0.5 0.6
Partial pressure (pa)

0.7

70 °C

0.8

0.9

Figure 39. Sorption isotherms of l-MCP on silica gel at 50, 60, 70, and 80°C

118

1.0

45.» -.

4o.

35-

30.

Ma (n mole/g)

25.

20

15‘

10

0.3

50°C

0.4 0.5 0.6
Partial pressure (pa)

60°C

0.7

70°C

80°C

0.9

Figure 40. Sorption isotherms of l-MCP on Tenax-T A at 50, 60, 70, and 80°C

119

45

4o .1

35.

30‘

25

Ma (n mole/g)

15

10

20~

0.0

0.2

0.4

50°C

0.6 0.8
Partial pressure (pa)

60°C

1.0

70°C
80°C ‘

1.2 1.4

Figure 41. Sorption isotherms of 1-MCP on activated clay at 50, 60, 70, and 80°C

120

Linear plots were derived from a least square fit of the log constant value (LnKs)
versus the reciprocal of the absolute temperature (1000/T, K) for each adsorbing agent as
shown in Figure 42. The linear equations were as follows: Y=5.5356X — 12.244 (silica
gel); Y=6.0074X — 14.159 (Tenax-TA); Y=3.443 8X — 7.9804 (activated clay), where X is

the 1000/T (°K) and Y is the —Log(Ks).

 

 

 

 

6.0
Silica gel
5.0
Tenax-TA

4.0
g 3.0 Activated clay
5

1.0

0.0 . . .

2.80 2.85 2.90 2.95 3.00 3.05 3.10 3.15

103rr (K“)

Figure 42. Linear plot of the log constant value (LnKs) versus the reciprocal of the
absolute temperature (lOOOfF, K)

121

As shown in Figures 39, 40, and 41, the sorption isotherms were linear functions

of the partial pressure of l-MCP, which indicates that they follow Henry’s law,

Ma: Ks-p (33)

where Ma is the concentration of l-MCP adsorbed, K5 is the Henry’s law solubility
coefficient, and p is the partial pressure of the l-MCP vapor entering the GC detector.

An increase in temperature decreased the amount of l-MCP absorbed on silica
gel. Similar results were obtained for the other two adsorbing agents. All correlation
coefficients for the isotherms were higher than 0.98. Ks values for l-MCP and each

adsorbing agent are summarized in Table 8.

Table 8. Solubility coefficients (K5) of l-MCP as a function of temperature for each
adsorbing agent

 

 

 

Ks
Temperature N mole/ g Pa
°C Silica gel Tenax-TA Activated clay
50 133.91 80.85 14.90
60 78.99 54.10 10.57
70 49.80 26.52 7.41
_ 80 30.97 17.69 6.14

 

l22

Silica gel had a much higher Henry’s law constant (K) than Tenax-TA and

activated clay. This implies that the amount of l-MCP adsorbed on the silica gel, at the

corresponding partial vapor pressure of l-MCP, was greater than that for Tenax-TA and

activated clay. Equation 33 was used to estimate the sorption isotherms of l-MCP at

23°C as shown in Figure 43. Ks values of at 23°C were 640 nmole/g-Pa for silica gel,

460 nmole/g-Pa for Tenax-TA, and 40 nmole/g-Pa for activated clay.

700

600 .

500

400

300

Me (n mole/g)

200

100

 

 

 

 

 

Silica gel
,, .
.’
/° Tenax-TA
/ I
1".) . ,,
.0/ I
. I ..
O,
, _ .1
x './
,,. '
/ fl '- Activated clay
,./.
C.
V ’1 A M... , a —-—a 3,... ~—#—A~ -—-fa— _, -~—a
o 0.2 0.4 0.6 0.8 1 12

Partial pressure (Pa)

Figure 43. Calculated sorption isotherms of l-MCP on each adsorbing agent at 23°C

123

Specific retention volume (V °g) values

Specific retention volumes (V°g) for each adsorbing agent at 50, 60, 70, and 80°C
are presented in Figures 44, 45, and 46. Table 9 shows that the specific retention volume
of l-MCP gas for the adsorbing agents depends on the temperature. The specific
retention volume of silica gel was higher than those for Tenax-TA and activated clay.
These values indicate that l-MCP gas has a stronger binding force with the silica gel than
the other adsorbing agents. V°g was not affected by the amount of l-MCP injected.

This assumes that l-MCP levels are relatively low and are not saturating the available
number of binding sites in the respective stationary phases. The specific retention
volume for all adsorbing agents increased with decreasing temperature because at lower
temperature, lower kinetic energy was necessary to bind l-MCP to the adsorbing agents’

binding sites. Therefore, more binding sites could be occupied.

Table 9. Specific retention volumes (V°g) of l-MCP with each adsorbing agent as a
function of temperature

 

 

 

Temperature Specific retention volume, V°g
°C mL/g
_ Silica gel Tenax-TA Activated clay
50 265i0.84 156d:0.22 25i0.28
60 16li1.66 91i0.15 17i0.19
70 992t0.62 52i0.16 12:1:0.31
80 6421:003 32i0.05 9i0.18

 

Data are the average of l-MCP uptakes of l, 2, 3, 4, 5, and 7nmole/g at infinite dilution.

124

Silica gel was found to strongly interact with l-MCP compared to Tenax-TA and
activated clay, based on the specific retention volume (V°g) for l-MCP and their
adsorbing agents, and on the thermodynamic interactions of adsorption of l-MCP
molecules, based on binding site theory. Specific retention volume of l-MCP gas for
silica gel, Tenax-TA, and activated clay at 50°C was approximately 265, 156, and
25mL/ g, respectively.

Values of water for unplasticized PVC and unplasticized P(VdC-VC) at 50°C
were approximately 24 and 18mL/g (Kalaouzis, 1992). V°g of VCM for resin E and
resin S of PVC polymers at 55°C for studying the effect of polyvinyl chloride (PVC)
polymer structure, monomer level and temperature on vinyl chloride monomer (V CM)
migration were approximately 12 and 0.13 mL/g, respectively (Apostolopoulos, 1988a).

The VCM specific retention volume for resins was dependent on the concentration and
temperature. The results showed that specific retention volume of 1-MCP gas for
adsorbing agents represented relatively higher value, compared to those of water and
VCM on PVC polymer.

Figure 47 shows a straight line for Log V°g against l/T between 50 and 80°C for
l-MCP and the respective adsorbing agents. Delarue (2000) observed retention behavior
using a linear plot of Log V°g vs UT of aroma molecules on carbohydrate matrixes such
as starches or maltodextrins, and evaluated the enthalpy of adsorption of these molecules
using IGC. Gauthier (1998) studied the physicochemical modifications of cellulose
using acyl chlorides having 5 and 16 carbon atoms. He found a linear relation between

Log V"g vs 1/T between 50 and 140°C for n-octane on a Cw-CE IGC column. The slope

of the linear line was used to determine the differential enthalpy adsorption value.

125

300

 

250

200

150

V°n (m l/g)

 

 

A

 

I»
I»
l.
I»

. . _.____.___.__. 80°C

50°C

70 °C

 

 

0.00

1.00 2.00 3.00 4.00 5.00 6.00
Amount of injected 1-NCP per gram of adsorbing agent (n mole/g)

7.00

Figure 44. Specific retention volume (V°g) for silica gel as a function of l-MCP
concentration at 50, 60, 70, and 80°C

126

8.00

300

 

 

 

 

 

 

 

 

% e A A g 50 0C
250
200
a 60 °C
> E— + + + __.
g 150
D
‘5
100 . g g : ; fi 70 °C
. . _+____+___§____, 80 °C
50
0
0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00

Amount of injected 1-NCP per gram of adsorbing agent (n mole/g)

Figure 44. Specific retention volume (V°g) for silica gel as a function of l-MCP
concentration at 50, 60, 70, and 80°C

126

300

 

 

 

 

 

 

250
200
’9 - , ,
g 150 ' ¢ 3 9 50°C
00!
>
100
k e a r + I 60°C
0
50 er a At a _._ 70C
o—o——o———o————o
80°C
0

 

0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00

Amount of injected 1-MCP per gram of adsorbing agent (n mole/g)

Figure 45. Specific retention volume (V°g) for Tenax-TA as a function of l-MCP
concentration at 50, 60, 70, and 80°C

127

 

100

 

 

 

 

 

 

 

80
60 -
’6»
2:
.5,
00
>
40
e e e : 050°C
20 o
n— I— + 4 460 C
m f f :r 4‘ “0°C
V v V V V 80°C
0
0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50

Amount of injected 1-MCP per gram of adsorbing agent (n mole/g)

Figure 46. Specific retention volume (V°g) for activated clay as a function of l-MCP
concentration at 50, 60, 70, and 80°C

128

3.00

 

 

 

 

 

2-50 Silica gel
Tenax-TA
2.00
or»
3 1.50 Activated clay
8’
_l
1.00
0.50
0.00 l
2.8 2.9 2.9 3.0 3.0 3.1 3.1 3.2
103/r (K")

Figure 47. Log specific retention volume (V°g) for l—MCP as a function of temperature

129

Thermodynamic parameters

Gibbs’s free energy, entropy, and enthalpy at the four temperatures are shown in
Tables 10 and 11. For these adsorbents, Data are from the average values of l-MCP

uptake at 1, 2, 3, 4, 5, and 7 nmole/g at infinite dilution.

Table 10. Gibbs’s free energy values of l-MCP at several temperatures

 

 

 

Temperature -AGs (kcal/mole)
oC Silica gel Tenax-TA Activated clay
50 3.36i0.02 2.46i0.01 1.60i0.07
60 3.16i0.04 2.20i0.01 l.41:l:0.08
70 2.94:l:0.05 1.91i0.02 1.27:t0.02
80 2.74i0.01 1.64i0.01 l.13i0.01

 

AGs values decrease with decreasing temperature as shown in Figures 48, 49, and
50. Low temperature favors the adsorption process. Gibbs’s free energy values for all
adsorbing agents were negative indicating that a spontaneous bonding process occurred
between l-MCP and the adsorbing agents. Silica gel had higher absolute values of AGs
as compared to Tenax-TA and activated clay. This indicates that silica gel is more
interactive than the other adsorbing agents tested. Gibbs’s free energy value for l-MCP
on silica gel at 50°C was approximately -3.36 kcal/mole at infinite dilution.

Gibbs’s free energy of water vapor for PVC-plasticizer mixtures and p(Vdc-VC)-
plasticizer mixtures at 50°C had the values of approximately -3.04 and -2.99 kcal/mole,
respectively (Kalaouzis, 1992). These values are similar to the free energy results for 1-
MCP on silica gel. However, AGS values for adsorption of vinylidiene chloride on VdC-

VC and VdC-AcN copolymers was approximately -1.57 and -1.63 kcal/mole,

130

respectively. Values increased with decrease in monomer concentration and
temperature, indicating a more favorable monomer-polymer interaction at low
temperatures and monomer concentrations (Demertzis, 1987). This pattern is similar to
the free energy value of vinyl chloride monomer on unplasticized resins E and S of the

PVC polymers as reported by Apostrolopoulos (1988a).

13]

3.60

 

 

 

 

 

 

 

3.40 4 o

3.20
75 k I 4 —I— H 60 °C
E
8 3.00
5 A A 0
(3 Ir a - - fi‘ 70 C
$1

2.80

o——+————+——o——o 80 °C
2.60
2.40
0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00

Amount of injected 1-MCP per gram of adsorbing agent (n mole/g)

Figure 48. Gibbs free energy of sorption (AG5) for silica gel as a function of 1-MCP
concentration and temperature.

132

3.60

 

3.20 ,

2.80

1P
o
l
0
l
D

O
2.40 ' T 50 C

 

L J I 1 l— —I 60°C

~AGs(kcal/mole)

2.00
a 70°C

H»
I»
l

I»

O
160 o—o———o——+——o 80 C

 

 

1.20
0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00

Amount of injected 1-MCP per gram of adsorbing agent (n mole/g)

 

Figure 49. Gibbs free energy of sorption (A6,) for Tenax-TA as a function of l—MCP
concentration and temperature.

133

2.60

 

 

 

 

 

 

 

 

2.20
16
o
E
8 1.80
as
‘2'}
- 9 = e as 4 50°C
1.40 ‘ # l— + H 60°C
'XJ- = A 70°C
\L A A A
' V f ' 80°C
1.00

 

0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00
Amount of injected 1-MCP per gram of adsorbing agent (n mole/g)

Figure 50. Gibbs free energy of sorption (A6,) for activated clay as a function of l-MCP
concentration and temperature.

134

As expected, the entropy values were negative. Tenax-TA had higher absolute

entropy values that silica gel and activated clay (Figures 51, 52, and 53). This indicates

that l-MCP is probably bonded more orderly to Tenax-TA than to the other two

adsorbing agents. A more regular binding pattern may indicate that Tenax-TA has a

more regular molecular architectural structure than the other two adsorbing agents.

Table 11. Entropy (A8,) and enthalpy (AHS) values of l-MCP at selected temperatures

 

 

 

 

Temperature -ASS (cal/mole K)
0C Silica gel Tenax-TA Activated clay
50 18.69i0.07 25.07zt0.02 14.96i0.05
60 18.74i0.08 25.0%0.01 1 5.09:1:0.03
7O 18.83zt0.09 25.22zt0.01 15.07:!20.O7
8O 18.85i0.07 25.26i0.01 15.02i0.05
-AHS (kcal/mole) 94010.03 10.56i0.01 6.44:!:0.01

 

 

 

 

Entropy values (ASS) appear to be independent of temperature as shown in Table

11. In addition, AS, values for the l-MCP/adsorbing agents were not affected by the 1-

MCP concentrations at infinite dilution. Within this temperature range, the spatial

structures of the adsorbing agents are not basically altered. This indication is in good

agreement with results reported by Kalaouzis (1992), Riganakos (1989), and

Apostolopoulos (1988a). However, the ASS values of freeze-dried coffee as a function of

water content and temperature varied depending on the temperature and water content.

Temperature dependence of the ASs values could be related to the higher kinetic energy

of water molecules at higher temperature.

135

 

20.00

19.50

19.00

 

 

18.50 50C

-ASs (canole K)
a 8
O O

18.00

 

 

17.50
0.00 1.00 2.00 3.00 4.00 5.00 6.00

Amount of injected 1-MCP per gram of adsorbing agent (n mole/g)

 

Figure 51. Entropy of sorption (A8,) for silica gel as a function of l—MCP concentration
and temperature.

136

26.50

 

 

 

 

 

 

 

 

26.00
9 25.50
52
0
g A A A
8 I I I 2 Sic
j; l 700
cc 4 a; :I 60C
<1 25.00 soc
24.50
24.00 .
2.00 4.00 6.00

Amount of injected 1-MCP per gram of adsorbing agent (n mole/g)

Figure 52. Entropy of sorption (A8,) for Tenax-TA as a function of l-MCP concentration
and temperature.

137

 

16.00 .

 

 

 

15.50 .
A k 509
X 15.00 70c
% fl 80:0 .
> :
“3 1
‘2] 14.50
14.00
13.50 3

 

 

0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00
Amount of injected 1-MCP per gram of adsorbing agent (n mole/g)

Figure 53. Entropy of sorption (A8,) for activated clay as a function of l-MCP
concentration and temperature

138

Enthalpy values of (AH,) for all the adsorbing agents were also negative (see
Table 11), which indicates that an exothermic interaction took place, between l-MCP and
the adsorbing agents. Tenax-TA had the lowest enthalpy values as shown in Figure 54.
This means that Tenax-TA not only adsorbs l-MCP more regularly, but also does it more
exothermically than silica gel despite the fact that silica gel has more adsorbing capacity
for l-MCP than that of Tenax-TA.

In the case of the enthalpy of the VCM-PVC system for the structure of the
polymer (Apostolopoulos, 198 8a), resin S of the PVC polymers showed more negative
AH, values than resin E. This suggests that resin S must sorb VCM more strongly than
resin E. The AH, values of sorption directly relate to the energy interaction between

sorbed water molecules and sorption sites in the food material (Riganakos, 1989).

Such thermodynamic interactions between l-MCP and the adsorbing agents may
have an important role with respect to the release of l-MCP molecules from the surface
phase of the adsorbing agent into the headspace phase of the produce. Factors such as
heat, moisture, and other interactive molecules can influence release of l—MCP molecules

from adsorbing agents.

139

11.00

 

 

 

 

 

 

 

T ' 4 Tenax-TA
10.00
g 9'00 Silica gel
E s
>
(U
U
as
i”
<1 8.00-
7.00
. . 4 Activated clay
6.00
0.00 1.00 2.00 3.00 4.00 5.00 6.00 7,00

Amount of injected 1-MCP per gram of each adsorbing agent (n mole/g)

Figure 54. Enthalpy of sorption (AH,) for each adsorbent as a function of l-MCP

concentration

140

CONCLUSION

Experimentation showed that IGC can be a useful method to study the interaction
relationship between l-MCP molecules and the surface of an adsorbing agent. IGC was
used to obtain 1-MCP sorption patterns for selected adsorbing agents. The values
determined can be used to predict the amount of l-MCP sorbed by a specific adsorbing
agent. Thermodynamic adsorption parameters were especially useful in characterizing
the specific interactions of l-MCP on the adsorbing agents.

However, IGC cannot be used to measure the sorption of l-MCP on activated
carbon due to its strong affinity which resulted in no measurable l-MCP in the infinite
concentration. Activated clay was not as effective in adsorbing 1-MCP as silica gel, or
Tenax-TA. While silica gel adsorbed more 1-MCP than Tenax-TA, its adsorption
pattern was less orderly and lower in enthalpy.

The relationship between 1-MCP and adsorbing agents must be known in order to
estimate the amount of 1-MCP needed under different environmental conditions, other
gases, and moisture levels. All must be considered in designing a l-MCP delivery
system. The interaction of another sorbate with sorbed 1-MCP on the surface of the
adsorbing agent can also be evaluated by defining the sorbate-sorbate interaction
relationship using IGC.

These time study results can be used to estimate the release rate of 1-MCP in a
packaging system. The information obtained in this work could be useful in designing a

device to control release of l-MCP in modified atmosphere packages.

141

APPENDIX C

300000 7. 7

250000

200000 .I

1 50000

Area Response

1 00000

50000 E

1-Butene (ug)

R2 = 0.9967

  
 

y = 380492x -29217

Figure 55. Standard calibration curve of l-butene on silica gel packing column.

142

35000 j
1

30000 .

  
 

25000,
1 y = 19594111 - 3025.7

I
20000 l R2 = 09998

Area Response

15000 1
10000 i

I;
5000 j
I

1-Butene (Hg)

Figure 56. Standard calibration curve of l-butene on Tenax-TA packing column.

143

 

50000 -,- 7A , , ,, fl 7‘77 -v7- 7, ,7, ,h,,_-_-_fl__.__.-_+#_mi_rlfl,r--AEA,

i 1
45000
40000

35000

  
 

y =185100x - 2319.8

25000 R2 = 0.9981

20000

Area Response

1 5000
1 0000

i
i
l
I
1
l
i
i
l
l
|
i
l
5000 l

i

i
i

i
i
i
l
i

1
30000 1
i
t
i
i
i
-i
i
i
‘i
i
i
1
!
i
i

i
j.

0 -.- w A _ _ A -_ -2 __,~_ ._--_--_-_.--_A__-__.__._--Ai

0.00 0.05 0.10 0.15 0.20 0.25 0.30
1-Butene (Hg)

Figure 57. Standard calibration curve of l-butene on activated clay packing column

I44

0.65

 

 

 

,.
‘9.
en
E
8 g ..
E "' U) 5
< '6
j.
10 N vi
1
(a) Silica gel (b) Tenax — TA (c) Activated Clay

Figure 58. Chromatographic peaks 1-butene on silica gel (a), T enax-TA (b), and
activated clay (c) stationary phase column for l-MCP standard calibration curve: Silica
gel (0.80g, 100°C), Tenax-TA (0.211g, 60°C), Activated Clay (0.,lug, 80°C).

145

SECTION VI: ADSORPTION AND RELEASE OF l-METHYL
CYCLOPROPENE (l-MCP) FROM SELECTED ADSORBING AGENTS

ABSTRACT

The partitioning of l-MCP between the gas/polymer matrix was determined for
several adsorbing agents, and in sachet materials to estimate the adsorption potential of 1-
MCP in dry air at 23°C. The l-MCP release study was performed using a closed system
under two different environmental conditions, dry air (0%RH) and 90%RH. Sachets
made from Tyvek®, paper, LDPE, and PVA materials were fabricated to contain silica gel
and activated carbon. The silica gel sachet had a relatively higher release rate at
90%RH, as compared to the release rate at the dry air condition. The activated carbon
sachet did not release l-MCP at either testing condition. Activated carbon had a very
strong affinity for l-MCP. The permeability coefficients of l-MCP and water in
polyethylene and polyvinyl acetate films were determined using a quasi-isostatic method.
LDPE sachets containing silica gel had a similar l-MCP release rate under both dry and
90%RH conditions due to its good water barrier properties. PVA sachets containing
silica gel had slow release of l-MCP. The amount of l-MCP released from PVA sachets
containing silica gel at 90%RH was larger than the amount of l-MCP released at the dry

air condition in a closed system.

146

INTRODUCTION

Among developing technologies used to prolong the storage life of fresh produce,
l-MCP is a novel compound which can inhibit the ethylene action on fresh produce
(Serek, 1994; Jiang, 1999; Abdi, 1998; Ku, 1999). Controlled exposure of l-MCP gas at
the surface of fresh produce can delay ripening and senescence.

To be used commercially with ripening produce during postharvest storage, 1-
MCP gas must have a delivery system. Two patents related to l-MCP for inhibiting the
ethylene response in the plants in order to extend their shelf life and developing the
delivery methods for the convenient and safe application with molecular encapsulation
agents such as cyclodextrin were issued in 1996 (Blankenship and Sisler, 1996) and 2000
(Daly, 2000). A l-MCP trapping system using a (Jr-cyclodextrin component was
marketed under the trade name Ethleloc®. This commercial product requires a gas
preparation procedure with solvent in order to release the l-MCP gas.

Use of an inert matrix material to release l-MCP may have application for fresh
produce without the l-MCP gas preparation step. In addition, use of a sachet may be
able to provide slow release of l-MCP gas in a fresh produce package.

The proposed release mechanism of l-MCP involves three stages. First, the 1-
MCP molecules are released into the free volume within the matrix of the adsorbing
agents in the sachet pouch. Second, l-MCP molecules pass through the film
microstructure into the headspace containing the fresh produce. Third, the l-MCP

molecules penetrate the surface of the fresh produce.

147

Release systems associated with drugs (Shao, 2001), pesticides (Fernandez-Perez,
1999), herbicides (Zhu, 2001), flavor (Greenblatt, 1993), food ingredients (Meyers,
1998), and antioxidants (Miltz, 1988) for use in the pharmaceutical, agricultural, and food
industries have been studied. The rate of release from these release systems depends on
the pH of the medium, the geometry, the size of the system structure, and environmental
conditions such as temperature, humidity, and air flow.

The present study focuses on l-MCP release using a delivery system that consists
of a sachet containing l-MCP sorbed onto selected adsorbing agents. The objectives of
this study were: (a) to determine the partition coefficient of l-MCP in adsorbing agents
and pouch materials; (b) to evaluate the sachet materials regarding their water and 1-
MCP gas barrier properties; (0) to determine the release of l-MCP from the sachets

containing the adsorbing agents.

148

MATERIALS AND METHODS

Materials

The selected materials used as adsorbing agents in these studies were silica gel,
grade 634-60A, 100-200 mesh (Aldrich Chemical Co., Milwaukee, WI), Tenax-TA, 60-
80 mesh (Alltech Associates, Inc., Deerfield, IL), activated clay (a modified
montmorillonite mineral, I-28E, Nanocor, Inc., Arlington Heights, IL), activated carbon,
20-40 mesh (Aldrich Chemical Co., Milwaukee, WI), and activated carbon, 80-100 mesh
(Alltech Associates, Inc., Deerfield, IL). The molecular encapsulation agent used was 01-
cyclodextrin (6 glucose monomers, Cerestar Co., Hammond, IN).

1-MCP was supplied by Biotechnologies for Horticulture, IL. Ethleloc® is a

commercial product. In its powder it has about 0.14% active ingredient by weight.

Sachet materials for l-MCP controlling release system

The materials used to fabricate the sachet pouches were filter paper (Walgreen
Co. Deerfield, IL), Tyvek® (Style 1073B, Dupont Co. Wilmington, DE), low density
polyethylene (LDPE, Dow Chemical Co., Midland, MI), and polyvinyl acetate (PVA,
Dow Chemical Co., Midland, MI). The average thickness of the test materials was
measured using the Model 549 micrometer from Testing Machines, Inc., (Amityville,

L.I., NY) and summarized in Table 12.

149

Table 12. The average thickness of the test materials used to fabricate the sachet.

 

 

Materials Thicknessa
(mil)
Filter paper 5.4:t0.06
Tyvek® (1073B) 5.1i0.11
LDPE 1 .3i0.06
PVA 1.3:t0.10

 

“ Mean values of three replicate results

Water vapor transmission rate of the paper and Tyvek® sachet materials

The water vapor transmission rates of the filter paper and Tyvek® materials were
determined using the dish method (ASTM Standard method E96-00). Three replicates
were tested at each of the following conditions, 25°C, 60%RH and 369°C, 89%RH using
a controlled environmental chamber (Hotpack Co., Philadelphia, PA). Approximately
20 grams of desiccant (anhydrous calcium sulfate, size 8 mesh, W.A. Hammond Drierite
Co. Xenia, OH) were placed into the bottom of an aluminum dish (1 .75cm depth). The
sample material was cut into a circular piece (8.50m diameter) and placed on top of the
test dish containing desiccant. A mask (9cm diameter) containing a small round hole
(2.5cm diameter) was then pasted onto the specimen’s top surface. The edges of the
mouth area in the test dishes were sealed using a melted paraffin wax, and the 2.50m
specimen area was exposed to the environmental conditions previously described. The
test dishes with desiccant were weighed using an analytical balance and placed in the
appropriate chambers. To determine the moisture sorption of the sample materials, a
control film specimen was treated in an identical manner (without desiccant) as the other

samples. The control and sample dish sets were reweighed at predetermined intervals.

150

The water vapor transmission (WVT) and permeance rate were calculated based on the

slope of the graph of weight gain as a function of time using equations 34 and 35.

WVTR= — (34)

WVT-€_ WVT-€
AP Sa'(Rl—R2)

 

 

Permeability = (35)

where SL=slope of the straight line for the weight gain versus time graph, g/min., A: test
area (0.0005m2), €= thickness (m), AP = vapor pressure difference (mmHg), S, =
saturated vapor pressure at 25°C (23.756mmHg) or 369°C (46.336mmHg), R1: relative
humidity at the test chamber (60%RH or 89%RH), R2 = relative humidity at the vapor
sink (0%RH).

Water vapor transmission rate of the LDPE and PVA sachet materials

The water vapor transmission rates of the LDPE and PVA materials were
measured using a Permatran-W® 3/31 apparatus (Mocon Inc, Minneapolis, Minnesota) in
accordance with ASTM F1249. The test film materials were evaluated at the test

temperatures of 23iO.S°C and 32i0.5°C, at a relative humidity of 90i3%.

151

Permeation measurements of l-MCP gas for sachet materials

l-MCP gas permeation experiments were carried out at 24:1:1°C, based on the
quasi-isostatic method. A schematic diagram of the permeation test apparatus is
presented in Figure 59. A constant concentration stream of l-MCP gas was supplied
through a 4 L glass reservoir using a gas circulation rotary pump (Universal Electric Co.,
Cowosso, MI) and then passed into the permeation cell. A constant concentration of
permeant vapor was then flowed through the center cell chamber at a continuous flow
rate of 45 to 50 mL/min. The unit was connected to a flow meter. The l-MCP gas that
passed through the films accumulated in the bottom chamber, and monitored as a
function of time. The permeation cell was comprised of two aluminum disc-shaped
plates and a hollow center ring. Both cell chambers were equipped with a sampling port.
The center ring contained an inlet and outlet port to accommodate the carrier gas stream.
The upper and lower cell chambers each had a volume of 50 cm3 ; the volume of the

center cavity was approximately 50 cm3

. The test films were placed in the cell so that
the center ring effectively isolated the upper and lower cell chambers. Hermetic
isolation of the chambers from each other and from the atmosphere was achieved by the
compression of overlapping Viton O-rings on the film specimen. Viton is a fluorocarbon
elastomer that is resistant to attack from most organic vapors. The concentration of 1-
MCP gas in the lower cell chambers was determined by gas chromatography using flame
ionization detection. At predetermined time intervals, 0.3 mL of headspace gas was

extracted from the upper and lower cell chambers with a gas-tight syringe and injected

directly into the gas chromatograph.

152

    

A—H
- a Aflowmetor

Figure 59. A schematic diagram of the l-MCP permeation test system.

GC instrumentation for l-MCP gas release analysis

The concentration of l-MCP gas was quantified using a Hewlett-Packard Model
5890A gas chromatograph equipped with dual flame ionization detectors and a fused
silica capillary SPB-5 nonpolar column (60m x 0.32mm ID) (Supelco Inc., Bellefonte,
PA). l-MCP gas was injected through the injection port of a thermal desorption unit and
into an empty glass tube. This allowed a sample to be thermally transferred, with carrier
gas flow into a capillary column in the gas chromatograph. The GC conditions were as
follows. The column temperature was 80°C for 10 minutes. Helium was used as a

carrier gas, at a flow rate of 2.2 mL/min. Injection and detector temperatures were set at

153

150 and 300°C. Standard calibration solutions were prepared using a serial dilution

procedure with l-butene gas (99.9% AGA Specialty Gas, Maumee, OH).

Partition coefficient

Partition coefficient studies were carried out using glass cells, 15 mL glass vials
were sealed with screw top hole caps and PTFE/Silicone septums (Supelco Inc,
Bellefonte, PA). The glass cells were purged using dry air (a compressed air, BOC
gases, Ann Arbor, MI) prior to adding the test materials at 23°C. The test material in
Petri dishes was conditioned in an open oven at 95°C for 24 hours, and cooled at room
temperature. Samples of each test material of about 0.5 g were transferred to the glass
vials which were sealed immediately. Glass vials containing 3 mm glass beads were
used as the controls for a non-interactive agent. A 250mL l-MCP stock gas was
generated by mixing about 1 gram of an Ethleloc® powder and 30 mL distilled water in
a glass vial, and sealing with PTFE/silicone septa and aluminum crimp caps (Fisher
scientific, Pittsburgh, PA) and holding at 23°C for 10 hours. 500 uL aliquots of l-MCP
gas were extracted through the stock cell septum and immediately transferred into the 15
mL sealed vials containing a known amount of test material. After 1 hour, the l-MCP
concentration in the headspace of the glass cell was determined by extracting a 40 uL

sample with a syringe through the septum of the 15 mL vial, and quantified using GC.

154

The l-MCP partition coefficient between the test materials and air was calculated as

follows:

a) Concentration of l-MCP in the headspace (mass of l-MCP/air mass)

_(R..><k><V,.)
W xV.

a 1

cs

 

(36)

where R, is the detector area response, k is the calibration factor from a standard
calibration curve (g/A.U.), Vn is net vial volmne (mL) representing the volume (a glass

vial volume — the test material volume), V, is an injection volume (mL), and W, is the air
mass (g) in the headspace.

b) Concentration of l-MCP in test materials (mass of l-MCP/mass of test material)

 

(37)

where 1101 is an amount of l-MCP injected (g) and W, is the test material mass (g).

c) Partition coefficient (K,/g)

Ks/g= —s' (38)

0‘3

155

Adsorption efficiency

About 0.4 g of adsorbing agents was conditioned at 95°C for 24 hours to activate
the binding sites on the surface. After cooling for two hours, samples of each test
material were placed into the 15 mL empty glass vials (Supelco Inc, Bellefonte, PA).
The vials were flushed with dry air and sealed immediately using screw caps and
PTFE/silicon covered septa (Supelco Inc, Bellefonte, PA). A known amount of l-MCP
was transferred through the septa and exposed to the surface of the adsorbing agents in a
dry air condition.

After exposing l-MCP gas to the surface of the adsorbing agents, 0.5 mL of
distilled water was added to the glass vial to obtain a high humidity environmental
condition. After one hour, 40 uL of gas in the headspace was extracted from the sealed
glass cell using a 100 uL gas tight syringe, (Hamilton Co., Reno, NY).

The gas sample was injected into the gas chromatograph to quantitatively
determine the amount of l-MCP at the dry air and high humidity conditions, respectively.

Each sample analysis was performed in three replicates.

156

 

 

Release of l-MCP gas through the sachets with adsorbing agents

Adsorbing agents were packaged in 3 cm x 4 cm test pouches, fabricated from the
four test materials. Each adsorbing agent was conditioned at 80°C for 24 hours and
placed into the sachets after cooling. Tyvek® (Style 10733) and LDPE materials were
sealed using an impulse heat sealer (Sencorp Systems Inc.) at 241.3 KPa with a 0.7
heating time and a 0.5 cooling time. PVA was sealed using an impulse heat sealer at
241.3 KPa with a 1 heating time and a 0.4 cooling time. Filter paper was sealed using
casein based white glue (Elmer’s Inc., Columbus, OH). For controls, each sachet

material was sealed in the absence of adsorbing agents.

Dynamic l-MCP gas trapping system for Tyvek® and filter paper sachets

The Tyvek® and paper sachets containing a known amount of silica gel
(approximately 1 gram) and activated carbon (approximately 0.5 grams) were placed into
a gas trapping glass cell and purged under dried nitrogen at a flow rate of 15 mL/min for
4 hours prior to exposing l-MCP gas to the adsorbing agents in the sachets. A dynamic
l-MCP trapping system was developed, which is described below. As shown in Figures
60 and 61, the gas trapping glass cell was designed to allow the adsorbing agents to trap
1 -MCP gas by the adsorbing agents at constant temperature. The glass cell was
equipped with one metal-link shelf with a stainless stand, to allow exposure of l-MCP
gas uniformly onto the surface of the sachets. The glass cell was also equipped with a

gas inlet and outlet port to provide continuous flow of the dried nitrogen gas stream and

157

 

l-MCP gas. Trapping l-MCP gas onto the adsorbent agents was carried out at 23i1°C
for 24hrs. A constant concentration of l-MCP vapor was flowed continually through the

test glass cell. When water reached the maximum level in the l-MCP generator cell, the

 

control valves at the gas inlet and outlet ports were closed. This allowed exposure of the

adsorbing agents to l-MCP gas for 24 hours.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

. 091
E R2 R1 C #8
C] Do
@ 15 D.
f v 1 0
Sachets 1-MCP 3
Yr. 9” _ ,1
a 66 _5W— :
_—1 ;
TH : — L '
,— _i
J TL
Nitrogen gas
1-MCP Trapping System
R1 :Rotometer for dried gas R2:Rotometer for 1-MCP gas
DV:Need|e valve DW:Distilled water tank
CzGas control valve S:Samp|ing port
M: magnetic bar GGz1-MCP gas generating container

Figure 60. A schematic diagram of the l-MCP gas trapping system used for sachets
containing adsorbing agents.

158

 

Figure 61. l-MCP gas trapping system for the sachets containing adsorbing agents.

l-MCP generator and dynamic trapping system

A l-MCP gas generator glass flask was interfaced to a water container, which
supported a constant concentration of l-MCP. A constant concentration of l-MCP gas
flowed continually through the adsorbent agent in the glass column by water pressure.
The gas generator system was interfaced to a flow meter and needle valve. The l-MCP
generator and gas trapping apparatus are shown in Figure 61.

l-MCP (3 grams) as Ethleloc® powder, Floralife, Inc., (Walterboro, SC), 0.14%
active ingredient, was weighed, and placed into a 320 mL glass flask holding 80 mL
distilled water. After mixing, the solution was stored in a glass flask for 6 hours at room

temperature to develop the appropriate concentration of l-MCP gas.

159

Direct injection of l-MCP gas for LDPE and PVA materials

Silicon tape (0.5 cm x 0.5 cm) was attached to the center surface of each sealed
sachet, and used to directly inject l-MCP gas into the sachet (Figure 62). 6 mL of a
known concentration of l-MCP gas was injected using a 10 mL polypropylene syringe
(B-D. Co., Franklin Lakes, NJ) through the silicon tape on the LDPE and PVA sachets

containing adsorbing agents, 0.5 g silica gel and 0.35 g activated carbon 20/40.

 

Figure 62. The sachets containing adsorbing agents ready for direct injection of l-MCP.

Controlled release of l-MCP gas from the adsorbing agents

After reaching equilibration at a constant concentration of l-MCP gas, the l-MCP
gas generator system was closed using the gas control valve in the outlet port of the glass
flask. A known amount of the l-MCP saturated adsorbing agent was then packed in
a 3 cm x 4 cm test sachet. The paper sachet was formed using glue to seal 3 sides. The

Tyvek® sachet was sealed using an impulse heat sealer (Sencorp Systems Inc., Hyannis,

160

MA) at 241.3KPa with a 0.8 heating time and a 0.6 cooling time. A test sachet was
positioned using stainless steel wire in an environmentally controlled glass cell which
was tightly closed with a glass cover. Release conditions were selected to maintain the
packaged sample in dried air (0% RH), and 90% RH using salt solution in a closed
system with no air flow at 23°C. A saturated salt solution was composed of 370 g/L
potassium nitrate in distilled water. The salt solution was prepared using the procedure
described in ASTM E-104 (1999) to obtain a relative humidity over 90%. Relative
humidity in the glass cell was then monitored using a hygrometer (Hygrodynamics Inc.,
Silver Springs, MA). A schematic diagram of the l-MCP gas release system is shown in
Figure 63. To determine l-MCP gas released from the pouches, an aliquot was
withdrawn using a 500 uL Hamilton microsyringe. An amount of this gas was injected

directly into the gas chromatograph as a function of time.

 

 

 

Figure 63. A schematic diagram of the closed system for l-MCP release study at
controlled humidity conditions. (SS: Salt solution, H: sachet holder, P: sachet sample)

161

The concentration of l-MCP released from the pouch was determined in duplicate
for each time interval. The l-MCP level in the closed system was calculated by

substitution into the following equation:

Rst.FxV,

l-MCPl 1 / =
mm“) VixleOOO

 

(39)

where V, is the injection volume (0.3mL), V, is the total headspace volume (3 80mL at
90%RH and 480mL at dried condition), CF. is the calibration factor from the external
standard calibration curve (ng/A.U.), R, is the detector response value for the sample
obtained using GC, and W is the amount of adsorbing agent (g)

162

 

 

RESULTS AND DISCUSSION

Partition coefficient

Partition coefficient (Kgg) values (Table 13) were determined as the ratio of the l-

MCP in the dried air headspace, to the concentration of the l-MCP in the solid matrix.

Table 13. Partition coefficients of l-MCP gas between solid matrix and air in a closed
system at 23°C.

 

 

Solid matrix C,,, (x 1 0°g/ g) C,,", (x10'°g/g) Partition coefficient (IQ/g)
Silica gel 704.21zt24.43 637.67i4.51 O.92i0.01
Tenax-TA 714.60i16.00 649.98i6.33 O.91i0.03
Activated clay 5582.52i68.76 204.33i4.45 0.04:t0.01
Activated carbon 52.79i12.52 698.69i2.45 l3.78:l:3.45
(20/40mesh)

Activated carbon 52.942t13.54 609.03i5.91 13.61i3.40

(80/ 1 OOmesh)

a-cyclodextrin 7753.63i1 13.31 10.68i10.10 2:0

(6 glucose monomers)

 

The air density at 23°C used to calculate the partition coefficient values was
0.001176 g/cm3 . The results were carried out in triplicate at a l-MCP concentration of
142ug. The partition coefficient values of the activated carbons were 13.78 (20/40mesh)
and 13.61 (80/100mesh), respectively. These values indicate that the activated carbon
has a very strong affinity for the l-MCP molecules at 23°C. l-MCP molecules also have
good affinity for silica gel and Tenax-TA, but less than the activated carbons. Partition
coefficients in silica gel and Tenax-TA were close to 1.00. The partition coefficient

indicates the attraction of l-MCP to the matrix. The affinity of l-MCP for the matrix is

163

low when the partition coefficient is close to zero. These data are in good agreement
with the results reported in chapter 5, which dealt with the interactions between l-MCP
and activated clay, and cyclodextrin materials. Low adsorption of l-MCP on the
cyclodextrin was expected (at dry condition) because l-MCP is trapped through
encapsulation by mixing with an aqueous solution (Bhandari, 1999).

The partition coefficient values for 1-MCP and the respective solid matrix at 23°C
in shown in Figure 64. The results indicate that the partition coefficient values at a 1-
MCP concentration of 142j.ig were not significant different from values at 86, 165, and
235ug of 1-MCP. These data demonstrate that the partition coefficients were not
significantly affected by the concentration of l-MCP in dried air. Biran (1979b) found
that partition coefficient values of vinyl chloride were fairly constant for vinyl chloride
concentrations between the headspace and tested food constituents. However, Halek
(1988) reported that partition coefficient values were dependent on the concentration in a
substrate between a cookie (or film) and air in a closed vial for each solvent.
Kieckbusch (1979) measured the partition coefficients of acetates in sucrose solutions at
different temperatures and concentrations. A sharp increase in the partition coefficient

values with increasing sucrose concentration was observed.

164

 

 

 

 

“1

l

l

1.60, l

l l

i l

g i l

is 1 l

c ' l

g, j Silica gel ‘

.5 0 80 j Tenax-TA l

1: .

t l
(U l
a. |

0.40 i j

l Activated clay l

j A A A A l

0% ”+7 -‘T.. i 7'.T ’— i -. "—"— '—‘ — _-T- A“ ‘___‘ 7———T a:,__~,_T “-— - _‘J

30 50 70 90 110 130 150

Amount of 1-IVK3P injected (9 x 10")

Figure 64. Partition coefficient of l-MCP gas between solid matrix and air in a closed

system as a function of the amount of l-MCP injected at 23°C.

165

Comparison of l-MCP gas partition coefficient values for sachet material and air

in a closed system is shown in Table 14.

Table 14. Partition coefficient values of l-MCP gas between sachet material and air in a
closed system at 23°C.

 

 

Sachet materials C,,, (x 1 Ofg/g) C,,.id (x10'°g/g) Partition coefficient (Kg/,)
Filter paperf 473.98:i:3.64 2.34:1:O.98 zO

Tyvek®1 433.47:L-15.65 6.40:1:O.44 0.01:1:0.00I
LDPE‘ 643.31:l:18.01 5.13i1.41 0.0l:h0.002
PVA’ 493 .30:t8.43 21 .45i0.69 0.04d:0.001

 

 

l and * Values represent the average of triple tests at 1-MCP concentrations of 8.1 1 pg and 11.62ug.

The partition coefficient of the PVA film/air system had a relatively higher value
in comparison to LDPE film or Tyvek®. These values indicate some affinity for l-MCP
by the PVA film. However, the partition coefficient values of paper, Tyvek®, and LDPE
were close to zero respectively, and thus no adsorption occurred. Halek (1988) indicated
that isotatic polypropylene film had a higher affinity for hexane and toluene, and a lower
affinity for oxygenated compounds in the film and air system. Gavara (1996) reported
that polystyrene film had high partition coefficient values for toluene in
water/polystyrene systems.

Partition coefficients can be affected by interaction of the l-MCP sorbate, the
solid matrix, and the packaging film, including polarity, molecular structure, solubility,
and active adsorption sites in the matrix. Biran (1979b) reported that the chemical nature
of the sorbent, the sorbate concentrations, and temperature are important factors affecting

the affinity of the migrant for the contacting phase. ‘

166

Permeability of 1-MCP gas and water vapor through the sachet materials

Water vapor and l-MCP gas permeability effect the selection of sachet materials

by impacting the slow release of the l-MCP molecules from the adsorbing agents.

Results for the water vapor permeability of the respective sachet materials are presented

in Table 15. These data showed the average of three samples for Filter paper and

Tyvek®, and the average of two samples for LDPE and PVA. Each value is expressed in

SI units.

Table 15. Water permeability of sachet materials used in the l-MCP release system.

 

 

 

 

 

 

 

 

 

Materials Transmission rate Permeability Conditions
coefficient

Filter paper 2.77:1:024T 5.25i0.46§ 25°C, 60%RH
9.41:1:O.17T 6.16d:0.11§ 37°C, 89%RH
Tyvek® (1073B) 2.012t0.28* 3.802t0.52§ 25°C, 60%RH
8.93:t0.29* 5.84d:O.19l 37°C, 89%RH
LDPE 0.26i0.01* 0.85i0.0f£ 23?, 90%RH
0.63zt0.03* 12210.05: 3TC, 90%RH
PVA 2.13i0.04* 6.96i0.09: 23°C, 90%RH
4.46i0.03* 8.60i0.25' 32°C, 90%RH

 

" and ‘ Transmission rate values represent in SI units of xkg/mz-day and x10'2kg/m2-day, respectively.
§ and " Permeability coefficient values represent in SI units of x10‘“kg-m/Pa- 5m2 and x 10"4kg-m/Pa- s-mz,

respectively.

Filter paper and Tyvek® materials exhibited relatively high permeability values,

due to their porous structures. Thus, moisture from the fresh produce could easily reach

the adsorbing agent through the sachet material. LDPE film showed good water vapor

barrier properties, compared to the filter paper and Tyvek® materials. The permeability

of water vapor through PVA film was approximately 10 times greater than the

permeability through LDPE film. The materials also exhibited an increase in

permeability of water vapor with an increase in temperature from 23°C to 32°C. The
results shown are similar to those for Tyvek® as reported by Peelrnaster packaging Co.
Niles, Illinois (Peelmaster Co., 2002) and data for LDPE published by Piergiovanni
(1995).

The transmission curves of l-MCP molecules through LDPE and PVA films at
23°C are presented graphically in Figures 71 and 72. The typical transmission profile
curve for the PVA film includes a large lag time while the LDPE film exhibits l-MCP
permeation early in the permeability test period. For the first 3 hours, no permeation
through the PVA film was detectable. Following this initial induction period, a
significant increase in the transmission was observed, after 9 hours steady state was
obtained. The straight line portion of the curve was used to approximate the steady state

portion by performing a linear regression analysis.

Table 16. l-MCP gas permeability of selected sachet materials

 

 

Materials Transmission Permeability Diffusion l-MCP
rate coefficient coefficient conc. (ng/mL)
LDPE 0.783c0.03T 2.94i0.29* l .3 3:1:O.09§ 25
PVA 0453:0031 3.47i0.25* 5.98i0.22‘ 10

 

1 Transmission rate values represent in SI units of x10°kglm2-day.
* Permeability coefficient values represent in SI units of x10"°kg-m/Pa-s-m2.

§ and . Diffusion coefficient values represent in SI units of x 10"2m2/sec and x10"4m2/sec, respectively.
The values were performed at 23°C, 43%RH condition.

l-MCP permeability coefficient values for LDPE and PVA films were determined
using a quasi-isostatic test method. Constant 1-MCP gas concentration flow of 25ng/mL
for LDPE film and 10ng/mL for PVA were used throughout the test period. The

permeability coefficient values of LDPE and PVA films at a given l-MCP concentration

168

 

(lat
resp
LDl
pen
male

for

Sin

P1

thc

re;

ICE

1';

Cl

C l

(Table 16) were determined to be 2.94x10'1° kg-m/Pa-s-m2 and 3.47x10'l6 kg-m/Pa-s-mz,
respectively. These results showed that the water vapor permeability coefficients for
LDPE and PVA films was approximately 200 times greater than the values for l—MCP
permeability coefficient through LDPE and PVA films at 23°C. This may impact the

release of l-MCP from the sachet pouch. Moreover, the diffusion coefficient of l-MCP

 

for the LDPE film was approximately 22 times greater than that for the PVA film. Thus,

l-MCP molecules can move faster through the LDPE structure, compared to the PVA

 

structure. The lower solubility coefficient of l-MCP for the LDPE film than that for the ~-
PVA film might be expected, because the permeability depends on both the solubility and
the rate at which permeant diffuses through the film (equation 20 in Chapter 2). It
represents the relative higher affinity of l-MCP molecules for the PVA structure. These
results are consistent with the partitioning of l-MCP between each film structure and air
(Table 13). These results are comparable to the data from Zobel (1985) regarding the
permeability of organic compounds through LDPE. The permeability of limonene and

linalool through LDPE was 1.90x10"°kg/m2-day and 1.80x10"° kg/mz-day at 10.67 Pa

 

vapor pressure, determined using a quasi-isostatic procedure. The diffusion coefficient
of the l-MCP gas through LDPE was higher than that for PVA. The diffusion
coefficients were affected by several factors, including the film barrier properties and
absorption properties of its molecular structure. Sadler (1991) mentioned that diffusion
coefficients relating to absorption of flavor volatiles by LDPE depended upon the amount

and volatility of the absorbed volatile. l

169

l-MCP adsorbing efficiency

The efficiency of l-MCP adsorption to the matrix agents was determined from the
difference between the initial added l-MCP, and that remaining in the headspace of the
matrix material in a closed system at 23°C. Table 17 shows the adsorbing efficiencies

for the different adsorbing agents.

Table 17. l-MCP adsorbing efficiency of selected adsorbing agents in a closed system.

 

 

 

Solid matrixes Average weight Adsorbing efficiency (%T
(grams) A dry air (0%RH) After treating with
distilled water
Silica gel 0.5023 88.57 70.56
Tenax-TA 0.4610 89.42 79.93
Activated carbon 0.5072 98.01 98.17
(20/40mesh)
Activated carbon 0.4712 98.86 98.21
£0/100mesh)

 

.. Adsorbing efficiency is the amount of 1-MCP in solid matrix x 100/ amount of l-MCP applied in a closed
system. The values are the results of duplicate analyses.

Activated carbon (80/ 100 mesh) had an adsorbing efficiency of 98.86% in dried air, in a
closed system. This result is in agreement with the partition coefficient values and that
obtained in Chapter 5, comparing l-MCP adsorbing efficiencies of silica gel, Tenax-TA,
and activated clay. After treating with distilled water, silica gel showed a relatively
lower adsorbing efficiency, compared to that under dry air. Activated carbon had a
strong affinity for l-MCP even when treated with distilled water. Smisek (1970) found
that activated carbon had high affinity for organic molecules, and other non-polar ‘
substances because most forms of activated carbon are non-polar. Zhou (2002)

measured the adsorption equilibria of nitrogen on silica gel over a large range of

170

 

reg

ca

212
L

temperatures. The transition of adsorption mechanisms from the sub— to the supercritical
region is considerably different for the adsorption of gaseous nitrogen by activated
carbon and silica gel.

Several studies were performed to determine the sorbate efficiency of the matrix
agents. Zhu (2001) determined the encapsulation efficiency of a starch-g-poly(viny1
alcohol) matrix for 2,4,5-trichlorophenoxyacetic acid herbicide as influenced by the
starch grafting ratio. Increasing the grafting ratio, increased the encapsulation efficiency
(%) and consequently reduced the release rate of the herbicide. J iang (2001) reported on
the preparation of a myoglobin (Mb) loaded polymethacrylic acid (PMAA)/gelatin
complex and the release characteristics of myoglobin in order to evaluate the
polyanion/ gelatin complexes as matrices. Mb entrapment efficiency in PMAA/gelatin
decreased with increase in the Mb/PMAA ratio. Even with a large Mb/PMAA ratio, an
entrapment efficiency of over 80% was achieved. Fernandez-Perez (1999) investigated
the controlled release of diuron from an alginate-bentonite formulation. The

encapsulation efficiency of diuron was higher than 98%.

l-MCP release

1-MCP release from the respective sachet materials containing either silica gel or
activated carbon was studied. The cumulative release of l-MCP gas from each sachet
pouch containing silica gel and/or activated carbon at two different environmental

conditions in dried air and 90% RH, is shown in Figures 65-67. The equilibrium

171

concentration of l-MCP was approximately 22.40x10'° g/mL for Tyvek® and paper
sachets in a gas trapping glass cell. As shown in Figures 65 and 66, most of the l-MCP
gas was rapidly released (less than 1 hour) in a closed system. The release of l-MCP
through the porous surface of the Tyvek® and filter paper was significantly faster than
that through the film structure. Tyvek® and filter paper sachet pouches, with no
adsorbing agents, were used as controls. No l-MCP molecules were released because
these sachet materials have low affinity for l-MCP molecules throughout a dynamic gas
trapping system. However, silica gel and/or activated carbon have high affinity for 1-
MCP molecules. Specifically, the Tyvek® and paper sachets containing activated carbon
released no 1-MCP at both dry air and 90%RH conditions during the storage study. The
Tyvek® and paper sachets containing silica gel released different amounts of l-MCP
between dry air and 90%RH conditions (Figures 65 and 66). This indicates that a certain
amount of moisture absorbed through the sachet materials can enhance the desorption of
the l-MCP molecules from the active binding sites of silica gel. l-MCP molecules may
then permeate through the sachet material.

Approximately 122x10’6 g/mL of l-MCP gas were directly supplied into each
LDPE and PVA sachet containing silica gel, activated carbon, and control (without
adsorbing agent). As shown in Figure 67, LDPE sachets released l-MCP at about the
same rate at 90% RH and dry air conditions, because LDPE has good barrier properties to
water vapor and l-MCP gas. The 1-MCP release rate through the films was mainly
dependent on l-MCP permeation through the film. However, PVA is a lower moisture
barrier film than LDPE film. Thus, the moisture can easily get through the film, and

help desorb l-MCP from the silica gel. Therefore, the release rate of 1-MCP from the

172

 

 

 

PVA sachets containing the silica gel at 90%RH is greater than that at 0%RH in a closed
system (Figure 70, C). Activated carbon did not release l-MCP gas regardless of the
sachet materials or storage conditions (Figure 65 and 67). Thus, water is not a strong
release factor for activated carbon. Activated carbons have high adsorptive capacity for

herbicides at low concentrations (Bosetto, 1992).

173

 

 

 

 

 

 

 

 

20
15 ‘ 0 Control. 90%RH
' 0 Control, 0%RH
A v Silica gel, 90%RH
g V V V Silica gel. 0%RH
3 v - Activated carbon, 90%RH
p.) 10 ‘ V V ' Cl Activated carbon. 0%RH
2 v
.-'- v v v v (A) A V
v
v v
5 ‘ (B)
O D
O 0 Cl 0 O 0 Cl C]
0 U—O—D—O—O—G . a ,1 o
0 2 4 6 8 10

Time (hours)

Figure 65. l-MCP gas release from the Tyvek® sachets in a closed system at 23°C.

174

 

20

 

 

 

15 —
0 Control. 90%RH
0 Control, 0%RH
3’ v Silica gel, 90%RH
2 v Silica gel. 0%RH
I 10 ~ I Activated carbon. 90%RH
LE) v D Activated carbon. 0%RH
# v v
V
V V
5 d V V V V
V V V V A)
V V
I (B)
C] I I
0 oc—o—o—o—oor . o t +
0 2 4 6 8

Time (hours)

Figure 66. l-MCP gas release from the paper sachets in a closed system at 23°C.

175

10

 

 

 

80

  
  
 
  

60 q Control, 0%RH

4W1, 90%RH

4° ‘ (B)

1-MCP (pg/g)

Silica gel, 90%RH
Silica gel, 0%RH

20*

Activated carbon, 90%RH

v Activated carbEn, 0%RH

0 5 1 0 1 5 20 25 30 35

 

 

   

 

 

Time (hours)

Figure 67. 1-MCP gas release from the LDPE sachets in a closed system at 23°C.

176

 

60

 

Control, 90%RH
Control, 90%RH
q

 

45 ~ A i A
O
‘ 0
§
,
E: 30 1 (B) (A)
o
5.
Silica gel, 90%RH j,
15“ §Iica gel, 0%RH Wk,
._. .. _ (
V V v

 

 

 

 

Activated carbon, 90%RH

JActivated cagbpn, 0%RH

 

 

I . 1 f

0 5 10 15 20

Time (hours)

25 30

35

Figure 68. l-MCP gas release from the PVA sachets in a closed system at 23°C.

177

CONCLUSION

Release of l-MCP from a sachet can be influenced by a large number of factors
such as, characteristics of the film materials, the adsorption abilities of the adsorbing
agents, the concentration of sorbate, the size of the sachet and environmental factors.
Sachets containing silica gel and activated clay can be adapted to trap l-MCP using a
dynamic adsorbing method for paper and Tyvek® sachets, and direct injection methods
for LDPE and PVA sachets in a closed system.

Silica gel is suitable for binding and release of l-MCP gas because the silica gel
has good binding efficiency at a dry air, and relatively lower binding efficiency of 1-
MCP at a high relative humidity condition.

l-MCP gas held by the adsorbing agent must release only under the
environmental conditions in the presence of the fresh produce. l-MCP gas held by the
adsorbing agent may desorb into the headspace containing the fresh produce at high
relative humidity because most fresh produce continues to lose moisture into the storage
or package atmospheres through transpiration. Thus, it is important to change the
binding efficiency of l-MCP on the adsorbing agent, depending on the relative humidity
during storage.

l-MCP release from filter paper and Tyvek® sachets was not affected by the
sachet materials but largely depended on the sobate adsorbing ability of the adsorbing
agents. The release rate of sachets made from LDPE and PVA were significantly
influenced by the permeation rate of the film structures. PVA is more suitable for slow

release of l-MCP gas than LDPE because PVA has higher water vapor permeability and

178

the same lower permeability to l-MCP gas, compared to LDPE. Therefore, PVA sachets
containing silica gel have potential use for slow release of l-MCP.

Activated carbon proved to be a very strong binding agent for l-MCP due to its
high adsorbing efficiency. Activated carbon did not release l-MCP molecules during the
test periods. However, multiple adsorbing agents, a combination of activated carbon
with silica gel, may have good potential for 1-MCP delivery in long term storage.

From the experimental results using a closed system, the selection of intermediate
water barrier films containing silica gel could be effective for slow release of l-MCP

from a sachet in an open environmental system.

179

APPENDIX D

The salt employed and its corresponding measured relative humidity is shown in Table

18.

Table 18. Salt solution and its corresponding relative humidity at 23°C

 

 

Headspace air condition Relative humidity at 23°C Source
Dried air gas 5 0% AGA Gas Inc., Maumee, OH
Potassium nitrate 90i2% EM Science, NJ

 

180

1200

1 000

800

Amount of 1-MCP (ng)

200

600 _

400 ,

 

 

 

 

0.0 1.0 2.0 3.0 4.0
Time (hours)

Figure 69. l-MCP gas transmission of LDPE film structure at 23°C

181

5.0

6.0

250

 

200

150 _

100

Amount of 1-MCP (ng)

50

 

 

 

0.0 2.0 4.0 6.0 8.0 10.0 12.0
Time (hours)

Figure 70. l-MCP gas transmission of PVA film structure at 23°C

182

._.-:5— 0.983

0.971

 

 

 

 

 

START
START

 

(a) l-MCP (b) 1-Butene

Figure 71. Chromatographic peak shapes of l-MCP and l-Butene on SPB-S nonpolar
capillary column at 80°C for l-MCP standard calibration curve: (a) l-MCP (0.14ug), (b)
1-Butene (0.06ug).

183

NOMENCLATURE

.> .>>~

11>
>.

06

'J)
U!

009 on

U‘.

9%

lcm

marmqsc

:9

€13"

xgwrh

(n

C?!
o}

ZBQT‘“

{D

Amount of sorbate adsorbed per unit mass of adsorbent (Eqn. 1)
Film surface area (Eqns. 16, 22, 23, 24, 25, 27)

Chart area bounded by the common curve along the peak height maxima; the gas
holdup distance (cmz) (Eqn. 2)

Calibration peak area (cmz) on the recorder chart when N moles of the sorbate gas
are injected into the column (Eqns. 2, 3)

Absorbance (OD units) at a given wavelength (Eqns. 30, 32)
Concentration of sorbate (Eqns. 1, 17)

Concentration of l-MCP in the headspace (mass of l-MCP/air mass) (Eqns. 36,
37, 38)

Amount of injected ethylene (ppm, v/v) (Eqn. 29)

Steady state concentration of permeant (Eqn. 25)

Concentration of 1-MCP in test materials (mass of l-MCP/mass of test material)
(Eqns. 37, 38)

Diffusion coefficient (Eqns. 20, 21, 22, 23, 26, 28)

SEC of the solution at wavelength A (Eqn. 30)

Specific extinction coefficient (SEC) of a lycopene (Eqn. 32)
Flux in the permeation process (Eqns. 16, 18, 21)

Rate of carrier gas flow (Eqns. 25)

Flux (Eqns. 16, 18, 21)

Correct gas flow rate (mL/s) (Eqns. 4, 6, 7, 8, 10, 11)

Measured flow rate (mL/s) (Eqn. 7, 10)

Recorder pen deflection (peak height, cm) (Eqn. 3)

Amount of l-MCP injected (Eqn. 37)

James and Martin compressibility factor (Eqns. 6, 7, 8, 9, 11)
Calibration factor from a standard calibration curve (Eqn. 36)
Kelvin degree temperature

Distribution constant (Eqns. 5, 13)

Henry's law solubility coefficient (or S) (Eqn. 33)

Partition coefficient (Eqn. 38)

Film thickness (Eqns. 21, 22, 23, 24, 25, 26, 27, 28, 35)
Beam-path (1cm) in the measuring cuvette (Eqn. 30)

Lag time (Eqn. 28)

Amount of l-MCP expressed in moles (Eqns. 2, 3)

Uptake moles of sorbate gas per unit weight of adsorbent (mole/g)
(Eqns. 2, 33)

Partial pressure of l-MCP vapor (Eqns. 3, 33)

Permeability coefficient (Eqns. 20, 24, 25, 27)

Column inlet pressure (Eqn. 9)

Column outlet pressure (Eqns. 9, 10)

Partial pressure of water at the room temperature (Eqn. 10)

Partial pressure gradient (Eqns. 24, 25, 27)

Chart speed (cm/min) (Eqn. 3)

184

71700

M

I“

"—lg-l—lm

The amount of the permeant molecules permeated (Eqn. 16, 22, 23, 24, 27)
Universal gas constant (Eqns. 3, 13, 14)

Detector area response (Eqn. 36)

Solubility coefficient (or K,) (Eqns. 17, 20)

Collecting time (hrs) of external ethylene produced in a tomato (Eqn. 29)
Column temperature (°K) (Eqns. 3, 10, 11, 12, 13, 14, 15)

Room temperature (°K) (Eqn. 10)

Unit time (Eqns. 16, 22, 23, 24)

Retention time of the l-MCP (Eqns. 4, 6, 7, 11)

Retention time of an unsorbed compound (air) (Eqns. 6, 8, 11)
Corrected carrier gas flow rate (mL/min) (Eqn. 3)

Retained volume of adsorbate at concentration, C (Eqn. 1)
Injection volume (mL) (Eqn. 36)

Specific retention volume (Eqns. 11, 12, 14)

Volume of an unsorbed compound (air) peak (Eqns. 5, 6)
Corrected retention volume of a non-retained compound (Eqns. 6, 8, 11)
Net retention volume (Eqns. 6, 12)

Net vial volume (Eqn. 36)

Retention volume of sorbate peak (Eqns. 4, 5, 6)

Corrected retention volume of a retained sorbate (Eqns. 6, 7, 11)
Adjusted retention volume excluding the void volume (Eqns. 5, 6)
Volume of the stationary phase (Eqns. 5)

Total headspace volume (Eqn. 29)

Total sample volume (Eqn. 32),

Adsorbent weight in the column (Eqns. 1, 2, 11, 12)

Air mass (g) in headspace (Eqn. 36)

Test material mass (Eqn. 37)

Tomato weight (g) (Eqns. 29, 32)

Free energy of sorption (kcal/mol) (Eqns. 13, 15)

Enthalpy changes (kcal/mole) (Eqns. 14, 15)

Entropy changes (kcal/°K mole) (Eqn. 15)

185

\

 

 

BIBLIOGRAPHY

Adams, D. 0., and Yang, S. F. (1979). Ethylene biosynthesis: Identification of 1-
aminocyclopropane-l-carboxylic acid as an intermediate in the conversion of methionine
to ethylene. Proc. Natl. Acad. Sci., 76, 170—174.

Amoore, J. E. and Buttery, R. G. (1978). Partition coefficients and comparative
olfactometry. Chem. Senses Flavoré, 57-71.

Apostolopoulos, D., and Gilbert, S. G. (1990). Water sorption of coffee solubles by
frontal inverse gas chromatography: Thermodynamic consideration. J. Food Sci., 55,
475-477, 487.

Apostolopoulos, D., and Gilbert, S. G. (19883). Study of the polyvinyl chloride polymer
structure in relation to migration of residual vinyl chloride monomer by inverse gas
chromatography. Packaging Technology and Science, 1, 177-183.

Apostolopoulos, D., and Gilbert, S. G. (1988b). Frontal inverse gas chromatography as
used in studying water sorption of coffee solubles. J. Food Sci., 53, 882-884.

 

Apostolopoulos, D., and Gilbert, 8. G. (1983). Water sorption of coffee solubles by
inverse gas chromatography. In G. Charalambous and G. Inglett (Eds.), Instrumental
Analysis of Foods. Academic press, Inc., New York, NY, vol. 2, 51-92.

 

 

ASTM (2000). E96 Standard testing method for water-vapor transmission of materials.
Annual book of ASTM standards, Philadelphia, PA.

ASTM (1999). E-104-85 (Reapproved 1996) Standard practice for maintaining constant
relative humidity by means of aqueous solutions. Annual book of ASTM standards,
Philadelphia, PA.

ASTM (1995). F1249 Standard test method for water vapor transmission rate through

plastic film and sheeting using a modulated infrared sensor. Annual book of ASTM
standards, Philadelphia, PA.

Atta-Aly, M. A. (1992). Effect of high temperature on ethylene biosynthesis by tomato
fruit. Postharvest Biology and Technology, A 19-24.

Bagreev, A. and Bandosz, T. J. (2000). Study of hydrogen sulfide adsorption on activated
carbons using inverse gas chromatography at infinite dilution. J. Phys. Chem. B, 104,
8841-8847.

Bansal, R. C., Donnet, J. B., and Stoeckli, F. (1988). Active carbon. Marcel Dekker, New
York.

 

186

Barr, C. D, Giacin, J. R., and Hernandez R. J. (2000). A determination of solubility
coefficient values determined by gravimetric and isostatic permeability techniques.
Packaging Technology Science, 13, 157-167.

Bhandari, B. R., D’Arcy, B. R., and Padukka, I. (1999). Encapsulation of lemon oil by

paste method using B—cyclodextrin: Encapsulation efficiency and profile of oil volatiles. L
Agric. Food Chem. 47, 5194-5197.

Biran, D., Giacin, J. R., Hayakawa, K., and Gilbert, S. G. (1979a). Vinylchloride sorption
by dry casein particles: Mechanical considerations. J. Food Sci., 44, 1, 59-61.

Biran, D., Gilbert, S. G., and Giacin, J. R. (1979b). Sorption of Vinylchloride by selected
food constituents. J. Food Sci., 44, 1, 56-58.

 

Blankenship, S. M. and Sisler, E. C. (1996). Method of counteracting an ethylene
response in plants. United States Patent, No. 5518988.

Block, G., Patterson, B., and Subar, A. (1992). Fruit, vegetables and cancer prevention: A
review of the epidemiological evidence. Nutritimnd Cancer,18, 1-29.

 

Boki, K., Kubo, M., Kawasaki, N ., Mori, H. (1992). Adsorption isotherms of pigments
from alkali-refined vegetable oils with clay minerals. JAOCS, 69 (4), 372-378.

Bosetto, M., Arfaioli, P., and Fusi, P. (1992). Adsorption of the herbicides alachlor and
metolachlor on two activated charcoals. The Science of the Total EnvironmentL123/124,
101-108.

Britton, G. (1983). Carotenoids. In G. Britton (Eds.), The Biochemistry of Natural
Pigments. Cambridge University Press, Cambridge, NY, 32-38.

Brody, A. L. (2002, February). Action in active and intelligent packaging. Food
Technology, 56, 2, 70-71.

Brody, A. L., Strupinsky E. R., and Kline, L. R. (2001). Oxygen scavengers. In A. L.
Brody, E. R. Strupinsky, and L. R. Kline (Eds), Active Packgging for Food Agplications.
chapter 2, 11-30.

Buttery, R. G., Guadagni, D. G., and Okano, S. (1965, November). Air-water partition
coefficients of some aldehydes. J. Sci. Food Agric., 16, 691-692.

Chaintreau, A., Grade, A., Munoz-Box, R. (1995). Determination of partition coefficients
and quantitation of headspace volatile compounds. Anal. Chem. 67, 3300-3304.

 

Chapman, D. M. and Pfannkoch, E. A. (1992). Thermodynamic limitations for pigment
adsorption during the bleaching of triglyceride oils. J AOCS, 69 (10), 1009-1014.

 

187

Clinton, S. K. (1998). Lycopene: chemistry, biology, and implications for human health
and disease. Nutrition Reviews, 56 (2), 35-51.

Crank, J. (1975). The mathematics of diffusion (2nd edition). Clarendon Press, Oxford,
England.

Daly, J. and Kourelis, B. (2000). Synthesis methods, complexes and delivery methods for
the safe and convenient storage, transport and application of compounds for inhibiting the
ethylene response in plants. United States Patent, No. 6017849.

Day, B. (2002). A fresh approach, Campden & Chorleywood food research association.
http://www.atalink.co.uk/pira/html/p128.htm (sponsored by UPM-kymmene Co.).

Davies, B.H. (1965). Analysis of carotenoid pigments. In T.W. Goodwin (Eds.),
Chemistry and BiochemistLv of Plant Pigments. Academic Press Inc., NY. Chapter 18,
489-532.

Delarue, J. and Giampaoli, P. (2000). Study of interaction phenomena between aroma
compounds and carbohydrate matrixes by inverse gas chromatography. J. Agric. Food
Chem. 48, 2372-2375.

 

 

DellaPenna, D., Alexander, D. C., and Bennett, A. B. (1986). Molecular cloning of
tomato fruit polygalacturonase: Analysis of polygalacturonase mRNA levels during
ripening. Proc. Natl. Acad. Sci. USA, 83, 6420-6424.

Demertzis, P. G., Kontominas, M. G., and Gilbert, S. G. (1987). Gas chromatographic
determination of sorption isotherms of vinylidiene chloride on vinylidene chloride
coplymers. J. Food Sci., 52, 747-750.

Demertzis, P. G., Riganakos, K.A., and Kontominas, M. G. (1989). Water sorption
isotherms of crystalline raffinose by inverse gas chromatography. International Journal of
Food Science and Technology, 24, 629-636.

Di Masio, P., Kaiser, 8., Sies, H. (1989). Lycopene as the most efficient biological
carotenoid singlet oxygen quencher. Arch. Biochem. Biophys. 274, 532-538.

 

Edwards, R. A. and Reuter, F. H. (1967, May). Pigment changes during the maturation of
tomato fruit. Food Technology in Australia, 352-357.

Efiuvwevwere, 3.1.0. and Thome, SN. (1988). Development of chilling injury
symptoms in stored tomato fruit (Lycopersicon esculentum Mill.). J. Sci. Food Agric.,
fl, 215-226.

 

Engewald, W. and Porscmann, J. (1991). Solid stationary phases. In H. Rotzsche (Eds),
Stationary Phases in Gas Chromatggraphy. Journal of chromatography library, Elsevier
Science Publishing Co., New York, NY, vol. 48, 103-141.

188

Fan, X., Mattheis, J. P., and Buchanan, D. (1998). Continuous requirement of ethylene
for apple fruit volatile synthesis. J. Agric. Food Chemu 46, 1959-1963.

 

Fan, X. and Matteis, J. P. (1999). Development of apple superficial scald, soft scald, core
flush, and greasiness is reduced by l-MCP. J. Agric. Food Chemfl 47, 3063-3068.

Femandez-Pérez, M., Villafranca-sanchez, M., Gonzalez-Pradas, E, and Flores-Cespedes,
F. (1999). Controlled release of diuron from alginate-bentonite formulation: water release
kinetics and soil mobility study. J. Agric. Food ChemL47, 791-798.

 

F loros, J. D. (1993). The shelf life of fruits and vegetables. In G. Charambous (Eds.),
Shelf Life Studies of Foods and Beveraggs. Elsevier Science Publishing Co., New York,
NY, 33: 195-216.

Garry, C., Klaus, B., Michelle, H., and Eberhard, S. (2001). Flavor delivery system
containing surfactants and alcohols. European Patent Application, EP1077034A1.

 

Gauthier, H., Coupas, A., Villemagne, P., and Gauthier, R. (1998). Physiochemical
modifications of partially esterified cellulose evidenced by inverse gas chromatography.
Journal of Applied Polymer Science, 69, 2195-2203.

Gavara, R., Catala, R., and Hemandez-Munoz, P. (1997). Study of aroma scalping
through thermosealable polymers used in food packaging by inverse gas chromatography.
Food Additives and Contaminants, 14, no. 6-7, 609-616.

Gavara, R., Hemendez, R. J ., and Giacin, J. R. (1996). Methods to determine partition
coefficient of organic compounds in water/polystylene systems. J. Food Sci., 61, 5, 947-
952.

 

Giacin, J. R. (1995). Factor affecting permeation, sorption, and migration processes in
package-product systems. In P. Ackennann, M. J agerstad, and T. Ohlsson (Eds.), Foods
and PackagingMaterials-Chemical Interactions. 12-22.

Golding, J .B., Shearer D., Wyllie, S.G., and McGlasson, W. B. (1998). Application of 1-
MCP and propylene to identify ethylene-dependent ripening processes in mature banana
fruit. Postharvest Biology and Technology, 14, 87-98.

Gorrnley, R. and Egan, S. (1978). F irrnness and colour of the fruit of some tomato
cultivars from various sources during storage. J. Sci. Food Agric. 29, 534-538.

Greenblatt, H. C., Dombroski, M., Klishevich, W., Kirkpatrick, J., Bajwa, 1., Garrison,
W., and Redding, B. K. (1993). Encapsulation and controlled release of flavours and
fragrances. Royal Society of Chemistry (Great Britafi, 138, 148-162.

Guillet, J. E., Romansky, M., Price, G. J., and Mark, R. V. D. (1989). Studies of polymer

189

structure and interactions by automated inverse gas chromatography. In D. R. Lloyd, T. C.
Ward, and H. P. Schreiber (Eds.), Inverse Gas Chromatography. (ACS Symposium series
391, American Chemical Society, Washington, DC, Chapter 3, 20-32.

 

Haard, N. F. and Salunkhe, D. K. (1975). Symposium: postharvest biology and handling
of fruits and vegetables. AVI publishing company, INC., Westport, Connecticut.

Habile, M., Barlow, P. J ., and Hole, M. (1992). Adsorptive bleaching of soybean oil with
non-montmorillonite Zambian clays. JAOCS 69 4, 379-383.

 

Halek, G. W. and Hatzidimitriu, E. (1988a). Partition coefficients of food packaging
printing ink solvents in soybean oil, chocolate liquor, and a high fat baked product. L
Food Sci., 53, 2, 568-596.

 

Halek, G. W. and Levinson, J. J ., (1988b). Partitioning behavior and off-flavor thresholds
in cookies from plastic packaging film printing ink compounds. J. Food Sci., 53,6, 1806-
1822.

Hall, C. B. ( 1961). The effect of low storage temperatures on the colour, carotenoid
pigments, shelf-life and firmness of ripened tomatoes. Proc Am Soc Hort Sci., 78, 480-
487.

Heaton, J. W. and Marangoni, A. G. (1996a). Chlorophyll degradation in processed and
senescent plant tissues. Trends in Food Science and Technology, 7, 8-15.

 

Heaton, J. W., Yada, R. Y., and Marangoni, A. G. (1996b). Discoloration of coleslaw is
caused by chlorophyll degradation. J. Agric. Food Chem. 44, 395-398.

Hemandez-Munoz, P., Gavara, R., Hernandez, R. J. (1998). Evaluation of solubility and
diffusion coefficients in polymer-film vapor systems by sorption experiments. Journal of
Membrane Science, 3993, 1-10.

Hernandez, R. J. and Gavara, R. (1994). Sorption and transport of water in nylon-6 films.
Journal of polymer science, Part B, Polymer Physics, vol. 32, 2367-2374.

Hernandez, R. J ., Giacin, J. R., and Baner, A. L. (1986). The evaluation of the aroma
barrier properties of polymer films. J. Plastic Film and Sheeting, 2, 3, 187-211.

 

Hobson, G. E. (1981). The short-tenn storage of tomato fruit. Journal of Horticultural
Science 56, 4, 363-368.

 

Hobson, G. E. and Davies, J. N. (1972). The tomato. In A. C. Hulme (Eds.),
Biochemistg of Fruits and Their Products. Academic press, NY, vol. 2, 437-482.

 

Ho, Y. C. and Yam, K. L. (1994). Comparison of vitamin E, Irganox 1010 and BHT as
antioxidants on release of off-flavor from HDPE bottles. Journal of plastic film &

190

sheeting, 10, 194-212.

 

Jaeger, C. R., Van der Sman, R. G. M., and Berkenboschi, A. C. (1999). Developing an
active packaging concept for perishable products-An integrated approach. VR Interpack

99 special, 56-58.

 

Jayaraman, K. S. and Raju, P. S. (1992, March-April). Development and evaluation of a
permanganate-based ethylene scrubber for extending the shelf life of fresh fruits and
vegetables. J. Food Sci. Technol. Qndia), 29, 2, 77-83.

Jenkins, W. A. and Harrington, J. P. (1991). Coffee. In W.A. Jenkins and J.P. Harrington
(Eds.), Packaging Foods with Plastics. Technomic Publishing, Lancaster, PA, 189-195.

 

J iang, H. L., and Zhu, K. J. (2001). Preparation and release characteristics of protein-
loaded polyanion/gelatin complex. Pharmaceutical Development and Technology, 6, 2,
231-240.

J iang, Y., Joyce, D. C., and Macnish, A. J. (1999). Extension of the shelf life of banana
fruit by 1-methylcyclopropene in combination with polyethylene bags. Postharvest
Biology and Technology, 16, 187-193.

Kalaouzis, P. J. and Demertzis, P. G. (1992). Water sorption and water vapour diffusion
in food-grade plastics packaging materials: Effect of a polymer plasticizer. Packaging
Technolggy Science, 5, 133-144.

Kalaouzis, P. J. and Demertzis, P. G., and Kontominas, M. G. (1993). Determination of
water uptake and moisture sorption isotherms of PVC-based food packaging materials:
Effect of plasticizer content and temperature. Packaging Technology and Science, 6, 261-
270.

Kende, H. (1993). Ethylene biosynthesis. Annual. Rev. Plant Physiol. Plant Mol. Biol. 44,
283-307.

Khachik, F., Beecher, G. R., Smith, J. C. Jr. (1995). Lutein, lycopene, and their oxidative
metabolites in chemoprevention of cancer. J. Cell Biochem. Suppl. 22, 236-246.

 

Kieckbusch, T. G. and King, C. J. (1979). Partition coefficients for acetates in food
systems. J. Agric. Food Chem., 27, 3, 504-507.

 

Kim, J ., Gross, K. C., and Solomos, T. (1991). Galactose metabolism and ethylene
production during development and ripening of tomato fruit. Postharvest Biology and
Technology, 1, 67-80.

 

Kim, H.O., Hewett, E.W., and Lallu, N. (2001). Softening and ethylene production of
kiwifruit reduced with 1-methyleyclopropene. Acta Hort., 553, 167-170.

 

191

Kim, J. and Solomos, T., and Gross, K. C. (1999). Changes in cell wall galactosyl and
soluble galactose content in tomato fruit stored in low oxygen atmospheres. Postharvest
Biology and Technology, 17, 33-38.

Kiselev, A. V. and Yashin, Y. I. (1969). Gas-chromatographic determination of
adsorption and specific surface for solids. In A. V. Kiselev and Y. I. Yashin (Eds.), Gas-
Adsorption Chromatograph. Plenum Press, New York, Chapter IV, 104-145.

Kolb, B., Welter, C., and Bichler, C. (1992). Determination of partition coefficients by
automatic equilibrium headspace gas chromatography by vapor phase calibration.
Chromatographia, 34, 235-240.

Kontominas, M. G., Gavara, R., and Giacin, J. R. (1994). Gas-solid chromatographic
study of polymer surfaces: Adsorption on polystyrene. Eur. Polym. J. Sci., 30, 2, 271-277.

Kontominas, M. G. and Giacin, J. R. (1993). Interaction of food grade polystylene with
styrene monomer using inverse gas chromatography: Migration aspects. In G.
Charalambous (Eds.), Food Flavors, Ingredients and Composition. Elservier Science
Publishers, 769-779.

Ku, V.V.V. and Wills, R.B.H. (1999). l-Methylcylopropene can differentially affect the
postharvest life of strawberries exposed to ethylene. HortScience, 34, 1, 119-120.

Laerrner, S. R, Young, S. S., and Zambetti, P. F. (1994, June). Could your packaging use
a dose of vitamin E? Food ProcessirygLSS, 35-36.

Lan, T. (1994). Polymer-clay nanocomposites. Doctoral dissertation, Michigan State
University, East Lansing.

Lee, D. S., Haggar, P. E., and Yam, K. L. (1992). Application of ceramic-filled polymeric
films for packaging fresh produce. Packaging Technology and Sciencej, 27-30.

 

Lee, S., Allen, H. E., Huang, C. P., Sparks, D. L. and Sanders, P. F., and Peijnenburg, W.
J. G. M. (1996). Predicting soil-water partition coefficients for cadmium. Environ. Sci.
Technol. 30, 3418-3424.

 

Leggott, N. L., Shephard, G. S., Stockenstrom, S., Staal, E., and van Schalkwyk, D. J.
(2001). The reduction of patulin in apple juice by three different types of activated carbon.
Food Additives and Contaminants, 18, 9, 825-829.

Lelievre, J. M., Latche, A., Jones, B., Bouzayen, M., and Pech, J.-C. (1997a). Ethylene
and fruit ripening. Phypsiolggia Plantarumpl 0L 727-739.

Lelievre, J-M., Tichit, L., Dao, P., F illion, L., Nam, Y-W., Pech, J-C., and Latche, A.
(1997b). Effects of chilling on the expression of ethylene biosynthesis genes in Passe-
Crasane pear (pyrus communis L.) fruits. Plant Molecular Biology, 33, 847-855.

 

192

Liang, C. and Pankow, J. F. (1996). Gas/particle partitioning of organic compounds to
environmental tobacco smoke: partition coefficients by desorption and comparison to
urban particulate material. Environ. Sci. Technol. 30, 2800-2805.

 

Liu, F. W. (1970). Storage of bananas in polyethylene bags with an ethylene absorbent.
Hortscience, 5, 1, 25-27.

Liu, K. J., Hernandez, R. J., and Giacin, J. R. (1991). The effect of water activity and
penetrant vapor activity on the permeation of toluene vapor through a two-side PVDC
coated opaque oriented polypropylene film. J. Plastic Film and Sheeting, 7, 56-67.

Liu, Y., Hua, X., and Guo, L. (1989). Effect of 2,5-norbomadiene on the ripening and the
production of ethylene in tomato fruits. In H. Clijsters et al. (Eds.), Biochemical and
Physiolggical Aspects of Ethylene Production in Lower and Higher Plants. Kluwer
Academic Publishers, 101-108.

Loffe, B.V. and Vitenberg, AG. (1984). Head-space Analysis and Related Methods in
Gas ChromatographLA Wiley-Interscience Publication.

 

Lépez-Garzon, F. J ., Pyda, M., and Domingo-Garcia, M. (1993). Studies of the surface

properties of active carbons by inverse gas chromatography at infinite dilution. Langmuir,
2, 531-536.

Lloyd, D. R., Ward, T. C., and Schreiber, H. P. (1989). Inverse gas chromatography:
Characterization of polymers and other materials, ACS Symposium Series,no. 39L
American Chemical Society, Washington, DC.

Lundqvist, A. And Odberg, L. (1997, June). Surface energy characterization of pigment
coatings by inverse gas chromatography. J. Pulp and Paper Science, 23, 6, J298-J303.

Male, C. J. (1999). 100 Origins of the tomato. In C. J. Male (Eds.), Heirloom Tomatoes
for the American Garden. Workman Publishing, 1-8.

 

Martinez-Valverde, I., Periago, M. J ., Provan, G., and Chesson, A. (2002). Phenolic
compounds, lycopene and antioxidnat activity in commercial varieties of tomato. J. Sci.
Food Agric. 8; 323-330.

Mathooko, F. M., Kubo, Y., and Inaba, A. (1995). Characterization of the regulation of
ethylene biosynthesis in tomato fruit by carbon dioxide and diszocyclopentadiene.
Postharv. Biol. Technol. 5, 221-233.

 

Mencarelli, F. and Saltveit, M. E. (1988). Ripening of mature-green tomato fruit slices. ._I_.
Amer. Soc. Hort. Sci. 113, 5, 742-745.

Meyers, M. A. (1998). Controlled release of food ingredients. International Sympfiosium

 

193

on Controlled Release of Bioactive Materialsp25th, Controlled Release Society, Inc.,
328-329.

McNair, H. M. and Miller, J. M. (1998). Basic Gas Chromatography. John Wiley & Sons,
Inc., New York, NY.

Miltz, J., Hoojjat, P., Han, J. K., Giacin, J. R., Harte, B. R., and Gray, 1. J. (1988). Loss of
antioxidants from high-density polyethylene. Its effect on oatmeal cereal oxidation. In J. J.
Hotchkiss (Eds.), Food and Packagipg Interactions. Washington, DC, American

Chemical Society, 83-93.

Mir, N. A. and Beaudry, R. M. (2001). Use of l-MCP to reduce the requirement for
refrigeration in the storage of apple fruit. Acta Hort., 553, 577-580.

Mitcham E. J. and Mcdonald, R. E. (1992). Effect of high temperature on cell wall
modifications associated with tomato fruit ripening. Postharvest Biology and Technology,
19 257'264.

Mizutani, Y., Nakamura, S., Kaneko, S., and Okamura, K. (1993). Microporous
polypropylene sheets. Ind. Eng. Chem. Res., 32, 221-227.

Moran, R. ( 1982). Formulae for determination of chlorophyllous pigments extracted with
N ,N -Dimethylformamide. Plant PhysioL. 69, 1376-1381.

Nahon, D. F ., Harrison, M., and Roozen, J. P. (2000). Modeling flavor release from
aqueous sucrose solution, using mass transfer and partition coefficients. J. Agric. Food
Chem. 48, 1278-1284.

 

N akatsuka, A., Shiomi, S., Kubo, Y., and Inaba, A. (1997). Expression and internal
feedback regulation of ACC synthase and ACC oxidase genes in ripening tomato fruit.
Plant Cell Physiol. 38, 10, 1103-1110.

 

Newman, J. P., Dodge, L. L. and Reid, M. S. (1998, January-March). Evaluation of
ethylene inhibitors for postharvest treatment of Gypsophila paniculata L. HortTechnology,
_8_, 1, 58-63.

 

Nguyen, M. L. and Schwartz, S. J. (1999). Lycopene: chemical and biological properties.
Food Technology, 53, 2, 38-45.

Nielsen, T. J. and Giacin, J. R. (1994). The sorption of limonene/ethyl acetate binary
vapour mixtures by a biaxially oriented polypropylene film. Packaging Technology and
Science 7, 247-258.

 

 

Okada, K., Inoue, Y., Satoh, K., and Katoh, S. (1992). Effects of light on degradation of
chlorophyll and proteins during senescence of detached rice leaves. Plant Cell Physiol. 33,
8,1183-1191.

194

Ooraikul, B. (1990). Modified atmosphere packaging of bakery products. In B. Ooraikul
and ME. Stiles (Eds.), Modified Atmosphere Packaging of Food. Ellis Harwood,
Chichester, 49—117.

Opperrnan, W,_J., Fourie, J._F., Sanderson, R._D., and Britz, T._J. (1999). A monolithic
device for in-package SO; generation for the control of post harvest Botrytis decay of
table grapes. J. Food Qual., 22, 4, 427-438.

Paik, SW. and Gilbert, S. G. (1986). Water sorption isotherms on sucrose and starch by '
modified inverse frontal gas chromatography. Journal of Chromatography, 351, 417-423.

Patwardhan, S. A. and Das, K. G. (1983). Chemical methods of controlled release. In K.
G. Das (Eds.), Controlled-Release Technology: Bioengineering Aspects. John Wiley &
Sons, Inc., New York, NY, 23-59. H

 

Pendyal, B., Johns, M. M., Marshall, W. E., Ahmedna, M., and Rao, R. M. (1999). The
effect of binders and agricultural by-products on physical and chemical properties of
granular activated carbons. Bioresource Technology, 68, 247-254.

Pinnavaia, T. J. (1983). Intercalated clay catalysts. Science, 220, 365-371.

 

Piringer, 0., Wolff, E., and Pfaff, K. (1993, November-December). Use of high
temperature-resistance sorbents as simulants for testing. Food Additive Contamiryation,
1_(_)_, 6, 621-629.

Peelmaster Co. (2002). Specification properties of Tyvek® 10733 by DuPont,
www.peelmaster.com/materials/ty1073.htm.

Piergiovanni, L., Fava, P, and Siciliano, A. (1995). A mathematical model for the
prediction of water vapour transmission rate at different temperature and relative
humidity combinations. Packaging Technology and Sciencg 8, 73-83.

Porat, R., Weiss, B., Cohen, L., Daus, A., Coren, R., and Droby, S. (1999). Effects of
ethylene and l-methylcyclopropene on the postharvest qualities of “Shamouti” oranges.
Postharvest Biology and Technology, 15, 155-163.

Ratanachinakom, B., and Klieber, A., and Simons, D. H. (1997). Effect of short-term
controlled atmospheres and maturity on ripening and eating quality of tomatoes.
Postharvest Biologyand Technology, 11, 149-154.

Riganakos, K. A., Demertzis, P. G., and Kontominas, M. G. (1989). Gas chromatographic
study of water sorption by wheat flour. J. Cereal Sciencefi, 261-271.

 

Risch, S. J. (1995). Review of patents for encapsulation and controlled release of food
ingredients. ACS symposium series, vol. 590, 196-203.

195

Rix, M. and Knight-Wilbum, K. (1994). Protecting packaged goods against moisture.
Clay and silica gel desiccation: An analysis of variance, Packag. (U.K.), 65,703, 10.

Rogers, C. E. (1985). Permeation of gases and vapors in polymers. In J. Comyn (Eds.),
Polymer Permeabiliyy. Elsevier Applied Science Publishers, New York, NY, 11-74.

Rooney, M. L. (1981). Oxygen scavenging from air in package headspace by singlet
oxygen reactions in polymer media. Journal of Food Science. 47, 291-294, 298.

Roy, G. M. (1995). Activated Carbon Applications in the Food and Pharmaceutical
Industries. Technomic Publishing Co., Lancaster, PA.

Rubatzky, V. E., and Yamaguchi, M. (1997). Tomato. In V. E. Rubatzky and M.
Yamaguchi (Eds.), World Vegetables: Principle, Production, and Nutrition Values.
Chapman & Hall, New York, NY, 532-553.

Ryall, A. L., and Lipton, W. J. (1979). Tomatoes. In A. L. Ryall and W. J. Lipton (Eds.),
Handling, Transportation and Storag of Fruits and Vegetables. AVI Publishing Co.,
Westport, CT, Chapter 8, 193-209.

Sadler, G. D. and Braddock, R. J. (1991). Absorption of citrus flavor volatiles by low
density polyethylene. Journal of Food Science, 56, 1, 35-37.

 

Sajiki, T. and Giacin, J. R. (1993). Permeation of ethylene acetate vapor through silica
deposited polyethylene terephthalate film and composite structures. J. Plastic Film and

Sheeting, 9, 97-111.

Saltveit, M. E. and Sharaf, A. R. (1992). Ethanol inhibits ripening of tomato fruit

harvested at various degrees of ripeness without affecting subsequent quality. J. Am. Soc.
Hort. Sci. 117, 793-798.

 

Satomi, K. (1988). Maintaining the freshness of foods by packaging techniques. JPI J.
(Japan), 26, 9, 74-78.

Serek, M. and Reid, M. S. (1993). Anti-ethylene treatments for potted flowering plants-
Relative efficacy of inhibitors of ethylene action and biosynthesis. HortScience 28, 1180-
1 181.

 

Serek, M., Sisler, E. C., and Reid, M. S. (1994). Novel gaseous ethylene binding inhibitor
prevents ethylene effects in potted flowering plants. J. Amer. Soc. Hort. Sci. 119, 6,
1230-1233.

Serek, M., Sisler, E. C., and Reid, M. S. (1995a). Effects of l-MCP on the vase life and
ethylene response of cut flowers. Plant Growth Regulation, 16, 93-97.

 

196

Serek, M., Sisler, E. C., and Reid, M. S. (1995b). l-Methylcyclopropene, A novel
gaseous inhibitor of ethylene action improves the life of fruits, cut flowers and potted
plants. Acta Hort.,394, 337-345.

Shao, Z. J., Farooqi, M. I., Diaz, S., Krishna, A. K., and Muhammad, N. A. (2001).
Effects of formulation variables and post-compression curing on drug release from a new
sustained-release matrix material: polyvinylacetate-povidone. Pharmaceutical
Development and Technology, 6, 2, 247-254.

Shorter, A. J ., Scott, K. J ., Ward, G., and Best, D. J. (1992). Effect of ethylene absorption
on the storage of Granny Smith apples held in polyethylene bags. Postharvest Biology
and Technology, 1, 189-194.

 

 

Sink, K. C., Hemer, R. C., Knowlton, L. L. (1974). Chlorophyll and carotenoids of the
fin tomato mutant. Can. J. 3% 52, 1657-1660.

 

Sirkar, K. K. Farrell, S., and Basu, R. (1999). Controlled release device and method
based on aqueous-organic partitioning in porous membranes. United States Patent, No.
5858385.

Sisler, E. C. and Blankenship, S. M. (1993). Effect of diazocyclopentadiene on tomato
ripening. Plant Growth Reg., 12, 155-160.

Sisler, E. C., Dupille, E., and Serek, M. (1996). Effect of 1-methylcyclopropene and
methylenecyclopmpane on ethylene binding and ethylene action on cut camations. Plant
Growth Regulation, 18, 79-86.

 

Sisler, E. C. and Lallu, N. (1994). Effect of diazocyclopentadiene (DACP) on tomato
fruits harvested at different ripening stages. Postharvest Biology and Technology, 4, 245-
254.

Sisler, E. C. and Serek, M. (1997). Inhibitors of ethylene responses in plants at the
receptor level: Recent developments. Physiologia Plantarum, 100, 577-582.

Sisler, E. C. and Yang, S. F. (1984). Anti-ethylene effect of cis-butene and cyclic olefins.
Phytochemistg, 23, 2765-2768.

 

Skog, L. J., Schaefer, B. H., and Smith, P. G. (2001). l-methylcyclopropene preserves the
firmness of plums during postharvest storage and ripening. Acta Hort., 553, 171- 172.

 

Smisek, M. and Cemy, S. (1970). Active Carbon: manufacture, properties and
applications. Elsevier Publishing Co., New York, NY.

 

Smith, D. L., Starrett, D. A., and Gross, K. C. (1998). A gene coding for tomato fruit -
galactosidase II is expressed during fruit ripening. Cloning, characterization, and
expression pattern. Plant Physiol. 117, 415-423.

 

197

Smith, J. P., Hoshino, J ., and Abe, Y. (1995). Interactive packaging involving sachet
technology. In M.L. Rooney (Eds.), Active Food Packaging; chapter 6, 143-173.

Sozzi, G. 0., Trinchero, G. D., and F raschina, A. A. (1999). Controlled-atmosphere
storage of tomato fruit: low oxygen or elevated carbon dioxide levels alter galactosidase
activity and inhibit exogenous ethylene action. J. Sci. Food Agric., 79, 1065-1070.

 

Teumac, F. N. (1995). The history of oxygen scavenger bottle closures In M. L. Rooney
(Eds.), Active Food Packaging. Blackie Academic and Profession, Chapman and Hall,
New York, NY, 193-201.

 

Throne, S. and Segurajauregui Alvarez, J. S. (1982). The effect of irregular storage
temperatures on firmness and surface colour in tomatoes. J. Sci. Food Agicu 33, 671-676.

Van Boekel, MAJ S. (2000). Kinetic modeling in food science: a case study on
chlorophyll degradation in olives (Anniversary review). J. Sci. Food Agric., 80, 3-9.

Viljanen, K., Sundberg, S., Ohshima, T., Heinonen, M. (2002). Carotenoids as
antioxidants to prevent photooxidation. Eur. J. Lipid Sci. Technol. 104, 353-359.

Vlachonasios, K. E., Kadyrzhanova, D. K., and Dilley, D. R. (2001). Heat treatment
prevents chilling injury of tomato fruits: Heat shock genes and heat shock proteins in the
resistance of tomato fruit to low temperatures. Acta Hort., 553, 543-547.

Vogt, J. A. (1985, June). Moisture in packaging. Can. Packag., 38, 6, 26-28, 34.

Vukov, A. J. and Gray, D. G. (1989), Properties of carbon fiber surfaces. In D. R. Lloyd,
T. C. Ward, and H. P. Schreiber (Eds.), Inverse Gas Chromatography. ACS Symposium
series 391, American Chemical Society, Washington, DC, Chapter 13, 168-184.

Watada, A. E., Norris, K. H., Worthington, J. T., and Massie, D. R. (1976a). Estimation
of chlorophyll and carotenoid contents of whole tomato by light absorbance technique. ._1_.
of Food Sci.,41, 329-332.

Watada, A. E., Aulenbach, B. B., and Worthington J. T. (1976b). Vitamins A and C in
ripe tomatoes as affected by stage of ripeness at harvest and by supplementary ethylene. .L
Food Scig 4L 856-858.

 

Watkins, C. B., Nock, J. F ., and Whitaker, B. D. (2000). Responses of early, mid and late
season apple cultivars to postharvest application of 1-methylcyclopropene (l-MCP)
under air and controlled atmosphere storage conditions. Postharvest Biology and
Technology, 19, 17-32.

 

Wills, R. B., Ku, VVV., and Warton, M. A. (2002). Use of l-methylcyclopropene to
extend the postharvest life of lettuce. J. Sci. Food Agric., 82, 1253-1255.

 

198

Wills, R. B. H., McGlasson, W. B., Graham, D., Lee, T., H., and Hall, E. G. (1989).
Physiology and biochemistry of fruit and vegetables. In R. B. H. Wills, et al. (Eds.),
Postharvest: An Introduction to the Physiology and Handling of Fruit and Vegetables.
chapter 3, 17-38.

Woods, J. L. (1990). Moisture loss from fruits and vegetables. Postharvest News and
Information, 1, 3, 195-199.

Wu, M. T., Jadhav, S. J ., and Salunkhe, D. K. (1972). Effects of sub-atmospheric
pressure storage on ripening of tomato fruits. J. Food Sci. 37, 952-956.

 

Yang, S. F. (1987). Regulation of biosynthesis and action of ethylene. Acta Hortic., 20L
53-59.

Yang, S. F ., Hoffman, N. E. (1984). Ethylene biosynthesis and its regulation in higher
plants. Ann. Rev. Plant Physio., 35, 155-189.

Yoshida, M., Kumakura, M., and Kaetsu, I. (1979). Controlled release of 5-fluorouracil
from adsorbents containing matrices polymerized by radiation. Polymer Journal, 11, 10,
775-779.

Zagory, D. (1995). Ethylene-removing packaging. In M.L. Rooney (Eds.), Active Food
Packaging. Blackie Academic & Professional, Glasgow, 38-54.

Zagory, D. and Kader, A. A. (1988, September). Modified atmosphere packaging of fresh
produce. Food technology, 70-77.

 

Zeoli, L. T. and Kydonieus, A. F. (1983). Physical methods of controlled release. In K. G.
Das (Eds.), Controlled-Release Technology: Bioengineering Aspects. John Wiley & Sons,
Inc., New York, NY, 61-120.

Ziegel, K. D., Frensdorff, H. K., and Blair, D. E. (1969). Measurement of hydrogen
isotope transport in poly(vinyl fluoride) films by permeation rate method. J. Polymer Sci.,
Part A-2 7,809.

 

 

Zhou, L., Bai, 8., Zhou, Y., and Yang, B. (2002). Adsorption of nitrogen on silica gel
over a large range of temperatures. Adsorption, 8, 79-87.

Zhu, Z. and Zhuo, R. (2001). Slow release behavior of starch-g-poly(vinyl alcohol)
matrix for 2,4,5-trichlorophenoxyacetic acid herbicide. European Polymer Journal, 37,
1913-1919.

Zobel, M. G. R. (1985). The odour permeability of polypropylene packaging film.
Polymer Testing, 5, 153-165.

 

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