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This is to certify that the

dissertation entitled

ALTERNATIVE STRATEGIES TO CONTROL SCALD OP APPLES
AND SOME BIOCHEMICAL BASES

presented by

ZHENYONG WANG

has been accepted towards fulfillment
of the requirements for

Ph. D. HORTICULTURE

degree in

 

 

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MAY BE RECALLED with earlier due date if requested.

 

DATE DUE

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ALTERNATIVE STRATEGIES TO CONTROL SCALD OF APPLES
AND SOME BIOCHEMICAL BASES

By

Zhenyong Wang

A DISSERTATION
Submitted to
Michigan State University

in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY
Department of Horticulture

1998

ABSTRACT
ALTERNATIVE STRATEGIES TO CONTROL SCALD OF APPLES
AND SOME BIOCHEMICAL BASES
By

Zhenyong Wang

Scald is a pervasive physiological disorder Of apples and pears induced by
storage at refrigerated temperatures in air or long term controlled atmosphere (CA).
Scald has the potential to destroy the market value and utility Of millions Of tons Of
apples and pears annually unless the fruits are treated with a postharvest drench
with diphenylamine (DPA) or ethoxyquin along with a fungicide. Numerous
countries have banned the use Of DPA and prohibited importation Of fruits SO
treated. The Objectives of these studies were tO develop alternative strategies to
control scald Of apples to avoid applying postharvest treatment with the scald
inhibitor DPA and to ensure fruit quality and food safety. The physiological and
biochemical bases Of apple scald were also investigated.

Granny Smith and Law Rome apples were placed under hypobaric storage
immediately after harvest or after 0.5, 1, 2, 3, 3.5, 4, 4.5, 5 or 6 months storage in
air at 1°C to determine the effects Of delaying imposition Of hypobaric storage on
ripening and scald development and production of OI-farnesene and its oxidation
product 6-methyl-5-heptene-2—one (MHO). Fruits did not scald during hypobaric
storage or afterwards when transferred to static air at one atmosphere continuously

for 4 months if they were placed under hypobaric conditions within one month after

harvest while held in air at 1°C; after 3 months delay, scald development was
Similarto that for fruits stored in air. MHO accumulated in the epicuticular wax when
fruits were placed under hypobaric storage after one month delay in air. MHO in the
epicuticular wax Of fruits stored hypobarically after 2 or more months delay was
released upon transfer Of fruits to 20°C; MHO accumulated in direct proportion to
the duration Of the delay to hypobaric storage. Hypobaric ventilation apparently
removes scald-related volatile substances including OI-famesene and MHO that
otherwise accumulates and partitions into the epicuticular wax of fruits stored in air
at atmospheric pressure.

Scald susceptible and not susceptible cvs. fruits were treated with different
concentrations Of ethanol vapor and different durations. Fruits were then stored in
3% 02 with 0% CO2 in flow-through CA and in air at 1 °C. The treatments with 6000
uL-L'1 ethanol vapor for 2 weeks were more effective for scald control than the other
treatments; higher levels Of ethanol for over 2 months caused fruit injury and Off-
flavor. The ethanol vapor treatment diminished the level of several low molecular
weight volatiles produced by the fruit. The rate and pattern Of OI-farnesene
production was Similar for fruits treated or not treated with ethanol vapor. Ethanol
vapor treatments reduced the rate of MHO production. This indicates that the CI-
famesene is not as closely related tO scald development as its oxidation product
MHO.

Fruits were treated with initial low 02 stress (ILOS) at different levels Of low
02 and various durations and then stored in different CA storage conditions and air

at 0.5-1°C. Superficial scald was markedly reduced by ILOS at 0.5% 02 for up to

two weeks followed by air storage. With CA storage at 3% 02 with 0% COZ,
following 0.5% and 0.25% ILOS for 2 weeks reduced scald; and with CA at 1.5%
02 with 3% 002. scald was prevented. ILOS at 0.25% 02 for two weeks or also
when followed with an additional two weeks Of low 02 stress after 2 months Of the
storage were the most effective treatments regimens for scald control. The
production Of OI-famesene and MHO was inhibited by ILOS and CA at 1.5% Oz.
The 0.25% 02 ILOS caused stronger inhibition on OI-farnesene and MHO
production than 0.5% 02 ILOS. Collectively, this results suggest that the
accumulation Of MHO is highly related to scald development of apples. A
commercial test Of initial low 02 stress confirmed the efficacy Of ILOS for controlling
scald.

The 'Scald-like' disorder Of Empire apples is a COz-linked physiological
disorder. The factors Of acclimatization Of preclimacteric fruits at 3°C in air or low
02 levels prior to elevating the CO2 concentration for subsequent long-term
controlled atmosphere were investigated. The results indicate that the Empire
‘Scald-like’ disorder can be effectively controlled by holding the fruits at 3°C for 3-4
weeks at 1.5 to 3% 02 without CO2 prior tO CA storage at 1.5% 02 + 3% C02.
Acclimatization Of fruits against the disorder was also achieved by storage in air but
this resulted in excessive ripening development with flesh firmness loss during

subsequent CA storage.

Dedicated tO my eternal companion Xiaozhu Pan,
whose faith in me encouraged me to achieve excellence in all I attempted to do,
and to our Children Shuyu and Brice Libai,

whose successful future I have strived to ensure.

ACKNOWLEDGMENTS

The timely completion Of this dissertation would not have been possible if not
for the support, mentoring, patience, and confidence Shown me by Dr. David R.
Dilley, my major professor and Chair Of my advisory committee. I sincerely
appreciate his advice for my personal as well as professional development.

I also appreciate the service Of Drs. Randolph M. Beaudry, Stephen A. Boyd,
Jerry N. Cash as members Of my advisory committee for their advice and
encouragement.

I 'm grateful tO Drs. Robert C. Hemer, Muraleedharan G. Nair, Jun Song,
Douglas Bermeister, John Everard, Dina Kadyrzhanova, Nazir Mir, and Manit
Kosttrakun for their assistance and encouragement.

I gratefully acknowledge the assistance of Weimin Deng, Torn J. McCully,
Konstantinos Vlachonasios, Hongying Jiang, Toni M. Warner and many other
colleagues in the lab for assistance in data collection and moral support to make it
possible for me to pursue this goal.

I am also grateful tO Lorri K. Busick, Joyce Jackson, Sherry Mulvaney, and
Gabrielle Seyka in the Department Office for their assistance.

I wish tO express Special thanks to my friend Xiaoyu Lin for his genuine
friendship and assistance, moral and spiritual support.

I owe Special thanks to my mother and brothers for their love, kindness,

sincerity, encouragement, moral and financial support.

vi

Finally, I express deep love and appreciation to my dear wife Xiaozhu and
our Children Shuyu and Brice Libai for their tireless support, understanding and

sacrifice, confidence, encouragement, and enthusiasm.

vii

TABLE OF CONTENTS

LIST OF TABLES .......................................................................................... xiv
LIST OF FIGURES ......................................................................................... xvi

INTRODUCTION .............................................................................................. 1

Overview Of the scald problem ..................................................... 1

Rationale for new research .......................................................... 3

Recent strategies tO control scald ................................................ 3

Current CA storage technology to control scald .......................... 4

New strategies to control scald .................................................... 5

Role Of volatiles in scald .............................................................. 5

Summary current knowledge about scald ................................... 6

Role Of active oxygen species ..................................................... 8

Cellular senescence and scald .................................................... 9
CHAPTER I ..................................................................................................... 10
LITERATURE REVIEW ................................................................................... 10
Scald of apples .................................................................................... 10
Superficial scald - a physiological disorder ................................. 10
Empire and Braebum disorders .................................................. 10

Factors related to susceptibility of superficial scald of apple ........ 11
Varieties ...................................................................................... 11
Preharvest factors ....................................................................... 1 1

viii

Maturity ...................................................................................... 1 2

Antioxidants ................................................................................ 1 2
Scald is a chilling injury-related disorder .................................... 13
Role of Volatiles ......................................................................... 14
6-methyl-5-heptene-2-one ......................................................... 1 6
Active oxygen species ............................................................... 16
Technologies to Control superficial scald Of apples ...................... 17
Storage conditions ..................................................................... 17
Low oxygen CA ................................... ‘ ....................................... 18
Low ethylene CA ........................................................................ 18
Low oxygen and low ethylene CA .............................................. 19
Hypoban'c storage ...................................................................... 19
Heat-treatment ........................................................................... 20
Chemical control ........................................................................ 20
Coating with food compatible antioxidant treatment ................... 21
Prediction of superficial scald ..................................................... 22
Alternative strategies to control scald are needed .......................... 22
Ethanol vapor treatment ............................................................. 25
Initial low oxygen stress .............................................................. 25
HYPOTHESES ...................................................................................... 26
OBJECTIVES ........................................................................................ 27
LITERATURE CITED ........................................................................... 28

ix

CHAPTER II ................................................................................................... 39

EFFECTS OF HYPOBARIC STORAGE ON THE CONTROL

OF APPLE SCALD ......................................................................................... 39
ABSTRACT .......................................................................................... 39
INTRODUCTION .................................................................................. 40
MATERIALS AND METHODS ............................................................ 43

Plant materials ........................................................................... 43

Hypobaric studies in 1995/96 .................................................... 43

Hypobaric studies 1996/97 ........................................................ 43

Hypobaric studies 1997/98 ........................................................ 44

RESULTS ............................................................................................ 45

Hypobaric studies in 1995—97 .................................................... 45

Hypobaric studies 1997/98 ........................................................ 47

DISCUSSION ....................................................................................... 52

Removing volatiles control scald ............................................... 52

Involvement of a—famesene and MHO ..................................... 54

LITERATURE CITED .......................................................................... 56

CHAPTER III ................................................................................................. 59
ETHANOL VAPOR CONTROLS SUPERFICIAL SCALD OF APPLES

DURING STORAGE ..................................................................................... 59

ABSTRACT ......................................................................................... 59

INTRODUCTION ................................................................................. 60

MATERIALS AND METHODS ............................................................. 62

Preliminary studies 1995 .......................................................... 62

Fruits .............................................................................. 62
Ethanol vapor treatments ............................................... 62
1996 studies .............................................................................. 64
RESULTS ............................................................................................ 65
MAP studies .............................................................................. 75
1996 studies .............................................................................. 78
DISCUSSION ....................................................................................... 84
DPA and a-famesene metabolism ............................................ 84
Ethanol vapor controls scald ..................................................... 85
Low oxygen induces ethanol production .................................... 87
LITERATURE CITED ........................................................................... 88
CHAPTER IV .................................................................................................. 91
EFFECTS OF INITIAL LOW OXYGEN STRESS ON CONTROLLING
SUPERFICIAL SCALD OF APPLES ................... . .......................................... 91
ABSTRACT .......................................................................................... 91
INTRODUCTION .................................................................................. 92
MATERIALS AND METHODS ............................................................. 94
Preliminary investigations (1996) ............................................... 94
Initial low oxygen stress studies( 1997) ...................................... 95
Continuous low 02 stress studies .............................................. 97

Initial low 02 stress to control scald; a commercial test at the MSU

xi

CHE 8 CA Storage Facility ............................................... 97

RESULTS ............................................................................................. 98
1996-97 studies ......................................................................... 98

Initial low oxygen stress followed by CA storage ............ 98

Initial low oxygen stress followed by air storage ............. 99

199 7/98 Studies ....................................................................... 1 03

Initial low oxygen stress studies .................................... 103

Continuous low or ultra low oxygen stress studies ........ 120

Initial low 02 stress studies at the MSU CHES CA Storage

Facility, a commercial test .................................. 124

DISCUSSION ..................................................................................... 127

Initial low oxygen stress controls scald .................................... 128

a—Famesene and MHO production .......................................... 128

Conclusions and implications ................................................... 129

Alternative strategies ................................................................ 131

LITERATURE CITED ......................................................................... 132

CHAPTER V .................................................................................................. 136
THE EFFECT OF ACCLIMATIZATION OF FRUITS ON THE CONTROL

OF A COZ-LINKED DISORDER OF EMPIRE APPLES ................................ 136

ABSTRACT ........................................................................................ 136

INTRODUCTION ................................................................................ 136

MATERIALS AND METHODS ........................................................... 137

xii

Preliminary experiments (1994) ................................................ 1 37

Carbonyl content determination ............................................... 138

Main experiment (1995) ............................................................ 138
RESULTS ........................................................................................... 139
Preliminary experiments .......................................................... 139

Main experiment ...................................................................... 142
DISCUSSION ..................................................................................... 147
The nature Of the disorder ....................................................... 147

The role of active oxygen species ........................................... 148

The acclimatization phenomenon ............................................ 151
LITERATURE CITED ......................................................................... 152
SUMMARY .................................................................................................... 154

xiii

LIST OF TABLES

CHAPTER II

Table 1. Effect Of pretreatment with hypobaric storage (0.05 atmosphere Of air)
followed by CA or air storage on scald development Of Granny Smith and Law
Rome apples at 1°C. Scald index (0=none; 1=Slight; 2=moderate; 3=severe) was
normalized tO 100. ............................................................................................ 46

Table 2. Effect of hypobaric storage (0.05 atmosphere Of air) and CA storage on
scald development Of Granny Smith and Law Rome apples at 1°C. Scald index
(0=none; 1=Slight; 2=moderate; 3=severe) was normalized to 100. ................. 46

CHAPTER III

Table 1 . Effect Of maintaining a constant ethanol vapor concentration for 2 months
on control Of scald Of Law Rome apples stored in air at 3°C for 3.5 months. ....76

Table 2. Effect Of initial ethanol vapor treatment on control Of scald of Law Rome

apples stored in air for 3.5 months at 3°C. ....................................................... 76

Table 3. Effect Of initial ethanol vapor treatment on control of scald Of Law Rome
apples stored in MAP with 2 mil LDPE for 3 months at 1°C. ............................ 77

Table 4. Ethanol vapor concentration Change in MAP with 2 mil LDPE at 1°C.
Ethanol at 0.25 g per fruit was added initially. .................................................. 77

CHAPTER IV

Table 1. Effect Of Initial low oxygen stress on control Of scald Of apples in the MSU
CHES CA storage trialz. .................................................................................. 124

CHAPTER V

Table 1. Effect Of ethanol vapor treatment on the incidence (%) Of the ‘Scald like'
disorder Of Empire apples stored at 0 or 5.5% CD2 + 1.5% 02 at 0.1 and 3°C with
and without pretreatment with diphenylamine (1000 mg-L"). ......................... 140

Table 2. The effect of carbon dioxide and diphenylamine on the development Of

xiv

Empire disorder at 3°C. Disorder index (0=none; 1=slight; 2=moderate; 3=severe)
was normalized to 100. ................................................................................... 141

Table 3. Ascorbate and dehydroascorbate content Of scalded and non-scalded
peel tissue Of Jonathan, Golden Delicious and Stayman apples. ................... 149

Table 4. Carbonyl content Of Law Rome apples Skin in relation tO scald

development of fruits stored for 6 months in air or CA at 3% CO2 + 3% 02 at 1 or
3°C. ................................................................................................................ 149

XV

LIST OF FIGURES

CHAPTER II

Figure 1. Effect Of delaying hypobaric storage on control Of scald Of Granny Smith
and Law Rome apples. Fruits were stored in air at 1°C prior to storing them
hypobarically at 0.05 atmosphere in air. Scald index (0=none; 1=slight;
2=moderate; 3=severe) was normalized to 100. ............................................... 48

Figure 2. Change in Ot-farnesene levels in headspace (A, B) and in the epicuticular
wax (C, D) Of Granny Smith (A, C) and Law Rome (B, D) apples stored in air for 0
to 6 months before hypobaric storage at 0.05 atmosphere in air. ..................... 49

Figure 3. Change in 6-methyl-5-heptene-2-one (MHO) levels in headspace (A, B)
and in the epicuticular wax (C, D) of Granny Smith (A, C) and Law Rome (B, D)
apples stored in air for 0 to 6 months before hypobaric storage at 0.05 atmosphere
in air. .................................................................................................................. 50

Figure 4. Change in OI-farnesene (A) and its oxidation product 6-methyl-5-
heptene-2-one (8) levels in headspace for different cultivars of apples after 7
months Of hypobaric storage at 1°C and then at 20°C in air. (Scald did not develop
on any fruits stored hypobarically but developed on all others except Golden
Delicious stored at atmosphere pressure.) ........................................................ 51

CHAPTER III

Figure 1. Changes in headspace ethanol vapor concentrations as a function Of
time. Ethanol vapors were applied to Law Rome apples in the experimental storage
chambers at 3°C by Circulating air through aqueous solutions Of ethanol at 0, 1, 2,
4 and 8% (v/v). Designation 1% w/o is for a Chamber without fruit. .................. 66

Figure 2. Changes in headspace ethanol vapor concentrations without (A) or with
(B) 20 fruits at 0 and 20°C. 59 of pure ethanol was added to 10 g Of vermiculite in
the experimental storage chamber. ................................................................... 67

Figure 3. Changes in headspace ethanol vapor concentrations. Ethanol was added
tO 10 g Of vermiculite in the experimental storage chamber containing McIntosh
apples at 3°C. .................................................................................................... 69

Figure 4. lntemal ethanol vapor (A) and ethylene (B) concentrations in Law Rome
and Red Delicious apple fruits as a function Of ethanol concentration (% v/v) in the

xvi

vapor generator. Ethanol concentrations were maintained for 2 months by
replenishing weekly with ethanol as necessary. ............................................... 70

Figure 5. The correlation between internal ethanol and ethylene from the fruits
exposed to the maintained ethanol concentrations for 2 months by replenishing
weekly with ethanol as necessary. .................................................................... 71

Figure 6. Changes in relative amounts of some volatile compounds in apple fruits
treated with ethanol vapors which were generated by Circulating air through various
concentrations (% v/v) Of ethanol in water: A1, 0%; A2, 5% and A3, 10% were
maintained for 2 months on Law Rome apples; B1, 0%; 82, 5% and 83, 10% were
maintained for 2 months on Red Delicious apples; C1, 0%; CZ, 2% and C3, 4%
were added initially to Law Rome apples. All fruits were stored in air for 3.5 months
at 3°C. P1-P6 stand for the peaks Of ethanol, hexylacetate, hexylbutanoate/
butylhexanoate, hexyl-2-methylbutanoate, hexylhexantoate, and OI-farnesene,
respectively. ...................................................................................................... 73

Figure 7. Effect Of oxygen levels on internal ethanol concentration for Granny
Smith, Law Rome, Red Delicious and ldared apples. Atmospheres were
equilibrated at the respective oxygen level for 2 days prior to sampling. .......... 74

Figure 8. Effect Of ethanol vapor on controlling apple scald for Granny Smith, Law
Rome and Red Delicious after 4 months of CA storage (3% 02 + 0% 002) at 1°C
plus 7 days at 20°C in air. Scald index (0=none; 1=slight; 2=moderate; 3=severe)
was normalized to 100. ..................................................................................... 79

Figure 9. Effect of ethanol vapor on controlling apple scald for Granny Smith, Law
Rome and Red Delicious after 7 months Of CA storage (3% 02 + 0% 002) at 1°C
after storage (A) and plus 7 days at 20°C in air (B). Scald index (0=none; 1=slight;
2=moderate; 3=severe) was normalized to 100. ............................................... 80

Figure 10. Effect Of ethanol vapor or diphenylamine (DPA) on controlling scald of
Granny Smith apples stored in air for 5 months at 1°C after storage (A) and plus 7
days at 20°C (B). Scald index (0=none; 1=slight; 2=moderate; 3=severe) was
normalized to 100. ............................................................................................. 81

Figure 11. Effect Of ethanol vapor on the production rates of OI-famesene for
Granny Smith apples during CA storage (3% 02 + 0% CO.) at 1°C. ................ 82

Figure 12. Effect Of ethanol vapor on the production rates of 6-methyl-5-heptene-2-

one for Granny Smith apples during CA storage (3% 02 + 0% C02) at 1°C. ....83

CHAPTER IV

xvii

Figure 1. Effect Of initial low oxygen stress and ultra low oxygen level continuously
on controlling apple scald of Granny Smith, Law Rome and Red Delicious apples
after 4 months Of CA storage (3% 02 + 0% 002) at 1°C plus 7 days at 20°C in air.
Scald index (0=none; 1=slight; 2=moderate; 3=severe) was normalized to 100.

.......................................................................................................................... 100

Figure 2. Effect Of initial low oxygen stress on controlling apple scald for Granny
Smith, Law Rome and Red Delicious after 7 months Of CA storage (3% 02 + 0%
C02) at 1°C on removal from storage (A) and plus 7 days at 20°C in air (B). Scald
index (0=none; 1=slight; 2=moderate; 3=severe) was normalized tO 100. ...... 101

Figure 3. Effect Of initial low oxygen stress on the production rates Of OI-farnesene
(A) and 6-methyl-5-heptene-2-one (B) for Granny Smith apples during CA storage
(3% 02 + 0% 00,.) at 1°C. .............................................................................. 102

Figure 4. Initial low oxygen stress attenuates scald Of Granny Smith apples stored
in air for 5 months at 1°C. Scald index (0=none; 1=slight; 2=moderate; 3=severe)

was normalized tO 100. (B after storage; I plus 7 days in air at 20°C). ........ 104

Figure 5. Effects of initial low oxygen stress, DPA and EtOH treatments on the
production rates Of Ot-famesene (A) and 6-methyl-5—heptene—2-one (B) for Granny
Smith apples after 5.5 months of air storage at 1°C. ........................... 105

Figure 6. Initial low oxygen stress attenuates scald Of Granny Smith and Law
Rome apples stored in CA (3% 02 + 0% CO2 or 1 .5% 02 + 3% 002) for 5 months(A)
and 8 months (B) at 05°C plus 7 days at 20°C in air. Scald index (0=none;
1=slight; 2=moderate; 3=severe) was normalized to 100. ............................... 106

Figure 7. Effects of initial low oxygen stress and oxygen levels in CA storage (3%
02 + 0% 002 or 1.5% 02 + 3% C02) on internal browning Of Law Rome (A) and
Granny Smith (B) apples stored for 8 months at 05°C plus 7 days at 20°C in air.
............................................................................................................................ 107

Figure 8. Effects Of initial low oxygen stress and oxygen levels in CA storage (3%
02 + 0% 002 or 1 .5% 02 + 3% 002) on core browning Of Law Rome (A) and Granny
Smith (B) apples stored for 8 months at 05°C plus 7 days at 20°C in air. ........... 109

Figure 9. Effects Of initial low oxygen stress and oxygen levels in CA storage (3%
02 + 0% CD2 or 1.5% 02 + 3% C02) on decay of Granny Smith (A) and Law Rome
(B) apples stored for 8 months at 05°C plus 7 days at 20°C in air. ................... 110

Figure 10. Effect of initial low oxygen stress and CA conditions (3% 02 + 0% 002
or 1.5% 02 + 3% C02) on flesh firmness retention Of Granny Smith apples stored
5 (AB) and 8 (0.0) months at 05°C. Flesh firmness on removal from storage (A,C)
and after 7 days at 20°C (B,D). ....................................................................... 111

xviii

Figure 11. Effect of initial low oxygen stress and CA conditions (3% 02 + 0% CO2
or 1.5% 02 + 3% 002) on flesh firmness retention of Law Rome apples stored 5
(AB) and 8 (CD) months at 05°C. Flesh firmness on removal from storage (A,C)
and after 7 days at 20°C (3.0). ........................................................................ 112

Figure 12. Effect Of initial low oxygen stress on Ot-famesene levels in the
headspace of Granny Smith (A, B) and Law Rome (C, D) apples stored in 1.5% (A,
C) and 3% (B, D) 02 CA for 5 months at 05°C. ............................................... 113

Figure 13. Effect of initial low oxygen stress on OI-farnesene levels in the
headspace of Granny Smith (A, B) and Law Rome (C, D) apples stored in 1.5% (A,
C) and 3% (B, D) 02 CA for 8 months at 05°C. ................................................. 114

Figure 14. Effect Of initial low oxygen stress on OI-famesene levels in the
epicuticular wax Of Granny Smith (A, B) and Law Rome (C, D) apples stored in
1.5% (A, C) and 3% (B, D) 02 CA for 8 months at 0.5°C.(Note different scale for C
and D). ............................................................................................................ 116

Figure 15. Effect Of initial low oxygen stress on 6-methyl-5-heptene-2-one (MHO)
levels in the headspace Of Granny Smith (A, B) and Law Rome (C, D) apples stored
in 1.5% (A, C) and 3% (B, D) 02 CA for 5 months at 05°C. ........................... 117

Figure 16. Effect of initial low oxygen stress on 6-methyl-5-heptene-2-one (MHO)
levels in the headspace of Granny Smith (A, B) and Law Rome (C, D) apples stored
in 1.5% (A, C) and 3% (B, D) 02 CA for 8 months at 05°C. ............................ 118

Figure 17. Effect of initial low oxygen stress on 6-methyl-5-heptene-2-one (MHO)
levels in the epicuticular wax of Granny Smith (A, B) and Law Rome (C, D) apples
stored in 1.5% (A, C) and 3% (B, D) 02 CA for 8 months at 05°C. .................. 119

Figure 18. Change in production rates Of ethanol (A), ethyl acetate (B) and
acetaldehyde (C) of Granny Smith at low levels of oxygen and air storage at 05°C.
.............................................................................................................................. 121

Figure 19. Change in production rates Of OI-farnesene (AB) and its oxidation
product MHO (C) of Granny Smith at low levels Of oxygen and air storage at 05°C.
(Fruits did not produce MHO when 02 lowerthan 1.25%) ................................... 123

Figure 20. Effects of initial low oxygen stress and oxygen levels in CA storage
(1.5% 02 + 0.5% 002) at the MSU CHES facility on controlling scald Of apples
stored for 8 months at 01°C plus 7 days at 20°C in air. Treatment designation: A
is for 1.5% 02 CA as control; B is for 0.55% 02 initial stress for 18 days then 1.5%
02 CA; C is for 0.61% 02 initial stress for 18 days then 1.5% 02 CA; D is for 0.7%
ultra-low 02 CA continuously. Scald index (0=none; 1=slight; 2=moderate;

xix

=severe) was normalized to 100. ................................................................... 126

CHAPTER V

Figure 1. (A) Effect Of acclimatization atmosphere on the incidence and severity
Of the scald-like disorder of Empire apples stored 6 months in CA at 3°C. Disorder
index on removal from storage. Disorder index (0=none; 1=slight; 2=moderate;
3=severe) was normalized to 100. .................................................................. 143

Figure 1. (B) Effect of acclimatization atmosphere on the incidence and severity
Of the scald-like disorder Of Empire apples stored 6 months in CA at 3°C plus 7 days
at 20°C in air. Disorder index (0=none; 1=slight; 2=moderate; 3=severe) was
normalized to 100. ........................................................................................... 144

Figure 2. (A) Effect Of acclimatization atmosphere on flesh firmness retention of
Empire apples stored 6 months in CA at 3°C. Flesh firmness on removal from
storage. Disorder index (0=none; 1=slight; 2=moderate; 3=severe) was normalized
to 100. The different letters at the top of the bars designate the difference at PS
0.01. ................................................................................................................. 145

Figure 2. (B) Effect of acclimatization atmosphere on flesh firmness retention Of
Empire apples stored 6 months in CA at 3°C plus 7 days at 20°C in air. Disorder
index (0=none; 1=slight; 2=moderate; 3=severe) was normalized to 100. The
different letters at the top of the bars designate the difference at Ps 0.01 . .......... 146

XX

INTRODUCTION

Overview of the scald problem:

Apples and pears are major US horticultural crops and are consumed as fresh fruits,
minimally processed Sliced fruits and after processing as canned or frozen, sauce,
bakery and juice items. The domestic and export market is large and growing for
these fresh ntits. The large Size Of the crops requires that more than 50 percent
Of the fruits grown be stored for extended periods Of up to one year to provide
orderly marketing Of fresh fruits and crop utilization for processing. Refrigerated air
storage and controlled atmosphere storage technologies are used for this purpose.
Scald is a devastating physiological disorder induced by storage of apples and
pears at refrigerated temperatures required to delay ripening and extend the
storage period Of these fruits. Scald has the potential tO destroy the market value
and utility of millions Of tons Of apples and pears annually unless the fruits are
treated with a postharvest drench with diphenylamine (DPA) or ethoxyquin (6-
ethoxy-1 ,2-dihyd rO-2,2,4-trimethquuinoline). The concentration Of these chemicals
can be as high as 2,000 mg-L'1 (w/v) for DPA and 2,700 mg-L‘1 for ethoxyquin which
leaves a considerable and detectable residue both on and within the fruit at near
maximal FDA tolerance levels. The postharvest drench solution must contain a
fungicide along with a wetting agent tO minimize decay development which may
arise from a long list of fruit pathogens (Hardenburg and Spalding, 1972). These

pathogens are carried on the fruit surface as Spores or decay lesions within the

fruits. The drench solutions become heavily loaded with inoculum unless a
fungicide is included. The only fungicide approved for postharvest application is the
thiabendazole (MerTect) and it is used tO kill the pathogens and prevent infection
through wounds in the fruit surface derived from mechanical damage in harvesting
and handling and through natural openings in the fruit surface. Captan has been
used for this purpose. Numerous countries have banned the use of DPA and
numerous fungicides including Captan and prohibit importation Of fruits so treated.
If the US is to market apples and pears world-wide and year-around, alternative
strategies must be developed to provide safe and economical control Of this
important disorder. Since the use Of DPA is diminishing globally because Of
environmental and health concerns and government mandates, this means that
apples and pears will generally not have any fungicides applied postharvest to
protect them against fungal pathogens during storage and marketing. Many Of the
fungicides once commonly used have been or may be withdrawn. The costs for
developing and registering new fungicides are prohibitive and contra-indicated by
market size and environmental concerns, respectively. Alternative strategies to
control pathological and physiological disorders of apples and pears are urgently
needed tO ensure that these important fruits which can be available from storage
will continue to be marketed on a year-around basis. Moreover, there is growing
concern among consumers, growers and processors of apples and pears that many
Of the pesticide chemicals employed to produce the crop and control postharvest

pathological and physiological disorders may be potentially harmful to humans.

Rationale for new research:

The possibility Of losing the use Of DPA for controlling apple scald in the future has
prompted renewed interest in developing alternative strategies to control this
potentially devastating disorder. A special symposium held in Yakima, Washington
in 1994 was dedicated to apple scald research needs. It was sponsored mainly by
the Washington State Tree Fruits Research Committee (WSTFRC). Researchers
from all over the world gathered tO summarize the current situation and to address
what must be done to control scald in the event that DPA was not re-registered for
use on apples and pears to control scald. At that time there was still a question as
to whether or not DPA would be approved. It was. In Spite of this reprieve, the
development of alternative strategies tO control scald has been a high priority

research Objective for many laboratories Since 1994.

Recent strategies to control scald:

Over the past several years, we and other researchers have found several non-
Chemical treatments that reduce or control scald incidence and severity. These
include: initial low 02 stress; initial high carbon dioxide stress; heat-treatment;
intermittent warming and initial ethanol vapor treatment. However, none Of these
treatments are yet practical to employ commercially for long-term controlled
atmosphere (CA) storage without placing the fruits and the industry at risk without
first confirming the validity and practicality of the most promising procedures to
employ. Decreasing the 02 level to ca. 0.7% during CA can effectively control scald

Of Red Delicious apples in some growing regions and seasons but not in others

(Lau, 1990; Yearsley et al., 1996). Moreover, some cvs. are susceptible to low 02
injury and all cvs. do not reach Optimum dessert quality subsequent to long-terrn CA
storage at 02 levels below 1% (Gran and Beaudry, 1993; Little, 1985; Truter et al.,
1994). This may have a negative effect on consumer acceptance Of apples in the
US as was found in Europe using ultra-low 02 CA. And this is now proving to be the

case in the US as well.

Current CA storage technology to control scald:

Our laboratory studies with CA atmosphere purging at 1.5% 02 have continued to
Show good control of apple scald. This has been demonstrated at the commercial
level at the Michigan State University Clarksville Horticulture Experiment Station
(MSU CHES) CA facility Since 1987 using 1.5% 02 with purging to scrub CO2
(Dilley, 1990). Numerous Michigan CA operators have verified this procedure with
good results for at least 6 months Of CA storage. Success using this procedure is
predicated on harvesting apples at the onset Of the ethylene climacteric, having the
fruits under CA within a week after harvest and employing a hollow fiber membrane
air separator such as the Perrnea system for CO2 scrubbing. These parameters are
not always readily achieved. For CA storage beyond 6 months, fruits generally
must be harvested at preclimacteric ethylene levels. Our laboratory studies Since
1960 employing continuous purging with low 02 atmospheres have shown good
control Of scald for at least 9 months. This suggests that atmosphere purging may
remove a fruit-produced volatile substance that otherwise accumulates and results

in scald. There is much precedence for this scenario dating back to the seminal

studies Of Brooks et al. (1919) more than 70 years ago showing that using mineral
Oil coated wraps can sometimes be effective to control scald. Fortunately, as a
direct result Of the recently expanded research activities the wor1d-over, much
information has been generated on fundamental aspects Of the scald disorder. This
is pointing the way towards understanding the basis Of the disorder and Offers
promise that new strategies to control the disorder may be developed to control

scald.

New strategies to control scald:

Two areas Of our research have been especially fruitful in this regard; ethanol vapor
treatments and initial low 02 stress followed by CA storage at 1.5% 02. We find that
the initial low 02 stress induces the fruits to produce their own ethanol and this may
explain the efficacy Of the exogenously applied ethanol vaportreatments in reducing
scald. The biochemical mechanism responsible for the beneficial effects on scald
control by low 02 stress and the ensuing metabolism of ethanol remain to be
elucidated. Extensive analyses Of the volatiles produced by apples receiving low
02 stress or ethanol vapor treatments support the premise that metabolism of or-
famesene in the pigment bearing cells is closely associated with development Of the

scald disorder.

Role of volatiles in scald:
We have found that an oxidation product Of OI-farnesene (Song and Beaudry, 1996;

Wang and Dilley, 1997), namely 6-methyl-5-heptene-2-one abbreviated as MHO,

is highly correlated with scald development. Moreover, DPA was found to inhibit
production Of MHO but not OI-famesene from which it iS derived. Our studies with
fruits stored with continuous hypobaric (subatmospheric pressure) ventilation at O2
partial pressures equivalent to low 02 CA indicate that apples (even the Granny
Smith cv.) do not scald even after a year Of such storage or after subsequent
removal to air at ambient temperature. Hypobaric storage favors the removal Of all
volatiles including ethylene produced by the fruits; although the fruits produce
normal levels Of OI-famesene and its oxidation product MHO following hypobaric
storage. This suggests that some metabolic pathway responsible for scald may be
affected. We suspect that the fruits capacity to metabolize Ot-famesene to produce
the MHO ketone in the scald development pathway has been down-regulated by
hypobaric ventilation. The same down-regulation effect may occur under ultra-low

02 CA, e.g., 0.7% 02, but this remains tO be proven.

Summary current knowledge about scald:

The mechanism whereby conventional CA storage at 1.5 to 2% O2 retards and
ultra-low 02 CA, e.g., 0.7% 02 prevents scald is not known. The results from a vast
body Of CA storage and physiological research spanning the past 60 years can be

succinctly summarized as follows.

Scald:
O is a manifestation Of membrane-based chilling injury,

0 is a dysfunction Of the pigment bearing cells Ofthe hypodermis, especially,

but not limited to, the non-red portion Of the fnJit surface,

becomes more prevalent as the fruits senescence or age during storage
and upon subsequent warming,

is caused by accumulation of a volatile by-product Of fruit metabolism in
the affected cells,

is a consequence Of oxidative metabolism involving reduced Species Of
Oz, e.g., active 02 Species (AOS) such as 0;, H202 and free radicals
derived from them such as the hydroxyl radical (~OH), peroxyl radical
(-OOH) and perhaps singlet (‘02),

may be controlled. in vivo by naturally occurring antioxidants such as
ascorbic acid, carotenoids, OI-tOCOpherOl and glutathione among others
which act tO scavenge free-radicals,

is largely controlled by exogenously applied anti-oxidants such as
diphenylamine (DPA), ethoxyquin, ascorbate—6-palmitate, butylated
hydroxy toluene (BHT), butylated hydroxyanisole (BHA), squalene and
others which scavenge free-radicals if applied Shortly after harvest,

is controlled by hypobaric storage if fruits are under hypobaric conditions
within one month,

is less prevalent with fruits harvested after exposure to cool temperatures
late in the growing season,

is a metabolic dysfunction likely caused as a consequence Of oxidation Of
OI-famesene to 6-methyl-5—heptene-2-one and its subsequent metabolism

via free-radical intermediates,

0 affects most but not all cvs. Of apples which suggests a genetic basis for
the disorder,

0 results in the oxidation Of essential amino acid functional groups Of
enzymes and Of membrane lipids to carbonyl derivatives disabling them
from functioning or targeting the proteins for proteolysis leading to cell

death.

Role Of active oxygen species:

All plant cells produce active 02 species (AOS) such as 0; and H202 as part of their
normal aerobic metabolism and this is augmented by biotic and abiotic stresses.
These AOS are normally dissipated in the cells by enzymatic reactions and by
naturally occurring antioxidants. Failure Of these defense systems to keep the titer
of A08 at low levels can result in AOS interacting non-enzymatically to produce
more highly reactive Species such as the hydroxyl radical (-OH), peroxyl radical
(OOH) and singlet O2 (‘02). Ubiquitious enzymes such as catalase, peroxidase,
ascorbic acid peroxidase and glutathione peroxidase metabolize H202 to water.
These enzymes exist in most Of the subcellular compartments even in the cell wall
(apoplast). Superoxide dismutase is likewise distributed and dismutates 0; to
water. The combined action Of these enzymes with naturally occurring antioxidants
maintains non-damaging levels Of 'OH radicals which otherwise may non-
discriminantly destroy membrane-bound lipids and proteins and cytosolic enzymes

essential for cellular homeostasis.

Cellular senescence and scald:

The normal progression Of plant cell senescence as occurs in ripening results in
lowering the cells ability tO cope with biotic and abiotic stresses. Consequently, as
the reserves Of naturally occurring antioxidants decline, the defense systems
decline. This is one reason plants lose the ability to counter microbial invasion. AS
enzymes and the lipo-protein membrane matrix become oxidatively and irreversibly
damaged, catastrophic events occur resulting in death. Cells Of the fruit hypodennis
affected by scald succumb in this manner and then display the well recognized
symptoms. However, the potential for the progression of cellular catabolism leading
to the eventual symptoms of scald develops within the first few weeks in storage
but symptoms remain unseen for many months by constrainment at low

temperature and low 02 levels during storage.

CHAPTER I

LITERATURE REVIEW

Scald of apples:

Superficial scald - a physiological disorder: Superficial scald iS a pervasive
physiological disorder of apples and pears that results in the browning of the Skin
during or after low temperature storage in air or after controlled atmosphere storage
which markedly extends the useful life span of these fruits. The affected cells Of the
hypodermis die and dehydrate (Bain, 1956, 1963). Most cvs. Of apples and pears
are susceptible tO scald (lngle and D’Souza, 1989). The disorder destroys the
appearance and therefore the fresh market value of the fruits. Affected fruits may
be processed as juice, sauce or sliced for bakery products but with product yield
reduction attending losses due to trimming. Product quality may be adversely
affected and costs are increased due to a higher labor input. NO evidence was
found tO involve flavanols, condensation products between flavonoid glycosides and
gallic acid or polymerisation Of flavan-3,4—diOlS in the disorder Of scald and the
oxidative coupling Of O-dihydroxyphenols in damaged tissue seems highly related
tO the browning (Piretti et al., 1994). The physiology and control Of scald has been
periodically reviewed (Smock 1961; Meigh, 1970; lngle and D’Souza, 1989;

Emongor et al., 1994).

Empire and Braebum disorders: Scald-like disorders on Empire and Braebum

apples have symptoms Similar to superficial scald and are also controlled by DPA

10

treatment (Burmeister and Dilley, 1995; Burrneister and Rowan, 1997). These
disorders Could occur only a few weeks after harvest while in storage and is highly
related tO the CO2 level in the storage (Burmeister and Dilley, 1995) while superficial
scald only occurs after a few months Of storage and is more severe when stored in
air. Johnson et al. (1998) concluded that fruits susceptible to COz-linked disorder
were known to have higher resistance to gas diffusion than cvs. not affected. Wang
et al. (1997b) found that Empire ‘Scald-Iike’ disorder could be controlled by
acclimatizating fruits in low 02 level and without CO2 for a few weeks at the
beginning Of storage. Braebum apples also have internal browning symptom
(Burmeister and Rowan, 1997) and an ultra-low CO2 CA and a Slow or 14-day delay
in establishing the CA conditions have been found to reduce the risk Of Braebum

browning disorder (Elgar et al., 1998; Lau, 1998)

Factors related to susceptibility of superficial scald of apples:

Varieties: Some cvs. Of apples are particulary susceptible to develop scald eg.
Law Rome, Granny Smith, Red Delicious, McIntosh and Cortland among others,
while other cvs. Show some natural resistance eg. Empire and Golden Delicious

(lngle and D’Souza, 1989; Emongor et al., 1994).

Preharvest factors: Environment during the growing season also influences scald
susceptibility as does mineral nutrition (Emongor et et., 1994; Fidler, 1957). Apples
with low calcium levels Often develop more scald than those with high levels

(Bramlage et al., 1974). Scald susceptibility was reported to be increased by high

11

application Of nitrogenous fertilizers in Delicious apples (Weeks et al., 1958). High
nitrogen may increase OI-famesene (2,6,10—trimethyI-2,6,9,11—dOdecatetraene)
and/or conjugated trienes indirectly in relation tO fruit maturity and/or the

development of wax on the fruit during storage (Emongor et et., 1994).

Maturity: Fruit maturity at harvest is a major factor; less mature fruits are prone to
scald while the more mature fruits are not when sampled from the same trees in the
same season (Anet, 1972b; Christophen, 1941; lngle and D’Souza, 1989). The fruit
industry has been driven to harvest apples at a relatively immature stage Of
development when attempting tO extend the storage season beyond six months in
CA storage. Delayed harvest increased the concentrations Of OI-tOCOpherOI and
carotenoids but not ascorbic acid and greatly reduced scald development (Barden
and Bramlage, 1994c). Early initiation Of ripening by preharvest applications Of 400
mg-L'1 ethephon 3-5 weeks before harvest reduced superficial scald and the content
Of lipid soluble antioxidants in the fruit peel Of ethephon-treated apples after eight
months in storage was increased (Curry, 1994). Similar results with ethephon found
by other researchers (Couey and Williams, 1973; Greene et al., 1977; Hammett,

1976; Lurie et al., 1989b; Windus and Shutak, 1977).

Antioxidants: Anet (1972b) proposed thatthe high scald susceptibility of immature
apples was due to an inefficient antioxidant system in their peel. Eleven
antioxidants from apple peel were isolated by using thin-layer Chromatography, but

only three tOCOpherOlS were identified (Anet, 1974). He concluded that scald did not

12

occur if the concentrations Of the antioxidants remained adequate to limit or-
famesene oxidation sufficiently. Lurie, et al. (1989a) concluded that DPA prevents
scald by its general antioxidant effect and not specifically by preventing the
oxidation Of OI-farnesene. An estimate of lipid-soluble antioxidant activity in apple
peel at harvest was negatively correlated with scald development (Meir and
Bramlage, 1988). Scalded tissue contained less OI-tocopherol than non-scalded
tissue in the same fruit (Gallerani et al., 1990). Antioxidant concentrations at
harvest were inversely related to maximum conjugated triene concentrations at the
end of storage and to scald development. However, no individual antioxidant was
associated consistently with conjugated triene accumulation or scald development.
And as conjugated triene concentrations increased, total lipid-soluble antioxidant
activity also increased but water-soluble antioxidants generally decreased during

storage (Barden and Bramlage, 1994b).

Scald is a chilling injury-related disorder: Superficial scald is considered to be
a chilling injury-related disorder (Watkins et al., 1995). Melville and Hardisty (1953)
found good control Of scald Of Granny Smith apples by storing them at ca. 5°C in
air for several weeks followed by storage at 0°C. This supports the premise that
scald is a chilling injury-related disorder. Apple scald does not occur on fruits stored
above 10°C. However, a disorder similar tO scald can be induced to develop at
20°C by an anaerobic atmosphere followed by returning the fruits to air (Dilley et al.,
1963). It tOO was prevented by DPA. Fruits gain potential tO scald by storing them

in air at 0 to 5°C while actual symptom development may appear many months

13

later. Evidence for this comes from studies with timing Of application of scald
inhibitors. DPA must be applied Shortly after harvest to be effective in controlling
scald (Shutak and Christopher, 1960). It becomes ineffectual once the fruits have
been in air storage for more than 3-4 weeks. This suggests that low temperature
storage in air potentiates scald development. This is consistent with scald being a

manifestation Of chilling injury.

lnterrnittant warming treatments are sometimes effective in preventing scald. Fruits
warmed to 20°C after 2 weeks in air at 0°C reduced scald while warming after 4
weeks was not effective (personal communication, Dr. C.B. Watkins, Cornell

University, Ithaca, NY ).

Role of Volatiles: Early investigations by Brooks et al. (1919) and studies by Fidler
(1950) suggested that volatile substances produced by the fruits were the cause of
the scald disorder. This was suggested since wrapping the fruits with mineral Oil
impregnated paper was partially successful in controlling scald SO they
hypothesized that some volatile organic substance produced by the fruit was
absorbed in the Oil and thus prevented its accumulation in the cuticle. Many
researchers believed that natural volatiles produced by apple fruits including OI-
farnesene and it oxidation products such as conjugated trienes, conjugated trienols
and 6-methyl-5-heptene-2-one were involved in apple scald development (Du and
Bramlage, 1993; Filmer and Meigh, 1971; Hall et al., 1953; Huelin and CoggiOla,

1970a; Meigh and Filmer, 1969; Murray et al., 1964; Murray, 1969; Spicer et al.,

14

1993; Song and Beaudry, 1996; Watkins et al., 1993; Whitaker et al., 1997). Fidler
(1950) concluded that scald involves a volatile as well as a non-volatile substance.
A volatile sesquiterpene, OI-famesene, was implicated in scald development more
than four decades ago (Meigh, 1969). OI-Famesene accumulates soon after harvest
(Meigh and Filmer, 1969) and it gradually autoxidizes at 1 °C due to being attacked
by a free radicals and the primary monomeric products are conjugated triene
hydroperoxides (Anet, 1969). The conjugated trienes accumulate during
subsequent storage (Anet, 1972a). Song and Beaudry (1996) found that MHO
applied tO apple fruits caused a scald-like disorder and this is a product Of or-
famesene oxidation. Scald was induced by the products Of OI-famesene oxidation,
and the concentration and time Of appearance Of these products determined the
severity of the disorder (Anet, 1972b). Scald development in Granny Smith, a cv.
very susceptible to scald, was correlated to the oxidation Of OI-famesene tO
conjugated triene hydroperoxides (Huelin and Coggiola, 19703; Filmer and Meigh,
1971 ). Extensive investigations in Australia (Huelin and Murray, 1966; Huelin and
Coggiola, 1968, 1970b, 1970c; Anet, 1972a; Anet and Coggiola, 1974) suggest that
scald results from the autooxidation Of a volatile sesquiterpene hydrocarbon OI-
famesene in the fruit skin (Meigh and Filmer,1969). The oxidation Of OI-famesene
yields conjugated triene hydroperoxide free-radicals and conjugated trienols
(Whitaker et al., 1997) which injure the cells and give rise to the symptoms Of scald
(Rowan et al.,1995). New techniques have been developed for rapid analysis of
volatile compounds (Matich et al., 1996; Song et al., 1997). Recently, Rupasinghe

et al. (1998) found that incorporation of radiolabel into OI-famesene from trans,

15

trans-[1-3H]-famesenepyrophosphate (FPP) was nearly 3-fOld lower in scald-
developing Skin tissue than in scald-free skin tissue Of the same apple. Ventilation
increases loss Of OI-famesene and reduces scald (Anet, 1972b). Whereas at the
same time Matich et al. (1998) suggested that reduction Of apple scald by ventilation
may not be explicable merely by enhanced evaporative depletion Of OI-famesene
since the rate Of loss Of OI-farnesene from the surface of the fruit depends more on
storage time than on its concentration in the wax (Matich et al., 1998). Scrubbing
CO2 with low 02 atmosphere during CA storage at 1.5% 02 controls scald (Dilley,
1990); this also removes other volatiles. Collectively, research strongly supports the

premise that some volatile(s) is closely related tO scald development.

6-methyl-5-heptene-2-one: Among the products Of the oxidation of OI-farnesene
are low-molecular-weig ht carbonyl compounds, some Of which were identified to no
effect on scald, however, 6-methyl-5-heptene-2-one, pyruvaldehyde, and
methylvinyl ketone require more study (Filmer and Meigh, 1971). The primary
volatile product during early stages Of OI-famesene oxidation is 6-methyl-5-heptene-
2-0ne (Anet, 1972a). Exogenous application Of 6-methyl-5—heptene-2-one vapor
can induced scald symptom on 9 cultivars Of apple; scald -Susceptible cultivar were

more sensitive than scald-resistant cultivars (Song and Beaudry, 1996).

Active oxygen species: All plant cells produce active oxygen species (AOS) such
as 0; and H202 as part of their normal aerobic metabolism and this is augmented

by biotic and abiotic stresses. These AOS are normally dissipated in the cells by

16

enzymatic reactions and by naturally occurring antioxidants. Failure of these
defense systems to keep the titer of A08 at low levels can result in AOS interacting
non-enzymatically tO produce more highly reactive Species (Halliwell and
Gutteridge, 1989) such as the hydroxyl radical (-OH), peroxyl radical (OOH) and
Singlet oxygen (‘02). Ubiquitious enzymes such as catalase, peroxidase, ascorbic
acid peroxidase and glutathione peroxidase metabolize H202 to water. These
enzymes exist in most Of the subcellular compartments and even in the cell wall
(apoplast). Superoxide dismutase is likewise distributed and dismutates 0; to
water. The combined action Of these enzymes with naturally occurring antioxidants
maintains non-damaging levels of OH radicals which otherwise may non-
discriminantly destroy membrane-bound lipids and proteins and cytosolic enzymes
essential for cellular homeostasis (Halliwell and Guttridge, 1989). The normal
progression of plant cell senescence as occurs in ripening results in lowering the
cells ability tO cope with biotic and abiotic stresses. Consequently, as the reserves
Of naturally occurring antioxidants declines, the defense systems decline. This is
one reason plants lose the ability to counter physiological disorders such as

superficial scald Of apples.

Technologies to Control superficial scald of apples:

Storage conditions: High 02 levels, low carbon dioxide levels, ethylene
accumulation and poor ventilation conditions during storage increase the scald
incidence and severity (Lau, 19853, 1985b; Little et al., 1985; Little and Peggie,

1987; Manseka and Vasilakakis, 1993; Porritt, 1966; Roberts et al., 1963).

17

Low oxygen CA: Ultra-low 02 levels in CA storage can also reduce scald (Little
and Peggie, 1987). Storage under ultra low CA conditions led to markedly lower
scald levels on the post-mature fruit, but did not greatly reduce scald on the pre-
mature and mature fruit (Truter et al., 1994). CA storage at 0.7 % 02 can be
effective in control Of scald in Red Delicious apples in the Northwest (Lau, 1990).
For other regions the results have been less promising and some cvs. Show
intolerance to the low 02 levels needed to control scald (Gran and Beaudry, 1993).
Low 02 or ultra low 02 also Showed Similar results in reducing or controlling apple
scald in other studies (Patterson and Workman, 1962; Roberts et al., 1963; Porritt,
1966; Sharples and Johnson, 1981; Chen et al., 1985; Little, 1985; Truter et al.,
1994; Yearsley et al., 1996). CA storage at 1.5 % 02 appears to maintain flesh
firmness and other quality attributes such as flavor for most of the cvs. we have
examined over many years and largely control scald whereas long-term CA storage

at higher 02 levels does not (Dilley, 1990).

Low ethylene CA: Low ethylene concentration in the storage atmosphere was
partially effective in reducing scald Of apples (Little et al., 1985; Liu, 1985; Skrzynski
et al., 1985). It was suggested that ethylene played a fundamental role in changes
associated with scald development based on Du and Bramlage (1994). Ethylene
was included because the delay in ripening by storage in a controlled atmosphere
and by modified atmosphere created by coatings are partially due tO alternation in

ethylene production and action (Kader, 1986). Removing ethylene from the storage

18

atmosphere can reduce scald (Knee and Hatfield, 1981 ). Our preliminary studies
(unpublished) with ethylene action inhibitors suggests that some aspect Of ethylene
action as a plant hormone is implicated in scald development. Our Observation was
recently confirmed by researchers in Washington State (Postharvest Physiology
Review by the Washington State Tree Fruits Research Committee, July 20-21 , 1998
in Wenatchee, WA). Low ethylene atmosphere during storage can Significantly
reduce scald incidence (Little et al., 1985; Liu, 1985; Skrzynski et al., 1985; Du and

Bramlage, 1994).

Low oxygen and low ethylene CA: Low 02 and low ethylene during storage have
been shown to reduce superficial scald development (Lau, 1989, 1990, 1993; Fica,
1991;). Ventilation of apples with low 02 atmospheres can reduce scald (Dilley,
1990). Hypobaric storage can completely control scald (Dilley, 1982). Low 02 and
low ethylene storage conditions can reduce or control scald development (Lau,
1983, 1985a, 1985b; Little, 1985; Johnson et al., 1989; Fica, 1991 ). Many Of these
studies suggest, as proposed by Brooks et al.(1919) more than 70 years ago and
extended by Fidler(1950), that scald development is caused by a volatile substance

produced naturally by the fruit.

Hypobaric storage: Refrigerated storage Of apples at reduced atmospheric
pressures Of 0.1 to 0.2 atmospheres prevents scald (Dilley, 1982). At 0.1
atmosphere Of air the partial pressure of O2 is 76 mm-Hg which is equivalent to

about 2% 02 at atmospheric pressure. Storage at 2% 02 can delay but not prevent

19

scald. Since hypobaric ventilation favors the removal Of volatiles produced by the

fruits including ethylene this may explain how it can prevent scald.

Heat-treatment: Early studies by Hardenburg and Anderson (1965) Showed that
prestorage heat-treatment could reduce the incidence Of superficial scald Of apples.
Prestorage heat-treatments Of apples for 4 days at 38°C provided control Of scald
on Granny Smith apples, but this affected the flavor due to a large reduction in
organic acids (Lurie et al., 1990, 1991). Moreover, heat-treatments are problematic

in commercial due tO large volumes Of fruits.

Chemical control: Partial control of scald was achieved by wrapping fruits in
mineral Oil impregnated paper. This labor intensive practice was in common use
until the 1960s when the antioxidant diphenylamine was introduced and
commercially developed as a postharvest control procedure (Smock, 1957). In this
procedure, fruits are drenched in an emulsified solution of 2,000 mg-L'1 (w/v) DPA
(Hall et al., 1961 ). DPA is known to prevent the autoxidation Of OI-famesene in vivo
and in vitrO (Anet and Coggiola, 1974) and control scald in vivo. Baker (1963)
reported that diphenylamine inhibited electron transport in plant mitochondria. DPA
also caused other physiological changes on stored apples in controlling scald (Lurie
et al., 1989a). Chen et al. (1990) reported that superficial scald Of ‘d’Anjou’ pears
was effectively controlled by ethoxyquin. Squalene is also known to reduce scald
(Cuny, 1998). Some amine-type antioxidants such as butylated hydroxytoluene

(BHT) and butylated hydroxyanisle (BHA) are less effective and some others are

20

potential carcinogenic compounds (Wills and Scott, 1977). Phorone (2,6-dimethyl-
2,5-heptadien-4-one) is known to control scald and limit the accumulation Of or-
famesene on fruits during storage and the amount of conjugated triene oxidation
products derived from OI-famesene. It was more effective than monoterpenes in
controlling scald (Scott et al., 1980) but taints the flavor Of the apples and no
toxicology data is available. Some monoterpenes are effective in controlling scald
(Wills et al., 1977). The antioxidant ethoxyquin (6-ethoxy-1 ,2-dihydrO-2,2,4-trimethyl
quinoline) has also been used to control scald Of apples (Johnson et al., 1980). It
is an additive commonly used to control oxidative rancidity in poultry feeds.
Ascorbate-6-palmitate has shown some control of scald (Bauchot and John, 1996).
However, there is great interest in the public sector and among research scientists
as well as in the fruit industry, to avoid using postharvest applied antioxidant

chemicals to apples and pears.

Coating with food compatible antioxidant treatment: Use Of chemicals such as
Semperfresh with food-compatible antioxidants can reduce superficial scald for a
few months but not in long term storage (Little and Barrand, 1989; Bauchot et al.,
1995b). Kallay (1994) found that Semperfresh and antioxidant combinations were
effective to control scald of Granny Smith apples. Bauchot et al. (1995a) concluded
that the limited control Of scald by ascorbyl palmitate plus Semperfresh is partially
related to the Observed modification Of the internal atmosphere Of the apple. A
sucrose ester coating has a potential to improve the storage quality Of apples (Smith

and Stow, 1994). Use of ascorbyl palmitate mixed with Oil resulted in marked

21

reduction in scald (DOdd and Bester, 1993). Ascorbic acid reduced scald incidence
and severity when apples were stored in CA for 43 weeks but did not reduce it
consistently when apples were stored in air (Chellew and Little, 1995). Vegetable
Oils reduced superficial scald probably because Of a physical effect but not related
to Chain length of fatty acids or degree Of unsaturation (Scott et al., 1995b; Little and
Barrand, 1989; Bauchot et al., 1995b; Kallay, 1994; Bauchot et al., 1995a; Smith

and Stow, 1994; DOdd and Bester, 1993; Chellew and Little, 1995).

Prediction Of superficial scald: There has been some success in predicting scald
susceptibility Of Cortland and Red Delicious apples in the Northeast (Barden and
Bramlage, 1994a) based upon the accumulation Of days or hours at temperature
below 10°C during the late maturation stage Of fruit development. For other regions

and cvs. this has given mixed results but they are still promising (Curry, 1998).

Alternative strategies to control scald are needed:

The issues of quality maintenance and control Of fruit and vegetable physiological
and pathological disorders have been addressed for over the past several decades
by postharvest researchers (Hall, 1957; Harvey, 1978). The storage technologies
which have been developed and implemented in commercial application by
postharvest researchers have addressed prolonging the useful shelf-life Of these
produce items (Blanpied, 1990; Curry, 1990). This research has largely addressed
the issues Of quality maintenance and control Of physiological disorders of fruits and

vegetables (Hintlian and Hotchkiss, 1986). The over-all goal has been to develop

22

and extend improved fruit storage technologies to provide high quality, wholesome
and safe foods to the consuming public at an affordable price while at the same
time assuring the producers and handlers Of the produce a sufficient profit margin

to encourage them tO adopt these technologies.

DPA and ethoxyquin have been used as described above for nearly forty years to
control superficial scald. Early research with DPA as a dip treatment Of fruits in bulk
bins was conducted in 1963 (Dilley and Dewey, 1963). This was quickly followed
by development Of over-head drench treatment facilities for treating entire truck
loads Of apples before they were put in CA storage (Dewey and Dilley, 1964); a
system still widely used today throughout the US and Canada. In Michigan alone
at least 5 million bushels (1x 10'5 tons) of apples are treated each year with a
postharvest drench treatment with DPA and the thiabendazole fungicide to control
scald and decay Of fruits during storage. Nationally, more than half of the apples
and pears grown are treated in a Similar manner with scald inhibitors and fungicide

prior to storage.

Disposal of the waste treating solutions poses a potential problem of contamination
of surface, stream and/or ground water. At least one instance Of fish kill has
occurred from DPA contamination from improperly disposed solutions. Disposal Of
DPA testing solutions to maintain the correct titer Of DPA in the treating solutions
also poses a serious problem because the Chemical reaction to analyze for DPA

forms a highly carcinogenic nitrOSO—derivative (personal communication with Scott

23

Ager Of Olympia, WA who developed the analytical procedure widely used
throughout the world while he was with Shieldbrite Corporation the major supplier

Of DPA formulated for use for scald control ).

DPA has been recently found to control some ‘scald-like’ COz-induced disorders
(Burmeister and Dilley, 1995) and internal breakdown disorders in apple fruits
(Burmeister and Rowan, 1997) and concern has been raised by researchers that
its use may be expanded for this purpose as well as for scald control. Scald affects

nearly all of the important apple and pear cvs.

A major goal Of the MSU postharvest research program for the past 6 years has
been to develop alternative strategies to control scald Of apples to avoid using any
postharvest-applied Chemicals. These studies (Burmeister et al. 1994; Wang et al.,
1995, 1997a, 1997b; Wang and Dilley, 1996, 1997; Dilley and Beaudry, 1998)
indicate that this may be achieved in the not-tO-distant future. These investigations
encompass fundamental studies Of the etiology and biochemistry Of the scald
disorder, controlled atmosphere (CA) storage experiments employing various O2
and carbon dioxide atmosphere regimens, initial low 02 stress regimens and semi-
commercial testing of CA regimens found from laboratory studies tO be potentially

applicable on commercial scale for the fruit industry.

Many researchers are searching alternatives to DPA for controlling superficial scald

of apples and searching effective methods to predict scald development. Two areas

24

Of our research include ethanol vapor treatments and initial low 02 stress followed
by CA storage at 1.5% 02 (Wang and Dilley, 1996,1997; Wang et al.,1997a) have

been especially fruitful in this regard.

Ethanol vapor treatment: Our studies (Wang and Dilley 1995,1997; Wang et al.,
1997a) on the efficacy Of ethanol vapor treatments to control scald were apparently
initiated at about the same time as those Of Scott’s group in Australia (Scott et al.,
1995a) who have extended their investigations (Ghahramani and Scott, 1998a) on

ethanol vapor treatments.

Initial low oxygen stress: We (Wang and Dilley, 1996,1997; Wang et al., 1997a)
and Ghahramani and Scott (1998b) find that the initial low 02 stress induces the
fruits to produce their own ethanol and this may explain the efficacy Of the
exogenously applied ethanol vapor treatments in reducing scald even when the
fruits are subsequently stored in air. The biochemical mechanisms responsible for
the beneficial effects on scald control by low 02 stress and the ensuing metabolism
Of ethanol remain to be elucidated. Extensive analyses Of the volatiles produced
by apples receiving low 02 stress or ethanol vapor treatments support the premise
that metabolism of OI-famesene in the pigment bearing cells is Closely associated
with development Of the scald disorder (Dilley and Beaudry, 1998; Wang et al.,
1997a; Wang and Dilley1996; 1997; Ghahramani and Scott, 1998b). Truter et al.,
(1994) reported that storage under ultra low CA conditions led tO markedly lower

scald levels on the post-mature fruit, but did not greatly reduce scald on the pre-

25

mature and mature fruit. Storage under the initial low 02 stress + ultra low 02 CA
regime, however, conferred low levels of superficial scald on pre-mature, mature
and post-mature fruit (Little et al., 1982). Recent studies by Van der Menrve et al.
(1997) indicate good control of scald by initial low 02 stress followed by CA storage

at 1% 02 with 3% CO2 at 1°C.

This dissertation focuses on investigations Of alternative strategies such as ethanol
vapor, initial low 02 stress, temperature and atmosphere, CA acclimatization and
hypobaric storage to control apple scald and their physiological and biochemical

bases.

HYPOTHESES:
1. The scald disorder is a manifestation Of chilling injury affecting the
pigment bearing cells of the fruit hypodennis. Fruits may acquire after
harvest resistance to scald. Symptoms Of the disorder are consequences Of
autooxidative processes in cell membranes brought about by low
temperature storage beyond the ability of naturally occurring antioxidants to

prevent the dysfunction.

2. Hypobaric storage controls scald by effectively decreasing the
accumulatiom Of scald-related volatile compounds in fruits which may affect
fruit metabolism such as ethylene, OI-famesene and its oxidation product

MHO.

26

3. Initial low 02 stress prior to CA storage controls scald by affecting the

metabolism including ethanol, OI-farnesene and its oxidation product MHO.

4. The Empire ‘scald-like’ disorder may be related to superficial scald since
the disorders Of both Empire and Braebum apples can be effectively

controlled by drenching with DPA before storage.

OBJECTIVES:
1. Determine and Optimize the level and duration Of ethanol vapor treatment

tO control scald Of apples and discuss the possible mechanism Of its action.

2. Determine and Optimize the level and duration Of initial low 02 stress and
subsequent CA conditions to safely and effectively control scald without

using postharvest- applied chemicals and without compromising fruit quality.

3. Determine the effective treatment regimens and gain insight into the
mechanism of scald control by identifying and quantifying scald related
volatile compounds in fruits or in their epicuticular wax substance in CA or
air storage and atmosphere purging through hypobaric storage through

GC/MS/SPME profile analysis.

4. Determine the pathway of formation Of 6-methyl-5-heptene—2-one which

has been implicated in scald development and the molecular mechanism Of

27

its action.

5. Determine the effects Of acclimatizing fruits in various gas atmosphere

regimens prior tO CA storage tO control Empire disorder.

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skin. J. Ag. FOOd Chem. 43: 2040-2045.

Rupasinghe, H.P.V., Paliyath, G. and Murr, DP. 1998. Biosynthesis of CI-
famesene and its relation to superficial scald development in ‘Delicious apples.
J. Amer. SOC. Hort. Sci. 123: 882-886.

Scott, K.J., Wills, R.B.H. and McBailey, W. 1980. The action of phorone and other
compounds in controlling superficial scald Of apples. Scientia Hort. 13: 9-14.

Scott, K.J., Yuen, C.M.C. and Ghahramani, F. 19953. Ethanol vapor- a new anti-
scald treatment for apples. Postharvest Biol. Technol. 6: 201-208.

Scott, K.J., Yuen, C.M.C. and Kim, G.H. 1995b. Reduction Of superficial scald Of
apples with vegetable oils. Postharvest Biol. and Technol. 6: 219-223.

Sharples, RC. and Johnson, OS. 1981. Storage Of cox in ultra low oxygen -
Sensitivity to low temperature injury. Rpt. E. Malling Res. Sta. pp. 133.

Shutak, V. and Christopher, ER 1960. Role Of the cuticle in development Of
storage scald on Corland apples. Pro. Amer. SOC. Hort. Sci. 76: 106-111.

Skrzynski, J., Fica, J. and Dilley, DR. 1985. The effect Of ethylene removal during
controlled atmosphere storage Of Red Delicious, Empire, Jonathan, ldared, and
Law Rome apples. In: S.M. Blankemship (ed.), Controlled Atmospheres for
Storage and Transport of Perishable Agricultural Commodities. Proc. 4th Natl.
CA Res. Conf., North Carolina State Univ., Hortic. Rep., 126: 115-126.

Smith, SM. and Stow, JR. 1994. The potential Of a sucrose ester coating material
for improving the storage and shelf-life qualities of Cox's Orange Pippin apples.
Ann. Appl. Biol. 104: 383-391.

Smock, RM. 1957. A comparison Of treatments for control Of the apple scald
disease. Proc. Amer. SOC. Hort. Sci. 69: 91-100.

Smock, RM. 1961. Methods of scald control on the apple. Cornell Univ. Agric.
Exp. Stn., Bull. NO. 970, pp. 55.

36

Song, J. and Beaudry, RM. 1996. Rethinking apple scald: new hypothesis on the
causal reason for development Of scald in apples. HortSCience 31: 605.

Song, J., Gardner, B.D., Holland, JP. and Beaudry, RM. 1997. Rapid analysis Of
volatile flavor compounds in apple fruit using SPME and GC/Time-of-Flight Mass
Spectrometry. J. Agri. Food Chem. 45: 1801-1807.

Spicer, J.A., Brimble, MA. and Rowan, DD. 1993. Oxidation Of OI-famesene. Aust.
J. Chem. 46: 1929-1939.

Truter, A.B., Combrink, J.C. and Burger, SA. 1994. Control Of superficial scald in
'Granny Smith' apples by ultra-low and stress levels Of oxygen as an alternative
to diphenylamine. J. Hort. Sci. 69: 581-587.

Van der Merwe, J.A., Combrink, J.C., Truter, AB. and Calitz, F .J. 1997. Effect Of
initial low oxygen stress treatment and CA storage at increased carbon dioxide
levels on post-storage quality Of South African-grown ‘Granny Smith’ and
‘Topred' apples. In: E.J. Mitchman (ed.) Apples and Pears. Proc. 7th lnt’l Nat.
CA Res. Conf., 2: 79-84.

Wang, Z., Kosittrakun, M. and Dilley, DR. 1995. Developments to control the
Empire ‘scaId-like’ disorder and superficial scald of apples. Proc. Mich. State
Hort. SOC. 125: 220-221.

Wang, Z. and Dilley, DR. 1996. Ethanol vapor treatment: A new alternative
approach to control superficial scald Of apples. Proc. Mich. State Hort. SOC. 126:
3540.

Wang, Z. and Dilley, DR. 1997. The relationship Of OI-farnesene production and
its oxidation product 6-methyl-5-heptene-2-one to superficial scald of Granny
Smith, Law Rome, Red Delicious and ldared apples during controlled
atmosphere and air storage. In: E.J. Mitchman (ed.) Apples and Pears. Proc.
7th lnt’l Nat. CA Res. Conf., 2: 98-104.

Wang, Z., McCully, T.J. and Dilley, OR. 19973. The effect of ultra low oxygen
storage, initial oxygen stress and ethanol vapor treatments on controlling
superficial scald Of Granny Smith, Law Rome, Red Delicious and ldared apples.
In: E.J. Mitchman (ed.) Apples and Pears. Proc. 7th lnt’l Nat. CA Res. Conf., 2:
105-1 11.

Wang, Z., Kosittrakun, M. and Dilley, D.R. 1997b. The effect of acclimatization of
fruits on the control Of a COz-linked disorder Of Empire apples. In: E.J.
Mitchman (ed.) Apples and Pears. Proc. 7‘h lnt’l Nat. CA Res. Conf., 2: 193-197.

Watkins, C.B., Barden, CL. and Bramlage, W.J. 1993. Relationships among CI-

37

famesene, conjugated trienes and ethylene production with superficial scald
development of apples. Acta Hort. 343: 155-160.

Watkins, C.B., Bramlage, W.J and Cregoe, BA. 1995. Superficial scald of granny
Smith apples is expressed as a typical chilling injury. J. Am. SOC. Hort. Sci. 120:
88-94.

Weeks, W.D., Southwick, F.W., Drake, M. and Steckel, J.E. 1958. The effect Of
varying rates Of nitrogen and potassium on the mineral composition of ‘Mclntosh’
foliage and fruit color. Prpc. Am. Soc. Hort. Sci. 71:11-19.

Whitaker, B.D., SolomOS, T. and Harrison, DJ. 1997. Quantification Of or-
famesene and its conjugated trienOl oxidation products from apple peel by C18-
HPLC with UV detection. J. Agric. Food Chem. 45: 760-765.

Wills, R.B.H. and Scott, K.J. 1977. Evaluation Of the use of butylated
hydroxytoluene to reduce superficial scald of apples. Scientia Hort. 6: 125-127.

Wills, R.B.H., Scott, K.J. and McBailey, W. 1977. Reduction of superficial scald in
apples with monoterpenes. Austral. J. Agr. Res. 28: 445-448.

Windus, ND. and Shutak, V.G. 1977. Effect of ethephon, diphenylamine and

daminozide on the incidence Of scald development on 'Cortland' apples. J.
Amer. SOC. Hort. Sci. 102: 715-718.

Yearsley, C.W., Banks, N.H., Ganesh, S. and Cleland, DJ. 1996. Determination
Of lower oxygen limits for apple fruit. Postharvest Biol. and Technol. 8: 95-109.

38

CHAPTER II
EFFECTS OF HYPOBARIC STORAGE ON THE CONTROL OF APPLE

SCALD

ABSTRACT
Law Rome and Granny Smith fruits were stored hypobarically at 0.05 atmosphere
pressure Of air with a ventilation rate Of one vessel void volume Change per hour at
97% RH and also in air and in CA storage at 1.5 and 3% 02 with 0 and 3% CO2 for
8 months at 1°C. Fruits were placed under hypobaric storage immediately after
harvest or after 0.5, 1, 2, 3, 3.5, 4, 4.5, 5 or 6 months storage in air at 1°C to
determine the effects of delaying imposition Of hypobaric storage on ripening and
scald development and production Of OI-famesene and its oxidation product 6-
methyl-5—heptene-2-one (M HO). Granny Smith and Law Rome apples did not scald
during hypobaric storage or afterwards when transfered to static air at one
atmosphere continuously for 4 months if they were placed under hypobaric
conditions within one month after harvest while held in air at 1°C. After 3 months .
delay, scald development was similar tO that for fruits stored in air. Fruits Of both
cvs. produced MHO and it accumulated in the epicuticular wax when placed under
hypobaric storage after one month delay in air at one atmosphere at 1°C. MHO
which had partitioned in the epicuticular wax of fruits stored hypobarically after 2 or
more months delay was released upon transfer Of fruits to 20°C; MHO accumulated
in direct proportion tO the duration Of the delay to hypobaric storage. In another

experiment with 5 apple cvs., the production rates of OI-famesene and MHO ketone

39

were low during hypobaric storage but increased upon removal from storage after
7 months over a 7 day period in air at 20°C and then Sharply decreased aftenrvard.
After storage, Ot-farnesene and MHO production rates were similar and high for Law
Rome, Mutsu, Red Delicious and Golden Delicious apples and were the lowest for
Granny Smith. Scald did not develop on any hypobarically stored fruits whereas it
did on all cvs. except Golden Delicious stored in air at one atmosphere. Hypobaric
ventilation apparently removes a scald-related volatile substance that othenrvise
accumulates and partitions into the epicuticular wax Of fruits stored in air at

atmospheric pressure.

INTRODUCTION
Hypobaric storage is a system Of storing commodities while ventilating with air at
less than atmospheric pressure. The commodity is placed in a vacuum-tight
container in which the absolute pressure, airtemperature and humidity are precisely
controlled and the rate Of air exchange is closely regulated. A vacuum pump
Operating continuously evacuates the Chamber tO the desired absolute pressure
(commonly to 0.05 to 0.1 atmosphere) at which time a vacuum regulator Opens and
admits air to the vessel. The partial pressure of each Of the component gases
comprising the air mixture is reduced in direct proportion tO the total pressure as
consequence Of Dalton’s Law. Thus at 0.1 atmosphere absolute total pressure, the
O2 partial pressure is equivalent to 2.1% (v/v) 02 at atmospheric pressure. And the
same is true for the other gases. Therefore, after pressure reduction, the partial

pressure Of water vapor in the ambient air is likewise reduced 10-fold. This

40

precisely why the air entering the vacuum vessel must be rehumidified and this is
done by passing the incoming air (after it comes through the vacuum regulator)
through a container of water which iS at the same pressure as the vacuum vessel.
This humidifies the rarified air which is then flushed through the vacuum vessel
containing the commodity. The temperature Of the water through which the air is
admitted determines its vapor pressure and thus the relative humidity Of the storage

vessel. NO other gas other than air is required.

The concept Of hypobaric storage of fruits was introduced by Burg and Burg (1966).
Since then it has been widely investigated (Dilley, 1972, 1977, 1982; Salunkhe and
Wu, 1973; Spalding and Reeder, 1976, 1977) and reviewed (Jamieson,1980;
Salunkhe and Wu,1975; TOlIe,1972). Hypobaric storage extends the useful life of
perishable commodities much beyond that achievable by CA storage at the
equivalent 02 levels. Preclimacteric apples at harvest remain preclimacteric during
hypobaric storage (Dilley,1972). This phenomenon is related to another basic
principle of hypobaric storage related to the diffusivity Of gases; ‘the rate Of gas
diffusion varies inversely with the absolute pressure of the gaseous medium through
which it diffuses’. Therefore at 0.1 atmosphere the rate Of gas diffusion is 10-fOld
greater than at 1 atmosphere. This has a direct bearing on the effective
concentration Of ethylene within plant tissues producing ethylene at a steady-state
rate. For example, the preclimacteric ethylene production rate of apples is fairly
constant at about 0.05 pL-L"-kg"-hr1 while the ethylene concentration in the

intercellular air Space is about 0.1 uL-L'1 at normal atmospheric pressure. If the

41

absolute pressure is decreased tO 0.1 atmosphere, the intemal ethylene
concentration is decreased 10-fOld which is much below the level to induce ripening
because it diffuses away from the fruits 10 times as fast. When this is coupled tO
a reduction in the rate at which ethylene is produced at the reduced 02 level
equivalent to 2.1%, an even further reduction in ethylene concentration may exist
within the fruit tissue. Moreover, the concentration Of CO2 produced in fruit
metabolism is likewise reduced. Additionally, the concentration of metabolic
products such aS ethanol, acetaldehyde and other compounds (eg. Ot-farnesene)
which have significant vapor pressures at fruit storage tempertures would likewise
be reduced under hypobaric ventilation compared to the concentration at
atmospheric pressure. OI-Farnesene and its oxidation products have been
implicated as factors in the superficial scald disorder (Anet, 1972; Song and
Beaudry, 1996). Apple fruits do not develop scald when stored hypobarically
(Dilley, 1972) while fruits stored at the equivalent partial pressure of O2 in CA
storage develop scald. The combined effects Of lower production rates of
oxidatively produced metabolites with significant vapor pressures at reduced 02
levels and reduced levels in the tissue under hypobaric storage conditions may at
least partially explain prevention Of scald. This indicates that fruits produce some
metabolite(s) or factor(s) related tO scald that can be attenuated by hypobaric
storage conditions. Hypobaric storage thus Offers an experimental system to

elucidate the nature Of these substances.

We hypothesized that hypobaric storage controls scald by effectively decreasing the

42

accumulatiom of scald-related volatile compounds in fruits which may affect fruit
metabolism such as ethylene, OI-farnesene and its oxidation product MHO.
Hypobaric storage was investigated to determine effective treatment regimens and

to gain insight into the mechanism Of scald control.

MATERIALS AND METHODS
Plant materials:
Law Rome, Granny Smith, Red Delicious, Mutsu and Golden Delicious apples were

harvested at the preclimacteric stage of maturity from the MSU CHES.

Hypobaric studies in 1995/96:

Apples were placed in two 1270-L vacuum vessels in a 1°C storage room. The
vessels were maintained hypobarically at 0.05 atmosphere pressure Of air with a
ventilation rate Of one vessel void volume change per hour at 97 RH (Dilley, 1982).
These conditions have proven to prevent apples from developing scald in more than
25 years of experimentation. After two months Of hypobaric storage at 1°C, fruits
were transferred to CA at 1.5% 02 plus 3% 002 and in air. After CA and air storage
for 6 months, scald index was measured based on the percentage Of fruit surface
area affected. Scald index was measured again after an additional 10 months Of

storage in air.

Hypobaric studies 1 996/9 7:

Fruits were stored hypobarically and also stored in air and in CA storage at 1.5 and

43

3% 02 with 0 and 3% CO2 for 8 months at 1°C. The hypobaric fruits were then
placed in air for an additional 4 or 10 months. Some fruits stored in CA for 2 months
were transferred to air. Scald index was measured as before at various intervals
of storage. OI-Famesene and MHO Of 5 cultivars were measured as described

below.

Hypobaric studies 1997/98:

Law Rome and Granny Smith fruits were stored hypobarically at 1°C and also
stored in air at the same temperature. Fruits were placed under hypobaric storage
immediately after harvest or after 0.5, 1, 2, 3, 3.5, 4, 4.5, 5 or 6 months storage in
air at 1°C to determine the effects Of delaying imposition Of hypobaric storage on
ripening and scald development and production Of (II-famesene and its oxidation
product 6-methyl-5-heptene-2-one (MHO). At appropriate intervals, fruits were
remOved from hypobaric storage and transferred tO 20°C in air in glass jars.
Subsamples were used for fruit firmness measurements and ripening and again
after 7 days. The headspace in the jars was sampled by SPME/GC/MS as
described by Song et al., (1997) to determine OI-famesene and MHO production
rates at daily or bi-daily intervals. Samples Of epicuticular wax were removed from
the fruits at each time interval and placed in glass vials to determine the amount Of

OI-famesene and MHO partioned in the cuticle by measuring the vial headspace.

RESULTS

Hypobaric studies in 1995-97:

Fruits stored hypobarically for 2 months then under CA at 1.5% 02 plus 3% CO2 for
6 months did not develop scald during storage (Table 1). A slight amount Of scald
developed on the Granny Smith and Law Rome cvs. for two months hypobarically
plus 6 months in CA and then in air continuous for 10 months. Fruits stored
hypobarically and then in air for 6 or 16 months had scald indices of 30 and 85,
respectively (Table 1). Fruits stored hypobarically for 8 months plus 4 months in air
did not develop scald (Table 2), whereas, scald developed after an additional 6
months Of storage in air. Granny Smith and Law Rome fruits stored under CA with
1.5% 02 for 8 months developed Slight scald with indices Of 8.3 and 10.3,
respectively; those stored for 2 months in CA then moved to air or those under CA
at 3% or more 02 scalded severely (Table 2). Collectively, the data indicate that
apples stored hypobarically do not scald during storage but may do so after
subsequent and extended periods Of time in a static air atmosphere (Table 2).
Moreover, CA storage is much less Of a deterrent to scald development than
hypobaric storage. Since hypobarically stored fruits eventually scald when
transferred to air at one atmosphere, this indicates that hypobaric storage does not
irreversibly inhibit scald development. And that only as little as 2 months of
hypobaric storage largely prevent scald suggesting that its effect is primarily
manifested during the first few months Of storage at low temperature by ameliorating
the effect Of a volatile substance produced by the fruits. This data is consistent with

our Observation that fruits do not scald when flow-through CA is employed at 1.5%

45

02 with 3% 002. Moreover, scald is largely controlled in commercial storages at
1 .5% 02 with 3% CO2 employing air separators tO remove CO2 by flushing. Flushing

also removes other volatile substances.

Table 1. Effect Of pretreatment with hypobaric storage (0.05 atmosphere Of air)
followed by CA or air storage on scald development Of Granny Smith and Law
Rome apples at 1°C.

 

 

 

Scald indexz
Treatments (1995) Granny Smith Law Rome
Hypobaric 2 mo. + CA (1 .5% 02 + 3% 00,.) 6 mo. 0 C 0 c
Hypobaric 2 mo. + CA (1 .5% 02 4.0 c 9.2 b
+ 3% 002) 6 mo. + 10 mo. in air
Hypobaric 2 mo. + 6 mo. in air 31.7 b 29.6 a
Hypobaric 2 mo. + 16 mo. in air 85.3 a -

 

z Scald index (0=none; 1=slight; 2=moderate; 3=severe) was normalized to 100.
Means within columns followed by different letters differ at ps.05.

Table 2. Effect of hypobaric storage (0.05 atmosphere Of air) and CA storage on
scald development Of Granny Smith and Law Rome apples at 1°C.

 

 

Scald indexz

Treatments (1996) Granny Smith Law Rome
Hypobaric 8 mo. 0 e 0 f
Hypobaric 8 mo. + 4 mO. in air 0 e 0 f
Hypobaric 8 mo. + 10 mo. in air 38.3 d 31.9 d
CA (1.5% 02 + 3% C02) 8 mo. 8.3 e 10.3 3
CA (3% 02 + 0% COZ) 8 mo. 87.6 b 77.3 b
CA (1 .5% 02 + 3% 002) 2 mO. + 6 mo. in air 61.7 c 49.9 C
Control (Air for 8 mo.) 100.0 a 100.0 a

 

z Scald index (0=none; 1=slight; 2=moderate; 3=severe) was normalized to 100.
Means within columns followed by different letters differ at ps.05.

46

Hypobaric studies 1997/98:

Hypobaric storage at 0.05 atmosphere prevents scald of Granny Smith and Law
Rome apples only if they are placed under hypobaric conditions within one month
after harvest and held in air at 1°C (Figure 1). After 3 months delay, scald

development is Similar tO that for fruits stored in air.

Progressively longer periods Of storage in air at 1°C before Granny Smith apples
were placed in hypobaric storage resulted in greater capacity Of the fruits to produce
a burst Of car-famesene production (Figure 2A) but not for Law Rome fruits for which
a nearly Opposite trend was found (Figure 2B). OI-Famesene production rates were
higher with Law Rome apples stored hypobarically within one month after harvest
but lower with Granny Smith apples treated similarly. The OI-famesene production
capacity of Granny Smith fruits placed in hypobaric storage within one month Of
harvest steadily increased after transferring the fruits tO air at 20°C eventually
reaching or exceeding that Of fruits stored after 2 months delay (Figure 2A). The
amount of OI-famesene partioned in the cuticle largely followed the pattern of its
production capacity for both cvs. (Figure 20,2D). Upon transfer to 20°C, the or-
famesene evolution rate from cuticle Of Law Rome apples was higher from apples
which were under hypobaric condition within two months than those more than two

months (Figure 2B,2D). This again mimicked the pattern of the production capacity.

MHO production rates were progressively greater as the delay tO hypobaric storage

increased with both cvs. (Figure 3A,3B). And, following a burst Observed one day

47

 

100
\ ~80
60

.40

Scald index

 

. ‘ 1' 20
Granny Smith

 

 

 

 

0 1 2 3 4 5 6
Months delay to hypobaric storage in air

Figure 1. Effect Of delaying hypobaric storage on control Of scald of Granny Smith

and Law Rome apples. Fruits were stored in air at 1°C prior to storing them

hypobarically at 0.05 atmosphere in air. Scald index (0=none; 1=slight;
=mOderate; 3=severe) was normalized to 100.

48

200TW- W. ___ -- _ -A A- __-_v.. -,__ W - - e- - - - .
160-11
120 i
aoi

 

a -F arn esene

)(units min"mg" at m/z 41 )(units min"mg‘1 at m/z 41)
on
o
oo

 

 

 

 

 

a -F arn esene

 

 

 

 

 
 
 
 
   
   
 

 

 

 

 

 

 

 

 

 

36000-1 . + 6 mo in air I C t
a) E i l -O— 4 mo delay (.
5 a *l + 3 mo delay i 1%
(0704000 ‘I = —3— 2mO delay 1
a) l
c E 1 l —e— 1 mo delay l l
5": 1 l —e— 6 mo in hypo t
u@2000 A T—
“.e i 2 . 4 i

'E .

a 0‘] l V l I r Mir—l _r

s i— 1 I i "“ "T "— ' '7‘ '7

#4000 ‘Q D f
a) E . .\

53030001: A i

83 '0: 1

5.520001
$5 I a.

E .! l
«£1000 l 4“?! .-

c I I”

a, 0.. ~ - — — . __. .

01 «(II

" o i 2 3 4
Days after removal from hypobaric storage at 20°C

Figure 2. Change in OI-farnesene levels in headspace (A, B) and in the epicuticular
wax (C, D) Of Granny Smith (A, C) and Law Rome (B, D) apples stored in air for 0
to 6 months before hypobaric storage at 0.05 atmosphere in air.

49

 

1.0,, .W W W. W W W W W W W WWWWWW WL __ W._ W W l_W,_. _._L. 2.- __.__J._- .._
I ‘ + 6moin air ' ,_
J 5 -o— 4modelay l A ‘
l g + 3modelay l l'

‘ + 2modelay . r
l -6— 1modelay l r
l -e— 6moin hypo_J L

 

 

 

MHO-ketone

 

 

 

ts min"g" atmlz 108) (units min"g'1 atm/z 108)

MHO-ketone

 

MHO-ketone

 

 

 

MHO-ketone

(units min"g“ atm/z 108) (units min"g“ atmlz 108) (uni

 

 

 

- 0 1 2 3 H4 5
Days after removal from hypobaric storage at 20°C

Figure 3. Change in 6-methyl-5-heptene-2-one (MHO) levels in headspace (A, B)
and in the epicuticular wax (C, D) Of Granny Smith (A, C) and Law Rome (B, D)
apples stored in air for 0 to 6 months before hypobaric storage at 0.05 atmosphere

in air.

50

,7 :I— OEnWS‘rfiiifi"

+ Red Delicious

60 1 l —O— Law Rome
; l

 
 
   

—3— Golden Delicious
' —A— Mutsu

 

 

 

. a-Famesene
(units mIn' mg“ at m/z108)

 

 

 

 

A

2-5 ' . + Granny Smith . s
I + Red Delicious 1 ;
, —0— Law Rome , O I

2'07 t —3— Golden Delicious l I-
I . —A— Mutsu l , ‘ l

MHO-ketone
(100 pmoles kg"hr“)
O'I

 

1.0.;
0'51
0.0-1 ' WWW . - . WWW-.. WWW
0 1 2 3 4
Days after removal from hyp5obaric6storag7e at 28°C 9

Figure 4. Change in OI-famesene (A) and its oxidation product 6-methyl-5-
heptene-2-one (B) levels in headspace for different cultivars Of apples after 7
months Of hypobaric storage at 1°C and then at 20°C in air. (Scald did not develop
on any fruits stored hypobarically but developed on all others except Golden
Delicious stored at atmospheric pressure.)

51

after transfer to 20°C, the rates gradually diminished. Fruits that were under
hypobaric storage within one month produced a nearly constant, low level Of MHO.
MHO which accumulated in the cuticle Of fruits delayed to hypobaric storage
gradually dissipated upon transfer Of fruits tO 20°C. Again, these fruits had the
lowest levels Of MHO production if the fruits were under hypobaric condition within

one month (Figure 3C,3D).

Based on the measurement Of 5 different cultivars including scald susceptible and
not susceptible cultivars, the production rates OfOI-famesene and MHO ketone were
low during hypobaric storage but quickly increased upon removal from storage after
7 months Of storage and held in air at 20°C; rates then, unexplicably, Sharply
decreased after about a week (Figure 4). The Granny Smith cv. had the lowest
rates among the 5 cvs. examined. All cvs. stored hypobarically did not scald but

they did when stored in air except Golden Delicious.

DISCUSSION
Removing volatiles controls scald:
Apple fruits stored hypobarically at O2 partial pressures equivalent to CA storage do
not scald during or after storage unless stored for prolonged periods of air storage
(Table 2). Hypobaric storage favors the removal Of ethylene and other volatile
substances produced by the fruits (Dilley, 1982). A low rate Of synthesis coupled
with continuous removal Of these substances by hypobaric ventilation results in

complete control Of scald during storage and even after the fruits are returned to a

52

static air atmosphere for a few months. Fruits so stored will eventually scald. This
indicates that the fruits produce some metabolites or factor(s) related to scald

development that are greatly attenuated by hypobaric storage.

Our hypobaric storage studies in the 1997/98 season have again confirmed that
hypobaric storage controls scald very effectively. But, to do so the fruits must be
under hypobaric storage within about one months of being harvested (Figure 1).
Fruits stored in air at 1°C for 2 months or longer prior tO placing them under
hypobaric storage at 0.05 atmosphere developed scald following storage while
those kept in air for progressively shorter times before hypobaric storage did not.
This is Significant because it indicates that the metabolism involved in scald
development becomes irreversible after 2 months storage in air. Moreover,
although hypobaric storage favors the removal Of all volatiles produced by the
fruits, none of these volatiles including OI-farnesene and its breakdown products can
be implicated as factors in scald beyond the initial 2 months Of storage. A trienOl
oxidation product Of OI-famesene has been implicated in scald (Whitaker, et al.,
1997) They may however be involved during this early period during which the
potential for scald development is gained by storage in air (Meigh, 1970; Shutak
and Christoffer, 1960; Watkins et al., 1995). These results are consistent with the
Observation that for DPA to be effective in controlling scald it must be applied within
a few weeks Of placing the fruits in air storage (Dilley and Dewey, 1963). In
addition, these studies reinforce the concept that scald is a chilling injury

phenomenon (Watkins et al., 1995).

53

It is apparent that some volatiles that can be removed by effective ventilation may
be involved in sCald development (Meigh and Filmer, 1969). DPA has been
shown to attenuate conversion Of OI-farnesene to triene hydroperoxides (Anet and
Coggiola, 1974). Apple fruits stored hypobarically at 02 partial pressures
equivalent to CA storage do not scald during storage but do after extended periods
in air storage upon removal (Tables 1,2). Since hypobaric storage favors the
removal of ethylene and other volatile substances produced by the fruits, this
suggests some involvement Of a volatile substance (Dilley, 1977, 1982; Jamieson,
1980). But if fruits were stored hypobarically only a couple Of months, scald still
developed after many months of subsequent storage in air (Table 1). This
indicates that short term hypobaric storage does not irreversibly inhibit scald
development. Continuous removal Of those volatile substances by hypobaric
ventilation for longer time results in complete control Of scald even after the fruits
are returned tO a static air atmosphere for up to 4 months (Table 2). This suggests
that the ability Of fruits to produce some metabolite(s) or factor(s) related to scald

development has been altered by hypobaric ventilation.

Involvement of a-famesene and MHO:

We also assessed the role of OI-farnesene and 6-methyl-5-heptene-2-one in scald
development. The pattern Of OI-farnesene production capacity following hypobaric
storage was similar for five cvs. examined in 1996 (Figure 4A). Moreover, the
production levels are similar to those Of fruits stored in CA and scald develops
during CA storage. The ability of the fruits to produce the car-famesene oxidation

54

product 6-methyl-5-heptene—2-one was also demonstrated. Fruits stored
hypobarically for 7 months did not develop scald; the production rates of or-
farnesene and MHO ketone were low but quickly increased upon removal from
storage and held in air at 20°C and then Sharply decreased afterward (Figure 4).
Hypobaric ventilation apparently removes a volatile substance that otherwise
accumulates perhaps by partitioning intO the epicuticular wax. The high level Of
the ketone production and absence Of scald strongly suggests that the sensitivity
Of response in cvs. to MHO ketone is much different or that the ketone may not be
the factor or only factor causing scald development. These data also indicate that
the ketone is not produced as a consequence Of scald development. The dramatic
increase then decrease in or-famesene production at 20°C was seen in all cvs.
and virtually all treatments (Figure 4). Very interestingly, OI-farnesene production
abruptly decreased after 7 days while the MHO production rate was still increasing.
Changes in the MHO followed the same trend except for a couple Of days’ delay
(Figure 4). This suggests that the trends are not due to abberancies Of analysis.
This data Clearly supports the notion that the 6-methyl-5-heptene-2-one is derived

from or-famesene.

Alternatively, the hypobarically stored fruit may lack the mechanism for the ketone
tO cause scald. There is ample precedence for this. Some portions Of the fruit
surface scald while adjoining portions do not scald. Yet, all cells would be expected
to produce Ot-famesene and MHO. Fruits lose susceptibility to scald as they mature

(Couey and Williams, 1973;Hammett, 1976) but they don’t lose ability to produce

55

OI-famesene and MHO. It is conceivable that the levels of natural antioxidants may
mitigate scald development (Anet, 1972,1974; Meir and Bramlage, 1988; Gallerani
et al., 1990). The levels Of the natural antioxidant ascorbic acid increase as apple
fruits mature whereas water-soluble antioxidants generally decrease during storage
(Barden and Bramlage, 1994a, 1994b); ascorbic acid and other natural antioxidants
may be protected from degradation when fruits are stored hypobarically. The red
portion of the red apple cvs. has higher levels Of ascorbic acid than the green
portions. and it is the green portion Of the fruit surface that is prone to scald
(Chapter V, Table 3). Moreover, this correlation of the disorder with the pigment
bearing cells Of the hypodermis implicates pathways of pigment biosynthesis. It is
apparent that susceptibility Of fruits to the scald disorder is developmentally
regulated by some aspect Of ethylene action since inhibition Of ethylene synthesis
or action can control scald (Personal communication with Meiseng Tian,
HortResearch Institute, New Zealand) but it is unlikely that this affects OI-famesene

or MHO production.

LITERATURE CITED

Anet, E.F.L.J. 1972. Superficial scald, a functional disorder Of stored apples: Vlll.
Volatile products from the autoxidation Of OI-farnesene. J. Sci. Food Agr. 23:
605-608.

Anet, E.F.L.J. 1974. Superficial scald, a functional disorder Of stored apples: Xl.
Apple antioxidants. J. Sci. FOOd Agric. 25: 299-304.

Anet, E.F.L.J. and Coggiola, l.M. 1974. Superficial scald, a functional disorder of
stored apples: X. Control Of OI-farnesene autoxidation. J. Sci. Food Agr. 25:
293-298.

56

Barden, CL. and Bramlage, W.J. 1994a. Relationships Of antioxidants in apple
peel tO Changes in OI-famesene and conjugated trienes during storage, and to
superficial scald development after storage. Postharvest Biol. Technology 4: 23-
33.

Barden, CL. and Bramlage, W.J. 1994b. Accumulation Of antioxidants in apple
peel as related to preharvest factors and superficial scald susceptibility Of the
fruit. J. Amer. SOC. Hort. Sci. 119: 264-269.

Burg, SP. and Burg, EA. 1966. Fruit storage at subatmospheric pressures.
Science 153: 314-315.

Couey, HM. and Williams, MW. 1973. Preharvest application Of ethephon on
scald and quality Of stored 'Delicious' apples. HortSCience 8: 56-57.

Dilley, DR. 1972. Hypobaric storage - a new concept for preservation Of
perishables. 102 Ann. Rpt., State Hort. Soc. Mich. 1972:82-89.

Dilley, DR. 1977. The hypobaric concept for controlled atmosphere storage. In:
D.H.Dewey (ed.) Horticultural Report. Proc. 2"d lnt’l Nat. CA Res. Conf., pp 29-
37.

Dilley, DR. 1982. Principles and effects Of hypobaric storage Of fruits and
vegetables. Am. SOC. Heating, Refrigeration and Air Conditioning Engineering
Transactions, 88: 1461-1478.

Dilley, D.R., Carpenter, W.J. and Burg, SP. 1975. Principles and application Of
hypobaric storage Of cut flowers. Acta Hort. 41: 249-268.

Dilley, DR. and Dewey, DH. 1963. Dip treatment Of apples in bulk boxes with
diphenylamine for control Of storage scald. Mich. Agr. Expt. Sta. Quart. Bul. 46:
73-79.

Dilley, DR. and Dewey, DH. 1973. Hypobaric storage Of apples. HortSCience 8:
273.

Gallerani, G., Pratella, GO. and Budini, R. A. 1990. The distribution and role Of
natural antioxidant substances in apple fruit affected by superficial scald . Adv.
Hort. Sci. 4: 144-146.

Ghahramani, F. and Scott, K.J. 1998a. The action of ethanol in controlling
superficial scald Of apples. Aust. J. Agric. Res. 49: 199-205.

Jamieson, W. 1980. Use of hypobaric conditions for refrigerated storage Of meats,
fruits, and vegetables. Food Technology. 1980: 64-71.

57

Meigh, OF. 1970. Apple scald. In: A.C. Hulme (Ed.), The Biochemistry Of Fruits
and Their Products. Vol. 1. Academic Press, London, pp. 555-569.

Meir, S. and Bramlage, W.J. 1988. Antioxidant activity in 'Cortland' apple peel and
susceptibility tO superficial scald after storage. J. Amer. Soc. Hort. Sci. 113:
412-418.

Salunkhe, OK. and Wu, MT. 1973. Effects of subatmospheric pressure storage on
ripening and associated chemical Changes Of certaindeciduous fruits. J. Amer.
SOC. Hort. Sci. 98: 113-116.

Salunkhe, OK. and Wu, MT. 1975. Subatmospheric storage Of fruits and
vegetables. In N.F.Haard and BK. Salunkhe (ed.) Postharvest Biology and
Handling Of Fruits and Vegetables. AVl.

Shutak, V. and Christopher, E.P. 1960. Role Of the cuticle in development Of
storage scald on Corland apples. Pro. Amer. SOC. Hort. Sci. 76: 106-111.

Song, J. and Beaudry, RM. 1996. Rethinking apple scald: new hypothesis on the
causal reason for development Of scald in apples. HortSCience 31: 605.

Spalding, DH. and Reeder, W.F. 1976. Low pressure (hypobaric) storage of limes.
J. amer. SOC. Hort. Sci. 10:367-370.

Spalding, OH. 1977. Current recommendations of atmospheres for transport and
storage Of tropical fruits. In: D.H.Dewey (ed.) Horticultural Report. Proc. 2”° lnt’l
Nat. CA Res. Conf., pp 242-249.

Tolle, W.E. 1969. Hypobaric storage of fresh produce. Year BOOk. United Fruit
and Veg. ASSOC. Pp. 27, 28, 30, 33, 34, 36, 38, 43.

Watkins, C.B., Bramlage, W.J and Cregoe, BA. 1995. Superficial scald Of granny
Smith apples is expressed as a typical chilling injury. J. Am. SOC. Hort. Sci. 120:
88-94.

Whitaker, B.D., Solomos, T. and Harrison, DJ 1997. Quantification Of or-

famesene and its conjugated trienol oxidation products from apple peel by C18-
HPLC with UV detection. J. Agric. FOOd Chem. 45: 760-765.

58

CHAPTER III
ETHANOL VAPOR CONTROLS SUPERFICIAL SCALD OF APPLES DURING

STORAGE

ABSTRACT
The Objective was to develop a new alternative strategy to control superficial scald

Of apple to avoid applying postharvest treatment with the scald inhibitor

diphenylamine (DPA).

Granny Smith, Law Rome, Red Delicious (scald susceptible) and ldared (not
susceptible) were harvested at the preclimacteric stage Of physiological
development. Fruits were treated at 1 °C with a) different concentrations of ethanol
vapor, b) different durations Of pretreatment with ethanol vapor, or C) without ethanol
treatment as control. Fruits were then stored in 3% 02 with 0% CO2 in flow-through
CA and in air at 1 °C. Scald was controlled when ethanol vapor was applied to Law
Rome and Red Delicious apples in the storage Chambers ventilated air with
enriched ethanol vapor and also in modified atmosphere packages by initially
adding ethanol. In CA at 3% 02 with 0% 002, fruits treated with 6000 IIL-L'1 Of
ethanol vapor for one week Showed Significantly less superficial scald. The
effectiveness Of ethanol vapor treatments decreased when the concentration or
exposure time to the ethanol vapor was decreased. All cultivars showed similar
responses to treatments. Even though apple fruits scalded when stored in air and

scald developed upon warming on the fruits from CA storage, the effects of ethanol

59

vapor pretreatments on reducing scald were evident in all treatments. The
treatments with 6000 pL-L'1 ethanol vapor were more effective for scald control than

the other treatments.

The ethanol vapor treatment diminished the level of several low molecular weight
volatiles produced by the fruit. The rate and pattern Of OI-famesene production were
Similar for fruits treated or not treated with ethanol vapor. The rate of MHO
production was much higher in control than for the ethanol vapor treatments. This
indicates that the OI-farnesene is not as Closely related to scald development as its

oxidation product MHO.

INTRODUCTION
Superficial scald is a pervasive physiological disorder Of apples and pears that
results in the browning Of the skin during or after low temperature storage in air or
after controlled atmosphere storage which markedly extends the useful life Span Of
these fruits. The affected cells Of the hypodermis die and dehydrate (Bain, 1956).
Most cvs. Of apples and pears are susceptible to scald. The disorder destroys the
appearance and therefore the fresh market value Of the fmits. The physiology and
control Of scald has been periodically reviewed (Smock 1961; Meigh, 1970; lngle
and D'Souza, 1989; Emongor et al., 1994). The disorder is prevented commercially
by postharvest drench treatment with diphenylamine (DPA). Numerous countries
have banned the use of DPA or importation Of DPA-treated fruits which mandates

development Of alternative methods to control scald. Fruits may possess at harvest

6O

or may acquire after harvest resistance to scald (lngle and D’Souza, 1989). The
skin browning symptoms of the disorder result from autooxidative processes in cell
membranes brought about by low temperature storage beyond the ability Of
endogenous antioxidants such as ascorbic acid or OI-tOCOpherOI to prevent the
dysfunction (Gallerani et al., 1990). Free-radicals such as hydroxyl (-OH),
hydroperoxyl (H02) and superoxide (02') may attack and destroy lipid and protein
macro-molecular structures essential for cellular function (Stadtman, 1993). The
scald inhibitor chemicals DPA and ethoxyquin are known to be effective inhibitors
of lipid peroxidation SO this may be their mechanism of action in controlling scald
(Huellin and Coggiola, 19703). Scott et al.(1995) found that ethanol vapor
treatment controlled scald of Granny Smith apples and we have confirmed this
(Wang and Dilley, 1996). Ethanol is known tO be a free-radical scavenger (Halliwell
and Gutteridge, 1989) and this may be a mechanism Of action in scald control. Little
et al.(1982) reported that scald Of Granny Smith apples was largely controlled by an
initial low 02 stress followed by CA storage. Ghahramani and Scott (1998a) found
that the initial low 02 stress induces the fruits tO produce their own ethanol and this
may be related to the efficacy Of the exogenously applied ethanol vapor treatments
in reducing scald even when the fruits are subsequently stored in air. The
biochemical mechanism responsible for the beneficial effects on scald control by
low 02 stress and the ensuing metabolism of ethanol remain tO be elucidated.
Extensive analyses of the volatiles produced by apples receiving low 02 stress or
ethanol vaportreatments support the premise that metabolism Of OI-farnesene in the

pigment bearing cells is closely associated with development Of the scald disorder

61

(Dilley and Beaudry, 1998; Wang et al., 1997; Wang and Dilley 1996; 1997;

Ghahramani and Scott, 1998b).

We hypothesize that the scald disorder is a manifestation of chilling injury affecting
the pigment bearing cells Of the fruit hypodermis. Symptoms of the disorder are
consequences Of autooxidative processes in cell membranes brought about by low
temperature storage beyond the ability Of naturally occurring antioxidants tO prevent
the dysfunction. We investigated initial ethanol vapor treatments to determine

effective treatment regimens and to gain insight into the mechanism Of scald control.

MATERIALS AND METHODS
Preliminary studies 1995:
Fruits: The apples used for these experiments came from the MSU CHES. They
were harvested at the preclimacteric stage of maturity approximately one week from
the onset Of the endogenous ethylene climactacteric. Fruits free Of defects were

randomized into treatment replicates comprised Of 40 to 60 fruits each.

Ethanol vapor treatments: Ethanol vapors were applied to Law Rome apples in 20-
L storage chambers at 3°C by Circulating air at a flow rate Of 80 mL min'1 through
aqueous solutions Of ethanol in flasks in series with the chambers. The initial
ethanol (ETOH) concentrations were 0, 1, 2, 4 and 8% (v/v). Law Rome and Red
Delicious apples were also treated at static ethanol vapor concentrations developed

by equilibration with 0, 5, 10, 20 and 40% (v/v) in the chambers. Law Rome apples

62

in modified atmosphere packages (MAP) with 2 mil low density polyethylene (LDPE)
were also treated by evaporating 0.25 9 ethanol per fruit within the package as was
done by Scott et al.(1995). The oxygen and carbon dioxide levels in the MAP
equilibrated tO 5% and 8%, respectively. Fruits from all treatments were stored at

1°C.

The ethanol concentration in the storage chamber or MAP headspaces and in fruits
was analyzed during storage. Ethanol was measured by gas Chromatography
employing a FlD and column Of Porapakt GTT 80/100. Volatile compounds
including OI-farnesene and 6-methyl-5-heptene-2-one were measured by SPME
combined with gas Chromatography/time Of flight mass spectrometer
(SPME/GC/T OF MS) as described by Song et al. (1997). Fruits were examined for
scald after storage and the data is expressed as a scald index based on the
percentage Of fmit surface area affected where no scald = 0, <25%=1, 25-50%=2

and >50%=3.

An experiment was set up to test the response for different cultivars Of apples to
produce ethanol after exposed to low levels Of 02. 02 concentrations Of 0.25, 0.5,
1.0, 2.0, and 21%, were employed in equal volume MAP with 5 mil LDPE at 1°C by
mixing N2 produced with a Permea air separator with air and distributing the gas
mixtures to the packages at an equal flow rate. Four cultivars, Granny Smith, Law
Rome, Red Delicious and ldared, five fruits each, were contained in each MAP for

2 days then the internal ethanol was measured on GC equipped with Porapak GTT

63

80/100 column and F ID detector.

1996 studies:

Granny Smith, Law Rome, Red Delicious and ldared fruits were harvested from
trees at the MSU CHES at the preclimacteric stage Of physiological development.
The maturity stage at harvest was about one week before the onset Of the
endogenous ethylene climacteric. Fruits were treated with a) different
concentrations Of ethanol vapor, b) different durations Of pretreatment with ethanol
vapor, or c) without ethanol vapor pretreatment as control. Fruits were then stored
in 3% 02 with 0% CO2 (dry lime added) in flow-through CA (0.12 atmosphere
turnovers per hr) for 4 to 7 months and in air at 1°C. Scald data is expressed as a
scald index based on the percentage Of fruit surface area affected described above
and scald index was normalized to 100. Scald index was measured after 4 and 7
months Of CA storage and 5 months of air storage and after 7 days in air at 20°C.
Three replicates Of 40-60 fruits each for each cv. were used to measure the scald

index for each treatment at each storage period.

TO determine the effects on ripening and scald development and production Of or-
famesene and its oxidation product 6-methyl-5-heptene-2-one (MHO) fruits were
removed from CA and air storage, at appropriate intervals, and transferred to 200
in air in 4-L glass jars. Headspace analysis Of 1.2 kilograms Of fruits in a jar was
done after a one hr enclosure period. OI-Farnesene and MHO in the headspace

were sampled by SPME and analyzed by SPME combined with GC/TOF MS. or-

64

Famesene production rate is expressed as relative units min'1 mg'1fw at 108 m/z
while the production rate Of MHO-ketone was expressed as anleS or 100 pmoles

kg‘1 hI’1 based on external standard.

RESULTS
The ethanol concentration in the headspace Of the fruit storage Chambers
decreased with time in storage from the initial levels established as Shown in Figure
1. The decrease in ethanol concentration iS a consequence Of the decrease in
ethanol in the solution because the solution was purged with air in a flow-through
system. Additionally, some Of the ethanol would be expected to be metabolized by
the fruit. An experiment was conducted to test this at 0 and 20°C. In chambers
without fruit (Figure 2A), the headspace ethanol concentration remained at near
initial levels for at least 7 days at 0°C and for 4 days at 20°C after which the level
declined. The higher ethanol vapor concentration at 20°C compared tO 0°C is a
direct consequence of the vapor pressure/temperature relationship. In Chambers
with fruits at 0°C (Figure 2B), the ethanol vapor concentration decreased slowly to
about one/half its initial level in 7 days. At 20°C ETOH decreased dramatically
within 2 days and then declined Slowly tO a minimum at 4 days and then slowly
increased. This pattern suggests that a considerable amount Of the applied ETOH
was dissolved in and metabolized by the fruit. The slow increase in ETOH after day

4 at 20°C may be a consequence Of ethanol toxicity.

A similar experiment was conducted with the McIntosh cv (Figure 3). Fruits were

65

 

 

 

 

—A—— Control .
1200,, —e— 1% ethanol solution , .
f. —a— 1% ethanol solution (w/o) ‘
l ‘ + 2% ethanol solution I .
i j + 4% ethanol solution
‘, l + 8% ethanol solution __ J ‘
'= I
1 ,
v 900 I
C
0
2D
(U
I: I
c l
(D
Q l
C l
8 6001
a l
c
m l
5 I
”’ l
l
3004‘
I I
I l
0} L

 

o 10 20 3'0 40 50 so 3' i0
Days in storage

Figure 1. Changes in headspace ethanol vapor concentrations as a function Of
time. Ethanol vapors were applied to Law Rome apples in the experimental storage
chambers at 3°C by Circulating air through aqueous solutions Of ethanol at 0, 1, 2,
4 and 8% (v/v). Designation 1% w/o is for a chamber without fruit.

66

 

 

Ethanol concentration (% v/v)

 

0W- WWWWW WW W _W-_
2.0.- WWWWWIW W--WW
i l
3 ' 0 v____ B '
\Z 16 I —e— 20°C I l
3- ‘ ‘ —EI— 0° ‘ I
8 I . i" 7"” T‘“”” J I
g 1.2.]
E I ~
a) I ' I
g 0.8‘ I I ‘.
8 I - - . ~
3 l I
g 0.4i ,
l . O
0.0W _ - W W’WW -- -W W. W...-W___
1 2 3 4 5 6

Days after treatment

Figure 2. Changes in headspace ethanol vapor concentrations without (A) or with
(B) 20 fruits at 0 and 20°C. 59 Of pure ethanol was added to 10 g Of vermiculite in
the experimental storage Chamber.

67

enclosed in chambers to which ETOH ( 0.2 to 12.8 g ) was added. The ETOH
concentration in the headspace was measured from 6 to 72 hours. The headspace
ethanol concentration remained at near initial level within 72 hrs in no fruit chamber
while in the chamber with fruits the ETOH concentration declined very fast (Figure
3A). The headspace ethanol concentration gradually decreased for the treatments
with initial amounts of ethanol at 1.6 g or below in a 20-L chamber while headspace
ethanol concentration of others increased continuously up to 24h then started to
decline (Figure 3B). This indicates that it requires some time for the ethanol to
vaporize at the low temperature condition when provided up to 3.2 g of liquid per
chamber. The data in Figure 3 clearly shows that fruits readily metabolize ETOH
but as the concentration increased beyond 1.6 9 administered the fruits’ ability to
utilize it diminished. Again, ETOH toxicity may explain the decrease in the rate of

ETOH consumption.

Direct evidence that fruit metabolism can be altered as a consequence of
metabolizing exogenously applied ETOH was obtained. Law Rome and Red
Delicious apples were ventilated with 0 to 8,000 pL-L‘1 ETOH vapor continuously for
2 months at 1°C. As the ETOH administered increased, the internal ethanol level
increased and the internal ethylene level decreased (Figure 4) indicating a reduction
in the ethylene production rate. The correlation between internal ethanol and
ethylene is given in Figure 5. This pertubation in ethylene production can be
interpreted as a metabolic response to ETOH stress.

In addition, the ETOH concentration in the fruit increased in direct proportion to the

68

 

 

’ —e— 0.49 ethanol w/f—rtlig fl
Lj— 0.4g ethanolw/o fruits

  

 

Ethanol concentration
(ull')
8:»
l_

 

  

101W WWWWWWWW WWzWWWW:

10000a‘ I a e 7 e s _# z _ _._ EW_W .

l e ~ . B a

. x —v— No ethanol l W

9000 ‘ ' + 0.29 ethanol 1 P-

1 l —69— 0.4g ethanol i r

j —03— 0.89 ethanol '

8000” + 1.69 ethanol ! l

'7" i + 3.29 ethanol l i

i 7000* J l + 6.49 ethanol ; .,

V l _ :1— 1§9Welhfl9t

8 6000; *
2: -l
£3 i

c 50004! g

G) L,
o l,
8 4000
0 ‘l

B .7 _

C 3000 _. /\ :

m j ‘ \ L

s ,4 T

“J 2000—9 V\' ‘ l

 

 

 

1 l
T’—"' “ T l ‘ l r '

0 10 20 30 40 50 60 70 80
Hours after treatment

Figure 3. Changes in headspace ethanol vapor concentrations. Ethanol was added
to 10 g of vermiculite in the experimental storage chamber containing McIntosh
apples at 3°C.

69

200 E Red Delicious ~
We Law Rome 1

—l

 

800 1

400.;

Internal ethanol concentration (ull 1)

. y" 2:._ ,. ‘
60 . r i "- 7 ”m 7 - 7 A W 7 _ W» I --— 7+

Red Delicious 1
Law Rome f

40

20 9

Internal ethylene concentration (pll")

 

0 800 100 H 3750 i 8000 ,
Ethanol vapor in headspace during treatment (“ll")

Figure 4. lntemal ethanol vapor (A) and ethylene (B) concentrations in Law Rome
and Red Delicious apple fruits as a function of ethanol concentration ("/0 v/v) in the
vapor generator. Ethanol concentrations were maintained for 2 months by
replenishing weekly with ethanol as necessary.

70

 

._ ,— T— . __

, _.__;__ Law Rome (
50» l —.—‘+- Red Delicious ,

.h
0

Y = 51.23 - 0.08817X + 5.1426-005X2

..L- ,W

*1 #1—
_ l

A

L.

Internal ethylene concentration (ul|")
N 00
o o

|
Y = 43.46 - 0.09628X + 8.261e-005)€" (
.,

 

0 200 400 600 800 1000 _
Internal ethanol concentration (pl|")

Figure 5. The correlation between internal ethanol and ethylene from the fruits
exposed to the maintained ethanol concentrations for 2 months by replenishing
weekly with ethanol as necessary.

71

ETOH vapor concentration (Figure 4A). In contrast, the ethylene concentration
decreased as the internal ETOH level increased up to 200 pL-L'1 and 250 pL-L‘1 for
Law Rome and Red Delicious fruits, respectively. (Figure 4B). This indicates that
ETOH directly affected ethylene production but the mechanism is not known. It is
clear that apple fruits readily absorb ETOH vapors and can metabolize a

considerable amount of applied ETOH without damage to the tissue.

Treatment with ETOH vapor generated by 5 and 10% ETOH (v/v) decreased the
production of volatile compounds such as hexylacetate, hexylbutanoate/
butylhexanoate, hexyl-2-methylbutanoate and hexylhexanoate by Law Rome
(Figure 6 A2,A3) and Red Delicious (Figure 6 32,33) apples when applied
continuously for 2 months compared to ventilation with air only. Whereas treatment
with ETOH vapor generated by initial 2 and 4% ETOH (v/v) was less effective
(Figure 6 02,03). The level of or-famesene was not markedly affected by ETOH
treatment. These results indicate that ETOH vaportreatment still significantly alters

fruit metabolism even though not affecting or-famesene production.

Apple cvs. vary in their response to low 02 levels. This is evident by ethanol
accumulation in the fruits as a function of 02 level (Figure 7). The internal ethanol
levels were higher for Red Delicious and ldared apples at 1% 02 level than for
Granny Smith and Law Rome apples. Whereas, when stored at 0.25% 02, Granny
Smith and Law Rome apples accumulated more ethanol than the others.

As a consequence of ETOH in the tissue or as a result of its metabolism or

72

I"
l

Bl 1

 

 

 

 

 

 

 

 

 

 

 

_.___.—.___

 

 

 

 

 

 

 

L. .l l.,.l..l

l . if c3 ‘1'
ll . . l

' l
ML,\LL_AM....J l... thJlWWJL #Ml.dlL—ln]\~kmzkj

 

 

 

 

 

 

 

 

 

 

 

K’

‘
«a...

 

 

 

Figure 6. Changes in relative amounts of some volatile compounds in apple fruits
treated with ethanol vapors which were generated by circulating airthrough various
concentrations (% v/v) of ethanol in water: A1, 0%; A2, 5% and A3, 10% were
maintained for 2 months on Law Rome apples; B1 , 0%; 82, 5% and B3, 10% were
maintained for 2 months on Red Delicious apples; C1, 0%; C2, 2% and C3, 4%
were added initially to Law Rome apples. All fruits were stored in air for 3.5 months
at 3°C. P1-P6 stand for the peaks of ethanol, hexylacetate, hexylbutanoate/
butylhexanoate, hexyl-2-methylbutanoate, hexylhexantoate, and or-famesene,
respectively.

73

 

 

 

 

__ —§— Granny Smith; I;
60 —§— Law Rome .
E— Red Delicious [
W§1daEdWW W-
a l
E
'
s A --
f3 40 i ‘ .
,3: .
8 l
0 l
C
o .l
0 l
5 l
C
m l
=5 201 L
LIJ l " .
0 .
1 l
w; “__T ,, ,_,,z._T ___ _, _ , , l ,‘ l
0.25 0.5 1.0 2.0 21

02 concentration (%)

Figure 7. Effect of oxygen levels on internal ethanol concentration for Granny
Smith, Law Rome, Red Delicious and ldared apples. Atmospheres were
equilibrated at the respective oxygen level for 2 days prior to sampling.

74

pertubation of normal tissue metabolism, the incidence and severity of scald is
reduced as shown in Tables 1 and 2. ETOH vapor treatment was found to diminish
or control scald of Law Rome apples stored in air (Tables 1, 2). As the ETOH vapor
concentration increased, the scald index for Law Rome apples stored in air was
reduced (Tables1 and 2). Fruits treated with ETOH vapor generated by an 8% (v/v)
ETOszater solution received an initial ETOH vapor level of about 1400 pL-L'1
ETOH and by 66 days the generator was delivering only 533 pL-L". These fruits
had the lowest amount of scald while those initially receiving 653 pL-L‘1 ETOH had
a higher scald index. Fruits treated with ETOH vapor at 800 and 1600 pL-L'1
continuously showed good scald control (Table 1). These data suggest that the

primary benefit of ETOH vapor treatment is derived early in the storage period.

MAP studies:

Ethanol vapor treatment in MAP also controlled scald (Table 3) when fruits received
an initial treatment with ETOH in MAP at about 9,000 pL-L'1 (Table 4). After 13 days
the ETOH concentration declined to about 200 pL-L‘1 (Table 4). During this time the
O2 and CO2 levels equilibrated to about 5 and 8%, respectively. The decrease in
the headspace ETOH level is a consequence of it becoming dissolved in the water
of the tissue and metabolized with adequate 02 being available. Scott et al., (1995)
found that ethanol vapor treatment was effective in controlling scald of Granny

Smith apples in MAP when ETOH was added initially.

75

Table 1. Effect of maintaining a constant ethanol vapor concentration for 2 months
on control of scald of Law Rome apples stored in air at 3°C for 3.5 months.

 

 

Ethanol conc. Ethanol vapor Scald indexz
(%(v/V)) 01”1 MW 0d +7d at 20°C
0 1.0 2.33
800 0 0.25
10 1600 0 0
20 3750 Slight ETOH Slight ETOH
Injury injury
40 8000 Severe ETOH Severe ETOH
Injury Injury

 

zScald index: 0=0%, 1=<25%, 2=25 to 50%, 3=>50% of surface scalded.

Table 2. Effect of initial ethanol vapor treatment on control of scald of Law Rome
apples stored in air for 3.5 months at 3°C.

 

 

Ethanol conc. Ethanol vapor Scald indexz
(%(V/V)) (IN I" (le))
0d 31d 66d 0d +7d at 20°C
0 0 12 0 2.63 2.93
1 1 70 89 53 2.67 2.83
2 339 169 1 10 2.40 2.79
4 653 320 196 1 .67 2.24
8 1394 737 533 0.83 1 .53

 

zScald index: 0=0%, 1=<25%, 2=25 to 50%, 3=>50% of surface scalded.

76

Table 3. Effect of initial ethanol vapor treatment on control of scald of Law Rome
apples stored in MAP with 2 mil LDPE for 3 months at 1°C.

 

 

Time exposed to Ethanol added Scald indexz
ethanol (g/fru it)
(weeks) 0d +7d at 20°C
0 (control) 0 0.98 1.66
1 0.25 0.38 0.58
2 0.25 0.27 0.54
4 0.25 0.40 0.62

 

zScald index: 0=0%, 1=<25%, 2=25 to 50%, =>50% of surface scalded.

Table 4. Ethanol vapor concentration change in MAP with 2 mil LDPE at 1°C.
Ethanol at 0.25 g per fruit was added initially.

 

Time Ethanol 02 CO2
(days) (pl l"1SD) (%1SD) (%1SD)
1 90681462 16.910.45 1 810.22
3 59271388 11211.8 4.110.55
5 32921311 66119 6.110.48
7 15381179 3.4125 7.710.66
9 645171 2813.3 8.311.03
1 1 346154 4912.6 8.210.88
13 220123 5.2120 8.410.58

 

77

1996 studies:

Our experiments in 1996-97 confirmed our earlier studies that ethanol vapor
treatment at the beginning of the storage period can prevent or largely diminish
superficial scald of apples as first reported by K. Scott et al. (1995) in Australia. We
observed that ethanol vapor treatment induced apples to accumulate ethanol or to
produce ethanol aerobically. Since apples produce ethanol when the 02 level is
below the extinction point of alcohol fermentation (ca. 1%) suggests that a storage
regimen of initial low 02 stress sufficient to induce the fruits to produce ethanol may

be employed to control scald.

We conducted extensive studies in 1996-97 to ascertain the effects of ethanol vapor
treatments on control of scald of Granny Smith, Red Delicious, Law Rome and

ldared apples. Fruits were stored in CA subsequent to receiving treatments.

Ethanol vapor treatment reduced scald incidence for all scald susceptible cvs.
(Granny Smith, Red Delicious, Law Rome) examined in a dose dependent manner
(Figure 8). The most effective treatment was one week exposure to 6000 pL-L‘1
ethanol vapor. Reducing the ethanol concentration and duration of exposure at a
given concentration reduced the effectiveness for scald control. Continuous
exposure of fruits to ethanol vapor at 800 to 1600 pL-L'1 for 8 weeks at 3% 02 with
subsequent storage at 3% O2 reduced but did not prevent scald. Likewise, exposure
to 2700 |JL°L'1 ethanol vapor for up to two weeks gave only marginal control of

scald. This suggests that it is the initial period in storage that ethanol vapor is

78

1001_A ng‘h—L 1 J l l

rwéiiéanny Smithfi
Law Rome |

804 1 i Red Delicious

 

 

Scald index

 

 

CA-contro
0 8 mill EtOH
8 wks

Treatments

Figure 8. Effect of ethanol vapor on controlling apple scald for Granny Smith, Law
Rome and Red Delicious after 4 months of CA storage (3% 02 + 0% 002) at 1°C
plus 7 days at 20°C in air. Scald index (0=none; 1=slight; 2=moderate; 3=severe)
was normalized to 100.

79

 

  

    

 

     

100 niAv 4 W 4_4__J J,,__W_ J
1 :{Qa‘nwsmm 1
807. 8818 Law Rome l ‘»
x 'l ‘ i Red Delicious l ‘
w l 7747117J,,W_.
1:: 60»« %
.E l t
.5 1
'5 4o. 1
0
en
20- L
. {A
0+* 74
14o-___1v,,._J ,1 _; g ,,
7 ‘ ‘1‘“ Granny Smith 1 1
1209 T Law Rome ‘ r
.100? ~‘ 1 iW wavy: t
s l 1 ** »
'5 5
E 604 l”
o l
f” 40 P
201 1
0 ‘ — — _ lW
:: :r: :r:
I O 0 Or» 0
o a “‘0 o-I on o-I
S no E” E E E“
E o. h .._ ,9
Q P d N
O
Treatments

Figure 9. Effect of ethanol vapor on controlling apple scald for Granny Smith, Law
Rome and Red Delicious after 7 months of CA storage (3% 02 + 0% C02) at 1°C
after storage (A) and plus 7 days at 20°C in air (B). Scald index (0=none; 1=slight;
2=moderate; 3=severe) was normalized to 100.

80

Scald index

 

l
L 1
x L L
_80 l
E5
'25
(U
o
(D l
_ "_r I I I I I I I
.2 519M319... 8.8: 31313:."
5 08 :E‘ :3, g; S‘i’ 55 S? s.- 53
0 2 E" g E” 500 E E" E EN
“2 .9 '9. ‘9. N. "- ‘9 ‘9
0 °- 1- ‘- N N
Treatments

Figure 10. Effect of ethanol vapor or diphenylamine (DPA) on controlling scald of
Granny Smith apples stored in air for 5 months at 1°C after storage (A) and plus 7
days at 20°C (B). Scald index (0=none; 1=slight; 2=moderate; 3=severe) was
normalized to 100.

81

    

 

1 —aa— 800 pll" EtOH 56d

a-Farnesene
(Units mg"min" at 108 mIz)

1 I”..-
1 1 —8— Control
1
l
|

 

 

L —B— 2700 ull'TfiEtOl-lif L
L —e— 2700 ull" EtOH 14d
1 + 6000 ull’1 EtOH 7d 1 1

    
 

a-Farnesene
(Units mg“min" at 108 mIz)

 

60 90 110 150 150
Days in storage

Figure 11. Effect of ethanol vapor on the production rates of or—farnesene for
Granny Smith apples during CA storage (3% 02 + 0% C02) at 1°C.

82

100T: ".fi_’w—:'_’_‘_f f f A ” 1 " ” ” A I ' 1 *’ —' I -

- , ée— Contro

1 1 —e— 800 pll‘1 EtOH 56d 1
1 1 -O- 1600 ull" EtOH 56d 1
1 _ , , ,_ W.

L .77'___i it

80

 

60$

MHO (pmoles kg"hr")
‘5

20-1

 

04- " _T" '7’ ~ T l ” ’ ’ ’_1 T ' — —1 —" "_ T"__ ”T" A ‘
100‘”: W ‘;—W:—_ _ f. *"'L‘ ”* AL I f — v
1' 1 —e— 2700011" EtOH 7d '
—®— 2700 pll" EtOH 14d '
80 4* '1—0— 6000 pll" EtOH 7d
60 - ,_

MHO (pmoles kg"hr")
‘5

 

 

60 90 fl ~1.20 f 150 1 80
Days in storage

Figure 12. Effect of ethanol vapor on the production rates of 6-methyl-5-heptene-2-
one for Granny Smith apples during CA storage (3% 02 + 0% COZ) at 1°C.

83

effective. The high initial ethanol vapor treatment of Red Delicious was quite

effective in controlling scald.

Whereas good control of scald was achieved during the first 4 months in CA by
ethanol vapor treatments, these treatments were not effective for longer CA storage
periods (Figure 9A, QB). Moreover, DPA treatment at 1000 mg-L’1 was also
ineffective for scald control for Granny Smith apples in this season (Figure 10A,
108). The fruits for these experiments were harvested quite immature to provide

high potential to scald.

The rate and pattern of or-famesene production were similar for fruits treated or not
treated with ETOH vapor (Figure 1 1A, 118). The rate of MHO production was much
higher in control than for the ethanol vapor treatments (Figure 12A, 123). This
indicates that the or-famesene is not as closely related to scald development as its

oxidation product MHO.

DISCUSSION
DPA and a—famesene metabolism:
After 5 months of air storage, DPA was found to promote or-famesene production
but inhibit production of the MHO ketone and this was associated with low incidence
of scald of Granny Smith apples (Figure 10; Chapter IV, Figure 5). In general,
treatments which resulted in low MHO production following storage were those

giving the best control of scald. The potential for scald to develop following many

84

months of storage is largely determined during the first 2 months of storage
(Meigh,1970; Shutak and Christopher, 1960; Watkins et al., 1995). If the
mechanism of DPA action is to inhibit the oxidation of or-famesene to the MHO
ketone, this must be demonstrated during the first few months of storage.
Production rate of MHO was lowerfor DPA-treated fruits than other treatments while
production rate of or-famesene was higher when measured after 5.5 months of
storage at 20°C (Chapter IV, Figure 5A,SB). Therefore, we conclude that the
mechanism of DPA in reducing scald may be related to inhibiting the oxidation of
car-famesene. Moreover, the conversion of or-famesene to the MHO appears to be
correlated to scald development but the role of MHO as a factor in causing scald
may be indirect; since a dramatic increase occurs in MHO production upon warming

the fruit of all cvs. including those which do not develop scald (Chapter I).

Ethanol vapor controls scald:

We have determined that ethanol supplied to fruits as a vapor is readily absorbed
and metabolized by the tissue; moreover, one of the consequences is reduced rate
of ethylene production (Wang and Dilley,1996). In addition, the production of low
molecular weight volatile compounds including hexylacetate, hexylbutanoate/
butylhexanoate, hexyl-2-methylbutanoate and hexylhexanoate is reduced by
ethanol vapor treatment (Figure 6). These data suggest that ethanol reduce the

effects of ethylene or ameliorate other metabolism to control scald.

Ethanol vapors controlled apple scald when applied to Law Rome and Red

85

Delicious apples in the storage chambers by ventilating air through aqueous
solutions of ethanol at different concentrations and in modified atmosphere package
by adding various initial concentrations of ethanol vapor. This confirms the
observation of Scott et al. (1995). Fruits in storage chambers treated with ethanol
vapor at 1,600 jJL-L'1 for about 2 months showed no scald when stored for an
additional period in air storage whereas the scald index for control fruits was up to
2.33 (Table 1). The similar results in the modified atmosphere experiments
confirmed that ethanol vapor could prevent apple scald (Scott et al., 1995). But,
when the concentrations of ethanol were up to 3750 or 8000 er-L'1 and were
applied to fruits continuously for 2 months, the fruits were injured as evidenced by
d arkening of the skin color and the fruits showed some fermentation damage (Table
1 ). Ethanol vapor treatments were not correlated with a reduction of or-famesene
production by the fruits. or-Famesene is an isoprenoid metabolite in the pathway
to carotenoid synthesis that has been implicated indirectly as a factor in scald
development (Meigh, 1970). Evidence for this is based on DPA reducing the level
of a conjugated terpene product of or-famesene oxidation (Anet and Coggiola,
1 974). Our results suggest that the control of scald by ethanol vapor treatment may
be related to a reduction of or-famesene conversion to its oxidation product MHO.
Ethanol vapor treatment resulted in accumulation of ethanol in the fruits in direct
proportion to the ethanol vapor concentration administered and reduced the rate of
ethylene production. The internal ethanol levels dropped rapidly when fruits were
returned to air without ethanol vapor except the fruits with high ethanol vapor 3750

or 8000 uL-L‘1 for 2 months continuously (Table 1). Those fruits were irreversibly

86

injured by the alcohol treatments and remained with off-flavors when returned to air.

Ethanol vapor treatment reduced apple scald, exposure to 6000 |JL'L'1 ethanol
vapor for one or two weeks was the most effective. The effectiveness of ethanol
vapor treatments diminished after long term CA storage at 3% 02 (Figure 9A,9B).
Perhaps better effectiveness of controlling scald may have been achieved if the CA

treatment had been at 1.2 to 1.5% 02 with 3% COZ.

Fruits did not show off-flavor after exposing to 6000 pL-L" ethanol vapor for two
weeks before storage but fruits did have an off-flavor after exposing to 3750 11L-L'1
or higher levels of ethanol vapor for 2 months. Newly harvested apples may have
a higher resistance to ethanol while after some time of storage the resistance may
decreased. Prolonged exposure to ethanol vapor concentrations at 3700 pL-L -1

apparently causes irreversible damage to cellular metabolism.

Low oxygen induces ethanol production:

Fruits held under ultra low levels of 02 resulted in ethanol production (Figure 7).
The internal ethanol levels were different since apple cvs. vary in their response to
low 02 levels. Ethanol accumulation at 0.5% 02 was the highest for Granny Smith
> Red Delicious > Law Rome > ldared; at 0.25% 02, Granny Smith and Law Rome
apples accumulated more ethanol than others. This suggest that apples may
become resistant to scald through producing ethanol. To achieve the maximum

resistance to scald each cv. of apples may require a specific and low 02 level for a

87

specific period of time. This remains to be elucidated.

Subjecting apple fruits to low 02 levels which induce ethanol production for a period
of two weeks at the beginning of the storage period was found to reduce the
incidence of scald on fruits subsequently stored at higher 02 levels. This suggests
that some aspect of ethanol metabolism may reduce scald. This may involve its
action as a free-radical scavenger, esterification of some intermediate, increasing
membrane fluidity or other means. The mechanism of ethanol action in reducing

scald needs further study.

LITERATURE CITED

Anet, E.F.L.J. and Coggiola, l.M. 1974. Superficial scald, a functional disorder of
stored apples: X. Control of or-farnesene autoxidation. J. Sci. Food Agr. 25:
293-298.

Bain, J.M. 1956. A histological study of the development of superficial scald in
‘Granny Smith’ apples. J. Hort. Sci. 31: 234-238.

Dilley, DR. and Beaudry, RM. 1998. NE-103 Postharvest Physiology of Fruits.
Annual Report Michigan Agric. Expt. Stat., Michigan State Univ., East Lansing,
MI.

Emongor, V.E., Murr, DP. and Lougheed, EC. 1994. Preharvest factors that
predispose apples to superficial scald. Postharvest Biol. and Technol. 4: 289-
300.

Gallerani, G., Pratella, CC. and Budini, R. A. 1990. The distribution and role of
natural antioxidant substances in apple fruit affected by superficial scald . Adv.
Hort. Sci. 4: 144-146.

Ghahramani, F. and Scott, K.J. 1998a. The action of ethanol in controlling
superficial scald of apples. Aust. J. Agric. Res. 49: 199-205.

Ghahramani, F. and Scott, K.J. 1998b. Oxygen stress of Granny Smith apples in

88

relation to superficial scald, ethanol, car-famesene and conjugated trienes. Aust.
J. Agric. Res. 49: 207-210.

Halliwell, B. and Gutteridge,l.C. 1989. Free radicals in biology and medicine.
Clarendon Press, Oxford. pp543.

Huelin, FE. and Coggiola, l.M. 1970a. Superficial scald, a functional disorder of
stored apples: V. Oxidation of or-famesene and its inhibition by diphenylamine.
J. Sci. Food. Agr. 21: 44-48.

lngle, M. and D’Souza, M. 1989. Physiology and control of superficial scald of
apples: A review. HortScience 24: 28-31.

Little, C.R., Faragher, JD. and Taylor, HS 1982. Effects of initial low oxygen
stress treatments in low oxygen modified atmosphere storage of Granny Smith
apples. J. Amer. Soc. Hort. Sci. 107: 320-323.

Meigh, DP 1970. Apple scald. In: A.C. Hulme (Ed.), The Biochemistry of Fruits
and Their Products. Vol. 1. Academic Press, London, pp. 555—569.

Scott, K.J., Yuen, C.M.C. and Ghahramani, F. 1995. Ethanol vapor— a new anti-
scald treatment for apples. Postharvest Biol. Technol. 6: 201-208.

Shutak, V. and Christopher, E.P. 1960. Role of the cuticle in development of
storage scald on Corland apples. Pro. Amer. Soc. Hort. Sci. 76: 106-111.

Smock, RM. 1961. Methods of scald control on the apple. Cornell Univ. Agric.
Exp. Stn., Bull. No. 970, pp. 55.

Song, J., Gardner, B.D., Holland, JP. and Beaudry, RM. 1997. Rapid analysis of
volatile flavor compounds in apple fruit using SPME and GC/Time-of-Flight Mass
Spectrometry. J. Agri. Food Chem. 45: 1801-1807.

Stadtman, ER. 1993*. Oxidation of free amino acids and amino acid residues in
proteins by radiolysis and by metal-catalyzed reactions. Annu. Rev. Biochem.
62: 797-821.

Wang, Z. and Dilley, DR. 1996. Ethanol vapor treatment: A new alternative
approach to control superficial scald of apples. Proc. Mich. State Hort. Soc. 126:
35-40.

Wang, Z. and Dilley, DR. 1997. The relationship of or-farnesene production and
its oxidation product 6-methyl-5-heptene-2-one to superficial scald of Granny
Smith, Law Rome, Red Delicious and ldared apples during controlled
atmosphere and air storage. In: E.J. Mitchman (ed.) Apples and Pears. Proc.

89

7th Int’l Nat. CA Res. Conf., 2: 98-104.

Wang, Z., McCully, T.J. and Dilley, D.R. 1997a. The effect of ultra low oxygen
storage, initial oxygen stress and ethanol vapor treatments on controlling
superficial scald of Granny Smith, Law Rome, Red Delicious and ldared apples.
In: E.J. Mitchman (ed.) Apples and Pears. Proc. 7th lnt’l Nat. CA Res. Conf., 2:
105-1 1 1.

Watkins, C.B., Bramlage, W.J and Cregoe, BA. 1995. Superficial scald of granny

Smith apples is expressed as a typical chilling injury. J. Am. Soc. Hort. Sci. 120:
88-94.

90

CHAPTER IV
INITIAL LOW OXYGEN STRESS CONTROLS SUPERFICIAL SCALD OF

APPLES

ABSTRACT
Alternative strategies to control apple scald to avoid the postharvest application of
scald inhibitors and fungicides were investigated. Granny Smith, Law Rome, Red
Delicious (scald susceptible) and ldared (not susceptible) were harvested at the
preclimacteric stage of physiological development. Fruits were treated at 1 °C with
a) initial low 02 stress (ILOS) at different levels of low 02 and various durations of
pretreatment, b) different CA storage conditions, c) ultra low 02 storage, d) without
any pretreatment as control. Fruits were then stored in 3% 02 with 0% C02, 0.7-
0.8% 02 with 3% COzand 1.5% 02 with 3% CO2 in flow-through CA and in air at 0.5-
1°C. Superficial scald was markedly reduced by initial low 02 conditioning at 0.5%
02 for up to two weeks followed by CA storage and with 0.8% 02 and 3% 002. With
CA storage at 3% 02 with 0% C02, 0.5% and 0.25% initial low 02 pretreatment for
2 weeks resulted in excellent control of scald, especially at 0.25% 02 for 2 weeks.
All cultivars showed similar responses to treatments. Even though apple fruits
scalded when stored in air and scald developed quickly upon warming after storage,
the effects on reducing scald by initial low 02 stress pretreatment were evident in
all treatments. The treatment with 0.25% 02 for two weeks or when this treatment
was followed with an additional two weeks of low 02 stress after 2 months of

storage, were more effective treatment regimens for scald control than the other

91

treatments. The production of or-farnesene and MHO was inhibited by initial low 02
stress treatments and 1.5% 02 CA condition. The 0.25% 02 initial stress treatment
caused stronger inhibition on or-Famesene and MHO production than 0.5% 02 initial
stress treament. This suggests that the accumulation of MHO is highly related to

scald development of apples.

A commercial test of initial low 02 stress confirmed the efficacy of ILOS for

controlling scald.

INTRODUCTION
Superficial scald of apples is a devastating physiological disorder which has the
potential to destroy the market value and utility of millions of tons of fresh apples
annually during long-term storage. The physiology and control of scald has been
periodically reviewed (Smock 1961; Meigh, 1970; Ingle and D’Souza, 1989;
Emongor et al., 1994). The disorder is prevented commercially by postharvest
drench treatment with diphenylamine (DPA) (Smock, 1957). The drench treatment
must include a fungicide to prevent decay development. Numerous countries have
banned the use of DPA or importation of DPA-treated fruits which mandates
development of alternative methods to control scald (Ke and Kadar, 1992).
Researchers (Barden and Bramlage, 1994a, 1994b; Gallerani et al., 1990)
proposed that fruits may possess at harvest or may acquire after harvest resistance
to scald; it is speculated that the skin browning symptoms of the disorder result

from autooxidative processes affecting cell membranes brought about by low

92

temperature storage beyond the ability of endogenous antioxidants such as
ascorbic acid or or-tocopherol to prevent the dysfunction. Free-radicals such as
hydroxyl (OH'), hydroperoxyl (H02) and superoxide (02') may attack and destroy
lipid and protein macro-molecular structures essential for cellular function (Anet,
1969; Halliwell and Gutteridge, 1989; Stadtman, 1993). Scald development in
Granny Smith, a cv. very susceptible to scald, was correlated to the oxidation of or-
famesene to conjugated triene hydroperoxides (Huelin and Coggiola, 1970a; Filmer
and Meigh, 1971). Extensive investigations in Australia (Huelin and Murray, 1966;
Huelin and Coggiola, 1968, 1970a, 1970b; Anet, 1972a, 1972b; Anet and Coggiola,
1974) suggest that scald results from the autooxidation of a volatile sesquiterpene
hydrocarbon or-famesene (2,6,1 0-trimethyl-2,6,9,1 1-dodecatetraene) in the fruit skin
( Meigh and Filmer,1969). The oxidation of or-famesene is hypothesized to yield
conjugated triene hydroperoxide free-radicals which injure the cells and give rise to
the symptoms of scald (Rowan et al.,1995). Song and Beaudry (1996) found that
MHO applied to apple fruits caused a scald-like disorder and this is a product of or-
famesene oxidation. Scald can be induced by the products of or-farnesene
oxidation, and the concentration and time of appearance of these products affects
the severity of the disorder (Anet, 1972b). The scald inhibitor chemicals DPA and
ethoxyquin are known to be effective inhibitors of lipid peroxidation so this may be
the mechanism of action in controlling scald. DPA is also an inhibitor of electron
transport in plant mitochondria (Baker, 1963). Scott et al.(1995) found that ethanol
vapor treatment controlled scald of Granny Smith apples and we have confirmed

this (Wang and Dilley, 1996, 1997; Wang et al., 1997) as has Ghahramani and

93

Scott (1998a). Ethanol is known to be a free-radical scavenger (Halliwell and
Gutteridge, 1989) and this may be a mechanism of action in scald control. Little et
al.(1982) reported that scald of Granny Smith apples was largely controlled by an
initial low 02 stress followed by CA storage. We hypothesized that initial low 02
stress prior to CA storage controls scald by affecting the metabolism including
ethanol, or-famesene and its oxidation product MHO. Initial low 02 stress
treatments were investigated to determine effective treatment regimens and to gain

insight into the mechanism of scald control.

MATERIALS AND METHODS

Preliminary investigations (1996):

Granny Smith, Law Rome, Red Delicious and ldared fruits were harvested from
trees at the MSU CHES at the preclimacteric stage of physiological development.
The maturity stage at harvest was about one week before the onset of the
endogenous ethylene climacteric. Three replicates of 40-60 fruits each for each
storage period evaluation for each cv. were treated with initial low 02 stress at 0.5%
02 for one or two weeks. Nitrogen gas prepared with a Permea air separator
adjusted to provide 0.5% 02 was employed to provide the low 02 atmosphere which
was distributed through a capillary flow board system for maintaining controlled
atmospheres in a flow-through system. Fruits were then stored in 3% 02 with 0%
CO2 (dry lime added) and 0.8% 02 with 3% 002 in the flow-through CA (0.12
atmosphere turnovers per hr) and in air for up to 7 months at 1°C. To bring out the

effect of treatments, CA at 3% 02 was employed since it is only partially effective

94

in controlling scald. Some fruits were treated with DPA 1000 mg- L'1 and also
placed in air at 1°C. Scald data is expressed as a scald index based on the
percentage of fruit surface area affected where no scald = 0, <25%=1, 25-50%=2
and >50%=3 and scald index was normalized to 100 by mutiplying 33.33. Scald
index was measured after 4 and 7 months of CA storage and 5 months of air

storage and after 7 days in air at 20°C.

To determine the effects on ripening and scald development and production of or-
famesene and its oxidation product 6-methyl-5-heptene-2-one (MHO). fruits were
removed from CA and air storage at appropriate intervals, and transferred to 20°C
in air in glass jars. Subsamples were used for fruit firmness measurements and
ripening and again after 7 days. The headspace in the jars was sampled by
SPME/GC/MS as described by Song et al. (1 997) to determine the car-famesene and
MHO production at daily or every other day intervals. Samples of epicuticular wax
were removed from the fruits at each time interval and placed in glass vials to

determine the amount of or-famesene and MHO partitioned in the epicuticular wax.

Initial low oxygen stress studies(1 99 7):

Preclimacteric Law Rome apples were obtained from the MSU CHES and Granny
Smith apples were obtained from a grower in Southwest Michigan. Maturity of both
cvs. was about two weeks prior to the onset of the endogenous ethylene climacteric
to assure high potential for scald development. Fruits were placed in 20-L CA

chambers employing about 40-60 fruits in each of three replicates for each storage

95

period evaluation. Two levels of initial low 02 stress were employed; 0.5 and
0.25% 02 for two weeks at 1°C. The control fruits did not receive low 02 stress.
The low 02 stress atmospheres were generated using a Permea air separator
adjusted to produce nitrogen which was blended with O2 in a gas mixing apparatus
and distributed to the chambers using a capillary flow board. After the low 02
stress, the CA chambers were stored in dynamic (slowly ventilated) CA at 3 or 1 .5%
02 at 1°C. A slow flow rate of 0.18-chamber atmospheres per hour was employed.
Some fruits so treated were again subjected to the same low 02 stress treatments
after two months in CA. Fruits were also stored in air and in CA without prior low

02 stress.

Examination of fruits for scald and quality parameters was made after 5 and 8
months of storage plus one week in air at 20°C. Scald data is expressed as a scald

index based on the percentage of fruit surface area affected as mentioned above.

Analysis of volatiles: SPME GC Mass spectrometry was employed to identify and
quantify the volatiles including or-farnesene and 6—methyl-5-heptene-2-one (MHO).
Five to 8 fruits from each treatment were enclosed in 4-L glass jars at 20°C for 1.5
hrs. and the headspace was sampled by SPME and analyzed employing a GC/MS
according to the procedure of Song et al.(1997). At the same time intervals, a
sample of the epicuticular wax scraped from the fruit surface was placed in small
glass vial at 20°C and the headspace gas was sampled by SPME and analyzed by

GC/MS to determine the nature of the compounds that had partitioned into the

96

epicuticular wax or cuticle.

Continuous low 02 stress studies:

Granny Smith apples as used for the initial low 02 stress studies were employed.
Fruits were placed in 10-L glass desiccators and ventilated with 0.13, 0.25, 0.4, 0.5,
0.75, 1.0, 1.25 and 1.5% 02 or with air at 1°C. The low 02 atmospheres were made
by mixing nitrogen generated by a Permea air separator with air employing a gas
mixing apparatus and a capillary flow board. The ventilation rate of the gas mixture
through the chambers was 54 mL-min‘1 (approximately 0.6 void volume exchanges
per hr). At weekly intervals throughout the 8 month storage period, the volatiles
produced by the fruits while under the storage atmospheres were sampled by
SPME directly from the headspaces of the storage chambers and analyzed by
GC/MS. The volatile profile produced by intact fruits of more than 60 compounds
was obtained. The volatile profile of fruits after transfer to 20°C was obtained in a
similar manner. Fruits were examined for scald and other physiolgical disorders

after removal from storage and again after 7 days at 200.

Initial low 02 stress to control scald; a commercial test at the MSU CHES CA
Storage Facility.

Apples from a hail-damaged area of Sparta, Michigan were employed. Fruits came
from five different orchards plus CHES. Red Delicious strains were Red Chief,
Starkrimson, NuRed, Ace and Super Spur. Romes were Law Rome and Galia

Beauty. Winesaps were Stayman and Turley. All fruits were of preclimacteric

97

maturity stage and were harvested and loaded into the rooms between October 6
and 13. Each room contained 126 bins (about 18 bu capacity per bin) and these
were loaded with an identical stacking pattern. CA rooms were sealed on October
13 and brought to 2.3% 02 by October 18. Dry lime was employed to scrub carbon
dioxide. A Permea nitrogen generator was used for initial 02 pull-down and as
needed to maintain 02 levels. CA room #1 was maintained at 0.7% 02 with 0.5%
carbon dioxide throughout the storage period at 0.1 °C. CA room #2 was maintained
at 1 .5% 02 with 0.5% carbon dioxide throughout the storage period at 01°C. Fruits
in CA room # 3 had initial low 02 stress at 0.55% +/- 0.19 for 18 days from October
20-November 6 and between Oct. 24-Nov. 4 (12 days) the 02 level was 0.48% +/-
0.11. Fruits in CA room # 4 had initial low 02 stress at 0.61% +/- .21 for 18 days
beginning on October 20th and between Oct. 27-Nov. 7 (12 days) the 02 level was
0.51% +/- 0.13. For CA rooms 3 and 4 the carbon dioxide level was 0.5%. At the
end of the initial low 02 stress, the temperature was raised in CA room 3 and 4 from
01°C to 2°C for three days then returned to 01°C and the 02 level was raised to
1.5% for the duration of the storage period. The rooms were opened on June 27‘h
after about 8 months in CA. Three forty-fruit samples were obtained from each lot
and examined for scald, internal disorders and flavor and again after a week at

20°C.

RESULTS
1996-97 studies:

Initial low oxygen stress followed by CA storage:

98

Granny Smith, Law Rome and Red Delicious apples without low 02 stress and
stored in air at 1°C were severely affected by scald and CA storage at 3% 02 gave
only partially control (Figure 1 ). Initial low 02 stress at 0.5% 02 for 1 or 2 weeks with
subsequent storage in CA at 3% O2 largely attenuated scald. Initial low 02 stress
reduced scald incidence and severity and 2 weeks at 0.5% 02 was more effective

than 1 week (Figure 1).

Whereas good control of scald was achieved during the first 4 months in CA by
initial low 02 stress treatments, these treatments were not effective for longer CA
storage periods (Figure 2A, 2B). Continuous CA storage at 0.8% 02 prevented
scald development but this treatment resulted in some damage from fermentation

resulting in low flavor development and off-flavors.

During 3% 02 CA storage, or-famesene production by fruits without initial low 02
stress treatment was similar to that from initial low 02 stress except the peak was
at 120 days of CA storage while or-famesene peaked at 150 days of storage in the
fruits with initial low 02 stress treatment (Figure 3A). Control fruits produced more

MHO than those fruits with 0.5% 02 initial treatment for 1 or 2 weeks (Figure 38).

Initial low oxygen stress followed by air storage:
For Granny Smith apples stored in air, all of the treatments including DPA gave
good apparent reduction in scald during storage at 1 °C but not after fruits were held

for 7 days at 20°C (Figure 4). Moreover, DPA treatment at 1000 mg-L'1 was also

99

1001 V V,1__,J_,_# _ _.1 A J1 ,1 WW 1

L l
1 1

,1 1

 

 

   

% 5: fiGrafiyTSTnith: 1
1 a 1 4.56- Law Rome 1 1
801 . I E Red Delicious 1 1
1 1,
1 l
x 60 7- L
a . l
1: . .
.5 1 1
1: L 1
a 1 l
u
(D 401 ,_
I
l
I
2.1
1
1
0,1 7 1:21-, ,J
Air CA 0.5° 0.5°/ O 0.8°/ 0
control control 1 wit OZ 2 wlzs 2 +3%°C62

Treatments

Figure 1. Effect of initial low oxygen stress and ultra low oxygen level continuously
on controlling apple scald of Granny Smith, Law Rome and Red Delicious apples
after 4 months of CA storage (3% 02 + 0% 002) at 1°C plus 7 days at 20°C in air.
Scald index (0=none; 1=slight; 2=moderate; 3=severe) was normalized to 100.

100

     
  
  

1*; @Wsmith T 1
”gm Law Rome 1
1 1839 0.1.1:... 1

Scald index

 

—— — +
171175151 semi 1
1 7 Law Rome 1 W.

m
a Red Deliciousil

ev—ir 1

Scald index

   

Air CA 0.5% O: 0.5% 02
control control 1 wk 2 wks
Treatments

Figure 2. Effect of initial low oxygen stress on controlling apple scald for Granny
Smith, Law Rome and Red Delicious after 7 months of CA storage (3% 02 + 0%
002) at 1°C on removal from storage (A) and plus 7 days at 20°C in air (B). Scald
index (0=none; 1=slight; 2=moderate; 3=severe) was normalized to 100.

101

_ _1._. 1___, W l

J 18:-“Control ' 1 A 1
25 _1 I —8— 0.5% 02 7d . 1
1: 95°12 02 W 7

a-Farnesene
(Units mg"min" at 108 mIz)

 

0-11191, . _. -1 W,r,,,,, 1v, 7 f, a 7__WJ_
120—1 — . ..__._ ._- _ .._ __ __...___.,_._

1 . e‘eoiuor ‘1 B 1
100771 I -'9'- 0.5% 02 7d ‘
l fl???” 9? 14d ..

.11 '— _- —

MHO (pmoles kg"hr°‘)

 

 

 

 

60‘ so 110 150 f 180 ‘-
Days in storage

Figure 3. Effect of initial low oxygen stress on the production rates of or-famesene
(A) and 6-methyl-5-heptene-2-one (B) for Granny Smith apples during CA storage
(3% 02 + 0% C02) at 1°C.

102

ineffective for scald control for Granny Smith apples. The fruits for these
experiments were harvested quite immature to provide high potential to scald. More
mature fruits with naturally lower scald potential would likely have shown good scald

control using similar treatments and storing them in CA at 1.5% 02.

After 5- and -a-half months of air storage without pretreatment at 1°C, a-famesene
production by Granny Smith fruits at 20°C was lower than for the other treatments.
Fruits treated with 1000 mg-L'1 DPA produced a higher level of a-famesene but a
lower level of MHO while control fruits produced less (Jr-famesene but more MHO
(Figure 5A, 5B). a-Famesene levels from fruits with initial low 02 stress or ethanol
vapor treatment were between those of the control and DPA treatments but MHO
levels were even higher than that in control (Figure 5). This indicates that the
production of (Jr-famesene and MHO for control apples has peaked out before

removal from storage.

199 7/98 Studies:

Initial low oxygen stress studies:

Increasing the intensity of low 02 stress progressively diminished the incidence and
severity of scald of Granny Smith and Law Rome fruits subsequently stored in CA
at 3% 02 whereas fruits stored at 1 5% 02 did not develop scald significantly (Figure
6A, 68). CA storage at 1.5% 02 of Oz-stressed fruits completely controlled internal
browning in both cvs. whereas CA storage at 3% 02 did not (Figure 7A, 7B). The

nature of internal browning was largely of minor intensity and was judged to be

103

1OOTL_r4_-_,;__L_#_1.‘NL_ k A A A l» #4? V; iL’HF 4_,~ilg,_,4A + V; _

l
.—
l
l
_T'"
D

l
Control 0.5% 02-1 wk 0. 5% 02-2 wks

? ‘
i l
25% i l E! l
*‘ l
l l
o , _ P
Treatments

N
m
l_._ __l W a.

0|
0

Scald index

!fl #4.?

4 ____ _ or 4 #

 

 

t,._44d

 

Figure 4. Initial low oxygen stress attenuates scald of Granny Smith apples stored
in air for 5 months at 1°C. Scald index (0=none; 1=slight; 2=moderate; 3=severe)

was normalized to 100. (El after storage; I plus 7 days in air at 20°C).

104

 

200

160-

_x
N
‘1’

(ac-Famesene
(units min‘ mg“ at m/z 108)

 

1 l L 1 L 1 A 4
A I + Control
( . I l —B—— DPA 1000 ppm r
+ 0.5% 0214d
' —9— EtOH vapor 6 mll' 14d H

 

 

 

 

 

 

 

 

T fi—T’_'¥—-¥T T T T i I

 

IF.(‘-

1 l - 1 1 l l l l

 

80*
401
10
8%
§
N
E
Ojig 6‘
I 'oo
2.:
.g 4__‘
."3
'E
3
2_.
O

 

 

+ Control

—8— DPA1000 ppm

+ 0.5% 0214d

-e— EtOH vapor 6 mil" 14d

 
 
 
  

 

 

 

 

    

 

     

 

  

T T T T

2 3 4 5 6 7
Days after removal from storage at 20°C

Figure 5. Effects of initial low oxygen stress, DPA and EtOH treatments on the
production rates of a-famesene (A) and 6-methyl-5-heptene-2-one (B) for Granny
Smith apples after 5.5 months of air storage at 1°C.

105

 

A .

i Granny Smith "_
@ Law Rome .
L

Scald index

 

 

 

 

 

 

W
I T T 7

i Granny Smith

 

 

 

 

 

 

@ Law Rome _
x .
o _
U )—
.E -
2 *
«I if”? b
o T
w i
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g *5 g g :8 t g :2 g t.
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O '1 5 " i o" " i (5 T5
as °* .2 5:: .2 5: 0:2
o' $3 :9, 55 3 “- t5 3 § § 3
Air Initial low oxygen stress Initial low oxygen stress
810F899 CA 0% C0: + 3.0% 0: CA 3.0% C0: + 1.5% 02

Figure 6. Initial low oxygen stress attenuates scald of Granny Smith and Law
Rome apples stored in CA (3% 02 + 0% CO2 or 1 .5% 02 + 3% C02) for 5 months(A)
and 8 months (B) at 05°C plus 7 days at 20°C in air. Scald index (0=none;
1=slight; 2=moderate; 3=severe) was normalized to 100.

106

 

100

 

 

 

 

 

 

 

 

 

 

1 A
S? 80~ _
.3 - i
= 60— ~
3 - .
2 .
1: ~ .
'3 40‘. T
c
a )-
§ 20~ _
0__
100
. B r
g 80~ ,
U) . .
.5 ~ .
C 60~ ~
3 -
e .
.o i .
—~ 40 . f
c
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0%;—u g
'2 g + 0 4. O .3 + a +
z “3 .. - s 0 s "‘ "
on N ~ on
32 O in O (5 on 3 (53 3C: (53
we 32‘; 3‘ w; to 32"; m 32“
a we a a. s we N. m 3
ch! 6 Non 9N 0 “EN
0 D
Air Initial low oxygen stress Initial low oxygen stress
storage CA 0% C0: + 3.0% 02 CA 3.0% CD: + 1.5% 0:

Figure 7. Effects of initial low oxygen stress and oxygen levels in CA storage (3%
02 + 0%-CO2 or 1.5% 02 + 3% 002) on internal browning of Law Rome (A) and
Granny Smith (B) apples stored for 8 months at 05°C plus 7 days at 20°C in air.

107

 

senescence-related in that it was most evident in the air stored fruits and in 3% 02
CA. This is supported by the observation that core browning (a slight discoloration
of tissue immediately adjacent to the seed cavity related to immaturity at harvest)
was most prevalent in Law Rome subjected to the most low 02 stress and only
when stored at 1.5% 02 CA (Figure 8A). This was less evident for Granny Smith
apples ( Figure 88) from all other CA atmosphere regimens which exhibited varying
degrees of core browning with no clear association with CA treatments. The
incidence of decay was generally inversely related to the degree of low 02 stress;
with more low 02 stress decay decreased. Decay was most prevalent in the air
control fruits for both cvs. (Figure 9). The lowest incidence of decay was generally

found for fruits subjected to two weeks of 0.25% 02.

Flesh firmness was retained better for both Granny Smith and Law Rome cvs after
receiving initial low 02 stress treatment than those without (Figures 10, 11).
Compared to the initial flesh firmness (82.5N for Granny Smith and 89.2N for Law
Rome) fruits of both cvs. had higher firmness when stored in 1.5% 02 CA than those

stored in 3% 02 CA (Figure 10, 11) as would be expected.

Apples without initial low 02 stress produced more a-famesene after 5 months of
storage in 1.5% 02 CA than those with initial low 02 stress treatments (Figure 12 A,
120) whereas fruits produced lower amounts of a-farnesene when removed from
3% 02 CA (Figure 123, 120). A similar pattern was seen for fruits held at the same

conditions for 8 months (Figure 13). Also, similar to the data in Figure 12, apples

108

 

 

100

T I‘T

604

40?

T j

Core browning (%)

 

No data

 

 

 

 

so?
609

40;

Core browning (%)

20?

 

 

 

h.

 

Air No data

None
None

0.5% O: 2 wks
0.5% O: 2 wks +
2 wks at 2 mo

0.25% Oz 2 wks
2 wks at 2 mo
0.5% O: 2 wks

0.5% O: 2 Wk! 4'
2 wks at 2 mo

0.25% O. 2 wks

2 wks at 2 mo

0.25% Oz 2 wks +
rum 0. 2 wks +

Air Initial low oxygen stress Initial low oxygen stress
storage CA 0% CO: + 3.0% 02 CA 3.0% C0: + 1.5% o.

 

 

Figure 8. Effects of initial low oxygen stress and oxygen levels in CA storage (3%
02 + 0% CO2 or 1 .5% 02 + 3% COZ) on core browning of Law Rome (A) and Granny
Smith (B) apples stored for 8 months at 05°C plus 7 days at 20°C in air.

109

 

 

 

 

 

 

 

 

20*
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N N" N. 3" N 3N N :N
0" 5‘6 0 ".36 6 3‘6 5 0" ‘6
.\° $32 :3? 9,3 3 .32 .\° \ofi
"a '0. 3 fig a a m. a} s a a, s
0 ON a ”_N ° 9'" 0' “l N
._ _ ~._*7 7.,..7J___ _ . _;
Air Initial low oxygen stress Initial low oxygen stress
storage CA 0% CO: + 3.0% 02 CA 3.0% C0: + 1.5% 01

Figure 9. Effects of initial low oxygen stress and oxygen levels in CA storage (3%
02 + 0% CO2 or 1.5% 02 + 3% C02) on decay of Granny Smith (A) and Law Rome
(B) apples stored for 8 months at 05°C plus 7 days at 20°C in air.

110

Initial FF= 82. SN

40‘ IIIII IIIII
Ill lllllB
80$ C,
60': . F
40. ‘ i,
20. E

Flesh firmness (N)oo
A N A O)
OO O 0 C30

 

D ,,
60 I 1::l I l I I I
+ o +
§ % t. E % 2 IE ii .32
z N i N "f. "N g N :0! N NN
\° 5 fl .\° 93 °\° 6.3 °\° '3
a. \o g 3 >3 In >3 a $3
6 Es N d j: ° 3N 6 SN
Initial low oxygen. stress Initial low ox gen stress
A0%C +30%02 CA3.0%C 2+1.5%02

Figure 10. Effect of initial low oxygen stress and CA conditions (3% 02 + 0% CO2
or 1.5% 02 + 3% 002) on flesh firmness retention of Granny Smith apples stored
5 (AB) and 8 (0.0) months at 0.5°C. Flesh firmness on removal from storage (A,C)
and after 7 days at 20°C (ED).

111

 

 

100- -_L__.M.

801 Initial FF= -98im .
60:

40

108i

IIILll
80:

604

J IIlLIIIJ
20:

108i ’ ' ”

L

j r Tfi—r>

I

 

Ifil

 

 

    

 

Flesh firmness (N)

 

 

 

 

 

802
602
40! I
20 A- .
0 1 ° '0 + O 0 T O o '1’ + £0 a o L
S i E i 32 E g E‘ ‘3‘ .35
z N g N N :N z N gN N NN
5 N '5 6 6.6 6 “:75 (5 6'5
x" C5 3 \° 33 \° 00° \° W
in e in .\’ i’o .\°-“ i’n .\°-"
:5 g 3 N. a 3 c “2 3 N g} 3
_ <5 N ° :5" °°' ° :5"
Initial low 0 gen stress Initial low 0 gen stress
CA 0% C 2+ 3. 0% 02 CA 3.0% C +1. 5% 02

Figure 11. Effect of initial low oxygen stress and CA conditions (3% 02 + 0% CO2
or 1.5% 02 + 3% C02) on flesh firmness retention of Law Rome apples stored 5
(AB) and 8 (CD) months at 0.5°C. Flesh firmness on removal from storage (A,C)
and after 7 days at 20°C (8.0).

112

 

 

 

 

a -Famesene

 

 

 

 

 

 

 

 

 

 

 

 

 

a:
C
a)
(D
a)
E
m
or
M

0 "L % LLT— ___7_ __‘ ' L“ I Y—L L L— L_"—T_ L ‘1-

I I ' _ i *L I I , l - __ _i_ __ ___L— _s

600 : I S: N8 initial 0. stress—m ‘—

   
 
 

+ 0.5% 02 for 2 wks
+ 0.5%022wks+2wksat2moCA .
—a— 0.25% Ozfor2wks I
~9- 0.25% o. 2wks +2wks at2 mo CA I

  
  

. . ., ., . afarnesiene . . ., _, . . .. .,
(units min mg atm/z 41)(unlts mIn mg atm/z 41)(unlts min mg atm/z 41)(un|ts min mg atm/z 41)

 

 

 

 

 

 

 

 

 

g 400 I ‘ I

g .4

LI- 200 I .

a l
0' LLT‘L‘LLL‘F"' “___L L

 

 

 

o 1 2 h 3 71 5 6 7
Days after removal from storage at 20°C

Figure 12. Effect of initial low oxygen stress on a-famesene levels in the
headspace of Granny Smith (A, B) and Law Rome (C, D) apples stored in 1.5% (A,

C) and 3% (B, D) 02 CA for 5 months at 0.5°C.

113

 

 

 

 

 

 

 

 

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+ No initial 0: stress I I
L
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T6 240I -e— 0.25% 02 for 2 wks

+ 0.5%022wks+2wksat2moCA '
‘_—e- 0.25%Oz2wks4-2wksa12moCAI

   

 

 
 

a-Famesene
mg
_k
on
‘P

 

 

 

'7 q ' 'A‘ I \A‘

E120—I n

g 60 j _ '4"%/ f"

5, O I l . “‘ “I " l :- "LT LL" 4:
1 2 3 4 5 6 7 8

Days after removal from storage at 20°C

Figure 13. Effect of initial low oxygen stress on a-famesene levels in the
headspace of Granny Smith (A, B) and Law Rome (C, D) apples stored in 1.5% (A,
C) and 3% (B, D) 02 CA for 8 months at 0.5°C.

114

with initial low 02 stress at 0.25% 02 level produced less a-famesene when fruits
were held at 1.5% 02 storage than those with 0.5% initial 02 stress treatment at 5
and 8 months (Figure 12A,C;13A,C), respectively. This indicates that the a-
famesene production of apples was inhibited by initial low 02 stress treatments and
also indicates initial 02 stress at the 0.25% 02 level and/or CA at the 1.5% 02 level
more strongly inhibits a-famesene production than was observed for fruits without

initial low 02 stress and/or CA at the 3% 02 level.

After transfer from 1.5% 02 CA to air at 20°C more or-famesene accumulated in the
epicuticular wax of Granny Smith apple fruits without initial low 02 stress treatment
than those transferred from 3% O2 storage (Figure 14A, 14B). Law Rome fruits with
initial low 02 stress had a higher potential to produce a-farnesene than Granny

Smith fruits after 8 months of storage at both 1.5% and 3% 02 (Figure 14C, 14D).

Fruits receiving 0.25% 02 initial stress treatments produced less MHO than those
with 0.5% 02 stress at all storage conditions for both Granny Smith and Law Rome
cvs. after 5 months of storage (Figure 15). Whereas, after an additional 3 months
of storage, apples moved from 3% 02 produced more MHO than those held at 1 .5%
02 (Figure 16). A similar pattern of MHO levels was seen under the various 02
levels of CA (Figure 17). In contrast, after 8 months of storage, some of the
potential to produce less MHO with 0.25% 02 initial stress treatments have been
decreased based on the analyses of headspace (Figures 16) and epicuticular wax

(Figure 17). This data indicate that both 0.25% 02 initial stress treatment and CA

115

 

1500"? I- II I I I- _ _ I I. I I I I- _III II II I
12001I A L
eooI I
600~

300;

 

 

a -Famesene

 

1508:

 

 

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a Famesene

 

 

 

 

 

 

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.2 I I
i O"1 T 'T”"" ' _I ' ‘I ' T I"— T T "TTT' ._I_.I_
1 2 3 4 5 6 7

Days after removal from storage at 20°C

Figure 14. Effect of initial low oxygen stress on a-famesene levels in the
epicuticular wax of Granny Smith (A, B) and Law Rome (C, D) apples stored in
1.5% (A, C) and 3% (B, D) 02 CA for 8 months at 0.5°C.(Note different scale for C

and D).

116

 

 

 

 

 

 

 

MHO-ketone

 

 

MHO;ketone
o

' at m/z 1 08) (units min' 9' at m/z 1 08) (units min"g“ at m/z 1 03)
—L

 

 

 

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f
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+ 0.5% 02 for 2 wks | C ‘
+ 0.5% 022wks+2wks at2mo CA I
, , —9— 0.25% 02 for 2 wks

—e— 0.25%022wks +2wksat2moCA!

///A: :6

+l

l

   

 

 

 

     

MHO;ketone
9
o
01

 

 

 

 

.o
.3;

MHO1-ketone
(units min' 9‘1 at mlz 1 08) (units min'

 

 

ITII

‘ o i 2 3 4 5 6 7
Days after removal from storage at 20°C

Figure 15. Effect of initial low oxygen stress on 6-methyl-5-heptene-2-one (MHO)
levels in the headspace of Granny Smith (A, B) and Law Rome (C, D) apples stored
in 1.5% (A, C) and 3% (B, D) 02 CA for 5 months at 0.5°C.

117

o o
4:. o:
>

 

 

 

 

MHO-ketone

 

 

 

 

MHO-ketone

(units min'1g'1 at mlz 1 08) (units min“g'1 at mlz 1 08) (units min"g" at mlz 1 08) (units min"g" at mlz 1 08)
co
m?
l
l
i

 

 

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l
1 2 3 4 5 6 7 8

Days after removal from storage at 20°C

Figure 16. Effect of initial low oxygen stress on 6-methyl-5-heptene-2-one (MHO)
levels in the headspace of Granny Smith (A, B) and Law Rome (C, D) apples stored
in 1.5% (A, C) and 3% (B, D) 02 CA for 8 months at 0.5°C.

118

 

MHO-ketone

 

 

 

l
308 i “‘ "‘ ’’’’’ ' ' 7 ' '“f "7 " f, :Tfi: E T;_ éi.‘_“ :_ _’ .,’;;.v‘ ‘7‘ ’
1 . + No initial 0, stress ‘2
. + 0.5% Ozfor2wks B '
250 i f-Av- 0.5%Oa2wks+2wksat2moCA '
. I —a— 0.25% o, for 2 wks

Lf‘ 0.25% O22m+2wksai2mocimji

 

 

MHO-ketone 1
(units min"g" at m/z 1 08) (units min' 9“ at mlz 1 03)

 

 

 

 

MHO-ketone
ts min"g" at mlz 1 08)

 

 

 

 

'E
3
66‘
S
E
£5
a,“
Afro:
g:
E
2,52 I
1 2 3 4 5 6 7

Days after removal from storage at 20°C

Figure 17. Effect of initial low oxygen stress on 6-methyl-5-heptene-2-one (MHO)
levels in the epicuticular wax of Granny Smith (A, B) and Law Rome (C, D) apples
stored in 1.5% (A, C) and 3% (B, D) 02 CA for 8 months at 0.5°C.

119

 

at1.5% 02 level has a stronger effect on inhibition of MHO production than does

0.5% initial 02 stress and CA at 3% 02 level.

Continuous low or ultra low oxygen stress studies:

GC/MS analyses of more than 60 volatiles produced by intact fruits and those which
accumulated in the cuticle were conducted throughout the 8-month storage period
at 0.5°C. Volatiles analyses of Granny Smith apples subjected to continuous low
02 stress levels ranging from 0.13 to 1.5% 02 and in air commenced at weekly
intervals beginning one week after the low 02 levels were established. The results
for acetaldehyde, ethanol and ethylacetate are shown in Figure 18 . The results for
fruits held at 0.75, 1.0, 1.25 and 1.5% 02 were similarto values for fruits held in air
and are ommitted from the Figure 18). Ethanol, ethylacetate and acetaldehyde
were detected during the storage only on 02 levels below 0.4% 02 stored fruits
(Figure 18A, 188, 18C). Very small amounts of ethanol and ethylacetate were
detected within the first 2 or 3 weeks of storage. Ethanol production was observed
by fruits held below 0.5% 02 beginning one week after commencing the low 02
regimens. At 0.13% 02. ethanol production rose to a peak after two weeks and then
declined to a steady-state value followed by an increase after 150 days. Fruits held
at 0.25% 02 produced ethanol at a lower level than those at 0.13% and rose to a
peak at 21 days followed by a gradual decline for the remainder of the storage
period (Figure 18A). A similar pattern was seen for fruits held at 0.4% 02 but the
level of ethanol production was much lower and declined to non-detectable levels

after a month of storage. The pattern of ethylacetate production was similar to that

120

 

50Ewsuu-us-fisuius_-u,dsusuuutwuu-u_-

 

       
 

 

 

fr? , v-———— w. i
V 40g l —e— 0.13% 02 ‘ F
a - 1+ 0.25%02 Ai-
_ E 30 i—e— 0.4%02 ,
O-H T i-v— 0.5%02
Cm 4 1
32 20i
“J x 7
:3 10“ I! AKA—AW L
a 0 WSW

 

 

V .44 r ——— tau— ‘11__—.i, _._

Ethyl acetate
(Units x105 at m/z 45)

Acetalqeh
(Units x 10 at m/z 43)
P r w .

:g .

 

 

 

 

yde

 

’M:

b 120 160 ' 200 ' 240
80'Days In storage

 

 

Figure 18. Change in production rates of ethanol (A), ethyl acetate (B) and
acetaldehyde (C) of Granny Smith at low levels of oxygen and air storage at 0.5°C.

121

of ethanol (Figure 188) but commenced slightly later. Acetaldehyde began to
progressively accumulate after 4 weeks at 0.4% 02 and below but was not
measurable until production of ethanol and ethylacetate reached their maximum
values (Figure 18C). Actaldehyde production at 0.13 and 0.25% 02 remained at
high values for several weeks before gradually declining. Near the end of the
storage period, fruits held at 0.13% 02 again increased in acetaldehyde production
and this again followed an increase in ethanol and ethylacetate production. This

may be related to irreversible damage to metabolism caused by 02 deprivation.

a-Farnesene was produced in copious amounts by fruits in air after 3 weeks and
was generally progressively lower as the 02 level decreased (Figure 19A,19B).
MHO was first detected from fruits held in air after about 45 days of storage and its
production peaked at 3 months then declined to half-peak values for the remainder
of the storage period (Figure 190). MHO was not detected at 02 levels below
1.25% and was generally detectable only at 1.5% 02 or above. The strong
relationship between the levels of O2 favoring ethanol production and reduced levels
of MHO suggest that ethanol metabolism may be directly involved in keeping MHO
at low levels. Alternatively, MHO production may involve an oxidative enzyme with
a fairly high affinity for molecular 02. The mechanism for scald control by initial low
02 stress remains to be resolved. Scald developed on the air-stored fruits after 4
months of storage and on the 1.5% 02 stored fruits after 10 months of storage.

(data not shown.)

122

 

N
O

 

     
 
     

 

 

 

 

 

 

 

 

 

:: L F __,___ ‘ .
:15, —o— Air , r
2E l +1.0%02 iA
3*, ; + 1.25% o. l .f
o‘“104 l+1.5%oz :
so . - .
‘— .
u. x 5.-.! i.
l , l
«3.52 i
'E 7 i
3 OJ L
3;:gL:-:1LA_L;L_.___I : LL LL; L LLL ;_":i“if_L
A , g + 0.13% 02 ‘
3 . 2 —e— 0.25% 02* B"
N —8- 0.40/o 02
EE 2 . i —e— 0.5% Oz % L
a)“ ( -e— 0.75% Oz ,
3’, co 1- + 1.0% 02 O 1
Cwo . + 1.25%02 .
5‘. 1 1 + 1.5% 02 e
u.- >< l
“‘59 i .. 1 " i
E i . .. ‘ \ ~ 5
3 07 "
eoLLL L :LLML ‘ _1 L; -L: L
8 i ! -0— Air
,_ g), +1.0%oz , _
u 40,: + 1.25% oz! ;
E . +1.5%o. ‘ ‘
OE '. __W_“ , J
L
20 20-. L
V a i
,‘~_’-_-’ 1 i
S o i 4 ' I L
V l

 

o" Shite—"‘66” 8o too—750“ 111—0 160 1210 260 220 2210
Days in storage

Figure 19. Change in production rates of or-farnesene (A,B) and its oxidation
product MHO (C) of Granny Smith at low levels of oxygen and air storage at 0.5°C.
(Fruits did not produce MHO when 02 lower than 1.25%)

123

 

Initial low 02 stress studies at the MSU CHES CA Storage Facility, 6 commercial
test:

No internal disorders or off-flavors were detected in fruits from any of the storage
regimens; scald was evident on fruits from the continuous 1.5% 02 CA upon
removal from storage but not for the other storage regimens and this intensified
after a week at 20°C. Fruits from ultra low CA (0.7% 02 throughout) were scald-free
(Table 1 and Figure 20). Some of the Red Delicious and Law Rome fruits exhibited
pigment purpling on removal from 0.7% 02 storage but this largely cleared after a
few days in air at 20°C. All cultivars from CA room #2 (1.5% 02 throughout)
exhibited scald of varying degrees with an over-all scald index average of 0.52.
Fruits from CA room 3 and 4 (initial low 02 stress for 18 days at 0.55 and 0.61 % 02)
had very low scald index values of 0.09 and 0.03, respectively. Galia Beauty Rome
was the only cv. with appreciable scald (last two entries in Table 1). These fruits

were very immature at harvest.

Table 1. Effect of initial low oxygen stress on control of scald of apples in the CHES
CA storage trial 2.

 

 

 

Scald index
No initial low oxygen stress initial low oxygen stress
Growery - 0.7% 02 1: 0.1 1.5% 02 :l: 0.1 0.55% 02 :l: 0.2 0.61% 02 i 0.2
Cultivar 18 days 18 days

 

Mean SE Mean SE Mean SE Mean SE
l- 0.00 0.00 0.04 0.05 0.00 0.00 0.00 0.00

 

Starkrimson

lV- 0.00 0.00 0.04 0.03 0.01 0.01 0.00 0.00
Starkrimson

V- 0.00 0.00 0.05 0.04 0.06 0.04 0.00 0.00
Starkrimson

l- 0.00 0.00 0.09 0.06 0.00 0.00 0.00 0.00
Nu Red

124

 

 

Table 1 continued

lll- 0.00 0.00 - - 0.06 0.04 0.00 0.00
SuperSpur

Vl- 0.00 0.00 - - - - 0.00 0.00
Red Chief

I- 0.00 0.00 0.01 0.01 0.00 0.00 0.00 0.00
Red Chief

V- 0.00 0.00 0.01 0.01 0.01 0.01 0.00 0.00
Red Chief

II- 0.00 0.00 0.15 0.13 - - 0.00 0.00
Red Chief

V-Winesap, 0.00 0.00 0.15 0.04 0.07 0.08 0.00 0.00
Tudey

l-Winesap, 0.00 0.00 0.18 0.10 0.04 0.05 0.00 0.00
Stayman

lll- 0.00 0.00 0.06 0.04 0.00 0.00 0.00 0.00
Rome, Law

IV- 0.00 0.00 0.34 0.10 0.01 0.01 0.00 0.00
Rome, Law

V- 0.00 0.00 0.46 0.25 0.01 0.02 0.00 0.00
Rome, Law

Vl- 0.00 0.00 1.10 0.25 0.07 0.04 0.00 0.00
Rome, Law

II- 0.00 0.00 1.22 0.11 0.12 0.09 0.00 0.00
Rome, Law

I- 0.00 0.00 1.51 0.34 0.04 0.03 0.00 0.00
Rome, Law

ll- 0.00 0.00 1.16 0.17 0.25 0.13 0.12 0.08
Rome, Galia

l- 0.00 0.00 2.22 0.37 0.71 0.23 0.37 0.16
Rome, Galia

Average 0.00 0.52 0.08 0.02

 

 

2 Harvest date October 7-13, 1997;Removal date of fruits from CA rooms June 27-
30, 1998. Analysis date July 4-6, 1998;

Scald index expressed as 0-none, 1-slight, 2-moderate, 3-severe;

Storage CA condition: Room #1 : 0.7% 02 + 0.5% CO2 continuous; Room #2: 1.5%
02 + 0.5% CO2 continuous; Room #3,#4: 1.5% 02 + 0.5% CO2 after initial low
oxygen stress.

Y 6 growers were designated as i, ll, lll, IV, V and VI.

125

 

Scald index

 

 

 

 

 

Starkrimson Winesap Galia Rome

Cultivars

Figure 20. Effects of initial low oxygen stress and oxygen levels in CA storage
(1.5% 02 + 0.5% C02) at the MSU CHES facility on controlling scald of apples
stored for 8 months at 01°C plus 7 days at 20°C in air. Treatment designation: A
is for 1.5% 02 CA as control; B is for 0.55% 02 initial stress for 18 days then 1.5%
02 CA; C is for 0.61% 02 initial stress for 18 days then 1.5% 02 CA; D is for 0.7%
ultra-low 02 CA continuously. Scald index (0=none; 1=slight; 2=moderate;
3=severe) was normalized to 100.

126

 

DISCUSSION
Our experiments in 1996-97 confirmed our earlier studies that initial low 02
treatment at the beginning of the storage period can largely diminish superficial
scald of apples as first reported by Little et al.(1982) in Australia and extended by
Ghahramari and Scott (1998b). We observed that initial low 02 stress treatment
induced apples to produce ethanol aerobically (Chapter ill). Moreover, ethanol
vaportreatment reduced scald (Chapter III). That apples produce ethanol when the
02 level is below the extinction point of alcohol fermentation (ca. 1%) suggests that
a storage regimen of initial low 02 stress may induce apples to produce ethanol and
be an effective means to control scald. Little et al.(1982) reported that scald of
Granny Smith apples was largely controlled by initial low 02 stress followed by low
02 CA storage. While some studies of initial low 02 stress indicate reduction of
scald others do not (Truter et al., 1994). Recent studies by Van der Menive et al.
(1997) indicate good control of scald by initial low 02 stress followed by CA storage

at 1% 02 with 3% CO2 at 1°C.

Apples receiving the 0.5% 02 initial stress produced ethanol during the low 02
stress as would be expected and has been reported (Ghahramari and Scott, 1998b;
Wang et al., 1997). This suggests that the reduction of scald incidence by the initial
low 02 stress treatment may be a consequence of the ethanol produced and/or
metabolism induced to reduce concentration of some metabolites or ameliorate their

action as related to scald development.

127

 

Initial low oxygen stress controls scald:

Initial low O,2 stress of 0.5% 02 for two weeks administered prior to CA storage at
3% O2 reduced the incidence and severity of apple scald; 0.5% 02 for 2 weeks gave
better control than for 1 week (Figure 1, 2). The effectiveness of initial 02 stress
treatments diminished after long term CA storage at 3% 02(Figure 4). Initial low 02
stress at 0.5 and 0.25% 02 for two weeks at 1°C followed by CA storage at 1.5%
02 at 1°C very effectively controlled scald of preclimacteric Law Rome and Granny
Smith apples not treated with DPA (Figure 6). These results confirm the
observation of Van der Menive et al. (1997). This treatment regimen resulted in
excellent retention of fruit firmness and over-all quality while CA storage at 3% 02
following low 02 stress did not (Figure 10, 11); the incidence of scald was however

progressively less as the degree of low 02 stress increased (Figure 6).

a—Famesene and MHO production:

Our results support the concept that ot-famesene and its breakdown product MHO
are related to the scald disorder (Anet, 1972b; Filmer and Meigh, 1971; Huelin and
Coggiola, 1970a; Rowan et al.,1995; Song and Beaudry, 1996). We find that
preclimacteric Law Rome and Granny Smith apples are remarkably tolerant of low
02 stress. 02 levels below 1.25% completely arrests MHO production (Figure 19)
and or-farnesene production decreased as 02 levels declined from 1.5 down to
0.13%. The data indicates that or-famesene is not the limiting factor but rather its

metabolism leading to MHO production and or metabolism may be.

128

 

MHO production by intact fruits and that which accumulated in the cuticle of both
Law Rome and Granny Smith apples as determined after 8 months in CA storage
revealed that initial low stress at 0.25% 02 for 2 weeks markedly lowered its
production/accumulation values (Figure 6). High MHO production/accumulation
values were strongly and directly proportional to the scald index. This supports a
putative role of MHO in the scald disorder but when it is a factor in subsequent scald
development must be early vs late during storage. This is because fruits stored
hypobarically within 2 months of being harvested do not scald whereas those stored
with longer delays do scald (Chapter II). Capacity for MHO production begins within

the first 2 months when fruits are stored in air.

Conclusions and implications:

We conclude that initial low 02 stress of ca. 0.25-0.5% 02 for 14 days at 0°C
followed by warming to 2°C for 3 days can effectively control scald of apples not
treated with DPA and subsequently stored at 1.5% 02 at 0°C employing a static CA
regimen. This confirms the results reported by Little and his colleagues (1982) and
others (Van der Menrve et al., 1997). Storage at 0.7% 02 throughout CA storage
completely controlled scald (Figure 20) confirming the results of Lau (1990).
However, some these fruits exhibited reversible evidence of anaerobic stress and
prolonging storage beyond 8 months at 0.7% 02 may have resulted in irreversible
damage and flavor quality impairment. Since the initial low 02 stress followed by
CA at 1.5% 02 controlled scald effectively and at commercially acceptable levels

this CA storage regimen may provide a safe and effective means to store apples not

129

 

treated with DPA. This also avoids the use of postharvest fungicides which is an
issue of growing concern. We previously found that an initial ethanol vapor
treatment of apples subsequently stored in air or CA at 3% 02 was partially effective
in controlling scald (Chapter III, Figure 8; Wang et al., 1997). Our present results
support the concept that the beneficial effects of initial low 02 stress may be related
to ethanol metabolism in some manner. The residual effect of ethanol vapor
treatment in reducing scald of apples subsequently stored in air (Wang and Dilley,
1996) suggests that ethanol may act apart from the effect of low CO2 alone. A
similar conclusion was recently reported by Ghahramani and Scott (1998b) who
suggested that control of scald by initial low 02 stress may be due to increased
production of ethanol and consequent reduction in cat-famesene and conjugated

trienes accumulation.

In our flow-through CA experiments we use gas mixtures at a flow rate that is just
sufficient to remove CO2 produced in respiration and provide a constant C02 and
02 level. This amount of fresh atmosphere flushing is sufficient to remove the trace
amounts of volatile substances that may partition into the epicuticular wax (Matich
et al., 1998). Such substances may include or-famesene and 6-methyl-5-heptene-2-
one derived from the biological oxidation of or-famesene (Filmer and Meigh, 1971;
Spicer et al., 1993) or perhaps a trienol derived from or-farnesene (Whitaker et al.,
1997). This has not been proven definitively. In theory removal of this volatile by
use of a scrubber is feasible. However, the efficacy of mineral oil impreguated

wraps (Brooks et al., 1919) and vegetable oil coatings (Scott, 1995b) to reduce

130

 

scald is thought to be as a consequence of volatiles being partioned into them.

Our data is consistent with our observation that fruits do not scald during 8-month
storage when flow-through CA is employed at 1.5% 02 with 3% 002. Moreover,
scald is largely controlled in commercial storages at 1.5% 02 with 3% CO2
employing air separators to remove CO2 by flushing (Dilley, 1990). Flushing the

atmosphere removes volatile substances.

Alternative strategies:

Several different strategies may be useful to control scald (Chellew and Little, 1995;
Kader, 1986; Kallay, 1994). These may depend on cv. and the duration of the
storage period. For fruits which are to be marketed after only 6 months in CA, using
an air separator to maintain 1.5% 02 with 3% CO2 has provided satisfactory control
of scald. Fruits for long-term CA for 7 or more months may require a more
demanding strategy. This may include initial low 02 stress at 0.25-0.5% 02 levels
for two weeks prior to CA at 1.5% 02 and 3% COZ. CA at 1 to 1.5% 02 using an air
separator to control CO2 at 1 to 2% may provide adequate scald control for mature
fruits not yet producing significant levels of ethylene. The commercial scale trial at
the MSU CHES CA Facility of initial low 02 stress demonstrated excellent control

of scald by employing this treatment regimen.

131

 

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In: E.J. Mitchman (ed.) Apples and Pears. Proc. 7th lnt’l Nat. CA Res. Conf., 2:
105-111.

Whitaker, B.D., Solomos, T. and Harrison, DJ. 1997. Quantification of or-

famesene and its conjugated trienol oxidation products from apple peel by Cm-
HPLC with UV detection. J. Agric. Food Chem. 45: 760-765.

135

 

CHAPTER V
THE EFFECT OF ACCLIMATIZATION OF FRUITS ON THE CONTROL OF A

COz-LINKED DISORDER OF EMPIRE APPLES

ABSTRACT
The 'scald-like' disorder of Empire apples is a COz-linked physiological disorder. In
this study the factors of acclimatization of preclimacteric fruits at 3°C in air or low
02 levels prior to elevating the CO2 concentration for subsequent long-term
controlled atmosphere were investigated. The results indicate that the Empire
“scald-like’ disorder can be effectively controlled by holding the fruits at 3°C for 3-4
weeks at 1.5 to 3% O2 without CO2 prior to CA storage at 1.5% 0,2 + 3% C02.
Acclimatization of fruits against the disorder was also achieved by storage in air but
this resulted in excessive ripening development with flesh firmness loss during

subsequent CA storage.

INTRODUCTION
The Empire 'scald-like' physiological disorder can occur during controlled
atmosphere (CA) storage (Burmeister and Dilley, 1995). It was concluded that it is
a COz-linked disorder that is (1) exacerbated by storage at 0°C at 02 levels below
1.5% with CO2 at or over 3%, (2) controlled by adding dry lime to the CA storage
chambers (0% 002), and (3) controlled by maintaining CO2 at or below 2%. The
disorder has many but not all of the attributes of superficial scald (Ingle and

D’Souza, 1989). The disorder is more prevalent with immature fruits; affects the

136

 

non-red surface of the fruits; is induced during the first few weeks of storage; and
is controlled by diphenylamine (DPA), an antioxidant employed to prevent scald.
This disorder has resulted in as much as a 20% reduction in packout by shippers
to meet US. and export standards. A similar physiological disorder has been
observed on immature Northern Spy (Burmeister and Dilley, 1995) and Braebum
apples (Burmeister and Rowan, 1997; Elgar et al., 1998). This prompted us to
investigate alternative methods to control the disorder. A recent report by Lau
(1998) suggests that the Braebum browning disorder is more prevalent with late-
harvested fruits. The Braebum browning can be controlled by DPA (Burmeister and
Rowan, 1997). We hypothesized that the Empire ‘scald-like’ disorder may be
related to superficial scald since the disorders of both Empire and Braebum apples
can be effectively controlled by drenching with DPA before storage. The two
disorders may not be the same since the Empire disorder does not happen in air
storage and was related to high 002 levels especially at the beginning of the
storage period. The Empire disorder can occur after only a few weeks in storage
while superficial scald occurs after several months. In this study, we investigated
the effects of acclimatizing fruits in various gas atmosphere regimens prior to CA

storage to control the disorder.

MATERIALS AND METHODS
Preliminary experiments (1994):
Preclimacteric Empire fruits with or without DPA at 1000 mg-L'1 were placed in 20-L

chambers ventilated with 0 or 5.5% C02 in humidified air at 0.1 and 3°C. Ethanol

137

 

vapors were applied to apples in some storage chambers by circulating 0 or 5.5%
CO2 through 1% aqueous solutions of ethanol in flasks in series with the chambers
to generate about 115 jJL-L'1 ethanol vapor into the chambers. The ventilation rate
was 0.4 atmosphere turnover per hr. Three replicates of 50 fruits were employed
each. Fruits were examined for the scald-like disorder incidence at weekly intervals

for 3 and half weeks. (See Table 1 for results).

Preclimacteric Empire apples were placed under CA at 1.5% 02 with 0, 3 and 5%
CO2 and in air at 3°C. Half the fruits for CA at 1.5% O2 +3% CO2 were added a
5009 dry lime-packet to each chamber to scrub C02, while half the fruits for CA at
1.5% 02 + 5% CO2 were treated with DPA 1000 mg-L“. The Empire disorder index
was measured after 1 and 5 months of storage and plus 7 days at 20°C in air. The
index was based on the percentage of fruit surface area affected where no disorder
= 0, <25%=1, 25-50%=2 and >50%=3 and disorder index was normalized to 100.

(See Table 2 for results).

Carbonyl content determination:
The carbonyl content of apple peel tissue was determined by the methods of Levine

et al. (1994) and Stadtman (1993).

Main experiment (1995):
Fruits from the MSU CHES were harvested at the preclimacteric stage of maturity

about 10 days before the onset of the endogenous ethylene climacteric. This was

138

 

done so the fruits would have a high potential to develop the ‘scald-like’ disorder
(Burmeister and Dilley, 1995). Fruits were randomized to fill 132 20-L chambers
with 45-65 fruits each. Atmospheres of 1.5 or 3% 02 were established within 1 day
of harvest and the CO2 level was brought up to 3 or 5% after 0, 0.5, 1, 2, 4, or 8
weeks in storage at 3C. The gas atmospheres were established by mixing nitrogen
produced by a Permea air separator with CO2 and air in a gas mixing apparatus.
The gas mixtures were humidified and distributed to the CA chambers using a
capillary flow-board system at a flow rate sufficient to provide 0.4 chamber void
volume changes per hour. Some fruits were kept at 1.5 or 3.0% 02 without C02,
some were held in air for 1 or 2 weeks prior to adjusting to the CA conditions while
others were held in air throughout the storage period of 6 months. A David Bishop
Oxystat ll Analyzer was employed to monitor the O2 and carbon dioxide levels on
a daily basis. Three replicates were used for each treatment. After 6 months of
storage, the disorder index was determined: no disorder = 0, <25%=1, 25-50%=2
and >50%=3 based on the percentage of the fruit surface area affected by the

disorder. Disorder index was normalized to 100 by multiplying 33.33.

RESULTS
Preliminary experiments:
Supplementing the CA atmosphere with ethanol vapor at 115 uL-L'1 (v/v) reduced
the disorder incidence during a 2 week period at 5% CO2 + 1.5% 02 but became
ineffective with longer storage durations (Table 1). Fruits at 3°C without CO2 in the

atmosphere were not affected with the disorder but with 3 or 5% 002 had serious

139

 

 

Table 1. Effect of ethanol vapor treatment on the incidence (%) of the ‘scald like'
disorder of Empire apples stored at 0 or 5.5% CO2 + 1.5% O2 at 0.1 and 3°C with
and without pretreatment with diphenylamine (1000 mg'L").

 

 

 

 

 

Treatments Disorder incidence (%)
Storage
tempfrature Ethanol co2 DPA 14d 20d 24d 24d + 2d
( C) vapor (%) (mg-L") at 20°C
(MN-'1)

0.1 0 5.5 0 62 71 65 79

0.1 115 5.5 0 15 37 43 83

0.1 115 5.5 1000 0 0 0 0

0.1 0 0 0 0 0 0 0

0.1 115 0 0 0 0 0 0

0.1 115 0 1000 0 0 0 0

3 0 5 5 0 58 60 69 82

3 115 5.5 0 23 40 50 80

3 115 5.5 1000 0 0 0 0

3 0 0 0 0 0 0 0

3 115 0 0 0 0 0 0

3 115 0 1000 0 0 0 0

 

140

 

disorder incidence (Table 2). Fruits at 3°C with 5009 of dry lime-packets to absorb
CO2 in the chambers with 3% 002 + 1.5% 02 significantly reduced the disorder
index. 1000 mg-L'1 DPA completely controlled the Empire disorder. Fruits stored
at 1.5% 02 without CO2 for 30d prior to CA with 3% CO2 + 1.5% 02 or 5% CD2 +

1.5% 02 did not show the disorder (Table 2).

Table 2. The effect of carbon dioxide and diphenylamine (DPA) on the
development of Empire disorder at 3°C. Disorder index (0=none; 1=slight;
2=moderate; 3=severe) was normalized to 100.

 

Disorder index

 

Treatments 30d+7d at 50d+7d at
20°C 20°C

 

Mean SE Mean SE

 

Control-air 0 0 0 0
0%COZ+1 .5%O2 0 0 0 0
3%COZ+1.5%O2 47.7 3.7 49.7 5.4
5%COZ+1.5%O2 71.3 6.8 73.0 9.2
0%COZ+1.5%O2 for 30d prior to 3%COz-I-1.5%O2 - - 0 0
0%COZ+1.5%O2 for 30d prior to 5%COz-I-1.5%O2 - - 0 0
3%COz-l-1.5%Oz+Lime-Package 1 1 .7 2.1 12.3 3.3

'5%co,+1.5%o,+oPA (1000 mg-L“) o o o o

 

141

 

 

Main experiment:

Establishing CA conditions of 5% CO2 + 1.5% O2 at 3°C within 1 day of harvest
resulted in the highest incidence and severity of the Empire disorder followed by 3%
CO2 + 1.5% 02. The incidence of the disorder was decreased when the level of 02
was increased from 1.5% O2 to 3% 02(Figure 1). After acclimatizating the fruit for
two weeks, the scald-like disorder index for fruits stored at 5% CO2 + 1.5% O2
reduced 86% while disorder index for fruits stored at 3% CO2 + 1.5% O2 reduced
94% comparing to the control (Figure 1). The incidence of the disorder did not
increase after removal from storage at 3°C to 20°C for 7 days (Figure 1A,1B).
Acclimatizating the fruit in air at 3°C before CA storage attenuated or prevented the
disorder. The fruits had to be acclimated at 3°C for 3-4 weeks at 1.5 or 3% O2 to
become insensitive to 5 or 3% C02. Holding the fruits in air for 1 week prior to
administering 3 or 5% CO2 with 1.5% 02 during storage was insufficient to control
the disorder whereas the fruits tolerated 3 or 5% CO2 after holding the fruits in air
for 2 weeks(Figure 1A,1B). But the 2 week delay to CA resulted in accelerating
flesh softening (Figure 2). Supplemental carbon dioxide was not necessary for at
least 1 month to maintain flesh firmness at initial firmness values when fruits were
kept at 1.5% 02. At 3% 02 the presence of 3 or 5% CO2 from the beginning of the
storage period was required to retard flesh firmness decrease. Fmits stored for the

entire storage duration without CO2 softened markedly (Figure 2A,ZB).

142

 

 

 

(A)

1oo____L___LL LL ,LL Lme,- I L I L , _ L,

80—7 3.0%C0,+1.5%02 7
604 I

40l
207 i
1081—44 ’ 47444——

4..I._.__4.J_ 1 g I

30; 5. 0%co, +1. 57.0

604 L
404 g
20 '- 1 I
708— 4444

% I i I __ ____.,L,I_,

 

4 4T, Tz—‘II‘ i—‘i‘ j' “m 7’ r

 

 

80 I 3.0%co,+3.0%o,
601
40 1
20 I

L,
F

‘ I

i _ I
_ ——T“——‘i——_T—‘ I Y r n “R

1 08 L 1 1 I I , I I ’1 ”I

Disorder index

  
   
   

 

JL 1 1._

303 5.0%co,+3.0%o,

604
404

20:].
OI—~ l-H——_— I

0 3.5 14 28 56 7 14 150/03 0% All‘
Days at lowO before CA DatyséCA In air 0 only Only

 

 

 

Figure 1. (A) Effect of acclimatization atmosphere on the incidence and severity
of the scald-like disorder of Empire apples stored 6 months in CA at 3°C. Disorder
index on removal from storage. Disorder index (0=none; 1=slight; 2=moderate;
3=severe) was normalized to 100.

143

 

 

 

 

 

 

 

 

 

 

 

 

 

 

100143144 s__7_--LLL_ L- - . L . ¢ 7
804 3.0%co,+1.5%o, I

60; L

407 i

20! I
8;L__L _éH T 7 ‘- I I;

10 1 1 1 LE
804 5. 0%CO, +1. 5%O, .

a: 607:
r: . .
-— 204 L
h 4 f
a: 8* 4f
'9 10 . L
8 8071 307.00, +3. mm, L
5 609 C
404 L

201 i L

I

1081 L L . L; . f‘I—TFT” _ : .“ T L f :
aoI 5.0%co,+3.0°/.o, L

60; L

40:

: L

207 L

0! I

 

053.5 7 14 28 56 7 141..5%30%Air

Days at low O, before CA goigncfl' 02 only O—NIY

 

Figure 1. (B) Effect of acclimatization atmosphere on the incidence and severity
of the scald-like disorder of Empire apples stored 6 months in CA at 3°C plus 7 days
at 20°C in air. Disorder index (0=none; 1=slight; 2=moderate; 3=severe) was
normalized to 100.

144

 

I L4__L L I 1 1_,+

A A8 A8 A8 C3.0%CO,-I-1.5%O,

Illllu...

5. 0%CO, +1 .,5%O

 

I 1

 

 

 

 

 

i
.7. AB AB E
V
(I) i
in t 1 .
«I .. ,
r: L
I. .. ..
.c ...-.115:
II)
2 A8 A8 AB AB
LI.

 

l I I I

 

5. 0%CO, +3. 0%0,

A
8 ABC 5CD BCD CDE CDE DE

6071

40?

It.
0:

0 35 7 14 28 56 141.5%3 0% Air
Days at lowO before CA SatySéCA In air 0 only Only

.--TfiTfidt.

I

I

 

l

 

Figure 2. (A) Effect of acclimatization atmosphere on flesh firmness retention of
Empire apples stored 6 months in CA at 3°C. Flesh firmness on removal from
storage. Disorder index (0=none; 1=slight; 2=moderate; 3=severe) was normalized
to 100. The different letters at the top of the bars designate the difference at P:
0.01.

145

 

 

 

15L- I

  
  

 

 

 

 

   

 

 

 

sci A A AB AB AB AB B 3.0%CO,+1.,5%O L
I
ii ;
A AB AB ABC Bc CS.,O%CO +1 .5%0, I
I
a ll I1] I D
in
on
o
c
i: 601 A A :0,.%CO+30%0, i
g L A8 A8 ABC I
2 40— l
u.
20
0". . ' I
I .
604’, A A8 ABC 5.,0°/.co +3. 0%0, L
L CDE BCDE DEA DABCD
407
I
20L
0.1
14 28 56 14 1.5%3 .0% Air
Days at lowO before CA gagfignm air 02 only 65W

Figure 2. (B) Effect of acclimatization atmosphere on flesh firmness retention of
Empire apples stored 6 months in CA at 3°C plus 7 days at 20°C in air. Disorder
index (0=none; 1=slight; 2=moderate; 3=severe) was normalized to 100. The
different letters at the top of the bars designate the difference at P3 0.01.

146

DISCUSSION
Fruits for long-term CA storage and those destined for export must be harvested at
the preclimacteric stage of maturity to ensure delay of ripening and excessive
firmness loss to meet market requirements. Moreover, the presence of CO2 during
CA storage helps to delay ripening. However, the presence of 002 during the first
few weeks of CA storage at the low 02 level of 1 .5% required to ensure good delay
of ripening during storage while the fruits are at the optimum temperature of 3°C can
lead to high incidence of the Empire ‘scald-like’ disorder (Burmeister et al., 1994;
Burmeister and Dilley, 1995). Storage at 0-1°C, is not advisable for Empire apples
since the disorder is exacerbated and can cause another disorder described as
lntemal flesh browning which is known to be a chilling-related and COz-linked
physiological disorder. This poses a restriction on the use of CA atmospheres and
low temperature to extend the storage period of Empire apples for certain markets
and/or potentially profitable marketing windows. While establishing CA conditions
of 3 to 5% CO2 with 1.5 or 3% 02 at 3°C within one week of harvest results in good
control of ripening with high retention of flesh firmness this storage regimen can

lead to serious incidence of the Empire disorder with preclimacteric fruits.

The nature of the disorder:

We confirmed that: (1) the disorder is caused by high CO2 levels; (2) this is
exacerbated at 02 levels below 1.5%; (3) the disorder is more prevalent with
immature fruits; and (4) this affects primarily the non-red fruit surface.

Diphenylamine (DPA) at 1000 mg-L’1 has been found to control the Empire disorder

147

 

Hg 1' g.

in laboratory experiments (Tables 1,2) and in commercial CA storage experience.

DAP also controls the Braeburm browning disorder (Burmeister and Rowan, 1997).

We do not recommend that Empires be treated with DPA. The fact that
diphenylamine applied as a postharvest drench treatment controlled the disorder
suggests the involvement of free-radicals as the mechanism responsible for the
dysfunction (Burmeister et al., 1994; Burrneister and Dilley, 1995; Burrneister and

Rowan, 1997).

The role of active oxygen species:

lntemal 002 may be high enough in the hypodermal cells to cause the disorder in
conjunction with low 02 in these fnJits. Ethanol is a free-radical scavenger and its
effectiveness in diminishing the disorder supports our hypothesis that high CO2
levels at low 02 levels foster the accumulation of free-radicals through a Fenton
reaction involving hydrogen peroxide (H202) and consequential oxidative
modification of protein amino groups (Halliwell and Gutteridge, 1989; Stadkman,
1993). The Fenton reaction is: H202 + Fe” -. Fe” + OH' + H0'. Ascorbic acid,
which can serve as an antioxidant in preventing the accumulation and reactions of
free-radicals thought to be involved in scald and the scald-like disorder, is much
lower in the non-red than in the red fruit skin colored with the anthocyanidin pigment
cyanidin (Table 3). Apple cultivars with high ascorbic acid levels are generally less

prone to scald than those with lower levels (Anet, 1972, 1974; Lurie

148

 

Table 3. Ascorbate and dehydroascorbate content of scalded and non-scalded
peel tissue of Jonathan, Golden Delicious and Stayman apples.

 

 

 

Cultivar Scald Ascorbate Dehyd roascorbate Total
(mg/1009 FW)z
Jonathan - 8.9a 1 1.9a 20.8a
+ 4.5b 6.3b 10.8b
Golden Delicious - 29.2a 9.9a 39.1a
+ 17.6b 10.6a 28.4b
Stayman (Green) - 13.4a 1.1a 14.5a
_(Qreen) + 6.99 3.79 10.6b__

 

Means within columns for the same cv. followed by different letters differ at ps.05.

Table 4. Carbonyl content of Law Rome apples skin in relation to scald
development of fruits stored for 6 months in air or CA at 3% CO2 + 3% 02 at 1 or
3°C.

 

 

 

Storage Treatments Scald incidence (%) Carbonyl content (out
of storage)

0 7d nmols/mg protein
1) 1°C 3:3 control 0 c 20 b 7.80 :l: 0.38 abc
2) 1°C 3:3 + DPA 0 c 0 c 7.33 :I: 0.07 bc
3) 1°C air control 13 b 53 a 7.40 :I: 0.42 abc
4) 1°C air control + DPA 0 c 0 c 6.91 :l: 0.56 c
5) 3°C 3:3 control 0 c 27 b 8.05 :l: 1.01 ab
6) 3°C 3:3 + DPA 0 c 0 c 7.25 :l: 0.52 bc
7) 3°C air control 20 a 47 a 8.50 :l: 0.21 a
8) 3°C air control + DPA 0 c 7 c 7.08 :l: 0.45 b

 

Means within columns followed by different letters differ at ps.05.

149

 

et al., 1989; Meir and Bramlage, 1988; Gallerani et al., 1990). However, ascorbate
levels generally do not change substantially as apple fruits mature and although
fruits become less prone to scald as they mature on the tree. The Empire ‘scald-
like’ disorder affects the non-red portion of the fruit and hardly ever the portion of
the surface that is highly pigmented with anthocyanins; the ascorbic acid levels in
the red portion are higher than in the green portion of the skin (Table 3). This is
consistent with the effect of ascorbic acid as an antioxidant. It is also consistent
with the observation that the antioxidant DPA can control the Empire disorder (Table

4) and the Braebum browning disorder (Burmeister and Rowan, 1997).

Protein oxidation may be related to scald development and also to the Empire
“scald-like’ disorder. The carbonyl content in a protein is a measure of the extent
of protein oxidation that has occurred. Protein oxidation can disable proteins from
functioning as active enzymes (Chen et al., 1993; Halliwell and Gutteridge, 1989).
The carbonyl content of Law Rome apples was 4 to 4.9 nmol mg" protein at harvest
and increased to 7 to 8 nmol mg" protein during 6 months in storage (Table 4).
DPA-treated apples had a lower carbonyl content than nontreated fruits. Fruits
which developed scald had a higher carbonyl content than the non-scalded fruits
(Table 4). Fruits stored in air at 3°C had a higher carbonyl content than those

stored at 1°C and the incidence of scald was about 50% at each temperature.

The carbonyl content of air stored fruits without DPA averaged over 1° and 3°C was

7.95 nmol mg" protein while that for DPA-treated fruits was 7.0 nmol mg" protein

150

 

(Table 4). This represents an actual increase of about 1% in carbonyl content per
mg of protein. This is a significant increase in actual carbonyl content as expressed
in relation to mg of protein. Collectively, the data indicate that: (1) scald is related
to the increase in carbonyl content and (2) DPA marginally lowers the carbonyl
content while it strongly inhibits scald development. This may indicate that if only
certain proteins are prevented from becoming oxidized by DPA scald may be

controlled.

The acclimatization phenomenon:

The results of the current studies indicate that Empire apples can be acclimatized
during the first few weeks of storage at 3°C to attenuate the incidence and severity
of the ‘scaId-like’ disorder. Storage in air or at 1 .5 or 3% 0, without CO, for several
weeks before CA storage at 1.5 or 3% O, with 3 or 5% CO, reduces or prevents the
disorder. Fruits susceptible to CO,-linked disorders are known to have higher
resistance to gas diffusion than cvs. not affected (Rajapakse et al., 1990; Park et
al., 1993; Johnson et al., 1998). This favors the accumulation of CO, in the internal
atmosphere and this may contribute to disorder incidence (Lau, 1998). Moreover,
fruits acclimate better in air than at low O, levels with respect to tolerating CO,
during subsequent CA storage. Elgar et al. (1998) found that rapid establishment
of CA increases the incidence of the Braebum browning disorder. This suggests
a requirement of oxidative metabolism in acclimatization for CO, tolerance. This
may involve some aspect of ethylene action. Storage of apples of all cvs. in air

beyond about 7 days induces preclimacteric fruits to produce ethylene

151

 

autocatalytically. This is associated with induction of the ripening processes and CA
storage is much less effective in delaying subsquent ripening development during
storage. Consequently, the fruits soften and marketing of such fruits becomes
problematic. Acclimatization in air at 3°C beyond 7 days before CA is established
results in excessive flesh softening even though it does prevent the disorder
incidence. However, acclimatization at 3°C at 1.5 or 3% 0, without CO, for up to 4
weeks and then raising the CO, to 3 or 5% to gain the ripening retardation effects
of CO, largely eliminates the Empire ‘scald-like’ disorder while providing good
control of ripening. This storage regimen fits well with current CA technology
employing the use of air separators for establishing and maintaining O, and CO,

levels during CA storage.

LITERATURE CITED

Anet, E.F.L.J. 1972. Superficial scald, a functional disorder of stored apples: Vlll.
Volatile products from the autoxidation of or-famesene. J. Sci. Food Agr. 23:
605-608.

Anet, E.F.L.J. 1974. Superficial scald, a functional disorder of stored apples: XI.
Apple antioxidants. J. Sci. Food Agric. 25: 299-304.

Burmeister, D.M., Wang, Z. and Dilley, DR. 1994. The Empire ‘scald-like’ disorder-
1994 studies. Proc. Mich. State Hort. Soc. 124: 207-209.

Burmeister, D.M. and Dilley, DR. 1995. A “scald-like’ controlled atmosphere
storage disorder of Empire apples- a chilling injury induced by C0,. Postharvest
Biol. Technol. 6: 1-7.

Burmeister, D.M. and Rowan, 8.1997. Physiological and biochemical basis for the
Braebum browning disorder (BBD). In: E.J. Mitchman (ed.) Apples and Pears.
Proc. 7th lnt’l Nat. CA Res. Conf., 2: 126-131.

Chen, Z., Silva, H. and Klessig, D.F. 1993. Active oxygen species in the induction

152

 

of plant systemic acquired resistance by salicylic acid. Science 262: 1883-
1886.

Elgar, H.J., Burmeister, D.M., Watkins, CB. 1998. Storage and handling effects
on a COZ-related internal browning disorder of ‘Braeburn’ apples. HortScience
33: 719-722.

Gallerani, G., Pratella, GO and Budini, R. A. 1990. The distribution and role of
natural antioxidant substances in apple fruit affected by superficial scald . Adv.
Hort. Sci. 4: 144-146.

Halliwell, B. and Gutteridge, LC. 1989. Free radicals in biology and medicine.
Clarendon Press, Oxford. pp543.

Ingle, M. and D’Souza, M. 1989. Physiology and control of superficial scald of
apples: A review. HortScience 24: 28-31.

Johnson, D.S., Dover, OJ. and Colgan, R.J. 1998. Effect of rate of establishment
of CA conditions on the development of ‘CO, injury’ in Bramley’s Seedling
apples. Acta Hort. 464: 351-356.

Lau, O.L. 1998. Effect of growing season, harvest maturity, waxing, low 0, and
elevated CO, on flesh browning disorders in ‘Braebum’ apples. Postharvest
Biol. and Technol. 14: 131-141.

vine, R.L., Williams, J.A. Stadtman, ER. and Shacter, E. 1994. Carbonyl assays
for determination of oxidatively modified proteins. Methods Enzymol. 233, Part
C: 346-363.

Lurie, 8., Klein, J. and Ben-Arie, R. 1989. Physiological changes in diphenylamine-
related 'Granny Smith' apples. Israel J. Botany 38: 199-207.

Meir, S. and Bramlage, W.J. 1988. Antioxidant activity in 'Cortland' apple peel and
susceptibility to superficial scald after storage. J. Amer. Soc. Hort. Sci. 113:
412-418.

Park, Y.M., Blanpied, G.D., Jozwiak, Z. and Liu, F.W. 1993. Postharvest studies
of resistance to gas diffusion in McIntosh apples. Postharvest Biol. Technol. 2:
329-339.

Rajapakse,N.C., Banks, N.H., Hewett, E.W. and Cleland, DJ 1990. Development
of oxygen concentration gradients in flesh tissues of bulky plant organs. J.
Amer. Soc. Hort. Sci. 115: 793-797.

Stadtman, ER. 1993. Oxidation of free amino acids and amino acid residues in

153

proteins by radiolysis and by metal-catalyzed reactions. Annu. Rev. Biochem.
62: 797-821.

SUMMARY

Scald is a pervasive physiological disorder of apples and pears induced by storage
at refrigerated temperatures in air or long term CA. Scald has the potential to
destroy the market value and utility of millions of tons of apples and pears annually
unless the fruits are treated with a postharvest drench with diphenylamine (DPA) or
ethoxyquin along with a fungicide. Numerous countries have banned the use of

DPA and prohibit importation of fruits so treated.

Hypobaric storage at 0.05 atmosphere prevents scald of Granny Smith and Law
Rome apples only if they are placed under hypobaric conditions within one month
after harvest and held in air at 1°C; after 3 months delay, scald development is
similar to that for fruits stored in air. MHO accumulated in direct proportion to the
duration of the delay in placing fruits in to hypobaric storage. Only as little as 2
months of hypobaric storage largely prevents scald. This suggests that the effect
of hypobaric storage is primarily manifested during the first few months of storage
at low temperature by ameliorating the effect of a volatile substance produced by
the fruits. Fruits stored hypobarically for 8 months plus 4 months in air did not
develop scald, whereas, scald developed after an additional 6 months of storage

in air. This results indicate that apples stored hypobarically do not scald during

154

 

storage but may do so after subsequent and extended periods of time in a static air
atmosphere. This is consistent with the observation that fruits remain preclimacteric
while in hypobaric storage. Fruits from scald non-susceptible cvs. stored
hypobarically for 7 months also produced similar amounts of or-farnesene and MHO
upon removal to 20°C for a few days. The levels of the natural antioxidant ascorbic
acid increase as apple fruits mature whereas water-soluble antioxidants generally
decrease during storage. It is conceivable that the levels of natural antioxidants
may mitigate scald development. Ascorbic acid and other natural antioxidants may

be protected from degradation when fruits are stored hypobarically.

Ethanol vapor treatment reduced scald incidence for all scald susceptible cvs.
(Granny Smith, Red Delicious, Law Rome) examined in a dose-dependent manner;
the most effective treatment was one week exposure to 6000 |.iL-L'1 ethanol vapor.
Reducing the ethanol concentration and duration of exposure at a given
concentration reduced the effectiveness for scald control. Ethanol supplied to fruits
as a vapor is readily absorbed and metabolized by the tissue and affected fruit
metabolism. The consequences are reduced rate of ethylene production, MHO
accumulation and the production of low molecular weight volatile compounds
including hexylacetate, hexylbutanoate/ butylhexanoate, hexyl-2-methylbutanoate
and hexylhexanoate but not or-farnesene since the rate and pattern of or-farnesene
production was similar for fruits treated or not treated with ETOH vapor. When the
concentrations of ethanol vapor were up to 3750 or 8000 pL-L" and were applied

to fruits continuously for 2 months, the fruits were injured as evidenced by darkening

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of the skin color and the fruits showed some fermentation damage. The rate of
MHO production was much higher in control than for the ethanol vapor treatments
indicating that or-farnesene is not as closely related to scald development as its
oxidation product MHO. Or or-farnesene is not the limiting factor but rather its

metabolism leading to MHO production and or metabolism may be.

Increasing the intensity of low 0, stress progressively diminished the incidence and
severity of scald of Granny Smith and Law Rome fruits subsequently stored in CA
at 3% 02 whereas fruits stored at 1.5% 0, did not develop scald significantly. Fruits
held under ultra low levels of 0, resulted in ethanol production. The lntemal ethanol
levels were different since apple cvs. vary in their response to low 0, levels.
Ethanol accumulation at 0.5% O, was the highest for Granny Smith > Red Delicious
> Law Rome > ldared; at 0.25% 0,, Granny Smith and Law Rome apples
accumulated more ethanol than others. This suggest that apples may become
resistant to scald through producing ethanol. Or that the reduction of scald
incidence by the initial low 0, stress treatment may be a consequence of the
ethanol produced and/or metabolism induced. It may reduce the concentration of
some metabolites or ameliorate their action as related to scald development. To
achieve the maximum resistance to scald each cv. of apples may require a specific

and low O, level for a specific period of time. This remains to be elucidated.

Subjecting apple fruits to low 0, levels which induce ethanol production for a period

of several weeks at the beginning of the storage period was found to reduce the

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incidence of scald on fruits subsequently stored at higher 0, levels. This suggests
that some aspect of ethanol metabolism may reduce scald and this needs further

study as an alternative strategy to control scald on apples.

car-Famesene production of apples was inhibited by initial low 0, stress treatments
and initial 0, stress at the 0.25% 0, level and/or CA at the 1.5% 0, level more
strongly inhibits or-farnesene production than was observed for fruits without initial
low 0, stress and/or CA at the 3% 0, level. Fruits receiving 0.25% 0, initial stress
treatments produced less MHO than those with 0.5% 0, stress at all storage
conditions for both Granny Smith and Law Rome cvs. after 5 months of storage.
This data indicate that the 0.25% 0, initial stress treatment followed by CA at 1.5%
0, level has a stronger effect on inhibition of MHO production than does 0.5% initial
0, stress and CA at 3% 0, level. The strong relationship between the levels of O,
favoring ethanol production and reduced levels of MHO suggest that ethanol
metabolism may be directly involved in keeping MHO at low levels. Alternatively,
MHO production may involve an oxidative enzyme with a fairly high affinity for
molecular 0,. The mechanism for scald control by initial low 0, stress remains to

be resolved.

High MHO production/accumulation values were strongly associated with scald
development. Capacity for MHO production begins within the first 2 months when
fruits are stored in air. This supports a putative role of MHO in the scald disorder

but when it is a factor in subsequent scald development must be early vs late during

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storage. This is because fruits stored hypobarically within 1 months of being

harvested do not scald whereas those stored with longer delays do scald.

We conclude that initial low 0, stress of ca. 0.25-0.5% O, for 14 days at 0°C
followed by warming to 2°C for 3 days can effectively control scald of apples not
treated with DPA when subsequently stored at 1.5% O, at 0°C employing a static
CA regimen. Storage at 0.7% 0, throughout CA storage completely controlled
scald. However, some these fruits exhibited reversible evidence of anaerobic stress
and prolonging storage beyond 8 months at 0.7% 02 may have resulted in
irreversible damage and flavor quality impairment. Since the initial low 0, stress
followed by CA at 1.5% 0, controlled scald effectively and at commercially
acceptable levels this CA storage regimen may provide a safe and effective means

to store apples not treated with DPA.

Several different strategies may be useful to control scald. These may depend on
cv. and the duration of the storage period. For fruits which are to be marketed after
only 6 months in CA, using an air separator to maintain 1.5% O, with 3% CO, has
provided satisfactory control of scald. Fruits for long-term CA for 7 or more months
may require a more demanding strategy. This may include initial low 0, stress at
0.25-0.5% 0, levels for two weeks prior to CA at 1.5% O, and 3% 00,. CA at 1 to
1.5% 0, using an air separator to control CO, at 1 to 2% may provide adequate
scald control for mature fruits not yet producing significant levels of ethylene. The

commercial scale trial at the MSU CHES CA Facility of initial low 0, stress

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llama-ha

 

 

demonstrated excellent control of scald by employing this treatment regimen.

Establishing CA conditions of 5% CO, + 1.5% O, at 3°C within 1 day of harvest
resulted in the highest incidence and severity of the Empire disorder of the
treatments tested followed by 3% CO, + 1.5% 0,. The incidence of the disorder
was decreased when the level of O, was increased from 1.5% O, to 3% O,. The
incidence of the disorder did not increase after removal from storage at 3°C to 20°C
for 7 days. Holding the fruits in air for 1 week prior to administering 3 or 5% CO,
with 1.5% 0, during storage was insufficient to control the disorder whereas the
fruits tolerated 3 or 5% CO, after holding the fruits in air for 2 weeks. But the 2
week delay to CA resulted in accelerating flesh softening. This suggests a

requirement of oxidative metabolism in acclimatization for CO, tolerance.

The results of the current studies confirmed that: (1 ) the Empire scald-like disorder
is caused by high CO, levels; (2) this is exacerbated at 0, levels below 1.5%; (3)
the disorder is more prevalent with immature fruits;(4) this affects primarily the non-
red fruit surface; and (5) DPA at 1000 mg-L" controls the Empire disorder. Our
results also indicate that Empire apples can be acclimatized during the first few
weeks of storage at 3°C to attenuate the incidence and severity of the ‘scald-like’
disorder to avoid using DPA. Acclimatization at 3°C at 1.5 or 3% 0, without CO, for
up to 4 weeks and then raising the CO, to 3 or 5% to gain the ripening retardation
effects of CO, largely eliminates the Empire ‘scald-like’ disorder while providing

good control of ripening. The susceptibility of fruits to CO,-linked disorders may be

159

related to differences in resistance to gas diffusion among cvs. This storage
regimen fits well with current CA technology employing the use of air separators for

establishing and maintaining O, and CO, levels during CA storage.

 

 

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